Hypoxia mimetic induces lipid accumulation through mitochondrial dysfunction and stimulates autophagy in murine preadipocyte cell line

BackgroundHypoxia occurs within adipose tissue of obese human and mice. However, its role in adipose tissue regulation is still controversial.MethodsWe used murine preadipocyte 3T3-L1 cells and hypoxia was induced by using hypoxia mimetic agents, as CoCl₂. To study adipocyte differentiation, we evaluated the adipocyte markers (PPARγ, C/EBPα and aP2), and a preadipocyte marker (pref-1) by qPCR, western blotting and immunofluorescence. Lipid accumulation was evaluated by Oil red-O assay and perilipin levels by western blotting and immunofluorescence. The effect of CoCl₂ in microRNA, miR-27a and miR-27b, levels was evaluated by qPCR. We also assessed the mitochondrial membrane potential and reactive oxygen species (ROS), superoxide and ATP production. The effect of hypoxia mimetic in autophagy was determined by LC3B and p62 level evaluation by western blotting.ResultsOur results show that the hypoxia mimetic cobalt chloride increases lipid accumulation with no expression of PPARγ2. Furthermore, using qPCR we observed that the hypoxia mimetic increases microRNAs miR-27a and miR-27b, which are known to block PPARγ2 expression. In contrast, cobalt chloride induces mitochondrial dysfunction, and increases ROS production and autophagy. Moreover, an antioxidant agent, glutathione, prevents lipid accumulation induced by hypoxia mimetic indicating that ROS are responsible for hypoxia-induced lipid accumulation.ConclusionsAll these results taken together suggest that hypoxia mimetic blocks differentiation and induces autophagy. Hypoxia mimetic also induces lipid accumulation through mitochondrial dysfunction and ROS accumulation.General significanceThis study highlights the importance of adipocyte response to hypoxia, which might impair adipocyte metabolism and compromise adipose tissue function.     

MicroRNA-302a inhibits adipogenesis by suppressing peroxisome proliferator-activated receptor γ expression

The present study explored the involvement of miR-302a in adipocyte differentiation via interaction with 3′-untranslated region of peroxisome proliferator-activated receptor gamma (PPARγ) mRNA. In differentiating 3T3-L1 adipocytes, expression of miR-302a was negatively correlated with that of the adipogenic gene aP2 and PPARγ. Overexpression of miR-302a inhibited adipogenic differentiation with lipid accumulation, and inversely anti-miR-302a increased the differentiation. In silico analysis revealed a complementary region of miR-302a seed sequence in 3′-UTR of PPARγ mRNA. Luciferase assay showed the direct interaction of miR-302a with PPARγ at the cellular level. The miR-302a inhibition of adipocyte differentiation was reversed by PPARγ overexpression. These findings suggest that miR-302a might be a negative regulator of adipocyte differentiation and that the dysregulation of miR-302a should lead to metabolic disorders.     

An Evi1-C/EBPβ complex controls peroxisome proliferator-activated receptor γ2 gene expression to initiate white fat cell differentiation

Fibroblastic preadipocyte cells are recruited to differentiate into new adipocytes during the formation and hyperplastic growth of white adipose tissue. Peroxisome proliferator-activated receptor γ (PPARγ), the master regulator of adipogenesis, is expressed at low levels in preadipocytes, and its levels increase dramatically and rapidly during the differentiation process. However, the mechanisms controlling the dynamic and selective expression of PPARγ in the adipocyte lineage remain largely unknown. We show here that the zinc finger protein Evi1 increases in preadipocytes at the onset of differentiation prior to increases in PPARγ levels. Evi1 expression converts nonadipogenic cells into adipocytes via an increase in the predifferentiation levels of PPARγ2, the adipose-selective isoform of PPARγ. Conversely, loss of Evi1 in preadipocytes blocks the induction of PPARγ2 and suppresses adipocyte differentiation. Evi1 binds with C/EBPβ to regulatory sites in the Pparγ locus at early stages of adipocyte differentiation, coincident with the induction of Pparγ2 expression. These results indicate that Evi1 is a key regulator of adipogenic competency.     

Contrary Effects of BMP-2 and ATRA on Adipogenesis in Mouse Mesenchymal Fibroblasts

This study evaluates the effects of bone morphogenetic protein 2 (BMP-2) and all trans retinoic acid (ATRA) on adipogenesis in primary mouse embryo fibroblasts (MEFs). In BMP-2-treated MEFs, lipid accumulation and substantial induction of the adipocyte specific marker 442-aP2 suggested the conversion of MEFs into adipocytes. Such adipogenesis was found to be mediated through sequential induction of C/EBPα, C/EBPβ, and PPARγ. Both the BMP/Smad and BMP/p38 pathways contributed to the adipocyte differentiation. Contrary to the effects of BMP-2, ATRA was demonstrated to inhibit adipocyte differentiation in MEFs. Semi-quantitative RT-PCR analysis revealed that ATRA caused a selective inhibition of both the basal and induction levels of C/EBPα and PPARγ, without altering the expression pattern of C/EBPβ. Taken together, these data suggest the roles of BMP-2 and ATRA in adipogenic differentiation of primary MEFs, and the possible molecular mechanism that involves the regulation of C/EBPα, C/EBPβ, and PPARγ.     

Role of C/EBPβ-LAP and C/EBPβ-LIP in early adipogenic differentiation of human white adipose-derived progenitors and at later stages in immature adipocytes

We investigated the role of the major isoforms of CCAAT enhancer binding protein β (C/EBPβ), C/EBPβ-LAP and C/EBPβ-LIP, in adipogenesis of human white adipose-derived stromal/progenitor cells (ASC). C/EBPβ gene expression was transiently induced early in adipogenesis. At later stages, in immature adipocytes, the C/EBPβ mRNA and protein levels declined. The C/EBPβ-LIP protein steady-state level decreased considerably stronger than the C/EBPβ-LAP level and the C/EBPβ-LIP half-life was significantly shorter than the C/EBPβ-LAP half-life. The turn-over of both C/EBPβ-isoforms was regulated by ubiquitin/proteasome-dependent degradation. These data suggest that the protein stability of the C/EBPβ-isoforms is differentially regulated in the course of adipogenesis and in immature adipocytes. Constitutive overexpression of C/EBPβ-LIP had antiadipogenic activity in human ASC. C/EBPβ-LAP, which promotes adipogenesis in mouse 3T3-L1 preadipocytes by directly activating expression of the adipogenic keyregulator PPARγ2, induced the expression of PPARγ2 and of the adipocyte differentiation gene product FABP4 in confluent ASC in the absence of adipogenic hormones. At later stages after hormone cocktail-induced adipogenesis, in immature adipocytes, constitutive overexpression of C/EBPβ-LAP led to reduced expression of PPARγ2 and FABP4, C/EBPα expression was downregulated and the expression of the adipocyte differentiation gene products adiponectin and leptin was impaired. These findings suggest that constitutive overexpression of C/EBPβ-LAP induces adipogenesis in human ASC and negatively regulates the expression of adipogenic regulators and certain adipocyte differentiation gene products in immature adipocytes. We conclude the regulation of both C/EBPβ gene expression and C/EBPβ-LIP and C/EBPβ-LAP protein turn-over plays an important role for the expression of adipogenic regulators and/or adipocyte differentiation genes in early adipogenic differentiation of human ASC and at later stages in human immature adipocytes.     

Resveratrol inhibits cell differentiation in 3T3-L1 adipocytes via activation of AMPK

Resveratrol (Res) is a natural polyphenolic compound with anti-inflammatory and antioxidant properties. Also, Res can inhibit lipogenesis and adipocyte differentiation. However, the underlying mechanisms of Res's functions remain largely unknown. AMP-activated protein kinase (AMPK) is a key player in adipocyte differentiation. Therefore, the purpose of our study was to determine the role played by AMPK in the Res-mediated regulation of adipocyte differentiation. Incubation of 3T3-L1 cells with Res confirmed that Res inhibited adipocyte differentiation. The phosphorylation of AMPKα was increased by Res in a dose-dependent manner, while total AMPKα levels were unchanged, and peroxisome proliferator-activated receptor γ (PPARγ), CCAAT-enhancer-binding protein α (C/EBPα), and sterol regulatory element-binding protein 1c (SREBP-1c) levels were decreased. Interestingly, pretreatment with AMPKα siRNA and Res promoted adipocyte differentiation, while the decrease of p-AMPKα increased PPARγ, C/EBPα, and SREBP-1c protein expression. Our study shows that Res is capable of inhibiting lipogenesis and differentiation of 3T3-L1 adipocytes via activation of AMPK, suggesting its potential therapeutic application in the treatment or prevention of obesity.     

SREBP-1c/MicroRNA 33b Genomic Loci Control Adipocyte Differentiation

White adipose tissue (WAT) is essential for maintaining metabolic function, especially during obesity. The intronic microRNAs miR-33a and miR-33b, located within the genes encoding sterol regulatory element-binding protein 2 (SREBP-2) and SREBP-1, respectively, are transcribed in concert with their host genes and function alongside them to regulate cholesterol, fatty acid, and glucose metabolism. SREBP-1 is highly expressed in mature WAT and plays a critical role in promoting in vitro adipocyte differentiation. It is unknown whether miR-33b is induced during or involved in adipogenesis. This is in part due to loss of miR-33b in rodents, precluding in vivo assessment of the impact of miR-33b using standard mouse models. This work demonstrates that miR-33b is highly induced upon differentiation of human preadipocytes, along with SREBP-1. We further report that miR-33b is an important regulator of adipogenesis, as inhibition of miR-33b enhanced lipid droplet accumulation. Conversely, overexpression of miR-33b impaired preadipocyte proliferation and reduced lipid droplet formation and the induction of peroxisome proliferator-activated receptor γ (PPARγ) target genes during differentiation. These effects may be mediated by targeting of HMGA2, cyclin-dependent kinase 6 (CDK6), and other predicted miR-33b targets. Together, these findings demonstrate a novel role of miR-33b in the regulation of adipocyte differentiation, with important implications for the development of obesity and metabolic disease.     

Dicer has a crucial role in the early stage of adipocyte differentiation, but not in lipid synthesis, in 3T3-L1 cells

Dicer is a rate-limiting enzyme for microRNA (miRNA) synthesis. To determine the effects of Dicer on adipogenesis, we performed stage-specific knockdown of Dicer using adenovirus encoding short-hairpin RNAi against Dicer in 3T3-L1 cells. When cells were infected with the adenovirus before induction of adipocyte differentiation, Dicer RNAi suppressed the gene expression of inducers of adipocyte differentiation such as PPARγ, C/EBPα, and FAS in 3T3-L1 cells during adipocyte differentiation. Concurrently, both adipocyte differentiation and cellular lipid accumulation were cancelled by Dicer RNAi when compared with control RNAi. Meanwhile, we addressed the roles of Dicer in lipid synthesis and accumulation in the final stages of differentiation. When the differentiated cells at day 4 after induction of differentiation were infected with adenovirus Dicer RNAi, cellular lipid accumulation was unchanged. Consistent with this, Dicer RNAi had no effects on the expression of genes related to cellular lipid accumulation, including PPARγ and FAS. Thus, Dicer controls proadipogenic genes such as C/EBPα and PPARγ in the early, but not in the late, stage of adipogenesis via regulation of miRNA synthesis.     

Regulation of osteoblast and adipocyte differentiation from human mesenchymal stem cells by conjugated linoleic acid

Conjugated linoleic acid (CLA) describes a group of isomers of linoleic acid and has variable effects on bone formation and adiposity in vivo and in vitro. The variability may be due to individual effects of the predominant bioactive 9cis,11trans (9,11) and 10trans,12cis (10,12) CLA isomers. Osteoblasts and adipocytes are derived from mesenchymal stem cells (MSCs), and bone loss is accompanied by an increase in marrow adiposity. Osteoblast differentiation from MSCs requires activation of Wnt/β-catenin signaling by Wnt10b, which inhibits adipocyte differentiation by suppressing CCAAT/enhancer-binding protein (C/EBP) α. The objective of this study was to determine if 9,11 and 10,12 CLA affect osteoblast and adipocyte differentiation from MSCs and to determine whether any effects are associated with changes in Wnt10b and C/EBPα expression. Osteoblast differentiation was assessed by calcium deposition, alkaline phosphatase (ALP) activity, and the expression of Wnt10b, runx2 and osteocalcin. Adipocyte differentiation was assessed by oil red O staining and C/EBPα, PPARγ and FABP4 expression. Compared to vehicle, 9,11 CLA decreased calcium deposition (～15%), increased oil red O staining (～21-28%) and increased FABP4 (AP2) expression (～58-75%). In contrast, 10,12 CLA increased calcium deposition (～12-60%), ALP activity (～2.1-fold) and the expression of Wnt10b (～60-80%) and osteocalcin (～90%), but decreased oil red O staining (～30%) and the expression of C/EBPα (～24-38%) and PPARγ (～60%) (P<.05). Thus, our findings demonstrate isomer-specific effects of CLA on MSC differentiation, and suggest that 10,12 CLA may be a useful therapeutic agent to promote osteoblast differentiation from MSCs.     

Fenoxycarb promotes adipogenesis in 3T3‐L1 preadipocytes

Lipophilic insect hormones and their analogs affect mammalian physiology by regulating the expression of metabolic genes. Therefore, we determined the effect of fenoxycarb, a juvenile hormone analog, on lipid metabolism in adipocytes. Here, we demonstrated that fenoxycarb dose‐dependently promoted lipid accumulation in 3T3‐L1 adipocytes during adipocyte differentiation and that its lipogenic effect was comparable to that of rosiglitazone, a well‐known ligand for peroxisome proliferator‐activated receptor gamma (PPARγ). Furthermore, fenoxycarb stimulated PPARγ activity without affecting other nuclear receptors, such as liver X receptor (LXR), farnesoid X‐activated receptor (FXR) and Nur77. In addition, fenoxycarb treatment increased the expression of PPARγ and fatty acid transporter protein 1 (FATP1) in 3T3‐L1 adipocytes, suggesting that fenoxycarb may facilitate adipocyte differentiation by enhancing PPARγ signaling, the master regulator of adipogenesis. Together, our results suggest that fenoxycarb promoted lipid accumulation in adipocytes, in part, by stimulating PPARγ.     

miR-27a is a negative regulator of adipocyte differentiation via suppressing PPARγ expression

microRNAs (miRNAs) are non-coding small RNAs regulating gene expression, cell growth, and differentiation. Although several miRNAs have been implicated in cell growth and differentiation, it is barely understood their roles in adipocyte differentiation. In the present study, we reveal that miR-27a is involved in adipocyte differentiation by binding to the PPARγ 3′-UTR whose sequence motifs are highly conserved in mammals. During adipogenesis, the expression level of miR-27a was inversely correlated with that of adipogenic marker genes such as PPARγ and adiponectin. In white adipose tissue, miR-27a was more abundantly expressed in stromal vascular cell fraction than in mature adipocyte fraction. Ectopic expression of miR-27a in 3T3-L1 pre-adipocytes repressed adipocyte differentiation by reducing PPARγ expression. Interestingly, the level of miR-27a in mature adipocyte fraction of obese mice was down-regulated than that of lean mice. Together, these results suggest that miR-27a would suppress adipocyte differentiation through targeting PPARγ and thereby down-regulation of miR-27a might be associated with adipose tissue dysregulation in obesity.     

Asiatic Acid Inhibits Adipogenic Differentiation of Bone Marrow Stromal Cells

Bone marrow mesenchymal stromal cells (BMSCs) are the common precursors for both osteoblasts and adipocytes. With aging, BMSC osteoblast differentiation decreases whereas BMSC differentiation into adipocytes increases, resulting in increased adipogenesis and bone loss. In the present study, we investigated the effect of asiatic acid (AA) on adipocytic differentiation of BMSCs. AA inhibited the adipogenic induction of lipid accumulation, activity of glycerol-3-phosphate dehydrogenase, and expression of marker genes in adipogenesis: peroxisome proliferation-activated receptor (PPAR)γ, adipocyte fatty acid-binding protein (ap) 2, and adipsin. Further, we found that AA did not alter clonal expansion rate and expression of C/EBPβ, upstream key regulator of PPARγ, and binding activity of C/EBPβ to PPARγ promoter was not affected by AA as well. These findings suggest that AA may modulate differentiation of BMSCs to cause a lineage shift away from the adipocytes, and inhibition of PPARγ by AA is through C/EBPβ-independent mechanisms. Thus, AA could be a potential candidate for a novel drug against osteoporosis.     

Effects of fermented rooibos (Aspalathus linearis) on adipocyte differentiation

Rooibos (Aspalathus linearis) contains a rich complement of polyphenols, including flavonoids, considered to be largely responsible for its health promoting effects, including combatting obesity. The purpose of this study was to examine the effect of fermented rooibos hot water soluble solids on in vitro adipocyte differentiation by using differentiating 3T3-L1 adipocytes. Hot water soluble solids were obtained when preparing an infusion of fermented rooibos at “cup-of-tea” strength. The major phenolic compounds (>5 mg/g) were isoorientin, orientin, quercetin-3-O-robinobioside and enolic phenylpyruvic acid-2-O-β-d-glucoside. Treatment of 3T3-L1 adipocytes with 10 μg/ml and 100 μg/ml of the rooibos soluble solids inhibited intracellular lipid accumulation by 22% (p < 0.01) and 15% (p < 0.05), respectively. Inhibition of adipogenesis was accompanied by decreased messenger RNA (mRNA) expression of PPARγ, PPARα, SREBF1 and FASN. Western blot analysis exhibited decreased PPARα, SREBF1 and AMPK protein expression. Impeded glycerol release into the culture medium was observed after rooibos treatment. None of the concentrations of rooibos hot water soluble solids was cytotoxic, in terms of ATP content. Interestingly, the higher concentration of hot water soluble solids increased ATP concentrations which were associated with increased basal glucose uptake. Decreased leptin secretion was observed after rooibos treatment. Our data show that hot water soluble solids from fermented rooibos inhibit adipogenesis and affect adipocyte metabolism, suggesting its potential in preventing obesity.