In mammals, adipose tissue, composed of BAT and WAT, collaborates in energy partitioning and performs metabolic regulatory functions. It is the most flexible tissue in the body, because it is remodeled in size and shape by modifications in adipocyte cell size and/or number, depending on developmental status and energy fluxes. Although numerous reviews have focused on the differentiation program of both brown and white adipocytes as well as on the pathophysiological role of white adipose tissues, the importance of mitochondrial activity in the differentiation or the dedifferentiation programs of adipose cells and in systemic metabolic alterations has not been extensively reviewed previously. Here, we address the crucial role of mitochondrial functions during adipogenesis and in mature adipocytes and discuss the cellular responses of white adipocytes to mitochondrial activity impairment. In addition, we discuss the increase in scientific knowledge regarding mitochondrial functions in the last 10 years and the recent suspicion of mitochondrial dysfunction in several 21st century epidemics (ie, obesity and diabetes), as well as in lipodystrophy found in HIV-treated patients, which can contribute to the development of new therapeutic strategies targeting adipocyte mitochondria.

Mitochondrial cytopathy has been associated with modifications of lipid metabolism in various situations, such as the acquisition of an abnormal adipocyte phenotype observed in multiple symmetrical lipomatosis or triglyceride (TG) accumulation in muscles associated with the myoclonic epilepsy with ragged red fibers syndrome. However, the molecular signaling leading to fat metabolism dysregulation in cells with impaired mitochondrial activity is still poorly understood. Here, we found that preadipocytes incubated with inhibitors of mitochondrial respiration such as antimycin A (AA) accumulate TG vesicles but do not acquire specific markers of adipocytes. Although the uptake of TG precursors is not stimulated in 3T3-L1 cells with impaired mitochondrial activity, we found a strong stimulation of glucose uptake in AA-treated cells mediated by calcium and phosphatidylinositol 3-kinase/Akt1/glycogen synthase kinase 3 ␤ , a pathway known to trigger the translocation of glucose transporter 4 to the plasma membrane in response to insulin. TG accumulation in AA-treated cells is mediated by a reduced peroxisome proliferator-activated receptor ␥ activity that downregulates muscle carnitine palmitoyl transferase-1 expression and fatty acid ␤ -oxidation, and by a direct conversion of glucose into TGs accompanied by the activation of carbohydrate-responsive element binding protein, a lipogenic transcription factor. Taken together, these results could explain how mitochondrial impairment leads to the multivesicular phenotype found in some mitochondria-originating diseases associated with a dysfunction in fat metabolism. The role of mitochondria in lipid homeostasis has been strongly emphasized in recent studies focusing on mitochondrial respiratory deficiency. Indeed, chronic mitochondrial dysfunction can lead to diseases characterized by lipid metabolism disorders and pathological triglyceride (TG) accumulation in several cell types (1-3). Genetic mitochondrial pathologies usually result from point mutations or deletions in mitochondrial DNA that finally impair oxidative phosphorylation capacity (4). Interestingly, some mitochondrial disorders affect lipid-metabolizing tissues such as muscular and adipose tissues. For example, the myoclonic epilepsy with ragged red fibers (MERRF) syndrome, commonly caused by a point mutation in the mitochondrial tRNA Lys -encoding gene (A8344G), is associated with myopathy, TG accumulation in muscles (5), and, in some cases, multiple symmetrical lipomatosis (MSL) (2, 6). MSL is a pathology characterized by the formation of lipomas containing abnormal white adipocytes smaller than normal adipocytes showing a multivesicular phenotype (1, 2, 7). Moreover, biochemical analyses have shown that cytochrome c oxidase activity is impaired in muscles from patients with MSL (8), supporting the fact that the disease is linked to mitochondrial dysfunction (6, 9). The role of mitochondria in the lipid metabolism of white adipose tissue was also strengthened in the pathogenesis of Abbreviations: AA,...

A controlled regulation of mitochondrial mass through either the production (biogenesis) or the degradation (mitochondrial quality control) of the organelle represents a crucial step for proper mitochondrial and cell function. Key steps of mitochondrial biogenesis and quality control are overviewed, with an emphasis on the role of mitochondrial chaperones and proteases that keep mitochondria fully functional, provided the mitochondrial activity impairment is not excessive. In this case, the whole organelle is degraded by mitochondrial autophagy or "mitophagy." Beside the maintenance of adequate mitochondrial abundance and functions for cell homeostasis, mitochondrial biogenesis might be enhanced, through discussed signaling pathways, in response to various physiological stimuli, like contractile activity, exposure to low temperatures, caloric restriction, and stem cells differentiation. In addition, mitochondrial dysfunction might also initiate a retrograde response, enabling cell adaptation through increased mitochondrial biogenesis.

Impairment of mitochondrial activity affects lipid-metabolizing tissues and mild mitochondrial uncoupling has been proposed as a possible strategy to fight obesity and associated diseases. In this report, we characterized the 3T3-L1-adipocyte `de-differentiation' induced by carbonyl cyanide (p-trifluoromethoxy)-phenylhydrazone (FCCP), a mitochondrial uncoupler. We found a decrease in triglyceride (TG) content in adipocytes incubated with this molecule. We next analyzed the expression of genes encoding adipogenic markers and effectors and compared the differentially expressed genes in adipocytes treated with FCCP or TNFα (a cytokine known to induce adipocyte de-differentiation). Furthermore, a significant decrease in the transcriptional activity of PPARγ and C/EBPα transcription factors was found in adipocytes with impaired mitochondrial activity. However, although these modifications were also found in TNFα-treated adipocytes, rosiglitazone and 9-cis retinoic acid (PPARγ and RXR ligands) were unable to prevent triglyceride loss in FCCP-treated cells. Metabolic assays also revealed that TG reduction could be mediated by a downregulation of lipid synthesis rather than an upregulation of fatty acid oxidation. Finally, lipolysis stimulated by the uncoupler also seems to contribute to the TG reduction, a process associated with perilipin A downregulation. These results highlight some new mechanisms that might potentially be involved in adipocyte de-differentiation initiated by a mitochondrial uncoupling.

Several mitochondrial pathologies are characterized by lipid redistribution and microvesicular cell phenotypes resulting from triglyceride accumulation in lipid-metabolizing tissues. However, the molecular mechanisms underlying abnormal fat distribution induced by mitochondrial dysfunction remain poorly understood. In this study, we show that inhibition of respiratory complex III by antimycin A as well as inhibition of mitochondrial protein synthesis trigger the accumulation of triglyceride vesicles in 3T3-L1 fibroblasts. We also show that treatment with antimycin A triggers CREB activation in these cells. To better delineate how mitochondrial dysfunction induces triglyceride accumulation in preadipocytes, we developed a low-density DNA microarray containing 89 probes, which allows gene expression analysis for major effectors and/or markers of adipogenesis. We thus determined gene expression profiles in 3T3-L1 cells incubated with antimycin A and compared the patterns obtained with differentially expressed genes during the course of in vitro adipogenesis induced by a standard pro-adipogenic cocktail. After an 8-day treatment, a set of 39 genes was found to be differentially expressed in cells treated with antimycin A, among them CCAAT/enhancer-binding protein α (C/EBPα), C/EBP homologous protein-10 (CHOP-10), mitochondrial glycerol-3-phosphate dehydrogenase (GPDmit), and stearoyl-CoA desaturase 1 (SCD1). We also demonstrate that overexpression of two dominant negative mutants of the cAMP-response element-binding protein CREB (K-CREB and M1-CREB) and siRNA transfection, which disrupt the factor activity and expression, respectively, inhibit antimycin-A-induced triglyceride accumulation. Furthermore, CREB knockdown with siRNA also downregulates the expression of several genes that contain cAMP-response element (CRE) sites in their promoter, among them one that is potentially involved in synthesis of triglycerides such as SCD1. These results highlight a new role for CREB in the control of triglyceride metabolism during the adaptative response of preadipocytes to mitochondrial dysfunction.

Alterations in mitochondrial activity resulting from defects in mitochondrial DNA (mtDNA) can modulate the biogenesis of mitochondria by mechanisms that are still poorly understood. In order to study mitochondrial biogenesis in cells with impaired mitochondrial activity, we used rho‐L929 and rho0143 B cells (partially and totally depleted of mtDNA, respectively), that maintain and even up‐regulate mitochondrial population, to characterize the activity of major transcriptional regulators (Sp1, YY1, MEF2, PPARgamma, NRF‐1, NRF‐2, CREB and PGC‐1α) known to control the expression of numerous nuclear genes encoding mitochondrial proteins. Among these regulators, cyclic AMP‐responsive element binding protein (CREB) activity was the only one to be increased in mtDNA‐depleted cells. CREB activation mediated by a calcium‐dependent pathway in these cells also regulates the expression of cytochrome c and the abundance of mitochondrial population as both are decreased in mtDNA‐depleted cells that over‐express CREB dominant negative mutants. Mitochondrial biogenesis in mtDNA‐depleted cells is also dependent on intracellular calcium as its chelation reduces mitochondrial mass. Despite a slight increase in mitochondrial mass in mtDNA‐depleted cells, the mitochondrial protein import activity was reduced as shown by a decrease in the import of radiolabeled matrix‐targeted recombinant proteins into isolated mitochondria and by the reduced mitochondrial localization of ectopically expressed HA‐apoaequorin targeted to the mitochondria. Decrease in ATP content, in mitochondrial membrane potential as well as reduction in mitochondrial Tim44 abundance could explain the lower mitochondrial protein import in mtDNA‐depleted cells. Taken together, these results suggest that mitochondrial biogenesis is stimulated in mtDNA‐depleted cells and involves a calcium‐CREB signalling pathway but is associated with a reduced mitochondrial import for matrix proteins.

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De Pauw A, Demine S, Tejerina S, Dieu M, Delaive E, Kel A, Renard P, Raes M, Arnould T. Mild mitochondrial uncoupling does not affect mitochondrial biogenesis but downregulates pyruvate carboxylase in adipocytes: role for triglyceride content reduction. Am J Physiol Endocrinol Metab 302: E1123-E1141, 2012. First published February 21, 2012 doi:10.1152/ajpendo.00117.2011In adipocytes, mitochondrial uncoupling is known to trigger a triglyceride loss comparable with the one induced by TNF␣, a proinflammatory cytokine. However, the impact of a mitochondrial uncoupling on the abundance/composition of mitochondria and its connection with triglyceride content in adipocytes is largely unknown. In this work, the effects of a mild mitochondrial uncoupling triggered by FCCP were investigated on the mitochondrial population of 3T3-L1 adipocytes by both quantitative and qualitative approaches. We found that mild mitochondrial uncoupling does not stimulate mitochondrial biogenesis in adipocytes but induces an adaptive cell response characterized by quantitative modifications of mitochondrial protein content. Superoxide anion radical level was increased in mitochondria of both TNF␣-and FCCP-treated adipocytes, whereas mitochondrial DNA copy number was significantly higher only in TNF␣-treated cells. Subproteomic analysis revealed that the abundance of pyruvate carboxylase was reduced significantly in mitochondria of TNF␣-and FCCP-treated adipocytes. Functional study showed that overexpression of this major enzyme of lipid metabolism is able to prevent the triglyceride content reduction in adipocytes exposed to mitochondrial uncoupling or TNF␣. These results suggest a new mechanism by which the effects of mitochondrial uncoupling might limit triglyceride accumulation in adipocytes.adipocytes; carbonyl cyanide p-trifluoromethoxyphenylhydrazone; tumor necrosis factor-␣; mitoproteome OBESITY RESULTS LARGELY FROM A CHRONICALLY POSITIVE energy balance. Therefore, reducing body energy intake and increasing energy expenditure are two approaches that have been translated into strategies to limit fat accumulation. Because mitochondria play a critical role in the process of energy expenditure, the alteration of oxidative phosphorylation (OX-PHOS) efficiency through OXPHOS uncoupling from ATP production in white adipocytes has been considered as an attractive approach to fight obesity (7, 69). In other words, uncoupling increases cellular metabolic demand since uncoupling OXPHOS lowers ATP production and increases the demand for reducing equivalents to restore/maintain mitochondrial membrane potential, thereby increasing substrate catabolism, which would theoretically decrease triglyceride (TG) stores in adipocytes. Indeed, although mitochondrial dysfunction could represent a major cause of lipid metabolism disorders and pathological triglyceride accumulation in several cell lines (50, 75), we and others have shown that mitochondrial uncoupling in adipocytes triggers lipolysis, limits fatty acid synthesis, and leads to a reduction in TG ...