This thesis has been approved by The Honors Tutorial College and Department of Biological Sciences ___________________________________ Dr. Darlene Berryman Professor, School of Applied Health Sciences and Wellness College of Health Sciences and Professions Thesis Adviser __________________________________ Dr. Soichi Tanda Director of Studies, Honors Tutorial College Biological Sciences _________________________________ Jeremy Webster Dean, Honors Tutorial College THE EFFECT OF GROWTH HORMONE ON THE MACROPHAGE CONTENT OF DIFFERENT ADIPOSE TISSUE DEPOTS A Thesis Presented to The Honors Tutorial College Ohio University _____________________________________________ In Partial Fulfillment Of the Requirements for Graduation From the Honors Tutorial College With the Degree of Bachelor of Science in Biological Sciences _____________________________________________ By Rachel D Munn June 2011 1 Table of Contents Acknowledgements…………………………………………………3 Abstract……………………………………………………………...4 Introduction…………………………………………………………6 Adipose Tissue………………………………………………6 Adipose Tissue Depots……………………………………...7 Obesity………………………………………………………11 Inflammation………………………………………………..12 Macrophages………………………………………………..14 Physiological Function……………………………...14 Adipose Tissue Macrophages……………………….15 Growth Hormone……………………………………………18 Function……………………………………………..19 Growth Hormone and Macrophages………………..23 Growth Hormone and Adipose……………………...24 Transgenic Mouse Models…………………………………..26 Significance of Research…………………………………….29 Materials and Methods…………………………………………….30 Results……………………………………………………………….36 Discussion……………………………………………………………46 References……………………………………………………………55 2 Acknowledgements This work was supported in part by the State of Ohio‟s Eminent Scholar Program that includes a gift from Milton and Lawrence Goll, by the AMVETS, by the Diabetes Research Initiative at Ohio University, by the Provost‟s Undergraduate Research Fund, and by NIH grants DK075436, AG019899, AG031736. Additionally I would like to personally thank Dr. Darlene Berryman, Dr. John Kopchick, and everyone working in Dr. Kopchick‟s lab at Edison Biotechnology Institute for their continued assistance and support in completing this project. I cannot imagine a better group of people with whom to work. I would also like to thank Dr. Soichi Tanda for his support throughout the entirety of my undergraduate career and for his belief in my abilities as a student. Finally, I would like my family and friends for providing such a support network. I am forever grateful. 3 Abstract The prominence of obesity has drastically increased during the last decades and is now approaching epidemic proportions. Obesity is highly associated with deleterious health conditions such as type 2 diabetes and atherosclerosis. The greatly increased amount of adipose tissue present in obese individuals exhibits a chronic, low-grade form of inflammation. This inflammation is hypothesized to be the underlying cause of the aforementioned obesity associated comorbidities. The source of this inflammation is thought to be a specific phenotype of macrophages, M1 macrophages, which infiltrate obese adipose tissue and secrete inflammatory cytokines as well as macrophage chemoattractants. Growth hormone drastically decreases the amount of fat mass by inducing lipolysis and prohibiting lipogenesis. Growth hormone also acts on macrophages; however, current research has yet to define the true effect. The purpose of this study is to elucidate the effect of growth hormone on macrophages within adipose tissue. This study uses three transgenic mouse models with varying levels of growth hormone signaling: bGH (bovine growth hormone), GHA (growth hormone antagonist), and GHR-/- (growth hormone receptor knockout). Two adipose tissue depots were dissected from mice of all three genotypes as well as wild type controls. mRNA expression analysis was performed on whole adipose tissue, via PCR Super Arrays. The expression of several macrophage markers was measured to determine the macrophage content and phenotype within adipose tissue. 4 The results obtained in this study showed no significant effect of growth hormone on the macrophage content of either adipose tissue depot studied. However, a differential content of macrophages between adipose tissue depots was seen, coinciding with previous research demonstrating the functional and metabolic differences between adipose tissue depots. 5 Introduction I. Adipose Tissue Adipose tissue is one of the many types of connective tissue. It is largely composed of adipocytes, or fat cells, but contains other cell types as well, including fibroblasts, macrophages, and endothelial cells. All of these cell types appear to be critical to the function of the tissue and may be responsible for certain molecules secreted by the tissue. Previously, it was thought that the only significant role of adipose tissue was to act as an energy reservoir via the storage of fat. Indeed, adipose functions as a dynamic reservoir to store or release energy depending on the needs of the body. If food and energy are plentiful, the body can deposit the excess energy in adipose tissue in the form of triglycerides (Sethi et al. 2007). During times of starvation or low energy, the body can access this energy by breaking down triglycerides into glycerol and free fatty acids, which are readily transported throughout the body (Sethi et al. 2007). More recently, research has shown that adipose tissue has an additional function as an important endocrine organ secreting hormones such as adiponectin and leptin, as well as many others. Leptin functions as a metabolic signal of energy sufficiency, and as obesity is a state of over-nutrition, high circulating leptin levels are associated with obesity (Kershaw et al. 2004). On the other hand, high levels of adiponectin are associated with lean states and increased insulin sensitivity (Yamauchi et al. 2009). Thus, the endocrine function of adipose tissue varies according to the 6 metabolic state of the tissue. Like all hormones, adipose tissue derived hormones not only affect the tissue itself, but many other tissues and organs as well. For example, leptin has far reaching effects and can dramatically alter energy balance by increasing appetite and decreasing energy expenditure when circulating leptin levels are too low (Kershaw et al. 2004). Equally, the dysfunction of adipose tissue, as in an obese or emaciated state, has far reaching effects on the body. For example, certain antiretroviral medicines used in the treatment of HIV can cause severe lipodystrophy, which is a deficiency in adipose tissue and is associated with metabolic dysfunction (Kershaw et al. 2004). The effects of obesity are discussed in detail below. II. Adipose Tissue Depots There are two distinct types of adipose tissue known as brown and white adipose, which differ in anatomical location, morphology, and function (Casteilla et al. 2001). White adipose tissue is present far more abundantly than brown adipose; however, brown adipose tissue contains more vasculature than white adipose tissue, and brown adipocytes contain more mitochondria and possess the unique ability to produce heat (Casteilla et al. 2001). Brown adipose is most prominent in newborn mice, as well as other mammals (Cinti et al. 2007). However, it has recently been shown that adult humans also have brown fat and that the amount of brown fat present is inversely correlated with body mass index (BMI) (Cypess et al. 2009). This suggests a role for brown fat in adult metabolism, and thus there is much current interest in elucidating the specific function of brown fat in adults. The largest brown adipose depots are located in the interscapular and perirenal areas. Because this study only 7 focuses on white adipose tissue, the remaining review of literature will focus on exclusively white adipose tissue. The white adipose depots can be simply classified as subcutaneous or visceral (Casteilla et al. 2001). There is some inconsistency with regards to the use of the term „visceral.‟ Some authors use visceral only to describe adipose tissue depots which drain into the portal vein, while others include all intra-abdominal depots in the visceral category. The view which categorizes intra-abdominal depots as visceral leads to an overly simplistic view of adipose tissue depots, as the true visceral and intra- abdominal depots have very distinct properties. Adipose tissue is distributed regionally throughout the body. The subcutaneous depot is located superficially beneath the skin in both the anterior and posterior regions (Cinti 2007). The visceral depots are located in the abdominal cavity and, in mice, include the perigonadal, retroperitoneal, and mesenteric depots (Cinti 2007). Using the strict definition of visceral, however, only the mesenteric depot is classified as visceral, while perigonadal and retroperitoneal are intra-abdominal depots. The perigonadal depot is connected to the ovaries in females and the epididymus in males. The retroperitoneal depot is located behind each kidney, while the mesenteric depot connects the intestines. These depots, as well as other depots not analyzed in this study, can be viewed in Figure 1. As can be seen here, adipose is a very anatomically heterogeneous tissue. 8 Figure 1. Representative adipose tissue depots (Sackmann Sala, 2010). The names of the individual depots are as follows: A: deep cervical, B: anterior subcutaneous (interscapular, subscapular, axillo-toracic, superficial cervical), C: visceral mediastinic, D: visceral mesenteric,