Modulation of glucose uptake in animal cells. Studies using plasma membrane vesicles isolated from nontransformed and simian virus 40-transformed mouse fibroblast cultures.

Plasma membrane vesicles isolated from nontransformed and Simian virus 40-transformed mouse fibroblast cultures catalyzed carrier-mediated D-glucose transport without detectable metabolic conversion to glucose 6-phosphate. Glucose transport activity was stereospecific, temperature-dependent, sensitive to inactivation by p-chloromercuriphenylsulfonate, and accompanied plasma membrane material during subcellular fractionation. D-Glucose efflux from vesicles was inhibited by phloretin, an inhibitor of glucose uptake in intact cells. Cytochalasin B, a potent inhibitor of glucose uptake when tested with the intact cells used for vesicle isolation did not inhibit glucose transport in vesicles despite the presence of high affinity cytochalasin binding sites in isolated membranes. The enhanced glucose uptake observed in intact cells after viral transformation was not expressed in vesicles: no significant differences in glucose transport specific activity could be detected in vesicle preparations from nontransformed and transformed mouse fibroblast cultures. These findings indicate that cellular components distinct from glucose carriers can mediate changes in glucose uptake in mouse fibroblast cultures in at least two cases: sensitivity to inhibition by cytochalasin B and the enhanced cellular sugar uptake observed after viral transformation.     

Potential mechanism of insulin action on glucose transport in the isolated rat diaphragm. Apparent translocation of intracellular transport units to the plasma membrane

[3H]Cytochalasin B binding and its competitive inhibition by D-glucose have been used to quantitate the number of functional glucose transport units in plasma and microsomal membranes prepared from intact rat diaphragm. In a series of three experiments, plasma membranes prepared from diaphragms which have not been incubated with insulin bind approximately 16 pmol of cytochalasin B/mg of membrane protein to the D-glucose-inhibitable binding site. If 280 nM (40,000 microunits/ml) insulin is present during the incubation, cytochalasin B binding to the plasma membranes is increased approximately 2-fold without alteration in the dissociation constant of this site. Membranes in the microsomal fraction prepared from diaphragms which have been incubated for 30 min in the absence of insulin contain 21 pmol of D-glucose-inhibitable cytochalasin B binding sites/mg of membrane protein. However, in the presence of insulin during the incubation period, the number of these sites in the microsomal fraction is decreased to 12 pmol/mg of membrane protein. These results suggest that insulin stimulates glucose transport in the isolated rat diaphragm primarily through a translocation of functional glucose transport units from an intracellular membrane pool to the plasma membrane. These results are similar to the results observed in rat adipose cells (Cushman, S. W., and Wardzala, L. J. (1980) J. Biol. Chem. 255, 4758-4762) and suggest that this mechanism of insulin-stimulated glucose transport activity may be general to other cell types.     

Insulin stimulates glucose transport in isolated human adipose cells through a translocation of intracellular glucose transporters to the plasma membrane: a preliminary report

Insulin's effect on glucose transport activity and the subcellular distribution of glucose transporters have been examined in isolated human abdominal adipose cells, by measuring 3-O-methylglucose transport and specific D-glucose-inhibitable cytochalasin B binding to plasma membranes and low-density microsomes, respectively. Insulin appears to stimulate glucose transport in isolated human adipose cell through the translocation of glucose transporters from a large intracellular pool to the plasma membrane as initially postulated for rat adipose and muscle cells.     

Identification of the D-glucose-inhibitable cytochalasin B binding site as the glucose transporter in rat diaphragm plasma and microsomal membranes

[3H]Cytochalasin B binding and its competitive inhibition by D-glucose have been used to identify, the glucose transporter in plasma and microsomal membranes prepared from intact rat diaphragm. Scatchard plot analysis of [3H]cytochalasin B binding yields a binding site with a dissociation constant of roughly 110 nM. Since the inhibition constant of cytochalasin B for D-glucose uptake by diaphragm plasma membranes is similar to this value, this site is identified as the glucose transporter. Plasma membranes prepared from diaphragms bind approx. 17 pmol of cytochalasin B/mg of membrane protein to the D-glucose-inhibitable site. If 280 nM (40000 microunits/ml) insulin is present during incubation, cytochalasin B binding is increased roughly 2-fold without alteration in the dissociation constant of this site. In addition, membranes in the microsomal fraction contain 21 pmol of D-glucose-inhibitable cytochalasin B binding sites/mg of membrane protein. In the presence of insulin during incubation the number of these sites in the microsomal fraction is decreased to 9 pmol/mg of membrane protein. These results suggest that rat diaphragm contain glucose transporters with characteristics identical to those observed for the rat adipose cell glucose transporter. In addition, insulin stimulates glucose transport in rat diaphragm through a translocation of functionally identical glucose transporters from an intracellular membrane pool to the plasma membrane without an alteration in the characteristics of these sites.     

Counter-regulation of insulin-stimulated glucose transport by catecholamines in the isolated rat adipose cell

The interaction between catecholamines and insulin in regulating glucose transport in isolated rat adipose cells has been evaluated. In the absence of insulin, 1 microM isoproterenol stimulates 3-O-methylglucose transport approximately 2-fold. However, isoproterenol in combination with adenosine deaminase inhibits glucose transport activity approximately 60%. N6-Phenylisopropyladenosine, a nonmetabolizable adenosine analogue, substantially reverses this inhibitory effect and actually stimulates glucose transport activity approximately 2-fold in the absence of isoproterenol. Dibutyryl cAMP inhibits glucose transport activity approximately 75% regardless of adenosine deaminase. While none of these agents significantly influences the basal concentration of plasma membrane glucose transporters, as assessed by specific D-glucose-inhibitable cytochalasin B binding, isoproterenol or dibutyryl cAMP in combination with adenosine deaminase reduces that in the low density microsomes 19 and 58%, respectively. In the presence of insulin, both isoproterenol and adenosine deaminase alone inhibit glucose transport activity approximately 25%. However, only the latter is accompanied by a corresponding decrease in the insulin-stimulated concentration of plasma membrane glucose transporters. Together, isoproterenol and adenosine deaminase inhibit insulin-stimulated glucose transport activity approximately 75%, even in the presence of 5 mM glucose to maintain cellular ATP levels. A similar inhibition is observed with dibutyryl cAMP. However, these agents decrease the insulin-stimulated concentration of plasma membrane glucose transporters only approximately 45%. Nevertheless, all of these inhibitory effects occur through decreases in the transport Vmax. In addition, N6-phenylisopropyladenosine partially reverses the inhibitory effects induced by the presence of adenosine deaminase. These results suggest that catecholamines counter-regulate basal and insulin-stimulated glucose transport in rat adipose cells through a cAMP-mediated mechanism, but only in part by modulating the translocation of glucose transporters.     

Reconstitution of the glucose transporter from bovine heart

Reconstitution of the glucose transporter from heart should be useful as an assay in its purification and in the study of its regulation. We have prepared plasma membranes from bovine heart which display D-glucose reversible binding of cytochalasin B (33 pmol sites/mg protein; Kd = 0.2 muM). The membrane proteins were reconstituted into liposomes by the freeze-thaw procedure. Reconstituted liposomes showed D-glucose transport activity which was stereospecific, saturable and inhibited by cytochalasin B, phloretin, and mercuric chloride. Compared to membrane proteins reconstituted directly, proteins obtained by dispersal of the membranes with low concentrations of cholate or by cholate solubilization showed 1.2- or 2.3-fold higher specific activities for reconstituted transport, respectively. SDS-polyacrylamide gel electrophoresis followed by electrophoretic protein transfer and labeling with antisera prepared against the human erythrocyte transporter identified a single band of about 45 kDa in membranes from both dog and bovine hearts, a size similar to that reported for a number of other glucose transporters in various animals and tissues.     

Regulation of glucose transport in Clone 9 cells by thyroid hormone.

Triiodothyronine (T3) is found to stimulate cytochalasin B-inhibitable glucose transport in Clone 9 cells, a 'non-transformed' rat liver cell line. After an initial lag period of more than 3 h, glucose transport rate is significantly increased at 6 h and reaches more than 3-times the control rate at 24 h. The enhancement of glucose transport by T3 is due to an increase in transport Vmax and occurs in the absence of a change in either the Km for glucose transport (approximately 3 mM) or the Ki for inhibition of transport by cytochalasin B ((1-2).10(-7) M). Consistent with the observed Ki for cytochalasin B, Northern blot analysis of RNA from control and T3-treated cells employing cDNA probes encoding GTs of the human erythrocyte/rat brain/HepG2 cell transporter (GLUT-1), rat muscle/fat cell transporter (GLUT-4), and rat liver transporter (GLUT-2) types indicates expression of only the GLUT-1 mRNA isoform in these cells. The abundance of GLUT-1 mRNA increases approx. 1.9-fold after 24 h of T3 treatment and is accompanied by an approx. 1.3-fold increase in the abundance of GLUT-1 in whole-cell extracts as demonstrated by Western blot analysis employing a polyclonal antibody directed against the 13 amino acid C-terminal peptide of GLUT-1. The more than 3-fold stimulation of glucose transport at 24 h substantially exceeds the fractional increment in transporter abundance suggesting that, in addition to increasing total GLUT-1 abundance, exposure to T3 may result in a translocation of transporters to the plasma membrane or an activation of pre-existing membrane transporter sites.     

Identification of the d-glucose-inhibitable cytochalasin B binding site as the glucose transporter in rat diaphragm plasma and microsomal membranes

[³H]Cytochalasin B binding and its competitive inhibition by d-glucose have been used to identify the glucose transporter in plasma and microsomal membranes prepared from intact rat diaphragm. Scatchard plot analysis of [³H]cytochalasin B binding yields a binding site with a dissociation constant of roughly 110 nM. Since the inhibition constant of cytochalasin B for d-glucose uptake by diaphragm plasma membranes is similar to this value, this site is identified as the glucose transporter. Plasma membranes prepared from diaphragms bind approx. 17 pmol of cytochalasin B/mg of membrane protein to the d-glucose-inhibitable site. If 280 nM (40 000 μunits/ml) insulin is present during incubation, cytochalasin B binding is increased roughly 2-fold without alteration in the dissociation constant of this site. In addition, membranes in the microsomal fraction contain 21 pmol of d-glucose-inhibitable cytochalasin B binding sites/mg of membrane protein. In the presence of insulin during incubation the number of these sites in the microsomal fraction is decreased to 9 pmol/mg of membrane protein. These results suggest that rat diaphragm contain glucose transporters with characteristics identical to those observed for the rat adipose cell glucose transporter. In addition, insulin stimulates glucose transport in rat diaphragm through a translocation of functionally identical glucose transporters from an intracellular membrane pool to the plasma membrane without an alteration in the characteristics of these sites.     

Long term regulation of glucose transporters by insulin in mature 3T3-F442A adipose cells. Differential effects on two glucose transporter subtypes

The question of a long term regulatory role of insulin on adipocyte glucose transporter content was addressed using the differentiating or fully mature 3T3-F442A adipocytes. Glucose transport was measured in intact cells. Glucose transporter content in plasma membranes and low density microsomes (LDM) was assessed by cytochalasin B binding and Western analysis. In insulin- versus spontaneously differentiated adipocytes, glucose transport and glucose transporters content of plasma membranes and LDM were increased 5-, 4-, and 2-fold, respectively. Insulin deprivation for 24 h induced a redistribution of glucose transporters in those cells which then displayed 2-fold higher glucose transport and glucose transporter content in plasma membranes than spontaneously differentiated cells and 3-fold more glucose transporters in LDM. When fully insulin-differentiated adipocytes were insulin-deprived for 4 days, there was a marked decrease in glucose transporters in both membrane fractions that was fully reversible by reexposing the cells to insulin for 4 days. Glucose uptake changes were closely proportionate to changes in glucose transporter content of plasma membranes as assessed by an antiserum to the C-terminal peptide of the erythrocyte/HepG2/brain-type glucose transporter. When Western blots were immunoblotted with 1F8 monoclonal antibody, specific for glucose transporter in insulin responsive tissues, an abundant immunoreactive protein was detected in both plasma membranes and LDM but the amount of this glucose transporter did not change with insulin exposure in any membrane fractions. In conclusion, insulin plays a long term regulatory role on cultured adipocyte glucose transporter content through a selective effect on the erythrocyte/HepG2/brain-type glucose transporter.     

Stimulatory effect of cold adaptation on glucose utilization by brown adipose tissue. Relationship with changes in the glucose transporter system

The effect of cold adaptation (4 degrees C) on the in vivo glucose utilization and on the number and properties of the glucose transporters has been studied in brown adipose tissue of normal rats. Glucose utilization was assessed in vivo by the 2-deoxyglucose method. Glucose transporters in plasma and microsomal membranes were quantified by the [3H]cytochalasin B-binding assay. After cold adaptation the in vivo glucose utilization by brown adipose tissue increased 21-fold compared to controls (22 degrees C). The number of glucose transporters in plasma membranes of brown adipose tissue increased from 75 to 436 pmol/g tissue and that of total glucose transporters (plasma + microsomal membranes) from 438 to 754 pmol/g tissue. In addition, cold adaptation increased the Hill coefficient of the plasma membrane transporter for cytochalasin B from 0.90 to 2.03 and decreased the Kd from 100 to 54 nM. This study shows that cold adaptation promotes: a translocation of glucose transporters from an intracellular pool to plasma membranes; an increased number of plasma membrane glucose transporters unaccounted for by the translocation process (e.g. "de novo" synthesis); an increase in the Hill coefficient for cytochalasin B that could also represent changes in the properties of the transporters vis-à-vis glucose, (e.g. positive cooperativity); and a decrease in the Kd value for cytochalasin B.     

Mechanism for markedly hyperresponsive insulin-stimulated glucose transport activity in adipose cells from insulin-treated streptozotocin diabetic rats. Evidence for increased glucose transporter intrinsic activity

The effects of insulin therapy in streptozotocin diabetic rats on the glucose transport response to insulin in adipose cells have been examined. At sequential intervals during subcutaneous insulin infusion, isolated cells were prepared and incubated with or without insulin, and 3-O-methylglucose transport was measured. Insulin treatment not only reversed the insulin-resistant glucose transport associated with diabetes, but resulted in a progressive hyperresponsiveness, peaking with a 3-fold overshoot at 7-8 days (12.1 +/- 0.3 versus 3.4 +/- 0.1 fmol/cell/min, mean +/- S.E.) and remaining elevated for more than 3 weeks. During the peak overshoot, glucose transporters in subcellular membrane fractions were assessed by cytochalasin B binding. Insulin therapy restored glucose transporter concentration in the plasma membranes of insulin-stimulated cells from a 40% depleted level previously reported in the diabetic state to approximately 35% greater than control (38 +/- 4 versus 28 +/- 2 pmol/mg of membrane protein). Glucose transporter concentration in the low-density microsomes from basal cells was also restored from an approximately 45% depleted level back to normal (50 +/- 4 versus 50 +/- 6 pmol/mg of membrane protein), whereas total intracellular glucose transporters were further increased due to an approximately 2-fold increase in low-density microsomal membrane protein. However, these increases remained markedly less than the enhancement of insulin-stimulated glucose transport activity in the intact cell. Thus, insulin treatment of diabetic rats produces a marked and sustained hyperresponsive insulin-stimulated glucose transport activity in the adipose cell with little more than a restoration to the non-diabetic control level of glucose transporter translocation. Because this enhanced glucose transport activity occurs through an increase in Vmax, insulin therapy appears to be associated with a marked increase in glucose transporter intrinsic activity.     

Substitution of leucine for tryptophan 412 does not abolish cytochalasin B labeling but markedly decreases the intrinsic activity of GLUT1 glucose transporter

GLUT1 glucose transporter cDNA was modified to introduce a single amino acid substitution of leucine for tryptophan 412, a putative cytochalasin B photo-affinity labeling site. Although the mutated transporter was expressed into plasma membranes of Chinese hamster ovary cells, glucose transport activity of the mutated transporter was observed to be only 15-30% of that of the wild-type GLUT1 when glucose transport activity was assessed by 2-deoxyglucose uptake at 0.1-10 mM concentrations. Analysis of glucose uptake kinetics depict that a mutation induced a 3-fold decrease in turnover number and a 2.5-fold increase in Km compared with the wild-type GLUT1. Importantly, cytochalasin B labeling was not abolished but decreased by 40%, and cytochalasin B binding was also decreased. In addition, the results obtained with side-specific glucose analogs suggested that the outer glucose binding site of the mutant appeared intact but the inner binding site was modulated. These results indicate 1) tryptophan 412 is not a cytochalasin B labeling site(s), although this residue is located in or close to the inner glucose binding site of the GLUT1 glucose transporter, 2) substitution of leucine for tryptophan 412 decreases the intrinsic activity of GLUT1 glucose transporter, which is definable as the turnover number/Km, to approximately 15% of that of the wild-type.     

Insulin-stimulated translocation of glucose transporters in the isolated rat adipose cells: Characterization of subcellular fractions

Insulin stimulates glucose transport in rat adipose cells through the translocation of glucose transporters from an intracellular pool to the plasma membrane. A detailed characterization of the morphology, protein composition and marker enzyme content of subcellular fractions of these cells, prepared by differential ultracentrifugation, and of the distribution of glucose transporters among these fractions is now described. Glucose transporters were measured using specific d-glucose-inhibitable [³H]cytochalasin B binding. In the basal state, roughly 90% of the cells' glucose transporters are associated with a low-density microsomal, Golgi marker enzyme-enriched membrane fraction. However, the distributions of glucose transporters and Golgi marker enzyme activities over all fractions are clearly distinct. Incubation of intact cells with insulin increases the number of glucose transporters in the plasma membrane fraction 4–5-fold and correspondingly decreases the intracellular pool, without influencing any other characteristics of the subcellular fractions examined or the estimated total number of glucose transporters (3.7·10⁶/cell). Insulin does not influence the K_d of the glucose transporters in the plasma membrane fraction for cytochalasin B binding (98 nM), but lowers that in the intracellular pool (from 141 to 93 nM). The calculated turnover numbers of the glucose transporters in the plasma membrane vesicles from basal and insulin-stimulated cells are similar (15·10³ mol of glucose/min per mol of transporters at 37°C), whereas insulin appears to increase the turnover number in the plasma membrane of intact cells roughly 4-fold. These results suggest that (1) the intracellular pool of glucose transporters may comprise a specialized membrane species, (2) intracellular glucose transporters may undergo conformational changes during their cycling to the plasma membrane in response to insulin, and (3) the translocation of glucose transporters may represent only one component in the mechanism through which insulin regulates glucose transport in the intact cell.     

Identification of an intracellular pool of glucose transporters from basal and insulin-stimulated rat skeletal muscle

The purpose of this study was to simultaneously isolate skeletal muscle plasma and microsomal membranes from the hind limbs of male Sprague-Dawley rats perfused either in the absence or presence of 20 milliunits/ml insulin and to determine the effect of insulin on the number and distribution of glucose transporters in these membrane fractions. Insulin increased hind limb glucose uptake greater than 3-fold (2.4 +/- 0.7 versus 9.2 +/- 1.0 mumol/g x h, p less than 0.001). Plasma membrane glucose transporter number, measured by cytochalasin B binding, increased 2-fold (9.1 +/- 1.0 to 20.4 +/- 3.1 pmol/mg protein, p less than 0.005) in insulin-stimulated muscle while microsomal membrane transporters decreased significantly (14.8 +/- 1.6 to 9.8 +/- 1.4 pmol/mg protein, p less than 0.05). No change in the dissociation constant (Kd approximately 120 nm) was observed. K+-stimulated-p-nitrophenol phosphatase, 5'-nucleotidase, and galactosyltransferase specific activity, enrichment, and recovery in the plasma and microsomal membrane fractions were not altered by insulin treatment. Western blot analysis using the monoclonal antibody mAb 1F8 (specific for the insulin-regulatable glucose transporter) demonstrated increased glucose transporter densities in plasma membranes from insulin-treated hind limb skeletal muscle compared with untreated tissues, while microsomal membranes from the insulin-treated hind limb skeletal muscle had a concomitant decrease in transporter density. We conclude that the increase in plasma membrane glucose transporters explains, at least in part, the increase in glucose uptake associated with insulin stimulation of hind limb skeletal muscle. Our data further suggest that these recruited transporters originate from an intracellular microsomal pool, consistent with the translocation hypothesis.     

Reconstitution of the glucose transporter from rat skeletal muscle

As a step in the purification and characterization of the glucose transporter from rat skeletal muscle, we have reconstituted glucose transport activity in liposomes. Plasma membranes were prepared from skeletal muscle which display D-glucose reversible binding of cytochalasin B (10 pmol sites/mg protein; KD = 0.3 microM). Older rats gave a slightly lower specific activity and much lower yield of sites per g muscle than young rats. Glucose transport activity was reconstituted into liposomes by the freeze-thaw procedure using either plasma membranes directly or cholate-extracted membrane proteins; the latter gave a 50% higher specific activity. The reconstituted transport activity was stereospecific, saturable, and inhibited by cytochalasin B, phloretin, and mercuric chloride. The optimum cholate concentration for extraction and reconstitution of transport activity was about 1.5%, and the highest specific activity of reconstituted transport was seen only at low ratios of protein to lipid in the reconstitution. Chromatography on agarose lentil lectin and agarose ethanethiol doubled both the specific activity of reconstituted transport and the fraction of glucose uptake which was stereospecific. In all of these respects the results were similar to our results with the bovine heart transporter (T. J. Wheeler and M. A. Hauck, Biochim. Biophys. Acta 818, 171-182 (1985)). Our findings suggest that further purification procedures developed for the heart transporter may be applicable to the skeletal muscle transporter as well.     

Effects of insulin on glucose transport and glucose transporters in rat heart.

The effect of insulin on glucose transport and glucose transporters was studied in perfused rat heart. Glucose transport was measured by the efflux of labelled 3-O-methylglucose from hearts preloaded with this hexose. Insulin stimulated 3-O-methylglucose transport by: (a) doubling the maximal velocity (Vmax); (b) decreasing the Kd from 6.9 to 2.7 mM; (c) increasing the Hill coefficient toward 3-O-methylglucose from 1.9 to 3.1; (d) increasing the efficiency of the transport process (k constant). Glucose transporters in enriched plasma and microsomal membranes from heart were quantified by the [3H]cytochalasin-B-binding assay. When added to normal hearts, insulin produced the following changes in the glucose transporters: (a) it increased the translocation of transporters from an intracellular pool to the plasma membranes; (b) it increased (from 1.6 to 2.7) the Hill coefficient of the transporters translocated into the plasma membranes toward cytochalasin B, suggesting the existence of a positive co-operativity among the transporters appearing in these membranes; (c) it increased the affinity of the transporters (and hence, possibly, of glucose) for cytochalasin B. The data provide evidence that the stimulatory effect of insulin on glucose transport may be due not to the sole translocation of intracellular glucose transporters to the plasma membrane, but to changes in the functional properties thereof.     

Insulin-responsive glucose transporters are concentrated in a cell surface-derived membrane fraction of 3T3-L1 adipocytes

The recently proposed mechanistic concept of a receptor-regulated entrance compartment for hexose transport formed by microvilli on 3T3-L1 adipocytes predicted a preferential localization of glucose transporters in these structures. The cytochalasin B-binding technique was used to determine in basal and insulin-stimulated cells the distribution of glucose transporters between plasma membranes, low density microsomes (LDM) and two cell surface-derived membrane fractions prepared by a hydrodynamic shearing technique. The shearing procedure applied prior to homogenization yielded a low density surface-derived vesicle (LDSV) fraction which contained nearly 60% of the cellular glucose transporters and the total insulin-sensitive transporter pool. The rest of the glucose transporter population was localized within the plasma membrane (5%) and the LDM fraction (37%). Pretreatment of the cells with insulin (20 mU/ml for 10 min) reduced the transporter content of the LDSV fraction by 40% and increased that of the plasma membrane fraction 4-fold. The transporter containing LDSV fraction was clearly differentiated from the LDM fraction by its low specific galactosyltransferase activity and its insulin-sensitivity. Scanning electron microscopy revealed that the LDSV fraction contained a rather uniform population of spherical vesicles of 100-200 nm in diameter.     

Effect of physical training on glucose transporters in fat cell fractions

Physical training increases maximally insulin-stimulated glucose assimilation and 3-O-methylglucose transport in epididymal fat cells. In the present report, glucose-inhibitable cytochalasin B binding in subcellular fractions of epididymal adipocytes was measured to assess changes in number of glucose transporters induced by training. Groups of rats trained by swimming were compared to control groups of the same age, matched with respect to body weight by restricted feeding. It was found that in trained rats the number of glucose transporters in the low density microsome fractions from non-insulin-stimulated fat cells was larger than in untrained rats. In both groups of rats, insulin stimulation of adipocytes decreased the number of glucose transporters in low-density microsomes by about 60% and increased the number of glucose transporters in the plasma membrane fractions. The number of glucose transporters in the plasma membrane fractions from maximally insulin-stimulated fat cells was larger in trained rats than in control rats. [U-¹⁴C]Glucose incorporation into lipids varied in proportion to plasma membrane cytochalasin B binding per cell under all conditions tested. The results explain the enhancing effect of training on insulin responsiveness transport of hexose in fat cells.     

Insulin-mediated translocation of glucose transporters from intracellular membranes to plasma membranes: sole mechanism of stimulation of glucose transport in L6 muscle cells

Plasma membranes and light microsomes were isolated from fused L6 muscle cells. Pre-treatment of cells with insulin did not affect marker enzyme or protein distribution in isolated membranes. The number of glucose transporters in the isolated membranes was calculated from the D-glucose-protectable binding of [3H]cytochalasin B. Glucose transporter number was higher in plasma membranes and lower in intracellular membranes derived from insulin-treated cells than in the corresponding fractions from untreated cells. The net increase in glucose transporters in plasma membranes was identical to the net decrease in glucose transporters in light microsomes (2 pmol/1.23 x 10(8) cells). The fold increase in glucose transporter number/mg protein in plasma membranes (2-fold) was similar to the fold increase in glucose transport caused by insulin. This suggests that recruitment of glucose transporters from intracellular membranes to the plasma membrane is the major mechanism of stimulation of hexose transport in L6 muscle cells. This is the first report of isolation of the two insulin-sensitive membrane elements from a cell line, and the results indicate that, in contrast to rat adipocytes, there is not change in the intrinsic activity of the transporters in response to insulin.