Biochemistry 1992, 31, 5386-5393
5386
Regulation of the Functional Expression of Hexose Transporter GLUT- 1 by Glucose in Murine Fibroblasts: Role of Lysosomal Degradation? Phillip A. Ortiz, Robert A. Honkanen, David E. Klingman, and Howard C. Haspel* Department of Physiology and Biophysics, State University of New York at Stony Brook, Stony Brook, New York I I794 Received October 10,1991;Revised Manuscript Received April 3, 1992 The nature of the membrane compartments involved in the regulation by glucose of hexose transport is not well defined. The effect of inhibitors of lysosomal protein degradation on hexose transport (Le., uptake of [3H]-2-deoxy-~-glucose)and hexose transporter protein GLUT-1 (Le., immunoblotting with antipeptide serum) in glucose-fed and -deprived cultured murine fibroblasts (3T3-C2 cells) was studied. The acidotropic amines chloroquine (20 pM) and ammonium chloride (10 mM) cause accumulation (both -4-fold) of GLUT-1 protein and a small increase (both -25%) in hexose transport in glucose-fed fibroblasts (24 h). The endopeptidase inhibitor, leupeptin (100 pM)causes accumulation (-4-fold) of GLUT-1 protein in glucose-fed fibroblasts (24 h) without changing hexose transport (55%).These agents do not greatly alter the electrophoretic mobility of GLUT- 1. Neither chloroquine nor leupeptin augment the glucose deprivation (24 h) induced increases in hexose transport (-4-fold) and GLUT-1 content (-7-fold). In contrast, chloroquine or leupeptin diminish the reversal by glucose refeeding of the glucose deprivation induced accumulation of GLUT-1 protein but fail to alter the return of hexose transport to control levels. These results with inhibitors of lysosomal function are consistent with a model in which for murine fibroblasts (i) degradation of GLUT-1 occurs by routing the carrier through acidified compartments to the lysosomes for proteolysis; (ii) inhibition of lysosomal degradation and buffering of acidified intracellular membrane compartments do not cause changes in the glycosylation of GLUT-1; (iii) degradation of GLUT-1 may be indirectly inhibited in glucose-deprived cells; (iv) degradation of GLUT- 1 does not directly control its functional expression; and (v) the ability of glucose to downregulate the functional expression of GLUT-1 may involve “internalization” into compartments which are independent of the pathways involved in its lysosomal degradation. ABSTRACT:
hereospecific glucose (Glc) transport across the plasma membrane of mammalian cells is mediated by a family of related proteins (Bell et al., 1990; Thorens et al., 1990; Kasanicki & Pilch, 1990). These structurally and functionally homologous proteins, designated GLUT-n (nomenclature of Bell and associates (1990)), differ in their tissue distribution, affinity for Glc, and regulation (Bell et al., 1990; Thorens et al., 1990; Kasanicki & Pilch, 1990). GLUT-1, a M , 55 000 transmembrane glycoprotein, was the first such transporter cloned and structurally identified (Mueckler et al., 1985; Birnbaum et al., 1986). It is found at high levels in brain microvessels, kidney, small intestine, placenta, mammalian fibroblasts, and human erythrocytes (Bell et ai., 1990). It has been suggested that GLUT-1 is the “basal” Glc transporter in many cell types (Thorens et al., 1990). The rate of hexose transport and the expression of GLUT-1 protein by mammalian fibroblasts can be acutely and/or chronically regulated by serum, growth factors, transformation, steroids, and nutrient deprivation (cf. Hiraki et al., 1988; Birnbaum et al., 1987; Homer et al., 1987; Rollins et al., 1988; Shawver et al., 1987; White & Weber, 1988; Haspel et al., 1986, 1991; Kalckar & Ullrey, 1984a,b; Flier et al., 1987; Yamada et al., 1983). In rodent fibroblasts Glc deprivation induced alterations in GLUT- 1 expression can be resolved into two components: accumulation of total GLUT-1 polypeptides and the appearance of the aglyco carrier of M, 38 000 (Haspel
et al., 1986). Total accumulation is believed to, either directly or indirectly, result from decreased “carrier inactivation” (Yamada et al., 1983) or degradation of GLUT-1 in response to alterations in the levels of hexose phosphates (Kalckar & Ullrey, 1984b). Aglyco GLUT-1 results from the inhibition of oligosaccharide biosynthesis observed during chronic Glc deprivation (Gershman & Robbins, 1981; Rearick et al., 1981) and is derived from the same core polypeptide as the M, 55 OOO carrier (Haspel et al., 1986). This aglyco form is not observed when cells are fed alternative hexoses, e.g., fructose (Fru), which support oligosaccharide biosynthesis (Haspel et al., 1986) and are not substrates for GLUT-1 (Carruthers, 1990; Gould et al., 1991). The aglyco form is distinct from the core glycosylated M, 42 000 polypeptide observed as a biosynthetic precursor or after treatment with inhibitors of oligosaccharide processing (Haspel et al., 1985, 1988a). In rodent fibroblasts, which express GLUT- 1 protein but not GLUT-2 or GLUT-4 (Haspel et al., 1991; Bell et al., 1990), the Glc deprivation induced increases in hexose transport and content of GLUT-1 protein are not paralleled by GLUT-1 protein synthesis or mRNA content and are not attenuated by treatment with blockers of protein synthesis (Haspel et al., 1986; White & Weber, 1988). In contrast to this posttranslational effect, Glc deprivation has been shown to increase the amount of GLUT-1 mRNA in rat L6 skeletal muscle cells, primary cultures of rat brain glial cells, and chick embryo fibroblasts (Walker et al.,
Supported by the Diabetes Research and Education Foundation, a Research and Development Award from the American Diabetes Association (H.C.H.), and a W. Burghardt Turner Dissertation Fellowship (P.A.O.). * Address correspondence to this author at Health Sciences Center/ T6-180, SUNY at Stony Brook, Stony Brook, NY 11794-8661.
Abbreviations: a-CT, antiserum specific to a carboxy-terminal synthetic peptide of type 1 hexose transporter; dGlc, 2-deoxy-~-glucose; DMEM, Dulbecco’s-modifiedEagle’s medium; ER, endoplasmic reticulum; Fru, fructose; Glc, glucose; Glc-6-Pi,glucose 6-phosphate; GLUT-I, type 1 or erythrocyte/HepG2/brain hexose transporter; M,,apparent molecular weight; PM, plasma membrane, SD, standard deviation;TGN, trans Golgi network.
0006-2960/92/0431-5386$03.00/0 0 1992 American Chemical Society
Biochemistry, Vol. 31, No. 23, 1992 5387
Lysosomal Inhibitors, GLUT- 1, and Glucose Deprivation
8 a n t h e s i s 81Core Glycosylatl9
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FIGURE 1: Model of regulation by glucose of the functional expression of GLUT-1 in murine fibroblasts. After synthesis and core glycosylation in the endoplasmic reticulum (ER), GLUT-1 (GT) undergoes oligosaccharide trimming in the Golgi. Most of the mature GLUT-1 of Glc-fed fibroblasts resides in unacidified intracellular membrane vesicles (small circles) of the trans Golgi network (TGN). From this site, it can cycle (double arrows) to and from the plasma membrane (PM). This process is regulated by Glc metabolism (+Glc (Glc-6-Pi?) and +IC). The majority of GLUT-1 that is routed (thick arrow versus dashed arrow, ?) through acidified (H+) intracellular compartments to (arrow heads) the lysosomes for degradation is derived from the intracellular pool of GLUT-1 present in the TGN. The model predicts that GLUT-1 ”internalization and/or externalization”, in contrast to GLUT-1 degradation, can be directly regulated by Glc metabolism.
1988, 1989, 1990; White & Weber, 1988; Koivisto et al., 1991; Shawver et al., 1987). Unlike the well-characterized insulin-induced recruitment of both G L U T 4 and GLUT-1 to the plasma membrane observed in adipocytes and muscle (Simpson & Cushman, 1986; Kasinicki & Pilch, 1990), a role for the subcellular redistribution of GLUT-1 in the regulation of carrier degradation during Glc deprivation had not been examined in detail. Recently, we have characterized the subcellular localization of GLUT-1 during Glc deprivation induced accumulation of the carrier in rodent fibroblasts by using immunoblotting of subcellular fractions and immunofluorescence microscopy (Haspel et al., 1991). We demonstrated that most GLUT-1 protein is located in intracellular membranes in Glc-fed fibroblasts and that Glc deprivation induces the selective accumulation of GLUT-1 in the plasma membrane. On the basis of these findings, a working model for the regulation of GLUT-1 accumulation and localization during Glc deprivation was formulated (Haspel et al., 1991) and is elaborated on in Figure 1. Klip and associates have recently suggested a similar mechanism for these phenomena in L6 muscle cells (Koivisto et al., 1991). A related hypothesis had previously been proposed by Christopher (1984), but at that time the molecular tools were not available to examine its mechanistic characteristics. Our model predicts that in the Glc-fed state intracellular GLUT-1 protein is constitutively produced and stored in unacidified membrane vesicles of the trans Golgi network (TGN). In this fed state, only small amounts of GLUT-1 cycle to the plasma membrane. Changes in the glycemic state, e.g., Glc deprivation, can alter intracellular hexose phosphate levels (Kalckar & Ullrey, 1984b) and this may regulate the cycling of GLUT-1 to and/or from the cell surface. In order to be degraded, the carrier must return to this intracellular site before it is routed through acidified intracellular compartments to the lysosomes. The model proposes that Glc deprivation induces “externalization” of intracellular GLUT- 1. This externalization may indirectly prevent GLUT- 1 degradation by removing the transporter from prelysosomal pathways and therefore led to its overall accumulation in the plasma membrane. Direct determinations of GLUT- 1 turnover are technically difficult (Haspel et al., 1985, 1986, 1991). This is because radiolabeling of relatively rare proteins, such as GLUT-1, to levels sufficient for detection requires amino acid deprivation, and this perturbation can itself alter GLUT- 1 turnover (Haspel et al., 1985). To examine the nature of the membrane compartments involved in the Glc deprivation effect, we now examine the
effects of inhibitors of lysosomal protein degradation on hexose transport and GLUT-1 protein in murine fibroblasts. We contrast the effects of these agents with those of Glc deprivation and other agents which alter membrane protein sorting. The role of lysosomal degradation on Glc regulation of hexose transport has been studied previously (Christopher & Morgan, 1981; Christopher, 1984). In these studies, an acidotropic amine, NH4+,led to inhibition of the loss of hexose transport activity in hamster fibroblasts following blockage of protein synthesis. However, the results were complex and difficult to interpret, and effects on transporter protein were not examined. Previous reports (Suzuki & Kono, 1979; Hammons & Jarret, 1980; Ezaki et al., 1986; Oka et al., 1987) have not demonstrated an acute (12 h) effect of acidotropic amines on insulin-responsive hexose transport and hexose transporters (both GLUT- 1 and GLUT-4) content/localization in rat adipocytes. These studies did not examine effects of these agents on the return of transport to basal levels following insulin removal and therefore did not preclude that recycling of hexose transporters involves an acidified compartment. Furthermore, chronic exposure to these agents was not examined. Recent ultrastructural studies on the immunolocalization of GLUT-4 in insulin-responsive tissues of the rat (Slot et al., 1991a,b) support the hypothesis that the “coated pit-endosome pathway” may be involved in the intracellular trafficking of some hexose transporters. In this report we show that, in contrast to Glc deprivation, chronic ( 2 6 h) exposure to lysosomal inhibitors causes the accumulation of GLUT- 1 protein without greatly altering hexose transport in murine fibroblasts. The roles of alterations in carrier degradation and localization in these phenomena are discussed. EXPERIMENTAL PROCEDURES Reagents. Leupeptin was purchased from Transformation Research (Boston, MA), and concentrated (1OOX) stocks were prepared in Glc-free Dulbecco’s-modified Eagle’s medium (DMEM) and stored at 4 OC for
