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thesis entitled

Sexual Dimorphisms of High-Fat or Glucose
Feeding in Rats: Differences in Body Composition

and the Hypertensive Response
presented by

Lauren Marie DeGrange

has been accepted towards fulfillment
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Masters degree in Biological Science

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I --—-——~————"—'—"— — --—~ ————~———-~—~'

 

SEXUAL DIMORPHISMS OF HIGH-FAT OR GLUCOSE FEEDING IN RATS:
DIFFERENCES IN BODY COMPOSITION AND THE HYPERTENSIVE RESPONSE

By

Lauren Marie DeGrange

A THESIS

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

MASTER OF SCIENCE

Department of Biological Science

1992

ABSTRACT
SEXUAL DIMORPHISMS OF HIGH-FAT OR GLUCOSE FEEDING IN RATS:
DIFFERENCES IN BODY COMPOSITION AND THE HYPERTENSIV E RESPONSE
By
Lauren Marie DeGrange
Female Sprague-Dawley rats resisted the hypertensive response which develops in males
fed either high-fat or glucose enriched diets. After 10 weeks of diet treatment systolic
blood pressure was 20% higher in males compared to females fed high-fat, and 15%
higher in males compared to glucose fed females (P<0.05). Females fed the high-fat diet
became obese(energy density = 6.21:0.3) and gained a higher percentage of body fat
compared to control females without exhibiting an increase in body weight. Females
failed to develop hyperinsulinemia or elevated norepinephrine levels. However
epinephrine excretion was elevated in females compared to males fed the high-fat diet.
Anatomical preference for fat accretion differed between males and females with females
depositing more fat in the interscapular and retroperitoneal regions. In both sexes
inguinal depot size seemed to parallel body growth while gonadal depot size paralleled
degree of adiposity. Differences in regional fat accretion and sympatho-adrenal system
response to diet manipulation may participate in the female ability to resist
hyperinsulinemia and hypertension. In conclusion females demonstrate a resistance to the

altered physiological state that males display in response to chronic high-fat or glucose

enriched diet feeding.

TABLE OF CONTENTS

I. INTRODUCTION

mewwpow?

Indications of the need for hypertension studies in females

Role of android obesity in the pathogenesis of metabolic aberrations
Rat models of obesity and fat topography

Macronutrient assimilation

Sympathetic nervous system

Obesity, sympatho-adrenal activity and hypertension

Effects of insulin on sympathetic activation and hypertension

Null Hypotheses

II. MATERIALS AND METHODS

F1909”?

General protocol

Blood pressure measurement
Measurement of sympatho-adrenal activity
Biochemical and physical analysis
Statistical analysis

111. RESULTS

A.

THEE-7‘0!”

Energy intake and body weight and energy density

a. High-fat versus control feeding
b. High-fat versus glucose feeding
c. Glucose versus control feeding

Fat localization

Blood pressure elevation

Sympathetic activation

Electrolyte excretion

Plasma insulin and glucose concentration

iii

IV. DISCUSSION

Obesity and macronutrient effects on hypertension

Insan stimulation of the sympathetic nervous system; indications of glucose
induction of a shift in sympathetic activity

High-fat induction of central and peripheral activation

Female resistance to hyperinsulinemic effects

Indications of catecholamine and gonadal effects on fat topography

Insulin, sympathetic nervous system and electrolyte handling

71919.0 P”?

V. SUMMARY AND CONCLUSIONS

iv

TABLE 1.

TABLE 2.

TABLE 3.

TABLE 4.

TABLE 5.

LIST OF TABLES

Composition of experimental diets.

Effect of experimental diets on energy consumption, body weight and
carcass energy, and fecal energy loss.

Effect of experimental diets on urinary catecholamines, sodium and
potassium, and fasting plasma insulin and glucose.

Percent differences in weights of right and left retroperitoneal, gonadal,
inguinal, and axillary fat pads from the same animal.

Effect of experimental diets on heart and liver weight and total fat weight
from five body fat depots.

FIGURE 1.

FIGURE 2.

FIGURE 3.

FIGURE 4.

FIGURE 5.

LIST OF FIGURES

Effects of high-fat or glucose feeding on systolic blood pressure, body
weight, and energy intake.

Effects of high-fat or glucose feeding on percentage body fat in five
regional fat depots.

Relationship between fasting plasma insulin and systolic blood pressure
during the tenth week of diet feeding.

Relationship between norepinephrine level and systolic blood pressure.

Relationship between epinephrine levels and retroperitoneal fat depot size
in female rats.

vi

INTRODUCTION

Indications of the need for hypertension stugies in females

Hypertension afflicts an estimated 35 million people in the United States(l).
Treatment of this disease is linked with reduced cardiovascular disease, cerebrovascular
and renal disease(1,15). Universally, there is a higher percentage of hypertension in men.
However, males and females develop hypertension at equal rates as the population
ages(1). Both men and women receive similar recommendations for treatment
hypertension, but investigative research has primarily focused on males. There is a need
for data on gender, like age and ethnic influences on hypertension and associated
complications(l). Research expanded to investigate hypertension in females would assist
in efficient treatment of women afflicted with hypertension as well as characterize the
disease process in general. This study was initiated to address the sex differences in the
hypertensive response to diet in male and female Sprague-Dawley rats. The specific aim
of this study was to elucidate sex differences in hemodynamic effects of macronutrients
and metabolic responses to these nutrients. Specifically, this approach examined lipid
accumulation, insulin sensitivity, sympathoadrenal activity, systolic blood pressure, and

electrolytes.

3

females from hyperinsulinemia and hyperglycemia( 10,19). Insulin functions in fat
metabolism by promoting lipid storage in adipose tissue. Adipose tissue, however, does
not react as a homogenous tissue to different hormonal stimuli(38). Specifically,
androgens and progesterone in conjunction with insulin cause hyperinsulinemia, and an
increase in body weight and fat(25,58). Conversely, estrogen improves peripheral insulin
utilization, lowers body fat( 18,58). Prenatal hormone organizational effect and
activational effects of hormones later in life contribute to the sex differences in body
weight and fat(6). Animal studies have shown these activational effects also include
androgen or progesterone stimulation of hyperphagia and insulin resistance and estrogen
suppression of food consumption as well as improved insulin sensitivity(6,33,35).

Factors responsible for location of body fat accretion are not completely known, yet sex
hormones appear to play an important role. Regional differences in adipocyte response
to insulin and sex hormones have been studied. Postmenopausal women with a
disproportionate amount of android fat show increased susceptibility to typically male
metabolic disorders such as diminished glucose tolerance and hyperinsulinemia(10,48).
Estrogen treatment has been shown to alleviate the effects of pharrnacologically induced
diabetes in male rats; testosterone treatment has been shown to worsen diabetes in female

rats.

Rat Models of Obesity and Fat Topography
In rats, strain, age, sex and diet all have an effect on the ability to produce

obesity(16,3l,45,51,54). Ventro-medial hypothalamic lesions and pharmacologic

4

manipulations are used in studies to induce obesity. Scientific investigations have
continually observed that dietary factors appear to play a substantial role in increasing
storage of body fat. Diet composition manipulations in rats best parallel human adiposity
changes.

High-fat or carbohydrate feeding results in obesity due to increased caloric intake
and feed efficiency(10,31). Increased palatability of the diet is the cause for the increased
intake, and the ability to convert diet energy into weight gain constitutes improved
efficiency(32,34). Animals fed diets of high energy content tend to lower consumption
to compensate for increased calorie density. This compensation of intake has been
demonstrated to be imperfect and animals fed high fat or glucose diets consume more
kilocalories over time due to an increase in palatability( 10,16). Feeding of high-fat,
highly palatable diets from weaning through adulthood produces the greatest tendency
toward obesity(54). In certain rat strains body weights can reach 900g and 35% fat after
20 weeks of high-fat feeding(45). During these 20 week or longer studies of ad libitum
feeding, females amass a greater percentage of fat, especially in gonadal, inguinal and
interscapular fat pads(31,51). Studies conducted over a shorter time period show an
increase of 16-32% in body weight of high-fat fed versus chow fed rats(31).

Body weight alone is not a reliable indicator of fat deposition(3l,45). As a rat
ages total body lipid shows a curvilinear relationship to body weight(45). Growth of
certain fat pads tightly correlate with body fat accretion, while others correlate with body
growth. Retroperitoneal depots grow exponentially compared to body growth(45). The

gonadal depots, on the other hand, are linearly related to body growth(45). Therefore,

5

retroperitoneal fat pads are a good representation of total body lipid, while gonadal
accumulation falls behind body lipid accretion(45). Retroperitoneal fat pad weighing in
Wistar rats allows for estimating total body fat to within 3—4% due to this tight
association of retroperitoneal fat deposition and body lipid(45). Specific metabolic and
morphologic characteristics effect changes in fat pad size as body weight increases.
Anatomical location as well as size of adipocytes are associated with differences in
insulin mediated lipid metabolism(36). Adipocytes from different anatomical sites, which
differ in lipid content will all adjust to one common size when transferred to the same

area of the body(3).

Microngtrient Assimilation

There are disparities in energy costs for nutrient assimilation. Eight to 20% of the energy
contained in a carbohydrate meal is used in the digestion and conversion to fat. Seven
percent of the total energy is used in the conversion of fat into adipose tissue(21,49).
Additionally, the conversion of carbohydrate and protein to fat ceases in diets containing
30% or more fat if the diet is not also high in carbohydrate. Therefore high fat diets can
lead to obesity through the increased efficiency of fat assimilation. Moreover, increased
adipose to lean body tissue ratio causes a decrease in basal metabolic rate,per unit body
weight, since adipose tissue has a slower metabolic rate(10,21). The resulting
disequilibrium between energy input and output due to efficient assimilation in obese
individuals will compound weight gain. The importance of diet composition in the

etiology of hypertension and related complications is realized, since the typical western

6

diet contains approximately 43% of its energy from fat(l6).
Sympathetic Nervous System

Peripheral autonomic innervation is profuse and intricate. Sympathetic and
parasympathetic nerves innervate many effector organs. Sympathetic outflow to different
organs has not been proven to be homogeneous(46). Norepinephrine is the major
catecholamine released at the effector tissues in response to increased sympathetic tone.
The adrenal medulla is the another source of peripherally released catecholamines.
Medullary secretions consists primarily of epinephrine with approximately 20%
norepinephrine. Plasma norepinephrine levels are generally six to seven times greater
than epinephrine(52). This sympatho—adrenal system is controlled by areas of the central
nervous system, including the ventromedial hypothalamus, pons, and medulla(42).

Alpha and B—adrenergic receptors mediate the response to norepinephrine or
epinephrine. Norepinephrine stimulatory effects on cardiac muscle (8 receptor), renal
function(B receptor), and smooth muscle of arterioles(0t receptor) are the primary inducers
of increase blood pressure(2,40,55).

Urinary catecholamine measurement is a good indicator of both central and
peripheral sympathetic activity, including adrenal medullary activity(50). Central control
of sympathetic activity is stimulated by diet and external influences like cold and
stress(24). The sympatho-adrenal effects can be uncoupled through demedullation. The
hypertensive response to high-fat or glucose feeding is significantly diminished in
demedullated anirnals(43). Females are suggested to have higher catecholamine levels

systemically than males. Uterine tissue especially is densely innervated(60). Estrogen

7

treatment causes elevated sympathetic activity while progesterone treatment reduced the
rise in norepinephrine in uterine tissue(60). These studies also demonstrated that
increased adrenergic activity in an organ does not always correlate with an increase in

plasma norepinephrine.

Obesity. Svmpamo-aWcuvitv and Hyperteniio_n

Chronic high fat or carbohydrate feeding results in an increased systolic blood
pressure in male rats. The sympathetic nervous system (SNS) is highly responsive to diet.
Overfeeding induces SNS activity, while fasting suppresses it(40). High-fat and simple
sugars (glucose, fructose, sucrose) are more effective than complex carbohydrates in
inducing sympatho-adrenal activity and hypertension(22,24,26,34).

An increase in SNS activity is apparent in male rats fed high-fat diets only when
it is associated with increased caloric intake and obesity(34,40,53). The hypertensive
response and increased SNS activity is not apparent when obesity is prevented by feeding
restricted amounts of a high-fat diet(34,40). This caloric restriction effect indicates the
important role of increased body weight in hypertension induced by fat. Weight loss in
obese, hypertensive humans lowers blood pressure and SNS activity (40,41).

Direct macronutrient effects should not be discounted as a factor involved in the
development of hypertension. Male rats chronically fed diets of 66% glucose became
hypertensive without obesity(22,26,34). Therefore, increased caloric intake as well as
macronutrient composition are involved in eliciting the hypertensive response. The

sympatho-adrenal system is thought to participate in mediating the consequence of obesity

8

and macronutrient effects on blood pressure(26,34).

In humans, established hypertensive subjects failed to show increased SNS activity,
yet borderline hypertensive individuals showed significantly increased plasma
catecholamine levels(14,28). There seems to be a point where sympatho-adrenal
activation ceases to play a role once hypertension is established. Once blood pressure is
established it is suggested that anatomical changes in arteriole smooth muscle override
the sympathoadrenal role in hypertension(14). In animal studies increased norepinephrine
levels have been demonstrated after five weeks of special diet feeding, approximately at
the onset of increased blood pressure(34).

Epinephrine may facilitate the action of the peripheral sympathetic nerves(2,7,13).
One hypothesis suggests that epinephrine is taken up by sympathetic nerve endings
peripherally and then released with norepinephrine as a co-transmitter during sympathetic
stimulation. Experiments using pharmacological doses of epinephrine have supported this
co-transmitter hypothesis. Determining if physiological concentrations of epinephrine
promote the same effects is less conclusive. A second hypothesis suggests that
epinephrine facilitates norepinephrine release by acting on the presynaptic B-adrenoceptor,
resulting in elevated norepinephrine release(2,7,l3). Additionally, the inhibitory
presynaptic Ot-adrenergic receptor may possibly be activated by epinephrine in addition
to [3 receptor stimulation(l3). Thus a balance exists between stimulation of these two
receptors which may change due to physiological status. Furthermore, epinephrine may
have trophic effects on cardiac muscle to heighten response to adrenergic stimulation(8).

Investigations of hypertension during its development will lead to uncovering the role of

9

the sympatho-adrenal system in the pathogenesis of hypertension.

Effects of Insulin on Sympathetic Activaytion and Hymrtension

 

The major function of insulin is glucose homeostasis. Release of insulin from B-
cells of the pancreas is stimulated by increased concentration of circulating metabolic
fuels. Overall, insulin functions at the level of the tissues to promote glucose utilization.
Fat storage in adipocytes is also induced by insulin, while fat mobilization is
inhibited(59). Insulin inhibits hepatic glucose production through reduced glycogenolysis
and gluconeogenesis.

Obese humans have been found to have 20% less binding capacity for insulin at
the level of the tissues due to a down regulation in the number of insulin receptors(47).
Post receptor deficiencies also attribute to reduced insulin action in obese individuals(47).
Non insulin dependent diabetics also demonstrate signal or regulatory protein defects
which culminate in reduced insulin action(47). The clinical sign of hyperglycemia in
NIDDM results when insulin production cannot meet high insulin needs due to reduced
peripheral sensitivity to insulin. In addition, hepatic glucose output is increased due to
deficient insulin effects on the liver. In NIDDM there is a positive feedback cycle of
hyperglycemia stimulating insulin release which cannot efficiently function, due to
receptor down regulation and postreceptor defects, thus more insulin is released and
hyperinsulinemia ensues.

Insulin may be the link between diet and SNS effects on blood
pressure(34,40,4l,50). Insulin is also a potent stimulator of SNS activity both centrally

and peripherally(37). Hyperinsulinemia has been associated with increased SNS activity

10

in hypertensive rats(34,40). Activation of the SN S through hyperinsulinemia is suggested
since plasma norepinephrine increases and clearance of circulating norepinephrine is not
altered during insulin infusion(50). Insulin can enter cerebral spinal fluid and insulin
receptors have been found in monkey and pig hypothalamus(39). Insulin may act as the
mediator for increased SN S activity by acting centrally at the ventromedial hypothalamus
to stimulate peripheral SN S activity. This central control mechanism is supported by
increases in SNS activity in a dose dependent relationship during hyperinsulinemic,
euglycemic clamping experiments in non diabetics humans(50). Additionally, subjects
with autonomic failure or diabetics with normal autonomic function do not produce the
increased response to insulin infusion or a glucose load, respectively(40).

The insulin mediated glucose hypothesis model explains the central control of SN S
activity in the ventromedial hypothalamus(VMH). Interruption of glucose metabolism
within the cells of the VMH, which occurs during fasting, causes inhibition of the
sympathetic center within the brain stem. Conversely, excess of glucose metabolism
allows sympathetic centers to increase central sympathetic outflow(37).

Insulin also acts centrally as a satiety signal to control food intake and body
weight(58). SNS stimulation and satiety functions support the importance of insulin as
a messenger between the brain and body metabolism.

The present investigation has attempted to compare gender differences in the
effects of high-fat and glucose diets on blood pressure, SNS activity, hyperinsulinemia,
energy retention and body composition. Disparities in the above parameters may assist

in elucidating sexual dimorphisms in the metabolic effects of specific nutrients.

ll

Null Hypotheses:

1.

Systolic blood pressure will be elevated equally in male and female rats
fed a high fat or refined sugar diet.

Occurrence of hyperphagia and hyperinsulinemia in hypertensive males
will be matched in females fed the same diet.

The degree of SNS activity and hyperinsulinemia paralleling hypertension
will be the same in males and females.

Energy intake and body energy accumulated throughout the experiment
will be equal between the sexes fed the same diets.

Macronutrient handling, and body fat distribution will be equivalent in

both sexes.

MATERIALS AND METHODS

General Protocol

Forty eight Spraque-Dawley rats (24 males and 24 females) were housed in a room
maintained at 25° C, 70% humidity and with a 12 hour light/dark cycle. The animals
were divided into 6 experimental groups consisting of eight rats per group. At the age
of 10-11 weeks of age each rat was allowed ad libitum access to one of three specially
formulaated diets. Protein, vitamin and mineral content were adjusted according to
expected food intake in order to provide equivalent amounts. (Table 1). The rats were
housed individually in order to measure individual food intake. Food intake was followed
three times weekly by measuring the amount given and subtracting the amount left in
cages (with corrections for moisture differences). Kilocalories of food consumed were
then calculated from amount and caloric value of the diet(Table 2). Body weight was

monitored three times per week.

Blood Pressure Mewmentp

Blood pressure(BP) was measured weekly in conscious animals using a
photoelectric sensor and tail cuff sphygmomanometer(Bunag & Butterfield, 1982). This
method is preferred over direct measurements of BP (via indwelling arterial catheter) for
this chronic feeding type of investigation. Rats were acclimated to the restraining
apparatus used for BP measurement for two weeks during which they were fed a stock

diet. After the acclimation period, experimental diet feeding was initiated and weekly

12

13

blood pressures for each rat were recorded between 0800 and 1200 hours. An equal
number of rats from each group were taken for BP measurement each time to keep
exercise and any other factors as constant as possible. A single, weekly measurement was
the average of four to five successive inflation-deflation cycles.
Mmment of sympatho-adrenal activity

Urine was collected approximately 7 weeks after diet initiation. Rats were
acclimated to metabolic cages for 36 hours prior to a 3 day collection period. Samples
were collected every 24 hours in the presence of 4.5 mls of 3M HCl and pH adjusted to
approx. 2.5 to deter catecholamine degradation. One ml aliquots where taken, prior to
pH adjustment, for Na+lK+ analysis. For each animal three 24 hour samples were
combined, diluted and stored at ~15 C. Catecholamines were purified using cation
exchange resin(Bio-Rex 70, 100-200 mesh: Bio-Rad Laboratories, Richmond, CA) and
concentrated using alumina-oxide (Bioanalytical Syatems, W. Lafayette, IN). Immediate
analysis followed by reverse phase column high pressure liquid chromatography (Bio-Sil,
ODS-SS, 250x4mm: Bio-Rad) and electrochemical detection (LC-4A: Bioanalytical

stems). Results were an average of duplicate sampling.

Biochemical and PhYSifll Analysis

At the conclusion of the 10 week diet feedings rats were sacrificed following a 5
hour fast. Blood was collected during decapitation for glucose and insulin analysis by the
glucose oxidase method(Beckman glucose analyzer) and RIA(Incstar, Stillwater, MN),

respectively. The heart, liver and 5 fat pads (gonadal, retroperitoneal, inguinal, axillary

14

and a single interscapular) were dissected and weighed to the nearest 0.0001 gram.
Comparisons of right and left pads were used to assess the reproducibility of the
dissection process.

Carcasses were autoclaved at 100° C for 2 hours and homogenized with the
addition of water (Polytron, Brinkman Instruments, Westbury, NY). Homogenates were
dried and 1 gram samples analyzed using an adiabatic calorimeter(Parr Instruments,
Moline, IL). Total body energy was calculated from averaged values of repeated
calorimetry trials of each rat homogenate. Fecal energy losses were also calculated from
adiabatic calorimetry of a 48 hour fecal sample collected during week 7 of the

experiment.

Statistical Analysis

Independent variables include diet offered, and gender. The experiment followed
a mixed factorial ANOVA design. Data were statistically analyzed by two-way
ANOVA(p=.05). A posteriori tests were conducted using the Student Neuman-Keuls

procedure.

RESULTS

Energy inta_keLbody weight and energy density

High-fat versus Control Feeding

Total energy intake in the female high-fat diet group exhibited no significant
difference compared to their control group (Figure 1). Energy intake increased
significantly by the sixth week in males fed the high-fat diet and continued to be higher
than control fed male rats throughout weeks seven through ten. Male controls consumed
23% more kcals and were approximately 45% heavier than control females(Table 2).
Body weight of the high-fat fed males at the end of 10 weeks of diet feeding was
approximately 6% greater than control males, and total kcals consumed was 10% greater
(Table 2). Energy density of the subjects at the end of ten weeks was higher in both
males and females fed high-fat diets compared to rats of the same gender fed the control
diet, (6% and 8%, respectively). Males and females fed high-fat did not significantly
differ in energy density, though the total body energy was 40% greater in males than in

females fed the high fat diet(Table 2).

High-fat males versus glucose feeding

Glucose fed male rats like the high-fat group consumed 10% more energy than the
control group over the ten week period (T able 2). However, body weights of glucose and
control males did not differ, (406.0 and 403.13, respectively). As shown previously,

energy density and total energy of the carcass of glucose fed males were significantly

15

16

lower than high-fat fed males and closely resembled control male values. (Figure 1)
Compared to female rats fed glucose, the female rats fed the high-fat diet had
approximately 20% greater total body energy, and 10% greater energy density of the

carcass, although body weights did not differ significantly (Table 2).

Glucose versus Control Feeding

Both gender groups showed no difference in total body energy or energy density
between their respective glucose and control fed groups. Energy density of male rats fed
glucose and control diets were both approximately 7% higher than female groups of the
same diet (T able 2).

Fecal energy losses represented less than 9% of daily energy intake in all

experimental groups(Table 2).

Fat Localization

Data discussed here as total percent body fat refers to the five fat depots that were
dissected, which are: gonadal, inguinal, retroperitoneal, axillary and interscapular.
Therefore, actual percent body adiposity is underestimated. In rats fed the same diet total
fat weight(g) in the five fat pads was greater in males than in femalesz able 5).
However, when expressed as a percentage of body weight significant differences were
evident between males and females on the same diet for all fat depot weights except
retroperitoneal and axillary(Figure 2). Even though retroperitoneal depots were bigger in

males, percent of the body weight that the two pads represented was not significantly

17

different between males and females fed the same diet. Females fed high-fat diets
actually had slightly greater representation of body fat by the retroperitoneal and
interscapular pads than fat fed males(Figure 2). Rats fed high-fat had a greater percentage
of fat than controls. The five pads contributed to a significantly lower percent body fat
in glucose fed males or females compared to the fat fed counterparts (22% and 30%
lower, respectively) Furthermore, glucose fed males had lower percentage of body fat in
all depots except retroperitoneal and interscapular compared to high-fat fed males. No
significant difference in depot weights existed between glucose fed rats and controls of
the same sex. Glucose fed females had significantly lower percentage fat in the gonadal,
retroperitoneal and inguinal pads than glucose fed males. Fat representing total body
weight of glucose females were significantly lower than females sustained on a high-fat

diet(Figure 2).

Blood Pressure Elevation

Baseline systolic blood pressures (wk 0) did not differ among all six groups of
rats. After five weeks of diet feeding systolic blood pressure of high-fat males increased
significantly when compared to male controls and females fed high-fat diets(Figure 1).
Systolic pressures of male rats fed the high-fat diet continued to increased throughout the
remaining five weeks. At the end of ten weeks the systolic pressure of the high-fat males
were 15% higher than male controls, and 25% higher than high-fat females. On the other
hand, blood pressures for females fed the high-fat diet remained constant and even

slightly decreased along with other female diet groups during weeks seven through ten

18
(Figure 1).

Systolic blood pressure of glucose fed male rats was significantly greater than
those fed the control diet between six and ten weeks of feedings. The elevation in BP
of glucose sustained male rats lagged slightly behind the increases measured in high-fat
males. At the tenth week of feeding systolic BP of glucose fed male rats was 15mm Hg
higher than male control rats and 25mm Hg higher than glucose fed females. The systolic
BP of females in all experimental groups were similar to that of control males throughout

the ten week period.

Sympathetic Activation

Urinary norepinephrine levels collected during week seven tended to be higher in
high-fat males (1377: 187) compared to glucose and control males (12123; 235 and
1201_+_ 268, respectively), although these differences are not statistically significant at
p<.05(Table 3). Norepinephrine excretion in all the females did not differ significantly

and was similar to control and glucose male norepinephrine excretion(Table 3).

Elecgolyw excretfl

High-fat and control males demonstrated significantly greater urinary levels of
sodium(Na) and potassium(K) compared to females fed the same diet(T able 3). However,
average daily Na/K intake were approximately 30% greater in males fed high-fat or
glucose diets compared to females fed the same diet, and 25% greater in control males

compared to control females(Table 2). The difference between intake and output of

19

sodium and potassium were estimated using concentrations of each electrolyte given in
the specific diets and amount of food consumed, to correct for intake differences(T able

3).

Plasma insulin and glucose concentgtjg
Fasting insulin levels in high-fat fed males, 1.95+0.11 ng/ml, were significantly

higher than females fed high-fat, 1.38+0.6ng/ml. Control male fasting insulin levels of
1.73+ 0.14 ng/ml, is slightly lower than high-fat or glucose fed males, although this
difference is not considered significantCI‘ able 4).

Plasma insulin levels of glucose fed males were significantly higher than those of
glucose fed females (1.94: 0.17 and 1.44: 0.24ng/ml, respectively, P<0.05)(T able 5).

Fasting plasma glucose levels did not differ among the six experimental groups at ten

weeks(Table 3).

DISCUSSION

Obesity and macrontmient effects on hyperter§i0_n

In an effort to broaden inquiry, I will propose two routes for macronutrient induction
of sympathetic nerve activation in the development of dietary induced hypertension. In
addition, I propose certain sex specific metabolic responses to dietary intake which allow
females to resist hyperinsulinemia and hypertension.

As in previous experiments, this investigation has demonstrated that male Sprague
Dawley rats exhibit hyperphagia, and subsequently become obese when fed a high-fat diet
ad libitum for ten weeks. Both males sustained on fat and glucose diets consumed more
energy(Figure 1). These rats develop hypertension with mean systolic blood pressure
levels of 181 and 169 mm Hg, respectively. Obesity, however, was absent in the
hyperphagic, hypertensive glucose sustained male rats (406g vs 403g for controls).
Incomplete compensation for increased energy content of diet as well as high efficiency
of dietary fat assimilation undoubtedly function to promote obesity from chronic high-fat
feeding. This energy efficiency is exhibited by females fed the same high-fat diet. (Table
2). This study suggests the hypertension in male rats can develop due to the obese state
or peripheral influences of the macronutrient glucose. Elevated systolic blood pressure in
male rats was induced through obesity as well as some direct or indirect influence of the
macronutrient glucose.

A possible scenario to account for the uncoupling of hyperphagia and obesity

observed in glucose fed males is increased energy expenditure due to the hightherrnogenic

20

21

effect of carbohydrate, discussed above. Less weight gain would result from glucose
feeding if this therrnogenic disparity actually occurs. A second possibility implements
direct effects of glucose on sympathetic nervous activity and energy expenditure in brown
adipose tissue(BAT). A shift or increase in sympathetic activity to the densely innervated
brown adipose tissue(BAT)is suggested. If a shift of SNS activity originates due to
glucose rather than fat feeding, more energy would be dissipated in glucose feeding
leading to less weight gain. The shift in metabolic pools resulting from chronic glucose
intake may function in the in shunt of SNS activity.

In contrast to males, females failed to exhibit hyperphagia in response to the
palatable diets. However, females fed high-fat in this experiment demonstrated obesity
through energy density of the carcass(Table 2). High feed efficiency is suggested to be
functioning here to increase body fat in females, and compensation of intake of the caloric
dense diet does seem to be more in-tune in these females. This indicates a sexual
dimorphism in the energy balance mechanism. Additionally, an uncoupling of obesity
and hypertension occurs in these females. Subsequently, no elevation of plasma insulin
was observed(Table 3). There seems to be a tendency in males toward altered metabolic
action of fat or glucose. The altered physiological state causing males to be

hyperinsulinemic.

Insulin stimulation of the sympaghetic nervous system; indications of glucose indaction
of a shift in sympathetic activity

The onset of hypertension in glucose fed rats lagged behind the rise in systolic

22

blood pressure of obese males(Figure 1). This suggests fats have a greater effect or more
direct route in the pathogenesis of hypertension. In this study insulin mediated
sympathetic promotion of increased blood pressure is less evident in the glucose fed
males. Norepinephrine excretion in high-fat males was slightly elevated although the
increase was not considered significant(34). In contrast to previous findings
norepinephrine excretion in glucose rats were unaffected(34).

Elevation of insulin in males fed glucose like males fed high—fat were slightly
elevated, although not considered significantly higher than compared to controls(T able 3).
However, male and female fed controls failed to exhibit differences in insulin levels while
levels were significantly higher in males compared to females fed high-fat or glucose.
With this in mind, it would seem that both glucose and fat postingestively induce insulin
secretion as a consequence of increased circulating metabolic fuels or endocrine
facilitation.

The non-significance of the elevation in insulin levels may be due to a limitation
in the method of taking one sample, rather than measurements of glucose and insulin
throughout a 24 hour period via indwelling catheterization. I suspect insulin resistance in
high-fat fed and glucose males would intensify if feeding was continued. The peripheral
state of hyperinsulinemia, I propose is functioning to stimulate sympatho-adrenal
norepinephrine release. Obesity compounds the peripheral pressor effects primarily by
promoting the insulin receptor down regulation/ insulin release cycle of insulin resistance.
Males fed glucose do not exhibit the protective uncoupling of dietary intake and increased

blood pressure mechanism of females. Direct or indirect effect through insulin may

23

function in glucose induced hypertension. Furthermore, a redistribution of SNS activity,
selectively stimulating the heart, blood vessels and kidney may be caused by glucose
effects and lead to hypertension in spite of reduced overall norepinephrine excretion
measured(T able 3).

Whether hyperinsulinemia precedes obesity is unclear. Obesity is necessary to
induce the hypertensive response in males, but not require in glucose induced
hypertension. It seems that increased anabolism of fat and increased circulating insulin
function synergistically, allowing a greater divergence of blood pressure from controls.
Furthermore, the sensitivity of the body to increased caloric intake and this positive
feedback cycle seems gender related(Figure 4). Females seem able to compensate energy
intake for energy dense diets as well as uncouple the insulin resistant effects of glucose
or high-fat feeding.

This investigation supports evidence of an overall correlation between hyperphagia
and hyperinsulinemia. Correlation of insulin and systolic blood pressure is relevant, and
stronger than norepinephrine, systolic blood pressure correlation,p=.38 and .33,
respectively(Figure 3& 4). It should be recalled that specific organ norepinephrine
turnover is not accurately measured by urinary means. Failure to measure increased
norepinephrine excretion in glucose stimulated hypertensive rats above controls may be
due a limitation the technique used to assess SNS activity. Urinary catecholamine
excretion is a final end pathway to central and peripheral(including adrenal
medullary)sympathetic activity. If glucose induced hypertension is mediated through

increased sympathetic turnover in a specific effector organ, this would explain the absence

24

of increased norepinephrine excretion. In the case of high percent glucose ingestion,
norepinephrine turnover would give a better indication of specific organ sympathetic
activation. These measurements would elucidate the possibility of a shift in sympathetic
activation to organs involved in response to glucose intake. Additionally, at seven weeks
of experimental feeding sympatho-adrenal activity proposed in the high-fat inducement
of hypertension may not be at peak function. Therefore, underestimation of sympathetic

activity in correlation to the blood pressure response observed.

High-fat induction of central and Eripheral activation

The results of this experiment supports the theory that insulin is the link between
diet and the pathological state of high blood pressure, either through direct effects on
peripheral organs or indirectly through stimulation of the sympatho-adrenal system.

Dietary fat may have a two fold effect on SNS activity by inducing central
sympathetic outflow and peripheral sympathetic activity. A mechanism that is suggested
involves differences in peripheral utilization of fats and glucose and the stimulation of
SNS outflow by the aforementioned insulin-glucose model of central control. It is
recalled that diets greater than 30% fat prevent the assimilation of carbohydrate to fat,
since ingested fat conversion to adipose is energetically favored. It can be assumed this
excess of fat is the physiological state of males sustained on high-fat in this study(T able
1). The carbohydrate that is ingested along with fat is subsequently free to be utilized
at the ventromedial hypothalamus to increase SNS outflow, rather than used for energy

peripherally. The increase in central outflow through insulin-mediated glucose

25

oxidation(discussed above) augments peripheral sympathetic activity. Overall, the diet
induced hyperinsulinemia state and central SNS activation result in an additive
hypertensive effect of increased cardiac output, vaso- and veno constriction and renal

sodium retention.

Female resistance to hyparinsulinemic effects

The maintenance of female blood pressure below 150 mm Hg, despite increased
body fat, shows a gender specific protection to the high-fat and glucose hyperinsulinemic
effects. Females fed high-fat diets in this experiment demonstrated obesity, increased
energy density of the carcass, without increased insulin levels. This maintenance of
insulin sensitivity suggests that the protective mechanism involves a shielding to insulin
resistance and insulin stimulated SNS activity. Overall, it seems females do not change
insulin sensitivity in response to increased obesity. Gonadal hormones undoubtedly play
a role indirectly and] or directly on these physiological parameters. Sex hormone
influence may increase insulin sensitivity of tissues in females, allowing insan elevation
to be avoided. Norepinephrine levels in females feed high-fat or glucose diets did have
slightly elevated norepinephrine compared to controls, although the difference was not
significant.(T able 3). This may be due to the aforementioned central stimulation of the
SNS by high-fat ingestion.

Elevated epinephrine levels of high-fat and glucose females compared to males fed
the same diet indicates a possible antihypertensive ability of this catecholamine in

females. In contrast to the ability of norepinephrine to induce pressor effects this study

26

suggest epinephrine is especially active(T able 2). The elevation in epinephrine could be
a consequence of an increased ratio of B to a-adrenergic receptors in peripheral organs.
Antihypertensive effects by the action of B-adrenergic stimulation may be functioning in
the gender protection from increased blood pressure, specifically gonadal effects on 0t :
[3 receptor ratio or affinity for catecholamines. The sympathetic stimulatory actions of
progesterone or inhibitory actions of estrogen would be a reasonable start point for further
investigation. Overall, a balance between the anabolic effects of insulin and lipolytic
effects of epinephrine may function in the uncoupling of dietary induced hypertension in
females. Determination of epinephrine correlation to sex hormones would clarify the

function of epinephrine in promoting or protecting the body from pressor effects.

Indications of catecholgine and gonadal effect_a on fat tonogpaLhy

Protection from the effects of obesity gives indication of sex specific disparities in
macronutrient sensitivity and handling. Location of fat accretion in the female may be
a contributing factor to the inability to induce hyperinsulinemia and obesity. The
retroperitoneal and interscapular areas seem to be a preferential area for fat deposition in
females (Figure 2). The correlation of epinephrine levels and retroperitoneal fat weight
is especially strong in females, p=.60(Figure 5). Males seem prone to accumulation in
the gonadal, inguinal and axillary regions. Estrogen and progesterone effects of fat

metabolism may allow fat topography consistent with reduced risk for insulin resistance.

It is suggest that certain anatomical areas in males the adipocytes are especially

27

sensitive to the anabolic effects of insulin. Thus fat accumulates easily, initially in the
inguinal area and sets the stage for obesity and subsequent insulin resistance. It would
be interesting to measure lipogenic enzyme activity in the inguinal depots in males.
Increased insulin sensitivity of adipocytes in the inguinal or gonadal area due to proximity
to gonadal blood flow is a possibility.

From this investigation it seems that the insulin sensitivity in females does not
change with obesity as easily as in males. Females demonstrate increased insulin
sensitivity compared to males throughout studies. The female physiological status may
be able to avoid the hyperphagic and hyperinsulinemic response by differential control
of insulin’s anabolic effect on adipose and other tissues of the body. Females may be
able to preferentially reduce adipose accumulation in high risk anatomical locations
without reducing sensitivity for insulin in other tissues of the body.

Gonadal and other neuroendocrine mechanisms undoubtedly participate in fat
topography differences between males and females. The characterization of gonadal
facilitated insulin mediated anabolism in different fat depots would delineate the particular
sex disparities in the mechanisms of lipid metabolism. Studies of gonadal effects on SNS
would assist in characterizing the mechanisms involved in cardiac, smooth muscle and
renal stimulation.

Inguinal depot size seem to parallel body growth best, while gonadal depot accretion
paralleled body lipid changes. Specifically, gonadal depot size followed carcass energy
increases, therefore, this fat pad was largest in the most obese rats and was progressively

smaller as body fat percentage declined(Figure 2). Inguinal pads were largest in the

28

heaviest (total body weight) animal and declined as weight of the animal declined
regardless of body lipid content. The interscapular area is the anatomical area for lipid
accretion in females regardless of diet composition in comparison to males which were
at least 37% heavier and had at least 30% more body fat. Notably, the percent fat in
retroperitoneal and axillary pads did not differ between sexes on the same diet. This
indicates, especially in high-fat fed animals, regional disparities in metabolic activity of
adipose tissue.

The preference for interscapular fat accumulation in females fed high energy diets
correlates with the Schemmel studies (51). The increased percent of fat in the
retroperitoneal rather than gonadal areas in females sustained on fat is in disagreement
with the Schemmel studies. Theses discrepancies may be due to a time factor. Rats in
the Schemmel study were sustained on the experimental diets approximately six times
longer than in the present study. Total fat from the five pads in this study representing
8% or less percentage of body fat(Figure 2). This is in contrast to the high percentage
of body fat(30-40%) attained in the Schemmel study. This time factor may indicate a
threshold response. For example, it is possible that females at a certain percent body fat
will cease fat deposition in the retroperitoneal depot and increase deposition in another.
Additionally, changes in deposition occur since females demonstrate a higher percentage
of body fat as obesity increases. The present study also indicates females may
preferentially accumulate subcutaneous or fat in another areas. This is suggested since
while fat fed females were equal to males in obesity, the percentage of body fat in the

five depots measured was about 1.5% less, indicating fat deposition elsewhere.

29

From this study it is interesting to note that energy density of the carcass parallels
percentage of fat attributed to the five pads dissected. Therefore it is suggested that in
Sprague-Dawley rats total weight of the retroperitoneal, gonadal, inguinal, axillary and
interscapular fat pads is a good indicator of carcass obesity, although total percentage of

fat will be underestimated.

Insulin, sympathetic nervous system and electrolyte hm

Insulin’s effect on renal sodium and potassium handling may function to induce
hypertension(17,35). Renal sympathetic innervation allows SNS stimulation and
subsequent renin secretion,. Additionally the SNS can increase sodium reabsorption by
direct action on renal tubules(41). Disparities in Na/K excretion may be related to
differences in body weight and percentage fat or dietary intake of sodium and potassium.
From estimations of daily sodium intake, adjusted for differences in daily intake and
specific diet content, differences between males and female sodium excretion can be
attribute to differences in intake(Table 2 & 3). Within gender groups the differences in
corrected excretion amounts of the studied electrolytes are negligible. Between gender
groups, however, males seem to excrete more sodium and potassium relative to females
indicating gender specific differences in electrolyte handling dispite changes in blood

pressure elevation(Table 3).

SUMMARY AND CONCLUSIONS

In summary the present study describes a female physiological state which protects
them from diet induced hyperinsulinemia and hypertension. Gonadal influences are most
likely the basis for improved insulin sensitivity demonstrated in females. High-fat or
glucose feeding in females seem to have a disparate action on sympath-adrenal system
stimulation with which indicate different physiological states between males and females
fed these experimental diets. Preference for interscapular and retroperitoneal fat accretion
in females in this study contrast the findings in other investigation. This contrast of
results may be due to differences in the degree of adiposity; other studies achieved body
fat percentages of 40% and above, typifying gross obesity. In this study the five
measured fat pads represented no greater than 7% of the total body weight. Additionally,
in Sprague-Dawley rats weights of five fat depots; retroperitoneal, inguinal, gonadal,
axillary and interscapular can be considered a good indicator of adiposity. Further
investigation is needed to characterize the hormonal protection of insulin sensitivity in
females in one of three hormonal conditions: ovariectomy with subsequent hormone
replacement, the hyperestrogenic state of pregnancy and chronic exposure to androgens.
Characterization of estrogen and progesterone effects on insulin receptor action in
different tissues and sympathetic action at specific tissues through studies in females will
lead to an understanding of the pathogenesis of cardiovascular disease and associated

diseases as well as improved treatment of these diseases in both sexes.

30

Conclusions:

1. Sex hormones are most likely the mediator of improved insulin sensitivity in females
and intensified insulin resistance in male Sprague-Dawley rats.

2. Anatomical site of fat accretion may be a factor in the ability of females to maintain
insulin sensitivity. Female hormonal environment may influence insulin affects at these
anatomical sites.

3. Increased adrenal medullary secretion of epinephrine in females suggests a need for
investigations in comparing adrenergic receptor ratios in specific tissues.

4. Females display a more efficient ability to balance energy intake and output which
allows for obesity and a higher percentage fat to occur without increased energy intake.
5. Insulin is associated with obesity and hypertension in male rats fed high-fat diets.
6. Insulin is associated with glucose induced hypertension in male rats.

7. Two routes of diet induced hypertension may involve direct effects of macronutrients
on sympatho-adrenal activating mechanisms and indirect effects through obesity.

8. The uncoupling of increased intake and obesity demonstrated in males fed glucose
enriched diets indicate glucose induction of an alternate physiological process which
dissipates the extra energy intake.

9. High-fat and glucose diet feeding may shift sympathetic activity to separate peripheral
organs to elicit the hypertensive response.

10. Glucose diets in both males and females tend to result in a deposition of less energy

in the body during chronic feeding compared to high-fat and starch fed animals

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42

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