The presence of brown adipose tissue (BAT) in human adults opens attractive perspectives to treat metabolic disorders. Indeed, BAT dissipates energy as heat via uncoupling protein (UCP)1. Brown adipocytes are located in specific deposits or can emerge among white fat through the so-called browning process. Although numerous inducers have been shown to drive this process, no study has investigated whether it could be controlled by specific metabolites. Here, we show that lactate, an important metabolic intermediate, induces browning of murine white adipose cells with expression of functional UCP1. Lactate-induced browning also occurs in human cells and in vivo. Lactate controls Ucp1 expression independently of hypoxia-inducible factor-1a and PPARa pathways but requires active PPARg signaling. We demonstrate that the lactate effect on Ucp1 is mediated by intracellular redox modifications as a result of lactate transport through monocarboxylate transporters. Further, the ketone body b-hydroxybutyrate, another metabolite that impacts redox state, is also a strong browning inducer. Because this redox-dependent increase in Ucp1 expression promotes an oxidative phenotype with mitochondria, browning appears as an adaptive mechanism to alleviate redox pressure. Our findings open new perspectives for the control of adipose tissue browning and its physiological relevance.

The product of the recently cloned mouse obese (ob) gene is likely to play an important role in a loop regulating the size of the adipose tissue mass. The hormonal regulation of the ob gene could affect adiposity. To investigate this point, the effect of insulin on ob gene expression was examined in cells of the 3T3-F442A preadipocyte clonal line. ob mRNA is absent from exponentially growing, undifferentiated cells as well as from confluent preadipose cells. Terminal differentiation of preadipose to adipose cells leads to the expression of ob mRNA detected by a sensitive and quantitative ribonuclease protection assay. In adipose cells, the level of ob mRNA is sensitive to insulin in the nanomolar range of concentrations with an increase from an average of 1 copy to 5-10 copies/cell. The effect of insulin was fully reversible and takes place primarily at a transcriptional level. The ob mRNA shows a rapid turnover, with a halflife of approximately 2 h in the absence or presence of insulin. The level of secreted Ob protein is also regulated by insulin. These results indicate that the ob gene is expressed in mature fat cells only and support the possibility that insulin is an important regulator of ob gene expression.The "lipostat" or "adipostat" theory postulates that the size of body fat stores is regulated by a feedback loop (1). This hypothesis is based upon the recovery of initial body weight following lipectomy (2) and parabiosis experiments between genetically obese and wild-type mice suggesting the existence of putative factor(s) regulating food intake (3). The recently cloned ob gene from mouse, rat, and human encodes a circulating factor of 16 kDa that is secreted from adipocytes from various adipose depots (4 -8). The OB protein, named leptin, appears to act at a distant site since injections of the leptin decrease food intake and body weight in ob/ob mice and their lean counterparts (9 -12). This phenomenon implicates directly or indirectly the hypothalamus since mice with chemical lesions of the ventromedial nucleus of the hypothalamus (VMH), 1 after becoming rapidly hyperinsulinemic, express a dramatic increase in the levels of ob mRNA (5, 13). A substantial fall in ob mRNA in the epididymal fat of lean mice has been observed after fasting; this phenomenon is rapidly reversed on refeeding (13-16). The correlation between insulin level and the levels of ob mRNA and plasma leptin suggests that insulin may have direct effects on ob gene expression (15)(16)(17)(18). In this paper, we present data using cultured adipocytes that support this hypothesis. EXPERIMENTAL PROCEDURESCell Culture-Culture conditions of cells of the 3T3-F442A clonal line have been described (19). Cells were plated at 10 3 cells/cm 2 in 60-or 100-mm dishes and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum. This medium was defined as standard medium. At confluence, standard medium was supplemented with 2 nM triiodothyronine (T 3 ) and 3 nM insulin, termed differentiation medium, for 10 ...

OBJECTIVEGrowth of white adipose tissue takes place in normal development and in obesity. A pool of adipose progenitors is responsible for the formation of new adipocytes and for the potential of this tissue to expand in response to chronic energy overload. However, factors controlling self-renewal of human adipose progenitors are largely unknown. We investigated the expression profile and the role of activin A in this process.RESEARCH DESIGN AND METHODSExpression of INHBA/activin A was investigated in three types of human adipose progenitors. We then analyzed at the molecular level the function of activin A during human adipogenesis. We finally investigated the status of activin A in adipose tissues of lean and obese subjects and analyzed macrophage-induced regulation of its expression.RESULTSINHBA/activin A is expressed by adipose progenitors from various fat depots, and its expression dramatically decreases as progenitors differentiate into adipocytes. Activin A regulates the number of undifferentiated progenitors. Sustained activation or inhibition of the activin A pathway impairs or promotes, respectively, adipocyte differentiation via the C/EBPβ-LAP and Smad2 pathway in an autocrine/paracrine manner. Activin A is expressed at higher levels in adipose tissue of obese patients compared with the expression levels in lean subjects. Indeed, activin A levels in adipose progenitors are dramatically increased by factors secreted by macrophages derived from obese adipose tissue.CONCLUSIONSAltogether, our data show that activin A plays a significant role in human adipogenesis. We propose a model in which macrophages that are located in adipose tissue regulate adipose progenitor self-renewal through activin A.

BackgroundIn severe obesity, as well as in normal development, the growth of adipose tissue is the result of an increase in adipocyte size and numbers, which is underlain by the stimulation of adipogenic differentiation of precursor cells. A better knowledge of the pathways that regulate adipogenesis is therefore essential for an improved understanding of adipose tissue expansion. As microRNAs (miRNAs) have a critical role in many differentiation processes, our study aimed to identify the role of miRNA-mediated gene silencing in the regulation of adipogenic differentiation.ResultsWe used deep sequencing to identify small RNAs that are differentially expressed during adipogenesis of adipose tissue-derived stem cells. This approach revealed the un-annotated miR-642a-3p as a highly adipocyte-specific miRNA. We then focused our study on the miR-30 family, which was also up-regulated during adipogenic differentiation and for which the role in adipogenesis had not yet been elucidated. Inhibition of the miR-30 family blocked adipogenesis, whilst over-expression of miR-30a and miR-30d stimulated this process. We additionally showed that both miR-30a and miR-30d target the transcription factor RUNX2, and stimulate adipogenesis via the modulation of this major regulator of osteogenesis.ConclusionsOverall, our data suggest that the miR-30 family plays a central role in adipocyte development. Moreover, as adipose tissue-derived stem cells can differentiate into either adipocytes or osteoblasts, the down-regulation of the osteogenesis regulator RUNX2 represents a plausible mechanism by which miR-30 miRNAs may contribute to adipogenic differentiation of adipose tissue-derived stem cells.

families are trans-acting nuclear factors playing a regulatory role in the differentiation of preadipocytes into adipocytes (1, 2). PPAR␥2 is predominantly expressed in adipose tissue and plays a critical role in the adipocyte differentiation process (3, 4). Expression of C/EBP␤ and C/EBP␦ genes is not restricted to adipose tissue but is induced early and transiently during the program of adipocyte differentiation. Expression of C/EBP␤ and C/EBP␦ decreases following adipocyte maturation, whereas expression of C/EBP␣ and PPAR␥2 gene is induced (5). C/EBP-binding sites have been identified in the PPAR␥2 promoter, and it has been recently shown that C/EBP proteins directly control transcription from the PPAR␥2 promoter (6). The adipogenic role of C/EBP␤ and C/EBP␦ has been previously demonstrated. Their ectopic expression in fibroblasts leads, in the presence of adipogenic hormones, to the adipocyte phenotype (7-9). Finally, the generation by homologous recombination of C/EBP␤ Ϫ/Ϫ ⅐␦ Ϫ/Ϫ mice has clearly established the essential role of these two C/EBPs for the acquisition of adipocytes both in vitro and in vivo (10). However, the extracellular factors and the intracellular signaling pathways involved in the regulation of C/EBP␤ and C/EBP␦ expression in preadipose cells are poorly defined. During the course of our investigation to identify extracellular factors that regulate early events in adipocyte differentiation, secretion of leukemia inhibitory factor (LIF) by preadipocytes was observed. LIF is known to induce differentiation of the murine myeloid leukemia cell line M1, to maintain pluripotent embryonic stem (ES) cells (11,12), and to modulate stem cell and differentiated cell type functions in vitro and in vivo (13). LIF and the related cytokines cardiotrophin (CT-1) and ciliary neurotrophic factor (CNTF) act through heterodimeric receptors comprised of LIF receptor and gp130 (14). We show in the current study that exogenous LIF stimulates terminal differentiation of Ob1771 and 3T3-F442A preadipose cells and induces adipocyte differentiation of multipotent mouse embryonic fibroblasts (MEF). Moreover, by using an antagonist of LIF receptor or genetically modified ES cells, we show that LIF receptor plays a key role during adipocyte differentiation. These results are at variance with previous reports showing an anti-adipogenic effect of LIF in 3T3-L1 preadipocytes (15,16). The differential response to LIF of 3T3-L1 cells versus other cells is discussed.The expression of C/EBP␤ and C/EBP␦ genes was rapidly induced in Ob1771 and 3T3-F442A preadipose cells after LIF addition. The role of the mitogen-activated protein kinase (MAPK) pathway in early events induced by LIF has been

ObjectiveThe present study was undertaken to characterize the remodeling phenotype of human adipose tissue (AT) macrophages (ATM) and to analyze their paracrine effects on AT progenitor cells.Research Design and MethodsThe phenotype of ATM, immunoselected from subcutaneous (Sc) AT originating from subjects with wide range of body mass index and from paired biopsies of Sc and omental (Om) AT from obese subjects, was studied by gene expression analysis in the native and activated states. The paracrine effects of ScATM on the phenotype of human ScAT progenitor cells (CD34+CD31−) were investigated.ResultsTwo main ATM phenotypes were distinguished based on gene expression profiles. For ScAT-derived ATM, obesity and adipocyte-derived factors favored a pro-fibrotic/remodeling phenotype whereas the OmAT location and hypoxic culture conditions favored a pro-angiogenic phenotype. Treatment of native human ScAT progenitor cells with ScATM-conditioned media induced the appearance of myofibroblast-like cells as shown by expression of both α-SMA and the transcription factor SNAIL, an effect mimicked by TGFβ1 and activinA. Immunohistochemical analyses showed the presence of double positive α-SMA and CD34 cells in the stroma of human ScAT. Moreover, the mRNA levels of SNAIL and SLUG in ScAT progenitor cells were higher in obese compared with lean subjects.ConclusionsHuman ATM exhibit distinct pro-angiogenic and matrix remodeling/fibrotic phenotypes according to the adiposity and the location of AT, that may be related to AT microenvironment including hypoxia and adipokines. Moreover, human ScAT progenitor cells have been identified as target cells for ScATM-derived TGFβ and as a potential source of fibrosis through their induction of myofibroblast-like cells.

Identification of molecular mechanisms involved in generation of different types of adipocytes is progressing substantially in mice. However, much less is known regarding characterization of brown (BAP) and white adipocyte progenitors (WAPs) in humans, highlighting the need for an in vitro model of human adipocyte development. Here, we report a procedure to selectively derive BAP and WAPs from human-induced pluripotent stem cells. Molecular characterization of APs of both phenotypes revealed that BMP4, Hox8, Hoxc9, and HoxA5 genes were specifically expressed in WAPs, whereas expression of PRDM16, Dio2, and Pax3 marked BAPs. We focused on Pax3 and we showed that expression of this transcription factor was enriched in human perirenal white adipose tissue samples expressing UCP1 and in human classical brown fat. Finally, functional experiments indicated that Pax3 was a critical player of human AP fate as its ectopic expression led to convert WAPs into brown-like APs. Together, these data support a model in which Pax3 is a new marker of human BAPs and a molecular mediator of their fate. The findings of this study could lead to new anti-obesity therapies based on the recruitment of APs and constitute a platform for investigating in vitro the developmental origins of human white and brown adipocytes.

Human induced pluripotent stem cells (hiPSCs) show great promise for obesity treatment as they represent an unlimited source of brown/brite adipose progenitors (BAPs). However, hiPSC-BAPs display a low adipogenic capacity compared to adult-BAPs when maintained in a traditional adipogenic cocktail. The reasons of this feature are unknown and hamper their use both in cell-based therapy and basic research. Here we show that treatment with TGFβ pathway inhibitor SB431542 together with ascorbic acid and EGF were required to promote hiPSCs-BAP differentiation at a level similar to adult-BAP differentiation. hiPSC-BAPs expressed the molecular identity of adult-UCP1 expressing cells (PAX3, CIDEA, DIO2) with both brown (ZIC1) and brite (CD137) adipocyte markers. Altogether, these data highlighted the critical role of TGFβ pathway in switching off hiPSC-brown adipogenesis and revealed novel factors to unlock their differentiation. As hiPSC-BAPs display similarities with adult-BAPs, it opens new opportunities to develop alternative strategies to counteract obesity.