Isolation of adipocyte plasma membrane antigens by immunoaffinity chromatography. Insulinomimetic antibodies do not bind directly to the insulin receptor or the glucose transport system (original) (raw)
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The Journal of biological chemistry, 1979
Antisera from rabbits injected with rat adipocyte plasma membranes or intrinsic proteins from such membranes, obtained by a dimethylmaleic anhydride extraction step, mimicked the action of insulin on both glucose transport and lipolysis in intact adipocytes. Biological activity in both types of antisera was mediated by immunoglobulin binding to one or more intrinsic proteins of the adipocyte plasma membrane since fat cells were unresponsive to all antisera absorbed with dimethylmaleic anhydride-extracted membranes. Acid treatment of immunoprecipitates released antibodies which activated glucose uptake and reacted with solubilized adipocyte membranes on immunodiffusion plates. The biologically active immunoglobulin preparations failed to form immunoprecipitin lines when tested against membranes from brain, liver, lung, muscle, kidney, and spleen. Insulin-sensitive glucose uptake in rat soleus muscle did not respond to the antisera. The antibodies activated hexose uptake into fat cell...
The Journal of biological chemistry, 1978
Plasma membrane vesicles from rat adipocytes were treated with dimethylmaleic anhydride to remove extrinsic proteins and then used to immunize rabbits. Immunodiffusion experiments performed in agarose containing Triton X-100 (0.1%) revealed a precipitin reaction between anti-membrane serum and the detergent-solubilized proteins from either intact adipocyte plasma membranes or membranes extracted with dimethylmaleic anhydride. Anti-membrane serum caused cytolysis of intact adipocytes and this effect could be eliminated by incubating the serum at 56 degrees C for 30 min to inactivate complement. Heat-inactivated anti-membrane serum caused a significant increase in 14CO2 production from D-[1-14C]glucose in intact fat cells and the partially purified immunoglobulin fraction from anti-membrane serum markedly stimulated 3-O-methylglucose transport. Maximum activation of transport occurred at a 1:5 dilution and was not additive to that achieved by a maximal dose of insulin. Under these con...
Biochimica Et Biophysica Acta - Biomembranes, 1972
Using methods previously described, we have isolated plasma membranes from insulin-treated adipocytes of the rat epididymal fat pad. Such membranes showed an accelerated uptake and release of D-glucose when compared with similar preparations from cells not exposed to the hormone. The only change observed was in the rate of D-glucose uptake; at equilibrium, D-glucose space was identical in both preparations. Vigorous alkaline hydrolysis of the insulin resulted in loss of its typical effect on both intact cells and derived plasma membranes. Anti-insulin antiserum, potent enough to inhibit the effect of the hormone when both were added to intact cells, did not prevent the insulin effect when added to plasma membranes prepared from insulin-treated cells. Addition of insulin directly to plasma membranes was without effect; exposure of cells prior to rupture was required. Studies of infrared spectra, native membrane protein fluorescence, and fluorescence of 8-anilino-I-naphthalene sulfonate added to the membranes, showed no differences between control plasma membranes and those prepared from insulin-treated cells. We conclude that: (I) plasma membranes can be prepared from insulin-treated fat cells which retain an enhanced glucose transport; (2) the effect of insulin on glucose transport does not involve large scale changes in the structure of the plasma membrane; (3) the insulin unresponsiveness of isolated plasma membranes, as well as the resistance to anti-insulin serum of membranes prepared from insulin-treated cells, appears to result from an uncoupling of insulin binding from glucose transport, the basis of which requires further exploration.
Internalization and intracellular processing of insulin and insulin receptors in adipocytes
Metabolism, 1982
I'orcinc I~UIII)L'OIII~O~~~~ IIIUIII~ wab gcnerousl~ auppl~cd b> Dr Ronald <'hawe of the Eli I.illy C II. \\;,I "'1 was purchaed frum the Yew t<ngland Nuclwr ('II., bovine xrum ,Ilbumin (i'rnction V 1 from Armour and Cu.. and collagcnasc l'rom Worthlngtun Binchemicalh. and chloroquinc. NaF. and 2.Gdinltrophenol from Sigma Male Sprague-Dawlq rats wcighlng I60 to 715 g \*crL: used lor alI cxpcrimenta. Rats were stunned bq :I blow to the head. decapitated. and epididymal fat pads removed. lbolated f31 cells wcrc prepared by shaking at 37OC l'or 60 min in Krebs-Rinpcr bicarbonate bulrer containing collagenase (3 mg/ml) and albumln (40 mg/ml) according to the method of Rodbell.' Cell\ were then liltcrcd through nylon mesh (350 PM). centrifuged at 400 rpm for 2 min and washed twice in Tris Butler. Adipocytc count5 wcrc performed according tu :i modification ol' method III of Hirsch and Gallian" in which the cells were fixed in 1% osmium tetroxide in 0.05 M collidinc buffer (made Isotonic with saline) l'or 24 hr al 37°C' and then (akcn up in a known volume of 0 I54 M NaC I l'or counting. Counting was performed with a model ZB Coulter Counter with 400 PM aperture. lrlsirlin Pretreutrwnt und l~issoc~iation Pr0wdurr.r Ten ml of adipocytes (4-h x IO' cells/ml) subpended in pH 7.(> bufer containing 35 mM Tris. I20 mM NaCI. I .7 mM MgSO4. 1.0 mM CaCI, 2.5 mM KCI. IO mM dextrose, and 1'~: bovine serum albumin (Tris-BSA bulrer) were incubated with insulin in 2 ml polypropylene flasks. Cells were then gently agitated In a bhaking water bath at the indicated times and temperatures. At the end of the incubation period, cells were transferred to I6 125 mm polystyrene tubes. centrifuged at 200 rpm for 7 min. and the insulin containing buffer removed and replaced with insulin free Tris-BSA butfer. pH 7.0. Adipocytes were again centrifuged, resuspended in
Journal of Supramolecular Structure, 1978
Plasma membrane vesicles prepared from adipocytes incubated with insulin exhibited accelerated D-glucose transport activity characteristic of insulin action o n intact fat cells. Both control and insulin-stimulated D-glucose transport activities were inhibited by cytochalasin B and thiol reagents. Extraction of plasma membranes with dimethylmaleic anhydride eluted 80% of the protein from plasma membrane vesicles. The two major glycoprotein bands (94,000 and 78,000 daltons) and small amounts of a 56,000-dalton band were retained in dodecyl sulfate gels of the extracted membranes. Both control and insulin-activated D-glucose transport activities were retained by plasma membrane vesicles extracted with dimethylmaleic anhydride. Cytochalasin B binding activity was also retained by extracted membrane vesicles and D-glucose uptake into extracted vesicles derived from untreated or insulin-treated fat cells was inhibited by cytochalasin B. These results suggest that the modification of the adipocyte hexose transport system elicited by insulin action is not altered by a major purification step which involves quantitative extraction of extrinsic membrane proteins.
The Biochemical journal, 1985
We examined the effects of the membrane-impermeant amino-group-modifying agent fluorescein isothiocyanate (FITC) on the basal and insulin-stimulated hexose-transport activity of isolated rat adipocytes. Pre-treatment of cells with FITC causes irreversible inhibition of transport measured in subsequently washed cells. Transport activity was inhibited by approx. 50% with 2 mM-FITC in 8 min. The cells respond to insulin, after FITC treatment and removal, and the fold increase in transport above the basal value caused by maximal concentrations of insulin was independent of the concentration of FITC used for pre-treatment over the range 0-2 mM, where basal activity was progressively inhibited. The ability of FITC to modify selectively hexose transporters accessible only to the external milieu was evaluated by two methods. (1) Free intracellular FITC, and the distribution of FITC bound to cellular components, were assessed after dialysis of the homogenate and subcellular fractionation on ...
Localization of the insulin receptor in caveolae of adipocyte plasma membrane
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 1999
The insulin receptor is a transmembrane protein of the plasma membrane, where it recognizes extracellular insulin and transmits signals into the cellular signaling network. We report that insulin receptors are localized and signal in caveolae microdomains of adipocyte plasma membrane. Immunogold electron microscopy and immunofluorescence microscopy show that insulin receptors are restricted to caveolae and are colocalized with caveolin over the plasma membrane. Insulin receptor was enriched in a caveolae-enriched fraction of plasma membrane. By extraction with beta-cyclodextrin or destruction with cholesterol oxidase, cholesterol reduction attenuated insulin receptor signaling to protein phosphorylation or glucose transport. Insulin signaling was regained by spontaneous recovery or by exogenous replenishment of cholesterol. beta-Cyclodextrin treatment caused a nearly complete annihilation of caveolae invaginations as examined by electron microscopy. This suggests that the receptor i...
Biochemical Pharmacology, 1987
Aspects of the mechanism by which insulin stimulates the membrane glucose transport system were examined by (1) assessing the influence of the bilayer lipid structure on transport stimulation characteristics, and (2) considering the form of the insulin dose-response curve. We tested the effects of membrane lipid perturbation on the insulin stimulation process. Benzyl alcohol, at concentrations (25mM) that grossly fluidize lipids forming the adipocyte membrane bilayer matrix, caused 50% inhibition of intrinsic transporter activity. However, this membrane perturbation had no significant effect on either the insulin dose-response curve (conducted at 37") or the time-course of the insulin stimulation of hexose transport (conducted at 32"). These data are difficult to rationalize in terms of a model in which transport stimulation involves interaction of transporters and hormone-bound receptors that is limited by lateral diffusion of these proteins in the fluid lipid bilayer. Curve-fitting experimental insulin dose-response data for stimulation of 2&0X)'-D-ghCOSe and D-glucose uptake provided an estimate of an insulin "association constant" for transport regulation that may be compared with recent insulin receptor binding data. Similar magnitude constants were obtained whether estimated directly from plots of transport velocity versus arithmetic hormone dose, or by extrapolation from linear segments of sigmoidal velocity versus log dose plots, or from inverse (Lineweaver-Burk-type) plots of the insulin dose-response data. Insulin apparently regulates transport by associating with a binding site, having an apparent dissociation constant which is determinable through kinetic measurements of hexose uptake (K,. This is in good agreement with the dissociation constant, KD, determined firn Scatchard plots of recent binding data to adipocytes, for a class of receptors representing the "high affinity" binding sites for insulin. Insulin dose-response. curve simulations also indicated that the stimulation process may be classified in pharmacologic terms as a typical graded biologic response and may involve insulin association with a site that regulates transport rates in a manner kinetically analogous to allosteric modulation of a V-series enzyme by a noncompetitive ligand. From the results we suggest that (1) a relatively close association occurs between transport and receptor proteins in the membrane, where the relative activation of transport depends on the fractional occupancy of functional high affinity receptors by insulin, and (2) the insulin stimulation of transport involves regions of the membrane that are not influenced significantly by disordering the membrane lipid matrix.