Lipoprotein metabolism plays an important role in the development of several human diseases, including coronary artery disease and the metabolic syndrome. A good comprehension of the factors that regulate the metabolism of the various lipoproteins is therefore key to better understanding the variables associated with the development of these diseases. Among the players identified are regulators such as caveolins and caveolae. Caveolae are small plasma membrane invaginations that are observed in terminally differentiated cells. Their most important protein marker, caveolin-1, has been shown to play a key role in the regulation of several cellular signaling pathways and in the regulation of plasma lipoprotein metabolism. In the present paper, we have examined the role of caveolin-1 in lipoprotein metabolism using caveolin-1-deficient (Cav-1−/−) mice. Our data show that, while Cav-1−/− mice show increased plasma triglyceride levels, they also display reduced hepatic very low-density lipoprotein (VLDL) secretion. Additionally, we also found that a caveolin-1 deficiency is associated with an increase in high-density lipoprotein (HDL), and these HDL particles are enriched in cholesteryl ester in Cav-1−/− mice when compared with HDL obtained from wild-type mice. Finally, our data suggest that a caveolin-1 deficiency prevents the transcytosis of LDL across endothelial cells, and therefore, that caveolin-1 may be implicated in the regulation of plasma LDL levels. Taken together, our studies suggest that caveolin-1 plays an important role in the regulation of lipoprotein metabolism by controlling their plasma levels as well as their lipid composition. Thus caveolin-1 may also play an important role in the development of atherosclerosis.
- high-density lipoprotein
caveolae are 50- to 100-nm cell surface plasma membrane invaginations that are found at the surface of terminally differentiated cells (31). They play important roles in the regulation of endocytosis, transcytosis, and cell signaling as well as cholesterol homeostasis. Caveolae are defined as plasma membrane domains, characterized by particularly high cholesterol and sphingolipid contents. The later properties are shared by caveolae and lipid rafts. In fact, caveolae are a subset of lipid rafts but differ from the latter in that they contain the protein caveolin. Several isoforms of caveolin have been identified (5). Caveolin-1 is the most common isoform, and its function has been extensively studied (31). The role of this protein has been more clearly characterized since the development of caveolin-1-deficient (Cav-1−/−) mice (8, 34). In particular, the role of caveolin-1 in breast and prostate cancer has been well documented (29, 39).
Caveolin-1 has also been suggested to play an important role in the regulation of plasma lipoprotein metabolism (11, 14). Cav-1−/− mice were shown to present increased plasma triglyceride (TG) levels compared with wild-type (WT) mice (33). Caveolin-1 has also been suggested to play a role in the regulation of high-density lipoprotein (HDL) metabolism. In particular, our studies have shown that caveolin-1 can act as a negative regulator of the scavenger receptor class B type I (SR-BI)-mediated HDL-CE uptake (17). These data are in agreement with the work of Matveev et al. (24). Other studies, however, have indicated that caveolin-1 may not affect this pathway (42). Further work is therefore required to evaluate this point. Studies by Fielding and Fielding (11) have also proposed an important role for caveolae/caveolin-1 in the regulation of cellular cholesterol efflux to HDL. In this model, caveolae may be the primary site where cholesterol efflux occurs. However, we and others have been unsuccessful in finding corroborating evidence (12, 13, 19, 24, 42, 43).
In the present study, we have examined the role of caveolin-1 in the regulation of lipoprotein metabolism. For this purpose, we have used the well-characterized caveolin-1-deficient mouse model (34). Our data suggest a complex role for caveolin-1 in the regulation of lipoprotein and lipid metabolisms.
Antibodies and their sources were the following: anti-caveolin-1 IgG (mAb 2297; gift of Dr. Roberto Campos-Gonzalez, BD Biosciences, San Diego, CA) (36); rabbit anti-SR-BI and anti-ATP-binding cassette transporter 1 (ABCA1, Novus Biological, Littleton, CO); rabbit anti-CD36, (Cayman Chemical, Ann Arbor, Mi); mouse anti-LDL-related protein receptor (LRP) clone 8B8 (Biodesign International, Saco, ME); and guinea pig pAb anti-adipose differentiation-related protein (ADRP, Research Diagnostics, Flanders, NJ). All other reagents were analytical grade.
Cav-1−/− mice have previously been described (34). All animals used in these studies were backcrossed at least six times into the C57Bl/6J genetic background and were genotyped by PCR, as previously described (34). Mice were kept on a 12-h light-dark cycle and, on a normal chow diet (LabDiet, Richmond, IN). All animal protocols used in this study were preapproved by the Albert Einstein College of Medicine Institute for Animal Studies and Thomas Jefferson University. Experiments were performed in 3-mo-old male mice, unless otherwise indicated.
Plasma lipoprotein analysis.
Blood samples were collected into EDTA-containing tubes following a laparotomy and subsequent clipping of the descending aorta. Plasma (200 μl) was isolated and loaded onto one Superose 6 column (analytical grade, GE Healthcare Bio-Sciences, Piscataway, NJ) to achieve a total bed volume of ∼24 ml and void volume of 8 ml. Plasma was passed over the columns at a flow rate of 0.25 ml/min, and 0.5-ml fractions were collected. Total cholesterol and TG content of each fraction was determined and plotted against elution volume [Wako Chemicals (Richmond, VA) and Sigma-Aldrich (St. Louis, MO) colorimetric kits, respectively].
Measurement of hepatic VLDL production.
The measurement of hepatic very low-density lipoprotein (VLDL) production was carried out after blocking VLDL catabolism with Triton WR-1339, an inhibitor of plasma TG lipolysis (30). Baseline plasma TG levels were first determined. Mice were then injected with 15% Triton WR-1339 (Sigma-Aldrich) in 0.9% NaCl solution (0.5 g/kg body wt). Finally, blood samples were collected after injection and plasma TG levels were assayed.
Liver lipid content determination.
Livers from 3-mo-old WT and Cav-1−/− mice were collected after a 4-h fasting period and snap-frozen in liquid N2. After solubilization of the tissue, tissue lipids were extracted by the method of Bligh and Dyer (2). TG and cholesterol concentrations were determined using colorimetric assays for cholesterol and TG (Wako Chemicals and Sigma-Aldrich colorimetric kits, respectively).
Western blot analysis of liver proteins.
Livers were harvested from 3-mo-old WT and Cav-1−/− mice and solubilized in lysis buffer, as previously described (17). Equivalent amounts of protein were then separated by SDS-PAGE and transferred to nitrocellulose. The expression levels of caveolin-1, ABCA1, LRP, CD36, and ADRP were assessed using specific antibodies.
In vitro 125I-labeled LDL uptake by aortic rings.
Aortic segments from three WT and three Cav-1−/− mice (3-mo-old animals) were generated and cut into 2- to 4-mm segments and placed into 24-well plates. Three segments were used per time point and were incubated with 5 μCi of 125I-labeled LDL (Biomedical Technologies, Stoughton, MA) in 0.5 ml of aerated serum-free DMEM for 15 min at 37°C. One set of aortic segments from both genotypes was also incubated at 4°C for 15 min. All samples were then washed four times in 0.2 M acetic acid and 0.5 M NaCl buffer, pH 2.5, to remove any 125I-labeled LDL that remained attached to the cell surface. The amount of radioactivity was determined for each set of aortic segments individually using an LKB 1282 CompuGamma scintillation counter. The samples were then dried and weighed. The final value for each sample was obtained by dividing the counts per minute value by the dry weight.
In vivo LDL clearance and tissue uptake.
One hundred microliters of 125I-labeled LDL (15 μCi diluted in a solution of 0.9% NaCl) were introduced into mice (3-mo-old Cav-1−/− and WT females) via tail vein injection. Blood samples were then collected from the tail at 2 and 45 min, and 2, 5, 7, and 24 h postinjection. Plasma was then isolated by centrifuging the blood at 6,000 rpm for 6 min at 4°C. The amount of TCA-precipitable radioactivity present in 10 μl of plasma was determined using a gamma counter. The rate of clearance for each mouse was determined by using the number of counts per minute obtained from the 2-min time point as the starting point, i.e., 100%.
Discontinuous gradient density ultracentrifugation and sample analysis.
Plasma samples were subjected to discontinuous gradient density ultracentrifugation, as previously described (27). After ultracentrifugation, fractions (1 ml) were collected from the top to the bottom, yielding a total of 11 fractions. The densities of the fractions were determined and their lipid content was determined using colorimetric reagent (Wako Chemicals and Sigma-Aldrich colorimetric kits).
Values are reported as the means ± SD. Comparisons between control and Cav-1−/− mice were performed using the Student t-test when appropriate.
Lipoprotein metabolism in Cav-1−/− mice.
We examined plasma cholesterol, TG, and lipoprotein distribution in WT and Cav-1−/− mice (Tables 1 and 2, Fig. 1). We found that plasma TG and cholesterol levels were significantly increased in 3-mo-old Cav-1−/− mice (Table 1). In addition, this increase in plasma TG was due to an increase in plasma VLDL/CM levels (Fig. 1A) in all cases. Moreover, very similar results were obtained in older animals (1-yr-old, see Fig. 1B). We also observed that plasma HDL levels in 3-mo-old mice were higher in Cav-1−/− mice than those in WT mice.
Effect of caveolin-1 deficiency on hepatic TGs production.
To further examine the regulation of plasma TG in Cav-1−/− mice, we examined the rate of liver VLDL-TG production in WT and Cav-1−/− mice. This experiment was performed by injecting mice with Triton WR-1339 (Fig. 2). This compound has the property of inhibiting TG hydrolysis (3), and the accumulation of TG in the plasma of the injected mice will reflect the rate of VLDL production (mice are fasted for 4 h before injection to prevent chylomicron secretion) (3). In WT and Cav-1−/− mice, the concentration of plasma TG rises linearly for 4 h after the initial injection of the compound. Interestingly, despite higher plasma TG levels at the time of injection, Cav-1−/− mice displayed reduced plasma TG levels 4 h after injection. These data suggest that Cav-1−/− mice present reduced VLDL production compared with WT animals.
Caveolin-1 deficiency is associated with reduced hepatic TG but increased hepatic cholesterol content.
The liver plays an important role in the regulation of lipoprotein and lipid metabolisms. As a consequence, liver lipid metabolism is often altered in cases of dyslipoproteinemia. Therefore, we decided to examine liver TG and cholesterol content. We found that liver TG and total cholesterol content were altered in Cav-1−/− mice. However, whereas liver TG content was reduced in Cav-1−/− mice, liver total cholesterol content was increased (Fig. 3). These findings suggest that liver TG (decreased by 35%) and cholesterol (increased by 30%) metabolism are affected differently in Cav-1−/− mice. In addition, these data are also consistent with a role for caveolin-1 in the regulation of cellular TG and cholesterol metabolism.
Expression of key hepatic proteins involved in lipoprotein metabolism in Cav-1−/− and WT mice.
An important regulator of plasma cholesterol levels is ABCA1. A deficiency in ABCA1 (in animal models or natural mutations found in Tangier's disease) leads to markedly reduced plasma HDL cholesterol levels. We show that liver ABCA1 protein levels were increased in Cav-1−/− mice (Fig. 4). This modification in ABCA1 protein levels may be associated with the increase in liver cholesterol content (Fig. 3) since previous studies have shown a positive correlation between cellular cholesterol levels and ABCA1 protein expression levels (21).
We have also examined other proteins involved in the regulation of TG and apoB-containing lipoproteins. Earlier studies have shown that caveolin-1-deficient mouse embryonic fibroblasts cannot accumulate large amounts of TG (6). Two main proteins are responsible for the formation of a lipid droplet within a cell and therefore play an important role in the accumulation of cellular TG. ADRP (or adipophilin) is expressed in most tissues, including the liver (4, 22). Perilipin is the adipose tissue-specific isoform. We now show that, in Cav-1−/− mice, liver ADRP protein levels are 50% reduced compared with those of WT animals (Fig. 4). This finding is in agreement with previous studies showing a correlation between ADRP protein levels and TG levels in hepatocytes (23). This reduction is consistent with the reduced liver TG content observed in Cav-1−/− mice. Conversely, we also find that the levels of two proteins involved in the uptake of remnant lipoproteins are increased. These proteins are LRP and CD36 (Fig. 4). Interestingly, these two proteins have been localized into caveolae. Their mislocalization in Cav-1−/− hepatocytes and other cell types may affect lipoprotein uptake and, therefore, affect lipoprotein and TG metabolisms. LRP is an important receptor for LDL and remnant lipoproteins, and its increased expression levels may be associated with increased hepatic uptake of these lipoproteins. Moreover, as caveolin-1 and CD36 have been suggested to play a role in the intracellular transport of fatty acids, caveolin-1 deficiency appears to be associated with altered metabolism of cellular fatty acids that may indirectly affect plasma lipoprotein levels. In fact, plasma levels of non-esterified fatty acid (NEFA) were found to be increased in the postprandial state in Cav-1−/− mice (33).
Cav-1−/− mice show defects in the aortic uptake of LDL particles, both in vitro and in vivo.
Many electron microscopy studies have now established that endothelial caveolae (a.k.a., plasmalemmal vesicles) are involved in the transcytosis of macromolecules, such as albumin, LDL, and oxidized LDL, from the blood vessel lumen to the subendothelial space (18, 20, 41). In support of this notion, we recently demonstrated that caveolin-1-deficient endothelial cells are indeed defective in the uptake and transport of serum albumin (37). However, it remains unknown whether the uptake and transport of LDL is affected in Cav-1−/− mice. To test this hypothesis directly, we examined the ability of isolated aortic segments to take up 125I-labeled LDL at 37°C in vitro (Fig. 5). Our results indicate that loss of caveolin-1 reduces 125I-labeled LDL uptake by ∼45–50%. In contrast, in vitro binding of 125I-labeled LDL at 4°C was not affected.
To establish the in vivo relevance of these findings, we next evaluated the clearance of 125I-labeled LDL in Cav-1−/− and WT control female mice, using tail vein injections (Fig. 6A). Interestingly, Cav-1−/− mice exhibited a reduced initial rate of clearance when compared with that of WT control animals (See the 45-min time point; percent initial plasma value; 9.18 ± 0.98% vs. 18.53 ± 5.81%). However, at 2 h postinjection, no significant differences in 125I-labeled LDL plasma levels were noted.
Twenty-four hours postinjection, Cav-1−/− and WT mice were euthanized, and the tissue distribution of 125I-labeled LDL was determined. Figure 6B shows the distribution of 125I-labeled LDL in the various tissues examined. Note that in the aortas of Cav-1−/− mice, the uptake of 125I-labeled LDL was reduced by >50% (331,351.9 ± 128,763.6 cpm/g vs. 155,525.1 ± 31,089.4 cpm/g). In contrast, the livers of Cav-1−/− mice showed a >30% increase in the uptake of 125I-labeled LDL (1,481,041.2 ± 440,051.3 cpm/g vs. 2,075,403.8 ± 272,991.9 cpm/g). However, no significant differences were noted in the other tissues examined.
Caveolin-1 deficiency is associated with altered lipoprotein composition.
We also present data indicating that plasma obtained from Cav-1−/− mice shows increased HDL-cholesterol levels as observed after separation of lipoproteins by gradient density ultracentrifugation (Fig. 7A). In addition, we demonstrate that these HDL particles are enriched in esterified cholesterol (Fig. 7B) and to a lesser extent in free cholesterol (Fig. 7C). However, the ratio of phospholipids to total cholesterol (Fig. 7D) is not affected in Cav-1−/− mice. These data suggest a defect in the metabolism of plasma HDL in Cav-1−/− mice and that this defect does not appear to be due to reduced cholesterol efflux from peripheral cells, as we have shown previously. Rather, this effect may be due to a defect in HDL catabolism, presumably in the liver.
The present study was performed to determine the role of caveolin-1 in the regulation of lipoprotein metabolism. Our data show that caveolin-1 controls both production and degradation of plasma TG. As a consequence, caveolin-1 directly regulates hepatic lipid metabolism. Additionally, we show that caveolin-1 is involved in the regulation of plasma cholesterol metabolism. In agreement with its role in the regulation of SR-BI function, we now show that a caveolin-1 deficiency is associated with altered cholesteryl ester metabolism.
Role of caveolin-1 in the regulation of plasma TG metabolism.
Our data demonstrate that the increase in plasma TG levels observed in Cav-1−/− mice is not due to an increase in VLDL production but rather due to reduced degradation of plasma TG. Since post-heparin lipoprotein lipase and hepatic lipase activity are not affected in Cav-1−/− mice (33), it follows that the tissue uptake and catabolism of NEFA is reduced in these animals. This finding is in agreement with the increased plasma NEFA levels observed in the postprandial state of Cav-1−/− mice (33). Concerning the catabolism of VLDL, our results suggest a reduction in VLDL degradation in Cav-1−/− mice. One possible mechanism by which caveolin-1 may regulate VLDL degradation might be via its ability to regulate insulin signaling in adipose tissue. Early studies have shown that caveolin-1 can act as an activator of insulin receptor signaling (44). Alterations in the regulation of the insulin signaling pathway in adipose tissue have indeed been demonstrated in Cav-1−/− mice. In fact, Cav-1−/− mice have recently been shown to be unresponsive to insulin compared with WT mice (7). For the present work, this observation is important since it is well documented that insulin can regulate lipoprotein lipase (LPL) activity and its release from adipocytes (32, 40). However, in our previous studies, we did not observe a reduced LPL activity in Cav-1−/− mice (33). In addition, increased levels of nonesterified fatty acids were also observed in the postprandial state in Cav-1−/− mice (33). Taken together, these results suggest that caveolin-1 deficiency may be associated with a reduced fatty acid uptake by adipose tissue. In support of this hypothesis, Ring et al. (35) have shown that caveolin-1 regulates the function of CD36, a fatty acid translocase. Overall, these findings suggest that caveolin-1 must play a key role in the regulation of fatty acid metabolism.
Caveolin and liver lipid metabolism.
We show that the rate of VLDL production is reduced in Cav-1−/− mice (compare slopes for the increase in plasma TG in Fig. 2). We also show that liver TG levels are decreased in Cav-1−/− mice. Accordingly, this decrease is associated with reduced ADRP protein levels. Since VLDL secretion is dependent on the availability of hepatocyte TG (38), we can hypothesize that the decrease in cellular TG levels in Cav-1−/− hepatocytes leads to reduced VLDL production. This finding is reminiscent of the one that was made for adipose tissue in Cav-1−/− mice (33) and suggest that these mice have a defective ability to store TG in the liver and adipose tissue.
Our results also suggest that liver TG (decreased by 35%) and cholesterol (increased by 30%) metabolism are differently regulated in Cav-1−/− mice. These findings are consistent with a role for caveolin-1 in the regulation of cellular TG and cholesterol metabolism, as previously suggested (12, 33). We have already shown that caveolin-1 deficiency is associated with reduced accumulation of cholesteryl ester in mouse embryonic fibroblasts and in macrophages (12). In this previous paper, we found that caveolin-1 is an important regulator of intracellular cholesterol metabolism, and the present study confirms this finding. This result may have important consequences as cellular cholesterol regulates important cellular signaling pathways and could have important impact on the development of various diseases. In addition, increase hepatic cholesterol levels may also be associated with the increased ABCA1 that we observed in the liver. As a consequence, a small increase in plasma HDL levels was observed in Cav-1−/− mice.
Liver regeneration studies have suggested an important role for caveolin-1 in the process (10, 15, 26). It was shown that caveolin-1 expression is upregulated after partial hepatectomy, and it participates in the regulation of fatty acid metabolism in hepatocytes, where it associates with lipid droplets. Therefore, the decreased TG levels observed in caveolin-1-deficient liver is consistent with these findings. The increased expression of CD36 that we observed in Cav-1−/− liver may be a compensatory mechanism to improve defective fatty acid uptake by this organ (9). In this model, CD36 may not be functional, because previous studies have shown that caveolin-1 is required for CD36 function using mouse embryonic fibroblasts (35). In agreement with this interpretation, CD36−/− and Cav-1−/− mice present very similar lipoprotein profiles and metabolisms (9). It is also important to note that CD36 deficiency has been associated with insulin resistance, defective fatty acid metabolism, and hypertriglyceridemia (1).
Caveolin-1 and atherosclerosis.
How does loss of caveolae and caveolin-1 protect against the development of atherosclerosis? One possibility is that loss of endothelial cell caveolae prevents the transcytotic movement of LDL particles from the blood to the subendothelial space, where they become trapped and accumulate. Here, we show that caveolin-1 deficiency is associated with a change in the clearance and the metabolism of LDL lipoproteins. This finding is important, because the transcytosis of LDL plays a critical role in the development of atherosclerosis. In fact, the transcytosis of LDL may lead to the accumulation of lipids in blood vessels and may accelerate the inflammation process that is a key step in the development of atherosclerosis. We have previously shown that, in the apolipoprotein E-deficient (apoE−/−) background, Cav-1−/− mice presented remarkable increases in the level of plasma apoB-containing lipoproteins. Our current data suggest that the clearance of LDL is reduced in Cav-1−/− mice, and, as a consequence, it may prevent the development of atherosclerosis in apoE−/− mice.
Taken together, our data indicate that caveolin-1 deficiency leads to reduced aortic uptake or accumulation of pro-atherogenic lipoproteins, such as LDL. This is consistent with the notion that endothelial caveolae normally transport LDL particles from the blood vessel lumen to the subendothelial space.
Role of caveolin-1 in the regulation of HDL metabolism.
Several studies have now suggested that caveolin-1 may regulate plasma HDL-cholesterol metabolism (17, 28). In fact, caveolin-1 may be involved in the regulation of key proteins implicated in lipoprotein metabolism. Some of the best targets include SR-BI. We and others (16, 24) have shown that caveolin-1 may negatively regulate selective HDL-CE uptake mediated by SR-BI. Whereas some authors have suggested that caveolin-1 may facilitate this process (25), others have proposed that it had no effect (42). We have shown that Cav-1−/− mice present increased plasma levels of HDL-CE and therefore, caveolin-1 may facilitate, on the whole, the uptake of HDL-CE. We cannot rule out that caveolin-1 may have different effect depending on the cellular context. However, the observed increase in plasma HDL-cholesterol levels may also be related to the increased hepatic ABCA1 expression that is observed in Cav-1−/− mice (Fig. 4).
Taken together, our data suggest that caveolin-1 plays an important role in the regulation of TG and cholesterol homeostasis, as well as in the regulation of lipoprotein metabolism (Fig. 8). In adipose tissue, it promotes TG storage but also its mobilization. In blood vessels and possibly in atherosclerotic lesions, it may promote cholesterol accumulation via LDL transcytosis across endothelial cells. The latter property may account for the pro-atherogenic properties of caveolin-1. Concerning the metabolism of lipoprotein, caveolin-1 has a direct impact on the regulation of VLDL production and is also involved in the regulation of plasma HDL levels.
P. G. Frank was supported by grants from the Elsa U. Pardee Foundation, the Jane Barsumian/Mary Lyons Trust, and the W. W. Smith Trust Fund. This research was also supported in part by a Pilot and Feasibility Grant (to P. G. Frank) from the Liver Research Center at Albert Einstein College of Medicine (Bronx, NY). M. P. Lisanti was supported by grants from the National Institutes of Health and the American Heart Association.
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