Bruce Spiegelman - US grants

The long range goals of this project are to improve our understanding of how the adipocyte regulates lipogenesis and lipolysis through the control of specific gene expression and the corresponding protein products. By improving our understanding of these processes, we may ultimately improve our ability to provide therapy for obesity obesity-linked diabetes and correlative cardiovascular disorders. In the previous funding period, we have isolated and characterized 3 genes which are transcriptionally activated during mouse adipocyte differentiation: glycerophosphate dehydrogenase, adipocyte P2 and adipsin. The DNA sequences necessary to activate these genes will be probed first by a transient transfection-expression assay. After demonstrating proper cell type specificity of expression, as has been done with the adipocyte P2 gene, these DNA segments will be further dissected to determine (1) which sequence elements play a role in activation of its own promoter, (2) whether it can direct transcription from other promoters, and (3) whether it can function in an enhancer-like fashion. Greatest attention will be focused on genetic elements which appear to represent common steps in the pathway of activation of different fat-specific genes. Also under study will be the sequence requirements to get proper responses to 2 hormones key in lipogenesis: cyclic AMP and tumor necrosis factor (cachectin). Experiments will be carried out to isolate and characterize those nuclear factors which bind to and may regulate adipocyte-specific promoters/enhancers/hormone-response elements. Included here will be the FSE2 binding protein which appears to be developmentally regulated and binds in sequence-specific fashion to an element found in at least 2 genes participating in adipocyte differentiation. The ultimate goal will be an in vitro reconstruction of a cell type-specific transcription pattern, using isolated DNA templates. In addition to transcriptional regulation, we will analyze the catalytic and physiological function of adipsin, the serine protease homologue which is produced and secreted by fat cells. This will be done by large scale expression of the cloned cDNA in a baculovirus vector and subsequent study of the proteolytic activity of this enzyme toward an variety of extracellular substrates of potential importance in adipose physiology. Biological activity will also be examined by neutralizing adipsin activity with monospecific antibodies and following subsequent effects on adipocyte differentiation and lipogenesis.

The goal of these investigations is to understand the regulation of cytoskeletal biosynthesis and function during mammalian cell development. The system utilized is the differentiation of mouse 3T3-adipocytes, where large and specific decreases in mRNA levels for actin and tubulin appear to play an important regulatory role in the differentiation process. The proposed experiments, which emphasize the regulation of cytoskeletal gene expression, cell structure and lipogenesis, are particularly relevant to human diseases involving fat cell number and function, such as obesity, diabetes and liposarcoma. Experiments underway use cloned cDNA probes for Beta-actin and Beta-tubulin to assay transcription during differentiation in both isolated nuclei and whole cells. If transcriptional changes cannot quantitatively account for changes in these mRNAs, we will study mRNA turnover and processing. The build-up and processing of nuclear RNAs will get particular attention in light of evidence suggesting the build-up of a putative actin mRNA precursor during differentiation. The actin gene active in adipocytes will be isolated and used to define the primary transcription unit and to ask if this putative actin precursor has a structure consistent with such a role. Further studies involve construction of in vitro systems to study relevant control mechanisms which are operating. Experiments will be performed to understand the normal physiological role of the cytoskeletal and morphological regulation of lipogenic gene expression shown to exist in adipocytes. In particular, the role of cyclic AMP's effects on cell adhesion and the cytoskeleton in the suppression of lipogenic gene expression by this key physiological agent will be examined by exposing differentiating cells to a variety of cyclic AMP agents while quantitatively varying substrate adhesiveness or treating with anticytoskeletal drugs. The subsequent expression of lipogenic protein and RNA will be studied with antibodies and cDNA clones previously constructed. Finally, the reversibility of cellular and molecular differentiation-dependent changes will be studied. This is now experimentally approachable by replating differentiated cells which have had lipid accumulation blocked and hence, retain firm attachment to the sub-stratum. Subsequent cell growth and cytoskeleton synthesis will be studied at the protein and RNA levels.

The goals of this project are (1) to understand the biochemical and molecular mechanisms that regulate preadipocyte determination and (2) to develop a specific marker or set of markers for the preadipocyte phenotype. Both of these goals are of great importance in human disease because abnormal preadipocyte formation may play a central role in massive obesity and lipodystrophy, life-threatening conditions involving adipose tissue. There are currently no molecular markers for the preadipocyte nor is there substantial data about how this cell type forms from mesenchymal precursors. cDNA clones specific for the preadipocyte phenotype will be isolated from a large cDNA phage library we have prepared from preadipose 3T3-F442A cells. This library will be screened differentially with cDNA from preadipocytes and non-preadipose 3T3 cells and by a subtractive-hybridization scheme using a modified cDNA labelling protocol we have developed. cDNA clones of interest will be further characterized by hybridization to mRNA from a battery of preadipose and non-preadipose mesenchymal cells. The identity of such sequences will be examined by cDNA sequence determination and computer assisted search of existing data banks. The intracellular localization of preadipose-specific proteins will be investigated by preparation of specific antibodies against the encoded protein using open-reading frame bacterial vectors, immunohistochemical procedures and Western blotting. The potential regulatory role of preadipose-specific mRNAs in cell determination will be investigated by the construction of viral- based vectors which can express these mRNAs in non-preadipose 3T3-cells and other cell types. The capacity of these cells to differentiate will subsequently be monitored at the morphological, RNA and protein levels. The effect of expression of anti-sense mRNA for preadipose-specific genes will be examined in fully determined preadipocytes. Because of the importance of the preadipocyte in human disease, the presence of preadipocyte-specific mRNA proteins will be quantitatively examined in cohorts of obese and lipodystrophic patients. Should these experiments indicate a possible defect in or overproduction of a particular gene product, we will examine the DNA of a cohort of such patients and normal subjects to assess the degree of allelic variation and restriction-fragments length polymorphisms in these populations.

This application requests funding for an international meeting entitled "Differentiation: New Perspectives" to be held September 20, 1988 in Oxford England. This meeting will be held under the sponsorship of the British Society for Cell Biology, the Imperial Cancer Research Fund (I.C.R.F.) and The Dana-Farber Cancer Institute (D.F.C.I.). It will be organized by Dr. Fiona Watt of the I.D.R.F. and Dr. Bruce Spiegelman (P.I.) of the D.F.C.I. The meeting will bring together noted experts on cell differentiation from different perspectives. The problem of cell differentiation is central to our understanding of a wide variety of diseased states such as obesity, diabetes and cancer. We will have sessions on extracellular factors inducing differentiation (J. Smith, E. Eichele), early decisions in differentiation and determination (C. Emerson, H. Weintraub), cis- and trans-acting factors (R. Tjian, B. Nadal-Ginard, R. Goodbourn) and transgenic mice (H. Westphal, D. Solter). Our keynote speaker discussing future perspectives in cell differentiation research will be Dr. David Baltimore of M.I.T. By bringing together this group we hope to accomplish 2 things: (1) to encourage people to become familiar with the broad range of tools (cellular, molecular, animal) available to study differentiation and (2) to encourage collaborations that permit rapid movement between these different levels. In addition, we hope to provide students attending with an appreciation that the problems of cell differentiation must be attacked at multiple levels. This meeting will have a decidedly international flavor as t has strong American and Euorpean participation, as well as British and American sponsorship.

The lab has previously shown that adipsin, a mouse adipocyte-derived serine protease, is secreted into the bloodstream and is markedly deficient in several models of genetic and acquired obesity, suggesting a possible functional role for this protein in the systemic or local regulations of energy balance. The overall goal of the proposed work is two-fold: to understand the role of adipsin in adipose physiology and obesity and to use the adipsin gene as a model to understand how obesity genes regulate the function of other gene promoters. Both of these goals are novel and will increase our understanding of the biochemical and genetic basis of obesity. Adipsin's direct role in the physiology of adipose cells will be approached in vitro by treating fat cells with adipsin and measuring changes in lipogenesis and lipolysis. Since adipsin activates the alternative pathway of complement via its intrinsic Factor D catalytic activity, the ability of various components of the alternative complement pathway to regulate adipose cell physiology will be studied, including complement-derived ligands such as C3a, C5a and Ba. In addition to an in vitro approach, adipsin will be administered acutely and chronically to obese and lean animals to ascertain the role of this protein and the alternative complement pathway in systemic energy balance in vivo. Multiple measures of systemic energy balance and hormonal regulation will be measured. Preliminary pharmacokinetic studies in lean and obese mice support the feasibility of altering adipsin levels in vivo by administration of the pure protein. New data in transgenic mice shows that a rodent obesity gene (db) suppresses adipsin expression through its action (direct or indirect) on the adipsin promoter. Complementary cellular and biochemical approaches will be used to study this pathway of signal transduction and identify the cis- and trans-acting factors involved in the "obesity-response element". Cultured adipocytes will be treated with serum from lean and obese mice and with various hormones to model the in vivo effects of obesity on the endogenous adipsin gene. By transfection of chimeric constructions and a series of deletions and mutations from the adipsin gene promoter into these cells, sequences involved in a putative "obesity-response element" will be mapped. A biochemical approach will involve mapping this response element in the adipsin promoter via DNA footprinting (and gelshift) analysis of the adipsin promoter DNA using nuclear extracts from lean and obese animals. The identity of putative "obesity-response elements" will be critically tested in vivo using point mutagenesis in the adipsin promoter and the construction of transgenic, obese mice. The trans-acting factor(s) binding to this response element and ultimately responsible for aberrant adipsin expression in obesity will be cloned and characterized by biochemical and new molecular genetic techniques.

The lab has previously shown that adipsin, a mouse adipocyte-derived serine protease, is secreted into the bloodstream and is markedly deficient in several models of genetic and acquired obesity, suggesting a possible functional role for this protein in the systemic or local regulations of energy balance. The overall goal of the proposed work is two-fold: to understand the role of adipsin in adipose physiology and obesity and to use the adipsin gene as a model to understand how obesity genes regulate the function of other gene promoters. Both of these goals are novel and will increase our understanding of the biochemical and genetic basis of obesity. Adipsin's direct role in the physiology of adipose cells will be approached in vitro by treating fat cells with adipsin and measuring changes in lipogenesis and lipolysis. Since adipsin activates the alternative pathway of complement via its intrinsic Factor D catalytic activity, the ability of various components of the alternative complement pathway to regulate adipose cell physiology will be studied, including complement-derived ligands such as C3a, C5a and Ba. In addition to an in vitro approach, adipsin will be administered acutely and chronically to obese and lean animals to ascertain the role of this protein and the alternative complement pathway in systemic energy balance in vivo. Multiple measures of systemic energy balance and hormonal regulation will be measured. Preliminary pharmacokinetic studies in lean and obese mice support the feasibility of altering adipsin levels in vivo by administration of the pure protein. New data in transgenic mice shows that a rodent obesity gene (db) suppresses adipsin expression through its action (direct or indirect) on the adipsin promoter. Complementary cellular and biochemical approaches will be used to study this pathway of signal transduction and identify the cis- and trans-acting factors involved in the "obesity-response element". Cultured adipocytes will be treated with serum from lean and obese mice and with various hormones to model the in vivo effects of obesity on the endogenous adipsin gene. By transfection of chimeric constructions and a series of deletions and mutations from the adipsin gene promoter into these cells, sequences involved in a putative "obesity-response element" will be mapped. A biochemical approach will involve mapping this response element in the adipsin promoter via DNA footprinting (and gelshift) analysis of the adipsin promoter DNA using nuclear extracts from lean and obese animals. The identity of putative "obesity-response elements" will be critically tested in vivo using point mutagenesis in the adipsin promoter and the construction of transgenic, obese mice. The trans-acting factor(s) binding to this response element and ultimately responsible for aberrant adipsin expression in obesity will be cloned and characterized by biochemical and new molecular genetic techniques.

protein protein interaction; physiology; gene induction /repression; developmental genetics; adipocytes; transcription factor; cyclin dependent kinase; thiazoles; hormone regulation /control mechanism; obesity; blood glucose; cell differentiation; enzyme inhibitors; hyperlipidemia; gene mutation; insulin; protein structure function; tissue /cell culture; polymerase chain reaction; genetically modified animals; laboratory mouse;

One of the major challenges facing molecular geneticists and developmental biologists is understanding the mechanisms which control tissue specific gene expression in higher animals. During the past few years, there has been extensive progress in this direction. A variety of novel in vivo and in vitro systems have been developed, a number of transcription factors involved in cell- type specific gene expression were identified and the genes encoding such factors were isolated. The hormonal and developmental mechanisms controlling the activity of these regulatory genes are subject to extensive investigation. This proposal requests funds to finance a summer FASEB conference on the topic of Transcription Regulation: Differentiation, Development, and Disease. This meeting will congregate researchers actively engaged in studying various aspects of tissue specific gene expression. In addition to discussing the basic mechanism of gene regulation, special emphasis will be placed on discussion of both normal and aberrant gene regulation in the following organ systems: liver and pancreas, muscle, blood and the nervous system. We will also have special sessions dealing with the regulation of development and embryogenesis. The emphasis will be on gene regulation in vertebrates and expecially in mammals. %%% The conference will be held at Copper Mountain, Colorado from June 14-19, 1992. The meeting is designed to have formal slide presentations, informal poster sessions, a workshop, periods of group discussions, and plenty of opportunity for individual interaction. The conference will summarize the current understandings of tissue specific gene expression and should lead to dissemination of new information, concepts, and methodologies.

Insulin resistance is a ubiquitous correlate of obesity and plays a crucial role in several important medical conditions associated with obesity: hypertension, hyperlipidemia and especially, non-insulin dependent diabetes mellitus (NIDDM). Recently, we have shown that the expression of TNF-alpha by adipose cells, particularly in the context of obesity, interferes with the tyrosine kinase activity of the insulin- receptor and causes insulin resistance. This competing renewal application suggests continuing our studies to understand the mechanisms of TNF-induced insulin resistance and its role in obesity-diabetes. Our preliminary data has suggested that IRS-1 is phosphorylated on non- tyrosine residues in response to TNF-a, and immunoprecipitated IRS-1 from TNF treated cells is associated with an inhibitory action on the insulin receptor. We will first map phosphorylation sites on the insulin receptor and IRS-I in response to TNF-alpha and the role of these modifications will be tested by in vitro mutagenesis. The ability of TNF-alpha-induced protein kinases to phosphorylate IRS-1 on key residues will be studied using purified recombinant IRS-1 as a target. Whether this modified IRS-1 can act as a direct inhibitor of the insulin receptor or must first associate with another inhibitory molecule from TNF-~ treated cells will be tested. If such an inhibitory molecule is identified, it will be purified and cloned. In addition to these biochemical studies, the genetic function of IRS-1 in TNF mediated inhibition will be investigated using cells from IRS-1 deficient mice. The extracellular components and intracellular regulators responsible for the expression of TNF-alpha from fat cells will be studied. Preliminary data suggests that cultured adipocytes normally express very little TNF-alpha mRNA but can be induced with high levels of lipids, insulin and dexamethasone. Analysis of the requirements for different lipids will be performed, along with studies of the requirement for certain hormones. Once a system with robust TNF-alpha expression is established, the intracellular regulatory components will be investigated. We will determine first whether the induction of TNF-alpha occurs at the transcriptional or post-transcriptional levels. Ultimately, this will lead to a delineation of required cis-acting nucleic acid sequences and the isolation and cloning of key trans-acting factors. Finally, we will create strains of otherwise normal transgenic mice that express TNF-alpha from fat tissue. The promoterienhancer of the aP2 gene will be linked to the TNF-a coding segment and the resulting mice will be compared to control mice in their insulin sensitivity, degree of adiposity and development of diabetes. Of course, it is possible that obesity is necessary to provide other molecules in addition to TN F- alpha in order to get insulin resistance. We will investigate this by inducing obesity in the transgenic strains by feeding high fat diets. Mice will again be studied for changes in glucose homeostasis and insulin sensitivity via glucose tolerance tests and stimulation of insulin receptor tyrosine phosphorylation in various tissues.

DESCRIPTION (provided by applicant): During the past several years, we have shown that the coactivator PGC-1 can activate and coordinate several key aspects of energy and glucose metabolism. It does this by binding to and coactivating a large number of nuclear receptors such as PPARgamma, HNF-4alpha, GR and other tissue-selective transcription factors such as MEF2C and NRF-1. Our emphasis in the next 5 years will be to determine in detail the physiological role of PGC-1 and key mechanisms that are used to activate its transcriptional properties. Our first Aim will be to determine with "state of the art" precision how PGC-1 alters respiration, mitochondrial uncoupling and the control of reactive oxygen species (ROS). Our second Aim will utilize "knock-out" mice (general and tissue-specific) for PGC-1 to determine the precise role of this protein in several processes where a function has been suggested - mitochondrial biogenesis, thermogenesis and glucose homeostasis. Physiological studies will utilize clamp technique to determine functions of specific tissues. Aim 3 investigates the molecular mechanisms whereby p38 MAP kinase can regulate both the degradation of PGC-1 and its transcriptional activity. In particular we will investigate whether and how p38 can modulate the ability of the APC ubiquitin ligase to interact with PGC-1. In a related Aim (4), we will use knock-out mice to investigate the rote of PGC-1 in the cachexia and hypermetabolism brought about in physiological states associated with elevated cytokines and p38 activation: infection and cancer. The last Aim (5) will begin studies of the biological role of a new, close homolog of PGC-1 we have termed PGC-1-beta. We will investigate the activities of this protein, which is expressed at very high levels in BAT and heart, on mitochondrial biogenesis, respiration and the determination of brown adiopytes. Disorders of energy balance and glucose homeostasis are key componenets of obesity and Type 2 diabetes, the most common metabolic disorders in the industrial world. These studies should elucidate key regulatory steps that may lead to new targeted therapies.

DESCRIPTION: ( applicant's abstract) PPAR-gamma is a member of the nuclear receptor gene family that has been shown to play a key role in adipogenesis. PPAR-gamma is also expressed at very high levels in the colonic epithelium. Since colon cancer is a leading cause of cancer deaths, it is of great interest to understand the role of this receptor in the development of the colonic epithelium. The investigator's previous work has indicated that ligand activation of PPAR-gamma in human colon cancer cells induces a differentiative-like response including the cessation of cell growth and expression of genes characteristic of mature colonic epithelium. Most recently, he has shown that human colon cancer is associated with loss of function mutations in PPAR-gamma. Paradoxically, others have shown that TZD ligands, when applied to the polyp-prone min mouse that are genetically deficient in APC, develop more colon polyps. The present proposal will critically evaluate the role of PPAR-gamma in normal and abnormal colon development, and study the transcriptional mechanisms that underlie these functions. Firstly, the investigator will determine how PPAR-gamma intersects pathways known to regulate colon cell biology. Preliminary data show a strong connection between PPAR-gamma and TGF-beta signaling; hence, biochemical and genetic studies will focus on how Smad proteins interact with PPAR-gamma, and the relative contributions these interactions make to both signaling systems in the colon. The investigator will also study the transcriptional mechanisms that enable PPAR-gamma to induce a distinct program of gene expression when it is activated in colon cells. This will utilize both yeast 2-hybrid screens and biochemical purification to isolate components which interact with PPAR-gamma in a colon- or epithelium-selective manner. The function of novel proteins will be studied to discern their transcriptional and biological properties by expressing them in various mammalian cells. The program of colon-selective genes regulated by PPAR-gamma will be better characterized through the use of SAGE analysis, applied to mRNA from colon cells treated with PPAR-gamma ligands. This work, done in collaboration with Dr. Ken Kinzler, will be used to characterize the expression of known genes, and to clone novel genes. Again, the function of downstream targets of PPAR-gamma will be characterized for their ability to regulate colon cell growth and differentiation. Finally, the role of PPAR-gamma in modulating colon cancer will be critically evaluated in transgenic mice, using potential dominant-negative alleles with a colon-selective promoter, and heterozygous PPAR-gamma KO mice that are already available. The initial studies will evaluate carcinogenesis using the chemical carcinogen DMH, though other transgenic methods of inducing colon cancer will also be evaluated. In summary, the investigator hopes that these studies will to provide a unique insight into the role of PPAR-gamma in normal and malignant colon development. Since PPAR-gamma ligands are clinically available now, this work may provide a framework in which to contemplate new therapeutic approaches to colon cancer.

DESCRIPTION (provided by applicant): The PGC-1 coactivators increase the transcription of target genes;they have emerged as major regulators of many pathways involved in energy metabolism important in diabetes, hyperlipidemia and obesity. The liver functions as a central integrator of energy homeostasis, particularly the switch between the fed and fasted states. The previous cycle of this grant has funded studies showing that PGC-1 a plays a major role in expression of the genes of hepatic gluconeogenesis, while PGC-1[unreadable] is a positive activator of a broad program of hepatic lipogenesis and lipoprotein secretion. The present application proposes studies aimed toward moving these studies into a physiological realm, including studies of an interesting new genetic variant of PGC-1[unreadable] found in many humans. Specific Aim 1 suggests detailed physiological studies of mice with a liver-specific mutation in PGC-1a. We will study glucose and lipid homeostasis during fasting and feeding in these mice, we will also challenge these animals with high fat-induced obesity and insulin resistance. Detailed analyses of metabolism will include glucose and insulin tolerance tests as well as hyperinsulinemic-euglycemic clamps. Aim 2 will perform proteomic analyses of PGC-1a and PGC-1[unreadable] holo- complexes from murine liver, using a new isolation procedure we have developed. Importantly, we will study changes in the molecular anatomy of these complexes during physiological and pathophysiological states such as fasting, high fat feeding and obesity. Aim 3 will be focused on the mechanisms by which fatty acids stimulate the PGC-1[unreadable] promoter;preliminary data suggests an important role for the PPAR nuclear receptors. Finally, Aim 4 will focus on biochemical and genetic studies of a novel allele of human PGC-1[unreadable] (A203P) that has been associated with a reduced risk of obesity. This variant has reduced coactivation function on ERRa and LXRa, key coactivation partners of PGC-1[unreadable]. Analyses of this allele will include studies of its ability to activate gene expression in hepatocytes and liver in vivo. We will also knock this allele into the germ-line of mice, and study the effects of this polymorphic variant on hepatic energy metabolism, and energy homeostasis more systematically. These studies will allow us to analyze the role of this human polymorphism in obesity, and will also allow us to generate new hypotheses concerning the role of PGC-1[unreadable] in human disease.

DESCRIPTION (provided by applicant): Heart disease is the leading cause of death in the industrialized world. Survival after myocardial infarction has improved considerably, but the cardiac remodeling that follows acute insults like ischemia/reperfusion injuries and chronic ones like hypertension and diabetes has led to a dramatic increase in the prevalence of heart failure. The failing heart displays numerous energetic abnormalities, including decreased expression of the transcriptional coactivator PGC-1a. The proven role of PGC-1a as a dominant regulator of mitochondrial biogenesis and respiration suggests that this molecule could represent a key control point for heart failure and open new therapeutic approaches. We and others have shown that PGC-1a -/- mice have important physiological cardiac abnormalities. Here, we will investigate the role of PGC-1a in cardiac disease. First, we will test if loss of PGC-1a exacerbates heart failure, by using transverse aortic constriction (TAG) in mice lacking PGC-1a. Preliminary data indicates a severe worsening of heart failure in the absence of PGC-1a. Conversely, we will also ask whether the development of heart failure can be ameliorated by mild, transgenic expression of PGC-1a in the heart. We also show in preliminary data that PGC-1a plays a key role in the suppression of reactive oxygen species (ROS) through expression of a broad program of ROS detoxification genes. Ischemia/reperfusion injury in the heart is thought to damage the heart in large part via generation of ROS. We will evaluate, using mice with gain or loss of PGC-1a, the role of this coactivator in ischemia/reperfusion injury in the heart. Analysis will be at both molecular and functional levels. Lastly, we will examine in detail the mechanisms by which PGC-1a plays a cardioprotective role by creating mutant alleles of this coactivator that selectively lose the ability to modulate either the ATP producing system or the ROS detoxification program. These alleles will be evaluated in a tissue culture setting and then knocked into the murine germline, and the subsequent effects on the heart will be examined. This proposed work, taken together, should critically evaluate the ability of the PGC-1 pathway to influence and perhaps ameliorate major forms of heart disease. This may lead to the development of a new class of therapeutics.

DESCRIPTION (provided by applicant): Mitochondria play a critical role in the energy homeostasis of the cell and the intact animal. Mitochondria oxidize substrates such as lipids and glucose, thereby serving as a central control point for fuels which are dysregulated in human type 2 diabetes. Recent data suggests that human type-2 diabetes is associated with lower expression of the mitochondrial OXPHOS system and their regulators, the PGC-1 coactivators, in muscle. Our recent unpublished genetic data clearly indicate that reduced PGC-1a levels in muscle can cause abnormal glucose tolerance, even in the heterozygous KO state. We propose here an integrated approach to develop chemical compounds that can modulate the PGC-1 coactivators and/or mitochondrial function in both cells and animals. The goal is to establish proof of concept that chemical manipulations of these systems can have a benefit in obesity and type-2 diabetes. We will utilize chemical libraries that include a large number of FDA-approved drugs and drug-like compounds, as well as novel chemical libraries produced at the Broad Institute. We will also investigate molecular targets of novel compounds where the target is not known, using a novel method involving the use of recombinant yeast strains. Specific Aim 1 will utilize mitochondrial-based screens to find compounds that can control mitochondria number, membrane potential and ATP synthesis. Specific Aim 2 will perform screens that will identify compounds that modulate the expression of PGC-1a and PGC-1beta, and their function in mitochondrial biology. Specific Aim 3 will utilize a novel method using recombinant yeast strain to identify targets that are being altered by novel chemical matter arising from Aims 1 and 2. In all cases, we will use cell-based screens of mitochondrial function and insulin-resistance. In addition, we will test the compounds arising from these screens in well-established animal models of obesity, insulin resistance and type-2 diabetes. All of these projects will make use of chemical screening at the Broad Institute and the animal resources at the DFCI. Together, this highly integrated and collaborative project has a high likelihood of providing proof of concept for a novel avenue to the therapeutics of obesity and diabetes.

DESCRIPTION (provided by applicant): This proposal is closely aligned with the Research Theme: "Translating Basic Science Discoveries into New and Better Treatments". The USA is in the midst of an epidemic of obesity that is causing a great deal of mortality and morbidity due to its associated conditions: hypertension, type 2 diabetes and certain cancers. It is also putting a great strain on our health care system. At the present time, there is no generally effective medical therapy for obesity that can augment ongoing efforts to educate the public about diet and exercise regimens. This proposal is aimed at driving therapeutics for human obesity forward, based on modulation of brown fat biology. The last several years have seen breakthroughs in the science of brown fat, a cell-type that plays a critical role in controlling metabolic rates and fighting obesity. Significant deposits of brown fat have been identified in normal healthy humans, and a major transcriptional regulator of brown fat, PRDM16, has been identified. This proposal is focused on developing the science and therapeutic approaches to human obesity, based on the control of brown fat formation and function. Our first Aim will investigate regulation of whole body energy homeostasis via transgenic manipulation of PRDM16, a transcriptional co-regulator that we discovered in 2007 as a dominant regulator of brown fat cell determination. A second Aim will isolate and characterize a second kind of brown fat cell that can reside in white adipose tissues and has substantial thermogenic capacity. Our third Aim will drive translation of our newly acquired knowledge about the role of the nuclear receptor PPAR3 in brown fat and thermogenesis directly into therapeutics. We will combine structure-based methods for chemical screening and compound optimization, with this new biochemical information, to develop PPAR3 ligands that have a preferential effect on brown fat development and function. We will develop compounds that have minimal properties of a classic PPAR3 agonist, but retain the ability to modulate the "browning" of certain white fat cells and stimulate brown fat-mediated energy expenditure in vivo. The ability to treat obesity in animal models will also be investigated with the new compounds. In our final Aim, we will utilize our extensive data concerning brown fat gene expression to investigate the secreted proteins of brown fat that are regulated during thermogenesis. We have already identified several such molecules and will examine their ability to control/affect brown fat cell differentiation and a thermogenic gene program in brown fat and subcutaneous white fat. PUBLIC HEALTH RELEVANCE: The rising tide of obesity in the USA presents a huge threat to the health of the American public, through its associated conditions: hypertension, diabetes and cardiovascular disease. This proposal is centered on therapeutic targeting of brown fat, a key part of the body's natural defense against obesity. Improvements in our ability to regulate pathways of energy expenditure mediated by brown fat promise to relieve the disease burden of the American population and lower healthcare costs in the USA.

DESCRIPTION (provided by applicant): We are in the midst of an epidemic of metabolic diseases such as obesity and diabetes, so a better understanding of the basic pathways of energy homeostasis is critical to the development of new medical therapies. Previous studies, including work funded under this grant, have shown that the PGC1 coactivators are major regulators of energy metabolism in mammalian systems. PGC1a regulates mitochondrial biogenesis and oxidative metabolism in many tissues, and links these genetic programs to the external environment. PGC1a controls important metabolic programs in the liver, brown fat, heart and skeletal muscle. In skeletal muscle, the PGC1a gene is induced during many kinds of exercise in mice and humans, and drives programs of mitochondrial biogenesis, fiber-type switching and resistance to atrophy/dystrophy. Most recently, we have shown that PGC1a initiates a novel program of angiogenesis that is potent and independent of the canonical HIF angiogenesis pathway. In preliminary data for this proposal, we show that the PGC1a gene gives rise to several novel proteins in muscle via alternative splicing. The original PGC1a (now called PGC1a1) and a new shorter form (termed PGC1a4) are both highly induced during exercise in mice. When expressed in primary culture or in vivo, PGC1a4 induced muscle cell hypertrophy but not mitochondrial biogenesis. This hypertrophy is associated with regulation of several genes of the myostatin and IGF1 pathways, both key signaling transduction systems of muscle growth. Our first Aim will test the hypothesis that PGC1a4 plays an important role in regulating physiological muscle hypertrophy and metabolic disease in vivo. To do this, we will create transgenic models of having both gain and loss of function of PGC1a4 in mice. We will combine these with different experimental models of muscle hypertrophy, atrophy and metabolic disease, including diet and age-induced obesity and diabetes. Our second Aim will investigate the mechanisms by which PGC1a4 can effect these changes in muscle cell growth and function. We will focus initially on the hypothesis that PGC1a4 works through the IGF1 and myostatin systems, since genes of these pathways are both robustly regulated by PGC1a4. Our last Aim will focus on the interaction between PGC1a1 and AMP kinase. We have created a murine strain with mutations in the AMPK sites in PGC1a1, and we will now critically test the hypothesis that this functional interaction contributes to the metabolic actions of both molecules in skeletal muscle and liver. Together, these studies promise to elucidate a major new pathway controlling skeletal muscle hypertrophy and muscle function. This has a direct impact on our ability to understand and provide new therapies in important diseases of aging, muscular dystrophies and obesity/diabetes.

DESCRIPTION (provided by applicant): Diabetes and obesity continue to represent major challenges to the health and health care systems of the USA and other countries. Current therapies are helpful, especially for type 2 diabetes, but they are still inadequate to prevent the negative effects of the Metabolic Syndrome on the cardiovascular system, cancer and other disorders. It has long been recognized that exercise is an excellent first line therapy for both diabetes and obesity [1]; however, many patients are unable to exercise sufficiently for a variety of reasons. A huge challenge has been to capture some of the benefits of exercise in a manner that can be useful medically for the very broad range of diseases for which exercise appears to provide benefit. Previous work on this grant has shown that the transcriptional coactivator PGC1¿ is induced with exercise in muscle and gives muscle many of the attributes seen in this tissue with chronic exercise. We show here that PGC1¿ expression in cultured muscle cells and transgenic mice stimulates the secretion of an activity that can drive the expression of UCP1 and other genes of the brown fat program in white adipose cells. Using global gene expression analyses, coupled with bioinformatics tools, we identify a key secreted protein as Fndc5, a muscle protein previously identified as an intracellular factor. We show here that Fndc 5 is cleaved and secreted as a novel 112 amino acid polypeptide termed irisin. Circulating irisin is increased with exercise in rodents and humans, and mildly elevated irisin in mice stimulates a browning of the white fat, with increased energy expenditure and improved glucose homeostasis. Aim 1 of this new application proposes purifying and cloning the cellular irisin receptor using an active fusion protein between irisin and the Fc moiety of human IgG. Alternatively, this tool can also be used to isolate the irisin receptor by expression cloning. Critical tests of the function of the irisin receptor will involve genetic manipulations in both cultured cells and in the adipose tissues of mice. Aim 2 examines the mechanism by which PGC1¿ controls irisin gene expression. We will employ a cultured muscle cell system to examine the chromatin marks at and near the Fndc5 locus to identify the key transcription factors through which PGC1¿ activates Fndc5 expression. We will also study whether manipulation of these key PGC1¿-docking factors can regulate irisin expression in vivo. Post-translation modifications (PTM) of PGC1¿ have been shown to be important modulators of its function but there have been no systematic examination of PTMs in any biological system. In Aim 3 we will purify PGC1¿ from skeletal muscle under sedentary and exercised conditions. In collaboration with Steven Gygi and Pere Puigserver, we will determine all covalent modifications by quantitative Mass Spectrometry. Mutant alleles will be utilized both in vitro and in vivo to ascertain the physiological significance of these PTMs. Together these studies should lend important insights into the basic science of muscle and exercise as well as open new avenues for the rapid development of new therapeutics. PUBLIC HEALTH RELEVANCE: Diabetes and obesity take a major toll on health in the US. We have found a new polypeptide hormone that is produced by muscle with exercise and we will explore the molecular mechanisms by which this hormone increases energy expenditure and improves abnormalities in glucose homeostasis. This work has exciting promise for the development of new treatments for metabolic diseases and other disorders that are improved with exercise.

DESCRIPTION (provided by applicant): We are in the midst of a worldwide epidemic in type 2 diabetes (T2D) that is exacerbated by an increasingly conservative pharmaceutical industry that is now desperate for new targets. A growing body of evidence implicates altered mitochondrial function in the pathogenesis of T2D and obesity. For example, mitochondrial metabolism is critical in the control of glucose stimulated insulin secretion, hepatic gluconeogenesis, and peripheral fuel oxidation. The only known direct target of metformin, one of the most useful agents for treating T2D, is a mitochondrial complex. Reduced mitochondrial mass/function have been documented in the skeletal muscle of humans with obesity and T2D, and during aging, and reversible with exercise. Brown fat, which expends chemical energy through mitochondrial uncoupling, has recently emerged as a possible therapeutic target for human obesity. Collectively, these observations raise the exciting hypothesis that modulating mitochondrial physiology may help prevent or reverse the pathophysiology of T2D and obesity. The goal of this R24 project is to discover a mechanistically diverse collection of small molecules with desirable pharmacologic properties that can modulate mitochondrial energetics in vivo by targeting transcriptional programs, translational programs, and direct mitochondrial physiology. Our highly integrated project brings together experts in mitochondrial biogenesis, bioenergetics, chemical screening, and medicinal chemistry, to build and pursue this bold therapeutic hypothesis. In Aim 1 we will follow-up on exciting preliminary data that has revealed a novel small molecule and its target, a plasma membrane ion channel that controls mitochondrial biogenesis via a transcriptional mechanism. Using this validated screening strategy, we will screen for additional novel small molecules acting via transcriptional mechanisms that promote brown fat differentiation. In Aim 2 we will follow-up on a large-scale chemical screen that is designed to discover small molecules that work at the level of post-translational modifications to influence mitochondrial biogenesis. In Aim 3 we will capitalize on the recent discovery of mitochondrial calcium channel subunits, enabled by the previous funding period of this grant, and screen for novel drugs that directly target mitochondrial physiology and energetics through targeting mitochondrial calcium flux. For all three aims we will collaborate closely with leading chemists at Broad Institute and Scripps to perform in-depth lead optimization and formulation and evaluate the novel drugs both in cultured cells as well as in rodent models. If successful, this collaborative project could result in the discovery of mechanistically diverse small molecules that will advance our fundamental understanding of the contribution of mitochondrial metabolism to the development of T2D, while also helping to launch a potentially brand new class of therapeutics for this growing epidemic.

? DESCRIPTION (provided by applicant): We are in the midst of a worldwide epidemic of obesity and related metabolic diseases. It is now known that the incidence type 2 diabetes, NASH, cardiovascular disorders and even cancer are increased with obesity. This represents a huge burden upon the health and health care system of the United States and much of the rest of the world. While education concerning diet and exercise are critical components to a solution for these problems, there is a huge need to improve both our basic understanding of and therapeutic options for all of these conditions. Two key parts of organismal energy balance are food intake and energy expenditure. The latter includes physical activity and brown fat-mediated thermogenesis. Our work under this grant (over several years) discovered the PGC1 transcriptional coactivators and their role in various pathways of mitochondrial biogenesis and oxidative metabolism. In the last grant cycle we identified 3 new proteins encoded by the PGC1? gene; PGC1?4, in particular, has interesting and powerful activities in muscle and brown fat. PGC1?4 does not stimulate mitochondrial biogenesis in muscle, but rather increases muscle hypertrophy, strength and resistance to cancer cachexia. Preliminary data here shows that it affects UCP1 and other themogenic genes in fat but again, does not stimulate mitochondrial biogenesis. PGC1?4 also increases expression of a novel myokine, meteorin-like (METRNL), that is a powerful stimulator of adipose tissue browning through modulation of the immune cells within fat tissue. The new cycle of this grant will explore the full range of biologicl functions of METRNL in cellular and systemic metabolism, using knock-out mice already breeding in our lab. Preliminary data has identified a clonal fat cell line that has a molecular response to METRNL and we will use these cells (3T3-F442A) to clone and characterize the METRNL receptor. Isolation of this receptor will be through protein purifications, using tagged MERTNL and advanced protein Mass Spectrometry methods, specifically isobaric tagging. It is expected that the receptor identification will also lead us to new tissues and pathways where METRNL may function in important ways. Lastly, we will return to a key mechanistic problem: how can PGC1?4, a shorter form of the canonical PGC1? protein, regulate such a different set of genes in both muscle and brown fat. Because the PGC1s are coregulatory proteins, this must involve, at least in part, differential interactions with partner transcription factors and other proteins. To investigate this, we will use purifications of the PGC1? complexes, followed by comparative and quantitative Mass Spectrometry. The function of these new factors in the PGC1 complexes will be studied by genetic and (potentially) pharmacological methods.

Abstract Post-translational modifications of proteins are a major mechanism for the modulation of protein activity. Among these, the most common is protein phosphorylation by a set of enzymes called protein kinases. Protein kinases (PKs) are involved in almost every pathway of biological significance in eukaryotes. Metabolic regulation requires protein kinase function in every system, including insulin and glucagon action, glycogenolysis, adipogenesis and adaptive thermogenesis. Excessive AKT protein kinase activity downstream of insulin action is also suspected of being a key link between obesity and cancer. The mammalian kinome includes some 560 known enzymes, about 0.2% of the entire coding genome. As far as it is known, every one of these enzymes utilizes ATP as a high-energy phosphate donor, transferring the ?-phosphate of ATP onto (mainly) serine, threonine or tyrosine residues. Nature has developed another high-energy phosphate containing molecule that is often more abundant than ATP: phosphocreatine (CrP). Because ATP is an inhibitor of ATP synthase, cells can't store ATP. Instead, the ?-phosphate of ATP can be transferred to creatine (Cr), regenerating ADP and allowing the electron transport chain to continue functioning in the ?forward? direction. Our recent work has demonstrated a non-canonical function of Cr and CrP in thermogenic adipose cells, which run a futile cycle of creatine phosphorylation and de-phosphorylation. This futile cycle expends energy without doing work and hence, results in the generation of heat. This work demonstrating a broader function of creatine than ?just? energy storage caused us to ask an unusual question: are there protein kinases that preferentially use CrP? In fact, we have demonstrated here, using high-resolution protein Mass Spectrometry, that brown fat cell extracts can utilize CrP to phosphorylate certain peptide sites (at both S/T and Y residues) that are not modified when ATP is used as a substrate. Our key goals moving forward are to (1) demonstrate that these phosphorylation events are direct phosphate transfer reaction from CrP to target proteins (2) to demonstrate that these phosphorylations are dependent on CrP in vivo, using murine models of Cr and CrP-deficient animals (3) purify and characterize the CrP-dependent PKs. These may be new members of the kinome or known PKs that alter peptide target specificity when they use CrP as a substrate (4) perform biochemical and biophysical studies to characterize the enzymatic reactions and identify the CrP binding sites on the PKs. (5) investigate the physiological importance of the CrP PKs, by mutating specific target sites in protein targets and ablating the CrP-dependent PKs themselves. This project will open up a potentially important new area in biochemistry and physiology, and represents a ?high-risk, high-reward type of project for which the Catalyst Award is intended.

a. Abstract The transcriptional coactivator PGC1? was discovered by my group in 1998. It functions as a dominant regulator of mitochondrial biogenesis and oxidative metabolism by coactivating several nuclear transcription factors that control the broad program of mitochondrial gene expression. PGC1? also has important tissue specific functions, including control of adipose thermogenesis, the fasting response in liver, and mitochondrial biology and resistance to atrophy in skeletal muscle. Mechanisms that activate thermogenesis in fat and prevent atrophy in muscle are of enormous importance in human metabolic diseases such as diabetes and obesity. Preliminary data illustrates a very robust and novel translational control of PGC1? mRNA in cultured cells and in vivo; this mRNA translation is regulated by insulin and IGF1 signaling through AKT and mTORC signaling. Moreover, it is negatively regulated by the presence of a very small open-reading frame (uORF) just upstream of the codon that begins translation of the canonical PGC1?1 (the canonical PGC1? isoform; hereafter just called PGC1?) mRNA. Loss of this uORF by deletion or mutation increases the translation of PGC1? mRNA while ablating the insulin/IGF1 effect. This uORF encodes a predicted peptide of 15 amino acids that is strongly conserved in all mammalian species. We will begin these studies by using several mouse models using CRISPR technology (now created) which increase or decrease expression of this uORF by altering the start codon of this small encoded peptide (Aim 1). Mice will be analyzed for effects on key aspects of animal metabolism and physiology (Aim 2). These will include energy expenditure and resistance to obesity-linked glucose intolerance via thermogenic fat, gluconeogenesis in liver and exercise tolerance in muscle. Since skeletal muscle and its atrophy is a critical component of aging and an important target of insulin action, we will examine atrophy in the muscle-selective models. Mechanisms by which the 5' UTR and uORF control translation of PGC1? mRNA will be examined in cells by determining if the uORF functions in cis or trans via 2 plasmid experiments and through use of molecular ?toeprint? and ?footprint? assays (Aim 3). The presence of the uORF peptide in cell extracts will be determined by Mass Spectrometry with the use of synthetic ?heavy? peptides as key internal standards. Moreover, we will set up an in vitro translation system and determine if this regulation can be recapitulated in vitro. Key regulatory components of this system will be isolated by established affinity chromatography methods using oligonucleotides. Finally, Aim 4 will address the critical question of how insulin/IGF1 signaling impacts this translational control through quantitative phosphoprotein Mass Spectrometry in insulin treated cells. Phospho-proteomic analyses will also be applied to components isolated through the affinity methods described above. Together, these data will provide crucial perspectives and potential new therapeutic targets through which mitochondrial biology, physiology and disease processes might be manipulated in in vivo settings.

PROJECT SUMMARY/ABSTRACT There is a great deal of interest in adipose biology, particularly in light of the world-wide epidemic in obesity and metabolic diseases, including type 2 diabetes, cardiovascular disease and cancer. While adipose tissues are best known as the major storage site for calories, certain fat tissues play a critical role in adaptive thermogenesis, the process whereby chemical energy is dissipated in the form of heat in response to external stimuli. Thermogenic adipose tissues, brown and beige, defend the body against hypothermia, obesity and other metabolic disorders. Critical unmet needs include understanding the detailed molecular pathways by which chemical energy is converted into heat and the discovery of human therapeutics that might increase amounts and function of thermogenic fat. Four years ago, we described a previously unknown thermogenic pathway in brown and beige fat that plays a major role in both energy expenditure and suppression of obesity in animal models. Disruptions of this futile creatine cycle causes levels of obesity not observed with ablations of any previously described thermogenic mechanisms, including UCP1; in response to these observations, I am focusing this grant entirely on further biochemical and physiological studies of this futile creatine pathway. One Aim will focus on the role of the creatine transporter (CrT) in fat tissues, where preliminary data with adipo-CrT KO mice shows that this exogenous pathway for creatine accumulation contributes significantly to whole body energy homeostasis. The physiological role of the CrT specifically in fat will be analyzed with metabolic cages to study mutant mice under several different physiological perturbations. This mutation will also be combined with our previous genetic model (adipo-GATM-KO), which is unable to synthesize creatine de novo, to create an animal model totally lacking adipose accumulation of creatine. A related Aim will be to study regulation of the CrT mRNA and protein; preliminary data shows mRNA to be down-regulated in fat cells from obese human subjects. Importantly, we will also use metabolomic studies (LC/MS) to follow the fate of phosphocreatine (CrP), as it is processed/hydrolyzed in mitochondria from thermogenic fat cells. Our last Aim will focus on a major unanswered biochemical question: exactly how is the high energy phosphate on CrP dissipated as part of this futile cycle. In this regard, we have exciting preliminary data using 31P NMR: mitochondrial preparations from thermogenic fat contain an activity that can hydrolyze CrP directly. We have purified this activity and have identified it as TNAP, an alkaline phosphatase. While not annotated as a mitochondrial protein, we find a substantial portion of this protein in the mitochondrial associated membrane (MAM) fraction. We will perform genetic and pharmacological manipulations of TNAP to determine its role in thermogenesis and the futile creatine cycle. We will also use protein Mass Spectrometry to determine how this protein may be modified to achieve its association with mitochondria. Together, these studies will advance basic knowledge of adaptive thermogenesis and provide potential new avenues to human therapeutics in metabolic diseases.