Preferential uptake of D-glucose by plasma membranes isolated from human adipose tissue.

A sensitive method for the measurement of the stereospecific uptake of D-glucose by plasma membranes isolated from human adipose tissue has been developed. The method is based on the difference in uptake of L-[14C]glucose and D-[3H]glucose as measured by the retention of radioactivity by the membrane preparation collected on Millipore filters. This D-glucose-uptake activity was reversible and did not involve any chemical alteration of the sugar. All uptake activity was lost upon boiling the membrane preparation for 5-10 min. All of the hydroxyl groups of D-glucose appear to be involved in a concerted fashion in the uptake reaction. The D-glucose-uptake activity was shown to be closely associated with glucose transport in adipose cells, since it exhibited the following properties characteristic of this carrier-mediated transport system. (a) The uptake was specific for the D-isomer of glucose. (b) Saturation of D-glucose uptake occurred with increasing concentrations of D-glucose, (c) The uptake activity was inhibited by N-ethylamleimide and phloretin, two reagents previously reported to inhibit D-glucose transport. We conclude that plasma membranes isolated from human adipose tissue retain the glucose transport activity of the intact cells and can be used in subsequent attempts at the isolation and characterization of this transport system.

D-Glucose uptake and insulin binding by the human adipose cell plasma membrane as a function of its polypeptide composition.

A preparation of plasma membranes isolated from human omental lipocytes is composed of about 15 major polypeptide components including three major glycoproteins with an apparent molecular weight range from 100000 to 23 000, as determined by sodium dodecyl sulfate - polyacrylamide gel electrophoresis. Extraction of this membrane preparation with sodium iodide or 2,3-dimethylmaleic anhydride solubilized 50 and 70% of the membrane protein, respectively, resulting from the extensive extraction of protein from all but two of the major membrane polypeptide components. This removal of protein did not affect the membrane's stereospecific D-glucose-uptake activity but did reduce its total specific [125I]insulin-binding activity by 46-67%. The binding of [125I]insulin to its specific receptor on lipocyte plasma membranes was detected at physiologic concentrations of the hormone and could be competitively displaced by increasing concentrations of native insulin. The kinetic behaviour of this reaction was approximated by Scatchard analysis, and both the affinity and binding capacity of the plasma membrane for insulin were increased at lower temperatures. These results suggest that D-glucose transport in human adipose tissue is mediated by an intrinsic component of the hydrophobic structure of the lipocyte plasma membrane, and represent a partial purification of this component. In addition, these studies demonstrate and characterize the binding of insulin to the plasma membrane isolated from human lipocytes. A quantitative study of this binding reaction may provide further understanding of the mechanisms underlying the decreased insulin responsiveness characteristic of human diabetes.

Involvement of membrane sulfhydryls in the activation and maintenance of nutrient transport in chick embryo fibroblasts.

At 5 microgram/ml, insulin stimulates hexose, A-system amino acid, and nucleoside transport by serum-starved chick embryo fibroblasts (CEF). This stimulation, although variable, is comparable to that induced by 4% serum. The sulfhydryl oxidants diamide (1-20 micrometer). hydrogen peroxide (500 micrometer), and methylene blue (50 micrometer) mimic the effect of insulin in CEF. PCMB-S,1 a sulfhydryl-reacting compound which penetrates the membrane slowly, has a complex effect on nutrient transport in serum- and glucose-starved CEF. Hexose uptake is inhibited by 0.1-1 mM PCMB-S in a time- and concentration-dependent manner, whereas A-system amino acid transport is inhibited maximally within 10 min of incubation and approaches control rates after 60 min. A differential sensitivity of CEF transport systems is also seen in cells exposed to membrane-impermeant glutathione-maleimide I, designated GS-Mal. At 2 mM GS-Mal reduces the rate of hexose uptake 80-100% in serum- and glucose-starved CEF; in contrast A-system amino acid uptake is unaffected. D-glucose, but not -L-glucose or cytochalasin B, protects against GS-Mal inhibition. These results are consistent with the hypothesis that sulfhydryl groups are involved in nutrient transport and that those sulfhydryls associated with the hexose transport system and essential for its function are located near the exofacial surface of the membrane in CEF.

Reconstitution of D-glucose transport in vesicles composed of lipids and intrinsic protein (zone 4.5) of the human erythrocyte membrane.

Elucidation of the mechanism of facilitated D-glucose transport in human erythrocytes is dependent on the identification and isolation of the membrane protein(s) mediating this process. Based on the fact that stereospecific D-glucose transport is reconstituted in liposomes prepared by sonication of a lipid suspension with ghosts or fractions derived from ghosts, a quantitative assay for the stereospecific D-glucose transport activity of these fractions was developed (Zala CA, Kahlenberg A: Biochem Biophys Res Commun 72:866, 1976). This assay was used to monitor the purification of ghosts. The solubilized membrane protein fraction was chromatographed on a column of diethylaminoethyl cellulose which was eluted stepwise with NaCl-phosphate buffers of increasing ionic strength. A fraction, eluted at an ionic strength of 0.1, displayed a 13- and 27-fold increase in reconstituted transport activity relative to ghosts and to the unfractionated Triton X-100 extract, respectively. This fraction, when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, consisted predominantly of the ghost proteins with an apparent molecular weight of 55,000, commonly designated as zone 4.5; periodic acid-Schiff-sensitive membrane glycoproteins 1-4 were absent. Transport reconstituted by this preparation of zone 4.5 membrane proteins was almost completely abolished by 1-fluoro-2,4-dinitrobenzene, mercuric chloride, and p-chloromercuribenzene sulfonate, but was unaffected by sodium iodoacetate. Extra- and intraliposomal phloretin and cytochalasin B, respectively, exhibited partial inhibition. The stereospecificity and inhibition characteristics of the reconstituted transport imply that all the components of the erythrocyte D-glucose transport system are contained in the zone 4.5 membrane protein preparation.

Partial puridication of a membrane protein from human erythrocytes involved in glucose transport.

In an attempt to determine which membrane proteins are essential to the stereospecific uptake of D-glucose, isolated human erythrocyte membranes were exposed to a variety of reagents capable of selectively extracting various membrane proteins. These reagents included EDTA, lithium 3,5-diiodosalicylate, sodium iodide, and 2,3-dimethylmaleic anhydride. Selective elution of spectrin and Components 2.1, 2.2, 2.3, 4.1, 4.2, 5, and 6 representing 65% of the ghost protein has no effect on the uptake of D-glucose. All of the sugar transport proteins are associated with a membrane residue consisting of the proteins of Bands 3, 4.5, and 7, the periodic acid-Schiff-sensitive glycoproteins, and ghost phospholipids. Specific cross-linking of the proteins of Band 3 of ghosts by the catalyzed oxidation of intrinsic sulfhydryl groups with the o-phenanthroline-cupric ion complex inhibits D-glucose uptake and alters the relative electrophoretic mobility of Band 3 proteins in sodium dodecyl sulfate-polyacrylamide-agarose gels. This uptake activity and the relative mobility of Band 3 proteins are recovered upon reversal of the cross-linking reaction by reduction with 2-mercaptoethanol. These results and other observations indicate that the D-glucose transport protein is an intrinsic component of the hydrophobic structure of the erythrocyte membrane and may be associated with the proteins of Band 3 which are glycoproteins spanning the membrane bilayer. It is proposed that D-glucose transport occurs through a water-filled channel formed by specific subunit aggregates of the transport proteins in the erythrocyte membrane rather than by rotation of the protein within the plane of the membrane.

Biochemical studies on the sulphated glycosaminoglycan fraction of skin fibroblasts cultured from a patient with the Hurler syndrome.

1. The metabolism of the sulphated glycosaminoglycan fraction in cultured skin fibroblasts derived from a patient with the Hurler syndrome and from a normal subject was studied. Two labelled precursors, Na(2) (35)SO(4) and d-[2-(3)H]glucose, were used and their intracellular fates during uptake and ;chase' periods were assessed after separation of sulphated glycosaminoglycans from hyaluronic acid. After 4 or 8h of exposure to culture medium containing both labels, [(35)S]sulphate incorporation into the sulphated glycosaminoglycan fraction was twofold greater in Hurler-syndrome cells than in normal cells. At the same time, the rate of incorporation of [(3)H]glucose into the sulphated glycosaminoglycan fraction was approximately the same for both cell types. Consequently, an increased (35)S/(3)H ratio (nmol of [(35)S]sulphate incorporated/nmol of [(3)H]glucose incorporated) was observed for Hurler-syndrome cells compared with normal cells. 2. The results of ;chase' experiments revealed that although the expected loss and relative retention of labelled sulphate occurred in the sulphated glycosaminoglycan fraction of normal and Hurler-syndrome cells, both cell types retained all of their radioactivity derived from [(3)H]glucose. 3. After 34h exposure to a ;corrective-factor' preparation from urine, the sulphated glycosaminoglycan content (as hexosamine and [(35)S]sulphate) of the Hurler-syndrome cells approached normal values. At the same time, there was an increase in specific radioactivity of ;corrected' Hurler-syndrome cells.

The disorder of hyaluronic acid metabolism in cultured skin fibroblasts derived from a patient with the Hurler syndrome.

The metabolism of hyaluronic acid in cultured skin fibroblasts derived from a patient with the Hurler syndrome and from a normal subject was examined. 1. An increased net incorporation of [(3)H]glucose into the hyaluronic acid fraction of the Hurler-syndrome cells occurred when compared with normal cells. 2. During a ;chase' period, approx. 35% of the radioactivity derived from glucose was lost from the hyaluronic acid fraction of the Hurler-syndrome cells, whereas the normal cells retained all their radioactivity. 3. Although the Hurler-syndrome cells contained a ninefold greater amount of hyaluronic acid than normal cells, simultaneous determination of the specific radioactivity derived from the label revealed a value for the Hurler-syndrome cells one-half that of normal cells. These results are taken to indicate that the Hurler cells synthesize hyaluronic acid de novo at a higher rate than do normal cells. 4. Exposure of Hurler-syndrome cultured fibroblasts to a crude urine corrective-factor preparation (Neufeld & Cantz, 1971), now known to contain alpha-l-iduronidase, the specific Hurler-syndrome corrective factor (Bach et al., 1972), decreased the hyaluronic acid content to near-normal values before any effect was observed on [(3)H]glucose incorporation into the hyaluronic acid fraction. 5. In addition, the hyaluronic acid content of the normal cells decreased after exposure to the corrective factor of urine. 6. The mobilization of hyaluronic acid in Hurler-syndrome and normal cells exposed to the crude corrective-factor preparation of urine caused a decrease in specific radioactivity in the ;corrected' Hurler-syndrome cells and an increase in specific radioactivity in the ;corrected' normal cells.