PI:Jodi L. Babitt, M.D. (Massachusetts General Hospital)
Abstract:Bone morphogenetic protein 6 (BMP6) as a regulator of pancreatic islet a -cell mass
Central to the pathogenesis of diabetes mellitus (DM) is the failure of pancreatic b-cells to produce adequate insulin to maintain glucose homeostasis. In type I DM, this is due to autoimmune destruction of b-cells, whereas in type II DM, this is due to loss of both b-cell number and function in the setting of insulin resistance, hyperglycemia, and oxidative stress. Pancreatic a-cells also play an important role in the pathogenesis of DM and as a potential therapeutic target. Long recognized as the source glucagon that stimulates hepatic glucose production and contributes to hyperglycemia in DM, a-cells have recently been reported to play an important role in the promotion and regeneration of b-cells, including the ability to transdifferentiate into b-cells. Understanding the factors that control a-cell mass therefore holds the promise to open up new therapeutic approaches for DM.
Bone morphogenetic proteins (BMPs) are a subfamily of the TGF- b superfamily of signaling molecules that were initially characterized by their ability to induce ectopic bone formation, but have subsequently been shown to regulate the development and homeostasis of many tissues. We will present preliminary data suggesting that BMP6 may function as a b -cell-derived secreted factor that regulates a - cell proliferation. Rat islets overexpressing the homeodomain transcription factor Pdx-1 under the control of an insulin promoter stimulated both rat and human a -cell proliferation. RNA-seq analysis identified BMP6 as one of the most highly upregulated transcripts. Treatment of human islets in culture with BMP6 stimulated a -cell proliferation, effects that were blocked by a small molecule BMP type I receptor inhibitor. Here, we propose to use our milligram quantities of recombinant BMP6 protein, Bmp6 global KO mice, and Bmp6 floxed mice to test the hypothesis that BMP6 is a b -cell derived factor that functions as a regulator of a -cell mass in vivo.
Public Health Relevance: Diabetes mellitus is a significant public health problem affecting more than 30 million people in the US alone. The long-term goals of this proposal are to understand the factors that govern islet cell mass and ultimately to develop new therapeutic avenues for diabetes.
PI: John Campbell, PhD (Beth Israel Deaconess Medical Center)
Abstract:Molecular and Functional Taxonomy of Vagal Motor Neurons
The parasympathetic nervous system controls glucose metabolism through neurons in the dorsal motor nucleus of the vagus (DMV). A subset of these neurons innervates the pancreas and controls its endocrine activity, including insulin release. Understanding how these DMV neurons function and what receptors and signaling molecules they express will reveal novel strategies for treating diabetes. Furthermore, since DMV provides most of the brain’s control of gut motility, including two DMV neuron populations that oppositely affect gastric muscle tone, its dysregulation in diabetes likely underlies gastroparesis. However, while the DMV is known to be functionally diverse, little is known about its genetic diversity. We therefore profiled gene expression in hundreds of cholinergic DMV neurons by single-nuclei RNA-Seq. Unsupervised clustering of these neurons based on their transcriptomes revealed 10 distinct neuron types and specific markers for each. We will now extend these studies by using a more comprehensive approach, Drop-seq, to profile genome-wide expression in thousands of neurons in and around the DMV and classify neuron types. In addition, using recombinase-based genetic technology, we will determine where each DMV neuron type projects by fluorescently labeling their axons and imaging innervated organs (e.g., stomach, pancreas). We will also determine the physiological role of each DMV neuron type by manipulating its activity in vivo while measuring gastrointestinal functions, including gut motility and blood insulin levels. By characterizing the gene expression, synaptic circuitry, and function of each DMV neuron type, these studies will yield unprecedented insight into neural control of glucose metabolism and digestion.
Public Health Relevance. This study is relevant to public health because it will provide mechanistic insight into how the brain controls glucose metabolism and digestion. While previous studies have shown that different types of motor neurons control different aspects of digestion and glucose metabolism through the vagus nerve, little is known about these motor neurons types, including how they signal to the target organs. Knowing what receptors and signaling molecules are expressed by the vagal motor neuron types that control digestion and glucose metabolism will reveal novel targets for treating gastroparesis and diabetes.
PI: Robert Levine, MD (Massachusetts General Hospital)
Abstract:Insights into diabetic cardiomyopathy from volume-overload stress
The nature of diabetic cardiomyopathy remains elusive, especially the reduction of systolic contractility that compounds fibrosis and diastolic stiffness. Some of the uncertainty relates to studies being done mainly under resting conditions. Systolic ventricular dysfunction in diabetes is most prominently expressed under stress. Ventricular dysfunction in metabolic and pressure-overload stress is associated with dysregulation of the transcription factor FoxO1, a central element in control of insulin signaling. A preliminary clinical database study has suggested that diabetic patients with moderate to severe mitral regurgitation have an accelerated increase in LV volume and mass compared with non-diabetic controls. To date, however, the interaction of diabetes and volume overload has not been studied in diabetic models. This pilot feasibility study would test the hypothesis that diabetes mellitus augments and accelerates the progression of systolic ventricular dysfunction and ventricular remodeling due to volume overload. This hypothesis will be tested in transgenic diabetic versus wild type rats, of a size most suitable for creation of an aortocaval fistula producing systemic volume overload. This procedure takes advantage of the skills of an experimental surgeon and laboratory resources at MGH for invasive hemodynamics and noninvasive imaging, in
collaboration with Dr. Joseph Hill, whose group has studied FoxO1 and diabetic cardiomyopathy extensively. Readouts will include studies of FoxO1 and other molecules in the insulin signaling pathway along with insulin resistance. This study can increase our understanding of the mechanisms and therapeutic targets in diabetic ventricular dysfunction.
The heart muscle becomes weakened in diabetes, especially when the heart is stressed. This has been studied in stress caused by high-fat diet and high blood pressure, but not in the volume overload caused by leaking heart valves and other conditions. We will study the molecular changes caused by diabetes in volume overload to understand the mechanisms of heart muscle weakening and develop improved treatments and preventive measures to maintain normal heart function in diabetes.
PI: James Markmann, MD., Ph.D. (Massachusetts General Hospital)
Abstract:The use of intrapleural pancreatic islet transplantation to enhance islet
Pancreatic islet cell transplantation provides a profound treatment for diabetic patients – improved health by avoidance of acute and chronic complications of diabetes, without the need for continued insulin injections. Yet, this treatment often requires multiple transplants due to poor islet survival and function, secondary to the suboptimal transplant tissue environment. In the setting of islet cells administered intravenously, there is a strong inflammatory response to the islets, resulting in decreased viability. In alternate transplantation sites, such as subcutaneous or intraperitoneal, the islets require passive diffusion of oxygen and nutrients from surrounding tissues for survival. Because of the limited oxygen concentration at these sites, islet viability is, again, dramatically decreased. To overcome these issues of oxygenation, recent technologies, e.g. BetaO2, utilize an external device with the significant downside of being cumbersome and requiring daily upkeep. An optimal strategy would be in vivo placement of islets in a well oxygenated environment that allows for survival and function. The work proposed in this application looks to address the issue of poor islet survival by determining the feasibility of intrapleural islet transplantation. The higher oxygen concentration and the greater surface area in the intrapleural space has the potential to provide a superior environment for the growth, survival, and function of the islet cells. Preliminary work in a murine system demonstrates the ability to place viable, functional islets in the pleural space, but the scalability of this procedure is yet to be determined. Therefore, we will investigate using the intrapleural approach to transplant islets to non-human primates, functioning as a preclinical model closely resembling human disease and physiology. The development of an optimal islet cell transplantation system in non-human primates will pave the way for more robust experimentation and in depth investigations of the physiologic and immunologic determinates of islet cell transplantation success and aid in transferring such technology to human patients.
PI: Thomas Michel, MD, PhD. (Brigham and Women’s Hospital)
Abstract:Exploiting novel biosensors to probe insulin-modulated signaling pathways in diabetic cardiomyopathy
For many years, the effects of diabetes on the heart were studied principally in the context of coronary artery and small vessel disease. By contrast, the pathogenesis of diabetic cardiomyopathy is less completely understood. The central aim of the proposed pilot studies is to identify mechanisms whereby insulin receptor-dependent modulation of intracellular reactive oxygen species (ROS) regulates cardiac myocyte signal transduction and physiological responses, with the ultimate goal of understanding the pathobiological basis of diabetic cardiomyopathy and identifying new drug targets.
Our long-standing studies on the biochemistry of endothelial nitric oxide synthase (eNOS) led to our recent discovery that protein phosphorylation pathways regulated by reactive oxygen species (ROS) are critical for NOS regulation. Insulin signaling has long been known to be modulated by ROS, but the molecular mechanisms are almost entirely unknown. We probed the roles of insulin-modulated ROS on cardiac myocyte function, and discovered that insulin-dependent signaling responses in cardiac myocytes are entirely dependent on the stable ROS hydrogen peroxide (H2O2). We found that cardiac myocytes isolated from mice fed a high-fat diet develop insulin resistance in cellular signaling responses involving H2O2. We cloned and characterized a series of novel differentially-targeted constructs expressing the H2O2 biosensor HyPer in the cardiotropic adeno-associated virus-9 (AAV9). We exploited these AAV9-based HyPer constructs in advanced cellular imaging approaches in mouse models in vivo to probe the roles of ROS in cardiac myocyte insulin signaling. We propose to exploit our novel H2O2 biosensors to characterize insulin-modulated ROS pathways in cardiac myocytes in a mouse model of type II diabetes.
The proposed studies will identify the physiological as well as pathophysiological pathways controlling insulin-modulated ROS responses in the heart, and may identify novel targets for the prevention and treatment of cardiac dysfunction in diabetes.
Lay summary: Many patients with diabetes suffer a form of heart failure called “diabetic cardiomyopathy”. With the increasing prevalence of diabetes in the USA, diabetic cardiomyopathy has become a public health problem without effective cures. The proposed studies will study heart cells (cardiac myocytes) using advanced imaging methods and novel biosensors to identify new therapeutic targets that may help to prevent and treat heart disease in diabetic patients.