| There were almost280million people suffering from diabetes mellitus all over the world. This number was expected be as high as48.3million by2050in America. Among this, type2debetes accounting for90%. In2007, diabetes is the seventh important cause of morality, with diabetic patients having twice the risk of death when compared with their age-matched counterparts. Meanwhile, diabetes increases the costs for the public health system. The medical cost for a diabetic patient is about twice or three times greater when compared with a non-diabetic patient. The estimated cost of diabetes in the USA is about US$174billion, and will reach as high as$192billion by2020, with about67%corresponding to direct costs. Patients with type2diabetic suffered from increasing risk of congestive heart failure and higher mortality rate due to myocardial infarction. Meanwhile, diabetic cardiomyopathy is now known to have a high prevalence in the asymptomatic type2diabetic patient. It was shown the prevalence of diastolic dysfunction to be60%in well-controlled type2diabetic patients. Type2diabetes is prevalent, expensive, and lethal that it becomes a worldwide concerning disease. However, the mechanisms and best treatment are still needed to be discerned.Bariatric surgery has been documented to improve glucose homeostasis and lead to improvement in glycemic control, resulting from caloric restriction and alternations in the gut hormones that control insulin secretion. The concept of ileal transposition (IT) was first described in rodent experiments. It has been confirmed that ileal transposition can improve glucose tolerance compared with other bariatric surgeries, was faster to perform and yielded a shorter recovery time in animal models of non-obese type2diabetes. These findings strongly suggested that an underlying hormonal mechanism involving the terminal ileum may participate in the resolution of type2diabetes after surgery. In addition, plasma glucose control often occurs before significant weight loss has been reached, suggesting that a direct effect of surgery rather than a secondary effect of weight loss and amelioration of obesity on glycemic control. Moreover, glucagon-like peptide-1(GLP-1), as a potent insulin secretagogue, secreted more in IT and has been shown to stimulate the myocardial glucose uptake in normal and postischemic isolated hearts. These findings raise the possibility that IT surgery involving switching a segment of proximal intestine from nutrient flow and a segment of distal intestine to earlier food exposure may play a role in myocardial glucose signaling cascade.To explore whether the myocardial glucose metabolism function can be improved after ileal transposition and the mechanisms underlying this, we used spontaneous non-obese Goto-Kakizaki (GK) type2diabetic rats as the animal model. First, we confirmed the changes of myocardial insulin signaling and glucose transport in GK rats. Then, followed by measurements of glucose homeostasis after IT surgery, we evaluated improvements of myocardial insulin resistance and signaling in GK rats. Also, we isolated cardiac myocytes and sitmulated with GLP-1and its agonist Ex-4to further explore the mechanism of resolution of myocardial glucose metabolism.Part1Changes of the myocardial glucose metabolism and insulin signaling in GK ratsAs a spontaneous non-obese type2diabetic rat model, GK rats exhibit mild hyperglycaemia and hyperinsulinaemia, impaired glucose-induced insulin secretion, marked glucose intolerance, hepatic glucose overproduction and peripheral insulin resistance. These changes may lead to impaired myocardial function. We first examined the function of myocardial glucose uptake, insulin signaling and glucose transport.1. Insulin stimulated myocardial glucose uptake was lower in GK rats.To investigate the myocardial glucose uptake, we collected the isolated rat hearts from8-10week GK rats or SD rats. We first perfused the heart to wash out the blood residue, incubated in Krebs-Ringer’s buffer with2D-3H-glucose for10min, and then added insulin to stimulate the uptake of glucose. The final results showing that the glucose uptake rates were similar without insulin stimulation in GK and SD rats. Meanwhile, insulin was capable of inducing the increase in glucose uptake. Glucose uptake in response to insulin was impaired in GK rats compared with SD rats.2. Insulin signaling in GK rat hearts.The process of autophosphorylation of a number of tyrosine residues can be initiated by binding of insulin with the insulin receptor. These tyrosine residues could be recognised by phosphotyrosine-binding (PTB) domains of adaptor proteins including members of the insulin receptor substrate family (IRS). This process led to the phosphorylation of important tyrosine residues on IRS proteins (IRS-1and IRS-2), some of which were then recognised by a lipid kinase: PI3-kinase (PI3K). In this study, we focused on the key proteins and their phosphorylations in insulin signaling pathway as well as IRS-1associated PI3K. We isolated total mRNA and protein from fresh hearts of GK and SD rats, using Western blot. RT-PCR and Co-IP to measure the expression level of IR-p, IRS-1. Akt and their phosphorylations. Moreover. IRS-1associated PI3K was measured after insulin sitmulation.2.1Expressions of total IR-β mRNA, total and phosphorylated IR-β protein were lower in GK rats. As a transmembrane high molecular protein, Insulin Recptor (IR) has2subunits:α and β. The activation of insulin signaling needs the conformational change of a subunit and leading to the tyrosine phosphorylation of β subunit, thus, resulting in the activation of downstream signals. Our study used Western blot, RT-PCR and Co-IP to confirm downregulation of IR-β mRNA, total and phosphorylated IR-β protein were lower in GK rats.2.2Expressions of total IRS-1mRNA, total and phosphorylated IRS-1protein were lower in GK rats.Insulin receptor substrates also related to the signaling induced by insulin receptor, among these IRS-1plays a significant role. Evidence has been shown that IRS-1knockout mice displayed severe insulin resistance, which confirmed the role of IRS-1in the insulin signaling. Our study used Western blot, RT-PCR and Co-IP techniques, confirmed the downregulation of IRS-1in the GK rat hearts.2.3Inhibition of insulin stimulated IRS-1/PI3K/Akt pathway.IRS-1associated PI3K is the major insulin signaling pathway. Retarded IRS-1/PI3K pathway has a negative impact on the downstream metabolism. We used Co-IP technique and showed the impaired IRS-1associated PI3K.Akt which is also called PKB, is a serine/theronine protein kinase. As an IRS/PI3K trarget protein, it can induce the cell cycle by activating CDK; it also can induce the activity of GLUT4to promote the uptake of glucose. Meanwhile, activated Akt can increase the glycogen synthesis by GSK3. In our study, we used Western blot and Co-IP to show the expression of total and phosphorylated Akt.3. Changes of glucose transport protein in GK rats.Glucose transport protein is the carrier of glucose. GLUT1expressed in most tissues, which can regulate the uptake of glucose, while GLUT4mainly expressed in adipose tissue and striated muscle. Under the stimulation of insulin, GLUT4can effectively transport the glucose from the cytosol to the cell membrane. It has been shown that the Akt and PI3K can induce the translocation of GLUT4. To further investigate the insulin signaling in GK rats, we collected the total and membrane protein from rat hearts and muscle, and measured the expressions of GLUT1/GLUT4. We found the downregulation of membrane associated GLUT4protein expression in rat hearts.Part2The effectiveness of ileal transposition on non-obese type2diabetic rats and myocardial glucose uptakeIt has been confirmed that ileal transposition can improve glucose tolerance compared with other bariatric surgeries, was faster to perform, and yielded a shorter recovery time in animal models of non-obese type2diabetes. These findings strongly suggested that an underlying hormonal mechanism involving the terminal ileum may participate in the resolution of type2diabetes after surgery. In addition, plasma glucose control often occurs before significant weight loss has been reached, suggesting a direct effect of surgery rather than a secondary effect of weight loss and amelioration of obesity on glycemic control. Bariatric surgery has been shown to improve left ventricular systolic dysfunction in young morbidly obese individuals with non-ischemic cardiomyopathy. The improvements ininsulin response and reduced direct toxic effects of adiposity on cardiomyocytes may contribute to myocardial function improvement. Meanwhile, partial regression of cardiac hypertrophy and reversal of both diastolic dysfunction and aortic distensibility impairment were found1year after bariatric surgery. To investigate whether the distal ileum has function in the glucose tolerance and glucose homeostasis. we used GK rats as our animal model and designed the following experiments.1. The evaluation of the ratsMale8-10week GK rats and age matched SD rat were housed in individual cages under constant room temperature (22±1℃) and relative humidity (45-55%) in a12-h light/dark cycle. In order to evaluate the stability of the type2diabetic rat models, we measured the body weight, food intake, and fasting glucose level before intervention. The GK mice were randomly divided into diabetic IT surgery group and diabetic sham surgery group, SD rats were treated as non-diabetic group.1.1Evaluation of the IT surgery group and Sham surgery group before surgeryNo difference was observed relating to the food intake, body weight, fast glucose, OGTT, ITT and serum insulin, peptide C, GLP-1, GIP, PYY in the IT group and sham group.1.2Evaluation of the IT surgery group and Sham surgery group after surgeryThere was no difference between the mean operative time and operative success rate within the two groups. All the rats were performed successfully, only2of them from each group were died due to obstruction and anastomotic leakage, the mortality rate was6.7%from each group. In total, ileal transposition is safe and reliable.1.2.1IT surgery on food intake, body weight, fast glucose, OGTT and ITTThe improvements of glucose level, OGTT and ITT occurred earlier than the decrease of food intake and body weight, suggesting the effect of IT on the glucose homeostasis is not dependent on changes of body weight and food intake. Meanwhile, the remission of type2diabetes sustained6months after IT, indicating the effect of IT is long and stable.1.2.2IT surgery on the function of pancreatic p cellThe function of pancreatic β cells refers to the ability of β cell in maintaining the stability of blood glucose level after stimulation of glucose. And the serum insulin and peptide C are considered important indicators of pancreatic β cell function. In this study, we collected the serum from fasting or glucose stimulated rats and used ELISA technique to test serum insulin and peptide C. Our findings indicate that IT is capable of inducing the secretion of serum insulin and peptide C.1.3IT surgery on secretion of gastrointestinal hormonesIt has been shown that gastric bypass surgery can induce the secretion of serum GLP-1and PYY. In the case of animal, GLP-1has been testified to induce the secretion of insulin, promote the proliferation of pancreatic β cell and inhibit its apoptosis. PYY also has been studied in the obese animals and been proved to be able to promote the function of pancreatic β cell. GIP also called glucose-dependent insulinotropic polypeptide, which can induce the secretion of insulin under hyperglycemia. By comparison experiments, we found the increase of GLP-1and PYY, while no change of GIP, suggesting GIP may not be a factor involving the remission of type2diabetes.2. IT surgery on glucose uptakeGlucose uptake is a important index of blood glucose homeostasis. In our study, we isolated fresh rat heart, perfused with insulin to stimulate the uptake of glucose. The results showed that the glucose uptake was not changed1month after ileal surgery. However, compared with sham group,6months after IT surgery increased the glucose uptake, suggesting IT surgery may up-regulate insulin signaling and transport of glucose.Part3The mechanism of ileal transposition on myocardial glucose metabolism in non-obese type2diabetic ratsPrevious studies have proved the impaired myocardial insulin signaling and glucose transport in non-obesed type2diabetic rats. Though many researchers reported the effectiveness of ileal transposition on improvements of blood glucose homeostasis. the detailed mechanism underlying this is unknown. Our study has found ileal transposition had systemic effect on the blood glucose homeostasis and myocardial glucose uptake. To further elucidate the mechanisms of IT on myocardial insulin signaling, we used isolated fresh rat hearts and muscle tissue. then applied Western blot, Co-IP to prove our hypothesis. In our study, we also isolated myocytes from GK and SD rat hearts, and stimulated with GLP-1peptide and its angonist Ex-4to see the effect of GLP-1on isolated cardiac myocytes insulin signaling.1. Ileal transposition on myocardial insulin signaling1.1The total and tyrosine phosphorylation of IR-β increasedIR-β increased6months after IT, the fold increase of phosphorylated IR-β was similar as IR-β, which indicates the increase of p-IR-β may be as a result of total IR-β.1.2The total and tyrosine phosphorylation of IRS-1increasedSimilarly, changes have been found in IRS-1between the IT and sham groups. However, expression of IRS-1and tyrosine phosphorylation of IRS-1showed an earlier difference between the IT group and sham-IT group1month after surgery. Meanwhile, time-dependent changes were also observed within the IT groups.1.3Improvements of insulin stimulated IRS-1/PI3K after ITIRS-1-associated PI3K is considered one of the important indicators of insulin resistance. We performed coimmunoprecipitation to explore the association of IRS-1and PI3K. In comparison with the IT-operated and sham-operated rats, there were significant differences in IRS-1-associated PI3K immunoprecipitation from the first month. Meanwhile, a time-dependent increase in IRS-1-associated PI3K complex was observed within the IT groups (p<0.05).2. Effect of it on myocardial and skeletal muscle glucose transport4expressionGLUT4is a key determinant of glucose homeostasis. Translocation of GLUT4. especially in the skeletal muscle, is considered to be the major mechanism responsible for the activity of insulin. Thus, we next examined total as well as the cell membrane GLUT4protein expression in the myocardium and skeletal muscle. As shown in, there was no significant difference between all groups relating to the protein expression of total GLUT4. However, the expression of membrane GLUT4was up-regulated by IT surgery in a short period (1month) after surgery, and this up-regulation was more drastic and reached statistical significance6months in comparison with1month after surgery.3. GLP-1and Ex-4have the insulin-mimetic effects on isolated GK rats cardiac myocytesBoth GLP-1and Ex-4were discovered that they can up-regulate the glucose uptake in isolated GK rats cardiac myocytes. Meanwhile, IRS-1associated PI3K increased in cardiac myocytes treated with either GLP-1or Ex-4, suggesting ileal transposition may act through the increased GLP-1to improve the insulin signaling and glucose uptake. |
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