The role of molecular energy sensors and metabolic changes in pancreatic beta cell differentiation

The aim of this thesis study is to evaluate the effects of metformin, which is commonly used in the treatment of insulin resistance in clinical practice, with natural sugar glucose or artificial sweetener aspartame on beta cell differentiation from pancreatic islet-derived progenitor cells (PID-PC) in vitro and to determine the possible molecular mechanisms. The signaling pathway associated with the differentiation of PID-PCs into beta cells in the presence of glucose and/or aspartame and/or metformin might be sweet taste receptor-stimulated, molecular energy sensors interacted and also eliminates the Warburg effect, which is known to be important in maintaining stem cell characters. We have focused our study on mitochondrial metabolic changes based on this idea. For the determination of beta cell differentiation, insulin and reactive oxygen species (ROS) producing cells were determined by flow cytometry. Insulin-producing cells were also immunohistochemically labeled. In order to demonstrate the role of GLUT1 in this differentiation mechanism, a spesific GLUT1 inhibitor WZB117 was applied to the PID-PCs. Expression levels of NGN3, PAX4, NKX2.2, PDX1, NKX6.1, INS2, GLUT2, Hexokinase and lactate dehydrogenase A genes were demonstrated by the reverse transcriptase-polymerase chain reaction (RT-PCR) method. Lactate, NAD+/NADH, NADP+/NADPH and glucose uptake levels were measured by colorimetric methods. Mitochondrial mass was determined fluorometrically with mitotracker green staining. Furthermore, GLUT1, MDH2, UCP2, Citrate synthase, T1R2, T1R3, PLCβ2, CAMKK2, LKB1, p-AMPK (Thr172), p-Akt (Th308)/(S473), Akt, p-mTOR (Ser2448) and FOXO1 levels were measured by western blotting technique. Changes in intracellular Ca2+ levels were determined by fluorometric method and the cells were visualized by a fluorescence microscope. The findings and results we have obtained as a result of all these procedures and findings are listed below: Aspartame and aspartame + glucose administration causes statistically significant beta cell differentiation, and metformin alone or in combination with other stimuli provides a higher rate of beta cell differentiation. The fact that GLUT1 inhibition supress beta cell differentiation in the groups metformin+aspartame and metformin+aspartame+glucose indicates that GLUT1 plays an important role in beta cell differentiation in the presence of aspartame and metformin. During beta cell differentiation, there is a metabolic reprogramming that takes place from anaerobic glycolysis to oxidative phosphorylation. The observation of a metabolic profile closer to the control in the groups especially glucose, aspartame and aspartame+glucose suggests that PID-PCs may be more numerous in these groups besides differentiated cells. Although the increase in mitochondrial mass was observed in all metformin treated groups, this increase was statistically significant only in the groups glucose+metformin and aspartame+glucose+metformin. The most significant increase occurred in the group aspartame+glucose+metformin and UCP2 levels were in parallel with the mitochondrial mass. Preservation of the levels of T1R2 subunit in the groups except of metformin+aspartame+glucose suggests that the signal is predominantly detected by this subunit. Although the cytoplasmic Ca+2 level in the group aspartame was lower compared to the control group, the Ca2+-induced CAMKK2 increased significantly. This finding suggested that cytoplasmic Ca2+ concentration was enough to improve the sweet taste signal. In this group, maintaining the PLCβ2 level is another indication that the sweet taste signal is not completely suppressed. The most significant change in metformin-treated groups is the marked decrease in cytoplasmic PLCβ2 and Ca2+ levels. However, increasing AMPK activation through CAMKK2 is noteworthy in only metformin-treated group. It is possible that the preserved actived AMPK levels and increased mTOR activation may occur through high glucose in the group glucose+metformin. FOXO1 levels increase in the groups metformin and metformin+glucose this indicates antioxidant defense.