Differentiation of Human Adipose-Derived Stem Cells into “Brite” (Brown-in-White) Adipocytes

It is well established now that adult humans possess active brown adipose tissue (BAT) which represents a potential pharmacological target to combat obesity and associated diseases. Moreover thermogenic brown-like adipocytes (“brite adipocytes”) appear also in mouse white adipose tissue (WAT) upon β3-adrenergic stimulation. We had previously shown that human multipotent adipose-derived stem cells (hMADS) are able to differentiate into cells which exhibit the key properties of human white adipocytes, and then to convert into functional brown adipocytes upon PPARγ activation. In light of a wealth of data indicating that thermogenic adipocytes from BAT and WAT have a distinct cellular origin, we have characterized at the molecular level UCP1 positive hMADS adipocytes from both sexes as brite adipocytes. Conversion of white to brown hMADS adipocytes is dependent on PPARγ activation with rosiglitazone as the most potent agonist and is inhibited by a PPARγ antagonist. In contrast to mouse cellular models, hMADS cells conversion into brown adipocytes is weakly induced by BMP7 treatment and not modulated by activation of the Hedgehog pathway. So far no primary or clonal precursor cells of human brown adipocytes have been obtained that can be used as a tool to develop therapeutic drugs and to gain further insights into the molecular mechanisms of brown adipogenesis in humans. Thus hMADS cells represent a suitable human cell model to delineate the formation and/or the uncoupling capacity of brown/brite adipocytes that could help to dissipate caloric excess intake among individuals

Characterization of human mesenchymal stem cell secretome at early steps of adipocyte and osteoblast differentiation

<p>Abstract</p> <p>Background</p> <p>It is well established that adipose tissue plays a key role in energy storage and release but is also a secretory organ and a source of stem cells. Among different lineages, stem cells are able to differentiate into adipocytes and osteoblasts. As secreted proteins could regulate the balance between both lineages, we aimed at characterizing the secretome of human multipotent adipose-derived stem cell (hMADS) at an early step of commitment to adipocytes and osteoblasts.</p> <p>Results</p> <p>A proteomic approach, using mono-dimensional electrophoresis and tandem mass spectrometry, allowed us to identify a total of 73 proteins at day 0 and day 3 of adipocyte and osteoblast differentiation. Analysis of identified proteins showed that 52 % corresponded to classical secreted proteins characterized by a signal peptide, that 37 % previously described in the extracellular compartment were devoid of signal peptide and that 11 % neither exhibited a signal peptide nor had been previously described extracellularly. These proteins were classified into 8 clusters according to their function. Quantitative analysis has been performed for 8 candidates: PAI-1, PEDF, BIGH3, PTX3, SPARC, ENO1, GRP78 and MMP2. Among them, PAI-1 was detected at day 0 and day 3 of osteoblast differentiation but never in adipocyte secretome. Furthermore we showed that PAI-1 mRNA was down-regulated in the bone of ovariectomized mice.</p> <p>Conclusion</p> <p>Given its regulation during the early events of hMADS cell differentiation and its status in ovariectomized mice, PAI-1 could play a role in the adipocyte/osteoblast balance and thus in bone diseases such as osteoporosis.</p

Fatty Acid Metabolite Profiling Reveals Oxylipins as Markers of Brown but Not Brite Adipose Tissue

Metabolites of omega-6 and omega-3 polyunsaturated fatty acids are important signaling molecules implicated in the control of adipogenesis and energy balance regulation. Some of these metabolites belonging to the group of oxylipins have been associated with non-shivering thermogenesis in mice mediated by brown or brite adipose tissue. We aimed to identify novel molecules with thermogenic potential and to clarify the relevance of these findings in a translational context. Therefore, we characterized and compared the oxylipin profiles of murine and human adipose tissues with different abundance of brown or brite adipocytes. A broad panel of 36 fatty acid metabolites was quantified in brown and white adipose tissues of C57BL/6J mice acclimatized to different ambient temperatures and in biopsies of human supraclavicular brown and white adipose tissue. The oxylipin profile of murine brite adipose tissue was not distinguishable from white adipose tissue, suggesting that adipose tissue browning in vivo is not associated with major changes in the oxylipin metabolism. Human brown and white adipose tissue also exhibited similar metabolite profiles. This is in line with previous studies proposing human brown adipose tissue to resemble the nature of murine brite adipose tissue representing a heterogeneous mixture of brite and white adipocytes. Although the global oxylipin profile served as a marker for the abundance of thermogenic adipocytes in bona fide brown but not white adipose tissue, we identified 5-HETE and 5,6-EET as individual compounds consistently associated with the abundance of brown or brite adipocytes in human BAT and murine brite fat. Further studies need to establish whether these candidates are mere markers or functional effectors of thermogenic capacity

Polyamine metabolism is involved in adipogenesis of 3T3-L1 cells

Polyamines spermidine and spermine are known to be required for mammalian cell proliferation and for embryonic development. Alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase (ODC) a limiting enzyme of polyamine biosynthesis, depleted the cellular polyamines and prevented triglyceride accumulation and differentiation in 3T3-L1 cells. In this study, to explore the function of polyamines in adipogenesis, we examined the effect of polyamine biosynthesis inhibitors on adipocyte differentiation and lipid accumulation of 3T3-L1 cells. The spermidine synthase inhibitor trans-4-methylcyclohexylamine (MCHA) increased spermine/spermidine ratios, whereas the spermine synthase inhibitor N-(3-aminopropyl)-cyclohexylamine (APCHA) decreased the ratios in the cells. MCHA was found to decrease lipid accumulation and GPDH activity during differentiation, while APCHA increased lipid accumulation and GPDH activity indicating the enhancement of differentiation. The polyamine-acetylating enzyme, spermidine/spermine N1-acetyltransferase (SSAT) activity was increased within a few hours after stimulus for differentiation, and was found to be elevated by APCHA. In mature adipocytes APCHA decreased lipid accumulation while MCHA had the opposite effect. An acetylpolyamine oxidase and spermine oxidase inhibitor MDL72527 or an antioxidant N-acetylcysteine prevented the promoting effect of APCHA on adipogenesis. These results suggest that not only spermine/spermidine ratios but also polyamine catabolic enzyme activity may contribute to adipogenesis

Comparative transcriptomics of human multipotent stem cells during adipogenesis and osteoblastogenesis

<p>Abstract</p> <p>Background</p> <p>A reciprocal relationship between bone and fat development in osteoporosis is clinically well established. Some of the key molecular regulators involved in this tissue replacement process have been identified. The detailed mechanisms governing the differentiation of mesenchymal stem cells (MSC) – the key cells involved – are however only now beginning to emerge. In an attempt to address the regulation of the adipocyte/osteoblast balance at the level of gene transcription in a comprehensive and unbiased manner, we performed a large-scale gene expression profiling study using a unique cellular model, human multipotent adipose tissue-derived stem cells (hMADS).</p> <p>Results</p> <p>The analysis of 1606 genes that were found to be differentially expressed between adipogenesis and osteoblastogenesis revealed gene repression to be most prevalent prior to commitment in both lineages. Computational analyses suggested that this gene repression is mediated by miRNAs. The transcriptional activation of lineage-specific molecular processes in both cases occurred predominantly after commitment. Analyses of the gene expression data and promoter sequences produced a set of 65 genes that are candidates for genes involved in the process of adipocyte/osteoblast commitment. Four of these genes were studied in more detail: <it>LXRα </it>and phospholipid transfer protein (<it>PLTP</it>) for adipogenesis, the nuclear receptor <it>COUP-TF1 </it>and one uncharacterized gene, <it>TMEM135 </it>for osteoblastogenesis. <it>PLTP </it>was secreted during both early and late time points of hMADS adipocyte differentiation. <it>LXRα</it>, <it>COUP-TF1</it>, and the transmembrane protein <it>TMEM135 </it>were studied in primary cultures of differentiating bone marrow stromal cells from healthy donors and were found to be transcriptionally activated in the corresponding lineages.</p> <p>Conclusion</p> <p>Our results reveal gene repression as a predominant early mechanism before final cell commitment. We were moreover able to identify 65 genes as candidates for genes controlling the adipocyte/osteoblast balance and to further evaluate four of these. Additional studies will explore the precise role of these candidate genes in regulating the adipogenesis/osteoblastogenesis switch.</p

Let-7i-5p represses brite adipocyte function in mice and humans

In response to cold or beta 3-adrenoreceptor stimulation brown adipose tissue (BAT) promotes non-shivering thermogenesis, leading to energy dissipation. BAT has long been thought to be absent or scarce in adult humans. The recent discovery of thermogenic brite/beige adipocytes has opened the way to development of novel innovative strategies to combat overweight/obesity and associated diseases. Thus it is of great interest to identify regulatory factors that govern the brite adipogenic program. Here, we carried out global microRNA (miRNA) expression profiling on human adipocytes to identify miRNAs that are regulated upon the conversion from white to brite adipocytes. Among the miRNAs that were differentially expressed, we found that Let-7i-5p was down regulated in brite adipocytes. A detailed analysis of the Let-7i-5p levels showed an inverse expression of UCP1 in murine and human brite adipocytes both in vivo and in vitro. Functional studies with Let-7i-5p mimic in human brite adipocytes in vitro revealed a decrease in the expression of UCP1 and in the oxygen consumption rate. Moreover, the Let-7i-5p mimic when injected into murine sub-cutaneous white adipose tissue inhibited partially beta 3-adrenergic activation of the browning process. These results suggest that the miRNAs Let-7i-5p participates in the recruitment and the function of brite adipocytes

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

International audienceAbstractObjective: 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 has been 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 C/EBPbeta-LAP and Smad2 pathway in an autocrine/paracrine manner. Activin A is expressed at higher levels in adipose tissue of obese patients compared to 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 which are located in adipose tissue regulate adipose progenitor self-renewal through activin A

miR-125b affects mitochondrial biogenesis and impairs brite adipocyte formation and function

Objective: In rodents and humans, besides brown adipose tissue (BAT), islands of thermogenic adipocytes, termed "brite" (brown-in-white) or beige adipocytes, emerge within white adipose tissue (WAT) after cold exposure or beta 3-adrenoceptor stimulation, which may protect from obesity and associated diseases. microRNAs are novel modulators of adipose tissue development and function. The purpose of this work was to characterize the role of microRNAs in the control of brite adipocyte formation.Methods/Results: Using human multipotent adipose derived stem cells, we identified miR-125b-5p as downregulated upon brite adipocyte formation. In humans and rodents, miR-125b-5p expression was lower in BAT than in WAT. In vitro, overexpression and knockdown of miR-125b-5p decreased and increased mitochondrial biogenesis, respectively. In vivo, miR-125b-5p levels were downregulated in subcutaneous WAT and interscapular BAT upon beta 3-adrenergic receptor stimulation. Injections of an miR-125b-5p mimic and LNA inhibitor directly into WAT inhibited and increased beta 3-adrenoceptor-mediated induction of UCP1, respectively, and mitochondrial brite adipocyte marker expression and mitochondriogenesis.Conclusion: Collectively, our results demonstrate that miR-125b-5p plays an important role in the repression of brite adipocyte function by modulating oxygen consumption and mitochondrial gene expression. (C) 2016 The Author(s). Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)