PET imaging of fatty acid metabolism
Fatty acid (FA) is an aliphatic hydrocarbon chain terminating in a carboxylic acid, CH3-(CH2)n-COOH, containing usually an even number, either 16 or 18, of carbon atoms. In unsaturated fatty acids the carbon chain contains one or more double bonds. Long-chain fatty acids (LCFAs) have more than 12 carbon atoms, medium-chain fatty acids (MCFAs) have 6-12 carbon atoms, and short-chain fatty acids (SCFAs) have 2-5 carbon atoms. FAs with more than 8 carbons are practically insoluble in water. Non-esterified fatty acids (NEFAs), or free fatty acids (FFAs), are associated with proteins, such as albumin in the circulation and interstitial space, and fatty acid binding proteins (FABPs) inside the cells. Esterified FAs (triacylglycerides, TAGs) in blood are bound to lipoproteins (HDL, LDL). In the blood, albumin is the main transport protein for FFAs, containing at least six binding sites for FAs. Fatty acids can readily be transported across plasma membranes because of their lipophilicity, but concentration is small in spaces devoid of FA binding proteins, such as inside red blood cells. Unbound free fatty acids in the plasma would be excreted in urine. Lipoproteins transport esterified triacylglycerol (triglycerides) into tissue capillaries, where endothelial lipases release the fatty acids. Free fatty acids are then transported into cells via protein carrier mediated pathway, including fatty acid translocase (CD36), fatty acid transport proteins (FATPs), and the plasma membrane isoform of fatty acid binding protein (FABPpm).
Fatty acids serve as source of energy and in storage of energy. Fatty acids are important fuels particularly in the liver, myocardium, kidneys, and skeletal muscle. FAs serve as substrates for synthesis of phospho- and glycolipids and lipoproteins, and as precursors of eicosanoids such as prostaglandins and leukotrienes. Fatty acids as such as non-reactive, but by converting ATP to AMP the acyl-CoA synthetases (ACSs) can "activate" FAs by thioesterification to CoA. The high-energy thioesther bond in the resulting CH3-(CH2)n-CO-S-CoA enables the participation of FA in metabolic pathways.
Circulating NEFAs (FFAs) mainly come from hydrolysis of esterified FAs (TAGs) in adipose tissue (Campbell et al., 1992; Ferrannini et al., 1997). Elevated plasma concentration of NEFAs and TAGs is associated with metabolic syndrome.
Liver and adipose tissue are the main lipogenic tissues. Saturated very long chain fatty acids are produced in the lungs and unsaturated very long chain fatty acids in the spleen. The FAs synthesized in adipose tissue are stored locally as triacylglycerol (esterified FAs). FAs synthesized in the liver are mainly transported to other tissues in lipoproteins (VLDL).
FAs are synthesized from cytoplasmic acetyl-CoA, which is produced from pyruvate in mitochondria, transported from mitochondria as citrate, and converted back to acetyl-CoA. Plasma membrane citrate transporter can transport citrate from plasma for FA synthesis, especially in the liver and brain, and in some tumours. Acetyl-CoA carboxylase 1 (ACC1) converts acetyl-CoA to malonyl-CoA, and fatty acid synthase (FASN) complex adds repeatedly malonyl-CoA to acetyl-CoA or acylCoA, consuming NADH and releasing CO2 in each step. Phospholipids are synthesized in endoplasmic reticulum from FAs in most cells. Fatty acid synthase expression is increased in many tumours, and can be assessed using [1-11C]acetate (Vāvere et al., 2008) and 2-[18F]fluoropropionic acid ([18F]FPA) (Zhao et al., 2019; Zhang et al., 2019 and 2021).
Fatty acids are transported to tissues either as triacylglycerol (TGA) in lipoproteins or as albumin-bound non-esterified ("free") fatty acids (FFAs). ACSs convert fatty acids to acyl-CoAs in the outer mitochondrial membrane, and a transport system (CPT1, CACT, and CPT2) on the inner mitochondrial membrane transports acyl-CoA into the mitochondria, with CPT1 (carnitine palmitoyl transferase 1) as the rate-limiting and tightly regulated step. Acyl-CoA is shortened by two carbons at a time, producing NADH and FADH2 for the electron transport chain, and acetyl-CoA, which may be further degraded to water and CO2 in the tricarboxylic acid cycle. Muscle cells oxidise fatty acids completely, but hepatocytes typically uses acetyl-CoA in synthesis of other substrates, such as ketone bodies.
Peroxisomes can also β-oxidate fatty acids, also those FAs that cannot be used by mitochondria. Peroxisomal β-oxidation is required for synthesis of docosahexanoic acid (DHA) and bile acids.
Shortening of FAs by one carbon (α-oxidation) is necessary for example in case of branched fatty acids. In ω-oxidation the terminal -CH3 group is oxidized to carboxylic acid in the endoplasmic reticulum, and the resulting dicarboxylic acid is then further β-oxidated in peroxisomes.
Labelled thia-substituted long-chain fatty acid analogues, which enter the cells by the same mechanisms as natural fatty acids, but are are trapped, can be used to assess total fatty acid utilization, including both storage and β-oxidation. The first-generation radiotracer [18F]FTHA (DeGrado et al., 1991) is commonly used for this purpose. (Z)-18-[18F]fluoro-4-thia-octa-dec-9-enoate ([18F]FTO) was found to be more specific tracer for fatty acid oxidation (Pandey et al., 2018; DeGrado et al., 2010 and 2018). Murakami et al (2023) have developed 7-[18F]fuoro-4-thia-oleate (7-[18F]FTO, [18F]AS3504073-00); based on preliminary evaluation, 7-[18F]FTO is not defluorinated in vivo.
Plasma FFA flux
The FFA rate of appearance in circulation (Ra), or plasma FFA flux, can be measured using [1-11C]palmitate. Arterial plasma samples are collected during the PET study, and measured radioactivity concentrations are corrected for circulating metabolites, mainly [11C]CO2, to be used in the analysis of the PET data. The metabolite-corrected plasma curve can also be used to calculate the whole body clearance of [1-11C]palmitate. Since appearance rate equals disappearance rate, and assuming that the clearance of palmitate is representative of the overall FFA clearance, the FFA rate of appearance (in units µmol/min) can be calculated as a product of [1-11C]palmitate clearance and plasma FFA concentration (Rigazio et al., 2008). FFA rate of appearance can be further normalized to subject's body weight. However, the result is different from that obtained with continuous infusion of [U-13C]palmitate (Han et al., 2017). Uncontrolled extravasation of injected PET tracer would lead to overestimation of FFA flux.
Non-PET methods for measurement of plasma FFA flux are time-consuming as they are performed applying steady-state method. Continuous intravenous infusion of palmitate labelled with either radioactive or non-radioactive isotope (such as 14C, 13C, or 3H) leads to steady plasma radioactivity level. Plasma clearance can then be calculated as the ratio of the intravenous infusion rate and the concentration of tracer in plasma. Methods for non-steady-state measurement have also been developed, and used in combination with PET (Bucci et al., 2015).
TGA turnover in tissue
[1-11C]palmitate PET data, combined with 1H-MRS measurement of tissue lipids, can be used to quantify myocardial triacylglyceride turnover (Bucci et al., 2012).
Bucci M, Borra R, Någren K, Maggio R, Tuunanen H, Oikonen V, Del Ry S, Viljanen T, Taittonen M, Rigazio S, Giannessi D, Parkkola R, Knuuti J, Nuutila P, Iozzo P. Human obesity is characterized by defective fat storage and enhanced muscle fatty acid oxidation, and trimetazidine gradually counteracts these abnormalities. Am J Physiol Endocrinol Metab. 2011; 301(1): E105-E112. doi: 10.1152/ajpendo.00680.2010.
Bucci M, Borra R, Någren K, Pärkkä JP, Del Ry S, Maggio R, Tuunanen H, Viljanen T, Cabiati M, Rigazio S, Taittonen M, Pagotto U, Parkkola R, Opie LH, Nuutila P, Knuuti J, Iozzo P. Trimetazidine reduces endogenous free fatty acid oxidation and improves myocardial efficiency in obese humans. Cardiovasc Ther. 2012; 30(6): 333-341. doi: 10.1111/j.1755-5922.2011.00275.x.
Canfora EE, Jocken JW, Blaak EE. Short-chain fatty acids in control of body weight and insulin sensitivity. Nat Rev Endocrinol. 2015; 11: 577-591. doi: 10.1038/nrendo.2015.128.
Cooper DE, Young PA, Klett EL, Coleman RA. Physiological consequences of compartmentalized Acyl-CoA metabolism. J Biol Chem. 2015; 290(33): 20023-20031. doi: 10.1074/jbc.R115.663260.
Dubikovskaya E, Chudnovskiy R, Karateev G, Park HM, Stahl A. Measurement of long-chain fatty acid uptake into adipocytes. Methods Enzymol. 2014; 538: 107-134. doi: 10.1016/b978-0-12-800280-3.00007-4.
Glatz JFC, Luiken JJFP, Bonen A. Membrane fatty acid transporters as regulators of lipid metabolism: implications for metabolic disease. Physiol Rev. 2010; 90: 367-417. doi: 10.1152/physrev.00003.2009.
Hagenfeldt L. Renal excretion of free fatty acids. Clin Chim Acta 1971; 32: 471-474. doi: 10.1016/0009-8981(71)90450-5.
Lopaschuk GD, Ussher JR, Folmes CDL, Jaswal JS, Stanley WC. Myocardial fatty acid metabolism in health and disease. Physiol Rev. 2010; 90: 207-258. doi: 10.1152/physrev.00015.2009.
Rambold AS, Cohen S, Lippincott-Schwartz J. Fatty acid trafficking in starved cells: regulation by lipid droplet lipolysis, autophagy, and mitochondrial fusion dynamics. Developmental Cell 2015; 32: 678-691. doi: 10.1016/j.devcel.2015.01.029.
Rigazio S, Lehto HR, Tuunanen H, Någren K, Kankaanpaa M, Simi C, Borra R, Naum AG, Parkkola R, Knuuti J, Nuutila P, Iozzo P. The lowering of hepatic fatty acid uptake improves liver function and insulin sensitivity without affecting hepatic fat content in humans. Am J Physiol Endocrinol Metab. 2008; 295(2): E413-E419. doi: 10.1152/ajpendo.00744.2007.
Rutkowski JM, Stern JH, Scherer PE. The cell biology of fat expansion. J Cell Biol. 2015; 208(5): 501-512. doi: 10.1083/jcb.201409063
Santosa S, Jensen MD. The sexual dimorphism of lipid kinetics in humans. Front Endocrinol (Lausanne) 2015; 6:103. doi: 10.3389/fendo.2015.00103.
Zhang L, Keung W, Samokhvalov V, Wang W, Lopaschuk GD. Role of fatty acid uptake and fatty acid β-oxidation in mediating insulin resistance in heart and skeletal muscle. Biochim Biophys Acta 2010; 1801: 1-22. doi: 10.1016/j.bbalip.2009.09.014.
Ying Q, Chan DC, Barrett PHR, Watts GF. Unravelling lipoprotein metabolism with stable isotopes: tracing the flow. Metabolism 2021; 124: 154887. doi: 10.1016/j.metabol.2021.154887.
Tags: Fatty acids, Acetyl-CoA, Lipoproteins
Updated at: 2023-05-11
Created at: 2015-10-13
Written by: Vesa Oikonen