Adipocyte Differentiation Markers Research Articles

Differentiation-inducing factor-1 potentiates adipogenic differentiation and attenuates the osteogenic differentiation of bone marrow-derived mesenchymal stem cells

Mesenchymal stem cells (MSCs) are an attractive cell source for tissue regeneration and repair. However, their low differentiation efficacy currently impedes the development of MSC therapy. Therefore, in this study, we investigated the effects of differentiation-inducing factor-1 (DIF-1) on the differentiation efficacy of bone marrow-derived MSCs (BM-MSCs) into adipogenic or osteogenic lineages. BM-MSCs, which were obtained from Sprague-Dawley rats, were positive for the MSC markers (CD29, CD73, and CD90). DIF-1 alone neither affected cell surface antigen expression nor induced adipogenic or osteogenic differentiation. However, DIF-1 significantly enhanced the effects of adipogenic differentiation stimuli, which were evaluated as the number of oil red-O positive cells and the expression of adipocyte differentiation markers (peroxisome proliferator-activated receptor gamma, adipocyte fatty acid-binding protein, and adiponectin). In contrast, DIF-1 significantly attenuated the effects of osteogenic differentiation stimuli, which were evaluated as alizarin red-S positive calcium deposition, and the expression of osteoblast differentiation markers alkaline phosphatase, runt-related transcription factor 2, and osteopontin. We further investigated the mechanism by which DIF-1 affects MSC differentiation efficacy and found that glycogen synthase kinase-3 was the main factor mediating the action of DIF-1 on the adipogenic differentiation of BM-MSCs, whereas it was only partially involved in osteogenic differentiation. These results suggest that DIF-1 supports MSC differentiation toward the desired cell fate by enhancing the differentiation efficacy.

The Effect of FOXC2-AS1 on White Adipocyte Browning and the Possible Regulatory Mechanism.

Obesity has become a worldwide epidemic, and obesity-related problems are becoming more severe in public health. Increasing brown adipose tissue (BAT) mass or/and activity in mice and humans has been demonstrated to help lose weight and improve whole-body metabolism. Studies on the conversion of white adipose tissue (WAT) to BAT under certain conditions have provided new possibilities for treating obesity and the related disorders. It has been established that long non-coding RNAs (lncRNAs) play an important role in the regulation of mouse adipocyte differentiation and thermogenic programs; however, the function and potential mechanism of lncRNA in the process of human white adipocyte browning remains unclear. In the present study, we identified a lncRNA called Forkhead Box C2 antisense RNA 1 (FOXC2-AS1), which was first identified in osteosarcoma, and it was highly expressed in human adipocytes but decreased during the white adipocyte differentiation program. FOXC2-AS1 expression was also induced by the thermogenic agent forskolin. Lentivirus-mediated overexpression of FOXC2-AS1 in human white adipocytes did not affect lipid drop accumulation, but significantly promoted the browning phenotype, as revealed by the increased respiratory capacity and the enhanced protein expression levels of brown adipocyte-specific markers. In contrast, inhibiting FOXC2-AS1 with small interfering RNA led to attenuated thermogenic capacity in human white adipocytes. RNA-sequencing analysis and western blot were used to identify a possible regulatory role of the autophagy signaling pathway in FOXC2-AS1 to mediate white-to-brown adipocyte conversion. The autophagy inhibitor 3-methyladenine restored the reduced UCP1 protein level and thermogenic capacity caused by inhibiting FOXC2-AS1. Overall, the present study characterized the potential role of FOXC2-AS1 and further identified a lncRNA-mediated mechanism for inducing browning of human white adipocytes and maintaining thermogenesis, further providing a potential strategy for treating obesity and related disorder.

Regucalcin enhances adipocyte differentiation and attenuates inflammation in 3T3-L1 cells.

Dysregulation of adipocyte differentiation and dysfunction play key roles in the pathogenesis of obesity and associated disorders such as diabetes and metabolic syndrome, and as such, a better understanding of the molecular mechanism of adipogenesis may help to elucidate the pathological condition of obesity and its associated disorders. Regucalcin (RGN) plays multiple regulatory roles in intracellular Ca2+ signaling pathways in mammalian cells. Here, we report that overexpression of RGN enhances lipid accumulation in 3T3‐L1 adipocyte cells after adipogenic stimulation, accompanied by upregulation of adipocyte differentiation marker proteins. In contrast, genetic disruption of RGN inhibited adipogenic stimulation‐induced differentiation of 3T3‐L1 cells. Furthermore, RGN overexpression in differentiated 3T3‐L1 adipocytes blocked inflammatory crosstalk between 3T3‐L1 adipocytes and RAW264.7 macrophages in a transwell coculture system. Knockdown of RGN expression in cocultured 3T3‐L1 adipocytes enhanced their susceptibility to RAW264.7 macrophage‐mediated inflammation. These results suggest that RGN is required for 3T3‐L1 adipocyte differentiation and that it exerts anti‐inflammatory activity against 3T3‐L1 adipocyte inflammation after coculture with RAW264.7 macrophages. Thus, RGN may be a novel regulator of adipocyte differentiation and act as a suppressor of inflammation in macrophage‐infiltrated adipocyte tissue.

Obesity-Induced Increase in Cystatin C Alleviates Tissue Inflammation.

We recently demonstrated that removal of one kidney (uninephrectomy [UniNx]) in mice reduced high-fat diet (HFD)-induced adipose tissue inflammation, thereby improving adipose tissue and hepatic insulin sensitivity. Of note, circulating cystatin C (CysC) levels were increased in UniNx compared with sham-operated mice. Importantly, CysC may have anti-inflammatory properties, and circulating CysC levels were reported to positively correlate with obesity in humans and as shown here in HFD-fed mice. However, the causal relationship of such observation remains unclear. HFD feeding of CysC-deficient (CysC knockout [KO]) mice worsened obesity-associated adipose tissue inflammation and dysfunction, as assessed by proinflammatory macrophage accumulation. In addition, mRNA expression of proinflammatory mediators was increased, whereas markers of adipocyte differentiation were decreased. Similar to findings in adipose tissue, expression of proinflammatory cytokines was increased in liver and skeletal muscle of CysC KO mice. In line, HFD-induced hepatic insulin resistance and impairment of glucose tolerance were further aggravated in KO mice. Consistently, chow-fed CysC KO mice were more susceptible to lipopolysaccharide-induced adipose tissue inflammation. In people with obesity, circulating CysC levels correlated negatively with adipose tissue Hif1α as well as IL6 mRNA expression. Moreover, healthy (i.e., insulin-sensitive) subjects with obesity had significantly higher mRNA expression of CysC in white adipose tissue. In conclusion, CysC is upregulated under obesity conditions and thereby counteracts inflammation of peripheral insulin-sensitive tissues and, thus, obesity-associated deterioration of glucose metabolism.

1698-P: Obesity-Induced Increase in Cystatin C Alleviates Tissue Inflammation and Hepatic Insulin Resistance

Removal of one kidney (uninephrectomy; UniNx) in mice reduced high fat-diet (HFD)-induced adipose tissue inflammation thereby improving adipose tissue and hepatic insulin sensitivity. Such finding was accompanied by increased circulating cystatin C levels in UniNx compared to sham-operated mice. Importantly, cystatin C may have anti-inflammatory properties, and circulating cystatin C levels were reported to positively correlate with obesity as well as type 2 diabetes. However, the causal relationship of such correlation remains unclear. Herein, HFD feeding of cystatin C-deficient (CysC KO) mice aggravated obesity-associated adipose tissue dysfunction and inflammation, as reflected by an accumulation of pro-inflammatory monocytes and elevated expression of pro-inflammatory mediators. Moreover, markers of adipocyte differentiation were decreased. Similarly to findings in adipose tissue, expression of pro-inflammatory cytokines was increased in liver and skeletal muscle of HFD-fed CysC KO mice. In line, HFD-induced hepatic insulin resistance and impairment of glucose tolerance were further aggravated in knockout mice. Consistently, chow-fed CysC KO mice were more susceptible to lipopolysaccharide (LPS)-induced adipose tissue inflammation. In people with obesity, circulating cystatin C levels correlated negatively with mRNA expression of pro-inflammatory cytokines such as Hif1α as well as IL-6. Moreover, healthy (i.e., insulin-sensitive) subjects with obesity depicted significantly higher mRNA expression of cystatin C in subcutaneous white adipose tissue. In conclusion, cystatin C is upregulated under obesity conditions and thereby counteracts inflammation of peripheral insulin-sensitive tissues and, thus, obesity-associated deterioration of glucose metabolism. Disclosure M.A. Dedual: None. S. Wueest: None. T. Challa: None. F. Lucchini: None. T. Aeppli: None. M. Borsigova: None. A.A. Mauracher: None. S. Vavassori: None. J. Pachlopnik Schmid: Advisory Panel; Self; Novartis AG, Swedish Orphan Biovitrum. M. Blüher: Advisory Panel; Self; AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Lilly Diabetes, Novo Nordisk A/S, Sanofi. Speaker’s Bureau; Self; Amgen, Novartis AG. D. Konrad: Research Support; Self; Novo Nordisk A/S. Funding Children’s Research Centre; University Children’s Hospital Zurich; University of Zurich (FK-18-027); Swiss National Science Foundation (310030-160129, 10030-179344); Wolfermann-Nägeli Foundation

Retinoids Augment Thiazolidinedione PPARγ Activation in Oral Cancer Cells

Head and neck squamous cell carcinoma affects nearly 500,000 people annually. Augmenting PPARγ functional activation is linked with multiple anti-carcinogenic processes in aerodigestive cell lines and animal models. PPARγ/RXRα heterodimers may be key partners in this activation. CA 9-22 and NA cell lines were treated with the PPARγ agonist ciglitazone and/or the RXRα agonist 9-cis-retinoic acid. PPARγ functional activation, cellular proliferation, apoptosis activity, and phenotype were subsequently analyzed. Ciglitazone and 9-cis-retinoic acid independently activated PPARγ and down-regulated the carcinogenic phenotype in vitro. Combination treatment significantly augmented these effects, further decreasing proliferation (p<0.0001), and increasing PPARγ functional activation (p<0.0001), apoptosis (p<0.05), and adipocyte differentiation markers (p<0.0001). The efficacy of the combination of ciglitazone and 9-cis-retinoic acid afforded lowering treatment concentrations while maintaining desired therapeutic outcomes, optimistically supporting the feasibility and practicality of this novel treatment option.

Indomethacin promotes browning and brown adipogenesis in both murine and human fat cells.

Indomethacin (Indo), a nonsteroidal antiinflammatory drug, has been shown to promote murine brown adipogenesis both in vitro and in vivo, possibly due to its peroxisome proliferator‐activated receptor gamma (PPARγ)‐agonist activities. However, it is unclear whether Indo induces browning of white adipocytes from both murine and human origins or induces human brown adipogenesis. To bridge the gap, this study investigated the effects of increasing concentrations of Indo on murine 3T3‐L1, human primary subcutaneous white adipocytes (HPsubQ), and human brown (HBr) adipocytes. The results show that Indo dose‐dependently enhanced 3T3‐L1 adipocyte differentiation and upregulated both mRNA and protein expression of brown and beige adipocyte markers, while simultaneously suppressing white adipocyte‐specific marker mRNA expression. mRNA and protein expression of mitochondrial biogenesis and structural genes were dose‐dependently enhanced in Indo treated 3T3‐L1 adipocytes. This was accompanied by augmented mitochondrial DNA, enhanced oxygen consumption, proton leak, and maximal and spare respiratory capacity. Dose‐dependent transactivation of PPARγ confirmed Indo's PPARγ‐agonist activity in 3T3‐L1 cells. Knockdown of PPARγ significantly attenuated Indo's activities in selective browning genes, demonstrating PPARγ dependence of these effects. Moreover, Indo enhanced mRNA and protein expression of brown markers in HPsubQ adipocytes. Interestingly, Indo‐induced differential effects on individual PPARγ isoforms with significant dose‐dependent induction of PPARγ‐2 and suppression of PPARγ‐1 protein expression. Finally, Indo significantly promoted brown adipogenesis in HBr cells. Taken together, these results demonstrate Indo to be a potent thermogenic compound in both murine and human fat cells and may be explored as a therapeutic agent for obesity treatment and prevention.

Breed difference and regulatory role of CRTC3 in porcine intramuscular adipocyte.

The cAMP responsive element binding protein (CREB)-regulated transcription coactivator 3 (CRTC3) is a member of the CRTC protein family and plays an important role in energy metabolism. The aim of this study was to determine if the expression of porcine CRTC3 is related to intramuscular fat (IMF) deposition and meat quality in Heigai pigs (a local fatty breed in China) and Duroc×Landrace×Yorkshire (DLY) pigs (a lean crossbred pig widely cultured in China). In addition, the effect of ectopic expression of CRTC3 on gene expression in porcine IMF adipocytes was also examined. Our results showed that Heigai pigs had lower lean percentage, thicker back fat thickness and smaller loin muscle area than DLY pigs. Compared with DLY pigs, Heigai pigs had higher marbling scores, better meat color and higher IMF contents and triglyceride concentrations. Higher levels of oxidative metabolic enzyme and expression of the slow oxidative muscle fiber-related genes were observed in longissimus dorsi muscle and psoas major muscle (P<0.05) from Heigai pigs. Notably, CRTC3 and adipocyte-specific marker genes were highly expressed in muscle tissues of Heigai pigs. The expression of lipolysis-related genes ATGL and HSL were lower in Heigai muscles. Moreover, forced expression of CRTC3 promoted lipid accumulation and increased the expression of PPARγ, C/EBPα, leptin and FABP4 (P<0.05), whereas it decreased the expression of ATGL and HSL in IMF adipocytes. These results suggest that CRTC3 expression is associated with lipid accumulation and IMF deposition in pigs.

Deer tongue tissue-derived cells are a novel type of cells that possess characteristics of mesenchymal stem cells

Background & Aim The ability of mesenchymal stem cells (MSCs) to differentiate into lineage specific tissue types, homing potential, and tissue repair potential has generated increasing interest in utilizing MSCs to study mechanisms in basic biology, as well as clinical therapeutics. MSCs have been isolated from several species, including human, canine, equine, feline and bovine. Deer MSCs have been isolated from antler and bone marrow. However, MSCs from deer adult tissue such as adipose, skin, tongue and etc. have not been studied. In this study, we performed isolation and characterization of deer tongue epithelium tissue-derived MSCs. Methods, Results & Conclusion Cells were isolated from tongue epithelium tissue according to the general method described in previous studies with minor modifications and further characterized for multipotent properties associated with MSCs. The cells were used for subsequent analyses (flow cytometric analysis, quantitative real-time RT-PCR, and differentiation potentials) at third passage (P3) or P4. We examined their cellular morphology, proliferation properties, expression of cell surface markers, and mesodermal differentiation potentials. The cells had a fibroblast-like spindle shape and formed a highly homogenous monolayer. The cells exhibited significant increase in proliferation during 20 passages under low-glucose DMEM culture condition. The cells expressed pluripotency markers such as Oct4, Sox2, and Nanog as well as cell surface markers such as CD73, CD90, and CD105, but did not express CD34 and CD45. The cells also exhibited the ability to undergo adipogenic, chondrogenic, and osteogenic differentiation. As a result, gene expression levels of collagen type II and PPAR-gamma as chondrocyte and adipocyte-specific markers, respectively, increased in expression in differentiated cells compared to those in undifferentiated cells based on quantitative real-time RT-PCR. Our results suggest that tongue epithelium tissue from deer can be used as a source of MSCs. This study suggests that tongues of deer could be used as a source of deer MSCs.

Transient receptor potential vanilloid 2 mediates the inhibitory effect of far-infrared irradiation on adipogenic differentiation of tonsil-derived mesenchymal stem cells

Background & Aim Far-infrared (FIR) irradiation inhibits adipogenic differentiation of tonsil-derived mesenchymal stem cells (TMSCs) by activating protein phosphatase 2B through enhancing intracellular Ca2+ mobilization, but its upsteam mechanism remains unclear. Transient receptor potential vanilloid (TRPV) is a well-known calcium-permeable channel located in the cell membrane. It has been reported that certain isotypes of TRPV could regulate adipogenesis in (pre)adipocytes. Here, we investigated whether or how the TRPV channel is involved in the inhibitory effect of FIR irradiation on the adipogenic differentiation of TMSCs. Methods, Results & Conclusion Methods TMSCs were exposed to FIR irradiation with or without TRPV inhibitors at room temperature for 30 min followed by adipogenic differentiation for 14 days. At the end of the differentiation, the degree of adipogenesis was assessed by staining lipid droplets with Oil red O solution, and by measuring the protein expressions of adipocyte-specific markers (peroxisome proliferator-activated receptor γ and fatty acid-binding protein 4) using Western blotting. Total RNA was extracted from TMSCs and mRNA transcript levels were measured by reverse transcript (RT)-PCR analysis. Results Exposure to FIR irradiation or a calcium ionophore ionomycin inhibited adipogenic differentiation of TMSCs. Treatment with ruthenium red, a pan-TRP channel inhibitor, reversed the FIR irradiation-inhibited adipogenesis. RT-PCR analysis revealed that TMSCs expressed all the TRPV channels except TRPV5. Based on the previous studies showing an involvement of TRPV1, 2, and 4 in adipogenesis, we tested specific inhibitors of these three TRPV isotypes and found that among those tested, only tranilast, a specific TRPV2 inhibitor, significantly attenuated the effects of FIR irradiation, such as increase of intracellular Ca2+ levels and inhibition of adipogenic differentiation of the TMSCs. When the TMSCs were transfected with small interfering RNA against TRPV2 to silence the TRPV2 gene, the observed effects of FIR irradiation on adipogenic differentiation were also completely reversed. Additionally, we also found that the probenecid, a TRPV2 activator, decreased the adipogenic differentiation of TMSCs. Conclusion Our data demonstrate that FIR irradiation increases intracellular Ca2+ levels through TRPV2 activation, thereby suppressing the adipogenic differentiation of TMSCs.

The Impact of Moderate-Intensity Continuous or High-Intensity Interval Training on Adipogenesis and Browning of Subcutaneous Adipose Tissue in Obese Male Rats.

This study compares the effect of two types of exercise training, i.e., moderate-intensity continuous training (MICT) or high-intensity interval training (HIIT) on the browning of subcutaneous white adipose tissue (scWAT) in obese male rats. Effects on fat composition, metabolites, and molecular markers of differentiation and energy expenditure were examined. Forty male Wistar rats were assigned to lean (n = 8) or obese (n = 32) groups and fed either a standard chow or high-fat obesogenic diet for 10 weeks. Eight lean and obese rats were then blood and tissue sampled, and the remaining obese animals were randomly allocated into sedentary, MICT, or HIIT (running on a treadmill 5 days/week) groups that were maintained for 12 weeks. Obesity increased plasma glucose and insulin and decreased irisin and FGF-21. In scWAT, this was accompanied with raised protein abundance of markers of adipocyte differentiation, i.e., C/EBP-α, C/EBP-β, and PPAR-γ, whereas brown fat-related genes, i.e., PRDM-16, AMPK/SIRT1/PGC-1α, were reduced as was UCP1 and markers of fatty acid transport, i.e., CD36 and CPT1. Exercise training increased protein expression of brown fat-related markers, i.e., PRDM-16, AMPK/SIRT1/PGC-1α, and UCP1, together with gene expression of fatty acid transport, i.e., CD36 and CPT1, but decreased markers of adipocyte differentiation, i.e., C/EBP-α, C/EBP-β, and plasma glucose. The majority of these adaptations were greater with HIIT compared to MICT. Our findings indicate that prolonged exercise training promotes the browning of white adipocytes, possibly through suppression of adipogenesis together with white to beige trans-differentiation and is dependent on the intensity of exercise.

Oligomers of the lipodystrophy protein seipin may co-ordinate GPAT3 and AGPAT2 enzymes to facilitate adipocyte differentiation

Seipin deficiency causes severe congenital generalized lipodystrophy (CGL) and metabolic disease. However, how seipin regulates adipocyte development and function remains incompletely understood. We previously showed that seipin acts as a scaffold protein for AGPAT2, whose disruption also causes CGL. More recently, seipin has been reported to promote adipogenesis by directly inhibiting GPAT3, leading to the suggestion that GPAT inhibitors could offer novel treatments for CGL. Here we investigated the interactions between seipin, GPAT3 and AGPAT2. We reveal that seipin and GPAT3 associate via direct interaction and that seipin can simultaneously bind GPAT3 and AGPAT2. Inhibiting the expression of seipin, AGPAT2 or GPAT3 led to impaired induction of early markers of adipocyte differentiation in cultured cells. However, consistent with normal adipose mass in GPAT3-null mice, GPAT3 inhibition did not prevent the formation of mature adipocytes. Nonetheless, loss of GPAT3 in seipin-deficient preadipocytes exacerbated the failure of adipogenesis in these cells. Thus, our data indicate that GPAT3 plays a modest positive role in adipogenesis and argue against the potential of GPAT inhibitors to rescue white adipose tissue mass in CGL2. Overall, our study reveals novel mechanistic insights regarding the molecular pathogenesis of severe lipodystrophy caused by mutations in either seipin or AGPAT2.

Characterization of different adipose depots in fattened buffalo: histological features and expression profiling of adipocyte markers.

Adipose tissue (AT) is a multi-depot organ in mammals. AT from various depots differs in composition and function. Revealing the composition feature of AT depots will provide valuable information for further research on the development and fat deposition patterns in buffalo. This study explored the cellular morphology and gene expression profiles of brown and beige markers in seven AT depots of fattened buffalo: three subcutaneous depots (back, sternum, and inguinal) and four visceral depots (perirenal, mesenteric, pericardial, and omental). Histological results showed unilocular adipocytes in all seven AT depots. Uncoupling protein 1 (UCP1) mRNA, a brown and beige adipocyte gene, was detected in all depots with the highest level in VAT depots, and a limited number of UCP1-positive unilocular adipocytes were observed in the three VAT depots. The mRNAs of PPARG coactivator 1 alpha (PGC1 ) and transmembrane protein 26 (TMEM26), brown or beige adipocyte markers, were identified in all seven depots and were mainly expressed in VAT depots. However, the mRNA of zinc finger protein of the cerebellum 1 (ZIC1), a brown adipocyte-specific marker, was almost undetectable. Our results demonstrated that all seven AT depots are white adipose tissue (WAT), with potential function of non-shivering thermogenesis in fattened buffalo. Beige adipocytes are more active in VAT depots than in WAT depots. These results improve our knowledge on the feature of different adipose tissue depots in buffalo, which will be useful for the research of fat deposition.

Resveratrol-induced brown fat-like phenotype in 3T3-L1 adipocytes partly via mTOR pathway.

BackgroundBrowning of white adipose tissues (WAT) is recognized as a novel way to combat obesity and its related comorbidities. Thus, a lot of dietary agents contributing to browning of WAT have been identified.ObjectiveIn this study, we try to explore the mechanism of the browning of WAT induced by resveratrol (Res) in 3T3-L1 adipocytes.MethodsThe levels of cell viability and lipid accumulation were evaluated under different concentrations of Res. Cell signaling pathway analysis was performed to investigate the possible mechanisms of the WAT browning effect of Res in 3T3-L1 cells.ResultsWe found that Res induced the brown fat-like phenotype by activating protein expressions of brown adipocyte-specific markers, such as peroxisome proliferator-activated receptor gamma (PPAR-γ), peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α), and uncoupling protein 1 (UCP1). Besides, Res reduced lipid accumulation, as shown by Oil Red O staining. The increased small lipid droplets implied that Res-treated 3T3-L1 adipocytes had some features of brown adipocytes. The brown fat-like phenotype in 3T3-L1 adipocytes induced by Res was possibly mediated by activation of mammalian target of rapamycin (mTOR), as brown adipocyte-specific markers were decreased by rapamycin, an inhibitor of mTOR and the MHY1485 treatment, an activator of mTOR, showed the similar effect of Res on browning markers.ConclusionsRes induced brown-like adipocyte phenotype in 3T3-L1 adipocytes partly via mTOR pathway, which provided new insights into the utilization of Res to prevent obesity and related comorbidities.

Matrix Gla protein regulates adipogenesis and is serum marker of visceral adiposity

ABSTRACT Objective Matrix Gla protein (MGP) is a potent calcification inhibitor. Mgp-/- mice display increased proportion of brown adipose tissue. However, whether MGP is involved in fat metabolism remains unclear. This study aims to investigate the involvement. Methods Expression of adipocyte differentiation markers was examined by RT-qPCR. Adipocyte formation was assessed by Oil Red staining. Serum triglyceride, cholesterol, and desphosphorylated-uncarboxylated MGP (dp-ucMGP) were quantified by ELISA. Visceral fat was detected by bioelectrical impedance analysis. Results MGP is highly expressed in visceral fat. MGP expression is induced during preadipocyte differentiation. Knockout of MGP leads to retardation of 3T3-L1 differentiation. Intracellular triglyceride amount is impaired while glycerol release is increased in MGP-depleted cells. Serum dp-ucMGP level is significantly increased in individual with higher visceral fat index (VFI) and waist height ratio (WHtR), but not body mass index (BMI). Additionally, dp-ucMGP positively correlates to low-density lipoprotein cholesterol (LDL-C) level. Conclusions MGP is involved in fat metabolism and serum inactive MGP level is associated with visceral fat. Our study uncovers for the first time the link between MGP and fat metabolism, and sheds light on the potential of dp-ucMGP as a novel serum marker.

Developmental programming: Adipose depot-specific changes and thermogenic adipocyte distribution in the female sheep

Prenatal testosterone (T)-treated female sheep exhibit an enhanced inflammatory and oxidative stress state in the visceral adipose tissue (VAT) but not in the subcutaneous (SAT), while surprisingly maintaining insulin sensitivity in both depots. In adult sheep, adipose tissue is predominantly composed of white adipocytes which favor lipid storage. Brown/beige adipocytes that make up the brown adipose tissue (BAT) favor lipid utilization due to thermogenic uncoupled protein 1 expression and are interspersed amidst white adipocytes, more so in epicardiac (ECAT) and perirenal (PRAT) depots. The impact of prenatal T-treatment on ECAT and PRAT depots are unknown. As BAT imparts a metabolically healthy phenotype, the depot-specific impact of prenatal T-treatment on inflammation, oxidative stress, differentiation and insulin sensitivity could be dictated by the distribution of brown adipocytes. This hypothesis was tested by assessing markers of oxidative stress, inflammation, adipocyte differentiation, fibrosis and thermogenesis in adipose depots from control and prenatal T (100 mg T propionate twice a week from days 30–90 of gestation) -treated female sheep at 21 months of age. Our results show prenatal T-treatment induces depot-specific changes in inflammation, oxidative stress state, collagen accumulation, and differentiation with changes being more pronounced in the VAT. Prenatal T-treatment also increased thermogenic gene expression in all depots indicative of increased browning with effects being more prominent in VAT and SAT. Considering that inflammatory and oxidative stress are also elevated, the increased brown adipocyte distribution is likely a compensatory response to maintain insulin sensitivity and function of organs in the proximity of respective depots.

Kbtbd11 contributes to adipocyte homeostasis through the activation of upstream stimulatory factor 1

The present study aimed to investigate the transcriptional regulation of Kbtbd11 in adipose tissue. To elucidate the physiological role of Kbtbd11 gene expression, adipose Kbtbd11 mRNA expression levels were estimated under various feeding states in wild-type mice. Kbtbd11 expression increased in a time-dependent manner in the adipose tissue in mice fed on chow diet, whereas the promotion of Kbtbd11 mRNA expression by refeeding was attenuated in mice fed on high-fat (HF) diet, suggesting the suppression of Kbtbd11 mRNA expression under HF diets and that changes in mRNA levels were associated with regulation of the transcription activity of Kbtbd11 by some transcription factors. To investigate the transcriptional regulation of Kbtbd11, the fragment upstream of either mouse Kbtbd11 or human KBTBD11 promoter was inserted into a luciferase vector. Luciferase reporter assays revealed that both mouse and human KBTBD11 promoter activity was increased by USF1. Direct USF1 binding to the Ebox in the Kbtbd11 promoter was confirmed by electrophoretic mobility shift and chromatin immunoprecipitation assays. In addition, the adipocyte differentiation marker levels increased instantly in Kbtbd11-overexpressing Usf1 knockdown cells than in Usf1 knockdown cells. These results imply an association of between Kbtbd11 with Usf1 expression and suggest the involvement of Kbtbd11 in a novel adipogenesis pathway.

A novel upstream transcription factor 1 target gene N4bp2l1 that regulates adipogenesis

N4BP2l1, which is highly expressed in oral squamous cell carcinoma, is associated with poor prognosis. However, N4bp2l1's role in adipogenesis remains unknown. We aimed to clarify the expression profile and transcriptional regulation of N4bp2l1 to elucidate the functions underlying the role of N4bp2l1 in adipocyte differentiation. Our results revealed that N4bp2l1 mRNA expression increased in 3T3-L1 cells in a differentiation-dependent manner. To investigate the transcriptional regulation of N4bp2l1, the 2-kb 5′ region upstream of the mouse N4bp2l1 promoter was cloned into a luciferase vector. Luciferase reporter assays indicated that USF1 induces the N4bp2l1 promoter activity. Electrophoretic mobility shift and chromatin immunoprecipitation assays confirmed that USF1 directly binds to the Ebox in the N4bp2l1 promoter. Furthermore, the expressions of adipocyte differentiation markers significantly decreased in N4bp2l1-knockdown cells compared with those in control cells. Our results demonstrated that N4bp2l1 is a novel USF1 target gene that may be involved in adipogenesis regulation.

Effect of ACSL3 Expression Levels on Preadipocyte Differentiation in Chinese Red Steppe Cattle

Domestic cattle are an important type of livestock, with beef production playing a major role in the agricultural economy. Adipocyte levels and fat content are interrelated, with meat quality being highly dependent on its fat content and distribution. Acyl-CoA synthetases of long-chain (ACSL) fatty acids (FAs) play an integral role in virtually every metabolic pathway in mammalian biochemistry, including complex lipid biosynthesis, protein modification, and β-oxidation processes. ACSL3 activity is also known to be associated with adipocyte differentiation; however, its biological mechanism of action is currently unclear. Gene expression in subcutaneous preadipocytes isolated from subcutaneous deposits of Chinese Red Steppe cattle has been studied using in vitro cell transfection, real-time polymerase chain reaction and western blot analysis. The lipid and triglyceride contents of lipid droplets have also been measured to verify the levels of gene expression. These combined studies show that ACSL3 is induced during adipocyte differentiation, with its overexpression promoting an increase in the triglyceride content of lipid droplets. Furthermore, mRNA and protein expression levels for adipocyte differentiation marker genes, such as peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer-binding protein alpha (C/EBPα), were markedly increased during mature adipocyte cell differentiation. Knockdown of ACSL3 expression using ACSL3 small interfering RNAs (siRNAs) resulted in a decrease in lipid content of cattle adipocytes, providing further evidence that ACSL3 plays a key role in the differentiation process.

Neuromedin B receptor disruption impairs adipogenesis in mice and 3T3-L1 cells.

Neuromedin B, a bombesin-like peptide, and its receptor, are expressed in white adipose tissue with undefined roles. Female mice with disruption of neuromedin B receptor (NB-R) exhibited partial resistance to diet-induced obesity leading to our hypothesis that NB-R is involved in adipogenesis. Here, we showed that adipose stem/stromal cells (ASC) from perigonadal fat of female NB-R-knockout mice, exposed to a differentiation protocol in vitro, accumulated less lipid (45%) than wild type, suggesting reduced capacity to differentiate under adipogenic input. To further explore mechanisms, preadipocytes 3T3-L1 cells were incubated in the presence of NB-R antagonist (PD168368) during the first 3 days in culture. Cells were analyzed in the end of the treatment (Day 3) and later when fully differentiated (Day 21). NB-R antagonist induced lower number of cells at day 3 and 21 (33-39%), reduced cell proliferation at day 3 (-53%) and reduced lipid accumulation at day 21 (-86%). The mRNA expressions of several adipocyte differentiation markers were importantly reduced at both days: Cebpb and Pparg and Fabp4, Plin-1 and Adipoq, and additionally Lep mRNA at day 21. The antagonist had no effect when incubated with mature 3T3-L1 adipocytes. Therefore, genetically disruption of NB-R in mice ASC or pharmacological antagonism of NB-R in 3T3-L1 cells impairs adipogenesis. The mechanisms suggested by results in 3T3-L1 cells involve reduction of cell proliferation and of early gene expressions, leading to decreased number of mature adipocytes. We speculate that NB-R antagonism may be useful to limit the increase in adiposity due to pre-adipocyte differentiation.