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目的探讨抵抗素对大鼠肝细胞糖异生代谢关键酶-磷酸烯醇式丙酮酸羧激酶(PEPCK)活性及mRNA表达的影响,了解抵抗素对肝脏糖代谢的影响并初步探讨抵抗素在肝脏胰岛素抵抗形成中的作用及机制。方法正常大鼠的肝细胞,不加抵抗素与加不同剂量的抵抗素(10、50、100 nmol/L)培养24 h。乳酸脱氢酶偶联比色法检测不同剂量抵抗素对PEPCK活性的影响;逆转录-聚合酶链反应(RT-PCR)检测PEPCK mRNA表达水平的变化。结果抵抗素浓度为10、50 nmol/L时,PEPCK活性[单位为mIU/(min.mg protein)]较空白对照组显著升高(31.8±2.4,32.0±2.3 vs 26.6±2.6,P<0.05),抵抗素浓度为100 nmol/L,PEPCK活性升高更明显(40.4±1.5 vs 26.6±2.6,P<0.01)。RT-PCR结果显示抵抗素浓度为10、50 nmol/L时,PEPCK mRNA表达水平(以β-actin校正)与空白对照组比较差异无统计学意义(P﹥0.05),抵抗素浓度为100 nmol/L,PEPCK mRNA表达水平显著升高(1.18±0.12 vs 0.74±0.08,P﹤0.01)。结论抵抗素浓度为10、50 nmol/L时,PEPCK活性显著升高,但对其mRNA表达水平无显著影响。抵抗素浓度为100 nmol/L时,PEPCK活性升高更明显,同时其mRNA表达水平也显著升高。表明该浓度的抵抗素至少部分通过促进PEPCK基因的表达实现对酶活性的调节。
Objective To investigate the effects of resistin on the activity of phosphoenolpyruvate carboxykinase (PEPCK), a key enzyme of gluconeogenesis in rat hepatocytes, and investigate the effect of resistin on hepatic glucose metabolism and to explore the role of resistin in liver The role of insulin resistance in the formation and mechanism. Methods Normal rat hepatocytes were incubated with different concentrations of resistin (10, 50, 100 nmol / L) for 24 h. The effects of different doses of resistin on the activity of PEPCK were detected by lactate dehydrogenase coupled colorimetric assay. The changes of PEPCK mRNA expression were detected by reverse transcription-polymerase chain reaction (RT-PCR). Results When the concentration of resistin was 10,50 nmol / L, the activity of PEPCK was significantly increased (mIU / min.mg protein) compared with the blank control group (31.8 ± 2.4,32.0 ± 2.3 vs 26.6 ± 2.6, P <0.05 ), The concentration of resistin was 100 nmol / L, the activity of PEPCK increased more obviously (40.4 ± 1.5 vs 26.6 ± 2.6, P <0.01). RT-PCR results showed that there was no significant difference in PEPCK mRNA expression (P-actin corrected) compared with blank control group (P> 0.05) at resistin concentration of 10 and 50 nmol / L, resistin concentration was 100 nmol / L, PEPCK mRNA expression was significantly increased (1.18 ± 0.12 vs 0.74 ± 0.08, P <0.01). Conclusion When the concentration of resistin is 10,50 nmol / L, the activity of PEPCK is significantly increased, but it has no significant effect on the mRNA expression level. When the concentration of resistin was 100 nmol / L, the activity of PEPCK increased more obviously, and its mRNA expression level also increased significantly. Resistin at this concentration indicated that modulation of enzyme activity was achieved, at least in part, by promoting the expression of the PEPCK gene.