Whole grain intake associated molecule 5-aminovaleric acid betaine decreases β-oxidation of fatty acids in mouse cardiomyocytes

Abstract

Despite epidemiological evidence showing that diets rich in whole grains reduce the risk of chronic life-style related diseases, biological mechanisms for these positive effects are mostly unknown. Increased 5-aminovaleric acid betaine (5-AVAB) levels in plasma and metabolically active tissues such as heart have been associated with consumption of diets rich in whole grains. However, biological effects of 5-AVAB are poorly understood. We evaluated 5-AVAB concentrations in human and mouse heart tissue (3-22 µM and 38-78 µM, respectively) using mass spectrometry. We show that 5-AVAB, at physiological concentration range, dose-dependently inhibits oxygen consumption due to β-oxidation of fatty acids, but does not otherwise compromise mitochondrial respiration, as measured with oxygen consumption rate in cultured mouse primary cardiomyocytes. We also demonstrate that this effect is caused by 5-AVAB induced reduction of cellular L-carnitine. Reduced L-carnitine levels are at least partly mediated by the inhibition of cell membrane carnitine transporter (OCTN2) as evaluated by in silico docking, and by siRNA mediated silencing of OCTN2 in cultured cardiomyocytes. 5-AVAB caused inhibition of β-oxidation of fatty acids is a novel mechanism on how diets rich in whole grains may regulate energy metabolism in the body. Elucidating potentially beneficial effects of 5-AVAB e.g. on cardiac physiology will require further in vivo investigations.

Figure 1

5-AVAB inhibits β-oxidation in cultured mouse cardiomyocytes. Effect of 5-AVAB treatment to mitochondrial respiration was assessed in cardiomyocytes. (a–d) After 24 h treatment in cardiomyocyte culture medium, which contains glucose as energy substrate, solution was changed to assay medium with palmitic acid as an energy source. (a) Treatment with different concentrations of 5-AVAB shows dose-dependent effect on palmitic acid oxidation as assessed by ratio of 6th and 1st measurement in individual assay wells (mean ± SEM). (b) This effect can be reversed by addition of glucose and pyruvate. (c) 5-AVAB has similar effect on the use of palmitic acid as meldonium, whereas glycine betaine shows no effect. (d) Addition of carnitine to the medium blocks the effect of 5-AVAB and meldonium. (e) In the mitochondrial stress test, none of the compounds had effect on the parameters of mitochondrial respiration in presence of glucose and pyruvate. CCM, cardiomyocyte culture medium; ETO, etomoxir; ANTI, antimycin A; MEL, meldonium; BET; glycine betaine; OLIGO, oligomycin; FCCP, carbonyl cyanide-4-trifluoromethoxy-phenylhydrazone. **p < 0.01; ***p < 0.001.

Figure 2

In silico docking of 5-AVAB to OCTN2. 5-AVAB docked into an in silico model of OCTN2 and predicted interactions between OCTN2 and 5-AVAB, L-carnitine and meldonium. TYR464, SER786 and GLN407 in the model correspond to TYR239, SER467 and GLN207 in the protein, respectively.

Figure 3

Effects of siRNA silencing of OCTN2 to intake of 5-AVAB. (a) Reduction of Slc22a5 expression (gene for OCTN2) in siRNA transfected neonatal cardiomyocytes (normalized to expression in the siCtrl (nonsense) transfected cells). (b) Silencing of OCTN2 decreased cellular intake of 5-AVAB in cultured mice cardiomyocytes when compared to control siRNA treated cells. (c,d) Silencing of OCTN2 reduces the L-carnitine and acetylcarnitine levels in mice cardiomyocytes similar to treatment with 100 µM 5-AVAB. *p < 0.05, **p < 0.01; ***p < 0.001.