RESUMO
The enzyme glutamine:fructose 6-phosphate amidotransferase (L-glutamine:D-fructose-6-phosphate amidotransferase; EC 2.6.1.16, GFAT) catalyzes the formation of glucosamine 6-phosphate from fructose 6-phosphate and glutamine. In view of the important role of GFAT in the hexosamine biosynthetic pathway, we have purified the enzyme from rat liver and characterized its physicochemical properties in comparison to those from the published microbial enzymes. The purified enzyme has a molecular mass of about 75 kDa as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. On a Sephacryl S-200 gel filtration column, the purified enzyme eluted in a single peak corresponding to a molecular mass of about 280 kDa, indicating that the active enzyme may be composed of four subunits. The N-terminal amino acid sequence of the purified enzyme was determined as X-G-I-F-A-Y-L-N-Y-H-X-P-R, where X indicates an unidentified residue. The K(M) values of the purified enzyme for fructose 6-phosphate and glutamine were 0.4 and 0.8 mM, respectively. The purified enzyme was inactivated by 4, 4'-dithiodipyridine, and the activity of the inactivated enzyme was restored by dithiothreitol. The inactivation followed pseudo first-order and saturation kinetics with the K(inact) of 5.0 microM. Kinetic studies also indicated that 4,4'-dithiodipyridine is a competitive inhibitor of the enzyme with respect to glutamine. Isolation and analysis of the cysteine-modified peptide indicated that Cys-1 was the modified site. Cys-1 has been suggested to play an important role in enzymatic activity of the Escherichia coli enzyme (M. N. Isupov, G. Obmolova, S. Butterworth, M. Badet-Denisot, B. Badet, I. Polikarpov, J. A. Littlechild, and A. Teplyakov, 1996, Structure 4, 801-810).
Assuntos
Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/isolamento & purificação , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/metabolismo , Fígado/enzimologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Dissulfetos/farmacologia , Ditiotreitol/farmacologia , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/antagonistas & inibidores , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/química , Humanos , Concentração de Íons de Hidrogênio , Cinética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Piridinas/farmacologia , Ratos , Ratos Sprague-Dawley , Alinhamento de Sequência , Análise de Sequência de Proteína , Homologia de Sequência de Aminoácidos , Relação Estrutura-AtividadeRESUMO
Insulin resistance can be induced in vivo by intravenous infusion of glucosamine or in cells by incubation with glucosamine. However, a publication (Hresko, R. C., et al. (1998) J. Biol. Chem. 273, 20658-20668) suggests a trivial explanation of glucosamine-induced insulin resistance whereby intracellular ATP pools are depleted presumably due to the phosphorylation of glucosamine to glucosamine 6-phosphate, a hexosamine pathway intermediate. The reduced ATP level impaired insulin receptor (IR) autophosphorylation and tyrosine kinase activity toward substrates. The present work describes the development and comparison of two methods for inducing insulin resistance, by treating 3T3-L1 adipocytes overnight using either 25 mM glucose/5 nM insulin or 2 mM glucosamine. Under these conditions basal glucose transport rates were comparable with controls. Insulin-stimulated 2-deoxyglucose uptake, however, was reduced by approximately 45% in response to both high glucose/insulin and glucosamine treatment, relative to control cells. The total relative amounts of the insulin-responsive glucose transporter, Glut4, remained constant under both treatment conditions. The relative phosphotyrosine (Tyr(P)) contents of the insulin receptor and its substrate 1 (IRS-1) were assessed in whole cell homogenates. With both methods to induce insulin resistance, IR/IRS-1 Tyr(P) levels were virtually indistinguishable from those in control cells. Insulin-stimulated phosphorylation of Akt on Ser(473) was not impaired in insulin-resistant cells. Furthermore, the relative Tyr(P) content of the PDGF receptor was comparable in high glucose/insulin- or glucosamine-treated 3T3-L1 adipocytes upon subsequent challenge with PDGF. Finally, the relative amounts of glutamine:fructose-6-phosphate amidotransferase and O-linked N-acetylglucosamine transferase, two important hexosamine pathway enzymes, were similar in both treatments when compared with controls. Thus, 3T3-L1 adipocytes can be used as a model system for studying insulin resistance induced by increased influx of glucose. Under appropriate experimental conditions, glucosamine treatment can mimic the effects of increased glucose flux without impairment of tyrosine phosphorylation-based signaling.
Assuntos
Adipócitos/metabolismo , Glucosamina/farmacologia , Glucose/metabolismo , Resistência à Insulina , Proteínas Musculares , Células 3T3 , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Transporte Biológico , Transportador de Glucose Tipo 4 , Insulina/farmacologia , Cinética , Camundongos , Modelos Biológicos , Dados de Sequência Molecular , Proteínas de Transporte de Monossacarídeos/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais , Transaminases/metabolismo , Tirosina/metabolismoRESUMO
Glutamine:fructose-6-phosphate amidotransferase (GFA) is the rate-limiting enzyme in hexosamine biosynthesis, an important pathway for cellular glucose sensing. Human GFA has two potential sites for phosphorylation by cAMP-dependent protein kinase A (PKA). To test whether GFA activity is regulated by cAMP-dependent phosphorylation, rat aortic smooth muscle cells were treated in vivo with cAMP-elevating agents, 10 micromol/l forskolin, 1 mmol/l 8-Br-cAMP, or 3-isobutyl-1-methylxanthine. All treatments resulted in rapid and significant increases (2- to 2.4-fold) in GFA activity assayed in cytosolic extracts. Maximal effects of forskolin were observed at 10 micromol/l and 60 min. Preincubation of cells with cycloheximide did not abolish the effect of forskolin. Incubation of cytosolic extracts at 37 degrees C for 10 min in a buffer without phosphatase inhibitors led to a 79% decrease of GFA activity. This loss of activity was inhibited by the addition of phosphatase inhibitors (5 mmol/l sodium orthovanadate, 50 mmol/l sodium fluoride, or 5 mmol/l EDTA, but not 100 nmol/l okadaic acid), suggesting that GFA undergoes rapid dephosphorylation by endogenous phosphatases. Purified GFA is phosphorylated in vitro by purified PKA, resulting in a 1.7-fold increase in GFA activity. Treatment of GFA with purified protein kinase C had no effect. We conclude that GFA activity may be modulated by cAMP-dependent phosphorylation.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , AMP Cíclico/metabolismo , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/metabolismo , Adenilil Ciclases/efeitos dos fármacos , Adenilil Ciclases/metabolismo , Sequência de Aminoácidos , Animais , Sistema Livre de Células/efeitos dos fármacos , Sistema Livre de Células/enzimologia , Células Cultivadas , Colforsina/farmacologia , AMP Cíclico/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/farmacologia , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/efeitos dos fármacos , Dados de Sequência Molecular , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Fosforilação/efeitos dos fármacos , Ratos , Homologia de Sequência de AminoácidosRESUMO
Lithium has been shown to increase glucose uptake in skeletal muscle and adipose tissues. The therapeutic effect of lithium on bipolar disease is thought to be mediated by its inhibitory effect on myo-inositol-1-monophosphatase (IMPase). We tested the hypothesis that the stimulatory effect of lithium on glucose uptake results from inhibition of IMPase and the resultant accumulation of inositol monophosphates (IP1) by comparing the effects of lithium and a selective IMPase inhibitor, L-690,488, on isolated rat adipocytes. Insulin produced a concentration-dependent stimulation of 2-deoxy-D-[14C]glucose (2-DG) transport (10 microU/ml caused half-maximal activation). Acute exposure to lithium stimulated basal glucose transport activity in a concentration-dependent manner, with a threefold stimulation at 30 mM lithium. Lithium also potentiated insulin-stimulated 2-DG transport. Lithium produced a concomitant increase in IP1 accumulation. In contrast, L-690,488 increased IP1 to levels comparable to those of lithium without stimulatory effects on 2-DG transport. These results demonstrate that stimulatory effects of lithium on glucose transport are not mediated by the inhibition of IMPase and subsequent accumulation of IP1 in rat adipocytes.
Assuntos
Adipócitos/metabolismo , Desoxiglucose/metabolismo , Glucose/metabolismo , Inosina Monofosfato/metabolismo , Cloreto de Lítio/farmacologia , Adipócitos/efeitos dos fármacos , Tecido Adiposo/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Células Cultivadas , Difosfonatos/farmacologia , Inibidores Enzimáticos/farmacologia , Epididimo , Inositol/metabolismo , Insulina/farmacologia , Cinética , Masculino , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Monoéster Fosfórico Hidrolases/metabolismo , Pró-Fármacos/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
It has been hypothesized that increased production of tumor necrosis factor-alpha (TNF-alpha) plays a role in causing the insulin resistance associated with obesity. Obesity with insulin resistance is associated with increased production of TNF-alpha by fat cells. Exposure of 3T3-L1 adipocytes to TNF-alpha for 3-4 days makes them insulin resistant. TNF-alpha has also been reported to rapidly (15-60 min) cause insulin resistance, with a decrease in insulin-stimulated tyrosine phosphorylation, in a number of cultured cell lines. Because skeletal muscle is the major tissue responsible for insulin-stimulated glucose disposal, we performed the present study to determine if acute exposure to TNF-alpha causes insulin resistance in muscle. We found that exposure of soleus muscles to 6 nmol/l TNF-alpha for 45 min in vitro had no inhibitory effect on insulin-stimulated tyrosine phosphorylation of the insulin receptor or insulin receptor substrate 1 (IRS-1) or on phosphatidylinositol 3-kinase association with IRS-1. Incubation of epitrochlearis and soleus muscles with 6 nmol/l TNF-alpha for 45 min or 4 h had no effect on insulin-stimulated 2-deoxyglucose (2-DG) uptake. Treatment of epitrochlearis muscles with 2 nmol/l TNF-alpha for 8 h also had no effect on insulin-stimulated 2-DG uptake. We conclude that in contrast to Fao hepatoma cells and 3T3-L1 fibroblasts, skeletal muscle does not become insulin resistant in response to short-term exposure to TNF-alpha.
Assuntos
Glucose/metabolismo , Hipoglicemiantes/farmacologia , Insulina/farmacologia , Músculo Esquelético/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Tirosina/metabolismo , Células 3T3 , Animais , Humanos , Técnicas In Vitro , Camundongos , Músculo Esquelético/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Ratos , Ratos Wistar , Proteínas Recombinantes/farmacologia , Estimulação Química , Fatores de Tempo , Células Tumorais CultivadasRESUMO
beta-Adrenergic stimulation has been reported to inhibit insulin-stimulated glucose transport in adipocytes. This effect has been attributed to a decrease in the intrinsic activity of the GLUT-4 isoform of the glucose transporter that is mediated by phosphorylation of GLUT-4. Early studies showed no inhibition of insulin-stimulated glucose transport by epinephrine in skeletal muscle. The purpose of this study was to determine the effect of epinephrine on GLUT-4 phosphorylation, and reevaluate the effect of beta-adrenergic stimulation on insulin-activated glucose transport, in skeletal muscle. We found that 1 microM epinephrine, which raised adenosine 3',5'-cyclic monophosphate approximately ninefold, resulted in GLUT-4 phosphorylation in rat skeletal muscle but had no inhibitory effect on insulin-stimulated 3-O-methyl-D-glucose (3-MG) transport. In contrast to 3-MG transport, the uptakes of 2-deoxyglucose and glucose were markedly inhibited by epinephrine treatment. This inhibitory effect was presumably mediated by stimulation of glycogenolysis, which resulted in an increase in glucose 6-phosphate concentration to levels known to severely inhibit hexokinase. We conclude that 1) beta-adrenergic stimulation decreases glucose uptake by raising glucose 6-phosphate concentration, thus inhibiting hexokinase, but does not inhibit insulin-stimulated glucose transport and 2) phosphorylation of GLUT-4 has no effect on glucose transport in skeletal muscle.
Assuntos
Epinefrina/farmacologia , Glucose/metabolismo , Insulina/farmacologia , Proteínas de Transporte de Monossacarídeos/metabolismo , Proteínas Musculares , Músculo Esquelético/metabolismo , 3-O-Metilglucose/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , AMP Cíclico/metabolismo , Desoxiglucose/metabolismo , Transportador de Glucose Tipo 4 , Glicogênio/metabolismo , Técnicas In Vitro , Cinética , Masculino , Músculo Esquelético/efeitos dos fármacos , Fosforilação , Ratos , Ratos WistarRESUMO
Enhanced glucose flux via the hexosamine biosynthetic pathway has been implicated in insulin resistance. We measured products of this pathway, UDP-N-acetyl hexosamines (UDP-HexNAc), and activity of the rate-limiting enzyme L-glutamine:D-fructose-6-phosphate amidotransferase (GFAT) in tissues of ob/ob mice and lean controls. Ob/ob mice were obese, hyperglycemic, and hyperinsulinemic. Resistance to the effect of insulin on glucose transport was demonstrated in isolated soleus muscles, although total GLUT-4 concentration was mildly increased in muscles from ob/ob mice. UDP-HexNAc concentrations in hindlimb muscles decreased between 8 and 17 wk but were always higher in ob/ob vs. controls (P < 0.001, mean increase 67%). Concentrations of UDP-hexoses and GDP-mannose were similar in ob/ob and control muscles. Muscle GFAT activity declined with age but was increased in ob/ob vs. controls at each age examined (P < 0.001, mean increase 108%). UDP-HexNAc concentrations and GFAT activity were similar in livers of ob/ob and controls. These data suggest that glucose flux via the hexosamine pathway is selectively increased in muscle but not liver of ob/ob mice and may contribute to muscle insulin resistance in this model of non-insulin-dependent diabetes mellitus.
Assuntos
Hexosaminas/metabolismo , Resistência à Insulina , Proteínas Musculares , Músculo Esquelético/metabolismo , Obesidade/metabolismo , Envelhecimento/metabolismo , Animais , Transporte Biológico , Glicemia/metabolismo , Glucose/metabolismo , Transportador de Glucose Tipo 4 , Insulina/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Proteínas de Transporte de Monossacarídeos/metabolismo , Desenvolvimento Muscular , Músculo Esquelético/crescimento & desenvolvimento , Obesidade/genética , Uridina Difosfato N-Acetilglicosamina/metabolismo , Açúcares de Uridina Difosfato/metabolismoRESUMO
The present study examined the effects of 6 wk of ovarian endocrine deficiency on skeletal muscle GLUT-4 glucose transporter protein and glucose transport activity in sedentary and endurance-trained rats. Female Wistar rats (10 wk old) underwent bilateral ovariectomy (OVX) or sham surgery followed by a 5-wk swim-training protocol. OVX resulted in no significant changes in glycogen or GLUT-4 glucose transporter concentration in the soleus, epitrochlearis, or flexor digitorum brevis (FDB) muscles or in basal and maximally insulin-stimulated 2-deoxy-D-[1,2-3H]glucose (2-[3H]DG) transport in the soleus or epitrochlearis, suggesting that moderate-duration ovarian hormone deficiency does not significantly impair insulin action in skeletal muscle. In contrast, OVX decreased the maximal activation of 2-[3H]DG transport in the FDB by in vitro electrical stimulation. OVX had no significant effect on the training-induced changes in oxidative enzyme activities, GLUT-4 protein expression, glycogen content, or insulin-stimulated 2-[3H]DG transport in the soleus or epitrochlearis. These findings provide the first evidence that ovarian hormone deficiency decreases contraction-stimulated glucose transport in skeletal muscle.
Assuntos
Glucose/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Músculo Esquelético/metabolismo , Ovariectomia , Animais , Feminino , Insulina/farmacologia , Condicionamento Físico Animal , Ratos , Ratos WistarRESUMO
Previous studies in animals and humans have shown that endurance exercise-training protocols of several weeks to many months in duration induce adaptive increases in skeletal muscle GLUT-4 protein concentration. It is generally assumed that the increase in GLUT-4 concentration is a long-term adaptation to training. The present study examined whether 7-10 days of cycle ergometer exercise could induce increases in skeletal muscle GLUT-4 levels. Eight healthy subjects (4 men, 4 women) aged 31 +/- 2 (SE) yr exercised 2 h daily at 65-70% of peak O2 uptake (VO2peak) for either 7 (n = 3) or 10 (n = 5) consecutive days. Muscle biopsies (vastus lateralis) were obtained before initiation of the exercise program and 36-48 h after the final bout of exercise. Glucose transporter protein was quantitated by Western blotting using antiserum specific for GLUT-4. VO2peak was increased by 10% (from 3.0 +/- 0.2 to 3.3 +/- 0.2 l/min; P < 0.01) in response to the training. Body weight did not change (74.3 +/- 4.6 before vs. 75.0 +/- 4.2 kg after) as a result of training. Muscle GLUT-4 immunoreactivity was increased 98% (from 584 +/- 50 to 1,154 +/- 40 counts per minute 125I/25 micrograms protein; P < 0.001) in response to training. Increase in VO2peak and GLUT-4 protein were similar for 7 and 10 days of training. These results suggest that, given an adequate training stimulus, adaptations in skeletal muscle GLUT-4 protein occur very rapidly. Furthermore, the increase in GLUT-4 after 7-10 days of exercise is as large as that reported in studies employing long-term training protocols.
Assuntos
Exercício Físico/fisiologia , Proteínas de Transporte de Monossacarídeos/metabolismo , Proteínas Musculares , Músculo Esquelético/metabolismo , Adulto , Western Blotting , Teste de Esforço , Feminino , Glucose/metabolismo , Teste de Tolerância a Glucose , Transportador de Glucose Tipo 4 , Humanos , Insulina/sangue , MasculinoRESUMO
There is evidence that an increase in sarcoplasmic Ca2+ stimulates glucose transport in muscle. Recent studies have provided the apparently conflicting finding that a sustained increase in cytosolic Ca2+ has little effect on basal glucose transport but inhibits insulin-stimulated transport. This study was done to try to explain this discrepancy. Continuous exposure of rat epitrochlearis and soleus muscles to the Ca2+ ionophore ionomycin (2 microM) had no effect on basal 2-deoxyglucose (2-DG) transport but blunted, by approximately 40%, stimulation of 2-DG transport by insulin. Decreasing Ca2+ in the medium to a very low level prevented this inhibition. Ionomycin induced a small increase in adenosine 3',5'-cyclic monophosphate (cAMP); however, studies with the protein kinase A (PKA) inhibitor HA-1004 provided evidence that activation of PKA by cAMP does not mediate the inhibition of glucose transport. When muscles were allowed to recover in the absence of ionomycin for 15 min, basal 2-DG transport was significantly increased. Our results agree with previous studies showing that a sustained influx of Ca2+ into the cytoplasm can inhibit insulin-stimulated glucose transport. They further show that stimulation of glucose transport by Ca2+ is also inhibited. A recovery period that allows this inhibition to wear off unmasks the stimulation of glucose transport by an increase in sarcoplasmic Ca2+.
Assuntos
Glucose/metabolismo , Insulina/farmacologia , Ionomicina/farmacologia , Músculos/metabolismo , Sulfonamidas , Animais , Transporte Biológico/efeitos dos fármacos , AMP Cíclico/metabolismo , Ativação Enzimática/efeitos dos fármacos , Epinefrina/farmacologia , Glucose/antagonistas & inibidores , Glicogênio/metabolismo , Isoquinolinas/farmacologia , Masculino , Fosforilase a/metabolismo , Fosforilação , Inibidores de Proteínas Quinases , Ratos , Ratos WistarRESUMO
Both the anabolic hormone insulin and contractile activity stimulate the uptake of glucose into mammalian skeletal muscle. In this study, we examined the role of phosphatidylinositol 3-kinase (PI 3-kinase), a putative mediator of insulin actions, in the stimulation of hexose uptake in response to hormone and contraction. Phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-triphosphate accumulate in skeletal muscle exposed to insulin but not hypoxia, which mimics stimulation of the contractile-dependent pathway of hexose transport activation. The fungal metabolite wortmannin, an inhibitor of PI 3-kinase, completely blocks the appearance of 3'-phospholipids in response to insulin. Moreover, wortmannin entirely prevented the increase in hexose uptake in muscle exposed to insulin but was without effect on muscle stimulated by repetitive contraction or hypoxia. These results support the view that PI 3-kinase is involved in the signaling pathways mediating insulin-responsive glucose transport in skeletal muscle but is not required for stimulation by hypoxia or contraction. Furthermore, these data indicate that there exist at least two signaling pathways leading to activation of glucose transport in skeletal muscle with differential sensitivities to wortmannin.
Assuntos
Androstadienos/farmacologia , Hexoses/metabolismo , Músculos/efeitos dos fármacos , Fosfotransferases (Aceptor do Grupo Álcool)/fisiologia , Animais , Transporte Biológico/efeitos dos fármacos , Hipóxia/metabolismo , Insulina/farmacologia , Masculino , Contração Muscular , Fosfatidilinositol 3-Quinases , Fosfatidilinositóis/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , WortmaninaRESUMO
Skeletal muscle glucose transport and metabolism were studied in a line of transgenic mice overexpressing the human Glut4 facilitative glucose transporter. Skeletal muscle Glut4 protein levels were increased 2-4-fold in transgenic animals relative to their nontransgenic litter mates. Glut4 overexpression increased total transport activity (measured with 1 mM 2-deoxy-D-glucose) in the isolated extensor digitorum brevis muscle in the presence of insulin; this increase was due to 1) an increase in basal glucose transport (0.8 +/- 0.1 versus 0.5 +/- 0.1 mumol.ml-1.20 min-1 in transgenic and control mice, respectively) and 2) an increase in insulin-stimulated transport (1.5 +/- 0.1 versus 0.8 +/- 0.1 mumol.ml-1.20 min-1 above basal transport in transgenic and control mice, respectively). Glut4 overexpression also increased glucose transport stimulated by muscle contractions. In addition, glycolysis and glucose incorporation into glycogen were enhanced in muscle isolated from transgenic mice compared to controls. These data demonstrate that Glut4 overexpression in skeletal muscle increases insulin- and contraction-stimulated glucose transport activity and glucose metabolism. These findings are consistent with the role of Glut4 as the primary mediator of transport stimulated by insulin or contractions.
Assuntos
Glucose/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Proteínas Musculares , Músculo Esquelético/metabolismo , Ácido 3-Hidroxibutírico , Animais , Transporte Biológico Ativo , Glicemia/metabolismo , Desoxiglucose/metabolismo , Estimulação Elétrica , Imunofluorescência , Glucagon/sangue , Transportador de Glucose Tipo 4 , Glicogênio/biossíntese , Glicólise , Humanos , Hidroxibutiratos/sangue , Insulina/sangue , Lactatos/sangue , Camundongos , Camundongos Transgênicos , Proteínas de Transporte de Monossacarídeos/biossíntese , Proteínas de Transporte de Monossacarídeos/genética , Músculo Esquelético/inervação , Músculo Esquelético/fisiologia , Valores de Referência , Nervo Isquiático/fisiologia , TrítioRESUMO
The calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) stimulates glucose transport in skeletal muscle, apparently by raising cytosolic Ca2+ (P. Palade. J. Biol. Chem. 262: 6142-6148, 1987; J.H. Youn, E.A. Gulve, and J.O. Holloszy. Am. J. Physiol. 260 (Cell Physiol. 29): C555-C561, 1991). This study was performed to describe the interactions between the effects of W-7 and those of hypoxia and of insulin on glucose transport. The effect on 3-O-methylglucose (3-MG) transport of 50 microM W-7 was additive to the effect of a maximal insulin stimulus (2,000 microU/ml) but not to the effect of maximal (60 min) hypoxic stimulus, suggesting that W-7 stimulates glucose transport via the same pathway as hypoxia, independent of the pathway activated by insulin. The effect of 50 microM W-7 was additive to that of a submaximal (20 min) hypoxia stimulus, indicating that W-7 does not interfere with the stimulation of glucose transport by hypoxia. In contrast, 50 microM W-7 had an inhibitory effect on stimulation of 3-MG transport by submaximally effective insulin levels, causing a fivefold increase in the concentration of insulin needed to produce a half-maximal stimulation of 3-MG transport, from approximately 70 to approximately 350 microU/ml (P < 0.05). Thus these data demonstrate that W-7 selectively inhibits insulin stimulation of glucose transport.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Glucose/metabolismo , Insulina/farmacologia , Metilglucosídeos/metabolismo , Músculos/metabolismo , Sulfonamidas/farmacologia , 3-O-Metilglucose , Aminacrina/farmacologia , Animais , Transporte Biológico/efeitos dos fármacos , Dantroleno/farmacologia , Hipóxia , Técnicas In Vitro , Cinética , Masculino , Músculos/efeitos dos fármacos , Ratos , Ratos Wistar , Trítio , Vasodilatadores/farmacologiaRESUMO
The maximal effects of insulin and muscle contractions on glucose transport are additive. GLUT-4 is the major glucose transporter isoform expressed in skeletal muscle. Muscle contraction and insulin each induce translocation of GLUT-4 from intracellular sites into the plasma membrane. The purpose of this study was to test the hypothesis that the incremental effect of contractions and insulin on glucose transport is mediated by additivity of the maximal effects of these stimuli on GLUT-4 translocation into the sarcolemma. Anesthetized rats were given insulin by intravenous infusion to raise plasma insulin to 2,635 +/- 638 microU/ml. The gastrocnemius-plantaris-soleus group was stimulated to contract via the sciatic nerve by using a protocol that maximally activates glucose transport. After treatment with insulin, contractions, or insulin plus contractions or no treatment, the gastrocnemius-plantaris-soleus muscle group was dissected out and was subjected to subcellular fractionation to separate the plasma membrane and intracellular membrane fractions. Insulin induced a 70% increase and contractions induced a 113% increase in the GLUT-4 content of the plasma membrane fraction. The effects of insulin and contractions were additive, as evidenced by a 185% increase in the GLUT-4 content of the sarcolemmal fraction. This finding provides evidence that the incremental effect of maximally effective insulin and contractile stimuli on glucose transport is mediated by additivity of their effects on GLUT-4 translocation into the sarcolemma.
Assuntos
Glucose/metabolismo , Insulina/farmacologia , Proteínas de Transporte de Monossacarídeos/metabolismo , Contração Muscular/fisiologia , Proteínas Musculares , Músculo Esquelético/metabolismo , Sarcolema/metabolismo , Animais , Western Blotting , Fracionamento Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Eletroforese em Gel de Poliacrilamida , Transportador de Glucose Tipo 4 , Insulina/sangue , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Masculino , Músculo Esquelético/efeitos dos fármacos , Ratos , Ratos Wistar , Sarcolema/efeitos dos fármacosRESUMO
Lithium is thought to have an insulin-like effect on glucose transport and metabolism in skeletal muscle and adipocytes. However, we found that lithium had only a minimal effect on basal glucose transport activity in rat epitrochlearis muscles. Instead, lithium markedly increased the sensitivity of glucose transport to insulin, so that the increase in glucose transport activity induced by 300 pM insulin was approximately 2.5-fold greater in the presence of lithium than in its absence. Lithium also caused a modest increase in insulin responsiveness. This enhancement of the susceptibility of the glucose transport process to stimulation was not limited to insulin, because lithium induced increases in the susceptibility of glucose transport to stimulation by contractile activity, hypoxia, a phorbol ester, and phospholipase C. Lithium also blunted the activation of glycogen phosphorylase by epinephrine. These effects were not mediated by inhibition of adenylate cyclase, because neither basal- nor epinephrine-stimulated muscle cAMP concentration was affected by lithium treatment. The effects of lithium on glucose transport and metabolism in skeletal muscle are strikingly similar to the persistent effects of exercise. These results support the possibility that lithium might be useful in the treatment of insulin resistance in patients with non-insulin-dependent diabetes mellitus.
Assuntos
Glucose/metabolismo , Insulina/farmacologia , Cloreto de Lítio/farmacologia , Músculos/metabolismo , 3-O-Metilglucose , Animais , Transporte Biológico/efeitos dos fármacos , Hipóxia Celular , Estimulação Elétrica , Ativação Enzimática , Epinefrina/farmacologia , Técnicas In Vitro , Masculino , Manitol/metabolismo , Metilglucosídeos/metabolismo , Contração Muscular/fisiologia , Músculos/efeitos dos fármacos , Músculos/fisiologia , Fosforilases/metabolismo , Ratos , Ratos WistarRESUMO
Glucose transport activity was examined in transgenic mice overexpressing the human GLUT1 glucose transporter in skeletal muscles. Basal transport activity measured in vitro with the glucose analog 2-deoxy-D-glucose (1 mM) was increased 2-8-fold in four different muscle preparations. Incubation of muscles from control nontransgenic littermates with a maximally effective concentration of insulin or with insulin-like growth factor-1 resulted in glucose transport rates that were 2-3-fold higher than basal. In contrast, insulin did not stimulate glucose transport activity in three different muscle preparations from transgenic animals; insulin-like growth factor-1 was similarly ineffective. Activation of System A amino acid transport activity (measured with the nonmetabolizable analog alpha-methylaminoisobutyrate) by insulin was not impaired in muscles from transgenic mice, indicating that the defect does not involve the insulin receptor. In skeletal muscle, glucose transport can be activated by muscle contractions or hypoxia via a pathway separate from that activated by insulin. Incubation of muscles under hypoxic conditions or stimulation of muscles to contract in situ did not increase glucose transport activity in muscles from GLUT1-overexpressing mice, in contrast to the stimulatory effects measured in muscles from control animals. These data suggest that increased glucose flux per se into skeletal muscle results in resistance of GLUT4 to activation by insulin and various other stimuli that activate glucose transport by mechanisms distinct from that of insulin. GLUT1-overexpressing mice thus provide a new model system for studying the effects of glucose-induced resistance to activation of glucose transport.
Assuntos
Glucose/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Proteínas Musculares , Músculos/metabolismo , Aminoácidos/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Expressão Gênica , Transportador de Glucose Tipo 1 , Transportador de Glucose Tipo 4 , Humanos , Insulina/farmacologia , Cinética , Camundongos , Camundongos Transgênicos , Proteínas de Transporte de Monossacarídeos/biossíntese , Proteínas de Transporte de Monossacarídeos/genética , Músculos/efeitos dos fármacos , Especificidade de Órgãos , Valores de Referência , beta-Alanina/análogos & derivados , beta-Alanina/metabolismoRESUMO
Muscle glycogen depletion by means of exercise is associated with increased insulin-stimulated glucose transport activity. To determine whether reduction in muscle glycogen content independent of muscle contractions would increase glucose transport activity, rats were injected with epinephrine (20 micrograms/100 g body wt) or saline. Two hours later, epitrochlearis muscles were removed, washed thoroughly to remove epinephrine, and assayed for glucose transport activity with 3-O-methyl-D-glucose (3-MG). Muscle adenosine 3',5'-cyclic monophosphate concentration was elevated 441% in muscles frozen immediately after removal from epinephrine-injected rats but had returned to control levels by the time 3-MG transport was measured. Prior exposure to epinephrine resulted in depletion of muscle glycogen [from 18.6 +/- 1.4 to 11.0 +/- 0.1 (SE) mumol glucose units/g wet wt] and a small increase in basal glucose transport activity (from 0.13 +/- 0.02 to 0.24 +/- 0.04 mumol 3-MG.ml-1 x 10 min-1, P < 0.05). A submaximally effective insulin concentration (30 microU/ml) induced a 70% greater increase in 3-MG transport in epinephrine-treated muscles than in controls (0.57 +/- 0.09 and 0.34 +/- 0.04 mumol.ml-1 x 10 min-1, respectively, P < 0.001). Response to a maximally effective concentration of insulin was unaltered by prior exposure to epinephrine. When epinephrine-induced glycogen depletion was prevented by prior injection with the beta-adrenergic antagonist propranolol, glucose transport activity was no longer enhanced by epinephrine.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Epinefrina/farmacologia , Glucose/metabolismo , Glicogênio/metabolismo , Insulina/farmacologia , Músculos/metabolismo , Antagonistas Adrenérgicos beta/farmacologia , Animais , AMP Cíclico/metabolismo , Técnicas In Vitro , Masculino , Músculos/efeitos dos fármacos , Músculos/enzimologia , Fosforilases/metabolismo , Esforço Físico/fisiologia , Ratos , Ratos WistarRESUMO
GLUT4 glucose transporter content and glucose transport capacity are closely correlated in skeletal muscle. In this study, we tested the hypothesis that a rapid increase in GLUT4 expression occurs as part of the early adaptive response of muscle to exercise and serves to enhance glycogen storage. Rats exercised by swimming had a approximately 2-fold increase in GLUT4 mRNA and a 50% increase in GLUT4 protein expression in epitrochlearis muscle 16 h after one prolonged exercise session. After a 2nd day of exercise, muscle GLUT4 protein was increased further to approximately 2-fold while there was no additional increase in GLUT4 mRNA. Muscle hexokinase activity also doubled in response to 2 days of exercise. Glucose transport activity maximally stimulated with insulin, contractions, or hypoxia was increased roughly in proportion to the adaptive increase in GLUT4 protein in epitrochlearis muscles. Treatment with insulin prior to subcellular fractionation of muscle resulted in a approximately 2-fold greater increase in GLUT4 content of a plasma membrane fraction in the 2-day swimmers than in controls. When epitrochlearis muscles were incubated with glucose and insulin, glycogen accumulation over 3 h was twice as great in muscles from 2-day swimmers as in control muscles. Our results show that a rapid increase in GLUT4 expression is an early adaptive response of muscle to exercise. This adaptation appears to be mediated by pretranslational mechanisms. We hypothesize that the physiological role of this adaptation is to enhance replenishment of muscle glycogen stores.
Assuntos
Glucose/metabolismo , Glicogênio/metabolismo , Insulina/farmacologia , Proteínas de Transporte de Monossacarídeos/biossíntese , Proteínas Musculares , Músculos/metabolismo , Condicionamento Físico Animal , RNA Mensageiro/metabolismo , 3-O-Metilglucose , Actinas/biossíntese , Animais , Desoxiglucose/metabolismo , Feminino , Transportador de Glucose Tipo 4 , Glicogênio/biossíntese , Hipóxia , Cinética , Metilglucosídeos/metabolismo , Proteínas de Transporte de Monossacarídeos/análise , Contração Muscular , Músculos/efeitos dos fármacos , Músculos/fisiologia , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Ratos , Valores de Referência , Fatores de TempoRESUMO
The insulin sensitivity of glucose transport is enhanced in skeletal muscle after a bout of exercise. In a previous study, stimulation of washed muscles to contract in vitro, in contrast to exercise, did not result in an increase in insulin sensitivity. The purpose of the present study was to explain this apparent discrepancy. We found that, although rat epitrochlearis muscles stimulated to contract in vitro after 15 min of incubation in Krebs-Henseleit buffer did not develop increased insulin sensitivity, muscles stimulated to contract immediately after being dissected showed a small but significant enhancement of the stimulation of 3-O-methyl-D-glucose transport by 30 microU/ml insulin. Furthermore, muscles stimulated to contract in situ and then allowed to recover in vitro showed as large an increase in insulin sensitivity as that which occurs after a bout of swimming. To follow up these findings suggesting involvement of a humoral factor, we incubated epitrochlearis muscles in serum before and during contractile activity in vitro. Epitrochlearis muscle insulin sensitivity was enhanced to as great an extent after in vitro contractile activity in serum as after swimming. Experiments involving charcoal treatment, ultrafiltration, or trypsin digestion provided evidence that the serum factor that interacts with contractions to enhance insulin sensitivity is a protein.
Assuntos
Fenômenos Fisiológicos Sanguíneos , Insulina/farmacologia , Contração Muscular , Músculos/efeitos dos fármacos , Animais , Proteínas Sanguíneas/metabolismo , Centrifugação com Gradiente de Concentração , Carvão Vegetal/farmacologia , Fator de Crescimento Insulin-Like I/farmacologia , Masculino , Ratos , Ratos Wistar , Albumina Sérica/farmacologia , Soroglobulinas/farmacologia , Natação , Tripsina/metabolismoRESUMO
The purpose of this study was to evaluate the suitability of the glucose analogue 2-deoxyglucose (2-DG) for measurement of glucose transport activity in rat skeletal muscles in vitro when transport rates are high. The goal was to determine whether glucose phosphorylation rather than transport becomes limiting under experimental conditions normally employed in muscle incubation experiments. The rate of 2-DG uptake assayed in the presence of 8 mM 2-DG and a maximally effective concentration of insulin remained linear for > or = 60 min in the split soleus and 120 min in the epitrochlearis. Hexokinase activity assayed in skeletal muscle homogenates was not inhibited appreciably by 2-deoxyglucose 6-phosphate (2-DG-6-P) concentrations in the range of those achieved intracellularly during the linear phase of 2-DG uptake (i.e., 2-DG-6-P below approximately 30 mM). During this linear phase of 2-DG uptake, total intracellular 2-DG concentrations did not exceed 30 mM. The combined effects of contractions plus a maximally effective concentration of insulin on glucose transport activity measured at a near-saturating concentration of 2-DG were additive in the epitrochlearis and the soleus. Our results indicate that, under the conditions employed in our isolated muscle preparations, 2-DG uptake accurately reflects glucose transport activity and that 2-DG is the most appropriate glucose analogue for measurement of glucose transport activity when transport rates are high.