Cellular origin of hexokinase in pancreatic islets

Transgenic or tumoral pancreatic islet beta cells with enhanced expression of low IS, hexokinases (HK) exhibit a leftward shift of the normal dose-res ponse curve for glucose-induced insulin release. Furthermore, HK catalyzes roughly 50% of total glucose phosphorylation measured in extracts from fres hly isolated rodent islets, suggesting that HK participates in the process of glucose sensing in beta cells, We previously observed that HK activity r epresents 20% of total glucose phosphorylation in purified rat beta cell pr eparations and that HK is not homogenously distributed over these cells. Th e present study provides several arguments for the idea that HH detected in freshly isolated rat islets or islet cell preparations originates mainly f rom contaminating exocrine cells. First, reverse transcriptase-polymerase c hain reaction using isoform-specific primers allowed detection of hexokinas e I and IV mRNA in rat beta cells, whereas the messenger levels encoding th e hexokinase II and III isoforms were undetectably low, However, immunoblot s indicated that hexokinase I protein was 10-fold more abundant in freshly isolated islets and flow-sorted exocrine cells than in purified rat beta ce ll preparations. Second, comparison of HII activity in the different pancre atic cell types resulted in 15-25-foId higher values in exocrine than in en docrine cells (acinar cells: 21 +/- 3 pmol of glucose 6-phosphate formed/h/ ng of DNA; duct cells: 30 +/- 8 pmol/h/ng of DNA; islet beta cells: 1.2 +/- 0.2 pmol/h/ng DNA; alpha cells: 0.9 +/- 0.4 pmol/h/ng of DNA), Since fresh ly purified beta cell preparations contain 3 +/- 1% exocrine cells, at leas t 50% of their HK activity can be accounted for by exocrine contamination, Third, after 5 days of culture of purified islet beta cells, both HK activi ty and the proportion of exocrine cells decreased by more than 1 order of m agnitude, while the ratio of glucokinase over hexokinase activity increased more than 10-fold, Finally, preincubating the cells with 50 mmol/liter 2-d eoxyglucose did not affect glucose stimulation of insulin biosynthesis and release, In conclusion, the observation that pancreatic exocrine cells are responsible for a major part of HK activity in islet cell preparations caut ions against the use of HK measurements in islet extracts in the study of t hese enzymes in glucose sensing by pancreatic beta cells.