Human Adipocyte Differentiation Research Articles

Profiling Analysis Reveals the Potential Contribution of Peptides to Human Adipocyte Differentiation.

Peptide drugs provide promising regimes in anti-obesity treatment. In order to identify potential bioactive peptides involved in adipogenesis. The intracellular peptides between preadipocytes and adipocytes are compared by liquid chromatography/mass spectrometry. The underlying biological function of the identified peptides are evaluated by gene ontology (GO) and pathway analysis of their precursors. To find more potential bioactive peptides, 50 peptide sequences are identified located in the functional domains with the use of the SMART and UniProt databases. Finally, the Open Targets Platform database is used to investigate the precursors related to metabolic diseases. A total of 181 downregulated peptides and 89 upregulated peptides after differentiation (fold change > 1.5 and p-value < 0.05) are identified. The GO and pathway analysis indicate that these differentially expressed peptides play a role in adipogenesis. A total of 10 putative peptides 6 to 26 amino acids in length are identified that might have bioactive effects in adipogenesis and metabolic diseases. On one hand, present preliminary research provides a better understanding of the intracellular peptides during adipocyte differentiation. On the other hand, it lays a foundation for discovering new peptide drugs in anti-obesity treatment.

Effects of Genistein on Differentiation and Viability of Human Visceral Adipocytes.

Obesity can lead to pathological growth of adipocytes by inducing inflammation and oxidative stress. Genistein could be a potential candidate for the treatment of obesity due to its antioxidant properties. Specific kits were used to examine the effects of genistein vs adiponectin on human visceral pre-adipocytes differentiation, cell viability, mitochondrial membrane potential, and oxidative stress in pre-adipocytes and in white/brown adipocytes. Western Blot was performed to examine changes in protein activation/expression. Genistein increased human visceral pre-adipocytes differentiation and browning, and caused a dose-related improvement of cell viability and mitochondrial membrane potential. Similar effects were observed in brown adipocytes and in white adipocytes, although in white cells the increase of cell viability was inversely related to the dose. Moreover, genistein potentiated AMP-activated protein kinase (AMPK)/mitofusin2 activation/expression in pre-adipocytes and white/brown adipocytes and protected them from the effects of hydrogen peroxide. The effects caused by genistein were similar to those of adiponectin. The results obtained showed that genistein increases human visceral pre-adipocytes differentiation and browning, protected against oxidative stress in pre-adipocytes and white/brown adipocytes through mechanisms related to AMPK-signalling and the keeping of mitochondrial function.

MiR-199a-3p regulates brown adipocyte differentiation through mTOR signaling pathway.

Recent discoveries of functional brown adipocytes in mammals illuminates their therapeutic potential for combating obesity and its associated diseases. However, on account of the limited amount and activity in adult humans of brown adipocyte depots, identification of miRNAs and characterization their regulatory roles in human brown adipogenesis are urgently needed. This study focused on the role of microRNA (miR)-199a-3p in human brown adipocyte differentiation and thermogenic capacity. A decreased expression pattern of miR-199a-3p was consistently observed during the differentiation course of brown adipocytes in mice and humans. Conversely, its level was induced during the differentiation course of human white pre-adipocytes derived from visceral fat. miR-199a-3p expression was relatively abundant in interscapular BAT (iBAT) and differentially regulated in the activated and aging BAT in mice. Additionally, miR-199a-3p expression level in human brown adipocytes was observed decreased upon thermogenic activation and increased by aging-related stimuli. Using primary pre-adipocytes, miR-199a-3p over-expression was capable of attenuating lipid accumulation and adipogenic gene expression as well as impairing brown adipocytes' metabolic characteristics as revealed by decreased mitochondrial DNA content and respiration. Suppression of miR-199a-3p by a locked nucleic acid (LNA) modified-anti-miR led to increased differentiation and thermogenesis in human brown adipocytes. By combining target prediction and examination, we identified mechanistic target of rapamycin kinase (mTOR) as a direct target of miR-199a-3p that affected brown adipogenesis and thermogenesis. Our results point to a novel role for miR-199a-3p and its downstream effector mTOR in human brown adipocyte differentiation and maintenance of thermogenic characteristics, which can be manipulated as therapeutic targets against obesity and its related metabolic disorders.

Interrogation of nonconserved human adipose lincRNAs identifies a regulatory role of linc-ADAL in adipocyte metabolism

Long intergenic noncoding RNAs (lincRNAs) have emerged as important modulators of cellular functions. Most lincRNAs are not conserved among mammals, raising the fundamental question of whether nonconserved adipose-expressed lincRNAs are functional. To address this, we performed deep RNA sequencing of gluteal subcutaneous adipose tissue from 25 healthy humans. We identified 1001 putative lincRNAs expressed in all samples through de novo reconstruction of noncoding transcriptomes and integration with existing lincRNA annotations. One hundred twenty lincRNAs had adipose-enriched expression, and 54 of these exhibited peroxisome proliferator-activated receptor γ (PPARγ) or CCAAT/enhancer binding protein α (C/EBPα) binding at their loci. Most of these adipose-enriched lincRNAs (~85%) were not conserved in mice, yet on average, they showed degrees of expression and binding of PPARγ and C/EBPα similar to those displayed by conserved lincRNAs. Most adipose lincRNAs differentially expressed (n = 53) in patients after bariatric surgery were nonconserved. The most abundant adipose-enriched lincRNA in our subcutaneous adipose data set, linc-ADAL, was nonconserved, up-regulated in adipose depots of obese individuals, and markedly induced during in vitro human adipocyte differentiation. We demonstrated that linc-ADAL interacts with heterogeneous nuclear ribonucleoprotein U (hnRNPU) and insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) at distinct subcellular locations to regulate adipocyte differentiation and lipogenesis.

Hydroxylated Metabolites of Common Airborne Polychlorinated Biphenyls and Their Potential for Disrupting Estrogen Homeostasis and Adipogenesis

Endocrine disruption, metabolic syndrome, diabetes, cancer, and other diseases have been associated with exposure to polychlorinated biphenyls. There is a growing interest in the lower chlorinated PCBs due to their presence in the indoor air of older buildings, their susceptibility to metabolism catalyzed by cytochrome P450 to form hydroxylated PCBs (OH‐PCBs), and their potential to disrupt endocrine function. OH‐PCBs are further metabolized in reactions catalyzed by cytosolic sulfotransferases (SULTs) to form PCB‐sulfates. Estrogen sulfotransferase (SULT1E1) is important in the regulation of cellular levels of active and inactive estrogens, with estradiol being a primary physiological substrate. Recent literature has suggested that decreased activity of SULT1E1 can impact adipocyte differentiation. We hypothesize that some lower‐chlorinated OH‐PCBs potently inhibit SULT1E1 causing increased cellular levels of estradiol and a decrease in human adipocyte differentiation. Our studies using purified recombinant human SULT1E1 (at 7.0 nM estradiol) found 4‐OH‐PCB 11 and 4′‐OH‐PCB 25 to be potent inhibitors, with IC50 values of 7.2 and 7.3 nM, respectively. 4′‐OH‐PCB 8 and 4‐OH PCB 52 inhibited SULT1E1 with IC50 values of 17.6 and 18.6 nM, respectively, and the least potent inhibitor, 4′‐OH‐PCB 3, had an IC50 of 1300 nM. We have exposed immortalized human adipocytes to these five OH‐PCBs and another known inhibitor of SULT1E1, triclosan, at concentrations of 1 uM and 10 uM for 72 hours followed by differentiation for 11 days. The cells were stained with AdipoRed to quantitate lipid accumulation. Exposure to 4′‐OH‐PCB 3 resulted in no significant reduction in lipid accumulation, while the remaining OH‐PCBs and triclosan showed significant reductions in adipocyte differentiation. This congener‐selective ability to inhibit SULT1E1 and adipocyte differentiation is consistent with the hypothesis that OH‐PCB‐mediated inhibition of SULT1E1 can inhibit human adipogenesis. Future studies will explore the role of this inhibition of SULT1E1 in adipogenesis by measuring mRNA expression of SULT1E1 and prominent adipogenic markers in these cells as well as cellular levels of estradiol and estradiol‐sulfate.Support or Funding InformationSupported by NIH grants P42 ES013661 and R25 GM058939, and a William Townsend Porter Fellowship from the American Physiological Society.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Integrated analysis of motif activity and gene expression changes of transcription factors.

The ability to predict transcription factors based on sequence information in regulatory elements is a key step in systems-level investigation of transcriptional regulation. Here, we have developed a novel tool, IMAGE, for precise prediction of causal transcription factors based on transcriptome profiling and genome-wide maps of enhancer activity. High precision is obtained by combining a near-complete database of position weight matrices (PWMs), generated by compiling public databases and systematic prediction of PWMs for uncharacterized transcription factors, with a state-of-the-art method for PWM scoring and a novel machine learning strategy, based on both enhancers and promoters, to predict the contribution of motifs to transcriptional activity. We applied IMAGE to published data obtained during 3T3-L1 adipocyte differentiation and showed that IMAGE predicts causal transcriptional regulators of this process with higher confidence than existing methods. Furthermore, we generated genome-wide maps of enhancer activity and transcripts during human mesenchymal stem cell commitment and adipocyte differentiation and used IMAGE to identify positive and negative transcriptional regulators of this process. Collectively, our results demonstrate that IMAGE is a powerful and precise method for prediction of regulators of gene expression.

Role of RNA-binding protein HuR in human adipocyte differentiation

Objective To investigate the expressions of RNA-binding protein human antigen R(HuR), fatty acid binding protein type 4(FABP4), fatty acid synthetase(FASN), and lipoprotein lipase(LPL)during the differentiation of human adipocytes, and to explore their possible roles. Methods Human adipose-derived mesenchymal stem cells were induced by adipogenic differentiation, and the adipogenesis of cells was observed by oil red O staining. The expressions of HuR, FABP4, FASN, and LPL mRNA and protein were detected by real-time PCR and Western blotting. After HuR was silenced by siRNA, the change of adipogenesis for human adipose-derived mesenchymal stem cells was observed and the expressions of adipogenic genes were detected. Results The expressions of HuR, FABP4, FASN, and LPL mRNA and protein were significantly increased after human adipose-derived mesenchymal stem cells were induced to differentiate into adipocytes(all P<0.01). After HuR expression was down-regulated by siRNA, the adipogenic level of human adipose-derived mesenchymal stem cells was reduced, with decreased protein levels of FABP4, FASN, and LPL(all P<0.05), which were without changes for their mRNA levels. Conclusion HuR promotes the differentiation of human adipocytes mainly via regulating the changes of FABP4, FASN, and LPL protein levels. (Chin J Endocrinol Metab, 2017, 33: 854-860) Key words: Adipocyte differentiation; RNA binding protein; Human antigen R; Adipocyte fatty acid binding protein; Fatty acid synthase; Lipoprotein lipase

Characterization of immortalized human brown and white pre-adipocyte cell models from a single donor.

Brown adipose tissue with its constituent brown adipocytes is a promising therapeutic target in metabolic disorders due to its ability to dissipate energy and improve systemic insulin sensitivity and glucose homeostasis. The molecular control of brown adipocyte differentiation and function has been extensively studied in mice, but relatively little is known about such regulatory mechanisms in humans, which in part is due to lack of human brown adipose tissue derived cell models. Here, we used retrovirus-mediated overexpression to stably integrate human telomerase reverse transcriptase (TERT) into stromal-vascular cell fractions from deep and superficial human neck adipose tissue biopsies from the same donor. The brown and white pre-adipocyte cell models (TERT-hBA and TERT-hWA, respectively) displayed a stable proliferation rate and differentiation until at least passage 20. Mature TERT-hBA adipocytes expressed higher levels of thermogenic marker genes and displayed a higher maximal respiratory capacity than mature TERT-hWA adipocytes. TERT-hBA adipocytes were UCP1-positive and responded to β-adrenergic stimulation by activating the PKA-MKK3/6-p38 MAPK signaling module and increasing thermogenic gene expression and oxygen consumption. Mature TERT-hWA adipocytes underwent efficient rosiglitazone-induced ‘browning’, as demonstrated by strongly increased expression of UCP1 and other brown adipocyte-enriched genes. In summary, the TERT-hBA and TERT-hWA cell models represent useful tools to obtain a better understanding of the molecular control of human brown and white adipocyte differentiation and function as well as of browning of human white adipocytes.

Heme Biosynthetic Pathway is Functionally Linked to Adipogenesis via Mitochondrial Respiratory Activity.

To investigate key enzymes of heme biosynthesis in human adipocytes and adipose tissue (AT). Heme biosynthesis-related gene expression (ALAS1, ALAD, HMBS) was investigated in whole AT from humans (n = 178 and n = 75) and rats according to obesity status and during adipogenesis of human preadipocytes. The effects of aminotriazole (an ALAD inhibitor) and of ALAD knockdown were also studied. Consistent heme biosynthesis-related gene expression was detected in both subcutaneous AT (SAT) and visceral AT (VAT) and was significantly increased in SAT. ALAS1, ALAD, and HMBS mRNAs were positively associated with adipogenic gene expression in human AT and significantly decreased in subjects with obesity. These results were replicated in an independent cohort. Both SAT and VAT heme levels were positively correlated with ALAS1, ALAD, and HMBS mRNAs. ALAD and HMBS were mainly expressed in adipocytes and increased during differentiation of human adipocytes in parallel to adipogenic genes. In rats, high-fat diet-induced weight gain resulted in decreased Alad and Hmbs mRNAs in a similar way to what was observed with Adipoq. Aminotriazole administration or ALAD knockdown attenuated adipogenesis in parallel with decreased glucose uptake and impaired mitochondrial respiratory function during human adipocyte differentiation. Current data suggest a possible role of heme biosynthesis in human adipogenesis.

Polychlorinated biphenyl 153 in lipid medium modulates differentiation of human adipocytes.

Emerging evidence indicates that polychlorinated biphenyls (PCBs) are involved in the development of diabetes mellitus in the obese. The purpose of this study was to determine mechanisms by which PCB 153 (2,2´,4,4´,5,5´-hexachloro-biphenyl) could influence diet-induced obesity and insulin resistance during adipogenesis. Lineage of h-ADMSCs was differentiated either as control (differentiation medium only), or with lipid vehicle modeling high fat nutrition (NuTRIflex) or lipid free vehicle (dimethylsulfoxide) for 28 days with or without PCB 153 daily co-exposure (in three concentrations 0.1, 1, and 10 microM). Gene expression analyses were performed using RT-qPCR at days 4, 10, 21, 24, 28; protein levels Akt and phosphorylated Akt (Phospho-Akt) by Western blot at days 4, and 21. PCB 153 treatment of h-ADMSCs only in lipid vehicle was associated with down regulation of key master genes of adipogenesis: PPARgamma, SREBP-1, PPARGC1B, and PLIN2 during the whole process of differentiation; and with increased Akt and decreased Phospho-Akt protein level at day 21. We have shown that PCB 153, in concentration 0.1 microM, has a potential in lipid rich environment to modulate differentiation of adipocytes. Because European and U.S. adults have been exposed to PCB 153, this particular nutrient-toxicant interaction potentially impacts human obesity and insulin sensitivity.

Growth hormone-releasing hormone is produced by adipocytes and regulates lipolysis through growth hormone receptor.

Growth hormone-releasing hormone (GHRH) has a crucial role in growth hormone (GH) secretion, but little is known about its production by adipocytes and its involvement in adipocyte metabolism. To determine whether GHRH and its receptor (GHRH-R) are present in human adipocytes and to study their levels in obesity. Also, to analyze the effects of GHRH on human adipocyte differentiation and lipolysis. GHRH/GHRH-R and GH/GH-R mRNA expression levels were analyzed in human mature adipocytes from non-obese and morbidly obese subjects. Human mesenchymal stem cells (HMSC) were differentiated to adipocytes with GHRH (10-14-10-8 M). Adipocyte differentiation, lipolysis and gene expression were measured and the effect of GH-R silencing was determined. Mature adipocytes from morbidly obese subjects showed a higher expression of GHRH and GH-R, and a lower expression of GHRH-R and GH than non-obese subjects (P<0.05). A total of 10-14-10-10 M GHRH induced an inhibition of lipid accumulation and PPAR-γ expression (P<0.05), and an increase in glycerol release and HSL expression (P<0.05) in human differentiated adipocytes. A total of 10-12-10-8 M GHRH decreased GHRH-R expression in human differentiated adipocytes (P<0.05). A total of 10-10-10-8 M GHRH increased GH and GH-R expression in human differentiated adipocytes (P<0.05). The effects of GHRH at 10-10 M on adipocyte differentiation and lipolysis were blocked when GH-R expression was silenced. GHRH and GHRH-R are expressed in human adipocytes and are negatively associated. GHRH at low doses may exert an anti-obesity effect by inhibiting HMSC differentiation in adipocytes and by increasing adipocyte lipolysis in an autocrine or paracrine pathway. These effects are mediated by GH and GH-R.

Low-dose exposure to bisphenols A, F and S of human primary adipocyte impacts coding and non-coding RNA profiles.

Bisphenol A (BPA) exposure has been suspected to be associated with deleterious effects on health including obesity and metabolically-linked diseases. Although bisphenols F (BPF) and S (BPS) are BPA structural analogs commonly used in many marketed products as a replacement for BPA, only sparse toxicological data are available yet. Our objective was to comprehensively characterize bisphenols gene targets in a human primary adipocyte model, in order to determine whether they may induce cellular dysfunction, using chronic exposure at two concentrations: a “low-dose” similar to the dose usually encountered in human biological fluids and a higher dose. Therefore, BPA, BPF and BPS have been added at 10 nM or 10 μM during the differentiation of human primary adipocytes from subcutaneous fat of three non-diabetic Caucasian female patients. Gene expression (mRNA/lncRNA) arrays and microRNA arrays, have been used to assess coding and non-coding RNA changes. We detected significantly deregulated mRNA/lncRNA and miRNA at low and high doses. Enrichment in “cancer” and “organismal injury and abnormalities” related pathways was found in response to the three products. Some long intergenic non-coding RNAs and small nucleolar RNAs were differentially expressed suggesting that bisphenols may also activate multiple cellular processes and epigenetic modifications. The analysis of upstream regulators of deregulated genes highlighted hormones or hormone-like chemicals suggesting that BPS and BPF can be suspected to interfere, just like BPA, with hormonal regulation and have to be considered as endocrine disruptors. All these results suggest that as BPA, its substitutes BPS and BPF should be used with the same restrictions.

Obesity Is Associated With Increased Expression of Very Low Density Lipoprotein Receptor in Adipose Tissue: A Possible Link to Increased VLDL Catabolism and Adiposity

BACKGROUNDVery‐low density lipoprotein receptor (VLDLR) is a member of the low‐density lipoprotein receptor family highly expressed in adipose tissue. It binds a variety of ligands including apolipoprotein E‐containing triglyceride‐rich lipoproteins, and plays a significant role in the catabolism of triglyceride‐rich lipoproteins. The role of VLDLR had been demonstrated in cell culture and animal models, but little is known about its regulation in human. The objective of this study was to assess the impact of obesity on VLDLR expression in adipose tissue, and to evaluate the role of VLDLR in human adipocyte differentiation and lipid deposition.SUBJECTS AND SAMPLESPaired subcutaneous and visceral adipose tissue aliquots, as well as blood samples, were obtained from a total of 76 subjects, 16 lean (body mass index: BMI ≤ 24.9 kg/m2), 18 overweight (25 ≤ BMI ≤ 29.9 kg/m2) and 42 obese and morbid obese (BMI ≥ 30 kg/m2) subjects. Concentration of plasma lipids (triglycerides, free fatty acids and cholesterol) and glucose was measured enzymatically. Blood insulin level was assayed by enzyme‐linked immunosorbent assay, and homeostasis model assessment of insulin resistance (HOMA‐IR) was determined. Adipocyte size was examined in hematoxylin and eosin‐stained sections, and VLDLR expression was analyzed in adipose tissue using Western blotting and RT‐PCR. In addition, VLDLR expression was examined in isolated pre‐adipocytes before differentiation and during differentiation to mature adipocytes.RESULTSCompared to lean subjects, VLDLR protein level and mRNA abundance in subcutaneous adipose tissue, was up‐regulated in overweight (+46%) and obese subjects (+119%). Similarly, VLDLR expression was up‐regulated in visceral fat but to lesser extent than subcutaneous fat. The abundance of VLDLR mRNA in subcutaneous adipose tissue was positively (p &lt; 0.05) associated with BMI and HOMA‐IR, but not with blood lipids. In addition, VLDLR expression in subcutaneous fat was significantly (p&lt;0.05) associated with increased adipocyte size and triglyceride content. In pre‐adipocytes, VLDLR protein level was virtually inexistent, but increased gradually during differentiation to reach a maximum level in mature adipocytes. Moreover, induction of VLDLR expression in differentiated adipocytes was associated with increased deposition of triglycerides derived mainly from VLDL.CONCLUSIONThe study indicates that obesity up‐regulates VLDLR expression in adipose tissue, and that induction of VLDLR expression during pre‐adipocyte differentiation is associated with increased lipid deposition suggesting a possible role of VLDLR in adipose tissue expansion in human.Support or Funding InformationHackensack University Medical Center

Human adipocyte differentiation and characterization in a perfusion-based cell culture device.

Adipocytes have gained significant attention recently, because they are not only functioning as energy storage but also as endocrine cells. Adipocytes secret various signaling molecules, including adiponectin, MCP-1, and IL-6, termed collectively as "adipokines". Adipokines regulate glucose metabolism, thereby play an important role in obesity, diabetes type 2, and other metabolic disorders. Conventionally, to study the secretory function, adipocytes are cultured in vitro in static conditions. However, static culturing condition falls short of mimicking the interstitial fluid flows in living systems. Here, we developed a perfusion device which allows dynamic culture of adipocytes under constant and mild flow using a double-layered fluidic structure. Adipocytes were cultured in the bottom layer while the culture media were constantly flown in the upper layer and perfused through a porous membrane that separate the two chambers. The porous membrane between the two chambers physically separates the cells from the flow stream while maintain a fluidic connection by diffusion. This setting not only provides continuous nutrient supply to adipocytes but also maintains a steady and mild shear stress on the cell membrane. It was found the perfusion-based culture conditions promoted faster growth of primary preadipocytes and stimulated greater adipogenesis compared to static culture condition. Adipocytes cultured under perfusion systems produced more MCP-1 and IL-6, but less adiponectin. When stimulated with TNF-α, adipocytes expressed higher level of MCP-1 and IL-6, but lower level of adiponectin. No significant glucose uptake regulation was observed after treating the adipocytes with insulin in both static and perfusion-based culture. Our results demonstrate that perfusion-base culture has played a role in the adipocyte function particularly the secretion of adipokines. More future studies are required to unveil the mechanisms behind perfusion's impact on adipocytes.

HMOX1 as a marker of iron excess-induced adipose tissue dysfunction, affecting glucose uptake and respiratory capacity in human adipocytes.

Iron excess in adipose tissue is known to promote adipose tissue dysfunction. Here, we aimed to investigate the possible role of haem oxygenase 1 (HMOX1) in iron excess-induced adipose tissue dysfunction. Cross-sectionally, HMOX1 gene expression in subcutaneous and visceral adipose tissue was analysed in two independent cohorts (n = 234 and 40) in relation to obesity. We also evaluated the impact of weight loss (n = 21), weight gain (in rats, n = 20) on HMOX1 mRNA; HMOX1 mRNA levels during human adipocyte differentiation; the effects of inflammation and iron on adipocyte HMOX1; and the effects of HMOX1-induced activity on adipocyte mitochondrial respiratory function, glucose uptake and adipogenesis. Adipose tissue HMOX1 was increased in obese participants (p = 0.01) and positively associated with obesity-related metabolic disturbances, and markers of iron accumulation, inflammation and oxidative stress (p < 0.01). HMOX1 was negatively correlated with mRNAs related to mitochondrial biogenesis, the insulin signalling pathway and adipogenesis (p < 0.01). These associations were replicated in an independent cohort. Bariatric surgery-induced weight loss led to reduced HMOX1 (0.024 ± 0.010 vs 0.010 ± 0.004 RU, p < 0.0001), whereas in rats, high-fat diet-induced weight gain resulted in increased Hmox1 mRNA levels (0.22 ± 0.15 vs 0.54 ± 0.22 RU, p = 0.005). These changes were in parallel with changes in BMI and adipose tissue markers of iron excess, adipogenesis and inflammation. In human adipocytes, iron excess and inflammation led to increased HMOX1 mRNA levels. HMOX1 induction (by haem arginate [hemin] administration), resulted in a significant reduction of mitochondrial respiratory capacity (including basal respiration and spare respiratory capacity), glucose uptake and adipogenesis in parallel with increased expression of inflammatory- and iron excess-related genes. HMOX1 is an important marker of iron excess-induced adipose tissue dysfunction and metabolic disturbances in human obesity.

Differentiation and Metabolic Interrogation of Human Adipocytes.

Adipocytes differentiated from preadipocytes provide a valuable model for the study of human adipocyte metabolism. We describe methods for isolation of human stromal vascular cells, expansion of preadipocytes, differentiation into mature adipocytes, and in vitro metabolic interrogation of adipocytes.

Human ADMC-Derived Adipocyte Thermogenic Capacity Is Regulated by IL-4 Receptor.

Type two innate immune system is anti-inflammatory and may play an important role as the means whereby “browning” is induced in subcutaneous adipocytes. It was shown that IL-4 may influence the fate of adipose cell precursors by promoting differentiation towards more thermogenic adipocytes in mice. Here, we investigated the influence of IL-4 and IL-4 receptor, a type two immune cytokine pathway, on the metabolic activity and thermogenic potential of human adipocytes differentiated from adipose-derived mesenchymal stem cells (ADMSCs) obtained from subcutaneous samples of healthy women undergoing abdominoplasty. Western blot analysis, qPCR, and biochemical analyses were performed 10 days after ADMSC differentiation into mature adipocytes was induced. IL-4 receptor was expressed in both precursor and differentiated adipocytes, and IL-4 treatment increased phosphorylation Y641 of signal transducer and activator of transcription 6 (STAT6) in both cell types. IL-4 treatment also increased expression of thermogenic proteins PGC-1α, UCP-1, and CITED1. In addition, IL-4 increased the secretion of adiponectin, leptin, and FGF21 and promoted lipolysis in differentiated adipocytes. In conclusion, IL-4 may directly modulate differentiation of human adipocytes towards a beige phenotype acting through IL-4 receptors on both adipose precursors and differentiated human adipocytes, metabolic effect that must be considered in some antiallergic drugs.

Persistent organic pollutants alter DNA methylation during human adipocyte differentiation

Ubiquitous persistent organic pollutants (POPs) can accumulate in humans where they might influence differentiation of adipocytes. The aim of this study was to investigate whether DNA methylation is one of the underlying mechanisms by which POPs affect adipocyte differentiation, and to what extent DNA methylation can be related to gene transcription. Adipocyte differentiation was induced in two human cell models with continuous exposure to different POPs throughout differentiation. From the seven tested POPs, perfluorooctanesulfonic acid (PFOS) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) decreased lipid accumulation, while tributyltin (TBT) increased lipid accumulation. In human mesenchymal stem cells (hMSCs), TCDD and TBT induced opposite gene expression profiles, whereas after PFOS exposure gene expression remained relatively stable. Genome-wide DNA methylation analysis showed that all three POPs affected DNA methylation patterns in adipogenic and other genes, possibly related to the phenotypic outcome, but without concomitant gene expression changes. Differential methylation was predominantly detected in intergenic regions, where the biological relevance of alterations in DNA methylation is unclear. This study demonstrates that POPs, at environmentally relevant levels, are able to induce differential DNA methylation in human differentiating adipocytes.

Transcriptional Dynamics During Human Adipogenesis and Its Link to Adipose Morphology and Distribution.

White adipose tissue (WAT) can develop into several phenotypes with different pathophysiological impact on type 2 diabetes. To better understand the adipogenic process, the transcriptional events that occur during in vitro differentiation of human adipocytes were investigated and the findings linked to WAT phenotypes. Single-molecule transcriptional profiling provided a detailed map of the expressional changes of genes, enhancers, and long noncoding RNAs, where different types of transcripts share common dynamics during differentiation. Common signatures include early downregulated, transient, and late induced transcripts, all of which are linked to distinct developmental processes during adipogenesis. Enhancers expressed during adipogenesis overlap significantly with genetic variants associated with WAT distribution. Transiently expressed and late induced genes are associated with hypertrophic WAT (few but large fat cells), a phenotype closely linked to insulin resistance and type 2 diabetes. Transcription factors that are expressed early or transiently affect differentiation and adipocyte function and are controlled by several well-known upstream regulators such as glucocorticosteroids, insulin, cAMP, and thyroid hormones. Taken together, our results suggest a complex but highly coordinated regulation of adipogenesis.

METRNL decreases during adipogenesis and inhibits adipocyte differentiation leading to adipocyte hypertrophy in humans.

Meteorin-like (METRNL) is a recently described circulating protein shown to be highly expressed in white adipose tissue and to beneficially affect energy metabolism in mice. We systematically evaluated the role of METRNL for human adipogenesis and its association with obesity, browning and hyperinsulinemia in children. In addition, we assessed the functional relevance of METRNL for human adipogenesis. METRNL expression decreased during human adipocyte differentiation in vitro. Coherently, METRNL expression was lower in isolated adipocytes compared with stromal vascular fraction (SVF) cells in human samples. Withdrawal of the peroxisome proliferator-activated receptor-γ (PPARγ) agonist rosiglitazone from adipogenic media partially preserved the METRNL downregulation during adipogenesis. METRNL expression was higher in adipocytes of obese compared with lean children and correlated with adipocyte size, whereas in SVF METRNL expression correlated with proliferation capacity. Concordantly, overexpression of METRNL inhibited human adipocyte differentiation as shown by decreased lipogenesis and lower expression of PPARγ and markers of adipogenesis, whereas experimental downregulation promoted adipogenesis. Proliferation, in contrast, was advanced by METRNL overexpression. These interactions with adipose tissue dynamics may contribute to the clinically observed body mass index-independent association of METRNL expression with hyperinsulinemia and adipose tissue inflammation in human samples. METRNL was not associated with UCP1 expression or induction of browning in white adipocytes. Taken together, the downregulation of METRNL during adipogenesis and functional induction of increased proliferation in SVF cells with concomitant inhibition of adipocyte differentiation may result in hypertrophic AT accumulation. This may also explain our observations of increased METRNL expression in adipocytes but not SVF cells in obese children compared with lean children and the subsequent hyperinsulinemia.