Choudhary Shanti, LaCasse Michelle, Choudhary Ratan Kumar, Rincon Mercedes, Beitz Donald C, Testroet Eric D
Department of Animal and Veterinary Sciences, University of Vermont, Burlington, VT 05446, USA.
Department of Immunology & Microbiology, University of Colorado Anschutz School of Medicine, Aurora, CO 80045, USA.
Animals (Basel). 2023 Mar 20;13(6):1101. doi: 10.3390/ani13061101.
Mitochondrial complex I inhibitor (iC1) is a methylation-controlled J protein (MCJ) that decreases cellular respiration by inhibiting oxidative phosphorylation. Recent rodent studies showed that loss or inhibition of iC1 was associated with preventing lipid accumulation. A common metabolic disorder of dairy cattle is a fatty liver disease (FLD), which often occurs during the periparturient period. In humans and rodents, iC1 is expressed in the liver and acts as a mitochondrial "brake". However, iC1 expression in bovine liver and its possible role in FLD development have not yet been characterized. We hypothesized that iC1 is expressed in the bovine liver and that the expression of iC1 is correlated with FLD in periparturient dairy cattle. To test this hypothesis, we collected bovine liver tissue samples from an abattoir and isolated primary hepatic cells immediately following harvest. Utilizing an in vitro model of bovine FLD developed in our laboratory, we cultured primary hepatic cells in low-glucose DMEM supplemented with 10% FBS. The basal media was made to induce lipid accumulation and cytotoxicity in the primary liver cells with three treatments. To the basal media (control) we added 0.4 mM palmitate (treatment 1) or 20 ng/mL TNFα (treatment 2), or both 0.4 mM palmitate and 20 ng/mL TNFα (treatment 3). Consistent with our hypothesis, we present the novel characterization of iC1 expression in primary bovine liver cells cultured with or without the addition of lipotoxic factors made to emulate bovine FLD. We demonstrate both in situ and in vitro expression of iC1 in bovine liver and mRNA expression in hepatic cells and in the precipitates of conditioned media. The results of RT-qPCR, IHC, and western blot all demonstrated the expression of iC1 in bovine liver. In addition, we isolated precipitates of conditioned media further demonstrated iC1 expression by RT-qPCR. The transcript of iC1 tended to be more concentrated (4-fold; > 0.05) in TNFα-treated conditioned media when compared with the control. Taken together, we present the novel finding that iC1 transcript and protein are expressed in liver tissue from dairy cattle, primary hepatic cells isolated from that liver tissue, and, finally, in the conditioned media derived from those cells. These novel findings and the prior findings on the role of iC1 in rodents and humans indicate that further investigation of the role of iC1 in the etiology and pathology of FLD in periparturient dairy cows is warranted.
线粒体复合体I抑制剂（iC1）是一种甲基化控制的J蛋白（MCJ），它通过抑制氧化磷酸化来降低细胞呼吸。最近的啮齿动物研究表明，iC1的缺失或抑制与预防脂质积累有关。奶牛常见的一种代谢紊乱疾病是脂肪肝（FLD），它通常发生在围产期。在人类和啮齿动物中，iC1在肝脏中表达，并作为线粒体的“刹车”。然而，iC1在牛肝脏中的表达及其在FLD发生发展中的可能作用尚未得到明确。我们假设iC1在牛肝脏中表达，并且iC1的表达与围产期奶牛的FLD相关。为了验证这一假设，我们从屠宰场收集牛肝脏组织样本，并在收获后立即分离原代肝细胞。利用我们实验室建立的牛FLD体外模型，我们在补充有10%胎牛血清的低葡萄糖DMEM中培养原代肝细胞。基础培养基通过三种处理来诱导原代肝细胞中的脂质积累和细胞毒性。在基础培养基（对照）中，我们添加0.4 mM棕榈酸（处理1）或20 ng/mL肿瘤坏死因子α（TNFα）（处理2），或同时添加0.4 mM棕榈酸和20 ng/mL TNFα（处理3）。与我们的假设一致，我们展示了在添加或不添加模拟牛FLD的脂毒性因子培养的原代牛肝细胞中iC1表达的新特征。我们证明了iC1在牛肝脏中的原位和体外表达以及在肝细胞和条件培养基沉淀物中的mRNA表达。逆转录定量聚合酶链反应（RT-qPCR）、免疫组织化学（IHC）和蛋白质印迹法（western blot）的结果均证明了iC1在牛肝脏中的表达。此外，我们分离的条件培养基沉淀物通过RT-qPCR进一步证明了iC1的表达。与对照相比，iC1转录本在TNFα处理的条件培养基中倾向于更集中（4倍；P>0.05）。综上所述，我们发现了一个新现象，即iC1转录本和蛋白质在奶牛的肝脏组织、从该肝脏组织分离的原代肝细胞以及最终从这些细胞衍生的条件培养基中均有表达。这些新发现以及之前关于iC1在啮齿动物和人类中作用的发现表明，有必要进一步研究iC1在围产期奶牛FLD病因学和病理学中的作用。
