High Glucose Induces Cellular Memory Mediated By ROS In INS-1 Cells During Subsequent Normoglycemia

[Background]The"metabolic memory", the idea that diabetic complication caused by high glucose persist even after glucose normalization, has been supported both in the laboratory and in the clinical trials. This concept emerged, clinically,when the results of a large type 1 diabetes clinical trial, the Diabetes Complications and Control Trial (DCCT) follow-up Epidemiology of Diabetes Interventions and Complications (EDIC) trial, came to light. In the EDIC trial, it was found that patients on the standard treatment regimen during the DCCT still had a higher incidence of diabetic complications such as retinopathy, nephropathy, hypertension, and neuropathy compared with their counter parts receiving intensive therapy throughout the trial several years after switching to intensive therapy, despite the fact that the mean glycated hemoglobin Alc levels of the groups were nearly equivalent. The metabolic memory has also been shown to be present in type 2 diabetes when the results from the UKPDS follow-up trial were published.These suggest that metabolic memory phenomenon exist in diabetic patients.The first investigation of such a memory was reported by the laboratory of Kern in the retina of diabetic dogs who were switched to good glucose control after either 2 months or 2.5 years of poor control and then analyzed at 5 years after beginning the study. The animals switched to good glucose control at 2 months had little evidence of retinopathy, as did their control counterparts receiving good glucose control throughout the study. In contrast, the animals switched to good control at 2.5 years had a similar incidence of retinopathy as their control counterparts who received poor glucose control throughout the 5-years study. Just after this first study was published, the laboratory of Lorenzi showed that there was a memory of basement membrane (collagenⅣ, fibronectin) mRNA induction in isolated endothelial cells and in the kidneys of streptozotocin (STZ)-induced diabetic rats several weeks after glucose normalization after several weeks of high glucose.In 2007, the metabolic memory phenomenon in endothelial and retinal cells was confirmed by laboratory of Ihnat. Using the similar design of the Lorenzi et al, namely 14 d of high glucose followed by 7 d of normal glucose, data in endothelial and retinal cells show that an overproduction of oxidative stress persists after the normalization of glucose levels. Furthermore, excess ROS is accompanied by accumulation of the oxidative stress markers, such as protein kinase C, Bax,3-NT and so on. For the first time, it was definitively shown in endothelial cells that reducing intracellular production of free radicals was capable of switching off the metabolic memory. A recent paper which was published in The Journal of Experimental Medicinehas in 2008, described how transient hyperglycemia (16 h) induces long-lasting activation of epigenetic changes in the promoter of the NF-κB subunit p65 in aortic endothelial cells,leading to increased p65 gene expression. Both the epigenetic changes and the gene expression changes persist for at least 6d of subsequent normal glycemia. Furthermore,hyperglycemia induced epigenetic changes and increased p65 expression were prevented by reducing mitochondrial superoxide production. That is mean, memory phenomenon could exist in cells and oxidative stress may be the important mechanism for its formation.Pancreaticβ-cell dysfuction and apoptosis are the keys of development of diabetes. So it is meaningful to do in-depth studies into the pathogeneses and mechanisms of pancreaticβ-cell dysfunction. Some past studies have indicated that long-term hyperglycemia led to pancreatic P-cell dysfunction and apoptosis, which was termed "glucose toxicity". Further study suggested that oxidative stress was an important mechanism underlying pancreaticβ-cell "glucose toxicity".Oxidative stress is the disorder between ROS generation and anti-oxidant systems,and the disorder which cause damage to cells.Levels of Gene expression of the antioxidant enzymes(SOD,Catalase,Glutathione peroxidase) in pancreaticβ-cell are lower compared with other tissue cells. So, pancreaticβ-cell are very sensitive to oxidative stress.As mentioned before,long-term hyperglycemia could lead to formation of multiple memory effects included persistent changes of inflammatory genes, continuous damages of mtDNA and so on in endothelial cells.Increasement of ROS generation may be the key of memory effect. The intrinsic antioxidant enzyme expression is low in the islet. Therefore, the islet cells are more sensitive to the damage of ROS. So it is interesting to see whether the oxidized stress damages caused by hyperglycemia could also induce the islet cell to have a long-lasting deleterious effect. Islet cell destruction is an important etiological factor in the development and progression of diabetes. Apoptosis induced by high glucose is important reason for function failure in islet cells and apoptosis in islet cells is mediated by apoptosis-related genes(bax,caspase-3, bcl-2).So, In this study, the rat insulinoma-derived cell line INS-1 is used as theβ-cell model, apoptosis-related genes(bax,caspase-3, bcl-2) and cell viability were used as detection factors, to investigate whether there were cellular memory in INS-1 (rat islet cell line) cells and related mechanisms.The research content includes two aspects:Chapter 1 Phenomenon of cellular memory in INS-1 cells[Objectives]To observe whether there were persistent injury of INS-1 cells during subsequent normoglycemia.[Methods]1. Cell culture:The rat insulinoma-derived INS-1 cells, a widely usedβ-cell surrogate, were cultured in 5% CO2 at 37℃in RPMI-1640 complete medium containing 10% fetal bovine serum(FBS),11.1 mmol/L D-glucose,1.0 mmol/L sodium pyruvate,50μmol/L 2-mercaptoethanol,100 U penicillin/mL and 100μg streptomycin/mL.2. There were four groups according to different concentrations of glucose and intervention ways:(1)The normal control group (Ctrl group, cells were cultured in RPMI-1640 complete medium containing 11.1 mmol/L glucose for 7 days);(2) The high glucose group (HG group, cells were cultured in RPMI-1640 complete medium containing 33.3 mmol/L glucose for 2 days);(3) 3 days memory group (3d memory group, cells were cultured in RPMI-1640 complete medium containing 33.3 mmol/L glucose for 2 days and then in RPMI-1640 complete medium containing 11.1 mmol/L glucose for 3 days);(4) 5 days memory group (5 d memory group, cells were cultured in RPMI-1640 complete medium containing 33.3 mmol/L glucose for 2 days and then in RPMI-1640 complete medium containing 11.1 mmol/L glucose for 5 days). The INS-1 cells of these four groups were cultured in basal medium for 24 hours before intervention to keep synchronized growth of cells.3,Cell viabilities were evaluated by MTT assay:The optical density (OD) value was read at 490nm in a plate reader. The reduction in optical density was used as a measurement of cell viability, normalized to cells incubated in the normal control group, which were considered 100% viable.4,The levels mRNA were measured by real-time PCR:Extract total cellular mRNA by Trizol from each group; determine its concentration and purity(purity must match condition:2>A260/280>1.8);synthetize cDNA by means of reverse transcription reaction; Synthetize target genes through PCR reaction; analyse solubility curve,amplification curve,CT valuses of target genes; calculate relative quantitative genes expression (RQ value). The value reflect level of celular mRNA expression.5. Statistical analysis:Statistical analyses were performed using the SPSS 13.0 software package. The data was expressed as the mean±S.D. If equal variances were achieved, one-way ANOVA was performed to evaluate the statistical significance of differences among groups and multiple comparisons were carried out using LSD method; If not, Welch approximate analysis of variance was performed to analyze the statistical significance of differences among groups and multiple comparisons were carried out using Dunnett's T3 method. P<0.05 was considered as significant.[Results]1. Cell viabilities:The OD values of the Ctrl group, HG group,3 d memory group,5 d memory group were 1.132±0.161,0.578±0.094,0.656±0.077,0.632±0.067 respectively. In contrast with the Ctrl group, the cell viabilities of the HG group and 3,5 days memory group were lower than Ctrl group (P=0.016, P=0.002, P=0.006).2. Levels of apoptosis-related genes mRNA:(1) Level of bax mRNA,:The RQ values of the Ctrl group, HG group,3 d memory group,5 d memory group were 1.000±0.000,1.886±0.100, 2.968±0.198,3.173±0.221, respectively. In contrast with the Ctrl group, the bax mRNA level of the HG group and 3,5 days memory group were higher than Ctrl group (P=0.002, P=0.001, P=0.001).(2) Level of caspase-3 mRNA:The RQ values of the Ctrl group, HG group,3 d memory group,5 d memory group were 1.000±0.000,1.692±0.212, 2.468±0.499,2.253±0.563 respectively. In contrast with the Ctrl group, the caspase-3 mRNA level of the HG group and 3,5 days memory group were were significantly increased.(P=0.002, P=0.004, P=0.013).(3) Level of bcl-2 mRNA:The RQ values of the Ctrl group, HG group,3 d memory group,5 d memory group were 1.000±0.000,0.474±0.131, 0.546±0.155,0.562±0.086. In comparison with the Ctrl group,the levels of bcl-2 mRNA in the HG group and 3,5 days memory group were significantly decreased (P=0.015, P=0.036, P=0.008).[Conclusions]1. High glucose could induce apoptosis of INS-1 cells,through changing expression of apoptosis gene.2. High glucose could induce INS-1 cells to get cellular memory which causes persistent changes of cell viability and altered expression about apoptosis gene during subsequent normoglycemia. Chapter 2 The preliminary mechanism of cellular memory induced by High glucose in INS-1 cells[Objective]To investigate related mechanisms of cellular memory induced by high glucose in INS-1 cells.[Methods]1. Cell culture were just the same as those of the chapter 1.2. There were four groups according to different concentrations of glucose and intervention ways:(1) The normal control group (Ctrl group, cells were cultured in RPMI-1640 complete medium containing 11.1 mmol/L glucose for 7 days);(2) The high glucose group (HG group, cells were cultured in RPMI-1640 complete medium containing 33.3 mmol/L glucose for 2 days);(3) Memory group (cells were cultured in RPMI-1640 complete medium containing 33.3 mmol/L glucose for 2 days and then in RPMI-1640 complete medium containing 11.1 mmol/L glucose for 5 days);(4) Intervention group (cells were cultured in RPMI-1640 complete medium containing 33.3 mmol/L glucose for 2 days and then in RPMI-1640 complete medium containing 11.1 mmol/L glucose plusa-lipoic acid 62.5umol/L for 5 days).3. Oxidative stress intervation:useα-Lipoic acid as antioxidant(yellow powder,molecular weight is 206.33).Prepare liquor ofα-Lipoic acid(final concentration is 62.5umol/L),add to medium of intervation group.4. Determination of ROS levels:Cells were resuspended in medium containing 10μmol/L DHE (molecular probes) and then incubated at 37℃for 40min. The mean fluorescence intensity (MFI), which was regarded as the ROS level, was detected by ELISA Reader at the excitation wavelength of 300nm and the emission wavelength of 610nm.5. Statistical analysis:Statistical analyses were performed using the SPSS 13.0 software package. The data was expressed as the mean±S.D. If equal variances were achieved, one-way ANOVA was performed to evaluate the statistical significance of differences among groups and multiple comparisons were carried out using LSD method; If not, Welch approximate analysis of variance was performed to analyze the statistical significance of differences among groups and multiple comparisons were carried out using Dunnett's T3 method. P<0.05 was considered as significant.[Results]1. Levels of apoptosis-related genes mRNA:(1) Level of bax mRNA:The RQ values of the Ctrl group, HG group, Memory group, Intervention group were 1.000±0.000,2.241±0.310,3.016±0.240, 1.427±0.189 respectively. In contrast with the Ctrl group, the bax mRNA level of the HG group and Memory group were higher than Ctrl group (P<0.001, P<0.001). The bax mRNA level of the Intervention group were markedly decreased, compared with Memory group(P<0.001).(2) Level of caspase-3 mRNA:The RQ values of the The RQ values of the Ctrl group, HG group, Memory group, Intervention group were 1.000±0.000, 1.774±0.119,2.403±0.427,1.218±0.177 respectively. In contrast with the Ctrl group, the caspase-3 mRNA level of the HG group and Memory group were were significantly increased(P=0.004,P=0.027). The caspase-3 mRNA level of the Intervention group were markedly decreased,compared with Memory group(P=0.029).(3) Level of bcl-2 mRNA:The RQ values of the Ctrl group, HG group, Memory group, Intervention group were 1.000±0.000,0.496±0.094,0.559±0.130, 0.903±0.093. In comparison with the Ctrl group,the levels of bcl-2 mRNA in the HG group and Memory group were significantly decreased (P＜0.001, P=0.002). The bcl-2 mRNA level of the Intervention group were markedly increased,compared with Memory group(P=0.003).2. ROS levels:The ROS levels of the Ctrl group, HG group, Memory group, Intervation group were 0.549±0.170,1.086±0.120,0.992±0.113,0.699±0.114 respectively. In comparison with the Ctrl group, the ROS levels in HG group and Memory group were increased significantly (P<0.001). Comparing to the Memory group, the ROS levels of Intervation group were decreased significantly (P=0.001).3. Cell viabilities:The OD values of the Ctrl group, HG group, Memory group, Intervation group were 1.126±0.166,0.554±0.088,0.592±0.084,1.083±0.181 respectively. In contrast with the Ctrl group, the cell viabilities of the HG group and Memory group were lower than Ctrl group (P<0.001). In contrast with the Memory group, the cell viabilities of the intervation group were increased significantly (P=0.014).[Conclusions]1. ROS may be the important mechanism to the formation of cellular memory in INS-1 cells.2. Reducing intracellular production of ROS may be capable of switching off the cellular memory partially.