Modulation of basal glucose transporter Km in the adipocyte by insulin and other factors

We have previously described experimental conditions where basal methylglucose transport in adipocytes exhibited an apparent Km of approximately 35 mM. Under those conditions insulin stimulated transport predominantly by decreasing the transport Km (Whitesell, R. R., and Abumrad, N. A. (1985) J. Biol. Chem. 260, 2894-2899). Our findings were in contrast with earlier reports that the Km of basal glucose transport was low (3-5 mM) and similar to that of transport in insulin-treated cells. In this study we have investigated the effect of different experimental conditions on the kinetics of basal glucose transport in adipocytes. When transport was assayed at 37 degrees C, cell agitation for 10 min prior to the transport assay decreased the basal Km from 35 to 12 mM. Deprivation of metabolic substrate produced a further reduction down to 2 mM. Refeeding starved cells with 1 mM glucose returned the Km back up to 12 mM in agitated cells and to 40 mM in stabilized cells. The effects of agitation to lower and of glucose to raise the basal Km were prevented by preincubating cells with dinitrophenol. Cell agitation or substrate lack did not alter the Vmax of basal transport and were without effect on both Km and Vmax in insulin-treated cells. The temperature dependencies of the kinetics of basal and stimulated transport were studied. A decrease in the assay temperature from 37 to 23 degrees C caused both basal Km and Vmax to drop proportionately from 25 to 5 mM, and 13 to 3.6 nmol/(microliter X min), respectively. In insulin-stimulated cells, only the Vmax was decreased (Km went from 3.5 to 3 mM, Vmax from 45 to 17 nmol/(microliter X min]. The results support the concept that experimental conditions can produce large changes in the Km of basal glucose transporters. Furthermore they explain why, under certain assay conditions (with temperatures around 23 degrees C or with deprivation of metabolic substrate), the effect of insulin on transport Km is not observed. Our data also suggest that basal transport characteristics do not persist in insulin-treated cells. We would propose that one of the actions of insulin (in addition to raising Vmax) is to change the characteristics of basal transporters by overriding metabolic factors which keep the Km high. Alternatively, insulin could cause the disappearance of basal transporters as new and different ones are recruited from intracellular stores.     

Characterization of threonine transport into a kidney epithelial cell line (BSC-1). Evidence for the presence of Na(+)-independent system asc [corrected

The transport routes for threonine in a primate kidney epithelial cell line (BSC-1) grown as monolayer in continuous cell culture were studied. We discovered at least four different transport systems for threonine uptake. The Na(+)-dependent route shows biphasic kinetics with a low and high affinity parameter. The apparent kinetic constants for Km1 and Km2 were 0.3 and 36 mM with apparent Vmax values of 6.3 and 90 nmol/mg protein/min, respectively. The high affinity, low Km component resembles system ASC activity, with respect to substrate selectivity. The Na(+)-independent route also exhibits biphasic kinetics. A high affinity component (apparent Km of 1.0 mM, and apparent Vmax of 7.2 nmol/mg protein/min) is sensitive to inhibition by leucine and the aminoendolevo-rotatory isomer of 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid, suggesting participation by system L. The low affinity component (apparent Km of 10.2 mM, and apparent Vmax of 71 nmol/mg protein/min) was specifically inhibited by threonine, serine, and alanine and could be assigned to system asc. The discrimination between system L and asc is based upon differences in pH sensitivity, trans stimulation, and Ki values. In addition, the effects of harmaline, a suspected sodium transport site inhibitor, have been studied. Harmaline noncompetitively inhibited Na(+)-dependent threonine uptake but had no effect on Na(+)-independent transport of threonine. This report is the first to present evidence for the presence of system asc in renal epithelial cells. The physiological and biochemical significance of our findings are discussed.     

Regulation of glucose transport in Candida utilis

The transport systems for glucose present in Candida utilis cells, growing in batch and continuous cultures on several carbon sources, have been studied. Two different systems were found: a proton symport and a facilitated diffusion system. The high-affinity symport (Km for glucose about 15 microM) transported one proton per mole of glucose and was partially constitutive, appearing in cells grown on gluconeogenic substrates such as lactate, ethanol and glycerol. It was also induced by glucose concentrations up to 0.7 mM and repressed by higher ones. The level of repression depended on the external glucose concentration at which cells had grown in a way similar to that shown by the maltose-uptake system, so both systems seem to be under a common glucose control. Initial uptake by facilitated diffusion, the only transport system present in cells growing at glucose concentrations higher than 10 mM, showed a complex kinetic dependence on the extracellular glucose concentration. This could be explained either by the presence of at least two different systems simultaneously active, one with a Km around 2 mM and the other with a Km of about 1 M, or by the allosteric or hysteretic behaviour of a single carrier whose apparent Km would oscillate between 2 and 70 mM.     

Infinite-cis kinetics support the carrier model for erythrocyte glucose transport

It has been claimed that the Km for infinite-cis uptake of glucose in human erythrocytes is so low that the carrier model for transport must be rejected. We redetermined this parameter for three experimental conditions and found instead that the Km values were in good agreement with the model. For each of a variety of cis glucose concentrations, cells were preequilibrated with various concentrations of glucose, and the apparent Km was determined as the intracellular concentration reducing the initial rate of net uptake by half. The dependence of the apparent Km values on the cis glucose was as predicted by the carrier model; the infinite-cis Km was determined from both this concentration dependence and the extrapolated value at infinite cis glucose. The resulting values were 15 mM for fresh blood at 0 degrees C, 39 mM for outdated blood at 0 degrees C, and 11 mM for outdated blood at 25 degrees C. Previous measurements of the Km at room temperature yielded values of 2-3 mM. These earlier studies used a time course procedure that indicated rapid changes in rates during the initial 10 s of uptake but did not directly measure such changes. We examined the uptake of 60 mM glucose at 20 degrees C into cells containing 0 and 5 mM glucose; rapid changes in rates were not observed in the first few seconds, and the time courses were more consistent with our higher Km values. Our new values, together with other initial rate measurements in the literature, support the adequacy of the carrier model to account for the kinetics of glucose transport in human erythrocytes.     

Simultaneous uptake of galactose and glucose by Azotobacter vinelandii.

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

Transport of 6-deoxyglucose in Saccharomyces cerevisiae.

The uptake of 6-deoxyglucose was measured in wild-type Saccharomyces cerevisiae, in a double mutant strain lacking activity for hexokinases A and B (hxkl hxk2), in a triple mutant strain lacking activity for both hexokinases and glucokinase (hxkl hxk2 glk), and in the triple mutant with high levels of activity of single kinases restored by introduction of the cloned genes. In the wild-type strain, uptake of the glucose analog showed two components, with Km values of ca. 20 mM ("high affinity") and 250 mM ("low affinity"), respectively. The double mutant also had high- and low-affinity components, but the triple mutant showed only low-affinity uptake. Reintroduction of the single kinases to the triple mutant restored high-affinity uptake. (Other experiments on 6-deoxyglucose uptake are also presented, including the apparent use of the galactose transport system when induced.) These results show that the recent implication of the kinases in transport of glucose (L.F. Bisson and D.G. Fraenkel, Proc. Natl. Acad. Sci. U.S.A. 80:1730-1734, 1983) applies equally to the nonmetabolized analog 6-deoxyglucose and suggests that the role of the kinases in transport is not merely a consequence of metabolism of the transported compound.     

Evidence for functionally distinct glucose transporters in basal and insulin-stimulated adipocytes

The activity and Km of glucose transport of rat adipocytes are quite variable in the basal state. This could be due to differing levels of highly saturable transport against a background of less saturable transport. Such heterogeneity could lead to differing conclusions as to the Km of basal cells compared to insulin-stimulated cells depending on the choice of substrate, the range of concentrations tested, and the rigor of data analysis. In the present work, we used a cell preparation which was stable and partially activated by constant agitation. We used a two-component model to fit the concentration dependence of D-glucose uptake. We defined two parallel pathways of glucose entry, a high-affinity/low-capacity pathway and a low-affinity/high-capacity pathway. Both pathways were stereospecific and were inhibited by cytochalasin B. The low-affinity pathway in basal cells had 97% of the total capacity (Vmax) with a high Km (greater than 50 mM). A second pathway had a very low Km (less than 1 mM) and only 3% of the total capacity, but contributed to 30-60% of glucose uptake at 8 mM glucose. In insulin-stimulated cells, a pathway with a Km of 4-5 mM dominated and contributed 85% of glucose transport. The low-affinity but not the very high affinity pathway persisted in stimulated cells, but its contribution was only 10-15% of transport at 8 mM glucose. These results suggest the presence of at least two functionally distinct transporters whose respective contributions can be characterized by nonlinear regression of data over a wide range of glucose concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sugar transport in Coprinus cinereus.

Two transport systems for glucose were detected: a high affinity system with a Km of 27 muM, and a low affinity system with a Km of 3.3 mM. The high affinity system transported glucose, 2-deoxy-D-glucose (Km = 26 muM), 3-O-methylglucose (Km = 19 muM), D-glucosamine (Km = 652 muM), D-fructose (Km = 2.3 mM) and L-sorbose (Km = 2.2 mM). All sugars were accumulated against concentration gradients. The high affinity system was strongly or completely inhibited by N-ethylmaleimide, quercetin, 2,4-dinitrophenol and sodium azide. The system had a distinct pH optimum (7.4) and optimum temperature (45 degrees C). The low affinity system transported glucose, 2-deoxy-D-glucose (Km = 7.5 mM), and 3-O-methylglucose (Km = 1.5 mM). Accumulation again occurred against a concentration gradient. The low affinity system was inhibited by N-ethylmaleimide, quercetin and 2,4-dinitrophenol, but not by sodium azide. The rate of uptake by the low affinity system was constant over a wide temperature range (30--50 degrees C) and was not much affected by pH; but as the pH of the medium was altered from 4.5 to 8.9 a co-ordinated increase in affinity for 2-deoxy-D-glucose (from 52.1 mM to 0.3 mM) and decrease in maximum velocity (by a factor of five) occurred. Both uptake systems were present insporelings germinated in media containing sodium acetate as sole carbon source. Only the low affinity system could initially be demonstrated in glucose-grown tissue, although the high affinity system was restored by starvation inglucose-free medium. The half-ti me for restoration of high affinity activity was 3.5 min and the process was unaffected by cycloheximide. Addition of glucose to an acetate-grown culture inactivated the high affinity system with a half-life of 5--7.5 s. Addition of cycloheximide to an acetate-grown culture caused decay of the high affinity system with a half-life of 80 min. Regulation is thus thought to depend on modulation of protein activity rather than synthesis, and the kinetics of glucose, 2-deoxy-D-glucose and 3-O-methylglucose uptake would be consistent with there being a single carrier showing negative co-operativity. Analysis of transport defective mutants revealed defects in both transport systems although the mutants used were alleles of a single gene. It is concluded that this gene (the ftr cistron) is the structural gene for an allosteric molecule which serves both transport systems.     

Role of microtubules in insulin and glucagon stimulation of amino acid transport in isolated rat hepatocytes

The effects of the microtubule inhibitor, colchicine, on insulin or glucagon stimulation of alpha-amino[1-14C]-isobutyric acid (AIB) transport were investigated in isolated hepatocytes from normal fed rats. Under all conditions tested, AIB uptake appeared to occur through two components of transport: a low affinity (Km approximately 50 mM) component and a high affinity (Km approximately 1 mM) component. Within 2 h of incubation, insulin and glucagon, at maximal concentrations, increase AIB (0.1 mM) uptake by 2- to 3-fold and 4- to 6-fold, respectively. Colchicine, at the low concentration of 5 X 10(-7) M, slightly reduces basal AIB transport, decreases by 80% the simulatory effect of insulin, and diminishes by 40% the stimulatory effect of either glucagon or dibutyryl cAMP. Kinetic analysis of AIB influx indicates that the drug inhibits the increase in Vmax of a high affinity (Km approximately 1 mM) component of transport stimulated by insulin or glucagon, without affecting the kinetic parameters of a low affinity component of transport (Km approximately 50 mM). Various short term hormonal effects of insulin and glucagon (changes in glucose, urea, and lactate production) were found not to be modified by the drug. Vinblastine elicits similar changes as colchicine on AIB uptake. Lumicolchicine, a colchicine analogue that does not bind to tubulin, has no effect. The concentration of colchicine (10(-7) M) required for half-maximal inhibition of hormone-stimulated AIB transport is in the appropriate range for specific microtubule disruption. These data suggest that microtubules are involved in the regulation of the insulin or glucagon stimulation of AIB transport in isolated rat hepatocytes.     

Evidence that protein kinase C may not be involved in the insulin action on cAMP phosphodiesterase: studies with electroporated rat adipocytes that were highly responsive to insulin.

Partially permeabilized rat adipocytes with a high responsiveness to insulin were prepared by electroporation and used to study the effect of 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) on insulin actions in adipocytes. H-7 is a well-documented inhibitor of several protein kinases, including protein kinase C; however, it does not rapidly enter adipocytes protected with the intact plasma membrane. The cells were suspended in Buffer X [4.74 mM NaCl, 118.0 mM KCl, 0.38 mM CaCl2, 1.00 mM EGTA, 1.19 mM Mg2SO4, 1.19 mM KH2PO4, 25.0 mM Hepes/K, 20 mg/ml bovine serum albumin, and 3 mM pyruvate/Na, pH 7.4] and electroporated six times with a Gene-Pulser (from Bio-Rad) set at 25 microF and 2 kV/cm. In cells electroporated as above, insulin stimulated (a) membrane-bound, cAMP phosphodiesterase approximately 2.6-fold when the hormone concentration was 10 nM and (b) glucose transport activity approximately 4.5-fold when the hormone concentration was raised to 100 nM. H-7 strongly inhibited the actions of insulin on both glucose transport (apparent Ki = 0.3 mM) and cAMP phosphodiesterase (apparent Ki = 1.2 mM) in electroporated adipocytes. H-7 also inhibited lipolysis in adipocytes; the apparent Ki value for the reaction in intact cells was 0.45 mM, and that in electroporated cells was 0.075 mM. It is suggested that a certain protein kinase or kinases that are significantly sensitive to H-7 may be involved in the insulin-dependent stimulation of glucose transport and that of phosphodiesterase. However, protein kinase C (or Ca2+/phospholipid-dependent protein kinase) may not be involved, at least, in the hormonal action on phosphodiesterase since the apparent Ki value of H-7 for the reaction is too high.     

Characterization of a novel variant of amino acid transport system asc in erythrocytes from Przewalski's horse (Equus przewalskii).

In thoroughbred horses, red blood cell amino acid transport activity is Na(+)-independent and controlled by three codominant genetic alleles (h, l, s), coding for high-affinity system asc1 (L-alanine apparent Km for influx at 37 degrees C congruent to 0.35 mM), low-affinity system asc2 (L-alanine Km congruent to 14 mM), and transport deficiency, respectively. The present study investigated amino acid transport mechanisms in red cells from four wild species: Przewalski's horse (Equus przewalskii), Hartmann's zebra (Zebra hartmannae), Grevy's zebra (Zebra grevyi), and onager (Equus hemonius). Red blood cell samples from different Przewalski's horses exhibited uniformly high rates of L-alanine uptake, mediated by a high-affinity asc1-type transport system. Mean apparent Km and Vmax values (+/- SE) for L-alanine influx at 37 degrees C in red cells from 10 individual animals were 0.373 +/- 0.068 mM and 2.27 +/- 0.11 mmol (L cells.h), respectively. As in thoroughbreds, the Przewalski's horse transporter interacted with dibasic as well as neutral amino acids. However, the Przewalski asc1 isoform transported L-lysine with a substantially (6.4-fold) higher apparent affinity than its thoroughbred counterpart (Km for influx 1.4 mM at 37 degrees C) and was also less prone to trans-stimulation effects. The novel high apparent affinity of the Przewalski's horse transporter for L-lysine provides additional key evidence of functional and possible structural similarities between asc and the classical Na(+)-dependent system ASC and between these systems and the Na(+)-independent dibasic amino acid transport system y+. Unlike Przewalski's horse, zebra red cells were polymorphic with respect to L-alanine transport activity, showing high-affinity or low-affinity saturable mechanisms of L-alanine uptake. Onager red cells transported this amino acid with intermediate affinity (apparent Km for influx 3.0 mM at 37 degrees C). Radiation inactivation analysis was used to estimate the target size of system asc in red cells from Przewalski's horse. The transporter's in situ apparent molecular weight was 158,000 +/- 2500 (SE).     

Repression and inhibition of transport systems for branched-chain amino acids in Salmonella typhimurium.

Kinetics of the transport systems common for entry of L-isoleucine, L-leucine, and L-valine in Salmonella typhimurium LT2 have been analyzed as a function of substrateconcentration in the range of 0.5 to 45 muM. The systems of transport mutants, KA203 (ilvT3) and KA204 (ilvT4), are composed of two components; apparent Km values for uptake of isoleucine, leucine, and valine by the low Km component are 2 muM, 2 to 3 muM, and 1 muM, respectively, and by the high Km component 30 muM, 20 to 40 muM, and 0.1 mM, respectively. The transport system(s) of the wild type has not been separated into components but rather displays single Km values of 9 muM for isoleucine, 10 muM for leucine, and 30 muM for valine. The transport activity of the wild type was repressed by L-leucine, alpha ketoisocaproate, glycyl-L-isoleucine, glycyl-L-leucine, and glycyl-L-methionine. That for the transport mutants was repressed by L-alanine, L-isoleucine, L-methionine, L-valine, alpha-ketoisovalerate, alpha-keto-beta-methylvalerate, glycyl-L-alanine, glycyl-L-threonine, and glycyl-L-valine, in addition to the compounds described above. Repression of the mutant transport systems resulted in disappearance of the low Km component for valine uptake, together with a decrease in Vmax of the high Km component; the kinetic analysis with isoleucine and leucine as substrates was not possible because of poor uptake. The maximum reduction of the transport activity for isoleucine was obtained after growing cells for two to three generations in a medium supplemented with repressor, and for the depression, protein synthesis was essential after removal of the repressor. The transport activity for labeled isoleucine in the transport mutant and wild-type strains was inhibited by unlabeled L-alanine, L-cysteine, L-isoleucine, L-leucine, L-methionine, L-threonine, and L-valine. D-Amino acids neither repressed nor inhibited the transport activity of cells for entry of isoleucine.     

Transport of glucose and fructose in rat hepatocytes at 37 degrees C

The kinetic parameters for transport of the nonmetabolizable glucose analogue 3-O-methyl-D-glucose and the relationship between transport and metabolism of D-glucose and D-fructose were determined in isolated rat hepatocytes at 37 degrees C and pH 7.4. 3-O-Methylglucose at a very low concentration (0.1 mM) equilibrated with the intracellular water with a rate constant of 0.41 s-1. Km for equilibrium exchange entry was 5.5 mM and Vmax was 2.2 mM X s-1 and similar results were obtained when using the zero-trans entry protocol. The rate constant for entry of tracer D-glucose was 0.15 s-1 and Km for glucose was about 20 mM. The phosphorylation rate for D-glucose was much slower than the transport rate. The rate constant for D-fructose entry was about 0.04 s-1, the apparent Km was about 100 mM and Vmax about 5 mM X s-1. The concentration dependence of 3-O-methylglucose inhibition of labelled fructose transport revealed biphasic kinetics indicating that fructose was transferred by both the glucose transporter and a fructose transporter. At concentrations lower than 1 mM, fructose metabolism appeared to be limited by the transport step.     

Glucose Transport in Astrocytes: Regulation by Thyroid Hormone

Primary cultures of astrocytes from newborn rat brain showed evidence of a substrate-saturable process for glucose transport. The system shows a relatively high affinity for the substrate, with an apparent Km of approximately 1 mM. Maintenance of the cells in medium containing thyroid-hormone-free serum for 3, 6, or 9 days resulted in significantly reduced rates of hexose transport. Addition of exogenous triiodothyronine to the transport incubation medium of these "hypothyroid" cells markedly increased the net rate of 2-deoxyglucose uptake within 60 s to values equal to or above those of control cultures (cells maintained in normal serum). These findings support a key role for thyroid hormone in the transport of glucose across plasma membranes of brain cells and demonstrate the presence of this regulatory system in astrocytes.     

Glucose-induced cAMP signalling in yeast requires both a G-protein coupled receptor system for extracellular glucose detection and a separable hexose kinase-dependent sensing process

In Saccharomyces cerevisiae, glucose activation of cAMP synthesis requires both the presence of the G-protein-coupled receptor (GPCR) system, Gpr1-Gpa2, and uptake and phosphorylation of the sugar. In a hxt-null strain that lacks all physiologically important glucose carriers, glucose transport as well as glucose-induced cAMP signalling can be restored by constitutive expression of the galactose permease. Hence, the glucose transporters do not seem to have a regulatory function but are only required for glucose uptake. We established a system in which the GPCR-dependent glucose-sensing process is separated from the glucose phosphorylation process. It is based on the specific transport and hydrolysis of maltose providing intracellular glucose in the absence of glucose transport. Preaddition of a low concentration (0.7 mM) of maltose to derepressed hxt-null cells and subsequent addition of glucose restored the glucose-induced cAMP signalling, although there was no glucose uptake. Addition of a low concentration of maltose itself does not increase the cAMP level but enhances Glu6P and apparently fulfils the intracellular glucose phosphorylation requirement for activation of the cAMP pathway by extracellular glucose. This system enabled us to analyse the affinity and specificity of the GPCR system for fermentable sugars. Gpr1 displayed a very low affinity for glucose (apparent Ka = 75 mM) and responded specifically to extracellular alpha and beta D-glucose and sucrose, but not to fructose, mannose or any glucose analogues tested. The presence of the constitutively active Gpa2val132 allele in a wild-type strain bypassed the requirement for Gpr1 and increased the low cAMP signal induced by fructose and by low glucose up to the same intensity as the high glucose signal. Therefore, the low cAMP increases observed with fructose and low glucose in wild-type cells result only from the low sensitivity of the Gpr1-Gpa2 system and not from the intracellular sugar kinase-dependent process. In conclusion, we have shown that the two essential requirements for glucose-induced activation of cAMP synthesis can be fulfilled separately: an extracellular glucose detection process dependent on Gpr1 and an intracellular sugar-sensing process requiring the hexose kinases.     

Absorption kinetics for leucine, cycloleucine and alpha-aminoisobutyric acid by rat small intestine in vivo

The intestinal absorption kinetics of three neutral amino acids, leucine, cycloleucine and alpha-aminoisobutyric acid, has been studied in rat jejunum in vivo, with luminal perfusion during successive periods, by measuring the passive component and the active transport. The mass-transfer coefficients of the passive process, are similar for the three amino acids and increase with the perfusion rate. The transport component, obtained from the difference between total absorption and passive diffusion, shows saturation kinetics and also increases with the perfusion rate. The apparent Michaelis constants, Km, and the maximal transport rates for the three amino acids have been determined. The Km values are greater than those reported for in vitro studies, a result imputable to greater thickness of the unstirred layers in vivo and to the unequal signification of the constant in both conditions. Passive flux has proved to be an important component for in vivo absorption, even at low substrate concentrations (1-5 mM), so that its evaluation cannot be neglected for the calculation ot the kinetic constants of the mediated transport.     

Glucose transport in vesicles reconstituted from Saccharomyces cerevisiae membranes and liposomes.

Glucose transport activity was reconstituted into liposomes by the freeze-thaw-sonication procedure from unextracted Saccharomyces cerevisiae membranes and preformed phospholipid liposomes. Fluorescence-dequenching measurements with octadecylrhodamine B chloride (R18)-labeled membranes showed that the yeast membrane lipids are diluted by the liposome lipids after the freeze-thaw-sonication procedure. At lipid-to-protein ratios greater than 75:1, vesicles with single transporters were formed. Reconstituted specific activity was increased at least twofold if the liposomes contained 50 mol% cholesterol. A further increase in specific activity, from 3- to 10-fold, was achieved by fractionation of the membranes on a Renografin gradient before reconstitution. Examination of the fractions from the Renografin gradient by sodium dodecyl sulfate-gel electrophoresis showed a parallel enrichment of glucose transport activity and a number of proteins including one with an apparent Mr of ca. 60,000, which might be the glucose transporter. Finally, preliminary kinetic analysis of glucose transport activity in vesicles reconstituted at a high lipid-to-protein ratio gave a Vmax of ca. 2.8 mumol/mg of protein per min at 23 degrees C and a Km of ca. 8 mM. The latter value corresponds to the kinase-independent, low-affinity component of glucose transport observed in wild-type cells.     

Vasopressin, insulin and peroxide(s) of vanadate (pervanadate) influence Na+ transport mediated by (Na+, K+)ATPase or Na+/H+ exchanger of rat liver plasma membrane vesicles

Uptake of 22Na+ by liver plasma membrane vesicles, reflecting Na+ transport by (Na+, K+)ATPase or Na+/H+ exchange was studied. Membrane vesicles were isolated from rat liver homogenates or from freshly prepared rat hepatocytes incubated in the presence of [Arg8]vasopressin or pervanadate and insulin. The ATP dependence of (Na+, K+)ATPase-mediated transport was determined from initial velocities of vanadate-sensitive uptake of 22Na+, the Na(+)-dependence of Na+/H+ exchange from initial velocities of amiloride-sensitive uptake. By studying vanadate-sensitive Na+ transport, high-affinity binding sites for ATP with an apparent Km(ATP) of 15 +/- 1 microM were observed at low concentrations of Na+ (1 mM) and K+ (1mM). At 90 mM Na+ and 60 mM K+ the apparent Km(ATP) was 103 +/- 25 microM. Vesiculation of membranes and loading of the vesicles prepared from liver homogenates in the presence of vasopressin increased the maximal velocities of vanadate-sensitive transport by 3.8-fold and 1.9-fold in the presence of low and high concentrations of Na+ and K+, respectively. The apparent Km(ATP) was shifted to 62 +/- 7 microM and 76 +/- 10 microM by vasopressin at low and high ion concentrations, respectively, indicating that the hormone reduced the influence of Na+ and K+ on ATP binding. In vesicles isolated from hepatocytes preincubated with 10 nM vasopression the hormone effect was conserved. Initial velocities of Na+ uptake (at high ion concentrations and 1 mM ATP) were increased 1.6-1.7-fold above control, after incubation of the cells with vasopressin or by affinity labelling of the cells with a photoreactive analogue of the hormone. The velocity of amiloride-sensitive Na+ transport was enhanced by incubating hepatocytes in the presence of 10 nM insulin (1.6-fold) or 0.3 mM pervanadate generated by mixing vanadate plus H2O2 (13-fold). The apparent Km(Na+) of Na+/H+ exchange was increased by pervanadate from 5.9 mM to 17.2 mM. Vesiculation and incubation of isolated membranes in the presence of pervanadate had no effect on the velocity of amiloride-sensitive Na+ transport. The results show that hormone receptor-mediated effects on (Na+, K+)ATPase and Na+/H+ exchange are conserved during the isolation of liver plasma membrane vesicles. Stable modifications of the transport systems or their membrane environment rather than ionic or metabolic responses requiring cell integrity appear to be involved in this regulation.     

Hexose transport in L6 rat myoblasts. I. Rate-limiting step, kinetic properties, and evidence for two systems

The hexose transport system of undifferentiated L6 rat myoblasts was investigated. 2-Deoxy-D-glucose (2-DOG) and 2-deoxy-2-fluoro-D-glucose (2FG) were used as analogues to investigate the rate-limiting step of hexose uptake into the cell. Virtually all of the 2-DOG or 2FG taken up into the cell was found to be in the phosphorylated form. No significant pool of intracellular free sugar could be detected. This demonstrates that hexose transport, not phosphorylation, is the rate-limiting step. The inhibitory effect of various glucose analogues on 2-DOG and 3-O-methyl-D-glucose (3-OMG) uptake revealed that these two sugars may be taken up into the cell by different carriers. In addition, kinetics analysis of the transport of both sugars also indicates that two hexose transport systems may be present in L6 cells. 2-DOG is transported by high and low affinity transport systems (Km 0.6 mM and 2.9 mM, respectively), whereas 3-OMG is transported by a low affinity system (Km 3.5 mM). Treatment of cells with ionophores or energy uncouplers results in inactivation of the high affinity system, but not the low affinity system.     

Insulin and glucagon stimulation of amino acid transport in isolated rat hepatocytes. Synthesis of a high affinity component of transport.

Insulin and glucagon stimulate amino acid transport in freshly prepared suspensions of isolated rat hepatocytes. The kinetic properties of alpha-amino[1-14C]isobutyric acid (AIB) transport were investigated in isolated hepatocytes following stimulation by either hormone in vitro. In nonhormonally treated cells (i.e. basal state), saturable transport occurred mainly through a low affinity (Km approximately equal to 40 mM) component. In insulin or glucagon-treated hepatocytes, saturable transport occurred through both a low affinity component (similar to that observed in the basal state) and a high affinity (Km approximately equal to 1 mM) component. At low AIB concentrations (less than 0.5 mM), insulin and glucagon at maximally stimulating doses increased AIB uptake about 2-fold and 5-fold, respectively. The high affinity component induced by either hormone exhibited the properties of the A (alanine preferring) mediation of amino acid transport. This component required 2 to 3 h for maximal expression, and its emergence was completely prevented by cycloheximide. Half-maximal stimulation was elicited by insulin at about 3 nM and by glucagon at about 1 nM. Dibutyryl cyclic AMP mimicked the glucagon effect and was not additive to it at maximal stimulation. Maximal effects of insulin and glucagon, or insulin and dibutyryl cyclic AMP, were additive. We conclude that insulin and glucagon can modulate amino acid entry in hepatocytes through the synthesis of a high affinity transport component.