舒林酸调节IKK通路对3T3-L1细胞IRS-1酪氨酸磷酸化的影响

Effect of sulindac on tyrosine phosphorylations of IRS-1 in 3T3-L1 adipocytes by regulating IKK pathway

目的探讨舒林酸通过调节IKK通路对分化成熟3T3-L1细胞胰岛素受体后信号转导蛋白胰岛素受体底物1(IRS-1)蛋白酪氨酸/丝氨酸(Tyr/Ser)残基磷酸化表达的影响。方法用地塞米松、IBMX和胰岛素三联培养诱导3T3-L1前脂肪细胞分化为成熟脂肪细胞,油红O染色观察脂肪细胞形态。诱导分化成熟的脂肪细胞如下分组干预,实时荧光定量PCR检测不同浓度炎症因子IL-1β(0,1,10,100 ng/ml)和(或)不同浓度IKK特异阻断剂舒林酸(0,0.1,1,10 mmol/L)对诱导分化成熟的脂肪细胞IKK通路激活状态的影响。Western Blot检测IL-1β和(或)舒林酸对诱导分化成熟的脂肪细胞IRS-1酪氨酸/丝氨酸残基磷酸化状态的影响。采用单因素方差分析进行统计学分析。结果实时荧光定量PCR和Western Blot结果显示,IL-1β10 ng/ml组诱导成熟脂肪细胞IKKβmRNA较对照组相对表达水平增加,分别为[(2.85±0.16)%,(1.00±0.12)%,P <0.01];而IRS-1酪氨酸的磷酸化相对表达量较对照组下降,分别为[(0.72±0.26)%,(1.00±0.24)%,P <0.01]。进一步予舒林酸(1 mmol/L、10 mmol/L)干预后较对照组显著逆转IL-1β诱导脂肪细胞IRS-1酪氨酸磷酸化的表达水平,分别为[(1.72±0.16)%,(1.90±0.08)%,(1.00±0.13)%,P <0.01],同时下调IRS-1丝氨酸磷酸化的表达水平[(0.79±0.16)%,(0.66±0.08)%,(1.00±0.10)%,P <0.05]。结论IL-1β通过促进诱导分化成熟脂肪细胞IKKβ的表达,激活脂肪细胞IKK炎症通路,抑制脂肪细胞IRS-1酪氨酸残基磷酸化的表达,舒林酸通过调节脂肪细胞IRS-1酪氨酸/丝氨酸残基磷酸化的表达,改善脂肪细胞胰岛素受体后信号转导。

Objective To investigate the effects of sulindac on tyrosine/serine phosphorylations of insulin postreceptor signal transducin IRS-1 in 3T3-L1 adipocytes by regulating the activity of IKK pathway in vitro. Methods 3T3-L1 pre-adipocytes were incubated with isobuthyl-methylxanthine, dexamethasone, insulin and differentiated into mature adipocytes as determined by Oil Red O staining. The differentiated maturate adipocytes were divided into the following groups with or without various concentrations of IL-1β(0, 1, 10, 100 ng/m1). Then the adipocytes of different groups were treated with different concentrations of sulindac(0, 0.1, 1,10 mmol/l)at the different time points and subjected to real-time RT-PCR and Western Blot analysis for IKK as well as the IRS-1 Tyr/Ser phosphorylation expression. Statistical analysis was performed using one-way ANOVA procedure. Results Compared with control group, the relative expression of mRNA expression of IKKβ was significantly increased in IL-1β 10 ng/ml treatment 3T3 L1 adipocytes[(2.85±0.16)% vs(1.00±0.12)%,P < 0.01] while IRS-1 tyrosine phosphorylation obviously decreased, the difference was statistically significant [(0.72±0.26)% vs(1.00±0.24)%,P < 0.01]. Further Western Blot results showed that the sulindac treatment significantly up-regulated the relative expression of IRS-1 impaired tyrosine phosphorylation induced by IL-1β induction[(1.72±0.16)%,(1.90±0.08)% vs(1.00±0.13)%,P < 0.01] but decreased IRS-1 serine phosphorylation[(0.79±0.16)%,(0.66±0.08)% vs(1.00±0.10)%, P < 0.05]. Conclusions These data suggest that IL-1β can promote the expression of IKKβ in adipocytes, activate the inflammatory pathway of IKK in adipocytes and inhibit the phosphorylation of IRS-1 tyrosine residues which may be one of the causes of insuliresistance, while sulindac can improve the insulin postreceptor signal transduction pathway in adipocytes by regulating the tyrosine/serine residues phosphorylation of IRS-1 in adipocytes.