Cycloheximide decreases glucose transporters in rat adipocyte plasma membranes without affecting insulin-stimulated glucose transport.

This study examines the relationship between insulin-stimulated glucose transport and insulin-induced translocation of glucose transporters in isolated rat adipocytes. Adipose cells were incubated with or without cycloheximide, a potent inhibitor of protein synthesis, for 60 min and then for an additional 30 min with or without insulin. After the incubation we measured 3-O-methylglucose transport in the adipose cells, and subcellular membrane fractions were prepared. The numbers of glucose transporters in the various membrane fractions were determined by the cytochalasin B binding assay. Basal and insulin-stimulated 3-O-methylglucose uptakes were not affected by cycloheximide. Furthermore, cycloheximide affected neither Vmax. nor Km of insulin-stimulated 3-O-methylglucose transport. In contrast, the number of glucose transporters in plasma membranes derived from cells preincubated with cycloheximide and insulin was markedly decreased compared with those from cells incubated with insulin alone (10.5 +/- 0.8 and 22.2 +/- 1.8 pmol/mg of protein respectively; P less than 0.005). The number of glucose transporters in cells incubated with cycloheximide alone was not significantly different compared with control cells. SDS/polyacrylamide-gel-electrophoretic analysis of [3H]cytochalasin-B-photolabelled plasma-membrane fractions revealed that cycloheximide decreases the amount of labelled glucose transporters in insulin-stimulated membranes. However, the apparent molecular mass of the protein was not changed by cycloheximide treatment. The effect of cycloheximide on the two-dimensional electrophoretic profile of the glucose transporter in insulin-stimulated low-density microsomal membranes revealed a decrease in the pI-6.4 glucose-transporter isoform, whereas the insulin-translocatable isoform (pI 5.6) was decreased. Thus the observed discrepancy between insulin-stimulated glucose transport and insulin-induced translocation of glucose transporters strongly suggests that a still unknown protein-synthesis-dependent mechanism is involved in insulin activation of glucose transport.     

Dexamethasone causes translocation of glucose transporters from the plasma membrane to an intracellular site in human fibroblasts

To investigate the mechanism by which glucocorticoids inhibit glucose transport in peripheral tissues, we have used a monoclonal antibody directed against the human glucose transporter to measure the relative amounts of glucose transporter polypeptide in various cell fractions of human foreskin fibroblasts after treatment with and without dexamethasone. In cells treated for 4 h with 100 nM dexamethasone, a decrease of 48% in glucose transport was accompanied by a decrease of 40% in the amount of glucose transporter polypeptide in a plasma membrane fraction enriched 10-fold in 5'-nucleotidase activity and a 78% increase in the amount of transporter polypeptide in a fraction of putative intracellular membranes, designated P2. There was no significant change in the amount of transporter polypeptide in whole cell lysates. Insulin (200 nM) stimulated glucose transport in basal fibroblasts by only 9%. However, addition of insulin for 30 min to cells that had been treated for 4 h with dexamethasone completely reversed the dexamethasone-induced decrease in glucose transport and also reversed the dexamethasone-induced changes in glucose transporter polypeptide content of the plasma membrane and P2 fractions. From these observations we conclude that dexamethasone decreases glucose transport by causing translocation of glucose transporters from the plasma membrane to an internal location and that insulin reverses the dexamethasone effect by reversing the translocation.     

Proposed mechanism of insulin-resistant glucose transport in the isolated guinea pig adipocyte. Small intracellular pool of glucose transporters

A marked resistance to the stimulatory action of insulin on glucose metabolism has previously been shown in guinea pig, compared to rat, adipose tissue and isolated adipocytes. The mechanism of insulin resistance in isolated guinea pig adipocytes has, therefore, been examined by measuring 125I-insulin binding, the stimulatory effect of insulin on 3-0-methylglucose transport and on lipogenesis from [3-3H]glucose, the inhibitory effect of insulin on glucagon-stimulated glycerol release, and the translocation of glucose transporters in response to insulin. The translocation of glucose transporters was assessed by measuring the distribution of specific D-glucose-inhibitable [3H]cytochalasin B binding sites among the plasma, and high and low density microsomal membrane fractions prepared by differential centrifugation from basal and insulin-stimulated cells. At a glucose concentration (0.5 mM) where transport is thought to be rate-limiting for metabolism, insulin stimulates lipogenesis from 30 to 80 fmol/cell/90 min in guinea pig cells and from 25 to 380 fmol/cell/90 min in rat cells with half-maximal effects at approximately 100 pM in both cell types. Insulin similarly stimulates 3-O-methylglucose transport from 0.40 to 0.70 fmol/cell/min and from 0.24 to 3.60 fmol/cell/min in guinea pig and rat fat cells, respectively. Nevertheless, guinea pig cells bind more insulin per cell than rat cells, and insulin fully inhibits glucagon-stimulated glycerol release. In addition, the differences between guinea pig and rat cells in the stimulatory effect of insulin on lipogenesis and 3-O-methylglucose transport cannot be explained by the greater cell size of the former compared to the latter (0.18 and 0.09 micrograms of lipid/cell, respectively). However, the number of glucose transporters in the low density microsomal membrane fraction prepared from basal guinea pig cells is markedly reduced compared to that from rat fat cells (12 and 70 pmol/mg of membrane protein, respectively) and the translocation of intracellular glucose transporters to the plasma membrane fraction in response to insulin is correspondingly reduced. These results suggest that guinea pig adipocytes are markedly resistant to the stimulatory action of insulin on glucose transport and that this resistance is the consequence of a relative depletion in the number of intracellular glucose transporters.     

Insulin and noradrenaline independently stimulate the translocation of glucose transporters from intracellular stores to the plasma membrane in mouse brown adipocytes.

The mechanism of the effect of noradrenaline on the transport of 3-O-methyl-D-[14C]glucose ([14C]-MG) was studied in mouse brown adipocytes. When cells were exposed to low concentrations (< 10(-8) M) of insulin, the [14C]-MG uptake by cells was enhanced by noradrenaline additively. The action of noradrenaline was mimicked by isoproterenol, and was completely blocked by propranolol. Exposing cells to noradrenaline induced both an increase in the transport activity of plasma membrane fractions and a decrease in that of microsomal fractions similar to insulin exposure, indicating that noradrenaline also induces the translocation of glucose transporters to the plasma membrane. The ratio of an increase in the transport activity of plasma membrane fraction to a decrease in the activity of microsomal fraction was lower in cells exposed to noradrenaline than in cells exposed to insulin. This quantitative disagreement suggests that there are at least two different modes involved in the regulation of the translocation of glucose transporters in mouse brown adipocytes.     

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.     

Quantitation of Na+/K+-ATpase and glucose transporter isoforms in rat adipocyte plasma membrane by immunogold labeling

We have quantitated and studied the topology of isoforms of the Na⁺/K⁺-ATPase and of the glucose transporter in rat adipocyte plasma membranes.Adipocytes were incubated with or without insulin for 15 min. Sheets of native plasma membrane, with the cytoplasmic face exposed, were prepared by adsorption to EM grids. Grids were incubated in parallel with monoclonal antibodies against the Na⁺/ K⁺-ATPase isoforms 1 and 2, and the glucose transporter isoforms GLUT1 and GLUT4, followed by immunogold labeling, negative staining and quantitation by counting of the gold particles in electron micrographs. In addition, the distribution of glucose transporters and Na⁺/K⁺-ATPase isoforms in subcellular membrane fractions prepared by an established fractionation procedure was monitored by Western blotting.We found that the Na⁺/K⁺-ATPases and the glucose transporters were confined to the planar part of the plasma membrane, without association to caveolar invaginations.The vast majority of the Na⁺/K⁺-ATPase molecules in the adipocyte plasma membrane were of the 2 isoform; GLUT4 was the dominating glucose transporter isoform.The total number of Na⁺/K⁺-ATPase molecules labeled in the plasma membrane was 3.5×10⁵ per cell, independent of insulin stimulation. Concomitantly, insulin increased GLUT4 labeling sevenfold to a value of 3.5×10⁵ per cell.The authors wish to thank Ulla Blankensteiner, Jonna Harpøth and Lisette Hansen for their skillful technical assistance and Birgit Risto for excellent work with the photographic prints. The 1 and 2 antibodies were kindly donated by K.W. Sweadner, Boston, and the F18 and F27 antibodies were granted by Peer N. Jørgensen, Novo-Nordisk, Bagsværd, Denmark. This work was supported by The NOVO Foundation, The Nordisk Insulin Foundation, The Danish Diabetes Foundation, The P. Carl Petersen Foundation and the Foundation of 17-12-81.     

Activation of rat adipocyte plasma membrane adenylate cyclase by sodium azide

The adenylate cyclase of rat adipocyte plasma membrane is stimulated by sodium azide with a half maximal activation of 100–150% occuring at 50 mM NaN₃. Studies of the effects of azide and fluoride indicate different mechanisms of stimulation of the enzyme by these ions. Comparable stimulation of the activity is obtained by 100 mM NaN₃ or 10 mM NaF but unlike azide, higher concentrations of fluoride cause inhibition of the enzyme. Fluoride activated adenylate cyclase is further stimulated by azide. Epinephrine stimulation of the enzyme is absent in the presence of fluoride but the hormone enhances the activity in the presence of azide. Reversal of the inhibitory action of GTP on adenylate cyclase by epinephrine is demonstrated even in the presence of azide but not in the presence of fluoride.     

Radiation inactivation of multispecific transport systems for bile acids and xenobiotics in basolateral rat liver plasma membrane vesicles.

The functional molecular mass of the cholate, phallotoxin, iodipamide, and ouabain transport proteins in isolated basolateral plasma membrane vesicles was determined by radiation inactivation. Purified basolateral plasma membrane vesicles were irradiated (-90 to -120 degrees C) with high energy electrons from a 10-MeV linear accelerator at doses from 0 to 30 megarads. After each dose, the initial uptake, the equilibrium binding, and the binding of the substrates at 4 degrees C were checked. The size of the transporting function was, for cholate, 107 +/- 8.9 kDa; for phallotoxin, 104 +/- 7 kDa; and for ouabain, 120 +/- 4.7 kDa. The target size for the binding proteins was 56 +/- 4.2, 57 +/- 5, and 47.2 +/- 1.95 kDa for cholate, phallotoxin, and taurocholate, respectively. In the case of iodipamide, the functional molecular mass for both the transport and binding proteins was 54 +/- 4.8 kDa.     

Distribution of glucose transporters and insulin receptors in the plasma membrane and transverse tubules of skeletal muscle

The distribution of glucose transporters and of insulin receptors on the surface membranes of skeletal muscle was studied, using isolated plasma membranes and transverse tubule preparations. (i) Plasma membranes from rabbit skeletal muscle were prepared according to Seiler and Fleischer (1982, J. Biol. Chem. 257, 13862-13871), and transverse tubules from rabbit skeletal muscle were prepared according to Rosemblatt et al. (1981, J. Biol. Chem. 256, 8140-8148) as modified by Hidalgo et al. (1983, J. Biol. Chem. 258, 13937-13945). The membranes were identified by the abundance of nitrendipine receptors in the transverse tubules, and their relative absence from the plasma membranes. (ii) Plasma membranes and transverse tubules were also isolated from rat skeletal muscle, according to a novel procedure that isolates both fractions from the same common homogenate. (iii) Glucose transporters were detected by D-glucose protectable binding of the specific inhibitor [3H]cytochalasin B, and insulin receptors were detected by saturable binding of 125I-insulin. The concentration of glucose transporters was about threefold (rabbit) or fivefold (rat) higher in the transverse tubule membrane compared to the plasma membrane, whereas the insulin receptor concentration was about the same in both membranes. These results indicate that the glucose transporters on the surface of the muscle are preferentially segregated to the transverse tubules, and this poses interesting consequences on the functional response of glucose transport to insulin in skeletal muscle.     

Photo-induced inactivation and uncoupling of gonadotropin receptors in rat ovarian plasma membrane

Extraction of membranes of Lactobacillus plantarum with Triton X-100/glycerol solubilized up to 80% of the undecaprenol kinase activity. Fractionation of the extract by gel chromatography separated endogenous phospholipid from the enzyme but simultaneously inactivated the enzyme. The kinase was reactivated by reconstitution with various synthetic phosphatidylcholines and purified L. plantarum phospholipids. Ditetradecanoylphosphatidylcholine and lysylphosphatidylglycerol were the best activators. Furthermore, the optimal environment for enzyme stimulation was provided by different defined molar ratios of Triton X-100/phospholipid. The ratios for the phospholipids tested ranged from 1.25 to 6.3. Similar substrate specificity and kinetic constants were observed for both the solubilized and reconstituted enzymes suggesting that no fundamental changes in the enzyme activity occurred during the delipidation-reconstitution process.     

High density lipoprotein stimulates sterol translocation between intracellular and plasma membrane pools in human monocyte-derived macrophages

Binding of high density lipoprotein (HDL) to its receptor on cultured fibroblasts and aortic endothelial cells was previously shown to facilitate sterol efflux by initiation of translocation of intracellular sterol to the plasma membrane. After cholesterol-loaded human monocyte-derived macrophages were incubated with either [3H]mevalonolactone or lipoprotein-associated [3H]cholesteryl ester to radiolabel intracellular pools of sterol, incubation with HDL3 led to stimulation of 3H-labeled sterol translocation from intracellular sites to the cell surface which preceeded maximum 3H-labeled sterol efflux. A similar pattern was demonstrated for macrophages that were preloaded with cholesterol derived from either low density lipoprotein (LDL), acetyl-LDL, or phospholipase C-modified LDL. However, in macrophages that were not loaded with cholesterol, HDL3 stimulated net movement of 3H-labeled sterol from the plasma membrane into intracellular compartments, the opposite direction from that seen for cholesterol-loaded cells. A similar influx pattern was found in nonloaded macrophages and fibroblasts that were labeled with trace amounts of exogenous [3H]cholesterol. Cholesterol translocation from intracellular pools to the cell surface of cholesterol-loaded macrophages appeared to be stimulated by receptor binding of HDL, since chemical modification of HDL with tetranitromethane (TNM), which abolishes its receptor binding, reduced its ability to stimulate 3H-labeled sterol translocation and efflux. In nonloaded cells, however, the ability of HDL3 to stimulate sterol efflux and movement of sterol from the plasma membrane into intracellular pools was unaffected by TNM modification. Thus, binding of HDL to its receptor on cholesterol-loaded macrophages appears to promote translocation of intracellular cholesterol to the plasma membrane followed by cholesterol efflux into the medium. However, in nonloaded macrophages, HDL stimulates sterol movement from the plasma membrane into intracellular pools by a receptor-independent process.     

Quantitative proteomic analysis of the adipocyte plasma membrane

The adipocyte is a key regulator of mammalian metabolism. To advance our understanding of this important cell, we have used quantitative proteomics to define the protein composition of the adipocyte plasma membrane (PM) in the presence and absence of insulin. Using this approach, we have identified a high confidence list of 486 PM proteins, 52 of which potentially represent novel cell surface proteins, including a member of the adiponectin receptor family and an unusually high number of hydrolases with no known function. Several novel insulin-responsive proteins including the sodium/hydrogen exchanger, NHE6 and the collagens III and VI were also identified, and we provide evidence of PM-ER association suggestive of a unique functional association between these two organelles in the adipocyte. Together these studies provide a wealth of potential therapeutic targets for the manipulation of adipocyte function and a valuable resource for metabolic research and PM biology.     

Inhibition by bacitracin of rat adipocyte plasma membrane degradation of 125I-insulin is associated with an increase in plasma membrane bound insulin and a potentiation of glucose oxidation by adipocytes

The present study demonstrated that at physiological concentrations of insulin bacitracin inhibited the degradation of specifically bound insulin by enzymes located in the rat adipocyte plasma membrane. Bacitracin increased the amount of intact insulin specifically bound to the plasma membrane and potentiated the stimulation of adipocyte glucose oxidation by submaximal concentrations of the hormone. In contrast to agents such as chloroquine, which inhibit lysosomal degradation of internalized insulin, bacitracin was shown by two approaches to inhibit a degradative process localized to the adipocyte plasma membrane. Cyanide and 2,4-dinitrophenol, agents which inhibit energy requiring endocytosis, had no effect on the bacitracin inhibition of cellular degradation of 125I-insulin. Bacitracin directly inhibited 125I-insulin degradation by isolated plasma membranes at similar concentrations and to a similar extent as found with cells. The degradative process inhibited by bacitracin accounted for the majority of cellular degradation of the hormone. The increased 125I-insulin bound to adipocytes was shown to be intact by gel chromatographic analysis and was localized to the plasma membrane by direct and indirect approaches. Bacitracin increased 125I-insulin specifically bound to isolated plasma membranes as early as 2 min. The 125I-insulin bound to adipocytes in the presence of bacitracin was completely dissociable by the addition of 8 microM unlabeled insulin whereas a significant portion of 125I-insulin bound to chloroquine-treated cells could not be dissociated. Bacitracin slowed dissociation of 125I-insulin from the cells. Bacitracin increased the 125I-insulin binding to cells in the presence and absence of cyanide and 2,4-dinitrophenol. Bacitracin potentiated the stimulation of adipocyte glucose oxidation at submaximal concentrations of insulin.     

Endogenous adipocyte apolipoprotein E is colocalized with caveolin at the adipocyte plasma membrane

Apolipoprotein (apo)E is well established as a secreted protein that plays an important role in systemic lipoprotein metabolism and vascular wall homeostasis. Recently, endogenous expression of apoE in adipocytes has been shown to play an important role in adipocyte lipoprotein metabolism and gene expression consistent with a nonsecreted cellular itinerary for apoE. We designed studies to evaluate if adipocyte apoE was retained as a constituent protein in adipocytes and to identify a cellular retention compartment. Using confocal microscopy, coimmunoprecipitation, and sucrose density cellular fractionation, we establish that endogenous apoE shares a cellular itinerary with the constituent protein caveolin-1. Altering adipocyte caveolar number by modulating cellular cholesterol flux or altering caveolin expression regulates the distribution of cellular apoE between cytoplasmic and plasma membrane compartments. A mechanism for colocalization of apoE with caveolin was established by demonstrating a noncovalent interaction between an aromatic amino acid-enriched apoE N-terminal domain with the caveolin scaffolding domain. Absent apoE expression in adipocytes alters caveolar lipid composition. These observations provide evidence for an interaction between two proteins involved in cellular lipid metabolism in a cell specialized for lipid storage and flux, and rationalize a biological basis for the impact of adipocyte apoE expression on adipocyte lipoprotein metabolism.     

Radiation inactivation analysis of the A1 adenosine receptor of rat brain. Decrease in radiation inactivation size in the presence of guanine nucleotide

Radiation inactivation analysis of the binding of the A1 adenosine receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine to rat brain membranes yielded a radiation inactivation size of 58 kDa. In the presence of GTP gamma S this was reduced to 33 kDa, in good agreement with the size of the ligand-binding subunit detected after photoaffinity labelling. The data indicate that the structural association of A1 adenosine receptors with G-protein components is altered in situ in the presence of guanine nucleotides.