Partly Ordered Synthesis and Degradation of Glycogen in Cultured Rat Myotubes

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 lastin-first-out principle, particularly in the initial period. Analysis of the size of the glycogen molecules and the ␤-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 ϳ53,000 glucosyl residues for the fully synthesized molecule (␤-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 (1, 2). The spherical form and branched structure of glycogen appear to be optimally suited to fast breakdown by the glycogen phosphorylase and the debranching enzyme (2). 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 ␣[134] glucosyl bonds and gives rise to two new B-chains by way of the ␣[136] branch points (3). 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 (2, 4). Further synthesis is not possible for steric reasons (2).For the first eight tiers, the ␣[134] glucosyl bonds are synthesized by proglycogen synthase and the ␣[136] branch points by branching enzyme, and the molecule is defined as...

Increases in glycogenin and glycogenin mRNA accompany glycogen resynthesis in human skeletal muscle

“…Using biochemical measurements of PG and MG in human skeletal muscle, we have shown that PG accumulation is the initial event during glycogen resynthesis (0 -4 h) followed by increases in MG (4 -24 h) (2). These data are consistent with the findings of Elsner et al (11), who have demonstrated using radiolabeled glucose a doubling of glycogen concentration in the early phases of glycogen resynthesis yet no increase in glycogen granule size in cultured rat myotubes. These findings have also been confirmed visually using transmission electron microscopy (TEM), a technique that allows the quantification of granule size, number, and subcellular distribution (20).…”

“…Given this, glycogen resynthesis likely involves 1) the synthesis of new GN-1 protein or 2) the addition of glucose residues to existing glycogen structures. Biochemical and electron microscopic data show both mechanisms to be involved, as there is an increase in granule size and number with glycogen resynthesis (11,21). New glycogen granule formation would theoretically require the synthesis of new GN-1 protein.…”

Blockades of mitogen-activated protein kinase and calcineurin both change fibre-type markers in skeletal muscle culture

“…Primary skeletal muscle cell culture Primary cultures of myotubes were prepared from the hindleg muscles of 21-day-old Wistar rat foetuses by a modification of the methods described by Elsner et al [20].…”

Glycogenin protein and mRNA expression in response to changing glycogen concentration in exercise and recovery

“…The initial phase of recovery is characterized primarily by an increase in PG followed by an increase in MG; this may reflect an initial increase in the number of small granules, followed by their enlargement and development into larger, mature granules (2, 28). Elsner et al (10) demonstrated an "ordered" pattern of granule synthesis using cultured rodent myotubes, in that existing granules appear to accept CHO initially, but the cell quickly begins to generate new granules even though the existing ones were only intermediate in size. However, it needs to be noted that the glycogen stores of the myotubes were only moderately compromised prior to the synthesis period.…”

“…Work with cultured myotubes (10) suggests that some glycogen granules may be completely catabolized when glycogen stores are decreased. Under these circumstances, it is unknown whether the protein is degraded or is liberated as apoprotein (apo)-GN-1 (i.e., unglucosylated) and/or is translocated.…”