RESUMEN
The stress-activated protein kinases (SAPKs) are differentially activated by a variety of cellular stressors in PC12 cells. SAPK activation has been linked to the induction of apoptotic cell death upon serum withdrawal from undifferentiated cells or following nerve growth factor (NGF) withdrawal of neuronally differentiated PC12 cells. However, withdrawal of trophic support from differentiated cells led to only a very modest elevation of SAPK activity and led us to investigate the basis of the relative insensitivity of these enzymes to stressors. NGF-stimulated differentiation of the cells resulted in the elevation of basal SAPK activity to levels four- to sevenfold greater than in untreated cells, which was correlated with an approximate fivefold increase in SAPK protein levels. Paradoxically, in NGF-differentiated PC12 cells, exposure to cellular stressors provoked a proportionately smaller stimulation of SAPK activity than that observed in naive cells, despite the presence of much higher levels of SAPK protein. The insensitivity of SAPK to activation by stressors was reflective of the activity of the SAPK activator SEK, whose activation was also diminished following NGF differentiation of the cells. The data demonstrate that SAPKs are subject to complex controls through both induction of SAPK expression and the regulation mediated by upstream elements within this pathway.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/biosíntesis , Proteínas Quinasas Activadas por Mitógenos , Factores de Crecimiento Nervioso/farmacología , Estrés Fisiológico/enzimología , Animales , Western Blotting , Diferenciación Celular/fisiología , Medio de Cultivo Libre de Suero , Calor , Proteínas Quinasas JNK Activadas por Mitógenos , Células PC12 , Proteínas Proto-Oncogénicas c-jun/biosíntesis , Ratas , Estimulación QuímicaRESUMEN
The spontaneously hypertensive rat (SHR) has been reported to be insulin-resistant compared to the Wistar-Kyoto (WKY) parent strain. Because insulin resistance usually reflects a defect in insulin action at the muscle, we compared the ability of muscle (gastrocnemius) to store glycogen in response to a standard oral glucose challenge in SHR to that in WKY. As a control, we examined the glycogen response in liver in these two rat strains. However, in vivo insulin action reflects both tissue responsiveness as well as substrate and hormone availability at the tissue level. To evaluate tissue responsiveness in vitro, we examined two parameters of insulin action: 1) muscle glycogen synthesis using 3H-glucose and 2) muscle glucose transport using 3H-2-deoxy-glucose (3H-2-DG). Thirteen-week-old male rats were studied after overnight fasting. Liver glycogen increased similarly (mean +/- SD shown) in response to glucose gavage feeding in both groups [WKY: 15.2 +/- 6.9 to 50.6 +/- 17.9 micromol/g wet wt (P < .05); SHR: 30 +/- 18 to 63.5 +/- 33.3 micromol/g wet wt (P < .01)]. On the other hand, muscle glycogen increased in WKY [13.7 +/- 2 to 17.8 +/- 1.1 micromol/g wet wt (P < .05)], whereas in SHR there was no significant change [14.6 +/- 2.1 to 15.3 +/- 2.99 micromol/g wet wt P = NS)]. Results of in vitro studies demonstrated that glycogen synthesis increased from 377 +/- 120 to 439 +/- 175 disintegrations per minute (dpm) 3H-glucose/mg extensor digitorum longus (EDL) in WKY when insulin increased from 0 to 1000 microU/mL (P < .05), whereas SHR the increase was from 289 +/- 89 to 565 +/- 187 (P < .05). Glucose transport increased from 483 +/- 74 to 785 +/- 369 dpm 3H-2-DG/mg EDL in WKY when insulin was increased from 0 to 500 microU/mL (P < .03), whereas in SHR the increase was 516 +/- 61 to 997 +/- 347 (P < .001). In summary, liver glycogen increased in response to feeding in a similar manner in both WKY and SHR, whereas muscle glycogen increased only in WKY. We conclude that in vivo muscle glycogen accumulation may represent an index of insulin resistance in SHR. In contrast, in vitro data suggest that both muscle glucose transport and glycogen synthesis were stimulated to a comparable degree by insulin in EDL strips from WKY and SHR; there were no significant differences between WKY and SHR. Further studies are needed to clarify these differences.
Asunto(s)
Hipertensión/metabolismo , Resistencia a la Insulina , Insulina/farmacología , Músculo Esquelético/efectos de los fármacos , Animales , Transporte Biológico , Glucosa/metabolismo , Glucógeno/metabolismo , Técnicas In Vitro , Masculino , Músculo Esquelético/metabolismo , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKYRESUMEN
Frequent coexistence of insulin resistance, central obesity, and hypertriglyceridemia in the same individual suggests an underlying common pathogenesis. Insulin resistance and hypertriglyceridemia can be induced by carbohydrate feeding in rats. Golden Syrian hamsters are believed to be resistant to the metabolic effects of dietary carbohydrates. We investigated the effects of diets containing 60% fructose or sucrose on glucose and lipid metabolism in hamsters, both in the fasting state and during an intravenous glucose tolerance test. Fructose caused obesity (weight after treatment: 131 +/- 7 gm in the control group, 155 +/- 5 gm in the fructose group, 136 +/- 7 gm in sucrose group, p < 0.04). Fructose also reduced glucose disappearance rate (KG: 2.69% +/- 0.39% in the control group, 1.45% +/- 0.18% in the fructose group, p < 0.02). Sucrose caused a marginal decrease in glucose disappearance (KG: 1.93% +/- 0.21%, p = 0.08 vs the control group). Only fructose feeding increased fasting plasma nonesterified fatty acids (0.645 +/- 0.087 mEq/L in the control group, 1.035 +/- 0.083 mEq/L in the fructose group, 0.606 +/- 0.061 mEq/L in the sucrose group, p < 0.002), plasma triglycerides (84 +/- 6 mg/dl in the control group, 270 +/- 65 mg/dl in the fructose group, 94 +/- 16 mg/dl in the sucrose group, p < 0.0002), and liver triglycerides (1.88 +/- 0.38 mg/gm liver weight in the control group, 2.35 =/- 0.24 mg/gm in the fructose group, 1.41 +/- 0.13 mg/gm in the sucrose group, p < 0.04). Previous studies in the rat have suggested that dietary carbohydrates induce insulin resistance by increasing plasma nonesterified fatty acids and triglycerides, which are preferentially used by the muscles. The present report shows that sucrose also can cause some decrease in glucose disappearance in the hamster without causing hypertriglyceridemia or increasing plasma nonesterified fatty acids. Thus other mechanisms may also contribute to the insulin resistance in the hamster. These findings suggest that hamsters provide a good model for investigation of hormonal and nutritional regulation of glucose and lipid metabolism.
Asunto(s)
Fructosa/toxicidad , Glucosa/metabolismo , Mesocricetus/metabolismo , Sacarosa/toxicidad , Triglicéridos/metabolismo , Animales , Glucemia/análisis , Peso Corporal/efectos de los fármacos , Cricetinae , Dieta , Ácidos Grasos no Esterificados/sangre , Fructosa/administración & dosificación , Glucógeno/análisis , Insulina/sangre , Glucógeno Hepático/análisis , Masculino , Músculos/química , Tamaño de los Órganos/efectos de los fármacos , Sacarosa/administración & dosificación , Triglicéridos/análisis , Triglicéridos/sangreRESUMEN
The ability of neonatal astrocytes to promote neurite outgrowth in vitro and in vivo diminishes as astrocytes mature. This property correlates with the developmental loss of the central nervous system's ability to regenerate after injury. Cell lines representative of immature and mature astrocytes would be useful for studies to determine differences between these two populations. Previous work on immortalization of bipotential neural/glial precursors and fully differentiated glial cells suggests that immortalization of astrocytes at timed intervals of culture may yield cell lines trapped in different maturation states. To test this, neonatal mouse cortical astrocytes were immortalized by retrovirus-mediated transfer of the SV40 T antigen (Tag) gene at 2, 6 and 17 days of culture. The clonal cell lines express Tag and are contact-inhibited. Three phenotypes that change as a function of astrocyte maturation were examined to determine the fidelity with which the cell lines represent immature and mature astrocytes. These were: (1) cell morphology, growth pattern and size, (2) level of glial fibrillary acidic protein (GFAP) expression, and (3) neurite outgrowth promotion. First, immature and mature lines resemble mortal type 1 astrocytes of corresponding ages with respect to morphology and growth pattern, and retain a quantitative difference in cell size (mature cells are larger). Second, the pattern of GFAP expression is preserved, with immature lines expressing lower levels than mature cell lines, but the overall GFAP levels are significantly lower in immortalized cell lines compared to mortal cells. Finally, promotion of neurite outgrowth from embryonic chick retinal ganglion cells on monolayers of the cell lines was examined. While all neurite outgrowth measures are significantly greater for the immortalized lines than for control 3T3 cells, they are attenuated relative to mortal astrocytes. The age-related pattern of stronger outgrowth support on immature astrocytes is retained for neurite initiation, but not retained for mean neurite length. Thus, SV40 Tag-immortalized astrocytes have a complex phenotype characterized by retention of age-related differences in morphology, growth pattern and cell size, and by a marked attenuation of some astrocyte-specific characteristics but retention of age-related differences in the expression level of these same characteristics, and finally, loss of the ability to support neurite extension at level characteristic of immature astrocytes.
Asunto(s)
Antígenos Transformadores de Poliomavirus/fisiología , Astrocitos/citología , Transformación Celular Viral , Células 3T3 , Animales , Animales Recién Nacidos , Diferenciación Celular , Línea Celular Transformada , Supervivencia Celular , Senescencia Celular , Embrión de Pollo , Inhibición de Contacto , Proteína Ácida Fibrilar de la Glía/biosíntesis , Ratones , Ratones Endogámicos C57BL , Neuritas/fisiología , Proteínas Recombinantes de Fusión , Células Ganglionares de la Retina/ultraestructura , TransfecciónRESUMEN
In previous studies, interleukin-1 (IL-1) or tumor necrosis factor (TNF) have been demonstrated to augment skeletal muscle protein breakdown in a manner similar to that induced by bacterial endotoxin. This response to IL-1 or TNF was elicited only after they were administered to animals for various periods, as their addition in vitro to incubated muscles from normal untreated rats was without effect. This suggested that IL-1 and TNF may augment muscle proteolysis in an indirect fashion. Serum levels of IL-1, TNF as well as interleukin-6 (IL-6) are all elevated during infection induced by bacterial endotoxin. Both IL-1 and TNF can induce the synthesis of IL-6 by a variety of cells. Because of this, in the present report, the ability of IL-6 to stimulate skeletal muscle protein breakdown was examined. Muscle protein breakdown was evaluated by measuring the release of both tyrosine and 3-methylhistidine by incubated muscles. Pretreatment of rats with IL-6 for 6 hr induced fever and increased the release of both tyrosine and 3-methylhistidine by the extensor digitorum longus muscle. However, IL-6 did not augment muscle proteolysis when muscles from normal untreated rats were incubated in the presence of the cytokine. The data suggest that the acute treatment of animals with IL-6 can augment muscle proteolysis. Whether this response is due to a direct effect of IL-6 on the myocyte or whether it is due to the production of other mediators remains unclear.
Asunto(s)
Interleucina-6/farmacología , Proteínas Musculares/metabolismo , Músculos/efectos de los fármacos , Animales , Temperatura Corporal/efectos de los fármacos , Técnicas In Vitro , Inyecciones Intraperitoneales , Inyecciones Intravenosas , Interleucina-6/administración & dosificación , Masculino , Metilhistidinas/metabolismo , Proteínas Musculares/efectos de los fármacos , Músculos/metabolismo , Ratas , Ratas Sprague-Dawley , Tirosina/metabolismoRESUMEN
Two glial cell types surround olfactory axons and glomeruli in the olfactory bulb (OB) and may influence synapse development and regeneration. OB astrocytes resemble type-1 astrocytes, and OB ensheathing cells resemble non-myelinating Schwann cells. We have produced clonal OB astrocyte and ensheathing cell lines from rat neonatal and adult OB cultures by SV40 large T antigen transduction. These cell lines have been characterized by morphology, growth characteristics, immunophenotype, and ability to promote neurite outgrowth in vitro. Neonatal and adult ensheathing cell lines were found to support higher neurite outgrowth than OB astrocyte lines. Neonatal OB astrocyte lines were of two types, high and low outgrowth support. The low support astrocyte lines express J1 and a chondroitin sulfate-containing proteoglycan as do astrocytes encircling the neonatal glomeruli in vivo. The adult OB astrocyte cell lines supported lower levels of outgrowth than adult ensheathing cell lines. These results are consistent with a positive role for ensheathing cells in OB synapse regeneration, in vivo. Further, based on our results, we hypothesize that ensheathing cells and high-outgrowth astrocytes facilitate axon growth in vivo, while low outgrowth astrocytes inhibit axon growth and may facilitate glomerulus formation.
Asunto(s)
Astrocitos/citología , Corteza Cerebral/citología , Neuritas/ultraestructura , Neuroglía/citología , Bulbo Olfatorio/citología , Células Ganglionares de la Retina/citología , Envejecimiento , Animales , Animales Recién Nacidos , Comunicación Celular , Línea Celular , Separación Celular/métodos , Embrión de Pollo , Células Clonales , Técnicas de Cultivo/métodos , Neuritas/fisiología , Neuroglía/ultraestructura , Bulbo Olfatorio/crecimiento & desarrollo , Ratas , Ratas Sprague-DawleyRESUMEN
A streptavidin-biotin-based three-step immunolabeling protocol for quantitative staining of intracellular antigens for flow cytometric analysis was evaluated using simian virus 40 (SV40) large T antigen. The concentration as well as the quantity of antibody used required optimization. The optimum labeling conditions varied moderately with cell lines that express T antigen levels over a 40-50-fold range. The procedure resulted in specific fluorescence 2.4 times higher than that using a comparable two-step indirect immunofluorescence technique. The gain in resolution was shown to be greater when staining cells with lower antigen levels. In the analysis of background fluorescence, the principal components were, as for the two-step technique, autofluorescence and propidium spectral overlap. While streptavidin does add to the background, the increase is relatively small. Decreasing the propidium concentration from 50 micrograms/ml to 5 micrograms/ml was found to reduce significantly the level of background from this source. Theoretical aspects of quantitative staining and of resolution versus quantification are discussed.
Asunto(s)
Antígenos/análisis , Proteínas Bacterianas , Citometría de Flujo/métodos , Coloración y Etiquetado , Células 3T3 , Animales , Antígenos Transformadores de Poliomavirus/análisis , Biotina , Ratones , Proteínas Recombinantes de Fusión , Sensibilidad y Especificidad , EstreptavidinaRESUMEN
The metabolic response to infection includes loss of lean tissue and increased nitrogen excretion. The loss of muscle tissue during infection results in large part from accelerated skeletal muscle protein breakdown. Recent studies suggest that macrophage-derived products secreted during infection may signal increased muscle proteolysis. To test this, in the present report the ability of interleukin (IL-1) and tumor necrosis factor (TNF) to enhance muscle proteolysis was examined. Young rats were injected intravenously with either recombinant human IL-1 or TNF. For comparison some rats were injected with bacterial endotoxin. Eight hours after each treatment, the extensor digitorum longus muscles were isolated and incubated in vitro to assess muscle proteolysis by measuring tyrosine and 3-methyl-L-histidine release by the incubated muscles. Treatment of rats with either IL-1, TNF, or endotoxin all induced fever, increased serum lactate, and reduced serum zinc levels. Despite similar metabolic changes, muscle proteolysis responded differently. As expected, endotoxin treatment enhanced muscle protein breakdown, whereas IL-1 treatment was without effect. On the other hand, TNF was effective in accelerating muscle protein breakdown. TNF addition in vitro failed to enhance muscle proteolysis by incubated muscles, suggesting that its effects may be mediated in an indirect manner; however, a direct mode of action cannot yet be ruled out. Overall, the data indicate that the acute administration of TNF can signal increased muscle proteolysis similar to that observed during infection.
Asunto(s)
Endotoxinas/farmacología , Interleucina-1/farmacología , Proteínas Musculares/metabolismo , Músculos/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Animales , Temperatura Corporal/efectos de los fármacos , Masculino , Metilhistidinas/análisis , Músculos/efectos de los fármacos , Ratas , Ratas Endogámicas , Proteínas Recombinantes/farmacologíaRESUMEN
Myofibrillar protein breakdown in skeletal muscle progresses through two distinct phases in response to chronic glucocorticoid administration in the rat, i.e., an early phase lasting 4-5 days, during which proteolysis increases followed by a later phase during which proteolysis decreases. The possible involvement of insulin and the iodothyronines in this phenomenon has now been examined. Diabetic, thyroidectomized, and normal rats were treated with corticosteroid for 10-11 days, and at timed intervals muscle proteolysis was evaluated by measuring the release of 3-methyl-L-histidine (3-MH) and tyrosine from the perfused hindquarter as well as the excretion of 3-MH in the urine. Corticosterone (CTC) administration to normal rats increased plasma insulin, whereas plasma 3,5,3'-triiodothyronine responded with an early rise followed by a fall after 4-5 days. However, the biphasic response of myofibrillar proteolysis to chronic glucocorticoid treatment was not abolished in CTC-treated diabetic or thyroidectomized rats. CTC treatment increased release of tyrosine by perfused muscle of diabetic rats but, unlike 3-MH release, did not diminish later. Thus the adaptation of myofibrillar proteolysis to chronic glucocorticoid treatment appears to be independent of insulin and thyroid hormones. However, insulin may play a role in curtailing glucocorticoid-induced breakdown of nonmyofibrillar proteins.
Asunto(s)
Corticosterona/farmacología , Diabetes Mellitus Experimental/fisiopatología , Proteínas Musculares/metabolismo , Músculos/fisiopatología , Miofibrillas/metabolismo , Tiroidectomía , Animales , Peso Corporal/efectos de los fármacos , Masculino , Metilhistidinas/metabolismo , Músculos/efectos de los fármacos , Músculos/metabolismo , Ratas , Ratas Endogámicas , Valores de Referencia , Tirosina/metabolismoRESUMEN
The goal of this study was to assess the effects of voluntary running activity in rats on various aspects of carbohydrate and protein metabolism. After 6 wk of exercise training, rats (ET) were rested for 24 h and their hindquarters, along with those from sedentary control (SC) and dietary control (DC) rats, were perfused with 0, 60, 250, or 6,000 microU/ml insulin. At 0 insulin, glucose clearance was similar for all groups, and it was increased with added insulin. However, the insulin effect was 20-40% greater for ET rats at all insulin concentrations (P less than 0.05). Muscle glycogen deposition also increased with added insulin but showed muscle-specific differences. Specifically, glycogen content of the plantaris muscle was significantly higher in ET compared with SC or DC rats, whereas this pattern was reversed in soleus muscle. In plantaris muscle, insulin stimulated glucose 6-phosphate (G-6-P)-independent (-G-6-P) glycogen synthase activity only in SC and DC rats and increased its affinity for G-6-P at 250 microU/ml in all groups. In contrast, the -G-6-P synthase activity was not increased in soleus muscle and was actually decreased in all groups at 6,000 microU/ml. Tyrosine release was suppressed by insulin in all groups, but this effect was significantly greater at insulin levels of 60 microU/ml (P less than 0.02) in hindquarters from ET rats compared with SC and DC rats. Neither insulin nor exercise training decreased 3-methylhistidine release from perfused hindquarters.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Metabolismo de los Hidratos de Carbono , Insulina/farmacología , Músculos/metabolismo , Esfuerzo Físico , Proteínas/metabolismo , Animales , Glucemia/metabolismo , Glucosa/metabolismo , Glucógeno/metabolismo , Glucógeno Sintasa/metabolismo , Miembro Posterior , Masculino , Metilhistidinas/metabolismo , Músculos/efectos de los fármacos , Perfusión , Ratas , Ratas Endogámicas , Tirosina/metabolismoRESUMEN
During the fasted-refed transition, hepatic glycogen repletion from glucose can occur by the direct and indirect pathway. In the indirect pathway, glucose is first metabolized to 3-carbon intermediates that then are converted in the liver to glucose 6-phosphate via the gluconeogenic pathway before conversion to glycogen. The present study evaluated whether skeletal muscle is a major source of 3-carbon intermediates (i.e., lactate, pyruvate, and alanine) during refeeding of 1-day fasted rats. Arteriovenous differences for lactate, pyruvate, and alanine across the anesthetized rat hindlimbs were used to evaluate muscle metabolism in the fed, fasted, and refed state. In the fasted state, liver glycogen was depleted, and muscle released 3-carbon intermediates. One hour after refeeding, hepatic glycogen was 30% repleted, and blood lactate, pyruvate, and alanine increased. Despite this, the release of alanine by muscle diminished at this time and lactate was removed. At 4 h after refeeding, 3-carbon intermediates were all released by hindlimb tissue but in an amount not greater than in the fasted state. Overall, these results suggest that skeletal muscle in the rat is not a major source of 3-carbon precursors for early postprandial hepatic glycogen repletion via the indirect pathway, nor is the rise in 3-carbon intermediates in blood during refeeding caused by their increased output by muscle.
Asunto(s)
Ingestión de Alimentos , Ayuno , Gluconeogénesis , Glucógeno/metabolismo , Músculos/metabolismo , Alanina/sangre , Animales , Glucemia/metabolismo , Lactatos/sangre , Hígado/metabolismo , Glucógeno Hepático/metabolismo , Masculino , Músculos/irrigación sanguínea , Piruvatos/sangre , Ratas , Ratas Endogámicas , Valores de Referencia , Flujo Sanguíneo RegionalRESUMEN
In order to clarify the cellular mechanisms of denervation atrophy of skeletal muscle, we have studied protein turnover in denervated and control rat soleus muscles in vitro under different conditions. By 24 h after cutting the sciatic nerve, overall protein breakdown was greater in the denervated soleus than in the contralateral control muscle, and by 3 days, net proteolysis had increased about 3-fold. Since protein synthesis increased slightly following denervation, the rise in proteolysis must be responsible for the muscle atrophy and the differential loss of contractile proteins. Like overall proteolysis, the breakdown of actin (as shown by 3-methyl-histidine production by the muscles) increased each day after denervation and by 3 days was 2.5 times faster than in controls. Treatments that block the lysosomal and Ca2(+)-dependent proteolytic systems did not reduce the increase in overall protein degradation and actin breakdown in the denervated muscles (maintained in complete medium at resting length). However, the content of the lysosomal protease, cathepsin B, increased about 2-fold by 3 days after denervation. Furthermore, conditions that activate intralysosomal proteolysis (incubation without insulin or amino acids) stimulated proteolysis 2-3-fold more in the denervated muscles than in controls. Also, incubation conditions that activate the Ca2(+)-dependent pathway (incubation with Ca2+ ionophores or allowing muscles to shorten) were 2-3 times more effective in enhancing overall proteolysis in the denervated muscle. None of these treatments affected 3-methylhistidine production. Thus, multiple proteolytic systems increase in parallel in the denervated muscle, but a nonlysosomal process (independent of Ca2+) appears mainly responsible for the rapid loss of cell proteins, especially of myofibrillar components.
Asunto(s)
Catepsina B/metabolismo , Desnervación Muscular , Proteínas Musculares/metabolismo , Músculos/metabolismo , Péptido Hidrolasas/metabolismo , Actinas/metabolismo , Animales , Atrofia , Insulina/farmacología , Lisosomas/enzimología , Masculino , Metilhistidinas/análisis , Músculos/inervación , Músculos/patología , Miofibrillas/metabolismo , Inhibidores de Proteasas/farmacología , Ratas , Ratas Endogámicas , Valores de Referencia , Retículo Sarcoplasmático/metabolismoRESUMEN
This study examined whether insulin stimulation of protein synthesis and inhibition of protein degradation is enhanced after exercise. The isolated perfused rat hindquarter preparation was used to evaluate net protein breakdown, myofibrillar protein degradation, and protein synthesis. Thirty minutes after treadmill exercise of high and moderate intensity, rates of tyrosine release were increased by 58 and 25%, respectively. Insulin at 75 microU/ml had no effect on these increases after intense exercise; however, 20,000 microU/ml of insulin totally inhibited this increase. Cycloheximide increased the tyrosine release in both control and exercised rat muscle. It also abolished the difference between them, suggesting that the increase in tyrosine release after exercise is caused by an inhibition of protein synthesis. Phenylalanine incorporation into protein was marginally depressed (22%, P = NS) in the white gastrocnemius muscle after intense exercise. Insulin at 200 microU/ml stimulated protein synthesis in these rats, but no more than it did in a nonexercised control group. Failure to observe a greater effect of insulin on protein metabolism was also noted when rat muscle was studied 150 min after intense exercise and after contractions induced by electrical stimulation of the sciatic nerve. These findings suggest that after exercise or electrically induced contractions the enhanced ability of insulin to stimulate hexose and amino acid transport is not paralleled by an increase in its ability to stimulate protein synthesis or inhibit protein degradation.
Asunto(s)
Insulina/farmacología , Músculos/fisiología , Esfuerzo Físico , Biosíntesis de Proteínas , Animales , Radioisótopos de Carbono , Masculino , Músculos/efectos de los fármacos , Músculos/metabolismo , Perfusión , Fenilalanina/metabolismo , Ratas , Ratas Endogámicas , Valores de Referencia , Tirosina/sangreRESUMEN
Deuterium labelled glucose has been used to study the pathway of hepatic glycogen synthesis during the fasted-refed transition in rats. Deuterium enrichment of liver glycogen was determined using nuclear magnetic resonance as well as mass spectroscopy. Sixty minutes after oral administration of deuterated glucose to fasted rats, the portal vein blood was fully enriched with deuterated glucose. Despite this, less than half of the glucose molecules incorporated into liver glycogen contained deuterium. The loss of deuterium label from glucose is consistent with hepatic glycogen synthesis by an indirect pathway requiring prior metabolism of glucose. The use of deuterium labelled glucose may prove to be a useful probe to study hepatic glycogen metabolism. Its use may also find application in the study of liver glycogen metabolism in humans by a noninvasive means.
Asunto(s)
Deuterio , Glucosa , Glucógeno/biosíntesis , Hígado/metabolismo , Animales , Glucemia/metabolismo , Espectroscopía de Resonancia Magnética , Masculino , Modelos Biológicos , Ratas , Ratas EndogámicasRESUMEN
Myofibrillar protein breakdown was evaluated by measuring the release of N tau-methylhistidine by isolated rat skeletal muscles or perfused rat muscles in the presence of a variety of agents known to affect Na+ flux. Total cell proteolysis was evaluated simultaneously by measuring tyrosine release by muscles after the inhibition of protein synthesis with cycloheximide. Treatment of muscles with the Na+ ionophore monensin or inhibitors of Na+-K+ ATPase (ouabain, digoxin or vanadate) decreased N tau-methylhistidine release by muscles by 21-35%. A phorbol ester (phorbol 12-myristate 13-acetate) as well as a synthetic diacylglycerol known to activate protein kinase C and a Na+/H+ antiport also decreased N tau-methylhistidine release by muscles. Removal of extracellular Na+ blocked the ability of these agents to attenuate N tau-methylhistidine release by muscles, suggesting that their effectiveness required a change in Na+ flux. In contrast with N tau-methylhistidine release by muscles, these agents, except for monensin, did not effect the release of tyrosine, suggesting that they attenuate specifically the breakdown of myofibrillar proteins. Overall these results indicate a link between Na+ and the regulation of protein breakdown in rat skeletal muscle, whereby an influx of Na+ can result in a decrease in myofibrillar proteolysis. Left unresolved is whether phospholipid hydrolysis is involved in this scheme.
Asunto(s)
Proteínas Musculares/metabolismo , Sodio/metabolismo , Animales , Diglicéridos/farmacología , Digoxina/farmacología , Masculino , Metilhistidinas/metabolismo , Monensina/farmacología , Músculos/efectos de los fármacos , Miofibrillas/metabolismo , Ouabaína/farmacología , Péptidos/farmacología , Ratas , Ratas Endogámicas , Acetato de Tetradecanoilforbol/farmacología , Vanadatos/farmacologíaRESUMEN
Previous studies have demonstrated that brief fasting augments and refeeding a complete diet diminishes the breakdown of myofibrillar proteins in rat skeletal muscle. The purpose of the present study was to determine which dietary component(s) was responsible for this effect and to determine the role of insulin and amino acids. Myofibrillar proteolysis was evaluated by measuring the release of 3-methylhistidine by perfused rat muscle of 1-day fasted rats and 1-day fasted rats refed for 4-24 h with a complete, protein-free, or lipid meal. For comparison, tyrosine release by perfused muscle was measured in the absence and presence of cycloheximide to evaluate net and total proteolysis, respectively. Refeeding of either diet increased plasma insulin. Despite this, myofibrillar proteolysis decreased only when protein or amino acids was included in the test meal. On the other hand, the complete or protein-free meal decreased tyrosine release in the absence but not in the presence of cycloheximide, suggesting that either diet enhanced muscle protein synthesis. Most amino acids in plasma and muscle decreased after refeeding the protein-free meal, whereas after the complete meal some amino acids in plasma and muscle increased, whereas other decreased or changed little. These results indicate that decreased myofibrillar proteolysis in muscle after refeeding of food-deprived rats requires dietary protein or amino acids. They also suggest that hormonal and/or nutritional factors other than insulin and amino acids may orchestrate this response. However, a role of amino acids cannot yet be excluded, because it is conceivable that changes in specific amino acids in plasma instead of muscle may signal diminished proteolysis.
Asunto(s)
Aminoácidos/farmacología , Proteínas en la Dieta/farmacología , Alimentos , Miofibrillas/metabolismo , Péptido Hidrolasas/metabolismo , Ácido 3-Hidroxibutírico , Aminoácidos/sangre , Aminoácidos/metabolismo , Animales , Glucemia/análisis , Peso Corporal , Grasas de la Dieta/farmacología , Miembro Posterior , Hidroxibutiratos/sangre , Insulina/sangre , Masculino , Músculos/metabolismo , Proteínas/metabolismo , Ratas , Ratas EndogámicasRESUMEN
Previous studies have reported that prolonged administration of pharmacological doses of glucocorticoids in young rats results in a rise in urinary 3-methyl-L-histidine (3-MH) excretion followed by a fall to initial levels by 8 days. To determine whether this response reflects events in skeletal muscle, protein breakdown in this tissue was evaluated using the perfused hindquarter preparation with rats treated with corticosterone (10 mg X 100 g-1 X day-1) for 2, 4, or 8 days. Myofibrillar and total cell proteolysis were evaluated by measuring the release of 3-MH and tyrosine, respectively, after inhibition of protein synthesis with cycloheximide. Corticosterone treatment resulted in an early increase (1-4 days) followed by a fall (4-8 days) in 3-MH excretion. 3-MH release by the perfused hindquarter of treated rats responded in a similar manner, in that its release increased at days 2 and 4 and decreased to control levels by day 8. On the other hand, corticosterone treatment did not affect the release of tyrosine by the perfused hindquarter. Corticosterone treatment diminished protein synthesis in muscle by 30-50% (P less than 0.01), which unlike 3-MH release by perfused muscle persisted throughout the treatment period. The data indicate that glucocorticoids specifically augment the breakdown of myofibrillar proteins in skeletal muscle. This response is attenuated with prolonged treatment and is unrelated to a loss of metabolic effectiveness of the steroid. Also our findings suggest that the breakdown of myofibrillar and nonmyofibrillar proteins might be regulated independently.
Asunto(s)
Corticoesteroides/farmacología , Proteínas Musculares/metabolismo , Miofibrillas/metabolismo , Animales , Peso Corporal , Masculino , Metilhistidinas/orina , Proteínas Musculares/biosíntesis , Músculos/anatomía & histología , Músculos/metabolismo , Tamaño de los Órganos , Perfusión , Ratas , Ratas Endogámicas , Tirosina/metabolismoRESUMEN
The decrease in plasma lactate during dichloroacetate (DCA) treatment is attributed to stimulation of lactate oxidation. To determine whether DCA also inhibits lactate production, we measured glucose metabolism in muscles of fed and fasted rats incubated with DCA and insulin. DCA increased glucose-6-phosphate, an allosteric modifier of glycogen synthase, approximately 50% and increased muscle glycogen synthesis and glycogen content greater than 25%. Lactate release fell; inhibition of glycolysis accounted for greater than 80% of the decrease. This was associated with a decrease in intracellular AMP, but no change in citrate or ATP. When lactate oxidation was increased by raising extracellular lactate, glycolysis decreased (r = - 0.91), suggesting that lactate oxidation regulates glycolysis. When muscle lactate production was greatly stimulated by thermal injury, DCA increased glycogen synthesis, normalized glycogen content, and inhibited glycolysis, thereby reducing lactate release. The major effect of DCA on lactate metabolism in muscle is to inhibit glycolysis.
Asunto(s)
Acetatos/farmacología , Ácido Dicloroacético/farmacología , Glucógeno/biosíntesis , Glucólisis/efectos de los fármacos , Insulina/farmacología , Músculos/metabolismo , Animales , Transporte Biológico Activo/efectos de los fármacos , Glucosa/metabolismo , Técnicas In Vitro , Cinética , Lactatos/metabolismo , Masculino , Músculos/efectos de los fármacos , Ratas , Ratas EndogámicasRESUMEN
The influence of Ca2+ on myofibrillar proteolysis was evaluated in the isolated extensor digitorum longus muscle incubated in vitro with agents previously shown to increase the intracellular concentration of Ca2+. Myofibrillar proteolysis was evaluated by measuring the release of N tau-methylhistidine, and total proteolysis was evaluated by measuring tyrosine release by incubated muscles after the inhibition of protein synthesis with cycloheximide. Incubated muscles released measurable quantities of N tau-methylhistidine, and muscle contents of the amino acids remained stable over 2 h of incubation. The release of N tau-methylhistidine by incubated muscles was similar to its release by perfused rat muscle in response to brief starvation, indicating the integrity of the incubated muscles. Ca2+ ionophore A23187, dibucaine, procaine, caffeine and elevated K+ concentration increased lactate release by incubated muscles and decreased tissue contents of ATP and phosphocreatine to varying degrees, indicating the metabolic effectiveness of the agents tested. Only A23187 and dibucaine increased total cell Ca2+, and they increased tyrosine release. Caffeine and elevated [K+] increased neither cell Ca2+ nor tyrosine release; however, only A23187 and dibucaine increased tyrosine release significantly. On the other hand, these agents were without effect on myofibrillar proteolysis as assessed by N tau-methylhistidine release by incubated muscles and changes in tissue contents of the amino acid. In fact, some of the agents tested tended to decrease myofibrillar proteolysis slightly. These results indicate that acute elevation of intracellular Ca2+ is associated with increased breakdown of non-myofibrillar but not myofibrillar proteins. Because of this, the role of elevated Ca2+ in muscle atrophy in certain pathological states is questioned. The data also indicate that the breakdown of myofibrillar and non-myofibrillar proteins in muscle is regulated independently and by different pathways, a conclusion reached in previous studies with perfused rat muscle.
Asunto(s)
Calcio/metabolismo , Proteínas Musculares/metabolismo , 2,4-Dinitrofenol , Adenosina Trifosfato/metabolismo , Animales , Dinitrofenoles/farmacología , Lactatos/metabolismo , Ácido Láctico , Masculino , Metilhistidinas/metabolismo , Fosfocreatina/metabolismo , Ratas , Ratas Endogámicas , Inanición/metabolismo , Tirosina/metabolismoRESUMEN
Previous studies indicated that protein sparing in skeletal muscle during prolonged starvation depends on the availability of lipid fuels. To test this relationship further, fasted rats conserving protein were treated in vivo for 6-8 h with the antilipolytic agent nicotinic acid (NA) or with tetradecylglycidate (TDGD), an inhibitor of long-chain fatty acid oxidation. After treatment, protein synthesis and degradation in skeletal muscle were evaluated with the perfused rat hindquarter. NA treatment decreased plasma 3-hydroxybutyrate and free fatty acids and increased plasma urea and urine urea excretion, indicating increased breakdown of body protein. TDGD produced similar metabolic effects, except that plasma free fatty acids were markedly increased as a result of inhibition of fatty acid oxidation. NA and TDGD also decreased plasma insulin and increased plasma corticosteroid. Inhibition of lipid metabolism in vivo resulted in accelerated loss of protein from skeletal muscle due to decreased protein synthesis and increased protein breakdown. NA increased both total (i.e., tyrosine release) and myofibrillar (i.e., 3-methylhistidine release) protein breakdown, whereas TDGD increased the breakdown of only nonmyofibrillar proteins (i.e., 3-methylhistidine release by perfused hindquarter was not altered). These data indicate that lipid fuels may directly modulate protein metabolism in muscle during prolonged starvation and may prevent a rise in catabolic hormones. They also indicate that free fatty acids may directly attenuate the breakdown of myofibrillar proteins in muscle during prolonged starvation and that this may be unrelated to their oxidation.