Cloning And Expression Of Human PTP1B And Partial Mechanism Study Of Tflc Therapeutic Actions On Type 2 Diabetes Mellitus

Protein tyrosine phosphatase 1B (PTP1B) belong to family of protein tyrosine phosphatases(PTPs), exists as two forms such as receptor-like and cytoplasmic signal transducing enzymes, dephosphorylate the phosphorylated tyrosine residues of substrate proteins and is the first identified and purified PTPs. PTP1B acted on the phosphorylated insulin receptor(IR), insulin receptors substrate-1 and -2(IRS-1, IRS-2), growth factor receptor binding protein 2(Grb-2) and phosphatidylinositol 3 kinase(PI-3K), which all related with insulin signal transduction, thus attenuated insulin signal transduction and resulted in post-receptor insulin resistance. Recently, someone imported siRNAs which targeted against PTP1B into mouse hepatoma and found that the insulin signal transduction was increased, thus proved the role of PTP1B in insulin signal transduction again.Overexpression of PTP1B could also cause leptin resistance, thus initiated the development of obesity and type 2 diabetes mellitus. Mice deficient with PTP1B exhibited increased insulin sensitivity as well as resistance to weight gain when fed with a high-fat diet, which resulted from an increased basal metabolic rate and total energy expenditure. They growed very well and had normal breeding capability and low cancer development ratio. All these results indicated that small molecular inhibitors of PTP1B would be potential effective anti-diabetic/anti-obese drugs and had no side effects. In fact, inhibitors against PTP1B had already become candicate drugs as one of insulin sensitizers; many laboratories in the world are screening inhibitors of PTP1B from Chinese traditional and herbal drugs or artificial systhesised drugs.PTP1B has close relationship with obesity, type 2 diabetes and tumors, so its large-scale induction expression is very important for studying these diseases. This research took PTP1B as an important candidate gene of T2DM, investigated the antidiabetic effects and possible mechanisms of TFLC on T2DM model rats. The main contents are divided into four sections as follows:1. Cloning and expression of human PTP1B cDNA in E.coliIn order to express the hPTP1B protein effectively in E.Coli, total RNA was isolated from the peripheral blood mononuclear cells of type 2 diabetes mellitus. PTP1B full length cDNA sequence was amplified using two-step RT-PCR. The cloning plasmid pMD-hPTP1B was constructed, then PTP1B was subcloned with primers containing restriction endonucleases recognition sites of BamHⅠand EcoRⅠand ligated into pET-28a(+), transducted into DE3, induced with IPTG and expressed proteins were detected with SDS-PAGE and Western blot. Induction conditions of high level expression were experimented. Human PTP1B was sequenced and the plasmid pET28a(+)-hPTP1B was cut with two enzymes. PTP1B was expressed successfully in the form of insoluble inclusion body in E.Coli, SDS-PAGE analysis showed the molecular weight was about 50 kD. Western blot result showed the highly expressed protein was really PTP1B. The best induction concentration of IPTG was 0.05 mmol·L-1, the best induction time was 5h and the best induction temperature was 37℃. The rhPTP1B and corresponding prokaryotic expression plasmids were obtained, thus established a basis for screening highly specific small molecular inhibitors.2. High expression of rhPTP1B and in vitro inhibition experiments of TFLCThe rhPTP1B was expressed effectively in E.coli DE3 transducted with pET28a(+)-hPTP1B, purified by affinity chromatography, then renatured, the activity was measured with different reactive conditions and inhibitors, such as Na3VO4 ,TFLC(a vulgar extract from traditional Chinese medicine called Laoying tea). The tests of enzyme activity and studies of the inhibitor were showed as following: 1. The activities of rhPTP1B increased with enzyme concentration and substrate concentration; 2. The optimal temperature was 35℃at which the enzyme activity was maximum; 3. Inhibitory manner of Na3VO4 was dependant on its concentration and possibly act as uncompetitive inhibitor. 4.TFLC had a definite inhibitory action on rhPTP1B, this maybe as one of mechanisms of its hypoglycemic effect. 5. Commonly used therapeutic drugs such as pioglitazone and metformin had no apparent inhibitory action on rhPTP1B, this result showed that the two drugs exhibited their hypoglycemic effect not through rhPTP1B.3. Expression and participation of human PTP1B in insulin signal transduction in human hepatoma HepG2 CellsHuman PTP1B full length cDNA was subcloned into pcDNA3.1/His-myc-B, then transfected into the HepG2 cells. After screening with 400mg·L-1G-418 for 2 weeks, a HepG2 cell line that stably expressed hPTP1B was obtained and testified by double enzyme cutting, RT-PCR and Western blot. The ectopically expressed human PTP1B cDNA was located in cytosol by immumofluorescence method, it had no influence on cellular proliferation by MTT assay. We also showed that expressed PTP1B affected the phosphorylation of known PTP1B targets such as insulin receptor substrate-1 (IRS-1) and insulin receptor (IR) after insulin stimulation, thus participated in insulin signal transduction pathway in HepG2 cells. We also constructed RNAi plasmid called pBI-hPTP1B and found out that inhibited expression of PTP1B extent could be reached about 50%. Maybe this RNAi plasmid was used to inhibit PTP1B expression in thearapy of type 2 diabetic rats in future. 4. Antidiabetic effect of total flavonoids from Litsea Coreana leve in high-fat emulsion/streptozotocin induced diabetic SD ratsA study was undertaken to evaluate the hypoglycemic activity and mechanism of TFLC on a new SD rat model of type 2 diabetes mellitus. Male Sprague–Dawley rats were fed high-fat emulsion via gavage for 22 weeks, then injected with 12 mg/kg streptozotocin (STZ) and followed by 0.5ml/kg CFA injection on next day to induce type 2 diabetes mellitus. The rat models were treated with TFLC (400 mg/kg) or Na3VO4 (10 mg/kg) via gavage for 6 weeks. HFE/STZ rats became obese, insulin resistant, exhibited increased levels of fasting blood glucose(FBG), glycated hemoglobin(HbA1c), fasting insulin(FINS), free fat acids (FFA), total cholesterol (TC), triglyceride (TG), malondialdehyde (MDA) and reduced superoxide dismutase (SOD) activities. Treatment with TFLC or Na3VO4 showed a significant decrease in FBG, HbA1c, FFA, TC, TG, MDA, HOMA-IR and a significant increase in ISI and SOD activities. Pancreatic immunohistochemistry indicated that TFLC or VAN could ameliorate auantic islets. Morphological changes in tissues of model rats included inflammation, mitochondrial lesions, steatosis, vaculous degeneration and hydropic degeneration in liver, heart and kidney while changes attenuated in two treatment groups. Expression of PTP1B were elevated in target tissues of model rats, but decreased in TFLC or Na3VO4 treatment rats. When used in a new T2DM rat model, TFLC could have anti-inflammation, anti-oxidation, insulin-sensitizing and hypoglycemic activity possibly by decreasing the elevated expression of PTP1B in the target tissues of T2DM rats.