scholarly journals Partial purification and reconstitution of the sarcolemmal l-lactate carrier from rat skeletal muscle

1994 ◽  
Vol 303 (1) ◽  
pp. 207-212 ◽  
Author(s):  
P J Allen ◽  
G A Brooks
Keyword(s):  
Skeletal Muscle ◽  
Sodium Phosphate ◽  
Partial Purification ◽  
Transport Activity ◽  
Rat Skeletal Muscle ◽  
Lactate Transport ◽  
Hindlimb Muscle ◽  
Sds Page ◽  
Transport Analysis ◽  
Lactate Exchange

Purified sarcolemmal membranes from mixed rat hindlimb muscle were solubilized with octylglucoside and the extract subjected to hydroxylapatite (HA) chromatography. Following protein elution with a sodium phosphate gradient and detergent removal by dialysis, the HA eluate was reconstituted into asolectin liposomes using a freeze-thaw procedure. Specific L-[14C]lactate transport activity eluting from the 0.2 M sodium phosphate fraction was 30-fold higher compared with native sarcolemmal vesicles (31.64 versus 1.06 nmol/min per mg). The reconstituted carrier exhibited Michaelis-Menten saturation kinetics with Km and Vmax. values of 46.2 +/- 6.6 mM and 498.7 +/- 17.2 nmol/15 s per mg respectively. L-Lactate transport activity was inhibited 57% by preincubation of proteoliposomes with 10 mM alpha-cyano-4-hydroxycinnamate, a known inhibitor of lactate transport. Analysis of the HA eluates by SDS/PAGE showed the presence of a 34 kDa band corresponding to lactate transport activity. Reconstitution of lactate transport activity eluting from the HA column, together with SDS/PAGE analysis suggests the presence of a 34 kDa polypeptide mediating sarcolemmal lactate exchange in rat skeletal muscle.

L(+)-Lactate Binding to a Protein in Rat Skeletal Muscle Plasma Membranes

1995 ◽  
Vol 20 (1) ◽  
pp. 112-124 ◽  
Author(s):  
Karl J. A. McCullagh ◽  
Arend Bonen
Keyword(s):  
Skeletal Muscle ◽  
Plasma Membranes ◽  
Mass Range ◽  
Dependent Manner ◽  
Rat Skeletal Muscle ◽  
Lactate Transport ◽  
Sds Page ◽  
Lactate And Pyruvate ◽  
Potential Inhibitors ◽  
Dose Dependent

Biochemical studies were conducted to determine the location of a putative lactate transport protein in rat skeletal muscle plasma membranes (PM). PM (50-100 μg protein) were incubated with [U-14C] L(+)-lactate, in the presence or absence of unlabeled monocarboxylates or potential inhibitors, after which proteins were separated by SDS-PAGE. Gel slices (2 mm) were cut and analyzed for14C. [U-14C] L(+)-lactate was bound to plasma membranes in the 30 to 40 kDa molecular mass range. Binding of [U-14C] L(+)-lactate was inhibited by N-ethylmaleimide, unlabeled L-lactate and pyruvate, and in a dose dependent manner by α-cyano-4-hydroxycinnamate (r = 0.995), but not by cytochalasin-B. The inhibition of [U-14C] L(+)-lactate binding was similar to the inhibition of lactate transport. Therefore the transport of L(+)-lactate across skeletal muscle plasma membranes involves a polypeptide of 30 to 40 kDa. Key words: transport, affinity labeling

scholarly journals Lactate transport activity in rat skeletal muscle sarcolemmal vesicles after acute exhaustive exercise

1999 ◽  
Vol 87 (3) ◽  
pp. 955-961 ◽  
Author(s):  
H. Dubouchaud ◽  
N. Eydoux ◽  
P. Granier ◽  
C. Préfaut ◽  
J. Mercier
Keyword(s):  
Skeletal Muscle ◽  
Time Course ◽  
Lactate Concentration ◽  
Treadmill Running ◽  
Exhaustive Exercise ◽  
Transport Activity ◽  
Rat Skeletal Muscle ◽  
Lactate Transport ◽  
Single Bout ◽  
Lactate Uptake

The effect of a single bout of exhaustive exercise on muscle lactate transport capacity was studied in rat skeletal muscle sarcolemmal (SL) vesicles. Rats were assigned to a control (C) group ( n = 14) or an acutely exercised (E) group ( n = 20). Exercise consisted of treadmill running (25 m/min, 10% grade) to exhaustion. SL vesicles purified from C and E rats were sealed because of sensitivity to osmotic forces. The time course of 1 mM lactate uptake in zero- trans conditions showed that the equilibrium level in the E group was significantly lower than in the C group ( P < 0.05). The initial rate of 1 mM lactate uptake decreased significantly from 2.44 ± 0.22 to 1.03 ± 0.08 nmol ⋅ min−1 ⋅ mg protein−1( P < 0.05) after exercise, whereas that of 50 mM lactate uptake did not differ significantly between the two groups. For 100 mM external lactate concentration ([lactate]), exhaustive exercise increased initial rates of lactate uptake (219.6 ± 36.3 to 465.4 ± 80.2 nmol ⋅ min−1 ⋅ mg protein−1, P < 0.05). Although saturation kinetics were observed in the C group with a maximal transport velocity of 233 nmol ⋅ min−1 ⋅ mg protein−1 and a Michealis-Menten constant of 24.5 mM, saturation properties were not seen after exhaustive exercise in the E group, because initial rates of lactate uptake increased linearly with external [lactate]. We conclude that a single bout of exhaustive exercise significantly modified SL lactate transport activity, resulting in a decrease in 1 mM lactate uptake and was associated with alterations in the saturable properties at [lactate] above 50 mM. These results suggest that changes in sarcolemmal lactate transport activity may alter lactate and proton exchanges after exhaustive exercise.

Effects of phenylarsine oxide on stimulation of glucose transport in rat skeletal muscle

1990 ◽  
Vol 258 (4) ◽  
pp. C648-C653 ◽  
Author(s):  
E. J. Henriksen ◽  
J. O. Holloszy
Keyword(s):  
Skeletal Muscle ◽  
Glucose Transport ◽  
Cytochalasin B ◽  
Contractile Activity ◽  
Transport Activity ◽  
Rat Skeletal Muscle ◽  
Phenylarsine Oxide ◽  
Muscle Glucose Transport ◽  
Glucose Analogue ◽  
Stimulation Of

The trivalent arsenical phenylarsine oxide (PAO) inhibits insulin-stimulated glucose transport in adipocytes and skeletal muscle through direct interactions with vicinal sulfhydryls. In muscle, glucose transport is also activated by contractile activity and hypoxia. It was therefore the purpose of the present study to investigate whether vicinal sulfhydryls are involved in the stimulation of glucose transport activity in the isolated rat epitrochlearis muscle by hypoxia or contractions. PAO (greater than 5 microM) caused a twofold increase in rate of transport of the nonmetabolizable glucose analogue 3-O-methylglucose (3-MG) that was completely prevented by cytochalasin B, the vicinal dithiol dimercaptopropanol, dantrolene, or 9-aminoacridine, both inhibitors of sarcoplasmic reticulum Ca2+ release, or omission of extracellular Ca2+. Although PAO treatment (greater than or equal to 20 microM) prevented approximately 80% of the increase in 3-MG transport caused by insulin, it resulted in only a approximately 50% inhibition of the stimulation of 3-MG transport by either hypoxia or contractile activity. PAO treatment (40 microM) of muscles already maximally stimulated by insulin, contractile activity, or hypoxia did not reverse the enhanced rate of 3-MG transport. These data suggest that vicinal sulfhydryls play a greater role in the activation of glucose transport by insulin than by muscle contractions or hypoxia. The finding that PAO inhibits the stimulation of glucose transport, but does not affect glucose transport after it has been stimulated, provides evidence that vicinal sulfhydryls are involved in the pathways for glucose transport activation in muscle, but not in the glucose transport mechanism itself.

scholarly journals Effect of 2-chloropropionate on initial lactate uptake by rat skeletal muscle sarcolemmal vesicles

1996 ◽  
Vol 81 (5) ◽  
pp. 1973-1977 ◽  
Author(s):  
P. Granier ◽  
H. Dubouchaud ◽  
N. Eydoux ◽  
J. Mercier ◽  
C. Préfaut
Keyword(s):  
Skeletal Muscle ◽  
Initial Rate ◽  
Lactate Concentration ◽  
Blood Lactate Concentration ◽  
Membrane Vesicles ◽  
Rat Skeletal Muscle ◽  
Lactate Transport ◽  
Sarcolemmal Membrane ◽  
Lactate Uptake ◽  
Sarcolemmal Vesicles

Granier, P., H. Dubouchaud, N. Eydoux, J. Mercier, and C. Préfaut. Effect of 2-chloropropionate on initial lactate uptake by rat skeletal muscle sarcolemmal vesicles. J. Appl. Physiol. 81(5): 1973–1977, 1996.—2-Chloropropionate (2-CP) is a halogenated monocarboxylic acid generally used to decrease blood lactate concentration in various metabolic states. To investigate whether it has an inhibitory effect on sarcolemmal lactate transport, we compared the initial rate of lactate transport in sarcolemmal membrane vesicles purified from 20 male Wistar rats with and without 2-CP. Transport by these vesicles was measured as uptake ofl-(+)-[U-14C]lactate under pH gradient-stimulated cisinhibition. The time courses of 1 mMl-(+)-lactate uptake into vesicles both with and without 10 mM 2-CP (l- ord-) displayed saturation kinetics. Lactate uptake values were lower with 10 mMl-2-CP and 10 mMd-2-CP in comparison to the control values. Both 10 mMl-2-CP and 10 mMd-2-CP significantly inhibited 1 mM l-(+)-lactate uptake (55.8 ± 9.1 and 53.5 ± 12.1%, respectively; P < 0.001), whereas a smaller inhibition was observed with a higher lactate concentration of 50 mM (40.2 ± 11.2 and 38.7 ± 12.4%; P < 0.001 and P < 0.05, respectively). However, a higher d-2-CP concentration (50 mM) increased the inhibition of pH-stimulated 1 mMl-(+)-lactate uptake (77.0 ± 9.4%; P < 0.001).d-2-CP had a trans-stimulation effect on the initial rate of lactate efflux of 1 mMl-(+)-lactate compared with baseline efflux (9.5 ± 0.8 vs. 5.1 ± 0.4 nmol ⋅ min−1 ⋅ mg protein−1; P < 0.05). 2-CP significantly inhibited the initial rate of lactate uptake in skeletal muscle sarcolemmal membrane vesicles. This result suggests that 2-CP is a nonstereoselective substrate of the lactate muscle carrier that impairs lactate transport.

T3 increases lactate transport and the expression of MCT4, but not MCT1, in rat skeletal muscle

2003 ◽  
Vol 285 (3) ◽  
pp. E622-E628 ◽  
Author(s):  
Yuxiang Wang ◽  
Mio Tonouchi ◽  
Dragana Miskovic ◽  
Hideo Hatta ◽  
Arend Bonen
Keyword(s):  
Skeletal Muscle ◽  
Protein Expression ◽  
Gastrocnemius Muscle ◽  
Muscle Metabolism ◽  
Monocarboxylate Transporters ◽  
Rat Skeletal Muscle ◽  
Lactate Transport ◽  
Expression Of Genes ◽  
Sarcolemmal Vesicles ◽  
White Gastrocnemius

Triiodothyronine (T3) regulates the expression of genes involved in muscle metabolism. Therefore, we examined the effects of a 7-day T3 treatment on the monocarboxylate transporters (MCT)1 and MCT4 in heart and in red (RG) and white gastrocnemius muscle (WG). We also examined rates of lactate transport into giant sarcolemmal vesicles and the plasmalemmal MCT1 and MCT4 in these vesicles. Ingestion of T3 markedly increased circulating serum T3 ( P < 0.05) and reduced weight gain ( P < 0.05). T3 upregulated MCT1 mRNA (RG +77, WG +49, heart +114%, P < 0.05) and MCT4 mRNA (RG +300, WG +40%). However, only MCT4 protein expression was increased (RG +43, WG +49%), not MCT1 protein expression. No changes in MCT1 protein were observed in any tissue. T3 treatment doubled the rate of lactate transport when vesicles were exposed to 1 mM lactate ( P < 0.05). However, plasmalemmal MCT4 was only modestly increased (+13%, P < 0.05). We conclude that T3 1) regulates MCT4, but not MCT1, protein expression and 2) increases lactate transport rates. This latter effect is difficult to explain by the modest changes in plasmalemmal MCT4. We speculate that either the activity of sarcolemmal MCTs has been altered or else other MCTs in muscle may have been upregulated.

scholarly journals Insulin stimulation of glucose transport activity in rat skeletal muscle: increase in cell surface GLUT4 as assessed by photolabelling

1994 ◽  
Vol 299 (3) ◽  
pp. 755-759 ◽  
Author(s):  
C M Wilson ◽  
S W Cushman
Keyword(s):  
Skeletal Muscle ◽  
Cell Surface ◽  
Glucose Transport ◽  
Fold Increase ◽  
Transport Activity ◽  
Insulin Stimulation ◽  
Rat Skeletal Muscle ◽  
Photoaffinity Label ◽  
Isolated Rat ◽  
Stimulation Of

We have used a photoaffinity label to quantify cell surface GLUT4 glucose transporters in isolated rat soleus muscles. In this system, insulin stimulated an 8.6-fold increase in 3-O-methylglucose glucose transport, while photolabelled GLUT4 increased 8-fold. These results demonstrate that the insulin-stimulated increase in glucose transport activity in skeletal muscle can be accounted for by an increase in surface-accessible GLUT4 content.

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