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

Abstract

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

Fig. 1.

Caveolin-3 modulates transmembrane FA movement. A: Parental cells were transiently transfected with caveolin-3 (or not) as outlined in “Experimental Procedures.” Whole-cell extracts were prepared and subjected to SDS-PAGE as was a rat muscle cell membrane preparation. B: The proteins were transferred and subjected to Western blotting, and detection was carried out with HRP-conjugated secondary antibody and chemiluminescence. A representative trace for BCECF fluorescence is shown in both parental (P) and high caveolin-1 expressing (B8) cells. C: The data are presented as the average of triplicates from four independent experiments comparing fluorescence values after 600 s for caveolin-3 transfected (P/cav3) and untransfected cells (P) as well as B8 cells (* = P ≤ 0.003, P/Cav3 versus P). BCECF, 2′7′-bis-(2-carboxyethyl)-5-(and6)-carboxyfluorescein.

Fig. 2.

Fluorescence imaging of HEK293 cells demonstrates localization of probes, binding, and transmembrane movement of oleic acid. HEK293 cells were labeled with either FPE (top panel) or loaded with BCECF (bottom panel) prior to the imaging with a two-photon confocal microscope (see “Methods”). The probes were excited at 780 nm and images were captured under a 40× objective (oil immersion) at a pixel resolution of 512 × 512 using an image acquisition time of ∼10 s. Image of multiple cells in a field were obtained before the addition of oleate (left panels) and confirmed the correct localization of each probe. Single cells (center panels) were chosen to monitor the response of each fluorophore to oleate. Addition of 20 μM oleate (right panels) results in a decrease of fluorescence intensity due to fatty acid binding (FPE) and transmembrane diffusion (BCECF). In these two independent experiments (FPE and BCECF), the images were taken 4 min after the addition of oleate. All images were pseudo-colored using the Image J software. BCECF, 2′7′-bis-(2-carboxyethyl)-5-(and6)-carboxyfluorescein; FPE, fluorescein-phosphatidylethanolamine; OA, oleic acid.

Fig. 3.

The binding and transmembrane movement of fatty acids, but not fatty amines, is modulated by caveolins. The binding of oleic acids (left panels) and fatty amines (right panels) to the cell membrane was assessed by measuring the changes of FPE intensity upon addition of lipid to either parent (top panels) or B8 cells (bottom panels). For all cell types, addition of oleic acid or oleoylamine (20 μM) to a rapidly stirred suspension of cells results in an initial rapid fluorescence change representing the binding of each lipid to the plasma membrane surface. The addition of oleic acid causes a FPE fluorescence decrease while the addition of 20 μM oleoylamine results in a FPE fluorescence increase in both cell types. For both lipids, no secondary slow phase of fluorescence change was observed upon addition to parental cells. Only in the case of oleic acid addition to B8 was a significant fluorescence recovery observed, indicating the sequestering of oleate, but not fatty amines, at the inner leaflet by caveolin. FPE, fluorescein-phosphatidylethanolamine.

Fig. 4.

Caveolin expression enhances cellular FA accumulation. A: After 48 h with 80 μM oleic acid, cells were fixed and stained with Oil Red O as described in “Experimental Procedures.” B: Triglyceride accumulation was determined and normalized to total cellular protein. The results are from 8 cell preparations with P ≤ 0.001 for high caveolin-1 expressing cells (B8) and caveolin-3 transfected (P/ Cav3) versus untransfected (P) cells at 48 h. C: Lipid droplets were isolated as described by Liu et al. (17). After SDS-PAGE and transfer, they were immuno­blotted for the indicated proteins as in Fig 1. ADRP, adipose differentiation-related protein; OA, oleic acid; TG, triglyceride.

Fig. 5.

Caveolins protect cells from FA-induced lipotoxicity. Cells were supplemented with the indicated concentration of oleate bound to BSA in a 4:1 ratio for 48 h. Control wells were incubated in BSA alone. At the end of the incubation time, media was aspirated. Cell survival was assessed as described in “Experimental Procedures.” The data are expressed as % cell survival normalized to unsupplemented cells. The data are the mean ± SD from 3–4 independent experiments, each done in triplicate. P/Cav3 (≠) versus Parent, P ≤ 0.016, 0.8 mM FA, 0.16, P ≤ 0.002. Cav1/B8 (*) versus Parent, P ≤ 0.015 and 0.004, respectively. OA, oleic acid.