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Evaluating the role of fibroblast and fibroblast growth factor 21 in growth hormone-induced adipose tissue fibrosis

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A Thesis

Presented to

The Honors Tutorial College

Ohio University

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In Partial Fulfillment of the Requirements for Graduation from the Honors Tutorial College with the degree of

Bachelor of Science in Biological Sciences

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by

Delaney Kate Geitgey

April 2020

TABLE OF CONTENTS

TABLE OF CONTENTS ...... 2 ABSTRACT ...... 4 INTRODUCTION...... 6 Characteristics of adipose tissue and adipocytes ...... 7 Adipocyte function...... 7 Adipose tissue depots ...... 8 Main classifications ...... 8 Subcutaneous depots ...... 9 Intra-abdominal depots ...... 10 Characteristics of fibrosis ...... 11 Extracellular matrix (ECM) and fibrosis ...... 11 Fibrotic disease states ...... 12 Development of fibrosis ...... 15 Contributing factors to fibrosis ...... 16 Fibroblasts in fibrosis ...... 16 Fibroblast activation protein ...... 17 Fibroblast growth factor 21 ...... 18 Epithelial-mesenchymal transition ...... 19 WAT fibrosis ...... 21 AT fibrosis mechanisms ...... 23 Characteristics of growth hormone ...... 26 Growth hormone mechanisms ...... 26 GH/IGF-1 axis ...... 27 Influence of GH on nutrient metabolism ...... 28 Clinical conditions associated with altered GH levels ...... 30 Laron Syndrome ...... 30 GH deficiency...... 32 GH excess...... 33 Growth hormone mouse lines ...... 34 GH and WAT fibrosis ...... 37 Research question and approach ...... 39 Pathway of interest ...... 39

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Specific aims ...... 40 METHODS ...... 42 Mice ...... 42 Body weight and composition analysis ...... 42 Plasma collection ...... 43 Dissection ...... 43 -linked immunosorbent assays (ELISAs) ...... 44 RNA isolation ...... 44 cDNA and qPCR ...... 45 Statistical analysis ...... 46 RESULTS ...... 47 Total body data ...... 47 Tissue-specific data ...... 48 Fat depot data ...... 48 Metabolic organ data ...... 50 Serum/plasma protein data ...... 51 RNA isolation ...... 54 DISCUSSION ...... 55 Fibroblast activation protein (FAP) ...... 56 Fibroblast growth factor 21 (FGF21) ...... 57 Beta-klotho (β-klotho) ...... 58 Procollagen peptides ...... 59 Procollagen 1 ...... 59 Procollagen 3 ...... 60 Conclusion ...... 61 Significance of results ...... 61 Limitations of findings ...... 61 Future directions ...... 63 REFERENCES ...... 66

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ABSTRACT

Adipose tissue (AT) is a unique energy storage tissue able to undergo significant hypertrophy and atrophy, dependent, in part, on nutrient status. When hypertrophy is in excess and sustained, the result is obesity, a common and widespread health problem.

However, when AT stores are selectively depleted or abnormally deposited, the resultant condition known as lipodystrophy can lead to comparable health outcomes as obesity.

That is, either extreme in AT mass results in similar metabolic outcomes (insulin resistance, increased immune cell infiltration, and increased production of inflammatory ). With either chronic obesity or lipodystrophy, AT can undergo major remodeling, which can ultimately result in unresolved chronic inflammation and AT fibrosis. Growth hormone radically alters AT mass, and mice that overexpress growth hormone (bGH mice) are lipodystrophic with a notable increase in AT fibrosis. Key players in AT fibrosis could include fibroblast activation protein (FAP) and fibroblast growth factor 21 (FGF21), among others. FAP has been positively correlated with ECM remodeling and its knockdown has been associated with improved metabolism in diet- induced obese mice, while FGF21 is associated with increased energy expenditure yet is cleaved and inactivated by FAP. Importantly, FGF21 and FAP levels were also recently assessed in patients with acromegaly, and these were suggested as a marker of tissue fibrosis and positively correlate with GH action. [1] These results provide compelling evidence that assessing the FAP/FGF21 interaction in our bGH mice will help elucidate the mechanisms underlying the relationship between GH and fibrosis. Thus, the purpose of this study was to determine if these molecules are altered in bGH mice. Using serum and plasma collected from bGH and littermate controls and ELISA assays, our

4 results indicate that FAP is not significantly altered in adult bGH mice, although sex- specific differences, with significantly higher FAP levels in female WT mice than in male

WT mice in two different cohorts, were apparent. Genotype-specific effects in other were evident only for males, who showed higher levels of both intact and total

FGF21 in bGH compared to WT. Procollagens, which are known fibrosis indicators and substrates of both GH and FAP, were evaluated to find higher levels of procollagen 1 in bGH males despite lower levels of procollagen 3 in the same mice compared to their WT controls. No significant difference in FGF21 among the female groups was apparent, and procollagen levels were not assessed in females. Levels of β-klotho, the co-receptor for

FGF21, did not differ significantly among any of the 4 groups. Collectively, these data provide evidence for significant variation in the regulation of these proteins in mice compared to humans, as humans exhibited lower levels of both procollagens associated with GH and showed no sex-specific effects among the other molecules. Results from this mouse study therefore prompt further investigation into the mechanisms by which

FGF21 is associated with increased GH levels.

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INTRODUCTION

In recent years, the prevalence of obesity, as defined by a body mass index of greater than 30kg/m2, is both widespread and on the rise. As of March

2019, the global incidence of this costly metabolic condition has doubled since

1980 and costs an annual $149.4 billion healthcare dollars in the United States alone. [2] The financial burden of obesity is largely exacerbated by its ability to initiate and enhance other disease states including hypertension, cardiovascular disease, metabolic syndrome, and diabetes. [3] These comorbidities often occur due to disruptions to the integrity of adipose tissue (AT) during the accumulation of fat mass. The hypertrophy in adipocytes that accompanies obesity frequently involves immune cell infiltration, increased production of inflammatory cytokines, and disruption in insulin receptor signaling. [4]

On the opposite end of the spectrum yet inducing similar results, lipodystrophy, or abnormal fat loss and distribution, can also cause metabolic disturbances throughout the body. Despite the reduced fat mass associated with a lipodystrophic state, the quality of the fat is altered in such a fashion that it can induce insulin resistance and resulting complications akin to those found in an obese state. [5] Collectivity, this shift in AT signaling and morphology in both lipodystrophy and obesity can cause a state of tissue remodeling that affects whole body metabolism and health parameters. [6] This tissue remodeling process is therefore being targeted as a key point of intervention to prevent the onset of other conditions and requires a thorough understanding of AT structure and function.

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Characteristics of adipose tissue and adipocytes

Adipocyte function

All dietary sources of energy are provided by combinations of three macromolecules: carbohydrates, proteins, and lipids. In necessary amounts, each macromolecule performs essential roles in the body and will be metabolized to fulfill that purpose. However, in excess, all three energy sources can surpass their functional capacity and will be processed for triglyceride (TG) storage in fat cells called adipocytes. Adipocytes retain stores of TGs that are increased with energy surplus and release energy when needed through the release of free fatty acids (FFAs). [7] Adipocytes reside in discrete depots within the body, collectively referred to as adipose tissue. Adipose tissue functions in numerous additional roles besides lipid storage and release, and these functions vary depending on location and other properties of the tissue. Adipose tissue is primarily classified as white adipose tissue (WAT) or brown adipose tissue

(BAT) according to its phenotypic and metabolic characteristics. While BAT is an important player in thermogenesis and has significance in different aspects of metabolism, WAT comprises a much greater portion of total body adipose tissue. [8] As WAT is the major focus in my thesis, WAT will also be the focus of this document.

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Adipose tissue depots

Main classifications

WAT contains multiple different depots found throughout the body, as illustrated in both Figure 1 and Figure 2. These depots primarily consist of two broad categories including the subcutaneous adipose tissue (SAT) and the intra- abdominal depots. Specific depot classifications vary among species and not all depots are found in both mouse and man. However, this general terminology provides a means to further differentiate fat types and locations in current model systems.

Figure 1. Distribution of white adipose tissue (WAT) depots in mouse models. Both posterior and anterior subcutaneous depots are referenced as subcutaneous adipose tissue (SAT). The right side of the figure depicts the term visceral as encompassing five intraabdominal depots according to the general definition of visceral adipose tissue (VAT). If using the strict visceral definition, only the mesenteric depot, which drains through the portal vein, would be considered visceral. However, the commonly accepted general definition of visceral includes all intra- abdominal depots as shown. [Figure used with permission [9]]

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Subcutaneous depots

SAT encompasses all anterior and posterior depots immediately beneath the skin, excluding the interscapular BAT. The superficial white fat layer is found in many areas of the body, including in gluteal, intramuscular, and abdominal regions for both mice and humans. For mice, the primary subcutaneous depot typically assessed is collected from the posterior region immediately beneath the skin and behind the quadriceps muscle. This depot is characterized by an inguinal lymph node and is referred to as the inguinal fat pad. [10] This SAT depot correlates with the gluteofemoral fat pad present in humans and provides a reliable location for subcutaneous collection and comparison.

Figure 2. Distribution of adipose tissue (AT) in humans. Brown adipose tissue (BAT) is located primarily in the interscapular region (j), with a small subset contained in a supraclavicular depot (i). Subcutaneous portions of white adipose tissue (SAT) are found in the abdominal (a), gluteofemoral (g), and intramuscular (h) regions. Other WAT depots contained intra- abdominally are the omental (b), retroperitoneal (d), gonadal (e) and pericardial (f) depots. The only true visceral adipose tissue (VAT) sources are the mesenteric (c) and omental (b) depots. [Figure used with permission [9]]

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Intra-abdominal depots

The intra-abdominal classification stems from an area of some debate, as this term is often used interchangeably with the word visceral as indicated in the mouse figure above (Figure 1). However, strictly defined visceral adipose tissue

(VAT) describes an intra-abdominal depot that drains through the portal vein; as such, the only true source of VAT shared in both mice and man is the mesenteric depot surrounding the intestines. [11] The other areas of deep internal fat, such as the retroperitoneal fat found behind the kidneys in both species, would then be considered intra-abdominal but not visceral. Likewise, the perigonadal (epididymal in males and periovarian in females) fat that encapsulates the reproductive organs in mouse models only would not be considered “true visceral.” Humans also possess a depot known as omental fat, which is located in the abdomen and extends inwards towards the mesenteric depot. [9] While this is a prominent visceral depot in man, the correlating depot in mice is present only in a small quantity when fed high fat diets and as such is not usually delimited. [12]

For the purpose of accurate comparison between different models, our lab routinely collects the following depots in our mouse studies: the subcutaneous inguinal depot, the visceral mesenteric depot, and the intra-abdominal retroperitoneal and perigonadal depots as shown in Figure 1. While the first three depots in mice have a correlation with human anatomy, the perigonadal depot does not. This depot is frequently studied due to the ease of its collection and mass, but any conclusions drawn from perigonadal samples alone are

10 limited. However, both perigonadal and the other intra-abdominal depots have gained clinical infamy from the frequent observation that disturbed AT morphology in these depots is often correlated with other disease states. This dysfunction was originally thought to cause enhanced metabolic disruption, and the accumulation of central obesity has often been regarded as more threatening to overall health than increased SAT. [13] However, recent discoveries about the physiological implications of SAT accumulation are raising questions about the role of each depot in contributing to global health and dysfunction. [14]

Further depot-specific differences in adipocyte structure and function are currently being investigated and will be described further in this document.

Characteristics of fibrosis

Extracellular matrix (ECM) and fibrosis

Adipose tissue alterations (expansion or contraction) rely largely on modifications to the extracellular matrix, or ECM. The ECM is a large network of various collagens, fibroblasts, proteoglycans, immune cells, and other fibrous molecules that surround the outside of cells and can alter properties of growth and expansion. This dynamic structure undergoes constant remodeling through processes of matrix degradation and rebuilding. The balance in remodeling dynamics can be upset in a state of stress or under different pathological conditions. A push towards positive matrix building, without compensatory degradation, can cause an excessive accumulation of ECM components – also referred to as tissue fibrosis – that may eventually become detrimental to tissue

11 health. [15] This shift from healthy tissue to fibrotic tissue is shown below in

Figure 3. Besides excessive accumulation of ECM, tissue fibrosis is also associated with the excessive production of cytokines, chemokines and growth factors as well as an influx of immune cells and alteration in resident cells.

Figure 3. Differences in the structure and function of the extracellular matrix in a state of fibrosis. Healthy tissue contains certain amounts of proteoglycans, fibroblasts, collagens, and elastins in the interstitial or extracellular matrix between cells. These components can undergo extreme remodeling with increased collagen connections, immune cell infiltration, and the increased presence of matrix proteins. This network shift can disrupt the basement membrane and cause greater mobilization of immune cells through the structural layer of the tissue. [Figure used with permission from [16]]

Fibrotic disease states

Tissue fibrosis has been implicated in many disease states and can affect the liver, kidney, lung, heart, and even tumor structure. Liver fibrosis commonly occurs in tandem with other hepatic conditions such as chronic hepatitis and is marked by increases in hyaluronic acid (HA), type IIII procollagen, type IV collagen, laminin (LN), transforming growth factor-β (TGF-β), matrix metalloproteinase (MMP)-2, and epidermal growth factor-receptor (EGF-R), as well as by other biochemical markers including total bilirubin, gamma-

12 glutamyltransferase, alpha-2 macroglobulin, haptoglobin, alanine aminotransferase, and apolipoprotein-A1. [17-21] Furthermore, liver fibrosis has been identified as a comorbid condition along with adipose tissue stiffness in obese states, prompting further investigation of the relationship of these mechanisms. [22]

Another organ commonly targeted in states of metabolic stress, the kidney, has fibrotic markers including WNT1-inducible-signaling pathway protein 1

(WISP-1), TGF-β, collagen 1, fibronectin, and α-smooth muscle actin (α-SMA).

Renal fibrosis is a common factor in the pathogenesis of chronic kidney disease and is a desirable point of targeted intervention to prevent further complications.

Identification of these biomarkers as noninvasive tools to assess fibrosis progression provides the potential for earlier clinical intervention and a promising strategy to combat this and other fibrotic diseases. [23]

Lung fibrosis often occurs in the form of an interstitial lung disease or pneumonia called idiopathic pulmonary fibrosis. Fibrosis in the lung can be associated with MMP-1 and MMP-7, as well as lysyl oxidase homolog 2

(LOXL2), the chemo-attractant CCL18, interleukin (IL)-8, insulin-like growth factor binding protein 2 (IGFBP2), intracellular adhesion molecules (ICAMs)-1 and -2, vascular endothelial growth factor (VEGF), leptin, and the YKL-40 protein, which is also implicated in liver fibrosis. [24]

The heart can also be affected by fibrosis and undergoes molecular damage through a similar process as other tissues. Myocardial fibrosis can initiate other forms of cardiovascular disease and can be identified by markers such as α-

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SMA, collagen 1a1, discoidin domain receptor 2 (DDR2), fibroblast-specific protein 1 (FSP1), periostin, platelet-derived growth factor α (PDGFα), transcription factor 21, thymus cell antigen 1, and vimentin. [25]

Even tumor metastasis may occur through an ECM remodeling. The proposed mechanism akin to the development of fibrosis in other tissues and is indicated by an elevated presence of fibronectin, VEGF receptor 1, lysyl oxidase

(LOX), hypoxia-inducible factor (HIF), type I collagen, and TGF-β. [26]

Examples of fibrosis in different disease states are shown below in Figure 4.

Figure 4. Fibrosis contributors in various disease states. Internal and external factors such as injury, inflammation, autoimmune disease, and genetic disorders can stimulate the alteration of cytokines and changes in the ECM. If these external stimuli are removed, the tissue will regress to a normal state. If the stimuli persist, extracellular proteins and inflammatory cells can build up and initiate fibrosis in the liver, kidney, lung, heart, and in tumors. Figure used with permission [27].

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Development of fibrosis

While the process of fibrosis has already been heavily implicated in disease states of other organs such as the lungs, liver, heart, and kidneys, only recently has fibrosis gained recognition as a poignant player in the metabolic conditions associated with adipose tissue alterations. [28] Fibrosis, regardless of tissue, is thought to be triggered by a variety of different mechanisms that eventually lead to alteration in immune cells, release of cytokines/growth factors and fibroblast changes with the outcome of ECM accumulation, as shown below in Figure 5.

Figure 5. General fibrosis schematic. External stimuli such as trauma, chemicals, and particle irritants as well as internal phenomena such as infection, diabetes, autoimmunity, and hypertension can all contribute to injury of various tissues. This tissue injury activates immune cells to release cytokines and growth factors. This immune response eventually recruitments fibroblasts that simultaneously proliferate, increase ECM synthesis, decrease ECM degradation, and recruit other cell types to stimulate fibrosis. [Figure used with permission from [29]]

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Triggers, such as infection or trauma, to a tissue will cause a stress response that results in an immune reaction to initiate repair. If sustained beyond the point of necessary repair, the original stressor along with the altered immune response can cause the location of injury to adopt a state of chronic inflammation. In this chronic state, the local damaged cells along with resident immune cells secrete mediators such as cytokines, chemokines, growth factors, and MMPs. These factors recruit additional immune cells to help remodel the tissue microarchitecture at the site of wound healing, while new cells of the same lineage are formed to replace the damaged ones. [30] The initial immune response, in which cells and secretion factors mediate wound healing, occurs during the regenerative phase of repair and leaves no notable damage. The potential second stage of repair, however, occurs when tissue remodeling surpasses the point of healing and begins the unhealthy accrual of matrix proteins during the process that we know as fibroplasia or fibrosis. [31]

Contributing factors to fibrosis

Fibroblasts in fibrosis

Fibroblasts have been identified as one of the most potent contributors to

ECM formation and regulation. Fibroblasts are found in connective tissues throughout the body and secrete structural and adhesive ECM components such as collagens, elastin, laminin, fibronectin, hyaluronan, and glycoproteins. [32]

In addition to supporting ECM building, fibroblasts can also respond to

16 paracrine and autocrine stimuli such as cytokines and growth factors that play a pivotal role in shifting the ongoing dynamics of ECM remodeling. Fibroblasts often respond to signals that initiate necessary wound healing processes and play a crucial role in tissue maintenance. The point at which fibroblasts surpass the point of wound healing and begin contributing to disproportionate matrix accumulation is the point at which fibrosis begins. [32] Because of their essential role in both healthy ECM structure and in fibrosis development, the regulation of fibroblasts is crucial to understanding the pathogenesis of fibrosis.

Fibroblast activation protein

Fibroblast mobilization can be initiated by the activity of a membrane- bound serine protease called fibroblast activation protein (FAP). This unique protein is capable of cleaving peptides or proteins through its dual endopeptidase and dipeptidyl aminopeptidase activities and is active in both its membrane-bound form and once shed into circulation. [33] The enzymatic activity of FAP is similar but distinct from its related family member DPP4, though both have been studied through similar degradomic techniques. FAP is present at low levels in adult organs and upregulated along with increased fibroblast activity in states of ECM remodeling; this enhanced activity also coincides with increased expression of FAP substrates procollagens 1 and 3.

Specifically, increased presence and activity of FAP has been associated with tumorigenesis, fibrotic conditions, and atherosclerosis. [33]

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Fibroblast growth factor 21

While the source of FAP during fibrotic states has not been identified, this protein is known to be shed from its location on the cell surface and to target fibroblast growth factor 21 (FGF21) for inactivation by cleavage. [34]

The endocrine peptide FGF21 is primarily produced in the liver but is also expressed in other tissues as shown below in Figure 6. FGF21 is secreted in an autocrine and paracrine fashion and binds with high affinity to the primary FGF family receptors FGFR, along with the essential co-receptor β-klotho, which is expressed on various cell types including adipocytes. [35] This peptide hormone specifically targets adipose tissue to increase the expression of

Transporter 1 (GLUT1) and increase lipolysis. [36] The ability of FGF21 to stimulate lipolysis and glucose uptake causes recognition of this protein as capable of increasing weight loss, energy expenditure, and insulin sensitivity in humans. [1]

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Figure 6. Regulation and effects of FGF21 throughout the liver, peripheral organs, and the central nervous system. The majority of circulating FGF21 is secreted from the liver, though it is also expressed in the liver, pancreas, WAT, BAT, and skeletal muscle. Liver FGF21 can be affected by regulation from outside factors such as fasting and through the alteration of specific proteins such as PPARα. Once circulating, hormonal FGF21 increases beneficial metabolic effects such as β-oxidation, ketogenesis, gluconeogenesis, insulin synthesis, WAT browning, and glucose uptake in peripheral organs. FGF21 also exerts various effects on the central nervous system and further contributes to hormonal regulation throughout the body. Collectively, the presence of FGF21 is protective against obesity, dyslipidemia, and insulin resistance or insensitivity. [Figure used with permission from [37]]

Epithelial-mesenchymal transition

Fibrosis development through the chronic buildup of ECM proteins can be initiated in part by a process known as epithelial-mesenchymal transition

(EMT) in which epithelial cells are altered to develop mesenchymal characteristics. Epithelial cells both cover the outside of the body and line the interior of cavities such as the lungs and gastrointestinal tract. They surround

19 the lumen of these internal organs with a layer of structured but selectively permeable cells that are anchored to the basement membrane and aid in the regulation of fluid passage and solute transport. Cells in a mesenchymal state are multipotent stromal cells that have greater variability in shape and structure with the ability to differentiate into numerous other cell types. Mesenchymal cells differentiated into fibroblasts or an adipocyte lineage also have a predisposition to secrete ECM components; therefore, the process of EMT may enable fibrosis development by enhancing the presence of cells capable of increasing the ECM while also dislocating inherent structural layers of the tissue. [38] The structural shift in cellular junctions is shown below in Figure 7.

Figure 7. Various molecules such as growth factors and extracellular matrix components exert an influence on the epithelial mesenchymal transition (EMT) process. Classic epithelial cells lose their traditional makers listed on the left and begin to express those of a mesenchymal cell as listed on the right. This alteration in expression also results in a phenotypic shift with greater potential for mobilization through the basement membrane and expansion. [Figure used with permission from [39]]

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Modified cells created in the progression of EMT often include fibroblasts and myofibroblasts, which produce MMPs and other wound closure proteins; these cell types and their secreted profibrotic cytokines have been strongly associated with the development of other fibrotic conditions such as pulmonary fibrosis. [40] Serum markers linked with EMT and liver fibrosis include HA, LN, TGF-β [19] and collagen IV [17] while serum WNT1- inducible signaling pathway protein-1 (WISP-1) is correlated with the presence of renal fibrosis and chronic kidney disease [23]. Collectively, these secreted cytokines and serum protein markers indicate a large network of signaling pathways that are involved in the development of fibrosis in many tissues.

Because the process of EMT is implicated in the recruitment of fibroblasts, an overlap between EMT and fibroblast regulation through FAP and FGF21 may play a role in fibrosis initiation. [41]

WAT fibrosis

Mature white adipocytes are mononucleated cells that contain large lipid droplets and can expand in response to increasing TG deposition. Adipocytes in all depots are capable of expansion both through hypertrophy and hyperplasia of these cells, though there are age, sex, and depot-specific differences that affect the extent to which a tissue can expand and be altered. [42] Both mechanisms allow for the healthy accumulation of fat and do not contribute negative metabolic consequences on their own. [7] However, WAT modification often

21 meets a point of restriction at which the adipocytes are no longer able to consistently grow or divide and to have sufficient vascularization. An important factor that contributes to the ability of adipocyte expansion and redistribution is the ECM.

The ECM, in excess, causes a state of rigidity that limits normal adipocyte dynamics. Excessive ECM accumulation, or WAT fibrosis, occurs along with a state of inflammation and cellular stress that can trigger a variety of downstream effects. [28] WAT fibrosis restricts the growth of adipocytes in such a way that the quality of the adipose tissue is altered as shown in Figure 8, which enhances the potential of obesity and lipodystrophy to lead to tissue dysfunction and symptoms of metabolic syndrome, insulin resistance, and diabetes. Importantly, not all depots appear equally susceptible to WAT fibrosis; that is, the subcutaneous fat depot appears to be the most greatly altered by fibrosis. [43] Therefore, understanding the mechanisms of specific fibrosis development in various WAT depots is key to better preventing the onset of many disease states.

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Figure 8. Metabolically healthy versus metabolically unhealthy obese states. An imbalance between energy intake and energy expenditure can lead to increased fat storage. Unhealthy mechanisms of storage in adipose tissue can result in hypertrophy and cause inflammation, fibrosis, hypoxia, and fat cell death or necrosis. Collectively, this dysfunction can lead to metabolically unhealthy obesity. Healthy fat storage, on the other hand, can lead to hyperplasia of adipocytes. If regulated properly, angiogenesis and adipose tissue expansion can occur in a manner that results in increased fat mass but a metabolically healthy state of obesity. [Figure used with permission from [44]]

AT fibrosis mechanisms

While the study of tissue-specific fibrosis is currently expanding to include adipose tissue, specific pathways or markers of the process have not yet been fully elucidated. Early studies of adipose tissue disruption in varying model organisms provide compelling evidence that key players in fibrosis could include TGF-β [45], collagen VI [46], hypoxia-inducible factor 1α (HIF1α)

[47], various peroxisome proliferator-activated receptor (PPAR) isoforms [48],

FAP [49], and FGF21 [50], among others. TGF-β is documented to be increased in subcutaneous depots of mice on a high-fat diet and is positively associated with mediators of inflammation and collagen turnover. [45] Collagen VI is a central component of the ECM in AT, and its knockout is associated with

23 improvements in whole-body energy homeostasis, due to a proposed mechanism of unrestricting adipocyte growth and fibrosis. [46] Obese mice have characterized hypoxic AT and an increase in HIF1α, which is associated with a decrease in the presence of beneficial adipokines such as adiponectin and plasminogen activator inhibitor type-1. [47] All PPAR isoforms are part of a large family of nuclear hormone receptors that can act as lipid sensors and can be activated by specific repressors or coactivators to influence their effects on processes such as inflammation, insulin resistance, lipid and glucose metabolism, and adipogenesis. Adipose tissue disruptions in obesity, insulin resistance, and lipodystrophy have been linked to defects in these receptors. [48]

FAP has been positively correlated with ECM remodeling and its knockdown has been associated with improved metabolism in diet-induced obese mice [49], while FGF21 is associated with increased energy expenditure and is upregulated by PPARγ agonist treatment [50].

Many of these molecules have already been mentioned previously and indicated in other disease states, suggesting a similar molecular pathogenesis in the fibrosis of different organs. Two crucial factors noted to contribute to the development of fibrosis specifically in adipose tissue are hypoxia and inflammation, as shown in Figure 9. Hypoxia occurs when adipocytes expand beyond the diffusional limit of oxygen, or blood vessels are unable to infiltrate the rigid network of ECM surrounding the cells, and the tissue suffocates.

Inflammation occurs as a natural response to any sort of stress or wound healing but can be increased in response to adipocyte hypertrophy and ECM

24 accumulation that further exacerbates the fibrotic response. [42] Many of the molecules of interest in the development of fibrosis, such as HIF1α and FAP, are also specifically involved in these processes. Another that has been documented to correlate with many of the fibrotic pathways listed above, as well as the observed induction of a general state of fibrosis in adipose tissue, is growth hormone (GH). [51]

Figure 9. Non-exclusive hypotheses that contribute to adipose tissue fibrosis. Adipocyte hypertrophy can cause hypoxia and dysfunction of the vasculature around adipocytes. Hypertrophy can also cause the recruitment of immune cells and initiate an inflammatory response. Both responses to adipocyte hypertrophy can lead to the development or exacerbation of fibrosis in adipose tissue. [Figure used with permission from [52]]

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Characteristics of growth hormone

Growth hormone mechanisms

Growth hormone (GH) is a 22kDa peptide hormone composed of 191 amino acids and secreted in a pulsatile manner from the somatotropic cells in the anterior pituitary gland. Release is controlled by the balance of two hypothalamic hormones: 1) growth hormone releasing hormone (GHRH), which promotes GH release; and 2) GH inhibitory hormone or somatostatin, which prevents GH release. When GH is released from the somatotrophs, it circulates throughout the body in an endocrine fashion. GH will then bind the growth hormone receptor (GHR), which is a dimeric extracellular receptor present on cells in many different organs and tissue systems including bone, muscle, adipose tissue, liver, and kidney. [53] As GH binds to the GHR, the receptor shifts in conformation to enable its two bound Janus-kinase 2 (JAK2) molecules to cross-activate one another. Each JAK2 will then activate the next step in the signaling cascade, most frequently by the phosphorylation of the Signal

Transducer and Activator of Transcription 5 (STAT5) a or b proteins. [54]

STAT5a/b will self-dimerize and translocate to the nucleus to initiate various downstream signaling events to perpetuate the stimulation of growth and cellular alterations. Dimerization of GHR can also initiate other signaling pathways, in addition to the central JAK/STAT cascade, as illustrated below in

Figure 10.

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Figure 10. Growth hormone (GH) signaling through receptor binding. GH binds the dimerized GH receptor (GHR), which undergoes a conformational shift to enable two bound Janus-kinase 2 (JAK2) molecules to activate one another. The activation of JAK2 is traditionally associated with the phosphorylation of STAT5a/b and initiation of further downstream signaling events; however, GHR dimerization can activate a variety of different signaling cascades with various effects on cellular organization, growth, and nutrient metabolism. [Figure used with permission from [54]]

GH/IGF-1 axis

One of the well-known effects of GH is its ability to stimulate the production of insulin-like growth factor 1 (IGF-1) in various tissues, with the majority of endocrine, circulating IGF-1 coming from the liver. [55] GHR and

IGF-1 receptors are present on a variety of organ systems and lead to differing tissue-specific effects especially in bone, muscle, and adipose tissue as shown below in Figure 11. Collectively, the action of these two related hormones is

27 referred to as the GH/IGF-1 axis in which both hormones influence growth and metabolism by acting on their target tissues. Because of the close association between GH and IGF-1 levels, it is often difficult to differentiate between the direct effects of GH and the direct effects of IGF-1. [56]

Figure 11. GH release from the pituitary exerts differential influences on tissues. Stimulation of GH release results in a catabolic glucose-sparing effect through lipolysis of adipocytes, while target tissues of anabolic growth include the bone, muscle, nervous system, and . Further growth effects are stimulated in the liver through glycogen breakdown for fuel. Liver stimulation also releases the majority of the body’s circulating IGF-1, which participates in a negative feedback loop on the hypothalamus to decrease further GH release. [Figure used with permission from [57]]

Influence of GH on nutrient metabolism

Growth hormone plays a global role in regulating growth by altering nutrient metabolism and by promoting anabolic processes in most tissues, like muscle and bone, while being catabolic in adipose tissue. Examples of these

28 variable but global roles of GH are described in Figure 11. GH affects the metabolism of all three macronutrients in order to cause these global effects.

Carbohydrate metabolism is primarily manipulated by the ability of GH to alter blood glucose homeostasis by decreasing the ability of insulin to promote the uptake of glucose in both muscle and adipose tissue [58]; this is sometimes referred to as the diabetogenic action of GH. The anabolic effects of GH in muscle-building and bone-building are speculated to occur primarily through adjustments in protein metabolism. GH and IGF-1 are thought to increase serum amino acid uptake in bone, muscle, nervous system, and immune cells; GH also decreases the oxidation of these amino acids to increase muscle protein synthesis. This protein accumulation leads to an increase in lean mass that is maintained in fasted states due to the tendency of GH to preferentially break down lipids or carbohydrates for fuel while maintaining protein stores. [59]

GH has a major influence on WAT both through direct and indirect mechanisms. One method by which GH increases lipolysis, or fat breakdown, is by binding to the GHR on adipocytes and activating the MEK-ERK pathway to cause downstream phosphorylation and deactivation of PPARγ. [60]

Deactivation of this receptor causes a downregulation in fat-specific protein 27

(FSP27) and increased lipolysis. Another mechanism of lipolysis occurs due to the activation of hormone-sensitive lipase (HSL) by GH activation of protein kinase C (PKC) and ERK. [60] HSL then catalyzes the hydrolysis of triglycerides (TGs) to form free fatty acids (FFA) and glycerol. [61]

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Collectively, GH binding results in increased lipid oxidation and increased circulation of FFA and ketone bodies in the bloodstream. [62]

GH simultaneously decreases lipogenesis, or the storage of new TGs, by suppressing the ability of adipocytes to uptake circulating lipids through inhibition of lipoprotein lipase. [63] Further, this suppression of lipogenesis occurs in part through GH’s contribution to insulin resistance by the uncoupling of insulin signaling through phosphatidylinositol (PI) 3-kinase and its downstream molecules [64], leading to an overall suppression of glucose uptake and decreased availability [63]. Together, the ability of GH to promote lipolysis and impede lipogenesis causes a decrease in adipocyte size and growth with a general decrease in total fat mass. This ability of GH to dramatically alter fat mass causes the influence of GH on AT to be a phenomenon of interest for clinical application.

Clinical conditions associated with altered GH levels

Laron Syndrome

The results of GH’s effect on different tissues lead to various phenotypes in a clinical setting. The first is a state of GH resistance, which has been termed

Laron Syndrome (LS). This condition arises due to a mutation in the GHR gene, which leads to a drastic decrease in GH signaling by cells and tissues. Patients with this condition are short in stature with a decrease in bone and muscle mass but a disproportionate increase in fat mass [65], with surprisingly variable

30 metabolic outcomes depending on their geographical location. The two primary cohorts of identified LS patients are found in Ecuador and in the Mediterranean, specifically in Israel. [66]

In the Ecuadorian cohort of about 90 individuals, the increased AT in these patients constitutes a counterintuitive “healthy obese” phenotype of GH resistance. Despite having an increase in the presence of fat mass, these individuals usually have improved metabolic parameters and the entire cohort reports decreased cancer development and diabetes incidence compared to unaffected populations. [67] In the Mediterranean cohort, on the other hand, some physical outcomes are different in these 60 individuals despite similar mutations causing GH resistance on the molecular level. [66] Individuals from this cohort also have an increase in fat mass to the point of morbid obesity, along with short stature. While they share a lifelong protection from cancer with the Ecuadorian cohort, patients from the Mediterranean cohort report impaired glucose metabolism and increased diabetes incidence compared to the

Ecuadorian cohort. [68] Together, the implications for GH resistance to decrease the incidence of two globally prevalent diseases makes the entire population of

LS patients a group of particular interest for studying chronic disease development and prevention.

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GH deficiency

A second clinical state that also results in decreased GH signaling is that of GH deficiency, or GHD. While LS is the direct result of a GHR mutation and ensuing GH resistance, the state of GHD is typically characterized by the disruption of either the GHRH-producing neurons in the hypothalamus or the

GH-releasing somatotrophs in the pituitary gland. [69] Genetic defects, tumors, or traumatic injury to either of these cell types can cause a decrease in the secretion of GH and result in a state of deficiency that can either be congenital, acquired, or idiopathic. [70] The congenital GHD state, similarly to LS, likely results from a genetic defect and causes clinical characteristics showing a decrease in lean mass with an increase in fat mass localized viscerally to the abdomen. Conversely to LS, most GH-deficient patients often have a decrease in metabolic health, especially in an acquired or idiopathic state. [71]

Individuals with GHD acquired later in life tend to experience more frequent hypoglycemic or low blood glucose episodes and altered cholesterol levels causing dyslipidemia [72], again pointing to the importance of understanding

GH signaling and its influence on metabolic health. Patients may be treated with

GH replacement therapy by injections in order to restore normal glucose metabolism, body composition, and fat mass and deposition across depots. [69]

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GH excess

The third clinical state of altered GH is that of GH excess from hypersecretion. This increase in GH typically arises due to an adenoma in the pituitary gland and either results in gigantism with childhood-onset or acromegaly if the adenoma develops later in life. Gigantism is characterized by increases in global bone and muscle mass and decreases in AT. Acromegaly differs from gigantism in that the onset of hypersecretion arises at a point in life in which overall height and weight are not as severely altered, but the extremities (hands, feet, facial features) are often enlarged and individuals have prominently increased bone structure and muscle density. [73] In addition to an increase in bone and muscle mass, acromegaly causes a decrease in both the

SAT and VAT fat depots. [74]

Both forms of GH excess can lead to other co-morbidities such as diabetes and cancer and often require treatment to alleviate the effects of overgrowth. The first treatment intervention usually involves the surgical removal of all or part of the pituitary adenoma. [75] Follow-up pharmaceutical interventions then target any remaining overgrowth by use of somatostatin analogs and/or GH antagonists (GHA) to combat further GH stimulation. [76]

Treatment to decrease GH levels results in increased fat mass, and acute treatment in mice has been found to cause AT increases localized to the mesenteric and inguinal subcutaneous depots with significant changes in adipocyte size and morphology. [77] Because both GH excess and GH deficient states lead to such interesting and drastically different phenotypes in clinical

33 populations, the underlying roles of this multifunctional hormone have been an area of interest in translational research for many years. In order to better understand the molecular mechanisms of GH and its effects in an invasive manner that cannot be performed in clinical settings, our lab has developed a variety of GH-altered mouse lines that recapitulate the clinical conditions.

Growth hormone mouse lines

Mice are commonly used for human-oriented scientific and medical research, due to their prevalent homology with the human genome and anatomy, fast reproduction rate, and ease of care in a laboratory setting. [78] It was not until 1974, though, that the technology necessary to generate transgenic mouse strains was created. Rudolf Jaenisch was the first person to insert a specific vector into a mouse embryo by microinjection during the blastocyst stage. The altered embryo was then re-implanted into a recipient female, who subsequently gave birth to pups that were expected to have expression reflective of genome alterations. [79] This technique has been altered and modified since this first experiment, but the basic principles remain the same and enable researchers to create mice with increased or decreased expression of certain genes in order to analyze their effects on development. [78]

34

Many conditional alterations and tissue-specific knockout mouse lines have been generated or used by our lab in the last 25 years and are useful for elucidating the effects of GH or specific related proteins in different target tissues. Examples include global GH receptor knockout (GHRKO), GH antagonist transgenic (GHA), and liver-specific GH receptor gene-disrupted

(LiGHRKO) mice, among others. Relevant to this thesis, we have generated a

C57BL/6J mouse line with an excess in GH action: bovine GH transgenic

(bGH) mice. [80] Transcription of the bGH cDNA, which is transfected into the embryo pronucleus, is driven by the mouse metallothionein I promoter and results in global GH overexpression in the developed mice. [81] An established colony of bGH mice has been generated in our lab and breeding procedures enable passage of the genotype and continuation of these GH studies without requiring transgenic manipulation on each mouse.

These bGH mice are often studied by comparison to wildtype (WT) controls and provide the opportunity to analyze the effects of GH overexpression in the bGH transgenic system. This system is similar to gigantism, though the GH overexpression in bGH mice is not specific to enhanced pituitary GH secretion as is found in acromegaly. [51] The bGH mice feature an increase in muscle, bone, and total mass with a decrease in fat mass at an adult age relative to WT littermates. [82] The multitude of GH effects on AT are still being teased out, though there are notable differences that vary both by age and by sex of the mice. For example, bGH mice have increased fat mass relative to WT littermates at a younger age, though this trend is alleviated after a

35 few months of growth, and older bGH mice have lower fat mass than controls.

[82] The AT of adult bGH mice also has an increase in T cells and M2 immune cells, as well as a decrease in peptide hormones primarily found in adipose tissue such as leptin and adiponectin. [83] The changes in both AT quality and quantity are also reportedly more significant in bGH males than in bGH females, with females showing a delay in the reduction of fat mass that occurs as bGH mice age. [82]

The smaller fat mass in both male and female bGH adults is correlated with smaller adipocyte size and with a greater accumulation of fibrosis through

ECM components, collagens, and inflammation. [43] This decreased fat mass with GH excess also correlates with a decrease in total glucose tolerance and insulin sensitivity, representative of an overall decrease in metabolic health in these mice. [60] This “unhealthy lean” paradox found in bGH mice usually leads to the greatest opportunity for significant findings in fibrosis studies and makes them an ideal archetype for studying AT morphology in all depots. Body composition, total mass, glucose tolerance, and insulin tolerance are frequently assessed during the normal lifespan of these mice. [84] These measurements routinely indicate that bGH mice undergo a process of premature aging and report decreased insulin tolerance, increased levels of IGF-1, and a predisposition to develop chronic diseases such as diabetes and cancer. Tissue- specific decline in functional capacity, such as reduced efficiency of the kidney and liver of bGH mice, has also been reported. [81]

36

GH and WAT fibrosis

GH has been documented to correlate with fibrosis states in varying tissues, significantly noted by collagen deposition in the liver, kidney, and muscle, as well as other tissues. [85, 86] Our lab has also found that an excess of

GH in bGH mice occurs alongside the development of fibrosis in adipose tissue.

[43] This pituitary hormone has therefore become pertinent in the investigation of AT fibrosis due to an observed correlation, and potential causation, of GH signaling and fibrosis development. While the exact mechanism of the influence of GH on AT fibrosis is not yet known, these data have prompted us to investigate this relationship. By using animal models to study the impact of GH on AT morphology and signaling mechanisms, we hope to elucidate the factors that lead to the onset of fibrosis, insulin resistance, and other condition

GH has been shown to specifically alter certain molecules associated with AT fibrosis; for example, it appears to be correlated with regulation of the expression of molecules such as TGF-β [87], different collagens [88], FAP, and

FGF21 [1]. In a recent clinical study, it was found that the treatment of patients with acromegaly resulted in reduced subcutaneous adipose tissue expression of

FAP and reduced collagen turnover. Furthermore, FGF21 levels were found to be reduced alongside GH reduction after surgical and pharmacological treatment for acromegaly, without any confounding correlation with IGF-1 levels. This group also deduced that GH induction caused the upregulation of FGF21, which then proceeded by a negative feedback loop to downregulate further GH expression. [1] Another group found that the mechanism by which FGF21

37 blunts GH signaling is through the reduction of STAT5, a GH-induced signal transduction factor, as measured in the epididymal fat of mice. [34] This relationship suggests a role for FGF21, and therefore FAP, in the regulation and response of GH signaling which has already been linked to fibrosis. [77] These clinical data provide a rationale to evaluate the levels of GH, FAP, and FGF21 in altered GH states in mouse models.

38

Research question and approach

Pathway of interest

In this study, the molecules of interest are fibrosis-related proteins that will be compared between two mouse lines with excess and normal levels of GH action. We know from previous data that GH action is positively correlated with adipose tissue fibrosis in a depot-specific manner, with the subcutaneous depot showing the greatest impact. [77] Furthermore, a recent study indicates that not only are the levels of circulating FAP [49] and FGF21 [50] correlated with

WAT fibrosis as previously reported, but both proteins are also correlated with

GH expression in humans [1]. In addition, we know that that the expression of these molecules is found to be significantly regulated by the treatment of acromegaly in the subcutaneous depot. [1] These data have not yet been established in other organisms or shown to be directly influencing WAT fibrosis; therefore, we propose to investigate whether the GH-induced changes in adipose tissue fibrosis seen in our bGH mice are associated with circulating levels of FAP and its related proteins (FGF21, β-Klotho, collagen fragments) or in tissue expression of these molecules.

Because FAP inactivates FGF21, and FGF21 is considered beneficial for metabolism, we expect that an increase in FAP would be associated with negative consequences, increased WAT fibrosis, and decreased levels of full length, active FGF21. [1] In order to fully understand all interactions involved in this signaling sequence, we will use a variety of methods to quantify both

39

FAP and FGF21; additionally, we will look at the expression of FAP substrates procollagens 1 and 3 and the FGF21 receptor β-klotho.

Specific aims

Our project investigates two different aims to establish evidence for a relationship between GH, FAP-related proteins, and adipose tissue fibrosis.

Aim 1: Determine if serum levels of FAP and FAP-related proteins [FGF21

(active and inactive), β-Klotho, and procollagens 1 and 3] are altered in mice with chronic excess in GH action.

For Aim 1, we have a cohort of age- and sex-matched bGH and WT mice.

Serum samples have been collected from the mice and are being analyzed by

ELISAs specific for each molecule of interest. Because these serum samples can reflect global fibrosis status and include circulating proteins from multiple different organs, we will also differentiate serum results by performing tissue- specific analysis.

Aim 2: Assess RNA expression of FAP, FGF21, β-Klotho, and procollagens 1 and 3 in bGH tissue samples from WAT (subcutaneous and perigonadal), liver, and kidney.

For Aim 2, we seek to corroborate any results found in serum by quantifying specific tissue expression of our molecules of interest. We are assessing a broad spectrum of tissues routinely collected in dissection and that are critical metabolic organs, including two primary WAT depots, the liver, and

40 the kidney. Each organ or tissue will be assessed separately to provide insight into its specific state of fibrosis as compared to global levels seen in serum. In order to look at RNA expression of these molecules for Aim 2, we are performing quantitative PCR using primers specific for each protein.

41

METHODS

Note: Due to COVID-19 concerns and restrictions placed on laboratory access, RNA quantification studies (Aim 2) were terminated prior to completion. All protein quantification in serum or plasma was completed in at least males.

Mice

bGH transgenic mice and their littermate controls in a pure C57BL/6J background were generated in the Kopchick laboratory as previously described and were used for all studies. [80] Genotyping revealed the generation of slightly uneven but comparable groups within the cohort: 8 WT males and 12 bGH males, along with 11 WT females and

6 bGH females. A separate cohort included 11 male WT, 10 male bGH, 9 female WT, and 10 female bGH mice all 5 months of age and housed under the same conditions to provide a secondary assessment of FAP in serum via ELISA. All mice were housed three to four per cage and given ad libitum access to water and rodent chow. The cages were maintained in a temperature (22°C) and humidity-controlled room and exposed to a 14- hour light, 10-hour dark cycle. Mice were killed at 7 months of age. All procedures were approved by the Ohio University Institutional Animal Care and Use Committee and fully complied with all federal, state, and local policies.

Body weight and composition analysis

Prior to dissection, the fat, fluid, and lean mass of the mice were assessed using a

Minispec mq Benchtop Nuclear Magnetic Resonance (NMR) analyzer (Bruker

Instruments, Minispec ND2506) according to the manufacturer’s recommendations and as previously described. [82] Body weight of each mouse was taken just prior to body

42 composition measurements using a Mettler Toledo PL 202-S balance. Body composition measurements were taken during a non-fasted state.

Plasma collection

Mice were fasted for 12 hours (11pm to 11am) prior to serum sample collection.

Whole blood was collected from the tip of the tail into heparinized collection tubes. After blood collection was finished, the blood samples were removed from ice and set to thaw at room temperature for 30 minutes. Tubes were centrifuged at 4°C at 12000 RPM for 10 minutes. After centrifugation, the plasma was collected and stored at -70°C for future use.

Dissection

At 7 months of age, the mice were fasted for 12 hours and then were anesthetized using carbon dioxide under approved conditions for a final blood collection. The final blood collection was made via retro-orbital technique immediately before euthanasia, and before proceeding with dissection of the remaining organs. The blood collected was kept on ice during dissection, placed for 30 minutes at room temperature immediately following dissection, and then centrifuged at 4°C at 12000 RPM for 10 minutes. The resultant serum was then collected from these samples for storage at -70°C and future use in two ELISA assays. After blood collection, mice were euthanized by cervical dislocation. White adipose tissue (inguinal subcutaneous, epdidymal (males) or periovarian (females), retroperitoneal, and mesenteric) depots were collected. The intrascapular brown adipose tissue was obtained next before subsequent dissection of all internal organs such as the liver and kidney. Each sample was immediately flash-frozen

43 in liquid nitrogen after collection and moved to storage in a -70°C freezer after the entire dissection process was completed.

Enzyme-linked immunosorbent assays (ELISAs)

To assess levels of proteins in serum or plasma, enzyme-linked immunosorbent assays (ELISAs) specific for each of our proteins of interest were performed. These assays were completed according to each manufacturer’s specific instructions and in accordance with the protocol contained in each kit. FAP was measured using the “Mouse

FAP DuoSet ELISA DY8647-05” and the “DuoSet ELISA Ancillary Kit 2

DY008” from R&D Systems for serum samples collected during dissection. FGF21 was measured in both its intact and total forms using the “Intact FGF-21 ELISA Kit SKU:

F2131-K01” and the “Total FGF-21 ELISA Assay SKU: T2131-K01” from Eagle

Biosciences using plasma samples collected during bleeding. Both procollagens were assessed in plasma using kits available from G-Biosciences, the “Immunotag™ Mouse

PICP (Procollagen I C-terminal Propeptide) ELISA Catalog No. IT5924” and the

“Immunotag™ Mouse PIIICP (C-terminal Procollagen Ⅲ Propeptide) ELISA Catalog

No. IT5925.” Finally, plasma β-Klotho quantities were measured using the “Klb elisa kit:

Mouse Beta-klotho ELISA Kit Catalog #MBS2880013” from MyBioSource, Inc. Capture and detection antibody binding procedures, as well as sample preparation and washing steps, were completed as outlined in each individual protocol.

RNA isolation

RNA isolation of both subcutaneous and perigonadal AT depots was performed using the “RNeasy Lipid Tissue Mini Kit (50) Cat No./ID: 74804” available from

44

Qiagen, while the liver samples were isolated using the “GeneJET RNA Purification Kit

Catalog number: K0731” from Thermo Fisher Scientific. Briefly, 30mg of each tissue was aliquoted into a new tube containing homogenization beads and the appropriate lysis buffer supplied in each kit type. The tubes were homogenized using the Precellys 24-

Dual homogenizer and then extraction was performed as specified by each manufacturer.

After isolation was complete, samples were assessed for purity using the Thermo Fisher

NanoDrop 2000c Spectrophotometer. RNA yields with a 260/280 nm ratio reading of

1.8-2.1 were considered pure enough for use in complimentary DNA (cDNA) generation. cDNA and qPCR

Purified RNA for each sample was diluted into a new tube with an appropriate amount of water so that each tube would contain the same concentration of RNA.

Reagents for cDNA generation were then added in accordance with specifications of the

“High-Capacity cDNA Reverse Transcription Kit, Catalog Number 4368814” from

Applied Biosystems, and the prepared 96-well reaction place was run through the

ThermalCycler under the following conditions: 25°C for 10 minutes, 37°C for 120 minutes, 85°C for 5 minutes, and 4°C at an infinite hold until ready for the next step.

Once the ThermalCycler reaction was finished, the generated cDNA was either stored at -

20°C or immediately prepared for qPCR. While initial qPCR runs with reference genes were performed using the Bio-Rad iCycler (Bio-Rad Laboratories), these runs were not successful, and further studies were not completed, as noted above, due to COVID-19 disruptions.

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Statistical analysis

All statistical analyses and graphs shown were generated using GraphPad Prism

8.4.1. Each set of measurements were subjected to the Shapiro-Wilk normality test and assumed a normal distribution if analysis indicated an insignificant alpha p-value greater than 0.05. If the normality test passed, then the four groups (male WT, male bGH, female

WT, and female bGH) were compared for interactions due to both sex and genotype by performing a two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons. In results that did not assume a normal distribution, the four groups were compared using the Kruskal-Wallis nonparametric one-way ANOVA followed by

Dunn’s multiple comparisons.

Both Procollagen 1 and Procollagen 3 were only assessed in males due to the limited availability of . Procollagen 1 samples were not normally distributed and therefore were compared using the nonparametric Mann-Whitney test. In contrast, the

Procollagen 3 data assumed a normal distribution and thus male WT and male bGH samples were compared using an unpaired t-test analysis. Similarly, an unpaired t-test was also used to compare the distributions of epididymal and periovarian fat due to sex differences that made direct comparison among all four groups inappropriate. Epididymal fat between male WT samples and male bGH samples were compared via one unpaired t- test, whereas the same method was applied separately to the periovarian depots of female samples. Significance was defined for all analyses as an alpha p-value < 0.05, with extremely significant values being denoted by p-value < 0.001.

46

RESULTS

Total body data

Body weight was significantly different among all 4 groups and was influenced by both sex and genotype with larger values seen both among males and in the bGH groups (Figure 12A). Similarly, total lean mass was also influenced by both variables and showed significant differences between all 4 groups with relative increases among males and bGH mice (Figure 12C). In contrast, both body length and total fluid mass for this group were found to exhibit significant differences between WT and bGH groups within males and females but did not appear to have a sex-specific difference, though both values were higher in the bGH groups (Figure 12B, 12E). Finally, no significant differences in total fat mass were found between any of the 4 groups when absolute mass was compared (Figure 12D). However, there were differences in relative fat mass (fat mass normalized to body weight); that is, percent fat mass decreased among both male and female bGH mice relative to their WT counterparts (male WT mice 7.3% fat mass vs. male bGH 4.9% fat mass; female WTs 12.3% fat mass vs. female bGH mice 7.9% fat mass).

47

Figure 12. Total Body Data of 7-month-old bGH Mice and Littermate Controls. A. Total body mass. B. Total body length. C. Lean mass. D. Fat mass. E. Fluid mass. Significant alpha values are indicated by the following: p < 0.05 one asterisk, p < 0.001 two asterisks **

Tissue-specific data

Fat depot data

Four primary WAT depots were measured independently for mass comparison between groups. The subcutaneous (subQ) depot showed no significant differences between males and females or between genotypes (Figure 13A). Likewise, no substantial variation in weight was apparent among the 4 groups in the mesenteric (mes) depot

(Figure 13D), or between the males in the epididymal depot and females in the periovarian depot (Figure 13B). The retroperitoneal (retro) depot, however, was the only fat depot to show a significant difference among any of the groups, exhibiting a sex-

48 specific difference that was only apparent between the male and female wildtypes with a larger mass seen in males but insignificant among the bGH mice (Figure 13C). In terms of relative fat mass or fat mass normalized to body weight, bGH mice had less fat depot mass for all depots compared to controls (subcutaneous depot: male WT mice 0.72% vs. male bGH 0.54% and female WTs 0.98%% vs. female bGH mice 0.59%; retroperitoneal depot: male WT 0.37% vs. male bGH mice 0.18% and female WT mice 0.22% vs. female bGHs 0.13%; epididymal depot: male WT 1.45% vs. male bGH 0.97%; periovarian depot: female WT mice 1.40% vs. female bGH mice 0.94%; mesenteric depot male WTs 0.84% vs. male bGH mice 0.66%; female WT mice 0.97% vs. female bGHs 0.78%).

Figure 13. Fat Depot Data for 7-month-old bGH mice and littermate controls. A. Subcutaneous depot mass. B. Perigonadal [ie., epididymal (males) and periovarian (females)] depot mass. C. Retroperitoneal depot mass. D. Mesenteric depot mass. Significant alpha values are indicated by the following: p < 0.05 one asterisk, p < 0.001 two asterisks **

49

Metabolic organ data

The two primary metabolic organs of interest in this study, the liver and kidney, were collected among others and weighed at the time of dissection. Liver mass was significantly different between genotypes, being enlarged in bGH mice, but did not vary substantially between the sexes (Figure 14A). A similar trend was seen in the measurements of kidney mass, where a genotype-specific effect resulted in larger kidneys for both male and female bGH groups, but a sex-specific effect was not apparent (Figure

14B).

Figure 14. Liver and Kidney Metabolic Organ Data for 7-month-old bGH mice and littermate controls. A. Liver mass. B. Kidney mass. Significant alpha values are indicated by the following: p < 0.05 one asterisk, p < 0.001 two asterisks **

50

Serum/plasma protein data

We assessed serum levels of FAP in two different cohorts of mice. The first cohort was the 7-month-old group of 37 mice used in other assays throughout the rest of this study; the second was a group of 40 mice approximately 5 months of age at the time of their dissection, which was used to confirm the 7 month-old data. Serum FAP levels in the 7-month-old cohort showed a sex-specific effect in WTs, with female WTs yielding a higher concentration of serum FAP than male WTs, but there were no significant difference in the bGH groups (Figure 15A). The 5-month-old cohort that was used to confirm this data gave similar results, but sex-specific differences were seen in between both WT and bGH groups. That is, both genotypes of females demonstrated significantly higher levels of FAP relative to their genotype-matched male counterparts (Figure 15B).

Figure 15. Serum FAP Levels. A. FAP concentration for standard 7-month-old cohort used for all other assays. B. FAP concentration for separate 5-month-old WT and bGH, male and female cohort. Significant alpha values are indicated by the following: p < 0.05 one asterisk, p < 0.001 two asterisks **

51

FGF21 was assessed in both its intact and total forms within the 7-month-old cohort of mice. Intact FGF21 levels revealed a genotype-specific difference that was only evident in males, with male bGH mice showing a significant increase in intact FGF21 levels relative to the male WT mice (Figure 16A).

Figure 16. Plasma FGF21 Levels in bGH mice and controls at 7 months of age. A. Intact FGF21. B. Total FGF21. Significant alpha values are indicated by the following: p < 0.05 one asterisk, p < 0.001 two asterisks **

No significant difference was evident within the female groups or between the females and males in levels of intact FGF21. The same trend was seen in the quantification of total FGF21 plasma concentration, with no difference seen within female groups or between females and males despite a significant increase in levels within male bGH mice as compared to the male WTs (Figure 16B). Quantification of the FGF21 co-receptor β-

Klotho revealed no significant difference was found between any of the 4 groups with respect to β-Klotho serum concentrations (Figure 17)

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Figure 17. Beta-Klotho ELISA Data for 7-month-old bGH mice and littermate controls. Significant alpha values are indicated by the following: p < 0.05 one asterisk, p < 0.001 two asterisks **

Known FAP substrates Procollagen 1 and Procollagen 3 were quantified in the 7- month-old cohort, though only male samples from this cohort were used due to limited kit reagents and more significant previous data in males than in females. [89, 90]

Quantification of the ELISA results in plasma revealed a particularly significant difference in procollagen 1 levels between the two male groups, with a decrease in the male bGH mice (p=0.0015) (Figure 18A). Procollagen 3 levels also significantly differed between the two groups but in the opposite direction, with an increase seen in the male bGH mice as compared to the male WT mice (p=0.015) (Figure 18B).

Figure 18. Plasma Procollagen Levels in 7-month-old bGH and littermate control mice. A. Procollagen 1 concentration. B. Procollagen 3 concentration. Significant alpha values are indicated by the following: p < 0.05 one asterisk, p < 0.001 two asterisks **

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RNA isolation

Extracted RNA was only selected for cDNA generation if it passed purity standards. 260/280 values of approximately 2 were considered ideal, with a range of 1.8–

2.1 considered acceptable. Samples that fell outside this range were re-extracted. Figure

19 summarizes all sample values used for the attempted qPCR reactions. Again, the subsequent qPCR attempts were halted due to COVID-19 disruptions.

Figure 19. RNA Isolation Purity Results. Subcutaneous and perigonadal fat depots, as well as the liver, were successfully isolated. Pure RNA was identified by a value of 1.8–2.1 for the 260/280 absorbance.

54

DISCUSSION

Our findings provide information both to confirm the previously reported phenotype of bGH mice and to evaluate the newer potential relationships between

FAP/FGF21 with this GH-overexpressing genotype. The bGH mice are larger than control mice both in length and weight and exhibit decreased fat mass and increased lean mass compared to WTs when normalized to total body weight. While absolute mass of all fat depots is not significantly different between genotypes, fat depot weights normalized to body weight are decreased in the bGH mice relative to controls. In contrast to the decreased fat mass associated with the bGH mice, both liver and kidney are significantly enlarged in both sexes of bGHs versus WTs. This giant lean phenotype of bGH mice and the enlarged mass of non-adipose tissues has been shown repeatedly in many other studies and thus will not be discussed further. [72, 82, 83, 91] Serum protein analysis of these mice did not show any genotype difference in FAP levels, but there is a fairly consistent sex-specific difference, with female mice having higher levels. The data show a genotype-specific difference in both intact and total FGF21 levels that is prevalent only in males, while no differences in β-klotho levels are seen among any of the groups.

Finally, the two procollagens assessed demonstrate an inverse relationship in that procollagen 1 is significantly downregulated in bGH mice, while procollagen 3 is upregulated. As will be discussed, results for the circulating protein levels differ in numerous aspects from expectations and require further investigation of mechanisms that may explain these phenomena.

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Fibroblast activation protein (FAP)

Assessments of FAP in serum reveal a sex-specific difference without any apparent influence of genotype on FAP levels. The insignificant differences in FAP levels between WT and bGH mice reflect previously published human data on FAP in patients with active acromegaly. [1] In the human study, FAP levels are assessed before and after treatment to control GH levels; in addition, this study compares FAP levels in serum of pre-treatment patients to a separate reference group with normal GH levels as a control. FAP levels are elevated in patients with active acromegaly and reduced when GH levels of patients with acromegaly are controlled and thus positively correlated with GH action in these individuals. However, FAP levels did not significantly differ between the pre-treatment and reference groups. [1] Comparisons made between the bGH and WT mice used in this study most closely reflect the pre-treatment and reference groups of the human study, as our bGH mice are not given any treatment to reduce GH levels.

In both mouse and man, extreme differences in GH action (normal versus acromegaly or bGH) are not associated with significant alterations in serum FAP, at least based on the only study to date that addresses FAP levels in GH-related conditions. [1]

Though the mice used in this study reflect human FAP data in terms of genotype-specific effects, the sex-specific effect seen in our mice has not been documented previously in humans. Previous results in mice include one documentation of sex-specificity, with the knockout of FAP inducing deleterious effects earlier in males than in females. [92] The use of two separate cohorts to confirm our findings solidifies their significance and the consistency of higher FAP levels in female versus male mice.

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Fibroblast growth factor 21 (FGF21)

Both intact and total FGF21 levels are significantly upregulated in male bGH mice versus male WT mice, without any apparent differences between the sexes or between female WT and bGH mice. Intact FGF21 is also referred to as active FGF21 and is the circulating form known to have metabolic benefits such as enhanced lipolysis; total

FGF21 measurements assess both intact FGF21 and the truncated version that has been inactivated by FAP. [34] Therefore, the upregulation of both intact and total FGF21 seen in the bGH males reveals that the increase is due to enhancement of both the active and inactive form of the protein. [34] Our observation of increased active FGF21 in mice overexpressing GH concurs with previous studies that document the ability of GH to induce FGF21 expression and upregulate both FGF21 mRNA and protein. [36] Motivated by the literature showing the effects of GH on FGF21, our lab also conducted research in this area in 2016 and assessed serum FGF21 levels in 7-month-old bGH mice. [90] This study also found an upregulation of FGF21 in bGH mice; however, only male mice are assessed in this previous study. [90] The results collected in our current study therefore reinforce previous data and also provide the opportunity to compare the two sexes.

One could postulate that the reason increased FGF21 activity is seen only in males is because of the female-specific enhancement of FAP shown in this study, and the known role of FAP to inactivate FGF21. [34] One study that used a dietary intervention to induce upregulation of FGF21 also documented an increase in the resulting FGF21 levels in male mice versus females, suggesting the possibility of a consistent sex-specific increase in FGF21 levels for males. [93] However, another study conducted in humans used a different dietary intervention to induce FGF21 and reported opposite results, with

57 an increased response of FGF21 in females versus males. [94] Varying accounts of sex- specific effects for both FAP and FGF21 indicate the need for further studies to be conducted, investigating the possible influence of sex hormones in these interactions.

Beta-klotho (β-klotho)

Evaluation via ELISA of the small molecule β-klotho reveals no significant differences, either sex-dependent or genotype-dependent, in its plasma concentration among any of our 4 groups. β-klotho is a co-receptor that works in tandem with the

FGF21 receptor, FGFR, to enable FGF21 signaling. [95] The β-klotho molecule allows binding of FGF21 to its receptor by overcoming the low affinity of FGF21 for the FGFR binding domain [96], and is assessed to determine the influence of FGF21 alterations on

β-klotho concentrations in serum. Because the bGH mice do not exhibit increased β- klotho concentration, our observation confirms previous data in which no significant differences are observed in β-klotho levels among WT and bGH mice with altered FGF21 levels. [90] The apparent upregulation of FGF21 therefore does not directly rely on, or induce, a corresponding upregulation of its co-receptor.

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Procollagen peptides

Both procollagen 1 and procollagen 3 were only measured in males and found to differ significantly between WT and bGH mice. Procollagen 1 is significantly decreased in bGH mice compared to WTs, while procollagen 3 is significantly increased in the bGH mice. The two procollagens have been documented to exhibit similar trends to one another in previous studies, with higher levels of both found after GH-increasing therapy

[97-99] and lower levels after GH-decreasing therapy [1]. An apparent discrepancy is therefore observed between our results and previous studies, because of the inverse relationship seen between procollagen 1 and procollagen 3 in these mice.

Procollagen 1

The functional unit of collagen 1 is formed from the cleavage of procollagen at both the N- and C-terminus. The C-terminal propeptide is removed by known as procollagen C-proteinases (PCPs) [100] and is a critical step in fibrillar collagen assembly [101]. Cleavage at the C-terminal to form the resulting procollagen I C-terminal propeptide (PICP) occurs in a more efficient manner than N-terminal cleavage [101], which is accomplished by 3 interchangeable enzymes to form the procollagen I N- terminal propeptide (PINP) [102]. Thus, both PICP and PINP are valuable biomarkers that can be measured to reflect collagen 1 assembly; because their concentration in serum is reported to be equimolar [103], we selected a PICP assay due to the more extensive literature supporting C-terminal procollagen measurements and because the assay kit was readily available [104-106]. However, further investigation of advancements in procollagen 1 measurements reveals that PICP may be more susceptible to hormonal

59 interference [104]; it reports more variable results such as positive regulation by GH in some studies [98, 99] and no GH-induced effect in others [104]. PINP is the propeptide hormone that has more reliably been reported to reflect changes in GH status [1, 107,

108] and as such may have been the more appropriate choice for our assay. Discrepancies both between our chosen method and between the reporting of procollagens in the literature may partially explain the unexpected downregulation of procollagen 1

(measured as PICP) in our bGH mice.

Procollagen 3

In a similar fashion to procollagen 1, procollagen 3 is also processed at both terminals and yields PIIICP and PIIINP products that can be used as biomarkers and are produced in equal quantities. [105] PIIICP was chosen for our assessment for the purpose of consistency with the PICP data, although other studies do report the historical use of

PICP with PIIINP. [97, 109] The preference for quantifying PIIINP rather than PIIICP appears apparent in many studies, as different groups report a positive correlation between GH manipulation and resulting PIIINP levels at the exclusion of PIIICP. [109-

112] However, one study does provide evidence of both PIIICP and PIIINP as valuable markers of atrial fibrosis [113] and reinforces the foundational notion that collagen markers are associated with fibrosis. Our data support the notion that even the C-terminal peptide of procollagen 3 is associated with both GH and fibrosis due to its significant upregulation in bGH mice and previous records of GH-induced upregulation of the

(distinct but similarly regulated) N-terminal procollagen 3 peptide. [109-112]

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Conclusion

Significance of results

Many conclusions drawn from the work and presented in this thesis contribute in an unexpected manner to this field of study. One globally relevant finding was the overwhelming evidence of sex-specific effects for different molecules, emphasizing the need for equal representation of both males and females in all studies. Males have frequently been the default for research in many different organisms [89], a habit which is even reinforced within our work assessing the procollagens. However, the broad nature of differences between males and females is well documented [114-117] yet all too frequently disregarded. Future studies in all fields need to advance to equally represent both males and females.

Limitations of findings

Though information obtained in this study still contributes to the larger body of work in the field of GH, AT, and fibrosis, there were limitations that must be addressed and considered in the interpretation of the results. First, the bGH mice are a valuable but imperfect model of acromegaly; bGH mice constitutively overexpress GH from an early embryonic stage and have high GH levels in all tissues [82], whereas acromegaly is an acquired condition due to a pituitary adenoma that can occur at any age and that results from excess GH secretion from the pituitary gland [118]. Moreover, though the excess secretion of GH seen in patients with acromegaly surpasses normal GH levels, it does not reach the extent of GH excess seen in the bGH mice. [91] These differences between the

61 two conditions must be acknowledged as potentially confounding to observed results. It is also important to acknowledge other key differences between mouse and man: the inherent genetic and anatomical differences, which include variations in fat depots as mentioned previously and which can also influence outcomes. [9]

Next, the use of both plasma and serum for ELISAs was an approach that could slightly alter results, and the same method of collection should have been used for all analyses. Plasma collection was mistakenly completed using the protocol for obtaining serum and as such the plasma used in the FGF21 and procollagen ELISAs was less than ideal. Serum is blood that has been coagulated and processed for the removal of clotting factors, while plasma is blood that has been treated with an anticoagulant and as such still has clotting factors present in the sample. [119] Each method of blood processing can yield usable results, but frequently one is better than the other for the assessment of certain molecules; each should be assessed in a trial run before making a selection in order to ensure optimal assay conditions. [120] Further, the measurement of our chosen molecules only in serum or plasma could reflect global fibrosis instead of WAT-specific fibrosis; while FAP, FGF21, β-klotho, and the procollagens are all known to be expressed in WAT, varying expression in other tissues as well could yield confounding results when assessing serum alone. [121-125] Serum results therefore need to be assessed along with tissue expression data that will be gathered moving forward.

Another limitation was the ELISA kit chosen for procollagens. The kits used for both procollagens assessed the C-terminal propeptide and not the N-terminal , which can yield confounding information due to varying definitions of “procollagen” in the literature and nonspecific references to one propeptide or the other. The reported

62 association of the N-terminal product with GH-related studies means the other propeptide could have been the ideal choice for procollagen 1 and procollagen 3. [1, 107-112]

Finally, the measurement of RNA in the attempted qPCR studies provides only one perspective of the quantity and activity of the molecules of interest. Because RNA is not the functional unit and does not reliably reflect abundance at the protein level, measurement of proteins in their active form would have provided better insight into in vivo dynamics and functional capacity of these molecules. [126] While some of these limitations were an unavoidable consequence of the methodology, there are improvements that can be made to the continuation of this work moving forward.

Future directions

There are a number of initial steps that should be undertaken to finalize the objectives of the current study. One of the first steps that should be finished moving forward is the completion of procollagen ELISAs in females. Both procollagen 1 and procollagen 3 were only assessed in males thus far, due to limited kit reagents and uncertainty about the dilution appropriate for our samples. Assessing these proteins in females as well is crucial to verify whether or not the inverse relationship between procollagen 1 and 3 in bGH mice is seen in both sexes. As procollagens are used as a marker of collagen turnover for a number of conditions [127-129], understanding their levels in bGH mice could be immensely helpful in our studies and could be indicative of

AT fibrosis. Additionally, both N-terminal procollagens (PINP and PIIINP) should be assessed and compared to the C-terminal results in order to ensure accurate measurements of functional collagen generation.

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The use of a second cohort for verification of ELISA results for all proteins, as done for the FAP analysis, would also enhance the significance of the data and will be considered moving forward. Second, the unfinished RNA isolation and qPCR work also needs to be continued; RNA needs to be isolated from kidney, BAT, and muscle to complete the preparation of cDNA from all metabolic tissues, and then qPCR needs to be run on these and preexisting cDNA preparations from subcutaneous and perigondal AT and liver. However, as said previously, a limitation of exclusively looking at RNA versus protein is problematic. Thus, additional analyses of these molecules at the tissue levels using Western blotting would be very informative.

In addition to the studies outlined in this thesis, there are additional experiments that could be undertaken to better understand the relationship between GH and

FAP/FGF21. For example, it would be beneficial to repeat the serum ELISA and qPCR assessments in other tissues and in mice with different phenotypes, such as bGH mice at different ages or other mouse strains with decreased GH action either by GH antagonism

(GHA) or GH receptor knockout (GHRKO). [14] Additionally, since we are mainly interested in AT fibrosis, our mice with adipose or adipocyte-specific disruption in GH signaling could be of interest to examine. [130, 131] Acute treatment of normal mice with exogenous GH could also be informative, and these procedures are well-established in our lab. [132] As mentioned above, western blotting or other assays that can assess proteins involved in FAP/FGF21 signaling could be helpful.

The goal moving forward should be to broaden the scope of this project by collecting data on the presence of these molecules and their downstream signaling components from varying approaches, whether by quantifying them at different stages of

64 synthesis (DNA, RNA, or protein), within different organs and tissues, or in mice of varying genetic backgrounds or with varying levels of GH action. This research may be implicated in different areas moving forward, specifically as it pertains to variations between males and females. Current studies in mice show sex-specific differences in the incidence of obesity and diabetes; males are more likely to develop diabetes while females are more predisposed to obesity and metabolic syndrome. [133] Ongoing human studies report similar results, with most reports indicating that males are more frequently affected by diabetes or by both conditions than females. [134-136] The sex-specific differences seen in FAP and FGF21 in our mice may underly sex-specific differences in the development of fibrosis and its associated metabolic conditions such as obesity and diabetes, though definitive conclusions about this relationship cannot yet be made.

Further analysis of these differences may reveal information for the future study of fibrosis, its related conditions, and their treatment or even prevention in both mouse and man. Although many questions remain unresolved, the work done for this thesis laid the foundation for a deeper investigation into the pathogenesis of GH-induced AT fibrosis and the role that FAP and FGF21 may play in this process.

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