Differentiation Of Human Preadipocytes Research Articles

Soluble Epoxide Hydrolase 2 Expression Is Elevated in Obese Humans and Decreased by Physical Activity.

Epoxide hydrolase 2 (EPHX2) is an emerging therapeutic target in several immunometabolic disorders. EPHX2 metabolizes anti-inflammatory epoxyeicosatrienoic acids into pro-inflammatory diols. The contribution of EPHX2 activity to human obesity remains unexplored. We compared the expression of EPHX2 between lean and obese humans (n = 20 each) in subcutaneous adipose tissue (SAT) and peripheral blood mononuclear cells (PBMCs) using RT-PCR, Western Blot analysis, immunohistochemistry, and confocal microscopy before and after a 3-month physical activity regimen. We also assessed EPHX2 levels during preadipocyte differentiation in humans and mice. EPHX2 mRNA and protein expression were significantly elevated in obese subjects, with concomitant elevated endoplasmic reticulum (ER) stress components (the 78-kDa glucose-regulated protein; GRP78, and the Activating transcription factor 6; ATF6) and inflammatory markers (Tumor necrosis factor-α; TNFα, and Interleukin 6; IL6) as compared to controls (p < 0.05). EPHX2 mRNA levels strongly correlated with adiposity markers. In obese individuals, physical activity attenuated EPHX2 expression levels in both the SAT and PBMCs, with a parallel decrease in ER stress and inflammation markers. EPHX2 expression was also elevated during differentiation of both human primary and 3T3-L1 mouse preadipocytes. Mediators of cellular stress (palmitate, homocysteine, and macrophage culture medium) also increased EPHX2 expression in 3T3-L1 preadipocytes. Our findings suggest that EPHX2 upregulation is linked to ER stress in adiposity and that physical activity may attenuate metabolic stress by reducing EPHX2 expression.

Three-dimensional printing of chemically crosslinked gelatin hydrogels for adipose tissue engineering

Despite their outstanding potential and the success that has already been achieved with three-dimensional (3D) printed hydrogel scaffolds, there has been little investigation into their application in the regeneration of damaged or missing adipose tissue (AT). Due to their macroscopic shape, microarchitecture, extracellular matrix-mimicking structure, degradability and soft tissue biomimetic mechanical properties, 3D printed hydrogel scaffolds have great potential for use in aesthetic, structural and functional restoration of AT. Here, we propose a simple and cost-effective 3D printing strategy using gelatin-based ink to fabricate scaffolds suitable for AT engineering. The ink, successfully printed here for the first time, was prepared by mixing gelatin and methylenebisacrylamide (a crosslinker) to initiate the crosslinking reaction. The solution was then loaded into the cartridge (temperature T = 35 °C) of a pneumatic extrusion-based 3D printer and printed on a cooled surface (T = 4 °C) in the appropriate time window for ink printability as verified by rheological tests. Subsequently, the printed gelatin hydrogels were crosslinked at different temperatures to optimize their stability and fix the printed structure. The gelatin scaffolds crosslinked at 20 °C remained stable for 21 days at physiological temperature, with compressive mechanical properties mimicking those of AT (i.e. elastic modulus = 20 kPa). The 3D printed scaffolds showed no indirect cytotoxic effects on a 3T3-L1 pre-adipocyte cell line. Moreover, the printed scaffolds successfully promoted adhesion and proliferation of primary human pre-adipocytes, as demonstrated by LIVE/DEAD staining and Alamar Blue assay. The differentiation of primary human pre-adipocytes isolated from three different donors according to adipogenic phenotype was demonstrated by an increase in PPARγ gene expression detected by real-time PCR and accumulated lipid droplets stained by Oil Red O, thus proving the potential of the 3D printed gelatin hydrogels as scaffolds for AT engineering.

Ghrelin Promotes Proliferation and Inhibits Differentiation of 3T3-L1 and Human Primary Preadipocytes.

ObjectiveGhrelin is a 28-amino-acid peptide that regulates energy hemostasis, glucose and lipid metabolism. We aimed to explore the effects of ghrelin on the proliferation and differentiation of 3T3-L1 and human primary preadipocytes.Methods3-(4,5-Dimethylthiazol-2-yl) 2,5-diphenyl tetrazolium bromide (MTT) spectrophotometry, Oil Red O staining, intracellular glycerol-3-phosphate dehydrogenase (G-3-PDH) assays and semiquantitative reverse transcription polymerase chain reaction were used to investigate the action of ghrelin.ResultsGhrelin (0.01–1000 ng/ml) significantly increased the numbers of 3T3-L1 cells, and the maximum stimulatory effect was observed with the 10 ng/ml ghrelin treatment for 24 h (p < 0.05). Ghrelin also promoted the proliferation of human primary preadipocytes from 24 h (p < 0.05) to 48 h (p < 0.05) at a concentration of 1000 ng/ml. Further investigation showed that IGF-1 levels were notably increased in ghrelin-treated 3T3-L1 and human preadipocytes, and IGF-1 antibody was capable to attenuate this stimulatory action of ghrelin (all p < 0.05). Additionally, ghrelin significantly suppressed the differentiation of 3T3-L1 and human primary preadipocytes; 10 ng/ml ghrelin notably downregulated G-3-PDH activities in 3T3-L1 cells on day 3 and in human cells from days 4 to 12 following differentiation (all p < 0.05), and the intracellular lipoprotein lipase mRNA levels were lower than that of the controls (p < 0.05). Further investigation showed that the mRNA levels of peroxisome proliferator-activated receptor γ2 (PPARγ2) and CCAAT/enhancer binding protein α (C/EBPs) were also suppressed in ghrelin-treated human differentiating adipocytes.ConclusionGhrelin promotes the proliferation of 3T3-L1 and human primary preadipocytes by increasing the expression of IGF-1. Ghrelin inhibits murine and human adipocyte differentiation by downregulating PPARγ2 and C/EBPα levels, consequently leading to decreased lipid accumulation and lipogenic enzymes expression.

Resveratrol Inhibits Human Visceral Preadipocyte Proliferation and Differentiation in vitro.

Visceral obesity is a high-risk factor for diabetes and metabolic syndrome. Resveratrol, a natural polyphenolic compound, has been reported to inhibit preadipocyte differentiation. However, the effect of resveratrol on human visceral preadipocyte (HPA-v) differentiation remains largely unknown. LIM domain only 3 (LMO3) promotes human preadipocyte differentiation by enhancing peroxisome proliferator-activated receptor γ (PPARγ) transcriptional activity, which is the master regulator of adipogenesis. The purpose of our study was to determine the effect of resveratrol (0-50 μM) on HPA-v proliferation and differentiation, and the role of LMO3 in resveratrol-mediated regulation of HPA-v differentiation. Resveratrol inhibited HPA-v proliferation and differentiation in a dose-dependent manner, and significantly decreased the mRNA expression levels of PPARG, CCAAT/enhancer-binding protein α (CEBPA), fatty acid-binding protein 4 (FABP4), acetyl-CoA carboxylase (ACC), and fatty acid synthase (FAS) (p < 0.05) at 10, 20, and 50 μM. The mRNA and protein levels of LMO3 were significantly reduced by ≥20 μM resveratrol (p < 0.05), and overexpression of LMO3 partially attenuated resveratrol-induced reduction of HPA-v differentiation by enhancing the PPARG transcriptional activity. Together, our study suggested that resveratrol reduced HPA-v proliferation and differentiation, as well as LMO3, which was partially responsible for the reduction of resveratrol-mediated adipocyte differentiation.

Impact of X-ray Exposure on the Proliferation and Differentiation of Human Pre-Adipocytes.

Radiotherapy is a widely used treatment option for cancer patients as well as for patients with musculoskeletal disorders. Adipocytes, the dominant cell type of adipose tissue, are known to constitute an active part of the tumor microenvironment. Moreover, adipocytes support inflammatory processes and cartilage degradation in chronic inflammatory diseases, i.e., rheumatoid and osteoarthritis. Since the production of inflammatory factors is linked to their differentiation stages, we set out to explore the radiation response of pre-adipocytes that may influence their inflammatory potential and differentiation capacity. This is the first study investigating the effects of X-ray irradiation on the proliferation and differentiation capacity of human primary pre-adipocytes, in comparison to Simpson–Golabi–Behmel Syndrome (SGBS) pre-adipocytes, an often-used in vitro model of human primary pre-adipocytes. Our results demonstrate a dose-dependent reduction of the proliferation capacity for both cell strains, whereas the potential for differentiation was mostly unaffected by irradiation. The expression of markers of adipogenic development, such as transcription factors (PPARγ, C/EBPα and C/EBPβ), as well as the release of adipokines (visfatin, adiponectin and leptin) were not significantly changed upon irradiation. However, after irradiation with high X-ray doses, an increased lipid accumulation was observed, which suggests a radiation-induced response of adipocytes related to inflammation. Our results indicate that pre-adipocytes are radio-resistant, and it remains to be elucidated whether this holds true for the overall inflammatory response of adipocytes upon irradiation.

Adipose tissue TSH as a new modulator of human adipocyte mitochondrial function.

Recent studies indicate a possible role of TSH/TSHR signalling axis on adipogenesis and adipose tissue physiology. Here, we aimed to investigate the relationship between adipose tissue TSHB and adipose tissue physiology-related gene expression. Subcutaneous and visceral adipose tissue TSHB gene expression was analysed in two independent cohorts [Cohort1 (N = 96) and Cohort2 (N = 45)] and after bariatric surgery-induced weight loss [Cohort3 (N = 22)]. Adipose tissue TSH protein expression was also analysed in a subgroup of participants from Cohort 1 (N = 16). The effects of recombinant TSH on human subcutaneous preadipocytes and adipocytes were investigated. In cohort 1, both visceral and subcutaneous adipose tissue TSHB gene expression was positively correlated with the expression of mitochondrial function (PPARGC1A, ISCA2, CISD1, SIRT1, NFE2L2, NRF1) and fatty acid mobilization (CAV1, ENGL1), but not with adipogenic-related genes. Of note, adipose tissue TSH protein levels were also associated with some of these markers of mitochondrial function and fatty acid mobilization. These associations were replicated in cohort 2. Bariatric surgery-induced weight loss resulted in increased subcutaneous adipose tissue TSHB in parallel to increased PPARGC1A. In human subcutaneous adipocytes, rh-TSH administration led to increased mitochondrial respiratory capacity in parallel to increased mitochondrial function- and adipogenic-related gene expression, but no significant effects were observed during differentiation of human preadipocytes. These data point to a possible role of adipose tissue TSH in the maintenance of adipocyte mitochondrial function.

Activation of brown adipocytes by placental growth factor

Gestational diabetes mellitus (GDM) is a type of diabetes and occurs during pregnancy. Brown adipose tissue (BAT) improves glucose homeostasis and mitigates insulin resistance, however, its activity is reduced in GDM. Placenta growth factor (PlGF) is an angiogenic factor produced by placental trophoblasts. Nevertheless, whether and how PlGF could affect BAT function in GDM are not defined. To investigate this question, 91 non-diabetic pregnant participants and 73 GDM patients were recruited to Gynaecology and Obstetrics Centre in Lu He hospital. Serum levels of PlGF were quantified by ELISA. Skin temperature was measured by far infrared thermography in the supraclavicular region where classical BATs were located. The direct effect of PlGF on BAT function was explored using the established human preadipocyte differentiation system. Thereby, we demonstrated that serum levels of PlGF were lower in GDM patients compared with controls, which was accompanied by decreased skin temperature in the supraclavicular region. By qPCR and western blot, mRNA and protein expression of UCP1 and OXPHOS were elevated in differentiated adipocytes treated with PlGF. PlGF stimulated mitochondrion transcription and increased copy number of mitochondrial. When subjected for respirometry, PlGF-treated differentiated adipocytes showed higher oxygen consumption rates than controls. PlGF induced AMPK phosphorylation and blockade of AMPK phosphorylation blunted UCP1 and OXPHOS expression in differentiated adipocytes. PlGF administration reduced cholesterol and triglyceride content in the liver and improved insulin sensitivity in db mice compared with control. In Conclusion, PlGF could activate BAT function. Downregulation of PlGF might contribute to the reduced BAT activity in GDM.

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.

Dechlorane Plus increases adipogenesis in 3T3-L1 and human primary preadipocytes independent of peroxisome proliferator-activated receptor γ transcriptional activity.

Polybrominated diphenyl ethers (PBDEs) are chemicals that were added to consumer products to reduce flammability but were deemed toxic and bioaccumulative and were phased out of commerce. Flame retardants (FRs) such as Dechlorane Plus (DP) were introduced as replacements. DP is being produced in high volumes and is detected in the environment, human milk, and human serum. Although human exposure to DP is evident, little is known about its potential effects on human health. We and others have shown that some FRs are potential obesogens, i.e., promote adipogenesis. However, the effects of DP on adipogenesis are not known. Murine 3T3-L1 and human primary subcutaneous (Sc) and omental (Om) preadipocytes were differentiated in the presence of DP (0.001-10 µM) and adipogenic effects were measured. Further, the ability of DP to activate the adipogenic transcription factor peroxisome proliferator-activated receptor γ (PPARγ) was also assessed. We show that treatment of murine preadipocytes with DP significantly (p < 0.05) increased lipid accumulation (2.5-fold) and the mRNA expression of adipogenic markers: fatty acid binding protein 4 (Fabp4), lipoprotein lipase (Lpl), perilipin (Plin), adipsin, and adiponectin. DP also significantly (p < 0.05) increased the protein levels of selected mature adipocyte markers. We further show using luciferase reporter assays that DP increased PPARγ transcriptional activity by threefold (p < 0.05). When the PPARγ agonist was replaced by DP in the human preadipocyte differentiation cocktail, DP significantly (p < 0.05) increased the mRNA levels of adipogenic markers, PPARγ, FABP4, and PLIN in human Sc as well as Om cultures. Finally, PPARγ antagonist studies revealed that DP-mediated upregulation of adipogenic markers Fabp4 and Lpl did not occur via PPARγ activation. The current study shows that DP can induce adipogenesis of murine and human preadipocytes. We show that, although DP can directly activate PPARγ, its adipogenic effects may be mediated via other pathways.

Role of 1α,25-Dihydroxyvitamin D3 in Adipogenesis of SGBS Cells: New Insights into Human Preadipocyte Proliferation

Background/Aims: Compared with non-obese individuals, obese individuals commonly store more vitamin D in adipose tissue. VDR expression in adipose tissue can influence adipogenesis and is therefore a target pathway deserving further study. This study aims to assess the role of 1,25(OH)₂D₃ in human preadipocyte proliferation and differentiation. Methods: RTCA, MTT, and trypan blue assays were used to assess the effects of 1,25(OH)₂D₃ on the viability, proliferation, and adipogenic differentiation of SGBS cells. Cell cycle and apoptosis analyses were performed with flow cytometry, triglycerides were quantified, and RT-qPCR was used to assess gene expression. Results: We confirmed that the SGBS cell model is suitable for studying adipogenesis and demonstrated that the differentiation protocol induces cell maturation, thereby increasing the lipid content of cells independently of treatment. 1,25(OH)₂D₃ treatment had different effects according to the cell stage, indicating different modes of action driving proliferation and differentiation. In preadipocytes, 1,25(OH)₂D₃ induced G1 growth arrest at both tested concentrations without altering CDKN1A gene expression. Treatment with 100 nM 1,25(OH)₂D₃ also decreased MTT absorbance and the lipid concentration. Moreover, increased normalized cell index values and decreased metabolic activity were not induced by proliferation or apoptosis. Exposure to 100 nM 1,25(OH)₂D₃ induced VDR, CEBPA, and CEBPB expression, even in the preadipocyte stage. During adipogenesis, 1,25(OH)₂D₃ had limited effects on processes such as VDR and PPARG gene expression, but it upregulated CEBPA expression. Conclusions: We demonstrated for the first time that 1,25(OH)₂D₃ induces changes in preadipocytes, including VDR expression and growth arrest, and increases the lipid content in adipocytes treated for 16 days. Preadipocytes are important cells in adipose tissue homeostasis, and understanding the role of 1,25(OH)₂D₃ in adipogenesis is a crucial step in ensuring adequate vitamin D supplementation, especially for obese individuals.

Proliferation, Metabolic Activity, and Adipogenic Differentiation of Human Preadipocytes Exposed to 2 Surfactants In Vitro

Fat grafting is a pivotal technique for tissue repair. Adipose stromal cells, including preadipocytes, play a major role in the regenerative effects attributed to fat grafting. But the benefits are impaired by the low survival of the graft due to mechanical stress during harvesting, hypoxia, and nutrient deprivation. Nonionic surfactant molecules demonstrated their efficacy in preventing and repairing mechanical damage on the cellular membrane, but it is poorly understood if and how they affect cellular viability, proliferation, and differentiation. We investigated the influence of 2 nonionic surfactants, Kolliphor®P188 and Kolliphor®EL, on cultured human preadipocytes. We analyzed their effects on metabolic activity, cell number, adipogenic differentiation, and secretion of growth factors. Kolliphor®P188 increased metabolic activity, while it did not influence proliferation and differentiation as well as growth factors release. Kolliphor®EL confirmed its cytotoxic effect at the highest concentrations applied. Contrariwise, treatment with lower concentrations significantly raised metabolic activity, induced adipogenesis, and increased insulin-like growth factor-1 and vascular endothelial growth factor secretion. The effect on differentiation was inhibited by blocking peroxisome proliferator-activated receptor gamma. Our results revealed important effects of surfactants on preadipocytes' survival, proliferation, death, and the interplay with their environment. Particularly Kolliphor®EL provides modes of action, which could recommend it for novel treatment to improve fat graft viability.

HO-1 inhibits preadipocyte proliferation and differentiation at the onset of obesity via ROS dependent activation of Akt2

Excessive accumulation of white adipose tissue (WAT) is a hallmark of obesity. The expansion of WAT in obesity involves proliferation and differentiation of adipose precursors, however, the underlying molecular mechanisms remain unclear. Here, we used an unbiased transcriptomics approach to identify the earliest molecular underpinnings occuring in adipose precursors following a brief HFD in mice. Our analysis identifies Heme Oxygenase-1 (HO-1) as strongly and selectively being upregulated in the adipose precursor fraction of WAT, upon high-fat diet (HFD) feeding. Specific deletion of HO-1 in adipose precursors of Hmox1fl/flPdgfraCre mice enhanced HFD-dependent visceral adipose precursor proliferation and differentiation. Mechanistically, HO-1 reduces HFD-induced AKT2 phosphorylation via ROS thresholding in mitochondria to reduce visceral adipose precursor proliferation. HO-1 influences adipogenesis in a cell-autonomous way by regulating events early in adipogenesis, during the process of mitotic clonal expansion, upstream of Cebpα and PPARγ. Similar effects on human preadipocyte proliferation and differentiation in vitro were observed upon modulation of HO-1 expression. This collectively renders HO-1 as an essential factor linking extrinsic factors (HFD) with inhibition of specific downstream molecular mediators (ROS & AKT2), resulting in diminished adipogenesis that may contribute to hyperplastic adipose tissue expansion.

Additional attention to combination antiretroviral therapy-related lipodystrophy.

The occurrence of lipodystrophy in patients taking anti-human immunodeficiency virus (HIV) medications is a serious problem as it is irreversible even after drug withdrawal. Although it was first recognized in patients taking proteinase inhibitors, other types of anti-HIV agents can also cause lipodystrophy. In a recent publication by Jones et al entitled “Highly active antiretroviral therapy dysregulates proliferation and differentiation of human pre-adipocytes” in World Journal of Virology, it was reported that simultaneous treatment of human subcutaneous adipocytes with anti-HIV drugs with different mechanisms of action synergistically exerted anti-adipogenesis effects in vitro, warning us to take utmost care in every case receiving combination antiretroviral therapy (cART). For elucidation of the molecular basis for cART-related lipodystrophy, multi-faceted approaches should be taken, based on a deeper understanding of the development and organization of adipose tissues.

Effect of N-(D-Ribopyranosyl) Taurine Sodium Salt on the Differentiation of Human Preadipocytes and Expression of Adipokines Through Inhibition of STAT-3 Signaling in Differentiated Human Adipocytes.

We investigated whether a taurine-ribose derivative, N-(D-ribopyranosyl)taurine sodium salt, inhibits the differentiation process of preadipocytes or modulates the expression of cytokines from adipocytes as does taurine chloramine (TauCl) in vitro. To know the inhibitory effects of taurine-ribose (Tau-Ribose) on differentiation process and adipokine expression, preadipocytes were incubated with Tau-Ribose in differentiation medium for 14 days. Differentiated adipocytes were also stimulated at the concentration of IL-1β 1 ng/ml with addition of Tau-Ribose. After 7 days of incubation, the levels of adiponectin, leptin, IL-6, and IL-8 were measured from the culture supernatants. At concentrations of 10-40 mM, Tau-Ribose dose-dependently inhibited the process of adipogenesis. The treatment of Tau-Ribose decreased the expression of transcription factors, which are necessary for adipogenesis and are known as adipocyte marker. Treatment with Tau-Ribose significantly modulated the production of IL-8 and IL-6. However, it did not modulate the production of adiponectin and leptin in IL-1β-activated adipocytes. As with taurine chloramine, Tau-Ribose also inhibited STAT-3 signaling, independent of MAPK signaling. In conclusion, Tau-Ribose inhibits the signaling pathway of STAT-3 and can change adipokines production; thus, it may have a potential as an agent for treating obesity-related diseases.

Highly active antiretroviral therapy dysregulates proliferation and differentiation of human pre-adipocytes.

AIMTo investigate the mechanism(s) by which potential effects of multi-drug highly-active antiretroviral therapy contributes to lipodystrophy syndrome.METHODSPreadipocytes from healthy donors were assessed for proliferation and differentiation in the presence of nucleoside reverse transcriptase inhibitors (NRTIs), nonnucleoside reverse transcriptase inhibitors (NNRTIs), and protease inhibitors (PIs) individually and in combination. Effects on proliferation were assessed with a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide assay and effects on differentiation were assessed from glycerol-3-phosphate dehydrogenase (GP DH) activity and quantitation of Oil Red O staining for intracellular lipid. Data were analyzed with a randomized block ANOVA with post-hoc Fisher’s Least Significant Difference test.RESULTSPreadipocyte proliferation was inhibited by a combination of NNRTI + NRTI (14% at 48 h, P < 0.001) and PI + NRTI (19% at 48 h, P < 0.001) with additional suppression when ritonavir (RTV) was added (26% at 48 h). The drug combination of atazanavir (ATV) + RTV + emtricitabine (FTC) + tenofovir (TDF) had the greatest inhibitory effect on proliferation at 48 h. Preadipocyte differentiation was most significantly reduced by the efavirenz + FTC + TDF assessed either by GPDH activity (64%) or lipid accumulation (39%), P < 0.001. Combining NRTIs with a PI (ATV + FTC + TDF) significantly suppressed differentiation (GPDH activity reduced 29%, lipid accumulation reduced by 19%, P < 0.01). This effect was slightly greater when a boosting amount of RTV was added (ATV + FTC + TDF + RTV, P < 0.001).CONCLUSIONAlthough combination antiretroviral therapy is clinically more efficacious than single drug regimens, it also has a much greater inhibitory effect on preadipocyte proliferation and differentiation.

Fibroblast growth factor-1 (FGF-1) promotes adipogenesis by downregulation of carboxypeptidase A4 (CPA4) – a negative regulator of adipogenesis implicated in the modulation of local and systemic insulin sensitivity

Fibroblast growth factor-1 (FGF-1) promotes differentiation of human preadipocytes into mature adipocytes via modulation of a BMP and Activin Membrane-Bound Inhibitor (BAMBI)/Peroxisome proliferator-activated receptor (PPARγ)-dependent network. Here, we combined transcriptomic and functional investigations to identify novel downstream effectors aligned with complementary analyses of gene expression in human adipose tissue to explore relationships with insulin sensitivity. RNA-Seq and qRT-PCR analysis revealed significant down-regulation of carboxypeptidase A4 (CPA4) following FGF-1 treatment or induction of differentiation of human preadipocytes in a BAMBI/PPARγ-independent manner. siRNA-mediated knockdown of CPA4 resulted in enhanced differentiation of human preadipocytes. Furthermore, expression of CPA4 in subcutaneous adipose tissue correlated negatively with indices of local and systemic (liver and muscle) insulin sensitivity. These results identify CPA4 as a negative regulator of adipogenesis that is down-regulated by FGF-1 and a putative deleterious modulator of local and systemic insulin sensitivity. Further investigations are required to define the molecular mechanism(s) involved and potential therapeutic opportunities.

Metformin-suppressed differentiation of human visceral preadipocytes: Involvement of microRNAs.

Visceral adipose tissue contributes to the pathophysiology of metabolic syndrome. Metformin has been reported to suppress lipogenesis in a murine preadipocyte cell line. However, the effect of metformin on the differentiation of human visceral adipose tissue remains unknown. MicroRNAs (miRNAs or miRs) have been suggested as therapeutic targets because of their involvement in the differentiation and maturation of fatty cells. The aim of this study was to determine whether metformin suppresses the differentiation of human preadipocytes and to identify miRNAs associated with the regulation of lipid metabolism. Human visceral preadipocytes (HPrAD-vis) were preincubated in growth media and then cultured with differentiation media containing metformin for 1 or 2 weeks. Adipogenic differentiation of the cells was assessed by Oil Red O staining, and soluble adiponectin in the culture media was measured using an enzyme-linked immunosorbent assay. Cell proliferation was assessed using a WST-8 assay, and the gene and protein expression of peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT-enhancer-binding protein α (C/EBPα) was determined by RT-qPCR and western blot analysis, respectively. miRNAs were profiled using human miRNA Oligo chips after total RNA was extracted and labeled. Oil Red O staining showed that metformin suppressed the accumulation of lipid droplets in HPrAD-vis cells. The adiponectin concentration in the culture media was also decreased in metformin-treated cells. The WST-8 assay revealed no effect on proliferation or growth inhibition following metformin treatment, although metformin suppressed the expression of PPARγ and C/EBPα. miRNA profiling further revealed differences between the metformin-treated group and control HPrAD-vis cells. Thus, the findings of the present study demonstrated that metformin suppressed the differentiation of human preadipocytes in vitro and altered the miRNA profile of these cells. Thus, the miRNAs whose expression levels were altered by metformin may contribute to the observed suppression of HPrAD-vis cell differentiation.

Influence of vitamin D on proliferation and differentiation of human preadipocytes in high-glucose condition

Objective To investigate the influence of 1, 25(OH)2D3(VD3) on proliferation and differentiation of human preadipocytes in high-glucose condition. Methods Primary culture of human preadipocytes were divided into 4 groups: the control group, high glucose group, control+ VD3 group, and high glucose+ VD3 group. Oil red O staining and extraction with isopropyl alcohol were employed to detect the total amount of fat in different groups. Real-time polymerase chain reaction was used to detect the mRNA levels of vitamin D receptor (VDR), CCAAT/enhancer binding protein a (C/EBPa) and peroxisome proliferator activated receptor gamma (PPARγ) in each group. Results Compared with the control group, the fat amount was significantly increased in the high glucose group(0.231±0.033 vs. 0.362±0.045, P=0.037) and high glucose+ VD3 group(0.231±0.033 vs. 0.358±0.057, P=0.033), decreased in the control+ VD3 group(0.231±0.033 vs. 0.174±0.029, P=0.016), but no significant difference in the glucose+ VD3 group(0.362±0.045 vs. 0.358±0.057, P=0.450); VDR mRNA level was decreased in the high glucose group(0.397±0.029 vs. 0.069±0.003, P=0.022), but VD3 increased the VDR mRNA levels in both the control+ VD3 group and the high glucose+ VD3 group(0.397±0.029 vs. 1.103±0.044, P=0.035; 0.069±0.003 vs. 0.152±0.021, P=0.041); the mRNA levels of C/EBPa and PPARγ were up-regulated in the high glucose group(0.135±0.048 vs. 0.788±0.063, P=0.018; 0.498±0.026 vs. 0.795±0.037, P=0.021), VD3 decreased the PPARγ mRNA level in the control+ VD3 group(0.498±0.026 vs. 0.237±0.011, P=0.029), but had no significant effect on C/EBPa mRNA(0.788±0.063 vs. 0.843±0.049, P>0.05) and PPARγ mRNA(0.795±0.037 vs. 0.813±0.041, P>0.05) expressions in the high glucose+ VD3 group or C/EBPa mRNA in the control+ VD3 group(0.135±0.048 vs. 0.156±0.022, P>0.05). Conclusion VD3 may lose the ability to inhibit the proliferation and differentiation of human preadipocyte due to reduced biological activity when exposed to high-glucose environment in vitro. Key words: Vitamin D; Vitamin D receptor; Obesity; Diabetes mellitus

MiR-1275 inhibits adipogenesis via ELK1 and its expression decreases in obese subjects.

Excessive adipocyte differentiation and proliferation are closely associated with the onset of obesity, which has been partially linked to microRNA expression. In previous studies, using miRNA microarray screening, we found that miR-1275 was significantly decreased in human mature adipocytes. In this study, we examined the role of miR-1275 in adipogenesis. Our results indicated that miR-1275 can inhibit the differentiation of human visceral preadipocytes without affecting their proliferation. ELK1, an E-twenty-six (ETS)-domain transcription factor associated with adipocyte differentiation, was strongly suppressed by miR-1275 in human visceral adipocytes. This was demonstrated via a dual-luciferase reporter assay and pointed to ELK1 as a direct target of miR-1275. Furthermore, miR-1275 expression was significantly diminished in the visceral adipose tissue of overweight and obese human subjects accompanied by a negative correlation with body mass index. These results suggest that miR-1275 could play a future role in the management of obesity, as a novel therapeutic target or biomarker.

Effect of High Glucose Concentration on Human Preadipocytes and Their Response to Macrophage-Conditioned Medium.

Adipose tissue expands via differentiation of preadipocytes into adipocytes (adipogenesis) and/or hypertrophy of existing adipocytes. A low adipogenic capacity promotes adipocyte hypertrophy, causing inflammatory macrophage accumulation and insulin resistance. Macrophage-conditioned medium (MacCM) inhibits adipogenesis and promotes adipocyte inflammation, but it is unknown whether these effects are altered by high glucose (HG) versus normal glucose (NG) concentrations. Our aim was to compare the effect of HG-MacCM versus NG-MacCM on human adipogenesis and adipocyte inflammation. Human monocyte-derived macrophages (MDMs) were placed in 5 mmol/L (NG) or 25 mmol/L (HG) glucose for 24 hours. MacCM was collected and its effect on differentiation of human subcutaneous abdominal preadipocytes and adipocyte inflammation was evaluated. HG-MacCM, but not NG-MacCM, inhibited triacylglycerol (TG) accumulation and protein expression of peroxisome proliferator-activated receptor γ (PPARgamma) during human adipogenesis. Preadipocytes differentiated in HG-MacCM displayed a more pro-inflammatory phenotype, as assessed by increased interleukin-6 and monocyte chemotactic protein-1 (MCP-1), as well as reduced adiponectin mRNA expression. In MDMs, HG increased phosphorylation of inhibitor of kappaB kinase (IKK)-beta and decreased protein expression of inhibitor of kappaB alpha. HG also reduced protein expression of PPARgamma in MDMs. However, no MDM changes in mRNA expression of MCP-1, interleukin-1beta or tumor necrosis factor-alpha were detected. The stimulatory effect of HG-MacCM on MCP-1 expression in adipocytes was partially inhibited when MDMs were treated with sc-514 (IKKbeta inhibitor). High glucose concentration accentuates the anti-adipogenic and pro-inflammatory effects of MacCM on human preadipocytes.