Glycogen Synthesis In Skeletal Muscle Research Articles

Hyperglycemia and insulin resistance: possible mechanisms.

Sustained hyperglycemia impairs insulin-stimulated glucose utilization and glycogen synthesis in human and rat skeletal muscles, a phenomenon referred to clinically as glucose toxicity. In rat extensor digitorum longus (EDL) muscle preparations preincubated for 2-4 h in a hyperglycemic medium (25 mM vs. 0 mM glucose), we have shown that the ability of insulin to stimulate glucose incorporation into glycogen is impaired. Interestingly, this was associated with a decreased activation of Akt/PKB, but not its upstream regulator, PI3-kinase. A similar pattern of signaling abnormalities has been observed in adipocytes, L6 muscle cells, C2C12 cells, and (as reported here) EDL incubated with C(2)-ceramide. On the other hand, no increase was observed in ceramide mass in EDL incubated with 25 mM glucose. Hyperglycemia-induced insulin resistance also has been described in adipocytes, where it has been linked to activation of novel and conventional protein kinase C isoforms that phosphorylate the insulin receptor and IRS. In addition, we have recently shown that hyperglycemia causes insulin resistance in cultured human umbilical vein endothelial cells (HUVEC). Here, it was associated with an increased propensity to apoptosis and, as in muscle, with an impaired ability of insulin to activate Akt. Interestingly, these effects of hyperglycemia and an increase in diacylglycerol synthesis, which is also caused, were prevented by adding AICAR, an activator of AMP-activated protein kinase (AMPK), to the incubation medium. These results suggest that hyperglycemia causes insulin resistance in cells other than those in classic insulin target tissues. Whether AMPK activation can reverse or prevent insulin resistance in all of these cells remains to be determined.

The Diabetic Phenotype Is Conserved in Myotubes Established From Diabetic Subjects

The most well-described defect in the pathophysiology of type 2 diabetes is reduced insulin-mediated glycogen synthesis in skeletal muscles. It is unclear whether this defect is primary or acquired secondary to dyslipidemia, hyperinsulinemia, or hyperglycemia. We determined the glycogen synthase (GS) activity; the content of glucose-6-phosphate, glucose, and glycogen; and the glucose transport in satellite cell cultures established from diabetic and control subjects. Myotubes were precultured in increasing insulin concentrations for 4 days and subsequently stimulated acutely by insulin. The present study shows that the basal glucose uptake as well as insulin-stimulated GS activity is reduced in satellite cell cultures established from patients with type 2 diabetes. Moreover, increasing insulin concentrations could compensate for the reduced GS activity to a certain extent, whereas chronic supraphysiological insulin concentrations induced insulin resistance in GS and glucose transport activity. Our data suggest that insulin resistance in patients with type 2 diabetes comprises at least two important defects under physiological insulin concentrations: a reduced glucose transport under basal conditions and a reduced GS activity under acute insulin stimulation, implicating a reduced glucose uptake in the fasting state and a diminished insulin-mediated storage of glucose as glycogen after a meal.

Human skeletal muscle expresses a glycogen-targeting subunit of PP1 that is identical to the insulin-sensitive glycogen-targeting subunit G(L) of liver.

Insulin has been previously shown to regulate the expression of the hepatic glycogen-targeting subunit, G(L), of protein phosphatase 1 (PP1) and is believed to control the activity of the PP1-G(L) complex by modulation of the level of phosphorylase a, which allosterically inhibits the activity of PP1-G(L). These mechanisms contribute to the ability of insulin to increase hepatic glycogen synthesis. Human G(L) shows >88% amino acid identity to its rat and mouse homologs, with complete conservation of the phosphorylase a binding site. G(L) is highly expressed in the liver and present at appreciable levels in heart tissue of all three species. Surprisingly, G(L) is highly expressed in human skeletal muscle while only being detected at very low levels in rat, mouse, and rabbit skeletal muscle. The amino acid sequence of G(L) predicted from the cDNA is identical in human liver and skeletal muscle and encoded by a gene on chromosome 8 at p23.1. The species-specific difference in the level of expression of G(L) mRNA and protein in skeletal muscle has important implications for understanding the mechanisms by which insulin regulates glycogen synthesis in human skeletal muscle and for questions regarding whether rodents are appropriate models for this purpose.

Islet Transplantation under the Kidney Capsule Corrects the Defects in Glycogen Metabolism in Both Liver and Muscle of Streptozocin-Diabetic Rats

Insulin-deficient rats are characterized by multiple defects in the pathway of glycogen synthesis and breakdown in both liver and skeletal muscle. The aim of this study was to clarify whether islet transplantation under the kidney capsule, which is associated with delivery of insulin into the peripheral circulation, is able to normalize glycogen metabolism in liver and muscle of streptozotocin-diabetic rats. Three groups of male Lewis rats were studied under fasting condition: controls, untreated diabetics, and islet transplanted diabetics. Glycogen content, glucose-6-phosphate concentration, and glycogen synthase activity were measured in both liver and skeletal muscle. Untreated diabetic rats were characterized by an increase in glycogen content of 178% and a reduction of glucose-6-phosphate level of 50%. Both glycogen and glucose-6-phosphate contents were restored to normal in transplanted diabetic rats. Active glycogen synthase (0.35 +/- 0.1 nmol/min/mg) and activity ratio (0.22 +/- 0.04) were significantly impaired compared with controls (0.99 +/- 0.2 nmol/min/mg and 0.43 +/- 0.06, respectively) and were normalized by islet transplantation. In the skeletal muscle, glycogen content was similar in the three groups of animals, whereas muscle glucose-6-phosphate level was reduced by 28% and glycogen synthase was in a less active state in the untreated diabetic rats. Both the glucose-6-phosphate concentration and the kinetic profile of glycogen synthase were normalized by islet transplantation. In conclusion, islet transplantation under the kidney capsule corrects the diabetes-induced abnormalities in glycogen and glucose-6-phosphate content and glycogen synthase activity in both liver and skeletal muscle.

Effects of diabetes, vanadium, and insulin on glycogen synthase activation in Wistar rats.

In vivo effects of insulin and vanadium treatment on glycogen synthase (GS), glycogen synthase kinase-3 (GSK-3) and protein phosphatase-1 (PP1) activity were determined in Wistar rats with streptozotocin (STZ)-induced diabetes. The skeletal muscle was freeze-clamped before or following an insulin injection (5 U/kg i.v.). Diabetes, vanadium, and insulin in vivo treatment did not affect muscle GSK-3beta activity as compared to controls. Following insulin stimulation in 4-week STZ-diabetic rats muscle GS fractional activity (GSFA) was increased 3 fold (p < 0.05), while in 7-week diabetic rats it remained unchanged, suggesting development of insulin resistance in longer term diabetes. Muscle PP1 activity was increased in diabetic rats and returned to normal after vanadium treatment, while muscle GSFA remained unchanged. Therefore, it is possible that PP1 is involved in the regulation of some other cellular events of vanadium (other than regulation of glycogen synthesis). The lack of effect of vanadium treatment in stimulating glycogen synthesis in skeletal muscle suggests the involvement of other metabolic pathways in the observed glucoregulatory effect of vanadium.

Obstructive Jaundice Results in Increased Liver Expression of Uncoupling Protein 2 and Intact Skeletal Muscle Glucose Metabolism in the Rat

Background: A majority of patients with pancreatic cancer have obstructive jaundice and diabetes with skeletal muscle insulin resistance. Surgery for these patients is associated with significant morbidity. Uncoupling protein 2 (UCP2) has been proposed to regulate energy expenditure and promote liver vulnerability. The effects of obstructive jaundice on muscle glucose metabolism and expression of UCP2 in liver and muscle are unknown. Methods: Rats were operated with bile duct ligation (BDL). After 7 days, UCP2 mRNA levels were determined in liver and muscle. Simultaneously, insulin-stimulated glucose transport and glycogen synthesis in skeletal muscle were analyzed in vitro. Results: The jaundiced rats lost more weight than pair-fed controls. UCP2 mRNA levels were increased 5-fold in liver but not in muscle in jaundiced rats compared to pair-fed controls. The jaundiced rats were hypoglycemic and hypoinsulinemic but demonstrated intact or enhanced insulin action on skeletal muscle glucose transport and glycogen synthesis in vitro. Muscle glycogen content was increased in the jaundiced rats. Conclusions: Experimental obstructive jaundice in the rat is associated with increased liver expression of UCP2, rapid weight loss, and intact insulin action on skeletal muscle glucose metabolism. Obstructive jaundice, by upregulated liver UCP2, may contribute to the cachexia and high surgical morbidity observed in these patients, but not to skeletal muscle insulin resistance in pancreatic cancer patients.

SH2-containing inositol phosphatase 2 negatively regulates insulin-induced glycogen synthesis in L6 myotubes

PI(3,4,5)P3 produced by PI3-kinase seems to be a key mediator for insulin's metabolic actions. We have recently cloned rat SHIP2 cDNA which is abundantly expressed in target tissues of insulin. Here, we clarify the role of SHIP2 possessing 5'-phosphatase activity toward PI(3,4,5)P3 in insulin signalling in the skeletal muscle. The role of SHIP2 in insulin-induced glycogen synthesis was studied by expressing wild-type (WT)-SHIP2 and a 5'-phosphatase defective (Delta IP)-SHIP2 into L6 myotubes by means of adenovirus mediated gene transfer. The early events of insulin signalling including tyrosine phosphorylation of the insulin receptor and IRS-1, IRS-1 association with the p85 subunit, and PI3-kinase activity were not affected by expression of WT- and Delta IP-SHIP2. Although PI(3,4,5)P3 and PI(3,4)P2 are known to possibly activate a downstream molecule of PI3-kinase Akt in vitro, overexpression of WT-SHIP2 inhibited insulin-induced phosphorylation and activation of Akt. Conversely, Akt activity was increased by expression of Delta IP-SHIP2. GSK3 beta located downstream of Akt is an important molecule to further transmit insulin signal for glycogen synthesis in skeletal muscles. In accordance with the results of Akt, insulin-induced phosphorylation and inactivation of GSK3 beta, subsequent activation of glycogen synthase and glycogen synthesis were decreased by expression of WT-SHIP2, whereas these events were increased by expression of Delta IP-SHIP2. Our results indicate that SHIP2 plays a negative regulatory role via the 5'-phosphatase activity in insulin signalling, and that PI(3,4,5)P3 rather than PI(3,4)P2 is important for in vivo regulation of insulin-induced Akt activation leading to glycogen synthesis in L6 myotubes.

Control of glycogen synthesis by glucose, glycogen, and insulin in cultured human muscle cells.

A key feature of type 2 diabetes is impairment in the stimulation of glycogen synthesis in skeletal muscle by insulin. Glycogen synthesis and the activity of the enzyme glycogen synthase (GS) have been studied in human myoblasts in culture under a variety of experimental conditions. Incubation in the absence of glucose for up to 6 h caused an approximately 50% decrease in glycogen content, which was associated with a small decrease in the fractional activity of GS. Subsequent reincubation with physiological concentrations of glucose led to a dramatic increase in the rate of glycogen synthesis and in the fractional activity of GS, an effect which was both time- and glucose concentration-dependent and essentially additive with the effects of insulin. This effect was seen only after glycogen depletion. Inhibitors of signaling pathways involved in the stimulation of glycogen synthesis by insulin were without significant effect on the stimulatory action of glucose. These results indicate that at least two distinct mechanisms exist to stimulate glycogen synthesis in human muscle: one acting in response to insulin and the other acting in response to glucose after glycogen depletion, such as that which results from exercise or starvation.

Lactate removal is not enhanced in nonstimulated perfused skeletal muscle after endurance training.

The effects of endurance training (running 40 m/min grade for 60 min, 5 days/wk for 8 wk) on skeletal muscle lactate removal was studied in rats by utilizing the isolated hindlimb perfusion technique. Hindlimbs were perfused (single-pass) with Krebs-Henseleit bicarbonate buffer, fresh bovine erythrocytes (hematocrit approximately 30%), 10 mM lactate, and [U-14C]lactate (30,000 dpm/ml). Arterial and venous blood samples were collected every 10 min for the duration of the experiment to assess lactate uptake. During perfusions, no significant differences in skeletal muscle lactate uptake were observed between trained (7.31 +/- 0.20 micromol/min) and control hindlimbs (6.98 +/- 0.43 micromol/min). In support, no significant differences were observed for [14C]lactate uptake in trained (22,776 +/- 370 dpm/min) compared with control hindlimbs (21,924 +/- 1,373 dpm/min). Concomitant with these observations, no significant differences were observed between groups for oxygen consumption (4.93 +/- 0.18 vs. 4.92 +/- 0.13 micromol/min), net skeletal muscle glycogen synthesis (7.1 +/- 0.4 vs. 6.5 +/- 0.3 micromol x 40 min(-1) x g(-1)), or 14CO2 production (2,203 +/- 185 vs. 2,098 +/- 155 dpm/min), trained and control, respectively. These findings indicate that endurance training does not affect lactate uptake or alter the metabolic fate of lactate in quiescent skeletal muscle.

In Vivo Insulin Regulation of Skeletal Muscle Glycogen Synthase in Calorie-Restricted and in Ad Libitum–Fed Rhesus Monkeys

Chronic calorie restriction in primates has been shown to have profound and unexpected effects on basal and on in vivo insulin action on skeletal muscle glycogen synthase (GS) activity. The decreased ability of insulin to activate skeletal muscle GS is a hallmark of insulin resistance and type 2 diabetes. The mechanism and role of in vivo insulin regulation of skeletal muscle GS are not fully understood. Two pathways for the activation of GS by insulin have been described by Larner and others: 1) insulin activates glucose transport that results in an increase in glucose-6-phosphate (G6P), thereby activating protein phosphatase-1, which in turn dephosphorylates and activates GS, therefore, pushing substrate into glycogen; and 2) insulin activates GS (perhaps by forming low-molecular-weight mediators which may activate protein phosphatase-1 and 2C) and activated GS subsequently pulls intermediates (e.g., G6P and uridine 5'-diphosphoglucose) into glycogen. To determine whether in vivo insulin regulates glycogen synthesis primarily via a push or pull mechanism and how this mechanism might be affected by long-term calorie restriction, skeletal muscle samples were obtained before and during a euglycemic hyperinsulinemic clamp from 41 rhesus monkeys. The monkeys varied widely in their degree of insulin sensitivity and age and included chronically calorie-restricted (CR) monkeys and ad libitum-fed monkeys. The ad libitum-fed monkeys included spontaneously type 2 diabetic, prediabetic and clinically normal animals. The apparent affinity of GS for the allosteric activator G6P (G6P Ka of GS) was measured and compared with G6P content in the muscle samples. Basal G6P Ka of GS was lower in the CR monkeys compared with the 3 ad libitum-fed groups (P: < or = 0.05). Only the normal ad libitum-fed monkeys had a decrease in the G6P Ka of GS with insulin (P: < 0.005). The insulin effect (insulin-stimulated minus basal) on the G6P Ka of GS was strongly positively related to the insulin effect on G6P content (r = 0.80, P: < 0.0001) across the entire group of monkeys. This finding supports the hypothesis that activation/dephosphorylation of GS by insulin is related to a decrease in G6P content and that paradoxical inactivation/phosphorylation of GS by insulin is related to an increase in G6P content (as demonstrated in 4 of 6 CR monkeys). Therefore, during a euglycemic hyperinsulinemic clamp, insulin regulates skeletal muscle glycogen synthesis primarily via a pull mechanism in both CR and in ad libitum-fed rhesus monkeys.

The lipid phosphatase SHIP2 controls insulin sensitivity.

Insulin is the primary hormone involved in glucose homeostasis, and impairment of insulin action and/or secretion has a critical role in the pathogenesis of diabetes mellitus. Type-II SH2-domain-containing inositol 5-phosphatase, or 'SHIP2', is a member of the inositol polyphosphate 5-phosphatase family. In vitro studies have shown that SHIP2, in response to stimulation by numerous growth factors and insulin, is closely linked to signalling events mediated by both phosphoinositide-3-OH kinase and Ras/mitogen-activated protein kinase. Here we report the generation of mice lacking the SHIP2 gene. Loss of SHIP2 leads to increased sensitivity to insulin, which is characterized by severe neonatal hypoglycaemia, deregulated expression of the genes involved in gluconeogenesis, and perinatal death. Adult mice that are heterozygous for the SHIP2 mutation have increased glucose tolerance and insulin sensitivity associated with an increased recruitment of the GLUT4 glucose transporter and increased glycogen synthesis in skeletal muscles. Our results show that SHIP2 is a potent negative regulator of insulin signalling and insulin sensitivity in vivo.

Glycogenin activity in human skeletal muscle is proportional to muscle glycogen concentration.

The de novo biosynthesis of glycogen is catalyzed by glycogenin, a self-glucosylating protein primer. To date, the role of glycogenin in regulating glycogen metabolism and the attainment of maximal glycogen levels in skeletal muscle are unknown. We measured glycogenin activity after enzymatic removal of glucose by alpha-amylase, an indirect measure of glycogenin amount. Seven male subjects performed an exercise and dietary protocol that resulted in one high-carbohydrate leg (HL) and one low-carbohydrate leg (LL) before testing. Resting muscle biopsies were obtained and analyzed for total glycogen, proglycogen (PG), macroglycogen (MG), and glycogenin activity. Results showed differences (P < 0.05) between HL and LL for total glycogen (438.0 +/- 69.5 vs. 305.7 +/- 57.4 mmol glucosyl units/kg dry wt) and PG (311.4 +/- 38.1 vs. 227.3 +/- 33.1 mmol glucosyl units/kg dry wt). A positive correlation between total muscle glycogen content and glycogenin activity (r = 0.84, P < 0.001) was observed. Similar positive correlations (P < 0.05) were also evident between both PG and MG concentration and glycogenin activity (PG, r = 0.82; MG, r = 0.84). It can be concluded that glycogenin does display activity in human skeletal muscle and is proportional to glycogen concentration. Thus it must be considered as a potential regulator of glycogen synthesis in human skeletal 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.

Regulation of glycogen synthesis in rat skeletal muscle after glycogen-depleting contractile activity: effects of adrenaline on glycogen synthesis and activation of glycogen synthase and glycogen phosphorylase

Regulation of glycogen synthesis in rat skeletal muscle after glycogen-depleting contractile activity: effects of adrenaline on glycogen synthesis and activation of glycogen synthase and glycogen phosphorylase

Regulation of glycogen synthesis in rat skeletal muscle after glycogen-depleting contractile activity: effects of adrenaline on glycogen synthesis and activation of glycogen synthase and glycogen phosphorylase

We investigated the effects of insulin and adrenaline on the rate of glycogen synthesis in skeletal muscles after electrical stimulation in vitro. The contractile activity decreased the glycogen concentration by 62%. After contractile activity, the glycogen stores were fully replenished at a constant and high rate for 3 h when 10 m-i.u./ml insulin was present. In the absence of insulin, only 65% of the initial glycogen stores was replenished. Adrenaline decreased insulin-stimulated glycogen synthesis. Surprisingly, adrenaline did not inhibit glycogen synthesis stimulated by glycogen-depleting contractile activity. In agreement with this, the fractional activity of glycogen synthase was high when adrenaline was present after exercise, whereas adrenaline decreased the fractional activity of glycogen synthase to a low level during stimulation with insulin. Furthermore, adrenaline activated glycogen phosphorylase almost completely during stimulation with insulin, whereas a much lower activation of glycogen phosphorylase was observed after contractile activity. Thus adrenaline does not inhibit contraction-stimulated glycogen synthesis.

Characterization of mouse glycogenin-1 cDNA and promoter region

Glycogenin-1 is an autocatalytic, self-glucosylating protein which acts as the primer for glycogen synthesis in mammalian skeletal muscle. In this study, we have cloned the glycogenin-1 cDNA from mouse skeletal muscle. Mouse glycogenin-1 has a predicted molecular mass of 37 399 Da, and the deduced amino acid sequence exhibited 87% homology with human glycogenin-1. Northern blot analysis specifically detected mouse glycogenin-1 transcript in skeletal muscle and heart, and to a lesser extent in kidney, lung and brain. 5′-RACE analysis revealed the major transcription start site to be localized 47 bp upstream of the initiation of translation codon. Sequence analysis of ∼2 kb of the 5′-flanking region revealed potentially important regions of homology between the mouse and human glycogenin-1 promoters. Several conserved but putative elements, including a TATA box, Sp1 site, and a cyclic AMP responsive element, were observed proximal to the transcription start site. Significantly, Northern blot analysis revealed dibutyryl-cAMP treatment of cultured mouse C2C12 myotubes markedly reduced the levels of glycogenin-1 mRNA in a dose-dependent manner.

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.

IAPP as a regulator of glucose homeostasis and pancreatic hormone secretion (review).

IAPP is a 37-amino acid peptide that is predominantly expressed in pancreatic beta cells. Despite co-secretion from islets the relative amounts of IAPP and insulin may vary. Since IAPP was first described as the major peptide constituent of amyloid in the islets of Langerhans of subjects with type 2 diabetes and insulinoma, many studies have been devoted to investigating the role of IAPP in formation of amyloid deposits and in diabetes pathogenesis. However, there is growing evidence for IAPP as an active islet hormone in addition to insulin and glucagon in glucose metabolic control. An inhibitory effect is seen by IAPP on gastric emptying, glycogen synthesis in skeletal muscle, islet insulin and glucagon secretion, whereas a stimulatory effect is seen on hepatic gluconeogenesis.

EFFECT OF EXERCISE ON GLYCOGENIN EXPRESSION IN HUMAN SKELETAL MUSCLE.

87 Glycogenin is a self-glycosylating protein which acts as a primer for glycogen synthesis in skeletal muscle. It has been shown recently that overexpression of glycogenin in L6 myoblasts enhances maximal glycogen storage (Hansen et al., Diabetologia 41(Suppl 1): A201, 1998 ). To investigate the effect of exercise on glycogenin expression in human skeletal muscle, eight untrained subjects (5F, 3M, 21.4 ± 1.2 yrs, 66.3 ± 5.0 kg, VO2peak = 2.30 ± 0.19 l.min−1, mean ± SEM) exercised for 60 min on a cycle ergometer at a power output requiring 60% VO2peak. Muscle samples were obtained by needle biopsy before and 3hr after exercise. Total RNA was extracted using the RNAzol technique and reverse transcribed to cDNA. Glycogenin cDNA (GeneBank U44131) was amplified by PCR and glycogenin gene expression was quantified, relative to 29S ribosomal protein cDNA, by gel electrophoresis and ethidium bromide staining. Following exercise, glycogenin mRNA had increased more than 2-fold (2.8 ± 0.6 vs. 1.2 ± 0.2 arb. units, P<0.05). The increase in skeletal muscle glycogenin gene expression may have an important role in facilitating post-exercise glycogen synthesis.

Mutational analysis of the coding regions of the genes encoding protein kinase B-alpha and -beta, phosphoinositide-dependent protein kinase-1, phosphatase targeting to glycogen, protein phosphatase inhibitor-1, and glycogenin: lessons from a search for genetic variability of the insulin-stimulated glycogen synthesis pathway of skeletal muscle in NIDDM patients.

The finding of a reduced insulin-stimulated glucose uptake and glycogen synthesis in the skeletal muscle of glucose-tolerant first-degree relatives of patients with NIDDM, as well as in cultured fibroblasts and skeletal muscle cells isolated from NIDDM patients, has been interpreted as evidence for a genetic involvement in the disease. The mode of inheritance of the common forms of NIDDM is as yet unclear, but the prevailing hypothesis supports a polygenic model. In the present study, we tested the hypothesis that the putative inheritable defects of insulin-stimulated muscle glycogen synthesis might be caused by genetic variability in the genes encoding proteins shown by biochemical evidence to be involved in insulin-stimulated glycogen synthesis in skeletal muscle. In 70 insulin-resistant Danish NIDDM patients, mutational analysis by reverse transcription-polymerase chain reaction-single strand conformation polymorphism-heteroduplex analysis was performed on genomic DNA or skeletal muscle-derived cDNAs encoding glycogenin, protein phosphatase inhibitor-1, phophatase targeting to glycogen, protein kinase B-alpha and -beta, and the phosphoinositide-dependent protein kinase-1. Although a number of silent variants were identified in some of the examined genes, we found no evidence for the hypothesis that the defective insulin-stimulated glycogen synthesis in skeletal muscle in NIDDM is caused by structural changes in the genes encoding the known components of the insulin-sensitive glycogen synthesis pathway of skeletal muscle.