| From thousand years, exploration in medical science has never ceased to find an effective way to cure human disease. Improving quality of our life and lengthening our lives have always been a holy mission for medical workers. The prevalence of diabetes in china is rising fast with the rapid economic development and the process of industrialization as well as life style changing and the acceleration of aging process. Diabetes has become another important chronic noncommunicable disease which can cause serious damage to people’s health following the disease of heart head blood-vessel and cancer. The WHO estimates that the Chinese economic losses will be at $557.7 billion because of diabetes and its related cardiovascular disease from 2005 to 2015.Howerve, whether developed countries or developing countries, a number of surveys show that diabetes control situation is still not optimistic.For a long time, colleagues at home and abroad take many efforts of the prevention and treatment of diabetes, such as carrying out a large number of propaganda and education on diabetes, epidemiological investigation, prevention and control research, as well as clinical and basic research. The constantly emerging new oral glucose-lowering drugs in the worldwide provide more choices for the treatment of diabetes. However, whether increasing insulin secretion or improving insulin sensitivity, or insulin pump replacement therapy, are mostly limited to the single target of "islet beta cells/insulin", which could not be entirely correct the pathological. The researches has began to pay more attention to the importance of glucagon in diabetes development and glucagon has become a new direction of research in the field of diabetes in recent years. For now, diabetes is considered to be due to decreased insulin secretion and excessive glucagon secretion. The glucagon levels of patients with type 2 diabetes(T2DM) is much higher than that of healthy population. Available data confirm that whether oral hypoglycemic drugs or insulin replacement therapy could improve glucagon levels of patients with T2DM, which suggest that glucagon is closely related to the occurrence and development of T2DM.Glucagon is a 29 amino acid peptide hormone, which is secreted from pancreatic a cells. As the major counterpart to insulin, glucagon plays an important role in promoting glycogen decomposition and sugar dysplasia as well as in blood glucose homeostasis. Pancreatic a-cell glucagon secretion is critically dependent on pancreatic β-cell insulin secretion. Normally, a decrease in the plasma glucose concentration causes a decrease in P-cell insulin secretion that signals an increase in a-cell glucagon secretion during hypoglycemia. In contrast, an increase in the plasma glucose concentration, among other stimuli, causes an increase in a-cell insulin secretion that signals a decrease, or at least no change, in a-cell glucagon secretion after a meal. T2DM is considered to be a direct consequence of insulin resistance and β-cell dysfunction. Pancreatic a cells and their secretary product, glucagon, are often overlooked. In recent years, several papers have focused on the signaling pathways ina cells that regulate glucagon secretion under physiological conditions, and have confirmed that insulin inhibit gene expression of pancreatic a cells and glucagon release through the insulin receptor substrate (IRS)- phosphatidyl inositol-3-kinase (PI3K) way. Impaired insulin signal transduction pathway after receptors on the a cell membrane could result in excess glucagon synthesis and secretion. Therefore, impaired insulin secretion weakened inhibition of glucagon secretion or decreased insulin sensitivity in the condition of diabetes causes inappropriate increase of glucagon secretion.Multiple lines of evidence have implacated that increased activation of the RAS is involved in the development of type 2 diabetes over the past few years. In agreement with that, a meta-analysis of comparative outcome trials has shown that RAS blockade, using either angiotensin Ⅱ type 1 receptor blockers (ARB) or angiotensin-converting enzyme inhibitors (ACEI), significantly reduced the incidence of new-onset type 2 diabetes in high-risk populations. However, the detailed mechanisms are still unknown, which might be explained by improved insulin secretion and insulin sensitivity. In addition to the systemic RAS, which is known for its classical effects on blood pressure, electrolyte and fluid homeostasis, RAS components have been identified in many tissues, including the kidney, heart, brain, nerve fibers, reproductive organs, blood vessels, liver, skeletal muscle, and pancreas. Thus, it may well be that the pancreatic RAS also affects islet function. Indeed, in vitro and in vivo studies performed in rodents have shown that the RAS induced islet fibrosis, oxidative stress, inflammation, and impaired insulin secretion, whereas RAS blockade with an ACEI or ARB improved islet morphology and function and increased glucose tolerance.Now, the research about intraislet RAS mostly focus on pancreatic P cell function. The characteristic change of pancreatic a cell function and glucagon secretion by intraislet RAS is yet not unclear. Current evidence demonstrate that the detrimental effects of the RAS on insulin secretion and insulin sensitivity are mediated by a reduction in pancreatic blood flow and induction of islet fibrosis, oxidative stress as well as inflammation. Our previous studies also found that candesartan treatment could not only significantly improve glucose tolerance and reduce insulin resistance,bus also have certain improvements of β cell function in db/db mice. Besides, the excess glucagon secretion has been also found reduced. Notwithstanding, it is unclear whether or not these benefits are dependent on the changes of circulating RAS components.Therefor, we utilized small interfering RNA (siRNA) to investigate the effects of ATIR knockdown on the expression of insulin signal transduction molecules and evaluate their dynamic characteristics of glucagon and insulin secretion in isolated islets of db/db mice and to explore the potential mechanisms invovled. Thus to further reveal the role of local RAS in the endocrine pancreas islet and relation with T2DM, and to expand the new idea of prevention and treatment of T2DM.Part I Expression of angiotensin II type 1 receptor in intro-islets of db/db miceObjectiveTo obtain isolated islets and observe the expression of angiotensin II type 1 receptor(AT1R) of islets in vitro in db/db mice.MethodFifteen eight-week old female db/db mice and fifteen age and gender matched nondiabetic littermates db/m mice were obtained from the Animal Experimentdetected by RT-PCR and western blotting.ResultsThe expression level of AT1R, both in mRNA and in protein, in isolated islets of db/db mice was nearly three times of db/m mice[mRNA:(320±25)% vs (100±8)%,P<0.05; protein:(310±20)% vs (100±8)%), both P＜0.05].ConclusionIt indicating that AT1R was overexpressed in diabetic pancreatic islets.Part Ⅱ Construction of recombinant adenoviruses containing small interfering RNA targeting AT1R of db/db miceObjectiveTo construct recombinant adenovirus containing small interfering RNA (siRNA) targetingATIR mRNA (Ad-siATIR) and amplify the adenovirus in 293 cells.MethodPlasmid vector containing mice AT1R siRNA was obtained from the company. Recombinant adenoviruses containing the siATIR sequences were constructed using AdEasy System. Briefly, for each pSilencerbased clone, the siRNA expression cassette was excised and ligated into linearized adenoviral shuttle vector pAdTrack. Subsequently,1 μg recombinant Pmel-linearized pAd-siAT1R was transfected into Escherichia coli BJ5183 cells with an adenoviralbackbone plasmid, pAdEasy-1. Recombinants were selected and successful recombination was determined by restriction endonuclease analysis. The linearized recombinant adenoviral construct was transfected into 293 cells and high-titer viral stocks were prepared. Viral titers were determined by plaque assay and expressed as plaque-forming units per mL (pfu/mL). No-load virus Ad-siControl was amplyficated and purificated to volume with the same way. The viral titers of Ad-siATRl and AdsiControl were 3.6 x 109 pfu/mL and 2.9 x 109 pfu/mL,respectively.ResultsRecombinant adenoviral vector pAdEasy-siRNA-ATIR was constructed successfully, which was confirmed by Pac I digestion. Green fluorescent protein(GFP) was expressed in 293 cells infected with recombinant adenovirus Ad-siAT1R. After being packaged in 293 cells and purified by CsCI banding, the liter of recombinant adenovirus Ad-siAT1R was as high as 3.6×109efu/ml.And the viral titers of Ad-siControl was 2.9 x 109 pfu/mL.ConclusionAdEasy-1 system is an efficient way to construct recombinant adenovirus containing siRNA targeting mice AT1R mRNA and this method would become a potent tool to investigate the biological function of intro-islets ATIR.Part Ⅲ Evaluation of kinetics of glucagon release in vitro Islet with adenovirus infected through islet perifusion systemObjectiveObservation of expression of ATIR as well as insulin receptor substrate (IRS), phosphatidyl inositol-3-kinases regulate subunits p85 [PI3K(p85)] and phosphorylation of serine/threonine protein kinase (p-Akt2) in islocated islet of db/db mice with adenovirus infected. And islet perifusion was performed to evaluate kinetics of insulin and glucagon release in vitro.MethodsIsolated islets of db/db mice were divided into three groups:1) Ad-siAT1R group, islets were transduced with Ad-siAT1R for 2h; 2) Ad-Control group, islets were transduced with Ad-siControl; 3) Control group, islets were not exposed to virus. Islets were cultured for 48 h. ATIR mRNA and protein levels were measured for each condition. IRS, PI3K (p85) and p-Akt2 were detected by western blot and the kinetics of insulin and glucagon release in vitro were evaluated through Islet perifusion.ResultsAfter virus transduced, Ad-siAT1R treatment resulted in significant decrease in ATIR mRNA and protein levels compared with islets treated with Ad-siControl (p< 0.05).Besides, western blot showed that islets treated with Ad-siATlR manifested significant increase of IRS-1, IRS-2,PI3K(p85)and p-Akt2 when compared with ones treated with Ad-siControl (P< 0.05).In addition, with down expression of ATIR, notable increased insulin secretion and decreased glucagon secretion levels were found by perifusion.ConclusionThe siRNA specific to AT1R can specifically suppress introislet ATIR expression, and thus improve the excessive secretion of glucagon. |
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