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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。
OBJECTIVE: To observe the effects of different concentrations of monounsaturated fatty acids (oleic acid) and saturated fatty acids (palmitic acid) on glucose transport in 3T3-L1 (pre) adipocytes and to explore the role of high free fatty acid (FFA) The significance of resistance formation and the mechanism of FFA-induced ASP resistance in 3T3-L1 (pre) adipocytes. Methods: The 3T3-L1 cells were cultured in vitro and induced to differentiate. The 3T3-L1 (pre) adipocytes were treated with different concentrations of FFA. After overnight incubation, cells were harvested and stained with 2-deoxy- [3H] -D-glucose. The basal state of 3T3-L1 mature adipocytes and preadipocytes and the glucose uptake rate of ASP stimulated state were observed. The basal state and ASP-stimulated guanylate binding protein alpha-q / 11 (Gαq / 11) were detected by Western blotting, (Gβ), phospho-protein kinase Calpha (p-PKCα) and phosphorylated protein kinase Czeta (p-PKCζ) protein expression. Results: After stimulated with ASP, glucose uptake rate of 3T3-L1 mature adipocytes and preadipocytes was 1.98 times (P <0.01) and 2.87 times (P <0.01) higher than basal state respectively. Low concentration of FFA did not affect ASP-stimulated glucose transport; while the glucose uptake rate of ASP stimulated by ASP in 1.0 mmol / L group was decreased by 47% (P <0.05) and 34% (P <0.05), respectively, , And preadipocyte glucose uptake decreased by 43% (P <0.05) and 62% (P <0.01), respectively. 1.0mmol / L oleate and palmitate inhibited the basal state of mature adipocytes and the expression of G|Á, G|Áq / 11, p-PKC|Áand p-PKC|Ä protein by ASP stimulation, while the protein expression of ASP stimulated by oleic acid group decreased by 47% (P <0.01), 44% Gαq / 11 (P <0.05), 39% p-PKCα and 20% p-PKCζ, respectively 0.05 or P <0.01). In preadipocytes, oleic acid only inhibited the protein expression of p-PKC|Á and p-PKC|Âaspeptide stimulated by ASP (all P <0.05), while palmitate down-regulated the expression of the above four signal proteins (P <0.05 or P <0.01). CONCLUSION: Oleic acid or palmitate inhibit ASP-stimulated glucose transport in 3T3-L1 mature adipocytes and preadipocytes, demonstrating that both ASP and FFA induce insulin resistance in adipocytes. The mechanism of FFA-induced ASP resistance is related to its interference with ASP-C5L2 signal transduction pathway. ASP resistance is involved in the pathophysiological process of “lipotoxicity” - insulin resistance / obesity.