Adenylyl cyclase 8 is central to glucagon-like peptide 1 signalling and effects of chronically elevated glucose in rat and human pancreatic beta cells.
OBJECTIVE
Glucose and incretins regulate beta cell function, gene expression and insulin exocytosis via calcium and cAMP. Prolonged exposure to elevated glucose (also termed glucotoxicity) disturbs calcium homeostasis, but little is known about cAMP signalling. We therefore investigated long-term effects of glucose on this pathway with special regard to the incretin glucagon-like peptide 1 (GLP-1).


METHODS
We exposed INS-1E cells and rat or human islets to different levels of glucose for 3 days and determined functional responses in terms of second messengers (cAMP, Ca(2+)), transcription profiles, activation of cAMP-responsive element (CRE) and secretion by measuring membrane capacitance. Moreover, we modulated directly the abundance of a calcium-sensitive adenylyl cyclase (ADCY8) and GLP-1 receptor (GLP1R).


RESULTS
GLP-1- or forskolin-mediated increases in cytosolic calcium, cAMP-levels or insulin secretion were largely reduced in INS-1E cells cultured at elevated glucose (>5.5 mmol/l). Statistical analysis of transcription profiles identified cAMP pathways as major targets regulated by glucose. Quantitative PCR confirmed these findings and unravelled marked downregulation of the calcium-sensitive adenylyl cyclase ADCY8 also in rat and in human islets. Re-expression of ADCY8, but not of the GLP1R, recovered GLP-1 signalling in glucotoxicity in INS-1E cells and in rat islets. Moreover, knockdown of this adenylyl cyclase showed that GLP-1-induced cAMP generation, calcium signalling, activation of the downstream target CRE and direct amplification of exocytosis by cAMP-raising agents (evaluated by capacitance measurement) proceeds via ADCY8.


CONCLUSIONS
cAMP-mediated pathways are modelled by glucose, and downregulation of the calcium-sensitive ADCY8 plays a central role herein, including signalling via the GLP1R.
Université de Bordeaux 1, Institut Européen de Chimie et Biologie, UMR CNRS 5248, 2 Av Robert Escarpit, 33607 Pessac, France.
B
B
Roger
J
J
Papin
P
P
Vacher
M
M
Raoux
A
A
Mulot
M
M
Dubois
J
J
Kerr-Conte
B H
BH
Voy
F
F
Pattou
G
G
Charpentier
J-C
JC
Jonas
N
N
Moustaïd-Moussa
J
J
Lang
D000262:Adenylyl Cyclases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism*; D003592:Cytophotometry; D004594:Electrophysiology; D052216:Glucagon-Like Peptide 1 / Q000494:pharmacology*; D000067757:Glucagon-Like Peptide-1 Receptor; D005947:Glucose / Q000494:pharmacology*; D006801:Humans; D050417:Insulin-Secreting Cells / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism*; D008954:Models, Biological; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction; D051381:Rats; D018027:Receptors, Glucagon / Q000235:genetics / Q000378:metabolism
C000593833:GLP1R protein, human; C000593835:Glp1r protein, rat; D000067757:Glucagon-Like Peptide-1 Receptor; D018027:Receptors, Glucagon; D005576:Colforsin; D052216:Glucagon-Like Peptide 1; D000242:Cyclic AMP; D000262:Adenylyl Cyclases; C118292:adenylyl cyclase 8; D005947:Glucose; D002118:Calcium