MOLECULAR PHYSIOLOGY AND BIOPHYSICS THE ROLE OF FOXM1 IN PANCREATIC BETA CELL MASS REGENERATION AND EXPANSION AMANDA ACKERMANN MISFELDT Dissertation under the direction of Maureen Gannon, Ph.D. The size of an organisms â cell population, or â cell mass, and its functional capacity are important determinants of glucose homeostasis. In normal individuals, â cell mass increases in response to obesity, pregnancy, and pancreatic injury by increasing â cell proliferation, â cell neogenesis, and/or â cell size. However, one or more of these processes does not occur properly or sufficiently in diabetic patients. Thus, identifying factors that regulate these processes could lend insight into the pathogenesis and potential treatment of diabetes. The forkhead box transcription factor FoxM1 activates expression of multiple genes that control cell cycle progression. Accordingly, FoxM1 is necessary for proper proliferation of postnatal â cells, and loss of FoxM1 in the pancreas results in reduced â cell mass and progressive diabetes. This dissertation examined â cell mass regeneration and expansion in response to pancreatic injury and diet-induced obesity in wild-type mice and littermates with pancreas-wide deletion of Foxm1. Regeneration of â cell mass after surgical removal of 60% of the pancreas was impaired in mice with pancreas-wide deletion of Foxm1 due to reduced â cell proliferation. Importantly, no defects in â cell neogenesis were observed. These studies highlight the similarities between â cell mass regeneration after injury and embryonic â cell mass development, as FoxM1 was not required for â cell proliferation during embryogenesis or in small (neogenic) â cell clusters during regeneration, but was required for proliferation of â cells within large (pre-existing) islets. When mice with pancreas-wide deletion of Foxm1 were fed a high-fat/high-carbohydrate diet, they were more susceptible to developing glucose intolerance and diabetes than were wild-type littermates, associated with impaired glucose-stimulated insulin secretion. These findings suggested that FoxM1 may play a role in â cell secretory function in addition to â cell mass compensation in response to diet-induced obesity. Together, these studies provide evidence that â cell proliferation is regulated differently during development versus adulthood, that regeneration after pancreatic injury mimics to some extent pancreas development, and that FoxM1 function may not be restricted to cell cycle regulation.

The finding that served as the foundation for this dissertation is that physiological stressors provoke the transition to an energy-depleted state in the mouse liver. This is important because adenine nucleotides define energy state and couple to nearly all metabolic processes in the liver. Change in ATP, ADP, and AMP levels also impacts metabolic enzymes in the liver governing substrate trafficking. The stress-induced increase in hepatic AMP:ATP, at least in acute circumstances, was hypothesized to be mediated by glucagon receptor activation and associated gluconeogenic flux through cytosolic phosphoenolpyruvate carboxykinase (PEPCK). This hypothesis was confirmed using in vivo experiments, including a newly developed hyperglucagonemic-euglycemic (glucagon) clamp, in mice lacking the glucagon receptor or PEPCK. The regulatory consequences of the glucagon-mediated fall in energy state included hepatic activation of AMP-activated protein kinase (AMPK). AMPK is a low energy sensor and appears to mediate regulatory adaptations linked to hepatic glucagon receptor activation. This conclusion is based on evidence that hepatic glucagon activation stimulates an AMPK-dependent pathway characterized by increased phosphorylation of acetyl-CoA carboxylase (ACC) and expression of peroxisome proliferator-activated receptor á and fibroblast growth factor 21 (FGF21) mRNA. These important factors modulate hepatic lipid oxidation and thus energy availability. The requirement for AMPK in this pathway was assessed using glucagon clamps in mice expressing an adenoviral-mediated dominant-negative form of AMPK. The potential long-term regulatory impact of the glucagon receptor-mediated transition to a hepatic energy-depleted state was based on studies in ob/+ and ob/ob mice chronically or acutely administered FGF21. These studies demonstrate that FGF21 has powerful hepato-centric effects to modulate glycemia and hepatic glucose flux. Acute FGF21 infusion results suggest a degree of resistance in obese, diabetic ob/ob mice that is overcome with chronic treatment. In conclusion, this dissertation introduces novel aspects of hepatic glucagon receptor activation and highlights the regulatory impact of adenine nucleotides. This work is important to understand metabolic regulation during stressors such as exercise and identify potential impairments in the liver associated with metabolic disease.

The protein complex composed of the cytoplasmic domain of band 3 (cdb3) and ankyrin forms one of the two major contact sites between the spectrin-based membrane skeleton and the lipid bilayer in human erythrocytes. This linkage is critical for maintaining the shape and viscoelastic properties of the membrane. Among the known membrane skeleton protein mutations, band 3 (anion exchanger 1 or AE1) variants account for about 20% of hereditary spherocytosis cases among Caucasians. The structure and function of cdb3 and ankyrin repeat domain 3-4 have been studied via site directed labeling methods in combination with conventional electron paramagnetic resonance (EPR), double electron electron resonance (DEER), and fluorescence spectroscopy. The central compact region (55-356) of the cdb3 dimer under physiological pH is indistinguishable from the crystal structure determined at pH 4.8. The N terminus (1-54) of cdb3 is dynamically disordered and capable of docking various cytoplasmic proteins. The similar disorder in the C terminus (357-359) of cdb3 is consistent with the weak motional coupling between the cytoplasmic domain and the transmembrane domain of band 3. The surface of the cdb3 peripheral domain that is opposite the dimerization arm interacts with the ankyrin groove. The á-helix 2, á-helix 3 and â strands 6, 7 (hairpin) of cdb3 may be directly involved in ankyrin binding. The band 3 Tuscaloosa mutation (P327R), which is located at a loop turning point of the dimerization arm, does not dissociate the cdb3 dimer but does perturb the region adjacent to position 327 and the downstream C terminus, thereby slightly destabilizing the dimer structure. These spectroscopic studies establish a structural model for cdb3 and its binding partner ankyrin in solution at neutral pH, which provides an important platform to further characterize protein-protein interactions that stabilize the membrane and naturally occurring mutations that cause human diseases.

Multiple sclerosis (MS) is a debilitating neuroimmunological and neuro-degenerative disease. Despite substantial evidence for polygenic inheritance, the MHC is the only region that clearly and consistently demonstrates linkage and association in MS studies. The goal of the work presented in this dissertation was to identify additional chromosomal regions harboring MS susceptibility genes. Our studies entailed a new genomic convergence approach incorporating information gained from positional (linkage and association) and functional (comparative sequence) studies. In conjunction with high-throughput genotyping and powerful new statistical analyses methods, this approach identified several regions suggesting the presence of MS loci. We began our investigation with a genomic linkage screen that identified seven chromosomal regions of interest in a data set of multiplex MS families. To narrow these regions, we developed an approach for more detailed linkage studies that capitalized on new methods for rapid and accurate genotyping of SNPs. In addition to increasing marker coverage in each region, we genotyped an expanded data set and devised covariate analyses schemes to account for genetic effect in the MHC. This method continued to provide evidence of linkage to several chromosomal regions and was successful in substantially narrowing two regions to only a few Mb. We then developed a systematic approach to expedite follow-up association studies in the positional candidate regions. In an attempt to increase the likelihood of detecting variants associated with MS, we employed a novel method to select SNPs located in multi-species conserved sequences. Use of this method on chromosome 1q44 resulted in the identification of four subregions demonstrating significant association with MS susceptibility. The work presented in this dissertation confirmed several regions warranting further investigation for genes conferring susceptibility to MS, including chromosomes 1q44, 2q35, 9q34, and 18p11. It is our hope that these studies will result in the discovery of several genes associated with MS and that our genomic convergence approach will provide researchers with a method for unraveling the genetic heterogeneity of MS and other complex genetic diseases.

Heart disease is the number one cause of death in the United States. There are many forms of heart disease including heart failure and arrhythmias. One underlying theme in heart disease and many other diseases is disrupted Ca2+ homeostasis. Calcium is a charge carrier and universal mediator of diverse cellular processes. In cardiac myocytes, these processes include excitation-contraction coupling, gene transcription and apoptosis. Ca2+ enters cardiac myocytes through L-type Ca2+ channels (LTCC) where it activates signaling molecules such as the multifunctional Ca2+/calmodulin dependent protein kinase II (CaMKII). CaMKII is one of many specialized proteins poised to respond to Ca2+ signaling in cardiac myocytes. Accumulating evidence links cardiac CaMKII activity to normal physiological regulation of several heart functions and to multiple pathological conditions. CaMKII is associated with cardiac LTCC complexes and increases channel open probability (PO) to dynamically increase Ca2+ current (ICa) and augment cellular Ca2+ signaling by a process called facilitation. I found that activated CaMKII binds to the LTCC b2a subunit close to a preferred CaMKII phosphorylation site, Thr498 and colocalizes with b2a in cardiomyocytes. Mutation of Thr498 to Ala (T498A) in b2a prevents CaMKII-mediated increases in the PO of recombinant LTCCs. Moreover, expression of b2a (T498A) in adult cardiomyocytes ablates CaMKII-mediated ICa facilitation, demonstrating that phosphorylation of b2a at Thr498 modulates native Ca2+ channels. In addition, I showed that binding requires CaMKII activation but phosphorylation at Thr498 inhibits binding. The b2a subunit also modulates CaMKII activity and enhances CaMKII autophosphorylation at a site other than Thr287 or Thr305/306. Analysis of the primary sequences of the four b isoforms reveal that the CaMKII binding/regulatory site is conserved in b1b but not in b3 nor b4 and CaMKII was shown to interact with b1b in a similar manner as b2a. Taken together these findings reveal a novel molecular mechanism for dynamic targeting of CaMKII to LTCCs and facilitating ICa that may modulate Ca2+ entry in diverse cell types co-expressing CaMKII and the b2a subunit. Future work based on these findings may identify a potential pharmacological target for the treatment of heart disease or other pathological conditions involving disrupted Ca2+ homeostasis.

The bed nucleus of the stria terminalis (BNST) is a region of the brain critical in mediating behavioral and physiological responses to stress and anxiety. Stress is a major cause of relapse for drug addicts attempting to abstain from drugs of abuse. The BNST receives a dense projection of the stress hormone norepinephrine (NE) from the nucleus tractus solitarus, and this projection is critical in stress-induced relapse to drug-seeking in rodent models. NE acts via adrenergic receptors (AR), including the alpha2A-AR subtype of the alpha2-ARs. This thesis describes work utilizing electrophysiology and immunohistochemistry approaches to characterize the function and localization of the alpha2A-AR in the dorsal BNST. We provide evidence supporting a heterosynaptic localization of the alpha2A-AR on presynaptic glutamate terminals where it functions to regulate glutamate release. We also demonstrate that activation of alpha2-ARs modulates GABAergic transmission in the dorsal BNST.

Adenoviruses (Ads) are a promising tool for gene and vaccine delivery. Though known for over 50 years, details about Ad structure and intermediates of its lifecycle remain elusive. CryoEM, a technique suitable for studying this large non-enveloped virus (>900Å in diameter), has increased our understanding of Ad structure, in particular of the components for which we do not have atomic resolutions structures. The cryoEM structure of a temperature-sensitive mutant, Ad2ts1, was determined to structurally characterize this intermediate in the Ad maturation process. Ad2ts1 fails to incorporate the viral protease and contains the precursor forms of multiple structural proteins when produced at non-permissive temperatures. It also has a cell entry defect and is defective in endosomal escape. The 10.5Å resolution cryoEM structure of Ad2ts1 shows that the core of the virus remains connected to the capsid via preproteins IIIa and VI, which are normally cleaved by the protease. In a second study, a cryoEM structure of an Ad vector, Ad35f, complexed with human ¦Á-defensin HD5, was determined to visualize the binding sites for defensin on the Ad capsid and to characterize the mechanism of neutralization. HD5 binds at negatively charged regions of the hexon, penton base, and fiber. Based on sequence analysis and information on which Ad types are neutralized by HD5, a critical binding site for neutralization is proposed, comprising 4 polar and negatively charged residues in the fiber N-terminal region and additional residues in the RGD-loop of the penton base. The mechanism of defensin neutralization may involve bridging the fiber and penton base and blocking programmed disassembly. These cryoEM studies highlight the importance of the exquisitely timed disassembly of the virion and release of viral proteins during cell entry.

This dissertation focused on effects of the biological mediators, nitric oxide (NO) and serotonin in the regulation of net hepatic glucose uptake (NHGU). This work demonstrated that NHGU was suppressed by an increase in hepatic NO or cGMP, resulting from intraportal infusion of an NO donor SIN-1 or a cGMP analog 8-Br-cGMP, respectively. Conversely, NHGU was enhanced by the decrease in hepatic cGMP resulting from the intraportal infusion of a soluble guanylate cyclase inhibitor ODQ. These data raise the possibility that under basal conditions, there is an inhibitory tone due to hepatic NO/cGMP that restrains NHGU, and that this inhibitory signal is removed in response to portal glucose delivery thereby enhancing NHGU. In addition, this work showed that NHGU was enhanced by the increase in hepatic serotonin which resulted from intraportal infusion of the selective serotonin reuptake inhibitor escitalopram. This raises the possibility that the serotonergic input to the liver could increase with portal glucose delivery thus explaining part of its ability to increase NHGU. Taken together, these data give rise to the possibility that portal glucose delivery results in removal of an inhibitory signal (NO) and the simultaneous augmentation of a stimulatory signal (serotonin), thereby augmenting glucose uptake by the liver. The studies described in this dissertation have a number of important implications. First, the data provide insight into mechanisms which can regulate NHGU in vivo. Second, they suggest a possible mechanism by which the portal glucose signal may work. Third, they suggest targets for pharmaceutical consideration as a means of correcting postprandial hyperglycemia in individuals with diabetes.

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.