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THE EFFECTS OF A TEPARY BEAN DIET AND EXERCISE
ON SERUM GLUCOSE, INSULIN, CHOLESTEROL, AND TRIGLYCERIDE
CONCENTRATIONS IN THE FATTY ZUCKER RAT
By

Pamela Elizabeth Burch

A THESIS

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

MASTER OF ARTS

Department of Physical Education and Exercise Science

1992

v”.

(z 5 7'

ABSTRACT

THE EFFECTS OF TEPARY BEAN DIET AND EXERCISE ON SERUM
GLUCOSE, INSULIN, CHOLESTEROL, AND TRIGLYCERIDE
CONCENTRATIONS IN THE FATTY ZUCKER RAT.

BY

Pamela E. Burch

Alterations in diet and activity level are standard recommendations in the
treatment of non-insulin dependent diabetes mellitus (NIDDM). The purpose of
this research was to examine the synergistic effects of a legume diet and
exercise on the NIDDM state. Forty, six-week old Fatty Zucker rats were
randomly assigned to tepary bean diet and exercise (TE), tepary bean diet only
(T), control diet and exercise (CE), and control diet only (C) groups. The
exercise groups participated in a 13 week treadmill training program. Fasting
blood glucose, insulin, cholesterol, and triglyceride concentrations were
assessed throughout the study. Body weight and food consumption were
measured weekly. Diet and/or exercise did not significantly effect serum insulin
or glucose concentration, although the tendancy was for both TE and T to
exhibit lower insulin and glucose levels than CE and 0. Cholesterol
concentrations were significantlyy reduced in TE and T versus C (p<.05), while
serum triglyceride levels were significantly reduced in TE versus 0 only (p<.05).
Thus. exercise training and legume diet function synergistically to lower semm
cholesterol and triglyceride concentrations in the Fatty Zucker rat while
minimally effecting fasting glucose and insulin levels.

In loving memory of
Mildred Hitzler
February 9, 1905-June 15, 1990

Though your heart is silent your words of encouragement
resound in my mind. I will dare to dream the impossible
dream and strive to beat the unbeatable odds.

ACKNOWLEDGEMENTS

This study was completed with the assistance and support of many
persons including professors, fellow students, friends, and family. Although it is
impossible to mention all those persons by name, some individuals deserve
special recognition for their contributions to this project. Thus, I express my
heartfelt thanks and appreciation.

The financial support of AURIG funds from HBO and BRSG and the
College of Education all of which enabled this research project to transpire.

Mom and Dad - For your emotional, spiritual, and financial support throughout
my long and arduous scholastic career. You have always been there for me
cheering on the sidelines and for that I am the most grateful.

To the chairperson of my Master's Thesis committee:

Dr. Carol Rodgers - An incredible investment of personal time and energy
was made in directing this research. Her input shaped the outline and data
analysis of this study. The personal encouragement and committment to the
work has made all the difference.

To the members of my thesis committee:

Dr. Rachel Schemmel - Her comprehensive knowledge in the area of
human nutrition enabled me to devise the nutrition portion of my research. It
was in HNF 841 that I was introduced to the Pima Indians and the tepary beans.
Her enthusiasm for the subject matter was an inspiration to me.

Jaci VanHeest - Her role as English teacher, devil's advocate, surgeon,
friend, and colleague played a definitive part in the evolution of this manuscript.
We may have small hoops but it will help to fulfill our big dreams.

Special thanks to:

Dr. Sally Walshaw and Dr. Susan Stein - the frequent house calls to the
animal room were very much appreciated. Their services were extended above
and beyond the call of duty.

Dr. Mark Uebersax - the use of his Fitzhammer Mill and Food lab.

Dr. M. Bennlnk - the use of his spectrophotometer.

Dr. I. Gray - the use of his spectrophotometer.

Dr. Khazuhara Suzuki - help in the analysis of the insulin samples.

Dr. W. Helferlch - the use of his lab and pH meter.

Jennifer Woudenberg - assistance in the training of the rats.

Krystyna Kras - protein analysis of the tepary bean.

Jill Robinson - assistance in the sacrificing procedure.

Kimberly Gold - body composition of the rats.

Joann Janes - making overheads and photocopies at the last minute; ordering
of the diagnostic kits.

Melinda Manore - Source of the tepary beans.

Michael Koerner - For his help in printing out this manuscript.

To all my friends in Chicago, Lansing, and Grand Rapids - Yes, “we are the
people our parents warned us about.”

Kristi, Marilee, and Lisa - Thanks for being so understanding, patient, and
catering to my inflexible schedule. Our monthly gatherings gave me something
to look fonIvard to and helped to keep me somewhat sane.

Cynthia - You were my roomate for the majority of this project and lived to tell
about it - whooda thought. I appreciate you being there to cook, counsel,
harass, run rats, and make an occasional vet visit.

Jayne Ward - Here's to ”Cajun martinis and playing afternoon golfl" You have
Biochemistry to thank for making you the official complaint box and Spring
Break rat runner. Your humorous cards always made things feel better. Thanks
for your moral support.

TABLE OF CONTENTS

List of figures ................................................................................................................ vi

List of Tables .................................................................................................................. vii

List of Appendices ........................................................................................................ viii
Chapter I. Introduction ................................................................................................. 1
Need for Study .................................................................................................. 2
Purpose .............................................................................................................. 3
Hypothesis ......................................................................................................... 3
Rationale ............................................................................................................ 3
Research Plan ................................................................................................... 5
Limitations .......................................................................................................... 5
Definitions .......................................................................................................... 6
Chapter II. Review Of Literature ................................................................................. 8
Diabetes Mellitus .............................................................................................. 8

Environmental Factors ..................................................................................... 10

Obesity .................................................................................................... 10

Nutrition .................................................................................................. 1 1

Legumes ................................................................................................. 12

Physical Activity ................................................................................................. 14

Triglycerides .......................................................................................... 1 5

Cholesterol ............................................................................................. 15

Insulin ...................................................................................................... 16

Glucose ................................................................................................... 17

Diet and Physical Activity ................................................................................ 18

Chapter III. Methods ..................................................................................................... 20

Animals ............................................................................................................... 20

Exercise Training .............................................................................................. 21

Food Intake and Weekly Weights .................................................................. 22

Blood and 89mm Assays ................................................................................ 22

MuscleExcision ................................................................................................. 24

Fat Depots .............. ., .......................................................................................... 24

Statistical Analysis ............................................................................................ 24

Chapter IV. Results ....................................................................................................... 25

Animal Characteristics ..................................................................................... 25

Food Consumption ........................................... ' ................................................ 2 8

Blood Serum Measures ................................................................................... 28

iv

Glucose .................................................................................... , ............... 28

insulin ...................................................................................................... 32
Cholesterol ............................................................................................. 32
Triglycerides .......................................................................................... 34
Chapter V. Discussion of Results ............................................................................... 37
Body Weight and Food Intake ........................................................................ 37
Glucose ............................................................................................................... 39
Insulin .................................................................................................................. 40
Cholesterol ......................................................................................................... 41
Triglycerides ...................................................................................................... 42
SDH and Muscle ............................................................................................... 43
Chapter VI. Summary, Conclusions, and Further Recommendations ................ 45
Summary ............................................................................................................ 45
Conclusions ....................................................................................................... 46
Recommendations ............................................................................................ 46
Appendices
Appendix A Pilot Study ............................................................................................. 48
Appendix 8 Procedure for Preparation of Tepary Beans ................................... 53
Appendix C Composition of Diets ........................................................................... 54
Appendix D Treadmill Running Protocol ............................................................... 56
Appendix E Kit Method for Glucose ....................................................................... 57
Appendix F Kit Method for Cholesterol ................................................................. 58
Appendix G Kit Method for Triglycerides ............................................................... 60
Appendix H Kit Method for Insulin .......................................................................... 62
Appendix I Method for Succinate Dehydrogenase Activity .............................. 64
List of References ......................................................................................................... 66

LIST OF FIGURES

Figure 1: THE EFFECT OF DIET AND EXERCISE ON BODY WEIGHT .......... 26
Figure 2: THE EFFECTS OF DIET AND EXERCISE ON FOOD INTAKE ......... 29
Figure 3: THE EFFECT OF DIET AND EXERCISE ON GLUCOSE
CONCENTRATION .................................................................................... 3 1
Figure 4: THE EFFECT OF DIET AND EXERCISE ON INSULIN
CONCENTRATION .................................................................................... 33
Figure 5: THE EFFECT OF DIET AND EXERCISE ON CHOLESTEROL
CONCENTRATION .................................................................................... 35

Figure 6: THE EFFECT OF DIET AND EXERCISE ON TRIGLYCERIDE
CONCENTRATION .................................................................................... 36

vi

LIST OF TABLES

Table 1: THE EFFECT OF DIET AND EXERCISE ON SELECTED TISSUE
WEIGHT AND SOLEUS MUSCLE SUCCINATE DEHYDROGENASE
ACTIVITY LEVEL AFTER 13 WEEKS ....................................................... 27

Table 2: THE EFFECT OF DIET AND EXERCISE ON MEAN RELATIVE FAT
DEPOTS WEIGHTS AFTER 13 WEEKS ................................................. 27

Table 3: THE EFFECT OF DIET AND EXERCISE ON FOOD INTAKE IN FATTY
ZUCKER RATS ............................................................................................. 3O

vii

LIST OF APPENDICES

Appendix A. Pilot Study ............................................................................................. 48
Appendix 8. Procedure for Preparation of Tepary Beans ................................... 53
Appendix C. Composition of Diets ............................................................................ 54
Appendix D. Treadmill Running Protocol ................................................................ 56
Appendix E. Ifit Method for Glucose ........................................................................ 57
Appendix F. Kit Method for Cholesterol .................................................................. 58
Appendix G. Kit Method for Triglycerides ................................................................ 60
Appendix H. Kit Method for Insulin ........................................................................... 62
Appendix l. Method for Succinate Dehydrogenase Activity ............................... 64

viii

CHAPTER I
INTRODUCTION

Non-insulin dependent diabetes mellitus (NIDDM) is a condition
characterized by hyperglycemia, significant insulin production, and insulin
resistant tissue [25]. it is often accompanied by obesity, coronary heart disease.
renal disorders and a variety of retinopathies [2,7,12,19,20.25.43.62]. Its
occurrence has been shown to be both genetically [2] and environmentally
linked [2.45.61]. Variables such as diet and activity level often play a role in the
maintenance of glucose homeostasis in this diseased state [6].

Among the Pima Indians. a population of native Americans. the highest
incidence of NIDDM in the world has been documented [5,33,61,69]. Diabetes
was almost unknown among the Pima's in the early 1900's but has been
recognized as a common disease since the 1950's [5]. Presently, it is a major
health problem afflicting half the adult Pima population over 35 years of age [3].
Examination of this society reveals that both obesity and NIDDM have become
common disorders in the Pima population in the 20th century while the
incidence of coronary heart disease (CHD) remains low [35]. Findings suggest
that the increased incidence of NIDDM and obesity is the result of lifestyle
changes experienced by the Pima's, such as the disruption in their traditional
lifestyle. and the introduction of a steady food supply [26.30] as a result of
urbanization during the mid-1900's [2.46.61]. As a population, the lifestyle of
this group of individuals has been studied by various researchers, in hopes of
identifying any unique characteristics which could potentially play a role in the

development of such a high incidence of diabetes. This study will examine the

2

effects of a moderate intensity exercise program and a diet high in a legume,
the tepary bean. which was specific to the traditional diet of the Pima Indians in
the beginning of the 20th century. on serum glucose. insulin, cholesterol and
triglyceride levels in the Fatty Zucker(fa/fa) rat. These blood serum parameters
were chosen since they represent traditional markers of the development and

progression of the NIDDM state in this particular animal model.

Will.

The need for this research lies in the potential identification of factors
which may effect the incidence. and severity of. N lDDM in the Fatty Zucker rat.
Such findings may facilitate the direction for human studies in regard to
diabetes mellitus and its relationship to diet and exercise, and diabetes mellitus
and coronaIy heart disease (CHD). especially among the Pima Indian
population. For instance. microvascular disease. particularly of the coronary
vessels. accounts for 75% of all deaths in diabetics [19]. However, the
prevalence of CHD among the Pima tribe is low [32,35] despite the high
prevalence of diabetes mellitus in the tribe [3.4.5.35]. The Pima Indians also
have a lower frequency of clinically apparent myocardial infarction than the
Caucasian population [35]. Serum cholesterol levels for Pima Indians over the
age of 40 years is low with readings below 200 mg/dl [35]. Although these
levels are higher than non-diabetic cholesterol levels the difference is not
statistically significant [35]. Several studies have confirmed that a deterioration
of glycemic control elevates lipid and lipoprotein levels and that
hypertriglyceridemia and hypercholesterolemia are common among diabetics
[29,56]. These observations support the fact that there is a need for further

investigation regarding lipid levels and NIDDM in order to gain further insight

3

into the reasons for the greater incidence of CHD among diabetic Caucasians
than among diabetic Pima Indians and non-diabetic Caucasians.

An overview of the literature reveals that there have been numerous
studies performed which examine the individual effects of diet and exercise on
blood glucose parameters of diabetes mellitus. However. the lack of studies
which examine synergistic effects of leguminous seeds and exercise on
glucose. insulin, triglycerides. and cholesterol levels in NIDDM rats reinforces

the need for this study

WW1.

The focus of this research is to examine the effect of a tepary bean diet
and a control diet in conjunction with exercise on glucose, insulin, triglyceride,

and cholesterol concentrations in the Fatty Zucker (fa/fa) rat.

Diwali.

There will be a significant difference between the blood glucose, insulin,
cholesterol, and triglyceride concentrations in the Fatty Zucker (fa/fa) rats in both
the tepary bean diet and exercise group and tepary bean and no exercise

versus both the control groups.

WWII.

The Fatty Zucker rat has been chosen as the model for this research
study since it is shown that its genetic predisposition for NIDDM characteristics
(hyperinsulinemia, hyperlipidemia. obesity. euglycemia and insulin resistance )
are such that it models the NIDDM state in humans [36,37,38,65,85]. Female

rats have been chosen for this study since they tend to maintain a normal body

4

weight in response to an increase in energy expenditure and therefbre are
more suitable for metabolic studies [64].

Dietary manipulation is the chief recommendation for NIDDM patients
[49]. A high complex carbohydrate diet is a treatment known to enhance insulin
stimulated glucose uptake by the skeletal muscle [37] and improve glucose
tolerance [57] thus preventing sharp rises in blood glucose and insulin levels
[44,57]. Findings Show that legumes retard rises in blood glucose and insulin
levels and are effective in controlling blood lipid levels [44.57]. In order to
examine the effect of a legume diet on the occurrence. and severity of NIDDM it
was necessary to determine the effect of this dietary manipulation on metabolic
factors known to be affected by the diabetic state. A diet containing a legume
(tepary bean) known to be consumed by the Pima Indian in the 1900's was
examined. Henceforth the tepary bean diet will be the experimental diet
referred to as (T). The control diet is designed to parallel the tepary bean diet
composition except for a variance in the source of kilocalories (kcal) derived
from carbohydrates (CHD) and protein (PRO). Although the source of the
carbohydrate and protein varies between the two diets. the present experiment
is designed to evaluate the effect of a tepary bean diet as a whole and then
subsequent reasearch will further assess isolated components of the tepary
bean if results from the first experiment show warrant.

It has been reported that exercise training can improve numerous cellular
functional parameters in diabetic animals [31.37.68] and, when combined with
diet manipulations. may reduce the extent of or need for drug therapy
recommended for some human diabetics [22.37]. Hence, the use of exercise as

a supporting treatment for the NIDDM rat is also a variable in this study.

WED.

Forty female Fatty Zucker rats were randomly assigned to one of four
experimental groups; tepary bean diet (T). control diet (C), exercise and tepary
bean diet (T E). exercise and control diet (CE). The animals in the two exercise
groups were subjected to a 13 week treadmill running program. It took the
animals five weeks to be able to run at the appropriate speed and duration.
During the last eight weeks they ran for sixty minutes per day five days per week
at 20 m/min [75]. All sedentary rats were handled in the same fashion as the
exercised animals except they did not run. Blood samples were drawn from the
tail artery weekly and analyzed for glucose and insulin at 0, 4, 8, and 13 weeks
and cholesterol and triglycerides at weeks 0, 6/7, and 13. Upon completion of
the 13 week experimental program animals were sacrificed using
methoxyflurane. Succinate dehydrogenase activity in the soleus muscle was
measured in order to assess the effectiveness of the training program [75].
Heart, liver, soleus, gastrocnemius and plantaris weights were measured in
addition to the assessment of the inguinal, parametrial, perirenal/retroperitineal
fat depots weights.

Mean values of control and experimental plasma insulin, glucose.
triglyceride, and cholesterol levels for each sampling period were compared
using a 1 x1 analysis of variance (group x week and week x group). Significant
interactive effects, as evidenced by repeated measures, were analyzed using a
post-hoe Scheffe' for between group comparisons. The level of significance

chosen was p< 0.05 in all instances.

Limitations.

If a difference between the two diets is found it will not be clear as to

whether it is the protein or carbohydrate source of the particular which diet was

6

responsible for the changes in blood serum parameters. In addition. any
Significant findings will be specific to a study with the same exercise program,
animal model. and diets.

Any benefits that may be revealed in this study for humans can only be

speculative due to the fact that an animal model was used.

Definitions.
Diabetes mellitus- chronic metabolic disorder characterized by

hyperglycemia, relative or absolute insulin deficiency, and deranged
carbohydrate, fat and protein metabolism.

DL-methionlone- essential amino acid.

Euglycemla- normal level of glucose in the blood.

Glycosuria- presence of glucose in urine.

Hypercholesterolemia- an excess of cholesterol in the blood. in the human
a value in excess of 240 mg/dl.

Hyperllpemia- elevated triglycerides in plasma.

Hyperlipidemla- elevated concentration of any or all lipids in plasma.
Hyperglycemia- abnormally increased content of sugar in the blood, fasting
venous blood levels of >140 mg/dl.

Hyperinsulinemla— excessive levels of insulin in the blood. A normal range
would be 5-20 uU/ml and a value in excess of that range would be considered
abnormal.

Hyperllpoproteinemla- excess of lipoproteins in the blood. usually a familial
disorder. Type IV involves a high level of VLDL and LDL values in the normal
range.

Hypertriglyceridemia- excess of triglycerides in the blood, values >500
mg/dl.

Insulin-dependent diabetes mellitus (IDDM)-usually develops before 25
years of age. Subjects are susceptible to ketosis. and are usually not obese; it
is characterized by absolute insulin deficiency, beta—cell lesions and necrosis.
Impaired glucose tolerance- non-diagnostic fasting plasma glucose levels
and levels during glucose tolerance test which lie between normal and diabetic
concentrations. An impaired glucose value would be >140mg/dl.

Insulin resistance- capacity for glucose transport into muscle cell affected.

7

Leguminous dlet- consisting of bean or pea foodstuffs.

Non-Insulin dependent diabetes mellitus (NIDDM)—common
characteristics are hyperinsulinemic, hyperglycemic, significant insulin
production and resistant to insulin-stimulated glucose disposal. and is not
susceptable to ketosis.

Pima Indian- group of Indians with the highest recorded incidence of NIDDM
in the world. They are located along the Gila River in Arizona .

Prediabetes- abnormal metabolic response to glucose loading before
diabetic signs or symptoms appear.

Succinate dehydrogenase- enzyme located in the mitochondrial matrix.
Catalyzes the reaction by which succinate is converted to fumerate. The activity
of succinate dehydrogenase in soleus muscle is used to examine changes in
aerobic capacity in response to training.

Zucker rat- the strains in the Zucker rat are Sherman and Merck stock. The
obesity of the fatty Zucker rat is autosomal recessive gene, fa.

CHAPTER II
REVIEW OF LITERATURE

SSW. Non-insulin dependent diabetes mellitus (NIDDM)
is a genetic and lifestyle disease strongly associated with obesity [7.22],
coronary artery disease, and various neurological impairments. The primary
control for NIDDM has been a high complex carbohydrate diet and exercise
coupled by weight loss [22,30]. It is the purpose of this review to provide the
reader with a synopsis of pertinent research specific to the nature of the
proposed study. The design of this chapter will be such that an overview of the
etiology of NIDDM will be discussed initially, followed by a review of the
environmental factors which have been documented as significant contributors
to the onset of NIDDM. The interrelationship between environmental factors,
particularly as they apply to the design of this study and their effect on blood
insulin, glucose, cholesterol, and triglyceride concentrations will be
emphasized.

W Diabetes mellitus can be described as a chronic
metabolic disorder characterized by hyperglycemia, relative or absolute insulin
deficiency. and deranged carbohydrate, fat and protein metabolism [2.25]. Over
time it will lead to the development of chronic complications such as

degeneration of vascular, organ and neuromuscular systems [11.25].

9

There are two primary types of diabetes mellitus: insulin dependent
(IDDM) and non-insulin dependent diabetes (NIDDM). Insulin dependent
diabetes, also known as juvenile diabetes, develops in youth or childhood.
These individuals are susceptible to ketosis. and are not usually obese. Insulin
dependent diabetes is also characterized by absolute insulin deficiency. beta-
cell lesions and necrosis [2,25]. Non-insulin dependent diabetes mellitus
(NIDDM) which is the more common form of diabetes. typically will occur in
obese (80% over the relative body weight) adult subjects. The common
characteristics of NIDDM are hyperinsulinemia, hyperglycemia, significant
insulin production and resistance to insulin-stimulated glucose disposal. and
lack of ketosis [2.25]. Non-insulin dependent diabetes mellitus is insidious and
has a strong familial pattern. The individual susceptible to NIDDM may remain
in a 'prediabetic" state before pathogenic environmental factors cause the
disease to surface in the form of abnormal glucose tolerance [2]. Alternatively
the genetic predisposition to NIDDM may not ever clinically express itself [2]. If
NIDDM does surface it progresses in three stages: 1) "prediabetes" with
abnormal glucose loading response. 2) impaired glucose tolerance and 3)
overt diabetes in which the classic diabetic symptoms are present [2]. In fact, it
is common for a person with a genetic predisposition for NIDDM to remain
asymptomatic for years before factors such as pregnancy or weight gain
catalyze the onset of the syndrome [2]. In summary. the progression of NIDDM
from stage to stage evolves slowly over time or in a rapid manner depending on
the genetic susceptibility and/or environmental factors involved [2].

The prevalence rate of NIDDM can vary from country to country and even
within a country. However. NIDDM has a greater incidence in the United States
than in Europe [2]. For example, the overall prevalence rate for diabetes in

Denmark is 0.4%, Norway 0.7%, England and Scotland 0.6%, versus an

10

estimated 6.6% of the individuals 20-74 years of age in the United States [2,27].
In several populations there has been a recent discovery of epidemic rates of
diabetic individuals. Examples of such populations include the Pima Indians in
Arizona [2], the Tamul speaking Eastern Indians in South Africa [39]. and some
Micronesian and Melanesian populations [86]. The frequency of diabetes
among the adults in these populations is greater than 20% [2.39.86]. In these
instances hereditary is not the sole contributor to the onset of diabetes but
instead, a combination of both genetic and environmental factors are implicated
in the onset of NIDDM [2,25]; thus supporting the evidence that NIDDM is a
group of syndromes with a variety of genetic and environmental causes [25].

Wars. The environmental factors which may
contribute to the evolution of NIDDM include. but are not limited to, obesity,
nutrition and physical inactivity [26.30.46]. all of which may be operative in
different proportions on muscle insulin resistance [37].

Obesity. Considerable evidence exists indicating a strong association
between obesity and NIDDM [22]. It has been shown that NIDDM occurs 2.9
times more in obese than non-obese individuals [69]. Subjects with a recent
onset of NIDDM are more obese than those in which NIDDM has not developed
[2.69]. Furthermore, 60-90% of all NIDDM subjects in the United States are
obese [2]. There is also a high correlation between the degree of obesity and
prevalence of NIDDM [2.69]. The hypothesis for the relationship between
obesity and development of NIDDM is that obese patients develop NIDDM due
to an increase in resistance to insulin [83]. Conversely, weight loss, especially
when in the early stages of obesity. leads to an improved glucose tolerance [2].
Obesity is associated with the deterioration of glucose tolerance and an
increase in tissue insulin resistance in both animals [53] and humans [2.55.84].

The effect of obesity on insulin resistance may be partially explained by the fact

11

that in obesity the muscle cells become enlarged. thus reducing the number of
access sites available to insulin [52,85]. In contrast, others have attributed
insulin resistance to the decreased effect of insulin on glucose metabolism in
adipocytes at the receptor and post-receptor level [84]. Other data indicates that
a decrease in glucose transport occurs in both adipocytes and muscle in
genetically obese and hyperinsulinemic rats and mice [47,53].

The most effective treatment for obesity is weight reduction. which can
ultimately improve or restore carbohydrate tolerance [2]. The ideal approach
toward weight loss is caloric restriction, dietary revision (e.g. increase fiber,
decrease fat consumption), and a chronic exercise program to help facilitate
caloric expenditure and enhance skeletal muscle insulin action [37]. Although
the evidence indicts obesity as the precipitating factor in the onset of NIDDM. it
is possible that genetic predisposition or other environmental factors
independently or synergistically result in both diabetes and obesity.

Nutrition. People predisposed to. or suffering from NIDDM, are advised
to avoid sharp rises in blood glucose and insulin levels [44.57]. A treatment
known to enhance the insulin action of skeletal muscle is a high complex
carbohydrate diet [37]. It has been shown that complex carbohydrates travel
more slowly through the small intestine [79], unlike other forms of carbohydrate
and nutrient sources which are digested rapidly in the upper gastrointestinal
tract. It is this reduced rate of digestion which is believed to cause the low blood
glucose response and subsequent decrease in insulin levels [40.41.42.80].
This is the basis for the recommendation that diabetics consume foods which
are high in complex carbohydrates and high in fiber as a means by which to
manage their diabetes [79]. One such form of complex carbohydrate which has
been given considerable attention in the past few years for its role in glycemic

control is leguminous fiber.

12

Legumes, In the past few years studies have examined the role of
leguminous fiber on metabolic control in IDDM and NIDDM [40-42,44,57.59].
The choice of legumes as a primary component for the diabetic diet is based on
the beneficial effect of legumes on glycemic control [40,41,42.57.77,79] and on
the reduction of serum cholesterol and triglyceride concentration in
hyperlipidemic individuals [8,77]. The mechanism behind this phenomenon is
believed to be the delay in absorption of glucose and fatty acids in the small
intestine which is caused by the leguminous fiber.

Karlstrom, Bengt, Asp, Boberg, Lithell and Berna [44] found that NIDDM
patients who consume a diet of beans and peas show an improved glucose
tolerance. Madar [57] in his study assesses soybean fiber versus rice fiber and
their respective effects on glucose control and lipid metabolism in diabetic rats.
He concludes that soybean as a fiber source is a potentially beneficial adjunct
to the treatment of diabetes due to its ability to delay glucose and triglyceride
absorption in the upper portion of the small intestine. Triglyceride and
cholesterol levels decreased in the rats fed the soybean diet but not in the
control rats. Jenkins and associates [40.41.42] in several studies, compare the
glycemic response of NIDDM patients to a variety of foods and found that the
glycemic response for the legumes is well below the mean of the other foods. In
particular, kidney beans. chick peas and lentils. all have glycemic effects which
are significantly below those of the non-leguminous foods. Jenkins, Wolever,
Taylor and Fielden conclude that the low glycemic response produced by these
leguminous foods is due to their slow digestion and the resultant malabsorption
[41]. Simpson, Lousley. Geekie. Simpson. Carter. Hockaday and Mann [77]
also performed a study comparing a high carbohydrate leguminous fiber diet
with a low carbohydrate diet on NIDDM and IDDM patients for six weeks. They

conclude that the high carbohydrate, high leguminous fiber diet results in

13

substantially greater blood glucose control in the NIDDM and IDDM Subjects
than subjects on the low carbohydrate diet [77]. In fact glucose. triglycerides.
and cholesterol were found to be significantly lower in the legume diet group.
The data also show that insulin levels were lower in the legume diet group, yet
not significantly [77]. The success of a legume as a source for glycemic control
is attributed to the gel-forming fibers which comprise the leguminous seed
because they are believed to retard the absorption and digestion of
carbohydrates [40.41.57.77].

In addition to the carbohydrates and gel-forming fiber component of
legumes. antinutrients. such as enzyme inhibitors, tannins. lectins, and phytic
acid, may also affect the rate of digestibility and glycemic response [79,80].
Thompson [79] conducted a study to test the hypothesis that antinutrients lower
the rate of starch digestion and blood glucose response to carbohydrate foods.
He analyzed several leguminous and non-leguminous foods for lectins, phytic
acid. tannins, and amylose and trypsin inhibitors. These foodstuffs were then
tested for in-vitro rate of starch digestion in diabetic and non-diabetic individuals
[79]. The researchers conclude that legumes with the highest phytic acid, lectin,
and tannin concentration are digested slowest and produced the flattest blood
glucose response [79]. Furthermore, in addition to these findings it is important
to recognize that legumes contain 510% more amylose starch than
amylopectin starch [73]. It has been shown that amylose starch is digested
more slowly than amylopectin starch [80]. Above-average proportions of
amylase in legumes is yet another proposed hypothesis for its' success in
glycemic control [16].

These findings suggest that starch digestibility and glycemic response
can be affected by factors such as gel-forming fibers. antinutrients, amylose and

amylopectin content. These factors may have some beneficial effect in the

14

management of diabetes in which the rate of glucose absorption is Critical.
Evidence also exists for the benficial effects of the nutritive components of
legumes on triglyceride and cholesterol levels [57,77]. Madar [57] found that
soybean fiber significantly decreases triglyceride and cholesterol
concentrations in diabetic rats when compared with rats fed rice fiber.
According to Simpson [77]. a leguminous fiber diet significantly reduces
cholesterol levels in NIDDM subjects.

Physical activity. Inactivity also has been identified as a potential risk
factor for adult-onset diabetes mellitus [6.7]. Chronic physical exercise
increases aerobic capacity and causes multiple adaptations in the
cardiovascular system [11]. It has also been noted as being a beneficial adjunct
to diet as a means of metabolic control in NIDDM subjects [13.22.77] and
animals [37.38.67]. The purpose of this section is to review the effects of
chronic exercise on serum triglycerides. cholesterol, insulin and glucose. Of
these parameters hypertriglyceridemia and hypercholesterolemia are the most
common in diabetes mellitus [19]. There is compelling evidence that
hyperlipidemia is causal or primary in the development of atherosclerosis [19].
Atherosclerosis seems to proceed at a more rapid rate and more extensively in
diabetics [19]. The basis for atherosclerosis being the leading cause of death in
the diabetic stems from the fact that diabetes intensifies most of the risk factors
for atherosclerosis [2]. Data shows that diabetics have a two times greater
chance than non-diabetics to develop CVD after the age of 40 [2.72]. Evidence
derived from clinical and autopsy data reveals that CVD accounts for 75% of all
deaths in diabetics versus 50% in the non-diabetic population [19]. This
provides a basis for examination of the effects of exercise on cholesterol and

triglycerides in addition to insulin and glucose.

15

W Several investigators have examined the effects of
endurance exercise programs of various lengths, and found that exercise
decreased total plasma triglycerides in non-diabetics [33.48.49.501 and NIDDM
subjects [68] when their baseline values are elevated. Data from several
studies reveal that triglyceride levels became lower as a result of exercise alone
in both human subjects [33.48.49,50.68] and animals [1.75.76]. More
specifically. Becker-Zimmerman. Berger. Berchtoid. Gries. Herberg. and
Schwenen [1] reports that a 12 week training program involving 7 week old
Fatty Zucker rats decreased their triglyceride levels. Huttunen, Lansimies.
Voutilainen, Ehnholm, Heitanen, Penttila. Siitonen. and Rauramaa [33]
postulate that chronic exercise effects VLDL synthesis or catabolism. Abnormal
insulin action is implicated as the cause for elevated triglyceride levels. This is
due to the fact that there is an insufficient increase in insulin production. a
decrease in peripheral glucose uptake, and a subsequent increase in free fatty
acid metabolism [66]. The result is VLDL-triglyceride production and an
increase in plasma triglyceride concentration [66]. Since exercise training
decreases insulin levels by increasing the peripheral sensitivity to insulin [51].
The increase in peripheral insulin sensitivity helps to regulate free fatty acid
metabolism which subsequently decreases the triglyceride production and
VLDL concentration [24.51].

mm Based on the variable results of several studies,
measurements on pre- and post-exercise training blood cholesterol levels do
not reveal such a clear consensus [1 ,33,49.50.68.81]. Lampman. Santinga,
Hodge. Block, Flora, and Bassett [50] found no appreciable decrease in
cholesterol levels in Type IV hyperlipoproteinemic men after a 10-week training
program. Further research by Lampman. Santinga, Bassett. Hydrick. Flora, and

Block [49] reveals that there is a significant decrease in cholesterol levels after

16

a 9 week training program Involving middle-aged hypertriglyceridemic men.
Tran and Brammell [81] found that physical training has a positive effect on
decreasing blood cholesterol levels especially in sedentary individuals and
those with high cholesterol levels. Ruderman et al. [68] conducted a 3-6 month
training program using NIDDM subjects and found a modest yet significant
decrease in cholesterol levels. Huttunen and associates [33] report a slight
decrease in cholesterol levels after a four month training program involving
non-diabetic males. Becker-Zimmerman et al. [1] conclude from their study that
a significantly lower serum cholesterol level in the fatty Zucker (fa/fa) rat can be
obtained after a 9-week training program. However. 25-week old rats from the
same study Showed no change in their cholesterol levels [1]. Becker-
Zimmerman et al. [1] attributes this phenomenon to the fact that by 25 weeks of
age the Fatty Zucker (fa/fa) rats already developed their metabolic syndrome
thus most treatments at this age are ineffective.

mm The effects of exercise on insulin levels is controversial. For
instance, Walberg, Mole. Sern [82] demonstrate that exercise swim training
significantly decreases the blood insulin levels in obese rats by half. Horton
[31] studied the effects of 6-week training program on fatty Zucker rats and also
found a significant decrease in insulin levels. Ten weeks of physical training
involving type IV hyperliproteinemic men resulted in a significant decrease in
blood insulin concentration [50]. Lampman et al. [49] conducted a subsequent
study and again discovered a significant decrease in insulin levels in
hypertriglyceridemic men after a 9-week training program. Crettaz. Horton,
Wardzala. Horton. and Renaud [17], however. demonstrate in their study that
physical training does not prevent the development of a hyperinsulinemic state.

Bogardus. Ravussin, Robbins. Wolfe, Horton, and Sims [10] examined

the effect of 12 weeks of a physcial training program and a hypocaloric diet in

17

NIDDM females. Improvements in both fasting plasma glucose and insulin are
shown in both the diet only group and the diet plus physical exercise group.
Lampman et al. [49] reports stable fasting glucose concentrations as a result of
9 week study using hypertriglyceridemic men. Lampman, Schteingart. and
Foss [52] report that with weight loss there is decrease in glucose and insulin
levels with the exercise groups showing the greatest decrease. Ruderman,
Ganda. and Johansen [68]. however, found no improvement in fasting glucose
and insulin concentrations after 14-34 weeks of physical exercise.

mum. Physical training has been shown to produce improved glucose
tolerance in NIDDM in both humans and rodents
[1 ,17.23,25.55.68.71 ,78.83.84]. Becker-Zimmerman et al. [1] found that insulin-
stimulated glucose uptake is significantly increased in 6-week-old rats trained
120 min/day, 12 m/min for 9 weeks on a treadmill versus an untrained age
matched group of rats. In addition. the researchers found marked deterioration
of glucose tolerance in a group of 25 week-old sedentary group which was not
evident in the exercise (12 m/min for 30 min/day for 6 weeks) group, although
both groups doubled their body weight during the course of the study. They
conclude that the prevention of glucose intolerance in the younger rats is
perhaps due to preservation of insulin sensitivity via the exercise program
during the rapid weight gain phase (7-16 weeks old). The 25 week old rats did,
however, experience significant improvement in insulin sensitivity and glucose
tolerance. Crettaz et al. [17] report that a treadmill training program in which the
rats ran for 2 hours/day at 20 m/min at an 8% grade also improves glucose
tolerance in obese Zucker rats. The researchers did. however, emphasize the
fact that training did not prevent the development of obesity, hyperinsulinemia,

or insulin resistance in the genetically obese animals [17].

18

Schneider. Amorosa. Khachadurian. and Ruderman [71] discovered after
a six week. 3 times a week training program with human NIDDM subjects. that
glycemic control and glucose disposal is improved. The researchers
emphasized the importance of exercising three or four times a week to achieve
continuous improvement in glycemic control [71]. Skor, Gavin, Hagberg.
Schuttz. Santiago. and Goldberg [78] conclude that chronic exercise training
does improve insulin sensitivity and increases muscle glucose utilization in
NIDDM subjects. Acute exercise, however, does not increase glucose
utilization. This improvement in insulin sensitivity may reflect post-receptor
changes or the effect of exercise on glucose transport [78]. Horton [31] went on
to support the participation of physical activity programs by NIDDM subjects as
a therapeutic means by which to manage the NIDDM and insulin resistant state.
Ruderman et al. [68] also concludes that physical activity enhances insulin
sensitivity in previously inactive NIDDM men. Thus. these studies support the
fact that chronic physical exercise increases muscle sensitivity to insulin action
and increases the rate of glucose disposal. Chronic exercise also has the
ability to prevent the impairment of glucose transport capacity in insulin deficient
animals and humans. These findings indicate that physiological adaptations
take place in response to chronic exercise. while acute muscular activity may
not produce the same long-term response. It is evident from the above
information that an exercise program helps to combat the high levels of
triglycerides. cholesterol. insulin. and glucose associated with NIDDM.

WW There are many studies examining the
Independent effects of training and dietary intervention on reducing muscle
insulin resistance. however there is little information on the combined effects of
these two factors. Ivy, Sherman. Cutler, and Katz [[37] examine the effects of a

high carbohydrate diet and exercise on muscle insulin resistance in obese

19

Zucker rats. They conclude that a treadmill training program at a 8% grade.
18m/min. 5 days week for 6 weeks significantly reduces insulin resistance due
to the increase in the rate of muscle glucose uptake after training regardless of
the diet. Ivy, Brozinick, Torgan. and Kastello [38] conducted another study
involving Fatty Zucker rats on a treadmill training program at 18 m/min, 1.5
hours/day. 5 days/week for 6-8 weeks. The investigators conclude that there is
an improvement in muscle insulin resistance associated with an improvement in
the glucose transport process. but the enhancement is specific to the red
gastrocnemius muscle and plantaris muscle.

An overview of the literature reveals that there have been numerous
studies performed which examine the individual effects of diet and exercise on
blood glucose parameters of diabetes mellitus. However, the lack of studies
which examine the synergistic effects of leguminous seeds and exercise on fatty
acid metabolism and glucose homeostasis in NIDDM rats reinforces the need
for this study. In addition. there is compelling evidence that hyperlipidemia is
causal or primary in the development of atherosclerosis [19]. In fact. it is shown
that increased triglyceride and cholesterol levels are positively correlated with
CVD [43]. However, hyperlipidemia combined with diabetes mellitus generally
can be treated with adequate diet, weight loss and exercise. Thus, the key to
controlling the emergence of diabetes mellitus and CVD is by monitoring these
factors; obesity, diet. and physical activity. One must realize the hazards of
inadequately controlled diabetes and the probable benefits of adequately
controlled diabetes mellitus. Such an investigation may potentially help to
control the incidence of diabetes mellitus in the native American population

where legumes were once an integral part of their diet.

CHAPTER III
METHODS

The focus of this research was to compare and contrast the effect of a
leguminous diet and a semipurified diet on glucose. insulin. triglyceride, and
cholesterol concentrations in sedentary and endurance trained Fatty Zucker
(fa/fa).rats

Anjmals. Since the Fatty Zucker (fa/fa) rat has previously been shown
to provide a good model for simulating NIDDM in humans [1.34.65] as well as to
exhibit compliance to exercise training [1 ,17.37,38,75.84] the Fatty Zucker rat
was chosen as the animal model for this research. The Fatty Zucker rat exhibits
NIDDM between 3-6 months of age [14.65]. The symptoms include
hyperglycemia. hyperinsulinemia, and hyperlipidemia [14.15.34]. The Fatty
Zucker (fa/fa) rat does not however exhibit signs of ketosis, infertility, weight
loss, or Beta-cell damage [14.15.34] which are also symptoms absent in the
diabetic state of the Pima Indian population [45.69].

Forty female Fatty Zucker (fa/fa) rats. six-weeks of age, were obtained
from Charles River Laboratory (Kingston, MA). Animals were housed in
individual cages in an animal room which was kept at 22 C :t 2 degrees and
maintained a 12-hour light/dark cycle. The animals were fed rat chow and

water ad Iibitum for 5 days so as to allow the animals to become familiarized to

20

21

their surroundings. On day six the animals were considered to be acclimatized
and onset of the experimental treatment was begun.

Animals were first matched according to body weight and then either
assigned to control (C; n=10); control exercise (CE; n=10); tepary (T; n=10); or
tepary exercise (TE; n=10) groups. The animals in the T and TE groups were
fed a tepary bean diet. groups C and CE received a purified control diet. The
composition of the tepary bean diet was based on the results of a previous pilot
study (Appendix A). The differentiating factor between the control diet and
tepary bean diet was the carbohydrate and protein sources. The tepary bean
being the primary source for protein and a contributing source of carbohydrates
in the T and TE diets versus casein and cornstarch in the control diet. Based on
kilocalorie (kcal) per gram the control diet was 19.0% protein, 45% CHO and 35
fat with total kcals equaling 454/1 009. The tepary bean diet was 17% protein,
46% CH0 and 37% fat with the total kcals equaling 450/1009. The preparation
procedure for the beans and a description of the two diets are contained in
Appendices B and C respectively. The groups were provided with food and
water ad Iibitum except when the food was removed 12 hours prior to a fasting
blood draw.

W The training for the exercise groups (CE and TE)
consisted of running on a motor driven treadmill at an initial speed of 15m/min
for 15 minutes. The speed and duration were increased gradually until the
animals were able to run at a speed of 20 m/min for 60 minutes. The animals
ran at 20 m/min for 60 min for 8 weeks, it took 5 weeks to train them to run at the
appropriate duration and speed. The running increment sequence is described
in Appendix D. This intensity and duration was selected since it has been
shown previously to elicit a training effect in Fatty Zucker rats [75]. The treadmill

structure was such that only 5 rats could run at any one time, each in her own

22

lane. The running lanes were covered with a screen but it was still possible to
nudge the rats if they would not run. No electrical shock was used. All
sedentary rats were handled in the same fashion as the exercised rats except
that they did not mn.

WW Food intake was assessed 2
times a week in conjunction with changing of the cage liners and food cups. In
order to measure the food that had spilled and fallen through the wire mesh
caging, paper toweling was placed under each cage. Food cups were weighed
and filled at the beginning of the week and then reweighed in the middle of the
week. The spilled food was collected by sorting through the animals feces and
adding that weight into the total food cup weight.

The animals were weighed once a week in a fasted state. prior to their
blood draw.

WW Fasted (12-15 hours) blood samples were
drawn from all animals at 0, 4, 8, and 13 weeks. In addition, blood was sampled
from one-half the animals from each group on alternate weeks. Care was taken
to ensure that sampling order was such that the average fasting time was
comparable across groups. Animals in the exercise groups (TE and CE) were
sampled 41-45 hours post their last exercise bout including week 13. This time
frame was chosen since in humans, it has been found that within 24 hours post-
exercise all exercise-induced changes in muscle glucose metabolism have
returned to normal [31]. Consequently any changes in blood glucose, insulin,
cholesterol and triglycerides which might be observed are the result of a chronic
not acute training affect. Animals were anesthetized using methoxyflurane.
Anesthesia was administered and maintained by a nose cone containing a
cottonball saturated with methoxyflurane. Once the animal was deemed

anesthetized by the absence of the toe pinch reflex and the animal exhibited

23

slow deep breaths the distance of the nose cone was adjusted to maintain a
stable level of anesthesia [9]. While in a dorsal recumbent position, ophthalmic
ointment was placed over the animal's eyes and the rat's tail was placed in a
container of warm water to assist in dilating the tail artery and to wash free any
urinary sugar that may be present on the tail [74]. A fcc syringe with a
heparinized 22 gauge x1 inch needle without the plunger was used to obtain 1
mL of blood. This volume was chosen since it was equivalent to approximately
1% of the rats' body weight and would not compromise the animals blood
volume. The bevel of the needle was kept up and the needle was guided into
the tail artery at a 20-30 degree angle [9]. Entrance into the artery was signaled
by a 'pop"[9]. Once in the artery the syringe was filled to the desired capacity.
Immediately upon withdrawal the blood was placed in a test tube and kept on
ice [9]. The needle was removed and pressure was then applied to the
puncture site with a bandage [9]. The tail bandage was removed 15-30 minutes
later [9]. The animal was placed alone in a recovery cage, lined with toweling
(not chips) until restoration of full motor coordination was exhibited by the
animal [9].

Upon completion of collections, all samples were centrifuged for 20-30
minutes at 1500xg. The total serum from the blood samples was removed and
stored at -20 C. Serum was later analyzed for glucose. insulin, triglycerides,
and cholesterol. Serum insulin and glucose were assessed from those
samples obtained at 0. 4. 8. 13 weeks. Serum triglyceride and cholesterol were
assessed from samples drawn at weeks 0. 6 or 7. and 13. Sigma R diagnostic
colormetric kits were used for glucose (Appendix E). cholesterol (Appendix F)
and triglyceride analysis (Appendix G). A Wako R diagnostic kit was used to

analyze insulin levels (Appendix H).

24

Muscle. Upon completion of the thirteen week experimental. period
animals were sacrificed using methoxyflurane. Following withdrawal of blood
from the tail artery. the left soleus. gastrocnemius and plantaris muscle were
excised from each animal, the connective tissue removed and the muscles
individually weighed. Additionally. a muscle sample from the belly of the soleus
muscle was flash frozen in liquid nitrogen and stored at -80 C. This sample was
then analyzed for succinate dehydrogenase enzyme activity level according to
the methods of Cooperstein et al. (Appendix I). The information obtained from
this analysis was used to indicate whether or not a training effect did indeed
occur as well as to determine the effect of diet and/or exercise on skeletal
muscle energy metabolism. The heart and liver were also excised from each
animal and weighed. Parametrial, perirenal and inguinal fat depots were also
removed from all animals and weighed.

W Mean values of TE. T, CE. C plasma insulin,
glucose. triglyceride and cholesterol levels for each respective blood sampling
period were initially compared using repeated measures analysis of variance
techniques. Main and interactive effects were further assessed using a one-
way ANOVA (group x time; time x group). When a significant F-ratio was
obtained (p <.05). Sheffe"s post-hoc test was used to determine statistical
significance between means. Heart, liver. muscle. and fat depot weights were
compared using a one-way ANOVA. The level of significance was chosen at p

< .05 in all instances.

CHAPTER IV
RESULTS

W193, Mean animal weight for each group increased
over the 13 week experimental period (Figure 1). Average body weight was not

significantly different between the four groups during the first three weeks of the
evaluation period. but during weeks 4-13 TE had a significantly lower average
body weight than C. TE had a significantly lower average body weight than CE
throughout weeks 4-13. Upon sacrifice (week 13) mean body weight was
significantly less in TE than in any of the other three groups (C. CE, T).

Average relative organ (heart. liver). muscle (soleus, gastrocnemius.
plantaris) and relative fat depot weights (inguinal, parametrial, retroperitoneal)
are presented in Tables 1 and 2 respectively. Relative weights were
determined by dividing the weight of the organ by the animals total body weight.
Average relative heart weight was significantly greater in TE (2.279) in
comparison to T (1.989) and C (1.899) (Table 1). Mean relative gatrocnemius
weight was found to be significantly greater in TE (.1989) versus CE (.1629) and
C (.154). In addition the plantaris weight was found to be significantly greater in
TE (.0389) versus C (.0279) (Table 1). Mean relative retroperitoneai fat weight
was significantly different between CE (3.659), TE (2.799), and T (2.81 g), with
CE being greater in both instances. No Significant differences were indicated

for relative liver.

25

26

 

700

Body Weight (grams)

 

 

 

 

 

+15
-9- C ., ‘
/"
-E-T . ’5’:
/§’
/:/'/ . . 2- 2 '-
él/ :- e a # # # a

 

 

O

r1'2fi3j415161718'911011 1213
Time(weeks)

Values are expressed as mean + s.e.m. (p (05)
‘vs.C;ffvs.CE;@vs.T

FIGURE 1: THE EFFECT OF DIETAND EXERCISE ON
BODY WEIGHT

27

Table 1. THE EFFECT OF DIET AND EXERCISE ON SELECTED TISSUE
WEIGHT AND SOLEUS MUSCLE SUCCINATE DEHYDROGENASE
ACTIVITY LEVEL AFTER 13 WEEKS.

1

 

Values are expressed as means i S.E.M.
‘vs.C (p<.05)

"vs.CE (p<.05)

“'vs. T (p<.05)

Table 2. THE EFFECT OF DIET AND EXERCISE ON MEAN RELATIVE FAT
DEPOTS WEIGHTS AFTER 13 WEEKS.

 

Values are expressed as means :1: SEM
‘vs.C (p<.05)
“vs.CE (p<.05)

28

inguinal. or parametrial weights. SDH activity was significantly increased only
in TE vs. T (5.68 vs. 2.68 respectively) (Table 1). Although the difference was
not significant CE had a higher activity level than C (3.22 vs. 2.89).

In addition to these alterations in physical characteristics all animals
displayed two common manifestations of diabetes known to the small animal
population. seborrheic skin disease and varying degrees of alopecia [60]. Both
conditions are due to protein catabolism and abnormal lipid metabolism
associated with diabetes [60].

Food consumption. Significant between group differences for average
weekly food consumption (grams) were observed for week 9, (T =122.5 g,
C=152.12g) and week 10 (C=150.44 g. TE=111.5 9) (Figure 2). Since the
tepary bean diet was higher in kcals than the control diet. the average amount
of kcals for each group was compared statistically (one-way ANOVA) to
examine whether the control groups also consumed more calories than the
tepary groups. The analyses revealed that there was a significant difference in
average total kcals consumed per week when comparing TE versus C and CE.
TE consumed significantly fewer total calories over the thirteen week period
(7444.3 kcals) versus C (8846.8 kcals) and CE (8438.6 kcals) (T able 3).

W. Cholesterol. triglyceride, glucose. and insulin
values were analyzed using repeated measures ANOVA techniques. In
instances where significant interactive effects were observed one-way‘Anova
(group x time and time x group) were conducted with post-hoc analysis
(Scheffe') where appropriate

Glucose. One way analysis of variance (group x time and time x group)
for glucose revealed that there was no significant between or within group
differences (Figure 3). The trend in the data over time did however show that

average CE, TE, and T blood glucose levels were maintained or declined

29

 

220

L _..L uOI_-.

 

 

 

 

1'2'3 [4'5'6r7'8'9110j11'12113
WEEK!
Valuesueexpressedascmeaniaem. (p<.05)

‘vs.

FlGUREZ:'I'I-IEEFFECTOFDIETANDEXERCISEON
FOODINTAKE

Table 3: THE EFFECT OF DIET AND EXERCISE ON FOOD INTAKE IN

30

 

 

 

 

 

FATTY ZUCKER RATS.
PARAMETER TE (n=9) T (n=10) CE (11:9) C (n=8)
Average Total Grams 1654.3 9 1828.2 9 1858.7 9 1948.6 9
:I: 54.42 4' :l: 46.61 :1: 47.28 :I: 46.25
Average Intake per 127.3 9 140.6 9 143.0 9 149.9 9
week 1 4.19 :t 3.59 :l: 3.64 :l: 3.56
Average Total Kcals 7444.3 8227.0 8438.6 8846.8
kcals kcals kcals kcals
1 244.9” 1 209.8 1 214.7 :1: 210.0
Average Weekly 572.6 kcals 632.9 kcals 649.1 kcals 680.5 kcals
Intake per Animal :l: 18.84" :I: 16.13 :I: 16.51 :1: 16.15

 

 

 

 

 

 

values are expressed as mean 1 S.E.M.

" vs. C (p<.05)
" vs. CE (p <.05)

 

31

 

   

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fifiififi

 

.........
..................
.......

 

..........................................................
Sia:::f;.>:c$fi;ca 94+; .....

u. an...”
cfik bSfii ....

 

 

m

mme SSSSSSSS
I mm D § ....... m
.imi.m.:mwzww.zm.:m

2295 zoF<E2mozoo 3830

Group

Values are expressed as mean + s.e.m. (p < .05)
§ vs. initial

FIGURE 3: THE EFFECT OF DIET AND EXERCISE ON
GLUCOSE CONCENTRATION

32

until week 8. then increased. while group C showed a gradual increase from
week 1 to week13. Although the values were not found to be significantly
different. average TE and T glucose values were lower than those observed in
both of the control groups (CE and C) by week 13. Moreover. by week 13. the
sedentary groups (C and T) had lower average glucose values than their
exercising counterparts (CE and TE respectively), although once again these
differences were not significant.

Insulin. One way analysis of variance (group x time and time x group) for
mean insulin concentration (u/L) revealed that the TE insulin levels increased
significantly from week 4 to week 8 and week 8 to week 13 (70.57 u/L - 114.75
p/L - 134.39 IIJL) (Figure 4). For T. insulin values increased significantly from
week 1 to week 13 (99.79 M - 161.12 p/L) and week 4 to week 13 (101.89 ulL
vs. 161.12 u/L). Group CE had significantly different insulin concentration
between week 1 and week 8 (93.02 u/L vs. 142.36 p/L). between week 1 and
week 13 (93.02 u/L vs. 182.45 ML). and also between week 4 and week 13
(108.97 p/L vs. 182.45 u/L). The insulin values for group C did not significantly
increase over the 13 week period.

Initial mean insulin concentration was found to be significantly lower in
TE than C (66.75 u/L vs. 131.43 u/L). By week 4 there was a significant
difference between TE and C (72.68 pJL vs. 134.55 u/L respectively), TE and CE
(72.68 M vs. 109.98 p/L respectively) were also observed. However. at the
end of the 13 week period. the mean insulin concentrations between the four
groups was not significantly different.

Cholesterol Repeated measures analysis indicated significant between
and within group differences in cholesterol concentration. Upon further
statistical analysis. the data showed that cholesterol (mg/dl) values increased

significantly from week 1 to week 13 for C (66.15 mg/dl vs. 242.95

INSULIN CONCENTRATION (U/L)

33

 

 

 

 

 

 

7//////////////////////////////////A""°2@

 

 

 

 

 

 

Group

Valuesareexpressedasmeanisem (p<.05)
*vs.C;#vs.CE;§vs.initiai;@vs.week4

FIGURE 4: THE EFFECT OF DIET AND EXERCISE ON
INSULIN CONCENTRATION

 

 

 

 

34
mg/dl). and for CE (74.91 mg/dl to 157.77 mg/dl) (Figure 5). Additionally. C

showed a significant increase from week 1 to week 6 (66.15 mg/dl to 196.04
mg/dl). Average values for group T did not change significantly throughout the
study.

At the onset of the study. there were no significant differences for
cholesterol values between the groups. By the midpoint of the study (week 6/7)
average cholesterol for TE was found to be significantly lower than C (90.78
mg/dl vs. 196.04 mg/dl). CE was significantly lower than C (121.1 mg/dl vs.
196.04 mg/dl). and T was significantly lower than C (100.37 mg/dl vs. 196.04
mg/dl). By week 13 the data analyses indicated that TE was still significantly
lower than C (106.81 mg/dl vs. 242.95 mg/dl). and T was significantly lower than
C (109.58 mg/di vs. 242.95 mg/dl). However. C and CE by week 13 were no
longer significantly different. By week 13 both the tepary groups (TE and T)
were significantly lower than C but not CE.

Triglycerides Initial triglyceride concentration (mg/dl) was not
significantly different across the four groups (figure 6). Triglyceride values
increased significantly from week 1 to the midpoint (week 6”). and week 1 to
week 13 in C. CE and T. while only increasing significantly from the onset to the
midpoint of the study in TE. Additionally. at the midpoint (week 6/7) group C
(1207.45 mg/dl) had significantly greater average triglyceride values than TE
(398.54 mg/dl). T (434.76 mg/dl), and CE (576.6 mg/dl). However, by week 13.
the triglyceride concentration for C (994.01 mg/dl) was only significantly greater
than TE (308.74 mg/dl).

35

 

 

 

 

 

 

 

 

‘ d I d. d I d d d «I d

_
m w
1

2295 2055:2828 beam—.8405

 

§vs. initial

I"vs.C

Values are expressed as mean :1; sem (p < .05)

THE EFFECT OF DIET AND EXERCISE ON
CHOLESTEROL CONCENTRATION

FIGURE 5

 

 

 

36

 

initial

P

 

 

 

I d

 

milmizm.

00 6

2395 ZOFéZmUZOO mammU>AOE

‘2 vs. C; § vs.

THE EFFECT OF DIET AND EXERCISE ON
TRIGLYCERIDE CONCENTRATION

 

Values are expressed as mean :t s.e.m. (p < .05)

FIGURE 6

 

CHAPTER V

Discussion of Results

It was the primary focus of this research to examine the synergistic effects
of diet (tepary bean vs. semipurified) and exercise (8 weeks at 20m/min; 1
hour/day) on the serum concentrations of glucose, insulin, triglyceride and
cholesterol in the genetically obese Fatty Zucker rat (fa/fa). This animal model
was chosen since in addition to obesity, fa/fa rats have been shown to exhibit
symptoms similar to the NIDDM state. It was thought that this factor, in
conjunction with the young age of the animals (6 weeks) would provide the
experimental condition necessary to gain further perspective on early
environmental manipulations (diet/exercise) and their long term effect on the
N IDDM state of the Fatty Zucker fa/fa rat.

W. The gain in body weight which was
observed in all animals over the thirteen week experimental period was
somewhat greater than that reported by others examining fa/fa rats of a similar
age [1,21,57,75]. However, it is important to the note that the difference in diet
between the studies could have accounted for much of the observed difference
in animal body weight. The lack of between group differences in body weight
prior to week three of the experimental period suggests that neither the diet or
activity manipulation affected the normal weight gain pattern of these animals

during the early growth phase. However, the data do indicate that by week four

37

38

the combination of the increased activity level and tepary diet did prove to
decrease animal weight gain. This trend persisted until the completion of the
study when average TE body weight was significantly less than the mean
animal body weight in all other groups. These data, in conjunction with the
observed (non-significant) lower body weight in both T and TE in contrast to the
counterpart control groups (C and CE) at all timepoints after experimental onset
does suggest some positive benefits of the leguminous diet over the
semipurified diet, with the benefits being magnified when an endurance
exercise program is added. The signficant difference in weekly food intake and
weekly kilocalorie consumption between TE, C, and CE would suggest that the
TE animals failed to compensate for their increased energy expenditure due to
the exercise program, a finding similar to that observed by Walberg et al. [82].
This difference may have been due, in part to the fiber content of the
leguminous diet which differed from the control diet in regards the source of
fiber (cellulose vs. non-nutritive fiber). Mazur, Remesy, and Demigne [58]
attributes the reduction in body weight gain to the fact that nutrient absorption
and caloric utilization differs in a fiber diet. Additionally, there is a greater
feeling of satiety and less hunger accompanied by a fiber diet [58].
Consequently, this may be the reason that the tepary diet groups weighed less
than the control diet groups. It is important to also note however that CE
animals had a signficantly greater body weight than TE animals, leading one to
speculate that the exercise training was also not the sole cause of the
differences in body weight. The difference in perirenal fat depots between the
groups and the increased abdominal fat depots which were observed in the
control animals suggests that consumption of the leguminous diet decreased
the amount of adipose tissue which was deposited, a factor which may have

been causal in the decreased body weight observed in the T and TE animals.

39

Schemmel, Mickelsen, and Mostosky [70] reported in their study that there is a
linear relationship with weight gain and perirenal fat deposition. Collectively
these data suggest that there is a potential positive effect of consuming a tepary
bean diet alone, and in conjunction with exercise, on body weight gain beyond
the initial growth phase.

Regardless of diet, both exercise groups tended to exhibit lower average
body weights than their counterpart sedentary group. This finding is typical of
exercise animals [1 ,21 ,57,75,82] and is indicative of the positive effects of
exercise training on body weight. The observed differences between sedentary
and exercised animals in hindlimb muscle weight and heart:body weight ratio is
characteristic of exercise-trained animals, and is supportive of a training effect
induced by the exercise program [63]. Although both heart:body weight ratio
and SDH were greater in TE and CE versus the counterpart groups (t and C),
the difference was only significant for TE versus T. The fact that training did not
significantly increase the heart:body ratio and SDH activity for CE versus C may
be partially due to the fact that CE weighed more than TE, and therefore the CE
animals were unable to maintain the same intensity during exercise that the TE
group did.

GIMME. In accordance with the findings of Simpson, Lousley. Geekie.
Simpson, Carter, Hockaday and Mann [77] there was no significant difference in
fasting blood glucose concentration between any of the four experimental
groups. Although this might suggest that the tepary diet did not enhance
glycemic control in the T and TE animals, it is important to note that only fasting
glucose levels were assessed in this study. Karlstrom, Vessby, Asp, Boberg,
Lithell, and Beme[44] have suggested that leguminous foods are successful in
controlling blood glucose levels, yet their influence is more prevalent on post-

prandial blood glucose rather than on fasting blood glucose concentration.

40

Similarly this limitation in glycemic control analysis may account for the lack of
difference in fasting glucose levels in sedentary (C and T) and exercised (CE
and TE) animals. The positive effects of exercise training on glycemic control is
typically best demonstrated through the use of a glucose tolerance test
[37,38,49,51,75]. Therefore, it appears that before any definitive conclusions
can be made on the role of the tepary bean on glycemic control in the fa/fa rat a
more inclusive assessment of glycemic control is necessary.

Insulin, The positive effects of a leguminous diet on reducing insulin
levels in diabetic rats has been previously demonstrated Madar et al. [57].
Although isolated significant differences in average serum insulin concentration
due to diet were noted in this study the initial between group differences and the
periodicity of these differences renders the findings relatively inconclusive. It is
however important to note that by week 13 the exercise groups (CE and TE)
exhibited a lower insulin concentration than the sedentary groups (C and T).
Both Walberg, Mole and Stern [82] and Crettaz, Horton, Wardzala. Horton, and
Renaud [17] showed similar findings in their studies examining exercised Fatty
Zucker rats and suggested that the depression of hyperinsulinemia is unique to
the exercised state. The primary factor for this difference is the observed
association between hyperinsulinemia and increasing body weight [54.55.83].
Leon, Conrad, and Casal [54] noted that without weight loss chronic endurance
exercise is ineffective in lowering fasting blood glucose, insulin and lipid levels
in NIDDM subjects. In this study only TE showed a significantly lower average
body weight, a factor which may have contributed to the lack of between group
significant difference in this parameter. It is also interesting to note that Walberg
[82] emphasized that exercise did not permanently lower insulin levels and
indicated that by 23 weeks of age insulin concentration no longer varied

between sedentary and exercised fa/fa groups. Since the duration of this study

41

was not equivalent to that of Walberg [82] it is impossible to determine whether
the difference observed in this study would have continued to persist over time.

Cholesterol. In accordance with the findings of Bingwen, Zhaofing, and
Rongjue [8] and Karlstrom et al. [44] cholesterol concentration in both tepary
groups (TE and T) at week 13 were significantly lower than that observed in the
control (C) group. In addition, the tendency was for average cholesterol to be
higher in both C and CE than in T and TE throughout the study. Collectively,
these data support the findngs of Haskell [28] who noted that decreases in
cholesterol appear to be more closely associated with dietary manipulation than
with increases in activity level. Since leguminous fiber has been shown to
interfere with cholesterol absorption and bile acid reabsorption in the small
intestine [57] one might speculate that the differences observed in this study
between the two dietary groups was, in part, due to the amount of fiber and the
fiber source of the tepary bean diet. Further assessment of the composition of
the tepary diet is necessary to further define their role in cholesterol control in
the NIDDM state, although these data do suggest that the effect is positive.

The mechanism associated with the cholesterol lowering effect of
exercise is thought to be related to the intensity and duration of the exercise
training [51]. Although both exercise groups (TE and CE) had lower cholesterol
concentrations than their counterpart control group (C and T) none of the
differences observed were significant. This finding is in contrast to the work of
Shepard, Durstine, and Davis [75] and Becker-Zimmerman et al. [1] who
reported significant differences in cholesterol concentration between their
exercise and sedentary groups, but is in agreement with the findings of others
[18.49-51.56] who have failed to observe any difference due to exercise training
such as was found in this study between C and CE SDH levels. However, since

lipoprotein concentrations were not measured in this study, there is question as

42

to whether HDL cholesterol levels were higher in the exercise group [18,75] or
the between group differences were masked, thus, definitive conclusions from
these data cannot be drawn.

W- It has been shown previously that soybean fiber delays
the absorption of fatty acids from the upper small intentine and thus provides
fewer substrates for triglyceride synthesis [57]. In this study, serum triglyceride
concentration was improved in those animals who consumed the tepary bean
diet with TE being significantly lower than C at the midpoint of the experiment
(TE 398.5, C 1207.5) and week 13 (TE 308.7, C 994.0). These data do however
tend to support the benefits of a legume diet in management of triglyceride
levels in the NIDDM state since, at all time points T and TE values were lower
than those observed for C and CE. Once again, the positive affects of a
leguminous fiber dietary component in the diet of NIDDM host is somewhat
supported by these findings. The role of exercise as a positive adjunct to
dietary manipulation in lowering triglyceride concentration in the NIDDM state is
somewhat controversial. Karlstrom et al. [44] reported an improvement in
triglyceride levels in their exercise group but emphasize that this improvement
did not result in significant differences between sedentary and exercising
animals. In contrast, Shepherd et al. [75] and Becker-Zimmerman et al. [1] both
reported that, in contrast to sedentary rats, there was a significant decrease in
triglyceride concentration. It should be noted, however, that initial triglyceride
concentration was appreciably higher in the control rats in Shepherd's study
than that observed in the animals used in the present study. Furthermore the
TE animals weighed significantly less than the other animal groups used in this
present study and since negative caloric balance has been shown to be
associated with a decrease in hepatic VLDL triglyceride production, resulting in

decreased blood triglyceride levels [75] it is impossible to conclude, without a

43

more defined lipid profile, whether the differences observed were directly due to
lower body weights, dietary manipulation or a combination of diet and exercise.
The trend in the data do support diet as a primary factor however since, at all
time points post experimental onset average triglyceride concentration was
higher in both groups which consumed the control diet (C and CE) than those
groups in which the animals consumed a leguminous diet (T and TE).

It was a primary intent of this study to examine the role of early
environmental changes (diet and activity level) on the development of the
NIDDM state in the fa/fa rat. The initial findings of this study suggest that
atthough the differences between the groups were not always signficant, in all
instances animals who consumed a leguminous diet exhibited positive
metabolic benefits, as demonstrated by the tendency of the data to demonstrate
the greatest decrease in blood serum glucose, insulin, cholesterol and
triglyceride concentration in those animals (T and TE). The most significant of
these findings being those observed in cholesterol and triglyceride
concentration and occurring during the later stages of the study as the animals
aged. Exercise tended to improve the NIDDM state, as measured by the blood
serum parameters of this study, regardless of diet, although once again the
differences were not always significant. The combination of a leguminous diet
and exercise tended to prove to be the most effective, although it is interesting
to note also that those sedentary animals consuming the leguminous diet
showed lower serum concentrations of triglyceride, cholesterol, and insulin than
those animals who exercised, yet consumed a semipurified diet. One may
speculate that any significant differences found in the triglyceride and
cholesterol concentrations for TE can be attributed to either the diet and/or the
training, whereas any lack of significance found for CBS blood serum measures

may be attributed to the fact that there was a limited training effect, as indicated

44

by.the lack of significant increase in SDH activity in the soleus muscle by those

animals in the CE group.

CHAPTER VI
SUMMARY, CONCLUSIONS and FURTHER
RECOMMENDATIONS

Summm The purpose of this study was to compare the effects of a
legume diet and a semipurified diet and exercise on glucose, insulin,
cholesterol, and triglyceride concentrations in sedentary and endurance trained
Fatty Zucker (fa/fa) rats. Forty female Fatty Zucker rats were randomly assigned
to one of four experimental groups; tepary bean diet (T), control diet (C),
exercise and tepary bean diet (TE), exercise and control diet (CE). The animals
in the two exercise groups were subjected to a 13 week training program. It
took the animals five weeks to be able to run at the appropriate speed and
duration. During the last eight weeks they ran for sixty minutes per day, five
days per week, at 20 m/min and 0% grade. Blood samples were drawn weekly
from the tail artery and analyzed for glucose and insulin at 0, 4, 8, and 13 weeks
and for cholesterol and triglycerides at weeks 0, 6/7, and 13. Weekly food
intake and body weights were also measured during the study. Upon
completion of the 13 week experimental program animals were sacrificed using
methoxyflurane. Additionally, succinate dehydrogenase activity in soleus
muscle was measured in order to assess the effectiveness of the training
program [75]. Heart, liver, plantaris, and gastrocnemius weights were
measured. In addition, inguinal, parametrial, and perirenal/retroperitineal fat

depots were also assessed.

45

46

Mean values of control and experimental plasma insulin, glucose,
triglyceride, and cholesterol levels for each sampling period were compared
using repeated measures and one-way ANOVA (group x week and week x
group) analysis of variance. Significant interactive effects were analyzed using
a post-hoc Scheffe' for between group comparisons. The level of significance
chosen was p 5.05 in all instances. Body weight increased as the study
progressed as was to be expected. The data revealed that glucose and insulin
concentrations in the rats were not affected in any of the four treatment groups
although the trend was that the tepary bean groups had lower serum
concentrations in all four of the parameters measured. Cholesterol
concentrations in TE and T were significantly lower than C by week 13.
Triglyceride concentrations for TE were also significantly lower than C at the
end of the experimental period.

W A tepary bean diet and exercise successfully reduces
cholesterol and triglyceride concentrations in the NIDDM Fatty Zucker rat.
Although none of the four treatment protocols significantly affected insulin and
glucose concentrations in the Fatty Zucker rat the trend was that the tepary bean
groups had lower glucose and insulin concentrations. Thus, the tepary bean
seems to be a beneficial adjunct to the NIDDM diet as a partial treatment for the
NIDDM metabolic state. The exercise groups in both the tepary bean and
control diets also had a lower insulin, cholesterol and triglyceride concentration
when compared with sedentary counterparts. Hence, exercise also seems to
be a positive addition to the NIDDM treatment regime. Logically, incorporation
of both a leguminous diet and exercise into the NIDDM subjects' regime would
serve as an optimal treatment.

Recommendations Due to the lack of significance in the glucose and

insulin concentration data an increase in sample size and an extension in the

47

duration of the experimental time frame would be beneficial to gain further
insight into long-term effects of the tepary diet on the NIDDM state. This
conclusion is based on the fact that the trend for glucose and insulin
concentrations was that the tepary groups had lower serum concentrations than
the control groups and in hindsight, it seems that a longer experimental period
and/or a more intense running protocol may have revealed significant results.
Additionally, in order to provide further insight into the precise effects that the
tepary bean has on blood glucose tolerance post-prandial glucose measures or
an OGTT should be performed since the influence of legumes on glucose
concentration is best demonstrated in post-prandial or OGTT measures [44].
Furthermore, HDL-cholesterol concentration would also be a useful parameter
to measure to further evaluate the effects of training. If similiar yet significant
results are found when this study is improved upon, then it would be warranted
that other legumes be compared with the tepary bean. Ultimately, the analysis
of the protein, fiber, and anti-nutrient components of the tepary bean should be
performed and compared with those components of other legumes to assess

whether the tepary bean nutrient composition varies from that of other legumes.

APPENDICES

APPENDIX A

Eiletstudx.

Experiment 1 A pilot study was conducted from 10/27/90-12/9/90 in
order to determine if the proposed tepary bean diet would allow normal growth
in the rats and the absence of diarrhea. The experimental animals used were 6
- six week-old SSB/PLRas rats, 3 males and 3 females, all litter mates. One
adult female SSB/PLRas rat was also used to determine if she would maintain
her body weight on the bean diet. The control animals were 2 male SSB/PLRas
rats similar in age but not from the same litter as the 6 experimental rats. The
experimental rats were randomly assigned to tepary bean and rat chow diets.
The intent of this experiment was to compare the growth rate of the
experimental rats with growth of the control group.

The animals were housed in the basement of the Food Science Building
in suspended cages. The room was kept on a 12 hour light/dark cycle and
maintained a constant temperature (70 F :2) and humidity (47-50%).

The tepary bean diet was as follows:
Tepary bean 76.55/100 gm
Minerals 4/100 gm
Vitamins 1/100 gm
DL-methionone 0.25/100 gm
Choline bitartrate 0.2/100 gm

Com oil 3/100 gm

Crisco 15/100 gm

The rats were weighed at the beginning and end of one week and the

food cups were measured daily. After one week on this diet the growth and
4 8

49

overall appearance of the experimental rats was deemed unsatisfactory due to

an inadequate growth rate for one week.

WW

Bali 8mm. titanium.
1 Female 53 69
2 Female 49 62
3 Female 46 59
5 Male 61 85
6 Male 60 82
7 Male 49 65

The diet was revised based on the assumption that inadequate growth was
related to the bean conent of the diet, thus the percentage of tepary beans

comprising the diet was reduced.

Experiment 2

W
Tepary bean 56.55/100 gm
Minerals 4/100 gm
Vitamins 1/100 gm

DL-methionione 0.40/100 gm
Choline bitartrate 0.2/100 gm

Corn oil 3/100 gm
Crisco 15/10 gm
Casein 5/100 gm
Sucrose 5/100 gm

Cornstarch 10/100 gm.

50

DL—methionione was increased from 0.25/100 to 0.4/100 on 11/1/90 to
improve the protein quality of the diet. The diet was known as Tepary diet Ila.

When the growth rates between Tepary bean diet I and Tepary diet Ila
were compared there was no significant difference between the growth rates of
the two diets, although the appearance of the rats had improved when they
were on Tepary Ila.

Experiment 3 The diet was further revised by decreasing the percent
of the tepary beans contained in the diet. The purpose of this dietary revision
was to assess if the rats gained more weight on the newly revised diet (Tepary
Ilb) versus weight gain when on Tepary diet Ila.

The seven rats were then paired according to weight and further subdivided into
two diet groups, Ila and Mb.

The revision on 11/7/90 was as follows:

Tepary beans 46.5/100 gm

Casein 5 -> 7.5/100

Sucrose 5 -> 7.5/100

Cornstarch 10 -> 15/100

Experiment 4 On 11/11/90 the design of the experiment was re-
evaluated and the two diet groups were matched according to weight and
divided into three groups, lla, MB, and rat chow. This change in the
experimental design enabled the investigators to compare the growth rate of the
two tepary bean diets with the growth rate of the rats on the rat chow. Since rat
chow is a common laboratory foodstuff the rats on this diet were expected to
gain weight, thus the weight gain by the rat chow rats would be considered as
the norm.

After one week the rat weights and daily food intake were compared.

51

Diet Rat If Gender mean wt. daily
gain (gm) Intake

(9m)

Rat chow 1 F 17 NA

7 M 22 NA

Tepary Ila 2 F 9 9.4
5 M 16 11.3

4 F 3 13

Tepary Ilb 3 F 10 7

6 M 22 13.2

Diet Ila and Nb were compared by converting the raw scores to 2 scores.
The data showed that there was no statistical difference between the female or
male rat on Ila vs. female or male rat on rat chow. Likewise there was no
statistical difference between the female or male rat on lIb vs. the female or
male rat chow rat. Based on the conclusion that there is no statistical difference
between Tepary Ila and Tepary Ilb. Tepary lIa will be used in the primary
investigation in order to incorporate the greatest percentage of tepary beans
possible yet still see an acceptable growth rate in the rats.

Experiment 5. Once the diet was chosen, the aim of the pilot study was
redirected toward examining the effect of Tepary lla on growth rate and physical
well being of exercised rats. Thus two rats on Tepary lla and an exercise
program were compared with 2 sedentary rats on rat chow and two sedentary

rats on Tepary lIa.

52

week 1 week 2 week 3 week 4
Diet Rat ff wt. galn wt.galn wt. gain wt. gain
(am) also!) (am) (om)

rat 1 17 1 17 12
chow
5 16 11 44 27
7 22 11 21 27
Ila 2 * 9 4 11 2
4 3 -10 6 6
3 * 10 1 10 6
6 22 13 24 19

* Denotes exercised rats

There was no statistical difference between the trained r ** and the
untrained rats in both the rat chow and Tepary Ila groups.

"On 11/25/90 rats 1, 2, 6 were afflicted with ringtail. A malady in which
the distal end of the tail is not receiving an adequate blood supply due to a
sudden drop in room temperature or humidity. In most cases the distal end of
the tail will fall off. The veterinarian recommended that the rats be moved to
cedar bedding cages where they might be warmer. She also advised that rat
#2 discontinue running on the treadmill as this might worsen the condition.

The pilot study enabled the investigators to establish a satisfactory diet

which allowed for normal growth of a rat on an exercise program.

APPENDIX B

W

1. Fr" 12 Mason jars one quarter full with tepary beans. Fill to the three quarter
mark with warm water. Soak 24 hours. Pour off water.

2. Add new water and autoclave the soaked beans for 1 hour.

3. Place the autoclaved beans in tin containers which are labeled. Place the
containers in the drying oven (72 F) until they reach a constant weight (about 60

hours).

Dried beans are ground into fine particles using The Fitz Hammer Mill (W.J.

Fitzpatrick Co., Chicago, IL).

53

APPENDIX C

Q 'I' I D' |
Ingedients

Minerals AIM-761511.2
Vitamins AOC1r3
dl-methlonone 1

choline bitartrate 1
Corn 0“ 4
Hydrogenated fat 5
Tepary Bean 5
Casein 1.7
Fiber 1.8
Cornstarch 9

Sucrose 4

1Purchased from Teklad Test Diets, Madison, WI.

Tepary Bean Diet
g/t 00g
4 9
1 9
0.4 g
0.2 g
3.0 g
15 g
56.40 g
5 9
0
10 g
5 9
104.00 9

2J. Nutr. 107:1340-1348, 1977.
3Association of Official Agricultural Chemists, Washington DC Official Methods
of Analyses of the Association of Official Agricultural Chemists 9th Ed., pg.680,

paragraph 39.133.
4Food Club, Skokie, IL.
5Crisco®

6Ramona Farms, AZ.

7High protein casein, 87.4% protein.

54

Control Dlet
g/1009
4 9
1 9
0.25 g
0.2 g
3.0 g
15 g
21.26 g
3.64 9
25.825 9
25.825 9
100.00 9

55

8Cellulose type.
9Argo®, Best Foods, Englewood Cliffs, N.J.

Component of tepary bean :
25% protein
64% carbohydrate
4.3% fiber
1.2% fat

APPENDIX D

I I'IIB . Ell

The goal of the treadmill training protocol was to have the rats run at a
speed of 20 m/min for 60 minutes for 8 weeks. The animals ran for 5
consecuetive days.

The training protocol began with the rats running at 12 m/min for 15
minutes. The meters per minute was increased by 1 meter every third day while
the duration was increased by 2 minutes every day except for the day that the
meters per minute was increased. The animals were now running for 35
minutes at 15 m/min and at this point in the training a more aggressive
approach was taken since the animals were now accustomed to the running
regimen. Increasing the duration to the 60 minute mark was considered the
more crucial of the two parameters (duration and speed). Thus, the duration
was increased by 5 minutes every day. By 4 weeks from the time of onset the
animals were running for 60 minutes. The speed was then increased by 1
meter a day over the next 2 weeks to get the animals to the point where they
were running at 20 m/min for 60 minutes. The intensity and duration was

maintained for the final 8 weeks of the experiment.

56

APPENDIX E

W9 [Procedure N05101-
Sigma Diagnostics, PO. Box 14508, St. Louis, MO 63178.

-Ouantitative enzymatic (glucose oxidase) determination in plasma
spectrophometrically analyzed at 450 nm.

-Prlnclple: Plasma sample is added to a mixture containing glucose oxidase,
perioxidase and o-dianisidine. The reaction is allowed to proceed to
completion in approximately 30 minutes at 37° C. The final color intensity is

proportional to the glucose concentration.

ELQQQQULQ

Prior to this point construct a calibration curve.

1. Label 3 or more tubes : BLANK, STANDARD, TEST 1, TEST 2, etc.
2. To BLANK, add 0.5 mL water.

To STANDARD, add 0.5 mL of a 20-fold dilution of Glucose Standard
Solution. .

To each TEST, add 0.5 mL of a 20-fold dilution of sample.

3. To each tube, add 5.0 mL of Combined Enzyme-Color Reagent Solution and
mix each tube thoroughly.

4. Incubate all tubes at 37 ° C for 30 min.

5. At the end of incubation period, remove all tubes from water bath. Read
absorbance of STANDARD and TEST, using BLANK as reference at 425-475 nm.
6. Calculate test values as follows:

Serum glucose [mg/dl] = A Test + A Standard x 100

57

APPENDIX F

W® [Procedure N03521-
Sigma Diagnostics, PO. Box 14508, St. Louis, MO 63178.

-Quantitative, enzymatic determination of total cholesterol in serum or plasma at
500 nm.

-PrInprIe: Cholesterol esters are first hydrolyzed by cholesterol esterase to
cholesterol. The cholesterol produced by hydrolysis is oxidized by cholesterol
oxidase to cholest-4-en-3-one and hydrogen peroxide. The hydrogen peroxide
produced is then coupled with the chromogen, 4-aminoantipyrine and p-
hydroxybenzenesulfonate in the presence of perioxidase to yield a
quinoneimine dye which has the absorbance maximum of 500 nm. The
intensity of the color produced is directly proportional to the total cholesterol

concentration in the sample.

Emaedure

1. Construct a calibration curve.

2. Prepare Cholesterol Reagent.

3. Set spectrophotometer wavelength to 500 nm.

4. Set up series of cuvetes for BLANK, CALIBRATOR, CONTROL, SAMPLE.
5. Warm reagent to assay temperature.

6. Pipet 1.0 mL reagent in each tube.

7. Add .01 mL deionized water [BLANK], Calibrator, Control and Sample to
appropriately labeled tubes. Mix by inversion.

8. Incubate cuvets for 5 min. at 37 ° C.

58

59

9. Read and record absorbance of all tubes at 500 nm. Complete readings
within 30 min. after end of incubation time.
10. To calculate cholesterolconcentration:
Serum cholesterol [mg/dl]
{[A test - A blank ] + [A calibrator-A blank]} + C Calibrator

APPENDIX G

W9 [Procedure No.336].
Sigma Diagnostics, PO. Box 14508, St. Louis, MO 63178.

-In vitro enzymatic colorimetric method for the quantitative determination of
triglyceride in serum and plasma.

-Princlple: Serum triglycerides are decomposed to gycerol and free fatty acids
by lipoprotein lipase (LPL). The gycerol thus produced is converted to glycerol-
3-phosphate by glycerol kinase (GK) in the presence of ATP. This G3P is then
oxidized by GSP oxidase to yield hydrogen peroxide.

The hydrogen peroxide thus produced yields a red color compound upon
oxidative condensation with p-chlorophenol and 4-aminoantipyrine in the
presence of perioxidase.

The amount of triglycerides contained in the original serum sample is
determined by measuring the absorbance of the developed color at 505 nm

and compared with the absorbance of the standard solution.

EmceduLe

1. Bring all reagents, samples, standard and control sera to room temperature
prior to use.

2. Label a series of 13 x 100 glass test tubes with SAMPLE, STANDARD,
CONTROL, BLANK.

3. Accurately pipette 20 ul of sample, and standard or control sera into the

appropriate test tube. DO NOT pipette serum into the tube labeled BLANK.

60

61

4. Using a dispenser or semi-automatic pipette, accurately add 3.0 mL of Color
Reagent Solution to all tubes, including the tube labelled BLANK.
5. Thoroughly mix tube contents.
6. Place all tubes in a 37° C :2 bath for 10 min.
7. Using an accurately calibrated spectrophotometer, measure the absorbance
of the sample, standard or control sera at 505 nm against the BLANK tube to
determine net absorbance.
8. Calculations: A Sample + A Standard x C Standard

A = Absorbance at 505 nm

C = Triglyceride Concentration

APPENDIX H

W9 [Catalogue No. 305-11511].
Wako Chemicals USA, Inc., 1600 Bellwood Rd., Richmond, VA 23237.

- In vitro enzyme-immunoassay method for the quantitative determination of

insulin in serum.

- Principle: The insulin test kit is a sandwich method using glass beads as a
solid phase. Insulin in the test sample is allowed to react with insulin antibody

attatched to the glass beads and enzyme (perioxidase) labelled insulin

antibody and then forms a complex. The amount of perioxidase bound to the

beads is directly proportional to the amount of insulin in the serum.

Emadura

1. Place 0.1 mL serum in test sample tubes in labelled glass test tubes with an
internal diameter of 11-12 mm in order to immerse the antibody beads
completely.

2. 0.5 mL enzyme-labelled antibody solution placed in the test tubes.

3. One anti-body bead per test tube and the tubes are incubated for 1 hour at
37° C.

4. Remove the reaction solution by using an aspirator, wash the antibody bead
three times with 3 mL of buffer solution and then transfer each bead to another
test tube.

5. Place 0.5 mL of substrate color solution in each test tube and incubate for

exactly 30 minutes.

62

63

6. Add 1.5 mL of Enzyme reaction solution, mix well, allow to stand at room
temperature for 5 minutes, and measure the absorbance against the blank

solution within 1 hour at 492 nm.

APPENDIX I

IIIIIIS °|D|I [SDIIIEIHI
C00perstein,_(L_Qj_B_i_Q|9_gjga|_Qb_e_mj§_tfl, 186:129-139.

Principle: Reduction of cytochrome c at 550 nm

Reagents:
Phosphate buffer 33mM Na2HPO4
33mM NaH2PO4
(mixed to pH 7.4 at 4° C)

4mM AICI2 in distilled H20
4mM Cacl2 in distilled H20
500mM Na succinate in P04 buffer (pH 7.4 at 30° C)
-store all stock solutions at 4° C
-stable for at least 6 weeks
-also require 0.03% BSA and .1 mM cytochrome 0 according to
working volume
Working Solutions:
according to volume required add:
10 parts PO4 buffer
4 parts AICI3
4 parts Cacl2
30 parts distilled H20
0.03% BSA
0.1 mM cytochrome c

adjust to pH 7.4 at 30° C
500 mM Na succinate (stock)
10 mM NaCN (stock)

Tissue Homogenate:
500 ug
dilution factor/1 mg x .5 mg = homogenate volume

64

65

Assay Procedure:

-lncubate 500 pg muscle with 100 pl Na succinate for two minutes in a
30° C water bath.

-Add 100 pl NaCN - vortex

-Add 2.8 ml of the working solution - vortex

-Set in a temperature controlled spectrophotometer (30° C) and

monitor the reduction of cytochrome c at 550 nm.

Blank:
Can use a blank with everything but the muscle sample or may use blank
with a solution containing everything but Na succinate.

Calculations:
SDH activity = OD/min x dilution factor

 

extinction coefficient (cyt c)

Notes:
-molar extinction coefficient for cytochrome c reduced at 550 nm is 29.5
-Since the amount of muscle used is constant (500 pg) and the
volume of homogenate added is negligible in the calculation of the
final volume there is a constant tissue dilution of:
-500 [.19 in 3 ml = 1 :6000
Hence: SDH activity = OD/min x 6000

 

29.5

units are u moles/g x min -1

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