Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118
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ABSTRACT |
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The major leptin-containing
membrane compartment was identified and characterized in rat adipose
cells by means of equilibrium density and velocity sucrose gradient
centrifugation. This compartment appears to be different from
peptide-containing secretory granules present in neuronal, endocrine,
and exocrine cells, as well as from insulin-sensitive GLUT-4-containing
vesicles abundant in adipocytes. Exocytosis of both leptin- and
GLUT-4-containing vesicles can be induced by insulin; however, only
leptin secretion is responsive to serum stimulation. This latter effect
is resistant to cycloheximide, suggesting that serum triggers the
release of a stored pool of presynthesized leptin molecules. We
conclude that regulated secretion of leptin and insulin-dependent
translocation of GLUT-4 represent different pathways of membrane
trafficking in rat adipose cells. NIH 3T3 cells ectopically expressing
CAAT box enhancer binding protein- and Swiss 3T3 cells expressing
peroxisome proliferator-activated receptor-
undergo differentiation
in vitro and acquire adipocyte morphology and insulin-responsive
glucose uptake. Only the former cell line, however, is capable of
leptin secretion. Thus different transcriptional mechanisms control the
developmental onset of these two major and independent physiological
functions in adipose cells.
regulated secretion; glucose transport; GLUT-4
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INTRODUCTION |
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ADIPOCYTES
PRODUCE AND SECRETE a variety of physiologically important
proteins (28), such as leptin (34), adipsin
(8), adipocyte complement-related protein (ACRP30)
(29), tumor necrosis factor- (14), and
lipoprotein lipase (11). Studies both in vivo and in vitro
have demonstrated that secretion of these proteins from adipose cells
has two components, constitutive and regulated. In other words,
adipocytes continuously release leptin (3, 5), adipsin
(18, 33), ACRP30 (4, 29), and lipoprotein lipase (11, 24) into the medium, but this process may be
acutely and substantially stimulated by insulin without any marked
changes in the constitutive secretory pathway (4). The
acute effect of insulin on secretion precedes major changes in the
biosynthesis of secreted proteins, and it is preserved, at least
partially, in the presence of cycloheximide (5, 24, 29).
This suggests that fat cells may possess regulatable pools of
presynthesized secreted proteins that may be discharged by insulin.
Secretory pathways in adipocytes, however, have not yet been
characterized at the molecular level, and intracellular membrane
structures that are responsible for accumulation and storage of
secreted proteins have not been identified. It is also not known
whether or not secretion from adipose cells may be regulated by any
secretagogue other than insulin.
In addition to secretion, adipose cells possess another pathway of intracellular protein trafficking that has been studied to a much greater extent. These cells translocate glucose transporter isoform 4 (GLUT-4) and several other co-localized proteins from intracellular vesicles to the cell surface in a strictly insulin-dependent fashion (9, 16, 23, 25). The role of GLUT-4-containing vesicles in the regulated secretion of soluble proteins from adipocytes has not been elucidated until recently, when two research groups demonstrated by immunofluorescence that GLUT-4 does not co-localize with either leptin (3) or ACRP30 (4). These results suggest that the "GLUT-4 pathway" may, in fact, be different from the regulated secretory pathway(s) in adipose cells.
Here, we studied compartmentalization of intracellular leptin in rat adipocytes and found that this protein is localized in a novel type of a secretory compartment that is different from both GLUT-4-containing vesicles and "classical" peptide-containing secretory granules present in endocrine cells. We have also demonstrated that, although both leptin secretion and glucose uptake in adipocytes are stimulated by insulin, only the former process is responsive to serum. This suggests that different signaling mechanisms may control regulated secretion and GLUT-4 translocation in fat cells. Finally, we show that different genetic programs are required for the developmental onset of leptin secretion and insulin-stimulated glucose uptake in differentiating adipose cells. Thus regulated secretion of leptin and translocation of GLUT-4-containing vesicles represent different pathways of membrane trafficking in adipose cells.
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EXPERIMENTAL PROCEDURES |
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Antibodies and cell lines.
In this study, we used monoclonal anti-GLUT-4 antibody 1F8
(15), rabbit polyclonal antibodies against calnexin
(StressGen) and secretogranin II (Biodesign International), goat
polyclonal antibody against chromogranin B (Research Diagnostics), and
mouse monoclonal antibodies against trans-Golgi network
marker 38 (TGN38, Affinity Bioreagents) and synaptophysin (Chemicon
International). NIH 3T3 cells ectopically expressing CAAT box enhancer
binding protein- (C/EBP
) and Swiss 3T3 cells expressing
peroxisome proliferator-activated receptor-
(PPAR
) were described
recently (10).
Isolation and fractionation of rat adipocytes. Adipocytes were isolated from epididymal fat pads of male Sprague-Dawley rats (150-200 g) by collagenase digestion (26) and were transferred to DMEM or to Krebs-Ringer phosphate (KRP) buffer (12.5 mM HEPES, 120 mM NaCl, 6 mM KCl, 1.2 mM MgSO4, 1 mM CaCl2, 0.6 mM Na2HPO4, 0.4 mM Na2PO4, 2.5 mM D-glucose, and 2% BSA, pH 7.4) for 15-20 min. Then, the medium was changed, and the cells were placed in the incubator (37°C, 5% CO2). Insulin (Eli Lilly) or fetal bovine serum (FBS, GIBCO) was administered to the cells where indicated for 1-2 h. The medium was saved for leptin determination by RIA, and the cells were washed twice with HES buffer [20 mM HEPES, 250 mM sucrose, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), 5 mM benzamidine, 1 µM pepstatin, 1 µM aprotinin, 1 µM leupeptin, pH 7.4] cooled to 18°C and homogenized with a Potter-Elvehjem Teflon pestle, and subcellular fractions were prepared by differential centrifugation (30).
Cell culture.
NIH 3T3 and Swiss 3T3 cells expressing retroviral C/EBP and PPAR
were grown in DMEM supplemented with 10% FBS. Differentiation was
induced (day 0) by changing the medium to DMEM containing 10% FBS, 0.39 µg/ml dexamethasone, 115 µg/ml IBMX, and 100 µg/ml insulin. After 48 h, the cells were transferred to DMEM containing 10% FBS and 25 µg/ml insulin. The cell medium was changed every 48 h. In the case of Swiss-PPAR
cells, 10 µM troglitazone was added to the medium (days 0-5) to promote
differentiation. Differentiation of C/EBP
-transfected NIH 3T3 cells
does not require troglitazone (10).
2-Deoxy-[3H]glucose uptake. Primary rat adipocytes were incubated in KRP without glucose in the absence or in the presence of insulin or FBS for 1 h at 37°C in the incubator with 5% CO2. An aliquot of fat cells (80 µl) was mixed with 40 µl of 2-deoxy-[3H]glucose (NEN) diluted with 2.5 mM of cold D-glucose to specific activity of 15 µCi/mM for 30 s in a long microfuge tube. Then, silicon oil (~40 µl) was added to the mixture, and the tube was centrifuged for 10 s in an Eppendorf microcentrifuge. The tube was cleaved in the middle of the oil layer, and floating fat cells were transferred to a scintillation vial. The amount of 2-deoxy-[3H]glucose taken by adipocytes was counted in an LKB scintillation counter. Each determination was done four times in parallel.
Fractionation of intracellular microsomes from rat adipocytes in sucrose gradients. For velocity fractionation, 1-2 mg of light and heavy microsomes (LM and HM, respectively) resuspended in 150-200 µl of HES or PBS were loaded on a 10-30% (wt/vol) sucrose gradient (in 10 mM HEPES, 150 mM NaCl, 0.1 mM MgCl2, 1 mM EGTA, pH 7.4) and centrifuged for 50 min at 48,000 rpm in a Beckman SW-50.1 rotor. For equilibrium density centrifugation, the same material was fractionated in a 10-50% (wt/vol) sucrose gradient for 16 h at 48,000 rpm. After each centrifugation, fractions were collected starting from the bottom of the tube and analyzed for the total protein content and for specific proteins by radioimmunoassay, Western blot, and dot blot assay.
Immunoadsorption of GLUT-4-containing vesicles.
Protein A-purified 1F8 antibody and nonspecific mouse IgG (Sigma) were
each coupled to acrylic beads (Reacti-gel GF 2000, Pierce) at a
concentration of 0.50 and 0.56 mg of antibody/ml of resin,
respectively, according to the manufacturer's instructions. Before
use, the beads were saturated with 2% BSA in PBS for 1 h and washed
with PBS. LM and HM (200-600 µg) from rat adipocytes were
incubated separately with 100-200 µl each of the specific and
nonspecific antibody-coupled beads overnight at 4°C. The beads were
washed five times with PBS and eluted with 1% Triton X-100 in PBS.
This fraction was used for leptin determination. The beads were then
washed again with PBS and with 10 mM Tris · HCl, pH 7.8, and
eluted with Laemmli sample buffer (19) for the analysis of
immunoadsorbed GLUT-4 by Western blotting.
Gel electrophoresis and immunoblotting. Proteins were separated by SDS-PAGE according to Laemmli (19) but without reducing agents and were transferred to Immobilon-P membrane (Millipore) in 25 mM Tris, 192 mM glycine, pH 8.3. After transfer, the membrane was blocked with 10% nonfat dry milk in PBS for 1 h at room temperature. Proteins were visualized with specific antibodies, horseradish peroxidase (HRP)-conjugated secondary antibodies (Sigma), and an enhanced chemiluminescent substrate kit (NEN). Autoradiograms were quantitated in a PhosphorImager (Molecular Dynamics).
Dot blot analysis. Nondenatured proteins were bound to Immobilon-P membrane with the help of a dot blot apparatus. The membrane was washed thoroughly and probed with anti-TGN38 antibody (1:500) in 5% BSA overnight at 4°C. Then, the membrane was washed and treated with HRP-conjugated secondary antibodies and chemiluminescent substrate similar to Western blot membranes. Resulting autoradiograms were quantitated in a computing densitometer (Molecular Dynamics).
Radioimmunoassay. Leptin content was determined with the help of a 125I-leptin radioimmunoassay kit (Linco) according to the manufacturer's instructions. In some experiments, when the leptin content was analyzed in the cell medium, samples were lyophilized and solubilized in water. Each determination (except for the gradient fractions) was done three to five times in parallel.
Protein content. Protein content was determined with a bicinchoninic acid kit (Pierce) according to the manufacturer's instructions.
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RESULTS |
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Intracellular pools of leptin and GLUT-4 are localized in different
vesicular carriers.
Rat adipocytes were separated into five crude subcellular fractions by
differential centrifugation according to Simpson et al.
(30). These fractions include: 1) first
low-speed pellet (mitochondria, nuclei, and lysosomes), 2)
plasma membrane (PM), 3) HM (intermediate fraction enriched
with endoplasmic reticulum, but also containing PM and LM markers),
4) LM, which contain Golgi apparatus, trans-Golgi
network, and endosomes, and 5) cytosol. Noteworthy, this
fractionation procedure allows the separation of the whole adipose cell
into subcellular fractions without any "leftover" with the
exception of the "lipid cake," which is discarded after the first
centrifugation. Of all these fractions, only HM and LM contained
significant amounts of leptin; therefore, they were fractionated
further in a 10-50% equilibrium density sucrose gradient. Under
these conditions, leptin-containing membranes form a distinct peak with
a buoyant density roughly corresponding to the density of other
microsomal structures in the cell (compare the distribution of leptin
and the total microsomal protein in Fig.
1).
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Leptin secretion but not glucose transport is stimulated by serum
at the posttranslational level.
As has been shown recently (3, 5), secretion of leptin
from adipocytes is acutely and substantially stimulated by insulin. Here, we confirm those results and demonstrate that serum also has an
analogous stimulatory effect on leptin secretion (Fig. 4A). In control experiments,
we have shown that leptin is stable in the adipocyte medium, even after
overnight incubation at 37°C (results not shown). Therefore, our
results should not be affected by leptin processing in the medium.
Cycloheximide does not prevent serum-induced acute increase in leptin
secretion (Fig. 4B, left); neither does it block
a concomitant decrease in intracellular leptin content (Fig.
4B, right). Noteworthy, the amount of
intracellular leptin in serum-treated adipocytes falls acutely below
the detection threshold. On the basis of this result and the data shown
in Figs. 1 and 3, we suggest that the major pool of intracellular
leptin in adipocytes is localized in a regulatable population of small storage vesicles. In our preliminary experiments, we checked several downstream secondary messengers to "shortcut" signaling pathways that have the potential to trigger acute leptin secretion. However, in
these experiments, the Ca2+ ionophore A-23187 and
the phorbol ester PMA (phorbol 12-myristate 13-acetate) were
without effect (data not shown).
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Developmental onset of leptin secretion and GLUT-4 translocation. It has been demonstrated previously (13, 20, 22) that GLUT-4 and leptin represent important markers of adipocyte differentiation. Because regulated secretion of leptin and GLUT-4 translocation are likely to represent different and independent pathways of intracellular membrane traffic, it is interesting to determine whether or not the same transcriptional mechanism is responsible for the developmental onset of these important physiological functions.
Differentiation of adipocytes is controlled mainly by two families of transcription factors, the C/EBP family and the PPAR family (21). It has been shown that, upon ectopic expression of PPAR
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DISCUSSION |
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We have compared the biochemical properties and regulation of leptin- and GLUT-4-containing vesicular carriers in adipose cells. All aspects of this study indicate that regulated secretion of leptin and insulin-dependent translocation of GLUT-4 represent different pathways of membrane trafficking. Also, the leptin-containing compartment from adipocytes is obviously different from the peptide-containing secretory granules present in neuronal, endocrine, and exocrine cells in buoyant density, sedimentational behavior, and kinetics of secretion. What, then, could be the nature of leptin-containing vesicles? A related question is whether this type of secretory vesicles is unique to adipocytes, or whether there are analogous compartments present in other cells.
Recent evidence suggests that cells that have previously been known as constitutive secretory cells may, in fact, possess regulated secretory pathways. Results obtained in Palade's laboratory (27) demonstrate that, in hepatocytes, different vesicular carriers are responsible for secretion of soluble proteins (albumin, apolipoprotein B, prothrombin, C3 component of the complement, and caeruloplasmin) and membrane proteins (polymeric IgA receptor, transferrin receptor, asialoglycoprotein receptor, etc.). These two types of vesicular carriers have different sizes but a similar buoyant density, which is much less than the buoyant density of classical dense core secretory granules. Thus hepatocytes sort their soluble secretory products away from the constitutive pathway of membrane protein delivery (as is the case with regulated secretion), but they discharge these products continuously without the involvement of a secretagogue.
A "real" regulated secretory pathway has recently been elucidated in other constitutive cell lines, L and CHO (7). In these cells, a fraction of newly synthesized glucosaminoglycans is retained inside the cell in a population of rab3D-containing post-Golgi storage vesicles with a buoyant density much less than that of classical secretory granules.
In our laboratory, we have recently found a novel type of secretory vesicles in brain and PC12 cells that can be separated from both secretory granules and synaptic vesicles (31, 32). These vesicles contain a novel enzyme, aminopeptidase B (6). Similar to adipocyte products, aminopeptidase B is constitutively released from cells, but its secretion can be substantially stimulated by various secretagogues (2).
Sedimentation properties and the buoyant density of leptin-containing vesicles in fat cells (Figs. 1 and 2), aminopeptidase B-containing vesicles from neurons (31, 32), regulated vesicles in CHO cells (7), and soluble protein-carrying secretory vesicles in hepatocytes (27) are all rather similar. In addition, adipocytes contain large amounts of rab3D (1) whose expression mimics that of leptin in differentiation (1, 20) and is upregulated along with leptin in adipocytes from obese rats (12). Thus adipocytes may secrete their physiologically important products via a novel regulated secretory pathway, the molecular aspects of which are just beginning to emerge.
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ACKNOWLEDGEMENTS |
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The authors would like to thank Dr. Tatyana Kupriyanova for the preparation of immunobeads.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-51586 to S. R. Farmer and DK-52057 and DK-56736 to K. V. Kandror and by a research grant from the American Diabetes Association to K. V. Kandror.
Address for reprint requests and other correspondence: K. V. Kandror, Boston Univ. School of Medicine, Dept. of Biochemistry, K121, 715 Albany St., Boston, MA 02118 (E-mail: kandror{at}biochem.bumc.bu.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 22 February 2000; accepted in final form 22 May 2000.
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