Stimulation of hexose transport in L6 rat myoblasts by antibody and by glucose starvation.
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ABSTRACT:
SUBMITTER: D'Amore T
PROVIDER: S-EPMC1147211 | biostudies-other | 1986 Sep
REPOSITORIES: biostudies-other
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Project description:A hexose-transport regulatory mutant (D1/S4) was isolated from L6 rat myoblasts on the basis of its resistance to detachment and cell lysis in the presence of antibody and complement. Growth studies indicated that D1/S4 cells had a slower doubling time (29 h) compared with the parental L6 cells (22 h). Furthermore, after 9 days growth, less than 1% cell fusion was observed with D1/S4 cells, whereas 95% cell fusion was observed with the L6 cells. When the parental L6 cells were starved of glucose or treated with anti-L6 antibody, a significant increase in the Vmax, of 2-deoxy-D-glucose (dGlc) and 3-O-methyl-D-glucose (MeGlc) transport was observed. Although glucose-grown D1/S4 cells possessed normal hexose-transport activity, the above treatments had no effect on dGlc and MeGlc transport in these cells. Electrophoresis and immunoblotting studies revealed that D1/S4 cells possessed decreased amounts of a 112 kDa plasma-membrane protein. It is conceivable that this protein may play a role in triggering the antibody- and glucose-starvation-mediated activation of hexose transport and in myogenic differentiation. Unlike D1/S4, mutant F72, a mutant defective in the high-affinity hexose-transport system, was found to possess normal amounts of the 112 kDa protein. Although glucose starvation has no effect on the hexose-transport activity in this mutant, its hexose transport activity can be increased by antibody treatment. These studies with mutants suggest the involvement of regulatory components in the activation of hexose transport.
| S-EPMC1148814 | biostudies-other
Project description:We have recently demonstrated that two hexose-transport systems are present in undifferentiated rat L6 myoblasts: D-glucose and 2-deoxy-D-glucose are preferentially transported by the high-affinity system, whereas 3-O-methyl-D-glucose is transported primarily by the low-affinity system. Mutant D23 is found to be defective only in the high-affinity hexose-transport system. The low-affinity transport system is much more sensitive to inhibition by cytochalasin B (CB). The present study examines the identity, properties and regulation of the CB-binding sites by measuring CB binding to both whole cells and plasma membrane. Scatchard analysis of the binding data revealed the presence of two CB-binding sites, namely CBH and CBL. These two sites differ not only in their affinity for CB, but their levels can also be differentially altered by various biochemical, physiological and genetic manipulations. CBL resembles the high-affinity hexose-transport system in that it is absent in mutant D23 and is present in larger quantities in glucose-starved cells. Moreover, CB binding to this site is inhibited by D-glucose and 2-deoxy-D-glucose, the preferred substrates of the high-affinity hexose-transport system. On the other hand, CBH is found to be unaltered in mutant D23, which also retains the normal low-affinity hexose-transport system. CBH also resembles the low-affinity transport system in that it is not elevated in glucose-starved cells. Furthermore, binding of CB to this site can be inhibited by 3-O-methyl-D-glucose, the preferred substrate of the low-affinity transport system. It should be noted that 2-deoxy-D-glucose does not have much effect on CBH, and vice versa. Studies with purified membrane preparations indicate that both CB-binding sites are present in similar ratios in the plasma membrane and the low-density microsomal fraction. Plasma-membrane studies also reveal that D-glucose 6-phosphate, but not 2-deoxy-D-glucose 6-phosphate, is very effective in activating CB binding. Data presented suggest that CB binding may be regulated by sugar analogues in an allosteric manner.
| S-EPMC1148964 | biostudies-other
Project description:The present investigation reports on the hexose transport properties of human myoblasts isolated from normal subjects and from patients with Duchenne muscular dystrophy (DMD). Similar to rat myoblast L6, normal human myoblasts possess a high- (HAHT) and a low- (LAHT) affinity hexose transport system. The non-metabolizable hexose analogue, 2-deoxyglucose, is preferentially taken up by HAHT. The transport of this analogue is the rate-limiting step in the uptake process. This human myoblast HAHT is also similar to that of the rat myoblast in its substrate specificity and in response to the energy uncouplers, cytochalasin B and phloretin. The human myoblast LAHT resembles that of rat myoblast in its insensitivity to energy uncouplers, and in its transport affinity and capacity for 3-O-methyl-D-glucose. Although DMD myoblasts resemble their normal counterpart in their ability to differentiate, they differ significantly in their hexose transport properties. In addition to HAHT and LAHT present in normal human myoblast, DMD myoblasts contain a super-high-affinity hexose transport system (SHAHT). SHAHT can be detected only at very low substrate concentrations. It differs from HAHT not only in its much higher transport affinity, but also in its response to the traditional hexose transport inhibitors. For example, SHAHT can be activated by cytochalasin B and phlorizin, whereas it is more sensitive to inhibition by phloretin. Unlike HAHT, energy uncouplers are found to be ineffective in inhibiting SHAHT. It should be mentioned that SHAHT cannot be detected in myoblasts isolated from patients with other types of myopathy. The present study serves to demonstrate that more than one hexose transport system is operating in human skeletal muscle cells, as found in other cell types.
| S-EPMC1133223 | biostudies-other
Project description:Methylglyoxal (MG), a highly reactive ?-dicarbonyl metabolite of glucose degradation pathways, protein and fatty acid metabolism, plays an important role in the pathogenesis of diabetic complications. Hyperglycemia triggers enhanced production of MG and increased generation of advanced glycation endproducts (AGEs). In non-enzymatic reactions, MG reacts with arginine residues of proteins to form the AGEs argpyrimidine and hydroimidazolone. Glyoxalase 1 (GLO1), in combination with glyoxalase 2 and the co-factor glutathione constitute the glyoxalase system, which is responsible for the detoxification of MG. A GLO1 specific knock down results in accumulation of MG in targeted cells. The aim of this study was to investigate the effect of intracellularly accumulated MG on insulin signaling and on the translocation of the glucose transporter 4 (GLUT4). Therefore, L6 cells stably expressing a myc-tagged GLUT4 were examined. For the intracellular accumulation of MG, GLO1, the first enzyme of the glyoxalase pathway, was down regulated by siRNA knock down and cells were cultivated under hyperglycemic conditions (25 mM glucose) for 48 h. Here we show that GLO1 knock down augmented GLUT4 level on the cell surface of L6 myoblasts at least in part through reduction of GLUT4 internalization, resulting in increased glucose uptake. However, intracellular accumulation of MG had no effect on GLUT4 concentration or modification. The antioxidant and MG scavenger NAC prevented the MG-induced GLUT4 translocation. Tiron, which is also a well-known antioxidant, had no impact on MG-induced GLUT4 translocation.
| S-EPMC3662699 | biostudies-literature
Project description:1. Leukaemia inhibitory factor (LIF) has been shown to have an important role during muscle regeneration. The regenerative capacity of muscles after contusion injury in LIF-knockout mice is impaired compared with that of wild-type mice. 2. To clarify whether LIF modulates muscle regeneration by regulating myogenic precursor cell activity, we studied LIF expression and myogenic precursor cell activity in gastrocnemius muscles from Wistar rats at various times after contusion injury using immunohistochemistry and the direct effect of LIF on a rat myoblast cell line (L6). 3. After contusion injury, transient upregulation of the mRNA expression of LIF, LIF receptors and signal transducer and activator of transcription (STAT) 3, downstream of LIF and involved in enhanced cell proliferation, was observed. A marked increase in LIF protein in the cytosol of damaged myofibres was strongly correlated with a significant increase in the number of myogenic precursor cells (MyoD-positive cells) by 12 h after contusion. In addition, coexpression of LIF and MyoD protein in control and injured muscles after contusion injury from 3 h to 7 days was evident. 4. Treatment of L6 cells with LIF (1 ng/mL) in serum-free medium enhanced proliferation (bromodeoxyuridine incorporation) by 24 h. This was accompanied by increased expression of c-Myc protein within 12 h and was abolished by short interference RNA against c-Myc mRNA. 5. Together, the results of the present study suggest that LIF acts via paracrine and autocrine actions to regulate myogenic precursor cell activity during muscle regeneration after contusion injury and that the proliferative effect of LIF on L6 cells occurs via c-Myc signalling.
| S-EPMC4550807 | biostudies-literature
Project description:Insulin at a concentration close to the physiological range (100 mu-units/ml) stimulated protein synthesis in L6 myoblasts by 17%. Pre-treatment with the phospholipase A2 inhibitors mepacrine or dexamethasone prevented this stimulation and decreased the release of prostaglandin F2 alpha, implicating the action of phospholipase A2 and the subsequent metabolism of arachidonic acid to prostaglandins in the stimulation of protein synthesis by physiological doses of insulin. Higher concentrations of insulin (500-1000 mu-units/ml) stimulated protein synthesis in the presence of mepacrine or dexamethasone, suggesting that an alternative pathway may become important in insulin action when phospholipase A2 is inhibited.
| S-EPMC1131793 | biostudies-other
Project description:All known D-xylose transporters are competitively inhibited by D-glucose, which is one of the major reasons hampering simultaneous fermentation of D-glucose and D-xylose, two primary sugars present in lignocellulosic biomass. We have set up a yeast growth-based screening system for mutant D-xylose transporters that are insensitive to the presence of D-glucose. All of the identified variants had a mutation at either a conserved asparagine residue in transmembrane helix 8 or a threonine residue in transmembrane helix 5. According to a homology model of the yeast hexose transporter Gal2 deduced from the crystal structure of the D-xylose transporter XylE from Escherichia coli, both residues are found in the same region of the protein and are positioned slightly to the extracellular side of the central sugar-binding pocket. Therefore, it is likely that alterations sterically prevent D-glucose but not D-xylose from entering the pocket. In contrast, changing amino acids that are supposed to directly interact with the C6 hydroxymethyl group of D-glucose negatively affected transport of both D-glucose and D-xylose. Determination of kinetic properties of the mutant transporters revealed that Gal2-N376F had the highest affinity for D-xylose, along with a moderate transport velocity, and had completely lost the ability to transport hexoses. These transporter versions should prove valuable for glucose-xylose cofermentation in lignocellulosic hydrolysates by Saccharomyces cerevisiae and other biotechnologically relevant organisms. Moreover, our data contribute to the mechanistic understanding of sugar transport because the decisive role of the conserved asparagine residue for determining sugar specificity has not been recognized before.
| S-EPMC3986176 | biostudies-literature
Project description:Insulin resistance and Type 2 diabetes are marked by an aberrant response in the insulin signaling network. The phosphoinositide-dependent serine/threonine kinase, Akt2, plays a key role in insulin signaling and glucose uptake, most notably within skeletal muscle. Protein-protein interaction regulates the functional consequence of Akt2 and in turn, Akt2's role in glucose uptake. However, only few insulin-responsive Akt2 interaction partners have been identified in skeletal muscle cells. In the present work, rat L6 myoblasts, a widely used insulin sensitive skeletal muscle cell line, were used to examine endogenous, insulin-stimulated Akt2 protein interaction partners. Akt2 co-immunoprecipitation was coupled with 1D-SDS-PAGE and fractions were analyzed by HPLC-ESI-MS/MS to reveal Akt2 protein-protein interactions. The pull-down assay displayed specificity for the Akt2 isoform; Akt1 and Akt3 unique peptides were not detected. A total of 49 were detected with a significantly increased (47) or decreased (2) association with Akt2 following insulin administration (n = 4; p<0.05). Multiple pathways were identified for the novel Akt2 interaction partners, such as the EIF2 and ubiquitination pathways. These data suggest that multiple new endogenous proteins may associate with Akt2 under basal as well as insulin-stimulated conditions, providing further insight into the insulin signaling network. Data are available via ProteomeXchange with identifier PXD002557.
| S-EPMC4605787 | biostudies-literature
Project description:Insulin resistance and Type 2 diabetes are marked by an aberrant response in the insulin signaling network. The phosphoinositide-dependent serine/threonine kinase, Akt2, plays a key role in insulin signaling and glucose uptake, most notably within skeletal muscle. Protein-protein interaction regulates the functional consequence of Akt2 and in turn, Akt2’s role in glucose uptake. However, only few insulin-responsive Akt2 interaction partners have been identified in skeletal muscle cells. In the present work, rat L6 myoblasts, a widely used insulin sensitive skeletal muscle cell line, were used to examine endogenous, insulin-stimulated Akt2 protein interaction partners. Akt2 co-immunoprecipitation was coupled with 1D-SDS-PAGE and fractions were analyzed by HPLC-ESI-MS/MS to reveal Akt2 protein-protein interactions. The pull-down assay displayed specificity for the Akt2 isoform; Akt1 and Akt3 unique peptides were not detected. A total of 330 proteins were significantly enriched as bonafide Akt2 protein interaction partners. Among them, 226 were novel for any cell types. Furthermore, 59 were detected with a significantly increased (57) or decreased (2) association with Akt2 following insulin administration (n=4; p<0.05). Multiple pathways were identified for the novel Akt2 interaction partners, such as the EIF2 and ubiquitination pathways. These data suggest that multiple new endogenous proteins may associate with Akt2 under basal as well as insulin-stimulated conditions, providing further insight into the insulin signaling network.
2015-09-30 | PXD002557 | Pride
Project description:BackgroundThyroid hormones (THs) act genomically to stimulate glucose transport by elevating glucose transporter (Slc2a) expression and glucose utilization by cells. However, nongenomic effects of THs are now emerging. Here, we assess how triiodothyronine (T(3)) acutely affects glucose transport and the content of GLUT4, GLUT1, and GLUT3 at the surface of muscle cells, and possible interactions between T(3) and insulin action.MethodsDifferentiated L6 myotubes transfected with myc-tagged Slc2a4 (L6-GLUT4myc) or Slc2a1 (L6-GLUT1myc) and wild-type L6 myotubes were studied in the following conditions: control, hypothyroid (Tx), Tx plus T(3), Tx plus insulin, and Tx plus insulin and T(3).ResultsGlucose uptake and GLUT4 content at the cell surface decreased in the Tx group relative to controls. T(3) treatment for 30 minutes increased glucose transport into L6-GLUT4myc cells without altering surface GLUT4 content, which increased only thereafter. The total amount of GLUT4 protein remained unchanged among the groups studied. The surface GLUT1 content of L6-GLUT1myc cells also remained unaltered after T(3) treatment; however, in these cells glucose transport was not stimulated by T(3). In wild-type L6 cells, although T(3) treatment increased the total amount of GLUT3, it did not change the surface GLUT3 content. Moreover, within 30 minutes, T(3) stimulation of glucose uptake was additive to that of insulin in L6-GLUT4myc cells. As expected, insulin elevated surface GLUT4 content and glucose uptake. However, interestingly, surface GLUT4 content remained unchanged or even dropped with T(3) plus insulin.ConclusionsThese data reveal that T(3) rapidly increases glucose uptake in L6-GLUT4myc cells, which, at least for 30 minutes, did not depend on an increment in GLUT4 at the cell surface yet potentiates insulin action. We propose that this rapid T(3) effect involves activation of GLUT4 transporters at the cell surface, but cannot discount the involvement of an unknown GLUT.
| S-EPMC3387762 | biostudies-literature