Ella S. Haugaard

Stimulation of glucose utilization by thioctic acid in rat diaphragm incubated in vitro

Abstract In rat diaphragm incubated in vitro thioctic acid increased the utilization of glucose. Time studies showed that, in contrast to the stimulatory effect of insulin on glucose uptake, the action of thioctic acid became apparent only after prolonged incubation. Thioctic acid did not interfere with the action of insulin and its effect was additive to that of insulin.

The role of sulfhydryl groups in oxidative phosphorylation and ion transport by rat liver mitochondria

Abstract The influence of the sulfhydryl reagent 5,5′-dithio-bis-(2-nitrobenzoic acid) (DNTB) on metabolism of liver mitochondria was studied under different experimental conditions. 1. 1. DTNB prevented the stimulation of respiration produced by ADP and inorganic phosphate in the presence of glutamate or succinate but had little effect on 2,4-dinitrophenol-stimulated respiration. 2. 2. The formation of ATP from ADP and phosphate was depressed by DTNB. The inhibition could be reversed by dithiothreitol. 3. 3. The increase in mitochondrial oxygen uptake produced by calcium and phosphate was depressed by DTNB. 4. 4. The uptake of calcium by mitochondria in the presence of ATP and glumate was only partially inhibited by DTNB. In contrast, the stimulatory effect of inorganic phosphate on calcium transport was completely prevented. 5. 5. In the presence of glutamate and no added ATP the uptake of calcium was associated with an entrance of phosphate. DTNB almost abolished the uptake of phosphate and inhibited calcium uptake by about 60%. The decrease in calcium uptake produced by DTNB was equal, mole for mole, to the decrease in phosphate uptake. 6. 6. DTNB completely prevented the extrusion of calcium exhibited by calcium-loaded mitochondria incubated in the presence of inorganic phosphate and a low concentration of magnesium. 7. 7. N-Ethylmaleimide had effects similar to DTNB but in addition severely inhibited 2,4-dinitrophenol-stimulated respiration with glutamate as substrate. 8. 8. It was concluded that a reactive site involving a sulfhydryl group is intimately involved in either the entrance of inorganic phosphate into the mitochondrion or in the formation of a phosphorylated intermediate essential for oxidative phosphorylation and ion transport.

Actions of lithium ions and insulin on glucose utilization, glycogen synthesis and glycogen synthase in the isolated rat diaphragm☆

Abstract The effects of lithium ions and insulin on carbohydrate metabolism of the isolated rat diaphragm were studied and compared. Like insulin, lithium ions caused a conversion of glycogen synthase D to the more active I form of the enzyme. Maximal activation of the enzyme was produced by about 5 mM LiCl. Lithium ions markedly increased glucose utilization and glycogen synthesis by the diaphragm, but the action of this ion appears to be exerted by a mechanism different from that of insulin, since the effects of maximal concentrations of the two agents are additive. In their action on glucose metabolism, lithium ions had a unique ability to direct the glucose taken up by the cell toward glycogen. Insulin and lithium ions had opposite effects on the tissue content of glucose 6-phosphate; insulin increased the tissue level of this metabolite, whereas lithium ions decreased it. ATP and creatine phosphate concentrations were not affected by insulin or lithium ions. The effects of lithium ions on carbohydrate metabolism are exerted at relatively low concentrations of Li, and our results indicate that significant alterations of carbohydrate metabolism may occur when therapeutic or toxic amounts of lithium salts are ingested by man.

Effects of a disulfide (Ellman's reagent) and thiols on oxidative phosphorylation and ion transport by rat liver mitochondria

Abstract The disulfide, 5,5′-dithio- bis -2-nitrobenzoic acid (DTNB), inhibits oxidative phosphorylation and uptake of calcium and phosphate ions by rat liver mitochondria. Half-maximal inhibition of oxidative phosphorylation is obtained with an amount of inhibitor of approximately 5 nmoles/mg of protein. Complete inhibition of oxidative phosphorylation and uptake of phosphate as well as abolition of phosphate-linked calcium uptake results from preincubation of mitochondria with DTNB followed by removal of excess inhibitor by washing. DTNB-treated mitochondria can be restored to normal function by additional incubation with dithiothreitol (DTT) followed by removal of excess DTT by washing. Oxidative phosphorylation during state 3 respiration can be completely stopped by DTNB and restored by DTT. It is concluded that one or more highly reactive -SH groups are essential for oxidative phosphorylation and ion transport in mitochondria.

Metabolic and electrolyte changes produced by lithium ions in the isolated rat diaphragm.

Abstract The effects of lithium ions on glucose metabolism and the tissue content of monovalent cations were studied in rat hemidiaphragms incubated in vitro . The entrance of lithium ions into the cell was associated with an increase in the rate of glucose utilization and glycogen synthesis. As lithium was taken up by the tissue, there was a concomitant loss of potassium. The loss of potassium could not account for the stimulation of glucose metabolism produced by lithium and it was concluded that lithium ions per se increased glucose uptake and particularly glycogen synthesis. Preincubation of hemidiaphragms in media containing different concentrations of lithium ions resulted in an increase in the rate of glycogen synthesis during a subsequent incubation in the absence of lithium. The stimulation of glycogen formation was directly related to the tissue content of lithium. Lithium ions did not significantly alter the cyclic AMP content of the tissues under conditions at which the rate of glucose metabolism was markedly enhanced.

Alteration by halothane of glucose and glycogen metabolism in rat skeletal muscle.

Exposure of resting rat diaphragm for one hour in vitro to halothane (1-1.5, 2-2.5 and 4-4.5 per cent in oxygen) produced significant alterations of intracellular glucose disposition. Glycolysis (as measured by Iactate production) increased, while glycogen formation was inhibited in a dose-related fashion. Net glucose uptake was unaffected by the anesthetic except during exposure to 4-4.5 per cent halothane, when 14 per cent depression of uptake was found. Total glycogen content decreased, due mainly to the inhibition of glycogen synthesis and to some extent to a stimulation of glycogenolysis. The anesthetic did not interfere with the effect of insulin on glucose uptake or the intracellular disposition of glucose. Creatine phosphate concentrations decreased following exposure of diaphragm to 1-1.5, 2-2.5 and 4-4.5 per cent halothane, while the adenosine triphosphate concentration declined after exposure to 4-4.5 per cent only. Although the mechanism(s) whereby halothane alters glucose and glycogen metabolism are unknown, it is possible that the anesthetic acts primarily by affecting membranes containing enzymes involved in the metabolism of glycogen.

Effects of epinephrine and cyclic AMP phosphodiesterase inhibitors on the glycogen synthetic pathway and glucose content in skeletal muscle

Abstract The actions on carbohydrate metabolism of epinephrine and the cyclic AMP phosphodiesterase inhibitors, theophylline and SQ 20009, were studied using rat hemidiaphragms incubated in vitro . The inhibition of glucose uptake produced by epinephrine was almost equal to the decrease in the incorporation of [ 14 C]glucosc into glycogen, while incorporation into lactate of 14 C from labeled glucose was not influenced by the hormone. Although epinephrine decreased total tissue glycogen, it had relatively little effect on the removal of radioactive glucose incorporated into glycogen during a preincubation in the absence of hormone when the tissue was subsequently incubated with nonradioactive glucose. This indicates that the decrease in the net incorporation of [ 14 C]glucose into glycogen produced by the hormone in vitro is caused predominantly by an inhibition of glycogen synthesis and cannot be accounted for by an increase in the turnover rate of glycogen. Theophylline and SQ 20009 produced a dose-dependent inhibition of glucose uptake and glycogen synthesis, similar to that of epinephrine. Incubation of hemidiaphragms with epinephrine or cyclic AMP phosphodiesterase inhibitors in the presence of glucose led to an increased intracellular accumulation of glucose. Possible explanations for this phenomenon are discussed. The uptake of the nonmetabolizable sugar. 3- O -methylgucose, by skeletal muscle was not affected by epinephrine, indicating that the hormone influences only the uptake of the natural substrate, glucose, which can be incorporated into glycogen. The alterations in muscle metabolism described here were associated with increases in the tissue content of cyclic AMP and it was concluded that an important action of epinephrine in resting muscle is a cyclic AMP-mediated inhibition of the glucose-to-glycogen synthetic pathway.

Effects of thiols and disulfides on glucose utilization and insulin action in the isolated rat diaphragm

Abstract The actions of 2,2′-dithiodipyridine (DTP) and dithiothreitol (DTT) on glucose utilization and lactate formation by rat diaphragm were studied in vitro . The disulfide DTP had no significant effect on glucose utilization in the absence of insulin but inhibited the effect of insulin on glucose utilization and increased lactate formation and breakdown of glycogen by the tissue. The interference by DTP with the action of insulin could be reversed by the sulfhydryl compound DTT. DTT stimulated glucose utilization and net lactate formation by rat diaphragm. This effect of the sulfhydryl compound was different from that of insulin in that it commenced not immediately but only after about 30 min of incubation. In contrast to insulin, DTT decreased rather than increased glycogen synthesis. When the muscle was under the influence of both insulin and a high concentration of DTT, the initial insulin effect was not abolished. However, after about 30 min of incubation, the rate of glucose utilization was the same with DTT alone as with DTT plus insulin, and the muscle appeared to be under the influence mainly of DTT.

Effect of diabetes and fasting on the uridine triphosphate content and uridine kinase activity of rat cardiac and skeletal muscle.

The influence of diabetes and starvation on uracil nucleotide metabolism in muscle was studied. It was found that the uridine triphosphate (UTP) content of heart and diaphragm muscle was decreased in fasted and streptozotocin-diabetic rats and that insulin treatment of diabetic animals restored the UTP concentration to normal levels. The ATP content of heart tissue was not altered under these conditions. It was also demonstrated that hemidiaphragms from streptozotocin-diabetic rats synthesized less UTP from uridine in vitro than hemidiaphragms from normal animals. Uridine kinase activity of extracts of cardiac and skeletal muscle from fasted and diabetic rats was lower than the activity found in extracts from control animals. It was concluded that uracil nucleotide synthesis by the salvage pathway is decreased in experimental diabetes and fasting.

Effects of epinephrine and the cyclic AMP phosphodiesterase inhibitor SQ 20009 on glucose and glycogen metabolism in skeletal muscle

Abstract In rat diaphragm incubated in vitro, epinephrine and isoproterenol inhibited glucose uptake and the incorporation of radioactive glucose into glycogen. The metabolic effects of epinephrine were inhibited by the beta-adrenergic blocking agents, propranolol and sotalol, but not by the alpha-adrenergic blocking drug, phentolamine. The phosphodiesterase inhibitor, 1-ethyl-4-(isopropylidine-hydrazino)-1H-pyrazolo-(3,4-b)-pyridine-5-carboxylic acid, ethyl ester, hydrochloride (SQ 20009), also decreased glucose uptake and glycogen synthesis. In contrast to the action of epinephrine, exogenous calcium ions were required for the effects of SQ 20009 on phosphorylase activation, increased tissue glucose- 6-phosphate levels and lactate production. It was concluded that the inhibition of glucose uptake that occurred when tissue cyclic AMP levels were increased by beta-adrenergic drugs or phosphodiesterase inhibitors was not due directly to accumulation of glucose-6-phosphate from glycogenolysis but was a result of a cyclic AMP-mediated inhibition of glycogen synthesis.