促酰化蛋白对3T3-L1脂肪细胞炎性反应的影响

Acylation Stimulating Protein Regulates the Inflammation in 3T3- L1 Adipocytes

目的观察3T3-L1成熟脂肪细胞胰岛素抵抗状态下促酰化蛋白(acylation stimulating protein,ASP)对炎性因子(IL-6、MCP-1、MIP-1α和TNF-α)分泌水平以及炎性信号因子(JNK1、IKKβ)蛋白表达的影响。方法 3T3-L1成熟脂肪细胞分别给予不同浓度(0、0.125、0.5、1.0mmol/L)油酸(C18∶1)或棕榈酸(C16∶0)温育过夜,诱导胰岛素抵抗,在此基础上给予ASP刺激,用ELISA测定IL-6、MCP-1、MIP-1α和TNF-α4种细胞炎性因子的分泌水平,采用Western blot法检测JNK1和KKβ蛋白表达。结果油酸诱导的胰岛素抵抗下,ASP刺激的脂肪细胞IL-6和MCP-1的分泌水平轻度下调,但差异无统计学意义;而ASP刺激的脂肪细胞MIP-1α和TNF-α的分泌水平显著下降,MIP-1α和TNF-α的分泌分别下调57%(P<0.05)和48%(P<0.05)(1.0mmol/L油酸组)。棕榈酸诱导的胰岛素抵抗下,ASP刺激的脂肪细胞IL-6分泌水平呈下降趋势,最大下调50%IL-6(P<0.05);22%MCP-1(P>0.05),35%MIP-1α(P>0.05)和38%TNF-α(P>0.05)的分泌。高浓度(1.0mmol/L)油酸和棕榈酸诱导的脂肪细胞胰岛素抵抗状态下,ASP刺激的炎性信号蛋白JNK1蛋白表达显著下调,分别为34%(P<0.05)和54%(P<0.01)。但IKKβ蛋白表达差异无统计学意义。结论在脂肪酸诱导的脂肪细胞胰岛素抵抗状态下,ASP在一定程度上调节炎性因子分泌,参与调节JNK、IKK/NF-κB信号通路中重要信号分子的功能,ASP参与了脂肪细胞脂毒性-炎性反应的调控。

Objective To study the changes of ASP on the inflammatory factors expression in 3T3 - L1 adipocytes under the conditions which produce insulin resistance by free fatty acids. Methods 3T3 - L1 preadipocytes were induced differentiated by cocktail hormones containing 10μg/ml insulin, 1 μmol/L dexamethasone and 0. 5mmol/L isobutylmethylxanthine. Then Ommol/L （ FFA - freeD- MEM/F12） , 0. 125mmol/L, 0.5mmol/L and 1.0mmol/L oleate or palmitate was added to cultured 3T3 -L1 adipocytes overnight. Both non - FFA treated and FFA treated 3T3 - L1 ceils were cultured with 1.0trmol/L ASP for 2 hours. Then the cultured media and cell proteins were extracted. Interlukin （IL） - 6, monocyte chemoattractant protein （MCP） - 1, macrophage inflammatory protein （MIP） - 1α, and tumor necrosis factor （TNF） -α concentrations were measured using mouse EL1SA kits according to the manufacturer＇s instructions. The c-Jun nterminal kinase （JNK） 1 and inhibitor of nuclear factor kappa - B kinase（IKK） β protein expression was measured by western blot. Results Oleate did not change ASP - stimulated IL - 6 and MCP - 1 secretion significantly in 3T3 - L1 adipocytes, but high dose of oleate effectively reduced the ASP - stimulated MIP - 1α and TNF - α secretions by 57% （ P 〈 0. 05 ） and 48% （ P 〈 0.05 ） at lmmol/L oleate in adipocytes. On palmitate groups, ASP - stimulated IL - 6 secretion was decreased by dose - response and reached a maximal suppression by 50% （P 〈 0.05,1. Ommol/L）. However, ASP- stimulated M CP- 1, MIP- 1α, and TNF-α secresions had no signifi- cant change. In 3T3 - L1 adipocytes, high dose of oleate or palmitate effectively reduced the ASP - induced JNK1 protein expression with a significant reduction by 34% （P 〈 0.05 for oleate）and 54% （ P 〈 0.01 for palmitate） at 1.0mmol/L FFA. However, overnight expo- sure of 3T3 - L1 adipocytes to FFA leaded to little inhibition（P 〉 0.05） of ASP - induced IKKβ protein expression. Conclusion ASP showed a function not only on inflammatory factors secretion, but also on some signal molecules changes in JNK signal pathway under the condition of insulin resistance induced by FFA in a cellular model. The study provides direct evidence of ASP involves in moderating the lipotocity -inflammation in adipocytes.