The Function Of Tph1 And Hsp90 In Islet β Cells Under High Glucose

Background and Objective Dysfunction of pancreatic β cell and insulin resistance of peripheral tissue are two main players in the pathogenesis of type 2 diabetes,while recent studies showed that the former plays a more important role.Glucose is the most essential regulator of pancreatic β-cell function.In the presence of high glucose,islet function is firstly enhanced,which increases insulin synthesis and secretion together with the subsequent β cells hypertrophy and proliferation,to keep glucose levels within normal range.Therefore,a better understanding of the molecular mechanisms of β-cell functional compensation under high glucose condition is significantly important for the prevention and treatment of type 2 diabetes.In the previous study,we found that under high glucose level,the expression of tryptophan hydroxylase 1(Tph1)in rat islets was significantly induced both in vitro and in vivo,which was accompanied by increased synthesis of serotonin.The regulatory effect of Tph1 is dose and time dependent.Overexpression of Tph1 in isolated rat islets can obviously increase insulin secretion.In this study,we intraperitoneally injected Tph1 inhibitor in high glucose-infused rat to further elucidate the effect of Tph1 on enhancing islet function under high glucose in vivo.We also built the rat model with pancreatic β cell-specific overexpression of Tph1.In addition,our previous microarray of rat islet under high glucose results revealed a marked reduction of heat shock protein 90(Hsp90)expression.As a consequence,we further explored the involvement of Hsp90 in the regulation of pancreatic β-cell functional compensation under high glucose condition.Materials and Methods 1.SD rats were intraperitoneally injected with Tph1 inhibitor(PCPA)or 0.45% saline on 2 consecutive days,and infused with 50% glucose or 0.45% saline for 24 h,then blood glucose and insulin levels were detected.Western blot,real-time PCR and immunohistochemistry were used to measure the mRNA and protein level of Tph1.Islets serotonin level was also measured.2.We generated the rat with pancreatic β cell-specific overexpression of Tph1,and performed the genotyping after 4 weeks of birth,which helped to establish a stable genetic background;3.We monitored the metabolic profile including body weight,food intake and random blood glucose in 10-week-old rats.The intraperitoneal glucose tolerance test(IPGTT)and insulin tolerance test(ITT)were performed.Blood glucose was determined from tail vein bleeds.Blood samples from tail vein were collected for insulin assay by a rat insulin ELISA kit.4.Isolated rat islets were treated with 3.3 and 16.7mmol/L glucose for 1h,insulin secretion was measured by ELISA;5.Western blot detected the expression of Tph1 protein extracted from different tissues of rats;6.After the isolated islets were pre-treatment with different concentrations of glucose,the mRNA and protein expression of Hsp90 were measured by real-time quantitative PCR and Western blot;7.The mRNA level of Hsp90 was detected after rat islets were treated with agonists and inhibitors of different signaling pathway;8.After rat islets were pretreated with Hsp90 inhibitors(17-DMAG and CCT018059),the secretion of insulin,as well as the expression levels of genes related to islets function were detected;9.Immunohistochemistry staining revealed the expression levels of Tph1,insulin,serotonin and Hsp90 in rat islets.Results 1.Tph1 inhibitor PCPA obviously inhibited the glucose stimulated serotonin synthesis and insulin secretion in vivo and further elevated the blood glucose;2.Tph1 was highly and specifically expressed in transgenic rats;3.Compared to wild-type rats,rats with Tph1 overexpression had no difference in body weight,food intake,fasting glucose and basal insulin secretion.However,the postprandial blood glucose was decreased in Tph1 transgenic rats,while fed serum insulin was raised;4.IPGTT revealed a significantly enhanced glucose tolerance in Tph1 transgenic rats,along with a parallel increase in glucose-stimulated insulin secretion(GSIS).ITT showed insulin sensitivity was comparable between transgenic rats and wild-type rats;5.The synthesis of serotonin was elevated in islets of Tph1 transgenic rats;6.The isolated islets from Tph1 transgenic rats released more insulin in response to high glucose concentrations compared with those from wild-type rats;7.Immunofluorescence staining of rat pancreatic sections indicated that Hsp90 was highly expressed in islet β cells;Microarray results showed that high glucose inhibited the mRNA expression of Hsp90 in rat islets,real-time quantitative PCR and Western blot confirmed the microarray results.Real-time PCR showed 5.6mmol/L glucose partly down-regulated Hsp90 expression in rat islets,and a glucose concentration of more than 8.3mmol/L had no further effect on Hsp90;8.Glucokinase(GK)agonist inhibited the expression of Hsp90,while calcium channel modulators,non-metabolic glucose analogs had no effect on Hsp90;9.Hsp90 inhibitor treatment significantly potentiated GSIS in rat islets,and the expression of genes related to islet function were changed.Conclusions 1.Pancreatic β cell-specific Tph1 overexpressed transgenic rats had an enhanced islet β cell secretory function as a result of the stimulated synthesis of serotonin,which improved glucose tolerance without affecting insulin sensitivity;2.Inhibiting Tph1 activity can reduce the glucose-induced insulin secretion in high glucose-infused rats;3.High glucose level down-regulates the expression of Hsp90 in pancreatic β cell through the activation of GK;4.Hsp90 inhibition enhanced GSIS in rat islets via the alteration of the expression of islet functional genes.