| Nonalcoholic fatty liver disease (NAFLD) consists of ectopic fat accumulation in the liver related not to alcohol consumption but to metabolic factors. Studies have shown that tye2diabetes (T2DM) is an independent risk factor leading the progress of Nonalcoholic fatty liver (NAFLD) to Nonalcoholic steatohepatitis (NASH) or liver fibrosis, but the mechanism is unclear. It has been recognized that oxidative stress and liver cell apoptosis are important events in the progress of NAFLD. Oxidative stress may induce synthesis of pro-inflammatory cytokines and death receptor expression, increase rates of hepatocyte apoptosis and mediate the progression to NASH and liver fibrosis. Mitochondrial permeability transition (MPT) is the swich of cell apoptosis.Glucose variability is one of important components of the dysglycemia. Clinical studies have shown that various markers of oxidative damage are significantly related to the mean amplitude of glycemic excursions. Improvement of glycemic control and inhibition of fluctuation in glucose concentrations may contribute to decrease oxidative stress reaction. Thus in this study, we suggest that glucose fluctuation-induced overproduction of superoxide may probably start the mitochondrial apoptosis pathway by inducing the occurance of MPT and lead the progress of NAFLD to NASH or liver fibrosis in T2DM.We establish liver cell models of steatosis and diabetic animal models with NAFLD (imitating glucose fluctuation in vitro and vivo by alternating glucose concentrations) and use technologies of transmission electron microscopy, flow cytometry, TUNEL, pathological staining, fluorescent probe and western blotting to investigate the effect of glucose fluctuation on liver cell apoptosis, necrosis and fibrosis of liver tissues, as well as the role of glucose fluctuation-induced superoxide in damaging the mitochondrial integrity and mitochondrial respiratory chain function and starting the mitochondrial apoptosis pathway of liver cells by inducing the occurance of MPT, and thus ultimately leading the progress of NAFLD to NASH or liver fibrosis in diabetes. Part One:The Effect of Intermittent High Glucose on Hepatocytes apoptosis in Diabetic Animal Models with NAFLDObjective:The aim was to establish diabetic animal models with NAFLD, and imitate glucose fluctuation in vivo. The research included the effect of glucose fluctuation on liver cell apoptosis, necrosis and fibrosis of liver tissues, and the role of glucose fluctuation-induced superoxide in damaging the mitochondrial function and starting the mitochondrial apoptosis pathway of liver cells.Methods:C57BL/6J mice maintained on high fat diet (45%fat, HFD) for2months, and then were injected with glucose (3.0g/kg) or saline twice a day by intraperitoneal injection to induce intermittent high glucose for3months continuously maintained on HFD. Firstly, observe the weight, glucose tolerance, insulin sensitivity, free fatty acid levels, hepatic enzyme levels in control group (mice treated with STD and NS), HFD+NS and HFD+F group. Secondly, liver lipid deposition, fibrosis and lipid peroxidation were determined by pathological staining. Thirdly, TUNEL and Western blotting were used to evaluated the influence of glucose fluctuation on hepatocyte apoptosis. Fourthly, the release of cytochrome c and production of ATP were evaluated by Western blotting and biochemical techniques.Results:1. Mice fed a HFD had higher body weight and more severe impaired glucose tolerance, insulin resistance, liver lipid deposition and fibrosis than those fed STD (P<0.01). However, the above parameters were comparable between HFD mice received glucose and NS injection (P>0.05).2. The percentage of TUNEL-positive stained cells and apoptosis related proteins, such as cleaved caspase-3, cleaved caspase-9and Bax were significantly increased in the liver of mice fed a HFD (P<0.01), which was further exacerbated by glucose injection (P<0.01vs.HFD+NS group).3. After20weeks of consuming a HFD,4-hydroxy-2-nonenal (HNE) and malondialdehyde (MDA) levels were significantly increased in the liver (P<0.01) and that these levels were further increased in mice exposed to IHG (P<0.05vs. HFD+NS group).4. Mice fed a HFD increased cytochrome c release from mitochondria, and ATP production compared with mice fed with STD (P<0.01). Furthermore, mice fed a HFD and injected with glucose had a higher release of cytochrome c (P<0.01), but an decreased ATP production (P<0.01vs. HFD+NS).Conclusion:1. Glucose tolerance, insulin sensitivity, hepatic enzyme levels, liver lipid deposition, fibrosis, hepatocyte apoptosis, lipid perpxidation (such as HNE and MDA), release of Cyt c and ATP production are significantly higher in mice fed a HFD compared to mice fed a STD mice.2. Glucose tolerance, insulin sensitivity, hepatic enzyme levels, liver lipid deposition and fibrosis are comparable between HFD mice injected with glucose or saline; however, glucose injected mice display marked hepatocyte apoptosis, lipid peroxidation (such as HNE and MDA), increase cytochrome c release and decrease ATP production. Our data suggests that intermittent high glucose induces mitochondrial oxidative stress and hepatocytes apoptosis. Part two:The Effect of Intermittent High Glucose on hepatocytes apoptosis in liver cell models of steatosisObjective:The aim was to establish liver cell models of steatosis, and imitate glucose fluctuation in vitro. And investigate the effect of glucose fluctuation on liver cell apoptosis, as well as the role of glucose fluctuation-induced superoxide in damaging the mitochondrial integrity and mitochondrial respiratory chain function and starting the mitochondrial apoptosis pathway of liver cells by inducing the occurance of mitochondrial permeability transition (MPT).Methods:Hepatocytes L02were incubated for72h in the medium containing different glucose concentrations with or without palmitic acid (PA,0.125mmmol/L):control (NG,5.5mmol/L), sustained high glucose (SHG,33.3mmol/L) and intermittent high glucose (IHG,5.5mmol/L and33.3mmol/L glucose alternating every12h). L02intracellular lipid droplets were observed with Oil Red O and Bodipy493/503staining, cell apoptosis, reactive oxygen species (ROS) production, mitochondrial superoxide and mitochondrial membrane potential (ΔΨm) were analyzed by flow cytometry. Apoptosis-related proteins Capase-3,9and PARP, and the release of cytochrome c were determined by Western blotting and the cell ultrastructure was monitored by electron microscopy. Cyclosporin A (CsA,1.5μmol/L) was adopted for MPT inhibition.Results:1. After72h experimentation, lipid accumulation was evident in cells exposed to PA. However, among the different glucose treatments, the lipid accumulation was similar.2. In the presence of PA, treatment with SHG induced higher apoptosis rate, with concomitant increases in the apoptosis related protein such as cleaved caspase-3,9and PARP compared with cells in NG (P<0.05); IHG further augmented the apoptosis (P<0.05vs SHG+PA). However, in the absence of, there was no significant effect of SHG or IHG on heptocyte apoptosis (P>0.05).3. L02cells exposed to PA and SHG produced increased levels of ROS and mitochondrial superoxide relative to control cells (P<0.05), with even higher ROS and mitochondrial superoxide levels detected in cells treated with IHG with PA (P<0.05vs. SHG+P group). However, in the absence of PA, SHG or IHG had no effect on oxidative stress in L02cells (P>0.05).4. In the L02cells with PA, compared with NG, SHG showed an increase in mitochondrial ultrastructural abnormalities (such as swollen mitochondrial and disappeared cristae) suggesting the opening of MPT pore, and induced more mitochondrial dysfunction presented as a decrease in ΔΨm and increase in cytochrome c release from mitochondria, which were exacerbated by exposure to PA and IHG. However, in the absence of PA, the different glucose treatments had no effect on mitochondrial morphology and function.5. Co-treatment with the MPT inhibitor CsA prevented both IHG and SHG with PA induced mitochondrial ultrastructural abnormalities, decreased cytochrome c release, ROS production and mitochondrial superoxide, as well as hepatocytes apoptosis.Conclusion:Glucose fluctuation together with lipotoxity might contribute to the progression of NAFLD by increasing ROS production, mitochondrial dysfunction and hepatocytes apoptosis, and MPT may act as a "central executioner". Treatment with the mitochondrial pore transition (MPT) inhibitor, cyclosporin A (1.5μmol/L), prevents mitochondrial dysfunction, oxidative stress, and hepatocyte apoptosis. |
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