Anomeric Specificity of the Stimulatory Effect ofd-Glucose on d-Fructose Phosphorylation by Human Liver Glucokinase

Abstract

(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...

Comparative Effects of Fructose and Glucose on Lipogenic Gene Expression and Intermediary Metabolism in HepG2 Liver Cells

“…For instance, it was shown that HK2 and HK4 are capable of phosphorylating fructose directly to F-6-P: Katzen et al determined the K m for glucose and fructose of rat HK2 to be 1.4×10 −4 M and 3.0×10 −3 M, respectively [29], and Malaisse et al reported that 25% of hepatic fructose in rats is phosphorylated to F-6-P by HK4 [30]. Studies have also shown that glucose confers kinetic cooperativity to human liver and β-cell hexokinase toward fructose phosphorylation to F-6-P [31], [32]. Finally, it is possible that active fructose metabolism to triose-phosphates could increase the intracellular G-6-P pool, and hence promote F-6-P generation and HBP flux through attenuation of glucose catabolism and not as a direct F-6-P precursor.…”

Fructokinase activity in rat liver, ileum, parotid gland, pancreas, pancreatic islet, B and non-B islet cell homogenates

“…In terms of metabolism, attention was drawn to the participation of different enzymes to the phosphorylation of the ketohexose, including fructokinase (1), the low-Km hexokinase (2), and the high-Km glucokinase with emphasis on the glucose-induced cooperativity of this enzyme towards D-fructose (3,4) and the anomeric specificity of such a process (5,6). The possible participation of D-fructose 1-phosphate in the regulation of glucokinase activity at the intervention of its regulatory protein (7) was also examined (Iwashiga K, et al, Diabetologia 38 (Suppl 1): abs.…”

Determination of the anomeric configuration in carbohydrates by longitudinal cross‐correlated relaxation studies: application to mono‐ and disaccharides

“…Since then, many studies of the role of anomeric specificity in D-glucose metabolism in human pancreatic islets and other cell types have appeared which suggest that there is an anomer-specific glucose receptor present in the beta cells in humans. 3 Recently it was shown that the˛-anomer of 4 Similarly, other metabolic actions involving disaccharides, glyco-conjugates, and nucleosides may also be anomer specific. Consequently, precise determination of the anomeric configuration in solution is critical to a better understanding of the biological function and metabolism of mono/polysaccharides and carbohydrates.…”