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Evidence for the Crabtree effect was first reported by H. Crabtree in 1929 and is defined as the glucose-induced decrease of cellular respiratory flux. This effect was observed in tumor cells and was not detected in most non-tumor cells. A number of hypotheses on the mechanism underlying the Crabtree effect have been formulated. However, to this day, no consensual mechanism for this effect has been described. In a previous study on isolated mitochondria, we have proposed that fructose-1,6-bisphosphate (F1,6bP), which inhibits the respiratory chain, induces the Crabtree effect. Using whole cells from the yeast Saccharomyces cerevisiae as a model, we show here not only that F1,6bP plays a key role in the process but that glucose-6-phosphate (G6P), a hexose that has an effect opposite to that of F1,6bP on the regulation of the respiratory flux, does as well. Thus, these findings reveal that the Crabtree effect strongly depends on the ratio between these two glycolysis-derived hexose phosphates. Last, in silico modeling of the Crabtree effect illustrated the requirement of an inhibition of the respiratory flux by a coordinated variation of glucose-6-phosphate and fructose-1,6-bisphosphate to fit the respiratory rate decrease observed upon glucose addition to cells. In summary, we conclude that two glycolysis-derived hexose phosphates, G6P and F1,6bP, play a key role in the induction of the Crabtree effect.

Glucose is required for cell growth and proliferation. The MondoA·Mlx transcription factor is glucose-responsive and accumulates in the nucleus by sensing glucose 6-phosphate. One direct and glucose-induced target of MondoA·Mlx complexes is thioredoxin-interacting protein (TXNIP). TXNIP is a potent negative regulator of glucose uptake, and hence its regulation by MondoA·Mlx triggers a feedback loop that restricts glucose uptake. This feedback loop is similar to the "hexose transport curb" first described almost 30 years ago. We show here that MondoA responds to the non-glucose hexoses, allose, 3-O-methylglucose, and glucosamine by accumulating in the nucleus and activating TXNIP transcription. The metabolic inhibitor 3-bromopyruvate blocks the transcriptional response to allose and 3-O-methylglucose, indicating that their metabolism, or a parallel pathway, is required to stimulate MondoA activity. Our dissection of the hexosamine biosynthetic pathway suggests that in addition to sensing glucose 6-phosphate, MondoA can also sense glucosamine 6-phosphate. Analysis of glucose uptake in wild-type, MondoA-null, or TXNIP-null murine embryonic fibroblasts indicates a role for the MondoA-TXNIP regulatory circuit in the hexose transport curb, although other redundant pathways also contribute.

Plant pathogenic fungi use a wide range of different strategies to gain access to the carbon sources of their host plants. The hemibiotrophic maize pathogen Colletotrichum graminicola (teleomorph Glomerella graminicola) colonizes its host plants, and, after a short biotrophic phase, switches to destructive, necrotrophic development. Here we present the identification of five hexose transporter genes from C. graminicola, CgHXT1 to CgHXT5, the functional characterization of the encoded proteins, and detailed expression studies for these genes during vegetative and pathogenic development. Whereas CgHXT4 is expressed under all conditions analyzed, transcript abundances of CgHXT1 and CgHXT3 are transiently up-regulated during the biotrophic phase, and CgHXT2 and CgHXT5 are expressed exclusively during necrotrophic development. Analyses of the encoded proteins characterized CgHXT5 as a low-affinity/high-capacity hexose transporter with a narrow substrate specificity for glucose and mannose. In contrast, CgHXT1 to CgHXT3 are high affinity/low capacity transporters that also accept other substrates, including fructose, galactose, or xylose. CgHXT4, the largest of the identified proteins, has only little transport activity and may function as a sugar sensor. Phylogenetic studies revealed hexose transporters closely related to the five CgHXT proteins also in other pathogenic fungi suggesting conserved functions of these proteins during fungal pathogenesis.

In numerous cell types, tumoral cells, proliferating cells, bacteria, and yeast, respiration is inhibited when high concentrations of glucose are added to the culture medium. This phenomenon has been named the "Crabtree effect." We used yeast to investigate (i) the short term event(s) associated with the Crabtree effect and (ii) a putative role of hexose phosphates in the inhibition of respiration. Indeed, yeast divide into "Crabtree-positive," where the Crabtree effect occurs, and "Crabtree-negative," where it does not. In mitochondria isolated from these two categories of yeast, we found that low, physiological concentrations of glucose 6-phosphate and fructose 6-phosphate slightly (20%) stimulated the respiratory flux and that this effect was strongly antagonized by fructose 1,6-bisphosphate (F16bP). On the other hand, F16bP by itself was able to inhibit mitochondrial respiration only in mitochondria isolated from a Crabtree-positive strain. Using permeabilized spheroplasts from Crabtree-positive yeast, we have shown that the sole effect observed at physiological concentrations of hexose phosphates is an inhibition of oxidative phosphorylation by F16bP. This F16bP-mediated inhibition was also observed in isolated rat liver mitochondria, extending this process to mammalian cells. From these results and taking into account that F16bP is able to accumulate in the cell cytoplasm, we propose that F16bP regulates oxidative phosphorylation and thus participates in the establishment of the Crabtree effect.

Whereas many reports substantiated the suitability of using [2-(13)C]glycerol and Mass Isotoper Distribution Analysis for gluconeogenesis, the use of [(13)C]glycerol had been shown to give lower estimates of gluconeogenesis (GNG). The reason for the underestimation has been attributed to asymmetric isotope incorporation during gluconeogenesis as well as zonation of gluconeogenic enzymes and a [(13)C]glycerol gradient across the liver. Since the cycling of glycerol carbons through the pentose cycle pathways can introduce asymmetry in glucose labeling pattern and tracer dilution, we present here a study of the role of the pentose cycle in gluconeogenesis in Fao cells. The metabolic regulation of glucose release and gluconeogenesis by insulin was also studied. Serum-starved cells were incubated for 24 h in Dulbecco's modified Eagle's media containing 1.5 mm [U-(13)C]glycerol. Mass isotopomers of whole glucose from medium or glycogen and those of the C-1-C-4 fragment were highly asymmetrical, typical of that resulting from the cycling of glucose carbon through the pentose cycle. Substantial exchange of tracer between hexose and pentose intermediates was observed. Our results offer an alternative mechanism for the asymmetrical labeling of glucose carbon from triose phosphate. The scrambling of (13)C in hexose phosphate via the pentose phosphate cycle prior to glucose release into the medium is indistinguishable from dilution of labeled glucose by glycogen using MIDA and probably accounts for the underestimation of GNG using (13)C tracer methods.

Infectious Neisseria gonorrhoeae make relatively large lipooligosaccharides (LOS) that structurally resemble human glycosphingolipids. MS11mkC is an LOS variant of N. gonorrhoeae strain MS11 which was isolated from men at the onset of dysuria (Schneider, H., Griffiss, J. M., Boslego, J. W., Hitchcock, P. J., Zahos, K. M., and Apicella, M. A. (1991) J. Exp. Med. 174, 1601-1605). Delayed extraction matrix-assisted laser desorption and ionization and electrospray ionization mass spectrometry of O-deacylated MS11mkC LOS produced ions consistent with known LOS which have lacto-N-neotetraose (Galbeta1--4GlcNAcbeta1--3Galbeta1--4Glc; paraglobosyl; monoclonal antibodies (mAbs) 1B2(+) and 06B4(+)) and GalNAc--lacto-N-neotetraose (gangliosyl; mAb 1-1-M+) oligosaccharides. Ion peaks for a larger LOS which also bound mAb 1B2 indicated the addition of a hexose (+162 Da) to gangliosyl LOS or the addition of a hexose and a N-acetylhexosamine (+365 Da) to paraglobosyl LOS. Analysis of HF-treated and O-deacylated LOS revealed three major components present in a phosphoethanolamine (PEA)0 and a PEA1 series. Digestion of MS11mkC LOS by beta-N-acetylhexosaminidase and beta-galactosidase, alone and sequentially, combined with mAb binding patterns, confirmed the presence of a nonreducing terminal repeating LacNAc ((Galbeta1--4GlcNAc)2) on the largest LOS, rather than a parallel oligosaccharide structure.

UhpT, the sugar phosphate transporter of Escherichia coli, acts to exchange internal inorganic phosphate for external hexose 6-phosphate. Because of this operational asymmetry, we studied variants in which right-side-out (RSO) or inside-out (ISO) orientations could be analyzed independently to ask whether the inward- and outward-facing UhpT surfaces have different substrate specificities. To study the RSO orientation, we constructed a histidine-tagged derivative, His10K291C/K294N, in which the sole external tryptic cleavage site (Lys294) had been removed. Functional assay as well as immunoblot analysis showed that trypsin treatment of proteoliposomes containing His10K291C/K294N led to loss of about 50% of the original population, reflecting retention of only the RSO population. To study the ISO orientation, we used a His10V284C derivative, in which a newly inserted external cysteine (Cys284) conferred sensitivity to the thiol-reactive agent, 3-(N-maleimidylpropionyl)biocytin. In this case, 3-(N-maleimidylpropionyl)biocytin treatment of proteoliposomes containing His10V284C gave about a 60% loss of activity, and immunodetection of biotin showed parallel modification of an equivalent fraction of the original population. Together, such findings indicate that the UhpT RSO and ISO orientations are in about equal proportion in proteoliposomes and that a single population can be generated by exposure of these derivatives to the appropriate agent. This allowed us to study proteoliposomes with UhpT functioning in RSO orientation (His10K291C/K294N) or ISO orientation (His10V284C) with respect to the kinetics of glucose 6-phosphate transport by phosphate-loaded proteoliposomes and also the inhibitions found with 2-deoxy-glucose 6-phosphate, mannose 6-phosphate, galactose 6-phosphate, fructose 6-phosphate, and inorganic phosphate. We found no significant differences in the behavior of UhpT in its different orientations, indicating that the transporter possesses an overall functional symmetry.

The dual lumenaly and vascularly perfused small intestine was used to determine the mechanism by which cholecystokinin octapeptide (CCK-8) decreases the rate of glucose absorption. With CCK-8 in the vascular perfusate the rate of 3-O-methyl-D-glucose absorption decreased, whereas the rate of D-fructose absorption was unaffected. The substrate pool size within the tissue during steady-state transport, in the presence and absence of CCK-8, was estimated by compartmental analysis of the 3-O-methyl-D-glucose washout into the vascular bed. When CCK-8 was included in the vascular perfusate, the absorptive cell pool size decreased when compared with untreated tissue. Both the steady-state hexose absorption data and the washout studies indicated that the locus of action of CCK-8 was the SGLT1 transporter located in the brush-border membrane. The SGLT1 protein abundance in isolated brush-border membranes, as quantified by Western blotting, showed a decrease that paralleled the decrease in the steady-state transport rate induced by CCK-8. These results indicate that CCK-8 diminishes the rate of intestinal hexose absorption by decreasing SGLT1 protein abundance in the brush-border membrane of the rat jejunum and therefore provides evidence for acute enteric hormonal regulation of the rate of glucose absorption across the small intestine.

Phosphoinositide 3-OH kinases and their products, D-3 phosphorylated phosphoinositides, are increasingly recognized as crucial elements in many signaling cascades. A reliable means to introduce these lipids into intact cells would be of great value for showing the physiological roles of this pathway and for testing the specificity of pharmacological inhibitors of the kinases. We have stereospecifically synthesized di-C8-PIP3/AM and di-C12-PIP3/AM, the heptakis(acetoxymethyl) esters of dioctanoyl- and dilauroylphosphatidylinositol 3,4,5-trisphosphate, in 14 steps from myo-inositol. The ability of these uncharged lipophilic derivatives to deliver phosphatidylinositol 3,4,5-trisphosphate across cell membranes was demonstrated on 3T3-L1 adipocytes and T84 colon carcinoma monolayers. Insulin stimulation of hexose uptake into adipocytes was inhibited by the kinase inhibitor wortmannin and was largely restored by di-C8-PIP3/AM, which had no effect in the absence of insulin. Thus phosphatidylinositol 3,4,5-trisphosphate or a metabolite was necessary but not sufficient for stimulation of hexose transport. In T84 epithelial monolayers, di-C12-PIP3/AM mimicked epidermal growth factor in inhibiting chloride secretion and potassium efflux, suggesting that phosphatidylinositol 3,4, 5-trisphosphate was sufficient to modulate these fluxes and mediate epidermal growth factor's action.

The mannose transporter of bacterial phosphotransferase system mediates uptake of mannose, glucose, and related hexoses by a mechanism that couples translocation with phosphorylation of the substrate. It consists of the transmembrane IIC(Man)-IID(Man) complex and the cytoplasmic IIAB(Man) subunit. IIAB(Man) has two flexibly linked domains, IIA(Man) and IIB(Man), each containing a phosphorylation site (His-10 and His-175). Phosphoryl groups are transferred from the phosphoryl carrier protein phospho-HPr to His-10, hence to His-175 and finally to the 6' OH of the transported hexose. Phosphate-binding sites and phosphate-catalytic sites frequently contain arginines, which by their guanidino group can stabilize phosphate through hydrogen bonding and electrostatic interactions. IIB(Man) contains five arginines which are invariant in the homologous IIB subunits of Escherichia coli, Klebsiella pneumoniae and Bacillus subtilis. The IIA domains have no conserved arginines. The five arginines were replaced by Lys or Gln one at a time, and the mutants were analyzed for transport and phosphorylation activity. All five IIB mutants can still be phosphorylated at His-175 by the IIA domain. R172Q is completely inactive with respect to glucose phosphotransferase (phosphoryltransfer from His-175 to the 6' OH of Glc) and hexose transport activity. R168Q has no hexose transport and strongly reduced phosphotransferase activity. R204K has no transport but almost normal phosphotransferase activity. R304Q has only slightly reduced transport activity. R190K behaves like wild-type IIAB(Man). Arg-168, Arg-172, and Arg-304 are part of the hydrogen bonding network on the surface of IIB, which contains the active site His-175 and the interface with the IIA domain (Schauder, S., Nunn, R.S., Lanz, R., Erni, B. and Schirmer, T. (1998) J. Mol. Biol. 276, 591-602) (Protein Data Bank accession code 1BLE). Arg-204 is at the putative interface between IIB(Man) and the IIC(Man)-IID(Man) complex.

Glucokinase is distinguished from yeast hexokinase and low Km mammalian hexokinases by its low affinity for glucose and its cooperative behavior, even though glucose binding residues and catalytic residues are highly conserved in all of these forms of hexokinase. The roles of Ser-151 and Asn-166 as determinants of hexose affinity and cooperative behavior of human glucokinase have been evaluated by site-directed mutagenesis, expression and purification of the wild-type and mutant enzymes, and steady-state kinetic analysis. Mutation of Asn-166 to arginine increased apparent affinity for both glucose and ATP by a factor of 3. Mutation of Ser-151 to cysteine, alanine, or glycine lowered the Km for glucose by factors of 2-, 26-, and 40-fold, respectively, decreased Vmax, abolished cooperativity for glucose, and also decreased Km for mannose and fructose. The Ser-151 mutants had hexose Km values similar to those of yeast hexokinase, hexokinase I, and the recombinantly expressed COOH-terminal half of hexokinase I. However, the Ki values for the competitive inhibitors, N-acetylglucosamine and glucose-6-P, were unchanged, suggesting that Ser-151 is not important for inhibitor binding. Mutation of Ser-151 also increased the Km for ATP about 5-fold and abolished the enzyme's low ATPase activity, which indicates it is essential for ATP hydrolysis. The substrate-induced change in intrinsic fluorescence of S151A occurred at a much lower glucose concentration than that for wild-type enzyme. The results implicate a dual role for Ser-151 as a determinant of hexose affinity and catalysis, exclusive of the glucose-induced conformational change, and suggest that the low hexose affinity of glucokinase is dependent on interaction of Ser-151 with other regions of the protein.

Both the anabolic hormone insulin and contractile activity stimulate the uptake of glucose into mammalian skeletal muscle. In this study, we examined the role of phosphatidylinositol 3-kinase (PI 3-kinase), a putative mediator of insulin actions, in the stimulation of hexose uptake in response to hormone and contraction. Phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-triphosphate accumulate in skeletal muscle exposed to insulin but not hypoxia, which mimics stimulation of the contractile-dependent pathway of hexose transport activation. The fungal metabolite wortmannin, an inhibitor of PI 3-kinase, completely blocks the appearance of 3′-phospholipids in response to insulin. Moreover, wortmannin entirely prevented the increase in hexose uptake in muscle exposed to insulin but was without effect on muscle stimulated by repetitive contraction or hypoxia. These results support the view that PI 3-kinase is involved in the signaling pathways mediating insulin-responsive glucose transport in skeletal muscle but is not required for stimulation by hypoxia or contraction. Furthermore, these data indicate that there exist at least two signaling pathways leading to activation of glucose transport in skeletal muscle with differential sensitivities to wortmannin.

We have investigated the steady-state and presteady-state kinetics of the cloned H+/hexose cotransporter from Arabidopsis thaliana (STP1) expressed in Xenopus oocytes using the two-electrode voltage-clamp method. Steady-state sugar-dependent currents were measured between -150 and +50 mV as a function of external [3-O-methyl-D-glucose] (3OMG) and [H+]. At pH 6.5 (316 nM H+) the maximal current for sugar, i3OMGmax, was voltage-dependent, increasing from 40 nA at -30 mV to 95 nA at -150 mV. The apparent affinity of sugar, K3OMG0.5, at pH 6.5 decreased from 30 microM at -30 mV to 11 microM at -70 mV and was then voltage-independent between -70 and -150 mV. Increasing the extracellular [H+] to 3160 nM (pH 5.5) increased i3OMGmax to 65 nA at -30 mV and 212 nA at -150 mV. K3OMG0.5 at pH 5.5 also increased and was voltage-dependent: 15 microM at -50 mV rising to 25 microM at -150 mV. At pH 6.5 and 5.5, the Hill coefficient n for 3OMG was voltage-independent and averaged 1.2 and 1.4, respectively. At saturating [3OMG] iHmax was voltage-dependent and KH0.5 was voltage-independent, averaging 370 nM (pH 6.4). The Hill coefficient n for H+ was voltage-independent and averaged 1. The sugar specificity of STP1 was D-mannose > or = 2-deoxyglucose > D-galactose > or = 3OMG > D-xylose > D-glucose > D-fucose > D-fructose > L-glucose > L-arabinose > D-arabinose, demonstrating that STP1 has a low substrate specificity. Transient currents recorded after rapid steps in membrane potential relaxed with time constants tau, between 3 and 14 ms. The charge movement Q (the integral of the current transients) fitted to a Boltzmann relation with maximal charge Qmax of 3.4 nanocoulombs and an apparent valence z approximately 1 corresponding to a transporter density of 2 x 10(10)/oocyte. Potential for 50% Qmax (V0.5) was -28 mV. At saturating 3OMG and at low external [H+] (pH 7.5), the transient STP1 currents were eliminated. The presteady-state data indicate that STP1 can bind H+ in the absence of sugar, and the steady-state data suggest that H+/hexose cotransport occurs via a sequential mechanism.

In rat adipocytes and soleus muscles, 2-hydroxypropyl-beta-cyclodextrin (CD) was found to have a relatively small or no effect on basal or insulin-stimulated hexose uptake, but markedly enhanced hexose uptake effects of phorbol esters and/or diacylglycerol. In rat adipocytes, the CD-induced enhancement of hexose uptake during concurrent phorbol ester treatment was not associated with an increase in GLUT4 glucose transporter translocation to the plasma membrane, which was stimulated comparably by insulin and phorbol esters. Moreover, CD appeared to activate or facilitate the activation of glucose transporters subsequent to their translocation to the plasma membrane during ongoing phorbol ester treatment. In rat adipocytes, CD also enhanced the translocation of protein kinase C (PKC)-beta to the plasma membrane during the action of phorbol esters, which alone had little or no effect on this specific PKC translocation. Although it is uncertain how CD alters the function of plasma membranes to enhance the translocation of PKC-beta to, and the activation of glucose transporters within, this subcellular fraction during phorbol ester treatment, our findings provide direct support for a two-step model in the activation of glucose transport. In addition, it seems clear that, at least in some cell types, simple phorbol ester treatment does not necessarily serve as a ubiquitous activator of all activable PKC pools and all potential PKC-mediated responses.

A presumably full-length cDNA clone of the mitochondrial ATP/ADP translocator (AAT) of Chlorella kessleri has been isolated and sequenced. The expression of the AAT gene is highly increased in the presence of D-glucose (14 mM). At least nine more genes are activated when autotrophically grown Chlorella cells switch to heterotrophic growth. Among these is the HUP1 gene coding for the hexose transporter (Sauer, N., Caspari, T., Klebl, F., and Tanner, W. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 7949-7952) and, as also shown in this paper, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. When glucose or the nonmetabolizable analogue 6-deoxyglucose is added to the cells, an increased expression of GAPDH or AAT is observed after 10 or 30 min, respectively. Hexose uptake mutants (HUP1-) do not respond to sugars in this way, which indicates that either the inducer has to be internalized or that the HUP1 translocator is part of the signal transduction mechanism.

Pentosidine is a recently discovered fluorescent protein cross-link from human extracellular matrix that involves lysyl and arginyl residues in an imidazo (4, 5b) pyridinium ring. Pentosidine could be synthesized in vitro by the reaction of ribose, lysine, and arginine. The potential biological significance of the molecule prompted us to investigate its mechanism of formation from D-ribose and key Maillard intermediates, as well as from other potential precursor sugars. The yield of pentosidine from N alpha-t-Boc-lysine, N alpha-t-Boc-arginine, and D-ribose was highest at pH 9.0 and 65 degrees C, but was unaffected by reactant ratios at alkaline pH suggesting an important role for base catalysis. Ribated Boc-lysine on incubation with N alpha-t-Boc-arginine afforded a fluorescent compound with UV, fluorescence, 1H NMR, and MS properties identical with those from native or synthetic pentosidine. 3-Deoxypentosone, however, was not a major pentosidine precursor. Pentosidine became slowly detectable in bovine serum albumin incubated with 0.25 M and 1.0 M glucose and reached, at 30 days, 13.2 and 17 pmol/mg bovine serum albumin, respectively. Spectroscopical properties of glucose-derived pentosidine were identical with those from ribose-derived pentosidine. Pentosidine formed from glucated Boc-lysine with N alpha-t-Boc-arginine in higher yields than from glucose under standard conditions. Fructose, and unexpectedly ascorbate, also formed pentosidine in similar yields as glucose. The discovery that pentosidine can form not only from pentoses but also from hexoses and ascorbate raises major new questions concerning biochemical pathways of the Maillard reaction in vivo.

Results of activity and spectral studies using fluorescence and circular dichroism show that AMP and fructose 2,6-bisphosphate (F-2,6-P2) activate Ascaris suum phosphofructokinase through specific and similar conformational changes. Inorganic compounds like (NH4)2SO4 and KH2PO4 also induce structural alterations in the enzyme in a manner different from those caused by AMP and F-2,6-P2. The enzyme is activated by both AMP and F-2,6-P2, in 20 mM phosphate buffer, pH 6.6, with 0.2 mM ATP and 1 mM F-6-P. The Kact values for AMP and F-2,6-P2 are 25 +/- 3 microM and 1.5 +/- 0.2 microM, respectively. Both effectors quench enzyme tryptophan fluorescence in phosphate, pH 6.6, in a concentration-dependent manner. The Kd values determined from the decrease in emission intensity at 342 nm as a function of effector concentration are 24 +/- 3 microM for AMP and 1.00 +/- 0.15 microM for F-2,6-P2, in excellent agreement with the values of Kact. Both effectors also produce dramatic changes in the CD spectrum of the enzyme, in the region from 240 to 190 nm representing the peptide backbone. Secondary structure calculations suggest an increase in the alpha-helical content of the enzyme in the presence of either effector. The Kd values obtained from the concentration dependence of the decrease in ellipticity at 210 nm are 22.8 +/- 5.3 microM and 1.3 +/- 0.2 microM, respectively, for AMP and F-2,6-P2, once again in close agreement with the Kact values for these effectors. The data imply that activation of phosphofructokinase by these effectors is concomitant with structural changes in the enzyme. Further, comparison of the difference CD spectra for the effects of AMP and F-2,6-P2 show that both of them produce similar conformational changes and probably stabilize a similar final activated state of the enzyme. Other hexose phosphate analogues such as fructose 6-phosphate, glucose 1,6-bisphosphate, and fructose 1,6-bisphosphate do not affect the CD spectrum of the enzyme. Ammonium sulfate has no effect on the CD spectrum of the enzyme in phosphate buffer but does cause a significant alteration in the spectrum obtained in Mes. Gel filtration high performance liquid chromatography using a Borosil TSK 400 column shows that the tetrameric state of the native enzyme is not affected by the presence of the effectors.

Operation of the phosphoglycerate transport protein (PgtP) of Salmonella typhimurium has been studied in proteoliposomes by using a technique in which membrane protein is solubilized and reconstituted directly from small volumes of cell cultures. When protein from induced cells was reconstituted into phosphate (Pi)-loaded proteoliposomes, it was possible to demonstrate a PgtP-mediated exchange of internal and external phosphate. For this homologous Pi:Pi antiport, kinetic analysis indicated a Michaelis constant (Kt) of 1 mM and a maximal velocity of 26 nmol/min mg of protein; arsenate inhibited with a Ki of 1.3 mM, suggesting that PgtP did not discriminate between these two inorganic substrates. Pi-loaded proteoliposomes also accumulated 3-phosphoglycerate and phosphoenolpyruvate, establishing for each of them a concentration gradient (in/out) of about 100-fold; phosphoenolpyruvate (Ki = 70 microM) rather than 3-phosphoglycerate (Kt = 700, Ki = 900 microM) was the preferred substrate for these conditions. We also concluded that such heterologous exchange was a neutral event, since its rate and extent were unaffected by the presence of a protonophore and unresponsive to the imposition of a membrane potential (positive or negative inside). In quantitative work, we found a stoichiometry of 1:1 for the exchange of Pi and 3-phosphoglycerate, and given an electroneutral exchange, this finding is most easily understood as the overall exchange of divalent Pi against divalent phosphoglycerate. These experiments establish that PgtP functions as an anion exchange protein and that it shares important mechanistic features with the Pi-linked antiporters, GlpT and UhpT, responsible for transport of glycerol 3-phosphate and hexose 6-phosphates into Escherichia coli.

We have proposed that glucose-6-phosphatase (EC 3.1.3.9) is a two-component system consisting of (a) a glucose-6-P-specific transporter which mediates the movement of the hexose phosphate from the cytosol to the lumen of the endoplasmic reticulum (or cisternae of the isolated microsomal vesicle), and (b) a nonspecific phosphohydrolase-phosphotransferase localized on the luminal surface of the membrane (Arion, W.J., Wallin, B.K., Lange, A.J., and Ballas, L.M. (1975) Mol. Cell. Biochem. 6, 75-83). Additional support for this model has been obtained by studying the interactions of D-mannose-6-P and D-mannose with the enzyme of untreated (i.e. intact) and taurocholate-disrupted microsomes. An exact correspondence was shown between the mannose-6-P phosphohydrolase activity at low substrate concentrations and the permeability of the microsomal membrane to EDTA. The state of intactness of the membrane influenced the kinetics of mannose inhibition of glucose-6-P hydrolysis; uncompetitive and noncompetitive inhibitions were observed for intact and disrupted microsomes, respectively. The apparent Km for glucose-6-P was smaller with intact preparations at mannose concentrations above 0.3 M. Mannose significantly inhibited total glucose-6-P utilization by intact microsomes, whereas D-glucose had a stimulatory effect. Both hexoses markedly enhanced the rate of glucose-6-P utilization by disrupted microsomes. The actions of mannose on the glucose-6-phosphatase of intact microsomes fully support the postulated transport model. They are predictable consequences of the synthesis and accumulation of mannose-6-P in the cisternae of microsomal vesicles which possess a nonspecific, multifunctional enzyme on the inner surface and a limiting membrane permeable to D-glucose, D-mannose, glucose-6-P, but impermeable to mannose-6-P. The latency of the mannose-6-P phosphohydrolase activity is proposed as a reliable, quantitative index of microsomal membrane integrity. The inherent limitations of the use of EDTA permeability for this purpose are discussed.

Direct measurement of 3-O-[3H]methyl-d-glucose uptake by brown fat cells with the use of a rapid filtration procedure was found to provide a suitable index of the d-glucose transport system activity. The rate of net 3-O-methylglucose influx across the fat cell membrane was 10 to 20 times greater than the uptake rate of l-[14C]glucose which enters the cells by simple diffusion. After equilibration the concentration of 3-O-methylglucose in the intracellular water space was the same as that in the medium, indicating that 3-O-methylglucose uptake occurs by facilitated diffusion. Detectable phosphorylation of this d-glucose analogue by brown fat cells under the conditions of these studies did not occur. 3-O-Methylglucose transport was consistently linear for 4 or more min at 23° and was several times greater at 37° and 23° than at 0°. Transport of 3-O-methylglucose was inhibited by d-glucose and by agents known to block d-glucose transport such as phlorizin and cytochalasin B. Both saturable and nonsaturable processes contributed to the initial velocity of 3-O-methylglucose uptake. The saturable component of net influx was estimated by subtraction of uptake rates monitored in the presence of cytochalasin B, which were used to approximate the nonsaturable component, from the initial velocities of 3-O-methylglucose uptake. The corrected values obtained followed simple Michaelis-Menten kinetics with an apparent Kt of about 4 mm. At high concentrations of the hexose, diffusion probably represented the major contribution to uptake since this portion of a Lineweaver-Burk plot of uncorrected values passed through the origin. Insulin markedly stimulated the initial rate of hexose transport, and this effect reflected an increase in apparent Vmax with no detectable difference in apparent Kt. Diamide, H2O2, vitamin K5, and spermine enhanced d-[1-14C]glucose conversion to 14CO2 to a greater extent than d-[6-14C]glucose oxidation, whereas in these experiments insulin increased oxidation of both sugars equally. Vitamin K5 and spermine had little effect on 3-O-methylglucose transport under conditions where insulin, diamide, and H2O2 significantly stimulated this process. The action of diamide and H2O2 to increase 3-O-methylglucose uptake was specific for the d-glucose transport system since, like insulin, they failed to alter l-glucose uptake. The data suggested that vitamin K5 and spermine stimulate fat cell glucose uptake and oxidation primarily by activating intra-cellular metabolic pathways while diamide and H2O2, in addition to an intracellular effect, also activate the d-glucose transport system. These results indicate that glucose uptake in brown fat cells is determined by both the activity of the membrane transport system and the rate of intra-cellular utilization and that alteration of either process can modulate glucose entry rates.

Cycloheximide, a potent inhibitor of protein synthesis, has been used to examine the relationship between recruitment of hexose carriers and the activation of glucose transport by insulin in rat adipocytes. Adipocytes were preincubated +/- cycloheximide for 90 min then +/- insulin for a further 30 min. We measured 3-O-methylglucose uptake in intact cells and in isolated plasma membrane vesicles. The concentration of glucose transporters in plasma membranes and low density microsomes was measured using a cytochalasin B binding assay. Cycloheximide had no affect on basal or insulin-stimulated 3-O-methylglucose uptake in intact cells or in plasma membrane vesicles. However, the number of glucose carriers in plasma membranes prepared from cells incubated with cycloheximide and insulin was markedly reduced compared to that from cells incubated with insulin alone (14 and 34 pmol/mg protein, respectively). Incubation of cells with cycloheximide alone did not change the concentration of glucose carriers in either plasma membranes or in low density microsomes compared to control cells. When isolated membranes were analyzed with an antiserum prepared against human erythrocyte glucose transporter, decreased cross-reactivity was observed in plasma membranes prepared from cycloheximide/insulin-treated cells compared to those from insulin cells. The present findings indicate that incubation of adipocytes with cycloheximide greatly reduces the number of hexose carriers in the plasma membrane of insulin-stimulated cells. Despite this reduction, insulin is still able to maximally stimulate glucose uptake. Thus, these data suggest an apparent dissociation between insulin stimulation of glucose transport activity and the recruitment of glucose carriers by the hormone.

The kinetics of the equilibrium exchange flux of 3-O-methylglucose (MeGlc) were examined in isolated rat adipocytes using a recently described technique (Whitesell, R. R., and Abumrad, N. A. (1985) J. Biol. Chem. 260, 2894-2899) in which the cells, under basal conditions, were reported to exhibit a high Km (35 mM) that was reduced (to 3 mM) upon treatment with insulin. When this technique was employed in the present study, the Km observed in basal adipocytes was 6.4 +/- 0.4 mM; insulin treatment did not affect this parameter (6.3 +/- 0.5 mM), although it increased the maximum velocity (phi max) 21-fold (from 3.0 +/- 0.3 to 63.7 +/- 1.1 nmol X min-1 X microliter of intracellular water-1). The large discrepancy in the basal Km values observed in the previous (35 mM) and the present (6.4 mM) studies is shown to be associated with relatively minor differences in basal MeGlc flux; these minor differences may reflect insufficient mixing of labeled MeGlc in the flux measurements of the previous study. In addition, the active phorbol ester, 12-O-tetradecanoylphorbol 13-acetate, at a concentration of 0.3 microM, caused a 2.8-fold elevation of phi max, with no modulation of Km. These results indicate that phi max, not Km, is the major kinetic parameter of hexose transport affected by insulin and phorbol esters, leading to enhancement of hexose uptake by the isolated rat adipocyte.

The presence of a reactive exofacial sulfhydryl on the human erythrocyte hexose carrier was used to test several predictions of the alternating conformation or one-site model of transport. The cell-impermeant glutathione-maleimide-I (GS-Mal) irreversibly inhibited hexose entry by decreasing the transport Vmax. This effect was potentiated by phloretin and maltose but decreased by cytochalasin B, indicating that under the one-site model the external sulfhydryl is on the outward-facing carrier but that it does not overlap with the exofacial substrate-binding site. Incubation of erythrocytes with maltose competitively inhibited the binding of [3H]cytochalasin B to the inward-facing carrier (Ki = 40 mM). Furthermore, both equilibrium cytochalasin B binding and its photolabeling of the band 4.5 carrier protein were decreased in ghosts prepared from GS-Mal-treated cells. Thus induction of an outward-facing carrier conformation with either maltose or GS-Mal caused the endofacial substrate-binding site to disappear. Dose-response studies of GS-Mal treatment of intact cells suggested that some functional carriers lack a reactive external sulfhydryl, which can be partially regenerated by pretreatment with excess cysteine. These data provide direct support for the one-site model of transport and further define the role of the external sulfhydryl in the transport mechanism.

A fucolipid that carried human blood group Lea activity was isolated from human small intestine. It contianed fucose, galactose, N-acetyl glucosamine, glucose, and ceramide in a molar ratio of 1:2:1:1:1. After periodate oxidation only 1 molecule of galactose and the N-acetylglucosamine remained. Permethylation of the lipid gave derivatives of a terminal fucose and galactose residue together with 2,4,6-tri-O-methylgalactose and 2,3,6-tri-O-methylglucose. After removal of fucose the lipid could be converted to a ceramide trihexoside with beta-galactosidase, and this, in turn, to ceramide lactoside by the action of beta-N-acetylhexosaminidase. Both enzymes converted the defucosylated derivative to a ceramide monohexoside. The methylated and the methylated and reduced derivatives of the intact lipid gave ions in mass spectrometry for a terminal hexose and deoxyhexose, a terminal trisaccharide of hexose, deoxyhexose and N-acetylhexosamine, and terminal tetra-and pentasaccharides. Ceramide fragments characteristic of hydroxy fatty acids with 16, 22, 23, and 24 carbons were found together with those of phytospingosine as the major long chain base. On the basis of these results and the immunologic activity of the fucolipid, the following structure is proposed: betaGal (1 leads to 3)betaGlcNAc (1 leads to 3)betaGal (1 leads to 4)Glc-ceramide alphaFuc (1 leads to 4).

[6-2H2]Glucose was used as a tracer for a comparative study on the metabolism of the neutral glycosylceramides in plasma of a control subject and a patient with Fabry's disease. The incorporation of the tracer into the glucosyl and galactosyl moieties of the glycosphingolipids was measured by gas chromatography-mass spectrometry of the tetra-0-acetyl methyl glycoside derivatives. Experiments on the precision and accuracy for measurements of [6,6-2H2]hexose in a sample demonstrated that incorporation of 0.2% or more of [6,6-2H2]glucose could be detected with a 95% confidence limit of +/-0.16%. The labeled substrate (35g) was infused into each subject with a 5-g priming dose and the remainder administered at a constant rate of 3 g/hour over a 10-hour period. During the infusion, the plasma glucose of each subject attained a concentration of about 30% [6,6-2H2]glucose which diminished rapidly after the administration of substrate was complete. A concentration of 0.8% [6,6-2H2]glucose was observed in glucosylceramide (GL-la) from plasma of both subjects between 48 and 72 hours after the infusion began. The label disappeared from this lipid at a logarithmic rate and 0.2% or less of the molecules were labeled 9 days after the experiment began. In contrast to the results with GL-la, the maximum incorporation of [6,6-2H2]hexose into lactosylceramide (galactosyl-(beta1 leads to 4)-glucosylceramide) was 2-fold higher in the Fabry patient (1.6%) than in the control (0.8%). The trihexosylceramide (galactosyl-(alpha1 leads to 4)-galactosyl-(beta1 leads to 4)-glucosylceramide, GL-3a) from plasma of the control reached a maximum of 0.4% [6,6-2H2]hexose in both the glucosyl and galactosyl moieties whereas the GL-3a from the Fabry patient was not significantly labeled. The maximal labeling of the GL-4a fraction (N-acetyl-galactosaminyl-galactosyl-galactosyl-glucosylceramide) was slightly depressed in the Fabry patient (0.4%) as compared to the control (0.7%). Turnover times for the glycosphingolipids of plasma were calculated to be from 4 to 8 days and the turnover rates were from 1 to 6 mumol/day.

The inhibitions of protein synthesis initiation in heme-deficient reticulocyte lysates and in GSSG-treated hemin-supplemented lysates are both characterized by the activation of heme-regulated eIF-2 alpha kinase, which phosphorylates the alpha-subunit of eukaryotic initiation factor (eIF-2). In both inhibitions, the accumulation of eIF phosphorylated in alpha-subunit (eIF-2(alpha P)) leads to the sequestration of reversing factor (RF) in a phosphorylated 15 S complex, RF.eIF-2(alpha P), in which RF is nonfunctional. A sensitive assay for the detection of endogenous RF activity in protein-synthesizing lysates indicates that, in GSSG-inhibited (1 mM GSSG) lysates, RF is more profoundly inhibited than in heme-deficient lysates. RF inactivation in GSSG-induced inhibition appears to be due to two separate but additive effects: (i) the formation of the phosphorylated 15 S RF complex, RF.eIF-2(alpha P), and (ii) the formation of disulfide complexes which inhibit RF activity. Both inhibitory effects are overcome by catalytic levels of exogenous RF which permits the resumption of protein synthesis. RF activity and protein synthesis in GSSG-inhibited lysates are efficiently restored by the delayed addition of glucose-6-P or 2-deoxyglucose-6-P (1 mM). The rescue of protein synthesis by hexose phosphate (1 mM) is proportional to the extent of RF recovery and is due in part to NADPH generation; even at levels of hexose phosphate (50 microM) too low to support protein synthesis, partial restoration of RF activity occurs due to increased NADPH/NADP+ ratios. The ability of dithiothreitol (1 mM) to restore RF activity in GSSG-treated but not heme-deficient lysates also provides evidence for a reducing mechanism which functions at the level of RF. The results suggest that NADPH plays a role in the maintenance of sulfhydryl groups essential for RF activity.

The addition of 0.167 to 4.0 mM cAMP to gel-filtered rabbit reticulocyte lysates stimulates the initial rate and the extent of polypeptide synthesis. The stimulation is at the initiation step of polypeptide synthesis as measured by the (i) increased dipeptide, methionyl-valine, accumulation in the presence of the specific initiation inhibitor, pactamycin, and (ii) increased formation of the 40 S and 80 S initiation complex when gel-filtered lysates are incubated with [35S]Met-tRNAFMet. Furthermore, a synergistic stimulation of protein synthesis is observed when cAMP and hexose phosphates (which alone elicit a 1.8-fold stimulation of protein synthesis) are added simultaneously to gel-filtered rabbit reticulocyte lysates. These results indicate that cAMP and hexose phosphates are both essential to maintain the high rate of initiation.

The effect of insulin and factors which have insulin-like activity on the kinetic parameters of 3-O-methyl-D-glucose (MeGlc) transport in rat adipocytes were assessed. Carrier-mediated uptake of MeGlc was estimated by the difference in the amounts of [14C]MeGlc and L-[3H]glucose taken up in cells under equilibrium exchange conditions at 37 degrees C. The Km and Vmax values in basal cells were 17.4 mM and 0.24 nmol/10(6) cells/s, respectively. Removal of endogenous adenosine by adenosine deaminase resulted in a 26% decrease in the basal rate due to a slight increase in the Km (19.6 mM) and a decrease in the Vmax value (0.20 nmol/10(6) cells/s). The maximum concentration (10 nM) of insulin decreased the Km to approximately one-half of the basal (7.1 mM) concomitant with an 8.5-fold increase in the Vmax value (2.04 nmol/10(6) cells/s). Submaximal concentrations (50 and 150 pM) of insulin, N6-phenylisopropyladenosine (1 microM), mechanical agitation of cells by centrifugal force (160 x g), low temperature (15 degrees C), 12-O-tetradecanoylphorbol-13-acetate (1 microM), and hydrogen peroxide (10 mM) all decreased the basal Km value to a range of 13.5-7.3 mM, concomitant with a 1.7-7.4-fold increase in the Vmax. A possible explanation for the alterations in the kinetic parameters may be that insulin and other factors cause the translocation of the mobile low-Km glucose transporters from an intracellular site to the cell surface, where the stationary high-Km transporters are located. Thus, when the Km and Vmax values of the hypothetical high-Km transporters were assumed to be 20 mM and 0.20 nmol/10(6) cells/s, respectively, and the Km of the low-Km transporters was assumed to be 7 mM, the theoretical Km decreased from 20 to 7.5 mM as the Vmax of the low-Km transporters increased from near 0 to 2.0 nmol/10(6) cells/s. The relation between empirical Km and Vmax values as affected by several agents and conditions followed closely the relation predicted by the above two-transporter model.

The effect of Glc deprivation (starvation) on hexose transporter (GT) polypeptide(s) (pp) was studied in 3T3-C2 murine fibroblasts. Cells deprived of Glc exhibit 5-fold increases in hexose transport and Glc-displaceable cytochalasin B binding. Immunoblots of membranes reveal a Mr 55,000 GT pp in fed (4 g of Glc/liter) cells and Mr 55,000 and Mr 42,000 GT pp in starved cells. A 10-40-fold increase in total GT pp occurs upon Glc deprivation; part of this accumulation (2-5-fold) is in the Mr 55,000 GT pp, and the remaining increase is in the Mr 42,000 GT pp. During the first 12 h of Glc deprivation only the Mr 55,000 GT pp accumulates. At later times (24-72 h) the Mr 42,000 GT pp appears and constitutes a larger fraction of the total accumulation. Similarly, the Glc concentration dependence of these phenomena reveals that the Mr 55,000 GT pp accumulates at higher concentrations of Glc (less than or equal to g/liter) than the Mr 42,000 GT pp (less than or equal to 0.5 g/liter). Using alternative nutrients, sugar analogs, and inhibitors we observed that the accumulation of total GT pp is dependent upon both hexose phosphate metabolism and the interaction of substrate with the GT. The role(s) of oligosaccharide biosynthesis, protein synthesis, and the transport process itself in the Glc deprivation-induced accumulation of GT pp were examined. The appearance of the Mr 42,000 GT pp but not the Mr 55,000 GT pp was dependent upon protein synthesis. The Glc deprivation-induced accumulation of GT pp is reversible upon refeeding with Glc (4 g/liter, 12 h). This reversal was dependent upon protein synthesis. The electrophoretic mobility of the Mr 42,000 GT pp is similar to the GT pp observed after tunicamycin treatment. The Mr 55,000 but not the Mr 42,000 GT pp binds specifically to agarose-bound wheat germ agglutinin and is sensitive to endoglycosidase F digestion. Oligosaccharide-stripped GT pp and the Mr 42,000 GT pp have the same Mr. The results suggest that the accumulation of total GT pp induced by Glc deprivation is partially independent of the effect of Glc deprivation on glycoprotein biogenesis. The appearance of the Mr 42,000 GT pp with aglyco characteristics is the result of the latter. The accumulation of total GT pp, however, is the result of a specialized and sensitive adaptation of the cell to Glc deprivation. The GT pp synthesized during chronic Glc deprivation has an Mr of 42,000; fed cells synthesize the Mr 55,000 GT pp. Neither the level of in vitro translatable GT mRNA nor the rate of GT pp synthesis are increased by Glc deprivation.(ABSTRACT TRUNCATED AT 400 WORDS)

The phlorizin binding properties of luminal membrane vesicles isolated from the LLC-PK1 cells, a continuous epithelial cell line derived from pig kidney, are studied. Scatchard analysis of this binding indicates the existence of a single high affinity sodium-dependent site with KD = 0.4 microM at 266 mM sodium. The specificity properties of this site indicate that it represents the binding of phlorizin to the hexose binding site of the sodium-dependent D-glucose transporter previously identified in this cell line. Both phlorizin equilibrium binding and the rate of phlorizin binding were found to be sigmoidal functions of sodium concentration. A Hill analysis of these data was consistent with a sodium:phlorizin stoichiometry of 2:1 in good agreement with the sodium:glucose stoichiometry already established in these cells. Phlorizin dissociation was also found to be sodium-dependent. On the basis of the phlorizin binding data presented here, a number of models of the binding of phlorizin and sodium to the transporter can be excluded. An analysis of a random binding model consistent with the data is presented. The significance of the LLC-PK1 sodium-dependent D-glucose transporter as a model system for related renal and intestinal transporters is discussed.

Spingosine, a naturally occurring inhibitor of protein kinase C, has recently been shown to have potent bioregulatory effects on a variety of cellular processes involving signal transduction mechanisms. In the present studies, we have investigated its effects on activation by insulin of hexose transport and glucose oxidation in isolated rat adipocytes. Preincubation of cells with this long-chain base blocked both the marked activation of these processes by insulin and the smaller activation by phorbol myristate acetate. Inhibition of both insulin and phorbol 12-myristate 13-acetate activation showed the same sphingosine concentration dependence, suggesting a common locus of action. The effectiveness of sphingosine was inversely proportional to the lipid content in the incubation (which was a function of both the age of the animal and the number of cells used) presumably due to dilution of the lipophilic long-chain base into the cellular triglycerides. Sphingosine did not affect either insulin binding to its receptor or the half-maximal concentration of the hormone required to activate hexose transport, but reduced the maximal responses. Thus, the inhibition was at a step distal to the binding of insulin to its receptor. Basal transport activity was not inhibited, suggesting a locus of action prior to the glucose transporter. The inhibitor was also effective when added following activation by insulin of hexose transport and resulted in a rapid reversal of activation (t 1/2 for inhibition was 2-4 min.). Sphingosine and its analogs showed a parallel potency for inhibition both of isolated protein kinase C and of insulin activation in adipocytes, consistent with an essential role for protein kinase C in the activation of hexose transport by insulin.

Maltosylisothiocyanate (MITC), synthesized as an affinity label for the hexose carrier, has been reported to label a Band 3 or Mr = 100,000 protein in human erythrocytes, in contradistinction to many studies showing the carrier as a Band 4.5 or Mr = 45,000-66,000 protein on gel electrophoresis. In this work the possibility that MITC interacts with the Band 3 anion transporter was studied. In intact human erythrocytes, MITC labeling was largely confined to Band 3 and was decreased by several competitive inhibitors of hexose transport. However, MITC also appeared to react with the anion transport protein, since MITC labeling of Band 3 was irreversibly decreased by the anion transport inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) and since MITC also irreversibly inhibited both tritiated dihydro-DIDS labeling of Band 3 and sulfate uptake in intact cells. Although 20 microM DIDS had little effect on hexose transport, the labeling of erythrocyte Band 3 by the dihydro analog was significantly diminished by competitive inhibitors of hexose transport. These data suggest that MITC labels in part the anion transporter as well as other DIDS-reactive sites on Band 3 which appear to be sensitive to competitive inhibitors of hexose transport.

In the present study we examined the role of glucose in the regulation of its own transport activity in the cultured 3T3 fat cell. A regulatory control of glucose became apparent after these cells were cultured in the absence of glucose. Glucose deprivation of the cells was accompanied by a specific time and protein synthesis-dependent increase in dGlc (2-deoxyglucose) uptake (up to 5-fold), which was due to an increase in the apparent Vmax of the transport system. Concomitantly, the stimulatory effect of insulin on hexose uptake almost completely disappeared. Addition of glucose to the glucose-deprived cells rapidly reversed the deprivation effects. Cycloheximide experiments revealed that the glucose deprivation-induced increase in hexose uptake required protein synthesis as well as a protein synthesis-independent response to glucose deprivation that retarded the turnover of hexose transport activity. Taken together, these data indicate that glucose deprivation is accompanied by retardation of the rate of degradation, internalization, or inactivation of hexose transporters while the increase in dGlc uptake requires at least the continuation of protein synthesis-dependent de novo synthesis, insertion, or activation of hexose transporters. Hexose competitively taken up with dGlc, including the nonmetabolizable glucose analogue 3-O-methylglucose, could replace glucose in the process of prevention and reversal of the deprivation effects, indicating that competitive transport but not the metabolism of hexose is a prerequisite for the regulatory effect of glucose on the activity of its own transport system. In conclusion, our results indicate that in cultured 3T3 fat cells glucose itself is involved in the regulation of the activity of its own transport system by influencing the rate of degradation, internalization, or inactivation of hexose transporters by a protein synthesis-independent mechanism.

Glucocorticoids inhibit glucose utilization by fat cells. The possibility that this effect results from altered glucose transport was investigated using an oil-centrifugation technique which allows a rapid (within 45 s) estimation of glucose or 3-O-methylglucose uptake by isolated fat cells. At high concentration (greater than 25 muM), dexamethasone inhibited glucose uptake within 1 min of its addition to fat cells. Efflux of 3-O-methylglucose was also impaired by 0.1 mM dexamethasone. However, diminished glucose uptake was not a specific effect of glucocorticoids; high concentrations (0.1 mM) of 17beta-estradiol, progesterone, and deoxycorticosterone produced a similar response in adipocytes. At a more physiologic steroid concentration (0.1 muM), glucocorticoids inhibited glucose uptake in a time-dependent manner (maximum effect in 1 to 2 hours). This effect was specific for glucocorticoids since, under these conditions, glucose uptake was not changed by the non-glucocorticoid steroids. Lineweaver-Burk analysis showed that 0.1 muM dexamethasone treatment produced a decrease in Vmax for glucose uptake but did not change the Ku. Hexokinase activity and ATP levels were not altered by this treatment, suggesting that processes involved in glucose phosphorylation were not affected. Dexamethasone treatment also caused a reduction in uptake of 3-O-methylglucose when assayed using a low sugar concentration (0.1 mM). At a high concentration (10 mM), uptake of the methyl sugar was only slightly less than normal in treated cells. Stimulation by insulin markedly enhanced uptake of glucose and 3-O-methylglucose by both treated and untreated cells. At a low hexose concentration (0.1 mM) and in the presence of insulin, sugar uptake by dexamethasone-treated cells was slightly less than control cells. Stimulation by insulin did however completely overcome the alteration in hexose uptake when larger concentrations of sugars (greater than 5 mM) were used. There was no detectable change in total protein synthesis during incubation of fat cells with dexamethasone. However, actinomycin C blocked the inhibitory effect of dexamethasone on glucose uptake. Cycloheximide, which caused a small inhibition in glucose uptake, prevented the full expression of the inhibitory effect of dexamethasone on glucose transport. These results indicate that dexamethasone alters the facilitated transport of glucose and, secondly, suggest that synthesis of RNA and protein is needed for glucocorticoid action.

Reaction of the phosphofructokinase from Ascaris suum with the reagent, diethylpyrocarbonate (DEPC), results in the loss of enzymatic activity. Treatment of the inactivated enzyme with hydroxylamine brings about the recovery of almost 80% of the original activity suggesting that the modified residues are histidines. Further evidence for the modification of histidines is that concomitant with the loss of activity, there is a change in A242 nm that corresponds to the derivatization of 5-6 histidines per subunit. There is no change in A278 nm during the derivatization process, thereby ruling out the modification of tyrosines by DEPC. Analyses of the first order inactivation rate constant for DEPC derivatization at different pH values resulted in the determination of a pKa of 6.4 +/- 0.1 for the group on the enzyme that reacts with DEPC. Derivatization of the enzyme with DEPC in the presence of fructose 6-phosphate (Fru-6-P) protected the enzyme against inactivation by 80%. ATP or MgATP gave no protection against DEPC inactivation. When the Fru-6-P-protected enzyme was further reacted with DEPC in the absence of Fru-6-P, a total of 2 histidines were modified per subunit, and the derivatization of one of these could be correlated with activity loss. When the phosphofructokinase that had been derivatized by DEPC in the presence of Fru-6-P was assayed, it was found that it no longer exhibited allosteric properties and appeared to be desensitized to ATP inhibition. This loss of ATP inhibition could be correlated with the modification of 2 histidines per subunit by DEPC. The first order rate constant for desensitization was determined at different pH values and a pKa value of 7.0 +/- 0.2 was obtained for the group(s) responsible for the desensitization. Regulatory studies with the desensitized enzyme revealed that the enzyme was not stimulated by AMP, NH4+, K+, phosphate, sulfate, or hexose bisphosphates. It is concluded that histidine may be involved both in the active site and the ATP inhibitory site of the ascarid phosphofructokinase.

We have previously shown that the sulfonylureas increase insulin-stimulated glucose transport in adipocytes mainly by enhancing the insulin-induced recruitment of glucose transporter from its intracellular storage pool to the plasma membrane (Jacobs, D. B., and Jung, C. Y. (1985) J. Biol. Chem. 260, 2593-2596). In order to determine if this sulfonylurea effect is mediated by a specific membrane-associated sulfonylurea-binding protein, in the present report we measured exact dose dependence of the transport enhancement activities of different sulfonylureas in adipocytes in primary culture and equilibrium binding affinities of these agents to various adipocyte membrane fractions. Glycuride was found to increase the insulin-stimulated, 3-O-methyl-D-glucose equilibrium exchange in cultured rat adipocytes by up to 60% with little effect in the absence of insulin. The effect developed gradually reaching the maximum level at 24 h of incubation. The effect was concentration dependent showing a simple, one-to-one stoichiometry and an apparent activation constant (Ka) of approximately 1 microM. Glypizide, tolazamide, and tolbutamide also enhanced the insulin-stimulated hexose transport by up to 60%, but with Ka of approximately 2, 11, and 25 microM, respectively. HB-699 and ciglitazone, non-sulfonylureas, were without effect under the same condition. In equilibrium binding experiments, [3H]glyburide was found to bind to adipocyte membranes at two or more protein-specific, saturable sites, with similar apparent dissociation constants (KD) ranging 1-3 microM. These protein-specific glyburide bindings were displaced not only by tolazamide and tolbutamide, but also by ciglitazone and HB-699, with indicated KD of 11-16, 80-85, 20-25, and 85-95 microM, respectively. However, with the plasma membrane fraction, the displacements by ciglitazone and HB-699 were partial and did not exceed 56-61% at maximum. Based on these findings, we propose that there is a sulfonylurea-specific-binding protein in the plasma membrane of adipocytes, and that this sulfonylurea-binding protein may play a key role in the enhancement of insulin-stimulated hexose transport by sulfonylureas, probably via potentiation of the insulin-induced recruitment of glucose transporter.

Exposure of insulin-responsive, differentiated 3T3-L1 cells (adipocytes) to 0.1 to 1.0 microgram/ml of insulin for 3 to 48 h resulted in a persistent state of enhanced 2-deoxy-D-glucose and 3-O-methyl-D-glucose uptake. Elevated basal transport activity was retained under conditions where 125I-insulin binding activity remained unchanged and exchangeable insulin was dissociated from cell surface receptors. The appearance of enhanced hexose transport activity was prevented by cycloheximide and could be distinguished from the activation of the glucose transport system observed in these and other cells during glucose deprivation. Rigorously washed adipocytes, exhibiting insulin-induced elevations in basal transport activity, were refractory to further stimulation by insulin during hexose uptake assays. Insulin-unresponsive 3T3-L1 preadipocytes failed to increase hexose uptake activity when treated under conditions that elicited an optimal response in adipocytes.

Kinetic studies of the rat adipocyte hexose transport system were performed using the integrated rate approach and these compared to the simple carrier model of transport. Equilibrium exchange 3-O-methylglucose entry and exit studies showed directional symmetry with Km = overall dissociation constants = 8-10 mM. Comparison of zero-trans and equilibrium exchange entry also revealed similar Km and Vmax values. Insulin pretreatment increased the maximal rate of transport at 20 mM 3-O-methylglucose about 5- to 6-fold with each procedure. Studies of glucose-induced steady state 3-O-methylglucose countertransport provided evidence that carrier permeability and not carrier-substrate dissociation was rate limiting for overall transport. These data, therefore, indicate equal mobility of the loaded and unloaded carriers. Network thermodynamic computer simulations of the simple carrier model using kinetic parameters derived from zero-trans experiments provided good fits of actual data. The effect of insulin was best represented by an increase in total number of carrier units. It is concluded that the adipocyte hexose carrier displays bidirectional symmetry, limitation of transport by carrier movement rather than substrate-carrier interaction, equal rates of movement of loaded and unloaded carriers, and adherence to a simple carrier model in which insulin increases the total number of carrier units.

Regulation of hexose transport was investigated in chicken embryo fibroblasts (CEF) which develop 4- to 8-fold enhanced hexose transport activity during glucose starvation. The presence of cycloheximide in low (0.5 micrograms/ml) concentrations during starvation largely blocked the enhancement of transport activity. Glucose refeeding of CEF in the starvation state led to a decline in transport to the basal level. This decline was either potentiated or blocked by the presence of cycloheximide in low or high (50 micrograms/ml) concentrations, respectively. Exposure of CEF in the fed state to low concentrations of cycloheximide resulted in a 70% decrease of transport within 6 h, whereas exposure to high concentrations of cycloheximide led to only a modest loss (35% decrease). In the glucose-starved state, CEF had no significant decline of transport when exposed to cycloheximide at either high or low concentrations. The uptake of 3-O-methylglucose by fed, starved, or cycloheximide-treated CEF correlated closely with D-glucose transport activity and [3H]cytochalasin B binding by plasma membranes prepared from CEF exposed to the same conditions. Hexose transport activity of CEF seems to largely depend on the number of functioning carriers in the plasma membrane, which apparently reflect the balance between carrier synthesis and inactivation. These two processes require protein synthesis, but are differentially sensitive to the effects of cycloheximide, such that low concentrations of cycloheximide appear to block primarily synthesis while high concentrations block both processes. Furthermore, during starvation the enhancement of transport appears largely due to decreased carrier inactivation in the face of continued carrier synthesis.

The reaction sequence for the biosynthesis of gangliosides by mouse neuroblastoma cells has been investigated by studying the pattern of incorporation of labeled precursors into sialoglycosphingolipids. Cultured NB41A cells incorporated N-[3H]acetylmannosamine into the sialic acid moiety of GM3 in less than 10 min. Labeled GM2 was not detected in cells incubated for less than 30 min, while measurable radioactivity did not appear in GM1 until after 60 to 90 min. Analogous experiments were carried out using [14C]galactose. No significant amount of labeled hexose was incorporated into asialo-GM2 during 60 min of culture. These studies are in accord with results of previous studies on glycosyltransferases of NB41A cells (Kemp, S. F., and Stoolmiller, A. C. (1976), J. Neurochem. 26, 723-732), and further support the concept that the pathway of synthesis of gangliosides proceeds via GM3 leads to GM2 leads to GM1.

The facilitated diffusion of D-glucose across human erythrocyte membranes requires an exofacial (outer surface) sulfhydryl group which can be alkylated by the impermeant reagents glutathione-maleimide and dextran-maleimide. The irreversible inhibition produced by these reagents is asymmetric; inhibition of glucose efflux considerably exceeds that of influx when transport is assayed in the absence of glucose on the opposite side of the membrane. Both D-glucose and cytochalasin B protect the exofacial transport site from alkylation by the impermeant maleimides. This masking effect provides the basis for a two-step procedure for differential labeling of the outer transport site with radioactive glutathione-maleimide. The method labels clearly and consistently a component of the membrane proteins which migrates in sodium dodecyl sulfate-polyacrylamide gels between Coomassie brilliant blue-stained Bands 4.2 and 5, corresponding to an apparent molecular weight of 65,000 to 70,000. Transport studies after inhibition with N-ethylmaleimide suggest that the hexose mechanism also requires a second sulfhydryl group which is not accessible at the cell surface.

Irradiation of intact rat adipocytes with high intensity ultraviolet light in the presence of 0.5 microM [3H] cytochalasin B results in the labeling of Mr 43,000 and 46,000 proteins that reside in the plasma membrane fraction. In contrast to the Mr 46,000 protein, the Mr 43,000 component is not observed in the microsome fraction and exhibits lower affinity for [3H]cytochalasin B. Photolabeling of the Mr 43,000 protein is inhibited by cytochalasin D, indicating it is not a hexose transporter component. The Mr 46,000 protein exhibits characteristics expected for the glucose transporter such that D-glucose or 3-O-methylglucose but not cytochalasin D inhibits its photolabeling with [3H] cytochalasin B. Furthermore, insulin addition to intact cells either prior to or after photoaffinity labeling of the Mr 46,000 protein causes a redistribution of this component from the low density microsomes to the plasma membrane fraction, as expected for the hexose transporter. Photolabeling of transporters in both the low density microsome and plasma membrane fractions is inhibited when intact cells are equilibrated with 50 mM ethylidene glucose prior to irradiation with [3H]cytochalasin B. Incubation of intact cells with 50 mM ethylidene glucose for 1 min at 15 degrees C leads to an intracellular concentration of only 2 mM. Under these conditions, the photoaffinity labeling in intact cells of hexose transporters that fractionate with the low density microsomes is unaffected, indicating these transporters are not exposed to the extracellular medium. In contrast, photolabeling in intact insulin-treated cells of hexose transporters that fractionate with the plasma membrane is inhibited under these incubation conditions. The results demonstrate that insulin action results in the exposure to the extracellular medium of previously sequestered hexose transporters.

In cultures of Escherichia coli W4597(K) and G34 under various nutritional conditions the rates of glucose utilization and cellular levels of fructose-1,6-P2 are quantitatively related by the Hill equation where the value of the Hill coefficient is approximately equal to 2. This is the first evidence that fructose-P2, or any metabolite which covaries with fructose-P2, modulates glucose utilization in E. coli. In light of previous observations from our laboratory this new observation and those in the succeeding report provide the first evidence that in E. coli glycolysis, glycogen synthesis and glucose utilization are coordinately regulated, thus providing for the coupling of ATP utilization and production under various metabolic circumstances. Alterations in the level of ATP apparently affect the velocity of phosphofructokinase, the rate-limiting enzyme in glycolysis, altering the cellular levels of glucose-6-P or fructose-P2. Changes in the levels of these hexose phosphates are quantitatively related to alterations in the rates of glucose utilization and glycogen synthesis in the intact E. coli cell.

Following the differentiation of 3T3-L1 preadipocytes insulin acutely activates the rate of 2-deoxy-[1-14C]glucose uptake in the mature 3T3-L1 adipocyte by 15- to 20-fold. Phenylarsine oxide, a trivalent arsenical that forms stable ring complexes with vicinal dithiols, prevents insulin-activated hexose uptake in a concentration-dependent manner (Ki = 7 microM) but has no inhibitory effect on basal hexose uptake. 2,3-Dimercaptopropanol at a level nearly stoichiometric to that of phenylarsine oxide prevents or rapidly reverses the inhibition of hexose uptake; 2-mercaptoethanol, even in high stoichiometric excess over the arsenical, does not reverse inhibition of hexose uptake. When phenylarsine oxide is added after adipocytes have been fully activated by insulin, 2-deoxy-[1-14C]glucose uptake rate decays slowly at a rate corresponding to that caused by the withdrawal of insulin (t1/2 = 10 min). Using the same conditions under which phenylarsine oxide blocked activation, the Km for deoxyglucose uptake, the rate at which 125I-insulin became cell-associated, and the 125I-insulin binding isotherm for solubilized insulin receptor were not affected by phenylarsine oxide. These results support the transporter translocation model for insulin-activated hexose transport and implicate vicinal sulfhydryl groups in a post-insulin binding event essential for the translocation of glucose transporters to the plasma membrane.

3-O-Methyl-D-glucose-resistant mutants were selected from Chinese hamster ovary cells after mutagenesis with ethyl methanesulfonate. A mutant, MegR24, was isolated which was significantly more resistant than the parent to 3-O-methylglucose. Uptake of 50 microM D-glucose by metabolizing MegR24 cells was 2- to 3-fold higher than the parental cells in the absence or presence of 100 mM 3-O-methylglucose. A study of transport of D-[3H]glucose in ATP-depleted cells indicated an apparent Km for D-glucose transport that was 3-fold lower for the mutant (2.7 +/- 0.3 mM) than for the parent (8.9 +/- 1.0 mM). The apparent Km for transport of 3-O-methylglucose by the mutant (10.9 +/- 2.4 mM) was almost 2-fold lower than that of the parent (21.4 +/- 4.7 mM). The Vmax values for transport of D-glucose or 3-O-methylglucose by the mutant and parental cell lines were not significantly different. The MegR24 mutant also exhibited enhanced countertransport of D-[3H]glucose following preloading of ATP-depleted cells with 100 mM 3-O-methylglucose. Simple diffusion of hexoses as measured by L-glucose uptake was not altered in the mutant. These results suggest that the MegR24 hexose carrier has an increased affinity for transport of hexoses and that the resistance to the cytotoxic effects of 3-O-methylglucose exhibited by MegR24 is due to its ability to transport D-glucose 2- to 3-fold more efficiently than the parental strain.

Incubation of chick embryo fibroblasts in glucose-free medium resulted in a dramatic increase in the rate of 2-deoxy-D-glucose transport. The greatest increase in rate occurred during the first 20 hours of incubation in glucose-free medium and was blocked by actinomycin D, dordycepin, or cycloheximide. The conditions of 2-deoxy-D-glucose concentration and time of incubation with the sugar were determined where transport rather than phosphorylation was rate-limiting in sugar uptake. These studies demonstrated that the transport of 2-deoxy-D-glucose was rate-limiting for only 1 or 2 min when the concentration of sugar in the medium was near the Km for transport, i.e. 2mM. No difference was found in the level of hexokinase activity in homogenates prepared from cells incubated glucose-free medium or standard medium when either 2-deoxy-D-[14C]glucose or D-glucose was used as substrate. A kinetic analysis of the initial rates of 2-deoxy-D-glucose transport by Lineweaver-Burk plots showed that the Vmax for sugar transport increased from 18 to 95 nmol per mg of protein per min when fibroblasts were incubated in glucose-free medium for 40 hours. The Km remained constant at 2 mM. Analysis of the initial rates of 3-omicron-methyl-D-glucose transport by Lineweaver-Burk plots further substantiated that the increase in sugar transport was due to an increase in the Vmax for transport with the Km remaining constant. The activation energy for the transport reaction calculated from an Arrhenius plot was 17.4 Cal per mol for cells cultured in the standard medium and 17.2 Cal per mol for cells cultured in the glucose-free medium. These results are consistent with the interpretation that the Vmax increase observed in hexose-starved cells is due to an increase in the number of transport sites.

Rapid kinetic techniques were employed to measure the transport of the nonmetabolizable hexose, 3-O-methyl-D-glucose, in suspensions of human HeLa cells, mouse L- and P388 leukemia cells, and Chinese hamster ovary cells in zero-trans entry and exit and equilibrium exchange procedures. The kinetic parameters of transport were computed by fitting appropriate integrated rate equations to time courses of transmembrane equilibration of radiolabeled substrate. Transport of all four lines, as in Novikoff rat hepatoma cells, conformed to a simple carrier model with directional symmetry but differential mobility of loaded and empty carrier. As was apparent from a comparison of influx and exchange flux, the loaded carrier of all cell types moved between 4 and 14 times faster than the empty carrier. ATP depletion of the cells by incubation in glucose-free medium containing KCN and iodoacetate had no significant effect on the kinetic properties of the transporter. ATP-depleted cells were used to measure the transport of D-glucose, 2-deoxyglucose, D-galactose, and D-glucosamine in the absence of intracellular metabolism. The differential mobilities of empty carrier and carrier loaded with these hexoses and the efficiency of their transport were equivalent to those observed with 3-O-methylglucose, but the Michaelis-Menten constants for the transport of D-galactose and D-glucosamine were 5-8-fold higher than those for D-glucose, 2-deoxy-D-glucose, and 3-O-methylglucose, which were about equivalent.

The effects of biotin upon the intracellular level of cGMP and the activity of glucokinase were examined in primary cultures of adult rat hepatocytes. The addition of biotin to the culture medium of hepatocytes increased their content of cGMP 3-fold within 1 h. A 4-fold increase in the activity of glucokinase was observed in response to the addition of the vitamin to the culture medium and the maximal response was observed 6 h following the addition to the medium. These maximum effects were noted when biotin was present in the culture medium at a concentration of 10(-6) M. The induction of glucokinase activity by biotin was preceded by an increase in the intracellular level of cGMP. The addition of 8-bromo-cGMP to the culture medium also increased the activity of glucokinase and its effects were not additive with respect to the effects of biotin. The induction of glucokinase by biotin or the cyclic nucleotide analog was not observed in the absence of insulin. The effects of biotin upon the activity of glucokinase could be mimicked by including glucose in the culture medium. When hexose utilization by the hepatocytes was blocked by the addition to the culture medium of N-acetylglucosamine, the induction of glucokinase by biotin was unaffected, whereas the induction brought about by glucose was not observed. The changes in the activity of the enzyme brought about by biotin or 8-bromo-cGMP was shown to arise as the result of changes in the rate of synthesis of the enzyme. In addition, using an in vitro translation assay and immunoprecipitation, it was found that biotin and 8-bromo-cGMP increased the amount of translatable mRNA coding for the enzyme.

Cytochalasin B inhibits phytomitogen-induced human lymphocyte proliferation with a Ki of approximately 6 X 10(-6) M. Cytochalasins A, C, D, E, and H are also inhibitory with varying degrees of potency, whereas cytochalasin G and chaetoglobosins A, B, C, E, F, and J are not at concentrations as high as 15 microM. Cytochalasin B also competitively inhibits carrier-mediated equilibrium exchange of hexose (Ki of approximately 7 X 10(-7) M), but cytochalasin E is ineffective. Cytochalasin B binds reversibly to the lymphocyte at three distinct sites: L, M, and H. The ligand binding at L site shows the apparent dissociation constant (Kd) of 1 to 3 X 10(-6) M and total binding sites (Bt) of 6 to 8 X 10(7)/cell, represents approximately 85% of the total saturable binding, displays a broad specificity interacting with cytochalasins C, D, and E, is not displaceable by D-glucose, is located mostly in a cytosol fraction, and exists in intimate relation to cytoskeletal actin. M site shows a Kd of 2 to 4 X 10(-7) M and Bt of 5 to 8 X 10(6)/cell, represents about 8% of the total saturable binding, shows stringent specificity not being displaced by cytochalasins C, D, and E, is competitively displaced by D-glucose and phloretin, and is quantitatively recoverable in the plasma membrane fraction. The binding to H site shows a Kd of 0.5 to 1.0 X 10(-7) M and Bt of 4 to 5 X 10(6)/cell, representing approximately 7% of the total saturable binding, shows a broad specificity, is insensitive to D-glucose, and is membrane bound. It is proposed that L site is actin and is involved in the inhibition of lymphocyte mitogenesis, whereas M site is associated with the hexose transport carrier. Structure-activity relationships of cytochalasin effects are also discussed.