Impaired Glycogen Synthesis Research Articles

Rapid normalization of hepatic glycogen metabolism in rats with long-term bile duct ligation after biliodigestive anastomosis

Background/Aims: Rats with chronic bile duct ligation have reduced hepatic glycogen stores and decreased activities of enzyme involved in glycogen metabolism. In the current studies, the reversibility of these changes following reversal of biliary obstruction by Roux-en-Y anastomosis (RY) was investigated.Methods: Rats were studied after bile duct ligation for 4 weeks (BDL rats), or 5 or 14 days after relief of biliary obstruction by RY. Control rats were pairfed to treated rats, and all rats were studied in the fed state.Results: The liver glycogen content was decreased in BDL rats (198±167 vs. 753±315 mg/liver in BDL vs. control rats) and normalized within 5 days after RY. The total activities of glycogen synthase and phosphorylase were both reduced by 51% in BDL as compared to control rats. Five days after RY, the activity of glycogen synthase had increased significantly in comparison to BDL rats, whereas glycogen phosphorylase had remained unchanged. Fourteen days after RY, both enzyme activities had completely normalized. Northern blots revealed reduced hepatic mRNA levels in BDL rats, for glycogen synthase and phosphorylase. While the mRNA level for glycogen synthase normalized within 5 days after RY, the level for glycogen phosphorylase increased but did not normalize completely within 14 days after RY.Conclusions: Hepatic glycogen stores are decreased in BDL rats but recover rapidly after relief of biliary obstruction. Reduced activity and mRNA levels of glycogen synthase suggest that impaired glycogen synthesis is the principal mechanism for decreased hepatic glycogen stores in BDL rats.

Five-hour fatty acid elevation increases muscle lipids and impairs glycogen synthesis in the rat

Insulin-mediated muscle glycogen synthesis is impaired after several weeks of high-fat feeding in rats, but not by short-term (2-hour) nonesterified fatty acids (NEFA) elevation induced by intravenous triglyceride/heparin infusion (TG/H). We examined whether a longer TG/H infusion induces defective glycogen synthesis. Five-hour hyperinsulinemic (700 pmol/L) euglycemic clamps with either TG/H or saline infusion were performed. TG/H-infused rats developed insulin resistance, but only after 2 to 3 hours. Red gastrocnemius glycogen synthesis rate decreased by 50% ( P < .01 v saline) associated with decreased glycogen synthase activity (GSa; assessed at several glucose-6-phosphate [G-6-P] levels; two-way ANOVA, P = .02) and increased muscle TG and total long-chain acyl coenzyme A (LCAC) content (twofold; P < .05 v saline). Thus a 3- to 5-hour NEFA elevation in the rat produced significant impairment of insulin-stimulated muscle glycogen synthesis, associated with muscle lipid accumulation. These effects were similar to those observed after several weeks of fat feeding. The 5-hour TG/H-infused rat is a useful model for studying lipid-induced muscle insulin resistance.

New variants in the glycogen synthase gene (Gln71His, Met416Val) in patients with NIDDM from eastern Finland.

Impaired glycogen synthesis after insulin stimulation accounts for most of the insulin resistance in patients with non-insulin-dependent diabetes mellitus (NIDDM). The glycogen synthase gene (GYS1), which encodes the rate-limiting enzyme for glycogen synthesis, is a promising candidate gene for NIDDM. Therefore, we screened all 16 exons of this gene by single-strand conformation polymorphism analysis in 40 patients with NIDDM (age 67 +/- 2 years, body mass index 28.2 +/- 0.6 kg/m2) from Taipalsaari, eastern Finland. The Gly464Ser variant (exon 11) and a silent polymorphism TTC342TTT (exon 7) have been reported previously. In addition, we found a new variant Gln71His (exon 2) and a new amino acid polymorphism Met416Val (exon 10). An additional sample of 65 patients with NIDDM and 82 normoglycaemic men (age 54 +/- 1 years, body mass index 26.3 +/- 1.4 kg/m2) were screened. The allele frequency of the TTC342TTT silent substitution was 0.29 in both NIDDM and normoglycaemic subjects. The Gln71His and Gly464Ser variants were found in 1 (1%) and 3 (3%) subjects, respectively, of the 105 NIDDM patients and in none of the 82 normoglycaemic men. The Met416Val polymorphism was found in 16 (15%) of the 105 NIDDM patients and in 14 (17%) of the 82 control subjects (all heterozygous). The Met416Val polymorphism was not associated with insulin resistance in two groups of normoglycaemic subjects. In conclusion, the new Gln71His and Met416Val substitutions and other variants of the glycogen synthase gene are unlikely to make a major contribution to insulin resistance and NIDDM in diabetic patients from eastern Finland.

Yeast PIG genes: PIG1 encodes a putative type 1 phosphatase subunit that interacts with the yeast glycogen synthase Gsy2p.

The biosynthesis of glycogen involves multiple proteins that associate with each other and the glycogen macromolecule. In efforts to understand the nature of these proteins, a two-hybrid screen was undertaken to detect proteins able to interact with Gsy2p, a major form of glycogen synthase in Saccharomyces cerevisiae. Two positives expressed proteins derived from genes designated PIG1 and PIG2, on chromosomes XIIR and IXL respectively. PIG1 codes for a protein with 38% identity over a 230 residue segment to Gac1p, a protein thought to be a type 1 protein phosphatase targeting subunit whose loss impairs glycogen synthesis. Pig2p has 30% identify to the protein corresponding to an open reading frame, YER054, on chromosome V. Deletion of PIG1 on its own had little effect on glycogen storage but, in combination with loss of GAC1, caused a more severe glycogen-deficient phenotype than seen in gac1 mutants. This result is consistent with Pig1p being functionally related to Gac1p and we propose that Pig1p may be a type 1 phosphatase regulatory subunit. Delection of PIG2, YER054, or both genes together caused no detectable change in glycogen metabolism under the conditions tested. Gac1p, Pig1p, Pig2p and the YER054p are the only four proteins coded by the yeast genome that share a conserved segment of approximately 25 residues, designated the GVNK motif, that is identifiable also in RGI, the mammalian type 1 phosphatase targeting subunit.

Increased hexosamine availability similarly impairs the action of insulin and IGF-1 on glucose disposal.

Prolonged glucosamine (GlcN) infusion increases the skeletal muscle hexosamine concentration and induces peripheral insulin resistance in conscious rats. IGF-1 and insulin share common steps in signal transduction, and the action of IGF-1 on carbohydrate metabolism is preserved in certain insulin-resistant states. In our study, we attempted to delineate whether increased GlcN availability also impairs the effects of IGF-1 on glucose uptake (Rd), glycolysis, and glycogen synthesis. We performed euglycemic IGF-1 (5 and 15 microg x kg(-1) x min(-1)) and insulin (3 and 18 mU mg x kg(-1) x min(-1)) clamp studies at 0-2 h and 5-7 h in conscious rats (n = 44) during saline or GlcN infusions. GlcN infusion raised plasma GlcN levels to approximately 2.0 mmol/l and skeletal muscle uridinediphospho-n-acetylglucosamine to 80-150 nmol/g (approximately three- to fivefold over basal). During physiological hyperinsulinemia (3 mU x kg(-1) x min(-1), plasma insulin approximately 50 microU/ml), GlcN infusion caused comparable decreases in Rd (15.7 +/- 1.0 [5-7 h] vs. 21.7 +/- 2.3 [0-2 h] mg x kg(-1) x min(-1); P < 0.01) and glycogen synthesis (5.4 +/- 0.5 [5-7 h] vs. 10.4 +/- 1.9 [0-2 h] mg x kg(-1) x min(-1); P < 0.005). Furthermore, GlcN markedly decreased Rd by 7.8 +/- 1.2 mg x kg(-1) x min(-1) (18.7 +/- 0.7 [5-7 h] vs. 26.5 +/- 1.3 [0-2 h] mg x kg(-1) x min(-1); P < 0.001 vs. control) during IGF-1 (5 microg x kg(-1) x min(-1)) clamp studies. This decline was associated with a 26% decrease in the steady-state concentration of skeletal muscle Glc-6-P (286 +/- 45 vs. 386 +/- 36 nmol/g; P < 0.01) and was primarily caused by impaired glycogen synthesis (6.7 +/- 0.5 [5-7 h] vs. 13.9 +/- 0.9 [0-2 h] mg x kg(-1) x min(-1); P < 0.005). The effects of GlcN infusion on glucose disposal (percentage decrease in Rd) were correlated (r2 = 0.803; P < 0.01) with the skeletal muscle concentration of UDP-GlcNAc. To investigate whether IGF-1 can overcome GlcN-induced insulin resistance, GlcN and insulin (18 mU x kg(-1) x min(-1)) were infused for 7 h during euglycemic clamps, and IGF-1 (15 microg x kg(-1) x min(-1)) was superimposed during the final 2 h. GlcN infusion induced severe impairment of insulin action on Rd (39.4 +/- 3.2 [4-5 h] vs. 49.8 +/- 3.6 [1-2 h] mg x kg(-1) x min(-1); P < 0.05), which the addition of IGF-1 failed to improve (35.9 +/- 2.3 [6-7 h] vs. 39.4 +/- 3.2 [4-5 h] mg x kg(-1) x min(-1); P > 0.1). In summary, GlcN induced severe resistance to the actions of both insulin and IGF-1 on glucose uptake and glycogen synthesis, and IGF-1 was unable to overcome GlcN-induced insulin resistance. Thus, it is likely that GlcN causes peripheral insulin resistance acting at a site common to both IGF-1 and insulin signaling pathways.

Chronic activation of protein kinase C in soleus muscles and other tissues of insulin-resistant type II diabetic Goto-Kakizaki (GK), obese/aged, and obese/Zucker rats. A mechanism for inhibiting glycogen synthesis.

We examined the possibility that protein kinase C (PKC) is chronically activated and may contribute to impaired glycogen synthesis and insulin resistance in soleus muscles of hyperinsulinemic type II diabetic Goto-Kakizaki (GK) rats. Relative to nondiabetic controls, PKC enzyme activity and levels of immunoreactive PKC-alpha, beta, epsilon, and delta were increased in membrane fractions and decreased cytosolic fractions of GK soleus muscles. In addition, PKC-theta levels were decreased in both membrane and cytosol fractios, whereas PKC-zeta levels were not changed in either fraction in GK soleus muscles. These increases in membrane PKC (alpha, beta, epsilon, and delta) could not be accounted for by alterations in PKC mRNA or total PKC levels but were associated with increases in membrane diacylglycerol (DAG) and therefore appeared to reflect translocative activation of PKC. In evaluation of potential causes for persistent PKC activation, membrane PKC levels were decreased in soleus muscles of hyperglycemic streptozotocin (STZ)-induced diabetic rats; thus, a role for simple hyperglycemia as a cause of PKC activation in GK rats was not evident in the STZ model. In support of the possibility that hyperinsulinemia contributed to PKC activation in GK soleus muscles, we found that DAG levels were increased, and PKC was translocated, in soleus muscles of both (1) normoglycemic hyperinsulinemic obese/aged rats and (2) mildly hyperglycemic hyperinsulinemic obese/Zucker rats. In keeping with the possibility that PKC activation may contribute to impaired glycogen synthase activation in GK muscles, phorbol esters inhibited, and a PKC inhibitor, RO 31-8220, increased insulin effects on glycogen synthesis in soleus muscles incubated in vitro. Our findings suggested that: (1) hyperinsulinemia, as observed in type II diabetic GK rats and certain genetic and nongenetic forms of obesity in rats, is associated with persistent translocation and activation of PKC in soleus muscles, and (2) this persistent PKC activation may contribute to impaired glycogen synthesis and insulin resistance.

Pho85p, a cyclin-dependent protein kinase, and the Snf1p protein kinase act antagonistically to control glycogen accumulation in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, nutrient levels control multiple cellular processes. Cells lacking the SNF1 gene cannot express glucose-repressible genes and do not accumulate the storage polysaccharide glycogen. The impaired glycogen synthesis is due to maintenance of glycogen synthase in a hyperphosphorylated, inactive state. In a screen for second site suppressors of the glycogen storage defect of snf1 cells, we identified a mutant gene that restored glycogen accumulation and which was allelic with PHO85, which encodes a member of the cyclin-dependent kinase family. In cells with disrupted PHO85 genes, we observed hyperaccumulation of glycogen, activation of glycogen synthase, and impaired glycogen synthase kinase activity. In snf1 cells, glycogen synthase kinase activity was elevated. Partial purification of glycogen synthase kinase activity from yeast extracts resulted in the separation of two fractions by phenyl-Sepharose chromatography, both of which phosphorylated and inactivated glycogen synthase. The activity of one of these, GPK2, was inhibited by olomoucine, which potently inhibits cyclin-dependent protein kinases, and contained an approximately 36-kDa species that reacted with antibodies to Pho85p. Analysis of Ser-to-Ala mutations at the three potential Gsy2p phosphorylation sites in pho85 cells implicated Ser-654 and/or Thr-667 in PHO85 control of glycogen synthase. We propose that Pho85p is a physiological glycogen synthase kinase, possibly acting downstream of Snf1p.

Genetic Interactions Between REG1/HEX2 and GLC7, the Gene Encoding the Protein Phosphatase Type 1 Catalytic Subunit in Saccharomyces cerevisiae

Mutations in GLC7, the gene encoding the type 1 protein phosphatase catalytic subunit, cause a variety of abberrant phenotypes in yeast, such as impaired glycogen synthesis and relief of glucose repression of the expression of some genes. Loss of function of the REG1/HEX2 gene, necessary for glucose repression of several genes, was found to suppress the glycogen-deficient phenotype of the glc7-1 allele. Deletion of REG1 in a wild-type background led to overaccumulation of glycogen as well as slow growth and an enlarged cell size. However, loss of REG1 did not suppress other phenotypes associated with GLC7 mutations, such as inability to sporulate or, in cells bearing the glc7Y-170 allele, lack of growth at 14 degrees. The effect of REG1 deletion on glycogen accumulation is not simply due to derepression of glucose-repressed genes, although it does require the presence of SNF1, which encodes a protein kinase essential for expression of glucose-repressed genes and for glycogen accumulation. We propose that REG1 has a role in controlling glycogen accumulation.

The effect of (steroid) immunosuppression on skeletal muscle glycogen metabolism in patients after kidney transplantation.

To examine the mechanisms by which immunosuppression by steroids impairs glycogen synthesis in human skeletal muscle, we measured glycogen synthase protein content and activity in muscle samples from 14 patients receiving corticosteroid therapy after kidney transplantation and in 20 healthy control subjects. A percutaneous muscle sample was taken before and at the end of a euglycemic hyperinsulinemic insulin clamp. Insulin-stimulated glucose disposal was reduced by 33% in kidney transplant patients compared with healthy controls (33.8 +/- 4.2 vs. 50.5 +/- 2.7 mumol (kg LBM)-1 min-1; P<0.01), primarily due to a decrease in nonoxidative glucose metabolism (14.2 +/- 3.3 vs. 32.3 +/- 2.7 mumol (kg LBM)-1 min-1; P<0.001). Glycogen synthase activity measured at both 0.1 mmol/L (17.6 +/- 2.6 vs. 24.0 +/- 2.2 nmol min-1 mg protein-1; P<0.05), and at 10 mmol/L glucose 6-phosphate (24.1 +/- 3.5 vs. 33.7 +- 2.4 nmol min-1 mg protein-1; P<0.05) and glycogen synthase protein concentrations (8.8 +/- 1.8 vs. 18.9 +/- 1.9 relative units per ng DNA; P<0.01) were lower in kidney transplant patients compared with controls. Glycogen synthase protein correlated with nonoxidative glucose metabolism (r=0.42; P=0.04). Alpha-actinin (used as a control of general protein degradation) was lower in kidney transplant patients compared with controls (4.4 +/- 0.8 vs. 9.6 +/- 1.1 cpm/ng DNA; P<0.01). In conclusion, corticosteroids cause insulin resistance, which correlates with impaired activation of glycogen synthase and decreased enzyme protein content. The decrease in glycogen synthase protein may reflect increased degradation rather than a defect in translation.

Defects in liver and muscle glycogen metabolism in neonatal and adult New Zealand Obese mice

Impaired glycogen synthesis is present in subjects at risk for developing non-insulin-dependent diabetes mellitus (NIDDM), suggesting that it is a primary defect in NIDDM. To examine whether defects in glycogen metabolism are present at birth in an animal model of NIDDM, glycogen synthase (GS), glycogen phosphorylase (GP), and total glycogen content were measured in liver and quadriceps muscle of 1-day- and 20-week-old insulin-resistant New Zealand Obese (NZO) mice and control (NZC) mice. In livers of both neonatal and adult NZO mice, active GS was reduced by 54% and 36%, respectively, as compared with that in NZC mice ( P < .03). Total liver GS activity was the same in neonates, but was 65% higher in adult NZO as compares with NZC mice ( P < .02). Liver glycogen was 28% lower at birth in NZO mice ( P < .03), but was 49% higher at 20 weeks of age. Active and total GP were the same in NZO and NZC animals, despite hyperinsulinemia in 20-week-old NZO mice. In muscle, active GS was reduced by 41 % in both 1-day- and 20-week-old NZO mice ( P < .02). Total GS was also lower in NZC mice at 1 day of age ( P < .01), but not at 20 weeks. No differences were detected in GP activity or in total glycogen content in muscle. Therefore, reduced GS activity is an early defect present at birth in the insulin-resistant NZO mouse in both liver and muscle. However, it is not the sole determinant of the amount of glycogen deposited in tissues: Elevated liver glycogen in adult NZO mice may contribute to hepatic glucose overproduction.

7 Insulin action and substrate competition

An increased supply of FFAs for oxidation leads to a reduced rate of glucose oxidation and interferes with the inhibitory action of insulin on hepatic glucose production. Available evidence indicates that in humans skeletal muscle is a site for such substrate competition, which involves both pyruvate oxidation and glycogen synthesis. The insulin resistance of obesity is thought to be mostly of metabolic origin, and fully reversible. A reduction in FFA supply by weight reduction can, however, reverse this defect. The insulin resistance associated with NIDDM is thought to be primary, with a strong genetic basis, and partially irreversible. Patients with NIDDM are unable to increase their glucose oxidation normally in response to insulin to meet the energy demands of the body. Increased oxidation of lipids represents a compensatory phenomenon to meet these demands. Therapeutic use of the glucose-FFA cycle to lower blood glucose levels has yielded conflicting results. Studies are in progress to develop agents that inhibit gluconeogenesis by interfering with FFA oxidation. Nicotinic acid derivatives seem to enhance glycogen synthesis acutely by activating glycogen synthetase. Whether these or similar agents can be used to restore impaired glycogen synthesis, the most characteristic genetic defect in NIDDM, cannot be answered until the effect has been proven in chronic studies. The existence of substrate competition between amino acids and glucose, and an intrinsic hypoaminoacidaemic property of amino acids, makes it possible to expand the Randel cycle into a glucose-FFA-amino acid cycle, which integrates control of substrate disposition at the whole body level.

Impaired Expression of Glycogen Synthase mRNA in Skeletal Muscle of NIDDM Patients

Based on recent studies of the abnormal physiology and biochemistry of the glycogen synthesis in skeletal muscle of non-insulin-dependent diabetes mellitus (NIDDM) patients and their first-degree relatives, the key enzyme of this pathway, glycogen synthase (GS), is considered a candidate gene in the pathogenesis of insulin resistance. Comparing matched groups of 14 NIDDM patients with 14 control subjects, we found that impaired insulin-stimulated nonoxidative glucose metabolism of peripheral tissue (P less than 0.02) and reduced total GS activity (P less than 0.05) of vastus lateralis muscle from patients with NIDDM were accompanied by a 39% reduction (P less than 0.02) in the steady state level of GS mRNA per microgram DNA of muscle. In both diabetic and control subjects, the mRNA expression of GS was unaffected after euglycemic-hyperinsulinemic clamp for 4 h. With single-stranded conformation polymorphism analysis of the entire coding sequence of the GS gene, we were unable to detect any genetic variants in a subset of eight NIDDM patients. We conclude that abnormal pretranslational regulation of the GS gene may contribute to impaired glycogen synthesis of muscle in NIDDM. Our studies give no evidence for structural changes in the coding region of the GS gene, and it is unknown if the decreased mRNA expression is due to impaired transcription or accelerated degradation of the transcript.

Fetal diabetes in rats and its effect on placental glycogen.

The role of fetal insulin in placental glycogen accumulation, which occurs despite insulin deficiency in maternal diabetes, was studied in rats. Streptozotocin was injected into fetuses of non-diabetic and streptozotocin-diabetic mothers on days 19.5 and 20.5 of gestation, causing fetal hypoinsulinaemia and pancreatic insulin depletion. Placental glycogen content of either 1.6 mg/g in non-diabetic rats or 6.5 mg/g in diabetic rats was not affected by fetal streptozotocin treatment. Glycogen distribution was also measured in the placenta to assess the effect of fetal hypoinsulinaemia on glycogen content in its fetal segment. The glycogen concentration ratio between the fetal and maternal segments in diabetic rats was approximately 0.3 and increased to approximately 0.5 in diabetic rats, without being affected by fetal hypoinsulinaemia. There was no significant effect of fetal hypoinsulinaemia on the activities of placental glycogen synthase or glycogen phosphorylase, both in non-diabetic and diabetic rats. Fetal hypoinsulinaemia was associated, however, with a marked decrease in fetal liver glycogen together with a decrease in fetal liver weight, which was more pronounced than the decrease in fetal body weight. Administration of insulin to the streptozotocin-treated fetuses restored the impaired glycogen synthesis (measured by incorporation of U-[14C]-glucose and 3H2O in the fetal liver) without affecting glycogen synthesis in the placenta.(ABSTRACT TRUNCATED AT 250 WORDS)

Control of Gluconeogenesis in Liver

The isolated perfused rat liver preparation was employed to study the effects of glucagon, fasting, and diabetes on the flow of exogenous lactate or endogenous substrates into the pathways of gluconeogenesis, glycogenesis, ketogenesis, lipogenesis, and the Krebs cycle in the liver. Measurements were made of the utilization of substrates and oxygen, the formation of glucose, glycogen, ketone bodies, and urea, and the incorporation of isotope from [14C]lactate into glucose, glycogen, protein, CO2, ketone bodies, cholesterol, and other lipids. Glucose synthesis from lactate was enhanced in livers treated with glucagon or from fasted or diabetic rats. The increased gluconeogenesis was associated with increased lactate utilization and was not the result of inhibition of alternative pathways of lactate metabolism. The disposition of isotope between glucose and glycogen indicated impaired glycogen synthesis in these livers. The changes in glucose and glycogen synthesis in livers from diabetic rats were reversed by insulin treatment in vivo. CO2 was the major product of lactate metabolism in livers from fed rats, whereas glucose was the major product in livers treated with glucagon or from fasted or diabetic rats. Ketone bodies, fatty acids, and cholesterol were minor products of lactate metabolism in all situations examined. Glucagon, fasting, and diabetes had negligible effects on the oxidation of lactate to CO2, but inhibited the synthesis of fatty acid and cholesterol from this substrate. Fasting and diabetes also reduced lactate ketogenesis. Urea production was increased about 2-fold by glucagon and diabetes and by about 30% by fasting. It was minimally changed by lactate. The alterations in ureogenesis indicated enhancement of gluconeogenesis from endogenous protein by glucagon, fasting, and diabetes. Ketone body production was increased about 10-fold in livers from fasted or diabetic rats perfused without lactate. The increase was apparently due to increased lipid utilization and decreased Krebs cycle activity. Glucagon produced a 2-fold increase in ketone bodies and it is suggested that these were mainly derived from protein. Lactate markedly suppressed ketogenesis and increased respiration in livers from fasted or diabetic rats. Both effects were attributable to an increase in the Krebs cycle. Lactate also replaced lipid as the major fuel oxidized by livers from fed rats and it is suggested that it acted by diverting endogenous fatty acid from oxidation to esterification. The metabolic changes observed in the present study are discussed from the viewpoints of underlying mechanisms and physiological implications. It is concluded that changes in the disposition of substrate (pyruvate or fatty acid) within the liver may be as important as changes in substrate supply in the alterations in gluconeogenesis and ketogenesis during fasting and diabetes.