Role Of Pten In The β-cell Defects Induced By Free Fatty Acid And The Protection Of Glp-1 Analogue Liraglutide

Obesity is a major risk factor in the development of type 2 diabetes and increases the risk of type 2 diabetes. The permanent elevation of plasma FFA is one of common characters of obesity. The elevation of plasma FFA can impairβ-cell function, inhibits glucose-stimulated insulin secretion (GSIS), decreasesβ-cell proliferation, inducesβ-cell apoptosis and decreasesβ-cell mass. These cause the development of type 2 diabetes. The prevention of lipotoxicity could represent a new strategy to preserve insulin secretion in type 2 diabetic patients.PTEN is a tumor suppressor gene encoding a phosphatase. Its mutations, deletions or dysregulation are found in many human tumors. Recent studies show that PTEN also plays an important role in theβ-cell disfunction. Akt is one of the major downstream targets of PTEN and is regulated by PI3K. And the insulin receptor substrate (insulin receptor 2/phosphoinositide 3-kinase [PI3K]) pathway plays a key role in regulatingβ-cell mass and function, while, PTEN can regulate the function ofβ-cell by inhibiting the PI3K pathway. In addition, PTEN, as a lipid phosphatase, dephosphorylates PIP₃ to PIP₂. PI₂ has been suggested to play an important role as an endogenous regulator of ATP-sensitive potassium (K_ATP) channels. PTEN may regulate insulin secretion by the activity of K_ATP channels regulated by PIP₂ .Liraglutide is a modified form of human GLP-1. GLP-1 plays an impotent role in protectingβ-cell. GLP-1 can stimulate glucose-induced insulin secretion, improveβ-cell function, promoteβ-cell proliferation and preventβ-cell apoptosis. These functions of GLP-1 have been reported to be associated with ATP-sensitive potassium (K_ATP) channels and Akt pathway. Therefore, Liraglutide may preventβ-cell dysfunction induced by FFA.Part 1 Role of PTEN in the FFA-inducedβcell apoptosisObjective: To explore the role of PTEN in the FFA-inducedβTC-3 cell apoptosis. Method: AfterβTC-3 cells were incubated with palmitic acid, cell viability was measured by MTT assay. the apoptotic cells were quanti?ed by ?ow cytometry and PTEN mRNA was detected by Real time PCR. Western blot was employed to observe the expression of PTEN, Akt and Active-Caspase3. Results: Exposure to the palmitic acid for 24 h resulted in significantly decreased viability and apoptosis ofβTC-3 cells. The result of western blot confirmed that palmitic acid decreased the expression of p-PTEN and p-Akt and increased the expression of Active-Caspase3. PTEN siRNA specifically suppressed PTEN expression and reversed the palmitic acid-induced inhibition of Akt phosphorylation and activation of Caspase3. Conclusion: Palmitic acid can induceβ-cell apoptosis by promoting PTEN dephosphorylation and increasing the activity of PTEN. PTEN plays an important role in FFA-inducedβ- cell apoptosis.Part 2 Effects of PTEN on ATP-sensitive potassium channels.Objective: To investigate the effect of PTEN on ATP-sensitive potassium channels. Methods: AfterβTC-3 cells were incubated with palmitic acid for 24 h, cells were incubated with glucose 2.8 mM or 22.4 mM for 1 h. The insulin level in the medium was determined by radioimmunoassy. The PIP2 level was detected by dot blot. Real time PCR was employed to observe the change of Kir6.2 and SUR1 mRNA. Cells were transfected with either Control siRNA or PTEN siRNA for 24 h and treated with palmitic acid for another 24 h, Kir6.2 and SUR1mRNA were analyzed by Real time PCR. Results: AfterβTC-3 cells were incubated with palmitic acid, GSIS, Kir6.2 and SUR1 mRNA level decreased and PIP₂ level increased in a palmitic acid concentration-dependent manner. PTEN siRNA enhanced the expression of Kir6.2 and SUR1 mRNA. Conclusion: PTEN involves in the activation of K_ATP by palmitic acid.Part 3 Protection of liraglutide in the FFA-inducedβcell defectObjective: To explore the role of GLP-1 analogue liraglutide in the FFA-inducedβcell defect. Methods:βTC-3 cells were treated with palmitic acid with or without liraglutide for 24 h. Kir6.2 mRNA were quanti?ed by Real time PCR. The expression of p-Akt, Akt and Active-Caspase3 were quanti?ed by Western blot. Results: Exposure ofβTC-3 cells to liraglutide caused an increase in the mRNA levels of Kir6.2. The result from western blot showed that liraglutide reversed the palmitic acid-induced inhibition of Akt phosphorylation and activation of Caspase3. Conclusion: Liraglutide can prevent FFA-inducedβcell defect by regulating the activity of K_ATP channels and Akt pathway.