Nutrient absorption by Aphidius ervi larvae

It is well documented that in the model system Aphidius ervi Haliday (Hymenoptera, Braconidae)/Acyrthosiphon pisum (Harris) (Homoptera, Aphididae) host regulation by the parasitoid larva induces in the aphid haemolymph major changes of the titer of nutritional compounds such as proteins, acylglycerols and free amino acids, in order to meet the stage-specific demands of the developing larva. Since little is known about how the larva absorbs these mobilized nutritional resources, nutrient absorption by larval stages of A. ervi was studied. In 2nd instar larvae, leucine was ten-fold accumulated in the haemocoel, and tyrosine and glutamine two-fold. Glucose and fructose were readily absorbed and fructose was extensively metabolized by larval tissues. In 3rd instars, the presence of a number of larvae that did not ingest the incubation medium enabled us to determine the respective amounts of substrate absorbed by the epidermis and the midgut. An accumulation of leucine in the haemocoel was observed only when midgut cells were involved in absorption, while the amino acid concentration within body fluids never exceeded that of the incubation medium when the uptake was performed only by epidermal cells. The immunofluorescence analysis, the mutual inhibition exerted on labeled glucose or fructose uptakes by a 100-fold excess of the sugars and the strong inhibition of uptakes induced by 0.2mM cytochalasin B support the expression of facilitative GLUT2-like transporters in the apical and basal cell membranes of midgut epithelial cells. Taken together, these results prove that both midgut and epidermis are involved in nutrient absorption throughout the parasitoid development, that GLUT2 transporters are responsible for glucose and fructose uptakes and that the chemical gradient that favors the passive influx of the two sugars is maintained by their conversion to other substrates.     

Polarity of transport of 2-deoxy-D-glucose and D-glucose by cultured renal epithelia (LLC-PK1).

At least two types of glucose transporter exist in cultured renal epithelial cells, a Na(+)-glucose cotransporter (SGLT), capable of interacting with D-glucose but not 2-deoxy-D-glucose (2dglc) and a facilitated transporter (GLUT) capable of interacting with both D-glucose and 2dglc. In order to examine the polarity of transport in cultured renal epithelia, 2dglc and D-glucose uptakes were measured in confluent cultures of LLC-PK1 cells grown on collagen-coated filters that permitted access of medium to both sides of the monolayer. The rates of basolateral uptake of both 1 mM glucose (Km 3.6 mM) and 1 mM 2dglc (Km 1.5 mM) were greater than apical uptake rates and the (apical-to-basolateral)/(basolateral-to-apical) flux ratio was high for glucose (9.4) and low for 2dglc (0.8), thus, confirming the lack of interaction of 2dglc with the apical SGLT. Specific glucose transport inhibitor studies using phlorizin, phloretin and cytochalasin B confirmed the polarised distribution of SGLT and GLUT in LLC-PK1 cells. Basolateral sugar uptake could be altered by addition of insulin (1 mU/ml) which increased 2dglc uptake by 72% and glucose uptake by 50% and by addition of 20 mM glucose to the medium during cell culture which decreased 2dglc uptake capacity at confluence by 30%. During growth to confluence, 2dglc uptake increased to a maximum, then decreased at the time of confluence, coincident with a rise in uptake capacity for alpha-methyl-D-glucoside, a hexose that interacts only with the apical SGLT. It was concluded that the non-metabolisable sugar 2dglc was a useful, specific probe for GLUT in LLC-PK1 cells and that GLUT was localised at the basolateral membrane after confluence.     

Cytochalasin B as a probe of protein structure and substrate recognition by the galactose/H+ transporter of Escherichia coli.

Cytochalasin B is a potent inhibitor of mammalian passive glucose transporters. The recent demonstration of sequence similarities between these proteins and several bacterial proton-linked sugar transporters suggested that cytochalasin B might be a useful tool for investigation of the galactose/H+ symport protein (GalP) of Escherichia coli. Equilibrium binding studies using membranes from a GalP-constitutive (GalPc) strain of E. coli revealed a single set of high affinity binding sites for cytochalasin B with a Kd of 0.8-2.2 microM. Binding was inhibited by D-glucose, but not by L-glucose. UV irradiation of the membranes in the presence of [4-3H]cytochalasin B photolabeled principally a protein of apparent Mr 38,000, corresponding to the GalP protein. Labeling was inhibited by greater than 80% in the presence of 500 mM D-glucose or D-galactose, the major substrates of the GalP system. The extent of inhibition of photolabeling by different sugars and sugar analogues showed that the substrate specificity of GalP closely resembles that of the mammalian passive glucose transporters. Structural similarity to the latter was revealed by tryptic digestion of [4-3H]cytochalasin B-photolabeled GalP, which yielded a radiolabeled fragment of apparent Mr 17,000-19,000, similar to that previously reported for the human erythrocyte glucose transporter.     

Absorption of sugars and amino acids by the epidermis of Aphidius ervi larvae

Aphidius ervi Haliday (Hymenoptera, Braconidae) is an endophagous parasitoid of several aphid species of economic importance, widely used in biological control. The definition of a suitable artificial diet for in vitro mass production of this parasitoid is still an unresolved issue that, to be properly addressed, requires a deeper understanding both of its nutritional needs and of the functional properties of the larval epithelia involved in nutrient absorption. The experimental evidence presented in this paper unequivocally demonstrates that the uptake of sugars and amino acids takes place through the body surface of the larval stages of A. ervi. These nutrients are efficiently absorbed by the larval epidermis, but the transport rate progressively declines over time. The epidermis exhibits a cross-reactivity to antibodies raised against the mammalian facilitative glucose transporter GLUT2 and the sodium cotransporter SGLT1. The analysis of sugar transport sensitivity to specific inhibitors indicates the involvement of GLUT2-like transporters, while a role for SGLT1-like transporters is not supported. The peculiar pathways of nutrient absorption in A. ervi larvae further corroborate the general idea that the pre-imaginal stages of endophagous koinobiont Hymenoptera, like Metazoan parasites, show a high degree of physiological integration with their hosts.     

Detection of two distinct transporter systems for 2-deoxyglucose uptake by the opportunistic pathogen Pneumocystis carinii.

Since the opportunistic pathogen Pneumocystis carinii grows only slowly in vitro, the mechanism of glucose uptake was investigated to better understand how the organism transports nutrients. Using the non-metabolizable analogue 2-deoxyglucose, two uptake systems were detected with Q(10) values of 2.12 and 2.09, respectively. One had a high affinity (K(m)=67.5 microM) and the other a low affinity (K(m)=5.99 mM) for 2-deoxyglucose uptake. Glucose or deoxyglucose phosphate products from transported radiolabeled substrates were not detected during the incubation times used in this study. Both systems were inhibited by mannose, galactose, fructose, galactosamine, glucosamine, and glucose but not by allose, 5-thioglucose, xylose, glucose 6-phosphate and glucuronic acid. Salicylhydroxamate, KCN, iodoacetate, and 2,4-dinitrophenol inhibited the high-affinity transporter, suggesting it required ATP. Ouabain, monensin, carbonyl cyanide m-chlorophenylhydrazone, and N,N'-dicyclohexylcarbodiimide also inhibited deoxyglucose uptake, as did the replacement of Na(+) in the incubation medium with choline, indicating requirements for Na(+) and H(+). The high-affinity system was also inhibited by the protein synthesis inhibitors cycloheximide and chloramphenicol. In contrast, the low-affinity system transported deoxyglucose by facilitated diffusion mechanisms. Unlike the human erythrocyte glucose transporter GLUT1, the P. carinii transporters recognized fructose and galactose and were relatively insensitive to cytochalasin B, suggesting that the P. carinii glucose transporters may be good drug targets.     

Transport of d-galactose by the gastrointestinal tract of the locust, Locusta migratoria

Due to exoskeleton, the absorption of nutrients in adult insects takes place across the gastrointestinal tract epithelium. In most physiological studies, sugar intestinal absorption has been described as a diffusional process and to date no sugar transporter has been cloned from the digestive tract of insects. In the present work, the existence of a saturable transport system for galactose in the gastric caeca of Locusta migratoria is clearly demonstrated. This transport shows a relatively high affinity for galactose (apparent K0.5=2-3 mM) and is inhibited by glucose, 2-deoxyglucose and with less potency by fructose and alpha-methyl-d-glucoside. The absence of sodium or the presence of phloridzin hardly affects galactose absorption, indicating that it is not mediated by a SGLT1-like transporter. The absence of K+, Cl-, Mg2+ and Ca2+ or changes in the pH do not modify galactose absorption either. Nevertheless, phloretin, cytochalasin B and theophylline (inhibitors of facilitative transporters) decrease sugar uptake around 50%. Xenopus laevis oocytes microinjected with poly A+ RNA isolated from gastric caeca show sodium-independent galactose uptake that is three times higher than in non-injected oocytes, further supporting the existence of a mRNA coding for at least one equilibrative sugar transporter in L. migratoria gastric caeca.     

SMIT2 mediates all myo-inositol uptake in apical membranes of rat small intestine

This study presents the characterization of myo-inositol (MI) uptake in rat intestine as evaluated by use of purified membrane preparations. Three secondary active MI cotransporters have been identified; two are Na(+) coupled (SMIT1 and SMIT2) and one is H(+) coupled (HMIT). Through inhibition studies using selective substrates such as d-chiro-inositol (DCI, specific for SMIT2) and l-fucose (specific for SMIT1), we show that SMIT2 is exclusively responsible for apical MI transport in rat intestine; rabbit intestine appears to lack apical transport of MI. Other sugar transport systems known to be present in apical membranes, such as SGLT1 or GLUT5, lacked any significant contribution to MI uptake. Functional analysis of rat SMIT2 activity, via electrophysiological studies in Xenopus oocytes, demonstrated similarities to the activities of SMIT2 from other species (rabbit and human) displaying high affinities for MI (0.150 +/- 0.040 mM), DCI (0.31 +/- 0.06 mM), and phlorizin (Pz; 0.016 +/- 0.007 mM); low affinity for glucose (36 +/- 7 mM); and no affinity for l-fucose. Although these functional characteristics essentially confirmed those found in rat intestinal apical membranes, a unique discrepancy was seen between the two systems studied in that the affinity constant for glucose was approximately 40-fold lower in vesicles (K(i) = 0.94 +/- 0.35 mM) than in oocytes. Finally, the transport system responsible for the basolateral efflux transporter of glucose in intestine, GLUT2, did not mediate any significant radiolabeled MI uptake in oocytes, indicating that this transport system does not participate in the basolateral exit of MI from small intestine.     

Sugar sensing by enterocytes combines polarity, membrane bound detectors and sugar metabolism

Sugar consumption and subsequent sugar metabolism are known to regulate the expression of genes involved in intestinal sugar absorption and delivery. Here we investigate the hypothesis that sugar-sensing detectors in membranes facing the intestinal lumen or the bloodstream can also modulate intestinal sugar absorption. We used wild-type and GLUT2-null mice, to show that dietary sugars stimulate the expression of sucrase-isomaltase (SI) and L-pyruvate kinase (L-PK) by GLUT2-dependent mechanisms, whereas the expression of GLUT5 and SGLT1, did not rely on the presence of GLUT2. By providing sugar metabolites, sugar transporters, including GLUT2, fuelled a sensing pathway. In Caco2/TC7 enterocytes, we could disconnect the sensing triggered by detector from that produced by metabolism, and found that GLUT2 generated a metabolism-independent pathway to stimulate the expression of SI and L-PK. In cultured enterocytes, both apical and basolateral fructose could increase the expression of GLUT5, conversely, basolateral sugar administration could stimulate the expression of GLUT2. Finally, we located the sweet-taste receptors T1R3 and T1R2 in plasma membranes, and we measured their cognate G alpha Gustducin mRNA levels. Furthermore, we showed that a T1R3 inhibitor altered the fructose-induced expression of SGLT1, GLUT5, and L-PK. Intestinal gene expression is thus controlled by a combination of at least three sugar-signaling pathways triggered by sugar metabolites and membrane sugar receptors that, according to membrane location, determine sugar-sensing polarity. This provides a rationale for how intestine adapts sugar delivery to blood and dietary sugar provision.     

Cloning and functional characterization of the human GLUT7 isoform SLC2A7 from the small intestine

Facilitated glucose transporters (GLUTs) mediate transport of sugars across cell membranes by using the chemical gradient of sugars as the driving force. Improved cloning techniques and database analyses have expanded this family of proteins to a total of 14 putative members. In this work a novel hexose transporter isoform, GLUT7, has been cloned from a human intestinal cDNA library by using a PCR-based strategy (GenBank accession no. AY571960). The encoded protein is comprised of 524 amino acid residues and shares 68% similarity and 53% identity with GLUT5, its most closely related isoform. When GLUT7 was expressed in Xenopus oocytes, it showed high-affinity transport for glucose (K(m) = 0.3 mM) and fructose (IC(50) = 0.060 mM). Galactose, 2-deoxy-d-glucose, and xylose were not transported. Uptake of 100 microM d-glucose was not inhibited by 200 microM phloretin or 100 microM cytochalasin B. Northern blotting indicated that the mRNA for GLUT7 is present in the human small intestine, colon, testis, and prostate. Western blotting and immunohistochemistry of rat tissues with an antibody raised against the predicted COOH-terminal sequence confirmed expression of the protein in the small intestine and indicated that the transporter is predominantly expressed in the enterocytes' brush-border membrane. The unusual substrate specificity and close sequence identity with GLUT5 suggest that GLUT7 represents an intermediate between class II GLUTs and the class I member GLUT2. Comparison between these proteins may provide key information as to the structural determinants for the recognition of fructose as a substrate.     

Fructose transporter in human spermatozoa and small intestine is GLUT5.

We recently reported that the glucose transporter isoform, GLUT5, is expressed on the brush border membrane of human small intestinal enterocytes (Davidson, N. O., Hausman, A. M. L., Ifkovits, C. A., Buse, J. B., Gould, G. W., Burant, C. F., and Bell, G. I. (1992) Am. J. Physiol. 262, C795-C800). To define its role in sugar transport, human GLUT5 was expressed in Xenopus oocytes and its substrate specificity and kinetic properties determined. GLUT5 exhibits selectivity for fructose transport, as determined by inhibition studies, with a Km of 6 mM. In addition, fructose transport by GLUT5 is not inhibited by cytochalasin B, a competitive inhibitor of facilitative glucose transporters. RNA and protein blotting studies showed the presence of high levels of GLUT5 mRNA and protein in human testis and spermatozoa, and immunocytochemical studies localize GLUT5 to the plasma membrane of mature spermatids and spermatozoa. The biochemical properties and tissue distribution of GLUT5 are consistent with a physiological role for this protein as a fructose transporter.     

Expression of facilitative glucose transporter in rat liver and choroid plexus

Summary Two isoforms of facilitative glucose transporters (GLUT), namely the erythroid/brain-type GLUT 1 and the liver-type GLUT 2, were demonstrated in native cryostat sections of normal rat liver and brain by immunofluorescence and a very sensitive immunoalkaline phosphatase reaction. Fixation with 0.1% alcoholic periodic acid resulted in an excellent localization of GLUT 2 in liver and GLUT 1 in brain. GLUT 1 in liver, however, could successfully be demonstrated after fixation with 1% alcoholic formaldehyde. GLUT 2 occurred in all hepatocytes as a basolateral membrane protein with a gradient of high expression in the periportal area and a lower one in the perivenous part. The first layer of hepatocytes adjacent to the hepatic vein coexpressed GLUT 1. In addition, GLUT 1 could be detected in the smooth muscle layer of the portal vein and in the apical and lateral plasma membrane of the bile duct epithelium. In brain, GLUT 1 showed a high expression in the microvessels, the ependym and in the basal plasma membrane of choroid plexus epithelial cells. The blood capillaries associated with the choroidal epithelium were, however, negative for GLUT 1. The importance of the new findings in this study for the physiological role of the respective facilitative glucose transport proteins is discussed.     

Hexose transport in preimplantation rabbit blastocysts

Transtrophectodermal 3-0-methyl glucose (3-0MG) transport in the rabbit blastocyst at Days 6 and 7 post coitum was investigated to understand better how the trophectoderm can regulate inner cell mass growth by controlling substrate availability. 3-0MG rapidly traversed the trophectoderm and displayed saturation kinetics (Km = 4.3 +/- 0.5 mM, Vmax = 79 +/- 3.8 nmol.cm-2). The flux of 3-0MG was inhibited nearly 95% by 10(-4) M-phloretin, and only 15% by 10(-4) M-phlorizin. Furthermore, 3-0MG influx was inhibited by cytochalasin B (5 microM) and was unaffected by removal of sodium. The transport system had a high specificity for 2-deoxy-D-glucose and glucose, and a very low specificity for fructose and 4-alpha-methyl glucoside. Western blots probed with a polyclonal antibody to the human erythrocyte glucose transport protein and also with a polyclonal antibody to the C-terminus of the glucose transport protein of the rat brain revealed a broad band with a molecular weight of 55,000. Using immuno-gold labelling techniques, Na(+)-independent glucose transporters were localized to both the apical and basolateral borders of the trophectodermal cell. These results suggest that the mechanism in the trophectoderm responsible for transport of glucose is similar to other sodium-independent glucose transport systems. In addition, 3-0MG influx was unaffected by short-term incubation with progesterone, the progesterone antagonist mifepristone (RU-486), PGF-2 alpha, PGE-2, insulin, or cAMP. Day-7 p.c. embryos also transported hexoses by a similar system because the influx rate and the phlorizin/phloretin sensitivity were the same as in the Day-6 p.c. embryo.     

Expression of facilitative glucose transporter in rat liver and choroid plexus. A histochemical study in native cryostat sections.

Two isoforms of facilitative glucose transporters (GLUT), namely the erythroid/brain-type GLUT 1 and the liver-type GLUT 2, were demonstrated in native cryostat sections of normal rat liver and brain by immunofluorescence and a very sensitive immunoalkaline phosphatase reaction. Fixation with 0.1% alcoholic periodic acid resulted in an excellent localization of GLUT 2 in liver and GLUT 1 in brain. GLUT 1 in liver, however, could successfully be demonstrated after fixation with 1% alcoholic formaldehyde. GLUT 2 occurred in all hepatocytes as a basolateral membrane protein with a gradient of high expression in the periportal area and a lower one in the perivenous part. The first layer of hepatocytes adjacent to the hepatic vein coexpressed GLUT 1. In addition, GLUT 1 could be detected in the smooth muscle layer of the portal vein and in the apical and lateral plasma membrane of the bile duct epithelium. In brain, GLUT 1 showed a high expression in the microvessels, the ependyma and in the basal plasma membrane of choroid plexus epithelial cells. The blood capillaries associated with the choroidal epithelium were, however, negative for GLUT 1. The importance of the new findings in this study for the physiological role of the respective facilitative glucose transport proteins is discussed.     

Acute and chronic signals controlling glucose transport in skeletal muscle.

Glucose transport into muscle cells occurs through facilitated diffusion mediated primarily by the GLUT1 and GLUT4 glucose transporters. These transporter proteins are controlled by acute and chronic exposure to insulin, glucose, muscle contraction, and hypoxia. We propose that acute responses occur through recruitment of pre-formed glucose transporters from an intracellular storage site to the plasma membrane. In contrast, chronic control is achieved by changes in transporter biosynthesis and protein stability. Using subcellular fractionation of rat skeletal muscle, recruitment of GLUT4 glucose transporters to the plasma membrane is demonstrated by acute exposure to insulin in vivo. The intracellular pool appears to arise from a unique organelle depleted of transverse tubule, plasma membrane, or sarcoplasmic reticulum markers. In diabetic rats, GLUT4 content in the plasma membranes and in the intracellular pool is reduced, and incomplete insulin-dependent GLUT4 recruitment is observed, possibly through a defective incorporation of transporters to the plasma membrane. The lower content of GLUT4 transporters in the muscle plasma membranes is reversed by restoration of normoglycemia with phlorizin treatment. In some muscle cells in culture, GLUT1 is the only transporter expressed yet they respond to insulin, suggesting that this transporter can also be regulated by acute mechanisms. In the L6 muscle cell line, GLUT1 transporter content diminishes during myogenesis and GLUT4 appears after cell fusion, reaching a molar ratio of about 1:1 in the plasma membrane. Prolonged exposure to high glucose diminishes the amount of GLUT1 protein in the plasma membrane by both endocytosis and reduced biosynthesis, and lowers GLUT4 protein content in the absence of changes in GLUT4 mRNA possibly through increased protein degradation. These studies suggest that the relative contribution of each transporter to transport activity, and the mechanisms by which glucose exerts control of the glucose transporters, will be key subjects of future investigations.     

Carbohydrate transport in Moniliformis dubius (Acanthocephala). I. The kinetics and specificity of hexose absorption.

The uptakes of 14C-glucose, -2-deoxyglucose, -mannose, -N-acetylglucosamine, -3-0-methylglucose, -fructose, and -galactose by female Moniliformis dubius were nonlinear, saturable functions of hexose concentration. Kinetic and inhibition studies indicated that glucose and 2-deoxyglucose were absorbed via a single common transport locus. Mannose, N-acetylglucosamine, 3-0-methylglucose, fructose, and galactose (in decreasing order of effectiveness) inhibited the uptake of glucose in a completely competitive manner; their absorptions appeared to be mediated by the glucose transport locus and, to some degree, by one or more additional transport systems. Kinetic studies suggested that the apparent inhibitions of 14C-glucose uptake by maltose and glucose-6-phosphate were due to free glucose liberated through the action of surface hydrolases. The uptake of 14C-glucose was also inhibited by salicin, alpha-methylglucoside, and beta-methylglucoside, but not by pentoses, L-hexoses, sugar alcohols, disaccharides (except maltose), gluconic acid, glucuronic acid, phlorizin, or ouabain. Glucose uptake was not Na+-dependent.     

Cellular expression of monocarboxylate transporters in the female reproductive organ of mice: implications for the genital lactate shuttle

The present study examined the cellular localization of monocarboxylate transporters (MCTs), glucose transporters (GLUTs), and some glycolysis-related molecules in the murine female genital tract to demonstrate existence of lactate/pyruvate-dependent energy systems. MCT1, a major MCT subtype, was localized selectively in the ovarian granulosa, oviductal-ciliated cells, and vaginal epithelium; all localizations were associated with intense expressions of glycolytic enzymes. MCT1 was localized in the cell membrane of granulosa cells, including fine processes extending from cumulus cells toward oocytes. The cumulus cells and oocytes showed intense signals for lactate dehydrogenase (LDH)-A and -B, respectively. The basolateral membrane of oviductal-ciliated cells expressed MCT4 as well as MCT1, while adjacent non-ciliated cells contained an intense immunoreactivity for aldolase-C, a glycolytic enzyme. The expression of GLUTs in the ovary was generally weak with an intense expression of GLUT1 only in some vascular endothelia. The oviductal epithelium expressed GLUT1 and GLUT3, respectively, in the basolateral and apical membrane of non-ciliated cells. In the vagina, the basal layers of epithelium were immunolabeled for MCT1 with the entire length of cell membrane, and expressed abundantly both GLUT1 and LDH-A. The findings correspond well with the rich existence of lactate in the genital fluids and strongly suggest the active transport of lactate/pyruvate in the female reproductive tract, which provides favorable conditions for oocytes, sperms, and zygotes.