Data Sheet 1_Dietary supplementation with L-carnitine elevates intracellular carnitine levels and affects gene expression of SLC25A20 and COX4I1, as well as non-mitochondrial respiration of bovine blood cells during systemic immune challenge.pdf

L-carnitine plays a key role in shuttling free fatty acids from the cytosol into the mitochondrial matrix. Fatty acids, among other substrates, are utilized by immune cells as an energy source. Therefore, L-carnitine, which is authorized as a feed additive in cattle, may influence the metabolism of peripheral blood mononuclear cells (PBMC) during an immune challenge. To test this hypothesis, a feeding trial was conducted with 53 German Holstein cows, comprising a control group (CON, n = 26) and an L-carnitine supplemented group (CAR, n = 27, 25 g rumen-protected L-carnitine/cow/d). On day 111 after calving, all cows were intravenously injected with lipopolysaccharides (LPS, 0.5 µg/kg body weight as bolus injection, E. coli) to induce a systemic immune challenge. Blood samples were collected on day 143 ante injectionem (ai), day 11 ai, 24 hours post injectionem (pi), and day 14 pi and PBMC were isolated. The used methods included high-performance liquid chromatography coupled with mass spectrometry, Alamar Blue assay, real-time qPCR, and the Mito Stress Test of the Seahorse Analyzer (Agilent, Santa Clara, California, USA). L-carnitine supplementation significantly increased intracellular concentrations of carnitine and its precursor γ-butyrobetaine in PBMC of dairy cows. The gene expression of carnitine-acylcarnitine translocase (SLC25A20) in PBMC remained stable in CAR, whereas it was upregulated in CON during the LPS challenge, suggesting an adaptation to increased energy demands in CON. A contrasting pattern was detected for the gene expression of cytochrome c oxidase subunit 4I1 (COX4I1), with stable levels in CON and a downregulation in CAR due to LPS injection. However, most of the investigated genes were unaffected by L-carnitine supplementation, and responded significantly to LPS injection. The same applied for PBMC mitochondrial functionality and metabolic activity as assessed by ex vivo approaches, whereas non-mitochondrial respiration rate was significantly affected by L-carnitine supplementation over time. In conclusion, dietary L-carnitine supplementation of 25 g per cow per day led to a balanced distribution of carnitine and γ-butyrobetaine between bovine blood cells and plasma, but there were only minor effects on gene level and cellular respiration.

L-肉碱（L-carnitine）在将游离脂肪酸从细胞质转运至线粒体基质的过程中发挥关键作用。脂肪酸与其他底物一同被免疫细胞用作能量来源。因此，作为牛饲料添加剂获批的L-肉碱，可能会在免疫应激期间影响外周血单个核细胞（peripheral blood mononuclear cells，PBMC）的代谢。
为验证这一假说，研究人员对53头德国荷斯坦奶牛开展了饲喂试验，设置对照组（CON，n=26）与L-肉碱添加组（CAR，n=27，每头奶牛每日饲喂25克瘤胃保护性L-肉碱）。在奶牛产后第111天，对所有奶牛静脉注射脂多糖（lipopolysaccharides，LPS，0.5微克/千克体重，大肠杆菌来源一次性推注）以诱导全身性免疫应激。
分别于注射前143天（ai）、注射前11天（ai）、注射后24小时（pi）以及注射后14天（pi）采集血样并分离外周血单个核细胞（PBMC）。本研究采用的实验方法包括高效液相色谱-串联质谱联用法、Alamar Blue检测法、实时定量聚合酶链反应（real-time qPCR）以及美国加利福尼亚州圣克拉拉市安捷伦（Agilent）Seahorse分析仪的线粒体应激测试（Mito Stress Test）。
L-肉碱添加可显著提高奶牛PBMC内肉碱及其前体γ-丁酸甜菜碱（γ-butyrobetaine）的胞内浓度。PBMC中肉碱-酰基肉碱转运酶（carnitine-acylcarnitine translocase，SLC25A20）的基因表达在CAR组中保持稳定，而在CON组的LPS应激期间出现上调，这提示CON组出现了应对能量需求增加的适应性变化。细胞色素c氧化酶亚基4I1（cytochrome c oxidase subunit 4I1，COX4I1）的基因表达则呈现相反模式：CON组水平保持稳定，而CAR组因LPS注射出现下调。
不过，大多数被检测基因的表达不受L-肉碱添加的影响，仅对LPS注射产生显著响应。这一规律同样适用于通过离体实验评估的PBMC线粒体功能与代谢活性，而非线粒体呼吸速率则随时间推移受到L-肉碱添加的显著影响。
综上，每头奶牛每日饲喂25克L-肉碱的日粮添加方案，可实现牛血细胞与血浆中肉碱及γ-丁酸甜菜碱的均衡分布，但对基因表达水平与细胞呼吸仅存在轻微影响。