Biomedical subjects

A Marette

Publications and source records attributed to A Marette.

At least 37 records · Page 2Linked to original sources

PubMed2000

Activation of p38 mitogen-activated protein kinase alpha and beta by insulin and contraction in rat skeletal muscle: potential role in the stimulation of glucose transport.

The stress-activated p38 mitogen-activated protein kinase (MAPK) was recently shown to be activated by insulin in muscle and adipose cells in culture. Here, we explore whether such stimulation is observed in rat skeletal muscle and whether muscle contraction can also affect the enzyme. Insulin injection (2 U over 3.5 min) resulted in increases in p38 MAPK phosphorylation measured in soleus (3.2-fold) and quadriceps (2.2-fold) muscles. Increased phosphorylation (3.5-fold) of an endogenous substrate of p38 MAPK, cAMP response element binder (CREB), was also observed. After in vivo insulin treatment, p38 MAPKalpha and p38 MAPKbeta isoforms were found to be activated (2.1- and 2.4-fold, respectively), using an in vitro kinase assay, in immunoprecipitates from quadriceps muscle extracts. In vitro insulin treatment (1 nmol/l over 4 min) and electrically-induced contraction of isolated extensor digitorum longus (EDL) muscle also doubled the kinase activity of p38 MAPKalpha and p38 MAPKbeta. The activity of both isoforms was inhibited in vitro by 10 micromol/l SB203580 in all muscles. To explore the possible participation of p38 MAPK in the stimulation of glucose uptake, EDL and soleus muscles were exposed to increasing doses of SB203580 before and during stimulation by insulin or contraction. SB203580 caused a significant reduction in the insulin- or contraction-stimulated 2-deoxyglucose uptake. Maximal inhibition (50-60%) occurred with 10 micromol/l SB203580. These results show that p38 MAPKalpha and -beta isoforms are activated by insulin and contraction in skeletal muscle. The data further suggest that activation of p38 MAPK may participate in the stimulation of glucose uptake by both stimuli in rat skeletal muscle.

Animals↗

PubMed2000

The transferrin receptor defines two distinct contraction-responsive GLUT4 vesicle populations in skeletal muscle.

Insulin and contraction increase glucose transport in an additive fashion in skeletal muscle. However, it is still unclear whether they do so by inducing the recruitment of GLUT4 transporters from the same or distinct intracellular compartments to the plasma membrane and the T-tubules. Using the transferrin receptor as a recognized marker of recycling endosomes, we have examined whether insulin and/or contraction recruit GLUT4 from this pool to either the plasma membranes or T-tubules, isolated by subcellular fractionation of perfused hindlimb muscles. Either stimulus independently increased GLUT4 translocation from an intracellular fraction to both the plasma membrane and T-tubules. The combination of insulin and contraction induced a marked (approximately threefold) and almost fully additive increase in GLUT4 content, but only in the plasma membrane. Insulin did not stimulate transferrin receptor recruitment from the GLUT4-containing intracellular fraction to either the plasma membrane or the T-tubules. In contrast, contraction stimulated the recruitment of the transferrin receptor from the same GLUT4-containing intracellular fraction to the plasma membrane but not to the T-tubules. Contraction-induced recruitment of the transferrin receptor was also observed from immunopurified GLUT4 vesicles. It is concluded that muscle contraction stimulates translocation of GLUT4 from two distinct intracellular compartments: 1) a population of recycling endosomes that is selectively recruited to the plasma membrane and 2) from GLUT4 storage vesicles that are also insulin-responsive and recruited to both the plasma membrane and the T-tubules. The lack of additive translocation of GLUT4 to the T-tubules may be linked to the failure of GLUT4-containing recycling endosomes to be recruited to these structures.

Animals↗

PubMed1999

Biochemical localisation of the 5-HT2A (serotonin) receptor in rat skeletal muscle.

The 5-HT2A receptor was recently shown to localise morphologically to the transverse tubules (TT) in rat foetal myoblasts. Receptor activation enhanced the expression of genes involved in myogenesis, and its TT localisation has led to the suggestion that it may participate in excitation-contraction coupling. In order to gain further insights into 5-HT2A receptor function in muscle we have (i) investigated its biochemical localisation in adult rat skeletal muscle and (ii) determined whether receptor expression is dependent upon muscle type. Immunoblot analysis of muscle membranes, isolated by subcellular fractionation, revealed that adult muscle expresses the 5-HT2A receptor and that it resides exclusively in plasma membranes and not in TT. No differences in 5-HT2A abundance were observed between red and white muscle, suggesting that receptor expression does not correlate with the metabolic or contractile properties of the muscle fibre. Our data indicate that 5-HT2A expression in skeletal muscle is maintained into adulthood and that its absence from TT make it an unlikely participant in the excitation-contraction coupling process.

Animals↗

PubMed1999

Mechanism of adipose tissue iNOS induction in endotoxemia.

The aim of the present study was to investigate the mechanism of adipose tissue inducible nitric oxide synthase (iNOS) induction in endotoxemia. Systemic administration of the bacterial endotoxin lipopolysaccharide (LPS) to rats for </=8 h markedly increased iNOS mRNA and protein levels in white and brown adipose tissues. This effect was comparable to or greater than the induction of iNOS in liver, kidney, or skeletal muscle. iNOS activity was also found to be greatly enhanced in both white and brown adipose tissues of LPS-treated rats (an approximately 12- to 20-fold increase). Treatment of cultured 3T3-L1 adipocytes with LPS, tumor necrosis factor-alpha (TNF-alpha), or interferon-gamma (IFN-gamma) alone failed to induce iNOS activity. However, when used in combination, TNF-alpha, IFN-gamma, and LPS markedly and synergistically increased iNOS activity in these cells. In conclusion, these results suggest that adipose tissue is a major site of iNOS expression in endotoxemia. Our data further indicate that iNOS induction can be reproduced in vitro in cultured adipocytes and that a concerted action of cytokines and endotoxin is needed for maximal activation of the enzyme.

3T3 Cells↗

PubMed1999

Insulin-like growth factor I circumvents defective insulin action in human myotonic dystrophy skeletal muscle cells.

Primary human skeletal muscle cell cultures derived from muscles of a myotonic dystrophy (DM) fetus provided a model in which both resistance to insulin action described in DM patient muscles and the potential ability of insulin-like growth factor I (IGF-I) to circumvent this defect could be investigated. Basal glucose uptake was the same in cultured DM cells as in normal myotubes. In DM cells, a dose of 10 nM insulin produced no stimulatory effect on glucose uptake, and at higher concentrations, stimulation of glucose uptake remained significantly lower than that in normal myotubes. In addition, basal and insulin-mediated protein synthesis were both significantly reduced compared with those in normal cells. In DM myotubes, insulin receptor messenger RNA expression and insulin receptor binding were significantly diminished, whereas the expression of GLUT1 and GLUT4 glucose transporters was not affected. These results indicate that impaired insulin action is retained in DM cultured myotubes. The action of recombinant human IGF-I (rhIGF-I) was evaluated in this cellular model. We showed that rhIGF-I is able to stimulate glucose uptake to a similar extent as in control cells and restore normal protein synthesis level in DM myotubes. Thus, rhIGF-I is able to bypass impaired insulin action in DM myotubes. This provides a solid foundation for the eventual use of rhIGF-I as an effective treatment of muscle weakness and wasting in DM.

Cells, Cultured↗

PubMed1999

Glucose rapidly decreases plasma membrane GLUT4 content in rat skeletal muscle.

We have previously demonstrated that chronic hyperglycemia per se decreases GLUT4 glucose transporter expression and plasma membrane content in mildly streptozotocin- (STZ) diabetic rats (Biochem. J. 284, 341-348, 1992). In the present study, we investigated the effect of an acute rise in glycemia on muscle GLUT4 and GLUT1 protein contents in the plasma membrane, in the absence of insulin elevation. Four experimental groups of rats were analyzed in the postabsorptive state: 1. Control rats. 2. Hyperglycemic STZ-diabetic rats with moderately reduced fasting insulin levels. 3. STZ-diabetic rats made normoglycemic with phlorizin treatment. 4. Phlorizin-treated (normoglycemic) STZ-diabetic rats infused with glucose for 40 min. The uniqueness of the latter model is that glycemia can be rapidly raised without any concomitant increase in plasma insulin levels. Plasma membranes were isolated from hindlimb muscle and GLUT1 and GLUT4 proteins amounts determined by Western blot analysis. As predicted, STZ-diabetes caused a significant decrease in the abundance of GLUT4 in the isolated plasma membranes. Normalization of glycemia for 3 d with phlorizin treatment restored plasma membrane GLUT4 content in muscle of STZ-diabetic rats. A sudden rise in glycemia over a period of 40 min caused the GLUT4 levels in the plasma membrane fraction to decrease to those of nontreated STZ-diabetic rats. In contrast to the GLUT4 transporter, plasma membrane GLUT1 abundance was not changed by the acute glucose challenge. It is concluded that glucose can have regulatory effect by acutely reducing plasma membrane GLUT4 protein contents in rat skeletal muscle. We hypothesize that this glucose-induced downregulation of plasma membrane GLUT4 could represent a protective mechanism against excessive glucose uptake under hyperglycemic conditions accompanied by insulin resistance.

Animals↗

PubMed1998

Insulin inhibits inducible nitric oxide synthase in skeletal muscle cells.

Recent studies have shown that cytokines and endotoxins impair insulin-stimulated glucose transport by activating the expression of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) production in skeletal muscle cells. In this study, we investigated whether iNOS induction is modulated by insulin in L6 myocytes. Long term exposure of muscle cells to tumour necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS) greatly increased iNOS mRNA expression and NO production. Addition of insulin to the cytokine/LPS-treated muscle cells reduced (by approximately 40%) NO production. This inhibition was similar to that observed with the synthetic glucocorticoid dexamethasone, a known inhibitor of iNOS in several cell types. The combination of insulin and dexamethasone was more effective than either agent alone in reducing NO production. Dexamethasone greatly inhibited the effect of cytokines/LPS to induce cellular iNOS mRNA expression. In strong contrast, insulin failed to reduce iNOS mRNA expression under similar conditions. These results show that insulin is a novel inhibitor of iNOS-mediated NO production in skeletal muscle cells. Furthermore, our data indicate that unlike glucocorticoids, insulin does not inhibit NO production by suppression of iNOS gene transcription.

Animals↗

PubMed1998

Insulin stimulation of glucose uptake in skeletal muscles and adipose tissues in vivo is NO dependent.

The purpose of this study was to investigate whether in vivo nitric oxide synthase (NOS) inhibition influences insulin-mediated glucose disposal in rat peripheral tissues. The NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME) or saline was infused constantly during a hyperinsulinemic-euglycemic clamp in normal rats. Glucose utilization rates of insulin-sensitive tissues (individual muscles, heart, and adipose tissues) were simultaneously determined using tracer infusion of 2-deoxy-D-[3H]glucose (2-[3H]DG). NOS blockade with L-NAME resulted in significant (P < 0.05) reduction in both whole body glucose disposal (-16%, P < 0.01) and plasma 2-[3H]DG disappearance rate (-30%, P < 0.05) during hyper-insulinemic-euglycemic clamp. L-NAME significantly decreased insulin-stimulated glucose uptake in heart (-62%, P = 0.01), soleus (-42%, P = 0.05), red (-53%, P < 0.001) and white (-62%, P < 0.001) gastrocnemius, tibialis (-57%, P < 0.01), and quadriceps (-33%, P < 0.05) muscles. The NOS inhibitor also decreased insulin action in brown interscapular (-47%, P < 0.01), retroperitoneal (-52%, P = 0.07), and gonadal (-66%, P = 0.06) adipose tissues. In contrast to in vivo NOS blockade, L-NAME failed to affect basal or insulin-stimulated 2-[3H]DG transport in isolated soleus or extensor digitorum longus muscles in vitro. These results support the hypothesis that the action of insulin to augment glucose uptake by skeletal muscles and other peripheral insulin-sensitive tissues in vivo is NO dependent.

Adipose Tissue↗

PubMed1998

Selective impairment in GLUT4 translocation to transverse tubules in skeletal muscle of streptozotocin-induced diabetic rats.

We previously reported that insulin induces the translocation of GLUT4 to both the plasma membrane and the transverse tubules (T-tubules) in rat skeletal muscle (Am J Physiol 270:E667-E676, 1996). The aim of the present study was to investigate whether the insulin-resistant glucose utilization of skeletal muscle from streptozotocin (STZ)-induced diabetic rats is linked to an impaired translocation of GLUT4 to the plasma membrane, the T-tubules, or both surface compartments. Whole-body insulin-mediated glucose disposal, assessed during a hyperinsulinemic-euglycemic clamp, was reduced by 48% (P < 0.01) in diabetic rats as compared with controls. Subcellular membrane fractions enriched with plasma membranes, T-tubules, or GLUT4-enriched intracellular membranes were isolated from hindlimb muscles of control and insulin-stimulated rats, and GLUT4 content was measured by Western blot analysis. In the absence of insulin (unstimulated), GLUT4 content in muscle of diabetic rats was markedly lower (by approximately 40%) in both the T-tubules and the intracellular membrane fraction as compared with controls. In contrast, the transporter protein levels were similar in the plasma membrane fraction. In skeletal muscle of control animals, the hyperinsulinemic clamp induced GLUT4 translocation from the intracellular membrane pool to both the plasma membrane and the T-tubule-enriched fractions (approximately 2.2-fold to approximately 2.5-fold). Surprisingly, insulin increased plasma membrane GLUT4 content to comparable levels in control and diabetic rat skeletal muscle. However, insulin-mediated GLUT4 translocation to the T-tubules was significantly reduced in the same muscle. Whole-body insulin action was significantly correlated with GLUT4 protein levels in the T-tubules, but not with the transporter content in either plasma membranes or intracellular membranes. These results strongly suggest that peripheral resistance to insulin action on glucose disposal in STZ-induced diabetic rats is caused by a selective impairment of GLUT4 translocation to skeletal muscle T-tubules.

Animals↗

PubMed1997

Cytokines modulate glucose transport in skeletal muscle by inducing the expression of inducible nitric oxide synthase.

The principal goal of the present study was to test the hypothesis that cytokines modulate glucose transport in skeletal muscle by increasing nitric oxide production. Cultured L6 skeletal muscle cells were incubated in the presence of tumour necrosis factor-alpha, interferon-gamma or lipopolysaccharide (LPS) alone or in combination for 24 h. Neither cytokines nor LPS alone induced NO production, as measured by nitrite concentrations in the medium. However, when used in combination, the two cytokines significantly stimulated NO production, and this effect was synergistically enhanced by the presence of LPS. Reverse transcriptase-PCR (RT-PCR) analysis revealed that NO release was associated with the induction of inducible (macrophage-type) NO synthase (iNOS). The increase in iNOS expression was confirmed at the protein level by Western-blot analysis and NADPH/diaphorase histochemical staining. Cytokines and LPS markedly increased basal glucose transport in L6 myocytes. Insulin also stimulated basal glucose transport, but significantly less in cells chronically exposed to cytokines/LPS. The sensitivity of L6 muscle cells to insulin-stimulated glucose transport was also significantly decreased by cytokines/LPS treatment. The NOS inhibitor NG-nitro-l-arginine methyl ester (l-NAME) inhibited nitrite production in cytokine/LPS-treated cells, and this prevented the increase in basal glucose transport and restored muscle cell responsiveness to insulin. Cytokines/LPS exposure significantly increased GLUT1 transporter protein levels but decreased GLUT4 expression in L6 cells. l-NAME treatment prevented the increase in GLUT1 protein content but failed to restore GLUT4 transporter levels. These results demonstrate that cytokines and LPS affect glucose transport and insulin action by inducing iNOS expression and NO production in skeletal muscle cells. The data further indicate that cytokines and LPS increase the expression of the GLUT1 transporter protein by an NO-dependent mechanism.

Animals↗

PubMed1997

Regional variation in adipose tissue insulin action and GLUT4 glucose transporter expression in severely obese premenopausal women.

Insulin action and GLUT4 expression were examined in adipose tissue of severely obese premenopausal women undergoing gastrointestinal surgery. Fat samples were taken from three different anatomical regions: the subcutaneous abdominal site, the round ligament (deep abdominal properitoneal fat), and the greater omentum (deep abdominal intraperitoneal fat). The stimulatory effect of insulin on glucose transport and the ability of the hormone to inhibit lipolysis were determined in adipocytes isolated from these three adipose depots. Insulin stimulated glucose transport 2-3 times over basal rates in all adipocytes. However, round ligament adipose cells showed a significantly greater responsiveness to insulin when compared to subcutaneous and omental adipocytes. Round ligament fat cells also displayed the greatest sensitivity and maximal antilipolytic response to insulin. We also investigated whether regional differences in fat cell insulin-stimulated glucose transport were linked to a differential expression of the GLUT4 glucose transporter. GLUT4 protein content in total membranes was 5 and 2.2 times greater in round ligament adipose tissue than in subcutaneous and omental fat depots, respectively. Moreover, GLUT4 mRNA levels were 2.1 and 3 times higher in round ligament than in subcutaneous or omental adipose tissues, respectively. Adipose tissue GLUT4 protein content was strongly and negatively associated (r = -0.79 to -0.89, p < 0.01) with the waist-to-hip ratio but not with total adiposity. In conclusion, these results demonstrate the existence of site differences in adipose tissue insulin action in morbidly obese women. The greater insulin effect on glucose transport in round ligament adipocytes was associated with a higher expression of GLUT4 when compared to subcutaneous abdominal and omental fat cells. Moreover, despite the regional variation in GLUT4 expression, an increased proportion of abdominal fat was found to be associated with lower levels of GLUT4 in all adipose regions investigated.

Adipocytes↗

PubMed1997

Electrical stimulation induces fiber type-specific translocation of GLUT-4 to T tubules in skeletal muscle.

Insulin and contraction independently stimulate glucose transport in skeletal muscle. Whereas insulin activates glucose transport more in muscles composed of type I and IIa fibers, electrical stimulation increases glucose transport at least as much in type IIb fiber-enriched muscles despite the fact that the latter fiber type contains less GLUT-4 glucose transporters. The aim of the present study was to test the hypothesis that a greater GLUT-4 translocation to the cell surface may underlie the higher contraction-stimulated glucose transport in type IIb myofibers. Leg muscles from rats were stimulated in situ at 100 Hz (200 ms) each 2 s via the sciatic nerve over a period of 20 min while the contralateral leg was kept at rest. Muscle 2-[3H]deoxy-D-glucose uptake (2-DG) was measured in separated red gastrocnemius (RG, type I and IIa fibers) and white gastrocnemius (WG, type IIb fibers) muscles. Resting 2-DG uptake was greater in RG than WG. Electrical stimulation increased 2-DG uptake over resting values similarly in WG and RG. Fractions enriched with either plasma membranes, transverse (T) tubules, triads, or GLUT-4-enriched intracellular membranes were isolated from RG and WG using a recently developed subcellular fractionation procedure. Electrical stimulation similarly increased GLUT-4 protein content in plasma membranes of RG and WG, whereas it stimulated GLUT-4 translocation more (approximately 50%) in T tubules of WG than in RG. GLUT-4 content was not changed in triads of both muscle types. The increments in cell surface GLUT-4 protein levels were paralleled by significant reductions in the amount of the transporter in the intracellular membrane fractions of both muscle types (by 60% in RG and 56% in WG). It is concluded that electrically induced contraction stimulates GLUT-4 translocation more in T tubules of WG than RG. The physiological implications of this finding for glucose uptake by contracting RG and WG muscles is discussed.

Animals↗

PubMed1997

Expression of nitric oxide synthase in skeletal muscle: a novel role for nitric oxide as a modulator of insulin action.

Previous studies have shown that nitric oxide synthase (NOS), the enzyme that catalyzes the formation of nitric oxide (NO), is expressed in skeletal muscle. The aim of the present study was to test the hypothesis that NO can modulate glucose metabolism in slow- and fast-twitch skeletal muscles. Calcium-dependent NOS was detected in skeletal muscle, and the enzyme activity was greater in fast-type extensor digitorum longus (EDL) muscles than in slow-type soleus muscles. Both the neuronal-type (nNOS) and endothelial-type (eNOS) enzymes are expressed in resting skeletal muscles. However, nNOS protein was only detected in EDL muscles, whereas eNOS protein contents were comparable in soleus and EDL muscles. NOS expression in muscle cryosections (diaphorase histochemistry) was located in vascular endothelium and in muscle fibers, and the staining was greater in type IIb than in type I and IIa fibers. The macrophage-type inducible NOS (iNOS) was not detected in resting muscle, but endotoxin treatment induced its expression, concomitant with elevated NO production. iNOS induction was associated with impaired insulin-stimulated glucose uptake in isolated rat muscles. In vitro, NOS blockade with specific inhibitors did not affect basal or insulin-stimulated glucose transport in EDL or soleus muscles. In contrast, the NO donors GEA 5024 and sodium nitroprusside induced dose-dependent inhibition (up to 50%) of maximal insulin-stimulated glucose transport in both muscles with minor effects on basal uptake values. GEA 5024 also blunted insulin-stimulated glucose transport and amino acid uptake in cultured L6 muscle cells without affecting insulin binding to its receptor. On the other hand, the permeable cGMP analogue dibutyryl cGMP did not affect muscle glucose transport. These results strongly suggest that NO modulates insulin action in both slow- and fast-type skeletal muscles. This novel autocrine action of NO in muscle appears to be mediated by cGMP-independent pathways.

Amino Acids↗

PubMed1996

Exercise induces the translocation of GLUT4 to transverse tubules from an intracellular pool in rat skeletal muscle.

Exercise stimulates glucose transport in skeletal muscle by increasing the number of GLUT4 glucose transporters at the cell surface. The effect of exercise on GLUT4 content in transverse tubules, which are deep extensions of the plasma membrane involved in myofiber contraction, has never been studied. In the present study, we have investigated whether acute exercise induces translocation of GLUT4 to the transverse tubules. Plasma membranes and transverse tubules were isolated from control and exercised rats by a newly developed membrane fractionation procedure. As expected, acute exercise increased GLUT4 content in plasma membranes (95%; p < 0.01). Importantly, exercise also significantly increased GLUT4 content in a transverse tubule-enriched fraction (60%; p < 0.05). Furthermore, acute exercise was found to decrease GLUT4 content in an intracellular membrane fraction (-40%; p < 0.001). In conclusion, this study demonstrates that GLUT4 is translocated not only to the plasma membranes but also to the transverse tubules in acutely exercised muscles. The results also indicate that GLUT4 is recruited from a novel intracellular membrane fraction.

5'-Nucleotidase↗

PubMed1996

Insulin-induced translocation of Na+-K+-ATPase subunits to the plasma membrane is muscle fiber type specific.

We have previously shown that an acute insulin treatment induces redistribution of the alpha 2- and beta 1- isoforms of the Na+-K+-ATPase from intracellular membranes to plasma membranes detected on subcellular fractionation of mixed muscles and immunoblotting with isoform-specific antibodies (H. S. Hundal et al. J. Biol. Chem. 267: 5040-5043, 1992). In the present study we give both biochemical and morphological evidence that this insulin effect is operative in muscles composed mostly of oxidative (red) fibers but not in muscles composed mostly of glycolytic (white) fibers. The redistribution of the Na+-K+-ATPase alpha 2- and beta 1-isoforms after insulin injection was detected in membranes isolated from and muscles (soleus, red gastrocnemius, red rectus femoris, and red vastus lateralis) but not in membranes from white muscles (white gastrocnemius, tensor fasciae latae, white rectus femoris, and white vastus lateralis). After insulin injection, the potassium-dependent 3-O-methylfluorescein phosphatase activity of the enzyme was higher by 22% in the plasma membrane-enriched fraction and lower by 15% in the internal membrane fraction isolated from red but not from white muscles. Quantitative immunoelectron microscopy of ultrathin muscle cryosections showed that in vivo insulin stimulation augmented the density of Na+-K+-ATPase alpha 2- and beta 1- isoforms at the plasma membrane of soleus muscle by 80 and 124%, respectively, with no change in white gastrocnemius muscle. The effect of insulin to increase the content of Na+-K+-ATPase alpha 2- and beta 1-subunits in isolated plasma membranes was still observed when glycemia was prevented from dropping by using hyperinsulinemic-euglycemic clamps. We conclude that the insulin-induced redistribution of the alpha 2- and beta 1-isoforms of the Na+-K+-ATPase from an intracellular pool to the plasma membrane in restricted to oxidative fiber-type skeletal muscles. This may be related to the selective expression of beta 1-subunits in these fibers and implies that the beta 2-subunit, typical of glycolytic muscles, does not sustain translocation of alpha 2 beta 2-complexes.

Animals↗

PubMed1996

A new procedure for the isolation of plasma membranes, T tubules, and internal membranes from skeletal muscle.

A new subcellular fractionation procedure for the simultaneous isolation of plasma membranes and transverse (T) tubule membranes from a rat skeletal muscle was developed. This new technique allows the isolation and separation of plasma membranes and T tubules in distinct subcellular fractions, as revealed by the membrane distribution of enzymatic and immunologic markers of both cell surface compartments. The procedure also yields a novel membrane fraction that is devoid of markers of both surface domains but is markedly enriched with GLUT-4 glucose transporters, thus strongly suggesting that it represents an intracellular pool of GLUT-4. Using this new procedure, we found that acute in vivo insulin administration (30 min) increased GLUT-4 protein content in the plasma membrane and a T tubule fraction (by approximately 80%), whereas a smaller elevation (35%) was observed in another fraction enriched with T tubules. Insulin induced a concomitant reduction (approximately 40%) in GLUT-4 abundance in the intracellular fraction. These results further support the hypothesis that T tubules are involved in the regulation of glucose transport in skeletal muscle. This novel fractionation method will be useful in investigating the regulation of muscle GLUT-4 transporters in other physiological and disease states such as diabetes, where defective translocation of the transporter protein to either one or both cell surface domains is suspected to occur.

Animals↗

PubMed1995

Marked depletion of GLUT4 glucose transporters in transverse tubules of skeletal muscle from streptozotocin-induced diabetic rats.

The principal goal of the present study was to determine the subcellular content of GLUT4 in diabetic rat muscle, and to test the hypothesis that a reduced abundance of the transporter protein in transverse tubules is responsible for impaired glucose utilization in that tissue. GLUT4 protein levels were measured in hindlimb muscle homogenates as well as in subcellular membrane fractions enriched with either plasma membranes, transverse tubules, or GLUT4-containing intracellular membranes from control and diabetic (streptozotocin-induced) rats. GLUT4 protein contents in diabetic muscle homogenates was reduced by 30% as compared to control rats. Subcellular fractionation experiments revealed that GLUT4 contents in transverse tubules-enriched fractions was markedly decreased (by 55-60%) in skeletal muscle of diabetic animals whereas no significant reductions in GLUT4 abundance was observed in the plasma membrane fraction. Moreover, GLUT4 was markedly depleted (by 45%) in the GLUT4-enriched intracellular membrane fraction. These results indicate that GLUT4 is markedly depleted in both the intracellular pool and in the cell surface membranes in muscle of STZ-diabetic rats. Most strikingly, this study demonstrates that transverse tubules and not the plasma membrane are the main sites of cell surface GLUT4 depletion in diabetic muscle.

Animals↗

PubMed1995

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.

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.

Animals↗