Perilipin 4 in human skeletal muscle: localization and effect of physical activity

Abstract

Perilipins (PLINs) coat the surface of lipid droplets and are important for the regulation of lipid turnover. Knowledge about the physiological role of the individual PLINs in skeletal muscle is limited although lipid metabolism is very important for muscle contraction. To determine the effect of long-term exercise on PLINs expression, 26 middle-aged, sedentary men underwent 12 weeks combined endurance and strength training intervention. Muscle biopsies from m. vastus lateralis and subcutaneous adipose tissue were taken before and after the intervention and total gene expression was measured with deep mRNA sequencing. PLIN4 mRNA exhibited the highest expression of all five PLINs in both tissues, and the expression was significantly reduced after long-term exercise in skeletal muscle. Moreover, PLIN4 mRNA expression levels in muscle correlated with the expression of genes involved in de novo phospholipid biosynthesis, with muscular content of phosphatidylethanolamine and phosphatidylcholine, and with the content of subsarcolemmal lipid droplets. The PLIN4 protein was mainly located at the periphery of skeletal muscle fibers, with higher levels in slow-twitch as compared to fast-twitch skeletal muscle fibers. In summary, we report reduced expression of PLIN4 after long-term physical activity, and preferential slow-twitch skeletal muscle fibers and plasma membrane-associated PLIN4 location.

Keywords: Endurance training; PLIN4; lipid droplets; phosphatidylcholine; phosphatidylethanolamine; sarcolemma; skeletal muscle fibre.

Figure 1

Gene expression of Perilipins (PLINs) in biopsies from m. vastus lateralis and adipose tissue before and after 12 weeks of training. mRNA expression was determined with high throughput sequencing and is shown as fragments per kilobase of transcript per million mapped reads (FPKM). (A) Skeletal muscle and (B) adipose tissue mRNA expression of the PLINs. Bars represent means ± SEM (n = 26 in muscle and n = 24 in adipose tissue). *P < 0.05, **P < 0.01, compared to values before training. Fold change (FC) = gene expression after 12 weeks of training/gene expression before the intervention.

Figure 2

Perilipin (PLIN)4 is close to the sarcolemma of skeletal muscle fibers whereas PLIN5 is spread uniformly. (A) Representative images (630X) from confocal fluorescence microscopy of semithin (5 μm) cross sections of paraffin-embedded human m. vastus lateralis biopsy (n = 10). Dystrophin (red, rabbit anti-dystrophin) binds to the outside of the sarcolemma, whereas PLIN4 (green, guinea pig anti-PLIN4) is located at or just beneath the sarcolemma. (B) The image (400X) was obtained by confocal fluorescence microscopy of sections of paraffin-embedded biopsies from m. vastus lateralis (n = 10), stained with antibodies against PLIN4 (red, rabbit anti-PLIN4) and PLIN5 (green, guinea pig anti-PLIN5).

Figure 3

Perilipin (PLIN)4 is associated with the membrane. (A) PLIN4 protein was analyzed by western blotting in different fractions (membrane/organelles, cytoskeleton, cytosol, and nuclear) from human muscle biopsies (n = 3). Each lane was loaded with 8 μg protein and in the membrane/organelles fraction a band was observed ∼ 120 kDa using a PLIN4-specific antibody (arrow). (B) It shows correlations between change in PLIN4 mRNA expression and change in liquid droplets (LDs) in different subcellular locations after 12 weeks of training. mRNA expression was determined with high throughput sequencing expressed as fragments per Kilobase per million fragments mapped (FPKM). The area of LDs was calculated from LD diameters using EM images. Bivariate correlation analysis was performed between change in PLIN4 mRNA expression levels and change in area of LDs in the SS (B) and IMF regions (C) after 12 weeks of training (n = 18).

Figure 4

Correlation analyses between the expression of PLIN4 and all other genes detected by mRNA sequencing in biopsies from m. vastus lateralis. The expression levels were correlated using Spearman’s test at before and after 12 weeks of training, and the change following long-term exercise (before 12 weeks – after 12 weeks). The Venn diagram shows the number of genes with correlation coefficients r > 0.5, and the overlapping areas represent the number of genes that are correlated in two or all data sets.

Figure 5

Perilipins (PLIN)4 is mostly expressed in slow muscle fibers. (A) Representative images (400X) from confocal fluorescence microscopy of sections of paraffin-embedded biopsies from m. vastus lateralis (n = 10), stained with antibodies against myosin heavy chain fast (red, mouse anti-myosin heavy chain; skeletal, fast) and PLIN4 (green, guinea pig anti-PLIN4). Fast muscle fibers are stained in bright red color, whereas slow fibers appear as dark. (B) M. soleus and m. gastrocnemius were dissected from male C57BL/6N mice (n = 5) and Plin4 mRNA expression was determined by quantitative RT-PCR. Plin4 mRNA was normalized to Tbp mRNA. Results are presented as means ± SEM, *P < 0.05 (two-tailed Student’s t-test).

Figure 6

Intramuscular adipocytes. (A) Sections of paraffin-embedded biopsies from m. vastus lateralis (n = 8) were stained with PLIN1 antibody (red, mouse anti-PLIN1) to detect adipocytes (A). Representative images (200X) were obtained by wide-field fluorescence microscopy. High magnification (1000X) of adipocytes is shown in the insert. (B) The sections were stained for PLIN4 (green, rabbit anti-PLIN4) and PLIN1 (red, mouse anti-PLIN1), showing partly co-localization in intramuscular adipocytes (n = 4). The merged image (200X) shows co-localization of the proteins (white arrow). The image was obtained by confocal fluorescence microscopy.