Separation and characterization of caveolae subclasses in the plasma membrane of primary adipocytes; segregation of specic proteins and functions ă ¨ ˚ Unn Ortegren1, Lan Yin1, Anita Ost1, Helen Karlsson2, Fredrik H Nystrom3 and Peter Stralfors1 Department of Cell Biology and Diabetes Research Centre, University of Linkoping, Sweden ă Department of Molecular and Clinical Medicine, University of Linkoping, Sweden ă Department of Medicine and Care and Diabetes Research Centre, University of Linkoping, Sweden ă Keywords cholesterol; FATP; GLUT4; insulin receptorSR-BI Correspondence P Stralfors, Department of Cell Biology, Faculty of Health Sciences, SE58185 Linkoping, Sweden ă Fax: +46 13 224314 Tel: +46 13 224315 E-mail: peter.stralfors@ibk.liu.se (Received April 2006, revised 27 April 2006, accepted 26 May 2006) doi:10.1111/j.1742-4658.2006.05345.x Caveolae are nearly ubiquitous plasma membrane domains that in adipocytes vary in size between 25 and 150 nm They constitute sites of entry into the cell as well as platforms for cell signalling We have previously reported that plasma membrane-associated caveolae that lack cell surface access can be identified by electron microscopy We now report the identification, after density gradient ultracentrifugation, of a subclass of very high-density apparently closed caveolae that were not labelled by cell surface protein labelling of intact cells These caveolae contained caveolin-1 and caveolin-2 Another class of high-density caveolae contained caveolin-1, caveolin-2 and specifically fatty acid transport protein-1, fatty acid transport protein-4, fatty acyl-CoA synthetase, hormone-sensitive lipase, perilipin, and insulin-regulated glucose transporter-4 This class of caveolae was specialized in fatty acid uptake and conversion to triacylglycerol A third class of low-density caveolae contained the insulin receptor, class B scavenger receptor-1, and insulin-regulated glucose transporter-4 Small amounts of these proteins were also detected in the high-density caveolae In response to insulin, the insulin receptor autophosphorylation and the amount of insulin-regulated glucose transporter-4 increased in these caveolae The molar ratio of cholesterol to phospholipid in the three caveolae classes varied considerably, from 0.4 in very high-density caveolae to 0.9 in low-density caveolae There was no correlation between the caveolar contents of caveolin and cholesterol The low-density caveolae, with the highest cholesterol concentration, were particularly enriched with the cholesterol-rich lipoprotein receptor class B scavenger receptor-1, which mediated cholesteryl ester uptake from high-density lipoprotein and generation of free cholesterol in these caveolae, suggesting a specific role in cholesterol uptake ⁄ metabolism These findings demonstrate a segregation of functions in caveolae subclasses Caveolae are defined by electron microscopy coated, omega- or flask-shaped invaginations plasma membrane and also by the presence structural protein caveolin (isoforms 1–3) [1] as unof the of the Caveo- lin-1 and caveolin-2 are found in most cell types, including adipocytes The caveolae membrane is rich in cholesterol and sphingomyelin [2–4] Adipocytes have a very high number of caveolae in their plasma Abbreviations FATP, fatty acid transport protein; GLUT4, insulin-regulated glucose transporter; HD-caveolae, high-density caveolae; HDL, high-density lipoprotein; LD-caveolae, low-density caveolae; LDL, low-density lipoprotein; SR-B1, class B scavenger receptor-1; sulfo-NHS-biotin, sulfo-Nhydroxysuccinimidyl-biotin; VHD-caveolae, very high-density caveolae; VLDL, very low-density lipoprotein FEBS Journal 273 (2006) 3381–3392 ª 2006 The Authors Journal compilation ê 2006 FEBS 3381 ă U Ortegren et al Identification of caveolae subclasses membranes About one-third of the plasma membrane of these cells constitutes caveolae membrane and the caveolae vary considerably in size, from about 25 to over 100 nm in diameter [5] In adipocytes the insulin receptor resides in caveolae [6,7], and insulin-stimulated uptake of glucose takes place in caveolae after insulin-regulated glucose transporter-4 (GLUT4) translocation to caveolae in the plasma membrane [8,9] We recently reported that a distinct class of caveolae in adipocytes harbour the enzymatic machinery for synthesis of triacylglycerol and represents a location for uptake of exogenous fatty acids and their conversion to triacylglycerol [10] We have earlier also reported that almost half of the roughly 106 caveolae in rat adipocytes are smaller than about 50 nm in diameter [5] By labelling intact cells with ruthenium red, which is electron dense and does not permeate the plasma membrane, and examination by electron microscopy we have demonstrated that these caveolae are not open to the cell surface and the surrounding medium [5] In contrast, the caveolae that are larger than about 50 nm are labelled by ruthenium red and are thus open cell surface caveolae [5] This indicates the presence of additional specialized subgroups or classes of caveolin-containing membranes in the plasma membrane We now report the separation and biochemical characterization of three classes of caveolae, which demonstrate a segregation of functions in caveolae subclasses Results Primary rat adipocytes were homogenized and plasma membranes isolated by differential and density gradient ultracentrifugation (Fig 1) The purified plasma membrane fraction contained less than 1% of the cell’s mitochondrial cytochrome oxidase and less than 7% of the cell Golgi TGN38 protein, indicating a very limited contamination by other membrane fractions of the purified plasma membrane [2] Purified plasma membranes were disrupted by sonication to release caveolae [2,11,12] and subjected to linear sucrose gradient ultracentrifugation Membranes in collected fractions were pelleted and analysed for caveolin content by SDS ⁄ PAGE and immunoblotting with anticaveolin-1 antibodies A broad peak with a shoulder of caveolin suggested heterogeneity and the partial separation of caveolin-containing membranes (Fig 2B) Analysis of the specific protein and lipid composition reported below demonstrated the presence of at least three subclasses of caveolae, each with its specific composition: a very high-density class of caveolae at about 31% sucrose (VHD-caveolae; corresponding to 3382 Fig Outline of the procedure used for isolation of caveolae subclasses Also shown is the step at which other cellular fractions are removed density ¼ 1.11 gỈmL)1), and two caveolae classes of lower density, a high-density class of caveolae at about 25% sucrose (HD-caveolae; 1.09 gỈmL)1) and a lowdensity class of caveolae at about 18% sucrose (LDcaveolae; 1.06 gỈmL)1), respectively (Fig 2B) This should be compared to intact plasma membranes with a density of 1.14 gỈmL)1 [13] It was not possible to attain complete separation of all three caveolae classes in a sucrose gradient Utilizing a step sucrose gradient, we demonstrated that the HD-caveolae and LD-caveolae can be separated and thus represent separate entities (Fig 3), although at the cost of the resolution from the caveolae at 31% sucrose The distribution of caveolin-2 coincided with that of caveolin-1 over all three caveolae (Fig 2C) Insulin did not affect the distribution between or amount of FEBS Journal 273 (2006) 3381–3392 ª 2006 The Authors Journal compilation ê 2006 FEBS ă U Ortegren et al Identication of caveolae subclasses A G B H C I D J E F K L Fig Linear density gradient ultracentrifugal separation of closed and open invaginated plasma membrane caveolae Purified adipocyte plasma membranes were disrupted in alkaline carbonate buffer and subjected to sucrose density gradient ultracentrifugation as detailed in Experimental procedures Equal volumes of fractions were collected from the bottom of the tube, and membranes were collected by centrifugation and analysed for the distribution of indicated protein or EZ-Link-sulfo-NHS-LC-biotin labelling by SDS ⁄ PAGE and immunoblotting or avidin-labelled HRP, respectively The amounts of indicated proteins are expressed in arbitrary densitometric units and normalized to percentage of maximum (A) Protein concentration (mgỈmL)1) (B) Caveolin-1 (C) Caveolin-2 (D) Cell surface biotin labelling of proteins (E) TGN38 (F) Insulin receptor (G) GLUT4 (H) Hormone-sensitive lipase (HSL) (I) FATP1 (J) FATP4 (K) Perilipin (L) SR-BI FEBS Journal 273 (2006) 3381–3392 ª 2006 The Authors Journal compilation ê 2006 FEBS 3383 ă U Ortegren et al Identification of caveolae subclasses membrane, before isolation of the caveolin-containing membranes After SDS ⁄ PAGE, blotting, and detection with horseradish peroxidase-labelled avidin, biotinylated A Fig Separation of HD-caveolae and LD-caveolae by two-step density gradient ultracentrifugation Purified adipocyte plasma membranes were disrupted in alkaline carbonate buffer and subjected to step sucrose, density gradient ultracentrifugation: a sample suspended in 45% sucrose was overlayered with 35%, 26% and 5% sucrose Equal volumes of fractions were collected from the bottom of the tube, membranes were collected by centrifugation, and analysed by SDS ⁄ PAGE and immunoblotting for the distribution of caveolin (——), perilipin ( ), and SR-BI (– — –) The HDcaveolae were collected at the 26 ⁄ 35% sucrose interphase and the LD-caveolae at the ⁄ 26% sucrose interphase caveolin-1 in the different caveolae (Fig 4A) The protein profile mirrored the caveolin profile (Figs 2A and 4A), indicating that the caveolae fractions are not contaminated by noncaveolar membranes [2] Noncaveolar membrane protein was found at the bottom of the sucrose gradient (Fig 2A) By immunogold labelling and transmission electron microscopy, membrane vesicles in all three classes of caveolae were found to contain caveolin-1 (Fig 5) In all three classes of caveolae, the same fraction of membrane vesicles were labelled against caveolin: VHD-caveolae 58 ± 3% (n ¼ 13); HD-caveolae 58 ± 5% (n ¼ 12); LD-caveolae 59 ± 5% (n ¼ 8) (mean ± SE, n ¼ number of grids examined from two separate preparations) We labelled the cell surface proteins of intact adipocytes with sulfo-N-hydroxysuccinimidyl-biotin (sulfo-NHS-biotin), which cannot penetrate the plasma B C Fig Effects of insulin on caveolin-1, insulin receptor autophosphorylation, and GLUT4 translocation Isolated adipocytes were incubated with (filled bars; insert lanes 4–6) or without (open bars; insert lanes 1–3) nM insulin for 20 min, after which caveolae were prepared and separated by density gradient ultracentrifugation as in Fig Fractions corresponding to the three caveolae peaks were analysed by SDS ⁄ PAGE and immunoblotting by loading equal amounts of protein (lanes and 4, VHD-caveolae; lanes and 5, HD-caveolae; lanes and 6, LD-caveolae) The amount of the analysed proteins is expressed as arbitrary densitometric units and normalized to percentage of maximum (A) Caveolin-1 (B) Tyrosinephosphorylated insulin receptor b-subunit (C) GLUT4 3384 FEBS Journal 273 (2006) 3381–3392 ª 2006 The Authors Journal compilation ê 2006 FEBS ă U Ortegren et al A B C Fig Immunogold labelling and electron microscopic visualization of caveolin-1 in caveolae fractions Caveolae fractions from density gradient ultracentrifugation were immunogold labelled against caveolin-1 and examined by transmission electron microscopy (A) VHDcaveolae (B) HD-caveolae (C) LD-caveolae proteins were detected in the HD-caveolae and LD-caveolae, particularly in the LD-caveolae, but there was no labelling coinciding with the VHD-caveolae (Fig 2D) As the sulfo-NHS-biotin reagent is negatively charged and this may interfere with access to caveolae and caveolar proteins, we repeated the experiment with the noncharged TFP-PEO-biotin protein-labelling Identification of caveolae subclasses reagent This produced protein labelling that was again limited to the HD-caveolae and LD-caveolae and especially the LD-caveolae (not shown) To examine whether the biotin labelling was restricted to the LD-caveolae, we separately subjected the higherand lower-density half of the sulfo-NHS-biotin-labelled peak to a second density gradient ultracentrifugal separation Each biotin-labelled density peak remained as a discrete entity, indicating that both HD-caveolae and LD-caveolae were subject to cell surface labelling (Fig 6) SDS ⁄ PAGE and silver staining of the three caveolin-containing membrane fractions showed that all three caveolae share a set of major proteins (Fig 7A) The major protein pattern also revealed that the indicated proteins with molecular masses of 75 kDa and 100 kDa were nearly absent from the VHD-caveolae and concentrated in the LD-caveolae Caveolin was enriched in all three caveolae fractions compared to the purified plasma membrane fraction (Fig 7B) TGN38, which is a trans-Golgi protein involved in trafficking to the plasma membrane, was present in the VHD-caveolae fractions (Fig 2E) The insulin receptor was present in both HD-caveolae and LD-caveolae fractions, but was mainly concentrated in the LD-caveolae (Fig 2F) The distribution of the insulin receptor was not affected by insulin (not shown), although the receptor was tyrosine phosphorylated in response to insulin, predominantly in the LD-caveolae (Fig 4B) The insulin-stimulated glucose transporter GLUT4 is present in low amounts in the plasma membrane, where it increases by translocation from intracellular locations in response to insulin stimulation of adipocytes [14,15] GLUT4 of the plasma membrane has been shown to mainly reside in caveolae under basal noninsulin-stimulated conditions and to translocate from intracellular stores to caveolae for glucose uptake in response to insulin [8,9] Under basal noninsulinstimulated conditions, GLUT4 was present mainly in the HD-caveolae (Fig 2G), but increased in response to insulin in both the LD-caveolae and HD-caveolae, with the increase being particularly pronounced in the LD-caveolae (Fig 4C) The presence of the triacylglycerol-hydrolysing enzyme hormone-sensitive lipase was identified specifically in the HD-caveolae (Fig 2H) The two fatty acid transport proteins FATP1 and FATP4 were largely confined to the HD-caveolae (Fig 2I,J) The triacylglycerol-synthesizing fatty acyl-CoA synthetase (not shown) [10] and the lipid-droplet protein perilipin (Fig 2K) were likewise confined to the HD-caveolae FATP1 has been found to translocate to the plasma FEBS Journal 273 (2006) 3381–3392 ª 2006 The Authors Journal compilation ê 2006 FEBS 3385 ă U Ortegren et al Identification of caveolae subclasses Fig Separate reseparation of cell surface biotin-labelled HD-caveolae and LD-caveolae by density gradient centrifugation Fractions containing HD-caveolae or LD-caveolae from the linear density gradient ultracentrifugation (corresponding to Fig fractions and 7, respectively) were separately subjected to a second round of linear density gradient ultracentrifugation, and analysed by SDS ⁄ PAGE and immunoblotting (A) HD-caveolae (B) LD-caveolae Caveolin-1 (——), cell surface biotin-labelled proteins (– – –) A B Fig Protein pattern and caveolin enrichment in caveolae fractions (A) VHD-caveolae from density gradient ultracentrifugation (lane a); HD-caveolae (lanes b1 and b2); LD-caveolae (lane c) a, b1 and b2, and c were separately subjected to SDS ⁄ PAGE (9% acrylamide) and silver staining Indicated are apparent molecular masses of reference proteins (B) SDS ⁄ PAGE and immunoblotting against caveolin-1 of VHD-caveolae (lane 1), HD-caveolae (lane 2), LD-caveolae (lane 3), and purified plasma membrane fraction (lane 4) Equal amounts of protein were subjected to analysis membrane in response to insulin stimulation of 3T3-L1 adipocytes, but much less so in primary rat adipocytes [16] We found, however, no significant effect of 3386 insulin on the amount of FATP1 in the plasma membrane or in caveolae (not shown) Cholesterol, which is a critical and major component of caveolae membranes [2,4], was, as expected, present in all caveolae fractions The molar ratio of cholesterol to phospholipid varied widely between the caveolae classes, however, from about 0.9 for the LD-caveolae to about 0.4 for the VHD-caveolae (Table 1) Likewise, the ratio of caveolin to cholesterol varied widely, being about six times lower in the LD-caveolae than in the closed VHD-caveolae (Table 1) Interestingly, the class B type scavenger receptor (SR-BI), which mediates uptake and efflux of cholesterol, was mainly found in the caveolae fraction with the highest concentration of cholesterol (Fig 2L) We confirmed the presence of SR-BI using different antibodies against the protein (see Experimental procedures), with the same results A functional role for the LD-caveolae in cholesterol metabolism was suggested by a time-dependent uptake and hydrolysis of radiolabelled cholesteryl ester from high-density lipoprotein (HDL) in the LD-caveolae membrane (Fig 8) Uptake was apparently mediated by SR-BI, as it was inhibited by the SR-BI inhibitor BLT-1 by 45% (not shown), which is similar to what has been reported for BLT-1 inhibition of SR-BI [17] Discussion Herein we have identified three caveolin-containing membrane fractions by their composition and cell surface accessibility, thus demonstrating the segregation of proteins and functions in different classes of caveolae We refer to all three as caveolae, because we have earlier used immunogold labelling and electron micro- FEBS Journal 273 (2006) 3381–3392 ª 2006 The Authors Journal compilation ê 2006 FEBS ă U Ortegren et al Identification of caveolae subclasses Table Constituent proteins and lipids of three subclasses of caveolae A compilation of components and properties Property Approximate density (gỈmL)1) Caveolin-1 Caveolin-2 Biotin cell surface labelling Insulin receptor TGN38 Perilipin Fatty acyl-CoA synthetase Enzymes of triacylglycerol synthesis from fatty acids FATP1 FATP4 Hormone-sensitive lipase GLUT4 SR-B1 Cholesterol (lmolặmg protein)1) Mean SE (n ẳ preparations)a Phospholipids (lmolặmg protein)1) Mean SE (n ẳ preparations)a Cholesterol ⁄ phospholipid ratio (mol ⁄ mol) Caveolin ⁄ cholesterol relative ratio VHDcaveolae HDcaveolae LDcaveolae 1.11 1.09 1.06 + + – – + – – – + + + + – ++ ++ ++ + + ++ ++ – – – – – – – – – 0.2 ± 0.01 ++ ++ ++ + + 0.4 ± 0.03 – – – + ++ 1.1 ± 0.1 0.4 ± 0.1 0.8 ± 0.1 1.2 ± 0.1 0.43 0.46 0.91 1⁄1 ⁄ 2.3 1⁄6 a Measured in the indicated caveolae subclasses, represented by fractions 3–4, 5–6, and 7, respectively, in Fig scopy to demonstrate that the localization of the caveolin in the plasma membrane of primary rat adipocytes is found in caveolar structures, with negligible amounts of caveolin in noninvaginated, nonvesicular structures [5] Moreover, we herein used immunogold labelling and electron microscopy to verify that membranes in the sucrose density gradient, corresponding to all three classes of caveolae, contained caveolin-1 The kinship between the three caveolae classes identified herein, which further justifies referring to all of them as caveolae, was demonstrated by their content of both caveolin-1 and caveolin-2 The coexistence of caveolin-1 and caveolin-2 in all three caveolae subclasses is in line with earlier findings that caveolin-1 expression is a prerequisite for proper expression of caveolin-2 [18] That at least three discrete classes of caveolae were indeed identified was demonstrated by both distinct subsets of specific proteins and substantially different concentrations of cholesterol in them Fig HDL cholesteryl ester uptake and conversion to free cholesterol Purified HDL with oleoyl-[3H]cholesterol were prepared as in Experimental procedures and incubated with isolated adipocytes for (open bars) or 10 (closed bars), after which cells were homogenized and caveolae classes fractionated as in Fig Fractions corresponding to the HD-caveolae or LD-caveolae were analysed for their content of free [3H]cholesterol or oleoyl-[3H]cholesterol by TLC In Table we have summarized the composition of the three subclasses of caveolae The three classes of caveolae were isolated from purified plasma membranes None of the three caveolae classes, which each represent about one-third of the caveolin, can for quantitative reasons therefore be explained as contamination by other membrane fractions containing caveolin, such as Golgi membranes or lipid bodies that contain minor fractions of total adipocyte caveolin It needs to be kept in mind that all cellular membrane compartments communicate through continuous membranes or by vesicular trafficking, and therefore biochemically isolated membranes represent fractions of a continuum This is especially true for fractions of the plasma membrane where any fractionation is artificial and a complete separation based on density is unlikely Indeed, the classes of caveolae with specific functions overlap in terms of their constituents as well as in the sucrose gradient It was not possible to completely separate the caveolae subclasses, but their distinct identities were ascertained by using a step sucrose gradient that clearly demonstrated the presence of HD-caveolae and LD-caveolae (Fig 3) Overlapping densities were indicated by a small amount of the LD-caveolae protein SR-B1 (Fig 2L) collecting at the higher sucrose density step together with the HD-caveolae and, likewise, by the small amount of HD-caveolae protein perilipin (Fig 2K) collecting at the low-density step together FEBS Journal 273 (2006) 3381–3392 ª 2006 The Authors Journal compilation ª 2006 FEBS 3387 ă U Ortegren et al Identication of caveolae subclasses with the LD-caveolae (Fig 3) It cannot be excluded that additional classes of caveolae are hidden in the partly separated peaks of caveolae defined herein Our findings nevertheless clearly demonstrate the segregation of different functions in different caveolae in the adipocyte plasma membrane This identification of caveolae subclasses can be compared with the initial isolation and separation of subclasses of lipoproteins from blood [e.g chylomicrons, very low-density lipoprotein (VLDL), low-density lipoprotein (LDL)], which were partially separated by gradient ultracentrifugation as a crucial step in their identification and definition This has been fundamental for understanding cardiovascular disease The concentration of cholesterol varied widely in the three identified caveolae classes Also, the ratio of caveolin to cholesterol varied widely, being about six times lower in the LD-caveolae than in the closed VHD-caveolae, demonstrating that factors other than caveolin control the concentration of cholesterol in the plasma membrane and its domains Others have noticed two bands after sucrose gradient ultracentrifugation of the detergent-resistant residue of whole 3T3-L1 adipocytes, but only one was found to represent caveolae [19] We have earlier shown that detergent extraction is a poor method for isolating caveolae from fat cells and that, for example, the insulin receptor [6] and lipids are extracted with the detergent [2], even at °C Two bands have also been found after discontinuous sucrose gradient centrifugation of a carbonate extract of rat adipocyte plasma membranes [20] VHD-caveolae lack cell surface access The biotin labelling of cell surface proteins indicates that the VHD-caveolae in particular, but to some extent also the HD-caveolae, are not accessible to the labelling reagents (negatively charged and uncharged) This is fully supported by our previous electron microscopic identification [5] of two morphologically distinct classes of caveolae at the plasma membrane ) canonical caveolae that are open to the extracellular space and caveolae lacking access from the cell surface Closed caveolae have restricted access through caveolae openings, and the presence of a ‘diaphragm’ over some caveolae openings has indeed been demonstrated by thin-section electron microscopy [21] However, we cannot rule out the possibility that the lack of labelling was due to these caveolae being void of cell surface proteins In addition to the high buoyant density, low cholesterol concentration, and low cholesterol to caveolin 3388 ratio of the VHD-caveolae, these differed from the open caveolae in that they contained no or very little insulin receptor, SR-BI or GLUT4 ) membrane proteins that interact with extracellular ligands or substrates ) or FATP1 and FATP4, which, presumably, bind fatty acids taken up in those caveolae [10] This again supports the interpretation that the VHD-caveolae are without cell surface access We cannot exclude the possibility that these VHDcaveolae membranes originate intracellularly TGN38 was found in the fractions corresponding to the closed VHD-caveolae, and this protein is found in highest amount in Golgi and is involved in vesicle transport between the plasma membrane and the trans-Golgi network [22,23] Golgi ⁄ microsomal contamination of the plasma membrane fraction may be the source of the small amounts of TGN38, but it is unlikely that the closed VHD-caveolae fraction is Golgi ⁄ microsomal membrane, for the following reasons: (a) the ratio of TGN38 to caveolin content was 15 times higher in the microsomal fraction than in the VHD-caveolae fraction (not shown); (b) it has been reported that Golgi predominantly contains caveolin-2 [24–26], which was present in the VHD-caveolae with the same relation to caveolin-1 as in the HD-caveolae and LD-caveolae; (c) the VHD-caveolae contained almost a third of the plasma membrane caveolin, and the majority of cellular caveolin is found in the plasma membrane of adipocytes; (d) the pattern of major proteins revealed by SDS ⁄ PAGE demonstrated a relation between the VHD-caveolae and HD-caveolae and LD-caveolae; and (e) closed caveolae without cell surface access have been demonstrated in the plasma membrane by electron microscopy [5] We not know the function of this class of closed caveolae, but obvious possibilities are vesicular transport between the Golgi and plasma membrane, and a readily available pool of membrane and caveolin for replenishment and formation of open caveolae, or potocytosis [27] A related possibility is the recently described caveolae that cycle between fused and free forms, which remain close to the plasma membrane in a volume limited by microfilaments [28] HD-caveolae as sites of fatty acid uptake and triacylglycerol synthesis We have previously used biochemical analysis, fluorescence confocal microscopy and electron microscopy to identify the HD-caveolae as specific sites of fatty acid uptake and conversion to triacylglycerol, including the unique presence of fatty acyl-CoA synthetase and perilipin in these caveolae [10] These findings are here FEBS Journal 273 (2006) 3381–3392 ª 2006 The Authors Journal compilation ª 2006 FEBS ¨ U Ortegren et al corroborated by the identification of the fatty acidbinding membrane proteins FATP1 and FATP4 in this specific class of caveolae FATP1 and FATP4 are the only FATP proteins expressed in adipocytes [16], although very low levels of FATP2 mRNA have been detected [16] Taken together, these findings strongly implicate the HD-caveolae as specific sites of fatty acid entry into the fat cells As previously pointed out [10], the role of caveolae as gateways for fatty acid entry is particularly relevant, since fatty acids are potent detergents that dissolve cell membranes and lyse cells [29] Owing to their relatively high detergent resistance, caveolae are adapted to cope with the detergent properties of fatty acids LD-caveolae as sites of cholesteryl ester uptake The very high concentration of cholesterol in the LD-caveolae, also when compared to the concentration of caveolin, indicates a specific function in cholesterol metabolism Such a notion is supported by the abundance in this caveolae subclass of SR-BI, which mediates binding of LDL and selective uptake of HDL cholesteryl esters and the efflux of cholesterol [30,31] SR-BI has previously been found in a caveolinenriched fraction of a cell line stably transfected with the protein [32] As SR-BI has been difficult to identify in adipocytes, despite very high levels of the corresponding mRNA [33,34], we confirmed its presence in the LD-caveolae peak with antibodies from two different sources We also demonstrated that these caveolae functioned in the uptake and hydrolysis of cholesteryl ester from HDL The involvement of SR-B1 in this uptake was indicated by the inhibition of the uptake by the SR-BI inhibitor BLT-1 Conclusions In conclusion, we demonstrate three subclasses of caveolae with compositions that suggest their involvement in specific processes at the plasma membrane The HD-caveolae are apparently specialized in fatty acid uptake and triacylglycerol synthesis [10], and the LD-caveolae appear to be specifically involved in cholesterol metabolism, while both classes have a role in insulin-stimulated glucose uptake Further investigations are needed to determine the genesis and function(s) of the apparently closed VHD-caveolae vesicles Obvious possibilities are Golgi–plasma membrane vesicular transport, potocytosis [27], or a readily available pool of membrane and caveolin for formation of open caveolae It will be important to elucidate the functional and dynamic relationships between closed Identification of caveolae subclasses and open caveolae, as well as how targeting and sequestration of the different proteins and lipids are regulated and maintained Experimental procedures Materials Harlan Sprague Dawley rats (130–160 g) were obtained from B & K Universal (Sollentuna, Sweden) The animals were treated in accordance with Swedish animal care regulations Antibodies against insulin receptor b-subunit (rabbit polyclonal) and caveolin-2 (mouse monoclonal) were from Santa Cruz Biotech (Santa Cruz, CA, USA), those against caveolin-1 (mouse monoclonal) were from Transduction Laboratories (Lexington, KY, USA), those against TGN38 (mouse monoclonal) were from Affinity Bioreagents Inc (Golden, CO, USA), those against GLUT4 (rabbit polyclonal) were from Biogenesis (Poole, UK) and those against SR-BI (rabbit polyclonal) were from Novus Biologicals (Littleton, CO, USA) Antibodies against FATP1 and FATP4 were a generous gift from A Stahl (Stanford University School of Medicine), those against SR-BI were from M Krieger (Massachusetts Institute of Technology), those against perilipin were from C Londos (National Institutes of Health), those against hormone-sensitive lipase were from C Holm (Lund University), and those against fatty acyl-CoA synthetase were from JE Schaffer (Washington School of Medicine) EZ-link sulfo-NHS-LC-biotin and EZ-Link TFP-PEO-biotin were from Perbio ⁄ Pierce (Tattenhall, UK), and HRP-linked streptavidin was from Amersham Bisosciences (Amersham, UK) Other chemicals were from Sigma-Aldrich (St Louis, MO, USA) or Boehringer Mannheim (Mannheim, Germany) or as indicated Isolation and incubation of adipocytes Adipocytes were isolated by collagenase digestion [35] Cells were kept in Krebs–Ringer solution (0.12 m NaCl, 4.7 mm KCl, 2.5 mm CaCl2, 1.2 mm MgSO4, 1.2 mm KH2PO4) containing 20 mm Hepes, pH 7.40, 1% (w ⁄ v) fatty acid-free BSA, 100 nm phenylisopropyladenosine, 0.5 mL)1 adenosine deaminase and mm glucose, at 37 °C on a shaking water bath When indicated, cells were incubated with nm insulin for 20 before homogenization To label plasma membrane proteins with biotin, adipocytes were incubated for 20 at 13 °C in the Krebs– Ringer solution with 0.1% (w ⁄ v) fatty acid-free BSA and 0.5 mgỈmL)1 EZ-Link Sulfo-NHS-LC-biotin or EZ-Link TFP-PEO-biotin, as indicated Labelling was terminated by incubation for 10 with 20 mm glycine Cells were washed three times with Krebs–Ringer solution, 1% (w ⁄ v) fatty acid-free BSA and 20 mm glycine FEBS Journal 273 (2006) 3381–3392 ª 2006 The Authors Journal compilation ª 2006 FEBS 3389 ă U Ortegren et al Identication of caveolae subclasses Preparation of subfractions of caveolae conjugated goat antirabbit antibodies (Aurion) for h at 37 °C After washing, membranes on the grids were further fixed in 2% glutaraldehyde for 10 and visualized with 1% (w ⁄ v) uranylacetate Transmission electron microscopy was performed with a Jeol EX1200 TEM-SCAN (Tokyo, Japan) To prepare caveolae fractions without detergent [2], adipocytes were homogenized in 10 mm Tris ⁄ HCl, pH 7.4, mm EDTA, 0.5 mm EGTA, 0.25 m sucrose, 25 mm NaF, mm Na2-pyrophosphate, with protease inhibitors, 10 lm leupeptin, lm pepstatin, lm aprotinin, mm iodoacetate, and 50 lm phenylmethylsulfonyl fluoride using a motor-driven Teflon ⁄ glass homogenizer at room temperature Subsequent procedures were carried out at 0–4 °C Cell debris and nuclei were removed by centrifugation (JA21, Beckman Instruments, Fullerton, CA, USA) at 1000 g for 10 A plasma membrane-containing pellet, obtained by centrifugation (JA21, Beckman) at 16 000 g for 20 min, was resuspended in 10 mm Tris ⁄ HCl, pH 7.4, mm EDTA and protease inhibitors Purified plasma membranes, obtained by sucrose density gradient centrifugation, were pelleted and resuspended in 0.5 m Na2CO3, pH 11, and sonicated with a probe-type sonifier (Soniprep 150, MSE, Crawley, UK) for · 20 s The homogenate was then adjusted to 40% (w ⁄ v) sucrose in 12 mm Mes, pH 6.5, 75 mm NaCl, 0.25 m Na2CO3, made into a linear sucrose gradient with 10% (w ⁄ v) sucrose in 12 mm Mes, pH 6.5, 75 mm NaCl, 0.25 m Na2CO3, and centrifuged at 200 000 g for 16–20 h in an SW41 rotor (Beckman) Fractions of mL were collected from the bottom of the tube Caveolae purity has been documented [2,12] A microsomal fraction was obtained as described [2] HDL3 was isolated from human blood as previously described [36,37] [1a,2a(n)-3H]Cholesteryl oleate (0.4 mCi) was dried on 20 mg of celite and incubated with HDL3 (3 mL, about mg of protein) overnight at 37 °C under N2, and then the mixture was filtered (pore size 0.22 lm) The [1a,2a(n)-3H]cholesteryl oleate-HDL was added to the cells and incubated at 37 °C Lipids were extracted from caveolae fractions using CHCl3 ⁄ CH3OH ⁄ sample (1 : : 0.9, v ⁄ v) Cholesterol and cholesteryl oleate were quantitated after separation by TLC developed in CHCl3 ⁄ CH3OH ⁄ H2O (65 : 35 : 2.5, v ⁄ v) for cm and, after drying, in hexane ⁄ diethylether ⁄ CH3COOH (70 : 30 : 1, v ⁄ v) to the top of the plate Silica acid was scraped off plates, suspended in 0.2 mL of methanol, and analysed for radioactivity by liquid scintillation To inhibit cholesteryl ester uptake, cells were incubated with 100 lm BLT-1 (#5234221, Chembridge, San Diego, CA, USA) for 30 at 37 °C prior to addition of HDL3, when radioactivity taken up by the cells was determined SDS ⁄ PAGE and immunoblotting Cholesterol determination Membranes were pelleted by centrifugation, and after SDS ⁄ PAGE, separated proteins were electrophoretically transferred to a polyvinylidene difluoride blotting membrane (Immobilone-P, Millipore, Bedford, MA, USA) and incubated with indicated antibodies When membranes were reprobed after stripping bound antibodies, stripping completeness was ascertained by incubation with secondary antibodies Bound antibodies were detected using ECL-plus with HRP-conjugated anti-IgG as secondary antibodies (Amersham Biosciences) Blots were quantitated by chemiluminiscence imaging (Las 1000, Fuji, Tokyo Japan) For determination of cholesterol content, membranes were pelleted by centrifugation and lipids extracted with 2propanol Cholesterol was then quantitated spectrofluorometrically by measuring the amount of H2O2 produced by cholesterol oxidase [38] Electron microscopy of immunogold-labelled caveolae membranes Membranes were collected on carbon formvar-covered nickel grids by spotting a drop of indicated caveolae-containing fractions from the ultracentrifugation sucrose gradient on the grids After washing and fixation in 3% paraformaldehyde for 15 min, grids were air-dried Membranes on grids were rehydrated and blocked in 5% (w ⁄ v) BSA (BSA-c, Aurion, The Netherlands), 1% (v ⁄ v) normal goat serum, and 0.1% (w ⁄ v) gelatine for h at 37 °C The grids were incubated with rabbit caveolin-1 polyclonal antibody overnight at °C and then with secondary 15 nm colloidal gold- 3390 Cholesteryl ester uptake from HDL and analysis of cholesteryl ester hydrolysis Phospholipid determination For determination of phospholipid content, membranes were pelleted by centrifugation and lipids extracted with CHCl3 ⁄ CH3OH ⁄ H2O (1 : : 0.9, v ⁄ v) Phospholipids were then determined as phosphate molybdate complexes after charring in perchloric acid, according to Fiske and Subarrow [39] and modified as in Svennerholm and Vanier [40] Protein determination Protein was quantitated using the protein quantitation kit Micro BCA from Pierce, with BSA as reference Acknowledgements We thank Drs A Stahl, M Krieger, C Londos, C Holm, and J E Shaffer for generously sharing their FEBS Journal 273 (2006) 3381–3392 ª 2006 The Authors Journal compilation ê 2006 FEBS ă U Ortegren et al antibodies The authors declare that they have no competing nancial interests Financial support was ă obtained from Lions Foundation, Ostergotland ă County Council, the Swedish Foundation for Strategic Research (Glycoconjugates in Biological Systems), the Novo Nordisk Foundation, the Swedish Diabetes Association, and the Swedish Research Council References Williams TM & Lisanti MP (2004) The caveolin proteins Genome Biol 5, 214.1214.8 ă Ortegren U, Karlsson M, Blazic N, Blomqvist M, ˚ Nystrom FH, Gustavsson J, Fredman P & Stralfors P (2004) Lipids and glycosphingolipids in caveolae and surrounding plasma membrane of primary rat adipocytes Eur J Biochem 271, 2028–2036 Razani B, Woodman SE & Lisanti MP (2002) Caveolae: from cell biology to animal physiology Pharmacol Rev 54, 431–467 Pike LJ, Han X, Chung K-N & Gross RW (2002) Lipid rafts are enriched in arachidonic acid and plasmenylethanolamine and their composition is independent of caveolin-1 expression: a quantitative electrospray ionization ⁄ mass spectrometric analysis Biochemistry 41, 20752088 ă Thorn H, Stenkula KG, Karlsson M, Ortegren U, ˚ Nystrom FH, Gustavsson J & Stralfors P (2003) Cell surface orifices of caveolae and localization of caveolin to the necks of caveolae in adipocytes Mol Biol Cell 14, 3967–3976 Gustavsson J, Parpal S, Karlsson M, Ramsing C, Thorn H, Borg M, Lindroth M, Peterson KH, Magnusson ˚ K-E & Stralfors P (1999) Localisation of the insulin receptor in caveolae of adipocyte plasma membrane FASEB J 13, 1961–1971 ˚ Parpal S, Karlsson M, Thorn H & Stralfors P (2001) Cholesterol depletion disrupts caveolae and insulin receptor signaling for metabolic control via IRS-1, but not for MAP-kinase control J Biol Chem 276, 9670– 9678 ˚ Gustavsson J, Parpal S & Stralfors P (1996) Insulinstimulated glucose uptake involves the transition of glucose transporters to a caveolae-rich fraction within the plasma membrane: implications for type II diabetes Mol Med 2, 367–372 ˚ Karlsson M, Thorn H, Parpal S, Stralfors P & Gustavsson J (2001) Insulin induces translocation of glucose transporter GLUT4 to plasma membrane caveolae in adipocytes FASEB J 16, 249251 ă ă 10 Ost A, Ortegren U, Gustavsson J, Nystrom FH & ˚ Stralfors P (2005) Triacylglycerol is synthesized in a specific subclass of caveolae in primary adipocytes J Biol Chem 280, 5–8 Identification of caveolae subclasses 11 Song KS, Li S, Okamoto T, Quilliam LA, Sargiacomo M & Lisanti MP (1996) Co-purification and direct interaction of ras with caveolin, an integral membrane protein of caveolae microdomains Detergent-free purification of caveolae membranes J Biol Chem 271, 9690–9697 ˚ 12 Aboulaich N, Vainonen J, Stralfors P & Vener AV (2004) Vectorial proteomics reveal targeting, phosphorylation and specific fragmentation of polymerase I and transcript release factor (PTRF) at the surface of caveolae in human adipocytes Biochem J 383, 237–248 13 McKeel DW & Jarett L (1970) Preparation and characterization of a plasma membrane fraction from isolated fat cells J Cell Biol 44, 417–432 14 Suzuki K & Kono T (1980) Evidence that insulin causes translocation of glucose transport activity to the plasma membrane from an intracellular storage site Proc Natl Acad Sci USA 77, 2542–2545 15 Cushman SW & Wardzala LJ (1980) Potential mechanism of insulin action on glucose transport in the isolated rat adipose cell J Biol Chem 255, 4758–4762 16 Stahl A, Evans JG, Pattel S, Hirsch D & Lodish HF (2002) Insulin causes fatty acid transport protein translocation and enhanced fatty acid uptake in adipocytes Dev Cell 2, 477–488 17 Nieland TJ, Penman M, Dori L, Krieger M & Kirchhausen T (2002) Discovery of chemical inhibitors of the selective transfer of lipids mediated by the HDL receptor SR-BI Proc Natl Acad Sci USA 99, 15422–15427 18 Razani B, Engelman JA, Wang XB, Schubert W, Zhang XL, Marks CB, Macaluso F, Russel RG, Li M, Pestell RG et al (2001) Caveolin-1 null mice are viable but show evidence of hyperproliferative and vascular abnormalities J Biol Chem 276, 38121–38138 19 Corley Mastick C, Brady MJ & Saltiel AR (1995) Insulin stimulates the tyrosine phosphorylation of caveolin J Cell Biol 129, 1523–1531 20 Muller G, Hanekop N, Wied S & Frick W (2002) Cholesterol depletion blocks redistribution of lipid raft components and insulin-mimetic signaling by glimperiride and phosphoinositolglycans in rat adipocytes Mol Med 8, 120–136 21 Simionescu N, Simionescu M & Palade GE (1981) Differentiated microdomains on the luminal surface of the capillary endothelium I Preferential distribution of anionic sites J Cell Biol 90, 605–613 22 Jones SM, Crosby JR, Salamero J & Howell KE (1993) A cytosolic complex of p62 and rab6 associates with TGN38 ⁄ 41 and is involved in budding of exocytotic vesicles from the trans-Golgi network J Cell Biol 122, 775–788 23 Reaves B, Horn M & Banting G (1993) TGN38 ⁄ 41 recycles between the cell surface and the TGN: brefeldin A affects its rate of return to the TGN Mol Biol Cell 4, 93–105 FEBS Journal 273 (2006) 3381–3392 ª 2006 The Authors Journal compilation ê 2006 FEBS 3391 ă U Ortegren et al Identification of caveolae subclasses 24 Breuza L, Corby S, Arsanto JP, Delgrossi MH, Scheiffele P & LeBivic A (2002) The scaffolding domain of caveolin is responsible for its Golgi localization in Caco-2 cells J Cell Sci 115, 4457–4467 25 Gargalovic P & Dory L (2001) Caveolin-1 and caveolin2 expression in mouse macrophages High density lipoprotein 3-stimulated secretion and a lack of significant subcellular co-localization J Biol Chem 276, 26164– 26170 26 Mora R, Bonilha VL, Marmorstein A, Scherer PE, Brown D, Lisanti MP & Rodriguez-Boulan E (1999) Caveolin-2 localizes to the golgi complex but redistributes to plasma membrane, caveolae, and rafts when co-expressed with caveolin-1 J Biol Chem 274, 25708– 25717 27 Anderson RGW, Kamen BA, Rothberg KG & Lacey SW (1992) Potocytosis: sequestration and transport of small molecules by caveolae Science 255, 410–411 28 Tagawa A, Mezzacasa, a Hayer A, Longatti A, Pelkmans L & Helenius A (2005) Assembly and trafficking of caveolar domains in the cell: caveolae as stable, cargo-triggered vesicular transporters J Cell Biol 170, 769–779 ˚ 29 Stralfors P (1990) Autolysis of isolated adipocytes by endogenously produced fatty acids FEBS Lett 263, 153–154 30 Acton SL, Scherer PE, Lodish HF & Krieger M (1994) Expression cloning of SR-BI, a CD36-related class B scavenger receptor J Biol Chem 269, 21003–21009 31 Connelly MA & Williams DL (2004) Scavenger receptor B1: a scavenger receptor with a mission to transport high density lipoprotein lipids Curr Opin Lipidol 15, 287–295 32 Babitt J, Trigatti B, Rigotti A, Smart EJ, Anderson RGW, Xu S & Krieger M (1997) Murine SR-B1, a high 3392 33 34 35 36 37 38 39 40 density lipoprotein receptor that mediates selective lipid uptake, is N-glycosylated and fatty acylated and colocalizes with plasma membrane caveolae J Biol Chem 272, 13242–13249 Acton S, Rigotti A, Landschulz KT, Xu S, Hobbs HH & Krieger M (1996) Identification of scavenger receptor SR-B1 as a high density lipoprotein receptor Science 271, 518–520 Landschulz KT, Pathak RK, Rigotti A, Krieger M & Hobbs HH (1996) Regulation of scavenger receptor, class B, type 1, a high density lipoprotein receptor, in liver and steroidogenic tissues of the rat J Clin Invest 98, 984–995 ˚ Stralfors P & Honnor RC (1989) Insulin-induced dephosphorylation of hormone-sensitive lipase Correlation with lipolysis and cAMP-dependent protein kinase activity Eur J Biochem 182, 379–385 Sattler W, Mohr D & Stocker R (1994) Rapid isolation of lipoproteins and assessment of their peroxidation by high-performance liquid chromatography postcolumn chemiluminescence Methods Enzymol 233, 469–489 Karlsson H, Leanderson P, Tagesson C & Lindahl M (2005) Lipoproteomics II: mapping of proteins in highdensity lipoprotein using two-dimensional gel electrophoresis and mass spectrometry Proteomics 5, 1431– 1445 Heider JG & Boyett RL (1978) The picomole determination of free and total cholesterol in cells in culture J Lipid Res 19, 515–518 Fiske CH & Subbarow YJ (1925) The colorimetric determination of phosphorus J Biol Chem 66, 375–400 Svennerholm L & Vanier MT (1972) The distribution of lipids in the human nervous system II Lipid composition of human fetal and infant brain Brain Res 47, 457–468 FEBS Journal 273 (2006) 3381–3392 ª 2006 The Authors Journal compilation ª 2006 FEBS ... in the HD -caveolae and LD -caveolae; (c) the VHD -caveolae contained almost a third of the plasma membrane caveolin, and the majority of cellular caveolin is found in the plasma membrane of adipocytes;. .. referring to all of them as caveolae, was demonstrated by their content of both caveolin-1 and caveolin-2 The coexistence of caveolin-1 and caveolin-2 in all three caveolae subclasses is in line... lower in the LD -caveolae than in the closed VHD -caveolae, demonstrating that factors other than caveolin control the concentration of cholesterol in the plasma membrane and its domains Others