Alberici 2014 Abstract MiP2014

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Effects of long-chain acyl-CoA synthetase 6 knockdown in primary skeletal muscle cells metabolism.

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Teodoro BG, Prado IM, Sampaio IH, Silveira LR, Alberici LC (2014)

Event: MiP2014

When long-chain fatty acids enter the cells, long-chain acyl-CoA synthases (ACSLs) convert them to acyl-CoAs in an ATP-dependent reaction. The resulting acyl-CoAs have numerous metabolic routes within cells, including incorporation into triacylglycerol (TAG) and membrane phospholipids. Acyl-CoAs are used as substrates for beta-oxidation and protein acylation and function as ligands for transcription factors. However, the function of ACSL6 in skeletal muscle cells has not been described. The aim of this study was to investigate the effects of ACSL6 knockdown on mitochondrial metabolism in skeletal muscle cells. Isolation of primary rat skeletal muscle cells from the lower limb was performed by collagenase II digestion [1]. Knockdown of ACSL6 was made by siRNA specific transfection. After the knockdown, the cells were collected for the following experiments: mRNA expression (RT-PCR), MS-MS lipid analyzes, cell viability (flow cytometry), oxygen consumption (Oroboros Oxygraph-2k) [2] and reactive oxygen species (ROS) production (Amplex UltraRed). ACSL6 siRNA transfection (20 nM) reduced the expression of ACSL6 mRNA by 70±8%. ACSL6 knockdown increased the free fatty acids C16:0 and C18:0 by 32±3% and 35±3%, respectively. siRNA transfection did not affect cell viability measured by propide iodate. ACSL6 genic silencing increased mitochondrial respiration in all states [pmol O2∙s−1∙10−6 cells]: ROUTINE respiration (297±30 vs 368±28), LEAK with oligomycin (91±5 vs 96±4) and noncoupled ET-pathway (610±45 vs 703±41), and decreased ROS production (P<0.05). ACSL6 genic silencing increased mRNA expression of oxidative genes PGC1 (~50%), UCP2 (~3 fold) and UCP3 (~5 fold), decreased mRNA expression of ACSL3 and had no effect on ACSL1 and β-hydroxyacyl-CoA dehydrogenase (β-HAD). ACSL6 knockdown increased the availability of free fatty acids, which are major regulators of UCP’s. This may reflect the action of signaling pathways which remodel the oxidative program of skeletal muscle cells, increasing mitochondrial respiration. These mechanisms may contribute to control metabolic diseases, such as insulin resistance and obesity.


Labels: MiParea: Respiration, Genetic knockout;overexpression  Pathology: Diabetes, Obesity  Stress:Oxidative stress;RONS  Organism: Rat  Tissue;cell: Skeletal muscle  Preparation: Intact cells 

Regulation: Fatty acid  Coupling state: LEAK, ROUTINE, ET 

HRR: Oxygraph-2k  Event: C3, Poster  MiP2014 

Affiliation

1-Dep Physics Chem, Fac Pharm Sci; 2-School Phys Edu Sport; Univ Sao Paulo, Ribeirão Preto, Brazil. - alberici@fcfrp.usp.br

References and acknowledgements

Supported by FAPESP.

  1. Barbosa MR, Sampaio IH, Teodoro BG, Sousa TA, Zoppi CC, Queiroz AL, Oliveira MP, Alberici LC, Teixeira FR, Manfiolli AO, Batista TM, Gameiro Cappelli AP, Reis RI, Frasson D, Kettelhut IC, Parreiras-E-Silva LT, Costa-Neto CM, Carneiro EM, Curi R, Silveira LR (2013) Hydrogen peroxide production regulates the mitochondrial function in insulin resistant muscle cells: effect of catalase overexpression. Biochim Biophys Acta 1832: 1591-604.
  2. Pesta D, Gnaiger E (2012) High-resolution respirometry. OXPHOS protocols for human cells and permeabilized fibres from small biopisies of human muscle. Methods Mol Biol 810: 25-58.