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J Biol Chem, Vol. 274, Issue 46, 32803-32809, November 12, 1999
From the Diabetes Research Center, Vrije Universiteit Brussel,
Brussels, B-1090 Belgium
Transgenic or tumoral pancreatic
islet beta cells with enhanced expression of low Km
hexokinases (HK) exhibit a leftward shift of the normal dose-response
curve for glucose-induced insulin release. Furthermore, HK catalyzes
roughly 50% of total glucose phosphorylation measured in extracts from
freshly isolated rodent islets, suggesting that HK participates in the
process of glucose sensing in beta cells. We previously observed that
HK activity represents 20% of total glucose phosphorylation in
purified rat beta cell preparations and that HK is not homogenously
distributed over these cells. The present study provides several
arguments for the idea that HK detected in freshly isolated rat islets
or islet cell preparations originates mainly from contaminating
exocrine cells. First, reverse transcriptase-polymerase chain reaction using isoform-specific primers allowed detection of hexokinase I and IV
mRNA in rat beta cells, whereas the messenger levels encoding the
hexokinase II and III isoforms were undetectably low. However,
immunoblots indicated that hexokinase I protein was 10-fold more
abundant in freshly isolated islets and flow-sorted exocrine cells than
in purified rat beta cell preparations. Second, comparison of HK
activity in the different pancreatic cell types resulted in 15-25-fold
higher values in exocrine than in endocrine 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
freshly purified beta cell preparations contain 3 ± 1% exocrine cells, at least 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 activity and the proportion of exocrine cells
decreased by more than 1 order of magnitude, while the ratio of
glucokinase over hexokinase activity increased more than 10-fold. Finally, preincubating the cells with 50 mmol/liter 2-deoxyglucose 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
cautions against the use of HK measurements in islet extracts in the
study of these enzymes in glucose sensing by pancreatic beta cells.
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