Biological Screening Of Antidiabetic Drugs With Dual Action On Both PPARα And PPARγ

Nowadays, obesity and type 2 diabetes induced by obesity have become diseases which endanger human beings seriously. Many studies show that the obesity and type 2 diabetes are co-exiting and their relationships are rather complicated. About 85% of type 2 diabetics victims are combined with obesity, and obesity has also been believed as an important danger inducement of type 2 diabetes. Obesity has been demonstrated to be associated with lipid metabolic disorders, which commonly increase the risk of insulin resistance. Type 2 diabetes is characterized with insulin resistance, and in most cases with hyperlipidemias. Therefore, new effective therapies for adjusting the insulin level and lipid metabolism are required to control type 2 diabetes.Peroxisome proliferator-activated receptors(PPARs) are ligand-activated transcription factors, which belong to the super family of the nuclear receptor family. The PPARs have specific tissue distributions and play a pivotal role in regulating the expression of a large number of genes involved in glucose and lipid metabolism. It has been shown that PPARa expressed highly in the liver, and mainly regulates lipid metabolism, While PPARγwhich mainly distributes in adipose tissue and skeletal muscle regulates glucose metabolism and improves the sensitivity of the tissues to the insulin. PPARs therefore constitute targets for the development of dual agonists which are useful in the prevention and treatment of obesity and type 2 diabetes.In this paper, 85 novel compounds were screened to find dual PPARa/γagonists using several screening models in vivo and vitro. And the regulation mechanism of the compounds to glucose and lipid were investigated in the levels of PPAR transcriptional regulation.The first screening, pM-hPPARa, pM-hPPARγand pB4-RES-tk-luc plasmids were developed by PCR and DNA clonal technologies. And a drug screening model containing pM-hPPARa, pM-hPPARγand pB4-RES-tk-luc plasmids was established. Then the pM-hPPARa or pM-hPPARγand pB4-RES-tk-luc plasmids were transiently cotransfected into HEK-293 cells, the dual PPARa/γactivated effects of 85 novel compounds were determined by examing the luciferase activity. The results showed that 18 compounds were found to be potent activators for both PPARa and PPARγ.The second screening, the two assays, including decrease of blood lipid levels with PPARa activation and preadipocyte differentiation with PPARγactivation were used to confirme the dual PPARa/γactivated effects of 18 compounds. The results showed that the 10 compounds cound decrease the blood lipid levels of hyperlipidaemia mice and promote 3T3-L1 cells differentiation. The dose-effect relationship was ensured. The EC₅₀ of 10 compounds were evaluated with the dual-luciferase reporter gene assay. The agonistic effects of compounds had been found to bc more potent agonists of PPARa, while had similar PPART activation effect comparing to resiglitazone.To preliminary assessment the toxicity of the compounds in vivo and vitor, the inhibit growth of HLF cells, teratogenesis of rat embryo midbrain cells and acute toxicity of mice assays were performed. The results showed that the 1 compound had great toxicity of cells and teratogenesis, the toxicity of other 6 compounds were minor in vivo and vitro.The drug screening assays based on pharmacodynamie action were preformed for ensuring the effects of the compounds on decreasing blood glucose and lipid. The glucose tolerant ability assay showed that the compounds had not effect on blood glucose and tolerant ability in normal KM mice, which indicated theβcell of pancreatic islet was not the target of the compounds. In type 2 diabetic mice assays, the 10mg/kg compounds had a great effect to reduce blood glucose, FFA, TG and T-CHO levels, and increase HDL-C levels. The 3mg/kg compounds had also regulatory effects on blood glucose, TG and FFA levels, which indicated the compound on high dose could cure the type 2 diabetes and the compound on low dose could improve the lipid metabolic disorders to prevent insulin resistance and type 2 diabetes.The last, RT-PCR and western blot were used to investigated the tissue distributions of PPARa and PPARγ. The results showed the PPARa expressed mainly in human hepatic cells and mouse liver tissue, and PPARγexpressed in human hepatic cells and mouse adipocytes, skeletal muscular cells, liver tissue, adipose tissue, skeletal muscle. The further researchs showed the compounds could increase the expressions of ACO, LPL, aP2 and GluT4 mRNA, indicating that the compounds had great effects on lowering blood glucose and lipid in type 2 diabetic patients.In summary, the novel dual PPARa/γagonists had been demonstrated by several screening models in vitro and vivo. They could not only control glucose and lipid metabolism, but also promote preadipocyte differentiation and improve insulin resistance. These results suggested that further studies should be carried out to develop these compounds as a novel therapy for metabolic disease, such as obesity, hyperlipidemia and type 2 diabetes.