Fish culture is conducted over fifty years in marine, brackish or freshwater. One of advantages of fish culture is the partial controllability of physiological conditions such as growth, lipid metabolism, body composition, stress responses and disease resistance. However, culture fish generally have low quality compared with wild fish. Actually lipid contents of culture fish are usually higher than those of wild fish. Lipids in fish muscle are one of the important factors for meat acceptability by human, but excessive lipid accumulation often deteriorates meat quality.

Fish, in special carnivorous fish, have the characteristics similar to mammals with type 2 diabetes in energy metabolisms. The metabolic syndrome including type 2 diabetes is a cluster of the most dangerous metabolic risk factors, including obesity, insulin resistance, cardiovascular diseases and hyperglycemia, etc. Hypertrophies of the adipocyte and insulin resistance are the major cause of being metabolic syndrome. Adipocyte is one of the most highly insulin-responsive cell. Adipocyte function not only as an energy reservoir but as an endocrine tissue. It produces and secretes adiponectin, one of the adipocytokines, which regulates energy metabolisms. The effects of adiponectin are mediated by adiponectin receptor, adiponectin receptor 1 (AdipoR1) and adiponectin receptor 2 (AdipoR2) and the signaling pathways via these receptors lead to the controlling systems of insulin sensitivity.

γ-Oryzanol (ORZ) is one of major bioactive components in rice bran oil. ORZ has been suggested to possess the effects of lowering serum cholesterol levels, the effects enhancing lipid and carbohydrate catabolisms, the anti-inflammatory effects, the anti-cancer effects and the antioxidative effects. Although mammals hardly accumulate ORZ in their skeletal muscles, it has been recently reported that fish accumulate the compounds in their skeletal muscle. However, there is very little information about the effects of ORZ administration of energy metabolism regulation in fish.

Previous studies verified that rainbow trout adiponectin transcripts were abundant in muscle and marginally detected in adipose tissue, in consistent with mammalian cases. The recent EST analysis for rainbow trout has suggested that heart fatty acid binding protein (H-FABP), a member of intracellular fatty acid binding proteins, is useful as a adipocyte marker together with adiponectin.

The aim of this study is to disclose the mechanisms for the physiological modulation of energy metabolism in rainbow trout. This research assessed the effects of ORZ administration on fish energy metabolism regulators, including lipid metabolism, glyconeogenesis and glycolysis. This study also pursued to reveal the distribution of the key cells, adipocytes, in rainbow trout skeletal muscle and liver in order to discuss about the modulation mechanisms for energy metabolisms.

1. The effect of γ-oryzanol administration on fish energy metabolism regulation systems

In this chapter, it was analyzed the effects of ORZ administration on energy metabolism of rainbow trout based on energy metabolism-related gene expressions. Rainbow trout (Oncorhynchus mykiss) were divided into three groups: group 1 was fed with control diet (CD), group 2 was fed with a low-concentration of ORZ diet (8 μg/kg body weight/day, LOD) and group 3 was fed with a high-concentration of ORZ diet (40 μg/kg body weight/day, HOD). After 4-week pre-feeding and 8-week ORZ-feeding, the greatest body weight gains were achieved in the HOD groups and the administration of ORZ improved feed conversion efficiency. Gene expressions related to lipid metabolism, gluconeogenesis and glycolysis were measured in skeletal muscle and liver of rainbow trout by semi quantitative real-time PCR. mRNA expression levels of adiponectin and adiponectin receptors were also analyzed. Gluconeogenesis was suppressed by low-concentration ORZ administration for 8 weeks in rainbow trout skeletal muscle. Adiponectin expression levels increased in both concentrations of ORZ for 4 weeks. AdipoR1 and adipoR2 gene expressions showed no significant difference in rainbow trout skeletal muscle. In liver, HOD administration would also enhance lipid catabolism pathways. Glyconeogenesis-related gene expressions were induced in the high ORZ administered group. Adiponectin and its receptor mRNA expression levels were also enhanced in the HOD group. From these resuls, ORZ administration would drive the lipid and carbohydrate metabolism equilibrium into the catabolism in rainbow trout.

2. Distribution of adipocyte-related cells in skeletal muscle and liver of rainbow trout

In mammals, adipose tissues, a bulk of adipocytes, have an important role in modulating energy metabolisms in whole body as stated above. However, there is no information about the physiologically active adipocytes corresponding to the mammalian counterpart in fish. In this chapter, anti-adiponectin and H-FABP antibodies were produced to investigate the distribution of physiologically active adipocytes in rainbow trout skeletal muscle and liver.

Antigenic peptides were designed through a prediction program (http://emboss.dbcls.jp/) and constructed 3D models using the SWISS-MODEL program (http://swissmodel.expasy.org/) and sybyl 8.1 software (Tripos, MO, USA) for the estimation of the distributions of the antigenic peptide sequences in the molecules. KLH-conjugated peptides were injected on the rabbit. After the third immunization, the rabbit was sacrificed and the sera were collected. Saturated ammonium sulfate is used to purification of antibodies. PD-10 column and HiTrap affinity column were used for desalted and purified, subsequently. Prior to investigation of the distribution of adiponectin and H-FABP proteins, the reactivity of each polyclonal antibody was confirmed by Western blot analysis.

Western blotting analysis with the anti-adiponectin antibody successfully detected the adiponectin signal around 75 kDa in a putative trimer in skeletal muscle proteins of rainbow trout. A protein signal of around 15 kDa in molecular mass was also successfully detected by the anti-H-FABP antibody. Subsequently, these antibodies were conducted immunohistochemistry in skeletal muscle and liver sections obtained with a vibratome (~500 micrometer in thickness). Adiponectin signals were detected in the small cells outside of skeletal muscle cells. H-FABP signals also showed the similar distributions. Higher magnification revealed that adiponectin and H-FABP signals were observed in adipocyte-like cells in rainbow trout skeletal muscle tissues, quite different from mammalian cases. Adiponectin and H-FABP signals were also spread in whole liver. These results suggest that the adiponectin gene expressions in skeletal muscle and liver of rainbow trout in the previous chapter are due to the adipocyte-related cells and that the adipocyte-related cells might have important roles in controlling energy metabolisms as mammalian adipose tissues. Taken together with the results of the previous chapter that the catabolizing compound ORZ enhanced the adiponectin expressions in skeletal muscle and liver, the adipocyte-related cells might physiologically function through not only endocrine but autocrine systems for individual organs.

The present study has provided that the administration of ORZ affects fish energy metabolisms, especially lipid metabolism, gluconeogenesis and glycolysis. Adiponectin and H-FABP-positive cells were present in skeletal muscle and liver of rainbow trout and these cells might have important roles for the modulation of energy metabolisms in rainbow trout. From the results of the previous studies that ORZ in diet is accumulated in rainbow trout skeletal muscle and liver, ORZ would directly and/or indirectly work on the adipocyte-related cells and consequently energy metabolism-related gene expressions in skeletal muscle and liver. Future investigations about the isolated and cultured adipocyte-related cells discovered in the present study will disclose the mechanisms controlling energy metabolisms of fish. The present study shows that ORZ is one promising tool for improving fish energy metabolisms like the case of human type 2 diabetes, poor in the catabolizing ability of lipid and carbohydrate and for improving culture fish meat quality.

韓 ユナ氏の提出論文Studies on mechanisms for physiological modulation of energy metabolism in rainbow trout Oncorhynchus mykiss (ニジマスのエネルギー代謝制御機構に関する研究)はニジマスのエネルギー代謝を制御する手法の確立とその結果重要であることが判明した筋組織中の脂肪細胞の存在を初めて明らかにしたものである.その概要を以下に示す.

現代の魚類養殖では生産コストと魚価の兼ね合いの中で,早期育成を目標にしていることに端を発する.そのために,飼料,給餌管理,防疫などの技術が進歩してきたが,一般に生簀内で飼育される魚の運動量が天然魚よりもはるかに少ないことと,飽食に近い環境であることから肥満状態にあることが天然魚に劣るという根本にあると言える.そこで本研究では,魚類特有のエネルギー代謝制御機構をコントロールすることによって,養殖魚の品質を改善する手法の確立に資することを目的とした.

近年の研究から,脂肪細胞が中性脂肪の蓄積によるエネルギー備蓄機能だけでなく,種々のシグナル分子「アディポサイトカイン」を分泌する内分泌器官として注目を集めている.肥満によって肥大した脂肪細胞からTNFα,レジスチン,FFAが大量に産生,分泌される.TNFαは骨格筋や肝臓でのインスリンシグナルに応答するインスリン受容体基質(IRS-1/IRS-2)のセリンリン酸化の増強をもたらす.このことでIRS-1/IRS-2のチロシンリン酸化が抑制され,インスリン受容体(IR)によるIRS-1/IRS-2下流へのインスリンシグナル伝達を阻害することでインスリン抵抗性を惹起させることが明らかにされている.このように脂肪細胞からはインスリンシグナルを抑制するアディポサイトカインが多く分泌される一方で,インスリン感受性改善に作用するアディポネクチンが発見された.アディポネクチンは内臓脂肪の蓄積と強い逆相関を示すアディポカインとして見出された.つまり哺乳類では,肥満によって糖質・脂質代謝を良好に保つアディポネクチンの分泌が低下することで,インスリン抵抗が生じ,糖尿病・メタボリックシンドロームを引き起こす可能性が示唆されている.さらに,米糠中に含まれるγ-オリザノールが哺乳類における血中アディポネクチンレベル改善作用を有することが発見された.一方,魚類ではこの分野の情報が皆無に等しい.最近のEST解析によってニジマスのアディポネクチンが筋組織で強く発現していることが明らかとなったことから,本研究ではアディポネクチンを分泌する細胞の分布解析も試みた.

第1章では,γ-オリザノールをニジマスに投与した際の影響を解析したところ,γ-オリザノールの投与によって有意に体重増加が促進されることが明らかとなった.また,筋肉や肝臓で脂質分解系が活性化され,血中のアディポネクチン量も増大することを明らかにした.糖の異化も促進される傾向が認められた.したがって,γ-オリザノールによるエネルギー代謝関連因子の発現が影響を受けることで,エネルギー産生におけるタンパク質への依存が改善され,体重増加につながったものと考えられた.

第2章ではニジマスのアディポネクチンおよび心筋型脂肪酸結合タンパク質(H-FABP)に対する特異的抗体を作製し,これらのマーカータンパク質を用いて筋組織および肝臓における脂肪細胞の分布を詳細に解析した.その結果,アディポネクチンおよびH-FABP陽性細胞は筋細胞間および肝実質細胞間に分散して存在することが明らかとなった.このような存在様式は哺乳類とは全く異なるものであり,さらに哺乳類幼若脂肪細胞はこれらのタンパク質を発現しないことが報告されていることから,魚類では機能的な脂肪細胞が,筋組織や肝臓などの末梢に分散し,末梢におけるエネルギー代謝を制御している可能性が示唆された.

以上,本研究はニジマスへのγ-オリザノール投与が早期養成を可能とすること,魚類脂肪細胞の筋組織における分布を初めて明らかにしたもので,基礎生物科学的な知見の提供だけでなく,産業上の応用にもつながるものとして,審査委員一同は,本論文が博士(農学)の学位論文として必要十分な条件を満たす,価値あるものと判定した.