Raghavendra G Mirmira, M.D./Ph.D. - US grants

Diabetes mellitus, a disorder resulting from the absolute (Type 1) or relative (Type 2) deficiency of insulin, affects approximately 6 percent of the U.S. population; the associated micro- and macrovascular complications makes this disease one of the leading causes of morbidity and mortality. The long term objective of this proposal is to understand the role that cell type specific gene transcription plays in the differentiation of the insulin-producing beta-cell of the pancreas. The information gathered from such studies should make it possible to reprogram various cell types in order to treat the cellular and genetic deficiencies occurring in diabetes. The general strategy to use the transcription factor Nkx6.1 as a paradigm for a beta-cell restricted transcription factor that is necessary to direct cell differentiation and proliferation. The focus of this proposal is to functionally characterize the transcription factor Nkx6.1 and to identify genes that are directly and indirectly regulated by this factor during the process of beta-cell differentiation. The specific aims of this proposal are: 1. Biochemically characterize Nkx6.1 with respect to its DNA binding properties 2. Characterize the functional activation domains of Nkx6.1 3. Identify and characterize target genes for Nkx6.1 action To achieve these aims, we will utilize in vitro biochemical assays of protein/DNA binding, transient mammalian cell transfections, transgenic mouse models, and cDNA subtraction/differential display. Taken together, the knowledge gained from these studies will provide insight into the mechanisms governing beta-cell differentiation in the developing pancreas. This information can eventually be applied to engineering new beta-cells for patients with diabetes.

Diabetes mellitus, a disorder resulting from the absolute (Type 1) or relative (Type 2) deficiency of insulin, affects approximately 6 percent of the U.S. population; the associated micro- and macrovascular complications makes this disease one of the leading causes of morbidity and mortality. The long term objective of this proposal is to understand the role that cell type specific gene transcription plays in the differentiation of the insulin-producing beta-cell of the pancreas. The information gathered from such studies should make it possible to reprogram various cell types in order to treat the cellular and genetic deficiencies occurring in diabetes. The general strategy to use the transcription factor Nkx6.1 as a paradigm for a beta-cell restricted transcription factor that is necessary to direct cell differentiation and proliferation. The focus of this proposal is to functionally characterize the transcription factor Nkx6.1 and to identify genes that are directly and indirectly regulated by this factor during the process of beta-cell differentiation. The specific aims of this proposal are: 1. Biochemically characterize Nkx6.1 with respect to its DNA binding properties 2. Characterize the functional activation domains of Nkx6.1 3. Identify and characterize target genes for Nkx6.1 action To achieve these aims, we will utilize in vitro biochemical assays of protein/DNA binding, transient mammalian cell transfections, transgenic mouse models, and cDNA subtraction/differential display. Taken together, the knowledge gained from these studies will provide insight into the mechanisms governing beta-cell differentiation in the developing pancreas. This information can eventually be applied to engineering new beta-cells for patients with diabetes.

DESCRIPTION: (Provided By Applicant) The incidence of both Type 1 and Type 2 diabetes mellitus is rising to alarming rates in the United States. With significant advances in molecular biology and gene therapy in recent years, new approaches for therapy of diabetes are coming from studies of pancreatic B-cell development. One very promising approach is to force precursor cell types to develop into insulin-producing cells by use of "gene regulators," or transcription factors. The long range objective of our laboratory is to understand the mechanisms by which transcription factors direct the differentiation of the insulin-producing 13-cells within the pancreatic islets of Langerhans. Our strategy for this proposal is to focus on Nkx6. 1, a transcription factor that controls the final step of B-cell differentiation. Targeted disruption of Nkx6. 1 in mice leads to embryos that lack B-cells, with no changes in the other cell types that comprise the islets of Langerhans. Based on our preliminary data, we hypothesize that the transcriptional function of Nkx6. 1 is specifically regulated, and to control 13-cell differentiation this factor undergoes a series of intramolecular structural adjustments and intermolecular protein interactions that serves to modulate its DNA binding and transactivation functions. Our specific aims are therefore directed toward a systematic analysis of the DNA binding and transactivation properties of Nkx6. 1, in order to form a model of how this factor functions at the molecular level to cause the final differentiation of B-cells. 1. Determine how the carboxyl terminus of Nkx6. I modulates DNA binding and sequence recognition. 2. Determine how the transcriptional repression and DNA binding activities of Nkx6.1 are modified by protein-protein interactions. 3. Determine the in vivo and in vitro distribution of target genes bound by Nkx6. 1. To achieve these aims, we will utilize in vitro and in vivo assays protein and DNA interactions, cell culture models of 13-cells, and chromatin immunoprecipitation assays. The studies proposed here will provide the framework for understanding the molecular events governing differentiation in the B-cell lineage, and can eventually be applied to engineering new B-cells for patients with diabetes. .

PROJECT ABSTRACT Diabetes is a debilitating and burdensome public health problem, affecting an estimated 29.1 million men, women, and children (9.3% of total population) in the U.S. alone. Patients with this and other related metabolic disorders are burdened with devastating quality of life and health consequences, making the need for proper disease management paramount. As a result, there is an urgent demand for transformative technologies that provide both physicians and patients with more options to prevent, diagnose, treat, and cure diabetes, metabolic diseases, and their complications. Furthermore, an interdisciplinary workforce is needed to accelerate development and delivery of these cutting-edge technologies to the clinic and market. The Bioengineering Interdisciplinary Training in Diabetes Research (BTDR) Program, which was initiated in 2013, was strategically designed to meet this need. A notable aspect of the program is that it integrates the strength in engineering education and technology development found at Purdue with excellence in diabetes research found at Indiana University School of Medicine, yielding an uncommon cross-fertilization. The mission of the BTDR program is to develop the next-generation interdisciplinary workforce that innovates, designs, and translates bioengineering technologies to advance the mechanistic understanding, prevention, and treatment of diabetes and its complications. The aims of the BTDR program are to: 1) recruit highly talented students in engineering, physical sciences, computational sciences, analytical chemistry, pharmacology, physiology, and endocrinology with strong research backgrounds and commitment to innovative technology development and diabetes related research; 2) ensure that the next generation of interdisciplinary educators, scientists, policy makers, and physicians includes individuals from diverse ethnic, social, economic, and regional backgrounds, maximizing the scope and breadth of societal impact; and 3) engage students in a novel training experience that integrates basic research, innovation, design, entrepreneurship, and translation, along with vertical mentoring, to develop an integrated workforce that is equipped to accelerate advanced diagnostics and therapeutics to market. The proposed training program provides training pathways for six predoctoral students (two years support per student) from various engineering, physical science, and medical science disciplines, culminating in PHD or MD-PHD degrees. BTDR offers an uncommon interdisciplinary curriculum and rigorous hands-on training focused on technology design and translation guided by faculty with expertise in the cross- cutting areas of Therapeutic Cell & Drug Delivery, Biosensing & Biomaging, Informatics & Modeling, and Molecular Mechanisms & Drug Targets. Trainee outcomes include the ability to operate beyond hypothesis- driven research, incorporating principles of engineering design, standardization and validation, regulatory policy, technology translation, and entrepreneurship.

Abstract This application is responsive to RFA-RM-19-011 and will focus on a poorly characterized GPCR in the Illuminating the Druggable Genome (IDG) database known as GPR31 in the context of diabetes. Over the past decade, our laboratory has focused on inflammation and the cellular response to inflammation as a central mechanism that contributes to ?-cell dysfunction and death type 1 diabetes (T1D) and type 2 diabetes (T2D). Specifically, our laboratory has demonstrated that 12-lipoxygenase (12-LOX), an enzyme involved in arachidonic acid metabolism and expressed in ?-cells, is activated in the context of ?-cell inflammation and produces the eicosanoid 12-S-hydroxyeicosatetraenoic acid (12(S)-HETE). 12(S)-HETE generates endoplasmic reticulum (ER) and oxidative stress in ? cells, but the mechanism through which this occurs has remained elusive. Deletion of the gene encoding 12-LOX in mice (Alox15), either conditionally in islets or globally, leads to protection from spontaneous type 1-like diabetes in NOD mice and obesity-induced type 2- like diabetes in high fat-fed mice. GPR31 has recently been de-orphaned and identified as the 12(S)-HETE receptor. We have generated congenic Gpr31b-/- mice on the C57BL6/J background through traditional ES cell cloning. Preliminary data suggest that Gpr31-/- mice are viable and metabolically normal, similar to Alox15- /- mice, however it remains to be determined if GPR31 mediates the effects of 12-LOX under diabetogenic conditions in ?-cells. In this proposal, we will generate preliminary data supporting the potential role of GPR31 as a key mediator of inflammation-induced ?-cell dysfunction and death, and the data and resources generated from this proposal will be made available to the Resource Dissemination and Outreach Center (RDOC) of the IDG program. This grant proposal continues a longstanding and productive collaboration between Drs. S. Tersey and R. Mirmira, who are co-located at the University of Chicago. The combined expertise of Dr. Mirmira in ? cell signaling cascades and Dr. Tersey in animal models of diabetes are synergistic towards the completion of this pilot proposal. Our Team will test the hypothesis that GPR31 promotes ?-cell inflammatory signaling and contributes to ?-cell dysfunction and death in the setting of diabetes. To test this hypothesis, the following two aims will be achieved within the timeframe allotted to this RFA: Aim 1: Elucidate the role of GPR31 in mediating the effects of diabetogenic inflammation and 12(S)-HETE in islets. Aim 2: Characterize the metabolic effects of GPR31 in vivo under normal and pro-inflammatory conditions. The primary impact of this proposal is the determination of whether GPR31 is a suitable target for drug development in the context of diabetic inflammation.

This project is inspired by the premise that signals and substrates extrinsic to the ?-cell trigger cell cycle entry, and that reactivation of the cell cycle in ?-cells would provide the potential for restoring ?-cell mass and function in diabetes. Our published and preliminary data suggest that the translation factor eIF5A functions as an acute response factor that catalyzes translation of specific mRNAs, enabling cellular replication. Strikingly, eIF5A is the only protein containing the unique, polyamine-derived amino acid hypusine, which is required for its function. Hypusine formation occurs posttranslationally and is governed by two rate-limiting enzymes, ornithine decarboxylase (ODC) and deoxyhypusine synthase (DHPS). ODC generates intracellular polyamines from arginine (and indirectly from glutamine, proline, methionine, phenylalanine, leucine), and DHPS utilizes the polyamine spermidine to form hypusine on eIF5A. Collectively, we refer to the ODC/DHPS/eIF5A proteins as belonging to the ?polyamine/hypusine pathway.? Because polyamine and hypusine productions can be manipulated by diet and/or small molecules, they represent real-world targets to intervene in ?-cell growth and replication. We hypothesize that the pathway that generates polyamines and hypusine links nutritional signals (amino acids, glucose) to mRNA translation to enable adaptive ?-cell replication. The strength of this Multi-PI R01 application is the collaborative effort between Drs. R. Mirmira (an expert in the polyamine/hypusine pathway in ?-cells) and L. Alonso (an expert in ?-cell replication); our Team is uniquely positioned with the relevant expertise, novel conditional knockout mice, and mRNA translation assessment tools to test this hypothesis. We propose the following 3 aims: Aim 1: Elucidate the mechanisms by which the polyamine/hypusine pathway governs adaptive ?-cell proliferation in models of obesity and hyperglycemia. Aim 2: Characterize how polyamine biosynthesis functions as a nutrient-activated gatekeeper for the proliferative signal induced by the UPR in ?-cells. Aim 3: Reveal an unusual function of eIF5A as a biosensor for amino acid and polyamine supply. We will use a comprehensive toolbox of state-of-the-art imaging and cell biology techniques and novel reagents, including the only collection of ODC/DHPS/eIF5A knockout mice, to reveal a role in ?-cell proliferation for an otherwise enigmatic pathway. Therefore, the primary impact of this proposal is the identification and mechanisms of the polyamine/hypusine pathway in rodent and human ?-cell replication.

Deficiencies in islet ?-cell function and/or mass are central in the transition from impaired glucose tolerance to frank diabetes in the setting of both type 1 and type 2 diabetes. The lipoxygenases (LOXs) represent a family of enzymes that catalyzes the oxygenation of cellular poly-unsaturated fatty acids to form lipid inflammatory mediators in ?-cells. The eicosanoid product of 12-LOX activity, 12-hydoxyeicosatetraenoic acid (12-HETE), imposes inflammatory and oxidative stress within ? cells. A challenge in lipoxygenase biology, however, is that humans and mice express different isoforms of 12-LOX in ? cells (encoded by ALOX12 and Alox15, respectively), with each isoform exhibiting different active-site characteristics. The strength of this application is the collaborative effort between Multi-PIs Drs. R. Mirmira (an expert in islet inflammation pathways) and R. Kulkarni (an expert in growth factor signaling in the islet), and coinvestigator J. Nadler (an expert in eicosanoid biology), who will collectively bring their expertise and unique reagents?including knockout and human gene knock-in mouse models that show human isoform activation, human 12-LOX-selective inhibitors, and primary human cells?to bear on the biology of 12-LOX and its inflammatory products. We hypothesize that during insulin resistance the activation of 12-LOX in ?-cells promotes ? cell dysfunction through receptor-mediated signaling by its downstream product 12-HETE. To test this hypothesis, we propose the following specific aims: Aim 1: Elucidate the molecular mechanisms linking ?-cell insulin resistance to 12-LOX activity and cellular dysfunction. Aim 2: Determine the contribution of 12-LOX activity to ? cell dysfunction in the setting of insulin resistance in vivo. Aim 3: Determine the role of the 12-HETE receptor GPR31 in mediating ?-cell dysfunction downstream of 12-LOX. Until now, tools and reagents to interrogate the biology of human 12-LOX and 12-HETE did not exist. The primary impact of this proposal will be to set the stage for the expectations of therapeutically targeting the human 12-LOX pathway in insulin resistance/?-cell dysfunction, and to identify a potential new target in the 12-HETE G protein-coupled receptor GPR31.

ABSTRACT The pathogenesis of type 1 diabetes (T1D) encompasses a spectrum ranging from aggressive autoimmunity toward islet ? cells to defects in ?-cell function that arise from inflammation. A perspective that has been gaining traction in recent years posits that intracellular signaling pathways arising from the ? cell response to inflammation can lead to the production of aberrant proteins that serve as neoantigens that initiate or exacerbate autoimmunity. This perspective has prompted our Team to identify and intervene in intracellular signaling pathways that affect ?-cell resilience as T1D progresses from the presymptomatic to symptomatic stages. This proposal takes a multidisciplinary Team Science approach that is responsive to RFA-DK-19-024 to define and intervene in early T1D disease processes affecting human islets. The integrated stress response (ISR) is a cytoprotective process whereby environmental stress signals are transduced intracellularly to activate a host of eIF2? kinases. The phosphorylation of eIF2? halts general mRNA translation initiation in an effort to redirect energy expenditure to mitigate the prevailing stress. The translationally inhibited mRNAs and their associated proteins are sequestered into intracellular stress granules (SGs), the formations of which are thought to divert cellular signaling toward an emergency response. Our preliminary data suggest that the ISR is activated in islets during early T1D, and that the pathway linking membrane-derived lipids to the production of proinflammatory lipid intermediates may trigger the ISR and the formation of SGs. We hypothesize that the activation of the ISR and formation of SGs is an early cellular response initiating ? cell stress in T1D that determines cell survival and can be monitored in pre- and early T1D individuals with minimal invasiveness. Our collaborative Team will test this hypothesis through the following aims: Aim 1: Define the mechanisms of stress granule formation and their fate upon activation of the integrated stress response in human islets. Aim 2: Determine the molecular events linking lipid metabolism, activation of the ISR, and stress granule formation in human islets. Aim 3: Identify protein, RNA, and lipid cargo in EVs as putative biomarkers of the human islet integrated stress response and T1D risk. This application leverages the expertise of 6 Multi-PIs in ?-cell biology, lipid and eicosanoid biology, functional genomics, proteomics, computational modeling, and clinical islet studies. The impact of this project will be to deliver new knowledge on an unstudied stress pathway in human islets and to identify and validate biomarker panels that reflect this stress state.