| Peroxisome proliferator activated receptors (PPARs) are transcriptional factors belonging to the ligand-activated nuclear receptor superfamily. Three isoforms of PPARs, encoded by different genes, have been identified such as PPARα, PPARβand PPARγ. All of the PPAR subfamily members heterodimerize with the receptor for 9-cis retinoic acid (RXR) and bind to target gene peroxisome proliferator elements (PPREs), a direct repeat of the sequence AGGTCA separated by one nucleotide (DR-1). From their discovery on, PPARs have being served as potential therapeutic targets for treating the metabolic syndrome and its related comlications.The fibrate class of hypolipidemic drugs acts through PPARαand the thiazolidinedione class of anti-diabetic agents acts through PPARγ, both of them have been used in clinic for many years. PPARγare involved in adipocyte proliferation and differentiation and its agonists improve insulin sensitivity by promoting fatty acid storage and inhibiting adipokine synthesis. The PPARγagonists are shown to increase adipogenesis and body weight, whereas PPARαagonists counteract these effects by decreasing food intake and fat deposits in animals. It was suggest that the side effect of the body weight increase accompanied by PPARγagonists may not be associated with PPARα/γdual agonists. Hence, the dual agonists with proper selectivities for both PPARα/γis now the important trend of the research and development of new drugs.The mechanism by which PAPRγdown-regulates gluconeogenesis in liver is still unclear. Since PPARγis a transcriptional activator,it requires a mediator to decrease the transcription of gluconeogenic genes. Recently, one molecule which was called Small heterodimer partner(SHP) has been shown to mediate the bile acid dependent down regulation of gluconeogenic gene expression in liver. This led us to explore the possibility that SHP may mediate the antigluconeogenic effect of PPARγ.According to the action mechanism of PPARs, we intend to establish a screening system based on the engineered cells for identifying the agonists from synthesized compounds, which activate both PPARαandγsubtypes. In addition, the molecular mechanism of pioglitazone on regulating liver gluconeogenesis with a rat model of typeⅡdiabetes was explored.The main methods and results are summarized as the following:Firstly, a report gene plasmid containing PPRE sequence was reconstructed. With the correponding primers, the segment of sequence containing PPRE from the promoter of acyl-CoA oxidase (ACO) gene was amplified by PCR, and then subcloned into pGL3 basic vector. Using PCR and squensing, the recombinant vector pGL3-PPRE was correctly identified.Secondly, a screening model based on HepG2 cells for PPARα/γdual agonists was established. In the frist step, the proper cell line was choosed for transfection among CHO, HeK293 and HepG2 cells according to their mRNA expression of PPARs.It was showed that HepG2 is the best, with the low mRNA expression of PPARs, highly efficient transfection and easily passenging. The recombnint pGL3-PPRE were transiently transfected into HepG2 cells with or without the presence of pSG5-PPARαor pSG5-PPARγindividual. Luciferase activities were measured using dual-luciferase reporter assay reagent according to the instruction in the kit. The results indicated the luciferase expression markedly increased in the co-transfected group than that in the non co-transfected group. With the stimulation of fenobrate or pioglitazone, the luciferase expression was also markedly increased in a concentration–dependent manner in both the conditions of co-transfection with and without pSG5-PPAR, as compared with the vehicle group. In addition, the transfected cells were tested with MK86 or GW9662, the specific antagonist for PPARαor PPARγrespectively. It was showed that the screening system is reliable and sensitive to reflect the activation of PPARs.Thirdly, the dual agonists for PPARαand PPARγwas screened among serial synthetic compounds using the above transfected cells. When HepG2 cells reached the exponential growth period, pGL3-PPRE and pRL-TK were co-transfected with pSG5-PPARαor pSG5-PPARγinto HepG2 cell according to the manufacture's protocol. Meanwhile, the cells not tobe cotransfected with pSG5-PPARαor pSG5-PPARγwere served as the control. The cells were separately stimulated for 24 hrs with five synthesized chemical compounds (LTD-1, 3, 4, 6 and 7) at different concentrations. Then the fluorescent potencies of the stimulated cells were measured with Veritas microplate luminometer. It was indicated that LTD-6 and LTD-7 could activate both of PPARαand PPARγ, of which LTD-7 was the most potent, LTD-4 could only activate PPARα,LTD-1 could only activate PPARγand LTD-3 could not activate either PPARαor PPARγ.Lastly, the molecular mechanism of pioglitazone on its anti-gluconeogenesis was investigated in a rat model with typeⅡdebetes.The idiopathic typeⅡdiabetes mellitus model of GK rats was used in the experiment. The rats were randomly divided into 3 groups: the model control group and pioglitazone treated groups with high(10mg/kg/d) and low(5mg/kg/d) doses. Meanwhile, Wistar rats are used as normal control group. The dosing period of the rats by intragastric administration was two weeks,once a day. The blood glucose and body weight were dynamicly measured before and duringthe dosing. At the next day of the last dosing day, the carbohydrate tolerance of the rats was tested, and the mRNA expression of PPARγ, SHP, glucose-6-phosphatase(G6P),fructose-1,6-bisphosphatase(FBP1),phosphor-enolpyruvate caboxykinase(PEPCK) was measured by RT-PCR in the liver. It was demonstrated that pioglitazone excerted its hypoglycemic activity and improvement on the carbohydrate tolerance accompanied by the body weight increasing. Furthermore, higher mRNA levels of PPARγand SHP, but lower G6P, FBP1, PEPCK in liver tissue were detected in the pioglitazone-treated rats.In conclusion, by the screening with the transfected cells, LTD-6, 7 were showed to posses the dual transactivation on PPARαand PPARγ. Furthermore, LTD-7 is the best candidate for PPARαand PPARγdual agonist. Pioglitazone has marked hypoglycemic activity, and can also improve the GK rats'carbohydrate tolerance, but with apparent weight increase. SHP was suggested to be involved in the acute antigluconeogenic effect of PPARγ. |
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