(Received for publication, October 12, 1995)
From the
A novel membrane aminopeptidase has been identified as a major protein in vesicles from rat adipocytes containing the glucose transporter isotype Glut4. In this study we have characterized this aminopeptidase, referred to as vp165, in 3T3-L1 adipocytes. The subcellular distributions of vp165 and Glut4 were determined by immunoisolation of vesicles with antibodies against both proteins, by immunofluorescence, and by subcellular fractionation and immunoblotting. Relative amounts of vp165 at the cell surface in basal and insulin-treated cells were assayed by cell surface biotinylation. These experiments showed that vp165 and Glut4 were entirely colocalized and that vp165 increased markedly at the cell surface in response to insulin, in a way similar to Glut4. When intact cells were assayed with a novel, membrane-impermeant fluorogenic substrate for vp165, we found that insulin stimulated aminopeptidase activity at the cell surface. This observation provides direct evidence for the functional consequence of vp165 translocation.
An important effect of insulin is to increase glucose transport into muscle and fat cells. The basis of this effect is an increase in the amount of the glucose transporter isotype Glut4 in the plasma membrane, which is probably largely due to insulin-elicited fusion of intracellular vesicles containing Glut4 with the plasma membrane(1, 2) . We and others have developed methods for isolating these Glut4 vesicles from fat and muscle cells and are analyzing the proteins in them(3, 4, 5, 6) . A major protein, of 165 kDa, in the Glut4 vesicles from rat adipocytes (designated vp165) has recently been characterized by the Pilch laboratory and ourselves(3, 7, 8) . Through cloning of the cDNA for vp165, we found that it is a novel membrane aminopeptidase, consisting of a 109 residue cytoplasmic amino-terminal domain that contains several potential sorting signals similar to those in Glut4, a single transmembrane segment, and a large lumenal domain that contains the active site(9) .
The distribution of vp165 in rat adipocytes has been determined for basal and insulin-treated cells by subcellular fractionation and immunoblotting(3, 7) . These earlier studies showed that vp165, like Glut4, is concentrated in the low density microsomes and redistributes to the plasma membrane in response to insulin. Moreover, they showed that intracellular vp165 is located in vesicles that also contain Glut4, since immunoadsorption of vesicles with antibodies against Glut4 also adsorbed most of the vp165(3, 7) . However, these earlier studies did not rigorously address the questions of whether vp165 and Glut4 are entirely colocalized and translocate in a quantitatively similar way in response to insulin. One reason for this is that subcellular fractionation provides only a crude indication of subcellular localization and typically underestimates translocation due to contamination of the plasma membrane fraction with intracellular membranes (see Results and Discussion). Also, since at the time antibodies that immunoadsorbed vp165 were not available, it was not possible to perform the complementary immunoadsorption of vesicles with antibodies against vp165, in order to determine whether all the Glut4 is in vesicles that also contain vp165. Another question not addressed by these earlier studies, which is especially important now that vp165 has been established to be an aminopeptidase, is whether the translocation of vp165 to the plasma membrane is, in fact, accompanied by the appearance of cell surface aminopeptidase activity.
In the present study, we have characterized vp165 in 3T3-L1 adipocytes, cultured cells that are very insulin-responsive and have been employed extensively for the investigation of insulin-stimulated Glut4 translocation, as well as other insulin actions. Through the use of better antibodies against vp165 and a membrane-impermeant aminopeptidase substrate, we have been able to answer the questions posed above.
Figure 4: Lys-AMC-glutathione as a membrane-impermeant substrate. Panel A, structure of Lys-AMC-glutathione, showing the state of protonation at pH 7. Panel B, substrate (0.6 mM in 400 µl) in 250 mM sucrose, 20 mM Hepes, pH 7.4, was incubated at 25 °C in the cuvette of the fluorescence spectrophotometer. At the indicated points, low density microsomes (LDM) from rat adipocytes (about 70 µg in 100 µl) and then the nonionic detergent (D) octaethylene glycol dodecyl ether (2.5 µl of 20%) were added. After subtraction of the background fluorescence change given by substrate alone, the rates without and with detergent were 0.36 and 5.3 arbitrary units/min.
Figure 5:
Cell surface aminopeptidase activity of
intact 3T3-L1 adipocytes. Hydrolysis of lysyl-AMC-glutathione by basal
() and insulin-treated (
) 3T3-L1 adipocytes at 37 °C was
measured as described under ``Experimental Procedures.'' The
fluorescence values (arbitrary units) given by 100-µl aliquots of
medium are plotted against the time of incubation of the substrate with
the cells. Each point is the average of three values given by the
aliquots from three 35-mm wells of cells ± the S.D. (error
bars). Four separate experiments of this type gave similar
results.
The membrane impermeability of the Lys-AMC-glutathione was tested with low density microsomes, freshly prepared (not frozen) from rat adipocytes as described in Keller et al.(9) , except for the omission of EDTA in the buffer. Activity in the absence and presence of a nonionic detergent was assayed at 25 °C in a recording fluorescence spectrometer (Hitachi model F-3010) with excitation at 365 nm and emission at 455 nm in a 0.5-ml cuvette with 5-mm path length.
For the assay of the aminopeptidase activity of intact 3T3-L1 adipocytes, the serum-free medium on 6-well plates was replaced with KRPB (1.4 ml/well), and the cells were either treated with 300 nM insulin for 15 min or left in the basal state, at 37 °C. Lysyl-AMC-glutathione was then added from a stock solution, such that the final concentration and volume were 0.5 mM and 1.5 ml/well. At various times aliquots (100 µl) were removed from each well, diluted with 400 µl of KRPB, and kept on ice until fluorescence was measured, as described above.
Figure 1: Colocalization of vp165 and Glut4 in 3T3-L1 Adipocytes. Panel A, vesicles were immunoadsorbed from the microsome/cytosol fraction of basal(-) and insulin-treated (+) 3T3-L1 adipocytes with antibodies against Glut4 (G), antibodies against vp165 (V), and irrelevant antibodies (C) on fixed S. aureus cells, as described under ``Experimental Procedures.'' Samples were also prepared in the same way without any adsorbent (N). The relative amounts of vp165 and GLUT4 in the adsorbed material (Adsorbed, lanes 1-8) and in the microsome/cytosol fraction after adsorption (Non-Adsorbed, lanes 9-16) were analyzed by immunoblotting. Each lane contains either 2.5% (lanes 1-8) or 0.3% (lanes 9-16) of the material derived from a 10-cm plate. Lanes 1-8 and 9-16 are separate immunoblots and so are not directly comparable. A repetition of this entire experiment gave similar results. Panel B, immunofluorescence of vp165 (left) and Glut4 (right) in 3T3-L1 adipocytes. Fixed permeabilized cells were labeled simultaneously with affinity-purified rabbit antibodies against the amino terminus of vp165 and a monoclonal mouse antibody against the carboxyl terminus of Glut4, which were then detected with fluorescein-conjugated antibodies against rabbit immunoglobulin and Texas Red-conjugated antibodies against mouse immunoglobulin, respectively. Most of the circles within each cell are fat droplets; the one partially surrounded by staining is the nucleus, which was identified by DAPI staining. The same staining pattern was seen in cells stained only for vp165 or Glut4, whereas no staining was observed with irrelevant rabbit immunoglobulin as the primary antibody.
The subcellular distributions of vp165 and Glut4 were also compared by double-label immunofluorescence (Fig. 1B). The patterns of staining for the two proteins were superimposable, with the most intense staining in vesicles surrounding a portion of the nucleus and additional staining in vesicles throughout the cell. This distribution is the same as the one previously described for Glut4(13) .
Figure 2: Insulin-stimulated translocation of vp165 and Glut4 to the plasma membrane. Plasma membrane (PM) and low density microsomes (LDM) fractions from basal(-) and insulin-treated (+) 3T3-L1 adipocytes were immunoblotted for vp165 and Glut4. Lanes 1-4 contain 10 µg of protein. The lanes labeled LDM 1/2 through 1/32 (lanes 5-9) were loaded with the indicated fraction of 10 µg of basal LDM. Similar results were obtained in three other separate subcellular fractionations.
Previous studies have indicated that the measurement of Glut4 translocation in 3T3-L1 adipocytes by subcellular fractionation and immunoblotting substantially underestimates the magnitude of the effect, probably because the plasma membrane fraction is contaminated by some intracellular vesicles containing Glut4. In our hands and others, the increase in Glut4 at the cell surface, when assessed by photoaffinity labeling, is 12-17-fold(10, 14) . This increase corresponds more closely to the increase in glucose transport, which is typically 10-20-fold(10, 14) . This criticism would also apply to the measurement of vp165 translocation by immunoblotting the plasma membrane fractions, and we were therefore led to develop a cell surface biotinylation method for assessment of vp165 translocation. This method was based on the finding, made by Kandror and Pilch(15) , that vp165 at the cell surface of rat adipocytes is susceptible to biotinylation.
Basal and insulin-treated 3T3-L1 adipocytes were cooled to 4 °C, in order to prevent membrane trafficking(16) , and then reacted with a membrane-impermeant reagent that biotinylates exposed amino groups. Subsequently vp165 was isolated by immunoprecipitation and its extent of biotinylation measured by blotting with streptavidin conjugated to horseradish peroxidase. The results in Fig. 3show that by this assay the increase in vp165 at the cell surface in response to insulin was approximately 8-fold (compare lane 1, with lanes 2-5). This higher value is closer to that expected on the basis of the results with Glut4 described above. In several of these experiments we also isolated Glut4 by immunoprecipitation and examined it for biotinylation, but did not detect any biotinylation (data not shown). This outcome is explicable by the fact that Glut4 has only one lysine in its predicted extracellular domain, whereas vp165 has 45 lysines in its extracellular domain(9, 17) . In the future, it should be possible to determine the kinetics of vp165 recycling in basal and insulin 3T3-L1 adipocytes by use of this cell surface biotinylation method, in an analogous way to the determination of the kinetics of Glut4 recycling by photoaffinity labeling Glut4 at the cell surface(18) .
Figure 3: Biotinylation of vp165 at the cell surface. Basal(-) and insulin-treated (+) 3T3-L1 adipocytes were surface biotinylated as described under ``Experimental Procedures.'' Samples of the cell lysates were then immunoprecipitated with antibodies against vp165 (V) and irrelevant antibodies (C). Biotinylation was detected by blotting with streptavidin conjugated to horseradish peroxidase. Lanes 1, 2, 6, and 7 contain immunoadsorbates derived from 10% of a 35-mm plate, and lanes 3-5 contain one-half, one-fourth, and one-eighth of this amount of the vp165 immunoadsorbate from insulin-treated cells. Similar results were obtained in a repetition of this entire experiment.
Intact 3T3-L1 adipocytes, in the basal and insulin-treated states, were assayed for activity toward the lysyl-AMC-glutathione (Fig. 5). In four separate experiments of this type, the activity of the insulin-treated cells ranged from 1.8- to 3.4-fold higher. Thus, as expected, insulin does increase cell surface aminopeptidase activity. However, the fold increase is less than predicted from the increase in vp165 at the cell surface as assayed by biotinylation (Fig. 3). A likely explanation for this difference is that there is another membrane aminopeptidase that is constitutively present at the cell surface and contributes significantly to the observed activity. A number of such aminopeptidases are known(19) . It is worth noting that this aminopeptidase assay provides a new way of monitoring insulin-stimulated translocation that does not require cell disruption.
Figure 6: Expression of vp165 and Glut4 upon differentiation of 3T3-L1 adipocytes. SDS samples were prepared from 10-cm plates of 3T3-L1 cells 2 days after reaching confluence (day 0 for differentiation) and at various days thereafter during the differentiation protocol. Samples containing 25 µg of protein were immunoblotted for vp165 and Glut4 (lanes 1-6). The standards (lanes 7-13) contain the stated fraction of 25 µg of protein of the day 8 sample. A repetition of this entire experiment yielded similar results.
The translocation of vp165 was accompanied by the predicted appearance of cell surface aminopeptidase activity. Thus, the characterization of this vesicle protein has led to the discovery of a hitherto unknown insulin effect on cell surface function. In order to understand the ramifications of this effect, it is now important to identify the physiological substrates for vp165. Finally, it should be noted that the Glut4 vesicles contain a number of unidentified major proteins (3, 4, 5) , and so it is possible that there are other proteins, like Glut4 and vp165, that translocate markedly in response to insulin and modify functions at the cell surface.