MOLECULAR PHYSIOLOGY AND BIOPHYSICS THE ROLE OF DIETARY FAT IN HYPOTHALAMIC INSULIN AND LEPTIN RESISTANCE AND THE PATHOGENESIS OF OBESITY KELLY ANN POSEY Dissertation under the direction of Kevin D. Niswender, M.D., PhD. Obesity has rapidly become a worldwide epidemic with a seemingly uncontrollable increase in prevalency. Yet, abundant evidence indicates that body weight is a tightly regulated physiological variable such that caloric intake is closely matched to energy expenditure over time to maintain a stable body weight and adiposity. The regulation of body adiposity can be modeled as a classical endocrine feedback loop in which the peripheral adiposity signals, insulin and leptin, convey the status of energy stores to the hypothalamus and coordinately regulate food intake and energy expenditure to promote the stability of adipose stores. Conversely, typical human obesity is characterized by hypothalamic resistance to the adiporegulatory effects of insulin and leptin and represents a state of dysregulated energy homeostasis. Although many factors are implicated in the development of obesity, dietary fat remains one of the most potent predictors of obesity. Therefore, I sought to elucidate potential mechanisms involved in the development of high-fat diet-induced hypothalamic insulin and leptin resistance and whether it contributes to the onset of obesity. My overall hypothesis is that dietary fat per se and not excess caloric intake contributes, either directly or indirectly, to the development of hypothalamic insulin and leptin resistance resulting in impaired regulation of body fat and the development of obesity. Results from these studies support a model in which cellular exposure to excess nutrients, particularly saturated fat, triggers cellular inflammation and insulin resistance that in turn contributes to impaired energy homeostasis and obesity. While insulin and leptin both function as adiposity negative feedback signals, studies performed at the onset of obesity suggest that the development of high-fat diet-induced hypothalamic insulin and leptin resistance are temporally and mechanistically distinct. This body of work extends previous findings and describes potential mechanisms involved in the development of high-fat diet-induced hypothalamic insulin and leptin resistance and obesity. Further elucidation of the mechanisms involved in hypothalamic resistance and the distinct functional roles of these adiposity hormones will aid in the development of therapeutic treatments to curb the obesity epidemic. Approved: ______________________________________ Date: _____________

MOLECULAR PHYSIOLOGY AND BIOPHYSICS The Role of Vascular Endothelial Growth Factor-A (VEGF-A) in Pancreatic Islet Function in Adults JEANNELLE A. KANTZ Thesis under the direction of Professor Dr. Alvin Powers Pancreatic islets are highly vascularized and this is important in insulin secretion in response to nutrients like glucose. The purpose of this research was to investigate the importance of vascular endothelial growth factor-A (VEGF-A) in adult islet function as well as the function and morphology of intraislet vasculature. To accomplish this I used a tamoxifen-inducible version of the Cre-loxP system (CreER). Two CreER transgenic lines, (RIP-CreER or Pdx1PB-CreER) allowed for β-cell or islet-specific inactivation, respectively, of VEGF-A following tamoxifen administration. RIP-CreER (Rat Insulin Promoter) is expressed in β-cells of the islet while Pdx1PB-CreER is expressed in all islet cell types. Cre-expressing mice were bred with Rosa26R (R26R) and Z/AP reporter mice in order to test (A) the administration method of tamoxifen and (B) the recombination efficiency. During these characterization studies we observed Cre recombination in the absence of tamoxifen treatment in islets of RIP-CreER;R26R mice, but not RIP-CreER;Z/AP mice. We found that Cre expression in RIP-CreER;R26R islets was ~ 4X higher than Cre expression observed in Pdx1PB-CreER;R26R. These findings indicate that Cre-mediated recombination of loxP sites depends both on the robustness of the promoter driving CreER expression and the susceptibility of the targeted allele to the Cre-mediated recombination. Based on these findings, we crossed Pdx1PB-CreER transgenic mice with VEGFfl/fl mice and measured the expression of VEGF-A in islets and the effects of VEGF-A reduction on islet function and intraislet vasculature. We found that administration of Tamoxifen effectively reduced VEGF-A levels in Pdx1PB-CreER; VEGFfl/fl mice. This reduction in islet VEGF-A expression reduced islet vasculature, and transiently impaired islet function. These findings are particularly important for experiments utilizing inducible systems. The results from our VEGF-A inactivation studies suggest that VEGF-A plays a role in the maintenance of intra-islet vasculature in adults, and that reductions in islet vascularization impact islet function.

Obesity is a strong risk factor for type 2 diabetes and cardiovascular disease partly because of the changes that occur in white adipose tissue (WAT) during weight gain. As obesity progresses, WAT expands, becomes dysfunctional, and increases its secretion of numerous adipokines, including two that will be highlighted in this dissertation, the hormone leptin and the chemokine macrophage inflammatory protein-1á (MIP-1á). Our underlying hypothesis was that elevated secretion of leptin and MIP-1á from WAT during obesity would influence development of obesity-related atherosclerosis, WAT inflammation, and systemic insulin resistance. To test the role of hematopoietic cell leptin receptor (LepR) in aortic root lesion formation, we conducted three different bone marrow transplantation studies in which bone marrow, with or without LepR, was transplanted into lethally irradiated: 1) low density lipoprotein receptor deficient (LDLR-/-) mice with moderate hyperleptinemia due to Western Diet (WD) feeding, 2) WD-fed LDLR-/- mice with pharmacologically induced hyperleptinemia (daily injection of 125 ìg leptin), or 3) obese, hyperleptinemic, LepR deficient LDLR-/- (LepRdb/db;LDLR-/-) mice. To test the role of MIP-1á in WAT inflammation, we fed WD to male and female MIP-1á deficient (MIP-1á-/-), heterozygous (MIP-1á+/-), and MIP-1á sufficient (MIP-1á+/+) mice for 16 weeks to induce obesity and examined their WAT. To identify the effects of hyperlipidemia on the metabolic role of MIP-1á, we fed WD to male and female MIP-1á-/-;LDLR-/-, MIP-1á+/-;LDLR-/-, and MIP-1á+/+;LDLR-/- mice and measured aortic root lesion area and fat mass. Removal of macrophage LepR did not influence atherosclerotic lesion formation. This suggests that non-hematopoietic cells, rather than macrophages, are potential mediators of leptins effects on aortic root lesion formation. We also demonstrated that although expression of MIP-1á increases as a consequence of weight gain this chemokine is not critical for the recruitment of monocytes and T-lymphocytes to WAT in mice with diet induced obesity. Furthermore, in the presence of hyperlipidemia, the absence of MIP-1á was not sufficient to attenuate fat mass gain or atherosclerosis during WD feeding. Taken together, these data demonstrate that although MIP-1á may be involved in WAT growth under some circumstances, the metabolic phenotype of MIP-1á-/- mice is similar to that of MIP-1á+/+ mice.

Small heat shock proteins (sHSPs) are a ubiquitous family of chaperones that protect unfolded proteins from irreversible aggregation in the cell. Human sHSPs are associated with the pathology of a variety of diseases. The high resolution structures of mammalian members of the sHSP family have not yet been achieved presumably because they form polydisperse oligomers. The engineered variants of monodisperse Hsp16.5 adapt to diverse quaternary structures, therefore serve as a model system for mammalian sHsps. In this work, we have combined biophysical approaches, cryoEM single particle reconstruction and spin labeling EPR spectroscopy to study various forms of engineered Hsp16.5 and their complexes with substrates. Our studies show that sequence variation in the N-terminal region of Hsp16.5 can dramatically influence its oligomeric structure. The oligomeric plasticity may result from the flexible linker region in the C-terminus, which allows two dimers to interact in a continuum of angles. A hypothetical model proposed for a polydisperse Hsp16.5 variant provides a feasible explanation for the polydispersity of human sHSPs. Our results suggest substrates are protected inside the sHSP oligomer through interactions with the N-termini and α-crystallin domain shell. For polydisperse and expanded oligomers, we hypothesize that increased volume and greater access to the substrate binding sites contribute to enhanced binding ability. Further, our knowledge from this study sheds light on the roles of sHSPs in human disease. Early onset cataractogenesis, related to an Arg mutation in human α-crystallin, may result from hyperactivity of the mutated α-crystallin, as Hsp16.5 with the analogous mutation shows conformational changes similar to those observed after substrate binding. From this study, we have gained insight into the structure and mechanism of polydisperse human sHSPs. The structural analysis of Hsp16.5 variants suggests that sequence divergence in the N-terminal region leads to the wide spectrum of quaternary structures in the sHSP family. We propose that the dynamic and polydisperse nature of sHSPs is important for chaperone function.

<p>Cells under stress accumulate misfolded and aggregated proteins which can be toxic and may lead to disease states. The induction of small heat shock protein (sHsp) expression enables cells to defend against protein aggregation. Human small heat shock protein 27 (Hsp27) undergoes equilibrium dissociation from an ensemble of large oligomers to a dimer. Equilibrium dissociation, to the dimer, plays an important role in Hsp27 chaperone activity and facilitates high affinity binding to destabilized proteins. In vivo, equilibrium dissociation is regulated by phosphorylation via stress-activated protein kinase MAPKAP kinase 2/3. Hsp27 phosphorylation induces changes in the size and mass distribution of the oligomer ensembles, which modulates Hsp27 chaperone activity. Phosphorylation occurs at multiple serine residues located within the N-terminal domain of Hsp27. <p>In this work, the structural changes that occur in the N-terminal domain after oligomer dissociation are examined, along with the N-terminal sequence determinants that modulate equilibrium dissociation. The equilibrium between Hsp27 oligomers and dimers was systematically analyzed through cysteine mutagenesis of selected N-terminal residues. The cysteines were derviatized with a sulfhydryl specific spin labels. An advantage of these labels is that they are sensitive to changes in their local environment, in the vicinity of the mutation site. Each spin labeled mutant was analyzed by electron paramagnetic resonance (EPR) for residue environment and solvent accessibility in three different contexts. The first context is in the large ensemble of oligomers (Hsp27-WT), after phosphorylation-induced dissociation, to the dimer, (Hsp27-D3), and the final context after complex formation with model substrate T4 Lysozyme (T4L). <p>Cysteine mutagenesis identified residues that modulate the Hsp27 dissociation equilibrium. EPR analysis revealed that oligomer dissociation disrupted subunit contacts, subsequently exposing the N-terminal domain to the aqueous environment. After T4L binding, the N-terminal domain transitions from a solvent exposed to a buried environment in the T4L/Hsp27 complex. Furthermore, the EPR data uncovered regions of the N-terminal domain that are highly dynamic. The results support a model of sHsp chaperone activity; in which, unstructured and highly dynamic flexible regions of the N-terminal domain are important for substrate binding.

Skeletal muscle is an important site for the maintenance of glucose metabolism. Under insulin-stimulated conditions it accounts for ~80% of whole body glucose disposal. Muscle glucose uptake (MGU) is a three step process involving delivery of glucose from the blood to the interstitium, facilitated transport into the intracellular space by glucose transporters, and irreversible phosphorylation of glucose to glucose-6-phosphate by hexokinase. Lipopolysaccharide (LPS) elicits a strong immune response and is known to impair insulin-mediated glucose disposal. This work aims to define the mechanism by which LPS acutely impairs MGU in the conscious mouse model in vivo. Our data demonstrates decreased cardiovascular function plays a central role in the impairment of skeletal MGU caused by LPS. Using mice overexpressing key proteins involved in MGU we sought to determine if increased glucose transport or phosphorylation capacity could improve glucose uptake in the setting of acute inflammatory stress. Our data demonstrate that enhanced glucose transport, but not phosphorylation capacity, ameliorates inflammation induced impairments in MGU. The observed improvements were also associated with improved cardiovascular function. Thus LPS-induced cardiovascular alterations likely play a dominant role in modulating insulin action.

The multidrug transporter EmrE is a proton coupled secondary transporter from Escherichia coli. EmrE confers resistance to a variety of positively charged hydrophobic cations by removing them out of the cell in exchange for protons. The mechanism of substrate transport has been well characterized. The conformational changes that initiate the movement of the substrate across the membrane have not been examined. The goal of this work was to investigate the structure and dynamics of EmrE using electron paramagnetic resonance (EPR) spectroscopy. The data set consists of the mobilities, solvent accessibilities of each spin labeled sites along the full length of the protein. These parameters were interpreted as constraints on the local steric environment, the orientation of helices in the lipid phase and packing of the transmembrane (TM) helices in the dimer. Dipolar coupling across the dimer interface of TM1, TM2 and TM3 reveal multiple spin label populations, suggesting different packing of EmrE monomers. Binding of tetraphenyl phosphonium (TPP+) to the protein reduces dipolar coupling between symmetry related spin label pairs located in the middle of TM1. In contrast, for sites located in the middle of TM2 and the N-terminal region of TM3 report increased dipolar coupling. In parallel, the N-terminus region of TM2 is slightly tilted and move away from the dimer interface. These changes are in support with the alternating access model. Moreover, upon substrate binding an increase in the structural order is observed in the C-terminus region of TM3 and loop connecting it to TM4. The EPR analysis suggests that in liposomes the structure of these regions deviate from the antiparallel static crystal structure in detergent micelles. A model to resolve these discrepancies is proposed. We also investigated the helix packing in EmrE monomer using double electron-electron resonance (DEER) to measure distance between two spin label sites in solution. The result reports the packing and orientation of TM2 and TM3 with respect to TM1 deviates from the crystal structure. Furthermore, multimodal distance distributions were observed for symmetry related sites specifically in TM3. The overall consensus of the distance measurements confirms the dynamic flexibility of EmrE backbone in the apo-state and decreases upon substrate binding.

Sirtuin 1 (Sirt1) is a NAD+-dependent deacetylase that exerts many of the pleiotropic effects of oxidative metabolism. Due to local hypoxia and hypertonicity, the renal medulla is subject to extreme oxidative stress. Here, we set out to investigate the role of Sirt1 in the kidney. Our initial analysis indicated that it was abundantly expressed in mouse renal medullary interstitial cells in vivo. Knocking down Sirt1 expression in primary mouse renal medullary interstitial cells substantially reduced cellular resistance to oxidative stress, while pharmacologic Sirt1 activation using either resveratrol or SRT2183 improved cell survival in response to oxidative stress. The unilateral ureteral obstruction (UUO) model of kidney injury induced markedly more renal apoptosis and fibrosis in Sirt1+/¨C mice than in wild-type controls, while pharmacologic Sirt1 activation substantially attenuated apoptosis and fibrosis in wild-type mice. Moreover, Sirt1 deficiency attenuated oxidative stress¨Cinduced COX2 expression in cultured mouse renal medullary interstitial cells, and Sirt1+/¨C mice displayed reduced UUO-induced COX2 expression in vivo. Conversely Sirt1 activation increased renal medullary interstitial cell COX2 expression both in vitro and in vivo. Furthermore, exogenous PGE2 markedly reduced apoptosis in Sirt1-deficient renal medullary interstitial cells following oxidative stress. Taken together, these results identify Sirt1 as an important protective factor for mouse renal medullary interstitial cells following oxidative stress and suggest that the protective function of Sirt1 is partly attributable to its regulation of COX2 induction. We therefore suggest that Sirt1 provides a potential therapeutic target to minimize renal medullary cell damage following oxidative stress.

Under hypoglycemic conditions, glucagon is secreted from α-cells, within pancreatic islets of Langerhans, to stimulate hepatic glucose output and, therefore, to restore proper glycemia. Once normoglycemia is reestablished, glucagon release is inhibited. Two general models have been proposed to account for this suppression: direct inhibition by glucose or indirect inhibition by paracrine factors released in the islet. To rigorously identify α-cells in the intact islet, we took advantage of transgenic mice expressing fluorescent proteins specifically in this cell-type. α-cell NAD(P)H responses to glucose demonstrate that α-cells metabolize glucose; glucokinase being the likely rate-limiting enzyme. Glucagon secretagogues such as arginine and pyruvate also enhance α-cell metabolic redox state, indicating that such an elevation is not sufficient to inhibit secretion. Importantly, glucose stimulates glucagon output from pure populations of flow-sorted α-cells. These observations argue against a direct effect of glucose and support the paracrine inhibition model. Pharmacological modulations of ion channels under low glucose conditions indicate that activation of L-type voltage-gated calcium channels is integral for α-cell calcium oscillations and glucagon secretion. In addition, α-cell [Ca2+]i and glucagon release are affected by KATP channel activity in a manner similar to insulin-secreting α-cells. Closure of KATP leads to greater [Ca2+]i and hormone output, whereas opening has the opposite effect. As a result, modulation of KATP channel activity could constitute a possible mechanism for regulating glucagon secretion. In particular, paracrine inhibitors could potentially suppress α-cell secretory activity by opening KATP channels and reducing [Ca2+]i. Because glucagon release from islets is inhibited by glucose, one would naively expect α-cell [Ca2+]i to drop concomitantly. However, our calcium imaging studies in intact islets reveal that glucose slightly elevates α-cell [Ca2+]i. Application of candidate paracrine inhibitors (insulin, zinc, GABA, and somatostatin) inhibits glucagon secretion but does not reduce α-cell calcium activity either. Taken together, the data indicate that [Ca2+]i and glucagon secretion are uncoupled at inhibitory concentrations of glucose, and that suppression occurs downstream from α-cell calcium signaling, presumably at the level of vesicle trafficking or exocytotic machinery.

All eukaryotic and some prokaryotic ClC anion transport proteins have extensive cytoplasmic C-termini containing two cystathionine-beta-synthase (CBS) domains. CBS domain secondary structure is highly conserved and consists of two alpha-helices and three beta-strands arranged as beta1-alpha1-beta2-beta3-alpha2. ClC CBS domain mutations cause muscle and bone disease and alter ClC gating. However, the precise functional roles of CBS domains and the structural bases by which they regulate ClC function are poorly understood. CLH-3a and CLH-3b are C. elegans ClC anion channel splice variants with strikingly different biophysical properties. Splice variation occurs at cytoplasmic N- and C-termini and includes several amino acids that form alpha2 of the second CBS domain (CBS2). We demonstrate that interchanging alpha2 between CLH-3a and CLH-3b interchanges their gating properties. The R-helix of ClC proteins forms part of the ion conducting pore and selectivity filter and is connected to the cytoplasmic C-terminus via a short stretch of cytoplasmic amino acids termed the R-helix linker. C-terminus conformation changes could cause R-helix structural rearrangements via this linker. X-ray structures of three ClC protein cytoplasmic C-termini suggest that alpha2 of CBS2 and the R-helix linker could be closely apposed and may therefore interact. We found that mutating apposing amino acids in alpha2 and the R-helix linker of CLH-3b was sufficient to give rise to CLH-3a-like gating and extracellular cysteine reactivity. We postulate that the R-helix linker interacts with CBS2 alpha2, and that this putative interaction provides a pathway by which cytoplasmic C-terminus conformational changes induce conformational changes in membrane domains that in turn modulate ClC function.