Alternate models for shared carriers or a single maturing carrier in hexose uptake into rabbit jejunum in vitro

The uptake (tissue accumulation) of three hexoses into rabbit jejunum was measured in a flux chamber in conditions of effective stirring. Glucose uptake was inhibited by galactose or 3-O-methylglucose: 1-40 mM galactose caused a progressive decline in glucose uptake; 1-5 mM 3-O-methylglucose inhibited glucose uptake but higher concentrations of 3-O-methylglucose had no further effect. When 1-40 mM 3-O-methylglucose was added to glucose plus galactose there was a further decrease in the uptake of glucose; adding 1-40 mM galactose to glucose plus 3-O-methylglucose also produced a decrease in glucose uptake. Both glucose and 3-O-methylglucose inhibited uptake of galactose but the pattern of inhibition varied between the two sugars. The uptake of 3-O-methylglucose was also inhibited by glucose and by galactose, but the uptake of 3-O-methylglucose in the presence of either galactose or glucose was no further reduced by adding the third hexose. Graphical analysis and analysis by non-linear regression both showed that neither the single Michaelis-Menten function, nor the single Michaelis-Menten-plus-competitive-inhibition function was appropriate for any of these data. The results are consistent with the hypothesis that either there are multiple (at least three) intestinal carriers for hexoses; alternatively that there is a single carrier whose transport properties for the three hexoses change differentially during cell maturation and migration up the villus.     

Ileal resection and dietary content of sucrose influence the intestinal uptake of monosaccharides

The intestinal uptake of 0.5 and 40 mM glucose, galactose, and 3-O-methyl glucose (3-O-MG) was examined in vitro in rabbits fed a high (HS) or a low (LS) sucrose diet. In animals with an intact intestinal tract, the jejunal uptake of 0.5 mM 3-O-MG was unaffected by the dietary content of sucrose, whereas the uptake of 40 mM 3-O-MG was lower in LS than HS. The uptake of 40 mM galactose was higher in LS than HS and the uptake of 0.5 mM galactose was similar in HS and LS, whereas the uptake of 0.5 mM but not 40 mM glucose was lower in LS than HS. In animals subjected 6 weeks previously to an ileal resection, the adaptive changes in the jejunal uptake of the hexoses in response to alterations in the dietary content of sucrose differed from the changes observed in rabbits with an intact intestinal tract. For example, feeding HS to ileal resected animals was associated with increased jejunal uptake of 40 mM galactose, decreased uptake of 40 mM glucose, and unchanged uptake of 40 mM 3-O-MG; whereas in control animals with an intact intestinal tract, feeding HS resulted in increased uptake of 40 mM 3-O-MG, decreased uptake of 40 mM galactose, and no change in the uptake of 40 mM glucose. A similar adaptive pattern was noted in the jejunum and ileum for the effect of dietary sucrose on the uptake of 0.5 and 40 mM glucose.(ABSTRACT TRUNCATED AT 250 WORDS)     

Galactose transport in Kluyveromyces lactis: major role of the glucose permease Hgt1

In Kluyveromyces lactis, galactose transport has been thought to be mediated by the lactose permease encoded by LAC12. In fact, a lac12 mutant unable to grow on lactose did not grow on galactose either and showed low and uninducible galactose uptake activity. The existence of other galactose transport systems, at low and at high affinity, had, however, been hypothesized on the basis of galactose uptake kinetics studies. Here we confirmed the existence of a second galactose transporter and we isolated its structural gene. It turned out to be HGT1, previously identified as encoding the high-affinity glucose carrier. Analysis of galactose transporter mutants, hgt1 and lac12, and the double mutant hgt1lac12, suggested that Hgt1 was the high-affinity and Lac12 was the low-affinity galactose transporter. HGT1 expression was strongly induced by galactose and insensitive to glucose repression. This could explain the rapid adaptation to galactose observed in K. lactis after a shift from glucose to galactose medium.     

Glucose effect and the galactose enzymes of Escherichia coli: correlation between glucose inhibition of induction and inducer transport

Adhya, Sankar (University of Wisconsin, Madison), and Harrison Echols. Glucose effect and the galactose enzymes of Escherichia coli: correlation between glucose inhibition of induction and inducer transport. J. Bacteriol. 92:601-608. 1966.-The inhibitory effect of glucose on the induction of the enzymes required for galactose utilization ("glucose effect") was studied in Escherichia coli. Experiments on the uptake into the cell of labeled inducers (d-galactose-C(14) and d-fucose-H(3)) pointed to inhibition at the level of inducer transport as the possible primary mechanism of the glucose effect in the case of the gal enzymes. This interpretation was supported by the finding that a mutant constitutive for the lac enzymes was resistant to glucose inhibition of galactose induction of the gal enzymes; the mutant had acquired a glucose-resistant alternative transport mechanism for galactose via the constitutively synthesized galactoside permease. Further support for the transport inhibition model was provided by the finding that glucose did not substantially inhibit induction of the gal enzymes when glucose and galactose were produced intracellularly by beta-galactosidase hydrolysis of lactose, even if excess glucose was added. The inducer uptake experiments also showed that d-galactose and d-fucose probably enter the cell via different transport systems, although uptake of both compounds was inhibited by glucose.     

Regulation of beta-D-galactosidase synthesis in Candida pseudotropicalis.

Regulation of lactose (beta-D-galactosidase) synthesis in the lactose-utilizing yeast Candida pseudotropicalis was studied. The enzyme was inducible by lactose and galactose. When grown on these sugars the enzyme level of the yeast was 20 times or higher than when grown on glycerol. The Km and optimal pH were similar for the lactase induced either by lactose or galactose. The hydrolysis of o-nitrophenyl-beta-D-galactopyranoside by the lactase was inhibited by galactose and several analogs and galactosides, but not by glucose. Lactose uptake activity observed in lactose-grown cells was very reduced in cells grown on glucose or galactose. Glucose repressed the induction of lactase, but not the metabolic system for galactose utilization. In continuous culture on lactose medium at dilution rates below 0.2 h-1 the specific lactase activity was higher than in batch cultures and decreased with increases in dilution rate. Lactase was induced by pulses of lactose and galactose in cells growing on glucose, but only at low dilution rates were the steady-state concentration of glucose was very low.     

The Effect of Nonanal on Dipodascus aggregatus IV. Studies with Different Carbon Sources

Nonanal (80 μ) in ethanolic solution stimulated the growth of Dipodascus aggregatus with fructose (55.5 mM) as carbon source (inoculum grown with fructose or glucose). If the inoculum had been grown with galactose, neither growth with galactose nor growth with glucose was affected by nonanal. If the inoculum had been grown with glucose, growth with galactose was weakly. stimulated. —Growth with galactose (galactose-grown inoculum) was strongly stimulated by nonanal if xylose at a low concentration (0.53 mM) was added. — The oxygen uptake of glucose grown cells with glucose as substrate was stimulated by 200 μM nonanal in the absence of ethanol. The respiratory activity of galactose-grown cells was also stimulated with galactose as well as with glucose as substrate. In the absence of exogenous substrate the oxygen uptake of glucose-grown cells was weakly stimulated by nonanal whereas that of galactose-grown cells was strongly stimulated.     

Coregulation of beta-galactoside uptake and hydrolysis by the hyperthermophilic bacterium Thermotoga neapolitana

Regulation of the beta-galactoside transport system in response to growth substrates in the extremely thermophilic anaerobic bacterium Thermotoga neapolitana was studied with the nonmetabolizable analog methyl-beta-D-thiogalactopyranoside (TMG) as the transport substrate. T. neapolitana cells grown on galactose or lactose accumulated TMG against a concentration gradient in an intracellular free sugar pool that was exchangeable with external galactose or lactose and showed induced levels of beta-galactosidase. Cells grown on glucose, maltose, or galactose plus glucose showed no capacity to accumulate TMG, though these cells carried out active transport of the nonmetabolizable glucose analog 2-deoxy-D-glucose. Glucose neither inhibited TMG uptake nor caused efflux of preaccumulated TMG; rather, glucose promoted TMG uptake by supplying metabolic energy. These data show that beta-D-galactosides are taken up by T. neapolitana via an active transport system that can be induced by galactose or lactose and repressed by glucose but which is not inhibited by glucose. Thus, the phenomenon of catabolite repression is present in T. neapolitana with respect to systems catalyzing both the transport and hydrolysis of beta-D-galactosides, but inducer exclusion and inducer expulsion, mechanisms that regulate permease activity, are not present. Regulation is manifest at the level of synthesis of the beta-galactoside transport system but not in the activity of the system.     

Remodeling of Oxidative Energy Metabolism by Galactose Improves Glucose Handling and Metabolic Switching in Human Skeletal Muscle Cells

Cultured human myotubes have a low mitochondrial oxidative potential. This study aims to remodel energy metabolism in myotubes by replacing glucose with galactose during growth and differentiation to ultimately examine the consequences for fatty acid and glucose metabolism. Exposure to galactose showed an increased [¹⁴C]oleic acid oxidation, whereas cellular uptake of oleic acid uptake was unchanged. On the other hand, both cellular uptake and oxidation of [¹⁴C]glucose increased in myotubes exposed to galactose. In the presence of the mitochondrial uncoupler carbonylcyanide p-trifluormethoxy-phenylhydrazone (FCCP) the reserve capacity for glucose oxidation was increased in cells grown with galactose. Staining and live imaging of the cells showed that myotubes exposed to galactose had a significant increase in mitochondrial and neutral lipid content. Suppressibility of fatty acid oxidation by acute addition of glucose was increased compared to cells grown in presence of glucose. In summary, we show that cells grown in galactose were more oxidative, had increased oxidative capacity and higher mitochondrial content, and showed an increased glucose handling. Interestingly, cells exposed to galactose showed an increased suppressibility of fatty acid metabolism. Thus, galactose improved glucose metabolism and metabolic switching of myotubes, representing a cell model that may be valuable for metabolic studies related to insulin resistance and disorders involving mitochondrial impairments.     

Simultaneous uptake of galactose and glucose by Azotobacter vinelandii.

Azotobacter vinelandii growing on galactosides induced two distinct permeases for glucose and galactose. The apparent Vmax and Km of the galactose permease were 16 nmol galactose/min per 10(10) cells and 0.5 mM, respectively. The apparent Vmax and Km of the glucose permease were 7.8 nmol glucose/min per 10(10) cells and 0.04 mM, respectively. Excess glucose had no effect on the galactose uptake. However, excess galactose inhibited glucose transport. The galactosides-induced glucose permease also exhibited different uptake kinetics from that induced by glucose.     

Evidence for critical-period programming of intestinal transport function: variations in the dietary ratio of polyunsaturated to saturated fatty acids alters ontogeny of the rat intestine

2-week isocaloric modifications in the dietary ratio of polyunsaturated/saturated fatty acids (P/S) alters intestinal transport in rats. This study was undertaken to test the hypotheses that (1) the fatty acid composition of a nutritionally adequate diet in early life has lasting consequences for active and passive intestinal transport processes; and (2) early life feeding experiences with diets of varying fatty acid composition influence the intestines' ability to adaptively up- or down-regulate intestinal transport in later life. Female Sprague-Dawley rats were weaned onto S or P and were maintained on these diets for 2, 10 or 12 weeks. An in vitro uptake technique was used in which the bulk phase was vigorously stirred to reduce the effective resistance of the intestinal unstirred water layer. P decreased and S increased the uptake of glucose, and this effect was progressive from 2 to 12 weeks. Switching from a P to an S diet decreased jejunal but increased ileal uptake of glucose, whereas switching from an S to a P diet was associated with a decline in both the jejunal and the ileal uptake of glucose. The ileal uptake of galactose increased as the animals grew on either P or S. Switching from P to S resulted in a decline in ileal uptake of galactose, whereas the opposite effect was observed when switching from S to P. The effect of feeding P or S on hexose uptake was influenced by the animals' dietary history: ileal glucose and galactose uptake was lower in animals fed P at an early age (PSP) than in animals fed P for the first time in later life (SSP). Jejunal glucose and galactose uptake was also lower in animals fed S at an early age (SPS) than in those fed S for the first time in later life (PPS). The alterations in the uptake of long-chain saturated and unsaturated fatty acids and cholesterol did not progress with longer periods of feeding, and in the jejunum, lipid uptake did not change when switching from P to S or S to P. Early feeding with P (PSP vs. SSP) was associated with lower jejunal uptake of 18:3 and lower ileal uptake of 12:0, whereas previous feeding with S (SPS vs. PPS) was associated with lower ileal uptake of cholesterol. The changes in uptake of hexoses and lipids was not explained by differences in the animals' food consumption, body or intestinal weight or mucosal surface area.(ABSTRACT TRUNCATED AT 400 WORDS)     

Comparative metabolic analysis of lactate for CHO cells in glucose and galactose

t-PA producing CHO cells have been shown to undergo a metabolic shift when the culture medium is supplemented with a mixture of glucose and galactose. This metabolic change is characterized by the reincorporation of lactate and its use as an additional carbon source. The aim of this work is to understand lactate metabolism. To do so, Chinese hamster ovary cells were grown in batch cultures in four different conditions consisting in different combinations of glucose and galactose. In experiments supplemented with glucose, only lactate production was observed. Cultures with glucose and galactose consumed glucose first and produced lactate at the same time, after glucose depletion galactose consumption began and lactate uptake was observed. Comparison of the metabolic state of cells with and without the shift by metabolic flux analysis show that the metabolic fluxes distribution changes mostly in the reactions involving pyruvate metabolism. When not enough pyruvate is being produced for cells to support their energy requirements, lactate dehydrogenase complex changes the direction of the reaction yielding pyruvate to feed the TCA cycle. The slow change from high fluxes during glucose consumption to low fluxes in galactose consumption generates intracellular conditions that allow the influx of lactate. Lactate consumption is possible in cell cultures supplemented with glucose and galactose due to the low rates at which galactose is consumed. Evidence suggests that an excessive production and accumulation of pyruvate during glucose consumption leads to lactate production and accumulation inside the cell. Other internal conditions such as a decrease in internal pH, forces the flow of lactate outside the cell. After metabolic shift the intracellular pool of pyruvate, lactate and H⁺ drops permitting the reversal of the monocarboxylate transporter direction, therefore leading to lactate uptake. Metabolic analysis comparing glucose and galactose consumption indicates that after metabolic shift not enough pyruvate is produced to supply energy metabolism and lactate is used for pyruvate synthesis. In addition, MFA indicates that most carbon consumed during low carbon flux is directed towards maintaining energy metabolism.     

UPTAKE OF ORGANIC,COMPOUNDS BY TWO FACULTATIVELY HETEROTROPHIC MARINE CENTRIC DIATOMS1,2

Glucose and galactose uptake ability in Cyclotella cryptica (clone WT-1-8) and glucose uptake ability in Coscinodiscus sp. develop rapidly in the dark. Induction of sugar uptake ability in the dark does not require the presence of sugar in the medium. The sugars are taken up by carrier-mediated systems. In C. cryptica glucose and galactose are probably taken up by the same system. The capacity of glucose uptake in this recently isolated clone of C. cryptica is nearly 5 times that of a previously studied clone (0-3A). Other organic compounds, which by themselves do not support heterotrophic growth, can be taken up and respired by both diatoms at considerable rates compared, to glucose and galactose. Therefore, in nature, these diatoms may be able to utilize a variety of dissolved organic compounds as sources of intermediary metabolites and as respiratory substrates.     

Regulation of sugar transport in Neurospora crassa

Sugar uptake systems in Neurospora crassa are catabolically repressed by glucose. Synthesis of a low K(m) glucose uptake system (system II) in Neurospora is derepressed during starvation for an externally supplied source of carbon and energy. Fasting also results in the derepression of uptake systems for fructose, galactose, and lactose. In contrast to the repression observed when cells were grown on glucose, sucrose, or fructose, system II was not repressed by growth on tryptone and casein hydrolysate. System II was inactivated in the presence of 0.1 m glucose and glucose plus cycloheximide but not by cycloheximide alone. Inactivation followed first-order kinetics with a half-time of 40 min. The addition of glycerol to the uptake medium had no significant effect on the kinetics of 3-0-methyl glucose uptake, suggesting that the system was not feedback inhibitable by catabolites of glycerol metabolism.     

Late effects of abdominal radiation on intestinal uptake of nutrients

The late effects of variable doses of abdominal irradiation on in vitro jejunal uptake were examined. The uptake of glucose, galactose, cholic acid, medium-chain length fatty acids, and decanol was studied 6 and 33 weeks following 300, 600, or 900 cGy abdominal irradiation. The intestinal morphological characteristics were similar 6 and 33 weeks after radiation. The uptake of cholic acid was unaffected by abdominal irradiation, but for glucose, galactose, and four fatty acids the direction and magnitude of the changes in uptake were influenced by the dose of irradiation and by the interval following exposure. The greater uptake of decanol at 6 weeks but lower uptake of decanol at 33 weeks reflected changes in the resistance of the intestinal unstirred water layer. These absorption changes suggest that the intestine may not be capable of correcting the transport abnormalities arising from sublethal doses of abdominal irradiation.     

beta-D-phosphogalactoside galactohydrolase of Streptococcus faecalis and the inhibition of its synthesis by glucose.

The lactose hydrolysing system of Streptococcus faecalis is described. It is closely related to that one of the group N streptocci as it consists of a beta-D-phosphogalactoside galactohydrolase (beta-Pgal). The uptake of methyl-beta-D-thiogalactoside (TMG), lactose, and glucose is maintained by the phosphoenolpyruvate-dependent phosphotransferase system (PTS) but the uptake of galactose is not. The induction time is 6--7 min. Inducers are lactose and galactose but not isopropyl-beta-D-galactoside (IPTG) and TMG. In the presence of glucose, mannose, and maltose no induction of beta-Pgal occurs but pyruvate and glycerol allow induction. The competitive inhibition of uptake of TMG by glucose suggests inducer exclusion by this sugar. TMG accumulates in the cells exclusively as a derivative.     

The Agrobacterium tumefaciens virulence gene chvE is part of a putative ABC-type sugar transport operon.

The Agrobacterium tumefaciens virulence determinant ChvE is a periplasmic binding protein which participates in chemotaxis and virulence gene induction in response to monosaccharides which occur in the plant wound environment. The region downstream of the A. tumefaciens chvE gene was cloned and sequenced for nucleotide and expression analysis. Three open reading frames transcribed in the same direction as chvE were revealed. The first two, together with chvE, encode putative proteins of a periplasmic binding protein-dependent sugar uptake system, or ABC-type (ATP binding cassette) transporter. The third open reading frame encodes a protein of unknown function. The deduced transporter gene products are related on the amino acid level to bacterial sugar transporters and probably function in glucose and galactose uptake. We have named these genes gguA, -B, and -C, for glucose galactose uptake. Mutations in gguA, gguB, or gguC do not affect virulence of A. tumefaciens on Kalanchoe diagremontiana; growth on 1 mM galactose, glucose, xylose, ribose, arabinose, fucose, or sucrose; or chemotaxis toward glucose, galactose, xylose, or arabinose.     

Taenia crassiceps: absorption of hexoses and partial characterization of Na + -dependent glucose absorption by larvae

The uptake of ¹⁴C-fructose by T. crassiceps larvae was linear with respect to concentration. Uptake of 0.05 mM¹⁴C-fructose was not inhibited by 5.0 mM unlabeled fructose, tagatose, or sorbose. Fructose appears to enter larvae by diffusion only. The uptake of radioglucose and radiogalactose was not linear with respect to concentration at low substrate concentrations; at high substrate concentrations, the uptake of both hexoses was linear with respect to concentration. Inhibitor studies indicated that both glucose and galactose enter larvae by a combination of diffusion and a mediated process, and that these hexoses are mutually competitive inhibitors of one another. The uptake of glucose and galactose was also inhibited by α-and β-methyl glucoside, fucose, and phlorizin, but not by several amino acids, certain sugar analogs, nor ouabain. Glucose transport is Na⁺ sensitive; K⁺ was demonstrated to be a competitive inhibitor of Na⁺ activation of glucose uptake. After a 90-min incubation in 5 mM unlabeled glucose, larvae accumulated glucose against an apparent concentration difference. Although larvae appear freely permeable to ouabain, this compound had no apparent effect on glucose accumulation. The results of this study are compared with previous studies on Hymenolepis diminuta, Calliobothrium verticillatum, Hydatigera (Taenia) taeniaeformis, and mammalian systems.     

Simultaneous utilization of galactose and glucose bySaccharomyces spp.

Summary The adaptation of yeast to galactose utilization allows the simultaneous uptake of galactose and glucose, during high cell density fermentation. Furthermore, yeast cells grown in galactose show higher rates of glucose uptake than those under derepressing conditions.     

Catabolite inhibition and sequential metabolism of sugars by Streptococcus lactis.

Growth of galactose-adapted cells of Streptococcus lactis ML(3) in a medium containing a mixture of glucose, galactose, and lactose was characterized initially by the simultaneous metabolism of glucose and lactose. Galactose was not significantly utilized until the latter sugars had been exhausted from the medium. The addition of glucose or lactose to a culture of S. lactis ML(3) growing exponentially on galactose caused immediate inhibition of galactose utilization and an increase in growth rate, concomitant with the preferential metabolism of the added sugar. Under nongrowing conditions, cells of S. lactis ML(3) grown previously on galactose metabolized the three separate sugars equally rapidly. However, cells suspended in buffer containing a mixture of glucose plus galactose or lactose plus galactose again consumed glucose or lactose preferentially. The rate of galactose metabolism was reduced by approximately 95% in the presence of the inhibitory sugar, but the maximum rate of metabolism was resumed upon exhaustion of glucose or lactose from the system. When presented with a mixture of glucose and lactose, the resting cells metabolized both sugars simultaneously. Lactose, glucose, and a non-metabolizable glucose analog (2-deoxy-d-glucose) prevented the phosphoenolpyruvate-dependent uptake of thiomethyl-beta-d-galactopyranoside (TMG), but the accumulation of TMG, like galactose metabolism, commenced immediately upon exhaustion of the metabolizable sugars from the medium. Growth of galactose-adapted cells of the lactose-defective variant S. lactis 7962 in the triple-sugar medium was characterized by the sequential metabolism of glucose, galactose, and lactose. Growth of S. lactis ML(3) and 7962 in the triple-sugar medium occurred without apparent diauxie, and for each strain the patterns of sequential sugar metabolism under growing and nongrowing conditions were identical. Fine control of the activities of preexisting enzyme systems by catabolite inhibition may afford a satisfactory explanation for the observed sequential utilization of sugars by these two organisms.     

Lack of protective effect of oral enprostil, a synthetic prostaglandin E2, on intestinal transport and morphology following abdominal irradiation in the rat

Previous studies have demonstrated that abdominal irradiation alters intestinal uptake of nutrients. The purpose of this study was to determine the effect of an orally administered synthetic prostaglandin E2, enprostil, given on three occasions shortly prior to a single exposure to 600 cGy external abdominal irradiation, on intestinal active and passive transport processes and villus morphology measured 7 days later. Animals were sham-irradiated (CONT) or were exposed to a single dose of 600 cGy external abdominal irradiation (RAD); two and one mornings before the day of irradiation or sham irradiation, and 1 h before irradiation or sham irradiation enprostil was administered. One half of CONT and RAD groups were dosed orally with enprostil, 5 micrograms/kg body weight, and the other half of the CONT and RAD groups were dosed with placebo. Seven days later the in vitro uptake of glucose, galactose, long-chain fatty acids, and cholesterol was determined in the four groups (CONT with and without enprostil, and RAD with and without enprostil). In CONT, enprostil was associated with increased jejunal uptake of glucose and ileal uptake of galactose. In RAD given enprostil, there was increased jejunal uptake of galactose but reduced ileal uptake of glucose and galactose. The expected radiation-associated decline in jejunal galactose uptake was prevented with enprostil. In CONT given enprostil, there was increased jejunal uptake of fatty acid (FA) 14:0 and 16:0 but reduced uptake of FA 18:0, 18:1, and 18:2; enprostil had no effect on lipid uptake in the ileum in CONT.(ABSTRACT TRUNCATED AT 250 WORDS)