Technologies for helping manage diabetes have proliferated in recent years and continue to develop at a rapid race. These include mobile health, patient portals, telehealth, and CGM/insulin pumps. While they provide increased convenience with self-management and are effective at improving behavioral and clinical outcomes, understanding who has access to and experience with these digital tools has implications for who can ultimately benefit. The digital divide has primary been focused on the gap in technology access across different groups; however, as access is becoming more widespread, there is evidence of a new type of digital inequality based on skills and confidence using digital tools. Unless we address differences in digital health literacy, there is a high potential for digital tools to exacerbate existing health disparities. This proposal aims to develop a brief, comprehensive, and structured intervention to improve digital health literacy among patients with diabetes (PWD) and evaluate its feasibility and acceptability. In Aim 1, we will employ a user-centered Design Sprint methodology to develop the intervention. This will include convening expert stakeholders to review, select, and adapt existing available resources. We will also examine the utility of a new screening tool to identify patients who can benefit from the intervention and identify relevant processes for delivering the intervention. In Aim 2, we will conduct a pre-post pilot study with PWD recruited from primary care clinics at Vanderbilt University Medical Center to evaluate the intervention’s feasibility and acceptability. As secondary outcomes, we will also explore preliminary effects of the intervention on patients’ confidence using and frequency accessing digital tools. This will be one of the first interventions designed to improve PWD’s digital health literacy. This project will provide the preliminary data to inform a large-scale RCT that evaluates effects of the intervention, as well as explore models for implementation when the intervention is integrated into routine clinical care.

Type 2 diabetes (T2D) is common in persons with HIV particularly among persons of African ancestry. Because of the importance of mitochondria in cellular bioenergetics, and the effects on mitochondria from both HIV infection and its treatment, our group has investigated the role of patterns of linked common mitochondrial DNA (mtDNA) variants, called haplogroups, in the development of T2D in persons with HIV. We have now replicated significant associations between the common African mtDNA haplogroup L3 (~30-40% of U.S. Non-Hispanic Black persons) and incident T2D in two different cohorts, showing that persons with HIV of African ancestry in haplogroup L3 have an approximately 50-60% increased incidence of T2D compared to those with other haplogroups. Significant associations were not found in persons without HIV, indicating that an interaction between the mitochondrial genetics and HIV is important to T2D development.

In order to develop our research from the initial stage of testing for genetic associations to the more advanced stage of investigating biochemical and cellular differences to elucidate mechanisms of effect, we propose here to analyze two independent datasets; one of various “omics” data and the second of physiological and adipocyte expression relevant to T2D. Both datasets include persons with and without HIV, diabetes information, and available genetic data that have already been or can be used to determine mitochondrial haplogroups. All data are currently available to the investigators. Support from the DRTC will allow us to carefully analyze this data to develop preliminary data for NIH grant applications for prospective studies.

Complications of overweight and obesity are responsible for an increasing burden of disease in an aging population of persons with HIV (PWH) in the era of modern ART. One of these complications, non-alcoholic fatty liver disease (NAFLD) is closely associated with visceral adiposity and is a leading cause of morbidity and mortality among PWH. PWH develop NAFLD at lower BMI and with greater physical activity relative to HIV-negative persons. Although the mechanisms underlying this difference are not well understood, intestinal barrier dysfunction is implicated in the pathogenesis of NAFLD and the accumulation of visceral adipose tissue in HIV-negative persons. HIV infection has been shown to alter the intestinal microbiome, change the plasma metabolome and promote intestinal barrier dysfunction; however, there are few data on the microbiome and metabolome among PWH with NAFLD and visceral adiposity. Our pilot preliminary data show that hepatic steatosis and visceral adiposity are associated with bacteria-related metabolites and lipid species [including trimethylamine N-oxide (TMAO) and phosphatidylcholine] among PWH on long-term ART. We hypothesize that intestinal dysbiosis in PWH contributes to NAFLD and visceral adiposity through two distinct mechanisms: 1) by impairment of IBF and 2) through alteration of the metabolome to promote hepatic and visceral lipid deposition. Our primary Aim for this project is to build upon the recent AIDS Clinical Trials Group study A5350 by testing the feasibility and limited efficacy of a multi-strain probiotic and prebiotic fiber on NAFLD and visceral adipose tissue markers in a prospective trial in PWH. This trial will leverage the well-developed infrastructure and large recruitment pool of the Tennessee Center for AIDS Research. I will be supported by mentors who have a history of multiple successful prior trainees and expertise in clinical and translational HIV research, the metabolome and the microbiome. The support provided by the DRTC P&F grant will allow me to advance my career and training goals, and the data generated with this project will form a foundation for the development of an R01-level grant focused on microbiome-based therapies for PWH with NAFLD and central obesity.

Obesity affects 40% of the U.S. population and rising, and is one of today’s biggest public health concerns. Losing weight is challenging, and maintaining weight loss even more so, owing to the many biochemical and hormonal changes that occur in the pathogenesis of obesity, some of which do not reverse after weight loss. Thyroid hormones (THs) have a large impact on whole-body energy balance. Their synthesis and release by the thyroid are finely regulated by central mechanisms to control serum TH levels and ensure adequate TH availability for target tissues. THs are also regulated by nutrient sensing, so that their levels modulate cellular energy expenditure to match energy availability. Given the reciprocal and fine-tuned relationship between THs and energy balance, one would expect strong upregulation of TH levels to counteract diet-induced weight gain (DIWG), but this is not the case. In fact, overnutrition induces mild hypothyroidism and impairs thyroid function, additionally causing significant histological changes. An adequate TH supply from the thyroid is critical for proper TH signaling, particularly in metabolic tissues that must generate higher levels of active T3 from the prohormone T4 than are available from the bloodstream. Moreover, substantial evidence links impaired thyroid function to DIWG and other metabolic dysfunctions, albeit with some controversy stemming mainly from poor study designs. Even so, this controversy underscores the importance of further investigation in this field, and neither the effects of overnutrition on thyroid histology and function nor the mechanism(s) underlying diet-induced TH abnormalities have been sufficiently investigated. We hypothesize that overnutrition impairs thyroid function, in part by perturbing intrathyroidal TSH signaling and inducing cellular stress responses, leading to an inadequate TH supply that further exacerbates DIWG.

Sedentary behavior (SB) is a strong modifiable risk factor for developing type 2 diabetes (T2D). Patients with T2D are particularly prone to high levels of SB (10-12 hours/day) and have a greater risk for the negative consequences of SB due to their low activity levels. There is a pressing need to develop novel strategies to reduce SB and improve daily activity. Reducing SB time with frequent standing/walking breaks is safe and offers cardiometabolic benefits. Wearable technology can prompt users to take breaks from sitting and subsequently reduce SB time. In addition, the use of a smart water bottle may be a promising strategy to naturally break sedentary time due to the impact of increased water intake on kitchen and restroom breaks. However, little is known about the feasibility and preliminary efficacy of a technology-based SB reduction program for T2D patients in a free-living setting. We proposed a pilot randomized control trial to develop a SB-reduction intervention focused on breaking sedentary time with standing/walking in CAD patients (n=70). Our 12-week intervention (n=35) will consist of 1) an instructional/goal-setting session, 2) an activity tracker (Fitbit) providing stand/walk prompts, 3) a smart water bottle (HidrateSpark) that syncs with the Fitbit, and 4) weekly tailored text messages for behavior reinforcement and weekly goal monitoring. The control group (n=35) will be given the American Diabetes Association (ADA) booklet, “Taking Care of Type 2 Diabetes”. This proposal will evaluate the feasibility and acceptability of the SB reduction intervention. We will also evaluate whether our intervention can reduce SB (primary outcome) and improve physical activity, cardiometabolic and patient-centered outcomes (secondary outcomes). Our intervention is significant because sedentary breaks can be easily and safely implemented into the T2D patients' daily routine and offer cardiometabolic benefits. This study will provide preliminary data for future fully-powered SB reduction studies.

Induced nephron progenitor cells mimic the nephron progenitor cells present only in the fetal kidney that go on to form most cell types of the adult nephron. Induced nephron progenitors are derived from adult human cells in tissue culture through transcription factor reprogramming with inducible piggyBac transposons. The reprogramming factors are SNAI2, EYA1, and SIX1. We have isolated and characterized exosomes from these induced nephron progenitor cells and find them to be abundant and protective in other settings. This proposal will evaluate the potential of induced nephron progenitor exosomes to treat diabetic kidney disease. Diabetic nephropathy (DN) results from glomerular podocyte dysfunction in response to a high glucose environment. Our lab has successfully derived podocytes from human induced pluripotent stem cells using an accelerated method. We found that treatment with high glucose induced actin rearrangement and apoptosis. In Aim 1, we hypothesize that the induced nephron progenitor cell-derived exosomes will protect human podocytes from apoptosis induced by high glucose by examining morphology, viability, and markers of apoptosis. In Aim 2, we hypothesize that the exosomes will reduce podocyte dysfunction in diabetic mouse kidneys. We will evaluate histology and markers of kidney function to determine the therapeutic effects on diabetic nephropathy. This pilot study will generate exciting new data in support of a larger study to identify the mechanism of induced nephron progenitor exosome protection from podocyte apoptosis.

Obesity is a risk factor for numerous diseases and a major cause of Type 2 diabetes (T2D). While chronic obesity leads to pathology, acute nutrient excess promotes a protective form of adipose tissue expansion through the generation of new adipocytes (adipogenesis). Severe obesity diminishes adipogenic capacity, which is proposed to worsen systemic metabolic health. We discovered a new regulation of adipogenesis through the mitochondrial sirtuin, (SIRT)-4, a NAD+-dependent protein deacylase and a regulator of global mitochondrial metabolism. We identified a critical role of SIRT4 in promoting adipogenesis through the induction of oxidation of leucine, a branched-chain amino acid (BCAA). This data aligns with previous observations that mitochondrial fuel oxidation and respiration are increased during adipocyte differentiation and are required to drive the process. While other BCAAs, isoleucine and valine, have negative effects on metabolic health, their role in adipogenesis has not been evaluated. Furthermore, BCAAs accumulate in the plasma of diabetic patients and enzymes in BCAA catabolic pathways are downregulated in diabetic adipose. Dysfunctional mitochondrial respiration in obese adipose tissue is also thought to exacerbate obesity, although the mechanistic link between decreased BCAA catabolism and decreased mitochondrial function has not been explored in this context. In this proposal we will test the hypothesis that BCAA catabolism promotes adipogenesis through the regulation of mitochondrial function. To this end, we will 1) dissect the role of leucine, isoleucine, and valine catabolism in adipogenesis, and 2) ascertain whether modulating BCAAs alters mitochondrial supercomplex formation and respiration in adipogenesis.

Clonal hematopoiesis of indeterminate potential (CHIP) is a newly recognized disorder characterized by the ontogenesis of a genetically distinct, proliferative clonal leukocyte population. The prevalence of CHIP increases with older age and is associated not only with risk of hematologic cancers, but with type 2 diabetes, fibrosis, systemic inflammation, and atherosclerotic cardiovascular diseases. Recapitulation of CHIP in mice by transplantation of clonal leukocytes results in accelerated insulin resistance, atherosclerosis, cardiac fibrosis and direct tissue infiltration of clonal macrophages and stimulation of interstitial fibrosis.

Age is a dominant risk factor for diabetic kidney disease (DKD), which is associated with accelerated cardiovascular disease, premature death, and progression to dialysis dependence. The biological mechanisms conferring this age-associated risk are incompletely understood. The final common pathologic process in progressive DKD is tubulointerstitial fibrosis, which is characterized by the accumulation of inflammatory infiltrates and fibroblasts within the kidney interstitium and permanent loss of tubular epithelial cells. Tubulointerstitial fibrosis also represents the central underlying lesion in the progression of acute kidney injury (AKI) to chronic disease.

Based on mechanistic links between CHIP and insulin resistance,atherogenesis, kidney interstitial inflammation, and fibrosis, we hypothesize that CHIP is a novel biological risk factor for DKD progression. To test this hypothesis, we propose to determine the associations of CHIP with kidney disease progression in established cohorts of DKD and AKI. The identification of clonal leukocytes as a novel mechanism of DKD progression would represent a new disease pathway that could motivate future targeted interventions.

The overall goal of this project is to perform preliminary studies exploring alterations in the lipidome, proteome, and secretome of adipocytes that are deficient in the autophagy-related protein PIK3C3. Adequate mass and function of adipose tissues (ATs) play an essential role in local and systemic energy homeostasis. Pathological expansion of ATs in obesity and pathological reduction of ATs in lipodystrophy lead to a similar array of metabolic diseases, highlighting the need to understand the underlying mechanisms as they can inform therapeutic interventions and reveal biomarkers. A number of cellular processes, including autophagy, play critical roles in maintaining healthy ATs. Recent studies on several autophagy-related proteins have revealed the impact that is specific to individual proteins. In this context, we have targeted PIK3C3 in murine adipocytes. We have found that PIK3C3 functions in all types of ATs with a crucial yet complex role in AT health and systemic energy homeostasis. Its absence disrupts adipocyte autophagy, hinders adipocyte differentiation and lipid handling, and impairs adipocyte survival. These pathologies are further exacerbated by high fat diet (HFD) feeding. Progressive loss of white ATs, whitening and eventual loss of brown ATs, and impaired browning of white ATs compromise thermogenesis and lead to metabolic disorders manifesting as dyslipidemia, hepatic steatosis, and insulin resistance that proceeds to overt type 2 diabetes. Several observations obtained during the study prompt us to pursue omic analyses to understand dynamically altered lipidome, proteome, and secretome in PIK3C3-deficient adipocytes. These include differential and age-dependent impact of PIK3C3 deficiency on different adipocytes, on different lipid metabolic pathways, on circulating small RNA pool, and on systemic insulin sensitivity and glucose clearance. Based on these striking preliminary studies, this application seeks support for lipidomic and proteomic analyses on the PIK3C3-deficient mouse and cell line models established during our study, utilizing the Center for Innovative Technology at Vanderbilt and Mass Spectrometry Research Center at VUMC. We will test the hypothesis that PIK3C3 plays an important role in shaping the lipidome, proteome, and secretome of adipocytes by performing 3 sets of experiments using primary and cell line models of brown and white adipocytes and extracellular fluids.

Diabetes is caused by the functional collapse of insulin-producing beta cells in the islet of Langerhans in the pancreas. Beta cell function is supported via paracrine signaling pathways from neighboring alpha and delta cells. Most alpha, beta and all delta cells in the healthy pancreas can be as old as cortical neurons and are classified as long-lived cells. During diabetes, beta cells become dysfunctional and/or lose their maturation via unknown mechanisms that are enhanced by aging and diabetes. Our recent study has linked the onset of islet cell longevity to early stages of postnatal life in mice between post-natal days 21 and 45 (P21 and P45, respectively). Further, data from our lab indicate that 25% of alpha and beta cells become long-lived and likely are functionally and transcriptionally mature prior to weaning at P21 – a time point that has been thought to be critical for functional maturation of all beta cells. This data suggests that a significant sub-set of beta cells makes the critical decision towards becoming a long-lived cell at a very early point in post-natal life and before all metabolic signaling cues associated with adulthood and sexual maturation are active. However, it is currently unknown what cellular adaptations and molecular pathways are associated with the early life onset of beta cell longevity. This project will establish the longevity and molecular signatures of beta cells at different post-natal developmental time points in the mouse pancreas through utilization of high-resolution single cell transcriptional sequencing and isotope tissue mapping. These profiling technologies are complementary and are expected to reveal the connection between beta cell transcriptome and epigenetic architecture and age during the critical window of time when beta cell longevity is determined. Understanding the pathways activated by maturing beta cells and associated with long-term maintenance of cell function and health will lead to new methods to promote and preserve beta cell function in diabetic patients.

Despite the fact that non-alcoholic fatty liver disease (NAFLD) affects nearly 30% of the US population, there is currently no approved pharmaceutical treatment to inhibit progression of NAFLD to its more severe stage of nonalcoholic steatohepatitis (NASH). Recent studies using isotope tracers to assess in vivo metabolism of human subjects and mouse models have uncovered that citric acid cycle flux is nearly 2-fold higher in NAFLD compared to controls, which was hypothesized to promote oxidative stress associated with NASH development. The liver X receptors (LXRs) are key regulators of cholesterol and fatty acid homeostasis in liver. When activated, LXRs induce a series of genes that are involved in fatty acid synthesis, absorption, transport and excretion. Activation of LXR has the potential to modulate membrane phospholipid (PL) composition, improve hypercholesterolemia, induce anti-inflammatory effects, regulate endoplasmic reticulum stress and increase insulin sensitivity, but may potentially increase liver hypertriglyceridemia. GW3965 is a selective, orally active non-steroidal agonist for the LXRs. It was shown to induce the expression of LXR target genes, modulate cellular PL composition and blunt the induction of downstream, unfolded protein response signaling pathways in primary mouse hepatocytes. However, the effects of LXR activation on in vivo liver metabolic fluxes during NASH development are not well understood. The overall objective of this proposal is to apply 2H/13C metabolic flux analysis (MFA) and molecular profiling to assess responses to pharmacologic LXR activation designed to inhibit progression from NAFLD to NASH in vivo. We hypothesize that even though LXR activation may worsen liver steatosis, it will reduce metabolic dysregulation by improving membrane fluidity and fatty acid trafficking through augmenting PL and cholesterol composition of the ER and mitochondrial membranes. This in turn may prevent overactivation of mitochondrial metabolic pathways and oxidative tissue injury leading to NASH. These studies will use a melanocortin-4 receptor knockout (MC4R-/-) mouse model that has been previously shown to rapidly and spontaneously develop characteristics of human NASH upon Western diet feeding. To enable a more comprehensive analysis of in vivo fluxes, we will apply a recently developed mass spectrometry (MS)-based MFA approach that explicitly accounts for Cori cycling between hepatic and extrahepatic compartments in order to relax several constraining assumptions of previous models. We also expect to utilize LC-MS based analysis of glycolytic intermediates to build more detailed models and further understand metabolic changes during NASH pathogenesis.

Genome-wide association studies (GWAS) have shown that the ‘T’ allele of the rs13266634 nonsynonymous single nucleotide polymorphism (SNP) in the SLC30A8 gene, which encodes the zinc transporter ZnT8, decreases susceptibility to type 2 diabetes (T2D). The ‘T’ allele changes ZnT8 amino acid 325 from an arginine to a tryptophan resulting in decreased ZnT8 activity. Similarly, SLC30A8 haploinsufficiency is markedly protective against the development of T2D. The SLC30A8 gene is predominantly expressed in pancreatic islet beta cells and both the rs13266634 ‘T’ allele and haploinsufficiency are associated with improved glucose-stimulated insulin secretion (GSIS). Reduced SLC30A8 expression also protects beta cells from both endoplasmic reticulum-associated and cytokine-mediated stress. These observations strongly suggest that ZnT8 inhibitors may prevent T2D through actions on beta cell function and clearly justify efforts to identify such compounds.

We have created a bacterial-based assay that enables us to quantitate ZnT8 activity in intact cells. This assay is based on the ability of ZnT8 to enhance the inhibitory effect of cadmium chloride on bacterial cell growth. The goal of Aim 1 is to compare two common inducible bacterial protein expression systems, in which ZnT8 expression is controlled by either isopropyl b-D-1-thiogalactopyranoside (IPTG) or L-arabinose, to identify one that is optimal for the induction of ZnT8 expression for use in a high-throughput screen (HTS). The goal of Aim 2 is to then use the optimized assay to perform a HTS using Vanderbilt’s HTS screening facility and compound libraries to identify compounds that affect bacterial growth specifically through an action on ZnT8. We hypothesize that that our novel ZnT8 transport assay will lead to the identification of ZnT8 inhibitors.

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 GBγ-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.

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.

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.

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.

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.

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.

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 T1D prone 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.

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.

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.

Diabetic Retinopathy (DR), a microvascular complication of Diabetes Mellitus, is the leading cause of blindness in working age adults.This work will explore the effects of manipulating the CXCL8:CXCR1/2 chemokine signaling system to reduce pathologic retinal angiogenesis in models of DR. It will also explore correlations between genetic variants in the CXCL8:CXCR1/2 chemokine signaling system and DR susceptibility, progression, and response to VEGF inhibitors (the gold standard drugs used to treat retinal angiogenesis in DR).

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 disease causing 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.

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 beta-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.

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.

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 pups at day E16.5. We will extract the hearts at different timepoints and use genome wide cytosine methylation profiling to delineate the difference between pups 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.

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.

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.

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.

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.