M. N. Goodman

Muscle Glucose Metabolism following Exercise in the Rat: INCREASED SENSITIVITY TO INSULIN

Muscle glycogen stores are depleted during exercise and are rapidly repleted during the recovery period. To investigate the mechanism for this phenomenon, untrained male rats were run for 45 min on a motor-driven treadmill and the ability of their muscles to utilize glucose was then assessed during perfusion of their isolated hindquarters. Glucose utilization by the hindquarter was the same in exercised and control rats perfused in the absence of added insulin; however, when insulin (30-40,000 muU/ml) was added to the perfusate, glucose utilization was greater after exercise. Prior exercise lowered both, the concentration of insulin that half-maximally stimulated glucose utilization (exercise, 150 muU/ml; control, 480 muU/ml) and modestly increased its maximum effect. The increase in insulin sensitivity persisted for 4 h following exercise, but was not present after 24 h. The rate-limiting step in glucose utilization enhanced by prior exercise appeared to be glucose transport across the cell membrane, as in neither control nor exercised rats did free glucose accumulate in the muscle cell. Following exercise, the ability of insulin to stimulate the release of lactate into the perfusate was unaltered; however its ability to stimulate the incorporation of [(14)C]glucose into glycogen in certain muscles was enhanced. Thus at a concentration of 75 muU/ml insulin stimulated glycogen synthesis eightfold more in the fast-twitch red fibers of the red gastrocnemius than it did in the same muscle of nonexercised rats. In contrast, insulin only minimally increased glycogen synthesis in the fast-twitch white fibers of the gastrocnemius, which were not glycogen-depleted. The uptake of 2-deoxyglucose by these muscles followed a similar pattern suggesting that glucose transport was also differentially enhanced. Prior exercise did not enhance the ability of insulin to convert glycogen synthase from its glucose-6-phosphate-dependent (D) to its glucose-6-phosphate-independent (1) form. On the other hand, following exercise, insulin prevented a marked decrease in muscle glucose-6-phosphate, which could have diminished synthase activity in situ. The possibility that exercise enhanced the ability of insulin to convert glycogen synthase D to an intermediate form of the enzyme, more sensitive to glucose-6-phosphate, remains to be explored. These results suggest that following exercise, glucose transport and glycogen synthesis in skeletal muscle are enhanced due at least in part to an increase in insulin sensitivity. They also suggest that this increase in insulin sensitivity occurs predominantly in muscle fibers that are deglycogenated during exercise.

Effect of Glucagon: Insulin Ratios on Hepatic Metabolism

Glucagon (1.7 × 10−9M) stimulated gluconeogenesis, ureogenesis, lactate production, ketogenesis, proteolysis and glycogenolysis in the isolated perfused rat liver. Insulin at relatively low concentrations (10-100 μU/ml.) suppressed these metabolic effects of glucagon. When a molar glucagon:insulin ratio (2.6 or 26) was selected, which partially suppressed the stimulatory effects of glucagon and the inhibitory effects of insulin, a 100-1,000 fold change in insulin and glucagon concentration at a constant glucagon:insulin ratio, did not alter the rate of gluconeogenesis, ketogenesis, ureogenesis, glycogenolysis or lactate production. These results indicate that glucagon and insulin are complete competative antagonists in the perfused liver. This suggests that it is the glucagon:insulin ratio and not the absolute concentration of either hormone that determines their metabolic events in liver.

Glucose Metabolism in Rat Skeletal Muscle at Rest: Effect of Starvation, Diabetes, Ketone Bodies and Free Fatty Acids

The influence of starvation, diabetic ketoacidosis, ketone bodies and free fatty acids on glucose metabolism in resting skeletal muscle was studied in the isolated perfused rat hindquarter perparation and in intact rats. In the hindquarter preparation, the provision of 1.3 mM oleate, 1 mM octanoate or 2 mM acetoacetate did not alter the uptake of glucose in the presence of insulin. In contrast, glucose uptake in the presence of insulin was significantly depressed in hindquarters of rats with diabetic ketosis. In fed, fasted and diabetic rats the distribution space of glucose in skeletal muscle in vivo ranged between 15 to 20 per cent (extracellular space, 18 to 20 per cent), indicating that transport into the cell and not phosphorylation was rate-limiting for glucose uptake. The administration of glucose and insulin did not increase the glucose distribution space in fed or fasted rats; however, it caused a marked increase in tissue lactate in the fasted group, suggesting inhibition of pyruvate oxidation. The concentrations of hexose monophosphates in skeletal muscle freeze-clamped in vivo were very similar in the three groups, indicating phosphofructokinase was not inhibited in the fasted or diabetic rats. There were also no major differences in the concentrations of citrate, glycerolphosphate, ATP, ADP and AMP: the concentration of acetyl CoA was increased in both forty-eight hour fasted and diabetic rats and free CoA was diminished in the diabetic rats. Tissue glycogen waslower in fasted and diabetic than in fed rats. The data suggest that in resting skeletal muscle there is no inhibition of glucose metabolism by exogenous fatty acids and ketone bodies anaiagous to that which occurs in heart and diaphragm. The rate limiting step in glucose uptake appears to be its transport into the cell which is inhibited in rats with diabetic ketosis. Glucose metabolism at the level of pyruvate dehydrogenase may be inhibited in skeletal muscle during fasting and diabetes as a result of changes in the concentrations of acetyl CoA and CoA.

Regulation of myofibrillar protein degradation in rat skeletal muscle during brief and prolonged starvation

Myofibrillar protein breakdown during brief and prolonged starvation was assessed in perfused rat skeletal muscle from 8-week-old fat-fed rats that conserve skeletal muscle protein during starvation and survive for 12 to 15 days and age-matched chow-fed rats that do not conserve protein and survive only five to six days. Following the inhibition of protein synthesis with cycloheximide, myofibrillar proteolysis was assessed by measuring the release of 3-methylhistidine from the perfused rat hindquarter while simultaneous measurement of total protein breakdown was assessed by measuring tyrosine release. Myofibrillar proteolysis progressed through three distinct phases during starvation: an early phase occurring within 24 hours in which proteolysis increased in all rats, a middle phase, which took three to five days to develop and during which proteolysis decreased and was present only in fat-fed rats, and a late phase in which proteolysis again increased. Total protein breakdown (ie, tyrosine release) changed little in phase I, decreased in phase II, and increased in phase III. The release of 3-methylhistidine from the perfused hindquarter reflected changes in muscle and urine of intact rats suggesting that data obtained with the perfused hindquarter reflected the in vivo situation. Insulin, amino acids, high concentrations of glucose, indomethacin, or epinephrine as well as adrenalectomy failed to attenuate the increase in 3-methylhistidine release from the perfused hindquarter during brief and late starvation. Free fatty acids and ketone bodies were also without effect in vitro. Refeeding fasting rats for four hours decreased myofibrillar proteolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein Sparing in Skeletal Muscle During Prolonged Starvation: Dependence on Lipid Fuel Availability

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.

Glucose and Amino Acid Metabolism in Perfused Skeletal Muscle: Effect of Dichloroacetate

Dichloroacetate lowers blood glucose and diminishes blood levels of lactate, pyruvate, and alanine in both starvation and diabetes. To determine the role of skeletal muscle in these changes, studies were carried out in intact rats, with the isolated perfused rat hindquarter and soleus muscle preparation. In hindquarters from fed, starved, and diabetic rats, dichloroacetate alone or in the presence of insulin did not augment glucose uptake. On the other hand, it dramatically curtailed the release of the gluconeogenic precursors lactate, pyruvate, and alanine. Dichloroacetate increased markedly the generation of 14CO2 from lactate-1-14C in starved and diabetic rats, suggesting activation of pyruvate dehydrogenase. The increment in lactate oxidation was stoichiometri-cally equivalent to the decrease in lactate plus alanine release. Glycolysis, as determined from the sum of lactate and alanine release plus lactate oxidation in the hindquarter and from the formation of 3H2O from 3-3H-glucose by the incubated soleus muscle, was not altered by dichloroacetate. Dichloroacetate curtailed the release of most amino acids in the perfused hindquarter of fed rats. In starved and diabetic rats, it caused an increase in the release of valine, leucine, and isoleucine, suggesting inhibition of their metabolism. As judged from lactate-pyruvate and 3-hydroxybutyrate-acetoacetate ratios and changes in tissue α-glycerol-phosphate, perfusion with dichloroacetate caused the cytosol of the muscle cell to become more reduced and the mitochondria more oxidized. Similar changes occurred when it was administered to intact animals. These findings suggest that the hypoglycemic effect of dichloroacetate is, at least in part, due to a decrease in the release of gluconeogenic precursors from skeletal muscle secondary to activation of pyruvate dehydrogenase.

Myofibrillar Protein Breakdown in Skeletal Muscle is Diminished in Rats With Chronic Streptozocin-Induced Diabetes

Previous reports have suggested that insulin may not regulate the breakdown of myofibrillar proteins in skeletal muscle. To further test the role of insulin, insulinopenia was produced by treating rats with streptozocin. After treatment, protein breakdown in skeletal muscle was evaluated with the isolated prefused rat hindquarter preparation. After the inhibition of protein synthesis with cycloheximide, total and myofibrillar protein breakdown were assessed by measuring the release of tyrosine and 3-methylhistidine, respectively, in the perfused hindquarters of diabetic and agematched control rats. Streptozocin-induced (65 mg/kg) diabetes (3- to 28-day duration) resulted in hyperglycemia, hypoinsulinemia, hyperphagia, increased plasma lipid levels, arrested body and muscle growth, and increased urea and 3-methylhistidine excretion. Despite this, protein breakdown in skeletal muscle diminished. The release of 3-methylhistidine by the perfused hindquarters of diabetic rats decreased, whereas the release of tyrosine remained unchanged, suggesting that the breakdown of myofibrillar proteins was affected specifically. 3-Methylhistidine (unbound) levels in skeletal muscle of unperfused diabetic rats as well as in skin decreased, whereas they increased twofold in the gastrointestinal tract. More severe diabetes (125 mg/kg streptozocin), which resulted in ketoacidosis, augmented protein breakdown in muscle; however, this response was due to a marked fall in food consumption (it was also evident when control rats were pair fed). These data reinforce previous conclusions that insulin does not play a major role in the regulation of myofibrillar protein breakdown in skeletal muscle. They also suggest that food consumption per se and/ or lipid availability may modulate myofibrillar proteolysis independent of insulin secretion. Lastly, increased 3-methylhistidine excretion in diabetic rats appeared to be of gastrointestinal origin. Because of this, caution must still be exercised in the use of 3-methylhistidine excretion as an index of myofibrillar proteolysis in skeletal muscle.

Insulin and exercise stimulate muscle alpha-aminoisobutyric acid transport by a Na+K+-ATPase independent pathway

Sodium ions are required for the active transport of amino acids such as alpha-aminoisobutyric acid (AIB) into skeletal muscle. To examine the role of Na+-K+-ATPase in this phenomenon, studies were carried out using the isolated perfused rat hindquarter preparation. Perfusion for 30 min with ouabain at a dose sufficient to inhibit the Na+-K+ pump (10(-4) M) inhibited the basal rate of AIB uptake in all muscles studied by up to 80%. However, it failed to inhibit the stimulation of AIB uptake, either by insulin (200 microU/ml) or electrically-induced muscle contractions. The increase in K+ release by the hindquarter in the presence of ouabain was the same under all conditions suggesting comparable inhibition of the Na+-K+ pump. These studies suggest that the basal, but not insulin or exercise-stimulated AIB transport into muscle is acutely dependent on a functional Na+-K+ pump. They also suggest that stimulated and basal uptake of AIB involve different mechanisms.

Determination of some l-amino acids in biological samples by aminoacylation of tRNA☆

Abstract A procedure involving the aminoacylation of tRNA for the measurement of l -amino acids is described. The procedure has been applied successfully to the measurements of amino acids in cold acid extracts from blood, plasma, as well as animal tissues. The slight reaction in the absence of tRNA was shown to be due probably to the tRNA content of the enzyme preparation. The requirements for aminoacylation (ionic strength, pH) of tRNA vary with individual amino acids. Nevertheless, the inclusion of a standard curve with each experiment allows the accurate measurement of amino acids under non ideal or even suboptimal conditions. The procedure has been found particularly useful for the study of serial changes in the concentrations of certain amino acids during organ perfusion.

INTERACTION BETWEEN KETONE-BODY AND CARBOHYDRATE METABOLISM IN PERIPHERAL TISSUE

ABSTRACT Ketone bodies inhibit glucose uptake and glycolysis in heart; however, they have not been demonstrated to alter these processes in the perfused rat hindquarter, a preparation composed predominantly of skeletal muscle. The hind-quarter contains a mixture of slow- and fast-twitch red and white muscle fibers. To explore the possibility that ketone bodies inhibit glucose utilization and glycolysis only in some of these fibers, the effect of acetoacetate on glucose metabolism was studied in incubated soleus (85% slow-twitch red) and extensor digitorum longus (50% white, 50% fast-twitch red) muscles. When the soleus was incubated with insulin and 5 mM glucose, acetoacetate inhibited glucose uptake, stimulated glycogen formation and caused an increase in tissue citrate, exactly as it does in heart. Likewise, glycolysis was inhibited at the level of phosphofructokinase. Except for a small increase in citrate, these effects were not observed in the extensor digitorum longus. Basal rates of glycolysis were five times greater in the incubated muscles than in either resting muscle in vivo or in the perfused hind-quarter. When glucose was omitted from the medium bathing the soleus, its rate of glycolysis was similar to that of the perfused hindquarter and acetoacetate failed to affect either the rate of glycolysis or the tissue concentration of citrate. Tissue malate was also diminished. In conclusion, the data suggest that (1) acetoacetate inhibits glucose uptake and glycolysis only in red muscle, and (2) that the accumulation of citrate by the soleus is linked to the rate of glycolysis, possibly through several mechanisms. In this respect, glycolysis appears to be autoregulatory.