Emilyn Alejandro - US grants

[unreadable] DESCRIPTION (provided by applicant): Diabetes is a major health problem in United States of America. Despite extensive study, the exact causes of this disease are not known. However, evidence suggests that all forms of diabetes are associated with reduced beta-cell function and survival. Our laboratory has recently shown that insulin itself controls beta-cell survival. Objective of research: The goal of the proposed research is to characterize the mechanisms through which insulin regulates beta-cell survival in human and mouse islets. We hypothesize that insulin may promote beta-cell survival via a kinase called Raf-1. Description of research project: We will utilize molecular biology, live-cell imaging and knockout mice to dissect the role of Raf-1 in anti-apoptotic beta-cell insulin signaling. Relevancy to diabetes: We expect our proposed experiments to yield new mechanistic information regarding insulin as a beta-cell survival factor. Greater understanding of the control of apoptosis in human islets is crucial to prevent beta-cell death in all types of diabetes and to improve clinical islet transplantation. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Type 2 diabetes (T2D) is a major health problem worldwide. Identifying modifiable risk factors is key in decreasing the incidence and associated economic burden of T2D. Both genetic and environmental factors contribute to the development of T2D. The fetal nutrient environment during pregnancy is as a major factor that modifies the risk for developing T2D. Studies from humans and animal models show robust associations between poor fetal growth and the development of T2D due to maternal malnutrition during pregnancy, which results from permanent changes in pancreatic ?-cell function and increases susceptibility to T2D. The overall research objective of this K01 proposal is to understand how maternal low-protein-diet during pregnancy (LP0.5) alters the offspring's ?-cell function and susceptibility to T2D and to identify the mechanistic link between LP0.5 and sensitivity to cellula stress dysregulation in chronic hyperglycemia and hyperlipidemia conditions (glucolipotoxicity). The roles of O-GlcNAc transferase (OGT), a nutrient-sensing protein and a key regulator of cellular stress responses, in LP0.5 ?-cell susceptibility to T2D will also be identified. Thus, th central hypothesis to be tested is that LP0.5 predisposes offspring to T2D by regulating OGT levels, which enhances the susceptibility of ?-cells to glucolipotoxicity-induced ER stress and cell death. Three specific aims will be carried out to test this hypothesis: 1) Identify the mechanisms of how LP0.5 ?-cell susceptibility to glucolipotoxicity; 2) Identify how OGT activity (enhanced O-GlcNAcylation) modulates ?-cell susceptibility to glucolipotoxicity-induced ER stress; and 3) Determine the extent to which gain of O-GlcNAcylation during pregnancy rescues the abnormalities induced by LP0.5. Based on preliminary data, the working hypothesis of these specific aims is that LP0.5 predisposes ?-cells to increased sensitivity to glucolipotoxicity by enhancing ER stress and cell death responses by regulating OGT activity. This hypothesis will be tested in vivo by subjecting LP0.5 mice to in vivo infusion of glucose and lipid. ?-cell functin, molecular markers of ER stress and cell death, and morphology of the ER will be assessed. The working hypothesis that enhancing O- GlcNAcylation during pregnancy will protect the offspring against T2D by inducing long-term gains in ?-cell mass and function will further show the importance of OGT in ¿-cell development and function. Thus, the metabolic profile and ?-cell phenotype of LP0.5 offspring exposed to a drug that enhances O-GlcNAcylation during gestation will be assessed in normal and diabetogenic conditions. These studies will fill significant gaps on the roles of OGT in ?-cell development and function and the pathogenesis of T2D and the results will have a significant impact on multiple levels. In the short-term, the generated data will identify the molecular mechanisms by which LP0.5 alters ?-cell development, impacting sensitivity to cellular stress and the susceptibility to T2D. The long-term objective is that these studies will aid in the development of drugs targeting OGT and dietary approaches to prevent T2D and other chronic diseases.

Project Summary: Type 2 diabetes (T2D) is a major public health concern worldwide. Chronic hyperglycemia and hyperlipidemia are causative factors for T2D by inducing pancreatic ?-cell failure. Two of the mechanisms causing ?-cell failure include mitochondrial dysfunction and disruption of protein quality control systems and pathological unfolded protein response (UPR) in the Endoplasmic Reticulum (ER) leading to death of insulin- producing ?-cells. O-GlcNAc modification (O-GlcNAcylation) onto proteins by the enzyme OGT (O-GlcNAc Transferase) is crucial for many important biological processes including mitochondrial function, ER stress response and metabolism. Global reduction of O-GlcNAcylation (by deleting OGT in ?-cells) causes T2D and ?-cell failure, in part, due to enhanced ER stress and hyperproinsulinemia. We hypothesize that OGT regulates survival and function by regulating the O-GlcNAcylation state of mitochondrial, ER-UPR, cytoplasmic, and nuclear proteins. Identification of O-GlcNAc modified proteins in ?-cells have not been done before and may lead to new targets for treatment for diabetes. Specific Aim1 will determine and quantify changes in proteome and to identify OGT targets in islets with conditional and inducible loss or gain-of-function OGT in ?- cells. Specific Aim2 will identify the mechanisms of hyperproinsulinemia and determine mitochondrial dysfunction in islets of mice with loss and gain-of-function OGT in ?-cells. Identification, site mapping, and quantification of O-GlcNAc proteins are a prerequisite to decipher their functions and to gain insights in the factors that determine structure- function relationship. In long term, this project will identify potential new pharmacological targets to improve ?-cell mass and function.

The rising prevalence of type 2 diabetes (T2D) is a major public health concern worldwide. It is clear that both genetic and environmental factors contribute to T2D. Known genetic variants account less than 10% of the risk for T2D predisposition. Evidence from human epidemiology and animal studies show that fetal nutrient environmental factors (e.g. placental insufficiency) provide additional susceptibility to T2D. We and others have shown that maternal low-protein diet throughout pregnancy (LP0.5) causes intrauterine-growth restriction (IUGR), a critical factor known to predispose offspring to T2D, by causing long-term consequences in ?-cell mass and function. Impaired placental growth and function are major causes of IUGR. mTOR, a nutrient-sensor kinase, couples signals from nutrients (e.g. amino acids)) and growth factors to promote cellular growth of an organism. Reduced mTOR signaling is correlated to decrease fetal placental function such as amino acid transport in human and rodent models of IUGR. By using multiple murine models, this R03 grant expands the K01 award and determine the requirement of fetal placental mTOR signaling in the developmental programming of ?-cell mass and function. We will test the hypothesis that changes in placental mTOR activity is sufficient to alter developmental programming of ?-cell dysfunction in the offspring with the following 2 aims: 1: Determine ?-cell mass and insulin secretion function in islets with placental loss or gain-of- function mTOR signaling; and 2: Identify the mechanisms of how placental mTOR signaling induces developmental programming of ?-cell mass and function in the offspring. The impact of these studies will show definitively the independent impact of placental mTOR signaling on ?-cell mass development and function programming in the offspring. We will identify specific placental factors that will yield novel insights into the mechanisms whereby mTOR regulates fetal nutrients. Finally, this grant will illustrate the translational relevance of placental mTOR as a biomarker to identify individuals at risk for T2D, thereby advancing clinical care.

Type 2 diabetes (T2D) is the most common chronic disease affecting human health. Recent longitudinal and genome-wide association studies provide strong evidence that the ability of pancreatic ?-cells to fulfill insulin demand through development, growth, survival, and function is a key determinant of whether an individual will develop T2D ! under various nutrient conditions. However, there are no effective clinical treatments that target ?-cell growth and maintenance of their differentiated identity as insulin producing-cells. We propose that OGT (O-GlcNAc Transferase), a nutrient-sensor expressed at a very high level in ?-cells, has key developmental regulatory properties and the ability to integrate signaling networks to regulate ?-cell plasticity in response to insulin demand and nutrient stress. OGT is the sole enzyme adding a single O-GlcNAc post-translational modification (O-GlcNAcylation) onto proteins to orchestrate and fine-tune glucose metabolism, and ?-cell growth and maintenance of identity under stress responses to nutrient changes and hormonal cues. We hypothesize that OGT tightly controls the O-GlcNAcylation state of downstream targets, including Pdx1, to promote ?-cell development and function. Thus, our long-term goal is to define the mechanisms of how OGT integrates signaling networks impinging on ?-cell plasticity (development and identity) to promote functional ?-cells. We will test our hypothesis with the following Aims: 1. To establish the molecular mechanisms of how OGT regulates ?-cell development and mass. 2. To delineate the mechanisms of how OGT regulates ?-cell mass and identity under metabolic stress. The impact of this grant will show the central role of OGT in ?-cell development and mass maintenance, and illustrate the translational relevance of OGT during time windows critical to metabolic health . Finally, these results will advance the field of ?-cell biology and will open new horizons for therapies for patients with diabetes.

Placental insufficiency during pregnancy causes intrauterine growth restriction (IUGR) and predisposes offspring to Type 2 diabetes with reductions in functional ?-cell mass. Understanding the mechanism of this decrease in ?-cell mass in utero is essential to developing treatment strategies to prevent or reverse it. The complement system is an innate immune amplification system essential for host defense, inflammation and fetal survival, but excessive complement activation is associated with pregnancy complications including preeclampsia with IUGR. In the reduced uteroplacental perfusion pressure (RUPP) model of placental insufficiency in rat, blood flow to uteroplacental unit is mechanically disrupted at embryonic day (e)14, beginning of third trimester, resulting in placental ischemia, high blood pressure in dam and IUGR. Using this model, our published studies demonstrated ? cell area is reduced and increased apoptosis evident in e19 islet of growth restricted fetus, and increased maternal complement activation is critical for hypertension in RUPP dam. Our preliminary studies demonstrate decreased C3 and increased macrophage marker in e19 islets of RUPP offspring. Long-term goal: Determine therapeutic utility of manipulating complement or macrophages in utero or postnatally to mitigate reduced ? cell mass in IUGR offspring. Objective: Assess contribution of C3, C3a and macrophages to ? cell mass and survival in RUPP model. Central hypothesis: Placental ischemia results in decreased C3/C3a and change in macrophage numbers or M1/M2 phenotype in islets of offspring, ultimately leading to reduced ? cell mass and increased apoptosis. Aim 1: Determine whether decrease in C3 and/or C3a (Aim 1) or change in macrophage numbers or phenotype (Aim 2) are required for placental ischemia-induced reduction in ? cell mass and apoptosis in developing fetal pancreas. Time course of changes in C3, C3a, C3a receptor, and macrophages will be determined from e15 to 12 wk postnatal. Effect of decreasing C3 by siRNA or antagonizing C3aR in normal pregnancy (in utero or postnatally) on ? cell area or mass, apoptosis and macrophages will be determined. Conversely, ability of increasing C3a by administration of a C3aR agonist in RUPP offspring to rescue ? cell mass will be evaluated. The effect of fetal macrophage depletion using clodronate liposomes in utero or postnatally on complement, ? cell mass and apoptosis in fetal islets with and without placental ischemia will be assessed. This research is innovative because it is the first to examine the mechanistic role of complement in pancreatic development and its response to IUGR stress, and combines intradisciplinary expertise in islet biology with expertise in complement and RUPP IUGR model. This contribution will be significant because it will determine if targeting complement system or macrophages early in pancreatic development is a feasible therapeutic strategy for preserving pancreatic ? cell mass in the face of placental ischemia and thus preventing long term consequences in adulthood.