In order to further evaluate cpt a function we determined
In order to further evaluate cpt1a function, we determined its tissue distribution in large yellow croaker. The mRNA levels of cpt1a were detected in all the tested tissues, but at varying levels. cpt1a mRNA expression was the highest in liver among different tissues, similar to reports in other fish species (Shi et al., 2017; Wu et al., 2016; Zheng et al., 2013a). Fish consume the stored lipids as a major source of metabolic energy when they are deprived of food. Hepatic lipid accumulation declined after fasting in large yellow croaker (Zheng et al., 2016a), red sea bream (Chatzifotis and Takeuchi, 1997), and rohu (Dar et al., 2018), in agreement with the present study. Considering the essential role of CPT1 in lipid metabolism, a molecular and enzymatic characterization of CPT1 in liver was conducted. This study is the first to show the alteration in the cpt1a mRNA levels, CPT1 activity, and CPT1 kinetics in fish as a response to starvation. In the present study, starvation up-regulated cpt1a mRNA expression in liver, in agreement with reports in other fish (Li et al., 2018; Lu et al., 2016; Shi et al., 2017). The present results indicated cpt1a could play an essential role in providing energy under fasting via fatty Atractyloside Dipotassium Salt β-oxidation. However, hepatic CPT1 activity was unchanged. Several studies reported that an increased mRNA expression of cpt1a was paralleled with an increase in its activity in fish exposed to starvation (Morash and McClelland, 2011; Zheng et al., 2013a). There is a time-lag effect between transcription and translation or protein modification changes enzyme kinetic properties (Zheng et al., 2013a). Km of CPT1 is a very useful index for evaluation of the substrate status in the tissues (Brown et al., 2001). In the present study, 4-day fasting significantly reduced Km values in liver of large yellow croaker. Carnitine availability is important for the optimal CPT1 activity. If free carnitine (FC) concentration in tissues becomes limiting, oxidation of fatty acids is markedly impaired (Bremer, 1983). Therefore, maintenance of tissue carnitine in support of CPT1 Km for optimal carnitine is an important part of regulation of CPT1 activity in mammals (Lin and Odle, 2003; Lyvers et al., 2007). To assess the metabolic demand for carnitine, we compared tissue FC concentrations to Km values of CPT1. In normal liver tissues of large yellow croaker, the FC concentrations are much lower than the respective Km, implying FC concentrations may be not enough to ensure the activity of CPT1. In fact, liver of a whitish colour that is characterized by increased hepatic lipid accumulation has commonly been observed in large yellow croaker fed on artificial food. Free carnitine concentration surpasses Km of CPT1 for carnitine in human skeletal muscle (Friolet et al., 1994), and in liver and skeletal muscle of the domestic felid (Lin et al., 2005). Interestingly, fasing for 4 days significantly increased the ratio of FC to Km and did not significantly affect catalytic capacity (Vmax), indicating that even low FC concentrations could support a high oxidative capacity of fatty acid. The present result was in agreement with the observed decrease in hepatic lipid content. Catalytic efficiency (Vmax/Km) relates total enzyme concentration to the interaction between the enzyme and the substrate. Our study showed that hepatic catalytic efficiency increased when fish fasted for 4 days. This result indicated that the fish had a high catalytic efficiency for fatty acid β-oxidation under short-term fasting. On the contrary, we found Vmax/Km and FC/Km values significantly declined during long-term fasting, perhaps involved in phosphorylation and dephosphorylation modifications of CPT1 that affect kinetic properties (Zheng et al., 2013a). Zheng et al. (2016a) suggested that long-term starvation induced membrane lipid peroxidation in large yellow croaker. Since CPT1 is a membrane protein, the oxidative damage may result in the reduced catalytic efficiency.