Triacylglycerol Is Synthesized in a Specific Subclass of Caveolae in Primary Adipocytes

Abstract

A principal metabolic function of adipocytes is to synthesize triacylglycerol (TG) from exogenous fatty acids. The level of fatty acids has to be tightly controlled in the adipocyte, as they can act as detergents that rapidly dissolve the plasma membrane, causing cell lysis if allowed to accumulate. Fatty acids therefore have to be efficiently converted to TG and stored in the central lipid droplet. We report that in intact primary adipocytes exogenous oleic acid was taken up and directly converted to TG in the plasma membrane, in a novel subclass of caveolae that specifically contains the protein perilipin. Isolated caveolae catalyzed de novo TG synthesis from oleic acid and glycerol 3-phosphate. Electron microscopy revealed the presence of caveolin and perilipin in caveolae and in lipid-laden bulbs in the plasma membrane, and fluorescence microscopy demonstrated colocalization of fatty acids/TG with caveolin and perilipin at the plasma membrane. A second caveolae fraction was isolated, which lacked perilipin and the triacylglycerol synthesizing enzymes. Both caveolae fractions contained caveolin-1 and the insulin receptor. The findings demonstrate that specific subclasses of caveolae carry out specific functions in cell metabolism. In particular, triacylglycerol is synthesized at the site of fatty acid entry in one of these caveolae classes. A principal metabolic function of adipocytes is to synthesize triacylglycerol (TG) from exogenous fatty acids. The level of fatty acids has to be tightly controlled in the adipocyte, as they can act as detergents that rapidly dissolve the plasma membrane, causing cell lysis if allowed to accumulate. Fatty acids therefore have to be efficiently converted to TG and stored in the central lipid droplet. We report that in intact primary adipocytes exogenous oleic acid was taken up and directly converted to TG in the plasma membrane, in a novel subclass of caveolae that specifically contains the protein perilipin. Isolated caveolae catalyzed de novo TG synthesis from oleic acid and glycerol 3-phosphate. Electron microscopy revealed the presence of caveolin and perilipin in caveolae and in lipid-laden bulbs in the plasma membrane, and fluorescence microscopy demonstrated colocalization of fatty acids/TG with caveolin and perilipin at the plasma membrane. A second caveolae fraction was isolated, which lacked perilipin and the triacylglycerol synthesizing enzymes. Both caveolae fractions contained caveolin-1 and the insulin receptor. The findings demonstrate that specific subclasses of caveolae carry out specific functions in cell metabolism. In particular, triacylglycerol is synthesized at the site of fatty acid entry in one of these caveolae classes. To gain knowledge of the biochemical pathways of fat metabolism in human physiology is central to understanding the derangements and pathology in the adipocyte handling of fatty acids associated with obesity and type 2 diabetes. The adipocyte stores large amounts of triacylglycerol (TG), 1The abbreviations used are: TG, triacylglycerol; Mes, 4-morpholineethanesulfonic acid. 1The abbreviations used are: TG, triacylglycerol; Mes, 4-morpholineethanesulfonic acid. which continually turn over during lipolytic hydrolysis and reacylation of exogenous fatty acids. Human adipocytes do not have the capacity to synthesize significant amounts of fatty acids de novo (1Goldrick R.B. J. Lipid Res. 1967; 8: 581-588Abstract Full Text PDF PubMed Google Scholar). The large part of triacylglycerol synthesis comes from fatty acids provided by lipoprotein lipase hydrolysis of chylomicron or very low density lipoprotein TG in the capillary blood vessels. Fatty acids act as detergents that rapidly dissolve the plasma membrane, causing cell lysis if allowed to accumulate (2Strålfors P. FEBS Lett. 1990; 263: 153-154Crossref PubMed Scopus (21) Google Scholar). The flow of fatty acids into the adipocyte can be high and these fatty acids have to be efficiently and rapidly converted to TG and stored away in the central lipid droplet. Several fatty acid-binding proteins that may mediate transfer of fatty acids have been identified (3Czech M.P. Mol. Cell. 2002; 9: 695-696Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Caveolae invaginations of the plasma membrane are typically 50-100 nm in diameter and are very abundant in adipocytes (4Thorn H. Stenkula K.G. Karlsson M. Örtegren U. Nystrom F.H. Gustavsson J. Strålfors P. Mol. Biol. Cell. 2003; 14: 3967-3976Crossref PubMed Scopus (112) Google Scholar). Caveolae are involved in vesicle-mediated uptake into the cell as well as in organizing signal transduction (5Razani B. Woodman S.E. Lisanti M.P. Pharmacol. Rev. 2002; 54: 431-467Crossref PubMed Scopus (841) Google Scholar, 6Isshiki M. Anderson R.G. Traffic. 2003; 4: 717-723Crossref PubMed Scopus (113) Google Scholar, 7Simons K. Toomre D. Nat. Rev. Mol. Cell. Biol. 2000; 1: 31-40Crossref PubMed Scopus (5134) Google Scholar). In adipocytes the insulin receptor is localized in caveolae (8Gustavsson J. Parpal S. Karlsson M. Ramsing C. Thorn H. Borg M. Lindroth M. Peterson K.H. Magnusson K-E. Strålfors P. FASEB J. 1999; 13: 1961-1971Crossref PubMed Scopus (316) Google Scholar, 9Karlsson M. Thorn H. Danielsson A. Stenkula K.G. Öst A. Gustavsson J. Nystrom F.H. Strålfors P. Eur. J. Biochem. 2004; 271: 2471-2479Crossref PubMed Scopus (85) Google Scholar) and in response to insulin glucose transporters GLUT4 are translocated to caveolae for glucose uptake (10Gustavsson J. Parpal S. Strålfors P. Mol. Med. 1996; 2: 367-372Crossref PubMed Google Scholar, 11Karlsson M. Thorn H. Parpal S. Strålfors P. Gustavsson J. FASEB J. 2001; 16: 249-251PubMed Google Scholar). Destruction of caveolae causes a reduced uptake of long-chain fatty acids in 3T3-L1 cells (12Pohl J. Ring A. Ehehalt R. Schulze-Bergkamen H. Schad A. Verkade P. Stremmel W. Biochemistry. 2004; 43: 4179-4187Crossref PubMed Scopus (83) Google Scholar), and the major structural protein of caveolae, caveolin, and the caveolae-localized protein, CD36, both bind fatty acids (13Trigatti B.L. Anderson R.G. Gerber G.E. Biochem. Biophys. Res. Commun. 1999; 255: 34-39Crossref PubMed Scopus (179) Google Scholar, 14Dorahy D.J. Lincz L.F. Meldrum C.J. Burns G.F. Biochem. J. 1996; 319: 67-72Crossref PubMed Scopus (115) Google Scholar). We hypothesized that also fatty acids are taken up and released via caveolae of the adipocyte (4Thorn H. Stenkula K.G. Karlsson M. Örtegren U. Nystrom F.H. Gustavsson J. Strålfors P. Mol. Biol. Cell. 2003; 14: 3967-3976Crossref PubMed Scopus (112) Google Scholar). Caveolae membranes are rich in cholesterol and sphingomyelin and are much more resistant to solubilization by detergents than are other parts of the plasma membrane and other cellular membranes. The adipocyte plasma membrane is covered by caveolae invaginations that enlarge the membrane area by 50% and potentially provide relatively detergent-resistant ports for fatty acid entry and exit (4Thorn H. Stenkula K.G. Karlsson M. Örtegren U. Nystrom F.H. Gustavsson J. Strålfors P. Mol. Biol. Cell. 2003; 14: 3967-3976Crossref PubMed Scopus (112) Google Scholar). Caveolin has been implicated in TG metabolism through its association with so called lipid bodies and with lipid droplets in 3T3-L1 adipocytes and other cell lines (15Liu P. Ying Y. Zhao Y. Mundy D.I. Zhu M. Anderson R.G.W. J. Biol. Chem. 2004; 279: 3787-3792Abstract Full Text Full Text PDF PubMed Scopus (435) Google Scholar, 16Pol A. Martin S. Fernandez M.A. Ferguson C. Carozzi A. Luetterforst R. Enrich C. Parton R.G. Mol. Biol. Cell. 2004; 15: 99-110Crossref PubMed Scopus (181) Google Scholar, 17Brasaemle D.L. Dolios G. Shapiro L. Wang R. J. Biol. Chem. 2004; 279: 46835-46842Abstract Full Text Full Text PDF PubMed Scopus (627) Google Scholar, 18Marchesan D. Rutberg M. Andersson L. Asp L. Larsson T. Boren J. Johansson B.R. Olofsson S-O. J. Biol. Chem. 2003; 278: 27293-27300Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Caveolin-1-/- mice that are deficient in caveolin, and caveolae have, moreover, been found to have deranged lipid metabolism and atrophied adipose tissue (19Cohen A.W. Razani B. Wang X.B. Combs T.P. Williams T.M. Scherer P.E. Lisanti M.P. Am. J. Physiol. 2003; 285: C222-C235Crossref PubMed Scopus (276) Google Scholar, 20Razani B. Combs T.P. Wang X.B. Frank P.G. Park D.S. Russel R.G. Li M. Tang B. Jelicks L.A. Scherer P.E. Lisant M.P. J. Biol. Chem. 2002; 277: 8635-8647Abstract Full Text Full Text PDF PubMed Scopus (461) Google Scholar). We now report that in primary adipocytes exogenous fatty acids are taken up and directly converted to triacylglycerol at the plasma membrane in a novel subclass of caveolae. Isolation and Incubation of Adipocytes—Adipocytes were isolated by collagenase digestion from Harlan Sprague-Dawley rats (130-160 g, B&K Universal, Sollentuna, Sweden) as described (21Strålfors P. Honnor R.C. Eur. J. Biochem. 1989; 182: 379-385Crossref PubMed Scopus (115) Google Scholar). Cells were preincubated with 5 nm insulin for 10 min. [3H]Oleic acid (Amersham Biosciences) or the fluorescent fatty acid analogue C1-BODIPY 500/510 dodecanoic acid (Molecular Probes, Eugene, OR) at 100 μm was added for 40 or 120 s. Incubations were terminated with 2 mm KCN. Cells incubated with [3H]oleic acid were homogenized and caveolae isolated from purified plasma membranes (22Örtegren U. Karlsson M. Blazic N. Blomqvist M. Nystrom F.H. Gustavsson J. Fredman P. Strålfors P. Eur. J. Biochem. 2004; 271: 2028-2036Crossref PubMed Scopus (122) Google Scholar). Isolation of Caveolae—To prepare caveolae fractions without detergent adipocytes were homogenized, and plasma membranes were isolated and disrupted by sonication in 0.5 m Na2CO3, pH 11 (22Örtegren U. Karlsson M. Blazic N. Blomqvist M. Nystrom F.H. Gustavsson J. Fredman P. Strålfors P. Eur. J. Biochem. 2004; 271: 2028-2036Crossref PubMed Scopus (122) Google Scholar). This was then adjusted to 45% sucrose in 15 mm Mes, pH 6.5, 100 mm NaCl, 0.3 m Na2CO3, with protease inhibitors, loaded under a continuous 10-40% sucrose density gradient, and centrifuged at 200,000 × g for 18 h. Fractions of 1 ml were collected from the bottom of the tube and membranes pelleted by centrifugation. Caveolae purity has been documented (22Örtegren U. Karlsson M. Blazic N. Blomqvist M. Nystrom F.H. Gustavsson J. Fredman P. Strålfors P. Eur. J. Biochem. 2004; 271: 2028-2036Crossref PubMed Scopus (122) Google Scholar, 23Aboulaich N. Vainonen J. Strålfors P. Vener A.V. Biochem. J. 2004; 383: 237-248Crossref PubMed Scopus (131) Google Scholar). A microsomal fraction was obtained as described (22Örtegren U. Karlsson M. Blazic N. Blomqvist M. Nystrom F.H. Gustavsson J. Fredman P. Strålfors P. Eur. J. Biochem. 2004; 271: 2028-2036Crossref PubMed Scopus (122) Google Scholar). For analysis of TG synthesis in isolated membrane fractions, as indicated, isolated caveolae, plasma membranes, or microsomal membrane fractions were transferred to 50 mm Tris-Cl, pH 7.5, 10 mm MgCl2, and 100 μm [3H]oleic acid or 100 μm [14C]glycerol 3-phosphate (Amersham Biosciences) with 200 μm CoA, 5 mm ATP were added. The mixtures were briefly sonicated with a probe-type sonifier and incubated for 30 min at 37 °C. Incubations were terminated by membrane pelleting and lipid extraction. Lipid Analyses—Membrane fractions were extracted with CHCl3/CH3OH/150 mm NaCl (1:1:0.9). The CHCl3 phase was applied to thin-layer chromatography (high performance TLC, silicic acid, 10 × 10 cm, Merck). For neutral lipids the plate was developed in CHCl3/CH3OH/H2O (65:35:2.5, by volume) for 2 cm and, after drying, in hexane/diethylether/CH3COOH (70:30:1, by volume) to the top of the plate (24Gustavsson J. Parpal S. Strålfors P. Exp. Cell Res. 1995; 221: 443-447Crossref PubMed Scopus (3) Google Scholar). Polar lipids were developed in CHCl3/CH3OH/H2O (65:25:4, by volume) (25Kates M. Burdon R.H. Knippenberg H.v.P Laboratory Techniques in Biochemistry and Molecular Biology. 3. Elsevier Science Publishers B. V., Amsterdam1986: 326-425Google Scholar). Genuine marker molecules (from Larodan, Malmo, Sweden) were added as internal standards. Silicic acid was scraped off plates, suspended in 0.2 ml of methanol, and analyzed for radioactivity by liquid scintillation in 2,5-diphenyloxazole/1,4-bis[2-(5-phenyloxazolyl)]benzene in toluene. SDS-PAGE and Immunoblotting—Separated proteins were detected with anti-caveolin-1, anti-insulin receptor β-subunit (Transduction Laboratories, Lexington, KY), anti-perilipin (generous gift of Dr. C. Londos, National Institutes of Health), or anti-fatty acyl-CoA synthetase (generous gift of Dr. J. E. Schaffer, Washington School of Medicine) antibodies. Bound antibodies were detected using ECL-plus with horse-radish peroxidase-conjugated anti-IgG as secondary antibody (Amersham Biosciences). Blots were quantitated by chemiluminiscence imaging (Las1000, Fuji). Electron Microscopy—Adipocytes were attached to poly-l-lysine-Formvar-coated nickel grids, which were flushed with ice-cold 150 mm KCl, 1.9 mm Tris-HCl buffer, pH 7.4. Plasma membranes remaining on the grids were washed, fixed, and treated for immunogold labeling as described (4Thorn H. Stenkula K.G. Karlsson M. Örtegren U. Nystrom F.H. Gustavsson J. Strålfors P. Mol. Biol. Cell. 2003; 14: 3967-3976Crossref PubMed Scopus (112) Google Scholar) using primary antibodies against caveolin-1 or perilipin. Secondary IgG were conjugated with colloidal gold (Aurion, Wageningen, The Netherlands). Grids were lyophilized and coated with 2-nm-thick tungsten in the freeze dryer (26Lindroth M. Fredriksson B-A. Bell P.B. J. Microsc. (Oxf.). 1991; 161: 229-239Crossref PubMed Scopus (19) Google Scholar). Transmission electron microscopy was with Jeol EX1200 TEM-SCAN (Jeol, Tokyo, Japan). Fluorescence Microscopy—Adipocytes were preincubated with 5 nm insulin for 10 min and the fluorescent fatty acid analogue C1-BODIPY 500/510 dodecanoic acid was added for 40 s when incubations were terminated with 2 mm KCN. Cells were rinsed and fixed with 3% paraformaldehyde and attached to poly-l-lysine coated coverslips. Cells were permeabilized with 0.1% saponin, or plasma membrane sheets were prepared by flushing as described above (9Karlsson M. Thorn H. Danielsson A. Stenkula K.G. Öst A. Gustavsson J. Nystrom F.H. Strålfors P. Eur. J. Biochem. 2004; 271: 2471-2479Crossref PubMed Scopus (85) Google Scholar). After blocking with bovine serum albumin cells were incubated with indicated primary antibody (anti-caveolin or anti-perilipin) and then fluorescent secondary antibodies (Alexa fluor 594 or 633). Confocal scanning microscopy was performed with a Nikon D-Eclipse CI (Nikon, Tokyo, Japan). To examine uptake of fatty acids and their conversion to TG by intact cells we incubated primary rat adipocytes with radiolabeled oleic acid, isolated the plasma membranes, and separated caveolae by gradient ultracentrifugation. Caveolae fractions were then analyzed for radiolabel incorporation into TG and other lipids. Caveolae were obtained as two separate milky bands after ultracentrifugation, both of which contained caveolin and the insulin receptor (fractions 6 and 8, Fig. 1a). A shoulder of caveolin suggested also a third caveolae fraction (fraction 4, Fig. 1a), which was not further analyzed herein. The pattern of major proteins was very similar in the two major caveolae fractions (Fig. 1c), demonstrating their close relation. The protein perilipin, which regulates lipolysis (27Clifford G.M. Londos C. Kraemer F.B. Vernon R.G. Yeaman S.J. J. Biol. Chem. 2000; 275: 5011-5015Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar), and the fatty acyl-CoA synthetase, which catalyzes the first step in the TG synthesis pathway (28Gargiulo C.E. Stuhlsatz-Krouper S.M. Schaffer J.E. J. Lipid Res. 1999; 40: 881-892Abstract Full Text Full Text PDF PubMed Google Scholar), were, however, confined to the higher density caveolae fraction (fraction 6, Fig. 1a). We extracted lipids and analyzed for metabolic conversion products of the [3H]oleic acid by thin-layer chromatography. A substantial fraction of the cellular radioactive fatty acid was recovered in the high density perilipin-containing caveolae as TG also as and as fatty acid (Fig. We not acid, or acid. be that bulbs have been to the large lipid fraction during and the ultracentrifugation. This fraction contained and more radiolabeled TG than the isolated plasma membrane after 40 and 120 TG the caveolae membrane the membrane has to to amounts of TG and a synthesis of may be We analyzed for conversion of the [3H]oleic to and found significant amounts of radioactivity as (Fig. and as that was in the high density caveolae. in intact adipocytes fatty acids are the cells and are converted to TG in a specific We also detected TG, fatty acid, and in the microsomal the of synthesized TG in the microsomal fraction was of that in the plasma membrane The of [3H]oleic acid conversion to TG in the cells was 100 of which is the of lipolytic hydrolysis of TG (4Thorn H. Stenkula K.G. Karlsson M. Örtegren U. Nystrom F.H. Gustavsson J. Strålfors P. Mol. Biol. Cell. 2003; 14: 3967-3976Crossref PubMed Scopus (112) Google Scholar, P. Fredriksson G. Strålfors P. H. B. Elsevier Science Publishers B. V., Scholar). was not to directly much of the TG synthesis of the adipocytes is localized to caveolae and therefore the plasma membrane, as synthesized TG was continually transferred to the central lipid droplet. We therefore the of the fatty acyl-CoA synthetase protein in a purified plasma membrane fraction with a using SDS-PAGE and of the protein was recovered in the purified plasma membrane fraction not a substantial for TG synthesis in the plasma membrane with its caveolae (28Gargiulo C.E. Stuhlsatz-Krouper S.M. Schaffer J.E. J. Lipid Res. 1999; 40: 881-892Abstract Full Text Full Text PDF PubMed Google Scholar). To that de novo TG synthesis in caveolae and of TG with caveolae during of the or of fatty acid with TG, we isolated the two caveolae fractions (high and low density and their to synthesize TG from the two of TG [3H]oleic acid and glycerol 3-phosphate or oleic acid and [14C]glycerol in the presence of and were preincubated with insulin for 10 min and of plasma membrane, caveolae, and microsomal Isolated membrane fractions were incubated with the indicated TG for 30 min at 37 when caveolae were pelleted and lipids extracted and analyzed by thin-layer chromatography. [3H]Oleic acid and [14C]glycerol 3-phosphate radioactivity was converted to TG by the high density caveolae and to a by the low density caveolae fraction (Fig. To the capacity of caveolae in the plasma membrane with that of the we the purified plasma membrane fraction with the microsomal The microsomal fraction synthesized TG to a similar as the plasma membrane with its caveolae (Fig. be that the TG capacity was by the to provide to the of plasma membrane by sonication and by the used to caveolae, which sonication in 0.5 at pH therefore large from to of both the oleic acid and glycerol 3-phosphate in TG demonstrated de novo TG synthesis in the isolated novo TG synthesis in isolated caveolae, plasma membrane, and microsomal and high and low density caveolae to fractions 6 and 8, in Fig. or plasma membrane with its caveolae and microsomal fractions were incubated with [3H]oleic acid 3-phosphate for 30 min. of the membrane fractions were a of were analyzed by thin-layer chromatography and liquid scintillation as of the high density caveolae or 2 as of the plasma membrane with its caveolae or The caveolae with its caveolin and associated perilipin to lipid which have been to caveolin and proteins when isolated and from other cell (15Liu P. Ying Y. Zhao Y. Mundy D.I. Zhu M. Anderson R.G.W. J. Biol. Chem. 2004; 279: 3787-3792Abstract Full Text Full Text PDF PubMed Scopus (435) Google Scholar, 16Pol A. Martin S. Fernandez M.A. Ferguson C. Carozzi A. Luetterforst R. Enrich C. Parton R.G. Mol. Biol. Cell. 2004; 15: 99-110Crossref PubMed Scopus (181) Google Scholar). described in primary they are plasma and do not in the to these caveolae be as lipid bodies and if in of lipid bodies electron microscopy of plasma membrane sheets with we identified lipid-laden bulbs over the membrane (Fig. labeling that both caveolin (Fig. a and and perilipin (Fig. and were associated with caveolae and with these lipid demonstrating their and to the isolated caveolae identified of plasma membrane sheets by electron microscopy on flushing the membranes to away cell and attached cell to membrane flushing of the membranes away the lipid which may they have The of fatty acid in the bulbs was by In cells with insulin fluorescent fatty acids in over the cell (Fig. 4, a and molecules accumulate or dissolve in the large lipid they are converted to a are therefore in the of triacylglycerol in the bulbs and in the lipid J. Biochem. Biophys. Res. Commun. PubMed Scopus Google Scholar). and very large fluorescent bulbs also at the plasma membrane. large bulbs were not covered with caveolin and to and dissolve into the large central lipid of the cell (Fig. 4, a and The of plasma membrane prepared from the cells incubated with fluorescent fatty acid, demonstrated fluorescent fatty bulbs in perilipin at the membrane (Fig. 4, a the plasma membrane and the lipid droplet. in the the of the 0.5 μm of the up to diameter of the is from the and from the of the bulbs that they the of the and that when TG in the bulbs these may with the central lipid droplet. therefore is that the TG can be transferred from their site of synthesis in caveolae at the plasma membrane to their site of by a This for a flow of TG from the plasma to the lipid as can be from the fluorescence findings are in with the structural for in the lipid from of adipocytes from mice A.W. Razani B. W. Williams T.M. Wang X.B. P. D.L. Scherer P.E. Lisanti M.P. 2004; PubMed Scopus Google Scholar). be to electron microscopy and of adipocytes to examine for the presence of the plasma membrane and the central lipid droplet. This report that caveolae are of fatty acid entry and of TG synthesis and may a novel for TG transfer to the central lipid in primary synthesis has been to in the G.F. J. Biol. Chem. Full Text PDF PubMed Google Scholar), which is for TG synthesis in and TG synthesis is with of chylomicron and very low density lipoprotein on the other are with a of potentially fatty acids from lipoprotein lipase catalyzed hydrolysis of lipoprotein TG in the these fatty acids are not they cell lysis (2Strålfors P. FEBS Lett. 1990; 263: 153-154Crossref PubMed Scopus (21) Google Scholar). In is that mice that caveolae a adipose tissue and which with B. Combs T.P. Wang X.B. Frank P.G. Park D.S. Russel R.G. Li M. Tang B. Jelicks L.A. Scherer P.E. Lisant M.P. J. Biol. Chem. 2002; 277: 8635-8647Abstract Full Text Full Text PDF PubMed Scopus (461) Google Scholar). findings now demonstrate that involved in the de novo synthesis of triacylglycerol from fatty acids and glycerol 3-phosphate are localized and in a novel specific subclass of caveolae in the plasma membrane of primary We are to Dr. C. Londos and Dr. J. E. Schaffer for their of antibodies.