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This is to certify that the
dissertation entitled

INVESTIGATIONS INTO THE TRANSLATIONAL EFFECTS
OF CYPROHEPTADINE

presented by

BELINDA SUE HAWKINS

has been accepted towards fulfillment
of the requirements for the

DOCTORAL degree in PHARMACOLOGY AND
TOXICOLOGY

 

 

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Major Professor’s Signature

397M 3/ 20-68

Date

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INVESTIGATIONS INTO THE TRANSLATIONAL EFFECTS OF
CYPROHEPTADINE

BY

Belinda Sue Hawkins

A DISSERTATION

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

DOCTOR OF PHILOSOPHY
Department of Pharmacology and Toxicology
2003

ABSTRACT

INVESTIGATIONS INTO THE TRANSLATIONAL EFFECTS OF
CYPROHEPTADINE

By

Belinda Sue Hawkins

The antihistaminic, antiserotonergic drug cyproheptadine (CPH) has been shown
to selectively and reversibly produce ultrastructural and biochemical alterations in
pancreatic B-cells. CPH has previously been shown to inhibit proinsulin synthesis and
deplete pancreatic insulin content in isolated rat pancreatic islets and in Rle5F cells.
This inhibition of insulin synthesis occurs without a commensurate loss of preproinsulin
mRNA (PPImRNA) levels, suggesting the drug is acting via one or more post-
transcriptional mechanisms to inhibit the synthesis of insulin. Experiments were
conducted to investigate the post-transcriptional mechanisms of CPH-induced inhibition
of insulin synthesis in Rle5F cells and to examine the hormone specificity of these
effects.

Using a subcellular fractionation technique followed by RT—PCR, CPH was found
to alter the subcellular localization of PPImRNA in Rle5F cells. This subcellular
dislocation was characterized by a decrease in the percentage of ribosome-associated
PPImRNA localized at the endoplasmic reticulum and increases in the percentages of
PPImRNA associated with various cytosolic ribosomal populations. This CPH-induced
PPImRNA subcellular dislocation was found to be concentration-dependent, chemical
structure-specific, and reversible. The time course of CPH-induced PPImRNA
dislocation was consistent with the time course of CPH-induced inhibition of insulin
synthesis. These findings are consistent with an effect of CPH on the translation of

PPImRNA and suggested an effect of the drug on the initiation stage of translation.

Further investigations were undertaken to examine the effect of CPH on
translation initiation. Polysome profile analysis after CPH treatment indicated an
increase in the monoribosome peak characteristic of an inhibition of initiation. In
addition, CPH treatment increased phosphorylation of the initiation factor elFZa and
decreased phosphorylation of the initiation factors elF4E and 4E-BP1. These results are
all consistent with a CPH-induced inhibition of initiation.

To investigate the specificity of the translational effects of CPH, the ability of the
drug to alter the subcellular localization of additional messages was examined. CPH
induced subcellular dislocation of preproglucagon, preproamylin, and B—actin mRNAs in
Rle5F cells suggesting that the translational effects of CPH are not specific to the
insulin message. In addition, the subcelluar dislocation of these mRNAs in Rle5F
cells occurred without alteration in the cellular content of these proteins, suggesting that
the depletion of cellular hormone content in response to CPH treatment may involve a
mechanism in addition to the inhibition of protein synthesis. In contrast to results from
Rle5F cells, no subcellular dislocation of preproglucagon mRNA was induced in the
clonal (1-06" line, aTC1.9. These findings are consistent with a B-cell specific effect of
the drug.

Taken together, these results suggest that the inhibition of insulin synthesis
elicited by CPH treatment in Rle5F cells involves alterations in initiation factor
phosphorylation leading to decreased initiation of PPImRNA. The mechanism
underlying the apparent specificity of CPH for the inhibition of insulin synthesis is not

elucidated in these results and remains unclear.

To my mother, for the love and support that made this possible.

ACKNOWLEDGMENTS

I would like to express sincere gratitude to my advisor, Dr. Lawrence J.
Fischer, for providing encouragement, direction, and insight throughout the
course of my graduate training. I would also like to thank the members of my
committee, Dr. K. Lookingland, Dr. L. K. Olson, and Dr. S. Watts, for their
excellent suggestions and guidance.

Finally, I would like to thank the Department of Pharmacology and
Toxicology and the Institute for Environmental Toxicology at Michigan State

University for financial support during my studies at MSU.

TABLE OF CONTENTS

LIST OF TABLES .................................................................................. ix
LIST OF FIGURES ................................................................................. x
ABBREVIATIONS ................................................................................ xiii
INTRODUCTION ................................................................................... 1

The Endocrine Pancreas ................................................................ 2

General Aspects of Peptide Hormone Synthesis,

Intracellular Conversion, and Storage ................................................ 4

Insulin Biosynthesis .............................................................. 7

Glucagon Biosynthesis ........................................................ 14

Amylin Biosynthesis ............................................................ 18

Secretory Protein Degradation .............................................. 18

Protein Translation ...................................................................... 19

Cyproheptadine .......................................................................... 26
Rle5F Cells as a Model for Investigations into

CPH-Induced B-Cell Toxicity ................................................. 33

Objectives and Rationale .............................................................. 34

MATERIALS AND METHODS ................................................................ 39

RESULTS .......................................................................................... 60

Effects of CPH on PPImRN_A in Rle5F Cells
Effect of CPH on PPImRNA Stability ...................................... 60

Effects of CPH on PPImRNA
Subcellular Localization ....................................................... 61

Effects of 2-DPMP and 4-DPMP on PPImRNA
Subcellular Localization ....................................................... 63

vi

Effects of CPH on Translation Initiation in RINm5F Cells

 

Effect of CPH on Polysome Distribution ................................... 71

Effect of CPH on the Phosphorylation States of

Initiation Factors elF2a, elF4E, and 4E-BP1 ............................ 71
Effecjt§ of CPH on Glucagon in Rle5F Cells

Effects of CPH on Preproglucagon mRNA Subcellular
Localization ....................................................................... 79

Effect of CPH on Cellular Glucagon Protein Levels .................... 79

Effect of Cycloheximide on Cellular Glucagon Protein

Levels .............................................................................. 80
Effects of CPH on PPAmRNA in Rle5F Cells

Effects of CPH on Preproamylin mRNA Subcellular

Localization ....................................................................... 86

Effects of CPH on B-Actin in Rle5F Cells

Effects of CPH on B-Actin mRNA Subcellular localization ............ 90
Effects of 2-DPMP and 4-DPMP on B-Actin mRNA

Subcellular Localization ....................................................... 90
Effect of CPH on Cellular B-Actin Protein Levels ....................... 91

Effects of CPH in a-Cells

Effect of CPH on Preproglucagon mRNA Subcellular
Localization in aTC1.9 Cells ................................................. 97

Effect of CPH on Cellular Glucagon Content
in aTC1.9 Cells .................................................................. 98

vii

Effects of CPH on Cellular Glucagon Content in Isolated

Rat Pancreatic Islets ........................................................... 98

DISCUSSION .................................................................................... 104

Effect of CPH on the stability of PPImRNA ...................................... 104

Effects of CPH on the subcellular localization of

PPImRNA in RINm5F cells .......................................................... 105

Effects of CPH on translation initiation in RINm5F cells ..................... 106

Effects of CPH on the subcellular localization of

PPGmRNA, PPAmRNA, and B-actin mRNA in Rle5F cells .............. 111

Lack of effect of CPH on cellular protein levels of glucagon

and B-actin in Rle5F cells ......................................................... 116

Lack of effect of CPH on PPGmRNA subcellular localization

and cellular glucagon content in a—cells .......................................... 119
CONCLUSIONS ................................................................................. 121
REFERENCES .................................................................................. 126

viii

LIST OF TABLES

1. Real-Time PCR Oligonucleotides ................................................... 48

10.

11.

12.

13.

14.

15.

LIST OF FIGURES

Preproinsulin .............................................................................. 1 1

Post-translational trafficking and processing of insulin

by the pancreatic B-cell ................................................................. 12
The proglucagon tree ................................................................... 17
Initiation of protein synthesis in mammalian cells ............................... 21
Assembly of the translation initiation complex .................................... 24
Chemical structures of CPH, 4-DPMP and 2-DPMP ........................... 32
Subcellular fractionation scheme .................................................... 45

Effects of CPH on PPImRNA in RINm5F Cells:

Lack of an effect of CPH on PPImRNA stability ................................. 64
Distribution of the ER in RINm5F subcellular fractions ........................ 65
Lack of an effect of CPH on total PPImRNA levels ............................. 66

Effects of CPH on the subcellular distribution of
PPImRNA .................................................................................. 67

Time course of CPH effects on the subcellular
distribution of PPImRNA ............................................................... 68

Reversibility of CPH effects on the subcellular
distribution of PPImRNA ............................................................... 69

Effects of 2-DPMP and 4-DPMP on the subcellular
distribution of PPImRNA ............................................................... 70
Effects of CPH on Translation Initiation iLRINm5F Cells

Polysome profile analysis of cellular lysates from
control and CPH-treated cells ........................................................ 72

16.
17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

Effect of CPH on elF2a (Ser51) phosphorylation ................................ 73
Effect of CPH on elF4E (Ser209) phosphorylation .............................. 75
Effect of CPH on 4E-BP1 (Ser65) phosphorylation ............................. 77

Effects of CPH on Glucaan in RINm5F Cells

Lack of an effect of CPH on total PPGmRNA levels ............................ 81
Effects of CPH on the subcellular distribution of

PPGmRNA ................................................................................. 82
Lack of an effect of CPH on cellular glucagon content ......................... 84

Effects of cycloheximide on cellular DNA and glucagon

content ...................................................................................... 85
Effects of CPH on PPAmRNA in RINm5F Cells

Lack of an effect of CPH on total PPAmRNA levels ............................ 87
Effects of CPH on the subcellular distribution of

preproamylin mRNA ..................................................................... 88

Effects of CPH on B-Actin in RINm5F Cells

Lack of an effect of CPH on total B-actin mRNA levels ........................ 92
Effects of CPH on the subcellular distribution of

B-actin mRNA ............................................................................. 93
Effects of 2-DPMP and 4-DPMP on the subcellular

distribution of B-actin mRNA .......................................................... 95
Lack of an effect of CPH on cellular B-actin content ............................ 96

Effects of CPH in (l-CeIISI

Distribution of the ER in aTC1.9 cell subcellular fractions ..................... 99

xi

30.

32.

33.

30.

31.

32.

33.

Lack of an effect of CPH on total PPGmRNA levels

in aTC1.9 cells .......................................................................... 100
Lack of an effect of CPH on the subcellular distribution of

PPGmRNA in aTC1.9 cells .......................................................... 101
Lack of an effect of CPH on cellular glucagon content in

aTC1.9 cells ............................................................................. 102
Lack of an effect of CPH on cellular glucagon content in

isolated rat pancreatic islets ......................................................... 103
Proposed site of CPH action to inhibit the synthesis of insulin ............. 125

xii

BCP
C-Peptide
CPH

CX

DEPC
DNA
DPMP
ECL
EDTA

eIF

ER

GLP
GPAIS
HPLC
HRP
mRNA
PABP
PCR

PNS
PPAmRNA
PPGmRNA
PPImRNA

ABBREVIATIONS

1-bromo-3-chloropropane
Connecting peptide
Cyproheptadine

Cycloheximide

Diethylpyrocarbonate

Deoxyribonucleic acid
Diphenylmethylpiperidine

Enhanced chemiluminescence
(Ethylenedinitrilo)tetraacetic acid
Eukaryotic initiation factor

Endoplasmic reticulum

Glucagon-like peptide

Guinea pig anti-insulin serum
High-performance liquid chromatography
Horseradish peroxidase

Messenger ribonucleic acid
Poly(A)-binding protein

Polymerase chain reaction

Post-nuclear supernatant

Preproamylin messenger ribonucleic acid
Preproglucagon messenger ribonucleic acid

Preproinsulin messenger ribonucleic acid

xiii

(n

(n

rn

PVDF
RIA
RNA
RT
808
SRP
SR
TCA
TLC
UPR
UTR

Polyvinylidene fluoride
Radioimmunoassay

Ribonucleic acid

Reverse transcription

Sodium dodecyl sulfate

Signal recognition particle

Signal recognition particle receptor
Trichloroacetic acid

Thin layer chromatography
Unfolded protein response

Untranslated region

xiv

INTRODUCTION

The endocrine pancreas, by secreting polypeptide hormones, both
positively and negatively regulates blood glucose levels. Though many peptides
are secreted by the endocrine pancreas, two peptides, insulin and glucagon, are
most involved in this regulation. The most well studied of these hormones,
insulin, functions to increase both tissue uptake and utilization of glucose.
Glucagon, with actions opposing that of insulin, promotes glycogenolysis and
gluconeogenesis. Abnormal regulation of blood glucose levels, producing
hyperglycemia, is a hallmark characteristic of diabetes. Diabetes is a
multifaceted disease containing a prominent genetic component that has yet to
be fully characterized. This genetic component of diabetes is thought to play a
permissive role, with some sort of environmental promotion, such as a viral or
chemical exposure, required for development of the disease [1]. Investigations
into the role of environmental factors in the development of the diabetic state are
widespread. In fact, certain exogenous chemical substances are known to
selectively damage the insulin-producing cells of the endocrine pancreas in both
laboratory animals and in humans [2]. It is widely believed that environmental
exposure to certain exogenous chemicals, including certain therapeutic
compounds, may play a role in the etiology of some cases of human diabetes.
Investigations into the mechanisms of xenobiotic compound toxicity in pancreatic

B-cells may provide valuable insight into the normal physiology of these cells as

well as into the etiology of certain types of diabetes mellitus. One B-cell toxicant

currently under investigation is the antihistaminic, antiserotonergic drug
cyproheptadine (CPH). CPH has been shown to inhibit proinsulin synthesis in
pancreatic B-cells leading to the depletion of pancreatic insulin content [3, 4].
This drug-induced inhibition of insulin synthesis has been shown to occur without
a commensurate decrease in preproinsulin mRNA (PPImRNA) levels [5, 6]. This
suggests a post-transcriptional mechanism of action for the compound. The
studies described within this thesis are directed towards elucidating the post-

transcriptional mechanism(s) involved in CPH-induced B-cell toxicity.

The Endocrine Pancreas

The endocrine pancreas is comprised of numerous groups of cells, known
as the islets of Langerhans, scattered throughout the pancreatic mass. The
islets of Langerhans are discrete clusters of hormone-secreting cells which
comprise approximately 1 to 2% of the total pancreatic volume [7] while the
remaining 98-99% is composed of exocrine, ductal, connective and neural
tissues. Islets are comprised of four major endocrine cell types: insulin-
producing B-cells, glucagon-producing a—cells, somatostatin-producing 8-cells,
and pancreatic polypeptide-producing PP- (or F-) cells. Ultrastructural and
immunocytochemical techniques have led to the identification of other minor islet
cell types while numerous other peptides and hormones have been localized to
the islet cells with the use of sensitive immunostaining techniques.

The B-cells are the most abundant islet cell type comprising 70 to 80% of

the total islet cell population in adult mammals; 6-cells comprise approximately

5% while the remaining 15 to 20% is either a- or PP-cells, depending on the
anatomic location of the islet within the pancreas [7]. The distribution of the
endocrine cells within the islet is nonrandom, with a core of B—cells surrounded by
a discontinuous mantle of non-B-cells 1 to 3 cells thick [8]. This organized
distribution of islet cell types is consistent with a role for paracrine influences in
the synthesis and secretion islet hormones. In addition, further evidence of cell-
to-cell communication comes from the findings that islets contain gap junctions
[9]. These gap junctions link different islet cell types and provide a means for the
transfer of ions, peptides, or bioelectric current between the various cell types
and are critical for normal B-cell secretory function [10].

The anatomy of the islet vascular supply is also consistent with paracrine
involvement in hormone secretion. The pancreas receives arterial blood from the
splenic, hepatic, and mesenteric arteries while venous drainage is into the
splenic and mesenteric veins. Afferent blood vessels penetrate nearly to the
core of the islet prior to branching out and returning to the islet surface. The
innermost cells of the islet therefore receive arterial blood, while those cells along
the periphery receive blood which contains molecules released from the inner
cells which secrete primarily insulin.

Islets cells are influenced by both sympathetic and parasympathetic
innervation. Sympathetic stimulation (via the splanchnic nerve) inhibits insulin
secretion and stimulates glucagon secretion while parasympathetic stimulation

(via the vagus nerve) stimulates insulin secretion and inhibits glucagon release.

The overall role of the endocrine pancreas is to coordinate the release of
hormones to direct both the storage and the use of fuels during times of nutrient
abundance and deficiency. Glucose is the primary physiologic regulator of
hormone secretion from the endocrine pancreas. Hyperglycemia stimulates the
release of insulin while hypoglycemia stimulates the release of glucagon. Insulin
serves to lower blood glucose levels by increasing the cellular uptake and
utilization of glucose throughout the body, with liver, skeletal muscle, and
adipose tissue being its primary targets. Glucagon raises blood glucose levels
by inducing the mobilization and release of glucose from the liver. Glucagon
stimulates insulin release while insulin inhibits glucagon secretion. Somatostatin
inhibits both insulin and glucagon secretion. The release of hormones from the
islet cells is highly regulated and these hormones act in concert to maintain blood

glucose levels in the normal physiologic range of 80-110 mgldL.

General Aspects of Peptide Hormone Synthesis, Intracellular ConversionI
and Storage

Peptide hormone synthesis begins with transcription of the hormone gene.
Transcription occurs in the nucleus with the newly transcribed messenger RNA
(mRNA) then being processed into a mature mRNA via the removal of non-
coding nucleotide sequences known as “introns” through a process known as
RNA splicing. Further mRNA posttranscriptional modifications include the
addition of a 7-methylguanosine residue at the 5’ end (known as the “cap”) and

the addition of a string of 40-200 adenosine nucleotides (known as the “poly(A)

tail”) at the 3’ end. The 5’ cap is thought to play an important role for efficient
translation while it has been suggested that the addition of the poly(A) tail serves
to stabilize the mRNA and thus prolong the survival of the message within the
cell. Once processed, the mature mRNA is exported from the nucleus into the
cytosol.

Translation of peptide hormone mRNA into the full-length polypeptide
begins in the cytosolic compartment on free cytoplasmic ribosomes. With few
exceptions, translation of the mRNA leads to the production of a polypeptide
chain which is considerably larger than the mature hormone. This extended form
of the peptide represents a precursor form of the exportable product with an NH2
terminal extension of 15-30 amino acid residues known as the “signal sequence”
or “signal peptide”. Signal peptides have been found in nearly all secretory
proteins of animal, plant, and bacterial origin and function to facilitate the
segregation of secretory proteins from the cytosolic compartment, where protein
synthesis is initiated, into the secretory pathway via a complex series of
molecular interactions resulting in the translocation of the nascent peptide across
the membrane and into the lumen of the ER [11]. At this early stage of synthesis,
the peptide precursor is known as a “preprohormone” or “prehormone”.
Following the onset of secretory protein translation, the signal peptide is bound
by a protein complex known as the signal recognition particle (SRP) as it
emerges from the translating ribosome [12-14]. SRP is a cytosolic
ribonucleoprotein complex [13] that binds the signal peptide, the ribosome, and

an ER-specific protein known as the “SRP receptor” (SR) or “docking protein”.

This SRP binding to the ribosome-associated nascent chain arrests polypeptide
chain elongation and concurrently induces an interaction with SR at the ER
membrane translocating the ribosome-associated nascent chain/SRP complex to
the ER [13, 15, 16]. The interaction of the translation-arrested ribosome/SRP
complex with SR results in the GTP—dependent release of SRP from both the
nascent chain and the ribosome [17, 18], thereby allowing peptide synthesis to
resume [19]. Released from SRP, the ribosome then engages with the protein
translocation machinery in the ER membrane and, concurrently with its
synthesis, the nascent peptide chain is translocated across the ER membrane.
As translation continues, the signal peptide is enzymatically removed by a signal
peptidase and released into the cisternal space. This cotranslational processing
of the nascent peptide occurs very rapidly thus the cellular concentrations of
preprohormone are very low.

After translation of the mRNA is completed, the peptide is released in its
entirety into the cisternae of the ER. After the removal of the signal peptide, the
remaining peptide is often still larger than the mature hormone and is designated
a “prohormone”. Full biologic activity of a hormone is achieved by limited
proteolysis of the prohorrnone into the mature secretory protein. This proteolysis
takes place in either the Golgi region or in the secretory granules themselves. In
certain situations, activation occurs extracellularly (e.g. zymogens). Intracellular
cleavage of the prohonnone is carried out by a variety of proteases having tryptic

and carboxypeptidase-like activities.

Intracellular transport of the newly formed prohorrnone from the cisternae
of the ER to the Golgi complex occurs via a vesicular system derived from the
transitional elements of the ER. This transport is energy dependent and can be
blocked by inhibitors of oxidative phosphorylation [20].

The Golgi complex provides both the enzymatic apparatus for the
conversion of certain prohonnones and the membranes which will form the
secretory granules themselves.

Secretory cells which release peptides in response to biological stimuli
require a system for the intracellular storage of these compounds. This is
accomplished through the formation of secretory granules. Secretory granules
typically consist of a central core of secretory material surrounded by a smooth
membrane. In certain cases, as with pancreatic B-cells, proteolytic processing of
the prohorrnone occurs within the granules themselves. In other cell types,
including those of the exocrine pancreas, the precursor is stored within the
secretory granules without further processing, which takes place extracellularly.
Some secretory cell types with a continuous secretion pattern lack a storage form

of their export product.

Insulin Biosynthesis

Insulin biosynthesis within the pancreatic B-cell begins with the
transcription of the insulin gene by RNA polymerase II into preproinsulin mRNA
(PPImRNA). The primary transcript is processed within the nucleus to form

mature PPImRNA. The posttranscriptional processing of PPImRNA includes the

addition of the 5’ cap and poly(A)tail and the splicing out of one or two introns
(depending on the insulin gene). The initial product of insulin gene translation is
a 109 amino acid peptide known as preproinsulin. Preproinsulin is rapidly
converted to proinsulin within the lumen of the endoplasmic reticulum (ER).
During the intracellular transport of proinsulin from its site of synthesis to the
storage granules, it is slowly cleaved by proteolysis to yield insulin and a 31-
residue peptide fragment known as the connecting peptide (C-peptide) (See
Figure 1).

Preproinsulin, discovered in 1975, is a larger form of proinsulin containing
a 24-residue signal peptide [21]. Once the signal peptide has been translocated
through the ER membrane, it is rapidly cleaved by signal peptidase located on
the inner surface of the ER membrane [22] converting preproinsulin to proinsulin.
In the case of preproinsulin, this cleavage occurs, for the most part, before
translation of the entire preproinsulin molecule has been completed [23].

Following the translocation of preproinsulin and the cleavage of the signal
peptide, proinsulin folds, undergoes rapid disulfide bond formation and is
transported from the ER to the cis region of the Golgi apparatus for further
processing and packaging [11]. Due to the rapid removal of the signal peptide in
the ER, preproinsulin is not a normal secretory product of B-cells [24].

Proinsulin, as is the case of other exportable proteins, is synthesized by
ribosomes associated with the rough endoplasmic reticulum [25] and constitutes
the major biosynthetic product found in the microsomal fraction [26]. As

mentioned above, proinsulin is derived from a larger precursor, preproinsulin,

which is rapidly cleaved to proinsulin in the ER. Proinsulin is then transported
from the ER to the cis Golgi apparatus in smooth microvesicles. It is then
transported from one Golgi stack to the next in a discrete population of vesicles
typified by the presence of a B-COP (a 110kD coat protein) coat on the cytosolic
face of their membrane [27]. The trans-most cisternae of the Golgi apparatus are
the site for sorting of products destined for either the constitutive or the regulated
secretory pathway [28]. A schematic of the post-translational trafficking and
processing of insulin in the pancreatic B—cell is shown in Figure 2. Pancreatic B-
cells normally sort more than 99% of newly synthesized proinsulin to secretory
granules for regulated release [29]. The earliest detectable form of an insulin
secretory granule carries a partial clatherin coat and contains proinsulin [30, 31].
This granule is known as the immature (or “coated”) granule and is formed by
pinocytosis of the clatherin coated domains of the trans Golgi [23]. Immature
insulin secretory granule formation can be inhibited by both ATP depletion [31]
and by the sodium ionophore monensin [32]. Three events occur in during the
maturation of the immature granule: progressive acidification of the intragranular
milieu, proinsulin to insulin conversion, and loss of the granule clatherin coat.
Granule acidification is due to the action of an ATP-dependent proton pump [33].
Immature granules are only mildly acidic and undergo gradual acidification as the
granules mature [31]. Proinsulin conversion occurs via the concerted activities
of two Ca”-dependent endoproteases, P01 and P02, and the exoprotease

carboxypeptidase H [34-37].

Proinsulin contains two pairs of basic residues at which cleavage must
occur to generate mature insulin: the Arg31Arg32 linkage at the B-chainlC-
peptide junction and the Lys64Arg65 linkage at the C-peptide/A-chain junction
[11]. PC1 cleaves almost exclusively on the carboxyl side of Arg31Arg32 at the
B-chainlC-peptide junction whereas PC2 prefers the Lys64Arg65 site at the C-
peptidelA-chain junction [38]. These enzymes display a narrow pH optima in the
range of pH 5-6 [39], highlighting the necessity of granule acidification for
proinsulin conversion. The complete conversion of proinsulin requires the
endoproteolytic cleavage at one or the other of these two sites followed by
trimming of residual C-terrninal basic residues by carboxypeptidase H [34]. A
second round of endoproteolysis followed by carboxypeptidase trimming then

generates mature insulin and C-peptide [11, 35].

10

C-PEPTIDE

 

Arg
Arg

SIGNAL PEPTIDE 2

Figure 1. Preproinsulin. The initial precursor of insulin contains four domains.
The signal peptide is cleaved off within the lumen of the ER to produce
proinsulin. Conversion of proinsulin to insulin occurs via the cleavage of two
pairs of basic amino acids to produce the A- and B-chains of insulin and C-
peptide. The A- and B-chains of insulin are joined together by two disulfide

bridges. Figure from [40].

11

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12

The combination of granule acidification and proinsulin to insulin
conversion provides the optimal environment for insulin crystallization within the
mature granule. Insulin is able to associate into dimers and, in the presence of
Zn”, these dimers can associate to form hexamers [41]. The Zn“ hexamers
then pack together to form a crystal lattice which forms the dense core of the
insulin secretory granule observed by electron microscopy [42]. C-peptide does
not co-crystallize with insulin and is therefore excluded from the insulin crystal.
C-peptide is found in the clear halo surrounding the dense core of the mature
secretory granules [43] and accumulates within the granules along with insulin.
Due to the localization and biochemical mechanism of proinsulin to insulin
conversion within the granule, C-peptide is secreted along with insulin in
equimolar amounts during exocytosis of the granule contents [44].

Interestingly, rats and mice synthesize two different insulins (insulins l and
II) that are coded for by two non-allelic insulin genes. All other mammals studied
to date produce a single type of insulin coded for by a single insulin gene [45].
The structures of both the genes [46, 47] and the corresponding insulins [48, 49]
have been well studied. In the rat, preproinsulin l and II differ by 7 amino acids:
3 in the signal peptide, 2 in the C-peptide, and 2 in the B-chain [46]. The mature
insulins differ at positions 9 and 29 of the B-chain where methionine replaces the
two lysines of insulin l [50]. In rats and mice, under normal physiologic
conditions, the ratio of preproinsulin l mRNA to preproinsulin ll mRNA is
approximately 60:40 [51, 52] while the expression of insulin Izinsulin ll follows the

same distribution [48, 53]. Interestingly, the rat insulinoma cell line RINm5F has

13

been shown to express only preproinsulin l mRNA [54], indicating an important

difference between primary B-cells and this clonal cell line.

Glucagon Biosynthesis

Though there is a great deal of information concerning the secretion of
pancreatic glucagon in response to various physiologic stimuli [55], relatively little
is known about the biosynthesis of glucagon. Although the processes of
hormone secretion and biosynthesis are closely related, further studies on the
biosynthesis of glucagon are needed to better understand the physiology and
pathophysiology of diabetes.

Glucagon biosynthesis begins with transcription of the glucagon gene into
preproglucagon mRNA. The preproglucagon gene is expressed in pancreatic on-
cells, the L-cells of the intestine, and in some specialized neurons in the CNS. A
single mRNA species exists, with no evidence of alternate splicing [56]. From
this mRNA, the 180-amino acid preproglucagon, which contains a 20-amino acid
signal sequence, is translated. Glucagon biosynthesis follows the same basic
mechanism as all other exportable products; synthesis begins in the cytosol and
the nascent peptide is translocated to the ER and shuttled into the secretory
pathway. After removal of the signal peptide, the 160-amino acid proglucagon
undergoes endoproteolytic processing, which varies according to the tissue in
question, leading to a series of active peptide fragments. Surprisingly, two
additional peptidic structures closely resembling glucagon are found in the C-

terrninal moiety of mammalian proglucagon [57-59]. The biological features of

14

these peptides, named “glucagon-like peptide l” and “glucagon—like peptide II”
(GLP-1 and GLP-2, respectively) have been subsequently studied and these
peptides, a truncated form of GLP-1 known as t-GLP-1 in particular, have been
found to play important roles in the regulation of insulin secretion.

The intestinal forms of glucagon-like peptides display immunological
features that differ from forms found in the pancreatic a-cell [60]. Two peptides,
both containing the 29-amino acid peptide glucagon, have been isolated from
porcine intestine, the 69-amino acid peptide glicentin [61] and the 37—amino acid
peptide oxyntomodulin [62, 63]. Both of these peptides contain a C-tenninal
octapeptide not found in pancreatic glucagons and this octapeptide is the main
feature distinguishing the intestinal type from the pancreatic type of proglucagon
processing. The final products from the first 69 amino acids of proglucagon are
essentially glucagon in the pancreatic a-cell and a mixture of glicentin and
oxyntomodulin in the intestinal L cells [64].

In addition to differential processing of the first 69 amino acids of
proglucagon, the C-terminal fragment also undergoes tissue specific processing.
Although small amounts of free GLP-1 (full length or truncated) and GLP-2 are
detectable in both porcine and human pancreas [65], the C-terrninal proglucagon
moiety remains virtually unchanged in the pancreas leading to the release of
fragment 72-158 [65], also known as “major proglucagon fragment” (MPGF) [66].
The role of MPGF is currently unknown. In contrast, intestinal L cells process the

proglucagon C-terminal in such a way as to generate and release biologically

15

active peptide fragments. A schematic of proglucagon processing is shown in
Figure 3.

The main proglucagon fragments are the result of cleavages at dibasic
residues though the specific prohorrnone convertases involved are still under
investigation. All but one dibasic site are loci where proglucagon is cleaved by
processing enzymes in both pancreatic a-cells and intestinal L-cells leading to

the production of biologically active peptides.

16

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Figure 3. The proglucagon tree. See text for details. Modified from [64].

17

Amylin Biosynthesis

Amylin (also known as islet amyloid polypeptide or IAPP) is a 37-amino
acid peptide that is produced by the pancreatic B—cell from the 89-amino acid
precursor preproamylin [67]. Amylin is found in the pancreatic B-cell but not the
or-cell [68-70] though in some transformed murine islet cell lines a large
percentage of cells contain both amylin and glucagon [71]. Amylin is colocalized
with insulin in the soluble component of [3-cell secretory granules and, as such, is
cosecreted with insulin [72-74]. Though both insulin and amylin are located
within insulin secretory granules, there is evidence for differential regulation of

the insulin and amylin genes [75].

Secretory Protein Degradation

The hormone content of secretory cells is a reflection of a closely
regulated balance between hormone biosynthesis, release, storage, and
degradation. While a wealth of information exists on the biosynthesis, release,
and storage of secretory proteins, relatively little is known of their cellular
degradation. In the case of insulin, the most common mechanism of cellular
degradation in the pancreatic B-cell is the fusion of secretory granules with
primary Iysosomes, a process known as granulolysis or crinophagy [76]. Though
B-cells have been shown to degrade a significant portion of their cellular insulin
[77, 78], it is degraded slowly even after its introduction into Iysosomes [76, 79,

80]. It has been suggested that this is due to the stability of the insulin crystal in

18

Iysosomes which have an acidic pH similar to that seen in the secretory granules
themselves.

For secretory proteins, it is suggested that the degradative pathway
ensures that aged, and possibly damaged, secretory material is disposed of. For
insulin, there is an apparent inverse correlation between the relative rates of

release and degradation of the hormone within the B-cell [77, 78, 81].

Protein Translation

Protein translation is conventionally divided into three separate stages:
initiation, elongation, and termination. Of these, initiation is generally regarded
as rate-limiting for protein synthesis and alterations in initiation are common
means of translational control [82]. Initiation itself can be divided into several
stages each of which is facilitated by proteins known as initiation factors
(eukaryotic initiation factors or elFs). The initiation machinery in eukaryotes is
highly complex and requires at least eleven initiation factors several of which are
comprised of multiple polypeptides [83].

The stages of initation are summarized below [84] and shown in Figure 4:
Stage I: the dissociation of the 808 ribosome into its component ribosomal
subunits, 408 and 60S, which requires the multimeric initiation factor elF3 and
probably elF1A as well; Stage II: the binding of the initiator methionyl-tRNA
(Met-tRNAiMe‘) to the small (40S) ribosomal subunit, which is mediated by elF2.
This stage also involves elF1A and results in the formation of the 43S

preinitiation complex; Stage III: the recognition of the 5’ cap of the mRNA by

19

elF4E. This step as well as the next one most likely occur concurrently with the
steps outlined above; Stage IV: the interaction of elF4E with the scaffolding
protein, elF4G and the RNA helicase elF4A to form an active elF4F complex;
Stage V: interaction of elF4F with the 43S preinitiation complex and the
subsequent scanning from the 5’ end of the mRNA to the AUG codon by the 408
subunit. The initiation codon of eukaryotic mRNAs is normally the first AUG
triplet downstream of the 5’-terminal cap and is usually separated from the cap
by a string of 50-100 nucleotides. This process also involves the RNA-binding
initiation factor elF4B; Stage VI: addition of the 608 subunit to form the 80S

initiation complex. This step also involves elF5.

20

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Changes in the activities of several translation initiation factors are thought
to play critical roles in the regulation of translation in mammalian cells [85, 86].
There are two sets of initiation factor interactions that have been extensively
studied and are generally regarded as the predominant regulators of translation
initiation: the interaction of elF2 with elF2B and the interactions between elF4E
and the 4E-BPs [87]. A schematic representation of the assembly of the
translation initiation complex and the interactions of these initiation factors is
shown in Figure 5.

The initiation factor elF2 is responsible for the binding of the Met-tRNAaM‘“
to the 408 subunit to form a stable elF2-GTP-Met-tRNA.""""t complex, the first step
in initiation. This elF2-dependent binding of the Met-tRNAaM‘“ to the 408 subunit
is required for subsequent mRNA binding [83, 88] thereby making the regulation
of this initiation factor an important target of translational control. At the end of
each round of initiation, elF2 is in an inactive, GDP-bound state that requires the
activity of a guanine-nucleotide exchange factor, elF2B, to form an elF2-GTP
complex capable of recruiting a new Met-tRNAiM‘”. This recycling step plays an
important role in the regulation of translation [89] and can be regulated by a
variety of mechanisms including phosphorylation of the a-subunit of elF2,
allosteric control, and phosphorylation of the 8 subunit of elF2B itself. The best
characterized regulator of elF2 activity is the phosphorylation of the ot-subunit of
elF2 at Ser51. Phosphorylation at this site increases the affinity of elF2 for
elF2B but renders the complex incapable of guanine-nucleotide exchange [85];

the overall effect is to decrease the amount of elF2B available to recycle the

22

remaining non-phosphorylated GDP-bound elF2 complexes thereby inhibiting
protein synthesis.

The binding of the 40S subunit to mRNA involves the interaction of several
initiation factors and is a potential site for overall translation regulation and for the
regulation of translation of specific mRNAs. The initiation of cap-dependent
translation involves the elF4F complex, which is comprised of the initiation
factors elF4E, elF4G and elF4A. The initiation factor elF4E binds to 5’-cap and
forms a complex with the scaffolding protein, elF4G, which recruits additional
initiation factors. The additional initiation factors include elF4A and elF4B, which
are involved in unwinding mRNA secondary structure, and the poly(A)-binding
protein (PABP), which interacts with the 3’-polyadenylated tail to circularize the
mRNA [90]. elF4E also binds to small regulatory proteins known as elF4E-
binding proteins (4E-BPs). There are two known 4E-BPs, 4E-BP1 and 4E-BP2
(also known as PHASI and PHASII). elF4G and the 4E-BPs compete for binding
to a conserved site on elF4E [90]. By preventing elF4G binding to elF4E, the
4E-BPs inhibit translation initiation. The binding of 4E-BP to elF4E is regulated
by phosphorylation of 4E-BP. Hyperphosphorylation of 4E-BP blocks its binding
to elF4E while in its hypophosphorylated state 4E-BP is bound to elF4E and
prevents elF4F assembly [90]. 4E-BP has been shown to be phosphorylated at
five to six sites and phosphorylation at several of these sites is required for elF4E

release [91, 92].

23

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9'}.
.-

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i '5

 

1‘-

Figure 5. Assembly of the translation initiation complex. The initiation factor
elF2 binds Met-tRNAiM‘” to the 408 ribosomal subunit. Initiation factor elF3
interacts with elF4G (though the particular elF3 subunit involved is not yet
known) while the C subunit of elF3 interacts with both elF1 and elF5. In yeast,
elF5 has been shown to bind directly to elF4G [93] (this interaction is depicted by
the dashed double-headed arrow). The initiation factors elF1, elF1A, elF2, elF3,
elF5, and elF5B bind to the 408 subunit at various times during the initiation
process. The factor elF4G serves as a scaffolding protein for the recruitment of
mRNA and contains binding sites for the cap-binding protein elF4E, elF4A, the
poly(A)-binding protein (PABP), and the elF4E kinase MNK1. The association of
elF4G, elF4E and elF4A is known as the elF4F complex. The 4E-BPs compete
with elF4G for binding to elF4E thereby preventing its interaction with elF4G.
Phosphorylation of 4E-BP disrupts elF4E binding which enables elF4E to interact
with elF4G and promotes translation. Phosphorylation of the a subunit of elF2
converts elF2 into an inhibitor of its multimeric guanine-nucleotide exchange
factor elFZB and inhibits translation. The initiation factors investigated in the
studies presented here are depicted in grey. “P”: phosphorylation. Figure

adapted from [87].

24

Fit

elF4E Kinag
I elF4F complex

    
   

glFZB

p

   

I

elF2 Kinase

Met-RNA,“

 

 

4E-BP Kinase(s)

 

 

 

Figure 5.

25

In addition to being regulated by the 4E-BPs, elF4E is also directly
regulated by phosphorylation at Ser209. Increased elF4E phosphorylation is
correlated with increased protein synthesis in response to various cellular stimuli,
including mitogens, serum, and growth factors [94-96] while reduced
phosphorylation is correlated with decreased protein synthesis in during heat
shock [97, 98], serum deprivation [99], mitosis [100], and in influenza and
adenovirus infected cells [101, 102]. While changes in the phosphorylation state
of elF4E have been directly correlated with alterations in translation rates in a
variety of cell types [103], the physiologic relevance of elF4E phosphorylation is

not fully understood.

Cyproheptadine

The involvement of environmental factors, including chemical exposures,
in the etiology of human diabetes has been under investigation for many years.
A small group of structurally diverse chemicals, including alloxan, streptozotocin,
chlorozotocin, vacor, and cyproheptadine, are known to produce adverse effects
in the insulin-secreting B-cells of the endocrine pancreas [104]. Exposure to
these chemicals can lead to a temporary or permanent diabetic state
characterized by a loss of glycemic control and hypoinsulinemia.

CPH (PERIACTIN, Merck, Sharp and Dohme, West Point, PA) is a drug
that was originally synthesized in the mid-1900’s in the quest for a novel
tranquilizer [105]. Though the structure of cyproheptadine is similar to that of the

tricyclic antidepressants, it possesses neither tranquilizing nor antidepressant

26

activities. However, the drug was found to be both a potent antihistaminic and
antiserotonergic agent [106]. It is the unique nature of CPH to act as both an
antihistamine and an antiserotonergic compound that has led to its wide variety
of clinical uses.

CPH was initially utilized for the treatment of seasonal allergies and
derrnatologic and allergic pruritus [107, 108] and in the treatment of migraine and
vascular headache [108, 109]. Due to the advent of the second generation
antihistamines and sumatriptan, the drug is no longer a preferred treatment for
these conditions.

Soon after its introduction, CPH was serendipitously found to stimulate
appetite and weight gain in asthmatic children [110]. Further studies confirmed
this finding in non-asthmatic children [111], adults [112, 113], and cats [114].
Though the exact mechanism of appetite stimulation is not known, the drug
continues to be used in both human and veterinary medicine as an appetite
stimulant.

In addition to its continued use as an appetite stimulant, CPH has been
found to be useful in the treatment of side effects associated with both selective
serotonin reuptake inhibitors (SSRls) utilized for the treatment of depression
[115, 116] and typical neuroleptics utilized for the treatment of schizophrenia
[117]. It is also utilized as a treatment for nightmares commonly associated with
post-traumatic stress disorder and dream anxiety disorder [118].

CPH is a member of a group of heterocyclic, nitrogen-containing

compounds possessing the ability to reversibly disrupt insulin cell function in

27

treated animals [119]. In rats, CPH treatment produces a loss in the number of
insulin secretory granules within 15 hours after a single oral dose of 45 mglkg
[120]. With continued treatment, the reduction in insulin secretory granules is
followed by dilation of the intercisternal space and vesiculation of the ER. The
small vesicles of ER appear to eventually coalesce to form large cytoplasmic
vacuoles which are visible within 4 days of treatment. No abnormalities in other
cellular organelles (mitochondria, nucleus, Golgi apparatus) are observed during
CPH treatment. The ultrastructural changes in islets from CPH-treated animals
are dose-dependent and B-cell specific. Glucagon-secreting a—cells,
somatostatin-secreting 5-cells and pancreatic acinar cells remain normal in
appearance after 2 weeks of treatment [120].

A striking characteristic of CPH-induced B-cell toxicity is that it is
reversible. In CPH-treated rats, very few insulin secretory granules are visible in
B-cells after several days of treatment, but upon withdrawal of the drug, a marked
increase in the number is observed within 24 hours [4]. The large cytoplasmic
vacuoles are more slow to disappear but the cells will become well granulated
with secretory granules despite their presence.

In addition to these morphologic alterations, there is a concurrent
depletion of pancreatic immunoreactive insulin content followed by a
hyperglycemic state in CPH-treated animals [4]. Pancreatic insulin levels are
reduced to 25% of control values after two daily oral doses of 45 mglkg [4].
Withdrawal of CPH treatment results in recovery of pancreatic insulin levels and

insulin secretory granules within 2-4 days [4].

28

The ability of CPH to inhibit insulin secretion has been demonstrated in
perfused rat pancreatic segments and intact pancreata [4, 121, 122], isolated rat
and mouse islets [123-126], and in the clonal insulinoma cell lines RINm5F and
HIT-T15 [127]. The CPH-induced inhibition of insulin release is thought to be
due to the inhibition of depolarization-dependent uptake of extracellular calcium
into insulin-secreting cells [125]. Insulin secretagogues that are dependent upon
voltage-dependent entry of extracellular calcium (e.g. high [K+], glucose, and
tolbutamide) are much more sensitive to CPH-induced inhibition than those not
dependent on extracellular calcium entry (e.g. veratridine, theophylline, and the
calcium ionophore A23187) [125].

The insulin-depleting activity of CPH in vivo has been demonstrated in
both isolated pancreatic islets and in the clonal insulinoma cell lines, RINm5F
and HIT-T15 [81, 127]. The effects seen in these cultured cell systems are dose
dependent, reversible, and follow approximately the same time course as those
seen in CPH-treated animals. The use of in vitm systems, including both isolated
pancreatic islets and clonal cell lines, has been shown to be particularly useful for
studies on the biochemical mechanisms of CPH-induced B-cell toxicity.

Using isolated pancreatic islets from naive rats, CPH has been shown to
inhibit [3H]Ieucine incorporation into proinsulin and insulin in a concentration-
dependent manner [3]. In these studies, pulse-chase experiments in the
absence or presence of CPH showed that the incorporation of [3H]leucine into
proinsulin was inhibited during a 30 min pulse and that a commensurate

reduction in labeled insulin occurred during a 120 min chase period [3]. At a

29

concentration of 16 [TM a 70% inhibition of proinsulin synthesis was
demonstrated with no effect on the synthesis of total TCA-precipitable islet
proteins [3]. These results show that CPH has a selective action on the inhibition
of insulin precursor synthesis. A similar concentration-dependent CPH-induced
inhibition of [3H]leucine incorporation into guinea pig anti-insulin serum (GPAIS)-
immunoprecipitable material in the two insulinoma cell lines, RINm5F and HIT-
T15, has also been documented [127]. It is likely that CPH is also inhibiting
insulin precursor synthesis in these two insulinoma cell lines though direct
evidence has not been provided as the antibody utilized for immunoprecipitation
in these studies binds both proinsulin and insulin.

A critical aspect of the CPH-induced inhibition of proinsulin and insulin
synthesis seen in both isolated pancreatic islets and in RINm5F cells is that
inhibition of (pro)insulin synthesis occurs without a commensurate loss in cellular
PPImRNA levels [5, 6]. lnforrnation from these studies suggests that the ability
of CPH to inhibit the synthesis of insulin and to lower the cellular insulin insulin in
primary or clonal cells does not involve a loss of PPImRNA; instead, a post-
transcriptional effect(s) appears to be involved in CPH-induced toxicity in both
cell systems.

As mentioned above, CPH is a member of a group of structurally-related
compounds possessing the ability to disrupt B-cell function in the endocrine
pancreas. Certain metabolites of CPH have been shown to be more potent than
the unchanged drug in inhibiting insulin synthesis in isolated pancreatic islets

[128] while other structurally related compounds lack CPH-like toxicity,

30

apparently due to a lack of sufficient structural similarity to CPH [119]. The
chemical 4-diphenylmethylpiperidine (4-DPMP) has been shown to produce
morphologic and biochemical changes in rat B-cells after in vivo and in vitro
treatment [119] and in RINm5F cells [6] similar to those seen with CPH treatment
while the structurally related compound 2-diphenylmethylpiperidine (2-DPMP)
does not. The structures of CPH and these DPMP isomers are shown in Figure
6. Through extensive testing of numerous structurally-related compounds, the
structure of 4-DPMP has been determined to be the simplest chemical structure
needed for CPH-like activity in the endocrine pancreas [129]. In addition, the
antihistaminic and antiserotonergic activities of CPH are not required for the toxic
effects in pancreatic B-cells because several active CPH analogs lack these
activities [105].

There is continued uncertainty regarding the susceptibility of humans to
the diabetogenic actions of CPH. Administration of CPH to children has been
associated with abnormal glucose tolerance [130] and CPH has been
successfully utilized in conjunction with diazoxide for the treatment of
hyperinsulinemia [131]. In addition, CPH has been shown to inhibit proinsulin

synthesis in isolated human islets treated in vitro [132].

31

N
e“,
CPH
(active)
”c ‘C’l
N- H
N
l
H
4 - DPMP 2 - DPMP
(active) (Inactive)

Figure 6. Chemical structures of CPH, 4-DPMP and 2-DPMP. The relative
activities of the compounds for producing functional alterations in insulin

producing cells in rat pancreatic islets are given in parentheses [6].

32

RINm5F Cells as a Model for Investigations into CPH-Induced B-Cell
Toxicity

The islets of Langerhans are small clusters of endocrine cells embedded
throughout the pancreatic mass. The isolation of islets is a labor-intensive
procedure requiring the selective digestion of pancreatic exocrine tissue followed
by purification of the islets [133]. A maximum of several hundred islets can
obtained from a rodent pancreas [134] mandating the use of many animals in
order to obtain sufficient quantities of tissue for experimentation. Further
isolation of pure B-cells from intact islets then requires specialized techniques. In
addition, B-cells are terminally differentiated and do not propagate in culture,
making long-tenn maintenance in culture very difficult. These difficulties
associated with the use of primary [I-cells can be overcome through the use of
insulin-secreting cell lines.

The RINm5F cell line was established from a radiation-induced
transplantable rat insulinoma [135, 136]. These cells have been used
extensively in studies of insulin cell function [137-140]. While RINm5F cells
have been widely utilized in diabetes research, they have lost some of the
functional characteristics of primary B-cells. Most importantly, RINm5F cells do
not release insulin in response to glucose [137]. Though the biochemical basis
of this glucose-unresponsiveness is not fully understood, it has been suggested
that abnormalities in glucose transport [141-143] and/or metabolism [144-146]
may account for the failure of glucose to stimulate insulin release from these

cells. In addition, in contrast to primary B-cells, RINm5F cells synthesize and

33

release both insulin and glucagon [137, 147] though these cells are not
commonly utilized for investigations into glucagon cell function.

RINm5F cells respond to CPH treatment with diminished insulin
biosynthesis and loss of cellular insulin content [6, 127]. These effects were
found to be consistent with the concentration-dependence, chemical structure-
specificity, and reversibility previously published on the effects of CPH in isolated
rat pancreatic islets and in treated animals. These results indicate that RINm5F

cells are adequate models for the actions of CPH in pancreatic B-cells [129, 148].

Objectives and Rationale

There were two overall objectives of these studies: to investigate the
post-transcriptional mechanisms of CPH-induced inhibition of insulin synthesis in
vitro and to investigate the hormone specificity of these post—transcriptional
effects. Prior to these studies, CPH was known to inhibit insulin synthesis and
deplete cellular insulin content in isolated islets in vitro [3, 5] and in the clonal
insulinoma cell lines RINm5F and HIT-T15 [127] with no apparent effect on the
synthesis of other cellular proteins or effect on other islet cell types. In addition,
the inhibition of insulin synthesis in these in vitro systems occurred without a
commensurate loss of PPImRNA levels as measured via Northern Analysis [5,

6]. These data led us to the following hypotheses:

Hypothesis 1: The inhibition of insulin synthesis in response to CPH treatment
in vitro is due to an effect of the compound on PPImRNA translation; specifically,
CPH inhibits the translocation of PPImRNA from the cytosol to the ER.

Specific Aim: To examine the effect of CPH on the subcellular
localization of preproinsulin mRNA in RINm5F cells. The synthesis of
secretory proteins begins in the cytosolic compartment. After the initiation of
translation, the ribosome-associated nascent chain is translocated to the ER
where synthesis continues and the protein is shuttled through the secretory
pathway. Any interruption of this process would prevent the movement of the
nascent peptide through the secretory pathway thereby inhibiting the formation of
the final protein product. Evaluation of the effects of CPH on the distribution of
PPImRNA associated with various translationally-relevant ribosomal populations
would provide direct evidence of a post-transcriptional effect of the compound on
insulin synthesis. Alterations in PPImRNA subcellular localization would suggest
an early translational effect such as an interference with the initiation stage of
translation or the translocation of the nascent chain to the ER after the onset of
translation.

CPH is known to inhibit insulin synthesis and to deplete cellular insulin
content in vivo and in vitro. This inhibition of insulin synthesis, characterized by a
decrease in [3H]leucine incorporation into (pro)insulin, leading to cellular insulin
loss, is known to be concentration—dependent, chemical structure-specific,
reversible, and rapid. If the inhibition of insulin synthesis seen in response to

CPH treatment involves alterations in the subcellular distribution of PPImRNA,

35

this subcellular dislocation will show the same concentration—dependence,
chemical structure-specificity, reversibility, and time course as the inhibition of

insulin synthesis.

Hypothesis 2: The CPH-induwd inhibition of PPImRNA translation in vitro
involves alterations in translation initiation.

Specific Aim: To examine the effects of CPH on translation initiation
in RINm5F Cells. Results from initial experiments examining the effects of CPH
on the subcellular distribution of PPImRNA suggested that CPH may be acting to
inhibit the initiation stage of translation. The initiation stage of translation
involves the coordinated interaction of several initiation factor complexes and is
dependent upon the regulatory phosphorylation of numerous proteins with the
factors elF2a, elF4E, and 4E-BP1 being the best characterized regulators of
initiation. To further examine the potential CPH-induced initiation block,
experiments were conducted to investigate the effect of the compound on
polysome distribution and phosphorylation of the initiation factors elF2a, elF4E,

and 4E-BP1.

Hypothesis 3: The CPH-induced inhibition of PPImRNA translocation from the

cytosol to the ER in vitro is specific to the insulin message and insulin-secreting

cells.

36

Specific Aims:

1. To examine the effect of CPH on the subcellular localization of
preproglucagon mRNA, preproamylin mRNA, and B-actin mRNA in RINm5F
cells. CPH has been shown to inhibit insulin synthesis without an effect on the
synthesis of total TCA-precipitable protein. The specificity of the post-
transcriptional effect of CPH on PPImRNA subcellular localization was
investigated by examining the effects of the compound on the subcellular
distribution of two additional secretory proteins mRNAs, preproglucagon mRNA
(PPGmRNA) and preproamylin mRNA (PPAmRNA), and the distribution of a
non-secretory protein mRNA, B-actin mRNA.

2. To determine if the subcellular dislocation of non-preproinsulin
mRNAs is associated with a loss of cellular protein content in RINm5F
cells. CPH has been shown to produce a marked loss of cellular insulin content
both in vivo and in vitro. The loss of cellular insulin content seen in response to
CPH treatment is known to involve the inhibition of insulin synthesis.
Experiments were conducted to examine the effects of CPH on the cellular
content of the secretory protein, glucagon, and the non-secretory protein, B-actin,
to investigate a correlation between subcellular mRNA dislocation and cellular
protein content.

3. To examine the effects of CPH in a-cells. CPH is a known B-cell
toxicant, causing both morphologic and biochemical changes. These
morphologic and biochemical alterations have been restricted to the pancreatic

B-cell, with both the a- and 8-cells remaining unaffected in vivo and in vitro. To

37

further characterize the effects of CPH on glucagon-producing cells, experiments
were conducted to examine the effects of the drug in the clonal glucagonoma cell
line, orTCl.9. This cell line is a pancreatic a-cell line cloned from the aTC1 cell
line. The aTC1 cell line was derived from an adenoma created in transgenic
mice expressing the SV40 large T antigen oncogene under the control of the rat
preproglucagon promoter [149]. Though the parental aTC1 cell line produces
both glucagon and insulin (and PPImRNA), the aTC1.9 cell line is terminally
differentiated and produces glucagon but not insulin or PPImRNA and has been
found to be an acceptable tool for investigations into the synthesis of glucagon
[150]. Experiments were performed in these cells to investigate the effects of
CPH on cell viability, cellular glucagon content, and subcellular PPGmRNA
localization. Experiments were also conducted to examine the effect of CPH on

cellular glucagon content in isolated rat pancreatic islets treated in vitro.

The continuous and varied clinical use of CPH warrants continued
investigation into the mechanisms of its toxicity. The B-cell toxicity of CPH is very
different from that of other diabetogenic agents in that it produces a reversible,
dose-dependent, and predictable response rather than permanent B-cell
destruction. Elucidation of the mechanisms by which CPH produces B-cell
toxicity may allow this agent to be a useful tool for investigations into the
synthesis, storage, and secretion of insulin under both normal and pathological

conditions.

38

MATERIALS AND METHODS

Materials
Animals

Male Sprague-Dawley rats weighing approximately 250 g were purchased
from Charles River Breeding Company (Portage, MI). Rats were housed two or
three animals per cage in the University Laboratory Animal Resources facility in
the basement of the Life Sciences Building, Michigan State University. Upon
arrival animals were allowed to acclimate for a minimum of 7 days prior to
experimentation. Animals received Purina Rodent Chow and clean tap water ad

Iibitum.

Clonal Cell Lines
RINm5F cells were the generous gift of Dr. Paolo Meda (Geneva,
Switzerland) while aTC1.9 cells were purchased from American Type Culture

Collection (ATCC; Manassas, VA).

Chemicals and Reagents

CPH (hydrochloride monohydrate) was obtained from the Merck Institute
for Therapeutic Research (West Point, PA). 4-diphenylmethylpiperidine (4-
DPMP) was obtained from Pfaltz and Bauer (Flushing, NY). 2-
diphenylmethylpiperidine (2-DPMP) was synthesized by Dr. H. Aboul-Enein
[119]. The purity of these compounds was verified by TLC as described by

Hintze et al. [151]. Roswell Park Memorial Institute (RPMI) 1640 culture medium

39

(180 mgldL D-glucose), F12K Nutrient Medium (Kaighn’s Modification), Cell
Dissociation Buffer (enzyme-free, Hank’s based), fetal bovine serum,
penicillin/streptomycin, trypsin/EDTA, ROX Reference Dye, and Platinum PCR
SuperMix were obtained from lnvitrogen (GIBCO; Carlsbad, CA). Hyperfilm was
obtained from Kodak Co. (Rochester, NY). Taqman EZ-RT PCR Core Reagents
were obtained from Applied Biosystems (Foster City, CA). Bisbenzimide
trihydrochloride (Hoescht No. 33258), TRl-Reagent, TRl-Reagent LS, Ficoll,
diethylpyrocarbonate (DEPC), actinomycin D, cycloheximide, calf thymus DNA,
antipain, aprotinin, Ieupeptin, bovine serum albumin, and chick egg ovalbumin
were obtained from Sigma Chemical Company (St. Louis, MO). Detergent
Compatible (DC) Protein Assay was purchased from Bio-Rad Laboratories
(Hercules, CA). 1-bromo-3-chloropropane (BCP) was purchased from Molecular
Research Center Inc. (Cincinnati, OH). lmProm-ll Reverse Transcription
System, RNasin Ribonuclease Inhibitor and DNase I were purchased from
Promega (Madison, WI). lmmobilon-P PVDF membranes were purchased from
Millipore, Inc. (Bedford, MA). Ribogreen RNA Quantitation Kit was purchased
from Molecular Probes (Eugene, OR). All other reagents were of the highest

quality commercially available.
Radioisotopes

Glucagon radioimmunoassay kits [125l] were purchased from Linco

Research Inc. (St. Charles, MO).

40

Methods
Islet preparation

Islets were isolated on Ficoll gradients from collagenase—digested
pancreata of male Sprague-Dawley rats weighing approximately 250 g in a
method modified from Lacy and Kostianovsky [133]. Islets were cultured
overnight free-floating in RPMI 1640 supplemented with 10% fetal bovine serum

[152] before experimentation.

Cell Culture

RINm5F cell were cultured as described by Gazdar et al. [136]. Cells
were grown at 37°C in an atmosphere of 5% C02 in RPMI 1640 medium
supplemented with 10% fetal bovine serum, 100 ug/mL penicillin and 100 mUlmL
streptomycin. These culture conditions were utilized for all experiments. Stock
cultures were passaged weekly and received fresh media every 2 days. The
cells used for these studies were between passages 40 and 60. Cells did not
reach confluence during the experimental period.

0LTC1.9 cells were grown at 37°C in an atmosphere of 5% 002 in F 12K
medium supplemented with 10% fetal bovine serum. These culture conditions
were utilized for all experiments. Stock cultures were passaged weekly and
received fresh media every 2 days. The cells used for these studies were
between passages 37 and 50. Cells did not reach confluence during the

experimental period.

41

Cellular Homogenization

Throughout all homogenizations, precautions were taken to prevent
ribonuclease contamination and the temperature was maintained at 4°C.

RINm5F cells were plated at 2 x 105 cells/dish in 100 mm culture dishes
and experimentation began on Day 5 after passage. After appropriate treatment
incubation as described in the Figure legends, the cells were washed 2X with
Ca“lMg” free buffer and harvested via gentle scraping. The cell harvests from
two 100 mm dishes were combined and homogenized in 4 mL standard
homogenization buffer (250 mM sucrose, 250 mM KCI, 10 mM MgCl2, 10 mM
Tris (pH 7.5), 2 mM dithiothreitol, 12 units of RNasin/mL, 5 uglmL leupeptin, 2
pg/mL antipain, and 2 uglmL aprotinin) with 10 strokes in a dounce homogenizer
followed by 4 passes through a 25 gauge needle attached to a 5 mL syringe.

(XTC1.9 cells were plated at 2 x 106 cells/dish in 100 mm culture dishes
and experimentation began on Day 5 after passage. After appropriate treatment
incubation as described in the figure legends, the cells were washed 2X with
Ca++lMg++ free buffer and harvested via gentle scraping. The cell harvest was
then homogenized in 4 mL standard homogenization buffer with 5 strokes in a
dounce homogenizer followed by 4 passes through a 25 gauge needle attached

to a 5 mL syringe.
Subcellular Fractionation

The subcellular fractionation procedure of Welsh et al. [153] was utilized to

separate various populations of ribosome-associated mRNA. Throughout

42

fractionation, precautions were taken to prevent ribonuclease contamination and
the temperature was maintained at 4°C. Cells were homogenized as described
above.

RINm5F Cells:

After an initial 1500 g centrifugation (5 minutes) (2700rpm in a Beckman
TJ—6 centrifuge) to pellet nuclei and cell debris, the supernatant (post-nuclear
supernatant; PNS) was centrifuged for 1 hour at 100,000 g (33,000rpm in a
Sorvall Discovery 90 centrifuge with a Beckman SW50.1 rotor). The pellet was
then resuspended in standard homogenization buffer and recentrifuged for 2
minutes at 750 g (2800rpm in a Sorvall Discovery 90 centrifuge with a Beckman
SW50.1 rotor) and then for 12 minutes at 130,000 g (37,000rpm in a Sorvall
Discovery 90 centrifuge with a Beckman SW50.1 rotor). The pellet, containing
membrane-bound (ER-bound) polyribosomes (polysomes), was resuspended in
standard homogenization buffer. The supernatant contains free polysomes and
some monoribosomes. The 100,000 g supernatant was then further centrifuged
for 2 hours at 255,000 g (48,000rpm for 161 min in a Sorvall Discovery 90
centrifuge with a Beckman SW50.1 rotor) to pellet most of the 808
monoribosomes.

aTC1.9 Cells:

After an initial 1500 g centrifugation (5 minutes) (2700rpm in a Beckman
TJ-6 centrifuge) to pellet nuclei and cell debris, the PNS was centrifuged for 1
hour at 100,000 g (38,200rpm in a Sorvall Discovery 90 centrifuge with a Sorvall

Ti1270 rotor). The pellet was then resuspended in standard homogenization

43

buffer and recentrifuged for 2 minutes at 750 g (1900rpm in a Beckman TJ-6
centrifuge) and then for 12 minutes at 130,000 g (43,500rpm in a Sorvall
Discovery 90 centrifuge with a Sorvall Ti1270 rotor). The pellet, containing ER-
bound polysomes, was resuspended in standard homogenization buffer. The
supernatant contains free polysomes and some monoribosomes. The 100,000 g
supernatant was then further centrifuged for 2 hours at 255,000 g (61 ,000mm in
a Sorvall Discovery 90 centrifuge with a Sorvall Ti1270 rotor) to pellet most of the
80S monoribosomes.

The subcellular fractionation schematic is shown in Figure 7.

RNA Extraction

Total RNA was extracted from whole cells using TRI Reagent and from all
subcellular fractions using TRI Reagent LS following manufacturer’s instructions.
For all RNA extractions, BCP was utilized as the phase separatant. RNA
samples were resuspended in 0.1% DEPC treated water and stored at —80°C
until analysis. Immediately prior to real-time RT-PCR analysis, RNA samples
were treated with DNase I (according to manufacturer’s instructions) to eliminate

any potential DNA contamination.

44

HOMOGENATE

 

 

 

 

 

 

 

 

 

 

 

 

 

1500 g 5 min
PELLET SUPERNATANT (PNS)
nuclei; cell debris; etc.
100,000 g 1 n
PELLET RESUSPENDED SUPERNATANT
750 g 2min
J 130,000 9 12min 255'000 ‘7 2 h
I
I
PELLET SUPERNATANT
ER-BOUND UNINITIATED
POLYSOMES mRNA
SUPERNATANT PELLET
FREE MONORIBOSOMES

POLYSOMES

Figure 7. Subcellular fractionation scheme. Adopted from [153].

45

Reverse Transcription

Total RNA extracted from all aTC1.9 cell fractions was quantitated using
Ribogreen Quantiation Kit according to manufacturer’s instructions. The RNA
samples were then subjected to reverse transcription using the lmProm-ll
Reverse Transcription Kit according to manufacturer’s instructions. Briefly, 4 [IL
of RNA sample (< 1 pg total RNA) was combined with 0.5 pg of Random Primers
and heated at 70°C for 5 minutes. After heating, the samples were immediately
chilled on ice for 5 minutes and spun for 10 seconds in a microcentrifuge. To
each sample, 4 [TL of lmProm-ll 5X Reaction Buffer, 1 [IL lmProm-ll Reverse
Transcriptase, 4.5 pL nuclease-free water, 20 U RNasin, 1 DL 10 mM dNTP (0.5
mM final concentration), and 4 [1L 25mM MgClz (5 mM final concentration) were
added. The samples were then transferred to a 96 well plate and reverse
transcribed to cDNA in a thermal cycler as follows: 25°C for 5 minutes, 42°C for
60 minutes, and 70°C for 15 minutes. After reverse transcription, cDNA samples
were stored on ice and immediately subjected to real-time PCR analysis. Total
RNA from na‘ive aTC1.9 cells was reverse transcribed with each sample set for

generation of an external cDNA standard curve for all real-time PCR assays.

Real-Time Quantitative PCR
Real-time PCR is a procedure for the detection of specific PCR products
based on the use of a fluorogenic probe which hybridizes within the PCR target

sequence and generates a fluorescent signal that accumulates during PCR

46

cycling in a manner that is proportional to the concentration of the amplified
product.

The 5' nuclease assay for detecting PCR amplification products in real-
time PCR employs a non-extendable oligonucleotide hybridization probe
containing a fluorescent reporter dye covalently linked to the 5' end and a
quencher dye, TAMRA (6-carboxy-tetramehtyl-rhodamine), covalently linked to
the 3' end. Because of the close proximity of the reporter dye and quencher dye,
the fluorescence of the reporter dye is suppressed, mainly through a F6rster-type
energy transfer [154]. During PCR cycling, the probe first hybridizes to the target
sequence, and is then cleaved via the 5'-3' exonuclease activity of the Taq DNA
polymerase. The cleavage of the probe results in an increase in fluorescence of
the reporter dye. This sequence of events occurs during each PCR cycle. The
exonuclease activity of the polymerase is only active if the fluorogenic probe is
annealed to the target; the enzyme cannot hydrolyze the probe when it is free in
solution. Therefore, the increase in fluorescence is proportional to the amount of
specific PCR product.

To ensure design success of the dual labeled hybridization probe and
primers, PrimerExpress (PE Applied Biosystems, Foster City, CA), was utilized to
design sequence specific oligonucleotides for use in these real-time PCR assays.
The oligonucleotides (hybridization probes and both fonrvard and reverse
primers) were synthesized and purified by HPLC by PE Applied Biosystems
(Foster City, CA). The specific oligonucleotides utilized in these experiments are

listed in Table 1.

47

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For all reactions, the fluorescent signal due to the DNA polymerase
cleavage of the hybridization probe and the release of the reporter dye was
monitored in real-time by the GeneAmp 5700 Sequence Detector (PE Applied
Biosystems, Foster City, CA). This instrument combines a conventional 96-well
therrnocycler, a laser for the excitation of the fluorescent dyes and a detection
and software system for the automatic collection and calculation of the
fluorescence released during PCR cycling. The fluorescence was detected by a
CCD camera detector. For each well, the camera collected the emission data
between 520 nm and 660 nm once every few seconds and an analysis algorithm
calculated the emissions of both the reporter (R) and the quencher dyes. The
ARn (change in reporter dye fluorescence) values represent the quantity of
fluorescent probe hydrolyzed and fit an exponential function generating an
amplification plot for each well while the algorithm simultaneously determines an
experimental threshold on the basis of the mean baseline signal in the first 15
cycles plus 10 standard deviations. The cycle number at which the fluorescent
signal of the well reaches this threshold (CT) is inversely proportional to the
number of target copies present in the initial sample. A CT value higher than the
total number of PCR cycles would indicate that the amount of mRNA template in
the well was insufficient to induce an increase in fluorescence reaching this
threshold and therefore quantitation of the sample cannot be performed. In the
experiments reported here, the CT of each sample was used to quantitate the
samples, using an external standard curve generated from total RNA extracted

from naive RINm5F or aTC1.9 cells [156] that was amplified in the same PCR

49

run. All standard and sample CT values for all experiments reported here were
between 16 and 35.

All aTC1.9 cell real-time PCR reactions were performed in a total volume
of 34.5 uL, containing 7 uL of standard or sample cDNA, 22.5 uL of Platinum
PCR SuperMix (final concentrations: 0.5 U Taq Platinum DNA polymerase, 20
mM Tris-HCI (ph 8.4), 50 mM KCI, 200 uM dNTP), 5 mM MQCIz, 0.7 pL of 50X
ROX Reference Dye (passive reference dye), and 100 nM of each primer and the
fluorescent probe. Each of these amplifications was performed in duplicate
wells, using the following conditions: 2 minutes at 50°C and 10 minutes at 94°C,

followed by a total of 45 cycles of 30 seconds at 95°C and 1 minute at 60°C.

Real-Time Quantitative RT-PCR

Real-time RT-PCR is an application of real-time PCR for the detection and
analysis of RNA. For these experiments, the TaqMan EZ RT-PCR assay system
(PE Applied Biosystems, Foster City, CA) was utilized for the measurement of
RINm5F cell mRNAs. TaqMan EZ RT-PCR utilizes the thh DNA polymerase,
which functions both as a thennoreactive reverse transcriptase and as a
therrnostable DNA polymerase. By using this assay system, samples were
reverse transcribed, amplified, and quantitated in a single well.

All TaqMan EZ RT-PCR reactions were performed in a total volume of 25
[IL containing 10|JL of standard or sample RNA (< 50 ng total RNA), 3 mM
Mn(OAc)2, 300 pM each deoxyATP, deoxyCTP, and deoxyGTP, 600 [M
deonyTP, 0.25 U AmpErase UNG, 0.5 U thh DNA polymerase, 200 nM of the

50

fluorogenic probe, 60 nM of the passive reference dye (ROX-AGTTGG), and 100
nM of each primer. Each of these amplifications was performed in duplicate
wells, using the following conditions: 2 minutes at 50°C, 30 minutes at 60°C, and
5 minutes at 95°C followed by a total of 40 cycles of 15 seconds at 94°C and 1

minute at 60°C.

Verification of Subcellular Fractionation Procedures

In order to verify the separation of the ER during the fractionation
procedure, the subcellular fractions were subjected to anti-calnexin
immunoblotting. Calnexin, an ER-specific protein [157], is only found in fractions
containing the ER. Protein from the subcellular fractions (including PNS) was
precipitated using the methanol/chloroform method as described by Wessel and
Flugge [158]. Protein samples were then mixed with SDS-sample buffer and
boiled for 5 minutes. Samples were then separated by SDS-PAGE on a 7.5%
gel. The proteins were transferred to Immobilon-P membranes (Millipore,
Bedford, MA), blocked for 4 hours with 5% nonfat milk in Tris-buffered saline
containing 0.1% Tween (TBST) containing 0.025% sodium azide at 4°C, and
incubated overnight at 4°C with primary antibody (anti-calnexin [Calbiochem, San
Diego, CA] 1212,500 dilution) in 5% nonfat milk in TBST containing 0.025%
sodium azide. The membranes were washed and then incubated for 1 hour at
4°C with secondary antibody (horseradish peroxidase-linked goat-anti rabbit lgG

[Jackson ImmunoResearch, West Grove, PA] 1:200,000 dilution) in TBST. Blot

51

detection was carried out using SuperSignal West Femto Maximum Sensitivity

Substrate (Pierce, Rockford, IL).

Determination of PPImRNA Stability

RINm5F cells were plated at 2 x 105 in 100 mm culture dishes.
Experimentation began on Day 5 after passage. Actinomycin D (5 pglmL), used
to prevent RNA synthesis, was added to the medium of RINm5F cells 30 minutes
prior to the addition of 0 or 10 [M CPH. The cells were then incubated for 0, 2,
6, or 12 hours. At the end of the incubation period, cells were washed 2X with
Ca““/Mg”+ free buffer and then lysed in TRI Reagent. Total RNA was then

extracted and quantitated as described above.

Polysome Profile Analysis

Efficient translation of insulin requires the assembly of polysomes.
Nascent preproinsulin is synthesized on polysomes comprised of 5-7 ribosomes
per PPImRNA [159]. Polysome profile analysis is a technique utilized to
measure polysome assembly or disaggregation. Increases in the
monoribosome/polysome ratio as detected via polysome profile analysis are
characteristic of the inhibition of translation initiation [160]. This technique is
extensively utilized for investigations into translational regulation in general [82]
and has been previously used in studies of insulin translation in particular [25,

159].

52

For these experiments, RINm5F cells were plated at 1 x 106 cells per dish
in 100 mm culture dishes and utilized on Day 5 after passage. Sucrose gradients
and cell extracts were prepared and analyzed using the method of Ruan et al.
[161]. Briefly, cells were incubated at 37°C for 105 minutes with either sterile
water (control) or 10 0M CPH. After incubation, the media was replaced with
fresh media containing 100 pglmL cycloheximide (CX) and appropriate treatment
(either sterile water or 10 [TM CPH) and incubation was continued for an
additional 15 minutes for a total treatment time of 2 hours. The media was then
removed and the cells were washed 2X with PBS (140 mM NaCl, 5 mM KCI, 8
mM NazHPO4, 1.5 mM KH2PO4, pH 7.2) containing 100 pglmL CX. The cells
were then removed via gentle trypsinization and harvested in PBS containing 100
uglmL CX. The cells were transferred to a conical test tube containing crushed
frozen PBS containing 100 ug/mL CX. The cells were then spun at 1000 g
(2200rpm in a Beckman TJ-6 centrifuge). The pellet was washed with PBS
containing 100 pg/mL CX. The supernatant was then removed and the tube
drained and placed on ice. The pellet was resuspended in 495 pL low salt buffer
(LSB; 20 mM Tris, 10 mM NaCl, 3 mM MgCl2, pH 7.4) and incubated on ice for 3
minutes. After incubation, 165 uL of 1.2% Triton N-101 (in LSB) was added to
the cells. The cell suspension was transferred to a 7 mL dounce homogenizer
and homogenized via 8 strokes. The homogenate was then transferred to a 1.5
mL Eppendorf tube (on ice) and spun at 10,000 g (11,000rpm in an Eppendorf
5415C centrifuge) for 1 minute. The supernatant (cell lysate) was transferred to

a fresh 1.5 mL Eppendorf tube containing 67 pL of 10 mg/mL heparin (in LSB

53

containing 1.5 M NaCl). This entire cell lysate was then layered onto a single
3.92 mL 0.5-1.5 M (continuous) sucrose gradient and spun at 41,800rpm (Sorvall
Discovery 90 centrifuge with a Beckman SW50.1 rotor) for 110 minutes.
Gradients were then unloaded using an Auto Densi—Flow (Labconco, Kansas
City, MO) and the A254 absorbance measured using an lsco UA-6 Absorbance

Detector (lsco, lnc., Lincoln, NE).

Determination of Initiation Factor Phosphorylation

For the determination of elFZa phophorylation, RINm5F cells were plated
at 5 x 105 cells per dish in 60 mm culture dishes and experimentation began one
day after passage. After appropriate incubation as noted in Figure legends,
treated and control cells were washed 2 X with Ca“lMg” free PBS, lysed in 300
uL sample buffer (62.5 mM Tris-HCI, pH 6.8, 50 mM DTT, 2% SDS, 10%
glycerol, and 0.01% bromophenol blue), heated at 100°C for 5 minutes and
centrifuged at 10,000 g (11,000rpm in an Eppendorf 54150 centrifuge) for 15
minutes. Following centrifugation, 15 DL of supernatant was separated via SDS-
PAGE on 10% gels followed by transfer to lmmobilon-P membranes.
Membranes were blocked with 5% nonfat milk in TBST at room temperature for
one hour followed by overnight incubation at 4°C with a 1:1000 dilution of rabbit
polyclonal antibody specific for elFZa phosphorylated at Ser51 or rabbit
polyclonal antibody specific for the nonphosphorylated form of the protein,

respectively (Cell Signaling, Beverly, MA) in 5% bovine serum albumin in TBST.

54

For determination of elF4E and 4E-BP1 phosphorylation, RINm5F cells
were plated at 1 x 106 cells per dish in 60 mm culture dishes and experimentation
began two days after passage. After appropriate incubation as noted in figure
legends, treated and control cells were processed as described above.
Supernatant samples were separated via SDS-PAGE on 15% gels followed by
transfer to Immobilon-P membranes. Membranes were blocked with 5% nonfat
milk in TBST at room temperature for one hour followed by overnight incubation
at 4°C with a 1:1000 dilution of rabbit polyclonal antibody specific for elF4E
phosphorylated at Ser209. 4E-BP1 phosphorylated at Ser65, or rabbit polyclonal
antibodies specific for the nonphosphorylated forms of the proteins, respectively
(Cell Signaling, Beverly, MA), in 5% bovine serum albumin in TBST. After
incubation with primary antibody, all membranes were washed and incubated for
one hour at room temperature with a 1:2000 dilution of HRP-linked goat anti-
rabbit lgG (Cell Signaling, Beverly, MA) in 5% nonfat milk in TBST.

lmmunoreactive bands were detected by enhanced chemiluminescence
(ECL, Amersham, Piscataway, NJ). After initial detection of phosphorylated or
nonphosphorylated initiation factors, the membranes were washed and incubated
overnight 4°C with a 1: 10,000 dilution of mouse monoclonal antibody specific for
B-actin in 4% chick ovalbumin in TBST (Novus Biologicals, Littleton, CO).
Membranes were then washed and incubated for one hour at 4°C with a
1:25.000 dilution of HRP-linked goat anti-mouse lgG (Jackson ImmunoResearch
Laboratories, West Grove, PA) in TBST. B-actin immunoreactive bands were

detected as described above.

55

To quantify the change in initiation factor phosphorylation, all blots were
subjected to densitometric scanning using NIH Image (NIH, Bethesda, MD).
Initiation factor protein levels (densitometric values) were normalized to B-actin

protein levels (densitometric values).

Determination of B-actin Protein Content

For these experiments, RINm5F were plated at 2 x 10‘ cells per dish in 35
mm culture dishes and experimentation began on Day 3 after passage. After
appropriate incubation as noted in Figure legends, cells were washed 2 X with 2
mL Ca”/Mg” free PBS. Cells were then washed 1X in L-RIPA (50 mM Tris-HCI,
pH 7.5, 150 mM NaCl, 2 mM EGTA, 0.1% Triton X-100, 100 uglmL PMSF, 100
ug/mL aprotinin, and 100 pg/mL leupeptin) then lysed in 200 uL L-RIPA. The
dish was then washed with 100 uL L-RIPA and the wash was combined with the
cell lysate. The lysate was passed 5X through a 25 gauge needle, incubated on
ice for 30 minutes, and then spun at 13,000 g (14,000rpm in an Eppendorf
5415C centrifuge) for 25 minutes. The supernatant was collected and stored at -
20°C until analysis. Supernatant samples were analyzed for protein content
using the Bio-Rad DC Protein Assay according to manufacturer's instructions.
Samples containing 8 ug protein were separated via SDS-PAGE on 7.5% gels
followed by transfer to lmmobilon-P membranes. Membranes were blocked for 4
hours with 4% chick ovalbumin in TBST containing 0.025% sodium azide at 4°C
followed by overnight incubation at 4°C with a 1:10,000 dilution of mouse

monoclonal antibody specific for B-actin in 4% chick ovalbumin in TBST (Novus

56

Biologicals, Littleton, CO) containing 0.025% sodium azide. Membranes were
then washed and incubated for one hour at 4°C with a 1:25,000 dilution of HRP-
linked goat anti-mouse lgG (Jackson ImmunoResearch Laboratories, West
Grove, PA) in TBST. lmmunoreactive bands were detected by enhanced

chemiluminescence (ECL, Amersham, Piscataway, NJ).

Determination of Cellular Hormone Content

For these experiments, RINm5F were plated at 2 X 104 cells per dish in 35
mm culture dishes (experimentation began on Day 3 after passage) while
aTC1.9 cells were plated at 1 X 105 cells per dish in 35 mm culture dishes
(experimentation began on Day 4 after passage). After appropriate incubation as
noted in Figure legends, cells were washed 2X with 2 mL versene (137 mM
NaCl, 2.7 mM KCI, 8.1 mM NazHPO4, 1.5 mM KHzPO4, 0.45 mM EDTA, 1%
phenol red) and harvested via gentle trypsinization. Cells were pelleted
(1200rpm in a Beckman TJ—6 centrifuge for 5 minutes) and resuspended in 1 mL
versene. Cells in 450 uL of this cell suspension were then pelleted again and
resuspended in either 450 pL of 1 N acetic acid or 450 DL of DNA assay buffer (2
M NaCl, 50 mM NazHPO4, and 2 mM EDTA) [162]. The cells that were
resuspended in 1N acetic acid were heated for 5 minutes at 95°C, sonicated (2X,
10 seconds) with a VirSonic 300 sonicator (Virtis Co., Inc, Gardiner, NY) at
setting 3 (microprobe), and stored at 4°C overnight to fully extract glucagon.
After the overnight extraction, the samples were then centrifuged at 1200rpm in a

Beckman TJ-6 centrifuge for 10 minutes at 4°C and the supernatant was stored

57

at -20°C until analyzed by radioimmunoassay. Cellular glucagon content was
quantitated via RIA using commercially available kits following manufacturer’s
guidelines. The cells that were resuspended in DNA assay buffer were sonicated
(2X, 10 seconds) with a VirSonic 300 sonicator (Virtis Co., Inc, Gardiner, NY) at
setting 3 (microprobe), stored at 4°C overnight, and centrifuged at 1200rpm in a
Beckman TJ-6 centrifuge for 10 minutes at 4°C. The supernatant was stored at -
20°C until assayed for DNA following the method of Labarca and Paigen [162].
For islet experiments, groups of 30 islets were incubated as noted in
Figure legends. After appropriate incubation, the islets were washed 2X with 1
mL Cah‘lMg++ free PBS. Islets were then resuspended in 1 mL Milli-Q water and
sonicated (2X, 10 seconds) with a VirSonic 300 sonicator (Virtis Co., Inc,
Gardiner, NY) at setting 3 (microprobe). 450 DL of this islet cell suspension was
added to both 450 uL of 2 N acetic acid and 450 uL of DNA assay buffer. Both
sets of samples were then stored at 4°C overnight. After the overnight
extraction, the samples were then centrifuged at 1200rpm in a Beckman TJ-6
centrifuge for 10 minutes at 4°C and the supematants were stored at -20°C until
analyzed by RIA or DNA assay [162]. Cellular glucagon content was quantitated
via radioimmunoassay using commercially available kits following manufacturer’s

guidelines.
Statistical Analysis

Results from all experiments are expressed as the mean 1 SEM. The n

refers to the number of individual treatments for all experiments. All experiments

58

were repeated with a minimum of two different passage numbers for all cell
experiments and two different islet preparations for primary cell experiments.
Statistical analyses were conducted by Student’s t-test. All percentile data was
transformed via arcsin square root transformation prior to statistical analysis.
Differences are considered to be significant only if the probability of error is less
than 5%.

Presentation of mRNA subcellular distribution data:

For all PCR reactions, an external standard curve derived from serial
dilutions of total RNA extracted from nai‘ve cells [156] was utilized for the
quantitation of all sample mRNA. A separate standard curve was run with each
PCR assay and all subcellular fraction samples from a single experiment were
analyzed in the same PCR run. To account for differences in the total amount of
RNA extracted between fractionation experiments, the data are presented as a
percentage of the sum total of mRNA recovered from all four subcellular fractions
per PCR run. This is a common means of reporting data on the distribution of
mRNA obtained from subcellular fractions [153, 163-165]. While the amount of
mRNA recovered from the subcellular fractions was variable between
fractionation experiments, the percent recovery of mRNA between experiments

was consistent.

59

RESULTS

Effects of CPH on PPImRNA in RINm5F Cells

Protein synthesis begins with the transcription of DNA to produce the
protein message. Once the message has been transcribed and processed, it is
exported from the nucleus into the cytosol where it is either stored, degraded or
translated. Results from previous studies have suggested that CPH inhibits
insulin synthesis and depletes cellular insulin content via one or more post-
transcriptional mechanisms. It was the aim of these studies to investigate the
effects of CPH on selected post-transcriptional events in order to elucidate
potential mechanisms by which the drug produces B-cell toxicity in the endocrine

pancreas.

Effect of CPH on PPImRNA Stability:

CPH is known to inhibit the incorporation of {3H]Ieucine into (pro)insulin in
RINm5F cells within 2 hours. This rapid inhibition of insulin synthesis has been
shown to occur without an alteration in PPImRNA levels, as measured via
Northern analysis, suggesting that a post-transcriptional effect is involved in the
CPH-induced inhibition of insulin synthesis. One possible post-transcriptional
effect is the destabilization of the insulin message. By decreasing the stability of
the message, there would be less message available for translation into protein
leading to a decrease in protein synthesis. In these experiments, actinomycin D,
a known inhibitor of RNA synthesis, was utilized. Cells were treated with

actinomycin D for a period of 30 minutes prior to beginning CPH treatment. By

60

inhibiting the synthesis of new PPImRNA, the decline of the message over time
can be monitored and any alteration in the rate of decay can be detected. The
effect of CPH on PPImRNA stability in RINm5F cells is shown in Figure 8. In
using an actinomycin D decay curve, a 10 uM CPH treatment over a 12 hour
period did not alter the stability of PPImRNA when compared to control values.
These results are in agreement with published reports indicating that acute CPH
treatment in vitro does not alter PPImRNA levels [6, 127] and provide further

support of an effect of the drug on translation.

Effects of CPH on PPImRNA Subcellular Localization:

It was the overall aim of these studies to examine the translocation of the
insulin message from the cytosol, where insulin synthesis begins, to the ER. As
the translocation of PPImRNA to the ER was of particular interest in these
experiments, it was necessary to utilize a subcellular fractionation procedure to
efficiently separate the ER from other subcellular compartments. This was
accomplished by modifying the procedure of Welsh et al. [153] for the separation
of RINm5F cell mRNA into four fractions: a free polysome fraction, an ER-bound
polysome fraction, a monoribosome fraction, and a fraction containing cytosolic,
uninitiated mRNA.

In order to determine the ER separation efficiency of the centrifugation
procedure used by Welsh et al. to produce subcellular fractions from isolated rat
pancreatic islets [153] in RINm5F cells, calnexin, an ER-specific protein [157],

was utilized to examine the subcellular fractions generated during the

61

fractionation procedure for the presence of the ER. As shown in Figure 9, by
using anti-calnexin Western Blotting, calnexin was found only in the fraction
reported to contain ER—bound polysomes, verifying the separation of the ER
during the fractionation procedure and demonstrating this to be a useful
technique for the determination of PPImRNA localized at the ER in this cell line.

The fractionation procedure was then utilized to examine the effects of
CPH treatment on the subcellular distribution of PPImRNA. As shown in Figure
10, the total amount of PPImRNA recovered from all subcellular fractions during
a 2 hour treatment of RINm5F cells with 10 pM CPH was 110.7 +I- 15.4% of
control values, indicating no treatment related alteration in PPImRNA levels.

The ability of CPH to alter the subcellular localization of PPImRNA is
shown in Figures 11 and 12. A 2 hour incubation with increasing concentrations
of CPH resulted in significant alterations in the percentages of recovered
PPImRNA associated with various ribosomal populations. These alterations in
PPImRNA subcellular localization were concentration dependent with the 10 pM
treatment group showing a 33% decrease in the percentage of PPImRNA found
at the ER and 56% and 52% increases in the percentages of uninitiated
PPImRNA and monoribosome-associated PPImRNA, respectively (Figure 11).
There was no effect on the subcellular distribution of PPImRNA at a
concentration of 1 pM CPH while both 5 [M and 10 uM CPH concentrations
induced subcellular PPImRNA dislocation. (Figure 11). This concentration
dependence is consistent with the concentration dependence of CPH-induced

inhibition of insulin synthesis in both isolated pancreatic islets and RINm5F cells

62

[6, 128]. The time course of CPH-induced PPImRNA subcellular dislocation is
shown in Figure 12. A 30 minute incubation with 10 pM CPH was sufficient to
induce these alterations in PPImRNA localization whereas no treatment-related
change occurred at 15 minutes.

Experiments were then conducted to determine whether the alterations in
PPImRNA subcellular distribution induced by CPH treatment were reversible. As
shown in Figure 13, a 24 hour recovery period following a 2 hour 10 [M CPH

treatment was sufficient to return the PPImRNA distribution to control values.

Effects of 2-DPMP and 4-DPMP on PPImRNA Subcellular Localization:
Results of experiments conducted to examine the chemical structure-
specificity of CPH-induced PPImRNA subcellular dislocation are shown in Figure
14. A 2 hour incubation with 10 [M 4-DPMP induced PPImRNA subcellular
dislocation nearly identical to that seen with CPH treatment while a 2 hour

incubation with 10 pM 2-DPMP showed no effect.

63

-e— CONTROL
120 - -o- 10 pM CPH

100 ‘
80 ‘
60 l

 

40-
20‘

PERCENT OF CONTROL

 

 

0 2 4 6 8 10 12

TIME (HOURS)
Figure 8. Lack of an effect of CPH on PPImRNA stability. RINm5F cells were
preincubated with actinomycin D (5 pg/mL) for 30 min prior to the addition of 10
pM CPH or 1% sterile water (control); incubation was then continued for 0, 2, 6,
or 12 h. PPImRNA was extracted and quantitated as described in Materials and
Methods. Data are expressed as mean :t SEM and are expressed as

percentages of non-actinomycin treated controls (n=4). No statistical differences

were noted between control and CPH treated values for any time point.

64

A B C DPNS

 

CALNEXIN
Mwsoko —> .

 

Figure 9. Distribution of the ER in RINm5F subcellular fractions. RINm5F cells
were homogenized then fractionated. Protein samples from the various fractions
were precipitated followed by separation via SDS-PAGE. Calnexin was detected
via immunoblotting as described in Materials and Methods. A: free polysome
fraction; B: ER-bound polysome fraction; C: uninitiated fraction; D:

monoribosome fraction; PNS: post-nuclear supernatant (positive control).

65

E] CONTROL

 

-10uMCPH

8

1204
E I
z 100-
O
0 80+
LI.
O 604
'—
z 401
|.I.|
g 20.
|.I.|
O. 0

 

 

 

 

 

Figure 10. Lack of an effect of CPH on total PPImRNA levels. RINm5F cells
were incubated with 10 pM CPH or 1% sterile water (control) for 2 h after which
they were homogenized and fractionated. PPImRNA was extracted and
quantitated as described in Materials and Methods. Values represent mean :I:
SEM (n=6) and data are expressed as percentage of controls. There are no

statistical differences between treated and control values.

66

 

 

 

 

 

<
E 30‘ :jCONTROL
E .
E 70
a:
'_LIJ 50-
20:
mm 40‘
0>
0:8 30-
UJLLI
mm 20‘
2' 10‘
*— o
O
I- A B C D

SUBCELLULAR FRACTIONS

Figure 11. Effects of CPH on the subcellular distribution of PPImRNA. RINm5F
cells were incubated with 1, 5, or 10 pM CPH or 1% sterile water (control) for 2 h
after which they were homogenized and fractionated. PPImRNA was extracted
and quantitated as described in Materials and Methods. A: free polysome
fraction; B: ER-bound polysome fraction; C: uninitiated fraction; D:
monoribosome fraction. Values represent mean 1: SEM (n=6). * denotes

statistical difference from control; p<0.05.

67

80

4O

0

0

PERCENT OF TOTAL RECOVERED PPImRNA

Figure

60‘

20‘

80*
60‘

20‘

W A

q

 

V :g

 

 

 

0 20 40 60 80 100120

C

 

 

 

* * *
W‘ ':8

0 20 40 60 80 100120

 

 

 

 

 

so-B

«c: 7"

40+ * * 4
*

201

0

0 20 40 60 80 100120

 

 

 

80«D

604

40‘ * * *

20‘ .4: T‘
—o

0

 

O 20 40 60 80 100120

TIME (MINUTES)

12. Time course of CPH effects on the subcellular distribution of

PPImRNA. RINm5F cells were treated with 10 pM CPH (+) or 1% sterile water

(control) (6) for 15, 30, 60, or 120 min after which the cells were homogenized

and fractionated. PPImRNA was extracted and quantitated as described in

Materials and Methods. A:

free polysome fraction; B: ER-bound polysome

fraction; C: uninitiated fraction; D: monoribosome fraction. Values represent

mean 1: SEM (n=6). * denotes statistical difference from control; p<0.05.

68

c: CONTROL
- 10 pM CPH

 

 

 

 

A B C D

PERCENT OF
TOTAL RECOVERED PPImRNA

SUBCELLULAR FRACTIONS

Figure 13. Reversibility of CPH effects on the subcellular distribution of
PPImRNA. RINm5F cells were incubated with 10 uM CPH or 1% sterile water
(control) for 2 h after which the treatment media was removed and replaced with
fresh media containing no CPH. The cells were allowed to recover for 24 h after
which they were homogenized and fractionated. PPImRNA was extracted and
quantitated as described in Materials and Methods. A: free polysome fraction;
B: ER-bound polysome fraction; C: uninitiated fraction; D: monoribosome
fraction. Values represent mean :I: SEM (n=6). No statistical differences were

noted between control and CPH treated values.

69

80 i [Z] CONTROL
:2: 10 pM 2-DPMP
7o . - 10 pM 4-DPMP

101

 

 

 

 

 

PERCENT OF
TOTAL RECOVERED PPImRNA
h
o

A B C D
SUBCELLULAR FRACTIONS

Figure 14. Effects of 2-DPMP and 4-DPMP on the subcellular distribution of
PPImRNA. RINm5F cells were incubated with 10 pM 2-DPMP, 10 [M 4-DPMP
or 0.1% ethanol (control) for 2 h after which they were homogenized and
fractionated. PPImRNA was extracted and quantitated as described in Materials
and Methods. A: free polysome fraction; B: ER-bound polysome fraction; C:
uninitiated fraction; D: monoribosome fraction. Values represent mean 1 SEM

(n=6). * denotes statistical difference from control; p<0.05.

70

_E_ff_e_ct_s_ of CPH on Translation Initiation in RINm5F Cells
Effect of CPH on Polysome Distribution:

In order to determine the effect of CPH on the assembly of polysomes in
RINm5F cells, polysome profile analyses were conducted. A representative
optical density profile is shown in Figure 15. A 2 hour 10 [M CPH treatment
induced an increase in the monoribosome peak when compared to control

tracings.

Effect of CPI-l on the Phosphorylation States of Initiation Factors elF2a,
elF4E, and 4E-BP1:

The phosphorylation states of the initiation factors elF2a, elF4E, and 4E-
BP1 after 10 [M CPH treatment were investigated to determine if the subcellular
mRNA dislocation induced by CPI-l treatment involves alterations in the
phosphorylation states of these initiation factors leading to decreased translation
initiation. A 24 hour 10 pM CPH treatment induced a 267% increase in the
phosphorylation of elF2a Ser51 while the level of nonphosphorylated
elF2a protein was unchanged (Figure 16). A 2 hour 10 pM CPH treatment
induced 34% and 35% decreases in elF4E Ser209 (Figure 17) and 4E-BP1
Ser65 (Figure 18) phosphorylation, respectively. As with elF2a, there were no
alterations in the levels of nonphosphorylated elF4E or 4E-BP1 seen in response

to CPH treatment.

71

 

 

 

 

 

 

A254 ABSORBANCE

 

ll

Control 10 pM CPH

Figure 15. Polysome profile analysis of cellular lysates from control and CPH-
treated cells. Cells were incubated with 10 uM CPH or 1% sterile water (control)
for 2 h after which they were lysed. Cell lysates were layered onto sucrose

gradients, centrifuged and the A254 absorbance measured as described in

Materials and Methods. The increase in the monoribosome peak (t) shown here

is representative of that seen in 3 separate experiments.

72

Figure 16. Effect of CPH on elF2a (Ser51) phosphorylation. RINm5F cells
were incubated with 10 pM CPH or 1% sterile water (control) for 24 h. Protein
samples were separated via SDS-PAGE and phosphorylated eIF2a (Ser51) (A)
and non-phosphorylated elF2a (B) were detected via immunoblotting as
described in Materials and Methods. Blots were quantitated via densitometric
scanning and normalized to B-actin protein. Representative Western Blots are
shown. Values represent mean :1: SEM (n=12) and data are expressed as a

percentage of control. * denotes statistical difference from control; p<0.05.

73

[:1 CONTROL
- 10 pm CPH

400 - *

 

PERCENT OF CONTROL

 

 

 

 

Phospho-eIF2a (Ser51) ... , .... ~

B-actin

 

400 '
350
300
250 '
200
150 .
100 _
50
0

Nonphosphorylated » - .-
eIF2a (Ser51)

B-actin - -

 

PERCENT OF CONTROL

 

 

 

 

 

Figure 16.

74

Figure 17. Effect of CPH on elF4E (Ser209) phosphorylation. RINm5F cells
were incubated with 10 pM CPH or 1% sterile water (control) for 2 h. Protein
samples were separated via SDS-PAGE and phosphorylated elF4E (Ser209) (A)
and non-phosphorylated elF4E (B) were detected via immunoblotting as
described in Materials and Methods. Blots were quantitated via densitometric
scanning and normalized to B-actin protein. Representative Western Blots are
shown. Values represent mean :t SEM (n=12) and data are expressed as a

percentage of control. * denotes statistical difference from control; p<0.05.

75

:3 CONTROL
- 10 pm CPH

120 I

100 4 T

 

80*

60‘

40‘

20-

PERCENT OF CONTROL

 

 

 

 

o
Phospho-eIF4E (Ser209) -- _—-—--

 

5'30““ —

 

 

 

 

 

 

 

_l 120 i
B O
E 100 « . T
c2)
0 80 «
3 so .
5 .0 .
I.I.I
g 20 -
l.l.|
D. 0
Nonphosphorylated .-.
elF4E (Ser209)

B-actin I—I-I—I

Figure 17.

76

Figure 18. Effect of CPH on 4E-BP1 (Ser65) phosphorylation. RINm5F cells
were incubated with 10 pM CPH or 1% sterile water (control) for 2 h. Protein
samples were separated via SDS-PAGE and phosphorylated 4E-BP1 (Ser65) (A)
and non-phosphorylated 4E-BP1 (B) were detected via immunoblotting as
described in Materials and Methods. Blots were quantitated via densitometric
scanning and normalized to B-actin protein. Representative Western Blots are
shown. Values represent mean :I: SEM (n=12) and data are expressed as a

percentage of control. * denotes statistical difference from control; p<0.05.

77

[:I CONTROL

 

- 10 [1M CPH
—| 120 -
A 8
'— 100 . T
2 *
O 30 .
U
B 60 I
E 40<
{'2‘
m 20 1
I.”
0.. 0

 

 

 

 

 

Phospho-4E-BP1 (Ser65) --—- al.—nun...

B-actin H

 

 

 

 

 

 

_| 120‘
B a I
'— 100 4 T
5
8 .
U 0
LL
0 60 I
5 .0.
‘3
m 20 -
I.”
o. 0
Nonphosphorylated.
4E-BP1(Ser65) .
B-actin W
Figure 18.

78

Effects of CPH on Glucagon in RINm5F Cells

 

Effects of CPH on PPGmRNA Subcellular Localization:

Experiments were conducted to determine the effects of CPH treatment
on the subcellular distribution of the secretory protein mRNA, PPGmRNA, in
RINm5F cells to examine the message specificity of CPH-induced PPImRNA
subcellular dislocation. As shown in Figure 19, the amount of total recovered
PPGmRNA after a 2 hour 10 pM CPH treatment was 87.5 :I: 7.8% of control
values, indicating no treatment related alteration in PPGmRNA levels.

Results from experiments conducted to examine the effect of CPH on
PPGmRNA subcellular localization are shown in Figure 20. A 2 hour 10 [TM CPH
treatment induced significant alterations in the distribution of PPGmRNA (Figure
20A). These alterations in PPGmRNA subcellular localization included a 20%
decrease in the percentage of PPGmRNA found at the ER and 50% and 33%
increases in the percentages of uninitiated PPGmRNA and monoribosome-
associated PPGmRNA, respectively. There was no significant change in the
percentage of PPGmRNA associated with free polysomes with CPH treatment.
This CPH-induced PPGmRNA dislocation induced by 2 hour 10 pM CPH
treatment was reversible after allowing the cells a 24 hour recovery period

following removal of the compound (Figure 208).

Effect of CPH on Cellular Glucagon Protein Levels:
Experiments were conducted to examine whether the CPH-induced

PPGmRNA dislocation seen in RINm5F cells was associated with a decrease in

79

cellular glucagon levels. Results from these experiments are shown in Figure 21.
Twenty-four, 48, and 72 hour treatments with various concentrations of CPH
produced no change in total cellular glucagon levels indicating that the
subcellular dislocation of PPGmRNA does not lead to a loss of cellular glucagon
with a time course similar to the loss of cellular insulin in these cells. In order to
ensure that CPH was not producing cytotoxicity during the 72 hour exposure
period, cytotoxicity analyses were conducted. Cells exposed to 10 pM CPH
showed no reduction in the ability to exclude trypan blue relative to control

indicating no cytotoxic action of the drug (data not shown).

Effect of Cycloheximide on Cellular Glucagon Protein Levels:

Experiments were conducted to determine the ability of cycloheximide, a
known inhibitor of protein synthesis, to deplete cellular glucagon content in
RINm5F cells after 24 or 48 hour exposures. The results of these studies are
presented in Figure 22. Increasing concentrations of cycloheximide were found
to inhibit cell growth and division, as shown by decreases in DNA content (Figure
22A). Interestingly, cycloheximide did not produce a depletion of cellular
glucagon content, even at concentrations that inhibited cell growth and division,
and, in fact, induced significant increases in cellular glucagon content after a 48

hour expoure (Figure 22B).

80

E CONTROL

 

 

 

 

 

 

-10uMCPH

o'

12 4
E o T
z 100‘
O
O 80‘
u.
0 60‘
I.—
Z 40«
UJ
g 20.
w
o. 0

Figure 19. Lack of effect of CPH on total PPGmRNA levels. RINm5F cells were
incubated with 10 pM CPH or 1% sterile water (control) for 2 h after which they
were homogenized and fractionated. PPGmRNA was extracted and quantitated
as described in Materials and Methods. Values represent mean :I: SEM (n=6).
Values are expressed as a percentage of controls. There are no statistical

differences between treated and control values.

81

Figure 20. Effects of CPH effects on the subcellular distribution of PPGmRNA.
RINm5F cells were incubated with 10 pM CPH or 1% sterile water (control) for 2
h. (A) After 2 h treatment, cells were immediately homogenized and fractionated
for determination of PPGmRNA subcellular distribution. (B) After 2 h treatment,
the treatment media was removed and replaced with fresh naive media. The
cells were allowed to recover for 24 h after which they were homogenized and
fractionated. PPGmRNA was extracted and quantitated as described in
Materials and Methods. A: free polysome fraction; B: ER-bound polysome
fraction; C: uninitiated fraction; D: monoribosome fraction. Values represent

mean :I: SEM (n=6). * denotes statistical difference from control. p<0.05.

82

 

 

 

 

 

 

 

 

g 30 - r_—] CONTROL
1 - 10 [1M CPH
A E 70 -
2 so -
3 a
r- 8 5° ‘
z r:
3': 4°‘
9% 3°‘
n: 20 ~
.1
. .. . I
f3 o .
A B C D
SUBCELLULAR FRACTIONS
g 80 -
B 5 70 *
3 a. 60 .
I- 8 50 I
2 a:
u: Lu 40 -
E 5
E a 30 -
n: 20 ‘
.1
IS 10 —
.9 o
A B C D
SUBCELLULAR FRACTIONS

Figure 20.

83

E3 CONTROL

 

 

 

 

 

 

 

 

 

 

< 1 pm CPH
z - 5 pM CPH
G 1.4 , - 10 pM CPH
a:
3 1.2 « E
g 1.0 ~ I
z 0. 1 I
O 8
(2 0.6 ‘
o 0.4 1
g 0.2 1
a, 0.0 , , -
= 24 48 72
HOURS OF TREATMENT

Figure 21. Lack of an effect of CPH on cellular glucagon content. RINm5F cells
were incubated with 1, 5, or 10 [M CPH or 1% sterile water (control) for 24, 48 or
72 h after which they were harvested. Glucagon and DNA were then extracted
and quantitated as described in Materials and Methods. Values represent mean
:I: SEM (n=9). There are no statistical differences between treated and control

values.

S CONTROL

 

 

 

 

 

 

 

 

 

 

 

 

 

 

3
o ] - 5 [1M cx
A E 100 L J— - 20 “M cx
Z .
O 80
< O
E u- 60 ‘
DO
:I- 40 ‘ *
z *
I.”
o 20 '
s [L
E, 0
24 48
HOURS OF TREATMENT
B 2 .
O < 1'6 *-
0 Z
< D 1-2 I *
s :2: 1
a a 0.8 1
a, a 0.4 1 H
C
0.0 , ,
24 48
HOURS OF TREATMENT

Figure 22. Effects of cycloheximide on cellular DNA and glucagon content.
RINm5F cells were incubated with 5 or 20 [TM cycloheximide (CX) or 0.1%
ethanol (control) for 24 or 48 h after which they were harvested. Glucagon and
DNA were analyzed as described in Materials and Methods. Values represent

mean :I: SEM (n=6). * denotes statistical difference from control p < 0.05.

85

 

Effects of CPH on PPAmRNA in RINm5F Cells
Effects of CPH on PPAmRNA Subcellular Localization:

Experiments were conducted to determine the effects of CPH treatment
on the subcellular distribution of the secretory protein mRNA, PPAmRNA, in
RINm5F cells to further examine the specificity of CPH-induced PPImRNA
subcellular dislocation. As shown in Figure 23, the amount of total recovered
PPAmRNA after a 2 hour 10 [TM CPH treatment was 71.2 :I: 4.7% of control
values, indicating no significant treatment related alteration in PPAmRNA levels.

Experiments were conducted to examine the effect of CPH on PPAmRNA
subcellular localization. As shown in Figure 24, a 2 hour 10 pM CPH treatment
induced significant alterations in the distribution of PPAmRNA (Figure 24A).
These alterations in PPAmRNA subcellular localization included a 34% decrease
in the percentage of PPAmRNA found at the ER and and a 38% increase in the
percentage of monoribosome-associated PPAmRNA. There was no significant
change in the percentage of PPAmRNA associated with free polysomes or the
percentage of uninitiated PPAmRNA after CPH treatment. This CPH-induced
PPAmRNA dislocation induced by 2 hour 10 11M CPI-I treatment was reversible
after allowing the cells a 24 hour recovery period following removal of the

compound (Figure 24B).

86

1:] CONTROL

 

 

 

 

 

 

-10pMCPH

o‘

120*
E
z 100- T
O
0 801
LL
0 60‘
|_.
Z 40.
LIJ
E!) 20‘
LIJ
0. 0

Figure 23. Lack of an effect of CPH on total PPAmRNA levels. RINm5F cells
were incubated with 10 pM CPH or 1% sterile water (control) for 2 h after which
they were homogenized and fractionated. PPAmRNA was extracted and
quantitated as described in Materials and Methods. Values represent mean 1
SEM (n=6). Values are expressed as a percentage of controls. There are no

statistical differences between treated and control values.

87

Figure 24. Effects of CPH effects on the subcellular distribution of PPAmRNA.
RINm5F cells were incubated with 10 [TM CPH or 1% sterile water (control) for 2
h. (A) After 2 h treatment, cells were immediately homogenized and fractionated
for determination of PPAmRNA subcellular distribution. (B) After 2 h treatment,
the treatment media was removed and replaced with fresh na'r've media. The
cells were allowed to recover for 24 h after which they were homogenized and
fractionated. PPAmRNA was extracted and quantitated as described in Materials
and Methods. A: free polysome fraction; B: ER-bound polysome fraction; C:
uninitiated fraction; D: monoribosome fraction. Values represent mean 1: SEM

(n=6). * denotes statistical difference from control. p<0.05.

88

30 ‘ 1:3 CONTROL

60- l
50‘

40-
30‘
20‘
10*

 

PERCENT OF
TOTAL RECOVERED PPAmRNA

 

 

 

 

A B C D
SUBCELLULAR FRACTIONS

801
704
60:
50+
40J
30~
20‘
10*

PERCENT OF
TOTAL RECOVERED PPAmRNA

 

 

 

 

A B C D
SUBCELLULAR FRACTIONS

Figure 24.

89

Effects of CPH on fi-Actin in RINm5F Qeflg
Effects of CPH on B-Actin mRNA Subcellular Localization:

Experiments were conducted to determine the effect of CPH on the
subcellular distribution of a non-secretory protein mRNA, B-actin mRNA, to
determine if the CPH-induced mRNA subcellular dislocation was specific to
secretory protein messages. As shown in Figure 25, the amount of total
recovered B-actin mRNA after a 2 hour 10 [TM CPH treatment was 98.3 :I: 7.8% of
control values, indicating no treatment related alteration in B-actin mRNA levels.

Results from experiments conducted to examine the effect of CPH on B-
actin mRNA subcellular localization are shown in Figure 26. A 2 hour 10 uM
CPH treatment induced significant alterations in the distribution of B-actin mRNA.
These alterations in B-actin mRNA subcellular localization included a 20%
decrease in the percentage of B-actin mRNA found at the ER, a 12% increase in
the percentage of B-actin mRNA associated with free polysomes and a 28%
increase in the percentage of monoribosome-associated B-actin mRNA (Figure
26A). There was no significant change in the percentage of uninitiated B-actin
mRNA with CPH treatment. This CPH-induced B-actin mRNA dislocation
induced by 2 hour 10 [TM CPH treatment was reversible after allowing the cells a

24 hour recovery period following removal of the compound (Figure 268).

Effects of 2-DPMP and 4-DPMP on B-Actin mRNA Subcellular Localization:
Experiments were conducted to examine the structure-specificity of CPH-

induced fl-actin mRNA subcellular dislocation. As with PPImRNA, a 2 h

90

 

incubation with 10 uM 4-DPMP induced B-actin mRNA subcellular dislocation

nearly identical to that seen with CPH treatment while a 2 h incubation with 10

pM 2-DPMP showed no effect (Figure 27).

Effect of CPH on Cellular B-Actin Protein Levels:

Experiments were conducted to examine whether the CPH-induced [3-
actin mRNA dislocation seen in RINm5F cells was associated with a decrease in
B—actin protein levels. Results from these experiments are shown in Figure 28.
A 48 hour 10 pM CPH treatment produced no change in total cellular B-actin
mRNA levels indicating that the subcellular dislocation of B-actin mRNA does not

lead to a loss of cellular B-actin protein with a time course similar to that of the

loss of cellular insulin in this cell line.

91

[:2] CONTROL

 

-10 all CPH
.1
O 120 1
m
E 100 ~ 7
O
o 80 <
u.
o 60 4
.—
z 40 1
u:
g 20 I
I.I.l
o. o

 

 

 

 

 

Figure 25. Lack of an effect of CPH on total B-actin mRNA levels. RINm5F cells
were incubated with 10 pM CPH or 1% sterile water (control) for 2 h after which
they were homogenized and fractionated. [s-actin mRNA was extracted and
quantitated as described in Materials and Methods. Values represent mean :t
SEM (n=6). Values are expressed as a percentage of controls. There are no

statistical differences between treated and control values.

92

Figure 26. Effects of CPH effects on the subcellular distribution of B-actin
mRNA. RINm5F cells were incubated with 10 pM CPH or 1% sterile water
(control) for 2 h. (A) After 2 h treatment, cells were immediately homogenized
and fractionated for determination of B-actin mRNA subcellular distribution. (B)
After 2 h treatment, the treatment media was removed and replaced with fresh
na‘r‘ve media. The cells were allowed to recover for 24 h after which they were
homogenized and fractionated. B-actin mRNA was extracted and quantitated as
described in Materials and Methods. A: free polysome fraction; B: ER—bound
polysome fraction; C: uninitiated fraction; D: monoribosome fraction. Values

represent mean i SEM (n=6). * denotes statistical difference from control.

p<0.05.

93

3° “ 1:: CONTROL

60~ *
50~
40* *
30‘
20*
10-

PERCENT OF
TOTAL RECOVERED B-ACTIN mRNA

 

 

 

 

A B C D
SUBCELLULAR FRACTIONS

801
70*
60‘
50‘
40*
30~
20*
10*

PERCENT OF
TOTAL RECOVERED B-ACTIN mRNA

 

 

 

A B C D

SUBCELLULAR FRACTIONS
Figure 26.

I: CONTROL
:2! 1
- 1

 

0 pM 2-DPMP
0 pm 4-DPMP

 

 

 

 

 

PERCENT OF TOTAL
RECOVERED B-ACTIN mRNA

 

A B

SUBCELLULAR FRACTIONS

Figure 27. Effects of 2-DPMP and 4-DPMP on the subcellular distribution of [3-
actin mRNA. RINm5F cells were incubated with 10 pM 2-DPMP, 10 pM 4-DPMP
or 0.1% ethanol (control) for 2 h after which they were homogenized and
fractionated. B-actin mRNA was extracted and quantitated as described in
Materials and Methods. A: free polysome fraction; B: ER-bound polysome
fraction; C: uninitiated fraction; D: monoribosome fraction. Values represent

mean 1: SEM (n=6). * denotes statistical difference from control; p<0.05.

95

E CONTROL

 

- 10 [1M CPH

-' 120*

9:

r— 100 « T
6

U 80 :

IL

0 60 ‘

E

m 40 "

32

I.l.| 201

O.

0

 

 

 

 

 

 

Figure 28. Lack of an effect of CPH on B-actin protein levels. RINm5F cells
were incubated with 10 pM CPH or 1% sterile water (control) for 48 h after which
they were harvested. Protein samples were separated via SDS-PAGE and B-
actin protein was detected via immunoblotting as described in Materials and
Methods. B-actin protein was quantitated via densitometric scanning. A
representative Western Blot is shown. Values represent mean :t SEM (n=8). No

statistical differences were noted between treated and control values.

96

_E_ffe_c_ts of CPH in a-Cellg

In order to determine if CPH induced cytotoxic effects in aTC1.9 cells,
experiments were conducted to examine the effect of CPH on the ability of these
cells to exclude trypan blue during various CPH treatments. A 10 uM CPH
treatment for up to 72 hours does not effect the ability of treated cells to exclude
trypan blue dye when compared to control, indicating no cytotoxic action of CPH

in this cell line (data not shown).

Effect of CPH on Preproglucagon mRNA Subcellular Localization in aTC1.9
Cells:

It was necessary to verify the ER separation efficiency of the
centrifugation procedure used by Welsh et al. to produce subcellular fractions
from isolated rat pancreatic islets [153] in aTC1.9 cells. Again, calnexin was
utilized to examine the subcellular fractions generated during the fractionation
procedure for the presence of the ER in these cells. As shown in Figure 29, by
using anti-calnexin Western Blotting, calnexin was found only in the fraction
reported to contain ER-bound polysomes, verifying the separation of the ER
during the fractionation procedure and demonstrating this to be a useful
technique for the determination of cellular mRNAs localized at the ER in this cell
line.

Experiments were then conducted to determine the effects of CPH
treatment on the subcellular distribution of PPGmRNA in aTC1.9 cells. As

shown in Figure 30, the amount of total recovered PPGmRNA after a 2 hour 10

97

pM CPH treatment was 79.9 1: 10.9% of control values, indicating no significant
treatment related alteration in PPGmRNA levels.

Results from experiments conducted to examine the effect of CPH on
PPGmRNA subcellular localization are shown in Figure 31. A 2 hour 10 pM CPH
treatment induced no significant alterations in the distribution of PPGmRNA in

these cells.

Effect of CPH on Cellular Glucagon Content in aTC1.9 Cells:

Experiments were conducted to examine the effect of CPH on cellular
glucagon levels. Results from these experiments are shown in Figure 32.
Twenty-four, 48, and 72 hour treatments with various concentrations of CPH

produced no significant change in total cellular glucagon levels.

Effects of CPH on cellular glucagon content in isolated pancreatic islets
Experiments were conducted to examine the ability of CPH to alter

the cellular glucagon content of isolated pancreatic islets treated in vitro. As

shown in Figure 33, there is no significant treatment related decrease in cellular

glucagon levels after a 48 hour treatment with 10 pM CPH.

98

CALNEXIN .
MW 90kD

   

Figure 29. Distribution of the ER in aTC1.9 cell subcellular fractions. aTC1.9
cells were homogenized then fractionated. Protein samples from the various
fractions were precipitated followed by separation via SDS-PAGE. Calnexin was
detected via immunoblotting as described in Materials and Methods. A: free
polysome fraction; B: ER-bound polysome fraction; C: uninitiated fraction; D:

monoribosome fraction; PNS: post-nuclear supernatant (positive control).

99

1;: CONTROL
- 10 pH CPH

120 <

100 1 T

 

80:

60:

20:

 

 

 

 

 

PERCENT OF CONTROL

Figure 30. Lack of an effect of CPH on total PPGmRNA levels in aTC1.9 cells.
aTC1.9 cells were incubated with 10 [M CPH or 1% sterile water (control) for 2 h
after which they were homogenized and fractionated. PPGmRNA was extracted
and quantitated as described in Materials and Methods. Values represent mean
1: SEM (n=6) and data are expressed as percentage of controls. There are no

statistical differences between treated and control values.

100

 

.14... tut? uA...

 

 

 

 

 

<
z
(r 100 1 1:3 CONTROL
E 90. -10 um CPH
O
o. 80 :
‘5 g 70 «
m 60 :
E 0:
I.I.l u>1 50 ‘
3:, O 4° ‘
a: 1: 3°-
05 20 ~
-I 10:
g o ._1:-_ 1-__
I- A B C D

SUBCELLULAR FRACTIONS

Figure 31. Lack of an effect of CPH on the subcellular distribution of PPGmRNA
in aTC1.9 cells. aTC1.9 cells were incubated with 10 [M CPH or 1% sterile
water (control) for 2 h after which they were homogenized and fractionated.
PPGmRNA was extracted and quantitated as described in Materials and
Methods. A: free polysome fraction; B: ER-bound polysome fraction; C:
uninitiated fraction; D: monoribosome fraction. Values represent mean :I: SEM

(n=6). There are no statistical differences between treated and control values.

101

E CONTROL

 

 

 

 

 

 

 

 

< 1pMCPH
D -10 pM CPH
3 14‘
3, 12 . I
z 10:
8 . . i
s . -
a 4‘
_l 2 .
G 0 __JJ _ -
Cl
C 24 48 72
HOURS OF TREATMENT

Figure 32. Lack of an effect of CPH on cellular glucagon content in orTC1.9
cells. orTC1.9 cells were incubated with 1, 5, or 10 pM CPH or 1% sterile water
(control) for 24, 48 or 72 hours after which they were harvested. Glucagon and
DNA were then extracted and quantitated as described in Materials and
Methods. Values represent mean :I: SEM (n=9). There are no statistical

differences between treated and control values.

102

E CONTROL

 

 

   

 

 

 

14‘ -10pMCPH
z .
O< ‘2
02 ml
<0
0 a) 31
D 3.
.1 a 6*
01°“ 4‘
O. 2,
0
24 48
HOURS OF TREATMENT

Figure 33. Lack of an effect of CPH on cellular glucagon content in isolated rat
pancreatic islets. Isolated rat islets were incubated with 10 pM CPH or 1% sterile
water (control) for 24 or 48 h after which they were harvested. Glucagon and
DNA were then extracted and quantitated as described in Materials and
Methods. Values represent mean 1: SEM (n=3-5). There are no statistical

differences between treated and control values.

103

DISCUSSION

Investigations into the toxicity of CPH in the endocrine pancreas have
been ongoing for the past 30 years. Prior to the present investigations it was
known that CPH induces morphologic and biochemical changes in the insulin-
producing B-cells of rats [120] with no apparent effect on the glucagon-producing
a-cells or somatostatin-producing S-cells of the islet. It was also known that CPH
inhibits insulin synthesis and depletes pancreatic insulin content in both isolated
rat pancreatic islets [3] and in clonal insulinoma cell lines [127] with no significant
effect on the synthesis of total TCA-precipitable proteins. This CPH-induced
inhibition of insulin synthesis occurred without a commensurate loss of PPImRNA
levels [5, 6], suggesting one or more post-transcriptional mechanisms of action
for the diabetogenic effects of the drug. Results presented in this thesis have
provided new information on the post-transcriptional mechanisms and the

specificity of CPH actions in insulin-producing cells.

Effect of CPH on the stability of PPImRNA

After export from the nucleus, a newly transcribed mRNA is stored,
translated, or degraded. The degradation of RNA in association with active
protein synthesis is a well known regulatory mechanism [166, 167]. Decreases
in PPImRNA stability would lead to increased degradation of the message
thereby leading to a decline in cellular PPImRNA levels. In the present studies,

using RT-PCR, there was no change in the levels of PPImRNA in response to

104

CPH treatment as compared with control during a 12 h actinomycin D treatment
to arrest RNA synthesis. These results indicated no treatment-related alteration
in PPImRNA decline indicating no CPH-related effect on PPImRNA stability.
These data are in agreement with published reports indicating that CPH does not
decrease PPImRNA levels (as measured via Northern Analysis) over a 12 h time

period in RINm5F cells [6].

Effects of CPH on the subcellular localization of PPImRNA in RINm5F cells
In the present studies, the CPH-induced inhibition of insulin synthesis was
further characterized by examining the effects of CPH and its analogs 4-DPMP
and 2-DPMP on the subcellular distribution of PPImRNA. The translation of
secretory proteins is a multi-step process beginning in the cytosolic compartment
with the association of the mRNA with several initiation factors to ultimately result
in the formation of a translationally-competent ribosome. The process then
involves the binding of the signal recognition particle to the emerging signal
sequence, inducing translational arrest, leading to the eventual translocation of
the ribosome-nascent chain complex to the ER where the nascent polypeptide is
finally shuttled through the secretory pathway. In these studies, using a
subcellular fractionation technique followed by real-time RT-PCR, the subcellular
location of PPImRNA associated with various ribosomal populations was
investigated in order to examine an effect of CPH on PPImRNA translocation in

the cell.

105

In the present studies, both CPH and 4-DPMP elicited a shift in the
percentage of PPImRNA localized at the ER to the monoribosome-associated
and uninitiated PPImRNA pools while 2-DPMP, a nontoxic CPH analog, was
ineffective in producing a change in PPImRNA subcellular localization. As the
synthesis of preproinsulin occurs on polysomes comprised of 5-7 ribosomes per
PPImRNA [159], this treatment-related increase in the percentages of
monoribosome-associated and uninitiated PPImRNA pools are indicative of a
possible inhibition of the initiation stage of translation. These translational effects
of CPH and its active analog 4-DPMP on the subcellular localization of the insulin
message were found to be consistent with previously published reports on the
concentration-dependence, chemical structure-specificity, and time course of the
inhibition of insulin synthesis and loss of cellular insulin content induced by these
compounds [3, 6, 81, 119, 127]. In addition, the PPImRNA subcellular
dislocation was found to be completely reversible upon removal of the drug.
Reversibility is a hallmark characteristic of CPH B-cell toxicity [4]. The inhibition
of insulin synthesis elicited by CPH treatment has been well characterized in
published reports [3, 6, 81, 119, 127] and the findings presented here show that
PPImRNA dislocation results from exposure to CPH and may be associated with

an inhibition of insulin synthesis.

Effects of CPH on translation initiation in RINm5F cells

Results from experiments examining the effects of CPH on the subcellular

distribution of PPImRNA indicated that the drug induces a dislocation of

106

PPImRNA from ER-bound polysomal pools to monoribosomal and uninitiated
pools. Since an effect of CPH on the initiation stage of translation would produce
such a PPImRNA dislocation, initial experiments were conducted to examine the
effects of the drug on the assembly of polysomes. These investigations revealed
an increase in the monoribosome peak in CPH-treated cells. An increase in the
monoribosome peak during polysome profile analysis is characteristic of an effect
at the level of translation initiation [160].

Further investigation into a potential effect of CPH on initiation was then
conducted by examining the effect of CPH on the phosphorylation state of
several key initiation factors known to be regulated via phosphorylation.

Most translational regulation of protein synthesis occurs at the level of
initiation. Translation is primarily regulated at the step of ribosomal binding to
mRNA [168]. This process requires the activity of a minimum of 10 different
initiation factors. The two best characterized initiation factor interactions are the
elF2-promoted binding of the Met-tRNA to the 408 ribosomal subunit, the
recycling of elF2, and the binding of mRNA to ribosomes promoted by the elF4F
complex [85, 169]. Translation is regulated in response to environmental stimuli
primarily via phosphorylation of the translational machinery [170]. In the present
study, the phosphorylation state of the initiation factors elF201, elF4E and 4E-BP1
were altered in response to CPH treatment in RINm5F cells. CPH was shown to
induce hyperphosphorylation of eIFZa (Ser51) and hypophosphorylation of both

elF4E (Ser209) and 4E-BP1 (Ser65). These alterations in initiation factor

107

phosphorylation are all consistent with a CPH-induced inhibition of translation
initiation (See Figure 5).

Electron micrographs of pancreatic islets isolated from CPH-treated rats
show marked ultrastructural changes. There is a loss of insulin secretion
granules, dilation and vesiculation of the ER, and the formation of large
cytoplasmic vacuoles. These vesicles and cytoplasmic vacuoles contain an
electron-dense material that has not yet been identified but has been speculated
to be abnormal secretory protein precursors [129, 171]. The accumulation of
abnormal (malfolded) proteins in the ER has been shown to trigger a multifaceted
cellular response, known as the unfolded protein response (UPR) that includes a
decrease in translation [172-174]. The translational inhibition induced by the
UPR is due to the activation of an ER kinase known as PERK (PKR-Like ER
Kinase) which phosphorylates elF2a at Ser51 [175, 176]. PERK is abundant in
the pancreas and has a very high level of basal activity [177, 178]. Interestingly,
PERK null mice exhibit B-cell ER ultrastructural changes, including the
vesiculation of the ER and loss of insulin secretory granules [177, 178], similar to
that seen in CPH-treated rats [120, 179], though there are significant
ultrastructural and biochemical differences between the -l- PERK phenotype and
CPH toxicity in the endocrine pancreas. First, there is no apparent involvement
of islet cell types other than the B—cell in CPH toxicity while the -/- PERK mice
exhibit biochemical and ultrastructural changes in both the endocrine and
exocrine cells of the pancreas. Secondly, B-cells of -l- PERK mice were shown

to undergo apoptosis [177, 178], while CPH treatment has not been associated

108

with cell death and its effects have been shown to be reversible [4, 127]. In this
study, increased phosphorylation of the initiation factor eIFZa was induced by
CPH treatment. While this CPH-induced increased elF2a phosphorylation can
contribute to the overall inhibition of insulin synthesis induced by CPH, further
experimentation is necessary to determine the significance of this
hyperphosphorylation in CPH-induced insulin synthesis inhibition. Additional
experimentation is also necessary to determine if this CPH-induced increase in
elF201 phosphorylation is due to activation of the UPR, activation of a gene-
specific mechanism of PPImRNA translational control, or to the activation of
some other mechanism. Further investigation into the potential involvement of
the UPR in CPH-induced inhibition of insulin synthesis may provide valuable
information on the role of PERK in the insulin biosynthetic pathway and may
provide additional insight into the Wolcott-Rallison syndrome, a rare genetic
disease typified by severe diabetes mellitus, mapped to a mutation in the human
PERK gene [180].

The role of elF4E phosphorylation in translational control is not fully
understood. elF4E has been shown to be phosphorylated by a number of
stimuli, including mitogens and growth hormones, though the physiologic
significance of elF4E phosphorylation has not been fully elucidated. Evidence
suggests that the phosphorylation of elF4E, while not necessary for elF4F
complex formation [181], does serve to enhance initiation [182].

The translation of PPImRNA has been under investigation for the past two

decades. The best characterized regulator of PPImRNA translation is glucose.

109

Glucose has been shown to elicit three translational effects on proinsulin
synthesis: an effect on the general translation machinery, including increases in
elF2B activity and 4E-BP1 phosphorylation [183-186]; an effect on the signal
peptide/SRP interaction [153, 187, 188]; and an effect involving the untranslated
regions (UTRs) of PPImRNA [189]. It has been proposed that the predominant
mechanism of glucose-regulated PPImRNA translation and proinsulin synthesis
is due to the latter mechanism and involves the cooperativity of both the 5’ and 3’
UTRs of PPImRNA [189]. The findings that both the 5‘ and 3’ UTRs are involved
supports the involvement of both the elF4F complex and PABP [190] and
suggests a possible role for translational regulation by hypophosphorylation of
elF4E or 4E-BP1. Hypophosphorylation, as seen with CPH treatment, of either
of these initiation factors could lead to decreased elF4F complex formation and
therefore inhibit the interaction between the 5’ and 3’ ends of PPImRNA leading
to decreased insulin synthesis. Further investigation into the role of CPH-
induced elF4E and 4E-BP1 hypophosphorylation in the inhibition of insulin
synthesis induced by this compound is necessary and experiments examining
elF4F complex formation and interaction with PABP will provide valuable
information on the regulation of PPImRNA translation.

Though the effects of CPH in RINm5F cells suggest an effect of the drug
on the general translation machinery, there is the potential for gene-specific
regulation of protein (possibly insulin) translation. Though alterations in initiation
factor phosphorylation have been shown to affect global rates of protein

synthesis, there are several reported cases where changes in the activity of elFs

110

induce gene-specific translational control [87]. While the inhibition of the
translation of specific mRNAs via alterations in global elF activity may seem
contradictory, examples of such gene-specific regulation have been steadily
accumulating over the past 25 years. The first example of mRNA specific
regulation was reported for B-globin, which is more efficiently initiated than a-
globin [191]. Conditions that reduce the efficiency of general translation for any
step in the initiation process disproportionately inhibit a—globin synthesis as
compared with that of B-globin [191]. Studies such as these have led to the
proposal that weaker mRNAs are more sensitive to small changes in initiation
efficiency and are therefore susceptible to gene-specific regulation when the
general translation machinery is perturbed [87]. This mechanism of regulation
may underlie the gene-specific regulation elicited by alterations in elF201 and 4E-
BP1 phosphorylation reported in several biological systems [87]. Whether the
genes investigated in the present research rank among these “weaker" genes

remains unknown.

Effects of CPH on the subcellular localization of PPGmRNA, PPAmRNA,
and B-actin mRNA in RINm5F cells

The apparent specificity of CPH action in the inhibition of insulin synthesis
and the involvement of the subcellular dislocation of PPImRNA in this effect
prompted investigations into the ability of CPH to induce subcellular dislocation of

two additional secretory protein messages, PPGmRNA and PPAmRNA.

111

Dislocation of these messages would not be expected if this treatment-related
dislocation was specific to PPImRNA.

As with PPImRNA, CPH-treatment in RINm5F cells reduced the
percentage of PPGmRNA and PPAmRNA associated with ER-bound polysomes
while increasing the percentages of these mRNAs associated with
monoribosomes. The percentages of uninitiated PPGmRNA and PPAmRNA
were also increased with CPH treatment. In addition, the subcellular dislocation
of PPGmRNA and PPAmRNA, like that of PPImRNA, was found to be reversible
upon removal of the compound.

The finding that CPH induced similar subcellular dislocation of two other
secretory protein messages, PPGmRNA, and PPAmRNA, in addition to
PPImRNA, suggested that CPH may act to inhibit the translocation of only
secretory protein messages to the ER. To further investigate this, the effect of
the CPH on the dislocation of a non-secretory protein message, B-actin mRNA,
was investigated.

As with the other messages examined, CPH induced a decrease in the
percentage of B-actin mRNA associated with ER-bound polysomes and an
increase in the percentage of the message associated with monoribosomes. In
the case of B-actin, the percentage of mRNA associated with free polysomes was
also significantly increased with CPH treatment, an effect not reported for any
other mRNA in this study. In addition, there was no effect on the percentage of

uninitiated B-actin mRNA with CPH treatment.

112

These results indicate that CPH alters the subcellular distribution of
PPImRNA, PPGmRNA, PPAmRNA and B-actin mRNA and demonstrate that this
translational effect of CPH is not specific to the insulin message or to secretory
protein messages in general in RINm5F cell.

While there is a great deal of information concerning the inhibition of
insulin synthesis in response to CPH treatment, there is no information available
on the effects of the drug on the synthesis of other B-cell secretory proteins.
CPH treatment does not significantly alter the synthesis of TCA-precipitable
material in isolated islets [5] or RINm5F cells [6]. Results presented here
indicate a CPH-related subcellular dislocation of both PPGmRNA and
PPAmRNA, in addition to PPImRNA, in RINm5F cells. As both PPGmRNA and
PPAmRNA must be translocated to the ER to complete translation, these data
suggest that CPH may be acting to inhibit the synthesis of these proteins as well.
RINm5F cells contain relatively low levels of glucagon [147] and amylin (B. S.
Hawkins, unpublished observation), as compared with insulin, and it is possible
that the TCA-precipitation method utilized by Miller et al. [127] may not be
sensitive enough to detect alterations in the synthesis of proteins that represent a
small fraction of total protein synthesis. Further investigation into the direct effect
of CPH on the synthesis (i.e. rate of incorporation of radiolabeled amino acid) of
glucagon and amylin in RINm5F cells is necessary to determine whether the
mRNA dislocation observed with CPH treatment is associated with an inhibition

of synthesis of these hormones.

113

The subcellular dislocation of B-actin mRNA was also induced by CPH
and 4-DPMP treatment. The dislocation of B-actin differed from that of the
secretory proteins in that there was an increase in the percentage of B-actin
mRNA associated with free polysomes and no increase in the percentage of
uninitiated B-actin mRNA. These differences may be due to the different
regulatory mechanisms involved in the synthesis of constitutive, as opposed to
regulated, proteins. In the case of B-actin, it is possible that synthesis of the
protein does indeed continue despite the dislocation of the message as it is not
necessary for B-actin mRNA to be translocated to the ER for translation into
protein. Though the translocation of the ribosome-nascent Chain complex to the
ER is not a mandatory step in the synthesis of non-secretory proteins such as [3-
actin, it has been reported that non-secretory proteins can be synthesized on ER-
bound ribosomes [192]. An effect of CPH on the binding of ribosomes to the
ER would result in a shift of B-actin mRNA from ER-bound pools to free cytosolic
pools where, in contrast to secretory protein synthesis, translation would
continue. It has also been reported that B-actin mRNA continues to be translated
under conditions where the synthesis of nearly all other proteins has been
inhibited, suggesting that translation of this protein may differ from that of most
other proteins [193]. Numerous potential explanations were provided by the
author for the continued synthesis of B-actin under conditions inhibitory to protein
synthesis; one suggestion being that the initiation of B-actin mRNA, as opposed
to other protein messages, may be able to continue under low levels of initiation

factors [193]. As is the case with glucagon and amylin, direct evidence on the

114

effect of CPH on the synthesis of B-actin is needed to further characterize the
specificity of CPH toxicity.

The ability of CPH to alter the subcellular localization of both secretory
and non-secretory protein messages and the ability of the compound to alter the
phosphorylation state of the initiation factors elF2a, elF4E, and 4E-BP1 are
consistent with an effect(s) of the drug on the general translation machinery.
Other potential effects of CPH could include alterations in the ability of ribosomes
to bind to the ER and/or changes in the concentration and/or activities of any of
the protein factors involved in the translation process. While there is convincing
evidence to support the role of PPImRNA subcellular dislocation in CPH-induced
inhibition of insulin synthesis, there is the possibility that the CPH-induced
subcellular PPGmRNA, PPAmRNA, and B-actin mRNA dislocation does not
result in decreased synthesis of these proteins.

The correlation between the characteristics of CPH-induced subcellular
PPImRNA dislocation and CPH-induced insulin synthesis inhibition strongly
support a role for the inhibition of PPImRNA translation in the drug-induced
inhibition of insulin synthesis. While this evidence is convincing, there is the
possibility that the subcellular fractionation technique employed in these studies
does not accurately reflect the actual mechanism underlying CPH toxicity in the
intact cell. The CPH-induced subcellular dislocation of PPImRNA may not be
involved in the inhibition of insulin synthesis but may simply be an event that is
correlated with or reflects the true mechanism of insulin synthesis inhibition. The

inhibition of polysome assembly and alterations in initiation factor

115

phosphorylation seen in response to CPH treatment support the results obtained
from the subcellular fractionation experiments making this an unlikely possibility.
Further experimentation to examine the effects of CPH on the synthesis of
glucagon and amylin will provide further information on the validity of this

experimental technique.

Lack of effect of CPH on cellular protein levels of glucagon and B-actin in
RINm5F cells

The inhibition of insulin synthesis in response to CPH treatment both in
vitro and in vivo is followed by a depletion of cellular insulin content [4, 81, 127,
171]. To examine the relationship between subcellular mRNA dislocation and
cellular protein content, experiments were conducted to examine the effects of
CPH on the cellular content of glucagon and B-actin to determine if the
subcellular dislocation of these messages is also associated with a loss of
cellular protein content. Results presented in this thesis show that the cellular
content of these proteins is not altered in response to CPH at concentrations and
time points known to produce a marked loss of cellular insulin content in RINm5F
cells. Due to the extremely low levels of amylin present in RINm5F cells,
consistent, reliable data on the effect of CPH on the cellular content of this
protein could not be obtained.

The cellular content of any secreted protein is maintained through a
balance of synthesis, release, and degradation. In isolated islets in culture, even

minor shifts in the relative rates of insulin biosynthesis and secretion noticeably

116

effect intracellular insulin degradation [77]. In addition, under certain
circumstances, the degradative pathway appears to contribute toward the
regulation of cellular insulin content to as great an extent as either insulin
biosynthesis or release [78]. Interestingly, in the present study, cycloheximide, a
general inhibitor of protein synthesis, was not capable of depleting cellular
glucagon content in RINm5F cells at concentrations which inhibited cell growth.
These data are complementary to the findings of C. P. Miller [148] which showed
that a 24 hour 5 pM cycloheximide treatment was incapable of inducing a decline
in cellular insulin content to an extent comparable to the loss of cellular insulin
content elicited by CPH treatment in this cell line. The inability of cycloheximide
to deplete the cellular content of insulin strongly suggests that a factor or factors
in addition to the inhibition of insulin synthesis, such as enhanced degradation
and/or secretion, is involved in the depletion of insulin content induced by CPH
treatment.

Insulin is degraded within the B-cell via the fusion of insulin secretory
granules with Iysosomes, a process known as crinophagy. However, the
existence of a non-lysosomal degradative pathway [194] in the degradation of
intracellular insulin cannot be ruled out, particularly as insulin-degrading activity
dissimilar to that of lysosomal proteases has been documented in islet
homogenates [195-197]. The kinetics of insulin degradation within the B-cell are
much slower than that of secretory proteins from other non-islet cell types, even
under conditions which induce extensive hormone degradation [76, 78]. The

slow rate of insulin degradation within the B-cell is due to crystallization of insulin

117

within the secretory granule. The insulin crystal requires an acidic milieu for
stability and both secretory granules and Iysosomes are acidic organelles [198].
CPH-treatment of isolated rat pancreatic islets has been shown to increase the
incorporation of insulin secretory granules into lysosomal bodies [81, 179, 199],
an event suggestive of a treatment-related increase in insulin degradation. It has
been previously suggested that enhanced insulin degradation is involved in CPH-
induced depletion of insulin content [81, 179], though no direct evidence of
enhanced degradation is available. A CPH-induced increase in the degradation
of insulin relative to that of glucagon or B-actin is a possible explanation for the
lack of effect of CPH on the cellular content of the other proteins investigated in
this study.

As mentioned previously, RINm5F cells differ from primary B-cells in their
ability to produce both insulin and glucagon. There have been no published
reports concerning the synthesis, storage, release, or degradation of amylin in
RINm5F cells. While the RINm5F cell line has been validated as a model for the
effects of CPH on both PPImRNA and insulin (synthesis, release, and cellular
content [6, 148]), it may not be useful for studies concerning the effects of CPH
on other proteins. The primary reason is that there is little information available
concerning the kinetics of biosynthesis or degradation of proteins other than
insulin in this cell line. It is also possible that, due to differences between the
kinetics and stimulation of insulin and non-insulin protein synthesis and cellular

turnover, the treatment conditions (culture conditions and/or time course) utilized

118

in these studies were not sufficient for the detection of CPH-induced alterations

in cellular protein content in these cells.

Lack of effect of CPI-l on PPGmRNA subcellular localization and cellular
glucagon content in a-cells

CPH produces both structural and biochemical changes in the insulin-
producing B-cells of rats [120] while the 01- or 8—cells of the islet are apparently
unaffected. Pancreatic acinar cells also remain normal in appearance and
contain normal numbers of zymogen granules after CPH treatment [120]. There
has been no report of a loss of glucagon in response to CPH treatment in vivo.
Halban et al. reported a loss of glucagon in isolated rat pancreatic islets treated
in vitro with 50 [M CPH for 6 days [81]. The loss of glucagon reported by Halban
et al. can most likely be attributed to a-cell death due to the high concentration of
CPH utilized and the extensive culture period. Consideration of all data support
a B-cell specific effect of CPH.

In the present study, the effects of CPH on the subcellular localization of
PPGmRNA and the cellular protein content of glucagon were investigated using
the clonal a-cell line, aTC1.9, to determine if the CPH-induced mRNA dislocation
reported in RINm5F cells could be reproduced in another cell type. In contrast to
results from RINm5F cells, there was no CPH-induced alteration in PPGmRNA
subcellular localization in aTC1.9 cells. There was also no decline in cellular
glucagon content in response to CPH treatment in these cells. Experiments were

then conducted in isolated rat pancreatic islets to determine if the results

119

obtained on the effect of CPH on the cellular glucagon content in aTC1.9 cells
could be reproduced in primary cells. As with the clonal cells, the cellular
glucagon content of isolated rat pancreatic islets treated with CPH in vitro was
not significantly altered. Though these data are consistent with a B-cell specific
effect of CPH, due to the limited number of experimental observations, they must
be considered preliminary findings. Further experimentation in both primary or-
cells and aTC1.9 cells is necessary to determine if these clonal cells are a valid
model for studies of the effects of CPH in a-cells.

There is no current information available to explain the cellular specificity
involved in the actions of CPH in insulin-producing cells. Studies on the relative
uptake of CPH by the different islet cell types have not been performed. It is
unlikely that there are differences in cellular CPH uptake among the islet cell
types as lipophilic amines generally cross membranes easily [129], however, this
possibility cannot be ruled out at this time. The mechanism underlying the

apparent B-cell specific toxicity of CPH remains to be elucidated.

120

CONCLUSIONS

Results presented in this thesis suggest that the CPH-induced inhibition of
insulin synthesis in RINm5F cells is due to an effect of the drug on PPImRNA
translation initiation. The initiation of translation is dependent upon the
coordinated activities of numerous initiation factors. The results presented here
suggest that the inhibition of insulin synthesis induced by CPH treatment results
from alterations in the phosphorylation states of three critical initiation factors,
elF201, elF4E, and 4E-BP1. By inducing Changes in the phosphorylation of these
initiation factors, the assembly of a translationally-competent ribosome is
prevented. This CPH-induced inhibition of PPImRNA initiation slows the
translation process at a stage preceding the translocation of the message to the
ER and ultimately results in the decrease in the percentage of PPImRNA found
at the ER during subcellular fractionation (See Figure 34). This treatment-related
inhibition of initiation also results in the increases in the uninitiated and
monoribosome-associated PPImRNA pools as reported here. The effect of CPH
on initiation is further supported through the data obtained from polysome profile
analysis. Increases in the monoribosome peak in these studies indicate an effect
of the drug characteristic of initiation inhibition.

The exact mechanisms underlying the CPH-induced alterations in initiation
factor phosphorylation remain to be elucidated. Further experimentation is
necessary to identify the specific kinases or phosphatases involved in the
reported CPH-induced initiation factor hypo- and hyperphosphorylation. In

addition, experimentation is necessary to determine if the CPH-induced

121

alterations in initiation factor phosphorylation are responsible for alterations in the
activity of these initiation factors. Though these factors are known to be
regulated via phosphorylation, it is not known whether the effect of CPH is
directly on the kinase(s) or phosphatase(s) involved or if these CPH-induced
changes in phosphorylation are due to an indirect action of the drug resulting
from the activation or inhibition of another mechanism.

The CPH-induced subcellular dislocation of PPGmRNA, PPAmRNA, and
B-actin mRNA indicates that this effect of the compound on translation initiation is
not insulin-specific. The alterations in phosphorylation of elF2a, elF4E, and 4E-
BP1 and the effects on polysome assembly support an effect of CPH on the
general translation machinery. CPH treatment has not been shown to result in
decreased synthesis of total protein though there is currently no specific
information available on the effect of CPH on the synthesis of glucagon, amylin
or B-actin. Further investigations into the effect of CPH on the synthesis on these
proteins will provide valuable information on the specificity of the translational
regulation of insulin synthesis and CPH-induced B-cell toxicity.

The loss of cellular insulin content in response to CPH treatment has been
demonstrated to result from the inhibition of insulin synthesis induced by the
drug. As CPH was shown to induce subcellular dislocation of PPGmRNA and B-
actin mRNA, the lack of effect of CPH on the cellular content of glucagon and [3-
actin suggests that measurements of cellular content do not necessarily reflect
alterations in protein synthesis. The inability of CPH or the general protein

synthesis inhibitor, cycloheximide, to reduce the cellular content of glucagon

122

suggests that a mechanism other than simply the inhibition of synthesis, such as
increased hormone degradation or secretion, is involved in the loss of cellular
hormone content seen in response to CPH treatment. This information is in
agreement with previous reports of the inability of cycloheximide to produce a
decline in the cellular insulin content in RINm5F cells. Again, information on the
effects of CPH on the synthesis of glucagon and B-actin is needed to fully
understand the relationship between the synthesis and cellular content of these
proteins. Also, further experimentation is necessary to understand the effects of
CPH on the degradation of insulin to determine the exact mechanism responsible
for the CPH-induced loss of cellular insulin content.

The inability of CPH to induce PPGmRNA subcellular dislocation or a loss
of cellular glucagon content in the clonal a-cell line, aTC1.9 supports previous
reports indicating that the adverse effects of CPH are specific to insulin-
producing cells. While this cell line has been utilized as a valid model for the
study of glucagon synthesis, further information on the effects of CPH both in
primary a-cells and in this cell line is needed to ensure that this cell line is a valid
model for studies of the effects of CPH in primary cells. The mechanism
underlying the apparent B-cell specificity of CPH toxicity remains to be
elucidated.

Though the CPH-induced inhibition of PPImRNA initiation described here
is well correlated with the inhibition of insulin synthesis induced by the drug, the
possibility that CPH-induced inhibition of insulin synthesis may be the result of

additional and/or unrelated mechanisms cannot be ruled out. These

123

mechanisms could include alterations in the elongation or termination stages of
translation, disruption of the signal recognition particle-mediated translocation of
PPImRNA to the ER, alteration of ribosome docking at the ER, disruption of
insertion of nascent preproinsulin into the lumen of the ER, and/or alteration of
preproinsulin or proinsulin processing.

CPH is a compound that possesses a variety of pharmacological and
toxicological activities and it is probable that the diabetogenic actions of the drug
are due to a combination of mechanisms. Further investigation is necessary to
fully understand the relationships that exist between the inhibition of insulin
synthesis, the inhibition of insulin secretion and the loss of cellular insulin content
induced by this drug. Further elucidation of the mechanisms by which CPH
produces B-cell toxicity may allow this agent to be a useful tool for the study of
insulin biosynthesis and regulation under both normal and pathological

conditions.

124

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REFERENCES

\erson, G.L. and SP. LeDoux, The role of chemicals in the etiology of
diabetes mellitus. Toxicol Pathol, 1989. 17(2): p. 357-63.

Fischer, L.J. and DE. Rickert, Pancreatic islet-cell toxicity. CRC Crit Rev
Toxicol, 1975. 3(2): p. 231-63.

Hintze, K.L., A.B. Grow, and L.J. Fischer, Cyproheptadine-induced
alterations in rat insulin synthesis. Biochem Pharmacol, 1977. 26(21): p.
2021-7.

Rickert, D.E., J. Burke, and L.J. Fischer, Cyproheptadine-induced
depletion of insulin in the rat. J Pharmacol Exp Ther, 1975. 193(2): p. 585-
93.

Miller, C.P., et al., Rapid alteration of pancreatic proinsulin and
preproinsulin messenger ribonucleic acid in rats treated with
cyproheptadine. Endocrinology, 1991. 128(6): p. 3040-6.

Miller, C.P., T.J. Reape, and L.J. Fischer, Inhibition of insulin production
by cyproheptadine in RINm5F rat insulinoma cells. J Biochem Toxicol,
1993. 8(3): p. 127-34.

Bonner-Weir, S. and FE. Smith, Islets of Langerhans: morphology and its
implications, in Joslin's Diabetes Mellitus, C.R. Khan and GC Weir,
Editors. 1994, Lea & Febiger: Philadelphia.

Orci, L. and RH. Unger, Functional subdivision of islets of Langerhans
and possible role of D cells. Lancet, 1975. 2(7947): p. 1243-4.

Orci, L., R.H. Unger, and AB Renold, Structural coupling between
pancreatic islet cells. Experientia, 1973. 29(8): p. 1015-8.

Meda, P., et al., Rapid and reversible secretion changes during
uncoupling of rat insulin- producing cells. J Clin Invest, 1990. 86(3): p.
759-68.

Steiner, D., G.l. Bell, and HS. Tager, Chemistry and biosynthesis of

pancreatic protein hormones. 2nd ed. Endocrinology, ed. L.J. DeGroot.
Vol. 2. 1989, Philadelphia: W. B. Saunders Company. 1263-1289.

126

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

Walter, P. and G. Blobel, Trans/ocation of proteins across the
endoplasmic reticulum III. Signal recognition protein (SRP) causes signal
sequence-dependent and site- specific arrest of Chain elongation that is
released by microsomal membranes. J Cell Biol, 1981. 91(2 Pt 1): p. 557-
61.

Nunnari, J. and P. Walter, Protein targeting to and translocation across the
membrane of the endoplasmic reticulum. Curr Opin Cell Biol, 1992. 4(4):
p. 573-80.

099, SC. and P. Walter, SRP samples nascent chains for the presence of
signal sequences by interacting with ribosomes at a discrete step during
translation elongation. Cell, 1995. 81(7): p. 1075-84.

Gilmore, R., P. Walter, and G. Blobel, Protein translocation across the
endoplasmic reticulum. II. Isolation and characterization of the signal
recognition particle receptor. J Cell Biol, 1982. 95(2 Pt 1): p. 470-7.

Meyer, D.l., E. Krause, and B. Dobberstein, Secretory protein
translocation across membranes-the role of the ”docking protein '. Nature,
1982. 297(5868): p. 647-50.

Connolly, T., P.J. Rapiejko, and R. Gilmore, Requirement of GTP
hydrolysis for dissociation of the signal recognition particle from its
receptor. Science, 1991. 252(5010): p. 1171-3.

Bacher, G., M. Pool, and B. Dobberstein, The ribosome regulates the
GTPase of the beta-subunit of the signal recognition particle receptor. J
Cell Biol, 1999. 146(4): p. 723-30.

Gilmore, R. and G. Blobel, Transient involvement of signal recognition
particle and its receptor in the microsomal membrane prior to protein
translocation. Cell, 1983. 35(3 Pt 2): p. 677-85.

Palade, G., Intracellular aspects of the process of protein synthesis.
Science, 1975. 189(4200): p. 347-58.

Chan, S.J., P. Keim, and D.F. Steiner, Cell-free synthesis of rat
preproinsulins: characterization and partial amino acid sequence
determination. Proc Natl Acad Sci U S A, 1976. 73(6): p. 1964-8.

Kreil, 6., Transfer of proteins across membranes. Annu Rev Biochem,
1981. 50: p. 317-48.

Halban, P., Proinsulin trafficking and processing in the pancreatic B cell.
Trends Endocrinol Metab, 1990. 1 (5): p. 261 -265.

127

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

35.

Patzelt, C., et al., Detection and kinetic behavior of preproinsulin in
pancreatic islets. Proc Natl Acad Sci U S A, 1978. 75(3): p. 1260-4.

Perrnutt, MA. and 0M. Kipnis, Insulin biosynthesis: studies of Islet
polyribosomes (nascent peptides- sucrose gradient analysis-gel filtration).
Proc Natl Acad Sci U S A, 1972. 69(2): p. 505-9.

Sorenson, R.L., M.W. Steffes, and AW. Lindall, Subcellularlocalization of
proinsulin to insulin conversion in isolated rat islets. Endocrinology, 1970.
86(1): p. 88-96.

Serafini, T., et al., A coat subunit of Golgi-derived non-cIathrin-coated
vesicles with homology to the cIathrin-coated vesicle coat protein beta-
adaptin. Nature, 1991. 349(6306): p. 215-20.

Orci, L., et al., The trans-most cisternae of the Golgi complex: a
compartment for sorting of secretory and plasma membrane proteins. Cell,
1987. 51(6): p. 1039-51.

Rhodes, C.J. and PA. Halban, Newly synthesized proinsulinfinsulin and
stored insulin are released from pancreatic B cells predominantly via a
regulated, rather than a constitutive, pathway. J Cell Biol, 1987. 105(1): p.
145-53.

Orci, L., Macro- and micro-domains in the endocrine pancreas. Diabetes,
1982. 31(6 Pt 1): p. 538-65.

Orci, L., The insulin factory: a tour of the plant surroundings and a visit to
the assembly line. The Minkowski lecture 1973 revisited. Diabetologia,
1985. 28(8): p. 528-46.

Gold, 6., et al., Effects of monensin on conversion of proinsulin to insulin
and secretion of newly synthesized insulin in isolated rat islets. Diabetes,
1984. 33(11): p. 1019-24.

Rhodes, C.J., et al., Stimulation by A TP of proinsulin to insulin conversion
in isolated rat pancreatic islet secretory granules. Association with the
ATP-dependent proton pump. J Biol Chem, 1987. 262(22): p. 10712-7.

Davidson, H.W. and JG. Hutton, The insulin-secretory-granule
carboxypeptidase H. Purification and demonstration of involvement in
proinsulin processing. Biochem J, 1987. 245(2): p. 575-82.

Davidson, H.W., C.J. Rhodes, and JG. Hutton, Intraorganellar calcium

and pH control proinsulin cleavage in the pancreatic beta cell via two
distinct site-specific endopeptidases. Nature, 1988. 333(6168): p. 93-6.

128

36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

46.

47.

Bailyes, E.M., et al., A member of the eukaryotic subtilisin family (PC3)
has the enzymic properties of the type 1 proinsulin-converting
endopeptidase. Biochem J, 1992. 285(Pt 2): p. 391-4.

Bennett, D. L., et al., Identification of the type 2 proinsulin processing
endopeptidase as P02, a member of the eukaryote subtilisin family. J Biol
Chem, 1992. 267(21): p. 15229-36.

Davidson, H.W., M. Peshavaria, and JG. Hutton, Pmteolytic conversion of
proinsulin into insulin. Identification of a Ca2+-dependent acidic
endopeptidase in isolated insulin-secretory granules. Biochem J, 1987.
246(2): p. 279-86.

Goodge, K.A. and JC. Hutton, Translational regulation of proinsulin
biosynthesis and proinsulin conversion in the pancreaticbeta-cell [In
Process Citation]. Semin Cell Dev Biol, 2000. 11(4): p. 235-42.

Shoelson, SE. and P. Halban, Insulin biosynthesis and chemistry, in
Joslin's Diabetes Mellitus, C.R. Khan and CC. Weir, Editors. 1994, Lea &
Febiger: Philadelphia.

Grant, P.T., T.L. Coombs, and EH. Frank, Difi'erences in the nature of the
interaction of insulin and proinsulin with zinc. Biochem J, 1972. 126(2): p.
433-40.

Greider, M.H., S.L. Howell, and PE. Lacy, Isolation and properties of
secretory granules from rat islets of Langerhans. II. Ultrastructure of the
beta granule. J Cell Biol, 1969. 41(1): p. 162—6.

Michael, J., et al., Studies on the molecular organization of rat insulin
secretory granules. J Biol Chem, 1987. 262(34): p. 16531-5.

Rubenstein, A.H., et al., Secretion of proinsulin C-peptide by pancreatic B
cells and its circulation in blood. Nature, 1969. 224.

Humble, R.E., Chemistry of insulin, in Handbook of Physiology, N.
Freinkel and D. Steiner, Editors. 1972, American Physiological Society:
Baltimore. p. 111-132.

Lomedico, P., et al., The structure and evolution of the two nonallelic rat
preproinsulin genes. Cell, 1979. 18(2): p. 545-58.

Cordell, B., et al., Isolation and characterization of a cloned rat insulin
gene. Cell, 1979. 18(2): p. 533-43.

129

48.

49.

50.

51.

52.

53.

54.

55.

56.

57.

58.

59.

60.

Clark, J.L. and D.F. Steiner, Insulin biosynthesis in the rat: demonstration
of two proinsulins. Proc Natl Acad Sci U S A, 1969. 62(1): p. 278-85.

Smith, L. F., Species variation in the amino acid sequence of insulin. Am J
Med, 1966. 40: p. 662-666.

Steiner, D.F., et al., The biosynthesis of insulin, in Handbook of
Physiology, N. Freinkel and D.F. Steiner, Editors. 1972, American
Physiological Society: Baltimore, MD. p. 175-198.

Koranyi, L., et al., Proinsulin I and II gene expression in inbred mouse
strains. Mol Endocrinol, 1989. 3(11): p. 1895-902.

Giddings, SJ. and LR. Camaghi, The two nonallelic rat insulin mRNAs
and pre-mRNAs are regulated coordinately in vivo. J Biol Chem, 1988.
263(8): p. 3845-9.

Kakita, K., K. O'Connell, and MA. Perrnutt, Pancreatic content ofinsulinsl
and II in laboratory rodents. Analysis by immunoelectrophoresis. Diabetes,
1982. 31(10): p. 841-5.

Fiedorek, F.T., Jr., L.R. Camaghi, and SJ. Giddings, Selective expression
of the insulin I gene in rat insulinoma-derived cell lines. Mol Endocrinol,
1990. 4(7): p. 990-9.

Lefebvre, P.J., Glucagon and its family revisited. Diabetes Care, 1995.
18(5): p. 715-30.

Novak, U., et al., Identical mRNA for preproglucagon in pancreas and gut.
Eur J Biochem, 1987. 164(3): p. 553-8.

Lopez, L.C., et al., Mammalian pancreatic preproglucagon contains three
glucagon-related peptides. Proc Natl Acad Sci U S A, 1983. 80(18): p.
5485-9.

Bell, 61., RF. Santerre, and GT. Mullenbach, Hamster preproglucagon
contains the sequence of glucagon and two related peptides. Nature,
1983. 302(5910): p. 716-8.

Bell, 61., et al., Exon duplication and divergence in the human
prepmglucagon gene. Nature, 1983. 304(5924): p. 368-71.

Valverde, I., et al., Characterization of glucagon-like immunoreactivity
(GLI). Diabetes, 1970. 19(9): p. 614-23.

130

61.

62.

63.

65.

66.

67.

68.

69.

70.

71.

72.

Thim, L. and A.J. Moody, The primary structure of porcine glicentin
(proglucagon). Regul Pept, 1981. 2(2): p. 139-50.

Bataille, D., et al., Isolation of glucagon-37 (bioactive
enteroglucagon/oxyntomodulin) from porcine jejunO-ileum.
Characterization of the peptide. FEBS Lett, 1982. 146(1): p. 79-86.

Bataille, D., et al., Isolation of glucagon-37 (bioactive
enteroglucagon/oxyntomodulin) from porcine jejuno-ileum. Isolation of the
peptide. FEBS Lett, 1982. 146(1): p. 73-8.

Bataille, D., Preproglucagon and its processing, in Handbook of
Experimental Pharmacology, P.J. Lefebvre, Editor. 1996, Springer-Verlag:
Heidelberg, Germany. p. 31-51.

Holst, J.J., et al., Proglucagon processing in porcine and human pancreas.
J Biol Chem, 1994. 269(29): p. 18827-33.

Patzelt, C. and E. Schiltz, Conversion of pmglucagon in pancreatic alpha
cells: the major endproducts are glucagon and a single peptide, the major
pmglucagon fragment, that contains two glucagon-like sequences. Proc
Natl Acad Sci U S A, 1984. 81(16): p. 5007-11.

Sanke, T., et al., An islet amyloid peptide is derived from an 89-amino acid
precursor by proteolytic processing. J Biol Chem, 1988. 263(33): p.
17243-6.

Johnson, K. H ., et al., lmmunolocalization of islet amyloid polypeptide
(IAPP) in pancreatic beta cells by means of peroxidase-antiperoxidase
(PAP) and protein A- gold techniques. Am J Pathol, 1988. 130(1): p. 1-8.

Lu kinius, A., et al., Co-Iocalization of islet amyloid polypeptide and insulin
in the B cell secretory granules of the human pancreatic islets.
Diabetologia, 1989. 32(4): p. 240-4.

Clark, A., et al., Localisation of islet amyloid peptide in Iipofuscin bodies
and secretory granules of human B-cells and in islets of type-2 diabetic
subjects. Cell Tissue Res, 1989. 257(1): p. 179-85.

Madsen, O. D., et al., Islet amyloid polypeptide and insulin expression are
controlled differently in primary and transformed islet cells. Mol Endocrinol,
1991. 5(1): p. 143-8.

Kanatsuka, A., et al., Secretion of islet amyloid polypeptide in response to
glucose. FEBS Lett, 1989. 259(1): p. 199-201.

131

73.

74.

75.

76.

77.

78.

79.

80.

81.

82.

83.

84.

Kahn, S.E., et al., Evidence of cosecretion of islet amyloid polypeptide and
insulin by beta-cells. Diabetes, 1990. 39(5): p. 634-8.

Mitsukawa, T., et al., Islet amyloid polypeptide response to glucose,
insulin, and somatostatin analogue administration. Diabetes, 1990. 39(5):
p. 639-42.

Cooper, G.J ., et al., Amylin and the amylin gene: structure, function and
relationship to islet amyloid and to diabetes mellitus. Biochim Biophys
Acta, 1989. 1014(3): p. 247-58.

Orci, L., et al., Insulin, not C-peptide (proinsulin), is present in crinophagic
bodies of the pancreatic B-cell. J Cell Biol, 1984. 98(1): p. 222-8.

Halban, RA, et al., Long-term exposure of isolated pancreatic islets to
mannoheptulose: evidence for insulin degradation in the beta cell.
Biochem Pharmacol, 1980. 29(19): p. 2625-33.

Halban, PA. and CB. Wollheim, Intracellular degradation of insulin stores
by rat pancreatic islets in vitro. An alternative pathway for homeostasis of
pancreatic insulin content. J Biol Chem, 1980. 255(13): p. 6003-6.

Halban, RA, et al., Resistance of the insulin crystal to lysosomal
proteases: implications for pancreatic B-cell crinophagy. Diabetologia,
1987. 30(5): p. 348-53.

Halban, RA, et al., Proinsulin modified by analogues of arginine and
lysine is degraded rapidly in pancreatic B-cells. Biochem J, 1984. 219(1):
p. 91 -7.

Halban, RA, et al., Perturbation of hormone storage and release induced
by cyproheptadine in rat pancreatic islets in vitro. Endocrinology, 1979.
104(4): p. 1096-106.

Mathews, M.B., N. Sonenberg, and J.W. Hershey, Origins and targets of
translational control, in Translational Control, J.W. Hershey, M.B.
Mathews, and N. Sonenberg, Editors. 1996, Cold Spring Harbor
Laboratory Press: Cold Spring Harbor, NY. p. 1-29.

Hershey, J.W. and WC. Merrick, Pathway and mechanism of initiation of
protein synthesis, in Translational Control of Gene Expression, N.
Sonenberg, J.W. Hershey, and MB. Mathews, Editors. 2000, Cold Spring
Harbor Laboratory Press: Cold Spring Harbor, NY. p. 33-88.

Proud, CG. and RM. Denton, Molecular mechanisms for the control of
translation by insulin. Biochem J, 1997. 328(Pt 2): p. 329-41.

132

85.

86.

87.

88.

89.

90.

91.

92.

93.

94.

95.

96.

Hershey, J.W., Translational control in mammalian cells. Annu Rev
Biochem, 1991. 60: p. 717-55.

Redpath, NT. and CG. Proud, Molecular mechanisms in the control of
translation by hormones and growth factors. Biochim Biophys Acta, 1994.
1220(2): p. 147-62.

Dever, T. E., Gene-specific regulation by general translation factors. Cell,
2002. 108(4): p. 545-56.

Hinnebusch, A.G., Translational control of GCN4: gene-specific regulation
by phosphorylation of elF2, in Translational Control, J.W. Hershey, M.B.
Mathews, and N. Sonenberg, Editors. 1996, Cold Spring Harbor
Laboratory Press: Cold Spring Harbor, NY. p. 199—244.

Price, N. and C. Proud, The guanine nucleotide-exchange factor, eIF-ZB.
Biochimie, 1994. 76(8): p. 748-60.

Gingras, A.C., B. Raught, and N. Sonenberg, elF4 initiation factors:
effectors of mRNA recnritment to ribosomes and regulators of translation.
Annu Rev Biochem, 1999. 68: p. 913-63.

Gingras, A.C., et al., Hierarchical phosphorylation of the translation
inhibitor 4E-BP1. Genes Dev, 2001. 15(21): p. 2852-64.

Mothe-Satney, l., et al., Multiple mechanisms control phosphorylation of
PHA S-l in five (S/T)P sites that govem translational repression. Mol Cell
Biol, 2000. 20(10): p. 3558-67.

Asano, K., et al., A multifactor complex of eukaryotic initiation factors,
elF 1, elF2, elF3, elF5, and initiator tRNA (Met) is an important translation
initiation intermediate in vivo. Genes Dev, 2000. 14(19): p. 2534-46.

Kaspar, R. L., et al., Simultaneous cytoplasmic redistribution of ribosomal
protein L32 mRNA and phosphorylation of eukaryotic initiation factor 4E
after mitogenic stimulation of Swiss 3T3 cells. J Biol Chem, 1990. 265(7):
p. 3619-22.

Marino, M.W., et al., Tumor necrosis factor induces phosphorylation of a
28-kDa mRNA cap- binding protein in human cervical carcinoma cells.
Proc Natl Acad Sci U S A, 1989. 86(21): p. 8417-21.

Morley, SJ. and J.A. Traugh, Phorbol esters stimulate phosphorylation of

eukaryotic initiation factors 3, 4B, and 4F. J Biol Chem, 1989. 264(5): p.
2401-4.

133

97.

98.

99.

100.

101.

102.

103.

104.

105.

106.

107.

108.

Duncan, R.F ., Translational control during heat shock, in Translational
Control, J.W. Hershey, M.B. Mathews, and N. Sonenberg, Editors. 1996,
Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY. p. 271-
294.

Duncan, R., 8.0. Milburn, and J.W. Hershey, Regulated phosphorylation
and low abundance of HeLa cell initiation factor elF-4F suggest a role in
translational control. Heat shock effects on elF-4F. J Biol Chem, 1987.
262(1): p. 380-8.

Duncan, R. and J.W. Hershey, Regulation of initiation factors during
translational repression caused by semm depletion. Covalent modification.
J Biol Chem, 1985. 260(9): p. 5493-7.

Bonneau, AM. and N. Sonenberg, Involvement of the 24-kDa cap-binding
protein in regulation of protein synthesis in mitosis. J Biol Chem, 1987.
262(23): p. 11134-9.

Feigenblum, D. and R.J. Schneider, Modification of eukaryotic initiation
factor 4F during infection by influenza virus. J Virol, 1993. 67(6): p. 3027-
35.

Huang, J.T. and R.J. Schneider, Adenovinrs inhibition of cellular protein
synthesis involves inactivation of cap-binding protein. Cell, 1991. 65(2): p.
271 -80.

Morley, S.J., in Protein Phosphorylation in Cell Growth Regulation, M.J.
Clemens, Editor. 1996, Hanrvood Academic Press: Amsterdam.

Yoon, J.W., et al., Efi'ects of environmental factors on the development of
insulin- dependent diabetes mellitus. Clin Invest Med, 1987. 10(5): p. 457-
69.

Engelhardt, E.L., et al., Structure-activity relationships in the
cyproheptadine series. J Med Chem, 1965. 8(6): p. 829-35.

Stone, C.A., et al., Antiserotonin-antihistaminic properties of
cyproheptadine. J Pharmacol Exp Ther, 1961. 131: p. 73-84.

Miller, J. and A. Fishman, A serotonin antagonist in the treatment of
allergic and allied disorders. Ann Allergy, 1961. 19: p. 164-171.

Miller, J., A serotonin antagonist, cyproheptadine: II. The treatment of
vascular type headache and pruritus. Ann Allergy, 1963. 21: p. 588-592.

134

109.

110.

111.

112.

113.

114.

115.

116.

117.

118.

119.

120.

121.

Diamond, S., B.J. Baltes, and H.W. Levine, A review of the pharmacology
of drugs used in the therapy of migraine. Headache, 1972. 12: p. 37-43.

Lavenstein, A.F., et al., Effect of cyproheptadine on asthmatic children:
Study of appetite, weight gain, and linear growth. J Am Med Assoc, 1962.
180: p. 90-94.

Bergen, J ., S. S., Appetite stimulating properties of cyproheptadine. Am J
Diseases Children, 1964. 108: p. 270-273.

Mainquet, P., Effect of cyproheptadine on anorexia and loss of weight in
adults. The Practitioner, 1972. 208: p. 797-800.

Stiel, J.N., G.W. Liddle, and W.W. Lacy, Studies of the mechanism of
cyproheptadine-induced weight gain in human subjects. Metabolism,
1970. 19: p. 192-200.

Chakrabarty, A.S., et al., Effect of cyproheptadine on the electrical activity
of the hypothalamic feeding centers. Brain Res, 1967. 6: p. 561-569.

Woodrum, ST. and CS. Brown, Management of SSRl-induced sexual
dysfunction. Ann Pharmacother, 1998. 32(11): p. 1209-15.

Frye, CB. and J.E. Berger, Treatment of sexual dysfunction induced by
selective serotonin-reuptake inhibitors. Am J Health Syst Pharrn, 1998.
55(11): p. 1167-9.

Akhondzadeh, S., et al., Cyproheptadine in treatment of chronic
schizophrenia: a double-blind, placebo-controlled study. J Clin Phann
Ther, 1999. 24(1): p. 49-52.

Brophy, M.H., Cyproheptadine for combat nightmares in post-traumatic
stress disorder and dream anxiety disorder. Mil Med, 1991. 156: p. 100-
101.

Hintze, K.L., H.Y. Aboul-Enein, and L.J. Fischer, lsomeric specificity of
diphenylmethylpiperidine in the production of rat pancreatic islet cell
toxicity. Toxicology, 1977. 7(2): p. 133-40.

Longnecker, D.S., J.S. Wold, and L.J. Fischer, Ultrastructural study of
alterations in beta cells of pancreatic islets from cyproheptadine-treated
rats. Diabetes, 1972. 21(2): p. 71-9.

Joost, H.G., W. Poser, and U. Panten, Inhibition of insulin release from the
rat pancreas by cyproheptadine and tricyclic antidepressants. Naunyn
Schmiedebergs Arch Pharmacol, 1974. 285(1): p. 99-102.

135

122.

123.

124.

125.

126.

127.

128.

129.

130.

131.

132.

133.

Joost, H.G., et al., Inhibition of insulin and glucagon release from the
perfused rat pancreas by cyproheptadine (Periactinol, Nuran).
Diabetologia, 1976. 12(3): p. 201—6.

Joost, H .G., et al., Inhibition of insulin release by cyproheptadine: effects
on 3', 5'- cyclic-A MP-content and 45Ca-accumulation of incubated mouse
islets. Acta Endocrinol (Copenh), 1976. 82(1): p. 121-6.

Richardson, B.P., M.L. McDaniel, and PE. Lacy, Effects of
cyproheptadine on insulin secretion by isolated perifused rat islets.
Diabetes. 1975. 24(9): p. 836-41.

Donatsch, P., et al., Mechanism by which cyproheptadine inhibits insulin
secretion. Br J Pharmacol, 1980. 70(3): p. 355-62.

Richardson, BF. and B. Berde, Proceedings: The effects of
cyproheptadine pretreatment on insulin release from isolated pancreatic
islets. Br J Pharmacol, 1976. 56(3): p. 356P-357P.

Miller, CF. and L.J. Fischer, Cyproheptadine-induced alterations in clonal
insulin-producing cell lines. Biochem Pharmacol, 1990. 39(12): p. 1983-
90.

Chow, S.A., J.L. Falany, and L.J. Fischer, Cyproheptadine metabolites
inhibit proinsulin and insulin biosynthesis and insulin release in isolated rat
pancreatic islets. Cell Biol Toxicol, 1989. 5(2): p. 129-43.

Fischer, L.J., Toxic effects of cyproheptadine and its analogs in insulin-
producing cells, in Endocrine Toxicology, J.A. Thomas and H.D. Colby,
Editors. 1997, Taylor & Francis: Washington.

Golander, A. and Z. Spirer, Inhibition of the insulin response to glucose
after treatment with cyproheptadine. Acta Paediatr Scand, 1982. 71(3): p.
485-7.

Rotenstein, D., S. Serbin, and T. Welsh, Palliative treatment of
hyperinsulinism with cypmheptadine and diazoxide. Pediatrics, 1992. 90(2
Pt 1): p. 212-5.

Jahr, H., et al., Cyproheptadine inhibits (pro)insulin biosynthesis and
secretion of isolated human pancreatic islets. Horm Metab Res, 1981.
13(6): p. 359.

Lacy, PE. and M. Kostianovsky, Method for the isolation of intact islets of
Langerhans from the rat pancreas. Diabetes, 1967. 16(1): p. 35-9.

136

134.

135.

136.

137.

138.

139.

140.

141.

142.

143.

144.

145.

Poitout, V., L.K. Olson, and RP. Robertson, Insulin-secreting cell lines:
classification, characteristics and potential applications. Diabetes Metab,
1996. 22(1): p. 7-14.

Chick, W.L., et al., A transplantable insulinoma in the rat. Proc Natl Acad
Sci U S A, 1977. 74(2): p. 628-32.

Gazdar, A. F., et al., Continuous, clonal, insulin- and somatostatin-
secreting cell lines established from a transplantable rat islet cell tumor.
Proc Natl Acad Sci U S A, 1980. 77(6): p. 3519-23.

Praz, G.A., et al., Regulation of immunoreactive-insulin release from a rat
cell line (RINm5F). Biochem J, 1983. 210(2): p. 345-52.

Welsh, M., et al., Control of insulin gene expression in pancreatic beta-
cells and in an insulin-producing cell line, RIN-5F cells. II. Regulation of
insulin mRNA stability. J Biol Chem, 1985. 260(25): p. 13590-4.

Wollheim, C.B., S. Ullrich, and T. Pozzan, Glyceraldehyde, but not cyclic
AMP-stimulated insulin release is preceded by a rise in cytosolic free
Ca2+. FEBS Lett, 1984. 177(1): p. 17-22.

Dobs, A.S., C. Broussolle, and MD. Lane, Regulation of insulin synthesis
in an insulin-producing cell line (RINm5F): long-tenn experiments. In Vitro
Cell Dev Biol, 1989. 25(2): p. 112-4.

Sener, A., M.H. Giroix, and W.J. Malaisse, Impaired uptake of D-glucose
by tumoral insulin-producing cells. Biochem Int, 1986. 12(6): p. 913-9.

Giroix, M.H., A. Sener, and W.J. Malaisse, D-glucose transport and
concentration in tumoral insulin-producing cells. Am J Physiol, 1986.
251(6 Pt 1): p. 0847-51.

Malaisse, W.J ., et al., 3-O-methyI-D-glucose transport in tumoral insulin-
producing cells. Am J Physiol, 1986. 251(6 Pt 1): p. 0841—6.

Halban, P.A., G.A. Praz, and CB. Wollheim, Abnormal glucose
metabolism accompanies failure of glucose to stimulate insulin release
from a rat pancreatic cell line (RINm5F). Biochem J, 1983. 212(2): p. 439-
43.

Giroix, M. H ., et al., Glucose metabolism in insulin-producing tumoral cells.
Arch Biochem Biophys, 1985. 241(2): p. 561-70.

137

146.

147.

148.

149.

150.

151.

152.

153.

154.

155.

156.

157.

Vischer, U ., et al., Hexokinase isoenzymes of RIN-m5F insulinoma cells.
Expression of glucokinase gene in insulin-producing cells. Biochem J,
1987. 241(1): p. 249-55.

Barreto, M., I. Valverde, and W.J. Malaisse, Glucagon storage, release
and degradation by tumoral islet cells (RINm5F line). Diabetes Res, 1989.
10(3): p. 121-3.

Miller, C. P., Investigations into the mechanism of cyproheptadine-induced
inhibition of proinsulin biosynthesis and depletion of pancreatic insulin
content, in Department of Pharmacology and Toxicology. 1990, Michigan
State University: East Lansing, MI.

Powers, A.C., et al., Proglucagon processing similar to normal islets in
pancreatic alpha- like cell line derived from transgenic mouse tumor.
Diabetes, 1990. 39(4): p. 406-14.

Hamaguchi, K. and EH. Leiter, Comparison of cytokine effects on mouse
pancreatic alpha-cell and beta- cell lines. Viability, secretory function, and
MHC antigen expression. Diabetes, 1990. 39(4): p. 415-25.

Hintze, K.L., J.S. Wold, and L.J. Fischer, Disposition of cyproheptadine in
rats, mice, and humans and identification of a stable epoxr'de metabolite.
Drug Metab Dispos, 1975. 3(1): p. 1-9.

Andersson, A., Isolated mouse pancreatic islets in culture: effects of
serum and different culture media on the insulin production of the islets.
Diabetologia, 1978. 14(6): p. 397-404.

Welsh, M., et al., Translational control of insulin biosynthesis. Evidence for
regulation of elongation, initiation and signal-recognition-particle-mediated
translational arrest by glucose. Biochem J, 1986. 235(2): p. 459-67.

Orlando, 0., P. Pinzani, and M. Pazzagli, Developments in quantitative
PCR. Clin Chem Lab Med, 1998. 36(5): p. 255-69.

Pelegri, C., et al., Islet endocrine-cell behavior from birth onward in mice
with the nonobese diabetic genetic background. Mol Med, 2001. 7(5): p.
31 1-9.

Held, C.A., et al., Real time quantitative PCR. Genome Res, 1996. 6(10):
p. 986-94.

Ahluwalia, N., et al., The p88 molecular chaperone is identical to the
endoplasmic reticulum membrane protein, calnexin. J Biol Chem, 1992.
267(15): p. 10914-8.

138

158.

159.

160.

161.

162.

163.

164.

165.

166.

167.

168.

169.

Wessel, D. and U1. Flugge, A method for the quantitative recovery of
protein in dilute solution in the presence of detergents and lipids. Anal
Biochem, 1984. 138(1): p. 141-3.

Perrnutt, MA. and D.M. Kipnis, Insulin biosynthesis and secretion. Fed
Proc, 1975. 34(7): p. 1549-55.

Pain, V.M. and EC. Henshaw, Initiation of protein synthesis in Ehriich
ascifes tumour cells. Evidence for physiological variation in the association
of methionyl- tRNAf with native 40-8 ribosomal subunits in vivo. Eur J
Biochem, 1975. 57(2): p. 335-42.

Ruan, H., C.Y. Brown, and DR. Morris, Analysis of Ribosome Loading
onto mRNA Species: Implications for Translational Control, in mRNA
Formation and Function. 1997, Academic Press. p. 305-321.

Labarca, C. and K. Paigen, A simple, rapid, and sensitive DNA assay
procedure. Anal Biochem, 1980. 102(2): p. 344-52.

Zambetti, G., et al., Differential association of membrane-bound and non-
membrane-bound polysomes with the cytoskeleton. Exp Cell Res, 1990.
191(2): p. 246-55.

Vedeler, A., l.F. Pryme, and J.E. Hesketh, Compartmenfalization of
polysomes into free, cytoskeletal-bound and membrane-bound
populations. Biochem Soc Trans, 1991. 19(4): p. 1108-11.

Fletcher, J.E., P.R. Copeland, and D.M. Driscoll, Polysome distribution of
phospholipid h ydroperoxide glutathione peroxidase mRNA: evrdence for a
block in elongation at the UGA/selenocysteine codon. Rna, 2000. 6(11): p.
1 573-84.

Graves, RA, et al., Translation is required for regulation of histone mRNA
degradation. Cell, 1987. 48(4): p. 615-26.

Yen, T.J., et al., Autoregulated changes in stability of polyribosome-bound
beta-fubulin mRNAs are specified by the first 13 translated nucleotides.

Mol Cell Biol, 1988. 3(3): p. 1224-35.

Kaufman, R.J., Control of gene expression at the level of translation
initiation. Curr Opin Biotechnol, 1994. 5(5): p. 550-7.

Merrick, W.C., Mechanism and regulation of eukaryotic protein synthesis.
Microbiol Rev, 1992. 56(2): p. 291-315.

139

 

170.

171.

172.

173.

174.

175.

176.

177.

178.

179.

180.

181.

Rhoads, R. E., Regulation of eukaryotic protein synthesis by initiation
factors. J Biol Chem, 1993. 268(5): p. 3017-20.

Rickert, D. E., et al., Cyproheptadine-induced insulin depletion in rat
pancreatic beta cells: demonstration by light and electron microscopic
immunocytochemistry. Horm Metab Res, 1976. 8(6): p. 430-4.

Bertolotti, A., et al., Dynamic interaction of BIP and ER stress transducers
in the unfolded- protein response. Nat Cell Biol, 2000. 2(6): p. 326-32.

Harding, H . P., et al., Perk is essential for translational regulation and cell
survival during the unfolded protein response. Mol Cell, 2000. 5(5): p. 897-
904.

Kaufman, R.J., Stress signaling from the lumen of the endoplasmic
reticulum: coordination of gene transcriptional and translational controls.
Genes Dev, 1999. 13(10): p. 1211-33.

Harding, H.P., Y. Zhang, and D. Ron, Protein translation and folding are
coupled by an endoplasmic-reticulum— resident kinase. Nature, 1999.
397(6716): p. 271-4.

Shi, Y., et al., Identification and Characterization of pancreatic eukaryotic
initiation factor 2 alpha-subunit kinase, PEK, involved in translatronal
control. Mol Cell Biol, 1998. 18(12): p. 7499-509.

Harding, H. P., et al., Diabetes mellitus and exocrine pancreatic
dysfunction in perk-l- mice reveals a role for translatronal control rn
secretory cell survival. Mol Cell, 2001 . 7(6): p. 1 153-63.

Zhang, R, et al., The PERK eukaryotic initiation factor 2 alpha kinase is
required for the development of the skeletal system, postnatal growth, and
the function and viability of the pancreas. Mol Cell Brol, 2002. 22(11): p.

3864-74.

Richardson, B.P., et al., Pancreatic beta cell changes induced by
cyproheptadine in vitro. Lab Invest, 1975. 33(5): p. 509-13.

Delepine, M., et al., EIF2A K3, encoding translation initiation factor 2-alpha
kinase 3, is mutated in patients with Wolcott-Rallison syndrome. Nat

Genet, 2000. 25(4): p. 406-9.

McKend rick, L., et al., Phosphorylation of eukaryotic initiation factor .4E
(elF4E) at Ser209 is not required for protein synthesrs rn vrtro and rn vrvo.
Eur J Biochem, 2001. 268(20): p. 5375-85.

140

 

182.

183.

184.

185.

186.

187.

188.

189.

190.

191.

192.

McKendrick, L., V.M. Pain, and SJ. Morley, Translation initiation factor
4E. Int J Biochem Cell Biol, 1999. 31(1): p. 31-5.

Gilligan, M., et al., Glucose stimulates the activity of the guanine
nucleotide-exchange factor eIF-ZB in isolated rat islets of Langerhans. J
Biol Chem, 1996. 271(4): p. 2121-5.

Xu, 6., et al., Insulin mediates glucose-stimulated phosphorylation of
PHA S-I by pancreatic beta cells. An insulin-receptor mechanism for
autoregulation of protein synthesis by translation. J Biol Chem, 1998.
273(8): p. 4485-91.

Patel, J., X. Wang, and CG. Proud, Glucose exerts a permissive effect on
the regulation of the initiation factor 4E binding protein 4E-BP1. Biochem
J, 2001. 358(Pt 2): p. 497-503.

Campbell, L.E., X. Wang, and CG. Proud, Nutrients differentially regulate
multiple translation factors and their control by insulin. Biochem J, 1999.
344 Pt 2: p. 433-41.

Guest, P.C., et al., Insulin secretory granule biogenesis. Co-ordinate
regulation of the biosynthesis of the majority of constrtuent proterns.
Biochem J, 1991. 274(Pt 1): p. 73-8.

Welsh, N., C. Oberg, and M. Welsh, GTP-binding proteins may stimulate
insulin biosynthesis in rat pancreatic islets by enhancing the Signal-
recognition-particle- dependent translocation of the rnsulrn mRNA poly-
/mono-some complex to the endoplasmic reticulum. Biochem J, 1991.

275(Pt 1): p. 23-8.

Wicksteed, B., et al., Cooperativity between the preproinsulin mRNA
untranslated regions is necessary for glucose-stimulated translatron. J BIOI

Chem, 2001. 276(25): p. 22553-8.

Sachs, AB, in Translational Control of Gene Expression, N. Sonenberg,
J.W. Hershey, and MB. Mathews, Editors. 1999, Cold Sprung Harbor

Laboratory Press: Cold Spring Harbor, NY.

Lodish, H.F., Translational control of protein synthesis. Annu Rev
Biochem, 1976. 45: p. 39-72.

Potter, MD. and CV. Nicchitta, Regulation of ribosome detachment from
the mammalian endoplasmic reticulum membrane. J Brol Chem, 2000.

275(43): p. 33828-35.

141

 

193.

194.

195.

196.

197.

198.

199.

Felipo, V. and S. Grisolia, Retention of actin synthesis in liver under
conditions that inhibit synthesis of almost all other proteins. F EBS Lett,
1987. 210(2): p. 173-6.

Goldberg, AL. and AC. St John, Intracellular protein degradation in
mammalian and bacterial cells: Part 2. Annu Rev Biochem, 1976. 45: p.
747-803.

Kohnert, K.D., et al., Breakdown of exogenous insulin by Langerhans
islets of the pancreas in vitro. Biochim Biophys Acta, 1974. 338(68-77).

Kohnert, K. D., et al., Demonstration of insulin degradation by thioI-protein
disulfide oxidoreductase (glutathione-insulin transhydrogenase) and
proteinases of pancreatic islets. Biochim Biophys Acta, 1976. 422(2): p.
254-9.

Zuhlke, H., et al., Proteolytic and transhydrogenolytic activities in isolated
pancreatic islets of rats. Acta Biol Med Ger, 1977. 36(11-12): p. 1695-703.

Orci, L., et al., Conversion of proinsulin to insulin occurs coordinately with
acidification of maturing secretory vesicles. J Cell Biol, 1986. 103(6 Pt 1):
p. 2273-81.

Zwahlen, R., B.P. Richardson, and RE. Hauser, The production and
elimination of myeloid bodies by cultured pancreatic islet cells. J
Ultrastruct Res, 1979. 67(3): p. 340-56.

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