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Enhanced cAMP Protein Kinase A Signaling Determines Improved Insulin Secretion in a Clonal Insulin-Producing ß-Cell Line (INS-1 832/13)

Departments of Cell and Molecular Biology (S.Y., U.F., L.F., L.S.H., H.M.), and Physiological Sciences (E.R.), Lund University, Lund SE-221 84, Sweden; and Sarah Stedman Center for Nutritional Studies and Duke Program in Diabetes Research (H.E.H.), Departments of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710

Address all correspondence and requests for reprints to: Hindrik Mulder, M.D., Ph.D, Department of Cell and Molecular Biology, Section for Molecular Signaling, Lund University, Biomedical Center C11, SE-221 84 Lund, Sweden. E-mail: hindrik.mulder{at}medkem.lu.se.

In type 2 diabetes, ß-cells become glucose unresponsive, contributing to hyperglycemia. To address this problem, we recently created clonal insulin-producing cell lines from the INS-1 insulinoma line, which exhibit glucose responsiveness ranging from poor to robust. Here, mechanisms that determine secretory performance were identified by functionally comparing glucoseresponsive 832/13 ß-cells with glucose-unresponsive 832/2 ß-cells. Thus, insulin secretion from 832/13 cells maximally rose 8-fold in response to glucose, whereas 832/2 cells responded only 1.5-fold. Insulin content in both lines was similar, indicating that differences in stimulus-secretion coupling account for the differential secretory performance. Forskolin or isobutylmethylxanthine markedly enhanced insulin secretion from 832/13 but not from 832/2 cells, suggesting that cAMP is essential for the enhanced secretory performance of 832/13 cells. Indeed, 8-bromoadenosine-3',5'-cyclic monophosphorothioate, rp-isomer (Rp-8-Br-cAMPS) an inhibitor of protein kinase A (PKA), inhibited insulin secretion in response to glucose with or without forskolin. Interestingly, whereas forskolin markedly increased cAMP in 832/2 cells, 832/13 cells exhibited only a marginal rise in cAMP. This suggests that 832/13 cells are more sensitive to cAMP. Indeed, the cAMP-induced exocytotic response in patch-clamped 832/13 cells was 2-fold greater than in 832/2 cells. Furthermore, immunoblotting revealed that expression of the catalytic subunit of PKA was 2-fold higher in 832/13 cells. Moreover, when the regulatory subunit of PKA was overexpressed in 832/13 cells, to reduce the level of unbound and catalytically active kinase, insulin secretion and PKA activity were blunted. Our findings show that cAMP-PKA signaling correlates with secretory performance in ß-cells.

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