P Wei1, M Shi, S Barnum, H Cho, T Carlson, J D Fraser. 1. Diabetes Biology Department, Pfizer Global Research and Development, La Jolla Laboratories, CA 92121, USA. ping.wei@pfizer.com
Abstract
AIMS/HYPOTHESIS: Glucokinase (GK), an enzyme that phosphorylates glucose to form glucose 6-phosphate, serves as the glucose sensor that regulates insulin secretion in beta cells. GK activators (GKAs) activate GK via binding to an allosteric site of the enzyme. GKAs increase glucose-stimulated insulin secretion and decrease blood glucose levels. Using the differentiated beta cell line INS-1, we investigated the role of GKAs in promoting beta cell growth and survival and preventing beta cell apoptosis induced by chronic exposure to high levels of glucose. METHODS: Proliferation was assessed using BrdU incorporation. Apoptosis was measured using caspase-3 activity. Immunoblot analysis was used to detect protein levels and the degree of phosphorylation. RESULTS: The GK agonists GKA50 and LY2121260 increased both cell replication and cell numbers when tested at basal levels of glucose (3 mmol/l) in INS-1 cells. GKAs promoted INS-1 cell proliferation via upregulation of insulin receptor substrate-2 and subsequent activation of protein kinase B phosphorylation. GKA50 also prevented the INS-1 cell apoptosis that was induced by chronic high glucose conditions, probably via an increase in GK protein levels and normalisation of the apoptotic protein BCL2-associated agonist of cell death (BAD) and its phosphorylation. As a result of the reduction in cell apoptosis, GKA50 prevented cell loss and maintained glucose-stimulated insulin secretion. In addition, the anti-apoptotic activity of GKA50 was significantly abrogated by other GKAs that do not inhibit apoptosis, suggesting that direct binding of GKA50 to GK is essential for its anti-apoptotic effect. CONCLUSION/ INTERPRETATION: Our results suggest novel roles of GKAs in promoting beta cell growth and preventing chronic-hyperglycaemia-induced beta cell apoptosis. Thus, GKAs may provide novel therapeutics that increase beta cell mass to maintain euglycaemia in diabetes.
AIMS/HYPOTHESIS: Glucokinase (GK), an enzyme that phosphorylates glucose to form glucose 6-phosphate, serves as the glucose sensor that regulates insulin secretion in beta cells. GK activators (GKAs) activate GK via binding to an allosteric site of the enzyme. GKAs increase glucose-stimulated insulin secretion and decrease blood glucose levels. Using the differentiated beta cell line INS-1, we investigated the role of GKAs in promoting beta cell growth and survival and preventing beta cell apoptosis induced by chronic exposure to high levels of glucose. METHODS: Proliferation was assessed using BrdU incorporation. Apoptosis was measured using caspase-3 activity. Immunoblot analysis was used to detect protein levels and the degree of phosphorylation. RESULTS: The GK agonists GKA50 and LY2121260 increased both cell replication and cell numbers when tested at basal levels of glucose (3 mmol/l) in INS-1 cells. GKAs promoted INS-1 cell proliferation via upregulation of insulin receptor substrate-2 and subsequent activation of protein kinase B phosphorylation. GKA50 also prevented the INS-1 cell apoptosis that was induced by chronic high glucose conditions, probably via an increase in GK protein levels and normalisation of the apoptotic protein BCL2-associated agonist of cell death (BAD) and its phosphorylation. As a result of the reduction in cell apoptosis, GKA50 prevented cell loss and maintained glucose-stimulated insulin secretion. In addition, the anti-apoptotic activity of GKA50 was significantly abrogated by other GKAs that do not inhibit apoptosis, suggesting that direct binding of GKA50 to GK is essential for its anti-apoptotic effect. CONCLUSION/ INTERPRETATION: Our results suggest novel roles of GKAs in promoting beta cell growth and preventing chronic-hyperglycaemia-induced beta cell apoptosis. Thus, GKAs may provide novel therapeutics that increase beta cell mass to maintain euglycaemia in diabetes.
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