Transport Pool Research Articles

Insulin-regulated Glucose Uptake in Rat Adipocytes Is Mediated by Two Transporter Isoforms Present in at Least Two Vesicle Populations

We have recently described a monoclonal antibody (1F8) that recognizes a form of glucose transporter unique to fat and muscle (James, D. E., Brown, R., Navarro, J., and Pilch, P. F. (1988) Nature 333, 183-185), tissues that respond acutely to insulin by markedly increasing their glucose uptake. Here, we report that rat adipocytes possess two immunologically distinct glucose-transporters: one recognized by 1F8, and one reactive with antibodies raised against the human erythrocyte glucose transporter. Immunoadsorption experiments indicate that these glucose transporters reside in different vesicle populations and that both transporter isoforms translocate from intracellular sites to the plasma membrane in response to insulin. The insulin-regulatable transporter resides in a unique vesicle that comprises 3% or less of the low density microsomes of fat cells and has a limited protein composition that does not include the bulk of another translocatable protein, the insulin-like growth factor II receptor. Immunoprecipitation with 1F8 of microsomal glucose transporters photoaffinity labeled with [3H]cytochalasin B brings down 90% of the label. Similarly, immunoprecipitation with 1F8 of glucose transporters from insulin-stimulated plasma membranes photolabeled with 3-[125I]iodo-4-azidophenethylamido-7-O-succinyldeacetyl-f ors kolin, another transporter-selective reagent, results in 75% of the labeled transporter localized in the immunoprecipitate. Thus, insulin action involves the combined effect of translocation from at least two vesicle pools each containing different glucose transporters. The 1F8-reactive transporter comprises the majority of the total transporter pool that is responsible for the insulin-induced increase in glucose transporter number.

Potential mechanism of insulin resistance in the aged Fischer rat adipocyte: depletion of the intracellular pool of glucose transporters with consequently fewer transporters in the plasma membrane in response to insulin

Potential mechanism of insulin resistance in the aged Fischer rat adipocyte: depletion of the intracellular pool of glucose transporters with consequently fewer transporters in the plasma membrane in response to insulin

Mechanism of mitogen-induced stimulation of glucose transport in human peripheral blood mononuclear cells. Evidence of an intracellular reserve pool of glucose carriers and their recruitment.

The present study examines the effects of phytohemagglutinin stimulation of a population of human (h) PBMC enriched in lymphocytes (hPBMC) on D-glucose displaceable cytochalasin B binding sites or medium-affinity sites (M-sites) in relation to glucose transport. Previously we have shown that M-sites are glucose transporters in hPBMC (Mookerjee, B.K., et al. 1981. J. Biol. Chem. 256:1290-1300). Equilibrium exchange of 3-O-methyl D-glucose in unstimulated cells revealed two populations with fast and slow flux rates. Phytohemagglutinin stimulates flux rates by converting part of the slow flux population to the fast flux population. M-sites occur in two distinct pools, one in plasma membrane and the other in microsomal fraction. Phytohemagglutinin treatment increases the plasma membrane pool size of M-sites with a concomitant reduction in the microsomal pool size without affecting the binding affinities or the total number of M-sites/cell. Data presented in this paper demonstrate that there are two pools of glucose transporters in these cells and phytohemagglutinin stimulation induces an energy-dependent net translocation of glucose transporters from an intracellular reserve pool to the plasma membrane, which accounts for greater than 60% of the increment in glucose transport.

Cycling of Amino-Nitrogen and other Nutrients between Shoots and Roots in Cereals—A Possible Mechanism Integrating Shoot and Root in the Regulation of Nutrient Uptake

A split root system was used to investigate the cycling of nitrogen between shoots and roots in young wheat and rye plants. 15N nitrate was supplied to one part of the root system for various periods, at the end of which these roots were excised. Xylem sap was then collected from the other roots which had not been supplied directly with 15N-nitrate. 15N detected in the xylem sap indicated cycling of nitrogen between shoots and roots. Calculations showed that over 60% of the amino-N flux in the xylem was cycling. Thus nitrate assimilation in the root could account for only a minor part of amino-N in the xylem sap. The specific activity of 15N in the total N of xylem sap was higher than in the total N of roots and shoots through which it had cycled. This is because exchange between amino-N in the transport pools and bulk tissue N is limited. It is proposed that there is, in effect, a single regulatory pool of amino-N, common to shoots and roots, and that this pool may be a key element in the control of N uptake at the level of the whole plant. The likely energy costs of cycling and implications for the partitioning of N between shoots and roots are discussed. In further investigations the cycling of 42K-potassium and 32S-sulphur was demonstrated.

Depression of Nitrate and Ammonium Transport in Barley Plants with Diminished Sulphate Status. Evidence of Co-regulation of Nitrogen and Sulphate Intake

When barley plants were grown in a solution with nitrate as the sole N-source but deprived of sulphate (−Splants) for 1 to 5 d, the capacity for sulphate transport by the roots increased very markedly; subsequent measurement of influx using 35S-labelled SO42- showed increases of > 10-fold compared to plants continuously supplied with sulphate (+S plants). There were only small effects on plant growth over a 5 d period and yet the influx of NO3-⁠, labelled with the short-lived tracer 13N, was diminished by approximately 30%. By contrast, the influx of phosphate was little affected by sulphate-deprivation. When a sulphate supply was restored to − S plants, the sulphate influx was quickly repressed over the subsequent 24 h and the nitrate influx was restored to >90% of the value in +S plants. When plants were grown in a solution with a mixed nitrate and ammonium supply and deprived of sulphate for 1 d or 5 d the depression of nitrate influx was more strongly marked (up to 55% depression). The influx of ammonium was also depressed after 5 d of sulphate-deprivation, but not at 1 d, nor where the concentration of ammonium in the uptake solution was lowered to 20 mmol m−3 or less. Additional measurements with 15N-labelled nitrate and ammonium over longer periods were used to determine net uptake. Net uptake of nitrate was depressed to a similar extent to efflux, but net ammonium uptake was depressed only in unbuffered uptake solution where the pH decreased to pH 4.9 during the uptake period. The 15N-tracer experiments showed that the translocation of label to the shoot, from both nitrate and ammonium, was depressed to a greater extent than net uptake in −S plants. The depression of nitrate influx, caused by 5 d of sulphate deprivation, could be relieved almost completely by providing plants with 1.0 mol m−3 L-methionine during the day prior to influx measurement. This treatment substantially decreased sulphate and potassium (86Rb-labelled) influx in both +S and −S plants, but greatly increased total S-status of the plants. This methionine treatment had no effect on ammonium influx or net uptake in − S plants but increased influx significantly in +S ones. When plants were grown with sulphate but deprived of nitrate for 4 d there was a marked depression of the sulphate influx (by 48–65%) but a smaller effect on phosphate influx (21–37% of +N). The results are discussed in relation to the effects of sulphate-deprivation on growth rate and the root: shoot weight ratio. It is concluded that the effects on influx and net uptake of nitrogen are more severe than could be accounted for by these factors. The decreased translocation of either nitrate, or the products of nitrate and ammonium assimilation from the roots, is suggested as a reason for the depression of influx. The restoration of nitrate influx and net uptake by methionine suggests that, for this ion at least, a shortage of S-amino acids within the plant may lead to the accumulation of inhibitory concentrations of non-S amino acids in the transport pool.

Exercise-induced increase in glucose transporters in plasma membranes of rat skeletal muscle.

A previously developed technique for the isolation of plasma and intracellular membrane fractions from rat skeletal muscle was used to investigate transporter migration after insulin treatment or a bout of exercise (45 min of treadmill). Glucose-inhibitable cytochalasin-B binding was used to estimate the number of glucose transporters. Insulin and exercise caused increases in glucose uptake into the hindlimb muscles of 5- and 3-fold, respectively. Each stimulus also caused a 2-fold increase in the number of glucose transporters in plasma membranes prepared from hindlimb muscles. The insulin-induced increase in plasma membrane transporters was accompanied by a concomitant decrease in transporters from the intracellular pool. In contrast to insulin, there was no concomitant decrease in the number of cytochalasin-B-binding sites in the intracellular membrane fraction from exercised muscles. The ability of both insulin and exercise to increase the number of transporters in the plasma membrane is in accordance with recruitment of transporters as one cause of increased transport activity. However, the inability of exercise to decrease the number of transporters in the insulin-sensitive intracellular pool suggests the existence of either a second recruitable transporter pool or masked glucose transporters in the plasma membrane that are unmasked by the muscle contractile activity.

Glucose transport into rat skeletal muscle: interaction between exercise and insulin.

This study was done to evaluate the effect of insulin on sugar transport into skeletal muscle after exercise. The permeability of rat epitrochlearis muscle to 3-O-methylglucose (3-MG) was measured after exposure to a range of insulin concentrations 30, 60, and 180 min after a bout of exercise. Thirty and 60 min after exercise, the effects of exercise and insulin on 3-MG transport were additive over a wide range of insulin concentrations, with no increase in sensitivity or responsiveness to insulin. After 180 min, when approximately 66% of the exercise-induced increase in sugar transport had worn off, both the responsiveness and sensitivity of the glucose transport process to insulin were increased. These findings appear compatible with the hypothesis that the actions of exercise and insulin result in activation and/or translocation into the plasma membrane of two separate pools of glucose transporters in mammalian skeletal muscle.

Relationship of 65Zn Absorption Kinetics to Intestinal Metallothionein in Rats: Effects of Zinc Depletion and Fasting

Intestinal 65Zn transport and metallothionein levels were examined in rats fed zinc-adequate and zinc-deficient diets and in rats subjected to an overnight fast. 65Zn uptake by intestines perfused with 1.5 microM 65Zn was greater in both zinc-deficient and fasted groups than in the control group. Mucosal retention of 65Zn was also greater in the zinc-deficient group but not in the fasted group. The greater 65Zn uptake in the fasted group was associated with a compartment that readily released 65Zn back into the lumen. Kinetic analysis of the rate of 65Zn transfer to the vascular space (absorption) showed that 65Zn absorption involved approximately 3% of mucosal 65Zn in a 40-min perfusion period. The half-life (t1/2) of this mucosal 65Zn rapid transport pool corresponded directly to changes in intestinal metallothionein levels. Both metallothionein and t1/2 were higher in the fasted group and lower in the zinc-deficient group than in controls. While the rate of 65Zn transport from this rapid transport pool decreased with increasing metallothionein level, the predicted pool size increased when the metallothionein level was elevated by fasting. These results indicate that the rate of zinc absorption is inversely related to intestinal metallothionein levels, but the portion of mucosal 65Zn available for absorption is directly related to intestinal metallothionein.

The action of phenylarsine oxide on the stereospecific uptake of D-glucose in basal and insulin-stimulated rat adipocytes

Phenylarsine oxide (PAO) has been used to inhibit the stereospecific uptake of D-glucose in basal and insulin-stimulated rat adipocytes. The inhibition is dose dependent and is partially reversed by dithiothreitol. The results are consistent with a direct interaction between the glucose transporter and PAO. By manipulating the sequence of exposure of cells to PAO and insulin it is possible to differentiate between the effects of PAO on transport into cells with receptor-rich and transporter-rich plasma membranes. PAO rapidly inhibits transport in insulin-stimulated adipocytes but at low concentrations inhibition is transient and recovery of stereospecific uptake takes place after approx. 20 min. The results can be interpreted in terms of the recruitment mechanism of insulin stimulation of transport and demonstrate that a relatively large intracellular pool of transporters exists after insulin stimulation. It also follows that sulphydryl groups probably play a critical role in the mechanism of glucose uptake.

Reversal of insulin resistance in diabetic rat adipocytes by insulin therapy. Restoration of pool of glucose transporters and enhancement of glucose-transport activity.

To determine the role of insulin in reversing the insulin resistance associated with depletion of the intracellular pool of glucose transporters, streptozocin-induced diabetic rats were treated with 5 U/day s.c. of insulin for 0, 8, or 14 days. At each time point, adipose cells were isolated, and 3-O-methylglucose transport was measured in the absence and presence of 1000 microU/ml insulin. With the cytochalasin B-binding assay, concentrations of glucose transporters in the plasma and the low-density microsomal membrane fractions were determined. Eight-day insulin therapy enhanced glucose transport rate (mean +/- SE) from 0.2 +/- 0.0 to 1.1 +/- 0.1 fmol X cell-1 X min-1 in the basal state and from 0.8 +/- 0.1 to 5.5 +/- 0.4 fmol X cell-1 X min-1 in the insulin-stimulated state in untreated and treated diabetic rats, respectively; this is a 3-fold increment of glucose transport rate in both states compared with control rats. After 14-day insulin therapy, glucose-transport activity declined toward normal but still remained approximately 1.5- and 4-fold higher than control and diabetic rats, respectively. Despite the persistent enhancement of glucose transport rate, concentration of glucose transporters in the intracellular pool was restored only to its prediabetic state. Likewise, the increased concentration of glucose transporters in the plasma membranes after insulin stimulation was similar to that of control rats. Thus, we suggest that 8-14 days of insulin therapy reversed the insulin resistance in diabetic rat adipocytes by at least two mechanisms: restoration of the intracellular pool of glucose transporters and enhancement of glucose-transport activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Reversal of insulin resistance in diabetic rat adipocytes by insulin therapy. Restoration of pool of glucose transporters and enhancement of glucose-transport activity

Reversal of insulin resistance in diabetic rat adipocytes by insulin therapy. Restoration of pool of glucose transporters and enhancement of glucose-transport activity

Accumulation and Conversion of Sugars by Developing Wheat Grains

The extent to which wheat grain growth is dependent on transport pool solute concentration was investigated by the use of illumination and partial grain removal to vary solute concentrations in the sieve tube and endosperm cavity saps of the wheat ear (Triticum aestivum L.). Short-term grain growth rates were estimated indirectly from the product of phloem area, sieve tube sap concentration, and (32)P translocation velocity. On a per grain basis, calculated rates of mass transport through the peduncle were fairly constant over a substantial range in other transport parameters (i.e. velocity, concentration, phloem area, and grain number). The rates were about 40% higher than expected; this probably reflects some unavoidable bias on faster-moving tracer in the velocity estimates. Sieve tube sap concentration increased in all experiments (by 20 to 64%), with a concomitant decline in velocity (to as low as 8% of the initial value). Endosperm cavity sucrose concentration also increased in all experiments, but cavity sap osmolality and total amino acid concentration remained nearly constant. No evidence was found for an increase in the rate of mass transport per grain through the peduncle in response to the treatments. This apparent unresponsiveness of grain growth rate to increased cavity sap sucrose concentration conflicts with earlier in vitro endosperm studies showing that sucrose uptake increased with increasing external sucrose concentration up to 150 to 200 millimolar.

Role of basolateral cell membranes in organic solute reabsorption in rabbit kidneys.

The present work explores the contributions of basolateral carrier systems in tubular reabsorption of organic solutes. Reabsorption of sugars and amino acids, as previously shown, can be represented by a three-compartment linear model that predicts that 1) if basolateral transport contributes to sugar reabsorption, alpha-methylglucoside reabsorption compared with that of glucose should be characterized by a longer transepithelial transit time (TET) and a correspondingly increased cellular transport pool (S), and 2) saturation of basolateral amino acid carriers, or presence of competing amino acids or other basolateral transport inhibitors, should prolong TET of a test amino acid, and increase S above the expected value. Both predictions were fully confirmed. Heavy metal intoxication not only inhibits transport of amino acids at the brush border, but also prolongs their TET and increases the size of S for a given reabsorbed load. Basolateral extrusion of amino acids is more sensitive to metals than is uptake across the brush border. Although basolateral carriers accelerate return of reabsorbed solute to blood, their contribution to reabsorption does not seem to be mandatory.

Sodium transport and the cellular sodium transport pool of colonic epithelium: effects of sodium loading, aldosterone and lithium

The cellular sodium transport pool and sodium transepithelial fluxes were investigated in vivo in rat distal colon in relation to sodium loading by intravenous infusion (3.5 h), and to short (4 h) and prolonged (72 h) i.v. administration of aldosterone. Considerable natriuresis and increase in body sodium content were produced by the sodium load but there was no significant effect on the transcellular sodium flux (active absorption from lumen to plasma) or on the sodium transport pool. Both short and prolonged aldosteronism produced similar increases in the transport pool and in the transcellular sodium flux, but the transepithelial electrical potential difference (p.d.) was significantly greater in rats given the prolonged infusion. Addition of amiloride to the solution in the lumen of the colon almost completely abolished the p.d., the transport pool and the transcellular sodium flux of the rats receiving prolonged infusion, but had much less effect in those given the short infusion. The time-course of recovery of p.d. following prolonged aldosteronism was similar to that described for the turnover rate of rat colonic epithelial cells. Lithium within the lumen had no significant effect in untreated rats but after prolonged aldosterone infusion lithium reduced the p.d. and the transcellular sodium flux although the transport pool was not reduced. These findings are consistent with the hypothesis that aldosteronism renders the apical membranes of the epithelial cells permeable to lithium and that intracellular accumulation of lithium depresses active sodium transfer.(ABSTRACT TRUNCATED AT 250 WORDS)

COMPARTMENTATION AND FLUXES OF SUCROSE IN INTACT LEAF BLADES OF BARLEY

SummarySucrose in barley leaf blades can be considered as being compartmented between two types of pool: transport (including mesophyll cytosol and apoplast, and vascular tissue) and vacuolar. The relative sizes of these pools, and their changes over a light/dark cycle, have been estimated using 14C labelling of the intact leaf and washout of 14C from leaf discs. Both types of pool increase during the photoperiod, the proportional distribution between them remaining the same; roughly one‐fifth of the sucrose is in the transport pool. The rate‐constants describing translocation, and loading and unloading of the vacuole, show diel changes, and fluxes of sucrose across the tonoplast are likely to be critical in controlling fluxes of photosynthetically fixed carbon within and out of barley leaves. The unloading of sucrose from vacuoles is probably the site where control is exercised; this conclusion is supported by a simple simulation model.

Modulation of glucose transport in hamster adipocytes by insulin and by?- and? 2-adrenoceptor agonists

Glucose transport in hamster adipocytes and its modulation by insulin and isoprenaline was characterized with the aid of the non-metabolizable hexose 3-0-methylglucose. Insulin stimulated the initial uptake rates by an increase in Vmax of the transport without any detectable change in Km. The hormone concentration producing half maximal stimulation was identical to that required in rat adipocytes. However, hamster adipocytes were much less responsive to insulin (3-fold stimulation as compared to a 12-fold stimulation in rat fat cells), and maximal transport rates were 10-fold lower than that observed in rat adipocytes. Accordingly, the number of glucose transporters, as assessed by glucose-inhibitable cytochalasin-B binding, was considerably lower in plasma membranes of hamster adipocytes. Moreover, no transporters were detected in the low-density microsomes which in insulin-sensitive cell types represent the intracellular pool of recruitable glucose transporters. The relative insulin resistance of the hamster fat cells may therefore be due to a depleted pool of intracellular glucose transporters. In the presence of adenosine, the beta-adrenoceptor agonist isoprenaline produced a moderate stimulation of the basal transport rate which was antagonized by the alpha 2-agonist clonidine. If adenosine deaminase was added in order to remove endogenous adenosine, isoprenaline inhibited the insulin-stimulated transport by 50%. In contrast to the stimulatory effects of insulin and isoproterenol, the inhibitory effect of the catecholamine was reversed by cooling the cells to 22 degrees. Glucagon produced a comparable inhibition, suggesting that the inhibitory effect was mediated by adenylate cyclase or its regulatory subunits.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin binding and insulin action in cultured fibroblasts: significant differences between a phosphoglucose isomerase-deficient mutant and the parental strain.

The interrelationships of hexose feeding and insulin action were studied in the Chinese hamster fibroblast cell lines 023 and DS-7. The latter, derived from 023 and deficient in phosphoglucose isomerase, has been used to map the metabolic requirements for aldohexose-mediated down-regulation or "curbing" of hexose transport. We have characterized insulin binding and the response to insulin in both cell lines to determine if the insulin-mediated stimulation of transport is similarly dependent on hexose metabolism. DS-7 cells exhibited 5-6 times as many high-affinity insulin binding sites as the parental strain. Apart from this difference, 023 and DS-7 cells showed comparable insulin binding characteristics, which are similar to those observed in other cell types. Insulin at a concentration of 1 microgram/ml (167 nM) was found to stimulate 3-O-methylglucose uptake by approximately equal to 50% in glucose-fed cells of both lines. In neither line did glucose starving significantly alter insulin binding or the insulin-induced stimulation of transport. Feeding with mannose or fructose was found to increase both parameters in 023 cells but had no effect on DS-7 cells. The increase in hexose uptake with the administration of insulin or with glucose starving was shown to be due to an increase in Vmax. Our studies suggest that insulin binding and effect are not regulated by hexose metabolism in the same manner as in the curbing process and insulin induces the recruitment of a transporter pool that is insensitive to hexose curbing.

Repeated exercise regulates glucose transport capacity in skeletal muscle.

The present study was undertaken to test the hypothesis that a muscle's habitual level of contractile activity might act as a long-term regulator of glucose transport capacity. Rats were rendered severely diabetic by injection of streptozotocin, 125 mg kg-1 body wt. Three days after the injection, insulin-stimulated and contraction-induced 3-methylglucose transport in the in vitro epitrochlearis muscle preparation was reduced to 25% and 52% of control capacity, respectively (P less than 0.001). However, when repeated 1-h swimming sessions (twice a day) were introduced during the 3 days of insulin deficiency, the reduction in contraction-induced glucose transport capacity was completely abolished. This indicates that the habitual level of contractile activity of a muscle regulates its capacity for exercise-stimulated glucose transport. Furthermore, one exercise session and a maximal insulin stimulus had an additive effect on 3-methylglucose transport, suggesting that there are two pools of intracellular glucose transporters or two mechanisms for mobilizing the same pool.

Regulation of intracellular sodium and pH by the electrogenic H+ pump in frog skin

We have investigated the role of the electrogenic hydrogen ion pump in the regulation of intracellular sodium ion activity (aiNa) and intracellular pH (pHi) in frog skin epithelial cells using double-barreled ion sensitive microelectrodes. When Rana esculenta skin is mounted in an Ussing chamber and bathed in 1 mM Na2SO4 buffered to pH 7.34 with imidazole on the apical side and in normal Ringer on the serosal side, the apical addition of the carbonic anhydrase inhibitor ethoxzolamide (10(-4)M) blocks net H+ ion excretion and Na absorption, producing a depolarization of 25-30 mV of the apical membrane potential (psi mc). We demonstrate that these changes are accompanied by a fall in aiNa from 6.2 +/- 0.5 mmol/l to 3.4 +/- 0.6 mmol/l and an increase in pHi from 7.20 +/- 0.03 to 7.38 +/- 0.08 (n = 12 skins). Voltage clamping psi mc to its control value in the presence of ethoxzolamide restored aiNa but the pHi remained alkaline. Furthermore, the fall in aiNa produced by ethoxzolamide could be mimicked by voltage clamping psi mc towards the value of the Nernst potential for Na at the apical membrane. These results indicate that the maintenance of the cellular Na+ transport pool is dependent on a favourable electrical driving force and counter-current generated by an electrogenic H+ pump at the apical membrane. Addition of amiloride (10(-5) mol/l) or substitution of external Na+ by Mg2+ or K+ caused a hyperpolarization of psi mc and a fall in aiNa.2+ e

Na+ and K+ transport at basolateral membranes of epithelial cells. III. Voltage independence of basolateral membrane Na+ efflux.

Na+ efflux across basolateral membranes of isolated epithelia of frog skin was tested for voltage sensitivity. The intracellular Na+ transport pool was loaded with 24Na from the apical solution and the rate of isotope appearance in the basolateral solution (JNa23) was measured at timed intervals of 30 s. Basolateral membrane voltage was depolarized by either 50 mM K+, 5 mM Ba++, or 80 mM NH+4. Whereas within 30 s ouabain caused inhibition of JNa23, depolarization of Vb by 30-60 mV caused no significant change of JNa23. Thus, both pump-mediated and leak Na+ effluxes were voltage independent. Although the pumps are electrogenic, pump-mediated Na+ efflux is voltage independent, perhaps because of a nonlinear relationship between pump current and transmembrane voltage. Voltage independence of the leak Na+ efflux confirms a previous suggestion (Cox and Helman, 1983. American Journal of Physiology. 245:F312-F321) that basolateral membrane Na+ leak fluxes are electroneutral.