Rat Hemidiaphragms Research Articles

Significance of the extracellular bicarbonate buffer system to anaerobic glycolysis in hypoxic muscle.

1. The influence of the carbon dioxide-bicarbonate buffer system on anaerobic energy production during severe hypoxia was studied in isolated right hemidiaphragms of rats.--2. When the tissue was incubated in a Ringer solution containing 25 mM HCO-3 aerated with 7% CO2 in N2 at pH 7.4, the lactate production and lactate content of the tissue increased.--3. At an extracellular (tissue bath) pH OF 6.9 the lactate production was stimulated when carbon dioxide and bicarbonate were changed to 19% and 25 mM, respectively. This stimulatory effect disappeared when these values were lowered to 7% and 7 mM.--4. At pH 7.4 the stimulatory effect of the carbon dioxide-bicarbonate system persisted when the buffer value was lowered from 60 to 3 mM by changing the system from an open (i.e. continuous gas equilibration) to a closed one (i.e. without any gas phase). Decreasing the glucose in the media from 22 to 0 mM reduced the lactate production and abolished the stimulatory effect of the carbon dioxide--bicarbonate system.--5. There was no direct effect of this system on the glycolytic enzymes (i.e. lactate production and activity of phosphofructokinase of homogenates).

A presynaptic effect of whole venom of Dendroaspis jamesoni on the hemidiaphragm of rat.

Dendroaspis jamesoni venom in a dose of 12.5 mug/ml restricts the uptake of Ca+ leading to inhibition of release of Ach from the motor nerve terminals of the hemidiaphragms of rats.

The effect of diabetes, insulin, and the redox potential on leucine metabolism by isolated rat hemidiaphragm.

The oxidation of leucine by hemidiaphragms of control and diabetic rats was studied in vitro. Rats were rendered diabetic with streptozotocin. Hemidiaphragms of diabetic rats produced approximately 50% more 14CO2 during incubation with 0.1 mM [1-14C]leucine than did control muscles. This was observed during incubation with or without glucose and in the presence or absence of a full complement of plasma amino acids. The concentration of leucine in the tissue water of hemidiaphragms from diabetic rats was greater than that in the control muscles before incubation. The specific activity of leucine at the end of 60 min incubation was not significantly different in diabetic and control muscles, indicating that the increased 14CO2 production represented stimulation of leucine oxidation. Hemidiaphragms of diabetic rats released more leucine into the medium during incubation than did control muscles. The stimulating effect of diabetes on leucine oxidation in vitro was reversible by insulin therapy prior to sacrifice. The addition of 5 mM pyruvate to a medium containing glucose inhibited 14CO2 production from [14C]leucine in control muscles, but stimulated leucine oxidation by hemidiaphragms of diabetic rats. Leucine oxidation by hemidiaphragms of diabetic rats was markedly stimulated by the addition of an electron acceptor, 0.02 mM methylene blue, suggesting that the NADH/NAD ratio may be rate-limiting for branched chain amino acid oxidation in muscles of diabetic rats, but not in muscles of controls. We suggest that the accelerated oxidation of branched chain amino acids by muscles may play a role in the acceleration of the muscle protein catabolism and gluconeogenesis which develop during insulin deficiency. The restraining effect of the cellular redox potential on branched chain amino acid oxidation may play a role in the eventual deceleration of protein catabolism during a prolonged fast.

Effect of parathion and its metabolites on calcium uptake activity of rat skeletal muscle sarcoplasmic reticulum in vitro

The insecticide parathion and its potent anticholinesterase metabolite paraoxon induce skeletal muscle necrosis when administered in vivo to rats. In the present study the effects in vitro of parathion and its metabolites on microsomal (sarcoplasmic reticular) calcium uptake activity in rat diaphragm skeletal muscle are examined. Parathion (0.05 mM) is a potent inhibitor of this calcium uptake. The inhibition is apparently competitive with calcium in the system. Parathion (0.05 mM) is also shown to inhibit calcium-dependent ATPase activity associated with the microsomal calcium uptake. Paraoxon, the active anticholineslerase metabolite of parathion, and p-nitrophenol, a hydrolytic metabolite of paraoxon, have no inhibitory effects at this level. At 1.5 mM levels they do inhibit the skeletal muscle microsomal calcium uptake. Eserine, a chemically unrelated anticholinesterase agent, also has inhibitory effects at 1.5 mM. When these same compounds are incubated with isolated rat hemidiaphragms they antagonize the muscle contraction elicited by direct stimulation of the muscle. The skeletal muscle necrosis caused by parathion and paraoxon appear to relate to the anticholinesterase activity in vivo. The relatively potent inhibition of calcium uptake activity of sarcoplasmic reticulum in vitro seen with parathion appears to be an independent action and not related to cholinesterase inhibition.

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

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 [ 14C]glucosc into glycogen, while incorporation into lactate of 14C 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 [ 14C]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.

Studies on the mechanism of centpiperalone-induced hypoglycemia.

The effect of centpiperalone [2-piperazino-4(3H)-quinazolinone monoacetate], a potent hypoglycemic compound on glucose and oxygen uptake, and lactic acid formation by rat hemidiaphragms suspended in glucose-phosphate buffer in Warburg bath at 37 degrees C and 80 shakes/min was studied in vitro. Although centpiperalone enhanced glucose and oxygen uptake by itself, no increment in the above parameters was seen when insulin was added simultaneously along with centpiperalone. Preincubation of hemidiaphragms with centpiperalone and their subsequent incubation with insulin did not indicate any potentiation of insulin action as indicated by uptake of oxygen and glucose. However when 16 times diluted serum from centpiperalone-treated rats was added to hemidiaphragms suspended in glucose-phosphate buffer, significant increase in glycogen content was observed compared to that seen in hemidiaphragms after addition of serum from normal untreated and alloxanized centpiperalone-fed rats. Definite degranulation of B-cells was seen in centpiperalone-fed rats using the aldehyde fuchsin staining technique. These data strongly suggest that centpiperalone induces hypoglycemia through liberation of insulin from B-cells.

Effects of intracellular lithium on epinephrine-induced accumulation of cyclic AMP in skeletal muscle

The effects of lithium and of magnesium ions on adenosine 3',5'-monophosphate (cyclic AMP) accumulation in isolated rat hemidiaphragms were investigated. Magnesium ions caused a small but significant increase in the basal concentration of cyclic AMP, whereas lithium ions had no effect. However, pretreatment of tissues with LiCl caused a significant dose-dependent reduction in the effect of epinephrine on the concentration of cyclic AMP. Magnesium ions enhanced the rise in tissue cyclic AMP caused by epinephrine, but this stimulatory effect of magnesium ions was reduced in tissues that had been pretreated with LiCl. It was concluded that lithium ions act at an intracellular site to inhibit hormone-induced accumulation of the cyclic nucleotide and was proposed that magnesium and lithium ions act at different cellular locations to produce their effects on hormone-induced cyclic AMP accumulation.

Leucine. A possible regulator of protein turnover in muscle.

Incorporation of radiolabeled precursors into muscle proteins was studied in isolated rat hemidiaphragms. A mixture of three branched-chain amino acids (0.3 mM each) added to media containing glucose stimulated the incorporation of [14C]lysine into proteins. When tested separately, valine was ineffective, isoleucine was inhibitory, but 0.5 mM leucine increased the specific activity of muscle proteins during incubation with [14C]lysine or [14C]acetate in hemidiaphragms from fed or fasted rats incubated with or without insulin. Preincubation with 0.5 mM leucine increased the specific activity of muscle proteins during a subsequent 30- or 60-min incubation with [14C]lysine or [14C]pyruvate without leucine. Preincubation with other amino acids (glutamate, histidine, methionine, phenylalanine, or tryptophan) did not exert this effect. When hemidiaphragms were incubated with a mixture of amino acids at concentrations found in rat serum and a [14C]lysine tracer, the specific activity of muscle proteins increased when leucine in the medium was raised from 0.1 to 0.5 mM. Experiments with actinomycin D and cycloheximide suggested that neither RNA synthesis nor protein synthesis are required for the initiation of the leucine effect. Leucine was not effective when added after 1 h preincubation without leucine. The concentration of lysine in the tissue water of diaphragms decreased during incubation with 0.5 mM leucine in the presence or absence of cycloheximide, suggesting that leucine inhibited protein degradation. During incubation with [3h]tyrosine (0.35 mM) the addition of 0.5 mM leucine increased the specific activity of muscle proteins, while the specific activity of intracellular tyrosine remained constant and its concentration decreased, suggesting that leucine also promoted protein synthesis. The concentration of leucine in muscle cells or a compartment thereof may play a role in regulating the turnover of muscle proteins and influence the transition to negative nitrogen balance during fasting, uncontrolled diabetes, and the posttraumatic state. Leucine may play a pivotal role in the protein-sparing effect of amino aicds.

Transport of peptide hormones across isolated rat mesentery: effect of human serum-bound insulin.

The exchange of 125I-insulin, 125I-glucagon, 125I-proinsulin, 125I-growth hormone, 131I-albumin, 14C-inulin, and 14C-dextran across isolated rat mesentery was studied in a diffusion cell. The passage of immunoprecipitable porcine 125I-insulin (0.88 ng./ml.) was not affected by porcine proinsulin (145 ng./ml.), crystalline porcine insulin (17.4 ng./ml.), human growth hormone (87 ng./ml.), bovine serum albumin (4.5 mg./ml.), or normal guinea pig serum (840 mug. protein/ml.). However, the rate of insulin exchange was reduced by guinea pig anti-insulin antiserum and partially purified human serum-bound insulin (175 mug. protein/ml.). Bound insulin at the same concentration did not affect the exchange of 125I-glucagon, 125I-growth hormone, 14C-inulin, or 14C-dextran. Further purification of bound insulin by Sephadex G-100 chromatography yielded an approximately 45,000-molecular-weight fraction that at 5 mug. protein permilliliter allowed essentially no insulin transport. This same fraction of bound insulin significantly inhibited the disappearance of immunoprecipitable porcine 125I-insulin from the incubation medium of isolated rat hemidiaphragms. Theses studies suggest that the transport of insulin across biologic membranes, mesothelium, and possible endothelium is specifically inhibited by bound insulin, a circulating macromolecule that possesses insulin-like activity.

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

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.

Synthesis of acetylcholine receptor by denervated rat diaphragm muscle.

Acetylcholine receptor was purified by affinity chromatography from denervated rat hemidiaphragms that had been incubated in organ culture for 24 hr in medium containing [35-S] methionine. Radioactive acetylcholine receptor was identified in purified preparations by zone sedimentation in a sucrose gradient, by isoelectric focusing, and by precipitation with an antiserum to the acetylcholine receptor from electric eel. When innervated and denervated hemidiaphragms were incubated with [35-S] methionine in organ culture, and the acetylcholine receptors from each were purified separately, only the preparation from denervated muscles contained radioactive receptor as determined by zone sedimentation. We conclude that newly synthesized receptor is accumulated as a result of muscle denervation.

The action of insulin of glycogen synthesis in rat diaphragm from intracellular and extracellular glucose

Rat hemidiaphragms were loaded with [U-14C] glucose at 2°C and subsequently incubated at 37°C with non-labeled glucose or [14C] glucose in the presence or absence of insulin. The incorporation of isotope into glycogen and lactate was determined. The results showed that insulin markedly stimulated the synthesis of glycogen from extracellular glucose while it had no effect on incorporation of isotope into glycogen from intracellular glucose. Lactate formation was not influenced by insulin. It was concluded that glucose transport in muscle is linked to glycogen sythesis and that insulin preferentially directs glucose entering the cell toward the formation of glycogen.

Action of Insulin on Transport of l-Alanine into Rat Diaphragm in Vitro: EVIDENCE THAT THE HORMONE AFFECTS ONLY ONE NEUTRAL AMINO ACID TRANSPORT SYSTEM

Abstract A study has been made of the ability of insulin to stimulate the transport of l-[14C]alanine into paired intact rat hemidiaphragms in vitro, and the results have been compared with those obtained with α-amino[14C]isobutyrate and l-[14C]leucine. A variety of agents and conditions have been tested for their relative abilities to alter total saturable uptake of the amino acids on the one hand, and the insulin stimulation of uptake on the other. Transport of alanine (0.5 mm) was increased about 20% by the presence of insulin (0.1 unit per ml) under the conditions of the experiment. A 10 to 20 mm level of α-aminoisobutyrate eliminated the insulin stimulation of alanine uptake while decreasing its total saturable uptake by only about 10%. Leucine, in contrast, could inhibit at least three-fourths of the total saturable alanine uptake without decreasing the increment in uptake produced by insulin. Omitting Na+ from the incubation medium decreased total alanine uptake by about 60%, but abolished insulin stimulation. Decreasing the pH of the incubation medium progressively from 7.4 to 5.4 brought a regular reduction in the insulin stimulation, but at the same time the total saturable alanine uptake was increased. Other agents and conditions gave less sharp distinction between the transport systems used by alanine. α-Aminoisobutyrate transport was more than doubled by 0.1 unit of insulin per ml in 1 hour. The pattern of its inhibition by different conditions suggests that this amino acid is transported in diaphragm nearly completely by one system, which is Na+-dependent as well as sensitive to insulin. Leucine transport, on the other hand, showed relatively little response to insulin, and was essentially unaltered by changes in buffer [Na+] or pH, or by the presence of competitive inhibitors other than leucine and isoleucine. The results suggest that l-alanine enters rat diaphragm by at least three saturable routes. One is Na+-independent; another is Na+-dependent and sensitive to insulin; while the third is Na+-dependent but not affected by insulin. The insulin-sensitive route accounts for no more than about 10 to 20% of the total saturable uptake at the alanine levels used. These facts support the hypothesis that insulin acts on only one neutral amino acid transport system in diaphragm; and that the hormone does not increase alanine transport greatly in this tissue because alanine does not use the insulin-sensitive system extensively.

The influence of sodium, potassium and lithium on the response of glycogen synthetase I to insulin and epinephrine in the isolated rat diaphragm.

Abstract 1. 1. Intact rat hemidiaphragms were incubated in media of different ionic composition in the absence and the presence of hormones i.e. insulin and epinephrine. After incubation, glycogen synthetase activity of diaphragm extracts was determined in the absence (I-form) and the presence of glucose 6-phosphate (D-form). 2. 2. Incubation of the diaphragms in media of high K+ concentration led to decreased glycogen synthetase I levels. The response of the synthetase system to insulin (conversion of the D-form to the I-form) was reduced under these conditions. Furthermore, the conversion of the I-form to the D-form of glycogen synthetase promoted by epinephrine was also reduced. 3. 3. Incubation of the diaphragm in lithium-containing media increased glycogen synthetase I. Insulin and lithium exerted additive effects in promoting D to I conversion of the synthetase. 4. 4. The response of glycogen synthetase of the diaphragm to insulin (conversion of the D-form to the I-form) was markedly reduced when (Na+-K+)-activated ATPase was blocked by 0.2 mM ouabain in the incubation medium. 5. 5. The insulin effect on glycogen synthetase was absent when active Na+ and K+ transport was blocked by incubation in a medium lacking K+. This was shown by: (a) incubation of the diaphragm in a K+-free medium to which tetraphenylboron, a K+-chelating agent, was added. (b) removal of K+ by preliminary soaking of the diaphragm in a medium containing Dowex 50 WNa. 6. 6. The response of glycogen synthetase to epinephrine was not affected by incubation of the diaphragm in a K+-free medium.

Stimulation of sensitivity of the glycogen phosphorylase-converting system in skeletal muscle by metabolic inhibitors

Intact rat hemidiaphragms were incubated in Krebs-Ringer bicarbonate media to which was added iodoacetate, fluoroacetate and/or pyruvate. The inhibitors increased the extent of conversion of phosphorylate b to a following addition of adrenalin or dibutyryl cyclic AMP and after tetanic electrical stimulation. Sensitivity to adrenalin was markedly increased. Pyruvate prevented the effects of these inhibitors and slightly depressed the response of the phosphorylase system to adrenalin in the absence of inhibitors. Metabolic blockade did not alter the response of the glycogen synthetase system to adrenalin. The data indicate that feedback regulation of the conversion of phosphorylase b to a occurs and that this may be indirectly mediated by the level of free intracellular calcium.

The Effect of Epinephrine, Glucagon, and the Nutritional State on the Oxidation of Branched Chain Amino Acids and Pyruvate by Isolated Hearts and Diaphragms of the Rat

Abstract The effect of epinephrine and glucagon on 14CO2 production from C-1-labeled branched chain amino acids, [1-14C]pyruvate, [1-14C]alanine, and [U-14C]glucose, was studied in isolated diaphragms and perfused hearts. The tissues were obtained from rats fed ad libitum or fasted for 48 hours. Epinephrine (10-4 m) stimulated 14CO2 production from leucine, valine, and alanine by hemidiaphragms of fasted rats incubated without substrate, but not by hemidiaphragms of rats fed ad libitum. The oxidation of histidine was not affected by epinephrine. Epinephrine (10-5 to 10-6 m) stimulated branched chain amino acid oxidation by hearts of fed or fasted rats during perfusion without substrate. The addition of 5.5 mm glucose to the perfusion medium abolished the stimulatory effect of epinephrine on branched chain amino acid oxidation. The addition of pyruvate markedly inhibited the oxidation of branched chain amino acids by perfused hearts, and abolished the stimulatory effect of epinephrine on this process. Glucagon (2 x 10-8 to 5 x 10-9 m) stimulated branched chain amino acid oxidation by hearts of fasted rats perfused without additional substrate. Insulin added to glucagon caused no further stimulation of leucine oxidation. Glucagon did not stimulate leucine oxidation by hearts of fed rats, nor by hearts of fasted rats perfused with glucose (5.5 mm) or pyruvate (20 mm). Glucagon did not affect branched chain amino acid oxidation by diaphragms. N6, O2'-Dibutyryl adenosine 3',5'-monophosphate (10-2 to 10-3 m) was mildly inhibitory. Hearts and diaphragms of fed rats oxidized pyruvate much faster than those of fasted rats. Epinephrine (10-5 to 10-6 m) stimulated the myocardial oxidation of pyruvate and alanine. Epinephrine (10-4 to 10-5 m) stimulated the uptake and oxidation of pyruvate by diaphragms of fed or fasted rats, but diaphragms of fed rats were more responsive to this effect of epinephrine than those of fasted rats. Insulin (1 milliunit per ml) mildly stimulated 14CO2 production from pyruvate by diaphragms of fed rats. Stimulation by 10-5 m epinephrine was greater than that by insulin. In the presence of epinephrine insulin caused no further stimulation. Epinephrine stimulated 14CO2 production from [U-14C]glucose by hemidiaphragms, but did not affect the uptake of glucose from the medium. 14CO2 production from [1-14C]acetate by diaphragms was not affected by epinephrine. Glucagon (2 x 10-8 m) stimulated 14CO2 production from [1-14C]pyruvate by hearts of fed rats, but not by hearts of fasted rats. Glucagon did not stimulate 14CO2 production from [2-14C]pyruvate. Glucagon and insulin stimulated 14CO2 production from [U-14C]glucose at high glucose concentrations (16.5 mm) and the effects of the two hormones were additive. The oxidation of pyruvate and that of the branched chain amino acids by skeletal and heart muscles is regulated by the nutritional state of the animal and is susceptible to hormonal regulation by glucagon, epinephrine, and insulin in hearts, and the latter two hormones in diaphragms. The data suggest that in muscles, the pyruvate dehydrogenase complex is activated in the fed state, and is more responsive to stimulation by epinephrine and glucagon than in the fasted state. On the other hand, branched chain amino acid oxidation by muscles, is more susceptible to stimulation by epinephrine or glucagon in the fasted than in the fed state, and the hormonal stimulation can be suppressed by pyruvate or glucose.

Motor and sensory reinnervation of fast and slow mammalian muscles.

1. The formation of endplates outside the original endplate region of a muscle fibre was studied in slow and fast rat muscles. It was found that such new endplates are readily formed on the soleus muscle, whereas hardly at all in the fast extensor digitorum longus. Most new endplates appear to be morphologically normal within 2 months after nerve implantation. 2. The time course of recovery of slow and fast cat muscles was followed after crushing the sciatic nerve. It was found that the slow soleus muscle recovers more rapidly than the fast flexor hallucis longus muscle. 3. The endplates of reinnervated cat muscles are more complicated than those of the control muscles, but have nevertheless fewer nerve terminals per endplate. Reinnervated muscles are more sensitive to curare and it is suggested that this is due to a decrease in transmitter release, for it was found that less acetylcholine is released from reinnervated rat hemidiaphragms than from control ones. 4. Motor and sensory reinnervation of spindles and tendon organs was studied. At the time when motor reinnervation is almost completed the sensory endings from spindles and tendon organs are highly abnormal. Thus sensory reinnervation proceeds much more slowly than motor.

Oxidation of Branched Chain Amino Acids by Isolated Hearts and Diaphragms of the Rat: THE EFFECT OF FATTY ACIDS, GLUCOSE, AND PYRUVATE RESPIRATION

Abstract Amino acid oxidation was studied in perfused hearts and incubated hemidiaphragms of rats fed ad libitum. Hearts were perfused with Krebs-Henseleit bicarbonate buffer containing glucose and 0.1 mm amino acids. The addition of 1 mm octanoate to the medium markedly stimulated 14CO2 production from [1-14C]leucine, isoleucine, and valine; inhibited 14CO2 production from [1-14C]alanine, pyruvate, and α-ketoglutarate, and did not affect 14CO2 production from [14C]histidine, threonine, glutamate, ornithine, and phenylalanine. Hemidiaphragms incubated with 1 mm hexanoate or 0.1 to 1.0 mm octanoate increased the oxidation of the branched chain amino acids by 80 to 200% and decreased 14CO2 production from and pyruvate. Octanoate stimulated the oxidation of isoleucine by hemidiaphragms at isoleucine concentrations between 0.01 to 1.0 mm; when isoleucine exceeded 2 mm, octanoate became inhibitory. Octanoate stimulated the oxidation of the branched chain amino acids in the presence or absence of glucose and in the presence or absence of insulin from the medium, in diaphragms and hearts. Bovine serum albumin did not affect branched chain amino acid oxidation in the absence of added fatty acids; it inhibited the stimulatory effect of the latter. The inhibition was proportional to the albumin concentration. Palmitate and oleate (1 mm) added to albumin (15 mg per ml) stimulated branched chain amino acid oxidation by hemidiaphragms; the effect was smaller than that observed with equimolar octanoate and albumin. In hearts, no effect of 1 mm palmitate was observed. Butyrate (2 mm) did not affect branched chain amino acid oxidation by hemidiaphragms; 4 mm dl-β-OH butyrate and 4 mm acetate were inhibitory. In hearts and diaphragms, the omission of glucose from the incubation medium stimulated the oxidation of the branched chain amino acids. Pyruvate added to media containing glucose caused further inhibition of branched chain amino acid oxidation. Insulin stimulated 14CO2 production from [14C]leucine in hearts obtained from rats fasted for 48 hours and perfused without glucose. In hearts perfused with [14C]leucine, the addition of octanoate to the perfusate did not affect the tissue concentration of free leucine nor the labeling of the leucine pool. Octanoate stimulated 14CO2 production from leucine by hemidiaphragms which were preloaded with [14C]leucine, indicating stimulation of leucine oxidation beyond the transport of leucine into muscle cells. The apparent inverse relationship between the oxidation of pyruvate and that of the branched chain amino acids suggests that regulation of the latter may occur at the oxidative decarboxylation of the branched chain α-keto acids in muscles. It is suggested that regulation of branched chain amino acid catabolism in muscles may compliment the alanine cycle, and may play a role in homeostasis under conditions of limited availability of glucose (e.g. fasting) or increased utilization (e.g. exercise).

Effects of diphenylhydantoin on the transport of Na + and K + and the regulation of sugar transport in muscle in vitro

1. 1. The distribution of the nonmetabolized glucose analog 3-O- methyl- d-[ 14C]-glucose and the intracellular concentration of Na + and K + were measured in “intact” rat hemidiaphragms, in vitro. 2. 2. 5,5-Diphenylhydantoin (DPH) at concentrations of 0.1 and 0.5 mM produced an increase in internal K + and a decrease in internal Na +, consistent with stimulation of the Na + pump. Higher concentrations, 1.0 and 5.0 mM had the opposite effect. Both the stimulatory and the inhibitory effects were potentiated by increased external K + (16 mM). 3. 3. Sugar transport was inhibited by DPH whenever internal K + was increased and Na + decreased and, conversely, sugar transport was stimulated following the opposite ionic changes. The changes in sugar transport were highly and significantly correlated to changes in internal Na + and K +. These effects were apparent on efflux, as well as on influx of sugar. Inhibition was present with and without insulin but was greater in the presence of the hormone. The inhibitory effect of DPH was abolished by 10 −5 M ouabain, which blocks the Na + pump but not by 10 −9 M ouabain. 4. 4. The ionic changes are evidence for a stimulatory effect of DPH on ion transport which changes to inhibition at higher drug concentrations. The effects on sugar transport provide additional evidence for inhibition of sugar transport by low internal Na + (or high K +) and support the postulated regulatory effect of internal Na + and/or K + on the sugar transport mechanism in skeletal muscle.

Evaluation of biological activity of chicken insulin in alloxan-diabetic rats

1. 1. Sensitivity curves were established in vivo employing equal amounts of crystalline chicken and beef insulin in alloxan-diabetic rats; the two insulins were compared also in vitro employing the rat hemidiaphragm bioassay. 2. 2. Chicken insulin had less hypoglycemic potential than did beef insulin in diabetic rats; plasma from diabetic rats severely inhibited the glucose-uptake activity of chicken insulin (more so than that of beef insulin) when incubated with normal rat hemidiaphragms. 3. 3. Chicken and beef insulin were equally active in promoting glucose uptake and glycogenesis in non-diabetic rat hemidiaphragms; however, the mammalian hormone was at least twice as potent as the avian hormone when assayed with diaphragm muscle of diabetic rats. 4. 4. The results obtained are discussed in relation to known plasma inhibitory factors of insulin and again emphasize the diabetic lesion existent at the membrane (glucose uptake) level.