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Expression of ob Gene in Adipose Cells REGULATION BY INSULIN

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 half-life 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.

Small RNA sequencing reveals miR-642a-3p as a novel adipocyte-specific microRNA and miR-30 as a key regulator of human adipogenesis

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.

Leukemia Inhibitory Factor and Its Receptor Promote Adipocyte Differentiation via the Mitogen-activated Protein Kinase Cascade

Extracellular factors and intracellular signaling pathways involved in early events of adipocyte differentiation are poorly defined. It is shown herein that expression of leukemia inhibitory factor (LIF) and LIF receptor is developmentally regulated during adipocyte differentiation. Preadipocytes secrete bioactive LIF, and an antagonist of LIF receptor inhibits adipogenesis. Genetically modified embryonic stem (ES) cells combined with culture conditions to commit stem cells into the adipocyte lineage were used to examine the requirement of LIF receptor during in vitro development of adipose cells. The capacity of embryoid bodies derived fromlifr −/− ES cells to undergo adipocyte differentiation is dramatically reduced. LIF addition stimulates adipocyte differentiation of Ob1771 and 3T3-F442A preadipocytes and that of peroxisome proliferator-activated receptor γ2 ligand-treated mouse embryonic fibroblasts. Expression of the early adipogenic transcription factors C/EBPβ and C/EBPδ is rapidly stimulated following exposure of preadipose cells to LIF. The selective inhibitors of mitogen-activated protein kinase kinase, i.e. PD98059 and U0126, inhibit LIF-induced C/EBP gene expression and prevent adipocyte differentiation induced by LIF. These results are in favor of a model that implicates stimulation of LIF receptor in the commitment of preadipocytes to undergo terminal differentiation by controlling the early expression of C/EBPβ and C/EBPδ genes via the mitogen-activated protein kinase cascade.

Browning of White Adipose Cells by Intermediate Metabolites: An Adaptive Mechanism to Alleviate Redox Pressure

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-1α and PPARα pathways but requires active PPARγ 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 β-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.

Activin A Plays a Critical Role in Proliferation and Differentiation of Human Adipose Progenitors.

OBJECTIVE Growth 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 METHODS Expression 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. RESULTS INHBA/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. CONCLUSIONS Altogether, 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.

Effects of GSK3 inhibitors on in vitro expansion and differentiation of human adipose-derived stem cells into adipocytes

BackgroundMultipotent stem cells exist within adipose tissue throughout life. An abnormal recruitment of these adipose precursor cells could participate to hyperplasia of adipose tissue observed in severe obesity or to hypoplasia of adipose tissue observed in lipodystrophy. Therefore, pharmacological molecules that control the pool of stem cells in adipose tissue are of great interest. Glycogen Synthase Kinase (GSK) 3 has been previously described as involved in differentiation of preadipose cells and might be a potential therapeutic target to modulate proliferation and differentiation of adipocyte precursors. However, the impact of GSK3 inhibition on human adipose-derived stem cells remained to be investigated. The aim of this study was to investigate GSK3 as a possible target for pharmacological inhibition of stem cell adipogenesis. To reach this goal, we studied the effects of pharmacological inhibitors of GSK3, i.e. lithium chloride (LiCl) and BIO on proliferation and adipocyte differentiation of multipotent stem cells derived from human adipose tissue.ResultsOur results showed that GSK3 inhibitors inhibited proliferation and clonogenicity of human stem cells, strongly suggesting that GSK3 inhibitors could be potent regulators of the pool of adipocyte precursors in adipose tissue. The impact of GSK3 inhibition on differentiation of hMADS cells was also investigated. Adipogenic and osteogenic differentiations were inhibited upon hMADS treatment with BIO. Whereas a chronic treatment was required to inhibit osteogenesis, a treatment that was strictly restricted to the early step of differentiation was sufficient to inhibit adipogenesis.ConclusionThese results demonstrated the feasibility of a pharmacological approach to regulate adipose-derived stem cell function and that GSK3 could represent a potential target for controlling adipocyte precursor pool under conditions where fat tissue formation is impaired.

Differentiation of Human Induced Pluripotent Stem Cells into Brown and White Adipocytes: Role of Pax3

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. Stem Cells 2014;32:1459–1467

Commitment of Mouse Embryonic Stem Cells to the Adipocyte Lineage Requires Retinoic Acid Receptor β and Active GSK3

Key events leading to terminal differentiation of preadipocytes into adipocytes have been identified in recent years. However, signaling pathways involved in the decision of stem cells to follow the adipogenic lineage have not yet been characterized. We have previously shown that differentiating mouse embryonic stem (mES) cells give rise to functional adipocytes upon an early treatment with retinoic acid (RA). The goal of this work was to identify regulators of RA-induced commitment of mES cells to the adipocyte lineage. First, we investigated the role of RA receptor (RAR) isotypes in the induction of mES cell adipogenesis. Using synthetic retinoids selective of RAR isotypes, we show that RARbeta activation is both sufficient and necessary to trigger commitment of mES cells to adipocytes. Then, we performed a small-scale drug screening to find signaling pathways involved in RARbeta-induced mES cell adipogenesis. We show that pharmacological inhibitors of glycogen synthase kinase (GSK) 3, completely inhibit RARbeta-induced adipogenesis in mES cells. This finding uncovers the requirement of active GSK3 in RARbeta-induced commitment of mES cells toward the adipocyte lineage. Finally, we investigated the role of the Wnt pathway, in which GSK3 is a critical negative regulator, in adipocyte commitment by analyzing Wnt pathway activity in RA- and RARbeta-induced mES cell adipogenesis. Our results suggest that although RARbeta and active GSK3 are required for RA-induced adipogenesis, they might be acting through a Wnt pathway-independent mechanism.

Development of Adipocytes from Differentiated ES Cells

Publisher Summary This chapter describes an in vitro system that allows access to the development of adipocytes from pluripotent mouse ES cells. Because there are no specific markers that can be used to identify adipoblasts and preadipocytes in vivo , the white adipose tissue can be identified only when it contains adipocytes (i.e., terminally differentiated cells) and cannot be detected macroscopically during embryogenesis. Therefore, very little is known about the ontogeny of adipose tissue. Mainly, the in vitro system used to study adipogenesis is immortal preadipocyte cell lines, which represent already determined cells. The use of in vitro differentiation of ES cells to analyze the effects of mutations on adipogenesis has begun recently and master genes that commit progression from pluripotent stem cells to the adipogenic lineage have not yet been identified. The chapter also describes a protocol for culture conditions in which ES cells undergo adipocyte differentiation at a high rate and in a highly reproducible fashion. This culture system should provide a powerful means for studying the first step of adipose cell development and a route to identify regulatory genes involved in adipogenesis.

Brown-like adipose progenitors derived from human induced pluripotent stem cells: Identification of critical pathways governing their adipogenic capacity.

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.