This decrease was time-dependent, with a significant effect being observed after 2 h. During this time period, the fractional activity of GS also fell slightly, decreasing after 5 h of glucose starvation from 0. This again caused a dramatic activation of GS to a fractional activity of 0.

To confirm that alterations in GS activity were a result of previous glucose withdrawal, cells were incubated with DMEM supplemented with 5. These data indicate that the stimulation of GS is predominantly due to the action of glucose and requires previous glycogen depletion because of glucose removal, but other components of the different media may modulate the magnitude of the response.

The increase in GS fractional activity in response to glucose was dependent on the duration of previous glucose deprivation and, hence, inversely related to the glycogen content of the cells.

In the absence of glycogen depletion, no significant activation of GS by glucose was observed Fig. Total GS activity remained essentially unchanged in all conditions, indicating that alteration in the expression of the GS polypeptide was not involved not shown.

Glucose treatment 6. The stimulation of GS was dependent on the concentration of glucose added, with significant stimulatory effects being observed in response to 1. The combined effects of glucose readdition and insulin on cells cultured in the absence of glucose was then examined.

In the absence of readministration of glucose, stimulatory effects of insulin on GS activity were minimal after glycogen depletion data not shown. This value further increased to 1, An increase in the rate of 2-deoxyglucose uptake was observed in myoblasts deprived of glucose for increasing time, reaching 1.

The effect of insulin treatment on 2-deoxyglucose uptake was examined in control and glucose-starved myoblasts Fig. To investigate the mechanisms by which glucose activates GS, selective inhibitors of signaling pathways known to be stimulated by insulin were used Fig.

Preincubation of cells with rapamycin, which selectively inhibits the activation of p70 s6k , failed to inhibit this stimulatory effect of glucose on GS activation.

Treatment of glycogen-depleted myoblasts with either the MEK inhibitor, PD, or the PI 3-kinase inhibitor wortmannin partly reduced the activity state of GS obtained in response to glucose readministration.

However, this is essentially attributable to these inhibitors reducing the basal activity state of GS, as previously observed Therefore, these findings indicate that the mechanisms involved in the activation of GS by glucose are independent of the activity of PI 3-kinase, the classical mitogen-activated protein kinase pathway, and the rapamycin-sensitive pathway leading to activation of p70 s6k.

Furthermore, during glucose readministration, there was no observable decrease in the activity of GSK-3 and no detectable phosphorylation of that protein, as detected by phosphospecific anti—GSK-3 antibodies Fig. No changes in the activity of protein kinase B were detected not shown.

Glucosephosphate G6P is a potent allosteric activator of GS 19 , whereas a number of small metabolites, such as ATP, ADP, and AMP, are capable of inhibiting GS activity After refeeding of glycogen-depleted cells, fractionation of extracts failed to significantly alter GS activity ratio 0.

The activity of endogenous PPs against GS was then measured in extracts from control and glycogen-depleted cells. Because it has been demonstrated that GS becomes a better substrate for PPs in the presence of G6P 21 , this was carried out over a range of G6P concentrations.

No differences were found in the rate of activation of GS by endogenous phosphatases in extracts prepared from control and glycogen-depleted cells. For example, at a physiological concentration of 0.

Therefore, glycogen depletion does not appear to stimulate phosphatase activity against GS, at least when measured subsequently in cell extracts. The results presented here demonstrate dramatic effects on both glycogen synthesis and glycogen synthase after glucose re-administration to human myoblasts that have previously been depleted of glycogen.

This is expressed as a fractional activity, i. Similar methodologies have been applied to studies of GS activity in muscle, where activity has been reported as a fractional velocity or as A0. The molecular mechanism responsible for this stimulatory effect of glucose is a primary concern.

Inhibition of GSK-3 is thought to be the key event in the activation of muscle GS by insulin; however, selective inhibitors of the known insulin-stimulated pathways leading potentially to GSK-3 inhibition failed to prevent the activation of GS during the refeeding of glucose to glycogen-depleted cells Fig.

Furthermore, no inhibition or phosphorylation of GSK-3 was observed in response to glucose Fig. However, the involvement of GSK-3 inhibition in this process cannot be completely ruled out, as GSK-3 could be inhibited by an alternative, yet unidentified, mechanism not involving phosphorylation of the single serine residue recognized by the phosphospecific antibodies and not being retained when the enzyme is assayed in cellular extracts.

In that regard, allosteric regulation of GSK-3 by glycogen or small metabolites must be considered. A recent study performed in rat skeletal muscle demonstrated an exercise-induced increase in GS activity accompanied by inactivation of GSK-3 The activity of GS is dependent on the relative contribution of GSK-3 and PP1 in determining the phosphorylation state of the enzyme.

Phosphorylation of the glycogen-binding subunit of PP1 has been postulated as a mechanism for activation of that enzyme in response to insulin; however, a growing body of evidence suggests that this is not the case rev. After exercise in human muscle, no increase in total or glycogen-associated PP activity was observed; however, the rate of reactivation of GS by endogenous phosphatases was increased, suggesting that GS became a better substrate for PP1 after exercise In the present work, no increase in the phosphatase activity directed toward GS was observed after the glycogen depletion of human myoblasts.

However, it is possible that PPs, principally PP1, can alter GS activity in vivo, independent of changes in the intrinsic activity of the phosphatase detected in in vitro assays. For example, effects of changes in intracellular G6P concentration to enhance the ability of PP1 to dephosphorylate GS 21 , or the effect of overexpression of a glycogen-binding subunit of PP1 in CHO cells, which results in an increase in basal and insulin-stimulated glycogen synthesis 25 , would not be detected as a change in phosphatase activity in cell extracts.

Work in rat adipocytes has previously shown that glucose can activate GS in the absence of insulin 26 , an effect attributed to increased glucose transport leading to accumulation of G6P, which can then increase PP activity toward GS. The extent to which such a mechanism contributes to the observations in muscle reported in this study remains to be established, although studies in mouse diaphragm have demonstrated a modest stimulatory effect of glucose on GS activity The model from rat adipocytes does not account for the effect of glycogen stores on the activation, and a recent study in human myotubes has indicated that glycogen repletion, after glucose administration to cells that have been depleted of glycogen by overexpression of glycogen phosphorylase, is not dependent on increases in intracellular G6P concentration However, metabolic flux through G6P has been suggested as the hub of coordinate regulation of muscle glycogen synthesis 29 ; changes in GS activity are a consequence of alterations in intracellular G6P concentration, a result of increased glucose uptake and hexokinase activity.

G6P is an allosteric activator of GS, but also serves to make GS a better substrate for dephosphorylation by PPs Low glycogen content in vivo could increase glucose uptake and hexokinase activity, resulting in an increase in intracellular G6P concentration and consequently in an increase in GS activity, although no obvious mechanism for this is known.

A further consideration is whether glycogen depletion followed by glucose refeeding alters the distribution of GS and its regulatory proteins within the myoblast. PP1, GSK-3, and GS are all capable of redistributing within the cell in response to metabolic and hormonal stimuli 5 , 30 , If glucose or one of its metabolites caused either increased interaction of GS and PP1 or decreased interaction of GS and GSK-3 in glycogen-depleted cells, this could explain the activation of GS in response to this nutrient.

An exercise-induced increase in glycogen resynthesis after glycogen depletion can only occur if adequate substrate is available. Indeed, after glucose deprivation of cultured human muscle cells, there was a twofold increase in the subsequent rate of 2-deoxyglucose uptake.

Increased glucose transporter numbers at the plasma membrane has been suggested as the mechanism for increased glucose uptake postexercise, and in rat skeletal muscle, a 1. The relationship between glycogen and membrane permeability to glucose is apparently reciprocal, with increasing glycogen content postexercise leading to a decrease in glucose uptake In summary, the work reported here has highlighted the important role of glucose in stimulating glycogen synthesis in muscle after glycogen depletion.

Recent elegant work using 31 P nuclear magnetic resonance to study glycogen metabolism in human muscle in vivo is yielding important information on the effects of glycogen on glycogen synthesis However, work with human muscle cells in culture has the benefit of providing a means of independently varying glycogen content, extracellular glucose concentration, and insulin levels.

The data reported here are consistent with work in human muscle in vivo showing activation of GS after exercise-mediated glycogen depletion 22 , leading to glycogen repletion in an insulin-independent phase The molecular mechanism leading to the activation of GS by glucose after glycogen depletion is as yet undefined, but is apparently distinct from the upstream signaling mechanisms used in the actions of insulin on this parameter.

Recent evidence suggests that AMP-activated protein kinase may play a role in insulin-independent exercise-stimulated glucose uptake The possibility that it is also involved in the stimulation of glycogen synthesis after glycogen depletion is an attractive one, although no evidence is currently available to support this.

Clearly, how the two different signaling pathways interact has implications for the overall control of glycogen synthesis in control subjects and patients with type 2 diabetes. Total glycogen content and GS activity in myoblasts cultured in the absence of glucose; effect of glucose readdition.

In the absence of glucose readdition, the GS activity was less, at every time point, then what was observed in nonstarved cultures. The fractional activity of glycogen synthase was determined in these extracts ——— , left y- axis. Concentration- and time-dependent effects of glucose on GS activity in cells cultured in the absence of glucose.

Extracts were prepared and the fractional activity of glycogen synthase determined. Combined effects of insulin and glucose on GS activity and glycogen synthesis in cells cultured in the absence or presence of glucose. The insulin-mediated fold-activation over basal values is also indicated.

At the times indicated, the rate of glucose uptake was determined A. Effect of selective inhibitors on activation of GS by glucose. Extracts were prepared and the activity of GS determined. Effects of insulin and glucose on GSK-3 activity and phosphorylation. Extracts were prepared and GSK-3 activity determined.

This work was supported by a grant from Diabetes U. held a Cooperative Awards in Science and Engineering studentship from the Biotechnology and Biological Sciences Research Council BBSRC , U. holds a studentship from BBSRC. We thank Dr.

Mark Walker and Prof. Doug Turnbull for obtaining the initial muscle biopsies and Dorothy Fittes for technical assistance with the cell culture. Address correspondence and reprint requests to Stephen J. Yeaman, School of Biochemistry and Genetics, the Medical School, University of Newcastle, Newcastle upon Tyne NE2 4HH, U.

E-mail: s. yeaman ncl. has received funds from Novo Nordisk. is employed by and holds stock in Novo Nordisk. holds stock in Xcellsyz, has received honoraria from Novo Nordisk and Glaxo Wellcome, and has received funds from Novo Nordisk and SmithKline Beecham.

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nature letters article. Abstract IT is well known that glycogen is utilized during muscular work, but there is very little information available about the resynthesis of glycogen after exhaustive exercise. Access through your institution.

Buy or subscribe. Change institution. Learn more. References Goldstein, M. Article Google Scholar Bergström, J. Google Scholar Bergström, J. Article ADS Google Scholar Hultman, E. Author information Authors and Affiliations Clinical Laboratory, St.

View author publications. Rights and permissions Reprints and permissions. About this article Cite this article BERGSTRÖM, J. Copy to clipboard. This article is cited by Sexual Dimorphism in Substrate Metabolism During Exercise Stéphanie M. Abo Elisa Casella Anita T.

Layton Bulletin of Mathematical Biology Analysis of sex-based differences in energy substrate utilization during moderate-intensity aerobic exercise Antonella Cano Lucia Ventura Andrea Manca European Journal of Applied Physiology A century of exercise physiology: key concepts in regulation of glycogen metabolism in skeletal muscle Abram Katz European Journal of Applied Physiology Do Sex Differences in Physiology Confer a Female Advantage in Ultra-Endurance Sport?

Nicholas B. Tiller Kirsty J. Elliott-Sale Guillaume Y. Millet Sports Medicine Enhanced skeletal muscle glycogen repletion after endurance exercise is associated with higher plasma insulin and skeletal muscle hexokinase 2 protein levels in mice: comparison of level running and downhill running model Yumiko Takahashi Juli Sarkar Hideo Hatta Journal of Physiology and Biochemistry

Our results are consistent with previous work showing that 4 days of hyperglycaemia reduces basal [ 37 ] and insulin-stimulated glycogen synthesis [ 37 , 38 ]. Whether high glucose treatment slows or decreases the differentiation of myocytes to myotubes is unknown. However, the morphological profile of the myotubes was unchanged following any of the treatments investigated here.

The increased glycogen synthesis and mRNA expression of PGC1, GLUT4 and MEF2C after long-term treatment with insulin may suggest enhanced myogenesis, although this is unlikely since the morphological pattern was unaltered. Marked changes in morphology and skeletal muscle cell differentiation have been reported following 5 days of exposure to high concentrations of troglitazone, another thiazolidinedione [ 39 ].

However, after rosiglitazone exposure, the morphology of the primary human skeletal muscle cells was unaltered. The difference between rosiglitazone and troglitazone in their effects on muscle morphology may be due to differences in the concentration of glitazone studied or a compound-specific effect.

Interestingly, short-term exposure 1 day of primary human muscle cells to troglitazone enhanced insulin action, similar to the results reported here for rosiglitazone [ 39 ]. Most in vitro studies conducted to dissect the effects of rosiglitazone and metformin on metabolic and gene-regulatory responses have been performed using adipocytes or hepatocytes.

Direct effects of troglitazone have previously been reported on cell lipid metabolism [ 40 ], gene expression [ 41 ] and glucose uptake [ 39 , 42 ] in human skeletal muscle.

Here we report that 8 days of rosiglitazone treatment increases glycogen synthesis in the absence or presence of insulin, and these changes are accompanied by increased expression of GLUT4 and PGC1 mRNA.

In our hands, GLUT4 and GLUT1 mRNA and protein expression are positively correlated in differentiating human skeletal muscle cells data available upon request. In contrast to these findings, in L6 myotubes rosiglitazone has been suggested to increase GLUT4 translocation, but not protein or mRNA content [ 43 ].

However, this particular conclusion was based on a much shorter rosiglitazone exposure 24 h , which may be insufficient to mediate gene-regulatory responses. Thus, in human muscle cells, part of the insulin-sensitising effect of rosiglitazone can be attributed to changes in mRNA and protein expression of glucose transporters.

AICAR activates AMPK and stimulates glucose uptake in skeletal muscle [ 45 ]. In the present study, acute AICAR treatment increased glycogen synthesis, while chronic 8 days AICAR treatment was without effect on insulin action, as assessed by glycogen synthesis, or expression of GLUT4 or PGC1 mRNAs.

The acute responses are consistent with findings in rat skeletal muscle [ 31 , 46 ] and perfused hindlimb [ 47 ], where AICAR treatment increased GLUT4 expression and translocation respectively. Furthermore, chronic AICAR treatment of Clone 9 cells increased glucose uptake [ 48 ].

Based on the observation that GLUT1 content was unchanged in the plasma membranes of AICAR-treated Clone 9 cells, the effects on glucose uptake were proposed to activate pre-existing plasma membrane GLUT1 transporters. Nevertheless, our present data reveal that chronic AICAR treatment in human myotubes selectively increases GLUT1, but not GLUT4, expression.

Moreover, in human myotubes exposed to AICAR for 8 days, mRNA expression of MEF2C was reduced. Whether AICAR has a negative effect on the regulation of myogenesis in this cell system remains to be demonstrated, but no such effects have previously been suggested.

However, MEF2C is involved in the regulation of differentiation-specific genes [ 16 ] and suppression of this gene may repress gene-regulatory responses. Metformin treatment of cells increases GLUT4 recruitment and increases glycogen synthesis [ 49 ].

Here we demonstrate that chronic metformin exposure increases the expression of GLUT4, PGC1 and all MEF2 isoforms A, C and D and increases the rate of basal and insulin-stimulated glycogen synthesis.

Metformin has been suggested to increase AMPK activity [ 29 ]. Interestingly, in human myotubes, metformin treatment was more potent than AICAR in increasing GLUT4 expression and in enhancing insulin sensitivity, suggesting that metformin activates additional AMPK-independent pathways.

In summary, exposure of human myotubes to metformin and rosiglitazone, two currently available antidiabetic agents, was associated with direct enhancement of the action of insulin in cultured skeletal muscle. The increase in insulin action correlated with increased mRNA expression of GLUT4.

Increased expression of PGC1, GLUT4 and MEF2 mRNA after antidiabetic treatment provides a molecular mechanism for increased carbohydrate metabolism, cellular survival and gene-regulatory responses that confer improved skeletal muscle metabolic function in insulin-resistant and diabetic subjects.

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Total glycogen synthase protein levels in homogenates, however, were unaffected by linoleate Fig. To address this discrepancy, and to compare the distribution of activity in L6 myotubes with that observed in rat muscles, we also assayed glycogen synthase activity in fractions from both control and linoleate-treated cells.

Total activities Fig. In this case, loss of activity in the cytosol was more comparable with the increased activity measured in the TIF, suggesting that the enzyme was indeed translocating in response to both insulin and linoleate. To determine the activation state of glycogen synthase in the different fractions, we also measured the activity in the presence of a more physiological G6P concentration Fig.

Insulin tended to increase the activity observed in the cytosol from control cells Fig. Increased activity was also observed in the TIF from insulin-stimulated control cells Fig.

In contrast, the glycogen synthase activity measured in the TIF from linoleate-pretreated cells Fig. In fact, lipid pretreatment significantly reduced the activation state of the enzyme observed in the TIF upon insulin stimulation relative to that in the TIF from control cells Fig.

The data presented in Fig. Because an insulin-sensitive pool from control cells was recovered in the same subcellular fraction, we examined potential causes of the lipid-dependent redistribution of the enzyme and attempted to distinguish biochemically between the pools.

Increased lipid availability can increase flux through the hexosamine pathway and promote protein glycosylation Hawkins et al. No evidence, however, for such a modification was observed in immunoprecipitates of the enzyme with an antibody specific for O -glycosylated protein in subsequent immunoblots not shown.

In addition, we were unable to determine any changes in proteins associated with glycogen synthase in immunoprecipitates, after SDS—PAGE and silver staining not shown. We also attempted to separate the two pools in the TIF by sequential solubilization involving Triton X, SDS- and urea-containing buffers.

No further fractionation, however, of the enzyme was achieved not shown. Immunoblotting using antibodies specific for the glycogen targeting protein PTG indicated that this was mostly recovered in the cytosol from L6 myotubes, with a minor proportion located in the TIF not shown , while the targeting protein R GL G M was not detected in L6 cell fractions, but was observed in the membrane fraction from skeletal muscle.

Both proteins failed to show alterations distribution in response to increased lipid availability not shown , suggesting that they did not mediate the change in the recovery of glycogen synthase through their own translocation.

Finally, because the translocation of glycogen synthase from a cytosolic fraction has been linked to alterations in cellular glycogen content in adipocytes Brady et al. Firstly, we determined the effect of linoleate on steady state glycogen levels. Upon fractionation of the cells, the greatest amount of glycogen was found in the TIF, but there was no effect of the FFA on total glycogen content in any fraction Fig.

Next, we examined the subcellular distribution of the glycogen synthesized over 1 h in response to insulin stimulation in control and lipid-pretreated myotubes previously measured in whole lysates Fig.

In this case, glycogen synthesized in response to insulin was recovered almost exclusively in the TIF, and this component was greatly reduced in linoleate-pretreated cells Fig.

Together with the data shown in Fig. The activity of glycogen synthase in muscle cells is dependent upon several factors. In addition to the importance of the phosphorylation state of the enzyme, the concentration of its allosteric activator G6P and the actions of the glycogen-targeting proteins, the role of cellular localization is becoming apparent Fernandez-Novell et al.

We here present evidence supporting the novel hypothesis that lipid-induced insulin resistance at the level of glycogen synthesis is explained at least in part by a repartitioning of glycogen synthase to an insulin-insensitive pool. The insulin-dependent translocation of glycogen synthase which we have observed in muscle of chow-fed rats and in control cells agrees with previous studies Brady et al.

An insulin-dependent association with glycogen is supported by the fact that the TIF contained the largest amount of total glycogen, and that glycogen synthesized in response to the hormone was mostly recovered in this fraction. Furthermore, while we observed translocation in rat muscle after a hyperinsulinaemic clamp, and in control cells upon 1-h insulin stimulation, as consistent with the co-sedimentation of glycogen synthase with newly synthesized, large, insoluble glycogen molecules, we did not observe such translocation after only min insulin stimulation data not shown , when smaller, nascent glycogen molecules might be recovered in the cytosol.

It should be noted that although we observed changes in the recovery of glycogen synthase in different subcellular fractions, these could indicate alterations in the association of the enzyme with protein or carbohydrate complexes, rather than in cellular localization, as previously reported in response to other stimuli Ferrer et al.

Thus, although immunoblotting and in vitro assays indicated that the major proportion of glycogen synthase is recovered in the TIF even in the absence of lipid, it is possible that in intact cells and muscle this pool is inhibited by association with glycogen Nielsen et al. Although fat-feeding, obesity or linoleate pretreatment appeared to cause a similar redistribution of glycogen synthase to the TIF, the pool of the enzyme increased in the TIF by lipid was probably distinct from that increased by insulin.

Firstly, glycogen synthase appearing in the TIF in a lipid-dependent manner exhibited a low fractional velocity and was resistant to insulin; second, the redistribution was associated with an inhibition of insulin-stimulated glycogen synthesis, in direct contrast to the repartitioning caused by insulin itself.

This is consistent with the sequestration of glycogen synthase in the TIF by lipid oversupply in such a way that it is no longer sensitive to activation by insulin. The amount of cytosolic glycogen synthase is thus reduced, and we hypothesize that this apparently minor pool of the enzyme is necessary for the full increase in glycogen synthesis to occur in response to the hormone.

Our observations are thus similar to those made with adipocytes Jensen et al. Insulin pretreatment, which was associated with diminished glycogen synthesis upon subsequent insulin stimulation, caused prior partitioning of the enzyme to the denser fraction.

It was concluded that a minor component of glycogen synthase was responsible for most of the newly synthesized glycogen in adipocytes, and that correct basal localization was required for activation of the enzyme by insulin Jensen et al.

Our data support a similar situation in muscle, in that the cytosolic component may contribute most to glycogen synthesis, and furthermore suggest that lipid oversupply, like insulin pretreatment, can lead to a sequestration of the enzyme that contributes to a reduction in subsequent, insulin-stimulated glycogen synthesis.

A possible explanation is that this redistribution reduces access by PP1 and hence limits activation normally promoted by dephosphorylation of the enzyme. Such a mechanism is supported by the lack of effect of linoleate on the insulin-stimulated activation of PKB, suggesting that the pathway downstream of this signalling component, which reduces further phosphorylation of glycogen synthase by inhibition of GSK-3, is still intact.

While we were unable to determine the mechanism by which lipid oversupply causes such a redistribution, we were able to discount a simple role for glycogen accumulation. Firstly, the glycogen content of rat skeletal muscle was not significantly increased by fat-feeding; second, linoleate pretreatment did not alter the steady-state levels of glycogen in any subcellular fraction from L6 myotubes, despite an increase in the partitioning of the enzyme in the TIF in each case.

We were also unable to find evidence for glycosylation or the association of a binding partner. We cannot, however, rule out a post-translational modification, such as phosphorylation of the enzyme or its targeting proteins, which might affect its binding. Indeed, a reason for the discrepancy between the amounts of glycogen synthase protein appearing in the TIF of lipid-treated cells and that translocating from the cytosol as determined by immunoblotting Fig.

Alternatively, direct association of glycogen synthase with lipid species in intact cells may promote its inhibition and sedimentation, as described for phosphatidic acid phosphohydrolase-1 Elabbadi et al. Furthermore, the lack of effect of insulin on steady-state G6P levels measured in these cells indicates that the observed increase in glycogen synthesis in response to insulin in control cells Fig.

Importantly, therefore, the inhibitory effect of linoleate on insulin-stimulated glycogen synthesis cannot be explained by a reduced effect of insulin on G6P concentration. Similarly, previous studies have also shown that G6P content is not reduced in muscle of fat-fed rats Kim et al.

In any case, the differing activation states of cytosolic and TIF-associated glycogen synthase we have observed in lipid-treated myotubes indicate that the cellular G6P concentration cannot be solely responsible for alterations in glycogen synthesis.

Taken together, these findings suggest that a primary defect at the level of glycogen synthase contributes to the diminished glycogen synthesis observed in the presence of lipid, strengthening the case for a role of compartmentalization.

The relative importance of the rate of glucose uptake and the activity of glycogen synthase in the determination of the rate of glycogen synthesis has been controversial, and it is likely that either factor can play an overriding role under specific physiological circumstances Fisher et al.

Thus, while NMR studies have indicated that glucose transport can be rate-limiting Roden et al. An impairment of glycogen synthase activity also contributes to the diminished glycogen synthesis seen in lipid-infused human subjects at higher free fatty acid levels Boden et al.

In summary, while previous work has suggested that exposure of muscle to increased lipid levels does not greatly affect the activity of glycogen synthase Johnson et al. While the myotubes are not an exact representation of muscle tissue, an advantage of the model we have employed is the ability to define conditions for the study of glycogen synthase under which the contribution of lipid effects on glucose transport and phosphorylation can be discounted.

This study has thus indicated a new aspect of lipid-induced insulin resistance at the level of glycogen synthesis, although further work is required to elucidate the mechanism of glycogen synthase repartitioning. The effect of high-fat feeding on glycogen synthase protein partitioning in rat skeletal muscle.

Rats which had been fed either a chow or a high-fat diet were subjected to a euglycaemic-hyperinsulinaemic clamp, or maintained under basal conditions, as indicated.

Skeletal muscle was fractionated and subjected to immunoblotting. a Immunoblots obtained with glycogen synthase and GSK-3 antibodies are shown. b The results of densitometry of glycogen synthase bands from muscle fractions of six animals per group are shown. c Immunoblots of glycogen synthase in the Triton-insoluble fraction TIF.

d Muscle fractions from chow- and fat-fed rats were assayed for glycogen synthase activity in the presence of 10 mM G6P to indicate the total amount of enzyme present in each fraction. The means of activities from muscle fractions of six animals per group are shown — note the discontinuous ordinate axis.

Citation: Journal of Endocrinology , 1; TIF fractions were diluted sixfold in comparison to cytosolic fractions. b The results of densitometry of glycogen synthase bands from muscle fractions of four animals per group, corrected for dilution, are shown. Effect of linoleate on glycogen synthesis and glucose uptake in L6 skeletal muscle cells.

a Basal and insulin-stimulated glycogen synthesis was determined in control myotubes and in myotubes pretreated for 16 h with linoleate. Results shown are means from three experiments, each carried out in triplicate.

b Basal and insulin-stimulated glucose uptake were determined in control and lipid-pretreated myotubes under the same conditions as for a. Results shown are means from two experiments, each carried out in triplicate. Effect of linoleate on glycogen phosphorylase and PKB. The activity of glycogen phosphorylase was determined in homogenates, in either the presence a or absence b of 5 mM AMP.

Results shown are combined means from six independent experiments, each carried out in duplicate. c Basal and insulin-stimulated PKB Ser phosphorylation was determined in lysates from control and lipid-pretreated myotubes by immunoblotting with a phospho-specific antibody.

The results of densitometry from three independent experiments, each carried out in triplicate, are shown. Effect of linoleate on glycogen synthase protein partitioning in L6 skeletal muscle cells. a Myotubes were pretreated in the absence or presence of linoleate for 16 h and incubated without or with insulin for 1 h.

Whole-cell lysates were fractionated to obtain cytosolic, solubilized-membrane and Triton-insoluble fractions. Samples were subjected to immunoblotting with either glycogen synthase high and low exposures of the same blot are shown , GSK-3 or β-actin antibodies as indicated.

b The results of densitometry of glycogen synthase bands from three independent experiments carried out in duplicate are shown. c Whole-myotube lysates were subjected to immunoblotting with antiglycogen synthase or anti-β-actin antibodies as indicated. The means from densitometry of three independent experiments done in triplicate are shown, after correction of glycogen synthase for β-actin loading lower panel.

Effect of linoleate on recovery of glycogen synthase activity in subcellular fractions from L6 cells. Myotubes were treated and fractionated as given in the legend for Fig. c The fractional velocity of glycogen synthase was calculated for each fraction as the ratio of activity measured at 0.

The means from three independent experiments are shown. Effect of linoleate on steady-state glycogen content and acute glycogen synthesis in subcellular fractions. a Myotubes were incubated for 48 h in media containing d -[U- 14 C]glucose before treatment with linoleate for 16 h, also in the presence of d -[U- 14 C]glucose, and fractionated as given in the legend for Fig.

Radiolabelled glycogen was determined in each fraction. Results shown are combined means from three independent experiments, each carried out in triplicate. b Myotubes were pretreated without or with linoleate for 16 h and incubated for 1 h in the absence or presence of insulin for the measurement of glycogen synthesis from d -[U- 14 C]glucose.

Radiolabelled glycogen was determined in each subcellular fraction. Results shown are means from five independent experiments, each carried out in triplicate. This work was funded by grants from the National Health and Medical Research Council of Australia CSP , and the Diabetes Australia Research Trust CSP, JMY , and by an Australian Postgraduate Award AJT.

The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work. American Journal of Physiology E —E Hormone and Metabolic Research 14 — Journal of Clinical Investigation 93 — Journal of Biological Chemistry — FEBS Letters 3 — Coleman DL Obese and diabetes: two mutant genes causing diabetes-obesity syndromes in mice.

Diabetologia 14 — Dresner A , Laurent D, Marcucci M, Griffin ME, Dufour S, Cline GW, Slezak LA, Andersen DK, Hundal RS, Rothman DL et al. Journal of Clinical Investigation — Biochimie 87 — Biochemical Journal — FEBS Letters — Biochemical Journal 17 — Analytical Biochemistry 47 20 — Journal of Clinical Investigation 99 — Mohamed A.

Omar, Lianguo Wang, Alexander S. Glycogen synthase kinase-3 GSK-3 is a multi-functional kinase that regulates signalling pathways affecting glycogen metabolism, protein synthesis, mitosis, and apoptosis.

GSK-3 inhibition limits cardiac ischaemia—reperfusion IR injury, but mechanisms are not clearly defined. Rates of glucose and palmitate oxidation were improved by SB. Glycogen synthase kinase-3 GSK-3 is a multi-functional kinase that regulates signalling pathways affecting glycogen metabolism, protein synthesis, mitosis and apoptosis.

It has 2 isoforms, α and β, that possess strong homology in their kinase domains. It actively inhibits hypertrophy and its inhibition stimulates development of cardiac hypertrophy. Tong et al. Furthermore, inhibition of GSK-3 was suggested as a mechanism explaining cardioprotection induced by postconditioning, 5 opioids, 6 bradykinin, 7 erythropoietin, 8 adenosine A 3 receptor activation, 9 isoflurane, 10 and PKCδ inhibition.

One proposed mechanism involves prevention of mitochondrial permeability transition pore mPTP opening 12 potentially due to effects on the voltage-dependent anion channel VDAC 13 or adenine nucleotide translocase ANT.

In addition, recent evidence from mitochondria that are deficient in all isoforms of VDAC shows that VDAC is dispensable in mPTP opening. Interestingly, although the initial function and naming of GSK-3 was related to its effects on glycogen synthase GS activity, the contribution of alterations in glycogen or glucose metabolism by GSK-3 inhibition to cardioprotection has not been investigated.

GSK-3 phosphorylates GS at Ser site 3a and Ser site 3b via a hierarchal mechanism and thereby inhibits GS activity. In this study, we test the hypothesis that inhibition of GSK-3 will stimulate glycogen synthesis, repartition glucose partially away from glycolysis, improve the coupling between glycolysis and glucose oxidation and reduce the potential for intracellular acidosis.

Male Sprague—Dawley rats — g were used in this study. All experiments were performed with accordance with the guidelines of the Animal Use and Care Committee, University of Alberta animal ethics protocol The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health NIH Publication No.

Rat hearts were cannulated for isolated working mode perfusion in a recirculating system under conditions of constant workload Rates of glycolysis, glucose oxidation, and palmitate oxidation were measured as described previously.

After an initial 45 min of baseline aerobic perfusion, hearts were subjected to 17 min of global ischaemia GI followed by 30 min of reperfusion. This was followed by 20 min of GI and 30 min of reperfusion. These durations of GI were sufficient to cause marked LV dysfunction that is reversible.

Longer periods of ischaemia resulted in the failure of the hearts to recover at reperfusion which would have hindered the measurement of the metabolic rates.

Once added, the drug remained in the recirculating system until the end of the perfusion protocol. This concentration of SB was shown previously to produce sufficient inhibition of GSK-3 and to induce cardioprotection in the isolated perfused rat heart.

Two series of hearts with different pre-treatment glycogen contents were studied. Results are expressed as means ± SEM of n observations. The significance of the differences for two group comparisons was estimated by Student's t -test.

The significance of difference in time-course experiments was estimated by two-way analysis of variance with repeated measures on time and a Newman—Keuls post-test.

Left ventricular mechanical function was stable during the initial period of baseline perfusion with no differences among experimental groups Figure 1 A. This cardioprotective effect of SB is similar to effects observed in previous studies.

SB improves recovery of LV function during reperfusion. A LV work during IR protocol in isolated working rat hearts treated with vehicle DMSO, 0.

Values are means ± SEM. SB-mediated alterations in energy substrate metabolism during reperfusion. This increase in ATP production by SB resulted from a higher contribution of palmitate and glucose oxidation, while there was a lower contribution from glycolysis Figure 2 I. This was associated with improved recovery of post-ischaemic LV function to In order to assess the exact role of the stimulation of glycogen synthesis induced by inhibition of GSK-3, it is important to delineate the cause and effect relationship between glycogen and glucose metabolism and improved LV function during reperfusion.

For this purpose, we studied the effects of SB in aerobically perfused hearts no ischaemia with normal G-replete or partially depleted glycogen stores G-depleted see Supplementary material online, Figure S1. SB had no effect on LV work in G-replete hearts 2.

SB elicited only a minor alteration in the rate of glycogen synthesis that was not significantly different from vehicle-treated hearts Figure 5 A. Effects of SB on glycogen and glucose metabolism in aerobic hearts.

SB had no effect on LV work in G-depleted hearts 2. SB did not affect glucose oxidation Figure 5 G. Effects of SB when administered at the onset of reperfusion. The first evidence for the role of GSK-3 in cardioprotection was obtained in studies showing that ischaemic preconditioning results in phosphorylation and inhibition of GSK-3β and that pharmacological inhibition of GSK-3 mimics the cardioprotective effects of preconditioning.

Specifically, our data indicate that inhibition of GSK-3 increases glycogen synthesis during reperfusion which partially repartitions glucosephosphate away from glycolysis. We also provide evidence that acceleration of glycogen synthesis is not a consequence of improved LV function, as similar metabolic alterations occur in glycogen-depleted aerobic hearts independent of changes in LV mechanical function.

In order to examine the relative rates of glycogen synthesis and glycolysis in the absence and presence of GSK-3 inhibition, studies were performed in isolated rat hearts that were perfused in working mode with both glucose and palmitate as energy substrates. These conditions ensure hearts are studied under conditions of physiological work load energy demand as well as adequate energy supply.

Moreover, aerobic perfusion conditions ensure the re-establishment of normal glycogen content previously severely depleted during deep anaesthesia and heart extraction , a key requirement for investigations of glucose and glycogen metabolism. While alteration of glycolysis may be involved, other mechanisms arising from GSK-3 inhibition during ischaemia may contribute, such as improved ionic homeostasis due to reduced mitochondrial ATP consumption, an effect possibly due to interaction of GSK-3 with VDAC.

However, as LV mechanical function energy demand and energy substrate metabolism are interdependent, additional experiments were performed in aerobic hearts in order to determine if alteration of glucose partitioning might simply be a consequence, rather than a cause, of enhanced recovery of LV function.

The ability of SB to produce a similar re-partitioning of glucose metabolism in aerobic hearts that are partially depleted of glycogen to levels similar to the end of GI confirms that the alterations in metabolism are not a consequence of changes in LV function.

Rather, it indicates that the enhanced recovery of LV function is due to the changes in metabolism. A well-described downstream consequence of GSK-3 inhibition is delayed opening of mPTP in response to reactive oxygen species.

However, direct interaction of GSK-3 with VDAC reduces adenine nucleotide transport across the outer mitochondrial membrane independent of mPTP opening, 17 thereby conserving ATP content by reducing mitochondrial ATP consumption.

However, it cannot explain cardioprotection when SB is administered only at the onset of reperfusion, a period when ATP generation returns close to pre-ischaemic levels.

Although the open probability of mPTP is reduced sharply in acidic pH in de-energized mitochondria, 51 , 52 exposure of respiring mitochondria to an acidic environment, such as in early reperfusion, will favour mitochondrial inorganic phosphate uptake that facilitates mPTP opening.

This may also explain the improved mitochondrial function, demonstrated by enhanced glucose and palmitate oxidation, during reperfusion in SB-treated hearts.

The stimulated mitochondrial oxidation may also arise due to the improved recovery of LV function and higher energy demand in SB-treated hearts. Furthermore, a direct interaction is unlikely as GSK-3 inhibition has no effect on mPTP opening in isolated mitochondria.

Although GSK-3 was initially discovered and named for its role in regulating glycogen metabolism, this is the first study to link this important effect on myocardial metabolism with cardioprotection. Our study highlights the ability of GSK-3 to regulate myocardial glycogen and glucose metabolism and demonstrates an additional mechanism linking GSK-3 inhibition with enhanced recovery of post-ischaemic mechanical function.