RESUMO
In skeletal muscle the Rab-GTPase-activating protein TBC1D1 has been implicated in the regulation of fatty acid oxidation by an unknown mechanism. We determined whether TBC1D1 altered fatty acid utilization via changes in protein-mediated fatty acid transport and/or selected enzymes regulating mitochondrial fatty acid oxidation. We also determined the effects of TBC1D1 on glucose transport and oxidation. Electrotransfection of mouse soleus muscles with TBC1D1 cDNA increased TBC1D1 protein after 2 wk (P<0.05), without altering its paralog AS160. TBC1D1 overexpression decreased basal palmitate oxidation (-22%) while blunting 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR)-stimulated palmitate oxidation (-18%). There was a tendency to increase fatty acid esterification (+10 nmol·g(-1)·60 min(-1), P=0.07), which reflected the reduction in fatty acid oxidation (-12 nmol·g(-1)·60 min(-1)). Concomitantly, basal (+21%) and AICAR-stimulated glucose oxidation (+8%) were increased in TBC1D1-transfected muscles relative to their respective controls (P<0.05), independent of changes in GLUT4 and glucose transport. The reductions in TBC1D1-mediated fatty acid oxidation could not be attributed to changes in the transporter FAT/CD36, muscle mitochondrial content, CPT1 expression or the expression and phosphorylation of AS160, acetyl-CoA carboxylase, or AMPK. However, TBC1D1 overexpression reduced ß-HAD enzyme activity (-18%, P<0.05). In conclusion, TBC1D1-mediated reduction of muscle fatty acid oxidation appears to occur via inhibition of ß-HAD activity.
Assuntos
3-Hidroxiacil-CoA Desidrogenases/metabolismo , Músculo Esquelético/enzimologia , Proteínas Nucleares/metabolismo , Palmitatos/metabolismo , 3-Hidroxiacil-CoA Desidrogenases/genética , Aminoimidazol Carboxamida/análogos & derivados , Animais , Ácidos Graxos/metabolismo , Proteínas Ativadoras de GTPase , Regulação da Expressão Gênica/fisiologia , Glucose/metabolismo , Peroxidação de Lipídeos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Nucleares/genética , Oxirredução , RibonucleotídeosRESUMO
AIMS/HYPOTHESIS: Little is known about the subcellular distribution of lipids in insulin-resistant skeletal muscle. However, it has recently been suggested that lipid accumulation in the subsarcolemmal region directly contributes to insulin resistance. Therefore we hypothesised that regional differences in lipid distribution in insulin-resistant muscle may be mediated by: (1) a reduction in fatty acid trafficking into mitochondria; and/or (2) a regional increase in the enzymes regulating lipid synthesis. METHODS: Transmission electron microscopy was used to quantify lipid droplet and mitochondrial abundance in the subsarcolemmal and intermyofibrillar compartments in red and white muscles from lean and obese Zucker rats. To estimate rates of lipid trafficking into mitochondria, the metabolic fate of radiolabelled palmitate was determined. Key enzymes of triacylglycerol synthesis were also determined in each subcellular region. RESULTS: Subsarcolemmal-compartmentalised lipids represented a small absolute fraction of the overall lipid content in muscle, as regardless of fibre composition (red/white) or phenotype (lean/obese), lipid droplets were more prevalent in the intermyofibrillar region, whereas insulin-resistant white muscles were devoid of subsarcolemmal-compartmentalised lipid droplets. While, in obese animals, lipid droplets accumulated in both subcellular regions, in red muscle of these animals lipids only appeared to be trafficked away from intermyofibrillar mitochondria, a process that cannot be explained by regional differences in the abundance of triacylglycerol esterification enzymes. CONCLUSIONS/INTERPRETATION: Lipid accumulation in the subsarcolemmal region is not necessary for insulin resistance. In the intermyofibrillar compartment, the diversion of lipids away from mitochondria in insulin-resistant animals probably contributes to lipid accumulation in this subcellular area.
Assuntos
Lipídeos/química , Obesidade/genética , Frações Subcelulares/metabolismo , Animais , DNA Mitocondrial/metabolismo , Modelos Animais de Doenças , Ácidos Graxos/química , Feminino , Glucose/metabolismo , Insulina/metabolismo , Microscopia Eletrônica de Transmissão/métodos , Mitocôndrias/metabolismo , Obesidade/metabolismo , Oxigênio/química , Ácido Palmítico/metabolismo , Ratos , Ratos Zucker , Triglicerídeos/químicaRESUMO
AIMS/HYPOTHESIS: We examined in skeletal muscle (1) whether fatty acid transport protein (FATP) 1 channels long-chain fatty acid (LCFA) to specific metabolic fates in rats; and (2) whether FATP1-mediated increases in LCFA uptake exacerbate the development of diet-induced insulin resistance in mice. We also examined whether FATP1 is altered in insulin-resistant obese Zucker rats. METHODS: LCFA uptake, oxidation and triacylglycerol esterification rates were measured in control and Fatp1-transfected soleus muscles to determine FATP1-mediated lipid handling. The effects of FATP1 on insulin sensitivity and triacylglycerol accumulation were determined in high-fat diet-fed wild-type mice and in muscle-specific Fatp1 (also known as Slc27a1) overexpressing transgenic mice driven by the muscle creatine kinase (Mck [also known as Ckm]) promoter. We also examined the relationship between FATP1 and both fatty acid transport and metabolism in insulin-resistant obese Zucker rats. RESULTS: Transient Fatp1 overexpression in soleus muscle increased (p < 0.05) palmitate transport (24%) and oxidation (35%), without altering triacylglycerol esterification or the intrinsic rate of palmitate oxidation in isolated mitochondria. In Mck/Fatp1 animals, Fatp1 mRNA and 15-(p-iodophenyl)-3-R,S-methylpentadecanoic acid uptake in skeletal muscle were upregulated (75%). However, insulin sensitivity and intramuscular triacylglycerol content did not differ between wild-type and Mck/Fatp1 mice following a 16 week high-fat diet. In insulin-resistant obese Zucker rats, LCFA transport and triacylglycerol accumulation were increased (85% and 24%, respectively), but this was not attributable to Fatp1 expression, as neither total cellular nor sarcolemmal FATP1 content were altered. CONCLUSIONS/INTERPRETATION: Overexpression of Fatp1 in skeletal muscle increased the rate of LCFA transport and channelled these lipids to oxidation, not to intramuscular lipid accumulation. Therefore, skeletal muscle FATP1 overabundance does not predispose animals to diet-induced insulin resistance.
Assuntos
Gorduras na Dieta/efeitos adversos , Proteínas de Transporte de Ácido Graxo/metabolismo , Ácidos Graxos/metabolismo , Resistência à Insulina/fisiologia , Músculo Esquelético/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Camundongos , Camundongos Transgênicos , Mitocôndrias Musculares/metabolismo , Obesidade/metabolismo , Obesidade/fisiopatologia , Oxirredução , Palmitatos/metabolismo , Ratos , Ratos Sprague-Dawley , Ratos Zucker , Triglicerídeos/metabolismoRESUMO
AIMS/HYPOTHESIS: Upon stimulation of insulin signalling or contraction-induced AMP-activated protein kinase (AMPK) activation, the glucose transporter GLUT4 and the long-chain fatty acid (LCFA) transporter CD36 similarly translocate from intracellular compartments to the plasma membrane of cardiomyocytes to increase uptake of glucose and LCFA, respectively. This similarity in regulation of GLUT4 traffic and CD36 traffic suggests that the same families of trafficking proteins, including vesicle-associated membrane proteins (VAMPs), are involved in both processes. While several VAMPs have been implicated in GLUT4 traffic, nothing is known about the putative function of VAMPs in CD36 traffic. Therefore, we compared the involvement of the myocardially produced VAMP isoforms in insulin- or contraction-induced GLUT4 and CD36 translocation. METHODS: Five VAMP isoforms were silenced in HL-1 cardiomyocytes. The cells were treated with insulin or the contraction-like AMPK activator oligomycin or were electrically stimulated to contract. Subsequently, GLUT4 and CD36 translocation as well as substrate uptake were measured. RESULTS: Three VAMPs were demonstrated to be necessary for both GLUT4 and CD36 translocation, either specifically in insulin-treated cells (VAMP2, VAMP5) or in oligomycin/contraction-treated cells (VAMP3). In addition, there are VAMPs specifically involved in either GLUT4 traffic (VAMP7 mediates basal GLUT4 retention) or CD36 traffic (VAMP4 mediates insulin- and oligomycin/contraction-induced CD36 translocation). CONCLUSIONS/INTERPRETATION: The involvement of distinct VAMP isoforms in both GLUT4 and CD36 translocation indicates that CD36 translocation, just like GLUT4 translocation, is a vesicle-mediated process dependent on soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex formation. The ability of other VAMPs to discriminate between GLUT4 and CD36 translocation allows the notion that myocardial substrate preference can be modulated by these VAMPs.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Antígenos CD36/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Insulina/farmacologia , Miócitos Cardíacos/metabolismo , Proteínas R-SNARE/metabolismo , Análise de Variância , Animais , Linhagem Celular , Células Cultivadas , Estimulação Elétrica , Insulina/metabolismo , Camundongos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Oligomicinas/metabolismo , Oligomicinas/farmacologia , Isoformas de Proteínas/metabolismo , Transporte ProteicoRESUMO
AIMS/HYPOTHESIS: Reductions in peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PGC-1alpha) levels have been associated with the skeletal muscle insulin resistance. However, in vivo, the therapeutic potential of PGC-1alpha has met with failure, as supra-physiological overexpression of PGC-1alpha induced insulin resistance, due to fatty acid translocase (FAT)-mediated lipid accumulation. Based on physiological and metabolic considerations, we hypothesised that a modest increase in PGC-1alpha levels would limit FAT upregulation and improve lipid metabolism and insulin sensitivity, although these effects may differ in lean and insulin-resistant muscle. METHODS: Pgc-1alpha was transfected into lean and obese Zucker rat muscles. Two weeks later we examined mitochondrial biogenesis, intramuscular lipids (triacylglycerol, diacylglycerol, ceramide), GLUT4 and FAT levels, insulin-stimulated glucose transport and signalling protein phosphorylation (thymoma viral proto-oncogene 2 [Akt2], Akt substrate of 160 kDa [AS160]), and fatty acid oxidation in subsarcolemmal and intermyofibrillar mitochondria. RESULTS: Electrotransfection yielded physiologically relevant increases in Pgc-1alpha (also known as Ppargc1a) mRNA and protein ( approximately 25%) in lean and obese muscle. This induced mitochondrial biogenesis, and increased FAT and GLUT4 levels, insulin-stimulated glucose transport, and Akt2 and AS160 phosphorylation in lean and obese animals, while bioactive intramuscular lipids were only reduced in obese muscle. Concurrently, PGC-1alpha increased palmitate oxidation in subsarcolemmal, but not in intermyofibrillar mitochondria, in both groups. In obese compared with lean animals, the PGC-1alpha-induced improvement in insulin-stimulated glucose transport was smaller, but intramuscular lipid reduction was greater. CONCLUSIONS/INTERPRETATIONS: Increases in PGC-1alpha levels, similar to those that can be induced by physiological stimuli, altered intramuscular lipids and improved fatty acid oxidation, insulin signalling and insulin-stimulated glucose transport, albeit to different extents in lean and insulin-resistant muscle. These positive effects are probably attributable to limiting the PGC-1alpha-induced increase in FAT, thereby preventing bioactive lipid accumulation as has occurred in transgenic PGC-1alpha animals.
Assuntos
Glucose/metabolismo , Insulina/farmacologia , Músculo Esquelético/metabolismo , Obesidade/metabolismo , Proteínas de Ligação a RNA/metabolismo , Magreza/metabolismo , Fatores de Transcrição/metabolismo , Animais , Western Blotting , Ácidos Graxos/metabolismo , Transportador de Glucose Tipo 4/genética , Transportador de Glucose Tipo 4/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Masculino , Músculo Esquelético/efeitos dos fármacos , Oxirredução/efeitos dos fármacos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Fosforilação/efeitos dos fármacos , Proto-Oncogene Mas , Proteínas de Ligação a RNA/genética , Ratos , Ratos Zucker , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/genéticaRESUMO
Skeletal muscle gene response to exercise depends on nutritional status during and after exercise, but it is unknown whether muscle adaptations to endurance training are affected by nutritional status during training sessions. Therefore, this study investigated the effect of an endurance training program (6 wk, 3 day/wk, 1-2 h, 75% of peak Vo(2)) in moderately active males. They trained in the fasted (F; n = 10) or carbohydrate-fed state (CHO; n = 10) while receiving a standardized diet [65 percent of total energy intake (En) from carbohydrates, 20%En fat, 15%En protein]. Before and after the training period, substrate use during a 2-h exercise bout was determined. During these experimental sessions, all subjects were in a fed condition and received extra carbohydrates (1 g.kg body wt(-1) .h(-1)). Peak Vo(2) (+7%), succinate dehydrogenase activity, GLUT4, and hexokinase II content were similarly increased between F and CHO. Fatty acid binding protein (FABPm) content increased significantly in F (P = 0.007). Intramyocellular triglyceride content (IMCL) remained unchanged in both groups. After training, pre-exercise glycogen content was higher in CHO (545 +/- 19 mmol/kg dry wt; P = 0.02), but not in F (434 +/- 32 mmol/kg dry wt; P = 0.23). For a given initial glycogen content, F blunted exercise-induced glycogen breakdown when compared with CHO (P = 0.04). Neither IMCL breakdown (P = 0.23) nor fat oxidation rates during exercise were altered by training. Thus short-term training elicits similar adaptations in peak Vo(2) whether carried out in the fasted or carbohydrate-fed state. Although there was a decrease in exercise-induced glycogen breakdown and an increase in proteins involved in fat handling after fasting training, fat oxidation during exercise with carbohydrate intake was not changed.
Assuntos
Carboidratos da Dieta/farmacologia , Exercício Físico/fisiologia , Jejum/fisiologia , Metabolismo/fisiologia , Aptidão Física/fisiologia , Adulto , Glicemia/metabolismo , Western Blotting , Peso Corporal , Gorduras/metabolismo , Hormônios/sangue , Humanos , Processamento de Imagem Assistida por Computador , Cinética , Masculino , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Consumo de Oxigênio/fisiologia , RNA/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Reversa , Succinato Desidrogenase/metabolismo , Fixação de TecidosRESUMO
IL-6 is a biologically active substance and is thought to contribute to the development of obesity. Recent findings suggest that susceptibility to intracellular lipid accumulation is to a large extent determined by changes in the expression of fatty acid transporters such as FAT/CD36, FABPpm and FATP-1. The aim of the present study was to determine the effect of IL-6 deficiency on the expression of fatty acid transporters, as well as, assess the concomitant changes in intracellular lipids. We found that Il-6 deficiency upregulated the myocardial expression of FAT/CD36 (+40%) and did not significantly affect the content of FABPpm and FATP-1 (+15% and +5% respectively). Although no change in the intramyocardial total lipid content was noted, there was a significant increase in the intracellular content of both free fatty acid (FFA), diacylglicerol (DG) and ceramide fractions (+45%, +37% and +48%, respectively) in hearts from IL-6 -/- mice. A trend for IL-6 deficiency to increase in saturated FA species in these fractions was also observed (+8%, +12% and +10%, respectively). In contrast, IL-6 deficiency has no effect on the content of monounsaturated fatty acid (MUFA) and polyunsaturated fatty acid (PUFA) species in each intramyocardial lipid fractions examined. These findings suggest that IL-6 deficiency results in 1) upregulation of myocardial content of FAT/CD36, 2) the increase in the content of biologically active lipid pools (FFA, DG and ceramide). This lipid accumulation with concomitant trend for increase in the saturation status of these lipid fractions may, at least in part, provide a factor related to the development of intramyocardial lipotoxicity, observed in obese individuals.
Assuntos
Proteínas de Transporte de Ácido Graxo/metabolismo , Interleucina-6/deficiência , Metabolismo dos Lipídeos , Miocárdio/metabolismo , Animais , Antígenos CD36/metabolismo , Ceramidas/metabolismo , Diglicerídeos/metabolismo , Ácidos Graxos não Esterificados/metabolismo , Feminino , Interleucina-6/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
The aim of this study was to determine the biochemical mechanism(s) responsible for enhanced FA utilization (oxidation and esterification) by perfused hearts from type 2 diabetic db/db mice. The plasma membrane content of fatty acid transporters FAT/CD36 and FABPpm was elevated in db/db hearts. Mitochondrial mechanisms that could contribute to elevated rates of FA oxidation were also examined. Carnitine palmitoyl transferase-1 activity was unchanged in mitochondria from db/db hearts, and sensitivity to inhibition by malonyl-CoA was unchanged. Malonyl-CoA content was elevated and AMP kinase activity was decreased in db/db hearts, opposite to what would be expected in hearts exhibiting elevated rates of FA oxidation. Uncoupling protein-3 expression was unchanged in mitochondria from db/db hearts. Therefore, enhanced FA utilization in db/db hearts is most likely due to increased FA uptake caused by increased plasma membrane content of FA transporters; the mitochondrial mechanisms examined do not contribute to elevated FA oxidation observed in db/db hearts.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Ácidos Graxos/metabolismo , Miocárdio/metabolismo , Animais , Membrana Celular/fisiologia , Diabetes Mellitus Tipo 2/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias/fisiologia , PerfusãoRESUMO
Uncoupling protein 3 (UCP3) expression is directly correlated to fatty acid oxidation in skeletal muscle. UCP3 has been hypothesized to facilitate high rates of fatty acid oxidation, but evidence thus far is lacking. Our aim was to investigate the effects of UCP3 overexpression and ablation on fatty acid uptake and metabolism in muscle of mice having congenic backgrounds. In mice constitutively expressing the UCP3 protein (human form) at levels just over twofold higher than normal (230% of wild-type levels), indirect calorimetry demonstrated no differences in total energy expenditure (VO2), but a shift toward increased fat oxidation compared with wild-type (WT) mice. Metabolic efficiency (gram weight gain/kcal ingested) was similar between Ucp3 overexpressors, WT and Ucp3 (-/-) mice. In muscle of Ucp3-tg mice, plasma membrane fatty acid binding protein (FABPpm) content was increased compared with WT mice. Although hormone-sensitive lipase activity was unchanged across the genotypes, there were increases in carnitine palmitoyltransferase I, beta-hydroxyacylCoA dehydrogenase, and citrate synthase activities and decreases in intramuscular triacylglycerol in muscle of Ucp3-tg mice. There were no differences in muscle mitochondrial content. High-energy phosphates and total muscle carnitine and CoA were also greater in Ucp3-tg compared with WT mice. Taken together, the findings demonstrate an increased capacity for fat oxidation in the absence of significant increases in thermogenesis in Ucp3-tg mice. Findings from Ucp3 (-/-) mice revealed few differences compared with WT mice, consistent with the possibility of compensatory mechanisms. In conjunction with our observed increases in CoA and carnitine in muscle of Ucp3 overexpressors, the findings support the hypothesized role for Ucp3 in facilitating fatty acid oxidation in muscle.
Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , Ácidos Graxos/metabolismo , Músculo Esquelético/metabolismo , 3-Hidroxiacil-CoA Desidrogenases/metabolismo , Difosfato de Adenosina/análise , Trifosfato de Adenosina/análise , Animais , Transporte Biológico , Calorimetria Indireta , Carnitina/análise , Carnitina O-Palmitoiltransferase/metabolismo , Proteínas de Transporte/análise , Citrato (si)-Sintase/metabolismo , Coenzima A/análise , Creatina/análise , Metabolismo Energético , Feminino , Expressão Gênica , Canais Iônicos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias Musculares/química , Proteínas Mitocondriais , Músculo Esquelético/ultraestrutura , Oxirredução , Consumo de Oxigênio , Fosfatos/análise , Triglicerídeos/análise , Proteína Desacopladora 3RESUMO
Insulin binding to crude plasma membranes derived from human skeletal muscle was characterized. Incubations were performed for 22 h at 4 degrees C. Typical insulin binding characteristics were found, i.e., (a) specificity for insulin, (b) pH sensitivity, (c) dissociation of insulin by the addition of excess insulin and (d) concave Scatchard curves. Half-maximal inhibition of 125I-labeled-insulin binding occurred at 1 X 10(-8) M. Affinity constants were 0.76 X 10(9) and 0.02 X 10(9) M-1 for the high- and low-affinity receptor (2-site model), respectively, and the corresponding receptor numbers were 89 and 1450 fmol/mg protein, respectively. The procedures employed permit the determination of insulin binding to small quantities of human muscle (approx. 250 mg).
Assuntos
Músculos/metabolismo , Receptor de Insulina/metabolismo , Adulto , Ligação Competitiva , Membrana Celular/metabolismo , Humanos , Insulina/análogos & derivados , Insulina/metabolismo , Cinética , Pessoa de Meia-IdadeRESUMO
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.
Assuntos
Proteínas de Transporte de Monossacarídeos/metabolismo , Contração Muscular/fisiologia , Proteínas Musculares , Músculo Esquelético/metabolismo , Receptores da Transferrina/metabolismo , Animais , Membrana Celular/metabolismo , Endossomos/metabolismo , Transportador de Glucose Tipo 4 , Membro Posterior , Insulina/farmacologia , Masculino , Ratos , Ratos Sprague-Dawley , Frações Subcelulares/metabolismoRESUMO
Insulin binding and 2-deoxy-d-glucose uptake were compared in the soleus, plantaris, and extensor digitorum longus (EDL) muscles, in vitro, since the known biochemical profile of the soleus differs markedly from the other two. The present study showed that insulin binding increased in all three muscles with increasing concentrations of insulin in the range of 0.2-30 nM. However, the increase in binding of insulin to soleus at each insulin concentration exceeded that observed in the other two muscles (P less than 0.05). Differences between the plantaris and EDL were not significant. The quantity of insulin bound at each concentration also increased more rapidly in the soleus than in either the plantaris (P less than 0.05) or EDL (P less than 0.05). Basal and insulin-stimulated uptake of 2-deoxy-d-glucose was also greater in the soleus than in the other muscles (P less than 0.05). Maximal 2-deoxy-d-glucose uptake occurred at 1 nM insulin in each of the three muscles. These results indicate that in metabolically distinct types of skeletal muscles glucose uptake can differ markedly, and this is related to differences in the insulin binding capacities of these muscles.
Assuntos
Desoxiaçúcares/metabolismo , Desoxiglucose/metabolismo , Insulina/metabolismo , Músculos/metabolismo , Animais , Relação Dose-Resposta a Droga , Insulina/farmacologia , Masculino , Camundongos , Músculos/efeitos dos fármacosRESUMO
We tested the hypothesis that increments in glucose metabolism in muscles from trained animals are caused by training adaptations in skeletal muscle and not by the residual effects of the last training session. The effects of a single bout of exercise on glucose metabolism (glycolysis and glycogenesis) were compared, against appropriate controls, in untrained (experiment 1) and trained (experiment 2) rat soleus muscles immediately (t = 0) and 3, 6, 24, 48, and 96 h after a standardized bout of exercise. [3H]Glucose incorporation into glycogen and glycolysis was measured in vitro in the absence and presence of insulin (0.1 and 10 nM). Experiment 1: A single bout of exercise provoked an increase in glycogenesis in the exercised, untrained muscles compared with the nonexercised, untrained muscles (0-96 h; P = 0.006). Glycolysis was not altered (0-96 h; P > 0.05). Experiment 2: In the exercised trained soleus, rates of glycolysis were greater than in the exercised, untrained soleus, at insulin concentrations of 0.1 nM (0-96 h; P = 0.005) and 10 nM (0-96 h; P = 0.01), but not in the absence of insulin (0-96 h; P > 0.05). No differences were observed in the rates of glycogenesis (0-96 h; P > 0.05). Therefore, acute exercise provokes increments in glycogenesis, whereas training increases glycolysis, in the presence of insulin, for some time after exercise. We speculate that insulin-dependent increments in glycolysis in trained muscles are a consequence of increased glucose transport caused by a greater pool of insulin-translocatable, intracellular glucose transporters.
Assuntos
Glucose/metabolismo , Insulina/farmacologia , Músculos/metabolismo , Condicionamento Físico Animal , Análise de Variância , Animais , Glicogênio/metabolismo , Glicólise/efeitos dos fármacos , Técnicas In Vitro , Cinética , Masculino , Músculos/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
OBJECTIVE: Alterations in myocardial metabolism occur early after the onset of diabetes suggesting that they may play a role in the development of cardiac dysfunction. Inhibition of myocardial pyruvate dehydrogenase (PDH), glucose transport and glycolysis have all been reported following diabetes. In vivo lactate is also a potential source of energy for the heart and its oxidation should not be affected by changes in glucose transport and glycolysis. Therefore, the objective of this study, was to test the hypothesis that following diabetes the inhibition of glucose oxidation would be greater than the inhibition of lactate oxidation. METHODS: Hearts from control and one-week-old diabetic rats were perfused with [1-13C]glucose (11 mmol/l) alone, [1-13C]glucose plus lactate (0.5 mmol/l) or glucose plus [3-13C]lactate (0.5 or 1.0 mmol/l) as substrates. Glucose and lactate oxidation rates were determined by combining 13C-NMR glutamate isotopomer analysis of tissue extracts with measurements of oxygen consumption. RESULTS: In diabetic hearts perfused with glucose alone, glucose oxidation was decreased compared to controls (0.31 +/- 0.08 vs. 0.71 +/- 0.11 mumoles/min/g wet weight; p < 0.05). Surprisingly, in hearts perfused with glucose plus 0.5 mmol/l lactate, there was no difference in glucose oxidation between control and diabetic groups (0.20 +/- 0.05 vs. 0.16 +/- 0.04 mumoles/min/g wet weight respectively). However, under these conditions lactate oxidation was markedly reduced in the diabetic group (0.89 +/- 0.18 vs. 0.24 +/- 0.05 mumoles/min/g wet weight; p < 0.05). At 1.0 mmol/l lactate oxidation was still significantly depressed in the diabetic group. CONCLUSION: There was a greater decrease in lactate oxidation relative to glucose oxidation in hearts from diabetic animals. These results demonstrate that diabetes leads to a specific inhibition of lactate oxidation independent of its effects on pyruvate dehydrogenase.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Glucose/farmacologia , Ácido Láctico/farmacologia , Miocárdio/metabolismo , Análise de Variância , Animais , Glucose/metabolismo , Ácido Láctico/metabolismo , Espectroscopia de Ressonância Magnética , Masculino , Oxirredução , Perfusão , Ratos , Ratos Sprague-DawleyRESUMO
OBJECTIVE: Previous studies have shown that chronic cardiac denervation impairs myocardial glucose oxidation. To investigate this further we tested whether the tissue content of glucose transporters, activity of glycolytic enzymes or metabolic capacity of pyruvate dehydrogenase were altered. Moreover, we investigated whether the decline in glucose utilization was associated with an upregulation of proteins and enzymes involved in fatty acid handling. Chronic cardiac denervation results also in decreased left ventricular efficiency. We explored whether alterations in mitochondrial properties could be held responsible for this phenomenon. METHODS: Twelve adult dogs were included in the study. In 6 of them chronic cardiac denervation was accomplished by surgical ablation of the extrinsic nerve fibers. The other 6 dogs were sham-operated. Biopsies were obtained from the left ventricle after 4-5 weeks of denervation. The content or enzymatic activity of proteins involved in fatty acid and glucose handling was assessed. Features of glutamate oxidation were measured in freshly isolated mitochondria. RESULTS: The content or activity of a set of fatty acid handling proteins did not change during chronic cardiac denervation. In contrast GLUT1 content significantly increased in the chronically denervated left ventricle, while the active form of pyruvate dehydrogenase declined (p < 0.05). Glutamate oxidation characteristics in freshly isolated mitochondria were not affected by chronic denervation. CONCLUSION: The impairment of glucose oxidation in the chronically denervated myocardium is most likely caused by a decline of pyruvate dehydrogenase in its active form. It is unlikely that the decrease in work efficiency is caused by alterations in mitochondrial properties.
Assuntos
Ácidos Graxos/metabolismo , Glucose/metabolismo , Proteínas Musculares , Miocárdio/metabolismo , Proteínas de Neoplasias , Simpatectomia , Animais , Carnitina Aciltransferases/metabolismo , Proteínas de Transporte/metabolismo , Cromatografia Líquida de Alta Pressão , Cães , Proteínas de Ligação a Ácido Graxo , Ácidos Graxos/sangue , Feminino , Transportador de Glucose Tipo 1 , Transportador de Glucose Tipo 4 , Ácido Glutâmico/metabolismo , Glicogênio/metabolismo , Coração/inervação , Metabolismo dos Lipídeos , Lipídeos/sangue , Masculino , Mitocôndrias Cardíacas/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Proteína P2 de Mielina/metabolismo , Oxirredução , Complexo Piruvato Desidrogenase/metabolismoRESUMO
The transport of long-chain fatty acids across cellular membranes most likely occurs to some extent by passive diffusion and additionally is facilitated by a number of membrane-associated and cytoplasmic proteins. In this overview we focus on the involvement of the membrane proteins fatty acid translocase (FAT/CD36), plasma membrane fatty acid-binding protein (FABPpm) and fatty acid-transport protein (FATP). Newly obtained evidence is presented that in skeletal muscle, fatty acid uptake is subject to short-term regulation by translocation of FAT/CD36 from intracellular stores to the plasma membrane, analogous to the regulation of muscular glucose uptake by GLUT-4 translocation. These new findings establish a significant role of membrane-associated proteins in the cellular fatty acid-uptake process. Possible implications for the uptake and transport of long-chain fatty acids by the brain are discussed.
Assuntos
Ácidos Graxos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras , Proteínas de Neoplasias , Proteínas Supressoras de Tumor , Albuminas/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Encéfalo/metabolismo , Antígenos CD36 , Proteínas de Transporte/metabolismo , Difusão , Estimulação Elétrica , Proteínas de Transporte de Ácido Graxo , Proteína 7 de Ligação a Ácidos Graxos , Proteínas de Ligação a Ácido Graxo , Humanos , Hipóxia-Isquemia Encefálica/metabolismo , Glicoproteínas de Membrana/metabolismo , Modelos Biológicos , Contração Muscular , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Palmitatos/metabolismo , Ratos , Sarcolema/metabolismoRESUMO
We hypothesized that glycogenesis increases in muscle during exercise before significant glycogen depletion occurs. Therefore, rats ran for 15 or 90 min at speeds of 8-22 m/min. D-[5-3H]glucose (10 microCi/100 g body wt) was administered 10 min before the end of exercise. Hindlimb muscles [soleus (SOL), plantaris (PL), extensor digitorum longus (EDL), and red (RG) and white gastrocnemius (WG)] and a portion of liver were analyzed for glycogen concentrations and rates of glycogen synthesis (i.e., D-[3H]glucose incorporated into glycogen). At rest, marked differences were observed among muscles in their rates of glucose incorporation into glycogen: i.e., SOL = 24.3 +/- 3.1, RG = 5.4 +/- 1.9, PL = 2.8 +/- 1.1, EDL = 0.54 +/- 0.10, WG = 0.12 +/- 0.02 (SE) dpm.micrograms glycogen-1.10 min-1 (P less than 0.05 between respective muscles). Compared with the glucose incorporation into glycogen at rest, increments in the PL (272%), RG (189%), WG (400%), EDL (274%), and liver (175%) were observed after 90 min of exercise (P less than 0.05, all data). In contrast, a decrease in glucose incorporation into glycogen (-62%) occurred in the SOL at min 15 (P less than 0.05), but this returned to the rates observed at rest after 90 min of exercise. This measure for rates of net glycogen synthesis (dpm.microgram glycogen-1.10 min-1) was weakly related to the ambient glycogen levels in most muscles; the exception was the SOL (r = -0.79; P less than 0.05). There was up to a 50-fold difference in glycogen synthesis among muscles.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Glicogênio/biossíntese , Glicogênio Hepático/biossíntese , Músculos/metabolismo , Condicionamento Físico Animal , Animais , Masculino , Ratos , Ratos EndogâmicosRESUMO
In the present study, we investigated the hypotheses that 1) skeletal muscle glyconeogenesis will increase after exercise, 2) greater changes in glyconeogenesis will be observed after exercise in fast-twitch muscles than in slow-twitch muscles, and 3) glycogen repletion will reduce the rates of glyconeogenesis. Mouse soleus and extensor digitorum longus (EDL) glycogen depots were reduced to the same levels by treadmill exercise (60 min) or epinephrine injection (75 micrograms/100 g body wt ip). Untreated animals were used as controls. We were able to prevent glycogen repletion by incubating muscles in vitro with sorbitol (75 mM) and to increase glycogen concentrations in vitro by incubating muscles with glucose (75 mM). The experimental results showed that glyconeogenesis was increased by exercise (EDL, +51%; soleus, +82%) when glycogen levels were kept low. When glycogen depots were increased, the rate of glyconeogenesis was lowered in the exercised EDL (P < 0.05) but not in the soleus (P > 0.05). Reductions in muscle glycogen by epinephrine did not change the rate of glyconeogenesis in EDL, either when glycogen depots were kept low or were repleted (P > 0.05). In contrast, in the soleus, epinephrine-induced reductions in glycogen did stimulate glyconeogenesis (P < 0.05). Analyses in EDL showed that in nonexercised muscles glycogen concentrations were minimally effective in altering the rates of glyconeogenesis. A 30% decrement in glycogen increased glyconeogenesis by 5% in resting muscles, whereas the same decrement increased glyconeogenesis by 51% in exercised muscles.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Glicogênio/biossíntese , Glicogênio/fisiologia , Músculos/metabolismo , Esforço Físico/fisiologia , Animais , Epinefrina/farmacologia , Glucose/metabolismo , Lactatos/metabolismo , Ácido Láctico , Masculino , Camundongos , Músculos/efeitos dos fármacosRESUMO
After 28 days of hindlimb-suspension, insulin binding, 2-deoxy-D-glucose (2-DG) uptake, and glucose metabolism (glycolysis and glycogenesis) were determined at various insulin concentrations (0.2-30 nM) in soleus muscle of young (18-day-old) and adult (150-day-old) rats. Compared with age-matched controls the young (YS) and adult suspended (AS) rats had lower soleus and body weights and insulin levels (P less than 0.05). Per milligram of protein, insulin binding, 2-DG uptake, and the rate of glycolysis were increased by approximately 200%, and the rate of glycogenesis was increased approximately 100% in the YS group (P less than 0.05). Except for a reduction in glycogenesis (P less than 0.05) all other parameters also increased in the AS rats (P less than 0.05). On the basis of the whole muscle the rate of glucose metabolism (glycogenesis + glycolysis) was reduced in the YS rats (P less than 0.05), but in the AS rats glucose metabolism was similar to the controls. Thus the increased glucose metabolism (i.e., per milligram of protein) in the YS and AS groups may represent a compensatory response by atrophied muscle to attempt to sustain glucose removal from the circulation. Because greater insulin binding occurred in YS muscle [35% slow-twitch (ST)] than in the control group (70% ST), and greater insulin binding occurred in the AS (81% ST) than in the control group (90% ST), higher insulin binding capacities are not always related to a high proportion of ST muscle fibers. In conclusion, after hindlimb suspension, marked increments in insulin binding and glucose metabolism occur in the soleus muscle.
Assuntos
Glucose/metabolismo , Membro Posterior , Imobilização , Insulina/metabolismo , Músculos/metabolismo , Envelhecimento/metabolismo , Animais , Desoxiglucose/metabolismo , Glicogênio/metabolismo , Glicólise , Cinética , Masculino , RatosRESUMO
The purpose of this study was to investigate whether epinephrine exerts an effect on glycogen metabolism in nonexercising (Non-Ex) as well as in exercising (Ex) skeletal muscle. Rats ran (15 m/min; 8% grade) on their forelimbs while their hindlimbs (Non-Ex) were suspended above the treadmill. Electromyographic records confirmed the lack of significant contractile activity in muscles during suspension. Plasma epinephrine levels were manipulated in three experimental groups (n = 20 for each group): adrenalectomized (ADX), intact adrenals (IA), and IA + epinephrine injection (+Ep). Another group of rats performed normal exercise on all four limbs (15 m/min; 8% grade). Muscle glycogen levels were measured in selected hindlimb muscles at t = 0 and after 90 min exercise (15 m/min; 8% grade) or suspended rest. In the absence of epinephrine (ADX), no glycogen loss was found (P greater than 0.05) in Non-Ex muscles during the exercise period. In the IA group (epinephrine levels elevated sixfold above basal at t = 90 min), glycogen levels in the nonexercising soleus, plantaris, and red and white gastrocnemius were significantly (P less than 0.05) depleted to 62 +/- 6, 67 +/- 6, 58 +/- 5, and 67 +/- 9% of control values, respectively. Similar decrements occurred in these muscles when exercise was performed on all four limbs (P greater than 0.05). We conclude that glycogenolysis occurs in nonexercising skeletal muscle independent of contractile activity, probably due to the effect of epinephrine. Furthermore, the present data strongly suggest that glycogen depletion patterns in muscles during exercise cannot be used as an index of motor unit recruitment.