Fig 1.
Accumulation and distribution of oleic acid in myotubes when co-incubated with fatty acids.
Human myotubes were grown and differentiated in 96-well ScintiPlate or 12-well tissue culture plates. On day 6 of differentiation the myotubes were treated with a mixture of 100 μM fatty acids for 24 h. The mixture was trace amounts of [14C]OA (9 μM) and non-labeled PA (16:0) or EPA (20:5, n-3) (91 μM). (A) Cell-associated radioactivity was measured during 24 h by SPA. Real-time accumulation of radiolabel was monitored as described in Methods. Results represent mean ± SEM (nmol/mg protein) for n = 3 donors. Significant increase for EPA vs. PA (all-over effect). p<0.05 for EPA vs. PA, LMM statistical test (SPSS). (B-C) Lipids were separated by thin layer chromatography and quantified by liquid scintillation. Results are shown as mean ± SEM for absolute values, nmol/mg protein (B) and related to PA, % (C) from 4 individual experiments. *p<0.05 for EPA vs. PA (t-test). EPA, eicosapentaenoic acid; PA, palmitic acid; SPA, scintillation proximity assay; LMM, linear mixed model; FFA, free fatty acid; DAG, diacylglycerol; TAG, triacylglycerol; CE, cholesteryl ester; PL, phospholipids.
Fig 2.
Lipolysis and re-esterification of oleic acid in myotubes after co-incubation with fatty acids.
Human myotubes were grown and differentiated in 96-well ScintiPlate tissue culture plates. On day 6 of differentiation the myotubes were treated with a mixture of 100 μM fatty acids for 24 h. The mixture was trace amounts of [14C]OA (9 μM) and non-labeled PA or EPA (20:5, n-3) (91 μM). (A) Total lipolysis (lipolysis measured in presence triacsin C, 10 μM) of cell-associated [14C]OA at 1, 2, 4, and 6 h after 24 h pretreatment. (B) Total lipolysis presented as relative decline (i.e. data normalized to cell-associated radioactivity at zero time) in cell-associated [14C]OA at 1, 2, 4, and 6 h after 24 h pretreatment. (C) Re-esterification of [14C]OA, calculated as the difference between lipolysis measured at 1, 2, 4, and 6 h by SPA in the presence or absence of triacsin C (10 μM). Results represent mean ± SEM as nmol/mg protein (A, C) and relative decline in cell-associated radioactivity (B) for n = 3 donors. Significant increased lipolysis, decline and re-esterification for EPA vs. PA. p<0.05 for EPA vs. PA (all-over effect), LMM statistical test (SPSS). EPA, eicosapentaenoic acid; PA, palmitic acid; SPA, scintillation proximity assay; LMM, linear mixed model.
Fig 3.
Oleic acid accumulation and oxidation after preincubation with fatty acids.
(A) OA β-oxidation during co-incubation with fatty acids. Human myotubes were grown and differentiated in 12-well tissue culture plates. On day 6 of differentiation the myotubes were treated with a mixture of 100 μM fatty acids for 24 h. The mixture was trace amounts of [14C]OA (9 μM) and non-labeled PA (16:0) or EPA (20:5, n-3) (91 μM). Figure shows formation of acid-soluble metabolites (ASMs) in myotubes after 24 h. Results are shown as mean ± SEM (related to PA, %) from 4 individual experiments. Absolute values (mean ± SEM); PA: 24.5 ± 4.8; EPA: 41.3 ± 8.7 nmol/mg cell protein. (B) OA accumulation and complete oxidation after preincubation with fatty acids. Human myotubes were treated with fatty acids (100 μM EPA or PA) for 24 h. Thereafter, accumulation (cell-associated) and complete oxidation (CO2 production) from added [14C]OA (100 μM) was measured for 4 h. Results represent mean ± SEM, n = 3. Absolute values (mean ± SEM) for PA; CO2: 15.9 ± 2.3; Cell-associated: 49.9 ± 10.9 nmol/mg cell protein. *p<0.05 for EPA vs. PA (t-test). CA, cell-associated; EPA, eicosapentaenoic acid; PA, palmitic acid.
Fig 4.
Mitochondrial function after preincubation with fatty acids.
Human skeletal muscle cells were grown in 96-well Seahorse tissue culture plates, and treated with 100 μM of PA or EPA for 24 h, before measurement of oxygen consumption rate (OCR) with the Seahorse XF96e analyzer. (A) OCR time curve from an experiment with one of two donors. (B) OCR parameter calculations by the Seahorse XF Mito Stress Test Report Generator from experiments with two different donors (n = 8). Results represent mean ± SEM. *p<0.05 for EPA vs. PA (t-test). EPA, eicosapentaenoic acid; PA, palmitic acid; Oligo, oligomycin; FCCP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; Rot, rotenone, AA, antimycin A.
Fig 5.
Myotubes were incubated with PA or EPA (100 μM) for 24 h and then harvested for RNA isolation. Affymetrix human NuGO GeneChip arrays was used to measure gene expression and analysis performed in Partek Genomics Suite 6.6 software. GO enrichment analysis revealed that the GO terms”Neutral lipid metabolic process” (GO:0006638, upper 3) and”Regulation of lipid storage” (GO:0010883, lower 4) were changed significantly (p<0.05) by EPA vs. PA. They are presented with % of genes up- or down-regulated in each child GO term and genes are presented in S1 Table. EPA, eicosapentaenoic acid; PA, palmitic acid.
Fig 6.
Effect of DGAT1 inhibitor on cell-associated and accumulated oleic acid.
(A) Human myotubes were treated with fatty acids (100 μM EPA or PA) for 24 h. Cell-associated [14C]OA (100 μM) was measured after 4 h with and without DGAT1 inhibitor (D1i, A922500, 1 μM). Results represent mean ± SEM, from experiments with 4 different donors and 4–8 parallels (n = 26). Cell-associated PA (100%): 53.8 ± 4.0 nmol/mg cell protein. (B) Human myotubes were grown and differentiated in 96-well ScintiPlate tissue culture plates. On day 6 of differentiation the myotubes were treated with a mixture of 100 μM fatty acids for 24 h and cell-associated radioactivity was measured during 24 h by SPA. The mixture was trace amounts of [14C]OA (9 μM) and non-labeled PA or EPA (91 μM). DGAT1 inhibitor was also added for 24 h. Results represent mean ± SEM from experiments with 3 different donors and 8 parallels (n = 24). Accumulated PA (100%): 156 ± 13.8 nmol/mg cell protein. #p<0.05 for D1i inhibitor vs. control in EPA-treated cells (t-test). EPA, eicosapentaenoic acid; PA, palmitic acid; D1i, diacylglycerol acyltransferase 1 inhibitor.