Perilipin 5, a lipid droplet-binding protein, protects heart from oxidative burden by sequestering fatty acid from excessive oxidation

Abstract

Lipid droplets (LDs) are ubiquitous organelles storing neutral lipids, including triacylglycerol (TAG) and cholesterol ester. The properties of LDs vary greatly among tissues, and LD-binding proteins, the perilipin family in particular, play critical roles in determining such diversity. Overaccumulation of TAG in LDs of non-adipose tissues may cause lipotoxicity, leading to diseases such as diabetes and cardiomyopathy. However, the physiological significance of non-adipose LDs in a normal state is poorly understood. To address this issue, we generated and characterized mice deficient in perilipin 5 (Plin5), a member of the perilipin family particularly abundant in the heart. The mutant mice lacked detectable LDs, containing significantly less TAG in the heart. Particulate structures containing another LD-binding protein, Plin2, but negative for lipid staining, remained in mutant mice hearts. LDs were recovered by perfusing the heart with an inhibitor of lipase. Cultured cardiomyocytes from Plin5-null mice more actively oxidized fatty acid than those of wild-type mice. Production of reactive oxygen species was increased in the mutant mice hearts, leading to a greater decline in heart function with age. This was, however, reduced by the administration of N-acetylcysteine, a precursor of an antioxidant, glutathione. Thus, we conclude that Plin5 is essential for maintaining LDs at detectable sizes in the heart, by antagonizing lipase(s). LDs in turn prevent excess reactive oxygen species production by sequestering fatty acid from oxidation and hence suppress oxidative burden to the heart.

FIGURE 1.

Generation of Plin5 knock-out mice. A, structures of wild-type and null Plin5 alleles. B, confirmation of Plin5 disruption by Southern blotting with genomic DNA prepared from the tail. Arrows indicate wild-type (WT) and knock-out (KO) bands corresponding to the fragments depicted in A. C, RT-PCR to reveal the absence of Plin5 mRNA in the hearts of Plin5^−/− mice. Plin4 mRNA was expressed at the normal level. D, confirmation of the absence of Plin5 protein in the hearts of Plin5^−/− mice. The graph on the right shows the Plin5/GAPDH ratios of mice of the three genotypes. ND, not detectable; ***, p < 0.001.

FIGURE 2.

Absence of LDs in the hearts of Plin5^−/− mice. A, ORO staining of heart cryosections of wild-type (panels a, b, e, and f) and Plin5^−/− (panels c, d, g, and h) mice in the fed (panels a–d) and fasted (panels e–h) states. Panels a, c, e, and g, low magnification (bar, 25 μm). Panels b, d, f, and h, high magnification (bar, 10 μm). No LD was observed even by close inspection of the sections of Plin5^−/− mice in either state. In wild-type mice, out of 20 randomly selected microscopic fields (each 86.4 × 60.5 μm), four in the fed state and all in the fasted state contained detectable LDs. The number of LDs in a field was 18.7 ± 11.0 in the fed mice and 966 ± 409 in the fasted mice, respectively (p = 4.3 × 10⁻⁹). The sizes of LDs were 0.22 ± 0.04 μm in the fed and 0.64 ± 0.11 μm in the fasted states, respectively (p = 2.3 × 10⁻⁷). B, representative electron microscopic images of the hearts of wild-type and Plin5^−/− mice fasted overnight. Left panels, low magnification (bar, 10 μm), and right panels, high magnification (bar, 2 μm). Note that LDs are observed as white ovals only in wild-type mice. C, ORO staining of soleus muscle cryosections. Panels a and b, wild-type, and panels c and d, Plin5^−/− mice, both fasted overnight. Panels a and c, low magnification. Bar, 100 μm. Panels b and d, high magnification of the area enclosed by a rectangle. Bar, 25 μm.

FIGURE 3.

TAG and FA contents in the tissues of fed and fasted wild-type and Plin5^−/− mice. A, TAG content in the heart (panel a), soleus muscle (panel b), liver (panel c), BAT (panel d), and inguinal WAT (panel e) (n = 5–8 per group). B, free FA content in the heart (panel a), soleus muscle (panel b), and liver (panel c) (n = 5–8 per group). Open bars, wild-type mice, and filled bars, Plin5^−/− mice. ***, p < 0.001; **, p < 0.01; *, p < 0.05; ###, p < 0.001; ##, p < 0.01; #, p < 0.05, against fed animals of the same genotypes.

FIGURE 4.

Energy metabolism of Plin5^−/− mice. A, food intake. Mice were housed individually for 10 days, and food intake per day was measured (n = 10 per group). B, oxygen consumption. C, respiratory quotient (n = 6 per group). D, locomotion of Plin5^−/− mice. Dot-plot demonstrates the time course of locomotor activity during 2 days. Each dot is the mean of locomotion of six mice. The graph on the right represents the total counts of locomotion during the experiment. *, p < 0.05.

FIGURE 5.

Subcellular fractionation of heart homogenates. A, TAG content in the fractions obtained by sucrose density gradient centrifugation of heart homogenates of wild-type and Plin5^−/− mice under fed and fasted conditions. Open bar, wild-type mice, and filled bar, Plin5^−/− mice. B, immunoblotting of Plin5 and Plin2 in the fractions of A. For fed animals, results of long exposure of the blots are also shown. Arrowhead, Plin5 and Plin2. Asterisk, nonspecific band, which was not recognized by another lot of anti-Plin5 antibody (Progen) raised against the same carboxyl-terminal epitope. C, protein contents in the same fractions. D, distribution of markers of mitochondria (cytochrome c oxidase subunit 3 (COX3)), endoplasmic reticulum (stearoyl-CoA desaturase 1 (SCD1)), and cytosol (glyceraldehyde-3-phosphate dehydrogenase (GAPDH)), and actin filament through the gradients. E, TAG content in the fractions after centrifugation through a wider sucrose-density gradient. Heart homogenates from fed wild-type (open bar) and Plin5^−/− (filled bar) mice were analyzed. Open circle, specific gravity (Sp. gr.) of each fraction. F, distribution of Plin5, Plin2, and marker proteins in the same fractions as in E. Calnexin was used as a marker of endoplasmic reticulum.

FIGURE 6.

Immunofluorescence staining of heart sections. A–D, immunofluorescence staining of Plin5 and Plin2 in heart sections from wild-type mice. A and C, fed mice; and B and D, starved mice. A and B, low magnification. Bar, 20 μm. C and D, high magnification. Bar, 5 μm. E–H, double staining with an anti-Plin5 antibody and Bodipy 493/503. Cryosections of the hearts of wild-type (E and F) and Plin5^−/− (G and H) mice under fed (E and G) and fasted (F and H) conditions were stained. I and J, immunostaining of Plin2 in Plin5^−/− mice under fed (I) and fasted (J) conditions. Bar, 5 μm.

FIGURE 7.

Expression of perilipin family proteins and lipases in the heart. A, immunoblotting of the proteins (three animals for each group). B, quantitative RT-PCR of mRNAs. Signal intensities were corrected based on that of Rplp0 (36B4) (n = 5 per group). Open bar, wild-type mice, and filled bar, Plin5^−/− mice. *, p < 0.05, against wild-type mice under the same feeding condition. Pnpla2 and Lipe denote the genes of ATGL and HSL, respectively.

FIGURE 8.

Plin5 maintains LDs by restricting lipases. A, decrease in heart lipase activities by the perfusion with BEL. Lipase activities were measured with the heart homogenates of mice perfused with saline or BEL (n = 3–4 per group). Assays were performed in the absence (open bar) or presence (filled bar) of 50 μm BEL. Relative enzyme activities taking the value of saline-treated wild-type mice measured without BEL (14.1 nmol of oleic acid released/h/mg of protein) as 1. *, p < 0.05; ##, p < 0.01; #, p < 0.05, against values obtained without BEL during the assay for the same genotypes of mice with the same treatment. B, effect of BEL perfusion on the occurrence of LDs in the hearts of wild-type and Plin5^−/− mice. Representative images of Nile Red-stained frozen sections are shown. Bar, 10 μm. Inset, high magnification view. Bar, 5 μm. Results were confirmed for several microscopic fields obtained from at least two animals for each group. C, effect of BEL on the protein content of Plin2 in the heart. Top, relative amount of Plin2 normalized with that of GAPDH in the heart lysates from the same animals as in A (n = 3), estimated from the result of immunoblotting (bottom). S, saline-treated, and B, BEL-treated. **, p < 0.01. D, effect of BEL on TAG content in the heart (n = 5–8 per group). ***, p < 0.001; **, p < 0.01, against saline-treated animals of the same genotype. ###, p < 0.001; #, p < 0.05, against wild-type mice with the same treatment. n. s., difference not significant.

FIGURE 9.

Lack of Plin5 promotes FA oxidation in cardiomyocytes. A, expression of perilipin family proteins and fatty acid oxidation enzymes in the hearts of wild-type mice before and after birth. VLCAD, very long-chain acyl-CoA dehydrogenase, and MCAD, medium-chain acyl-CoA dehydrogenase. B, expression of perilipin family proteins, lipases, and fatty acid-oxidizing enzymes in cardiomyocytes after the culture for 2 days in the presence of oleic acid (see “Experimental Procedures”). Cardiomyocytes prepared from the hearts of eight wild-type and Plin5^−/− mice 3 days after birth were seeded in three culture dishes, respectively. Arrowhead, correct band of Plin5 and HSL. *, nonspecific band. C–E, FA oxidation by cardiomyocytes as assessed by conversion of [¹⁴C]palmitic acid to CO₂ (C), intracellular ASM (D), and extracellular ASM (E). F, total incorporation of FA. Open bar, wild-type mice, and filled bar, Plin5^−/− mice. The assay was performed with or without etomoxir. n = 8–9 representing the number of independent experiments per group. In each experiment, values obtained with three culture dishes were averaged. ***, p < 0.001; **, p < 0.01; ##, p < 0.01; #, p < 0.05, against etomoxir-untreated cells of the same genotype.

FIGURE 10.

Plin5 deficiency enhances oxidative burden in the heart, causing a functional decline. A, evaluation of ROS generation by TBARS assay. Malondialdehyde (MDA) was quantified colorimetrically to monitor lipid peroxidation. Measurements were performed on the hearts from mice at 16–18 weeks (n = 9), 30–38 weeks (n = 10), and mice continuously treated with NAC from 16 to 18 weeks of age to the day of the experiment at 30–32 weeks of age (n = 5 for wild-type and 4 for Plin5^−/− mice, respectively). B, representative images of echocardiography in age-matched mice (16–18 or 30–38 weeks old). Long and short vertical lines indicate LVID;d and LVID;s, respectively. C, values of LVID;d (top panel), LVID;s (middle panel), and FS (bottom panel) were evaluated from images of echocardiography, at heart rates of 440–450 beats/min. n = 6 for both genotypes at 16–18 weeks, 23 and 16 for wild-type and Plin5^−/− mice at 30–38 weeks, respectively, and 5 and 4 for NAC-treated wild-type and Plin5^−/− mice, respectively. Open bar, wild-type mice, and filled bar, Plin5^−/− mice. ***, p < 0.001; **, p < 0.01; *, p < 0.05; ##, p < 0.01; #, p < 0.05, against the values of young mice of the same genotype. D, regression analysis of the correlations between the heart MDA content and LVID;d (top panel), LVID;s (middle panel), and FS (bottom panel). The first-order regression line was obtained by the least squares method from the data for individual mice. Open and closed circles, wild-type and Plin5^−/− mice at 30–38 weeks, and open and closed triangles, NAC-treated wild-type and Plin5^−/− mice, respectively.