Metabolic and mitogenic effects of IGF-I and insulin on muscle cells of rainbow trout

The relative function of IGF-I and insulin on fish muscle metabolism and growth has been investigated by the isolation and culture at different stages (myoblasts at day 1, myocytes at day 4, and myotubes at day 10) of rainbow trout muscle cells. This in vitro model avoids interactions with endogenous peptides, which could interfere with the muscle response. In these cells, the effects of IGF-I and insulin on cell proliferation, 2-deoxyglucose (2-DG), and l-alanine uptake at different development stages, and the use of inhibitors were studied and quantified. Insulin (10-1,000 nM) and IGF-I (10-100 nM) stimulated 2-DG uptake in trout myocytes at day 4 in a similar manner (maximum of 124% for insulin and of 142% for IGF-I), and this stimulation increased when cells differentiated to myotubes (maximum for IGF-I of 193%). When incubating the cells with PD-98059 and especially cytochalasin B, a reduction in 2-DG uptake was observed, suggesting that glucose transport takes place through specific facilitative transporters. IGF-I (1-100 nM) stimulated the l-alanine uptake in myocytes at day 4 (maximum of 239%), reaching higher values of stimulation than insulin (100-1,000 nM) (maximum of 160%). This stimulation decreased when cells developed to myotubes at day 10 (118% for IGF-I and 114% for insulin). IGF-I (0.125-25 nM) had a significant effect on myoblast proliferation, measured by thymidine incorporation (maximum of 170%), and required the presence of 2-5% fetal serum (FBS) to promote thymidine uptake. On the other hand, insulin was totally ineffective in stimulating thymidine uptake. We conclude that IGF-I is more effective than insulin in stimulating glucose and alanine uptake in rainbow trout myosatellite cells and that the degree of stimulation changes when cells differentiate to myotubes. IGF-I stimulates cell proliferation in this model of muscle in vitro and insulin does not. These results indicate the important role of IGF-I on growth and metabolism of fish muscle.     

Stimulation of glucose transport in L6 muscle cells by long-term intermittent stretch-relaxation.

Skeletal muscle stretch increases resting metabolism and causes hypertrophy. We have examined the effect of mechanical stretch in vitro on glucose transport activity and transporter contents in L6 muscle cells. Long-term (24-48 h) stretch-relaxation (25% maximal elongation at 30 cycles per min) of cell monolayers significantly increased glucose uptake by 1.6- to 2-fold in myotubes but not in myoblasts. The presence of serum was required for the stretch-relaxation induced increase in glucose uptake. Cycloheximide inhibited the mechanical stimulation of glucose uptake, and the latter response was not additive to the stimulatory effect of long-term exposure to insulin. GLUT1 and GLUT4 glucose transporter contents were not changed in total cell membranes from mechanically stimulated cells relative to controls. These results indicate that mechanical stimulation through passive stretch may be an important regulation of nutrient uptake in fetal myotubes independent of innervation.     

Possibility of autocrine beta-adrenergic signaling in C2C12 myotubes

Levodopa reportedly inhibits insulin action in skeletal muscle. Here we show that C2C12 myotubes produce levodopa and that insulin-stimulated glucose transport is enhanced when endogenous levodopa is depleted. Exogenous levodopa prevented the stimulation of glucose transport by insulin (P < 0.05) and increased cAMP concentrations (P < 0.05). The decrease in insulin-stimulated glucose transport caused by levodopa was attenuated by propranolol (a beta-adrenergic antagonist) and prevented by NSD-1015 (NSD), an inhibitor of DOPA decarboxylase (DDC; converts levodopa to dopamine). Propranolol and NSD both prevented levodopa-related increases in [cAMP]. However, the effects of levodopa were unlikely to be dependent on the conversion of levodopa to catecholamines because we could detect neither DDC in myotubes nor catecholamines in media after incubation of myotubes with levodopa. The data suggest the possibility of novel autocrine beta-adrenergic action in C2C12 myotubes in which levodopa, produced by myotubes, could have hormone-like effects that impinge on glucose metabolism.     

cDNA cloning and functional characterization of a novel splice variant of c-Cbl-associated protein from mouse skeletal muscle

c-Cbl-associated protein (CAP) is an SH3-containing adapter protein that binds to the proto-oncogene c-Cbl. Recent work suggests that signaling through these molecules is involved in the regulation of insulin-stimulated glucose uptake in 3T3-L1 adipocytes. Skeletal muscle is the major site of insulin-stimulated glucose disposal but there have been no reports of CAP function in this tissue. Using RT-PCR of mouse skeletal muscle RNA, we discovered a novel splice variant of CAP (CAPSM; GenBank Accession No. AF521593) that is different from the adipocyte form by inclusion of a novel 168 bp fragment. This fragment encodes a peptide sequence that shows very high similarity with exon 25 of the human homologue of CAP (SORBS1). To understand the function of CAPSM in glucose uptake regulation, L6 myotubes were transfected with either CAPSM or a truncated CAPSM devoid of all three SH3-binding domains (CAPDeltaSH3), which prevents CAP association with c-Cbl. Transfection with CAPDeltaSH3 decreased insulin-stimulated 2-deoxyglucose (2-DG) uptake and reduced c-Cbl phosphorylation. In contrast, transfection of L6 myotubes with CAPDeltaSH3 had no effect on dinitrophenol (DNP)- or hypoxia-stimulated glucose uptake, stimuli that work through insulin-independent mechanisms for the regulation of glucose uptake. These data demonstrate the existence of a novel CAP isoform expressed in skeletal muscle, and suggest the involvement of the CAP/Cbl pathway in the regulation of insulin-stimulated glucose uptake in L6 myotubes.     

Mechanical signals and IGF-I gene splicing in vitro in relation to development of skeletal muscle

It has been shown that the insulin-like growth factor (IGF-I) gene is spliced in response to mechanical signals producing forms of IGF-I which have different actions. In order to study how mechanical signals influence this gene splicing in developing muscle, C2C12 cells were grown in three-dimensional (3D) culture and subjected to different regimens of mechanical strain. IGF-IEa which initiates the fusion of myoblasts to form myotubes was found to be constitutively expressed in myoblasts and myotubes (held under endogenous tension) and its expression upregulated by a single ramp stretch of 1-h duration but reduced by repeated cyclical stretch. In contrast, mechano growth factor (MGF), which is involved in the proliferation of mononucleated myoblasts that are required for secondary myotube formation and to establish the muscle satellite (stem) cell pool, showed no significant constitutive expression in static cultures, but was upregulated by a single ramp stretch and by cycling loading. The latter types of force simulate those generated in myoblasts by the first contractions of myotubes. These data indicate the importance of seeking to understand the physiological signals that determine the ratios of splice variants of some growth factor/tissue factor genes in the early stages of development of skeletal muscle.     

Voluntary exercise training enhances glucose transport in muscle stimulated by insulin-like growth factor I

Hokama, Jason Y., Ryan S. Streeper, and Erik J. Henriksen.Voluntary exercise training enhances glucose transport in muscle stimulated by insulin-like growth factor I. J. Appl. Physiol. 82(2): 508-512, 1997.Skeletal muscle glucosetransport can be regulated by hormonal factors such as insulin andinsulin-like growth factor I (IGF-I). Although it is well establishedthat exercise training increases insulin action on muscle glucosetransport, it is currently unknown whether exercise training leads toan enhancement of IGF-I-stimulated glucose transport in skeletal muscle. Therefore, we measured glucose transport activity [by using 2-deoxy-D-glucose (2-DG)uptake] in the isolated rat epitrochlearis muscle stimulated bysubmaximally and maximally effective concentrations of insulin (0.2 and13.3 nM) or IGF-I (5 and 50 nM) after 1, 2, and 3 wk of voluntary wheelrunning (WR). After 1 wk of WR, both submaximal andmaximal insulin-stimulated 2-DG uptake rates were significantly(P < 0.05) enhanced (43 and 31%)compared with those of sedentary controls, and these variables werefurther increased after 2 (86 and 57%) and 3 wk (71 and 70%) ofWR. Submaximal and maximal IGF-I-stimulated 2-DG uptakerates were significantly enhanced after 1 wk of WR (82 and 61%), andthese increases did not expand substantially after 2 (71 and 58%) and3 wk (96 and 70%) of WR. This enhancement of hormone-stimulated 2-DGuptake in WR muscles preceded any alteration in glucose transporter(GLUT-4) protein level, which increased only after 2 (24%) and 3 wk(54%) of WR. Increases in GLUT-4 protein were significantly correlated (r = 0.844) with increases in citratesynthase. These results indicate that exercise training can enhanceboth insulin-stimulated and IGF-I-stimulated muscle glucose transportactivity and that these improvements can develop without an increase inGLUT-4 protein.

     

Regulation of amino acid transport and protein metabolism in myotubes derived from chicken muscle satellite cells by insulin-like growth factor-I

The effects of insulin and insulin-like growth factor-I (IGF-I) on amino acid transport and protein metabolism were compared in myotubes derived from chicken breast muscle satellite cells. Protein synthesis was assessed by continuous labelling with [³H]-tyrosine. Protein degradation was estimated by the release of trichloroacetic acid (TCA) soluble radioactivity by cells which had been previously labelled with [³H]-tyrosine for 3 days. Amino acid transport was measured in myotubes incubated in Dulbecco's modified Eagle's medium (DMEM) 0.5% bovine serum albumin (BSA) with or without insulin or IGF-I. Subsequent [³H]-aminoisobutyric acid (AIB) uptake was then measured in amino acid-free medium. IGF-I was more efficient than insulin at equimolar concentration (3.2 nmol/l) in stimulating protein synthesis (127 and 113% of basal, respectively) and inhibiting protein degradation (32% and 13% inhibition of protein degradation following 4 h incubation). Half maximal effective concentrations for stimulation of AIB uptake were 0.27 ± 0.03 nmol/l and 34.8 ± 3.1 nmol/l for IGF-I and insulin respectively, with maximal stimulation of about 340% of basal. Cycloheximide (3.6 μmol/l) diminished IGF-I-stimulated AIB uptake by 55%. Chicken growth hormone had no effect on basal AIB uptake in these cells and neither glucagon nor dexamethasone had an effect on basal or IGF-I-stimulated AIB uptake. This study demonstrates an anabolic effect for IGF-I in myotubes derived from primary chicken satellite cells which is mediated by the type I IGF receptor, since the cation-independent mannose 6-phosphate receptor does not bind IGF-II in chicken cells. © 1993 Wiley-Liss, Inc.     

Regulation of glucose transporters by insulin and extracellular glucose in C2C12 myotubes

It is well established that insulin stimulation of glucose uptake in skeletal muscle cells is mediated through translocation of GLUT4 from intracellular storage sites to the cell surface. However, the established skeletal muscle cell lines, with the exception of L6 myocytes, reportedly show minimal insulin-dependent glucose uptake and GLUT4 translocation. Using C(2)C(12) myocytes expressing exofacial-Myc-GLUT4-enhanced cyan fluorescent protein, we herein show that differentiated C(2)C(12) myotubes are equipped with basic GLUT4 translocation machinery that can be activated by insulin stimulation ( approximately 3-fold increase as assessed by anti-Myc antibody uptake and immunostaining assay). However, this insulin stimulation of GLUT4 translocation was difficult to demonstrate with a conventional 2-deoxyglucose uptake assay because of markedly elevated basal glucose uptake via other glucose transporter(s). Intriguingly, the basal glucose transport activity in C(2)C(12) myotubes appeared to be acutely suppressed within 5 min by preincubation with a pathophysiologically high level of extracellular glucose (25 mM). In contrast, this activity was augmented by acute glucose deprivation via an unidentified mechanism that is independent of GLUT4 translocation but is dependent on phosphatidylinositol 3-kinase activity. Taken together, these findings indicate that regulation of the facilitative glucose transport system in differentiated C(2)C(12) myotubes can be achieved through surprisingly acute glucose-dependent modulation of the activity of glucose transporter(s), which apparently contributes to obscuring the insulin augmentation of glucose uptake elicited by GLUT4 translocation. We herein also describe several methods of monitoring insulin-dependent glucose uptake in C(2)C(12) myotubes and propose this cell line to be a useful model for analyzing GLUT4 translocation in skeletal muscle.     

Contraction- and hypoxia-stimulated glucose transport is mediated by a Ca2+-dependent mechanism in slow-twitch rat soleus muscle

Increases in contraction-stimulated glucose transport in fast-twitch rat epitrochlearis muscle are mediated by AMPK- and Ca2+/calmodulin-dependent protein kinase (CAMK)-dependent signaling pathways. However, recent studies provide evidence suggesting that contraction-stimulated glucose transport in slow-twitch skeletal muscle is mediated through an AMPK-independent pathway. The purpose of the present study was to test the hypothesis that contraction-stimulated glucose transport in rat slow-twitch soleus muscle is mediated by an AMPK-independent/Ca2+-dependent pathway. Caffeine, a sarcoplasmic reticulum (SR) Ca2+-releasing agent, at a concentration that does not cause muscle contractions or decreases in high-energy phosphates, led to an approximately 2-fold increase in 2-deoxyglucose (2-DG) uptake in isolated split soleus muscles. This increase in glucose transport was prevented by the SR calcium channel blocker dantrolene and the CAMK inhibitor KN93. Conversely, 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR), an AMPK activator, had no effect on 2-DG uptake in isolated split soleus muscles yet resulted in an approximately 2-fold increase in the phosphorylation of AMPK and its downstream substrate acetyl-CoA carboxylase. The hypoxia-induced increase in 2-DG uptake was prevented by dantrolene and KN93, whereas hypoxia-stimulated phosphorylation of AMPK was unaltered by these agents. Tetanic muscle contractions resulted in an approximately 3.5-fold increase in 2-DG uptake that was prevented by KN93, which did not prevent AMPK phosphorylation. Taken in concert, our results provide evidence that hypoxia- and contraction-stimulated glucose transport is mediated entirely through a Ca2+-dependent mechanism in rat slow-twitch muscle.     

Increased IGFR activity and glucose transport in cultured skeletal muscle from insulin receptor null mice

We have studied the role of the insulin receptor (IR) in metabolic and growth-promoting effects of insulin on primary cultures of skeletal muscle derived from the limb muscle of IR null mice. Cultures of IR null skeletal muscle displayed normal morphology and spontaneous contractile activity. Expression of muscle-differentiating proteins was slightly reduced in myoblasts and myotubes of the IR null skeletal muscle cells, whereas that of the Na+/K+ pump appeared to be unchanged. Insulin-like growth factor receptor (IGFR) expression was higher in myoblasts from IR knockout (IRKO) than from IR wild-type (IRWT) mice but was essentially unchanged in myotubes. Expression of the GLUT-1 and GLUT-4 transporters appeared to be higher in IRKO than in IRWT myoblasts and was significantly greater in myotubes from IRKO than from IRWT cultures. Consistent with GLUT expression, both basal and insulin or insulin-like growth factor I (IGF-I)-stimulated glucose uptakes were higher in IR null skeletal myotubes than in wild-type skeletal myotubes. Interestingly, autophosphorylation of IGFR induced by insulin and IGF-I was markedly increased in IR null skeletal myotubes. These results indicate that, in the absence of IR, there is a compensatory increase in basal as well as in insulin- and IGF-I-induced glucose transport, the former being mediated via increased activation of the IGF-I receptor.     

p38gamma MAPK regulation of glucose transporter expression and glucose uptake in L6 myotubes and mouse skeletal muscle

Skeletal muscle expresses at least three p38 MAPKs (alpha, beta, gamma). However, no studies have examined the potential regulation of glucose uptake by p38gamma, the isoform predominantly expressed in skeletal muscle and highly regulated by exercise. L6 myotubes were transfected with empty vector (pCAGGS), activating MKK6 (MKK6CA), or p38gamma-specific siRNA. MKK6CA-transfected cells had higher rates of basal 2-deoxy-d-[3H]glucose (2-DG) uptake (P < 0.05) but lower rates of 2,4-dinitrophenol (DNP)-stimulated glucose uptake, an uncoupler of oxidative phosphorylation that operates through an insulin-independent mechanism (P < 0.05). These effects were reversed when MKK6CA cells were cotransfected with p38gamma-specific siRNA. To determine whether the p38gamma isoform is involved in the regulation of contraction-stimulated glucose uptake in adult skeletal muscle, the tibialis anterior muscles of mice were injected with pCAGGS or wild-type p38gamma (p38gammaWT) followed by intramuscular electroporation. Basal and contraction-stimulated 2-DG uptake in vivo was determined 14 days later. Overexpression of p38gammaWT resulted in higher basal rates of glucose uptake compared with pCAGGS (P < 0.05). Muscles overexpressing p38gammaWT showed a trend for lower in situ contraction-mediated glucose uptake (P = 0.08) and significantly lower total GLUT4 levels (P < 0.05). These data suggest that p38gamma increases basal glucose uptake and decreases DNP- and contraction-stimulated glucose uptake, partially by affecting levels of glucose transporter expression in skeletal muscle. These findings are consistent with the hypothesis that activation of stress kinases such as p38 are negative regulators of stimulated glucose uptake in peripheral tissues.     

Hepatocyte growth factor is a novel stimulator of glucose uptake and metabolism in skeletal muscle cells

Skeletal muscle plays a major role in glucose and lipid metabolism. Active hepatocyte growth factor (HGF) is present in the extracellular matrix in skeletal muscle. However, the effects of HGF on glucose and lipid metabolism in skeletal muscle are completely unknown. We therefore examined the effects of HGF on deoxyglucose uptake (DOGU), glucose utilization, and fatty acid oxidation (FAO) in skeletal muscle cells. HGF significantly enhanced DOGU in mouse soleus muscles in vitro. Furthermore, HGF significantly increased: (i) DOGU in a time- and dose-dependent manner; (ii) glucose utilization; and (iii) plasma membrane expression of Glut-1 and Glut-4 in the rat skeletal muscle model of L6 myotubes. HGF-mediated effect on DOGU was dependent on the activation of phosphatidylinositol 3-kinase signaling pathway. On the other hand, HGF markedly and significantly decreased FAO in L6 myotubes without affecting the activities of carnitine palmitoyltransferase I and II. Collectively, these results indicate that HGF is a potent activator of glucose transport and metabolism and also a strong inhibitor of FAO in rodent myotubes. HGF, through its ability to stimulate glucose transport and metabolism and to impair FAO, may participate in the regulation of glucose disposal in skeletal muscle.     

IGF-I and insulin regulate glucose transport in mouse blastocysts via IGF-I receptor

The roles of glucose deprivation, insulin, and insulin-like growth factor I (IGF-I) in the regulation of glucose transport in the mouse blastocyst were examined. Glucose transport, measured by uptake of 3-O-methyl glucose (3-OMG), was increased by 19% (P < 0.01) in response to glucose deprivation. Both IGF-I and insulin stimulated uptake, but IGF-I was 1,000-fold more potent than insulin, increasing uptake by 51% at 1.7 pM (P < 0.001). These effects began to appear after 20 min of incubation with growth factors, and required the simultaneous presence of glucose. The relative potencies of insulin and IGF-I suggest that the actions of IGF-I and insulin were both mediated via the IGF-I receptor. The inactivity of a specific agonistic insulin receptor antibody (B10) confirms this and suggests that this action may be independent of signalling through IRS-1. Cycloheximide decreased growth factor-stimulated transport by about 40%, indicating that both protein synthesis and transporter recruitment from cytoplasmic stores are responsible for maximal stimulation. These characteristics are consistent with GLUT1-facilitated glucose uptake and suggest that GLUT1 is the regulatable transporter in mouse blastocysts. Stimulation of GLUT1 may be a ubiquitous feature of the autocrine/paracrine activity of IGF-I in cell growth and proliferation. © 1996 Wiley-Liss, Inc.     

Autocrine Phosphorylation of p70S6k in Response to Acute Stretch in Myotubes

Phosphorylation of 70-KDa S6 kinase (p70^S6k) is correlated with in vivo skeletal muscle hypertrophy. Experiments tested whether mechanical stretch is sufficient to increase p70^S6k phosphorylation in skeletal myotubes. Immediately following stretch, there was a small increase in p70^S6k phosphorylation (63.2 ± 8.5%) with maximal phosphorylation at 3 h (129.5 ± 22.2%) and it remained elevated through 24 h (46.0 ± 17.2%). To test whether an autocrine mechanism is involved, unstretched myotubes were incubated with medium from the stretch group for 10 min. Conditioned medium resulted in the phosphorylation of p70^S6k in unstretched myotubes (92.8 ± 28.9%) to levels comparable to the 3-h stretch group. These data indicate that p70^S6k is phosphorylated in stretched myotubes via a mechanism that most likely involves an autocrine signaling pathway.     

An analysis of the effects of stretch on IGF-I secretion from rat ventricular fibroblasts

Mechanical force can induce a number of fundamental short- and long-term responses in myocardium. These include alterations in ECM, activation of cell-signaling pathways, altered gene regulation, changes in cell proliferation and growth, and secretion of a number of peptides and growth factors. It is now known that a number of these autocrine/paracrine factors are secreted from both cardiomyocytes and ventricular cardiac fibroblasts (CFb) in response to stretch. One such substance is IGF-I. IGF-I is an important autocrine/paracrine factor that can regulate physiological or pathophysiological responses, such as hypertrophy. In this study, we addressed the possible effects of mechanical perturbation, biaxial strain, on IGF-I secretion from adult rat CFb. CFb were subjected to either static stretch (3-10%) or cyclic stretch (10%; 0.1-1 Hz) over a 24-h period. IGF-1 secretion from CFb in response to selected stretch paradigms was examined using ELISA to measure IGF-I concentrations in conditioned media. Static stretch did not result in any measurable modulation of IGF-I secretion from CFb. However, cyclic stretch significantly increased IGF-I secretion from CFb in a frequency- and time-dependent manner compared with nonstretched controls. This stretch-induced increase in secretion was relatively insensitive to changes in extracellular [Ca(2+)] or to block of L-type Ca(2+) channels. In contrast, thapsigargin, an inhibitor of sarco(endo)plasmic reticulum Ca(2+) ATPase, remarkably decreased stretch-induced IGF-I secretion from CFb. We further show that IGF-I can upregulate mRNA expression of atrial natriuretic peptide in myocytes. In summary, cyclic stretch can significantly increase IGF-I secretion from CFb, and this effect is dependent on a thapsigargin-sensitive pool of intracellular [Ca(2+)].     

Protein kinase Cdelta mediates insulin-induced glucose transport in primary cultures of rat skeletal muscle

Insulin activates certain protein kinase C (PKC) isoforms that are involved in insulin-induced glucose transport. In this study, we investigated the possibility that activation of PKCdelta by insulin participates in the mediation of insulin effects on glucose transport in skeletal muscle. Studies were performed on primary cultures of rat skeletal myotubes. The role of PKCdelta in insulin-induced glucose uptake was evaluated both by selective pharmacological blockade and by over-expression of wild-type and point-mutated inactive PKCdelta isoforms in skeletal myotubes. We found that insulin induces tyrosine phosphorylation and translocation of PKCdelta to the plasma membrane and increases the activity of this isoform. Insulin-induced effects on translocation and phosphorylation of PKCdelta were blocked by a low concentration of rottlerin, whereas the effects of insulin on other PKC isoforms were not. This selective blockade of PKCdelta by rottlerin also inhibited insulin-induced translocation of glucose transporter 4 (GLUT4), but not glucose transporter 3 (GLUT3), and significantly reduced the stimulation of glucose uptake by insulin. When overexpressed in skeletal muscle, PKCdelta and PKCdelta were both active. Overexpression of PKCdelta induced the translocation of GLUT4 to the plasma membrane and increased basal glucose uptake to levels attained by insulin. Moreover, insulin did not increase glucose uptake further in cells overexpressing PKCdelta. Overexpression of PKCdelta did not affect basal glucose uptake or GLUT4 location. Stimulation of glucose uptake by insulin in cells overexpressing PKCdelta was similar to that in untransfected cells. Transfection of skeletal myotubes with dominant negative mutant PKCdelta did not alter basal glucose uptake but blocked insulin-induced GLUT4 translocation and glucose transport. These results demonstrate that insulin activates PKCdelta and that activated PKCdelta is a major signaling molecule in insulin-induced glucose transport.     

Distinct regulation of glucose transport and GLUT1/GLUT3 transporters by glucose deprivation and IGF-I in chromaffin cells

Effects of prolonged metabolic (glucose deprivation) and hormonal [insulin-like growth factor I (IGF-I)] challenge on regulation of glucose transporter (GLUT) expression, glucose transport rate and possible signaling pathways involved were studied in the neuroendocrine chromaffin cell. The results show that bovine chromaffin cells express both GLUT1 and GLUT3. Glucose deprivation and IGF-I activation led to an elevation of GLUT1 and GLUT3 mRNA, the strongest effect being that of IGF-I on GLUT3 mRNA. Both types of stimulus increased the GLUT1 protein content in a cycloheximide (CHX)-sensitive manner, and the glucose transport rate was elevated by 3- to 4-fold after 48 h under both experimental conditions. IGF-I-induced glucose uptake was totally suppressed by CHX. In contrast, only approximately 50% of transport activation in glucose-deprived cells was sensitive to the protein synthesis inhibitor. Specific inhibitors of mTOR/FRAP and p38 MAPK each partially blocked IGF-I-stimulated glucose transport, but had no effect on transport rate in glucose-deprived cells. The results are consistent with IGF-I-activated transport being completely dependent on new GLUT protein synthesis while the enhanced transport in glucose-deprived cells was partially achieved independent of new synthesis of proteins, suggesting a mechanism relying on preexisting transporters.     

Mechanical signal transduction in skeletal muscle growth and adaptation.

The adaptability of skeletal muscle to changes in the mechanical environment has been well characterized at the tissue and system levels, but the mechanisms through which mechanical signals are transduced to chemical signals that influence muscle growth and metabolism remain largely unidentified. However, several findings have suggested that mechanical signal transduction in muscle may occur through signaling pathways that are shared with insulin-like growth factor (IGF)-I. The involvement of IGF-I-mediated signaling for mechanical signal transduction in muscle was originally suggested by the observations that muscle releases IGF-I on mechanical stimulation, that IGF-I is a potent agent for promoting muscle growth and affecting phenotype, and that IGF-I can function as an autocrine hormone in muscle. Accumulating evidence shows that at least two signaling pathways downstream of IGF-I binding can influence muscle growth and adaptation. Signaling via the calcineurin/nuclear factor of activated T-cell pathway has been shown to have a powerful influence on promoting the slow/type I phenotype in muscle but can also increase muscle mass. Neural stimulation of muscle can activate this pathway, although whether neural activation of the pathway can occur independent of mechanical activation or independent of IGF-I-mediated signaling remains to be explored. Signaling via the Akt/mammalian target of rapamycin pathway can also increase muscle growth, and recent findings show that activation of this pathway can occur as a response to mechanical stimulation applied directly to muscle cells, independent of signals derived from other cells. In addition, mechanical activation of mammalian target of rapamycin, Akt, and other downstream signals is apparently independent of autocrine factors, which suggests that activation of the mechanical pathway occurs independent of muscle-mediated IGF-I release.     

ATP stimulates glucose transport through activation of P2 purinergic receptors in C(2)C(12) skeletal muscle cells

Extracellular ATP acts as a signal that regulates a variety of cellular processes via binding to P2 purinergic receptors (P2 receptors). We herein investigated the effects and signaling pathways of ATP on glucose uptake in C(2)C(12) skeletal muscle cells. ATP as well as P2 receptor agonists (ATP-gamma S) stimulated the rate of glucose uptake, while P2 receptor antagonists (suramin) inhibited the stimulatory effect of ATP, indicating that P2 receptors are involved. This ATP-stimulated glucose transport was blocked by specific inhibitors of Gi protein (pertusiss toxin), phospholipase C (U73122), protein kinase C (GF109203X), and phosphatidylinositol (PI) 3-kinase (LY294002). ATP stimulated PI 3-kinase activity and P2 receptor antagonists blocked this activation. In C(2)C(12) myotubes expressing glucose transporter GLUT4, ATP increased basal and insulin-stimulated glucose transport. Finally, ATP facilitated translocation of GLUT1 and GLUT4 into plasma membrane. These results together suggest that cells respond to extracellular ATP to increase glucose transport through P2 receptors.     

PPARdelta activator GW-501516 has no acute effect on glucose transport in skeletal muscle

It has been reported that treatment of cultured human skeletal muscle myotubes with the peroxisome proliferator-activated receptor-delta (PPARdelta) activator GW-501516 directly stimulates glucose transport and enhances insulin action. Cultured myotubes are minimally responsive to insulin stimulation of glucose transport and are not a good model for studying skeletal muscle glucose transport. The purpose of this study was to evaluate the effect of GW-501516 on glucose transport to determine whether the findings on cultured myotubes have relevance to skeletal muscle. Rat epitrochlearis and soleus muscles were treated for 6 h with 10, 100, or 500 nM GW-501516, followed by measurement of 2-deoxyglucose uptake. GW-501516 had no effect on glucose uptake. There was no effect on insulin sensitivity or responsiveness. Also, in contrast to findings on myotubes, treatment of muscles with GW-501516 did not result in increased phosphorylation or increased expression of AMP-activated protein kinase (AMPK) or p38 mitogen-activated protein kinase (MAPK). Treatment of epitrochlearis muscles with GW-501516 for 24 h induced a threefold increase in uncoupling protein-3 mRNA, providing evidence that the GW-501516 compound that we used gets into and is active in skeletal muscle. In conclusion, our results show that, in contrast to myotubes in culture, skeletal muscle does not respond to GW-501516 with 1) an increase in AMPK or p38 MAPK phosphorylation or expression or 2) direct stimulation of glucose transport or enhanced insulin action.