BBA - Molecular and Cell Biology of Lipids 1866 (2021) 158887 Available online 14 January 2021 1388-1981/© 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Plasma 3-hydroxyisobutyrate (3-HIB) and methylmalonic acid (MMA) are markers of hepatic mitochondrial fatty acid oxidation in male Wistar rats Mona Synnøve Bjune a , Carine Lindquist a , Marit Hallvardsdotter Stafsnes b , Bodil Bjørndal a, 1 , Per Bruheim b , Thomas A. Aloysius a , Ottar Nygård a, c , Jon Skorve a , Lise Madsen d , Simon N. Dankel a, * , Rolf Kristian Berge a, c, * a Department of Clinical Science, University of Bergen, Bergen, Norway b Department of Biotechnology and Food Science, Faculty of Natural Sciences, NTNU Norwegian University of Science and Technology, N-7491 Trondheim, Norway c Department of Heart Disease, Haukeland University Hospital, Bergen, Norway d Institute of Marine Research, NO-5817 Bergen, Norway A R T I C L E INFO Keywords: BCAA 3-Hydroxisobutyrate Methylmalonic acid TCA cycle Fatty acid β-oxidation Insulin resistance ABSTRACT Objective: Discovery of specifc markers that refect altered hepatic fatty acid oxidation could help to detect an individual’s risk of fatty liver, type 2 diabetes and cardiovascular disease at an early stage. Lipid and protein metabolism are intimately linked, but our understanding of this crosstalk remains limited. Methods: In male Wistar rats, we used synthetic fatty acid analogues (3-thia fatty acids) as a tool to induce hepatic fatty acid oxidation and mitochondrial biogenesis, to gain new insight into the link between fatty acid oxidation, amino acid metabolism and TCA cycle-related intermediate metabolites in liver and plasma. Results: Rats treated with 3-thia fatty acids had 3-fold higher hepatic, but not adipose and skeletal muscle, expression of the thioesterase 3-hydroxyisobutyryl-CoA hydrolase (Hibch), which controls the formation of 3- hydroxyisobutyrate (3-HIB) in the valine degradation pathway. Consequently, 3-thia fatty acid-stimulated he- patic fatty acid oxidation and ketogenesis was accompanied by decreased plasma 3-HIB and increased methyl- malonic acid (MMA) concentrations further downstream in BCAA catabolism. The higher plasma MMA corresponded to higher MMA-CoA hydrolase activity and hepatic expression of GTP-specifc succinyl-CoA syn- thase (Suclg2) and succinate dehydrogenase (Sdhb), and lower MMA-CoA mutase activity. Plasma 3-HIB corre- lated positively to plasma and hepatic concentrations of TAG, plasma total fatty acids, plasma NEFA and insulin/ glucose ratio, while the reverse correlations were seen for MMA. Conclusion: Our study provides new insight into TCA cycle-related metabolic changes associated with altered hepatic fatty acid fux, and identifes 3-HIB and MMA as novel circulating markers refective of mitochondrial β-oxidation in male Wistar rats. 1. Introduction Altered mitochondrial function in liver, adipose and muscle tissue has been suggested to underlie the development of insulin resistance and increased risk of developing several diseases [1–4]. In situations of excess hepatic lipid uptake, an adaptive increase in mitochondrial oxidative capacity can prevent fatty liver and related conditions including insulin resistance and type 2 diabetes [5]. A better under- standing of the molecular mechanisms underlying reduced hepatic fatty acid oxidation could help identify individuals with developing insulin resistance and facilitate early prevention and treatment. Metabolomic studies have revealed an elevation of fasting branched- chain amino acid (BCAA) concentrations in the circulation of people with obesity, insulin resistance and type 2 diabetes [6–9]. Circulating BCAA and metabolite concentrations depend on the net metabolism of BCAAs in several different tissues including liver, skeletal muscle, pancreas and adipose tissue [10]. Insulin resistance is associated with reduced BCAA catabolism in adipocytes [11,12] and a concomitant in- crease in BCAA catabolism in skeletal muscle [10]. BCAAs provide substrates for the TCA cycle. Impaired oxidation of BCAAs may result in * Corresponding authors at: Department of Clinical Science, University of Bergen, PO Box 7804, 5020 Bergen, Norway. E-mail addresses: simon.dankel@uib.no (S.N. Dankel), rolf.berge@uib.no (R.K. Berge). 1 Present address: Department of Sport, Food and Natural Sciences, Western Norway University of Applied Sciences, 5020 Bergen, Norway. Contents lists available at ScienceDirect BBA - Molecular and Cell Biology of Lipids journal homepage: www.elsevier.com/locate/bbalip https://doi.org/10.1016/j.bbalip.2021.158887 Received 3 December 2020; Received in revised form 11 January 2021; Accepted 13 January 2021