肿瘤坏死因子α诱导3T3-L1脂肪细胞胰岛素抵抗的机制

Mechanisms of TNF-α-induced insulin resistance in 3T3-L1 adipocytes

目的探讨内源性硫化氢(H2S)在肿瘤坏死因子-α(TNF-α)诱导的3T3-L1脂肪细胞胰岛素抵抗中的作用。方法 3T3-L1前脂肪细胞诱导分化成熟后,分别加入0、10 ng/ml、50 ng/ml、100 ng/ml TNF-α培养24 h,干预组在加入50 ng/ml TNF-α前30 min分别加入胱硫醚γ-裂解酶(CSE)抑制剂炔丙基甘氨酸(PPG,10 mmol/L)和二辛可宁酸(BCA,2 mmol/L)。上述处理后各组细胞用胰岛素刺激。另设无TNF-α和胰岛素作用的对照组。用葡萄糖氧化酶法检测培养液中葡萄糖消耗,2-脱氧-[3H]-D-葡萄糖摄入法检测葡萄糖摄取,亚甲基蓝法测定检测细胞H2S含量。结果与对照组比较,3T3-L1脂肪细胞胰岛素刺激的葡萄糖消耗[(32.43±2.60)%vs(5.31±1.87)%,P<0.01]和摄取[(7.91±0.31)cpm vs(1.38±0.13)cpm,P<0.01]明显增加,10 ng/ml、50 ng/ml、100 ng/ml TNF-α作用后脂肪细胞葡萄糖消耗[(23.27±1.41)%、(14.73±3.76)%、(7.81±1.56)%vs(32.43±2.60)%,P<0.01)和摄取[(5.86±0.95)cpm、(2.60±0.35)cpm、(1.87±0.17)cpm vs(7.91±0.31)cpm,P<0.01)呈剂量依赖性减少;而H2S生成量呈浓度依赖性增加[(0.06±0.002)mmol/(g·protein)、(0.11±0.003)mmol/(g·protein)、(0.14±0.005)mmol/(g·protein)vs(0.17±0.001)mmol/(g·protein),P<0.01)]。PPG和BCA预处理分别使脂肪细胞对胰岛素刺激的葡萄糖消耗[(24.52±2.63)%、(27.84±1.82)%vs(14.73±3.76)%,P<0.01]和葡萄糖摄取[(7.22±0.27)cpm、(7.96±0.19)cpm vs(2.60±0.35)cpm,P<0.01]显著增加。结论内源性H2S在TNF-α诱导的脂肪细胞胰岛抵抗中发挥介导作用,抑制内源性CSE/H2S体系可能是治疗胰岛素抵抗的新靶点。

Objective To demonstrate the roles of hydrogen sulfide （H2S） on TNF-α-induced insulin re-sistance in 3T3-L1 adipocytes. Methods The differentiated 3T3-L1 adipocytes were incubated without or with 100 nmol/L insulin for 30 min periods. For experiments with TNF-αtreatment, different concentrations of TNF-α（0, 10 ng/ml, 50 ng/ml, 100 ng/ml） were added for 24 h and then treated with 100 nmol/L insulin for 30 min. For the inter-vention group, 3T3-L1 adipocytes were pretreated with the cystathionineγ-lyase （CSE） inhibitor DL-propargylgly-cine （PPG, 10 mmol/L） or beta-cyano-L-alanine （BCA, 2 mmol/L） for 30 min prior to treatment with 50 ng/ml of TNF-α for 24 h and insulin stimulation （100 nmol/L, 30 min）. The glucose consumption in the medium was mea-sured by glucose oxidase method, and the glucose uptake were detected by 2-[3H]-DG method. The content of H2S in culture supernatant was measured by the methylene blue method. Results Compared with the control group, in-sulin-induced glucose consumption [（32.43 ± 2.60）% vs （5.31 ± 1.87）%, P〈0.01）] and uptake [（7.91 ± 0.31） cpm vs （1.38±0.13） cpm, P〈0.01）] in 3T3-L1 adipocytes were significantly higher. After treatment with TNF-α（10 ng/ml, 50 ng/ml, 100 ng/ml）, the insulin-induced glucose consumption [（23.27 ± 1.41）%, （14.73 ± 3.76）%, （7.81 ± 1.56）% vs （32.43±2.60）%, P〈0.01] and uptake [（5.86±0.95） cpm, （2.60±0.35） cpm, （1.87±0.17） cpm vs （7.91±0.31） cpm, P〈0.01）] were significantly decreased in a dose-dependent manner, while endogenous H2S generation increased in a dose-de-pendent manner [（0.06 ± 0.002） mmol/（g·protein）, （0.11 ± 0.003） mmol/（g·protein）, （0.14 ± 0.005） mmol/（g·protein） vs （0.17±0.001） mmol/（g·protein）, P〈0.01）]. Pretreatment with the CSE inhibitor PPG （10 mmol/L） or BCA （2 mmol/L） for 30 min respectively resulted in significant increase in insulin-induced glucose consumption [（24.52 ± 2.63）%, （27.84±1.82）%vs （14.73±3.76）%, P〈0.01] and uptake [（7.22±0.27） cpm, （7.96±0.19） cpm vs （2.60±0.35） cpm, P〈0.01] in 3T3-L1 adipocytes. Conclusion Endogenous H2S mediates TNF-α-induced insulin resistance in 3T3-L1 adipocytes, and inhibition of endogenous CSE/H2S system may be a novel therapeutic target for insulin resistance.