Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

L6 myoblasts spontaneously undergo differentiation and cell fusion into myotubes. These cells express both GLUT1 and GLUT4 glucose transporters, but their expression varies during myogenesis. We now report that the subcellular distribution and the protein processing by glycosylation of both glucose transporter isoforms also change during myogenesis. Crude plasma membrane and light microsome fractions were isolated from either myoblasts or myotubes and characterized by the presence of two functional proteins, the Na+/K(+)-ATPase and the dihydropyridine receptor (DHPR). Immunoreactive alpha 1 subunit of the Na+/K(+)-ATPase was faint in the crude plasma membrane fraction from myoblasts, but abundant in both membrane fractions from myotubes. In contrast, the alpha 1 subunit of the DHPR, which is expressed only in differentiated muscle, was detected in crude plasma membrane from myotubes but not from myoblasts. Therefore, crude plasma membrane fractions from myoblasts and myotubes contain cell surface markers, and the composition of these membranes appears to be developmentally regulated during myogenesis. GLUT1 protein was more abundant in the crude plasma membrane relative to the light microsome fraction prepared from either myoblasts or myotubes. The molecular size in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the GLUT1 transporters in myotubes was smaller than that in myoblasts (Mr 47,000 and 53,000, respectively). GLUT4 protein (Mr 48,000) was barely detectable in the crude plasma membrane fraction and was almost absent in the light microsome fraction prepared from myoblasts. However, GLUT4 protein was abundant in myotubes and was predominantly located in the light microsome fraction. Treatment with endoglycosidase F reduced the molecular size of the transporters in all fractions to Mr 46,000 for GLUT1 and Mr 47,000 for GLUT4 proteins. In myotubes, acute insulin treatment increased the crude plasma membrane content of GLUT1 marginally and of GLUT4 markedly, with a concomitant decrease in the light microsomal fraction. These results indicate that: (a) the subcellular distribution of glucose transporters is regulated during myogenesis, GLUT4 being preferentially sorted to intracellular membranes; (b) both GLUT1 and GLUT4 transporters are processed by N-linked glycosylation to form the mature transporters in the course of myogenesis; and (c) insulin causes modest recruitment of GLUT1 transporters and marked recruitment of GLUT4 transporters, from light microsomes to plasma membranes in L6 myotubes.
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PMID:Development regulation of the subcellular distribution and glycosylation of GLUT1 and GLUT4 glucose transporters during myogenesis of L6 muscle cells. 131 24

Unlike glucose transport, where translocation of the insulin-responsive glucose transporter (GLUT4) from an intracellular compartment to the plasma membrane is the principal mechanism underlying insulin stimulation, no consensus exists presently for the mechanism by which insulin activates the Na+/K(+)-ATPase. We have investigated (i) the subunit isoforms expressed and (ii) the effect of insulin on the subcellular distribution of the alpha beta isoforms of the Na+/K(+)-ATPase in plasma membranes (PM) and internal membranes (IM) from rat skeletal muscle. Western blot analysis, using isoform-specific antibodies to the various subunits of the Na+/K(+)-ATPase, revealed that skeletal muscle PM contains the alpha 1 and alpha 2 catalytic subunits and the beta 1 and beta 2 subunits of the Na+ pump. Skeletal muscle IM were enriched in alpha 2, beta 1, and beta 2; alpha 1 was barely detectable in this fraction. After insulin treatment, alpha 2 content in the PM increased, with a parallel decrease in its abundance in the IM pool; insulin did not have any effect on alpha 1 isoform amount or subcellular distribution. The beta 1 subunit, but not beta 2, was also elevated in the PM after insulin treatment, but this increase originated from a sucrose gradient fraction different from that of the alpha 2 subunit. Our findings suggest that insulin induces an isoform-specific translocation of Na+ pump subunits from different intracellular sources to the PM and that the hormone-responsive enzyme in rat skeletal muscle is an alpha 2:beta 1 dimer.
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PMID:Insulin induces translocation of the alpha 2 and beta 1 subunits of the Na+/K(+)-ATPase from intracellular compartments to the plasma membrane in mammalian skeletal muscle. 131 81

Skeletal muscle surface membrane is constituted by the PM domain and its specialized deep invaginations known as TTs. We have shown previously that insulin induces a rapid translocation of GLUT4s from an IM pool to the PM in rat skeletal muscle (6). In this study, we have investigated the possibility that insulin also stimulates the translocation of GLUT4 proteins to TTs, which constitute the largest area of the cell surface envelope. PM, TTs, and IM components of control and insulinized skeletal muscle were isolated by subcellular fractionation. The TTs then were purified further by removing vesicles of SR origin by using a Ca-loading procedure. Ca-loading resulted in a five- to sevenfold increase in the purification of TTs in the unloaded fraction relative to the loaded fraction, assessed by immunoblotting with an anti-DHP-receptor monoclonal antibody. In contrast, estimation of the content of Ca(2+)-ATPase protein (a marker of SR) with a specific polyclonal antibody revealed that most, if not all, SR vesicles were recovered in the Ca-loaded fraction. Western blotting with an anti-COOH-terminal GLUT4 protein polyclonal antibody revealed that acute insulin injection in vivo (30 min) increased the content of GLUT4 (by 90%) in isolated PMs and markedly enhanced (by 180%) GLUT4 content in purified TTs. Importantly, these insulin-dependent changes in GLUT4 content of PM and purified TTs were seen in the absence of changes in the alpha 1-subunit of the Na(+)-K(+)-ATPase, a surface membrane marker.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Insulin induces the translocation of GLUT4 from a unique intracellular organelle to transverse tubules in rat skeletal muscle. 144 97

D-Glucose protectable cytochalasin B (CB) binding to subcellular membrane fractions was used to determine glucose transporter number in red (quadriceps-gastrocnemius-soleus) and white (quadriceps-gastrocnemius) rat muscle. CB binding was twofold higher in isolated plasma membranes of red than of white muscle. In contrast, the number of transporters in an isolated insulin-sensitive intracellular membrane organelle was similar in the two muscle groups. Immunoblotting and immunofluorescence microscopy with anti-GLUT4 and anti-GLUT1 antibodies indicated that both GLUT1 and GLUT4 transporter isoforms account for the higher abundance of CB binding sites in plasma membranes of red than of white muscle. Immunofluorescence localized GLUT4 to both the surface and the interior of the muscle cell and demonstrated that type I (slow twitch oxidative) and type IIa (fast twitch oxidative-glycolytic) fibers are enriched in GLUT4 protein compared with type IIb (fast twitch glycolytic) fibers. In contrast, GLUT1 reactivity was restricted to the surface of the muscle cell and was also highly enriched in the perineurial sheaths of peripheral nerves and the capsules of muscle spindles present in both red and white muscles. Insulin caused a twofold increase in CB binding in isolated plasma membranes of red or white muscles with a corresponding 40-50% decrease in CB binding in isolated intracellular membranes. These changes in CB binding were paralleled by similar changes in the membrane distribution of the GLUT4 glucose transporter isoform and in glucose transport activity measured after insulin perfusion of hindquarter muscles. In contrast, insulin did not change the distribution of either GLUT1 glucose transporters or Na(+)-K(+)-ATPase alpha 1-subunits. The molar ratio of GLUT4 to GLUT1 in red and white muscle plasma membranes was found to be 4:1 in the basal state and 7:1 in the insulin-stimulated state. These results indicate that red muscle contains a higher amount of GLUT1 and GLUT4 transporters at the plasma membrane than white muscle in the basal and insulin-stimulated states but that GLUT4 translocation does not differ between muscle types. In addition, GLUT4 expression correlates with the metabolic nature (oxidative vs. glycolytic) of skeletal muscle fibers, rather than with their contractile properties (slow twitch vs. fast twitch).
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PMID:Abundance, localization, and insulin-induced translocation of glucose transporters in red and white muscle. 151 90

In obesity, impaired glucose tolerance (IGT), non-insulin-dependent diabetes mellitus (NIDDM), and gestational diabetes mellitus (GDM), defects in glucose transport system activity, contribute to insulin resistance in target tissues. In adipocytes from obese and NIDDM patients, we found that pretranslational suppression of the insulin-responsive GLUT4 glucose transporter isoform is a major cause of cellular insulin resistance; however, whether this process is operative in skeletal muscle is not clear. To address this issue, we performed percutaneous biopsies of the vastus lateralis in lean and obese control subjects and in obese patients with IGT and NIDDM and open biopsies of the rectus abdominis at cesarian section in lean and obese gravidas and gravidas with GDM. GLUT4 was measured in total postnuclear membrane fractions from both muscles by immunoblot analyses. The maximally insulin-stimulated rate of in vivo glucose disposal, assessed with euglycemic glucose clamps, decreased 26% in obesity and 74% in NIDDM, reflecting diminished glucose uptake by muscle. However, in vastus lateralis, relative amounts of GLUT4 per milligram membrane protein were similar (NS) among lean (1.0 +/- 0.2) and obese (1.5 +/- 0.3) subjects and patients with IGT (1.4 +/- 0.2) and NIDDM (1.2 +/- 0.2). GLUT4 content was also unchanged when levels were normalized per wet weight, per total protein, and per DNA as an index of cell number. Levels of GLUT4 mRNA were similarly not affected by obesity, IGT, or NIDDM whether normalized per RNA or for the amount of an unrelated constitutive mRNA species. Because muscle fibers (types I and II) exhibit different capacities for insulin-mediated glucose uptake, we tested whether a change in fiber composition could cause insulin resistance without altering overall levels of GLUT4. However, we found that quantities of fiber-specific isoenzymes (phopholamban and types I and II Ca(2+)-ATPase) were similar in all subject groups. In rectus abdominis, GLUT4 content was similar in the lean, obese, and GDM gravidas whether normalized per milligram membrane protein (relative levels were 1.0 +/- 0.2, 1.3 +/- 0.1, and 1.0 +/- 0.2, respectively) or per wet weight, total protein, and DNA. We conclude that in human disease states characterized by insulin resistance, i.e., obesity, IGT, NIDDM, and GDM, GLUT4 gene expression is normal in vastus lateralis or rectus abdominis. To the extent that these muscles are representative of total muscle mass, insulin resistance in skeletal muscle may involve impaired GLUT4 function or translocation and not transporter depletion as observed in adipose tissue.
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PMID:Gene expression of GLUT4 in skeletal muscle from insulin-resistant patients with obesity, IGT, GDM, and NIDDM. 153 55

In skeletal muscle insulin induces the translocation of both the GLUT4 glucose transporter and the alpha 2 subunit of the Na,K-ATPase from an intracellular membrane (IM) compartment to the plasma membrane (PM). Fractionation studies of rat skeletal muscle using a discontinuous sucrose gradient have indicated that the insulin-induced loss of both proteins occurs from a fraction containing intracellular membranes (IM) of common density. This raises the possibility that both proteins may be colocalized in a single intracellular compartment or are present in separate membrane vesicles that are of similar buoyant density. In this study we report the membrane vesicles from the insulin-responsive IM fraction can in fact be separated on the basis of differences in their sedimentation velocities; immunoblot analyses of fractions collected from a sucrose velocity gradient revealed the presence of two separate peaks for GLUT4 and the alpha 2 subunit of the Na,K-ATPase. One of these peaks representing a fast sedimenting population of vesicles (with a sedimentation coefficient of 2697 +/- 57 S) reacted against antibodies to the alpha 2 subunit of the Na,K-ATPase, whereas, the second peak contained a population of much slower sedimenting vesicles (with a sedimentation coefficient of 209 +/- 4 S) were practically devoid of the alpha 2-subunit. By contrast, the slow sedimenting vesicles were enriched by approximately 32-fold in GLUT4 relative to the starting IM fraction when the fractional protein content was taken into account. Immunoprecipitation of GLUT4-containing vesicles from the insulin-sensitive IM fraction revealed that no immunoreactivity towards either the alpha 2 or the beta 1 subunits of the Na,K-ATPase could be observed, signifying that the insulin-responsive subunits of the Na,K-ATPase and GLUT4 were present in different membrane vesicles and that it was unlikely, therefore, that the insulin-induced redistribution of these proteins to the PM occurs from a common intracellular pool.
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PMID:Sedimentation and immunological analyses of GLUT4 and alpha 2-Na,K-ATPase subunit-containing vesicles from rat skeletal muscle: evidence for segregation. 861 24

The expression of sodium-potassium pumps and glucose transporters in pure adipocyte plasma membranes from a hyperthyroid animal model was studied. Hyperthyroidism was induced by enteral administration of five doses of 90 micrograms of triiodothyronine every second day to 8-week-old rats. Following isolation of epididymal adipocytes, 3-O-methylglucose transport was measured and the number of Na/K-ATPase-(alpha 1- and alpha 2-isoforms) and glucose transporter (GLUT1 and GLUT4) molecules in sheets of adipocyte plasma membrane were determined by quantitative immunoelectron microscopy, using gold labelling. Maximal in vitro insulin stimulation of adipocytes increased the glucose transport rate and the amount of GLUT4 in the plasma membrane 15-fold, whereas the amount of alpha 2 was unaffected. In adipocytes from hyperthyroid rats, mean adipocyte volume was decreased by 18% and the quantities of GLUT4 per unit area of plasma membrane (maximal insulin stimulation) and of alpha 2 were decreased by 19% and 15%, respectively. Thus, hypotrophia of fat tissue in the hyperthyroid state is associated with a decreased expression in the plasma membrane of the glucose transporter GLUT4 and the alpha 2-isoform of Na/K-ATPase.
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PMID:Quantity of Na/K-ATPase and glucose transporters in the plasma membrane of rat adipocytes is reduced by in vivo triiodothyronine. 758 95

The GLUT4 glucose transporter and the alpha 2 subunit of the Na+,K(+)-ATPase of rat skeletal muscle are two proteins which redistribute from intracellular membranes to plasma membranes following in vivo insulin stimulation. Here we show that although both proteins co-segregate after subcellular fractionation of unstimulated rat hindlimb muscles, they do not share the same intracellular residence inside the muscle fibre. By immunogold single- and double-labeling on ultrathin muscle cryosections with specific antibodies, the GLUT4 glucose transporter and the Na+,K(+)-ATPase alpha 2 subunit were observed on different vesicular structures within the cell. GLUT4 was detected on subsarcolemmal and perinuclear membranes, and at the junction between myofibrillar A and I bands where triads are localized. The alpha 2 subunit of the Na+,K(+)-ATPase was observed at the plasma membrane and in distinct subsarcolemmal vesicles and intermyofibrillar membranes. Quantitative analysis of double-labeling of GLUT4 and Na+,K(+)-ATPase alpha 2 subunit revealed that less than 6% of the two proteins co-localize in the same continuous vesicular structures. The differential intracellular localization of the two proteins was further confirmed by immunopurification of GLUT4-containing membranes from muscle homogenates, in which the alpha 2 subunit of the Na+,K(+)-ATPase was found only at the same extent as the alpha 1 subunit of the enzyme, a protein exclusively present at the plasma membrane.
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PMID:The GLUT4 glucose transporter and the alpha 2 subunit of the Na+,K(+)-ATPase do not localize to the same intracellular vesicles in rat skeletal muscle. 778 25

GLUT4 translocation and activation of glucose uptake in skeletal muscle can be induced by both physiological (i.e., insulin, nerve stimulation, or exercise) and pharmacological (i.e., phorbol ester) means. Recently, we demonstrated that high glucose levels may mimic the effects of phorbol esters on protein kinase C (PKC) and insulin receptor function (J Biol Chem 269:3381-3386, 1994). In this study, we tested whether the previously described effects of phorbol esters on translocation of GLUT4 in myotubes in culture and also in rat skeletal muscle might be mimicked by glucose. We found that stimulation of C2C12 myotubes with both insulin (10(-7) mol/l, 5 min) and glucose (25 mmol/l, 10 min) induces a comparable increase of the GLUT4 content in the plasma membrane. To test whether this effect occurs in intact rat skeletal muscle as well, two different model systems were used. As an in vitro model, isolated rat hindlimbs were perfused for 80 min with medium containing 6 mmol/l glucose +/- insulin (1.6 x 10(-9) mmol/l, 40 min) or 25 mmol/l glucose. As an in vivo model, acute hyperglycemia (> 11 mmol/l glucose, 20 min) was induced in Wistar rats by intraperitoneal injection of glucose under simultaneous suppression of the endogenous insulin release by injection of somatostatin. In both models, subcellular fractions were prepared from hindlimb skeletal muscle, and plasma membranes were characterized by the enrichment of the marker enzyme alpha 1 Na(+)-K(+)-ATPase.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Acute hyperglycemia provides an insulin-independent inducer for GLUT4 translocation in C2C12 myotubes and rat skeletal muscle. 778 29

Murine models of insulin resistance and diabetes are versatile and have been used to investigate genetic and metabolic disorders. However, the principal assays to assess insulin action, i.e., the euglycemic-hyperinsulinemic clamp and subcellular distribution of glucose transporters, have not been implemented in this species. Here we describe procedures which allow these methods to be adapted to mice. When normal C57bl/6j mice were infused with graded doses of insulin (1, 3, 10 or 30 mU/kg/min) during a euglycemic-hyerinsulinemic clamp, the glucose infusion rate necessary to maintain euglycemia increased in a dose-dependent manner (7.4 +/- 1.7, 13.1 +/- 3.6, 24.1 +/- 2.3 or 34.8 +/- 7.5 mg/kg/min), respectively. Hindlimb muscles were isolated and samples of 2-3 g were subjected to subcellular fractionation finalizing on 25%, 30% and 35% sucrose gradients. Fraction F25 (plasma membranes) was enriched in alpha 2 Na+/K(+)-ATPase and GLUT1 glucose transporters, whereas fraction F35 (intracellular membranes) was enriched in Ca(2+)-ATPase and GLUT4 glucose transporters. Following insulin treatment, GLUT4 increased in F25 and decreased in F35. Insulin treatment had no effect on GLUT1 in F25. However, unlike in rat skeletal muscle, GLUT1 was detectable in F35 and its content decreased in this fraction following insulin treatment. The results demonstrate that whole-body glucose utilization can be assessed in mice using euglycemic-hyperinsulinemic clamps and demonstrate how subcellular fractionation procedures can be applied to murine muscle. Murine muscle GLUT4 translocates from an intracellular storage site to the plasma membrane in response to insulin.
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PMID:Insulin action on whole body glucose utilization and on muscle glucose transporter translocation in mice. 813 7


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