IGF-I、GH和CLA调控脂肪细胞增殖分化和基因表达

为探讨IGF-I、GH、CLA对不同部位来源脂肪前体细胞的增殖和分化程度的影响，本研究以大鼠为试验模型，分别从附睾及皮下脂肪组织分离得到脂肪前体细胞，并在不同浓度的IGF-I、GH、 CLA处理后，用MTT法检测脂肪前体细胞的增殖，用油红O染色法检测脂肪前体细胞的分化聚脂程度结果表明：各剂量IGF-I和CLA均能够显著促进大鼠附睾及皮下脂肪前体细胞的增殖与分化，但GH对大鼠附睾及皮下脂肪前体细胞的增殖与分化均无明显作用。为进一步观察IGF-I和CLA对皮下脂肪组织的作用机制，试验还采用相对定量RT-PCR法检测细胞中PPARγ和C/EBPα的基因表达水平。研究发现，100ng/ml IGF-I可显著上调大鼠脂肪前体细胞中PPARγ 与C/EBPα的基因表达水平； 48ng/ml GH和100μM CLA可显著促进PPARγ的基因表达，但对C/EBPα mRNA表达无显著影响。上述结果提示IGF-I可能主要通过上调PPARγ 与C/EBPα基因的高水平表达进而促进大鼠脂肪前体细胞的分化，而CLA对脂肪前体细胞的作用在上调PPARγ的表达的同时，还作为PPARγ的配体激活该受体而发挥作用     

Effects of capsaicin on induction of apoptosis and inhibition of adipogenesis in 3T3-L1 cells

Currently, at the beginning of the 21st century, obesity has become the leading metabolic disease in the world. It is a serious health problem in industrialized countries. Previous research has suggested that decreased preadipocyte differentiation and proliferation and decreased lipogenesis are mechanisms to reduce obesity. In the present study, the effects of capsaicin on the induction of apoptosis and inhibition of lipid accumulation in 3T3-L1 preadipocytes and adipocytes were investigated. The results demonstrated that capsaicin decreased cell population growth of 3T3-L1 preadipocytes, assessed with the MTT assay. Flow cytometric analysis of 3T3-L1 preadipocytes exposed to capsaicin showed that apoptotic cells increased in a time- and dose-dependent manner. Treatment with capsaicin decreased the number of normal cells and increased the number of early apoptotic and late apoptotic cells in a dose-dependent manner. The treatment of cells with capsaicin caused the loss of mitochondria membrane potential (delta psi m). The induction of apoptosis in 3T3-L1 preadipocytes by capsaicin was mediated through the activation of caspase-3, Bax, and Bak, and then through the cleavage of PARP and the down-regulation of Bcl-2. Moreover, capsaicin significantly decreased the amount of intracellular triglycerides and glycerol-3-phosphate dehydrogenase (GPDH) activity in 3T3-L1 adipocytes. Capsaicin also inhibited the expression of PPARgamma, C/EBPalpha, and leptin, but induced up-regulation of adiponectin at the protein level. These results demonstrate that capsaicin efficiently induces apoptosis and inhibits adipogenesis in 3T3-L1 preadipocytes and adipocytes.     

Effect of eriodictyol on glucose uptake and insulin resistance in vitro

Eriodictyol [2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-2,3-dihydrochromen-4-one] is a flavonoid with anti-inflammatory and antioxidant activities. Because inflammation and oxidative stress play critical roles in the pathogenesis of diabetes mellitus, the present study was designed to explore whether eriodictyol has therapeutic potential for the treatment of type 2 diabetes. The results show that eriodictyol increased insulin-stimulated glucose uptake in both human hepatocellular liver carcinoma cells (HepG2) and differentiated 3T3-L1 adipocytes under high-glucose conditions. Eriodictyol also up-regulated the mRNA expression of peroxisome proliferator-activated receptor γ2 (PPARγ2) and adipocyte-specific fatty acid-binding protein (aP2) as well as the protein levels of PPARγ2 in differentiated 3T3-L1 adipocytes. Furthermore, it reactivated Akt in HepG2 cells with high-glucose-induced insulin resistance. This response was strongly inhibited by pretreatment with the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002, indicating that eriodictyol increased Akt phosphorylation by activating the PI3K/Akt pathway. These results imply that eriodictyol can increase glucose uptake and improve insulin resistance, suggesting that it may possess antidiabetic properties.     

Regulation effects of Crataegus pinnatifida leaf on glucose and lipids metabolism

The leaf of Crataegus pinnatifida (Rosaceae) is commonly consumed either raw or cooked to improve digestion and promote blood circulation in China. To investigate the regulation effects of it on glucose and lipid metabolism, the flavonoids fraction was prepared and analyzed by HPLC and LC-MS. In vivo, at doses of 250 and 500 mg/kg, the flavonoids fraction showed inhibitory effects on TG and glucose absorption, accelerating effects on gastrointestinal transit but no effect on gastric emptying. In vitro, treatment of 3T3-L1 preadipocytes with 30 μg/mL flavonoids fraction significantly suppressed the accumulation of TG and free fatty acid. It also suppressed the gene expressions of C/EBPα, PPARγ, SREBP 1c, aP2 and adiponectin but did not affect that of leptin. C. pinnatifida leaf may be useful for type 2 diabetics and hyperlipidemics as a foodstuff.     

Intracellular mediators of procyanidin-induced lipolysis in 3T3-L1 adipocytes

We have previously reported that grape seed procyanidins stimulate long-term lipolysis on 3T3-L1 fully differentiated adipocytes. To unravel the molecular mechanism by which procyanidins exert this effect, we checked the involvement of two main cellular targets in adipose cells: protein kinase A (PKA) and peroxisome proliferator-activated receptor-gamma (PPAR-gamma). Procyanidin treatment increased intracellular cAMP levels in 3T3-L1 adipocytes, and their lipolytic effect was inhibited by simultaneous treatment with H89, a PKA specific inhibitor. BRL49653, a very highly specific ligand of PPAR-gamma, totally abolished the lipolytic effect of procyanidins. Simultaneous to this long-term lipolytic effect, the mRNA levels of some differentiation adipocyte markers decreased, although there were no changes in the triglyceride content of the cells. BRL49653 did not antagonize the decrements of differentiation markers. These results support a mediation of PPAR-gamma and PKA on the lipolytic effects of procyanidins on 3T3-L1 adipocytes.     

Inhibitory effect of α-lipoic acid on platelet aggregation is mediated by PPARs

Peroxisome proliferator-activated receptors (PPARs) isoforms (α, β/δ, and γ are present in human platelets, and activation of PPARs inhibits platelet aggregation. α-Lipoic acid (ALA), occurring naturally in human food, has been reported to exhibit an antiplatelet activity. However, the mechanisms underlying ALA-mediated inhibition of platelet aggregation remain unknown. The aim of this study was to investigate whether the antiplatelet activity of ALA is mediated by PPARs. ALA itself significantly induced PPARα/γ activation in platelets and increased intracellular amounts of PPARα/γ by blocking PPARα/γ secretion from arachidonic acid (AA)-activated platelets. Moreover, ALA significantly inhibited AA-induced platelet aggregation, Ca(2+) mobilization, and cyclooxygenase-1 (COX-1) activity, but increased cyclic AMP production in rabbit washed platelets. Importantly, ALA also enhanced interaction of PPARα/γ with protein kinase Cα (PKCα) and COX-1 accompanied by an inhibition of PKCα activity in resting and AA-activated platelets. However, the above effects of ALA on platelets were markedly reversed by simultaneous addition of selective PPARα antagonist (GW6471) or PPARγ antagonist (GW9662). Taken together, the present study provides a novel mechanism by which ALA inhibition of platelet aggregation is mediated by PPARα/γ-dependent processes, which involve interaction with PKCα and COX-1, increase of cyclic AMP formation, and inhibition of intracellular Ca(2+) mobilization.     

鸡C/EBPα抗血清制备及组织表达的特性

制备鸡（Gallus gallus）CAAT增强子结合蛋白α（CAAT/enhancer-binding protein α,C/EBPα）的多克隆抗血清,并分析其组织表达特性。通过限制性酶切,从T-C/EBPα质粒中获得C/EBPα基因的编码区序列（coding sequence,CDS）,构建原核表达载体,以大肠杆菌（Escherichia coli）为宿主,用IPTG诱导重组pGEX6p/C/EBPα的表达,经Glutathione Sepharose 4B柱层析纯化后,以其为免疫原制备兔抗鸡C/EBPα多抗。ELISA方法测定抗体效价达1∶12800,Western blot方法显示抗体具有较高的特异性。用此抗血清分析了鸡C/EBPα的组织表达特性,结果表明,鸡的回肠、肾脏、肌胃、腺胃、心脏、肝脏、腹脂、脾脏等组织中C/EBPα蛋白表达量较高,而在胸肌、腿肌中表达量较低。同时分析C/EBPα基因在高低脂系公鸡肝脏组织中的表达差异,结果显示该基因在低脂系公鸡肝脏中表达量较高。     

Cinnamaldehyde prevents adipocyte differentiation and adipogenesis via regulation of peroxisome proliferator-activated receptor-γ (PPARγ) and AMP-activated protein kinase (AMPK) pathways

Cinnamaldehyde (CA), one of the active components of cinnamon, has been known to exert several pharmacological effects such as anti-inflammatory, antioxidant, antitumor, and antidiabetic activities. However, its antiobesity effect has not been reported yet. This study investigated the antidifferentiation effect of CA on 3T3-L1 preadipocytes, and the antiobesity activity of CA was further explored using high-fat-diet-induced obese ICR mice. During 3T3-L1 preadipocytes were differentiated into adipocytes, 10-40 μM CA was treated and lipid contents were quantified by Oil Red O staining, along with changes in the expression of genes and proteins associated with adipocyte differentiation and adipogenesis. It was found that CA significantly reduced lipid accumulation and down-regulated the expression of peroxisome proliferator-activated receptor-γ (PPAR-γ), CCAAT/enhancer-binding proteins α (C/EBPα), and sterol regulatory element-binding protein 1 (SREBP1) in concentration-dependent manners. Moreover, CA markedly up-regulated AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC), and these effects were blunted in the presence of AMPK inhibitor, compound C. In the animal study, weight gains, insulin resistance index, plasma triglyceride (TG), nonesterified fatty acid (NEFA), and cholesterol levels in the 40 mg/kg of CA-administered group were significantly decreased by 67.3, 55, 39, 31, and 23%, respectively, when compared to the high-fat diet control group. In summary, these results suggest that CA exerts antiadipogenic effects through modulation of the PPAR-γ and AMPK signaling pathways.     

Conjugated linoleic acid regulates phosphorylation of PPARγ by modulation of ERK 1/2 and p38 signaling in human macrophages/fatty acid-laden macrophages

Stimulation of macrophages by a variety fatty acids causes activation of MAP kinases (MAPKs). The consequences arising from down-regulation of MAPKs may be a limitation in the activity of PPARγ, which is modulated by a modification catalyzed by these kinases. Phosphorylation of MAP kinases-ERK1/2 and p38 as well as PPARγ was determined by real-time polymerase chain reaction and Western blotting in human macrophages cultured with conjugated linoleic acids (CLAs). We demonstrated that CLA isomers alter MAP kinase phosphorylation and PPARγ activation. Phosphorylation of ERK1/2 was diminished in cells cultivated with cis-9,trans-11 CLA, whereas phosphorylation of p38 was reduced by trans-10,cis-12 CLA. PPARγ was phosphorylated mainly by ERK1/2, and consequently, PPARγ phosphorylation was suppressed mainly by cis-9,trans-11 isomer. In human adipocytes, cis-9,trans-11 C 18:2 raised the activation of PPAR and several of its downstream target genes. We suggest that a similar process may also occur in human macrophages.     

Effects of flavonoids and phenolic acids on the inhibition of adipogenesis in 3T3-L1 adipocytes

Obesity has become a global epidemic in both developed and developing countries, and it is a significant risk factor for various diseases such as diabetes, cancer, heart disease, and hypertension. In the present study, the effect of naturally occurring antioxidants (flavonoids and phenolic acids) on the inhibition of adipogenesis in 3T3-L1 adipocytes was investigated. The results showed that o-coumaric acid and rutin had the highest inhibition on intracellular triglyceride (61.3 and 83.0%, respectively) among 15 phenolic acids and 6 flavonoids tested. However, the oil red o stained material (OROSM) showed that cell number in 3T3-L1 adipocytes was not influenced by those compounds. For glycerol-3-phosphate dehydrogenase (GPDH) activity, the data indicated that o-coumaric acid and rutin had the highest inhibition on GPDH activity (54.2 and 66.8%, respectively) among the compounds tested. o-Coumaric acid and rutin also inhibited the expression of PPARgamma, C/EBPalpha and leptin and then up-regulated expression of adiponectin at the protein level. Some naturally occurring antioxidants efficiently suppressed adipogenesis in 3T3-L1 adipocytes. These results suggest that o-coumaric acid and rutin targeted for adipocyte functions could be effective in improving the symptoms of metabolic syndrome.     

AGE-induced interference of glucose uptake and transport as a possible cause of insulin resistance in adipocytes

The purpose of this study was to investigate the distinct roles of advanced glycation end products (AGEs) on insulin-mediated glucose disposal in 3T3-L1 adipocytes and C2C12 skeletal muscle cells. AGE-modified proteins, namely, GO-AGEs, were prepared by incubating bovine serum albumin (BSA) with glyoxal (GO) for 7 days. Glucose utilization rates and the expression of insulin signaling-associated proteins, including Akt, insulin receptor substrate-1, and glucose transporter 4, were determined. GO-AGEs caused insulin resistance (IR) by suppressing insulin-stimulated glucose uptake both in 3T3-L1 adipocytes and C2C12 muscle cells. Interestingly, an unexpected finding was that insulin-stimulated glucose transport in adipocytes was affected by GO-AGEs in a biphasic manner, with an initial steep increase (168%) during the first 8 h of incubation followed by a significantly impaired uptake after extended culture times (24-48 h, p < 0.05). Treatment with GO-AGEs for 24 h markedly accelerated lipid droplet formation compared to the BSA control; however, it was blocked by incubation with an anti-RAGE antibody. Our study suggests that GO-AGEs induce an early dramatic elevation of glucose transport in adipocytes that may be related to the activation of insulin signaling; however, subsequent IR may result from increased oxidative stress and proinflammatory TNF-α production.     

慢病毒介导shRNA干扰颗粒体蛋白前体(GRN)促进猪前体脂肪细胞分化

为研究颗粒体蛋白前体(granulin,GRN)在脂肪细胞发育过程中的作用,本实验构建了GRN短发夹RNA(short hairpin RNA,shRNA)慢病毒干扰载体,包装并感染猪前体脂肪细胞,采用油红O染色、油红O提取比色法检测猪前体脂肪细胞分化情况,采用Real-time PCR法检测成脂关键基因mRNA的表达变化情况。结果显示,GRN慢病毒干扰载体病毒滴度在5×107TU/mL以上,病毒感染前体脂肪细胞后显著降低了GRN的表达,shRNA2干扰效率最高,达到76%,沉默GRN后能够促进猪前体脂肪细胞分化;脂肪细胞分化标志基因过氧化物酶体增殖物激活受体γ(PPARγ)、固醇调节原件结合蛋白(SREBP-1c)、脂肪细胞型脂肪酸结合蛋白(ap2)和脂蛋白酯酶(LPL)基因mRNA表达量均升高。结果表明,降低GRN基因表达促进了猪前体脂肪细胞分化,揭示GRN在猪前体脂肪细胞分化过程中可能起到抑制作用。