LAUSR

Purpose Despite emerging evidence suggesting requisite roles for protein kinases in islet beta-cell function, protein phosphatases remain an under studied area in islet biology. The PP2A, which accounts for 80% of total serine/threonine phosphatases, has been implicated in many facets of cell regulation including proliferation, survival, and apoptosis. We recently reported hyperactivation of PP2A in islet beta-cells following exposure to high glucose (metabolic stress) conditions, and proposed that hyperactivation of PP2A could lead to dephosphorylation of key proteins necessary for islet function. Herein, we studied putative regulatory roles for alpha4, a non-canonical adaptor regulatory subunit of PP2A, in the sustained activation of PP2A under metabolic stress. Methods Pancreatic islets were isolated by the collagenase digestion method. Human islets were from Prodo Labs. INS-1 832/13 cells, rat and human islets were cultured under basal (2.5 mM glucose) or metabolic stress (20-30 mM glucose) conditions for 24-48 hrs. PP2A activity was determined by immunoprecipitation phosphatase assay. Degree of cell death was detected by Annexin V/Propidium fluorescence staining. Results Metabolic stress significantly increased PP2A activity in INS-1 832/13 cells (~ 3 fold), rodent islets (~ 1.8 fold) and human islets (~ 2.2 fold). Sustained activation of PP2A was also seen in islets derived from pre-diabetic (7 weeks; ~1.4 fold) and diabetic (13 weeks; ~ 2 fold) ZDF rats. Western blot analysis indicated that alpha4 is expressed in INS-1 832/13 cells, rat islets and human islets. Metabolic stress also increased the expression of alpha4 in INS-1 cells and human islets. siRNA-mediated suppression of alpha4 expression markedly (-60%) inhibited high glucose-induced PP2A activity and cell death in INS-1 832/13 cells. Conclusions Alpha4 promotes PP2A activation and dysfunction of pancreatic beta-cells under high glucose-exposure conditions. We conclude that alpha4 could represent a novel target to impede PP2A-derived signaling events leading to beta-cell demise under metabolic stress.