Saudi Cultural Missions Theses & Dissertations
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Item Restricted Coordinated Oscillations In Glucose-Stimulated Insulin Secretion And Protein Phosphorylation In Clonal Pancreatic Β-Cells: Exploring Metabolic Control Of Exocytosis(2023) Alnuwaiser, Mohammad; Deeney, Jude T; Torentheim, Keith; Corkey, Barbara E.Introduction: Diabetes Mellitus affects 415 million people worldwide. It causes hyperglycemia due to impaired insulin production or action. It has been known for a long time that insulin secretion oscillates in vivo and in vitro. These oscillations in insulin release are impaired in diabetic patients. Oscillations in insulin secretion are driven by oscillations in metabolic coupling factors including the ATP/ADP ratio and intracellular Ca2+. The Aim of this thesis is to determine whether phosphorylation of proteins regulating β-cell lipid metabolism correlates with oscillations in insulin secretion. Methods: INS-1 cells were cultured in 4 and 11 mM glucose in 48-well plates. Insulin secretion was initiated with 12 mM glucose at timed intervals to generate an oscillation profile over 22 min. Media was collected and insulin was assayed by fluorescence based HTRF insulin assay. Cell protein was extracted with SDS-PAGE sample buffer, separated by electrophoresis and transferred to PVDF membrane for western blotting after SDS PAGE electophoresis. Phosphorylated and unphosphorylated acetyl-CoA carboxylase (ACC) and AMP-activated protein kinase (AMPK) were detected with specific rabbit antibodies (Cell Signaling). Protein bands were detected on a GE LAS-4000 gel imager using enhanced chemiluminescence. Bands were analyzed using ImageJ software (Schneider, Rasband and Eliceiri 2012). Results: Insulin oscillations were detected over the 22 min time course with at least three resolved peaks of insulin secretion for cells cultured in either 4 or 11 mM glucose. The oscillations were of a 5 min period under both culture conditions while the amplitude was 10-20 fold higher in 4 mM glucose cells. The amplitude was dependent on the insulin content of the cells such that when normalized to insulin content the average insulin secretion was well matched between the high and low glucose conditions. Oscillations in pACC/ACC and pAMPK/AMPK ratios were detected in cells cultured in both 4 mM and 11 mM glucose. In cells cultured at 4 mM glucose the pACC/ACC ratio oscillated with a similar period to insulin but was slightly left shifted such that pACC peaked before insulin. This correlation was not as strictly adhered to in cells cultured at high glucose. Oscillations in pAMPK/AMPK tracked well with those of pACC/ACC in cells cultured at both 4 and 11 mM. pAMPK/AMPK peaks were left-shifted relative to peaks in insulin secretion in cells cultured at 4 mM glucose while they seemed to be coincident with insulin peaks in cells cultured at 11 mM glucose. Conclusion: Oscillations in insulin secretion are accompanied by oscillations in ACC and AMPK phosphorylation to regulate lipid signals that amplify normal glucose-stimulated insulin secretion. Chronic excess nutrients may alter changes in ACC and AMPK phosphorylation resulting in impaired oscillations in insulin secretion. Regulation of lipid signals in the pancreatic β-cell may provide therapeutic benefit in the treatment of hyperinsulinemia, insulin resistance and Type 2 diabetes.10 0