| Epidemiological survey showed there existed a U-typed relationship between alcohol and type 2 diabetes whereby moderate alcohol intake counteract the genesis and development of type 2 diabetes while long-term excessive alcohol intake increased the risk of type 2 diabetes. High-fat diet can increase serum free fatty acids, decrease glucose oxidation and uptake, lead to insulin resistance, reduce insulin secretion, thereby evoke the "lipotoxicity" of diabetes. So, long-term heavy drinking and high fat diet both are two common independent risk factors of type 2 diabetes. Home and abroad go into paying close attention to the impact of drinking or high grease alone on diabetes but seldom put them together in comparison. However, in our daily life, people also ingest many fats, while drink. So, while these two factors exist together what impact will emerge?AMP-activated protein kinase (AMPK)—the body's energy monitor, whose activity is regulated by the AMP/ATP ratio, at the same time it is believed to be the important substrate of controlling insulin release and insulin content.Pancreatic duodenal homeobox factor-1 (PDX-1) is the important transcription factor of insulin promoter and pancreas'growth, which can enter the cell nucleus and tie with insulin gene to urge insulin mRNA's transcription. It also can normally activate the expression of insulin and GLUT2, which plays an important regulatory role on endocrine function of pancreatic beta cells. Glucose transporter protein 2 (GLUT2) is belong to glucose transporter protein family (GLUTs), which can catalyze plasma glucose transport, promote glucose into pancreatic beta cells and form glucoreceptors with glucokinase (GCK), regulating pancreatic beta cell insulin secretion. It is mainly controlled by PDX-1 transcriptional regulation.This experiment aims to observe the influence of alcohol, saturated free fatty acids (palmitic acid) and their combination (Wine plus fat) on INS-1 cells secretory function, and to explore the mechanism by detecting the level of its function Indicators:pancreatic duodenal homeobox factor -1 (PDX-1), the downstream regulatory factor—Glucose transporter 2 (Glut2) and "cellular energy detector"—AMPK.OBJECTIVES:1. To observe the effect of alcohol, palmitic acid and their combination (alcohol plus fat) on the isletβcells (INS-1 cells) insulin release function.2. To observe the effect of alcohol, palmitic acid and their combination (alcohol added fat) on the isletβcells (INS-1 cells) in the key molecule in regulation of insulin gene PDX-1, its downstream molecules of glucose transporter GLUT2 and the energy sensor AMPK protein levels.Methods:1. INS-1 cells were grown in RPMI1640 medium containing 10%(v/v) fetal bovine serum,100 U/ml penicillin,0.1 mg/mL streptomycin,50μM (3-mercaptoethanol,1 mmol/1 sodium pyruvate. Cell were cultured in dishes and maintained at 37℃in a humidified atmosphere of 5% CO2 and passaged every 7-10 days by mild trypsinization after 80%-90% confluence. Cells were seeded in dishes (106 cells per dish) or 24-well plates (2×105 cells per well) and left to attach for 48 h before treatment. After that, cells were incubated at 37℃for 48 h in the absence or presence of 20 mM ethanol,100 mM ethanol,0.2 mM palmatic acid,20 mM ethanol plus 0.2 mM palmatic acid or 100mM ethanol plus 0.2 mM palmatic acid.2. Insulin secretion assay: After incubation and treatment in 24-well plates, cells (two wells each group) were pre-cultured for 30 min with KRB buffer (500 ul/well) containing 0.2% BSA and 3.3 mmol/L glucose, and then respectively swapped with KRB buffer (500 ul/well). For each group, one contained 3.3 mmol/L glucose (basic glucose stimulation, BIS), another contained 27.8 mmol/L glucose (glucose-stimulated insulin secretion, GSIS). The supernatant was collected, Insulin secretion levels were determined by radioimmunoassay(RIA).3. Detection of expression of PDX-1, GLUT2 and AMPK:the protein expression of PDX-1, GLUT2 and AMPK were detected by Westernblotting.Results:1.The effect of different treatment factors on INS-1 cells insulin releaseWe first observed the effect of ethanol or/and palmatate on insulin secretion function of INS-1 cells. As shown in Figure 1 A, compared to control group (BIS,0.9±0.094μIU/μg; GSIS,1.975±0.254μIU/μg), there was no obviously differences in 20 mM EtOH group and 0.2 mM PA+20 mM EtOH group; 0.2 mM PA significantly increased BIS to 1.332±0.29μIU/μg (p<0.05) and decreased GSIS to 1.156±0.13μIU/μg (p<0.05) in INS-1 cells. However,100 mM EtOH group evidently reduced GSIS to 1.126±0.138μIU/μg (p< 0.05) without BIS significantly alter. Meanwhile, the GSIS of 0.2 mM PA+100 mM EtOH group (BIS,0.91±0.282μIU/μg,*p>0.05; GSIS,1.028±0.338μIU/μg,*p< 0.05) was much more lowered than control and slightly lowered than 0.2 mM PA group. Relative to 0.2 mM PA group,0.2 mM PA+ 20 mM EtOH group markedly rosed the GSIS (1.944±0.68μIU/μg,#p<0.05) and reduced the BIS to 1.135±0.237μIU/μg (but there is no statistical significance, p>0.05)Accordingly, The ISI value of 100 mM EtOH reduced by 36.3%(*p<0.05) while,0.2 mM PA group was depressed to 53% compared with control (*p<0.05), simultaneously, the ISI value was restored to normal in 0.2 mM PA+20 mM EtOH cultured cells (Fig. 1B). We also can find that The ISI value of 100 mM EtOH group, 0.2 mM PA group and 0.2 mM PA plus 100 mM EtOH co-treated group were all much more lower than 20 mM EtOH group (&p<0.05). The results suggest that single heavy ethanol or palmitate alone can impair insuin secretion, while moderate ethanol mainly increase the GSIS (and also slightly decrease BIS) to ameliorate insulin secretion function impaired by palmatate in INS-1 cells.2. Effects of different treatment factors on PDX-1 expression in INS-1 cells. To gain insight into the functional changes in INS-1 cells treated in different groups, we performed Western blotting analyses for PDX-1 expression. Compared with the control group:①consistent with insullin secretion, PDX-1 expression level was no significant bias in 20 mM ETOH treatment group and 0.2 mM PA+20 mM EtOH treatment group. (Fig.2) PDX-1 protein levels in 100 mM EtOH group,0.2 mM PA treatment group and 0.2 mM PA+100 mM EtOH co-treatment group were obviously reduced (78.5% of control,*p<0.05; 70% of control,*p<0.05; 77% of control,*p<0.05). And, in contrast to the two ethanol treatment groups (20 mM EtOH group and 100 mM EtOH group),0.2mM PA treatment group displayed a greater impact (#p<0.05) in INS-1 cells.And 20 mM EtOH blunted the suppressant effects of palmitate on GSIS was also associated with the increase of expression of PDX-1 protein (#p<0.05) in 0.2 mM PA plus 20 mM EtOH co-treated INS-1 cells.3. Effects of different treatment factors on GLUT2 expression in INS-1 cells.To further investigate the mechanism, we also monitored changes of GLUT2 protein expression. As the downstream regulatory factor of PDX-1, GLUT2 showed similar trends:Compared to control, Glut2 expression levels of 100 mM EtOH group,0.2 mM PA treatment group and 0.2 mM PA+20 mM EtOH group were significantly depressed to 82.4%(*p< 0.05),62.5%(*p< 0.05) and 69.1%(*p< 0.05), respectively, and, there was no discrepancy between them. Meanwhile, in Figure 3, we can find that in contrast to ethanol treatment groups, palmitate treatment group exhibited a greater influence in INS-1 cells, too. Compared with 0.2 mM PA treatment group, Glut2 protein levels of 0.2 mM PA+20 mM EtOH was increased by 35.4%(#p<0.05), These results suggest that moderate ethanol can up-regulate lower Glut2 protein expression inducedby palmitate in INS-1 cells.4. Effects of different treatment factors on AMPK protein expression in INS-1 cellsAMP-activated protein kinase (AMPK) is an cellular energy detector that controls systemic glucose homeostasis and is closely associated with insulin secretion of (3-cells. So, we determine the changes of AMPK (T-AMPK and phospho-AMPK) expression in INS-1 cells treated with ethanol, palmitate and ethanol plus palmitate to further observe the functional diversity.As shown in Fig.4, except the P-AMPK/T-AMPK ratio of 0.2 mM PA group was lowered by 18%(*p< 0.05), there was no outstandingly change in cells with different treatment in contrast to control. At the same time, we can find that cells treated with 0.2 mM PA and 20 mM EtOH together for 48 hours enhanced the decrease of P-AMPK/T-AMPK ratio caused by 0.2 mM PA (#p< 0.05).20 mM ETOH treatment and 0.2 mM PA plus 20 mM EtOH treatment both did not remarkably affect T-AMPK protein expression, while 100 mM EtOH treatment,0.2 mM PA treatment and 0.2 mM PA plus 100 mM EtOH treatment obviously decreased T-AMPK protein level by 17.5%, (*p< 0.05),26.7%(*p<0.05) and 20.7% (*p<0.05), respectively. Furthermore, theT-AMPK protein level of 0.2 mM PA plus 20 mM EtOH group was 1.27 times of 0.2 mM PA treatment group (#p< 0.05).CONCLUSIONS:1. Both high alcohol and palmitate(PA) inhibited highglucose-stimulated insulin release and reduced PDX-1 and GLUT2 protein levels; high alcohol decreased the expression of T-AMPK but has no effect on AMPK activation (P-AMPK/T-AMPK), while PA has not only reduced T-AMPK level but also damaged AMPK activity.when ethanol and palmatic acid were applicated together,moderate alcohol (20 mmol/L ethanol) can obviously improve the pancreaticβcell damage caused by palmitic acid, evoking notable recovery ofβ-cell function. But, high alcohol did not have the effect... |
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