| Sirtuins (SIRT) are NAD-dependent histone deacetylases, belonging to the silentinformation regulator factor family, involved in energy production, fatty acid metabolism,cell cycle and the regulation of the stress response, closely related with the life span ofcells.. There are7sirtuins in mammals (SIRT1-7) whose functions are postulated to belargely determined by their intracellular localization. SIRT3is a crucial regulator ofmitochondrial function, controlling global acetylation of the organelle. SIRT3is importantin mammalian mitochondrial deacetylase and highly expresses in skeletal muscle. SIRT3can reduce the level of intracellular ROS and, more importantly, the SIRT3greatlyenhanced superoxide dismutase-2, reducing cells radicals and promoting the tolerancecapacity of oxidative stress.Oxidative stress (OS) can be defined as the steady state condition where the freeradical/reactive oxygen species (ROS) flux is balanced by antioxidant defenses. Theupregulation of OS, which mainly occurs in conditions of increased flux of free radicals/ROS and/or reduced antioxidant levels, is now accepted to play a critical role in thepathogenesis of obesity, atherosclerosis, T2D and IR. Skeletal muscle is the major organinvolved in postprandial glucose uptake and metabolism and peripheral insulin sensitivity.Changes in mitochondrial mass and oxidative phosphorylation have been shown to occurin muscle of individuals with diabetes and insulin resistance.Jinlida granules consist of ginseng, sophora, ophiopogon, cornus, poria, perrin,berberine, Anemarrhena Epimedium (Sunburn) Salvia, arrowroot, litchi seed and so on.Clinical studies have shown that jinlida granules have protection on the treatment ofdiabetes and its complications, reducing the insulin resistance of type2diabetes andprotect β-cell function, but its pharmacological mechanism remains to be further clear.Metformin is currently the drug of first choice for the treatment of T2D, in which itsmechanisms are to reduce hepatic glucose output and to increase the insulin mediatedutilization of glucose. Metformin is also described as an insulin-sensitizer, leading toreduction in insulin resistance and a significant decrease in plasma fasting insulin levels,been reported to have numerous cellular effects in multiple tissues. Both experimental andclinical studies indicate that oxidative stress plays a major role in the development andcomplications of type2diabetes. Previous studies demonstrated that metformin attenuatedperipheral insulin resistance and decreased plasma and brain oxidative stress ininsulin-resistant rats caused by long-term HFD consumption. Metformin not only influences mitochondrial biogenesis but also the function of mitochondrial oxidativemachinery, but the mechanism still needs further research. Therefore, to explore the role ofSIRT3in skeletal muscle tissue diabetic rats and the mechanisms of improving insulinresistance in skeletal muscle, which elucidate the mechanism of insulin resistance, andprovide a solid theoretical basis for finding new therapeutic targets in clinical andexpanding the application of metformin.The aim of this study is to clarify the exact role for SIRT3in insulin resistance ofskeletal muscle in diabetic rats. This study is consisted of three main parts：The first part isto investigate the effect of jinlida granules and metformin on oxidative stress instreptozotocin induced diabetic rats, the second is to see the effect of jinlida granules andmetformin on SIRT3and oxidative stress of skeletal muscle in diabetic rats. Finally, theeffect of SIRT3on glucose uptake and ROS production in L6cells, further investigationunderlying molecules mechanism of insulin resistance.In this study, we found that SIRT3mRNA and protein expression is decreased in HFDand STZ induced T2DM animal model; Jinlida granules and metformin had a certainprotective effect on diabetic rats skeletal muscle, could improve the oxidative stress levelsin diabetic rats, and could increase SIRT3expression of skeletal muscle in diabetic rats. Invitro, over expression of SIRT3alleviated insulin resistance of L6and L6-IR cells,Metformin increased the expression of SIRT3and SOD2, as well as down regulation ofNF-κBP65and JNK1. When SIRT3knocked down, could reverse the decrease ofNF-κBP65and JNK1, and increase of SOD2that induced by metformin. While, overexpression of SIRT3promoted the effect of metformin on NF-κB P65, JNK1and SOD2.Metformin increased glucose uptake of L6-IR cells, Knocked down expression of SIRT3diminished the glucose uptake effect that induced by metformin, while over expression ofSIRT3increased the glucose uptake induced by metformin. Accordingly, ROS productionwas decreased by metformin, and ROS production was increased when SIRT3wasknocked down, and ROS production was decreased when SIRT3was over expressed.These results demonstrated the pharmacological effect of metformin in promoting glucoseuptake may partly through SIRT3. Part I Effects of jinlida granules and metformin onoxidative stress and skeletal muscle in HFD andstreptozotocin-induced diabetic ratsObjective: To observe the impact of jinlida granules and metformin on oxidativestress indicators and its protective effect on skeletal muscle tissue ultrastructure in HFDand streptozotocin-induced diabetic rats.Methods: Eighty male Sprague-Dawley (SD) rats were divided into diabetic modelgroup (70) and normal control group (NC)(10) by using a random number table. Diabetesrats were induced a single intraperitoneal injection of streptozotocin (STZ) at a dose of30mg kg-1body weight dissolved in citrate buffer after four weeks. Type2diabetic ratmodels were tested by HOME-IR3days after injection were included in the study andwere randomly divided into seven groups：DM control group, JL, JM, JH, ALA, INS, MTgroups. Rats’ plasma SOD, ROS, GSH-Px activity and MDA content were detected byELISA after eight weeks. The cellular ultrastructure of the skeletal muscle was observedthrough light microscope.Results: Compared with NC group, FBG, FINS, TC, TG, LDL, ROS and MDA wereincreased in DM group,(p<0.05), while serum GSH, SOD, HDL and ISI were decreased(p<0.05); Compared with DM group, in the rats of MT and JH groups, FBG, FINS, TC,TG, LDL, ROS and MDA were decreased (p<0.05), but serum GSH, SOD, HDL and ISIwere increased (p<0.05). Obvious chromosome aberrations, dilatate, widened synapticclefts as well as disappeared mitochondriales crista were also observed in the diabetic rats’skeletal muscle by TEM.Conclusion: Jinlida granules and metformin significantly improves fasting bloodglucose, fasting insulin, and insulin sensitivity index, blood lipids, and oxidative stressindex levels in type2diabetes rats, having a protective effect on skeletal muscle in diabeticrats. Part II Effect of jinlida granules and metformin onSIRT3and oxidative stress of skeletal muscle in HFDand streptozotocin-induced diabetic ratsObjective: To observe the effects of jinlida granules and metformin on oxidativestress and SIRT3of skeletal muscle in diabetic rats, explore the possible mechanism ofmetformin improves skeletal muscle insulin resistance.Methods: Rats’ skeletal muscle superoxide dismutase (SOD), reactive oxygenspecies (ROS) and glutathione peroxidase (GSH-Px) activity and malondialdehyde (MDA)content were detected by ELISA after eight weeks. Real-time quantitative PCR andWestern Blotting were used to detect the expression of SIRT3mRNA and SIRT3protein ofskeletal muscle in diabetic rats.Results: Compared with NC group, the expression of SIRT3mRNA (p<0.01) andSIRT3protein (p<0.05) of skeletal muscle were decreased in DM group rats; comparedwith DM group rats, the expression of SIRT3mRNA (p<0.01) and SIRT3protein (p<0.05)of skeletal muscle were increased in MT and JH group (p<0.05). Compared with NC group,GSH and SOD of skeletal muscle were decreased in DM group rats (p<0.05)，MDA andROS increased. Compared with DM group, MDA and ROS of skeletal muscle weredecreased in MT and JH groups (p<0.05), but GSH and SOD of skeletal muscle wereincreased (p<0.05).Conclusion: Metformin and jinlida granules are effective in improving oxidativestress in skeletal muscle in diabetic; the mechanism may be through increasing theexpression of SIRT3mRNA and protein in skeletal muscle, therefore enhancing insulinsensitivity in rats with diabetes, and delaying the development of diabetes. Part Ⅲ Impact of metformin on SIRT3in L6insulinresistance cellsObjective: To study the role of SIRT3in insulin resistance of L6skeletal muscle cells,explore possible signaling pathway metformin to improve insulin resistance in skeletalmuscle.Methods: Differentiated L6skeletal muscle cells were treated with750μM ofpalmitic acid for L6insulin resistance cell model. A plasmid with Flag tagged SIRT3wasobtained from Addgene (plasmid13814). L6cells in24-well plates were transfected with200ng of SIRT3plasmid or with empty pcDNA3.1control plasmid using Fugene HD(Roche) according to the manufacturer’s protocol. For knocking down human SIRT3,specific siRNA (5’-GCC CAA CGT CAC TCA CTA CTT-3’) targeting SIRT3as published.The transfection of siRNA into L6cells using Fugene HD according to the manufacturer’sinstructions (Roche). Glucose uptake and ROS were measured in the L6cells, L6insulinresistance cells, SIRT3-/-cells and SIRT3+/+cells. Anti-SIRT3, anti-NF-κB P65, anti-JNK1,anti-SOD2and anti-GAPDH were analyzed by Western blot.Results: Glucose uptake was significantly decreased in insulin resistant L6cells. ROSlevel in insulin resistant L6cells were significantly increased than L6cells. Overexpression of SIRT3could dramatically decreased ROS production in L6-IR cells,metformin increased the expression of SIRT3and SOD2, as well as downed regulation ofNF-κB P65and JNK1, when SIRT3knocked down, could reverse the decrease of NF-κBP65and JNK1, and increase of SOD2that induced by metformin.Conclusion: SIRT3significantly improve glucose absorption rate and reducegeneration of ROS in L6insulin resistance cells, metformin may improve cellularoxidative stress state by upregulating SIRT3expression, thereby strengthening insulinsensitivity of cells. |
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