Induction Of Glucokinase Research Articles

Insulin-induced cytokine production in macrophages causes insulin resistance in hepatocytes.

Overweight and obesity are associated with hyperinsulinemia, insulin resistance, and a low-grade inflammation. Although hyperinsulinemia is generally thought to result from an attempt of the β-cell to compensate for insulin resistance, there is evidence that hyperinsulinaemia itself may contribute to the development of insulin resistance and possibly the low-grade inflammation. To test this hypothesis, U937 macrophages were exposed to insulin. In these cells, insulin induced expression of the proinflammatory cytokines IL-1β, IL-8, CCL2, and OSM. The insulin-elicited induction of IL-1β was independent of the presence of endotoxin and most likely mediated by an insulin-dependent activation of NF-κB. Supernatants of the insulin-treated U937 macrophages rendered primary cultures of rat hepatocytes insulin resistant; they attenuated the insulin-dependent induction of glucokinase by 50%. The cytokines contained in the supernatants of insulin-treated U937 macrophages activated ERK1/2 and IKKβ, resulting in an inhibitory serine phosphorylation of the insulin receptor substrate. In addition, STAT3 was activated and SOCS3 induced, further contributing to the interruption of the insulin receptor signal chain in hepatocytes. These results indicate that hyperinsulinemia per se might contribute to the low-grade inflammation prevailing in overweight and obese patients and thereby promote the development of insulin resistance particularly in the liver, because the insulin concentration in the portal circulation is much higher than in all other tissues.

Role of Pregnane X Receptor in Obesity and Glucose Homeostasis in Male Mice

Clinical obesity is a complex metabolic disorder affecting one in three adults. Recent reports suggest that pregnane X receptor (PXR), a xenobiotic nuclear receptor important for defense against toxic agents and for eliminating drugs and other xenobiotics, may be involved in obesity. Noting differences in ligand specificities between human and mouse PXRs, the role of PXR in high fat diet (HFD)-induced obesity was examined using male PXR-humanized (hPXR) transgenic and PXR-knock-out (PXR-KO) mice in comparison to wild-type (WT) mice. After 16 weeks on either a control diet or HFD, WT mice showed greater weight gain, whereas PXR-KO mice gained less weight due to their resistance to HFD-induced decreases in adipose tissue peroxisome proliferator-activated receptor α and induction of hepatic carnitine palmitoyltransferase 1, suggesting increased energy metabolism. Interestingly, control-fed PXR-KO mice exhibited hepatomegaly, hyperinsulinemia, and hyperleptinemia but hypoadiponectinemia and lower adiponectin receptor R2 mRNA levels relative to WT mice. Evaluation of these biologic indicators in hPXR mice fed a control diet or HFD revealed further differences between the mouse and human receptors. Importantly, although HFD-fed hPXR mice were resistant to HFD-induced obesity, both PXR-KO and hPXR mice exhibited impaired induction of glucokinase involved in glucose utilization and displayed elevated fasting glucose levels and severely impaired glucose tolerance. Moreover, the basal hepatic levels of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase 1 were increased in hPXR mice compared with WT mice. Altogether, although the mouse PXR promotes HFD-induced obesity, the hPXR mouse carries a genetic predisposition for type 2 diabetes and thus provides a model for exploring the role of human PXR in the metabolic syndrome.

Metabolism and fatty acid profile in fat and lean rainbow trout lines fed with vegetable oil: effect of carbohydrates.

The present study investigated the effect of dietary carbohydrates on metabolism, with special focus on fatty acid bioconversion and flesh lipid composition in two rainbow trout lines divergently selected for muscle lipid content and fed with vegetable oils. These lines were chosen based on previously demonstrated potential differences in LC-PUFA synthesis and carbohydrate utilization. Applying a factorial study design, juvenile trout from the lean (L) and the fat (F) line were fed vegetable oil based diets with or without gelatinised starch (17.1%) for 12 weeks. Blood, liver, muscle, intestine and adipose tissue were sampled after the last meal. Feed intake and growth was higher in the L line than the F line, irrespective of the diet. Moderate postprandial hyperglycemia, strong induction of hepatic glucokinase and repressed glucose-6-phosphatase transcripts confirmed the metabolic response of both lines to carbohydrate intake. Further at the transcriptional level, dietary carbohydrate in the presence of n-3 LC-PUFA deficient vegetable oils enhanced intestinal chylomicron assembly, disturbed hepatic lipid metabolism and importantly elicited a higher response of key desaturase and elongase enzymes in the liver and intestine that endorsed our hypothesis. PPARγ was identified as the factor mediating this dietary regulation of fatty acid bioconversion enzymes in the liver. However, these molecular changes were not sufficient to modify the fatty acid composition of muscle or liver. Concerning the genotype effect, there was no evidence of substantial genotypic difference in lipid metabolism, LC-PUFA synthesis and flesh fatty acid profile when fed with vegetable oils. The minor reduction in plasma glucose and triglyceride levels in the F line was linked to potentially higher glucose and lipid uptake in the muscle. Overall, these data emphasize the importance of dietary macro-nutrient interface in evolving fish nutrition strategies.

Glucokinase contributes to glucose phosphorylation in d-lactic acid production by Sporolactobacillus inulinus Y2-8

Sporolactobacillus inulinus, a homofermentative lactic acid bacterium, is a species capable of efficient industrial D-lactic acid production from glucose. Glucose phosphorylation is the key step of glucose metabolism, and fine-tuned expression of which can improve D-lactic acid production. During growth on high-concentration glucose, a fast induction of high glucokinase (GLK) activity was observed, and paralleled the patterns of glucose consumption and D-lactic acid accumulation, while phosphoenolpyruvate phosphotransferase system (PTS) activity was completely repressed. The transmembrane proton gradient of 1.3-1.5 units was expected to generate a large proton motive force to the uptake of glucose. This suggests that the GLK pathway is the major route for glucose utilization, with the uptake of glucose through PTS-independent transport systems and phosphorylation of glucose by GLK in S. inulinus D-lactic acid production. The gene encoding GLK was cloned from S. inulinus and expressed in Escherichia coli. The amino acid sequence revealed significant similarity to GLK sequences from Bacillaceae. The recombinant GLK was purified and shown to be a homodimer with a subunit molecular mass of 34.5kDa. Strikingly, it demonstrated an unusual broad substrate specificity, catalyzing phosphorylation of 2-deoxyglucose, mannitol, maltose, galactose and glucosamine, in addition to glucose. This report documented the key step concerning glucose phosphorylation of S. inulinus, which will help to understand the regulation of glucose metabolism and D-lactic acid production.

Disruption of HIF-1α in hepatocytes impairs glucose metabolism in diet-induced obesity mice

The liver plays a central role in glucose homeostasis in the whole-body by responding to environmental factors including nutrients, hormones, and oxygen. In conditions of metabolic overload such as diabetes mellitus and obesity, coordinated regulation between oxygen supply and consumption has been reported to be disrupted and subsequently cause tissue hypoxia, although pathological significance of the disease-related hypoxia remains elusive. To investigate the role of tissue hypoxia in the liver on systemic glucose homeostasis, mice lacking HIF-1α gene, a critical component of a master regulator of hypoxic response, in hepatocytes were exposed to high fat/sucrose diet (HFSD). Exposure to HFSD for 5 weeks elicited liver hypoxia with a transient increase in HIF-1α protein expression in the liver of control mice. Glucose disposal was marginally impaired in control mice when challenged oral glucose tolerance test, but such impairment was enhanced in the mutant mice. This alteration was accompanied by a complete inhibition of glucokinase induction with a significant reduction of hepatic glucose uptake. Mice fed HFSD for 20 weeks exhibited fasting hyperglycemia and glucose intolerance, whereas these metabolic phenotypes deteriorated considerably with severe insulin resistance in skeletal muscles and adipose tissues in the mutant mice. These findings suggest that HIF-1 in hepatocytes plays protective roles against the progression of diabetes mellitus.

Oncostatin M produced in Kupffer cells in response to PGE2: possible contributor to hepatic insulin resistance and steatosis

Hepatic insulin resistance is a major contributor to hyperglycemia in metabolic syndrome and type II diabetes. It is caused in part by the low-grade inflammation that accompanies both diseases, leading to elevated local and circulating levels of cytokines and cyclooxygenase (COX) products such as prostaglandin E2 (PGE2). In a recent study, PGE2 produced in Kupffer cells attenuated insulin-dependent glucose utilization by interrupting the intracellular signal chain downstream of the insulin receptor in hepatocytes. In addition to directly affecting insulin signaling in hepatocytes, PGE2 in the liver might affect insulin resistance by modulating cytokine production in non-parenchymal cells. In accordance with this hypothesis, PGE2 stimulated oncostatin M (OSM) production by Kupffer cells. OSM in turn attenuated insulin-dependent Akt activation and, as a downstream target, glucokinase induction in hepatocytes, most likely by inducing suppressor of cytokine signaling 3 (SOCS3). In addition, it inhibited the expression of key enzymes of hepatic lipid metabolism. COX-2 and OSM mRNA were induced early in the course of the development of non-alcoholic steatohepatitis (NASH) in mice. Thus, induction of OSM production in Kupffer cells by an autocrine PGE2-dependent feed-forward loop may be an additional, thus far unrecognized, mechanism contributing to hepatic insulin resistance and the development of NASH.

Induction of glucokinase in chicken liver by dietary carbohydrates

We recently provided evidence of the presence of glucokinase (GCK) in the chicken liver [Berradi, H., Taouis, M., Cassy, S., Rideau, N., 2005. Glucokinase in chicken ( Gallus gallus). Partial cDNA cloning, immunodetection and activity determination. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 141, 129–139]. In the present study we addressed the question of whether nutritional regulation of GCK occurs. Several nutritional conditions were compared in chickens (5 weeks old) previously trained to meal-feeding. One group was left in the fasted state (F: 24 h) and one was tested at the end of the 2 h meal (refed: RF). Two other 2 h meal-refed groups received an acute oral saccharose load (6 ml/kg BW) just before the 2 h meal and were sacrificed either at the end of the meal (Saccharose refed, SRF) or 3 h later (SRF+3). Liver GCK mRNA and protein levels did not differ between F, RF and SRF chickens but were significantly increased in SRF+3 chickens (2-fold, p < 0.05). GCK activity did not differ between F and RF chickens but increased significantly in SRF and SRF+3 chickens (1.7-fold, p < 0.05). Chicken liver GCK expression (mRNA and protein) and activity were therefore inducible in these chickens by feeding a meal with acute oral administration of carbohydrate. These and recent findings demonstrating insulin dependency of the liver GCK mRNA and protein strongly suggest that GCK may have an important role in carbohydrate metabolism, including that of the chicken. However, even in these highly stimulatory conditions, liver GCK activity remained relatively low in comparison with other species. The latter result may partly explain the high plasma glucose level in the chicken.

Insulin induction of glucokinase and fatty acid synthase in hepatocytes: analysis of the roles of sterol-regulatory-element-binding protein-1c and liver X receptor

Insulin induction of glucokinase and fatty acid synthase in hepatocytes: analysis of the roles of sterol-regulatory-element-binding protein-1c and liver X receptor

Role of Sp1 and SREBP-1a in the insulin-mediated regulation of glucokinase transcription in the liver of gilthead sea bream ( Sparus aurata)

Insulin induction of glucokinase (GCK) transcription in the liver is essential for maintaining glucose homeostasis. To study the molecular mechanism underlying the regulation of hepatic GCK expression in the carnivorous fish gilthead sea bream ( Sparus aurata), we analysed the role of sterol regulatory element binding protein-1a (SREBP-1a) and specificity protein (Sp) 1 in insulin-dependent GCK transcription. Transient transfection experiments performed in HepG2 cells and electrophoretic mobility shift assays allowed us to identify a cis-element in the proximal region of GCK promoter implicated in transactivation by SREBP-1a. Consistently, mutations in the SRE binding site completely abolished the enhancing effect of SREBP-1a. These results and previous findings suggest that SREBP-1a plays a role in the transcriptional regulation of key enzymes in glycolysis–gluconeogenesis. Since SREBP-1a and Sp1 may mediate insulin action on S. aurata GCK transcription, we analysed the effect of insulin on HepG2 cells transfected with GCK promoter reporter constructs carrying intact or mutated SRE or Sp boxes. Insulin transactivated GCK irrespective of the presence of an intact or mutated SRE box. However, insulin failed to induce GCK transcription when using reporter constructs that had either a mutated Sp site or no Sp site. Our findings indicate that Sp1, rather than SREBP-1a, mediates the insulin-dependent induction of S. aurata GCK.

NeuroD and reaggregation induce β‐cell specific gene expression in cultured hepatocytes

Our goal was to convert adult mouse hepatocytes to pancreatic beta-cells. To facilitate conversion, cultured primary hepatocytes were dedifferentiated by the removal of dexamethasone (Dex) from the culture media. Removal of Dex caused detachment of hepatocytes from the culture dish, but the addition of betacellulin prevented this from happening. With the combination of lack of Dex and addition of betacellulin, albumin mRNA levels decreased. Cultured hepatocytes had a faint expression of insulin 2 mRNA, Nkx 6.1 and Pax 6 mRNA. Dedifferentiated hepatocytes were transduced with adenoviruses expressing NeuroD1, Ngn 3, or Pax 4. NeuroD1 transduction increased the insulin 2 mRNA but caused detachment of cells. However, when hepatocytes were allowed to reaggregate for 4 and 6 days in hydrophobic plates after transduction with NeuroD1, further increases of insulin 2 mRNA were found along with induction of PDX-1, IAPP, NeuroD1, Ngn3, Pax 4, Isl-1, PC1, PC2 and islet glucokinase. Additionally, glucagon, pancreatic polypeptide and somatostatin expression were induced, but neither elastase 1 nor insulin 1 mRNA could be detected. Ngn 3 and Pax 4 had effects similar to NeuroD1, but did not increase insulin 2 mRNA as much as NeuroD1. We conclude that the combination of NeuroD1 and reaggregation promotes cultured dedifferentiated hepatocytes to differentiate towards a pancreatic beta-cell phenotype.

Insulin induction of glucokinase and fatty acid synthase in hepatocytes: analysis of the roles of sterol-regulatory-element-binding protein-1c and liver X receptor

The transcription activator SREBP-1c (sterol-regulatory-element-binding protein-1c) is induced by insulin in the liver and is considered a master regulator of lipogenic genes such as FASN (fatty acid synthase). The question of whether SREBP-1c is also a mediator of insulin action on the regulatory enzyme of glucose metabolism GCK (glucokinase) is controversial. In the present paper, we induced SREBP-1c to various levels with insulin or the liver X receptor ligand T0901317 in primary hepatocytes and asked if these levels correlated with those of GCK or FASN mRNA expression, using the latter as positive control. Insulin and T0901317 triggered the accumulation of precursor and processed forms of SREBP-1c to similar levels and with comparable kinetics, and both effectors together caused synergistic increases in SREBP-1c protein levels. These effects were accompanied by commensurate elevation of FASN mRNA, notably by a synergistic response to both effectors. By contrast, GCK mRNA was unresponsive to T0901317 and was induced only by insulin. Treatment of hepatocytes with insulin and/or T0901317 resulted in the recruitment of SREBP-1c to the FASN promoter as shown by chromatin immunoprecipitation, whereas SREBP-1c did not bind to the GCK promoter. Lastly, we observed that the glycogen synthase kinase-3 inhibitor SB216763 produced a small increase in SREBP-1c protein level, which was further augmented in the presence of T0901317. The level of FASN mRNA varied in parallel with SREBP-1c, while GCK mRNA was unaffected. Collectively, these results showed that increases in SREBP-1c were neither necessary nor sufficient for GCK induction in hepatocytes, while at the same time they underscored the role of SREBP-1c as a key regulator of FASN.

Hepatic glucokinase turnover in intact and adrenalectomized rats in vivo.

Regulation of synthesis and degradation of glucokinase, key enzyme of glucose metabolism in liver, was investigated in intact and adrenalectomized rats in vivo, using pulse-labeling experiments and a specific antibody against the enzyme. Refeeding glucose in starved rats resulted in a marked rise in glucokinase activity (from the starvation value 4.8 mU/mg protein to 9.6 mU/mg protein at 4 h, and to 21.8 mU/mg protein at 8 h), which closely correlated to the increase in enzyme synthesis by factor 1.7 at 4 h and 4.1 at 8 h. Similar alterations in enzyme activity and synthesis were observed after glucose refeeding in adrenalectomized/glucocorticoid-restored rats. In contrast, refeeding glucose in adrenalectomized rats led within 8 h only to a small elevation in enzyme activity (from the starvation value 4.2 mU/mg protein to 9.6 mU/mg protein) and a minor rise in enzyme synthesis (factor 1.7). Glucocorticoids per se were without effect on glucokinase activity and synthesis in starved rats. When adapted to pure glucose diet, intact, adrenalectomized and adrenalectomized/glucocorticoid-restored rats showed highly elevated levels in glucokinase (27, 23, 28 mU/mg protein, respectively). However, enzyme synthesis was elevated significantly only in intact and adrenalectomized/glucocorticoid-restored rats. Under these conditions glucokinase degradation was estimated by the double-pulse-labeling technique, applying [14C]leucine and [3H]leucine. From the 3H/14C ratios the apparent half-lives were calculated: 17 h in intact and adrenalectomized/glucocorticoid-restored rats, and 35 h in adrenalectomized rats. It is concluded that in vivo glucocorticoids not only exert a 'permissive' action on glucokinase induction, but also enhance the degradation of the enzyme.

Molecular cloning of hepatic glucose-6-phosphatase catalytic subunit from gilthead sea bream ( Sparus aurata): response of its mRNA levels and glucokinase expression to refeeding and diet composition

To examine the relationship between structure and function of glucose-6-phosphatase (G6Pase) in fish, we undertook molecular cloning and modulation of G6Pase expression by starvation and refeeding on diets with different nutrient composition in the liver of the carnivorous fish, Sparus aurata. A cDNA encoding the full-length G6Pase catalytic subunit from the liver of S. aurata was isolated. This cDNA encodes a 350-amino acid protein, with low homology to the mammalian G6Pase, although it contains most of the key residues required for catalysis. Based on hydrophobicity and membrane structure prediction, we propose a model containing nine-transmembrane regions for S. aurata G6Pase. Northern blots showed that refeeding after a prolonged starvation rapidly reverses the glucose/glucose-6-phosphate substrate cycle flux in the fish liver through decreased G6Pase expression and strong glucokinase (GK) induction. The effect of refeeding different diets on G6Pase and GK expression, indicated that hepatic intermediary metabolism of fish fed diets with low protein/high carbohydrate diets is impelled towards utilization of dietary carbohydrates, by means of modulation of GK mRNA levels rather than G6Pase expression. These findings challenge the role attributed to dysregulation of G6Pase or GK expression in the low ability of carnivorous fish to metabolise glucose.

Insulin-independent induction of sterol regulatory element-binding protein-1c expression in the livers of streptozotocin-treated mice.

Insulin and glucose together have been previously shown to regulate hepatic sterol regulatory element-binding protein (SREBP)-1c expression. We sought to explore the nutritional regulation of lipogenesis through SREBP-1c induction in a setting where effects of sugars versus insulin could be distinguished. To do so, mice were insulin depleted by streptozotocin (STZ) administration and subjected to a fasting-refeeding protocol with glucose, fructose, or sucrose. Unexpectedly, the insulin-depleted mice exhibited a marked induction of SREBP-1c on all sugars, and this increase in SREBP-1c was even more dramatic than in the non-STZ-administered controls. The time course of changes in SREBP-1 induction varied depending on the type of sugars in both control and STZ-administered mice. Glucose refeeding gave a peak of SREBP-1c induction, whereas fructose refeeding caused slow and gradual increments, and sucrose refeeding fell between these two responses. Expression of various lipogenic enzymes were also gradually increased over time, irrespective of the types of sugars, with greater intensities in STZ-administered than in nontreated mice. In contrast, induction of hepatic glucokinase and suppression of phoshoenolpyruvate carboxykinase were insulin dependent in an early refed state. These data clearly demonstrate that nutritional regulation of SREBP-1c and lipogenic genes may be completely independent of insulin as long as sufficient carbohydrates are available.

Liver glucokinase can be activated by peroxisome proliferator-activated receptor-gamma.

Thiazolidinediones (TZDs), synthetic ligands of peroxisome proliferator-activated receptor (PPAR)-gamma, are known to decrease hepatic glucose production and increase glycogen synthesis in diabetic animals. Recently it was reported that glucokinase (GK) expression was increased by TZDs in the liver of diabetic ZDF rats. However, the mechanism whereby TZDs increase GK expression is not yet studied. We have assumed that liver type glucokinase (LGK) induction by TZDs could be achieved by direct transcriptional activation. Thus, we have dissected the LGK promoter to explore the presence of a PPAR response element (PPRE) in the promoter. From this study, we were able to localize a PPRE in the -116/-104 region of the rat LGK gene. The PPAR-gamma/retinoid X receptor-alpha heterodimer was bound to the element and activated the LGK promoter. The LGK promoter lacking the PPRE or having mutations in the PPRE could not be activated by PPAR-gamma. Furthermore, troglitazone increased endogenous GK mRNA in primary hepatocytes. These results indicate that PPAR-gamma can directly activate GK expression in liver and may contribute to improving glucose homeostasis in type 2 diabetes.

Analysis of the role of protein kinase B (cAKT) in insulin-dependent induction of glucokinase and sterol regulatory element-binding protein 1 (SREBP1) mRNAs in hepatocytes.

Previous work showed that acute stimulation of a conditionally active protein kinase B (PKB or cAKT) was sufficient to elicit insulin-like induction of GCK (glucokinase) and SREBP1 (sterol regulatory element-binding protein 1) in hepatocytes [Iynedjian, Roth, Fleischmann and Gjinovci (2000) Biochem. J. 351, 621-627; Fleischmann and Iynedjian (2000) Biochem. J. 349, 13-17]. The objective of the present study was to determine whether activation of PKB during insulin stimulation of hepatocytes was a necessary condition for the induction of the two genes. Activation of PKB by insulin was inhibited by pretreatment of the hepatocytes with C2 ceramide. This resulted in the inhibition of insulin-dependent increases in GCK and SREBP1 mRNAs. A triple mutant of PKB failed to interfere with insulin activation of PKB in hepatocytes even at high overexpression levels achieved after adenovirus transduction. A PKB-CaaX fusion protein, which can act as a dominant-negative inhibitor of PKB activation in other cells, was shown to be constitutively activated in hepatocytes and to trigger insulin-like induction of GCK and SREBP1. In addition, constitutive PKB-CaaX activity caused refractoriness of the hepatocytes to insulin signalling at an upstream step resulting in the inhibition of both extracellular-signal-regulated kinase 1/2 and endogenous PKB activation. The stimulation of gene expression by constitutively active PKB-CaaX and inhibition of the insulin effect by ceramide are compatible with a role for PKB in the insulin-dependent induction of GCK and SREBP1.

Activation of protein kinase B/cAkt in hepatocytes is sufficient for the induction of expression of the gene encoding glucokinase

Inhibitors of signalling pathways were used to dissect the mechanism of insulin action on expression of the gene encoding glucokinase in cultured rat hepatocytes. Wortmannin and LY 294002 completely prevented the insulin-induced increase in glucokinase mRNA seen in unhibited cells, indicating that the phosphoinositide 3-kinase module has a key role. A ligand inducible protein kinase B (PKB, also termed cAkt) fusion protein was expressed by using adenoviral transduction of hepatocytes in primary culture. The PKB activity of this protein was shown to be activated in transduced hepatocytes within 30 min of the addition of 4-hydroxytamoxifen and to stay high for 8 h, as a result of serine phosphorylation at position 473 of PKB. The increase in PKB activity was reflected in the hyperphosphorylation of phosphorylated, heat and acid stable regulated by insulin protein (PHAS-I; also termed 4E-BP1, for eukaryotic initiation factor 4E-binding protein 1), a protein involved in the regulation of translation initiation. These effects were comparable to the insulin-induced activation of endogenous PKB and phosphorylation of PHAS-I in non-transduced hepatocytes. The addition of tamoxifen to transduced hepatocytes resulted in an induction of glucokinase mRNA with kinetics and magnitude similar to those of insulin-induced mRNA accumulation. The effect of tamoxifen depended on stimulated PKB activity because it did not occur in hepatocytes that were transduced with a mutant PKB fusion protein that was refractory to activation with tamoxifen. These results establish that acute activation of PKB is sufficient to produce an insulin-like induction of glucokinase in isolated hepatocytes. Together with the inhibition by phosphoinositide 3-kinase inhibitors, they suggest that the activation of PKB might be critical in mediating the induction of glucokinase by insulin. In addition, experiments showed that PD98059 decreased by half the increase in glucokinase mRNA brought about by insulin, suggesting a contributory role of the mitogen-activated protein kinase cascade.

Regulatory role of glucose-6 phosphatase in the repletion of liver glycogen during refeeding in fasted rats

We analyzed the biochemical mechanisms involved in the liver glycogen repletion upon refeeding for 360 min in 48 and 96 h-fasted rats. In 48 h-fasted rats, the glycogen synthesis involved a rapid and further sustained induction of glucokinase (GK) (increased twice from 90 min) and a rapid but transient activation of glycogen synthase a (GSa) (maximal increase by 150% at 90 min). It did not involve the inhibition of glycogen phosphorylase a (GPa). In 96 h-fasted rats, the glycogen repletion did not involve the induction of GK for the first 180 min of refeeding. It involved a slow activation of GSa (maximal 150% increase at 180 min) and a rapid inhibition of GPa (significant from 90 min, maximal 50% inhibition by 180 min). In both groups of rats, there was a progressive inhibition of the glucose-6 phosphatase (Glc6Pase) activity (maximal suppression by 30% in both groups at 360 min). These results highlighted a key role for the inhibition of Glc6Pase activity in the liver glycogen repletion upon refeeding.

Mutants of glucokinase cause hypoglycaemia- and hyperglycaemia syndromes and their analysis illuminates fundamental quantitative concepts of glucose homeostasis.

Mutations of the glucokinase gene cause hyperglycaemia or hypoglycaemia. A quantitative understanding of these defects of glucose homeostasis linked to the glucokinase gene was lacking. Therefore a database of kinetic variables of wild-type and 20 missense mutants of glucokinase was developed and used in mathematical modelling to predict the thresholds for glucose-stimulated insulin release. Recombinant human glucokinase was generated in E. coli. The k(cat), glucose S(0.5), ATP K(m), and Hill number of glucokinase were determined. Inhibition by Stearoyl CoA and glucokinase regulatory protein and thermal stability were assayed for all mutants kinetically similar to wild-type glucokinase. A mathematical model predicting the threshold for glucose-stimulated insulin release was constructed. This model is based on the two substrate kinetics of glucokinase and the kinetic variables of the database. It is assumed that both glucokinase gene alleles are equally expressed in beta-cells and that induction of glucokinase occurs as a function of basal blood glucose. Large changes, varying greatly between mutants were found in nearly all variables. Glucokinase flux at threshold for glucose-stimulated insulin release was about 25 % of total phosphorylating potential in the normal beta-cell and this was used to predict thresholds for the mutant heterozygotes. Clinical data for maturity onset diabetes of the young type linked to the glucokinase gene and familial hyperinsulinaemic hypoglycaemia linked to the glucokinase gene and the glucokinase kinetic data of this study were used to test the model. The model predicts fasting blood glucose between 3 and 7 mmol/l in these cases. A kinetics database of wild-type and 20 mutants of glucokinase was developed. Many kinetic differences were found for the mutants. The mathematical model to calculate the threshold for glucose-stimulated insulin release predicts fasting blood glucose between 3 and 7 mmol/l in subjects with glucokinase gene mutations. [Diabetologia 42: 1175-1186]

Dietary Glycemic Index May Influence Cancer Risk by Modulating IGF-I Activity: A Hypothesis

A high-glycemic-index diet increases hepatocyte exposure to insulin, and thus should up-regulate IGF-I activity—both by stimulating IGF-I synthesis and by suppressing hepatic production of IGFBP-1. The resulting increase in IGF-I activity might be expected to have a significant cancer promotional impact in light of IGF-I's role as a progressional growth factor and anti-apoptotic agent in a great many normal and neoplastic tissues. Although direct epidemiological data linking the glycemic index of habitual diets to cancer risk are currently scarce, the profusion of reports suggesting that legumes—one of the lowest-glycemic-index staple foods—are cancer preventive is consistent with the thesis that a low-glycemic-index diet is protective in this regard; the apparent protection associated with high intakes of fruit and of fiber-rich foods may also be partially traceable to reduced glycemia. Slowly digested starch may be stored preferentially in the liver, accounting for its favorable impact on satiety and glycemic regulation; through induction of hepatic glucokinase, high-dose biotin may enhance hepatic uptake of portal glucose and thus reduce the effective glycemic index of meals. Choosing low-glycemic index starchy foods is only one of a number of measures—which include exercise training, a very-low-fat vegan diet, appropriate weight reduction, caloric restriction, and intake of chromium, biguanides, and possibly high-dose biotin—that tend to minimize diurnal insulin secretion in the context of good glycemic control, thereby decreasing risk for atherothrombotic disease, diabetes, obesity, and cancer, and perhaps even slowing the aging process.