| Part1:Construction and evaluation of non-obese type2diabetic rat model using sleeve gastrectomyBackgroundThe incidence of type2diabetes(T2DM) in the past few decades had a rapid rising. Currently there are over300million people worldwide suffering from T2DM, including25million patients in the United States,55million patients in India, and about80million patients in China, which results in a big economic and social burden. The traditional treatment such as restricted diet, exercise, hypoglycemic drugs and insulin can control blood glucose and obviously reduce the incidence of complications of T2DM. However, it only delays the onset of diabetes while it can’t prevent late complications of systemic vital organs.In recent years, more and more clinical and basic research showed that bariatric surgery such as sleeve gastrectomy not only induces weight loss, but also significantly improves glucose metabolism, achieving good diabetic control in obese combining with T2DM. However, the mechanism of bariatric surgery in improving glucose metabolism is still unclear. Studies have shown that the anti-diabetic effects of bariatric surgery not only depends on weight loss, but also relates with the change of gastrointestinal anatomy and physiology post operation. At present, the main hypotheses to explain the anti-diabetic effect of sleeve gastrectomy is "Ghrelin hypothesis", but there are much controversial and the hypothesis is currently inconclusive. So in addition to the effect of Ghrelin, is the remission of T2DM by sleeve gastrectomy associated with other mechanisms? Does sleeve gastrectomy lead to physiological signalings changing in the small intestine? Meanwhile, as the incidence of the non-obese T2DM is increasing, does sleeve gastrectomy induce genuine improvement in glucose metabolism? Is this procedure safe and effective? In order to study diabetic control of sleeve gastrectomy, evaluate its safety and efficacy, and explore the underlying mechanism, this study constructed three non-obese type2diabetic animal models of metabolic surgery. By comparing with each operation model, the study aims to confirm the diabetic control by sleeve gastrectomy. Furthermore, by comparison with the analysis of glucoregulatory hormones changes, the study further explores the mechanism of diabetic improvement by sleeve gastrectomy.ObjectivesThis study aims to apply the non-obese type2diabetic rats-Goto-Kakizaki (GK) rats to establish three metabolic surgery models including sleeve gastrectomy for studying of the following questions:(1) assessment about the safety and efficacy of sleeve gastrectomy inducing diabetes remission in obese type2diabetic rat model;(2) comparing with other metabolic surgery to further clarify the diabetic control of sleeve gastrectomy;(3) analysis of glucoregulatory hormones and other indicators of metabolism to explore the diabetic improvement mechanism of sleeve gastrectomy.MethodsOur study constructed three metabolic surgery models inculding sleeve gastrectomy, duodenal-jejunal bypass, and sleeve gastrectomy with duodenal-jejunal bypass. The sleeve gastrectomy with duodenal-jejunal bypass surgery is a procedure which includes sleeve gastrectomy and duodenal-jejunal bypass.In this stud, the non-obese type2diabetic GK rats were subjected to SG, DJB, SGDJB and sham surgery: Sham-operated SG (SOSG), Sham-operated DJB (SODJB) and Sham-operated SGDJB (SOSGDJB). The indicators of the above groups were compared with those of the control group. The indicators includes:(1) the operative time and successful rate of surgery, postoperative recovery, and surgical complications;(2) body weight and food intake at each time point;(3) fasting blood glucose at each time point (before operations and at2,4,8,12, and16weeks after surgery);(4) oral glucose tolerance test at each time point(before operations and at2,4,8,12, and16weeks after surgery);(5) the insulin tolerance test at each time point(before operations and at2,4,8,12, and16weeks after surgery);(6) glucose-stimulated insulin secretion and fasting plasma C-peptide levels at2and16weeks after surgery;(7) fasting ghrelin levels at2and16weeks after surgery;(8) glucose-stimulated glucagon-like peptide-1, peptide YY, and glucose-dependent insulinotropic peptide levels at2and16weeks after surgery.Results1. There was no rats death in all groups including SG group, DJB group, SGDJB group, and the sham group within24hours after surgery. The operating time of SG and DJB procedures was shorter than that of SGDJB surgery (P<0.05), however, all surgery were completed successfully.2. At2weeks post operation, there was no significant difference in food intake among all groups (P>0.05). But at4to16weeks postoperatively, the SG and SGDJB rats showed less food intake than those of DJB rats, the sham operation rats, and the control rats (P<0.05), but there was no significant difference in food intake among DJB rats, the sham operation rats, and the control rats (P>0.05). At2weeks after surgery, there was no significant difference in body weight of all rats (P>0.05). At6to16weeks postoperatively, the SG and SGDJB rats showed lower body weight than those of other three groups (P<0.05), but there was no significant difference in body weight among DJB rats, the sham operation rats, and the control rats (P>0.05). SG and SGDJB groups did not differ from each other both in food intake and body weight(P>0.05).3. The data of fasting blood glucose and oral glucose tolerance test showed that, at2to16weeks postoperatively, the operated groups had lower fasting plasma glucose levels and AUCOGTT values (P<0.05). But since8weeks post operation, the rats in SG and SGDJB groups exhibited lower fasting blood glucose levels than those of DJB rats (P<0.05). Since12weeks post operation, the rats in SG and SGDJB groups exhibited lower AUCOGTT values than those of DJB rats (P<0.05). No difference in fasting glucose and AUCOGTT values were observed between SG and SGDJB rats (P>0.05).4. The data of insulin tolerance test showed that, at2to16weeks postoperatively, the operated groups had lower fasting plasma glucose levels and AUCOGTT values (P<0.05). But at16weeks post operation, the SG and SGDJB rats exhibited lower AUCITT values than those of DJB rats (P<0.05). No difference in AUCITT values were observed between SG and SGDJB rats (P>0.05).5. At2and16weeks post operation, the fasting serum C-peptide levels of the operated groups was higher than those of the sham and control groups (P<0.05). Compared with the rats in the sham and control groups, the operated groups showed higher insulin secretion2weeks postoperation at measuring time of60and120min after oral glucose gavage (P<0.05). Moreover, at16weeks postoperation, higher insulin levels were observed at all measuring times in the operated groups than those in all other groups (P<0.05). But at16weeks postoperation, DJB rats showed lower fasting serum C-peptide, and serum insulin levels than those of SG and SGDJB groups (P<0.05). At2and16weeks post operation, the operated groups exhibited higher AUCinsulin values (P<0.05). At16weeks postoperation, DJB rats showed lower AUCinsulin values than those of SG and SGDJB groups (P<0.05). No difference in AUCinsulin values were observed between SG and SGDJB rats (P>0.05).6. At postoperative week2and16, the SG and SGDJB rats showed lower fasting ghrelin levels than those of DJB, the sham operation, and the control rats (P<.05). In addition, the fasting ghrelin levels of the SG and SGDJB groups16weeks after operation were lower than those2weeks after operation (P<0.05). No difference in fasting ghrelin levels were observed among the DJB, sham operation, and control groups (P>0.05). No difference in fasting ghrelin levels were observed between SG and SGDJB rats (P>0.05).7. At2weeks post operation, compared with the rats in the sham and control groups, the operated groups showed higher GLP-1levels at measuring time of30and60min after oral glucose gavage (P<0.05).But the rats in SG group showed lower GLP-1levels than the DJB and SGDJB groups2weeks postoperation at measuring time of30and60min after oral glucose gavage (P<0.05). Moreover, at16weeks postoperation, higher GLP-1levels were observed at all measuring times in the DJB and SGDJB groups than those of SG, sham, and the control rats (P<0.05). The rats in SG group showed higher GLP-1levels than the sham and control groups16weeks postoperation at measuring time of30and60min after oral glucose gavage (P<0.05). At postoperative week2and16, compared with the rats in the sham and control groups, the operated groups showed higher PYY levels at all measuring times (P<0.05).Furthermore, At postoperative week2and16, the SG and SGDJB rats showed higher PYY levels at all measuring times than those of DJB rats (P<0.05). No difference in GIP levels and AUCGIP values were observed among all rats (P>0.05). At postoperative week2and16, the operated rats showed higher AUCGLP-1and AUCPYY values than the sham and control groups(P<0.05).At2and16weeks post operation, the SG rats exhibited lower AUCGLG-1values than the DJB and SGDJB rats(P<0.05). At2and16weeks post operation, the DJB rats exhibited lower AUCPYY values than the SG and SGDJB rats(P<0.05).Conclusions1. The present study showed that the SG surgery was safe and effective in improving type2diabetes, which can effectively induce long-term low blood glucose levels post operation in non-obese diabetic rats.2. The SG surgery exhibited better diabetic improvement than that of DJB and showed comparable diabetic improvement to that of SGDJB.3. The SG rats showed reduction of fasting ghrelin(time-dependent), and elevation of glucose-stimulated GLP-1and PYY post operation. And postoperative changes in these hormones were related to glucose metabolism improvement.4. Improved insulin resistance and enhanced β-cell function both contributed to the remission of diabetes by SG. The early diabetic improvement after SG surgery is independent of weight loss.These findings showed that the sleeve gastrectomy was safe and effective in diabetic control on non-obese T2DM subjects. The SG surgery obviously improved the insulin sensitivity, however, its effect on insulin sensitivity and mechanisms needs further research. Part2:The effectiveness of sleeve gastrectomy on insulin sensitivity in non-obese type2diabetic ratsBackgroundImpaired insulin sensitivity (insulin resistance) means the decreased sensitivity of organs respond to insulin, leading to decreased insulin-mediated glucose utilization As one of the main mechanisms for type2diabetes, it accompanies throughout the pathophysiological process of type2diabetes, and plays an important role in the pathogenesis of a variety of metabolic syndromes (obesity, dyslipidemia, etc.). Insulin resistance is mainly divided into hepatic insulin resistance (Decreased hepatic insulin sensitivity and increased hepatic glucose output) and peripheral tissue insulin resistance (Decreased peripheral insulin sensitivity, which mainly includes decreased skeletal muscle glucose utilization). Our previous studies successfully constructed a non-obese type2diabetic rat model of SG and found that the SG surgery was safe and effective in improving glucose homeostasis in non-obese type2diabetic rats, and could significantly improve insulin sensitivity. So does the sleeve gastrectomy have effect on peripheral and hepatic insulin sensitivity in non-obese type2diabetic rats? What about the early and long-term changes in insulin sensitivity post operation? There is no relevant studies. Therefore, this study successfully performed sleeve gastrectomy in non-obese type2diabetic rat model to study the effect of sleeve gastrectomy on hepatic and peripheral insulin sensitivity at early and long-term stages post operation. This study also provided evidence for in-depth study of pathogenesis and the mechanisms in improved glucose metabolism by SG.ObjectivesThis study aims to conduct research on the following questions:(1) evaluate the effect of sleeve gastrectomy on insulin sensitivity at early and long-term stages post operation;(2)study the effect of sleeve gastrectomy on hepatic and peripheral tissue insulin sensitivity at early term postoperatively in non-obese type2diabetic rats;(3) explore the effect of sleeve gastrectomy on hepatic and peripheral tissue insulin sensitivity in long-term post operation;(4) comparing the early term with long-term in postoperative hepatic and peripheral tissue insulin sensitivity to further explore the mechanism of diabetic improvement by SG.MethodsIn this study, non-obese type2diabetic GK rats were randomly divided into SG, sham group (SOSG), and control group. The key indicators for monitoring:(1) insulin tolerance test (ITT), and homeostasis model of assessment for insulin resistance index(HOMA-IR) were performed before operations, and at2and36weeks after surgery for preliminary assessment of the early and late insulin sensitivity improvement;(2) pyruvate challenge test (PCT) was administrated to assess the gluconeogenesis capability in order to reflect hepatic insulin sensitivity before operation, and at2and36weeks post operation;(3) extended hyperinsulinemic euglycemic clamp studies(EHECS) was done before operations, and at2and36weeks post operation. calculating the endogenous glucose production (Hepatic glucose production, HGP) at the basis and steady-state for evaluation of hepatic insulin sensitivity, calculating the exogenous glucose infusion rate (Glucose infusion rate, GIR) at the steady-state for evaluation of peripheral tissue insulin sensitivity.Results1. No difference in parameters including AUCITT, HOMA-IR, AUCPCT, HGP,and GIR were observed among SG, sham, and control group before surgery (P>0.05).2. Tolerance test showed that, at2and36weeks post surgery, AUCITT values of SG rats were significantly lower than the sham and control group(P<0.05). And at36weeks post surgery, the difference is more drastic.3. Insulin resistance index results showed that, post operation, HOMA-IR values of SG rats were significantly lower than the sham and control group(P<0.05). And at36weeks post surgery, the difference is greater.4. Pyruvate tolerance test showed that, after at2and36weeks post operation, SG rats exhibited lower AUCPCT values than other two groups(P<0.05), And at36weeks post surgery, the difference is more drastic.5. The data of the extended hyperinsulinemic euglycemic clamp test showed that, at2weeks post surgery, the basal HGP of SG rats was lower than other two groups(P<0.05). While no difference in GIR at steady-state were observed among three groups(P>0.05); At36weeks post surgery, there were statistically significant difference in the baseline and steady-state HGP, and the steady-state GIR beteen SG group and other two groups(P<0.05). No difference were observed between the sham and control group at2weeks post operation (P>0.05).6. Compared with the baseline and steady-state HGP, and the steady-state GIR of SG rats at2weeks post operation, those of SG rats at36weeks post operation exhibited significant difference (P<0.05).Conclusions1. The non-obese type2diabetic rats underwent sleeve gastrectomy showed significant improvement in early and long-term insulin sensitivity post operation.2. The SG rats exhibited improved hepatic insulin sensitivity at early stage post surgery. While the improvement of peripheral insulin sensitivity is not obvious at early stage post surgery. At long term post operation, rats in SG group showed improved hepatic and peripheral insulin sensitivity dually.3. Long-term insulin sensitivity was improved more obviously after SG surgery. Compared with the early term after operation, the amelioration of hepatic and peripheral insulin sensitivity at long term after SG surgery was better, and the improvement was time-dependent.This study jointly tested insulin sensitivity using a variety of techniques to explore the important mechanism in remission of type2diabetes--improved insulin sensitivity. Our study investigated the improvement of the hepatic and peripheral insulin sensitivity at early and long term post SG surgery. It provides a new direction for the study of type2diabetes and the mechanism of remission of type2diabetes after metabolic surgery Part3:The mechanisms of sleeve gastrectomy on improving insulin sensitivity in non-obese type2diabetic ratsBackgroundStudies suggested that just by gastrointestinal hormone changes to explain the rapid improvement in insulin resistance and blood glucose after metabolic surgery was partial. It needs further study. The liver is the vital organ of insulin resistance in T2DM, whose insulin resistance appearing as increased hepatic glucose output. One important reason is that the hepatic gluconeogenesis(liver is the main gluconeogenesis organ) and glycogenolysis function disorders occur, resulting in increased blood glucose levels. Furthermore, increased hepatic gluconeogenesis has a prominent role in hepatic insulin resistance. Recent studies have found that the small intestine not only is a "digestive tract", but also maintains glucose homeostasis through gluconeogenesis. Intestinal gluconeogenesis may regulate hepatic gluconeogenesis through the "enterohepatic axis" and thus participates in the improvement of glucose metabolism. On the other hand, recent studies found that hepatic and peripheral insulin signaling transduction pathway also plays an important role in the process of insulin resistance in diabetes. Our previous studies have shown that SG surgery improved hepatic insulin sensitivity, while did not improve peripheral insulin sensitivity at early stage post surgery. At long term post operation, rats in SG group showed improved hepatic and peripheral insulin sensitivity dually. However, the mechanisms to improve insulin sensitivity after SG surgery has not been elucidated. Does the improvement of insulin sensitivity by SG in non-obese type2diabetic rats have association with insulin signaling pathway and gluconeogenesis? There is no relevant reports.To further elucidate the mechanism by which SG improves insulin sensitivity on non-obese type2diabetic rats, this study successfully established a SG rat model. First, we investigated the change of peripheral and hepatic insulin signaling transduction pathways at early and long terms after SG. On the other hand, we also explored the change of intestinal and hepatic gluconeogenesis at early and long term after SG. The aim is to provide new ideas and methods for studying mechanisms of diabetic control by metabolic surgery, and to provide new ideas for the treatment of type2diabetes.ObjectivesThis study aims to conduct research on the following questions:(1) the expression of key genes in the hepatic insulin signaling transduction pathway at early and long term post operation;(2) the expression of key genes in the skeletal muscle insulin signaling transduction pathway at early and long term post operation;(3) the activity and expression of key enzymes in intestinal gluconeogenesis at early and long term post operation;(4) the activity and expression of key enzymes in hepatic gluconeogenesis at early and long term post operation;(5) comparing the early-term change with the long-term change in the insulin signaling transduction pathway and gluconeogenesis to further explore the mechanisms to improve insulin sensitivity by sleeve gastrectomy.MethodsIn this study, the non-obese type2diabetic GK rats were randomly divided into SG, sham group (SOSG) and control group.At2and36weeks post operation,10mice in each group were sacrificed to retain the fresh skeletal muscle, liver, and small intestine, respectively. We explored the insulin signaling pathway and gluconeogenesis using western blot, real-time polymerase chain reaction, and other molecular biology techniques. The key indicators for monitoring:(1) fasting blood glucose and ITT at2and36weeks after surgery to confirm the effect of SG surgery on improvement of insulin sensitivity;(2) the key genes of hepatic insulin signaling transduction pathway, detect the mRNA, protein and protein phosphorylation expression of the hepatic insulin receptor-β(IR-β), and insulin receptor substrate-2(IRS-2) at2and36weeks after surgery;(3) the key genes of skeletal musle insulin signaling transduction pathway. detect the mRNA, protein and protein phosphorylation expression of the skeletal musle insulin receptor-β(IR-β), and insulin receptor substrate-1(IRS-1) at2and36weeks after surgery;(4) the key enzymes in the intestinal gluconeogenesis. detect the small intestine [Four segments of intestine of rats in three groups were removed and separated. The length of each segment was10cm. proximal jejunum:10cm distance from Treitz ligament, distal jejunum:30cm distance from Treitz ligament, proximal ileum:20cm distance from the ileocecal ileum, distal ileum:10cm distance from the ileocecal ileum]. detect the activity, mRNA, and protein expression of the intestinal phosphoenoylpyruvate carboxykinase (PEPCK), and glucose-6-phosphatase (G6Pase) at2and36weeks after surgery;(5) detect the activity, mRNA, and protein expression of the hepatic phosphoenoylpyruvate carboxykinase (PEPCK), and glucose-6-phosphatase (G6Pase) at2and36weeks post operation.Results1. At2and36weeks after surgery, fasting blood glucose and AUCITT values of SG rats were lower than those of the sham and control group (P<0.05), once more providing evidence for the improvement of insulin sensitivity by SG surgery.2. At2and36weeks after surgery, compared to the sham and control group, SG rats exhibited increased expression of hepatic IR-β, and IRS-2mRNA levels, total protein and phosphorylated protein (P<0.05). Compared to at2weeks post surgery, the difference is more drastic at36weeks post surgery.3. At postoperative week2, No difference were found in the expression of skeletal muscle IR-β, and IRS-1mRNA, total protein and phosphorylated protein among all three groups (P>0.05); while at postoperative week36, SG rats showed up-regulated expression of skeletal muscle IR-P, and IRS-1mRNA levels, total protein, and phosphorylated protein (P<0.05).4. At postoperative week2and36, compared to the sham and control group, SG rats exhibited increased intestinal PEPCK and G6Pase enzymes activity(P<0.05), and up-regulated PEPCK and G6Pase mRNA and protein expression(P<0.05). Compared to at2weeks post surgery, the difference in mRNA and protein expression is more obvious at36weeks post surgery.5. At postoperative week2and36, compared to the sham and control group, SG rats exhibited decreased hepatic PEPCK and G6Pase enzymes activity(P<0.05), and down-regulated PEPCK and G6Pase mRNA and protein expression(P<0.05). Compared to at2weeks post surgery, the difference in mRNA and protein expression is more obvious at36weeks post surgery.Conclusions1. SG is effective in up-regulating the expression of the key proteins in the hepatic insulin signalinging pathway(IR-β,IRS-2), while the expression of the key proteins in the peripheral muscle insulin signalinging pathway(IR-β, IRS-1) was unchanged at early stage post operation, suggesting enhanced early postoperative hepatic insulin signaling transduction pathways is involved in improving hepatic insulin sensitivity. No change in the early postoperative peripheral muscle insulin signaling transduction pathway was consistent with no improvement in the early postoperative peripheral insulin sensitivity.2. SG is effective in up-regulating the expression of the key proteins in the hepatic insulin signalinging pathway(IR-β, IRS-2), and the expression of the key proteins in the peripheral muscle insulin signalinging pathway(IR-β, IRS-1) was also increased. Enhanced long-term postoperative hepatic and peripheral muscle insulin signaling transduction pathways were involved in the long-term postoperative insulin sensitivity improvement. This is consistent with improved long-term postoperative hepatic and peripheral insulin sensitivity by SG.3. SG down-regulated the expression and decreased the activity of the key regulatory enzymes of hepatic gluconeogenesis (PEPCK and G6Pase) at early term post operation, and the down-regulation was more significantly at long term post operation, suggesting SG surgery resulted in decreasing hepatic gluconeogenesis and reducing hepatic glucose output to achieve improvement of hepatic insulin sensitivity. 4. SG up-regulated the expression and increased the activity of the key regulatory enzymes of intestinal gluconeogenesis (PEPCK and G6Pase) at early term post operation, and the up-regulation was more significantly at long term post operation,, suggesting that the down-regulated hepatic gluconeogenesis is closely related to the enhanced intestinal gluconeogenesis.Our findings indicated that the improvement of insulin sensitivity was closely related to insulin signaling transduction pathway and hepatic gluconeogenesis. Early postoperative enhancement of hepatic insulin signaling transduction pathway and down-regulation of hepatic gluconeogenesis contributed to improving insulin sensitivity at early stage post operation. Whereas enhanced hepatic and peripheral insulin signaling transduction pathway and decreased hepatic gluconeogenesis jointly contributed to improving insulin sensitivity at long term post operation. The study also found that the down-regulated hepatic gluconeogenesis was closely related to the enhanced intestinal gluconeogenesis, providing new ideas for studying the role and mechanism of metabolic surgery in the treatment of diabetes. |
PDF Full Text Request