黄芩素对猪前体脂肪细胞增殖分化的影响

研究黄芩素(BAI)对猪前体脂肪细胞增殖分化的影响,并探讨其可能的作用机制。原代培养猪前体脂肪细胞,采用油红O染色观察细胞分化的形态学变化;MTT检测细胞增殖状况;油红O染色提取定量分析细胞内脂肪生成及细胞分化程度;分光光度法测定脂肪酸合酶(FAS)的活性;逆转录-聚合酶链反应(RT-PCR)检测分化特异基因过氧化物酶体增殖物激活受体γ2(PPARγ2)mRNA表达变化。结果显示,前体脂肪细胞在分化成脂肪细胞的过程中,其形态由梭形变成椭圆形、圆形,细胞内充满大小不一的脂滴;BAI浓度在160～640μmol/L时显著抑制其增殖(P<0.05)、BAI浓度为40～320μmol/L时显著抑制PPARγ2mRNA表达和FAS的活性,并抑制细胞分化(P<0.05)。以上结果说明,BAI对前体脂肪细胞增殖分化均有一定抑制作用,BAI可能通过抑制PPARγ2mRNA表达和降低FAS活性,从而抑制猪前体脂肪细胞分化。     

Ginsenoside Rb1 promotes adipogenesis in 3T3-L1 cells by enhancing PPARgamma2 and C/EBPalpha gene expression

Evidence has accumulated that ginseng and its main active constituents, ginsenosides, possess anti-diabetic and insulin-sensitizing properties which may be partly realized by regulating adipocyte development and functions. In the present study, we explored the effect of ginsenoside Rb(1), the most abundant ginsenoside in ginseng root, on adipogenesis of 3T3-L1 cells. We found that with standard differentiation inducers, ginsenoside Rb(1) facilitated adipogenesis of 3T3-L1 preadipocytes in a dose-dependent manner; 10 microM Rb(1) increased lipid accumulation by about 56%. Treatment of differentiating adipocytes with 10 microM Rb(1) increased the expression of mRNA and protein of PPARgamma(2) and C/EBPalpha, as well as mRNA of ap2, one of their target genes. After the treatment of differentiating adipocytes with Rb(1), basal and insulin-mediated glucose uptake was significantly augmented, accompanied by the up-regulation of mRNA and protein level of GLUT4, but not of GLUT1. In addition, ginsenoside Rb(1) also inhibited the proliferation of preconfluent 3T3-L1 preadipocytes. Our data indicate that anti-diabetic and insulin-sensitizing activities of ginsenosides, at least in part, are involved in the enhancing effect on PPARgamma2 and C/EBPalpha expression, hence promoting adipogenesis.     

Rohitukine inhibits in vitro adipogenesis arresting mitotic clonal expansion and improves dyslipidemia in vivo

We developed a common feature pharmacophore model using known antiadipogenic compounds (CFPMA). We identified rohitukine, a reported chromone anticancer alkaloid as a potential hit through in silico mapping of the in-house natural product library on CFPMA. Studies were designed to assess the antiadipogenic potential of rohitukine. Rohitukine was isolated from Dysoxylum binacteriferum Hook. to ⬧95% purity. As predicted by CFPMA, rohitukine was indeed found to be an antiadipogenic molecule. Rohitukine inhibited lipid accumulation and adipogenic differentiation in a concentration- and exposure-time-dependent manner in 3T3-L1 and C3H10T1/2 cells. Rohitukine downregulated expression of PPARγ, CCAAT/enhancer binding protein α, adipocyte protein 2 (aP2), FAS, and glucose transporter 4. It also suppressed mRNA expression of LPL, sterol-regulatory element binding protein (SREBP) 1c, FAS, and aP2, the downstream targets of PPARγ. Rohitukine arrests cells in S phase during mitotic clonal expansion. Rohitukine was bioavailable, and 25.7% of orally administered compound reached systemic circulation. We evaluated the effect of rohitukine on dyslipidemia induced by high-fat diet in the hamster model. Rohitukine increased hepatic expression of liver X receptor α and decreased expression of SREBP-2 and associated targets. Rohitukine decreased hepatic and gonadal lipid accumulation and ameliorated dyslipidemia significantly. In summary, our strategy to identify a novel antiadipogenic molecule using CFPMA successfully resulted in identification of rohitukine, which confirmed antiadipogenic activity and also exhibited in vivo antidyslipidemic activity.     

Transient suppression of PPARgamma directed ES cells into an osteoblastic lineage

Osteoblasts and adipocytes are believed to share a common progenitor. Peroxisome proliferator-activated receptor gamma (PPARgamma) plays a key role in the switching of these two cell lineages. Here, we demonstrated the differentiation of ES cells into an osteoblastic lineage using siRNA against PPARgamma without the addition of any osteogenic factors. We found that PPARgamma-siRNA downregulated the expression of aP2 mRNA and lipid accumulation, whereas it upregulated the expression of osteocalcin and calcium deposition. These results suggested that ES cells were directed into an osteoblastic lineage. Therefore, transient suppression using PPARgamma-siRNA may be a novel tool to induce differentiation of ES cells into osteoblasts.     

4-Hydroxynonenal and PPARgamma ligands affect proliferation, differentiation, and apoptosis in colon cancer cells

PPARgamma ligands inhibit growth and induce apoptosis of various cancer cells. 4-Hydroxynonenal (HNE), a product of lipid peroxidation, inhibits proliferation and induces differentiation or apoptosis in neoplastic cells. The aim of this work was to investigate the effects of PPARgamma ligands (rosiglitazone and 15-deoxy-prostaglandin J2 (15d-PGJ2)) and HNE, alone or in association, on proliferation, apoptosis, differentiation, and growth-related and apoptosis-related gene expression in colon cancer cells (CaCo-2 cells). PPARgamma ligands inhibited cell proliferation (IC50 was 37.47+/-6.6 microM, for 15d-PGJ2, and 170.34+/-20 microM for rosiglitazone). HNE (1 microM) inhibited cell growth by 70%. Apoptosis was induced by 15d-PGJ2 and HNE and, to a minor extent, rosiglitazone. Differentiation was induced by rosiglitazone and by 15d-PGJ2, but not by HNE. PPARgamma ligands inhibited c-myc expression. HNE induced a transitory increase in c-myc expression and a subsequent down-regulation. HNE induced p21 expression, whereas PPARgamma ligands did not. Expression of the bax gene was increased by HNE and 15d-PGJ2, but not by rosiglitazone. No synergism or antagonism was found between HNE and PPARgamma ligands. Both apoptosis and differentiation induction may be responsible for the inhibition of proliferation by PPARgamma ligands; apoptosis and c-myc and p21 expression seem to be involved in the inhibition of proliferation by HNE.     

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.     

Emodin with PPARgamma ligand-binding activity promotes adipocyte differentiation and increases glucose uptake in 3T3-Ll cells

Emodin, one of the main active components in the root and rhizome of Rheum palmatum L, promoted the conversion of 3T3-L1 fibroblasts to adipocytes, as evidenced by increased glycerol-3-phosphate dehydrogenase (GPDH) activity and the expression of adipocyte aP2 mRNA, as well as accelerated triacylglycerol (TG) accumulation, which was associated with increased mRNA expression levels of both C/EBPalpha and PPARgamma2. By using surface plasmon resonance (SPR) experiment, it was showed that emodin exhibited a very high binding affinity to PPARgamma. In differentiated 3T3-L1 adipocytes, emodin induced a time- and dose-dependent increase in glucose uptake as well as GLUT1 and GLUT4 mRNA expression, and the rate of uptake was partly abrogated by wortmannin (phosphoinositide 3-kinase inhibitor). Meanwhile, insulin-stimulated glucose uptake was increased significantly after treatment with low doses of emodin, and the degree of potentiation was decreased thereafter in response to increasing concentrations. Furthermore, 50 microM emodin profoundly inhibited insulin-stimulated glucose uptake by 25%. These data suggest a new role for emodin as a PPARgamma agonist in 3T3-L1 cells. Besides, it is possible that emodin may also possess other properties contribute to glucose utilization in the adipocytes.     

A combination of octanoate and oleate promotes in vitro differentiation of porcine intramuscular adipocytes

To understand the relationship between intramuscular adipogenesis in the pig and the supply fatty acids, we established a clonal porcine intramuscular preadipocyte (PIP) line from the marbling muscle tissue of female Duroc pig. Confluent PIP cells exhibited a fibroblastic appearance. Their adipogenic ability was investigated using confluent PIP cells after exchanging growth medium for adipogenic medium containing 50 ng/mL insulin, 0.25 microM dexamethasone, 2 mM octanoate, and 200 microM oleate. Appropriate concentrations of octanoate and oleate for the induction of adipogenesis were determined from the ability of cells to accumulate lipid and the toxicity of fatty acids. When cells were cultured in differentiation medium for 8 days, large numbers of lipid droplets were observed in differentiated PIP cells, and their cytosolic TG content increased in a time-dependent manner. While oleate only induced the expression of PPARgamma mRNA, but not that of C/EBPalpha, octanoate significantly induced the expression of both PPARgamma and C/EBPalpha mRNA. Octanoate and oleate accelerated the inducing effect of insulin and dexamethasone on the expression of aP2 mRNA. These results indicate that a combination of octanoate and oleate synergistically induced PIP adipogenesis, and that the stimulation of octanoate was essential to the trigger for the adipogenesis in PIP cells.     

PPARgamma2 regulates lipogenesis and lipid accumulation in steatotic hepatocytes

Peroxisome proliferator-activated receptor-gamma (PPARgamma) is considered to be one of the master regulators of adipocyte differentiation. PPARgamma2 is abundantly expressed in mature adipocytes and is elevated in the livers of animals that develop fatty livers. The aim of this study was to determine the ability of PPARgamma2 to induce lipid accumulation in hepatocytes and to delineate molecular mechanisms driving this process. The hepatic cell line AML-12 was used to generate a cell line stably expressing PPARgamma2. Oil Red O staining revealed that PPARgamma2 induces lipid accumulation in hepatocytes. This phenotype is accompanied by a selective upregulation of several adipogenic and lipogenic genes including adipose differentiation-related protein (ADRP), adipocyte fatty acid-binding protein 4, sterol regulatory element-binding protein-1 (SREBP-1), fatty acid synthase (FAS), and acetyl-CoA carboxylase, genes whose expression levels are known to increase in steatotic livers of ob/ob mice. Furthermore, the PPARgamma2-regulated induction of both SREBP-1 and FAS parallels an increase in de novo triacylglycerol synthesis in hepatocytes. Triacylglycerol synthesis and lipid accumulation are further enhanced by culturing hepatocytes with troglitazone in the absence of exogenous lipids. These results correspond with an increase in the lipid droplet protein, ADRP, and the data demonstrate that ADRP functions to coat lipid droplets in hepatocytes as observed by confocal microscopy. Taken together, these observations propose a role for PPARgamma2 as an inducer of steatosis in hepatocytes and suggest that this phenomenon occurs through an induction of pathways regulating de novo lipid synthesis.     

Glucosamine regulates hepatic lipid accumulation by sensing glucose levels or feeding states of normal and excess

Dose-dependent lipid accumulation was induced by glucose in HepG2 cells. GlcN also exerted a promotory effect on lipid accumulation in HepG2 cells under normal glucose conditions (NG, 5 mM) and liver of normal fed zebrafish larvae. High glucose (HG, 25 mM)-induced lipid accumulation was suppressed by l-glutamine-d-fructose 6-phosphate amidotransferase inhibitors. ER stress inhibitors did not suppress HG or GlcN-mediated lipid accumulation. HG and GlcN stimulated protein expression, DNA binding and O-GlcNAcylation of carbohydrate-responsive element-binding protein (ChREBP). Furthermore, both HG and GlcN increased nuclear sterol regulatory element-binding protein-1 (SREBP-1) levels in HepG2 cells. In contrast to its stimulatory effect under NG, GlcN suppressed lipid accumulation in HepG2 cells under HG conditions. Similarly, GlcN suppressed lipid accumulation in livers of overfed zebrafish. In addition, GlcN activity on DNA binding and O-GlcNAcylation of ChREBP was stimulatory under NG and inhibitory under HG conditions. Moreover, GlcN enhanced ChREBP, SREBP-1c, ACC, FAS, L-PK and SCD-1 mRNA expression under NG but inhibited HG-induced upregulation in HepG2 cells. The O-GlcNAc transferase inhibitor, alloxan, reduced lipid accumulation by HG or GlcN while the O-GlcNAcase inhibitor, PUGNAc, enhanced lipid accumulation in HepG2 cells and liver of zebrafish larvae. GlcN-induced lipid accumulation was inhibited by the AMPK activator, AICAR. Phosphorylation of AMPK (p-AMPK) was suppressed by GlcN under NG while increased by GlcN under HG. PUGNAc downregulated p-AMPK while alloxan restored GlcN- or HG-induced p-AMPK inhibition. Our results collectively suggest that GlcN regulates lipogenesis by sensing the glucose or energy states of normal and excess fuel through AMPK modulation.     

PPARgamma is not a critical mediator of primary monocyte differentiation or foam cell formation.

In the present report we clarify the role of PPARgamma in differentiation and function of human-derived monocyte/macrophages in vitro. Rosiglitazone, a selective PPARgamma activator, had no effect on the kinetics of appearance of monocyte/macrophage differentiation markers or on cell size or granularity. Depletion of PPARgamma by more than 90% using antisense oligonucleotides did not influence accumulation of oxidized LDL or prevent the upregulation of CD36 that normally accompanies oxLDL treatment. In contrast, PPARgamma depletion reduced the expression of ABCA1 and LXRalpha mRNAs. Metalloproteinase-9 expression, a marker of atherosclerotic plaque vulnerability, was suppressed by rosiglitazone. We conclude that activation of PPARgamma does not affect monocyte/macrophage differentiation. In addition, PPARgamma is not absolutely required for oxLDL-driven lipid accumulation, but is required for full expression of ABCA1 and LXRalpha. Our data support a role for rosiglitazone as a potential directly acting antiatherosclerotic agent.     

Inhibitory effects of tannic acid on fatty acid synthase and 3T3-L1 preadipocyte

Tannic acid is a hydrolyzable tannin that exists in many widespread edible plants with a variety of biological activities. In this study, we found that tannic acid potently inhibited the activity of fatty acid synthase (FAS) in a concentration-dependent manner with a half-inhibitory concentration value (IC₅₀) of 0.14 μM. The inhibition kinetic results showed that the inhibition of FAS by tannic acid was mixed competitive and noncompetitive manner with respect to acetyl-CoA and malonyl-CoA, but uncompetitive to NADPH. Tannic acid prevented the differentiation of 3T3-L1 pre-adipocytes, and thus repressed intracellular lipid accumulation. In the meantime, tannic acid decreased the expression of FAS and down-regulated the mRNA level of FAS and PPARγ during adipocyte differentiation. Further studies showed that the inhibitory effect of tannic acid did not relate to FAS non-specific sedimentation. Since FAS was believed to be a therapeutic target of obesity, these findings suggested that tannic acid was considered having potential in the prevention of obesity.     

Liver X receptor preferentially activates de novo lipogenesis in human preadipocytes

The liver X receptor (LXR) was demonstrated to play a key role in cholesterol metabolism in liver, intestine and macrophage. However, its function on the regulation of preadipocyte differentiation remains unclear since contradictory results were reported. The objective of the present study was to unravel the functionality of LXR in human preadipocytes. We show that the LXR agonist T0901317 strongly stimulated the expression of SREBP-1c and the lipogenic enzymes ACC-1, FAS and SCD-1 in both the human preadipose cell line Chub-S7 as well as human primary stromal vascular fraction (SVF) cells. The effects on gene expression were associated with the stimulation of de novo lipogenesis in both cell models, resulting in the induction of lipid accumulation. In contrast with a PPARgamma agonist (BRL49653), T0901317 enhanced only slightly the expression of PPARgamma dependent genes (PPARgamma, aP2 and adiponectin) in Chub-S7 cells and failed to change their expression in human SVF cells. These results show that LXR stimulated preferentially triglyceride accumulation in human preadipocytes via the induction of de novo lipogenesis, rather than activating the differentiation process through PPARgamma activation.