Objective To study the effect of acylation stimulating protein(ASP) on lipid droplets associated protein TIP47 during the differentiation process of adipocytes and the change of this effect after blocking PLC signal pathway.Methods(1)To treat 3T3-L1 adipocytes at indicated time point during differentiation with ASP、U73122、U73122 and ASP respectively,including control group in the same time.(2)To examine the mRNA and protein expression levels of TIP47 by RT-PCR and Western blot technique respectively.Results(1) Exposure to ASP for certain time resulted in an obvious increase of TIP47 gene and protein expression in 3T3-L1 adipocytes;(2)Exposure to U73122 which is a blocking agent of PLC signal pathway for certain time resulted in the decrease of TIP47 gene and protein expression in 3T3-L1 adipocytes;(3)Gene and protein level of TIP47 in adipocytes treated with both U73122 and ASP was significantly lower than that in adipocytes treated only with ASP,while was higher than that in adipocytes treated only with U73122.Conclusion PLC signal pathway plays a role in signal transduction of regulation of ASP on expression of TIP47.These results improve the understanding of adipogenesis of ASP on molecular level,and offer some new thinking in etiology,prevention and treatment of obesity.

AIM: To investigate the role of individual protein isoforms in the regulation of 3T3-L1 adipocyte development through detecting temporal patterns of Gβ,Gα/q,phosphorylated phosphoinositide 3-kinase IA and IB isoforms and activated protein kinase C α and ζ subtypes involved in Gβ/Gαq-PI3K-PKC signaling pathway during 3T3-L1 preadipocyte differentiation.METHODS: The cells were induced by 1-methyl-3-isobutylxanthine,dexamethasone and insulin in vitro,and harvested at indicated time points(0 d,6 h,12 h,1 d,3 d,6 d,9 d),then the total proteins of these cells were extracted.The expressions of Gβ,Gα/q,p101,phosphorylated p85,p55,p110γ,PKCα and ζ were assayed by Western blotting.RESULTS: (1) Gαq/11 and Gβ increased after induction of differentiation,reaching maxima respectively at 3 d and 1 d when cellular level of Gβ was 1.97±0.16-fold higher and expression of Gαq/11 was 2.34±0.22-fold higher than that in just-confluent(0 d) cells.Subsequently the expression declined.(2) Compared to 0 d,phosphorylated p110γ,p55 and p85 elevated slightly at 12 h,and decreased significantly by the end of the treatment period(9 d) which coincided with maximal differentiation.While the expression of p101 elevated slightly at 12 h,no statistical significance through differentiation was found.(3) Phosphorylated PKCα increased significantly,peaking at 3 d of differentiation.Expression of phosphorylated PKCζ decreased during differentiation and its level 45.52% lower in adipocytes than that in preadipocyte was observed.CONCLUSION: The protein levels elevating at the early stage of differentiation correlate with the time point at which clonal expansion of cells is observed.Phosphorylated p55 and PKCζ decrease in adipocytes than that in preadipocyte,indicating an inhibitory influence upon the late differentiation process.

To study the effect of acylation stimulating protein(ASP) on the expression of lipid droplets associated protein TIP47 during preadipocyte differentiation and evaluate its significance in adipogenesis.The localization of TIP47 were analyzed by immunofluorescent staining in 3T3-L1 preadipocytes.3T3L1 preadipocytes were differentiated with the traditional hormone cocktail,then incubated with ASP and insulin respectively at indicated time point,the mRNA and protein levels of TIP47 were determined by semiquantitive-PT-PCR and Western blot analysis.The results showed that TIP47 located in the cytoplasm;and its expression decreased during the differentiation process in 3T3-L1 adipocytes.Exposure to ASP resulted in an increase of TIP47,but the effect of ASP was not significant after 48 hours of differentiation.Insulin could elevate the expression of TIP47 in 3T3-L1 adipocytes at 0 day of differentiation,but not lasting after then.Taken together,this study provided evidence that the promotion of adipogenesis by ASP might be associated with the up-regulation of lipid droplets associated protein TIP47 expression.

Perilipin and adipophilin, two significant lipid droplet (LD)-specific proteins, participate in storing fat or ectopic lipid deposition and fat mobilization in many types of mammalian cells. Acylation stimulating protein (ASP) is a novel adipocyte-derived hormone known for a major determinant for triglyceride synthesis (TGS) and lipid metabolism. The present study was aimed to investigate: (1) whether ASP, rather than insulin, is a powerful potentiator which could physiologically and directly influence TGS during 3T3-L1 preadipocyte differentiation; (2) whether ASP exposure at indicated time points during 3T3-L1 preadipocyte differentiation could influence the gene/protein expression of adipophilin and perilipin. 3T3-L1 preadipocytes were differentiated by traditional hormone cocktail and divided into control, ASP and insulin groups according to the treatment of ASP (1 mmol/L) or insulin (100 nmol/L). ASP-stimulated and insulin-stimulated TGS rate at indicated time points (0 d, 3 d, 6 d, 9 d) were assayed by measuring the incorporation of [(3)H]-oleic acid into TG, and the corresponding glucose transport was assayed by [(3)H]-2-DG uptake. The effects of ASP or insulin on gene/protein expression of adipophilin and perilipin at indicated time points were evaluated by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. The results obtained were as follows: (1) on the 3rd and 6th day of differentiation, ASP dramatically enhanced TGS rate compared with control group (P<0.05, P<0.01); There was no significant difference in TGS rate between insulin group and control group; (2) on the 6th and 9th day of differentiation, both ASP and insulin promoted glucose uptake (P<0.05, P<0.01), and the promoting effect in ASP group was greater than that in insulin group; (3) ASP elevated adipophilin gene and protein expression at the very early stage of differentiation (P<0.05, P<0.001) and had no significant effect from the 4th day of differentiation. Perilipin gene and protein expression increased throughout preadipocyte differentiation and its expression was up-regulated following ASP stimulation from the 3rd day of differentiation (P<0.05, P<0.001) to the end of differentiation (P<0.05); (4) Insulin did not affect gene and protein variation pattern of adipophilin and perilipin. Taken together, this study provides evidence that ASP-evoked changes in gene and protein expression of adipophilin and perilipin correlate with ASP-stimulated TGS acceleration, and adipophilin and perilipin are involved in the molecular mechanism of ASP-induced adipogenesis and LD formation.

原核生物、真核生物、植物体的非神经细胞和组织中,尤其是多种免疫活性细胞中,均证实乙酰胆碱酯酶（acetylcholinesterase,AChE）、胆碱乙酰转移酶（choline acetyltransferase,ChAT）和乙酰胆碱受体（acetylcholine receptor,AChR）各亚型在内的胆碱能系统组分的存在,其中烟碱样乙酰胆碱受体α7（nicotinic acetylcholine receptor α7,nAChRα7）是烟碱样胆碱能抗炎通路（nicotinic anti-inflammatory pathway）中重要的分子核心机制,同时也是机体限制宿主防御反应扩大的内源性抗炎机制之一.本文旨在探讨（前）脂肪细胞上非神经元型胆碱能系统是否存在及初步揭示烟碱样胆碱能受体α7对前脂肪细胞功能的影响.以体外培养的3T3-L1前脂肪细胞为研究对象,采用免疫组化和蛋白质免疫印迹技术,分别检测前脂肪细胞和成熟脂肪细胞中乙酰胆碱酯酶、胆碱乙酰转移酶和烟碱样乙酰胆碱受体α7的3种胆碱能系统主要组分的蛋白表达.另将前脂肪细胞分为给予广谱烟碱样乙酰胆碱受体激动剂尼古丁、特异性烟碱样乙酰胆碱受体α7激动剂氯化胆碱及特异性烟碱样乙酰胆碱受体α7拮抗剂甲基牛扁亭碱干预12 h、24 h、36 h,并设立相应处理时间的空白对照组,逆转录聚合酶链反应检测前脂肪细胞visfatin mRNA表达情况.免疫组化染色可见前脂肪细胞中AChE、ChAT及AChRα7均有阳性表达;蛋白免疫印迹检测进一步半定量证实了前脂肪细胞和成熟脂肪细胞中AChE、ChAT及AChRα7的蛋白表达;拮抗剂甲基牛扁亭碱（10-6~10-4mol/L）时间、剂量依赖性上调前脂肪细胞visfatin mRNA表达（1.3~1.55-fold,P＜0.01）,与对应空白对照组相比,存在显著性统计学差异;加入不同剂量的尼古丁和氯化胆碱,则前脂肪细胞中visfatin mRNA表达水平与对应空白对照组相比,均不同程度地下降,其中以氯化胆碱的抑制效应更为显著.前脂肪细胞与成熟脂肪细胞中均存在有独立的胆碱能体系,其中AChRα7很可能在调节脂肪细胞因子分泌及肥胖相关的病理生理过程中发挥重要作用.

Aim To investigate the effect of the functional state of nAChRα7 on chemerin and chemerinR gene expression and to initially explore the molecular influencing mechanism of non-cholinergic system to lipolytic hydrolysis in preadipocytes and mature adipocytes. Method 3T3-L1 preadipocytes were differentiated with 1-methyl-3-isobuthylxanthine+Insulin+Dexamethasone+fetal bovine serum+DMEM/F12.The relatively quantitative RT-PCR was used to detect the expression of chemerin and chemerinR at indicated time point including 0 h,6 h,12 h,1 d,3 d,6 d and 9 d during 3T3-LI cells differentiation,and the gene expression change of chemerin and chemerinR in preadipocytes and fully differentiated adipocytes were observed when cells treated by various concentration(10-8 mol/L,10-6 mol/L and 10-4 mol/L) of selective nAChRα7 agonist choline chloride or selective nAChRα7 antagonist methyllycaconitine. Results ①The expression of chemerin and chemerinR mRNA was low but detectable in confluent 3T3-L1 preadipocytes.When hormonally stimulated by IBMX+DEX+Insulin,their expression both increased markedly until day 9.②The results showed that the mRNA level of chemerin in both preadipocytes and adipocytes and that of chemerinR in preadipocytes were significantly increased by methyllycaconitine and down-regulated by choline.However,the above nAChRα7 ligands had no obvious effect on chemerinR expression in mature adipocytes.③The above nAChRα7 ligands have stronger effect on preadipocytes than adipocytes. Conclusion nAChRα7 is probably the important factor that allows non-neuronal cholinergic system play biological effect.nAChRα7 mediated lipolysis,in part by directly activating a nicotinic cholinergic receptor located in adipocyte,in part by regulating other lipolysis-associated adipokines.