Hypoxia Induces Saturated Fatty Acids Accumulation and Reduces Unsaturated Fatty Acids Independently of Reverse Tricarboxylic Acid Cycle in L6 Myotubes

Abstract

Obstructive sleep apnea syndrome, characterized by repetitive episodes of tissue hypoxia, is associated with several metabolic impairments. Role of fatty acids and lipids attracts attention in its pathogenesis for their metabolic effects. Parallelly, hypoxia-induced activation of reverse tricarboxylic acid cycle (rTCA) with reductive glutamine metabolism provides precursor molecules for de novo lipogenesis. Gas-permeable cultureware was used to culture L6-myotubes in chronic hypoxia (12%, 4% and 1% O₂) with ¹³C labelled glutamine and inhibitors of glutamine uptake or rTCA-mediated lipogenesis. We investigated changes in lipidomic profile, ¹³C appearance in rTCA-related metabolites, gene and protein expression of rTCA-related proteins and glutamine transporters, glucose uptake and lactate production. Lipid content increased by 308% at 1% O_2, predominantly composed of saturated fatty acids, while triacylglyceroles containing unsaturated fatty acids and membrane lipids (phosphatidylcholines, phosphatidylethanolamines, phosphatidylinositol) decreased by 20-70%. rTCA labelling of malate, citrate and 2-hydroxyglutarate increased by 4.7-fold, 2.2-fold and 1.9-fold in 1% O₂, respectively. ATP-dependent citrate lyase inhibition in 1% O₂ decreased lipid amount by 23% and increased intensity of triacylglyceroles containing unsaturated fatty acids by 56-80%. Lactate production increased with hypoxia. Glucose uptake dropped by 75% with progression of hypoxia from 4% to 1% O₂. Protein expression remained unchanged. Altogether, hypoxia modified cell metabolism leading to lipid composition alteration and rTCA activation.

Keywords: L6 myotubes; glutamin; hypoxia; lipids; obstructive sleep apnea; reverse TCA.

Figure 1

The Effect of Hypoxia and ACLY Inhibitor on Intracellular Lipid Content. The effect of hypoxia and ACLY inhibitor (SB-204990) on lipid accumulation in cells. Differentiated L6 myotubes were exposed to hypoxia for 7 days, subsequently treated with 40 μM SB-204990 for 24 hours and lipid content data compared to respective vehicle-treated (DMSO) cells. N = 6; ^*p < 0.01 for comparison with control exposures (12% O₂, 2-way ANOVA); ^$p < 0.01 for comparison with 4% O₂ exposures (2-way ANOVA); ^#p < 0.05 for comparsion with vehicle treated cells (2-way ANOVA).

Figure 2

The Effect of Hypoxia on Lipidomic Profile. The effect of hypoxia on intracellular lipidomic profile based on ANOVA FDR p-value < 0.01 and PLS-DA VIP score > 1. Data are presented as HCA map with colorful visualization of log₂FC (Fold change) – (A). The effect of hypoxia and ATP-dependent citrate lyase inhibition (40 μM SB-204990) on clustering of cell samples in 1% O₂. Data are presented as PCA Score plot (B). Lipids with increased intensity with progression of hypoxia (C, p < 0.01). Lipids with “up and down” pattern (increased at 4_% O2, decreased at 1% O₂) (D, p < 0.01). Lipids affected by ACLY inhibitor at 1% O₂ (E, p < 0.01).

Figure 3

The Effect of Hypoxia on Incorportaion of 13C to rTCA Metabolites and Fatty Acids. The effect of hypoxia on quantity of metabolites generated in rTCA (A). Incoporation of ¹³C derived form 1-¹³C-labelled glutamine to metabolites generated in rTCA, subtracted from the effect of non-labelled glutamine (B) and percentage of ¹³C carbon derived from 5-¹³C-labelled glutamine in intracellular palmitate (C). ^*p < 0.05 for comparison with 12% O₂ (ANOVA, A - N = 3, B, C – N = 6); ^#p < 0.05 for comparsion with 4% O₂ exposures (ANOVA, A - N = 3, B, C – N = 6); ^$p < 0.05 for comparsion with DMSO - Control (ANOVA, N = 6).

Figure 4

The Effect of Hypoxia and rTCA Inhibitors on Glucose Uptake and Lactate Production. The effect of hypoxia (A), glutamine mitochondrial transport inhibitors (C) and ATP-dependent citrate lyase inhibition (D) on glucose uptake or lactate production (B). (A, B) Glucose uptake and 24-h lactate production was measured after exposure to hypoxia for 7 days. Differentiated L6 myotubes were exposed to hypoxia for 7 days and subsequently treated with 500 nM CB-839, 1 mM AOA, 2 μM BPTES (C) or 40 μM SB-204990 (D) for 24 hours. ^*p < 0.05 for comparison with control exposures (ANOVA for A, B or 2-way ANOVA for C, D, N = 6); ^#p < 0.05 for comparsion with 4% O₂ exposures (ANOVA, N = 6).

Figure 5

The Effect of Hypoxia on Expression of Genes and Proteins Involved in Glutamine Transport and Lipid Synthesis. The effect of hypoxia on gene (A) and protein (B) expression. Differentiated L6 myotubes were exposed to hypoxia for 7 days and gene and protein expression analysis was quantified. ^*p < 0.05 for comparison with control exposures (12% O₂, ANOVA, N = 6), ^#p < 0.05 for comparison with 4% O₂ exposures (ANOVA, N = 6).