脂肪酸诱导脂肪细胞促酰化蛋白抵抗及机制研究

Mechanism of acylation stimulating protein resistance induced by fatty acid in 3T3-L1 adipocytes and preadipocytes

目的:观察不同浓度单不饱和脂肪酸(油酸)和饱和脂肪酸(棕榈酸)对3T3-L1(前)脂肪细胞葡萄糖转运的影响,探讨高游离脂肪酸(FFA)负荷在促酰化蛋白(ASP)抵抗形成中的意义,及FFA诱导的3T3-L1(前)脂肪细胞ASP抵抗的机制。方法:体外培养3T3-L1细胞,诱导细胞分化,用不同浓度FFA作用于3T3-L1(前)脂肪细胞,孵育过夜后收获细胞,采用2-脱氧-[3H]-D-葡萄糖掺入法,观察3T3-L1成熟脂肪细胞和前脂肪细胞基础状态和ASP刺激状态的葡萄糖摄取率;采用Western blotting法检测基础状态和ASP刺激的鸟苷酸结合蛋白alpha-q/11(Gαq/11),鸟苷酸结合蛋白beta(Gβ),磷酸化蛋白激酶Calpha(p-PKCα)和磷酸化蛋白激酶Czeta(p-PKCζ)蛋白表达。结果:ASP刺激后,3T3-L1成熟脂肪细胞和前脂肪细胞葡萄糖摄取率分别是基础状态的1.98倍(P<0.01)和2.87倍(P<0.01)。低浓度FFA不影响ASP刺激的葡萄糖转运;而1.0mmol/L时油酸组和棕榈酸组ASP刺激的成熟脂肪细胞葡萄糖摄取率分别减少47%(P<0.05)和34%(P<0.05),前脂肪细胞葡萄糖摄取率分别减少43%(P<0.05)和62%(P<0.01)。1.0mmol/L油酸和棕榈酸抑制成熟脂肪细胞基础状态和ASP刺激的Gβ、Gαq/11、p-PKCα和p-PKCζ蛋白表达,油酸组ASP刺激的蛋白表达分别减少了47%Gβ(P<0.01),44%Gαq/11(P<0.05)、39%p-PKCα(P<0.05)和20%p-PKCζ(P>0.05);棕榈酸组也可观察到类似现象(P<0.05或P<0.01)。在前脂肪细胞,油酸仅抑制ASP刺激的p-PKCα和p-PKCζ(均P<0.05)蛋白表达;而棕榈酸下调上述4种信号蛋白的表达(P<0.05或P<0.01)。结论:油酸或棕榈酸抑制3T3-L1成熟脂肪细胞和前脂肪细胞ASP刺激的葡萄糖转运,证明FFA诱导脂肪细胞产生胰岛素抵抗状态下同时存在ASP抵抗。FFA诱导的ASP抵抗的发生机制与其干扰ASP-C5L2信号转导途径有关。ASP抵抗参与了"脂毒性"-胰岛素抵抗/肥胖症的病理生理过程。

AIM：To evaluate the potential acylation stimulating protein （ASP） resistance in both adipocytes and preadipocytes under the conditions by which insulin resistance is produced by the stimulation of free fatty acids （FFA）,and to explore the mechanism of ASP resistance on post-receptor level. METHODS： 3T3-L1 preadipocytes were induced to differentiate. Then the cells were treated with oleate or palmitate at concentration of 0 mmol/L （FFA-free DMEM/F12）,0.125 mmol/L,0.5 mmol/L or 1.0 mmol/L overnight. Glucose transport was assessed by [3H] 2-deoxyglucose uptake to evaluate insulin resistance and ASP resistance. Both non-FFA treated and FFA treated 3T3-L1 cells were cultured with ASP at concentration of 5.0 μmol/L for 4 h,then the cell proteins were extracted,and the expressions of guanine nucleotide binding protein beta （Gβ）,guanine nucleotide-binding protein alpha-q/11（Gαq/11）,phosphorylated-protein kinase Cα （p-PKCα） and phosphorylated-protein kinase Cζ （p-PKCζ） were measured by Western blotting. RESULTS： Both adipocytes and preadipocytes were responsive to ASP. ASP stimulation increased glucose transport by 198% in adipocytes and by 287% in preadipocytes （P〈0.01 vs PBS）. FFA at concentration of 0.125 mmol/L did not change ASP-stimulated glucose transport significantly,but high dose of oleate or palmitate effectively reduced the ASP response with a significant reduction by 47% （P〈0.05 for oleate） and 34% （P〈0.05 for palmitate） at 1 mmol/L FFA in adipocytes. Similarly in preadipocytes,glucose uptake rates were decreased by 43% （P〈0.05 for oleate） and 62% （P〈0.01 for palmitate） at 1 mmol/L FFA. Effects were comparable to those obtained with insulin. After overnight incubation with oleate or palmitate in adipocytes and preadipocytes,Gβ,Gαq/11,p-PKCα and p-PKCζ were downregulated both in the absence of ASP treatment and in the presence of ASP treatment in adipocytes. At concentration of 1.0 mmol/L,oleate inhibited the expressions of ASP-induced Gβ,Gαq/11,p-PKCα and p-PKCζ in adipocytes by 47%,44%,39% （P〈0.05,P〈0.01） and 20% （P〈0.05）,respectively. Palmitate also effectively blocked the expressions of ASP （at concentration of 1.0 mmol/L）-induced Gβ,Gαq/11,p-PKCα and p-PKCζ by 50%,43%,44% and 43% （P〈0.05,P〈0.01） in adipocytes. In preadipocytes,oleate only inhibited ASP-induced p-PKCα and p-PKCζ significantly by 39% and 19%,respectively （P〈0.05）. However,overnight exposure of 3T3-L1 preadipocytes to 1 mmol/L palmitate leaded to 45%,50%,52% and 21% （P〈0.05,P〈0.01） inhibition of ASP-induced expressions of Gβ,Gαq/11,p-PKCα and p-PKCζ,respectively. CONCLUSION： Oleate and palmitate inhibit ASP-mediated stimulation of glucose transport both in adipocytes and preadipocytes. The study provides direct evidence of ASP resistance under the condition of insulin resistance induced by FFA in a cellular model. The mechanism of action involves both changes in expression of C5L2 as well as signaling parameters. Fatty acid-induced ASP resistance may contribute to the physiological abnormalities associated with insulin resistance and obesity phenotype.