Effects of 4-nonylphenol on adipogenesis in 3T3-L1 preadipocytes and C3H/10T1/2 mesenchymal stem cells

Obesogens are a subset of endocrine disruptor chemicals (EDCs) that cause obesity. The typical EDC 4-nonylphenol (4-NP) has been identified as an obesogen. However, the in vitro effects of 4-NP on adipogenesis remain unclear. In this study, 3T3-L1 preadipocytes and C3H/10T1/2 mesenchymal stem cells (MSCs) were used to investigate the influence of 4-NP on adipogenesis. The differentiation protocols for 3T3-L1 preadipocytes and C3H/10T1/2 MSCs took 8 and 12 days, respectively, beginning at Day 0. In differentiated 3T3-L1 preadipocytes, 20 μM 4-NP decreased cell viability on Days 4 and 8. Exposure to 4-NP inhibited triglyceride (TG) accumulation and adipogenic marker expression on Days 0–8, but the inhibitory effects were weaker on Days 2–8. The protein expression of pSTAT3 or STAT3 decreased on Days 0–8 and 2–8. Conversely, 4-NP promoted TG accumulation and the adipogenic marker expression in C3H/10T1/2 adipocytes. The opposing effects were attributed to physiological differences between the two cell lines. The 3T3-L1 preadipocytes are dependent on mitotic clonal expansion (MCE) to drive differentiation, while C3H/10T1/2MSCs and human preadipocytes are not. Additionally, 4-NP downregulated β-catenin expression in C3H/10T1/2 adipocytes. Accordingly, we hypothesized that 4-NP promotes adipogenesis. The role of the canonical Wnt pathway in the promotion of adipogenesis by 4-NP requires further validation. This study provides new insights into the mechanisms and appropriate risk management of 4-NP.     

Ultrasound stimulates cyclooxygenase-2 expression and increases bone formation through integrin, focal adhesion kinase, phosphatidylinositol 3-kinase, and Akt pathway in osteoblasts

It has been shown that ultrasound (US) stimulation accelerates fracture healing in animal models and in clinical studies. Here we found that US stimulation transiently increased the surface expression of alpha2, alpha5, beta1, and beta3 integrins in cultured osteoblasts, as shown by flow cytometric analysis and immunofluorescence staining. US stimulation increased prostaglandin E(2) formation and the protein and mRNA levels of cyclooxygenase-2 (COX-2). At the mechanistic level, anti-integrin alpha5beta1 and alphavbeta3 antibodies or rhodostomin, a snake venom disintegrin, attenuated the US-induced COX-2 expression. Phosphatidylinositol 3-kinase (PI3K) inhibitors 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002) and wortmannin also inhibited the potentiating action of US. US stimulation increased the phosphorylation of focal adhesion kinase (FAK), extracellular signal-regulated kinases (ERK), p85 subunit of PI3K, and serine 473 of Akt. COX-2 promoter activity was enhanced by US stimulation in cells transfected with pCOX2-Luc. Cotransfection with dominant-negative mutant of FAK(Y397F), p85(Deltap85), Akt(K179A), or ERK2(K52R) inhibited the potentiating action of US on COX-2 promoter activity. Expression of mineralized nodule was lower in dominant-negative mutants of FAK, p85, and Akt-transfected clones than in vector-transfected control cells. Taken together, our results provide evidence that US stimulation increases COX-2 expression and promotes bone formation in osteoblasts via the integrin/FAK/PI3K/Akt and ERK signaling pathway.     

Inhibition of mitotic clonal expansion mediates fisetin-exerted prevention of adipocyte differentiation in 3T3-L1 cells

Adipocytes are the key player in adipose tissue inflammation and subsequent systemic insulin resistance and its development involves complex process of proliferation and differentiation of preadipocytes. Fistein, a polyphenol flavonoid, is known to exert anti-inflammatory, anti-carcinogenic and anti-diabetic effects. In this study, we aimed to investigate the effect of fisetin on adipocyte proliferation and differentiation in 3T3-L1 preadipocyte cell line and its mechanism of action. We found that fisetin inhibits adipocyte differentiation in a concentration dependent manner, which were evidenced by Oil Red O staining and the protein expression of mature adipocyte marker genes fatty acid synthase and peroxisome proliferator-activated receptor γ. Moreover, the proliferation of preadipocytes was also markedly suppressed by treatment of fisetin for 24 and 48 h in the differentiation medium. We also found that fisetin inhibition of adipocyte differentiation was largely due to the effect on mitotic clonal expansion. Fisetin suppression of preadipocyte proliferation at early stage of differentiation was accompanied by the changes of expression of a series of cell cycle regulatory proteins. Altogether, our results suggest that the inhibition of adipocyte differentiation by fisetin may be at least in part mediated by cell cycle arrest during adipogenesis.     

Direct adipotropic actions of atorvastatin: differentiation state-dependent induction of apoptosis, modulation of endocrine function, and inhibition of glucose uptake

Statins exert anti-inflammatory, anti-atherogenic actions. The mechanisms responsible for these effects remain only partially elucidated. Diabetes and obesity are characterized by low-grade inflammation. Metabolic and endocrine adipocyte dysfunction is known to play a crucial role in the development of these disorders and the related cardiovascular complications. Thus, direct modulation of adipocyte function may represent a mechanism of pleiotropic statin actions. We investigated effects of atorvastatin on apoptosis, differentiation, endocrine, and metabolic functions in murine white and brown adipocyte lines. Direct exposure of differentiating preadipocytes to atorvastatin strongly reduced lipid accumulation and diminished protein expression of the differentiation marker CCAAT/enhancer binding protein-beta (CEBP-beta). In fully differentiated adipocytes, however, lipid accumulation remained unchanged after chronic atorvastatin treatment. Furthermore, cell viability was reduced in response to atorvastatin treatment in proliferating and differentiating preadipocytes, but not in differentiated cells. Moreover, atorvastatin induced apoptosis and inhibited protein kinase B (AKT) phosphorylation in proliferating and differentiating preadipocytes, but not in differentiated adipocytes. On the endocrine level, direct atorvastatin treatment of differentiated white adipocytes enhanced expression of the pro-inflammatory adipokine interleukin-6 (IL-6), and downregulated expression of the insulin-mimetic and anti-inflammatory adipokines visfatin and adiponectin. Finally, these direct adipotropic endocrine effects of atorvastatin were paralleled by the acute inhibition of insulin-induced glucose uptake in differentiated white adipocytes, while protein expression of the thermogenic uncoupling protein-1 (UCP-1) in brown adipocytes remained unchanged. Taken together, our data for the first time demonstrate direct differentiation state-dependent effects of atorvastatin including apoptosis, modulation of pro-inflammatory and glucostatic adipokine expression, and insulin resistance in adipose cells. These differential interactions may explain variable clinical observations.     

The cannabinoid CB1 receptor antagonist rimonabant (SR141716) inhibits cell proliferation and increases markers of adipocyte maturation in cultured mouse 3T3 F442A preadipocytes

Adipocyte cell proliferation is an important process in body fat mass development in obesity. Adiponectin or Acrp30 is an adipocytokine exclusively expressed and secreted by adipose tissue that regulates lipid and glucose metabolism and plays a key role in body weight regulation and homeostasis. Adiponectin mRNA expression in adipose tissue and plasma level of adiponectin are decreased in obesity and type 2 diabetes. In obese rodents, the selective CB(1) receptor antagonist rimonabant reduces food intake and body weight and improves lipid and glucose parameters. We have reported previously that rimonabant stimulated adiponectin mRNA expression in adipose tissue of obese fa/fa rats, by a direct effect on adipocytes. We report here that rimonabant (10-400 nM) inhibits cell proliferation of cultured mouse 3T3 F442A preadipocytes in a concentration-dependent manner. In parallel to this inhibitory effect on preadipocyte cell proliferation, rimonabant (25-100 nM) stimulates mRNA expression and protein levels of two late markers of adipocyte differentiation (adiponectin and glyceraldehyde-3-phosphate dehydrogenase) with a maximal effect at 100 nM, without inducing the accumulation of lipid droplets. Furthermore, treatment of mouse 3T3 F442A preadipocytes with rimonabant (100 nM) inhibits basal and serum-induced p42/44 mitogen-activated protein (MAP) kinase activity. These results suggest that inhibition of MAP kinase activity by rimonabant may be one of mechanisms involved in the inhibition of 3T3 F442A preadipocyte cell proliferation and stimulation of adiponectin and GAPDH expression. The inhibition of preadipocyte cell proliferation and the induction of adipocyte late "maturation" may participate in rimonabant-induced antiobesity effects, particularly the reduction of body fat mass.     

The anti-angiogenic herbal extracts Ob-X from Morus alba,Melissa officinalis,and Artemisia capillaris suppresses adipogenesis in 3T3-L1 adipocytes

Context: Growing adipose tissue is thought to require adipogenesis, angiogenesis, and extracellular matrix (ECM) remodeling. Close examination of developing adipose tissue microvasculature reveals that angiogenesis often precedes adipogenesis. Since our previous study demonstrated that Ob-X, the anti-angiogenic herbal composition composed of Melissa officinalis L. (Labiatae), Morus alba L. (Moraceae), and Artemisia capillaris Thunb. (Compositae), reduced adipose tissue mass in obese mice, we hypothesized that adipogenesis can be inhibited by Ob-X.

Objective: To investigate the effects of the anti-angiogenic herbal extracts Ob-X on adipogenesis in 3T3-L1 adipocytes.

Materials and methods: After differentiated 3T3-L1 adipocytes were treated with Ob-X, we studied the effects of Ob-X on triglyceride accumulation and expression of genes involved in adipogenesis, angiogenesis, and ECM remodeling.

Results: Treatment of cells with Ob-X inhibited lipid accumulation and adipocyte-specific gene expression caused by troglitazone or monocyte differentiation-inducing (MDI) mix. Ob-X reduced mRNA levels of angiogenic factors (vascular endothelial growth factor-A, -B, -C, -D, and fibroblast growth factor-2) and matrix metalloproteinases (MMPs; MMP-2 and MMP-9), whereas it increased mRNA levels of angiogenic inhibitors [(thrombospondin-1, tissue inhibitor of metalloproteinase-1 (TIMP-1), and TIMP-2)] in differentiated cells. MMP-2 and MMP-9 activities were also decreased in Ob-X-treated cells.

Discussion and conclusion: These results suggest that the anti-angiogenic herbal composition Ob-X inhibits differentiation of preadipocytes into adipocytes. These events may be mediated by changes in the expression of genes involved in lipogenesis, angiogenesis, and the MMP system. Thus, by reducing adipogenesis, anti-angiogenic Ob-X provides a possible therapeutic approach for the prevention and treatment of human obesity and its related disorders.     

Anti-obesity effect of sulfated glucosamine by AMPK signal pathway in 3T3-L1 adipocytes

In this study, we investigated the effect of sulfated glucosamine (SGlc) on adipogenesis of 3T3-L1 adipocytes during differentiation of preadipocytes into adipocytes by measuring lipid accumulation and adipogenesis related factors. Treatment with SGlc reduced the triglyceride content in Oil-Red O staining and enhanced glycerol secretion in adipocytes in a dose-dependent manner. In addition, SGlc induced the down-regulation of adipogenesis related factors and adipocyte specific gene promoters. Moreover, treatment of 3T3-L1 adipocytes with SGlc activated the phosphorylated adenosine monophosphate-activated protein kinase (AMPK) α and β along with their substrate, acetyl-CoA carboxylase (ACC). These results suggest that inhibitory effect of SGlc on adipocyte differentiation might be mediated through the up-regulation of AMPK pathway.     

Enhanced CCAAT/enhancer-binding protein beta-liver-enriched inhibitory protein production by Oltipraz, which accompanies CUG repeat-binding protein-1 (CUGBP1) RNA-binding protein activation, leads to inhibition of preadipocyte differentiation

The CCAAT/enhancer-binding protein (C/EBP) beta-isoforms liver-enriched activator protein (LAP) and truncated dominant-negative liver-enriched inhibitory protein (LIP) differentially regulate adipogenesis. We previously demonstrated that oltipraz (5-[2-pyrazinyl]-4-methyl-1,2-dithiol-3-thione), a cancer-chemopreventive agent, promotes C/EBPbeta-LAP activation in hepatocytes. This study investigated whether oltipraz affects adipocyte differentiation and, if so, the molecular basis for the alterations in adipogenesis. The expression of LIP notably increased 6 to 48 h after oltipraz treatment of 3T3-L1 preadipocytes, whereas that of LAP was minimally changed. Oltipraz treatment approximately 3-fold elevated the ratio of LIP to LAP. Immunoblot, gel-shift, and Southwestern analyses revealed that oltipraz enhanced the levels of nuclear LIP and LAP and their binding to the C/EBP-binding site. Cotransfection of predipocytes with the plasmid encoding LIP interfered with LAP-mediated luciferase expression, confirming the inhibitory role of LIP in gene expression. Likewise, LAP-mediated luciferase gene transactivation was inhibited by oltipraz, as was observed by cotransfection of a dominant-negative mutant form of C/EBP. Oltipraz enhanced cytoplasmic translocation and RNA binding of CUG repeat-binding protein-1 (CUGBP1) but not calreticulin, another RNA-binding protein that interacts with C/EBPbeta mRNA. When 3T3-L1 preadipocytes were induced to differentiate by exposure to 3-isobutyl-1-methylxanthine, dexamethasone, and insulin, oltipraz markedly inhibited hormone-induced adipocyte differentiation. In primary cultured rat preadipocytes, oltipraz enhanced LIP production and inhibited adipocyte differentiation. In conclusion, oltipraz inhibits adipogenesis by promoting LIP production and activation, and the enhanced LIP production accompanies cytoplasmic translocation of CUGBP1 and its binding to the GC-rich region of C/EBPbeta mRNA. Our finding holds significance in that adipogenesis can be pharmacologically controlled by LIP production.     

Expression of multiple Transient Receptor Potential channel genes in murine 3T3-L1 cell lines and adipose tissue

BackgroundCalcium and its signaling have a role in adipogenesis. Transient Receptor Potential (TRP) channels are non-selective cation channels with a high permeability to calcium.MethodsIn the present study the expression of multiple TRP channels on mouse 3T3-L1 preadipocyte and adipocyte cells, white (WAT) and brown (BAT) adipose tissues was investigated using real time PCR (RT-PCR).ResultsTRPV1, TRPV3, TRPM8, TRPC4, TRPC6 were differentially expressed in preadipocytes and adipocytes suggesting their significance in adipogenesis. Genes for multiple TRP channels were also expressed in murineWAT and BAT, out of which TRPV4, TRPV6 and TRPC6 showed differential expression.ConclusionPresent study demonstrates the expression of TRP channels in mouse cell lines and adipose tissues.     

树大网膜前脂肪细胞原代培养及诱导分化

目的探讨树大网膜前脂肪细胞原代培养的方法,优化其诱导分化的方法。方法采用酶消化的方法对树大网膜组织进行原代培养,待其传至第三代时,用含0.5mmoL/L3-异丁基-1-甲基黄嘌呤、10μmol/L胰岛素、0.2mmoL/L吲哚美辛、1μmoL/L地塞米松的高糖DMEM诱导配方对树大网膜前脂肪细胞进行成脂诱导,14d后见到细胞内聚集脂滴,对其进行油红O染色。结果树大网膜脂肪细胞在体外成功培养,细胞形态良好,呈梭形。诱导后的脂肪细胞第3d出现少数出现小脂滴,14d左右,大部分细胞胞质中出现圆而大的脂滴。结论树大网膜脂肪组织可在体外进行培养,稳定传代并能诱导分化为成熟的脂肪细胞。     

白藜芦醇对小鼠3T3-L1细胞增殖与分化的影响及其机制

目的探讨白藜芦醇(resveratrol,Res)对小鼠3T3-L1细胞增殖、分化的影响及其机制。方法培养3T3-L1前脂肪细胞,添加不同剂量的白藜芦醇,用WST-1法检测细胞的增殖;用油红O染色后以染色比色法分析脂肪细胞的分化程度;采用RealtimePCR技术检测各组脂联素和瘦素的mRNA表达;采用Westernblot技术检测相关位点,包括沉默信息调节因子1(silent information regulator 1,Sirt1)、过氧化物酶增殖物激活受体γ(peroxisome proliferator activated re-ceptor γ,PPARγ)、CCAAT/增强子结合蛋白α(CCTT Aenhan-cerbinding proteinα,C/EBPα)的蛋白质表达。结果Res明显抑制3T3-L1前脂肪细胞增殖,且呈一定的时间-剂量依赖关系;与对照组比较,Res组脂联素和瘦素的mRNA表达水平下降(P<0.01),Res抑制3T3-L1前脂肪细胞向成熟脂肪细胞分化;添加Res导致脂肪细胞中Sirt1蛋白表达水平上升,PPARγ和C/EBPα蛋白表达水平下降。结论白藜芦醇能抑制3T3-L1前脂肪细胞增殖和分化,其机制可能是通过增加Sirt1表达,抑制脂肪细胞分化相关蛋白PPARγ、C/EBPα的表达。     

番石榴叶天然成分抑制3T3-L1细胞成脂分化的研究

目的观察番石榴叶天然成分槲皮素、槲皮素-3-O-(6″-芥子酸)-β-D-吡喃半乳糖苷(Quercetin-3-O-(6″-eru-coyl)-β-D-galactopyranoside,QEG)及槲皮素-3-O-(6″-阿魏酸)-β-D-吡喃半乳糖苷(Quercetin-3-O-(6″-feruloyl)-β-D-ga-lactopyranoside,QFG)对小鼠3T3-L1细胞成脂分化的影响。方法诱导3T3-L1细胞成脂分化,油红O染色法测定脂滴含量,实时定量PCR(Q-PCR)检测丝氨酸蛋白酶脂肪因子adipsin,CCTTA增强子结合蛋白α(C/EBPα)、过氧化物酶体增殖物激活受体γ(PPARγ)、雌激素受体α(ERα)和雌激素受体β(ERβ)mRNA表达,Western blot检测C/EBPα和PPARγ的蛋白表达。结果 QFG和槲皮素都能抑制3T3-L1细胞成脂分化,而QEG对此没有作用。QFG能够剂量依赖性地抑制成脂分化,并且降低adipsin、C/EBPα和PPARγmRNA及后两者蛋白表达,而对ERα和ERβmRNA的表达没有影响。结论 QFG抑制细胞成脂分化的能力比槲皮素强,其作用主要是通过抑制C/EBPα和PPARγ的表达而实现的,而且可能与雌激素受体途径无关。     

Honokiol enhances adipocyte differentiation by potentiating insulin signaling in 3T3-L1 preadipocytes

Adipose tissue plays an essential role in energy homeostasis as a metabolic and endocrine organ. Accordingly, adipocytes are emerging as a major drug target for obesity and obesity-mediated metabolic syndrome. Dysfunction of enlarged adipocytes in obesity is involved in obesity-mediated metabolic syndrome. Adipocytokines, such as adiponectin released from small adipocytes, are able to prevent these disorders. In this study, we found that honokiol, an ingredient of Magnolia officinalis used in traditional Chinese and Japanese medicines, enhanced adipocyte differentiation in 3T3-L1 preadipocytes. Oil Red O staining showed that treatment with honokiol in the presence of insulin dose-dependently increased lipid accumulation in 3T3-L1 preadipoyctes although its activity was weak compared with rosiglitazone. During adipocyte differentiation, the expression of peroxisome proliferator-activated receptor γ2 (PPARγ2) mRNA and PPARγ target genes such as adipocyte protein 2 (aP2), adiponectin, and GLUT4 was induced by treatment with 10?μM honokiol. However, honokiol failed to show direct binding to the PPARγ ligand-binding domain in vitro. In preadipocytes, treatment with honokiol in the presence of insulin increased the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 protein and Akt protein, early insulin signaling pathways related to adipocyte differentiation, compared with insulin-only treatment. Taken together, our results suggest that honokiol promotes adipocyte differentiation through increased expression of PPARγ2 mRNA and potentiation of insulin signaling pathways such as the Ras/ERK1/2 and phosphoinositide-3-kinase (PI3K)/Akt signaling pathways.