| The amount of alcohol beverage consumed in the world has being increased in recent decades. The total amount of alcohol consumption of the globe was nearly 200million tons and the nation that had the largest consumption was China. Alcohol is a two-edged sword. Light to moderate alcohol intake can reduce the incidence of atherosclerosis and coronary heart disease, thus the mortality rates due to these diseases are low. But. at the same time, the harmful effect of heavy alcohol consumed should not be neglected and the alcohol abuse has being an important public health problem. Because of the rapid increase in the incidence of diabetes worldwide, the relationship between alcohol intake and the risk of diabetes has been noticed widely. Many epidemiological researches suggested there exists a U-shaped relationship between the amount of alcohol consumption and the risk of diabetes. Chronic regular mild to moderate alcohol consumption among healthy people may be associated with increased insulin sensitivity and a reduced risk of type 2 diabetes, while excessive consumption can impair glycacmic control and increase the risk of type 2 diabetes. But until now. the mechanism of the diabetogenic action of alcohol remains unexplored. Type 2 diabetes is characterized by insulin resistance and impaired insulin secretion. Although the relative contribution of insulin resistance versus insulin deficiency remains a matter of controversy, there is considerable evidence that deficient β-cell function plays a dominating role in the establishment of overt diabetes. So, it is necessary to study the effect of alcohol on β-cell function and the mechanism involving it. Diabetes is believed to occur due to absolute or relative deficiency of insulin when β-cell function is impaired. In the past, too much researches focused on β-cells destroyed in type 1 diabetes and it is generally accepted that β-cell destruction occurs mainly by apoptosis. Recently, more and more evidence demonstrated that the failure of β-cell function is also related to apoptosis, so the role of β-cell apoptosis during the development of type 2 diabetes spurred the interest of research in this area. Another aspect of this research is whether alcohol could increase the risk of diabetes due to inducing apoptosis of pancreatic islet β-cells.In the present study, we examined the direct effect of alcohol on insulin secretion and apoptosis of pancreatic islet β-cells in vitro. At the same time, we investigated the potential mechanism involving. From this study, we hope to elucidate some understanding about the mechanism between the alcohol and diabetes and provide scientific advice for healthy drinking. The experiments were designed and the main results were summarized as follows:Part I Establishment of culturing methods of primary rat pancreaticislet and mouse insulinoma cells (NIT-1 cells)Primary culture of rat pancreatic islet and identificationObjective To establish the method of primary rat pancreatic islet in order to provide enough purified, survival and functional rat islets for next experiments in this study.Methods Islets were isolated from male Wistar rats by ductal injection of pancreas with KRBB, then collagenase digestion and a one-step single-layer Histopaque-1077 purification. Evaluate the islet quality by dithizone stain, AO/PI stain, electron microscope and determination the function of glucose stimulated insulin secretion (GSIS).Results 540 ± 84 islets and 335 ± 81 islets were obtained in 1 rat pancreas after digestion and purification individually, with purity over 90% and living islets 99%. The results of GSIS demonstrate a functional profile of glucose sensitivity. Electron microscope of the isolated islet showed a healthy, well-preserved intracellular structure containing insulin granules in the β-cell cytoplasm.Conclusion This is a simple and effective method to gain enough and functional integrity rat islets. Therefore, the situation was capable of further research. Culture of mouse insulinoma cells (NIT-1 cells) and identification of insulinsecretion functionObjective Insulin is secreted by pancreatic islet β-cells. So, we use a kind of β-cell line (NIT-1 cells, established from mouse insulinoma) for further study the influence of alcohol on β-cells function and apoptosis. The aim of this part of study is to identify the function and establish the suitable culture condition of NIT-1 cells.Methods Choose the culture medium through MTT assay. Identify the insulin producing capability of NIT-1 cells by immunocytochemical method and Identify the responsiveness to secretagogues with glucose stimulated insulin secretion method.Results NIT-1 cells grew well when the glucose concentration in the medium was between 2.8 and 11.2mmol/L. But the growth of cells was inhibited when the glucose concentration exceeded 11.2mmol/L. Using immunocytochemical method, almost all of the cells showed brown staining pattern over the cytoplasm, thus implying the good capability of insulin biosynthesis in NIT-1 cells. Insulin secretion is responsive to glucose concentration well in the medium. The stimulation was increased with increasing glucose concentration and began to dwindle when the glucose concentration was higher than 11.2mmol/LConclusion The suitable concentration of glucose in DMEM culture medium was 5.6mmol/L for NIT-1 cells. NIT-1 cells used in this study showed satisfactory function of insulin secretion and responsiveness to GSIS.Part II Effect of alcohol on insulin secretion of islet β-cells and study of related mechanismEffect of alcohol on insulin secretion of islet β-cellsObjective To examine primary cultured rat pancreatic islet (β cells) and NIT-1 cells insulin secretion in response to a glucose challenge following exposure to different concentrations of alcohol. Therefore, we can conclude how the alcohol influence the β-cells function., Methods1. To observe alcohol-induced changes in cell morphology, NIT-1 cells were examined by phase-contrast microscopy after cells were treated with alcohol atconcentrations of 0, 50, 100, 200 and 400 mmol/L for 24 h.2. Effect of alcohol on insulin secretion in NIT-1 cells(1) NIT-1 cells were treated with 100mmol/L alcohol for 24h and insulin release wasmeasured by radioimmunoassay after 2h incubation in KRBB containing 0, 1.4, 2.8, 5.6, 11.2 and 22.4mmol/L glucose.(2) NIT-1 cells were exposed to various concentrations of alcohol (0, 50, 100, 200, 400mmol/L) for 6, 12 and 24 h and insulin release was measured by radioimmunoassay after 2h incubation in KRBB containing 5.6 and 22.4mmol/L glucose.3. Effect of alcohol on insulin secretion in primary cultured rat pancreatic islet (β-cells):(1) Islets (β-cells) were exposed to various concentrations of alcohol (0, 10, 50, 100, 200mmol/L) for 12 h and insulin release was measured by radioimmunoassay after 2h incubation in KRBB containing 5.6 and 22.4mmol/L glucose.(2) Islets (β-cells) were exposed to various concentrations of alcohol (0,100 mmol/L) for 6, 12 and 24h and insulin release was measured by radioimmunoassay after 2h incubation in KRBB containing 5.6 and 22.4mmol/L glucose.Results1. Morphological changes induced by low concentrations of alcohol were little. But NIT-1 cells of high concentrations (especially 400mmol/L) of alcohol treated groups presented with cell shrinkage, irregularity in shape and cellular detachment.2. Effect of alcohol on insulin secretion in NIT-1 cells(1) The tendency of GSIS in both untreated and treated group was similar and the insulin secretion was correlated with the concentration of glucose. With increasing glucose concentration, the insulin release was increased first and reached the peak at 5.6 mmol/L glucose, then began to dwindle. But at the same concentrations of glucose, alcohol inhibited insulin secretion compared to the untreated control group. During incubation with 2.8, 5.6 and 22.4mmol/L individually, the inhibitory effects were significant. So, in the next GSIS experiments, the concentrations of glucose were 5.6 and 22.4 mmol/L.(2) At 6h, alcohol increased the glucose-stimulated insulin secretion. At 12h and 24h, alcohol decreased the insulin secretion. When alcohol concentration and exposure time were same, insulin release response to 22.4 mmol/L glucose was less than those to 5.6mmol/L glucose. The insulin secretion was correlated with the alcohol concentration. The insulin secretion was decreased after alcohol exposure time prolonged even if the dosage of alcohol was identical.3. Effect of alcohol on insulin secretion in primary cultured rat pancreatic islet (β-cells)(1) There was a reverse U-shaped association between GSIS (stimulated with both 5.6 and 22.4 mmol/L glucose) and alcohol dosage. Compared to untreated group, insulin release was increased significantly after exposed to low dosage of alcohol, but decreased markedly after exposed to high dosage of alcohol.(2) The effect of alcohol on insulin secretion (incubated with 5.6 and 22.4 mmol/L glucose) in islet (β-cells) was time dependency. At 6h, alcohol increased the GSIS, but alcohol decreased the GSIS at 24h. The effect of alcohol on GSIS at 22.4 mmol/L glucose was more significant than those at 5.6 mmol/L glucose.Conclusion: Depending on the timing and concentrations, alcohol can exert both stimulatory and inhibitory effects on insulin secretion.Effect of alcohol on INS and Cytochrome oxidase II mRNA expression in NIT-1 cells and INS mRNA expression in primary cultured rat pancreatic islets (β-cells)Objective To investigated the mechanism of the effect of alcohol on β-cell function, INS mRNA expression and Cytochrome oxidase II mRNA expression were observed because they are the key points related to the procedure of insulin secretion.Methods1. After NIT-1 cells were exposed to various concentrations of alcohol (0, 50, 100, 200, 400mmol/L) for 6, 12 and 24h, the levels of insulin and COXII mRNA expression were analyzed by RT-PCR method. 2. After primary cultured rat pancreatic islets were treated with various concentrations of alcohol (0, 50, 100, 200 mmol/L) for 12h, the levels of insulin mRNA expression were analyzed by RT-PCR method. After islets were treated with alcohol (0, 100 mmol/L) for 6, 12 and 24h, the levels of insulin mRNA expression were analyzed by RT-PCR method.Results1. The effect of alcohol on INS and COX II mRNA expression in NIT-1 cells were depended on the time and concentration. INS and COX II mRNA expression were upgraded at 6h and began to decrease at 12h. At 24 h, high alcohol group decreased COX II mRNA expression significantly.2. The effect of alcohol on INS mRNA expression in islets (β-cells) were depended on the time and concentrations. INS mRNA expression was upgraded at low concentration (50mmol/L), but began to decrease with the concentrations increasing. INS mRNA expression was upgraded (P<0.05) at 6h and began to decrease at 12h. At 24 h, the expression decreased significantly compared to untreated group (p<0.05).Conclusion: Influence of alcohol on INS mRNA, COX II mRNA expression in β-cells may be involved in the effect of alcohol on insulin secretion.Part III Influence of alcohol on apoptosis in NIT-1 cellsEffect of alcohol on apoptosis in NIT-1 cellsObjective To investigate whether alcohol induces apoptosis in NIT-1 cells.Methods After NIT-1 cells were treated with alcohol at concentrations of 0, 50, 100, 200 and 400 mmol/L for 6h, 12h and 24h, the LDH activity released in the media was measured in order to determine the cytotoxicity of alcohol. After NIT-1 cells were exposed to various concentrations of alcohol (same as above), single cell gel electrophoresis (SCGE), agarose gel electrophoresis and AnnexinV/PI methods were performed to detect the apoptosis induced by alcohol.Results1. At 6h, alcohol induced no significant cytotoxic effect in NIT-1 cells. At 12h, 24h, high concentration alcohol damaged the plasma membrane and the LDH activity increased. At 6h, 12h and 24h, the activity of LDH was positive correlation to the dosage of alcohol. At the same dosage of alcohol, the LDH activity was increased after the exposure time prolonged.2. SCGE results showed that low concentration alcohol induced no obviously DNA damage in NIT-1 cells, but the rate of DNA migration, degree of DNA damage and average length of DNA migration of 200 and 400 mmol/L alcohol-treated groups were significantly changed compared to those of untreated group .3. The results of DNA fragmentation assay showed that 400 mmol/L alcohol treatment induced a classic "DNA ladder" pattern. 200 mmol/L alcohol-treated group had a few "DNA ladder" pattern and the other groups appeared no "DNA ladder" pattern.4. The results of Annexin V/PI assay indicated that the apoptosis rates of NIT-1 cellsin 200 and 400mmol/L alcohol-treated groups were higher than that of untreated group. Conclusion Alcohol induced apoptosis in NIT-1 cellsMechanism of the effect of alcohol on apoptosis in NIT-1 cells Objective To study the mechanism of alcohol induced apoptosis via oxidative stress Methods After NIT-1 cells were exposed to various concentrations of alcohol (0,50, 100, 200,400mmol/L) for 6, 12 and 24h, the experiments were performed as follows: 1 .Malondialdehyde (MDA) content, Glutathione (GSH) level, Superoxide dismutase(SOD) and Glutathione peroxidase (GSH-Px) were measured in NIT-1 cells to evaluatethe oxidative damage degree.2. The levels of Bcl-2, Bax mRNA expression were determined by RT-PCR method.3. The level of Caspase-3 mRNA expression was measured by RT-PCR.4. Caspase-3 enzyme activity was measured using Ac-DEVD-pNa which is the substrate of Caspase-3 enzyme.Results1. The MDA content was higher, the SOD activity, GSH-Px activity and GSH content were lower in alcohol group than those of control group. The MDA content, SOD activity, GSH-Px activity and GSH content were correlated with the concentrations of alcohol and the correlation coefficients were 0.9034, -0.7258, -0.6201, -0.6222 (p<0.01) respectively.2. At 6h, the ration of Bcl-2/Bax expression was increased first, then decreased with the increasing of alcohol dosage. At 12h and 24h, the ration was decreased. By treatment at 400mmol/L, the decrease was dramatically compared to control group.3. Alcohol increased the caspase-3 mRNA expression at high concentrations of alcohol and long exposure time. At 24h, the caspase-3 mRNA expression was increased significantly by treatment at 400mmol/L.4. Alcohol increased the caspase-3 enzyme activity by a dose and time dependent manner. After 6h exposure to alcohol, the enzyme activity changed little. After 12h, the enzyme activity was seen to be increased dramatically by treatment with alcohol at 200 and 400 mmol/L respectively. After 24h, the enzyme activity was increased markedly by treatment with alcohol at 100, 200 and 400 mmol/L respectively.Conclusion Higher concentrations of alcohol induced imbalance of oxidative and antioxidative ability in NIT-1 cells, then oxidative stress further leads to apoptosis. Based on the results, alcohol appears to activate specific intracellular death-related pathways leading to bax-dependant caspase-3 activation and the induction of apoptosis in NIT-1 cells. From the results of the present study, it could be suggested that alcohol-induced apoptosis in NIT-1 cells is a possible pathologic mechanism of alcohol-related diabetes.
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