Glycogen Metabolism In Skeletal Muscle Research Articles

MicroRNA-92b in the skeletal muscle regulates exercise capacity via modulation of glucose metabolism.

Exercise mimetics is a proposed class of therapeutics that specifically mimics or enhances the therapeutic effects of exercise. Muscle glycogen and lactate extrusion are critical for physical performance. The mechanism by which glycogen and lactate metabolism are manipulated during exercise remains unclear. This study aimed to assess the effect of miR-92b on the upregulation of exercise training-induced physical performance. Adeno-associated virus (AAV)-mediated skeletal muscle miR-92b overexpression in C57BLKS/J mice, and global knockout of miR-92b mice were used to explore the function of miR-92b in glycogen and lactate metabolism in skeletal muscle. AAV-mediated UGP2 or MCT4 knockdown in WT or miR-92 knockout mice was used to confirm whether miR-92b regulates glycogen and lactate metabolism in skeletal muscle through UGP2 and MCT4. Body weight, muscle weight, grip strength, running time and distance to exhaustion, and muscle histology were assessed. The expression levels of muscle mass-related and function-related proteins were analysed by immunoblotting or immunostaining. Global knockout of miR-92b resulted in normal body weight and insulin sensitivity, but higher glycogen content before exercise exhaustion (0.8538±0.0417 vs. 1.043±0.040, **P=0.0087), lower lactate levels after exercise exhaustion (4.133±0.2589 vs. 3.207±0.2511, *P=0.0279), and better exercise capacity (running distance to exhaustion, 3616±86.71 vs. 4231±90.29, ***P=0.0006; running time to exhaustion, 186.8±8.027 vs. 220.8±3.156, **P=0.0028), as compared with those observed in the control mice. Mice skeletal muscle overexpressing miR-92b (both miR-92b-3p and miR-92b-5p) displayed lower glycogen content before exercise exhaustion (0.6318±0.0231 vs. 0.535±0.0194, **P=0.0094), and higher lactate accumulation after exercise exhaustion (4.5±0.2394 vs. 5.467±0.1892, *P=0.01), and poorer exercise capacity (running distance to exhaustion, 4005±81.65 vs. 3228±149.8, ***P<0.0001; running time to exhaustion, 225.5±7.689 vs. 163±6.476, **P=0.001). Mechanistic analysis revealed that miR-92b-3p targets UDP-glucose pyrophosphorylase 2 (UGP2) expression to inhibit glycogen synthesis, while miR-92b-5p represses lactate extrusion by directly target monocarboxylate transporter 4 (MCT4). Knockdown of UGP2 and MCT4 reversed the effects observed in the absence of miR-92b in vivo. This study revealed regulatory pathways, including miR-92b-3p/UGP2/glycogen synthesis and miR-92b-5p/MCT4/lactate extrusion, which could be targeted to control exercise capacity.

Anti-diabetic potential of Psidium guajava leaf in streptozotocin induced diabetic rats

BackgroundPsidium guajava is a medicinal plant with antidiabetic properties and can be found growing in tropical and sub-tropical countries around the world. PurposeThe aim of the present study was to investigate the anti-diabetic potential of Psidium guajava leaf in streptozotocin-induced diabetic rats, with particular focus on key enzymes of glycogen metabolism in skeletal muscle. Study designThe study design involved preparation of an aqueous Psidium guajava leaf extract; investigation of its effect on muscle glycogen synthase and phosphorylase activities and possible protective effect on pancreas in streptozotocin induced diabetic male Sprague-Dawley rats and phytochemical study of the extract to identify further compounds with anti-diabetic potential. MethodDiabetes was induced in male Sprague-Dawley rats with a single intraperitoneal injection of 40 mg/kg streptozotocin. Normal and diabetic animals were treated with 400 mg/kg body weight of Psidium guajava leaves aqueous extract for a period of 14 days. ResultsThe treatment of diabetic animals with the Psidium guajava extract ameliorated damage to the pancreatic islets and enhanced lowering of blood glucose following a glucose load. Diabetes significantly decreased (P < 0.05) glycogen synthase activity by 31% compared to normal animals this being associated with a decrease in enzyme expression. Psidium guajava treatment returned the enzyme activity to near normal levels. In diabetic animals, glycogen phosphorylase activity was elevated but not significantly (P > 0.5) and treatment of both diabetic and normal animals with the extract reduced activity of the enzyme. The Psidium guajava extract also reduced glycogen phosphorylase expression in diabetic animals. Skeletal muscle glycogen content reduced by diabetes was increased as a result of treatment of diabetic animals with the Psidium guajava extract. Phytochemical analysis of the aqueous extract of Psidium guajava using Gas Chromatography-Mass Spectroscopy indicated the presence of triterpenes and phenolic compounds and we also report what we believe are three new compounds. ConclusionThe results from this study suggest that the antidiabetic effects of Psidium guajava may be due to modulation of glycogen metabolism mediated by phenolic compounds and triterpenes present in the extract.

Effects of Thiamin Restriction on Exercise-Associated Glycogen Metabolism and AMPK Activation Level in Skeletal Muscle

This study aimed to investigate the direct influence of a decrease in the cellular thiamin level, before the onset of anorexia (one of the symptoms of thiamin deficiency) on glycogen metabolism and the AMP-activated protein kinase (AMPK) activation levels in skeletal muscle at rest and in response to exercise. Male Wistar rats were classified as the control diet (CON) group or the thiamin-deficient diet (TD) group and consumed the assigned diets for 1 week. Skeletal muscles were taken from the rats at rest, those that underwent low-intensity swimming (LIS), or high-intensity intermittent swimming (HIS) conducted immediately before dissection. There were no significant differences in food intake, locomotive activity, or body weight between groups, but thiamin pyrophosphate in the skeletal muscles of the TD group was significantly lower than that of the CON group. Muscle glycogen and lactate levels in the blood and muscle were equivalent between groups at rest and in response to exercise. The mitochondrial content was equal between groups, and AMPK in the skeletal muscles of TD rats was normally activated by LIS and HIS. In conclusion, with a lowered cellular thiamin level, the exercise-associated glycogen metabolism and AMPK activation level in skeletal muscle were normally regulated.

McArdle disease and pregnancy: A case report and scoping review of pregnancy outcomes.

McArdle disease is an autosomal recessive disorder affecting skeletal muscle glycogen metabolism. Limited data are available regarding pregnancy outcomes with this genetic condition. We present a recent case of a woman with McArdle disease, along with a scoping review of all published literature regarding pregnancy and delivery outcomes for women with McArdle disease. A total of 35 cases are summarised. Overall, pregnancy does not worsen or increase the risk for disease flare. Women can successfully deliver vaginally, with consideration of an assisted second stage recommended to reduce the risk of postpartum rhabdomyolysis.

Effects of Centella asiatica on skeletal muscle structure and key enzymes of glucose and glycogen metabolism in type 2 diabetic rats

BackgroundDysregulation of glucose and glycogen metabolism are crucial mechanisms implicated in type 2 diabetes mellitus (T2DM). Centella asiatica (L.) Urban (Apiaceae) has been utilized as a traditional medicine in Africa and Asia for centuries and is commercially available as a dietary supplement. AimWe explored for the first time, the possible efficacy of Centella asiatica (CA) extract in ameliorating T2DM-induced changes in key enzymes involved in glucose and glycogen metabolism in the rat skeletal muscle. MethodsDiabetic rats were orally treated with vehicle, CA (500 and 1000 mg/kg) or metformin (300 mg/kg) daily for 14 days. Skeletal muscle activities of hexokinase (HK), phosphofructokinase (PFK) and fructose 1,6-bisphosphatase (FBPase) were determined by spectrophotometric assays while those of glycogen synthase (GS) and glycogen phosphorylase (GP) were assayed radio-chemically. Histological examination of skeletal muscle was also performed. ResultsRats with induced T2DM had reduced activities of HK (25%), PFK (88%), and GS (38%) when compared to non-diabetic rats. Treatment of diabetic rats with CA500 increased the activities of PFK (7-fold), and FBPase (23%). Further, treatment of diabetic rats with CA1000 also increased the activities of GS (27%) and GP (50%) with little change in these parameters for diabetic rats treated with CA500. These effects probably led to the reduced blood glucose level and elevated skeletal muscle glycogen content observed in CA-treated rats relative to diabetic controls. Furthermore, CA treated rats had reduced the morphological damage of skeletal muscle fibres compared to the non-treated diabetic control rats. ConclusionOur findings strongly suggest that the anti-diabetic effects of CA in part target muscle glucose and glycogen metabolism and hence supporting its folkloric medical use as an anti-diabetic remedy.

GNIP1 E3 ubiquitin ligase is a novel player in regulating glycogen metabolism in skeletal muscle

BackgroundGlycogenin-interacting protein 1 (GNIP1) is a tripartite motif (TRIM) protein with E3 ubiquitin ligase activity that interacts with glycogenin. These data suggest that GNIP1 could play a major role in the control of glycogen metabolism. However, direct evidence based on functional analysis remains to be obtained. ObjectivesThe aim of this study was 1) to define the expression pattern of glycogenin-interacting protein/Tripartite motif containing protein 7 (GNIP/TRIM7) isoforms in humans, 2) to test their ubiquitin E3 ligase activity, and 3) to analyze the functional effects of GNIP1 on muscle glucose/glycogen metabolism both in human cultured cells and in vivo in mice. ResultsWe show that GNIP1 was the most abundant GNIP/TRIM7 isoform in human skeletal muscle, whereas in cardiac muscle only TRIM7 was expressed. GNIP1 and TRIM7 had autoubiquitination activity in vitro and were localized in the Golgi apparatus and cytosol respectively in LHCN-M2 myoblasts. GNIP1 overexpression increased glucose uptake in LHCN-M2 myotubes. Overexpression of GNIP1 in mouse muscle in vivo increased glycogen content, glycogen synthase (GS) activity and phospho-GSK-3α/β (Ser21/9) and phospho-Akt (Ser473) content, whereas decreased GS phosphorylation in Ser640. These modifications led to decreased blood glucose levels, lactate levels and body weight, without changing whole-body insulin or glucose tolerance in mouse. ConclusionGNIP1 is an ubiquitin ligase with a markedly glycogenic effect in skeletal muscle.

Muscle molecular adaptations to endurance exercise training are conditioned by glycogen availability: a proteomics-based analysis in the McArdle mouse model.

Although they are unable to utilize muscle glycogen, McArdle mice adapt favourably to an individualized moderate-intensity endurance exercise training regime. Yet, they fail to reach the performance capacity of healthy mice with normal glycogen availability. There is a remarkable difference in the protein networks involved in muscle tissue adaptations to endurance exercise training in mice with and without glycogen availability. Indeed, endurance exercise training promoted the expression of only three proteins common to both McArdle and wild-type mice: LIMCH1, PARP1 and TIGD4. In turn, trained McArdle mice presented strong expression of mitogen-activated protein kinase 12 (MAPK12). McArdle's disease is an inborn disorder of skeletal muscle glycogen metabolism that results in blockade of glycogen breakdown due to mutations in the myophosphorylase gene. We recently developed a mouse model carrying the homozygous p.R50X common human mutation (McArdle mouse), facilitating the study of how glycogen availability affects muscle molecular adaptations to endurance exercise training. Using quantitative differential analysis by liquid chromatography with tandem mass spectrometry, we analysed the quadriceps muscle proteome of 16-week-old McArdle (n=5) and wild-type (WT) (n=4) mice previously subjected to 8weeks' moderate-intensity treadmill training or to an equivalent control (no training) period. Protein networks enriched within the differentially expressed proteins with training in WT and McArdle mice were assessed by hypergeometric enrichment analysis. Whereas endurance exercise training improved the estimated maximal aerobic capacity of both WT and McArdle mice as compared with controls, it was ∼50% lower than normal in McArdle mice before and after training. We found a remarkable difference in the protein networks involved in muscle tissue adaptations induced by endurance exercise training with and without glycogen availability, and training induced the expression of only three proteins common to McArdle and WT mice: LIM and calponin homology domains-containing protein 1 (LIMCH1), poly (ADP-ribose) polymerase 1 (PARP1 - although the training effect was more marked in McArdle mice), and tigger transposable element derived 4 (TIGD4). Trained McArdle mice presented strong expression of mitogen-activated protein kinase 12 (MAPK12). Through an in-depth proteomic analysis, we provide mechanistic insight into how glycogen availability affects muscle protein signalling adaptations to endurance exercise training.

Nrf2-Mediated Regulation of Skeletal Muscle Glycogen Metabolism.

Nrf2 (NF-E2-related factor 2) contributes to the maintenance of glucose homeostasis in vivo Nrf2 suppresses blood glucose levels by protecting pancreatic β cells from oxidative stress and improving peripheral tissue glucose utilization. To elucidate the molecular mechanisms by which Nrf2 contributes to the maintenance of glucose homeostasis, we generated skeletal muscle (SkM)-specific Keap1 knockout (Keap1MuKO) mice that express abundant Nrf2 in their SkM and then examined Nrf2 target gene expression in that tissue. In Keap1MuKO mice, blood glucose levels were significantly downregulated and the levels of the glycogen branching enzyme (Gbe1) and muscle-type PhKα subunit (Phka1) mRNAs, along with those of the glycogen branching enzyme (GBE) and the phosphorylase b kinase α subunit (PhKα) protein, were significantly upregulated in mouse SkM. Consistent with this result, chemical Nrf2 inducers promoted Gbe1 and Phka1 mRNA expression in both mouse SkM and C2C12 myotubes. Chromatin immunoprecipitation analysis demonstrated that Nrf2 binds the Gbe1 and Phka1 upstream promoter regions. In Keap1MuKO mice, muscle glycogen content was strongly reduced and forced GBE expression in C2C12 myotubes promoted glucose uptake. Therefore, our results demonstrate that Nrf2 induction in SkM increases GBE and PhKα expression and reduces muscle glycogen content, resulting in improved glucose tolerance. Our results also indicate that Nrf2 differentially regulates glycogen metabolism in SkM and the liver.

Dysfunctional Muscle and Liver Glycogen Metabolism in mdx Dystrophic Mice

BackgroundDuchenne muscular dystrophy (DMD) is a severe, genetic muscle wasting disorder characterised by progressive muscle weakness. DMD is caused by mutations in the dystrophin (dmd) gene resulting in very low levels or a complete absence of the dystrophin protein, a key structural element of muscle fibres which is responsible for the proper transmission of force. In the absence of dystrophin, muscle fibres become damaged easily during contraction resulting in their degeneration. DMD patients and mdx mice (an animal model of DMD) exhibit altered metabolic disturbances that cannot be attributed to the loss of dystrophin directly. We tested the hypothesis that glycogen metabolism is defective in mdx dystrophic mice.ResultsDystrophic mdx mice had increased skeletal muscle glycogen (79%, (P<0.01)). Skeletal muscle glycogen synthesis is initiated by glycogenin, the expression of which was increased by 50% in mdx mice (P<0.0001). Glycogen synthase activity was 12% higher (P<0.05) but glycogen branching enzyme activity was 70% lower (P<0.01) in mdx compared with wild-type mice. The rate-limiting enzyme for glycogen breakdown, glycogen phosphorylase, had 62% lower activity (P<0.01) in mdx mice resulting from a 24% reduction in PKA activity (P<0.01). In mdx mice glycogen debranching enzyme expression was 50% higher (P<0.001) together with starch-binding domain protein 1 (219% higher; P<0.01). In addition, mdx mice were glucose intolerant (P<0.01) and had 30% less liver glycogen (P<0.05) compared with control mice. Subsequent analysis of the enzymes dysregulated in skeletal muscle glycogen metabolism in mdx mice identified reduced glycogenin protein expression (46% less; P<0.05) as a possible cause of this phenotype.ConclusionWe identified that mdx mice were glucose intolerant, and had increased skeletal muscle glycogen but reduced amounts of liver glycogen.

Nuclear receptors and epigenetic signaling: Novel regulators of glycogen metabolism in skeletal muscle

Glycogen is an energy storage depot for the mammalian species. This review focuses on recent developments that have identified the role of nuclear hormone receptor (NR) signaling and epigenomic control in the regulation of important genes that modulate glycogen metabolism. Specifically, new studies have revealed that the NR4A subgroup (of the NR superfamily) are strikingly sensitive to beta-adrenergic stimulation in skeletal muscle, and transgenic studies in mice have revealed the expression of these NRs affects endurance and glycogen levels in muscle. Furthermore, other studies have demonstrated that one of the NR coregulator class of enzymes that mediate chromatin remodeling, the histone methyltransferases (for example, protein arginine methyltransferase 4) regulates the expression of several genes involved in glycogen metabolism and glycogen storage diseases in skeletal muscle. Importantly, NRs and histone methyltransferases, have the potential to be pharmacologically exploited and may provide novel targets in the quest to treat disorders of glycogen storage.

Physiological aspects of the subcellular localization of glycogen in skeletal muscle

Glucose is stored in skeletal muscle fibers as glycogen, a branched-chain polymer observed in electron microscopy images as roughly spherical particles (known as β-particles of 10-45 nm in diameter), which are distributed in distinct localizations within the myofibers and are physically associated with metabolic and scaffolding proteins. Although the subcellular localization of glycogen has been recognized for more than 40 years, the physiological role of the distinct localizations has received sparse attention. Recently, however, studies involving stereological, unbiased, quantitative methods have investigated the role and regulation of these distinct deposits of glycogen. In this report, we review the available literature regarding the subcellular localization of glycogen in skeletal muscle as investigated by electron microscopy studies and put this into perspective in terms of the architectural, topological, and dynamic organization of skeletal muscle fibers. In summary, the distribution of glycogen within skeletal muscle fibers has been shown to depend on the fiber phenotype, individual training status, short-term immobilization, and exercise and to influence both muscle contractility and fatigability. Based on all these data, the available literature strongly indicates that the subcellular localization of glycogen has to be taken into consideration to fully understand and appreciate the role and regulation of glycogen metabolism and signaling in skeletal muscle. A full understanding of these phenomena may prove vital in elucidating the mechanisms that integrate basic cellular events with changing glycogen content.

Involvement of Skeletal Muscle Protein, Glycogen, and Fat Metabolism in the Adaptation on Early Lactation of Dairy Cows

During early lactation, high-yielding dairy cows cannot consume enough feed to meet nutrient requirements. As a consequence, animals drop into negative energy balance and mobilize body reserves including muscle protein and glycogen for milk production, direct oxidation, and hepatic gluconeogenesis. To examine which muscle metabolic processes contribute to the adaptation during early lactation, six German Holstein cows were blood sampled and muscle biopsied throughout the periparturient period. From pregnancy to lactation, the free plasma amino acid pattern imbalanced and plasma glucose decreased. Several muscle amino acids, as well as total muscle protein, fat, and glycogen, and the expression of glucose transporter-4 were reduced within the first 4 weeks of lactation. The 2-DE and MALDI-TOF-MS analysis identified 43 differentially expressed muscle protein spots throughout the periparturient period. In early lactation, expression of cytoskeletal proteins and enzymes involved in glycogen synthesis and in the TCA cycle was decreased, whereas proteins related to glycolysis, fatty acid degradation, lactate, and ATP production were increased. On the basis of these results, we propose a model in which the muscle breakdown in early lactation provides substrates for milk production by a decoupled Cori cycle favoring hepatic gluconeogenesis and by interfering with feed intake signaling.

Human skeletal muscle glycogen utilization in exhaustive exercise: role of subcellular localization and fibre type

Although glycogen is known to be heterogeneously distributed within skeletal muscle cells, there is presently little information available about the role of fibre types, utilization and resynthesis during and after exercise with respect to glycogen localization. Here, we tested the hypothesis that utilization of glycogen with different subcellular localizations during exhaustive arm and leg exercise differs and examined the influence of fibre type and carbohydrate availability on its subsequent resynthesis. When 10 elite endurance athletes (22 ± 1 years, VO2 max = 68 ± 5 ml kg-1 min-1, mean ± SD) performed one hour of exhaustive arm and leg exercise, transmission electron microscopy revealed more pronounced depletion of intramyofibrillar than of intermyofibrillar and subsarcolemmal glycogen. This phenomenon was the same for type I and II fibres, although at rest prior to exercise, the former contained more intramyofibrillar and subsarcolemmal glycogen than the latter. In highly glycogen-depleted fibres, the remaining small intermyofibrillar and subsarcolemmal glycogen particles were often found to cluster in groupings. In the recovery period, when the athletes received either a carbohydrate-rich meal or only water the impaired resynthesis of glycogen with water alone was associated primarily with intramyofibrillar glycogen. In conclusion, after prolonged high-intensity exercise the depletion of glycogen is dependent on subcellular localization. In addition, the localization of glycogen appears to be influenced by fibre type prior to exercise, as well as carbohydrate availability during the subsequent period of recovery. These findings provide insight into the significance of fibre type-specific compartmentalization of glycogen metabolism in skeletal muscle during exercise and subsequent recovery. .

New emerging role of protein-tyrosine phosphatase 1B in the regulation of glycogen metabolism in basal and TNF-α-induced insulin-resistant conditions in an immortalised muscle cell line isolated from mice

Protein-tyrosine phosphatase 1B (PTP1B) negatively regulates insulin action, promoting attenuation of the insulin signalling pathway. The production of this phosphatase is enhanced in insulin-resistant states, such as obesity and type 2 diabetes, where high levels of proinflammatory cytokines (TNF-α, IL-6) are found. In these metabolic conditions, insulin action on glycogen metabolism in skeletal muscle is greatly impaired. We addressed the role of PTP1B on glycogen metabolism in basal and insulin-resistant conditions promoted by TNF-α. We studied the effect of TNF-α in the presence and absence of insulin on glycogen content and synthesis, glycogen synthase (GS) and glycogen phosphorylase (GP) activities and on glycogen synthesis and degradation signalling pathways. For this purpose we used immortalised cell lines isolated from skeletal muscle from mice lacking PTP1B. Absence of PTP1B caused activation of GS and GP with a net glycogenolytic effect, reflected in lower amounts of glycogen and activation of the glycogenolytic signalling pathway, with higher rates of phosphorylation of cyclic adenosine monophosphate-dependent kinase (PKA), phosphorylase kinase (PhK) and GP phosphorylation. Nevertheless, insulin action was strongly enhanced in Ptp1b (also known as Ptpn1)(-/-) cells in terms of glycogen content, synthesis, GS activation rates and GS Ser641 dephosphorylation. Treatment with TNF-α augmented the activity ratios of both GS and GP, and impaired insulin stimulation of glycogen synthesis in wild-type myocytes, whereas Ptp1b (-/-) myocytes restored this inhibitory effect. We report a glycogenolytic effect of TNF-α, as demonstrated by greater activation of the degradation signalling cascade PKA/PhK/GP. In our model, this effect is mediated by the activation of PKA. We provide new data about the role of PTP1B in glycogen metabolism and confirm the beneficial effect that absence of the phosphatase confers against an insulin-resistant condition.

Intracellular compartmentalization of skeletal muscle glycogen metabolism and insulin signalling

The interest in skeletal muscle metabolism and insulin signalling has increased exponentially in recent years as a consequence of their role in the development of type 2 diabetes mellitus. Despite this, the exact mechanisms involved in the regulation of skeletal muscle glycogen metabolism and insulin signalling transduction remain elusive. We believe that one of the reasons is that the role of intracellular compartmentalization as a regulator of metabolic pathways and signalling transduction has been rather ignored. This paper briefly reviews the literature to discuss the role of intracellular compartmentalization in the regulation of skeletal muscle glycogen metabolism and insulin signalling. As a result, a hypothetical regulatory mechanism is proposed by which cells could direct glycogen resynthesis towards different pools of glycogen particles depending on the metabolic needs. Furthermore, we discuss the role of skeletal muscle transverse tubules as potential modulators of tissue insulin responsiveness.

Uniaxial Cyclic Stretch-Stimulated Glucose Transport Is Mediated by a Ca2+-Dependent Mechanism in Cultured Skeletal Muscle Cells

Objective: Mechanical stimuli such as stretch increase glucose transport and glycogen metabolism in skeletal muscle. However, the molecular mechanisms involved in the mechanotransduction events are poorly understood. The present study was conducted in order to determine whether the signaling mechanism leading to mechanical stretch-stimulated glucose transport is similar to, or distinct from, the signaling mechanisms leading to insulin- and contraction-stimulated glucose transport in cultured muscle cells. Methods: Cultured C2C12 myotubes were stretched, after which the 2-deoxy-D-glucose (2-DG) uptake was measured. Results: Following cyclic stretch, C2C12 myotubes showed a significant increase in 2-DG uptake, and this effect was not prevented by inhibiting phosphatidylinositol 3-kinase or 5′-AMP-activated protein kinase and by extracellular Ca²⁺ chelation. Conversely, the stretch-stimulated 2-DG uptake was completely prevented by dantrolene (an inhibitor of Ca²⁺ release from sarcoplasmic reticulum). Furthermore, the stretch-stimulated 2-DG uptake was prevented by the Ca²⁺/calmodulin-dependent kinase inhibitor KN93 which did not prevent the insulin-stimulated 2-DG uptake. Conclusions: These results suggest that the effects of stretch-stimulated glucose transport are independent of the insulin-signaling pathway. By contrast, following mechanical stretch in skeletal muscle, the signal transduction pathway leading to glucose transport may require the participation of cytosolic Ca²⁺ and Ca²⁺/calmodulin kinase, but not 5′-AMP-activated protein kinase.

Expression of Human Protein Phosphatase-1 in Saccharomyces cerevisiae Highlights the Role of Phosphatase Isoforms in Regulating Eukaryotic Functions

Human (PP1) isoforms, PP1alpha, PP1beta, PP1gamma1, and PP1gamma2, differ in primary sequences at N and C termini that potentially bind cellular regulators and define their physiological functions. The GLC7 gene encodes the PP1 catalytic subunit with >80% sequence identity to human PP1 and is essential for viability of Saccharomyces cerevisiae. In yeast, Glc7p regulates glycogen and protein synthesis, actin cytoskeleton, gene expression, and cell division. We substituted human PP1 for Glc7p in yeast to investigate the ability of individual isoforms to catalyze Glc7p functions. S. cerevisiae expressing human PP1 isoforms were viable. PP1alpha-expressing yeast grew more rapidly than strains expressing other isoforms. On the other hand, PP1alpha-expressing yeast accumulated less glycogen than PP1beta-or PP1gamma1-expressing yeast. Yeast expressing human PP1 were indistinguishable from WT yeast in glucose derepression. However, unlike WT yeast, strains expressing human PP1 failed to sporulate. Analysis of chimeric PP1alpha/beta subunits highlighted a critical role for their unique N termini in defining PP1alpha and PP1beta functions in yeast. Biochemical studies established that the differing association of PP1 isoforms with the yeast glycogen-targeting subunit, Gac1p, accounted for their differences in glycogen synthesis. In contrast to human PP1 expressed in Escherichia coli, enzymes expressed in yeast displayed in vitro biochemical properties closely resembling PP1 from mammalian tissues. Thus, PP1 expression in yeast should facilitate future structure-function studies of this protein serine/threonine phosphatase.

The skeletal muscle-specific glycogen-targeted protein phosphatase 1 plays a major role in the regulation of glycogen metabolism by adrenaline in vivo

Adrenaline and insulin are the major hormones regulating glycogen metabolism in skeletal muscle. We have investigated the effects of these hormones on the rate-limiting enzymes of glycogen degradation and synthesis (phosphorylase and glycogen synthase respectively) in GM−/− mice homozygous for a null allele of the major skeletal muscle glycogen targeting subunit (GM) of protein phosphatase 1 (PP1). Hyperphosphorylation of Ser14 in phosphorylase, and Ser7, Ser640 and Ser640/644 of GS, in the skeletal muscle of GM−/− mice compared with GM+/+ mice indicates that the PP1-GM complex is the major phosphatase that dephosphorylates these sites in vivo. Adrenaline caused a 2.4-fold increase in the phosphorylase (−/+AMP) activity ratio in the skeletal muscle of control mice compared to a 1.4 fold increase in GM−/− mice. Adrenaline also elicited a 67% decrease in the GS (−/+G6P) activity ratio in control mice but only a small decrease in the skeletal muscle of GM−/− mice indicating that GM is required for the full response of phosphorylase and GS to adrenaline. PP1-GM activity and the amount of PP1 bound to GM decreased 40% and 45% respectively, in response to adrenaline in control mice. The data support a model in which adrenaline stimulates phosphorylation of phosphorylase Ser14 and GS Ser7 in GM+/+ mice by both kinase activation and PP1-GM inhibition and the phosphorylation of GS Ser640 and Ser640/644 by PP1-GM inhibition alone. Insulin decreased the phosphorylation of GS Ser640 and Ser640/644 and stimulated the GS (−/+G6P) activity ratio by ∼2-fold in the skeletal muscle of either GM−/− and or control mice, but the low basal and insulin stimulated GS activity ratios in GM−/− mice indicate that PP1-GM is essential for maintaining normal basal and maximum insulin stimulated GS activity ratios in vivo.

Effect of acute activation of 5′-AMP-activated protein kinase on glycogen regulation in isolated rat skeletal muscle

5'-AMP-activated protein kinase (AMPK) has been implicated in glycogen metabolism in skeletal muscle. However, the physiological relevance of increased AMPK activity during exercise has not been fully clarified. This study was performed to determine the direct effects of acute AMPK activation on muscle glycogen regulation. For this purpose, we used an isolated rat muscle preparation and pharmacologically activated AMPK with 5-aminoimidazole-4-carboxamide-1-beta-D-ribonucleoside (AICAR). Tetanic contraction in vitro markedly activated the alpha(1)- and alpha(2)-isoforms of AMPK, with a corresponding increase in the rate of 3-O-methylglucose uptake. Incubation with AICAR elicited similar enhancement of AMPK activity and 3-O-methylglucose uptake in rat epitrochlearis muscle. In contrast, whereas contraction stimulated glycogen synthase (GS), AICAR treatment decreased GS activity. Insulin-stimulated GS activity also decreased after AICAR treatment. Whereas contraction activated glycogen phosphorylase (GP), AICAR did not alter GP activity. The muscle glycogen content decreased in response to contraction but was unchanged by AICAR. Lactate release was markedly increased when muscles were stimulated with AICAR in buffer containing glucose, indicating that the glucose taken up into the muscle was catabolized via glycolysis. Our results suggest that AMPK does not mediate contraction-stimulated glycogen synthesis or glycogenolysis in skeletal muscle and also that acute AMPK activation leads to an increased glycolytic flux by antagonizing contraction-stimulated glycogen synthesis.

Glycogen synthase binds to sarcoplasmic reticulum and is phosphorylated by CaMKII in fast-twitch skeletal muscle

We investigated the subcellular localization of glycogen synthase (GS) in the adductor muscle of anesthetized rabbits injected intravenously with propranolol. Under these experimental conditions, glycogen content was about 10 mmol/kg of fresh tissue. Immunofluorescent and fractionation studies showed that GS associated with sarcoplasmic reticulum (SR) membranes. Glycogen and GS always co-sedimented, suggesting a predominant role of glycogen in targeting of GS to SR. SR-associated GS was phosphorylated in vitro by SR-bound Ca 2+-calmodulin dependent protein kinase (CaMKII) and dephosphorylated by endogenous protein phosphatase 1 (PP1c). Based on measurements of GS activity ratio, in vitro phosphorylation of GS by CaMKII did not significantly affect GS activity per se. However, GS activity ratio was slightly reduced, when SR membranes were further incubated with ATP after prior phosphorylation by CaMKII, suggesting that CaMKII might act sinergistically with other protein kinases. We propose that SR-bound CaMKII plays a role in regulation of glycogen metabolism in skeletal muscle, when intracellular Ca 2+ is raised.