scholarly journals Glutamine is a good substrate for glycogen synthesis in isolated hepatocytes from 72 h-starved rats, but not from 24 h- or 48 h-starved rats

1992 ◽  
Vol 288 (3) ◽  
pp. 795-799 ◽  
Author(s):  
O Mouterde ◽  
S Claeyssens ◽  
A Chedeville ◽  
A Lavoinne
Keyword(s):  
Glycogen Synthase ◽  
Glycogen Synthesis ◽  
Lactate Production ◽  
Isolated Hepatocytes ◽  
Cell Swelling ◽  
Starvation Period ◽  
Glutamate Concentration ◽  
Glutamine Transport ◽  
Gluconeogenic Substrate

In isolated hepatocytes from 24 h-starved rats, no glycogen synthesis was observed in the presence of glutamine. By contrast, glutamine was the best gluconeogenic substrate to induce glycogen synthesis in isolated hepatocytes from 72 h-starved rats. The effect of glutamine on glycogen synthesis was not accompanied by parallel changes in glucose or lactate production. Glutamine activated glycogen synthase independently of the starvation period; however, the extent of synthase activation was 2-fold higher in isolated hepatocytes from 72 h-starved rats than in hepatocytes from 24 h-starved rats. This increase in synthase activation was associated with increased cell swelling. The rate of glutamine transport was not significantly different in hepatocytes from 24 h- and 72 h-starved rats. By contrast, the intracellular glutamate concentration was 1.5-fold higher after 3 days of starvation in hepatocytes incubated with 5 mM-glutamine. We propose that glutamine may play a key role in the glycogen synthesis observed in vivo after 3 days of starvation.

scholarly journals Inhibition of autophagic proteolysis by inhibitors of phosphoinositide 3-kinase can interfere with the regulation of glycogen synthesis in isolated hepatocytes

2002 ◽  
Vol 368 (3) ◽  
pp. 827-833 ◽  
Author(s):  
Peter F. DUBBELHUIS ◽  
Daphne A. VAN SLUIJTERS ◽  
Edward F.C. BLOMMAART ◽  
Lori A. GUSTAFSON ◽  
George M. VAN WOERKOM ◽  
...  
Keyword(s):  
Amino Acids ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Proteinase Inhibitors ◽  
Glycogen Synthase ◽  
Glycogen Synthesis ◽  
Regulatory Volume Decrease ◽  
Isolated Hepatocytes ◽  
Cell Swelling ◽  
Phosphoinositide 3 Kinase

Amino acid-induced cell swelling stimulates conversion of glucose into glycogen in isolated hepatocytes. Activation of glycogen synthase (GS) phosphatase, caused by the fall in intracellular chloride accompanying regulatory volume decrease, and activation of phosphoinositide 3-kinase (PI 3-kinase), induced by cell swelling, have been proposed as underlying mechanisms. Because PI 3-kinase controls autophagic proteolysis, we examined the possibility that PI 3-kinase inhibitors interfere with glycogen production due to their anti-proteolytic action. The PI 3-kinase inhibitor wortmannin inhibited endogenous proteolysis, the production of glycogen from glucose and the activity of active (dephosphorylated) GS (GSa) in the absence of added amino acids. The stimulation by amino acids of glycogen production and of GSa was only slightly affected by wortmannin. These effects of wortmannin could be mimicked by proteinase inhibitors. A combination of leucine, phenylalanine and tyrosine, which we showed previously to stimulate PI 3-kinase-dependent phosphorylation of ribosomal protein S6, did not stimulate glycogen production from glucose. In contrast with wortmannin, LY294002, another PI 3-kinase inhibitor, strongly inhibited both glycogen synthesis and GSa activity, irrespective of the presence of amino acids. Inhibition of glycogen synthesis by LY294002 could be ascribed in part to increased glycogenolysis and glycolysis. It is concluded that, in hepatocytes, activation of PI 3-kinase may not be responsible for the stimulation of glycogen synthesis by amino acids; LY294002 inhibits glycogen synthesis and stimulates glycogen breakdown by a mechanism that is unrelated to its action as an inhibitor of PI 3-kinase.

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

2008 ◽  
Vol 294 (1) ◽  
pp. E28-E35 ◽  
Author(s):  
Michale Bouskila ◽  
Michael F. Hirshman ◽  
Jørgen Jensen ◽  
Laurie J. Goodyear ◽  
Kei Sakamoto
Keyword(s):  
Skeletal Muscle ◽  
Glucose Uptake ◽  
Muscle Glycogen ◽  
Glycogen Content ◽  
Glycogen Synthase ◽  
Glycogen Synthesis ◽  
Wild Type ◽  
Muscle Glycogen Synthesis ◽  
Mutant Forms

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 GSK3α and -β genes are replaced with mutant forms (GSK3α/βS21A/S21A/S9A/S9A), which are nonresponsive to insulin. Although insulin failed to promote dephosphorylation and activation of GS in GSK3α/βS21A/S21A/S9A/S9Amice, 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.

scholarly journals Transgenic overexpression of protein targeting to glycogen markedly increases adipocytic glycogen storage in mice

2007 ◽  
Vol 292 (3) ◽  
pp. E952-E963 ◽  
Author(s):  
Michael J. Jurczak ◽  
Arpad M. Danos ◽  
Victoria R. Rehrmann ◽  
Margaret B. Allison ◽  
Cynthia C. Greenberg ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Fatty Acid ◽  
Glycogen Synthase ◽  
Glycogen Synthesis ◽  
Transgenic Animals ◽  
Glycogen Metabolism ◽  
Glycogen Storage ◽  
Fatty Acid Binding ◽  
Fat Depots

Adipocytes express the rate-limiting enzymes required for glycogen metabolism and increase glycogen synthesis in response to insulin. However, the physiological function of adipocytic glycogen in vivo is unclear, due in part to the low absolute levels and the apparent biophysical constraints of adipocyte morphology on glycogen accumulation. To further study the regulation of glycogen metabolism in adipose tissue, transgenic mice were generated that overexpressed the protein phosphatase-1 (PP1) glycogen-targeting subunit (PTG) driven by the adipocyte fatty acid binding protein (aP2) promoter. Exogenous PTG was detected in gonadal, perirenal, and brown fat depots, but it was not detected in any other tissue examined. PTG overexpression resulted in a modest redistribution of PP1 to glycogen particles, corresponding to a threefold increase in the glycogen synthase activity ratio. Glycogen synthase protein levels were also increased twofold, resulting in a combined greater than sixfold enhancement of basal glycogen synthase specific activity. Adipocytic glycogen levels were increased 200- to 400-fold in transgenic animals, and this increase was maintained to 1 yr of age. In contrast, lipid metabolism in transgenic adipose tissue was not significantly altered, as assessed by lipogenic rates, weight gain on normal or high-fat diets, or circulating free fatty acid levels after a fast. However, circulating and adipocytic leptin levels were doubled in transgenic animals, whereas adiponectin expression was unchanged. Cumulatively, these data indicate that murine adipocytes are capable of storing far higher levels of glycogen than previously reported. Furthermore, these results were obtained by overexpression of an endogenous adipocytic protein, suggesting that mechanisms may exist in vivo to maintain adipocytic glycogen storage at a physiological set point.

scholarly journals Overexpression of SH2-Containing Inositol Phosphatase 2 Results in Negative Regulation of Insulin-Induced Metabolic Actions in 3T3-L1 Adipocytes via Its 5′-Phosphatase Catalytic Activity

2001 ◽  
Vol 21 (5) ◽  
pp. 1633-1646 ◽  
Author(s):  
Tsutomu Wada ◽  
Toshiyasu Sasaoka ◽  
Makoto Funaki ◽  
Hiroyuki Hori ◽  
Shihou Murakami ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Glucose Uptake ◽  
Phosphatase Activity ◽  
Insulin Signaling ◽  
Glycogen Synthase ◽  
Glycogen Synthesis ◽  
Dominant Negative ◽  
Inositol Phosphatase

ABSTRACT Phosphatidylinositol (PI) 3-kinase plays an important role in various metabolic actions of insulin including glucose uptake and glycogen synthesis. Although PI 3-kinase primarily functions as a lipid kinase which preferentially phosphorylates the D-3 position of phospholipids, the effect of hydrolysis of the key PI 3-kinase product PI 3,4,5-triphosphate [PI(3,4,5)P3] on these biological responses is unknown. We recently cloned rat SH2-containing inositol phosphatase 2 (SHIP2) cDNA which possesses the 5′-phosphatase activity to hydrolyze PI(3,4,5)P3 to PI 3,4-bisphosphate [PI(3,4)P2] and which is mainly expressed in the target tissues of insulin. To study the role of SHIP2 in insulin signaling, wild-type SHIP2 (WT-SHIP2) and 5′-phosphatase-defective SHIP2 (ΔIP-SHIP2) were overexpressed in 3T3-L1 adipocytes by means of adenovirus-mediated gene transfer. Early events of insulin signaling including insulin-induced tyrosine phosphorylation of the insulin receptor β subunit and IRS-1, IRS-1 association with the p85 subunit, and PI 3-kinase activity were not affected by expression of either WT-SHIP2 or ΔIP-SHIP2. Because WT-SHIP2 possesses the 5′-phosphatase catalytic region, its overexpression marked by decreased insulin-induced PI(3,4,5)P3 production, as expected. In contrast, the amount of PI(3,4,5)P3 was increased by the expression of ΔIP-SHIP2, indicating that ΔIP-SHIP2 functions in a dominant-negative manner in 3T3-L1 adipocytes. Both PI(3,4,5)P3 and PI(3,4)P2 were known to possibly activate downstream targets Akt and protein kinase Cλ in vitro. Importantly, expression of WT-SHIP2 inhibited insulin-induced activation of Akt and protein kinase Cλ, whereas these activations were increased by expression of ΔIP-SHIP2 in vivo. Consistent with the regulation of downstream molecules of PI 3-kinase, insulin-induced 2-deoxyglucose uptake and Glut4 translocation were decreased by expression of WT-SHIP2 and increased by expression of ΔIP-SHIP2. In addition, insulin-induced phosphorylation of GSK-3β and activation of PP1 followed by activation of glycogen synthase and glycogen synthesis were decreased by expression of WT-SHIP2 and increased by the expression of ΔIP-SHIP2. These results indicate that SHIP2 negatively regulates metabolic signaling of insulin via the 5′-phosphatase activity and that PI(3,4,5)P3 rather than PI(3,4)P2 is important for in vivo regulation of insulin-induced activation of downstream molecules of PI 3-kinase leading to glucose uptake and glycogen synthesis.

Glycogen Metabolism in Psoriatic Epidermis and in Regenerating Epidermis

1984 ◽  
Vol 67 (3) ◽  
pp. 291-298 ◽  
Author(s):  
C. S. Harmon ◽  
P. J. R. Phizackerley
Keyword(s):  
Glycogen Content ◽  
Glycogen Synthase ◽  
Quantitative Estimation ◽  
Glycogen Synthesis ◽  
Pentose Phosphate ◽  
Normal Epithelium ◽  
Glycogen Synthase Activity ◽  
Psoriatic Lesions ◽  
Wound Epithelium

1. The observation that the glycogen content of epidermis from psoriatic lesions and from regenerating wound epithelium is increased has been confirmed by quantitative estimation. 2. In epidermis from psoriatic lesions, although the proportion of glycogen synthase in the I form is only about 5% of the total and similar to control values, total glycogen synthase activity is increased approximately 4-fold and hence glycogen synthase I activity is increased to the same extent. In contrast, total phosphorylase activity is only slightly increased and, since the proportion of the enzyme in the a form is reduced, phosphorylase a activity is similar to control values. 3. In epidermis from psoriatic lesions, the concentration of UDP-glucose is approximately doubled, and the concentrations of fructose 1,6-bisphosphate and of 6-phosphogluconate are increased approximately 5-fold. It is concluded that rates of glycogen synthesis, of glycolysis and of the pentose phosphate pathway are all enhanced in vivo and in consequence the rate of glucose uptake by psoriatic epidermis must be increased. 4. In the non-involved epidermis of psoriatic patients the glycogen content is within normal limits, and although total glycogen synthase activity is increased the ratio of glycogen synthase I to phosphorylase a is maintained at normal levels by the appropriate phosphorylation of both enzymes. 5. In regenerating wound epithelium in the pig, the changes in enzyme activity and in metabolite concentration closely resemble those found in epithelium from psoriatic lesions except that in wound epithelium the proportion of phosphorylase in the a form is increased relative to normal epithelium.

scholarly journals Validating GSK3 as an in vivo target of lithium action

2009 ◽  
Vol 37 (5) ◽  
pp. 1133-1138 ◽  
Author(s):  
W. Timothy O'Brien ◽  
Peter S. Klein
Keyword(s):  
Glycogen Synthase ◽  
Insulin Signalling ◽  
Lithium Treatment ◽  
Glycogen Synthesis ◽  
Model Systems ◽  
Glycogen Synthase Kinase 3 ◽  
Therapeutic Effects ◽  
Inositol Monophosphatase ◽  
Direct Target

Lithium is widely used to treat bipolar disorder, but its mechanism of action in this disorder is unknown. Lithium directly inhibits GSK3 (glycogen synthase kinase 3), a critical regulator of multiple signal transduction pathways. Inhibition of GSK3 provides a compelling explanation for many of the known effects of lithium, including effects on early development and insulin signalling/glycogen synthesis. However, lithium also inhibits inositol monophosphatase, several structurally related phosphomonoesterases, phosphoglucomutase and the scaffolding function of β-arrestin-2. It is not known which of these targets is responsible for the behavioural or therapeutic effects of lithium in vivo. The present review discusses basic criteria that can be applied to model systems to validate a proposed direct target of lithium. In this context, we describe a set of simple behaviours in mice that are robustly affected by chronic lithium treatment and are similarly affected by structurally diverse GSK3 inhibitors and by removing one copy of the Gsk3b gene. These observations, from several independent laboratories, support a central role for GSK3 in mediating behavioural responses to lithium.

scholarly journals The activity of glycogen synthase phosphatase limits hepatic glycogen deposition in the adrenalectomized starved rat

1983 ◽  
Vol 214 (2) ◽  
pp. 539-545 ◽  
Author(s):  
M Bollen ◽  
G Gevers ◽  
W Stalmans
Keyword(s):  
Phosphatase Activity ◽  
Protein Phosphatase ◽  
Glycogen Synthase ◽  
Glycogen Synthesis ◽  
Hepatic Glycogen ◽  
Complete Inactivation ◽  
Glycogen Deposition ◽  
Initial Recovery ◽  
No Activation

Hepatocytes from adrenalectomized 48 h-starved rats responded to increasing glucose concentrations with a progressively more complete inactivation of phosphorylase. Yet no activation of glycogen synthase occurred, even in a K+-rich medium. Protein phosphatase activities in crude liver preparations were assayed with purified substrates. Adrenalectomy plus starvation decreased synthase phosphatase activity by about 90%, but hardly affected phosphorylase phosphatase activity. Synthase b present in liver extracts from adrenalectomized starved rats was rapidly and completely converted into the a form on addition of liver extract from a normal fed rat. Glycogen synthesis can be slowly re-induced by administration of either glucose or cortisol to the deficient rats. In these conditions there was a close correspondence between the initial recovery of synthase phosphatase activity and the amount of synthase a present in the liver. The latter parameter was strictly correlated with the measured rate of glycogen synthesis in vivo. The decreased activity of synthase phosphatase emerges thus as the single factor that limits hepatic glycogen deposition in the adrenalectomized starved rat.

scholarly journals Cyclin Partners Determine Pho85 Protein Kinase Substrate Specificity In Vitro and In Vivo: Control of Glycogen Biosynthesis by Pcl8 and Pcl10

1998 ◽  
Vol 18 (6) ◽  
pp. 3289-3299 ◽  
Author(s):  
Dongqing Huang ◽  
Jason Moffat ◽  
Wayne A. Wilson ◽  
Lynda Moore ◽  
Christine Cheng ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acid Phosphatase ◽  
Protein Kinase ◽  
Substrate Specificity ◽  
Glycogen Synthase ◽  
Glycogen Synthesis ◽  
Glycogen Accumulation ◽  
Acid Phosphatase Gene

ABSTRACT In Saccharomyces cerevisiae, PHO85 encodes a cyclin-dependent protein kinase (Cdk) with multiple roles in cell cycle and metabolic controls. In association with the cyclin Pho80, Pho85 controls acid phosphatase gene expression through phosphorylation of the transcription factor Pho4. Pho85 has also been implicated as a kinase that phosphorylates and negatively regulates glycogen synthase (Gsy2), and deletion of PHO85 causes glycogen overaccumulation. We report that the Pcl8/Pcl10 subgroup of cyclins directs Pho85 to phosphorylate glycogen synthase both in vivo and in vitro. Disruption of PCL8 and PCL10 caused hyperaccumulation of glycogen, activation of glycogen synthase, and a reduction in glycogen synthase kinase activity in vivo. However, unlikepho85 mutants, pcl8 pcl10 cells had normal morphologies, grew on glycerol, and showed proper regulation of acid phosphatase gene expression. In vitro, Pho80-Pho85 complexes effectively phosphorylated Pho4 but had much lower activity toward Gsy2. In contrast, Pcl10-Pho85 complexes phosphorylated Gsy2 at Ser-654 and Thr-667, two physiologically relevant sites, but only poorly phosphorylated Pho4. Thus, both the in vitro and in vivo substrate specificity of Pho85 is determined by the cyclin partner. Mutation ofPHO85 suppressed the glycogen storage deficiency ofsnf1 or glc7-1 mutants in which glycogen synthase is locked in an inactive state. Deletion of PCL8and PCL10 corrected the deficit in glycogen synthase activity in both the snf1 and glc7-1 mutants, but glycogen synthesis was restored only in the glc7-1mutant strain. This genetic result suggests an additional role for Pho85 in the negative regulation of glycogen accumulation that is independent of Pcl8 and Pcl10.

Effect of hepatic nerves on disposition of an intraduodenal glucose load

1993 ◽  
Vol 265 (3) ◽  
pp. E487-E496 ◽  
Author(s):  
M. C. Moore ◽  
G. I. Shulman ◽  
A. Giaccari ◽  
M. J. Pagliassotti ◽  
G. Cline ◽  
...  
Keyword(s):  
Glycogen Synthase ◽  
Glycogen Synthesis ◽  
Lactate Production ◽  
Glucose Infusion ◽  
Hepatic Glycogen ◽  
Indirect Pathway ◽  
Hepatic Glucose ◽  
Hepatic Glucose Uptake ◽  
Hepatic Nerves ◽  
Glycogen Synthase Activity

We examined the disposition of a continuous 4-h intraduodenal glucose infusion (8 mg.kg-1 x min-1, labeled with [1-13C]glucose and [3-3H]glucose) in nine conscious hepatic-denervated dogs. Cumulative net hepatic uptakes (in grams of glucose equivalents) were 13.7 +/- 2.5 glucose, 3.1 +/- 0.6 gluconeogenic amino acids, and 0.8 +/- 0.1 glycerol. Net hepatic glycogen synthesis totalled 11.0 +/- 0.9 g, 55-62% via the direct pathway. All values were similar to those in hepatic-innervated dogs. Glycogen synthase activity and rate of glycogen synthesis were positively correlated (r2 = 0.913, P < 0.05). Variability in net hepatic glycogen synthesis and the mass of glycogen synthesized via the indirect pathway was reduced in hepatic-denervated dogs (P < 0.05). In conclusion, the glycemic response and rate of net glycogen synthesis during an intraduodenal glucose infusion was no different in hepatic-denervated and -innervated dogs. Net hepatic glucose uptake was sufficient to account for all net hepatic glycogen synthesis and lactate production, consistent with an intrahepatic source of gluconeogenic precursors for glycogen synthesis via the indirect pathway. Hepatic nerves appear responsible for much of the variability in net hepatic glycogen synthesis and in the mass of glycogen synthesized via the indirect pathway in normal dogs.

Measurement of myocardial glycogen synthesis in diabetic and fasted rats

1990 ◽  
Vol 258 (1) ◽  
pp. E184-E190 ◽  
Author(s):  
M. R. Laughlin ◽  
W. A. Petit ◽  
R. G. Shulman ◽  
E. J. Barrett
Keyword(s):  
Insulin Infusion ◽  
Glycogen Synthase ◽  
Glycogen Synthesis ◽  
Alpha Activity ◽  
Tissue Extracts ◽  
13C Nuclear Magnetic Resonance ◽  
Average Activity ◽  
Rate Limiting ◽  
Fasted Rats

Diabetes and fasting provoke an increase in heart glycogen content, despite a decline in the amount of active glycogen synthase present. To determine if the activity of glycogen synthase i is still rate limiting for glycogen synthesis, we used 13C-nuclear magnetic resonance to measure the in vivo rate of glycogen synthesis and compared this with the activity of glycogen synthase and phosphorylase measured in tissue extracts using physiological concentrations of substrates and activators. In the basal state the activity of glycogen synthase i was depressed in the diabetic and fasted hearts (P less than 0.01). The rate of heart glycogen synthesis was measured during a 50-min infusion of D-[1-13C]-glucose (10 mg/min) and insulin (1 U/min) and averaged 0.32 +/- 0.04 mumol.min-1.g wet wt-1 in controls and was diminished in both the diabetic (0.18 +/- 0.04 mumol.min-1.g wet wt-1) and fasted (0.16 +/- 0.03 mumol.min-1.g wet wt-1) and fasted (0.16 +/- 0.03 mumol.min-1.g wet wt-1) rats (P less than 0.05 for each). During the glucose and insulin infusion the average activity of glycogen synthase i was greater in control than diabetic or fasted hearts (P less than 0.01 for each) and approximated the rates of net glycogen synthesis in each group. In contrast, there were no significant differences in phosphorylase alpha activity, measured in tissue extracts, among the three groups. Furthermore, although this phosphorylase alpha activity greatly exceeded synthase activity, it did not appear to be expressed in vivo. We conclude that in normal, diabetic, and fasted rats, glycogen synthase is rate limiting for glycogen synthesis.

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