Caudatin suppresses adipogenesis in 3T3-L1 adipocytes and reduces body weight gain in high-fat diet-fed mice through activation of hedgehog signaling

BackgroundThe regulative effects of caudatin, a C-21 steroid that is identified from Cynanchum bungee roots, on adipogenesis and obesity have not been studied. Many studies have demonstrated that the activation of hedgehog (Hh) signaling can help prevent obesity. Therefore, we hypothesized that caudatin can inhibit adipogenesis and obesity via activating the Hh signaling pathway.MethodsTo investigate the effects of caudatin on adipogenesis in 3T3-L1 preadipocytes and high-fat diet induced obesity in C57BL/6 mice, in vitro and in vivo experiments were performed. For in vitro evaluation, Oil red O staining were used to represent lipid accumulation in differentiated 3T3-L1 adipocytes. For in vivo assessment, male 5 week-old C57BL/6 mice were fed with standard chow diet, high-fat diet (HFD), HFD with 25 mg/kg caudatin, HFD with 1mg/kg purmorpharmine for 10 weeks, respectively. Hh signaling and key adipogenic marker involved in adipogenesis were evaluated by real-time PCR and western blot. The adipocyte size of white adopose tissue and lipid storage of liver were visualized by hematoxylin and eosin staining. In addition, the expression of Gli1 and peroxisome proliferator-activated receptor γ (PPARγ) in white adipose tissue were investigated by immunohistochemistry staining.ResultsCaudatin suppressed the accumulation of lipid droplets and downregulated the expression of key adipogenic factors, i.e., peroxisome proliferator-activated receptor γ PPARγ and CCAAT-enhancer binding protein α (C/EBPα), through activating Hh signaling in differentiated 3T3-L1 cells. Furthermore, caudatin and the Hh activator purmorpharmine significantly decreased body weight gain and white adipose tissue (WAT) weight in HFD-induced mice and affected adipogenic markers and Hh signaling mediators in WAT, which were in line with the in vitro experimental results.ConclusionTo our best knowledge, it is the first report to demonstrate that caudatin downregulated adipocyte differentiation and suppressed HFD-induced body weight gain through activating the Hh signaling pathway, suggesting that caudatin can potentially counteract obesity.     

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

Polyamines spermidine and spermine are known to be required for mammalian cell proliferation and for embryonic development. Alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase (ODC) a limiting enzyme of polyamine biosynthesis, depleted the cellular polyamines and prevented triglyceride accumulation and differentiation in 3T3-L1 cells. In this study, to explore the function of polyamines in adipogenesis, we examined the effect of polyamine biosynthesis inhibitors on adipocyte differentiation and lipid accumulation of 3T3-L1 cells. The spermidine synthase inhibitor trans-4-methylcyclohexylamine (MCHA) increased spermine/spermidine ratios, whereas the spermine synthase inhibitor N-(3-aminopropyl)-cyclohexylamine (APCHA) decreased the ratios in the cells. MCHA was found to decrease lipid accumulation and GPDH activity during differentiation, while APCHA increased lipid accumulation and GPDH activity indicating the enhancement of differentiation. The polyamine-acetylating enzyme, spermidine/spermine N ¹-acetyltransferase (SSAT) activity was increased within a few hours after stimulus for differentiation, and was found to be elevated by APCHA. In mature adipocytes APCHA decreased lipid accumulation while MCHA had the opposite effect. An acetylpolyamine oxidase and spermine oxidase inhibitor MDL72527 or an antioxidant N-acetylcysteine prevented the promoting effect of APCHA on adipogenesis. These results suggest that not only spermine/spermidine ratios but also polyamine catabolic enzyme activity may contribute to adipogenesis.     

LMO4 modulates proliferation and differentiation of 3T3-L1 preadipocytes

Previous microarray analyses revealed that LMO4 is expressed in 3T3-L1 preadipocytes, however, its roles in adipogenesis are unknown. In the present study, using RT-PCR sequencing and quantitative real-time RT-PCR, we confirmed that LMO4 gene is expressed in 3T3-L1 preadipocytes and its expression peaks at the early stage of 3T3-L1 preadipocyte differentiation. Further analyses showed that LMO4 knockdown decreased the proliferation of 3T3-L1 preadipocytes, and attenuated the differentiation of 3T3-L1 preadipocytes, as evidenced by reduced lipid accumulation and down-regulation of PPARγ gene expression. Collectively, our findings indicate that LMO4 is a novel modulator of adipogenesis.     

Blueberry Peel Extracts Inhibit Adipogenesis in 3T3-L1 Cells and Reduce High-Fat Diet-Induced Obesity

This study examined the anti-obesity effect and mechanism of action of blueberry peel extracts (BPE) in 3T3-L1 cells and high-fat diet (HFD)-induced obese rats. The levels of lipid accumulation were measured, along with the changes in the expression of genes and proteins associated with adipocyte differentiation in 3T3-L1 cells. Evidenced by Oil-red O staining and triglyceride assay, BPE dose-dependently inhibited lipid accumulation at concentrations of 0, 50, and 200 µg/ml. BPE decreased the expression of the key adipocyte differentiation regulator C/EBPβ, as well as the C/EBPα and PPARγ genes, during the differentiation of preadipocytes into adipocytes. Moreover, BPE down-regulated adipocyte-specific genes such as aP2 and FAS compared with control adipocytes. The specific mechanism mediating the effects of BP revealed that insulin-stimulated phosphorylation of Akt was strongly decreased, and its downstream substrate, phospho-GSK3β, was downregulated by BPE treatment in 3T3-L1 cells. Together, these data indicated that BP exerted anti-adipogenic activity by inhibiting the expression of PPARγ and C/EBPβ and the Akt signaling pathway in 3T3-L1 adipocytes. Next, we investigated whether BP extracts attenuated HFD-induced obesity in rats. Oral administration of BPE reduced HFD-induced body weight gain significantly without affecting food intake. The epididymal or perirenal adipose tissue weights were lower in rats on an HFD plus BPE compared with the tissue weights of HFD-induced obese rats. Total cholesterol and triglyceride levels in the rats fed BPE were modestly reduced, and the HDL-cholesterol level was significantly increased in HFD plus BP-fed rats compared with those of HFD-fed rats. Taken together, these results demonstrated an inhibitory effect of BP on adipogenesis through the down-regulation of C/EBPβ, C/EBPα, and PPARγ and the reduction of the phospho-Akt adipogenic factor in 3T3-L1 cells. Moreover, BPE reduced body weight gain and inhibited fat accumulation in an HFD-induced animal model of obesity.     

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.     

Anti-adipogenesis and metabolism-regulating effects of heat-inactivated Streptococcus thermophilus MN-ZLW-002

Metabolic syndrome and obesity have become serious threats to public health worldwide. This study was conducted to evaluate the anti-adipogenesis and metabolism-regulating effects of heat-inactivated Streptococcus thermophilus MN-ZLW-002 (MN-ZLW-002), which can be used as a yogurt starter. In vitro study suggested that MN-ZLW-002 stimulated the RAW264.7 macrophages to produce significant amounts of interleukin (IL)-6, IL-10 and tumour necrosis factor (TNF)-α and induced intense phosphorylation of P38, p44/42 MAPK and nuclear factor κB. MN-ZLW-002-stimulated RAW264.7-conditioned medium (CM) notably suppressed the differentiation and adipogenesis of 3T3-L1 pre-adipocytes. The 12-week in vivo study suggested that orally administered MN-ZLW-002 significantly reduced the weight gain of mice caused by the high-fat diet (HFD) at weeks 3–8; decreased fasting blood glucose levels at week 4 and week 8; decreased serum total triglyceride level at week 12. MN-ZLW-002 also reduced serum IL-1β and chemokine ligand 3 levels in the HFD-fed mice. These findings suggest that heat-inactivated MN-ZLW-002 can suppress adipocytes differentiation and lipid accumulation by regulating the immune response, possibly via the release of cytokines, particularly TNF-α; MN-ZLW-002 can improve metabolism-related indicators in the early stage of HFD intervention and regulate the related pro-inflammatory immune response.     

miR-128-3p regulates 3T3-L1 adipogenesis and lipolysis by targeting <Emphasis Type="Italic">Pparg</Emphasis> and <Emphasis Type="Italic">Sertad2</Emphasis>

Differentiation of adipocytes and their aggregation to adipose tissue are critical for mammalian growth and development. MicroRNAs (miRNAs) are a class of endogenous small non-coding RNAs that play important roles in adipogenesis and lipid metabolism. miR-128-3p may contribute to adipose tissue development according to the previous studies. However, the role of miR-128-3p in the process of preadipocyte differentiation and lipid metabolism is not yet understood. The purpose of this research was to investigate the biological function and molecular mechanism of miR-128-3p in 3T3-L1 cells. In the present study, we found that miR-128-3p was downregulated during the process of 3T3-L1 preadipocyte differentiation. Overexpression of miR-128-3p obstructed the expressions of adipogenic marker genes as well as the lipid droplets accumulation and triglyceride content, suggesting the importance of miR-128-3p for adipogenesis. Moreover, miR-128-3p could lead to the retardation of cell proliferation in 3T3-L1 preadipocytes. Further evidences showed that, as a negative regulator of adipogenesis, miR-128-3p could directly target peroxisome proliferator-activated receptor γ (Pparg) which resulted in the suppression of 3T3-L1 preadipocyte differentiation, and miR-128-3p could also bind with SERTA domain containing 2 (Sertad2) which drove triglyceride hydrolysis and lipolysis. In addition, inhibition of Sertad2 with siRNA displayed the same effects as overexpression of miR-128-3p. Our research demonstrated that miR-128-3p impeded 3T3-L1 adipogenesis by targeting Pparg and Sertad2, resulting in the obstruction of preadipocyte differentiation and promotion of lipolysis. Taken together, this study offers profound insight into the mechanism of miRNA-mediated adipogenesis and lipid metabolism.     

N-Caffeoyltryptophan enhances adipogenic differentiation in preadipocytes and improves glucose tolerance in mice

Coffee consumption has been shown to reduce the risk of developing type 2 diabetes mellitus (T2DM) in humans; however, the exact mechanism is not completely understood. Here, we demonstrate that N-caffeoyltryptophan (CTP), an ingredient of coffee, enhances adipogenic differentiation and promotes glucose uptake into adipocytes. CTP increased lipid accumulation and adipogenic markers (PPARγ, C/EBPα, and FABP4) expression in mouse 3T3-L1 preadipocyte cell lines and primary preadipocytes. In addition, CTP promoted glucose uptake in 3T3-L1 cells. In the oral glucose tolerance test, daily administration of CTP (30 mg/kg/day, i.p.) for a week reduced blood glucose levels in mice. In 3T3-L1 cells, adipogenic differentiation and increased adipogenic markers expression induced by CTP were inhibited by U0126, a selective MEK1/2 inhibitor. Furthermore, mRNA induction of Pparg by CTP was abrogated in SIRT1 siRNA-transfected 3T3-L1 cells. These results suggest the involvement of the MEK/ERK signaling and SIRT1 in the mechanism of adipogenic function of CTP. Taken together, CTP might contribute to the reduction in postprandial glycemia and a subsequent reduction in onset risk for T2DM.     

Obesity Resistance and Enhanced Insulin Sensitivity in Ahnak-/- Mice Fed a High Fat Diet Are Related to Impaired Adipogenesis and Increased Energy Expenditure

ObjectiveRecent evidence has suggested that AHNAK expression is altered in obesity, although its role in adipose tissue development remains unclear. The objective of this study was to determine the molecular mechanism by which Ahnak influences adipogenesis and glucose homeostasis.DesignWe investigated the in vitro role of AHNAK in adipogenesis using adipose-derived mesenchymal stem cells (ADSCs) and C3H10T1/2 cells. AHNAK-KO male mice were fed a high-fat diet (HFD; 60% calories from fat) and examined for glucose and insulin tolerances, for body fat compositions, and by hyperinsulinemic-euglycemic clamping. Energy expenditures were assessed using metabolic cages and by measuring the expression levels of genes involved in thermogenesis in white or brown adipose tissues.ResultsAdipogenesis in ADSCs was impaired in AHNAK-KO mice. The loss of AHNAK led to decreased BMP4/SMAD1 signaling, resulting in the downregulation of key regulators of adipocyte differentiation (P<0.05). AHNAK directly interacted with SMAD1 on the Pparγ2 promoter. Concomitantly, HFD-fed AHNAK-KO mice displayed reduced hepatosteatosis and improved metabolic profiles, including improved glucose tolerance (P<0.001), enhanced insulin sensitivity (P<0.001), and increased energy expenditure (P<0.05), without undergoing alterations in food intake and physical activity.ConclusionAHNAK plays a crucial role in body fat accumulation by regulating adipose tissue development via interaction with the SMAD1 protein and can be involved in metabolic homeostasis.     

Phoenixin-14 stimulates differentiation of 3T3-L1 preadipocytes via cAMP/Epac-dependent mechanism

Phoenixin-14 (PNX) is a newly discovered peptide produced by proteolytic cleavage of the small integral membrane protein 20 (Smim20). Previous studies showed that PNX is involved in controlling reproduction, pain, anxiety and memory. Furthermore, in humans, PNX positively correlates with BMI suggesting a potential role of PNX in controlling fat accumulation in obesity. Since the influence of PNX on adipose tissue formation has not been so far demonstrated, we investigated the effects of PNX on proliferation and differentiation of preadipocytes using 3T3-L1 and rat primary preadipocytes. We detected Smim20 and Gpr173 mRNA in 3T3-L1 preadipocytes as well as in rat primary preadipocytes. Furthermore, we found that PNX peptide is produced and secreted from 3T3-L1 and rat primary adipocytes. PNX increased 3T3-L1 preadipocytes proliferation and viability. PNX stimulated the expression of adipogenic genes (Pparγ, C/ebpβ and Fabp4) in 3T3-L1 adipocytes. 3T3-L1 preadipocytes differentiated in the presence of PNX had increased lipid content. Stimulation of cell proliferation and differentiation by PNX was also confirmed in rat preadipocytes. PNX failed to induce AKT phosphorylation, however, PNX increased cAMP levels in 3T3-L1 cells. Suppression of Epac signalling attenuated PNX-induced Pparγ expression without affecting cell proliferation. Our data show that PNX stimulates differentiation of 3T3-L1 and rat primary preadipocytes into mature adipocytes via cAMP/Epac-dependent pathway. In conclusion our data shows that phoenixin promotes white adipogenesis, thereby may be involved in controlling body mass regulation.     

A water-soluble extract of Petalonia binghamiae inhibits the expression of adipogenic regulators in 3T3-L1 preadipocytes and reduces adiposity and weight gain in rats fed a high-fat diet

We previously showed that an ethanolic extract of the edible brown algae Petalonia binghamiae promotes the differentiation of 3T3-L1 preadipocytes and decreases hyperglycemia in streptozotocin-induced diabetic mice. Here, we report that a water-soluble extract of P. binghamiae thalli, prepared by enzymatic digestion, inhibits preadipocyte differentiation and adipogenesis in a dose-dependent manner. In differentiating 3T3-L1 preadipocytes, the extract (designated PBEE) decreased the expression of peroxisome proliferator-activated receptor γ, CCAAT/enhancer-binding proteins α and β, and fatty acid-binding protein aP2. It also inhibited the mitotic clonal expansion process of adipocyte differentiation, and it inhibited insulin-stimulated uptake of glucose into mature 3T3-L1 adipocytes by reducing phosphorylation of insulin receptor substrate-1. In rats with high-fat diet (HFD)-induced obesity, PBEE exhibited potent anti-obesity effects. In this animal model, increases in body weight and fat storage were suppressed by the addition of PBEE to the drinking water at 500 mg/L for 30 days. PBEE supplementation reduced serum levels of glutamic pyruvic and glutamic oxaloacetic transaminases and increased the serum level of high-density lipoprotein cholesterol. Moreover, it significantly decreased the accumulation of lipid droplets in liver tissue, suggesting a protective effect against HFD-induced hepatic steatosis. Taken together, these data demonstrate that PBEE inhibits preadipocyte differentiation and adipogenesis in cultured cells and in rodent models of obesity.     

Trans10, cis12 conjugated linoleic acid inhibits 3T3-L1 adipocyte adipogenesis by elevating β-catenin levels

BackgroundTrans-10, cis-12 (t10-c12) CLA treatment reduces lipid accumulation in differentiating mouse and human adipocytes, and decreases fat mass in mice, yet the mechanism of action remains unknown.ObjectiveThis study investigated the effect of the cis-9, trans-11 (c9-t11) and t10-c12 CLA isomers on the Wnt/β-catenin pathway, which has been reported to inhibit adipogenesis by down-regulating PPARγ.ResultsWe observed that t10-c12 CLA treatment of 3T3-L1 adipocytes increases the levels of β-catenin and Ser-675 phosphorylated β-catenin due to inhibition of its degradation. These changes in β-catenin were not linked to either the Wnt/β-catenin agonist Wnt10b or other upstream effectors such as SFRP-5. Paradoxically, the presence of higher amounts of β-catenin did not elevate cyclin D1 levels, which is recognized as a critical target gene. Neither of the CLA isomers affected the localization of β-catenin in the cytosol and nucleus as determined by immunofluorescence microscopy. However, subcellular fractionation suggested the level of cytosolic β-catenin was reduced in t10-c12 CLA treated cells. Immunoprecipitation revealed that t10-c12 CLA increased the interaction of β-catenin and PPARγ.Conclusionst10-c12-CLA inhibits adipocyte differentiation by increasing β-catenin stability in 3T3-L1 adipocytes, thus enhancing sequestration of PPARγ in an inactive complex, which prevents progression of adipogenesis.     

Benzyl isothiocyanate ameliorates lipid accumulation in 3T3-L1 preadipocytes during adipocyte differentiation

ABSTRACT

Benzyl isothiocyanate (BITC) is an organosulfur compound derived from cruciferous vegetables and papaya seeds. In this study, we investigated the effect of BITC on the lipid accumulation in 3T3-L1 preadipocytes during adipocyte differentiation. The treatment of BITC during the differentiation-inducing stage significantly ameliorated the lipid accumulation, whereas it had no inhibitory effect during the differentiation-maintaining stage. BITC also significantly suppressed the mRNA expression of the adipocyte-specific markers, such as CCAAT/enhancer-binding protein α (C/EBPα), C/EBPβ, C/EBPδ and peroxisome proliferator-activated receptor γ. BITC significantly inhibited the phosphorylation of extracellular signal-regulated kinase phosphorylation, whereas it enhanced that of AMP-activated protein kinase. Furthermore, BITC significantly suppressed the intracellular 2-deoxyglucose uptake as well as glucose transporter 4 expression. These results suggest that inhibition of the adipocyte differentiation and glucose uptake may mainly contribute to the inhibitory effect of BITC on the lipid accumulation in 3T3-L1 preadipocytes.

Abbreviations: PPARγ: peroxisome proliferator-activated receptor γ; CEBP: CCAAT/enhancer-binding protein; GLUT4: glucose transporter 4; AMPK: AMP-activated protein kinase; ERK1/2: extracellular signal-regulated kinase 1/2; MAPK: a mitogen-activated protein kinase; ITCs: isothiocyanates; BITC: benzyl isothiocyanate; FBS: fetal bovine serum; CS: calf serum; AITC: allyl ITC; IBMX: 3-isobutyl-1-methylxanthine; LDH: lactate dehydrogenase; KRH: Krebs-Ringer-Hepes-bicarbonate; 2-DG: 2-deoxy-d-glucose     

Regulation of adipocytokine secretion and adipocyte hypertrophy by polymethoxyflavonoids, nobiletin and tangeretin

AimsThe polymethoxyflavonoids nobiletin and tangeretin possess several important biological properties such as neuroprotective, antimetastatic, anticancer, and anti-inflammatory properties. The present study was undertaken to examine whether nobiletin and tangeretin could modulate adipocytokine secretion and to evaluate the effects of these flavonoids on the hypertrophy of mature adipocytes.Main methodsAll experiments were performed on the murine preadipocyte cell line 3T3-L1. We studied the formation of intracellular lipid droplets in adipocytes and the apoptosis-inducing activity to evaluate the effects of polymethoxyflavonoids on adipocyte differentiation and hypertrophy, respectively. The secretion of adipocytokines was measured using ELISA.Key findingsWe demonstrated that the combined treatment of differentiation reagents with nobiletin or tangeretin differentiated 3T3-L1 preadipocytes into adipocytes possessing less intracellular triglyceride as compared to vehicle-treated differentiated 3T3-L1 adipocytes. Both flavonoids increased the secretion of an insulin-sensitizing factor, adiponectin, but concomitantly decreased the secretion of an insulin-resistance factor, MCP-1, in 3T3-L1 adipocytes. Furthermore, nobiletin was found to decrease the secretion of resistin, which serves as an insulin-resistance factor. In mature 3T3-L1 adipocytes, nobiletin induced apoptosis; tangeretin, in contrast, did not induce apoptosis, but suppressed further triglyceride accumulation.SignificanceOur results suggest that nobiletin and tangeretin are promising therapeutic candidates for the prevention and treatment of insulin resistance by modulating the adipocytokine secretion balance. We also demonstrated the different effects of nobiletin and tangeretin on mature adipocytes.