Muscle Glycogen Synthesis Research Articles

Restoration of muscle functionality by genetic suppression of glycogen synthesis in a murine model of Pompe disease

Glycogen storage disease type II (GSDII) or Pompe disease is an autosomal recessive disorder caused by acid alpha-glucosidase (GAA) deficiency, leading to lysosomal glycogen accumulation. Affected individuals store glycogen mainly in cardiac and skeletal muscle tissues resulting in fatal hypertrophic cardiomyopathy and respiratory failure in the most severe infantile form. Enzyme replacement therapy has already proved some efficacy, but results remain variable especially in skeletal muscle. Substrate reduction therapy was successfully used to improve the phenotype in several lysosomal storage disorders. We have recently demonstrated that shRNA-mediated reduction of glycogen synthesis led to a significant reduction of glycogen accumulation in skeletal muscle of GSDII mice. In this paper, we analyzed the effect of a complete genetic elimination of glycogen synthesis in the same GSDII model. GAA and glycogen synthase 1 (GYS1) KO mice were inter-crossed to generate a new double-KO model. GAA/GYS1-KO mice exhibited a profound reduction of the amount of glycogen in the heart and skeletal muscles, a significant decrease in lysosomal swelling and autophagic build-up as well as a complete correction of cardiomegaly. In addition, the abnormalities in glucose metabolism and insulin tolerance observed in the GSDII model were corrected in double-KO mice. Muscle atrophy observed in 11-month-old GSDII mice was less pronounced in GAA/GYS1-KO mice, resulting in improved exercise capacity. These data demonstrate that long-term elimination of muscle glycogen synthesis leads to a significant improvement of structural, metabolic and functional defects in GSDII mice and offers a new perspective for the treatment of Pompe disease.

Hypothalamic Orexin Stimulates Feeding-Associated Glucose Utilization in Skeletal Muscle via Sympathetic Nervous System

Hypothalamic neurons containing orexin (hypocretin) are activated during motivated behaviors and active waking. We show that injection of orexin-A into the ventromedial hypothalamus (VMH) of mice or rats increased glucose uptake and promoted insulin-induced glucose uptake and glycogen synthesis in skeletal muscle, but not in white adipose tissue, by activating the sympathetic nervous system. These effects of orexin were blunted in mice lacking beta-adrenergic receptors but were restored by forced expression of the beta(2)-adrenergic receptor in both myocytes and nonmyocyte cells of skeletal muscle. Orexin neurons are activated by conditioned sweet tasting and directly excite VMH neurons, thereby increasing muscle glucose metabolism and its insulin sensitivity. Orexin and its receptor in VMH thus play a key role in the regulation of muscle glucose metabolism associated with highly motivated behavior by activating muscle sympathetic nerves and beta(2)-adrenergic signaling.

Where Does Insulin Resistance Start?

During the last two decades, many studies have focused on the pivotal role of the hypothalamus in the control of energy metabolism (1). Hypothalamic nuclei, particularly the arcuate and the ventromedial, receive numerous peripheral inputs from adipokines (leptin, adiponectin, and resistin) and free fatty acids. These signals of “nutrient abundance” lead to the activation of multiple hypothalamic pathways that overlap and generate a plethora of central and peripheral responses (2). In this context, the central neuronal signals generated by insulin have been the most extensively studied (3). The pivotal experiment performed by Woods et al. (4) in the late 1970s showed that intracerebral infusion of insulin in baboons reduced food intake and body weight and paved the way for intense scientific investigation over subsequent years by highlighting the key role of insulin at the central nervous system (CNS) level. Insulin receptors (5) and components of the insulin signaling pathway (6) are widely distributed in the brain. Insulin interacts with its binding sites by crossing the blood-brain barrier through a receptor-mediated and saturable transport mechanism (7), although it has been hypothesized that insulin is also synthesized in the brain (7). Considerable evidence has been generated to indicate that insulin can modulate the expression of neuropeptides involved in the regulation of food intake within the CNS (8) and also can influence glucoregulation via CNS connections that regulate hepatic glucose production (9), glycogen synthesis in the skeletal muscle (10), and fat metabolism in adipocytes (11). By activating its receptors, insulin directly suppresses prepro-NPY mRNA transcription in the arcuate nucleus, leading to a reduction in NPY and a decrease in food intake (12), whereas intracerebroventricular delivery of insulin increases the expression of pro-opiomelanocortin (POMC) (13) (Fig. 1). This hypothalamic mode of insulin action resembles the well-known effect of leptin, suggesting that these …

Magnetic resonance spectroscopy shows an inverse correlation between intramyocellular lipid content in human calf muscle and local glycogen synthesis rate

Intramyocellular lipid (IMCL) content of skeletal muscle, as measured with (1)H MRS, is inversely correlated with insulin sensitivity as determined by whole body glucose uptake. The latter, however, does not necessarily represent the actual glucose uptake in the corresponding skeletal muscle. In this study, we examined whether IMCL content in human calf muscle correlated with local glucose uptake assessed by measurement of glycogen synthesis rate within the same muscle compartment. We studied 20 subjects belonging to four subgroups of five persons each: young lean, elderly lean, young obese and elderly obese. IMCL content in the soleus and gastrocnemius muscle was determined using (1)H MR spectroscopic imaging and local glycogen synthesis rate in the calf muscle was measured by (13)C MRS during a euglycaemic hyperinsulinaemic clamp with 20% w/v 30% (13)C-1-labelled glucose infusion. Significantly higher IMCL contents were found in elderly (soleus: p < 0.0001 and gastrocnemius: p < 0.01) and obese subjects (p < 0.01 for both muscles). Local glycogen synthesis rate decreased significantly with obesity (p < 0.01). The principal finding of this study was that the mean IMCL content of the soleus and gastrocnemius muscles was indeed inversely correlated with the local glycogen synthesis rate in the calf muscle (r(s) = -0.50, p < 0.05), with a very similar dependency as the inverse correlation between mean IMCL content and total body glucose uptake (r(s) = -0.54, p < 0.05). We conclude that IMCL content of the soleus and gastrocnemius muscles reflects a measure for local insulin resistance within the same muscle compartment as determined by glycogen synthesis rate. Although the inverse correlation suggests that insulin sensitivity is affected by the local amount of fat present, it remains to be determined if this is a cause or a consequence.

Glycogen content regulates insulin‐ but not contraction‐mediated glycogen synthase activation in the rat slow‐twitch soleus muscles

The aim of this study was to investigate the effect of glycogen content on glycogen synthase (GS) activation and phosphorylation in the slow-twitch soleus muscles after contraction, during insulin stimulation and when these two stimuli were combined. Glycogen content was manipulated in vivo with 24 h fasting and fasting followed by 24 h refeeding. Soleus strips were electrically stimulated for 30 min in vitro, and GS activation and phosphorylation were investigated after an additional 30 min incubation with or without insulin. Fasting reduced glycogen content in soleus muscle by 40% and refeeding enhanced by 40%, compared to rats with free access to chow. Insulin-stimulated GS fractional activity was inversely correlated with glycogen content (R = -0.95, P < 0.001, n = 24) and rate of glycogen synthesis was also inversely correlated with glycogen content (R = -0.70, P < 0.001, n = 36). After contraction, GS fractional activity was increased to similar levels in muscles with low, normal and high glycogen content; rate of glycogen synthesis after contraction was also similar. After contraction, insulin additively increased GS activation at all glycogen contents. Group means of GS fractional activity was inversely correlated with GS Ser(641) (R = -0.93, P < 0.001) and Ser(645,649,653,657) (R = -0.85, P < 0.001) phosphorylation, but not with Ser(7) phosphorylation. Glycogen content regulates insulin- but not contraction-stimulated GS activation and glycogen synthesis in soleus muscles. Furthermore, phosphorylation of GS Ser(641) and Ser(645,649,653,657) seems to regulate GS activity in soleus.

Stimulatory effect of apigenin-6-C-β- l-fucopyranoside on insulin secretion and glycogen synthesis

In vivo and in vitro treatments were carried out to investigate the effects of apigenin-6-C-β- l-fucopyranoside ( 1), isolated from Averrhoa carambola L. (Oxalidaceae), on serum glucose and insulin levels in hyperglycemic rats as well as its effect on glycogen synthesis in normal rat soleus muscle. Apigenin-6-C-β- l-fucopyranoside showed an acute effect on blood glucose lowering in hyperglycemic rats and stimulated glucose-induced insulin secretion. A stimulatory effect of 1 on glycogen synthesis was observed when muscles were incubated with this flavonoid and also its effect was completely nullified by pre-treatment with insulin signal transduction inhibitors. Taking this into account, the MAPK–PP1 and PI3K–GSK3 pathways are involved in the apigenin-6-C-β- l-fucopyranoside-induced increase in glycogen synthesis in muscle. This study provides evidence for dual effects of apigenin-6-C-β- l-fucopyranoside as an antihyperglycemic (insulin secretion) as well as an insulinomimetic (glycogen synthesis) agent.

The second-meal phenomenon is associated with enhanced muscle glycogen storage in humans

The rise in blood glucose after lunch is less if breakfast has been eaten. The metabolic basis of this second-meal phenomenon remains uncertain. We hypothesized that storage of ingested glucose as glycogen could be responsible during the post-meal suppression of plasma NEFAs (non-esterified fatty acids; 'free' fatty acids). In the present study we determined the metabolic basis of the second-meal phenomenon. Healthy subjects were studied on two separate days, with breakfast and without breakfast in a random order. We studied metabolic changes after a standardized test lunch labelled with 3 g of 13C-labelled (99%) glucose. Changes in post-prandial muscle glycogen storage were measured using 13C magnetic resonance spectroscopy. The rise in plasma glucose after lunch was significantly less if breakfast had been taken (0.9+/-0.3 compared with 3.2+/-0.3 mmol/l, with and without breakfast respectively; P<0.001), despite comparable insulin responses. Pre-lunch NEFAs were suppressed after breakfast (0.13+/-0.03 compared with 0.51+/-0.04 mmol/l) and levels correlated positively with the maximum glucose rise after lunch (r=0.62, P=0.001). The increase in muscle glycogen signal was greater 5 h after lunch on the breakfast day (103+/-21 compared with 48+/-12 units; P<0.007) and correlated negatively with plasma NEFA concentrations before lunch (r=-0.48, P<0.05). The second-meal effect is associated with priming of muscle glycogen synthesis consequent upon sustained suppression of plasma NEFA concentrations.

N-3 Fatty acids and rosiglitazone improve insulin sensitivity through additive stimulatory effects on muscle glycogen synthesis in mice fed a high-fat diet

n-3 Fatty acids and rosiglitazone improve insulin sensitivity through additive stimulatory effects on muscle glycogen synthesis in mice fed a high-fat diet

Signaling pathways of kaempferol-3-neohesperidoside in glycogen synthesis in rat soleus muscle

Kaempferol 3-neohesperidoside is one of the several compounds that have been reported to have insulin-like properties in terms of glucose lowering. We studied the effect of kaempferol 3-neohesperidoside in glycogen synthesis in rat soleus muscle through the incorporation of 14C- d-glucose in glycogen. Kaempferol 3-neohesperidoside stimulates glycogen synthesis in rat soleus muscle by approximately 2.38-fold. Insulin at 100 nM showed a stimulatory effect on glycogen synthesis when compared with the control group. The stimulatory effect of kaempferol 3-neophesperidoside on glycogen synthesis was inhibited by wortmannin, the phosphatidylinositol 3-kinase (PI3K) inhibitor, and enhanced by lithium chloride, a glycogen synthase kinase 3 (GSK-3) inhibitor. Moreover, the stimulatory effect of kaempferol 3-neohesperidoside was also nullified by PD98059, a specific inhibitor of mitogen-activated protein kinase (MEK) and by calyculin A, an inhibitor of protein phosphatase 1 (PP1) activity. It was concluded that the PI3K – GSK-3 pathway and MAPK – PP1 pathway are involved in the stimulatory kaempferol 3-neohesperidoside effect on glycogen synthesis in rat soleus muscle.

Lipid and insulin infusion-induced skeletal muscle insulin resistance is likely due to metabolic feedback and not changes in IRS-1, Akt, or AS160 phosphorylation

Type 2 diabetes is characterized by hyperlipidemia, hyperinsulinemia, and insulin resistance. The aim of this study was to investigate whether acute hyperlipidemia-induced insulin resistance in the presence of hyperinsulinemia was due to defective insulin signaling. Hyperinsulinemia (approximately 300 mU/l) with hyperlipidemia or glycerol (control) was produced in cannulated male Wistar rats for 0.5, 1 h, 3 h, or 5 h. The glucose infusion rate required to maintain euglycemia was significantly reduced by 3 h with lipid infusion and was further reduced after 5 h of infusion, with no difference in plasma insulin levels, indicating development of insulin resistance. Consistent with this finding, in vivo skeletal muscle glucose uptake (31%, P < 0.05) and glycogen synthesis rate (38%, P < 0.02) were significantly reduced after 5 h compared with 3 h of lipid infusion. Despite the development of insulin resistance, there was no difference in the phosphorylation state of multiple insulin-signaling intermediates or muscle diacylglyceride and ceramide content over the same time course. However, there was an increase in cumulative exposure to long-chain acyl-CoA (70%) with lipid infusion. Interestingly, although muscle pyruvate dehydrogenase kinase 4 protein content was decreased in hyperinsulinemic glycerol-infused rats, this decrease was blunted in muscle from hyperinsulinemic lipid-infused rats. Decreased pyruvate dehydrogenase complex activity was also observed in lipid- and insulin-infused animals (43%). Overall, these results suggest that acute reductions in muscle glucose metabolism in rats with hyperlipidemia and hyperinsulinemia are more likely a result of substrate competition than a significant early defect in insulin action or signaling.

Maintenance of contractile properties despite impaired skeletal muscle oxidative metabolism in diet‐induced obesity.

Diet‐induced obesity (DIO) is increasing in prevalence among youth; a time where robust muscle growth occurs. We hypothesized that consumption of a high fat diet (HFD) in early adulthood would negatively impact mouse muscle metabolism and contractile function. HFD (~60% kcal fat for 8 weeks) induced an obese and insulin resistant phenotype with reduced relative muscle mass (72 ± 1.9%) compared to control.Using isolated single muscle fibers for analysis, HFD induced a significant impairment in palmitate and glucose oxidation (72.8 ± 6.6% and 61.8 ± 9.1% of control respectively). This novel technique was validated in intact muscle. Impaired insulin responses were noted for glucose metabolism and glycogen synthesis in HFD muscle vs. control, while glucose and palmitate uptake was not altered between groups. Preliminary RT‐PCR results demonstrate no difference in CPT‐1, PGC‐1α, PPARα or UCP‐2 expression. Further gene and protein analysis into the mechanisms underlying changes in fat uptake and oxidation are currently underway. Despite metabolic impairments, HFD muscle exhibited no loss of maximal contractile force or alteration in fatigue rates.Our data suggest that HFD skeletal muscle is highly adaptive in the face of impaired substrate oxidation, as demonstrated by the preservation of contractile properties. These findings contribute to our understanding of the impact of DIO on developing skeletal muscle.

N-3 Fatty acids and rosiglitazone improve insulin sensitivity through additive stimulatory effects on muscle glycogen synthesis in mice fed a high-fat diet

Fatty acids of marine origin, i.e. docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) act as hypolipidaemics, but they do not improve glycaemic control in obese and diabetic patients. Thiazolidinediones like rosiglitazone are specific activators of peroxisome proliferator-activated receptor gamma, which improve whole-body insulin sensitivity. We hypothesised that a combined treatment with a DHA and EPA concentrate (DHA/EPA) and rosiglitazone would correct, by complementary additive mechanisms, impairments of lipid and glucose homeostasis in obesity. Male C57BL/6 mice were fed a corn oil-based high-fat diet. The effects of DHA/EPA (replacing 15% dietary lipids), rosiglitazone (10 mg/kg diet) or a combination of both on body weight, adiposity, metabolic markers and adiponectin in plasma, as well as on liver and muscle gene expression and metabolism were analysed. Euglycaemic-hyperinsulinaemic clamps were used to characterise the changes in insulin sensitivity. The effects of the treatments were also analysed in dietary obese mice with impaired glucose tolerance (IGT). DHA/EPA and rosiglitazone exerted additive effects in prevention of obesity, adipocyte hypertrophy, low-grade adipose tissue inflammation, dyslipidaemia and insulin resistance, while inducing adiponectin, suppressing hepatic lipogenesis and decreasing muscle ceramide concentration. The improvement in glucose tolerance reflected a synergistic stimulatory effect of the combined treatment on muscle glycogen synthesis and its sensitivity to insulin. The combination treatment also reversed dietary obesity, dyslipidaemia and IGT. DHA/EPA and rosiglitazone can be used as complementary therapies to counteract dyslipidaemia and insulin resistance. The combination treatment may reduce dose requirements and hence the incidence of adverse side effects of thiazolidinedione therapy.

Knockdown of endogenous SKIP gene enhanced insulin-induced glycogen synthesis signaling in differentiating C2C12 myoblasts

PI(3,4,5)P(3) produced by the activated PI3-kinase is a key lipid second messenger in cell signaling downstream of insulin. Skeletal muscle and kidney-enriched inositol phosphatase (SKIP) identified as a 5'-inositol phosphatase that hydrolyzes PI(3,4,5) P(3) to PI(3,4)P(2), negatively regulates the insulin-induced glycogen synthesis in skeletal muscle. However the mechanism by which this occurs remains unclear. To elucidate the function of SKIP in glycogen synthesis, we employed RNAi techniques to knockdown the SKIP gene in differentiating C2C12 myoblasts. Insulin-induced phosphorylation of Akt (protein kinase B) and GSK-3beta (Glycogen synthase kinase), subsequent dephosphorylation of glycogen synthase and glycogen synthesis were increased by inhibiting the expression of SKIP, whereas the insulin-induced glycogen synthesis was decreased by overexpression of WT-SKIP. Our results suggest that SKIP plays a negative regulatory role in Akt/ GSK-3beta/GS (glycogen synthase) pathway leading to glycogen synthesis in myocytes.

Insulin Resistance in Striated Muscle-specific Integrin Receptor β1-deficient Mice

Integrin receptor plays key roles in mediating both inside-out and outside-in signaling between cells and the extracellular matrix. We have observed that the tissue-specific loss of the integrin beta1 subunit in striated muscle results in a near complete loss of integrin beta1 subunit protein expression concomitant with a loss of talin and to a lesser extent, a reduction in F-actin content. Muscle-specific integrin beta1-deficient mice had no significant difference in food intake, weight gain, fasting glucose, and insulin levels with their littermate controls. However, dynamic analysis of glucose homeostasis using euglycemichyperinsulinemic clamps demonstrated a 44 and 48% reduction of insulin-stimulated glucose infusion rate and glucose clearance, respectively. The whole body insulin resistance resulted from a specific inhibition of skeletal muscle glucose uptake and glycogen synthesis without any significant effect on the insulin suppression of hepatic glucose output or insulin-stimulated glucose uptake in adipose tissue. The reduction in skeletal muscle insulin responsiveness occurred without any change in GLUT4 protein expression levels but was associated with an impairment of the insulin-stimulated protein kinase B/Akt serine 473 phosphorylation but not threonine 308. The inhibition of insulin-stimulated serine 473 phosphorylation occurred concomitantly with a decrease in integrin-linked kinase expression but with no change in the mTOR.Rictor.LST8 complex (mTORC2). These data demonstrate an in vivo crucial role of integrin beta1 signaling events in mediating cross-talk to that of insulin action.

Coingestion of protein with carbohydrate during recovery from endurance exercise stimulates skeletal muscle protein synthesis in humans

Coingestion of protein with carbohydrate (CHO) during recovery from exercise can affect muscle glycogen synthesis, particularly if CHO intake is suboptimal. Another potential benefit of protein feeding is an increased synthesis rate of muscle proteins, as is well documented after resistance exercise. In contrast, the effect of nutrient manipulation on muscle protein kinetics after aerobic exercise remains largely unexplored. We tested the hypothesis that ingesting protein with CHO after a standardized 2-h bout of cycle exercise would increase mixed muscle fractional synthetic rate (FSR) and whole body net protein balance (WBNB) vs. trials matched for total CHO or total energy intake. We also examined whether postexercise glycogen synthesis could be enhanced by adding protein or additional CHO to a feeding protocol that provided 1.2 g CHO x kg(-1) x h(-1), which is the rate generally recommended to maximize this process. Six active men ingested drinks during the first 3 h of recovery that provided either 1.2 g CHO.kg(-1).h(-1) (L-CHO), 1.2 g CHO + 0.4 g protein x kg(-1) x h(-1) (PRO-CHO), or 1.6 g CHO x kg(-1) x h(-1) (H-CHO) in random order. Based on a primed constant infusion of l-[ring-(2)H(5)]phenylalanine, analysis of biopsies (vastus lateralis) obtained at 0 and 4 h of recovery showed that muscle FSR was higher (P < 0.05) in PRO-CHO (0.09 +/- 0.01%/h) vs. both L-CHO (0.07 +/- 0.01%/h) and H-CHO (0.06 +/- 0.01%/h). WBNB assessed using [1-(13)C]leucine was positive only during PRO-CHO, and this was mainly attributable to a reduced rate of protein breakdown. Glycogen synthesis rate was not different between trials. We conclude that ingesting protein with CHO during recovery from aerobic exercise increased muscle FSR and improved WBNB, compared with feeding strategies that provided CHO only and were matched for total CHO or total energy intake. However, adding protein or additional CHO to a feeding strategy that provided 1.2 g CHO x kg(-1) x h(-1) did not further enhance glycogen resynthesis during recovery.

Postexercise Muscle Glycogen Synthesis with Combined Glucose and Fructose Ingestion

To evaluate the efficacy of using combined glucose and fructose (GF) ingestion as a means to stimulate short-term (4 h) postexercise muscle glycogen synthesis compared to glucose only (G). On two separate occasions, six endurance-trained men performed an exhaustive glycogen-depleting exercise bout followed by a 4-h recovery period. Muscle biopsy samples were obtained from the vastus lateralis muscle at 0, 1, and 4 h after exercise. Subjects ingested carbohydrate solutions containing G (90 g x h(-1)) or GF (G = 60 g x h(-1); F = 30 g x h(-1)) commencing immediately after exercise and every 30 min thereafter. Immediate postexercise muscle glycogen concentrations were similar in both trials (G = 128 +/- 25 mmol x kg(-1) dry muscle (dm) vs GF = 112 +/- 16 mmol x kg(-1) dm; P > 0.05). Total glycogen storage during the 4-h recovery period was 176 +/- 33 and 155 +/- 31 mmol x kg(-1) dm for G and GF, respectively (G vs GF, P > 0.05). Hence, mean muscle glycogen synthesis rates during the 4-h recovery period did not differ between the two conditions (G = 44 +/- 8 mmol x kg(-1) dm x h(-1) vs GF = 39 +/- 8 mmol x kg(-1) dm x h(-1), P > 0.05). Plasma glucose and serum insulin responses during the recovery period were similar in both conditions, although plasma lactate concentrations were significantly elevated during GF compared to G (by approximately 0.8 mmol x L(-1), P < 0.05). Glucose and glucose/fructose (2:1 ratio) solutions, ingested at a rate of 90 g x h(-1), are equally effective at restoring muscle glycogen in exercised muscles during the recovery from exhaustive exercise.

Paying the Price for Eating Ice Cream: Is Excessive GLP-1 Signaling in the Brain the Culprit?

Paying the Price for Eating Ice Cream: Is Excessive GLP-1 Signaling in the Brain the Culprit?

Role of fatty acids in the development of insulin resistance and type 2 diabetes mellitus

Insulin resistance is defined as the reduced responsiveness to normal circulating levels of insulin. It is the basic condition of type 2 diabetes mellitus, in which both experimental animals and humans accumulate lipids intracellularly in skeletal muscle and liver. Measurement of these lipids in humans, using nuclear magnetic resonance spectroscopy after lipid infusion, indicated they could cause inhibition of the glucose transporter GLUT4, thereby suppressing glucose entry into cells and inhibiting glucose oxidation and glycogen synthesis in muscle. Furthermore, it is known that the enzyme acetyl-CoA carboxylase2 (ACC2) suppresses the oxidation of fatty acids by inhibiting the entry of fatty acids into mitochondria. Further support for the lipocentric hypothesis of the pathogenesis of insulin resistance was provided by knocking out the gene coding for ACC2 in mice; this led to greater fatty acid oxidation, reduced fat mass and, in consequence, greatly enhanced insulin sensitivity. These studies suggest that a specific inhibitor of ACC2 would have therapeutic potential for type 2 diabetes mellitus.

Modulation of glycogen synthesis by RNA interference: towards a new therapeutic approach for glycogenosis type II

Glycogen storage disease type II (GSDII) or Pompe disease is an autosomal recessive disorder caused by defects in the acid alpha-glucosidase gene, which leads to lysosomal glycogen accumulation and enlargement of the lysosomes mainly in cardiac and muscle tissues, resulting in fatal hypertrophic cardiomyopathy and respiratory failure in the most severely affected patients. Enzyme replacement therapy has already proven to be beneficial in this disease, but correction of pathology in skeletal muscle still remains a challenge. As substrate deprivation was successfully used to improve the phenotype in other lysosomal storage disorders, we explore here a novel therapeutic approach for GSDII based on a modulation of muscle glycogen synthesis. Short hairpin ribonucleic acids (shRNAs) targeted to the two major enzymes involved in glycogen synthesis, i.e. glycogenin (shGYG) and glycogen synthase (shGYS), were selected. C2C12 cells and primary myoblasts from GSDII mice were stably transduced with lentiviral vectors expressing both the shRNAs and the enhanced green fluorescent protein (EGFP) reporter gene. Efficient and specific inhibition of GYG and GYS was associated not only with a decrease in cytoplasmic and lysosomal glycogen accumulation in transduced cells, but also with a strong reduction in the lysosomal size, as demonstrated by confocal microscopy analysis. A single intramuscular injection of recombinant AAV-1 (adeno-associated virus-1) vectors expressing shGYS into newborn GSDII mice led to a significant reduction in glycogen accumulation, demonstrating the in vivo therapeutic efficiency. These data offer new perspectives for the treatment of GSDII and could be relevant to other muscle glycogenoses.

Glimepiride as insulin sensitizer: increased liver and muscle responses to insulin

Glimepiride, a low-potency insulin secretagogue, is as efficient on glycaemic control as other sulphonylureas, suggesting an additional insulin-sensitizer role. The aim of the present study was to confirm the insulin-sensitizer role of glimepiride and to show extra-pancreatic effects of the drug. Three-month-old monosodium glutamate (MSG)-induced obese insulin-resistant rats were treated (OG) or not treated (O) with glimepiride for 4 weeks and compared with age-matched non-obese rats (C). Insulin sensitivity in whole body, glucose transporter 4 (GLUT4) protein content, glucose uptake and glycogen synthesis in oxidative skeletal muscle and phospho-glycogen synthase kinase (p-GSK3) and glycogen content in liver were analysed. Insulin sensitivity, analysed by the insulin tolerance test, was 30% lower in O than in C rats (p < 0.05), and OG rats recovered this parameter (p < 0.05). In oxidative muscle, glimepiride increased the GLUT4 protein content (50%, p < 0.001) and recovered the obesity-induced reduction ( approximately 20%) of the in vitro insulin-stimulated glucose uptake and incorporation into glycogen. In liver, glimepiride increased p-GSK3 (p < 0.01) and glycogen (p < 0.05) contents. The increased GLUT4 protein expression and glucose utilization in oxidative muscle and the increased insulin sensitivity and glycogen storage in liver evidence the insulin-sensitizer effect of glimepiride, which must be important to enable the glimepiride drug to promote an efficient glycaemic control.