| Background and objectiveDiabetes has become a global epidemic, and its incidence increased year by year and isoften accompanied with a variety of complications, which seriously threatens human health.Type2diabetes is the most commonly observed among adult diabetic patients, mainlybecause of the metabolic disorders caused by insufficient insulin produced in the body and/or glucose-induced insulin resistance, leading to high levels of blood sugar. Thepathogenesis of diabetes is complex, and it has been reported that high concentrations ofglucose oxidase, a food additive, can also induce insulin resistance, leading to diabetes.Glucose oxidase is a key enzyme in the pathway of glucose metabolism to produce gluconicacid and hydrogen peroxide (H2O2) via catalyzeing the β-D-glucose, playing an importantrole in the pathogenesis of diabetes. It has been revealed that the glucose oxidase caninduce apoptosis in a variety of cells including pancreatic β cells, neuronal cells andfibroblasts, possibly through the generation of H2O2. However, the more in-depthtoxicological mechanisms have not been reported. To better clarify the pathogenesis ofdiabetes, diabetic animal models were generated for studies. Streptozotocin (STZ) is one ofthe most commonly drugs used in inducing and establishing the animal models of diabetes,as STZ has exerts selective toxicity on the islet β cells. In experimental models of diabetes,different STZ treatment induces different diabetic responses. Currently, the treatment oftype2diabetes mainly depends on drugs based on the change of lifestyles, but the effectsare far from satisfactory. In recent years, a large number of clinical researches demonstratedthat surgery is effective for diabetes treatment.This study aims:1) to investigate the toxic effect of glucose oxidase on islet β cells,trying to provide more evidence on the toxic mechanism of glucose oxidase in diabetes.2)to provide further support for the application of STZ in diabetes, by comparing the effectsof multiple low-dose STZ (MLD) and single high-dose STZ (SHD) on ROS production andultrastructure of islet β cells.3) to evaluate the outcomes and safety of LRYGB surgery in type2diabetes therapy.MethodsRIN-m5F islet β cells were treated with glucose oxidase. MTT assay was used toexamine the cell survival rate. DCFH fluorescence assay was performed to measure theintracellular ROS levels. Flow cytometry was used to determine the apoptosis and cell cycle.The subcellular structure was observed with the transmission electron microscope. Theexpression levels of mitofussins were measured by Real-time RT-PCR.High fat diet-fed rats were given i.p. MLD (30mg/kg·d,3d) or SHD (120mg/kg).After a12h-fasting following the last injection, the blood and pancreas were obtained.Blood insulin was measured using ELISA, and hydroxyl radical and hydrogen peroxideusing the chemical spectrometric method. The ultrastructure of islet β cells was observedunder an electro microscope.The clinical data of33patients suffered the T2DM who had received the LRYGBsurgery during the last2years in our hospital were retrospectively analyzed.Diabetes-related factors were monitored before and1,3and6months after the surgery,including the body mass index (BMI), waistline, oral glucose tolerance test (OGTT), Cpeptide and insulin release tests, glycosylated hemoglobin (HbA1c), blood lipids. In thefollow-up at12month after surgery, relative diabetes-related factors of patients such asBMA, waistline, fasting blood glucose and the level of HbA1c were examined.ResultsGlucose oxidase dose-dependently decreased the cells survival rate. In contrast,glucose oxidase dose-dependently increased the intracellular ROS level and apoptosis. Inthe subcellular structure, glucose oxidase revealed potent damage effect on mitochondria,appearing as reduced mitochondria quantity, reduced mitochondria size, and mitochondrialfussion and fission. The further data showed that glucose oxidase stimulated the expressionof mitofussin1, mitofussin2and optic atrophy protein1.Compared with the control rats fed with the normal chow diet, the fasting plasmaglucose increased significantly in both MLD and SHD rats, with higher increase in MLDgroup. The blood insulin level increased significantly and insulin sensitive index decreasedsignificantly in MLD rats. In SHD rats, no marked changes were observed in the insulinlevels, and insulin sensitive index decreased slightly. Both MLD and SHD treatment induced mitochondria swell of islet β cells. MLD significantly reduced the number ofmitochondria, compared to the moderate decrease by SHD treatment. MLD increased thelevels of hydroxyl radical and hydrogen peroxide, while SHD almost had no effect.All33patients recovered successfully, no dead case. Three patients with complicationafter surgery (9%), including1anastomotic leakage and2anastomosic ulcer, were curedthrough conservative treatment. Compared to preoperative values, the BMI, waistline,HbA1c, total cholesterol, triglyceride, fasting blood glucose, insulin and C-peptidesignificantly decreased in1,3and6months after surgery (P <0.05). BMI, waistline, fastingblood glucose and HbA1c were further decreased at the12month after surgery. In OGTTtest, blood glucose decreased at60,120,180min in postoperative groups, in comparisonwith preoperative group. In insulin and C-peptide release tests, the peak release of seruminsulin and C-peptide was earlier in postoperative groups than in preoperative group. Allthe changes were the most significantly different in6months after surgery, compared to1and3months. In all18patients with1-year follow-up, diabetes condition was relieved in11patients (61.1%). The blood glucose level was controlled in5patients (27.8%). Twopatients were improved (11.1%).ConclusionGlucose oxidase expresses potent toxic effect on islet β cells. The related mechanismis involved in intracellular H2O2acculuamtion, which damages the mitochondria, and theninduces mitochondria-related apoptosis.Compared to the sole cytotoxic effect of SHD on islet β cells, MLD reveals a moresevere and dual diabetes induction mechanism: β cells damage and insulin resistanceinduction, which may be due to the ROS accumulation.Laparoscopic Roux-en-Y gastric bypass improved glycometabolism and insulinsecretion, and is an effective treatment on type2diabetes. |
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