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 (gk(lox)). 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 metabolic responses of individual islet beta cells from gk(lox/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 similar to 5 mM glucose, In contrast, the normal response cells showed a sigmoidal response with 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 within intact islets and that intercellular coupling within the islet plays little or no role in glucose-stimulated metabolic responses.