The rate of protein degradation in isolated skeletal muscle does not correlate with reduction-oxidation status.
ABSTRACT: It has been suggested that the cytoplasmic reduction-oxidation state correlates with, and may regulate, rates of protein breakdown in skeletal muscle. To test whether an increased lactate/pyruvate ratio is in fact generally associated with low proteolytic rates, this ratio was measured in rat extensor digitorum longus muscles incubated under conditions that rates of protein breakdown. Treatment with the calcium ionophore A23187 caused similar large increases in the lactate/pyruvate ratio at 2 microM, where proteolysis did not change, and at 20 microM, where proteolysis was greatly accelerated. Omission of Ca2+ from the medium slowed proteolysis, but decreased the lactate/pyruvate ratio. In muscles incubated at 40 degrees C, rates of proteolysis were faster, but the lactate/pyruvate ratios were higher than 37 degrees C. Thus alterations in the redox status do not necessarily correlate with, and can occur independently of, changes in proteolysis. Furthermore, insulin and inhibitors of lysosomal proteinases decreased proteolysis but, in contrast with previous reports, failed to alter the lactate/pyruvate ratio. In addition, protein breakdown decreased in muscles maintained under tension, although redox state did not change. Thus protein degradation can fall without a concomitant change in the reduction-oxidation state.
Project description:The role of prostaglandins in the regulation of muscle protein breakdown is controversial. We examined the influence of arachidonic acid (5 microM), prostaglandin E2 (PGE2) (2.8 microM) and the prostaglandin-synthesis inhibitor indomethacin (3 microM) on total and myofibrillar protein breakdown in rat extensor digitorum longus and soleus muscles incubated under different conditions in vitro. In other experiments, the effects of indomethacin, administered in vivo to septic rats (3 mg/kg, injected subcutaneously twice after induction of sepsis by caecal ligation and puncture) on plasma levels and muscle release of PGE2 and on total and myofibrillar protein breakdown rates were determined. Total and myofibrillar proteolysis was assessed by measuring production by incubated muscles of tyrosine and 3-methylhistidine respectively. Arachidonic acid or PGE2 added during incubation of muscles from normal rats did not affect total or myofibrillar protein degradation under a variety of different conditions in vitro. Indomethacin inhibited muscle PGE2 production by incubated muscles from septic rats, but did not lower proteolytic rates. Administration in vivo of indomethacin did not affect total or myofibrillar muscle protein breakdown, despite effective plasma levels of indomethacin with decreased plasma PGE2 levels and inhibition of muscle PGE2 release. The present results suggest that protein breakdown in skeletal muscle of normal or septic rats is not regulated by PGE2 or other prostaglandins.
Project description: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.
Project description:Nitric oxide activates guanylate cyclase to form cGMP, comprising a signalling system that is believed to be a distinct mechanism for increasing glucose transport and metabolism in skeletal muscle. The effects of a selective cGMP phosphodiesterase inhibitor, zaprinast, on basal glucose utilization was investigated in incubated rat soleus muscle preparations isolated from both insulin-sensitive (lean Zucker; Fa/?) and insulin-resistant (obese Zucker; fa/fa) rats. Zaprinast at 27 microM significantly increased cGMP levels in incubated soleus muscle isolated from lean, but not obese, Zucker rats. Muscles were incubated with 14C-labelled glucose and various concentrations of zaprinast (3, 27 and 243 microM). Zaprinast (at 27 and 243 microM) significantly increased rates of net and 14C-labelled lactate release and of glycogen synthesis in lean Zucker rat soleus muscle; glucose oxidation was also increased by 27 microM zaprinast. In addition, regardless of concentration, the phosphodiesterase inhibitor failed to increase any aspect of 14C-labelled glucose utilization in soleus muscles isolated from obese Zucker rats. The maximal activity of nitric oxide synthase (NOS) was significantly decreased in insulin-resistant obese Zucker muscles. Thus the lack of effect of zaprinast in insulin-resistant skeletal muscle is consistent with decreased NOS activity. To test whether there is a defect in insulin-resistant skeletal muscle for endogenous activation of guanylate cyclase, soleus muscles were isolated from both insulin-sensitive and insulin-resistant Zucker rats and incubated with various concentrations of the NO donor sodium nitroprusside (SNP; 0.1, 1, 5 and 15 mM). SNP significantly increased rates of net and 14C-labelled lactate release, as well as glucose oxidation in muscles isolated from both insulin-sensitive and insulin-resistant rats. A decreased response to SNP was observed in the dose-dependent generation of cGMP within isolated soleus muscles from insulin-resistant rats. A possible link between impaired NO/cGMP signalling and abnormal glucose utilization by skeletal muscle is discussed.
Project description:Denervated (1-10 days) rat epitrochlearis muscles were isolated, and basal and insulin-stimulated protein and glucose metabolism were studied. Although basal rates of glycolysis and glucose transport were increased in 1-10-day-denervated muscles, basal glycogen-synthesis rates were unaltered and glycogen concentrations were decreased. Basal rates of protein degradation and synthesis were increased in 1-10-day-denervated muscles. The increase in degradation was greater than that in synthesis, resulting in muscle atrophy. Increased rates of proteolysis and glycolysis were accompanied by elevated release rates of leucine, alanine, glutamate, pyruvate and lactate from 3-10-day-denervated muscles. ATP and phosphocreatine were decreased in 3-10-day-denervated muscles. Insulin resistance of glycogen synthesis occurred in 1-10-day denervated muscles. Insulin-stimulated glycolysis and glucose transport were inhibited by day 3 of denervation, and recovered by day 10. Inhibition of insulin-stimulated protein synthesis was observed only in 3-day-denervated muscles, whereas regulation by insulin of net proteolysis was unaffected in 1-10-day-denervated muscles. Thus the results demonstrate enhanced glycolysis, proteolysis and protein synthesis, and decreased energy stores, in denervated muscle. They further suggest a defect in insulin's action on protein synthesis in denervated muscles as well as on glucose metabolism. However, the lack of concurrent changes in all insulin-sensitive pathways and the absence of insulin-resistance for proteolysis suggest multiple and specific cellular defects in insulin's action in denervated muscle.
Project description:1. The effect of hypocaloric feeding (25% of normal food intake for 21 days) of rats on the enzymic and metabolic adaptations in the gastrocnemius, plantaris and soleus muscles was studied. 2. In control and hypocaloric rats the muscle relaxation rates at 100 Hz were 35.76 and 11.38% force loss/10 ms respectively. Control rats exhibited enhanced force of muscle contraction as the frequency of stimulation increased from 10 to 100 Hz, with maximum force being at 100 Hz. Hypocaloric rats exhibited a decrease in the increment of force being exerted at high frequencies, with maintenance of force at lower stimulatory frequencies. 3. In muscles of hypocaloric rats, there were significant decreases in the maximal activities of hexokinase (17.6-37.0%), 6-phosphofructokinase (22.7-34.2%), pyruvate kinase (21.2-36.0%), citrate synthase (34.1-41.5%), oxoglutarate dehydrogenase (29.4-52.4%) and 3-hydroxyacyl-CoA dehydrogenase (26.7-32.1%), whereas the activities of glycogen phosphorylase increased (23.8-43.4%) compared with control values. 4. In soleus-muscle strip preparations of hypocaloric rats, there were significant decreases in the rates of lactate production (28.1%) and glucose oxidation (32.6%) compared with control preparations. 5. Mitochondrial preparations from muscles of hypocaloric rats incubated with various substrates exhibited decreased rates of oxygen uptake compared with control preparations. 6. In muscles of hypocaloric rats (gastrocnemius and soleus), there were significant decreases in the concentrations of glycogen (P less than 0.001) and phosphocreatine (P less than 0.001) and increases in those of pyruvate (P less than 0.001), lactate (P less than 0.001) and ADP (P less than 0.001), whereas those of ATP and AMP remained unchanged. 7. Calculated [lactate]/[pyruvate] and [ATP]/[ADP] ratios exhibited significant increases (P less than 0.05) and decreases (P less than 0.05) in muscles of hypocaloric rats respectively. 8. The results are discussed in relation to the genesis of muscle dysfunction caused by malnutrition.
Project description:The present study characterized total and myofibrillar protein breakdown rates in a muscle preparation frequently used in vitro, i.e. incubated extensor digitorum longus (EDL) and soleus (SOL) muscles of young rats. Total and myofibrillar protein breakdown rates were assessed by determining net production by the incubated muscles of tyrosine and 3-methylhistidine (3-MH) respectively. Both amino acids were determined by h.p.l.c. Both total and myofibrillar protein breakdown rates were higher in SOL than in EDL muscles and were decreased by incubating the muscles maintained at resting length, rather than flaccid. After fasting for 72 h, total protein breakdown (i.e. tyrosine release) was increased by 73% and 138% in EDL muscles incubated flaccid and at resting length respectively. Net production of tyrosine by SOL muscle was not significantly altered by fasting. In contrast, myofibrillar protein degradation (i.e. 3-MH release) was markedly increased by fasting in both muscles. When tissue was incubated in the presence of 1 munit of insulin/ml, total protein breakdown rate was inhibited by 17-20%, and the response to the hormone was similar in muscles incubated flaccid or at resting length. In contrast, myofibrillar protein breakdown rate was not altered by insulin in any of the muscle preparations. The results support the concepts of individual regulation of myofibrillar and non-myofibrillar proteins and of different effects of various conditions on protein breakdown in different types of skeletal muscle. Thus determination of both tyrosine and 3-MH production in red and white muscle is important for a more complete understanding of protein regulation in skeletal muscle.
Project description:Pyruvate kinase activity and the rates of gluconeogenesis and glycolysis in rat hepatocytes were evaluated by production of glucose and lactate + pyruvate from dihydroxyacetone during a feeding cycle or progressive starvation. In fed rats, during daylight (low food intake) and until darkness, gluconeogenesis progressively increased and glycolysis decreased slightly, but gluconeogenesis never exceeded glycolysis. During nocturnal feeding, gluconeogenesis and glycolysis returned to their morning rates. After 8 h starvation, an equal proportion of dihydroxyacetone was converted into glucose and into lactate + pyruvate. When glycogen was depleted (11 h of starvation), gluconeogenesis was maximal and glycolysis minimal. In fed and starved rats, the concentration of fructose 1,6-bisphosphate was the same. The activity ratio of pyruvate kinase (ratio of velocity at 0.5 mM-phosphoenolpyruvate to the maximum catalytic activity obtained with 4mM-phosphoenolpyruvate) was high in crude extracts of cells incubated with dihydroxyacetone and low in (NH4)2SO4-treated extracts, but remained unchanged during the whole experiment. There was no correlation between the rates of gluconeogenesis and glycolysis from dihydroxyacetone and the activity ratio of pyruvate kinase.
Project description:1. The effects of changes in the cytoplasmic [NADH]/[NAD+] ratio on the efficacy of glucagon to alter rates of metabolism in isolated rat hepatocytes were examined. 2. Under reduced conditions (with 10mM-lactate), 10nM-glucagon stimulated both gluconeogenesis and urea synthesis in isolated hepatocytes from 48h-starved rats; under oxidized conditions (with 10mM-pyruvate), 10nM-glucagon had no effect on either of these rates. 3. The ability of glucagon to alter the concentration of 3':5'-cyclic AMP and the rates of glucose output, glycogen breakdown and glycolysis in cells from fed rats were each affected by a change in the extracellular [lactate]/[pyruvate] ratio; minimal effects of glucagon occurred at low [lactate]/[pyruvate] ratios. 4. Dose-response curves for glucagon-mediated changes in cyclic AMP concentration and glucose output indicated that under oxidized conditions the ability of glucagon to alter each parameter was decreased without affecting the concentration of hormone at which half-maximal effects occurred. 5. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.05 mM) significantly reversed the inhibitory effects of pyruvate on glucagon-stimulated glucose output. 6. For exogenously added cyclic [3H]AMP(0.1 mM), oxidized conditions decreased the stimulatory effect on glucose output as well as the intracellular concentration of cyclic AMP attained, but did not alter the amount of cyclic [3H]AMP taken up. 7. The effects of lactate, pyruvate, NAD+ and NADH on cyclic AMP phosphodiesterase activities of rat hepatocytes were examined. 8. NADH (0.01--1 MM) inhibited the low-Km enzyme, particularly that which was associated with the plasma membrane. 9. The inhibition of membrane-bound cyclic AMP phosphodiesterase by NADH was specific, reversible and resulted in a decrease in the maximal velocity of the enzyme. 10. It is proposed that regulation of the membrane-bound low-Km cyclic AMP phosphodiesterase by nicotinamide nucleotides provides the molecular basis for the effect of redox state on the hormonal control of hepatocyte metabolism by glucagon.
Project description:The metabolism of lactate, pyruvate and glucose was studied in epididymal adipose tissue of starved, normally fed and starved-re-fed rats. Lactate conversion into fatty acid occurred at an appreciable rate only in the adipocyte of starved-re-fed animals. NNN'N'-Tetramethyl-p-phenylenediamine, an agent that transports reducing power from the cytoplasm to the mitochondria, caused large increments of fatty acid synthesis from lactate and a smaller one from glucose but a decrease in that from pyruvate. Glucose (1.0mm) increased fatty acid synthesis from lactate 4.3-fold but only 1.67-fold from pyruvate in adipocytes from normally fed animals. 2-Deoxyglucose decreased fatty acid synthesis from lactate to a greater degree (threefold) compared to that from pyruvate in adipocytes from starved-re-fed animals. l-Glycerol 3-phosphate contents were approximately equal in epididymal fat-pads, incubated in the presence of lactate or pyruvate, from normally fed animals, whereas the addition of 1mm-glucose resulted in a tenfold increase in l-glycerol 3-phosphate content only in the presence of lactate. The l-glycerol 3-phosphate content was tenfold higher in adipose tissue from starved-re-fed animals incubated in the presence of lactate than in the presence of pyruvate. 2-Deoxyglucose caused these values to be slightly lowered in the presence of lactate. We suggest that lactate metabolism is limited by the rate of NADH removal from the cytoplasm. In the starved-re-fed state, this occurs by reduction of dihydroxyacetone phosphate formed from glycogen to produce l-glycerol 3-phosphate, thus permitting lactate conversion into fatty acid. When glucose is the substrate, and rates of transport are not limiting, the rate of removal of cytoplasmic NADH limits glucose conversion into fatty acid.
Project description:By use of different inhibitors, we distinguished three proteolytic processes in rat skeletal muscle. When soleus muscles maintained under tension were exposed to the calcium ionophore A23187 or were incubated under no tension in the presence of Ca2+, net protein breakdown increased by 50-80%. Although leupeptin and E-64 inhibit this acceleration of protein breakdown almost completely, other agents that prevent lysosomal function, such as methylamine or leucine methyl ester, did not inhibit this effect. A similar increase in net proteolysis occurred in muscle fibres injured by cutting, and this response was also inhibited by leupeptin, but not by methylamine. In contrast, all these inhibitors markedly decreased the 2-fold increase in protein breakdown induced by incubating muscles without insulin and leucine, isoleucine and valine. In addition, the low rate of proteolysis seen in muscles under passive tension in complete medium was not affected by any of these inhibitors. Thus the basal degradative process in muscle does not involve lysosomes or thiol proteinases, and muscle can enhance protein breakdown by two mechanisms: lack of insulin and nutrients enhances a lysosomal process in muscle, as in other cells, whereas Ca2+ and muscle injury activate a distinct pathway involving cytosolic thiol proteinase(s).