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• W2051567796 abstract "The following questions concerning glycogen synthesis and degradation were examined in cultured rat myotubes. 1) Is synthesis and degradation of the individual glycogen molecule a strictly ordered process, with the last glucosyl unit incorporated into the molecule being the first to be released (the last-in-first-out principle), or is it a random process? 2) Are all glycogen molecules in skeletal muscle synthesized and degraded in phase (simultaneous order) or out of phase (sequential order)? Basal glycogen stores were minimized by fasting and were subsequently replenished in two intervals, the first (0–0.5 h) with tritium-labeled and the second (0.5–3 h) with carbon-labeled glucose as precursor. Glycogen degradation was initiated by addition of forskolin. The kinetics of glycogen accumulation as well as degradation could be approximated by monoexponential equations with rate constants of 0.81 and 1.39 h−1, respectively. The degradation of glycogen largely followed the last-in-first-out principle, particularly in the initial period. Analysis of the size of the glycogen molecules and the β-dextrin limit during glycogen accumulation and degradation showed that both synthesis and degradation of glycogen molecules are largely sequential and the small deviation from this order is most pronounced at the beginning of the accumulation and at the end of the degradation period. This pattern may reflect the number of synthase and phosphorylase molecules and fits well with the role of glycogen in skeletal muscle as a readily available energy store and with the known structure of the glycogen molecule. It is emphasized that the observed nonlinear relation between the change in glycogen concentration and release of label during glycogen degradation may have important practical consequences for interpretation of experimental data. The following questions concerning glycogen synthesis and degradation were examined in cultured rat myotubes. 1) Is synthesis and degradation of the individual glycogen molecule a strictly ordered process, with the last glucosyl unit incorporated into the molecule being the first to be released (the last-in-first-out principle), or is it a random process? 2) Are all glycogen molecules in skeletal muscle synthesized and degraded in phase (simultaneous order) or out of phase (sequential order)? Basal glycogen stores were minimized by fasting and were subsequently replenished in two intervals, the first (0–0.5 h) with tritium-labeled and the second (0.5–3 h) with carbon-labeled glucose as precursor. Glycogen degradation was initiated by addition of forskolin. The kinetics of glycogen accumulation as well as degradation could be approximated by monoexponential equations with rate constants of 0.81 and 1.39 h−1, respectively. The degradation of glycogen largely followed the last-in-first-out principle, particularly in the initial period. Analysis of the size of the glycogen molecules and the β-dextrin limit during glycogen accumulation and degradation showed that both synthesis and degradation of glycogen molecules are largely sequential and the small deviation from this order is most pronounced at the beginning of the accumulation and at the end of the degradation period. This pattern may reflect the number of synthase and phosphorylase molecules and fits well with the role of glycogen in skeletal muscle as a readily available energy store and with the known structure of the glycogen molecule. It is emphasized that the observed nonlinear relation between the change in glycogen concentration and release of label during glycogen degradation may have important practical consequences for interpretation of experimental data. Glycogen is a branched polymer of glucose of spherical geometry composed of ∼53,000 glucosyl residues for the fully synthesized molecule (β-glycogen). Glycogen plays a very dynamic role in the energy metabolism of skeletal muscle by serving as a readily available energy source during muscle contraction of high intensity (1Suarez R.K. Staples J.F. Lighton J.R.B. West T.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7065-7069Crossref PubMed Scopus (65) Google Scholar, 2Menléndez-Hevia E. Waddell T.G. Shelton E.D. Biochem. J. 1993; 295: 477-483Crossref PubMed Scopus (123) Google Scholar). The spherical form and branched structure of glycogen appear to be optimally suited to fast breakdown by the glycogen phosphorylase and the debranching enzyme (2Menléndez-Hevia E. Waddell T.G. Shelton E.D. Biochem. J. 1993; 295: 477-483Crossref PubMed Scopus (123) Google Scholar). The basic structure of glycogen consists of layers (tiers) of branched glucosyl chains with the protein glycogenin as the core. Each B-chain consists of 13 glucosyl residues linked by α[1→4] glucosyl bonds and gives rise to two new B-chains by way of the α[1→6] branch points (3Goldsmith E. Sprang S. Fletterick R. J. Mol. Biol. 1982; 156: 411-427Crossref PubMed Scopus (123) Google Scholar). This design principle is repeated until 11 layers of B-chains are formed and the synthesis is completed by the addition of one tier of A-chains of the same length as the B-chain, but without branch points (2Menléndez-Hevia E. Waddell T.G. Shelton E.D. Biochem. J. 1993; 295: 477-483Crossref PubMed Scopus (123) Google Scholar, 4Devlin T.M. Textbook of Biochemistry. 4th Ed. Wiley-Liss, New York1997: 312-331Google Scholar). Further synthesis is not possible for steric reasons (2Menléndez-Hevia E. Waddell T.G. Shelton E.D. Biochem. J. 1993; 295: 477-483Crossref PubMed Scopus (123) Google Scholar). For the first eight tiers, the α[1→4] glucosyl bonds are synthesized by proglycogen synthase and the α[1→6] branch points by branching enzyme, and the molecule is defined as proglycogen. For the last four tiers, the α[1→4] glucosyl bonds are synthesized by glycogen synthase and the α[1→6] branch point by branching enzyme, and the molecule is defined as β-glycogen. It is assumed that glycogen synthesis and degradation under dynamic physiological conditions involve only the four outer tiers (tiers 9–12) (5Lomako J. Lomako W.M. Whelan W.J. FEBS Lett. 1991; 279: 223-228Crossref PubMed Scopus (76) Google Scholar, 6Lomako J. Lomako W.M. Whelan W.J. Dombro R.S. Neary J.T. Norenberg M.D. FASEB J. 1993; 7: 1386-1393Crossref PubMed Scopus (84) Google Scholar, 7Alonso M. Lomako J. Lomako W.M. Whelan W.J. FASEB J. 1995; 9: 1126-1137Crossref PubMed Scopus (203) Google Scholar, 8Meléndez R. Meléndez-Hevia E. Canela E.I. J. Mol. Evol. 1997; 45: 446-455Crossref PubMed Scopus (70) Google Scholar), which, however, account for some 96% of the glucosyl residues of the full sized β-glycogen molecule. Proglycogen can therefore be considered a scaffold for the metabolically active four outer tiers of the β-glycogen molecule. For a full sized β-glycogen molecule, 35% of the glucosyl residues may be removed before the first debranching occurs (2Menléndez-Hevia E. Waddell T.G. Shelton E.D. Biochem. J. 1993; 295: 477-483Crossref PubMed Scopus (123) Google Scholar). Cognate with this highly ordered structure of the individual glycogen molecule (defined as intramolecular order), there may also be an intermolecular order, in the sense that all glycogen molecules are degraded/synthesized simultaneously. This has been investigated in adipocytes and in the liver (9Devos P. Hers H.G. Eur. J. Biochem. 1979; 99: 161-167Crossref PubMed Scopus (50) Google Scholar, 10Devos P. Hers H.G Biochem. Biophys. Res. Commun. 1980; 95: 1031-1036Crossref PubMed Scopus (15) Google Scholar), but not in skeletal muscles. The aim of the present work was to investigate: 1) whether synthesis and degradation in skeletal muscle of the individual glycogen molecule is a strictly ordered process with the last glucosyl unit incorporated into the molecule being the first to be released (the “last-in-first-out” principle), or if it is a random process; and 2) whether all glycogen molecules are synthesized and degraded in phase (simultaneous order) or out of phase (sequential order). One important practical consequence of this work is related to the interpretation of data, where the rate of glycogen degradation is estimated from the release of label from glycogen. With such data it is assumed that incorporation/release of label is proportional to actual incorporation/release of glucosyl units. Depending on the mechanism of synthesis and degradation, however, this may not have to be the case. Fetal calf serum (FCS) 1The abbreviations used are:FCSfetal calf serumDMEMDulbecco's modified Eagle's mediumAraCcytosine-1-β-d-arabinofuranosideCMfDPBSDulbecco's phosphate-buffered saline with 1% glucose and without Ca2+and Mg2+EMelectron microscopy and horse serum were purchased from Biological Industries. Penicillin was obtained from Løvens Kemiske Fabrik, Ballerup, Denmark. Culture dishes were obtained from Nunc, Roskilde, Denmark. [6-3H]Glucose and [U-14C]glucose were purchased from Amersham Biosciences, Inc. Glycogen were obtained from Roche Molecular Biochemicals. Trypsin was purchased from Invitrogen. Dulbecco's modified Eagle's medium (DMEM), gentamycin, cytosine-1-β-d-arabinofuranoside (AraC), and all other chemicals were obtained from Sigma. fetal calf serum Dulbecco's modified Eagle's medium cytosine-1-β-d-arabinofuranoside Dulbecco's phosphate-buffered saline with 1% glucose and without Ca2+and Mg2+ electron microscopy Primary cultures of myotubes were prepared from the hindleg muscles of 21-day-old rat fetuses of Wistar rats by a modification of the method described by Kühl et al. (11Kühl U. Timpl R. von der Mark R.K. Dev. Biol. 1982; 93: 344-354Crossref PubMed Scopus (131) Google Scholar) and Daniels (12Daniels M.P. J. Cell Sci. 1990; 97: 615-627PubMed Google Scholar). Muscles from 10–15 fetuses were finely cut with a pair of scissors for 10 min in 2 drops of Dulbecco's phosphate-buffered saline with 1% glucose and without Ca2+ and Mg2+(CMfDPBS), and transferred to a centrifuge glass with 10 ml of 0.1% collagenase I + 0.15% trypsin with EDTA + 0.01% DNase I in CMfDPBS. After incubation on a water bath at 37 °C for 30 min with shaking every 5 min for the first 20 min and every 2 min for the last 10 min, the digest was triturated 15 times at room temperature with a wide bore pipette tip, diluted with 10 ml (80% DMEM with 25 mmNaHCO3 and 25 mm glucose + 20% FCS), and centrifuged for 8 min at 270 × g. The cell pellet was suspended in 5 ml (80% DMEM with 25 mm NaHCO3and 25 mm glucose + 20% FCS) and filtered by syringe pressure through a 20-μm filter. The centrifuge tube was washed six times with 5 ml (80% DMEM with 25 mm NaHCO3and 25 mm glucose + 20% FCS), which was also passed through the filter. The filtrates were collected on a 150-mm uncoated culture dish and incubated for 45 min in 5% CO2 + 95% atmosphere air at 37 °C. The nonadhering cells were diluted to 1 × 106 cells/ml, and 25 ml were plated on a 150-mm gelatin-coated culture dish, prepared by coating the dish with 10 ml of 0.1% gelatin for 2–3 h at 37 °C and removed by suction twice immediately before plating. The myoblasts were cultured to confluence for 48 h at 37 °C in 5% CO2 + 95% atmosphere air, and were released from the dish by dispase treatment; the medium was removed by suction and 10 ml of 37 °C 0.025% dispase in CMfDPBS was added. After 1 min at room temperature, the solution was removed, 10 ml of the same solution was added, and the dishes incubated at 37 °C for 5 min. The dispase solution was replaced by 10 ml (80% DMEM with 25 mm NaHCO3 and 25 mmglucose + 20% FCS) and the cells incubated for 25 min at 37 °C in 5% CO2 + 95% atmosphere air. The medium was carefully removed and the myoblasts loosened by tapping the dish against the edge of a table for 1 min while rotating the dish. The cells were washed into a centrifuge tube by 5 ml (80% DMEM with 25 mmNaHCO3 and 25 mm glucose + 20% FCS). The empty dishes were tapped against the edge of a table and washed into the centrifuge tube with 5 ml (80% DMEM with 25 mmNaHCO3 and 25 mm glucose + 20% FCS). After centrifugation for 5 min at 70 × g, the cell pellets were combined in a single centrifuge tube and incubated with gentle shaking every minute for 5 min at 37 °C with 1/7 volume of 1% dispase in CMfDPBS. One volume (80% DMEM with 25 mmNaHCO3 and 25 mm glucose + 20% FCS) was added, and the suspension was centrifuged for 5 min at 70 ×g. The cell pellet was suspended in 5 ml (80% DMEM with 25 mm NaHCO3 and 25 mm glucose + 20% FCS), triturated 15 times with a wide bore pipette tip, and filtered by syringe pressure through a 20-μm filter. The centrifuge tube was washed three times with 5 ml (80% DMEM with 25 mmNaHCO3 and 25 mm glucose + 20% FCS), which was also passed through the filter. The myoblast suspension was diluted with (80% DMEM with 25 mm NaHCO3 and 25 mm glucose + 20% FCS). 2 ml of 0.4 × 106cells/ml was plated on 35-mm (8.8 cm2) Matrigel®-coated culture dishes. Dishes were coated with 0.6 ml of 1% Matrigel® (13Funanage V.L. Smith S.M. Minnich M.A. J. Cell. Physiol. 1992; 150: 251-257Crossref PubMed Scopus (29) Google Scholar) in DMEM (4 °C), incubated for 24 h at 37 °C in 5% CO2 + 95% atmosphere air and removed by suction twice immediately before plating. The day of myoblast plating was defined as day 0. The myoblasts were confluent with beginning myotube formation 48 h after plating, and the medium was changed to 90% DMEM with 25 mmNaHCO3 and 25 mm glucose + 10% horse serum. Thereafter, half of the medium was changed three times per week, on day 6 with addition of 20 μm AraC and from day 9 with 10 μm AraC to prevent further proliferation of nonfused cells (14Turo K.A. Florini J. Am. J. Physiol. 1982; 243: C278-C284Crossref PubMed Google Scholar). From day 12 to day 21, the cultures consisted mainly of well differentiated cross-striated and spontaneously contracting myotubes as judged by light and electron microscopy and by a creatine kinase activity at ∼10 units/mg protein. The dishes were placed on ice, the medium was removed by suction, and the cells washed three times at 1–2 °C with Hanks' balanced salt solution. 1000 ml of KF buffer (15Baque S. Guinovart J.J. Gómez-Foix A.M. J. Biol. Chem. 1996; 272: 2594-2598Abstract Full Text Full Text PDF Scopus (29) Google Scholar) with 1 mm dithiothreitol instead of 15 mmβ-mercaptoethanol was added to each dish, and the cells were scraped loose with a rubber policeman and homogenized on ice by ultrasonication (Branson Sonifier B-12) at 50 watts for 5 s. The homogenates were kept frozen at −80 °C until analyses. Protein content was measured by the method of Lowry et al. (16Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) with bovine serum albumin as standard. The dishes were placed on ice, the medium was removed from the dishes by suction and 1000 μl of 0.4n KOH was added to each dish. After 5 min at room temperature, aliquots of the homogenates were stored frozen at −20 °C until analysis. Glycogen was determined as glucose after treatment with amyloglucosidase as described by Katz et al. (17Katz J. Rognstad R. Curr. Top. Cell. Regul. 1976; 10: 237-289Crossref PubMed Scopus (199) Google Scholar). Aliquots for determination of β-dextrin limits were acidified to pH 4.8 by 1m citric acid and digested with β-amylase (9Devos P. Hers H.G. Eur. J. Biochem. 1979; 99: 161-167Crossref PubMed Scopus (50) Google Scholar). The β-dextrin digest were precipitated with 2 volumes of absolute ethanol for 1 h on ice, centrifuged, and washed two times with 70% ethanol. The final precipitate was hydrolyzed in 1 m HCl for 2 h at 80 °C and neutralized with 1 m NaOH. 3 ml of Ultima Gold liquid scintillation solution was added, and the radioactivity was determined in a Packard Scintillator with external counting efficiency determination. The procedure for determination of radioactivity in glycogen originating from [3H]- and [14C]glucose and3H2O was identical, but treatment with β-amylase was omitted and, for the determination of radioactivity in glycogen originating from 3H2O, two additional washing procedures with 70% ethanol were included. Homogenates in 0.4m KOH were heated on a boiling water bath for 10 min, cooled, and centrifuged for 2 min at 10,000 × g. The supernatant was applied on a 5-cm Dowex-50 (H+) on top of 7-cm Dowex-1 (acetate) and the columns eluted with 25 ml water. The eluate was lyophilized and dissolved in 100 μl of water and kept at −20 °C until EM analysis. Glycogen granules were negatively stained (1% uranyl acetate in water) on 200-mesh formvar and carbon-coated nickel grids previously subjected to glow discharge just before use. Electron microscopy was carried out using a Zeiss EM 900 electron microscope operated at 80 kV. The mechanism of glycogen synthesis and degradation was elucidated by the following experimental design, as illustrated in Fig. 1 A. From day 14 to day 15, cells were depleted of glycogen for 24 h. On day 15, at t = 0 h, the cell cultures were incubated under conditions favoring glycogen synthesis. Glycogen accumulation reached a maximum after ∼3 h; the first interval of glycogen accumulated occurred from 0 to 0.5 h and was carried out in the presence of [6-3H]glucose; the second interval of glycogen accumulated from 0.5 to 3 h was carried out with [U-14C]glucose. At 3 h, incubation conditions were changed so glycogen degradation was favored, i.e. low glucose and forskolin. The medium composition for the periods was as follows: depletion period, DMEM with 25 mm NaHCO3, 0 mm glucose, 10% horse serum; accumulation period, first interval, DMEM (19 mm NaHCO3, 19 mmHepes, pH 7.2, and 0.1 mm creatine), 10 mmglucose, and 1.6 μCi/ml [6-3H]glucose; second interval, DMEM (same composition as for first interval), 10 mmglucose, and 0.4 μCi/ml [U-14C]glucose; and degradation period, DMEM (same composition as for first interval, but without pyruvate), 1 mm glucose, and 10 μmforskolin. At the time points indicated in Fig. 1 B (0, 0.5, 1.5, and 3 h for the glycogen accumulation period and 3, 3.25, 3.5, 4, 5, 6, and 7 h for the degradation period) total glycogen and radioactivity in β-dextrin (for the estimation of degree of sequential synthesis/degradation, see “β-Dextrin Limit” below) and glycogen (for estimation of degree of the last-in-first-out principle, see “Calculations” below) were determined. The molecular size distribution of glycogen (for the estimation of degree of sequential synthesis/degradation) was determined at 0, 0.5, 3, 3.5, and 7 h. Recovery of the 3H radioactivity, which was incorporated in glycogen from 0 to 0.5 h, was estimated by determination of the3H content at t = 1.5 and 3 h. For14C radioactivity, incorporated in glycogen fromt = 0.5 to 3 h, recovery was determined att = 4 h in parallel dishes where the accumulation period was extended to 4 h in the absence of radioactivity fromt = 3 to 4 h. Glycogen net synthesis during the degradation period from 3 to 7 h was determined in parallel dishes, which were treated as for glycogen accumulation in two intervals as described above, but without addition of radioactive tracers. At the start of the degradation period att = 3 h, 500 μCi of3H2O/ml of medium was added and 3H radioactivity in glycogen was determined at t = 4, 5, 6, and 7 h. The β-dextrin limit was determined as the amount (or radioactivity) of glycogen remaining after treatment with β-amylase divided by the total amount (or radioactivity) of glycogen. In glycogen particles with full sized A-chains, i.e. 13 glucosyl units, the size of the β-dextrin relative to the untreated glycogen molecule is (2(t − 1) − 1)/(2t − 1), where t is the number of glucosyl tiers, 2(t − 1) − 1 is the number of glucosyl chains left in glycogen (β-dextrin) after treatment with β-amylase and 2t − 1 is the total number of glucosyl chains in glycogen (2Menléndez-Hevia E. Waddell T.G. Shelton E.D. Biochem. J. 1993; 295: 477-483Crossref PubMed Scopus (123) Google Scholar, 18Manners D.J. Adv. Carbohydr. Chem. 1957; 12: 262-298Google Scholar) resulting in a β-dextrin limit of 48–50% for 5 ≤ t ≤ 12. For A-chains shorter than 13 glucosyl residues, the β-dextrin limit was calculated as (13(2(t − 1) − 1)/(13(2(t − 1) − 1) +n(2t − 1)), where n is the number of glucosyl residues in the A-chains. During glycogen synthesis, the β-dextrin limit will range from 65 to 50%, assuming a minimum A-chain length of 7 glucosyl residues, and during degradation the range will be from 50 to 76%, assuming a minimum A-chain length of 4 glucosyl residues. If all full sized glycogen particles behave similarly during degradation, i.e. a fully simultaneous degradation pattern, it follows from the definition of the β-dextrin limit that the relative amount of glycogen at a given time point can be estimated as the β-dextrin limit in the full sized particles divided by the β-dextrin limit at that time point: glycogent= β-dextrin limitt = 0/β-dextrin limitt. The accumulation and degradation of both labeled and total glycogen could be described by monoexponential equations (see “Results,” Figs. 1 and 2, and Table I).Table IKinetics of glycogen accumulation and degradationGlycspanGlyct= 0Glyct→ ∞Glycmaxk accuk degh−1h−1Glycogen accumulation0.944 ± 0.0450.166 ± 0.0231.109 ± 0.0370.81 ± 0.10Glycogen degradation0.913 ± 0.0160.095 ± 0.0181.008 ± 0.0111.39 ± 0.14Values are means ± S.E. (n = 8) and calculated by best curve fit using Equations 1 and 2 after normalization of glycogen content at 3 h to 1. Open table in a new tab Values are means ± S.E. (n = 8) and calculated by best curve fit using Equations 1 and 2 after normalization of glycogen content at 3 h to 1. Glycaccu=Glycspan­accu×(1−e−kaccu×t)+Glyct=0Equation 1 andGlycdeg=Glycspan­deg×(e−kdeg×t)+Glyct→∞Equation 2 Glycaccu and Glycdeg are the glycogen concentration during the synthesis and degradation period, respectively; Glycspan-accu and Glycspan-degare the span of glycogen concentrations for the synthesis and degradation period, respectively; t is time; andk accu and k deg are rate constants for the synthesis and degradation, respectively. The maximal glycogen concentration Glycmax equals Glycspan-accu + Glyct = 0 or Glycspan-deg + Glyct→∞. The glycogen content was normalized to 1 at t = 3 h. To quantify the degree of last-in-first-out, the value of the following expression was calculated for the time intervals where release of tracer is estimated to take place if 100% degree of order is assumed (cf. Fig. 1 A, Table II).fraction of tracer released from labeled glycogen−fraction of glycogen degraded1−fraction of glycogen degradedEquation 3 The degree of order ranges from zero to one, corresponding to completely random and fully ordered degradation of glycogen, respectively.Table IICalculated degree of last-in-first-outInterval (isotope)Estimated degradation interval if 100% degree of last-in-first-outEstimated fraction of tracer released from glycogen in the degradation intervalEstimated fraction of glycogen degraded in the time intervalDegree of last-in-first-outFirst (3H)3.75 ± 0.07–5.38 ± 0.430.33 ± 0.050.29 ± 0.02−0.03 ± 0.03Second (14C)3–3.75 ± 0.070.71 ± 0.050.57 ± 0.060.36 ± 0.03*Values are means ± S.E. (n = 8). The degree of last-in-first-out was calculated as described under “Calculations.” Statistical test for significance was performed by Student’st test. *, p < 0.001. Open table in a new tab Values are means ± S.E. (n = 8). The degree of last-in-first-out was calculated as described under “Calculations.” Statistical test for significance was performed by Student’st test. *, p < 0.001. The degradation time intervals where release of tracer is estimated to take place if 100% degree of order is assumed were calculated from Equations 1 and 2, and the release of 3H or 14C label was calculated from Equation 2 using these time points and the degradation constants for 3H and 14C, respectively. The glycogen pool in the muscle cells was labeled over two intervals during the accumulation of glycogen, first with [6-3H]glucose for 0.5 h and then from 0.5 to 3 h with [U-14C]glucose (cf. Fig.1 A and “Experimental Design”). The subsequent release of radioactivity during glycogen degradation was intended to show the degree to which the synthesis and degradation of glycogen followed the last-in-first-out-principle. Prior to the initiation of glycogen accumulation, the myotubes contained 47 ± 8 nmol of glycogen/mg of protein. Glycogen accumulation was initiated by changing the medium to 10 mm glucose (3H-labeled from t = 0–0.5 h and14C-labeled from 0.5–3 h) and after 0.5 and 3 h, glycogen content had increased to 151 ± 18 and 310 ± 35 nmol/mg of protein (n = 8), respectively. The glycogen accumulation for the 3-h interval could be well fitted by the monoexponential Equation 1 (see “Calculations”), with a rate constant k accu of 0.81 ± 0.10 h−1, Glycspan-accu = 0.944 ± 0.045, Glyct = 0 = 0.166 ± 0.023, and Glycmax = 1.109 ± 0.037 (TableI). Glycogen degradation was followed for 4 h and initiated by a medium containing 1 mm glucose and 10 μmforskolin, which is supposed to activate glycogen phosphorylase (15Baque S. Guinovart J.J. Gómez-Foix A.M. J. Biol. Chem. 1996; 272: 2594-2598Abstract Full Text Full Text PDF Scopus (29) Google Scholar). Analogous to the synthesis, the degradation process could be well approximated by the monoexponential Equation 2 (see “Calculations”) with a rate constant k deg of 1.39 ± 0.14 h−1, Glycspan-deg = 0.913 ± 0.016, Glyct→∞ = 0.095 ± 0.018, and Glycmax = 1.008 ± 0.011 (Table I). Interpretation of the results would be complicated if significant glycogen recycling took place during the experiments. Glycogen degradation during the accumulation period and glycogen synthesis during the degradation period was therefore estimated. In the [14C]glycogen accumulation interval (from 0.5 to 3 h), the 3H content of glycogen decreased linearly by 4 ± 1% h−1 (p < 0.001). The14C content in glycogen decreased by 6 ± 6% h−1 (p > 0.05), as determined in parallel experiments where the glycogen accumulation period was extended to 4 h with unlabeled glucose in the period from 3 to 4 h. Recycling of radioactive tracer may therefore have occurred. However, this is probably not a problem because released labeled glucose 1-phosphate and glucose would be diluted by the much higher concentration of labeled precursors (10 mm, see “Experimental Procedures”). During the degradation period (from 4 to 7 h), net glycogen synthesis was determined as incorporation of 3H2O into glycogen. The synthesis rate of glycogen synthesis under degradation conditions was extremely low and nonsignificant. Because the 3H label was introduced first during the glycogen synthesis, it could be expected to be released last and vice versa for the 14C label if the last-in-first-out principle applies and if all glycogen molecules are in the same phase of degradation. The data show that there was no significant release of 3H-labeled glucose from glycogen during the first 15 min of the degradation period, during which 25% of the glucosyl units was released (Fig. 2). Thereafter, however, significant 3H release took place, although at a lower rate than 14C release as reflected in the increase in the 3H/14C ratio from 3 to 7 h (Fig. 3). With the ratio normalized to 1 at t = 3 h, it increased linearly to ∼7 at t = 7 h. The release of [14C]glucose started immediately upon establishing degradation conditions and followed first order kinetics, as did the total glycogen pool, but the rate constant was ∼40% higher (Fig.2). The degree to which the last-in-first-out principle applies to glycogen synthesis and degradation in myotubes was calculated as described under “Calculations” and is shown in TableII. The time interval in which all14C or 3H was expected to be released if a strict last-in-first-out principle applied was calculated from the time cause of isotope incorporation in glycogen as described under “Calculations” and is given in Table II. The estimated release of isotope in these intervals allowed calculation of the degree of last-in-first-out to 0.03 ± 0.03 (mean ± S.E. (n = 8)) for 3H-labeled glycogen and 0.36 ± 0.03 (mean ± S.E. (n = 8)) for14C-labeled glycogen (p < 0.001), respectively. These results indicate a completely random degradation of the first synthesized (3H-labeled) glycogen and a partially ordered degradation of the last synthesized (14C-labeled) glycogen. To check for possible procedural effects, the order in which the two tracers were administered was reversed, although with no effect (data not shown). The mean diameter of glycogen particles increased from 24.9 ± 1.8 nm (mean ± S.E. (n = 3)) at t = 0 h to 28.1 ± 1.1 nm at t = 0.5 h, remained constant during accumulation from t = 0.5–3 h (29.4 ± 1.6 nm att = 3 h) and during degradation fromt = 3–3.5 h (29.1 ± 1.1 nm at t= 3.5 h), and then declined to the initial value at the end of the degradation period (24.4 ± 0.1 nm at t = 7 h) (Fig. 4). β-Dextrin limits during the glycogen accumulation period (Fig. 5 A) were determined to 61 ± 6%, 50 ± 4%, and 48 ± 1% (mean ± S.E. (n = 8)) at t = 0.5, 1.5, and 3 h, respectively. This means that the glycogen molecules must have a mean A-chain length of ∼13 glucosyl residues at 1.5 and 3 h. From 3 to 7 h, the β-dextrin limit increased from approximately 48 to 67%, suggesting a decrease in the average A-chain length. The [3H]β-dextrin limit (Fig. 5 B) increased gradually in the accumulation period from ∼0.61 at t= 0.5 h to 0.75 at t = 3 h. This increase was not the result of release of outer, tritium-labeled glucosyl residues because the decrease of 3H in β-dextrin and in total glycogen from 0.5 to 3 h occurred at the same rate (results not shown). In the degradation period, the [3H]β-dextrin limit increased linearly to ∼0.87, but the S.E. of the values betweent = 3–7 h were big. In th" @default.
• W2051567796 created "2016-06-24" @default.
• W2051567796 creator A5008019188 @default.
• W2051567796 creator A5033971995 @default.
• W2051567796 creator A5034101869 @default.
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• W2051567796 date "2002-02-01" @default.
• W2051567796 modified "2023-09-27" @default.
• W2051567796 title "Partly Ordered Synthesis and Degradation of Glycogen in Cultured Rat Myotubes" @default.
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