Noradrenaline and glycogen content and the activity of several enzymes of carbohydrate metabolism in normal, embryonic, and partly denervated livers and in hepatomas of the rat]

The noradrenaline and glycogen contents as well as hexokinase, glucokinase and glucose-6-phosphatase activities were determined in normal, embryonic and partially denervated (bilateral dissection of the Nervus splanchnicus or Nervus vagus) rat liver and in two transplantable hepatomas. In embryonic liver and hepatomas a strong decrease or complete loss of noradrenaline and glycogen levels and glucokinase and glucose-6-phosphatase activities is demonstrable as compared to the livers of adult animals, while the hexokinase activity is enhanced. Following bilateral splanchnicotomy the glycogen content and hexokinase activity are enhanced; the glucose-6-phosphatase activity is reduced, and the liver does not contain any noradrenaline. Bilateral vagotomy causes decrease of the glycogen content, of the hexokinase and glucokinase activities and an enhancement of glucose-6-phosphatase activity. The results lend support to the idea of antagonistic action of the sympathetic and parasympathetic nervous systems upon several partial reactions of carbohydrate metabolism of liver. In addition, it can be assumed that the alterations of the carbohydrate metabolism demonstrable in hepatomas as compared to normal liver are not solely attributable to disturbance or breakdown of the nervous regulation.     

The role of glucose 6-phosphate in mediating the effects of glucokinase overexpression on hepatic glucose metabolism

Pharmacological activation or overexpression of glucokinase in hepatocytes stimulates glucose phosphorylation, glycolysis and glycogen synthesis. We used an inhibitor of glucose 6-phosphate (Glc6P) hydrolysis, namely the chlorogenic derivative, 1-[2-(4-chloro-phenyl)-cyclopropylmethoxy]-3, 4-dihydroxy-5-(3-imidazo[4,5-b]pyridin-1-yl-3-phenyl-acryloyloxy)-cyclohexanecarboxylic acid (also known as S4048), to determine the contribution of Glc6P concentration, as distinct from glucokinase protein or activity, to the control of glycolysis and glycogen synthesis by glucokinase overexpression. The validity of S4048 for testing the role of Glc6P was supported by its lack of effect on glucokinase binding and its nuclear/cytoplasmic distribution. The stimulation of glycolysis by glucokinase overexpression correlated strongly with glucose phosphorylation, whereas glycogen synthesis correlated strongly with Glc6P concentration. Metabolic control analysis was used to determine the sensitivity of glycogenic flux to glucokinase or Glc6P at varying glucose concentrations (5-20 mm). The concentration control coefficient of glucokinase on Glc6P (1.4-1.7) was relatively independent of glucose concentration, whereas the flux control coefficients of Glc6P (2.4-1.0) and glucokinase (3.7-1.8) on glycogen synthesis decreased with glucose concentration. The high sensitivity of glycogenic flux to Glc6P at low glucose concentration is consistent with covalent modification by Glc6P of both phosphorylase and glycogen synthase. The high control strength of glucokinase on glycogenic flux is explained by its concentration control coefficient on Glc6P and the high control strength of Glc6P on glycogen synthesis. It is suggested that the regulatory strength of pharmacological glucokinase activators on glycogen metabolism can be predicted from their effect on the Glc6P content.     

The role of the regulatory protein of glucokinase in the glucose sensory mechanism of the hepatocyte

Glucokinase has a very high flux control coefficient (greater than unity) on glycogen synthesis from glucose in hepatocytes (Agius et al., J. Biol. Chem. 271, 30479-30486, 1996). Hepatic glucokinase is inhibited by a 68-kDa glucokinase regulatory protein (GKRP) that is expressed in molar excess. To establish the relative control exerted by glucokinase and GKRP, we applied metabolic control analysis to determine the flux control coefficient of GKRP on glucose metabolism in hepatocytes. Adenovirus-mediated overexpression of GKRP (by up to 2-fold above endogenous levels) increased glucokinase binding and inhibited glucose phosphorylation, glycolysis, and glycogen synthesis over a wide range of concentrations of glucose and sorbitol. It decreased the affinity of glucokinase translocation for glucose and increased the control coefficient of glucokinase on glycogen synthesis. GKRP had a negative control coefficient of glycogen synthesis that is slightly greater than unity (-1.2) and a control coefficient on glycolysis of -0.5. The control coefficient of GKRP on glycogen synthesis decreased with increasing glucokinase overexpression (4-fold) at elevated glucose concentration (35 mM), which favors dissociation of glucokinase from GKRP, but not at 7.5 mM glucose. Under the latter conditions, glucokinase and GKRP have large and inverse control coefficients on glycogen synthesis, suggesting that a large component of the positive control coefficient of glucokinase is counterbalanced by the negative coefficient of GKRP. It is concluded that glucokinase and GKRP exert reciprocal control; therefore, mutations in GKRP affecting the expression or function of the protein may impact the phenotype even in the heterozygote state, similar to glucokinase mutations in maturity onset diabetes of the young type 2. Our results show that the mechanism comprising glucokinase and GKRP confers a markedly extended responsiveness and sensitivity to changes in glucose concentration on the hepatocyte.     

Factors underlying significant underestimations of glucokinase activity in crude liver extracts: physiological implications of higher cellular activity

M. Kuwajima, C. B. Newgard, D. W. Foster, and J. D. McGarry (1986, J. Biol. Chem. 261, 8849-8853) have concluded that the reason postprandial hepatic glycogenesis occurs primarily from gluconeogenic precursors rather than glucose is because glucokinase activity is insufficient to support the observed rates of glycogen synthesis. F. L. Alvares and R. C. Nordlie (1977, J. Biol. Chem. 252, 8404-8414) have concluded that the combined activities of glucokinase and hexokinase are less than the apparent rates of hepatic glucose uptake. We have identified several factors in the assays used in these studies which lead to substantial underestimations of glucokinase activity. Glucokinase was assayed either by allowing glucose 6-phosphate to accumulate over 10 min (discontinuous assay) or by coupling the formation of glucose 6-phosphate with its oxidation by Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase and NAD (continuous assay). Accurate determinations of glucokinase at 37 degrees C with subsaturating glucose require both 100 mM KCl and 2.5 mM dithioerythritol in the assay medium; 2-mercaptoethanol will not substitute for dithioerythritol. When both KCl and dithioerythritol are absent (Kuwajima et al.) glucokinase activity is underestimated by 3- to 5-fold. The discontinuous assay as used previously (Alvares and Nordlie) underestimates glucokinase activity in crude extracts by 2- to 2.5-fold, due in part to the hydrolysis of glucose 6-phosphate and its transformation to other hexose monophosphates. Under optimized conditions at 37 degrees C both assays yield similar results in extracts from fed rats, i.e., 2-3 and 4-5 units/g liver at 10 and 100 mM glucose, respectively. Some implications of the finding that total hepatic glucose phosphorylating capacity at physiological concentrations significantly exceeds the observed rates of postprandial glycogen synthesis are discussed.     

Distribution profile of glucokinase and hexokinase in parenchymal and sinusoidal cells of rat liver during development

Activities of glucokinase and hexokinase were measured in whole liver, in isolated parenchymal cells and in sinusoidal cells from neonatal, suckling and weanling rats. Hexokinase activity was found to be high in fetal and newborn livers, decreased gradually and attained a plateau in 21 days. Glucokinase activity, on the other hand, was very low in fetal and newborn livers, but increased 40 fold in 21 days after birth. Measurement of the enzyme activities in isolated cells revealed a gradual increase in glucokinase activity in parenchymal cells and a parallel decrease of this enzyme in sinusoidal cells as a function of age. Hexokinase activity in parenchymal or sinusoidal cells did not change significantly at different stages of growth. The relative contribution by parenchymal cells to the liver glucokinase activity increased gradually during maturation whereas that by the sinusoidal cells decreased during this period. The evidence presented suggests that sinusoidal cells may be playing a vital role in the metabolic activities of liver in the early stages of postnatal development.     

A kinetic analysis of glucokinase and glucose-6-P phosphatase in dictyostelium

Summary Using a mathematical model of carbohydrate metabolism in Dictyostelium discoideum, the kinetic expressions describing the activities of glucokinase and glucose-6-P phosphatase have been analyzed. The constraints on the kinetic mechanisms and relative activities of these two enzymes were investigated by comparing computer simulations to experimental data. The results indicated that, (1) glucose-6-P is compartmentalized with respect to the enzymes involved in glucose-6-P, trehalose and glycogen metabolism, (2) a differences of approximately 0.6 mm/min in maximum specific activity of glucokinase compared to glucose-6-P phosphatase is required in order for the model to produce end product carbohydrate levels consistent with those observed experimentally, (3) the Km of glucokinase for glucose strongly influences the steady state levels of glucose in the absence of external glucose, and (4) changing the order of product removal in the reaction catalyzed by glucose-6-P phosphatase influences the level of glycogen and trehalose.     

Induction and suppression of the key enzymes of glycolysis and gluconeogenesis in isolated perfused rat liver in response to glucose, fructose and lactate

1. Measurements were made of the activities of the four key enzymes involved in gluconeogenesis, pyruvate carboxylase (EC 6.4.1.1), phosphoenolpyruvate carboxylase (EC 4.1.1.32), fructose 1,6-diphosphatase (EC 3.1.3.11) and glucose 6-phosphatase (EC 3.1.3.9), of serine dehydratase (EC 4.2.1.13) and of the four enzymes unique to glycolysis, glucokinase (EC 2.7.1.2), hexokinase (EC 2.7.1.1), phosphofructokinase (EC 2.7.1.11) and pyruvate kinase (EC 2.7.1.40), in livers from starved rats perfused with glucose, fructose or lactate. Changes in perfusate concentrations of glucose, fructose, lactate, pyruvate, urea and amino acid were monitored for each perfusion. 2. Addition of 15mm-glucose at the start of perfusion decreased the activity of pyruvate carboxylase. Constant infusion of glucose to maintain the concentration also decreased the activities of phosphoenolpyruvate carboxylase, fructose 1,6-diphosphatase and serine dehydratase. Addition of 2.2mm-glucose initially to give a perfusate sugar concentration similar to the blood sugar concentration of starved animals had no effect on the activities of the enzymes compared with zero-time controls. 3. Addition of 15mm-fructose initially decreased glucokinase activity. Constant infusion of fructose decreased activities of glucokinase, phosphofructokinase, pyruvate carboxylase, phosphoenolpyruvate carboxylase, glucose 6-phosphatase and serine dehydratase. 4. Addition of 7mm-lactate initially elevated the activity of pyruvate carboxylase, as also did constant infusion; maintenance of a perfusate lactate concentration of 18mm induced both pyruvate carboxylase and phosphoenolpyruvate carboxylase activities. 5. Addition of cycloheximide had no effect on the activities of the enzymes after 4h of perfusion at either low or high concentrations of glucose or at high lactate concentration. Cycloheximide also prevented the loss or induction of pyruvate carboxylase and phosphoenolpyruvate carboxylase activities with high substrate concentrations. 6. Significant amounts of glycogen were deposited in all perfusions, except for those containing cycloheximide at the lowest glucose concentration. Lipid was found to increase only in the experiments with high fructose concentrations. 7. Perfusion with either fructose or glucose decreased the rates of ureogenesis; addition of cycloheximide increased urea efflux from the liver.     

Acute inhibition of hepatic glucose-6-phosphatase does not affect gluconeogenesis but directs gluconeogenic flux toward glycogen in fasted rats. A pharmacological study with the chlorogenic acid derivative S4048

Effects of acute inhibition of glucose-6-phosphatase activity by the chlorogenic acid derivative S4048 on hepatic carbohydrate fluxes were examined in isolated rat hepatocytes and in vivo in rats. Fluxes were calculated using tracer dilution techniques and mass isotopomer distribution analysis in plasma glucose and urinary paracetamol-glucuronide after infusion of [U-(13)C]glucose, [2-(13)C]glycerol, [1-(2)H]galactose, and paracetamol. In hepatocytes, glucose-6-phosphate (Glc-6-P) content, net glycogen synthesis, and lactate production from glucose and dihydroxyacetone increased strongly in the presence of S4048 (10 microm). In livers of S4048-treated rats (0.5 mg kg(-1)min(-)); 8 h) Glc-6-P content increased strongly (+440%), and massive glycogen accumulation (+1260%) was observed in periportal areas. Total glucose production was diminished by 50%. The gluconeogenic flux to Glc-6-P was unaffected (i.e. 33.3 +/- 2.0 versus 33.2 +/- 2.9 micromol kg(-1)min(-1)in control and S4048-treated rats, respectively). Newly synthesized Glc-6-P was redistributed from glucose production (62 +/- 1 versus 38 +/- 1%; p < 0.001) to glycogen synthesis (35 +/- 5% versus 65 +/- 5%; p < 0.005) by S4048. This was associated with a strong inhibition (-82%) of the flux through glucokinase and an increase (+83%) of the flux through glycogen synthase, while the flux through glycogen phosphorylase remained unaffected. In livers from S4048-treated rats, mRNA levels of genes encoding Glc-6-P hydrolase (approximately 9-fold), Glc-6-P translocase (approximately 4-fold), glycogen synthase (approximately 7-fold) and L-type pyruvate kinase (approximately 4-fold) were increased, whereas glucokinase expression was almost abolished. In accordance with unaltered gluconeogenic flux, expression of the gene encoding phosphoenolpyruvate carboxykinase was unaffected in the S4048-treated rats. Thus, acute inhibition of glucose-6-phosphatase activity by S4048 elicited 1) a repartitioning of newly synthesized Glc-6-P from glucose production into glycogen synthesis without affecting the gluconeogenic flux to Glc-6-P and 2) a cellular response aimed at maintaining cellular Glc-6-P homeostasis.     

Effect of dichlorvos on hepatic and pancreatic glucokinase activity and gene expression, and on insulin mRNA levels

Several studies have shown that organophosphate pesticides affect carbohydrate metabolism and produce hyperglycemia. It has been reported that exposure to the organophosphate pesticide dichlorvos affects glucose homeostasis and decreases liver glycogen content. Glucokinase (EC 2.7.1.1) is a tissue-specific enzyme expressed in liver and in pancreatic beta cells that plays a crucial role in glycogen synthesis and glucose homeostasis. In the present study we analyzed the effect of one or three days of dichlorvos administration [20 mg/kg body weight] on the activity and mRNA levels of hepatic and pancreatic glucokinase as well as on insulin mRNA abundance in the rat. We found that the pesticide affects pancreatic and hepatic glucokinase activity and expression differently. In the liver the pesticide decreased the enzyme activity; on the contrary glucokinase mRNA levels were increased. In contrast, pancreatic glucokinase activity as well as mRNA levels were not affected by the treatment. Insulin mRNA levels were not modified by dichlorvos administration. Our results suggest that the decreased activity of hepatic glucokinase may account for the adverse effects of dichlorvos on glucose metabolism.     

Antihyperglycemic effect of aqueous and ethanolic extracts of Gongronema latifolium leaves on glucose and glycogen metabolism in livers of normal and streptozotocin-induced diabetic rats

The present study was designed to investigate the antihyperglycemic effects of aqueous and ethanolic extracts from Gongronema latifolium leaves on glucose and glycogen metabolism in livers of non-diabetic and streptozotocin-induced diabetic rats. To investigate the effects of aqueous or ethanolic leaf extracts of G. latifolium, non-diabetic and STZ diabetic rats were treated twice daily (100 mg/Kg) for two weeks. Diabetic rats showed a significant decrease in the activities of hepatic hexokinase (HK), phosphofructokinase (PFK) and glucose-6-phosphate dehydrogenase (G6PDH) and an increase in glucokinase (GK) activity. The levels of hepatic glycogen and glucose were also increased in diabetic rats. However, there were no significant differences in the activities of glucose-6-phosphatase (G6Pase) in treated and untreated diabetic rats. The ethanolic extract significantly increased the activities of HK (p<0.01), PFK (p<0.001) and G6PDH (p<0.01) in diabetic rats, decreased the activity of GK (p<0.05) and the levels of hepatic glycogen (p<0.01) and both hepatic (p<0.001) and blood glucose (40%). The aqueous extract of G. latifolium was only able to significantly increase the activities of HK and decrease the activities of GK but did not produce any significant change in the hepatic glycogen and both hepatic and blood glucose content of diabetic rats. Our data show that the ethanolic extract from G. latifolium leaves has antihyperglycemic potency, which is thought to be mediated through the activation of HK, PFK, G6PDH and inhibition of GK in the liver. The ethanolic extract is under further investigation to determine the chemical structure of the active compound(s) and its/their mechanism of action.     

Kinases and phosphatases of hepatic glycogen metabolism during fasted to refed transition in normal and streptozotocin-induced diabetic rats.

Normal and streptozotocin-induced diabetic rats were fasted for 24 hours and refed for 4 hours. Changes in the activities of glycogen metabolizing enzymes in liver were followed during this period. In normal rats, hepatic glycogen content increased gradually after the onset of food intake. The percent of active glycogen synthase increased to a peak value at 1h which coincided with a significant (P less than 0.02) increase in synthase phosphatase activity. Phosphorylase alpha and the percent of alpha increased significantly (P less than 0.01) after the meal which correlated with similar increases in cAMP-dependent protein kinase and phosphorylase kinase activities. Activation of enzymes involved in both synthesis and degradation of glycogen during fasted to refed transition indicate a probable substrate cycling. In diabetic livers, there was marked decrease in the activities of glycogen metabolizing enzymes and their levels did not alter significantly in response to the meal indicating a poor turnover of glycogen.     

The development of hepatic glucokinase in the neonatal rat

1. Glucokinase and hexokinase activities have been determined in the livers of newborn rats and attempts made to influence in vivo the development of the glucokinase. 2. Glucokinase first appears in rat liver about 16 days after birth and adult activities are reached 10–12 days later. Evidence is presented which indicates that this represents synthesis of new protein. Hexokinase activities remain constant throughout the period of glucokinase development. 3. Both exogenous glucose and insulin are necessary for the natural development of glucokinase, for this is retarded in starved and alloxan-diabetic neonatal rats. 4. The absence of glucokinase during the first 2 weeks of extrauterine life in the rat is not due to lack of insulin. 5. Attempts to advance the time at which glucokinase first appears by infusions of glucose, insulin and chlorpropamide alone and in various combinations have resulted in marginal effects only. 6. When rats are starved for 3 days during the period of glucokinase development and then re-fed, glucokinase is more rapidly synthesized, indicating that the potential ability to synthesize glucokinase continues to develop throughout the period of starvation. 7. Some possible reasons for the comparatively late development of glucokinase are discussed.     

The control of hepatic glycogen metabolism in an in vitro model of sepsis

Culturing hepatocytes with a combination of LPS, TNF-α, IL-1β and IFN-γ resulted in an inhibition of glucose output from glycogen and prevented the repletion of glycogen in freshly cultured cells. The reduced glycogen mobilisation correlated with the lower cell glycogen content and reduced rate of glycogen synthesis from [U-¹⁴C]glucose rather than alterations in either total phosphorylase or phosphorylase a activity. There was no change in the percentage of glycogen exported as glucose nor the production of lactate plus pyruvate indicating that redistribution of the Gluc-6-P cannot explain the failure of the liver to export glucose. Although changes in glycogen mobilisation correlated with NO production, inhibition of NO synthase by inclusion of L-NMMA in the culture medium failed to prevent the inhibition of either glycogen accumulation or mobilisation by the proinflammatory cytokines, precluding the involvement of NO in this response. LPS plus cytokine treatment had no effect on total glycogen synthase activity although the activity ratio was lowered, indicative of increased phosphorylation. The inhibition of glycogen synthesis correlated with a fall in the intracellular concentrations of Gluc-6-P and UDP-glucose and in the absence of measured changes in kinase activity, it is suggested that the fall in Gluc-6-P reduces both substrate supply and glycogen synthase phosphatase activity. The fall in Gluc-6-P coincided with a reduction in total glucokinase and hexokinase activity within the cells, but no significant change in either the translocation of glucokinase or glucose-6-phosphatase activity. This demonstrates direct cytokine effects on glycogen metabolism independent of changes in glucoregulatory hormones.     

Biochemical and ultrastructural hepatic changes during vitamin B12 deficiency in animals and man.

Vitamin B12 deficiency has been shown to result in an increase in content and activity of the hepatic cytosolic enzymes of fatty acid synthesis. The present study demonstrated that ATP citrate lyase, an enzyme whose activity has been positively correlated with rates of fatty acid biosynthesis, also increased in the livers of B12-deficient animals. Total and specific activity of hepatic citrate synthase, an enzyme whose activity is unaffected by a variety of dietary and hormonal changes, also was found to be increased in the B12-deprived state. By contrast, the activity of hepatic succinate-cytochrome c reductase, a portion of a multicomponent enzyme complex synthesized in part within the mitochondria, was unchanged in B12 deficiency. Vitamin B12 deprivation resulted in an increase in hepatic mitochondrial cristae membranes in both animals and man. Histochemical and chemical analysis demonstrated increased glycogen in the liver cells from B12-deficient animals and man. Thus, in the livers from vitamin B12-deficient animals there is an increased activity of the otherwise highly constant Krebs cycle enzyme citrate synthase, and in both animals and man there are increased mitochondrial cristae membranes.     

The influence of diurnal rhythms of carbohydrate metabolism in adult rat liver on the metabolic characteristics of isolated liver parenchymal cells

Rats trained to the “8 + 16” controlled feeding cycle where food is only available for the first 8 h of the 12 h dark period exhibit a pronounced diurnal rhythm of hepatic glycogen metabolism. Glycogen is stored within the liver parenchymal cells during the dark period and subsequently mobilized for energy production during the light period. Hepatocytes, isolated by collagenase perfusion, from livers of such animals have differing capacities for glycogen synthesis when incubated with glucose. Cells prepared at the end of the 16 h period without food have very little capacity for synthesis compared with much higher rates obtained in cells obtained during the feeding period. Cells obtained from livers containing a large glycogen concentration produce a net breakdown of glycogen during incubations with glucose, however experiments using radioactively labelled glucose indicate that synthesis does occur in these cells. The changes in the capacity of the cells for glycogen synthesis appear to be due, in part, to changes in the percentage of the cell population involved in synthesis and in the activity of glycogen synthetase $\text{a}$. Attempts to influence the rate of glycogen synthesis at any time of day with insulin or dexamethasone were unsuccessful.     

Glucokinase in bird liver: a membrane bound enzyme

There have been numerous reports that liver of birds contain only isoenzymes of the low K_M hexokinases, but lack the high K_M glucokinase. We describe here the presence of glucokinase in livers of chicken and Japanese quail. The enzyme is membrane bound and is solubilized by vigorous mechanical disruption of the tissue. With gentle homogenization the glucokinase is recovered upon centrifugation in the 1000g pellet, from which it may be liberated by prolonged sonication. It appears to be localized in the cell plasma membrane. The activities of hexokinase and glucokinase appear to be about equal in liver parenchyma of fed chicken, but in that of Japanese quail the activity of glucokinase exceeds greatly that of hexokinase.     

Hepatic glycogen synthesis is highly sensitive to phosphorylase activity: evidence from metabolic control analysis

We used metabolic control analysis to determine the flux control coefficient of phosphorylase on glycogen synthesis in hepatocytes by titration with a specific phosphorylase inhibitor (CP-91149) or by expression of muscle phosphorylase using recombinant adenovirus. The muscle isoform was used because it is catalytically active in the b-state. CP-91149 inactivated phosphorylase with sequential activation of glycogen synthase. It increased glycogen synthesis by 7-fold at 5 mm glucose and by 2-fold at 20 mm glucose with a decrease in the concentration of glucose causing half-maximal rate (S(0.5)) from 26 to 19 mm. Muscle phosphorylase was expressed in hepatocytes mainly in the b-state. Low levels of phosphorylase expression inhibited glycogen synthesis by 50%, with little further inhibition at higher enzyme expression, and caused inactivation of glycogen synthase that was reversed by CP-91149. At endogenous activity, phosphorylase has a very high (greater than unity) negative control coefficient on glycogen synthesis, regardless of whether it is determined by enzyme inactivation or overexpression. This high control is attenuated by glucokinase overexpression, indicating dependence on other enzymes with high control. The high control coefficient of phosphorylase on glycogen synthesis affirms that phosphorylase is a strong candidate target for controlling hyperglycemia in type 2 diabetes in both the absorptive and postabsorptive states.     

Phosphorylation by liver glucokinase of D-glucose anomers at anomeric equilibrium

The relative contribution of each anomer of D-glucose to the overall phosphorylation rate of the hexose tested at anomeric equilibrium was examined in rat liver postmicrosomal supernatants under conditions aimed at characterizing the activity of glucokinase, with negligible interference of either hexokinase, N-acetyl-D-glucosamine kinase or glucose-6-phosphatase (acting as a phosphotransferase). Both at 10 degrees and 30 degrees C, the relative contribution of each anomer was unaffected by the concentration of D-glucose. At both temperatures, the alpha/beta ratio for the contribution of each anomer was slightly, but significantly, lower than the alpha/beta ratio of anomer concentrations. These findings, which are consistent with the anomeric specificity of glucokinase in terms of affinity, cooperativity and maximal velocity, reveal that the preferred alpha-anomeric substrate for both glycogen synthesis and glycolysis is generated by glucokinase at a lower rate than is beta-D-glucose-6-phosphate.     

The influence of diurnal rhythms of carbohydrate metabolism in adult rat liver on the metabolic characteristics of isolated liver parenchymal cells.

Rats trained to the "8 + 16" controlled feeding cycle where food is only available for the first 8 h of the 12 h dark period exhibit a pronounced diurnal rhythm of hepatic glycogen metabolism. Glycogen is stored within the liver parenchymal cells during the dark period and subsequently mobilized for energy production during the light period. Hepatocytes, isolated by collagenase perfusion, from livers of such animals have differing capacities for glycogen synthesis when incubated with glucose. Cells prepared at the end of the 16 h period without food have very little capacity for synthesis compared with much higher rates obtained in cells obtained during the feeding period. Cells obtained from liver containing a large glycogen concentration produce a net breakdown of glycogen during incubations with glucose, however experiments using radioactively labelled glucose indicate that synthesis does occur in these cells. The changes in the capacity of the cells for glycogen synthesis appear to be due, in part, to changes in the percentage of the cell population involved in synthesis and in the activity of glycogen synthetase a. Attempts of influence the rate of glycogen synthesis at any time of day with insulin or dexamethasone were unsuccessful.     

Transforming growth factor beta 2 differentially modulates interleukin-1 beta- and tumour-necrosis-factor-alpha-stimulated phospholipase A2 and prostaglandin E2 synthesis in rat renal mesangial cells.

Isolated hepatocytes from streptozotocin-diabetic rats failed to respond to a glucose load with an activation of glycogen synthase. This lesion was associated with severely decreased activities of glycogen-synthase phosphatase and of glucokinase. All these defects were abolished after consumption for 13-18 days of drinking water containing Na3VO4 (0.7 mg/ml), and they were partially restored after 3.5 days, when the blood glucose concentration was already normalized. In all conditions the maximal extent of activation of glycogen synthase in cells closely parallelled the activity of glycogen-synthase phosphatase.