Enhanced expression of hexokinase I in pancreatic islets induced by sucrose administration.

Administration of a sucrose-rich diet (SRD) to normal hamsters induces an insulin-resistant state and a significant increase of insulin secretion and beta-cell mass. Islets isolated from these animals had a marked increase in glucose metabolism and glucose-induced insulin secretion, at both low and high glucose concentrations. They also presented increased hexokinase (HK) activity, without measurable changes in glucokinase (GK) activity. In this study we measured HK and GK activity in homogenates of islets isolated from normal control and SRD-fed hamsters, as well as in their particulate and cytosolic fractions. We also measured transcription rate (mRNA by reverse transcriptase PCR) and expression levels (Western blotting) of both enzymes in these islets. We found an increase in HK activity and expression levels, without measurable changes in HK mRNA level in SRD-fed animals. Whereas a similar GK activity was measured in homogenates of islets isolated from both groups, such activity was significantly higher in the cytosolic fraction of SRD islets. On the other hand, GK transcription rate and expression level were similar in both experimental groups. Our results suggest that the increased beta-cell secretory response to low glucose can be partly ascribed to an increased activity of islet HK consecutive to an enhanced expression of the enzyme, while the enhanced response to high glucose could be due to changes in GK compartmentalization.

Changes induced by sucrose administration on glucose metabolism in pancreatic islets in normal hamsters.

We correlated the changes in glucose-induced insulin secretion with those observed in glucose metabolism and hexokinase/glucokinase activity in islets from normal sucrose-fed hamsters. Blood glucose and insulin levels were measured in normal male hamsters fed with (S5) or without (C5) 10% sucrose in the drinking water for 5 weeks. Isolated islets (collagenase digestion) from both groups of animals were used to study insulin secretion, (14)CO(2) and (3)H(2)O production from D-[U-(14)C]-glucose and D-[5-(3)H]-glucose respectively, with 3.3 or 16.7 mM glucose in the medium, and hexokinase/glucokinase activity (fluorometric assay) in islet homogenates. Whereas S5 and C5 animals had comparable normal blood glucose levels, S5 showed higher insulin levels than C5 hamsters (2.3+/-0.1 vs 0.6+/-0.03 ng/ml, P<0.001). Islets from S5 hamsters released significantly more insulin than C5 islets in the presence of low and high glucose (3.3 mM glucose: 0.77+/-0.04 vs 0.20+/-0.06 pg/ng DNA/min, P<0.001; 16.7 mM glucose: 2.77+/-0.12 vs 0.85+/-0.06 pg/ng DNA/min, P<0.001) and produced significantly higher amounts of (14)CO(2) and (3)H(2)O at both glucose concentrations ((14)CO(2): 3.3 mM glucose: 0.27+/-0.01 vs 0.18+/-0.01, P<0.001; 16.7 mM glucose: 1.44+/-0.15 vs 0.96+/-0.08, P<0.02; (3)H(2)O: 3.3 mM glucose: 0.31+/-0.02 vs 0.15+/-0.01, P<0.001; 16.7 mM glucose: 1.46+/-0.20 vs 0.76+/-0.05 pmol glucose/ng DNA/min, P<0.005). The hexokinase K(m) and V(max) values from S5 animals were significantly higher than those from C5 ones (K(m): 100.14+/-7.01 vs 59.90+/- 3.95 microM, P<0.001; V(max): 0.010+/-0.0005 vs 0.008+/- 0.0006 pmol glucose/ng DNA/min, P<0.02). Conversely, the glucokinase K(m) value from S5 animals was significantly lower than in C5 animals (K(m): 15.31+/-2.64 vs 35.01+/-1.65 mM, P<0.001), whereas V(max) figures were within a comparable range in both groups (V(max): 0.048+/-0.009 vs 0.094+/-0.035 pmol glucose/ng DNA/min, not significant). The glucose phosphorylation ratio measured at 1 and 100 mM (hexokinase/glucokinase ratio) was significantly higher in S5 (0.26+/-0.02) than in C5 animals (0.11+/-0.01, P<0.005), and it was attributable to an increase in the hexokinase activity in S5 animals. In conclusion, sucrose administration increased the hexokinase/glucokinase activity ratio in the islets, which would condition the increase in glucose metabolism by beta-cells, and in beta-cell sensitivity and responsiveness to glucose. These results support the concept that increased hexokinase rather than glucokinase activity causes the beta-cell hypersensitivity to glucose, hexokinase being metabolically more active than glucokinase to up-regulate beta-cell function.

Possible relationship between the B-cell threshold for glucose-induced insulin secretion and blood glucose concentrations in the normal toad.

The aim of this work was to gain information on the possible relationship between basal glycemia in the toad and the B-cell threshold for glucose-induced insulin release. Hence, pieces of pancreas from Bufo arenarum were incubated with 2 to 20 mM glucose or preincubated with 2 mM glucose plus the hexokinase and glucokinase inhibitors (50 mM of 2-deoxyglucose and mannoheptulose, respectively) followed by an incubation with different glucose concentrations. The maximal rate of insulin release occurred at 8 mM glucose, while 50% of the release (K(s50)) was observed at 7 mM glucose. Regardless of the glucose concentration employed, pancreas pieces preincubated with 2-deoxyglucose released less insulin than the corresponding controls. On the other hand, mannoheptulose significantly inhibited the release of insulin at high glucose concentrations, having no effect at low glucose concentrations. The blocking effect of these two inhibitors is the first indirect evidence of the existence of the hexokinase/glucokinase enzymic system in the toad pancreas. Since the activity ratio of this system determines the glucose sensitivity of the insulin secretory mechanism, it is concluded that the possible existence of a higher ratio of these enzymes in toad B cells could explain the particular characteristics of glucose sensitivity in this animal, which in turn may explain its low blood glucose concentration.