Nitrate enhances skeletal muscle fatty acid oxidation via a nitric oxide-cGMP-PPAR-mediated mechanism.

Tom Ashmore,James A West,Kieran Clarke,Martin Feelisch,Randall S Johnson,Bernadette O Fernandez,Andrew S Cowburn,Andrew J Murray,John Finnerty,Andrea J Morash,Steven A Murfitt,Cristina Branco,Lee D Roberts,Julian L Griffin,Aleksandra O Kotwica

doi:10.1186/s12915-015-0221-6

Abstract

BackgroundInsulin sensitivity in skeletal muscle is associated with metabolic flexibility, including a high capacity to increase fatty acid (FA) oxidation in response to increased lipid supply. Lipid overload, however, can result in incomplete FA oxidation and accumulation of potentially harmful intermediates where mitochondrial tricarboxylic acid cycle capacity cannot keep pace with rates of β-oxidation. Enhancement of muscle FA oxidation in combination with mitochondrial biogenesis is therefore emerging as a strategy to treat metabolic disease. Dietary inorganic nitrate was recently shown to reverse aspects of the metabolic syndrome in rodents by as yet incompletely defined mechanisms.ResultsHerein, we report that nitrate enhances skeletal muscle FA oxidation in rodents in a dose-dependent manner. We show that nitrate induces FA oxidation through a soluble guanylate cyclase (sGC)/cGMP-mediated PPARβ/δ- and PPARα-dependent mechanism. Enhanced PPARβ/δ and PPARα expression and DNA binding induces expression of FA oxidation enzymes, increasing muscle carnitine and lowering tissue malonyl-CoA concentrations, thereby supporting intra-mitochondrial pathways of FA oxidation and enhancing mitochondrial respiration. At higher doses, nitrate induces mitochondrial biogenesis, further increasing FA oxidation and lowering long-chain FA concentrations. Meanwhile, nitrate did not affect mitochondrial FA oxidation in PPARα−/− mice. In C2C12 myotubes, nitrate increased expression of the PPARα targets Cpt1b, Acadl, Hadh and Ucp3, and enhanced oxidative phosphorylation rates with palmitoyl-carnitine; however, these changes in gene expression and respiration were prevented by inhibition of either sGC or protein kinase G. Elevation of cGMP, via the inhibition of phosphodiesterase 5 by sildenafil, also increased expression of Cpt1b, Acadl and Ucp3, as well as CPT1B protein levels, and further enhanced the effect of nitrate supplementation.ConclusionsNitrate may therefore be effective in the treatment of metabolic disease by inducing FA oxidation in muscle.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-015-0221-6) contains supplementary material, which is available to authorized users.

Highlights

Insulin sensitivity in skeletal muscle is associated with metabolic flexibility, including a high capacity to increase fatty acid (FA) oxidation in response to increased lipid supply
Dietary nitrate counteracts the hypoxic suppression of PPARγ coactivator 1α (PGC-1α) in skeletal muscle and enhances FA oxidation in both normoxic and hypoxic skeletal muscle Since nitrate supplementation prevented the hypoxic suppression of FA oxidation in rat cardiac muscle [23], we investigated whether nitrate was protective in the skeletal muscle of rats exposed to hypoxic conditions (13 % O2) for 14 days compared with rats housed in a normoxic environment
Hypoxia decreased total NOx, and this was unaffected by dietary nitrate supplementation (Fig. 1a)

Summary

Introduction

Insulin sensitivity in skeletal muscle is associated with metabolic flexibility, including a high capacity to increase fatty acid (FA) oxidation in response to increased lipid supply. Can result in incomplete FA oxidation and accumulation of potentially harmful intermediates where mitochondrial tricarboxylic acid cycle capacity cannot keep pace with rates of β-oxidation. Enhancement of muscle FA oxidation in combination with mitochondrial biogenesis is emerging as a strategy to treat metabolic disease. Promotion of muscle FA oxidation emerged as a strategy for the treatment of insulin resistance [6], with the peroxisome proliferator-activated receptor (PPAR) transcription factors representing key targets [7]. In high-fat feeding models, enhanced PPAR target gene expression was associated with increased rates of incomplete FA oxidation and intra-mitochondrial accumulation of potentially harmful intermediates, e.g. acylCoAs and acyl-carnitines [11]. In mice lacking malonyl-CoA decarboxylase (MCD), decreased mitochondrial FA import and catabolism were associated with protection against glucose intolerance [11]

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