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1.
The characteristics and regulatory nature of sugar transport in freshly isolated bovine adrenal chromaffin cells were investigated. Transport was measured by following the cell/medium distribution of non-metabolizable glucose analogue, 3-O-methyl-D-glucose. The uptake of 3-O-methyl-D-glucose was was mediated by a saturable transport system with a Km of 8.2 mM and a Vmax of 0.69 nmol/mg protein per min. Basal 3-O-methyl-D-glucose transport was competitively inhibited by D-glucose and a countertransport effect was demonstrated. Cytochalasin B and phloretin, which are specific inhibitors of carrier-mediated glucose transport, significantly decreased basal 3-O-methyl-D-glucose uptake. Basal transport was stimulated by 50 mU/ml insulin, an effect associated with an increase in Vmax. The stimulatory effect of insulin was depressed in medium lacking external Ca2+, or containing the Ca2+-antagonistic ion, La3+, or the Ca2+ channel blocker, methoxyverapamil (D-600). The data suggest that the uptake of 3-O-methyl-D-glucose in freshly isolated bovine adrenal chromaffin cells is mediated by a specific facilitated diffusion mechanism, and is subject to regulation by insulin, thus resembling sugar transport in muscle. In addition, the insulin effect appears to depend on the presence of extracellular Ca2+. 相似文献
2.
Sarcolemmal glucose transport in Ca2+-tolerant myocytes from adult rat heart. Calcium dependence of insulin action 总被引:1,自引:0,他引:1
Cardiac myocytes were isolated from adult rat ventricles by a method which preserves their functional integrity, including long survival in physiological concentrations of Ca2+. Sarcolemmal glucose transport was assessed by measuring linear initial uptake rates of the nonmetabolized glucose analog 3-O-methyl-D-glucose. Transport was saturable and showed competition by D-glucose and other features of chemical and stereo-selectivity. Transport was stimulated by insulin in a dose-dependent manner, resulting in an almost 5-fold increase in Vmax, with little change in Km. Stimulation of 3-methylglucose transport by insulin was largely Ca2+-dependent. Omission of Ca2+ from the incubation medium caused a minor rise in basal 3-methylglucose uptake but the insulin-stimulated rise in Vmax was only 30%. The Ca2+ antagonist D600 also antagonized stimulation of hexose transport by insulin. In all the above respects, 3-methylglucose transport in myocytes is identical to that in intact heart muscle. In addition, the decrease in insulin response by Ca2+ omission was partially reversed by subsequent return to a Ca2+-containing medium. ATP levels remained stable in the absence of Ca2+, showing that the Ca2+ dependence did not reflect nonspecific cell damage. 相似文献
3.
Modulation of basal glucose transporter Km in the adipocyte by insulin and other factors 总被引:3,自引:0,他引:3
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. 相似文献
4.
Glucose uptake in human and animal muscle cells in culture 总被引:5,自引:0,他引:5
Human muscle cells were grown in culture from satellite cells present in muscle biopsies and fusion-competent clones were identified. Hexose uptake was studied in fused myotubes of human muscle cells in culture and compared with hexose uptake in myotubes of the rat L6 and mouse C2C12 muscle cell lines. Uptake of 2-deoxyglucose was saturable and showed an apparent Km of about 1.5 mM in myotubes of all three cell types. The Vmax of uptake was about 6000 pmol/(min.mg protein) in human cells, 4000 pmol/(min.mg protein) in mouse C2C12 muscle cells, and 500 pmol/(min.mg protein) in L6 cells. Hexose uptake was inhibited approximately 90% by cytochalasin B in human, rat, and mouse muscle cell cultures. Insulin stimulated 2-deoxyglucose uptake in all three cultures. The hormone also stimulated transport of 3-O-methylglucose. The sensitivity to insulin was higher in human and C2C12 mouse myotubes (half-maximal stimulation observed at 3.5 X 10(-9) M) than in rat L6 myotubes (half-maximal stimulation observed at 2.5 X 10(-8) M). However, insulin (10(-6) M) stimulated hexose uptake to a larger extent (2.37-fold) in L6 than in either human (1.58-fold) or mouse (1.39-fold) myotubes. It is concluded that human muscle cells grown in culture display carrier-mediated glucose uptake, with qualitatively similar characteristics to those of other muscle cells, and that insulin stimulates hexose uptake in human cells. These cultures will be instrumental in the study of human insulin resistance and in investigations on the mechanism of action of antidiabetic drugs. 相似文献
5.
T Y Wong C A Murdock S P Concannon T D Lockey 《Biochimie et biologie cellulaire》1991,69(10-11):711-714
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. 相似文献
6.
Activation of glucose transport in muscle by prolonged exposure to insulin. Effects of glucose and insulin concentrations 总被引:5,自引:0,他引:5
D A Young J J Uhl G D Cartee J O Holloszy 《The Journal of biological chemistry》1986,261(34):16049-16053
Glucose transport activity was found to increase over 5 h in rat epitrochlearis muscle in response to a moderate concentration (50-100 microunits/ml) of insulin. This process was examined using 3-methylglucose. The increase in permeability to 3-methylglucose was 2- to 4-fold greater after 5 h than after 1 h in muscles incubated with 50 microunits/ml of insulin and 1 or 8 mM glucose. The increase in permeability to 3-methylglucose during the period between 1 and 5 h of exposure to 50 microunits/ml of insulin and 1 mM glucose was due to an increase in the apparent Vmax of sugar transport. There were two components to this activation of glucose transport. One, which was not influenced by inhibition of protein synthesis, resulted in activation of sugar transport to the same extent by 50 microunits/ml as by 20,000 microunits/ml of insulin; however, this activation took approximately 20 times longer with 50 microunits/ml insulin. The other, which was blocked by cycloheximide, resulted in a further activation of sugar transport to a level higher than that attained in response to 20,000 microunits/ml of insulin. Glucose had no effect on activation of sugar transport during the first hour, but a high concentration (20-36 mM) of glucose prevented the further activation of glucose transport during prolonged treatment with 50 microunits/ml of insulin. It appears from these results that prolonged exposure to a moderate concentration of insulin has previously unrecognized effects that include: a progressive activation of glucose transport over a long time that eventually results in as great a response as a "supramaximal" insulin concentration, and in the presence of low glucose concentration, further activation of glucose transport by an additional, protein synthesis-dependent mechanism. The results also show that a high concentration of glucose can, under some conditions, inhibit stimulation of its own transport. 相似文献
7.
Induction of sugar transport in chick embryo fibroblasts by hexose starvation. Evidence for transcriptional regulation of transport. 总被引:5,自引:0,他引:5
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. 相似文献
8.
We showed earlier that insulin stimulated sugar transport in adrenal chromaffin cells (Bigornia, L. and Bihler, I. Biochim. Biophys. Acta 885, 335-344). Transport regulation and its Ca2+ -dependence was further investigated in isolated bovine adrenal chromaffin cells, serving as a model of a homogeneous neuronal cell population. Uptake of the nonmetabolizable glucose analogue, 3-O-methyl-D-glucose was stimulated by hyperosmolar medium, and this effect was abolished in the absence of external Ca2+, or depressed in the presence of La3+ or the slow Ca2+ channel blocker methoxyverapamil. Basal transport was also stimulated by factors (acetylcholine, carbamylcholine, low-Na+ medium), which cause Ca2+ -dependent catecholamine release, and these effects were abolished in Ca2+ -free medium. In addition insulin, acetylcholine, hyperosmolar and low-Na+ medium significantly increased 45Ca uptake. Thus, glucose transport in adrenal chromaffin cells was stimulated by insulin and hyperosmolarity in a Ca2+ -dependent manner, as in muscle. Sensitivity to secretory stimuli, a regulatory feature perhaps characteristic of this cell type, was also demonstrated. In contrast to muscle, sugar transport was not affected by Na+ -pump inhibition, metabolic inhibitors or the Na+ ionophore monensin, suggesting that Ca2+ influx by Na+/Ca2+ exchange does not play a significant role in the activation of sugar transport in chromaffin cells. 相似文献
9.
The effect of insulin on glucose entry has been studied in monolayer cultures of human diploid fibroblastic cells. Influence of insulin on total cell glucose incorporation was evaluated using [14C] glucose. Glucose incorporation was increased up to two-fold in the presence of insulin. Insulin action occurred within 30 minutes and could be observed with insulin concentrations as low as 10(-10) M (10 microU)ml). The action of insulin was enhanced by preincubation in glucose-free medium. After glucose starvation the cells converted glucose primarily to glycogen and nucleotides, and the stimulation by insulin was observed equally in both fractions. Influence of insulin on the kinetics of hexose transport was studied using 2-deoxyglucose and 3-0-methyl glucose. A large diffusion component was corrected using rho-chloromercuribenzoic acid or phloridzin. Km for facilitated diffusion averaged 1.9 mM for 2-deoxyglucose and 5.3 mM for 3-O-methyl glucose, and Vmax ranged from 10-24 nmoles/min/mg cell protein. Insulin resulted in a 150% increase in Vmax with no significant change in Km. The data suggest that human diploid fibroblasts can be a useful system for the study of insulin's glucoregulatory action. 相似文献
10.
11.
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. 相似文献
12.
Cytochalasin B does not serve as a marker of glucose transport in rabbit erythrocytes 总被引:2,自引:0,他引:2
S G Albert 《Biochemistry international》1984,9(1):93-103
Glucose inhibitable cytochalasin B binding to erythrocyte membranes has been used as a marker of the glucose transporter. Glucose transport and cytochalasin B binding in rabbit erythrocytes differ from those activities found in human erythrocytes. We evaluated the uptake of 3-0-methylglucose and found similar Km (4.81 +/- 1.20 mM (SEM) and 6.59 +/- 0.72 mM) though significantly different Vmax (5.2 +/- 0.7 nM . min-1/10(9) cells and 234 +/- 13 nM X min -1/10(9) cells, p less than 0.001) for rabbit and human erythrocytes, respectively. Equilibrium binding of cytochalasin B to human erythrocyte membranes demonstrates a high affinity cytochalasin B binding site (Kd 38.6 +/- 22.7 nM) which is displaced by glucose. No comparable glucose inhibitable cytochalasin B site exists for rabbit erythrocyte membranes. Photoaffinity labeling of cytochalasin B confirms the presence of a glucose inhibitable cytochalasin B binding site in human, but not rabbit erythrocyte membranes. Cytochalasin B binding is a useful method in the identification of the glucose transporter in human cells, but the technique may be less useful in other species. 相似文献
13.
3-O-Methyl-D-glucose transport across the plasma membrane of isolated rat hepatocytes was followed for net entry of the sugar into sugar-free cells (zero trans entry), net exit of sugar into sugar-free medium (zero trans exit) and for unidirectional entry and exit fluxes when cells had been equilibrated with sugar in the extracellular medium (equilibrium exchange entry and exit). These measurements were performed at 20 degrees C and pH 7.4 by the use of simple manual methods. Initial rates of transport showed a Michaelis--Menten dependency on the sugar concentration at the cis side of the membrane over the range of concentrations tested (100 microM to 100 mM). Transport was found to be symmetrical with no evidence of substrate stimulation of transport from the trans side of the membrane. Parameters (mean values +/- S.E.M.) of transport were estimated as Vmax. 86.2 +/- 9.7 mmol/litre of cell water per min and Km 18.1 +/- 5.9 mM for exchange entry, Vmax. 78.8 +/- 5.3 mmol/litre of cell water per min and Km 17.6 +/- 3.5 mM for exchange exit, Vmax. 84.1 +/- 8.4 mmol/litre of cell water per min and Km 16.8 +/- 4.6 mM for zero trans exit. 相似文献
14.
At least two types of glucose transporter exist in cultured renal epithelial cells, a Na(+)-glucose cotransporter (SGLT), capable of interacting with D-glucose but not 2-deoxy-D-glucose (2dglc) and a facilitated transporter (GLUT) capable of interacting with both D-glucose and 2dglc. In order to examine the polarity of transport in cultured renal epithelia, 2dglc and D-glucose uptakes were measured in confluent cultures of LLC-PK1 cells grown on collagen-coated filters that permitted access of medium to both sides of the monolayer. The rates of basolateral uptake of both 1 mM glucose (Km 3.6 mM) and 1 mM 2dglc (Km 1.5 mM) were greater than apical uptake rates and the (apical-to-basolateral)/(basolateral-to-apical) flux ratio was high for glucose (9.4) and low for 2dglc (0.8), thus, confirming the lack of interaction of 2dglc with the apical SGLT. Specific glucose transport inhibitor studies using phlorizin, phloretin and cytochalasin B confirmed the polarised distribution of SGLT and GLUT in LLC-PK1 cells. Basolateral sugar uptake could be altered by addition of insulin (1 mU/ml) which increased 2dglc uptake by 72% and glucose uptake by 50% and by addition of 20 mM glucose to the medium during cell culture which decreased 2dglc uptake capacity at confluence by 30%. During growth to confluence, 2dglc uptake increased to a maximum, then decreased at the time of confluence, coincident with a rise in uptake capacity for alpha-methyl-D-glucoside, a hexose that interacts only with the apical SGLT. It was concluded that the non-metabolisable sugar 2dglc was a useful, specific probe for GLUT in LLC-PK1 cells and that GLUT was localised at the basolateral membrane after confluence. 相似文献
15.
Reassessment of the translocation hypothesis by kinetic studies on hexose transport in isolated rat adipocytes 总被引:2,自引:0,他引:2
K Suzuki 《The Journal of biological chemistry》1988,263(25):12247-12252
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. 相似文献
16.
Intracellular ATP has been reported either to stimulate [Jacquez, J.A. (1983) Biochim. Biophys. Acta 727, 367-378] or to inhibit [Hebert, D. N., & Carruthers, A. (1986) J. Biol. Chem. 261, 10093-10099] human erythrocyte sugar transport. This current study provides a rational explanation for these divergent findings. Protein-mediated 3-O-methyl-alpha-D-glucopyranoside (3OMG) uptake by intact human red blood cells (lacking intracellular sugar) at ice temperature in isotonic KCl containing 2 mM MgCl2, 2 mM EGTA, and 5 mM Tris-HCl, pH 7.4 (KCl medium), is characterized by a Km(app) of 0.4 +/- 0.1 mM and a Vmax of 114 +/- 20 mumol L-1 min-1. Lysis of red cells in 40 volumes of EGTA-containing hypotonic medium and resealing in 10 volumes of KCl medium increase the Km(app) and Vmax for uptake to 7.1 +/- 1.8 mM and 841 +/- 191 mumol L-1 min-1, respectively. Addition of ATP (4 mM) to the resealing medium restores Michaelis and velocity constants for zero-trans 3OMG uptake to 0.42 +/- 0.11 mM and 110 +/- 15 mumol L-1 min-1, respectively. Addition of CaCl2 to extracellular KCl medium (calculated [Ca2+]o = 101 microM) reduces the Vmax for zero-trans 3OMG uptake in intact cells and ATP-containing ghosts by 79 +/- 4% and 61 +/- 9%, respectively. Intracellular Ca2+ (15 microM) reduces the Vmax for 3OMG uptake by ATP-containing ghosts by 38 +/- 12%. In nominally ATP-free ghosts, extracellular (101 microM) and intracellular (11 microM) Ca2+ reduce the Vmax for 3OMG uptake by 96 and 94%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
17.
Mechanisms of the ability of insulin to activate the glucose-transport system in rat adipocytes. 总被引:15,自引:6,他引:9
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J M Olefsky 《The Biochemical journal》1978,172(1):137-145
Isolated rat adipocytes were used to assess the mechanisms of the ability of insulin to accelerate glucose transport. Glucose transport was determined by measuring the initial rates of 2-deoxyglucose uptake, and at 24 degrees C insulin increased the Vmax. of transport from 7.3 +/- 1 to 23.1 +/- 2 nmol/min per 10(6) cells, but the Km value remained unchanged (2.5, cf. 2.4 mM). When the Vmax. of basal and insulin-stimulated transport was measured as a function of temperature (15-37 degrees C), parallel Arrhenius plots were obtained yielding equal activation energies of approx. 59kJ/mol. Since both processes have equal activation energies the data indicate that insulin increases Vmax. by increasing the number of available carriers rather than enhancing intrinsic activity of already functioning carriers. Since the ability of insulin to activate glucose transport did not decrease with temperature (whereas plasma-membrane fluidity declines), it is suggested that lateral diffusion of insulin receptors within the plasma-membrane bilayer is not a rat-determining step in insulin action. 相似文献
18.
Insulin (0.1 microM) and 1 microM epinephrine each increased the uptake and phosphorylation of 2-deoxyglucose by the perfused rat heart by increasing the apparent Vmax without altering the Km. Isoproterenol (10 microM), 50 microM methoxamine and 10 mM CaCl2 also increased uptake. Lowering of the perfusate Ca2+ concentration from 1.27 to 0.1 mM Ca2+, addition of the Ca2+ channel blocker nifedipine (1 microM) or addition of 1.7 mM EGTA decreased the basal rate of uptake of 2-deoxyglucose and prevented the stimulation due to 1 microM epinephrine. Stimulation of 2-deoxyglucose uptake by 0.1 microM insulin was only partly inhibited by Ca2+ omission, nifedipine or 1 mM EGTA. Half-maximal stimulation of 2-deoxyglucose uptake by insulin occurred at 2 nM and 0.4 nM for medium containing 1.27 and 0.1 mM Ca2+, respectively. Maximal concentrations of insulin (0.1 microM) and epinephrine (1 microM) were additive for glucose uptake and lactate output but were not additive for uptake of 2-deoxyglucose. Half-maximal stimulation of 2-deoxyglucose uptake by epinephrine occurred at 0.2 microM but maximal concentrations of epinephrine (e.g., 1 microM) gave lower rates of 2-deoxyglucose uptake than that attained by maximal concentrations of insulin. The addition of insulin increased uptake of 2-deoxyglucose at all concentrations of epinephrine but epinephrine only increased uptake at sub-maximal concentrations of insulin. The role of Ca2+ in signal reversal was also studied. Removal of 1 microM epinephrine after a 10 min exposure period resulted in a rapid return of contractility to basal values but the rate of 2-deoxyglucose uptake increased further and remained elevated at 20 min unless the Ca2+ concentration was lowered to 0.1 mM or nifedipine (1 microM) was added. Similarly, removal of 0.1 microM insulin after a 10 min exposure period did not affect the rate of 2-deoxyglucose uptake, which did not return to basal values within 20 min unless the concentration of Ca2+ was decreased to 0.1 mM. Insulin-mediated increase in 2-deoxyglucose uptake at 0.1 mM Ca2+ reversed upon hormone removal. It is concluded that catecholamines mediate a Ca2+-dependent increase in 2-deoxyglucose transport from either alpha or beta receptors. Insulin has both a Ca2+-dependent and a Ca2+-independent component. Reversal studies suggest an additional role for Ca2+ in maintaining the activated transport state when activated by either epinephrine or insulin. 相似文献
19.
Amino acid transport was studied in primary cultures of parenchymal cells isolated from adult rat liver by a collagenase perfusion technique and maintained as a monolayer in a serum-free culture medium. These cells carried out gluconeogenesis from three carbon precursors (alanine, pyruvate, and lactate) in response to glucagon addition. Amino acid transport was assayed by measuring the uptake of the nonmetabolizable amino acid, alpha-aminoisobutyric acid (AIB). Addition of insulin or glucagon to culture rat liver parenchymal cells resulted in an increased influx of AIB transport. The glucocorticoid, dexamethasone, when added alone to cultures did not affect AIB transport. However, prior or simultaneous addition of dexamethasone to glucagon-treated cells caused a strong potentiation of the glucagon induction of AIB transport. Kinetic analysis of the effects of insulin and glucagon demonstrated that insulin increased the Vmax for transport without changing the Km while glucagon primarily decreased the Km for AIB transport. The effect of dexamethasone was to increase the Vmax of the low Km system. 相似文献
20.
Transport of sugars in chick-embryo fibroblasts. Evidence for a low-affinity system and a high-affinity system for glucose transport. 总被引:5,自引:0,他引:5
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The rate of D-glucose uptake by cells that had been deprived of sugar for 18-24h was consistently observed to be 15-20 times higher than that in control cells maintained for the same length of time in medium containing glucose. This increased rate of glucose transport by sugar-starved cells was due to a 3-5-fold increase in the Vmax. value of a low-affinity system (Km 1 mM) combined with an increase in the Vmax of a separate high-affinity system (Km 0.05-0.2 mM). The high-affinity system, which was most characteristic of starved cells, was particularly sensitive to low concentrations of the thiol reagent N-ethylmaleimide; 50% inhibition of uptake occurred at approx. 0.01 mM-N-ethylmaleimide. In contrast with the high-affinity system, the low-affinity system of either the fed cells or the starved cells was unaffected by N-ethylmaleimide. In addition to the increases in the rate of D-glucose transport, cells deprived of sugar had increased rates of transport of 3-O-methyl-D-glucose and 2-deoxy-D-glucose. No measurable high-affinity transport system could be demonstrated for the transport of 3-O-methylgucose, and N-ethylmaleimide did not alter the initial rate. Thus the transport of 3-O-methyglucose by both fed and starved cells was exclusively by the N-ethylmaleimide-insensitive low-affinity system. The low-affinity system also appeared to be the primary means for the transport of 2-deoxyglucose by fed and starved cells. However, some of the transport of 2-deoxyglucose by starved cells was inhibited by N-ethylmaleimide, suggesting that 2-deoxyglucose may also be transported by a high-affinity system. The results of experiments that measured transport kinetics strongly suggest that glucose can be transported by a least two separate systems, and 3-O-methylglucose and 2-deoxyglucose by one. Support for these interpretations comes from the analysis of the effects of N-ethylmaleimide and cycloheximide as well as from the results of competition experiments. The uptake of glucose is quite different from that of 2-deoxyglucose and 3-O-methylglucose. The net result of sugar starvation serves to emphasize these differences. The apparent de-repression of the transport systems studied presents an interesting basis for further studies of the regulation of transport in a variety of cells. 相似文献