Xylose Uptake Research Articles

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 insullin- 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 nM) 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 H 2O 2 (5 mM), diamide (0.2 mM) and vitamin K-5 (0.05 mM). This effect of N-ethylmaleimide on insulin 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 blockae 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 effet of sulphydryl blockade but no protective effect was observed with H 2O 2. None of the oxidants protected against the inhibitory effect of 3 nM 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.

Effect of cadmium and zinc on intestinal absorption of xylose and tryptophan in the fresh water teleost fish, [formula omitted][formula omitted

The effect of cadmium and of zinc on the rate of uptake of a pentose sugar xylose and an aminoacid tryptophan by the intestine of a teleost fish, Heteropneustes fossilis was studied under two experimental conditions. In the first, four concentrations of cadmium or zinc (1.0 mM, 0.1 mM, 0.01 mM and 0.001 mM) mixed with the nutrient solution were filled in the intestinal sacs, and the rate of absorption was recorded after 1 h at 23°C. In the second experiment fish were exposed by bath to a sublethal concentration of cadmium (0.26 mg/1) or zinc (4 mg/1) for 15 and 30 days and the rate of absorption of the two nutrients was measured. The activity of intestinal Na +, K + activated adenosine triphosphatase was also assayed. The two heavy metals at all the four concentrations decreased the rate of intestinal transport of nutrients. Increase in the concentration of each of the heavy metals decreased the uptake of nutrients, but the decreases were not linear. The rate of intestinal absorption of the two nutrients was also reduced by exposure of fish to the heavy metals in vivo . The activity of Na +, K + ATPase decreased in vitro with all four concentrations of cadmium and zinc and was diminished in fish exposed for 15 and 30 days. Of the two heavy metals, cadmium was more effective in reducing the rate of transport of xylose and tryptophan.

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.

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

These experiments examined the stimulatory effects of H 2O 2, 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 H 2O 2 (5–10 mM); at these centrations the oxidant did not affect muscle ATP levels. Cytochalasin B (5 μM) abolishe doxidant-stimulated xylose uptake. Catalase (20 U/ml) abolished the stimulatory effect of H 2O 2, but did not affect diamide- or vitamin K-5-stimulated transport. The ability of oxidant to stimulate sugar transport in anaerobic muslce could be demonstrated in shor 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 concentrations; at these higher concentrations muscle ATP levels were lowered. All three oxidant 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) H 2O 2, diamide and vitamin K-5 stimulate stereospecific sugar transport in sloleus muscle by some mechanisms other than lowering of ATP levels; (2) stimulation of sugar transport by oxidants in an ATP-dependent process; (3) some oxidant-sensitive sulphydryl group is critically involved in the process which activates muscle sugar transport.

Glycosyl acceptors in intact and permeabilized normal and cystic fibrosis fibroblasts.

Using cell permeabilization, a technique which allows addition of exogenously supplied radiolabeled sugar nucleotides to serve as direct glycosyl donors, oligosaccharide biosynthesis was examined in fibroblasts obtained from normal and cystic fibrosis (CF) subjects. Incubation of logarithmically growing cells with either radiolabeled leucine or xylose has indicated that there was a difference in the synthetic rate between the cell types. Protein synthesis in normal cells made permeable with 50 micrograms/ml lysolecithin (LL) was demonstrated to be absent, and could not be induced to take place by adding exogenous components, including energy sources and amino acids, normally required for protein synthesis. Thus radiolabeled sugars were being added to peptide acceptors which were already present at the time of LL addition. Both permeable and intact fibroblasts were exposed to labeled UDP-xylose, UDP-galactose, and UDP-glucuronic acid, all donors of mucopolysaccharide precursors. The uptake of xylose into protein was the same for both normal and CF cells, but permeable CF fibroblasts incorporated statistically greater amounts of sugar from UDP-galactose and UDP-glucuronic acid. Intact CF cells were also labeled using these two sugar nucleotides. Trypan blue exclusion indicated CF and normal fibroblasts were equally intact. This and the fact that preincubation of CF cells with the appropriate cold sugar nucleotide eliminated the differences in incorporation between the normal and CF cells suggested that CF fibroblasts had more cell surface acceptor than the normal cells.

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.

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.

Uptake and Metabolic Fate of Glucose, Arabinose, and Xylose by Zea mays Coleoptiles in Relation to Cell Wall Synthesis

According to the acid-growth hypothesis, auxin-induced secretion of hydrogen ions activate "wall loosening" enzymes that change the rheological properties of the cell wall. The wall loosening process may yield monosaccharides by the enzymic cleavage of load-bearing polysaccharides. Our study was initiated to determine the metabolic fate of such sugars when released from the major hemicellulosic polysaccharides of the cell walls of Zea mays coleoptiles.Excised coleoptile sections accumulated radioactive glucose, arabinose, and xylose supplied in an incubation medium, and the radioactivity from these sugars was incorporated into polysaccharides. At least 50% of the radioactivity from glucose accumulated in the soluble neutral sugar fraction regardless of external concentrations. The distribution of radioactivity from xylose into all subcellular fractions was similar to that from glucose, indicating that xylose was converted to glucose before being used by the coleoptile. IAA increased the incorporation of glucose into cell wall polysaccharide and neutral sugar pools when the exogenous concentration was higher than 1 millimolar.Over 80% of the radioactivity from arabinose accumulated by the coleoptile sections was incorporated into soluble and noncellulosic polymers; IAA induced an increase in the incorporation of arabinose into noncellulosic polymers by 22%. Accumulation of radioactivity from arabinose into polysaccharide was enhanced by IAA at concentrations of exogenous arabinose up to 33 millimolar.IAA promoted the incorporation of both arabinose and glucose into cell wall polysaccharides even when elongation was inhibited by CaCl(2), indicating that the influence of IAA was not a consequence of the growth response.

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.

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.

Stimulatory and inhibitory effects of EDTA on sugar transport by rat soleus muscle

The uptake of D-[ 14C]xylose by rat soleus muscle was stimulated rapidly and transiently by brief exposure to EDTA (0.1–20 mM). EDTA also stimulated xylose uptake in the presence of insulin (0.1 U/ml). Prolonged exposure to EDTA (60 min) inhibited insulin-stimulated xylose uptake and depressed 125I-insulin binding; these effects were associated with the lowering of muscle ATP. The stimulatory effect was abolished by the substitution of Ca-EDTA (or Mg-EDTA) for EDTA; Ca-EDTA did not eliminate the inhibitory effect. There was no inhibitory effect when Ca 2+ (5 mM) was added along with Ca-EDTA, or when Zn-EDTA was used instead. There was no effect of EGTA (5 mM) on xylose uptake measured in the presence or absence of insulin. It is concluded (1) that the stimulatory effect of EDTA is most likely due to the chelation of Mg 2+, (2) that the inhibitory effects of EDTA are due to the chelation of some metal ion whith a higher affinity for the chelator than either Ca 2+ or Mg 2+.

D-xylose transport in isolated skeletal muscle of chickens: Effects of insulin and tolbutamide

1. Cellular uptake of sugar by an incubated avian skeletal muscle (M. extensor metacarpi radialis) was estimated by measuring the uptake of D-[U- 14C]xylose. 2. Xylose uptake (1–14 μmol/g/hr) and the time courses for penetration of the muscles by xylose and by the extracellular marker D_[1- 3H]-sorbital, were similar to those reported for mammalian muscles 3. Bovine insulin produced modest increases (usually <50%) in xylose uptake and was effective at a concentration in the medium (500 μU/ml)-lower than in most earlier in vitro studies of avian muscles. 4. Tolbutamide (0.5–2 mg/ml) caused similar increases in xylose uptake, both in the presence and absence of exogenous insulin. 5. The results provide further evidence for a direct extrapancreatic action of tolbutamide on avian muscle.

Effect of protein synthesis inhibitors on xylose uptake and 125I-insulin binding by rat soleus muscle

Prolonged exposure (90–180 min) to cycloheximide (0.2 mg/ml), puromycin (0.2 mg/ml) or chloramphenicol (0.1 mg/ml) did not affect 125I-insulin binding by rat soleus muscle. Chloramphenicol (2 mg/ml) depressed insulin binding and insulin-stimulated xylose uptake; these effects were attributed to the “toxic” effect of chloramphenicol on muscle ATP levels. Cycloheximide and puromycin inhibited insulin-stimulated xylose uptake without affecting ATP. Puromycin and chloramphenicol, but not cycloheximide, also inhibited basal sugar transport. This difference, and the rapid onset of all these inhibitory effects, suggest that they are not due to the inhibition of protein synthesis, but rather to some more direct effect on sugar transport itself.

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.

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.

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

1. 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°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. 2. Xylose uptake, measured in freshly isolated soleus muscles, was approximately 3.5–4.0 μmol/g per h. When the muscles were preincubated at 37°C, this rate fell by 50% during the first 30 min and then slowly increased. 3. 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 μmol/g per h) after 20 min. 4. 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 to 27°C. The stimulatory effect of anoxia was promptly reversed when muscles were transferred from anaerobic to aerobic conditions. 5. 5. There was a 5-min lag period before xylose uptake was stimulated by 2,4-dinitrophenol (0.05 mM) or by sodium salicylate (5 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. 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°C, or when xylose uptake was restored to basal values by transferring muscles from anaerobic to aerobic conditions. 7. 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. 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.

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°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 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°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.

Effect of hydrocortisone and dexamethasone on xylose uptake by isolated rat soleus muscle

To determine the effects of glucocorticoids on sugar uptake, xylose uptake by isolated rat soleus muscle of bilaterally adrenalectomized animals was studied. The results indicate that in vitro addition of 10 −4 M hydrocortusine, dexamethasone or hydrocortisone sodium succinate had no inhibitory effect on basal xylose uptake. In the presence of both low and high medium insulin, the above steroids failed to inhibit insulin-stimulated uptake. When the concentration of hydrocortisone sodium succinate was increased to 10 −2 M, insulin-stimulated uptake was decreased. The results thus indicate that glucocorticoids at concentrations observed under physiological or pathological conditions do not inihibit basal or insulin-stimulated sugar uptake.

Studies on membrane transport—VIII. Absorption of monosaccharides by Fasciola hepatica

1. 1. Ligated and unligated Fasciola absorb the non-metabolized sugar 3-O-methylglucose at equal rates. Uptake is a non-linear function of concentration but the sugar is not accumulated. 2. 2. Glucose absorption is a non-linear function of concentration and is inhibited by 3-0-methylglucose, mannose, glucosamine and galactose. Phlorizin was without effect. 3. 3. Fructose, mannose, glucosamine, and ribose absorption were non-linear functions while xylose uptake was a linear function of concentration. 4. 4. Interactions of the above sugars were examined. Sugar absorption was not affected by the absence of sodium nor by the presence of ouabain. 5. 5. Sugar transport in Fasciola is discussed in terms of numbers of mechanisms and compared with other parasitic animals.

Gastric Biopsies in Metabolic Investigations: In vitro studies on potassium loss and glycolysis in human gastric mucosal biopsies

By gastroscopy human gastric mucosal biopsies are obtained and incubated in a nutrient medium at room temperature. The potassium loss from the cells, the uptake of glucose and xylose, and the production of lactate are examined. The studies indicate that human gastric mucosal biopsies display certain characteristic metabolic activities, and may thus be considered acceptable tissues for further metabolic investigations.