Effects Of Different Kinds Of Free Fatty Acids On TXNIP Expression In INS-1 Islet Cells

Background:The prevalence of diabetes has increased year after year,which has become a serious threat to human health.Pancreatic ?-cell apoptosis is the key factor in the development of diabetes.Recent studies have shown that long-term high levels of free fatty acids(FFAs),especially saturated fatty acids,can cause insulin resistance,islet?-cell dysfunction,and the increase in beta-cell apoptosis in type 2 diabetic patients.Thioredoxin interacting protein(TXNIP),called VDUP1(Vitamin D(3)up-regulated protein 1)and TBP2(thioredoxin-binding protein-2),belongs to the protein family of a-arrestins,which was found to be the only endogenous Thioredoxin(Trx)binding protein.It was highly expressed in diabetes tissues.There were a large number of studies shown that TXNIP expression was significantly increased in response to high glucose,and it play an important role in inducing P-cell apoptosis.However,FFAs as one of the major causes of diabetes,its effect on TXNIP expression and the possible differences are unclear.Therefore,the aim of this study is to research the effects of different free fatty acids on TXNIP expression in INS-1 cells and its possible mechanism.Objective:1.The different saturation of free fatty acids were used to culture INS-1 cells,observing the effects of FFAs on INS-1 cell apoptosis and TXNIP expression.2.To analyze the mechanisms of FFAs affecting TXNIP expression.Methods:1.Groups:INS-1 cells were divided into four groups:normal control group(RPMI1640 medium containing 0.55%fatty acid free-BSA),saturated fatty acid-palmitate group(0.5 mmol/L palmitate+0.55%fatty acid free-BSA),monounsaturated fatty acids-palmitoleic acid(0.5 mmol/L palmitoleic acid+0.55%fatty acid free-BSA)and polyunsaturated fatty acid-DHA group(0.5 mmol/L DHA+0.55%fatty acid free-BSA).After cultured for 24 h,cells were collected for further assay.2.Real-time PCR was used to measured TXNIP mRNA expression in INS-1 cells incubated with FFAs.3.Western blot was used to test the expression of TXNIP,caspase-3,cleaved caspase-3,ChREBP,FOXO1,p-NF-?B in INS-1 cells incubated with FFAs.4.The expression of ChREBP in INS-1 cells incubated with FFAs were measured by immunofluorescence assay.5.Flow cytometry which measured with Annexin V-FITC Apoptosis Detection Kit were used to analysis the apoptosis of INS-1 cells.Results:1.Differences in the saturation of fatty acids have different effects on TXNIP expression.Compared with the control group,TXNIP mRNA and protein expression were significantly increased in saturated fatty acid-palmitate group.While,the expression of TXNIP in unsaturated fatty acid palmitoleic acid group and DHA group was not significantly changed(figure 1).As shown in Fig.2,the expression of TXNIP induced by palmitate was increased at 18 h,and peaked at 24 h.At 36 h,the TXNIP expression was decreased to the baseline.While,there was no significant difference in the expression of TXNIP between palmitoleic acid and DHA at 12h,18h,24h,36h.2.The effect of different saturation of free fatty acids on INS-1 cells apoptosis.As shown in Fig.3,the ratio of cleaved caspase-3/caspase-3 in palmitate group was significantly increased when compared with that in control group.But,there was no difference in palmitoleic acid group and DHA group.According to the flowcytometry results,the apoptotic index of INS-1 cells in palmitate not in palmitoleic acid group and DHA group was significantly higher than in control group.(figure 4)3.Effects of palmitate on the expression of transcription factors ChREBP and FOXO1 protein.ChREBP expression in palmitate-treated INS-1 cells was examined by western blot and immunofluorescence,the results showed that palmitate increased ChREBP expression.The expression of FOXO1 was decreased in palmitate group compared with control group.(figure 5-6)4.NF-?B mediates palmitate induced TXNIP expression.Compared with the control group,the expression of p-NF-?B p65 was significantly increased in palmitate group(figure 7).As shown in Fig.8,INS-1 cells were incubated with palmitate in the presence or absence of NF-?B inhibitor PDTC.The inhibitor significantly reduced TXNIP mRNA and protein expression compared with the non-inhibited group.Similarly,a NF-?B permeable inhibitory peptide,SN50,also reduced the upregulation of palmitate-induced TXNIP expression(figure 9).At the same time,we found that PDTC significantly reduced the up-regulation of ChREBP induced by palmitate(figure 10),but had no effect on the expression of FOXO1(figure 11).5.p38 MAPK is relative to palmitate induced TXNIP expression.Compared with the non-inhibited,p38 MAPK inhibitor SB203580 significantly decreased the TXNIP mRNA and protein expression up-regulated by palmitate(figure 12).And the p38 inhibitor SB203580 completely abolished the effect of palmitate on NF-?B phosphorylation(figure 13),blocked the palmitate regulation on ChREBP and FOXO1 expression(figure 14,15).Conclusion:1.The saturated fatty acid-palmitate not the unsaturated fatty acid-palmitoleic acid or DHA promoted INS-1 islet cell apoptosis by up-regulating TXNIP expression.2.Palmitate regulation of TXNIP expression may be mediated by activating of the p38MAPK/NF-?B/ChREBP signaling and FOXO1 also maybe participate in the regulation of TXNIP expression.