Louis H. Philipson - US grants

The focus of this revised application is to elucidate the roles of voltage- dependent K+ channels in the regulation of pancreatic beta-cell membrane potential, [Ca2+]i, and insulin secretion. Several types of K+ channels are involved in controlling both depolarization and repolarization of the beta-cell membrane. These oscillatory changes in membrane potential regulate Ca2+ influx via voltage-dependent Ca2+ channels and the subsequent release of Ca2+ from intracellular stores. This application proposes to study these events in mouse transgenic models in which delayed rectifier K+ channel expression is enhanced by overexpression of a tagged human channel, or diminished by the expression of a dominant-negative channel construct. The first specific aim is to characterize the expression of the hPCN1/Kv1.5 channel and a truncated dominant-negative channel in transgenic mice. Transgenic mice over-expressing the Kv1.5 channel have been constructed and the initial characterization of one pedigree is discussed. Preliminary experiments in one pedigree have verified the presence of the transgene and the associated occurrence of age-dependent hyperglycemia. betaTC3 insulinoma cells have also now been constructed which overexpress the same transgene, and preliminary results show profound effects on glucose- stimulated [Ca2+]i transients. These effects are substantially reversed by K+channel blockers. An additional model will be constructed which expresses a similar but truncated transgene, which contains only the amino terminal binding element for channel assembly and the first membrane spanning domain. The predicted effect will be to prevent normal association of Shaker-like ion channels in the beta-cell, attenuating delayed rectifier expression. The second aim is to determine the effects of these transgenes on beta-cell or islet whole cell current, membrane repolarization, and [Ca2+]. Building on preliminary data on the betaTC3- cells which overexpress Kv1.5, isolated islets and dispersed beta-cells will be studied by patch clamp techniques in the whole cell configuration to measure outward currents, and by loading with fura-2 to measure [Ca2+]i transients. Study of this model will reveal what, if any, other regulatory mechanisms exist to control repolarization. The third specific aim is to characterize insulin secretion in these novel transgenic ion channel models. These experiments will test the hypothesis that oscillations in membrane potential and [Ca2+]i are functionally related to oscillations in insulin secretion and may serve as a direct demonstration of the importance of repolarization in regulating hormone secretion in unique models of defective glucose signalling. It is anticipated that the research performed under this proposal will lead to an increased understanding of the relationships between the beta-cell membrane potential, [Ca2+]i, and insulin secretion and may lead to novel therapeutic approaches in non- insulin dependent diabetes mellitus.

NOD mouse; laboratory mouse; tissue /cell culture

DESCRIPTION ( Adapted from applicant's abstract): This renewal application of an award in the third and final year aims to elucidate the roles of repolarizing delayed rectifier K channels (Kv) in pancreatic B-cell excitation-secretion coupling, specifically examining electrical activity, (Ca2+)i, and insulin secretion. Major goals of the first funding period have been met with the conclusion that Kv1 channels are unlikely to play a major role in the B-cell. Significant progress included demonstration of Kv2 and Kv3 expression in B-cells and insulinoma cells. In the next period we will test th hypothesis that regulation of membrane repolarization by Kv2 and Kv3 govern B-cell glucose responsiveness through these interlocking Specific Aims: Specific Aim 1: To define the factors that influence the expression of Kv channels in the B-cells of transgenic mice. Dominant negative transgenics for Kv2 and Kv3 will be compared with similar constructs that suppress related Ca2+-activated K channels. Factors that determine transgenic and endogenous Kv channel expression will be defined. Specific Aim 2: To define the effects of K isoform suppression on glucose tolerance and insulin secretion. Glucose induce secretion will be studied in whole animals, the isolated perfused pancreas, an perifused islets and compared with antagonist blockade of Kv channels. These experiments, in combination with those of the next aim, will test the hypothesis that Kv regulation of membrane repolarization is functionally related to insulin secretion, and will determine whether alterations in B-cell excitability lead to compensatory changes or hyperinsulinemia. Specific Aim 3:_To define the effects of Kv isoform suppression on whole cell currents, electrical activity and (Ca2+)i transients in B-cells and islets, and correlat them with changes in insulin secretion. The B-cells of delayed rectifier-deficient mice are predicted to have slowed repolarization, increase excitability, and enhanced (Ca2+)i transients. The amplitude and pharmacology of the outward currents and the characteristics of electrical bursting will be determined. Experiments performed under these specific aims will allow us to (1) test the overall hypothesis that Kv channels play a key role in membrane repolarization; (2) test the degree of coupling of glucose stimulation to membrane events and insulin secretion in vivo models and (3) define the functional importance of specific delayed rectifier isoforms expressed in B-cells. The differences in sensitivities of these isoforms to Kv blockers may lead to the development of novel therapeutic approaches in NIDDM.

DESCRIPTION (provided by applicant): The overall goal of this proposal is to understand the calcium (Ca2+)-dependent stimulus-secretion coupling mechanisms that regulate beta-cell function in vivo from the perspectives of biophysics, physiology and molecular biology. This goal will be achieved by studying intracellular Ca 2+ concentration, mitochondrial function, glucose metabolism, and insulin secretion in mouse and human islets in which the beta-cells have been engineered specifically for functional imaging. Ca2+-dependent signal transduction mechanisms that regulate insulin secretion have been well-defined in vitro using intact islets, primary cultures of beta-cells, and insulinoma cells. Notwithstanding the important contributions of the in vitro approaches, knowledge of the mechanisms underlying beta-cell function in intact islets in vivo remains incomplete. Several technical limitations of the current imaging methodologies do not permit the study of beta-cell function within the complex multicellular environment of the pancreas in situ. The central focus of the experiments described in this proposal is to develop and characterize a novel imaging approach, which will facilitate the study of islet cell function in situ. In Specific Aim 1, viral gene transfer vectors will be used to transduce mouse cells with genetically targeted biosynthetic fluorescent Ca 2+ sensors. Their effectiveness in studying a-cell biophysical and physiological responses following secretagogue stimulation will be evaluated. In Specific Aim 2, the utility of these sensors as functional imaging indicators in intact mouse islets will be assessed. In Specific Aim 3, beta-cells within intact human islets will be engineered to express Ca 2+ biosensors and human islet function studied in vitro with comparison to mouse islets. In Specific Aim 4, transgenic mouse models in which the beta-cells have been genetically enhanced to express Ca 2+ biosensors will be developed and characterized in vitro and in vivo by confocal microfluorometry and by measurements of insulin secretion. The studies will provide new understanding of islet cell biology that will benefit clinical strategies to preserve and maintain functional beta-cell mass.

The Islet Cell Biology Core provides services and hands-on training to independently funded investigators in the isolation and functional characterization of pancreatic islets from normal and diabetic humans and mice. It also maintains a repository of insulinoma cell lines as well as other endocrine cell lines. The primary emphasis of the Core is to facilitate studies of primary islet cells, and it has developed many unique tools and techniques for carrying such studies including novel animals models, biophysical methods and a library of adenovirus-based expression constructs for studying beta-cell function. The long-range objectives and goals of the Islet Cell Biology Core are to provide state-of-the-art technology and know-how to understanding the beta cell in health and disease. It has the following Specific Aims and Objectives: Provide advice, service and training in the isolation of pancreatic islets from normal and diabetic mice and humans; Provide insulinoma and other endocrine cell lines; Provide advice, service and training in the characterization of mouse and human pancreatic islets and beta cell (insulin biosynthesis and secretion; biophysical methods for studying islet and beta-cell function (e.g. calcium imaging, electrophysiology, total internal reflection fluorescent microscopy); biochemical methods for studying beta-cell function (e.g. phosphorylation, proliferation, apoptosis); and immunohistochemical analysis of pancreatic islets) and Provide advice, service and training on the use of adenovirus-based expression constructs to study protein function in beta cells and other cell types in vitro and in vivo. Drs. Philipson, Rhodes and Witkowski are Directors of this Core. They will be advised by experts in pancreatic islets and beta cells (Prince and Steiner), cellular and physiological imaging (Bindokas, Chen and Glick), ion channels (Hanck and Nelson), spectroscopy (Halpern and Scherer) and cell dynamics (Dinner) to ensure that the Core services are at the forefront of technology and thus able to anticipate and quickly repond to users needs.

DESCRIPTION (provided by applicant): This application Chicagoland TrialNet Clinical Center is in response to the RFA -DK-13-010 Type 1 Diabetes TrialNet Clinical Centers (U01). Here we describe a joint effort from the University of Chicago and Advocate Children's Hospital (ACH) sites (Park Ridge and Oak Lawn, Illinois) and Advocate Health Care (AHC) to create a new Clinical Center in the Chicagoland area. These organizations along with our other affiliate (Lurie Children's Hospital) and partner sites saw over 9000 patients with type 1 diabetes (T1D) in 2012. Together, these practice sites along with other participating key adult and pediatric diabetes practitioners from Chicago reach over 12 million individuals within a two-hour drive of a screening location. The Chicago community, strong supporters of the Juvenile Diabetes Research Foundation (JDRF) and American Diabetes Association (ADA), has been under-represented in clinical trials for Type 1 Diabetes (T1D). This partnership will bring together the resources of major medical centers committed to T1D research and clinical care to reach more of the estimated 100,000 individuals with T1D in the Chicagoland area (about 0.8% of the area population) and their families, as well as the 2000 projected new cases of T1D and their families each year. This outreach will involve diverse communities from some of the wealthiest to some of the most underserved in the United States. We will build on our success in creating the largest database for monogenic diabetes in the United States. We will engage the greater Chicagoland community through innovative social media and direct communications combining media support, direct visits to local practices, community events supported by our institutions and partners and outreach to large networks of faith communities. The University of Chicago has been committed to diabetes research for over 100 years, and together with Advocate Children's Hospital and other partners we will bring a new sense of energy, organization, and purpose to TrialNet recruitment and screening in the Chicagoland region.

DESCRIPTION (provided by applicant): The Pediatric Endocrinology Research Training Program at the University of Chicago will train pediatric physician-scientist leaders in the investigation of endocrine diseases. This training program will help fill the national shortage of physician-scientists in the area of pediatric endocrinology. Two research training slots are requested in this competitive renewal application so that two trainees can be entered annually into a two-year research training program. Thus, this program is for trainees who have had introductory laboratory and course work in their initial year of pediatric endocrinology training and have developed a research project to which they are prepared to dedicate 80% effort for the two-year training period. The training is based in a combined Pediatric-Adult Endocrinology, Diabetes, and Metabolism unit, the Institute for Endocrine Discovery and Clinical Care which is now 6 years old. We are unique in the country with a fully integrated section that is a major training institution of dual boarded physician scientists in pediatric and adult endocrinology. The Senior Training Faculty numbers 15 investigators from four University departments (Medicine, Pediatrics, Human Genetics, Health Studies) who carry out a broad range of endocrine-related clinical and basic research supported by a substantial base of NIH and other peer-reviewed research grants including 27 R-awards, 2 K- awards, 5 P-awards and 6 U-awards. Each is an established investigator. In addition, the program is aided by the participation of 10 Associate Training Faculty and 5 additional resource training faculty from the Departments of Medicine, Pediatrics, Ob/Gyn and Health Studies. The University provides a rich environment of other faculty and physical resources. Trainees are selected on the basis of prior individual accomplishments including prior research training and experience as well as commitment to an academic research career. Trainees select a basic and clinical research mentor and an advisor, and the trainee and mentors jointly identify a primary and secondary research project. Trainees then undergo at least 3 years of training in the research laboratory of the preceptor(s), during which time they assume a progressively greater responsibility for developing research hypotheses, designing experiments, analyzing the data and preparing abstracts and scientific manuscripts. This research training occurs within the framework of a required core curriculum consisting of courses that describe and review current research methodology and research advances as well as statistical analysis of research data. The setting is one that emphasizes translational research and timely monitoring of trainee progress.

? DESCRIPTION (provided by applicant): Single gene mutations cause approximately 1% of all cases of diabetes and up to 10% of cases diagnosed at less than 35 years of age. However, the vast majority of cases of monogenic diabetes are misdiagnosed as type 1 or type 2 diabetes. Here we propose to evaluate the consequences of a diagnosis of monogenic diabetes and to develop and validate new cost-effective methods to improve diagnosis and treatment. We and others have suggested that a precise genetic diagnosis of diabetes enables targeted therapy, leads to improved quality of life, and aids in diagnosis of diabetes in other family members including earlier diagnosis in children. Despite this, the overwhelming majority of patients with these forms of diabetes remain undiagnosed and most often inappropriately treated. Those with diabetes due to mutations in KCNJ11, ABCC8, HNF1A and HNF4A may be effectively managed with sulfonylurea therapy instead of suboptimal insulin injections, while individuals with GCK-related diabetes generally do not require drug treatment. However, the best treatment of several other forms of monogenic diabetes remains poorly understood. Furthermore, the variables predicting failure of monotherapy in sulfonylurea-responsive forms and the best choices for second-line agents is not known. Using a simulation model of diabetes costs and complications, we have demonstrated the potential cost-effectiveness of genetic testing for the diagnosis of neonatal diabetes and MODY. The development of targeted next-generation sequencing approaches that facilitate testing of the more than 40 genes known to cause monogenic diabetes will allow us to efficiently examine the true cost-effectiveness of genetic testing. With genetic testing for diabetes slowly becoming more readily available, the impact of testing results - positive, negative and inconclusive - have not been fully assessed. We propose to 1) Determine the efficiency of next-generation sequencing in monogenic diabetes and impact on the cost-effectiveness of genetic testing leading to targeted treatment and 2) Determine the impact of positive genetic testing results on treatment and quality of life while also assessing th impact of indeterminate results. The overall goal of this proposal is to determine the benefits of routine testing for monogenic forms of diabetes in appropriately selected individuals and promote the resulting improvements in treatment through guidance and support of patients, families and physicians.

PROJECT SUMMARY ? ADMINISTRATIVE CORE ABSTRACT The Administrative Core oversees the National Center for Identification and Study of Individuals with Atypical Diabetes Mellitus. The purpose of the Center is to characterize the phenotypic and genetic spectrum of atypical diabetes. In order to do so, we will bring together internationally recognized diabetes investigators with expertise in the pathophysiology and genetics of diabetes to: 1. Foster the study of individuals with rare/atypical forms of diabetes mellitus; 2. Identify and analyze rare phenotypic and genotypic variants of diabetes that may ultimately provide insights into more prevalent, heterogeneous forms of type 2 diabetes mellitus (T2DM) in the general population; and 3. Develop a community resource to advance research in this area through the collection and dissemination of phenotypic data and biological samples for access by the diabetes research community. The lines of authority for this Center includes a Director, Louis H. Philipson (PI), a co-Director, Jose Florez (co-PI), and two Associate Directors, Siri Atma W. Greeley and Graeme I. Bell, who are responsible for the day-to-day and longer-term administration, coordination and evaluation of Center activities. They are supported by a Research Advisory Committee comprised of the Directors and Co-Directors of the Core and Projects, a Project Manager, Project Administrator, and members of an Internal Advisory Committee: Rudy Leibel (Columbia University), John B. Buse (University of North Carolina), Steven E. Kahn (University of Washington), and Alvin Powers (Vanderbilt University). An expert in the ethical conduct of medical research, Lainie Ross, will also assist the Administrative Core in its duties. With the support of the NIDDK, an External Evaluation Committee will be created. In summary, the Administrative Core will provide leadership, strategic and programmatic vision, oversight and management of a program that promotes collaboration, synergy and excellence in diabetes research. The main goals of the Administrative Core will be to: 1. Create an administrative structure to provide research and financial management of all Center activities to ensure optimal utilization of resources including the Center?s database; 2. Provide a platform for promoting maximum communication and exchange of ideas among Center members and colleagues; 3. Use the IRB at the University of Utah Trial Innovation Center (TIC), funded by the National Center for Advancing Translational Sciences (NCATS), as the central IRB for clinical research studies; and 4. Coordinate outreach to raise awareness of research efforts, disseminate research progress, and foster translation and application of research findings to the broader community.

PROJECT SUMMARY/ABSTRACT RADIANT [Rare and Atypical DIAbetes NeTwork] is a nationwide network of researchers seeking to identify new rare, atypical forms of diabetes. To discover novel forms of atypical diabetes, the RADIANT investigators will perform genetic testing and deep phenotyping which may include some or all of the following: a physical exam, psychological and cognitive screening tests, laboratory data, and a variety of other clinical tests. This deep phenotyping will enable the investigators to look for patterns amongst patient-participants presenting with atypical diabetes. It will generate a significant amount of participant-specific health data, some of which will have unknown significance. RADIANT investigators are developing policies about which results to return and how to return them. In this supplement we focus on the return of non-genetic clinical results. Returning all results may not be beneficial (information overload); and yet deciding which results to return is not inherently value neutral. Returning results is further complicated by the fact that not all of the results are fully interpretable; leaving participants with information of unknown significance and investigators unsure of how to explain the uncertainty. We aim to explore what participants think they are signing up for when they agree to the return of results and whether investigators are prepared to return these results. The controversies being faced by the RADIANT consortium are five-fold: 1) which results to share; 2) how to disclose abnormal results; 3) whether and to what extent investigators have an obligation to provide an interpretation of research findings to participants; 4) whether participants can refuse to get research results that may be clinically actionable; and 5) what are the obligations of RADIANT investigators if participants elect not to receive results and/or elect not to allow the results to be shared with their HCPs. Given that these types of findings may become more common as discovery research continues to expand, it is important to understand how best to serve the needs and goals of both participants and investigators in an era where reporting back results is also becoming more commonplace. We aim to explore these questions with individuals who have atypical diabetes and the RADIANT investigators who study them. The timing of this project is ideal given that the 21st Century Cures Act goes into effect on April 5, 2021. The Cures Act gives patients fast and free virtual access to their clinical laboratory records, imaging data, and physician notes. We will explore the challenges raised by blurring practices in clinical care and research from the perspective of RADIANT participants and investigators, and the ability of physician-investigators and patient-participants to distinguish between the two. The outcome of the proposed study is highly generalizable to many other studies outside of diabetes genetics research given broad support for (patient and) participant access to their individual (clinical and) research results.

PROJECT SUMMARY ? ENRICHMENT PROGRAM The Enrichment Program promotes knowledge, the exchange of ideas, training, and education in a culture of multidisciplinary interaction, collaboration, and synergy. It serves as a forum to present and discuss diabetes research across the translational spectrum through the Annual Chicago Diabetes Day held on the University of Chicago campus in May and Diabetes & Obesity Research Day held on the University of Illinois at Chicago campus in October. These meetings provides an opportunity for investigators with Pilot and Feasibility Studies to present their findings, informs its membership of new trends, discoveries, programs, grant opportunities, and development in diabetes research. The Enrichment Program also supports the Midwest Islet Club and Diabetes Research Center leadership organizes the Kroc Lecture, the Lydia J. Roberts Lecture and the Max Miller Lecture in Diabetes Research. The Enrichment program supports training and educational opportunities for undergraduate and medical students, house-staff and postdoctoral fellows as well as for patients with diabetes. The program has been highly successful and we believe is a ?crown jewel? of this Diabetes Research Center. However, we believe there is also room to improve and in the new five years will implement new initiatives in the area of diabetes and bioengineering as well as provide direct support for a diabetes seminar program at the Medical College of Wisconsin.

PROJECT SUMMARY ? DISCOVERY AND ANALYSIS PROGRAM ABSTRACT Here we describe the Discovery and Analysis Program for the National Center for the Identification and Study of Individuals with Atypical Diabetes Mellitus. The purpose of the project is to bring together internationally recognized diabetes investigators with expertise in the pathophysiology and genetics of diabetes to: A) Foster the study of individuals with rare/atypical forms of diabetes mellitus and B) Identify and analyze rare phenotypes and genotypic variants of diabetes that may ultimately provide insights into more prevalent, heterogeneous forms of type 2 diabetes mellitus (T2DM) in the general population. The central hypothesis of the entire center is that the identification and study of new cases of rare/atypical forms of diabetes will yield greater insights into the etiology and genetic heterogeneity of T2DM. To reach the goals of this project, building on the track records of the participating groups over the last three decades, we will seek to accomplish the following Specific Aims: (1) Identify and enroll individuals/families (children and adults) with rare/atypical forms of diabetes utilizing a unified ascertainment protocol supported by the resources and expertise from participating diabetes centers; (2) Characterize novel diabetes genes/variants in the cohort associated with new rare/atypical forms of diabetes and identify pre-symptomatic at-risk members of the family; and (3) Characterize the cardinal features and phenotypic spectrum associated with the identified novel genes/variants and evaluate age-related disease penetrance. The general approach is to promulgate a strategy for identification of rare/atypical forms, filter out primary autoimmune and known monogenic forms, and further characterize the remainder. Deeper phenotyping and genomic characterization of these individuals and their families in subsequent studies should help to characterize milder or otherwise atypical subtypes present in the spectrum of T2DM in the general population and reveal novel mechanistic pathways involved in the pathogenesis of diabetes. Identification of these novel genes and pathways may ultimately point to novel strategies for the diagnosis, treatment, and prevention of T2DM.