The Effect of Varying Levels of GH Treatment on the Body Composition of Obese Mice

Home , Bovine somatotropin

The Effect of Varying Levels of GH Treatment on the Body Composition of Obese and

Diabetic Mice

A thesis presented to

the faculty of

the College of Health and Human Services of Ohio University

In partial fulfillment

of the requirements for the degree

Master of Science

Amanda J. Palmer

March 2008

This thesis titled

The Effect of Varying Levels of GH Treatment on the Body Composition of Obese and

Diabetic Mice

AMANDA J. PALMER

has been approved for

the School of Human and Consumer Sciences

and the College of Health and Human Services by

Darlene E. Berryman

Associate Professor of Human and Consumer Sciences

Gary S. Neiman

Dean, College of Health and Human Services

ABSTRACT

PALMER, AMANDA J., M.S., March 2008, Food and Nutrition

The Effect of Varying Levels of GH Treatment on the Body Composition of Obese Mice

(89 pp)

Director of Thesis: Darlene E. Berryman

This study examined the effects of various doses of Growth Hormone (GH) on obesity and diabetes in mice. Male C57Bl/6J mice were either placed on a low-fat (LF) or high-fat (HF) diet at 3 weeks of age and maintained on that diet for 16 weeks. The HF group was divided into five injection groups and received either daily saline injections or increasing doses of bGH, while the LF group was injected with saline. Injections were performed daily for 6 weeks. Obese mice injected with the highest dose of GH (5 µg/g

BW) had the greatest increase in lean mass and the greatest decrease in fat mass.

Remarkably, these animals did not differ significantly from the LF saline-injected controls after 6 weeks of GH treatment and had virtually no change in overall body weight while continuing on a HF diet. Furthermore, the highest dose of GH improved blood glucose levels to near LF control levels, despite the known diabetogenic effect of

GH, but was unable to return plasma insulin to control levels suggesting some level of insulin resistance.

Approved: ______

Darlene E. Berryman

Associate Professor of Human and Consumer Sciences

ACKNOWLEDGMENTS

I would like to thank first and foremost my advisor, Darlene Berryman for the many opportunities to continue my research and education that she has given me. I would also like to thank Ed List, who entrusted this project to me, to everyone else on the second floor of the Edison Biotechnology Institute (which are too many to name) and Dr.

John Kopchick who has allowed me to work in his lab. Also, I would like to thank Diana

Manchester for her help and support through this program and her invaluable editing advice. Additionally, the support from the Diabetes Research Initiative at Ohio

University, an OURC grant and a grant from the Monsanto Company were paramount in completing this research.

I would like to also thank all of my friends and family members who have continued to be a system of support.

TABLE OF CONTENTS

Page

Abstract...... 3

Acknowledgments ...... 4

List of Tables ...... 8

List of Figures...... 9

Chapter 1: Introduction...... 10

Statement of the Problem...... 12

Research Questions...... 13

Purpose of the Study...... 13

Limitations/Delimitations...... 14

Definition of Terms ...... 14

Chapter 2: Review of Literature ...... 16

Obesity...... 16

Type 2 Diabetes ...... 17

Growth Hormone ...... 19

GH Secretion...... 19

GH Receptor Binding and Signal Transduction ...... 22

Biological Effects of GH ...... 24

GH Treatment in Humans...... 25

Mouse Models in GH, Obesity and Diabetes Research...... 31

Conclusions...... 33

Chapter 3: Materials and Methods...... 34

Animals...... 34

Growth Hormone Preparation...... 35

GH Injections...... 35

Weight and Body Composition Measurements ...... 36

Food Intake Measurements...... 38

Blood Glucose Measurements and Collection of Plasma...... 38

Plasma Measurements ...... 39

Glucose Tolerance Test ...... 39

Tissue Collection ...... 39

Statistical Analysis...... 40

Chapter 4: Results...... 41

Weight Gain...... 41

Weight Accumulation...... 41

Fat Mass Accumulation ...... 44

Lean Mass Accumulation ...... 46

Blood Glucose Levels...... 49

Serum Insulin Levels ...... 51

Serum IGF-1 Levels ...... 53

Organ Weights...... 55

Chapter 5: Discussion...... 59

Future Studies ...... 65

Conclusions...... 67

References...... 69

Appendix A...... 87

Appendix B...... 88

Appendix C...... 89

LIST OF TABLES

Page

Table 1: Summary of Human GH Injection Studies on Treatment of Obesity………….27

Table 2: Assignment of Treatment Groups……………………………….……………..36

Table 3: Organ Weights………………………………………………………...……….56

LIST OF FIGURES

Page

Figure 1: Timeline and summary of GH injection study……………………………..…37

Figure 2: Weight measurements over phase 2…………………………………………..42

Figure 3: Weight accumulation over phase 2……………………………………...……43

Figure 4: Fat mass accumulation over phase 2……………………………………….....45

Figure 5: Lean mass accumulation over phase 2………………………………………..48

Figure 6: Blood glucose measurements, week 6…………………………………….…..50

Figure 7: Serum insulin measurements, week 6………………………………….……..52

Figure 8: Serum IGF-1 measurements, week 6……………………………………..…..54

Figure 9: Adipose depot weights………………………………………………………..57

CHAPTER 1: INTRODUCTION

The rates of obese and overweight individuals globally have reached epidemic

proportions. The World Health Organization (WHO) estimates that as of 2005 more than

1.6 billion adults are clinically overweight (BMI > 25 kg/m2), with 400 million considered clinically obese (BMI > 30 kg/m2) (WHO, 2006b). Continuing with this

current trend, by the year 2015 approximately 2.3 billion adults will be overweight and

700 million of them will be obese (WHO, 2006b). Obesity-related complications are also

occurring at alarming rates. Specifically, type 2 diabetes mellitus is estimated to affect

nearly 6% of the world’s population (Adeghate, Schattner, & Dunn, 2006). In the United

States, the Centers for Disease Control and Prevention (CDC) estimates 20.8 million

people have diabetes (approximately 7.0% of the population) (CDC, 2005). These alarming disease rates have prompted researchers to search for new methods to treat obesity.

Type 2 diabetes mellitus comprises approximately 90% of all diabetes cases

(WHO, 2006a). Obesity is considered a major risk factor, present in about 80% of

patients (Adeghate et al., 2006). A schematic illustrating the prevalence of obesity and

diabetes as of 2001 is shown in Appendix A. The progression of type 2 diabetes includes

insulin resistance, hyperglycemia and eventual pancreatic β-cell dysfunction (Kahn, Hull,

& Utzschneider, 2006). Body fat distribution is critical in the development of insulin

resistance. Fat tissue located centrally has been associated with a greater risk of insulin

resistance while individuals with fat tissue located more peripherally have a lower risk of

developing insulin resistance (Kahn et al., 2006).

Growth hormone (GH) is secreted from the anterior pituitary in a pulsatile

manner. GH is of interest in the treatment of obesity due to its lipolytic/antilipogenic

effects as well as its anabolic effect on the growth of lean muscle. GH levels have also

been shown to decrease in an obese state, which further supports the argument to use GH

as a potential therapeutic agent in the treatment of obesity (Shadid & Jensen, 2003).

However, GH has also been shown to have diabetogenic effects by antagonizing insulin’s

actions (Kopchick & Andry, 2000). Mouse models that express bovine GH (bGH) and

therefore have higher GH activity, have been shown to have a significantly shorter

lifespan, decreased fat mass, increased lean muscle mass, develop insulin resistance and

increased renal damage (Berryman et al., 2006; Chen et al., 1995). Similarly, studies

involving acromegalic patients, who secrete excess GH, also experience similar

complications including hypertension, diabetes, sleep apnea, cardiovascular disease,

cancer and a mortality rate 2-3 times higher than that of the normal population (Molitch,

1992). These negative consequences of GH may make it a poor option for treatment of

obesity.

Recent studies have been conducted using human GH (hGH) injections to treat

obese human subjects. Currently, there are few studies that report significant and favorable body composition changes when daily standard doses of hGH are administered to humans (Attallah, Friedlander, & Hoffman, 2006). Due to the possible harmful effects of excess GH, especially related to the health risks associated with diabetes, it would be useful to use an animal model first to study the effects of daily GH injections not only on

body composition, but also in regard to other physiological parameters related to obesity

and diabetes.

The C57Bl/6J mouse model has evolved as a useful model for obesity and

diabetes-related studies. This model has been shown to progress from obesity to insulin

resistance and eventual type 2 diabetes when fed a high-fat diet, and remain lean on a

low-fat diet. Furthermore, they display the same distribution and deposition of fat as seen in humans with high-fat feeding (Rebuffe-Scrive, Surwit, Feinglos, Kuhn, & Rodin,

1993; Surwit, Kuhn, Cochrane, McCubbin, & Feinglos, 1988). The ability to induce obesity through diet makes this mouse model very useful for studies involving obesity and its subsequent progression to diabetes.

Statement of the Problem

The high rate of overweight and obese individuals and the associated health

problems are of great concern. GH treatment has been used in an attempt to decrease

adiposity and increase lean muscle mass. However, there are few studies that have

demonstrated a significant effect of GH injections on body composition. Due to its

diabetogenic effect, the addition of GH to an obese, possibly insulin resistant state may

be more harmful causing exaggeration of the diabetic state. It is also unclear what dosage

of GH would be effective in changing body composition in an obese state. Obese animal

models are excellent for studying these effects because of the possible health risks

involved with administering high doses of GH to obese humans. Also, research with

animal models allows for the collection of many different parameters that would be

difficult if not impossible to obtain using humans. Compared to human studies, animal

13 models also offer the researcher more ability to control factors pertaining to their external and internal environment such as diet, temperature and humidity, light/dark cycles, time of procedures and genetic similarity.

Research Questions

1. Does the body composition (lean mass and fat mass) of male, obese C57Bl/6J mice change with varying dosages of daily GH injections compared to male, obese or lean C57Bl/6J mice injected with a saline solution?

2. Are the plasma levels of glucose, insulin and IGF-1 altered in male, obese

C57Bl/6J mice when given various dosages of daily GH injections when compared to lean and obese saline-injected controls?

3. Do organ weights change in male, obese C57Bl/6J mice when given various dosages of daily GH injections as compared to lean and obese saline-injected controls?

Purpose of the Study

This study aims to determine changes in an obese/diabetic state that are related to daily injections of GH. Most of the previous studies have used obese human subjects and injected varying concentrations of GH. There are few studies using mice which assess whole body composition changes with daily GH injections. In this study, not only will whole body composition be measured to assess the obese state, but also physiological parameters, such as glucose and insulin to assess the diabetic state. Tissue collection at the conclusion of the study will also allow for further investigations. This study will also help to determine if an effective and safe dosage of GH exists. GH injections may serve

as an aid to weight loss. However, because of GH’s diabetogenic effects, it is still unclear as to whether this type of treatment would be beneficial or harmful to current health status. This research will provide valuable insight for possible outcomes of this

type of treatment on obese and diabetic human subjects.

Limitations/Delimitations

1. This study uses mice and may not be fully generalized to the human

population.

2. Mice in this study are bred to be genetically equivalent; however, some variations may exist.

3. Multiple animals (2-3) will be housed in one cage possibly leading to

hierarchical changes that affect food consumption and possible variations in adiposity.

4. Stress levels of the animals during the study may fluctuate due to procedures.

This may cause variations in results unrelated to the GH injections.

5. The injection of the foreign protein, bGH, into the mice may result in an

increased immunological response and possible production of antibodies.

6. The nuclear magnetic resonance (NMR) technology used to measure body

composition is very sensitive; however, due to movement of the animals, variations may

exist.

Definition of Terms

Adipose tissue: An organ of the body made up of various depots in which excess

energy is stored as fat.

Growth hormone (GH): A 191-amino-acid protein, secreted by somatotroph cells

located in the anterior pituitary gland, which promotes growth of tissues (Kopchick &

Andry, 2000).

IGF-1: Insulin-like growth factor 1, belongs to a family of molecules including

IGF-2 and insulin, that can bind to one of the IGFBP (IGF binding protein) as well as the

IGF1R and insulin receptor. It is involved mainly in growth, development and

differentiation (LeRoith & Yakar, 2007).

Insulin resistance: A physiological condition in which the tissues become

insensitive to insulin’s actions and are no longer able to respond to insulin signals

properly. Insulin secretion from the pancreas is increased in an attempt to maintain

normal glucose levels (Kahn et al., 2006).

Lipolysis: Hydrolysis of triglycerides stored in adipocytes to free fatty acids

(FFA) and glycerol (Wang & Fotsch, 2006).

Obesity: Accumulation of fat tissue in which body weight becomes above what is

healthy for a given height or increases the likelihood of certain diseases. Body Mass

Index (BMI) is a measurement used to compare weight and height, where a BMI > 30 is considered obese (CDC, 2006).

Type 2 diabetes mellitus: An endocrine disorder characterized by insulin

resistance and eventual pancreatic β-cell loss leading to increased plasma glucose levels

and several serious complications. Most commonly associated with obesity, older age

and genetics (CDC, 2005).

CHAPTER 2: REVIEW OF LITERATURE

The high rates of overweight and obese individuals present many social and

economic problems. With the proportion of obese adults approaching epidemic proportions, research has focused on ways to treat and prevent obesity. GH is a logical therapeutic target because of its ability to decrease fat mass and increase lean muscle mass. However, there are potentially harmful consequences that may accompany this

type of treatment. This review will include an overview of obesity, the interaction of GH

with adipose tissue as well as potential uses for and consequences of this type of

treatment.

Obesity

The prevalence of obesity worldwide has increased dramatically over the past 20

years. The CDC reports that in the United States obesity among adults age 20-74 years

has increased from 15% in 1976-1980 to 32.9% in 2003-2004 (CDC, 2006). The WHO

estimates that of adults age 15 and older, approximately 1.6 billion are overweight and

400 million are obese globally (WHO, 2006b). Furthermore, the WHO estimates that by

the year 2015, 2.3 billion adults will be overweight and 700 million will be obese (WHO,

2006b). Equally alarming are the growing rates of childhood obesity. In the United

States, the CDC estimates that in the same 20-year time frame (1976-1980 through 2003-

2004) the number of overweight children increased from 5.0% to 13.9% in ages 2-5,

6.5% to 18.8% in ages 6-11, and 5.0% to 17.4% in ages 12-19 (CDC, 2006). These

alarming statistics indicate a growing public health concern of epidemic proportions.

Obesity is defined as having excess body fat. Clinically, this is assessed by

measuring body mass index (BMI), which takes into account both weight and height. A

BMI greater than 30 is considered obese (CDC, 2006). Many health problems have been

identified as comorbidities with obesity. The incidence of type 2 diabetes, cardiovascular

disease, hypertension, dyslipidemia, metabolic syndrome, gallbladder disease,

osteoarthritis, sleep apnea and certain cancers has been shown to increase with obesity

(Wyatt, Winters, & Dubbert, 2006). It is estimated that in the United States, the annual

medical expenses for the treatment of overweight, obesity and attributable diseases is

between $51.5- $78.5 billion (Finkelstein, Fiebelkorn, & Wang, 2003). The annual

estimates for obesity alone are between $26.8-47.5 billion (Finkelstein et al., 2003). Due

to the prevalence of obesity, its related complications and the economic strain it

contributes, a new urgency is driving the need for research of obesity prevention and

treatment.

Type 2 Diabetes

The incidence of diabetes has increased drastically, affecting nearly 6% of the

world’s population (Adeghate et al., 2006). The WHO estimates that more than 180 million people worldwide are affected, with the number expected to double by the year

2030 (WHO, 2006a). Type 2 diabetes is estimated to make up nearly 90% of all cases

(WHO, 2006a). A link has been established between obesity and the development of

type 2 diabetes. Of all reported cases, nearly 80% of type 2 diabetic patients are obese

(Adeghate et al., 2006).

The progression of type 2 diabetes includes the development of insulin resistance,

impaired glucose tolerance and eventual pancreatic β-cell dysfunction (Kahn et al., 2006).

A 1963 study showed that when obese nondiabetic individuals were given an increased glucose load, their circulating insulin increased, while their circulating glucose remained constant (Karam, Grodsky, & Forsham, 1963). Another study involving the Pima Indian population showed that impairment of both insulin secretion and insulin action are present prior to the development of type 2 diabetes, indicating that dysfunction of pancreatic β-cells and insulin resistance may occur early in the disease progression, potentially before impaired glucose tolerance (Weyer, Bogardus, Mott, & Pratley, 1999).

Studies with the ob/ob mouse (a mouse that lacks the gene for leptin and causes marked obesity) (Malik & Young, 1996) showed a marked increase in insulin resistance (Mayer,

Andrus, & Silides, 1953). The development of glucose intolerance may develop later in the stages of type 2 diabetes. Studies have shown that in male rhesus monkeys, the development of glucose intolerance occurred several years after the development of insulin resistance (Hansen & Bodkin, 1986).

Potentially the distribution of adipose tissue is a more important indicator of risk

for type 2 diabetes and insulin resistance than overall obesity. Studies involving both lean and obese individuals have shown that an increase in central obesity, or intra- abdominal adipose, is related to increased insulin resistance regardless of BMI. Android obesity, or obesity localized to the midsection, is considered to be related to diabetes in

93% of males and 60% of females (Vague, 1956). In lean individuals, increased insulin

resistance is related to an increase in subcutaneous and intra-abdominal fat of 45% and

70%, respectively (Cnop et al., 2002). Regression analysis showed that intra-abdominal

fat explained 54% of the variance in insulin sensitivity between lean groups (Cnop et al.,

2002). High rates of both obesity and type 2 diabetes have led to increased research into

the relative importance of adipose tissue from various regions in the development of

insulin resistance.

Growth Hormone

Growth hormone is a 191-amino-acid polypeptide secreted from the somatotrophs

of the anterior pituitary. GH, as its name implies, is a pivotal hormone in longitudinal bone growth. However, GH also has a significant impact on nutrient metabolism, including carbohydrate, fat, protein and mineral metabolism. For example, it promotes both lipolysis and lean muscle accretion. This important effect has made GH treatment a

new and important area of obesity research. However, it also has potentially harmful

effects which may or may not be exaggerated in obese individuals, such as increased

insulin resistance. The danger of treating obese and potentially type 2 diabetic patients

with a hormone known to increase the diabetic state is not yet understood in an animal

model.

GH Secretion

The release of GH from the anterior pituitary is controlled mainly by two

peptides, growth hormone-releasing hormone (GHRH) and somatostatin, both of which are secreted by the hypothalamus (Tannenbaum & Ling, 1984). GH is secreted in a

pulsatile manner, with increased secretion occurring during sleep cycles (Takahashi,

Kipnis, & Daughaday, 1968). In humans, GH levels are high after birth and then decline until puberty. During puberty, GH levels as well as secretion rates are increased during both sleep and wake periods (Finkelstein, Roffwarg, Boyar, Kream, & Hellman, 1972).

In adulthood, GH secretion decreases between the ages of 20 and 70, with eventual levels in the elderly reaching 12-20% of those seen in puberty (Ho & Hoffman, 1993).

Differences in secretion are also seen in obesity, with levels of GH decreased compared to normal-weight individuals. However, these concentrations have been shown to increase in obese, nondiabetic subjects when in a fasted, hypoglycemic state (Herrold &

Tzagournis, 1970).

The release of GH is controlled by a negative feedback loop, with ultrashort-loop, short-loop and long-loop feedback mechanisms. A diagram of the feedback mechanism of GH is shown in Appendix B. GH has been suggested to act directly on the pituitary to inhibit GH release in the ultrashort-loop mechanism. As concentrations of GH in the blood rise, the release of somatostatin and GHRH from the hypothalamus are increased and decreased, respectively, to further decrease the release of GH from the anterior pituitary, resulting in a short-loop feedback. The long-loop feedback mechanism results from insulin-like growth factor 1 (IGF-1) in the serum released in response to GH, acting at the hypothalamus and anterior pituitary to inhibit GH secretion (Yamasaki, Prager,

Gebremedhin, Moise, & Melmed, 1991). However, other hormones and compounds in the body have also been shown to impact GH secretion and inhibition. Leptin, a hormone which regulated metabolism of nutrients and food intake, decreased overall spontaneous

GH secretion in both fed and fasted rats, with the fasted animals exhibiting an amplified

response (Carro, Senaris, Considine, Casanueva, & Dieguez, 1997). Various other hormones and neurotransmitters also affect the stimulation and inhibition of GH, either

by themselves or through regulation of somatostatin and GHRH.

Alterations in GH secretion and concentrations are also seen with exogenous

stresses. In humans, the greatest amplitude and frequency of GH secretion occurs during

sleep (Obal, Payne, Kapas, Opp, & Krueger, 1991). Light/dark cycles have less of an

effect on GH secretion when compared to slow-wave sleep phases (Obal et al., 1991).

Psychological stress may also be related to GH secretion, though the relationship between

stress and GH secretion in humans is not clear. For example, exercise and undernutrition

increase GH secretion in humans, depending on the intensity and duration of exercise as

well as muscle mass (Weltman, Wideman, Weltman, & Veldhuis, 2003). However, in

individuals with decreased GH levels, such as the obese or the elderly, exercise fails to

stimulate the release of excess GH (Kanaley, Weatherup-Dentes, Jaynes, & Hartman,

1999; Pyka, Wiswell, & Marcus, 1992). In humans, a 2-day fast results in a fivefold

increase in the 24-hour secretion of GH, a twofold increase in the number of daily pulses

and in the overall mass released during each pulse (Hartman et al., 1992).

Certain nutritional states also have an impact on GH secretion. Glucose, free fatty

acids (FFA) and amino acids have all been shown to impact the release of GH in various

models and disease states. In primates, a hyperglycemic state is related to decreased

levels of GH, while hypoglycemia results in increased secretion of GH (Himsworth,

Carmel, & Frantz, 1972). In humans, FFA have been related to decreased secretion of

GH (Imaki et al., 1985). Certain amino acids, especially essential and branched chain

amino acids, stimulate GH release in various models (Giustina et al., 1992; Stewart,

Koerker, & Goodner, 1984). Thus, GH secretion and nutrient status are intimately

related: GH has a significant impact on nutrient status, and nutrient status is a significant regulator of GH secretion.

GH Receptor Binding and Signal Transduction

In order to promote its various effects, GH must bind to the GH receptor on the

target tissue. In serum, GH is found either free or bound to the GH binding protein

(GHBP), a soluble portion of the GHR. It has been shown that approximately 40-60% of

GH is bound to GHBP in normal serum (Baumann, Amburn, & Shaw, 1988). The GHBP

is a truncated portion of the GHR, that corresponds to the extracellular domain (Hadden

& Prout, 1964). In some species, including humans, the GHBP is formed by proteolytic cleavage of the GHR (Dastot et al., 1996). In other species, including monkeys, mice and rats, the GHBP is formed by alternative splicing of the GHR mRNA (Baumbach, Horner,

& Logan, 1989; Martini et al., 1997; Talamantes, 1994). Regardless of the mechanism, most species appear to have GHBP, suggesting an important evolutionarily conserved function for this binding protein. An important role of GHBP appears to be increasing the half-life of GH by decreasing both its clearance and degradation rate (Baumann et al.,

1988).

The GHR is a membrane spanning protein consisting of intracellular,

transmembrane and extracellular domains. The presence of GHR has been shown to be

abundant in liver tissue. However, other tissues such as muscle, bone, adipose, kidney and embryonic stem cells have also been shown to express the receptor to some extent

(Kelly, Djiane, Postel-Vinay, & Edery, 1991; Ohlsson et al., 1993). There are two important sites on the GH molecule that are necessary for correct binding to the receptor, often referred to as site-1 and site-2. When binding occurs, site-1 is bound first by GH, followed by site-2 binding. The GH molecule binds to a preformed receptor dimer, resulting in signal transduction (Brown et al., 2005). Binding of the GHR leads to tyrosine phosphorylation of the GHR and phosphorylation of the JAK2 (janus kinase 2) molecule, followed by phosphorylation and signal transduction by a host of other molecules (Zhu, Goh, Graichen, Ling, & Lobie, 2001), most notably STAT 5 (signal transducer and activator of transcription). Ultimately, these second messenger systems have the ability to alter the expression of various genes related to GH function (Kopchick

& Andry, 2000).

Disruption of GH binding to the receptor is demonstrated by the GH antagonist

(GHA) model. This molecule is able to bind at site-1, but because of specific mutations, site-2 is unable to bind to the receptor properly. Glycine located at position-119 for bGH and -120 for hGH, is critical for proper GH binding and action. When substituted with any other amino acid other than alanine, site-2 binding is disrupted and no signal transduction occurs (Chen, Chen, Yun, Wagner, & Kopchick, 1994; Chen, Wight, Mehta,

Wagner, & Kopchick, 1991; Ross et al., 2001). Further studies of this model have shown that GHA binds the GHR but is not able to promote intracellular signaling (Clackson,

Ultsch, Wells, & de Vos, 1998; Harding et al., 1996).

Biological Effects of GH

The binding of GH to GHR results in altered expression of numerous proteins.

The end result is that GH impacts various pathways. As mentioned previously, the main

impact of GH is on longitudinal bone growth. This is illustrated by the impact of an excess of GH leading to gigantism or acromegaly. However, GH has other functions,

impacting other tissues and processes of the body, like nutrient metabolism and

proportion of lean versus fat mass (or body composition). In humans, pulses of GH are

followed by an increase in circulating FFA, indicating an increase in lipolysis (Moller,

Jorgensen, Alberti, Flyvbjerg, & Schmitz, 1990). The deposition and storage of lipids in

adipose tissue, or lipogenesis, has also been shown to decrease with GH. GH has also

been shown to impact carbohydrate metabolism. The diabetogenic effect of GH has been

described in numerous studies. When GH was introduced to both normal and diabetic

rats an increase in insulin resistance was reported (Milman & Russell, 1950). Although

not clearly understood, the mechanism in which GH interferes with glucose metabolism

is thought to be due in part to the cross talk between insulin and GH (Dominici et al.,

2005). More recently, this cross talk has been found in adipose tissue to be related to

p85α-subunit of PI-3 (phosphoinositide-3) kinase, a protein involved in mediating the

insulin effects on adipose tissue (del Rincon et al., 2007). When p85α is disrupted in

mice, an increase in insulin sensitivity and hypoglycemia is observed (Terauchi et al.,

1999). In relation to GH status, animals with low GH levels have reduced levels of p85α

and decreased insulin levels (del Rincon et al., 2007). Conversely, animals with excess

GH have increased levels of p85α and are hyperinsulinemic (del Rincon et al., 2007).

Additionally, the ability of insulin to activate PI-3 kinase activity is decreased in animals

with excess GH (del Rincon et al., 2007). The increase in the p85α subunit by GH

negatively effects the insulin pathway in adipocytes by decreasing PI-3 kinase activity

(del Rincon et al., 2007). This evidence supports the cross talk theory and provides

evidence that the p85α subunit and PI-3 kinase are central to anti-insulin effects of GH.

Various animal models have been used to demonstrate the effects of chronic, high

levels of GH. The bGH transgenic mouse model has been shown to have

hyperinsulinemia (Quaife et al., 1989) and increased insulin resistance (Valera et al.,

1993). In humans, acromegalic patients, who have high levels of GH, have also been

shown to have insulin resistance, impaired glucose tolerance and increased risk of

developing diabetes (Ezzat et al., 1994). The use of the GHA in acromegalic patients has

shown an improvement of both fasting blood glucose and insulin resistance with no

change in body weight (Rose & Clemmons, 2002). These results suggest a possible role of GHA in the treatment of diabetes.

GH also has a significant impact on body composition, in part due to its impact on

nutrient metabolism. When GH-deficient adults were treated with GH, a significant

increase in lean body mass and a significant decrease in fat mass were observed

(Salomon, Cuneo, Hesp, & Sonksen, 1989). These studies support the importance of GH

on the regulation of body composition.

GH Treatment in Humans

GH has been used to treat various diseases in humans. Because of its lipolytic/

antilipogenic effects, GH has been considered to be a potential treatment for obesity.

However, due to the diabetogenic effect of GH, using this treatment in an obese group

may lead to further complications. Despite these risks, multiple studies have addressed

the effectiveness of GH treatment on the development of obesity. A summary of these

studies is shown in Table 1. For example, when rhGH at 0.1 mg/kg IBW was given to

obese subjects under dietary restrictions, the loss of fat mass was not found to differ from

the control group, while the loss of lean body mass was attenuated (Clemmons, Snyder,

Williams, & Underwood, 1987). This same study also found no difference in fasting

blood glucose or serum insulin levels (Clemmons et al., 1987). In a similar study, obese,

diet-restricted subjects were injected with GH at 0.1 mg/kg IBW for 11 weeks with no

significant difference in the rate of fat loss or difference in glucose tolerance (Snyder,

Clemmons, & Underwood, 1988). Additionally, these subjects had increased serum IGF-

1 throughout the study, even after anabolic effects had ceased (Snyder et al., 1988).

Another study measuring the effect of low-dose (0.02 U/kg/day) GH continuous infusion

in obese men found no change in insulin or IGF-1 levels (Oscarsson, Lindstedt,

Lundberg, & Eden, 1996).

Additional studies have examined the lipolytic action of GH. When obese women

were injected with GH at 0.03 mg/kg IBW for 5 weeks, an ~50% decrease in lipoprotein

lipase (LPL, a hormone involved in the hydrolysis of lipids) was observed in both subcutaneous and intra-abdominal adipose tissues (Richelsen et al., 1994). Additionally,

FFA concentrations increased after treatment, indicating an increase in lipolysis

(Richelsen et al., 1994). The decrease in LPL activity was also examined and it was found that the decrease was in both the adipose tissue-specific and muscle-specific forms

Table 1

Effect of GH Injections SummaryReference of HumanSubjects GH Injection Length of StudyStudies Age ofon Participants Treatment GH of dosage Obesity Diet

Weight Fat Mass Lean Mass IGF-1 Insulin Glucose Clemmons, Snyder, Hypocaloric (24 Williams, & Underwood,8, obese (5 women, 23-50 years (mean 3 weeks, 0.1 mg/kg Cal/kg, 1 g 1987 3 men) 11 weeks 34.4) IBW every other day protein/kg IBW) NS NS NR Increased NS NS Hypocaloric (18 Snyder, Clemmons, & 20, obese (16 0.1 mg/kg IBW every Cal/kg IBW, 1.2 g Underwood, 1988 women, 4 men) 13 weeks 20-54 years other day, weeks 2-12 protein/ kg IBW) NS NS NR Increased NS NS

Hypocaloric (high Increased with Snyder, Clemmons, & 0.1 mg/kg IBW every carbohydrate vs high carbohydrate, Underwood, 1989 11, obese women 10 weeks 21-49 years other day, 3 weeks high fat) NS Decreased NR Increased NS NS with high fat Hypocaloric (25 24, obese (22 kcal/kg IBW, 1.2 g Decreased (greater Kim et al., 1999 women, 2 men) 12 weeks 22-46 years 0.18 U/kg IBW/weekprotein/kg IBW) NS visceral fat loss) Increased Increased NS NS 0.03 IU/kg IBW-0.08 Hypocaloric (3135 Norrelund et al., 2000 15, obese women 4 weeks mean 36.6 yearsIU/kg IBW kJ/day) NS NR NR Increased Increased NR Increased Increased Increased (significance not Oscarsson, Lindstedt, (significance not (significance not reported) Lundberg, & Eden, 1996 8, overweight men 2 weeks 42-59 years 0.02 IU/kg/day No restrictions NR NR NR reported) reported)

0.15 IU/kg twice daily Berneis, Vosmeer, & 30, normal weight + methylprednisolone Keller, 1996 men 1 week mean 25.4 years 0.5 mg/kg/day No restrictions NR Decreased NR NR Increased NR

Decreased Snyder, Underwood, & 0.05 mg/kg IBW for 28Hypocaloric (15 (remained greater Clemmons, 1995 11, obese 38 days 25-49 (mean 39)days kcal/kg IBW) than controls) Decreased NR Increased Increased Increased Kamel, Norgren, Elimam, Danielsson, & 0.033 mg/kg/day for 6 Median BMI Marcus, 2000 10, obese boys 12 months 10-12 years months No restrictions decreased Decreased NS NR NS NS

Thompson et al., 1998 33, obese women 12 weeks mean 67.1 years 0.025 mg/kg BW, daily Hypocaloric NS Decreased Increased Increased NR NR 24, obese, T2DM Decreased (12 women, 12 1-1.5 U/day, 5 No change in (significant visceral fat loss) Increased Increased Decreased NS Ahn et al., 2006 men) 12 weeks mean 53.7 years days/week Hypocaloric BMI

Table 1 (continued). Effect of GH Injections Reference Subjects Length of Study Age of Participants GH dosage Diet

Weight Fat Mass Lean Mass IGF-1 Insulin Glucose Decreased (decreased intra- Richelsen et al., 1994 9, obese women 5 weeks 24-46 years 0.03 mg/ kg IBW daily No restrictions Increasedabdominal fat) Increased NR Increased Increased No change in Johannsson et al., 1997 30, obese men 9 months 48-66 years 9.5 µg/kg, daily No restrictionsBMI Decreased NS Increased NS NS Hypocaloric (12 Snyder, Underwood, & kcal/kg IBW, 1 g/kg Clemmons, 1990 8, obese women 15 weeks 18-37 years 0.1 mg/kg IBW, IBW)daily NS NS NS Increased Increased Increased

1 U/kg IBW/week, Hypocaloric (41.86 Significantly Tagliaferri et al., 1998 20, obese women 4 weeks mean 25.4 yearsdaily injections kJ/kg) NS NS more retained Increased Increased NS 0.03 IU/kg-0.08 IU/kg,Hypocaloric (740 Richelsen et al., 2000 18, obese women 4 weeks ~35 years daily kcal/day) NS NS NR Increased NS NR 0.1 IU/kg/week, daily Hypocaloric (1100- Sartorio et al., 2004 20, obese women 3 weeks 61-75 years injections 1500 kcal/day) NS NS NS Increased NR NR Increased first 6 9.5 µg/kg/day + 850 months, returned mg Metformin twice to baseline at Herrmann et al., 2004 25, obese men 18 months mean 55 years daily No restrictions NS Decreased NS Increased NS conclusion 24, obese (11 men, No change in Tomlinson et al., 2003 13 women) 8 months mean 41 years 0.4 mg/day No restrictions BMI NS NS Increased NS NS

0.13-0.67 mg/day Franco et al., 2005 40, obese women 12 months 51-63 years (began after 1 month) No restrictions NS NS NS Increased Increased NS Lucidi, Parlanti, Piccioni, Santeusanio, & De Feo, 6, overweight, 2002 obese men 1 week 33-50 years 3.3 µg/kg, daily No restrictions NS NR NRNR Increased NS Increased Skaggs & Crist, 1991 12, obese women 4 weeks 29-50 years 0.08 mg/kg IBW No restrictions NS Decreased (NS) NS NR NR Albert & Mooradian, 59, obese (44 2004 women, 15 men) 6 months 20-45 years 200-600 µg/day Hypocaloric Decreased Decreased NS Increased NS NS Pasarica, Zachwieja, 30, overweight Decreased Dejonge, Redman, & (visceral obesity) 0.95 mg, daily for 24 (significant visceral Smith, 2007 men 50 weeks 40-70 years weeks No restrictions Increased fat loss) Increased Increased Increased Increased

Note. Only statistically significant changes are reported. NS=No significant difference from placebo or control; NR= Not reported.

of LPL (Richelsen et al., 2000). Lipid kinetic studies have shown that treatment with GH

is associated with an approximate 25% increase in the rate of lipolysis with changes in

protein or carbohydrate metabolism (Lucidi, Parlanti, Piccioni, Santeusanio, & De Feo,

2002). Additional studies have also reported increases in FFA concentrations, serum

glycerol levels, triglycerides and LPL activity following GH injections (Johannsson et al.,

1997; Richelsen et al., 2000; Snyder, Underwood, & Clemmons, 1995).

The location of adipose tissue loss has also been examined. When daily doses of

GH at 0.03 mg/kg IBW were injected into obese women, a significant amount of fat mass

was lost and a significant decrease in the visceral fat (7%) was observed (Richelsen et al.,

1994). A 12-week study using daily injections of GH at 0.03 IU/kg IBW and a hypocaloric diet reported a significant decrease in fat mass and a significant decrease in the visceral fat area (Kim et al., 1999). Injections of 0.33 mg GH/day and a calorie restricted diet caused significant fat mass loss and reduced visceral fat area (Ahn et al.,

2006). A 6-month study using overweight males with adipose tissue localized in the

abdominal region concluded that 0.95 mg GH/day for 24 weeks caused significant fat

mass loss, with significant decreases in visceral adipose tissue (Pasarica, Zachwieja,

Dejonge, Redman, & Smith, 2007).

Several studies have also examined the effect of diet restriction and various diet

compositions. When the composition of a restricted diet was altered to contain either

more lipid or more carbohydrate, it was found that GH was more responsive with the

high-carbohydrate diet, leading to an increase in IGF-1 and urinary C-peptide excretion

(Snyder, Clemmons, & Underwood, 1989). More recent studies have shown that GH

treatment at 0.18 U/kg IBW/wk in obese, diet restricted subjects was related to an

increase in lean body mass with a decrease in visceral fat mass (Kim et al., 1999). When

obese subjects were placed on a hypocaloric diet which was 500 kcal/day less than what

was needed for normal weight maintenance, both weight and fat mass decreased with GH

injections (Albert & Mooradian, 2004). The administration of GH over 4 weeks at 0.03

IU/kg IBW - 0.08 IU/kg IBW with dietary restriction helped to preserve protein stores

when compared to controls (Norrelund et al., 2000). Additionally, an increase in lipid

oxidation was observed, while the serum insulin levels remained unchanged in the GH

treated group (Norrelund et al., 2000). Additional studies have shown the benefits of

both a hypocaloric diet and exercise. When patients were placed on a diet of 500

kcal/day less than needed for weight maintenance and followed an exercise regimen, fat

mass was significantly decreased (Thompson et al., 1998).

Additional studies have involved using GH in conjunction with other therapies. A

study involving the injection of both GH and glucocorticoids found that when injected

together, resting energy expenditure and serum insulin both increased when compared to

the control group. Similarly, the estimated fat mass of the combination group decreased

significantly during the study (Berneis, Vosmeer, & Keller, 1996). The concurrent use of

GH and metformin, an insulin-sensitizing agent, decreased fat mass, but had no

significant effect on glucose metabolism or lean mass (Herrmann et al., 2004).

These studies have shown varying results, sometimes with little effect on the treatment of obesity. In most cases, the change in body composition was not significant, and the report was based on the trend of data. Of the 24 studies examined, 13 reported

significant decreases in fat mass (Ahn et al., 2006; Albert & Mooradian, 2004; Berneis et

al., 1996; Herrmann et al., 2004; Johannsson et al., 1997; Kamel, Norgren, Elimam,

Danielsson, & Marcus, 2000; Kim et al., 1999; Pasarica et al., 2007; Richelsen et al.,

1994; Skaggs & Crist, 1991; Snyder et al., 1989; Snyder et al., 1995; Thompson et al.,

1998). Only six studies report either a significant increase or significant retention of lean mass (Ahn et al., 2006; Kim et al., 1999; Pasarica et al., 2007; Richelsen et al., 1994;

Tagliaferri et al., 1998; Thompson et al., 1998). The amounts of GH injected (0.0033-

0.95 mg/kg) as well as the length of studies (1 week to 18 months) also varied in these studies. Additionally, the level of GH used to treat patients in most cases was fairly low, suggesting that an effective dosage needs to be determined. GH, specifically hGH, is also able to bind to the prolactin (PRL) receptor (Somers, Ultsch, De Vos, & Kossiakoff,

1994). Recently, studies have shown that PRL inhibits lipolysis (Brandebourg, Bown, &

Ben-Jonathan, 2007) and animals that lack PRL receptors have decreased adipose tissue

(Flint, Binart, Boumard, Kopchick, & Kelly, 2006). The confounding effects of GH

binding to the PRL receptor may explain the discrepancies between many of these

studies.

Mouse Models in GH, Obesity and Diabetes Research

An important model for studying both diet-induced obesity and type 2 diabetes is

the C57Bl/6J mouse. For obesity and diabetes, this model has been shown to progress

through the same stages of obesity and type 2 diabetes as seen in humans. Previously, it

has been reported that on a diet consisting of 20.5% protein, 35.8% fat and 36.8%

carbohydrate, C57Bl/6J mice became obese and gained weight faster than a similar strain,

C57Bl/AJ (Surwit et al., 1988). When glucose and insulin were measured in these animals, the obese C57Bl/6J mice were found to be significantly more hyperglycemic and hyperinsulinemic than lean C57Bl/6J as well as lean and obese C57Bl/A/J mice

(Surwit et al., 1988).

GH has been shown to affect body composition in animals with altered GH function. When mice expressing excess bGH (excess GH action), GHA (expression of the GH antagonist) and GHR -/- (GH function absent) mice were compared, bGH animals were found to have a leaner phenotype than the other two models (Berryman et al., 2004).

When various adipose depots of these models were compared, the majority of the excess adipose tissue in the GHA and GHR -/- models was found in the subcutaneous depot

(Berryman et al., 2004). Furthermore, when the bGH and GHR -/- models were placed on a high-fat diet, GHR -/- mice showed a greater susceptibility to gaining fat mass

(Berryman et al., 2006). A figure of these animal models and their relevant phenotypes is shown in Appendix C. These effects can be explained by previous studies observing the effect of GH on both lean and adipose tissues. In mouse models with altered GH states, a

HF (high-fat) diet resulted in less accumulation of adipose tissues in animals with excess

GH as compared to animals with an absence of GH signaling (GHR -/-) and controls

(Berryman et al., 2006). In this same group of animals, accumulation of subcutaneous adipose tissue was significantly greater in animals that lacked GH signaling, indicating a possible depot-specific effect.

Few studies have examined the effects of administration of GH to treat obesity in animal models. To measure the acute response, GH was administered to both control and

obese mice. Glucose tolerance was found to be impaired to a greater extent in the obese

animals after 12 hours (Shull & Mayer, 1956).

Conclusions

Obesity and related comorbidities have become a major health concern in the

United States and worldwide. The search for a viable treatment has led many to consider

the use of GH. This is an attractive choice due to the lipolytic and antilipogenic effect on adipose tissue. Additionally, GH has an anabolic effect on lean muscle mass. However,

GH is diabetogenic causing an insulin resistant state and decreased glucose tolerance.

The treatment of obesity, a state that is high risk for developing diabetes, with a known diabetogenic hormone needs to be further evaluated.

Previous studies have shown a possible benefit to treatment with GH. However,

the dosages administered have not all shown significant effects. A dosage gradient

administered to an obese animal model would help to identify an optimal GH dose as

well as possible complications that accompany this treatment.

CHAPTER 3: MATERIALS AND METHODS

Previous studies have shown the potential of GH as a therapeutic treatment of

obesity. In this study, various dosages of GH were used to evaluate its effectiveness on

improving body composition of obese mice. Measurements of physiological parameters

such as glucose, insulin and IGF-1 levels as well as organ weights will help to determine

possible harmful effects of this type of treatment.

Animals

Mice in the C57Bl/6J background were used for this study. Previous studies have characterized this model as useful for the study of diet-induced obesity and diabetes due to their human-like progression from obesity, insulin resistance and type 2 diabetes when fed a high-fat diet (Surwit et al., 1988). A total of 49 male C57/Bl6J mice at 21 days of age were received from Jackson Laboratories (Bar Harbor, ME) and divided into two different experimental groups. The first group, consisting of 42 mice, was placed on a high-fat (HF) diet produced by Research Diets Inc. (New Brunswick, NJ), while the second group, consisting of the remaining 8 mice, was placed on a standard laboratory rodent chow (ProLab RMH 3000). The HF diet supplied 20% of total kilocalories from

carbohydrates, 20% kilocalories from protein and 60% kilocalories from fat. The

standard low-fat (LF) diet supplied 60% of total kilocalories from carbohydrates, 26% of

kilocalories from protein and 14% of kilocalories from fat.

Mice were housed 2-3 per cage and food and water were supplied ad libitum. The

cages were kept in a temperature- and humidity-controlled room and exposed to a 12-

hour light/dark cycle. All procedures were approved by the Ohio University Institutional

Animal Care and Use Committee and fully complied with federal, state and local policies.

Growth Hormone Preparation

Bovine growth hormone (recombinant bovine somatotropin, rBST) was received as a generous gift from the Monsanto Company (St. Louis, MO). The GH was first dissolved in 1% acetic acid solution to a concentration of 10 mg/mL. This solution was then added to filtered and sterilized phosphate buffered saline (PBS) to a final concentration of 500

μg/mL. Six mL were aliquoted into conical tubes and frozen at -80º C until use.

Prior to injections, 6 mL of GH were placed in a 37º C water bath. The remaining doses of GH were completed using serial dilutions of 1:10 of the 500 μg/mL solution.

Sterilized 1X PBS was used to complete all dilutions and for use as a control. A total of four doses were freshly prepared each day to the following concentrations: 500 µg/mL,

50 µg/mL, 5.0 µg/mL and 0.5 µg/mL.

GH Injections

The first group of mice (n=42) was split into five different groups of 8-9 mice after 16 weeks of HF diet treatment. Weight measurements and glucose measurements prior to the assignment were used to ensure that the groups did not differ from one another. Each group was assigned to one of four GH injection concentrations or the PBS control group. One set of eight mice were placed in the 1X PBS saline control group.

The remaining groups were arranged as follows to receive various dosages of bGH daily based on body weight: 8 mice received 0.005 µg/g; 9 mice, 0.05 µg/g; 8 mice, 0.5 µg/g;

and 9 mice, 5.0 µg/g. The second group, consisting of 8 mice on a LF diet, was also used as controls. All mice in this group were placed in the 1X PBS saline-injected group. A summary of the assignment of mice to treatment groups is shown in Table 2.

Table 2

Assignment of Mice to GH Injection Groups

Diet LF HF HF HF HF HF Number of Mice788989

1X PBS 1X PBS GH Dose (0.01 mL/g) (0.01 mL/g) 0.005 μg/g 0.05 μg/g 0.5 μg/g 5.0 μg/g

Subcutaneous GH injections were performed daily for six weeks using the assigned GH dose for each group. An amount of 0.01 mL/g body weight was injected and the most current body weights were used. Injection sites were rotated weekly between the neck and the lower back to minimize possible exaggerated local effects and skin irritations.

The injections occurred at 3:00 PM ± 3 hours daily.

Weight and Body Composition Measurements

Total body mass was assessed weekly throughout the study using a standard scale to the tenth of a gram. After 16 weeks on either a HF or LF diet, weekly body composition measurements were performed using the Bruker Minispec (The Woodlands,

TX). This machine uses NMR technology to assess the total fat mass, lean mass, and fluid mass in grams of the animals (Barac-Nieto & Gupta, 1996). The measurements were performed at the same time each week, reducing potential variations caused by the

daily injections. Total lean mass and weight were used to calculate percent lean mass.

Total fat mass and weight were also used to calculate the percent fat mass. The total

accumulation of lean mass and fat mass was assessed beginning with the second week of

measurements. A summary of all measurements and time points is represented in

Figure 1.

Phase 1 (not shown): Induce obesity and type 2 diabetes •Place 50 mice on a HF diet for 16 weeks •Measure weights weekly

Phase 2: GH treatment •Daily GH injections •Measure weights, body composition and food consumption weekly •Glucose (G), Insulin (I) and IGF-1 measured periodically

Subcutaneous GH Injection

0 1 2 3 4 5 6

G I G I G G I G G I IGF-1 IGF-1 IGF-1 IGF-1 GTT

Weigh, Measure Body Composition and Feed Mice Dissect Mice

Figure 1. Timeline, in weeks, and summary of phase 1 and phase 2 of the GH injection study.

Food Intake Measurements

Total food intake/cage was assessed weekly throughout the study by subtracting

the total grams of food remaining in the cage from the total grams of food added the previous week. These measurements are reported as the average number of kilocalories consumed by each animal in each experimental group. Food was measured on a standard scale.

Blood Glucose Measurements and Collection of Plasma

Blood glucose measurement and plasma collection began after 16 weeks of diet

treatment and continued every other week to the end of the study. The measurements and

collections were completed between 9:00 a.m. and 12:00 p.m. Prior to glucose

measurements and collection, the mice were fasted for 12 hours. An infrared heating

lamp was used to enhance blood collection. To collect blood, the tip of the tail was

removed and blood glucose measurements were taken using a LifeScan OneTouch

glucometer (Milpitas, CA) with the first drop of blood. Immediately after the blood

glucose measurements, blood was collected using Chase Natelson heparanized capillary

tubes (VWR International, Bridgeport NJ) and stored on ice. It was then spun at 4° C for

10 minutes at 7,000 x g, to prevent lysis of the red blood cells. Plasma was collected and

stored at -80° C until further analysis.

Glucose measurements were also taken in triplicate after the third week (19 weeks

of age) of injections and continued every other week to the end of the study. The mice

were not placed under an infrared lamp in order to reduce stress levels. The tip of the tail

was cut and the first three drops of blood were used for plasma glucose measurements

using a LifeScan OneTouch Glucometer.

Plasma Measurements

Several plasma metabolite concentrations associated with diabetes, GH and

obesity were measured using ELISA (enzyme linked immunosorbent assay) kits. Free

IGF-1 concentration was measured using the Free IGF-1: DSL-10-9400, Coated Well

ELISA (Diagnostic Systems Laboratories, Webster TX). Insulin concentrations were

determined using the ultra sensitive mouse Insulin ELISA kit (ALPCO Diagnostics,

Windham, NH) as per the instructions of the manufacturer.

Glucose Tolerance Test

Glucose tolerance tests were performed during the final week (week 7) of the

injections. Mice were fasted for 12 hours prior to the measurements, which began at 9:00 a.m. For all mice, an interperitoneal injection of a 25% glucose solution was administered at 0.01 mL/g body weight. Glucose measurements were performed in duplicate using a OneTouch LifeScan Glucometer beginning before the injection and

continuing 60-, 120-, 180-, 240- and 360-minutes after the injection.

Tissue Collection

Animals were sacrificed by cervical dislocation after 22 total weeks of diet

treatment and 6 weeks of daily GH injections. The following tissues were collected: skin,

inguinal adipose tissue, epididymal adipose tissue, retroperitoneal adipose tissue,

mesenteric adipose tissue, kidney, heart, liver, muscle, pancreas and bone. All tissues

collected were weighed using a standard laboratory scale and flash frozen in cryogenic

vials using liquid nitrogen. They were then stored at -80° C until further use.

Statistical Analysis

Statistics were performed on all body composition measurements, glucose measurements, insulin and IGF-1 concentrations, and tissue weights using SPSS version

14.0 software (Chicago, IL). Groups are reported as LF-PBS (LF diet, PBS injected),

HF-PBS (HF diet, PBS injected), HF-0.005 (HF diet, 0.005 µg GH/g BW injected), HF-

0.05 (HF diet, 0.05 µg GH/g BW injected), HF-0.5 (HF diet, 0.5 µg GH/g BW injected)

and HF-5 (HF diet, 5 µg GH/g BW injected). Tissue weights were analyzed using

analysis of variance (ANOVA) and group means are reported. The remaining

measurements were analyzed using repeated measures analysis to determine variances

within groups. Group means were then be compared using ANOVA and planned contrasts were used to determine statistical significance between groups. A value of p<0.05 was regarded as statistically significant.

CHAPTER 4: RESULTS

Weight Gain

The weight of mice on a HF diet increased as compared to controls on a LF diet.

When placed on a HF diet, C57Bl/6J mice had increased body weight that became

exaggerated over time compared to mice fed a LF diet (see Figure 2). During phase 2 of the study, the weights of all mice on a HF diet, regardless of GH dosage, remained

greater than the LF-PBS group (see Figure 2).

Weight Accumulation

The weight change over the course of phase 2 (GH injections) of the study varied

within treatment groups. Weight accumulation data from all time points in phase 2 of the

study (GH injections) is shown in Figure 3. After 1 week of injections, there was no significant difference in weight change between groups. After 2 weeks of injections, the

HF-5 group had a significant decrease in weight as compared to the HF-PBS and the HF-

0.05 groups (F(5,43)=3.546, p>.05). After 3 weeks of injections, the weight increase of

the HF-0.5 group was significantly less than the HF-0.05 group and the weight decrease

of the HF-5 group was significantly different than all groups (F(5,43)=10.521, p>.05).

After 4 weeks of injections, the weight decrease of the HF-5 group was significantly different from the HF-PBS, HF-0.005 and HF-0.05 groups (F(5,43)=11.477, p>.05).

Similarly, the weight increase in HF-PBS and HF-0.05 groups was not significantly

different from the HF-0.005 group, but was significantly different from the remaining

treatment groups (F(5,43)=11.477, p>.05). After 5 weeks of treatment, the weight gain

Phase 1 Phase 2 50

25 LF-PBS Weight (g) HF-PBS 20 HF-0.005 15 HF-0.05 HF-0.5 10 HF-5 5 0 1 2 3 4 0 1 2 3 4 5 6

Months Weeks

Figure 2. Weight (grams) measurements of mice on a HF or LF diet injected with PBS,

0.005 µg GH/g BW, 0.05 µg GH/g BW, 0.5 µg GH/g BW or 5 µg GH/g BW during phase 1 and phase 2 of the study. Data are expressed as mean ± SEM.

Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 6 b b b

b b,c 4 b b b

a,b a a a,c 2 a a a,b a a a a,b a a a a a a a,b a,b a,b a,c a,c a,b b a b c c 0 Change inWeight (g)

-2

-4

Injection PBS 0.005 0.05 0.5 5 PBS 0.005 0.05 0.5 5 PBS 0.005 0.05 0.5 5 PBS 0.005 0.05 0.5 5 PBS 0.005 0.05 0.5 5 PBS PBS PBS PBS PBS 0.005 0.05 0.5 5 (µg/g BW) PBS PBS

Diet LF HF LF HF LF HF LF HF LF HF LF HF

Figure 3. Weight accumulation (grams) of mice on a HF or LF diet injected with PBS,

0.005 µg GH/g BW, 0.05 µg GH/g BW, 0.5 µg GH/g BW or 5 µg GH/g BW during phase 2 of the study. Means within each week with a common letter do not differ, p>.05.

Data are expressed as mean ± SEM.

of the LF-PBS, HF-0.5 and HF-5 groups were significantly less than the HF-PBS, HF-

0.005 and HF-0.05 groups (F(5,43)=12.068, p>.05). At the conclusion of the study, after

6 weeks of injections, the overall weight gain was significantly less in the LF-PBS and

HF-5 groups compared to HF-PBS, HF-0.005 and HF-0.05 groups (F(5,43)=13.761,

p>.05). The HF-0.5 group had significantly less weight accumulation than both the HF-

PBS and HF-0.05 groups (F(5,43)=13.761, p>.05). The total weight accumulation after 6

weeks of injections was 0.06 ± 0.34 g in the LF-PBS, 4.75 ± 0.53 g in the HF-PBS, 3.69

± 0.50 g in the HF-0.005, 4.12 ± 0.67 g in the HF-0.05, 1.46 ± 0.57 g in the HF-0.5 and

0.43 ± 0.66 g in the HF-5 groups (Figure 3). Using the HF-PBS as the standard control

group, the total weight accumulation after 6 weeks of injections for the LF-PBS, HF-

0.005, HF-0.05, HF-0.5 and HF-5 groups were 1.2%, 77.8%, 86.8%, 30.7% and 9.0%, respectively.

Fat Mass Accumulation

The accumulation of fat mass over the course of phase 2 of the study was altered

with GH injections. Fat accumulation data from all time points in phase 2 are shown in

Figure 4. After one week of injections, the increase in fat mass of the LF-PBS, HF-PBS,

HF-0.005 and HF-0.05 groups did not significantly differ from one another. The fat

accumulation in the HF-0.5 group did not differ from the LF-PBS group, but was

significantly less than the HF-PBS, HF-0.005 and HF-0.05 groups and significantly

greater than the HF-5 group. The HF-5 group had a significant decrease in fat

accumulation as compared to all other treatment groups (F(5,43)=6.905, p>.05). After 2

weeks of injections, the accumulated fat mass of the HF-0.5 group was significantly less

Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 6

b 4 b b b a,b b a,b a,b a,b 2 a a a a a a a a,c a a,b a a,b a,b a,c a b a,b d 0 c b c b c c cd c

-2 Accumulatedmass fat (g) -4

-6

Injection PBS 0.005 0.05 0.5 5 PBS 0.005 0.05 0.5 5 PBS 0.005 0.05 0.5 5 PBS 0.005 0.05 0.5 5 PBS 0.005 0.05 0.5 5 PBS PBS PBS PBS 0.005 0.05 0.5 5 PBS PBS (µg/g BW) PBS Diet LF HF LF HF LF HF LF HF LF HF LF HF

Figure 4. Fat mass accumulation (grams) of mice on a HF or LF diet injected with PBS,

0.005 µg GH/g BW, 0.05 µg GH/g BW, 0.5 µg GH/g BW or 5 µg GH/g BW during phase 2 of the study. Means within each week with a common letter do not differ, p>.05.

Data are expressed as mean ± SEM.

than the HF-PBS and HF-0.005 groups, and again, the accumulated fat mass of the HF-5

group was significantly less than all other treatment groups (F(5,43)=31.325, p>.05).

After 3 weeks of injections, the HF-0.5 group had significantly less fat mass

accumulation than the HF-PBS, HF-0.005 and HF-0.05 groups. The HF-5 group had

significantly decreased fat mass accumulation compared to all other treatment groups

(F(5,43)=40.667, p>.05). After 4 and 5 weeks of injection, fat accumulation of the HF-

0.5 group was significantly less than the HF-PBS, HF-0.005 and HF-0.05 groups. The

HF-PBS injected group had significantly greater fat mass accumulation than the LF-PBS,

HF-0.5 and HF-5 groups. As before, the HF-5 group continued to have significantly less

fat mass accumulation compared to all other treatment groups (F(5,43)=42.770, p>.05).

By 6 weeks of injections, the LF-PBS and HF-0.5 groups had significantly less

accumulated fat mass than the HF-PBS, HF-0.005 and HF-0.05 groups. The HF-5 group

had significantly decreased fat mass compared to all other treatment groups

(F(5,43)=32.516, p>.05). The total fat mass accumulation after 6 weeks of injections was

0.47 ± 0.09 g in the LF-PBS, 3.65 ± 0.40 g in the HF-PBS, 3.1 ± 0.42 g in the HF-0.005,

2.87 ± 0.49 g in the HF-0.05, 0.20 ± 0.66 g in the HF-0.5 and -4.09 ± 0.81g in the HF-5

groups (see Figure 4). Using the HF-PBS as the comparison group, the total fat

accumulation after six weeks of injections for the LF-PBS, HF-0.005, HF-0.05, HF-0.5 and HF-5 groups were 12.9%, 84.9%, 78.7%, 5.5% and -112.0%, respectively.

Lean Mass Accumulation

The lean mass accumulation over the course of phase 2 of the study was altered

with GH treatment. Lean mass accumulation data from phase 2 of the study is shown for

all time points in Figure 5. After 1 week of injection treatment the lean mass

accumulation of the HF-0.5 group was significantly greater than the LF-PBS, HF-PBS,

HF-0.005 and HF-0.05 groups. The accumulation of lean mass in the HF-5 group was

significantly greater than all other treatment groups (F(5,43)=26.253, p>.05) and this

significant increase was maintained throughout the study (F(5,43)=41.076, p>.05),

(F(5,43)=56.361, p>.05), (F(5,43)=58.552, p>.05), (F(5,43)=33.262, p>.05),

(F(5,43)=46.620, p>.05). After 2 weeks of injection treatment, the accumulation of lean

mass in the LF-PBS injected group was significantly less than the HF-0.05, HF-0.5 and

HF-5 groups. The lean mass accumulation was significantly greater in the HF-0.5 group

than the LF-PBS, HF-PBS and HF-0.005 groups (F(5,43)=41.076, p>.05). After 3 weeks

of injection treatment, the accumulation of lean mass was significantly less in the LF-

PBS injected group than the HF-0.05, HF-0.5 and HF-5 groups. The lean mass

accumulation in the HF-0.5 group was significantly greater than the LF-PBS, HF-PBS

and HF-0.005 groups (F(5,43)=56.361, p>.05). After 4 weeks of injections, the

accumulation of lean mass in the LF-PBS injected group was significantly less than the

HF-PBS, HF-0.05, HF-0.5 and HF-5 groups. The HF-0.5 group gained significantly

more lean mass than the LF-PBS and HF-0.005 groups (F(5,43)=58.552, p>.05). After 5 weeks of injections, the LF-PBS group gained significantly less weight than the HF-0.5 and HF-5 groups. The HF-PBS, HF-0.005 and HF-0.05 groups did not differ significantly in lean mass accumulation from either the LF-PBS or the HF-0.5 groups

(F(5,43)=33.262, p>.05). After 6 weeks of injections, theLF-PBS injection group lost

significantly more lean muscle mass than all other treatment groups. The HF-PBS,

Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 5

c 4 c d

d 3 d

c 2

c b b c b 1 b c b,c b,c a,b b a b a,b a,b b,c b aaa a a,b a a,b a 0 a,b a a,b a a,b Lean Mass Accumulation (g)

-1

-2 PBS 0.005 0.05 0.5 5 PBS PBS 0.005 0.05 0.5 5 PBS 0.005 0.05 0.5 5 PBS 0.005 0.05 0.5 5 PBS 0.005 0.05 0.5 5 PBS PBS PBS PBS 0.005 0.05 0.5 5 PBS PBS

LF HF LF HF LF HF LF HF LF HF LF HF

Figure 5. Lean mass accumulation (grams) of mice on a HF or LF diet injected with

PBS, 0.005 µg GH/g BW, 0.05 µg GH/g BW, 0.5 µg GH/g BW or 5 µg GH/g BW during phase 2 of the study. Means within each week with a common letter do not differ, p>.05.

Data are expressed as mean ± SEM.

HF-0.005, HF-0.05 and HF-0.5 groups did not differ significantly from one another

(F(5,43)=46.620, p>.05). The total lean mass accumulation after 6 weeks of injections was -1.35 ± 0.22 g in the LF-PBS, 0.03 ± 0.29 g in the HF-PBS, 0.02 ± 0.12 g in the HF-

0.005, 0.73 ± 0.28 g in the HF-0.05, 0.87 ± 0.29 g in the HF-0.5 and 3.87 ± 0.36 in the

HF-5 groups (see Figure 5).

Blood Glucose Levels

Blood glucose levels during phase 2 of the study were altered significantly upon

treatment with GH. After 3 weeks of GH injections (data not shown), the LF-PBS

injected group had significantly decreased blood glucose levels than all other treatment

groups. The blood glucose levels of the HF-PBS, HF-0.005, HF-0.05 and HF-0.5 groups

did not differ significantly from one another. The HF-5 group had significantly

decreased blood glucose levels from the HF-PBS, HF-0.005, HF-0.05 and HF-0.5 groups

and significantly greater blood glucose than the LF-PBS treatment group

(F(5,43)=34.160, p>.05). After 4.5 weeks of injections (data not shown), the LF-PBS

and HF-5 groups did not differ significantly from one another and had significantly lower

glucose levels than the other treatment groups. The HF-PBS, HF-0.005 and HF-0.05

groups had significantly greater blood glucose levels than the LF-PBS, HF-0.5 and HF-5

groups. The HF-0.5 group had significantly lower blood glucose levels compared to the

HF-PBS, HF-0.005 and HF-0.05 groups and significantly greater blood glucose levels

than the LF-PBS and HF-5 groups (F(5,43)=34.074, p>.05). After 6 weeks of injections

(see Figure 6), both the LF-PBS and HF-5 groups had significantly lower blood glucose levels than the other treatment groups and did not differ significantly from one another.

220

b b 200

b,c

180 c

160

140 a Glucose (mg/dL) Glucose

120

100

Injection PBS PBS 0.005 0.05 0.5 5 (µg/g BW)

Diet LF HF

Figure 6. Week 6 blood glucose measurements of mice (mg/dL) on a HF or LF diet injected with PBS, 0.005 µg GH/g BW, 0.05 µg GH/g BW, 0.5 µg GH/g BW or 5 µg

GH/g BW during phase 2 of the study. Means shown with a common letter do not differ, p<.05. Data are expressed as mean ± SEM.

The HF-0.5 group had significantly lower blood glucose than the HF-PBS and HF-0.05

groups and significantly higher blood glucose levels than the LF-PBS and HF-5 groups.

The HF-PBS, HF-0.005 and HF-0.05 groups did not differ significantly from one another

(F(5,43)=24.719, p>.05). After 6 weeks of injections, the blood glucose levels were

121 ± 7.9 mg/dL in the LF-PBS, 194 ± 5.0 mg/dL in the HF-PBS, 181 ± 6.8 mg/dL in the

HF-0.005, 195 ± 5.6 mg/dL in the HF-0.05, 166 ± 4.3 mg/dL in the HF-0.5 and 137 ± 8.2

mg/dL in the HF-5 groups (see Figure 6).

Serum Insulin Levels

Serum insulin levels during phase 2 of the study remained unchanged upon

treatment with GH. Prior to beginning GH injections, the insulin levels of the LF-PBS

treated group were significantly lower than all other treatment groups (data not

shown). The serum insulin levels of the HF-PBS, HF-0.005, HF-0.05, HF-0.5 and HF-5

groups were not significantly different from one another (F(5,43)=5.595, p>.05). After 2 weeks of injection treatment, the LF-PBS injected group had significantly lower serum insulin levels than the HF-0.005 and HF-0.05 treatment groups (data not shown).

(F(5,43)=3.538, p>.05). After 4 weeks of injections, the LF-PBS injected group had significantly lower serum insulin levels than the HF-PBS, HF-0.005, HF-0.05 and HF-0.5 treatment groups (F(5,43)=3.835, p>.05) (data not shown). After 6 weeks of injections, the LF-PBS injected group had significantly lower plasma insulin levels than the HF-

PBS, HF-0.005, HF-0.05 and HF-5 groups (see Figure 7). The HF-PBS, HF-0.005, HF-

0.05, HF-0.5 and HF-5 treatment groups did not differ significantly from one another

(F(5,43)=4.891, p>.05). After 6 weeks of injections the serum insulin levels were

b b 4

b b a,b 3

2 Insulin (ng/mL) Insulin 1 a

Injection PBS PBS 0.005 0.05 0.5 5 (µg/g BW)

Diet LF HF

Figure 7. Week 6 serum insulin measurements (ng/dL) of mice on a HF or LF diet injected with PBS, 0.005 µg GH/g BW, 0.05 µg GH/g BW, 0.5 µg GH/g BW or 5 µg

GH/g BW during phase 2 of the study. Means shown with a common letter do not differ, p>.05. Data are expressed as mean ± SEM.

0.65 ± 0.04 ng/mL for the LF-PBS, 3.42 ± 0.40 ng/mLfor the HF-PBS, 2.87 ± 0.37

ng/mL for the HF-0.005, 3.47 ± 0.53 ng/mL for the HF-0.05, 2.56 ± 0.57 ng/mL for the

HF-0.5 and 2.69 ± 0.57 ng/mL for the HF-5 groups (see Figure 7).

Serum IGF-1 Levels

Serum IGF-1 levels during phase 2 of the study were altered significantly upon

treatment with GH. The serum IGF-1 levels prior to beginning GH injections were

significantly less in the LF-PBS (217.9 ± 12.5 ng/mL) injected group compared to HF-

PBS (294.6 ± 7.2 ng/mL), HF-0.005 (374.2 ± 14.1 ng/mL), HF-0.05 (353.6 ± 18.5

ng/mL) and HF-5 (307.3 ± 19.5 ng/mL) groups. The serum IGF-1 levels of the HF-0.005

group were significantly greater than the LF-PBS, HF-PBS and HF-0.5 groups. The HF-

0.05 group had significantly greater serum IGF-1 levels than the LF-PBS and HF-0.5 groups (F(5,43)=10.950, p>.05). After 2 weeks of GH treatment, the LF-PBS injected group had significantly lower serum IGF-1 levels than the HF-PBS, HF-0.005, HF-0.05 and HF-5 groups (data not shown). The HF-0.5 group had significantly lower serum

IGF-1 levels than the HF-0.005 and HF-0.05 groups. The HF-5 group had significantly greater serum IGF-1 levels than all other treatment groups (F(5,43)=26.635, p>.05) and

this was maintained throughout the study (F(5,43)=22.260, p>.05), (F(5,43)=27.373, p>.05). The LF-PBS, HF-PBS, HF-0.005, HF-0.05 and HF-0.5 groups did not differ significantly from one another (data not shown) (F(5,43)=22.260, p>.05). After 6 weeks of injections (see Figure 8), the serum IGF-1 levels of the LF-PBS injected group were significantly lower than the HF-PBS, HF-0.005, HF-0.05 and HF-5 groups

(F(5,43)=27.373, p>.05). After 6 weeks of injections the serum IGF-1 levels were

500 c

400

300 bbba,b

a 200 IGF-1 (ng/mL) IGF-1

100

Injection PBS PBS 0.005 0.05 0.5 5 (µg/g BW)

Diet LF HF

Figure 8. Week 6 serum IGF-1 measurements (ng/dL) of mice on a HF or LF diet injected with PBS, 0.005 µg GH/g BW, 0.05 µg GH/g BW, 0.5 µg GH/g BW or 5 µg

GH/g BW during phase 2 of the study. Means shown with a common letter do not differ, p>.05. Data are expressed as mean ± SEM.

184.5 ± 14.4 ng/mL for the LF-PBS, 269.5 ± 17.5 ng/mL for the HF-PBS, 270.7 ± 10.1

ng/mL for the HF-0.005, 257.1 ± 15.0 ng/mL for the HF-0.05, 253.0 ± 16.0 ng/mL for the

HF-0.5 and 435.3 ± 24.2 for the HF-5 groups (see Figure 8).

Organ Weights

The weight of four adipose depots was altered significantly upon treatment with

GH. Mean organ weights are shown in Table 3. The weights of heart (F(5,43)=1.644,

p>.05), liver (F(5,43)=1.560, p>.05) and spleen (F(5,43)=1.721, p>.05) were not altered

among any of the treatment groups. The kidney weight of the HF-0.005 group was significantly less than the HF-5 group; however, these groups were not significantly

different from the remaining treatment groups (F(5,43)=2.914, p>.05). The weight of the

inguinal (subcutaneous) adipose depot (see Figure 9) was decreased significantly in the

LF-PBS and HF-5 groups as compared to the HF-PBS, HF-0.005, HF-0.05 and HF-0.5

groups (F(5,43)=17.490, p>.05). Using the HF-PBS group as the standard control group, the percent change in weight of the inguinal adipose tissue of the HF-0.005, HF-0.05,

HF-0.5 and HF-5 groups were 6.25%, 7.35%, -14.25% and -59.36%, respectively. The

weight of the epididymal adipose depot (see Figure 9) was significantly decreased in the

LF-PBS injected group compared to all other treatment groups. The HF-5 group had a significantly smaller epididymal adipose depot compared to the HF-PBS, HF-0.005 and

HF-0.05 groups (F(5,43)=26.933, p>.05). Using the HF-PBS group as the standard control group, the percent change in weight of the epididymal adipose tissue of the HF-

0.005, HF-0.05, HF-0.5 and HF-5 groups were -9.12%, -7.17%, -12.27% and -35.81%,

Table 3

Organ Weight Measurements (grams) of Mice on a HF or LF Diet Injected with PBS,

0.005 µg GH/g BW, 0.05 µg GH/g BW, 0.5 µg GH/g BW or 5 µg GH/g BW

Diet Injection Heart Liver Kidney Spleen

LF PBS 0.14 ± 0.12 a 1.49 ± 0.12 a 0.34 ± 0.02 a,b 0.19 ± 0.02 a

HF PBS 0.15 ± 0.01 a 1.64 ± 0.10 a 0.36 ± 0.01 a,b 0.19 ± 0.02 a

HF 0.005 µg/g BW 0.17 ± 0.13 a 1.55 ± 0.14 a 0.31 ± 0.03 a 0.19 ± 0.03 a

HF 0.05 µg/g BW 0.14 ± 0.005 a 1.53 ± 0.12 a 0.33 ± 0.01 a,b 0.19 ± 0.03 a

HF 0.5 µg/g BW 0.15 ± 0.01 a 1.24 ± 0.11 a 0.33 ± 0.02 a,b 0.24 ± 0.03 a

a a b a HF 5.0 µg/g BW 0.18 ± 0.01 1.44 ± 0.08 0.38 ± 0.01 0.26 ± 0.02

Note. Means within each tissue group shown with a common letter do not differ, p>.05.

Data are expressed as mean ± SEM.

Adipose Depot: Subcutaneous (Inguinal) Epididymal Retroperitoneal Mesenteric 4

b b 3 b b b b b b 2 c Weight (g) (g) (g) Weight Weight Weight b b a b b b,c 1 bb b,c a,c a a a,c a a 0

Injection PBSPBSPBSPBS PBS PBS PBS PBS 0.0050.0050.0050.005 0.050.050.050.05 0.50.50.50.5 5.05.05.05.0 PBSPBSPBSPBS PBSPBSPBSPBS PBSPBSPBSPBS 0.0050.0050.0050.005 0.050.050.050.05 0.50.50.50.5 5.05.05.05.0 PBSPBSPBSPBS 0.0050.0050.0050.005 0.050.050.050.05 0.50.50.50.5 5.05.05.05.0 PBSPBSPBSPBS (µg/g BW) PBSPBSPBSPBS 0.005 0.005 0.005 0.005 0.050.050.050.05 0.50.50.50.5 5.05.05.05.0

Diet LF LF HF LF HF LF HF HF

Figure 9. Mean weights (gram) of four adipose depots of mice on a LF or HF diet injected with PBS, 0.005 µg GH/g BW, 0.05 µg

GH/g BW, 0.5 µg GH/g BW or 5.0 µg GH/g BW during phase 2 of the study. Means within each tissue group shown with a common letter do not differ, p>.05. Data are expressed as mean ± SEM.

respectively. The weight of the retroperitoneal adipose depot (see Figure 9) in the LF-

PBS injection group was significantly smaller than the HF-PBS, HF-0.005, HF-0.05, and

HF-0.5 groups. The HF-5 group had a significantly smaller retroperitoneal adipose depot

than the HF-PBS, HF-0.005 and HF-0.05 groups (F(5,43)=16.663, p>.05). Using the

HF-PBS group as the standard control group, the percent change in weight of the

retroperitoneal adipose tissue of the HF-0.005, HF-0.05, HF-0.5 and HF-5 groups were -

15.73%, -8.65%, -24.82% and -47.29%, respectively. The weight of the mesenteric

adipose depot (see Figure 9) in the LF-PBS injection group was significantly smaller than

the HF-PBS, HF-0.005, HF-0.05 and HF-0.5 groups. The mesenteric adipose depot in

the HF-5 group was significantly smaller than the HF-PBS, HF-0.005 and HF-0.05

groups (F(5,43)=12.959, p>.05). Using the HF-PBS group as the standard control group, the percent change in weight of the mesenteric adipose tissue of the HF-0.005, HF-0.05,

HF-0.5 and HF-5 groups were -7.64%, -0.44%, -28.40% and -51.02%, respectively.

CHAPTER 5: DISCUSSION

The increasing incidence of obesity and related complications has prompted

intensive research into prevention and treatment of this condition, now considered to be a

major epidemic. GH has become a logical target for therapy because of its effects on

body composition, namely increasing lean mass and decreasing adipose tissue. However,

the possible accompanying diabetogenic effects of GH may further enhance the

complications associated with obesity. In this study, mice were placed on a HF or LF

diet and then injected with saline or increasing levels of bGH. Body composition, weight

and several metabolic parameters were measured periodically to assess the effectiveness

of GH on the treatment of obesity. In terms of body composition, obese mice injected

with the highest dose of GH (5 µg/g BW) had the greatest increase in lean mass and the

greatest decrease in fat mass. Remarkably, these animals did not differ significantly in weight change from the LF-PBS controls after 6 weeks of GH treatment and had virtually no change in overall body weight while continuing on a HF diet. Additionally, the greatest impact on decreasing fat mass and increasing lean mass was observed with the

highest dose of GH. Furthermore, the highest dose of GH improved blood glucose levels

to near LF control levels, despite the known diabetogenic effect of GH, but was unable to

return serum insulin to control levels suggesting some level of insulin resistance.

Recently, GH has been used as a potential therapeutic agent to treat obesity.

Human studies have reported mixed results as to its effectiveness in reducing fat mass

(Richelsen et al., 1994; Richelsen et al., 2000; Sartorio et al., 2004; Skaggs & Crist,

1991; Snyder, Underwood, & Clemmons, 1990; Tagliaferri et al., 1998). In calorically-

60 restricted patients receiving GH, there appears to be no effect on the loss of fat mass; however, the loss of lean mass is attenuated (Clemmons et al., 1987). Additional studies have shown small reductions in fat mass and gains or retention of lean mass in obese patients (Berneis et al., 1996; Kim et al., 1999; Norrelund et al., 2000). Few animal studies have been conducted assessing the effectiveness of GH on obesity, increasing concern over the safety of using this treatment in humans. GH is a known diabetogenic agent, having well documented anti-insulin effects. The use of this type of compound in obese, possibly insulin resistant or diabetic patients may cause additional diabetic complications.

Diet is an important variable when conducting studies on obesity. In most human studies involving GH injections, neither the caloric intake nor the caloric macronutrient content of patients is well controlled (Franco et al., 2005; Herrmann et al., 2004;

Tomlinson et al., 2003). Additionally, in human studies the patient’s adherence to a specific diet is more difficult to control. In the present study, animals were placed on either a HF or a LF diet where diet composition and macronutrient content was controlled. The injection of GH, especially in the highest dose, caused a loss in fat mass and increase in lean mass even as these animals were maintained on a HF diet. The lower doses of GH had little to no effect as compared to the highest dose administered. As HF diets were used for all animals treated with GH, additional studies may provide insight as to the effectiveness of these low doses of GH in combination with a LF or reduced calorie diet.

GH treatment also caused changes in body composition. In the HF-PBS group,

there was an accumulation of fat mass with no apparent change in lean mass leading to an

overall increase in body weight. As the amount of GH injected increased, the loss of fat

mass also increased as well as the accumulation of lean mass. The lowest doses of GH

(HF-0.005 and HF-0.05) did not appear to effect body composition as these groups followed the same trend as the HF-PBS group. In the HF-0.5 group, there was no apparent gain or loss of fat mass with an overall increase in the amount of lean mass.

The highest dose of GH (HF-5) showed a trend approaching no change in overall body weight. This dose of GH had the greatest effect on overall body composition. In this group, the loss of fat mass was accompanied by a dramatic increase in lean mass to almost the same degree, a trend that would be favorable for the treatment of obesity. This positive impact on lean and fat mass with GH treatment has been observed previously

(Berneis et al., 1996; Kim et al., 1999; Norrelund et al., 2000). There was no change in overall body weight due to this complete reversal in body composition. Therefore, measuring body weight as opposed to body composition may not provide a clear picture

of the effectiveness of GH on reducing fat mass.

The effectiveness of GH on both adipose tissue and lean tissue also seemed to

vary with time. During the first 3 weeks of injections, the HF-5 group lost an average of

4.4 grams of fat mass. However, during the last 3 weeks of injections, there was no

additional loss in fat mass. The lean mass accumulation appeared to be more consistent

over the 6 weeks of injections. The HF-5 group continued to gain lean mass consistently

over phase 2 of the study. After 3 weeks of injections, this group had gained 2.6 grams

of lean mass and after 6 weeks had gained 3.9 grams of lean mass. These results indicate

that the injection of GH appears to have a more immediate impact on adipose tissue and

lipolysis that diminished over time. The effect on lean mass does not appear to be as

dramatic; however, the effect was consistent throughout the injections and may have continued to increase if the injections continued for longer than 6 weeks. Additional studies may provide additional information as to how long the anabolic effect on lean mass will continue and whether the catabolic effect on adipose tissue would return.

High GH levels usually result in high IGF-1 levels. The levels of GH in this

study were related to the levels of IGF-1, with higher doses of GH causing increases in

IGF-1. The HF-5 group had significantly higher IGF-1 levels than all other treatment

groups, beginning at week 2 of phase 2 and continuing throughout the study.

Interestingly, the animals on the HF diet had significantly higher IGF-1 levels than the

LF-control group at the start of phase 2, suggesting that obesity or the intake of a high fat

diet may contribute to increased IGF-1 levels. With the exception of week 4 during

phase 2, the IGF-1 levels of the HF animals remained higher than those of the LF control

group. It has been reported previously that increases in IGF-1 occur in an obese state,

more specifically in the agouti mouse model (Martin et al., 2006). GH levels in these

mice also tend to be increased, but the levels were not significant. The hyperinsulinemic state of the animals in this study may also contribute to the increased IGF-1 levels in the

HF animals. Studies have shown that increased insulin levels are associated with increased IGF-1 gene transcription (Kaytor, Zhu, Pao, & Phillips, 2001; Phillips, Harp,

Goldstein, Klein, & Pao, 1990). Additionally, studies in mice have shown an increase in

IGF-1 levels in animals fed a high fat, high carbohydrate diet (Venkateswaran et al.,

2007). However, in humans both IGF-1 and GH levels have been reported to decrease

with obesity (De Marinis et al., 2004; Eden Engstrom et al., 2006; Rasmussen et al.,

1995; Rasmussen, Juul, Kjems, & Hilsted, 2006). The discrepancies between these

reports and the connection of IGF-1 levels with high fat intake and obesity warrant

further investigation.

The diabetogenic and antiinsulin effects make GH a less than ideal therapeutic

agent for the treatment of obesity. To assess the progression of diabetes, glucose and

insulin measurements were assessed every other week during phase 2 of the study.

Additionally, triplicate glucose measurements were taken at various time points and a

glucose tolerance test was performed at the end of the study. Glucose levels overall

decreased as the amount of GH injected increased. Additionally, the HF-5 group had

decreased blood glucose such that after 4.5 weeks of GH treatment their blood glucose

levels did not significantly differ from those of the LF-PBS group. This was maintained

throughout the conclusion of phase 2 of the study. The HF-0.5 group also showed a

significant decrease in blood glucose compared to the HF-PBS group after 4.5 weeks;

however, the levels were still significantly higher than the LF-PBS group. These

decreases in blood glucose levels suggest that the loss of overall fat mass is able to overcome the diabetogenic effects of GH. Serum insulin measurements were also assessed to measure the diabetic status of the animals. Insulin levels were found to be higher in all HF groups when compared to the LF-PBS group. Specifically, in the HF-0.5 and HF-5 groups, which both had decreased blood glucose, the insulin levels were not

64 found to be different from the HF-PBS group. This indicates that while the blood glucose levels approached normal levels, the animals remained hyperinsulinemic and insulin resistant. While the diabetogenic effect of GH may have been overcome by the decrease in fat mass, the anti-insulin effects of GH may cause the hyperinsulinemic state to persist, suggesting that these properties of GH are distinct and can be separated from one another.

GH has been shown previously to influence organ size and weight. In addition,

GH appears to have depot-specific effect on adipose tissue, with previous studies showing a decrease in the size of the subcutaneous, retroperitoneal and epididymal adipose depots in mice expressing excess GH (Berryman et al., 2004; Berryman et al.,

2006). Models with reduced GH signaling (GH antagonist) and lack of GH signaling

(GH receptor knockout) have increased adipose tissue, with the majority located in the subcutaneous region, suggesting that this depot may be more responsive to the absence of

GH (Berryman et al., 2004; Berryman et al., 2006). Taking into account the differences in size between these animals, it appears that the depots affected by GH, in order from most to least responsive are subcutaneous (inguinal), retroperitoneal and epididymal, with no data provided on the size of the mesenteric depot (Berryman et al., 2004; Berryman et al., 2006). Significant differences in weights of adipose depots were found in this study after the injection of GH. The HF-5 group had the greatest decrease in the adipose depots measured, with varying decreases seen in the remaining groups. The inguinal

(subcutaneous) depot appeared to be the most responsive to GH injections with a 59.4% decrease in size. However, the remaining adipose depots were also highly affected by the

GH treatment, with all depots in the HF-5 group decreasing significantly in size. In this

study it appears that the depots are affected differently, with (in order of most to least

change) the subcutaneous (-59.36%), mesenteric (-51.02%), retroperitoneal (-47.29%)

and epididymal (-35.81%) depots decreasing in size by different amounts.

No significant differences were found in the weights of the other organs collected.

However, trends were observed with some tissues suggesting that additional treatment

with GH or higher doses may cause significant differences. Specifically, livers showed a

decreasing trend with increasing concentrations of GH injections. The group with the

highest doses of GH had livers that, while not significant, tended to be smaller than the

LF-PBS group as well as all the HF groups. The spleen size of the HF-5 group tended to

be larger than any of the other groups, a finding that has been reported previously for

transgenic animals expressing excess GH (Debeljuk, Steger, Wright, Mattison, & Bartke,

1999). This indicates a possible inflammatory response to the bGH used for injections.

Serum measurements of inflammatory markers as well as measurements of bGH antibody

production would provide further insight on the inflammatory status of these animals.

Spleen size has also been reported to increase in animals expressing excess GH (Debeljuk

et al., 1999). Due to the possible immune response to the exogenous GH, it cannot be

concluded that the increase in spleen size is due to the effects of GH signaling.

Future Studies

Additional studies are needed to resolve several findings in the present study.

One issue that remains troubling is the increased insulin levels in the GH treated animals even when the fat mass was decreased and glucose levels normalized. The combined therapy of GH with oral hypoglycemic drugs used to treat diabetes may be helpful to

66 resolve the hyperinsulinemic state that is still present with the treatment of the highest

GH dose. For example, combined therapy of GH with IGF-1, which is known to have insulin like action, may produce a more normal insulin and glucose state while maintaining the positive body composition effects. Previous studies have shown the positive effects of IGF-1 treatment on glucose metabolism in patients with type 1 and type 2 diabetes (Yuen & Dunger, 2007). Weight and, specifically, inguinal fat pad mass were also decreased upon the treatment of GH and IGF-1 in obese rats (Clark et al., 1996;

Dubuis, Deal, Tsagaroulis, Clark, & Van Vliet, 1996). These studies indicate that a positive effect on diabetes and obesity may be achieved when using the hormones in tandem. Additionally, the true diabetogenic effects of the GH treatment can not be fully understood because of the lack of a GH injected LF control. Further studies injecting LF control animals with various levels of GH would show the overall insulin resistant and diabetogenic effect of GH, independent of obese status.

In the highest dose of GH, the accumulation of lean mass with the loss of fat mass may allow these animals to maintain their decreased fat mass state longer if GH injections are stopped. In effect, they may become more resistant to weight regain.

Further studies may provide information on the attenuation of fat mass once injections are stopped and how long this effect persists. Other studies that manipulate diet, such as macronutrient content and caloric intake would also be helpful. The loss of lean muscle as well as fat mass when on a LF diet has been reported previously (Forbes, 1970).

Related to this study, making the animals obese on the HF diet and then reversing their diet to LF either in the presence of injections of GH or PBS would be useful to determine

67 the extent of this lean mass loss with diet versus GH action. Due to the possible dose dependent trend of lean mass accumulation and fat mass loss, a longer study may also show a beneficial effect of the lower doses of GH and/or other, more dramatic consequences of all GH doses. Additionally, the most effective GH doses appeared to be

0.5 µg/g BW and 5 µg/g BW in this study. Future studies may use doses in this range to see if the positive body composition outcomes are maintained and if the negative anti- insulin effects are diminished. The timing of doses may also play a role in the effectiveness of GH. By altering the timing of injections to multiple injections per day or reducing injections to once every 2 days, body composition, glucose and insulin measurements would indicate positive or negative effects of the timing.

Conclusions

1. GH injections caused gain of lean mass and loss of fat mass in mice maintained on a HF diet. The gain of lean mass and loss of fat mass occurred to a greater extent with higher doses of GH.

2. No change in overall body weight was observed with the highest dose of GH.

The gain in lean mass roughly equaled the loss in fat mass, accounting for the stable weights observed in these animals.

3. The catabolic effect of GH on fat mass declined over time, while the anabolic effect on lean mass was maintained.

4. IGF-1 levels were higher with the highest dose of GH; however, IGF-1 levels were significantly elevated in all HF groups.

5. Blood glucose levels decreased with the two highest doses of GH. Glucose

levels with the highest dose of GH were not different from the LF controls after 4.5 weeks of treatment.

6. Plasma insulin levels were elevated in all HF treatment groups. High doses of

GH lowered glucose levels, however; these animals remained hyperinsulinemic.

7. The weight of all four adipose depots measured was decreased with GH

treatment. The subcutaneous depot was most responsive with a decrease of 59.4% in the

HF-5 group.

8. No significant weight differences were observed in the heart, liver, kidney or

spleen. Trends of decreasing liver size and increasing spleen size were observed in the

HF-5 group.

9. Future studies may help elucidate and minimize the insulin resistant effect of

GH as well as the long term effect of GH treatment on body composition.

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APPENDIX A: STATE BY STATE ANALYSIS OF OBESITY AND DIABETES RATES IN THE UNITED STATES

(MOKDAD ET AL., 2003)

Note. Adapted from the CDC at http://www.cdc.gov/diabetes/statistics/maps/index.htm

APPENDIX B: GROWTH HORMONE RELEASE, CONTROL AND TISSUE

EFFECTS (KOPCHICK & ANDRY, 2000)

APPENDIX C: PHENOTYPIC AND METABOLIC PARAMETERS OF TRANSGENIC MOUSE MODELS WITH

ALTERED GH SIGNALING