| Now there has been a tragedy increase in diabetes across the world, paralleling the overweight and obesity epidemic. There are 95 percent of those people belonging to type 2 diabetes. Therefore, finding better treatments and novel prevention strategies for type 2 diabetes is a matter of great urgency. To accomplish this goal, appropriate experimental models are considered as essential tools for understanding the molecular basis, pathogenesis of the vascular and neural lesions, actions of therapeutic agents and genetic or environmental influences that increase the risks of type 2 diabetes.Although there are numerous animal models (natural as well as developed) available for the study of type 2 diabetes, the pattern of disease establishment and progress in most of them did not appear to be similar to the clinical situation in humans. Thus, there is a continued quest among the investigators with respect to establishment of better animal model for type 2 diabetes either by adjusting the existing methods or/and by developing new methodologies or a combination of both.Many studies have reported that the rats fed with high-fat diet (HFD) develop insulin resistance but not frank hyperglycemia or diabetes. Suggesting that the HFD might be a better way to initiate the insulin resistance which is one of the important features of type 2 diabetes. At the same time, Streptozotocin (STZ) is widely used to reproducibly induce both insulin-dependent and non- insulin-dependent diabetes mellitus presently by inducingβcell death through alkylation of DNA. Although high-dose STZ severely impairs insulin secretion mimicking type 1 DM, low dose STZ has been known to induce a mild impairment of insulin secretion which is similar to the feature of the later stage of type 2 diabetes. Therefore, investigators have started to develop a rat model by feeding the animal with high-fat diet following low-dose STZ that would closely mimic the natural history of the disease events (from insulin resistance toβcell dysfunction) as well as metabolic characteristics of human type 2 diabetes. The successful establishment of such a model would be cheaper, easily accessible and practical for the investigation as well as testing of various compounds for the treatment of type 2 diabetes. Although the appearance of the type 2 diabetes pattern was achieved by combining the feeding of HFD and low dose of STZ treatment in non-genetic, out-bred rats, the injection dose of STZ and its methodologies were not consistent in those studies. Others reported that STZ may also be given in multiple low doses. It has been extensively used in the development of type 1 diabetes in rats and mice to study immune response in pancreas, since the multiple low-dose injections of STZ could induce a gradual, autoimmune destruction ofβcells instead of the rapid destruction induced by single high dose injection. However, it has not been reported whether the high fat diet has synergistic effect on speedying the development of type 2 diabetes with multiple low doses STZ.Glucocorticoid has been documented for a long time as a key regulator of salt and water metabolism, blood pressure, immune function and metabolism. Recent evidence suggests that the action of glucocorticoid is regulated by glucocorticoid receptors and 11β-Hydroxysteroid dehydrogenase (11β-HSD), both of which play key roles in the pathophysiology of type 2 diabetes and metabolic syndrome. At present, a great deal of research is focused on the management of local glucocorticoid action as a therapeutic strategy. It has been reported that the most classical actions of glucocorticoids were regulated principally via the activation of intracellular receptors (glucocorticoid receptor). The interaction of intracellular cortisol/corticosterone with glucocorticoid receptor is mainly mediated by 11β-HSD1, which offers a key advantage for regulating glucocorticoid action. It reduces glucocorticoid action selectively without influencing the central negative feedback. A substantial data have demonstrated that inhibition of 11β-HSD1 activity can increase insulin sensitivity without the consequences of changing the circulating glucocorticoid levels in liver and adipose tissues. Beneficial effects of inhibiting 11β-HSD1 in adipose tissue have been estimated from in vitro studies.Skeletal muscle is an important target tissue for insulin stimulated glucose consumption. The molecular mechanisms of insulin-dependent glucose uptake in skeletal muscle have been broadly explored. Normally, circulating insulin binding to the insulin receptor (IR), phosphorylatesβ-subunit and causes insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation, which binds to the p85 subunit of phosphatidyl inositol 3-kinase (PI3K). This in turn activates Akt, leading to glucose transporter-4 (GLUT-4) translocation. It has been proved that this insulin signaling pathway present abnormally in the skeletal muscle of obesity and diabetic patient. The change on the insulin signaling pathway related protein has been verified to be the key point in the pathogenesis of insulin resistance and diabetes. Therefore, it is really important to investigate theeffect of alteration of 11β-HSD1 in the skeletal muscle of type 2 diabetes.Present study attempt to develop an ideal type 2 diabetes animal models, explore the optame dosage of STZ injection and method, observed the successful rate of diabetic model. Meanwhile the IPGTT and ITT were also performed, find the level of insulin resistance, and adjust the times of STZ injection. High fat diet combined once STZ injection with moderate low dose to induce diabetic and no damage on the pancreas. However, in the first section, we found that 45 mg/kg STZ is too much to induce type 2 diabetes. The 25, 30, 35 mg/kg are not enough to induce type 2 diabetes with lower diabetic successful rate, therefore we examined in the second section with multiple low dose STZ injection (25, 30 mg/kg, twice i.p.). In the second section, we found 25mg/kg twice ip has low successful rate, so we focus on the 30mg/kg twice injection group, the IPGTT and ITT have been carried out. Following this section, we continued the third section experiments. We use multiple low dose STZ injection to destroy the pancreaseβcell, combined with insulin resistance induced by high fat diet. Our study demonstrates that a combination of HFD and multiple low dose of STZ (30mg/kg, twice) injection could be effectively used to generate a rat model that mimics the natural history and metabolic characteristics of type 2 diabetes in humans. This method will successfully produce type 2 diabeteic rat models, as well as provide the enough number of diabetic rats once which will make the investigation more convincing. It was also useful in exploring the pathogensis of type 2 diabetes and evaluating the effect of therapeutic compounds on the treatment of type 2 diabetes.The present study also observe the pancrease morphology, the corticosterone level in the blood, the alteration of 11β-HSD1 and GR in this type 2diabetic model in skeletal muscle, and study the abnormality of insulin signal transduction pathway related protein. RT-PCR was used to evaluate the mRNA level of 11β-HSD1 and GR,western blot was used to study the protein level of 11β-HSD1, GR, insulin receptor, AKT and GLUT4. Spectrofluorometric determination analysis examined the alteration of corticosterone level in the serum. We found that corticosterone level in the serum was no changed in the model and control group, the protein level of 11β-HSD1 and glucocorticoid receptor was significantly increased. The mRNA level of 11β-HSD1 was also elevated. The mRNA level of glucocorticoid receptor was decreased. After insulin stimulation, diabetic rats had no significant changes in the level of the insulin receptorβ-subunit (IR-β), AKT, as in phosphorylated AKT in the gastrocnemius muscle compared to its basal state. Similar results were observed in the protein expression level of glucose transporter 4 (GLUT4). Our data indicate that the alteration of 11β-HSD1 at protein and mRNA level may be related to the abnormality of insulin signal pathway in skeletal muscle.This effect may be mediated by glucocorticoid receptor.Conclusively, based on the others'study, the present study develop a type 2 diabetes animal model by a combination of HFD and multiple low dose of STZ injection could be effectively used to generate a rat model that mimics the natural history and metabolic characteristics of type 2 diabetes in humans, which present high diabetic incidency, low expence and enough number of models can be developed at the same time.Meanwhile, the insulin signaling pathway related protein present abnormally in this model which indicate the decrease of insulin sensitivity, and the key enzyme of glucocorticoid(11β-HSD1) and glucocorticoid receptor were changed in the skeletal muscle as well. It may provide theory basis to produce 11β-HSD1 inhibitory drugs interfering with the pathogenesis of type 2 diabetes.
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