The Effect Of PPARδ On Lipid Metabolism And Insulin Release Of Pancreatic β Cells

Background and ObjectivePPAR, which contains three isotypes of PPARα, PPARδ(β) and PPARγ, belongs to the nuclear receptor superfamily. By now, the structure and function of PPARαand PPARγare clear, but as the lastly identified isotype, the function of PPARδremains obscure. PPARδis widly expressed in the islet, cardiac muscle, skeletal muscle, adipose tissue, skin and brain. In the cardiac muscle, skeletal muscle and adipose tissue, PPARδis an important regulator of fatty acid oxidation. The up-regulation of PPARδor activation of PPARδby its agonists could enhance the oxidation and utilization of fatty acids, so PPARδis believed to be the upper regulator of fatty acids oxidase and a potential target for the treatment of metabolic syndrome. By contrast, some specialist reported that PPARδpromoted cholesterol influx and lipid accumulation in macrophage, which is opposite to the result in other tissues. These results indicate that the role of PPARδin lipid metabolism may be tissue-specific.Impairment of isletβcells by 1ipotoxicity plays a central role during the development of type 2 diabetes mellitus, and the improvement of lipid metabolism may contribute to the prevention and treatment of metabolic syndrom. By now, there are much indirect data shows that PPARδmay play an important role in the regulation of lipid metabolism and insulin release inβcells. But we don't find any direct report of the function of PPARδinβcells. So we adopt the Adeasy system to get the adenovirus vector of PPARδ, the dominant negative mutant of PPARδ(PPARδ-DN) and PPARδ-shRNA, by which we could observe the effect of PPARδon the expression of fatty acids oxidase, TG concentration, insulin content and insulin-related genes. Finally we could get the effect of PPARδon lipid metabolism, insulin release of pancreaticβcells and their elementary mechanism.Methods1. The INS-1 cells were incubated in the media containing 3 mmol/L, 11 mmol/L and 20 mmol/L glucose for 24 hours, respectively, then the PPARδmRNA was detected by RT-PCR and the PPARδprotein level was determined by Western blot.2. The INS-1 cells were incubated in the media containing 0.125 mmol/L, 0.25 mmol/L and 0.5 mmol/L PA for 24 hours, respectively, then the PPARδmRNA was detected by RT-PCR and PPARδprotein level was determined by Western blot.3. The Adeasy system was used to construct the PPARδand PPARδdominant negative mutant (PPARδ-DN) adenovirus vector.4. The PPARδ-shRNA adenovirus vector was constructed after the PPARδsiRNA plasmid pGensil/PPARδwas constructed successfully.5. The INS-1 cells were infected by PPARδadenovirus, PPARδ-DN adenovirus and PPARδ-shRNA adenovirus, then the mRNA level of ACO, CPT1, FATP1 and LCAD was detected by RT-PCR. At the same time, the intracellular content of TG was determined.6. The INS-1 cells was infected by PPARδadenovirus, PPARδ-DN adenovirus and PPARδ-shRNA adenovirus, then the mRNA level of preproinsulin was detected by RT-PCR and the insulin content was determined by RIA.7. The INS-1 cells was infected by PPARδadenovirus, PPARδ-DN adenovirus and PPARδ-shRNA adenovirus, then the mRNA level of GLUT2, PDK4 and UCP-2 was detected by RT-PCR.Results1. The expression of PPARδmRNA and protein were both down-regulated by glucose.2. The expression of PPARδmRNA and protein were both up-regulated by PA.3. The PPARδand PPARδ-DN adenovirus vectors were constructed successfully.4. The pGensil/PPARδ-shRNA was constructed successfully, and then the PPARδ-shRNA adenovirus vector was constructed.5. The mRNA level of ACO, CPT1, FATP1 and LCAD was higher in INS-1 cells infected by PPARδadenovirus than that in the controls. And the level of them was much lower in INS-1 cells infected by PPARδ-DN and PPARδ-shRNA adenovirus. By contrast, the TG content in INS-1 cells infected by PPARδwas much lower than the controls and opposite results were observed in INS-1 cells infected by PPARδ-DN and PPARδ-shRNA adenovirus.6. In INS-1 cells infected by PPARδadenovirus, the insulin content and the mRNA level of preproinsulin were lower than the contols. By contrast, the insulin content and the mRNA level of preproinsulin were much higher than the contols in INS-1 cells infected by PPARδ-DN and PPARδ-shRNA adenovirus.7. PPARδadenovirus upregulated the mRNA level of PDK4, UCP-2 and down-regulated the expression of GLUT2. But the PPARδ-DN and PPARδ-shRNA adenovirus down-regulated the mRNA level of PDK4, UCP-2 and up-regulated the expression of GLUT2.Conclusion1. The glucose is a negative regulator and fatty acids is a positive regulator of the PPARδexpression in INS-1 cells, which suggest that PPARδmay play an important role in lipid and glucose metabolism of pancreaticβcells.2. PPARδcould enhance the fatty acids oxidation and alleviate the lipid deposition in INS-1 cells, which contribute to lighten the 1ipotoxicity of INS-1 cells.3. PPARδcould depress insulin release of INS-1 cells, and its mechanism may involve the upregulation of PDK4,UCP-2 and down-regulation of GLUT2.4. The effect of PPARδon islet may be complicated: First, PPARδenhances the fatty acids oxidation and alleviates the lipid deposition, whichi may lighten the 1ipotoxicity-induced apoptosis. Second, PPARδabate the hyperinsulinemia in condition of hyperlipemia to prevent exhausting insulin release capacity. Third, the attenuation of insulin release by PPARδmay aggravate the glucotoxicity on islet. So the prolonged effect of PPARδonβcells need to be studied in the future.