Fawaz Haj - US grants

Type 2 diabetes mellitus is one of the most prevalent metabolic diseases that is characterized by hyperinsulinemia, insulin resistance, and defect(s) in islet secretory function. Insulin[unreadable]s action is mediated by a complex network of signaling events that are initiated upon insulin binding to the insulin receptor (IR). The precise mechanism underlying insulin resistance remains incompletely understood but it is thought that tyrosine phosphorylation plays an important role. Tyrosine phosphorylation is tightly controlled by the opposing actions of protein-tyrosine kinases and protein-tyrosine phosphatases (PTPs). Protein-tyrosine phophatase 1B (PTP1B) has been implicated as a major physiological regulator of glucose homeostasis and adiposity. Recent insights into the physiological role of PTP1B, using tissue-specific deletion approach, revealed a diverse and complex function in various insulin-responsive tissues. However, the role of PTP1B in regulating pancreatic function remains largely unexplored. Insulin signaling plays an important role in -cell function and mass, thus it is important to understand the physiological role of PTP1B, a major regulator of insulin signaling, in the endocrine pancreas. We previously demonstrated a role for PTP1B in regulating -cell homeostasis and showed that PTP1B deficiency can partially compensate for -cell failure in insulin receptor substrate 2 (IRS2) knockout (KO) mice. In preliminary studies we show that (i) PTP1B is expressed in primary islets, (ii) is a regulator of -cell function, insulin secretion and systemic glucose homeostasis, and (iii) identified putative substrates that are involved in cell-cell communication. These preliminary findings are the first to directly implicate PTP1B in the regulation of islet function in vivo. To fully assess the physiological role of PTP1B in pancreatic islets, we will employ three complimentary approaches. We will generate pancreas-specific PTP1B knockout (panc- PTP1B KO) mice to study the direct consequences of PTP1B loss in the pancreas in vivo. Equally important are the dissection of the molecular mechanisms mediating PTP1B[unreadable]s function, and characterization of PTP1B substrates in -cells. The broad goals of this proposal are to investigate the physiological role of PTP1B in pancreas endocrine function with the long-term aim of generating therapies for the treatment of diabetes. It is envisioned that the successful completion of these studies will lead to major insights into the regulation of pancreatic -cell signaling, and aid in identifying targets for therapeutic intervention for both type 1 and 2 diabetes.

DESCRIPTION (provided by applicant): Disorders of body mass regulation such as obesity are increasingly common causes of morbidity and mortality. Despite significant progress, many cellular and molecular details of energy homeostasis remain unknown. Increased understanding of the molecular signaling mechanisms will aid in the development of more effective therapies for obesity and its associated disorders, such as type 2 diabetes. Protein tyrosine phosphorylation and dephosphorylation is a fundamental signaling mechanism in cells, and is tightly controlled by the opposing actions of protein-tyrosine kinases and protein-tyrosine phosphatases (PTPs). Protein-tyrosine phophatase 1B (PTP1B) is an important physiological regulator of metabolism. PTP1B whole-body knockout (KO) mice are hypersensitive to insulin and leptin, and are resistant to high fat diet-induced obesity. Additional studies using tissue-specific deletion, revealed a diverse and complex function of PTP1B in various peripheral insulin-responsive tissues (liver and muscle) and brain. However, the metabolic role of adipose PTP1B remains largely unexplored. To fully assess the physiological role of adipose PTP1B, we will employ three complementary approaches. We will generate adipose-specific PTP1B KO mice which will enable assessment of consequences of PTP1B loss in adipose tissue in vivo. Equally important are the dissection of molecular mechanisms mediating PTP1B function, and characterization of the dynamic PTP1B-substrate(s) interaction with high spatial and temporal resolution. In preliminary studies, we show that: (i) mice with adipose-PTP1B deletion are resistant to high fat diet-induced obesity, (ii) this is a result, at least in part, of increased energy expenditure in these mice. (iii) In addition, adipose PTP1B deletion improves systemic insulin sensitivity and glucose homeostasis. (iv) Indentify pyruvate kinase M2 as a novel substrate of PTP1B. Thus, our preliminary studies indicate that adipose PTP1B is a major regulator of body mass and glucose homeostasis. The broad goals of this proposal are to investigate the metabolic role of adipose PTP1B. PTP1B is an attractive target for therapy of obesity and diabetes with many pharmaceutical companies developing PTP1B inhibitors. Therefore, understanding how adipose PTP1B acts is vital for its evaluation of use as a target for therapeutic intervention. PUBLIC HEALTH RELEVANCE: Obesity and type 2 diabetes are very prevalent metabolic diseases affecting millions of people in the United States and worldwide. The goal of this proposal is to utilize genetically engineered mouse models and advanced biochemical and imaging approaches to determine the physiological role of adipose protein-tyrosine phosphatase 1B in glucose homeostasis and body mass regulation. Data generated from this proposal will aid in the identification of therapeutic targets for prevention and treatment of obesity and type 2 diabetes.

DESCRIPTION (provided by applicant): Disorders of body mass regulation such as obesity are increasingly common causes of morbidity and mortality. Despite significant progress, many cellular and molecular details of energy homeostasis remain unknown. Increased understanding of the molecular signaling mechanisms will aid in the development of more effective therapies for obesity and its associated disorders, such as type 2 diabetes. Protein tyrosine phosphorylation and dephosphorylation is a fundamental signaling mechanism in cells, and is tightly controlled by the opposing actions of protein-tyrosine kinases and protein-tyrosine phosphatases (PTPs). Protein-tyrosine phophatase 1B (PTP1B) is an important physiological regulator of metabolism. PTP1B whole-body knockout (KO) mice are hypersensitive to insulin and leptin, and are resistant to high fat diet-induced obesity. Additional studies using tissue-specific deletion, revealed a diverse and complex function of PTP1B in various peripheral insulin-responsive tissues (liver and muscle) and brain. However, the metabolic role of adipose PTP1B remains largely unexplored. To fully assess the physiological role of adipose PTP1B, we will employ three complementary approaches. We will generate adipose-specific PTP1B KO mice which will enable assessment of consequences of PTP1B loss in adipose tissue in vivo. Equally important are the dissection of molecular mechanisms mediating PTP1B function, and characterization of the dynamic PTP1B-substrate(s) interaction with high spatial and temporal resolution. In preliminary studies, we show that: (i) mice with adipose-PTP1B deletion are resistant to high fat diet-induced obesity, (ii) this is a result, at least in part, of increased energy expenditure in these mice. (iii) In addition, adipose PTP1B deletion improves systemic insulin sensitivity and glucose homeostasis. (iv) Indentify pyruvate kinase M2 as a novel substrate of PTP1B. Thus, our preliminary studies indicate that adipose PTP1B is a major regulator of body mass and glucose homeostasis. The broad goals of this proposal are to investigate the metabolic role of adipose PTP1B. PTP1B is an attractive target for therapy of obesity and diabetes with many pharmaceutical companies developing PTP1B inhibitors. Therefore, understanding how adipose PTP1B acts is vital for its evaluation of use as a target for therapeutic intervention.

DESCRIPTION (provided by applicant): Type 2 diabetes mellitus is one of the most prevalent metabolic diseases that is characterized by hyperinsulinemia, insulin resistance, and defect(s) in islet secretory function. Pancreatic ¿-cells dynamically respond to fluctuations in blood glucose with the regulated secretion of insulin. Increased understanding of the molecular signaling mechanisms that underlie the function of these dynamic, insulin-producing cells will aid in the development of more effective therapies. TCPTP is a ubiquitously expressed non-receptor protein-tyrosine phosphatase. Mice with whole-body TCPTP deletion exhibit hematopoietic defects and die after birth thus hampering detailed assessment of their metabolic phenotype. The function of TCPTP in the pancreas remains largely unresolved, but a growing body of evidence suggests a role in ¿-cell function. Genome-wide association screens identified PTPN2 as a susceptibility gene involved in the pathogenesis of type 1 diabetes. In addition, TCPTP regulates cytokine-induced ¿-cell apoptosis. Moreover, TCPTP modulates endoplasmic reticulum stress signaling in the glucose-responsive MIN6 ¿-cells. Further, TCPTP is a key regulator of insulin, leptin, and c-Src signaling pathways which play important roles in ¿-cell function. To determine the physiological role of TCPTP in pancreatic islets, we will employ two complementary approaches. We will generate pancreas TCPTP knockout (KO) mice to study the direct consequences of TCPTP loss in the pancreas in vivo. In addition, we will determine the effects of TCPTP knockdown and pharmacological inhibition on ¿-cell function. Equally important is the dissection of the molecular mechanisms mediating TCPTP actions. In preliminary studies we demonstrated that: (i) pancreas TCPTP KO mice exhibited impaired glucose tolerance and attenuated glucose-stimulated insulin secretion (GSIS). (ii) The secretory defect in GSIS was confirmed in ex vivo studies indicating that the effects were cell autonomous. (iii) TCPTP knockdown and pharmacological inhibition in MIN6 ¿-cells attenuated GSIS. (iv) Identified STAT3 as a TCPTP substrate in ¿-cells. The broad goals of this proposal are to investigate the physiological role of TCPTP in pancreas endocrine function with the long-term aim of generating therapies for the treatment of diabetes.

ABSTRACT: PROJECT 5 Hazardous waste sites contain complex mixtures of a wide variety of toxic chemicals that contaminate and linger in the environment. The acute toxicities of numerous Superfund (SF) chemicals have been extensively investigated; however, further studies are needed to determine their chronic effects on human health. Several SF chemicals (e.g. naphthalene, PCBs, and CCl4), which are found in environmental samples from the Yurok Tribe of the Klamath River basin, and in California air, have been shown to induce endoplasmic reticulum (ER) stress in cultured cells. Furthermore, in animal models, long-term exposure to CCl4 leads to ER stress in tissues, resulting in fibrosis and organ damage. Thus, the central hypothesis for this project is that chronic exposure to xenobiotics leads to ER stress that then contributes to chronic inflammation, tissue fibrosis and eventual organ failure. Based on the novel concept that the magnitude of ER stress is proportional to the amount of chronic exposure to chemicals, and monitoring ER stress will help predict resulting biological effects, the long term goal of this project is to develop a high-content and medium throughput bioassay to test the potential of SF chemicals to induce ER stress (Aim IIIA), and a biomarker of ER stress-associated biological effects for bio-fluid analysis (Aim IIIB). Toward these objectives, cell-based assays (Aim I) will be used to understand the mechanisms by which exposure to environmental toxins leads to ER stress. In addition, in animal models (Aim II, with Project 4), Project 5 will evaluate the effects of chronic exposure to hazardous chemicals on ER stress, and test if seric oxylipids are surrogate biomarkers for ER stress (with Cores A and B). The methodology developed and data obtained from the cell cultures and animal models will be directly translated in developing biomarker assays (Aim IIIA and B; with Projects 2 and 3, and Cores A and B). Finally, research findings will be utilized to serve the community at large by testing field samples collected from the Klamath River basin, the Central Valley and the Sierra Nevada foothills in California, and at or around SF-sites across the U.S. (Aim IIIC with Project 1, Cores B, C and D), as well as transferring to the scientists of the Yurok Tribe Environmental Program (with Core E). Accordingly, the overall goals of this project are to: 1) test lipid mediators as potential diagnostic biomarkers for the magnitude of ER stress response that often contributes to organ damage, 2) develop fast, inexpensive and reliable new cell-based bioassays to detect, assess and quantitate the effects of hazardous substances on ER stress, 3) provide new mechanistic insights into the effects of chronic exposure to SF chemicals on ER stress and fibrotic diseases, 4) develop biomarkers assays for bio-fluids for the quantification of tissue-specific effects of xenobiotics on ER stress, and 5) translate our findings by assessing risk on human health, by analyzing field samples.