PPARα, δ and FOXO1 Gene Silencing Overturns Palmitate-Induced Inhibition of Pyruvate Oxidation Differentially in C2C12 Myotubes

The molecular mechanisms by which free fatty acids (FFA) inhibit muscle glucose oxidation is still elusive. We recently showed that C2C12 myotubes treated with palmitate (PAL) presented with greater protein expression levels of PDK4 and transcription factors <i>PPARα</i> and <i>PPA...

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Autores principales:	Hung-Che Chien, Despina Constantin, Paul L. Greenhaff, Dumitru Constantin-Teodosiu
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	free fatty acids metabolic inflexibility muscle cell fuel selection Biology (General) QH301-705.5
Acceso en línea:	https://doaj.org/article/dd0e320f408a417c96cb06e994deeb33
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spelling	oai:doaj.org-article:dd0e320f408a417c96cb06e994deeb332021-11-25T16:46:58ZPPARα, δ and FOXO1 Gene Silencing Overturns Palmitate-Induced Inhibition of Pyruvate Oxidation Differentially in C2C12 Myotubes10.3390/biology101110982079-7737https://doaj.org/article/dd0e320f408a417c96cb06e994deeb332021-10-01T00:00:00Zhttps://www.mdpi.com/2079-7737/10/11/1098https://doaj.org/toc/2079-7737The molecular mechanisms by which free fatty acids (FFA) inhibit muscle glucose oxidation is still elusive. We recently showed that C2C12 myotubes treated with palmitate (PAL) presented with greater protein expression levels of PDK4 and transcription factors <i>PPARα</i> and <i>PPARδ</i> and lower <i>p</i>-<i>FOXO</i>/<i>t</i>-<i>FOXO</i> protein ratios when compared to control. This was complemented with the hallmarks of metabolic inflexibility (MI), i.e., reduced rates of glucose uptake, PDC activity and maximal pyruvate-derived ATP production rates (MAPR). However, the relative contribution of these transcription factors to the increase in PDK4 and reduced glucose oxidation could not be established. Therefore, by using a similar myotube model, a series of individual siRNA gene silencing experiments, validated at transcriptional and translation levels, were performed in conjunction with measurements of glucose uptake, PDC activity, MAPR and concentrations of metabolites reflecting PDC flux (lactate and acetylcarnitine). Gene silencing of <i>PPARα</i>, <i>δ</i> and <i>FOXO1</i> individually reduced PAL-mediated inhibition of PDC activity and increased glucose uptake, albeit by different mechanisms as only <i>PPARδ</i> and <i>FOXO1</i> silencing markedly reduced PDK4 protein content. Additionally, <i>PPARα</i> and <i>FOXO1</i> silencing, but not <i>PPARδ</i>, increased MAPR with PAL. <i>PPARδ</i> silencing also decreased FOXO1 protein. Since <i>FOXO1</i> silencing did not alter PPARδ protein, this suggests that <i>FOXO1</i> might be a <i>PPARδ</i> downstream target. In summary, this study suggests that the molecular mechanisms by which PAL reduces PDC-mediated glucose-derived pyruvate oxidation in muscle occur primarily through increased <i>PPARδ</i> and <i>FOXO1</i> mediated increases in PDK4 protein expression and secondarily through PPARα mediated allosteric inhibition of PDC flux. Furthermore, since <i>PPARδ</i> seems to control FOXO1 expression, this may reflect an important role for <i>PPARδ</i> in preventing glucose oxidation under conditions of increased lipid availability.Hung-Che ChienDespina ConstantinPaul L. GreenhaffDumitru Constantin-TeodosiuMDPI AGarticlefree fatty acidsmetabolic inflexibilitymuscle cellfuel selectionBiology (General)QH301-705.5ENBiology, Vol 10, Iss 1098, p 1098 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	free fatty acids metabolic inflexibility muscle cell fuel selection Biology (General) QH301-705.5
spellingShingle	free fatty acids metabolic inflexibility muscle cell fuel selection Biology (General) QH301-705.5 Hung-Che Chien Despina Constantin Paul L. Greenhaff Dumitru Constantin-Teodosiu PPARα, δ and FOXO1 Gene Silencing Overturns Palmitate-Induced Inhibition of Pyruvate Oxidation Differentially in C2C12 Myotubes
description	The molecular mechanisms by which free fatty acids (FFA) inhibit muscle glucose oxidation is still elusive. We recently showed that C2C12 myotubes treated with palmitate (PAL) presented with greater protein expression levels of PDK4 and transcription factors <i>PPARα</i> and <i>PPARδ</i> and lower <i>p</i>-<i>FOXO</i>/<i>t</i>-<i>FOXO</i> protein ratios when compared to control. This was complemented with the hallmarks of metabolic inflexibility (MI), i.e., reduced rates of glucose uptake, PDC activity and maximal pyruvate-derived ATP production rates (MAPR). However, the relative contribution of these transcription factors to the increase in PDK4 and reduced glucose oxidation could not be established. Therefore, by using a similar myotube model, a series of individual siRNA gene silencing experiments, validated at transcriptional and translation levels, were performed in conjunction with measurements of glucose uptake, PDC activity, MAPR and concentrations of metabolites reflecting PDC flux (lactate and acetylcarnitine). Gene silencing of <i>PPARα</i>, <i>δ</i> and <i>FOXO1</i> individually reduced PAL-mediated inhibition of PDC activity and increased glucose uptake, albeit by different mechanisms as only <i>PPARδ</i> and <i>FOXO1</i> silencing markedly reduced PDK4 protein content. Additionally, <i>PPARα</i> and <i>FOXO1</i> silencing, but not <i>PPARδ</i>, increased MAPR with PAL. <i>PPARδ</i> silencing also decreased FOXO1 protein. Since <i>FOXO1</i> silencing did not alter PPARδ protein, this suggests that <i>FOXO1</i> might be a <i>PPARδ</i> downstream target. In summary, this study suggests that the molecular mechanisms by which PAL reduces PDC-mediated glucose-derived pyruvate oxidation in muscle occur primarily through increased <i>PPARδ</i> and <i>FOXO1</i> mediated increases in PDK4 protein expression and secondarily through PPARα mediated allosteric inhibition of PDC flux. Furthermore, since <i>PPARδ</i> seems to control FOXO1 expression, this may reflect an important role for <i>PPARδ</i> in preventing glucose oxidation under conditions of increased lipid availability.
format	article
author	Hung-Che Chien Despina Constantin Paul L. Greenhaff Dumitru Constantin-Teodosiu
author_facet	Hung-Che Chien Despina Constantin Paul L. Greenhaff Dumitru Constantin-Teodosiu
author_sort	Hung-Che Chien
title	PPARα, δ and FOXO1 Gene Silencing Overturns Palmitate-Induced Inhibition of Pyruvate Oxidation Differentially in C2C12 Myotubes
title_short	PPARα, δ and FOXO1 Gene Silencing Overturns Palmitate-Induced Inhibition of Pyruvate Oxidation Differentially in C2C12 Myotubes
title_full	PPARα, δ and FOXO1 Gene Silencing Overturns Palmitate-Induced Inhibition of Pyruvate Oxidation Differentially in C2C12 Myotubes
title_fullStr	PPARα, δ and FOXO1 Gene Silencing Overturns Palmitate-Induced Inhibition of Pyruvate Oxidation Differentially in C2C12 Myotubes
title_full_unstemmed	PPARα, δ and FOXO1 Gene Silencing Overturns Palmitate-Induced Inhibition of Pyruvate Oxidation Differentially in C2C12 Myotubes
title_sort	pparα, δ and foxo1 gene silencing overturns palmitate-induced inhibition of pyruvate oxidation differentially in c2c12 myotubes
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/dd0e320f408a417c96cb06e994deeb33
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PPARα, δ and FOXO1 Gene Silencing Overturns Palmitate-Induced Inhibition of Pyruvate Oxidation Differentially in C2C12 Myotubes

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