Susanna Baqué

Muscle glycogen synthase translocates from the cell nucleus to the cytosol in response to glucose

We have studied the intracellular localization of muscular glycogen synthase by fusing the green fluorescent protein (GFP) of the jelly‐fish Aequorea victoria to the N‐terminus of human muscle glycogen synthase (HMGS), and expressing the chimeric protein in C2C12, COS‐1 cells, and primary cultured rat hepatocytes. In contrast to what we have recently found for the hepatic glycogen synthase (Fernández‐Novell et al. (1997) Biochem. J. 321, 227–231), the GFP/HMGS fusion protein is localized to the nucleus of the cell in the absence of glucose, and in the presence of the sugar it is essentially found in the cytosol. Insulin is not required for the translocation of the enzyme.

Glucose has to be phosphorylated to activate glycogen synthase, but not to inactivate glycogen phosphorylase in hepatocytes

2‐Deoxyglucose and 5‐thioglucose, in the same fashion as glucose, cause the inactivation of the rat hepatocyte glycogen phosphorylase and the activation of glycogen synthase. However, 6‐deoxyglucose and 1,5‐anhydroglucitol inactivate phosphorylase without increasing the activation state of glycogen synthase. With 3‐O‐methylglucose no changes in the activity or these enzymes occurred. These results prove that while glucose is the molecule that triggers the inactivation of phosphorylase, glucose 6‐phosphate is the signal for glucose synthase activation and that a metabolite control of the activation state of glycogen synthase is operative in hepatocytes.

The antidiabetic agent sodium tungstate activates glycogen synthesis through an insulin receptor-independent pathway.

Sodium tungstate is a powerful antidiabetic agent when administered orally. In primary cultured hepatocytes, tungstate showed insulin-like actions, which led to an increase in glycogen synthesis and accumulation. However, this compound did not significantly alter the insulin receptor activation state or dephosphorylation rate in cultured cells (CHO-R) or in primary hepatocytes, in either short or long term treatments. In contrast, at low concentrations, tungstate induced a transient strong activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) after 5–10 min of treatment, in a similar way to insulin. Moreover, this compound did not significantly delay or inhibit the dephosphorylation of ERK1/2. ERK1/2 activation triggered a cascade of downstream events, which included the phosphorylation of p90rsk and glycogen synthase-kinase 3β. Experiments with a specific inhibitor of ERK1/2 activation and kinase assays indicate that these proteins were directly involved in the stimulation of glycogen synthase and glycogen synthesis induced by tungstate without a direct involvement of protein kinase B (PKB/Akt). These results show a direct involvement of ERK1/2 in the mechanism of action of tungstate at the hepatic level.

Glycogenin, the primer of glycogen synthesis, binds to actin

We have studied the intracellular localization of glycogenin by fusing green fluorescent protein (GFP) to the N‐terminus of rabbit muscle glycogenin and expressing the chimeric protein in C2C12, COS‐1 and rat hepatic cells. The fusion protein showed a nuclear and cytosolic distribution and partially co‐localized with actin in the cytosol. Disruption of the actin cytoskeleton with cytochalasin D led to a change in the pattern of green fluorescence, which coincided with that observed for the remaining non‐depolymerized actin. The distribution of the single point mutant K324A was completely uniform and was not affected by this drug. These findings indicate that rabbit muscle glycogenin binds to actin through the heptapeptide 321DNIKKKL327, a common motif found in other actin‐binding proteins, which is located at the C‐terminal end of this protein, and suggest that the actin cytoskeleton plays an important role in glycogen metabolism.

Overexpression of Muscle Glycogen Phosphorylase in Cultured Human Muscle Fibers Causes Increased Glucose Consumption and Nonoxidative Disposal

The effect of increased expression of glycogen phosphorylase on glucose metabolism in human muscle was examined in primary cultured fibers transduced with recombinant adenovirus AdCMV-MGP encoding muscle glycogen phosphorylase. Increments of 20-fold in total enzyme activity and of 14-fold of the active form of the enzyme were associated with a 30% reduction in basal glycogen levels. Total glycogen synthase activity was doubled in AdCMV-MGP-transduced cells even though the activity ratio was decreased. Incubation with forskolin, which inactivated glycogen synthase and activated glycogen phosphorylase, induced greater net glycogenolysis in engineered cells. In unstimulated fibers, lactate production was three times higher in AdCMV-MGP fibers as compared with controls, despite similar rates of glycogenolysis. In transduced fibers incubated with 2-deoxyglucose, the level of 2-deoxyglucose 6-phosphate was about 8-fold elevated over the control even though hexokinase activity was unmodified in AdCMV-MGP fibers. Overexpression of glycogen phosphorylase also led to enhancement of [U-14C]glucose incorporation into glycogen, lactate, and lipid. Accordingly, determination of lipid cell content revealed that engineered cells were accumulating lipids. Furthermore, 14CO formation from [U-14C]glucose was 1.6-fold higher, whereas 14CO formation from [6-14C]glucose was unmodified, in AdCMV-MGP fibers. Our data show that in human skeletal muscle cells in culture, the increase in glycogen phosphorylase activity is able to up-regulate glycogen synthase activity indicating the enhancement of glycogen turnover. We suggest that the increase in glycogen phosphorylase and, thereby, in glycogen metabolism, is sufficient to enhance glucose uptake in the muscle cell. Glucose taken up by engineered muscle cells is essentially disposed of through nonoxidative metabolism and converted into lactate and lipid.

Determinants of the nucleocytoplasmic shuttling of muscle glycogen synthase

Muscle glycogen synthase (MGS) presents a nuclear speckled pattern in primary cultured human muscle and in 3T3‐L1 cells deprived of glucose and with depleted glycogen reserves. Nuclear accumulation of the enzyme correlates inversely with cellular glycogen content. Although the glucose‐induced export of MGS from the nucleus to the cytoplasm is blocked by leptomycin B, and therefore mediated by CRM1, no nuclear export signal was identified in the sequence of the protein. Deletion analysis shows that the region comprising amino acids 555–633 of human MGS, which encompasses an Arg‐rich cluster involved in the allosteric activation of the enzyme by Glc6P, is crucial for its nuclear concentration and aggregation. Mutation of these Arg residues, which desensitizes the enzyme towards Glc6P, interferes with its nuclear accumulation. In contrast, the known phosphorylation sites of MGS that regulate its activity are not involved in the control of its subcellular distribution. Nuclear human MGS colocalizes with the promyelocytic leukaemia oncoprotein and p80‐coilin, a marker of Cajal bodies. The subnuclear distribution of MGS is altered by incubation with transcription inhibitors. These observations suggest that, in addition to its metabolic function, MGS may participate in nuclear processes.

Overexpression of Glycogen Phosphorylase Increases GLUT4 Expression and Glucose Transport in Cultured Skeletal Human Muscle

Skeletal muscle glucose utilization, a major factor in the control of whole-body glucose tolerance, is modulated in accordance with the muscle metabolic demand. For instance, it is increased in chronic contraction or exercise training in association with elevated expression of GLUT4 and hexokinase II (HK-II). In this work, the contribution of increased metabolic flux to the regulation of the glucose transport capacity was analyzed in cultured human skeletal muscle engineered to overexpress glycogen phosphorylase (GP). Myocytes treated with an adenovirus-bearing muscle GP cDNA (AdCMVMGP) expressed 10 times higher GP activity and exhibited a twofold increase in the Vmax for 2-deoxy-D-[3H]glucose (2-DG) uptake, with no effect on the apparent Km. The stimulatory effect of insulin on 2-DG uptake was also markedly enhanced in AdCMVMGP- treated cells, which showed maximal insulin stimulation 2.8 times higher than control cells. No changes in HKII total activity or the intracellular compartmentalization were found. GLUT4, protein, and mRNA were raised in AdCMV-MGP-treated cells, suggesting pretranslational activation. GLUT4 was immunodetected intracellularly with a perinuclear predominance. Culture in glucose-free or high-glucose medium did not alter GLUT4 protein content in either control cells or AdCMV-MGP-treated cells. Control and GP-overexpressing cells showed similar autoinhibition of glucose transport, although they appeared to differ in the mechanism(s) involved in this effect. Whereas GLUT1 protein increased in control cells when they were switched from a high-glucose to a glucose-free medium, GLUT1 remained unaltered in GP-expressing cells upon glucose deprivation. Therefore, the increased intracellular metabolic (glycogenolytic-glycolytic) flux that occurs in muscle cells overexpressing GP causes an increase in GLUT4 expression and enhances basal and insulin-stimulated glucose transport, without significant changes in the autoinhibition of glucose transport. This mechanism of regulation may be operative in the postexercise situation in which GLUT4 expression is upregulated in coordination with increased glycolytic flux and energy demand.

Intracellular Distribution of Glycogen Synthase: Another Regulatory Mechanism of Glycogen Metabolism?

As Joseph Larner (1990) pointed out, glycogen has been a major catalyst in the formulation of many concepts of modern biology. A number of broad principles have emerged from studies of glycogen metabolism. Thus, covalent regulation by phosphorylation was originally described as the mechanism of activation of glycogen phosphorylase. Phosphorylase kinase was the second enzyme described as being activated by this mechanism. The third enzyme known to be controlled by phosphorylation was glycogen synthase, although in this case the regulation was inverse since phosphorylation caused the inactivation of the enzyme. The discovery of cyclic AMP and the development of the intracellular messenger concept emerged from the study of the regulation of glycogen phosphorylase by glucagon and adrenaline. These studies also led to the notion of the cascade of phosphorylation reactions. The concept and emerging significance of multiple phosphorylation also originated in the research of the control of glycogen synthase. Later, this evolved into the theory of hierarchical phosphorylation. Parallel studies on the dephosphorylation of glycogen synthase showed the ability of insulin to modify the activity of protein kinases and protein phosphatases. In Larner’s words, “glycogen has sparked its fair share of major discoveries in biology”.