Effect of 25-hydroxyvitamin D3 on Na+-dependent D-glucose transport in rachitic rats

We have previously reported that feeding rats on Steenbock and Black's rickets-inducing diet, deficient in vitamin D and with an altered Ca/P ratio, leads to metabolic consequences and a marked decrease of Na+-dependent D-glucose uptake at the jejunum-ileum level. To clarify the relationship between experimental rickets and D-glucose uptake, 25-hydroxyvitamin D3 (25-OH-D3) was given to rats fed on the rickets inducing diet. In the jejunum-ileum of these animals Na+-dependent D-glucose uptake returned to the values of the controls while the decrease in D-glucose uptake in the brush-border membrane vesicles prepared from kidney cortex of rachitic animals was not corrected by the administration of 25-OH-D3.     

Effect of vitamin D deficiency on D-glucose and citrate transport in rat renal basolateral membrane vesicles.

We had previously reported that feeding rats on Steenbock and Black's rickets-inducing diet markedly influences the metabolic picture of the kidney and the transmembrane transport systems of D-glucose and citrate in renal brush-border membrane vesicles. We have now studied D-glucose and citrate transport into basolateral membrane vesicles prepared from kidney cortex of control and rachitic rats and the effect of 1,25-dihydroxyvitamin D3 on these transport systems was also investigated. D-glucose and citrate uptake, determined in the presence of a Na(+)-gradient, was lowered in rachitic animals and 1,25-dihydroxyvitamin D3 administration proved to be ineffective in restoring normal values. Citrate transport, determined in the presence of a K(+)-gradient, was not influenced by both rickets and 1,25-dihydroxyvitamin D3 supply. The in vitro addition to vesicle preparations of calcium or phosphate or citrate or 1,25-dihydroxyvitamin D3 did not show a selective influence on D-glucose and citrate uptake.     

Hexose metabolism in pancreatic islet cells: the coupling between hexose phosphorylation and mitochondrial respiration

The possible relevance of D-glucose phosphorylation by mitochondria-bound hexokinase to the control of respiration was examined in mitochondria prepared from either tumoral pancreatic islet cells (RINm5F line) or normal rat liver. In both systems, ATP generated by mitochondria exposed to ADP and succinate could serve as a substrate for the phosphorylation of D-glucose. However, after exposure to exogenous ADP in the presence of succinate, only mitochondria isolated from RINm5F cells displayed a sizeable increase in O2 consumption in response to a subsequent administration of D-glucose. In this respect, the discrepancy between mitochondria from islet cells and liver, respectively, was found to be attributable to the much lower hexokinase activity, relative to respiratory rate, in liver than in RINm5F cell mitochondria. It is speculated that the coupling between hexose phosphorylation and respiration in islet cells may prime the mitochondria to generate ATP during the early metabolic and secretory response to a rise in extracellular D-glucose concentration.     

Glucose intolerance associated to insulin resistance and increased insulin secretion in rats depleted in long-chain omega3 fatty acids.

An intragastric D-glucose tolerance test was performed, after overnight starvation, in female rats depleted in long-chain polyunsaturated omega3 fatty acids (omega3D rats) and control rats of same age and gender. The plasma D-glucose and insulin concentrations, insulinogenic index, and HOMA for insulin resistance were all higher, after overnight starvation, in omega3D rats than in control animals. Over the 120-minute period following the intragastric administration of D-glucose, the area under the curve for the same four variables was also higher in omega3D rats than in control animals. In addition to visceral obesity, liver steatosis, hypertension, and cardiac hypertrophy, the omega3D rats thus display further features of the metabolic syndrome, namely glucose intolerance and insulin resistance, despite hyperinsulinemia.     

32P, 86Rb+ and 45Ca2+ handling by tumoral insulin-secreting cells (RINm5F line)

In perifused tumoral islet cells (RINm5F line), which were prelabelled with either [32P]orthophosphate, 86Rb+ or 45Ca2+, the administration of D-glucose (1.4, 2.8 or 16.7 mM) increased the efflux of 32P, decreased the outflow of 86Rb, increased slightly the efflux of 45Ca from cells perifused in the presence of Ca2+, and decreased modestly the outflow of 45Ca from cells perifused in the absence of Ca2+. D-glucose also stimulated the net uptake of 45Ca2+. When Ba2+ (2 mM) was used, in the absence of Ca2+, instead of D-glucose as an insulin secretagogue, the efflux of 32P was little affected, but the outflow of 45Ca was dramatically increased. These changes are qualitatively similar to those occurring in normal islet cells. Nevertheless, the ionic response to D-glucose appeared, as a rule, less marked in tumoral than normal islet cells. Moreover, the concentration-response relationship was shifted to a lower range of hexose concentrations in the RINm5F cells.     

Glucose-dependent regulation of glucose transport activity, protein, and mRNA in primary cultures of rat brain glial cells

D-Glucose deprivation of primary rat brain glial cell cultures, by incubation with 25 mM D-fructose for 24 h, resulted in a 4-5-fold induction of D-glucose transport activity. In contrast, 24-h D-glucose starvation of primary rat brain neuronal cultures had only a marginal effect (1.5-2-fold) on D-glucose transport activity. Northern blot analysis of total cellular RNA demonstrated that under these conditions the rat brain glial cells specifically increased the steady-state level of the D-glucose transporter mRNA 4-6-fold, whereas Northern blot analysis of the neuronal cell cultures revealed no significant alteration in the amount of D-glucose transporter mRNA by D-glucose deprivation. These findings demonstrated that the D-glucose-dependent regulation of the D-glucose transporter system occurred in a brain cell type-specific manner. The ED50 for the D-glucose starvation increase in the D-glucose transporter mRNA, in the glial cell cultures, occurred at approximately 3.5 mM D-glucose with maximal effect at 0.5 mM D-glucose. Readdition of D-glucose to the starved cell cultures reversed the increase in the D-glucose transporter mRNA levels and D-glucose transport activity to control values within 24 h. The increase in the D-glucose transporter mRNA was relatively rapid with half-maximal stimulation at approximately 2 h and maximal induction by 6-12 h of D-glucose deprivation. A similar time course was also observed for the starvation-induced increase in D-glucose transport activity and D-glucose transporter protein, as determined by Western blot analysis. These results document that, in rat brain glial cells, D-glucose transport activity, protein, and mRNA are regulated by the extracellular D-glucose concentration. Further, this suggests a potential role for hyperglycemia in the down-regulation of the D-glucose transport system in vivo.     

Study on the absorption and utilization of D-galactose and D-glucose by liver and muscular tissue in the albino rat

In order to compare the disposition of D-galactose and D-glucose in various organs of the rat, we have measured the amount of 14C-galactose and 14C-glucose present in the enteric canal, blood, muscle and liver, 2h. after the oral administration of the labelled esoses. Our results show that the two esoses are ebsorbed almost completely and to the same extent by two hours after their administration, Galactose has a longer half-life than glucose, being more slowly utilized in the synthesis of glycogen in liver and muscle.     

The acute effects of ethanol on protein synthesis in isolated rat liver cells are pH-dependent

Acute administration of ethanol to isolated rat liver cells induced a pH-dependent inhibition of protein synthesis. The effect of the alcohol was highest at pH 7.0 and nil at pH 7.8. 4-methyl-pyrazole partially reversed the action of ethanol only below pH 7.4. Time-course experiments suggested that ethanol could act preventing the initiation of new polypeptide chains stimulated by D-glucose, and that this effect is abolished at pH 7.8.     

Environmental modulation of the inhibitory action of D-mannoheptulose upon D-glucose metabolism in isolated rat pancreatic islets

In pancreatic islets prepared from fed rats and incubated at a low concentration (1.7 mM) of D-glucose, D-mannoheptulose (10.0 mM) virtually fails to affect the metabolism of the hexose. Likewise, in islets from starved rats, the relative extent of the inhibitory action of D-mannoheptulose upon D-glucose metabolism is much more marked at high (16.7 mM) than low (1.7 mM) hexose concentration. Nevertheless, despite decreasing the metabolism of D-glucose, starvation augments the sensitivity to D-mannoheptulose in the islets incubated at a low concentration of the hexose, D-galactose, but not D-fructose, also augments the inhibitory action of D-mannoheptulose upon D-glucose metabolism in islets prepared from fed rats and exposed to the low concentration of D-glucose. A comparable situation prevails in islets exposed to 2-ketoisocaproate. Forskolin, however, which decreases D-glucose catabolism in the islets from fed rats exposed to 1.7 mM D-glucose, fails to affect significantly the inhibitory action of D-mannoheptulose on D-glucose metabolism. It is proposed that hexoses transported by the same carrier as D-glucose and non-glucidic nutrient secretagogues somehow increase D-mannoheptulose uptake by the islet cells. The latter two conditions may be operative in islets exposed to a high concentration of D-glucose, this accounting for the exquisite sensitivity to D-mannoheptulose of glucose-stimulated islets.     

Lactation-induced changes in calcium handling by rat pancreatic islets

Glucose-stimulated insulin release occurred at a lower rate in pancreatic islets removed from lactating than non-lactating rats. This defect was corrected in the presence of either gliclazide or a calcium-agonist. With both agents present, insulin release from islets of lactating rats was greater. When islets were prelabelled with 45calcium, gliclazide stimulated to the same extent 45Ca outflow in islets from lactating and non-lactating rats, respectively. However, when the islets were prelabelled with 45Ca in the presence of gliclazide, the administration of Ba2+ increased effluent radioactivity more markedly in islets from non-lactating than lactating rats. This suggests that lactation favours, in gliclazide-stimulated islets, the sequestration of 45Ca in non-labile subcellular pools. When D-glucose was used instead of Ba2+, the greater lability of 45Ca in islets from non-lactating animals was apparently masked by a lesser efficiency in the metabolism and cationic effects of D-glucose in the non-lactating rats. The calcium-ionophoretic effect of islet extracts was higher in lactating than non-lactating rats. These results support the view that a depletion of endogenous calcium stores accounts, in part at least, for the decreased insulin secretory responsiveness to D-glucose in lactation, since the latter apparently favours the function of those systems involved in either the entry of calcium into or its sequestration within the islet cells.     

Evaluation of the bioconversion of genetically modified switchgrass using simultaneous saccharification and fermentation and a consolidated bioprocessing approach

Background

In mixed sugar fermentations with recombinant Saccharomyces cerevisiae strains able to ferment D-xylose and L-arabinose the pentose sugars are normally only utilized after depletion of D-glucose. This has been attributed to competitive inhibition of pentose uptake by D-glucose as pentose sugars are taken up into yeast cells by individual members of the yeast hexose transporter family. We wanted to investigate whether D-glucose inhibits pentose utilization only by blocking its uptake or also by interfering with its further metabolism.

Results

To distinguish between inhibitory effects of D-glucose on pentose uptake and pentose catabolism, maltose was used as an alternative carbon source in maltose-pentose co-consumption experiments. Maltose is taken up by a specific maltose transport system and hydrolyzed only intracellularly into two D-glucose molecules. Pentose consumption decreased by about 20 - 30% during the simultaneous utilization of maltose indicating that hexose catabolism can impede pentose utilization. To test whether intracellular D-glucose might impair pentose utilization, hexo-/glucokinase deletion mutants were constructed. Those mutants are known to accumulate intracellular D-glucose when incubated with maltose. However, pentose utilization was not effected in the presence of maltose. Addition of increasing concentrations of D-glucose to the hexo-/glucokinase mutants finally completely blocked D-xylose as well as L-arabinose consumption, indicating a pronounced inhibitory effect of D-glucose on pentose uptake. Nevertheless, constitutive overexpression of pentose-transporting hexose transporters like Hxt7 and Gal2 could improve pentose consumption in the presence of D-glucose.

Conclusion

Our results confirm that D-glucose impairs the simultaneous utilization of pentoses mainly due to inhibition of pentose uptake. Whereas intracellular D-glucose does not seem to have an inhibitory effect on pentose utilization, further catabolism of D-glucose can also impede pentose utilization. Nevertheless, the results suggest that co-fermentation of pentoses in the presence of D-glucose can significantly be improved by the overexpression of pentose transporters, especially if they are not inhibited by D-glucose.     

Transport of D-glucose and 3-O-methyl-D-glucose in the cyanobacteria Aphanocapsa 6714 and Nostoc strain Mac.

1. The cyanobacterium Aphanocapsa 6714 which grow in the dark on D-glucose, will take up D-glucose and the analogue 3-O-methyl-D-glucose; uptake of each of these compounds was inhibited competitively by the other and by 6-deoxy-D-glucose. 2. This cyanobacterium accumulated 3-O-methyl-D-glucose up to 100-fold relative to the medium but did not modify or metabolize it to a significant degree. 3. Intracellular 3-O-methyl-D-glucose was rapidly displaced from Aphanocapsa 6714 by exogenous D-glucose and 3-O-methyl-D-glucose. 4. Although not characterized to the same extent, D-glucose and 3-O-methyl-D-glucose uptake by Nostoc strain Mac, another cyanobacterium capable of growth in the dark on D-glucose, was similar. 5. Other cyanobacteria that do not grow on D-glucose take up this compound at much lower rates which were unaffected by analogues of D-glucose that greatly reduced carbohydrate uptake by Aphanocapsa 6714 and Nostoc strain Mac. 6. It is therefore proposed that Aphanocapsa 6714 and Nostoc strain Mac possess a mechanism for the active transport of D-glucose. The absence of this transport mechanism is suggested as the reason why other strains fail to grow in the dark on this substrate. These latter organisms are therefore naturally cryptic with respect to D-glucose as a growth substrate.     

Glucose transfer across the intact guinea-pig placenta

Experiments were carried out in anaesthetized pregnant guinea-pigs. Following the maternal injection of a bolus containing 14C-hexose and 3H2O, blood was sampled from the fetal umbilical vein during a single circulatory transit. A placental transfer index was calculated from the ratio of the tracers in the fetal whole blood divided by that in maternal plasma. The transfer index for D-glucose, 0.66 +/- 0.03 (SEM), greatly exceeded that for L-glucose, 0.013 +/- 0.004. Elevation of the maternal plasma D-glucose concentration, with unlabelled D-glucose, resulted in saturation of D-glucose transfer with an apparent Km of 1.2 x 10(-2) mol/l mean maternal plasma D-glucose. Phlorizin at maternal plasma concentrations of approximately 10(-3) mol/l inhibited D-glucose transfer by 40%. Phloretin did not affect D-glucose transfer at levels estimated to be 10(-4) mol/l. Specificity studies with substituted D-glucose analogues showed that alpha-methyl-D-glucoside is not transported by a facilitated pathway; 2-deoxy-D-glucose and 3-O-methyl-D-glucose share the D-glucose carrier and D-galactose has a partial affinity for the D-glucose carrier.     

Inhibition of intestinal sugar transport by phenformin.

The effect of phenformin on the absorption of D-glucose and D-galactose by hamster and rat intestine, was studied. Phenformin did not affect D-glucose absorption by rat intestine, but it inhibited at 10(-3) to 10(-2) M the absorption of D-glucose and D-galactose by hamster intestine. The inhibition was higher when D-glucose was tested. Phenformin also inhibited active accumulation of these sugars by rings of hamster small intestine, in vitro; this effect was greater when D-glucose was utilized. The drug inhibits the oxygen uptake in the tissue in the absence or in the presence of added substrate. Phenformin, as previously suggested, does not seem to act as a specific inhibitor on D-glucose transport, but most likely by its inhibitory effect on mitochondrial respiration.     

Inhibition of glucose-induced insulin release by 3-O-methyl-D-glucose: Enzymatic,metabolic and cationic determinants

The analog of D-glucose, 3-O-methyl-D-glucose, is thought to delay the equilibration of D-glucose concentration across the plasma membrane of pancreatic islet B-cells, but not to exert any marked inhibitory action upon the late phase of glucose-stimulated insulin release. In this study, however, 3-O-methyl-D-glucose, when tested in high concentrations (30-80 mM) was found to cause a rapid, sustained and not rapidly reversible inhibition of glucose-induced insulin release in rat pancreatic islets. In relative terms, the inhibitory action of 3-O-methyl-D-glucose was more marked at low than high concentrations of D-glucose. It could not be attributed to hyperosmolarity and appeared specific for the insulinotropic action of D-glucose, as distinct from non-glucidic nutrient secretagogues. Although 3-O-methyl-D-glucose and D-glucose failed to exert any reciprocal effect upon the steady-state value for the net uptake of these monosaccharides by the islets, the glucose analog inhibited D-[5-3H]glucose utilization and D-[U-14C]glucose oxidation. This coincided with increased 86Rb outflow and decreased 45Ca outflow from prelabelled islets, as well as decreased 45Ca net uptake. A preferential effect of 3-O-methyl-D-glucose upon the first phase of glucose-stimulated insulin release was judged compatible with an altered initial rate of D-glucose entry into islet B-cells. The long-term inhibitory action of the glucose analog upon the metabolic and secretory response to D-glucose, however, may be due, in part at least, to an impaired rate of D-glucose phosphorylation. The phosphorylation of the hexose by beef heart hexokinase and human B-cell glucokinase, as well as by parotid and islet homogenates, was indeed inhibited by 3-O-methyl-D-glucose. The relationship between insulin release and D-glucose utilization or oxidation in the presence of 3-O-methyl-D-glucose was not different from that otherwise observed at increasing concentrations of either D-glucose or D-mannoheptulose. It is concluded, therefore, that 3-O-methyl-D-glucose adversely affects the metabolism and insulinotropic action of D-glucose by a mechanism largely unrelated to changes in the intracellular concentration of the latter hexose.     

Influence of S9 mix on the expression of mutants in the L-arabinose resistance test of Salmonella typhimurium

S9 mix produces an effect similar to that of D-glucose in the L-arabinose resistance test of Salmonella typhimurium, releasing the growth inhibition exerted by L-arabinose. Two elements are responsible for this effect: the glucose-6-phosphate present in the cofactors of the S9 mix and the S9 fraction itself. UV light was used as a mutagen to compare the efficiency of S9 mix and D-glucose in allowing phenotypic expression of mutants in selective plates with L-arabinose; 0.5 ml of S9 mix per plate showed and efficacy similar to that of 0.5 mg of D-glucose per plate. To verify that the S9 mix is equivalent to D-glucose traces in selective plates with respect to the number of induced mutants in compounds requiring metabolic activation, we utilized 2 direct-acting nitrofurans. Our conclusion is that activation of agents could be erroneously attributed to the S9 mix, when plates with 0.5 mg of D-glucose are compared to plates with 0.5 ml of S9 mix plus 0.5 mg of D-glucose. Our results suggest that D-glucose traces be omitted in experiments requiring the presence of the S9 mixture.     

Distribution of tritium in D-glucose and starch labeled by tritium-atom bombardment

Samples of D-glucose and starch were labeled by tritium-atom bombardment. Up to 51% incorporation into D-glucose as non-labile tritium was achieved for crystalline, anhydrous D-glucose and 41% for the amylose-butyl alcohol complex. Distribution of tritium in the carbon skeleton of D-glucose was calculated by comparing the specific molar activity of D-glucose with that of its derivatives. Derivatives prepared were D-gluconic acid, D-arabino-hexulose phenylosotriazole, 4-formyl-2-phenyltriazole, 2-phenyltriazole-4-carboxylic acid, D-arabino-hexulose phenylflavazole, 3-formyl-1-phenylflavazole, and formaldehyde dimedone. The tritium distribution showed definite structural effects. Generally, the products from films of D-glucose and the amylose-butyl alcohol complex had nearly uniform distribution of tritium in D-glucose. The product from crystalline α-D-glucose monohydrate had zero tritium at C-2 and twice the expected amount of tritium at C-5, and that from starch granules had zero or near zero tritium at C-3 and close to twice the expected amount of tritium at C-2.     

Vanadate stimulates D-glucose transport into sarcolemmal vesicles from rat skeletal muscles.

Vanadate is known to have various insulin-like actions including activation of D-glucose uptake into the skeletal muscle and adipose tissue. In this study, we examined the effect of orthovanadate on D-glucose uptake into sarcolemmal vesicles prepared from rat hind limb skeletal muscles. In the presence of 10 mM vanadate, the initial rate of D-glucose uptake into sarcolemmal vesicles was enhanced 4-5 times above the basal value. Half-maximal concentration for this effect of vanadate was 3 mM. The D-glucose uptake was also stimulated by metavanadate, but not by selenite, selenate, or molybdate. When vanadate was removed from the vesicles by dilution and centrifugation, D-glucose uptake into the vesicles returned to the basal level, indicating that the effect of vanadate was reversible. Saturation curves showed that the Vmax value for the D-glucose uptake was enhanced more than 4-fold by 10 mM vanadate. Therefore, the activation of D-glucose uptake was due, at least in part, to a large increase in the Vmax value. These results suggest that vanadate increases the intrinsic activity (turnover number) of skeletal muscle glucose transporters in a reversible manner.     

Aboral changes in D-glucose transport by human intestinal brush-border membrane vesicles.

D-Glucose transport was investigated in isolated brush-border membrane vesicles from human small intestine. Characteristics of D-glucose transport from the jejunum were compared with that in the mid and terminal ileum. Jejunal and mid-ileal D-glucose transport was Na+-dependent and electrogenic. The transient overshoot of jejunal D-glucose transport was significantly greater than corresponding values in mid-ileum. The terminal ileum did not exhibit Na+-dependent D-glucose transport, but did exhibit Na+-dependent taurocholate transport. Na+-glucose co-transport activity as measured by tracer-exchange experiments was greatest in the jejunum, and diminished aborally. We conclude that D-glucose transport in man is Na+-dependent and electrogenic in the proximal intestine and directly related to the activity of D-glucose-Na+ transporters present in the brush-border membranes. D-Glucose transport in the terminal ileum resembles colonic transport of D-glucose.     

The Na+ gradient-dependent transport of D-glucose in renal brush border membranes.

The Na+-dependent transport of D-glucose was studied in brush border membrane vesicles isolated from the rabbit renal cortex. The presence of a Na+ gradient between the external incubation medium and the intravesicular medium induced a marked stimulation of D-glucose uptake. Accumulation of the sugar in the vesicles reached a maximum and then decreased, indicating efflux. The final level of uptake of the sugar in the presence of the Na+ gradient was identical with that attained in the absence of the gradient, suggesting that equilibrium was established. At the peak of the overshoot the uptake of D-glucose was more than 10-fold the equilibrium value. These results suggest that the imposition of a large extravesicular to intravesicular gradient of Na+ effects the transient movement of D-glucose into renal brush border membranes against its concentration gradient. The stimulation of D-glucose uptake into the membranes was specific for Na+. The rate of uptake was enhanced with increased concentration of Na+. Increasing Na+ in the external medium lowered the apparent Km for D-glucose. The Na+ gradient effect on D-glucose transport was dissected into a stimulatory effect when Na+ and sugar were on the same side of the membrane (cis stimulation) and an inhibitory effect when Na+ and sugar were on opposite sides of the membrane (trans inhibition). The uptake of D-glucose, at a given concentration of sugar, reflected the sum of the contributions from a Na+-dependent transport system and a Na+-independent system. The relative stimulation of D-glucose uptake by Na+ decreased as the sugar concentration increased. It is suggested, however, that at physiological concentrations of D-glucose the asymmetry of Na+ across the brush border membrane might fully account for uphill D-glucose transport. The physiological significance of the findings is enhanced additionally by observations that the Na+-dependent D-glucose transport system in the membranes in vitro possessed the sugar specificities and higg phlorizin sensitivity characteristic of more intact preparations. These results provide strong experimental evidence for the role of Na+ in transporting D-glucose across the renal proximal tubule luminal membrane.