Role of caveolin-1 in the regulation of lipoprotein metabolism

Abstract

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.

Fig. 1.

Lipoprotein profile observed in caveolin-1-deficient (Cav-1−/−) mice. Mice fed a chow diet. Fasting plasma samples isolated from 3 mice from each group [wild-type (WT) ▪, and Cav-1−/−, □] were pooled and loaded atop one Superose 6 column. Fractions were then collected and analyzed for cholesterol content. Profiles shown were obtained for 3-mo-old (A) or 1-yr-old (B) animals. VLDL, low-density lipoprotein; HDL, high-density lipoprotein; LDL, low-density lipoprotein; IDL, intermediate-density lipoprotein.

Fig. 2.

Effect of caveolin-1 deficiency on hepatic triglycerides (TGs) production. The measurement of hepatic VLDL production was carried out after blocking VLDL catabolism with Triton WR-1339, an inhibitor of plasma TG lipolysis (30). Baseline murine plasma TG were first determined. Mice were then injected with 15% Triton WR-1339 (Sigma-Aldrich, St. Louis, MO) in 0.9% NaCl solution (0.5 g/kg body wt). Blood samples were collected after injection and plasma TG were assayed. *Linear regression was performed and an F-test was used to compare slope values, which were found to be significantly different (P < 0.001).

Fig. 3.

Liver lipid content observed in 3-mo-old male WT and Cav-1−/− mice. Livers 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 cholesterol and TG assays. *Statistical significance (P < 0.05) and n = 5 for each group of animals.

Fig. 4.

Expression of key hepatic proteins involved in lipoprotein metabolism in Cav-1−/− and WT mice. Livers were harvested from 3-mo-old male WT and Cav-1−/− mice and solubilized. Equivalent amounts of total protein were then separated by SDS-PAGE and transferred to nitrocellulose. The expression levels of caveolin-1, ATP-binding cassette Transporter 1 (ABCA1), LDL-related protein receptor (LRP), CD36, and adipose differentiation-related protein (ADRP) were assessed using specific antibodies. Results with two representative animals are shown for each genotype (3-mo-old male Cav-1−/− and WT mice).

Fig. 5.

Cav-1−/− mice show defects in the aortic uptake of LDL particles in vitro. Aortic ring segments were collected from Cav-1−/− and WT control mice and incubated with ¹²⁵I-labeled LDL for a period of 15 min at either 37°C (left, for internalization) or 4°C (right, for binding only). Note that despite normal binding activity, caveolin-1-deficient aortic segments cannot internalize ¹²⁵I-labeled LDL as efficiently as aortic rings obtained from WT control animals. Thus our results indicate that loss of caveolin-1 reduces ¹²⁵I-labeled LDL uptake by ∼45–50%. *Statistical significance (P < 0.05).

Fig. 6.

Cav-1−/− mice show defects in the aortic uptake of LDL particles in vivo. A: LDL clearance in vivo. ¹²⁵I-labeled LDL was injected into the tail veins of 3-mo-old female mice (WT vs. Cav-1−/− animals). Note that Cav-1−/− mice exhibited a reduced initial rate of clearance when compared with WT control animals (See * at 45 min time point; % initial plasma value; 9.18 ± 0.98% vs. 18.53 ± 5.81%). However, at 2 h postinjection, no significant difference in ¹²⁵I-labeled LDL plasma levels was noted. B: LDL uptake in vivo. Twenty-four hours postinjection, WT and Cav-1−/− mice were euthanized, and the tissue distribution of ¹²⁵I-labeled LDL was determined. Note that in the aortas of Cav-1−/−, 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; See also the right inset). These data indicate that a caveolin-1 deficiency leads to reduced aortic uptake or accumulation of pro-atherogenic lipoproteins, such as LDL. In contrast, the livers of Cav-1−/− mice showed an 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). *Statistical significance (P < 0.05).

Fig. 7.

Lipid content of plasma lipoproteins isolated by gradient density ultracentrifugation. Fasting plasma samples were isolated from five WT and Cav-1−/− mice (2-mo-old male) and applied to discontinuous gradient density ultracentrifugation as described by McManus et al. (27). After ultracentrifugation, fractions (1 ml) were collected from the top to the bottom of the gradient, yielding a total of 12 fractions. The lipid content of each fraction was determined and plotted as a function of the fraction number. HDL-containing fractions were identified as fractions 5 and 6 (characterized by their density and apolipoprotein A-I content). Note that for plasma obtained from Cav-1−/− mice, the HDL fraction was especially enriched in esterified cholesterol (see B).

Fig. 8.

Role of caveolin-1 in the regulation of lipid and lipoprotein metabolism. Our results and those of others suggest that caveolin-1 plays various functions in different organs. 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.