利用核磁共振為基礎的代謝體學探討綠色鱘魚在食物限制下的影響

Abstract

食物限制是環境壓力的其中一種。這種環境壓力可能會嚴重影響生物體並導致族群的下降或滅絕。魚類的生命週期中可能常會接觸到食物限制的狀態，因為魚類較容易暴露於極端的環境或是特殊的情況，例如：氣候變遷、季節性的溫度差異、暴露於環境汙染物、棲息地破壞或是處於洄游產卵的狀態。 綠色鱘魚在2006年被美國瀕臨絕種動物保護法給編列為受到威脅的物種，過度的捕魚和棲息地破壞是導致其族群下降的因素，鱘魚對於環境壓力影響是易感受性的，從溫度、氣候因子、化學汙染或是營養狀態的細微改變都可能會導致鱘魚的生理狀態受到影響。 代謝體學是一種高通量檢測生物系統內的小分子代謝物的方法，用以了解表現型態的發展與機制，而核磁共振光譜儀是一個常被用來研究代謝體學的方法，它提供快速的取得圖譜、不會破壞樣本、高通量的資訊以及較少量的樣本前處理步驟等優點。核磁共振為基礎的代謝體學配合多變量分析(例如:主成分分析)可用於檢測暴露於環境壓力的生物體內之代謝物改變。 本研究的目的是利用核磁共振光譜儀為基礎的代謝體學探討綠色鱘魚暴露在環境壓力下的影響，綠色鱘魚的幼年期和青年期在不同的食物限制下(100%, 50%, 25%, 12.5%)分別暴露2週與4週。分別取得肌肉、肝臟與腎臟萃取親水性與疏水性代謝物並且運用核磁共振光譜儀和多變量分析的方法分析食物限制的影響。 我們從主成分分析的結果發現，不同年齡的綠鱘當受到食物限制時，各個組織皆會發生代謝物改變。我們的研究結果顯示，肌肉、肝臟與腎臟組織中的胺基酸或乳酸會因食物限制而產生顯著的變化，這些代謝物和糖質新生有關；在幼年綠鱘的腎臟組織和青年綠鱘的肌肉、肝臟以及腎臟組織，與糖解作用有關的葡萄糖濃度也顯著下降；此外，我們也發現在幼年綠鱘的肝臟組織與青年綠鱘的肌肉、肝臟和腎臟組織中皆發現了脂肪酸與甘油濃度顯著降低，可能是用來補充不足的能量；除了能量的擾動，我們也發現與滲透壓調節和抗氧化壓力相關代謝物的濃度變化。這些結果顯示在食物限制下，可能會影響滲透壓平衡與產生氧化壓力。核磁共振光譜儀為基礎的代謝體對了解暴露在環境壓力下的生物之代謝物變化有很大的助益。

Food restriction, one of the environmental stresses, could produce serious effects to organisms and may cause to population decline and extinction. In different life stages, wild fish often encounter to food restriction because they usually expose to extreme environment or special status, such as climatic change, low temperature, environmental pollution, habitat destruction or spawning migration. Green sturgeon was listed as ‘‘threatened’’ in 2006 under the U.S. Endangered Species Act. Overfishing and habitat degradation lead to green sturgeon population decline. Since green sturgeon is susceptible to environmental stress, subtle changes from temperature, climatic factors, chemical exposure and nutritional situation will affect its physiological status. Metabolomic approach is a high-throughput screening method to examine changes of metabolic profiles in biological systems to understand possible mechanisms for phenotype development. Nuclear magnetic resonance (NMR) spectroscopy, a common instrument applied in the field of metabolomics, supplies a fast, non-disruptive, high-throughput method that requires the least sample preparation. NMR coupled with multivariate statistical analysis such as principal components analysis (PCA) can be used to examine metabolic effects of environment stress in organisms. The purpose of this study is to study effects of feed restriction in different tissues and life stages of green sturgeon by using NMR-based metabolomics. Green sturgeon fingerling and juvenile were fed four feed restriction (100%, 50%, 25% and 12.5%) for 2 and 4 weeks, respectively. Both hydrophilic and hydrophobic metabolites from the muscle, liver, and kidney were extracted and analyzed by NMR followed PCA. The PCA results showed feed-restriction dependant trends from the analysis of all tissues types of different life stages, except the analysis of hydrophobic muscle metaolome from the fingerling. We found a lot of energy-related metabolites changed in different tissues of sturgeon after feed restriction. Our results showed that numerous amino acids or lactate were altered which are relate to imbalanced glycogenesis in muscle, liver and kidney of different stages of sturgeon. We also found decreased glucose probably due to glycolysis in all tissues of different stages of sturgeon except for the muscle and liver of fingerling. In addition, we also found fatty acids and glycerol degraded in the liver of fingerling and all tissues of juvenile for supplemental energy supply. Besides energy disturbance after feed restriction, we also discovered changes of osmolyte-related metabolites and antioxidants. These results suggested feed restriction may cause changes of osmotic balance and oxidative stress. We conducted that NMR-based metabolomics is useful in understanding the metabolic changes due to environmental stresses.