Liver glycogen synthase but not the muscle isoform differentiates between glucose 6-phosphate produced by glucokinase or hexokinase

Using adenovirus-mediated gene transfer into FTO-2B cells, a rat hepatoma cell line, we have overexpressed hexokinase I (HK I), glucokinase (GK), liver glycogen synthase (LGS), muscle glycogen synthase (MGS), and combinations of each of the two glucose-phosphorylating enzymes with each one of the GS isoforms. FTO-2B cells do not synthesize glycogen even when incubated with high doses of glucose. Adenovirus-induced overexpression of HK I and/or LGS, two enzymes endogenously expressed by these cells, did not produce a significant increase in the levels of active GS and the total glycogen content. In contrast, GK overexpression led to the glucose-dependent activation of endogenous or overexpressed LGS and to the accumulation of glycogen. Similarly overexpressed MGS was efficiently activated by the glucose-6-phosphate (Glc-6-P) produced by either endogenous or overexpressed HK I and by overexpressed GK. These results indicate the existence of at least two pools of Glc-6-P in the cell, one of them is accessible to both isoforms of GS and is replenished by the action of GK, whereas LGS is excluded from the cellular compartment where the Glc-6-P produced by HK I is directed. These findings are interpreted in terms of the metabolic role that the two pairs of enzymes, HK I-MGS in the muscle and GK-LGS in the hepatocyte, perform in their respective tissues.     

Glucose 6-phosphate produced by gluconeogenesis and by glucokinase is equally effective in activating hepatic glycogen synthase

Glucose 6-phosphate (Glc-6-P) produced in cultured hepatocytes by direct phosphorylation of glucose or by gluconeogenesis from dihydroxyacetone (DHA) was equally effective in activating glycogen synthase (GS). However, glycogen accumulation was higher in hepatocytes incubated with glucose than in those treated with DHA. This difference was attributed to decreased futile cycling through GS and glycogen phosphorylase (GP) in the glucose-treated hepatocytes, owing to the partial inactivation of GP induced by glucose. Our results indicate that the gluconeogenic pathway and the glucokinase-mediated phosphorylation of glucose deliver their common product to the same Glc-6-P pool, which is accessible to liver GS. As observed in the treatment with glucose, incubation of cultured hepatocytes with DHA caused the translocation of GS from a uniform cytoplasmic distribution to the hepatocyte periphery and a similar pattern of glycogen deposition. We hypothesize that Glc-6-P has a major role in glycogen metabolism not only by determining the activation state of GS but also by controlling its subcellular distribution in the hepatocyte.     

Upregulation of hepatic glucose 6-phosphatase gene expression in rats treated with an inhibitor of glucose-6-phosphate translocase

The multicomponent hepatic glucose 6-phosphatase (Glc-6-Pase) system catalyzes the terminal step of hepatic glucose production and plays a key role in the regulation of blood glucose. We used the chlorogenic acid derivative S 3483, a reversible inhibitor of the glucose-6-phosphate (Glc-6-P) translocase component, to demonstrate for the first time upregulation of Glc-6-Pase expression in rat liver in vivo after inhibition of Glc-6-P translocase. In accordance with its mode of action, S 3483-treatment of overnight-fasted rats induced hypoglycemia and increased blood lactate, hepatic Glc-6-P, and glycogen. The metabolic changes were accompanied by rapid and marked increases in Glc-6-Pase mRNA (above 35-fold), protein (about 2-fold), and enzymatic activity (about 2-fold). Maximal mRNA levels were reached after 4 h of treatment. Glycemia, blood lactate, and Glc-6-Pase mRNA levels returned to control values, whereas Glc-6-P and glycogen levels decreased but were still elevated 2 h after S 3483 withdrawal. The capacity for Glc-6-P influx was only marginally increased after 8.5 h of treatment. Prevention of hypoglycemia by euglycemic clamp did not abolish the increase in Glc-6-Pase mRNA induced by S 3483 treatment. A similar pattern of hypoglycemia and possibly of associated counterregulatory responses elicited by treatment with the phosphoenolpyruvate carboxykinase inhibitor 3-mercaptopicolinic acid could account for only a 2-fold induction of Glc-6-Pase mRNA. These findings suggest that the significant upregulation of Glc-6-Pase gene expression observed after treatment of rats in vivo with an inhibitor of Glc-6-P translocase is caused predominantly either by S 3483 per se or by the compound-induced changes of intracellular carbohydrate metabolism.     

A potential new role for muscle in blood glucose homeostasis

The breakdown of tissue glycogen into glucose is critical for blood glucose homeostasis between meals. In the final steps of glycogenolysis, intracellular glucose 6-phosphate (Glc-6-P) is transported into the endoplasmic reticulum where it is hydrolyzed to glucose by glucose-6-phosphatase (Glc-6-Pase). Although the majority of body glycogen is stored in the muscle, the current dogma holds that Glc-6-Pase (now named Glc-6-Pase-alpha) is expressed only in the liver, kidney, and intestine, implying that muscle glycogen cannot contribute to interprandial blood glucose homeostasis. Recently we reported a second Glc-6-P hydrolase, Glc-6-Pase-beta. Glc-6-Pase-beta shares kinetic and structural similarities to Glc-6-Pase-alpha and couples with the Glc-6-P transporter to form an active Glc-6-Pase complex (Shieh, J.-J., Pan, C.-J., Mansfield, B. C., and Chou, J. Y. (2003) J. Biol. Chem. 278, 47098-47103). Here we demonstrate that muscle expresses both Glc-6-Pase-beta and Glc-6-P transporter and that they can couple to form an active Glc-6-Pase complex. Our data suggest that muscle may have a previously unrecognized role in interprandial glucose homeostasis.     

Deficiency of a Glycogen Synthase-associated Protein,Epm2aip1, Causes Decreased Glycogen Synthesis and Hepatic Insulin Resistance

Glycogen synthesis is a major component of the insulin response, and defective glycogen synthesis is a major portion of insulin resistance. Insulin regulates glycogen synthase (GS) through incompletely defined pathways that activate the enzyme through dephosphorylation and, more potently, allosteric activation. We identify Epm2aip1 as a GS-associated protein. We show that the absence of Epm2aip1 in mice impairs allosteric activation of GS by glucose 6-phosphate, decreases hepatic glycogen synthesis, increases liver fat, causes hepatic insulin resistance, and protects against age-related obesity. Our work identifies a novel GS-associated GS activity-modulating component of insulin resistance.     

Glucose 6-phosphate plays a central role in the regulation of glycogen synthesis in a glycogen-storing liver cell line

Two substrains of the epithelial liver cell line C1I, one storing large amounts of glycogen, the other one being very poor in glycogen were used as a model for studying glycogen synthesis. The glycogen content of glycogen-rich cells doubled during the proliferative phase and remained high in plateau phase although glycogen synthase I activity was not significantly altered during growth cycle and was too low to account for the increase in glycogen. However, the activity of the glucose 6-phosphate (Glc6-P)-dependent synthase rose continuously during growth cycle, and intracellular Glc6-P-concentration increased about 10-fold in log phase cells to 0.72 mumol g-1 wet weight. A0.5 of synthase for Glc6-P was 0.79 mM. It was also found that in contrast to the enzyme from normal liver, glycogen phosphorylase a from C1I cells was inhibited by Glc6-P, the apparent Ki being 0.45 mM. It was concluded that glycogen accumulation in C1I cells was due to stimulation of synthase and inhibition of phosphorylase by Glc6-P. Findings from the glycogen-poor cell line which revealed similar specific activities of synthase and phosphorylase but only low Glc6-P (0.056 mumol g-1 wet weight) supported this conclusion. Addition of glucose to starved cells resulted in a transient activation of synthase in both cell lines. Net glycogen synthesis, was, however, only observed in the cells with a high Glc6-P-content. Thus, modulation of synthase and phosphorylase by Glc6-P and not activation/inactivation of the enzymes seems to play a predominant role in glycogen accumulation in this cell line.     

Insulin promotes glycogen synthesis in the absence of GSK3 phosphorylation in skeletal muscle

Insulin promotes dephosphorylation and activation of glycogen synthase (GS) by inactivating glycogen synthase kinase (GSK) 3 through phosphorylation. Insulin also promotes glucose uptake and glucose 6-phosphate (G-6-P) production, which allosterically activates GS. The relative importance of these two regulatory mechanisms in the activation of GS in vivo is unknown. The aim of this study was to investigate if dephosphorylation of GS mediated via GSK3 is required for normal glycogen synthesis in skeletal muscle with insulin. We employed GSK3 knockin mice in which wild-type GSK3 alpha and -beta genes are replaced with mutant forms (GSK3 alpha/beta S21A/S21A/S9A/S9A), which are nonresponsive to insulin. Although insulin failed to promote dephosphorylation and activation of GS in GSK3 alpha/beta S21A/S21A/S9A/S9A mice, glycogen content in different muscles from these mice was similar compared with wild-type mice. Basal and epinephrine-stimulated activity of muscle glycogen phosphorylase was comparable between wild-type and GSK3 knockin mice. Incubation of isolated soleus muscle in Krebs buffer containing 5.5 mM glucose in the presence or absence of insulin revealed that the levels of G-6-P, the rate of [14C]glucose incorporation into glycogen, and an increase in total glycogen content were similar between wild-type and GSK3 knockin mice. Injection of glucose containing 2-deoxy-[3H]glucose and [14C]glucose also resulted in similar rates of muscle glucose uptake and glycogen synthesis in vivo between wild-type and GSK3 knockin mice. These results suggest that insulin-mediated inhibition of GSK3 is not a rate-limiting step in muscle glycogen synthesis in mice. This suggests that allosteric regulation of GS by G-6-P may play a key role in insulin-stimulated muscle glycogen synthesis in vivo.     

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.     

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.     

Mitochondrial hexokinase from differentiated and undifferentiated HT29 colon cancer cells: effect of some metabolites on the bound/soluble equilibrium

1. Solubilization of mitochondrial bound hexokinase (HK), which represents 75-80% of the total enzyme activity in the cells, was investigated in freshly isolated mitochondria from undifferentiated (Glc+) or differentiated (Glc-) HT29 adenocarcinoma cells. In both models, the bound HK is almost completely released in vitro by 100 microM glucose 6-P (G 6-P). 2. Free ATP (5 mM) or palmitate (800 microM) produce a partial solubilization of bound HK, more markedly in the case of Glc- mitochondria. 3. Glucose or glucose 1-P are found unable to solubilize bound HK. Glucose 1,6-P2, 2-deoxyglucose 6-P or glucosamine 6-P can solubilize the enzyme but are less efficient than G 6-P. 4. Mg2+ and Pi are found to counteract the glucose 6-P induced solubilization of HK in both types of mitochondria. Taking into account the intracellular concentrations of these ions, this could in part explain why, in HT29 cells, HK is predominantly bound to the mitochondria.     

Effects of high-fat diet and exercise training on intracellular glucose metabolism in rats

We examined the effects of high-fat diet (HFD) and exercise training on insulin-stimulated whole body glucose fluxes and several key steps of glucose metabolism in skeletal muscle. Rats were maintained for 3 wk on either low-fat (LFD) or high-fat diet with or without exercise training (swimming for 3 h per day). After the 3-wk diet/exercise treatments, animals underwent hyperinsulinemic euglycemic clamp experiments for measurements of insulin-stimulated whole body glucose fluxes. In addition, muscle samples were taken at the end of the clamps for measurements of glucose 6-phosphate (G-6-P) and GLUT-4 protein contents, hexokinase, and glycogen synthase (GS) activities. Insulin-stimulated glucose uptake was decreased by HFD and increased by exercise training (P < 0.01 for both). The opposite effects of HFD and exercise training on insulin-stimulated glucose uptake were associated with similar increases in muscle G-6-P levels (P < 0.05 for both). However, the increase in G-6-P level was accompanied by decreased GS activity without changes in GLUT-4 protein content and hexokinase activities in the HFD group. In contrast, the increase in G-6-P level in the exercise-trained group was accompanied by increased GLUT-4 protein content and hexokinase II (cytosolic) and GS activities. These results suggest that HFD and exercise training affect insulin sensitivity by acting predominantly on different steps of intracellular glucose metabolism. High-fat feeding appears to induce insulin resistance by affecting predominantly steps distal to G-6-P (e.g., glycolysis and glycogen synthesis). Exercise training affected multiple steps of glucose metabolism both proximal and distal to G-6-P. However, increased muscle G-6-P levels in the face of increased glucose metabolic fluxes suggest that the effect of exercise training is quantitatively more prominent on the steps proximal to G-6-P (i.e., glucose transport and phosphorylation).     

Brain contains a functional glucose-6-phosphatase complex capable of endogenous glucose production

Glucose is absolutely essential for the survival and function of the brain. In our current understanding, there is no endogenous glucose production in the brain, and it is totally dependent upon blood glucose. This glucose is generated between meals by the hydrolysis of glucose-6-phosphate (Glc-6-P) in the liver and the kidney. Recently, we reported a ubiquitously expressed Glc-6-P hydrolase, glucose-6-phosphatase-beta (Glc-6-Pase-beta), that can couple with the Glc-6-P transporter to hydrolyze Glc-6-P to glucose in the terminal stages of glycogenolysis and gluconeogenesis. Here we show that astrocytes, the main reservoir of brain glycogen, express both the Glc-6-Pase-beta and Glc-6-P transporter activities and that these activities can couple to form an active Glc-6-Pase complex, suggesting that astrocytes may provide an endogenous source of brain glucose.     

Hepatic glycogen synthesis in the absence of glucokinase: the case of embryonic liver

Glucokinase (GK, hexokinase type IV) is required for the accumulation of glycogen in adult liver and hepatoma cells. Paradoxically, mammalian embryonic livers store glycogen successfully in the absence of GK. Here we address how mammalian embryonic livers, but not adult livers or hepatoma cells, manage to accumulate glycogen in the absence of this enzyme. Hexokinase type I or II (HKI, HKII) substitutes for GK in hepatomas and in embryonic livers. We engineered FTO2B cells, a hepatoma cell line in which GK is not expressed, to unveil the modifications required to allow them to accumulate glycogen. In the light of these results, we then examined glycogen metabolism in embryonic liver. Glycogen accumulation in FTO2B cells can be triggered through elevated expression of HKI or either of the protein phosphatase 1 regulatory subunits, namely PTG or G L. Between these two strategies to activate glycogen deposition in the absence of GK, embryonic livers choose to express massive levels of HKI and HKII. We conclude that although the GK/liver glycogen synthase tandem is ideally suited to store glycogen in liver when blood glucose is high, the substitution of HKI for GK in embryonic livers allows the HKI/liver glycogen synthase tandem to make glycogen independently of the glucose concentration in blood, although it requires huge levels of HK. Moreover, the physiological consequence of the HK isoform switch is that the embryonic liver safeguards its glycogen deposits, required as the main source of energy at birth, from maternal starvation.     

Glycogen synthase- "R": the occurrence and significance of a previously unknown form of GS, found in metabolically active human leucocytes.

1. Glycogen synthesis in un-starved human leucocytes cannot be explained for by the known D- and I-forms of glycogen synthase (GS) (E.C. 2.4.1.11). 2. It can be accounted for by the conversion of GS-D into a non-GS-I species, distinguished by an activation constant for Glc-6-P which is compatible with in vivo activity, and by its markedly less susceptibility to inhibition by ATP and P_i. 3. The new enzyme, denoted GS-R, has been obtained in the partially purified state and kinetically characterized. 4. The presence/absence of GS-R in intact cells can be related to their metabolic status. GS-R can be induced by a glucose load. 5. Preliminary studies indicate GS-D to GS-R conversion involves a partial dephosphorylation, and is blocked by NaF in vitro.     

Effects of altered thyroid status on beta-adrenergic actions on skeletal muscle glycogen metabolism

The effects of hypothyroidism on glycogen metabolism in rat skeletal muscle were studied using the perfused rat hindlimb preparation. Three weeks after propylthiouracil treatment, serum thyroxine was undetectable and muscle glycogen and Glc-6-P were decreased. Basal and epinephrine-stimulated phosphorylase a and phosphorylase b kinase activities were also significantly reduced, as were epinephrine-stimulated cAMP accumulation and cAMP-dependent protein kinase activity. Conversely, basal and epinephrine-stimulated glycogen synthase I activities were significantly higher while the Ka of the enzyme for Glc-6-P was lower in hypothyroid animals. Propylthiouracil-treated rats also had increased phosphoprotein phosphatase activities towards phosphorylase and glycogen synthase and decreased activity of phosphatase inhibitor 1. beta-Adrenergic receptor binding and basal and epinephrine-stimulated adenylate cyclase activities were reduced in muscle particulate fractions from hypothyroid rats. Administration of triiodothyronine to rats for 3 days after 3 weeks of propylthiouracil treatment restored the altered metabolic parameters to normal. It is proposed that the decreased beta-adrenergic responsiveness of the enzymes of glycogen metabolism in hypothyroid rat skeletal muscle is due to increased activity of phosphoprotein phosphatases and to reduced beta-adrenergic receptors and adenylate cyclase activity.     

Effect of feeding aqueous extract of Pterocarpus marsupium on glycogen content of tissues and the key enzymes of carbohydrate metabolism

The Indian traditional system of medicine prescribed plant therapies for diseases including diabetes mellitus called madhumeh in Sanskrit. One such plant mentioned in Ayurveda is Pterocarpus marsupium (PM). In the present study, aqueous extract of PM (1 g/kg PO) was assessed for its effect on glycogen levels of insulin dependent (skeletal muscle and liver), insulin-independent tissues (kidneys and brain) and enzymes such as glucokinase (GK), hexokinase (HK), and phosphofructokinase (PFK). Administration of PM led to decrease in blood glucose levels by 38 and 60% on 15th and 30th day of the experiment. Liver and 2-kidney weight expressed as percentage of body-weight was significantly increased in diabetics (p < 0.0005) vs. normal controls and this alteration in the renal weight (p < 0.0005) but not liver weight was normalized by feeding of PM extract. Renal glycogen content increased by over 10 fold while hepatic and skeletal muscle glycogen content decreased by 75 and 68% in diabetic controls vs. controls and these alteration in glycogen content was partly prevented by PM. Activity of HK, GK and PFK in diabetic controls was 35, 50 and 60% of the controls and PM completely corrected this alteration in PFK and only partly in HK and GK.     

Regulation of insulin binding and glycogenesis by insulin and dexamethasone in cultured rat hepatocytes

Regulation of insulin-binding and basal (insulin-independent) as well as insulin-stimulated glycogen synthesis from [¹⁴C]glucose, net glycogen deposition and glycogen synthase activation by insulin and dexamethasone were studied in primary cultures of adult rat hepatocytes maintained under chemically defined conditions. (1) Insulin receptor number was increased in a dose-dependent fashion by dexamethasone added to the medium between 24 and 48 h of culture and reduced by insulin, whereas ligand affinity remained unaltered. Insulin-induced down-regulation of insulin receptors was not affected by the glucocorticoid. (2) Although the changes in the sensitivity to insulin of glycogen synthesis from glucose and net glycogen deposition paralleled the modulation of the number of insulin receptors, postbinding events appear to be implicated also in the regulation of insulin-sensitivity. (3) Alterations of the responsiveness of glycogen synthesis to insulin caused by the glucocorticoid and/or insulin and by variation between individual rats were inversely related to cellular glycogen contents, suggesting that hepatocellular glycogen content participates in the regulation of insulin-responsiveness of this metabolic pathway. (4) Regulation of insulin-independent glycogenesis in response to an increase from 5 to 10 mM glucose, and of insulin-dependent glycogen synthesis were different. Since the effects of this ‘physiological’ increase in exogenous glucose were small compared to the acute action of insulin, insulin rather than portal venous glucose is considered to represent the prime stimulator of hepatic glycogen synthesis.     

Glycogen synthesis from glucose and fructose in hepatocytes from diabetic rats

In rat hepatocytes, the basal glycogen synthase activation state is decreased in the fed and diabetic states, whereas glycogen phosphorylase a activity decreases only in diabetes. Diabetes practically abolishes the time- and dose-dependent activation of glycogen synthase to glucose especially in the fed state. Fructose, however, is still able to activate this enzyme. Glycogen phosphorylase response to both sugars is operative in all cases. Cell incubation with the combination of 20 mM glucose plus 3 mM fructose produces a great activation of glycogen synthase and a potentiated glycogen deposition in both normal and diabetic conditions. Using radiolabeled sugars, we demonstrate that this enhanced glycogen synthesis is achieved from both glucose and fructose even in the diabetic state. Therefore, the presence of fructose plays a permissive role in glycogen synthesis from glucose in diabetic animals. Glucose and fructose increase the intracellular concentration of glucose 6-phosphate and fructose reduces the concentration of ATP. There is a close correlation between the ratio of the intracellular concentrations of glucose 6-phosphate and ATP (G6-P/ATP) and the activation state of glycogen synthase in hepatocytes from both normal and diabetic animals. However, for any given value of the G6-P/ATP ratio, the activation state of glycogen synthase in diabetic animals is always lower than that of normal animals. This suggests that the system that activates glycogen synthase (synthase phosphatase activity) is impaired in the diabetic state. The permissive effect of fructose is probably exerted through its capacity to increase the G6-P/ATP ratio which may partially increase synthase phosphatase activity, rendering glycogen synthase active.     

Elucidation of the role of fructose 2,6-bisphosphate in the regulation of glucose fluxes in mice using in vivo (13)C NMR measurements of hepatic carbohydrate metabolism.

Fructose 2,6-bisphosphate (Fru-2,6-P2) plays an important role in the regulation of major carbohydrate fluxes as both allosteric activator and inhibitor of target enzymes. To examine the role of Fru-2,6-P2 in the regulation of hepatic carbohydrate metabolism in vivo, Fru-2,6-P2 levels were elevated in ADM mice with adenovirus-mediated overexpression of a double mutant bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (n = 6), in comparison to normal control mice (control, n = 6). The rates of hepatic glycogen synthesis in the ADM and control mouse liver in vivo were measured using new advances in 13C NMR including 3D localization in conjunction with [1-13C]glucose infusion. In addition to glycogen C1, the C6 and C2-C5 signals were measured simultaneously for the first time in vivo, which provide the basis for the estimation of direct and indirect synthesis of glycogen in the liver. The rate of label incorporation into glycogen C1 was not different between the control and ADM group, whereas the rate of label incorporation into glycogen C6 signals was in the ADM group 5.6 +/- 0.5 micro mol.g-1.h-1, which was higher than that of the control group of 3.7 +/- 0.5 micro mol.g-1.h-1 (P < 0.02). The rates of net glycogen synthesis, determined by the glycogen C2-C5 signal changes, were twofold higher in the ADM group (P = 0.04). The results provide direct in vivo evidence that the effects of elevated Fru-2,6-P2 levels in the liver include increased glycogen storage through indirect synthesis of glycogen. These observations provide a key to understanding the mechanisms by which elevated hepatic Fru-2,6-P2 levels promote reduced hepatic glucose production and lower blood glucose in diabetes mellitus.