Evaluation of the effects of L-carnitine on medaka (Oryzias latipes) fatty liver

Abstract

Lifestyle-related diseases have become a major issue in recent years. The increasing incidence of fatty liver underlines the urgency with which the issues of non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) need to be addressed. L-carnitine is a compound known to transport fatty acids into the mitochondria to enhance β-oxidation-mediated metabolism of fats. In this study, the effects of L-carnitine administration on fatty liver of medaka (Oryzias latipes) were analysed, to check for disease improvement and metabolic changes. Additionally, the effects of the concomitant administration of L-carnitine and eicosapentaenoic acid (20:5n-3) (EPA) were investigated. Findings indicated reduced lipid deposition, increase in metabolites associated with β-oxidation, and significant reduction in fatty acid levels in the liver, implying improvement in fatty liver condition. Concomitant administration of L-carnitine and EPA resulted in further benefits, via changes in fatty acid composition in the medaka fatty liver model.

Figure 1

Comparison of body weight, liver size, tissue staining, and protein expression between the HFD group and the HFD + L-carnitine group. (a) A schematic diagram of the methods of feeding and agent administration. Medakas (n = 20 in a tank) were fed either a normal diet or HFD and raised for 4 weeks. Following this period, 1 mM L-carnitine was administered, and for 2 weeks the animals were raised and the water was replaced every 3 days until the medakas were sacrificed for analysis. (b) Change in body weight following L-carnitine administration. There was no clear difference in body weight among the groups before L-carnitine administration. On Day 42, body weight significantly increased in HFD group compared to normal diet group, but there was no difference between the HFD group and the HFD + L-carnitine group. *Represents p < 0.05 (HFD group compared to normal diet group). n.s represents not significant (normal diet group compared to normal diet + carnitine group, HFD + carnitine group compared to HFD group). (c) A photograph of isolated livers on Day 42. The livers in the HFD group are significantly enlarged compared to those in the normal diet group. (d) Staining of liver sections on Day 42. Top: Haematoxylin and eosin staining. Bottom: Oil-red O staining. (e) The expression analysis of SOD2 by western blotting. The extracted protein samples from the liver in the HFD group (left five lanes) and the HFD + L-carnitine group (right five lanes) were subjected to electrophoresis. Top: anti-SOD2 antibody. Bottom: β-actin antibody (loading control). The corrected signal intensity of SOD2 compared with that of β-actin can be seen in the graph. SOD2 expression is significantly higher in the HFD + L-carnitine group.

Figure 2

Comparison of metabolites identified by CE-TOF-MS. (a) The metabolites are superimposed on a metabolite pathway map involving the metabolism of L-carnitine and ketone bodies. (b) The metabolites are superimposed on a metabolite pathway map involving the metabolism of glycolysis, TCA cycle and energy carriers. Blue: HFD group. Red: HFD + L-carnitine group. The metabolites showing a significant difference between groups were indicated with *<0.05.

Figure 3

GC-MS analysis of changes in lipid metabolism. (a) A schematic diagram of the methods of feeding and agent administration. Medakas (n = 15 in a tank) were fed HFD or HFD + EPA (HFD + 5 wt.% EPA) and raised for 4 weeks. Following this, 1 mM L-carnitine was administered and they were raised for 2 weeks while the water was replaced every 3 days. At the end of the period, they were sacrificed. (b) Change in body weight due to L-carnitine administration. There was no clear difference in body weight among the groups before L-carnitine administration. On Day 42, the body weight significantly increased in the normal diet group and HFD group, but there was no difference between the HFD group and HFD + L-carnitine group. *Represents p < 0.05. (c) Comparison of serum triglyceride level. **Represents p < 0.01 compared to HFD group. (d) Changes in gene expression of Srebf1, Acc1, and Lcad in the liver. Data are means ± SD. *Represents p < 0.05, **Represents p < 0.01 compared to HFD group. (e) Principal component analysis normalized fatty acid data obtained from medaka livers. Percentage values indicated on the axes represent the contribution rate of the first (PC1), second (PC2), and third (PC3). Every group was clearly separated.