Stimulation of glucose phosphorylation by fructose in isolated rat hepatocytes (original) (raw)
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Fructose 1-phosphate and the regulation of glucokinase activity in isolated hepatocytes
European journal of biochemistry / FEBS, 1990
Fructose 1-phosphate kinase was partially purified from Clostridium difficile and used to develop specific assays of fructose 1-phosphate and fructose. The concentration of fructose 1-phosphate was below the detection limit of the assay (25 pmol/mg protein) in hepatocytes incubated in the presence of glucose as sole carbohydrate. Addition of fructose (0.05-1 mM) caused a concentration-dependent and transient increase in the fructose 1-phosphate content. Glucagon (1 microM) and ethanol (10 mM) caused a severalfold decrease in the concentration of fructose 1-phosphate in cells incubated with fructose, whereas the addition of 0.1 microM vasopressin or 10 mM glycerone, or raising the concentration of glucose from 5 mM to 20 mM had the opposite effect. All these agents caused changes in the concentration of triose phosphates that almost paralleled those of the fructose 1-phosphate concentration. Sorbitol had a similar effect to fructose in causing the formation of fructose 1-phosphate. D...
Biochemical Journal, 1984
Hepatocytes from overnight-starved rats were incubated with 1-20 mM-fructose, -dihydroxyacetone, -glycerol, -alanine or -lactate and -pyruvate with or without 0.1 microM-glucagon. The production of glucose and lactate was measured, as was the content of fructose 2,6-bisphosphate. The concentrations of fructose (below 5 mM) and dihydroxyacetone (above 1 mM) that gave rise to an increase in fructose 2,6-bisphosphate were those at which a glucagon effect on the production of glucose and lactate could be observed. Glycerol had no effect on fructose 2,6-bisphosphate content or on production of lactate, and glucagon did not stimulate the production of glucose from this precursor. With alanine or lactate/pyruvate as substrates, glucagon stimulated glucose production whether the concentration of fructose 2,6-bisphosphate was increased or not. The extent of inactivation of pyruvate kinase by glucagon was not affected by the presence of the various gluconeogenic precursors. The role of fructo...
Journal of Biological Chemistry, 2003
The anomeric specificity of the wild-type recombinant forms of human liver and B-cell glucokinase was investigated using radioactive anomers of D-glucose as tracers. With D-glucose at anomeric equilibrium and at 30°C, the maximal velocity, Hill number, and K s amounted, respectively, to 16 mol min ؊1 mg ؊1 , 1.8 and 6.9 mM in the case of liver glucokinase, and 7.3 mol min ؊1 mg ؊1 , 2.0 and 7.1 mM in the case of B-cell glucokinase. Whether at 20 -22 or 30°C, the maximal velocity, Hill number, and K m were significantly lower with ␣-D-glucose than with -D-glucose in both liver and B-cell glucokinase. As a result of these differences, the reaction velocity was higher with ␣-D-glucose at low hexose concentrations, while the opposite situation prevailed at high hexose concentrations. In the presence of 0.2 mM D-fructose 6-phosphate, the glucokinase regulatory protein caused a concentrationrelated inhibition of D-glucose phosphorylation, such an effect fading out at high concentrations of either D-glucose or glucokinase relative to that of its regulatory protein. The phosphorylation of ␣-D-glucose by liver glucokinase appeared more resistant than that of -D-glucose to the inhibitory action of D-fructose 6-phosphate, as mediated by the glucokinase regulatory protein. Such a phenomenon failed to achieve statistical significance in the case of the B-cell glucokinase. It is proposed that this information, especially the novel findings concerning the anomeric difference in both Hill number and sensitivity to the glucokinase regulatory protein, should be taken into account when considering the respective contributions of ␣and -Dglucose to the overall phosphorylation of equilibrated D-glucose by glucokinase.
Biochemical Journal, 1993
The release of glucokinase from digitonin-permeabilized hepatocytes shows different characteristics with respect to ionic strength and [MgCl2] from the release of other cytoplasmic enzymes. Release of glucokinase is most rapid at low ionic strength (300 mM sucrose, 3 mM Hepes) and is inhibited by increasing concentration of KCl [concn. giving half-maximal inhibition (I50) 25 mM] or Mg2+ (I50 0.5 mM). Release of phosphoglucoisomerase, phosphoglucomutase and glucose-6-phosphate dehydrogenase is independent of ionic strength, but shows a small inhibition by MgCl2 (20%, versus > 80% for glucokinase). Lactate dehydrogenase release increases with increasing ionic strength [concn. giving half-maximal activation (A50) 10 mM KCl] or [MgCl2]. The rate and extent of glucokinase release during permeabilization in 300 mM sucrose, 5 mM MgCl2 or in medium with ionic composition resembling cytoplasm (150 mM K+, 50 mM Cl-, 1 mM Mg2+) depends on the substrate concentrations with which the hepatocy...
The Biochemical journal, 1980
1. Recycling of metabolites between fructose 6-phosphate and triose phosphates has been investigated in isolated hepatocytes by the randomization of carbon between C((1)) and C((6)) of glucose formed from [1-(14)C]galactose. 2. Randomization of carbon atoms was regularly observed with hepatocytes isolated from fed rats and was then little influenced by the concentration of glucose in the incubation medium. It was decreased by about 50% in the presence of glucagon. 3. Randomization of carbon atoms by hepatocytes isolated from starved rats was barely detectable at physiological concentrations of glucose in the incubation medium, but was greatly increased with increasing glucose concentrations. It was nearly completely suppressed by glucagon. These large changes can be attributed to parallel variations in the activity of phosphofructokinase. 4. The main factors that appear to control the activity of phosphofructokinase under these experimental conditions are the concentration of fructo...
Biochemical Journal, 2000
Glucokinase translocates between the cytoplasm and nucleus of hepatocytes where it is bound to a 68 kDa protein. The mechanism by which glucose induces translocation of glucokinase from the nucleus was investigated using glucose analogues that are not phosphorylated by glucokinase. There was strong synergism on glucokinase translocation between effects of glucose analogues (glucosamine, 5-thioglucose, mannoheptulose) and sorbitol, a precursor of fructose 1-phosphate. In the absence of glucose or glucose analogues, sorbitol had a smaller effect than glucose on translocation. However, sorbitol potentiated the effects of glucose analogues. In the absence of sorbitol the effect of glucose on glucokinase translocation is sigmoidal with a Hill coefficient of 1n9 suggesting involvement of two glucose-binding sites. The effects of glucosamine and 5-thioglucose were also sigmoidal but with lower Hill Coefficients. In the presence of sorbitol, the effects of glucose, glucosamine and 5-thioglucose were hyperbolic. Mannoheptulose, unlike the other glucose
Endocrinology, 2004
The effects of fructose-2,6-bisphosphate (F-2,6-P2) on hepatic glucokinase (GK) and glucose-6-phosphatase (G-6-Pase) gene expression were investigated in streptozotocin-treated mice, which exhibited undetectable levels of insulin. Hepatic F-2,6-P2 levels were manipulated by adenovirus-mediated overexpression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Streptozotocin treatment alone or with infusion of control adenovirus leads to a dramatic decrease in hepatic F-2,6-P2 content compared with normal nondiabetic mice. This is accompanied by a 14-fold decrease in GK and a 3-fold increase in G-6-Pase protein levels, consistent with a diabetic phenotype. Streptozotocin-treated mice that were infused with adenovirus-overexpressing an engineered 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase with high kinase activity and little bisphosphatase activity showed high levels of hepatic F-2,6-P2. Surprisingly, these mice had a 13-fold increase in GK protein and a 2-fold decrease...