P&F Awards

Sheila Collins, PhD

Biochemistry of Metabolic Disease

Modulating Gbg -SNARE interaction for adipose tissue metabolism and energy expenditure. 

Obesity  is  now  an  epidemic  in  the  United  States  –  more  than  40%  of  the  population obese  or  severely obese (2016 data from CDC). The associated risk for type 2 diabetes, cardiovascular disease, hypertension, and  certain  cancers  continue  to  escalate.    Given  that this  epidemic  is  driven  by  an  overall  positive  energy balance,  and  since  current pharmacological  treatments  for  obesity  are  largely  ineffective,  agents  acting through peripheral mechanisms to increase energy expenditure (EE) should be particularly valuable. We have discovered  a  mechanism  that  is  unexpectedly  effective  at  increasing  energy expenditure  (EE)  and improving glucose homeostasis.  Disabling the GBy-mediated inhibition of hormone and neurotransmitter secretion  through  its  interaction  with  the  exocytotic  fusion machinery  has  led  to  metabolic  phenotypes  of enhanced  insulin  action,  protection  against diet-induced  obesity  (DIO)  despite  similar  food  intake,  and increased brown adipose tissue (BAT) thermogenesis and ‘beiging’/’browning’ in adipose tissue.  Our long- term  goal  is  to understand  the  signaling  mechanisms  that  control  body  fat  metabolism  and  its consequences for glucose/insulin homeostasis and cardiovascular disease.  

Danielle Dean, PhD

Diabetes, Endocrinology, & Metabolism
Molecular Physiology and Biophysics

Glucagon Resistance in Obesity 

Glucagon and its partner insulin are dually linked in both their secretion from islet cells and action in the liver. Glucagon signaling increases hepatic glucose output, and hyperglucagonemia is partly responsible for the hyperglycemia in diabetes making glucagon an attractive target for therapeutic intervention. Interrupting glucagon signaling lowers blood glucose, but also results hyperglucagonemia and alpha cell hyperplasia. Our investigation of the mechanism for alpha cell proliferation led to the description describe a conserved liver-
alpha cell axis where glucagon is a critical regulator of amino acid homeostasis. In return, amino acids regulate alpha cell function and proliferation. New evidence suggests that dysfunction of the axis in humans may result in the hyperglucagonemia observed in diabetes. This proposal outlines important but often overlooked roles for glucagon that extend beyond glycemia and investigating its role in ureagenesis/amino acid homeostasis. We propose that hyperaminoacidemia observed in the obese and diabetic states are due to dysregulation of the 
liver-alpha cell axis. We will leverage transcriptomics from liver of obese mice with mouse clamping studies to understand if glucagon resistance is a feature of the obese liver and will form the basis for future funding applications.  

Dawn Newcomb, PhD

Allergy, Pulmonary, and Critical Care Medicine
Pathology, Microbiology and Immunology

Obesity and estrogen signaling upregulate Th17 cell metabolism in asthma 

Obese women have the highest prevalence of severe, uncontrolled asthma leading to increased asthma- related morbidity, exacerbations, and health care costs.1 New therapeutic options are critically needed for patients, particularly obese women, with severe asthma. Asthma is not a uniform disease with mild to severe phenotypes that is driven by increase airway inflammation, mucus production, and airway hyperreactivity. Milder phenotypes of asthma have increased airway eosinophils and CD4+ Th2 cell cytokine production where more severe phenotypes of asthma, that do not respond well to corticosteroids, have increased airway neutrophils and IL17-producing T cells (Th17). T cell metabolism plays a critical role to in T cell differentiation and inflammation, thereby influencing airway inflammation in asthma. Dr. Rathmell’s laboratory (co-I on this application) showed glutamine metabolism is critical for Th17 cell differentiation and allergen-induced airway inflammation in a mouse model. Glutamine is metabolized to glutamate by glutaminase (GLS), and GLS inhibition with CB839 impaired development of Th17 cells and protected against lung inflammation in a mouse model of neutrophilic asthma. Preliminary data for this application showed 1) patients with severe asthma have increased Th17 cells and glutamine metabolism compared to healthy controls, 2) Women with severe asthma have increased Th17 cells compared to men with severe asthma, 3) high fat diet (HFD, 45%) fed female, but not male, mice had increased frequencies of Th17 cells, and 4) 17β-estrogen signaling through estrogen receptor alpha (ER-α) increased Th17 cell differentiation, IL-17A production, and Th17 cell metabolism. However, it remains unclear if ER-α signaling and obesity alter T cell glutamine metabolism for preferential differentiation of Th17 cells and IL-17A production.  Based on these data, we hypothesize that obesity and ER-α signaling increase glutamine metabolism in activated T cells, leading to increased Th17 cell differentiation and IL-17A production in severe asthma. To test our hypothesis, we have assembled an impressive team of experts and utilize many of Vanderbilt University Medical Center’s core facilities and propose the following aims:

Aim 1: Test if T cells from healthy and severe asthma human donors that are normal weight or obese have different regulation of glutamine metabolism that may be targeted.  
Aim 2: Determine if ER-α signaling enhances glutamine metabolism in T cells in obese female mice.

Todd Graham, Ph.D.

Biological Sciences

Influence of Atp10a and Atp10d on diet induced obesity and insulin resistance
 

Nearly 40% of US adults and 19% of US children are clinically obese with Hispanic and non-Hispanic black populations being particularly overrepresented. This growing obesity epidemic leads to increased incidences of type 2 diabetes, heart disease, stroke and some cancers. At least 10% of the US population has diabetes with most of these cases being type 2 diabetes. In addition to the personal trauma associated with obesity-related diseases, annual costs of treatment are estimated to be $147 billion per year in the US (2008 dollars) (https://www.cdc.gov/obesity; https://www.cdc.gov/diabetes). It is essential to gain a better understanding of underlying genetic risk factors for obesity and type 2 diabetes in order to manage and ultimately resolve this major public health problem. ATP10A and ATP10D have been linked to diet-induced obesity and insulin resistance in mice and humans, but how these type IV P-type ATPases (P4-ATPases) contribute to metabolic disease is unknown. Recent studies indicate ATP10A and ATP10D transport specific sphingolipid species across the plasma membrane of cultured cells. However, the physiological consequences of this lipid transport activity are poorly understood. For example, existing mouse models are inadequate to determine if Atp10a/10d deficiency is sufficient to cause insulin resistance and dyslipidemia in mice fed a high fat diet. Therefore, new murine models bearing precise deletions or mutations have been generated. The current pilot and feasibility study proposes to test whether these Atp10a and Atp10d deficient mice display diet-induced obesity, insulin resistance and dyslipidemia (perturbations on serum lipid profiles) when fed a high fat diet.

Hassane S. Mchaourab, Ph.D.

Molecular Physiology and Biophysics

Biophysical Analysis of G6PC1, a Key Factor Driving Elevated Hepatic Glucose Production in Diabetes 

Glucose-6-phosphatase is a multi-component enzyme system located in the endoplasmic reticulu that plays a critical role in maintaining interprandial blood glucose homeostasis. Mediating the terminal ste of gluconeogenesis and glycogenolysis in the liver, the membrane-bound catalytic subunit, G6PC1, work in concert with accessory transport proteins to catalyze the hydrolysis of glucose-6-phosphate to glucos and inorganic phosphate. Previous studies have suggested that dysregulation of G6PC1 gene expressio contributes to elevated hepatic glucose production (HGP) associated with diabetes. Furthermore, mutations in G6PC1 that compromise activity lead to severe hypoglycemia, forming the clinical basis of glycogen storage disease (GSD). Importantly, the lack of high-resolution structural models precludes a complet understanding of G6PC1 function and impairment. The major bottleneck for a detailed structural analysis of G6PC1 is the absence of efficient heterologous expression and purification strategies that isolate the enzyme in a functional form. This proposal describes logical, time-tested approaches to express and purif G6PC1 for functional and biophysical experiments, which sets the stage for detailed structural analysis by crystallography and/or cryo-electron microscopy. The methodology employs a small-scale screenin approach utilizing the highly sensitive technique of fluorescence detection size exclusion chromatograph to rapidly identify optimal gene constructs and expression conditions, which will inform large-scal expression and purification protocols from insect cells to obtain milligram quantities of wild type and mutan G6PC1. Global structural properties of purified G6PC1 determined from a suite of biophysical tools will be correlated with in vitro hydrolase activity. The results of this work will support progress toward a detailed description of the structural basis of G6PC1 activity and the relationship to disease-associated mutations.
 

Shelia Collins, Ph.D.

Cardiovascular Medicine

Augmentation of natriuretic peptide signaling: developing a screen for the inhibition of a novel receptor heterodimer

The ‘second messenger’ signaling molecule cyclic guanosine monophosphate (cGMP) is a well-established therapeutic target for lowering blood pressure, being exemplified by nitrovasodilators (such as nitroglycerin, isosorbide dinitrate, etc.) and PDE5 inhibitors (such as sildenafil and tadalafil). Nitrovasodilators activate the so-called soluble guanylate cyclases (sGCs), and the resulting cGMP can then activate protein kinase G (PKG). The other important class of blood pressure lowering GCs are the membrane-bound, ‘particulate’ GCs. The major receptor in this class is NP receptor-A (NPRA; also called GC-A). It binds the cardiac peptide hormones atrial natriuretic peptide (ANP1-28) and B-type natriuretic peptide (BNP1-32). Upon ANP/BNP binding to the homodimeric NPRA, the
intracellular domains that form the GC generate cGMP. NPs also bind to NP receptor-C (NPRC), another homodimeric receptor that lacks GC activity; rather, it is referred to as the ‘NP clearance receptor’, because it has been shown to endocytose the NPs, contributing to their removal from circulation. Despite the important physiology of the cardiac NPs, unlike the nitrates and sGCs there are no small molecules which can activate or augment NPRA, and this is a limiting factor in the clinical utilization of this arm of the cGMP producing pathway.

In addition to lowering blood pressure, ANP and BNP are also important regulators of fuel metabolism. They activate
adipocyte lipolysis as well as the amount and activity of brown adipocytes (Collins, 2014), and skeletal muscle fatty acid oxidation (Engeli et al., 2012). We and others have shown that the level of NPRC is dynamically regulated, and the relative ratio of NPRA to NPRC dictates the signaling strength through NPRA (Bordicchia et al., 2012; Kuhn et al., 2004; Wu et al., 2017).

The Scientific Premise of this project is that, contrary to the conventional view of NP receptors functioning as homodimers, these receptor proteins also form heterodimers that cannot produce cGMP. Therefore, this finding represents a paradigm-shifting new mechanism, with clear implications for better understanding the biology of these receptors and screening for compounds that can specifically block heterodimer formation, to thus improve NPRA signaling.

The central hypothesis is that the formation of an NPRA:NPRC heterodimer is a major mechanism by which NPRC inhibits NP-cGMP signaling. This is a somewhat heretical concept because it challenges the ‘competition for ligand’ model, which is the universally accepted mechanism for how NPRC can blunt hormone binding to NPRA and cGMP levels. The objective of this application is to work with the Vanderbilt Institute of Chemical Biology to generate receptor-FRET pairs of NPRA and NPRC to develop an assay that can be used to screen for molecules that will disrupt heterodimer formation, possibly enhancing NPRA activity.
 

Rachel H. Bonami, Ph.D.

Division of Rheumatology & Immunology

Interrogating the Molecular Origins of B Lymphocyte Autoimmunity for Insulin

Patients who present with two or more islet autoantibodies have an 80% likelihood of symptomatic type 1 diabetes (T1D) onset within 20 years. Islet autoantibodies are produced by autoreactive B lymphocytes that defy normal immune regulation to differentiate into antibody-secreting cells. Our preliminary data in T1Dprone non-obese diabetic mice suggest that whereas most islet-reactive B lymphocytes are capable of driving beta cell attack through autoantigen presentation to T cells, only a minority successfully differentiate further into autoantibody-secreting cells. Furthermore, insulin-reactive antibodies predict disease risk, yet little is known about insulin-binding B lymphocytes in human T1D patients. This proposal seeks to interrogate this critical, understudied population of antigen-presenting B lymphocytes in human pre-symptomatic and new-onset T1D subjects to improve our understanding of how the disease process unfolds and facilitate the development of new immune-targeted therapeutic strategies. We hypothesize that B lymphocyte transcriptional and repertoire perturbations exist in the T1D prodrome that correlate with early disease stages. The following aims will test this hypothesis: Aim 1: To transcriptionally identify aberrant B lymphocyte population states in pre-symptomatic T1D patients and correlate immunoglobulin repertoire shifts at the single cell level, and Aim 2: To elucidate how the anti-insulin B lymphocyte repertoire evolves during the pre-symptomatic period ofT1D. The proposed aims will employ cutting-edge technologies possessed by few other institutions to advance the T1D field by identifying BCR repertoire shifts and dysregulated pathways by which B lymphocytes escape normal immune control, as well as by tracking the evolution of anti-insulin responses in pre-symptomatic and new-onset T1D patients. These studies will generate paired, single cell transcriptomic and B cell receptor (BCR) repertoire data amongst pre-symptomatic T1D and islet autoantibody-negative controls to synergistically enhance our understanding of how autoreactive B lymphocytes breach normal immune regulation barriers to enter the circulating repertoire of patients to drive T1D (Aim 1). BCR repertoire data, including V gene usage and mutation analysis, along with antibody isotype and affinity studies will be conducted to capture and characterize insulin-binding B lymphocytes as hybridomas from pre-symptomatic vs. new onset T1D patient PBMC (Aim 2). These studies hold potential to identify new biomarkers of early disease stages and enhance our knowledge about how B lymphocyte islet autoimmunity develops to drive beta cell attack.

Erkan Karakas, Ph.D.

Department of Molecular Physiology & Biophysics

Structural characterization of inositol-1,4,5-triphosphate receptors

Mitochondria are dynamic and complex organelles that are essential for a wide range of metabolic and signaling processes. Mitochondrial dysfunction is strongly associated with a growing number of metabolic and neurodegenerative diseases and cancer. A crucial regulator of mitochondrial functions is mitochondrial Ca2+ influx, which relies on synapse-like contact sites with the endoplasmic reticulum (ER). Sustained Ca2+ transfer into mitochondria at these contact sites is necessary to maintain synthesis of ATP and mitochondrial substrates, whereas excessive or reduced Ca2+ transfer leads to initiation of apoptotic cell death or autophagy, respectively. Recent studies link aberrant Ca2+ signaling at ER-mitochondria contact sites to the onset and progression o many metabolic diseases including diabetes-2 and obesity related illnesses. Despite recent identification of components of the Ca2+ transfer machinery at ER-mitochondria contact sites and their emergence as potential therapeutic targets, the molecular mechanisms underlying regulation of mitochondrial Ca2+ influx is poorly understood, hampering development of tools to fine tune Ca2+ flux in diseased states. One of the long-term goals of my research program is to uncover the molecular mechanisms underlying the activity and regulation of inosito1-1,4,5-triphosphate receptors (IP3Rs), the key regulator of Ca2+ transfer into mitochondria, using cutting edge structural biological methods including X-ray crystallography and cryo-electron microscopy (cryo-EM) together with biophysical, biochemical and functional methods. The aim of this proposal is to i) perform identification and biochemical, pharmacological and structural characterization of IP3R domains that can be expressed as isolated entities, and ii) express and purify intact recombinant IP3R suitable for the subsequent structural and functional analysis. These studies will facilitate the necessary tools to address more specific questions regarding to IP3R activity and regulation and would pave the path for discovery of novel tools to control the receptor activity in diabetes and obesity as well as other diseases where aberrant IP3R activity is involved.

Ethan Lippman, Ph.D.

Department of Chemical & Biomolecular Engineering

Mechanisms of leptin transport across the human blood-brain barrier

Novel strategies are needed to treat obesity and its associated metabolic detriments. This research proposal seeks to clarify the mechanisms by which leptin, an integral hormone that regulates energy utilization and metabolism, is transported through the blood-ˇbrain barrier (BBB) and into the brain where it exerts its primary effects. Paradoxically, obese individuals have increased levels of leptin in the bloodstream, but leptin transport across the BBB is reduced. The mechanisms behind this so-ˇcalled “peripheral leptin resistance” are unknown, mainly because the receptors and transporters that shuttle leptin across the BBB are also unknown. We hypothesize that if this leptin transport machinery is properly characterized, it can be directly manipulated to increase leptin brain uptake in obese individuals, thereby providing a novel route for treating obesity and obesity-ˇrelated behaviors. To identify this leptin transport machinery, we will utilize: (1) robust human brain endothelial cell sources that appropriately mimic BBB behavior, and (2) cutting-ˇedge targeted and high throughput CRISPR techniques. Aim 1 will assess leptin uptake in BBB endothelial cells derived from human induced pluripotent stem cells (iPSCs) after treatment with a library of small molecule agonists. After identification of compounds that increase leptin uptake, RNA sequencing will be conducted to profile upregulated genes that are potentially responsible for this increase. Gene knockouts will then be generated in the iPSC-ˇderived BBB model to validate their role in leptin transport. Aim 2 will assess leptin uptake in an immortalized BBB endothelial cell line after it is transduced with a genome-ˇwide CRISPR activation library, such that every cell overexpresses a single gene. Positive selection and RNA sequencing will be used to determine genes whose upregulation consequently increases leptin uptake. The relevance of each gene for leptin transport will then be confirmed in the iPSC-ˇderived BBB model. The combination of targeted and high throughput assays maximizes the odds of success for identifying novel contributors and regulators of leptin transport into the brain. This work is expected to have an important positive impact on the field of obesity research by providing insight into a central metabolic question that has remained unanswered for over 20 years. Furthermore, outcomes from this research will motivate exciting in vivo studies to validate the identified mechanisms, as well as create immediate opportunities for drug screening campaigns to specifically target the leptin transport machinery.

Maulik Patel, Ph.D.

Department of Biological Sciences

Mechanisms Underlying Metabolic Regulation of Mutant Mitochondrial Genome Dynamics

Mitochondrial dysfunction underlies a variety of metabolic diseases, as well as age-related decline in health. Common sources of mitochondrial dysfunction include mutations in the mitochondrial genome (mtDNA), a small circular chromosome that encodes several genes essential for mitochondrial respiration. Mutant mtDNA (ΔmtDNA) has been implicated in a number of diseases characterized by metabolic dysfunction, including obesity and diabetes. However, ΔmtDNA do not follow the inheritance patterns that are typical of the nuclear genome, making it difficult to predict the risk of developing a ΔmtDNA-associated disease and highlighting the importance of investigating the cellular mechanisms governing the propagation of ΔmtDNA. We have preliminarily found metabolic signals, namely insulin signaling and the ability to metabolize glucose, to be important regulators of ΔmtDNA proliferation in a Caenorhabditis elegans model of mitochondrial disease. Specifically, insulin signaling promotes the proliferation of ΔmtDNA while glycolysis inhibition suppresses it. While these findings are consistent with previous reporting that energy metabolism is an important regulator of mitochondrial networks, the underlying mechanisms are not known. We propose to follow up on these exciting findings by investigating the role of insulin signaling, as well as the utilization of dietary nutrients, on the maintenance and propagation of a pathogenic deletion-bearing mitochondrial genome in the simple animal model, C. elegans. Specifically, our first aim seeks to characterize the downstream mechanisms by which insulin signaling modulates ΔmtDNA levels. Our second aim will characterize the mechanisms underlying the shift in ΔmtDNA levels upon altered utilization of dietary nutrients. This study will yield key insights on the mechanisms governing the propagation of diseasecausing mutations in mtDNA, as well as provide a framework for further research on the potential usage of nutritional and metabolic interventions in combating diseases associated with mitochondrial mutations.

Year 2 Grant Awardees

Rolanda Lister, M.D.

Department of Obstetrics & Gynecology

Carrie A. Grueter, Ph.D.

Department of Anesthesiology

Sean Davies, Ph.D.

Department of Pharmacology

Ayush Giri, M.S., Ph.D.

Department of Obstetrics and Gynecology

Dolly Padovani-Claudio, M.D. Ph.D.

Department of Opthamology & Visual Sciences

Rolanda Lister, M.D.

Department of Obstetrics & Gynecology

I hypothesize that maternal hyperglycemia induces changes in DNA methylation in the developing heart thus increasing the risk of congenital heart defects due to abnormal mRNA expression of cardiac important genes. I have three specific aims to test this hypothesis. Aim 1: To analyze the cardiac phenotypes of embryos born to dams with hyperglycemia compared with pups of euglycemic pregnancies. Aim 2: To identify methylationsensitive DNA loci within developing fetal hearts that are more vulnerable to maternal hyperglycemia using a
high-resolution genome-wide cytosine methylation profiling assay. Aim 3: To juxtapose differential mRNA expression in developing fetal hearts with changes in genome wide DNA methylation.

Research strategy:
Diabetes will be induced in standard 8 week old CD-1 WT female mice with a one time intraperitoneal injection of 150 mg/kg of streptozotocin (STZ). Histological analysis of fetal cardiac morphology will be performed on the hearts of the hyperglycemic and the euglycemic pupps at day E16.5. We will extract the hearts at different timepoints and use genome wide cytosine methylation profiling to delineate the difference between pupps of hyperglycemic mothers versus controls. We will then juxtapose differential mRNA expression. With this information, we can then in the future obtain gene candidates that may be the targets for
intervention.

Expected Results:
We anticipate that maternal diabetes will alter DNA methylation of specific genes related to cardiac development. We believe that by sequencing the entire genome of the extracted DNA after creating our library following the modified HELP-tagging assay and sequence, we will identify candidate genes that are implicated in dysregulation of the methylation patterns that ultimately predispose these embryos to congenitally acquired cardiac lesions.

David Jacobson, Ph.D.

Department of Molecular Physiology & Biophysics

The overall objective of this study is to identify selective activators and inhibitors of the two-pore-domain potassium channel, TALK-1. TALK-1 channels are key regulators of pancreatic -cell electrical excitability, Ca2+ homeostasis and insulin secretion. KCNK16 the gene that codes for TALK-1 is the most abundant K+ channel transcript of the islet and TALK-1 is the most islet-restricted ion channel. Moreover, a mutation in KCNK16 results in neonatal diabetes and a nonsynonymous polymorphism in KCNK16 causes an increased predisposition for type-2 diabetes. However, our understanding of the role(s) of TALK-1 channel in human islets remains obscure due to a lack of specific and potent pharmacology. Therefore, this project will utilize a robust thallium (Tl+) based fluorescent assay in a high throughput screen (HTS) to identify pharmacological probes of the human TALK-1 channel. The assay will be performed on a tetracycline inducible TALK-1 cell line, which was selected for based on its performance in the Tl+ assay. The TALK-1 Tl+ assay was validated with primary screens of two small molecule libraries including the Spectrum Collection (~2320 molecules) and a bioactive lipid library (~928 molecules), which identified a small cohort of activators and inhibitors of TALK-1. The primary screens were utilized to optimize the Tl+ assay for use with the TALK-1 cell line in a large HTS. Building on these preliminary studies, this proposal plans to perform a HTS on the human TALK-1 channel with 35,000 structurally diverse small molecules from the Vanderbilt HTS small molecule library. This will be accomplished using 1. A Tl+ flux based HTS, which will be followed by 2. Secondary assays utilizing Tl+ flux, electrophysiology and Ca2+ imaging to support rapid hit-to-lead progression. This will be followed by assays to determine the mechanism of action of TALK-1 regulation including TALK-1 in single
channel inside out membrane patches as well as inhibition of G-protein-coupled receptor signaling, and finally 3.Molecular regulators of TALK-1 activity identified in this HTS will be utilized to test the influence of TALK-1 channels on human and mouse islet cell electrical activity, Ca2+ homeostasis and insulin secretion.

Anne Kenworthy, Ph.D.

Department of Molecular Physiology & Biophysics

Macroautophagy (hereafter referred to as autophagy) is an intracellular catabolic pathway involved in recycling of cellular components. The normal functioning of a variety of cell types important in both diabetes and obesity, including pancreatic beta cells and adipocytes, depends heavily on autophagy. Thus, it is critically important to understand the mechanisms that underlie this process. MAP1LC3B (LC3) is one of the central proteins involved in autophagy, playing essential roles in both the formation of autophagosomes as well as the capturing cargo for degradation by autophagy. Emerging evidence suggests nuclear forms of LC3 are also important in autophagy. However, the functions of nuclear LC3 are still largely unexplored and little information is currently available regarding LC3’s interacting partners in the nucleus. Work from our group has shown that in the nucleus, LC3 is contained within ~1000 kDa complexes. We also identified an initial list of candidate LC3-interacting proteins using mass spectrometry. These studies have revealed a number of intriguing candidate interacting partners of nuclear LC3, including ribosomal subunits and tubulin. These findings lead us to hypothesize that additional functions of nuclear LC3 remain to be discovered, and that the identification of bona fide nuclear LC3 interacting proteins could provide new insights into how autophagy maintains cellular and organismal homeostasis. Here, we propose to leverage and expand on our initial findings by i) determining how the nuclear LC3 interactome is physiologically regulated and ii) validating key interacting partners of nuclear LC3 and exploring the functional significance of their interactions. These studies will thus set the stage for obtaining external funding for future work focused on identifying new functions of nuclear LC3. Ultimately, these studies should provide a framework that will inform our understanding of how autophagy functions as a general regulator of metabolism and how this pathway can be exploited to help prevent and treat diabetes and obesity.

Nathan C. Bingham, M.D., Ph.D.

Department of Pediatrics, Division of Pediatric Endocrinology

Mitigation of hypothalamic inflammation via ablation of microglial IKKbeta

Rodents fed a high-fat diet (HFD), upregulate proinflammatory cytokines within the mediobasal hypothalamus (MBH), an important center of neuronal control of appetite and metabolism. This metabolically-induced inflammation, or ‘metaflammation,’ contributes to central leptin resistance, increased caloric intake, and obesity. These results run contrary to a large body of work that has identified hypothalamic inflammatory signaling as a mediator of the sickness-induced cachexia response triggered in response to classic inflammatory stimuli such as injury, infection, cancer, or autoimmunity.

As the innate immune cells of the CNS, microglia have been implicated as potential effector cells of both metabolic and classical inflammatory stimuli within the hypothalamus. Here, we propose strategies for defining the role of microglial NF-kB signalling, a key molecular of the microglial inflammatory response, in the development of hypothalamic inflammation. We have developed an inducible Cre-Lox mouse line to ablate NF-kB signaling specifically in microglia. Study of these mice will provide a better understanding of the mechanisms whereby microglia contribute to hypothalamic inflammation and could uncover novel targets for the treatment of diet-induced obesity and cachexia.

Justin M. Gregory, M.D.

Department of Pediatrics, Division of Pediatric Endocrinology

Peripheral Insulin Delivery's Contribution to Insulin Resistance in Type 1 Diabetes

The goal of this pilot and feasibility proposal is to determine the pathophysiologic mechanisms underpinning insulin resistance (IR) in type 1 diabetes (T1DM), a consistent but under-recognized problem in this condition and a major predisposing factor to macrovascular disease, the leading cause of death in these patients. My research will test the hypothesis that IR in T1DM is predominantly a consequence of iatrogenic hyperinsulinemia in the peripheral circulation (as opposed to an effect of chronic hyperglycemia, as is commonly thought). I will test this hypothesis using a novel cross-sectional study design evaluating IR in 3 groups: subjects with T1DM, glucokinase mutations, and non-diabetic controls. I will utilize the

hyperinsulinemic, euglycemic clamp to exploit key metabolic differences between these 3 groups and determine the etiology of T1DM IR at whole-body and tissue-specific levels. These studies will increase our understanding of IR in T1DM and how novel therapeutic approaches could alleviate this obstacle to optimal cardiovascular health for patients who live with this condition.

Carrie A. Grueter, Ph.D.

Department of Anesthesiology

DGAT1 as a central regulator of diet-induced obesity

Evidence indicates an essential role for the central nervous system (CNS), particularly lipid-sensing neurons in the hypothalamus, in the regulation of whole-body energy balance. It is suggested that different classes of lipids are used by lipid-sensing neurons, not as nutrients, but as cellular messengers which relay information regarding whole-body energy status. Even though the enzymes responsible for TG synthesis, acyl-CoA:diacylglycerol acyltransferase-1 and -2 (DGAT-1 and -2), are expressed in the brain and are known to regulate of whole-body EB, their function in the CNS has yet to be investigated. The long-term objective of my research program is to understand the physiological relevance(s) of intracellular TG and how it impacts CNS processes. The overall objectives for this proposal, which will establish the platform for achieving my long-term goal, are 1) to identify the neuroanatomical expression and distribution of Dgat1, 2) to elucidate the impact of central DGAT1 on the regulation of whole-body energy balance. I hypothesize that intracellular TG metabolism in the CNS, mediated by DGAT1, impacts lipid-sensing in the brain and thus regulates of whole-body energy balance. The rationale for this proposal is that identification of specific cell-types and lipid messengers mediated by DGAT1 in the CNS will provide mechanistic insight into how and where intracellular TG metabolism impacts the regulation of whole-body energy balance. These data will open the door for discovery of new therapeutic approaches for the prevention and treatment of obesity, type 2 diabetes and mechanistically related disorders such as depression and anxiety.

Raymond D. Blind, Ph.D.

Department of Medicine

Novel anti-diabetic therapeutics by nuclear receptor competitive displacement

The exogenous plant phospholipid DLPC was recently found to activate the nuclear receptor NR5A2 and have dramatic anti-diabetic effects in the mouse liver. DLPC is thought to activate NR5A2 the same way all nuclear receptors are thought to be activated - as an allosteric switch. However, we recently discovered that NR5A2 can act as scaffod for endogenous phospholipid ligands, and these phospholipids themselves mediate new interactions. This new paradigm suggests that past drug screening platforms that searched for allosteric modulators of NR5A2 were misguided, explaining why those efforts failed. Here, we propose a new type of nuclear receptor screen designed to detect endogenous phospholipid competitive displacement, not

allostery. This screen is ready for anti-diabetic therapeutic development as all the in vivo models, used to validate DLPC in vivo, are ready and await a new DLPC-like molecule. The NR5A2 target is exceptionally well validated, and the novel nature of our screen, based on new mechanistic information, suggests our new screen has an excellent chance to lead to novel & pharmacologically tractable DLPC-like anti-diabetes therapeutics.

David M. Aronoff, M.D.
Associate Professor, Department of Medicine, Division of Infectious Diseases

Gestational diabetes mellitus (GDM) impacts up to 1 in 10 pregnancies in the US and significantly increases the risk of complications, including infant macrosomia, neonatal hypoglycemia, preeclampsia, premature labor, and Cesarean delivery. It also increases the risk of postpartum complications in both mother and child including late onset diabetes and cardiovascular disease. Causal mechanisms explaining how GDM predisposes to these adverse outcomes are poorly defined, but the placenta is increasingly appreciated to be a target organ of diabetes. This proposal addresses a potential cause of exaggerated placental inflammation in GDM related to increased iron accumulation by macrophages within this vital organ. Placental macrophages (PMs) play an important role in normal placental development and govern maternal-fetal tolerance and tissue inflammatory tone. Notably, PM numbers increase and they take on a more pro-inflammatory phenotype in GDM. We recently found that healthy human PMs accumulate iron through undefined mechanisms. Using a mouse model of GDM, we also found increased tissue iron staining and greater macrophage infiltration in diseased placentae, along with increased fetal resorption (fewer live pups). A potential explanation for increased placental iron in GDM is the previously described higher expression of the hemoglobin-haptoglobin receptor (CD163) by PMs affected by GDM. Thus, our central hypothesis is that PMs exhibit an exaggerated proinflammatory phenotype in GDM, related to an increased accumulation of intracellular iron and enhanced CD163 expression.

This proposal brings together a new multidisciplinary team of investigators to test our hypothesis through three specific aims: In Aim 1 we will characterize the cellular immunophenotype of the mouse placenta in GDM, with a special focus on PMs, CD163 expression, and intracellular iron storage. Immunophenotyping studies in a robust mouse model of GDM will assess leukocyte phenotypes including macrophages (both M1 and M2 subtypes), CD4+ cells, CD8+ cells, NK cells (NK1.1+ or CD335+), and neutrophils (Neu7/4+). Tissue inflammation will be assessed by histology and flow cytometry and macrophage expression of CD163 and accumulation of iron will be assessed. In Aim 2 we will determine the cellular inflammatory phenotype of human placental tissues affected by GDM using archived tissue specimens linked to clinical meta-data from the electronic medical record, with a special focus on PMs, CD163 expression, and intracellular iron storage. Case and control placentae will be analyzed histologically for inflammation. Macrophage abundance, polarization, and iron accumulation will be determined by specific tissue immunostaining (and iron staining). These studies will help validate a mouse model of GDM using human tissues, while advancing our understanding of the impact that GDM has on placental biology. They will also provide critical preliminary data for externally-supported grants that can expand institutional efforts to prevent and treat GDM.

Leslie J. Crofford, M.D.
Professor, Department of Medicine, Division of Rheumatology

Patients with chronic inflammatory diseases, including rheumatoid arthritis (RA), have an increased prevalence of obesity and metabolic syndrome (MetS).  Little is known about the phenotype of adipose tissue (AT) in patients with RA.  It is also unknown if experimental inflammatory arthritis stimulates changes in AT and metabolic derangement.  An important characteristic of inflammation in both arthritis and AT is activation of the prostaglandin (PG) biosynthetic pathway characterized by markedly increased expression of cyclooxygenase -2 and microsomal PGE synthase-1 (mPGES-1).  In inflamed tissues, expression of PGE2 increases disproportionately to other PGs because of coordinated regulation of these two biosynthetic enzymes.  When mPGES-1 is genetically deleted in mice (KO), PG synthesis is shunted toward other terminal PG in a cell and tissue specific manner.  In adipose tissues, shunting is towards alternate species that may lead to changes in AT phenotype, specifically in the capacity to develop brown-in-white or brite adipocytes.  Increased brown/brite adipocyte activity is inversely associated with obesity, age, and type II diabetes.  Our preliminary data demonstrate that mPGES-1 KO mice are resistant to weight gain when being fed a high-fat (HF) diet.  In addition, we showed that mPGES-1 KO mice exhibit markedly increased expression of UCP-1 mRNA, the marker of brite adipocytes, in white AT and increased energy expenditure.  Thus, our preliminary data suggests that mPGES KO mice could be resistant to arthritis-induced MetS due to their ability to promote “browning” of white AT.  In this pilot project, we will test the hypotheses that (1) mPGES-1 deficiency reduces weight gain by stimulating brite adipocyte phenotype during HF feeding, (2) inflammatory arthritis increases AT inflammation and induces MetS, and (3) mPGES-1 deficiency blocks arthritis-associated changes in AT and MetS.  This will be accomplished by determining the effect of mPGES-1 deficiency on differentiation to the brite adipocyte phenotype, obesity, and energy metabolism during HF feeding and determining if a murine model of RA increases inflammation in AT and alters energy metabolism and test whether the AT phenotype in mPGES-1 deficient mice is modulated by arthritis.

Takamune Takahashi, M.D., Ph.D.
Associate Professor, Department of Medicine, Division of Nephrology

Diabetic nephropathy (DN) is a major diabetic complication that determines the morbidity and mortality of the diabetic patients. Although clinical indicators or risk factors of this disease have been described, the currently available tests do not reliably assess its severity or progression in individual patients, making it difficult to do the targeted and intensified treatment to high-risk patients. Renal fibrosis is a hallmark of progressive DN; therefore, it is critical to evaluate the presence and extent of renal fibrosis in the diabetic kidney to treat the patients as well as to predict their long-term outcome. However, the current clinical tests lack the sensitivity and specificity to measure renal fibrosis in diabetic kidney. Although renal biopsy can diagnose fibrosis, it is invasive and prone to sampling errors, and does not reliably measure renal fibrosis in the affected kidney. Thus, a non-invasive test that better evaluate renal fibrosis would greatly improve the assessment of this disease. In recent decade, a variety of magnetic resonance imaging (MRI) methods have been developed and applied to human disease including cancer and brain disorders. These techniques have enabled us to assess the pathological changes in disease organ at molecular and cellular levels. Magnetization transfer (MT) imaging is a MRI technique that evaluates large and immobile macromolecules distributed within the tissue and could provide a means to evaluate the pathological events that are accompanied by the changes of macromolecular components, such as fibrosis and apoptosis. However, this method is poorly applied to kidney disease including DN. Therefore, here we will evaluate the utility of MT imaging in measuring renal fibrosis in diabetic kidney using a mouse model of progressive DN (db/db eNOS -/- mice). The aims of this study are: 1) To optimize and establish the MT protocol for mouse kidney imaging; 2) To examine the correlation between MT data and histological or biochemical measures of renal fibrosis. Thus, this application explores a new MRI test to assess renal fibrosis in DN. Given the fact that this MRI technique can be translated to clinics, the present work should efficiently improve the outcome of the DN patients.

Heidi E. Hamm, Ph.D.
Professor, Department of Pharmacology

Role of a2A adrenergic receptors and Gbg-SNARE interaction in impaired insulin secretion in T2D

One of the earliest and most important pathophysiological signs of type 2 diabetes (T2D) is impaired insulin release. A number of therapies used in the clinic, such as GLP1- agonists and DPP-4 inhibitors, act to enhance insulin release from the beta cell. Gi/o-coupled G-protein coupled receptors (GPCRs) inhibit insulin release. One mechanism through which these receptors inhibit insulin release is through liberating G protein βγ subunits. We have demonstrated that Gβγ can directly inhibit exocytosis at a point distal to Ca2+ entry by binding to soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. It has been shown that beta cell α2A adrenergic receptors (α2AAR) profoundly inhibit insulin release by this mechanism. This proposal aims to investigate the role of the α2AAR in the inhibition of insulin release in animal models of T2D. We hypothesize that overactive inhibition of insulin secretion by Gi/o coupled GPCRs is a part of the impaired insulin secretion in the pathogenesis of T2D. In Aim 1, we will test whether α2AARmediated inhibition is enhanced in islets isolated from animal models of T2D. In Aim 2, we will test whether small molecule inhibitors of the Gβγ-SNARE interaction are able to overcome the inhibitory effect of α2AAR agonists upon insulin release. Finally, in Aim 3, we will determine whether the inhibitors synergize with α2AAR antagonists or compounds that enhance insulin secretion such as sulfonylureas, agonists of Gs or Gqcoupled receptors (e.g. GLP-1 agonists). These studies should allow us to evaluate both the role of α2A adrenergic receptor-mediated inhibitory signaling and the Gβγ-SNARE interaction in impaired insulin secretion in T2D.

Jerod S. Denton, Ph.D.
Assistant Professor, Division of Anesthesiology, Department of Medicine

Development of small-molecule Kir4.2 modulators for treatment of type 2 diabetes

Type 2 Diabetes Mellitus (T2DM) imposes a substantial burden on society through increased healthcare costs, loss of productivity, and reduced quality of life. The number of diagnosed T2DM patients is projected to increase dramatically in the coming decades. Therefore, developing improved treatment strategies will be essential to lessen the economic and societal impact of T2DM. KCNJ15, which encodes the inward rectifier potassium (Kir) channel Kir4.2, was recently identified as a T2DM susceptibility gene. Kir4.2 is expressed in glucose-responsive, insulin-secreting beta-cells of the pancreas, where recent studies suggest that its up-regulation in T2DM leads to diminished glucose-induced insulin secretion. Importantly, siRNA-mediated knock-down of Kir4.2 expression in vivo increases insulin secretion and lowers blood glucose in diabetic mice. Taken together, these studies raise important questions regarding the physiology of Kir4.2 in beta cells and suggest the intriguing possibility that Kir4.2 represents a novel drug target for T2DM. There are currently no specific pharmacological modulators of Kir4.2. Therefore, the goal of this proposal is to develop, validate, and implement a high-throughput screening (HTS) assay to enable the discovery of the first small-molecule probes of Kir4.2 function. In Aim 1, the investigators will develop a thallium flux-based fluorescence assay to monitor Kir4.2 activity in a 384-well plate format. The robustness of the assay will be determined by meeting a series of performance benchmarks and running a pilot screen of 3,655 compounds in the Vanderbilt HTS center. In Aim 2, the investigators will perform a 30,000 compound screen (15,000 compounds each in funding years 1 and 2) of the Vanderbilt Institute of Chemical Biology Library. The potency and selectivity of Kir4.2 modulators will be characterized in Aim 3 using a panel of established high-throughput thallium flux assays for 9 different Kir channels. These assays dramatically shorten the time from hit discovery in a primary screen to lead compound identification and optimization. The successful outcome of the proposed work will be the development of pre-clinical tool compounds for exploring the physiology of Kir4.2 in beta cells and its therapeutic potential in T2DM.

Kasey C. Vickers, Ph.D.
Assistant Professor, Medicine and Molecular Physiology & Biophysics

Mechanisms and consequences of HDL microRNA communication in diabetes

We have previously reported that HDL transports and delivers beta-cell specific miRNAs to recipient hepatocytes. Therefore, we aim to determine if beta cells export specific miRNAs to HDL to be delivered to theliver as part of a novel endocrine-like communication network. Furthermore, we aim to determine if this cell-tocellcommunication is altered in hyperglycemia and corrected by Colesevelam treatment, a diabetes drug thathas been found to improve beat cell function and insulin secretion. Results from this pilot and feasibility studywill provide a foundation of basic understanding into a new paradigm of cell communication that likely

contributes to systemic glucose homeostasis.Extracellular RNA and HDL-mediated intercellular communication has tremendous applicability to manydiseases. As new investigator to Vanderbilt, I have a strong interest in expanding my research program toinclude diabetes, and have a significant interest in a research career in diabetes. Nevertheless, myinvestigation into diabetes is just beginning; therefore, this award will provide the necessary funds to obtainsufficient data for individual grant support in the field of diabetes.

Danny G. Winder, Ph.D.
Professor, Molecular Physiology & Biophysics

BNST CRF and maintenance of weight loss

Maintenance of diet-induced weight loss is a major problem in the treatment of type II diabetes. Animal model studies have suggested an important role for corticotropin releasing factor (CRF) neurons in the bed nucleus of the stria terminalis (BNST) in stress-reward interactions, and more recently in feeding behavior. In particular, CRF signaling within the BNST produces anorexia. Recent studies demonstrate that caloric restriction in mice produces a profound decrease in CRF levels in the BNST that persists even after a return to ad libitum feeding.

This may represent the removal of an important brake on stress-induced feeding behavior that contributes to diet relapse. Currently, however, very little is understood regarding the CRF system in the BNST. We have recently successfully utilized a genetic reporter strategy to isolate CRF neurons within the BNST for morphological and electrophysiological analysis. Here, we propose to use optogenetic and fluorescent-reporterbased animal models to determine inputs and outputs of CRF neurons in the BNST, to determine the specific inputs to the BNST that are opposingly regulated by CRF and orexin, and to examine the long-term impact of

caloric restriction on the excitability of BNST CRF neurons and neuropeptide function in the region. The successful completion of these studies will delineate a microcircuit potentially involved in diet relapse and set the stage for 1) further ex vivo studies testing for means of controlling this circuit and 2) in vivo analysis of the impact of activation and inhibition of this circuit on feeding behavior.

David A. Jacobson, Ph.D.
Assistant Professor
Molecular Physiology and Biophysics

Two pore-domain potassium channels: Pancreatic islet expression and function

Pancreatic islet hormone secretion is imperative to maintaining glucose homeostasis and becomes perturbed in 7.8% of the United States population that develops diabetes. Thus, identifying the mechanisms that regulate islet hormone secretion may reveal new therapeutic targets for treating diabetes. Glucose stimulated insulin secretion results from calcium entry that occurs due to beta-cell membrane potential depolarization, which activates voltage dependent calcium channels (VDCCs). The activity of two-pore domain potassium (K2P) channels regulates the membrane potential in neurons, which influences calcium entry and electrical activity. However, a role of K2P channels in regulating the pancreatic β-cell membrane potential is unknown. We have determined that K2P channels are expressed in pancreatic islet cells where their currents modulate the membrane potential and influence glucose stimulated calcium influx. Therefore, this project plans to test the hypothesis that K2P channels modulate the membrane potential of mouse and human islet cells regulating calcium influx and hormone secretion. The specific objectives are to: A. Define the K2P channels of mouse and human islet cells, B. Identify the biophysical regulation of islet K2P channels and their influence on membrane potential and hormone secretion, and C. Characterize the pharmacology of islet K2P channels and utilize this pharmacology to address the influence of K2P channels on human and mouse islet cell electrical activity and hormone secretion. These goals will significantly contribute to our understanding of the ion channels that influence islet hormone secretion and provide important insights of potential therapeutic targets relevant to diabetes.

Anne K. Kenworthy, Ph.D.
Associate Professor
Molecular Physiology and Biophysics

Small molecule modulators of the Y4 receptor for treatment of obesity

Pancreatic beta cells faced a variety of ongoing stresses that lead to the accumulation of damaged proteins and organelles. Recent evidence indicates that a housekeeping process known as autophagy plays a critical role in clearing these damaged components, as genetic disruption of autophagy in pancreatic beta cells leads to beta cell dysfunction and loss of beta cell mass in mice. In addition, ubiquitinated protein aggregates accumulate in stressed beta cells, suggesting that a form of autophagy known as selective autophagy becomes compromised in response to conditions that lead to beta cell stress. However, very little is known about the molecular mechanisms that lead to the formation of these potentially toxic protein aggregates, what steps in selective autophagy are rate limiting for their clearance, or how their presence impacts beta cell function under conditions where selective autophagy fails. As a first step toward addressing these questions, in this pilot project we will test the hypothesis that selective autophagy is responsible for the clearance of both cytoplasmic and nuclear protein aggregates that form in response to beta cell stress, and that failure of this process is associated with beta cell dysfunction. To do so, we will utilize a combination of live cell imaging and immunocytochemical approaches to 1) test the hypothesis that two key components of the selective autophagy pathway, LC3 and p62, cooperate to remove ubiquitinated protein aggregates from the nucleus as well as the cytoplasm, and that this pathway functions to clear aggregates formed in response to oxidative stress in pancreatic beta cells in vitro and 2) determine if the accumulation of cytoplasmic and nuclear ubiquitinated protein aggregates is a general characteristic of mouse models of beta cell dysfunction and human islets from type 2 diabetics. In addition to testing a novel role for selective autophagy in nuclear quality control, these pilot studies will enable us to develop model systems to study how selective autophagy contributes to beta cell homeostasis, determine how this process becomes compromised in type 2 diabetes, and identify steps in this pathway that could serve as potential targets for therapeutic intervention in order to prevent or reverse beta cell damage in humans.

Jens Meiler, Ph.D.
Assistant Professor Chemistry
Pharmacology and Biomedical Informatics

AND 

C. David Weaver, Ph.D.
Assistant Professor
Molecular Pharmacology

The 375 amino acid G-protein coupled receptor (GPCR) neuropeptide Y4 receptor (Y4) is expressed both in the periphery, including the gastrointestinal tract, and in the central nervous system. The Y4 subtype is the only receptor of the Y-receptor family with a very high affinity for the 36-residue pancreatic polypeptide (PP). Selective small molecule modulators of this receptor would not only be valuable probe molecules to study its pharmacology, they also present attractive strategies for treatment of obesity. In fact, variants of PP are currently in phase II clinical trials for treatment of obesity (TM30339, 7TM Pharma). However, as PP is a peptide, issues of stability and bioavailability remain. The development of subtype-selective orthosteric ligands has failed in the past hampering development of effective probe molecules and therapeutics6. It is the objective of the present study to identify small molecule allosteric modulators of Y4 as tools for pharmacological research and to test their potential in a therapeutic strategy. We argue that allosteric modulators of GPCRs have a higher chance to be selective as allosteric binding sites tend to be evolutionary less conserved between subtypes. The therapeutic potential of allosteric modulators is further increased by their ability to fine-tune the receptor instead of turning it entirely on or off. Side effects are reduced not only through subtype selectivity but also as the therapeutic acts only at times when the receptor is engaged by its native ligand. It is the central hypothesis of the present proposal that identification of small molecule allosteric modulators selective to Y4 will allow the development of probe molecules to study subtype-selective pharmacology and can seed drug discovery programs in obesity. Preliminary data demonstrate the adaptation of an Y4 functional assay for high-throughput screening (HTS). A pilot screen of 2,000 compounds of the Spectrum Collection (MicroSource) yielded several hit compounds. Among those, Niclosamide displayed a robust, selective allosteric potentiation with an EC50-value of 410 nM. Cell lines for orthogonal GIRK-based assay for hit validation and to establish the initial selectivity profile through testing allosteric modulation of Y1/2/5 have been established.

Craig L. Duvall, Ph.D.
Assistant Professor
Biomedical Engineering

siRNA Delivery Scaffold for Altering Cytokine Signaling in Diabetes Skin Wounds

Impaired wound healing is a hallmark of diabetes, and chronic diabetic skin ulcers are the number one cause of non-traumatic limb amputations in the United States. Impaired wound closure in this setting is characterized by a cytotoxic, hyperinflammatory state and insufficient proliferation and migration of keratinocytes, fibroblasts, and vascular cells. Here, studies are proposed for development of a novel tissue scaffold for sustained siRNA delivery targeting the suppressor of cytokine signaling (SOCS) family member SOCS3. SOCS3 overexpression has been linked to hypoproliferative keratinocyte phenotype and cytotoxic (M1) activation of macrophages in the diabetic wounds. The central hypothesis of this proposal is that knockdown of SOCS3 using a controlled siRNA delivery system will induce reparative cellular phenotypes (M2 macrophage activation and keratinocyte proliferation) and promote diabetic wound healing. Aim 1 involves development of the delivery system and characterization of its siRNA release kinetics and bioactivity in vitro and in vivo. Aim 2 is to assess the functional effects of SOCS3 knockdown on macrophages and keratinocytes in vitro and on healing in a mouse diabetic skin wound model. The expected outcomes for the proposal include validation of a new platform technology for siRNA delivery to skin wounds and establishing SOCS3 as a potential target for wound therapies, with the long-term goal of reducing wound related morbidity and mortality in diabetic patients.

Daniel J. Moore, M.D., Ph.D.
Assistant Professor of Pediatrics
Assistant Professor of Pathology, Microbiology and Immunology

The Microbial Ecology of Immune Tolerance in Type 1 Diabetes

Type 1 diabetes is a relentless autoimmune disorder that afflicts over 2 million Americans and will be diagnosed in over 15,000 new children in the next year. The mechanisms that trigger this disease are unknown, but numerous epidemiologic studies point to environmental factors that may be both protective and provocative. The concept of environmental triggers is also supported by genetic studies; long-term studies on identical twins show that fewer than 60% of twins become concordant for disease even after decades of follow-up. While the circumstantial evidence for environmental effects is large, their role has not been described with mechanistic precision. Thus, interventions aimed at changing the effects of the environment remain haphazard. Recent data suggest that the human microbiome, or metagenome, may determine susceptibility to autoimmune disorders. These studies have been extended to demonstrate that the gut microbiome plays a deterministic role in diabetes susceptibility in the NOD mouse, the primary preclinical model of diabetes (Wen et al, Nature, 2008). Moreover, the microbiome can be modulated by genetic disruption of innate immune function to produce a tolerogenic state, the composition of which is not yet specified. We hypothesize that shifts in the microbiome between tolerogenic and non-tolerogenic states dictate diabetes progression and that these states can be specified and harnessed for diabetes prevention and reversal. In this proposal, we will pursue the first detailed analysis of diabetes-protective and -promoting microbiomes (Aim 1). We will further unravel the mechanisms by which innate and adaptive immunity modifies its composition by capitalizing on our recent description of a diabetes-preventing peptide inhibitor of proinflammatory signaling to the nucleus (Aim 2). These studies will lead to clinically translatable interventions that will enhance immunomodulatory therapies for T1D by identifying and correcting the autoimmunity-promoting state of the resident microbiota in genetically-prone individuals.

Kate L.J. Ellacott, Ph.D.
Assistant Professor of Molecular Physiology and Biophysics

The Effect of Obesity on Cerebral Endothelial Cell Function

Obesity is associated with chronic low-grade inflammation in the periphery, which in turn is linked to the development of metabolic syndrome and cardiovascular disease. Endothelial cells of the peripheral vasculature are negatively affected by inflammatory stimuli and this dysfunction contributes to the development of atherosclerosis in obese individuals. In addition to the peripheral vasculature, endothelial cells are also a critical component of the blood-brain barrier (BBB) and cerebral vasculature. Mounting evidence from clinical studies suggests that obesity is associated with increased vulnerability of the central nervous system (CNS); for example, obese individuals have a 6% higher incidence of stroke for every unit increase in body mass index greater than 30. Furthermore, obese individuals have an 11% increase in mortality following traumatic brain injury and a staggering 74% increased risk of dementia. Obesity and diabetes are known to affect transport across the BBB and alterations in function at this critical interface may contribute to the increased susceptibility of the CNS to damage. The molecular mechanisms underlying this phenomenon have not been examined. We hypothesize that obesity promotes alterations in cerebral endothelial cells, which lead to dysfunction, thus altering BBB and cerebral vascular physiology. This pilot grant proposal will address the following specific aims: 1) To identify specific molecular targets in cerebral endothelial cells regulated by chronic diet-induced obesity in mice; 2) To examine the regulation of expression of these targets over time with the development of obesity and metabolic syndrome. As a new independent investigator a DRTC P&F grant award would provide invaluable funding to allow my laboratory to generate preliminary data on this novel area of study for a National Institutes of Health (NIH) R01 application that we aim to submit in 2011.

Pandu R. Gangula, Ph.D.
Associate Professor, Obstetrics and Gynecology, Meharry Medical College

Oxidative Stress and Gastric Nitrergic Motility in Diabetics

Gastroparesis is a disabilitating disease affecting predominantly young women. The biological basis of this disorder and its associated gender bias remains poorly understood. Our recent data suggests that gastric motility is mainly regulated by nitrergic system and interstitial cells of Cajal (ICC) in healthy females and diabetes induction by STZ significantly impaired this system and leading to delayed gastric emptying associated with elevated reactive oxygen species (ROS). However, the underlying biochemical mechanisms by which ROS contributes to gastroparesis are not well understood. Nrf2 (NF-E2-related factor 2) is a transcriptional factor that regulates the expression of Phase II genes involved in regulating levels of reactive oxygen species. Heme Oxygenase I and glutathione (GSH) biosynthesis enzyme, Gluamate Cysteine Ligase subunits (Gclm and Gclc) are two examples of Phase II genes regulated by Nrf2. GSH is a major soluble antioxidant in cells and is involved in many aspects of cellular metabolism, including detoxification of peroxides and xenobiotics. Nrf2 KO and Gclm KO mice have low GSH levels and are highly susceptible to oxidative stress. Our recent novel data demonstrated that gastric nitrergic relaxation is impaired in Nrf2 KO and in Gclm KO mice. These data suggest that chronic depletion of GSH may be one of the detrimental factors in the pathogenesis of diabetic gastroparesis due to increased oxidative stress. We have, therefore, designed this proposal to investigate the mechanisms responsible for the disturbances in the nitrergic control of gastric motility in Nrf2 KO and Gclm KO females with diabetic or non-diabetic gastroparesis. Based on our preliminary data, we hypothesize that reactive oxygen species is an important component in the pathogensis of gastroparesis. To test this hypothesis we propose the following specific aim: Specific Aim: To determine the biochemical mechanism responsible for impaired gastric nitrergic relaxation, gastric emptying and ICC in Nrf2 KO and Gclm-KO mouse models of diabetes. Preliminary data generated from DRTC funds will provide a basis to prepare a major grant application and to investigate in detail with regards to the protective role of GSH on gastric motility functions and mechanisms involved in this setting.

Irina Kaverina, Ph.D.
Assistant Professor, Cell and Developmental Biology

Role of Distinct Microtubule Populations in Beta Cell Insulin Secretion

Impairment of insulin secretion contributes to the development of type 2 diabetes. During the extended second phase of glucose-stimulated secretion insulin granules residing in the cell interior are transported toward the plasma membrane by microtubule-dependent transport. Organization of microtubule tracks is an important factor in efficiency of secretion but its regulation is poorly understood. The goal of this pilot project is to determine major pathways that define configuration of microtubule network in pancreatic beta cells through regulation of nucleation and stabilization of microtubules. While microtubules in vertebrate cells are known to nucleate at the centrosomes, most microtubules detected in beta cells are associated with membrane structures such as the Golgi rather than the centrosome. We have recently identified the Golgi complex as an alternative microtubule-organizing center and now propose to test whether Golgi-derived microtubules serve as major tracks for insulin traffic (Aim 1). Microtubules are also needed to support Golgi integrity. We propose to test whether Golgi-derived microtubules support Golgi reorganization triggered by glucose to facilitate fast insulin processing (Aim 2). Glucose stimulation not only induces insulin secretion but also increases amounts of tubulin polymer, suggesting that this stimulus may increase number of stable microtubules, which are preferred tracks for granule delivering motor kinesin-1. Based on our preliminary data, we propose to test whether microtubules that serve as tracks for insulin granules are specifically stabilized by Rab6A-dependent mechanism (Aim 3). Research strategy includes cutting-edge microscopy and cell manipulation approaches, as well all molecular and biochemical tools. The results of this pilot project will potentially lead to an extensive mechanistic study that could, in turn, contribute to diabetes treatment strategies by revealing new molecular therapeutic targets regulating efficiency of insulin secretion.

Chandra Y. Osborn, Ph.D., M.P.H.
Assistant Professor, Center for Health Services Research

Leveraging Patient Portals to Provide Medication Adherence Support in Diabetes

Antihyperglycemic, antihypertensive, and lipid lowering medications have enormous promise for reducing morbidity and mortality in patients with T2DM, but adherence is often suboptimal. Readily available health information technologies (HIT) can provide real-time information and feedback to patients and providers to support medication safety, identify adverse drug events, and improve patient adherence. This proposal describes a research plan that will result in pilot data for the candidate's K award resubmission to NIDDK, in which a patient portal delivered medication adherence will be evaluated among patients with diabetes. As a social/health psychologist who has already received a fair amount of training in behavioral health research, this DRTC Pilot & Feasibility mechanism is crucial to providing Dr. Osborn with experience using HIT delivery systems, pilot data on the feasibility of her proposed intervention, and a jump start on her career as an independent investigator in the Prevention & Control Core of the Vanderbilt DRTC. Dr. Osborn’s immediate goal is to develop and test the usability of a medication adherence intervention for patients with T2DM and co-morbid hypertension and/or dyslipidemia that will be delivered through the My Health at Vanderbilt patient portal. This will be achieved by conducting focus groups to inform intervention content and collecting usability data from diabetes patients once the intervention is developed. The pilot study aims are twofold to 1) identify the optimal structure and content of a patient portal delivered medication adherence intervention for diabetes patients and 2) design, test for usability and refine this intervention for future testing in a three-arm randomized trial (i.e., NIDDK K Award proposal). Leveraging technology in the proposed research will augment the candidate’s existing training in the design and evaluation of behavior change interventions. Most importantly, it will accelerate her career as a successful independent investigator well equipped for significant contributions to designing cutting-edge, evidence-based interventions that have broad application and are effective at improving the care of patients with diabetes.

Malcolm J. Avison, Ph.D.
Professor, Department of Radiology and Radiological Sciences

Functional Brain Imaging in Mouse Genetic Models of Obesity and Diabetes

Functional neuroimaging is now widely employed in human studies to identify functional phenotypic correlates of genetic polymorphisms and epi-genetic variability. Conversely, genetically manipulated mouse models are increasingly used to test (and to generate) novel hypotheses related to the genetic basis of variability in behavior. Both approaches are gaining increased attention in the area of obesity and diabetes research, as recognition of the importance of understanding the central mechanisms regulating food intake (and behaviors therein) in developing effective strategies for managing obesity. However significant gaps remain in linking molecular and cellular consequences of genetic manipulations to systems-level effects on CNS circuitry in mice, and in identifying the specific molecular and cellular pathways mediating the (epi-)genetic influences on brain function in humans. The present proposal seeks to address this by bringing together experts in neuroimaging, obesity and diabetes research, and mouse genetic models of obesity and diabetes. Specifically, we propose to address a significant gap in technology (human and rat imaging have progressed rapidly), to develop core methodology for functional imaging of brain circuitry in genetically modified mice. The research will focus on developing a toolbox of techniques for acquisition and analysis of the neural correlates of neuro-genetic manipulations, and will validate them in well characterized mouse models of obesity and diabetes. Specifically, we will: 1. Translate and extend existing functional imaging methods developed in rats to mice; 2. Develop novel computational tools for the identification of systems/circuit level effects of cell-type specific genetic manipulation.

Wenbiao Chen, Ph.D.
Assistant Professor, Department of Molecular Physiology and Biophysics

Nutrient Regulation of Beta Cell Mass in the Zebrafish

Intrauterine malnutrition, manifested as low birth weight, is strongly associated with type 2 diabetes. Another hall marker of intrauterine malnutrition is the decreased mass of pancreatic beta cell. In rodent models, this decrease of beta cell mass is irreversible late in life, predisposing the animal to type 2 diabetes. The mechanism of malnutrition-induced decrease of beta cell mass is not clear. We have demonstrated that zebrafish fries experience a marked expansion of beta cell mass during the first 4 days of free feeding and food withholding during the period completely abolish the expansion. In the proposed pilot and feasibility studies, we will determine whether the diminishment of beta cell mass is irreversible as in rodents, and whether the nutrient-sensing mTOR pathway is involved in food-induced beta cell growth in this model. Lastly, we will develop a transgenic fish that fluorescently labels the membrane and nucleus of beta cells for precise measurement of beta cell size and number. The transgenic line should facilitate screens for the identification of genes and compounds that alter this nutrient response. Since nutrient sensing and beta cell biology are largely conserved in vertebrates, studies in the zebrafish should provide relevant insights in humans.

Charles R. Flynn, Ph.D.
Assistant Professor

Diacylglycerols and Insulin Action in Skeletal Muscle upon Caloric Restriction

Insulin resistance in skeletal muscle is a characteristic abnormality of obesity and type 2 diabetes (T2DM). Although the mechanisms responsible for this pathophysiology remain unclear, the intramuscular accumulation of lipids is crucial. Current evidence points towards dyslipidemia-induced DAG accumulation and inflammation as a consequence of a high fat diet as contributory. In this project, we will test the hypothesis that in skeletal muscle, molecular species of DAGs enriched in palmitate, a reactive oxygen species
(ROS) promoting fatty acid will be decreased preferentially in both short- and long-term caloric restriction, but that the decrement will be greater in the morbidly obese cohort. We also anticipate that DAG species rich in oleate, a protective fatty acid, will remain unchanged in both groups during the short-term but will increase with long term caloric restriction in the morbidly obese. Lipid extracted from skeletal muscle biopsies obtained both before and after short-term and long-term caloric restriction will be analyzed using mass spectrometry-based lipidomics approaches to monitor molecular DAG species accumulation. The hyperinsulinemic-euglycemic clamp technique will be used to assess insulin-stimulated glucose disposal activity. The differential expression of lipids in muscle will be correlated with circulating and tissue resident markers of inflammation, fibrosis and macrophage infiltration. We expect data from our proposed studies will provide new insight into mechanisms underlying insulin resistance.

Sabina B. Gesell, Ph.D.
Research Assistant Professor
Department of Pediatrics

Building Social Networks to Prevent Postpartum Weight Retention

Obesity is highly associated with type 2 diabetes. Inspired by the seminal work of Christakis and Fowler that demonstrated the power of social network phenomena on the development of obesity and cessation of smoking, this project sets the groundwork for examining whether social networks can play a significant role in preventing postpartum weight retention on a population-level. The goal of this proposal is to conduct feasibility pilot work to 1) establish recruitment and retention rates of pregnant Latinas who are recent immigrants and at high risk of developing obesity; 2) develop and manualize a culturally-tailored intervention program that increases ties among participants and with community resources; and 3) test tools to accurately capture social network information that allow for social network analysis, and parameter estimation to inform future power calculations. A within-subjects repeated measure design will be used investigate to the effect of a social network enhancement intervention on change in social network attributes. We estimate being able to recruit 30 lower income pregnant Latinas > 18 years who will receive a 12-week social networking intervention during pregnancy with the goal of influencing postpartum outcomes. Primary outcomes of interest are various network attributes; secondary outcomes are postpartum body mass index and body composition. Data will be collected at baseline, at completion of the intervention, at 1 moth postpartum and 6 months postpartum. Recruitment and retention rates will be calculated (Aim 1) and patterns of missing data will be examined (Aim 2). Stage one of the social network analysis will include assessing the degree and nature of network formation by examining the creation of dyadic ties, the formation of subgroups in the network, and by measuring changes in the overall density of the network (Aim 3). Stage two will treat individual-level network-related characteristics as independent variables predicting intervention-related outcomes.

Todd M. Hulgan, M.D., M.P.H.
Assistant Professor
Department of Medicine

Adipokines and Oxidant Stress in Diabetic and Non-Diabetic HIV-Infected Subjects

Human immunodeficiency virus (HIV) infection and the resulting acquired immunodeficiency syndrome (AIDS) is one of the greatest public health challenges in history. Antiretroviral therapy (ART) has dramatically reduced morbidity and mortality due to HIV/AIDS, but remains limited by long-term complications. These complications include mitochondrial toxicities due primarily to nucleoside reverse transcriptase inhibitors (NRTI), and metabolic complications attributed to the non-NRTI (NNRTI) and protease inhibitor (PI) drug classes. These ART complications mirror the metabolic syndrome, with insulin resistance (and overt type 2 diabetes mellitus), dyslipidemia
(predominant hypertriglyceridemia), and lipodystrophy (including abdominal obesity). Not surprisingly, excess cardiovascular disease (CVD) and myocardial infarction rates have been reported in association with ART exposure. Mechanisms of metabolic complications in HIV infection and its treatment are poorly understood, and appear to differ by drug class. NRTI have adverse effects on mitochondrial and cellular energetics. Some NRTI have direct effects on insulin sensitivity, and have been associated with development of insulin resistance and diabetes in cohort studies. PI influence cellular lipid and glucose metabolism, leading to hypoleptinemic and hypoadiponectinemic states. The additional influence of chronic inflammation due to HIV infection is unknown. Much remains to be learned about the effects of HIV and ART on fundamental processes of oxidant stress, inflammation, and adipogenesis in HIV-infected patients, especially as they relate to ART-associated fat redistribution, obesity, insulin resistance, and diabetes. The overarching hypothesis of the proposed studies is that in HIV-infected persons, adiponectin will be negatively correlated with F2-isoprostanes (a urine biomarker of oxidant stress and CVD risk), but this correlation will be influenced by ART exposure, fat content (total, trunk, limb) by dual-energy X-ray absorptiometry, and by the presence of insulin resistance by homeostasis model assessment and/or diabetes mellitus. This hypothesis will be tested through the determination of fasting adipokine levels in stored specimens from an ongoing cohort study of HIV-infected, chronically ART-treated, non-diabetic subjects (N=50), and in prospectively enrolled, chronically HIV-infected, ART-naïve, non-diabetic (N=30) and ART-treated diabetic subjects (N=30). We will then determine correlations between adipokines and urine eicosanoid biomarker data, adjusted for regional fat content and the presence of insulin resistance or diabetes mellitus.

Stacey S. Huppert, Ph.D.
Assistant Professor
Department of Cell and Developmental Biology

Requirement of Notch Signaling for Beta Cell Neogenesis

Development of safe and effective therapeutic options for diabetes mellitus requires a thorough understanding of the genetic components governing pancreatic development, maintenance, and injury responses. Transplantation of islet cells has been successfully performed, relieving patients with type I diabetes of symptoms for extended periods of time. This suggests that diabetes can be treated by replenishing deficient ß cells. ß cell mass is normally dynamic, responding to meet endocrine demands throughout life. Increases in ß cell mass can occur via self-duplication of existing ß cells, but is also proposed to occur via ß cell neogenesis from adult stem/progenitor cells. Notch signaling is a critical molecular component for lineage commitment decisions of pancreatic progenitors during embryonic development, regulating multiple steps of cell maturation relative to neighboring cells. While this embryonic role has been described, it is unknown if Notch signaling plays a role during activation of adult multipotent progenitors in response to injury, regulating new islet formation. We hypothesize that Notch signaling is crucial for lineage commitment and/or cell fate decisions of the facultative progenitor cells within the adult pancreatic ductal epithelium following partial duct ligation. The aims within this proposal will take advantage of pre-existing mouse models that enable lineage tracing and inducible lineage-specific ablation of Notch signaling within the keratin 19-positive ductal epithelium.

Patricia A. Labosky, Ph.D.
Associate Professor
Department of Cell and Developmental Biology

Control of Beta Cell Proliferation during Pregnancy

Diabetes affects an estimated 150 million people worldwide and the disease carries with it a myriad of associated health problems. Unfortunately, most treatments for diabetic patients are inadequate because they do not regulate blood glucose levels precisely enough to eliminate complications. The insulin producing cells of the pancreas, beta cells, do not normally proliferate extensively in adults. However, under certain conditions of metabolic challenge (pregnancy and obesity), beta cells undergo proliferation and beta cell mass expands up to two-fold. One hope for developing promising diabetes treatments is based on manipulating beta cell proliferation. A critical piece of information needed for this strategy is an understanding of the molecular mechanisms controlling beta cell proliferation. Our goal is to understand how normal beta cell proliferation in response to metabolic demands is regulated with the long-term expectation that this information could then be used to increase beta cell mass in diabetic patients. The transcription factor Foxd3 is required for the survival, selfrenewal and multipotent nature of several disparate progenitor cell types, embryonic stem (ES) cells, trophoblast and neural crest progenitor cells. Foxd3 is also expressed in the pancreatic primordium beginning at 10.5 dpc and becomes localized predominantly to beta cells after birth. Mice carrying a tissue specific deletion of Foxd3 in the pancreatic epithelium have normal glucose homeostasis. However, pregnant mutant mice exhibit gestational diabetes. Here we propose a series of experiments to test the hypothesis that Foxd3 is required for altered beta cell function and/or proliferation during pregnancy with the expectation that a better understanding of the molecules controlling these compensatory beta cell changes will augment the development of improved therapies for diabetes.

John M. Stafford, M.D., Ph.D.
Assistant Professor
Department of Medicine

Portal Glucose as a Driver of Diabetic Dyslipidemia

In recent decades, death from coronary heart disease
(CHD) has declined by 40% in the US population. By stark contrast, for patients with diabetes, risk of death from CHD continues to rise. This difference in CHD risk between diabetic patients and the general population may result from additional CHD risk factors associated with elevations in serum triglyceride (TG). Hyperglycemia is a major driver of TG production in diabetes. Several key control points in hepatic TG production are independently controlled by both glucose and insulin - In normal physiology, glucose and insulin signals are coordinated to control the production and utilization of TG, the body’s main energy source. Much of this coordination may be due to the functional organization of the liver and portal venous system. Remarkably, after a mixed-meal gut enterocytes shuttle meal-lipid into gut lymphatics, which drain to the systemic circulation, avoiding the liver. By contrast, mealrelated glucose is directed into the portal venous system, which drains directly to the liver. This anatomic organization is mirrored in the structure of liver acini, which compartmentalize glucose production from lipid production. Portal delivery of glucose also activates a “portal signal†mediated by the autonomic nervous system to promote hepatic glucose uptake, and may limit lipid production. Diabetic patients have abnormalities in both postprandial glucose and lipid metabolism. We propose that with diabetes, the anatomic organization of the portal system contributes to augmented glucose-mediated lipid production. Our pilot data using clamp and tracer techniques combined with molecular dissection of the insulin-signaling pathway demonstrate that peripheral delivery of glucose promotes VLDL production only with impaired insulin action. In this proposal, we test the hypothesis that portal glucose delivery will further augment hepatic VLDL production (AIM1) and that portal glucose more potently promotes VLDL secretion when insulin action is disrupted (AIM2). This situation is analogous to the physiology of diabetes where either insulin deficiency (DM1) or insulin resistance (DM2) contribute to postprandial glucose and lipid abnormalities. These studies are highly collaborative with Dr. Masa Shiota, director of the Rat Metabolic Physiology Core. Collectively these studies will provide detailed information about the contribution of hyperglycemia to altered VLDL in diabetes, a major contributor to CHD in diabetic patients. The molecular mechanisms of how this metabolic control is coordinated will be the subject of future experiments, and will undoubtedly serve as the roots of many years of future studies for the lab. Support of the DRTC Pilot and Feasibility Program will be an important contributor to my prolonged success in diabetes research.