| 【Background】Type 2 Diabetes Mellitus(T2DM) is an important chronic noncommunicable disease which is seriously harmful to human health. The main characteristics of T2 DM include insulin resistance(IR), dysfunction of β cells and hyperglycemia. T2 DM patients are easily to develop various complications of mutiple organs such as diabetic retinopathy, diabetic nephropathy, failures of heart and vessels, as well as disorders of nervous system. The pathogenesis of T2 DM is complicated. Genetic factors, environmental influences and behaviors contribute to it. IR is the main pathogenesis, and is difficult for treatment. According to a number of literature and previous studies of our laboratory, oxidative stress plays an important role in insulin resistance occurrence and development. Besides, mitochondrial damage is critical mechanism of insulin resistance occurring. Mitochondria are major sources of cellular ROS and also the main targets attacked by ROS. However, the interactions and molecule mechanisms between oxidative stress and mitochondrial damage during occurrence and development of IR are still not clear.Nrf2 is a critical transcription factor regulating a series of antioxidative enzymes expression. Nuclear respire factor 1(NRF-1) plays an important role in mitochodrial biogenesis through regulating transcription of mitochondrial transcription factor A(mt TFA). It has been reported that Nrf2 partly regulated transcription of NRF-1, mitochondrial biogenesis and myocardial preservation. However, the interaction between Nrf2 and NRF-1 during insulin resistance occurring and development needs further research.Based on a high-fat-diet induced IR animal model, features of changing in oxidative stress, mitochondrial structure and function, as well as key molecules including Nrf2, NRF-1 and mt TFA were investigated. Expressions changes of Nrf2, NRF-1 and mt TFA were also observed in T2 DM model db/db mice. L02 cells were transfected with p CDNA3-Myc3-Nrf2 to investigate regulating mechanisms of Nrf2 on NRF-1 and mt TFA. This research provides an important experimental basis for identifying pathogenesis of IR and developing interventions against it. 【Aims】1. To investigate the dynamic changes of insulin resistance, liver steatosis, oxidative stress and insulin resistance induced by high-fat-diet.2. To investigate the dynamic changes of Nrf2, NRF-1 and mt TFA in insulin resistance occurrence and development induced by high-fat-diet.3. To investigate the molecule mechanisms of insulin resistance induced by dynamic changing of Nrf2, NRF-1 and mt TFA. 【Methods】1. Establishing IR model by high-fat-diet: The mice were fed with high-fat-diet containing 10% fat. IPGTT/IPITT were conducted at 8th and 12 th week. IPGTT/IPITT and other molecular biological indicators were tested every 4 weeks between 16 th and 28 th week.2. Indicators related to insulin sensitivity were tested: After fasting overnight, blood glucose of mice were tested by Roche glucometer and strips. IPGTT/IPITT were conducted through intraperitoneal injection of glucose(1 g·kg-1) and insulin(0.75 IU·kg-1). Plasma was collected for determination of insulin using ELISA kit. HOMA-IR was calculated for estimating IR. Insulin(10 IU·kg-1) was intraperitoneally injected for insulin signal testing.3. Changes of hepatic morphology observing: Oil Red O staining was used for testing liver steatosis. Sirus red-picric acid staining was used for testing liver fibrosis.4. Oxidative stress related indicators testing: Fluorescent probe DHE was used for testing ROS level. MDA content, GSSG/GSH and activities of antioxidative enzymes were tested using commercial kits.5. Mitochondria related indicators detecting: Changes of mitochondrial structure were observed by transmission electron microscope. Oxytherm liquid-phase oxygen measurement system was used for detecting oxygen consumption.6. Cell model establishing: Up-regulating Nrf2 expression through transfecting p CDNA3-Myc3-Nrf2 plasmids into L02 cells.7. Critical molecules detecting: Western Blot was used to detect expression of Nrf2, NRF-1, mt TFA, P-Akt, SREBP1 c and a series of antioxidative enzymes.【Results】1. High-fat-diet induced IR mice showed impaired glucose tolerance at 12 th week, then blood glucose and insulin increased in turn. Impaired insulin tolerance was shown at 24 th week. Insulin signal molecule P-Akt decreased from 20 th week. The HOMA-IR of high-fat-diet fed mice was always higher than control. Blood glucose and HOMA-IR of db/db mice increased while insulin decreased.2. Liver steatosis and liver fibrosis deteriorated gradually in high-fat-diet induced IR mice. Lipid synthesis critical transcription factor SREBP1 c expression increased between 20 th and 24 th week, and then decreased to control at 28 th week. Serious liver steatosis was exhibited and SREBP1 c increased significantly in db/db mice.3. Hepatic ROS level of high-fat-diet induced IR mice was increased at 16 th week and reached peak at 20 th week, and then decreased after 24 th week. MDA increased gradually. Antioxidative enzymes expressions were increased at 16 th week, then decreased at 28 thweek. MDA content and GSSG/GSH were elevated in db/db mice. Activities of CAT and SOD were increased. Activity of GPx was decreased while its expression was increased. Expressions of other antioxidative enzymes were not changed. 80 h after transfecting plasmids, expressions of antioxidative enzymes were increased in L02 cells.4. Structure of mitochondria became abnormal induced by high-fat-diet from 16 th week and then aggravated. Oxygen consumption was increased at 16 th week and decreased after 20 th week.5. In high-fat-diet induced IR mice, Nrf2(100 k D) was increased between 16 th and 20 th week, and then decreased from 24 th week while Nrf2(61 k D), NRF-1 and mt TFA expressions were decreased. Nrf2(100 k D) was not changed significantly while Nrf2(61 k D), NRF-1 and mt TFA expressions were decreased in db/db mice. 80 h after transfected plasmids, expression of Nrf2(100 k D) was increased significantly while Nrf2(61 k D), NRF-1 and mt TFA expressions were not significantly changed. 【Conclusions】1. Liver steatosis, oxidative stress and mitochondrial damage worsen in insulin resistance occurrence and development induced by high-fat-diet.2. Nrf2, NRF-1 and mt TFA expressions dynamically changed in insulin resistance occurrence and development induced by high-fat-diet, and had closely relationship with liver steatosis, oxidative stress, as well as mitochondrial damage.3. Expressions of Nrf2, NRF-1 and mt TFA were changed in T2 DM model animal db/db mice compared with wild type mice.4. Excess ROS impaired antioxidative defense systemand induced oxidative stress through down-regulating Nrf2(100 k D), and then resulted in insulin resistance.5. Excess ROS inhibited NRF-1, mt TFA and induced mitochondrial damage through down-regulating Nrf2(61 k D), and then accelerated insulin resistance. |
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