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M K Gould

Publications and source records attributed to M K Gould.

At least 37 records · Page 2Linked to original sources
PubMed1984

Inhibitory effects of N-ethylmaleimide on insulin- and oxidant-stimulated sugar transport and on 125I-labelled insulin binding by rat soleus muscle.

These experiments examined the effects of N-ethylmaleimide on insulin- and oxidant-stimulated sugar transport in soleus muscle in terms of the Thiol-Redox model for insulin-stimulated adipocyte sugar transport (Czech, M.P. (1976) J. Cell. Physiol. 89, 661-668). Brief exposure (1 min) to N-ethylmaleimide (0.3-10 mM) inhibited the stimulatory effect of insulin (0.1 U/ml) on D-[U-14C]xylose uptake by rat soleus muscle. N-Ethylmaleimide also inhibited the stimulatory effects of H2O2 (5 mM), diamide (0.2 mM) and vitamin K-5 (0.05 mM). This effect of N-ethylmaleimide on insulin action was paralleled by the inhibition of 125I-labelled insulin binding by the muscle. N-ethylmaleimide lowered muscle ATP; however, its effects on sugar transport and 125I-labelled insulin binding could be dissociated from its effect on ATP. Exposing muscles to insulin prior to N-ethylmaleimide did not abolish the inhibitory effect of sulphydryl blockade on insulin-stimulated sugar transport, but did reduce the effect of the inhibitor by 20-30%. Conversely, when muscles were first allowed to bind 125I-labelled insulin and then exposed to the inhibitor, there was no effect of N-ethylmaleimide on pre-bound insulin. Exposure to diamide or vitamin K-5 before N-ethylmaleimide (1 mM) attenuated the inhibitory effect of sulphydryl blockade but no protective effect was observed with H2O2. None of the oxidants protected against the inhibitory effect of 3 mM N-ethylmaleimide. It is concluded that there are two N-ethylmaleimide-sensitive sites involved in the activation of muscle sugar transport at the post-receptor level. One of these would appear to be similar to the Thiol-Redox site described in the adipocyte; the other site appears to be an essential sulphydryl group whose function does not involve oxidation to a disulphide.

Animals↗
PubMed1983

ATP depletion promotes deactivation of insulin-stimulated sugar transport in rat soleus muscle.

It has been reported that deactivation of insulin-stimulated sugar transport in adipocytes is an energy-dependent process (F. V. Vega, R. J. Key, J. E. Jordan, and T. Kono (1980) Arch. Biochem. Biophys. 203, 167-173). The stimulatory effect of insulin (0.1 U/ml) on the uptake of D-[U-14C]xylose by rat soleus muscle was rapidly reversed when muscle ATP was depleted by exposure to 2,4-dinitrophenol (0.5 mM). Insulin action was not completely eliminated by ATP depletion; there was a small, residual stimulatory effect of the hormone which persisted for about 30 min after muscle ATP had been lowered to an unmeasurable level. The extent of deactivation was not altered when the rate of ATP depletion was accelerated, either by increasing the 2,4-dinitrophenol concentration, or by inducing leakiness by incubating muscles for 90 min at 37 degrees C prior to the addition of the uncoupler. 2,4-Dinitrophenol lowered steady-state 125I-insulin binding. These differences between the effect of ATP depletion on insulin-stimulated sugar transport in muscle and adipose tissue may be related to the action of the uncoupler in lowering steady-state insulin binding in muscle. Such a fall in bound insulin could be expected to promote deactivation during the period of ATP depletion. However, at present the possibility that these differences may represent some more fundamental difference in deactivation between muscle and adipose tissue cannot be excluded.

2,4-Dinitrophenol↗
PubMed1983

Stimulatory and inhibitory effects of hydrogen peroxide, diamide and vitamin K-5 on sugar transport in rat soleus muscle.

The experiments examined the stimulatory effects of H2O2, diamide and vitamin K-5 on the uptake of D-[U-14C]xylose by rat soleus muscle. All three oxidants stimulated sugar transport to the same extent, which was 30-40% of the maximal stimulatory effect of insulin (0.1 U/ml). Maximum stimulation was achieved with vitamin K-5 (0.01-0.1 mM), diamide (0.3 mM) and H2O2 (5-10 mM); at these concentrations the oxidants did not affect muscle ATP levels. Cytochalasin B (5 microM) abolished oxidant-stimulated xylose uptake. Catalase (20 U/ml) abolished the stimulatory effect of H2O2, but did not affect diamide- or vitamin K-5-stimulated transport. The ability of oxidants to stimulate sugar transport in anaerobic muscle could be demonstrated in short term (30 min) experiments, where muscle contained about 50% of its original ATP, but not after 120 min, when ATP levels were depleted. Oxidant-stimulated sugar transport was diminished and even abolished at supra-optimal oxidant concentrations; at these higher concentrations muscle ATP levels were lowered. All three oxidants inhibited the stimulatory effect of 2,4-dinitrophenol on sugar transport; this effect could be demonstrated using those oxidant concentrations which induced maximal stimulation of basal xylose uptake and which did not affect muscle ATP. It is concluded that: (1) H2O2, diamide and vitamin K-5 stimulate stereospecific sugar transport in soleus muscle by some mechanism other than lowering of ATP levels; (2) stimulation of sugar transport by oxidants is an ATP-dependent process; (3) some oxidant-sensitive sulphydryl group is critically involved in the process which activates muscle sugar transport.

Adenosine Triphosphate↗
PubMed1983

Inhibition of insulin-stimulated xylose uptake in rat soleus muscle by cycloheximide.

Cycloheximide (20-200 mg/l) did not affect basal D-[U-14C]xylose uptake by rat soleus muscle (2.4 +/- 0.2 mumol . g-1 . h-1). However, the stimulatory effect of insulin on sugar transport was progressively reduced from 375% above basal in control muscles to 170% in muscles exposed to 200 mg cycloheximide/l but above this concentration cycloheximide inhibited basal xylose uptake without further effect on the incremental effect of insulin. Cycloheximide affected the insulin dose-response curve both by depressing insulin sensitivity and by reducing the maximum stimulatory effect of the hormone. In contrast to the inhibition of insulin action, which increased progressively over the range 20-200 mg cycloheximide/l, muscle protein synthesis was inhibited maximally at a concentration of 10 mg/l. Cycloheximide also inhibited the insulinomimetic effects of anoxia, 2:4-dinitrophenol, salicylate, cooling, hydrogen peroxide, diamide, vitamin K5, hyperosmolarity and EDTA, but did not affect concanavalin A-stimulated xylose uptake. It is concluded that cycloheximide inhibits insulin-stimulated sugar transport at some late post-receptor step, and that this effect of cycloheximide is not secondary to the inhibition of protein synthesis.

Animals↗
PubMed1983

ATP-dependence of 125I-insulin binding by rat soleus muscle.

Muscle ATP levels were lowered by incubating rat soleus muscles under anaerobic conditions, or in the presence of 2:4-dinitrophenol (0.5 mM), EDTA (5 mM) or mannitol (400 mM). 125I-insulin binding, measured under equilibrium conditions at 25 degrees C, was reduced by 49-71% in ATP-depleted muscles. Insulin binding was also determined using two other procedures which minimized internalization of 125I-insulin: these were (a) 5 min at 25 degrees C, and (b) 24 h at 3 degrees C. Under these conditions, 125I-insulin binding was reduced by 28-55% in ATP-depleted muscles. These results confirm that in soleus muscle the effect of ATP-depletion on 125I-insulin binding is actually concerned with the binding step itself and not merely a reflection of ATP-dependent internalization of the bound hormone.

2,4-Dinitrophenol↗
PubMed1982

Multiple effects of sulphydryl reagents on sugar transport by rat soleus muscle.

Iodoacetate, over the range 0.2-2 mM, stimulated the uptake of D-xylose by rat soleus muscle and inhibited anaerobic lactate production by soleus muscle. Stimulation of sugar transport is considered to be due to the resultant fall in ATP. p-Chloromercuribenzene sulphonate (0.5-2 mM) stimulated xylose uptake to a lesser extent than iodoacetate and induced a proportionately smaller fall in ATP, consistent with the inhibitory effect of p-chloromercuribenzene sulphonate on lactate production. Under certain conditions, p-chloromercuribenzene sulphonate stimulated sugar transport without affecting the ATP level. This suggests that whereas p-chloromercuribenzene sulphonate can be expected to stimulate sugar transport through the lowering of muscle ATP, it may also act through some other mechanism. No stimulatory effect on xylose uptake was observed when muscles were exposed to N-ethylmaleimide (0.02-2 mM) either for brief (1 min) or more prolonged (30 min) periods. Because N-ethylmaleimide induced a marked fall in muscle ATP, it is surprising that N-ethylmaleimide did not stimulate sugar transport; in most experiments this inhibitor actually inhibited sugar transport. N-Ethylmaleimide inhibited the stimulation of sugar transport by 2,4-dinitrophenol and anoxia; this inhibitory effect appears to explain why N-ethylmaleimide itself did not stimulate sugar transport. p-Chloromercuribenzene sulphonate also inhibited 2,4-dinitrophenol-stimulated xylose uptake by a mechanism which seems similar to that of N-ethylmaleimide; this could explain in part the modest stimulatory effect of this inhibitor on muscle sugar transport.

2,4-Dinitrophenol↗
PubMed1982

Inhibition of insulin-stimulated xylose uptake in denervated rat soleus muscle: a post-receptor effect.

Insulin (100 U/l) stimulated xylose uptake in rat soleus muscle from a basal value of 2.3 +/- 0.5 to 11.6 +/- 2.1 mumol . g-1 . h-1. Denervation (section of the sciatic nerve) markedly reduced the stimulatory action of insulin (basal 1.3 +/- 0.4 mumol . g-1 . h-1; insulin-stimulated 4.5 +/- 0.6 mumol . g-1 . h-1). This effect appeared 3 days after denervation and was maximal after 5 days. Denervation also affected the insulin dose response curve; there was no effect of insulin in denervated muscle until the concentration exceeded 1 U/l. Denervation inhibited insulin-stimulated alpha-aminoisobutyrate uptake by 77% but did not affect 125I-insulin binding or glucose-independent activation of glycogen synthase by insulin. There was no effect of denervation on the insulinomimetic effects of concanavalin A, hydrogen peroxide, vitamin K5, anoxia, 2:4-dinitrophenol, cooling, hyperosmolarity or EDTA, but the effect of diamide was inhibited. It is concluded [1] that denervation inhibits insulin-stimulated sugar transport at some early post-receptor step, and [2] that the mechanism whereby insulin activates glycogen synthase is different from the activation of the membrane transport of sugars and amino acids.

Aminoisobutyric Acids↗
PubMed1982

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+.

Adenosine Triphosphate↗
PubMed1981

Stimulation of sugar transport in rat soleus muscle by prolonged cooling at 0 degrees C.

The uptake of D-xylose by isolated rat soleus muscle (measured at 37 degrees C) was stimulated by prolonged cooling at 0 degrees C. The effect of cooling reached a maximum value after 3 h and was reversed on rewarming; reversal was temperature-dependent. Cooling stimulated xylose uptake sub-maximally compared with the effect of insulin (100 U/l). Xylose uptake in cooled muscle was further stimulated by insulin, but not by anoxia. The effect of cooling and its reversal were still demonstrable in the presence of ouabain (1 mmol/l), or when unidirectional efflux of calcium and magnesium from the muscle was induced by EDTA (5 mmol/l). The ionophore, A23187 (2.5 mg/l), depressed the effect of cooling in the presence of EDTA but not in the presence of EGTA. It is concluded that cooling disrupts and intracellular magnesium-p]ump and that muscle sugar transport is consequentially stimulated through an increase in cytoplasmic magnesium.

Animals↗
PubMed1981

Effects of hyperosmolarity on basal and insulin-stimulated muscle sugar transport.

Xylose uptake was stimulated rapidly when rat soleus muscles were incubated in Krebs-bicarbonate medium made hyperosmolar by the addition of 200 mM mannitol. The stimulatory effect of hyperosmolarity increased as the concentration of mannitol was raised from 50 to 200 mM; above 200 mM, the effect tended to decline. Mannitol stimulated sugar transport submaximally compared with the effect of insulin. The stimulatory effect of hyperosmolarity persisted over a 60-min incubation period, but could be reversed by transferring the muscles to an isotonic incubation medium. Xylose uptake measured in the presence of insulin (0.1 U/ml) was depressed when muscles were exposed to concentrations of mannitol greater than 100 mM. This effect was not reversible; xylose uptake remained depressed when these muscles were transferred to an isotonic medium. Hyperosmolarity also depressed the binding of 125I-insulin irreversibly. These inhibitory effects of hyperosmolarity were associated with the lowering of muscle ATP. This effect on ATP provides an explanation for the inhibitory effects of hyperosmolarity on insulin-stimulated sugar transport and on insulin binding, in terms of the ATP-dependence of these two processes.

Adenosine Triphosphate↗
PubMed1979

Isolation of plasma membrane vesicles, derived from transverse tubules, by selective homogenization of subcellular fractions of frog skeletal muscle in isotonic media.

A new technique for isolating fragmented plasma membranes from skeletal muscle has been developed that is based on gentle mechanical disruption of selected homogenate fractions. (Na+ + K+)-stimulated, Mg2+-dependent ATPase was used as an enzymatic marker for the plasma membrane, Ca2+-stimulated, Mg2+-dependent ATPase as a marker for sarcoplasmic reticulum, and succinate dehydrogenase for mitochondria. Cell segments in an amber low-speed (800 x g) pellet of a frog muscle homogenate were disrupted by repeated gentle shearing with a Polytron homogenizer. Sarcoplasmic reticulum was released into the low-speed supernatant, whereas most of the plasma membrane marker remained in a white, fluffy layer of the sediment, which contained sarcolemma and myofibrils. Additional gentle shearing of the white low-speed sediment extracted plasma membranes in a form that required centrifugation at 100,000 x g for pelleting. This pellet, the fragmented plasma membrane fraction, had a relatively high specific activity of (Na+ + K+)-stimulated ATPase compared with the other fractions, but it had essentially no Ca2+-stimulated ATPase activity and only a small percentage of the succinate dehydrogenase activity of the homogenate. Experimental evidence suggests that the fragmented plasma membrane fraction is derived from delicate transverse tubules rather than from the thicker, basement membrane-coated sarcolemmal sheath of muscle cells. Electron microscopy showed small vesicles lined bu a single thin membrane. Hydroxyproline, a characteristic constituent of collagen and basememt membrane, could not be detected in this fraction.

Adenosine Triphosphatases↗
PubMed1978

Permissive effect of ATP on insulin-stimulated sugar transport by rat soleus muscle.

The stimulatory effect of insulin (0.1 U/ml) on D-xylose uptake was progressively lost when rat soleus muscles were preincubated at 37 degrees C under anaerobic conditions for longer than 30 min; after 90 min these muscles were completely insensitive to insulin. This effect was associated with the loss of muscle ATP. When the breakdown of ATP was retarded either by lowering the preincubation temperature or by preincubation with 5 mM glucose, the effect of insulin in anaerobic muscle was correspondingly prolonged. Under certain conditions, externally added ATP promoted an effect of insulin in otherwise insulin-unresponsive muscles. This effect was small in magnitude and was complicated by the degradation of the added ATP in the incubation medium and by the fact that ATP also tended to inhibit insulin-stimulated xylose uptake. These results indicate that there is a relationship between insulin-stimulated sugar transport and muscle ATP levels. This supports the proposal that there may be some ATP-dependent reaction(s) involved in the mechanism whereby insulin promotes the process of muscle sugar transport.

Adenosine Triphosphate↗
PubMed1978

Effect of prolonged anaerobiosis on 125I-insulin binding to rat soleus muscle: permissive effect of ATP.

The specific binding of 125I-insulin by rat soleus muscle was depressed when muscle ATP was depleted, either by prolonged anoxia or more rapidly with 2,4-dinitrophenol. Insulin binding was not eliminated in ATP-depleted muscle, but was reduced by 70--80%. Insulin binding by aerobic muscle could be resolved into two components; a high-affinity, low-capacity site (KD = 7.8 nM) and a low-affinity, high-capacity site (KD = 390 nM). The stimulatory effect of insulin on xylose uptake could be correlated with binding to the high-affinity site. These results indicate that there is some ATP-dependent process involved in the regulation of insulin binding by soleus muscle. It is suggested that this could be a phosphorylation-dephosphorylation system, acting either on the receptor itself or on some closely related membrane protein.

Adenosine Triphosphate↗
PubMed1977

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.

Adenosine Triphosphate↗
PubMed1976

Short-term measurement of D-xylose uptake by isolated rat soleus muscle.

The uptake of D-[U-14C]xylose by isolated rat soleus muscle was studied, using D-[1-3H]-sorbitol as an extracellular marker. Xylose uptake was limited by the diffusion of the sugar into and through the extracellular water. This could be overcome in part by allowing the test sugars to pre-equilibrate in the extracellular water at 0 degrees C, before measuring xylose uptake. It was not necessary to fill the extracellular water with the test sugars to obtain maximum rates of xylose uptake. From this it was concluded that the sugar carrier sites were located in a specific region on the plasma membrane, readily accessible to sugar carrier sites were located in a specific region on the plasma membrane, readily accessible to sugars entering the interstitial water. Pre-equilibration was more effective in the absence of insulin than in the presence of the hormone. This suggested that insulin may influence sugar uptake at some site prior to the cell membrane. Pre-incubation at 0 degrees C itself stimulated sugar transport. This effect of cooling was not influenced by insulin, nor did it appear to affect the stimulatory action of insulin on xylose transport.

Animals↗
PubMed1975

The effects of exogenous gonadotropins on ovarian adenylate cyclase activity.

Adenylate cyclase was assayed in pellets prepared by centrifuging for 10 min at 600 x g homogenates of ovaries of immature rats treated with PMS and hCG. Activity was detected in the absence of any stimulatory agents and increased markedly in the presence of fluoride. Dose-dependent activity occurred in vitro in response to LH ranging from 0.2 to 10 mug/ml of incubate but not at higher concentrations. Marked changes in adenylate cyclase activities were observed with preparations from ovaries excised at various times after gonadotropin treatment. These changes, measured as the response to both fluoride and LH were observed to occur in four main stages. An initial decrease occurred 1/2 to 1 day after administration of hCG. Activity then increased and remained significantly elevated from day 3 to 7. A second but more dramatic rise was observed on day 8 and this enhanced level of activity remained elevated until day 13. A decreased level of activity occurred on day 15. Unstimulated activity remained low for the 16 day period studied although significant rises were observed on day 3 and days 8 to 15 after the administration of hCG. We have suggested that these changes in the adenylate cyclase activity modulate the level of cyclic AMP in the luteal cells and thereby induce changes in the activity of enzymes involved in progestin biosynthesis.

Adenylyl Cyclases↗