Seasonal variations in photoperiod affect hepatic metabolism of medaka (Oryzias latipes)

Abstract

Organisms living in temperate regions are sensitive to seasonal variations in the environment; they are known to accumulate energy as fat in their livers during the winter when days are shorter, temperatures are lower, and food is scarce. However, the effect of variations in photoperiod alone on hepatic lipid metabolism has not been well studied. Therefore, in this study, we analyzed lipid metabolism in the liver of medaka, Oryzias latipes, while varying the length of days at constant temperature. Larger amounts of fatty acids accumulated in the liver after 14 days under short-day conditions than under long-day conditions. Metabolome analysis showed no accumulation of long-chain unsaturated fatty acids, but showed a significant accumulation of long-chain saturated fatty acids. Short-day conditions induced a reduction in the levels of succinate, fumarate, and malate in the tricarboxylic acid cycle, decreased expression of PPARα, and decreased accumulation of acylcarnitine, which suggested inhibition of lipolysis. In addition, transparent medaka fed on a high-fat diet under short-day conditions exhibited greater amounts of fat accumulation and developed fatty liver. The findings of our study will be useful for creating a medaka hepatic steatosis model for future studies of hepatic steatosis-related diseases.

Keywords: fatty liver; medaka; metabolome; photoperiod; tricarboxylic acid cycle.

Fig. 1

Photoperiod‐dependent changes in the liver of medaka fed with a normal diet. (A) Schematic illustration of experiments conducted on medaka reared under long‐day and short‐day conditions. (B) Changes in the medakas' body weight at day 14 after exposure to variations in photoperiod. Data are represented as mean ± SD (n = 17). NS indicates not significant, *Indicates significant difference (P ≤ 0.05) (Student’s t‐test). (C) Hematoxylin and eosin staining of medaka liver reared under long‐day and short‐day conditions. (D) Oil‐red‐O staining of medaka liver reared under long‐day and short‐day conditions. The bars indicate 100 µm.

Fig. 2

Metabolomic analysis of medaka liver. (A) Principal component analysis of normalized metabolic data obtained from the liver of medaka reared under long‐day and short‐day conditions. Percentage values indicated on the axes represent the contribution rate of the first (PC1) and second (PC2) principal components to the total amount of variation. Identical photoperiods are encircled by dotted lines of the same color. (B) Changes in metabolites involved in the ketone body. The vertical axis represents relative concentration. (C) Changes in metabolites involved in TCA cycle. (D) Changes in metabolites involved in the glycolytic pathway. The vertical axis represents relative concentration. The vertical axis represents relative concentration. NS indicates not significant. *Indicates significant difference (P ≤ 0.05) (Mann–Whitney U‐test). The error bars indicate SD (n = 3).

Fig. 3

Changes in gene expression related to glycolysis, TCA cycle, and fatty acid oxidation by real‐time RT–PCR. Real‐time PCR analysis of the gene expression was evaluated using six samples in each group. ef1α was used as internal control. gapdh: glyceraldehyde‐3‐phosphate dehydrogenase, ald: fructose 1,6‐bisphosphate aldolase, mdh: malate dehydrogenase, cs: citrate synthase, cpt1a: carnitine palmitoyltransferase 1, acox1: acyl‐CoA oxidase 1. Data are represented as mean ± SD. *Indicates significant difference (P ≤ 0.05) (Mann–Whitney U‐test), n.s indicates not significant. The error bars indicate SD (n = 6).

Fig. 4

Metabolomic changes involved in glutathione biosynthesis and taurine biosynthesis. (A) Changes in metabolites involved in glutathione biosynthesis. (B) Changes in metabolites involved in taurine biosynthesis. Data are represented as mean ± SD (n = 3). *indicates significant difference (P ≤ 0.05) (Mann–Whitney U‐test). Blue color indicates downregulated metabolites. Red color indicates upregulated metabolites. The error bars indicate SD (n = 3).

Fig. 5

Evaluation of hepatic steatosis using a transparent medaka model fed with a high‐fat diet. (A) Changes in the body weights of medaka on day 28 after exposure to variations in photoperiod. (B) WT medaka (cab strain). The liver is invisible. (C) T5 transparent medaka fed with a normal diet. The heart and the liver are visible. (D) Magnified photograph of the heart and liver of T5 transparent medaka fed with a HFD. (E) T5 transparent medaka on day 28 after exposure to variations of photoperiod. Left: long‐day; right: short‐day (F) HE‐stained image of the liver on day 28 after exposure to variations in photoperiod. (G) Oil‐red‐O‐stained image of the liver on day 28 after exposure to variations of photoperiod. Data are represented as mean ± SD. Statistical significance was calculated using t‐test. * indicates significant difference (P ≤ 0.05) (Student’s t‐test). The bars indicate 100 µm. The error bars indicate SD (n = 10).