Body Composition and Adipokine Levels in Growth Hormone Antagonist 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
Vishakha Magon
June 2009
© 2009 Vishakha Magon. All Rights Reserved.
2
This thesis titled
Body Composition and Adipokine Levels in Growth Hormone Antagonist Mice
by
VISHAKHA MAGON
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 3
ABSTRACT
MAGON, VISHAKHA, M.S., June 2009, Food and Nutrition
Body Composition and Adipokine Levels in Growth Hormone Antagonist Mice (173 pp.)
Director of Thesis: Darlene E. Berryman
The study examined age- and gender-related changes in body weight, body
composition (6 to 80 weeks of age), fasting blood glucose level, and plasma leptin level
(8, 13, 26, 52, and 72 weeks) in male and female growth hormone antagonist (GHA) transgenic mice in comparison to their wild-type (WT) littermate mice. Tissue and organ weights were also assessed at the conclusion of the study. Results showed that male GHA mice approached the weight of male WT mice during the later phase of life but female
GHA mice always weighed significantly less than their controls. GHA mice of both genders exhibited lower lean mass, higher fat mass, and higher serum leptin level with
advancing age. Euglycemia was exhibited in GHA mice and this trend was evident throughout the period of study. Higher subcutaneous (absolute and percent) and
retroperitoneal (absolute) fat pad and lower organ weights (absolute: all organs; percent:
heart, kidney, muscle) were found in GHA mice when compared to controls.
Approved: ______
Darlene E. Berryman
Associate Professor of Human and Consumer Sciences
4
ACKNOWLEDEMENT
I would like to acknowledge and thank my advisor, Dr. Darlene Berryman for her
support, time, encouragement, and advice throughout the course of my Master’s program.
My heartfelt gratitude to my advisory committee, Dr. Fang Meng and Dr. Shigeru Okada,
for their invaluable guidance and timely insights.
A special thank you to Dr. John Kopchick and everyone in his lab at Edison
Biotechnology Institute. Additionally, I would like to thank the School of Human and
Consumer Sciences and the Diabetes Research Initiative for funding my education and providing me the opportunity to work as a graduate assistant.
Last but not least, I thank my family and friends for their endless love and encouragement. Things would not have been easy without their support.
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TABLE OF CONTENTS
Page
ABSTRACT ...... 3
ACKNOWLEDEMENT ...... 4
LIST OF TABLES ...... 10
LIST OF FIGURES ...... 11
LIST OF ABBREVIATIONS ...... 12
CHAPTER 1: INTRODUCTION ...... 14
Statement of the Problem ...... 18
Research Questions ...... 19
Purpose of the Study ...... 20
Limitations/Delimitations ...... 21
Definitions of Terms ...... 22
CHAPTER 2: REVIEW OF LITERATURE ...... 24
Overview of Growth Hormone ...... 24
GH Gene and Protein ...... 25
Structure of GH ...... 25
GH Secretion ...... 26
Biological Effects of GH ...... 28
GH Receptor and GH Binding Protein ...... 29
GH Receptor (GHR) ...... 29 6
GH Binding Protein ...... 31
Mechanism of Action of GH to GHR ...... 32
GH-GHR Interaction ...... 32
Insulin-like Growth Factor ...... 34
Adipose Tissue ...... 36
Types of Adipose Tissue ...... 38
Adipokines ...... 40
Leptin ...... 43
Leptin Receptor ...... 44
Functions of Leptin ...... 46
Obesity and Serum Leptin ...... 48
Leptin Resistance ...... 49
Regulation of Leptin ...... 50
GH and Adipose Tissue ...... 51
The Effects of GH on Adipocyte Differentiation...... 51
GHR in Adipose Tissue ...... 52
GH and Body Composition ...... 54
Alteration in Body Composition due to Variation in GH Secretion ...... 54
Mouse Models with Altered GH Function ...... 57
Growth Hormone Antagonist (GHA) Mice ...... 57
Biological Effects of Growth Hormone Antagonist (GHA) ...... 61
Effects of GHA as a Therapy ...... 61 7
Measurement Techniques to Assess Body Composition ...... 64
Carcass Analysis ...... 64
Dual-energy X-ray Absorption (DEXA)...... 65
Magnetic Resonance Imaging (MRI ...... 67
Nuclear Magnetic Resonance (NMR) ...... 68
Summary ...... 69
CHAPTER 3: METHODOLOGY ...... 71
Animals ...... 71
Production of Genetically Modified Mice ...... 71
Animal Description and Care ...... 72
Measurements ...... 72
Weight Gain Profiles ...... 72
Body Composition Analysis ...... 73
Measurement of Fasting Blood Glucose Levels and Plasma Leptin Levels ...... 73
Tissue/Organ Weights ...... 74
Statistical Analysis ...... 75
CHAPTER 4: RESULTS ...... 77
Body Weight ...... 77
Fat Mass ...... 80
Lean Mass ...... 86
Fluid Mass ...... 90
Body Composition Profile ...... 91 8
Blood Glucose Levels ...... 93
Plasma Leptin Concentrations ...... 95
Tissue/Organ Weights ...... 96
Note ...... 97
CHAPTER 5: DISCUSSION AND CONCLUSION ...... 102
Summary ...... 110
Future Studies ...... 111
REFERENCES ...... 112
APPENDIX A: PHENOTYPE OF GROWTH HORMONE ANTAGONIST AND
WILD-TYPE MICE ...... 164
APPENDIX B: TWO-WAY ANOVA RESULTS OF BODY WEIGHTS IN GHA AND
WT MICE ...... 165
APPENDIX C: TWO-WAY ANOVA RESULTS OF ABSOLUTE FAT MASS IN GHA
AND WT MICE ...... 166
APPENDIX D: TWO-WAY ANOVA RESULTS OF PERCENT FAT MASS IN GHA
AND WT MICE ...... 167
APPENDIX E: TWO-WAY ANOVA RESULTS OF ABSOLUTE LEAN MASS IN
GHA AND WT MICE ...... 168
APPENDIX F: TWO-WAY ANOVA RESULTS OF PERCENT LEAN MASS IN GHA
AND WT MICE ...... 169
APPENDIX G: TWO-WAY ANOVA RESULTS OF ABSOLUTE FLUID MASS IN
GHA AND WT MICE ...... 170 9
APPENDIX H: TWO-WAY ANOVA RESULTS OF PERCENT FLUID MASS IN
GHA AND WT MICE ...... 171
APPENDIX I: TWO-WAY ANOVA RESULTS OF FASTING BLOOD GLUCOSE
LEVELS IN GHA AND WT MICE ...... 172
APPENDIX J: TWO-WAY ANOVA RESULTS OF PLAMA LEPTIN
CONCENTRATIONS IN GHA AND WT MICE ...... 173
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LIST OF TABLES
Page
Table 1: Adipokines and Their Metabolic Functions ...... 41
Table 2: Statistical Tests Used to Analyze the Research Questions ...... 76
Table 3: Two-way ANOVA Results of Absolute Tissue/Organ Weights in GHA and WT
Mice ...... 99
Table 4: Two-way ANOVA Results of Percent Tissue/Organ Weights in GHA and WT
Mice ...... 100 11
LIST OF FIGURES
Page
Figure 1: Body weights of GHA mice and WT mice over time...... 79
Figure 2: Absolute fat mass of GHA mice and WT mice over time ...... 82
Figure 3: Percent fat mass of GHA mice and WT mice over time ...... 85
Figure 4: Lean mass of GHA mice and WT mice over time ...... 87
Figure 5: Percent lean mass of GHA mice and WT mice over time ...... 89
Figure 6: Absolute weight and body composition profile over time in GHA and WT
mice ...... 92
Figure 7: Fasting blood glucose levels in GHA and WT mice over time ...... 94
Figure 8: Plasma leptin level in GHA and WT mice over time ...... 96
Figure 9: Tissue/organ weights (absolute and when normalized to body weight) of GHA
and WT mice over time ...... 101
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LIST OF ABBREVIATIONS
ATP : Adenosine Triphosphate
BAT : Brown Adipose Tissue
bGH : Bovine Growth Hormone
cAMP : Cyclic Adenosine Monophosphate
CNS : Central Nervous System
Cys : Cystine
DNA : Deoxyribonucleic Acid
GH : Growth Hormone
GHA : Growth Hormone Antagonist
GHBP : Growth Hormone Binding Protein
GHR : Growth Hormone Receptor
GHRH : Growth Hormone Releasing Hormone
GHRP : Growth Hormone Releasing Peptide
ICV : Intracerebroventricular
IGF-I : Insulin-like Growth Factor-I
IGFBP : Insulin-like Growth Factor Binding Protein
IGF-IR : Insulin-like Growth Factor-I Receptor
IL : Interleukin
IRS : Insulin Receptor Substrate
JAK-2 : Janus Kinase-2 13
NMR : Nuclear Magnetic Resonance
PAI-1 : Plasminogen Activator Inhibitor-1
PCR : Polymerase Chain Reaction
SOCS : Supressors of Cytokine Signaling
SREBP-1c : Sterol Response Element Binding Protein-1c
SRIH : Somatotropin Release Inhibiting Hormone
STAT : Signal Transduction and Activator of Transcription
TNF-α : Tumor Necrosis Factor-α
WAT : White Adipose Tissue
WT : Wild Type
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CHAPTER 1: INTRODUCTION
Adipose tissue was earlier viewed as a simple repository for storing energy. But the growing concern with obesity and insulin resistance has led to closer examination of this tissue. An increase in adipose tissue deposition, especially the white adipose tissue
(WAT) is generally associated with profound metabolic and biochemical changes in the body. In obese individuals, the capacity to store excess fat in adipose tissue substantially increases which leads to the abnormal lipid deposition in other tissues as well. The accumulation of lipids in nonadipose tissues consequently contributes to the development of a number of chronic ailments like type 2 diabetes (Caprio, et al., 1995), high blood pressure, dyslipidemia (Brambilla, et al., 1994; Caprio, et al., 1995), coronary artery diseases (Brochu, Poehlman, & Ades, 2000), and some cancers (Calle & Kaaks, 2004).
Thus, excessive fat deposition in adipose tissue eventually leads to increased mortality and morbidity rates. The alarming growth rate of obesity-related disorders in the current era has precipitated the need to acquire a better understanding of the biology of adipose tissue. Recent research efforts have emphasized on evaluating the physiological role of adipose tissue based upon the location of individual fat depots (subcutaneous, visceral, and interstitial adipose tissue) and pathological conditions due to abnormal fat deposition.
An increased understanding of the molecular and biological aspects of adipose tissue has proved it to be an essential organ. Some of the vital functions of adipose tissue are:
1. Storing energy in the form of triglycerides and mobilizing stored lipid in the
form of free fatty acids and glycerol during energy crisis; 15
2. Enhancing thermogenesis (in brown adipose tissue [BAT] with its uncoupling
protein-1) especially in neonates and small mammals in cold environments to
survive the cold stress (Klaus, 2004; Lafontan, 2005);
3. Providing thermal insulation to the body;
4. Participating in inflammatory processes (Cousin, et al., 1999);
5. Secreting variety of protein hormones called as adipokines that act in either
endocrine, autocrine, or paracrine manner.
Collectively, adipose tissue is regarded as an important metabolic organ that plays
a significant role in coordinating number of functions. Most importantly, it is the active
release of a variety of adipokines that has the ability to influence body’s biological
processes, such as energy balance, thermoregulation, and immune function.
Growth hormone (GH) is a hormone released from pituitary gland. As the name
implies, GH promote longitudinal growth of skeletal and soft tissues (Isaksson, Eden, &
Jansson, 1985). In addition, GH is also known to govern a variety of other functions. For
example, GH is one of the key regulators of adipose tissue metabolism. GH promotes its effect by acting as an anabolic agent. Any alteration in GH level induces significant
changes in the body due to its ability to influence carbohydrate, lipid, and protein
metabolism (Davidson, 1987). Excessive GH levels promote a lean phenotype by
increasing muscle mass and reducing accretion of adipose tissue (B. A. Bengtsson,
Brummer, Eden, Bosaeus, & Lindstedt, 1989; Davidson, 1987; Strobl & Thomas, 1994).
On the other hand, deficiency in the GH levels lead to obesity due to significant increase in body fat and reduction of lean body mass (Donahue & Beamer, 1993; Rosen, Bosaeus, 16
Tolli, Lindstedt, & Bengtsson, 1993). Number of related research in both humans and
laboratory animals has revealed that GH plays a profound role in regulating body composition (Carrel, Myers, Whitman, & Allen, 1999, Gause & Eden, 1985; Knapp,
Chen, Turner, Byers, & Kopchick, 1994, Searle, Murray, & Baker, 1992; Turner, Knapp,
Byers, & Kopchick, 1998; S. Yang, Bjorntorp, Liu, & Eden, 1996).
Many studies have utilized animal models, specifically mice. The basis behind
using mice models for laboratory studies is the close resemblance of mouse GH structure
and function to human GH (Chene, Martal, de la Llosa, & Charrier, 1989). In order to
gain in-depth knowledge regarding the effect of altered GH levels on body composition,
genetically modified mice have always been a very useful tool.
In this project, growth hormone antagonist (GHA) mice were utilized to assess the relationship of GH deficiency on adipose tissue. GHA mice are a type of transgenic
mouse line that are generated by substituting the amino acid glycine-119 with lysine at
site-2 of the receptor binding region in the third helix of GH (W. Y. Chen, White,
Wagner, & Kopchick, 1991). Due to close resemblance of GHA with GH, GHA
specifically competes with the endogenous GH’s ability to bind to the receptor, thus
acting as a competitive inhibitor (W. Y. Chen, Wight, et al., 1991). Because of this
antagonistic effect, there is reduction of intracellular GH signaling that eventually leads
to the development of dwarf GHA mice (W. Y. Chen, Wight, Mehta, et al., 1991; W. Y.
Chen, Wight, Wagner, & Kopchick, 1990; W. Y. Chen, White, et al., 1991; Coschigano
et al., 2003; see Appendix A). A previous study compared GHA male mice with age-
related control littermates for 11 months and found that GHA male mice while dwarf in 17 early life have a tendency to eventually catch up to the weight of wild-type (WT) littermate mice (Coschigano et al., 2003). Futhermore, GHA male mice maintained normal fasting blood glucose level, serum insulin level, and lifespan (Coschigano et al.,
2003). Another study showed that GHA male mice exhibit marked obesity at the age of 6 months (Berryman et al., 2004).
In humans, many therapeutic agents are currently available to modify GH function. For instance, GH replacement in GH deficient obese adults and children is recognized as a therapy for the treatment of body composition by decreasing visceral adiposity and promoting lean phenotype (Binnerts, Swart, et al., 1992; Johannsson, et al.,
1997; Kuromaru et al., 1998; Salomon, Cuneo, Hesp, & Sonksen, 1989). Likewise, treatment of excessive GH levels in the body, a condition known as acromegaly, is important because high levels of GH have numerous negative effects, such as cardiovascular problems, type 2 diabetes, and kidney problems. Treatments for acromegaly and its related problems are also of prime interest due to the underlying impact of excess of GH on the sufferer. GHA is a commercially available Food and Drug
Administration approved drug, marketed under the name pegvisomant, for treatment of acromegaly. The use of pegvisomant has been shown to normalize serum insulin-like growth factor-I (IGF-I) level in over 90% of the patients all over the world, but the efficiency of long-term administration of GHA drug is still debatable. Hence, the determination of body composition in GHA mice is of primary interest for two reasons.
First, GHA mice represent a good model to study the long-term effects of antagonizing
GH function on body composition, as this animal model seems to mirror the effect of this 18
drug on humans. Secondly, GHA is an approved therapeutic agent and its long-term
impact on body composition has never been assessed. Previous studies have established
that male GHA mice develop obesity at the age of 6 months (Berryman et al., 2004).
However, no one has tracked the alterations in the body composition of these mice over their entire lifespan or studied the root cause of the weight gain characteristic of these mice later in their life. In addition, adipokine levels over the lifespan in GHA mice and the body composition of the female GHA mice have never been assessed. Therefore, this thesis attempts to fill the gap in the literature by investigating the impact of reduced GH signaling on body composition and adipokine levels in male and female GHA mice over
their lifespan (youth to late adulthood).
Statement of the Problem
GH deficiency enhances fat mass and reduces lean body mass (Donahue &
Beamer, 1993; Rosen et al., 1993) in almost all vertebrates. Likewise, previous research
on GHA mice, a genetically engineered mouse model with GH deficiency, revealed that
these male mice were obese at the age of 6 months (Berryman et al., 2004). Interestingly,
male GHA mice were reported previously to weigh significantly less in their early life but
eventually caught up with littermate control mice in body weight in their later life
(Coschigano et al., 2003). However, the exact contribution of lean mass versus fat mass
in this eventual weight gain has not been assessed. Furthermore pegvisomant, a FDA-
approved drug for treating acromegaly in humans, is currently being tested for treatment
of various GH related conditions, such as cancer and diabetes. Because studying body 19
composition over time in humans treated with this drug would be challenging, research
conducted on GHA mice provides a means to test the long-term impacts of GH antagonist on accumulation of adipose tissue, which is also relevant to the onset of diabetes and
cancer.
Research Questions
In this study, the following research questions were addressed:
1. How does reduced GH signaling in GHA mice affect body composition (fat and
lean mass) over time?
H1: The male GHA mice exhibit increase in fat mass and decrease in lean mass
that contribute to weight gain at the later phase of life.
H 2: The male GHA mice exhibit increase in lean mass and decrease in fat mass
that contribute to weight gain at the later phase of life.
H3: The male GHA mice exhibit increase in both fat mass and lean mass that
contribute to weight gain at the later phase of life.
2. How does plasma leptin level alter with advancing age in GHA male mice?
H1: The male GHA mice exhibit higher plasma leptin levels with advancing age.
3. Are there significant gender differences in body weight, body composition,
fasting blood glucose levels, plasma leptin levels, and tissue/organ weights
between male GHA mice and female GHA mice?
H1: The male GHA mice weigh more than female GHA mice. 20
H2: The male GHA mice have higher total and percent fat mass than female GHA
mice.
H3: The male GHA mice have higher total and percent lean mass than female
GHA mice.
H4: The male GHA mice have higher fasting blood glucose levels than female
GHA mice.
H5: The male GHA mice have higher plasma leptin levels than female GHA
mice.
H6: The tissue/organ weight of male GHA mice is greater than female GHA mice.
Purpose of the Study
Previous research in our laboratory has shown that 6 month old male GHA mice are obese (Berryman et al., 2004). These mice have also been reported to exhibit normal lifespan and relatively normal insulin sensitivity, at least as demonstrated in 11 month old male mice (Coschigano et al., 2003). An interesting observation in male GHA mice was that, while they remain dwarf and weigh significantly less in early life, they eventually catch up to littermate controls in body weight around 11 months of age (Coschigano et al., 2003). The purpose of this study is to determine the cause of the weight gain in later life via body composition measurements in male GHA mice. Additionally, the analysis of plasma leptin levels paired with body composition in GHA mice might provide an insight to a few of the unanswered questions. First, the cause of eventual weight gain with advancing age in the male GHA mice. Second, the reason as to why GHA mice do not 21 exhibit the improved insulin sensitivity or improved lifespan as seen with other mouse models with repression in GH signaling. Third, to address the role of GHA on adipose tissue in female mice and finally, to provide an insight into the role of GHA, an FDA approved drug, for treatment of body composition and several disorders like diabetes and cancer.
Limitations/Delimitations
1. Mice placed in one cage may develop a hierarchy system that can result in altered
weight gains and body composition.
2. The Bruker nuclear magnetic resonance (NMR) analyzer may not reflect the exact
body composition, either due to its own limitations or due to manual error.
3. As the mouse models do not fully represent humans, the data collected from the
mouse models may not represent the human condition.
4. Physical activity and amount of food eaten by mice was not monitored or
measured. 22
Definitions of Terms
Acromegaly. A disfiguring condition caused by GH hypersecretion due to pituitary somatotroph adenoma that leads to premature death and increased morbidity
(Melmed, 1998). Acromegalic patients can be visually diagnosed due to the enlargement and thickening of bones including hands, feet, and face. Other associated problems are hypertension, headache, sleep apnea, visual disturbance, and amenorrhea (Nabarro,
1987).
Adipocyte. Cells that primarily compose adipose tissue and extensively store energy in the form of fat.
Adipose tissue. It is an anatomical term used for a specialized connective tissue and is mainly composed of adipocytes (Large, Peroni, Letexier, Ray, & Beylot, 2004).
Adipose tissue is mainly viewed as an energy storage depot, a thermal insulator, and a mechanical cushion in mammals although more recently it has been demonstrated to have an endocrine function.
Adipokines. Adipokines are defined as the factors that are specifically expressed in the adipocyte which are secreted and delivered into the systemic circulation.
Adipokines can affect both central and peripheral organs like skeletal muscles, immune system, adrenal cortex, brain, and sympathetic nervous system (Ahima, 2006).
Leptin. Adipocytes secrete leptin in direct proportion to the adipose tissue. The primary function of leptin is to regulate food intake and energy expenditure that are mediated by satiety signals via hypothalamic pathways (Bjorbaek & Kahn, 2004). In vitro studies have also shown that leptin inhibit lipogenesis (Ramsay, 2003) and stimulate 23 lipolysis (Frubeck, Aguado, & Martinez, 1997). Other functions of leptin include improving insulin sensitivity (Lam, et al., 2004) and promoting angiogenic activity (Park, et al., 2001).
Lipolysis. It is defined as breakdown of triglycerides or fatty acid mobilization from adipose tissue. GH is well-known to triggers lipolysis in adipose tissue (Dietz &
Schwartz, 1991).
Growth hormone antagonist (GHA) mice. GHA mice are transgenic and display dwarf phenotype as a result of the endogenous competition of GHA with GH (W. Y.
Chen, White, et al., 1991). GHA mice are generated by mutating bovine GH gene and are characterized by increased fat mass in the subcutaneous region (Berryman et al., 2004).
Nuclear magnetic resonance (NMR) analyzer. NMR is a noninvasive equipment used to assess the body composition, i.e., whole-body fat, total body water, and total lean mass in live unanesthetized mice (Mystkowski, et al., 2000).
. 24
CHAPTER 2: REVIEW OF LITERATURE
Overview of Growth Hormone
Growth hormone (GH), also called somatotropin, is a protein that is produced and released by somatotrophic cells of the anterior pituitary gland. Somatotrophs possesses secretory granules that store and later secrete mature GH into the circulation. Released
GH interacts and activates the receptors present on other tissues and exhibits diverse biological effects. For instance, GH plays a pivotal role in promoting postnatal longitudinal growth by increasing both cell size and number in most of the body cells and tissues (Isaksson et al., 1985). For this reason, certain imbalances of GH levels before puberty in young vertebrates brings about physiological changes like gigantism (casued due to excess of GH activity) or dwarfism (brought about by GH deficiency; Donahue &
Beamer, 1993; Zhou et al., 1997). Moreover, GH exerts profound effect on nutrient metabolism affecting protein, carbohydrate, and lipid content in the body (Davidson,
1987; Strobl & Thomas, 1994). The mechanism of GH action on substrate metabolism is brought about by stimulating protein accretion in muscle, redirecting glucose intended for lipid deposition to other tissues, and promoting lipid mobilization from adipose stores
(Davidson, 1987). Thus, GH action is multifaceted and promotes diverse effects in the body.
25
GH Gene and Protein
GH is a member of a cytokine superfamily that includes group of homologues proteins like prolactin, placental lactogens (Forsyth, 1986), mouse proliferin (Linzer, Lee,
Ogren, Talamantes, & Nathans, 1985), rat decidual PRL-like protein (Roby, et al., 1993), and somatolactin (Ono, et al., 1990). These hormones encode similar primary genes, share highly related primary structures (Corbacho, Martinez de la Escalera, & Clapp,
2002) and physiological functions, such as growth promotion, lactation, cell development, and cell differentiation (Isaksson et al., 1985).
Structure of GH
GH is a four-helical bundle protein. The three-dimensional structure was first established for porcine GH (Abdel-Meguid et al., 1987) and then few years later for human GH (A. M. de Vos, Ultsch, & Kossiakoff, 1992). Closer analysis of the crystalline
structure of porcine and human GH revealed that both contain four alpha (α) helices with
around 21-30 amino acids in each, two disulfide bridges, and four antiparallel alpha
helices that are arranged in an up-up-down-down manner (Watahiki, Yamamoto,
Yamakawa, Tanaka, & Nakashima, 1989). Structurally, half of the amino acids reside in
four α-helices and ~20 hydrophobic residues bunch at the core of the GH protein (Abdel-
Meguid et al., 1987). The clustered hydrophobic amino acids are responsible to tightly pack the four-helix bundle in a stable configuration (A. M. de Vos et al., 1992).
Human GH is a single chain polypeptide containing ~191 amino acids and molecular mass of ~22 kDa. The alpha helical structure and the disulfide bridges of human GH (Cys 35-Cys165 and Cys 182-Cys 169) present in helix 1 and 2 are extremely 26
important for sustaining GH’s biological activity and for promoting proper interaction with GH receptors present on the surface of various target tissues (X. Z. Chen et al.,
1992).
GH Secretion
There are many factors upon which GH secretion depends. First, GH secretion varies throughout the day. GH, which is secreted from the anterior pituitary gland, is released in the systemic circulation in a pulsatile manner that peaks at night during sleep in both normal men and women (Fowelin, Attvall, von Schenck, Smith, & Lager, 1990;
Tannenbaum & Ling, 1984). Second, different phases of life also impact GH secretion and action. For instance, GH level peaks during puberty, decreases in adulthood, and then further declines with the age (Korbonits & Besser, 1996). Most importantly, GH
secretion is regulated by different hormones such as GH releasing hormone (GHRH),
somatotropin release-inhibiting hormone (SRIH), insulin-like growth factor-I (IGF-I),
and GH releasing peptides (GHRPs). Out of these, the two opposing hypothalamic
hormones, GHRH and SRIH predominantly regulate GH release from the pituitary.
GHRH stimulates GH synthesis and release; whereas, SRIH or somatostatin attenuates
GH secretion (Frohman & Kineman, 1998; Muller, 1987). In brief, GHRH exerts its
activity by binding with GHRH receptors, a G-protein-coupled receptor (Mayo, 1992).
GHRH-GHRH receptor binding leads to an increase in the concentration of cyclic
adenosine monophosphate (cAMP) in somatotrophs (Mayo, 1992) that consequently
stimulates GH secretion (McCormick, Brady, Theill, & Karin, 1990). On the contrary, 27
SRIH decreases the level of cAMP in somatotrophs (Harwood, Grewe, & Aguilera,
1984), thereby inhibits the release of GH (McCormick et al., 1990).
GH release is also controlled by GH itself (Goth, Makara, & Gerendai, 2001) and
by systemic produced IGF-I from liver, bone, and other types of tissues (Salmon &
Daughaday, 1957). GH directly acts on the hypothalamus by a short-loop mechanism;
stimulates the release of SRIH and inhibits GHRH. On the other hand, IGF-I acts through
long-loop mechanism and inhibits the stimulatory effect of GHRH via negative feedback
(Goth et al., 2001). But there are certain conditions where circulating IGF-I are low, for
e.g., in cases of Laron syndrome (defective GH receptor) that consequently inhibits the
stimulation of SRIH and elevates GH concentration in the blood (Goth et al., 2001).
GH releasing peptides (GHRPs) also act as potent stimulators of GH secretion
(Bowers, 1998). Among GHRPs, the most recognizable form is ghrelin, which was first isolated from rat stomach (Kojima et al., 1999). Since then, a number of other tissues has been discovered that produce considerable amount of ghrelin such as intestine, pancreas, gonads, kidneys, heart, vasculature, and adrenal (Andreis, et al., 2003; Gnanapavan, et al., 2002; Muccioli, Papotti, Locatelli, Ghigo, & Deghenghi, 2001; Tortorella et al., 2003;
Volante, et al., 2002). Ghrelin binds to ghrelin receptors (also known as GH secretagogue
receptors) that are expressed in the normal pituitary gland and promotes various functions
(Pong et al., 1996). For example, ghrelin is known to stimulate appetite and prolactin
release, to promote adipogenesis, energy metabolism, to increase osteoblastic activity,
and to regulate homeostasis (Broglio, et al., 2002; Fukushima, et al., 2005; Thompson, et 28
al., 2004). The significance of ghrelin in the release of GH lies in the fact that ghrelin is
capable of independently regulating the GH secretion (Yamazaki, et al., 2002).
Many other factors have also been identified that influence GH secretion. They
can be broadly categorized under nutritional status and physical status. Nutritional status includes obesity, malnutrition, fasting, and diabetes mellitus state (Muller, Locatelli, &
Cocchi, 1999). For instance, hypoglycemia and increased levels of amino acids in the blood stimulates the release of GH (Muller et al., 1999); whereas, hyperglycemia and free fatty acids suppress GH secretion (Lanzi, Losa, Mignogna, Caumo, & Pontiroli, 1999).
Physical factors like sleep, stress, and exercise also have a profound impact on the GH serum level (Bertherat, Bluet-Pajot, & Epelbaum, 1995; Holl, Hartman, Veldhuis, Taylor,
& Thorner, 1991; Kraemer, et al., 1991).
Biological Effects of GH
GH has a profound effect throughout the body. One of the most significant roles of GH is the enlargement of various tissues, thus GH is well-recognized as a vital hormone that can significantly affect various aspects of postnatal longitudinal growth
(Isaksson, Lindahl, Nilsson, & Isgaard, 1987). The GH action is mediated by its ability to
bind to specific GH receptors (GHRs) present on the surface of target tissues. The GH-
GHR binding triggers various signaling pathways that eventually vary the transcription
levels of many genes; most importantly that of IGF-I expression which is dramatically
increased (Okada & Kopchick, 2001). Like GH, IGF-I once released also has the ability
to regulate animal growth and tissue differentiation (Daughaday & Rotwein, 1989;
Froesch, Schmid, Schwander, & Zapf, 1985). Thus, GH both directly and indirectly 29
(through IGF-I action) influences the production of various metabolites (Green,
Morikawa, & Nixon, 1985) that promote longitudinal growth in the body.
GH is one of the prime regulators of substrate metabolism (i.e., carbohydrate, lipid, nitrogen, and mineral metabolism) within a cell (Casanueva, 1992; Davidson,
1987). As mentioned in brief earlier, GH stimulates protein accretion in muscle, redirects glucose intended for lipid deposition to other tissues, and promotes lipid mobilization from adipose stores (Davidson, 1987). In carbohydrate metabolism, GH has two opposing actions: an insulin-like effect and an anti-insulin effect. Initially, there is early insulin-like effect that lasts for around 2 h and involves lipogenesis and increased glucose and amino acid metabolism (Casanueva, 1992). Thereafter, the anti-insulin effect comes into play after 3 h of GH exposure and induces lipolysis, hyperglycemia, and hyperinsulinemia
(Campbell, Davidson, & Lei, 1950; Davidson, 1987; Houssay & Anderson, 1949; Moller et al., 1991). The anti-insulin effect appears to be more relevant to the human condition where cells are consistently exposed to some level of GH. In addition, GH is known to promote anabolic effects in the body by enhancing lean body mass and reducing fat mass
(Corpas, Harman, & Blackman, 1993). Nitrogen retention, mineral metabolism, and electrolyte balance are some additional functions of GH (Strobl & Thomas, 1994).
GH Receptor and GH Binding Protein
GH Receptor (GHR)
The ability of GH to promote variety of biological effects is dependent upon its affinity to bind to specific receptors known as growth hormone receptors (GHRs) that are 30 present on the surface of target tissues. GHR is a type I transmembrane glycoprotein that belongs to class-I hematopoietin/cytokine-growth hormone/prolactin receptor superfamily (Kelly, et al., 1994) and are ubiquitously expressed throughout the body
(Lobie, Garcia-Aragon, Wang, Baumbach, & Waters, 1992). High levels of GHR are expressed especially in liver and adipose tissue (D. W. Leung, et al., 1987) together with muscles (Kolle, et al., 1998), mammary glands (D. W. Sinowatz et al., 2000), bones
(Barnard, Ng, Martin, & Waters, 1991), kidneys (Menon, et al., 1994), and embryonic stem cells (Ohlsson et al., 1993). Immune cells especially T-cells, B-cells, and monocytes of both human and mouse tissue have also been shown to localize GHRs (Hull,
Thiagarajah, & Harvey, 1996).
Structure of GH receptor. GHR is a single pass transmembrane polypeptide having molecular mass of 70 kDa and ~620 amino acids in length depending on the species (Postel-Vinay & Finidori, 1995). Structurally, GHR contains an extracellular hormone binding region along with 246 amino acids at the amino terminus (ectodomain) and an intracellular signaling region spanning ~350 amino acids at its carboxyl terminus
(D. W. Leung, et al., 1987). There are some species specific differences in the gene and the protein. For instance, size and sequence of mouse GHR are similar to the human
GHR gene with the exception of the presence of two more exons- 4B and 8 in mouse
GHR gene (Talamantes, 1994).
A closer analysis of the crystalline structure of human GHR revealed that extracellularly GHR contains two specific domains termed as domain 1 (amino acids 1-
123) and domain 2 (amino acids 128-238), which are connected by four amino acids 31
(124-127; A. M.de Vos et al., 1992). Each domain in the extracellular region is composed
of seven β-strands that are divided into two antiparallel β sheets (A. M. de Vos et al.,
1992). These β sheets include three disulfide bonds (Cys 38-48, Cys 83-94, and Cys 108-
122) that brings stability to the structure of GHR (A. M. de Vos et al., 1992). Moreover,
hydrogen bonds (between arginine 43 and glutamic acid 169) and salt bridge (between
arginine 39 and aspartic acid 123) further adds stability to GHR structure (Wells, et al.,
1993). Domain 2 of GHR is characterized by the presence of two regions: an extracellular
dimerization domain (indirectly involved in ligand binding) and a conserved YXXFS
motif (tyrosine; where X is glycine, serine, lysine, or glutamic acid, phenylalanine; and
serine; Kopchick & Andry, 2000). The YXXFS motif is consistent with a conserved
WSXWS sequence (tryptophan, serine, any amino acid, tryptophan, serine) that are
present in all the members of cytokine superfamily, except GHR (Kelly, et al., 1994).
The intracellular region of the GHR is predominantly responsible for inducing
secondary transduction pathways. The transduction pathways are discussed in detail in
the subsequent section entitled mechanism of action of GH to GHR.
GH Binding Protein
The extracellular domain of GHR releases growth hormone binding protein
(GHBP) into the extracellular space. GHBP is a glycoprotein that has ~255-273 amino
acids and a molecular weight of 55 kDa (Bass, Mulkerrin, & Wells, 1991). The released
GHBP is produced from GHR by two mechanisms, namely, alternative splicing of the
GHR precursor mRNA in rodents (Baumbach, Horner, & Logan, 1989) and targeted
proteolysis in humans, rabbits, monkeys, chickens, and other species (Dastot, Duquesnoy, 32
Sobrier, Goossens, & Amselem, 1998; Smith, Kuniyoshi, & Talamantes, 1989). The net effect of resultant GHBP is difficult to predict owing to some of its contradictory functions. Several studies have revealed the inhibitory effect of GHBP, for e.g., GHBP competes with GH at the local level for binding to the GHRs, thereby, inhibiting GH action as clearly shown the in vitro studies (Mannor, Winer, Shaw, & Baumann, 1991).
In contrast, several other studies have demonstrated some beneficial effects of GHBP. It is believed that nearly 60% of circulating GH is bound to GHBP (Baumann, Amburn, &
Shaw, 1988). Hence, under normal circumstances, GHBP may act as a means to reserve
GH in the serum, allowing considerable quantity of GH to be accessible at all times
(Kopchick & Andry, 2000). In vitro studies have also shown that if GHBP is given in large doses, it enhances GH activity, which is in contrast to its inhibitory action in vivo
(R. G. Clark, et al., 1996). The GH-GHBP complex also decreases the clearance or chemical degradation of GH that occurs through GHR mediated pathway such as cellular internalizations (G. Bauman, 2005). Furthermore, in humans, GH-GHBP complex increases the molecular weight and the plasma half-life of free GH from 4-9 min to 25-29 min (Baumann et al., 1988; Veldhuis, Johnson, Faunt, Mercado, & Baumann, 1993). By and large, due to the controversial functions, the significance of circulating GHBP is yet to be resolved.
Mechanism of Action of GH to GHR
GH-GHR Interaction
GH binds to GHRs present on target tissues with high affinity. The biological activities of GH are mediated only when GH binds perfectly with GHR that initiate 33
various signal cascades within the cell (Silva, Weber, & Thorner, 1993). Generally, GHR
exists as a preformed homodimer (two structurally similar receptors linked together) on
the outer membrane of the target tissue (Gent, van Kerkhof, Roza, Bu, & Strous, 2002).
The GH-GHR interaction is a sequential process, in which one GH molecule binds with
two preformed GHRs (Cunningham et al., 1991; A. M. de Vos et al., 1992). This
interaction brings conformational changes in the GHR that is required for promoting
signaling transduction (Mellado, et al., 1997; Rowlinson, et al., 1998). In brief, GH
contains two binding sites: site-1 and site-2 (W. Y. Chen, Wight, Mehta, et al., 1991;
Cunningham et al., 1991). Site-1 of GH binds to GHR; the same GH molecule then binds to the second monomer of the GHR homodimer through a low affinity binding site-2
(Argetsinger, et al., 1993). On binding of GH to site-2 of GHR, the second GHR molecule slightly rotates and moves vertically with respect to first GHR molecule that consequently brings about a conformational change in the intracellular signaling region
GHR; thereby, activating various signal transduction pathways within the cell (Brown-
Borg, Borg, Meliska, & Bartke, 1996). Predominantly, the GH-GHR induced signaling
cascade has two requirements: (a) GH must interact with two GHRs and (b) GH at helix
three (site-2) must bind to the second monomer of GHR, in particular with aspartic acid
152, tyrosine 200, and serine 201 of GHR (Gent, Van Den Eijnden, Van Kerkhof, &
Strous, 2003).
Janus Kinases and Other Signaling Pathways
When GH binds to GHR, then 121-kDa tyrosine kinase JAK-2 (Janus-kinase)
present in intracellular signaling region of GHR gets activated via phosphorylation. GH- 34 induced JAK2 activation further initiates various intracellular signaling pathways within the cell (Argetsinger ,et al., 1993) such as activation of signal transducer and activator of transcription (STAT) family, i.e., STAT 1, 3, and 5 (Silva et al., 1993; X. Wang, Xu,
Souza, & Kopchick, 1994), mitogen-activated protein kinase pathway (Winston &
Bertics, 1992), and insulin receptor substrate (IRS) family, i.e., IRS-1 and 2 (Argetsinger, et al., 1995). All these activated signal transduction pathways have their unique actions and regulates transcription of variety of genes. However, depending on the substrates available in the particular cell type, any one of these pathways would predominate.
Example of one such gene that is transcribed and is of considerable importance is IGF-I, though the mechanism of its production is difficult to delineate. All major organs in the body, such as liver, bone, muscle, and adipose tissue produce significant amount of IGF-I due to GH action (Casanueva, 1992). These molecules (GH and IGF-I) together affect cell metabolism, differentiation, and proliferation (Daughaday & Rotwein, 1989; Froesch et al., 1985). Thus, GH-GHR signaling cascades are complex and their impacts are widespread, complex, and diverse.
Insulin-like Growth Factor
The IGF system consists of two IGFs (IGF-I and IGF-II), two receptors, along with a minimum of six IGF-binding proteins (IGFBPs; Zapf & Froesch, 1986). Numerous studies using genetically engineered mice (J. P. Liu, Baker, Perkins, Robertson, &
Efstratiadis, 1993), human IGF-I gene mutations (Woods, Camacho-Hubner, Savage, & 35
Clark, 1996), and infants (Kajantie, et al.,