The Role And Mechanisms Of Txnip In The Effect Of S-Equol On Insulin Secretion In High Glucose Cultured INS-1 Cells

Diabetic mellitus(DM), one of the most common chronic metabolic diseases, is serious harm to human health, which brings heavy mental and financial burden to individuals and their families.The pathogenesis of DM is extremely complex, and it has not yet been fully elucidated. Until now, the insulin resistance and insulin secretory dysfunction have been considered as the two main factors in the pathogenesis of DM, which have become important targets for prevention and treatment of DM. However, most of the clinical drugs used have many adverse reactions. Therefore, constantly looking for new ways for DM prevention and treatment has become the focus of today’s medical research, particularly through dietary means. Equol(Eq) is an important microbial metabolized product of soy isoflavones(SI) which is secondary metabolites formed during soybean growth. Eq includes two isomers S, R and in vivo of animal is S-type[1]. Numerous studies showed that SI intervention could increase insulin secretion, promote glucose uptake and utilization, thus effectively control blood glucose levels, improve diabetes symptoms. Further study found that the biological activity of SI is mainly achieved by equol[2, 3]. Eq has a higher bioavailability and more effective bioacivities than those of SI, including estrogen-like, anti-inflammatory and anti-oxidation effects. It has been demonstrated that Eq has protective effects on osteoporosis, menopausal syndrome, cardiovascular diseases, breast and prostate cancer[4]. Moreover, studies have shown that Eq significantly increases glucose tolerance[5], promotes glucose uptake and utilization, improves insulin sensitivity[6]. But its specific mechanism needs further study.The latest study found that thioredoxin-interacting protein(Txnip) plays a key role in the regulation of insulin secretion. Insulin sensitivity decrease, hyperglycemia, glucocorticoids changes and β cells apoptosis can induce the expression of Txnip, thereby inhibiting glucose uptake and utilization. Txnip expression is regulated by a number of external glucose regulatory pathways, among which the carbohydrate response element binding protein(ChREBP) and Max-like protein(MLx) are the most important regulation factors. It has been found that hyperglycemia can promote dephosphorylation of ChREBP and activate ChREBP. When activated ChREBP penetrated into the nucleus, it combines with the heterodimer partner MLX, thereafter binding to histone acetylation P300, resulting in histone H4 acetylation, thereby performing chromatin modification, while promoting RNA polymerase Ⅱ(PolⅡ) transferred to the promoter region of Txnip, ultimately inducing Txnip gene transcription[7]. Thus, whether S-Eq regulates pancreatic β cell insulin secretory function through ChREBP-Txnip signaling pathway needs to been further elucideted.In the present study, we treated rat insulinoma(INS-1) cells with high glucose to induce pancreatic β cell injury model in vitro. And CCK-8 for cell viability assay, ELISA assay for glucose-stimulated insulin secretion(GSIS) function, TUNEL method combined with Annexin V-FITC / PI flow cytometry apoptosis were used to detect the apoptosis of INS-1 cell caused by high glucose. We also used realtime PCR to detect the expression of preproinsulin(PPI), glucose transporter 2(Glut2), mitochondrial uncoupling protein 2(UCP2) mRNA. Western blot was used to measure the Glut2 and UCP2 protein expression. In addition, PKA and PP2 A activity were detected by PKA and PP2 A kit, siRNA transfection along with Western blot were used to detected ChREBP and Txnip protein expression, Chip and dual luciferase reporter gene technology were used to furhher investigate the possible role of ChREBP-Txnip signaling pathway in the effect of S-Eq on pancreatic cell insulin secretory function.The main results and conclusions of this study are as follows:(1)S-Eq significantly increased cell viability and reduced apoptosis in high glucose treated INS-1 cells(P < 0.05).(2)S-Eq significantly up-regulated the expression level of PPI mRNA, improved GSIS function, enhanced Glut2 and reduced UCP2 transcriptional expression level in high glucose treated INS-1 cells(P < 0.05).(3)S-Eq significantly reduced intracellular PP2 A activity, increased PKA activity, and inhibited the expression of ChREBP in high glucose treated INS-1 cells(P < 0.05).(4)S-Eq significantly decreased the ChREBP accumulation on ChoRE cis-acting elements, and downregulated Txnip promoter transcriptional activity after ChREBP(wt) transfection, thereby inhibiting Txnip expression. And the promoter transcriptional activity of Txnip was significantly decreased after transfecting ChREBP(dm) plasmid in INS-1 cells. Furthermore, ChREBP and Txnip expression were markedly reduced after using siRNA silencing ChREBP gene expression. And S-Eq had a significant inhibitory effect on Txnip expression induced by high glucose(P < 0.05).Conclusions: S-Eq could regulate the activity of ChREBP and inhibit Txnip signaling pathways, thereby reducing apoptosis, enhancing Glut2 and UCP2 expression in high glucose-treated pancreatic INS-1 β cell, ultimately improving insulin secretion function.