Effects And Mechanisms Of Testosterone On Glucose Stimulated Insulin Secretion And Cell Apoptosis In β-cells

Part ⅠClinical analysis of glucose metabolic data in women with PCOS Background:Polycystic ovary syndrome (PCOS) a disorder with complex etiology and heterogeneity manifestations. The syndromen is featured with chronic anovulation, hyperandrogenemia and polycystic ovarian changes under ultrasound. It oftern combined with insulin resistance (IR) and glucose-lipid-abnormal, which is similar to T2DM. The incidence of abnomal glucose metabolism is increasing with age in PCOS women. One of the most important clinical manifestations of PCOS is obesity, most of which are abdominal obesity. Obesity not only participates in the pathogenesisi of PCOS, but also is the risk factor for IR and T2DM. However, normal weight PCOS women still have higher incidence of IR than control. Revelent analysis showed that IR was closely related to testosterone.Objective:In this study, PCOS women aged20-40years old, matched body mass index were grouped according to testosterone level to study the endocrine and metabolic features.Methods:3342PCOS women were selected form outpatients of Reprocuctive Hospital Affiliated to Shandong University from September2008to October2011. The cases were divided and analyzed endocrine and glucose metabolism according to whether they had hyperandrogememia.Results:1. There were1680PCOS women (50.27%) had normal testosterone (T<60ng/dl); and49.73%had hyperandrogenemia.2. PCOS with hyperandrogenemia showed higher FSH, LH and E2level than the other group, which had significant differences (P<0.05); BMI, waist to hip ratio and prolactin leves had no differences between the two groups.3. The glucose and insulin level were higher in hyperandrogenemia group except for G0, FINS, INS30, INS60(P<0.05).4.△I30/△G30, modified β-cell function index (MBCI) and disposal index (DI) were lower in hyperandrogenemia group (P<0.05). Insulin sesitive index (ISI) and HOMA-β had no statistical differencs (P>0.05).5. Hyperandrogenemia group had higher prevalenc of glucose disorder than normal testosterone group (P<0.05).Conclusions:PCOS with hyperandrogenemia showed more obvious endocrine and glucose disorders than normal testosterone group. β-cell secretion and DI were lower in hyperandrogenemia PCOS group. Part IIRegulation in the Production and Secretion of Insulin from INS-1Cells by TestosteroneBackground:Coexistence of hyperinsulinemia and hyperandrogenism are often seen in women with polycystic ovary syndrome (PCOS). And many studies have found that hyperandrogenemia is closely related with hyperinsulinemia (impaired glucose tolerance). It is found that the incidence of IR is up to70-80%in PCOS patients, but only a small part of them will enentually develop to T2DM. Therefore, it is necessary to study the reasons for the defects of insulin secretion. More and more evidence showed that when the pancreatic β-cell function is normal, abnormal glucose metabolism will not appear. So, pancreatic β-cell function is the key factor to determine whether diabetes will occur. If β-cell maintains their compensatory ability, T2DM does not occur.Pancreatic/duodenal hormobox (PDX-1) gene has an important role in early pancreas development, insulin gene expression and GSIS. PDX-1can promote insulin gene transcription and activate a series of genes involved in the maintenance of β-cell function and survival. Johson et al found that β-cell apoptosis, insulin secretion defect and diabetes are partly caused by PDX-1defect. PDX-1can regulate insulin secretion through up-regulation insulin gene, glucokinase (GCK) and glucose transporter2(Glut-2). Glut-2was a transmembrane protein and mainly responsible for glucose transporter and insulin secretion.MAPKs are highly conserved extracellular signaling molecules that regulate kinase cascade. Abnormal MAPKs can lead to inflammation, cancer, obesity and diabetes. ERK1/2belong to MAPKs family, which is involved in insulin gene expression. Blocking ERK1/2can stop insulin promoter activation. MAPKs are downstream molecules of androgen receptor. We therefore tested the impairment effect of testosterone on glucose-stimulated insulin secretion in INS-1cells.Objective:1. To determine the effects of testosterone on insulin gene expression and insulin secretion.2. To make sure whether androgen receptor/ERK1/2/PDX-1signaling pathway envolved in these phenomenons.Methods:1. INS-1cells were treated with different concentrations of testosterone (from1.7×l0-10M to1.7×10-6M) and examined at different time points.1×10-7M flutamide was pre-treated half an hour before1.7×10-7M testosterone treatment. Detect MDA content in each group.2. After testosterone treatment,2h GSIS were performed. With RT-PCR method to test PDX-1mRNA, Glut-2mRNA and insulin mRNA, androgen receptor mRNA expression; with western blotting to detect PDX-1, Glut-2and ERK1/2protein expression.Results:1. The response of INS-1cell to glucose:Under regular culture condition, INS-1cells had well GSIS performance.16.7mM glucose was the best stimulate concentration.2. The regulatory effect of testosterone to INS-1cells:In contrast to control,1.7×10-8M and1.7×10-7M testosterone treatment for48h could promote GSIS.1.7×10-6M testosterone treatment could decrease GSIS. Alternatively, long-time and high-concentration testosterone treatment significantly impaired GSIS. Flutamide could partly block testosterone induced insulin secretion.3. The effect and mechanism of testosterone to INS-1cells:AR mRNA was first confirmed in INS-1cells and its expression were upregulated with testosterone concentration.1.7×10-/M testosterone treatment for48h could enhance AR mRNA, PDX-1mRNA and Glut2mRNA expression up to2-fold. Alternatively, long-time and high-concentration testosterone treatment significantly impaired insulin mRNA levels.Testosterone could promote PDX-1, Glut2protein expression and ERK1/2phosphorylation. But1.7×10-6M testosterone decreased these protein expressions. Total ERK1/2expression had no difference between groups. As expected, long term testosterone treatment decreased PDX-1, Glut-2and p-ERK1/2protein expression.Testosterone treatment could add MDA content and flutamide, as a blocker of AR, could attenuate the increase of ERK1/2phosphorylation and MDA content.Conclusions:Testosterone could affect insulin gene expression and secretion at least in part through AR/ERK1/2/PDX-1signaling pathway, which may participate in glucose metabolic disorder in PCOS. Part ⅢEndoplasmic Reticulum Stress Contributes to Testosterone Inducted apoptosis in INS-1CellsBackground:Endoplasmic Reticulum (ER) is the main site of protein synthesis, folding and Ca2+storage. It has close relationship with nuclear signal transduction and can continuously sense intracellular changes. Many cell stress conditions, such as ischemia, hypoxemia, protein synthesis and peroxide could triger endoplamic reticulum stress (ERS). When the damage is minor, β-cells can maintain survival through ERS. But long-term and excessive ERS can cause cell damage and lead to unique apoptotic pathway or integrate other apoptotic signaling. Under normal conditions, ER modified protein through oxidation of cysteine residues in the new polypeptide chain to form a disulfide bond. This process could produce reactive oxygen species (ROS). And the ROS could be cleared by glutathione (GSH)-antioxidant defense mechanism. The condition of ERS could increase th load of ER, excessibe ROS exceeds the antioxidant defense capacity leading to oxidative stress. Meanwhile, oxidative stress chould induce ERS through Ca2+balance disorder. ERS and oxidative stress interact with each other and consititute a complex system.In women with PCOS, hyperandrogenemia and hyperinsulinemia/IR are often co-presented, which indicates there is a close relationship between hyperandrogenemia and β-cell damage. Testosterone might cause β-cell apoptosis through protein synthesis and ROS. Medina MC found that DHEA could enlarge β-cell volume and promote GSIS. Androgen/androgen receptor pathway could impair liver function and cause liver cell apoptosis through ERS pathway. Pancreas and liver are belongs to endocrine glands, which play important roles in maintain glucose homeostasis. So, we determined to find whether testosterone could cause β-cell apoptosis through ERS. Objective:The relationship of testosterone and insulin are mostly concentrated in insulin secretion. Few reports are concerning cell proliferation/apoptosis in β-cells. This experiment wanted to observe the relationship of ER stress pathway and INS-1cell apoptosis.Methods:1. INS-1cells were treated with different concentrations of testosterone and examined at different time points.1×10-7M flutamide was pre-treated half an hour before1.7×10-7M testosterone treatment.2. After testosterone treatment, MTT cell viability detection, flow cytometry and fluorescence were adopted to detect cell apoptosis and cell cycle. Electron microscopy to see morphological observation of INS-1cell organelles. With RT-PCR method to test Bip, ATF4, CHOP, SERAC2b and Bax mRNA expression in INS-1cells; with Western blotting to detect Bip, p-eIF2a, ATF4and CHOP protein expression.Results:1. The effect of testosteroen to INS-1cell proliferation and apoptosis:MTT showed testosterone treatment could decrease cell proliferation and cause cell apoptosis. After1.7×10-7M testosterone treatment, INS-1cell apoptosis was2.83folds to control group. With testosterone treating time adding to72h, cell apoptosis rate raised to3.62folds compared to control group(P<0.05).1×10-7M flutamide could partly reduce testosterone induced INS-1cell apoptosis. Fluorescence results support that testosterone had concentration-and time-dependent effects on INS-1cell apoptosis.2. The effect of testosterone to cell cycle:24h and48h testosterone treatment can promote cell proliferation. After72h treatment, cell proliferation capacity began to weaken. 3. Morphological observation of INS-1cell organelles under electron microscopy: INS-1cell body is round with a number of small protrusions stretched out. The nucleus is round, too. Nuclea chromatin is dispersed so the picture showed low electrol density. Cytoplasm contains a large number of rough ER. After1.7×10-7M testosterone treated for48h, part of INS-1cells showed cell volume reduction, nuclear condensation, electron density increasing and rough ER pool expansion.4. The mechanism of testosteroen to INS-1cell apoptosis:1.7×10-7M testosterone treated for48h could significantly enhance Bip mRNA, ATF4mRNA and CHOP mRNA expression.1.7×10-7M testosterone treated for72h could promote CHOP mRNA level to4.75times compared to control group. While SERCA2b mRNA didn't change much between testosterone concentrations.Testosterone could promote ERS protein expression in concentreation-and time-dependent manner. p-eIF2a and CHOP protein were obviously increased after1.7×10-7M testosterone treatment. Protein expression of Bip, p-eIF2α, ATF4and CHOP were increasing with treating time. Fluamide could partly block testosterone induced ERS and cell apoptosis.Conclusions:Long-term and super-physiological concentration of testosterone could inhibit cell proliferation and induce INS-1cell apoptosis. p-eIF2α/ATF4/CHOP pathway might play an important role in this impairment.