The Expression And Significance Of Perk And P-perk In The Livers Of Insulin-resistance Rats

PrefaceDiabetes is a serious public health problem of common diseases, the current 7 million worldwide,100 million DM patients, DM in the next 25 years will reach 300 million patients with 6 million. DM consists of clinical type 1 diabetes and type 2 diabetes, and T2DM accounted for DM 90% to 95%. T2DM is a skeletal muscle, liver and fat insulin target tissues such as the main features of insulin resistance. The pathogenesis of T2DM is not entirely clear, recent research found that endoplasmic reticulum stress induced insulin resistance in obesity occupy an important position, and the target tissue insulin resistance and pancreatic B cell dysfunction is closely related; so ER stress in obesity led to the role of insulin resistance in diabetes has become a hot research field. Endoplasmic reticulum is important organelles within cells, to regulate protein synthesis and folded after synthesis and gather, is to regulate cell stress response and cell calcium level places, but also cholesterol and lipid synthesis of steroids and many places. In hypoxia, oxidative stress, abnormal glycosylation reactions and the imbalance of calcium homeostasis such as the role of various factors, the nascent polypeptide chain folding and assembly modification interference, will lead to unfolded protein in the ER in the accumulation causes cells to produce endoplasmic reticulum stress, and activates the unfolded protein response signaling pathway, leading to cell itself by changing its transcription and translation process, to reduce protein synthesis, decreased protein into the endoplasmic reticulum content, while increases in the endoplasmic reticulum molecular chaperone and folded protein, increased endoplasmic reticulum protein folding functions. Cells also can be by up-regulating endoplasmic reticulum protein degradation pathway related genes Biaoda, Jia Su unfolded protein degradation process, in order to increase cell Zaiyou Hai factors of survival. ERS is under stress, to maintain cell homeostasis, to maintain cell survival, cell activated response mechanisms to protect themselves. Study found that after 16 weeks of high fat diet compared to mice and mice with normal diet, fatty tissue in the ERs of the signs, such as PREK phosphorylation and JNK activity increased significantly, ob/ob mice and wild-type mice compared, adipose tissue of phosphorylated PERK and IRE-la was significantly higher, and JNK activity and the splicing of XBP-1 also increased significantly, these are tips Obesity can lead to fat cells ERs. But the occurrence of obesity in the fatty tissue of the reason ERs are still unclear. However, the following hypothesis:①In the case of nutrition and endoplasmic reticulum in protein synthesis increased significantly induced UPR;②excess of nutrients can be a signal for induction of ERs in obese state under, FFA levels were significantly increased. Study found that in liver cells and pancreatic islet B cells, FFA can induce UPR, but in the fat cells, FFA internal ER function remains unclear, in 3T3-L1 adipocytes, FFA can activate JNK, leading to IR. The UPR can also activate JNK, so FFA and ERs may be a link; Obesity occurs when ERs③One reason is the lack of glucose status, which in many cells, including fat cells are considered to be the incentive for ERs. Fat cells in obesity, because cells IR, insulin signaling pathway is inhibited, reducing sugar intake, cells in the absence of glucose or unstable situation, prone to stress. Hosogai so found that obese adipose tissue in mice and wild type mice, hypoxia appears obvious signs of fat cells in culture, hypoxia induced UPR, showed increased expression of Bip, etc., eIF2a phosphorylation and increase in XBP-1 splicing. The role of these various factors, mutual adjustment, leading to obesity fat cells ERs.In order to cope with and adapt to ERS. Cells need a nucleus from the ER to the signal transduction pathway to increase the ER protein folding capacity, this approach is the UPR. UPR includes three signaling pathways. By 3 types of ER resident proteins start:type-lER transmembrane protein kiIlase IRE1, PKR like ER kinase PERK) and activating transcription factor 6 ATF6. Another. Recent reports from different cells were isolated from other types of signal receptors and pathways. UPR not only increased the number of ER membranes also increased with protein folding and protein chaperone related to the number of modifying enzymes. Also reduced by blocking protein translation Quwang ER's protein. Finally. Unfolded peptides and return to the cytoplasm by means of the proteasome dependent degradation. This process is called ER-Associated degradation ERAD. However, if the final does not restore the normal function of ER. For example, the UPR may lead to prolonged severe apoptosis. Therefore, once the loss function is not correctly folded proteins in the cell surface. The cells may be organized very quickly removed. In recent years, the study found, ERS and islet B cell survival, apoptosis and related to the incidence of diabetes, while diabetes, high ERS appropriate state intervention may become a new means of treating diabetes. At present, although the ERS has gradually become involved in diabetes research focus, but the ERS-mediated islet B cell dysfunction and insulin resistance in the exact mechanism, ERS onset in the different types of diabetes and diabetic complications in the role of the endoplasmic reticulum chaperones, diabetes therapy, ERS and the common pathway of diabetic complications, the relationship between oxidative stress, need to be further clarified.Materials and methodsGrade 4 week-old male SPF Wistar 30 rats weighing 80-120g, were purchased from Experimental Animal Center of China Medical University. Adaptive feeding rats after 1 week, were randomly divided into normal diet group (NC group, n=15) and high-fat diet group (HF group, n=15). NC group received basic diet, as a control group. HF group received high fat diet, the establishment of insulin resistance in rats. After 10 weeks, each group randomly selected five rows high plasma insulin-normal glucose clamp to assess insulin resistance in rat model.Left common carotid artery access heparin saline syringe, the right femoral vein access after Tee who were connected with 40mU/mL Insulin Injection (Dan Mainuo Nordisk products) and 10% glucose injection of 50 mL syringe, Afterwards micro-infusion pump. Stand for 30 min, common carotid artery blood test based on blood glucose and 1 mL of basal insulin. First, insulin infusion, infusion rate 1.67 mU·kg-1·min-1), every 5 min 1 drop of carotid blood glucose test, when blood sugar "(based on the value of±0.5) mmol/L glucose infusion begins, lose Note rate from 4-6 mg·kg-1·min-1 start. Blood sugar test once every 5min, glucose infusion rate adjusted to maintain blood glucose (baseline±0.5) mmol/L, for 3 times in the range of blood glucose, clamp formation. Experimental total of about 2 h,60-120 min calculated the average GIR, evaluation of insulin sensitivity. Carotid arterial blood sample 4℃,3000×g, centrifugation 15 min, separating serum frozen home-80℃refrigerator. NC group and HF group,20 rats without making clamp fasting 16 h, by 10% chloral hydrate (0.3mg/kg) intraperitoneal anesthesia, the abdominal aorta puncture and blood of about 4 mL,4℃,3000×g,15 min after centrifugation, separated serum frozen home-80℃refrigerator. Cut into approximately 100 mg liver tissue the size of the deletion of blood vessels and other connective tissue, dip dry after washing, placed in 1.5 ml EP tube and frozen at-80℃refrigerator.The liver tissue 100 mg, by adding 1 ml protein lysis buffer after 30 min standing ultrasonic homogenizer,4℃,12000×g, centrifugation 30 min, the supernatant obtained, BCA protein quantitation method. Taken with 30μg of total protein samples were SDS-PAGE electrophoresis, the wet transfer method protein bands transferred to PVDF membrane, containing 5% skim milk TBST closed after 1h followed by adding an anti-(rabbit anti-rat PERK single cloned antibody,1:500 dilution), secondary antibodies (horseradish peroxidase labeled goat anti-rabbit monoclonal antibody,1:5 000 dilution), after washing the membrane with enhanced chemiluminescence imaging, using gel image analysis system analysis. Detection of p-PERK protein level of an antibody is rabbit anti-rat p-PERK antibody (1:1000 dilution), secondary antibodies to horseradish peroxidase labeled goat anti-rabbit polyclonal antibody (1:5000 dilution).Take 100μL of serum frozen, applications insulin ELISA kit (R & D company), operate according to specifications, testing based on state serum insulin levels.ResultCompared with NC group,the glucose infusion rate (GIR60-120) of HF group was significantly lower (0.90±0.15 mg·kg·min-1 vs 4.97±0.68 mg·kg-1·min-1, P<0.01). Compared with NC group,the level of p-PERK in the liver of HF group was significantly higher(192.89±25.16 vs 63.04±15.61, P<0.05) and p-PERK/PERK was also significantly higher(0.99±0.12 vs0.33±0.08, P<0.05)DiscussIn this study, HF rats showed insulin levels higher than the NC group basis (P 0.05) and HF rats GIR60-120 were significantly lower than NC group (P<0.01), confirmed the successful establishment of rat model of IR; Liver PERK and p-PERK protein expression, HF rats, liver expression of PERK compared with NC group no significant difference (P> 0.05), and p-PERK expression was significantly increased (P <0.05). And rat liver p-PERK/PERK HF ratio was significantly higher (P<0.05). Confirmed that high-fat diet-induced rat model of IR there ERS. In this study, the expression of PERK was no significant difference between the two groups may be due to PERK also expressed in physiological conditions. ERS as part of PERK when activated by phosphorylation, thus activation of downstream pathways. In this study, fresh lard fat diet formula is the main component of saturated fatty acids, after 10 weeks of high fat diet, compared with NC group, HF rats based on body weight and blood glucose was no significant difference (P> 0.05), The serum TG level was significantly higher (P<0.01), HF rats, liver p-PERK protein levels were significantly increased (P<0.05), and basal insulin levels were significantly increased (P<0.05). These results show that the high fat diet may cause liver toxicity through lipid ERS, causing IR. Nutritional signals and ERS can activate PERK, activated PERK kinase through activation of an important inflammatory activity of JNK and regulation of insulin. PERK directly on the IRS could change the activity of insulin.ConclusionThe ascensus of p-PERK protein and p-PERK/PERK in the livers of insulin-resistance rats indicate that endoplasmic reticulum stress possibly participates in the genesis of insulin resistance.