Energetic requirement of carbachol-induced Ca2+ signaling in single mouse beta-cells.
Insulin secretion is under multifactorial control by glucose and neurohumoral factors like acetylcholine (ACH), which activate the Ca2+/phospholipase C signaling pathway. All insulin secretagogues elevate cytosolic free Ca2+ ([Ca2+]i) that is central to the stimulation of insulin secretion. The actions of ACH on [Ca2+]i are glucose dependent but the metabolic steps involved are only partly understood. Here we have characterized the metabolic steps by which glucose exerts its synergistic effects on ACH-linked Ca2+-signals. [Ca2+]i was measured in single fura-2 loaded beta-cells. The ACH analog carbachol (3 microM) caused rise in [Ca2+]i that was strongly dependent on the extracellular glucose concentration ranging from 0-10 mM. Iodoacetate, which blocks glycolysis, thereby preventing the generation of NADH and ATP from glucose metabolism, and rotenone or antimycin, which inhibit complex 1 and 2 of the mitochondrial respiratory chain, respectively, inhibited in glucose (6 mM) the carbachol-induced Ca2+ signal to a similar extent as glucose deprivation. This demonstrates that glucose metabolism and generation of ATP through oxidative phosphorylation of energy rich substrates like NADH and FADH2 are required for carbachol-induced Ca2+ signals. While sodium arsenate, which prevents net glycolytic production of ATP without inhibiting glycolysis, had no significant effect on the carbachol-induced Ca2+-signal, the mitochondrial pyruvate transport inhibitor alpha-cyano-4-hydroxycinnamate and the Krebs cycle inhibitor monofluoroacetate strongly suppressed the rise in [Ca2+]i elicited by carbachol. While pyruvate was ineffective, methyl pyruvate, a membrane-permeant pyruvate analog, and alpha-ketoisocaproate in combination with glutamine, which are both substrates for mitochondrial ATP production, could restore the carbachol-induced Ca2+ signal in glucose-free medium. These data demonstrate for the first time that Krebs cycle metabolism of glucose and ATP formation through oxidative phosphorylation is critical for the glucose dependency of ACH-linked Ca2+-signals in mouse beta-cells, and they suggest that mitochondrial metabolism plays a key role in the interactive regulation of beta-cells by neurohumoral factors activating the Ca2+/phospholipase C signaling pathway.
Schöfl
C
C
Abteilung Klinische Endokrinologie, Medizinische Hochschule Hannover, Germany. schoefl.christof@mh-hannover.de
Börger
J
J
Lange
S
S
von zur Mühlen
A
A
Brabant
G
G
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology*; D002478:Cells, Cultured; D018679:Cholinergic Agonists / Q000494:pharmacology*; D003470:Culture Media; D004734:Energy Metabolism; D005260:Female; D005947:Glucose / Q000378:metabolism / Q000494:pharmacology; D005973:Glutamine / Q000494:pharmacology; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000187:drug effects / Q000378:metabolism*; D007651:Keto Acids / Q000494:pharmacology; D051379:Mice; D009243:NAD / Q000378:metabolism; D011773:Pyruvates / Q000494:pharmacology; D015398:Signal Transduction; D010738:Type C Phospholipases / Q000378:metabolism
D018679:Cholinergic Agonists; D003470:Culture Media; D007328:Insulin; D007651:Keto Acids; D011773:Pyruvates; D005973:Glutamine; D009243:NAD; C104813:methyl pyruvate; C013082:alpha-ketoisocaproic acid; D000255:Adenosine Triphosphate; D002217:Carbachol; D010738:Type C Phospholipases; D005947:Glucose; D002118:Calcium