Photoaffinity labeling of insulin-sensitive hexose transporters in intact rat adipocytes. Direct evidence that latent transporters become exposed to the extracellular space in response to insulin

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

Exofacial photolabelling of the human erythrocyte glucose transporter with an azitrifluoroethylbenzoyl-substituted bismannose.

The synthesis of 2-N-[4-(1'-azitrifluoroethyl)benzoyl]-1,3-bis-(D-mannos-4-++ +yloxy)-2- propylamine (ATB-BMPA) is described. This compound was used as an exofacial probe for the human erythrocyte glucose-transport system. A new method is described for directly estimating the affinity for exofacial ligands which bind to the erythrocyte glucose transporter. By using this equilibrium-binding method, the Ki for ATB-BMPA was found to be 338 +/- 37 microM at 0 degrees C and 368 +/- 59 microM at 20 degrees C. This was similar to the concentration of ATB-BMPA required to half-maximally inhibit D-galactose uptake (Ki = 297 +/- 53 microM). The new photoaffinity reagent labelled the glucose transporter in intact cells but, because of its improved selectivity, was also used to label the glucose transporter in isolated erythrocyte membranes. The ATB-BMPA-labelled glucose transporter was 80% immunoprecipitated by anti-(GLUT1-C-terminal peptide) antibody, which shows that the GLUT1 glucose transporter is the major isoform present in erythrocytes. The labelling of the glucose transporter at its exofacial site, and the adoption of an outward-facing conformation, renders the transport system resistant to thermolysin and trypsin treatment. Trypsin treatment of the unlabelled glucose transporter in erythrocyte membranes produced an 18 kDa fragment which was subsequently labelled by ATB-BMPA, but had low affinity for this exofacial ligand. This suggests that the trypsin-treated transporter adopts an inward-facing conformation. The ability of D-glucose to displace ATB-BMPA from the native transporter and from the 18 kDa trypsin fragment have been compared. The D-glucose concentration which was required to obtain half-maximal inhibition of ATB-BMPA labelling was 6-fold lower for the 18 kDa tryptic fragment.     

Exofacial photoaffinity labelling of the human erythrocyte sugar transporter

The 4-azidosalicylate derivative of 1,3-bis(D-mannos-4'-yloxy)-2-[2-3H]propylamine (ASA-[2-3H]BMPA) has been tested as a photoaffinity label for the sugar transporter in human erythrocytes. When photolysed in the presence of intact erythrocytes, ASA-[2-3H]BMPA covalently binds to the exofacial surface of the transporter. This labelled protein appears as a broad band in the 4.5 region in sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis. The peak of radiolabel incorporation gives an apparent Mr of approx. 50 000 on 5-20% acrylamide gels. The binding is 80% inhibitable by 320 mM 4,6-O-ethylidene-D-glucose, by 320 mM D-glucose and by 50 microM cytochalasin B. Photoirradiation of a saturating concentration of ASA-BMPA in the presence of erythrocytes results in a 25-30% loss of D-galactose transport activity. From transport inactivation data and estimations of the amount of ASA-[2-3H]BMPA binding to the transporter it is calculated that there are approx. 220 000 exofacial hexose-transport binding sites per erythrocyte. The labelling of the transporter has been carried out using freshly drawn blood and 4-weeks-old transfusion blood. No change in the binding profile on SDS-polyacrylamide gel electrophoresis was observed. Proteolytic digestion of the ASA-[2-3H]BMPA-labelled transporter with either trypsin or alpha-chymotrypsin results in the appearance of a labelled 19 kDa fragment on SDS-polyacrylamide gel electrophoresis.     

The barley scutellar peptide transporter: biochemical characterization and localization to the plasma membrane

Thiol-affinity labelling was used to identify and characterize components of the peptide transport system in the barley (Hordeum vulgare) scutellar epithelium. SDS-PAGE and 2D-PAGE in conjunction with fluorography were used to study derivatized proteins. Membrane proteins of 42 kDa and 66 kDa were identified using a strategy devised to label substrate protectable protein with the thiol specific reagent [14C] N-ethylmaleimide (NEM). The scutellar plasma membrane is the anticipated site of transporters involved in the mobilization of endosperm storage reserves in the germinating barley grain. The subcellular localization of these proteins to the plasma membrane was demonstrated by thiol-affinity labelling of high purity plasma membrane vesicles isolated from barley scutellar tissue. A peptide transporter, HvPTR1, specific to the barley scutellum has recently been cloned in this laboratory. A 66 kDa protein, comparable to the predicted molecular mass of HvPTR1, was identified by [14C]NEM labelling studies of Xenopus laevis oocytes expressing HvPTR1 cRNA, but not water injected controls. Peptide antiserum raised to HvPTR1 also cross-reacted with a 66 kDa membrane protein in barley scutellar tissue. This confirms that the 66 kDa protein identified here by thiol-affinity labelling studies is the barley scutellum peptide transporter HvPTR1, and demonstrates that this protein is localized to the plasma membrane of scutellar epithelial cells during germination.     

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.     

Insulin-stimulated translocation of the HepG2/erythrocyte-type glucose transporter expressed in 3T3-L1 adipocytes

Insulin stimulates glucose transport into adipocytes, at least in part, via the translocation of intracellular transporters to the plasma membrane. The human HepG2-type transporter, which is not insulin-responsive in its native cell type, was expressed in 3T3-L1 adipocytes by infection with recombinant retrovirus harboring the HepG2 transporter cDNA in order to determine whether glucose transporter translocation in adipocytes is restricted to a distinct insulin-sensitive transporter species. The distributions of the endogenous murine and the HepG2 transporters were estimated by quantitative immunoblot analysis of subcellular fractions probed with either a monoclonal antibody that recognized only the human transporter or a polyclonal antibody that recognized both transporter species. In the basal state, the intracellular membrane fraction comprised approximately 50% of the total of each transporter type. Insulin decreased the content of both transporter species in the intracellular membranes by approximately 50% and increased the plasma membrane content of both species by approximately 1.5-2-fold. The similar insulin-mediated increase in the plasma membrane content of endogenous murine and HepG2 glucose transporters was verified by labeling of cell surface glycoproteins with [3H]NaBH4 followed by immunoprecipitation with glucose transporter antibodies. These data indicate that insulin-mediated translocation in 3T3-L1 adipocytes is not restricted to a tissue-specific insulin-responsive glucose transporter species and suggest that other tissue-specific factors regulate the translocation process.     

Biochemical and functional characterization of the rat liver glucose-transport system. Comparisons with the adipocyte glucose-transport system.

The properties of the glucose-transport systems in rat adipocytes and hepatocytes were compared in cells prepared from the same animals. Hormones and other agents which cause a large stimulation of 3-O-methylglucose transport in adipocytes were without acute effect in hepatocytes. Hepatocytes displayed a lower affinity for 3-O-methylglucose (20 mM) and alternative substrates than adipocytes (6 mM), whereas inhibitor affinities were similar in both cell types. The concentration and distribution of glucose transporters were determined by Scatchard analysis of D-glucose-inhibitable [3H]cytochalasin B binding to subcellular fractions. In liver, most of the transporters were located in the plasma membrane (42 +/- 5 pmol/mg of protein) with a small amount (4 +/- 3 pmol/mg) in the low-density microsomal fraction ('microsomes'), the reverse of the situation in adipocytes. Glucose transporters were covalently labelled with [3H]cytochalasin B by using the photochemical cross-linking agent hydroxysuccinimidyl-4-azidobenzoate and analysed by SDS/polyacrylamide-gel electrophoresis. A single D-glucose-inhibitable peak with a molecular mass of 40-50 kDa was seen in both plasma membrane and low-density microsomes. This peak was further characterized by isoelectric focusing and revealed a single peak of specific [3H]cytochalasin B binding at pI 6.05 in both low-density microsomes and plasma membrane, compared with peaks at pI 6.4 and 5.6 in adipocyte membranes. In summary: the glucose-transport system in hepatocytes has a lower affinity and higher capacity than that in adipocytes, and is also not accurately modulated by insulin; the subcellular distribution of glucose transporters in the liver suggests that few intracellular transporters would be available for translocation; the liver transporter has a molecular mass similar to that of the adipocyte transporter; the liver glucose transporter exists as a single charged form (pI 6.05), compared with the multiple forms in adipocytes. This difference in charge could reflect a functionally important difference in molecular structure between the two cell types.     

Glucose transporters in chromaffin cells: subcellular distribution and characterization

The glucose transporter was identified and characterized by cytochalasin B binding in subcellular membrane fractions of chromaffin tissue. The binding was saturable with Kd of about 0.3 microM for each subcellular fraction. The Bmax capacity was 12-16 pmol/mg protein for enriched plasma membrane fractions, 6.3 pmol/mg protein for microsomal membrane preparations and 5.4 pmol/mg protein for chromaffin granule membranes. Irreversible photoaffinity labelling of the glucose-protectable binding sites with [3H]cytochalasin B followed by solubilization and polyacrylamide gel electrophoresis from enriched plasma membrane preparations demonstrated the presence of three molecular species: 97 +/- 10, 51.5 +/- 6 and 30 +/- 4 kDa. The chromaffin granule membranes showed only a molecular species of 80 +/- 10 kDa.     

Purification of insulin-dependent exocytic vesicles containing the glucose transporter

In muscle and fat, insulin causes the cellular redistribution of glucose transporters and insulin-like growth factor II receptors from an intracellular pool of membranes (low density microsomes) to the plasma membrane. This translocation is a major mechanism by which insulin stimulates cellular glucose uptake. Our aim was to purify and characterize the insulin-regulatable exocytic intracellular membranes that are enriched in glucose transporter. Low density microsome and plasma membrane fractions were isolated from basal and insulin-stimulated rat adipocytes by differential centrifugation. In cells exposed to insulin, glucose transporters were decreased in the low density microsomes and correspondingly increased in the plasma membranes as determined by immunoblotting and cytochalasin B binding. Low density microsomes were further fractionated by sucrose density gradient centrifugation. Membranes containing glucose transporters were separated from the major protein-containing peaks and from plasma membranes, Golgi, and endoplasmic reticulum. Further fractionation was achieved by agarose gel electrophoresis. Overall, the intracellular membranes enriched in transporter were purified 9-fold compared to low density microsomes. These purified membranes had the following characteristics: 1) uniformly sized vesicles, diameter 60-100 nm; 2) insulin-regulatable protein composition, one constituent being an Mr 43,000 protein that co-migrated with immunoblotted glucose transporters; 3) enrichment in insulin-like growth factor II receptors, but of a lesser degree than the enrichment in transporters. Thus, using a three-step procedure, insulin-sensitive translocatable vesicles from adipocytes have been highly purified. These are similar in size and density to endosomes, and the glucose transporter is a major constituent of this distinct vesicle population.     

The D-glucose transporter is tissue-specific. Skeletal muscle and adipose tissue have a unique form of glucose transporter

Using isotopic equilibration with [3H]D-glucose and measurement of D-glucose inhibitable cytochalasin B binding, I show that the erythrocytes of embryonic and newborn rats contain D-glucose transporters. On the basis of cytochalasin B binding and the time course of isotopic exchange, the number of transporters in rat embryonic erythrocytes is only 5% of that in human erythrocytes. Antibodies raised against the human erythrocyte glucose transporter were used as a probe to investigate the structural similarity between transporters. On this basis, the polypeptides of the glucose transporter of human erythrocytes and of embryonic rat erythrocytes are similar but not identical; in addition, certain antibodies showed similar reactivity toward the transporter of rat embryonic erythrocytes and that of rat brain. These antibodies, however, react with brain transporters 5 to 10 times better than with those of skeletal muscle and adipocytes suggesting that insulin responsive tissues may have a different type of glucose transporter. The cellular location of glucose transporters in skeletal muscle, determined by immunofluorescence, is on the plasma membrane or very close to the plasma membrane.     

Photoaffinity labelling of the ATP-binding site of the epidermal growth factor-dependent protein kinase.

Epidermal growth factor (EGF), after binding to its receptor, activates a tyrosine-specific protein kinase which phosphorylates several substrates, including the EGF receptor itself. The effects of a photoaffinity analogue of ATP, 3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl)adenosine 5'-triphosphate (arylazido-beta-alanyl-ATP) on the EGF-dependent protein kinase in A431 human tumour cell plasma membrane vesicles was investigated. This analogue was capable of inactivating the EGF-receptor kinase in a photodependent manner. Partial inactivation occurred at an analogue concentration of 1 microM and complete inactivation occurred at 10 microM when a 2 min light exposure was used. Arylazido-beta-alanine at 100 microM and ATP at 100 microM were incapable of inactivating the enzyme with 2 min of light exposure. The photodependent inactivation of the enzyme by the analogue could be partially blocked by 20 mM-ATP and more effectively blocked by either 20 mM-adenosine 5'-[beta gamma-imido]triphosphate or 20 mM-guanosine 5'-[beta gamma-imido]triphosphate, indicating nucleotide-binding site specificity. Arylazido-beta-alanyl-[alpha-32P]ATP was capable of labelling membrane proteins in a photodependent manner. Numerous proteins were labelled, the most prominent of which ran with an apparent Mr of 53000 on polyacrylamide-gel electrophoresis. A band of minor intensity was seen of Mr corresponding to the EGF receptor (170000). Immunoprecipitation of affinity-labelled and solubilized membranes with an anti-(EGF receptor) monoclonal antibody demonstrated that the Mr 170000 receptor protein was photoaffinity labelled by the analogue. The Mr 53000 peptide was not specifically bound by the anti-receptor antibody. The affinity labelling of the receptor was not enhanced by EGF, suggesting that EGF stimulation of the kinase activity does not result from changes in the affinity of the kinase for ATP. These studies demonstrate that arylazido-beta-alanyl-ATP interacts with the ATP-binding site of the EGF-receptor kinase with apparent high affinity and that this analogue is an effective photoaffinity label for the kinase. Furthermore, these studies demonstrate that the EGF receptor, identified by using monoclonal antibodies, contains an ATP-binding site, providing further confirmation that the EGF receptor and EGF-dependent protein kinase are domains of the Mr 170000 protein.     

In situ photolabelling of the human androgen receptor

In situ photoaffinity labelling of the human androgen receptor has been performed in the LNCaP (Lymph Node Carcinoma of the Prostate) cell line. The covalently labelled receptors were identified by SDS-PAGE. Intact LNCaP cells, incubated with [3H]-R1881 and subsequently irradiated with u.v. light and directly solubilized in SDS-buffer, revealed two photolabelled protein bands at 110 and 50 kDa. Irradiation of intact cells and subsequent isolation of nuclei followed by extraction with 0.5 M NaCl resulted in one major photolabelled protein band at 110 kDa. The labelling of this band could be completely suppressed by a 100-fold molar excess of non-radioactive R1881. Photolabelling of androgen receptors in a cytosolic preparation of LNCaP cells after anion exchange chromatography resulted in a much lower labelling efficiency compared with the in situ labelling procedure, although the androgen receptor was purified 100-fold. The steroid binding domain of the human androgen receptor has been partially mapped with chymotrypsin and S. aureus V8 protease digestion. Proteolytic digestion with chymotrypsin of purified photoaffinity-labelled 110 kDa human androgen receptor resulted in the generation of a 15 kDa peptide which still contains the covalently linked hormone. It is concluded that the in situ photoaffinity labelling technique can be applied successfully for characterization of the steroid binding domain of androgen receptors in prostate cancer cells and in other androgen target cells. Furthermore, it was demonstrated that the human androgen receptor is a monomer with a molecular mass of 110 kDa, of which the steroid binding site is confined to a 15 kDa domain.     

Development regulation of the subcellular distribution and glycosylation of GLUT1 and GLUT4 glucose transporters during myogenesis of L6 muscle cells.

L6 myoblasts spontaneously undergo differentiation and cell fusion into myotubes. These cells express both GLUT1 and GLUT4 glucose transporters, but their expression varies during myogenesis. We now report that the subcellular distribution and the protein processing by glycosylation of both glucose transporter isoforms also change during myogenesis. Crude plasma membrane and light microsome fractions were isolated from either myoblasts or myotubes and characterized by the presence of two functional proteins, the Na+/K(+)-ATPase and the dihydropyridine receptor (DHPR). Immunoreactive alpha 1 subunit of the Na+/K(+)-ATPase was faint in the crude plasma membrane fraction from myoblasts, but abundant in both membrane fractions from myotubes. In contrast, the alpha 1 subunit of the DHPR, which is expressed only in differentiated muscle, was detected in crude plasma membrane from myotubes but not from myoblasts. Therefore, crude plasma membrane fractions from myoblasts and myotubes contain cell surface markers, and the composition of these membranes appears to be developmentally regulated during myogenesis. GLUT1 protein was more abundant in the crude plasma membrane relative to the light microsome fraction prepared from either myoblasts or myotubes. The molecular size in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the GLUT1 transporters in myotubes was smaller than that in myoblasts (Mr 47,000 and 53,000, respectively). GLUT4 protein (Mr 48,000) was barely detectable in the crude plasma membrane fraction and was almost absent in the light microsome fraction prepared from myoblasts. However, GLUT4 protein was abundant in myotubes and was predominantly located in the light microsome fraction. Treatment with endoglycosidase F reduced the molecular size of the transporters in all fractions to Mr 46,000 for GLUT1 and Mr 47,000 for GLUT4 proteins. In myotubes, acute insulin treatment increased the crude plasma membrane content of GLUT1 marginally and of GLUT4 markedly, with a concomitant decrease in the light microsomal fraction. These results indicate that: (a) the subcellular distribution of glucose transporters is regulated during myogenesis, GLUT4 being preferentially sorted to intracellular membranes; (b) both GLUT1 and GLUT4 transporters are processed by N-linked glycosylation to form the mature transporters in the course of myogenesis; and (c) insulin causes modest recruitment of GLUT1 transporters and marked recruitment of GLUT4 transporters, from light microsomes to plasma membranes in L6 myotubes.     

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.     

One transporter per vesicle: determination of the basis of the insulin effect on glucose transport

The basis for insulin stimulation of glucose transport in rat adipocytes has been investigated by determining the relative number of functional glucose transporters in the plasma and microsomal membranes from basal and insulin-treated cells. Each fraction was solubilized with cholate and then reconstituted into vesicles of about 500 A in diameter through removal of the cholate by dialysis. This procedure distributed the glucose transporters into the vesicles at a density of either one or none per vesicle. Consequently the fraction of the intravesicular volume that rapidly equilibrated with D-glucose provided an estimate of the relative number of functional transporters. By means of this one-transporter-per-vesicle method, it was found that insulin increased the number of transporters in the plasma membrane by a factor of 2.4 and decreased the number in the microsomes to 68% of the original value. These results provide independent evidence for the hypothesis that insulin causes the translocation of functional transporters from an intracellular location to the plasma membrane.     

Derivatization of the human erythrocyte glucose transporter using a novel forskolin photoaffinity label

An iodinated photoaffinity label for the glucose transporter, 3-iodo-4-azidophenethylamido-7-O-succinyldeacetyl-forskolin (IAPS-forskolin), has been synthesized, purified, and characterized. The I50 for inhibition of 3-O-methylglucose transport in red blood cells by IAPS-forskolin was found to be 0.05 microM. The carrier free radioiodinated label is a highly specific photoaffinity label for the human erythrocyte glucose transporter. Photolysis of erythrocyte membranes (ghosts) and purified glucose transporter preparations with 1-2 nM [125I]IAPS-forskolin and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed specific derivatization of a broad band with an apparent molecular mass of 40-70 kDa. Photoincorporation into erythrocyte membranes using 2 nM [125I]IAPS-forskolin was protected with D-glucose (I50 400 mM), cytochalasin B (I50 0.5 microM), and forskolin (I50 10 microM). No protection was observed with L-glucose (600 mM). Endo-beta-galactosidase digestion of [125I] IAPS-forskolin-labeled ghosts and purified transporter resulted in a dramatic sharpening of the specifically radiolabeled transporter to 40 kDa. Trypsinization of [125I]IAPS-forskolin-labeled ghosts and purified transporter reduced the specifically radiolabeled transporter to a sharp peak at 18 kDa. [125I]IAPS-forskolin will be a useful tool to study the structural aspects of the glucose transporter.     

Dissociation of insulin-stimulated glucose transport from the translocation of glucose carriers in rat adipose cells

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

Activation of cell surface glucose transporters measured by photoaffinity labeling of insulin-sensitive 3T3-L1 adipocytes.

Several studies have demonstrated that the intrinsic catalytic activity of cell surface glucose transporters is highly regulated in 3T3-L1 adipocytes expressing GLUT1 (erythrocyte/brain) and GLUT4 (adipocyte/skeletal muscle) glucose transporter isoforms. For example, inhibition of protein synthesis in these cells by anisomycin or cycloheximide leads to marked increases in hexose transport without a change in the levels of cell surface glucose transporter proteins (Clancy, B. M., Harrison, S. A., Buxton, J. M., and Czech, M. P. (1991) J. Biol. Chem. 266, 10122-10130). In the present work the exofacial hexose binding sites on GLUT1 and GLUT4 in anisomycin-treated 3T3-L1 adipocytes were labeled with the cell-impermeant photoaffinity reagent [2-3H]2-N-[4-(1-azitrifluoroethyl)benzoyl]-1,3-bis- (D-mannos-4-yloxy)-2-propylamine [( 2-3H] ATB-BMPA) to determine which isoform is activated by protein synthetic blockade. As expected, a 15-fold increase in 2-deoxyglucose uptake in response to insulin was associated with 1.7- and 2.6-fold elevations in plasma membrane GLUT1 and GLUT4 protein levels, respectively. Anisomycin treatment of cultured adipocytes for 5 h produced an 8-fold stimulation of hexose transport but no increase in the content of glucose transporters in the plasma membrane fraction as measured by protein immunoblot analysis. Cell surface GLUT1 levels were also shown to be unaffected on 3T3-L1 adipocytes in response to anisomycin using an independent method, the binding of an antiexofacial GLUT1 antibody to intact cells. In contrast, anisomycin fully mimicked the action of insulin to stimulate (about 4-fold) the radiolabeling of GLUT1 transporters specifically immunoprecipitated from intact 3T3-L1 adipocytes irradiated after incubation with [2-3H] ATB-BMPA. Photolabeling of GLUT4 under these conditions was also significantly enhanced (1.8-fold) by anisomycin treatment, but this effect was only 15% of that caused by insulin. These results suggest that: 1) the photoaffinity reagent [2-3H]ATB-BMPA labels those cell surface glucose transporters present in a catalytically active state rather than total cell surface transporters as assumed previously and 2) inhibition of protein synthesis in 3T3-L1 adipocytes stimulates sugar transport primarily by enhancing the intrinsic catalytic activity of cell surface GLUT1, and to a lesser extent, GLUT4 proteins.     

An inhibitor of p38 mitogen-activated protein kinase prevents insulin-stimulated glucose transport but not glucose transporter translocation in 3T3-L1 adipocytes and L6 myotubes

The precise mechanisms underlying insulin-stimulated glucose transport still require investigation. Here we assessed the effect of SB203580, an inhibitor of the p38 MAP kinase family, on insulin-stimulated glucose transport in 3T3-L1 adipocytes and L6 myotubes. We found that SB203580, but not its inactive analogue (SB202474), prevented insulin-stimulated glucose transport in both cell types with an IC50 similar to that for inhibition of p38 MAP kinase (0.6 microM). Basal glucose uptake was not affected. Moreover, SB203580 added only during the transport assay did not inhibit basal or insulin-stimulated transport. SB203580 did not inhibit insulin-stimulated translocation of the glucose transporters GLUT1 or GLUT4 in 3T3-L1 adipocytes as assessed by immunoblotting of subcellular fractions or by immunofluorescence of membrane lawns. L6 muscle cells expressing GLUT4 tagged on an extracellular domain with a Myc epitope (GLUT4myc) were used to assess the functional insertion of GLUT4 into the plasma membrane. SB203580 did not affect the insulin-induced gain in GLUT4myc exposure at the cell surface but largely reduced the stimulation of glucose uptake. SB203580 had no effect on insulin-dependent insulin receptor substrate-1 phosphorylation, association of the p85 subunit of phosphatidylinositol 3-kinase with insulin receptor substrate-1, nor on phosphatidylinositol 3-kinase, Akt1, Akt2, or Akt3 activities in 3T3-L1 adipocytes. In conclusion, in the presence of SB203580, insulin caused normal translocation and cell surface membrane insertion of glucose transporters without stimulating glucose transport. We propose that insulin stimulates two independent signals contributing to stimulation of glucose transport: phosphatidylinositol 3-kinase leads to glucose transporter translocation and a pathway involving p38 MAP kinase leads to activation of the recruited glucose transporter at the membrane.