3-OMG Transport Research Articles

Insulin signaling in rabbit preimplantation embryos

Addition of insulin during In-vitro-culture improves embryonic development by increasing cell proliferation and reducing apoptosis. The insulin receptor (IR) signaling involves two major pathways, the mitogenic-activated protein kinase (MAPK) and the phosphatidylinositol 3-kinase (PI3-K). In muscle and adipocyte cells insulin stimulates glucose uptake mainly by activation of the PI3-K pathway and translocation of glucose transporter (GLUT) 4. We have currently shown the expression of two insulin-responsive GLUT (4, 8) in rabbit embryos. The mechanism of insulin action on embryonic cells, however, is still not clear. In the present study the expression of IR-isoforms (IR-A, IR-B) and the activation of the PI3-kinase and the MAPK signaling pathways were studied in rabbit preimplantation embryos. The rabbit mRNA sequences for the coding region of IR-A and -B were cloned and sequenced. Using specific primer combinations for RT-PCR the expression of both isoforms was analyzed. The IR expression was detectable from the blastocyst stage onwards, with an IR-isoform specific pattern for embryoblast (Em) and trophoblast (Tr) cells. Whereas IR-A was found in both Em and Tr cells, the IR-B-isoform was detectable only in the trophoblast. In cultured blastocysts the effect of insulin stimulation (170 nM) was studied by analysis of Akt and Erk phosphorylation, GLUT4 translocation and measurement of 3-OMG glucose transport. Insulin stimulated the phosphorylation of the mitogenic activated protein kinase Erk1/2, but not the phosphorylation of the PI3-K dependent protein kinase Akt. Glucose uptake and GLUT4 localization were not influenced by insulin stimulation. Activation of the MAPK signaling pathway in the absence of GLUT4 translocation and of a glucose transport response suggest that in the rabbit preimplantation embryo insulin is acting as a growth factor rather than a component of glucose homeostatic control. The possible function of insulin-responsive GLUT isoforms and the cell specific expression of IR-A and -B in rabbit preimplantation embryos need further investigation.

Effects of anticonvulsants on GLUT1-mediated glucose transport in GLUT1 deficiency syndrome in vitro.

Facilitative type-1 glucose transporter (GLUT1) deficiency syndrome is caused by a defect of glucose transport into brain, resulting in an epileptic encephalopathy. Seizures respond effectively to a ketogenic diet, but a subgroup of patients require add-on anticonvulsant therapy or do not tolerate the diet. With the exception of barbiturates, which have been shown to inhibit GLUT1 function, no anticonvulsants have been investigated for possible interactions with GLUT1. Kinetic analyses of (14)C-labeled 3-O-methyl glucose (3OMG) uptake into erythroctes were performed in 11 patients and 30 controls. For in vitro inhibition studies, zero-trans influx of 3OMG (5 mmol/L) into erythrocytes was determined following preincubation with diazepam, carbamazepine, phenytoin, and chloralhydrate. In addition, the effects of ethanol on cell lysis and 3OMG transport into erythrocytes were determined. In patients, mean 3OMG influx was 53% of controls. Ethanol, diazepam, and chloralhydrate significantly inhibited GLUT1 function. Erythrocyte cell lysis was evident at concentrations of 2.5% ethanol. Diazepam, chloralhydrate, and ethanol are inhibitors of GLUT1 function in vitro and might potentiate the effects of GLUT1-mediated glucose transport in patients with GLUT1 deficiency syndrome. In contrast, no inhibitory effects were observed for carbamazepine and phenytoin, indicating that these substances might be preferable for additional seizure control in this disorder.

Influence of food deprivation on the transport of 3-O-methyl-alpha-D-glucose across the isolated ruminal epithelium of sheep.

Recent studies provided evidence that the ruminal epithelium is able to absorb D-glucose even at physiologically low intraruminal concentrations. To elucidate whether ruminal D-glucose transport shows adaptive responses during food deprivation, transport of 3-0-methyl-alpha-D-glucose (3-OMG), a hardly metabolizable D-glucose analogue, was measured in isolated ruminal epithelia obtained from hay-fed or food-deprived adult sheep. In both groups, a significant net absorption of 3-OMG to the serosal side (in vivo: blood side oriented) could be detected at 3-OMG concentrations between 0.25 mM and 5 mM. Net absorption of 3-OMG was abolished by mucosal (in vivo: lumen side oriented) addition of phlorizin, an inhibitor of the sodium glucose-linked transporter 1 (SGLT-1). Net absorption of 3-OMG followed Michaelis-Menten kinetics, but apparent affinity and maximal transport capacity were lower in epithelia obtained from food-deprived sheep. In contrast to the decrease of the (secondary) active 3-OMG transport, serosal-to-mucosal permeation of 3-OMG increased after food deprivation, suggesting an elevated passive 3-OMG transfer. It is concluded that the altered transport characteristics are either part of a global energy-sparing process during food deprivation (i.e., a lowered activity of the Na+/K+-ATPase) or result from specific down-regulation of SGLT-1.

MRNA Expression and Transport Characterization of Conditionally Immortalized Rat Brain Capillary Endothelial Cell Lines; a New in vitro BBB Model for Drug Targeting

Brain capillary endothelial cell lines (TR-BBB) were established from a recently developed transgenic rat harboring temperature-sensitive simian virus 40 (ts SV 40) large T-antigen gene (Tg rat) and used to characterize the endothelial marker, transport activity, and mRNA expression of transporters and tight-junction strand proteins at the blood-brain barrier (BBB). These cell lines expressed active large T-antigen and grew well at 33°C with a doubling-time of about 22-31 hr, but did not grow at 39°C. TR-BBBs expressed the typical endothelial marker, von Willebrand factor, and exhibited acetylated low-density lipoprotein uptake activity. Although the γ-glutamyltranspeptidase activity in TR-BBBs was ~13% of that of the brain capillary fraction of a normal rat, it was localized in the apical side, suggesting that it reflects the functional polarity of the in vivo BBB. The mRNA of tight-junction strand proteins such as claudine-5, occludin, and junctional adhesion molecule are expressed in TR-BBB 13. Drug efflux transporter, P-glycoprotein, with a molecular weight of 170 kDa was expressed in all TR-BBBs and mdr 1a, mdr 1b, and mdr 2 mRNA were detected in TR-BBBs using RT-PCR. Moreover, mrp1 mRNA was expressed in all TR-BBBs. Influx transporter, GLUT-1, expressed at 55 kDa was revealed by Western blot analysis. It had 3-O-memyl-D-glucose (3-OMG) uptake activity which was concentration-dependent with a Michaelis-Menten constant of 9.86 ± 1.20 mM. The mRNA of large neutral amino acid transporter, which consists of LAT-1 and 4F2hc was expressed in TR-BBBs. In conclusion, the conditionally immortalized rat brain capillary endothelial cell lines (TR-BBB) had endothelial makers, expressed mRNA for tight-junction strand proteins and the influx and efflux transporters and produced GLUT-1, which is capable of 3-OMG transport activity.

Comparative study of the small intestinal monosaccharide and amino acid transport as a function of age

Total proline and 3-oxy-methyl-D-glucose transport across jejunum and ileum of chicken aged 1 day to 100 days has been studied using a perfusion method in vivo and was determined as a function of body weight in the 7 age groups studied. The jejunum and ileum showed a progressive decrease in the 3-OMG transport with age. The total transport of L-proline increased in the first week after birth and decreased thereafter. The jejunal transport of proline is smaller than 3-OMG in the first day of life but shows a similar behaviour thereafter. The ileum and jejunum showed similar capacity of transport of L-proline.

Glucose transport in carp erythrocytes: individual variation and effects of osmotic swelling, extracellular pH and catecholamines

The characteristics of the uptake of 3-O-methyl-d-glucose (3-OMG) by carp (Cyprinus carpio) erythrocytes were studied in vitro with tracer methods. There is large individual variation in the permeability of the carp erythrocyte membrane to 3-OMG. Although transport is inhibited by cytochalasin B and phloretin, the lack of saturation kinetics for transport in a physiologically relevant concentration range suggests either that a glucose transporter does not exist or that its affinity for glucose is extremely low. The marked increase in transport after osmotic swelling and the inhibition of swelling-induced glucose transport by cytochalasin B suggest that the glucose transport pathway in carp erythrocytes (both in isotonic and hypotonic conditions) may be similar to the volume-activated channel described for flounder erythrocytes. 3-OMG transport across the carp erythrocyte membrane is increased by catecholamines by a mechanism independent of the catecholamine-induced cell swelling.

Pathways for glucose transport in type II pneumocytes freshly isolated from adult guinea pig lung.

Previous in vivo studies of sugar transport across the mature pulmonary epithelium have provided evidence for the existence of a specific phlorizin-inhibitable, sodium-dependent transport process for D-glucose, although no direct evidence for the cellular location of this transport system in fresh cells has been shown to date. With the use of elastase digestion and lectin agglutination, a pure preparation of type II alveolar epithelial cells was isolated from adult guinea pig lung. This preparation always contained >90% type II cells and typically showed approximately 85% cell viability 2-3 h after the isolation procedure had begun. At 37 degrees C, cells showed specific [3H]phlorizin binding that was attenuated by D-glucose and completely abolished by sodium replacement. Substantial accumulation of the hexose [14C]methyl alpha-D-glucopyranoside (14C-labeled AMG), a substrate specific for the sodium-dependent glucose cotransporter was found in the presence of extracellular sodium; this accumulation above equilibrium was abolished on removal of sodium, addition of phlorizin, or in the presence of a saturating concentration (69 mM) of D-glucose. The apparent inhibition constant (Ki) for glucose inhibition of AMG uptake was 0.4 mM and for phlorizin, 0.5 microM. The Hill plot of sodium activation of AMG uptake gave a coefficient of 2.8, suggesting cooperativeness between sodium and AMG transport. 3-O-[14C]methyl-D-glucose (3-OMG) transport was also blocked by phlorizin. Phloretin, in the presence of phlorizin, slowed the initial rate of entry but did not affect the equilibrium that was attained in the presence of phlorizin alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Maintenance of intestinal nutrient transport during hibernation.

We studied nutrient absorption across the brush-border membrane in jejunal tissues from active 13-lined ground squirrels and in hibernating squirrels that had not eaten for at least 6 wk. Body weights and jejunal wet weights per centimeter were significantly reduced in the hibernators. Rates of total and carrier-mediated uptake of 3-O-methylglucose (3-OMG) per centimeter intestinal length were greater in the active squirrels, but 3-OMG uptakes per milligram intestinal weight were greater in the hibernators, due to a significantly greater maximum rate of uptake (Jmax) per milligram with no differences in apparent Michaelis constant (Km). Passive permeabilities to L-glucose were similar in both groups. Total uptake of L-proline per centimeter was greater in active squirrels, but total proline uptake per milligram was greater in the hibernators due to a significantly greater Jmax per milligram with no difference in apparent Km. Na(+)-independent proline uptake accounted for a greater proportion of total proline uptake in active compared with hibernating squirrels. As a consequence, Na(+)-dependent proline uptake was greater in the hibernators when uptake was normalized either to intestinal length or intestinal weight. Thus hibernation is associated with an increase in the Jmax per milligram for 3-OMG and proline transport, as well as a shift in the Na+ dependency of proline uptake. We conclude that nutrient absorption is selectively retained in mammalian hibernators to maintain transport function after the extended winter fast.

Inhibitors of protein synthesis cause increased hexose transport in cultured human fibroblasts by a mechanism other than transporter translocation.

We have investigated the effect of various inhibitors of protein synthesis on hexose transport in human skin fibroblasts using 2-deoxy-D-glucose (2-DG) and 3-0-methyl-D-glucose (3-OMG) to measure hexose transport. Exposure of glucose-fed, serum-free cultures to cycloheximide (CHX) (50 micrograms/ml) for 6 h resulted in increased 2-DG transport (3.81 +/- .53 vs. 6.62 +/- .88 nmoles/mg protein/2 min; n = 9) and 3-OMG transport (1.36 +/- .66 vs. 3.18 +/- .83 nmoles/mg protein/30 sec; n = 4) in the CHX exposed group. Under these conditions inhibition of protein synthesis was greater than 90%. This CHX induced transport increase was time dependent (approaching maximum within 1 h of exposure to CHX) and related to an increase in the Vmax of hexose transport in the CHX exposed group (18.4 +/- 2.4 vs. 4.8 +/- 1.1 nmoles 2-DG/mg protein/min) with no difference in the transport Km (1.55 +/- .63 vs. 2.92 +/- .59 mM). Further, the CHX induced increase in hexose transport was reversible. Exposure of human fibroblasts to inhibitors of protein synthesis with different mechanisms of action (e.g., puromycin, pactamycin, or CHX) all generated hexose transport increases in a concentration-dependent fashion correlating with their increasing inhibitory effects on protein synthesis. Nucleotidase enriched (i.e., plasma membrane) fractions of control and CHX-exposed cells showed no differences in D-glucose inhibitable cytochalasin B binding activity. Further, quantitative Western analysis of nucleotidase enriched fractions indicated CHX exposure resulted in no significant increase in glucose transporter mass compared with control plasma membrane fractions. Glucose deprived cells, however, which exhibited increased sugar transport comparable to the CHX-exposed group, did show increased glucose transporter mass in the plasma membrane fraction. The data indicate that inhibitors of protein synthesis can cause a significant elevation in hexose transport and that the hexose transporter mass in the isolated plasma membrane fractions did not reflect the whole cell transport change. It is suggested that a mechanism other than glucose transporter translocation to the plasma membrane may be involved in causing this sugar transport increase.

Transport of 1,5-anhydro-D-glucitol into human polymorphonuclear leukocytes.

1,5-Anhydro-D-glucitol (AG) is one of the main polyols and its structure resembles glucose. It has been proposed that decreased serum AG concentrations in diabetic patients are a novel indicator of diabetic metabolic derangement. However, the pathway of AG metabolism still remains to be clarified. In this study we investigated the transport of AG into human polymorphonuclear leukocytes (PMNLs) isolated from healthy volunteers and found that 0.1 mM 3-O-methy-D-glucose (3OMG) was equilibrated with a half saturation time of 10 s, while the uptake rate of AG was much slower. The concentration dependence of AG uptake revealed that the AG transport velocity reached a plateau, with a Km of about 50 mM and Vmax of about 25 nmol/min/10(7) cells. Transport of 14C-labeled 3OMG was inhibited by unlabeled D-glucose or AG in a dose-dependent manner. The mean inhibition constant (Ki) for D-glucose and for AG were 1.06 and 4.93 mM, respectively. Cytochalasin B (20 microM) inhibited 3OMG transport by 90% but AG transport by only 50%. S/V for 14C-labeled AG transport plotted against the concentration of unlabeled 3OMG showed a non-linear and biphasic pattern. These results suggest that AG influx into PMNLs is mediated not only by the cytochalasin B-sensitive glucose transport system but also via another facilitated transport system.

Enhancement of Glucose transport in clone 9 cells by exposure to alkaline pH: Studies on potential mechanisms

Incubation of a nontransformed rat liver cell line, Clone 9, at pH 8.5 resulted in an approximately 16-fold stimulation of cytochalasin B-inhibitable 3-O-methylglucose (3-OMG) transport, an effect that was independent of the presence of serum. Exposure to 100 ng/ml 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulated 3-OMG uptake, and the enhancement was not additive to that produced by incubation at pH 8.5. In cells "depleted" of protein kinase C activity by a 20-hr exposure to TPA, however, the stimulation of 3-OMG transport in response to incubation at alkaline pH was still fully demonstrable. In control and alkaline pH-exposed cells, the inhibition of 3-OMG uptake by cytochalasin B was consistent with a single-site ligand binding model (K1 approximately 10(-7) M). Northern blot analysis demonstrated the presence of only the human erythrocyte/rat brain/HepG2 cell glucose transporter-mRNA isoform (EGT), and the abundance of this mRNA was unchanged following exposure to alkaline pH. Immunoblot analysis, using polyclonal antibodies directed against the carboxy-terminal dodecapeptide of EGT, demonstrated an approximately 2.0-fold increase in the abundance of transporters in partially purified plasma membrane fractions following incubation at pH 8.5, while EGT abundance was unchanged in whole-cell extracts. It is concluded that the stimulation of glucose transport in response to incubation of Clone 9 cells at alkaline pH does not require the presence of serum or activation of protein kinase C, and that the response is at least in part mediated by an increase in the number of glucose transporters in the plasma membrane.

D-Glucose permeability in river lamprey ( lampetra fluviatilis) and carp ( cyprinus carpio) erythrocytes

1. 1. The transport of 3-O- methyl- d- glucose (3-OMG) in lamprey and carp erythrocytes was studied. 2. 2. In lamprey erythrocytes the half-time for the equilibration of 3-OMG was fast, approx. 8 min. In contrast, the erythrocytes of carp were almost impermeable to 3-OMG, with a half-time for equilibration of 14.2 hr. 3. 3. 3-OMG was taken up by lamprey erythrocytes both by facilitated diffusion and simple diffusion. The presence of carrier-mediated transport was indicated by saturation kinetics and by inhibition by phloretin. The K m and V m of the saturable component of 3-OMG transport were 1.6mmol/l and 12.4mmol/kg packed cells/hr. 4. 4. Since the 3-OMG transport in carp erythrocytes showed no sign of saturation kinetics, it appears to proceed by simple diffusion only. 5. 5. These results suggest that, as for most other teleost fish so far studied, low glucose permeability is insufficient to maintain normal energy metabolism in carp erythrocytes. In contrast, in agnathans facilitated glucose transport seems to be quite effective.

Glucose transport in fish erythrocytes: variable cytochalasin-B-sensitive hexose transport activity in the common eel (Anguilla japonica) and transport deficiency in the paddyfield eel (Monopterus albus) and rainbow trout (Salmo gairdneri)

Erythrocytes from individual common eels (Anguilla japonica Temminck and Schlegel) exhibited widely variable initial rates of cytochalasin-B-sensitive 3-O-methyl-D-glucose (3-OMG) zero-trans influx, in the range of 0-19.5 mmol l-cells-1 h-1 (5 mmol l-1 extracellular concentration at 20 degrees C, 50 animals tested). Storage of cells at 4 degrees C in a glucose-containing medium for up to 72 h had no effect on 3-OMG uptake, and there was no correlation between the sugar permeabilities of erythrocytes from different fish and intracellular ATP levels. Adrenaline and noradrenaline increased cytochalasin-B-sensitive 3-OMG transport activity; half-maximal stimulation occurred at catecholamine concentrations in the region of 1 mumol l-1. This catecholamine-induced stimulation of sugar transport appeared to be independent of the basal cytochalasin-B-sensitive 3-OMG permeability of the cells. Kinetically, catecholamines increased the Vm of transport without changing the apparent Km (approx. 1.4 mmol l-1). Saturable 3-OMG influx was inhibited by phloretin, D-glucose, D-deoxyglucose and D-galactose, but not by D-fructose and L-glucose. Transporter stereoselectivity was confirmed by direct measurements of D- and L-glucose uptake. Erythrocytes from two other fish species, Monopterus albus Richardson (paddyfield eel) and Salmo gairdneri Richardson (rainbow trout), unlike those from the common eel, were uniformly deficient with respect to cytochalasin-B-sensitive 3-OMG and D-glucose transport activity. Catecholamines had no effect on sugar uptake in these species.

Relationship between insulin stimulation and endogenous regulation of 2‐deoxyglucose uptake in 3T3‐L1 adipocytes

The occurrence of the endogenous regulatory response to high rates of 2-deoxyglucose (2-DG) uptake, as previously described for C6 glioma cells during incubation with 2 mM 2-DG (Lange et al.: J. Cell. Physiol., 1989), was studied in 3T3-L1 preadipocytes and adipocytes, and the influence of insulin on this endogenous uptake regulation was examined. In contrast to 3T3-L1 preadipocytes, insulin-sensitive differentiated 3T3-L1 adipocytes displayed the time-dependent cyclic pattern of 2-DG uptake rates characteristic of the membrane-limited and endogenously regulated cellular state of hexose utilization. Although insulin induced a threefold stimulation of 2-DG tracer uptake in adipocytes, the hormone did not additionally stimulate the uptake rates or affect the periodic response: maximum and minimum levels of uptake remained unchanged. Scanning electron microscopy (SEM) revealed that the acquirement of the differentiated state is accompanied by a conspicuous transformation of the smooth surface of undifferentiated 3T3-L1 cells into a surface covered by numerous microvilli of uniform size and appearance. Treatment with insulin (10 mU/ml; 10 minutes) converted these microvilli into voluminous saccular membrane protrusions of the same type as had been formed during incubation of 3T3-L1 adipocytes with 2 mM 2-DG, and which have previously been shown to be involved in the endogenous uptake regulation of C6 glioma cells (Lange et al.: J. Cell. Physiol., 1989). These insulin-induced saccated membrane areas appeared to become integrated into the cell surface. Accordingly, insulin treatment caused a twofold increase of the intracellular distribution space of 3-O-methylglucose (3-OMG) in 3T3-L1 adipocytes. This insulin-induced increase of the 3-OMG distribution space exhibited the same time (t1/2 = 2-2.5 minutes) and dose dependence (EC50 = 20 nM) as the insulin-induced stimulation of 3-OMG transport. Glucose deprivation during the differentiation period inhibited the outgrowth of microvilli from the cell surface. Glucose starvation (18 hours at less than 0.5 mM) induced a conspicuous reduction of the length of microvilli on differentiated 3T3-L1 cells. In this state, the stalks of the microvilli are almost invisible and the enlarged spherical tips of the microvilli (with an average diameter of 370 nm compared to 230 nm of fed cells) appeared to protrude directly out of the cell surface. Starvation-induced shortening of microvilli was accompanied by a threefold increase of the basal 3-OMG transport rate and a greater than twofold increase of the intracellular 3-OMG distribution space as compared to fed cells (10 mM; 18 hours).(ABSTRACT TRUNCATED AT 400 WORDS)

Skeletal muscle glucose transport in obese Zucker rats after exercise training.

Exercise training has been found to reduce the muscle insulin resistance of the obese Zucker rat (fa/fa). The purpose of the present study was to determine whether this reduction in muscle insulin resistance was associated with an improvement in the glucose transport process and if it was fiber-type specific. Rats were randomly assigned to a sedentary or training group. Training consisted of treadmill running at 18 m/min up an 8% grade, 1.5 h/day, 5 days/wk, for 6-8 wk. The rate of muscle glucose transport was assessed in the absence of insulin and in the presence of a physiological (0.15 mU/ml), a submaximal (1.50 mU/ml), and a maximal (15.0 mU/ml) insulin concentration by determining the rate of 3-O-methyl-D-glucose (3-OMG) accumulation during hindlimb perfusion. The average 3-OMG transport rate of the red gastrocnemii (fast-twitch oxidative-glycolytic fibers) was significantly higher in the trained compared with the sedentary obese rats in the absence of insulin and in the presence of the three insulin concentrations. Significant improvements in 3-OMG transport were also observed in the plantarii (mixed fibers) of trained obese rats in the presence of 0, 0.15, and 15.0 mU/ml insulin. Training appeared to have little effect on the insulin-stimulated 3-OMG transport of the soleus (slow-twitch oxidative fibers) or white gastrocnemius (fast-twitch glycolytic fibers). The results suggest that the improvement in the muscle insulin resistance of the obese Zucker rat after moderate endurance training was associated with an improvement in the glucose transport process but that it was fiber-type specific.

Analysis of protein-mediated 3-O-methylglucose transport in rat erythrocytes: rejection of the alternating conformation carrier model for sugar transport

3-O-Methylglucose (3OMG) transport in rat erythrocytes (RBCs) is mediated by a low-capacity, facilitated diffusion-type process. This study examines whether the characteristics of sugar transport in rat RBCs are consistent with the predictions of two diametric, theoretical mechanisms for sugar transport. The one-site carrier describes a transport mechanism in which sugar influx and efflux substrate binding sites are mutually exclusive. The two-site carrier describes a transport mechanism in which sugar influx and efflux substrate binding sites can exist simultaneously but may interact in a cooperative fashion when occupied by substrate. Michaelis and velocity parameters for saturable 3OMG transport in rat erythrocytes at 24 degrees C were obtained from initial rate measurements of 3OMG transport. The results are incompatible with the predictions of the one-site carrier but are consistent with the predictions of a symmetric two-site carrier, displaying negligible cooperativity between substrate binding sites. This allows reduction of the two-site carrier transport equations to a form containing fewer constants than the one-site carrier equations without limiting their predictive success. While the available evidence does not prove that rat erythrocyte sugar transport is mediated by a two-site mechanism, we conclude that adoption of the formally more complex one-site model for sugar transport in rat erythrocytes is unnecessary and unwarranted. Counterflow experiments have also been performed in which the time course of radiolabeled 3OMG uptake is measured in cells containing saturating levels of 3OMG. The results of these experiments are consistent with the hypothesis [Naftalin et al. (1985) Biochim. Biophys. Acta 820, 235-249] that exchange of sugar between intracellular compartments (cell water and hemoglobin) can be rate limiting for transport under certain conditions.

Intestinal adaptation to diabetes. Altered Na-dependent nutrient absorption in streptozocin-treated chronically diabetic rats.

To examine the pattern and mechanisms of enhanced intestinal nutrient absorption in diabetes, we measured intestinal transport of 3-O-methylglucose (3OMG), l-alanine (ALA), and SO4 in male Lewis rats made diabetic with streptozocin. Diabetes enhanced 3OMG absorption fivefold in ileum and threefold in jejunum; ALA absorption increased twofold in ileum but not at all in jejunum; ileal SO4 transport was unaffected. Increases in 3OMG and ALA transport were due solely to increases in maximum velocity. The enhancement of ileal glucose absorption was half-maximal in 40-45 d, could be reversed by 10 d of treatment with insulin and did not result from adrenergic denervation. The density of glucose carriers per milligram brush border protein (measured as [3H]phlorizin binding sites) was not altered but there was a sixfold increase in the number of glucose-inhibitable [3H]phlorizin-binding sites in the intact epithelium. Generalized mucosal hypertrophy accounted for less than 30% of this increase. We conclude that the intestine adapts to streptozocin-induced diabetes through recruitment of additional brush border carriers for sugar, probably in the midvillus-to-crypt region.

Hexose transport in L6 rat myoblasts. II. The effects of sulfhydryl reagents

The importance of sulfhydryl groups for hexose transport in undifferentiated L6 rat myoblasts was investigated. N-ethylmaleimide (NEM) and p-chloromercuribenzenesulfonic acid (pCMBS) inhibited 2-deoxy-D-glucose (2-DOG) transport in a time and concentration-dependent manner. The inhibition produced by both reagents was virtually complete within 5 min, although neither reagent inhibited transport more than 70-80% regardless of the concentrations or incubation times used. Furthermore, the inhibition of 2-DOG transport by pCMBS or NEM could not be prevented by simultaneous preincubation of cells with 20 mM D-glucose or 20 mM 2-DOG. This suggests that sulfhydryl groups required for transport are separate from the hexose binding and transport site. By comparing the effects of the membrane impermeant pCMBS to those of the membrane permeant NEM, cell surface sulfhydryl groups were shown to be essential for hexose binding and transport. In contrast to the inhibition of 2-DOG transport, pCMBS and NEM had much less of an effect on 3-O-methyl-D-glucose (3-OMG) transport. For example, 1 mM NEM inhibited 2-DOG transport by 66%, whereas 3-OMG transport was inhibited by only 7%. This supports the suggestion that these hexose analogues may be transported by different carriers. Kinetic analysis of transport shows that treatment of cells with 1 mM NEM or 1 pCMBS results in inactivation of the high affinity 2-DOG transport system, whereas the low affinity transport system is unaffected. 3-OMG is preferentially transported by the low affinity system.

Isolation and characterization of hexose transport mutants in L6 rat myoblasts.

A method for the selection and isolation of hexose transport mutants in undifferentiated rat myoblast L6 cells is reported; 2-deoxy-D-glucose (2-DOG)-and 2-deoxy-2-fluoro-D-glucose (2FG)-resistant mutants were selected after mutagenization of L6 cells with ethyl methanesulfonate. Of these, D18 and D23 (selected with 0.1 mM 2-DOG) and F72 and F76 (selected with 0.1 mM 2FG) exhibited the lowest hexose transport activity. Uptake of 0.06 mM 2-DOG, 2FG, or 3-O-methyl-D-glucose (3-OMG) by mutants grown in fructose medium supplemented with 0.05 mM 2FG was about four- to five-fold lower than the parental L6 cells. These mutants contain normal levels of ATP and glycolytic enzyme activities. They also exhibit normal transport activities for alpha-aminoisobutyric acid and fructose. Furthermore, hexose transport was observed to be decreased in plasma membrane vesicles prepared from these mutants. Kinetic analysis of 2-DOG and 3-OMG transport in mutant F72 demonstrated that the Vmax for 2-DOG uptake was significantly reduced, whereas the Vmax for 3-OMG transport was not affected. In all cases, the affinity for these hexose analogues was unaffected. In addition mutant F72 was found to be only slightly affected by treatment with various energy inhibitors and sulfhydryl reagents. The results suggest that this mutant is defective in, or has low levels of, a plasma membrane component(s) involved in the high-affinity hexose transport system.

Monosaccharide transport into hemocytes of a sipunculan worm Themiste dyscrita.

The hemerythrin-containing blood cells, or hemocytes, of the sipunculan worm Themiste dyscrita were found to have a stereospecific and nonconcentrative monosaccharide transport system. The transport system transferred both D-glucose and 3-O-methyl-D-glucose (3-OMG), and transport into cells by this system was rapid, reaching 50% equilibrium in approximately 20 s at 10 degrees C with an initial concentration gradient of 0.1 mM; the contribution to total uptake by simple diffusion was very small. 3-OMG uptake showed saturation kinetics with a low half-saturation constant (Km less than or equal to 0.1 mM). The uptake of labeled 3-OMG by the hemocytes was strongly inhibited by unlabeled 3-OMG, 2-deoxy-D-glucose, alpha- and beta-D-glucose, D-galactose, and D-mannose. It was moderately inhibited by D-xylose, only slightly by alpha-methyl-D-glucoside and D-fructose, and uninhibited by sucrose, L-glucose, or D-sorbitol. Phloretin was more potent than phloridzin in blocking entry of 3-OMG. Cytochalasin B did not bind tightly to the T. dyscrita transporter and was not a potent inhibitor of transport; it half-maximally inhibited 3-OMG transport at 0.1 mM. Therefore, despite some differences the data suggest functional similarities in the mechanism of monosaccharide transport into blood cells of mammals and this invertebrate.