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

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.

with NASH are at risk of developing cirrhosis/liver cancer and new therapies must be developed to combat the illness. The efficacy of L-carnitine administration in the treatment of fatty liver has been reported in humans and mice. Although in some studies on the effects of L-carnitine added in the feed of relatively large edible fish, better fish growth and changes in lipid utilization were reported, no effect was observed in other such studies. Factors including administration period, amino acid intake, dosage, and carnitine palmitoyl transferase (CPT) activity are considered associated with these discrepancies 3,4 , suggesting that the possible use of the compound in this context is yet to be fully explored.
In recent years, small fish such as medakas and zebrafish, have gained attention as new model organisms. These fish are smaller than rodents such as mice, which reduces required space as well as cost. In addition, methods for preparing transgenic and knockout fish are well established based on the genome project. They also tend to grow and proliferate rapidly, and large-scale screening can easily be performed. Among small fish, especially, the medaka possesses an ability to hibernate, has high prolificacy, grows rapidly, is omnivorous and possesses carbohydrate/lipid metabolism functions similar to those found in mammals 5 .
Of significance to our study, a high-fat diet (HFD)-fed medaka model has been established 6,7 . It has previously been reported that drug assessment using a HFD-fed medaka model is feasible 8 . However, no studies of the effects of L-carnitine on fatty liver found in this model have been reported. In the present study, where metabolomic analysis and lipid analysis were performed, the effects of L-carnitine administration on fatty acid metamorphosis in the medaka model were first shown. In addition, since eicosapentaenoic acid (EPA) is known to decrease upon administration of L-carnitine, we analysed the change in lipid profile caused by concomitant administration of EPA with L-carnitine, regarding which only a few detailed reports have been found.

Results
The effects of L-carnitine on body weight, liver morphology and enzyme profile. Adult medakas were fed either a normal diet or an HFD for a month, followed by additional L-carnitine administration for a further 2 weeks (Fig. 1a). There was a significant increase in body weight in the HFD group compared to that in the normal diet group. No significant change in body weight was observed following L-carnitine administration in either group (Fig. 1b). The isolated medaka livers were whitish and enlarged in the HFD group. Macroscopically, no clear changes were detected following L-carnitine administration in either group (Fig. 1c). The hematoxylin and eosin (H&E) staining of liver sections revealed a greater deposition of lipid droplets in the normal diet group (Fig. 1d upper). Oil-red-O staining of liver sections revealed more lipid accumulation in the HFD group than in the normal diet group. Additionally, a decrease in lipid accumulation was observed following L-carnitine administration to the medaka in the HFD group (Fig. 1d lower). Furthermore, western blot analysis was performed and an increased expression of an antioxidant enzyme, superoxide dismutase 2 (SOD2) following L-carnitine administration, was seen (Fig. 1e).
Comparison of lipid metabolism. Since lipids could not be analysed in detail by capillary electrophoresis-time-of-flight/mass spectrometry (CE-TOF-MS), we performed gas chromatography-mass spectrometry (GC-MS) to analyse lipid molecular changes. We also analysed the effects of concomitant administration of L-carnitine and EPA. We also evaluated the effect of EPA on body weight by titrating EPA. After 2 months feeding of EPA, the body weight was decreased in 10% and 20% EPA administration groups ( Supplementary  Fig. S1). We examined the change in liver by HE staining and found that fat deposition in liver increased in all EPA administration groups. Medakas were divided into the HFD group, HFD + L-carnitine group, HFD + EPA group, and the HFD + EPA + carnitine group (number of fish was 15 in each group) to analyse changes in lipid metabolism in each of them (Fig. 3a). No significant changes in body weight were observed among the four groups (Fig. 3b). We also evaluated the triglyceride level in serum, and found that triglyceride level was greatly decreased in EPA and EPA + L-carnitine group (Fig. 3c). Furthermore we examined the mRNA expression level of fat related genes including sterol regulatory element-binding factor 1 (Srebf1), Acetyl-CoA carboxylase (Acc1), and long-chain acyl-CoA dehydrogenase (Lcad). Expressional level of these genes were decreased in EPA and EPA + L-carnitine group (Fig. 3d). We then evaluated fatty acid composition by GC-MS and separated the four groups by principal component analysis (PCA) (Fig. 3e). The lipid profiling of the HFD + L-carnitine group when compared to that of the HFD group showed significant decreases in pentadecanoate (15:0), margarate (17:0), linoleate (18:6n-6), ganma-linolenate (18:3n-6), and arachidonate (20:4n-6) ( Table 2). In contrast, the comparison of the lipid profiles between the HFD group and the HFD + EPA group showed significant increases in EPA, Docosahexaenoic acid (22:6n-3) (DHA), and docosapentaenoate (DPA) (22:5n-3), and significant decreases in stearate and arachidonate (20:4n-6) in the latter group (Table 3). Comparative analysis of lipid metabolism between the HFD + EPA + L-carnitine group and the HFD group showed a significant increase in EPA (EPA was Scientific RepoRts | 7: 2749 | DOI:10.1038/s41598-017-02924-5 below detection limits in the HFD group), DHA and DPA and a significant decrease in arachidonate in the former group (20:4n-6) ( Table 4). Other fatty acids, which were found to be decreased by L-carnitine administration, were not decreased by the concomitant administration of L-carnitine and EPA.

Discussion
The role of L-carnitine in weight reduction in humans and mouse models has been reported in a number of studies. In these, weight reduction was the result of a combination of L-carnitine administration and walking in overweight women 9 or L-carnitine administration for 14 days in a model involving endurance training 10 . In humans, most studies do not show a positive effect on the weight reduction facilitated by L-carnitine. In the present study also, there was no clear weight change in the HFD + L-carnitine group when compared with the HFD group, following 2 weeks of L-carnitine administration.
The improvement of fatty liver in humans and mice has been reported in several studies. Malaguarnera et al. reported an L-carnitine-mediated improvement in glucose plasma levels, lipid profiles, and histological manifestation in patients with NASH 11 . Xia et al. reported that L-carnitine supplements improved the fatty liver in type 2 diabetic mice, and that the increase in fatty acid oxidation and decrease in the L-carnitine/Actyl-L-carnitine ratio are important 12 . With regard to fish models, there is a report showing that L-carnitine administration in catfish resulted in a change in the lipid metabolism that caused lipid deposition in the liver and muscles 13 . In the current study, tissue staining showed a decrease in lipid deposition in the liver via L-carnitine administration. It was thereby demonstrated that L-carnitine administration may be useful in the improvement of fatty liver. In addition, as was previously reported, we observed an increase in the expression of SOD2, which is known to be involved in the mitochondrial oxidative stress pathway 14 . The result supports the idea that a suppression of oxidative stress in the liver by L-carnitine prevent NASH development.

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).     The metabolomic analysis, which was performed to assess the mechanisms of the improvement of fatty liver by L-carnitine in more detail, demonstrated a 4.4-fold increase in the amount of L-carnitine found in the liver following L-carnitine administration. A 1.7-fold increase in physiological L-carnitine levels was previously reported in a mouse model of NASH 15 . The reason for the greater increase in our study is presumably that excessive L-carnitine is excreted into the urine in mice, while in the medaka model, the compound was dissolved in the water in the tank, which allowed medakas to maintain higher L-carnitine levels. Increases in levels of the L-carnitine metabolites, butyrylcarnitine (ratio: 7.7) and O-acetylcarnitine (ratio: 14.0), and a decrease in the LC/ALC ratio were observed ( Table 2). In addition, although statistical significance could not be calculated for values below detection limits, acetyl-CoA and ATP levels can be considered to have increased upon L-carnitine administration, which suggests that β oxidation of lipids was enhanced to produce acetyl-CoA, resulting in ATP production (Table 1). There was an increase in ADP (ratio: 2.0) as well, but the ratio of the increase was higher in ATP (ratio: 3.4). That is, ATP was elevated more than ADP, as can be seen from the ATP/ADP ratio, which suggests that an increase in the adenine nucleotide pool activates the production of more ATP. The increase in ATP production by L-carnitine has also been reported in a study showing that in human primary chondrocytes, ATP production increased at 24 hours following 1 mM L-carnitine administration.
Among other metabolites that exhibited significant changes in the metabolomic analysis, spermidine (ratio: 1.3), a polyamine having strong anti-oxidant activity, acts as an intrinsic free radical scavenger. Spermidine is synthesized from spermine by spermidine/spermine N1-acetyltransferase using acetyl-CoA as a substrate.
An increase in dihydroxyacetone phosphate (DHAP) (ratio: 1.5) was also observed. Glycerin is converted to DHAP via glycerol-3-phosphate and enters the glycolytic pathway to be metabolized. An increase in DHAP consequently suggests advancement of lipolysis 16 .
There was a decrease in 3-methylhistidine (ratio: 0.6). Blood level of this molecule is reported to be useful as an index of protein catabolism 17 . The result suggests that because lipids are used as an energy source, the requirement for energy from protein catabolism is less.
Acetylated L-carnitine crosses the blood-brain barrier via membrane receptors to enter the brain and be converted into GABA. In fact, a study reported that L-carnitine acted to decrease ammonia levels in patients with liver cirrhosis and improved feelings of fatigue 11 .   Table 2. The comparison of fatty acid profiles of the HFD group and HFD + L-carnitine group. The relative areas of the peaks are shown. The ratio represents the values for the HFD + L-carnitine group to the values for the HFD group. A significant difference (p < 0.05) or (p < 0.01) is shown by one underline or two underlines. The ratio column is indicated by font that is underlined (increased; p < 0.05), bold (increased; p < 0.01), italic and underlined (decreased; p < 0.05) and italic and bold (decreased; p < 0.01).  Table 3. The comparison of fatty acid profiles of the HFD group and HFD + EPA group. The relative areas of the peaks are shown. The ratio represents the values for the HFD + EPA group to the values for the HFD group. A significant difference (p < 0.05) or (p < 0.01) is shown by one underline or two underlines. The ratio column is indicated by font that is underlined (increased; p < 0.05), bold (increased; p < 0.01), italic and underlined (decreased; p < 0.05) and italic and bold (decreased; p < 0.01).
2-Aminoadipic acid (2-AAA) (ratio: 0.7), a degradation product of lysine that has not been fully characterized, is a potential modulator of glucose homeostasis in humans. 2-AAA is an important marker in assessment of risk for development of diabetes in humans. A study reported that individuals with 2-AAA levels in the top quartile have a 4-fold risk of developing diabetes compared to the rest of the population 18 . Therefore, it is interesting that 2-AAA was decreased in the group with L-carnitine administration, as it suggested a possible decrease in risk for diabetes.
In the liver, acyl-CoA, generated from free fatty acids, is transferred into the mitochondria via the carnitine shuttle and is converted to acetyl-CoA, which is metabolized in the TCA cycle. Ketone bodies are known to be generated from acetyl-CoA when a large amount of acyl-CoA remains unmetabolised following the TCA cycle. However, no increase in ketone bodies was observed following L-carnitine administration in the current study (Fig. 3a). This suggests efficient transport of acyl-CoA into the mitochondria and subsequent metabolism.
Furthermore, a fact that has attracted attention in recent years is that dietary L-carnitine appears to cause the acceleration of atherosclerosis, owing to its conversion in the body to trimethylamine (TMA) and trimethylamine-N-oxide (TMAO) by enteric bacteria 19 . In the current study, neither TMA nor TMAO was found at elevated levels in either the HFD or HFD + L-carnitine group. The fact that TMA and TMAO were detected in the normal diet group suggested that the amount of TMA or TMAO synthesized in our medaka model in response to L-carnitine administration was small.
In general, L-carnitine is not required for short-and medium-chain fatty acids to enter the mitochondrial matrix as described before, but is necessary for long-chain fatty acids to do so. In order to analyse lipid profiles in more detail, we performed lipid analysis by GC-MS. Although the changes were not significant, most of the fatty acids measured generally decreased in the L-carnitine group. Linoleate (18:6n-6), margarate (17:0), ganma-linolenate (18:3n-6), arachidonate (20:4n-6), and pentadecanoate (15:0) levels were significantly lower in the HFD + L-carnitine group compared to those in the HFD group. This acyl-chain length preference probably depends on the substrate specificity of acyl-coenzyme A synthetase enzymes (ACSs;EC 6.2.1.x), the "activators" of fatty acids, which form a thioester with CoA 20 . However, much is still unknown about this mechanism and the details remain to be investigated. Further study on the detailed relationship between chain length of fatty acids and β oxidation of fatty acids by L-carnitine may be undertaken.
Although n-3 polyunsaturated fatty acids such as EPA and DHA are known to have anti-inflammatory and anti-cancer activity, their detailed mechanisms of action have not been fully understood. EPA is converted by an elongation enzyme to DPA, which is converted to DHA by a desaturation enzyme. Upon stimulation of the cells, the polyunsaturated fatty acid arachidonate (20:4n-6) is released from the cell membrane by phospholipase A2. Subsequently, prostaglandin and thromboxane generated by cyclooxygenase as well as leukotrienes generated by lipoxygenase from arachidonate (20:4n-6) function as lipid mediators. Since EPA and DHA have been shown  Table 4. Comparison of fatty acid profiles of the HFD group and HFD + EPA + L-carnitine group. The relative areas of the peaks are shown. The ratio represents the values for the HFD + EPA + L-carnitine group to the values for the HFD group. A significant difference (p < 0.05) or (p < 0.01) is shown by one underline or two underlines. The ratio column is indicated by font that is underlined (increased; p < 0.05), bold (increased; p < 0.01), italic and underlined (decreased; p < 0.05) and italic and bold (decreased; p < 0.01). n.d.: not detected, n.a.: not available.
to decrease in case of L-carnitine administration in Pagrus major 21 , we analysed the effects of the concomitant administration of L-carnitine and EPA. When EPA alone was administered, significant increases in fatty acids derived from EPA, DHA and DPA, and significant decreases in stearate and arachidonate (20:4n-6) were observed (Table 3). We also found that fat deposition in liver increased in each EPA groups (Supplemental Fig. S1), suggesting that most deposited fat is EPA derived fatty acid, and change in fatty acid composition is effective in improving fatty liver. There is a report showing that EPA alone increases mitochondrial fatty acid oxidation, while DHA increases peroxisomal fatty acid oxidation 22 . The results of the present study, however, showed that β oxidation of some fatty acids was not changed much by EPA administration, suggesting that EPA is less efficient in decreasing fatty acid levels than L-carnitine. In spite of this, decreases in omega-6 fatty acid levels were observed in our study, as previously reported, which support the usefulness of EPA administration. The analysis of lipid metabolism in case of concomitant administration of HFD, L-carnitine and EPA compared to that in case of HFD administration demonstrated a significant increase in EPA and DHA levels and a decrease in arachidonate levels (20:4n-6) ( Table 4). Other fatty acids that were decreased by L-carnitine administration were not decreased by concomitant administration of L-carnitine and EPA. This data indicates that elevated EPA prevents β oxidation of other fatty acids. A significant decrease in arachidonate levels (20:4n-6) may be the cause of anti-inflammatory effects. It may be that under L-carnitine administration, EPA was preferentially utilised for β oxidation over other fatty acids. The increase in omega 3 fatty acids, known to decrease liver fat based on reported clinical studies 23 , and decrease in arachidonate levels (20:4n-6) are the most important benefits of concomitant administration of EPA and L-carnitine. Only a few detailed studies on the effects of the concomitant administration of EPA and L-carnitine have been reported so far, but the important results from our study warrant future further investigation to assess potential human application. L-carnitine is a highly convenient agent, with no significant side effects in humans reported in clinical studies. Additionally, no side effects were observed in the medakas in our study. In conclusion, we evaluated the usefulness of L-carnitine using a medaka model of fatty liver. The current study showed that the administration of L-carnitine alone is effective in improving fatty liver, and that concomitant administration of L-carnitine and EPA elevates this effectiveness. The detailed metabolome-level effects of L-carnitine on lipid metabolism were analysed based on results obtained using a small fish, medaka, in the current study, and the findings are expected to be valuable in developing treatment for fatty liver-related diseases in the future.

Methods
Animals and reagents. An inbred medaka strain (Kyoto-Cab) was used in this study 24 . The protocol was approved by the Committee on the Ethics of Animal Experiments, University of Yamaguchi. All methods were performed in accordance with the relevant guidelines and regulations by the Committee on the Ethics of Animal Experiments, University of Yamaguchi. All surgery for medaka was performed under tricaine anaesthesia. All efforts were made to minimize their suffering during the course of this study. Medakas were sacrificed via overdose of anaesthesia. The proportions of protein, fat and carbohydrate, as well as the fatty acid compositions of the control HFD diets that were used in this study are elucidated in a previous report. The control diet (Hikari Crest) was purchased from Kyorin Co. Ltd, Hyogo, Japan. The HFD was purchased from CLEA Japan Inc., Tokyo, Japan. For EPA treatment, eicosapentaenoic acid (EPA, purity >99%; Mochida Pharmaceutical Co. Ltd, Tokyo, Japan) was mixed with HFD32 at a concentration of 5% by weight.
Western blot analysis. Protein lysates were obtained by homogenizing tissues or cell pellets in sample buffer containing 62.5 mM Tris-HCl (pH 6.8), 4% SDS, 200 mM dithiothreitol (DTT), 10% glycerol, and 0.001% bromophenol blue using a tissue to buffer ratio of 1:10 (w/v), followed by boiling. Western blot analysis was performed using purified polyclonal anti-human SOD2 rabbit IgG. Antibodies against β-actin (Sigma) were purchased from the indicated suppliers.
Histology. Euthanized fish were slit open from the anal vent to the gills, and the entire body was fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (Muto, Tokyo, Japan). The liver was dissected, dehydrated in alcohol, and embedded in paraffin according to standard procedures. Serial sections (3 μm thick) were cut and stained with hematoxylin and eosin (H&E). Intracellular lipids were stained with Oil Red O to analyse fat accumulation in the liver. Blood analysis. Blood samples were collected as described 8 . Fish were kept on ice for 1-2 minutes and then bled by cutting a ventral portion of the tail fin. Blood was collected in a microcapillary tube and the volume measured. Blood samples were kept at room temperature for 1 hour before centrifugation at 1200 × g for 30 minutes at 4 °C.TG contents were analysed using Fujifilm drychem 3500 (Japan).
Real-time RT-PCR. Livers were isolated and total RNA was extracted and purified using the RNeasy kit (Qiagen, Hilden, Germany). cDNAs were synthesized using purified RNA plus random hexamers and the Transcriptor First Strand cDNA synthesis kit (Roche, Indianapolis, IN). Quantitative real-time RT-PCR was performed as described, and primers sequences were written in the report 7 .

Metabolome analysis.
Approximately 50 mg of frozen tissue was plunged into 1500 µL of 50% acetonitrile/ Milli-Q water containing internal standards (Solution ID: 304-1002, Human Metabolome Technologies, Inc., Tsuruoka, Japan) at 0 °C in order to inactivate the enzymes present in the cells. The tissue was homogenized thrice at 1500 rpm for 120 s using a tissue homogenizer (Micro Smash MS100R, Tomy Digital Biology Co., Ltd., Tokyo, Japan) following which the homogenate was centrifuged at 2300 × g and 4 °C for 5 min. Subsequently, 800 µL of upper aqueous layer was centrifugally filtered through a Millipore 5-kDa cutoff filter at 9100 × g and 4 °C for 120 min to remove proteins. The filtrate was centrifugally concentrated and re-suspended in 50 µL of Milli-Q water for CE-MS analysis. Metabolome measurements were carried out through a facility service at Human Metabolome Technologies Inc., Tsuruoka, Japan.

GC-MS analysis.
After methanol was added, samples were shredded and subjected to sonication by adding an internal standard (C19 methylester). Chloroform was added and the chloroform phase was collected. After samples were dried by blowing a stream of nitrogen gas, hexane and then methanol were added and samples were incubated for 120 min. After water was added, the hexane phase was collected to use in GC-MS. We used the SHIMADZU QP-2010 Ultra and GC-MS Metabolic Components Database Ver. 2 software. In the statistical analysis, we applied data alignment by Fragment Align using the Pirouette software to perform PCA. The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Statistical Analysis.
To determine statistical significance, Welsh's two-factor t-tests were performed in ArrayStudio (Omicsoft) or "R" to compare protein-normalized data between experimental groups; P < 0.05 was considered significant.