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THE ROLE OF FATTY ACID ELONGASES IN GENE
EXPRESSION AND LIPID METABOLISM

By
YUN WANG

A DISSERTATION

Submitted to
Michigan State University
in partial fulﬁllment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Department of Physiology
2007

ABSTRACT

ROLE OF FATTY ACID ELONGASES ON HEPATIC GENE

EXPRESSION AND LIPID METABOLISM

By

Yun Wang

Fatty acid elongation and desaturation represent key metabolic steps that
determine the fatty acyl composition of complex lipids in cells. Of these two metabolic
pathways, factors controlling fatty acid elongase expression in mammalian tissues remain
poorly deﬁned. My studies established that of the 7 elongase subtypes expressed in
mammals, adult rat liver expresses mRN A encoding 4 elongase (Elovl) subtypes; ElovlS
> Elovll = Elov12 = Elovl6.

Once the hepatic proﬁle for elongase subtype expression was established, I
examined the nutritional and developmental regulation of hepatic fatty acid elongase as
well as the role of key transcription factors in the control of hepatic elongase and
desaturase expression in rat primary hepatocytes and in liver in vivo. These studies
established that hepatic elongase expression is controlled by several transcription factors;
including PPARa, SREBP-l, LXR, ChREBP and MLX. The in vivo studies show that
changes in hepatic lipid composition induced by diabetes or obesity correlated with
changes in elongase and desaturase expression.

Even though individual elongase subtypes have different substrate preferences,

tissue speciﬁc expression, and are regulated differently, the signiﬁcance of this diversity

is unknown. In an effort to better understand the impact of altered elongase expression on
hepatic physiology, I over expressed Elov12 and ElovlS in primary hepatocytes using
recombinant adenovirus technology. Overexpression of both Elov12 (Ad-Elov12) and
ElovlS (Ad-ElovlS) enhanced 20:5,n-3 conversion to 22:5,n-3, but only Elov12 stimulated
the formation of 24:5,n-3. The enhanced rate of 20:5 ,n—3 to elongated fatty acids
attenuated 20:5-mediated induction on PPARa regulated transcripts. Over expressed
Elov12, but not over expressed Elov15, also enhanced 20:5-mediated suppression of
SREBP-l nuclear content.

To gain a better understanding of the function of these enzymes in vivo, I used the
recombinant adenovirus to over express ElovlS in livers of C57BL/6 male mice. When
compared to a control virus, overexpression of Ad-ElovlS suppressed hepatic glycogen
content and lowered blood glucose. I attributed this response to suppressed expression of
several genes involved in glucose metabolism, i.e., Glut2, L-PK, PepCK. As in primary
hepatocytes, overexpressed ElovlS also suppressed several PPARa target genes. Over
expressed ElovlS also promoted changes in both the hepatic and plasma lipid proﬁle
consistent with ElovlS overexpression. Over expressed Elovl5 also signiﬁcantly
suppressed blood triglyceride during refeeding. Taken together, these studies indicate that
changes in hepatic Elov15 activity, alone, are sufﬁcient to impact both hepatic and plasma
glucose and lipid composition.

In conclusion, these studies establish a role for speciﬁc transcriptional regulatory
networks in the control of hepatic elongase gene expression and hepatic lipid
composition. Based on my overexpression studies, fatty acid elongases play an important

role in hepatic and whole body glucose and lipid metabolism.

iv

To my parents

and'
my ﬁus5anJ

ACKNOWLEDGEMENTS

This thesis is the result of four and half years of work in Michigan State
University where I have been accompanied and supported by many people. It is pleasant
that, now, I have the opportunity to express my gratitude to all of them.

First a very special thank to my major advisor Dr. Donald B. Jump. His
enthusiasm and integral view on science has made a deep impression on me. I owe him
lots of gratitude for having shown me this way of research. He could not even realize
how much I have learned from him. Besides being an excellent supervisor, Don was as
close as a family member and a good ﬁiend to me. It is my pleasure to get to know him in
my life.

I would also like to gratefully acknowledge my committee members, Dr. Dale
Romsos, Dr. Kathy Gallo, Dr. Laura McCabe and Dr. L. Karl Olson, for their monitoring
of my work and took effort in providing me with valuable suggestions and ideas on some
experiments of this thesis. I extend my thanks to Dr. Olson, Dr. Jeannine Scott and Dr.
Andrea Amalﬁtano who provide helpful guidance about the construction and application
of adenovirus and share their equipment with me.

I warmly thank Dr. Busik for her valuable advice and friendly help. Her extensive
discussions around my work have been very helpful for my study.

I owe my thanks to my lab mates, Dr. J inghua Xu, Dr. Daniela Botolin, Barbara
Christian and Dr. Olivier Demeure, for their kind help in daily research work and warm
friendship in life.

During this thesis work I have collaborated with many colleagues for whom I

have great regard, I thank Dr. Kate Claycomb and Dale Romsos for their generous supply

of liver from lean and obese mice; thank Dr. Howard C. Towle at the University of
Minnesota for generously providing recombinant adenovirus expressing SREBP-lc
nuclear form and the dominant negative MLX. For the liver histology reported in this
thesis, I would like to acknowledge technical support by: Amy S. Porter (HT) ASCP
QIHC, Kathleen A. Campbell (HT) ASCP QIHC, Ricky A. Rosebury (HT) ASCP QIHC
of the Investigative HistoPathology Laboratory, Department of Physiology. I wish to
extend my warmest thanks to all those who have helped me with my work in the
Department of Physiology. I thank Dr. Heidemann and Dr. Weber for sharing their
microscopes; thank the help from Dr. Kreulen’s lab for the use of real time PCR
equipment; thank Chris Green, Katrina Linning, Carol Hall, Dr. Xiaopeng Li for their
kind help in use of the equipment in their labs.

I also appreciate the precious friendship from Dr. Diana Ye, Dr. Weiqin Chen, Dr.
Xiaoling Dai, Dr. Wei Ni, Hui-yuan Tang, Wei Li, and Xiaohong Wang and his husband
Hui Wang. I thank them for their kind help in my life in Michigan.

I am very grateful for my dear husband Fengyuan, for his love and patience
during my PhD study. One of the best experiences during this period was the birth of our
lovely daughter Anji, who provided a joyful time to our life.

Finally, I have a deep sense of gratitude to my dear parents for their 30 years’

support without any reservation and persistent inspiration in my journey to PhD.

vi

TABLE OF CONTENTS

LIST OF FIGURES x
LIST OF TABLES xi
LIST OF ABBREVIATIONS xii
INTRODUCTION .................................................................................... 1
Chapter 1. Literature Review ........................................................................ 3
1.1 Signiﬁcance of fatty acid metabolism .......................................................... 3
1.1.1 Fatty acid metabolism ..................................................................... 3
1.1.1.1 General features of fatty acid ................................................... 3

1.1.1.2 Dietary fatty acid metabolism ................................................... 4

1.1.1.3 De novo synthesis of fatty acids ................................................ 6
1.1.1.4 Modiﬁcation of dietary fatty and endogenous fatty acids: elongation and
desaturation .................................................................................. 8

1.1.2 Fatty acid regulation of gene transcription ............................................ 15
1.1.2.1 PPARa .................................................................................................... 16

1.1.2.2 SREBP-lc ........................................................................ 18

1.1.2.3. ChREBP/MLX .................................................................. 19
1.1.2.4 Other transcriptional factor regulated by fatty acids ........................ 20

1.1.3. Fatty acid and chronic disease ......................................................... 22
1.1.3.1 .Cardiovascular disease ......................................................... 22
1.1.3 .2.Diabetes ........................................................................... 23
1.1.3.3.Inﬂammation ..................................................................... 24
1.1.3.4.Cancer ............................................................................ 25

1.2 Fatty acid elongases ............................................................................ 27
1.2.1 Elovll ...................................................................................... 31
1.2.2 Elov12 ...................................................................................... 32
1.2.3 Elovl3 ...................................................................................... 32
1.2.4 Elovl4 ...................................................................................... 34
1.2.5 ElovlS ...................................................................................... 35
1.2.6 Elovl6 ...................................................................................... 35
1.2.7 Elovl7 .................................................................................... 36

1.3 Statement of the problem and Hypothesis ................................................ 36
Chapter2. Tissue-Speciﬁc, nutritional and Development Regulation of Rat Fatty Acid
Elongases ............................................................................................. 39
2.1 Introduction ...................................................................................... 39
2.2 Methods and Materials ......................................................................... 41
2.3 Results ............................................................................................ 47
2.3.1. Tissue-speciﬁc expression of rat fatty acid elongases .............................. 47

vii

2.3.2. Dietary Effects on Hepatic Elongase and Desaturase Gene Expression ......... 47
2.3.3. Development regulation of Hepatic fatty acid elongase and desaturase gene
expression ........................................................................................... 56
2.4. Disccussion ..................................................................................... 60

Chapter 3. Regulation of Hepatic Fatty Acid Elongase and Desaturase Expression in

Diabetes and Obesity ............................................................................... 68
3.1 Introduction ...................................................................................... 68
3.2 Methods and Materials ........................................... ' .............................. 69
3.3 Results ............................................................................................ 76

3.3.1. Elongase and desaturase expression in rat and mouse liver ....................... 76

3.3.2. Role of PPARa in the control of hepatic elongase and desaturase expression..76
3.3.3. Regulation of elongase and desaturase expression in primary rat hepatocytes.79

3.3.4. Metabolism of fatty acids by fatty acid elongases .................................. 86
3.3.5. Regulation of elongase and desaturase expression in animal models of
metabolic disease ................................................................................ 90
3.4. Disccussion ..................................................................................... 103

Chapter 4. Effect of Fatty Acid Elongases on Gene Expression and Lipid Metabolism in

Rat Primary Hepatocytes ......................................................................... 112
4.1 Introduction ..................................................................................... 1 12
4.2 Methods and Materials ........................................................................ 113
4.3 Results .......................................................................................... 1 18

4.3.1. Efﬁcacy of Ad-Elov12 and Ad-ElovlS infection in rat primary hepatocytes .118
4.3.2. Effect of Elov12 and Elov15 on fatty acid metabolism in rat primary
hepatocytes ...................................................................................... 123
4.3.3. Effect of Elov12 and Elov15 on gene expression in rat primary hepatocytes 123
4.3.4. Effect of Elov12 and Elov15 on SREBPs nuclear content in rat primary

hepatocytes .................................................................................... 125
4.4. Disccussion ..................................................................................... 129
Chapter 5. Effect of ElovlS on Hepatic Function and Lipid metabolism in
Vivo .................................................................................................. 133
5.1 Introduction ..................................................................................... 133
5.2 Methods and Materials ........................................................................ 134
5.3 Results .......................................................................................... 139

53.1 .Generation of the mice overexpressing ElovlS in liver ............................ 139

5.3.2. Metabolic characterization of the mice overexpressing ElovlS in liver. . . 141

5.3.3. Alteration of glucose metabolism in Ad-ElovlS injected mice .................. 143

5.3.4. Decreased expression if genes involved in lipid metabolism in Ad-Elov15

injected mice ................................................................................... 148

5.3.5. Effect of ElovlS overexpression in hepatic and plasma fatty acid proﬁle. . 1 51
5 .4. Disccussion ..................................................................................... 15 1
Chapter 6. Conclusions and Future Directions ................................................ 160
APPENDIX ......................................................................................... 165

viii

PUBLICTIONS .................................................................................... 165
BIBLIOGRAPHY ................................................................................. 167

ix

LIST OF FIGURES

Figure 1.1 .............................................................................. 5
Figure 1.2 .............................................................................. 7
Figure 1.3 .............................................................................. 10
Figure 1.4 ............................................................................ 11
Figure 2.1 ............................................................................ 48
Figure 2.2 ............................................................................ 49
Figure 2.3 ............................................................................ 53
Figure 2.4 ............................................................................ 55
Figure 2.5 ............................................................................ 57
Figure 2.6 ............................................................................ 59
Figure 2.7 ............................................................................ 61
Figure 3.1 ............................................................................ 77
Figure 3.2 ............................................................................ 80
Figure 3.3 ............................................................................ 82
Figure 3.4 ............................................................................ 84
Figure 3.5 ............................................................................ 87
Figure 3.6 ............................................................................ 89
Figure 3.7 ............................................................................ 91
Figure 3.8 ............................................................................ 94
Figure 3.9 ............................................................................ 96
Figure 3.10 .............................................................................. 100
Figure 3.11 .......................................................................... 104
Figure 4.1 ........................................................................... 119
Figure 4.2 ........................................................................... 124
Figure 4.3 ........................................................................... 127
Figure 4.4 ........................................................................... 128
Figure 4.5 ........................................................................... 130
Figure 5.1 ........................................................................... 140
Figure 5.2 ........................................................................... 142
Figure 5.3 ........................................................................... 144
Figure 5.4 ........................................................................... 147
Figure 5.5 .......................................................................... 149
Figure 5.6 .......................................................................... 152
Figure 5.7 .......................................................................... 154
Figure 5.8 .......................................................................... 155
Figure 5.9 .......................................................................... 156

LIST OF TABLES

Table 1.1 ............................................................................................. 29
Table 1.2 ............................................................................................. 30
Table 2.1 ............................................................................................. 46
Table 3.1 ............................................................................................. 73
Table 3.2 ............................................................................................ 107
Table 4.1 ............................................................................................ 117
Table 5.1 ............................................................................................ 138

Images in this dissertation are presented in color

xi

LIST OF ABBREVIATIONS

AA: arachidonic acid

ACC: acetyl CoA Carboxylase

ACS: acyl-CoA synthetase

ACAT: acyl-CoA cholesterol acyltransferase

Ad: Adenovirus

ALA: Alpha-linolenic acid

AOX: Acyl CoA oxidase

bHLH/ZIP: basic helix-loop—helix/leucine zipper

ChoRE: carbohydrate regulatory elements

ChREBP: Carbohydrate regulatory element-binding protein
Cig 30: Cold induced glycoprotein with molecular weight about 30kDa
CNS: central nerve system

CPT: camitine palmitoyltransferase

CVD: cardiovascular disease

CYP4A: cytochrome P450

DGAT: diacylglycerol acyltransferase

DHA: docosahexaenoic acid

ASD: fatty acid desaturase 5

A6D: fatty acid desaturase 6

A9D: fatty acid desaturase 9

EFAD: essential fatty acid deﬁciency

xii

Elovl-l: fatty acid elongase family member 1
Elovl-2: fatty acid elongase family member 2
Elovl-3: fatty acid elongase family member 3
Elov1-4: fatty acid elongase family member 4
Elovl-S: fatty acid elongase family member 5
Elov1-6: fatty acid elongase family member 6
Elovl-7: fatty acid elongase family member 7
EPA: eicosapentaenoic acid

ER: endoplasmic reticulum

FAS: fatty acid synthase

GK: glucokinase

GLUTZ: glucose transporter 2

HNF-4 a: hepatic nuclear factor-4a

H&E: hepatoxylin and eosin

HMGCSZ: 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 2
LA: linoleic acid

LBD: ligand binding domain

LPK: L-pyruvate kinase

LTB4; leukotriene B4

LUC: luciferase

LXR: liver X receptor

MLX: MAX-like factor

MTE-I: mitochondial thioesterase I

xiii

MUFA: monounsaturated fatty acid

NEFA: non-esteriﬁed fatty acid

NFKB: nuclear factor KB

ORO: oil red O

PAS: Periodic acid shift

PEPCK: phosphoenolpyruvate carboxykinase
PPAR: peroxisome proliferator-activated receptor
PGEz; prostaglandin E2

PUFA: polyunsaturated fatty acids

RXRa: retinoid X receptor a

SCAP: SREBP-cleavage activating protein

SCD: Stearoyl-CoA desaturase

SREBP: sterol regulatory element-binding protein
SREs: sterol —regulatory elements

SIP: Site 1 protease

SZP: Site 2 protease

VLCFA: very long chain fatty acid

xiv

INTRODUCTION

Fatty acids affect many structural, metabolic, and regulatory components of cells.
It has become increasingly apparent that fatty acids are capable of regulating, either
directly or indirectly, the expression of numerous genes, both positively and negatively
[1-4]. The impact of fatty acids on regulatory systems is determined by the type and
quantity of fatty acid. The enzymes involved in the modiﬁcation of fatty acid structure
include fatty acyl CoA synthetases and thioesterases, fatty acid elongases and
desaturases, peroxisomal 0 oxidation (chain shortening) and phospholipid and
triglyceride lipases. Our lab has an interest in how the fatty acid elongases lead to
structural changes in fatty acids that control transcription via the control of fatty acid-
regulated transcription factors, peroxisomal proliferate activator a (PPARa) and sterol
regulatory element binding protein-1 (SREBP-l) and carbohydrate regulatory element
binding protein (ChREBP).

Four fatty acid elongases (Elovll, 2, 5 and 6) and 3 desaturases [A5 desaturase
(A5D), A6 desaturase (A6D) and A9 desaturase (A9D)] are expressed in rat, mouse and
human liver. Among these enzymes, the 3 desaturases are well studied, especially A9D.
These enzymes are regulated by hormones, development and dietary lipids [5-10]. A9D
knockout mice are resistant to dietary fat-induced hepatic steatosis and have improved
insulin sensitivity in muscle [1 1-13]. As such, this enzyme has been recognized as a drug
target for obesity treatment recently. Compared to the desaturases, however, we are still
in the early stages of understanding how fatty acid elongases are regulated and what their

roles are in physiology. Moreover, fatty acid structure plays a key role in the regulation

of transcriptional factors [14]. As such, enzymes that modify fatty acid structure are also
likely to impact gene expression. The goal of my research has been to gain information
about the regulation of the fatty acid elongase family. In addition, I wanted to determine
if there was any evidence in support or against the notion that fatty acid elongases play a

role in the control of hepatic gene transcription and lipid metabolism in vitro and in vivo.

Chapter 1

Literature Review

1.1. Signiﬁcance of fatty acid metabolism

1.1.1. Fatty acid metabolism

1.1.1.1. General features of fatty acid

1.1.1.1.1. Classification of fatty acids

Fatty acids are biochemical molecules that are composed of a hydrocarbon chain
and a carboxyl group. According to the number of double bonds in the polycarbon chain,
fatty acids can be classiﬁed to saturated fatty acid and unsaturated fatty acid. Saturated
fatty acid, such as palmitic acid (16:0), contains no double bonds in the carbon chain.
Unsaturated fatty acids, like oleic acid (18:1,n-9) contain one double bond while linoleic
(18:2,n-6) and arachidonic acid (20:4,n-6) contain more than 1 double bonds. Therefore,
unsaturated fatty acids are further classified into monounsaturated fatty acid (MU FA),
such as oleic acid, and polyunsaturated. fatty acid (PUF A) (Fig. 1.1). Depending on the
position of the ﬁrst double bond relative to the methyl end of fatty acid, PUFA can be
classiﬁed into two major groups, n-3 and n-6. Sometimes the symbol to is subtituted for

the lowercase letter 11, making it (0-3 and (0-6. These two groups of PUFA are not

convertible and have different biological ﬁmctions. Alpha(a)-linolenic (ALA, 18:3,n-3),

docosahexaenoic (DHA, 22:6, n-3) and eicosapentaenoic (EPA, 20:5, n-3) acids are
representative n-3 fatty acids. Linoleic acid (LA, 18:2, n-6) and arachidonic acid (AA,

20:4, n-6) are n-6 fatty acids (Fig. 1.1).

1.1.1.1.2. Essential fatty acids

The human body can produce all but two of the fatty acids it needs. These two
essential fatty acids are 18:2,n-6 and 18:3,n-3; both are widely distributed in plant and
ﬁsh oils. Since these fatty acids can not be made in the body from other fatty acid
substrates and must be supplied by the diet, they are called essential fatty acids. Essential
fatty acids are parent compounds of the n-3 and n-6 fatty acid series, respectively. In the
body, essential fatty acids are assimilated into membranes as complex phospholipids;
they are also used to produce hormone-like substances that regulate a wide range of
functions, including blood pressure, blood clotting, blood lipid levels, the immune

response, and the inﬂammation response [15].

1.1.1.2. Dietary fatty acid metabolism

Dietary lipids are absorbed from the small intestines and transported in the blood
to various tissues. The transport vehicles for these lipids are large lipoprotein complexes
called chylomicrons. The liver removes chylorrricron remnants ﬁ'om the circulation;
cholesterol and triglycerides are repackaged into very low-density lipoproteins (V LDLs)

and secreted from the liver for delivery to various tissues.

16:0
CH3
HOOC /\/\/\/\/\/\/\/

HOOC CH3

 

HOOC

       

  

$3311 COOH 20:511'3

Figure 1.1. Structure of fatty acids. According to the number of double bond in
the carbon chain, fatty acid can be classiﬁed as a saturated fatty acid (SFA) [Palmitic
acid,l6:0], monounsaturated fatty acid (MUFA) [Oleic acid, 18:1,n-9], or
polyunsaturated fatty acid (PUFA), such as linoleic acid (18:2,n-6), arachidonic acid
(20:4,n-6) and eicosapentaenoic acid (20:5,n-3).

Plasma lipids can be derived from the diet (chylorrricrons), liver (V LDL) or
mobilized from storage depots (adipose tissue) as non-esteriﬁed fatty acids (NEFA).
Fatty acids in complex lipids, like triglycerides, are excised by the action of lipoprotein
lipase. Plasma NEFAs are transported in the blood, loosely bound to albumin; NEFA are
taken up by multiple tissues, including the liver, adipose tissue and muscle. Fatty acid can

also be synthesized de novo ﬁom glucose [16].

When plasma fatty acids reach cell membrane, they enter cells via diffussion or
by fatty acid transport proteins, e.g., FATP or CD36 [17]. Once inside the cell, fatty
acids are rapidly converted to fatty acyl CoA by the action of fatty acyl CoA synthetases
(ACS); some members of the FATP family also can convert NEFA to Fa-CoA during the
transport process [18]. The formation of fatty acyl CoA is the rate-limiting step for nearly
all subsequent metabolic transformations of fatty acid, such as elongation, desaturation,
[i-oxidation (mitochondrial or peroxisomal), or assimilation into complex lipids.
Microsomal mono-oxidation and eicosanoid synthesis, however, utilizes the NEFA form

of the fatty acid as substrate [14](Fig. 1.2).

1.1.1.3. De novo synthesis of fatty acids

Fatty acid synthesis occurs primarily in the cytoplasm of liver, adipose tissue,
central nervous system and lactating mammary gland [19]. Among these tissues, liver has
the highest rate for fatty acid synthesis. During caloric excess, the liver converts glucose
to pyruvate. Pyruvate is converted to citrate in the mitochondrial (tricarboxylic acid
cycle), Excessive citrate (not used in aerobic respiration) is transported out of the

mitochondria to the cytosol where ATP citrate lyase converts citrate to acetyl-CoA.

    
   
    

Intracellular

   
 

   
 

 

 

 

 

 

   

[High] {FABP} ACS {ACBP} Eioosanoids‘
[N EF A] [NEFA]—->[FA-CoA] *
TE .
I LPL Elongation
(W cm Desaturation

   

 

 
 

 

I
Iﬂ-Oxidation l«——’

P i .l Neutral Lipid
'0‘” "'— Stora evs Export
Acyiation ~ g

 
 

 

 

 

 
 

 

TAG

 

 

   

 

 

   
 

HSL

  

Extracellular VLDl.

Chylomlcron

NEFA

Figure l. 2. Overview of cellular fatty acid metabolism. When the plasma fatty acid
reach the cell membrane, they are either via diffussion or by fatty acid transprot proteins
(F ATP or CD3 6) to enter into the cell. Inside, fatty acid must be activated before
proceeding through metabolism. Activation consists of conversion of the NEFA to its
CoA derivative under the control of acyl CoA synthetases (ACS). The formation of fatty
acyl CoA is the rate-limiting step for fatty acid further modiﬁcation, including
elongation/desaturation, B-oxidation (mitochondrial or peroxisomal), or assimilation into
complex lipids.

Acetyl-CoA is a substrate for acetyl CoA carboxylase 1 (ACCl) which converts acetyl-
CoA to malonyl CoA. Malonyl CoA is the rate-limiting substrate for fatty acid synthesis.
A multi-enzyme complex, called fatty acid synthase (FAS), then uses malonyl-CoA,
acetyl-CoA, and NADPH to elongate fatty acids in two-carbon increments in the cytosol
[20]. The usual end product for this reaction in rodents is palmitate (16:0) [21]. Insulin,
triiodothyronine (T3), glucocorticoids and glucose induce, while 2C18 PUFA, glucagon,

and epinephrine suppress de novo fatty acid synthesis [14].

1.1.1.4. Modification of dietary and endogenous fatty acids: elongation

and desaturation

The liver plays a central role in whole body glucose, fatty acid and cholesterol
metabolism. Once fatty acids enter hepatocytes, they are converted to their active form,
acyl CoA. They enter pathways that lead to their elongation, desaturation, B-oxidation or
assimilation into complex lipids (phospholipids, triglycerides and cholesterol esters).
Fatty acid elongation, desaturation and B-oxidation are three chemical modiﬁcations that
alter fatty acid structure. Each of these reactions occurs in most tissues. Our focus in the

following discussion is on hepatic fatty acid elongation and desaturation.

Fatty acid elongation occurs in both the endoplasmic reticulum (ER) and the
mitochondria. The elongation in ER represents the predominant pathway and malonyl
CoA is the source of the added carbons. The process resembles the elongation steps of de
novo fatty acid synthesis, but involves separate enzymes. These reactions consists of four
sequential and independent steps: 1) a condensation between fatty acyl CoA and malonyl

CoA to form 3-ketoacyl-CoA mediated by a fatty acid elongase (Elovl) [also known as [3-

acylketo CoA synthase] ; 2) a reduction of the 3-ketoacyl-CoA to form 3-hydroxyacyl-
CoA using NADPH, mediated by B-acylketo CoA reductase; 3) a dehydration of 3-
hydroxyacyl-CoA to trans-2,3-enoyl-CoA mediated by (B-hydroxyacyl CoA dehydrase);
4) a reduction of trans-2,3-enoyl-COA to acyl CoA, mediated by trans-2-enol CoA
reductase [22]. The overall reactions are shown schematically in Fig. 1.3 [23]. The
condensation step catalyzed by the fatty acid elongase (Elovl) is the regulated and rate-
limiting step for fatty acid elongation [24]. Unlike de novo fatty acid synthesis, these

reactions are separable and are not catalyzed by a multifunctional enzyme.

Mitochondrial elongation is a minor pathway and acetyl CoA is the source of the
added carbons. In the mitochondria, fatty acid elongation occurs by a reversal of [3-
oxidation, except that one NADPH and one NADH are required (IS-oxidation yields two
NADH). Mitochondrial fatty acid elongation acts primarily on fatty acyl CoA substrates
shorter than 16 carbons [25]. Desaturation of fatty acids introduces a double bond into the
fatty acid; this reaction also occurs in ER of many tissues. This reaction requires an
electron transport system involving: cytochrome b5, desaturase and NADPH- cytochrome

b5 reductase [9].

In mammals, there are 3 distinct fatty acid desaturases (A5, A6 and A9 desaturase).
Each has a different substrate requirement and generates a double bond at speciﬁc carbon
bonds relative to the carboxyl end. For example, A5-desaturase inserts a double bond
between the 5th and 6th carbon bond from the carboxyl end. A minimum chain length of
16-18 carbons is required for the desaturation, mammals can not synthesize n-6 and n-3

fatty acids de novo. Desaturation and 2-carbon elongation ﬁinction together in metabolic

Fatty acyl-CoA

 

Condensation

 

 

 

Elongase

3-Ketoacyl-CoA

 

Reduction

 

 

 

KAR

3-HydroxyacyI-CoA

 

 

Dehydration

 

 

Dehydratase

Trans-2,3-enoyl-CoA

 

 

 

Reduction I TER

Elongated Fatty acyl-CoA

Figure 1. 3. Microsomal fatty acyl elongation. The elongation in ER represents the
predominant pathway and malonyl CoA is the source of the added carbons. The process
consists of four sequential but independent reactions: 1) a condensation between fatty
acyl CoA and malonyl CoA to form 3-ketoacyl-CoA ; 2) a reduction of the 3-ketoacyl-
CoA to form 3-hydroxyacyl-CoA using NADPH; 3) a dehydration of 3-hydroxyacyl-COA
to trans-2,3-enoyl-COA; 4) a reduction of trans-2,3-enoyl-CoA to acyl CoA KAR, 3-
ketoacyl-CoA reductase. TER, trans-2,3-enoyl-CoA reductase.

10

Elovl6

1.4A q 1620 —> 1830

491;] I

16:1 —" 18:1
1-43 Elov15&6

Diet

N6; 18:2 —>18:3 ->20:3 ->20:4 ->22:4 ->24:4 ->24:5 ->22:5
A60 Elovl5 A50 Elov12 Elov12 A‘D pﬁ-Ox
N3: 18:3 —> 18:4 ->20:4 ->20:5 ->22:5 ->24:5 ->24:6 ->22:6

 

 

 

Essential
Fatty Acids

Figure 1. 4. Scheme of endogenous fatty acid synthesis pathway. 4A described the de
novo fatty acid synthesis from glucose. ElovlS, Elovl6 and A9-desaturase (A9D) are
involved in this pathway. The end products of this pathway (16:0, 16:1 and 18: l)
accumulate in tissues. 4B illustrates the polyunsaturated fatty acid synthesis pathway
from essential fatty acids. 18:2n6, and 18:3n3 are converted to long chain unsaturated
fatty acids in vivo by a series of elongation (Elov12 & -5) and desaturation (As-desaturase
[A5 D] and A6 -desaturase [A6D] reactions that take place in the endoplasmic reticulum and
the peroxisome via ﬂ-oxidation (pﬂ-Ox)

11

pathways to generate polyunsaturated fatty acids. As illustrated in Fig. 1.4, these two

chemical modiﬁcations often alternate in the synthesis of long chain PUFA.

The predominant saturated and monounsaturated fatty acids synthesized in
mammals are long chain fatty acids (Fig. 4A). The major products of this pathway are
palmitate (16:0), palmitoleate (16:1,n-7), oleate (18:1,n-9) and vaccinate (18:1,n-7).
Mead acid (20:3,n-9) can arise from this pathway when essential fatty acids (18:2,n-6 and
18:3,n-3) are deﬁcient or when animals are fed olive oil and treated with peroxisome

proliferators, like WY14,643 [26].

Stearoyl-CoA desaturase (SCD), also known as A9 desaturase, is the rate-limiting
enzyme catalyzing the synthesis of 18:1 and 16:1. 18:1 is the preferred substrate for acyl-
CoAzcholesterol acyltransferase (ACAT) and diacylglycerol acyltransferase (DGA'I), the
enzymes responsible for cholesteryl ester and triglyceride synthesis, respectively [27]. In
addition, 18:1 is the major MUFA in human adipose tissue and in the membrane
phospholipid of most non-neuronal cells. The ratio of 18:0 to 18:1 has been implicated in
the regulation of cell growth and differentiation through effects on membrane ﬂuidity and
signal transduction [10]. MUFAs also inﬂuence apoptosis and may have some role in
mutagenesis of some tumors [28-30]. Overall, these fatty acids are important components
of phospholipids; they also represent the largest energy storage reservoir in the form of

triglycerides, and are the preferred fatty acids used for the esteriﬁcation of cholesterol.

A second pathway is for synthesis of unsaturated fatty acids utilizes the essential

fatty acid precursors (LA, 18:2,n-6 and ALA, 18:3,n—3) to form the long-chain derivatives

12

arachidonic acid (AA, 20:4,n-6) and docosahexaenoic acid (DHA, 22:6,n-3), respectively
(Fig. 1.4B). The ﬁrst part of this metabolic pathway takes place in the endoplasmic
reticulrun and consists of sequential alternating elongation and desaturation steps
catalyzed by fatty acid elongase-2 (Elov12) and -5 (Elov15) and the desaturases, A5-
desaturase (A5D) and A‘s-desaturase (A6D). The A6-desaturase is the rate-limiting step of
the pathway. A part of the pathway that applies only to the synthesis of DHA (22:6,n-3)
involves the elongation of eicosapentaenoic acid (EPA, 20:5,n-3) to 22:5,n-3 and 24:5,n-
3 by Elov12, followed by desaturation of 24:5,n—3 by A6D to form 24:6,n-3. 24:6,n-3 is
then chain shortened in the peroxisome, i.e., peroxisomal B-oxidation, to form 22:6,n-3.
These pathways, deﬁned by Sprecher and his colleagues [31], are the only metabolic
routes to generate AA (20:4, n6) and DHA (22:6, n3) from dietary precursors, 18:2,n-6
and 18:3,n-3 (Fig. 1.4 B). 18:2,n-6, 20:4,n-6 and 22:6,n-3 are the major PUFA

accumulating in membranes of all cells.

Of the two essential fatty acids (18:2,n-6 and 18:3,n-3), linoleic acid (18:2,n-6) is
the predominant essential fatty acid in the diet. Dietary abundance, coupled with the fact
that much of dietary 18:3,n-3 is B-oxidized limits the availability of 18:3,n-3 for PUFA

synthesis [32]. As a result, little 18:3,n—3 accumulates in tissues, appears in the blood or

is converted to long-chain PUFA.

The products of these reactions provide substrates for complex lipid synthesis;
such as: 1) incorporation into phosphoglycero- and phospho-ether lipids in membranes
(AA and DHA), neural tissue is enriched in DHA. Little AA and DHA appear in

sphingolipids; 2) substrates for eicosanoid (prostaglandins, thromboxanes, leukotrienes &

13

prostacyclins are derived from 20:4,n-6) and docosanoid synthesis (resolvins &
protectins are derived from 22:6,n-3); 3) B-oxidation for energy metabolism; 4) Stored as
neutral lipids, triglycerides and cholesterol esters. AA, but little DHA, appears in
cholesterol esters (unpublished observation). Cunnane’s laboratory used isotope tracer
and other methods to show that membrane lipids in liver and brain preferentially use
exogenous long chain PUFAs rather than those produced endogenously [33]. AA that is
not incorporated into membranes may be used by eicosanoid metabolism which requires
a readily available supply at all times. AA is the major precursor for the synthesis of
eicosanoids, which are powerful cellular regulatory substances and mediators of
inﬂammation, platelet aggregation, T-cell proliferation, lymphocyte migration,
vasoconstriction and dilation, and the production of several immune and inﬂammatory
substances [34]. EPA (20:5,n—3) is a poor substrate for cyclooxygenase-l and -2.
Eicosanoids derived from 20:5,n-3 typically have weak activity when compared to the
AA-derived eicosanoids [17,35,36]. Thus, EPA can moderate the production and activity
of AA-derived eicosanoids. It is believed that eicosanoids derived from n-6 fatty acids
contribute to the development and deterioration of several chronic diseases [3 7-40]. DHA,
which is the end product of the n-3 PUFA pathway, is the most abundant n-3 PUFA in all
tissues. It is critical for the development of central neural system and retina [41-43].
Deﬁciency of DHA causes a similar phenotype to essential fatty acid deﬁciency,

including severe macular dystrophy and impaired cognitive function.

14

1.1.2. Fatty acid regulation of gene transcription

Fatty acids regulate the expression of numerous genes, both positively and
negatively [14,44-46]. Fatty acids directly bind to and regulate several nuclear receptors,
including members of the peroxisome proliferator-activated receptor (PPAR) family
(PPARa, 0/8, 71 & 72), liver X receptor (LXRu, but not LXRB), retinoid X receptor a
(RXRu) , hepatic nuclear factor-4a (HNF-4 a) [47-55]. In this case, fatty acids act like
hydrophobic hormones to control the activity of these key transcriptional factors. Of
these, however, the PPAR family are generally considered bona ﬁde in vivo targets of

fatty acids [14].

In contrast, fatty acids also regulate other transcriptional factors through indirect
mechanism by changing their nuclear abundance or activity. These transcription factors
include sterol regulatory element binding protein-1 (SREBP-l, a & c) [56], MAX-like
factor-X (MLX), carbohydrate regulatory element-binding protein (ChREBP) complex
[55,57] and nuclear factor KB (NFicB) [14]. In vivo studies with rats and mice have
established that hepatic PPARa, SREBP-l, ChREBP and MLX are target for control by

dietary fatty acids [5 8-61 ].

PPARa induces fatty acid oxidation, whereas SREBP-l induces fatty acid
synthesis. ChREBP/MLX induce glucose utilization for fatty acid synthesis [55,57,62].
These mechanisms control, not only hepatic lipid metabolism, but also whole body lipid
metabolism by affecting VLDL composition and secretion. As such, these regulatory

pathways may contribute to the onset and progression of chronic disease, such as

15

atherosclerosis, diabetes and obesity [44,63]. This review will mainly focus on the three

transcriptional factors, PPARoc, SREBP-l and the ChREBP/MLX heterodimer.

1.1.2.1 PPARa

PPARa is the predominant PPAR subtype expressed in liver. It can be activated
by PUFA and their oxidized derivatives as well as by hypoliporrric drugs of the ﬁbrate
family, including fenoﬁbrate or gemﬁbrozil [51]. PPARa plays a role in the regulation of
an extensive network of genes involved in glucose and lipid metabolism [58,64-67].
PPARa, as well as other PPARs, bind DNA as a heterodimer with RXR at direct repeat in
the 5’ ﬂanking regions of many genes, such as camitine palmitoyltransferase (CPT), acyl-
CoA oxidase (AOX), and fatty acyl-CoA synthetase-1 (ACSl) [68-70]. Like other nuclear
receptors, ligand binding recruits co-regulators to the promoter and changes gene

transcription [71].

PPARa was the ﬁrst transcription factor identiﬁed as a prospective fatty acid
receptor [51]. PUFAs are recognized as the PPARa activators [53]. This ﬁnding parallels
the metabolic ﬁndings that PUFA induce fatty acid oxidation [72]. In cells, non-esteriﬁed
fatty acids (NEF A) serve as PPARa ligands. Since NEFA bind PPARa with different
afﬁnities, the composition of intracellular NEFA is an important determinant in the
control of PPAR activity. Intracellular NEFA composition is affected by the concentration
of exogenous fatty acids entering cells, their rate of removal via acyl CoA-synthetase—
dependent and -independent mechanisms, e.g. microsomal mono-oxygenation, and the
return of NEF A or oxidized lipids to the NEFA pool as a result of lipid metabolism [14].

The relative abundance of several putative fatty acid ligands for PPARa in rat primary

l6

hepatocytes is 20:4,n-6 = 18:2,n-6 = 18:1,n-9 > 22:6,n-3 > 18:3,n-3 or n-6 = 20:5,n-3 [53].
Challenging cells with 18:1,n-9, 18:2,n-6 or 20:4,n-6 has little impact on the mass of
these fatty acids in the NEFA fraction of cells. In contrast, the addition of 20:5,n-3,
22:5,n-3 and 22:6,n-3 to hepatocytes signiﬁcantly enriches the NEFA fraction of cells
with these fatty acids. Of the 3 n-3 PUFA, 20:5,n-3 signiﬁcantly induced PPARa activity
and PPARa target genes, like AOX and CYP4A [53]. Structural analysis of PPARB
indicated that the carboxyl end of fatty acid inserts into the hydrophobic pocket of ligand
binding domain (LBD) [73]. 20:5,n-3 and 22:6,n-3 have a similar structure near the
carboxyl end of carbon chain. However, elongation of 20:5,n-3 to 22:5,n-3 increases the
rigidity of the fatty acid, which may inﬂuence its binding to PPARa [14]. This indicates
that major changes in intracellular NEFA composition and mass alone may not be
sufficient to trigger a PPARa response. Fatty acid structure may be a key factor in the

control of PPARa activity.

Although PUFA are weak agonists of PPAR compared with pharmacological
agonists (e.g., ﬁbrates and thiazolidinediones), PUFA have signiﬁcant effects on cell
physiology. PUFA-mediated induction of PPARa-regulated genes shifts hepatic
metabolism away from lipid synthesis and storage toward lipid oxidation [74,75]. It also
affects insulin sensitivity in various tissues, particularly skeletal muscle [76]. As such,
PUFA composition of cells will likely impact gene expression and sensitivity to key

hormonal regulatory systems.

17

1.1.2.2. SREBP-l

SREBPs are basic helix-loop-helix-leucine zipper (bHLH-LZ) transcription
factors involved in cholesterol, fatty acid and complex lipid synthesis [77]. SREBPs bind
DNA cis-regulatory elements called sterol-regulatory elements (SREs) as dimers. There
are two separate genes encoding the SREBP-1 and SREBP-2. SREBP-1 regulates the
expression of genes involved in fatty acid and triglyceride synthesis, while SREBP-2
regulates the expression of genes involved in cholesterol synthesis [78]. Two isoforrns of
SREBP-1 have been described. These isoforms, SREBP-1a and SREBP-1c, arise from
differential promoter and exon usage. SREBP-1c is the predominant SREBP-l isoforrn
expressed in rodent and human liver. Despite its greater abundance, SREBP-1c is
reported to be the weaker of the two transcription factors in activating gene transcription.

This is because SREBP-lo lacks a 29 acidic rich arrrino acid region present in SREBP-1a.

When SREBPs are ﬁrst translated as large precursor proteins (~125 kd). These
proteins are tethered to the endoplasmic reticulum (ER). SREBP binds a second
membrane protein SREBP-cleavage activating protein (SCAP) at its C-tenninal end. A
third family of proteins, INSIGs, interacts with SCAP in the endoplasmic reticulum.
Depletion of sterol from cells leads to the movement of the SCAP-SREBP complex ﬁom
the endoplasmic reticulum to the Golgi. SCAP plays an escort role for this movement,
while INSIG interacts with SCAP and prevent this movement when intracellular sterols
are high [79,80]. Two proteases, called Site 1 protease (SIP) and Site 2 protease (SZP)
are present in the Golgi. These proteases cleave SREBP precursors to release the mature

nuclear form of SREBP (nSREBP, ~65 kd) from the Golgi. SREBPs dirnerized and bind

l8

to importin-B for transport to the nucleus. Once in nuclei, SREBP bind as dimers at SRE
in promoters of target genes. SREBP binding to SREs promotes recruitment of co-
activators, e.g., CBP, RNA polymerase II and initiates gene transcription. There are no
reports of SREBP functioning as receptors for either cholesterol or fatty acids. As such,
genes regulated by SREBPs are controlled by factors affecting the nuclear abundance of

SREBP.

In the liver, SREBP-2 is more sensitive to the cholesterol mediated inhibition than
SREBP-1. In contrast to SREBP-2, the nuclear content of SREBP-1 is suppressed by
unsaturated fatty acids. This phenomenon is observed in livers of animals fed PUFA-
containing diets as well as in primary hepatocytes and some, but not all, SREBP-1c-
expressing cells lines treated with PUFA [74,81]. The effects of PUFA on SREBP-1
levels are very complex. It has been attributed to inhibition of transcription of the
SREBP-1c gene as well as enhanced turnover of the mRNA encoding SREBP-1,
interference with the SREBP-1 processing, and enhanced proteasomal degradation
[61,82]. The effect of 20-22 carbon PUFA on SREBP-1 mRNA and gene transcription is
similar. 22:6,n-3, however, has an added impact on controlling SREBP-1 nuclear content.
22:6,n-3enhances 26S proteasomal degradation of nuclear SREBP-1, but not the SREBP-
1 precursor or nuclear SREBP-2 [83]. As such, 22:6,n-3 is the most potent fatty acid

suppressor of SREBP-1 nuclear abundance and SREBPl-mediated gene expression.

1.1.2.3. ChREBP/MLX

ChREBP and MLX are bHLH-LZ transcription factors. They bind as

heterodimers to carbohydrate regulatory elements (ChoRE) located in promoters of

19

glucose-responsive genes, like L-type pyruvate kinase (LPK), ACC, FAS, 814, SCD-l
and GLUT2 [62,84-86]. Both our group [55] and the Postic group [57] have reported that
components in the ChREBP/MLX heterodimer are regulated by PUFA in liver and
primary hepatocytes. However, the results from these two groups are not identical.
Postic’s group indicated that PUFA suppressed ChREBP activity by enhancing ChREBP
mRNA decay as well as inhibiting its nuclear translocation. Our group reported that n-3
PUFA have no effect on ChREBP nuclear abundance, but suppressed MLX nuclear
abundance. This controversy may come from the different animal species as well as the
experiment design. Whatever the case, ﬁnding that PUFA control ChREBP/MLX
nuclear abundance provides an explanation for those fatty acid-regulated hepatic genes

that were independent of PPARa and SREBP-l [5 5].

1.1.2.4. Other transcription factors regulated by fatty acids

1.1.2.4.1 Liver X receptor (LXRa)

LXRs were ﬁrst identiﬁed as fatty acid regulated transcriptional factors by Brown
and Goldstein group [87]. LXRs, like PPARs, belong to the steroid-thyroid receptor
superfarnily. There are two subtypes, LXRa and LXR B in this family. LXRa is the
predominant one expressed in liver. LXRs bind oxysterols and regulate the expression of
genes involved in hepatic bile acid synthesis, reverse cholesterol synthesis, lipogenesis
and glucose uptake [81,88,89]. PUFA antagonize oxysterol activation of LXRu in HEK
293 and hepatoma cell lines by interfering with oxysterol binding [54,87]. However, a
recent study from our group showed that LXR-regulated transcripts, such as CYP7A,

ABCAl, ABCGS and ABCG8, were not sensitive to PUFA suppression in rat primary

20

hepatocytes and in liver [52]. The condition used to access PUFA control of
transcriptional factors in this study was sufficient to suppress SREBP-1c and induce
PPARa targeted genes. These observations raised doubt as to whether LXRa were bona

ﬁde targets for PUFA control in vivo.

1.1.2.4.2 Hepatic nuclear factor-4 (HNF4)

HNF 4 is an orphan nuclear receptor regulating an extensive network of genes
controlling glycolysis and lipogenesis in liver and other tissues [14]. Few of these genes
are regulated by dietary PUFA in vivo except L-PK, glucose 6 phosphatases,
apolipoproteins A1 and C111. Our laboratory ﬁrst reported that HN F -4 recognition
sequence is a component of the PUFA-response region of L-PK gene [90]. Later, the Bar-
tana group showed evidence that HNF4a bound fatty acyl CoAs. They suggested that
fatty acyl CoA might account for the effect of fatty acids on transferring and Apo CIII
promoter activity [47]. However, structural studies on HNF-4 protein indicated that fatty
acids and not fatty acyl CoA co-crystallized with HNF-4 ligand binding domain. These
ﬁndings triggered controversy over HNF-4 ligands. Recent studies from our laboratory
showed that 20:5,n-3 treatment in rat primary hepatocytes promotes transient change
HNF-4u—LPK promoter interaction, but no effect on HNF4 transactivation on LPK
promoter [55]. 20:5, n-3 is a minor fatty acid in the pool of NEFA and esteriﬁed lipid
fraction of liver and hepatocytes [83]. Greater than 99% of intracellular 20:5,n-3 was
esteriﬁed after hepatocytes were treated. Intracellular non-esteriﬁed 20:5,n-3 is highest at
1.5 and lowest at 24 hours after challenging cells with 20:5,n-3 [55]. This suggest that

non-esteriﬁed 20:5,n-3 can regulate HNF-4 binding activity to LPK promoter. The

21

mechanism for this control, however, is unknown. In the view of the Jump lab, this

control does not involve direct binding of 20:5,n-3 by I-INF-4.

This discussion of fatty acid regulated transcription factors highlights those
factors that are well-established targets for PUFA control, i.e., PPARa and SREBP-1,
and those that are either recently identiﬁed (ChREBP/MLX) or there is some controversy
(LXRor and HNF-4a). An underlying fact is that the type and quantity of intracellular
fatty acid controls these transcription factors and their regulatory networks. This thesis
focuses on a metabolic pathway (fatty acid elongation) that alters the type of fatty acid in
the cells. I am predicting that changes in fatty acid elongation will impact certain

transcriptional regulatory networks.

1.1.3. Fatty acid and chronic disease

1.1.3.1. Cardiovascular disease

Numerous studies have revealed the beneﬁts of n-3 PUFA on cardiovascular
disease (CVD) [91-93]. N-3 PUFA can be obtained ﬁom two dietary sources: seafood
and certain nut and plant oils. Fish or ﬁsh oil contains EPA and DHA, whereas canola
and walnut oil contain ALA. ALA is less potent than EPA and DHA. Mechanisms by
which n-3 fatty acids reduce CVD risk are under active investigation. Research to date,
however, suggests there are several mechanisms involved in the protective effect of

PUFA on cardiovascular health.

22

Evidence ﬁ'om epidemiological studies and clinical trials suggest that n-3 PUF A
have an important anti-arrhythmic effect in patients with CVD [94,95]. However, the
details of the anti-arrhythmic action of n-3 PUFA remain to be elucidated. In addition, n-
3 PUFA is reported to decrease risk for thrombosis due to decreases in platelet
aggregation. A decrease in thromboxane (TXAz) coupled with increases in prostaglandin
(PG12 and PGI3) production, decrease in whole blood viscosity and an increase in
bleeding time [96]. As described above, eicosanoid metabolic products from AA (n-6
PUFA) are more biologically active than those derived from EPA (n-3 PUFA) [14].
Supplementation of n-3 PUFA will replace n-6 PUFA as the precursor of eicosanoids,
while increasing the amount of n-6 PUFA consumption contributes to the development of
thrombi and athomas. PUFA is also reported to decrease triglyceride and remnant
lipoprotein levels by replacing saturated fat in diet. Additional beneﬁt is shown by n-3
PUFA which consistently lowers blood triacylglycerol levels in hypertriglyceridemic
patients. In contrast, n-6 fatty acids either have no effect or increase blood triglycerides
[97]. In addition, PUFA can decrease rate of growth of the atherosclerotic plaque by

improving endothelial function and reducing inﬂammatory responses [98].

1.1.3.2 Diabetes

Type 2 diabetes (non-insulin dependent diabetes melittus, NIDDM) is the most
prevalent form of diabetes in western societies. NIDDM is a metabolic disorder
characterized by hyperglycemia in the presence of insulin resistance and
hypertriglyceridemia. Skeletal muscle is the principal site of insulin-mediated glucose

disposal. Campbell and colleagues showed that insulin resistance and hyperinsulinemia

23

are inversely associated with the amount of C20 and C22 fatty acids in the phospholipids
of muscle cell membranes in patients with coronary heart disease and normal volunteers
[99]. Increasing the amount of C20 and C22 PUFA in membrane leads to an increase in
membrane ﬂuidity, insulin receptor and insulin action [100]. High dietary intake of
saturated and trans fat interferes with the elongation and desaturation of essential fatty
acids to AA, EPA and DHA. These fatty acids also contribute to the development of
insulin resistance [100,101]. Skeletal muscle triglyceride levels are also inversely related
to insulin action [102]. Clinical trials of dietary supplemention of n-3 PUFA to type 2
diabetic patients lowers triacylglycerol concentrations with no adverse effects on glucose

control [103].

Leptin is a hormone that suppresses appetite and enhances basal metabolic rate.
As such, it has a remarkable effect on the regulation of body fat [104]. It is reported that
serum leptin levels are inﬂuenced in patients with type 1 diabetes by the type of fat in the
diet [105]. In particular, n-3 PUFA has been found to decrease leptin gene expression

both in vivo and in vitro [106].

1.1.3.3 Inflammation

Inﬂammation is characterized symptomatically by pain, redness, and swelling.
The disordered or excessive inﬂammation also contributes to the loss of function. This
results from the release of inﬂammatory mediators, predominantly from activated
leukocytes that migrate into the target area. Among the key inﬂammatory mediators are
prostaglandin E2 (PGEz) and leukotriene B4 (LTB4), which are derived from the n-6

PUFA, AA [107]. Eicosanoids derived from AA and EPA have very similar molecular

24

structures, but very different biologic effects. Those derived ﬁom EPA are generally
much less potent in inﬂammation than those derived from AA. As such, a majority of n-6
fatty acids will result in a proinﬂarrrmatory status by producing prostaglandins of the :2
series and leukotrienes of the 4 series. As the ratio of n-3 to n-6 increases, m0re
prostaglandins of the 3 series and leukotrienes of the 5 series are produced. As such, n-3
fatty acids exert anti-inﬂammatory effects via competitive inhibition of the n-6-derived

proinﬂammatory eicosanoids [108].

The immune response also plays a role in the development of inﬂammation by
producing immunologic mediators such as cytokines. PUFA have been shown to have
effects on immune response [109]. Dietary supplementation of n-3 fatty acids inﬂuences
the cytokine production and lymphocyte proliferation. This effect is more dramatic on

older versus younger women.
1.1.3.4 Cancer

Cancer is a disease characterized by abnormal cell division without any control.
Many cancers have a high mortality rate. Epidemiological studies indicate that women in
countries with high-fat diets have a higher risk (5-fold) of breast cancer than women in
countries with low-fat consumption. Such observations strongly suggest that a high intake
of dietary fat could increase breast cancer risk [110,111]. This also provides evidence that
dietary or exogenously derived fatty acids may play an important role in the
carcinogenesis, evolution and/or progression of breast cancer. Several studies also
showed that diets containing corn oil, with high levels of n-6 PUFA, enhance breast and

colon tumorgenesis in rodents. In contrast, diets enriched in n-3 PUFA, e.g., ﬁsh oil,

25

reduces carcinogenesis [112]. Saturated fatty acids have no discernible effect on
mammary carcinogenesis or progression. Several mechanisms were proposed to explain
the mechanism by which the intake of n-3 PUFA might lower the risk of cancer.
Inhibition of eicosanoid biosynthesis from AA is the most important one among these
mechanisms. High intake of n-6 PUFA experimentally induces various physiological and
metabolic effects [112,113]. It can increase ornithine decarboxylase activity in colon
mucosa which leads to the enhanced epithelial polyamine levels and increase colon crypt
cell proliferation. It can also up-regulate activities of protein kinases, such as protein
kinase C in rodent mammary gland and increases number of estrogen receptor binding
sites. Prostaglandins, thromboxanes, leukotrienes and hydroxy and hydroxyperoxy fatty
acids are involved in tumor initiation and promotion, cell proliferation, tissue invasion
and metastatis. Tumor cells produce more eicosanoids than their normal cell counterparts;
oxidized lipids ultimately derived from linoleic acid have been linked to increased cell
growth and tissue metastasis. The ﬁnding that oleic acid and n-3 PUFA, speciﬁcally EPA,
inhibits the desaturation reaction, the ﬁrst step from linoleic acid to arachidonic acid and
eventually to eicosanoid production, may partially explain their inhibitory effects on

turnorigenesis.

Clearly, the impact of dietary fat on chronic disease is signiﬁcant. What is less
clear is how chronic disease might impact enzymes involved in fat metabolism. Equally
unclear is how changes in these enzymes impact cellular fat composition and impact the
onset and progression of these diseases. This thesis will begin to address these important

medical issues.

26

1.2. Fatty acid elongases

The focus of this thesis is on fatty acid elongases. This section will review our

current understanding to the role these enzymes play in lipid metabolism.

Fatty acid elongases (Elovls) are the condensing enzymes that interact with 3-keto
acyl-CoA reductase, a dehydratase and trans-2,3-enoyl-COA reductase to elongate fatty
acids (Fig. 1.3). The rate of fatty acid elongation is determined by the activity of elongase
(condensing enzyme). There are 7 distinct elongase subtypes (Elovl-l, -2, -3, -4, -5, -6 &
-7) present in the mouse, rat and human genomes. Each has a speciﬁc substrate
preference in terms of chain length and the degree of fatty acid unsaturation. The
members of this family are predicted to have 5 or 6 transmembrane spanning domain that
traverse the endoplasmic reticulum. Since the third transmembrane stretch is very long, it
is not clear whether 5 or 6 transmembrane domains in this family protein [114]. Also, this
protein family share a common structure of a single histidine-box motif (HXXHH), and

an ER retention signal (KKXX-like) [115].

Elovl3 was the ﬁrst elongase to be identiﬁed by Jacobsson group in 1997 [116]. It
was originally named cig30 (Cold induced glycoprotein with molecular weight about
30kDa) because the gene can be induced signiﬁcantly in brown adipose tissue when the
mice are exposed to cold environment. In silica search identiﬁed two homologues of
cig30, which were ﬁrst termed Sscl and Ssc2 (sequence similarity to cig30) [114]. The
ofﬁcial names for these two enzymes are Elovll and Elov12, respectively. Shortly after

that, a murine long chain fatty acid elongase was identiﬁed by gene array analysis from

27

SREBP] transgenic mice liver [117]. This enzyme was called Elovl6. ElovlS was the ﬁrst
human elongase identiﬁed [118]. Later, the rat ElovlS was reported to have similarity to
human Elov15 [119]. Mutation in the human Elovl4 gene were described as being
responsible for an autosomal dominant macular degeneration [120] (Table 1.1). Because
of their separate isolations and cloning over the years, fatty acid elongases have been
given many names, e.g, Cig30, SSCl, etc. With the discovery of multiple subtypes in
human and rodent genomes, the nomenclature was revised to the following. Elovl,

e_lgngase of yery long fatty acid. ( Table 1.2).

28

nm_019422
bab31310
af170908
nm_134255
nm_148941
bc016468
ay053453

nm_019422
bab31310
af170908
nm_134255
nm_14894l
bc016468
ay053453

nm_019422
bab31310
af170908
nm_134255
nm_148941
bc016468
ay053453

nm_019422
bab31310
af170908
nm_134255
nm_148941
bc016468
ay053453

nm_019422
bab31310
af170908
nm_134255
nm_14894l
bc016468
ayos3453

nm_019422
bab31310
af170908
nm_134255
nm_148941
bc016468
ay053453

1
7
2
5
4
3
6

\Jk‘ 01W p m N d H m w 9 m M q H
Oioa.> Uina~u p. 9“” *‘UIh°

air» much nr-q F‘

Table 1. 1. Alignment of mouse fatty acid elongases
Accession# Elov1#

.................. MEAVVNLYHELMKHADPRIQSYPLMGSPLLITSILLTYVYFI
............ MAFSDLTSRTVRFYDNWIKDADPRVEDYLLMSSPLPQTIILGLYVYFV
----------- MEQLKAFDNEVNAFLDNMFGPRDSRVRGWFLLDSYLPTFILTITYLLSI
.............. MEHFDASLSTYFKAFLGPRDTRVKGWFLLDNYIPTFVCSVIYLLIV
MGLLDSEPGSVLNAMSTAFNDTVEFYRWTWTIADKRVADWPLMQSPWPTISISTLYLLFV
-------- MDTSMNFSRGLKMDLMQPYDFETFQDLR---PFLEEYWVSSFLIVVVYLLLI

............ MNMS----VLTLQEYEFEKQFNENEAIQWMQENWKKSFLFSALYAAFI
. . *

LSLGPRIMANRKPFQLRGFMIVYNFSLVILSLY-IVYEFLMSGWLSTYTWRCDPIDFSNS
TSLGPKLMENRKPFELKKAMITYNFFIVLFSVY-MCYEFVMSGWGTGYSFRCDIVDYSQS
W-LGNKYMKNRPALSLRGILTLYNLAITLLSAY-MLVELILSSWEGGYNLQCQNLDSAGE
W-LGPKYMKNRQPFSCRGILQLYNLGLTLLSLY-MFYELVTGVWEGKYNFFCQGTRSAGE
W-LGPKWMKDREPFQMRLVLIIYNFGMVLLNLF-IFRELFMGSYNAGYSYICQSVDYSND
V-VGQTYMRTRKSFSLQRPLILWSFFLAIFSILGTLRMWKFMATVMFTVGLKQTVCFAIY
F-GGRHLMNKRAKFELRKPLVLWSLTLAVFSIFGALRTGAYMLYILMTKGLKQSVCDQSF

* * i :.

ooooo

nil-Box
PEALRMVRVAWLFMLSKVIELMDTVIFILRKKDGQVTFLHVFHHSVLPWSWWWGIKIAPG
PRAMRMVHTCWLYYFSKFIELLDTIFFVLRKKNSQVTFLHVFHHTIMPWTWWFGVKFAAG
G-DVRVAKVLWWYYFSKLVEFLDTIFFVLRKKTNQITFLHVYHHASMFNIWWCVLNWIPC
S-DMKIIRVLWWYYFSKLIEFMDTFFFILRKNNHQITVLHVYHHATMLNIWWFVMNWVPC
VNEVRIAAALWWYFVSKGVEYLDTVFFILRKKNNQVSFLHVYHHCTMFTLWWIGIKWVAG
TDDAVVRFWSFLFLLSKVVELGDTAFIILRKR--PLIFVHWYHHSTVLLFTSFGYKNKVP
YNGPVSKFWAYAFVLSKAPELGDTIFIILRKQ--KLIFLHWYHHITVLLYSWYSYKDMVA

.I'* i it :::*i*. : l:* :i* :

GMGSFHAMINSSVHVVMYLYYGLSALGPVAQPYLWWKKHMTAIQLIQFVLVSLHISQYYF
GLGTFHAFLNTAVHVVMYSYYGLCAMGPAYQKYLWWKKHLTSLQLVQFVLVTIHIGQIFF
GQSFFGPTLNSFIHILMYSYYGLSVF-PSMHKYLWWKKYLTQAQLVQFVLTITHTLSAVV
GHSYFGATLNSFIHVLMYSYYGLSSI-PSMRPYLWWKKYITQGQLVQFVLTIIQTTCGVF
GQAFFGAQMNSFIHVIMYSYYGLTAFGPWIQKYLWWKRYLTMLQLVQFHVTIGHTALSLY
SGGWF-MTMNFGVHSVMYTYYTMKAA--KLKHPNLLPMVITSLOILQMVLGTIFGILNYI
GGGWF-MTMNYGVHAVMYSYYALRAA--GFRVSRKFAMFITLSQITQMLMGCVINYLVFN

* :i :* :a+ it :i t: a:
MP8 ----- CNYQYPIIIHLIWMYGTIFFILFSNFWYHSYTKG ----- KRLPRAVQQ----
MED ----- CNYQYPVFLYIIMSYGCIFLLLFLHFWYRAYTKG ----- QRLPKTLE -----
KP ------ CGFPFGCLIFQSS-YMMTLVILFLNFYIQTYRKK---PVKKELQEKE -----
WP ------ CSFPLGWLFFQIG-YMISLIALFTNFYIQTYNKKGASRRKDHLKGHQNGSVA
TD ------ CPFPKWMHWALIA-YAISFIFLFLNFYTRTYNEP ----- KQSKTGKTATNGI
WRQEKG--CHTTTEHFFWSFMLYGT-YFILFAHFFHRAYLRP ----- KGKVASKSQ""
WMQHDNDQCYSHFQNIFWSSLMYLS-YLVLFCHFFFEAYIG ------ KVKKATKAE----
* a . it :t: _:* :
--NGAPATTKVKAN ------------

--NGN---CKSKRH ------------
VKNGFPKAHLIVANGMTDKKA -~--
AVNGHTNSFPSLENSVKPRKQRKD--
SSNGVNKSEKALENGKPQKNGKPKGE

29

Elongase

Sub e lovl

1 Genbank #
Alias

Chromosome #
Location, mb

Genbank #
Alias
Chromosome#
Location, mb

Genbank#
Alias
Chromosome #
Location mb

Genbank #
Alias
Chromosome #
Location, mb

Genbank #
Alias
Chromosome #
Location, mb

Genbank #
Alias
Chromosome #
Location, mb

Genbank #
Alias
Chromosome #
Location, mb

Mouse, Rat and Human.

Table 1. 2. Chromosomal Location of Elongase Subtypes in

 

Mme Rat HIM
NM_019422 AAL71993
SSCl CGI-88, SSCl
4 1 1
117.3 138.9 43.2
AF 1 70908 AAH60809
SSC2 SSC2
13 17 6
41.2 29.5 11.1
BC016468 AAH34344
c1030 BLOB
19 1 10
46.3 251.6 103.7
NM_148941 Q9GZR5 AAH3 8506
ELO4
9 8 6
86.5 88.9 80.6
NM_134255 NM_134382 AAF70631
FAEl RELOl HELOl
9 s 5
80.5 83 53.2
AY053453 NM_134383 NPO76995
LCE, FACE E102
3 2 4
133.1 226.9 111.4
BAB31310, AKA050441
9130013K24RIK Q9NT66
13 2 5
106.8 39.5 60.1

30

1.2.1 Elovll

Elovll was ﬁrst discovered by its homology to Elov1—3. Complementation studies
with yeast mutants with defective fatty acid elongation with the murine Elovll indicated
that Elovl-l was involved in elongation of very long chain fatty acids (up to 26 carbons)
and sphingolipid formation [121]. Sphingolipids are essential for cell proliferation.
Abnormal sphingolipid synthesis hinders cell growth [122]. Numerous studies show that
sphingolipids, as well as very long chain fatty acid synthase (V LCFAS) are important for
the formation of functional lipid microdomains (lipid rafts) in plasma membrane [123].
Disruption of the yeast Elovll homologue, E103, leads to a broad range of defects
including altered expression of the plasma membrane ATPase and lower glucose uptake
[124]. The overlapping functions of yeast E103 and murine Elovll indicates that
mammalian enzymes are also important in membrane related functions associated with

the lipid rafts.

Elovll is ubiquitously expressed in mouse tissues. This means most mammalian
tissues are endowed with the capacity to synthesize VLCF A up to C26. Interestingly, the
mRNA level of Elovll is particularly high in stomach, lung, kidney, skin and intestine.
The common feature of these tissues is that they have separate compartments with widely
different water content. Therefore, it is conceivable that higher Elovll levels are needed
for VLCFA synthesis in order to reduce water permeability of the epithelial barrier [125].
Quaking and J irnpy are two mouse mutant strains that have dramatically decreased
Elovll mRNA level. Both strains are deﬁcient in nerve myelination throughout the

central nerve system (CNS), which results in severe motor problems and early death.

31

Moreover, both strains show decreased elongation activity towards 20:0-CoA and 22:0-
CoA and lower levels of VLCFA in the brain [126]. The severity of elongation defect is

proportional to Elovll mRNA expression.

1.2.2 Elov12

When Elov12 (SSC2) gene was ﬁrst described in 2000, there was no functional
characterization for this enzyme. Investigators hypothesized that it may play a role in
PUFA synthesis based on the complementation studies from yeast system [114]. Later on,
both mouse Elov12 and human Elov12 were shown to use arachidonic acid (20:4,n-6),
eicosapentaeonic acid (20:5,n-3), docosatetraenoic acid (22:4,n-6) and docosapentaenoic
acid (22:5,n-3) in yeast and mammalian HEK 293 cells when they are transfected with
the vector expressing Elov12 gene [117,127]. In contrast, Elov12 did not elongate
saturated and monounsaturated fatty acids. Northern blot analysis revealed that the
highest level of expression of Elov12 was found in mouse liver and testis, followed by

brain and kidney [127].

1.2.3 Elovl3

Compared to other mammalian fatty acid elongases, Elovl3 is the one most
studied. Elovl3 was ﬁrst cloned from brown adipose tissue (BAT) of mice exposed to
cold temperature [116]. When mice were exposed to cold temperature for 3 days, the
rrricrosomal fraction from BAT of Elovl3 ablated mice has decreased ability to elongate
saturated C16-C22 substrates. There was, however, little difference in elongation activity

after mice were exposed to low temperatures for 3 weeks [128,129]. Further analysis of

32

fatty acid proﬁle in these mice revealed a transient alteration of several different fatty
acid species in the mice aﬁer 3 days of cold exposure but not in the mice exposed to cold
for 3 weeks cold [129]. Moreover, there are almost no lipid droplets in BAT of Elovl3
ablated mice when they are under therrnoneutral conditions (3 0°C). Such studies support
a role for short term, but not long term, adaptive changes in BAT lipid metabolism

following cold exposure.

Jacobsson’s group showed that cold exposure induced Elovl3 gene expression
required PPARa regulation [130]. In cultured brown adipocytes, a mixture of
norepinephrine, dexarnethasone, and the PPARa ligand Wy14,643, which rendered the
adipocytes a high oxidative state, was required for substantial induction of Elovl3
expression. In contrast, this treatment suppressed Elovll mRNA levels. Interestingly, the

LXR agonist, T0901317, repressed Elovl-3 expression [130].

A most prominent phenotypic feature in Elovl3 -/- mice was found in the skin.
Skin ﬁmctions as a water barrier and very long chain fatty acids are important for this
physiological function. Elov13-/- mice showed an impaired water barrier function which
was attributed to a change of VLCFA synthesis [128]. Although most of identiﬁed fatty
acid elongases are expressed in murine skin [114,127], nothing was known about the
speciﬁc roles of the individual enzymes in skin. In situ hybridization indicated that
Elovl3 has a distinct expression in the skin that was restricted to the sebaceous glands and

the epithelial cells of the hair follicles [128].

Recent studies from the Baler group revealed circadian regulation of Elovl3 in

liver that involved RevErba and SREBP-1. This regulation was sex- and tissue-speciﬁc

33

[131]. The reason for circadian regulation of Elovl3 can be explained by the needs for the
liver to synthesize long-chain fatty acids. The main fraction of intracellular VLCFAs in
liver is esteriﬁed in various lipids, predominantly in Sphingolipids. As such, circadian
expression of Elovl3 as well as other lipogenic enzymes may contribute to maintenance

membrane ﬂuidity, which consequently regulates a rhythm of the membrane-linked

signaling pathways [13 1].
1.2.4 Elovl4

Tissue expression of Elovl4 is one of the most restricted amongst the elongases; it
is expressed predominantly in the retina and to a less extent in brain, and testis [120]. The
high expression of Elovl4 in the retina suggest an involvement in the synthesis of
docosahexaenoic acid, 22:6,n-3. Nearly 50% of all acyl chains in phospholipids are DHA,

the highest content of all mammalian tissues.

Mutation of Elovl-4 was the ﬁrst mutation of a fatty acid elongase linked to
human disease. Mutation of the Elovl4 locus is found in patients with the genetic
autosomal dominant disease, Stargardts-like macular dystrophy [120]. Stargardts-macular
dystrophy is one of several forms of macular dystrophy. This disease is characterized by
decreased visual acuity, macular atrophy and extensive fundus ﬂecks. Genetic mapping
by Zhang’s group identiﬁed a 5-bp deletion in the Elovl4 gene that may be responsible
for this for of inherited macular degeneration [120]. About the same time, Allikmets, et al
[132] found another mutation in Elovl4 gene associated with this disease. This mutation
results in a frame shiﬁ and the truncation of the ELOVL4 protein, similar to the effect of

5-bp deletion.

34

In vitro studies show that the mutant Elovl4 protein exerts a dominant-negative
effect by recruiting wild-type protein into perinuclear cytoplasmic inclusions [133]. The
interaction between the wild-type and mutant forms of Elovl4 and the resultant alteration
in the trafﬁcking of the wild-type ELOVL4 protein suggest a mechanism for the
pathology associated with in patients harboring the autosomal dominant Stargardts-like

macular dystrophy genotype.

1.2.5 ElovlS

The Elov15 gene encodes a protein of 299 amino acids, which shares 56.4%
amino acid sequence identity with Elov12. Elov15 is involved in the synthesis of mono
and polyunsaturated fatty acids of C16-C22, but not polyunsaturated fatty acids beyond
C22 [127]. Elov15 is widely expressed with high levels in testis, adrenal gland, lung and
brain [115]. This is consistent with the fact that these tissues contain very high levels of

docosapentanoic acid, 22:5,n-3 and adrenic acid, 22:4, n-6.

1.2.6. Elovl6

The identiﬁcation of Elovl-6 was described by Moon et al. in 2001. It was
discovered in livers of transgenic mice overexpressing SREBP-1 [117]. This enzyme is
ubiquitously expressed. High fat-containing tissues such as white and brown adipose
tissue, liver, and brain, displayed high levels of Elovl6 transcripts, whereas the low fat-
containing tissues like spleen, skeletal muscle, and heart exhibit low levels of the Elovl6
transcript. In vitro microsomal fatty acid elongation assays demonstrated that Elovl6

possessed elongase activity speciﬁc for C1 2-C16 saturated and monounsaturated fatty

35

acids. The enzyme is induced by SREBP]. Its expression is also induced in the liver and
adipose tissue in the refed state after fasting, but suppressed by the presence of dietary
PUFAs [117,134]. It encodes 267 amino acid residues and has 96% identity with the

human homologue of 265 amino acids [115].
1.2.7 Elovl7

In silica analysis of the human and mouse genomes revealed the existence of the
seventh member in elongase family. This gene is located on chromosome #13 in mouse,
and #2 in rat. It encodes 281 amino acids residues and contains a histidine-rich motif
(HXXHH), and ER retension signal (KKXX-like), both typical for members of fatty acid
elongase family. The histidines in the HXXHH motif may act with aspartate and
glutamate to coordinate an Fe-O-F e cofactor to receive electrons from cytochrome b5 or
a cytochrome b5-like domain in an NAD(P)H-dependent manner [114,117]. The
similarities in sequence organization amongst the 7 proteins suggest Elovl7, i.e.,
BAB31310, mouse; Q9NT66, human, is a fatty acid elongase. Unfortunately, there is no

experimental data on Elovl-7 substrate speciﬁcity or its regulation.

1.3. Statement of the Problem and Hypothesis.

Multiple mammalian fatty acid elongases and fatty acid desaturases play a major
role in determining cellular lipid content. Of these enzymes, the most extensively studied
is A9D. Multiple factors regulate A9D expression. Studies with the D9D -/- mouse

implicate this enzyme in the management of hepatic and whole body lipid metabolism,

36

energy expenditure and tissue sensitivity to insulin [12,135]. These studies have targeted
A9D as a candidate enzyme for the development of inhibitors of its activity to manage of

whole body lipid metabolism.

In contrast to desaturases like D9D, we are in the early stage of understanding the
role fatty acid elongases play in cell function and composition. We have little information
on their regulation; we are unclear of their substrates and products. We do not know how
they are regulated in chronic disease or which transcriptional regulatory networks control
their expression. Studies in our laboratory have established a strong link between fatty
acid structure and the control of key transcriptional regulatory networks controlling
glucose and lipid metabolism [83,136]. Elongase activity alters fatty acid structure. As
such, these enzymes have the potential to impact cellular lipid composition and affect key
transcriptional regulatory networks. If so, then, elongases might join with A9D as targets

for drug development in the management of fatty acid-linked chronic diseases.

The goal of this study is to gain information about the regulation of the elongase
family of enzymes. A second goal is to gain evidence in support or against the notion that
fatty acid elongases play a dual role in hepatic physiology: to control hepatic lipid
composition and to impact hepatic gene transcription both in cells (in vitro) and in the

mouse (in vivo).

I hypothesized that fatty acid elongases play a dual role in hepatic physiology: a) to
regulate cellular levels of long chain unsaturated fatty acids, and b) to regulate cellular

levels of ligands controlling hepatic gene expression. The aims are:

37

Aim 1: To identify substrates of elongases in rat primary hepatocytes.

To identify regulatory factors controlling fatty acid elongase family expression.

Aim 2: To elucidate the role of Elovl-2 & Elov15 on hepatic gene expression in rat

primary hepatocytes.

Aim 3: To determine how over expression of speciﬁc fatty acid elongases affect hepatic

function and blood lipid composition.

The outcome of these studies will provided new information on the role fatty acid

elongases play in hepatic physiology.

38

Chapter 2

Tissue-Speciﬁc, Nutritional and Developmental

Regulation of Rat Fatty Acid Elongases.

2.1. Introduction

Most cells have the capacity to synthesize fatty acids from glucose, de novo. This
pathway utilizes products from glycolysis and along with the two enzymes, acetyl CoA
carboxylase and fatty acid synthase, generates palmitate (16:0). Insulin, T3,
glucocorticoids and glucose induce, while C20 PUFA, glucagon and epinephrine suppress
de novo lipogenesis [14,46,137]. Many cells also have the capacity to modify fatty acid
structure through metabolic pathways that include desaturation, elongation, mono-
oxidation and peroxisomal B-oxidation (chain shortening). Such modiﬁcations occur to
fatty acids generated de novo as well as fatty acids derived from the diet. These
metabolic pathways play an important role in the maintenance of membrane lipid
composition, the generation of precursors for certain signaling molecules, like
eicosanoids. These pathways may also contribute to the control of cellular fatty acids
impacting speciﬁc nuclear receptors, e.g., PPARa [14,53,54].

Of the various pathways known to affect fatty acid structure, physiological control
of fatty acid elongation remains poorly deﬁned. The predominant pathway for fatty acid
elongation occurs in the endoplasmic reticulum and uses malonyl-CoA and fatty acyl-
CoA as substrates for C2 additions to fatty acids. Elongases are condensing enzymes that

interact with 3-keto acyl-CoA reductase, a dehydratase and trans 2,3-enoyl CoA

39

reductase to elongate fatty acids [24,115,138]. The rate of fatty acid elongation is
determined by the activity of the elongase (condensing enzyme) and not the reductases or
the dehydratase. Six distinct fatty acid elongase subtypes (Elovll through Elovl6) are
present in the mouse, rat and human genomes. Elovll (Sscl) and Elovl6 (LCE, FACE,
rE102) elongate saturated and monounsaturated fatty acids. Disorders of sphingolipid-
metabolism, like the Quaking and J impy phenotypes [114] have been associated with
impaired Elovll activity. Elovl6 is induced in transgenic mice over expressing SREBP-1
[117,134,139]. Elov12 (Ssc2) substrates include, C2o.22 PUFA, while Elov15 (FAEl,
Relol, Helol) may utilize a broad substrate array, C1622 [117,119]. Elov12 and Elov15
likely play a role in endogenous PUF A synthesis; i.e., the conversion of essential fatty
acid precursors, linoleic acid (18:2n6) and a-linolenic acid (18:3n3) to arachidonic acid
(20:4n6) and docosahexaenoic acid (22:6n3) [139]. Elovl3 (Cig30, E103) and Elovl4
(E104) are expressed in the skin [140] and retina [141], respectively. Both Elovl3 and
Elovl4 elongate a broad array of fatty acids 5C26. Elovl3 is induced in brown adipose
tissue following exposure of animals to the cold [142]. Stargardt-like macular dystrophy
and autosomal dominant macular dystrophy is associated with defective Elovl4
expression [120].

Many studies have examined the regulation of mammalian elongases at the level
of enzyme activity [25,116,117,119,127,134,138,143-156]. Overlapping
substrate/product proﬁles for these enzymes has made it difficult to ascertain which
enzyme subtypes are regulated under speciﬁc physiological conditions. More recent
studies have examined nutritional and tissue-speciﬁc regulation of elongase gene

expression [116,117,134,140,153,155]. We are unaware of any study that has examined

40

the expression of all elongase subtypes in speciﬁc tissues. We have cloned 5 of the 7
fatty acid elongases known to be expressed in mammals and examined their expression in
rat liver. Elovl4 was not examined because of its retina-speciﬁc expression. Elovl7 was
not studies because it is poorly expressed in liver (unpublished observation). Our studies
included an analysis of the tissue-speciﬁc expression of elongases as well as the effect of
starvation, ﬁsh oil and PPARa agonist feeding on hepatic elongase expression and
elongase enzymatic activity. Developmental regulation of hepatic elongase expression
and activity is also included. Of the 4 fatty acid elongases expressed in rat liver, Elov15
emerges as the predominant elongase expressed, based on mRNA abundance. Elov15 may
account for much of the developmental and nutritional regulation of rat hepatic fatty acid

elongation.

2.2. Methods and Materials

Cloning of mouse and rat fatty acid elongases and desaturases. cDNAs for fatty acid
elongases and desaturases were cloned by reverse transcriptase-polymerase chain
reaction (RT-PCR) methods. Primers corresponding to the open reading frame of each
elongase and desaturase (Table 2.1) were designed based on sequences obtained from
Genebank and the location of the open reading frame as determined using DNA-Star.
Mouse liver was used as template to clone all desaturases and elongases, except Elov15;
Elov15 was cloned ﬁ'om rat liver. The RT-PCR products were inserted into TOPO-
cloning-plasmids (Invitrogen, Carlsbad, CA) and screened by blue-white selection.
Positive clones were selected and sequenced at the Genomic Technology Support Facility

at Michigan State University. Sequences were veriﬁed by alignment to sequences in

41

Genebank (Blast, http://www.ncbi.nlm.nih.gov/blast/ blZseq/bl2.html). Rat and mouse
elongases are 285% homologous at the protein sequence level.

Animals. All procedures for the use and care of animals for laboratory research have
been approved by the All University Committee for Animal Use and Care at Michigan
State University. Rats are maintained on Harlan-Teklad laboratory chow (#8640) and
water ad lib. Tissues for the analysis of elongase expression were derived ﬁom 60 day
old Spraque-Dawley male rats maintained on a chow diet ad lib. Nutritional regulation of
elongases used a meal-feeding protocol [53,157,158]. Brieﬂy, rats were acclimated to
meal-feeding Of a high carbohydrate (glucose) diet (HiCHO) [ICN Biochemicals, Aurora,
OH] supplemented with olive (Pompiean, Baltimore, MD) at 10% w/w for 7 days. The
meal began at 8 AM and ended at noon. After the acclimation period, rats were either
maintained on the olive oil diet or switched to a HiCHO diet supplemented with ﬁsh oil
(Dyets, Inc., Bethlehem, PA) at 10% w/w or a HiCHO diet supplemented with
WY14,643 at 0.1% w/w [158]. Animals were maintained on the olive oil, ﬁsh oil or
WY14, 643 diets for 7 days. Fasted rats were meal fed the HiCHO-olive oil diet 7 days,
but were fasted for 24 hrs before euthanasia. Two hours after completion of the ﬁnal meal
animals were euthanized for tissue and blood collection. The developmental studies used
timed pregnant female Spraque-Dawley rats from Charles River Laboratories
(Kalamazoo, MI). Fetal livers were obtained at 18-19 days post coitum and pooled for
analysis. Male suckling pups (1-20 days of age) and weaned male animals (30 days of
age) were used as a source of liver. Rats were weaned onto Harlan-Teklad laboratory

chow at 21 days postpartum.

42

Primary hepatocytes. Rat primary hepatocytes ﬁ'om Teklad chow-fed (ad lib) male
Spraque-Dawley rats were prepared and treated with fatty acids or WY14,643 as
previously described [52,136].

In vitro fatty acid elongation assay. Rat liver microsomes were isolated by differential
centrifugation [159]. Elongation reactions were carried out with modiﬁcations to the
procedure described by Moon et al [117]. Brieﬂy, reaction mixtures contained 50 pg
microsomal proteins in a total reaction volume of 100 pl. The elongation assay used fatty
acyl CoA as substrates. The reaction constituents are: 50 mM potassium phosphate
buffer- pH 6.5, 5 pM rotenone (Sigma), 40 pM fatty acyl CoA (Avanti Polar Lipids,
Alibaster, Al and Sigma, St. Louis, MO), 60 pM malonyl CoA (Sigma) [6.5 dpm/pmol]
[2-‘4C]-malonyl CoA (Perkin Elmer), 1 mM NADPH (Sigma) 20 pM BSA (fatty acid
free). Reactions (at 37°C) were initiated with the addition of NADPH. Reactions were
terminated after 20 minutes with the addition of 100 pl 5 N KOH + 10% methanol; lipids
were saponiﬁed for 1 hr at 65°C. The saponiﬁcation reaction was acidiﬁed with 100 pl 5
N HCL; 100 pl ethanol was added to aid hexane extraction of fatty acids. Elongated fatty
acids were collected by 2 independent extractions with hexane (800 pl). Hexane extracts
were pooled and l4C-radioactivity was quantiﬁed by B -scintillation counting. Restrlts are
expressed as Elongase Activity Units, mnoles l4C-malonyl CoA incorporated/mg
protein/2O mins. Formation of reaction products was dependent on the presence of
NADPH and the fatty acid CoA. Fatty acid elongation products were veriﬁed by reverse-
phase-HPLC chromatography using a ﬂow-through B—scintillation counter [136].

RNA extraction, northern analysis and RT -PCR. RNA was extracted and separated in

denaturing (formaldehyde) agarose gels, transferred to nitrocellulose and hybridized with

43

32P-cDNAs. The cDNAs for the various transcripts has been described previously
[52,136,159]. Hybridization was visualized by phosphoimager analysis (Molecular
Dynamics, Sunnyvale, CA). To ensure an accurate assessment of all elongases, reverse
transcriptase-polymerase chain reaction (RT-PCR) was used; transcript-speciﬁc primers
are described in Table 2.1.

Immunoblotting. Hepatic nuclear and rrricrosomal proteins were separated by SDS-
PAGE and transferred to nitrocellulose. SREBP-1, SREBP-2 and CYP4A were detected
as previously described [159]. PPARa was measured using antibodies obtained from
Santa Cruz Biotechnology. Anti-goat antibodies were also obtained from Santa Cruz. The
detection system employed the Super Signal West Pico chemiluminescence kit (Pierce).
Quantitation of hepatic and plasma mead acid (20:3,n-9) levels. Total lipid was
extracted from liver or plasma in chloroform:methanol (2:1) plus 1 mM butylated
hydroxytoluene [136]. 7-Nonadecenoic acid (19:1) was added as a recovery standard at
the time of extraction. Protein (Bio-Rad) was measured in extracts after the initial
homogenization step. Total lipids were saponiﬁed, fractionated and quantiﬁed by reverse
phase HPLC (RP-HPLC) using a YMC J-Sphere (ODS-H80) column and a sigrnoidal
gradient starting at 86.5% acetonitrile + acetic acid (0.1%) and ending at 100 %
acetonitrile + acetic acid (0.1%) over 50 mins with a ﬂow rate of 1.0 ml/min using a
Waters 600 controller. Fatty acids were detected using both UV absorbance at 192 nm
(Waters model 2487) and evaporative light scatter (Waters model 2420). Fatty acid
composition and structures were conﬁrmed at the MSU Mass Spectrometry facility by

GC/MS (wwwbchmsuedu/facilities/massspec/index.htnrl). Fatty acid standards for RP-

44

HPLC were obtained from Nu-Chek Prep (Elysian, MN). Mead acid (20:3,n-9) was
obtained from Sigma-Aldrich (St. Louis, MO).
Statistical Analysis. Statistical analysis involved Student’s t-test; p values were

calculated using Microsoft-excel t-test for 2 samples with unequal variance.

45

 

Table 2.1 Primers used to clone Elovls and desaturases from mouse or rat

liver.

 

 

 

 

 

 

 

 

 

 

Enzyme Accession
No. Primer Sequence
Elovll BC006735 Sense:ATGGAGGCTGTTGTCAACTTG
Antisense: TCAGTTGGCCTTGACCTTGGT
Elov12 NM_019423 Sense:ATGGGCGGCCGCATGGAGCAGCTGAAGGCCTTT
Antisense: TTATTGAGCCTTCTTGTCCGT
Elovl3 AF 054504 Sense: ATGGACACATCCATGAATTTC
Antisense: TCATTGGCTCTTGGATGCAAC
Elov15 NM_1343 82 Sense: ATGGAACATTTCGATGCGTCA
Antisense: TCAATCCTTCCGCTGCTTCCT
Elovl6 AY053453 Sense: ATGAACATGTCAGTGTTGACT
Antisense: TCTAGACTACTCAGCCTTCGTGGCTTTCTT
ASD NM_146094 Sense: ATGGCTCCCGACCCGGTGCCG
Antisense: CTATTGGTGAAGGTAAGCGTC
A6D BC057189 Sense: AGTCGACATGGGGAAGGGAGGTAACCAG
Antisense: TCATTTATGGAGGTAAGCATC
A9D NM_009127 Sense: ATGCCGGCCCACATGCTCCAA

 

 

Antisense: TCAGCTACTCTTGTGACTCCC

 

46

 

 

2.3. Results

2.3.1. Tissue-speciﬁc expression of rat fatty acid elongases.

Northern and RT-PCR approaches were used to determine the proﬁle of fatty acid
elongase expression in several rat tissues (Fig. 2.1). Elovll was well expressed in lung,
brain, kidney and heart, but is barely detectable in liver, brown adipose tissue and skin.
Because northern analysis of hepatic Elov12 indicates this transcript is expressed at low
levels (see Fig. 2.2), a RT-PCR approach was used to examine its expression in various
rat tissues. Elov12 is expressed in liver, lung, brain and kidney; Elov12 expression was not
detected other tissues. Elovl3 was only detected in skin. Elov15 expression was readily
detected in all tissues examined. Elovl6 mRNA is also expressed at low levels in rat liver
(Fig. 2.2). RT-PCR was used to show that Elovl6 is expressed in all tissues; its highest
expression is in brain. The tissue distribution of Elovll [155], Elov12 [155], Elovl3
[140,155], Elov15 [119] and Elovl6 [117,119] is similar to previous reports. Rat liver
expresses 4 distinct fatty acid elongases, Elovll, Elov12, Elov15 and Elovl6. The relative
abundance of the elongase mRN As in livers of adult male rats fed Teklad chow diets ad

lib is: Elov15 > Elovll = Elov12 = Elovl6 (Fig. 2.1 and 2.2).

2.3.2. Dietary Effects on Hepatic Elongase and Desaturase Gene
Expression.

To examine the effects of diet on hepatic elongase expression, adult male rats
were meal fed high carbohydrate diets supplemented with olive oil (10% w/w), ﬁsh oil

(10%w/w) or the PPARa agonist, WY14,643 (0.1% w/w) for 7 days. Because elongases

47

Elongase Liver Lung Skin Heart Method

1 NB
2

Retina Specific

Out

 

Figure 2. 1. Tissue speciﬁc expression of rat fatty acid elongases. RNA was extracted
from rat liver, lung, brain, brown adipose tissue (BAT), kidney, skin and heart. These
tissues were obtained from a 60 day old male rat maintained on a Teklad-Harlan chow.
diet, ad lib. Elovll, Elovl3, and Elov15 mRNAs were detected by northern analysis using
cDNAs cloned as described 1n Materials and Methods. Elov12 and Elovl6 were detected
by RT—PCR (35 cycles).

48

Figure 2. 2. Effect of diet on rat hepatic elongase and desaturase expression.
Male Spraque-Dawley rats (50 days of age) were meal-fed a high carbohydrate
diet (HiCHO) supplemented with olive (10% w/w) [Olive], ﬁsh oil (10% w/w)
[Fish] or olive plus WY14, 643 (0.1% w/w) [WY14, 643] for 7 days. Fasted
animals were meal fed the HiCHO-olive oil diet as described above, but were
fasted for 24 hrs prior to euthanasia. A: Northern blot of elongase and desaturase
mRNAs. A rat liver standard (S) derived ﬁom a 90 day old rat fed Teklad chow
(82403 was included in all northern blots for Elovll, Elov12, Elov15, Elovl6, A5, A6
and A desaturase mRN A measurement. Single transcripts were detected for each
mRNA, except Elovl6. Two transcripts have been reported for Elovl6 [117]. In
all cases, the size of the mRNA corresponded with the reported size of the
transcript. A representative etlridium bromide stain of 18 S and 28 S mRNA
documents equal loading of the RNA samples. Elovl3 was not detected in any
treatment (not shown). B: Fold changes in fatty acid elongase, fatty acid
desaturase and fatty acid synthase (F AS) mRNA abundance following dietary
challenge. Top panel: Effect of refeeding fasted animals on mRNA expression.
Results are expressed as the Mean : SD; N=3. *p50.05; fasted vs fed. Middle
panel: Effect of ﬁsh oil feeding on mRNA expression. Results are expressed the
Mean i SD,n=6/ group. *p30.05, olive vs ﬁsh. Bottom panel: Effect of WY14,
643 feeding on mRNA expression. Results are expressed as the Mean :
SD,n=6/group. *p50.05; olive vs olive + WY14, 643.

49

2.2A

S Fasted Olive

EIOVI-1

Elovl-S
Elovl-6

 

2.213. _
Fasted V8 Fed o

2.38

_A
O
l

O .-‘
01-0-0

Fold Change in mRNA Abundance
o

 

 

OM50)

ElovI-1 EIovl-Z ElovI-S EIovI-G (150 660 d90 FAS

50

act in concert with desaturases for PUFA synthesis, we examined the hepatic expression
of 4 elongases(Elov11, -2, -5 and -6), 3 desaturases (A5, A6 and A9) and fatty acid
synthase (FAS). Our goal was to determine whether there is any evidence for coordinate
regulation of pathways involved in fatty acid elongation, fatty acid desaturation and de
novo lipogenesis (DNL). Figure 2.2 (A and B) illustrates the effect of these treatments on
mRN A abundance of the various transcripts.

Fasting and refeeding: Fasting suppresses the expression of enzymes involved in
DNL, fatty acid desaturation [46,137,160] and at least one elongase, Elovl6 [119,134]. To
determine if other hepatic fatty acid elongases are regulated by fasting, rats meal-fed a If
high carbohydrate diet supplemented with olive oil (10 % w/w) were fasted for 24 hrs
and refed the same diet for 4 hrs (Figs. 2.2). mRNAs encoding Elovll and Elov15 were
induced marginally (1.5- and 1.9-fold) by refeeding fasted rats. Only Elovl6 mRNA
displayed a robust (14-fold) induction in response to refeeding fasted rats. In contrast, all
of the desaturase (A5, A6, A9) mRNAs, as well as mRNA FAS, were induced 5.2-, 2.6-, 18-
and 6.5-fold, respectively. Unlike the desaturases, the elongases do not display a uniform
response to fasting and refeeding.

Fish oil Feeding. Feeding rats diets enriched in N3-PUFA, i.e., ﬁsh oil, inhibits
DNL and the expression of all fatty acid desaturases [2,5,160]. While ﬁsh oil suppressed
FAS and each desaturase mRN A by 250%, ﬁsh oil feeding had no signiﬁcant effect on
Elovll and Elov12 mRNA abundance. Only mRNAElovls and mRNAElovm were
signiﬁcantly (_>_50%) suppressed by ﬁsh oil (Fig. 2.2). Fish oil feeding does not

uniformly suppress expression of all hepatic fatty acid elongases.

51

WY14,643 Feeding. All hepatic fatty acid desaturases are induced by PPARa
agonists, like WY14,643 [134,160,161]. To determine if the elongases are regulated by
WY14,643, rats were fed WY14,643 (0.1% w/w) in the high carbohydrate-olive oil diet
for 7 days (Fig. 2.2). mRNAs encoding all of the desaturases and 3 of the elongases were
induced by WY14, 643. Elovll and Elov15 were induced 2- and 2.5-fold; Elovl6 was
induced nearly 4-fold. Elov12 mRNA was not affected by WY14,643 feeding. A5, A6 and
A9 desaturase mRNAs were induced 4.1-, 2.3- and 4.8-fold respectively, while mRNApAs
was not signiﬁcantly affected by WY14,643 feeding. The effect of WY14,643 on both
elongase and desaturase expression suggest PPARa agonist may induce long chain ‘
polyunsaturated synthesis.

Dietary Effects on Hepatic Elongase Activity. We next determined if changes in
hepatic elongase mRN A abundance is consistent with hepatic fatty acid elongase activity.
Elongase assays used 7 substrates, i.e., 16:0-CoA, 18:0-CoA, 18:1,n-9-COA, 20:0-COA,
20:4,n-6-COA, 22:0-C0A and 24:0-COA, in separate assays (Fig. 2.3). Based on reports
by others, these fatty acyl-CoAs likely serve as substrates for l or more of the hepatic
elongases. Speciﬁcally, 16:0-C0A is a substrate for Elovll and Elovl6, but not Elov12
[117,134,155]. 18:1-CoA is not a substrate for Elovl6 [134], but is a substrate for other
elongases." 20:4,n-6 is a substrate for Elov12 and Elov15, but not Elovl6 [117,119,134].
20:0-C0A, 22:0-CoA and 24:0-COA are substrates for Elov11[155].

When compared to the olive oil-fed rats, hepatic microsomes isolated from fasted
rats had a signiﬁcantly lower (~50%) elongase activity when using 16:0-COA and 20:4-
CoA as substrate. Elongation of other fatty acyl CoAs was not signiﬁcantly affected by

fasting and refeeding. This effect correlates with the suppression of Elovll , Elov15 and

52

 

-L
“J
I

[1:1 Fast I Olive 1:: Fish 1:: WY14,643 in Chow]

 

A

CD CD
I l

r--—-4

  

A
I

 

     

’2222222332:

0

 

Elongase Activity, Units
N O)

 

:zzzzz

  

 

 

 

 

 

 

 

 

 

 

 

 

 

I

16:0-00A 18:0-00A 18:1-00A 20:0-CoA 20:4-CoA 22:0-COA 24:0-CoA
Elongase Substrates

C)

Figure 2. 3. Effect of diet on hepatic fatty acid elongase activity. In vitro assays for
elongase activity used microsomes isolated from livers of rats fasted or fed a high
carbohydrate diet containing olive oil, ﬁsh oil or WY14, 643 as described in the Methods.
Separate reactions were run using 16:0-CoA, 18:0-CoA, 18:1n9-COA, 20:0-COA, 20:4n6-
CoA, 22: O-CoA and 24: 0- CoA as substrate. Results are expressed as Elongase Activity,
Units (nmoles l4C-Malonyl CoA incorporated/mg protein), Mean + SD ,n=3/group. *p<
0. 05, Olive oil fed vs Fasted, Fish oil fed or WY14, 643 fed.

53

Elovl6 mRNA in starved animals (Fig. 2.2). A comparison of the elongase activity in
microsomes isolated from olive and ﬁsh oil fed animals indicates that ﬁsh oil feeding
suppressed (~60%) the elongation of both 16:0-C0A and 20:4-C0A. Fish oil suppressed
only Elovl6 and Elov15 mRNA, respectively (Fig. 2.2). Feeding rats WY14,643 increased
elongation of all saturated and monounsaturated fatty acyl-CoA substrates, except 16:0-
CoA, 2- to 3-fold. Increased elongase activity following WY14,643 feeding correlates
with increased hepatic abundance of mRNAs encoding Elovll , Elov15 and Elovl6 (Fig.
2.2). Increased Elovll expression in livers of WY14,643-fed rats is likely involved in
the increased elongation activity toward saturated and monounsaturated fatty acids.

Effect of ﬁsh oil and WY14,643 on hepatic and plasma fatty acid composition.
We next determined if changes in elongase gene expression and elongase activity had any
effect on hepatic or plasma lipid composition (Fig. 2.4). For this analysis, we focused on
mead acid (20:3,n-9) levels, because: 1) 20:3,n-9 is not a dietary fat; its presence in cells
is due to elongation and desaturation of 18:1,n-7 or 18:1,n-9 [162]; 2) dietary n-3 and n-6
PUFA suppress tissue levels of 20:3,n-9, by competing with 18:1,n-9 for A6 desaturase
[163] and suppressing the expression of mRNAs encoding A5 and A‘5 desaturases. The
contribution of elongases or WY14, 643 to 20:3,n-9 synthesis is unknown.

Fish oil and WY14,643 affect tissue and plasma levels of 20:3,n-9 and 20:4,n-6
(Fig. 2.4). In olive oil-fed rats, the mole % of 20:3,n-9 and 20:4,n-6 in the plasma is 0.7
and 6.4. In liver, the mole % of 20:3,n-9 and 20:4,n-6 is 1.8 and 14.2. The ratio of 20:3,n-
9/20:4,n-6 in plasma and liver is 0.1 and 0.13. These values are in line with essential fatty
acid sufﬁciency [164]. Rats fed the ﬁsh oil diet have a > 95% reduction in plasma and

hepatic 20:3,n-9. In contrast, olive oil-WY14,643 fed animal have 4- and 5-fold increased

54

Plasma
10 - IDZO:3,n-9 I20:4,n-6|

 

 

 

 

 

 

Liver

20"

Fatty Acid, Mole °/o

15'

10'

   

 

 

 

 

75 Olive .Flshép

Figure 2.4. Effect of diet on plasma and liver mead acid levels. Total lipids extracted
from rat liver and plasma of animals fed olive oil, ﬁsh oil or WY14,643 were saponiﬁed,
separated by RP-HPLC and quantiﬁed (Methods and Materials). The. identity of all fatty
acids was conﬁrmed by gas chromatography-mass spectrometry. Levels of mead acid
(20:3,n-9) and arachidonic acid (20:4,n-6) were quantiﬁed and the results are presented as
Mole % of the total saponiﬁed lipid fraction, Mean + SD, n= 3. The mole % of 20: 3 ,n-9
in olive oil vs ﬁsh oil- fed animals and m olive oil and olive oil + WY14, 643- fed animals
was signiﬁcantly different, *p<0. 01.

55

in 20:3,n-9 levels in plasma and liver, respectively. The 20:3,n-9/20:4,n-6 ratio in these
animals is 0.8 and 0.6 in plasma and liver, a value found in essential fatty acid deﬁciency
(EFAD). The elevation in 20:3,n-9 levels can be correlated to increased elongation of
18:1-CoA (Fig. 2.3) and elevated Elovll, Elov15, A5 and A6 desaturase mRNA abundance

(Fig. 2.2) plus the increased dietary intake of 18:1,n-9 in the olive oil diet.

2.3.3. Developmental regulation of hepatic fatty acid elongase and

desaturase gene expression.

SREBP-lo, a key transcription factor for controlling hepatic lipid synthesis [78],
has been implicated in the regulation of at least one fatty acid elongases (Elovl6)
[117,134,139]. In an effort to determine if changes in SREBP-lo nuclear content
correlate with elongase expression, we have used the model of early postnatal
development. In this model, SREBP-1 is present in fetal hepatic nuclei (Fig. 2.5, upper
panel). Shortly after birth, however, nuclear SREBP-1 levels fall and do not rise again
until the animals are weaned at 21 days postpartum [159]. We examined the expression
of hepatic elongases and desaturases in rat livers derived from fetal animals (18 days post
coitum), at 10 days postpartum (suckling) and 30 days postpartum (weaned). Fatty acid
synthase (F AS) was included for comparison (Fig. 2.5 lower panel). Expression of all 4
elongases (Elovll, -2, -5 and -6), all three desaturases (A5, A6, and A9) and FAS were
detected in fetal liver. Elovll decreased to adult values after birth. Elov12 remains
unchanged from 3 days prepartum to 30 days postpartum. Fetal Elovl 5 expression is
<10% of the 30 day value and increased 5-fold by 10 days and 10-fold by 30 days
postpartum. Elovl6 mRNA fell after birth to undetectable levels, but increased to adult

values after weaning. The developmental regulation of Elovl6, A9 desaturase and F AS

56

A Immunoblot
Days Postpartum

Fetal 1 7 15 30

 

Nuclear SREBP-1 . .
Nuclear SREBP-2 "' n. *1
mRNA Abundance

 

 

 

[3 Fetal .10 Days Old I] 30 Days Old

 

 

* *

 

   

Relative mRNA Abundance W
O -l N 00 h 01 O)

Elovl-1 Elovl-2 Elovl-5 Elovl-6 d5 D dSD d9 D FAS

Figure 2.- 5. DevelOpmental regulation of rat hepatic SREBP-l and -2 nuclear
content and mRNA abundance of fatty acid elongase, fatty acid desaturase and fatty
acid synthase. A. Immunoblot: Nuclear proteins isolated ﬁom livers at 18 days
postcoitum (Fetal) , 1, 7, 15 and 30 days postpartum were prepared, separated by SDS-
PAGE, transferred to nitrocellulose and probed with antibodies for SREBP-1 and
SREBP-2 [159]. The inclusion of SREBP-2 shows that hepatic SREBP—l and SREBP-2
nuclear content is regulated differently during development. B_. mRN A Abundance: Rat
liver mRNA from 18 days postcoitum (Fetal), 10 and 30 days postpartum was used to
quantify levels of fatty acid elongases (Elovll, Elov12, Elov15, Elovl6), fatty acid
desaturases (ASD, A6D and A9D) and fatty acid synthase (F AS). The results are
represented as “Relative RNA Abundance” and are normalized to the level of expression
in a 90 day old male rat. Mean : SD,number of animals (N)/group = 3. *p50.05, Fetal vs
10 or 30 days.

57

parallels changes in SREBP-1 nuclear content during early development. However, at no
time in this analysis was Elovl6 mRN A found to be an abundant transcript. In contrast,
mRNAs encoding Elov15, A5 and A6 desaturase are very low in fetal liver and increase
after parturition. Developmental changes in Elov15, A5 and A6 desaturase do not parallel
changes in SREBP-l nuclear content.

At parturition, animals ingest a high fat milk diet where 65% of the calories are
fat. The ingestion of the high fat milk diet is associated with the activation of PPARa-
regulated transcripts [165,166]. Nuclear PPARa content changes little in livers of fetal,
neonatal or weaned animals (Fig. 2.6). The PPARa-regulated transcript, CYP4A, is 5
induced (~10-fold) within 1 day of birth; both mRNAcyp4A and microsomal CYP4A
protein increase >10-fold. The induction of mRNAcyp4A parallels the induction of
mRNAElovls aﬁer birth. Both A5 and A6 desaturase mRNAs rapidly increase after birth (not
shown). This observation, coupled with the fact that mRNAEIovls is induced by WY14,643
(Fig. 2.2), suggest that PPARa and the high fat milk diets contribute to the postnatal
increase in mRNA Elov15-

Developmental regulation of hepatic fatty acid elongase activity. Fatty acid '
elongation activity was examined in microsomes isolated from fetal liver and livers of 10
and 30 days old male rats (Fig. 2.7). Fatty acid elongation activity for all substrates is low
in fetal liver and increased 5- to 20-fold by 10 days postpartum. However, elongation of
€22-24 saturated fatty acids is ~2 to 3-fold higher than €16-13 saturated fatty acids.
Elongation activity for each substrate was induced signiﬁcantly by 10 days postpartum.

The level of elongase activity at 10 days postpartum was at or near adult (90 days) values.

58

Days Postpartum

 

Fetal 3 7 10 15 18 21 90

IB: PPAR-alpha

 

NB: CYP4A

 

 

 

IB: CYP4A

 

 

NB: Elovl-5

 

 

Figure 2. 6. Rapid induction of hepatic Elov15 and CYP4A mRNA following
parturition. NB___ Elov15: mRNA was extracted from livers of fetal rats and from male rats
3, 7, 10, 15,18, 21 and 90 days old. A northern blot illustrating the induction of 1.310145 18
shown. IB CYP4A and IB PPARa: nuclear (PPARa) or microsomal (CYP4A) proteins
were isolated from livers at 18 days postcoitum (fetal) and 3, 7, .10, 15, 18,21 and 30
days postpartum. Proteins were separated by SDS}PAGE, transferred-to nitrocellulose
and probed with antibodies for PPARa or'CYP4A, Imm'uriob'ldt (1B) [159]. TotalRNA
was extracted from rat livers at 18 days post coitum (fetal) and 1, 7, 15, 21 and 30 days
postpartum (3 animals/group); S, RNA from 90 day old rat liver. RNA was separated and
probed for CYP4A [75].

59

of the substrates used, 2-3 times more 20:4-CoA was converted to its elongation product
(22:4,n-6) than any other fatty acyl CoA tested.

The low elongase activity in fetal livers correlates with the low level of
expression of all hepatic elongases. Note that Elovll , Elov12 and Elovl6 are expressed at
low levels in both fetal and adult livers (Figs. 2.1, 2.2 & 2.5). The postnatal increase in
hepatic elongase activity correlates with only one elongase transcript, i.e., Elov15. I
mRNAElovls was induced (5-fold) by 10 days postpartum (Fig. 2.5 and 2.6). Since no
other elongase was significantly induced by 10 days postpartum, our studies suggest that
Elov15 is the likely elongase responsible for a major fraction hepatic fatty acid elongation.
If so, then Elov15 is capable of elongating a broad range of fatty acid substrates, including
saturated (16:0, 18:0, 20:0, 22:0 & 24:0), monounsaturated (18:1n9) and polyunsaturated
(20:4n6) fatty acids (Fig. 2.7). Elov15 mRNA is expressed at higher levels than all other
elongases. If changes in elongase mRNA parallel changes in elongase protein and
elongase activity, then much of the increase in hepatic fatty acid elongase activity that

occurs during postnatal development can be attributed to Elov15.

2.4. Discussion

Liver (Fig. 2.7), primary hepatocytes [53], Hek293 [54] and FTC-2b hepatoma
cells display robust elongation activity toward C20 PUFA. C2022 PUFA fatty acids ‘
regulate the activity of fatty acid-regulated nuclear receptors, like PPARa and LXRa
[53,54]. As such, changes in C20 PUFA elongation might impact PPARa and LXRa
action. In an effort to determine which elongase might be responsible for C20 elongation,

we examined the expression and activity of rat liver fatty acid elongases. These studies

60

 

 

 

 

.2
c .
3 4 ' Days Postpartum
E Baum-mum
.2 3 -
‘6
< 2
i
3’ 1
2
m
0 .

 

 

 

 

 

 

 

 

16:0-00A 18:0-00A 18:1-GOA 20:0-CoA 20:4—CoA 22:0-00A 24:0-CoA
Elongase Substrates

Figure 2. 7. Developmental regulation of hepatic fatty acid elongase activity. In
vitro assays for elongase activity used microsomes isolated from livers of fetal rats (18
days post coitium), 10, 30 and 90 days postpartum. Separate reactions were run using
16:0-CoA, 18:0-CoA, 18:1,n-9-CoA, 20:0-CoA, 20:4,n-6-CoA, 22:0-COA and 24:0-CoA
as substrate. Results are expressed as Elongase Activity, Units (”C-Malonyl CoA nmoles
incorporated/mg protein), Mean j; SD,n=3/ group. Elongase activity in fetal liver was
signiﬁcantly lower than postpartum liver for all substrates examined,i:p5 0.05.552,

“.;t
a
..

61

have revealed new information on the regulation of elongase expression and which
elongase likely contributes to C20 PUFA metabolism in liver.

Of the 4 fatty acid elongases (Elovll, Elov12, Elov15 and Elov16) expressed in
liver (Figs. 2.1 and 2.2), only Elovll, Elov12 and Elov15 use C20 fatty acid substrates
[117,119,155]. The mRN As encoding Elovll and Elov12 are expressed at very low levels
relative to mRNAErovls in adult rat liver. Moreover, Elovll and Elov12 mRNAs do not
increase during postnatal development, a time in which the liver substantially increases
its capacity for C20 PUFA elongation (Fig. 2.7). Elov15 is the only fatty acid elongase
readily detected in primary hepatocytes. Based on this observation, we suggest that
Elov15 is responsible for elongation of C20 PUFA to C22 PUFA in primary hepatocytes.
Finding that Elov15 mRNA is expressed in broad array of tissues (Fig. 2.1) [119],
regulated during postnatal development (Fig. 2.5), by dietary fat and PPARa agonist
(WY14,643) (Fig. 2.2), suggest changes in Elov15 expression and activity may be
important for the maintenance of cellular long chain fatty acids. Whether changes in
Elov15 expression and activity affect fatty acid-regulated nuclear receptor function is an
issue addressed in a later chapter.

It is unclear if changes in elongase activity during postnatal development are due
solely to changes in elongase expression. Elongases are condensing enzymes that interact
with 3-keto acyl-CoA reductase, a dehydratase and trans 2,3-enoyl CoA reductase to
elongate fatty acids [24,138,153]. The rate of fatty acid elongation is generally viewed as

being determined by the activity of the elongase (condensing enzyme) and not the

reductases or dehydratase. However, the low elongase activity in fetal liver may be due to

 

 

deﬁcient expression of the reductases and dehydratase. Additional studies will be
required to examine the expression of these enzymes during postnatal development.

These developmental and nutritional studies have implicated 2 transcription
factors in control of elongase expression, i.e., PPARa and SREBP-1. During postnatal
development two distinct patterns of fatty acid elongase and desaturase expression are
evident. These two patterns of expression can be linked to changes in SREBP-l nuclear
content or the activation of PPARa. Both SREBP-l and PPARa play major roles in
whole body and hepatic lipid metabolism [51,78]. Changes in SREBP-1 nuclear content
closely parallel changes in FAS, A9 desaturase and Elovl6 expression (Fig. 2.5). SREBP-
1 is expressed in fetal livers at levels near adult values. During the suckling period,
however, SREBP-1 is absent from the nucleus. FAS, A9 desaturase and Elovl6 increase
after weaning when SREBP-1 nuclear content increases. SREBP-1 nuclear abundance
has been linked, directly or indirectly, to the transcriptional control of each of these genes
[78,139,167]. The regulation of SREBP-1 nuclear content during postnatal development
may be mediated by changes in proteolytic conversion of the SREBP-1c precursort(~125
kd) to the mature form (~65 kd) and its transport to the nucleus [159]. Ingestion of the
high fat milk diet (~65% calories as fat) begins at parturition. The ingestion of the high
fat milk diet, coupled with low blood insulin levels [168] likely contributes to this
regulatory scheme.

The expression of the other elongases, particularly Elov15, does not parallel
changes in SREBP-1 nuclear content during postnatal development (Fig. 2.5). Treatment
of primary hepatocytes with T1317, an LXR agonist, signiﬁcantly elevates SREBP-1

nuclear content as well as several SREBP-l regulated transcripts [52]. T1317 only

63

modestly induced mRNAglovls (<3 0%) in primary hepatocytes (not shown). Unlike Elovl6
[117,134], Elov15 does not appear to be a target for either SREBP-1 or LXRa.

Despite the ﬁnding that the postnatal induction of Elov15, A5 and A6'desaturase
closely parallels the induction of PPARa-regulated transcripts, e.g., Cyp4A (Fig. 2.6)
[75 ,158], the role PPARa plays in [144] this regulatory scheme is less clear. PPARa
agonist, like WY14,643 (Fig. 2.2) and ﬁbrates induce hepatic elongase activity toward a
broad range of fatty acids. Our studies show that WY14,643 induced Elovll, -5 and -6
mRNA (Fig. 2.2). Studies with primary hepatocytes, however, have indicated that while
PPARa target genes, like CYP4A and AOX, are well induced in primary hepatocytes
[136,158], transcripts encoding elongases, e.g., Elov15, are only marginally (<25%)
induced (not shown). The lack of a signiﬁcant WY14,643 effect on Elov15 (or other
elongases) expression in primary hepatocytes suggest these enzymes are not a direct
target of PPARa. Clearly, many factors change during postnatal development, e.g.,
glucocorticoids, leptin, T3 [169,170], that could impact hepatic elongase expression.
These and other factors were evaluated for their contribution to elOngase expression in
the next chapter.

' Our studies reveal two regulatory patterns for elongase and desaturase expression
during postnatal development. These different patterns of expression likely have
physiological signiﬁcance. Blood of pregnant female rats fed a Teklad chow diet contains
levels of 20:4,n-6 and 22:6,n-3 at 10 and 5 mole %, respectively (not shown). In contrast,
rat milk is deﬁcient in 20:4,n-6 and 22:6,n-3, i.e., 0.5 and 0.1 mole % respectively.
Analysis of hepatic fatty acid proﬁles in fetal, 10 and 30 day old liver shows that the

mole % of 20:4,n-6 and 22:6,n-3 remains essentially unchanged, at 15-20 mole% (not

64

shown). Since neonates grow very rapidly, the only mechanism available to sustain the
constant level of hepatic 20:4,n-6 and 22:6,n-3 in the absence of an extrahepatic source of
€20-22 PUFA is to induce PUFA synthesis. The capacity of the neonatal liver to synthesize
20:4,n-6 and 22:6,n-3 from dietary precursors (18:2,n-6 and 18:3,-n3) is achieved, at least
in part, by inducing mRNAs encoding A5 and A6 desaturase and Elov15 (Fig. 2.6 and 2.7).
Fetal liver has high blood levels of 20:4,n-6 and 22:6,n-3 and low elongase
activity, and low levels of mRNAs encoding Elov15, A5 and A6 desaturase. Adult animals
fed ﬁsh oil (a rich source of 20:5,n-3, 22:5,n-3 and 22:6,n-3), have low 20:4,n-6 and
20:5,n-3 elongation activity (Fig. 2.5) and suppressed expression of mRNAs encoding
Elov15, A5 and A6 desaturase (Fig. 2.2). Thus, changes in Elov15, A5 and A6 desaturase
expression respond to changes in dietary PUFA composition and tissue requirements for
C2o.22 PUFA. Elov12 has also been implicated in PUFA synthesis [118,127]. Yet
mRNAglom remains unresponsive during postnatal development, following
fasting/refeeding and ﬁsh oil treatment. Based on these ﬁndings, Elov15, and not Elov12,
appears to play the major role, along with A5 and A6 desaturase, in the adaptiVe response
of PUFA synthesis following changes in dietary lipid composition. This response is
mediated, at least in part, through the regulation of the cellular abundance of the mRNAs
encoding these enzymes. Additional studies will be needed to further deﬁne the roles
Elov12 and Elov15 play in the control of hepatic PUFA synthesis. In this regard, raising
rats on n3—PUF A deﬁcient diets leads to changes in blood and tissue lipid proﬁles as well
as diminished cognitive function [171]. Whether similar effects occur in null mutations of

speciﬁc elongases or desaturases remains to be determined.

65

Feeding rats ﬁsh oil suppresses lipogenic gene expression [52,157,172] and
mRNAglovls (250%) (Fig. 2.2). Primary hepatocytes treated with 20:5,n-3 showed ~50%
reduction in Elov15 mRNA (not shown). Many of the effects of ﬁsh [oil on hepatic gene
expression can be attributed to suppression of nuclear levels of SREBP-1c or activation
of PPARa [14]. Our ﬁndings indicate that neither factor induces major changes in Elov15
expression in primary hepatocytes. Hepatic L-pyruvate kinase gene transcription is
suppressed by 20:5,n-3 through mechanisms that are independent of PPARa and SREBP-
lc [157,158]. The target for 20:5,n-3 action on L-PK is a region that binds a carbohydrate
regulatory element binding protein (ChREBP), Mlx and HNF-4 [90,173]. Elov15 mRNA
is unresponsive to changes in glucose treatment of primary hepatocytes suggesting that '
CHREBP and Mlx are not involved in Elov15 expression (not shown). The role of HNF-4
in Elov15 expression is under investigation.

Finally, ﬁnding that WY14,643 elevated hepatic and plasma levels of mead acid
(20:3,n-9) in olive oil-fed rats was a surprise (Fig. 2.4). Mead acid is a elongation and
desaturation (A5 and A6 desaturase) product of vaccenic (18:1,n-7) and oleic (18:1,n-9) '
acid [162]. The effect of dietary n3 and n6 PUFA on tissue and plasma levels of 20:3‘,n'-9
has been well described [164]. It is generally accepted that much cf the PUFA-mediated
suppression of 20:3,n-9 content in cells is due to competition of PUFA for A6 desaturase
(n3 > n6 > n9) [163]. The elevated tissue content of €20-22 PUFA, coupled with the
decline in both elongation and desaturation capacity (Fig. 2.2-2.4), likely accounts for the
low levels of 20:3,n-9 in ﬁsh oil-fed animals. Contrary to the effects of ﬁsh oil,
WY14,643 signiﬁcantly increased hepatic and plasma levels of 20:3,n—9. The level of

18:2,n-6 in the olive oil diet (3.3mole %) is sufﬁcient to prevent EF AD. Ingestion of a

66

18:1n9-enriched diet, however, coupled with the induction of hepatic Elovll, Elov15 and
Elovl6 plus A5 and A6 desaturases by WY14,643, is apparently sufficient to increase
20:3,n-9 synthesis and its accumulation in tissues and plasma. The consequence of
elevated 20:3,n-9 production is an increase in the hepatic 20:3,n-9/20:4,n-6 to 0.62, a
value consistent with essential fatty acid deﬁciency (EFAD) [164]. Clearly, the elevated
expression of key enzymes involved in PUF A synthesis, plus ingestion of a 18:1,n-9

enriched diet, is sufﬁcient to shift the tissue balance of 20:3,n-9 and 20:4,n-6.

67

Chapter 3

Regulation of Hepatic Fatty Acid Elongase and

Desaturase Expression in Diabetes and Obesity

3.1 Introduction

The liver plays a central role in whole body lipid metabolism. Fatty acids are
synthesized de novo from glucose. This pathway utilizes products from glycolysis and
along with the two enzymes, acetyl CoA carboxylase and fatty acid synthase, generates
palmitate (16:0). Insulin, triiodothyronine (T3), glucocorticoids and glucose induce, while
C20 polyunsaturated fatty acids (PUFA), glucagon and epinephrine suppress de novo
lipogenesis [14,137,174]. The liver also modiﬁes fatty acid structure through metabolic
pathways that include desaturation, elongation, mono-oxidation and peroxisomal [3-
oxidation (chain shortening). Such modiﬁcations occur to fatty acids generated de novo,
as well as fatty acids derived from the diet. These pathways are particularly critical for
the generation of end products of PUFA synthesis. Arachidonic acid (20:4,n-6) and
docosahexaenoic acid (22:6,n-3) are the main C20-22 PUFA accumulating in membranes
of all tissues [31]. Together, these metabolic pathways play an important role in the
maintenance of membrane lipid composition and lipid storage, the generation of
precursors for signaling molecules, like eicosanoids, and the control of fatty acid-

regulated transcription factors [14,52-54].

68

Of the various pathways known to affect fatty acid structure, physiological control
of fatty acid elongation remains poorly deﬁned. We recently reported on the tissue-
speciﬁc, nutritional and developmental regulation of fatty acid elongases in the rat [175].
The outcome of those studies suggested that hepatic elongase expression might be
controlled by at least two transcription factors, peroxisome proliferator-activated receptor
(PPAR; and sterol regulatory element binding protein-1 (SREBP-1). This report extends
those ﬁndings by examining SREBP-1, PPARa, liver X receptor (LXR), carbohydrate
regulatory element binding protein (ChREBP) and MAX-like factor X (MLX) control of
elongase and desaturase expression. Our analysis also includes in. vivo studies to
determine if changes in hepatic lipid composition induced by diabetes or obesity
correlated with changes in elongase and desaturase expression. Overall, these studies
establish a role for speciﬁc transcriptional regulatory networks in the control of hepatic

desaturase and elongase gene expression and hepatic lipid composition.

3.2 Materials and Methods

Animals. All procedures for the use and care of animals for laboratory research have
been approved by the All University Committee for Animal Use and Care at Michigan
State University.

Streptggotocin-Induced Diabetes: Male Spraque-Dawley rats (200-250 g) (Charles River
Laboratories, Kalamazoo, M1) were maintained on Harlan-Teklad laboratory chow
(#8640) and water ad lib. Rats were injected, intraperitoneally with streptozotocin (7.5
mg/ 100 g. BW) and 3 ml of 25% glucose [176]. Three weeks later, blood glucose was
measured on animals receiving no streptozotocin (control) or streptozotocin (diabetic,

blood glucose 3120 mg/dl). Blood glucose was measured in isoﬂurane anesthetized rats

69

using a glucose meter (Freestyle Flash, Thera Sense, Inc, Alameda, CA). Control and
diabetic rats were euthanized (isoﬂurane anesthesia and exsanguinations) for recovery of
blood (plasma) and liver.

High Fat Feeding of C5 7BL/6 Mice: Male C57BL/6 mice (Jackson Laboratories, Bar
Harbor Maine), 2 months of age, were fed diets containing 10% lard (D12450B) or 60%
lard (D12492), Research Diets, Inc.) ad lib for 10 weeks. Four days prior to euthanasia,
mice were subjected to a glucose tolerance test. Brieﬂy, mice were injected with glucose
(2 g/kg, IP). Blood was withdrawn from the tail vein prior to and after glucose treatment.
Blood glucose was measured using a hand-held glucose meter (Freestyle Flash, Thera
Sense, Inc, Alameda, CA). Mice were euthanized using isoflurane and exsanguinated;
blood and liver was recovered. Livers were used for RNA and lipid extraction.

Lean amL Obese Mice. Livers from lean and obese C57BL/6 mice were obtained from
Drs. Romsos and Claycomb (Department of Food Science and Human Nutrition, MSU).
Male lean C57BL/6J-Lep0b/+ and obese (C57BL/6J-LepOb/0b) mice (B6.V-Lep ob/J' ,
#000632, Jackson Labs) were maintained on a Harlan-Teklad laboratory chow (#8640)
diet and water ad lib. Livers were used for RNA, lipid and protein extraction.

Wild type and PPARa [-/-Lmice. Homozygous wild type and PPARa-null (-/-) mice on
an Sv/129 genetic background [177,178]were fed either a control diet or one containing
WY14643 (at 50 or 500 ppm: Bio-Serv, Piscataway, NJ) for 1 week. Mice were
euthanized, livers were removed and RNA was isolated for analysis of gene expression.

2, 3, 7, 8-tetracliorodibenzo-p-dioxin (T CDD) treated mice. Female C57BL/6 mice were
treated by gavage with 0.1 ml of sesame oil for a nominal dose of 0 (vehicle control)lor

30 rig/kg body weight of TCDD. Mice were sacriﬁced 168 h aﬁer dosing. Livers were

70

removed and RNA was isolated for analysis of gene expression and fatty acid
composition.

Primary Rat Hepatocytes: Male Spraque Dawley rats (Charles River Laboratories,
Kalamazoo, M1) were maintained on Harlan-Teklad laboratory chow (#8640) and water
ad lib. Rat primary hepatocytes were prepared from Teklad chow-fed (ad lib) male
Spraque-Dawley rats, cultured on BioCoat (type I collagen) plates (Beckon Dickinson,
Belford, MA.[179]. For RNA and protein extraction, cells were plated onto 100 mm type I
collagen-coated plates (BD Bioscience, Bedford, MA) at 107 cells/plate in Williams E
(Gibco/Invitrogen, Carlsbad CA), 10 mM lactate, 10 nM dexamethasone (Sigma, St. Louis),
and 10 % fetal bovine serum (Gibco/Invitrogen). For adenoviral infection studies, cells
were plated in the same media onto 6 well type 1 collagen-coated plates at 1.5 x 106
cells/well. The ratio of culture media to cell number was maintained constant for the
diﬁerent plating conditions. For treatments, hepatocytes were incubated in medium
(\Vrlliams E + 10 nM dexamethasone without or with 100 nM insan and/or 25 mM
glucose. .

RNA extraction, northern analysis and quantitative real time-PCR (qR T -PCR). Total
RNA was extracted from primary hepatocytes or liver samples and used for template for
qRT-PCR or northern analysis as previously described [175]. SpeCiﬁc primers for each
gene (Table 3.1) were designed using Primer Express software (Applied Biosystems,
Foster City, CA). First strand cDNA was synthesized using the SuperScript II RNase H-
Reverse Transcriptase (Invitrogen Carlsbad, CA). Synthesized cDNA was mixed with 2x
SYBR Green PCR Master Mix (Applied Biosystems) and various sets of gene-speciﬁc

forward and reverse primers and subjected to real-time PCR quantiﬁcation using the ABI

71

PRISM 7700 Sequence Detection System (Applied Biosystems). All reactions were
performed in triplicate. The relative amounts of mRNAs were calculated by using the
comparative CT method (User Bulletin #2, Applied Biosystems). Cyclophilin was used as
a control and all results were normalized to the abundance of cyclophilin mRNA.
Primers used for quantitative real time PCR are listed in Table 3.1.

Lipid attraction and quantitation of hepatic fatty acids composition. Total lipid was
extracted from liver in chloroforrnzmethanol (2: 1) plus 1 mM butylated hydroxytoluene
[175]. 7-nonadecenoic acid (19:1) was added as a recovery standard at the time of
extraction. Protein (Bio-Rad, Hercules, CA) was measured in extracts after the initial
homogenization step. Total lipids were saponiﬁed, fractionated and quantiﬁed by reverse
phase HPLC (RP-HPLC) using a YMC J-Sphere (ODS-H80) column and a gradient
starting at 77.5% acetonitrile + acetic acid (0.1%) and ending at 100 % acetonitrile +
acetic acid (0.1%) over 90 mins with a ﬂow rate of 1.0 ml/min using a Waters 600
controller. Fatty acids were detected using both UV absorbance at 192 nm (Waters model
2487) and evaporative light scatter (Waters model 2420). Fatty acid composition and
structures were conﬁrmed at the MSU Mass Spectrometry facility by GC/MS
(wwwzbchmsucdu/facilities/massspec/index.html). Fatty acid standards for RP-HPLC
were obtained ﬁom Nu-Chek Prep (Elysian, MN).

Immunoblotting. Liver microsomal and nuclear extracts were prepared as described
previously [159,175]. Proteins (SO-100 11g) extracted ﬁom microsomal or nuclear
ﬁactions were separated electrophoretically by SDS-polyacrylamide gel electrophoresis

(NuPAGE 4-12% polyacrylamide Bis-Tris, Invitrogen) and transferred to nitrocellulose

72

 

 

Table 3. 1. Primers for quantitative reverse transcriptase-polymerase chain reaction

 

 

 

 

Gene Forward Reverse

. Rat
Elovll CCCTACCTTTGGTGGAAGAA TCCAGATGAGGTGGATGATG
Elov12 TTTGGCTGTCTCATCTTCCA GGGAAACCGTTCTTCACTTC
Elov15 TACCACCATGCCACTATGCT GACGTGGATGAAGCTGTTGA
Elovl6 CAACGGACCTGTCAGCAA GTGGTACCAGTGCAGGAAGA
ASD TGGAGAGCAACTGGTI‘TGTG GTTGAAGGCTGACTGGTGAA
A6D TGTCCACAAGTTTGTCATTGG ACACGTGCAGGCTCT'ITATG
A9D ACA'I‘TCAATCTCGGGAGAACA CCATGCAGTCGATGAAGAAC
LXRa TCAGCATCTTCTCTGCAGACCGG TCATTAGCATCCGTGGGAACA
LXRB AAGCTGGTGAGCCTGCGC CGGCAGCTTCTTGTCCTG
SREBP-1c GATTGCACATTTGAAGACATGCTT GTCCCAGGAAGGCTTCCAGAGA
L-PK AGGAGTC'ITCCCCTTGCTCT ACCTGTCACCACAATCACCA
Cyclophilin CTTCTTGCTGGTCTTGCCATTCCT GGATGGCAAGCATGTGGTCTTTG
B-Actin ACTAT‘TGGCAACGAGCGGTT TGTCAGCAATGCCTGGGTACA

Mouse
Elovll CCCTACCTTTGGTGGAAGAA ATCCAGATGAGGTGGATGATG
Elov12 ACGCTGGTCATCCTGTTCTT GCCACAATTAAGTGGGCTTT
Elov15 GGTGGCTG'ITCTTCCAGATT CCCTTCAGGTGGTCTTTCC
ElOVl6 ACAATGGACCTGTCAGCAAA GTACCAGTGCAGGAAGATCAGT
ASD TGTGTGGGTGACACAGATGA GTTGAAGGCTGATTGGTGAA
A6D CCACCGACATTTCCAACAC GGGCAGGTATTTCAGCTTC'IT
A9D TCAACTTCACCACGTTCTTCA CTCCCGTCTCCAGTTCTCTT
Cyclophilin CTTCTTGCTGGTCTTGCCATTCCT GGATGGCAAGCATGTGGTCTTTG
B-Actin GACGGCCAGGTCATCACTAT CGGATGTCAACGTCACACT’I‘

Human
Elovl 1 GCTGGCTGAGCACCTATACC TCAGCTCAATGAACTTGGAGAA
Elov12 CCCTTCGG'ITGTCTCATC'IT CAGGTGGCTCTTGCATATCTT
Elov15 GTGCACATI‘CCCTCTTGGTT TGGTCCTTCAGGTGGTCTI‘T
Elovl6 CTAAGCAAAGCACCCGAACT GGCAACCATGTCT’ITGTAGGA
ASD TTGGCCTGGATGATTACCTT CTGTGTCACCCACACAAACC
A61) ATCCCTTTCTACGGCATCCT TAGGCCTCCTGGTCAATCTC
A9D CACCCAGCTGTCAAAGAGAA GATGAAGCACATCATCAGCAA
[3-Actin CTCTTCCAGCC'ITCC’ITCCT TGTI‘GGCGTACAGGTCTTI‘G

 

73

 

membranes. Membranes were incubated with antibodies for SREBP-1 (IgG-2A4, sc-
13551) (Santa Cruz Biotechnology, San Cruz, CA). HNF-4a (C-19), MLX (N-l7), anti-
goat and anti-rabbit antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz,
CA). ChREBP antibody was obtained from (Novus Biologicals, Littleton, CO). Anti-
mouse and anti-rabbit secondary antibodies were obtained from Bio-Rad (Hercules, CA);
anti-goat antibodies were obtained from Santa Cruz Biotechnology. The SuperSignal
West Pico chemiluminescence kit (Pierce) detection system was used.
Recombinant adenovirus. Cloning of cDNA for Elov12, Elov15 and Elovl6 was described
previously [175]. The coding region for each transcript was ligated into Ad-Easy XL
adenoviral vector system (Stratagene), recombined in BJ 5183 cells and propagated in
XL10 Gold ultra-competent cells. Ad-DNA was packaged into adenoviral particles Ad-
293 cells. The resultant adenovirus was ampliﬁed in l-IEK293 cells.

Recombinant adenovirus expressing dominant negative MLX (Ad-anLm and
the doxycycline inducible nuclear SREBP-1c (Ad-nSREBPl) were obtained from H.
Towle, University of Minnesota, Minneapolis, MN [62,180]. Adenovirus was propagated
and ampliﬁed Hek293 cells. Viral lysates were titered using the Adeno-X Rapid Titer Kit,
Clontech. Confluent primary hepatocytes were infected (5-10 plaque forming units
[PFU]/cell). Using Ad-GFP as a control for infection, greater than 80% of primary
hepatocytes expressed functional protein at the 5-10 PFU/cell level.
In vitro fatty acid elongation assay. Rat liver microsomes were isolated by differential
centrifugation [159]. Elongation reactions were carried out with modiﬁcations to the
procedure described by Moon et al [117]. Brieﬂy, reaction mixtures contained 50 ug

microsomal proteins in a total reaction volume of 100 pl. The reaction constituents are:

74

50 mM potassium phosphate buffer- pH 6.5, 5 11M rotenone (Sigma), 40 11M fatty acyl
CoA (Avanti Polar Lipids, Alibaster, Al and Sigma, St. Louis, MO), 60 11M malonyl
CoA (Sigma) [6.5 dpm/pmol] [2-14C]-malonyl CoA (Perkin Elmer), 1 mM NADPH
(Sigma) 20 uM BSA (fatty acid free). Reactions (at 37°C) were initiated with the
addition of NADPH. When fatty acids were used as substrate, NaOH neutralized fatty
acid (40 uM) replaced fatty acyl CoA. CoASH (at 100 11M), MgC12 (1 mM) and ATP (1
mM) were added to the reaction to generate fatty acyl-CoA. Elongase reactions were
terminated after 20 minutes with the addition of 100 pl 5 N KOH + 10% methanol; lipids
were saponiﬁed for 1 hr at 65°C. The saponiﬁcation reaction was acidiﬁed with 100 pl 5
N HCl; 100 pl ethanol was added to aid hexane extraction of fatty acids. Elongated fatty
acids were collected by 2 independent extractions with hexane (800 pl). Hexane extracts
were pooled and l4C-radioactivity was quantiﬁed by B-scintillation counting. Results are
expressed as Elongase Activity Units, mnoles l4C-malonyl CoA incorporated/mg
protein/20 mins. F ormation of reaction products was dependent on the presence of
NADPH and the fatty acid CoA. Fatty acid elongation products were veriﬁed by reverse?

phase-HPLC chromatography using a ﬂow-through B-scintillation counter [53].

Statistical Analysis. Statistical analysis used Student’s t-test and ANOVA plus post hoc

Tukey HSD test (http://faculty.vassar.edu/lowry/VassarStatshtml).

 

75

 

 

3.3 Results

3.3.1. Elongase and desaturase expression in rat and mouse liver.

Our ﬁrst objective was to compare fatty acid elongase and desaturase expression
in rat and mouse (Fig. 3.1A). Of the 7 elongases identiﬁed in the genomes of these
species, qRT-PCR analysis indicated that only 4 elongases are expressed in liver, Elovll,
Elov12, Elov15 and Elovl6. Based on relative mRNA abundance, the hierarchy of
elongase expression in rat and mouse liver is: Elov15 > Elovll = Elov12 = Elovl6. The
hierarchy of desaturase expression is: A9D (stearoyl CoA desaturase-1, SCD-l) >
Asdesaturase (A5 D) = A6 desaturase (A6D).

Fatty acid elongase activity was assessed using 3 substrates. 16:0-CoA is a
substrate for Elovll and Elovl6; 20:4-CoA is a substrate for Elov12 and Elov15; and 24:0-
CoA is a substrate for Elovll [153]. Elongation of 16:0-CoA to 18:0-CoA was highest in
mouse liver, while elongation of 24:0-COA to 26:0-CoA was comparable amongst speCies.
Differences in elongation activity can be attributed to elongase subtype expression

amongst species.

3.3.2. Role of PPARa in the control of hepatic elongase and desaturase

expression.

Feeding rats the PPARa agonist, WY14643, induces certain hepatic fatty acid
elongases and desaturases and promotes changes in hepatic and plasma lipid composition
[26]. Herein, we evaluate further the role PPARa plays in the control of hepatic elongase
and desaturase expression. The effect of WY14643 on elongase and desaturase
expression in wild type and PPARa null (-/-) mice was examined. Mice were fed a

control diet or one containing WY14643 (at 50 or 500 mg WY14643/kg of diet) for l

76

 

 

 

Figure 3. 1. Fatty acid elongase and desaturase expression in rat, mouse and
human liver. A. Liver from male Spraque-Dawley rats (2-3 months of age) and
C57BL/6 mice (2-4 months of age) were used for RNA extraction and
measurement of elongase and desaturase expression as described in Materials and
Methods. Rats and mice were maintained on chow diets ad lib. mRN A abundance
for each elongase and desaturase was measured by qRT-PCR. Results are
expressed relative to an internal standard, Bactin (transcript/Bactin) mean :
SD,n=4. B. Rat and mouse were extracted for microsomes to assay elongase
activity using three separate substrates, 16:0-CoA, 20:4-CoA and 24:0-COA.
Results are expressed as Elongase Activity (nmoles l4C-malonyl CoA assimilated
into fatty acids/mg protein). Mean : SD,n=4. *p50.05 vs rat liver, ANOVA.

77

3.1A 0.41 *

.°
to
on

C1 rat
I mouse

ogzuiﬁlﬂiﬂ

Elovl1 Elovl2 Elovl5 Elovls DGD D90

.°
MP
01:»

O
L;
0|

Transcript/Actin
O
N

0
d

 

3.18

A

N -h
1 1

X’

D rat
I mouse

—L ‘
l |

Elongase activity

 

onhmmo
l

16:0-GOA 20:4-CoA 24:0-CoA

78

week (Fig. 3.2). Of the 4 elongases expressed in mouse liver, WY14643 induced only
Elov15 and Elovl6, 14- and 4-fold, respectively. Previous studies established that the
PPARa agonist increased desaturase expression [134]. Our results indicate thatiAéD‘and
A9D transcript levels were induced ~6-fold by WY14643, while A5 D was weakly induced.
Elovll and Elov12 mRNA abundance was unresponsive to WY14643 treatment. The
absence of a WY14643 effect on elongase and desaturase expression in PPARa (-/-) mice
indicates that PPARa plays a role in controlling both elongase (Elov15 and Elovl6) and

desaturase (A6D and A9D) expression.

3.3.3. Regulation of elongase and desaturase expression in primary rat

hepatocytes.

Insulin, SREBP-1c [134,181], LXR agonist (T0901317, T1317), glucose,
ChREBP and MLX [62] control desaturase expression [9]. The following studies
determined if these same transcriptional regulatory systems control elongase expression
in rat primary hepatocytes.

Regulation of elongases and desaturases by insan and LXR agonist.

Insulin regulates lipid synthesis, at least in part, by controlling SREBP-l nuclear
abundance [179,182]. LXR agonist stimulate lipogenesis through direct and indirect
mechanisms [183]. LXR/RXR heterodimers bind LXRE in promoters of responsive
lipogenic genes. LXR agonist also induce lipogenic gene expression through the
induction of SREBP-1c gene transcription [182,183]. The effect of insulin and T1317 on
hepatocyte elongase and desaturase expression was examined.

In the absence of insulin or T1317, SREBP-1 nuclear abundance in hepatocytes is

low (Fig. 3.3 insert). Treatment of rat primary hepatocytes with insulin or T1317 induced

79

16

12

(P

 

[00 I50 0500 no 350 msoo

 

 

 

 

Elovl-1 Elovl-2 Elovl-5 Elovl-6 DSD 06D D9D

Figure 3. 2. The role of PPARa in the control of hepatic elongase and desaturase
expression. Homozygous wild type and PPARa-null (-/-) mice on a Sv/129 genetic
background [177,178] were fed either a control diet or one containing WY14643 (at 50 or
500 mg/kg of diet; Bioserv, Piscataway, NJ) for 1 week. Liver RNA was extracted and
used as template for qRT-PCR. Results are reported as Fold Change in mRNA abundance
(transcript/cyclophilin) for each enzyme, mean : SD,n=4. *p50.05, Students T-T est vs
wild type on a control diet.

80

nuclear SREBP-1 ~4-fold, but had no effect on SREBP-2 nuclear abundance. T1317 (1
uM) had no signiﬁcant effect on Elovll, -2 or -5 expression in rat primary hepatocytes
and only modestly induced Elovl6, ~l .5-fold (Fig. 3.3). In contrast, all 3 desaturases were
induced between 2- and 15-fold; A9D was most responsive. Insulin induced Elovl6 and
A51) 51.5-fold, while A6D and A9D were induced >3-fold. Co-treatment with insulin and
T1317 had no additive effect on SREBP-1 nuclear abundance or the expression of any
elongase or desaturase.

These studies suggest that the induction of Elovl6, ASD, A6D and A9D by insulin
and T1317 likely involves control of SREBP-1 nuclear abundance. Although others have
reported that insulin induces LXRa in primary hepatocytes [184], we found no evidence
for an insulin effect on either LXRa or LXRa mRNA abundance (not shown).
Glucocorticoids, T3 and leptin had no effect on elongase expression in primary rat
hepatocytes. None of the hormones tested induced hepatic Elovl3, Elovl4 or Elovl7 (not
shown).

Effect of over expressed nuclear SREBP-1c on elongase and desaturase expression.

To further evaluate the SREBP-1 control of elongase and desaturase expression,
primary hepatocytes were infected with a recombinant adenovirus containing a
doxycycline-inducible nuclear form of SREBP-1c [Ad-nSREBP-lc] [180]. These cells
received no insulin or LXR agonist. Treatment of primary hepatocytes with doxycycline
signiﬁcantly induced the nuclear form of SREBP-1c (not shown) as well as the
endogenous SREBP-1 transcript (Fig. 3.4). The endogenous SREBP-1c promoter

contains a SRE and is activated by elevated levels of nuclear SREBP [182]. Over

81

Veh Ins T1317 Both

H—ir-A-y—L-‘r-L-s
InSREBP-1 | W]
|nSREBP-2Ip~———~|

    

 

lEIVehicle I Insulin 1:1 T1317 8 Both]

Fold Change
3 a

01

 

 

Elovl-2 Elovl-5 Elovl-6 D5D D6D DQD

Elovl-1

Figure 3. 3. Effect of insulin and LXR agonist (Tl317) on fatty acid elongase and
desaturase expression in rat primary hepatocytes. Rat primary hepatocytes were
maintained in Williams E medium containing 10 mM lactate, 10 nM dexamethasone .

(DEX) with no insulin overnight. The next day, cells were switched to Williams E
medium containing 25 mM glucose, 10 nM DEX (Vehicle) in the presence or absence of

insulin (100 nM) or the LXR agonist, T0901317 [T13l7, Cayman Chemical Co. Ann
Arbor, MI] (1 11M). After 24 hours of treatment, cells were harvested for RNA extraction

and the measurement of nuclear SREBP-1 and SREBP-2 by irnmunoblot (insert,
duplicate samples/treatment). Elongase and desaturase mRN A abundance was quantiﬁed

by qRT-PCR. Results are expressed as the Fold Change in mRNA versus Vehicle
(transcript/cyclophilin); mean : SD,n=4. These results are representative of 2 separate

studies. *p_<_0.05 vs vehicle, ANOVA.

82

 

expressed nSREBP-lc induced transcripts encoding Elov12, Elovl6, A5 D, A6D and A9D
~3-fold at the maximal doxycycline dose.

In a second study, Ad-nSREBP-lc infected cells were treated with insulin or
T1317 (Fig. 3.4B). The goal of this study was to determine if insulin or T1317 had effects
on elongase or desaturase expression independent of SREBP-1c. Over expressed SREBP-
1c (Ad-SREBP-lc) induced Elov12 (2.6-fold) and Elovl6 (6-fold), while ASD, A6D and
A9D transcripts were induced 3.6-, 4.8- and 3.1—fold, respectively. Addition of insulin had
no additional effect on the expression of any elongase or desaturase. Addition of T1317
induced Elovll 50%, while A9D was induced 4-fold. The expression of no other elongase
or desaturase was affected by T1317. Based on these results, the induction of Elovl6, ASD,
A6D by insulin and T1317 (Fig. 3.4A) is due to elevated SREBP-1 nuclear abundance.
Only A9D expression is induced by elevated SREBP-1 nuclear abundance and
independently by LXR agonist. Elovll was modestly responsive to LXR agonist only
when SREBP-1 was over expressed. Elov15 mRNA abundance was unresponsive to
changes in insulin, LXR agonist and SREBP-1 nuclear abundance (Fig. 3.4B).

Glucose, ChREBP & MLX regulation of elongase and desaturase expression.

Insulin stimulated glucose metabolism induces the translocation of ChREBP to
the nucleus where ChREBP/MLX heterodimers bind to carbohydrate regulatory elements
(ChoRE) in promoters of glucose responsive genes involved in glycolysis and lipogenesis
[185,186]. The effect of glucose on elongase and desaturase expression in primary
hepatocytes was examined. Primary hepatocytes were maintained in medium containing

lactate (20 mM) and insulin (100 nM) or switched to medium containing 25 mM glucose

83

Figure 3. 4. Effect of over expressed nuclear SREBP-1c on elongase and
desaturase expression. A. Primary rat hepatocytes in Williams E medium + 10
mM lactate + 10 nM DEX, but no insulin, were infected with a recombinant
adenovirus expressing the nuclear form of SREBP-1c under the control of
doxycycline [180]. Cells were switched to Williams E medium + 25 mM glucose
with no insulin in the absence or presence of doxycycline (25, 200 or 250 ng/ml).
Cells were harvested 24 hrs afterward for RNA extraction and measurement of
elongase and desaturase mRN A abundance by qRT-PCR (transcript/cyclophilin).
Endogenous SREBP-1 expression was quantiﬁed by northern analysis. Results are
represented as Fold Induction by SREBP-1c, mean i SD,n=4. These results are
representative of 2 separate studies. *p<0.05 vs vehicle, ANOVA. B. Primary rat
hepatocytes in Williams E medium + 10 mM lactate + 10 nM DEX, but no
insulin, were infected with a recombinant adenovirus expressing the nuclear form
of SREBP-1c under the control of doxycycline [180]. Cells were switched to
Williams E medium + 25 mM glucose with doxycycline at 250 ng/ml, in the
absence or presence of insulin (100 nM) or T1317 (1 uM). Cells were harvested
24 hrs afterward for RNA extraction and measurement of elongase and desaturase
mRN A abundance by qRT-PCR (transcript/cyclophilin). Results are represented
as Fold Induction by insulin or T1317, mean : SD,n=3. These results are
representative of 1 study. *p<0.05 vs vehicle, ANOVA.

84

9‘
A
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a: 5 . DOIZ5D1OOI25O . ,
0) ﬁg
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= 2 - =3: :3: ' i it '~:
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volllslzslsi iil ilsils
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3'43“ 0.7 2.6 1.6 6 3.8 4.6 3.1,
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0

LI.
“II II III I III
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Elovl-1 Elovl-2 Elovl- 5 Elovl-6 D5D

85

plus insulin. This treatment induced the accumulation of ChREBP in hepatocyte nuclei
with no effect on MLX nuclear abundance Fig. 3.5 (insert). L—pyruvate kinase (LPK), a
glucose-responsive transcript, is controlled at the transcriptional level by binding the
ChREBP/MLX heterodimer to its promoter [62]. Switching hepatocytes from lactate to
glucose induced mRNAL-pK ~60-fold (Fig. 3.5). Elovl6 and A9D mRNAs were induced
7.6- and 10-fold, respectively. No other elongase or desaturase was affected by glucose.

To verify the role ChREBP/MLX heterodimers play in this regulatory process,
primary hepatocytes in lactate-containing medium were infected with recombinant
adenovirus expressing luciferase (Ad-Luc) or a dominant negative version of MLX (Ade
anLX). MLX is required for ChREBP to bind ChoRE in glucose-responsive promoters.
Overexpressed dn-MLX attenuates the glucose induction of L-PK [173].

Infecting cells with Ad-Luc had no effect on the glucose induction of Elovl6, A9D
or L-PK (Fig. 3.5). Infection of cells with Ad-anLX completely attenuated the glucose
induction of L-PK, Elovl6 and A9D. Ad-Luc or Ad-anLX expression in primary
hepatocytes had no effect on Elovll, Elov12, Elov15, ASD or A6D. These studies indicate
that glucose regulates both Elovl6 and A9D expression by mechanisms that control the
nuclear abundance of ChREBP and MLX. ChREBP and MLX play no role in the control

of Elovll, Elov12, Elov15, A5 D or A6D expression.

3.3.4. Metabolism of fatty acids by fatty acid elongases.

Fatty acid elongases have overlapping substrate speciﬁcities [153,187]. Herein,
the substrate speciﬁcity of 3 fatty acid elongases was examined. Recombinant adenovirus
expressing luciferase (Ad-Luc), Elov12 (Ad-Elov12), Elov15 (Ad-Elov15) and Elovl6 (Ad-

Elovl6) were used to over express these enzymes in rat primary hepatocytes. Substrate

86

Glucose
Lac 1.5 6 24 Hr.

I ChREBP [. a... «I-v «Iil DNolnfection-Lactate -

I Mlx P - 9: INolnfection-Glucose

D Ad-Luc + Glucose
lAd-anLX + Glucose

I
G
1
G
4
c

 

 

N

.3
0|
I

.0
01
I

 

 

.31
35 .
D
D
D
>0

Elovl-1 Elovl-2 Elovl-5 Elovl-6 DSD L-PK

mRNA Abundance
Relative to Glucose Treatment
A
L

 

 

O
I

Figure 3. 5. Effect of glucose on elongase and desaturase expression. Primary rat
hepatocytes in Williams E medium + 10 mM lactate + 10 nM DEX + 10 nM insulin were
switched to Williams E medium + 25 mM glucose + 10 nM DEX + 10 nM insulin. After
24 hrs of treatment cells were harvested for extraction of nuclear proteins and RNA. The
nuclear abundance of ChREBP and MLX was measured by irnmunoblotting at the times
indicated [59] (Insert). Elongase, desaturase and L-pyruvate kinase (L-PK) mRNA i -
abundance was quantiﬁed by qRT-PCR (transcript/cyclophilin). White bars, cells
maintained in lactate; black bars, cells switched to glucose.

, In a second experiment, primary hepatocytes in Williams E medium + 10 mM
lactate + 10 nM DEX + 10 nM insulin were infected with recombinant adenovirus
expressing luciferase (Luc, gray bars) or dominant negative MLX (dn-MLX, checked
bars). After 24 hours of treatment, cells were switched to Williams E medium + 25 mM
glucose + 10 nM DEX + 10 nM insulin. After 24 hrs of glucose treatment, cells were
harvested for RNA extraction and elongase, desaturase and L-pyruvate kinase (L-PK)
mRN A expression was quantiﬁed by qRT-PCR (transcript/cyclophilin). Results are
represented as mRN A Abundance, Relative to Glucose Treatment, mean i SD,n=4.
These results are representative of 2 separate studies. *p<0.001 vs glucose treated cells,
ANO VA.

87

speciﬁcity was examined using saturate (16:0), monounsaturated (16:1,n-7), and
polyunsaturated (18:3,n-6, 20:5,n-3 and 22:5,n—3) fatty acids. Ad-Luc infected cells
served as a control for basal elongase activity using the various substrates. Ad-Elov12
infected hepatocytes elongate only 20:5,n-3 and 22:5,n-3 (Fig. 3.6). Ad-ElovlS infected
cells elongated 16:1,n-7, 18:3,n-6 and 20:5,n-3. Ad-Elovl6 infected cells elongated only
16:0 and 16:1,n-7 (Fig. 3.6). 3

Expression of hepatic elongases and desaturases is controlled by PPARa, SREBP-
] and ChREBP/MLX (Fig. 3.2-3.5). To determine if changes in elongase and desaturase
activity impact hepatocyte fatty acid composition, primary hepatocytes incubated with
insulin (induces SREBP-1), PPARa agonists and glucose (induces ChREBP) were
examined for the capacity to elongate and desaturate l4C-16:0. Primary hepatocytes were
maintained in Williams E media with lactate and no insulin overnight. This treatment
effectively lowers SREBP-1 and ChREBP nuclear abundance [59,179]. The next day
cells were treated with lactate or glucose containing media in the absence or presence of
insulin and WY14643. All cells received 100 uM l4C-16:01 After 24 hrs of treatment,
cells were harvested for lipid extraction and analysis of elongation and desaturation
products by RP-HPLC.

Cells maintained in lactate media with no insulin or WY14643 do not desaturate
16:0 (Fig. 3.7B, C & D), but elongate 16:0 to 18:0. Addition of insulin or WY14643 to
lactate-treated cells induces formation of 16:1,n-7, while only WY14643 induces
formation of 18:1 (n-7/n-9). The combination of these treatments has no additive effect.

In cells switched to glucose-containing media, 16:0 desaturation to 16:1,n-7 and

the elongation and desaturation of 16:0 to 18:1,n-7/n-9 is induced when compared‘to the

88

 

D Ad-Luc D Ad-Elovl-Z
I Ad-Elovl-5 I] Ad-Elovl-6

0)
O
I

 

 

 

01
T

   

_L —L N N
01 O
I I

O
I

XXXXXXXXXXXXXXXX

 

 

 

Elongase Activrty
(nmolelmg protein)

 

 

 

 

 

Klil lALALLIiLAKilLiILLI

our

Palmitate 16:1,n—7 18:3,n-6 20:5,n-3 22:5,n—3

Fatty Acid Substrate

Figure 3. 6. Substrate specificity of hepatic fatty acid elongases. Recombinant
adenovirus expressing luciferase (Luc) [white bar], Elov12 [checked bar], Elov15 [black
bar] and Elovl6 [gray bar] were used to infect primary hepatocytes (5 PFU/cell). After 24
hrs, cells were harvested for elongase activity using various fatty acid substrates (see
Methods and Materials). Results are expressed as Elongase Activity (nmoles 4C-malonyl
CoA assimilated into fatty acid/mg protein). Results are represented as mean 1; SD, n=3.
Results are representative of 2 separate studies. - . -

89

lactate-treated cells. The combination of glucose + insulin or glucose + WY14643
induced formation of both 16:1,n-7 and 18:1,n-7/n-9 synergistically. Glucose, insulin and
WY14643 have no apparent effect on the elongation of 16:0 to 18:0. Since 18:0 is a
substrate for A9D, measuring changes in 18:0 may not accurately reﬂect effects of these
treatments on elongation activity. In an effort to reveal an effect on elongation, we
examined the effect of glucose, insulin and WY14643 on the 18:1 to 16:1 ratio (Fig.
3.7E). This ratio would remain constant if these treatments did not regulate elongation.
The most impressive effect on the 18:1/16:1 ratio is seen in cells maintained in lactate-
containing medium supplemented with WY14643. Such studies indicate that hepatocyte
levels of 18:1 (n-7/n-9) are controlled by both elongation and desaturation pathways. The
elongation pathway is not a constitutive pathway. Cellular levels of 18:1 are not

determined solely by regulating A9D expression.

3.3.5. Regulation of elongase and desaturase expression in animal

models of metabolic disease.

PPARa [188], SREBP-1 [189] and glucose metabolism, ChREBP & MLX [186]

and LXR [190] play an important role in metabolic diseases like diabetes and obesity .
Herein, we will determine if changes in hepatic lipid metabolism and composition
induced by diabetes and obesity can be attributed to changes in elongase and desaturase
expression. Three metabolic disorders were examined, streptozotocin-induced diabetes,
glucose intolerance induced by high fat diets, obesity induced by leptin deﬁciency.
Nuclear levels of SREBP-1, ChREBP, MLX and HNF-4a were monitored to correlate
changes in nuclear content of these transcription factors with changes in elongase and

desaturase expression.

90

14 Elovl-6 A’D
3.7A. C-16:0 ——>18:O —>18:1,n-9

 

 

 

 

 

 

 

A9D
Elovl5
16:1,"..7 —18:1,n-7
Elovl-6
3.73. D Lactate I Glucose
_ 10 -
0
g ‘- 8 '
.2 to
a "' 6
‘1’ 3
E o 4
0 co
0 ‘- 2
.\°
0 .
Veh lnsulin WY14643 Both
3.76. CI Lactate I Glucose

 

 

 

d

ON-FOQO

 

% Conversion of
16 0 to 181

 

Veh Insulin WY14643 Both

91

3.7D.

 

 

 

 

 

 

 

 

   

 

 

 

 

 

 

 

 

 

El Lactate I Glucose

'8 10 -

E: 8 -

i1; 5 '

2° 4 '

8:2 2 '

:2 0 L—

Veh Insulin WY14643 Both
3.7E.
El Lactate I Glucose

21.5 -

z; 1 .

“0.5 - l I ~

0

33 o

"‘ Veh Insulin WY14643 Both

Figure 3. 7. Effects of insulin and WY14643 on monounsaturated fatty acid
synthesis. A. The pathway for conversion of 16:0 to 16:1,n-7, 18:1,n-7 and 18:1,n-9 by
Elov15, Elovl6 and A9D is diagrammed. B-E. Primary rat hepatocytes in cultured in
Williams E + 10 nM Dex + 10 mM lactate overnight were maintained in the same
medium or switched to Williams E + 10 nM Dex + 25 mM glucose in the absence or
presence of insulin (100 nM) or WY14643 (50 uM). Cells received 100 uM l4C-l6:0 +
20 uM bovine serum albumin for 24 hrs. After 24 hrs of treatment, cells were extracted
for total lipid and the lipid was saponiﬁed and the distribution of 14C-amongst 16:0,
16:1,n-7, 18:0 and 18:1,n-7 and n-9 was measured by RP-HPLC and a ﬂow-through B-
scintillation counter. Technical limitations preclude resolution of 18:1,n-7 and 18:1,n-9
by RP-I-IPLC. As such, the results are reported as 18:1. Results are reported as (B) %
Conversion of l4C-16:0 to l4C-16:1; (C) % Conversion of l4C-16:0 to l4C-18:1; (D) %
Conversion of l4C-l6:0 to l4C-l8:0; (E) ratio of l4C-18:1 to l4C-l6:l, an index of
elongation. Results are the mean of duplicate sample. Results are representative of 2
separate studies.

92

Streptozotocin-Indueed Diabetes.

Rats made diabetic using streptozotocin have elevated blood glucose (378 i 21
mg/dl) when compared to control animals (77.9 i 5.2 mg/dl). Liver nuclei from diabetic
rats contained little detectable nuclear SREBP-1 and suppressed levels of MLX, but no
signiﬁcant change in ChREBP or HNF-4a (Fig. 3.8A). Diabetes suppressed the
expression of lipogenic and glycolytic genes, e.g., fatty acid synthase and L-PK, by 370%
[176], while expression of the gluconeogenic enzyme, phosphoenolpyruvate
carboxykinase (Peka) was induced 3-fold (Fig. 3.8). The decline in the nuclear
abundance of SREBP-1 and MLX correlated with a ~45% decline in Elovl6 and a >95%
decline in A9D mRN A abundance. Other elongases and desaturases remained unaffected.
Examination of hepatic lipid composition revealed a signiﬁcant 30% decline in 16:0 in
diabetic animals, but no change in other saturated, mono- or polytursaturated fatty acids.
The decline in 16:0 is consistent with the decline in de novo lipogenesis in livers of
diabetic rats [176].

Glucose-Intolerance Induced by High Fat Diets.

High fat diets induce glucose intolerance, insulin resistance, fatty liver and altered
hepatic metabolism [191,192]. To examine the effects of diet induced diabetes on
elongaSe and desaturase expression, male C57BL/6 mice were fed a diet with 10% of the
calories as fat (low fat, lard diet) or 60% of the calories as fat (high fat, lard diet).
Animals fed the low fat diet are 29 g and have blood glucose and insulin levels within the
normal range, 121 mg/dl and 0.5 ng/ml (Table 2). Animals fed the high fat diet are ~44 g,
with elevated blood glucose (152 mg/dl) and insulin (3.9 ng/ml). When compared to the

low fat fed group, high fat fed animals were glucose intolerant (Fig. 3.9A) and have fatty

93

e
#3
6°
\6

3.8A Control Diabetic «0

Value

     

"

Microsomal SREBP—1 0.7

3.8B

N (a) h-
1

Fold Change

 

 

O

Elovl1 Elovl2 Elovl5 Elov|6 D5D 06D DQD Peka

Figure 3. 8. Effect of streptozotocin-induced diabetes on hepatic elongase and
desaturase expression. Male Spraque-Dawley rats were made diabetic using
streptozotocin as described in Materials and Methods. Livers from control and diabetic
animals were used for the isolation of nuclear and microsomal proteins for
immunoblotting (A) and RNA extraction for qRT-PCR (B). [A] Effect of diabetes on
SREBP-l (microsomal [pSREBP-l] and nuclear [nSREBP-1]), and nuclear ChREBP,
MLX and HNF-4a abundance. Protein levels were measured by immunoblot (Materials
and Methods). Duplicate samples for each treatment are shown. The effect of diabetes on
the abundance of these proteins was quantiﬁed and expressed as Fold Change (mean,n=5)
induced by diabetes. Statistical signiﬁcance (p-value) was assessed by Student’s T-test.
[B] Effect of diabetes on elongase, desaturase and phosphoenolpyruvate carboxykinase
(Peka) expression. Transcripts encoding fatty acid elongases, fatty acid desaturases and
cyclophilin were quantiﬁed by qRT-PCR, while Peka was quantiﬁed by northern blot
analysis. Results are expressed as Fold Change in expression (transcript/cyclophilin)
induced by diabetes. *p<0.05 vs control; N=5/group, Students T-test.

94

livers [191]. Nuclear levels of SREBP-l and MLX were suppressed 60% and 50% (Fig.
3.9B), while ChREBP and HNF-4u nuclear levels remained unchanged.

Expression levels of Elovll and Elov12 were not signiﬁcantly affected by high
dietary fat (Fig. 3.9C). Elov15 and Elovl6 mRNA abundance, however, was suppressed
by 50% and 75% with the high fat intake. The high fat diet had no effect on A5 D or A6D,
but signiﬁcantly (280%) suppressed A9D expression. High fat diets signiﬁcantly
suppressed fatty acid elongation of 16:0-CoA and 20:4-CoA, but not 24:0-CoA (Fig.
3.9D).

Palmitic (16:0), stearic (18:0), oleic (18:1,n-9) and linoleic (18:2,n-6) acids
represent the major fatty acids in both diets. Analysis of hepatic lipid composition
indicates that 18:2,n-6 accumulates relative to 20:4,n-6 in livers of high fat fed animals
(Fig. 3.9E). 18:2,n-6, an essential fatty acid, is converted to 20:4,n-6. Since neither diet
contains 20:4,n-6, the appearance of 20:4,n-6 in the livers requires conversion from
18:2,n-6 to 20:4,n-6 by elongation (Elov12 and Elov15) and desaturation (A5 D and A6D).
In the low fat diet, the ratio of 20:4,n-6 to 18:2,n-6 is 0.75. In the high fat diet the ratio is
0.3. Failure to convert 18:2,n-6 to 20:4,n-6 is consistent with a decline an Elov15
expression, a key enzyme involved in PUFA synthesis.

Elongase and Desaturase Expression in Livers of Lean (Lep°b’+) and Obese (Lep°w°b)
Mice.

Defective leptin expression in C57BL/6J-Lep0b’0b

mice leads to hyperphagia,
hyperinsulinemia, insulin resistance and obesity [193]. When compared to the lean
(LepOb/U littennates, obese animals are heavier and have elevated blood levels of glucose

and insulin (Table 3.2). The livers of obese mice are massively engorged with lipid,

95

Figure 3. 9. Effect of high fat diets on hepatic elongase and desaturase
expression. Male C57BL/6 mice were fed 10% or 60% lard diets for 10 weeks as
described in (Materials and Methods) [191]. [A] After nine weeks on the low and
high fat diets, mice were assessed for glucose tolerance (Materials and Methods).
Blood glucose was measured at the times indicated: 10% Cal (as fat), solid line-
ﬁlled circle, 60% Cal (as fat), dashed line-ﬁlled square. Results are expressed as
Blood Glucose (mg/d1), mean : SD,n=8 in each group. [B]. Effect of dietary fat
on SREBP (nricrosomal [pSREBP-l] and nuclear [nSREBP-1]), ChREBP, MLX
and HNF-4a. After ten weeks on the diet, livers were recovered for nuclear and
microsomal protein and RNA extraction as in Fig. 8. Nuclear and microsomal
proteins were measured as described in Materials and Methods. Representative
immunoblots from duplicate samples for each treatment are shown. The effect of
dietary fat on the abundance of these proteins was quantiﬁed and expressed as
Fold Change (mean,n=4 animals/group) induced by the high fat diet. Statistical
signiﬁcance (p-value) was assessed by Student ’s T-test. [C] Effect of dietary fat
on elongase and desaturase expression. RNA was extracted and used for qRT-
PCR analysis of elongase and desaturase expression. Results are expressed as
Fold Change (transcript/cyclophilin), mean : SD,n=8. [D] Effect of dietary fat on
elongase activity. Hepatic microsomal preparations were used for fatty acid
elongase assays (Materials and Methods). Results are expressed as Elongase
Activity, (nmoles l4C-malonyl CoA assimilated into fatty acids/mg protein).
Mean :1; SD,n=8. *p50.05 vs 10% Calories as fat, Student’s T -Test. [B] Effect of
dietary fat on hepatic lipid composition. Total lipids were extracted and
saponiﬁed; fatty acid levels were quantiﬁed by RP-HPLC (Materials and
Methods). Results are expressed as Fatty Acid Mole%, mean : SD,n=8/ group;
*p50.05 vs 10% lard diet, Students T -test. [10% Calories as Fat (open bars) and
60% Calories as Fat (ﬁlled bars)].

96

3.9A

coo
E o

a) i“ 60A: Cal
E 400- \

v \
a, / i
w

8 300—

2

(D

'8 200—

a": 10% Cal

 

 

 

 

§

0 20 40 60 80 100
Minutes after Glucose Administration

393 10% Cal 60% Cal

Fold Change
.°
0. .5

O

 

 

 

Elovl-1Elovl-2 Elovl-5 Elovl-6 D5D D60 D90

97

010% Calories as Fat

D
3
3

 

 

 

 

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F
s
a
s I
.m
b
a
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24:0-CoA

20:4-CoA

16:0-COA

 
 

010% Calories as Fat
I 60% Calories as Fat

0 0 0 0 0
5 4 3 2 1

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s
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98

predominantly as neutral lipid. Hepatic nuclei derived from obese mice have a 2.6- and 2-
fold increase in SREBP-1 and MLX nuclear abundance, but no change in ChREBP or
HNF-4a when compared to the lean littermates. Expression levels of Elov15, Elovl6, A5 D,
A6D and A9D are increased in livers of obese mice when compared to the lean littermates.
Obesity also results in the induction of lipogenic gene expression (ACC, F AS) and
PPARa target genes, e.g., Cyp4A and AOX (not shown). Fatty acid elongation of 16:0-
CoA is increased 2-fold, while elongation of 20:4-CoA and 24:0-CoA was not affected
(Fig. 3.10C).

Fatty acid analysis of livers from lean and obese mice reveals a ~9-fold increase
in total esteriﬁed fatty acid. Much of this increase is in the form of neutral lipid esteriﬁed
with 18:1 (n-7 and n-9) (Fig. 3.10D). While monounsaturated fatty acid abundance
increased, hepatic PUFA content, i.e., 18:2,n-6, 20:4,n-6 and 22:6,n-3, declined. This
might appear inconsistent with the observed increase in fatty acid elongase activity and

elongase and desaturase expression. LepOb/Ob

mice, however, are hyperphagic; ingestion of
excessive calories as carbohydrate elevates plasma insulin (Table 2) and enhances de
novo lipogenesis and monounsaturated fatty acid synthesis. Induction of Elov15, Elovl6
and A9D expression by activated PPARa and elevated SREBP-1 and MLX nuclear
content facilitates monounsaturated fatty acid (18:1,n-7 and 18:1,n-9) synthesis which is
assimilated into neutral lipid. Elongation, as well as desaturation, are key steps for
converting the end products of de novo lipogenesis to monounsaturated fatty acids. These
studies indicate that fatty acid elongation is induced along with de novo lipogenesis and
Ag-desaturation. Induction of these pathways contributes to the fatty liver phenotype

ob/ob

characteristic of Lep mice.

99

Figure 3. 10. Effect of leptin deﬁciency on hepatic elongase and desaturase
expression. Male lean C57BL/6J-Lep0b * and obese (C57BL/6J-Lep0b/0b) mice
(B6.V-Lep ob/J , #000632, Jackson Labs) were maintained on a Harlan-Teklad
laboratory chow (#8640) diet and water ad lib. Livers from these mice were used
for the isolation of nuclear and microsomal protein for immunoblotting (A) and
RNA extraction for qRT-PCR (B). [A] Effect of obesity on SREBP-1
(microsomal [pSREBP-l] and nuclear [nSREBP-1]) and nuclear ChREBP, MLX
and HNF-4a abundance. Protein levels were measured by immunoblot (Materials
and Methods). Triplicate samples for each phenotype are shown. The effect of
leptin deﬁciency on the abundance of these proteins was quantiﬁed and expressed
as Fold Change (mean,n=4) induced by leptin deﬁciency. Statistical signiﬁcance
(p-value) was assessed by Student’s T-test. [B] Effect of leptin deﬁciency on
elongase and desaturase expression. RNA was extracted and used for qRT-PCR
analysis of elongase and desaturase expression. Results are expressed as Fold
Change (transcript/cyclophilin), mean : SD,n=4. *p<0.001 vs Lean; Students T -
test. [C] Effect of leptin deﬁciency on elongase activity. Hepatic nricrosomal
preparations were used for fatty acid elongase assays (Materials and Methods).
Results are expressed as Elongase Activity, (nmoles l4C-malonyl CoA assimilated
into fatty acids/mg protein). Mean j; SD,nﬂ. *p_<_0.01 vs Lean, Student is T -Test.
D. Effect of leptin deﬁciency on hepatic lipid composition. Total lipids were
extracted and saponiﬁed; fatty acid levels were quantiﬁed by RP-HPLC
(Materials and Methods). Results are expressed as Fatty Acid Mole%, mean i
SD,n=4/group; *p50.01 vs Lean animals, Students T -test. [Lean (open bars) and
Obese (ﬁlled bars)].

100

 

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102

Elongase and Desaturase Expression in Livers of TCDD treated mice.

TCDD is a toxic compound causing controversial human health effects by altering
gene expression via the activation of the aryl hydrocarbon receptor (AhR), a member of
the basic-helix-loop-helix-PAS (bHLH-PAS) family [194]. Hepatotoxicity is the severe
result of TCDD exposure and is characterized by hepatomegaly accompanied by
hepatocyte hypertrophy, fat accumulation, immune inﬁltration, necrosis, and alterations
in liver enzymes [195]. Expression level of Elov15 is increased, whereas Elovl-2, Elovl-6
and A5 D are suppressed by 50% to 60% in livers of TCDD treated mice when compared
to the control mice (Fig. 3.11 A). The TCDD treatment had no effect on Elovll, A6D or
A9D expression. Moreover, TCDD treatment signiﬁcantly decreases fatty acid elongation
of 16:0-CoA, but not 20:4-COA and 24:0-COA (Fig. 3.11 B). This is due to the
suppressed expression of Elovl6 by TCDD treatment.

Fatty acid analysis of livers from TCDD treated mice reveals a ~2-fold increase in
total esteriﬁed fatty acid. Much of this increase is due to the accumulation of 16:0, 18:1n—
9 and 18:2,n-6 (Fig. 3.11C). Conversion of 18:2,n-6 to 20:4,n-6 is suppressed by 50% in
TCDD treated mice, which is not consistent with an increase in Elov15 expression (Fig.

3.11D). The decreased expression of A5 D may account for it.

3.4. Discussion

F atty acid elongation and desaturation are two key metabolic routes for the
synthesis of saturated, mono- and polyunsaturated fatty acids. Of these, fatty acid
desaturases have received considerable attention for their regulation by hormones and

nutrient and their capacity to generate speciﬁc unsaturated fatty acids. The outcome of

103

Figure 3. 11. Effect of TCDD treatment on hepatic elongase and desaturase
expression. Male C57BL/6 mice mice were treated by gavage with 0.1 ml of
sesame oil for a nominal dose of 0 (vehicle control) or 30 ug/kg body weight of
TCDD for 168hr as described in (Materials and Methods) [200]. [A] Effect of
TCDD on elongase and desaturase expression. RNA was extracted and used for
qRT-PCR analysis of elongase and desaturase expression. Results are expressed
as Fold Change (TCDD/V EH), mean : SD,n=4. [B] Effect of TCDD on elongase
activity. Hepatic microsomal preparations were used for fatty acid elongase
assays (Materials and Methods). Results are expressed as Elongase Activity,
(nmoles l4C-malonyl CoA assimilated into fatty acids/mg protein). Mean :
SD,n=4. *p50.05 vs Veh, Student ’3 T -Test. [C.D] Effect of TCDD on hepatic
lipid composition. Total lipids were extracted and saponiﬁed; fatty acid levels
were quantiﬁed by RP-HPLC (Materials and Methods). Results are expressed as
Fatty Acid Mole%, mean i SD,n=4/group.

104

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these studies indicates that desaturases are well-regulated enzymes that play an important
role in cellular and whole body lipid composition [9,160]. One of these enzymes, A9D
(stearoyl CoA desaturase-1), has emerged as a key enzyme in the control of whole body
lipid composition [196].

In contrast to the desaturases, fatty acid elongase have only recently been
recognized as proteins regulated at the pretranslational level [139,187,197]. Our previous
studies indicated that fatty acid elongases are regulated by tissue-speciﬁc and nutritional
factors and during postnatal development [26]. Those studies implicated certain
transcription factors, like SREBP-1 and PPARa, as regulators of both elongase and
desaturase expression. The current report extends those previous observations by
evaluating the role of several hormones (insulin, T3, glucocorticoids and leptin),
transcription factors (SREBP-1c, PPARa, LXR, ChREBP and MLX) and nutrients
(glucose and fat) in the control of hepatic elongase and desaturase gene expression, fatty
acid elongase activity and lipid composition. Finding the involvement of these factors in
the control of elongase expression prompted studies to evaluate how these enzymes were
regulated in metabolic disease. The outcome of these studies has provided new
information on how changes in both elongase and desaturase expression in metabolic
disease contributes to hepatic lipid composition.

Seven fatty acid elongase subtypes (Elovll to Elovl7) have been identiﬁed in the
genomes of the rat, mouse and human genomes (www.cnsembl.org). Of these, 4 elongase
subtypes are expressed in rat & mouse liver (Fig. 3.1). The hierarchy for hepatic

expression of these enzymes is sirrrilar in these two species:

106

 

Table 3. 2. Dietary composition and physiological parameters of C57BL/6 mice

 

 

Low Fat versus High Fat Lean versus Obese
Parameter Low Fat High Fat Chow Chow
Calories as fat (%) 10 60 20.3 20.3
Calories as carbohydrate 70 20 55.4 55.4
(%)
Calories as protein (%) 20 20 24.4 24.4
Body weight (g) 29.0 i 1.0 44.0 :t 23.7 d: 1.5 55.1 i 2.8

1.0

Fasting blood glucose 121.0 :1: 1.0 152.0 :t 1 12.0 i 11 266.0 i
(mg/d1) 0.3 69.0
Plasma insulin (ng/ml) 0.5 :t 0.05 3.9 i 0.4 0.2 + 0.06 15.4 d: 1.04
Animals per group 8 8 4 4

 

107

 

Elov15>Elovl1=Elov12=Elovl6. Analysis of elongase activity indicates that mouse liver
has the highest elongase activity.

Elovll. Elovll is a low abundance elongase in liver of rat and mouse. Based on
studies in yeast, Elovll elongates a broad array of saturated and monounsaturated fatty
acids. Elovll expression, however, is not regulated by any physiological manipulation
used so far in this or our previous study [26]. Thus, changes in hepatic lipid composition
induced during postnatal development, fasting and refeeding, diabetes, obesity, dietary fat,
LXR or PPARa. agonist can not be attributed to changes in Elovll activity. Hepatic
Elovll appears to be expressed constitutively.

Elov12. Elov12 is also a low abundance elongase in liver of rat and mouse. In
contrast to other elongases, Elov12 has a very narrow substrate preference; it elongates
C20 and C22 PUFA (Fig. 3.6). As such, Elov12 participates in the conversion of essential
fatty acid precursors to end products of PUFA synthesis, i.e., 20:4,n-6 and 22:6,n-3. Like
Elovll, Elov12 is not regulated by any factors examined in this report or our previous
report. The exception to this is the induction of Elov12 mRNA following over expression
of SREBP-1c (Fig. 3.3). Since insulin, LXR agonist and glucose fail to induce this
transcript, we feel the induction of Elov12 by overexpressed SREBP-lo may have limited
physiological signiﬁcance in viva.

Elov15. Elov15 is the most abundant elongase transcript in rat and mouse. It also
is expressed in many tissues, induced during postnatal development and suppressed by
feeding rats n-3 PUFA enriched diets [26]. Several hormones (insulin, T3,
glucocorticoids and leptin) and transcription factors (SREBP-1c, LXR, ChREBP and

MLX) have no impact on hepatic Elov15 expression. Only PPARa, n-3 PUFA-enriched

108

[26], high fat diets (Fig. 3.9) and obesity (Fig. 3.10) affect Elov15 expression. Regulation
of Elov15 has physiological signiﬁcance. Feeding rats a high carbohydrate diet
supplemented with olive oil plus WY14643 signiﬁcantly increased mead acid (20:3,n-9)
production [26]. Mead acid is an elongation and desaturation product of 18:1,n-9, the
predominant fatty acid in olive oil. It is our view that WY14643 induction of Elov15
contributes to the formation of 20:3,n-9. Elov15 also converts 16:1,n-7, but not 16:0, to an
18-carbon monounsaturated fatty acid (18:1,n-7) (Fig. 3.6) as well as elongating an
intermediate (18:3,n-6) in the pathway for n-6 PUFA synthesis (20:4,n-6) (Fig. 3.6).
Suppression of Elov15 in high fat-fed mice correlates with decreased hepatic 20:4,n-
6/18:2,n-6 ratio (Fig. 3.9). Enhanced Elov15 expression correlates with the elevated
content of l8-carbon monounsaturated fatty acids in livers of obese rrrice (LepOM’b) (Fig.
3.10). Many PPARa-regulated transcripts, like acyl CoA oxidase and CYP4A, are

”/0” mice (not shown). Induction of Elov15 in livers of obese mice

induced in livers of Lep
is likely due to PPARa activation. Mice exposed to TCDD, leading to fatty liver, have
elevated Elovl-5 expression and enzyme activity (Fig. 3.11).

Despite the role Elov15 plays in PUFA synthesis and its elevation in livers of
obese mice, hepatic lipids in obese animals are not enriched in PUFA. In fact, obese
livers are depleted of PUFA relative to other fatty acids like 18:1 (Fig. 3.10). ASD, A6D
and A9D are induced in livers of obese mice, but to differing extents (Fig. 3.10). A5 D and
A6D are induced in obese liver as a result of elevated nuclear abundance of SREBP-1 and
activation of PPARa. A9D is induced by these same transcription factors, but also by

elevated nuclear ChREBP/MLX (Fig. 3.10). Thus, hyperphagia resulting from defective

leptin production, coupled with the ingestion of the high carbohydrate diet stimulates de

109

novo lipogenesis and monounsaturated fatty acid synthesis. In this instance, Elov15
substrates, in particular 16:1,n-7 (Fig. 3.6) are end products of de novo lipogenesis and
A9D. Elevated expression of Elov15, Elovl6 and A9D, coupled with enhanced production
of end products of de novo lipogenesis yields elevates 18:1,n-7 and 18:1,n-9 production.

Elovl6. Elovl6 is a low abundance enzyme in livers of rat and mouse. Like Elov12,
Elovl6 has a narrow substrate preference, i.e., C12 and C16 saturated and unsaturated
fatty acids (Fig. 3.6) [117]. In contrast to the other elongases, Elovl6 is regulated by
multiple regulatory factors. Insulin and LXR agonist induce SREBP-1 which induce
Elovl6 expression (Fig. 3.3 & 3.4). Insulin-induced glucose metabolism induces ChREBP
nuclear content. ChREBP and MLX regulate glucose-regulated genes including L-PK,
FAS and A9D. Elovl6 is amongst these glucose-regulated genes (Fig. 3.5). Activation of
PPARa also induces Elovl6 (Fig. 3.6). Elovl6 is regulated during postnatal development,
but unlike Elov15, Elovl6 expression falls at birth and is induced at weaning. Elovl6
expression during early postnatal development parallels SREBP-1 nuclear abundance
[26]. Finding that both Elovl6 and ND are induced along with L-PK and FAS (Fig. 3.10)
indicates that these enzymes play a role in the hepatic response to excess carbohydrate
consumption.

Excess carbohydrate is channeled to de novo lipogenesis via enhanced L-PK
activity. Insulin-stimulated glucose metabolism induces ChREBP translocation into
hepatic nuclei [186,198,199]. ChREBP and MLX heterodirnerize and bind ChoREs in
promoters of responsive genes, like LPK, ACC and FAS. Insulin also increases SREBP-
1 nuclear abundance leading to increased promoter occupancy of SREBP-1 on SRE in

target genes, e.g., ACC, FAS and A9D. Consistent with this scenario is the increased

110

nuclear abundance of SREBP-1 and MLX in livers derived from obese animals (Fig.
3.10). The end product of de novo lipogenesis, 16:0, is elongated (Elovl6) and
desaturated (A9D) to yield 18:1, the fatty acid accumulating in livers of obese mice. In
this metabolic scheme, there appears to be a tight coordination between glycolysis, de
novo lipogenesis, fatty acid elongation (Elovl6) and desaturation (A9D) that involves 3
transcription factors, ChREBP, MLX and SREBP-lo.

Although these studies provide a link between ChREBP, MLX, SREBP-l and
PPARa in the control of elongase expression, the mechanism(s) for this control remains
undefrned. Whether this control involves direct interaction of these transcription factors
with regulatory elements in the promoters of the elongases or indirect control through
other mechanisms will require detailed analysis of the promoters for Elov15 and Elovl6.
These studies are beyond the scope of this report.

In conclusion, we have established that speciﬁc hepatic fatty acid elongases,
Elov15 and Elovl6 are regulated in liver by nutrients (glucose and fat), hormones (insulin)
and nuclear receptor agonist, i.e., LXR and PPARa agonist. ChREBP, MLX, SREBP-1,
PPARa and LXR control elongase, as well as desaturase expression. Only A9D is
independently regulated by LXR. Metabolic diseases, like diabetes and obesity, induce
changes in hepatic lipid composition by controlling the function of key transcription
factors that impact elongase and desaturase expression. These studies support the notion
that regulation of both fatty acid elongase and desaturase expression may play an
important role in managing hepatic lipid composition in response to changes in dietary

and hormonal status.

111

Chapter 4

Effect of Fatty Acid Elongases on Gene Expression and

Lipid Metabolism in Rat Primary Hepatocytes

4.1. Introduction

Fatty acid elongases catalyze the regulated and rate limiting step in long chain
fatty acid elongation. Fatty acid elongases work in concert with desaturases to produce
fatty acids longer than 16 carbons. The fate of these fatty acids is to serve as substrate for:
1) phospholipids and membranes synthesis; 2) eicosanoid synthesis (prostaglandins,
thromboxanes and leukotrienes); 3) B-oxidation and energy metabolism; 4) triglycerides
and lipid storage. Disturbance in any of these metabolic pathways is linked to metabolic
disorders such as hepatic steatosis, hyperlipiderrria, inﬂammation and insulin resistance.

Seven fatty acid elongase (Elovl) isoforms, each the product of a separate gene,
have been cloned and characterized in mammals [115]. Evidence for the importance of
Elovl in physiology comes from mutant, knockout and transgenic mice. For example,
Quaking and J impy are two mouse mutant strains that have dramatically decreased Elovl-
] mRNA level [126]. Elovl3 is induced in brown adipose tissue following exposure of
animals to the cold [116]. The water barrier function was impaired in Elovl3 null rrrice.
Elovl6 is induced in transgenic mice over expressing SREBP-l [24,78,134].

Among the 7 isoforms, four expressed in rat and mouse liver (Elovl-l, -2, 1-5 and -
6); Elovl-5 is the most abundant based on its mRNA abundance. Elovl-5 utilizes a broad

array of fatty acids as substrates (€16-20) for MU FA and PUFA synthesis [24,115,201]. It

112

 

is induced during postnatal development and suppressed by feeding rats n-3 PUFA
enriched diets [175]. Recently, we reported PPARa agonist (WY14,643) and leptin-

deﬁcient obesity (LepOb/Ob

) induce Elovl-5 mRNA and enzyme activity leading to changes
in hepatic and blood lipid composition [201]. In unpublished studies, we have also found
that mice exposed to TCDD, leading to fatty liver, have elevated Elovl-5 expression and
enzyme activity5 .

C20 PUF A are the most potent fatty acids activating PPARa and its target genes
[53]. Both Elovl-2 and Elovl-5 elongate C20 fatty acids. Compared to Elovl-5, however,
Elovl-2 is expressed at lower levels in liver; its hepatic expression is constitutive in all
physiological and pathological conditions tested. Elovl-2 also has a narrow substrate
preference, €20.22 PUFA. As such, we hypothesize that changes in hepatic Elovl-2 or
Elovl-5 might affect PUFA control PPARa-mediated gene expression. To test this
hypothesis, we developed recombinant adenovirus harboring the Elovl-2 (Ad-Elovl-Z) or
Elovl-5 (Ad-Elovl-S). We used recombinant adenovirus technology to over express
Elovl-2 and Elovl-5 in rat primary hepatocytes. The outcome of these studies show that

altered expression of these enzymes signiﬁcantly impacts hepatocyte lipid compoSitiOn

and PPARa-regulated gene expression.

4.2 Materials and Methods

Recombinant adenovirus. Cloning of cDNA for Elovl-2 and Elovl-5 was described
previously [201]. Cloning of cDNA for ACS4 is performed as described [175]. Primers
of acyl CoA synthetase-4 (AC S4) corresponding to its open reading frame (Forward:
atggcaaagagaataaaagctaagc; Reversezttatttgcccccatacatc) were designed based 0n

Sequences obtained ﬁ'om Genbank and the location of the open reading frame was

113

determined using DNA-Star. Rat liver was used as template. The coding region for each

transcript was ligated into Ad-Easy XL adenoviral vector system (Stratagene),

recombined in B] 5183 cells and propagated in XL10 Gold ultra-competent cells. Ad-

DNA was packaged into adenoviral particles in Ad-293 cells (Strategene). The resultant

adenovirus was titered using Adeno-XTM rapid titer kit (BD Biosciences, Palo Alto, CA)

and used to infect rat primary hepatocytes.

Rat primary hepatocyte isolation and adenovirus infection. Male Spraque Dawley rats

(Charles River Laboratories, Kalamazoo, M1) were maintained on Harlan-Teklad

laboratory chow (#8640) and water ad lib. Rat primary hepatocytes were prepared from

Teklad chow-fed (ad lib) male Spraque-Dawley rats, cultured on BioCoat (type I

collagen) plates (Beckon Dickinson, Belford, MA.[201]. For RNA and protein extraction,

cells were plated onto 100 mm type I collagen-coated plates (BD Bioscience, Bedford, MA)
at 107 cells/plate in Williams E (Gibco/Invitrogen, Carlsbad CA), 10 mM lactate, 10 nM

dexamethasone (Sigma, St. Louis), and 10 % fetal bovine serum (Gibco/Invitrogen). For
adenoviral infection studies, cells were plated in the same media onto 6 well type 1

collagen-coated plates at 1.5 x 106 cells/well. The ratio of culture media to cell number was

maintained constant for the different plating conditions. For treatments, hepatocytes were

incubated in medium (Mlliams E + 10 nM dexamethasone without or with 100 nM insulin
and/or 25 mM glucose. Conﬂuent primary hepatocytes were infected (5-10 plaque

forming units [PF U]/cell). Using Ad-green ﬂuorescent protein (Ad-GFP) as a control for
infection, greater than 80% of primary hepatocytes expressed functional protein at'the 5-

10 PFU/cell level. The control virus used routinely for these studies, however, was Ad-

114

luciferase (Ad-Luc). Ad—Luc was obtained from C. Rhodes, Paciﬁc Northwest Research
Institute, Seattle, WA.

Acyl-CoA synthetase 4 (ACS4) activity assay Acyl-CoA synthetase activity for ACS4
was determined using an isotopic method [202]. The assay mix for the isotopic method
included 300 mM Tris—HCl, pH 7.4, 150 mM MgC12, 2.25mM glutatlrione, 20 mM ATP,
0.2 mM CoA, 200 [1M fatty acid, and 10 nCi [14C]fatty acid in 0.1% Triton X-100 in a
total volume of 100 [11. The reaction was started by adding 5 pg postnuclear homogenate
protein and the mixture was incubated for 5 min at 37 °C. Reactions were terminated
with 0.2 ml of Dole's solution, isopropanol: heptane: 0.5 M H2 804 (40/10/1; v/v). water
(0.15 ml) was added and unreacted palmitate was removed in the organic phase by
extracting 2-3 times with 0.3 ml heptane. [14C]fatty acyl-CoA formed during the reaction
was measured by scintillation counting. ‘
RNA extraction, northern analysis and quantitative real time-PCR (qR T -PCR). Total
RNA was extracted from primary hepatocytes and used for northern blot and qRT-PCR
analysis as previously described [74,201]. 32P[cDNA] far LPK, SREBPl, thMG-CoA
Synthase or CYP4A2 were as described previously [74,179]. Hybridization was
quantiﬁed by Phosphorlmager analysis (Molecular Dynamics). Speciﬁc primers for real
time PCR for each gene (Table 4.1) were designed using Primer Express software
(Applied Biosystems, Foster City, CA). First strand cDNA was synthesized using the
SuperScript II RNase H- Reverse Transcriptase (Invitrogen Carlsbad, CA). Synthesized
cDNA was rrrixed with 2x SYBR Green PCR Master Mix (Applied Bibsystems) and
various sets of gene-speciﬁc forward and reverse primers and subjected to real-time PCR

quantiﬁcation using the ABI PRISM 7700 Sequence Detection System (Applied

115

Biosystems). All reactions were performed in triplicate. The relative amounts of mRNAs
were calculated by using the comparative CT method (User Bulletin #2, Applied
Biosystems). Cyclophilin was used as a control and all results were normalized to the
abundance of cyclophilin mRNA. Primers used for quantitative real time PCR are listed
in Table 4.1.

Lipid extraction and quantitation of hepatic fatty acids composition. Total lipid was
extracted from rat primary hepatocytes in chloroformzmethanol (2: 1) plus 1 mM
butylated hydroxytoluene [175]. 7-nonadecenoic acid (19:1) was added as a recovery
standard at the time of extraction. Protein (Bio-Rad, Hercules, CA) was measured in
extracts after the initial homogenization step. Total lipids were saponiﬁed, fractionated
and quantiﬁed by reverse phase HPLC (RP-HPLC) using a YMC J-Sphere (ODS-H80)
column and a gradient starting at 77.5% acetonitrile + acetic acid (0.1%) and ending at
100 % acetonitrile + acetic acid (0.1%) over 90 mins with a ﬂow rate of 1.0 ml/min using
a Waters 600 controller. Fatty acids were detected using both UV absorbance at 192 nm
(Waters model 2487) and evaporative light scatter (Waters model 2420). Fatty acid
composition and structures were confrnned at the MSU Mass Spectrometry facility by
GC/MS (wumzbchmsuedu/facilities/massspec/indcx.html). Fatty acid standards for RP-
I-IPLC were obtained from Nu-Chek Prep (Elysian, MN).

Immunoblotting. Total, cytosolic, microsomal and nuclear protein extracts from rat
primary hepatocytes were prepared as described previously [159,175]. Proteins (50-100
pg) extracted from microsomal or nuclear fractions were separated electrophoretically by
SDS-polyacrylamide gel electrophoresis (NuPAGE 4-12% polyacrylamide Bis-Tris,

Invitrogen) and transferred to nitrocellulose membranes. Membranes were incubated with

116

Table 4. l.Primer sequences used for gene expression by northern blot and
quantitative reverse transcription-PCR.

 

 

 

 

 

Gene Forward Reverse

INSIG l TGCAGATCCAGCGGAATGT CCAGGCGGAGGAGAAGATG
INSIG 2A GACGGATGTGTTGAAGGATTTCT TGGACTGAAGCAGACCAATGTC
INSIG ZB CCGGCAGAGCTCAGGATTT AACTGTGGACTGAAGCAGACCAA
SCAP ATGAGGAGCTGTGGAGGAAA CCGTGGATTCAGGTGTAGTGT

 

 

CYCLOPHILIN

 

CTTCTTGCTGGTCTTGCCATTCCT

 

GGATGGCAAGCATGTGGTCTTTG

 

117

 

 

antibodies for, SREBP-1 (IgG-2A4, sc-13551), SREBP-2(Santa Cruz Biotechnology, San
Cruz, CA). Anti-mouse and anti-rabbit secondary antibodies were obtained from Bio-Rad
(Hercules, CA). The SuperSignal West Pico chemiluminescence kit (Pierce) detection
system was used.

Statistical Analysis. Statistical analysis used Student’s t—test and AN OVA plus post hoc

Tukey HSD test (http://faculty.vassar.edu/lowrv/VassarStats.html).

4.3 Result

4.3.1. Efficacy of Ad-Elov12 and Ad-ElovlS infection in rat primary

hepatocytes.

Rat primary hepatocytes were infected with Ad—Elole, Ad-ElovlS, Ad-Elovl6
Ad-Luc (Luciferase) or Ad-BGal (B-galactosidase) at 10 pfu/cell for 4 hrs. After infection,
medium was replaced with fresh medium containing 14C-20:5,n-3, 14C-20:4,n-6 or 14C-
16:0 at 100 uM for an overnight incubation. The total lipid was extracted and saponiﬁed
fatty acids were fractionated by RP-HPLC as described in Methods. The results showed
that the cells infected with Ad-BGal or Ad-Elov12 and treated with 14(2 labeled 16:0
converted 25% of 16:0 to 18:0 (Fig 4.1 B). However, cells infected with Ad-Elovl6i
converted 55% of 16:0 to 18:0 (Fig 4.1 B) and cells infected with Ad-ElovlS converted
35% of 16:0 to 18:0 (Fig. 4.1 C). Primary hepatocytes typically converted 40% of
exogenous C20 to C22. Infecting cells with Ad-BGal, or Ad—Luc did not affect this
reaction. The cells infected with Ad-Elovl-2 and treated with 14C labeled 20:5,n-3
converted 70% of 20:5,n—3 to 22:5,n-3 and 24:5,n-3 (Fig 4.1 A). The cells infected with

Ad-Elovl-S and treated with 14C labeled 20:4,n-6 convert 60% of 20:4,n-6 to 22:4,n-6,

118

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Figure 4. 1. Fatty acid analysis of hepatocytes overexpressed Elov12, Elov15, Elovl6,
BGal and Luc. Primary hepatocytes were infected with 10 pfu/cell Ad-Elov12, Ad-ElovlS,
Ad-Elovl6, Ad-BGal and Ad-Luc respectively. Then, either ”(2-16:0 or 1“c-2o:5n-3 or
l4C-20z4n6 (at 250 uM, 1.7 Ci/mole) were treated to these 4 group of cells. Total lipids
were saponiﬁed, fractionated, and quantiﬁed by RP-HPLC. Distribution of l4C-16:0 (B
and C) and 14C-20 (A and D) derivatives were fractionated by RP-HPLC. The level of
radioactivity was quantiﬁed using a ﬂow-through scintillation counter in line with the
HPLC unit.

122

but not 24:4,n-6 (Fig. 4.1 D). This study shows that overexpression of Elov12 and Elov15
by recombinant adenovirus leads to increased conversion of their substrates to longer
fatty acids. C20 and C22 are the substrates for the Elov12. C16 and C20 unsaturated fatty
acids are the substrates for Elov15. This study indicates that recombinant adenovirus can

be used to over express elongases in primary hepatocytes and change the lipid proﬁle.—

4.3.2. Effect of Elov12 and Elov15 on fatty acid metabolism in rat

primary hepatocytes.

Further analysis of lipid proﬁle in hepatocytes indicated that overexpression of
either Elov12 or Elov15, reduced 20:5,n-3 level signiﬁcantly, which is the most potent
fatty acid activator of PPARa. Additionally, Elovl-2 increased 24:5,n-3 and Elovl-5

increased 22:5,n-3 level (Fig. 4.2 A and B).

4.3.3. Effect of Elov12 and Elov15 on gene expression in rat primary

hepatocytes.

Prior to the addition of fatty acids to primary hepatocytes, intracellular non-
esteriﬁed unsaturated fatty acid levels are very low, representing 50.1% of the total fatty
acyl chains in the cell. The relative abundance of putative PPARa ligands in the NEFA
pool is 20:4,n-6 = 18:2,n-6 = 18:1,n-9 > 22:6,n-3 > 18:3,n-3/6 = 20:5,n-3. When
compared to all PUFA in primary hepatocytes, 20:5,n-3 is the most active ligand for
activating PPARa [136]. Since both Elovl-2 and Elovl-5 can convert C20 fatty acids'to
C22 fatty acids, overexpression of Elovl-2 or Elovl-5 might attenuate 20:5,n-3 effects on

PPARa-regulated gene transcript by rapidly reducing 20:5,n-3 mass in hepatocytes.

123

A. Total Fatty Acid

   
     

 

       

    
 
 
 

  

 

 

 

 

 

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Figure 4. 2. Fatty acids analysis of rat primary hepatocytes overexpressing Elov12
and Elov15. Primary rat hepatocytes were infected with 10pfu/cell adenovirus expressing
Elov12, Elov15 and luciferase (control) followed by 250uM 20: 5n3 treatment. Cells were ,
harvested at 24 hr after 20: 5n3 treatment. A. Total lipids were extracted and saponiﬁed '
and analyzed by RP-HPLC. Each fatty acid was expressed as percentage of total fatty

acid amount. B: NEFA were separated from total lipid. Each fatty acid was expressed as... 0
percentage of total fatty acid amount. 0

124

To test this hypothesis, hepatocytes were infected with either 10 pfu/cell Ad-
Elovl-2, ad-Elovl-S and Ad-acyl CoA synthetase-4 [Ad-ACS4 a control virus] Overnight
and treated with 250 0M 20:5,n-3 for 24 hr. Total RNA was isolated and used for -
northern analysis to examine the expression of PPARa target gene i.e., cytochrome P450-
4A (CYP4A), cytosolic thioesterase I (CTE-I) and mitochondrial HMG-CoA synthase 2
(HMGCSZ). The efﬁcacy of Ad-ACS4 was indicated by enzyme assay as shown in Fig
4.4 A. The results show that overexpression of Elovl-2 signiﬁcantly suppressed 20:5 ,n-3
induction on PPARa target genes CYP4A, CTE-I and HMGCS2, but did not affect
20:5,n-3 effects on the expression of mRNASREBp-1 or mRNApr (Fig. 4.3).
Overexpression of Elovl-5 has the similar effect as Elovl-2 on PPARa target genes (Data

not shown).

4.3.4. Effect of Elov12 and Elov15 on SREBPs nuclear content in rat
primary hepatocytes.

The nuclear abundance of SREBP-l, but not SREBP-2, is suppressed by PUFA.
Of the C18-22 PUFA found in primary hepatocytes, 22:6,n-3 is the most potent
suppressor. The mechanism for this control involves 22:6-mediated activation of SREBP-
1 26S proteasomal degradation [159]. Based on the differential effects of PUFA on
SREBP-1 content, 1 next determined if changes in elongase activity affected SREBP-l
nuclear abundance. The experiment was designed as the gene expression study. Instead of
RNA analysis, however, nuclear and microsomal protein extracts were isolated ﬁom
infected primary hepatocytes to measure SREBP-1 and SREBP-2 by immunoblot
analysis. Elevated Elovl-2 activity in the absence of exogenous PUFA had no effect on

SREBP-1 or SREBP-2 nuclear abundance. Addition of 20:5,n-3, however, enhanced the

125

suppression of SREBP-1 nuclear content, without effects on SREBP-2 nuclear content.
Over expressing Elov15 or Elovl6, however, did not alter the 20:5,n-3 effect on SREBP-1
nuclear content (Fig. 4.4). Elovl6 did not utilize 20:5,n-3 as substrate. Although Elov15
converts 20:5,n-3 to 22:5,n-3, Elov15 does not convert 22:5,n-3 to 24:5,n-3. This study
suggests that excessive formation of 24:5,n—3 impacts SREBP-1 nuclear abundance.
Unfortunately, 24:5,n—3 is not commercially available, thus limiting a direct test of
whether 24:5,n-3 or some other action of Elov12 over expression affects SREBP-1
nuclear abundance.

We examined possible mechanisms for this effect of Elov12 over expression on
20:5-mediated suppression of SREBP-l nuclear abundance. As shown in Fig.4.3,
infection of hepatocytes with Ad—Elov12 did not enhance the effect of 20:5,n-3 on
mRNASREBm abundance. 22:6,n-3 is a robust suppressor of hepatocyte SREBP-l nuclear
abundance [179]. The mechanism for this suppression involved 22:6-mediated 26S
proteasomal degradation of nuclear SREBP-1 [179]. Treating Ad-Elov12 infected
hepatocytes with 26S proteasomal inhibitors (MG132 or lactacystin) did not block the
enhance 20:5,n-3 suppression of mature SREBP-1 (Data not shown). I next examined the
effects of these treatments on proteins involved in proteolytic maturation of SREBP.
Analysis of Insig-l and Insig-Z mRNA and SCAP protein revealed mRNA of Insigl and
Insig2a were induced by overexpression of Elov12 in the cells treated with 20:5,n-3
compared to Ad-Luc group. There is no change of mRNA of Insin and SCAP by

overexpression of Elov12 (Data not shown). However, SCAP protein was suppressed by

126

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Figure 4. 3. Effect of Elov12 on hepatic gene expression. A. Efﬁcacy of Ad-ACS4 in
rat primary hepatocytes. Postrruclear fraction was prepared by differential centrifugation
from rat liver and the rat primary hepatocytes infected with different dosage of Ad-ACS4.
Fractions (Sug protein) were assayed for ACS activity. B. Primary rat hepatocytes were
infected with 10pfu/cell Ad-Elov12 or Ad-ACS4 (control) followed by 250uM 20:5n3
treatment. RNA was harvested at 24 hours after treatment. Northern blot was performed
to exam PPARa targeted genes (Cyp4A, CTE-l, HMG CoA Synthase) and SREBP-1c
and L-PK gene expression. Mean : SD,n=3. These results are representative of 3 separate
studies.

127

20:5,n—3 - + -

+
nSREBP-1 -
nSREBP-2

pSREBP4
pSREBP-Z

 

Figure 4. 4. Overexpression of Elov12 effect on 20:5,n-3 regulation of rat hepatic
sterol regulatory element binding protein 1 (SREBP-l) and SREBP-2 nuclear
content. Primary hepatocytes were prepared and infected with adenovirus expressing.
Elov12, Elov15, Elovl6 and luciferase 24 hours. Followed by treatment of 250 uM 20:5,n-
3 described before. Nuclear and microsomal proteins were isolated from cells after 24
hours fatty acid treatment. Western blot was performed and probed with antibodies fOr
SREBP-1 and SREBP-2.

128

overexpression of Elov12 followed by 20:5,n-3 treatment (Fig 4.5 C). Based on the
outcome of these studies, elevated expression of Elov12 coupled with the addition of
20:5,n—3 conversion to 24:5,n-3 inhibits the proteolytic maturation of SREBP-1, but not

SREPB-2 in rat primary hepatocytes.

4.4. Discussion

My previous studies established that certain fatty acid elongases, like fatty acid
desaturases, are regulated during development, by nutritional status and by hormones
[175,201]. My goal in this study was to determine the effect of altered elongase
expression on rat primary hepatocyte lipid metabolism. Since changes in hepatic lipid
content are known to control two major transcriptional regulatory networks, 1 determined
if altered elongase expression affected these networks, i.e., PPARa and SREBP-1. The
outcome of these studies supports the hypothesis that fatty acid elongases affect both
hepatocyte lipid content and gene expression.

Fatty acid metabolism in the liver is tightly regulated. Fatty acids are oxidized to
meet energy needs, or esteriﬁed to produce triglycerides, which are either stored in the
liver or exported to peripheral tissue by VLDL particles. Two classes of transcription
factors, namely, SREBPs and PPARs play a major role in lipid metabolism and energy
homeostasis. Our studies suggest that overexpression of hepatic Elov12 or Elov15 can
alter fatty acid structure, which will affect the composition of intracellular NEFA. As
such, these enzymes contribute to the availability of fatty acid metabolites controlling
SREBP-l and PPARa functions. Overexpression of both Elov12 and Elov15 enhance
20:5,n-3 conversion to 22:5 ,n-3 or 24:5,n-3, which leads to reduction of 20:5,n-3 and thus

attenuates 20:5,n-3 mediated induction on PPARa regulated transcripts. Moreover,

129

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Figure 4. 5. Effect of overexpression of Elov12 on mRNA of Insigs and SCAP protein.
Primary rat hepatocytes were infected with 10pfu/cell Ad-Elov12 or Ad-Luc (control)
followed by 250uM 20:5n3 treatment. RNA and microsome protein were harvested at 24
hours aﬁer treatment. Real time PCR was performed to exam mRNA of insigl (A),
insig2a (B) and SCAP protein level (C). These results are representative of 3 separate
studies.

130

overexpression of Elov12, but not Elov12, can enhance 20:5,n-3 mediated suppression of
SREBP-1c nuclear content.

Over expressed Elov12: In liver, Elov12 is constitutively expressed at low levels.
Its substrate preference is limited to €20.22 PUFA. Over expression of Elovl-2 enhanced
the capacity of hepatocytes to convert 20:5,n-3 to 22:5,n-3 and 24:5,n-3. Interestingly,
this does not lead to elevated 22:6,n-3 formation. The likely explanation for this is the
suppression of A6-desaturase activity in cultured rat primary hepatocytes (unpublished
observation). Over expression of Elov12 lowers total and non-esteriﬁed 20:5,n-3 levels in
hepatocytes. A consequence of this is the attenuation of 20:5-mediated induction of
PPARa target genes, i.e., CYP4A, CTEl and I-IMGCSZ.

A second outcome of our analysis was frnding that over expressed Elov12
signiﬁcantly enhanced the 20:5-mediated suppression of mature (nuclear) SREBP-1 in
hepatocytes. The mechanism for this effect is attributed to effects on proteolytic
maturation of SREBP-1. This interpretation is based on the following observations. First,
over expressed Elov12 did not enhance 20:5,n-3 suppression of mRNASREBp-1 in primary
hepatocyte. Second, 26S proteasomal inhibitors did not block the 20:5,n-3/Ad-Elov12
suppression of mature SREBP-1. Third, over expressed Elov12 suppressed hepatic levels
of SCAP and induce INSIG 1 or 2. Taken together, these frnding support the notion that
Elov12 alters 20:5,n—3 metabolism in such a fashion to impact SREBP—l maturation to the
nuclear form.

Over expressed Elov15: Like Elov12, over expression of Elov15 signiﬁcantly
lower hepatic levels of 20:5,n-3 (Fig. 4.1D) and attenuated 20:5,n-3 induction of PPARa

target genes. Unlike Elov12, Elov15 does not elongate 20:5,n-3 to 24:5 ,n-3. Elov15 also

131

does not enhance 20:5-mediated suppression of SREBP-l nuclear abundance. Elov15, in
contrast to Elov12, is the most abundant elongase in liver; it is also well regulated during
post-natal develop, by diet, hormones drugs and toxins. As such, changes in Elov15
activity in these various physiological and pathophysiological states will likely affect
hepatic lipid composition. Elov15 effects on gene expression, however, are likely to be
not as widespread as those seen with Elov12. Elov15 effects may be restricted to the
control of PPARa signaling. More studies will be required to verify this notion.

In summary, fatty acid elongases have the capacity to signiﬁcantly impact hepatic
lipid composition. In doing so, elongase-mediated changes in hepatocyte lipid
composition affect at least two transcriptional regulatory networks known to be
controlled by exogenous fatty acids, viz. PPARa and SREBP-1. Such frndings reveal the
importance of fatty acid metabolism on the control of these transcription factors. These
studies, however, are in primary hepatocytes. Studies in the next chapter reveal effects of
elongase over expression in vivo. These studies reveal even more effects of elongases on

hepatic metabolism and gene expression.

132

Chapter 5

Effect of Elov15 on Hepatic Function and Lipid

Metabolism in Vivo

5.1. Introduction-

Seven fatty acid elongase(Elovl) isoforms, each the product of a separate gene,
have been idendiﬁed in the mammalian genome; 7 of these isoforms have been cloned
and characterized in mammals [115]. Evidence for the importance of Elovls in
physiology comes from transgenic mice studies. For examples, Quaking and Jimpy are
the two mouse mutant strains have dramatically decreased Elovl-l mRNA level [126].
Elovl-3 is induced in brown adipose tissue following exposure of animals to the cold
[116]. The water barrier function was impaired in Elovl-3 null mice. Elovl-6 is induced in
transgenic mice over expressing SREBP-1 [24,78,134]. Among these isoforms, there are
four expressed in rat liver (Elovl-l, -2, -5 and -6), in which Elovl-5 is the most abundant;
Elovl-5 utilizing a broad substrate array, €16-22, involved in PUF A synthesis [24,115,201].
It is induced during postnatal development and suppressed by feeding rats n-3 PUFA
enriched diets [175]. Recently, we reported PPARa, high fat diets and obesity affect
Elovl-5 expression [201]. Over expressing Elovl-5 in rat primary hepatocytes alters
hepatic gene expression, at least in part, by controlling ligand availability for key

transcriptional factors, like PPARa.

133

Even though individual Elovl isoforms have different substrate preferences, tissue
speciﬁc expression, and are regulated differently [115,133,175,201,203], the signiﬁcance
of this diversity is unknown. The regulation of elongase paralleling the change of hepatic
fatty acid composition under pathological conditions leads us to test the speciﬁc role of
individual elongase in hepatic function and onset of chronic disease. Here, I over eXpress
Elovl-5 in mouse liver by recombinant adenovirus and test the hypothesis that Elovl-5

will affect hepatic function by manipulating fatty acids structure.

5.2 Materials and Methods

Recombinant adenovirus. Cloning of cDNA for Elovl-5 was described previously [201].
The coding region for Elov15 transcript was ligated into Ad-Easy XL adenoviral vector
system (Stratagene), recombined in BJ 5183 cells and propagated in XL10 Gold ultra-
competent cells. Ad-DNA was packaged into adenoviral particles Ad-293 cells. The
resultant adenovirus was ampliﬁed in HEK293 cells and puriﬁed by cesium chloride
gradient centrifuge.

Animals. All animal procedures were done in accordance with Michigan State Universtiy
Institutional Animal Care and Use committee approved guidelines. Six-week-old male
C57BL/6 mice were purchased from Charles River, Kalamazoo and adapted to the
environment for 1 week before study. All rrrice were housed in a speciﬁc pathogen-free
environment with a 12-h light/dark cycle in a temperature-controlled environment. Mice
had free access to water and regular chow diet. The adenovirus was intravenously
administrated (via the retro-orbital sinus) into 3-4 groups animal: saline (n=8), Ad-
LUC(n=8), Ad-Elovl-5(n=8). All treated animals were injected with 2 X 1011 virus

particles/animal in a ﬁnal volume of 200 pl sterile PBS + 1% sucrose; saline injected

134

mice received the PBS + 1% sucrose alone. Mice were given TekLad chow and water, ad
lib, and were weighed daily. The 3rd day after injection, all mice were fasted overnight.
The next day, 4 mice from each group were then refed with chow diet for 4 hrs before
anesthesia. All animals were euthanized at noon — 2 PM on the 4th day post injection.
Immunohistology: At euthansia, a sample of liver was taken for histology: a) formalin-
ﬁxed, parafﬁn-embedded for hematoxylin & eosin (H&E) stain and periodic acid-Schiff
(PAS) staining to detect glycogen; a second sample was ﬁxed in OCT, ﬁ'ozen (liquid
nitrogen/-80°C) and stained with Oil Red 0 (ORO) to detect lipid. Embedding,
sectioning, staining and slide preparation was done in the Investigative HistoPatholOgy
Laboratory Staff, Department of Physiology-MSU.

Blood glucose measurement and plasma preparation: Blood was rapidly drawn from
the vena cava (~1 ml) into a heparin syringe. Glucose measurement was performed at the
time of blood collection using a hand-held glucose meter (One touch 11, Life Scan Inc.).
The remaining blood was centrifuged at 800 rpm for 10min, and the plasma was stored in
-80°C for subsequent assays for cholesterol, triglycerides and saponiﬁed fatty acid proﬁle.
Measurement of liver glycogen content: Liver (~0.1 g) was homogenized in PBS,
adjusted to 0.5 M KOH, and incubated at 95°C for 1 hr. The homogenate was cooled,
neutralized with 0.1X vol. 6% Nasta; glycogen was precipitated with 3 volumes of
ethanol. The precipitate was collected by centrifugation, washed with 7 0% ethanol and
resuspended in 1 ml water, adjusted to 50 mM Na-acetate, pH 5.0. Glycogen was
hydrolyzed with amyloglucosidase and the released glucose measured using a Wako
Glucose assay kit; Standards were glucose (200 mg/dl) and glycogen (1 mg/ml) [204]

Absorbance was read in a spectrophotometer (DU 640; Beckrnan) at 340 nm wavelength. ‘

135

In vitro fatty acid elongation assay. Rat liver microsomes were isolated by differential
centrifugation [201]. Elongation reactions were carried out with modiﬁcations to the
procedure described by Moon et al [117]. Brieﬂy, reaction mixtures contained 50 pg
microsomal proteins in a total reaction volume of 100 pl. The reaction constituents are:
50 mM potassium phosphate buffer- pH 6.5, 5 pM rotenone (Sigma), 40 pM fatty acyl
CoA (Avanti Polar Lipids, Alibaster, Al and Sigma, St. Louis, MO), 60 pM malonyl
CoA (Sigma) [6.5 dpm/pmol] [2-14C]-malonyl CoA (Perkin Elmer), 1 mM NADPH
(Sigma) 20 pM BSA (fatty acid free). Reactions (at 37°C) were initiated with the
addition of NADPH. When fatty acids were used as substrate,n-aOH neutralized fatty
acid (40 pM) replaced fatty acyl CoA. CoASH (at 100 pM), MgC12 (1 mM) andiATP 0(1
mM) were added to the reaction to generate fatty acyl-CoA. Elongase reactions were
terminated after 20 minutes with the addition of 100 pl 5 N KOH + 10% methanol; lipids
were saponiﬁed for 1 hr at 65°C. The saponiﬁcation reaction was acidiﬁed with 100 pl 5
N HCl; 100 pl ethanol was added to aid hexane extraction of fatty acids. Elongated fatty
acids were collected by 2 independent extractions with hexane (800 p1). Hexane extracts
were pooled and l4C-radioactivity was quantiﬁed by B-scintillation counting. ReSults are
expressed as Elongase Activity Units,n-moles l4C-malonyl CoA incorporated/mg
protein/20 mins. Formation of reaction products was dependent on the presence of
NADPH and the fatty acid CoA. Fatty acid elongation products were veriﬁed by reverse-
phase-HPLC chromatography using a ﬂow-through B-scintillation counter.

RNA extraction, northern analysis and quantitative real time-PCR (qRT-PCR). Total
RNA was extracted from liver samples and used for template for qRT-PCR analysis as

previously described [201]. Speciﬁc primers for each gene (Table 5.1) were designed

136

using Primer Express software (Applied Biosystems, Foster City, CA). First strand cDNA
was synthesized using the SuperScript II RNase H- Reverse Transcriptase (Invitrogen
Carlsbad, CA). Synthesized cDNA was mixed with 2x SYBR Green PCR Master Mix
(Applied Biosystems) and various sets of gene-speciﬁc forward and reverse primers and
subjected to real-time PCR quantiﬁcation using the ABI PRISM 7700 Sequence
Detection System (Applied Biosystems). All reactions were performed in triplicate. The
relative amounts of mRN As were calculated by using the comparative CT method (User
Bulletin #2, Applied Biosystems). Cyclophilin was used as a control and all results were
normalized to the abundance of cyclophilin mRN A. Primers used for quantitative real
time PCR are listed in Table 5.1.

Lipid extraction and quantitation of hepatic fatty acids composition. Total lipid was
extracted from liver in chloroformzmethanol (2: 1) plus 1 mM butylated hydroxytoluene
[175]. 7-nonadecenoic acid ( 19:1) was added as a recovery standard at the time of
extraction. Protein (Bio-Rad, Hercules, CA) was measured in extracts after the initial
homogenization step. Total lipids were saponiﬁed, fractionated and quantiﬁed by reverse
phase HPLC (RP-HPLC) using a YMC J-Sphere (ODS—H80) column and a gradient
starting at 77.5% acetonitrile + acetic acid (0.1%) and ending at 100 % acetonitrile +
acetic acid (0.1%) over 90 mins with a ﬂow rate of 1.0 ml/min using a Waters 600
controller. Fatty acids were detected using both UV absorbance at 192 nm (Waters model
2487) and evaporative light scatter (Waters model 2420). Fatty acid composition and
structures were conﬁrmed at the MSU Mass Spectrometry facility by GC/MS
(www.bch.msu.edu/facilities/massspec/index.html). Fatty acid standards for RP-HPLC

were obtained from Nu-Chek Prep (Elysian, MN).

137

Table 5. l.Primer sequences used for gene expression by quantitative
reverse transcription-PCR for mouse.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Gene Forward Reverse

ACC TGTCCGCACTGACTGTAACC ATTTCCATAGCCGACTTCCA

F AS CAAATACAATGGCACCCTGA TGGCGAAGCCGTAGTTAGTT
SREBP-1 AGGAGAACCTGACCCTACGA GGTAAGCGTCTCCACCACTT
CYP4A10 TGTTTGACCCTTCCAGGTTT CAATCACCTI‘CAGCTCACTCA
HMGCSZ CCTTGAACGAGTGGATGAGA CAGATGCTGTTTGGGTAGCA
AOX-1 CCCAACTGTGACTTCCATCA ACGGATAGGGACAACAAAGG
L-PK AGGAGTCTTCCCCTTGCTCT ACCTGTCACCACAATCACCA
PEPCK AC ATTGCCTGGATGAAGTTTG GGCA'I'I'I‘GGATI‘TGTCTTCAC
GLUT2 GAGGAAGTCAGGGCAAAGAA AAGAGCTGGATCACGGAGAC
G6PC TCGTGGCTGGAGTCTTGTC GCGAAACCAAACAAGAAGATG
GCK GAGATGGATGTGGTGGCAAT ACCAGCTCCACATTCTGCAT
CHREBP CTGGGGACCTAAACAGGAGC GAAGCCACCCTATAGCTCCC
A9 D TCAACTTCACCACGTTCTTCA CTCCCGTCTCCAGTTCTCTT

A5 D TGTGTGGGTGACACAGATGA GTTGAAGGCTGATTGGTGAA
A6 D CCACCGACATTTCCAACAC GGGCAGGTATTTCAGCTTCTT
ELOVLS GGTGGCTGTTCTTCCAGATT CCCTTCAGGTGGTCTTTCC
ELOVLZ ACGCTGGTCATCCTGTTCTT GCCACAATTAAGTGGGCTTT
CYCLOPHILIN CTTCTTGCTGGTCTTGCCATTCCT GGATGGCAAGCATGTGGTCTTTG

 

138

 

Immunoblotting. Liver rrricrosomal and nuclear extracts were prepared as described
previously [159,175]. Proteins (50-100 pg) extracted ﬁ'om microsomal or nuclear
fractions were separated electrophoretically by SDS-polyacrylamide gel electrophoresis
(NuPAGE 4-12% polyacrylamide Bis-Tris, Invitrogen) and transferred to nitrocellulose
membranes. Membranes were incubated with antibodies for ChREBP (Novus Biologicals,
Littleton, CO); MLX(N-l7), SREBP-l (IgG-2A4, sc-l3551), SREBP-2, ERKl (so-94),
and phosphor-ERK (SC-7383) (Santa Cruz Biotechnology, San Cruz, CA); GSKBB,
phosphor-GSKBB (Ser9) and caspase 9 (Cell signaling technology, Beverly, MA). Anti-
mouse, anti-goat and anti-rabbit secondary antibodies were obtained ﬁom Santa Cruz
Biotechnology (Santa Cruz, CA). The SuperSignal West Pico chemiluminescence kit

(Pierce) detection system was used.

Statistical Analysis. Statistical analysis used Student’s t-test and AN OVA plus post hoc

Tukey HSD test (htg)://facultv.vassar.edu/louryNassarStats.html).

5.3 Result

5.3.1. Generation of the mice overexpressing Elov15 in liver.

C57BL/6 mice were injected with saline, Ad-Luc (control virus) and Ad-Elovl-S,
respectively. Liver and kidney RNA were examined for Elovl-5 gene expression in these
mice. Elovl-5 mRNA level was elevated ~30 fold compared to Ad-luciferase and saline
groups (data not shown) in mouse liver. There is no signiﬁcant difference of Elovl-5
expression in kidney, heart and spleen among the 3 treatments (data not shown). This

outcome indicated that the adenovirus were sequestered by the liver, not other tissues.

139

 

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Figure 5. 1. Effect of overexpressing Elovls on elongase activity in mice liver. Male
C57BL/6 mice were injected with Ad—Luc and Ad-Elov15 and fed with chow diet for 4
days as described in (Materials and Methods). Hepatic rrricrosomal preparations were
used for fatty acid elongase assays (Materials and Methods). Results are expressed as
Elongase Activity, (nmoles 14C-malonyl CoA assimilated into fatty acids/mg protein).
Mean 1: SD,n=8. *p50.01 vs Ad-Luc, Student’s T -T est.

140

Liver elongase assay showed that enzyme activity of Elovl-5 was increased by 2-3
fold as compared to Ad-Luc and saline groups ( Fig. 5.1). This level of Elovl-5 activity is

ob/ob

seen in animals fed the peroxisome proliferator, WY14643, or in livers of Lep mice

[26,205].

5.3.2. Metabolic characterization of the mice overexpressing Elov15 in

liver.

On a normal chow diet, all Ad injected mice lose body weight and have decreased
food intake in the ﬁrst day aﬁer injection compared to the saline group (data not shown).
Two days after injection, food intake resumes and body weight increases. By day 4, post
injection, body weight and food intake between the saline and Ad-infected groups is not
signiﬁcantly different. All the Ad infected animals developed a modest, but insigniﬁcant
increase in liver weight when compared to the saline group (data not shown). Livers of
the Ad-Elovl-S group have small white lesions compared to Ad-Luc infected groups (Fig.
5.2 A).

Surprisingly, the blood glucose measured at the time of euthansia decreased 30%-
40% in the Ad-Elovl-5 groups compared to the control group (Ad-luc and saline groups)
in both fasting and refeeding state (Fig. 5.3 A). Hepatic glycogen stores, determined by
Periodic acid shiﬁ (PAS) staining, was signiﬁcantly decreased in Ad-Elovl-S groups
compared to Ad-Luc group (Fig. 5.2 B). Direct measure of hepatic glycogen content
(Methods) conﬁrmed the PAS staining results (Fig. 5.3 B). The fold change of hepatic
glycogen levels during fast and refeeding of the Ad-Elovl-S infected mice was decreased
4 fold compared to Ad-Luc group. Hepatic lipid content, determined by oil red O staining

(ORO), mirrored the quantitative hepatic triglyceride in all Ad groups (Fig. 5.2 B); Ad-

141

 

A. Ad-Luc Ad-Elovl5

 

 

B- Ad-Luc Ad-Elovl5

 

 

Figure 5. 2. Liver morphology and histology for Ad mice. A. Liver morphology of Ad
injected mice. B. Liver histology of Ad mice. Liver sections from the mice
overexpressing Luc and Elov15 were stained with hepatoxylin and eosin (H&E) and oil
red O (ORO) and (PAS), magniﬁcation 20X. Arrows indicated necrosis in Ad-Elov15
liver. Sections are representive of several animals for each condition. Image in this ﬁgure
is presented in color.

142

Elov15 had not major effect on hepatic neutral lipid content. Blood triglyceride levels,
however, were signiﬁcantly decreased in the fed state of Ad-Elovl-S infected animal. (Fig.
5.3 C). No change of blood cholesterol level was observed in Ad-Elovl-S injected mice

(Data not shown).

5.3.3. Alteration of glucose metabolism in Ad-ElovlS injected mice.

Stores of glycogen in the liver are considered the main buffer of blood glucose
levels. Since glycogen level is suppressed in Ad-Elovl-S injected mice, we ﬁrst
hypothesized that overexpression of Elovl-5 altered hepatic glucose metabolism. Fasting
induces glycogenolysis and gluconeogenesis, whereas refeeding induces glycolysis and
glycogenesis. We ﬁrst examined the mRNA expression and nuclear content of ChREBP,
which is required for most glucose-regulated hepatic genes [55,62]. Results showed that
mRNAChREBp was decreased in the refed group overexpressing Elovl-5, but not in Ad-Luc
group (Fig. 5.4 A). Immunoblotting of ChREBP and MLX in fed mice showed a decline
in nuclear ChREBP, but no change of nuclear MLX, the ChREBP heterodimer partner.
(Fig. 5.4 B). L-PK, a target of ChREBP/MLX and a major glycolytic enzyme, was
signiﬁcantly suppressed in livers of Ad-ElovlS infected animals when compared to Ad-
Luc infected animals (Fig. 5.5). A second ChREBP/MLX target gene was examined.
Glut2 is a key hepatic glucose transporter. Like L-PK, Glut2 expression was also
suppressed in Ad-Elovl-5 infected animals in both the fasted and fed state. The
gluconeogenic enzymes, phosphoenolpyruvate carboxykinase (Peka) and glucose-6-
phosphatase (G6Pase) were examined. Elovl-5 signiﬁcantly suppressed mRNApcka and

mRNAmsmc in both fasting and refeeding (Fig. 5.5).

143

 

 

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Figure. 5.3. Effect of overexpressing Elov15 on blood glucose, triglyceride and
hepatic glycogen level. A. Blood glucose level was measured by blood glucose meter
(One touch 11, Life Scan Inc.). The open bar represents the fast group; the ﬁlled bar
represents the refeeding group; Mean j; SD,n=4. #P<0.05 vs fast group, anova. B. Liver
glycogen level was measure as described in the method. Results are expressed as Fold
Change (refeeding/fast), mean 1 SD, n=4. C. Effect of overexpressing Elov15 on blood
triglyceride level. Blood triglyceride level was measured by Wako L—Type TG H test kit.
The open bar represents the fast group; the ﬁlled bar represents the refeeding group;
Mean :1; SD,n=4. #P<.01,##P<.05.

145

Glycogen synthase kinase-30 (Gsk-3B) is a serine/threonine kinase that plays a
key role in controlling glycogen synthesis by regulating the phosphorylation status of
glycogen synthase [206]. Gsk-3B is a highly-regulated kinase; its activity is controlled by
its phosphorylation status. Insulin suppresses Gsk-3B activity by inducing its
phosphorylation through the PIBK-Akt pathway [207]. Inhibition of GSK3|3 releases
glycogen synthase from inhibition and stimulate glycogen synthesis. I examined total and
phospho-Gsk3B in livers of fed Ad-infected animals only. Contrary to my expectation,
the phosphorylation status of Gsk-3B (ser-9) was signiﬁcantly increased in Ad-Elovl-5
injected when compared to the saline or Ad-Luc infected groups (Fig. 5.6). As such,
Elovl-5 over expression does not suppress hepatic glycogen content by activating Gsk-3B.

I also examined a second target of insulin action, Erk. Insulin rapidly, but
transiently induces Erk phosphorylation in rat primary hepatocytes [83]. Over expressed
Elovl-5 increased phospho-Erk with little effect on total Erk (Fig. 5.6). No adenovirus
infection increased the formation of caspase 9 (37.5 kd), an indicator of intrinsic
apoptosis [208](Fig. 5.6).

Taken together, these results indicate that elevated hepatic activity of Elovl-5
mimics some, but not all of the effects of insulin: a) induces Gsk-3B phosphorylation; b)
induces Erk phosphorylation; c) suppresses Peka gene expression. Unfortunately,
however, there are contradictory outcomes; such as the suppression of hepatic glycogen
content and expression levels for ChREBP, L-PK and Glut-2, with little effect on
glucokinase. While my studies do not provide a complete explanation for the effects of
over expressed Elovl-5 on hepatic glucose metabolism, these studies provide clear

evidence that change in fatty acid elongase activity, alone, are sufﬁcient to promote

146

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relative abundance,
ChREBP/cyclophilin

 

 

 

 

 

 

Ad- Luc Ad-Elovi-5

 

 

Figure. 5.4. Effect of overexpression of Elov15 on hepatic ChREBP gene expression
and nuclear content. A. Effect of overexpression of Elov15 on hepatic ChREBP gene
expression. RNA was extracted and used for qRT-PCR analysis of ChREBP gene.
Results are expressed as Fold Change (transcript/cyclophilin), mean 1 SD, n=4. B. Effect
of overexpression of Ad-ElovlS on nuclear ChREBP and MLX abundance. Livers from
Ad-Luc and Ad-ElovlS injected mice were used for the isolation of nuclear proteins for
immunoblotting. Duplicate samples for each treatment are shown.

147

major changes in hepatic glucose metabolism and key signaling pathways that impact

hepatic glucose metabolism.

5.3.4. Decreased expression of genes involved in lipid metabolism in Ad-

ElovlS injected mice.

Lipid metabolism is a second major hepatic metabolic pathway that is affected by
dietary lipid composition. Herein, I examined changes in hepatic expression of genes
involved in hepatic lipid metabolism. Elevated expression of Elov15 (Ad-Elov15) in fasted
mice suppressed expression of PPARa target genes, CYP4A and HMGCSZ levels, but
not AOX-1, by 70% (Fig. 5.7). There was no change in SREBP-1 gene expression,
SREBP-1 nuclear content or SREBPl-target genes, ACC and FAS, in Ad-ElovlS injected
mice comparing Ad-Luc injected mice (Data not shown). However, these three genes
responded nicely to the fast-refeeding. In addition, mRNA levels for SCD-l, the
desaturase responsible for the production of monounsaturated fatty acids was
signiﬁcantly suppressed in liver of Ad-ElovlS injected mice compared with Ad-Luc
injected mice in refed states (Fig. 5.7). SCDl expression is controlled by PPARa,
SREBP-l and ChREBP/MLX [205]. The decline in SCDl expression in Ad-ElovlS
infected animals is likely due to disruption of the PPARa and ChREBP/MLX regulatory

pathways.

148

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Figure. 5.5. Effect of overexpressing Elov15 on gene expression involved in glucose
metabolism. RNA was extracted and used for qRT-PCR analysis of gene involved in
glucose metabolism. Results are expressed as Fold Change (transcript/cyclophilin), mean
1 SD, n=4. #P<0.01.

150

5.3.5. Effect of Elov15 overexpression in hepatic and plasma fatty acid

proﬁle.

Elov15 utilizes a broad array of fatty acid substrates, €16.20, to impact both MUFA
and PUFA synthesis. I hypothesized that overexpression of Elov15 can promote
endogenous PUFA synthesis. To eliminate the contribution of dietary lipids from the
absorptive phase, I examined hepatic and plasma fatty acid of fasted mice. The outcome
of these studies provided unexpected results. The ratio of 20:4,n-6 to 18:2,n-6 is
decreased in Ad-ElovlS injected mice (Fig. 5.8). 18:2,n-6, an essential fatty acid, is
converted to 20:4,n-6 by elongation (Elov15) and desaturation (A5 D and A°D). There was
no increase in 22:4,n-6 in livers of Ad-ElovlS infected animals. Since other enzymes
involved in PUFA synthesis pathway, i.e., ASD and A°D desaturase, did not change, I
speculate that over expressed Elov15 stimulated the conversion of 20:4, n-6 to 22:4,n-6
and its peroxisomal degradation. PUFA of 322 carbons are preferred substrates for
peroxisomal oxidation [209].

Two interesting observations were made in my analysis of the plasma fatty acid
proﬁle. Mice receiving Ad-ElovlS had a higher (by 40%) level of PUFA in the plasma
(Fig. 5.9). This is due to the accumulation of 18:2,n-6 in the plasma. Second, the level of

18:0, relative to 18:1, was increased (Fig. 5.9).

5.4. Discussion

The data from these in vivo studies support the hypothesis of this thesis. Altered
expression of an elongase, alone, is sufﬁcient to change hepatic gene expression. The

outcome of these studies, however, revealed a more complicated change in hepatic

151

 

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Figure. 5.6 Effect of overexpression of Elov15 on regulating GSK30 and ERK. Total
protein was extracted from liver as methods described. Protein levels were measured by

immunoblot (Materials and Methods). Duplicate samples for each treatment are shown.

152

metabolism affecting both glucose and lipid metabolism. As such, these studies may have
revealed novel linkages between fatty acid elongation and other facets of hepatic
metabolism.

Several reports in the literature have demonstrated that infection of adenovirus
into the retro-orbital sinus of mice results in >99% of the virus infecting liver [210,21 1],
and as expected, we did not observe Elov15 expression in other tissues, including kidney,
heart, or spleen (data not shown). Overexpression of Elov15 in mouse liver, by the
recombinant adenoviral approach, elevated elongase activity 3-fold when compared
to liver of animals infected with the Ad-Luc control or control (saline injected) animals.

°°/°° obese mice

This level of expression is seen in other physiological states, e.g., Lep
[205], TCDD-treated mice (Fig 3.11 A) and WY14,643 treated rats [26]. As such, the
change in elongase activity is within a physiological range.

Several key observations were made. First, there was ~40% decrease in blood
glucose level in fasted and fed Ad-ElovlS infected mice. The likely explanation for this is
the decline in hepatic glycogen content and expression of genes involved in
gluconeogenesis (PEPCK and Glc-6-P), coupled with a decline in genes involved in
glucose transport (Glut2) and glycolysis (LPK). The outcome of these results implicated
some change in hepatic insulin signaling. Analysis of two downstream targets of insulin
action, namely, Gsk3B and Erk indicated that elevated Elovl-5 activity as associated with
increased phosphorylation of these proteins. This same effect is observed in acute
treatment of hepatocytes with insulin [83]. As such, elevated Elov15 activity appears to

mimic insulin action. Defects in liver and muscle glycogen synthesis are major factors

contributing to postprandrial hyperglycemia in patients with type 2 diabetes [212]. Our

153

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154

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Figure. 5.7. Effect of overexpressing Elov15 on PPARa target gene expression. RNA
was extracted and used for qRT-PCR analysis of PPARa target gene, Cyp4A and
HMGCSZ as well
(transcript/cyclophilin), mean 1 SD, n=4. #p<.01, *p<.05.

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Figure. 5.8. Effect of overexpressing Elov15 on hepatic lipid composition in the
fasted mice. Total lipids were extracted and saponiﬁed; fatty acid levels were quantiﬁed
by RP-HPLC (Materials and Methods). Results are expressed as Fatty Acid Mole%,
mean 1 SD, n=4/group.

155

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fasted mice. Total lipids were extracted and saponiﬁed; fatty acid levels were quantiﬁed
by RP-HPLC (Materials and Methods). Results are expressed as Fatty Acid Mole%,
mean : SD, n=4/ group.

156

results suggest that elevated Elov15 lowers blood glucose levels. We observed in high fat-
fed animals, both glucose-intolerance and lower hepatic Elov15 expression [205]. Perhaps
alterations in hepatic Elov15 levels might improve blood glucose levels in high-fat-fed
mice. More analysis will be required to fully understand the underlying mechanism(s) by
which elevated Elov15 impacts hepatic glucose metabolism.

A second key observation is the effect of elevated hepatic Elov15 activity on
hepatic and plasma lipid composition. While overexpressed Elov15 did not signiﬁcantly
impact SREBP-1, and its target genes F AS and ACC during fast and refeeding, over
expressed Elov15 did impact the expression of several PPARa target genes, CYP4A and
HMGCS2. The effect of over expressed Elov15 was most apparent in the fasted state.

Although total hepatic lipid content did not change, the type of fatty acids seen in V
liver and plasma did change. In liver, overexpressed Elov15 resulted in a reduction of 20-
22 carbon PUF A. We speculate this is due to enhanced degradation in the peroxisome.
The oxidation reaction of fatty acyl-CoA is the initial and rate-limiting step of the
peroxisomal beta-oxidation pathway. AOXs and pristanoyl-CoA oxidase (PCOX) can
oxidize the CoA esters of straight-chain fatty acids. Interestingly, the expression of AOX-
1, which is responsible for peroxisomal B-oxidation, was not affected by Elovl-5
overexpression. However, we need additional data on how overexpression of Elov15
affects other subtypes of AOX and PCOX. Hepatic content of 18:1,n-9 increased in livers
of mice receiving Ad-ElovlS. 18:1 is the preferred substrate for triglyceride and
cholesterol ester synthesis [27]. The ratio of 18:1 to 18:0 has been implicated in the

regulation of cell growth and differentiation through effects on membrane ﬂuidity and

157

signal transduction [10]. With this outcome, 1 expected to see an elevation in plasma
triglyceride content.

Overexpressed Elov15 had three effects on plasma lipids; a) decreased triglyceride
levels; b) decreased ratio of 18:1,n-9 to 18:0; and c) change in PUFA composition,
mainly due to the accumulation of 18:2,n-6. Since 18:2,n-6 is only available from the
diet, we speculate the accumulation of 18:2,n-6 in plasma is due to the decreased uptake
of this fatty acid by liver or the increased efflux of 18:2,n-6 from the liver if the uptakcd
18:2,n-6 is not converted to its CoA form rapidly. While my studies do not fully explain
the mechanisms for these observations, they point out that change in hepatic lipid
metabolism lead to changes in blood lipid composition. In studies not shown here,
animals infected with Ad-Elov12 show a remarkable decline in hepatic 20:4,n-6 levels.
Whereas, in animals infected with Ad-A°-desaturase show a signiﬁcant increase in
plasma 20:4,n-6 levels (data not shown). Taken together, these studies highlight the fact
that hepatic PUF A synthesis has a major effect on blood lipids and likely will
signiﬁcantly impact lipid composition in other tissues.

Since both fatty acid and glucose metabolism are altered by Elov15
overexpression, this led us to question how the early disruption of the fatty acid balance
in the liver or plasma may inﬂuence subsequent abnormalities in glucose metabolism.
Skeletal muscle also plays a major role in the development of metabolic disorders,
through the regulation of insulin-induced glucose disposal. The increased PUF A amoumt
in plasma may change the fatty acid composition in the membrane of skeletal muscle,
which may modify insulin signaling pathway in skeletal muscle to impact blood glucose.

More studies are needed to test this hypothesis.

158

In conclusion, the liver plays a central role in the regulation of whole body lipid
and glucose homeostasis. My ﬁndings indicate that overexpression of Elov15, alone, can
alter hepatic function which leads to changes in whole body and hepatic glucose and lipid
metabolism. These results strongly suggest that fatty acid elongases play an important
role in management of glucose and fatty acid metabolism. Whether control of hepatic
Elov15 activity can be used to impact blood glucose and triglyceride in metabolic diseases

remains an important unanswered question.

159

Chapter 6

Conclusions and Future Directions

In the last two decades, a growing number of articles emphasized the role of fatty
acid in the control of gene expression. Fatty acids can directly bind to and regulate the
activity of some transcriptional factors, such as, PPAR family [51,53]. In this case, fatty
acids act like hydrophobic hormones to control the activity or abundance of key
transcriptional factors. In contrast, fatty acids also regulate other transcriptional factors
through indirect mechanism by changing their nuclear abundance and activity, such as
SREBP-1c [14]. Whatever mechanisms are involved in fatty acid regulation of gene
expression, the structure of fatty acid is the key determinant for the regulation of
transcriptional factors [14,53,159].

During fatty acid metabolism, several steps are involved in the modiﬁcation of
fatty acid structure, including elongation/desaturation, 0 oxidation (mitochondrial or
peroxisomal), or assimilation into complex lipids [14]. This suggests the enMes in
these metabolic pathways may inﬂuence gene expression by modifying fatty acid
structures. My studies focus on the enzyme involved in the fatty acid elongation, which
are called fatty acid elongase.

Four fatty acid elongases (Elovll, 2, 5 and 6) and 3 desaturases (A5D, A6D and
A9D) are expressed in liver. Among these enzymes, the desaturases are well studied,
especially A9D. These enzymes are regulated by hormones, development and dietary
lipids [5-10]. A9D knockout mice have shown low possibility to develop hepatic steatosis

and improved insulin sensitivity in muscle [1 1-13]. As such, A9D had been recognized as

160

drug target for obesity treatment recently. Compared to the desaturases, we are still in the
early stage of understanding how fatty acid elongases are regulated and what their roles
are in physiology. My studies ﬁlled this gap with new information.

In the ﬁrst part of these studies, I examined the nutritional and developmental
regulation of rat hepatic Elovl expression and correlated changes in elongase expression
with elongase enzymatic activity. Of the four elongase subtypes expressed in rat liver,
Elov15 is the most abundant elongase transcript. Rat hepatic Elov15 expression is
regulated at the pretranslational level by dietary n3 PUFAs and PPARa agonist and
during postnatal development. These same factors regulate hepatic Elovl activity.
Changes in the expression of the elongases (and their activity) and desaturases are
sufficient to induce changes in tissue and plasma levels of speciﬁc fatty acids (e.g.,
20:3,n-9). Unlike A5, A°, and A9 desaturases, the elongases do not display a uniform
response to fasting-refeeding, ﬁsh oil, or PPARa agonist. In this regard, changes in
nuclear SREBP-l levels correlate with changes in Elovl-6 expression but not other
elongases. Although PPARa agonists induce all desaturases and three elongases (Elovll,
5, and 6), the mechanism for this control remains unresolved.

Next, we further show the evidence that Elov15 and Elovl6 are regulated in liver
by nutrients (glucose and fat), hormones (insulin) and nuclear receptor agonist, i.e., LXR
and PPARa agonist. From these studies, we conclude that ChREBP, MLX, SREBP-1,
PPARa and LXR control elongase, as well as desaturase expression.

Metabolic diseases, like diabetes and obesity, induce changes in hepatic lipid
composition by controlling the function of key transcription factors that impact elongase

and desaturase expression. These studies support the notion that regulation of fatty acid

161

elongase, as well as desaturases, may play an important role in managing hepatic lipid
composition in response to changes in dietary and hormonal status.

Individual Elovl isoforms have different substrate preferences, tissue speciﬁc
expression, and are regulated differently [115,133,175,201,203]. The signiﬁcance of this
diversity is unknown. To get more information about the role of individual hepatic
elongases, we amplify the individual elongase activity in hepatocytes and liver by
recombinant adenovirus technology. The recombinant adenovirus approach elevated
elongase expression and activity to 3 to 5 folds in rat primary hepatocytes. In addition,
overexpression of Elov12 and Elov15 can suppress 20:5,n-3 PPARa regulated gene
expression. I speculate this is due to the suppression of hepatocyte 20:5,n-3 levels and
accumulation of 22:5,n-3 or 24:5,n—3. 22:5,n-3, and possibly 24:5,n-3, are weak activators
of PPARa. Moreover, the overexpression of Elov12, but not Elov15, can enhance 20:5 ,n-
3 suppression of nSREBP-l content without affecting nSREBP-Z. This strongly supports
the notion that fatty elongase have the capacity to alter gene expression by controlling
cellular content of fatty acids that impact transcription factor activity or nuclear
abundance.

To broaden our view about the function of individual elongases in hepatic
function and whole body lipid metabolism, 1 investigated the effect of overexpression of
Elov15 in mice in vivo. Elov15 is the most abundant elongase expressed in rat liver.
Moveover, it has broad substrate speciﬁcity. Overexpression of Elov15 in mouse liver was
achieved by use of adenoviral intravenous transfer to obtain liver-speciﬁc overexpression.
Signiﬁcant reduction in blood glucose in both fasting and refeeding as well as deﬁciency

of glycogen storage during refeeding in Elov15 overexpressed mice suggests Elov15 play

162

an important role in glucose homeostasis. The suppressed gene expression of PEPCK
during fasting can partly explain this observation; it suggests that gluconeogenesis is
suppressed. Effects on GSK30 and ERK phosphorylation status were observed by
overexpressing Elovl-5. These results mimic insulin effects on these signaling pathways.

Overexpression of Elov15 also signiﬁcantly reduced PPAR 0. target gene
expression while having no signiﬁcant effect on SREBP-l and its targets genes.
Moreover, overexpression of Elov15 alters the fatty acid composition both in liver and in
plasma. Most important, overexpression of Elov15 decreased blood triglyceride level
during refeeding. Taken together, these ﬁndings suggest that Elov15 is also an important
factor controlling both hepatic and whole body lipid metabolism.

In summary, this thesis provides novel information about hepatic fatty acid
elongases, in particular Elov15. The results indicate that Elov15 plays an important role in
hepatic function as well as whole body glucose and lipid metabolism. Abnormal
expression of Elov15 in pathological conditions can account for the fatty acid proﬁle in
both liver and plasma. These studies also suggest Elov15 may be the potential target for
treating common diseases such as hypertriglyceridemia, obesity, and insulin-resistant
diabetes.

In future, the use of microarray methods will help expand the effects of Elov15
overexpressed on liver function and may provide new clues to identify speciﬁc molecular
events/pathways inﬂuenced by Elov15. Moreover, modulating Elov15 expression via
siRNA knock down and adenoviral transduction will let us understand better and

comprehensively the role of Elov15 plays in glucose and lipid metabolism.

163

To investigate the role of Elov15 on the onset of chronic disease, we can
overexpress or knockdown Elov15 in diabetic or other pathological animal models, such
as the Zucker rat, Lep°°/°° mouse or the high fat-fed C57BL/6 (glucose intolerant) mouse,
to test if changes in Elov15 inﬂuence the onset and progression of metabolic disease.

We can also use the same methods to test special role of other hepatic elongases.
By this, we will have comprehensive understanding about fatty acid elongase family. The
discovery of the effect of fatty acid elongase on glucose homeostasis may identify novel

pathways to combat diabetes, beneﬁting millions of such patients worldwide.

164

APPENDIX
PUBLICATIONS

Journal Articles

1.

Wang Y, Botolin D, Xu J, Christian B, Mitchell E, J ayaprakasam B,n-air M,
Peters J, Busik J, Olson LK, Jump DB. Regulation of hepatic fatty acid elongase
and desaturase expression in diabetes and obesity. J Lipid Res. 47: 2028-2041
(2006)

Botolin D, Wang Y, Christian B, Jump DB. Docosahexaneoic acid (22:6,n-3)
regulates rat hepatocyte SREBP-1 nuclear abundance by Erk- and 26S
proteasome-dependent pathways. J Lipid Res. 2006 Jan;47: 1 81-92 (2005).

Jump, D.B.; Botolin, D.; Wang, Y.; Xu, J .; Christian, B. & Demeure, 0. Fatty
Acid regulation of hepatic gene transcription. J Nutr, 2005, 135, 2503-2506

Wang Y, Botolin D, Christian B, Busik J, Xu J, Jump DB. Tissue-
speciﬁc,nutritional, and developmental regulation of rat fatty acid elongases. J
Lipid Res. 46:706—15 (2005).

Wang Y, Botolin D, Christian B, Jump DB. Role of fatty acid elongases in ,
hepatic lipid and glucose metabolism. Manuscript in preparation for cell
metabolism.

Conference Abstracts

1. Regulation of rat hepatic elongase and desaturase gene expression. Yun

Wang, Botolin D, Barbara Christian and Donald B. Jump.Regulation of hepatic
elongase and desaturase gene expression. Poster presentation, EB 2006, San
Francisco, California

The impact of hepatic metabolism on fatty acid-regulated PPARa activity
and SEEBP-l nuclear content. D.B. Jump and Y. Wang. FASEB Summer
Conference: Nutrient Control of Gene Expression, July 30-Aug. 5, 2005, Tuscon,
AZ

. Gene regulation in response to polyunsaturated fatty acids. D.B. Jump and

Y.Wang, and D. Botolin, J. Xu, 0. Demeure, J. Busik, W. Chen and B. Christian.
International Conference on the Bioscience of Lipids, September 20-24, 2005 in
Ajaccio, Corsica.

165

4. The impact of hepatic metabolism on fatty acid-regulated PPARa activity.
D.B. Jump and Y. Wang. Fatty acids and cell signaling conference, Paris, France,
Sept. 28, 2005.

166

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