Enhanced cAMP protein kinase A signaling determines improved insulin secretion in a clonal insulin-producing beta-cell line (INS-1 832/13).
In type 2 diabetes, beta-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 glucose-responsive 832/13 beta-cells with glucose-unresponsive 832/2 beta-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 beta-cells.
Yang
Shumin
S
Department of Cell and Molecular Biology, Lund University, SE-221 84 Lund, Sweden.
Fransson
Ulrika
U
Fagerhus
Lillian
L
Holst
Lena Stenson
LS
Hohmeier
Hans E
HE
Renström
Erik
E
Mulder
Hindrik
H
D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000502:physiology; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000502:physiology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000032:analysis / Q000378:metabolism / Q000502:physiology*; D025802:Electric Capacitance; D005089:Exocytosis / Q000502:physiology; D005947:Glucose / Q000494:pharmacology / Q000502:physiology; D006801:Humans; D007328:Insulin / Q000032:analysis / Q000378:metabolism*; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D015398:Signal Transduction
D007328:Insulin; D005576:Colforsin; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D005947:Glucose