| Background:Type 2 diabetes mellitus is a serious and chronic non-infectious metabolic disease. It belongs to the category of "diabetes" in traditional Chinese medicine, also be called as"lung elimination" or "GE Consumer" name. Its prevalence is rapidly increasing in China, becoming an important health problem. The total number of diabetic patients in 2014 will be over 100 million. The prevalence rate of diabetes is up to 11.6% of people over the age of 18, of which more than 95% are type 2 diabetes mellitus. Insulin resistance and/or insufficient insulin secretion are the major causes of type 2 diabetes mellitus. Despite extensive investigations into the type 2 diabetic entity, to date the precise mechanism of this disease is still poorly understood. Numerous factors are implicated in the pathogenesis of type 2 diabetes, and the defect of GLUT4 function is an important one that may increase insulin resistance and reduce glucose uptake in insulin-independent diabetes. Skeletal muscle and adipose tissue are quantitatively the most important targets for insulin-stimulated glucose disposal that predominantly depends on GLUT4. Only at the cell surface can GLUT4 transport glucose into cells. The maximal glucose clearance activity by skeletal muscle and adipose tissue is directly proportional to the GLUT4 protein concentration in plasma membranes, which represents and reflects insulin sensitivity. Although only 10% of insulin-stimulated glucose uptake occurs in adipose tissue, this process is specially important for controlling whole-body energy homeostasis and glucose metabolism. Adipose tissue essentially contributes to the obesity-driven insulin resistance syndrome as it can buffer excess energy and control metabolic homeostasis. In particular, obesity is now recognized as a risk factor for the development of insulin resistance and type 2 diabetes. Adipose tissue is also an important target for studying glucose clearance and diabetes.Based on different strategy, the treaments of diabetes with Chinese medicine have obtained the certain effect, such as treatment with heat Sheng Jin, Qi Yin and Jianpi Huatan to promote blood circulation, in order to remove phlegm and blood stasis and to alleviate liver and clearing heat, as well as to purge fire detoxification. The Chinese medicine attaches aim to the overall adjustment via varies ways and polycyclic festival, including reducing blood glucose, improving lipid and metabolic disorder in the diabetoc states. Therefore, Chinese medicine has a great advantage in treatment of diabetes mellitus.Numerous studies have shown that berberine may improve insulin resistance, decrease blood glucose, regulate lipid disorder and insulin sensitivity, not induce obesity and water retention. However, galanin can increase food intake and body weight of subjects. Our past studies found that administration of M35, a galanin antagonist, is reduced insulin sensitivity in both normal and type 2 diabetic rats, suggesting that endogenous galanin elevates insulin sensitivity in skeletal muscles and adipose tissues. However, it is unclear whether central galanin regulates insulin sensitivity, and whether berberine in coordination with galanin improve the enhancement effect of insulin sensitivity.This research explores the central role of galanin in regulation of insulin sensitivity in adipose tissue, and the combinative effect of berberine with galanin, type 2 galanin receptor (GALR2) specific agonists and antagonists respectively on amelioration of insulin resistance in type 2 diabetic rats. This study is helpful to understanding whether berberine in coordination with galanin reduces insulin resistance in the adipose tissue of type 2 diabetes. This exploration may develop a novel therapeutic approach to prevent and treat type 2 diabetes mellitus.Objectives:Understanding the central impact of galanin on decreasing insulin resistance and judging whether berberine cooperates with galanin and GALR2 agonists to reduce insulin resistance in adipose tissue of type 2 diabetic rats and their signal pathway in the regulation of insulin sensitivity.Methods:1. In order to understand the central role of galanin, we investigated the changes of insulin resistance after intracerebroventricular administration of M35 in adipocytes of type 2 diabetic rats. In this experiment animal exercise was taken as a physiological stimulus to increase central GAL mRNA expression and GAL secretion. Euglycaemic hyperinsulinaemic clamp and 2DG tests were used to determine insulin sensitivity. Plasma levels of insulin, triglyceride and cholesterol were monitored with each ELISA test case respectively. GLUT4 expressed level in adipose tissue and GAL mRNA expression in the hypothalamus of rats by Western Blot and real-time PCR respectively. These contribute to our understanding of central role for GAL in regulation of insulin sensitivity in type 2 diabetic rats.2. In order to judge whether synergy of berberine with galanin reduces insulin resistance in adipose tissue of type 2 diabetic rats and benefites the signal transduction, the experimental rats were individually and jointly given berberine, galanin and Akt inhibitor MK-2206. Insulin sensitivity was determined by 2DG content in adipocytes of type 2 diabetic rats. The levels of GLUT4, VAMP2, Akt, pAkt, pAkt2Thr308, pAkt2Ser473, pAS160Thr462, pFoxO1 and pGSK-30 in adipose tissue of rats by Western Blot method. The GLUT4 mRNA and Akt2 mRNA expression in adipose tissue of rats by real time PCR. These contribute to our understanding of central role for GAL in regulation of insulin sensitivity in type 2 diabetic rats and whether berberine cooperates with galanin further to reduce insulin resistance and to promote phosphorylation of Akt and its downstream signaling proteins in adipose tissue of type 2 diabetic rats.3. In order to judge whether synergy of berberine and GALR2 agonists reduce insulin resistance in adipose tissue of type 2 diabetic mice, the experimental diabetic mice were injected with berberine, the GALR2 agonist M1896, GALR2 antagonist M871 and saline respectively and jointly. Body weight and food intake of the mice were measured before and after the experiment. The plasma glucose levels of all mice were inspected with a Glucose meter. Plasma levels of insulin, c-reactive protein, adiponectin, galanin and leptin with each ELISA test case respectively. The GLUT4 expressed level in adipose tissue of mice by Western Blot method. The GLUT4 mRNA quantity in adipose tissue by real time PCR. Based on body weight and blood glucose changes before and after the experiment and changes in insulin sensitivity index, we investigated whether synergy of berberine and GALR2 agonists further reduced insulin resistance in adipose tissue of type 2 diabetic mice, whereas not affecting food intake and body weight of animals.Results:1. Exercise significantly increaced the expression of central GAL mRNA in both healthy and type 2 diabetic rats. After central injection of M35, the body weight of rats, glucose infusion rate,2-DG levels, total optical density of GLUT4 in myocytes as also as the translocation of GLUT4 from endomembrane to plasma membrane were significantly decreased, while plasma glucose levels, the secretion of insulin, cholesterol and triglyceride levels were significantly increased.2. After combined administration of berberine and galanin,2-DG levels in adipocytes, total GLUT4, GLUT4 and VAMP2 levels in plasma membrane of rats were significantly higher than administration either one only in the type 2 diabetic rats. In addition, pAkt2Thr308 and pAkt2Ser473 levels, pAkt/Akt ratio and Akt2 mRNA levels in adipocytes of rats were significantly higher too. In the downstream protein of Akt2, joint injection of berberine and galanin enhanced the pAS160Thr462 levels in adipocytes of rats, while the expression of FoxO1 and GSK-3β were significantly reduced compared with treatment of berberine or galanin only. But there were no statistical change of GLUT4 mRNA levels in adipocytes and optical density of GLUT4 in endomembrane. Akt inhibitors MK-2206 could effectively antagonize above changes resulting from the synergistic effect of berberine and galanin.3. The central injection of GALR2 agonists into diabetic mice significantly decreased insulin and CRP secretion, blood glucose levels and insulin resistance, while significantly increased adiponectin and leptin secretion, glucose clearance, GLUT4 mRNA levels as well as total optical density of GLUT4 in adipocytes, translocation of GLUT4 from endomembrane to plasma membrane, but not food intake and body weight. Compared with administration of either berberine or galanin, the mice combined administered of berberine and galanin were significantly decreased the food intake, body weight, blood glucose, CRP secretion and insulin resistance, while significantly increased adiponectin and leptin secretion, glucose clearance, GLUT4 mRNA levels and total optical density of GLUT4, as well as translocation of GLUT4 from endomembrane to plasma membrane in adipocytes.Conclusions:1. The central injection of M35 decreased the GLUT4 translocation and glucose uptake in the fat cells of diabetic rats as well as increased insulin resistance, suggesting that endogenous galanin could increase the number of GLUT4 and promote its membrane translocation. It also suggested that galanin is one of the most important hormones to promote glucose transport across membrane. Galanin is an indispensable factor to maintain the balance of glucose, without which the role of insulin in stimulation of glucose uptake is decreased.2. Berberine could increase the role of galanin to mitigate insulin resistance through Akt2 signal protein pathways, but could not further increase the number of galanin in fat cells. By blocking the action of Akt with Akt inhibitors MK-2206, the elevated role of berberine was blocked. Therefore, berberine is Akt-dependent to enhance the effect of galanin on alleviation of insulin resistance.3. Combination of berberine and GalR2 agonists could significantly improve the insulin sensitivity compared with either berberine or GalR2 agonist in diabetic mice. Due to the activation of GalR2 has no effect on weight gain, combination of berberine and GalR2 agonists provides a new way to effectively prevent and treat insulin resistance and type 2 diabetes mellitus.without increase of boy weight. |
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