The goal of my dissertation project was to characterize interactions between the transactivator Rap1 and Taf subunits of the TFIID complex. This experimental problem falls under a larger umbrella of gene regulation, since TFIID makes widespread contributions to Pol II transcription. The precise mechanisms underlying TFIID function remain obscure. A major clue to orient dissection of TFIID function is that transactivator proteins are known to directly interact with Tafs. However, there is little information on the physiological relevance of these interactions much less about precise effects on TFIID activity; this relates to difficulties in undertaking a multi-faceted experimental investigation in the metazoan organisms where transactivator-Taf interactions were first discovered. No such limitations exist in the bakers yeast Saccharomyces cerevisiae, although at the time our studies were initiated the best characterized yeast transactivators were known to function by TFIID-independent mechanisms. A breakthrough occurred when it was shown that the yeast Rap1 activator and TFIID physically occupy and regulate common target genes, and that there exist regulatory interactions between these factors. Subsequently our lab identified and characterized physical interactions between Rap1 and TFIID, studies that I participated in. My initial involvement grew into this dissertation. To narrow my focus, I identified three Taf proteins that Rap1 interacts with. These three Tafs co-localize in the three-dimensional TFIID structure, an observation that persuaded me to consider that each Rap1-Taf interaction is physiologically relevant. To narrow my focus further, I defined the Rap1-binding domains within each Taf. I found that two of the three Taf domains are required for cellular viability, and that amino acid substitutions within either domain confer reduced cellular growth. Such cells exhibit deficiencies in transcription of most genes co-regulated by Rap1 and TFIID, but not at the expense of Taf or TFIID stability. Consistent with compromised Rap1-TFIID interaction, we were able to show that Tafs containing amino acid substitutions bind Rap1 with reduced affinity. I obtained evidence regarding specific Rap1 domains involved in Taf interaction. Finally, I conducted a molecular dissection of the Taf4 Rap1 binding domain, and identified two essential regions at amino acid resolution.

Re-uptake of neurotransmitters through membrane-bound transport proteins is the primary mechanism of terminating synaptic transmission in the central nervous system. Biogenic monoamine transporters couple the movement of substrate to the pre-existing Na+ gradient that provides the thermodynamic driving force for translocation. However, the conspicuous absence of high resolution structural details precludes a fundamental understanding of the molecular basis for energy coupling to the conformational motion that underlies the transport process. The crystal structure of LeuT, a bacterial amino acid transporter homolog, provided the first molecular glimpse into the architecture of these transporters. Site-directed spin labeling and EPR spectroscopy was used to characterize the structural features of conformational intermediates sampled by LeuT under biochemical conditions that mimic distinct steps of a transport cycle. Spin labels reported local and global conformational changes on the extracellular side of LeuT, showing that ion binding produces an outward-facing state that exposes the substrate permeation pathway. In contrast, substrate binding reverses this effect and establishes an occluded conformation. These results, combined with molecular dynamics simulations and functional analysis, provide a unique dynamic perspective on an emerging model of Na+-coupled transport.

The goal of this dissertation was to elucidate the metabolic and hepatocellular consequences associated with chronic consumption of a high-fat, high-fructose diet (HFFD), focusing on perturbations in the regulation of HGU and glycogen synthesis (GSYN) by hyperglycemia (HG), hyperinsulinemia (HI), and portal vein glucose (PoG) delivery. We demonstrated that consumption of a HFFD results in impaired glucose tolerance after 4 weeks of feeding, and renders the liver incapable of switching from net glucose output to uptake despite the presence of HI, HG, and PoG delivery. These findings were replicated in a physiologic mixed-meal setting, in which HFFD-fed dogs exhibited excessive postprandial hyperglycemia in association with accelerated gastric emptying and glucose absorption, impaired suppression of lipolysis, and diminished net HGU. These data prompted us to investigate the molecular explanation for impaired HGU associated with HFFD feeding. In normal dogs, PoG delivery in the presence of HI and HG triggered a coordinated cellular response involving an increase in the activity of hepatic glucokinase (GK) and glycogen synthase (GS), which was associated with further augmentation in HGU and GSYN in vivo. In contrast, 4 weeks of HFFD feeding resulted in biochemical insulin resistance, a marked decrease in GK protein content, and loss of the stimulatory effects of PoG delivery on GK and GS activity. These mechanistic defects correlated with diminished HGU and GSYN in vivo. Finally, we determined that both high dietary fat and fructose (in isoenergetic quantities) impair HGU, GSYN, and GK activity, but the defects were significantly greater in high-fructose-fed than in high-fat-fed dogs. In fact, a selective increase in dietary fructose recapitulated nearly all of the metabolic and cellular defects evident in the combination HFFD group. Altogether, our findings suggest that impaired HGU is one of the early metabolic consequences associated with glucose intolerance induced by consumption of a Western diet, and raise the possibility that nutritional modulation of hepatic GK might be causally linked to impaired regulation of HGU in the early stages of diabetes development.

Recent genome wide association (GWA) studies have linked single nucleotide polymorphisms (SNPs) in the G6PC2 gene with elevated fasting plasma glucose (FPG) and in the SLC30A8 gene with altered susceptibility to type 2 diabetes. To demonstrate that changes in G6PC2 and SLC30A8 expression affect FPG and T2D risk, respectively, I characterized the effect of global deletion of G6pc2 and Slc30a8 in mice.<p>G6PC2 is expressed in insulin producing ß-cells and encodes a glucose-6-phosphatase catalytic subunit. My studies demonstrated that G6pc2 is an inhibitory component of the ß-cell glucose sensor, acting in a futile cycle to oppose glucokinase and modulate the S0.5 of glucose-stimulated insulin secretion (GSIS). I also found that sequestration of calcium into the ER, an important event in GSIS, is impaired in the absence of G6pc2, indicating that G6pc2 plays dual roles in the regulation of GSIS. These observations are consistent with human GWA study data which revealed that SNPs within the G6PC2 gene are not only associated with variations in fasting glycemia but also a reduction in insulin secretion during glucose tolerance tests.<p>The SLC30A8 gene is expressed in islets and encodes the zinc transporter ZnT-8. On a C57BL/6J x 129SvEV genetic background, I demonstrated that Slc30a8 KO mice have reduced fasting plasma insulin levels and islets isolated from these mice have impaired GSIS. Slc30a8 KO mice displayed no differences in fasting blood glucose levels or in glucose tolerance. On the pure C57BL/6J genetic background, however, female, but not male, Slc30a8 KO mice displayed reduced fasting plasma insulin levels with no change in fasting blood glucose or GSIS from isolated islets. These observations demonstrates that both 129SvEv-specific modifier genes and gender can modulate the impact of Slc30a8 deletion,. My data therefore suggest that, despite the marked reduction of zinc in Slc30a8 KO mouse islets, the absence of ZnT-8 does not have a substantial impact on normal mouse physiology. In contrast to my studies on G6pc2, my data on Slc30a8 do not provide strong support for the GWA study results suggesting a connection between this gene and T2D risk.

This project and the experiments discussed in this document sought to contribute a functional basis for SNPs within G6PC2 significantly associated with variations in fasting blood glucose by genome-wide association study. Through a combination of binding, reporter gene expression, and in vitro minigene splicing analyses, the roles of a number of SNPs were examined, including rs573225, rs13431652, rs2232316, and rs560887. It was demonstrated that rs13431652 and rs2232316 affect binding of NF-Y and Foxa2, respectively, with changes seen in fusion gene assays correlating these data with the genetic associations of the GWAS. Variations in Foxa2 binding were also observed with allelic variation at rs573225, although fusion gene analysis of this SNP failed to support the binding and genetic data, as well as the function of G6PC2. Because of this inconsistency, future utilization of cell lines and transient transfection for this nature of experiment must be heavily scrutinized. Additionally, splicing variation of G6PC2 exons, based on allelic variation at a number of SNPs, was assessed. By utilizing a minigene plasmid capable of forming a spliced RNA product in vitro, it was found that rs560887 significantly affects splicing of exon 4. The magnitude of this result was found to be based in part on the non-consensus 5 exon 4 splice junction present in humans. These experiments as a whole serve to corroborate and lend a functional basis to the SNPs of G6PC2 associated with variations in fasting blood glucose.

The ability to learn, memorize and recall information is a prerequisite for the full and comprehensive life of any individual. Understanding molecular mechanisms underlying memory is fundamental to neuroscience research. Excitatory glutamatergic synapses are the sites of changes in the strength of neuronal connectivity that are believed to underlie memory. Synaptic transmission is often modulated by calcium signals through N-methyl D-Aspartate-type glutamate receptors (NMDARs) and/or voltage-gated Ca2+ channels. These signals are detected by calmodulin (CaM), which regulates effector proteins such as calcium-calmodulin-dependent protein kinase II (CaMKII). Activation of CaMKII elicits multiple responses at excitatory synapses. Neuronal postsynaptic densities contain several CaMKII-associated proteins, such as NMDAR GluN2B subunits, α-actinin, and densin, that may collaborate to dynamically regulate CaMKII targeting and/or activity during synaptic activation. The CaMKII holoenzyme can simultaneously interact with GluN2B, densin and α-actinin in vitro and the complex can be isolated from mouse brains. My dissertation has explored the roles of α-actinin and densin in modulating the localization and/or activity of CaMKII. I showed that a novel domain in densin is a potent CaMKII inhibitor when using GluA1 glutamate receptor subunits as the substrate, but is ineffective when using GluN2B as the substrate, both in vitro and in cells. I also found that α-actinin mimics CaM in binding to the CaMKII regulatory domain and targets CaMKIIα to F-actin in cells. The interaction with α-actinin and CaM is differentially regulated by CaMKII autophosphorylation in the regulatory domain. In vitro, α-actinin inhibits Ca2+/CaM-dependent phosphorylation of certain substrates by CaMKII, but can activate phosphorylation of certain substrates. Similarly, in intact cells, α-actinin inhibits CaMKII phosphorylation of glutamate receptor GluA1 subunits, but stabilizes CaMKII association with GluN2B-containing NMDARs and enhances phosphorylation of GluN2B. Taken together these data show that α-actinin and densin can collaborate to inhibit the phosphorylation of GluA1-containing AMPARs, but precisely target CaMKII to phosphorylate GluN2B-containing NMDARs. We postulate that these interactions fine-tune the downstream actions of CaMKII in response to synaptic activity.

Obesity is closely linked to many metabolic diseases such as insulin resistance (IR), type 2 diabetes mellitus and cardiovascular disease. In the past decade, it has been established that immune cells are recruited to adipose tissue (AT), triggering an inflammatory response characterized by abnormal cytokine production and activation of inflammatory signaling pathways that are temporally associated with IR. Nonetheless, there is a gap in our understanding of what triggers and regulates this inflammatory response. During the completion of this dissertation I investigated several factors involved with AT dysfunction, inflammation and IR during obesity: the leptin receptor (LepR), CC-chemokine receptor 2 (CCR2) and complement factor 5 (C5). First, I showed that hematopoeitic LepR deficiency does not affect macrophage recruitment to AT or insulin sensitivity during high fat diet induced obesity. Second, I discovered the aberrant accumulation of eosinophils in the AT of CCR2 deficient mice and showed that these are important in regulating macrophage polarization, AT inflammation and systemic insulin sensitivity. Third, I found that C5 deficiency leads to an inflammation-independent downregulation of the insulin receptor in the liver, AT and muscle, promoting severe systemic IR. Overall, this dissertation made important contributions to the field of immunometabolism by adding to our current knowledge of macrophage recruitment and polarization, eosinophil function in metabolism, and the novel concept of an inflammation-independent modulation of glucose metabolism by the immune system. Approved: Professor Alyssa H. Hasty

Metabotropic glutamate receptors (mGluRs) are important modulators of excitatory transmission throughout the central nervous system, and have been implicated in a variety of neurological disorders. This work focuses primarily on two mGluR subtypes, mGluR5 and mGluR8, both of which act to depress excitatory transmission in the bed nucleus of the stria terminalis (BNST). The BNST is an integral component of the brains stress/anxiety and addiction circuitry, and excitatory transmission in this region is thought to be recruited by both in vivo stress and in vivo drug exposure. <p> The first part of this thesis focuses on mGluR5, the function of which is altered by in vivo cocaine. Using biochemical techniques, I characterized mGluR5 activation of the ERK/MAPK cascade and examined several potential mechanisms by which cocaine could regulate mGluR5 function in vivo. I found no change in gross surface levels of mGluR5 in the BNST after in vivo cocaine and that mGluR5 antagonism failed to block cocaine-induced ERK activation. This suggests cocaine-induced disruption of mGluR5 function is not targeting the receptor itself, but rather occurs upstream of mGluR5-induced ERK activation. Thus, the mechanism of cocaine-induced disruption of mGluR5 function remains unknown. <p> The second half of this thesis focuses on mGluR8, unique among mGluR subtypes for its distinct expression patterns. Mice lacking mGluR8 exhibit a naïve anxiety phenotype, suggesting this receptor may function as a brake to prevent activation of anxiety circuitry. Using electrophysiology, I utilized converging pharmacological and genetic approaches to identify an important role for mGluR8 in regulating excitatory transmission in the BNST. Furthermore, I found that mGluR8 function is regulated by adrenergic receptor signaling in the BNST, and is disrupted by in vivo stress exposure. Overall, this thesis highlights the importance of the BNST in stress and addiction circuitry, and identifies mGluR8 as a critical regulator of excitatory transmission in this region that is selectively disrupted by noradrenergic signaling and in vivo stress.

Diet-induced obesity (DIO) is associated with low-grade hypothalamic inflammation, which contributes to insulin and leptin resistance leading to impaired energy balance regulation and obesity. Diets rich in monounsaturated fat reduce inflammation, improve insulin sensitivity and are less obesogenic than those high in saturated fat. The nuclear receptor Peroxisome proliferator-activated receptor delta (PPARδ) regulates the expression of genes involved in metabolism and inflammation in response to unsaturated fatty acids. This work tested the hypothesis that PPARδ functions as a fatty acid sensor which mediates the protective effects of dietary monounsaturated fats on DIO associated inflammation and insulin resistance in hypothalamic neurons. We began our investigation by feeding rats eucaloric high-fat diets enriched in saturated or monounsaturated fat. A clonal hypothalamic neuronal cell line was also treated with combinations of the saturated fatty acid palmitate or monounsaturated fatty acid oleate and with the PPARδ agonist GW0742 or antagonist GSK0660. Dietary or exogenous exposure to palmitate increased mRNA expression of the inflammatory cytokine interleukin-6 (IL-6). Activation of PPARδ in vivo by feeding rats an oleate-enriched diet or in vitro by oleate or GW0742 reduced expression of IL-6. These anti-inflammatory effects were reversed in neuronal cells exposed to physiologically relevant concentrations of palmitate and oleate in the presence of GSK0660, which also reduced insulin activation of Akt. Cre-LoxP technology was used to investigate the role of neuronal PPARδ in energy balance and DIO. On a chow diet, knockout (KO) mice have increased fat mass, despite reduced body weight and lean mass. Elevated hypothalamic inflammation was accompanied by leptin resistance and abnormal feeding and neuroendocrine responses to fasting. KO mice gained significantly more fat mass on a high-fat diet, but were resistant to diet-induced elevations in CNS inflammation and lipid accumulation. Gene expression analysis indicated increased expression of genes involved in fatty acid oxidation and increased expression/compensation by other PPARs. Collectively, this work has identified PPARδ as a protective fatty acid sensor, and revealed a role for neuronal PPARδ in the regulation of body composition, hypothalamic gene expression and feeding responses.

Pluripotent stem cells, because of their ability to differentiate into any cell type, have been widely advocated as a means of producing a nearly unlimited source of new insulin-producing β cells for the treatment of diabetic diseases. However, while there has been remarkable progress in learning how to direct the differentiation of human embryonic stem (hES) cells towards pancreatic endocrine cell fates, insulin-expressing cells made in this manner are often polyhormonal and lack a normal response to glucose, thereby suggesting a need for a deeper understanding of the gene regulatory networks that are established in a stepwise manner during pancreas development. My thesis studies explored three main topics, each of which holds potential for the development of improved hES cell directed pancreatic differentiation protocols and the discovery of genes that may specifically affect β cell development. First, we used mice that contained a fluorescent reporter allele and fluorescence-activated cell sorting (FACS) to isolate several discrete pancreatic cell populations which were then analyzed using whole transcriptome sequencing (RNA-Seq). By doing so, we were able to examine the genetic requirement and temporal changes of cells expressing pancreas specific transcription factor 1a (Ptf1a), a marker of the pancreatic multipotent progenitor cells (MPCs) and of acinar-specified cells, during pancreas development. By comparing the transcriptional profiles, we identified five gene clusters, each of which provides insights into the dynamics of gene expression during specific aspects of pancreas development. Second, my studies revealed that Nephrocan, an inhibitor of the TGFβ signaling pathway, was expressed in pancreatic MPCs. Thus, to explore the role of Nepn further, we generated mice containing a single copy insertion of a Nepn-Cherry transgene. Finally, to facilitate the combinatorial sorting of Pdx1- and Ptf1a-expressing cells during early pancreas development, we generated a mouse line expressing a cyan fluorescent protein under control of the endogenous pancreatic and duodenal homeobox 1 (Pdx1) gene. The research that I have performed is part of a larger project focused on generating and characterizing a series of high quality transcriptional profiles representing key stages in the generation of pancreatic endocrine cells that occur naturally in the mouse. We anticipate that further analysis of the datasets I have generated for specific developmental stages, in combination with similarly generated datasets at other developmental stages, will facilitate identification of signaling pathways and gene clusters essential for formation of functional pancreatic β cells in the mouse, thereby stimulating new hypotheses for identifying pro-β cell signals necessary to direct the differentiation of pluripotent stem cells into pancreatic β cells.