Altered glucose 1,6-bisphosphate and fructose 2,6-biphosphate levels in low-frequency stimulated rabbit fast-twitch muscle

Glucose 1,6-bisphosphate (Glc-1,6-P2) and fructose 2,6-bisphosphate (Fru-2,6-P2) concentrations display pronounced increases in rabbit fast-twitch muscle during chronic low-frequency stimulation. These increases are first seen after stimulation periods exceeding 3 h and reach maxima after 12-24 h of stimulation (approximately 3-fold for Glc-1,6-P2 and 5-fold for Fru-2,6-P2). Both metabolites regress to normal values after stimulation periods longer than 4 days. The fact that their increases coincide with the replenishment of glycogen after its initial depletion, could point to a role of Glc-1,6-P2 and Fru-2,6-P2 in glycogen metabolism.     

Phosphorylation-dependent translocation of glycogen synthase to a novel structure during glycogen resynthesis

Glycogen metabolism has been the subject of extensive research, but the mechanisms by which it is regulated are still not fully understood. It is well accepted that the rate-limiting enzymes in glycogenesis and glycogenolysis are glycogen synthase (GS) and glycogen phosphorylase (GPh), respectively. Both enzymes are regulated by reversible phosphorylation and by allosteric effectors. However, evidence in the literature indicates that changes in muscle GS and GPh intracellular distribution may constitute a new regulatory mechanism of glycogen metabolism. Already in the 1960s, it was proposed that glycogen was present in dynamic cellular organelles that were termed glycosomas but no such cellular entities have ever been demonstrated. The aim of this study was to characterize muscle GS and GPh intracellular distribution and to identify possible translocation processes of both enzymes. Using in situ stimulation of rabbit tibialis anterior muscle, we show GS and GPh intracellular redistribution at the beginning of glycogen resynthesis after contraction-induced glycogen depletion. We identify a new "player," a new intracellular compartment involved in skeletal muscle glycogen metabolism. They are spherical structures that were not present in basal muscle, and we present evidence that indicate that they are products of actin cytoskeleton remodeling. Furthermore, for the first time, we show a phosphorylation-dependent intracellular distribution of GS. Here, we present evidence of a new regulatory mechanism of skeletal muscle glycogen metabolism based on glycogen enzyme intracellular compartmentalization.     

Disturbances of the sarcoplasmic reticulum and transverse tubular system in 24-h electrostimulated fast-twitch skeletal muscle

Chronic low-frequency stimulation of rabbit tibialis anterior muscle over a 24-h period induces a conspicuous loss of isometric tension that is unrelated to muscle energy metabolism (J.A. Cadefau, J. Parra, R. Cusso, G. Heine, D. Pette, Responses of fatigable and fatigue-resistant fibres of rabbit muscle to low-frequency stimulation, Pflugers Arch. 424 (1993) 529-537). To assess the involvement of sarcoplasmic reticulum and transverse tubular system in this force impairment, we isolated microsomal fractions from stimulated and control (contralateral, unstimulated) muscles on discontinuous sucrose gradients (27-32-34-38-45%, wt/wt). All the fractions were characterized in terms of calcium content, Ca2+/Mg2+-ATPase activity, and radioligand binding of [3H]-PN 200-110 and [3H]ryanodine, specific to dihydropyridine-sensitive calcium channels and ryanodine receptors, respectively. Gradient fractions of muscles stimulated for 24 h underwent acute changes in the pattern of protein bands. First, light fractions from longitudinal sarcoplasmic reticulum, enriched in Ca2+-ATPase activity, R1 and R2, were greatly reduced (67% and 51%, respectively); this reduction was reflected in protein yield of crude microsomal fractions prior to gradient loading (25%). Second, heavy fractions from the sarcoplasmic reticulum were modified, and part (52%) of the R3 fraction was shifted to the R4 fraction, which appeared as a thick, clotted band. Quantification of [3H]-PN 200-110 and [3H]-ryanodine binding revealed co-migration of terminal cisternae and t-tubules from R3 to R4, indicating the presence of triads. This density change may be associated with calcium overload of the sarcoplasmic reticulum, since total calcium rose three- to fourfold in stimulated muscle homogenates. These changes correlate well with ultrastructural damage to longitudinal sarcoplasmic reticulum and swelling of t-tubules revealed by electron microscopy. The ultrastructural changes observed here reflect exercise-induced damage of membrane systems that might severely compromise muscle function. Since this process is reversible, we suggest that it may be part of a physiological response to fatigue.     

A novel role of neuregulin in skeletal muscle. Neuregulin stimulates glucose uptake, glucose transporter translocation, and transporter expression in muscle cells

Neuregulins regulate the expression of acetylcholine receptor genes and induce development of the neuromuscular junction in muscle. In studying whether neuregulins regulate glucose uptake in muscle, we analyzed the effect of a recombinant neuregulin, (r)heregulin-beta1-(177-244) (HRG), on L6E9 muscle cells, which express the neuregulin receptors ErbB2 and ErbB3. L6E9 responded acutely to HRG by a time- and concentration-dependent stimulation of 2-deoxyglucose uptake. HRG-induced stimulation of glucose transport was additive to the effect of insulin. The acute stimulation of the glucose transport induced by HRG was a consequence of the translocation of GLUT4, GLUT1, and GLUT3 glucose carriers to the cell surface. The effect of HRG on glucose transport was dependent on phosphatidylinositol 3-kinase activity. HRG also stimulated glucose transport in the incubated soleus muscle and was additive to the effect of insulin. Chronic exposure of L6E9 cells to HRG potentiated myogenic differentiation, and under these conditions, glucose transport was also stimulated. The activation of glucose transport after chronic HRG exposure was due to enhanced cell content of GLUT1 and GLUT3 and to increased abundance of these carriers at the plasma membrane. However, under these conditions, GLUT4 expression was markedly down-regulated. Muscle denervation is associated with GLUT1 induction and GLUT4 repression. In this connection, muscle denervation caused a marked increase in the content of ErbB2 and ErbB3 receptors, which occurred in the absence of alterations in neuregulin mRNA levels. This fact suggests that neuregulins regulate glucose transporter expression in denervated muscle. We conclude that neuregulins regulate glucose uptake in L6E9 muscle cells by mechanisms involving the recruitment of glucose transporters to the cell surface and modulation of their expression. Neuregulins may also participate in the adaptations in glucose transport that take place in the muscle fiber after denervation.     

Differences between glycogen biogenesis in fast- and slow-twitch rabbit muscle

Skeletal muscle glycogen is an essential energy substrate for muscular activity. The biochemical properties of the enzymes involved in de novo synthesis of glycogen were analysed in two types of rabbit skeletal muscle fiber (fast- and slow-twitch). Glycogen concentration was higher in fast-twitch muscle than in slow-twitch muscle, but the latter contained many more small intermediate-acceptor molecules that could act as glycogen synthase substrates. The enzymes involved in de novo synthesis of glycogen in fast-twitch muscle were strongly stimulated by Glc-6-P, but those in slow-twitch muscle were not.     

Contractile activity modifies Fru-2,6-P(2) metabolism in rabbit fast twitch skeletal muscle.

Modification of muscular contractile patterns by denervation and chronic low frequency stimulation induces structural, physiological, and biochemical alterations in fast twitch skeletal muscles. Fructose 2,6-bisphosphate is a potent activator of 6-phosphofructo-1-kinase, a key regulatory enzyme of glycolysis in animal tissues. The concentration of Fru-2,6-P(2) depends on the activity of the bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2), which catalyzes the synthesis and degradation of this metabolite. This enzyme has several isoforms, the relative abundance of which depends on the tissue metabolic properties. Skeletal muscle expresses two of these isoforms; it mainly contains the muscle isozyme (M-type) and a small amount of the liver isozyme (L-type), whose expression is under hormonal control. Moreover, contractile activity regulates expression of muscular proteins related with glucose metabolism. Fast twitch rabbit skeletal muscle denervation or chronic low frequency stimulation can provide information about the regulation of this enzyme. Our results show an increase in Fru-2,6-P(2) concentration after 2 days of denervation or stimulation. In denervated muscle, this increase is mediated by a rise in liver PFK-2/FBPase-2 isozyme, while in stimulated muscle it is mediated by a rise in muscle PFK-2/FBPase-2 isozyme. In conclusion, our results show that contractile activity could alter the expression of PFK-2/FBPase-2.     

The changes in the energy metabolism of human muscle induced by training

The biochemical effects of training programmes have been studied with a kinetic model of central metabolism, using enzyme activities and metabolite concentrations measured at rest and after 30 s maximum-intensity exercise, collected before and after long and short periods of training, which differed only by the duration of the rest intervals. After short periods of training the glycolytic flux at rest was three times higher than it had been before training, whereas during exercise the flux and energy consumption remained the same as before training. Long periods of training had less effect on the glycolytic flux at rest, but increased it in response to exercise, increasing the contribution of oxidative phosphorylation.     

Effect of chronic electrostimulation of rabbit skeletal muscle on calmodulin level and protein kinase activity

(a) Chronic electrostimulation of fast-twitch skeletal muscles makes them resemble slow-twitch muscles. The involvement of second-messenger cascades in this muscle reprogramming is not well understood. The goal of this study was to examine protein kinase activities and calmodulin levels as a function of the duration of electrostimulation. (b) Fast-twitch rabbit muscle was subjected to continuous low-frequency electrostimulation for 2 weeks. The extensor digitorum longus was taken and examined for calmodulin concentration and cAMP-dependent (PKA). Ca(2+)-phospholipid-dependent (PKC) and Ca(2+)-calmodulin-dependent (CaM kinase or PKB) protein kinase activities. (c) Electrostimulation for 14 days led to a significant increase in total calmodulin level and PKB activity, both rising in the cytosolic fraction. Protein kinase C translocated to the membrane fraction, although total activity did not change. (d) These changes could be related with electrostimulation-induced changes in excitation-contraction coupling.     

Glycogen depletion and resynthesis during 14 days of chronic low-frequency stimulation of rabbit muscle

Electro-stimulation alters muscle metabolism and the extent of this change depends on application intensity and duration. The effect of 14 days of chronic electro-stimulation on glycogen turnover and on the regulation of glycogen synthase in fast-twitch muscle was studied. The results showed that macro- and proglycogen degrade simultaneously during the first hour of stimulation. After 3 h, the muscle showed net synthesis, with an increase in the proglycogen fraction. The glycogen content peaked after 4 days of stimulation, macroglycogen being the predominant fraction at that time.Glycogen synthase was determined during electro-stimulation. The activity of this enzyme was measured at low UDPG concentration with either high or low Glu-6-P content. Western blots were performed against glycogen synthase over a range of stimulation periods. Activation of this enzyme was maximum before the net synthesis of glycogen, partial during net synthesis, and low during late synthesis. These observations suggest that the more active, dephosphorylated and very low phosphorylated forms of glycogen synthase may participate in the first steps of glycogen resynthesis before net synthesis is observed, while partially phosphorylated forms are most active during glycogen elongation.