| Objective:Type 2 diabetes mellitus（T2D）is a chronic metabolic disease characterized by relative insufficient insulin secretion and insulin resistance caused by obesity,smoking,and lack of exercise.Loss of isletβ-cell function is a determinant of the transition from an obese and insulin resistant state to T2D.Serum amyloid protein 1（SAA1）is a stress protein with pro-inflammatory properties,which promotes the occurrence of obesity,diabetes,rheumatoid arthritis and metabolic syndrome related diseases by promoting the activation of immune cells and endothelial cell damage.SAA1 can induce the translocation of p65 from the cytoplasm to the nucleus to activate the NF-κB signaling pathway,and the abnormal activation of NF-κB can lead to the loss of pancreaticβcell function.The aim of this study was to investigate whether SAA1 contributes to the loss of isletβfunction in HFD-induced T2D via NF-κB signaling.Methods:1.After C57 mice were fed with high-fat diet for 24 weeks,body weight,fasting glucose and glucose tolerance were measured,and whether the T2D mouse model was successfully established was judged according to the test results.2.ELISA and Real time-PCR were performed to detect the expression level of SAA1 in serum and liver of T2D mice and to observe the changes of SAA1 in T2D.3.After MIN6 cells and isolated mouse islets were treated with SAA1,the m RNA expression levels of insulin,IL-1β,TNF-α,Bax and Bcl-2 were detected by Real time-PCR to verify whether SAA1 affected the function of MIN6 cells and islets.4.Western blot was used to detect the protein expression levels of My D88,P-IKKα/β,IKKα/β,P-IKBα,Ik Bα,P-p65 and p65 in MIN6 cells after treatment with SAA1,SAA1+BAY and BAY.To verify whether SAA1 could activate NF-κB signaling in MIN6 cells.5.After MIN6 cells were treated with SAA1,SAA1+BAY,or BAY,the m RNA expression levels of insulin,IL-1β,TNF-α,Bax,and Bcl-2 in MIN6 cells were detected by Real time-PCR.The insulin secretion function of MIN6 cells stimulated by glucose was detected by ELISA.Western blot was used to detect the protein expression levels of apoptosis-related molecules Bax,caspase 3 and cleaved-caspase 3 in MIN6 cells to verify whether SAA1 affected the function of MIN6 cells through NF-κB signaling pathway.6.The isolated mouse islets were treated with SAA1,SAA1+BAY,and BAY,and the protein expression levels of My D88,P-IKKα/β,IKKα/β,P-IKBα,Ik Bα,P-p65,and p65 in the NF-κB signaling pathway were detected by Western blot.To verify whether SAA1 could activate NF-κB signaling in MIN6 cells.7.The isolated mouse islets were treated with SAA1,SAA1+BAY,or BAY,and the m RNA expression levels of insulin,IL-1β,TNF-α,Bax,and Bcl-2 were detected by Real time-PCR.The insulin secretion of islets stimulated by glucose was detected by ELISA.Western blot was used to detect the protein expression levels of apoptosis-related molecules Bax,caspase 3 and cleaved-caspase 3 in islets to verify whether SAA1 affected islet function through NF-κB signaling pathway.8.SAA1-/-mice and WT mice were fed with high-fat diet for 24 weeks to establish T2D models.The body weight and fasting blood glucose of the mice were detected to determine the effect of SAA1 gene deletion on T2D progression.9.SAA1-/-mice and WT mice were fed with high-fat diet for 24 weeks,and islets were collected.The m RNA expressions of insulin,IL-1β,TNF-α,Bax and Bcl-2 in islets were detected by Real time-PCR.Western blot was used to detect the protein expression levels of apoptosis-related molecules Bax,caspase 3 and cleave-caspase 3in islets to verify the effect of SAA1 deficiency on islet dysfunction induced by high-fat diet.10.SAA1-/-mice and WT mice were fed with high-fat diet for 24 weeks.Islets were collected,and the expression of P-p65 in islets was detected by immunofluorescence staining.The protein expression levels of My D88,P-IKKα/β,IKKα/β,P-IKBα,Ik Bα,P-p65 and p65 in pancreatic islets were detected by Western blot to observe the effect of SAA1 deletion on the activation of NF-κB signaling pathway in pancreatic islets induced by high-fat diet.Results:1.High-fat diet mice had increased body weight,increased fasting glucose and decreased glucose tolerance,and a high-fat diet-induced T2D mouse model was successfully established.2.The expression of SAA1 in the serum and liver of high-fat diet mice was significantly increased,and T2D resulted in increased SAA1 levels in mice.3.After SAA1 stimulation,the expression of insulin and Bcl-2 was decreased,and the expression of IL-1β,TNF-αand Bax was increased.SAA1 aggravated the dysfunction and inflammatory response of MIN6 cells and islets.4.After MIN6 cells were treated with SAA1,SAA1+BAY,and BAY,the levels of My D88,P-IKKα/β,P-IKKα,and P-p65 in SAA1-induced MIN6 cells were higher than those in untreated MIN6 cells.However,BAY inhibited this effect of SAA1,indicating that SAA1 could induce activation of NF-κB signaling in MIN6 cells.5.Real time-PCR results showed that SAA1+BAY treatment resulted in increased expression of insulin and Bcl-2 and decreased expression of IL-1β,TNF-αand Bax in MIN6 cells compared with SAA1 stimulation alone.In addition,SAA1-treated MIN6cells significantly inhibited glucose-stimulated insulin secretion,which was alleviated by BAY.Moreover,BAY inhibited the expression of cleaved caspase-3 and Bax induced by SAA1.These results indicated that SAA1 affected the function of MIN6cells through NF-κB signaling pathway.6.The levels of My D88,P-IKKα/β,P-IKKαand P-p65 in SAA1-induced islets were higher than those in untreated islets after treatment with SAA1,SAA1+BAY and BAY.However,this effect of SAA1 was inhibited by BAY,indicating that SAA1 could induce NF-κB signaling activation in pancreatic islets.7.Real time-PCR results showed that SAA1+BAY treatment resulted in increased expression of insulin and Bcl-2 and decreased expression of IL-1β,TNF-α,and Bax in pancreatic islets compared with SAA1 stimulation alone.In addition,SAA1-stimulated insulin secretion was significantly inhibited by BAY,which was alleviated by SAA1-stimulated insulin secretion.Moreover,the expression of cleaved caspase-3 and Bax induced by SAA1 was inhibited by BAY.These results indicate that SAA1 affects islet function through NF-κB signaling pathway.8.Real time-PCR results showed that SAA1+BAY treatment resulted in increased expression of insulin and Bcl-2 and decreased expression of IL-1β,TNF-α,and Bax in pancreatic islets compared with SAA1 stimulation alone.In addition,SAA1-stimulated insulin secretion was significantly inhibited by BAY,which was alleviated by SAA1-stimulated insulin secretion.Moreover,the expression of cleaved caspase-3 and Bax induced by SAA1 was inhibited by BAY.These results indicate that SAA1 affects islet function through NF-κB signaling pathway.9.SAA1-/-mice and WT mice were fed with normal diet or high-fat diet,respectively.After 24 weeks,the body weight and fasting blood glucose of SAA1-/-mice and WT fed with high-fat diet increased significantly,indicating that SAA1 gene deletion T2D mouse model was successfully established.10.After HFD feeding,the expression of P-p65 in islets of SAA1-/-mice was lower than that of WT mice,and the protein expression levels of My D88,P-IKKα/β,P-IKBαand P-p65 in islets of WT mice were higher than those of SAA1-/-mice.These results indicate that HFD-induced activation of NF-κB signaling in islets of T2D mice is regulated by SAA1.Conclusions:In this study,we found that high-fat diet-induced T2D mice significantly increased the expression of SAA1 in both serum and liver.In MIN6 cells and isolated mouse islets,SAA1 inhibits insulin secretion and promotes apoptotic molecule expression in MIN6 cells and islets.The NF-κB signaling pathway inhibitor BAY can reduce SAA1’s effect.At the same time,SAA1 deletion can improve the high-fat diet-induced pancreaticβcell dysfunction and reduce the expression of NF-κB signaling molecules.Our results suggest that high-fat diet-induced SAA1 may exacerbate T2D production by enhancing pancreatic beta cell dysfunction,which may depend on activation of the NF-κB signaling pathway. |
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