Adenovirus-mediated knockout of a conditional glucokinase gene in isolated pancreatic islets reveals an essential role for proximal metabolic coupling events in glucose-stimulated insulin secretion.
The relationship between glucokinase (GK) and glucose-stimulated metabolism, and the potential for metabolic coupling between beta cells, was examined in isolated mouse islets by using a recombinant adenovirus that expresses Cre recombinase (AdenoCre) to inactivate a conditional GK gene allele (gklox). Analysis of AdenoCre-treated islets indicated that the gklox allele in approximately 30% of islet cells was converted to a nonexpressing variant (gkdel). This resulted in a heterogeneous population of beta cells where GK was absent in some cells. Quantitative two-photon excitation imaging of NAD(P)H autofluorescence was then used to measure glucose-stimulated metabolic responses of individual islet beta cells from gklox/lox mice. In AdenoCre-infected islets, approximately one-third of the beta cells showed markedly lower NAD(P)H responses. These cells also exhibited glucose dose responses consistent with the loss of GK. Glucose dose responses of the low-responding cells were not sigmoidal and reached a maximum at approximately 5 mM glucose. In contrast, the normal response cells showed a sigmoidal response with an KcatS0.5 of approximately 8 mM. These data provide direct evidence that GK is essential for glucose-stimulated metabolic responses in beta cells within intact islets and that intercellular coupling within the islet plays little or no role in glucose-stimulated metabolic responses.
Piston
D W
DW
Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37232, USA. dave.piston@mcmail.vanderbilt.edu
Knobel
S M
SM
Postic
C
C
Shelton
K D
KD
Magnuson
M A
MA
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D005941:Glucokinase / Q000235:genetics* / Q000378:metabolism; D005947:Glucose / Q000494:pharmacology*; D066298:In Vitro Techniques; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009249:NADP / Q000378:metabolism
D007328:Insulin; D009249:NADP; D005941:Glucokinase; D005947:Glucose