M.K. Gould

Kinetics of glucose uptake in isolated soleus muscle

Abstract 1. 1. Rat soleus muscle can be removed from the animal without cutting the muscle fibres, and so it is a very suitable preparation for in vitro experimentation. 2. 2. Measurement of the basic parameters of glucose uptake showed that soleus muscle behaved similarly to other in vitro muscle preparations with one important difference, namely soleus muscle did not accumulate glucose intracellularly in the presence of insulin. It was thus possible to study the effects of insulin and anoxia on the kinetics of sugar transport. 3. 3. Whereas both insulin and anoxia stimulated the entry of glucose into the muscle cell, the present experiments indicated that the effect of insulin was to lower the apparent K m of glucose uptake, while anoxia increased the ν max .

Evidence for the uptake of ATP by rat soleus muscle in vitro.

1. 1. Rat soleus muscle was incubated in the presence of [8-14C] ATP, [8-14C]ADP or [8-14C]adenosine. Samples of incubation medium and muscle extract were subjected to electrophoretic separation and radioactivity present in ATP, ADP, AMP, IMP, adenosine, inosine and hypoxanthine was counted. 2. 2. Extensive degradation of the added nucleotides was observed. Whereas adenylate kinase and adenosine deaminase activities appeared in the incubation medium, the other enzymes responsible for ATP degradation did not, suggesting that they were located on the exterior surface of the muscle cell. 3. 3. The concentrations of 14C-labelled ATP and ADP found in the muscle indicated that these compounds were present within the fibres. Evidence is presented which suggests that ATP, and to a lesser extent ADP, entered the muscle as such and were not synthesized within the muscle from 14C-labelled adenosine.

Effect of anoxia, 2,4-dinitrophenol and salicylate on xylose transport by isolated rat soleus muscle.

1. These studies examined the theory that ATP served to regulate muscle sugar transport by a feedback mechanism. Xylose uptake by isolated rat soleus muscle was determined over a 5-min period following preincubation at 37 degrees C for various times in the presence of insulin (0.1 unit/ml), 2,4-dinitrophenol (0.5 or 0.05 mM) or salicylate (5 mM) or under anaerobic conditions. 2. Xylose uptake, measured in freshly isolated soleus muscles, was approximately 3.5--4.0 mumol/g per h. When the muscles were preincubated at 37 degrees C, this rate fell by 50% during the first 30 min and then slowly increased. 3. The stimulatory effect of insulin was evident within 2 min in freshly isolated soleus muscle and increased on preincubation, reaching a maximum value (approx. 14 mumol/g per h) after 20 min. 4. There was a 10-min lag period before xylose uptake was stimulated by anoxia. This lag period was approximately doubled when the incubation temperature was lowered from 37 degrees C. The stimulatory effect of anoxia was promptly reversed when muscles were transferred from anaerobic to aerobic conditions. 5. There was a 5-min lag period before xylose uptake was stimulated by 2,4-dinitrophenol (0.05 mM) or by sodium salicylate (mM). At a concentration of 0.5 mM, 2,4-dinitrophenol stimulated xylose uptake in freshly isolated muscle. Whereas the stimulatory effects of insulin, anoxia and salicylate all tended to plateau with time, the effect of 2,4-dinitrophenol tended to peak and then decline. 6. There was no obvious relationship between total muscle ATP levels and xylose uptake. The stimulatory effect of anoxia, 2,4-dinitrophenol or salicylate on xylose uptake was not preceded by the fall in muscle ATP. Similarly, ATP levels did not change when xylose uptake was stimulated by anoxia at 27 degrees C, or when xylose uptake was restored to basal values by transferring muscles from anaerobic to aerobic conditions. 7. It was argued that the presence of the myofibrils could act as a permeability barrier, which would limit the access of ATP produced within the interior of the cell to a regulatory site on, or close to, the sarcolemma. On the other hand, it is conceivable that the ATP produced on the periphery of the fibre by the subsarcolemmal mitochondria could play a more specific role in the feedback regulation of sugar transport. 8. Insulin stimulated xylose uptake in the presence of 2,4-dinitrophenol (0.5 mM) when this was measured in freshly isolated muscle, but not after a period of preincubation. This suggested that there may be some ATP-dependent process involved in the stimulatory effect of insulin.

The action of insulin on glucose uptake by isolated rat soleus muscle I. Effects of cations

Abstract 1. 1. The effects of monovalent cations on glucose uptake by isolated rat soleus muscle could be shown, simply by varying the concentrations of these cations in the incubation medium. Glucose uptake was stimulated by Na + and inhibited by K + . Stimulation of glucose uptake by insulin did not depend on the presence of monovalent cations in the incubation medium. 2. 2. In order to demonstrate the effect of divalent cations, it was necessary to “strip” the endogenous Ca 2+ and Mg 2+ from the muscle, by preincubation for 15 min in Krebs-Henseleit HCO 3 - medium containing 5 mM EDTA. Glucose uptake by these EDTA-stripped muscles, measured in Ca 2+ /Mg 2+ -free medium, was depressed, and could not be increased by the addition of insulin. 3. 3. The basal uptake rate could be restored to normal by the addition of either Ca 2+ (2.5 mM) or Mg 2+ (1.1 mM); at a concentration of 0.1 mM, Ca 2+ , but not Mg 2+ , was still effective. Conversely, Mg 2+ , but not Ca 2+ , was required for the stimulation of glucose uptake by insulin. 4. 4. It is proposed that Ca 2+ is somehow required for the structural integrity of the cell membrane, whereas Mg 2+ probably participates in the mechanism whereby insulin promotes the binding of glucose to the sugar transport carrier.

Effect of externally added ATP on glucose uptake by isolated rat soleus muscle

1. 1. Glucose uptake by isolated rat soleus muscle was stimulated by insulin, anoxia, 2,4-dinitrophenol and salicylate. Anoxia and salicylate both stimulated glucose uptake by increasing the vmax of this process. 2. 2. Anoxia, 2,4-dinitrophenol and salicylate, but not insulin, depleted the ATP content. When muscles were incubated at 21° anoxia stimulated glucose uptake, but did not affect the ATP concentration. 3. 3. ATP (5 mM) added to the incubation medium inhibited glucose uptake under anaerobic conditions, but not aerobically. In the presence of 2,4-dinitrophenol, salicylate or insulin, ATP was without effect. 4. 4. Inhibition of anaerobic glucose uptake was specific to ATP; neither GTP, ITP nor the breakdown products of ATP were effective in this respect. ATP inhibited glucose uptake competitively. This inhibition was not due to chelation of divalent cations. 5. 5. From these experiments it is concluded that the effect of anoxia on sugar transport is not directly mediated via ATP.

Effect of Ionophore A23187 on Basal and Insulin-stimulated Sugar Transport by Rat Soleus Muscle

The ionophore A23187 (10 micrograms/ml) did not affect the uptake of D-[U-14C]xylose by rat soleus muscle incubated under basal conditions. When muscles were incubated in a Ca2+/Mg2+-free (CMF) medium, A23187 promoted the efflux of intracellular Mg2+ and the efflux of 45Ca from preloaded muscles. Under these conditions, conditions, A23187 inhibited insulin-stimulated sugar transport without affecting 125I-insulin binding by the muscle. A23187 induced a slight fall in muscle ATP (16–18%); this does not appear to be responsible for the inhibitory effect of the ionophore on sugar transport. The inhibitory effect of A23187 was completely abolished when the CMF medium was supplemented with Mg2+ and partially reversed by Mn2+ or Zn2+; supplementation with Ca2+ did not reverse the inhibitory effect of the ionophore. These results suggest that insulin stimulates muscle sugar transport through a mechanism that involves intracellular Mg2+.

Synthesis of glycogen from glucose and lactate in isolated rat soleus muscle

Abstract Glycogen synthesis was studied in isolated rat soleus muscle following a period of anaerobic preincubation to deplete the glycogen level. Synthesis was unaffected by varying either the anions or divalent cations present in the incubation medium, however, monovalent cations had a marked effect. Sodium ions promoted glycogenesis while potassium ions led to glycogen depletion. In contrast to the synthesis observed using glycogen-depleted muscle, synthesis in freshly isolated muscle appeared to be limited by the availability of glucose. It was also found that incorporation of 14C-labelled glucose into muscle glycogen always exceeded net synthesis of glycogen. From this it was concluded that net synthesis represented the difference between the amount of glycogen formed in the peripheral fibers of the tissue where glucose and oxygen were readily available, and the amount of glycogen used in the innermost fibers where the availability of glucose and oxygen was limited. Lactate stimulated the formation of glycogen in the presence of glucose, but was itself ineffective as a precursor for glycogen synthesis. The incorporation of 14C-labelled lactate into muscle glycogen is considered to represent a functional reversal of glycolysis, however, as glycogen was synthesized from glucose approximately ten times faster than labelled lactate was incorporated, direct reversal of glycolysis does not appear to be a major source of muscle glycogen.

Pituitary peptides with direct action on the metabolism of carbohydrates and fatty acids

Abstract An improved method for preparing biologically active polypeptides from the anterior pituitary is described. Two active fractions have been obtained. One fraction accelerates fatty acid synthesis from acetate by liver slices. The other fraction inhibits this synthesis in slices, homogenates and a soluble fraction from liver. This fraction also inhibits the enzymic activity of glyceraldehyde-3-phosphate dehydrogenase and α-glycerophosphate dehydrogenase. It has no effect on the activity of hexokinase, phosphofructokinase, aldolase, lactate dehydrogenase, glucose-6-phosphate dehydrogenase, alcohol dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase, malic enzyme or aspartate aminotransferase.

The action of insulin on glucose uptake by isolated rat soleus muscle II. Dissociation of a priming effect of insulin from its stimulatory effect

Abstract 1. 1. Glucose uptake was determined in soleus muscle after preincubation for 15 min in Krebs-Henseleit HCO3- medium containing 5 mM EDTA. Anoxia did not stimulate glucose uptake by these EDTA-treated muscles, unless insulin, together with Ca2+ and Mg2+, were added to the incubation medium. 2. 2. Whereas the permissive effect of insulin could be shown at concentrations below 10 μunits/ml, it was only above this concentration that insulin stimulated glucose uptake in normal muscle. 3. 3. When normal muscle was incubated aerobically in the absence of insulin, the initial rate of glucose uptake was maintained for only 1 h. Insulin at a concentration of 10 μunits/ml, which did not affect this rate, prolonged the linearity for the entire 2-h incubation period. 4. 4. From this it is proposed that insulin at low concentrations exerts a “priming” effect on the cell, which would correspond to the stabilizing action often attributed to the hormone. Stimulation of sugar transport by higher concentrations of insulin thus represents a secondary action of the hormone.

Insulin-stimulated sugar transport and 125I-insulin binding by rat soleus muscle: Permissive effect of ATP

Summary The effect of insulin (0.1 U/ml) on the uptake of D-[U- 14 C] xylose by rat soleus muscle was abolished by preincubation for 90 min under anaerobic conditions. This loss of insulin sensitivity was associated with decreased binding of 125 I-insulin. Prolonged anoxia also depleted muscle ATP. These results are in accord with the proposal that the action of insulin on sugar transport involves some ATP-dependent step. Aerobic incubation (90 min) with cycloheximide (200 μg/ml) did not affect 125 I-insulin binding. This suggests that the effect of prolonged anoxia was not due simply to any effect on the synthesis of the insulin receptor, but rather to some more direct effect of ATP on the interaction of insulin with its receptor.