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 β 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 ∼30% of islet cells was converted to a nonexpressing variant (gk del). This resulted in a heterogeneous population of β 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 β cells fromgk lox/lox mice. In AdenoCre-infected islets, approximately one-third of the β 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 ∼5 mmglucose. In contrast, the normal response cells showed a sigmoidal response with an K catS0.5 of ∼8 mm. These data provide direct evidence that GK is essential for glucose-stimulated metabolic responses in β cells within intact islets and that intercellular coupling within the islet plays little or no role in glucose-stimulated metabolic responses.

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