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”I : LIBRARY
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I Vivo Metabolite Regulation of

migrlnositol Biosynthesis

 

presented by

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;y_VIvo METABOLITE REGULATION OF
mxgrlNOSITOL BIOSYNTHESIS

By

Thomas Paul Rancour

A THESIS

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

MASTER OF SCIENCE

Department of Biochemistry

1978

ABSTRACT

l_ VIVO METABOLITE REGULATION OF
mygeINOSITOL BIOSYNTHESIS

 

By

Thomas Paul Rancour

lg vi g_regulation of mygeinositol biosynthesis by the
cytosolic NADf/NADH ratio and glucose-6-phosphate levels were
investigated in mammalian systems.

Ethanol feeding of pregnant rats was utilized in an
attempt to lower the fetal liver cytosolic NADf/NADH ratio.
A significantly reduced redox state was expected to inhibit
mygeinositol biosynthesis since the rate limiting enzyme,
g—mygeinositol-l-phosphate synthase, has an absolute require-
ment for NAD+. However, ethanol consumption by dams did not
significantly alter the redox state of the fetal liver, nor
were mygrinositol levels in fetal liver significantly changed.

Streptozotocin diabetes resulted in a two fold increase
in testicular glucose-6-phosphate levels and a three fold
increase in testicular mygrinositol. Further results

suggested that i__vivo testicular mygeinositol levels were

 

primarily regulated by the size of the glucose-6-phosphate
pool and that increases in mygrinositol content were not due
to increases in the specific activities of the biosynthetic
enzymes or mygeinositol transport. Elevated testicular
glucose-6-phosphate levels were attributed to an increased
flux of glucose into the testis, rather than due to increased
hexokinase activity. The effects of elevated testicular
gyg-inositol levels on testicular sperm counts and tubulin

polymerization were also examined.

DEDICATION

To my wife, Nancy, for her constant love and encouragement.

ii

ACKNOWLEDGEMENTS

I wish to express my sincere appreciation to
Dr. William W. Wells for his enthusiasm, guidance, patience,
and financial support throughout these studies. I would
also like to thank my committee members, Dr. Loran L. Bieber,
Dr. Dale R. Romsos, and Dr. Charles C. Sweeley for helping
me in this endeavor.

Special thanks to Nancy for her love, patience,

understanding, and for making it all worthwhile.

iii

TABLE OF CONTENTS

Page
LIST OF TABLES . . . . . . . . . . . . . . . . . . . . .vii'
LIST OF FIGURES. . . . . . . . . . . . . . . . . . . . . ix
LIST OF ABBREVIATIONS. . . . . . . . . . . . . . . . . . X
INTRODUCTION
Objectives and Rationale. . . . . . . . . . . . . . 1
Literature Survey . . . . . . . . . . . . . . . . . 3

myo o-Inositol Occurence and Role in Growth

and Development. . . . . . . . . . . . . . g

Biosynthesis of my_-Inositol . . . . .

Review of Male Reproductive Tract Physiology . 6

Role of myg— Inositol in the Male

Reproduct1ve Tract . . . . . . 9

Additional Proposed Roles and Metabolism

of m o-Inositol. . . . . . . . . . . . . . . . 12
References. . . . . . . . . . . . . . . . . . . . . 14

Chapter

I. CHARACTERIZATION OF m1_rINOSITOL CONTENT OF
FETAL TISSUES FROM ETHANOL- FED AND m o-INOSITOL

DEPRIVED, ETHANOL- FED DAMS . . . . . 20
Abstract. . . . . . . . . . . . . . . . . 20
Introduction. . . . . . . . . . . . . 21
Materials and Methods . . . . . . . . . . 23

Reagents . . . . . . . . . . . . . 23
Animal Diets and Handling. . . . . . 23
Experiment A . . . . . . . . . . . . 23
Experiment B . . . . . . . . . . . 25
Experiment C . . . . . . . 27
Tissue and Plasma Free my_-Inositol

and Glucose Determinations . . . . . 27

Perchloric Acid Tissue Extract
Preparation. 0 O O O O O O I I O O O 28

L-Lactate Determinations . . . . . . 28
Pyruvate Determinations. . . . . 29
Calculation of NADt/NADH Ratios. . . 30
Statistics . . . . . . . . . . . . . 30

iv

Chapter Page

Results . . . . . . . . . . . . . . . . . 31
Experiment A . . . . . . . . . . . . 31
Experiment B . . . . . . . . . . . . 34
Experiment C . . . . . . . . . . . . 39

Discussion. . . . . . . . . . . . . . . . 43

References. . . . . . . . . . . . . . . . 47

II. myo-INOSITOL BIOSYNTHESIS IN THE RAT TESTIS

DURING HYPERGLYCEMIA OR ETHANOL FEEDING. . . . 49
Abstract. . . . . . . . . . . . . . . . . 49
Introduction. . . . . . . . . . . . . 50
Materials and Methods . . . . . . . . . . 52

Reagents . . . . . . . . . . . . . . 52
Animals and Diets. . . . . . . . 53
Administration of Streptozotocin . . 53
Administration of Insulin. . . . . 53

Collection of Tissue and Plasma

for m o-Inositol and Metabolite
Analysis . . . . . . . . . . . . 54
Plasma and Tissue Glucose Assays . . 54
Perchloric Acid Tissue Extract
Preparation and Assays

for Metabolites. . . . . . . . 54
Calculation of C tosolic and
Mitochondrial NAl‘IIE /NADH Ratios . . . 55
Plasma and Tissue m o-Inositol
Determinations . . . . . . . . . . . 55
Preparation of m o-Inositol Bio-
synthesizing Enzymes from Rat Testis 55
Q-Glucose-é-Phosphate:erygelnositol
-1-Phosphate Synthase Assay. . . . . 56
L-myg—Inositol-1-Phosphate

Phosphatase Assay. . .1. . . . . . . 56
Determination of Inorganic

Phosphate. . . . . . . . . . . . . . 57
Protein Determinations . . . . . 57
Pair Feeding of Ethanol Fed and

Control Rats . . . . . . . . . . . 57
Hexokinase Preparation . . . . . . . 58
Hexokinase Assay . . . . . . . . . . 59
Kinetic Studies. . . . . . . . . . . 59
Sperm Counts . . . . . . . . . . . 59

Determination of Total and Polymer-
ized Tubulin in Testes . . . . . . . 59

Statistics . . . . . . . . . . . . . 60

Page
Results 0 O O I O O O O O O 0 O O O O O O 61

Effects of Streptozotocin Induced
Diabetes and Subsequent Intraperi-
toneal Insulin Injection on Testes

m o-Inositol Levels. . . . . . . . . 61
Kinetics of Streptozotocin Induced
Diabetes and Effects on Testis and
Plasma mygelnositol Levels . . . . . 63
Synthase and Phosphatase Activities,
Exp-Inositol Content, and Cyto-

plasmic Redox State of Testes from
Normal, Diabetic, and Ethanol
Fed Rats . . . . . . . . . . .
Effects of Chronic Ethanol Con-
sumption on Redox State and

m o-Inositol Content of Liver and
Testes . . . . . . . . . . . . . . . 71
Intracellular Hexokinase Distribution
in the Testis of Normal, Diabetic,

and Alcohol Intoxicated Rats . . . . 76
Investigation of the Effects of
Diabetes and Concurrent Elevated

Testis myg-Inositol Levels on
Testicular Sperm Count and Tubulin

. . . 7O

Polymerization . . . . . . . . . . . 79
Discussion. . . . . . . . . . . . . . . . 83
Acknowledgements. . . . . . . . . . . . . 90
References. . . . . . . . . . . . . . . . 91

vi

Table

Chapter
I.

II.

III.

IV.

VI.

VII.

Chapter
I.

II.

III.

II

LIST OF TABLES

Page

Experiment A Diet Composition . . . . .

Experiment A: Litter Size, Diet
Consumption, and Weight Profile of
Pregnant Ethanol Fed and Pair Fed

Control Rats. . . . . . . . . . . . . . .

Experiment Ar Selected Fetal Tissue
myQ-Inositol and Plasma Glucose
Concentrations. . . . . . . . . . .

Experiment B: Pregnancy Rate, Litter Size,
and Diet Composition for Ethanol Fed and
contrOl Rats I I I I I I I I I I I I I I I

Experiment B: Selected Fetal Tissue
o-Inositol Levels and Fetal Liver
Cytosolic NAD /NADH Ratios. . . . . . . .

Experiment C: Ethanol Consumption by
Pregnant Rats, Litter Size, Fetal Body
Weights, and Fetal Liver Cytosolic

NAD /NADH Ratios. . . . . . . . . . . . .

Experiment C: Fetal and Maternal Tissue
and Plasma o-Inositol Levels. . . . . .

myo-Inositol Contents of Testes and Plasma
of Normal, Diabetic, and Insulin
Injected Diabetic Rats. . . . . . . .

Specific Activities of the Exp-Inositol
Biosynthesizing Enzymes, m o—Inositol and
Selected Metabolite Levels, and the
Cytosolic Redox State pf Testes from
Normal, Diabetic, and Ethanol Fed Rats.

Exp-Inositol and Selected Metabolite

Levels and Redox State of Liver from
Control and Ethanol Fed Rats. . . . . .

vii

24

32

33

35

38

4O

42

62

73

75

IV.

VI.

VII.

Page

myg-Inositol and Selected Metabolite
Levels, Redox State and Sperm Count of
Testes from Ethanol Fed and Control Rats. 77

Effects of Metabolic State on In Vivo
Distribution of Testis Hexokinase . . . . 78

Effects of Diabetes and Elevated Testes
mygelnositol Levels on Sperm Counts . . . 80

Effects of Diabetes and Elevated Testis

myg-Inositol Levels on Testis Tubulin
Content and Tubulin Polymerization. . . . 82

viii

LIST OF FIGURES

Figure Page
Chapter I
1. Maternal Weight Profile (Experiment B). - 37
Chapter II
1. Effects of Streptozotocin Injection
on Plasma Glucose Concentration and
Testes Glucose Content in Adult Rats. . . 65
2. Effects of Streptozotocin Injection on
Testes Glucose-6-Phosphate Levels and
Testes m o-Inositol Content . . . . . . . 68
3. Effect of Streptozotocin Injection on
Plasma m o-Inositol Concentration . . . . 69

ix

ATP

DMSO

EGTA

EtOH

G6P

GTP

HEPES

NAD , NADH

NADP+, NADPH

Tris

max

LIST OF ABBREVIATIONS

Adenosine-5'-triphosphate
Dimethylsulfoxide

Ethylene glycol bisOQ-aminoethyl ether)N,
N, N', N' tetraacetic acid

Ethanol
QgGlucose-6-phosphate
Guanosine-5'-triphosphate

N-2-Hydroxyethylpiperazine-N'-2-ethane
sulfonic acid

Michaelis constant

Oxidized and reduced nicotinamide adenine
dinucleotide

Oxidized and reduced nicotinamide adenine
dinucleotide phosphate

Trishydroxymethylaminomethane

Maximum velocity

INTRODUCTION

Objectives and Rationale

mygelnositol has been proposed to serve several
functions in nature including that of an essential growth
factor for microorganisms (1-4) and mammalian cell lines
(5,6), a potential vitamin in animals (7). an agent important
in the maturation of spermatozoa in the mammalian male repro-
ductive tract (8-11), and a component of phosphoinositides,
important membrane constituents (12). A pathway has been
described in several mammalian species for the biosynthesis
of mygeinositol consisting of two enzymes, Drglucose-6-
phosphates Lymygeinositol-1-phosphate synthase (EC 5.5.1.4),
an enzyme having an absolute requirement for NAD+, and eryg:
inositol-l-phosphate phosphatase (EC 3.1.3.25) (13-18). It
was the purpose of this research to investigate the ;n_yiyg
regulation of the mammalian mygrinositol biosynthetic path-
way by selected metabolites.

Chapter I deals with the regulation of g§_ngyg,myg-
inositol biosynthesis in fetal rat tissues by the cytosol
NADf/NADH ratio. A fetal rat system was chosen since
previous work in this laboratory demonstrated that fetal
rat liver contained high activities of the mygeinositol
biosynthetic enzymes (18). Since the rate limiting step

of Exp-inositol biosynthesis is at the level of the NAD+-

1

2

linked synthase (19), a lowered cytosol NADf/NADH ratio
was expected to result in decreased Exp—inositol biosynthesis.
Ethanol was fed to pregnant rats in an attempt to lower the
fetal cytosol NADf/NADH ratio as a result of ethanol oxi-
dation through the NAD+-linked alcohol dehydrogenase of

fetal rat liver. The approach used to evaluate i vivo

 

changes in fetal m o-inositol biosynthesis was to quantitate
the concentration of the free cyclitol in various fetal tis-
sues. In order to minimize error due to mygrinositol con-
tributions from the diet and enteric microorganisms, mygr
inositol free diets and inclusion of the antibiotic phthalyl-
sulfathiazole in the diet were utilized in some experiments.
Lowered fetal myg-inositol biosynthesis in the rat fetus

may be deleterious to normal growth and development.

Chapter II characterizes the in vivo regulation of

 

mygeinositol biosynthesis in the rat testis by elevated
glucose and glucose—6—phosphate levels. The rat testis was
chosen since it has been shown to contain a high capacity
for mygrinositol biosynthesis (13). Hyperglycemia was
induced in male rats through injection of streptozotocin,

a diabetogenic agent. Since the testis is not an insulin
dependent tissue, hyperglycemia was expected to elevate
glucose and glucose-6-phosphate levels in the testis. In
response to an increase of testis glucose-6—phosphate, the
substrate of the NAD+ dependent synthase of the mygsinositol
biosynthesizing pathway, increased production of mygrinositol

was predicted. Elevated testicular mygeinositol levels in

diabetic animals may reveal information on the role of

3

mygeinositol in the male reproductive tract.

Literature Survey

myg—Inositol, a cyclitol first discovered by Scherer in 1850
(20), is a ubiquitous compound in nature. It exists in
several forms including free mygyinositol, phosphoinosi—
tides, phytic acid and other inositol phosphate esters, 6-O-
,8-Drgalactopyranosyl mygrinositol (676-galactinol): it is a
constituent of glycerides, and is found as mono- and di-O-
methylated derivatives (7). Phosphoinositides or gyp-
inositol containing phospholipids are the most predominant
form of Exp-inositol, serving as major components of
membranes (12).

In addition to its importance in phosphoinositides,
mygeinositol has been proposed to have a role in nutrition.
mygrlnositol has been shown to be an essential growth factor
for yeast and fungi (1-4), as well as for numerous normal
and cancerous cell lines (5,6). Woolley demonstrated in
1940 that mice maintained on a mygeinositol deficient diet
failed to thrive, displayed alopecia, and died within two
to three weeks (21). However, conflicting results have
been obtained by numerous investigators concerning the
existence of an essential nutrient role for mygyinositol in
mammalian systems (22-31)- Absolute mygrinositol deficiency
has been difficult to achieve. In yiyg_mygeinositol bio-
synthesis, degradation of phosphatidylinositol, mygrinos-

itol production by intestinal microorganisms, and transport

L,

of the cyclitol between tissues have all complicated
efforts to obtain evidence that dietary mygrinositol is

required for growth and development of mammals.

Biosynthesi§_g§ Exp-Inositol. An endogenous biosynthetic
pathway for .mygeinositol in mammalian systems has been
suspected since the original observation by Vohl (32) in
1858 that significant quantities of m o-inositol were found
in the urine of diabetics. Several years later Needham (33)
detected mygrinositol in the urine of rats maintained for
eight months on a myg—inositol free diet. Other investigators
supported the argument for endogenous biosynthesis in mam-
mals by demonstrating that tissue levels of Exp-inositol
were not depleted during dietary inositol deprivation (34)
even after intestinal flora suspected of supplying the
cyclitol were eliminated (35).

The suggestion that m o-inositol was synthesized from
glucose was originally postulated by Maquenne (36) as early
as 1887 after the structural similarities between the cyclitol
and the monosacoharide were realized. This hypothesis was
further developed by Fischer (37) and gained support with
the elucidation of the configurational similarities of
glucose and mygeinositol (38). Daughaday and others (39)
supplied the first direct evidence for glucose as a precur-
sor for mygrinositol in mammals by demonstrating the incor-

14

poration of randomly labelled C-glucose into mygeinositol.

14

Using 6- C-glucose, Hauser (40) subsequently showed that

incorporation of label into mygeinositol was faster in young

rats than in adults and that label was initially incorporated
into the free mygrinositol pool and then into phospholipids.
Using a rat testis homogenate system, Eisenberg, gt_aly(13)
studied the incorporation of glucose labelled at specific
carbons into mygrinositol and deduced from the labelling
patterns that mygsinositol biosynthesis might occur by a
direct internal aldolization of glucose. Subsequent studies
in yeast (41-43), Neurospora crassa (44), rat testis (13—16),
rat mammary gland (17,18), and fetal rat liver (18) resulted
in the discovery of erygrinositol-1-phosphate synthase

(EC 5.5.1.4), an enzyme which catalyzes the NAD+ requiring
internal aldolization of glucose-6-phosphate to yield eryge
inositol-l-phosphate. mygrlnositol-l-phosphate formed in
the synthase reaction is hydrolyzed specifically by eryg:
inositol-l-phosphate phosphatase (EC 3.1.3.25) to yield
erygeinositol (43,16).

Following the discovery of these biosynthetic enzymes,
investigations were conducted to elucidate the enzyme
mechanisms involved. Mechanistic work concentrated on the
synthase since this enzyme was determined to be rate limiting
in mygeinositol biosynthesis (19). The synthase from mam—
malian systems was shown by a number of investigators to
be a cycloaldolase of the type I class (19, 45-48). The
postulated mechanism for the synthase involves an NAD+ depen-
dent oxidation of the C-5 carbon of glucose-6-phosphate to
a keto group, followed by Schiff base formation and an aldol
condensation of the C-6 and C-1 carbons to yield 2-mygr
inosose-1-phosphate,and reduction to form.1—ery2:inositol-

1-phosphate. However, recent evidence presented by Sherman,

6

gt al.argues against the proposed Schiff base intermediate

in the synthase mechanism (49). It was demonstrated using

18

testis synthase that O was retained in mygeinositol syn-

thesized from De(5-180)-glucose-6-phosphate and further, no

180 into product was observed when the

incorporation of
synthase reaction was carried out in a medium enriched in
H2180. Sherman, gt al.8uggested that the synthase is

neither a type I or II cycloaldolase and that the cyclization
process may proceed through general base catalysis. Synthase
from N. crassa (50) and yeast (51) has been shown to be a

2+

type II cycloaldolase in which Zn is the participating

metal cation.

Review 9: Male Reproductive Tragt_Physiology. Spermatozoa
are formed in the seminiferous tubules of the testis and
are the end product of the spermatogenic function of this
tissue (52). In addition to its spermatogenic function,
the testis possesses an endocrine androgenic activity.
Testosterone is secreted from Leydig cells, cells located
in the intertubular tissue of the testis, and this hormone
determines the output of seminal fluids by the accessory
organs as well as secondary sex characteristics (53). Ser-
toli cells or "nurse cells" located in the intertubular
spaces, are cells containing prominent nucleoli and nuclei.
They secrete androgen binding protein in response to fol-

licle stimulating hormone and/or testosterone (54,55). Since
certain steps of spermatogenesis are dependent on testosterone

(56), androgen binding protein may have an essential role

7

in concentrating testosterone within the seminiferous tubule
fluid for binding by receptors on germ cells (54). Sertoli
cells have also been demonstrated to form aggregates surrounding
the apical end of the developing spermatid (57) and engage
in other interactions with the developing germ cells (58).
In addition to their role in spermatogenesis, Sertoli cells
adjacent to each other have been shown to form tight junctions
which are believed to be the basis for the blood-testis
barrier (55. 57-59)-

The epididymis, attached to the testis through a group
of ducts (ductuli efferentes), serves as a site for the
concentration, maturation, and storage of sperm. Sperm
passage from the testis into the epididymis varies from a
few days to several weeks, depending on the species, and
depends on the peristaltic movement of the seminiferous
tubules (60). There is no direct evidence that the spermatozoa
move into the epididymis under their own locomotion. Sperm
are concentrated within the epididymis as a result of reab-
sorption of as much as ninety-nine percent of the fluid
produced by the testis. In this process sodium chloride
is absorbed; however, glutamic acid and mygrinositol are not
appreciably taken up by the epididymis(61). Phosphatidyl-
choline, sialic acid, and carnitine are secreted into the
lumen of the epididymis. These substances are most likely
incorporated into the maturing sperm as they undergo changes
in membrane characteristics and increase in specific gravity
and fertilizing capacity (52). Sperm remain in the epididy-

mis in a non-motile state until ejaculation. The substrate

used by sperm for basal metabolism during this non-motile
period is not known with certainty, but is thought to be
endogenous phospholipid (62).

After travelling from the epididymis by way of the
ductus deferens, sperm sequentially reach the seminal vesicles,
prostate gland, and bulbourethral glands (Cowper's glands),
known collectively as the accessory glands of the male repro-
ductive tract. While the physiology of the accessory glands
is not well understood, all are known to secrete fluids into
the lumen of the sperm-carrying ducts which both dilute
and facilitate the transport of sperm.

The seminal vesicle contributes substantially to the
volume of the ejaculate by a thick secretion rich in fructose,
the primary energy source of the sperm, and containing
considerable amounts of mygrinositol (52). In addition, the
vesicular secretion contains riboflavin and other flavins
which give the secretion a yellowish appearance.

The secretion from the prostate is slightly acidic and
contains amylase, -glucuronidase, several proteolytic
enzymes and fibrinolysin, enzymes thought to be involved in
the mechanism of semen coagulation (52). It is also the
main source of citric acid and acid phosphatase of the semen.
The secretion from the Cowper's glands is rich in sialoproteins
in some species which serve to coagulate sperm after
ejacultion. In rodents, coagulating glands located on the
surface of the seminal vesicles are the source of a

secretion which is believed to be important in the coagulation

9

process. In addition, in rodents, the coagulating gland
secretion is a main source of fructose, together with the
prostate secretion, rather than the seminal vesicles as in

other mammals.

Role _§ myo-Inositol i t e Male Reproductive Tract. Interest

-——

 

in the function of myg-inositol in the male reproductive
tract followed the observation by Mann (63,64) that the
dialyzate from boar seminal vesicle secretion contained

from 40 to 70 percent mygeinositol, thus making it the richest
source of the free cyclitol in nature. Semen from the boar
was also found to have a high mygsinositol content (382-625
mg/dl) (65). Semen from other species has considerable
concentrations of mygeinositol including human (50-60 mg/dl)
(65), bull (25-46 mg/dl) (65), and ram (14-20 mg/dl) (66).
Other investigators showed that mygyinositol occurs in the
testis fluid of the ram at a level of over 100 times that
found in the peripheral plasma (67,68). These high levels

of mygeinositol in the seminal vesicle secretion, semen,

and testis fluid were originally suggested to play a role

in the maintenance of osmotic equilibrium in the seminal
plasma, a fluid which generally contains less sodium chloride
than other body fluids (64,69).

Subsequently Eisenberg and Bolden characterized an
enzyme system in the testis responsible for the biosynthesis
of mygrinositol (13). However, they demonstrated that the
epididymis, seminal vesicle, and seminal fluid had only a

slight capacity for the biosynthesis of the cyclitol. In

10

addition, the testis was found to store little mygrinositol,
while the accessory organs, having only slight biosynthetic
ability, maintained higher concentrations of the cyclitol.
These authors noted that myg-inositol levels increased from
testis to epididymis to seminal vesicle and based on this
evidence, it was postulated that mygrinositol may play a role
in the maturation of spermatozoa as they migrate from the
testis through the epididymis, finally mixing with the
seminal fluid at ejaculation.

The high levels of mygeinositol found in the accessory
organs,in spite of low biosynthetic capabilities, suggested
an ability of these tissues to concentrate inositol from
the blood. Lewin and Sulimovioi (70) demonstrated in rats
that intraperitoneally injected radioactive mygrinositol was
accumulated from the blood into the coagulating gland and
seminal vesicle within two hours, and the prostate, epidid-
ymis, and ductus deferens concentrated mygeinositol at a lower
rate. In addition, mygeinositol uptake by the accessory
organs is thought to be under androgen control (71). In
contrast to the accessory organs, mygrinositol is synthesized
in the testis from glucose in preference to the utilization
of preformed inositol present in the blood plasma (70,72);
therefore, a testis-blood barrier for mygeinositol is
thought to exist.

The site of mygeinositol synthesis within the testis was
originally suggested by Eisenberg (16) to be at the level
of the seminiferous tubule. Subsequent studies by Voglmayr

and White (73) indicated that testicular spermatozoa have

11

a capacity for endogenous biosynthesis of the cyclitol and
suggested that this process may occur in the cytoplasmic
droplet of the testicular spermatozoan. However, the bio-
synthetic activity of these sperm is unlikely to entirely
account for the inositol concentration observed in the testis
fluid (68) since inositol levels considerably in excess of
blood plasma have been observed when sperm numbers were

less than 0.01% of normal (74). In contrast to testicular
spermatozoa, epididymal spermatozoa are unable to synthesize
inositol (16,73); the regulatory or developmental process

by which inositol biosynthesis in spermatozoa is controlled
has not been elucidated. In addition, the metabolic role

of mygrinositol synthesized by testicular spermatozoa is not
clear. The endogenous inositol may be involved in resynthesis
of phosphatidylinositol which is utilized by the spermatozoa
during their period of maturation in the epididymis (18),
presumably after hydrolysis and oxidation of the fatty acid
components (9). Another possible role is that endogenous and/
or extracellular mygeinositol servesas a possible source of
glucose for the spermatozoa since Posternak, gt al-(lO)
demonstrated that there is partial conversion of radio-
actively labelled inositol into labelled glucose. Also, extra-
cellular mygeinositol has been found to be oxidized in

small but significant amounts by both testicular (73) and
ejaculated spermatozoa (75). Exp-Inositol has also been
proposed to be essential to the maintenance of the cellular

integrity of testicular spermatozoa (11) since it was ob-

served that mygrinositol biosynthesis was significantly

12

depressed three to four days after cryptorchidism of the
testes; lowered o-inositol levels in the testes were
paralleled by germ cell loss.

Although the exact biochemical role of myprinositol has
yet to be clearly defined in the male reproductive system,
recent evidence has suggested that gypsinositol levels in
human seminal fluid may be of diagnostic importance. Lewin
and Beer (76) have shown that if the myprinositol content of
seminal fluid drops from its normal value of about 500 ug/ml
to a level of 100 ug/ml or less, this may be indicative of
prostatitis. In contrast, if the seminal fluid contains
1000 ug/ml myg—inositol or more, it may indicate that the
ejaculate consists mainly of prostatic fluid, a condition

which may be associated with low fertility.

Additional Proposed Rplg§_apg Metabolism p§_mypelnositol.

In addition to the above roles of myg-inositol in nutrition
and the male reproductive tract, a number of other functions
have been proposed for this cyclitol. myprlnositol has
been shown to be a substrate for the synthesis of 61fl-
galactinol in rat mammary gland and milk (77), certain
indole acetic acid esters in plants (78), phytic acid and
specific uronic acids and pentoses in plants (79) , and

the antibiotics streptomycin (80) and bluenomycin (81).

In addition, o-inositol has been identified as a cofactor
in the biosynthesis of the polysaccharides verbascose (82)
and stachyose (83), functioning as an acceptor of galactosyl

residues. Several investigators have demonstrated that

13

that mypeinositol functions as a lipotrophic agent in
certain types of fatty livers (84-88). Other proposed phy-
siological roles of myg—inositol include an inhibitor of
mitotic poisons (89), a stabilizer of microtubules (90), and
an effector of cell morphogenesis and cytogenesis (91).
In addition, the myprinositol containing phospholipid, phos-
phatidylinositol, has been suggested to be involved in
post-synaptic events in the sympathetic nerves of the brain
(92,93) and in the contractile mechanism of mitochondria (94).
Several investigators have studied the catabolism of pyp:
inositol. Charalampous originally suggested that two EMS:
inositol oxygenases, one producing Q: and the other producing
Leglucuronate existed in rat kidney homogenates (95,96).
Thonet and Hoffmann—Ostenhof later refuted the claim that
two oxygenases were present in the kidney and determined
that only the enzyme producing Qrgluronate from Exp-inositol
was present (97). It is now generally accepted that pyg-
inositol is catabolized by Deglucuronate formation through
mypeinositol oxygenase, followed by formation of stylulose-
5-phosphate production through the glucuronate—xylulose
pathway (98,99). Xylulose-5-phosphate can be transformed
to glycolytic intermediates through pentose phosphate
interconversions and subsequently to glucose, C02 or other

products (98).

10.

11.

12.

13.

14.

15.

16.
17.
18.

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CHAPTER I

CHARACTERIZATION OF myo—INOSITOL CONTENT
OF FETAL TISSUES FROM
ETHANOL-FED AND myg—INOSITOL DEPRIVED, ETHANOL-FED DAMS

ABSTRACT

The effects of ethanol consumption by pregnant rats
on fetal myp—inositol levels both during adequate and pyg-
inositol deprived dietary states were investigated. Ethanol
feeding was used in an attempt to decrease the fetal liver
cytosol NADf/NADH ratio to determine the effect of redox
state on 3p_y3y6 regulation of fetal myprinositol biosynthesis.
However, ethanol consumption by dams did not significantly
alter the redox state of the fetal liver under the conditions
and feeding intervals tested, nor were m o-inositol levels
in the fetal liver significantly changed.

Dams fed a myg—inositol adequate diet containing 10%
(v/v) ethanol for a seven day interval during their gestation
periods, did bear fetuses having blood plasma glucose and
mypeinositol concentrations significantly lower (P<.05) than
control levels. However, no alterations in fetal tissue
mypeinositol levels were observed. In another experiment,
fetal brain pyprinositol levels were significantly lower
(P<.05) than control values in fetuses from rats maintained
on 28% (v/v) ethanol and a myprinositol free diet for a
period of three to four weeks before pregnancy and throughout
gestation, but no perturbationscf fetal pypeinositol contents

were observed in either blood plasma or other tissues analyzed.

20

21

Pregnant rats fed a pypeinositol free diet and either 30%
(v/v) or 35% (v/v) ethanol during the last fourteen days

of gestation had a significantly elevated (P<L05) plasma
mypeinositol concentration (two-fold and four-fold, respec-
tively) compared to control dams. However, in this experi-
ment no alterations in fetal tissue or plasma pyg—inositol
contents were detected. The increased maternal plasma
inositol levels were also paralleled by increased maternal
liver free m o-inositol content, significantly higher (P<.05)

than control values.

INTRODUCTION

The role of myQ-inositol in the mammalian fetus and
neonate has been under investigation by several laboratories
since the original observation by Offergeld (1) that the

o-inositol concentration of human fetal blood exceeded that
of maternal blood by nearly ten-fold, suggesting an
important function for mypeinositol in development. Fetal
blood plasma of rats (2) and other mammals (3,4) have also
been shown to be from seven to eight fold higher in inositol
than maternal plasma, supporting this original finding. In
addition, plasma m o-inositol levels have been found to
remain up to four fold higher than maternal plasma concen-
trations for up to sixteen days postpartum (2). Fetal mype
inositol biosynthesis was suggested as responsible for
elevated fetal blood pypeinositol levels after it was observed
that the cyclitol was present in the perfusate from fetal

sheepljver (5) and rat fetuses or their isolated livers (6).

22

Later studies revealed that fetal rat liver contained high
activities of Qrglucose-6-phosphate: Lemyprinositol-l-phos-
phate synthase (EC 5.5.1.4) and erygeinositol-1-phosphates
Lrpyprinositol phosphatase (EC 3.1.3.25) (2).

Although little is known about the 3p_y3y6_regu1ation
of the mygeinositol biosynthetic enzymes in fetal tissues,
regulation may in part depend on the redox state of the cell
cytosol. The redox state may play a role since the synthase
from a number of sources has been shown to have an absolute

requirement for NAD+ 3p vitro, including rat mammary gland

 

(Km=0.5 mM) (7), rat testis (Km=0.5 mM) (8), N. crassa
(Km=1.90 mM) (9,10), and yeast (Km=1.50 mM) (11,12).

Since the role of the cell redox state in mypr inositol
biosynthesis has not been established 3p,y3y6, especially
in terms of the developing fetus and its apparently high
requirement for pygeinositol, studies were conducted to
alter the cytosol NADf/NADH ratio of the fetus. Maternal
dietary ethanol was utilized in an attempt to lower the
fetal cytosol NADf/NADH ratio and thus, the rate of fetal
pygeinositol biosynthesis.

These experiments may also be of interest in the
study of the biochemistry and metabolism in the clinically
observed "fetal alcohol syndrome" (13). This syndrome
has been recently detected in infants born to alcoholic

mothers and is manifested by fetal growth retardation,

morphological and cardiac anomalies, and brain dysfunction (13).

23

MATERIALS AND METHODS

Reagents. The following materials were obtained from the
indicated sources. Choline hydrochloride, phthalylsulfa-
thiazole, oxidized and reduced nicotinamide adenine dinuc-
leotide (NAD+ and NADH), and beef heart lactate dehydrogenase
(EC 1.1.1.27) from Sigma Chemical Company; vitamin-free
casein, m o-inositol, 0(-cellulose, Wesson mineral salt

mix and vitamin fortification mixes from Nutritional Bio-
chemical Corporation; lactalbumin hydrolysate from Gibco
Diagnostics; Mazola pure corn oil from Best Foods; U.S.P.
quality 100% ethanol from IMC Chemical Group, Inc.r*-methyl
mannoside from General Biochemicals, Inc.; 3% OV-l (w/w)

on Chromosorb W (100-200 mesh) from Applied Science Labor-
atories, Inc., and trimethylchlorosilane and hexamethyl-

disilazane from Pierce Chemical Co.

Animal Q363§_666_Handling. For all experiments, animals
were housed individually at 22 degrees in polycarbonate
cages with wood shavings unless otherwise noted. The
animal environment had a light cycle of 12 hours (6:00 a.m.
to 6:00 p.m.).

Experiment 2, In Experiment A timed pregnant Holtzman
(Madison, WI) rats (250-300 g) were fed a low fat, nutri-
tionally adequate liquid diet containing 10% ethanol (v/v)
and 0.5% (w/w) pyp—inositol during days 13 through 19 of
their gestation periods. This diet (Table I) was a modifi-
cation of the low fat liquid diet introduced by Rietz (15).

A control diet, identical with the ethanol containing diet

TABLE I

Experiment A Diet Composition

 

 

Component ancentration
o by welght)
Dextrose 39-4
Lactalbumin Hydrolysate 26.6
Ethanola 22.8
Salt Solutionsb 5.1
Mazola Corn Oil 2.3
Tween - 80 1.6
Vitamin MixC 1.2
Citric Acid 0.5
myp-Inositol 0.5

 

aControl diets did not contain ethanol; a total of

138 g of dextrose was substituted for 100 ml of
ethanol per liter of diet. Both ethanol containing
and control diets had a caloric value of 1.58 kcal/ml.

bThe salt solutions together consisted of (% by weight):
49.363% CaClZ'ZHZO, 39.420% KH2P04, 8.890% NaCl. 1.138%
MgSOu-7H20, 0.709% ferric ammonium citrate, 0.278%
CuS04-5H20, 0.137% Mnsou-Hzo, 0.0580% ZnCl 0.006%

KI, and 0.0005% (NH4)6M0 4-4H20.

2.
702
0The vitamin mix consisted of (g/kg): Vitamin A ester
(palmitate and acetate) concentrate (200,000 U/g), 4.5;
Vitamin 03 (400,000 u/g). 0.25;¢(-tocopherol, 5.0;
ascorbic acid, 45.0; choline chloride, 75.0; and men-
aquinone, 2.25. Additional vitamins (mg/kg):
p—aminobenzoic acid, 5.0; niacin, 4.5; riboflavin, 1.0;
pyridoxine-HCl, 1.0; thiamine-H01, 1.0; calcium
pantothenate, 3.0; biotin, 20.0; folic acid, 90.0;

and Vitamin B12, 1.35.

24

25

with the exception that dextrose was isocalorically sub-
stituted for ethanol, was fed to a second group of timed
pregnant rats. All rats were in synchronous pregnancy,
having been determined to be sperm positive on the same day
by the Holtzman Company. Control rats were initially paired
with alcohol fed rats on the basis of body weight and each
pair was fed isocalorically throughout the experiment. Total
calories consumed per day by each alcohol fed rat were fed
to the corresponding control dam on the following day. On
day 20 of gestation, (1 day prepartum), fetuses were excised
by Caesarean section from ether anesthetized animals and
examined for gross anatomical anomalies. Fetal livers

from each litter were collected, pooled, and stored at ~80
degrees. Fetal brains were similarly pooled and frozen.
Maternal and fetal blood samples were collected in heparinized
hematocrit tubes, centrifuged, and the plasma stored at -80
degrees.

Experiment 2. In the second experiment 15 Holtzman virgin
rats (200-220 g) were given 30 g/100 ml drinking water
(27.5% ethanol v/v) and an otherwise nutritionally adequate
solid diet containing no myg—inositol1 and 0.5% (w/w)
phflmdylsulfathiazole for a period of 3 to4vweeks before
pregnancy in a modification of the protocol described by

Tze and Lee (16). The solid diet2 contained phthalylsulfa-

 

1Contained less than 0.001 mg free myo-inositol per 100 g

diet by gas chromatographic analysis.

The diet consisted of (% by weight): 62.4% dextrose, 20.0%
casein, 10.0% corn oil, 4.0% Wesson mineral salts, 2.0% mype

Exxfitol free vitamin preparation, 1.0% -cellulose, 0.5%
phthalylsulfathlazole and 0.1% chollne hydrochlorlde. The
caloric value was 4.2 kcaL/g.

2

26

thiazole in order to prevent the possible contribution of
of pypyinositol to the diet by intestinal flora. An iso-
calorically fed control group of 8 rats received the same
solid diet and water. The average number of total calories
consumed by the alcohol fed rats per day was fed to each
of the rats in this control group on the following day.

In addition a second control group of 7 rats was fed the
same solid diet and water 66 libitum. After 3 weeks on their
respective diets, rats were mated. At mating three females
were placed into a standard polycarbonate breeding cage
overnight with one male of the same strain and age. Only
water was available to the rats during mating. Vaginal
smears were taken the following morning and no later than
12 hours after introduction to the breeding cages. The
presence of spermatozoa in vaginal smears was taken as

the positive criterion for pregnancy. All rats were
returned to their respective cages and diets during the day
and sperm negative rats were again placed into breeding
cages at night for up to six successive nights or until
pregnant. On day 20 of gestation, rats were ether anes-
thetized, fetuses quickly examined, and the fetal livers
from each litter rapidly excised, frozen by immersion

in liquid nitrogen, pooled, and stored at -80 degrees.
Fetal brain and blood samples were collected and stored

as before. Maternal blood samples were taken by heart
puncture using a heparinized syringe and blood was cen-

trifuged and plasma frozen at -80 degrees.

27

Experiment 6, Holtzman timed pregnant rats (250-300 g) were
maintained for 14 days of their gestation periods (days 6
through 19) on a solution of either 30% (v/v) or 35% (v/v)
ethanol and the same myg—inositol free, phthalylsulfathia-
zole containing diet utilized in Experiment B. A group of
control dams received the solid diet and were isocalorically
pair fed with ethanol fed rats. On day 20 of gestation
fetal brains, blood, and liquid nitrogen frozen fetal

livers were collected and stored as before, after rapid
weighing and morphological observation of each fetus.
Maternal blood was collected by heart puncture and plasma
obtained was stored as previously described. In addition

maternal livers were removed and stored at -80 degrees.

Tissue 666 Plasma EEEE myo-Inositol afli Glucose Determinations.
Up to 0.1 g tissue or 40 ul plasma were deproteinized by

the method of Somogyi (17) after prior addition of 30 ug or

14 ug of the internal standard.«amethylmannoside, respectively.
In all deproteinizations the ratio of tissue or plasma to

the balanced 5.0% ZnSOu and 0.3 N Ba(0H)2 Somogyi reagents

was 1 to 7. Following centrifugation at 3200 rpm for 5 min

to remove precipitated protein, supernatants were deionized

by filtration through approximately 1 g of MB-3 resin, and
then taken to dryness using a rotary evaporator. Tissue or
plasma samples were trimethylsilylated with 150 or 100 ul
respectively, using a mixture of pyridine, hexamethyldi-
silazane, and trimethylchlorosilane (5s 2: 0.5, v/v/v). After

overnight incubation at room temperature, the sugar and

28

cyclitol derivatives were quantitated by gas-liquid chrom-

atography according to the method of Wells, 63 63.(18).

Perchloric A636 Tissue Extract Preparation. Tissue extracts
were prepared according to the method of Williamson, 63 23.
(19) with several modifications. Liquid nitrogen frozen
tissue was pulverized using a mortar and pestle at dry ice
temperature; 1.0 g of frozen tissue was weighed into a 15 x
100 mm plastic centrifuge tube standing on dry ice and con-
taining 1.75 ml of frozen 30% (w/v) H0104. The tubes were
then placed on ice and allowed to slowly thaw until all
tissue had been extracted (20 min). Following homogeniza—
tion at 4 degrees using a motor driven teflon pestle, 0.69 ml
of water were added and the homogenate was centrifuged at
15,000 rpm in a Sorvall SS34 rotor for 15 min to remove
precipitated protein. The resulting supernatant was ad-
justed to pH 5-6 with 20% (w/v) KOH and the resulting KClO“
precipitate removed by centrifugation as above. The extract
was shaken with acid washed Florisil (activated magnesium
silicate, 0.1 g/ml extract) for 30 seconds to remove en-
dogenous fluorescence due to flavins, recentrifuged, and

frozen immediately at —80 degrees.

L-Lactate Determinations. Lactate content of perchloric

acid tissue extracts was determined fluorometrically through
a modification of the method of Gutmann and Wahlefeld (20).
Assays were conducted in 3 ml, 10 x 75 mm borosilicate Dispo
culture tubes. Each reaction mixture contained the following

components at the indicated final concentrations in a total

29

volume of 1.5 ml: 0.1 M glycine buffer, pH 10.0; 50 mM
hydrazine, pH 10.0; 133 uM NAD+, and up to 6.7 uM lactate.
Initial fluorescence measurements were taken at room tem-
perature with a Farrand Ratio Fluorometer 2 equipped with

a primary filter having an excitation optimum at 340 nm

and a secondary filter with an emission maximum at 460 nm.
Reactions were initiated by the addition of beef heart
lactate dehydrogenase (60 ug protein). Samples were
incubated at room temperature for 15 minutes and the increase
in fluorescence due to NADH production determined. Linearity
of the assay was verified by using two different volumes

of each extract. Fluorescence changes were calibrated by
measuring the increase in fluorescence with the addition

of known amounts of lactate and NADH.

Pyruvate Qeterminations. Pyruvate levels of perchloric

acid tissue extracts were determined fluorometrically

by the method of Passonneau and Lowry (21) with minor
modifications. Each reaction tube contained the following
components at the indicated final concentrations in a total
volume of 1.5 ml: 150 mM imidazole buffer, pH 7.0; 3.33 uM
NADH, and up to 2.0 uM pyruvate. After determining the
initial fluorescence using the same conditions as described
in the lactate assay, reactions were initiated by the addition
of beef heart lactate dehydrogenase (5.75 ug protein). After
incubating at room temperature for 20 minutes, the decrease
in NADH fluorescence was measured. Fluorescence changes

were calibrated by measuring both the decrease in fluores—

cence with the addition of a known amount of pyruvate and

30

the increase in fluorescence with the addition of a known

quantity of NADH.

Calculation 63 NADIZNADH Ratio. The cytosolic NADi/NADH
ratio was calculated by the method of Krebs, §£.al'(22)-
Upon rearranging the equilibrium constant expression for
the cytosolic lactate dehydrogenase system, the following
relationship is obtained:

NAD+/NADH = pyruvate/lactate x 1/K,

where K is equal to 1.11 x 10'“

at pH 7.0, ionic strength
0.25, and 38 degrees, and takes into account the hydrogen

ion concentration.

tatistics. Statistical analyses were carried out using

r

either two-tailed t-tests or paired t-tests as indicated (23).

RESULTS

Expg§3ment 2, Table II presents data on the litter size,
maternal diet consumption, and maternal weight profile for
the 10% (v/v) ethanol fed and pair fed control dams of
Experiment A. Caloric intake for both groups was comparable.
During the 7 day experiment (diets were fed during days 13
through 19 of gestation), ethanol fed rats consumed 25.7 i
3.1 ethanol calories/day, which accounted for 34.9 i 0.4%
of the total calories consumed. No significant differences
were noted for either the average maternal weight increase
during the experiment or for the average litter size of

the two groups. In addition, 10% (v/v) ethanol feeding
for the 7 day interval did not appear to affect the number
of successful pregnancies; 9 out of 12 sperm positive
ethanol fed rats and 8 out of 12 control diet fed sperm
positive rate actually were pregnant. There was no evi-
dence of resorptions from both groups not bearing fetuses;
most likely these rats were not pregnant initially.

Table III shows the myprinositol levels of selected
fetal tissues and blood plasma, as well as fetal plasma
glucose concentrations for the 10% (v/v) ethanol fed and
control diet fed groups. Plasma pyp-inositol concentrations
were 1.6 fold lower (P(0.1) in fetuses from the ethanol
fed dams than for fetuses from control dams. However, no
significant differences existed between the two fetal

groups with respect to free myo-inositol levels in brain

31

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32

TABLE III

Experiment A: Selected Fetal Tissue

and Plasma Glucose Concentrations

o-Inositol

a

 

Fetuses of

Fetuses of

 

 

 

Tissue 10% Ethanol Control Diet

Fed Dams Fed Dams
Brain 3.38 i 1.04 3.42 i 1.49
Liver 0.410 3 0.140 0.360 3 0.080
Plasma 0.194 : 0.084b 0.304 1 0.130
Plasma b

 

aBrain and liver myo-inositol values are expressed

as umoles/g wet weight.

glucose levels are expressed as mmoles/l.
values are the mean 1 standard deviation.
and glucose assays were as outlined in the Methods

Plasma pyp-inositol and

All
Inositol

section. n = 8, except for plasma glucose where

11:70
b

test.

33

Significantly different from controls (P<.01) by
two-tailed paired t-

34

and liver. Fetal plasma glucose concentrations were 1.9
fold lower (P<.01) in the ethanol fed group than for the
control diet fed group, paralleling the difference in
plasma mypeinositol concentrations. Although not shown

in Table III maternal plasma m o-inositol and plasma glucose
concentrations were not significantly different for the
ethanol fed and control diet fed dams. Ethanol fed and
control diet fed maternal plasma pypyinositol levels were
68.2 : 18.1,uM (n=9) and 74.3 i 20.4 nM (n=8), respectively.
Plasma glucose from ethanol fed dams was 8.50 i_1.60 mM
(n=9) and from control diet fed rats 7.36 i 2.3 mM (n=8).

Experiment 2. Table IV presents data concerning the
pregnancy rate, litter size, and diet consumption of ethanol
fed and control rats of Experiment B. In this experiment
the effects of a long-term, chronic ethanol consumption

on fetal myp-inositol metabolism were determined. As shown,
rats maintained on 27.5% (v/v) ethanol and a mygrinositol
free diet (as determined by gas—liquid chromatography) had
a lower percentage of successful pregnancies as compared

to group fed and 66 libitum control rats under the same
mating conditions. In addition ethanol fed'dams had a
significantly smaller (P(.05) litter size than the group
fed control dams. Although daily caloric intakes by the
alcoholic and the isocalorically fed control groups were
comparable, the average weight gain during the experiment
for the ethanol fed rats was only 55% of that recorded for

the group fed controls, probably a reflection of dehydration

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35

36

and the smaller average litter size of the ethanol fed dams.
The ethanol fed rats consumed an average of 23.2 i 2.7
ethanol calories/day or 24.9 i 2.9% of their average total
daily calories.

The maternal body weight profile for the three groups
of experimental animals used in Experiment B is shown in
Figure 1. Ethanol fed rats lost weight during the first
five days of their diet; however, after this initial loss,
they gained weight at approximately the same rate as did
the isocalorically fed control rats, both before and during
pregnancy. 22 libitum fed control rats showed a rate of
weight gain slightly larger than the control group iso-
calorically fed with the ethanol fed rats.

The free myprinositol content of several fetal tissues
and plasma from ethanol fed and control dams is presented
in Table V. Fetal brain mypeinositol levels were signifi-
cantly lower (P<.05) in fetuses from ethanol fed dams than
in fetuses from isocalorically fed control dams. No sig-
nificant differences were found between the experimental
groups with respect to fetal liver or fetal plasma pyg-
inositol levels.

Also shown in Table V are the cytosolic ratios of
NADI/NADH for fetal livers from the three experimental
groups, as determined by the fetal hepatic substrate levels
of the NAD+-linked lactate dehydrogenase system. Fetal
liver from ethanol fed dams showed a slight reduction in
the NADfi/NADH ratio as compared to the isocaloric control

group; however, differences were not significant at the

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38

39

P=.05 level.

Although fetuses were not weighed prior to dissection,
it was generally observed that fetuses from the ethanol fed
dams were smaller than control fetuses from either group.

In addition, fetuses from alcohol fed mothers appeared

more emaciated than control fetuses. No gross malformations
or resorptions were observed in fetuses from any of the
experimental animals although some ethanol fed dams exhibited

fatty livers.

2xperiment 2. The effects of ethanol consumption (either

30% (v/v) or 35% (v/v)) on fetal tissue myp—inositol metabolism
were investigated during the latter part of pregnancy (days

6 through 19). Table VI presents data on ethanol consump-
tion and litter size of pregnant rats in this experiment,
along with the corresponding average body weights and hepatic
cytosolic NADf/NADH ratios of fetuses from these dams. As
shown, ethanol consumption was comparable for both-the 30%
and the 35% ethanol fed dams. Ethanol calories amounted

to 33.5% and 30.7% of the total calories consumed per day

by the 30% and 35% ethanol fed dams, respectively. No
significant differences were observed in the average litter
sizes of ethanol fed dams in comparison to those of iso-
calorically pair fed or 22 libitum fed control dams. However,
alcohol fed dams bore fetuses having significantly lower
(P<.01) body weights than fetuses for either pair fed or

22 libitum fed control dams. In addition, fetuses from
alcohol fed dams appeared to be of smaller proportions

and generally emaciated compared to control fetuses: however,

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41

no gross morphological anomalies or resorptions were noted.
It was also observed that alcohol fed dams had both fatty
livers and fatty placentas in most cases. As shown in
Table VI, no significant differences were found between
the liver cytosolic redox states of fetuses from ethanol
fed dams and those fetuses from control dams.

The pypeinositol content of maternal and fetal tissues
are presented in Table VII. Maternal plasma from 30% (v/v)
and 35% (v/v) ethanol fed dams was two fold and four fold
higher in pyp-inositol content, respectively, than plasma
from either pair fed or 22 libitum fed control rats.
Maternal liver pypeinositol was significantly higher (P<L05)
in 30% (v/v) ethanol fed dams compared to isocalorically
fed control dams. Liver myp—inositol was also significantly
higher (P<.02) for 35% (v/v) ethanol fed dams than either
pair fed or 62 libitum fed control dams. No significant
differences were observed, however, in fetal plasma, liver,
or brain myp—inositol contents between alcohol fed and con-

trol groups.

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49

 

DISCUSSION

Feeding of ethanol to pregnant rats at the dosages
and durations used in this study had a minimal effect on
the fetal liver cytosolic NADt/NADH ratio and fetal liver
m o-inositol content. However, ethanol consumption was
shown to perturb fetal brain and plasma myg—inositol levels
under certain conditions.

The absence of an ethanol effect on the fetal cytosolic
redox state does not imply a placental barrier to ethanol
since this drug has been demonstrated to be present in
fetal blood at the same levels as in maternal blood (24-28).
However, it is possible that the majority of ethanol consumed
was metabolized by the maternal liver, thereby significantly
decreasing ethanol exposure to the fetus. Furthermore,
alcohol dehydrogenase activity of fetal rat liver is reported
to be low; approximately 150 nmoles/min/g at 3 days before
term, or one-sixth the activity found in adult rat liver (29).
Therefore, depletion of the cytosolic NAD+ pool through the
conversion of NAD+ to its reduced form during the alcohol
dehydrogenase catalyzed oxidation of ethanol to acetaldehyde
probably does not occur to a significant extent in fetal liver

or other fetal tissues i vivo. Although the NAD+ linked

 

acetaldehyde dehydrogenase activity of fetal rat liver homOg-
enates is reported to be fairly high (#9% of the activity
found in adult rat liver), several studies have shown that

the majority of maternal plasma acetaldehyde is oxidized

43

4#

by the placenta (30-32). Since the oxidation of acetal-
dehyde in fetal liver is limited, a significant decrease
in the cytosolic NADfi/NADH ratio due to the action of
acetaldehyde dehydrogenase would not be expected. Also,
since acetaldehyde dehydrogenase is mainly a mitochondrial
enzyme, acetaldehyde oxidation would be expected to have
only almhflmal effect on the cytosolic redox state.

It has been demonstrated in 22122 by Wells, g: _l.
that the activity of Q-glucose-o-phosphate: eryg-inositol-
1-phosphate synthase from adult rat brain is decreased by
10% when the NADfi/NADH ratio is reduced two fold from its
optimal ratio (33). Further, it was shown that a ten fold
reduction in the NADf/NADH ratio from the optimal ratio
resulted in only a 30% inhibition of mygeinositol formation.
While no direct comparison can be drawn between the adult
rat brain synthase and the fetal liver synthase, it is
probable that a considerable reduction in the NADfi/NADH
ratio is required for a significant decrease in in yizg,mygf
inositol biosynthesis.

Plasma mygeinositol concentrations were observed to
be significantly lower than control levels in fetuses from
rats fed 10% (v/v) ethanol and a nutritionally adequate
diet containing 0.5% (w/w) myg—inositol during 7 days of
their pregnancy (Experiment A). However, this reduction in
fetal plasma inositol was not accompanied by a decrease in
the mygrinositol content of any of the fetal tissues
measured. Plasma myg—inositol levels were not different

from control concentrations in fetuses from rats fed 27.5%

45

(v/v) ethanol for 3 to 4 weeks prior to and throughout
pregnancy and a solid diet free of myg—inositol (Experiment
B). However, fetuses from ethanol fed dams had lower myg:
inositol levels in the brain compared to control animals.
In a similar experiment in which either 30% (v/v) or 35% (v/v)
ethanol was fed to pregnant rats for 1# days during their
gestation periods along with a myg—inositol free diet, no
significant differences were observed in the fetal plasma
or tissue mygrinositol levels (Experiment C). However,
maternal plasma and liver free m o-inositol concentrations
were significantly greater than control values. These
observations may possibly be explained in terms of ethanol
effects on placental transport of mygyinositol, transport
of m o-inositol from fetal tissues to fetal plasma, phos-
phatidylinositol metabolism, or mygfinositol catabolism.
Further work in this research area might emcompass
defining the effects of ethanol on each of these processes
above. In addition, the mechanism of mygeinositol transport
across the placenta remains to be elucidated. Placental
transfer of mygrinositol from maternal to fetal blood is
probably of major importance since it has been estimated
to supply approximately #0% of the myg—inositol present
in the fetal plasma, the remaining 60% originating from
fetal tissue biosynthesis of the cyclitol (34). Since
placental transport of myg-inositol must occur across a
concentration gradient, such a process would most likely

involve active transport.

Investigation of the redox state regulation of myo-

46

inositol biosynthesis in fetal tissue will have to be
approached through means other than ethanol feeding due

to several complications. These include low fetal ethanol
exposure due to metabolism of ethanol by the maternal
liver, low ethanol oxidizing capacity of the fetal liver,
cytotoxic effects of ethanol, and alteration of a

significant number of metabolic processes with ethanol feeding.

10.

11.

12.

13.

14.
15.

16.
17.
18.

19.

REFERENCES

Offergeld, H., z. Geburtsh. Gynockol. 5Q, 189 (1906).

Burton, L. E. and Wells, W. W., DeveloE. Biol. 21,
35 (1974)-

Campling, J. D. and Nixon, D. A., J. Physiol. 1 6,
71 (1954)-

Nixon, D. A., g. Physiol. 112, 700 (1952).

Andrews, W. H. H., Ritton, H. G., Hugget, A. St. G.,
and Dixon, P. A., J, Physiol. 153, 199 (1960).

Nixon, D. A., Biol. Neonat. 12, 113 (1968).

Naccarato, W. F., Ray, R. E., and Wells, W. W.,
Arch. Biochem. Biophys. 164I 194 (1974).

Barnett, J. E. G., Rasheec, A., and Corina, D. L.,
Biochem. J. 131,21 (1973).

Mogyoros, M., Brunner, A., and Pina, E., Biochim.
Biophys. Acta 2&9, 420 (1972).

Pina, E., Saldana, Y., Brunner, A., and Chagoya, V.,
Ann. N.Y. Acad. Sci. 165, 541 (1968).

Chen, I. W. and Charalampous, F. C., J. Biol. Chem.
240. 3507 (1965)-

Charalampous, F. and Chen I. W., Meth. Enzymol. 9,
698 (1966)-

Jones, K. L., Smith, D. W., Ulleland, C. N., and
Streissguth, A. P., Lancet 1,1267 (1973).

 

 

Streissguth, A. P., Ann. N.Y. Acad. Sci. 223, 140 (1976).

Rietz, R., personal communication, University of
Nevada, Reno, Nevada 89557.

Tze, W. J. and Lee, M., Nature 252, 479 (1975).
Somogyi, M., J, Biol. Chem. 16g, 69 (1945).

Wells, W. W., Pittman, T. A., and Wells, H. J., Anal.
Biochem. 19, 450 (1965).

Williamson, D. H., Lund, P., and Krebs, H. A., Biochem.
J. 103, 514 (1967).

47

20.

21.

22.

23.

24.

25.

26.

27.

28.
29.

30.

31.

32.

33.

34.

as

Gutmann, I. and Wahlefeld, A. W. in Methods 9:
Enzymatic Analysis (Bergmeyer, H. U., ed.), Vol. 3,
p. 1464, Academic Press, Inc., New York (1974).

Passonneau, J. V. and Lowry, 0. H. in Methods 2:
Enzymatic Analysis (Bergmeyer, H. U., ed.), Vol. 3,
p. 1452, Academic Press, Inc., New York (1974).

Williamson, D. H., Lund, P., and Krebs, H. A., Biochem.

Zar, J. H., Biostatistical Analysis, Prentice Hall,
Inc., Englewood Cliffs, NJ (1974).

Kesaniemi, Y. A. and Sip el, H. W., Acta Pharmacol.
gt Toxicol. 31, 43 (1975).

Belinkoff, S. and Hall, 0., Amer. J. Obstet. Gynec.
52; 429 (1950)-

Chapman, E. R. and Williams, P. T” Amer. J. Obstet.
Gynec. 6;, 662 (1951).

Fetchko, A. M., Weber, J. E., Carrol, J. H., and
Thomas, G. J., Amer. J. Obstet. Gynec. 62, 662 (1951).

Dilts, P. V., Amer. J. Obstet. gynec. 102, 1018 (1970).

Raika, N. C. R., Koskinen, M., and Pekkarainen, P.,
Biochem. J. 103, 623 (1967).

Sippel, H. W. and Kesaniemi, Y. A., Acta Pharmacol. gt
Toxicol. 32. 49 (1975)-

Kesaniemi, Y. A., J. Obstet. Gynec. Brit. Commonwealth
81. 84 (1974b).

McIsaac, W. H. and Creaven, P. J. in Biolo ical Aspects
Q;_Alcohol (Roach, M. K., ed.), pp. 233-266, University

of Texas Press, Austin, TX (1971

Wells, W. W., McIntyre, J. P., Schlichter, D. J.,
Wacholtz, M. C., and Spieker, S. E., Ann. N.Y. Acad.
Sci. 165, 559 (1969).

Burton, L. E., Ph. D. Thesis, Michigan State University,
p. 38 (1976).

CHAPTER II

gyngNOSITOL BIOSYNTHESIS IN THE RAT TESTIS
DURING HYPERGLYCEMIA OR ETHANOL FEEDING

ABSTRACT

The effects of streptozotocin induced hyperglycemia and
prolonged ethanol feeding on gg,ngxg,mygrinositol biosynthesis
in the rat testis were examined. Acute hyperglycemia was evi-
dent in rats 72 hours after streptozotocin administration.
Testis m o-inositol contents were 2.7 fold higher in dia-
betic animals than in control rats injected with citrate buf-
fer. No changes were observed in the specific activities
of the m o-inositol biosynthesizing enzymes: however, hyper-
glycemic rats displayed elevated testis glucose and glucose-
6-phosphate levels, approximately 4 fold and 2 fold in excess
of control values, respectively. Insulin treatment of
diabetic rats resulted in the lowering of plasma glucose,
testis glucose, and testis glucose-6-phosphate to normal or
subnormal levels, but mygrinositol levels remained signifi-
cantly elevated compared to control animals, although slightly
lower than that observed for untreated diabetic rats. Testis
glucose and glucose-6-phosphate levels increased significantly
10 hours and 12 hours after streptozotocin injection, respec—
tively. However, testis myg—inositol content lagged and did
not increase appreciably until 24 hours following injection
of the drug. Streptozotocin diabetic rats had a significantly
decreased testis cytosolic NADf/NADH ratio compared to control

49

50

animals 72 hours after injection.

Feeding of 40% ethanol to male rats for a three day
period resulted in a small but significant decrease in testis
glucose, glucose-6-phosphate, and mygrinositol levels compared
to control animals, but no significant alterations in the
testis cytosolic redox state were observed. Subsequent work
utilizing an isocaloric paired feeding model failed to show
perturbations in the testis mygrinositol level or testis redox
state of rats fed 40% ethanol for two weeks.

The potential role of testis hexokinase distribution in
the regulation of mygrinositol biosynthesis in normal, dia-
betic, and ethanol intoxicated rats was investigated. No
significant differences in testis hexokinase distribution or
in the kinetic characteristics of the soluble and particulate
hexokinase activities were observed.

Testicular sperm counts and tubulin polymerization
in streptozotocin diabetic rats were not significantly dif-

ferent from control values.

INTRODUCTION

Eisenberg and Bolden (1) were the first to describe
an active system in the mammalian testis for the synthesis
of myg—inositol and postulated that the process occurred at
the level of the seminiferous tubule. Later Voglmayr and
White demonstrated that testicular spermatozoa were capable
of synthesizing Exp-inositol but indicated that it was unlikely
that spermatozoa were the only site in the testis for the

biosynthesis of the cyclitol (2). In spite of a high bio-

51

synthetic rate the testis stores little myg—inositol, while
the epididymis, which possesses only a slight capacity for
synthesis, maintains a much higher concentration of myg—inosi-
tol (3). Subsequent workers described a testis-blood barrier
for myg—inositol which did not exist for the epididymis or
accessory sex organs (4,5).

The synthase catalyzed conversion of glucose-6-phosphate
to inositol-l-phosphate has been identified as the rate
limiting step in Exp-inositol biosynthesis (6) and therefore,
a probable site for metabolic regulation of the pathway. In
fact, several in zitgg inhibitors of the synthase from several
sources have been reported including NADH (7-9). PYrophos-
phate (10), 2-deoxy-Qeglucose-6-phosphate (11), and 5-thio-Q-
glucose-6-phosphate (12). ;n ygzg studies with 2-deoxy-Q:
glucose demonstrated that this glucose analog was converted
to the 6-phosphate in mouse testis and liver:and myg—inositol
levels were significantly decreased in both tissues (12).
While mygeinositol has been detected in the urine of diabetics
(13,14), little is known of the effects of hyperglycemia on
in zizg mygrinositol biosynthesis in the testis, a tissue in
which glucose uptake is insulin independent (15). Therefore,
experiments were conducted using streptozotocin diabetic rats
to determine the effects of hyperglycemia on testicular
glucose, glucose-6-phosphate, and mygeinositol levels. In
addition, the effects of ethanol induced hypoglycemia and
a decreased cytosolic NADf/NADH ratio on mygrinositol metab-
olism in the rat testis were investigated. These studies are

of interest since alteration of testicular myq—inositol bio-

52

synthesis may be related to impairment of male reproductive
function in mammals during diabetes mellitus (16-22) and tes-

ticular atrophy and infertility in man during alcoholism (23-28).
MATERIALS AND METHODS

Reagents. The following reagents were obtained from the indi-
cated sources. Streptozotocin was the generous gift of the
Upjohn Co., Kalamazoo, MI. Iletin (bovine zinc-insulin) was
obtained from the Eli Lilly Co.; colchicine (3 Ci/mmole) from
New England Nuclear; dimethylsulfoxide (DMSO) from Fisher
Scientific Co.; 3. peroides p-hydroxybutyrate dehydrogenase
(EC 1.1.1.30), beef heart lactate dehydrogenase (EC 1.1.1.27),
yeast glucose-6-phosphate dehydrogenase (EC 1.1.1.49), E. ggrgr
ggngs citrate lyase (EC 4.1.3.6), pig heart malate dehydrogenase
(EC 1.1.1.37), yeast hexokinase (EC 2.7.1.1), oxidized and
reduced nicotinamide adenine dinucleotide (NAD+, NADH), oxi-
dized and reduced nicotinamide adenine dinucleotide phosphate
(NADP+, NADPH), ethylene glycol bis (fl-aminoethyl ether) N, N,
N',N'-tetra—acetic acid (EGTA), sodium guanosine triphosphate
(GTP), and N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic

acid (HEPES) from Sigma Chemical Co.: USP quality 100% ethanol
from IMC Chemical Group Inc.;«K-methylmannoside from General
Biochemicals Inc.; 3% 0V-1 (w/w) on Chromosorb W (100-200 mesh)
from.Applied Science Laboratories Inc.; and trimethylchloro-
silane and hexamethyldisilazane from Pierce Chemical Co.
eryg-inositol-1-phosphate was previously prepared in this

laboratory.

53

Animals Eng Qigtg. Male rats (200-225 g) of the Holtzman
strain (Madison, WI) were used in all studies. Animals

were fed a standard solid diet of Wayne Lab Blox (Allied
Mills, Chicago, IL) and water gg libitum.unless otherwise
noted. Exceptions included an experiment in which a group
of rats was maintained on the standard solid diet and a solu-
tion of 40% (v/v) ethanol for 3 days. In another ethanol
study control rats receiving the standard solid diet and
water were pair fed with rats receiving the same solid diet

and a solution of 40% (v/v) ethanol for 14 days.

Administration 2; Streptozotocin. Within 20 min before use,

a stock solution of 65 mg/ml streptozotocin in 100 mM ace-
tate buffer, pH 4.5, was prepared and kept on ice. Hyper-
glycemia was induced in male rats by injection of strep-
tozotocin (65 mg/kg) into the saphenous vein of ether anes-
thetized animals using a 1 ml plastic syringe equipped with
a30.‘gauge needle. Control animals received injections of
citrate buffer (1 ml/kg) under otherwise identical conditions.
Leg incisions were closed with metal wound clips following

all injections.

Administration 9: Insulin. Streptozotocin diabetic rats to
be injected with insulin were initially proven hyperglycemic
by analysis of blood glucose in samples collected in hep-
arinized capillary tubes from tail vein incisions (see below
for glucose assay). Exactly 72 hours after streptozotocin
injection, diabetic rats were divided into two groups; one
group receiving 40 units Iletin/kg (i.p.) and a second group

receiving an i.p. injection of an equal volume of physiological

54

saline. Following an interval of 4 hours after insulin
or saline injection, all rats were sacrificed and tissues

collected as described below.

Collection of Tissue Eng Plasma £9; myo-Inositol agg,Mgtgb~
9;; g Analysis. Rats were weighed and placed under light
ether anesthesia. The right testis of each animal was
rapidly excised, the epididymis and connective tissue de-
tached, and the testis immersed in liquid nitrogen within
15 seconds. The left testis was removed and placed on ice.
In some experiments, a portion of the liver was freeze-
clamped using liquid nitrogen cooled tongs and the tissue
was immersed immediately in liquid nitrogen. Blood was
collected by heart puncture using a syringe previously
rinsed with heparinized saline. Plasma was prepared
from whole blood kept at 4 degrees by centrifugation at

3200 rpm in a Brinkman desk top centrifuge.

Plasma and Tissue Glucose Assays. Plasma glucose levels
were determined on 10 ul samples using a Gilford 3500 Semi-
Automated Computer-Directed Analyzer and a Worthington/
Gilford reagent kit containing the enzymes hexokinase and
glucose-6-phosphate dehydrogenase. Tissue glucose deter-
minations were conducted on perchloric acid extracts of
liquid nitrogen frozen tissue using the same method and
including suitable blanks to compensate for endogenous

light absorption at 340 nm by the extracts.

Perchloric Acid Tissue Extract Preparation and Assays for

Metabolites. Perchloric acid extracts of liquid nitrogen

 

55

frozen tissues were prepared as previously described in
Chapter I. Glucose-6-phosphate, ATP, and citrate were ana-
lyzed by the methods of Lowry and Passonneau (29). Q-(-)-3-
hydroxybutyrate and acetoacetate were determined by the method
of Williamson and Mellanby (30). Pyruvate and Lelactate were

analyzed as previously described in Chapter I.

Calculation 2i Cytosolic gag Mitochondrial NADIZNADH Ratigg.
The calculation of the cytosolic and mitochondrial NAD+/NADH
ratios was done according to the method of Krebs, ££.§l-(31)
employing the lactate dehydrogenase and fl-hydroxybutyrate
dehydrogenase systems. In general,

NADf/NADH = (oxidized substrate)/(reduced substrate) x l/K,
where K = 1.11 x 10‘“ for the lactate dehydrogenase system,
and K = 4.93 x 10'2 for the fi-hydroxybutyrate dehydrogenase

system at 38 degrees, pH 7.0, and ionic strength 0.25.

Plasma agg_Tissue,myo-Ino§itql Determinations. The 919:
inositol content of plasma and tissue samples was determined
by gas-liquid chromatography by the method of Wells (32).
¢erethylmannoside was used as the internal standard in all

determinations.

Preparation 2: myo-Inositol Biosynthesizing Enzymes from

3;: Testis. Synthase was prepared according to the procedure
of Barnett, gt gl.(33) with minor modifications. Heat treat-
ment of the homogenate at 60 degrees was extended from

2 minutes to 10 minutes to insure inactivation of non-
specific phosphatases and phosphoglucoisomerase. The ad-

ditional 8 minutes of heat treatment was found to give

56

approximately 2 fold greater yields of the cyclase and phos-
phatase. This step was conducted prior to centrifugation

at 105,000 x g. The resolubilized 40% ammonium sulfate
precipitate was dialyzed overnight at 4 degrees against 4 l
of 50 mM Tris-acetate, pH 7.5, and 1 mM fl-mercaptoethanol,
and then centrifuged at 14,500 x g for 15 minutes. The super-
natant was assayed immediately for synthase activity. The
phosphatase was obtained from the supernatant fraction of
synthase preparations by adjusting the ammonium sulfate con-
centration from 40% to 60% saturation, dialyzing the resolu-
bilized precipitate against 4 liters of the buffer above, and

recentrifugation of the dialysate.

D-Glucose-6-Phosphate: L-pyo-Inositol-l-Phosphate Sypthase

 

Apsgy. The synthase assay was performed according to the
method of Barnett, pp al_(33). Inorganic phosphate liberated
from Lrgyg-inositol-1-phosphate by sodium periodate treat-
ment was assayed as described below. One unit of synthase
activity was defined as 1 nmole of mypeinositol-l-phosphate

produced per hour at 37 degrees.

L—myo-Inositol-1-Phosphate Phosphatase Assay. The phos-
phatase was assayed using the method of Eisenberg (6). The

reaction mixture was incubated at 37 degrees for 30 minutes
and was contained in a final volume of 0.5 ml 40 mM Tris-
acetate, pH 7.4; 100 mM KCl, 3 mM MgClz-6H20, 0.2 mM erypr
inositol-l-phosphate, and 15 ug of protein. The reaction
was terminated after 30 minutes by addition of 0.25 ml 20%

TCA. After removal of precipitated protein by centrifugation,

57

a 0.5 ml aliquot was analyzed for inorganic phosphate as
described below. A blank in which TCA was added prior to
the addition of the enzyme preparation was employed to com-
pensate for inorganic phosphate released by the action of
TCA and other background inorganic phosphate. One unit of
phosphatase activity was defined as 1 umole of phosphate

liberated from L-pyp—inositol-1-phosphate per hour at 37 degrees.

Determination pf_lnorganic Phosphate. Inorganic phos-

phate was assayed using a modification of the method of Ames
(34). A total of 2.1 ml of phosphate color reagent con-
taining one part 10% (w/v) ascorbic acid and six parts of
0.42% ammonium molybdate-4H20 in 1.0 N H2804 were added to
up to 0.9 ml of the sample to be assayed. After incubation
at 45 degrees for 20 min and cooling to room temperature,
absorbance due to the reduced phosphomolybdate complex

was measured at 700 nm using a Gilford Model 300 spectro—

photometer.

Protein Determinations. Protein was determined by the method

of Lowry, pp a;.(35) using bovine serum albumin as the standard.

2p;p_Feeding pf Ethanol Egg apd Control Raps. In order to
insure a comparable caloric intake for ethanol fed and con-
trol rats, a pair feeding model was implemented in one exper-
iment. Rats were fed a 40% (v/v) solution of ethanol (2.23
kcal/ml) and a commercial solid diet (Wayne Lab Blox, Chicago,
IL) (2.97 kcal/g) gg_libitum for two weeks and total and

ethanol caloric intakes were calculated daily. Ethanol solu-

tions were made available to animals through pint-sized

58

drinking bottles and suitable control bottles were included
to compensate for spillage and evaporation. Control rats
were initially paired with ethanol fed rats on the basis

of body weight and maintained on water and the same commer-
cial solid diet. However, caloric intake was restricted
for control animals to the equivalent number of calories
consumed on the previous day by the corresponding ethanol

fed rat.

Hexokinase Preparation. The preparation of hexokinase from
rat testes was a modification of the method of Wells, gt 3;.
(36). Testes from ether anesthetized rats were rapidly
removed, submerged in cold 0.25 M sucrose containing 1mM
,fi-mwrcaptoethanol, immediately minced with scissors, and
completely homogenized for 30 seconds at 65 volts using a
Tekmar homogenizer. The time between excision of tissue

and the initiation of homogenization was less than 20 seconds.
Samples were taken from either normal, streptozotocin dia-
betic or ethanol injected rats. For diabetic rats, strep-
tozotocin was administered 72 hours before sacrifice. Eth-
anol injected rats received 10% (v/v) ethanol at a dosage

of 2 g EtOH/kg intraperitoneally and were sacrificed 25
minutes after injection. An aliquot of the crude homogenate
was assayed for hexokinase activity and the remainder was
centrifuged at 40,000 x g for 10 minutes which provided

two fractions: the supernant containing soluble hexokinase
and the particulate,which was calculated from the difference

between soluble and crude homogenate activities.

59

Hexokinase Assay. Determination of hexokinase activity

was by a modification of the procedure of Hernandez and Crane
(37). Each reaction mixture contained 3.3 mM glucose, 6.7 mM
ATP, 6.? mM MgClZ, 40 mM potassium HEPES, pH 7.5: 10 mM 1-
thioglycerol, 0.64 mM NADP+, and 1 unit of glucose-6-phos—
phate dehydrogenase in a total volume of 1.0 ml. The reaction
was initiated by adding an aliquot of sample. NADPH formation
was followed at 340 nm using a Gilford 2400-S recording spec-
trophotometer at 30 degrees. One unit of hexokinase was

defined as 1 umole of NADPH formed per minute.

Kinetic Studies. Kinetic studies were conducted on solu—
ble and particulate hexokinase preparations from control
rat testis. Rates were determined by monitoring the forma-
tion of NADPH produced through the coupled hexokinase and
glucose-6-phosphate dehydrogenase reactions with time.

The K11? for ATP and glucose of the hexokinase preparations
were calculated from the appropriate kinetic data using

computer generated Hill plots.

Sperm Counts. Sperm counts were conducted according to

the method of Kirton, gp_a;.(38).

Determination pi Total and Polymerized Tubulin ip Testes.

 

Polymerized and depolymerized forms of tubulin in testes
were quantitated through the colchicine-binding assay of
Pipeleers, pp g;.(39). The depolymerized tubulin fraction
was prepared by homogenizing 0.3 g testes in 5 ml of a
microtubule stabilizing solution containing 50% glycerol,

5% DMSO, 0.5 mM GTP, 0.5 mM MgCl and 0.5 mM EGTA in 10 mM

2!
phosphate buffer, pH 6.95, followed by centrifugation at

60

100,000 x g for 45 min at 30 degrees. The amount of free
depolymerized tubulin was then determined by assaying an
aliquot of the resulting supernatant. The polymerized
tubulin fraction was obtained by resuspending the high

speed pellet in a tubulin depolymerizing solution containing

0.25 M sucrose, 0.50 mM MgCl and 0.50 mM GTP in 10 mM

2!
phosphate buffer, pH 6.95, followed by centrifugation at
100,000 x g for 45 minutes at 4 degrees. The resulting
supernatant was assayed by the colchicine-binding method

to determine polymerized tubulin content.

Statistics. Unless otherwise noted, statistical analyses

were performed using the two—tailed t—test (40).

RESULTS

Effects p§_Streptppotoc;p Induced Diabetes gpg Subsequent
Intraperitoneal Insulin Injection pp Testes pyg71nositol Levels.
The effects of streptozotocin induced hyperglycemia and
insulin-compensated hyperglycemia on the o-inositol content
of adult rat testes was examined. As shown in Table I, 72
hours after intravenous injection of streptozotocin (65mg/kg).
rats had blood plasma glucose concentrations significantly
higher (P(.001) than control animals which had been injected
with citrate buffer in sham operations. In addition, dia-
betic rats gained significantly less (P(.05) weight during
the 3 day experiment than did control animals. Although
caloric intakes for both groups were not measured, this
discrepancy in weight gains was probably not due to dietary
factors but was a result of dehydration caused by polyuria
observed in the streptozotocin injected rats.

Plasma glucose levels in diabetic rats decreased to a
final value of 95.4 mg%, significantly lower (P(.001) than
control plasma glucose concentration, 4 hours after insulin
injection. Testes glucose was significantly higher (P(.02)
and glucose-6-phosphate levels were lower (P(.Ol) than control
values in diabetic and insulin treated diabetic animals
respectively.

Testes pyprinositol content in diabetic animals was
approximately 2.7 fold greater than control levels. Insulin
treatment of diabetic rats for 4 hours lowered testes pypr

inositol levels; however, the mpg-inositol content remained

61

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62

63

greater than control values (approximately 1.9 fold).
Plasma o-inositol levels in streptozotocin diabetic rats
were unexpectedly lower (P<101) than levels observed in

control or insulin injected diabetic animals.

Kinetics pf Streptozotocin Induced Diabetes apd Effects pp
Testis apg Plasma pyo-Inositol Levels. The time course of
streptozotocin induced diabetes in male rats, and its effect
on testes glucose, glucose-6-phosphate, Exp-inositol, and
plasma myp—inositol levels were determined. As depicted

in Figure 1A, plasma glucose levels rose sharply from a
value of 200 mg% at 10 hours after injection of streptozo-
tocin to 340 mg% at 12 hours. Plasma glucose continued to
rise steadily after this point and plateaued 24 hours after
injection at a value of 454 mg%, approximately 3 fold
higher than control levels. Hyperglycemia was maintained
at this level in streptozotocin injected rats for at least
an additional 48 hours. Plasma glucose concentrations in
control rats did not significantly deviate from approx-
imately 150 mg% throughout the 72 hour experiment.

Testes glucose levels (Figure 1B) were observed to
generally parallel plasma glucose concentrations in both
control and diabetic rats. Control rats had a testes glu-
cose content of approximately 1.0 umole/g throughout the
experiment. Streptozotocin injected animals displayed a
gradual increase in testis glucose 10 hours after injection
which continued until approximately 24 hours after injection,
reaching a final level of 3.91 umoles/g or 4 fold greater

than control values. This hyperglycemia observed in the

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64

PLASMA GLUCOSE (mg%)

TESTES GLUCOSE (umoles/g)

600

500

p—
C)
<3

300

200

100

 

 

 

 

 

 

 

 

té—txfx-P ~I~4 H
l I | I “it I
O 8 16 24 48 72
HOURS AFTER INJECTION
4t

 

 

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HOURS AFTER INJECTION

65

 

 

66

testes 24 hours after injection was maintained at nearly
the same level for at least 48 additional hours.

The glucose-6-phosphate content of testes from control
and streptozotocin injected rats as a function of time after
injection is presented in Figure 2A. Glucose-6-phosphate
levels increased sharply at 12 hours after injection of
streptozotocin or approximately 2 hours following the
initial elevation of testes glucose levels. Glucose-6-
phosphate concentration in diabetic animals plateaued between
16 and 18 hours after streptozotocin injection to a value
of 40 nmoles/g. After this plateau, glucose-6-phosphate
in testes decreased within 6 hours to a steady state level
of approximately 30 nmoles/g or 1.5 fold greater than con-
trol values observed throughout the experiment.

The testes pyprinositol levels observed in the strep-
tozotocin injected and control rats throughout the 72 hour
interval are shown in Figure 2B. No significant differences
were noted between the myprinositol contents of the two
groups during the first 24 hours. However, pyprinositol
levels increased slowly at 24 hours after injection to
levels of 1.762 1 0.16 and 1.795 i 0.40 umoles/g after 48
and 72 hours respectively. Thus, myprinositol levels in
the testes were not observed to increase until 12 and 14
hours after elevation of testes glucose-6-phosphate and
testes glucose in streptozotocin injected rats.

In Figure 3 the profile of plasma m o-inositol con—
centrations in control and streptozotocin injected rats

during the 72 hour interval following streptozotocin in-

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67

 

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72

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16

 

 

 

 

 

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48

16

HOURS AFTER INJECTION

68

(uM)
K0
0

20

10

PLASMA MYO-INOSITOL

 

 

 

 

 

11
’ll
a.
__ J.
1 1 I Jpl
0 8 16 24 48 72
HOURS AFTER INJECTION
Figure 3: Effect of Streptozotocin Injection on

Plasma pyp-Inositol Concentration.
Conditions were as described in Figure 1.
Each point represents the mean : stan-
dard deviation of 4 streptozotocin in-
jectedaf,or 3 control—o animals.

69

7O

jection is shown. No significant differences were observed
between the two groups. In addition plasma inositol levels
did not show any diurnal variations but remained relatively
constant at approximately 20 uM during the 3 day interval.
Although not shown in Figure 3, testes citrate levels
were measured 24, 48, and 72 hours after injection of
both diabetic and control animals. No significant dif-
ferences were noted between the testes citrate levels of
the two groups at these time points. In addition, no
diurnal variations were noted; testes citrate levels remained
unchanged during the experiment for both diabetic (0.218 1
0.024 umoles/g, n = 12) and control (0.200 i 0.011 umoles/g,

n = 7) groups.

Synthase apg Phosphatase Activities, myo—Inppitol Content
apg Ethanol Fed Rats. The effects of streptozotocin in-
duced diabetes as well as 40% (v/v) ethanol feeding on the
activities of the mpg-inositol biosynthetic enzymes in rat
testes were investigated. In addition, the effects of these
metabolic states on the pypeinositol, glucose, glucose-6-
phosphate, and ATP contents and the cytosolic redox state
of the testes were determined.

During the 72 hour experiment, control and diabetic
rats consumed a comparable number of calories per day
(82.2 i 3.4 and 72.0 i 6.9 respectively). Rats fed the
same standard rat chow diet and a solution of 40% (v/v)
ethanol pg libitum consumed 22.3 i 9.0 ethanol calories/

day and 55.2 i 11.4 total calories/day, significantly less

71

(P(.05) than the other experimental groups. The plasma
glucose level of streptozotocin injected rats was 505.1 :
51.9 mg%, significantly higher (P(.Ol) than levels observed
in either control (177.6 : 13.0 mg%) or ethanol fed (184.5 1
12.9 mg%) animals. No significant differences were observed
between the plasma pyp-inositol concentrations of control
(21.0 i 7.2 uM), diabetic (29.5 i 13.6 uM) and ethanol fed
(17.2 i 5.6 uM) rats.

As shown in Table II, no significant differences were
observed in either the synthase or phosphatase activities
in the testes of streptozotocin diabetic or 40% (v/v) eth-
anol fed rats compared to control animals. However, free
pypeinositol and glucose-6-phosphate levels in the diabetic
testes were elevated approximately 1.5 fold over control
values. Also, Exp-inositol levels in testes from ethanol
fed rats were only 85% of control values. This reduced
mypeinositol level in the ethanol fed rat was also paralleled
by a comparable reduction in the glucose-6-phosphate content
of the testes. Testes glucose concentrations were approx-
imately 400% higher in diabetic and 15% lower in ethanol
fed rats compared to control animals. While no significant
differences were noted in the testes ATP levels between the
three experimental groups, the testes cytosolic NADf/NADH
ratio in diabetic rats was significantly lower than either

control (P<301) or ethanol fed (P(.05) rats.

Effects pf Chronic Ethanol Consumption pp_Redox State and
pyo-Inositol Content pf Liver and Testes. The effects of

ethanol consumption on testes pyp-inositol content were

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72

 

 

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73

72+

reinvestigated under conditions in which ethanol fed and

control animals were isocalorically pair fed and ethanol
feeding was extended to a two week interval. In addition
the redox states of both the liver and testes were characterized.
During the experiment ethanol fed rats consumed 56.7 i 13.8
kcal/day (n = 6), ethanol calories accounting for 53.0 i 12.8%
of all calories. In comparison pair fed controls consumed
57.3 i 11.7 kcal/day. Body weight of ethanol fed rats
decreased significantly (P(.05 by the two-tailed paired t-
test) from 343.0 i 7.5 g to 261.0 1 26.8 g for a net change
of -82.7 i 27.7 g. However, the body weight of pair fed
control rats did not significantly change; the initial
weight was 341.0 1 7.3 g and final weight 350.1 i 28.0 g.
The differences between the final body weights of the eth-
anol fed and pair fed control rats was 91.0 i 11.5 g (sig-
nificant at P<5001 by the two-tailed paired t-test). The
discrepancy was most likely due to dehydration of the
ethanol fed rats.

Table III presents pyp-inositol and selected metabolites
and the redox states of the liver from normal and ethanol
fed rats. As shown ethanol fed rats were hypoglycemic
compared to control animals, having a plasma glucose con-
centration approximately 28% lower (P(.Ol) than control
levels. No significant alterations were observed in the
cytosolic or mitochondrial redox states of the liver with
ethanol feeding: however, lactate and citrate content of
livers from alcohol fed rats were both about 50% lower than

control levels (significant at P<LO5 abd P<J02 respectively).

TABLE III

myo-Inositol and Selected Metabolite Levels and Redox State
of Liver from Control and Ethanol Fed Ratsa

 

 

 

 

Metabolite Ethanol Fed Control Pairs
Glucose-6-Phos hate
(umoles/g)3 0.312 .1 0.059 0.300 : 0.056 6
o-Inositol
m(Emoles/g) 0.263 1 0.107 0.217 i 0.087 4
Citrate (nmoles/g) 85.54 112.71C 176.7 : 8.2 5
Lactate (umoles/g) 0.253 i 0.061(1 0.529 : 0.107 4
Pyruvate (umoles/g) 0.047 .t 0.009 0.091 i 0.017 4
Cytosolic NAD+/NADH 1998 _t 375 1574 i 263 4
Hydroxybutyrate
fiL (nmoles/g) 114.7 : 20.8 116.4 : 24.3 4
Acetoacetate
(nmoles/g) 26.1 i 10.9 31.9 i 6.3 4
Mitocpondrial
NAD /NADH 4.67 3: 2.07 6.24 i 3.27 4
Plasma Glucose b
(mg%) 99.9 i 21.0 138.3 1 11.8 6

 

aAll male rats initially wei hed 330-350 g. Ethanol fed rats
received a solution of 40% Tv/v) ethanol and a commercial
solid diet for a two week period. Control rats were pair fed
to ethanol fed rats as described in the Methods section. All
values represent the mean :_standard deviation for the in-
dicated number of pairs. The data was analyzed using two-
tailed paired t-tests.

bSignificantly different from control values at P<.01.

CSignificantly different from control values at P<.02.

dSignificantly different from control values at P<.05.

75

76

Liver mpg-inositol content was not different for the eth-
anol fed (0.263 :_O.107 umoles/g) animals compared to pair
fed controls (0.217 1 0.087 umoles/g). In addition, liver
glucose-6-phosphate levels were unchanged upon ethanol feeding
and remained at approximately 0.3 umoles/g.

The cytosolic redox state of the testes (Table IV) was
not altered with ethanol feeding. No decrease in testes
glucose levels in ethanol fed rats was observed in spite
of the previously noted hypoglycemia of these animals.

In addition, glucose-6-phosphate and pyprinositol levels

of the testes of ethanol fed rats remained unchanged com-
pared to control levels for these metabolites. Also shown

in Table IV, no significant differences were observed between
alcohol fed and control rats with respect to testes weight,

the testes to body weight ratio, and testicular sperm count.

Intracellular Hexokinase Distribution ip;ppp Testis pi

Normal, Diabetic, gpg AJQohol Intoxicated_Rat§. The dis-
tribution of hexokinase activity between the cytosol and
mitochondria of the rat testis was investigated in normal,
streptozotocin diabetic and ethanol injected rats. Table V
presents the soluble hexokinase activity of rat testis as

a percentage of total hexokinase activity during the different
metabolic states. As shown, neither ethanol intoxication

nor streptozotocin induced hyperglycemia significantly

altered the intracellular distribution of hexokinase in the

testis; approximately 65% of the total activity was soluble
and 35% particulate.

TABLE IV

myprInositol and Selected Metabolite Levels, Redox State
and Sperm Count of Testes from Ethanol Fed and Control Ratsa

 

 

Parameter Ethanol Fed Control
Glucose-6-Phosphate

(nmoles/g) 21.08 i 3.64 22.61 i 2.35
Glucose (umoles/g) 1.14 i 0.26 1.07 i 0.08
Lactate (umoles/g) 0.950 : 0.343 0.845 1 0.069
Pyruvate (umoles/g) 0.019 t 0.005 0.026 :t 0.002
Cytosolic NADfi/NADH 199 i 69 275 i,26
pyp—Inositol (umoles/g) 1.22 i 0.15 1.00 i 0.14
Testis Weight (g) 1.72 i 0.11 1.72 i 0.09
Sperm/testis x 10'8 1.98 :_0.13 2.01 i 0.16
Sperm/g x 10'8 1.16 i 0.06 1.17 i 0.06

 

aAll conditions were as described in the legend to Table III.
All values represent the mean : standard deviation of 6 pairs

of animals.

77

No statistically significant differences for any
of the parameters measured were observed.

TABLE V

Effects of Metabolic State on Lg Vivo Distribution

of Testis Hexokinase

 

 

. a % Soluble Plasma Glucose
Anlmals Hexokinase Activity (mg%)
EtOH Injected 62.26 i 6.05 N.D.C
Control 67.95 i 5.74 N.D.
Diabetic 70.54 i 6.02 551.1 i 25.2
Control 69.75 i 5.24 182.3 1 12.8

 

a'All rats were initially 200-225 g.
received 10% (v/v) ethanol at a dosage of 2 g/kg 25 min
Diabetic rats were injected with 65 mg

before sacrifice.
streptozotocin/kg 72 hr before sacrifice.

Ethanol injected rats

Hexokinase

preparations and assay were as described in the Methods
section. All values represent the mean 1 standard deviation
for the indicated number of animals.

b% soluble hexokinase activity =

CN.D.---not determined

78

hi h s eed su ernatant activit x 10‘
whole homogenate activity

79

Although not shown in Table V, kinetic analyses were
conducted on hexokinase preparations from rat testis cyto-
solic and mitochondrial fractions. The Kms for ATP in
the soluble and particulate enzymes were 0.361 1 0.030 mM
(n = 2) and 0.409 i.0'052 mM (n = 2) respectively. The Kms
for glucose were 0.066 1: 0.011 mM (n = 3) and 0.020 is. 0.007 mM
(n = 3) respectively, significantly different at P<1002 by

the two-tailed t-test.

Investigation pf ppp Effects p§_Diabetes apg Concurrent
Elevated Testis myo-Ipositol Levelp pp Testicular §pppp
Qpppp Egg Testis Tubulin Polymerization. The effects of

a two week period of streptozotocin diabetes on testicular
myp-inositol levels and sperm count are depicted in Table VI.
Streptozotocin injected rats had plasma glucose levels ap-
proximately 5 fold greater (P(.01) than control animals

and testes pyp-inositol levels 2.5 fold (P(LOl) in excess
of control rats. Diabetic rats did not display increased
plasma pyp-inositol levels, however. No significant dif-
ferences were observed in the number of sperm/gram testis
or in the number of sperm/testis between the diabetic and
control animals. Although the testicular sperm count
remained unaffected during diabetes, results from this
study do not preclude any changes in the viability or
fertilizing capacity of the sperm. Although the average
testis weight from diabetic animals was significantly lower
(P(.005) than control animal testis weight, the ratio of
testis to body weight was significantly higher (P(.001) for

streptozotocin injected rats compared to control animals.

TABLE VI

Effects of Diabetes and Elevated Testes pyo-Inositol Levels
on Sperm Countsa

 

Parameter Control Diabetic

 

Plasma Glucose

(mg%) 155.1 1 15.2 766.9 : 105.8b
Plasma m o-Inositol

(uM 21.21 i 8.45 30.75 .1 13.86
Testes pyp-Inositol

(umoles/g) 1.64 i 0.21 4.27 i 0.95b
Sperm/g testis x 10’8 1.08 i 0.09 1.24 1 0.08b
Sperm/testis x 10’8 1.68 i 0.21 1.71 i 0.21
Testes wt/body wt x 103 5.35 .t 0.47 7.58 i: 1.28(1
Testis wt (g) 1.52 i 0.06 1.01 : 0.37°

 

aRats initially weighed between 175-185 g and were 6 weeks of
age. Diabetic animals were injected 2 weeks prior to sac-
rifice with streptozotocin as previously described in the
Methods section. All values represent the mean 1 standard
deviation of 5 control and 9 diabetic animals. Statistical
analyses were performed using the two-tailed t-test.

bSignificant at P(.01.

0Significant at P(.005.

dSignificant at P<2001.

80

81

Therefore, no conclusive evidence was found for testicular
atrophy in diabetic rats.

Presented in Table VII are data concerning the effects
of hyperglycemia on total testis tubulin and testes tubulin
polymerization. Rats injected with streptozotocin 84 hours
before sacrifice displayed plasma glucose levels approximately
2.7 fold higher (P(.001) and testes pyp-inositol levels
2.1 fold higher (P(LOl) than control levels. However, no
significant differences were noted between the total tubulin
content or percentage of tubulin polymerized in the testes

of control and diabetic rats.

TABLE VII

Effects of Diabetes and Elevated Testis myp—Inositol
Levels on Testis Tubulin Content and Tubulin Polymerizationa

 

Parameter Control Diabetic

 

Plasma Glucose

(mg%) 185.9 1 11.9 499.8 _+_ 44.00
Testis pyp-Inositol c

(umoles/g) 1.093 i 0.103 2.327 1 0.849
Total Testis Tubulin

(pmoles/mg) 3.86 : 0.42 4.25 i 0.32
Testis Tubulin
Percent Polymerized 38.9 :_4.1 41.4 + 2.9

 

aRats initially weighed between 200-225 g. Diabetic animals
were injected 84 hours prior to sacrifice with streptozoto-
cin as previously described in the Methods section. All

values represent the mean 1 standard deviation of 6 animals.

bSignificantly different from controls, P<.001, by the two-
tailed t-test.

0Significantly different from controls, P<.01, by the two-

82

DISCUSSION

Administration of streptozotocin, a diabetogenic
antibiotic from Streptomyces achromogenes which specifically
and irreversibly causes a defect in the degranulation of
pancreatic fi-cells (41,42), resulted in hyperglycemia and
subsequent elevation of testicular pyp-inositol levels in
the rat. These elevated pypeinositol levels were not due to
increased specific activities of the biosynthetic synthase
and phosphatase enzymes in the diabetic testis. In addition,
testicular uptake of pyprinositol from blood is not likely
to have been responsible for the increased cyclitol levels
since a testis-blood barrier has been described for
pyp-inositol (4,5) and no significant alterations in plasma
pyg—inositol concentrations were noted during streptozotocin
dflflxfies in this study. However, elevated testicular glucose
and glucose-6-phosphate levels were observed to accompany
increased pyp-inositol content in the testis of rats 72 hours

after streptozotocin injection, suggesting that l vivo

 

elevation of glucose-6-phosphate, substrate of the synthase,
led to increased myp—inositol biosynthesis. This hypothesis
was strengthened by the observation that 4 hours after insulin
treatment of diabetic rats glucose-6—phosphate levels
decreased to normal and concurrently pyprinositol in the
testis decreased toward normal levels. The failure of
testicular pyp-inositol levels to return completely to

normal following insulin administration to diabetic rats may

be attributed to the slow turnover of the cyclitol in the testis (4).

83

84

In order to establish a direct correlation between elevated
glucose-6-phosphate levels and increased pyp-inositol content
of the testis the concentration of these metabolites were
determined during the induction of streptozotocin hyper-
glycemia. Investigation of the kinetics of streptozotocin
effects revealed that hyperglycemia was apparent between
10 and 12 hours after injection of the drug. Testis glucose
closely paralleled changes in plasma glucose concentrations
while testicular glucose-6-phosphate levels increased
significantly 12 hours after injection. pyp-Inositol content
of the testis did not increase appreciably until approximately
24 hours after streptozotocin administration. The observed
lag in the elevation of the testicular pyp-inositol level-
with respect to the initial increase in the level of glucose-
6-phosphate may be due to utilization of jglucose-6-phosphate
primarily in other pathways. Another possible explanation
rests in the observation that the cytosolic NADf/NADH ratio
of the diabetic testis is below normal. A significantly
reduced cytosolic redox-state of the testis would be
expected to partially inhibit the NAD+ requiring synthase
catalyzed formation of myp-inositol-l-phosphate. Thus,
although elevated glucose-6-phosphate levels favor increased
pyp-inositol biosynthesis, a concurrent decrease in the
testis cytosolic NADf/NADH ratio could have an antagonistic
effect and temporarily inhibit the formation of the cyclitol.

The basis for a reduced NADf/NADH ratio in the testis
cytosol during diabetes is not well understood. The changes

in the redox-state may be related to increased levels of

85

both acetoacetate and/fiLhydroxybutyrate associated with an
increase in/B-oxidation of fatty acids, as has been proposed
for the case of the diabetic liver (43). Another proposal
has been that alterations of the redox-state may be due to

a disturbance in the hormonal balance between insulin and
glucocorticoids during diabetes (44). However. it is
concluded from these studies that the most important mode

for l vivo control of mype inositol biosynthesis is regulation

 

of the size of the cytosolic glucose-6-phosphate pool.

1.:

The 'n vivo regulation of testicular mypginositol

 

levels by glucose-6-phosphate pools was evaluated in an
ethanol-fed model. Ethanol feeding of male rats was expected
to result in hypoglycemia and possibly lowered testicular
glucose-6-phosphate levels. In addition, a reduced testis
cytosolic NAD+/NADH ratio was expected due to the pyridine
nucleotide-linked oxidation of ethanol. These metabolic
conditions would be predicted to synergistically act to
decrease pyp-inositol biosynthesis in the testis. Although
a mild hypoglycemia was established during ethanol
consumption by rats, no significant decreases in testicular
glucose or glucose-6-phosphate were noted. In addition,
the testis and liver cytosolic NADf/NADH ratios were
unaltered by ethanol feeding under the conditions employed,
suggesting that the various systems for transporting NADH
from the cytosol to the mitochondria were capable of
handling the NADH formed during ethanol oxidation.

However, it is more probable that ethanol was predominantly

86

oxidized by the NADP+ linked microsomal ethanol oxidizing
system of the liver (45) and therefore little change in

the cytosolic NADfi/NADH ratiorncurred in either the liver
or testis. Testicular gyg-inositol levels were also un-
altered during ethanol feeding. Thus this model was in-
conclusive in corroborating any regulatory role of
glucose-6-phosphate levels or the cytosolic NADfi/NADH ratio
on in xiyg_testicular mygeinositol biosynthesis.

Regulation of glucose-6-phosphate pools might involve
hexokinase and/or phosphofructokinase activities.
Testicular citrate levels were monitored in diabetic rats
in order to indirectly ascertain the effects of phosphofructo-
kinase activity on glucose-6-phosphate levels. Citrate is
a known allosteric inhibitor or phosphofructokinase, thus
citrate levels should be inversely proportional to phosph—

fructokinase activity 1 vivo and inhibition of this enzyme

 

might lead to a significantly increased glucose-6-phosphate
pool. No significant differences were noted between the
citrate levels of diabetic and control rats, indicating
that regulation of glucose-6-phosphate levels through
phosphofructokinase activity is not likely during strepto-
zotocin diabetes.

Hexokinase equilibrium between soluble and mitochondrial
forms has been proposed as a mechanism of importance for
controlling glucose phosphorylation in chick brain since
the kinetic parameters of the bound and soluble enzymes
are significantly different (36,46). During ischemia for

example, a higher percentage of total brain hexokinase

87

activity exists in the more active particulate fraction (46).
The hexokinase distributions of rat testes were therefore
measured during diabetic and alcohol-intoxicated metabolic
states in order to evaluate their possible role in the
regulation of the cytosolic glucose-6-phosphate pool. No
significant differences in the distribution of rat testis
hexokinase activity were observed in the metabolic states
tested compared to the equilibrium distribution of the
control animals. In addition, no differences were observed
in the Km for ATP of soluble and bound hexokinase activites
from control animals. While the particulate hexokinase
activity had a significantly smaller Km for glucose

compared to the soluble form in the normal testis, both
enzyme forms would be expected to be saturated with glucose
during most metabolic states and thus this kinetic difference
is probably not important with respect to regulation of
glucose-6-phosphate pools.

Normal adult mammalian testis is reported to contain
predominantly Types I and II hexokinase in equal amounts (47).
Testis from rat (47,48) and man(49) have also been shown to
contain an unusual isoenzyme of hexokinase designated
sperm type since it has been found in developing germinal
cells and Sertoli cells. Thus, the testis contains a complex
collection of hexokinase isoenzymes compared to the brain,
for example, which contains approximately 98% Type I
hexokinase(50).

Although no changes were observed in the distribution

of total hexokinase activity during different metabolic

88

states, these results do not preclude the possibility that
redistribution of specific hexokinase isoenzymes does occur.
Katzen et al.(51) have shown that streptozotozin induced
diabetes in rats results in the virtual dissappearance of
Type II hexokinase from the supernatants of crude homogenates
of heart and fat pad, while Type I hexokinase was unaffected.
Total hexokinase specific activity may also be altered
during metabolic states. It has been demonstrated that a
60% decrease in the hexokinase specific activity of rat
heart muscle occurs during streptozotocin diabetes (51).
Testicular degeneration and decreased spermatogenesis
have been reported in genetically selected rats after
eight months of uncontrolled diabetes (22). In addition,
ten months of a diabetic state resulted in hyperplasia
of Leydig cells in these rats. Reduced sexual behavior (16),
reduced accessory sex organ weights (17-19), and degen-
erative changes in seminiferous tubules (20-22) have also
been described in diabetic rats. Hypogonadism (23,24) and
germinal cell injury (25-28) have been reported during
chronic alcoholism in experimental animals and man.
Whether mygeinositol is involved either directly or indirectly
in these fertility dysfunctions has not been elucidated.
Preliminary results from this study indicate that sperm
counts are unaffected by significantly elevated testicular
ng-inositol levels in streptozotocin injected diabetic rats
after two weeks. In addition, sperm counts were not
significantly changed in rats fed 40% (v/v) ethanol for

two weeks, however, Exp-inositol levels were not significantly

89

altered in this system. Although not presented here, current
work in this laboratory suggests that streptozotocin diabetic
male mice are unable to impregnate normal female mice, but
impotence is not displayed in these male mice.

While the exact role of mygrinositol in the testis is
unknown, the cyclitol may be important in mitotic processes.
Early reports by Chargaff et al. (52,53) proposed that
mygrinositol counteracted the antimitogenic activity
of colchicine. The basis for this antimitogenic activity
may be due to the disruption of microtubules (54). Therefore,
the effect of elevated testicular mygrinositol in diabetic
rats on microtubule formation through tubulin polymerization
in the testis was investigated. However, no significant
differences were noted between the percentages of total
tubulin polymerized in the testis of normal and strepto-
zotocin diabetic rats. These results neither prove nor
disprove the existence of an important role for mygrinositol
in fertility and further work is required to resolve this
question.

;n_yizg myg-inositol biosynthesis in the rat testis
system has been shown to be directly and primarily regulated
by the size of the glucose-6-phosphate pool during normal
and diabetic metabolic states. Elevated glucose-6-phosphate
in the diabetic testis appears to result simply from an
increased flux of glucose into the testis and subsequent
conversion to glucose-6-phosphate. Whether testis hexokinase
isoenzymes become saturated with glucose or whether a

specific.redistribution of theses isoenzymes occurs during

9O

diabetes which subsequently affects the glucose-6-phosphate
pool remains to be determined. mygrlnositol levels in the
testis increase approximately three fold after 72 hours of
hyperglycemia and remained at this level for at least two
weeks after injection of streptozotocin. Although elevated
gyg-inositol levels caused no observed adverse effects on
testicular sperm counts, further research will be required
to determine the effects of mygeinositol on fertility and

its role in the testes and accessory sex organs.

ACKNOWLEDGEMENTS
The author wishes to thank Mrs. E. Dale HaWkins for
her skillful technical assistance in the hexokinase work and
Mr. Larry Fishel fer his expert assistance in the determination

of the sperm counts.

10.

11.

12.

13.

14.

15.

16.

17.

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