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PubMed1982

Glycogen-containing lysosomes and glycogen loss in the cardiocytes of embryonic and neonatal mice.

In the mouse embryo the transformation of mesenchymal cells into cardiac myocytes was seen to be indicated by the appearance of glycogen and myosin filaments. In the early force-producing muscle cells, but particularly in the specialized cells destined to become part of the A-V conduction system, the glycogen became abundant. With maturation of the embryo and differentiation of the cardiocytes, the glycogen content decreased in both the force-producing and "conducting" muscle fibers. In late fetal life, and spectacularly after parturition, the loss of glycogen content in the specialized cells was accompanied by the appearance of glycogen-containing lysosomes. Changes in the structure of such lysosomes indicated ongoing dissolution and uncommon use of metabolizable glycogen.

Animals↗

PubMed1975

Glycogen, its chemistry and morphological appearance in the electron microscope.11. The complex formed in the selective contrast staining of glycogen.

Selective contrast staining of glycogen in untreated ultrathin sections of aldehyde-fixed tissues, double-fixed with 1% osmium tetroxide containing 0.05 M K3Fe(CN)6 as reported previously (De Bruijn, 1973), may also be obtained by the addition of either K4Fe(CN)6,K3Co(CN)6,K2Ru(CN)6, or K4Os(CN)6. On the other hand, addition of K3Cr(CN)6, K2Ni(CN)4, K3Mn(CN)6, K3Rh(CN)6, K2Pd(CN)4, K2Pt(CN)4, or K3Ir(CN)6 produces no effect. Hexavalent osmium oxide compounds, such as K2OsO4 and OsO3-2 pyridine, react selectively with a native (or acquired) ligand in the aldehyde-fixed glycogen, but do not render it more electron dense than its immediate surroundings. The presence of these osmium oxides is detected and they are rendered more electron dense by an accumulation reaction by the application on ultrathin sections of phosphotungstic acid (PTA) or a mixture of K2OsO4 and K4Fe(CN)6. As selective contrast staining of glycogen is also obtained by double fixation of the aldehyde-fixed tissue with 0.05 M K2OsO4 solutions containing 0.05 M K4Fe(CN)6 or 0.05 M K4Os(CN)6, it is postulated that in such tissue, both the selective reaction of K2OsO4 with the ligand in the aldehyde-fixed glycogen, and the accumulation of heavy metal at the sites occupied by the K2OsO4, occur simultaneously. A proposal for the constitution of this heavy metal osmium/cyanide complex is formulated and arguments are presented that both compounds are formed in the selective contrast stained glycogen areas of such treated tissues. The relative contribution of the components to the final contrast and its complex character is demonstrated by staining ultrathin glutaraldehyde sections intermittently with 0.05 M solutions of K2OsO4 and K4Fe(CN)6; it is shown that after at least three intermittent reactions with both K2OsO4 and K4Fe(CN)6, the glycogen areas in such sections became contrast stained.

Animals↗

PubMedDate not supplied

Purkinje fibre glycogen. A morphologic and biochemical study of glycogen particles isolated from the cow conducting system.

Glycogen from the cow conducting system was extracted by crude and mild methods. For comparison similar extractions were also performed on cow ordinary ventricular tissue. Glycogen particles from the conducting system, isolated by a mild method, were characterized by a low molecular weight (3-5 X 10(6)) and small dimensions (average diameter about 30 nm). 3.5-7% protein was firmly bound to the glycogen. The glycogen, based on spectrophotometric analysis, appeared to be in a native state. Glycogen as a polysaccharide-protein complex can thus be obtained from the cow conducting system and is proposed to be useful for analysis of the structure and function of glycogen in the conducting system.

Animals↗

PubMed1977

Glycogen estimation by a rapid enzymic method in very small samples of human endometrium: glycogen content in the endometrium of infertile patients during the menstrual cycle.

An enzymic method using alpha-glucosidases was adapted for measuring glycogen in very small samples (3 mg) of human endometrium. The method is useful as a clinical test of the physiologic function of human endometrium. The glycogen content of the endometrium of normal and infertile patients was measured during the menstrual cycle. The maximal content in both groups was observed between the 16th and 23rd days of the cycle, but the glycogen content of the infertile group was significantly lower (P less than 0.005). These results confirm the reports of others. Endometrial glycogen and urinary pregnanediol levels in 32 infertile patients were measured on day 7 after ovulation. The glycogen content of the endometrium of 21 of these patients, who showed normal excretion of urinary pregnanediol (greater than or equal to 2 mg/day), was significantly higher than that of the other 11 patients who showed low excretion of urinary pregnanediol (less than 2 mg/day) (P less than 0.005). This finding suggests that there is a high correlation between the function of the corpus luteum and endometrial glycogen deposition.

Clinical Enzyme Tests↗

PubMed2002

Glycogen levels and glycogen catabolism in livers from arthritic rats.

Hepatic glycogen catabolism and glycogen levels in rats with chronic arthritis were investigated. At 9:00 a.m., the hepatic glycogen contents of ad libitum fed arthritic and normal rats were 225.5+/-17.7 and 332.1+/-28.6 micromol glucosyl units x (g liver)(-1), respectively. Food intake of arthritic and normal rats was equal to 100.1+/-6.7 and 105.0+/-3.1 mg x (g body w)(-1) x (per 24 h)(-1), respectively. In isolated perfused livers from normal and arthritic rats the rates of glucose, lactate and pyruvate release were the same when substrate- and hormone-free perfusion was performed. During an infusion period of 20 min glucagon caused an increment in glucose release of 35.3+/-4.7 micromol x (g liver)(-1) in livers from arthritic rats; in the normal condition the corresponding increment was 69.6+/-5.7 micromol x (g liver)(-1). Lactate and pyruvate productions (indicators of glycolysis) were diminished by glucagon in livers from normal rats; in the arthritic condition an initial stimulation was found, followed by a slow decay, which did not result in significant inhibition at the end of the glucagon infusion period (20 min). The actions of cAMP and dibutyryl-cAMP were similar to those of glucagon. It was concluded that livers from arthritic rats show an impaired capacity of releasing glucose under the stimulus of glucagon. This can be partly due to the lower glycogen levels and partly to a smaller capacity of inhibiting glycolysis. Reduction in glycogen levels was not associated with reduction in food intake or failure in the energetic state of the hepatic cells. These changes in glycogen metabolism may be related to reduced gluconeogenic capacity of the livers and/or to production of inflammatory mediators observed in the arthritis disease.

Adenosine Diphosphate↗

PubMed1972

Effects of denervation on the glycogen content and on the activities of enzymes of glucose and glycogen metabolism in rat diaphragm muscle.

1. Changes in the content and concentration of glycogen and in the activity of a number of enzymes involved in glucose and glycogen metabolism were studied in the rat hemidiaphragm after unilateral denervation. 2. After nerve section the tissue hypertrophies; this hypertrophy is said to be confined to the smaller red fibres and not to the white. 3. The total hexokinase activity increases, whereas that of total glycogen phosphorylase decreases. The specific activity of phosphorylase a, determined after Halothane anaesthesia, remains fairly constant. 4. In fed animals the denervated tissue stores less glycogen, but in the early stages its glycogen content does not fall on starvation. 5. The effect of denervation on the specific activities of several other characteristically white-fibre enzymes are not consistent with the response of glycogen phosphorylase; the increase in content of glyceraldehyde 3-phosphate dehydrogenase and lactate dehydrogenase is thought to be related to proliferation of the sarcoplasmic reticulum. 6. The ratio of lactate dehydrogenase M/H subunits increases at the height of the hypertrophy, but then declines as the mass of the tissue falls. 7. The chronology of these changes in enzyme activities suggests a multiplicity of distinct responses after nerve section not consistent with any one model, either specific fibre development or reversion to de-differentiated, foetal-type metabolism.

Animals↗

PubMed1970

Two forms of yeast glycogen synthetase and their role in glycogen accumulation.

The glycogen content of yeast rises dramatically just before the onset of the stationary phase of growth. Concomitantly, a rapid increase was found in the glucose 6-phosphate-independent (I) activity of glycogen synthetase, as well as in the total amount of enzyme. A mutant (GS 1-36) was obtained, which did not accumulate glycogen during growth. The synthetase from this strain was in the glucose 6-phosphate-dependent (D) form at all times. The total enzymatic activity of the mutant also increased sharply at the end of the logarithmic phase, although its maximal value was only one third that of the parent strain. Incubation with glucose of wild type resting cells from the logarithmic phase resulted in a slow accumulation of glycogen, which was accelerated after 20 min. At the same time a transformation from the D to I form of the enzyme was detected. The same slow initial rate of glycogen deposition was found with stationary cells of mutant GS 1-36, but the rate gradually declined to zero, rather than accelerating. The interconversion of the I and D forms was obtained with extracts from cells harvested during different phases of growth. Examination of the properties of the I and D forms showed that the latter was much more strongly inhibited by ATP at low glucose 6-phosphate concentration. These findings clearly establish the fundamental role of the I form in glycogen accumulation. When taken together with previous results, they also show that the physiological significance of the I-D interconversion depends on the concentration of glucose 6-phosphate. Under certain conditions glucsoe 6-phosphate appears to regulate directly the activity of the predominant form of the enzyme.

Adenosine Triphosphate↗

PubMed1968

Glycogen in human peripheral blood leukocytes. II. The macromolecular state of leukocyte glycogen.

Glycogen of normal human blood leukocytes was studied in cell suspensions containing chiefly neutrophiles. In electron micrographs of neutrophiles stained with lead the glycogen particles appear to be relatively uniform with a diameter of 20 mmu. At high magnification the 20 mmu particle appears to be composed of at least eight subunits. Leukocyte glycogen released by lysis or homogenization sediments as a single peak of high molecular weight material. The great majority of the cell glycogen can be accounted for in the large molecular weight material. The large molecular weight material is degraded to small fragments by alpha-amylase and partially degraded by beta-amylase. Purification of cell glycogen by alkali extraction and ethanol precipitation produces a relatively uniform particle smaller than the original native macromolecule. Native glycogen was prepared in pure form by a sucrose density gradient technique and its purity demonstrated by its susceptibility to purified alpha-amylase and by analytical ultracentrifugation.

Adult↗

PubMed1999

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.

3-Phosphoinositide-Dependent Protein Kinases↗

PubMed1975

Evidence for the allosteric regulation of glycogen synthesis in the intact Escherichia coli cell. Agreement of the values of the parameters of the Hill equation fitted to data for glycogen synthesis in vivo with the abailable values obtained in vitro with adenosine diphosphoglucose synthetase.

In various nutrient-limited cultures of either Escherichia coli W4597(K) or G34 a 10-fold range of rates of glycogen synthesis is observed while the energy charge values (0.86 plus or minus 0.01) and glucose 6-phosphate levels are essentially the same in each condition. The steady state level of fructose 1,6-diphosphate in these cultures varies from experiment to experiment as a function of the observed rate of glycogen synthesis. These data were fitted to the Hill equation by a nonlinear regression analysis and the statistically most probable values obtained for the Hill coefficient (n), A0.5, and V were, respectively, 2.08, 0.82mM, and 1030 mumol/g of protein per hour. The values of the first two parameters agree well with values available at energy charge 0.85 for the in vitro synthesis of ADPG by the ADPG synthetase of E. coli. When the difference in the glucose 1-phosphate concentration used in the studies in vitro from the apparent glucose 1-phosphate concentration in vivo (estimated from the glucose 6-phosphate levels) is considered, the in vitro value of V (1140 mumol of ADPG synthesized per g of protein per hour) is quite similar to the value of V (1030 mumol of glucose incorporated into glycogen per g of protein per hour) for glycogen synthesis in vivo. The close agreement of the values of the parameters of the Hill equation for glycogen synthesis in vivo to the values obtained for ADPG synthesis in vitro provides the most quantitative evidence yet obtained that allosteric regulation of bacterial glycogen synthesis functions in vivo.

Adenosine Diphosphate↗

PubMed1975

Glycogen metabolism in white and red muscle or normal and diabetic rats. Degradation of glycogen by adrenaline.

The author studied the effect of adrenaline (500 mug/kg s.c.) on the glycogen content of white (extensor digitorum longus -- EDL) and red (soleus -- SOL) muscle of normal and alloxan-diabetic rats. In normal rats, whose nutritional state varied at the time of adrenaline administration (after a 24 hours' fast, fed ad libitum or given 5 g glucose/kg as a 20% solution intragastrically 2 hours before injecting adrenaline), no marked post-adrenaline differences were found between the size of the decrease in the amount of glycogen in white and red muscle. In addition, no significant differences were found between the three groups of animals in glycogen concentration in the EDL (0.3+/-0.05, 0.35+/-0.03 and 0.26+/-0.02 mg/g) or in the SOL, apart from one exception (0.23+/-0.02, 0.2+/-0.01, and 0.51+/-0.03 mg/g), after adrenaline. The glycogen concentration in the white and red muscle of diabetic rats fed ad libitum fell to values similar to those in normal rats after adrenaline (0.32+/-0.05 mg/g in the EDL and 0.18+/-0.02 mg/g in the SOL). These results supoort the view of authors who hold that glycogenolysis is possible without pre-activation of phosphorylase; they also support the idea, expressed by Krebs, of the existence of a reciprocal relationship between phosphorylase activity and the glycogen concentration, according to which glycogen itself may influence its own degradation.

Animals↗

PubMed1990

In vivo portal-hepatic venous gradients of glycogenic precursors and incorporation of D-[3-3H]glucose into liver glycogen in the awake rat.

Male Wistar fed rats were chronically cannulated and fed ground chow for 2 h for 6 days. On the 7th post-operative day, blood was simultaneously drawn from the portal and hepatic veins over a 2-h feeding period. The position of the hepatic vein cannula was verified using a tritiated water washout technique. In separate experiments, 200 microCi of [3-3H]glucose was added to the food in order to determine the relative contribution of D-glucose and 3-C precursors to newly synthesized glycogen. The 22-h fasting plasma portal vein concentrations of D-glucose, L-lactate, and L-alanine were 4.8 +/- 0.03, 0.81 +/- 0.06, and 0.20 +/- 0.03 mM, respectively (n = 5). The fasting hepatic vein plasma concentrations were 5.1 +/- 0.2, 0.70 +/- 0.15 and 0.19 +/- 0.03 mM, respectively. The portal-hepatic vein gradients after 22 h were -0.24, +0.16, and +0.01 mM for D-glucose, L-lactate, and L-alanine, respectively. At 20 min after beginning the meal, the respective gradients were +2.2, +0.53, and +0.44 mM, indicating hepatic uptake of all glycogen precursors. Of the total carbon from the three major precursors entering the liver as C-6, D-glucose contributed 82%, while alanine and lactate contributed 18% at 20 min. As portal vein D-glucose and L-alanine levels exceeded 6.65 +/- 0.69 and 0.32 +/- 0.07 mM, respectively, the portal-hepatic venous gradient became positive and increased linearly with portal concentrations. The glycogen concentration in the liver increased from a 22-h fast value of 5 mumol of glucosyl units/g wet weight to 101 +/- 7 mumol/g 2 h after the meal. The mean specific activity of portal vein plasma of [3-3H]glucose was 11,490 +/- 1,180 dpm/mumol (+/- S.E.) and that in the glycogen isolated from liver was 8,175 +/- 785 dpm/mumol of glycosyl units 2 h after the meal. The specific activity of liver [3H]glycogen relative to glucose after the meal was 0.73 +/- 0.08. It was concluded that a minimum of 73% of the newly synthesized glycogen was formed from the uptake and direct phosphorylation of portal blood D-glucose by the liver without prior conversion of glucose to 3-C units.

Alanine↗

PubMed1979

Regulation of glycogen synthesis and glucose utilization in Escherichia coli during maintenance of the energy charge. Quantitative correlation of changes in the rates of glycogen synthesis and glucose utilization with simultaneous changes in the cellular levels of both glucose 6-phosphate and fructose 1,6-diphosphate.

Treatment of nitrogen-starved cultures of Escherichia coli W4597(K) with sodium azide results in simultaneous changes in both glucose 6-phosphate and fructose 1,6-diphosphate as well as in the rate of glycogen synthesis. Based on these observations, a comprehensive equation was developed which relates the cellular levels of both of these hexose phosphates with the rate of glycogen synthesis. This relationship apparently represents the interaction in vivo between the rate-limiting enzyme of bacterial glycogen synthesis, glucose 1-phosphate adenylyltransferase (adenosine diphosphoglucose synthetase, EC 2.7.7.27), and its substrate glucose 1-phosphate (reflected by glucose 6-phosphate) and its major allosteric activator fructose diphosphate. The form of the equation that describes this relationship was determined from studies presented here of the kinetic properties of the E. coli W4597(K) enzyme in the presence of physiological concentrations of its substrates and modulators. We show here and in subsequent reports of this series that the comprehensive relationship between glycogen synthesis and hexose phosphates can serve as a reference to evaluate the possible participation of new factors in the regulation of glycogen synthesis. Treatment with NaN3 did not change the cellular level of glucose 1-phosphate adenylyltransferase. The value of the adenylate energy charge, (ATP + 1/2 ADP)/(ATP + ADP + AMP), was maintained despite losses of up to 35% in cellular adenylates. The quantitative co-variance between hexose phosphates and the cellular rate of glucose utilization that we previously described for other metabolic conditions was also observed in the azide-treated cultures. We integrate the new information into the system of coordinated regulation of glycogen synthesis, glycolysis, and glucose utilization that we proposed previously.

Adenosine Diphosphate Glucose↗

PubMed1982

Rabbit liver glycogen synthase kinases. Characterization of a protein kinase (PC0.7) able to phosphorylate glycogen synthase and phosvitin.

A rabbit liver protein kinase (PC0.7), able to phosphorylate glycogen synthase and phosvitin, has been extensively purified. The enzyme had apparent Mr = 170,000-190,000 as judged by gel filtration and was associated with two major polypeptide species, alpha (Mr = 43,000) and beta (Mr = 25,000). Two other polypeptides, Mr = 38,000 and Mr = 35,000, were also detected. Treatment with trypsin led to an enzyme composed only of polypeptides of Mr = 35,000 and Mr = 25,000. The beta-polypeptide underwent autophosphorylation when incubated with Mg2+ and ATP or GTP. The protein kinase was effective in utilizing both ATP and GTP as the phosphoryl donor (apparent Km values 5-11 microM and 9-19 microM, respectively). The enzyme phosphorylated phosvitin, casein, and glycogen synthase but not histone or phosphorylase and was inhibited by heparin. Phosphorylation of glycogen synthase proceeded to approximately 0.5 phosphate/subunit with little inactivation of the glycogen synthase. The phosphorylation occurred predominantly in a 21,000-dalton CNBr fragment of glycogen synthase that had been previously shown to reside toward the COOH terminus of the molecule. The liver PC0.7 appeared very similar to an analogous enzyme isolated from rabbit muscle (DePaoli-Roach, A. A., Ahmad, Z., and Roach, P. J. (1981) J. Biol. Chem. 256, 8955-8962). The present work, therefore, provides a point of contact between the Ca2+ and cyclic nucleotide-independent glycogen synthase kinases of rabbit liver and muscle.

Animals↗

PubMed1984

Glycogen synthase kinases. Classification of a rabbit liver casein and glycogen synthase kinase (casein kinase-1) as a distinct enzyme.

A protein kinase, able to phosphorylate casein, phosvitin, and glycogen synthase, was purified approximately 9000-fold from rabbit liver, and appeared analogous to an enzyme studied by Itarte and Huang (Itarte, E., and Huang, K.-P. (1979) J. Biol. Chem. 254, 4052-4057). This enzyme, designated here casein kinase-1, was shown to be a distinct glycogen synthase kinase and in particular to be different from the protein kinase GSK-3 (Hemmings, B.A., Yellowlees, D., Kernohan, J.C., and Cohen, P. (1981) Eur. J. Biochem. 119, 443-451). Casein kinase-1 had native molecular weight of 30,000 as judged by gel filtration. The enzyme phosphorylated beta-casein A or B better than kappa-casein or alpha s1-casein, and modified only serine residues in beta-casein B and phosvitin. The apparent Km for ATP was 11 microM, and GTP was ineffective as a phosphoryl donor. The phosphorylation of glycogen synthase by casein kinase-1 was inhibited by glycogen, half-maximally at 2 mg/ml, and by heparin, half-maximally at 0.5-1.0 microgram/ml, but was unaffected by Ca2+ and/or calmodulin, or by cyclic AMP. Phosphorylation of muscle glycogen synthase proceeded to a stoichiometry of at least 6 phosphates/subunit with reduction in the +/- glucose-6-P activity ratio to less than 0.4. Phosphate was introduced into both a COOH-terminal CNBr fragment (CB-2) as well as a NH2-terminal fragment (CB-1). At a phosphorylation stoichiometry of 6 phosphates/subunit, 84% of the phosphate was associated with CB-2 and 6.5% with CB-1. The remainder of the phosphate was introduced into another CNBr fragment of apparent molecular weight 16,500. Phosphorylation by casein kinase-1 correlated with reduced electrophoretic mobilities, as analyzed on polyacrylamide gels in the presence of sodium dodecyl sulfate, of the intact glycogen synthase subunit, as well as the CNBr fragments CB-1 and CB-2.

Adenosine Triphosphate↗

PubMed1998

Glycogen-rich malignant melanomas and glycogen-rich balloon cell malignant melanomas: frequency and pattern of PAS positivity in primary and metastatic melanomas.

OBJECTIVE: After identifying a metastatic glycogen-rich balloon cell malignant melanoma, originally thought to be a benign clear cell tumor of the lung, we investigated the extent of positive reactions, or "positivity," of malignant melanoma to periodic acid-Schiff (PAS) staining. METHODS: Frequency, intensity, and distribution of PAS positivity was studied in 61 excisional biopsy specimens from 58 patients with malignant melanoma. For comparison, 17 benign nevi from 10 patients were examined. RESULTS: Positivity for PAS was seen in all cases. All malignant melanomas and benign nevi were characterized by weak, diffuse, diastase-resistant PAS positivity. Additionally, focal or diffuse, strong diastase-sensitive PAS positivity was observed in 9 of 61 melanomas (15%); 7 were metastatic and 2 were primary invasive melanomas. Strong diastase-sensitive PAS positivity was seen in all lesions with 30% or more balloon cell features and only in advanced primary or metastatic lesions. The presence of glycogen was confirmed by transmission electron microscopy. CONCLUSIONS: Cutaneous malignant melanomas have weak, diastase-resistant PAS positivity. Strong diastase-sensitive PAS positivity, consistent with the presence of intracytoplasmic glycogen, is seen in many primary and metastatic melanomas with balloon cell features. Depending on the content of the balloon cells, these melanomas are best categorized as either glycogen-rich malignant melanomas or glycogen-rich balloon cell malignant melanomas. Because many tumors with clear cell features contain glycogen, such content often is an unreliable differential feature.

Biopsy↗

PubMed1988

Liver and muscle insulin sensitivity, glycogen concentration and glycogen synthase activity in a rat model of non-insulin-dependent diabetes.

Mild diabetes was induced in adult rats with streptozotocin (45 mg/kg body weight), and insulin sensitivity, glycogen deposition and glycogen synthase activity assessed in liver and muscle 5 weeks later. Diabetic rats had significantly elevated fasting blood glucose concentrations (5.6 +/- 0.1 versus 3.6 +/- 0.1 mmol/l, p less than 0.001), and blood glucose concentrations 2 h after a 1 g/kg glucose load (12.0 +/- 0.6 versus 3.7 +/- 0.2 mmol/l, p less than 0.001). After a 20-h fast hepatic glucose output was significantly elevated (58 +/- 3 versus 47 +/- 3 mumol.min-1.kg-1, p less than 0.05), and failed to suppress at high insulin concentrations during a euglycaemic clamp (hepatic glucose output 21 +/- 4 versus 2 +/- 4 mumol.min-1.kg-1, p less than 0.02). Liver glycogen was lower in the diabetic rats by the end of the clamp (16 +/- 3 versus 38 +/- 6 mumol/g wet wt, p less than 0.05). At the end of the clamp total glucose turnover was lower in the diabetic rats (107 +/- 4 versus 161 +/- 17 mumol.min-1.kg-1, p less than 0.01), as was skeletal muscle glycogen synthase activity (0.46 +/- 0.04 versus 0.67 +/- 0.05 U/g wet wt, p less than 0.01) and glycogen concentration (22 +/- 2 versus 33 +/- 3 mumol/g wet wt, p less than 0.05). Blood lactate and pyruvate responses suggested that glycolytic pathways were similarly affected. Thus, insulin insensitivity develops in both liver and skeletal muscle after 5 weeks of mild streptozotocin-induced diabetes.

Animals↗

PubMed1993

Inactivation of rabbit muscle glycogen synthase by glycogen synthase kinase-3. Dominant role of the phosphorylation of Ser-640 (site-3a).

Rabbit skeletal muscle glycogen synthase, a rate-limiting enzyme for glycogen biosynthesis, is regulated by multisite phosphorylation. The protein kinase glycogen synthase kinase 3 (GSK-3) phosphorylates 4 Ser residues (Ser-640, Ser-644, Ser-648, and Ser-652; also known as sites 3a, 3b, 3c, and 4, respectively) at the COOH terminus of the subunit. Phosphorylation of these sites by GSK-3 is sequential, from COOH- to NH2-terminal, and is wholly dependent on prior phosphorylation by casein kinase II at Ser-656 (site 5). Expression in Escherichia coli was used to generate mutant forms of glycogen synthase, S640A, S644A, and S648A, in which site 3a, site 3b, or site 3c was changed to Ala, respectively. The purified enzymes had -/+ glucose-6-P activity ratios in the range of 0.8-0.9. Phosphorylation by casein kinase II and GSK-3 gave results consistent with the model of obligate sequential action of GSK-3. Phosphorylation at site 5, sites 4 + 5, or sites 3c + 4 + 5 had no measurable effect on activity. When sites 3b + 3c + 4 + 5 were phosphorylated, modest inactivation resulted. Additional phosphorylation at site 3a, however, was potently inactivating, reducing the -/+ glucose-6-P activity ratio to 0.1 and increasing the glucose-6-P concentration needed for half-maximal activation by an order of magnitude. Introduction of each additional phosphate, in the order site 4, 3c, 3b, and 3a, caused an incremental reduction in the mobility of the subunit when analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The results of this study demonstrate that GSK-3 phosphorylation of site 3a (Ser-640), and to a lesser extent, site 3b, correlates with inactivation of glycogen synthase by GSK-3. Evidence is also presented for an allosteric mechanism of inactivation whereby modification of one subunit influences the activity state of adjacent subunits.

Amino Acid Sequence↗