Cellular origin of hexokinase in pancreatic islets.
Transgenic or tumoral pancreatic islet beta cells with enhanced expression of low K(m) 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.
Schuit
F
F
Diabetes Research Center, Vrije Universiteit Brussel, Brussels, B-1090 Belgium. fschuit@minf.vub.ac.be
Moens
K
K
Heimberg
H
H
Pipeleers
D
D
D000818:Animals; D003847:Deoxyglucose / Q000494:pharmacology; D005434:Flow Cytometry; D015971:Gene Expression Regulation, Enzymologic; D005941:Glucokinase / Q000032:analysis; D005947:Glucose / Q000378:metabolism; D006593:Hexokinase / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D007328:Insulin / Q000378:metabolism; D007515:Islets of Langerhans / Q000201:enzymology*; D007527:Isoenzymes / Q000032:analysis / Q000378:metabolism; D007700:Kinetics; D008297:Male; D008356:Mannoheptulose / Q000494:pharmacology; D011384:Proinsulin / Q000096:biosynthesis; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction
D007328:Insulin; D007527:Isoenzymes; D012333:RNA, Messenger; D008356:Mannoheptulose; D011384:Proinsulin; D003847:Deoxyglucose; D006593:Hexokinase; D005941:Glucokinase; D005947:Glucose