White adipose tissue is an important endocrine organ involved in the control of whole-body metabolism, insulin sensitivity, and food intake. To better understand these functions, 3T3-L1 cell differentiation was studied by using combined proteomic and genomic strategies. The proteomics approach developed here exploits velocity gradient centrifugation as an alternative to isoelectric focusing for protein separation in the first dimension. A 20-to 30-fold increase in the concentration of numerous mitochondrial proteins was observed during adipogenesis, as determined by mass spectrometry and database correlation analysis. Light and electron microscopy confirmed a large increase in the number of mitochondrion profiles with differentiation. Furthermore, mRNA profiles obtained by using Affymetrix GeneChips revealed statistically significant increases in the expression of many nucleus-encoded mitochondrial genes during adipogenesis. Qualitative changes in mitochondrial composition also occur during adipose differentiation, as exemplified by increases in expression of proteins involved in fatty acid metabolism and of mitochondrial chaperones. Furthermore, the insulin sensitizer rosiglitazone caused striking changes in mitochondrial shape and expression of selective mitochondrial proteins. Thus, although mitochondrial biogenesis has classically been associated with brown adipocyte differentiation and thermogenesis, our results reveal that mitochondrial biogenesis and remodeling are inherent to adipose differentiation per se and are influenced by the actions of insulin sensitizers.

Adipose tissue plays a central role in the control of energy homeostasis through the storage and turnover of triglycerides and through the secretion of factors that affect satiety and fuel utilization. Agents that enhance insulin sensitivity, such as rosiglitazone, appear to exert their therapeutic effect through adipose tissue, but the precise mechanisms of their actions are unclear. Rosiglitazone changes the morphological features and protein profiles of mitochondria in 3T3-L1 adipocytes. To examine the relevance of these effects in vivo, we studied white adipocytes from ob/ob mice during the development of obesity and after treatment with rosiglitazone. The levels of approximately 50% of gene transcripts encoding mitochondrial proteins were decreased with the onset of obesity. About half of those genes were upregulated after treatment with rosiglitazone, and this was accompanied by an increase in mitochondrial mass and changes in mitochondrial structure. Functionally, adipocytes from rosiglitazone-treated mice displayed markedly enhanced oxygen consumption and significantly increased palmitate oxidation. These data reveal mitochondrial remodeling and increased energy expenditure in white fat in response to rosiglitazone treatment in vivo and suggest that enhanced lipid utilization in this tissue may affect whole-body energy homeostasis and insulin sensitivity. IntroductionThe ability of brown adipose tissue to affect whole-body metabolism upon thermogenic stress has been known for many years (1, 2). More recently, the important contribution of white adipose tissue (WAT) to the control of energy homeostasis has been recognized with the study of tissue-specific knockout models and the discovery of adipocyte-specific secreted factors that have powerful effects on fuel metabolism (3, 4). These observations have raised many as-yetunanswered questions about the mechanisms by which adipocytes maintain their energy homeostasis as well as sense and respond to the metabolic requirements of the organism.Brown adipocytes contain a large complement of mitochondria that serve as a source of heat generated as a consequence of flow through the electron transport chain under uncoupled conditions elicited by uncoupling protein 1 (UCP-1). Recently we reported that adipogenesis of the 3T3-L1 cell line, representative of white adipocytes, is also accompanied by a stimulation of mitochondrial biogenesis (5). The need for a large mitochondrial mass in white fat can be linked to key functions of the adipocyte that require mitochondrial function. For example, adipocytes must generate glycerol 3-phosphate at a rate sufficient to sustain triglyceride synthesis, and for this the glyceroneogenic pathway and

Adipose tissue plays a central role in the control of energy homeostasis through the storage and turnover of triglycerides and through the secretion of factors that affect satiety and fuel utilization. Agents that enhance insulin sensitivity, such as rosiglitazone, appear to exert their therapeutic effect through adipose tissue, but the precise mechanisms of their actions are unclear. Rosiglitazone changes the morphological features and protein profiles of mitochondria in 3T3-L1 adipocytes. To examine the relevance of these effects in vivo, we studied white adipocytes from ob/ob mice during the development of obesity and after treatment with rosiglitazone. The levels of approximately 50% of gene transcripts encoding mitochondrial proteins were decreased with the onset of obesity. About half of those genes were upregulated after treatment with rosiglitazone, and this was accompanied by an increase in mitochondrial mass and changes in mitochondrial structure. Functionally, adipocytes from rosiglitazone-treated mice displayed markedly enhanced oxygen consumption and significantly increased palmitate oxidation. These data reveal mitochondrial remodeling and increased energy expenditure in white fat in response to rosiglitazone treatment in vivo and suggest that enhanced lipid utilization in this tissue may affect whole-body energy homeostasis and insulin sensitivity. IntroductionThe ability of brown adipose tissue to affect whole-body metabolism upon thermogenic stress has been known for many years (1, 2). More recently, the important contribution of white adipose tissue (WAT) to the control of energy homeostasis has been recognized with the study of tissue-specific knockout models and the discovery of adipocyte-specific secreted factors that have powerful effects on fuel metabolism (3, 4). These observations have raised many as-yetunanswered questions about the mechanisms by which adipocytes maintain their energy homeostasis as well as sense and respond to the metabolic requirements of the organism.Brown adipocytes contain a large complement of mitochondria that serve as a source of heat generated as a consequence of flow through the electron transport chain under uncoupled conditions elicited by uncoupling protein 1 (UCP-1). Recently we reported that adipogenesis of the 3T3-L1 cell line, representative of white adipocytes, is also accompanied by a stimulation of mitochondrial biogenesis (5). The need for a large mitochondrial mass in white fat can be linked to key functions of the adipocyte that require mitochondrial function. For example, adipocytes must generate glycerol 3-phosphate at a rate sufficient to sustain triglyceride synthesis, and for this the glyceroneogenic pathway and

Apoptosis is a regulated cell death program controlled by extrinsic and intrinsic signaling pathways. The intrinsic pathway involves stress signals that activate pro-apoptotic members of the Bcl-2 family, inducing permeabilization of mitochondria and release of apoptogenic factors. These proteins localize to the outer mitochondrial membrane. Ian4, a mitochondrial outer membrane protein with GTP-binding activity, is normally present in thymocytes, T cells, and B cells. We and others have recently discovered that a mutation in the rat Ian4 gene results in severe T cell lymphopenia that is associated with the expression of autoimmune diabetes. The mechanism by which Ian4 controls T cell homeostasis is unknown. Here we show that the absence of Ian4 in T cells causes mitochondrial dysfunction, increased mitochondrial levels of stress-inducible chaperonins and a leucine-rich protein, and T cell-specific spontaneous apoptosis. T cell activation and caspase 8 inhibition both prevented apoptosis, whereas transfection of T cells with Ian4-specific small interfering RNA recapitulated the apoptotic phenotype. The findings establish Ian4 as a tissuespecific regulator of mitochondrial integrity. Apoptosis is a regulated cell death program that can be initiated by two different signaling pathways. The extrinsic pathway involves ligation of cell surface death receptors and recruitment of proteins to a death-inducing signaling complex (1). The intrinsic pathway is death receptor-independent and involves stress signals that activate pro-apoptotic members of the Bcl-2 family; this action in turn induces permeabilization of mitochondria and the release of apoptogenic factors (2). These proteins are localized to the outer mitochondrial membrane, but the mechanisms responsible for regulating mitochondrial homeostasis localize to the inner membrane (3). How the two systems interact is unknown.Immune-associated nucleotide-binding protein 4 (Ian4) was originally identified as a highly expressed protein in Bcr͞Abl-transformed 32D cells (4). It localized to the mitochondrial outer membrane and displayed GTP-binding activity in vitro (4). Cells transfected with mutated Bcr͞Abl constructs lacked oncogenic potential and displayed lower Ian4 expression. Homologues of Ian4 are present in mouse, rat, and human (5). More recently, Ian4 was found to be disrupted in diabetes-prone BB (BBDP) rats (5, 6), which develop severe T lymphopenia due to apoptosis of recent thymic emigrants (7). We hypothesized that mitochondrial Ian4 (also known in the rat as Ian4l1 and Ian5) plays an important role in regulating T cell survival through control of T cell apoptosis. MethodsAnimals. Diabetes-resistant BB (BBDR) rats and BBDP rats were obtained from Biomedical Research Models (Worcester, MA). Approximately 90% of BBDP rats develop spontaneous autoimmune diabetes; they are Ian4 Ϫ/Ϫ and congenitally lymphopenic (8). BBDR rats are Ian4 ϩ/ϩ and never become spontaneously diabetic (8). Wistar Furth (WF) rats were obtained from Harlan-Sprague-Dawley. A congenic, no...

Mice with a fat-specific insulin receptor knock-out (FIRKO) exhibit a polarization of white adipose tissue into two populations of cells, one small (diameter <50 m) and one large (diameter >100 m), accompanied by changes in insulin-stimulated glucose uptake, triglyceride synthesis, and lipolysis. To characterize these subclasses of adipocytes, we have used a proteomics approach in which isolated adipocytes from FIRKO and control (IR lox/lox) mice were separated by size, fractionated into cytosolic and membrane subfractions, and analyzed by sucrose gradient, SDS-PAGE, and mass spectrometry. A total of 27 alterations in protein expression at key steps in lipid and energy metabolism could be defined, which were coordinately regulated by adipocyte cell size, impaired insulin signaling, or both. Nine proteins, including vimentin, EH-domain-containing protein 2, elongation factor 2, glucose-regulated protein 78, transketolase, and succinyl-CoA transferase were primarily affected by presence or absence of insulin signaling, whereas 21 proteins, including myosin non-muscle form A, annexin 2, annexin A6, and Hsp47 were regulated in relation to adipocyte size. Of these 27 alterations in protein expression, 14 changes correlated with altered levels of mRNA, whereas the remaining 13 were the result of changes in protein translation or turnover. These data suggest an intrinsic heterogeneity in adipocytes with differences in protein expression patterns caused by transcriptional and post-transcriptional alterations related to insulin action and cellular lipid accumulation.Adipose tissue plays a central role in the pathogenesis of diabetes and obesity. White adipose tissue provides the primary site of energy storage in the body and also serves as an important endocrine cell through secretion of hormones such as leptin, adiponectin (ACRP30), tumor necrosis factor ␣, resistin, and other cytokines (1). Brown adipose tissue is the major site of energy expenditure through expression of uncoupling protein 1 and its role in thermogenesis (2).Insulin signaling plays an important role in lipid storage and the process of adipogenesis for both white and brown adipocytes. The loss of insulin action selectively in adipose tissue in mice with a fat-specific insulin receptor knock-out (FIRKO) 1 leads to profound changes in adipocyte function, including changes in glucose metabolism, lipid metabolism, and protein expression (3). FIRKO mice have reduced fat mass and are protected against age-and diet-related obesity and its associated metabolic abnormalities, including glucose intolerance. In addition, these mice have increased longevity, despite normal or increased food intake (4). Adipose tissue-specific insulin receptor knock-out in FIRKO mice also causes heterogeneity of white adipose tissue with polarization into small (diameter Ͻ50 m) and large (diameter Ͼ150 m) subclasses of adipocytes (3). Western blot analysis of candidate molecules reveals changes in the expression of several key adipocyte proteins, such as ACRP30, fatty acid synthase...