ACSL4 is a target for β-hydroxybutyrate–induced increase in fatty acid content and lipid droplet accumulation in bovine mammary epithelial cells

doi:10.1016/j.jia.2024.12.004

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Ming Li¹, Jingjing Wang², Jianan Wen², Juan J. Loor³, Qianming Jiang³, Jingyi Wang¹, Huijing Zhang¹, Yue Yang¹, Wei Yang⁴, Bingbing Zhang², Chuang Xu¹^#

¹College of Veterinary Medicine, China Agricultural University, Beijing 100193, China

²College of Life Science and Technology, Heilongjiang Bayi Agricultural University, Daqing 163319, China

³Mammalian NutriPhysio Genomics, Department of Animal Sciences and Division of Nutritional Sciences, University of Illinois, Urbana 61801

⁴College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing 163319, China

Highlights
1. ACSL4 regulates fatty acid synthesis and accumulation in BMECs during ketosis.
2. BHB leads to an increase in ACSL4 expression, which in turn increases fatty acid content and lipid droplet accumulation, and inhibiting ACSL4 would reduce the effects of BHB.
3. ACSL4 suppression decreases ROS, improves mitochondrial function, and increases fatty acid oxidation.

Abstract
References

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摘要奶牛酮病是一种常见的代谢性疾病，通常发生在泌乳早期，并且与血液中高浓度β-羟丁酸（BHB）有关。部分到达乳腺的BHB会被用作合成脂肪酸的前体。最近，非反刍动物研究表明，长链脂酰辅酶A合成酶4（ACSL4）可能在细胞脂肪酸代谢的调节中发挥作用，但ACSL4在响应BHB时如何介导细胞脂质代谢的机制尚不清楚。为了明确这一问题，我们对牛乳腺活检组织和永生化的牛乳腺上皮细胞系（MAC-T）进行了体内和体外分析。体内研究（n = 6头牛/组）对健康牛（血浆BHB < 0.60 mmol L^-1）或患有酮病的奶牛（血浆BHB > 2.0 mmol L^-1）进行乳腺组织活检。在体外实验中，MAC-T细胞分别接受了0、0.3、0.6、1.2或2.4 mmol L^-1 BHB处理24小时，以确定BHB最佳处理剂量。随后，MAC-T细胞接受1.2 mmol L^-1 BHB处理0、3、6、12、24或48小时。此外，MAC-T细胞还分别进行了ACSL4小干扰RNA（siACSL4）处理24小时或ACSL4过表达质粒（pcACSL4）处理36小时，之后用1.2 mmol L^-1 BHB处理24小时。研究结果表明，BHB处理后，乳腺组织和体外细胞实验中的脂肪合成相关的mRNA和蛋白质表达量均增加。BHB通过激活ACSL4表达增加了脂肪酸含量，而抑制ACSL4则减少了BHB引起的活性氧（ROS）过度产生、线粒体膜电位增强、脂肪酸含量增加以及脂滴积累。此外，通过过表达质粒提高ACSL4表达进一步明确了其在BHB刺激中的分子作用。ACSL4过表达增强了BHB引起的脂肪酸含量的增加和脂滴的积累。总体而言，研究表明ACSL4在BHB调控的脂肪酸合成过程中起关键作用。减少ACSL4表达会降低脂肪酸含量和脂滴积累，改善线粒体功能，促进脂肪酸氧化。本文首次报道了在外周血BHB浓度显著增加的时期，改变ACSL4丰度有助于调节乳腺上皮细胞中异常的脂肪酸代谢，从而为调控产后能量负平衡下乳脂代谢异常提供理论依据。

Abstract

Ketosis, a common metabolic disease during early lactation, is associated with high circulating levels of β-hydroxybutyrate (BHB). A portion of BHB that reaches the mammary gland is utilized as precursor for synthesis of fatty acids. Recent findings from nonruminant studies revealed that long chain fatty acyl-CoA ligase 4 (ACSL4) could play a role in the regulation of cellular fatty acid metabolism, but the mechanisms by which ACSL4 mediates cellular lipid metabolism in response to BHB remains unclear. To achieve the aims, we conducted in vivo or in vitro analyses using bovine mammary gland biopsies and the immortalized mammary epithelial cell line (MAC-T). The in vivo study (n = 6 cows group^-1) involved healthy cows (plasma BHB < 0.60 mmol L^-1) or ketotic cows (plasma BHB > 2.0 mmol L^-1) from which mammary gland tissue was biopsied. In vitro, MAC-T cells were challenged with 0, 0.3, 0.6, 1.2, or 2.4 mmol L^-1 BHB for 24 h to determine an optimal dose. Subsequently, MAC-T were incubated with 1.2 mmol L^-1 BHB for 0, 3, 6, 12, 24, or 48 h. Furthermore, MAC-T cells were treated with small interfering ACSL4 (siACSL4) for 24 h or ACSL4 overexpression plasmid (pcACSL4) for 36 h followed by a challenge with 1.2 mmol L^-1 BHB for 24 h. Results showed that increased mRNA and protein abundance of lipogenic genes were linked to both mammary gland and in vitro challenge with BHB. BHB increased fatty acid content by activating ACSL4 expression, whereas inhibition of ACSL4 reduced BHB-induced reactive oxygen species (ROS) overproduction, enhancement of mitochondrial membrane potential, increase in fatty acid content, and lipid droplet accumulation. Furthermore, we also elevated ACSL4 expression with an overexpression plasmid to clarify its molecular role in response to BHB challenge. ACSL4 overexpression enhances BHB-induced lipid droplet accumulation by increased fatty acid content. Overall, the information showed that ACSL4 is crucial for the process of producing fatty acids from exogenous BHB. Reduced ACSL4 decreased fatty acid content and lipid droplet accumulation, improved mitochondrial function, directed more fatty acids towards oxidation. Thus, ACSL4 plays an important role in determining the fate of intracellular fatty acids and BHB in BMECs.

Keywords: bovine mammary epithelial cells fatty acid contents ketosiss BHBs ACSL4

Online: 05 December 2024

Fund:

The study was supported by grants from the National Key Research and Development Program of China (2023YFD1800804 and 2023YFD1801100), The National Natural Science Foundation of China (32172926), China Agriculture Research System (CARS-36).

About author: Ming Li, E-mail: liming5697@163.com; #Correspondence Chuang Xu, E-mail: xuchuang7175@163.com

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Ming Li, Jingjing Wang, Jianan Wen, Juan J. Loor, Qianming Jiang, Jingyi Wang, Huijing Zhang, Yue Yang, Wei Yang, Bingbing Zhang, Chuang Xu. 2024. ACSL4 is a target for β-hydroxybutyrate–induced increase in fatty acid content and lipid droplet accumulation in bovine mammary epithelial cells. Journal of Integrative Agriculture, Doi:10.1016/j.jia.2024.12.004

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