Caveolins sequester FA on the cytoplasmic leaflet of the plasma membrane, augment triglyceride formation, and protect cells from lipotoxicity.

Ectopic expression of caveolin-1 in HEK293 cells enhances FA sequestration in membranes as measured by a pH-sensitive fluorescent dye (1). We hypothesized that sequestration of FA is due to the enrichment of caveolin in the cytosolic leaflet and its ability to facilitate the formation of lipid rafts to buffer high FA levels. Here we show that ec-topic expression of caveolin-3 also results in enhanced FA sequestration. To further discriminate the effect that caveolins have on transmembrane FA movement and distribution, we labeled the outer membrane leaflet with fluorescein-phosphatidylethanolamine (FPE), whose emission is quenched by the presence of FA anions. Real-time measurements made with FPE and control experiments with positively charged fatty amines support our hypothesis that caveolins promote localization of FA anions through interactions with basic amino acid residues (lysines and arginines) present at the C termini of caveolins-1 and -3.


Cell lines
HEK293 cells transfected with or without caveolin-1 were established and grown as described ( 1,31 ). Transient transfection of caveolin-3 was done using FuGENE 6 in accordance with the manufacturer's instructions. The caveolin-3 gene was cloned into the PcB7 vector and transfected into HEK293 parental cells. Total cell extracts were prepared from 60 mm plates of HEK293 cells after transient transfection with 1-3 µg of DNA for 24 h. Cells were washed with cold PBS and scraped into 100 µl of lysis buffer (50 mM Tris pH 7.4, 100 mM NaCl, 1% sodium deoxycholate, 4% Nonidet, 0.4% SDS) containing a protease inhibitor mixture from American Bioanalytical (Natick, MA). Soluble protein concentration was determined from the supernatant after microcentrifugation using the BCA protein assay from Pierce (Rockford, IL). Following SDS-PAGE, Western blotting was performed to confi rm protein expression. Caveolin-3 expression levels from each transfection were confi rmed by Western blotting and found to be comparable. A crude rat skeletal muscle membrane preparation ( 32 ) was also analyzed in this way as a control for the level of expression. Primary antibody against caveolin-3 was obtained from BD Transduction Laboratories (Lexington, KY) and was subsequently detected using a secondary antibody conjugated to horseradish peroxidase from Sigma and a chemiluminescence substrate from Perkin Elmer Life Sciences (Boston, MA).

Sphingomyelinase and M ␤ CD treatment of cells
Sphingomyelinase from Bacillus cereus was added at a fi nal concentration of 100 mU/ml for 3 h at 37°C to digest plasma membrane sphingomyelin (SM). To extract plasma membrane cholesterol, cells were incubated with DMEM supplemented with or without 10 mM M ␤ CD for 20 min at 37°C. In each case, the cells were then washed following pretreatment and prepared for fl uorescence and cholesterol measurements as previously described ( 1 ).

Quantifi cation of sphingomyelin
Lipids from HEK293 cells were extracted by the Folsch method ( 33 ). Briefl y, the lipid phase of cellular extracts was dried and spotted onto Uniplate Silica Gel G TLC plates from Analtech Inc. (Newark, DE), and the lipids were separated using droplet function remain unclear, wild-type and mutant caveolins are either associated with or targeted to lipid droplets in a variety of cell types (13)(14)(15)(16)(17). It has been suggested that caveolae may be sites for the initiation of triglyceride synthesis in adipocytes ( 18 ). Caveolin-1 has been shown to contain FA binding sites ( 19 ), but as this protein family is imbedded within the caveolae membranes, FA binding has not been well characterized, as is the case for all membrane bound proteins which bind FA when compared with soluble FA binding proteins ( 20 ). Our studies aimed at characterizing FA binding in vivo have shown that caveolin-1 expression modulates FA movement and transmembrane distribution in a model system of HEK293 cells overexpressing this protein ( 1 ).
Caveolins/caveolae could play a protective role in cells, particularly adipocytes, by buffering the cell against transient and very high levels of FA or even chronic, high circulating levels of FA that are associated with insulin resistance and diabetes ( 21 ). The cytotoxic effects of FA have been termed lipotoxicity and are evident in insulin-producing cells of the pancreas and in liver and muscle ( 22,23 ). The molecular mechanisms of these lipotoxic actions remain unclear. They could represent localized disruptions of membranes (detergent effects) or metabolic effects. They may also be tissuedependent ( 24,25 ), but at the cellular level, concentrations of FA in the high physiological range cause cell death in pancreatic ␤ cells ( 26 ), cardiomyocytes ( 27 ), and hepatocytes ( 28 ) as well as in commonly used cultured cells, such as Chinese hamster ovary (CHO) fi broblasts ( 29 ).
We have previously described the selection of HEK293 cell lines expressing caveolin-1. These cells exhibit slow metabolism of FA into esterifi ed products and are therefore ideal for observing both rapid and slow phases of FA fl ux into cells. We showed that levels of caveolin-1 above a certain threshold result in an approximately 2-fold increase in FA partitioning to the inner leafl et of the plasma membrane ( 1 ). We hypothesized that caveolin expression could lead to the formation of lipid raft domains that are able to sequester higher levels of FA compared with cells lacking these domains. Structural features of caveolin-1, specifi cally the C terminus which contains numerous arginines and lysines, may serve as a "reservoir" to sequester and stabilize high levels of FA anions. Here we investigate whether caveolin-3 and its similar structural features functions in the same way. To add precision to our fl uorescence measurements, we employ a new fl uorescent phospholipid to demonstrate that the uptake modulated by caveolins involves movement of FA from the outer leaflet of the plasma membrane as it is sequestered more abundantly into the inner leafl et. As a control experiment, we showed that caveolins do not cause redistribution of positively charged fatty amines in the leafl ets of the plasma membrane. Finally, we show that expression of caveolin-1 and 3 enhance TG storage and protect HEK293 cells from the cytotoxic effects of high FA concentrations.
isopropanol and four parts water). The stain was removed, and cells were washed with water.

Determination of triglyceride content
TG content was determined in cell lysates using a colorimetric assay (Triglycerides reagent) as previously described according to the manufacturer's instructions (Point Scientifi c Inc., Canton, MI). Cell lysates were prepared using 1% NP-40 in PBS, and results were normalized to 100 µg of total cellular protein.

Cytotoxicity assay
HEK293 cells ( ‫ف‬ 2.5 × 10 4 ) were seeded in 96 well plates and grown overnight to confl uence. FA complexed to BSA at different concentrations were then added, and cells were incubated for 48 h. Cell viability was then assessed using a CellTiter 96 AQ One Solution Cell Proliferation Assay according to the manufacturer's instructions (Promega, Madison, WI).

Caveolin-3 modulates transmembrane FA movement
We proposed a mechanistic model whereby caveolin-1 at the inner leafl et of the bilayer enhances the amount of FA anions at this locus by virtue of its multiple positive charges, particularly in the C terminus region, which is expected to lie close to the inner leafl et of the plasma membrane ( 1 ). Like caveolin-1, caveolin-3 contains multiple lysine and arginine residues in the same region of its structure and, therefore, would be expected to enhance partitioning of FA into the cytosolic leafl et.
To test this hypothesis, parental HEK293 cells were transiently transfected with caveolin-3 cDNA. After 24 h, cells were harvested for Western blotting and for real-time fl uorescence assays of FA uptake by measurement of intracellular pH using BCECF as we have done previously ( 1 ). As shown in Fig. 1A , transfected caveolin-3 was expressed in HEK293 cells (P-cav3) at a level that is slightly lower than that found naturally in rat skeletal muscle. FA uptake was then assessed in these and parental cells by monitoring BCECF fl uorescence ( Fig. 1B ). Addition of 20 µM oleic acid (arrow) resulted in a very rapid (t 1/2 р 2 s) decrease in fl uorescence, which was equal in magnitude in both cell lines. However, an additional slow decrease in pH (minutes) was seen only in cells expressing high levels of caveolin-3 so that the total fl uorescence drop of BCECF, which is linearly related to pH ( 35 ), was twice that of the parental cells. This altered and unique response is the same as that previously observed in cells expressing high levels of caveolin-1 ( 1 ). Fig. 1C shows the total change in BCECF fl uorescence from parental and B8 cells in four independent experiments with different cell preparations.
The expression of caveolin in HEK293 cells signifi cantly increases the levels of membrane cholesterol ( 1 ). Since cholesterol and sphingolipids levels are coordinately regulated ( 36 ), the observed effects of the caveolins on transmembrane FA movement could also be due to the elevated levels of these lipid species. Cholesterol can be rapidly extracted from cells by methyl-␤ -cyclodextrin (M ␤ CD) ( 37 ), and SM can be digested with sphingomyelinase. Accord-a chloroform:methanol:water solvent system (65:25:4). Bands were quantifi ed by densitometry using the TotalLab program (nonlinear dynamics).

Labeling cells with FPE and BCECF for fl uorescence measurements of FA binding and movement across the membrane
HEK293 cells were grown in DMEM supplemented with 5% fetal bovine serum and 5% calf serum and loaded with the pH probe BCECF as described previously ( 1 ). To label the outer leaflet of the plasma membrane, the desired amount of FPE stock was dried under nitrogen, resuspended in DMSO, and added to a separate batch of cells at a fi nal concentration of 9 µM. Cells were incubated for 1 h in the dark, washed in 20 mM MOPS-KRB buffer (pH 7.4), and resuspended in buffer after treatment with 0.1% Trypsin-EDTA for 10 s.

Fluorescence measurements
Fluorescence measurements were made using a Spex ® Fluoromax-2 from Jobin Yvon (Edison, NJ). The fl uorescence of BCECF was measured using a ratiometric signal of excitation at 439 nm and 505 nm ( R = ex505/ex439) with an emission of 535 nm. The emission of FPE was measured at 520 nm after exciting the probe at 490 nm. Experiments monitoring the binding and transmembrane movement of FA into cultured HEK293 cells were carried out at 37°C as described previously ( 1 ). Because FPE and BCECF are not compatible for use in the same cells (overlapping excitation wavelengths), separate cell batches were used to measure each probe. Briefl y, the fl uorescence signals of each probe (ratiometric excitation or single emission) were measured as unbound oleate (20 M fi nal concentration) was added through the injection port above a rapidly stirred cuvette to ensure instantaneous mixing of FA with cells. Vehicle alone, DMSO, or KOH added at 1:500 dilution to the external buffer did not affect intracellular pH or FPE fl uorescence. When monitoring the fl uorescence of a single probe, changes in fl uorescence can be resolved to within 2 s.
For single-cell imaging experiments, the desired amount of FPE stock was dried and resuspended in DMSO for addition to cells that were attached to the bottom of cover slips and grown as described above (4.2 M fi nal FPE concentration). In the case of BCECF, cells were incubated in culture medium containing 2 mM BCECF-AM. After an incubation time of 30-40 min in the dark with gentle agitation, the culture medium was replaced by MOPS buffer (pH 7.4), and cells were incubated for 40 min at 37°C prior to microscopic imaging. For imaging changes in FPE and BCECF fl uorescence in single cells, a concentration of 20 M OA was added to 2 ml of MOPS buffer in a collagen-coated glass bottom culture dish from MatTek Corporation (Ashland, MA) containing HEK293 cells. Cells were allowed to incubate with FA for several minutes before images were obtained with a two-photon laser scanning confocal microscope (excitation wavelength = 780 nm). Unlike the online fl uorescence measurements described above, kinetic measurements with this approach are limited by the inability to mix the FA rapidly with the cells. Therefore, detection of FA binding and transmembrane movement by each probe is limited by the time required for the FA to diffuse through the buffer and reach the cell surface.

Oil Red O staining
Oil Red O staining was performed essentially as described ( 34 ). Cells supplemented with FA for the indicated time were washed with PBS and fi xed with 3.7% formaldehyde (Sigma) in PBS for 15 min. Cells were stained for 1.5 h in freshly diluted Oil Red O solution ( six parts 0.5% Oil Red O stock solution in performed new measurements with the fl uorescent probe FPE. This phospholipid molecule is labeled with a fl uorescein group that lies at the polar interface with the aqueous phase and is very sensitive to the membrane surface poten-ingly, we applied these reagents to parental and high caveolin-1 expressing cells and then monitored the binding of FA to the membrane surface and change in intracellular pH (as in Fig. 1B ). Quantitative responses of BCECF in each cell type are given in Table 1 . Within 5 min, M ␤ CD removed 56% of the cholesterol in B8 cells but had no signifi cant effect on the rate or magnitude of BCECF fl uorescence. There was no effect of M ␤ CD on any parameter in the parental cells. The same result was found after 20 min of M ␤ CD exposure (data not shown). Sphingomyelinase reduced SM levels in B8 cells by 46 ± 9% and did not affect caveolin-1 expression. Additionally, caveolin-1 remained localized only at the cell surface in appreciable abundance, as was the case following M ␤ CD treatment (data not shown). However, net FA movement from the extracellular medium across the plasma membrane was modestly but signifi cantly ( ‫ف‬ 25%, P р 0.05) decreased as assessed by BCECF fl uorescence. These data indicate that SM present in the outer leafl et may play a partial role in modulating the acute uptake of FA into cells, possibly by modulating raft formation, and that the signifi cant increase in FA uptake into B8 cells is largely SM-independent.

FPE reports the binding (and loss) of FA anions in the extracellular leafl et of the plasma membrane
Our measurements of FA movement into and across the plasma membrane in the experiments described above detect the arrival of FA at the inner leafl et. Some ( ‫ف‬ 50%) of the protonated FA lose a proton, which is detected by the pH-sensitive probe BCECF ( 1,38,39 ). To discriminate the steps of FA binding to the extracellular leafl et of the membrane from its translocation across the lipid bilayer, we  Total cholesterol, total sphingomyelin, and BCECF fl uorescence were measured in parental and high caveolin-1 expressing cells (B8) treated with or without incubation with 10 mM M ␤ CD for 5 min at 37°C. BCECF data are the mean ± SD of 3-4 independent experiments. Statistical signifi cance for cholesterol levels in B8 cells after 5 min incubation with M ␤ CD as compared with untreated cells was determined by Student's t -test. Cholesterol was not reduced in parental cells. There was no signifi cant difference in FA partitioning due to this treatment in either cell preparation as determined by BCECF fl uorescence. Similarly, total sphingomyelin and BCECF fl uorescence were measured in parental and high caveolin-1 expressing cells (B8) treated for 3 h with 100 mU/ml sphingomyelinase (SMase), which signifi cantly decreased SM levels ( P р 0.01) in both parental and B8 cells. However, a SMasedependent decrease in the BCECF signal, compared with the vehicle, was observed only in the B8 cells and was signifi cantly different from the fl uorescence values in the parental cell line.
To test our hypothesis that the positive charges of caveolins mediate these effects on FA uptake by binding the FA anion, we performed parallel experiments with fatty amines, which have a net positive charge in the membrane interface ( 43 ). Thus, binding of the amine is expected to cause an increase in FPE fl uorescence. These results were indeed observed for C18:1 (oleoyl) amine, as illustrated for the FPE ( Fig. 3B, D ). In the parent cells, both the FA and the fatty amine showed a fast initial change and no subsequent changes in fl uorescence over a period of ten minutes. In the B8 cells, the FA resulted in a fast decrease followed by a slower increase, signifying a slow decrease in FA anions in the extracellular leafl et. Alternatively, caveolin-1 expressing cells showed only a small recovery (fl uorescence decrease) of the signal upon addition of the fatty amine, an effect which was not at all comparable to the effect of a much stronger attraction of caveolin for the FA anion than for the positively charged amine.

Caveolin expression enhances FA accumulation and protects against FA-induced lipotoxicity
Mice lacking caveolae as a consequence of caveolin-1 ( 12 ) or cavin/PTRF ( 7 ) gene deletion have smaller fat depots and are hyperlipidemic. These changes suggest that adipocytes from these animals have either compromised triglyceride storage or enhanced constitutive lipolysis or both. Our HEK293 cell model is not suitable for studies of lipolysis because these cells do not express necessary members of the perilipin family ( 44 ). However, they are useful for determining the effects of caveolin expression on lipid accumulation. As shown by Oil Red O staining of lipids ( Fig. 4A ), expression of caveolin-1 and caveolin-3 results in substantially more lipid accumulation than in the parental cells, and quantitative analysis reveals an approximate doubling of triglyceride accumulation ( Fig.  4B ). Interestingly, we isolated the lipid droplets from the caveolin-1 expressing cells and found a substantial association of this protein in the droplet along with the usual lipid droplet protein ADRP (adipose differentiationrelated protein) ( Fig. 4C ), consistent with data from other cell types ( 16,17 ).
From these novel results, we postulate that caveolin expression may protect cells from lipotoxicity by enabling them to tolerate high levels of FA within the plasma membrane as well as by enhancing storage. Accordingly, we incubated the indicated cell lines with 0.4, 0.8, and 1.6 mM of albumin-bound OA for 48 h, after which cell viability was assessed ( Fig. 5 ). At all three concentrations of FA, cells expressing caveolin 1 were resistant to OA-induced cell death as compared with the parental line. The decrease in survival of parental cells was already signifi cant ( P < 0.05) at 0.4 mM albumin-bound OA compared with cells expressing high levels of caveolin-1, and the protective effect of caveolin-1 was more pronounced at 0.8 and 1.6 mM albumin-bound FA. Similarly, caveolin-3 cells were tial ( 40,41 ). Thus, changes in its fl uorescence are directly proportional to the number of FA anions found in the lipid bilayer. Furthermore, when added to the extracellular leafl et, FPE remains in this leafl et because of its inability to translocate to the inner leafl et ( 42 ).
We fi rst examined single cells by confocal microscopy to verify that loading FPE into HEK293 cells resulted in labeling of the plasma membrane and to verify that it is not internalized into the cells (Fig. 2). Representative fi elds of HEK293 cells labeled with FPE confi rmed that the fl uorescence is restricted to the cell rim ( Fig. 2A ), whereas cells with entrapped BCECF exhibited the expected uniform distribution of the probe in the cytosol ( Fig. 2B ). Furthermore, addition of OA to the external medium resulted in a decrease in fl uorescence in the individual cells ( Fig. 2C ) as well as in populations monitored by online fl uorescence measurements ( Fig. 3 ). In these experiments, the addition of exogenous OA also caused a reduction in BCECF fl uorescence, which coincides with a reduction in intracellular pH as in Fig. 1 .
Addition of oleic acid to a cuvette containing FPE-loaded parental cells (P) and cells expressing high caveolin-1 (B8) revealed identical rapid decreases (t 1/2 р 2 s) in FPE fl uorescence in both cell lines ( Fig. 3 , left panels). After this initial fast phase, a second slower kinetic phase was evident only in the B8 (high-caveolin) cells. Within minutes, the FPE fl uorescence increased again by ‫ف‬ 50%, whereas the parental line showed minimal recovery of fl uorescence (<5%) during the time of the measurement. This change (i.e., fl uorescence recovery) refl ects a decrease in concentration of negative charges at the outer leafl et, or more simply, a loss of FA from the outer membrane leafl et with Fig. 2. Fluorescence imaging of HEK293 cells demonstrates localization of probes, binding, and transmembrane movement of oleic acid. HEK293 cells were labeled with either FPE (top panel) or loaded with BCECF (bottom panel) prior to the imaging with a two-photon confocal microscope (see "Methods"). The probes were excited at 780 nm and images were captured under a 40× objective (oil immersion) at a pixel resolution of 512 × 512 using an image acquisition time of ‫ف‬ 10 s. Image of multiple cells in a fi eld were obtained before the addition of oleate (left panels) and confi rmed the correct localization of each probe. Single cells (center panels) were chosen to monitor the response of each fl uorophore to oleate. Addition of 20 µM oleate (right panels) results in a decrease of fl uorescence intensity due to fatty acid binding (FPE) and transmembrane diffusion (BCECF). In these two independent experiments (FPE and BCECF), the images were taken 4 min after the addition of oleate. All images were pseudo-colored using the Image J software. BCECF, 2 ′ 7 ′ -bis-(2-carboxyethyl)-5-(and6)carboxyfl uorescein; FPE, fl uorescein-phosphatidylethanolamine; OA, oleic acid. of proteins known to modulate these processes in a variety of different ways.
The very high level of caveolae in adipocytes ( 11 ) is suggestive of a role for caveolins/caveolae in FA movement across the plasma membrane. It is further supported by the lipodystrophic lean phenotype of mice lacking caveolin in their adipocytes ( 7,12 ). We previously obtained direct data for FA transport in the plasma membrane with a real-time fl uorescence assay that supports a role of caveolin-1 in modulating transmembrane FA movement; we postulated that the C terminus region of this protein provides a juxtamembrane sink of positive charge that can sequester FA and "buffer" the membrane from high concentrations of FA ( 1 ). We showed in this study that ectopic caveolin expression increases membrane cholesterol levels, which in turn are known to increase membrane sphingomyelin ( 36 ), raising the possibility that these lipids could also be involved in modulating FA movement. However, we ruled out any effect of cholesterol on FA movement and showed that sphingomyelin had a small effect ( Table 1 ). Thus we obtained further support for our original model by three independent means. signifi cantly resistant to the cytotoxic effects of 0.8 and 1.6 mM albumin-bound oleate.

DISCUSSION
Dysregulation of adipocyte lipid metabolism leading to chronically elevated levels of free FA is a major contributor to the development of insulin resistance and type 2 diabetes ( 21 ). Fundamental biophysical and metabolic processes that can affect circulating FA concentrations include its cellular uptake and transport across the plasma membrane, its incorporation into triglyceride, and its release there from by hormonally regulated lipolysis. Here we address the former two processes in a model cell system ( 1 ) without some of the complications inherent in a more complex cell, such as the adipocyte. For example, in addition to high levels of caveolin-1, fat cells highly express CD36 ( 45 ) and fatty acid transport proteins (FATP) 1 and 4 ( 46 ), proteins whose expression substantially affects adipocyte FA uptake and metabolism. Thus, the effect of caveolins on FA uptake and storage in HEK293 cells can be studied under more defi ned conditions and in the absence in the case of pancreatic islet cells, protects them from lipotoxicity ( 47 ). Here we show that caveolin expression enhances triglyceride storage perhaps by directing caveolins ( Fig. 4C ) to the lipid droplet, although the mechanism of how this enhances lipid storage is not yet known. As noted previously, caveolins have been seen to associate with lipid droplets in a variety of other cell types (13)(14)(15)(16)48 ), again by an unknown mechanism. Although caveolae are not generally thought to be highly dynamic structures, we see association of caveolin with lipid droplets in 5 h, the fi rst time that we can see appreciable lipid accumulation in the HEK293 cells (data not shown).
Finally, caveolins protect against FA-induced cell death ( Fig. 5 ). In addition to the above-noted observation that lipid accumulation has this effect, we postulate that caveo-First, we showed that caveolin-3 behaved identically to caveolin-1 with regard to FA fl ux ( Fig. 1 ), as would be predicted from our model. There is a slow recovery of fl uorescence change refl ecting proton release to the cytosol as FA anions accumulate and are stabilized by caveolin in the inner membrane leafl et.
Second, we used a new assay for assessing transmembrane FA movement using FPE ( Figs. 2 and 3 ) to show that movement from the outer to the inner leafl et is the step affected by caveolin expression. Single-cell imaging ( Fig. 2 ) is an important complement to our fl uorescence measurements in cell suspensions because it ( i ) reveals the behavior of individual cells as opposed to the average of many cells and ( ii ) shows that the fl uorescent probes are properly localized, which can only be assumed in measurements obtained from cell suspensions. Thus our results ( Fig. 2 ) demonstrated both the localization of FPE in the plasma membrane and the expected decrease in fl uorescence after addition of OA. The cell suspension data ( Fig. 3 ) show that, following the initial rapid binding of FA at the outer leafl et (and simultaneous diffusion across the bilayer), the slow change in FPE fl uorescence refl ects a slow decrease in the number of FA anions in the outer leafl et. Thus the second slow phase of transmembrane movement inferred from the internal pH [ Fig.  1 and ( 1 )] is due to the slow movement of FA from the outer leafl et to the inner leafl et.
Third, we used a fatty amine, which would be positively charged, and showed that caveolin expression had little or no effect on transmembrane FA movement ( Fig. 3 ) as would be predicted by our model.
What then would be the purpose of caveolins modulating the transmembrane FA fl ux and increasing their levels in the cytosolic leafl et of the plasma membrane? Two possibilities that come to mind are to regulate FA storage in some way and to protect cells from the cytotoxic effects of high FA concentrations. Here we show data that supports both possibilities ( Figs. 4 and 5 ), and indeed, these two roles are complementary. That is, it has previously been suggested that increased storage of triglycerides, Fig. 4. Caveolin expression enhances cellular FA accumulation. A: After 48 h with 80 M oleic acid, cells were fi xed and stained with Oil Red O as described in "Experimental Procedures." B: Triglyceride accumulation was determined and normalized to total cellular protein. The results are from 8 cell preparations with P р 0.001 for high caveolin-1 expressing cells (B8) and caveolin-3 transfected (P/ Cav3) versus untransfected (P) cells at 48 h. C: Lipid droplets were isolated as described by Liu et al. ( 17 ). After SDS-PAGE and transfer, they were immunoblotted for the indicated proteins as in Fig 1 . ADRP, adipose differentiation-related protein; OA, oleic acid; TG, triglyceride. lins serve as positively charged "buffers" to accommodate high levels of membrane-associated FA anions. Indeed, all aspects of FA dynamics involve abundant proteins that bind them and modulate their concentration and metabolism. In circulation, FA are bound to albumin and in cells, to FABPs. A reason that caveolin expression is so high in adipocytes could be to protect against the very high levels of FA that can be released upon hormonally activated lipolysis, which under certain conditions, can be shown to result in adipocyte autolysis ( 49 ). We are in the process of looking at adipocytes that do or don't express caveolins to address this and other issues concerning the role of caveolins/caveolae in adipocyte FA fl ux and metabolism.