Jerrold M. Olefsky - US grants

Insulin is the major hormone involved in overall glucose homeostasis, and abnormalities of insulin action are central to the pathogenesis of a variety of disease states. Consequently, detailed knowledge of the basic events underlying insulin's ability to interact with cells will lead to a better understanding of the mechanisms of disease states in which insulin action is abnoral. Therefore, the overall goals of this research proposal are to study the relationships between insulin binding to its cell surface receptors and the events which occur following this initial step. 1) To accomplish this, we plan studies of the insulin receptor intinerary. This includes studies of internalization of insulin receptor complexes, dissociation of insulin from receptors within endosomal vesicles, physical segregation of insulin from receptor and then recycling, degradation, and sequestration of the internalized receptor. Concomitant studies of intracellular insulin degradation will also be conducted. We will also examine subcellular localization to determine where in the cell these events occur. 2) Secondly, we will turn our attention to studies of adipocyte glucose transport. We plan experiments to isolate and purify glucose transporters (GT), and to raise polyclonal and monoclonal antibodies against GT. We plan to test the hypothesis that insulin induces covalent modification of GT (phosphorylation) and that this subserves an important physiologic role. Additionally, GT number and structure will be assessed in various physiologic conditions, in hepatocytes compared to adipocytes, and in plasma membrane versus low density membrane compartments. 3) Studies of the recently identified insulin receptor kinase activity will also be carried out. We plan to examine autophosphorylation as well as exogenous substrate phosphorylation in cell free systems and in intact cells, in an attempt to further understand the potential role of this kinase activity in normal insulin action as well as pathophysiologic conditions. 4) Finally, we will examine the mechanisms of abnormal insulin action in three models of post-binding cellular insulin resistance. These include studies of cells with down regulated insulin receptors and decreased maximal glucose transport activity, studies of cells with normal insulin binding but decresed insulin sensitivity, and studies of neuraminidase treated cells with normal insulin binding and decreased insulin stimulated glucose transport. The studies outlined in this proposal should provide needed information which will further our understanding of the cellular actions of this important hormone.

The central theme of this research proposal is to further elucidate the mechanism of insulin resistance in obesity and NIDDM, with particular emphasis on post-receptor defects in insulin's action on glucose disposal. To accomplish this, we plan a broadly based in vitro and in vivo approach. We will determine the in vivo Km and Vmax for overall glucose disposal in both obesity and NIDDM and will combine this with in vitro studies of adipocytes in which glucose transport Km and Vmax is assessed along with the number and distribution of cellular glucose carriers, the level of insulin mediator substance, and insulin receptor kinase activity. Detailed studies of insulin receptor structure, internalization, processing, and degradation will be conducted in cells from obese and NIDDM patients will also be treated with a 3 week period of CSII, to normalize glucose levels. After this, the effects of insuliln therapy on the various in vivo and vitro aspects of insulin resistance will be assessed by repeat studies. In this proposal we will also examine the mechanisms of various therapies such as insulin treatment, sulfonylurea treatment, and weight reduction in obese or NIDDM patients. We also propose that abnormalities in the kinetics of insulin action play a role in insulin resistance and plan to test this by measuring the rate of activation and deactivation of insulin's effects to stimulate glucose disposal and inhibit hepatic glucose production. The role of intermittant versus sustained hyperinsulinemia in causing post-receptor defects will also be assessed, and new methods to measure the rate of non-insulin mediated glucose disposal in vivo are proposed for NIDDM. We also plan to re-examine the Randle hypothesis by measuring the effects of elevated FFA levels on in vivo insulin action and the potential beneficial effects of medium chain triglycerides. Finally, the influence of CSII treatment on the therapeutic effects of sulfonylureas in NIDDM will be explored. Since insulin resistance is a major characteristic feature of obese and NIDDM patients, it is hoped that a better understanding of the in vivo and in vitro mechanisms underlying this metabolic abnormality will lead to better knowledge of the pathogenesis of these disorders, and a more rationale of therapeutic modalities.

DESCRIPTION (provided by applicant): The overall purpose of this program is the training of new scientists capable of performing high quality biomedical research in the areas of diabetes, metabolism and insulin and growth factor action. The program is centered within the Division of Endocrinology and Metabolism of the Department of Medicine and includes 17 faculty participants. The faculty represent a range of interests, skills and approaches, ranging from basic research to clinical investigation, all converging on the common themes of diabetes, metabolism and signal transduction. The proposed program will provide training opportunities at the postdoctoral level in either basic or clinical research. The disciplines range from clinical diabetes, to clinical research, biochemistry, molecular biology, cell biology and animal physiology. The program will include: (1) intensive laboratory and/or clinical research training, (2) seminars and other conferences, and (3) formal course instruction. The primary focus on the training program is the research undertaken by each trainee in association with a member of the training grant faculty. Under close supervision by the faculty member, the trainee will be encouraged, and expected, to assume an increasingly independent scientific role in all aspects of the research. In addition, the training program will foster and encourage a scholarly exchange of ideas and intellectual cross fertilization. Weekly seminars are held where ongoing research is presented for discussion by the group as a whole. Another weekly meeting is devoted to discussions by training grant faculty and trainees of recent scientific advances. In part, this conference functions as a Journal Club. Finally, all trainees are required to take 4 quarters of formal course work, including a required course entitled "Biomedical Research Ethics" offered through the School of Medicine. Thus, this training program expands and brings together various activities encompassing the training and education of postdoctoral fellows in the fields of diabetes, metabolism and signal transduction at the University of California, San Diego School of Medicine.

Non-insulin dependent diabetes mellitus (NIDDM) is a common, chronic disease with increasing incidence in the United States, responsible for an enormous burden of morbidity and mortality. Most of the morbidity, mortality, and financial burden of NIDDM is related to the development of complications. Consequently, prevention of NIDDM, or even a substantial delay in disease onset, will have a major financial and health impact in this country. Compelling evidence exists to indicate that insulin resistance is an early defect present in the pre-diabetic state. Consequently, it is logical to direct primary prevention interventions at improving this metabolic defect. It is well established that physical exercise is a lifestyle intervention which can ameliorate insulin resistance, and therefore, might prevent NIDDM. Troglitazone is a newly avail-able oral hypoglycemic agent, from the Thiozoladinedione class of drugs. This agent exerts its effects primarily by improving insulin resistance, and this has been demonstrated in NIDDM and IGT populations. Consequently, we propose a 2 X 2 factorial design in which patients will be randomly assigned in a double blind fashion to either Troglitazone, placebo, exercise, or Troglitazone plus exercise groups. The study will be performed in patients with IGT, as well as in those with NIDDM who do not have fasting hyperglycemia. Power calculations indicate that a total of 4,000 patients in this multi-center cooperative trial will be sufficient to detect positive outcomes, and this will incorporate approximately 200 patients/Clinical Center. This study cohort will consist of patients with IGT, former gestational diabetes mellitus, NIDDM without fasting hyperglycemia, and approximately 50% of all subjects should be from minority populations. This study will be conducted over 7 years, including a planning phase, recruitment phase, 4-5 year treatment phase, and close out phase. This study should provide a rigorous test of the hypothesis that the proposed interventions will prevent NIDDM.

To determine whether hyperglycemia will lead to the secondary component of insulin resistance in niddm by decreasing insulin receptor kinase activity through a pkc mediated mechanism.

Designed to evaluate patients with unusual forms of in vivo insulin resistance. The underlying pathophysiology will be defined by evaluating endogenous insulin secretion, searching for the presence of hormonal insulin action and immune insulin antagonists, and examining peripheral insulin action in detail.

polycystic ovary syndrome; hormone regulation /control mechanism; insulin sensitivity /resistance; adipocytes; weight loss; phorbols; insulin receptor; pyruvate dehydrogenase; lipolysis; hydrogen peroxide; insulinlike growth factor; aminoacid transport; androgens; glucose transport; cellular pathology; glycogen synthase; glycogenolysis; SDS polyacrylamide gel electrophoresis; fibroblasts; bioassay; cell free system; polymerase chain reaction; nucleic acid sequence; human tissue; western blottings; tissue /cell culture;

Assess the physiologic significance of kinetic defects in insulin secretion and glycemic excursion in various groups of insulin resistant subjects by adding to the functional insulin resistance in vivo.

insulin receptor; protein tyrosine kinase; insulin sensitivity /resistance; hyperglycemia; enzyme activity; serine; phosphorylation; clinical research; tissue /cell culture; human subject;

hexokinase; noninsulin dependent diabetes mellitus; insulin sensitivity /resistance; glucose metabolism; phosphorylation; glycogenesis; glucose transport; insulin; gene expression; enzyme biosynthesis;

glucose clamp technique; diagnosis design /evaluation; noninsulin dependent diabetes mellitus; glucose metabolism; liver metabolism; blood glucose; veins; clinical research; diagnostic catheterization; human subject;

glucose tolerance; noninsulin dependent diabetes mellitus; glucose metabolism; muscle metabolism; liver metabolism; glucose transport; clinical research; human subject;

Study to determine whether blocking growth hormone's effects can improve the action of insulin and help to lower the blood sugar level in people with noninsulin dependent (type II) diabetes mellitus.

Compare efficacy of troglitazone and metformin in reducing the daily requirements for exogenous insulin to maintain strict glycemic control in insulin treated subjects with NIDDM. Define the time course of the drug induced insulin dose reduction.

troglitazone; receptor sensitivity; insulin sensitivity /resistance; striated muscles; glucose metabolism; drug screening /evaluation; clinical research; human subject;

Investigate activation and deactivation of insulin action on hepatic glucose production and peripheral glucose utilization. There are two separate mechanisms which may account for changes in activation kinetics, namely, changes in regional blood flow in response to insulin and transit of insulin from the circulation to the intersttial space.

Determine the incidence of arterial compression of the right vagus nerve and right lateral medulla oblongata in patients with NIDDM in order to evaluate the hypothesis that NIDDM is a vascular compression syndrome. Show increase in left sided vascular compression upon the lower brain stem in subjects with neurogenic hypertension.

Determine if Fluasterone leads to an enhancement of insulin action on whole body glucose utilization and hepatic glucose production and hence better glycemic control in NIDDM, and determine the effective dose range of oral Fluasterone in NIDDM, and to gather additional safety and pharmacokinetics data for oral Fluasterone.

An novel gonadotrope cell line (LbetaT) has been recently developed which has many of the features of a mature gonadotrope including the expression of the GnRH receptor and both the glycoprotein alpha- subunit and the LH Beta-subunit proteins. We plan to use these cells to investigate GnRH signaling pathways. These experiments will involve the micro injection of antibodies and recombinant proteins into a single LbetaT cells, the use recombination adenoviruses, or cell-permeable inhibitor peptides to block or stimulate selective pathways. A number of end-points to monitor GnRH signaling will be used including LH secretion, activation of MAPKinase, induction of c- fos and jun-B, stimulation of BrdU incorporation, and inositol phosphate and diacylglygerol production. Aim1. To test the hypotheses that Gq mediates GnRH signaling the LbetaT cells. We will determine whether the GnRH receptor couples to Gq. Signaling by the Galphaq subunit and Gbetagamma subunits will be blocked by specific inhibitors. Constitutively active versions will be introduced into cells to mimic GnRH stimulation. Aim 2. To test the hypothesis that tyrosine phosphorylation of Gq associated proteins is required for GnRH signaling in LbetaT cells. We will investigate whether proteins associated with Gq are tyrosine phosphorylated and whether phosphorylation is required for GnRH signaling. A mutant Galphaq will be introduced into cells to determine whether signaling is impaired. Aim 3. To test the hypothesis that GnRH -stimulated MAPKinase activation is ras dependent in LbetaT cells. We will investigate whether activation of MAPKinase by GnRH requires activation of ras. Is activated by GnRH. If ras independent, then we will determine whether PKC activates the pathway downstream of ras. Aim 4. To test the hypothesis that GnRH utilizes multiple pathways to stimulate gene expression through distinct response elements in LbetaT cells. Reporter genes containing multimeric GnRH-response elements from the mouse and human alpha- and rat LH Beta-promoter will be constructed. These will be injected into LbetaT cells along with antibodies or recombinant proteins to block selective transcription factors or kinases.

Determine whether troglitazone treatment when initiated in participants during early stages of Type II diabetes can reduce postprandial glucose levels assessed by oral glucose tolerance testing and improve peripheral insulin sensitivity assessed by frequently sampled intravenous glucose tolerance testing. Assess whether troglitazone treatment alters the rate and quantity of insulin secreted by pancreatic beta cells in response to increasing concentrations of glucose assessed by graded glucose infusion testing preceded and followed by arginine stimulation.

human therapy evaluation; disease /disorder prevention /control; disease /disorder proneness /risk; noninsulin dependent diabetes mellitus; metformin; glucose metabolism; chemoprevention; high risk behavior /lifestyle; clinical research; human subject;

DESCRIPTION (provided by applicant): There are genetic and environmental causes of insulin resistance, and clearly these two inputs can be additive and interactive. A high fat intake is an important environmental factor which can cause, or exacerbate, insulin resistance and enhance the risk for the development of Type II diabetes. Our recent studies have shown that lipid/heparin infusions lead to insulin resistance in men, but not in pre-menopausal women. We also have preliminary data showing that post-menopausal women are fully susceptible to fat-induced insulin resistance and that estrogen replacement therapy re-establishes the protective state. In addition, we have conducted a series of studies in rats, demonstrating that estrogenization (endogenous or exogenous) will protect females from fat -induced insulin resistance. Based on these findings, we propose that men and non-replaced post-menopausal women will exhibit fat-induced insulin resistance, whereas, adequately estrogenized women will be protected. We will test these ideas, not only by employing the lipid/heparin infusion glucose clamp technique, but also by placing experimental subjects on control and high fat diets. It is also possible that adequate estrogen can ameliorate the effects of other physiologic causes of insulin resistance. Thus, we also will conduct studies to determine whether estrogenization can protect women from the insulin resistance induced by obesity and aging. Using muscle biopsy samples collected during the glucose clamp studies, we will conduct experiments aimed at identifying cellular mechanisms for these protective effects of estrogens. We also propose an extensive series of animal studies, in which we will explore in more detail the mechanisms of estrogen protection from fat-induced insulin resistance. We will conduct studies in normal male and female rats, ovariectomized rats, and old estrogen deficient female rats+/- treatment with estradiol, an estrogen antagonist, or estrogen receptor isoform specific agonists. Studies in mice with deletion of the alpha or beta forms of the estrogen receptor, as well as muscle specific estrogen receptor specific knockout animals are also proposed. We will also determine whether the fat cell secreted protein ACRP3O is modulated by estrogen status, and whether the insulin sensitizing effects of ACRP3O are responsible for the estrogen induced protection from insulin resistance. If the concepts contained in this application prove correct, then these findings could have significant implications concerning the mechanisms of insulin resistance as well as the treatment and possibly prevention of this disorder.

gender difference; free fatty acids; insulin sensitivity /resistance; enzyme activity; long chain fatty acid; phosphorylation; muscle metabolism; gene expression; acyl coA; menopause; postmenopause; hexosamines; protein kinase; clinical research; human subject;

DESCRIPTION (provided by applicant): High fat intake is a major environmental factor leading to decreased insulin sensitivity contributing to the rising incidence of interrelated insulin resistant diseases such as Syndrome X, PCOS, Type 2 diabetes mellitus, and obesity. We have demonstrated that estrogenized women and female rodents are protected from fat induced insulin resistance, whereas, males, and estrogen deficient females are fully susceptible to these adverse effects of fat. In this application, we plan a broad based, in vivo and in vitro approach to elucidate the mechanisms of fat induced insulin resistance and the protective effects of estrogens, using various novel animal model systems, 3T3-L1 adipocytes in vitro, and the non-classical insulin target tissue ovarian granulose cells (GCs) An underlying hypothesis in this application is that excess fat metabolism due to elevated FFA levels, or high fat diets, leads to activation of the "inflammatory pathway" and that specific serine/ threonine kinases in this pathway such as PKC theta, IKK beta, or JNK, or genes induced as a result of NfkappaB activation, feedback on the insulin signaling system to cause insulin resistance. Our preliminary data show that treatment of 3T3-L1 adipocytes in vitro with FFAs leads to a marked state of cellular insulin resistance, and we will exploit this novel system to conduct new studies aimed at elucidating the molecular mechanisms of FFA induced insulin resistance and estrogen's protection against these effects. Finally, since we hypothesize that GCs from insulin resistant animals and women (particularly PCOS) can be insulin/ IGF-I resistant with functional consequences, we propose an extensive series of studies in GCs prepared from normal rats and insulin resistant rodents, to determine whether FFA treatment causes insulin resistance in these cells, as it dos in insulin target cells, and to identify the underlying mechanisms. We will also study the basic signaling systems for insulin, IGF-I and FSH in these cells. Taken together the results of these studies should greatly enhance our understanding of the mechanisms of fat induced insulin resistance, in classic and non-classic insulin target tissues, and also elucidate the mechanisms underlying the protective effects of estrogens. These studies should also highlight the role of inflammatory pathway activation in these pathophysiologic events and this may have potential therapeutic implications for new treatment approaches.

insulin; dietary carbohydrates; blood lipid; obesity; blood glucose; caloric dietary content; gender difference; diet therapy; clinical research; human subject; nutrition related tag;

troglitazone; human therapy evaluation; drug screening /evaluation; insulin sensitivity /resistance; inflammation; antiinflammatory agents; pancreatic islet function; glucose metabolism; fatty acid metabolism; free fatty acids; adipose tissue; clinical research; human subject;

peroxisome proliferator activated receptor; troglitazone; drug interactions; inhibitor /antagonist; human therapy evaluation; combination chemotherapy; insulin sensitivity /resistance; diabetes mellitus therapy; noninsulin dependent diabetes mellitus; lipid metabolism; drug screening /evaluation; clinical research; human subject;

There are a number of physiologic situations in which metabolic signals, particularly those related to glucoregulatory and lipid metabolism, impinge on reproductive function. The networks that control metabolic homeostasis and the hypothalamic (H)/pituitary (P)/gonadal (G) axis are highly complex, reflecting a large set of intersecting feedback regulatory circuits. In this proposal, we will undertake a molecular and biochemical dissection of selected elements of the metabolic signaling system to determine their impact on HP function. Thus, insulin, adiponectin, and PPARgamma signaling are all key elements in major metabolic regulatory networks, and we will test a number of hypotheses related to how these pathways affect HP function. The program proposed is a broad-based approach involving genetic, in vivo physiology, and in vitro studies. In specific, based on genetic deletion of insulin receptors (IR), it is clear that absence of IRs in the brain leads to infertility. Our own in vitro data show substantial effects of insulin on HP function in cultured cells. We will use a variety of genetic approaches, including knockout and transgenic methodology as well as stereotaxic gene manipulation to create novel animal models for investigation. In vitro studies in immortalized GnRH neuronal cells (GT1-7) and pituitary cells (LbetaT2) are also proposed. Adiponectin is an adipose-derived factor that has major effects on glucose and lipid metabolism. Transgenic hyperadiponectinemic mice are infertile and hyperadiponectinemic rats display decreased LH secretion. These findings will be pursued at genetic, physiologic, and in vitro levels to understand the effects of this adipocytokine on reproduction. Lastly, stimulation of the PPARgamma receptor can restore fertility in PCOS women, and we have now found that PPARgamma modulates GnRH signaling and LH release in LbetaT2 cells. In addition, we have initial evidence showing that TZD treatment of PCOS women lowers the elevated LH levels. Taken together, this approach should allow us to parse out specific metabolic signaling pathways to deduce their individual and combined effects on HP function. These studies should provide new insights into the interactions between metabolic homeostasis and reproductive function allowing us to begin unraveling these multi-faceted interacting pathways. The results will also improve our understanding of reproductive function in metabolic disorders such as PCOS, Type 2 diabetes, and obesity, in which abnormalities of HP function have been well described.

DESCRIPTION, OVERALL (provided by applicant): The overall mission of the UCLA-UCSD DERC is to foster research in the prevention and treatment of diabetes and its complications and ultimately to improve the lives of patients with diabetes. This unique Center crossed institutional boundaries to harness the energy and excitement of research in diabetes, metabolism, endocrinology, and cardiovascular disease in both institutions and to serve the needs of diabetes/endocrinology researchers at UCSD and UCLA, which comprises the major component of research in these fields in Southern California. The DERC has fostered interaction and collaboration between talented researchers at both institutions and has played an important role in bringing outstanding scientists only peripherally involved in diabetes research to focus their efforts in the diabetes arena. Our membership has grown from 93 to 136 with combined current NIH, ADA and JDRF funding of nearly $ 50 M. Biomedical Research Bases consist of: Nuclear Receptors, Cell Signaling, Metabolism Complications, Microvascular Complications, and p-cell with leaders in all fields as members of these Bases. The highlights during the first four years of the DERC include: 1) A marked expansion with the acquisition of state-of-the-art technology for all Cores, 2) Establishment of the UCLA Hillblom Islet Research Center (2004), Director Peter Butler, who has also just assumed the editor-in-chief position of Diabetes, 3) Designation of the Lasker Award to Ronald Evans, one of our Senior DERC faculty, 4) Substantial recruitment of Diabetes/Endocrinology based faculty at both UCLA-UCSD (collectively 8 physician scientists, 8 basic scientists and 6 clinicians), 5) Achievement of the highest ranking P&F score in national P&F competition by Steven Chessler (USCD), 6) Establishment of a new Program Project grant among Drs. Glass, Olefsky, and Rosenfeld, focused on the study of inflammation, macrophages, and insulin resistance, entitled "Gene Networks Controlling Macrophage-Adipocyte Interactions and Insulin Resistance." The Cores: Transgenic and Knockout Mouse, Mouse Phenotyping, Transcriptional Genomics, Human Genetics have been heavily used by DERC members as judged by the extensive number of publications supported by the Cores. Because of scientific rationale and interest of our members, the Inflammation Core was recently established. The UCLA-UCSD DERC has emerged as a key focal point and important catalyst of diabetes/endocrinology research, as well as a resource for education, training, raising awareness of diabetes care research and promoting translational research. Future directions will continue to strive for seamless integration of researchers at both institutions, to enhance technology and research capability in the DERC, to promote translational research activity with involvement of health services research, and to potentially partner with nanotechnology.

Reproduction is very energy expensive. The energy requirement for a pregnancy is estimated at 160,000 Real with an additional 500-1000 kcal/day for lactation. Because of this requirement, it is likely that mechanisms have evolved to suppress reproduction under periods of famine. Our goal is to understand the ntegration of metabolism in the regulation of fertility and how abnormalities in metabolic homeostasis, both over and under-nutrition, can lead to infertility. The adipose-derived peptide hormone adiponectin, the ongevity-associated protein SirT1, and the PPARy receptor all intersect to regulate insulin sensitivity and exert multiple biological effects in various tissues. Therefore, the role of these proteins in controlling GnRH and gonadotropin gene expression, synthesis, secretion, and fertility in the insulin-resistant prenatal androgenized mouse will be addressed utilizing a combined in vivo and in vitro approach. In the first specific aim, we will test the hypothesis that SirT1 and adiponectin suppress the central HPG axis during times of nutritional deprivation. This will be tested using tissue-specific SirT1 knockouts and transgenics in the brain and pituitary gonadotrope, and adiponectin null mice. In the second specific aim, we will test the hypothesis that a high fat diet increases inflammatory signaling in the hypothalamus and pituitary leading to dysregulation of gonadotropin secretion, and that PPARy suppresses inflammatory signaling to normalize LH levels. This will be tested using tissue-specific deletion of the PPARv receptor in gonadotropes or in the whole brain to identify the site of PPARy action. In the third specific aim, we will test the hypothesis that the prenatally androgenized mouse has increased tissue inflammation that contributes to the insulin resistance. We will determine the site of insulin resistance and will test whether improving insulin resistance rescues the infertility in these mice. Each aim will feature a series of in vivo animal studies paired with a complementary series of in vitro experiments to elucidate molecular mechanisms.

DESCRIPTION (provided by applicant): Insulin resistance, hepatic inflammation, and NAFLD are interlocking pathophysiologic events, but the mechanisms of these abnormalities, and the ways in which these different processes interact, are poorly understood. This is a broad, collaborative application in which the four participating PI's and laboratories will concentrate their focus on the etiology and pathophysiology of hepatic inflammation, steatosis, and insulin resistance. The scale of this application is substantial and will focus on four overall specific aims. In the first aim, an ambitious, large scale time course will be undertaken in high fat diet (HFD)/obese mice, coupled with systematic in vitro and in vivo measurements to uncover the dynamic temporal time course and key transition points enabling the development of hepatic insulin resistance/inflammation/steatosis. The second aim explores a novel hypotheses which proposes that changes in intestinal microflora and gut permeability to bacterial products triggers inflammatory signals directed to the liver. These inflammatory stimuli then interact with immune cells in the liver, generating the chronic hepatic inflammatory state. The third aim encompasses several new ideas and hypotheses aimed at delineating the molecular mechanisms underlying the metabolic disturbances in the liver. These studies will involve tracking the itinerary of immune cells to the liver, identifying the phenotypic function of the different liver cell types, studies of biochemical pathways involved in insulin signaling, lipogenesis/fat oxidation, inflammation, and the identification of transcription factor cistromes and epigenetic changes in genomic loci induced by obesity. In vivo and in vitro studies in a number of knockout mice will be heavily used in the pursuit of these studies. The final aim proposes translational studies in which liver biopsies will be obtained from obese NAFLD subjects before and after weight loss. Cellular, biochemical, and genomic studies will be performed in these biopsies and correlated with the in vivo clinical data on these patients. In this way, we will be able to test the ideas and concepts learned from the basic studies in the first three aims for relevance to human pathophysiology. PUBLIC HEALTH RELEVANCE: NAFLD is closely associated with hepatic insulin resistance and inflammation and is the most common liver disease in the US. The pathophysiologic mechanisms underlying the interactions between hepatic insulin resistance, inflammation, and steatosis are poorly understood, and this project should lead to a greatly improved basic understanding of this disorder with the potential to lead to new therapeutic opportunities.

Obesity confers increased risk for various forms of cancer. Breast, colon, and liver cancer are all increased in obese populations and the epidemiologic evidence for the obesity - breast cancer connection is compelling. One in eight women will be diagnosed with breast cancer during her lifetime. Breast cancer is strongly associated with age as incidence increases 10-fold for women age 260 compared to women age S50. Increased risk with age seems related to post-menopausal hormone levels as both obesity and hyperinsulinemia are associated with increased breast cancer risk only in women not on hormone replacement therapy. Metabolic Syndrome is associated with a higher incidence of aggressive triple negative breast tumors (ER-/PR-/HER2-) which is likely accelerated by ovarian hormone decline after menopause, as post-menopausal women are more susceptible to the deleterious metabolic effects of obesity including chronic inflammation and insulin resistance. Rodent studies have confirmed this relationship, showing that diet-induced obesity and high fat diets lead to increased incidence and growth of tumors in various breast cancer models. Despite this body of correlative evidence, the mechanisms of obesity-induced breast cancer risk remain poorly understood. Diet composition is an important factor as diets rich in saturated and omega 6 (w6) fatty acids (FAs) are pro-inflammatory and increase breast cancer risk, but diets rich in omega 3 (w3) FAs are anti-inflammatory and decrease cancer risk. The clinical data is less clear but meta-analyses of multiple human breast cancer risk studies suggest that the ratio of oo6 to w3 FAs is a critical factor. We have found that the beneficial anti-inflammatory and insulin-sensitizing effects of 0)3 FAs are mediated by the G-protein coupled receptor GPR120. Due to the potential link between obesity, insulin resistance and breast cancer risk in post-menopausal women, we hypothesize that GPR120 is the critical mediator of the protective effects of w3 FAs in breast cancer. We will test this in four specific aims that combine 1) studies using orthotopic tumor cell transplants and 2) spontaneous tumors in obese wild type (WT) and GPR120 knockout (KO) mice, ¿ w3 FA supplementation, 3) studies using orthotopic mouse and human tumor cell transplants into RAG2 KO mice, and 4) studies of metastasis using genetically marked tumor cells in obese WT and GPR120 KO mice. We hypothesize that u)3 FAs will attenuate tumorigenesis and metastasis in WT but not GPR120 KO mice through their anti-inflammatory/insulin-sensitizing actions This project aims to provide mechanistic depth that is complementary to aims of Projects 2 & 3.

Chronic tissue inflammation is an important contributor to the decreased insulin sensitivity associated with obesity/type 2 diabetes and that the macrophage adipocyte axis is a key effector causing this metabolic defect. We have recentiy taken a new approach to this problem and have generated adipocyte-specific NCoR KO mice (AKO mice). In AKO animals, PPARy becomes constitutively active, leading to a robust anti- inflammatory insulin sensitive phenotype. We also find that phosphorylation of PPARy at serine 273 is markedly blunted when NCoR is deleted in adipocytes. In this application, we propose several new hypotheses to explain the insulin sensitivity in our AKO mice and these lead to a number of studies to examine the regulation of serine 273 serine PPARy phosphorylation and the functional propoerties of this non-phosphorylated form of the receptor. We will also conduct a series of molecular studies to identity the global gene expression patterns in primary adipocytes from WT and AKO mice, as well as the global DNA binding sites (cistromes) of PPARy, NCoR and SMRT. We also hypothesize that the central physiologic mechanism leading to the insulin resistance in the AKO mice is that deletion of NCoR leads to cell autonomous activation of PPARy. Thus, causes reduced chemotactic signaling, with decreased adipose tissue macrophage content, decreased inflammation and improved insulin sensitivity. In this context, we have made new observations indicating that the leukotriene chemokine, LBT4, and its receptor BLT1, may play a dominant role in macrophage migration into adipose tissue. Thus, we have compelling new data showing that treatment of macrophages with a BLT1 inhibitor markedly reduces macrophage chemotaxis in vitro and, that treatment of obese mice with the BLT1 inhibitor causes a robust improvement in glucose tolerance and insulin sensitivity. A combined in vitro and in vivo approach is proposed to test the hypotheses generated from these new data. These latter studies have strong translational implications since BLT1 could emerge as an important new target for insulin sensitizing drug discovery. RELEVANCE (See instmctions): The proposed studies will directly contribute to our understanding of mechanisms that regulate the initiation, amplification and resolution of pathogenic forms of inflammation that contribute insulin resistance and the development of type 2 diabetes.

The project focuses on the concept that chronic activation of infiammatory pathways and that the macro- phage/adipocyte nexus provides a l

PROJECT SUMMARY (See instructions): The Overall mission of the UCSD/UCLA/Salk/Cedars Sinai DRC continues to center on fostering research in the prevention and treatment of diabetes and its complications to ultimately improve the lives of patients. For the past decade, our DRC has been unique in linking together the diabetes/metabolism research activities of two major universities within the UC system and two outstanding Institutes in Southern California. We believe this has been a novel, and a very successful effort. The DRC has fostered new collaborations and interactions between outstanding scientists within and across these institutions. Our research base is comprised of the following focus areas: Nuclear Receptors, Cell Signaling, Metabolism, Diabetes Complications, and Islet/Beta Cell Biology. Each of these areas has outstanding leaders who facilitate interactions and sharing of resources. The DRC has played an important role in promoting the careers of young scientists as they move on to the status of independent investigators by awarding pilot and feasibility grants. As an acknowledgement of our success in this effort, UCSD and. UCLA have agreed to provide over $100,000/year in additional unrestricted funds to augment our P&F program The DRC will continue to advance scientific and intellectual interactions by organizing and facilitating meetings, lectures, and mentoring efforts that are part of our enrichment core. We will further accelerate diabetes research at the four DRC Institutions by providing state-of-the-art services through five cutting edge cores: A) Transgenic and Knock-out Mouse Core, B) Metabolic and Molecular Physiology Core, C) Epigenetics and Genomics Core, D) Human Genetics Core, and E) Novel Target Discovery and Assay Development Core. All of our research cores have been updated with new services and latest technologies in the upcoming project period to reflect the many advances in this field as they relate to diabetes and metabolism research. Our cores are heavily used by our DRC faculty that have been exceptionally successful as can be judged by the numerous publications in high impact journals (665) and the substantial peer review grant support that they have accrued ($154,601,201). The current competitive renewal application includes many new scientific and technological advancements, including the incorporation of novel genomic, proteomic, and metabolomics services that are now available to our members. As future directions, we will continue to strive for seamless integration of research at all participating institutions to enhance technology and research capability within the DRC and to promote translational research activity and collaborations with the CTSI programs at each institution.

The overall mission ofthe UCSD/UCLA DRC confinues to center on fostering research in the prevention and treatment of diabetes and its complicafions to ultimately improve the lives of patierits. For the past decade, our DRC has been unique in linking together the diabetes/metabolism activifies of two major universities within the UC system. We believe this has been a novel, and a very successful effort; Thus, we have been able harness the collective energy and scientific excitement at UCSD/Salk and UCLA/Cedars-Sinai, which comprise the major proportion of research in diabetes/metabolism in Southern California. The DRC has fostered new collaborations and interactions between outstanding scienfists within and across these insfitutions and has played an important role in promofing the careers of young scienfists as they move on to the status of independent invesfigators. Our research base faculty membership now includes 109 outstanding scienfists who have been excepfionally successful as can be judged by the numerous publicafions in high impact journals and the generous peer review grant support that they have accrued ($155 million dollars). Some selective highlights over the past four years include: (1) Organizafion of the Pediatric Diabetes Research Center (PDRC) at UCSD along with the addifion of the La Jolla Allergy and Immunology Institute (LIAI) on the UCSD campus, which brings a number of outstanding new faculty into the DRC, all focused exclusively on type 1 diabetes research. (2) Establishment and occupancy of the Institute for Regenerative Medicine on the UCSD campus which has a focus on human stem cell research and beta cell generafion (3) Major recruitments and addifion of diabetes/metabolism-based scientists at both UCLA and UCSD. For example, this includes Richard Bergman and Marilyn Ader, and their respecfive groups, at Cedars-Sinai, Kumar Sharma at UCSD, and Matthias von Herrath at LIAI. (4) Outstanding success of our P&F awardees as they move fonward and upward in their scientific careers. An acknowledgement of this, UCSD and UCLA have agreed to provide $100,000/year in unrestricted funds to augment our P&F program. (5) Establishment and confinuafion of several program project grants and center among our collaborafing DRC faculty, headed by Drs. Chris Glass, Pam Mellon, Susan Taylor, Aldons Lusis, Joe Witztum, and Steve Young. All of our Research cores have been updated with new services and state-of-the-art technologies in the upcoming project period. In addifion, we have added a new Novel Target Discovery and Assay Development Core to reflect the many advances in this field as they relate to diabetes and metabolism research.

DESCRIPTION (provided by applicant): The prevalence of Type 2 diabetes has risen dramatically in the United States and globally for the past few decades and has now reached epidemic proportions. The etiology of this disease involves both insulin resistance and decreased ? cell function, and one typically needs both defects (2 hit hypothesis) in order to develop the full hyperglycemic diabetic state. Current anti-diabetic therapeutics is available, but is inadequate to control the disease in most patients and there is a large unmet medical need for better methods of treating diabetes to prevent morbidity and mortality. Our recent work has led to the discovery that Fractalkine (FKN) (CX3CL1) working exclusively by signaling through its cognate receptor CX3CR1 in ? cells, leads to enhanced glucose, arginine, and GLP-1 stimulated insulin secretion with markedly improved glucose tolerance in obese and diabetic mouse models. Thus, CX3CR1 KO mice are glucose intolerant due to decreased insulin secretion. Furthermore, neutralization of circulating FKN by administration of anti-FKN antibodies leads to an abrupt decrease in insulin secretion with glucose intolerance in WT mice. Furthermore, in vivo FKN administration leads to increased insulin secretion with improved glucose tolerance in WT mice, but is completely without effect in CX3CR1 KO animals. In vitro, FKN administration directly causes increased insulin secretion in ? cell lines, isolated islets and perfused islets, but it is without any effect when CX3CR1 is deleted from the ? cells. This led to the conclusion that FKN is a novel potentiator of ? cell insulin secretion. This proposal seeks to build on this newly identified FKN/CX3CR1 ? cell regulatory system to identify the underlying cellular and molecular mechanisms of FKN-induced insulin secretion. We will also test the hypothesis that long-term FKN treatment will have beneficial effects on glucose metabolism and insulin secretion in a series of hyperglycemic mouse models. In addition, we will test the overall hypothesis that FKN will have beneficial effects on ? cell health. This is based on our current findings that FKN inhibits ? cell apoptosis and stimulates the ? cell differentiation gene program. Finally, we will test the additional hypothesis that FKN administration in vivo will inhibit the development of atherosclerosis in the LDLR KO mouse model. If the ideas incorporated into this application are supported by the proposed experiments, then this would strongly support the concept that a FKN-based biotherapeutic could be administered in vivo to potentiate glucose stimulated insulin secretion in man. This therapeutic strategy could be used for the treatment of patients with Type 2 diabetes mellitus to augment their ability to secrete insulin in response to nutrients and other stimuli and to prevent the decline in ? cell mass which characterizes this disease. This would lead to improved glycemic control adding a new component in our therapeutic armamentarium for the treatment of this widespread disease.