AG-1024 br Material and methods br Results br
Material and methods
Conclusions To conclude, although CPT2 and CPT1 must have co-evolved to establish the carnitine shuttling, these enzymes are the most distantly related carnitine transferases, split early in evolution, during which a switch occurred in their location, from the mitochondrial matrix to the mitochondrial outer membrane and vice versa. ChAT is most closely related to CrAT and has evolved to a further extend than CrAT to gain its choline specific function. CPT1 has duplicated several times during evolution, resulting in the isoforms CPT1A, CPT1B and CPT1C, and, in C. elegans, in five extra CPT1-like genes. CPT1C is the AG-1024 specific isoform that resulted from positive and/or relaxed selection in the mammalian lineage. Our evolutionary delineation of the mammalian carnitine/choline acyltransferases fits current knowledge on their functions. It extends this knowledge and provides tangible leads for further experimental research into the functioning of this fascinating enzyme family with a key role in metabolism and signalling.
Acknowledgements This work is part of a research program, which is financed by the Province of Fryslân (01120657), the Netherlands and Alfasigma Nederland B.V (direct contribution to grant number 01120657).
Introduction Carnitine palmitoyltransferase 1 (CPT1) is a rate-limiting enzyme in mitochondrial β-oxidation of long-chain fatty acids (Mcgarry and Brown, 1997). Therefore, understanding of functional and regulatory properties of CPT1 is of importance. There are three genes encoding CPT1 protein with specific tissue distribution in mammals: a liver isoform (cpt1a) (Britton et al., 1995), a muscle isoform (cpt1b) (Yamazaki et al., 1996), and a brain isoform (cpt1c) (Price et al., 2002). Similarly, multiple genes of cpt1 were cloned in fish (Boukouvala et al., 2010; Morash et al., 2008, Morash et al., 2010; Shi et al., 2017; Wu et al., 2016; Zheng et al., 2013a, Zheng et al., 2013b). Effects of fasting on expression patterns of cpt1 have been well elucidated in fish (Boukouvala et al., 2010; Lu et al., 2016; Morash et al., 2009; Shi et al., 2017). These data showed that mRNA expression of different cpt1 genes was responsive to fasting, thus suggesting that each cpt1 gene may play a functional role in fatty acid β-oxidation. However, changes of cpt1 at a transcriptional level could not always reflect its function. For example, some of pseudogenes can be detected at a transcriptional level, but they are unable to produce protein products (Mighell et al., 2000). Further evidence should be necessary to confirm whether all cpt1 genes have the biological function. Regulation of CPT1 is complicated, possibly involving changes in enzymatic activity, protein kinetic property, and gene transcription. CPT1 activity, to some extent, depends on tissue carnitine availability because carnitine is a necessary substance for CPT1 catalytic activity. Carnitine plays an essential role in supporting optimal CPT1 activity (Lin and Odle, 2003). Studies reported that CPT1 activity increased with the increasing carnitine concentrations in the mitochondrial matrix CPT1 (Pande and Parvin, 1980). It has been well known that dietary l-carnitine supplements reduced the lipid content of liver and muscle in fish (Santulli and d'Amelio, 1986; Ozorio et al., 2001; Ma et al., 2008). The regulation of CPT1 activity relates change in the amount of the enzyme notably by modifying its expression level. Elucidation of the kinetic responsiveness of CPT1 to fasting will improve our understanding of CPT1 in fish. However, little is known about the responsiveness of carnitine status and CPT1 kinetics to fasting in fish. Fasting is one of the most important environmental stressors, which could influence lipid content and related enzymatic activities in fish (Araújo (Araújo et al., 2016; Kjær et al., 2009). Thus, it is possible starvation would affect the regulation of CPT1, however, these warrant investigation.