Although once considered a simple energy storage depot, the adipose tissue is now known to be a powerful regulator of whole body insulin sensitivity and energy metabolism. This metabolically dynamic organ functions to safely store excess fatty acid as triglyceride, thereby preventing lipotoxicity in peripheral tissues and the development of insulin resistance. In addition, the adipose tissue acts as an endocrine organ and secretes factors, called adipokines, which influence whole body insulin sensitivity and glucose homeostasis. Therefore, understanding adipose tissue development and biology is essential to understanding whole body energy metabolism. A master regulator of adipose tissue development and whole body insulin sensitivity is the nuclear receptor, PPARγ. Due to the importance of this nuclear receptor in maintaining adipocyte function, disruptions in PPARγ activity result in severe metabolic abnormalities, such as insulin resistance and type 2 diabetes. Conversely, PPARγ activation by synthetic agonists ameliorates these conditions, demonstrating the potent control this nuclear receptor has on whole body metabolism. Therefore, understanding how PPARγ expression and activity are regulated, particularly in the adipose tissue, is paramount to understanding the pathogenesis of type 2 diabetes. While there are several synthetic PPARγ agonists available, identifying the endogenous ligand or ligands is still an area of intense investigation. Since fatty acids can induce PPARγ activation, in the first part of this thesis, I screened several fatty acid metabolizing enzymes present in the adipocyte to identify novel modulators of PPARγ activity. These studies revealed that the fatty acid Δ9 desaturase, Stearoyl CoA Desaturase 2 (SCD2), is absolutely required for 3T3-L1 adipogenesis and to maintain adipocyte-specific gene expression in fully differentiated cells. Although SCD2 does not appear to regulate PPARγ ligand production, it does potently regulate PPARγ activity by maintaining the synthesis of PPARγ protein. Surprisingly, this effect was found only with SCD2 and not with the highly homologous protein, SCD1. Therefore, these findings identify separate cellular functions for these SCD isoforms and reveal a novel and essential role for fatty acid desaturation in the adipocyte. Equally important to understanding PPARγ regulation is identifying the downstream mechanisms by which PPARγ activation improves insulin sensitivity. Evidence suggests that the PPARγ target gene, Cidea, is involved in mediating insulin sensitivity by binding to lipid droplets and promoting lipid storage in the adipocyte. Therefore, the second part of thesis provides mechanistic detail into Cidea function by showing that the carboxy terminal 104 amino acids is necessary and sufficient for lipid droplet targeting and the stimulation of triglyceride storage. However, these studies also identified a novel function for Cidea, which requires both the carboxy and amino termini: to induce larger and fewer droplets from smaller dispersed droplets, indicating the possible fusion of droplets. Perhaps this striking change in lipid droplet morphology allows tighter packing and more efficient storage of triglyceride and identifies a novel role for Cidea in lipid metabolism. The results presented in this thesis elucidate key aspects of lipid metabolism that maintain adipocyte function: SCD2 is required to maintain PPARγ protein expression in the mouse; Cidea is a downstream effector of PPARγ activity by promoting efficient triglyceride storage. Therefore, these findings enhance our understanding of adipocyte biology.

The aim of this investigation was to determine the effect of a nicotine-conditioned context on locomotor hyperactivity in an animal model of D2-priming, and whether conditioned hyperactivity could be blocked by the D2 antagonist eticlopride or the D3 antagonist nafadotride. D2-primed male rats showed enhanced nicotine sensitization as evidenced by statistically significant differences in horizontal activity. D2-primed female rats administered nicotine demonstrated an increased hypoactive response after initial sensitization and increased stereotypy. Eticlopride and nafadotride blocked sensitization to nicotine in both D2-primed and non D2-primed males and females. Eticlopride blocked conditioned hyperactivity in females but not in males. D2-primed female rats administered nicotine demonstrated significantly higher conditioned-hyperactivity as compared to non D2-primed females and controls, and this increase was more effectively blocked by nafadotride as compared to eticlopride. These results suggest differential roles of the dopamine D2 and D3 receptors in both adolescent nicotine sensitization and conditioned activating effects of nicotine.

As the prevalence of vitamin D deficiency continues to grow, mounting evidence supporting its link with chronic disease strengthens suggesting vitamin D’s candidacy in the prevention and treatment of multiple disease states and their complications. Dietary guidelines, however, do not take sun exposure into account. The present study sought to explore the impact of sun exposure on vitamin D status (serum 25(OH)D), and identify other significant determinants of serum levels which may have the greatest effects on overall health. Participants (n = 34) were pre-menopausal women aged 18 to 50 years (mean age 39 ± 6 years), who had their blood drawn at a local pathology lab and a follow-up appointment at a health assessment lab for the collection of other measurements. Mean serum 25(OH)D level was 64 ± 18 nmol/L, and mean dietary vitamin D intake was approximately 327 ± 229 IU/day. Although 82% of participants were below the RDA guidelines (600 IU/day for females ages 9-50 years) for dietary vitamin D intake, only 32% had serum 25(OH)D levels < 50 nmol/L (the recommended level of sufficiency for bone health) reflecting deficiency. While serum 25(OH)D levels were significantly correlated to dietary vitamin D intake (r = 0.42, p = 0.0139), it is reasonable to assume that participants obtained adequate vitamin D from sun exposure. Fasting serum insulin levels were significantly, positively correlated with BMI (r = 0.83, p < 0.0001), and sun exposure index (Body Surface Area x Minutes of Direct Sunlight) was significantly, positively correlated with serum 25(OH)D levels (fall weekend SEI: r = 0.47, p = 0.0059; spring weekend SEI: r = 0.43, p = 0.0135; average weekend SEI: r = 0.43, p = 0.013; and average overall SEI: r = 0.39, p = 0.0247). Reported sun exposure appeared to be least during winter weekdays and the most during summer weekends. Regression analysis was used to determine the strongest predictors of serum 25(OH)D levels, which were found to be sun exposure, dietary vitamin D intake, skin reflectance, age, BMI, and ethnicity (R2 = 0.58 , p = 0.0031), demonstrating that simple questionnaires, such as those employed in this study, can help to predict serum 25(OH)D status and thus be considered in the future treatment of vitamin D deficiency.

Vitamin A metabolites and retinoids may slow the progression of breast cancer and elicit anti-neoplastic properties similar to those of omega-3 fatty acids. Studies using animal models show a decrease in the incidence, growth and metastisis of mammary tumors in the presence of specific fatty acids. This effect is also seen with use of retinoids, specifically all-trans retinoic acid (AtRA). Thus, fatty acids may also alter retinoid homeostasis in mammary carcinoma cells (MCF-7s). The potential for inter/co dependency among fatty acids and retinoids is considerable, and here it has been hypothesized that a decrease in cancer progression will occur in the presence of both compounds. MCF-7’s were seeded in a 48 well plate at 5,000 cells per well. After 24 hr, cells were treated with either 1 µM AtRA alone, fatty acids alone, or AtRA + fatty acids. Fatty acid treatments (Linoleic, and Linolenic) were administered at 2.5 uM concentrations. Each fatty acid treatment was also combined with 1 µM AtRA to determine if there is a synergistic effect on slowing cell growth. Both culture media and treatments were changed at 24 hour intervals over a 3 day trial. When compared to the controls, cells treated with 1 µM AtRA or 2.5 µM Linolenic acid both inhibited cell growth. Interestingly, when combined with Linolenic acid, AtRA treatment resulted in a significant (nearly 50%) additional growth inhibition when compared to treatment with AtRA alone. Our results suggest that AtRA and Linolenic acid have a inter/co dependency that significantly inhibits breast cancer cell growth in vitro by 73.4 % compared to control, and 49.7% compared to AtRA alone over 72 hours. We conclude that AtRA and linolenic acid have a combined effect in breast cancer cell proliferation in-vitro and their role in dietary prevention warrants further investigation.

The primary purpose of this research was to examine the effects of HRT in women with acute estrogen deficiency from surgically induced menopause. The secondary purpose was to evaluate how HRT improves symptoms of acute estrogen deficiency and quality of life (QOL) in women using hormone supplementation. Peer reviewed articles published from 2000 to 2017 that were written in the English language with a focus on the use of HRT in women with acute estrogen deficiency after surgical menopause were evaluated for relevance. Evidence suggests the primary reason for decreased use of HRT is the associated risks outweighing the benefits; however, this is not reflected in health care provider's (HCP's) clinical experience. HCP's were more likely to prescribe HRT for themselves or family members if they were experiencing the negative side effects of estrogen deficiency due to surgical menopause, but not to women in their care with similar clinical manifestations of menopause. Additionally, serious risks associated with HRT for acute estrogen deficiency remain incongruent with HRT for women experiencing natural menopause; although risk for breast cancer due to HRT was a universal concern. Risks of HRT related to thromboembolism, stroke and heart disease, were discussed with comparison to the undesirable clinical manifestations of menopause. Results indicate further education and research is needed that explores the risks and benefits for HRT in women with sudden onset of estrogen deficiency from surgical menopause.

The insulin-like growth factor-1 receptor (IGF-1R) and many of its downstream signaling components have long been implicated in tumor progression and resistance to therapy. The insulin receptor substrate-1 (IRS-1) and IRS-2 adaptor proteins are two of the major downstream signaling intermediates of the IGF-1R. Despite their considerable homology, previous work in our lab and others has shown that IRS-1 and IRS-2 play divergent roles in breast cancer cells. Signaling through IRS-1 promotes cell proliferation, whereas signaling through IRS-2 promotes cell motility and invasion, as well as glycolysis. Moreover, using a mouse model of mammary tumorigenesis, our lab demonstrated that IRS-2 acts as a positive regulator of metastasis, while IRS-1 cannot compensate for this function. The focus of my thesis research is to understand how IRS-2, but not IRS-1, promotes breast carcinoma cell invasion and metabolism to support metastasis. In preliminary studies, I have found that IRS-1 and IRS-2 exhibit different expression patterns in both cell lines and human tumors with correlations to patient survival, which provides a potential mechanism for their distinct functions. The localization of IRS-1 and IRS-2 within separate intracellular compartments would determine their access to downstream effectors and substrates, and this would result in unique cellular outcomes. Specifically, I have observed that IRS-2, but not IRS-1, co-localizes with microtubules in breast carcinoma cell lines with implications for signaling through AKT and mTORC2. The goal of this research is to determine how the localization of IRS-2 contributes to its regulation of breast cancer progression and response to therapy and how this information could be used to better predict patient outcomes.

The insulin/IGF-1 signaling (IIS) was initially identified in C. elegansto control a developmental phenotype called dauer. Subsequently, it was realized that lifespan was extended by mutations in this pathway and became an intense focus of study. The IIS pathway regulates growth, metabolism and longevity across phylogeny and plays important roles in human disease such as cancer and diabetes. Given the large number of cellular processes that this pathway controls, understanding the regulatory mechanisms that modulate insulin/IGF-1 signaling is of paramount importance. IIS signaling is a very well-studied kinase cascade but few phosphatases in the pathway are known. Identification of these phosphatases, especially those that counteract the activity of the kinases, would provide a better insight into the regulation of this critical pathway. Study of serine/threonine phosphatases is hampered by the lack of appropriate reagents. In Chapter II, we discuss the design and results of an RNAi screen of serine/threonine phosphatases performed in C. elegans using dauer formation as a phenotypic output. We identified several strong regulators of dauer formation and in Chapter III, proceed to characterize one of the top candidates of our screen, pptr-1. We show that pptr-1 regulates the IIS and thereby affects lifespan, development and metabolism in C .elegans. pptr-1gene encodes a protein with high homology to the mammalian B56 family of PP2A regulatory subunits. PP2A is a ubiquitously expressed phosphatase that is involved in multiple cellular processes whose specificity determined by its association with distinct regulatory subunits. Our studies using C. elegans provides mechanistic insight into how the PP2A regulatory subunit PPTR-1 specifically modulates AKT-1 activity by regulating its phosphorylation status in the context of a whole organism. Furthermore, we show that this mechanism of regulation is conserved in mammals.

Background: The endocrine pancreas contains multiple cell types co-localized into clusters called the Islets of Langerhans. The predominant cell types include alpha and beta cells, which produce glucagon and insulin, respectively. The regulated release of these hormones maintains whole body glucose homeostasis, essential for normal metabolism and to prevent diabetes and complications from the disease. Given the heterogeneous nature of islet composition and absence of unique surface markers, many previous studies have focused on the whole islet. Sorting islet cells by intracellular hormone expression overcomes this limitation and provides pure populations of individual islet cell subsets, specifically alpha and beta cells. This technique provides the framework for characterizing human islet composition and will work towards identifying the genetic changes alpha and beta cells undergo during development, growth, and proliferation. Methods: Human islets obtained from cadaveric donors are dissociated into a single cell suspension, fixed, permeabilized, and labeled with antibodies specific to glucagon, insulin, and somatostatin. Individual alpha, beta, and delta cell populations are simultaneously isolated using fluorescence activated cell sorting. Candidate gene expression and microRNA profiles have been obtained for alpha and beta cell populations using a quantitative nuclease protection assay. Thus far, RNA has been extracted from whole islets and beta cells and subjected to next generation sequencing analysis. Results: The ratio of beta to alpha cells significantly increases with donor age and trends higher in female donors; BMI does not appear to significantly alter the ratio. Further, we have begun to investigate the unique gene expression profiles of alpha and beta cells versus whole islets, and have characterized the microRNA profiles of the two cell subsets. Conclusions: By establishing methods to profile multiple characteristics of alpha and beta cells, we hope to determine how gene, miRNA, and protein expression patterns change under environmental conditions that lead to beta cell failure or promote beta cell development, growth, and proliferation.

Protein folding in the endoplasmic reticulum (ER) is essential for proper cellular function. However, the sensitive environment in the ER can be perturbed by both pathological processes as well as by physiological processes such as a large biosynthetic load placed on the ER. ER stress is a specific type of intracellular stress caused by the accumulation of immature or abnormal misfolded or unfolded proteins in the ER. Simply defined, ER stress is a disequilibrium between ER load and folding capacity. Cells have an adaptive response that counteracts ER stress called the "Unfolded Protein Response” (UPR). The ability to adapt to physiological levels of ER stress is especially important for maintaining ER homeostasis in secretory cells. This also holds true for pancreatic β-cells, which must fold and process large amounts of the hormone insulin. Pancreatic β-cells minimize abnormal levels of glycemia through adaptive changes in the production and regulated secretion of insulin. This process is highly sensitive, so that small degrees of hypo- or hyperglycemia result in altered insulin release. The frequent fluctuation of blood glucose levels in humans requires that β-cells control proinsulin folding in the ER with exquisite sensitivity. Any imbalance between the load of insulin translation into the ER and the actual capacity of the ER to properly fold and process the insulin negatively affects the homeostasis of β-cells and causes ER stress. In this dissertation, we show that Inositol Requiring 1 (IRE1), an ER-resident kinase/endoribonuclease and a central regulator of ER stress signaling, is essential for maintaining ER homeostasis in pancreatic β-cells. Importantly, IRE1 has a crucial function in the body’s normal production of insulin in response to high glucose. Phosphorylation and subsequent activation of IRE1 by transient exposure to high glucose is coupled to insulin biosynthesis, while inactivation of IRE1 by siRNA or inhibition of IRE1 phosphorylation abolishes insulin biosynthesis. IRE1 signaling under these physiological ER stress conditions utilizes a unique subset of downstream components of IRE1 and has a beneficial effect on pancreatic β-cell homeostasis. In contrast, we show that chronic exposure of β-cells to high glucose causes pathological levels of ER stress and hyperactivation of IRE1, leading to the degradation of insulin mRNA. The term “glucose toxicity” refers to impaired insulin secretion by β-cells in response to chronic stimulation by glucose and is characterized by a sharp decline in insulin gene expression. However, the molecular mechanisms of glucose toxicity are not well understood. We show that hyperactivation of IRE1 caused by chronic high glucose treatment or IRE1 overexpression leads to insulin mRNA degradation in pancreatic β-cells. Inhibition of IRE1 signaling using a dominant negative form of the protein prevents insulin mRNA degradation in β-cells. Additionally, islets from mice heterozygous for IRE1 retain expression of more insulin mRNA after chronic high glucose treatment than do their wild-type littermates. This work suggests that the rapid degradation of insulin mRNA could provide immediate relief for the ER and free up the translocation machinery. Thus, this mechanism may represent an essential element in the adaptation of β-cells to chronic hyperglycemia. This adaptation is crucial for the maintenance of β-cell homeostasis and may explain in part why the β-cells of diabetic patients with chronic hyperglycemia stop producing insulin without simply undergoing apoptosis. This work implies that prolonged activation of IRE1 signaling is involved in the molecular mechanisms underlying glucose toxicity. This work therefore reveals two distinct activities elicited by IRE1 in pancreatic β-cells. IRE1 signaling activated by transient exposure to high glucose enhances proinsulin biosynthesis, while chronic exposure of β-cells to high glucose causes hyperactivation of IRE1, leading to the degradation of insulin mRNA. Physiological IRE1 activation by transient high glucose levels in pancreatic β cells has a beneficial effect on insulin biosynthesis. However, pathological IRE1 activation by chronic high glucose or experimental drugs negatively affects insulin gene expression. In the future, a system to induce a physiological level of IRE1 activation, and/or reduce the pathological level of IRE1 activation could be used to enhance insulin biosynthesis and secretion in people with diabetes, and may lead to the development of new and more effective clinical approaches to the treatment of this disorder.

The "RNA world" hypothesis makes two predictions that RNA should have been able both to catalyze RNA replication and to direct protein synthesis. The evolution of RNA-catalyzed protein synthesis should be critical in the transition from the RNA world to the modem biological systems. Peptide bond formation is a fundamental step in modem protein biosynthesis. Although many evidence suggests that the ribosome is a ribozyme, peptide bond formation has not been achieved with ribosomal RNAs only. The goal of this thesis is to investigate whether RNA could catalyze peptide bond formation and how RNA catalyzes peptide bond formation. Two systems have been employed to approach these questions, the ribozyme system and the ribosome system. Ribozymes have been isolated by in vitro selection that can catalyze peptide bond formation using the aminoacyl-adenylate as the substrate. The isolation of such peptide-synthesizing ribozymes suggests that RNA of antiquity might have directed protein synthesis and bolsters the "RNA world" hypothesis. In the other approach, a novel assay has been established to probe the ribosomal peptidyltransferase reaction in the presence of intact ribosome, ribosomal subunit, or ribosomal RNA alone. Several aspects of the peptidyltransfer reaction have been examined in both systems including metal ion requirement, pH dependence and substrate specificity. The coherence between the two systems is discussed and their potential applications are explored. Although the ribozyme system might not be a reminiscence of the ribosome catalysis, it is still unique in other studies. The newly established assay for ribosomal peptidyltransferase reaction provides a good system to investigate the mechanism of ribosomal reaction and may have potential application in drug screening to search for the specific peptidyltransferase inhibitors.