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The characteristics of glucose transport across the blood brain barrier and its relation to cerebral glucose metabolism.

The evidence suggests that glucose transport across the blood brain barrier (BBB) in the dog is normally not a rate-limiting step in cerebral metabolism; however, transport may become rate-limiting under conditions of extreme hypoglycemia or anoxia. Studies on the mechanism of glucose transport from blood to brain do not at this time permit us to distinguish between active transport and facilitated diffusion; however, a decrease in the rate of unidirectional transport during anoxia suggests that an energy-dependent process may be involved. In spite of this evidence, glucose transport across the BBB is similar to the facilitated diffusion of glucose into the red cell in terms of the structural requirements of the glucose molecule, the pattern of inhibition by phlorizin, phloretin and cytochalasin B, and the lack of sensitivity to Na+ or insulin.

Animals↗

Caecal and colonic uptake of both linoleic acid and cholesterol in rats following intestinal resection.

Caecal and colonic uptake of both linoleic acid and cholesterol were studied in rats after distal small bowel resection (DSBR). The results showed that the surgical operation increased the caecal and colonic uptake of linoleic acid. Supplementation with linolenic acid inhibited caecal and colonic uptake of linoleic acid. Experiments carried out in the presence of rotenone and ouabain suggest that facilitated diffusion is the predominant mechanism of caecal and colonic linoleic acid absorption, at least at low concentrations. An increase in caecal and colonic uptake of cholesterol was observed after the surgical operation. The study showed that facilitated diffusion seems to be the mechanism of linoleic acid absorption in the caecum and colon, and that both organ growth and changes in transport function of the epithelial cells of caecum and colon appear to be involved in the adaptive response of the bowel to intestinal resection.

Adenosine Triphosphate↗

Interconversion and glucose-induced inactivation of glucose transport systems in Candida shehatae.

During starvation (derepression) glucose-grown cells of Candida shehatae IGC 3607 displayed total interconversion of facilitated diffusion of glucose into a glucose-proton symport, dependent on de novo protein synthesis (proteosynthetic interconversion). The reverse process, inactivation of the proton symport induced by glucose or 2-deoxyglucose, was not accompanied by reemergence of the facilitated diffusion function. The inactivation process had a rapid initial and a slow second phase. The rapid inactivation depended on the external sugar concentration and was reversible while the subsequent slow inactivation was irreversible and independent of the external concentration of the signalling sugar. Interaction of the latter with a surface receptor was indicated by the range of sugar concentrations that affected rapid inactivation.

Biological Transport↗

Glucose uptake and metabolism in the Trichinella spiralis nurse cell.

Isolated Trichinella spiralis nurse cells transport a significantly greater amount of glucose/mg of protein than the normal skeletal muscle cell line (L6). V(max) and K(m) estimations revealed that nurse cells have a much higher saturation point than L6 cells for glucose. The effects of numerous physiological conditions (Na(+) concentration, pH, and temperature) on nurse cell glucose uptake were investigated. It was determined that sodium concentration had no effect on glucose uptake. Low (<6.5) and high (>7.3) pH and low (5 degrees C) temperatures significantly effected glucose uptake. The two hormones, insulin and epinephrine, appeared to have little, if any, influence on the rate of glucose uptake by nurse cells. Glucose uptake was inhibited in the presence of 6-carbon carbohydrates. The H(+)/glucose symport inhibitors, dicyclohexylcarbodiimide (DCCD) and Carbonyl cyanide 4-trifluoromethoxyphenlhydrazone (FCCP), and the facilitated diffusion inhibitor phloretin also inhibited glucose uptake. Oubain, a Na(+)/glucose symport inhibitor, did not inhibit glucose uptake. These data, in conjunction with Western blot analyses, revealed that the transport of glucose occurs via H(+)/glucose symport and facilitated diffusion, perhaps through the glucose transport proteins GLUT 1 and/or 4. It was also demonstrated that nurse cells are capable of synthesising glycogen. It appears that glycogen is in a constant state of flux and physiological conditions, such as glucose concentration, significantly influence the synthesis of this macromolecule. We conclude that these results are consistent with the hypothesis that nurse cells, at least maintained in vitro, are metabolically highly active but show significant divergence from normal muscle cells in several fundamental aspects of sugar metabolism.

Animals↗

Bacterial zinc transporters and regulators.

Zn2+ homeostasis in bacteria is achieved by export systems and uptake systems which are separately regulated by their own regulators. Three types of Zn2+ export systems that protect cells from high toxic concentrations of Zn2+ have been identified: RND multi-drug efflux transporters, P-type ATPases, and cation-diffusion facilitators. The RND type exporters for Zn2+ are only found in a few gram-negative bacteria; they allow a very efficient export across the cytoplasmic membrane and the outer membrane of the cell. P-type ATPases and cation-diffusion facilitators belong to protein families that are also found in eukaryotes. The exporters are regulated in bacteria by MerR-like repressor/activators or by ArsR-like repressors. For the high-affinity uptake of Zn2+, several binding-protein-dependent ABC transporters belonging to one class have been identified in different bacteria. Zn2+ ABC transporters are regulated by Zur repressors, which belong to the Fur protein family of iron regulators. Little is known about low-affinity Zn2+ uptake under zinc-replete conditions. One known example is the phosphate uptake system Pit, which may cotransport Zn2+ in Escherichia coli. Similarly, the citrate-metal cotransporter CitM in Bacillus subtilis may help to supply Zn2+.

ATP-Binding Cassette Transporters↗

Glucose uptake by Trypanosoma brucei. Rate-limiting steps in glycolysis and regulation of the glycolytic flux.

Glucose uptake and metabolism in the bloodstream form of the glycosome-containing protozoan parasite Trypanosoma brucei was measured using 14C-labeled glucose in combination with the silicone oil centrifugation technique in short term (5-60 s) incubations. Glucose rather than glucose analogues was used to study the interrelation between the uptake process and the subsequent metabolic steps. Glucose uptake over the plasma membrane occurred by facilitated diffusion, which limited the overall glycolytic rate at external glucose concentrations (glcout) below 5 mM. At higher glcout another step, either transport over the glycosomal membrane or phosphorylation by hexokinase became rate-limiting. Mathematical modeling assuming that glucose uptake occurs by facilitated diffusion followed by an enzymatic step accurately predicts the experimental data. As predicted by the model, the internal concentration of non-metabolized glucose remains low till glcout = 5 mM and increases at higher external concentrations. In contrast to glucose, glycerol entered the cell by simple diffusion. Externally supplied glycerol did not affect glucose metabolism but externally added glucose interfered with glycerol metabolism in a way that suggests that the rate-limiting step is at the level of glycerol kinase. Our observations suggest that the bloodstream form of T. brucei adapts its glucose transport in a way that gives maximum yield at minimum expense.

Animals↗

COMPARISON OF THE ACTIVE TRANSPORT SYSTEMS FOR ALPHA-THIOETHYL- D-GLUCOPYRANOSIDE AND MALTOSE IN SACCHAROMYCES CEREVISIAE.

Okada, Hirosuke (University of Wisconsin, Madison), and H. O. Halvorson. Comparison of the active transport systems for alpha-thioethyl-d-glucopyranoside and maltose in Saccharomyces cerevisiae. J. Bacteriol. 86:966-970. 1963.-Inducible systems for the active transport of both maltose and alpha-thioethyl-d-glucopyranoside (alpha-TEG) have been described in strains of Saccharomyces cerevisiae. The properties of induction of the alpha-TEG-accumulating system were similar to other inducible systems in yeast: the differential rate of synthesis was constant, and induction was inhibited by amino acid analogues. At temperatures below 20 C, the temperature dependence of alpha-TEG accumulation in induced cells was identical to that in facilitated diffusion. From a survey of various inducers, a coordinate induction of both active-transport systems was observed. These findings led to the conclusion that in active transport a common inducible enzyme is coupled to both the alpha-TEG and maltose facilitated-diffusion systems.

Biological Transport↗

Transport and metabolism of vitamin B6 in lactic acid bacteria.

Streptococcus faecalis 8043 concentrates extracellular [3H]pyridoxal or [3H]pyridoxamine primarily as the corresponding 5'-phosphates. Accumulation of pyridoxamine requires an exogenous energy source and is inhibited by glycolysis inhibitors. A membrane potential is not required for transport of pyridoxamine, and an artificially generated potential does not drive uptake in this organism. Based on this and other evidence, it is concluded that S. faecalis accumulates pyridoxamine by facilitated diffusion in conjunction with trapping by pyridoxal kinase. Pyridoxamine-P is not concentrated, but equilibrates with that provided externally. Lactobacillus casei 7469 concentrates radioactivity only from pyridoxal, which appears internally as pyridoxal-P, suggesting that it too absorbs the vitamin by facilitated diffusion plus trapping. The specificity of the growth requirement of S. faecalis and L. casei for vitamin B6 parallels the specificity of the transport systems for this vitamin in these organisms. Lactobacillus delbrueckii 7469, however, which specifically requires pyridoxamine-P or pyridoxal-P for growth, accumulates both these compounds and pyridoxine-P from the medium, apparently by active transport, but not pyridoxine, pyridoxamine, or pyridoxal. While pyridoxal-P and pyridoxamine-P are interconvertible in this organism, pyridoxine-P is not further metabolized, thus accounting for the specificity of the growth requirement. These and previous results show (a) that different organisms may employ quite different transport machinery in utilization of a given external nutrient, and (b) that the specificity of the growth requirement for a given form of a vitamin frequently arises from the specificity of transport, but that internal metabolism of the compounds also plays a significant role in some organisms.

Biological Transport, Active↗

Ethanolamine transport in human placental brush-border membrane vesicles.

Pathways for transport of ethanolamine by human placental epithelia were investigated by measurement of [3H]ethanolamine uptake in brush-border membrane vesicles isolated by divalent cation precipitation. The presence of a conductive uptake pathway for ethanolamine was suggested by the marked stimulation of ethanolamine uptake to levels exceeding equilibrium induced by an inside-negative potassium diffusion potential. Evidence to suggest conductive ethanolamine uptake resulted from a mediated transport process included 1) the concentration-dependent inhibition by choline; 2) trans-stimulation of choline and ethanolamine uptake by ethanolamine; and 3) substrate-specific inhibition by chemically related analogs. Transport of both choline and ethanolamine by a common facilitated diffusion mechanism is suggested by 1) trans-stimulation of choline uptake by ethanolamine; 2) mutual inhibition of conductive choline and ethanolamine uptake by ethanolamine and choline; 3) the effect of ethanolamine on the kinetics of conductive choline uptake; and 4) the rank order inhibition of choline and ethanolamine uptake by the same panel of chemical analogs. The present study identifies the presence of a facilitated diffusion mechanism as a brush-border membrane transport pathway for choline and ethanolamine accumulation by human placenta.

Adult↗

The high Km glucose transporter of islets of Langerhans is functionally similar to the low affinity transporter of liver and has an identical primary sequence.

The liver has been shown to contain a facilitated diffusion glucose transporter with high Km for glucose that is structurally distinct from the low Km glucose transporters found in most other tissues. We find that 3-O-methyl glucose is greater than 90% equilibrated across dispersed islet cells within 60 s, consistent with a facilitated diffusion transport mechanism. L-Glucose uptake was minimal throughout the time course, indicating stereospecificity. Measurement of glucose transport over a range of 3-O-methyl glucose concentrations from 0.05 to 60 mM revealed the presence of a component of glucose transport with an apparent Km of 17 mM, a value essentially identical to that previously reported for liver. Interestingly, a second component of glucose transport was also observed with an apparent Km of 1.4 mM, as has been reported for other tissues such as erythrocytes that are known to contain the "HepG2" or "erythroid/brain" type glucose transporter. Further evidence for the existence of two transport components is provided by the observation that a low concentration of cytochalasin B (0.4 microM) completely inhibits the low Km transport activity but has no effect on the high Km transporter. The kinetic similarity of high Km glucose transport in liver and islets is readily understood in light of our structural analysis. Sequence analysis of cDNA clones indicates that the liver and islet glucose transporters have identical sequences and, thus, are the products of the same gene.

3-O-Methylglucose↗

The origin of cells: a symbiosis between genes, catalysts, and membranes.

The gap between early molecular evolution and the origin of the first cell may have been bridged by a photoheterotrophic obcell, consisting of genes and ribosomes attached to the outer surface of a phospholipid vesicle containing a light-driven proton pump and a proton-driven pyrophosphate synthase. I argue that the obcell was the substratum for the origin of DNA replication; DNA segregation by the growth and division of the peptidoglycan murein; periplasmic solute-binding proteins; bioenergetics, including the F0F1 proton-driven ATP synthase; active transport of calcium; and facilitated diffusion of nutrients across membranes, and that it played the major role in the replacement of ribozymes by protein catalysts. Curved growth of the peptidoglycan and a mutation causing septum formation produced the first true cell. Evolution of porins, sodium extrusion and potassium import, conversion of the facilitated diffusion proteins to active pumps, and the evolution of intermediary metabolism, carbon and nitrogen fixation, and of substrate level phosphorylation, completed the origin of the first negibacterial eubacterium, from which all other cells evolved, and from which they have inherited most of their major catalytic properties--with the notable exceptions of reverse transcriptase, RNA splicing, and methanogenesis, all of which I believe evolved very much later.

Animals↗

Analysis of the transport of valproic acid into saliva from serum.

A study was conducted to explore whether a positive correlation between serum and salivary concentrations of the well-known antiepileptic drug, valproic acid (VPA), in epileptic patients could be explained by facilitated diffusion. The total concentration in saliva (Cs) would be related to the apparent ratio (R(app)++ = 100.Cs/Ct) of Cs to the total concentration in serum (Ct) as follows: Cs = A.R(app)B. This equation can be illustrated with microcomputer-simulated figures by assuming a process of facilitated diffusion for the transport of VPA into saliva from blood by the mechanism of monocarboxylic acid absorption through the intestinal brush-border membrane vesicles. The above equation has been proved to be valid when applied to the data reported separately by Gugler and coworkers and by Nitsche and Mascher, who evaluated the pharmacokinetics of VPA. Moreover, we can estimate the serum concentration with the salivary concentration using the above equation.

Adolescent↗

Concentrative uridine transport by murine splenocytes: kinetics, substrate specificity, and sodium dependency.

A previous report from this laboratory indicated that the concentration of free uridine (Urd) in many normal murine tissues greatly exceeds that in plasma. We now report that Urd uptake by isolated murine splenocytes is concentrative, and that the rate of uptake from medium containing 10 to 500 microM [3H]Urd conforms to a process that is saturable with a Km of 38.0 +/- 4.1 (SE) microM and Vmax of 2.70 +/- 0.27 pmol/s/microliter cell water. Other ribosyl and deoxyribosyl pyrimidine nucleosides or their analogues were not concentrated by splenocytes; however, ribosyl and deoxyribosyl purine nucleosides and, to a lesser extent, deoxyuridine did inhibit Urd uptake. In this system Urd uptake was not inhibited by 1 microM nitrobenzylthioinosine or 10 microM dipyridamole but was significantly inhibited by 5 mM NaN3 or 250 microM KCN. Transport of Urd involves Na+ cotransport as evidenced by complete inhibition when Na+ is replaced by Li+ in the incubation medium, and it is also inhibited by 3 mM ouabain. Active Urd transport coexists with the nonspecific, carrier mediated, facilitated diffusion of nucleosides as demonstrated by the inhibition of Urd efflux and thymidine influx in splenocytes by nitrobenzylthioinosine and dipyridamole. Under identical conditions, Urd entry into L1210 leukemia cells was nonconcentrative and non-Na+ dependent but inhibited by nitrobenzylthionosine. That nucleosides enter most cultured neoplastic cell lines by facilitated diffusion and not the active transport mechanism for Urd confirms earlier findings and may represent an exploitable target for chemotherapy.

Animals↗

Sodium-dependent nucleoside transport in rabbit intestinal epithelium.

Cellular uptake of formycin B, a poorly metabolized analog of inosine, by the isolated epithelium of rabbit jejunum is three times higher in the presence of Na+ than without this cation. The Na(+)-dependent nucleoside transport system is located in the apical membrane of the enterocytes and is capable of uphill transport, as shown for formycin B and adenosine with brush border membrane vesicles. According to present and earlier evidence, nucleoside transport across the basolateral membrane appears to have the properties of facilitated diffusion. Na(+)-dependent formycin B transport activity in intestinal epithelium decreases from jejunum to ileum and is absent in descending colon. As with Na(+)-coupled cotransport systems for other organic solutes, apical entry of formycin B is driven by the electrochemical Na+ gradient into the cell. In contrast to the facilitated diffusion system for nucleosides, Na(+)-dependent formycin B transport is not inhibited by nitrobenzylthioinosine, but both carrier systems are sensitive to inhibitors of D-glucose transport. Natural purine nucleosides and uridine are strong inhibitors of Na(+)-dependent formycin B transport. Transepithelial flux measurements substantiated that the Na(+)-dependent transport mechanism for formycin B functions as an absorptive system.

Animals↗

A mathematical model of the P-glycoprotein pump as a mediator of multidrug resistance.

Cells displaying the classic multidrug resistant (MDR) phenotype possess a transmembrane protein (p170 or P-glycoprotein) which can actively extrude cytotoxic agents from the cytoplasm. A mathematical model of this drug efflux pump has been developed. Outward transport is modeled as a facilitated diffusion process. Since energy-dependent efflux of cytotoxic agents requires that ATP also bind to p170, the model includes a dynamic calculation for efflux rate which considers Michaelis-Menten kinetics for both the substrate agent and ATP. The final system consists of one partial differential equation (PDE) for the facilitated diffusion of substrate agents out of the cell, a 2 x 2 ordinary differential equation (ODE) system for the dynamic calculation of the ATP-ADP pool, and a dynamic algebraic calculation of the efflux rate given substrate levels at the interior cell membrane interface and ATP levels in the cell. A stability analysis of the ATP-ADP pool distribution and a simplistic closed form solution of the linearized PDE are included. Numerical simulations are also provided.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

Direct measurement of lactose/proton symport in Escherichia coli membrane vesicles: further evidence for the involvement of histidine residue(s).

Addition of lactose to Escherichia coli ML 308-225 membrane vesicles under nonenergized conditions induces transient alkalinization of the medium, and the initial rate of proton influx is stimulated by valinomycin and abolished by nigericin or carbonyl cyanide m-chlorophenylhydrazone. A functional lac y gene product is absolutely required as the effect is not observed in ML 308-225 vesicles treated with N-ethylmaleimide nor with vesicles from uninduced Escherichia coli ML 30. Furthermore, the magnitude of the phenomenon is enhanced about 3-fold in vesicles from Escherichia coli T206, which contain amplified levels of the lac carrier protein. Kinetic parameters for lactose-induced proton influx are the same as those determined for lactose-facilitated diffusion, and quantitative comparison of the initial rates of the two fluxes indicates that the stoichiometry between protons and lactose is 1:1. Treatment of ML 308-225 vesicles with diethyl pyrocarbonate causes inactivation of lactose-induced proton influx. Remarkably, however, treatment with the histidine reagent enhances the rate of lactose-facilitated diffusion in a manner suggesting that the altered lac carrier catalyzes lactose influx without the symport of protons. The results are consistent with the hypothesis that acylation of a histidyl residue(s) in the lac carrier protein dissociates lactose influx from proton influx and indicate that this residue(s) play(s) an important role in the pathway of proton translocation.

Biological Transport↗

Molecular and functional characterization and tissue localization of 2 glucose transporter homologues (TGTP1 and TGTP2) from the tapeworm Taenia solium.

Tapeworms absorb and consume large quantities of glucose through their syncytial tegument, storing the excess as glycogen. Although some studies on the metabolism of glucose in several tapeworms are available, the proteins that mediate its uptake and distribution in their tissue have not been identified. We describe the isolation and characterization of cDNA clones encoding 2 facilitated diffusion glucose transporters (TGTP1 and TGTP2) from Taenia solium, the causal agent of human and porcine cysticercosis. Radio-isotope labelled hexose uptake mediated by TGTP1 expressed in Xenopus oocytes is inhibited by the natural stereoisomers D-glucose and D-mannose but not by L-glucose. Transport by TGTP1 is sensitive to classical inhibitors of facilitated diffusion such as phloretin and cytochalasin B, and insensitive to ouabain. TGTP2 did not function in Xenopus oocytes. Localization studies using specific anti-TGTP1 and anti-TGTP2 antibodies show that TGTP1 is abundant in a number of structures underlying the tegument in adult parasites and larvae, whereas TGTP2 appears to be localized only on the tegumentary surface of the larvae and is not detected in adults.

Amino Acid Sequence↗

Rapid three-dimensional diffusion MRI facilitates the study of acute stroke in mice.

MRI studies using mouse brain models of ischemia are becoming a valuable tool for understanding the mechanism of stroke, since transgenic models are now available. However, the small size of the mouse brain and the surgical complexity of creating ischemia in mice make it technically challenging to obtain high-quality MRI data. Therefore, there are few reports of MRI studies in murine cerebral ischemia. In this project a newly developed rapid 3D diffusion-weighted imaging (DWI) technique was applied to study experimental stroke in a mouse model of reversible middle cerebral artery occlusion (MCAO). Ischemic volumes were successfully delineated using this 3D whole-brain imaging technique with high spatial (0.34 x 0.5 x 1.0 mm(3) before zero-filling) and temporal (7 min) resolution. The 3D observation revealed the characteristic evolution of stroke after transient MCAO. There was a temporarily high diffusion constant in the cortex during early reperfusion, followed by a secondary energy failure in the cortex and caudate-putamen at 6 and 21 h of reperfusion. Magn Reson Med 46:183-188, 2001.

Animals↗