Muscle glycogen synthase isoform is responsible for testicular glycogen synthesis: Glycogen overproduction induces apoptosis in male germ cells

Glycogen is the main source of glucose for many biological events. However, this molecule may have other functions, including those that have deleterious effects on cells. The rate‐limiting enzyme in glycogen synthesis is glycogen synthase (GS). It is encoded by two genes, GYS1, expressed in muscle (muscle glycogen synthase, MGS) and other tissues, and GYS2, primarily expressed in liver (liver glycogen synthase, LGS). Expression of GS and its activity have been widely studied in many tissues. To date, it is not clear which GS isoform is responsible for glycogen synthesis and the role of glycogen in testis. Using RT‐PCR, Western blot and immunofluorescence, we have detected expression of MGS but not LGS in mice testis during development. We have also evaluated GS activity and glycogen storage at different days after birth and we show that both GS activity and levels of glycogen are higher during the first days of development. Using RT‐PCR, we have also shown that malin and laforin are expressed in testis, key enzymes for regulation of GS activity. These proteins form an active complex that regulates MGS by poly‐ubiquitination in both Sertoli cell and male germ cell lines. In addition, PTG overexpression in male germ cell line triggered apoptosis by caspase3 activation, proposing a proapoptotic role of glycogen in testis. These findings suggest that GS activity and glycogen synthesis in testis could be regulated and a disruption of this process may be responsible for the apoptosis and degeneration of seminiferous tubules and possible cause of infertility. J. Cell. Biochem. 114: 1653–1664, 2013. © 2013 Wiley Periodicals, Inc.     

Role of the PDK1-PKB-GSK3 pathway in regulating glycogen synthase and glucose uptake in the heart

In order to investigate the importance of the PDK1-PKB-GSK3 signalling network in regulating glycogen synthase (GS) in the heart, we have employed tissue specific conditional knockout mice lacking PDK1 in muscle (mPDK1-/-), as well as knockin mice in which the protein kinase B (PKB) phosphorylation site on glycogen synthase kinase-3alpha (GSK3alpha) (Ser21) and GSK3beta (Ser9) is changed to Ala. We demonstrate that in hearts from mPDK1-/- or double GSK3alpha/GSK3beta knockin mice, insulin failed to stimulate the activity of GS or induce its dephosphorylation at residues that are phosphorylated by GSK3. We also establish that in the heart, both GSK3 isoforms participate in the regulation of GS, with GSK3beta playing a more prominent role. This contrasts with skeletal muscle where GSK3beta is the major regulator of insulin-induced GS activity. Despite the inability of insulin to stimulate glycogen synthesis in hearts from the mPDK1-/- or double GSK3alpha/GSK3beta knockin mice, these animals possessed normal levels of cardiac glycogen, demonstrating that total glycogen levels are regulated independently of insulin's ability to stimulate GS in the heart and that mechanisms such as allosteric activation of GS by glucose-6-phosphate and/or activation of GS by muscle contraction, could operate to maintain normal glycogen levels in these mice. We also demonstrate that in cardiomyocytes derived from the mPDK1-/- hearts, although the levels of glucose transporter type 4 (GLUT4) are increased 2-fold, insulin failed to stimulate glucose uptake, providing genetic evidence that PDK1 plays a crucial role in enabling insulin to promote glucose uptake in cardiac muscle.     

Postexercise glucose uptake and glycogen synthesis in skeletal muscle from GLUT4-deficient mice.

To determine the role of GLUT4 on postexercise glucose transport and glycogen resynthesis in skeletal muscle, GLUT4-deficient and wild-type mice were studied after a 3 h swim exercise. In wild-type mice, insulin and swimming each increased 2-deoxyglucose uptake by twofold in extensor digitorum longus muscle. In contrast, insulin did not increase 2-deoxyglucose glucose uptake in muscle from GLUT4-null mice. Swimming increased glucose transport twofold in muscle from fed GLUT4-null mice, with no effect noted in fasted GLUT4-null mice. This exercise-associated 2-deoxyglucose glucose uptake was not accompanied by increased cell surface GLUT1 content. Glucose transport in GLUT4-null muscle was increased 1.6-fold over basal levels after electrical stimulation. Contraction-induced glucose transport activity was fourfold greater in wild-type vs. GLUT4-null muscle. Glycogen content in gastrocnemius muscle was similar between wild-type and GLUT4-null mice and was reduced approximately 50% after exercise. After 5 h carbohydrate refeeding, muscle glycogen content was fully restored in wild-type, with no change in GLUT4-null mice. After 24 h carbohydrate refeeding, muscle glycogen in GLUT4-null mice was restored to fed levels. In conclusion, GLUT4 is the major transporter responsible for exercise-induced glucose transport. Also, postexercise glycogen resynthesis in muscle was greatly delayed; unlike wild-type mice, glycogen supercompensation was not found. GLUT4 it is not essential for glycogen repletion since muscle glycogen levels in previously exercised GLUT4-null mice were totally restored after 24 h carbohydrate refeeding.-Ryder, J. W., Kawano, Y., Galuska, D., Fahlman, R., Wallberg-Henriksson, H., Charron, M. J., Zierath, J. R. Postexercise glucose uptake and glycogen synthesis in skeletal muscle from GLUT4-deficient mice.     

Preservation of glucose metabolism in hypertrophic GLUT4-null hearts

GLUT4-null mice lacking the insulin-sensitive glucose transporter are not diabetic but do exhibit abnormalities in glucose and lipid metabolism. The most striking morphological consequence of ablating GLUT4 is cardiac hypertrophy. GLUT4-null hearts display characteristics of hypertrophy caused by hypertension. However, GLUT4-null mice have normal blood pressure and maintain a normal cardiac contractile protein profile. Unexpectedly, although they lack the predominant glucose transporter in the heart, GLUT4-null hearts transport glucose and synthesize glycogen at normal levels, but gene expression of rate-limiting enzymes involved in fatty acid oxidation is decreased. The GLUT4-null heart represents a unique model of hypertrophy that may be used to study the consequences of altered substrate utilization in normal and pathophysiological conditions.     

p38γ and p38δ regulate postnatal cardiac metabolism through glycogen synthase 1

During the first weeks of postnatal heart development, cardiomyocytes undergo a major adaptive metabolic shift from glycolytic energy production to fatty acid oxidation. This metabolic change is contemporaneous to the up-regulation and activation of the p38γ and p38δ stress-activated protein kinases in the heart. We demonstrate that p38γ/δ contribute to the early postnatal cardiac metabolic switch through inhibitory phosphorylation of glycogen synthase 1 (GYS1) and glycogen metabolism inactivation. Premature induction of p38γ/δ activation in cardiomyocytes of newborn mice results in an early GYS1 phosphorylation and inhibition of cardiac glycogen production, triggering an early metabolic shift that induces a deficit in cardiomyocyte fuel supply, leading to whole-body metabolic deregulation and maladaptive cardiac pathogenesis. Notably, the adverse effects of forced premature cardiac p38γ/δ activation in neonate mice are prevented by maternal diet supplementation of fatty acids during pregnancy and lactation. These results suggest that diet interventions have a potential for treating human cardiac genetic diseases that affect heart metabolism.

This study elucidates the role of the protein kinases p37γ and p38δ in regulating the metabolic switch that occurs in early postnatal development, revealing that they inhibit glycogen synthase 1 and glycogen metabolism. Deregulation of this mechanism results in cardiac defects and metabolic alterations which can be prevented by maternal fatty acid diet supplementation during pregnancy and lactation.     

Molecular characterization of glycogen synthase 1 and its tissue expression profile with type II hexokinase and muscle-type phosphofructokinase in horses

Muscle glycogen synthase (GYS1) is the rate-limiting enzyme in glycogen synthesis, and its activity is regulated by the phosphorylation states of certain amino acid residues encoded by the GYS1 gene. In the present study, the authors molecularly characterized the full-length equine GYS1 (eGYS1) cDNA and found that it contains a less common polyadenylation signal (AATACA). An amino acid alignment with other mammalian GYS1 showed that the phosphorylation sites in eGYS1 are completely conserved. Genomic DNA analysis revealed that the equine-specific substitutions (Glu 16 Asp and Ala 252 Thr) were completely conserved among six equine species. The tissue expression profiles of eGYS1, equine type II hexokinase (eHKII) and muscle-type phosphofructokinase (ePFKM) were determined by real-time PCR and western blot analysis. The mRNA expression level of eGYS1 was significantly higher in the cervical muscle as compared to other tissues. The cervical muscle and heart tissue samples contained a broad range of eGYS1 protein bands that appeared to reflect multiple phosphorylation states. eHKII was predominately expressed only in the cervical muscle; unlike its expression in other mammals, eHKII was not substantially expressed in the insulin-responsive heart or adipose tissue of horse. The expression level of ePFKM mRNA was significantly higher in the heart than in the cervical muscle, which differs from the PFKM expression pattern of other mammals. These tissue expression profiles are fundamental for the understanding of equine glucose metabolism.     

Mass spectrometric quantification of glycogen to assess primary substrate accumulation in the Pompe mouse

Glycogen storage in the α-glucosidase knockout((6neo/6neo)) mouse recapitulates the biochemical defect that occurs in the human condition; as such, this mouse serves as a model for the inherited metabolic deficiency of lysosomal acid α-glucosidase known as Pompe disease. Although this model has been widely used for the assessment of therapies, the time course of glycogen accumulation that occurs as untreated Pompe mice age has not been reported. To address this, we developed a quantitative method involving amyloglucosidase digestion of glycogen and quantification of the resulting free glucose by liquid chromatography/electrospray ionization-tandem mass spectrometry. The method was sensitive enough to measure as little as 0.1 μg of glycogen in tissue extracts with intra- and interassay coefficients of variation of less than 12%. Quantification of glycogen in tissues from Pompe mice from birth to 26 weeks of age showed that, in addition to the accumulation of glycogen in the heart and skeletal muscle, glycogen also progressively accumulated in the brain, diaphragm, and skin. Glycogen storage was also evident at birth in these tissues. This method may be particularly useful for longitudinal assessment of glycogen reduction in response to experimental therapies being trialed in this model.     

Insulin control of glycogen metabolism in knockout mice lacking the muscle-specific protein phosphatase PP1G/RGL

The regulatory-targeting subunit (RGL), also called GM) of the muscle-specific glycogen-associated protein phosphatase PP1G targets the enzyme to glycogen where it modulates the activity of glycogen-metabolizing enzymes. PP1G/RGL has been postulated to play a central role in epinephrine and insulin control of glycogen metabolism via phosphorylation of RGL. To investigate the function of the phosphatase, RGL knockout mice were generated. Animals lacking RGL show no obvious defects. The RGL protein is absent from the skeletal and cardiac muscle of null mutants and present at approximately 50% of the wild-type level in heterozygotes. Both the level and activity of C1 protein are also decreased by approximately 50% in the RGL-deficient mice. In skeletal muscle, the glycogen synthase (GS) activity ratio in the absence and presence of glucose-6-phosphate is reduced from 0.3 in the wild type to 0.1 in the null mutant RGL mice, whereas the phosphorylase activity ratio in the absence and presence of AMP is increased from 0.4 to 0.7. Glycogen accumulation is decreased by approximately 90%. Despite impaired glycogen accumulation in muscle, the animals remain normoglycemic. Glucose tolerance and insulin responsiveness are identical in wild-type and knockout mice, as are basal and insulin-stimulated glucose uptakes in skeletal muscle. Most importantly, insulin activated GS in both wild-type and RGL null mutant mice and stimulated a GS-specific protein phosphatase in both groups. These results demonstrate that RGL is genetically linked to glycogen metabolism, since its loss decreases PP1 and basal GS activities and glycogen accumulation. However, PP1G/RGL is not required for insulin activation of GS in skeletal muscle, and rather another GS-specific phosphatase appears to be involved.     

Cardiac glycogen accumulation after dexamethasone is regulated by AMPK

Glycogen is an immediate source of glucose for cardiac tissue to maintain its metabolic homeostasis. However, its excess brings about cardiac structural and physiological impairments. Previously, we have demonstrated that in hearts from dexamethasone (Dex)-treated animals, glycogen accumulation was enhanced. We examined the influence of 5'-AMP-activated protein kinase (AMPK) on glucose entry and glycogen synthase as a means of regulating the accumulation of this stored polysaccharide. After Dex, cardiac tissue had a limited contribution toward the development of whole body insulin resistance. Measurement of glucose transporter 4 (GLUT4) at the plasma membrane revealed an excess presence of this transporter protein at this location. Interestingly, this was accompanied by an increase in GLUT4 in the intracellular membrane fraction, an effect that was well correlated with increased GLUT4 mRNA. Both total and phosphorylated AMPK increased after Dex. Immunoprecipitation of Akt substrate of 160 kDa (AS160) followed by Western blot analysis demonstrated no change in Akt phosphorylation at Ser(473) and Thr(308) in Dex-treated hearts. However, there was a significant increase in AMPK phosphorylation at Thr(172), which correlated well with AS160 phosphorylation. In Dex-treated hearts, there was a considerable reduction in the phosphorylation of glycogen synthase, whereas glycogen synthase kinase-3-beta phosphorylation was augmented. Our data suggest that AMPK-mediated glucose entry combined with the activation of glycogen synthase and a reduction in glucose oxidation (Qi et al., Diabetes 53: 1790-1797, 2004) act together to promote glycogen storage. Should these effects persist chronically in the heart, they may explain the increased morbidity and mortality observed with long-term excesses in endogenous or exogenous glucocorticoids.     

Inhibitor-1 is not required for the activation of glycogen synthase by insulin in skeletal muscle.

Glycogen synthase is an excellent in vitro substrate for protein phosphatase-1 (PP1), which is potently inhibited by the phosphorylated forms of DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, M(r) = 32,000) and Inhibitor-1. To test the hypothesis that the activation of glycogen synthase by insulin is due to a decrease in the inhibition of PP1 by the phosphatase inhibitors, we have investigated the effects of insulin on glycogen synthesis in skeletal muscles from wild-type mice and mice lacking Inhibitor-1 and DARPP-32 as a result of targeted disruption of the genes encoding the two proteins. Insulin increased glycogen synthase activity and the synthesis of glycogen to the same extent in wild-type and knockout mice, indicating that neither Inhibitor-1 nor DARPP-32 is required for the full stimulatory effects of insulin on glycogen synthase and glycogen synthesis in skeletal muscle.     

Exercise capacity of mice genetically lacking muscle glycogen synthase: in mice, muscle glycogen is not essential for exercise

The glucose storage polymer glycogen is generally considered to be an important source of energy for skeletal muscle contraction and a factor in exercise endurance. A genetically modified mouse model lacking muscle glycogen was used to examine whether the absence of the polysaccharide affects the ability of mice to run on a treadmill. The MGSKO mouse has the GYS1 gene, encoding the muscle isoform of glycogen synthase, disrupted so that skeletal muscle totally lacks glycogen. The morphology of the soleus and quadriceps muscles from MGSKO mice appeared normal. MGSKO-null mice, along with wild type littermates, were exercised to exhaustion. There were no significant differences in the work performed by MGSKO mice as compared with their wild type littermates. The amount of liver glycogen consumed during exercise was similar for MGSKO and wild type animals. Fasting reduced exercise endurance, and after overnight fasting, there was a trend to reduced exercise endurance for the MGSKO mice. These studies provide genetic evidence that in mice muscle glycogen is not essential for strenuous exercise and has relatively little effect on endurance.     

Glycogen synthase (GYS1) mutation causes a novel skeletal muscle glycogenosis

Polysaccharide storage myopathy (PSSM) is a novel glycogenosis in horses characterized by abnormal glycogen accumulation in skeletal muscle and muscle damage with exertion. It is unlike glycogen storage diseases resulting from known defects in glycogenolysis, glycolysis, and glycogen synthesis that have been described in humans and domestic animals. A genome-wide association identified GYS1, encoding skeletal muscle glycogen synthase (GS), as a candidate gene for PSSM. DNA sequence analysis revealed a mutation resulting in an arginine-to-histidine substitution in a highly conserved region of GS. Functional analysis demonstrated an elevated GS activity in PSSM horses, and haplotype analysis and allele age estimation demonstrated that this mutation is identical by descent among horse breeds. This is the first report of a gain-of-function mutation in GYS1 resulting in a glycogenosis.     

Sex difference in neural tube defects in p53-null mice is caused by differences in the complement of X not Y genes

To shed light on the biological origins of sex differences in neural tube defects (NTDs), we examined Trp53-null C57BL/6 mouse embryos and neonates at 10.5 and 18.5 days post coitus (dpc) and at birth. We confirmed that female embryos show more NTDs than males. We also examined mice in which the testis-determining gene Sry is deleted from the Y chromosome but inserted onto an autosome as a transgene, producing XX and XY gonadal females and XX and XY gonadal males. At birth, Trp53 nullizygous mice were predominantly XY rather than XX, irrespective of gonadal type, showing that the sex difference in the lethal effect of Trp53 nullizygosity by postnatal day 1 is caused by differences in sex chromosome complement. At 10.5 dpc, the incidence of NTDs in Trp53-null progeny of XY* mice, among which the number of the X chromosomes varies independently of the presence or absence of a Y chromosome, was higher in mice with two copies of the X chromosome than in mice with a single copy. The presence of a Y chromosome had no protective effect, suggesting that sex differences in NTDs are caused by sex differences in the number of X chromosomes.     

Exercise training does not correct abnormal cardiac glycogen accumulation in the db/db mouse model of type 2 diabetes

Substrate imbalance is a well-recognized feature of diabetic cardiomyopathy. Insulin resistance effectively limits carbohydrate oxidation, resulting in abnormal cardiac glycogen accumulation. Aims of the present study were to 1) characterize the role of glycogen-associated proteins involved in excessive glycogen accumulation in type 2 diabetic hearts and 2) determine if exercise training can attenuate abnormal cardiac glycogen accumulation. Control (db(+)) and genetically diabetic (db/db) C57BL/KsJ-lepr(db)/lepr(db) mice were subjected to sedentary or treadmill exercise regimens. Exercise training consisted of high-intensity/short-duration (10 days) and low-intensity/long-duration (6 wk) protocols. Glycogen levels were elevated by 35-50% in db/db hearts. Exercise training further increased (2- to 3-fold) glycogen levels in db/db hearts. Analysis of soluble and insoluble glycogen pools revealed no differential accumulation of one glycogen subspecies. Phosphorylation (Ser(640)) of glycogen synthase, an indicator of enzymatic fractional activity, was greater in db/db mice subjected to sedentary and exercise regimens. Elevated glycogen levels were accompanied by decreased phosphorylation (Thr(172)) of 5'-AMP-activated kinase and phosphorylation (Ser(79)) of its downstream substrate acetyl-CoA carboxylase. Glycogen concentration was not associated with increases in other glycogen-associated proteins, including malin and laforin. Novel observations show that exercise training does not correct diabetes-induced elevations in cardiac glycogen but, rather, precipitates further accumulation.     

Appearance of a nonfunctional isozyme of hepatic glycogen synthase in late gestation

Glycogen levels, glycogen synthase activities, and glycogen synthase protein levels were determined in liver tissues obtained from 14- to 19-day-old fetal mice, newborn mice, and adult mice. The results of these experiments demonstrate a significant increase in the quantity of hepatic glycogen synthase beginning at Day 17 of gestation and reaching adult levels at birth. However, during the same time period, there is a dramatic decrease in total glycogen synthase activity suggesting that the accumulating glycogen synthase molecules are unable to transfer UDP-glucose to glycogen. These inversely coordinated changes in the quantity and activity of glycogen synthase are consistent with the suggestion that glycogen synthesis in the near-term fetal mouse is being maintained by preexisting enzyme, while accumulating enzyme molecules may represent a quiescent isozyme.     

A GYS1 gene mutation is highly associated with polysaccharide storage myopathy in Cob Normand draught horses

Glycogen storage diseases or glycogenoses are inherited diseases caused by abnormalities of enzymes that regulate the synthesis or degradation of glycogen. Deleterious mutations in many genes of the glyco(geno)lytic or the glycogenesis pathways can potentially cause a glycogenosis, and currently mutations in fourteen different genes are known to cause animal or human glycogenoses, resulting in myopathies and/or hepatic disorders. The genetic bases of two forms of glycogenosis are currently known in horses. A fatal neonatal polysystemic type IV glycogenosis, inherited recessively in affected Quarter Horse foals, is due to a mutation in the glycogen branching enzyme gene ( GBE1 ). A second type of glycogenosis, termed polysaccharide storage myopathy (PSSM), is observed in adult Quarter Horses and other breeds. A severe form of PSSM also occurs in draught horses. A mutation in the skeletal muscle glycogen synthase gene ( GYS1 ) was recently reported to be highly associated with PSSM in Quarter Horses and Belgian draught horses. This GYS1 point mutation appears to cause a gain-of-function of the enzyme and to result in the accumulation of a glycogen-like, less-branched polysaccharide in skeletal muscle. It is inherited as a dominant trait. The aim of this work was to test for possible associations between genetic polymorphisms in four candidate genes of the glycogen pathway or the GYS1 mutation in Cob Normand draught horses diagnosed with PSSM by muscle biopsy.     

Heparan sulfate expression in the neural crest is essential for mouse cardiogenesis

Impaired heparan sulfate (HS) synthesis in vertebrate development causes complex malformations due to the functional disruption of multiple HS-binding growth factors and morphogens. Here, we report developmental heart defects in mice bearing a targeted disruption of the HS-generating enzyme GlcNAc N-deacetylase/GlcN N-sulfotransferase 1 (NDST1), including ventricular septal defects (VSD), persistent truncus arteriosus (PTA), double outlet right ventricle (DORV), and retroesophageal right subclavian artery (RERSC). These defects closely resemble cardiac anomalies observed in mice made deficient in the cardiogenic regulator fibroblast growth factor 8 (FGF8). Consistent with this, we show that HS-dependent FGF8/FGF-receptor2C assembly and FGF8-dependent ERK-phosphorylation are strongly reduced in NDST1^−/− embryonic cells and tissues. Moreover, WNT1-Cre/LoxP-mediated conditional targeting of NDST function in neural crest cells (NCCs) revealed that their impaired HS-dependent development contributes strongly to the observed cardiac defects. These findings raise the possibility that defects in HS biosynthesis may contribute to congenital heart defects in humans that represent the most common type of birth defect.