(2) analyzed the cooperativity of human B-cell glucokinase through such a stimulatory effect of D-glucose on D-fructose phosphorylation. They concluded that the effect of the aldohexose on D-fructose phosphorylation indeed reflects the positive cooperativity for D-glucose, as mediated by its binding to the catalytic site. Further experiments conducted in isolated rat pancreatic islets have documented that D-glucose also causes a concentration-related increase in the oxidation of D-[U-14 C]fructose (3). A comparable situation was observed in pancreatic islets prepared from either Goto-Kakizaki rats or adult rats that had been injected with streptozotocin during the neonatal period, i.e. in two animal models of non-insulin-dependent diabetes mellitus (4, 5).The major aim of the present study was to investigate whether the stimulatory effect of D-glucose on D-fructose phosphorylation by human liver glucokinase displays anomeric specificity. The effects of the two anomers of D-glucose upon14 C]fructose conversion to 14 CO 2 and 14 C-labeled acidic metabolites and upon the cationic and insulin secretory responses to D-fructose were also examined in isolated rat pancreatic islets. The experiments were conducted over 10 min of incubation at 25°C (D-fructose phosphorylation), 60 min of incubation at 4°C (D-fructose metabolism in islets) or with D-glucose anomers maintained for 90 min or less at 4°C (perifused islets) to minimize the interconversion of the glucose anomers (6). Under these conditions, the fraction of ␣-D-glucose converted to -D-glucose, expressed relative to the equilibrium value, is close to 5.4 and 9.0% after 60 and 90 min of incubation at 4°C and to 17.8% after 10 min incubation at 25°C (6). The mean value for the fractional conversion of each anomer during the incubation period used for the measurement of biological variables is close to only half of these percentages. C]glucose (PerkinElmer Life Sciences), and its purity assessed as previously reported (7). Recombinant liver glucokinase was kindly provided by Prof. E. Van Schaftingen (Université Catholique de Louvain, Brussels, Belgium).
EXPERIMENTAL PROCEDURES
Materials-D-Fructose
D-[U-14 C]Fructose Phosphorylation-The phosphorylation of D-fructose (10 mM; mixed with a tracer amount of D-[U-14 C]fructose) was conducted over 10 min of incubation at 25°C in a reaction mixture (0.1 ml) consisting of a Hepes-NaOH buffer (50 mM, pH 7.5) containing 6 mM MgCl 2 , 60 mM KCl, 10 mM KH 2 PO 4 , 0.2 mg/ml bovine serum albumin, 5 mM ATP, human liver glucokinase (about 25 g/ml or 0.3 units/ml) and, when required, freshly dissolved ␣-or -D-glucose. Labeled Dfructose 6-phosphate was then separated from its precursor by ion exchange chromatography (8). Blank values were measured in the absence of glucokinase.
Metabolism of D-[U-14 C]Fructose in Pancreatic Islets-Groups of 30 pancreatic islets each, prepared by the collagenase procedure from fed Wistar rats (9), were incubated for 60 min at 4°C in 0.1 ml of a Hepesand bicarbonate-buffered medium (10) containing b...