Role of Caveolin-1 in the Modulation of Lipolysis and Lipid Droplet Formation

Antibodies and their sources were as follows: polyclonal rabbit anti-perilipin was as previously described (25); guinea pig anti-perilipin (polyclonal antibody [pAb]; Research Diagnostics, Flanders, NJ); anti-PKA RI (monoclonal antibody [mAb]; BD Pharmingen, San Diego, CA); anti-PKA RIIα (pAb; Santa Cruz Biotech, Santa Cruz, CA); anti-PKA RIIβ (mAb; BD Pharmingen); anti-PKA α-catalytic (pAb; Santa Cruz, CA); anti-PKA catalytic (mAb; BD Pharmingen); anti-cAMP response element binding (CREB) protein (pAb), anti-phospho-CREB (pAb), and anti-phospho-ser/thr-PKA-specific phosphosubstrate [P-RXX(S/T)] (all pAbs; Cell Signaling, Beverly, MA); anti-β-tubulin (mAb; Sigma, St. Louis, MO); and anti-β₃-adrenergic receptor (pAb; Alpha Diagnostics, San Antonio, TX). Anti-caveolin-1 (mAb, clone 2234) was a gift from Dr. Roberto Campos-Gonzalez (BD Pharmingen). Anti-HSL (pAb) was a generous gift from Dr. Constantine Londos (National Institutes of Health, Bethesda, MD). The β₃-specific adrenoceptor agonist, CL-316,243 (CL), was from Sigma.

Animals were housed at the Institute for Animal Studies, Albert Einstein College of Medicine, as previously described (26,27). Importantly, all of our studies were carried out using mice between the ages of 8 and 12 weeks, at which age we have previously shown that the body weights of wild-type and caveolin-1 null mice are indistinguishable and that the perigonadal fat pads of caveolin-1 null mice do not exhibit reduced adipocyte size, changes in lipid droplet size, or other histological abnormalities that occur in older caveolin-1 null mice (28).

The perigonadal fat pads of wild-type mice were analyzed by Western blot for caveolin-1 expression. Mice were killed in the fed state (8 h after the onset of the dark cycle), the fasted state (48 h of food deprivation beginning at the onset of the dark cycle), or 24 h after refeeding (48 h of fasting, followed by 24 h of access to food). The results shown are representative of three independent experiments.

Baseline serum samples were collected from 4-h-fasted wild-type (n = 17) and caveolin-1 null (n = 15) mice by bleeding the tail of each mouse. A cohort of these mice was then given an intraperitoneal injection of the β₃-specific agonist CL-316,243 (0.1 mg/kg), and serum samples were collected after 15 min. The remaining mice (n = 6 for each group) were further fasted for 48 h, at which time serum was again collected. Free glycerol content (GPO-Trinder; Sigma) and nonesterified fatty acid (NEFA) levels (Half-Micro tests; Roche) were determined colorimetrically

Adipocyte isolation and lipolysis measurements were conducted essentially as described by Carpéné (29) and Tansey et al. (8), with minor modifications. Briefly, perigonadal fat pads from fasted wild-type (n ≥5) and caveolin-1 null (n ≥5) mice were placed in Krebs-Ringer bicarbonate HEPES buffer (KRBH; Sigma) supplemented with 3% BSA, 500 nmol/l adenosine, and 2 mg/ml collagenase type I (Worthington Biochemical, Lakewood, NJ) and agitated for 60 min at 37°C. The cells were washed and resuspended in KRBH. Aliquots were placed in new vials containing 1 unit/ml adenosine deaminase and 100 nmol/l N⁶-phenylisopropyladenosine for basal activity, or they were placed in 5 μmol/l CL-316,243 for stimulated activity. After 60 min, glycerol release was measured using a colorimetric assay (GPO-Trinder; Sigma). Data are normalized to mean mass of cellular lipid, as described by Carpéné (29).

Western blot analysis was performed essentially as previously described (30). Briefly, for tissue Western blots, wild-type (n ≥6) and caveolin-1 null (n ≥6) perigonadal fat pads were harvested and homogenized in radioimmunoprecipitation assay (RIPA) buffer (30). For Western blot analysis of cells in culture, confluent 60-mm dishes were lysed in buffer (10 mmol/l Tris, pH 7.5, 50 mmol/l NaCl, 1% Triton X-100, 60 mmol/l n-octyl glucoside, 2 nmol/l sodium orthovanadate, 0.1 μg/ml okadaic acid, and 40 nmol/l bis-peroxovanadium 1,10-phenanthroline) containing protease inhibitors (Boehringer Mannheim). In some experiments, β-tubulin is shown as a control for equal loading.

A PepTag PKA activity assay (Promega) was used according to the manufacturer’s instructions, with minor modifications. Wild-type (n ≥6) and caveolin-1 null (n ≥6) perigonadal fat pads were removed and homogenized in extraction buffer (25 mmol/l Tris pH 7.4, 0.5 mmol/l EDTA, 0.5 mmol/l EGTA, 10 mmol/l β-mercaptoethanol, and 0.5 mmol/l phenylmethylsulfonyl fluoride) containing protease inhibitors (Boehringer Mannheim). The lysates were centrifuged, and the supernatants were used to determine PKA activity. Some samples were pretreated with the cAMP-dependent protein kinase peptide inhibitor. A positive control (active PKA catalytic subunit) and a negative control (extraction buffer) were included.

3T3-L1 preadipocytes from the American Type Culture Collection (Manassas, VA) and immortalized MEFs (wild-type and caveolin-1 knockout) were grown in Dulbecco’s modified Eagle’s media supplemented with glutamine, antibiotics (penicillin and streptomycin), and 10% FCS. 3T3-L1 cells were differentiated according to standard protocols.

The cDNA for mouse perilipin A (19) was subcloned into the pEF6/V5-His TOPO expression vector (Invitrogen). MEFs were transfected with the Lipofectamine Plus reagent (Invitrogen), and stable clones were selected in 10 μg/ml blasticidin (Invitrogen). At least six wild-type and six caveolin-1 null clones were isolated and analyzed for stable expression of perilipin A.

Peri-MEFs (wild-type, n = 6 independent clones; caveolin-1 null, n = 6 independent clones) were loaded with 400 μmol/l of oleic acid for 24 h to promote the formation of lipid droplets. The cells were washed in PBS and fixed in 70% ethanol for 1 min, followed by staining with Sudan IV (Sigma) for 5 min. In parallel experiments, the cells were either imaged or scraped into DMSO, and the absorbance was read.

Peri-MEFs were loaded with oleic acid, fixed in 2% paraformaldehyde, permeabilized in 0.02% Triton X-100, and immunostained with anti-perilipin antibodies. Bodipy 493/503 (5 μmol/l; Molecular Probes) was included during the secondary antibody incubation to specifically stain neutral lipids.

Immunoprecipitation was performed as previously described (31). Briefly, 3T3-L1 adipocytes (day 10 differentiated) were washed twice with ice-cold PBS and scraped into 1 ml of lysis buffer. For immunoprecipitation from perigonadal fat, mouse tissue was harvested, homogenized in a Polytron tissue grinder, and solubilized in RIPA buffer. Precleared lysates were incubated for 4 h with anti-caveolin-1 IgG or anti-perilipin IgG. Immune complexes were then resolved by SDS-PAGE and processed for immunoblotting. For 3T3-L1 cells, each experiment was repeated at least three times independently, with virtually identical results. For tissue immunoprecipitation, each condition of each experiment was repeated in at least three mice for each genotype.

Wild-type and caveolin-1 null perigonadal fat pads were subjected to high-pressure freezing, using a Leica EMpact High Pressure Freezer apparatus (Leica Microsystems, Vienna, Austria). Frozen samples were then transferred to a Leica electron microscopy AFS Freeze Substitution Unit and freeze substituted in 1% osmium tetroxide in acetone. They were brought from −90°C to room temperature over 2–3 days, rinsed in acetone, and embedded in LX112 epoxy resin (Ladd, Burlington, VT). Ultrathin sections of ∼70–80 nm were cut on a Reichart Ultracut UCT, stained with uranyl acetate followed by lead citrate, and viewed using a Jeol 1200EX transmission electron microscope at 80 kV. High-pressure freeze electron microscopy was performed at the Analytical Imaging Facility of the Albert Einstein College of Medicine.

Several factors, including 1) the high levels of caveolin-1 expression in adipocytes, 2) the ability of caveolin-1 to associate with lipid droplets, and 3) the previously described obesity-resistant phenotype of caveolin-1 null mice (4,5,28,30,32) all suggest that caveolin-1 may play a critical role in modulating lipid storage and/or breakdown. Therefore, we investigated the effect of prolonged fasting (i.e., maximal physiological lipid breakdown) on caveolin-1 expression in the perigonadal fat pads of wild-type mice.

Interestingly, Western blot analysis revealed that caveolin-1 protein levels were greatly increased by approximately threefold in mice subjected to a 48-h fast, when compared with the fed state (Fig. 1A). Additionally, in fasted mice, caveolin-1 expression returned to baseline levels 24 h after food was re-administered.

To determine whether caveolin-1 plays a physiological role in lipid mobilization, we next explored the effects of prolonged fasting in caveolin-1 (−/−) null mice. Interestingly, we found that although serum NEFA levels, which are products of lipolysis, increased significantly in wild-type mice fasted for 48 h, serum NEFA levels of 12-week-old caveolin-1 null mice remained at baseline during fasting (Fig. 1B). Although these results could be attributable to an increased re-esterification rate and/or increased fatty acid utilization in caveolin-1 null mice, they may also suggest that loss of caveolin-1 results in an inability to properly mobilize stored triglycerides during fasting.

Because the above findings suggest that caveolin-1 may be a necessary cofactor in the lipolytic response, we next investigated the in vitro rate of lipolysis in isolated adipocytes from 12-week-old wild-type and caveolin-1 null mice. Glycerol release to the media in baseline and stimulated (60 min, 5 μmol/l CL-316,243) adipocytes was determined as a correlate of lipolytic activity. Before treatment, glycerol levels did not differ statistically between wild-type and caveolin-1 null adipocytes (Fig. 2A). However, after treatment with the β₃-specific adrenergic receptor agonist CL-316,243, glycerol levels increased ∼30-fold in wild-type cells, but they increased only about sixfold in caveolin-1 null cells. Thus, caveolin-1 null adipocytes show an approximately fivefold reduction in their lipolytic response to a β₃-specific agonist. This finding indicates that, similar to fasting, pharmacological adrenergic stimulation fails to elicit lipolysis in caveolin-1-deficient adipocytes.

These findings were next substantiated in vivo by measuring serum NEFA and glycerol levels at baseline and after intraperitoneal administration of CL-316,243 (0.1 mg/kg). Again, baseline NEFA and glycerol levels were not significantly different between wild-type and caveolin-1 null mice, although caveolin-1 null levels tended to be lower. After 15 min of CL-316,243 treatment, wild-type mice showed a normal increase in glycerol (∼3.5-fold) and NEFA (∼2-fold) levels, indicative of increased lipolysis, whereas caveolin-1 null mice remained near baseline (Fig. 2B and C). Taken together, these findings indicate that caveolin-1 is normally required for a proper physiological or agonist-induced lipolytic response.

Because PKA has been shown to be a major regulatory element of lipolytic signals in adipocytes (11) and our findings thus far have demonstrated a perturbation in lipolysis in caveolin-1 deficient animals, we next examined the expression levels of the various PKA subunits in adipose tissue. Western blot analysis of wild-type and caveolin-1 null perigonadal fat pads revealed that the expression of the RI and RII-α subunits was clearly elevated in caveolin-1 null mice, whereas the levels of RII-β and the catalytic-α subunits remained unchanged (Fig. 3A). Although these changes do not provide a clear mechanistic basis for the lipolytic alterations observed in caveolin-1 null mice, it is possible that caveolin-1 serves a similar function as PKA R subunits, i.e., binding to and stabilizing the PKA catalytic subunit. Indeed, the PKA catalytic subunit has been shown to interact with caveolin-1 in cultured cells (1). Thus, one would expect that, similar to the loss of any regulatory subunit, an increase in other R subunits would follow the loss of caveolin-1.

PKA activity was next directly assessed in adipose tissue extracts derived from wild-type and caveolin-1 null mice. As seen in Fig. 3B, loss of caveolin-1 leads to markedly increased PKA-mediated peptide phosphorylation. Spectrophotometric quantification of these findings revealed that approximately fivefold more PKA activity was detected in caveolin-1 null adipose tissue extracts, as compared with wild-type extracts (Fig. 3C). In addition, this kinase activity was indeed PKA-specific because preincubation with the cAMP-dependent protein kinase peptide inhibitor completely abolished peptide phosphorylation (Fig. 3B). Thus, PKA activity is dramatically increased in caveolin-1 null adipocytes.

Although many kinases can phosphorylate the CREB protein, its phosphorylation generally correlates with PKA activity in vivo (33). Therefore, to determine whether pharmacologically induced lipolysis was hyperactivated in vivo, we next assessed the phosphorylation status of CREB in caveolin-1 null adipose tissue after an intraperitoneal injection of CL-316,243 (15 min, 0.1 mg/kg).

Figure 3D shows that CREB phosphorylation is dramatically increased in adipose tissue derived from caveolin-1 null mice, whereas total CREB levels remain unchanged. This indicates that β₃-agonist stimulation results in greater PKA activity in caveolin-1 null mice than in wild-type mice. Although this finding provides evidence that caveolin-1 is a negative regulator of PKA, it also seemingly contradicts our lipolysis studies described above, in that, if PKA is hyperactivated, lipolysis should be maximally activated. This prompted us to look for additional PKA substrates that may be alternatively regulated in caveolin-1 null adipose tissue.

Because PKA is hyperactivated in caveolin-1 null mouse adipocytes, under conditions at which lipolysis occurs at submaximal levels, we sought to determine the mechanism behind this paradoxical phenomenon. One of the major targets for PKA-mediated phosphorylation in the adipocyte is perilipin, which, in the unphosphorylated state, inhibits lipolysis by coating the lipid droplet and providing a barrier between HSL and the lipid core (8,9,11,19,34). Because several studies have shown that caveolins can target to lipid droplets (3–5,35), we postulated that caveolin-1 might play a functional role in lipid droplet homeostasis via modulation of perilipin phosphorylation.

To test this hypothesis directly, we next examined β₃-agonist-treated perigonadal fat pads from caveolin-1 null and wild-type mice for perilipin protein content, as well as its phosphorylation status by Western blot. Using a perilipin-specific antibody and a phospho-PKA motif-specific antibody [P-RXX(S/T)], we found that perilipin phosphorylation is dramatically decreased (∼90%) in caveolin-1 null mice, without any changes in perilipin protein content (Fig. 3E). Therefore, one hypothesis is that caveolin-1 is a necessary coupling factor that is normally required for PKA-mediated phosphorylation of perilipin. A prediction of this hypothesis is that caveolin-1, PKA, and perilipin may form a stable complex in vivo.

We also examined the protein levels of the β₃-adrenergic receptor by Western blot analysis to exclude the possibility that the changes in signaling are secondary to changes in β₃-adrenergic receptor expression levels. As shown in Fig. 3F, β₃-adrenergic receptor levels are unchanged in caveolin-1 null fat pads as compared with wild-type fat pads.

To explore the notion that caveolin-1 normally couples PKA activity to perilipin phosphorylation, we next performed a series of coimmunoprecipitation experiments in 3T3-L1 adipocytes. 3T3-L1 adipocytes were treated with or without CL-316,243 (5 μmol/l, 60 min) and subjected to coimmunoprecipitation with anti-caveolin-1 and anti-perilipin antibodies. The results, shown in Fig. 4A, demonstrate that β₃-agonist treatment leads to the phosphorylation of perilipin, as predicted, and the formation of a protein complex containing perilipin, caveolin-1, and importantly the catalytic subunit of PKA (Fig. 4B and C). In contrast, the PKA RII-β subunit was found to coimmunoprecipitate with perilipin under both baseline and stimulated conditions.

Because caveolin-1 is known to interact with the catalytic subunit of PKA, these results suggest that, under conditions of lipolytic stimulation, caveolin-1 serves to bring PKA into close proximity with perilipin, allowing PKA-mediated phosphorylation. In further support of this idea, we found that coimmunoprecipitation of 3T3-L1 adipocyte lysates with antibodies directed against caveolin-1 provided results similar to those detailed above (Fig. 4D). More specifically, caveolin-1 and perilipin were found to coimmunoprecipitate only after β₃-agonist treatment, and the majority of the perilipin in this complex was phosphorylated.

The above results in 3T3-L1 adipocytes suggest that caveolin-1 serves to recruit the catalytic subunit of PKA to perilipin. To test this hypothesis further, we undertook a similar study in wild-type and caveolin-1 null mice (+/− CL-316,243 treatment, 0.1 mg/kg, 30 min). In accordance with the results obtained in 3T3-L1 cells, coimmunoprecipitation of perigonadal fat pad lysates with anti-perilipin antibodies revealed that caveolin-1 and the catalytic subunit of PKA were found in a tight complex with perilipin after β₃-agonist treatment in wild-type mice (Fig. 5B and C). However, in caveolin-1 null mice, complex formation was defective, as predicted. Additionally, perilipin and phospho-perilipin were only detected in wild-type lysates when coimmunoprecipitated with anti-caveolin-1 antibodies (Fig. 5D). In addition, we did not find HSL in complexes immunoprecipitated with anti-perilipin or anti-caveolin-1 antibodies from wild-type or caveolin-1 null mice, under baseline or stimulated conditions (data not shown).

Figure 5A shows that perilipin levels were equal in wild-type and caveolin-1 null mice and that phosphorylated perilipin was found mainly in wild-type β₃-agonist-stimulated mice. These results directly support the hypothesis that caveolin-1 brings the catalytic subunit of PKA into close contact with perilipin, thus allowing perilipin to be phosphorylated. In addition, these results provide a novel mechanism by which a lack of caveolin-1 results in decreased perilipin phosphorylation, even in the context of increased PKA activity.

Although perilipin expression is limited to adipocytes and steroidogenic cells, the overexpression of perilipin in other cell types has been shown to result in lipid droplet accumulation (23). Therefore, to address the role of caveolin-1 in de novo lipid droplet formation, we constructed perilipin-overexpressing cell lines from wild-type and caveolin-1-deficient MEFs (Peri-MEFs). After isolation of several clones for each genotype, perilipin expression was assessed by Western blot analysis. Similar to endogenous perilipin expression in perigonadal fat pads, we found that caveolin-1 was not required for stable expression of perilipin in caveolin-1-deficient MEFs (Fig. 6A). Both wild-type and caveolin-1 null MEFs expressed equivalent levels of perilipin.

To assess the ability of the Peri-MEFs to take up and store triglycerides, these cells were loaded with oleic acid, as described by Tansey et al. (23). Interestingly, we found that after 24 h of lipid loading, Peri-MEFs lacking caveolin-1 stored dramatically less lipid as compared with wild-type Peri-MEFs (Fig. 6B and C). Note that wild-type Peri-MEFs exhibit many bright red fluorescent Sudan IV-staining lipid droplets, whereas in caveolin-1 null Peri-MEFs, it is relatively difficult to find any Sudan IV-positive cells (Fig. 6B). Spectrophotometric analysis of numerous samples confirmed that wild-type Peri-MEFs contained ∼4.5-fold more Sudan IV-staining lipid droplets than caveolin-1 null Peri-MEFs treated identically (Fig. 6C).

Lipid-loaded Peri-MEFs were also fixed, costained with anti-perilipin antibodies (red stain) and Bodipy 493/503 (specific for neutral lipids, green stain), and analyzed by immunofluorescence microscopy (Fig. 6D). Interestingly, there were many more lipid droplets in wild-type Peri-MEFs, and these lipid droplets tended to be much larger than those observed in caveolin-1 null Peri-MEFs. This is despite the fact that both were outlined by a ring of perilipin, consistent with previous reports (17,19,23). These results provide the first molecular genetic evidence that caveolin-1 is either functionally required for or greatly facilitates lipid droplet accumulation.

Conventional fixation methodologies used by electron microscopists fail to preserve both cytoplasmic structures and lipid droplets in adipocytes in the same preparation (36,37). Thus, we searched for new methodologies to visualize both cytoplasmic structures and the lipid droplet membrane within the same adipocyte.

Using a relatively new technique with high pressure and rapid tissue freezing, we were able to obtain high-quality electron micrographs of intact lipid droplets and other adipocyte cellular constituents. Examination of the contact area between lipid droplets and adipocyte cytoplasm revealed that an electron-dense material surrounds the lipid droplet in wild-type adipocytes (Fig. 7A, arrowheads) that most likely represents a conglomeration of various proteins, such as TIP47, adipophilin, perilipin, and S3–12 (34,38–43). Embedded within this material are numerous organelles that resemble mitochondria (Fig. 7A, arrows). A fibrous network is found juxtaposed to this electron-dense area, reported previously to contain intermediate fibers, such as vimentin (36,44). In a similar analysis of caveolin-1 null adipose tissue, we found a dramatic and near-complete absence of both mitochondria and electron-dense staining in contact with the lipid droplet (Fig. 7B, arrowheads). Thus, these dramatic defects in the lipid droplet architecture of caveolin-1 null perigonadal adipocytes are consistent with the notion that caveolin-1 normally plays a critical structural role in lipid droplet accumulation in adipocytes.

In the present study, we have demonstrated that caveolin-1 is involved in both lipid storage and breakdown in vivo. Analysis of lipolysis established a major role for caveolin-1 in both fasting and agonist-induced triglyceride hydrolysis. These results were somewhat surprising considering the known role of caveolin-1 in modulating PKA activity and the role of PKA in mediating lipolysis. Our group has previously shown that caveolin-1 negatively regulates PKA activity through a direct interaction with the catalytic subunit (1). Although these results were obtained in cell culture, we extrapolated their relevance to the whole animal. Therefore, we would predict that caveolin-1 null mice would show an increase in PKA activity. Additionally, because activation of PKA normally leads to the phosphorylation of HSL and perilipin, and thus the activation of lipolysis, it would also be expected that caveolin-1 null mice would show increased lipolysis.

A thorough experimental evaluation of this hypothesis demonstrated that PKA activity was indeed greatly increased in caveolin-1 null adipose tissue. How, then, does increased PKA activity fail to translate into increased lipolysis? To investigate this question, we examined the phosphorylation state of perilipin. Mutational analysis has shown that the phosphorylation of one or more of the three NH₂-terminal PKA phosphorylation sites within perilipin is necessary for maximal lipolysis (17,21–23). With this knowledge in hand, we then analyzed the phosphorylation status of perilipin after lipolytic stimulation in wild-type and caveolin-1 null mice. Interestingly, we found that despite increased PKA activity, perilipin does not become significantly phosphorylated in caveolin-1-deficient fat pads, and thus its inhibitory hold on lipolysis is not released. In addition, we found that the protein levels of HSL were unchanged in caveolin-1 null animals (data not shown). Although this finding does not directly assess the activity of HSL, recent evidence has indicated that the phosphorylation of perilipin is a more accurate correlate of the rate of lipolysis. For instance, perilipin null MEFs have elevated basal lipolysis; however, they fail to respond maximally to lipolytic stimulation. Perilipin and, more specifically, fully phosphorylated perilipin must be present in adipocytes to elicit HSL translocation to the lipid droplet and subsequent hydrolysis (17).

It is thought that the compartmentalization of PKA, via an association with the regulatory subunits, allows for the local activation of PKA specifically within a micro-environment, rather than diffusely throughout the cell (6). Recombinant expression of caveolin-1 in cultured cells alters the distribution of PKA, and these two proteins colocalize by immunofluorescence microscopy (1). In light of these findings, our data suggest that caveolin-1 may function as a necessary coupling factor to drive the PKA-mediated phosphorylation of perilipin. To address this issue, we performed numerous coimmunoprecipitation experiments in 3T3-L1 adipocytes and mouse fat pads. Our results in 3T3-L1 adipocytes clearly demonstrate that lipolytic stimulation results in a ligand-induced association between perilipin and caveolin-1. Additionally, this complex contains the catalytic subunit of PKA, which is known to be responsible for the phosphorylation of perilipin (23,45).

Although these results strongly suggest that caveolin-1 is necessary to physically couple PKA to perilipin, experiments in caveolin-1 null mice provided definitive proof for the caveolin-dependence of these interactions. More specifically, we demonstrate that in caveolin-1 null fat pads, the catalytic subunit of PKA fails to coimmunoprecipitate with perilipin, clearly showing that caveolin-1 couples PKA activity to perilipin phosphorylation. However, how can caveolin-1, which is known to inhibit PKA activity, be necessary for PKA-mediated phosphorylation? In searching the literature, one finds that this dual role of caveolin-1 has been previously characterized in the case of eNOS activation. Here, caveolin-1 acts as potent inhibitor of eNOS activity; however, it also tethers eNOS to plasma membrane caveolae so that it can be activated by G protein-coupled receptors, which also reside in caveolae (46–50). A similar mechanism could occur in the case of perilipin and PKA, where caveolin-1 is necessary to bring PKA into close proximity with perilipin, where it is released upon activation. Nevertheless, our results clearly indicate that caveolin-1 is necessary for PKA-mediated phosphorylation of perilipin.

In this study, we also identify a novel role for caveolin-1 in lipid droplet accumulation. Our findings indicate that perilipin-transfected caveolin-1 null fibroblasts show ∼4.5-fold less de novo lipid droplet accumulation than similarly transfected wild-type cells. Thus, caveolin-1 plays a dual role in lipid droplet metabolism, affecting both lipid droplet accumulation and breakdown. This dual role is consistent with our previous findings showing that caveolin-1 null mice are obesity resistant (28), and it suggests a mechanistic explanation for this phenotype. Overall, loss of caveolin-1 leads to a decrease in lipid accumulation and, thus, progressive white adipose tissue atrophy.

This work was supported by grants from the National Institutes of Health (NIH) and the Susan G. Komen Breast Cancer Foundation (to M.P.L); by NIH grant R01-DK55758 and the American Diabetes Association (to P.E.S.). A.W.C., P.I., and T.M.W. were supported by an NIH Medical Scientist Training Grant (T32-GM07288). M.P.L is the recipient of a Hirschl/Weil-Caulier Career Scientist Award.

The authors thank Dr. Charles S. Rubin, Albert Einstein College of Medicine, for his helpful discussions regarding PKA. They also thank the staff of the Analytical Imaging Facility, Albert Einstein College of Medicine, for their excellent intellectual and technical assistance.