| Part I To construct T2DM rat model and evaluate the effects of duodenal-jejunal bypass surgery on glucose metabolism in the type 2 diabetic rat model BackgroundWith the development of social economy and the changes of the life style, diabetes has become one of chronic diseases that do great harm to people’s health worldwide, and about 90% of which are type 2 diabetes mellitus (T2DM). The number of people affected diabetes is steadily increasing. According to the data released by the International Diabetes Federation,387 million people worldwide had diabetes in 2014, and this number is expected to rise to 592 million by 2035. Although diet,exercise,oral hypoglycemic drugs and insulin injection can control the hyper-glycemia in short time, however,the compliance rate is very low for these treatments are life-long.The long-term glycemic control is not ideal with the conventional therapies. Therefore, searching for new and efficient treatments for T2DM has become the focus of global attention.From the 1960s,clinical investigations have shown that many obesity patients with type 2 diabetes had undergone bariatric surgery, and their blood glucose quickly returned to normal before significant weight loss.Thereafter,these patients no longer need antidiabetic drugs to maintain blood glucose.So, We find such a new way to treat diabetes through surgery. Roux-en-Y gastric bypass (RYGB),as one of the standard bariatric surgery, can improve or even complete remission the diabetes of morbid obesity patients.However, there is significant weight loss after surgery,so it is not recommended to non-obese diabetic patients. Non-obese type 2 diabetes patients are most common in China,so searching for surgical procedures for these patients is of great importance.Duodenal-jejunal bypass (DJB) is a new surgical model,which preserves the gastric volume intact while bypassing the entire duodenum and the proximal jejunum.Currently,duodenal-jejunal bypass (DJB) has been shown to be an effective surgical treatments for type 2 diabetes mellitus (T2DM). However, the underlying mechanisms are poorly understood.So,it is necessary to study further on the mechanism of DJB. For these reasons, developing a diabetic animal model and conducting DJB in this animal model is the basis for further research.ObjectiveIn this part of study, we aimed to develop a T2DM rat model through high-fat diet (HFD) feeding and intraperitoneal injection of low dose streptozotocin (STZ). Afterwards, DJB was conducted in the diabetic rats to evaluate its effects on glucose metabolism in the type 2 diabetic rat model.MethodsIn this part of study, insulin resistance was firstly induced in male SD rats by HFD feeding, then a low dose of STZ (35mg/kg) was injected intraperitoneally to induce a diabetic state. Afterwards,DJB and sham surgery were conducted in the T2DM rats. Then at indicated time points postoperatively,we evaluated the body weight, caloric intake, fasting blood glucose, random blood glucose, glucose tolerance, insulin tolerance,insulin resistance, glucose stimulated insulin secretion.Results1. The T2DM rat model was successfully induced by HFD and low dose of STZ.and the random glucose and AUCITT of this rat model were all increased.2. No significant difference was observed between rats in the DJB and SHAM group in terms of body weight and caloric intake at any observational time (*P>0.05).3. There was no significant difference in fasting blood glucose between the DJB and the SHAM group preoperatively (P>0.05); The DJB group exhibited lower values of fasting blood glucose than the sham group at all measured time points between 2 and 16 weeks postoperatively (P<0.05). This result demonstrated that DJB could significantly improve the fasting blood glucose in the T2DM rats.4. There was no significant difference in random blood glucose between the DJB and the SHAM group preoperatively (P>0.05); At1、2、4、6、8、10、12、14、 16 weeks postoperatively, the DJB group showed lower values of random blood glucose than the sham group(P<0.05). This result demonstrated that DJB could significantly improve the random blood glucose in the T2DM rats.5.A5 2、4、8、12、16 weeks postoperatively, the values of AUCPGTT of DJB group were all statistically lower than the sham group (P<0.05). This result demonstrated that DJB could significantly improve the blood glucose tolerance in the T2DM rats.6. At 2、4、8、 16 weeks postoperatively, the values of AUCITT of DJB group were all statistically lower than the sham group (P<0.05). This result demonstrated that DJB could significantly improve the insulin sensitivity in the T2DM rats.7. There was no significant difference in HOMA-IR between the DJB and the SHAM group preoperatively (P>0.05); The DJB group exhibited lower values of HOMA-IR than the sham group at 2 and 16 weeks postoperatively (P<0.05). This result demonstrated that DJB could significantly lower the insulin resistance in the T2DM rats.8. At 2 and 16 weeks postoperatively, no significant difference was observed with respect to the glucose stimulated insulin secretion levels between rats in the DJB and SHAM groups(P>0.05).Conclusions1. The T2DM rat model was successfully induced.and DJB surgery were successfully conducted in the T2DM rats,and all the data confirmed that DJB could significantly improve glucose metabolism (fasting blood glucose, random blood glucose and blood glucose tolerance) in type 2 diabetic rats,which was independent on the loss of caloric intake and body weight.2. DJB could significantly improve the insulin sensitivity of T2DM rats and no significant increase of insulin secretion was observed in the short term.Part Ⅱ The effects of DJB on the endoplasmic reticulum (ER) stress state in the liver tissue and the involving signaling of T2DM rats BackgroundInsulin resistance is one of the features of type 2 diabetes mellitus (T2DM) and is characterized by impaired uptake and utilization of glucose in insulin sensitive target organs and by impaired inhibition of hepatic glucose output. Although insulin resistance is well characterized, the mechanisms by which insulin resistance develops are still inconclusive.Accumulating evidences suggest that endoplasmic reticulum (ER) stress plays an important role in the development of insulin resistance.The ER is a critical intracellular organelle that coordinates the synthesis, folding and transporting of proteins.Ribosomes attached to the ER membranes release newly synthesized peptides into the ER lumen, where protein chaperones and foldases assist in the proper posttranslational modification and folding of these peptides.The properly folded proteins are then released to the Golgi complex for final modification before they are transported to their final destination. If the influx of misfolded or unfolded peptides exceeds the ER folding and/or processing capacity, ER stress ensues. Three proximal ER stress sensors have been identified.They are inositol-requiring enzyme-1 (IRE-1), PKR-like ER protein kinase(PERK), and activating transcription factor-6(ATF-6).These sensors trigger activation of pathways, termed the unfolded protein response (UPR), which act to alleviate ER stress. Previous studies have demonstrated that ER stress plays a central role in the development of insulin resistance and diabetes by triggering JNK activity via inositol-requiring enzyme-1(IRE-1).The activation of JNK contributes to insulin resistance by direct phosphorylation of Ser307 in IRS-1.The phosphorylation of Ser307 in IRS-1 blocks interactions with the insulin receptor and inhibits insulin receptor signaling,so insulin resistance and type 2 diabetes mellitus ensues. Under normal conditions, the binding of insulin to its receptor can cause some changes in insulin receptor, and these changes can cause phosphorylation of tyrosine residues in insulin receptor, thereby can cause the aggregation of insulin receptor substrate-1.The phosphorylation of tyrosine residues is necessary for insulin receptor substrate activation and insulin sensitivity. The phosphorylation of Ser307 in IRS-1 can inhibit the phosphorylation of tyrosine residues and this results in insulin resistance and type 2 diabetes mellitus.However, to our knowledge, there are not yet any reports investigating the effects of DJB on ER stress and the involving signaling.ObjectiveThis part of study was designed to investigate the effects of DJB on ER stress state in the liver tissue as well as related signaling pathways which associated with insulin resistance. We hope that the results of this investigation might help to provide new insight in elucidating the underlying mechanisms of glucose metabolism amelioration after DJB surgery.Methods 16 weeks postoperatively, liver tissues in each group were collected.Then Western blotting was performed to examine the changes of p-PERK、IRE-1、p-IRE-1、JNK、 p-JNK、IRS-1 and p-IRS1ser307 protein in each group.Results1. In the liver tissue, the protein of p-PERK was expressed in both the DJB group and the SHAM group; compared with the SHAM group, the expression of p-PERK protein in the DJB group was significantly reduced (*P<0.05).2. The expression of IRE-1 was not significantly different between the DJB group and the SHAM group(P>0.05), while the ratio of p-IRE-1 to IRE-1 was significantly reduced in the DJB group compared with the SHAM group (*P<0.05).3. The total JNK was not significantly different between the DJB group and the SHAM group (P>0.05), while the ratio of p-JNK to total JNK was significantly reduced in the DJB group compared with the SHAM group (*P<0.05).4. The expression of IRS-1 in liver was not significantly different between the DJB group and the SHAM group (P>0.05), while the ratio of p-IRS1Ser307 to IRS-1 was significantly decreased in the DJB group compared with the SHAM group (*P<0.05).Conclusions1. As the protein of p-PERK was the iconic protein of endoplasmic reticulum stress and it was expressed in both the DJB group and the SHAM group in the liver tissue,So endoplasmic reticulum stress developed in both groups.2.16 weeks postoperatively, the p-PERK and p-IRE-1 in liver tissue was markedly reduced in the DJB group, indicating that DJB surgery can alleviate ER stress.3.16 weeks postoperatively, the p-JNK in liver tissue was markedly reduced in the DJB group, indicating that DJB surgery can decrease the activation of JNK.4.16 weeks postoperatively, the p-IRSlser307 in liver tissue was markedly reduced in the DJB group, indicating that DJB surgery can significantly improve the insulin signaling pathway. |
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