| Diabetes mellitus(DM) is seriously endangering human's health because of its high incidence, death and disability. Type 2 diabetes makes up most part of the incidence of DM. Insulin resistance(IR) is very common in type 2 diabetes. In recent years, pathogenesis of IR, especially different factors caused attenuation of insulin signaling, has caused attention. Free fatty acid(FFA) takes an important role in the pathogenesis of IR. Studies in vitro and in vivo showed that FFA decreased tyrosin phosphorylation of insulin receptor(InsR), insulin receptor substrate-1 and -2(IRS-1 and IRS-2) in liver, skeletal muscle and fat. Lately researchers hypothesized that IKK- 3 is a key mediator of attenuating insulin signaling and showed that high dose of salicylates, which inhibit IKK- β activity, prevented IR by sensitizing insulin signaling. FFA activate IKK- β and also cause IR. The aim of this study is to examine weather or not aspirin can ameliorate attenuation of insulin signaling caused by FFA in primary cultured rat hepatocytes, study the mechanism of IKK- β mediated IR and aspirin's protective effects.Materials and methodsHepatocytes were isolated with a single two-step perfusion method from Wistarrats' livers and cultured in culture-medium. When cells covered all over the bottom of the culture bottles, they were serum starved for 16 hours and then divided into 4 groups. Group â… was control group, cells were incubated with none-serum-medium. Group â…¡ was palmitic acid group, cells were incubated with none-serum-medium including 25uM palmitic acid. Groupâ…¢ was palmitic acid and aspirin group, cells were incubated with none-serum-medium including 25uM palmitic acid and 5mM aspirin. Group â…£ was aspirin group, cells were incubated with none-serum-medium including 5mM aspirin. When cells were incubated for 2 and 6 hours, they were stimulated with 200 nM insulin for 10 min at each time point, some of these cells were not stimulated by insulin for control. Then cells were chilled and lysed. Proteins were immunoprecipated and separated by SDS-PAGE, and then detected by Western blotting with anti-IRS-2 and phosphotyrosine antibody respectly. Experiments were repeated for 9 times in each group. Results1. Palmitic acid(25μM ) treatment decreased tyrosin phosphorylation of IRS-2 in cultured rat hepatocytes at 2- and 6-hour time point (Western blot Amount were 29.49±3.62 and 20.18±1.57, respectively) compared with control(Western blot Amount were 90.35±3.85 and 90.34±3.66, respectively),p<0.05.2. Palmitic acid(25μM ) treatment decreased protein abundance of IRS-2 in cultured rat hepatocytes at 6-hour time point(Western blot Amount was 55.17±2.48) compared with control(Western blot Amount was 78.95±3.37), p<0.05. But no significant difference was found at 2-hour tune point between palmitic acid group(Western blot Amount was 77.88±3.57) and control(Western blot Amount was 78.95±3.37), p>0.05.3. Decreased tyrosin phosphorylation of IRS-2 induced by palmitic acid(25μM) treatment at 2- and 6-hour time point (Western blot Amount were 29.49 3.62 and 20.18 1.57, respectively) were ameliorated by co-incubating rat hepatocytes with palmitic acid(25μM ) and aspirin(5mM) (Western blot Amount were 79.76±3.91 and 76.02 2.37, respectively). (p<0.05).4. Decreased IRS-2 protein abundance induced by palmitic acid(25μM ) at 6-hour time point(Western blot Amount was 55.17±2.48) was reversed by co-incubating rat hepatocytes with palmitic acid(25μM) and aspirin(5mM) (Western blot Amount was77.67±3.24),p<0.05.5. There was no significant difference in tyrosin phosphorylation of IRS-2 between aspirin group(Western blot Amount were 91.19±3.11 and 92.05±3.74, respectively) and control group(Western blot Amount were 90.35±3.85 and 90.34±3.66, respectively) at 2- and 6-hour time point ,p>0.05.6. No difference was found in protein abundance of IRS-2 between aspirin group(Western blot Amount were 78.39±2.99 and 80.61±3.64, respectively) and control group(Western blot Amount were 78... |
PDF Full Text Request