Skeletal muscle perilipin 3 and coatomer proteins are increased following exercise and are associated with fat oxidation

Abstract

Lipid droplet-associated proteins such as perilipin 3 (PLIN3) and coatomer GTPase proteins (GBF1, ARF1, Sec23a, and ARFRP1) are expressed in skeletal muscle but little is known so far as to their regulation of lipolysis. We aimed here to explore the effects of lipolytic stimulation in vitro in primary human myotubes as well as in vivo following an acute exercise bout. In vitro lipolytic stimulation by epinephrine (100 μM) or by a lipolytic cocktail (30 μM palmitate, 4 μM forskolin, and 0.5 μM ionomycin, PFI) resulted in increases in PLIN3 protein content. Coatomer GTPases such as GBF1, ARF1, Sec23a, and ARFRP1 also increased in response to lipolytic stimuli. Furthermore, a long duration endurance exercise bout (20 males; age 24.0 ± 4.5 y; BMI 23.6 ± 1.8 kg/m(2)) increased PLIN3 protein in human skeletal muscle (p = 0.03) in proportion to ex vivo palmitate oxidation (r = 0.45, p = 0.04) and whole body in vivo fat oxidation (r = 0.52, p = 0.03). Protein content of ARF1 was increased (p = 0.04) while mRNA expression was increased for several other coatomers (GBF1, ARF1, and Sec23a, all p<0.05). These data provide novel observational insight into the possible relationships between lipolysis and PLIN3 along with these coatomoer GTPase proteins in human skeletal muscle.

Figure 1. Epinephrine and lipolytic cocktail (PFI) in human primary myotubes increases perilipin 3 and coatomer proteins.

A) Primary human skeletal muscle myotubes (n = 5) were stimulated with 100 uM of epinephrine. PLIN3 expression as assessed by densitometry increased continuously with epinephrine. Additionally, GTPases ARF1 and GBF1 increased 30 minutes following epinephrine stimulation and ARFRP1 increased 1 hour following epinephrine stimulation. B) PLIN3, ATGL ARFRP1, GBF1 and ARF1 protein content increased after in vitro lipolytic stimulus (PFI) in primary human skeletal muscle myotube (n = 5).

Figure 2. Coatomer GTPase gene expression changes with lipolytic cocktail (PFI) in human primary myotubes.

mRNA level of Sec23a, ARF1 and GBF1 in cultured human myotubes before and after PFI treatment (0, 30 min and 1 h) (n = 5). *p<0.05

Figure 3. Changes in protein and gene expression relating to lipolysis with a single bout of endurance exercise in human skeletal muscle.

A) Representative blots of PLIN3, ATGL, ARF1, ARFRP1 and loading control GAPDH. B) Quantitative bar graph of skeletal muscle PLIN3 protein after an acute exercise bout (n = 19). C) Quantitative bar graph of skeletal muscle ATGL protein after an acute exercise bout (n = 19). D) mRNA levels of lipid droplet coatomer genes (n = 19), and (E) mRNA levels of oxidative genes, in skeletal muscle of healthy subjects in response to an acute exercise bout (n = 14–19). *p<0.05.

Figure 4. Associations between change in perilipin 3 (PLIN3) protein in skeletal muscle tissue and fat oxidation.

A) Correlation between the change in PLIN3 protein expression and the change in ex vivo palmitate oxidation measured from skeletal muscle tissue homogenates (n = 18). B) Correlation between the percent change in PLIN3 protein expression and whole body cumulative fat oxidation measured by indirect calorimetry adjusted for fat free mass (n = 18). C) Correlation between the change in total glycogen content and the change in PLIN3 protein during exercise (n = 18).