Long-chain acyl-CoA synthetase 6 regulates lipid synthesis and mitochondrial oxidative capacity in human and rat skeletal muscle

Abstract

Key points: Long-chain acyl-CoA synthetase 6 (ACSL6) mRNA is present in human and rat skeletal muscle, and is modulated by nutritional status: exercise and fasting decrease ACSL6 mRNA, whereas acute lipid ingestion increase its expression. ACSL6 genic inhibition in rat primary myotubes decreased lipid accumulation, as well as activated the higher mitochondrial oxidative capacity programme and fatty acid oxidation through the AMPK/PGC1-α pathway. ACSL6 overexpression in human primary myotubes increased phospholipid species and decreased oxidative metabolism.

Abstract: Long-chain acyl-CoA synthetases (ACSL 1 to 6) are key enzymes regulating the partitioning of acyl-CoA species toward different metabolic fates such as lipid synthesis or β-oxidation. Despite our understanding of ecotopic lipid accumulation in skeletal muscle being associated with metabolic diseases such as obesity and type II diabetes, the role of specific ACSL isoforms in lipid synthesis remains unclear. In the present study, we describe for the first time the presence of ACSL6 mRNA in human skeletal muscle and the role that ACSL6 plays in lipid synthesis in both rodent and human skeletal muscle. ACSL6 mRNA was observed to be up-regulated by acute high-fat meal ingestion in both rodents and humans. In rats, we also demonstrated that fasting and chronic aerobic training negatively modulated the ACSL6 mRNA and other genes of lipid synthesis. Similar results were obtained following ACSL6 knockdown in rat myotubes, which was associated with a decreased accumulation of TAGs and lipid droplets. Under the same knockdown condition, we further demonstrate an increase in fatty acid content, p-AMPK, mitochondrial content, mitochondrial respiratory rates and palmitate oxidation. These results were associated with increased PGC-1α, UCP2 and UCP3 mRNA and decreased reactive oxygen species production. In human myotubes, ACSL6 overexpression reduced palmitate oxidation and PGC-1α mRNA. In conclusion, ACSL6 drives acyl-CoA toward lipid synthesis and its downregulation improves mitochondrial biogenesis, respiratory capacity and lipid oxidation. These outcomes are associated with the activation of the AMPK/PGC1-α pathway.

Keywords: ACSL6; human skeletal muscle; long-chain acyl-CoA synthetase; mitochondria; primary skeletal muscle cells; triacylglycerol synthesis; β-oxidation.

Figure 1. ACSL6, SREBP‐1c and DGAT‐1 mRNA expression at different metabolic conditions in skeletal muscle of rats and humans

Rat muscle ACSL6, SREBP‐1c and DGAT1 mRNA expression after 48 h of fasting (A) (n = 5); ACSL6 mRNA expression after 6 weeks of aerobic exercise (B) (n = 6); ACSL6 (C), SREBP‐1c (D) and DGAT1 (E) mRNA expression in a time course after acute lipid ingestion (n = 5); and ACSL6 mRNA expression after 6 weeks of a HFD (F) (n = 6). Lean and obese human muscle ACSL6 mRNA 4 h after acute ingestion of a high‐fat meal (G) and lean human ACSL6 mRNA 7 days after ingestion of a HFD (H) (n = 6 for lean and n = 5 for obese). ^*p < 0.05; ^**p < 0.01.

Figure 2. Effects of specific ACSL6 siRNA tritation on ACSL6 mRNA expression and cell viability in primary cells of rat skeletal muscle

ACSL6 mRNA at 20, 40 and 80 nm ACSL6 siRNA or scrambled siRNA (A) (n = 6), representative plots from flow cytometry acquisition (B) and percentage of cell viability (C) in cells at 30 nm ACSL6 siRNA, 10% dimethyl sulphoxide (necrose condition) or scramble (n = 6). ^* P < 0.05; ^** P < 0.01. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 3. Effects of ACSL6 knockdown in primary cells of rat skeletal muscle

Representative fluorescence microscopy images of LD staining using BODIPY 493/503 in scramble (left) and siRNA ACSL6 transfected (right) cells (A) (n = 3 independent culture and seven images of each culture); LD density per cell (B); I _LIP/I ₅₅₀ = absolute intensity of the ion peaks of lipids/absolute intensity of the ion peak of m/z 550, DAG, phosphatidylcholine (PC) and TAG (C) (n = 5); and I _LIP/I ₂₂₇ = absolute intensity of the ion peaks of lipids/absolute intensity of the ion peak of m/z 227, fatty acid (FA) (D) (n = 5) analysed by MS, mRNA expression DGAT1, DGAT2 and SREBP‐1c (E) (n = 6) in scramble and siRNA ACSL6 transfected cells. ^* P < 0.05; ^** P < 0.01. [Colour figure can be viewed at wileyonlinelibrary.com]

Effects of ACSL6 knockdown on oxidative metabolism in primary cells of rat skeletal muscle

Figure 4

Typical traces of O₂ consumption (A) and rates (pmol s⁻¹ mg protein⁻¹) (B) in ROUTINE (State R), LEAK (State L) and NONCOUPLED (State E) states by scramble and siRNA ACSL6 transfected cells. Where indicated, cells (10⁶), oligomycin (oligo, 1 μg ml^–1) and CCCP (2 μm) were added to the medium (n = 12). Immunoblotting (C) and blot densitometry (D) of p‐AMPK and AMPK (n = 4), mRNA expression of PGC1α, UCP2, UCP3, ACSL1, ACSL3 and HAD (E) (n = 6), citrate synthase activity (F) (n = 4), time‐scan (G) and rates (H) of H₂O₂ production (n = 6) and radiolabelled ^c14palmitate oxidation (I) (n = 4) in scramble and siRNA ACSL6 transfected cells.^* P < 0.05; ^** P < 0.01.

Figure 5. Efficiency of ACSL6 plasmid transfection in cells of human skeletal muscle

Representative fluorescence microscopy images of ACSL6/GFP transfection in myoblast (A, upper) and myotubes (A, lower), mRNA expression (B) in cells transfected with the ACSL6 plasmid or empty vector (n = 4 different cultures). ^* P < 0.05; ^** P < 0.01. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 6. Effects of ACSL6 overexpression in cells of human skeletal muscle

Representative fluorescence microscopy images of LD staining using BODIPY 493/503 in empty vector (left panel) and ACSL6 transfected (right panel) cells (A) and LD density (B) (n = 3 independent culture and seven images of each culture), relative ion abundances of phospholipids (725:901; 756:901 and 782:901) and TAGs (808:901; 881:901) analysed by MS in empty vector (upper) and ACSL6 transfected (lower) cells (C) and quantification (D) (n = 4), immunoblotting of DGAT1 (E) (n = 4), radiolabelled C¹⁴palmitate oxidation in the presence or not of carnitine palmitoyltransferase‐1 (CPT1) inhibitor etomoxir (F) (n = 6), and PGC1α mRNA expression (G) (n = 4) in empty vector and ACSL6 transfected cells. ^* P < 0.05; ^** P < 0.01. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 7. Representative diagram of ACSL6 mechanism

Under physiological conditions (A), ACSL1 drives acyl‐CoA to fatty acid oxidation (Li et al. 2015). We propose that ACSL6 drives acyl‐CoA toward lipid synthesis because physical exercise or fasting decreased mRNA ACSL6, whereas feeding increased its expression. Under ACSL6 knockdown (B), an increased content of free fatty acid could induce UCP expression. UCP activity could reduce ATP/ADP, resulting in the increased AMPK activation, which in turn increases mitochondrial biogenesis and fatty acid oxidation through PGC1α. Under ACSL6 overexpression (C), ACSL6 drives acyl‐CoA away from mitochondria, increasing phospholipid synthesis and decreasing PGC1α expression and fatty acid oxidation. Black arrows indicating an increase (↑) or decrease (↓) evaluated in the present study, whereas grey arrows indicate the proposed mechanisms. [Colour figure can be viewed at wileyonlinelibrary.com]