Screening Of Molecular Targets Involved In Antagonistic Effects Of Natural Antioxidants On Insulin Resistance

The differential expressions of almost 100 genes from transcription and translation level and their abnormal biological functions are involved in the progress of molecular pathogenesis of diabetes. It is especially notable that oxidative assault is a core mechanism involved in the occurrence and development of diabetes mellitus and its complications, which has been one of the frontier in the field of diabetes research in recent years. Diabetes is a chronic degenerative disease caused by genetic factors and environmental factors. It needs a potential clinical condition for a long time from beta cell damage to diabetes onset. Under the condition that the original cause of diabetes is not clear and diabetes incidence is rising continuously, screening the molecular targets of protective effects of Natural antioxidants (NA)on islet beta cells may play a crucial role in the prevention and control of diabetes from the core mechanism of diabetes onset.Diabetes mellitus patients are mostly belonging to type 2 diabetes and insulin resistance is the main cause in type 2 diabetes. More and more data demonstrate that ROS produced by oxidative stress. On the one hand, it can activate a series of stress signaling pathway within beta cells as a signal molecule, induce beta cells to oxidative stress reaction, which result in the damage of beta cells and the decrease of the secretion of insulin. On the other hand, it can block insulin singnaling pathway and lead to insulin resistance as well. Insulin resistance signaling pathway under oxidative stress: (1) Oxidative stress activates JNK, IKK and PKC, and they all can increase phosphorylation of serine in 307site of IRS and also restrain phosphorylation of normal tyrosine in the site, which can eventually lead to a block in insulin conduction. (2) After the inhibition of phosphorylation in tyrosine of IRS1, Oxidative stress can hinder insulin signal in transmitting to PI3K, and decrease the transposition of GLUT2 from cytoplasm to cell membrane after the treatment of insulin, which can eventually lead to insulin resistance. (3) Oxidative stress activates IKK and make NFκB enter nucleus after get acitivity, which can also increase insulin resistance and lead to apoptosis.Natural antioxidants mainly refer to a variety of vitamins, trace elements, and the other antioxidants in food, which can prevent oxidative stress and remove the free radicals in the body. Research shows that Natural antioxidants have an important role in antagonism to oxidative stress and in prevention and cure diabetes. In recent years we have made progress in antioxidant and control of diabetes research about NA. We has selected many species, such as Se, VE, VC, lipoic acid, niacinamide, soybean isoflavone, etc. The antidiabetic mechanism of NA maybe as follows: (1) to play a insulin-like role or to promote insulin release; (2) to clear free radicals and restrain lipid peroxidation in order to prevent islet beta cells from oxidative injury; (3) to improve the sensitivity to insulin; (4) to reduce DNA damage or promote DNA repair: such as niacinamide, soybean isoflavone, etc. Niacinamide can directly add NAD pool in the body, promote DNA repair, and it also can ensure the normal operation of the TCA cycle in order to avoid cell death. Soybean isoflavone can protect DNA from oxidative attack. At present, the molecular targets of natural antioxidants on protecting beta cells and antagonisting insulin resistance in beta cells are still not clear. We have optimized out five kinds of natural antioxidants which are selenium, vitamin E, thioctic acid, niacinamide and soybean isoflavone based on literature evidence and accumulation of long-term research in order to observe molecular targets, function characteristics, biological effects and molecular mechanism of their functions against insulin resistance. From transcription and translation level, we observed the expression of related transcription factors and genes in insulin resistance signaling pathway in order to elaborate the molecular mechanism of NA against insulin resistance and discover their molecular targets. This study consists of three parts: the first part, improvement on oxidative stress model in rat beta cell line and establishment insulin resistance in HepG2 cell line: the study on oxidative stress in rat beta cells induced by H2O2 with different concentrations or different reaction time and cell proliferation, ROS content and MDA content, SOD activity and GSH-PX activity detected by flow cytometry etc; The doses or reaction time of selenium, vitamin E, thioctic acid, niacinamide and soybean isoflavone were selected on the basis of oxidative stress model optimization; The insulin resistance liver cell model was established through HepG2 cells transfected with pcDNA3.1-human-resistin, as a high level cell model to observe molecular mechanism of NA against insulin resistance; The second part, the study on effects of natural antioxidants on proliferation and apoptosis of rat beta cells under oxidative stress; cell cycle and apoptosis of rat beta cells detected by flow cytometry to explain whether natural antioxidants have protective effects on rat islet beta cells and antagonist the key link of diabetes which is insulin absolute or relative inadequacy; The third part, effects of natural antioxidants on the expression of transcription factors and genes in insulin resistance signaling pathway: mRNA expression of FOXO1, PDX1, insulin2, IRS1 ,MafA, GLUT2 and AKT2 genes in rat beta cells were detected by semi-quantitative RT-PCR or real-time quantitatie PCR; mRNA expression of JNK1, IRS1, PI3K, GLUT2, PKC genes in HepG2 cells were detected by real-time PCR; Protein expression of FOXO1, PDX1, NFκB, insulin, IRS1, JNK1 and AKT2 genes in rat beta cells were detected by immunocytochemistry staining and Westernblot; Protein expression of IRS1 and JNK1 genes in HepG2 cells were detected by immunocytochemistry staining and Westernblot.Results:1. The oxidative stress model , optimized doses and reaction time of NA were improved. This model was proved successfully through testing antioxidant enzymes vitality and radical level. On the basis of this oxidative stress model, we optimized the dose and reaction time of NA combined with the other NA. The Optimal Doses of antioxidants were selected, that was 0.1 mg/L sodium selenite, 5 mg/L vitamin E, 10 mg/L lipoic acid, 122 mg/L niacinamide and 0.432 mg/L soybean isoflavone and the Optimal reaction time was 24 h. Under this condition, NA can obviously improve antioxidant biological markers and reduce the generation of free radical.2. The insulin resistance model was established by constructing HepG2 cells transfected with pcDNA3.1-human-resistin. On the basis of recent research progress, we constructed eukaryotic expression vector with pcDNA3.1-human-resistin through genetic engineering restructuring technology and mediated HepG2 cells by liposomes to further confirm antagonistic effects of NA on insulin resistance singnaling pathway. It's the first time to establish insulin resistance cell model based on HepG2 cells transfected with resistin gene. It was proved successfully through the glucose uptake experiment and glycogen staining experiment. This model provided brand-new scientific tools and research means for us to further study on mechanism of antagonistic effects of NA on insulin resistance in peripheral tissue.3. To the best of our knowledge, this is the first report on confirmed antagonistic effects of NA on insulin resistance caused by transfecting resistin gene based on insulin resistance cell model transfected with pcDNA3.1-human-resistin. Its features were that both glucose uptake ratio and glycogen synthesis decreased obviously in HepG2 cells transfected with resistin gene. Adding different kinds of antioxidants obviously increased glucose uptake ratio and glycogen synthesis in HepG2 cells transfected with resistin gene,Which significantly reversed insulin resistance effects caused by resistin gene. Because resistin and insulin resistance are newly concerned and they become one of hot issues in diabetes research field. The main link of the effect of resistin on insulin resistance is through insulin receptor. we are the first to confirm antagonistic effects of NA on key link of resistin to action, which provided experiment basis for us to put forward key targets about NA against insulin resistance.4.The protective effects of NA on islet beta cells were confirmed through antagonizing apoptosis and promoting proliferation indirectly. Cell cycle results detected by flow cytometry showed that combined use of sodium selenite, vitamin E, thioctic acid, niacinamide and soybean isoflavone can obviously increase the percentage of S phase cells and significantly decrease the percentage of G0/G1 phase cells. Cell apoptosis results detected by flow cytometry showed that combined use of NA can significantly decrease the percentage of early apoptosis cells, late apoptosis cells and death cells. These results suggested that NA had protective effects on islet beta cells against oxidative stress and antagonized the key intermediate link in diabetes.5. Many molecular targets about antagonistic effects of NA on insulin resistance singnaling pathway were elaborated. We have found that NA regulated the expression of main transcription factors and genes in insulin resistance singnaling pathway on the basis of oxidative stress model of islet beta cell line. These included that NA reduced the mRNA expression of FOXO1, and raised the mRNA expression of PDX1, IRS1, insulin2, MafA,GLUT2 and AKT2. They also reduced the protein expression of FOXO1, JNK1 and NFκB and raised the protein expression of PDX1,IRS1, insulin2 and AKT2. We confirmed that NA regulated the expression level of transcription and translation of many molecular targets about insulin resistance in islet beta cells. Therefore, we preliminarily screened molecular targets about protective effects on islet beta cells and antagonistic effects of NA on insulin resistance.6. We further elaborated some molecular targets about antagonistic effects of NA on insulin resistance singnaling pathway in HepG2 cells transfected with resistin gene. On the basis of insulin resistance singnaling pathway under oxidative stress, we studied on the effects of NA on the expression of related genes in this pathway. The results showed that NA reduced the mRNA expression of JNK1 and PKC and raised the mRNA expression of IRS1 and GLUT2 .They also reduced the protein expression of JNK1 and raised the protein expression of IRS1. We further confirmed that NA regulated the expression level of transcription and translation of many molecular targets about insulin resistance in peripheral tissue. Therefore, we preliminarily screened molecular targets about antagonistic effects of NA on insulin resistance in peripheral tissue.This study discussed the regulating effects of NA on expression of related transcription factors and genes from the level of transcription and translation in insulin resistance singnaling pathway. We also studied molecular mechanism of the improvement of NA on insulin resistance in beta cells and the protective effects of NA on beta cells. We preliminarily screened FOXO1,NFκB,JNK1,PDX1,IRS1,insulin2,MafA,GLUT2 and AKT2 genes, which might be molecular targets about protective effects on islet beta cells and antagonistic effects of NA on insulin resistance. And NFκB,JNK1,PKC and GLUT2 genes might be molecular targets about antagonistic effects of NA on insulin resistance in peripheral tissue. We have found the above targets and putforward molecular targets for improvement of insulin resistance and developmentⅡlevel prevention of diabetes for people in nutrition field.