Adenovirus-mediated knockout of a conditional glucokinase gene in isolatedpancreatic 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 (gk(l ox)). Analysis of AdenoCre-treated islets indicated that the gk(lox) allele in similar to 30% of islet cells was converted to a nonexpressing variant (gk(del)). 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 metabo lic responses of individual islet beta cells from gk(lox/lox) mice. In Aden oCre-infected islets, approximately one-third of the beta cells showed mark edly lower NAD(P)H responses. These cells also exhibited glucose dose respo nses consistent with the loss of GK. Glucose dose responses of the low-resp onding cells were not sigmoidal and reached a maximum at similar to 5 mM gl ucose, In contrast, the normal response cells showed a sigmoidal response w ith an KcatS0.5 of similar to 8 mM. These data provide direct evidence that GK is essential for glucose-stimulated metabolic responses in beta cells w ithin intact islets and that intercellular coupling within the islet plays little or no role in glucose-stimulated metabolic responses.