Biomedical subjects
T T Aoki
Publications and source records attributed to T T Aoki.
Leucine meal increases glutamine and total nitrogen release from forearm muscle.
To assess the consequences of elevated branched chain amino acid levels on alanine, glutamine, and ammonia metabolism in muscle, L-leucine meals (14.7 g) were consumed by six normal postabsorptive individuals. Bilateral forearm studies were performed, and the dominant arm was subjected to 15 min of light exercise, using a calibrated dynamometer, beginning 45 min after the ingestion of the meal. Large uptakes of leucine were seen across both forearm muscle beds within 30 min of the meal. After exercise, blood flow in the dominant arm increased from 3.1 +/- 0.4 to 5.2 +/- 0.9 ml/100 ml forearm per minute (mean +/- SEM, P less than 0.005). Glutamine flux out of the dominant forearm increased threefold after the ingestion of the leucine meal and increased eightfold over base line after exercise. Less marked changes (significant only at 90 min) in the nonexercised, nondominant arm were also seen. Alanine flux out of the dominant forearm muscle bed increased modestly at 75 and 90 min. No significant change in ammonia flux across either forearm muscle bed was noted. Unexpectedly, large and significant net nitrogen loss from both forearm muscle beds was documented. Thus, following the ingestion of a leucine meal and light exercise, the primary means by which excess nitrogen is routed out of muscle is via glutamine formation and release with alanine and ammonia pathways playing relatively minor roles. More importantly, the ingestion of significant amounts of leucine by normal subjects, presumably in optimal nitrogen balance, results in a net loss of nitrogen from muscle.
Metabolic adaptations to starvation, semistarvation, and carbohydrate restriction.
The metabolic adaptation in man to starvation, semistarvation, and carbohydrate restriction is complex and involves a number of hormones, substrates, and tissues. In particular, however, the need for the ketoacids beta-hydroxybutyric acid and acetoacetic acid to replace glucose as the primary fuel for the brain of fasting man appears to be the key to maximum protein conservation. That is, ketogenesis is necessary to provide the brain with a fat-derived, water-soluble, insulin-independent, glucose-equivalent fuel. This adaptation is associated with a small loss of the ketoacids into the urine (100-150 mM/day or 40-60 calories/day). The ketonuria, in turn, necessitates increased renal utilization of muscle-derived glutamine. Synthesis of glutamine by muscle requires muscle proteolysis. Administration of glucose in amounts needed to meet the requirements of the brain results in suppression of ketogenesis in fasting man and a significant diminution in nitrogen mobilization and utilization as well.
Ornithine decarboxylase activity in insulin-deficient states.
The activity of ornithine decarboxylase, the rate-controlling enzyme in polyamine biosynthesis, was determined in tissues of normal control rats and rats made diabetic with streptozotocin. In untreated diabetic rats fed ad libitum, ornithine decarboxylase activity was markedly diminished in liver, skeletal muscle, heart and thymus. Ornithine decarboxylase was not diminished in a comparable group of diabetic rats maintained on insulin. Starvation for 48h decreased ornithine decarboxylase activity to very low values in tissues of both normal and diabetic rats. In the normal group, refeeding caused a biphasic increase in liver ornithine decarboxylase; there was a 20-fold increase in activity at 3h followed by a decrease in activity, and a second peak between 9 and 24h. Increases in ornithine decarboxylase in skeletal muscle, heart and thymus were not evident until after 24-48h of refeeding, and only a single increase occurred. The increase in liver ornithine decarboxylase in diabetic rats was greater than in normal rats after 3h of refeeding, but there was no second peak. In peripheral tissues, the increase in ornithine decarboxylase with refeeding was diminished. Skeletal-muscle ornithine decarboxylase is induced more rapidly when meal-fed rats are refed after a period without food. Refeeding these rats after a 48h period without food caused a 5-fold increase in ornithine decarboxylase in skeletal muscle at 3h in control rats but failed to increase activity in diabetic rats. When insulin was administered alone or together with food to the diabetic rats, muscle ornithine decarboxylase increased to activities even higher than in the refed controls. In conclusion, these findings indicate that the regulation of ornithine decarboxylase in many tissues is grossly impaired in diabetes and starvation. They also suggest that polyamine formation in vivo is an integral component of the growth-promoting effect of insulin or some factor dependent on insulin.
Pyrroline-5-carboxylate synthase activity in mammalian cells.
Although glutamic acid is known to be a precursor for proline biosynthesis, the enzymatic conversion of glutamic acid to pyrroline-5-carboxylic acid, the immediate precursor of proline, has not been demonstrated in cell-free systems. By providing appropriate concentrations of ATP and NADPH and blocking further metabolism of pyrroline-5-carboxylic acid, we have developed a method for measuring the formation of pyrroline-5-carboxylic acid from glutamic acid in homogenates of mammalian cells. We have designated this activity pyrroline-5-carboxylate synthase. To confirm that our assay is a valid measure of the initial step in proline biosynthesis from glutamic acid, we have compared two mutant lines of Chinese hamster ovary cell. Proline prototrophic cells, which can synthesize proline from glutamic acid, have easily measurable pyrroline-5-carboxylate synthase activity (5.97 nmol of pyrroline-5-carboxylic acid per hr per mg of homogenate protein). In contrast, proline auxotrophic cells, which are unable to synthesize proline from glutamic acid, have no detectable pyrroline-5-carboxylate synthase activity.
Starvation in the rat. II. Effect of age and obesity on protein sparing and fuel metabolism.
Sixteen-week-old control and obese rats survive longer than 8-wk-old control rats. In addition, unlike the 8-wk-old group, they conserve tissue RNA and protein. To evaluate the basis for this, the effects of starvation on circulating fuels and hormones and the urinary excretion of nitrogen and 3-methylhistidine (3MH) were compared in the three groups. Urinary nitrogen and 3MH diminished during prolonged starvation in 16-wk-old obese and control rats, suggesting that both groups are able to conserve protein and curtail muscle proteolysis. In contrast, urine nitrogen and 3MH did not decrease in 8-wk-old control rats. Protein conservation in the older rats was associated with diminished blood levels of alanine and increased levels of lipid fuels, ketone bodies, and free fatty acids. Although ketone bodies and free fatty acids were also increased during the first few days of starvation in 8-wk-old rats, there was no evidence of protein sparing. In all groups, as fat stores became exhausted terminally, blood lipid levels decreased and protein catabolism increased. Starvation caused insulin to decrease to comparable levels in all rats; however, minimal levels were reached later in the older groups. Thyroxine and triiodothyronine (T3) decreased during the fast in both control groups; however, T3 did not decrease in the obese rats. These findings support the contention that the conservation of protein during prolonged starvation requires the continued availability of lipid fuels. The role of insulin and thyroid hormone in modulating these adaptations is unclear.
Pancreatic alpha cell response to alanine during and after normal and diabetic pregnancies.
Pancreatic alpha cell response to oral alanine was assessed in the third trimester of pregnancy and in the puerperium in 16 insulin-dependent diabetic and 7 normal pegnant women. Insulin response was also measured in the nondiabetic subjects. The nondiabetic subjects had higher basal glucagon and insulin levels as well as a greater response to oral alanine stimulation at 34 weeks' gestation than at 6 weeks post partum. In addition, basal levels of both hormones remained low at a time remote from pregnancy (9 months post partum), indicating both hyperinsulinemia and hyperglucagonemia in the postabsorptive state in normal human pregnancy. The secretory response of glucagon and insulin or oral alanine was blunted at 6 weeks post partum in the nondiabetic subjects. This suggests that the late puerperium may not be an appropriate "nonpregnant control period" for metabolic studies. During pregnancy, basal and stimulated glucagon levels were not significantly different in diabetic and normal women. Despite higher concentrations of blood glucose in diabetic women, basal and stimulated glucagon secretion was equivalent in the 2 groups. No pegnancy-induced increment in glucagon secretion was evident in insulin-treated diabetic subjects. Thus hyperglucagonemia does not contribute to the increased requirements for insulin during pregnancy in these women.
Dimethyltriazenoimidazole carboxamide therapy of malignant glucagonoma.
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The effects of glucagon on protein metabolism in normal man.
Plasma glucagon rises after major injury and could act to increase gluconeogenesis and ureagenesis in the post-traumatic state. This study documents the effect of prolonged glucagon infusion on ureagenesis and nitrogen excretion, as well as possible sources of the increased ureagenesis, in normal man. Four healthy men fasted for 6 days during intravenous infusion of glucose (750 gmday), establishing a steady state of minimal ureagenesis. Glucagon (1 mg/day) then was added to the infusion for 5 days. Glucose alone was given for the final 2 days. Forearm muscle flux of metabolites was determined by standard arterial-deep venous sampling and capacitance plethysmography. Glucagon concentration was suppressed during glucose infusion (11 +/- 13 pg/ml) and rose to levels seen in subjects with major trauma during glucagon infusion (669 +/- 138 pg/ml). Glucose infusion stabilized urine nitrogen excretion at 1.54 +/- 0.42 gm of N/sq m/day. Nitrogen excretion increased to 2.40 +/- 0.53 gm of N/sq m/day with glucagon infusion, with urea accounting for the increased excretion. Excretion of 3-methylhistidine was unchanged. Plasma amino acid concentration was strikingly reduced on the first day of glucagon infusion, where it stabilized. Forearm flux showed a slight net release of amino acid nitrogen during glucose infusion. Addition of glucagon to the glucose infusion resulted in a net uptake of nitrogen by forearm skeletal muscle. These evidences strong suggest that glucagon infusion in normal man increases ureagenesis, not only at the expense of the free amino acid pool, but by the hydrolysis of visceral protein as well, with muscle protein being maintained.
Mechanisms of protein sparing in carbohydrate loaded man.
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Induction of urea cycle enzymes of rat liver by glucagon.
All five urea cycle enzymes of rat liver increased in activity 48 h after subcutaneous administration of crystalline zinc glucagon to male rats and remained elevated after 7 days of continuous glucagon infusion. The maximum ratios of enzyme activities over those of controls were 2.0 for carbamyl phosphate synthetase, 1.3 for ornithine transcarbamylase, 2.7 for argininosuccinate synthetase, 3.2 for argininosuccinase, and 2.2 for arginase. Actinomycin D or puromycin prevented these responses to glucagon. The increase in arginase activity after zinc glucagon treatment was matched by an increase in immunoprecipitable enzyme. All five enzymes were induced by physiological plasma levels of glucagon. Tube feeding of casein hydrolysate for 2 days increased all five enzyme activities 1.5- to 2.2-fold and resulted in plasma glucagon levels similar to those required for induction by exogenous glucagon. Thus, glucagon is an inducer of the entire urea cycle in rat liver and plays a role in the induction of the cycle by protein feeding.
Glucose and amino acid metabolism in perfused skeletal muscle. Effect of dichloroacetate.
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The redox state and regulation of amino acid metabolism in man.
Traditionally, regulation of amino acid metabolism in both postabsorptive and prolonged-fasted man has been generally regarded as being hormonal in nature. In particular, insulin, and to a lesser extent glucagon, have been nominated for key roles in this process. More recently, however, reconsideration of previous studies involving insulin, glucagon, and protein meals as well as previously unreported studies (cortisol and tri-iodothyronine) from this laboratory, have suggested another means of regulating amino acid metabolism in fasting man. This new hypothesis is centered on the redox state of muscle of fasting man, which is remarkably reduced in both cytosolic and mitochondrial compartments. It was found that insulin, and to a lesser extent glucagon, when infused into fasting subjects (1) rendered muscle significantly more reduced, and (2) resulted in a diminution in urinary nitrogen excretion. In contrast, when either tri-iodothyronine or cortisol were administered to fasting individuals (1) muscle was found to become more oxidized when compared with the control period, and (2) increased urinary nitrogen excretion was observed in both cases. It was noteworthy that the ingestion of a protein meal by a nitrogen-depleted individual was followed by a dramatic change in muscle redox state (the muscle became more reduced), together with marked uptakes of a variety of amino acids. It is therefore proposed that the protein conservation evidenced by fasting man may be dependent on the reduced state of muslce tissue.
Protein conservation during prolonged fast: a function of triiodothyronine levels.
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Effects of exogenous glucagon and epinephrine in physiological amounts on the blood levels of free fatty acids and glycerol in dogs.
Exogenous glucagon or epinephrine were infused into normal overnight fasted dogs to raise circulating hormone levels to concentrations within the physiologic range. Plasma levels of glycerol and free fatty acids remained unchanged during the glucagon infusion, but rose significantly during the administration of epinephrine. Plasma insulin in the systemic circulation remained unchanged during the glucagon infusion and increased slightly during the infusion of thecatecholamine. The data suggest that in normal dogs glucagon in physiological amounts has no lipolytic effect. The importance of the sympathetic nervous system in regulating lipolysis in normal mammals is stressed.
Metabolic and endocrine studies in a case of lipoatrophic diabetes.
A 20-yr-old female with congenital lipoatrophic diabetes was studied, with the following findings: (1) Serum insulin levels increased after both oral glucose and intravenous arginine administration; there was no growth hormone response to the latter. (2) The infusion of insulin (0.1 units and 0.5 units/kg) during the fed state and following at 110-hr fast produced only minimal changes of various fuels measured, with the exception of a decrease in the branched-chain amino acids. (3) There was a minimal production of ketones during the 110-hr fast. (4) Matabolic expenditure was markedly increased during the postabsorptive state (65-75 kcal/hr/sg m); it fell into the normal range during the 110-hr fast (31-35 kcal/hr/sq m). (5) Following meals, the patient experienced complaints ranging from cold and shivering to feeling hot with gross diaphoresis. These findings were associated with intermittent lability of her skin temperature, which varied 1 degree - 2 degrees F during a 3-hr period. (6) Progressive increases in doses of regular insulin before each meal resulted in up to a total of 9000 units/day being required before normal blood glucose levels were achieved. (7) A 2-wk therapeutic trial of pimozide provided no significant changes in a variety of hormones and fuels in the basal state or following insulin perturbations. (8) A variety of pituitary hormones and pituitary target organ hormones were studied in both the hypothyroid (Hashimoto's thyroiditis) and euthyroid state (following thyroid replacement). All the hormone responses were normal except that growth hormone did not rise during the slow wave sleep in either thyroid state.
Secretion by glucagonomas of a possible glucagon precursor.
Five patients with glucagonomas had elevated plasma levels of total glucagon immunoreactivity. Gel filtrations of these plasma samples on Bio-Gel P30 columns showed that most of the immunoreactivity eluted in the 3,500-(true glucagon) and 9,000-dalton fractions. After the administration of alpha cell effectors, changes in total glucagon immunoreactivity were seen which were accounted for primarily by the 3,500-dalton species, but there were also changes in the 9,000-dalton moiety. Venous effluent plasma from tumors of two subjects contained elevated concentrations of glucagon immunoreactivity in both fractions. When material from both the 3,500- and 9,000-dalton peaks were serially diluted in a glucagon immunoassay, parallel displacement curves were found, suggesting that both have similar or identical antigenic determinants. Thus, with conversion to a neoplastic state, alpha cells of glucagonomas, much like beta cells of insulinomas, may secrete an increased amount of a larger, 9,000-mol wt glucagon species which may be a prohormone.
Effects of beta-hydroxybutyrate, glycerol, and free fatty acid infusions on glucagon and epinephrine secretion in dogs during acute hypoglycemia.
The importance of glucagon in the regulation of carbohydrate metabolism is clearly established. However, the role played by this hormone in the regulation of the overall fuel economy is less certain, particularly with respect to such nonglucose fuels as free fatty acids, glycerol, and ketoacids. In order to elucidate glucagon's role with respect to the latter substrates, dogs were infused with solutions of these three fuels, and their A-cell responses to concomitant insulin-induced hypoglycemia were studied. In addition, epinephrine levels were also monitored. It was found that while these infusions failed to suppress glucagon release, the ketoacid infusion did significantly reduce epinephrine secretion during the insulin-induced hypoglycemic period. It was therefore concluded that glucagon secretion under these experimental conditions is not responsive to prevailing non-glucose fuel levels. Indeed, these data suggest that the sympathetic nervous system may play an important role in the regulation of the over-all fuel economy.