Research Of Effect Resistin Gene Transfection On The Expression Of Transcription Factors Sp1 And PPARγ MRNA In Vitro

The mechanism of diabetes involves insulin resistance and absolute or relative deficiency of insulin secretion from pancreatic islet beta cells. The formation of insulin resistance is related with complicated genetic and environmental factors, especially with life style. The aetiology of insulin resistance referred to dietary factor, cytotoxic effect of high glucose, smoking, obesity, pregnancy and motion. The mechanism of insulin resistance resulted from different etiological factors all concerned with impairment of structure and function in molecular levels of insulin receptor and post-receptor in the insulin target tissues, and with disorder of hormones that regulating insulin action.The current data showed that insulin resistance is related with a variety of cytokines secreted from adipose tissue, resistin is one of the most important cytokines discovered in recent years. Resistin was originally discovered in mice white adipose tissue, because resistin cause insulin resistance, elevate blood glucose level and promote adipose cell proliferation, at the beginning of its identification it was considered to serve as an important link between obesity and insulin resistance, so it was referred to as resistin. Up to present time, the exact molecular mechanism of insulin resistance induced by resistin remained to be explored, though it has been reported that there was intimate relationship between insulin resistance and obesity in patients with diabetes and in rodents, but subsequently performed basic and clinic researches presented diverse results.In the development process from obesity to insulin resistance and to diabetes, It was proposed that inflammatory reaction initiated by cytokines secreted from adipose tissue is the most important initiating agent, and which affects the generation and development of the disease's course. Adipose tissue secrets a variety of cytokines (or called pro-inflammatory factor),including tumour necrosis factor- alpha, interleukin-6,adiponectin,resistin and other different kind inflammatory markers. All these cytokines affect the activity of molecule in the insulin receptor signal transmission system through cellular signal transmission system, block the signal transmission and decrease the sensitivity of insulin receptor and cause insulin resistance.The insulin target tissue in the human body include adipose tissue, muscular tissue and hepatic tissue, all of these tissues could be induced insulin resistance. Because resistin was originally identified in the adipose tissue of mice, the researchers usually adopt adipose cells derived from mice to do experiments that related with resistin, however,at present,in human resistin was thought mainly secreted from monocytes in the blood, so to investigate the mechanism of insulin resistance in human, it is advisable to investigate the resistin's effects on the three kinds of tissues respectively. Though HepG2 cell derived from human hepatoma cell strain, since it possesses entire hepatocyte energy metabolism system,so usually it was adopted to perform research related with hepatocyte glucose and fat metabolism in vitro.Sp1 is a well known transcription factor, it has been reported that Sp1 was activated in the presence of high glucose, high insulin content and oxidative stress, the activated Sp1 could promote target gene transcription. It was identified that Sp1 participated in the regulation of resistin gene expression in the adipose tissue, the activated Sp1 could enhance resistin gene expression and production.Peroxisome Proliferator-activated Receptorγbelonged to the super-family of nuclear hormone receptor, it is attributed to the transcription factors of ligand-activation dependent. The initiate data about functions of PPARγmainly involved in regulation of adipose cell differentiation and metabolism of glucose and fat. The recent data showed that PPARγagonists possess anti-inflammation and anti- oxidative activity and it was proposed that it is independent of PPARγactiviation, the mechanism of which referred to the inhibition of transcription factor nuclear factor-kappa B.As it was reported in the previous study rosiglitazone exert protective effects on the oxidative stress-mediated ishchemia- reperfusion damage in different tissues. Oxidative stress is associated with insulin resistance and is thought to contribute to development and progression towards type 2 diabetes.It has been found increased levels of reactive species in animal experiment and clinic research associated with diabetes, ROS could enhance the activity of cellular signal transmission system and transcription factor system, such as NF-kB and active protein-1, which is common transcriptions factors activated by ROS, Sp1 is also could be activated by oxidative stress, ultimately, ROS caused blockage of insulin signal transmission and production of a large amount of cytokines of different kinds, some of them are closely associated with induction of insulin resistance.As an agent of insulin receptor sensitizer, the main mechanism of rosiglitazone's pharmacological actions is traditionally believed to be associated with activation of PPARγ, through which regulating expression of a series of genes responsible for the glucose and fat metabolism,improving insulin receptor sensitivity, however, in a recent study it showed that rosiglitazone decreased resistin gene expression in fully differentiated 3T3-L1 adipocytes and improved the sensitivity of insulin receptor, the mechanism of which was to decrease the O-glycosylation status of Sp1 and this process maybe involved in the repression of Sp1 promoting resistin expression. The other reports proposed that rosiglitazone improved insulin receptor sensitivity through mechanism of activation of adiponectin pathway.In the states of obesity, insulin resistance and diabetes there is a lot of ROS production and transcription factor Sp1 could be activated in the states of high levels of ROS and insulin resistance , while rosiglitazone could improve insulin receptor sensitivity and exert anti-inflammatory and antioxidative actions, in addition to this, in a recent study it showed that rosiglitazone failed to activate PPARγexpression in the presence of increased level of ROS, all these suggest that high ROS level could activate Sp1, throught which resulted in increased resistin expression, and high level of ROS could also resist the TZDs'action, or the expression of PPARγwas inhibited when in the presence of high level ROS.As described above, resistin plays a pivotal role in the formation of insulin resistance, transcription factor Sp1 enhance the activity of resistin promoter, PPARγregulate the expression of resistin, increased ROS content contribute to the development of insulin resistance, and in the presence of elevated ROS levels TZDs′action could be suppressed. So it is important to elucidate the relationship among resistin, Sp1, PPARγand ROS during the formation of insulin resistance, this may supply a correct guidance for the reasonable administration of TZDs and antioxidant to the diabetes patients.The present study make the academic progress as followed:1. Successfully performed the transfection of a recombinant eukaryotic expression vector pcDNA3.1-Retn encoding human resistin gene in HepG2 cells, Both for the protein and gene expression of resistin was assessed through way of RT-PCR and immunocytochemistry, the detection results showed that stable transfection was successfully performed and over-expressing resistin HepG2 cell lines was constructed, which is very necessary and important for the further investigation of the mechanism of insulin resistance induced by resistin, and it provided a scientific and dependable manner and tool of study.2. The results of glucose uptake experiment indicate the glucose uptake ability of resistin gene transfected cells decreased when cultured in high glucose and high insulin medium, over-expressed resistin induced insulin resistance. The present study demonstrated that the induction of insulin resistace by resistin is specific and potent.3. The Sp1 mRNA expression was detected by RT-PCR and the results suggest resistin gene transfection caused significant up-regulation of Sp1 mRNA expression. There has not been any reports on the same research areas up to now, This is a new exploration of resistin effect on the HepG2 cells.4.The PPARγmRNA expression was assessed by RT-PCR and the results suggest PPARγmRNA expression present no significant differences in resistin gene transfected cells when compared with control,but appears a decline tendency. When treatment with rosiglitazone there presented significant down-regulation expression of PPARγmRNA in the resistin gene transfected cells, this provided new experimental data for the correct administration of TZDs with diabetes,there has not been any reports on the same research areas up to present.5.The content of reactive oxygen species was detected by flow cytometry and the results indicate that resistin gene transfecting elevated the ROS level and treatment with rosiglitazone caused the elevated ROS level to decline, but it showed no significant difference compared with control. This result suggest that rosiglitazone possess antioxidant action and which provide new insight for the clinical application of antioxidant combined with TZDs to the patients with diabetes. The antioxidative action of rosiglitazone may be independent of PPARγactivation, but it is beneficial to the improvement insulin receptor sensitivity.