临床酮症是奶牛的一种有害代谢疾病,常伴有脂肪组织严重的脂解和炎症。我们先前的研究表明,患有临床酮症的奶牛脂肪组织中钙调蛋白(CaM)水平上调了2.401倍。因此,我们假设CaM可能调节临床酮症奶牛的脂解和炎症反应。为了验证假设,我们对临床症状和血清β-羟丁酸(BHB)浓度进行了全面的兽医评估.随后,我们在产后17±4天收集了6头健康奶牛和6头临床酮症荷斯坦奶牛的皮下脂肪组织样本。商业试剂盒用于测试BHB的丰度,非酯化脂肪酸(NEFA),肝功能指数(LFI),白细胞介素-6(IL-6),IL-1β,和肿瘤坏死因子-α(TNF-α)。我们发现患有临床酮症的奶牛表现出更高水平的BHB,NEFA,LFI,IL-6,IL-1β,TNF-α,葡萄糖水平低于健康奶牛。此外,丰富的CaM,toll样受体4(TLR4),核因子κB激酶β亚基(IKK)抑制剂,磷酸化核因子κBp65/核因子κBp65(p-NF-κBp65/NF-κBp65),脂肪三酰甘油脂肪酶(ATGL),磷酸化激素敏感脂肪酶/激素敏感脂肪酶(p-HSL/HSL)增加,而临床酮症奶牛的脂肪组织中perilipin-1(PLIN1)的含量降低。为了调查这些反应的潜在机制,我们从健康奶牛的脂肪组织中分离了原代牛脂肪细胞,并通过脂多糖(LPS)诱导了TLR4/IKK/NF-κBp65介导的炎症反应。此外,我们用CaM过表达腺病毒和CaM小干扰RNA处理原代牛脂肪细胞。体外,LPS上调TLR4、IKK、p-NF-κBp65,ATGL,p-HSL/HSL,和CaM和下调的PLIN1。此外,CaM沉默下调了LPS激活的p-HSL/HSL的丰度,TLR4,IKK,牛脂肪细胞中p-NF-κBp65和PLIN1上调,除了ATGL。然而,CaM过表达上调了LPS激活的p-HSL/HSL的丰度,TLR4,IKK,牛脂肪细胞中p-NF-κBp65和PLIN1表达下调。这些数据表明,CaM通过HSL和PINL1促进脂肪细胞的脂解,同时激活TLR4/IKK/NF-κB炎症途径以刺激炎症反应。CaM之间存在正反馈回路,脂解,和炎症。抑制CaM可能是减轻脂肪组织代谢失调的适应性机制。从而缓解脂解和炎症反应。
Clinical ketosis is a detrimental metabolic disease in dairy cows, often accompanied by severe lipolysis and inflammation in adipose tissue. Our previous study suggested a 2.401-fold upregulation in the
calmodulin (CaM) level in the adipose tissue of cows with clinical ketosis. Therefore, we hypothesized that CaM may regulate lipolysis and inflammatory responses in cows with clinical ketosis. To verify the hypothesis, we conducted a thorough veterinary assessment of clinical symptoms and serum β-hydroxybutyrate (BHB) concentration. Subsequently, we collected subcutaneous adipose tissue samples from six healthy and six clinically ketotic Holstein cows at 17 ± 4 days postpartum. Commercial kits were used to test the abundance of BHB, non-esterified fatty acid (NEFA), the liver function index (LFI), interleukin-6 (IL-6), IL-1β, and tumor necrosis factor-α (TNF-α). We found that cows with clinical ketosis exhibited higher levels of BHB, NEFA, LFI, IL-6, IL-1β, TNF-α, and lower glucose levels than healthy cows. Furthermore, the abundance of CaM, toll-like receptor 4 (TLR4), inhibitor of nuclear factor κB kinase subunit β (IKK), phosphorylated nuclear factor κB p65/nuclear factor κB p65 (p-NF-κB p65/NF-κB p65), adipose triacylglycerol lipase (ATGL), and phosphorylated hormone-sensitive lipase/hormone-sensitive lipase (p-HSL/HSL) was increased, while that of perilipin-1 (PLIN1) was decreased in the adipose tissue of cows with clinical ketosis. To investigate the mechanism underlying the responses, we isolated the primary bovine adipocytes from the adipose tissue of healthy cows and induced the inflammatory response mediated by TLR4/IKK/NF-κB p65 with lipopolysaccharide (LPS). Additionally, we treated the primary bovine adipocytes with CaM overexpression adenovirus and CaM small interfering RNA. In vitro, LPS upregulated the abundance of TLR4, IKK, p-NF-κB p65, ATGL, p-HSL/HSL, and CaM and downregulated PLIN1. Furthermore, CaM silencing downregulated the abundance of LPS-activated p-HSL/HSL, TLR4, IKK, and p-NF-κB p65 and upregulated PLIN1 in bovine adipocytes, except for ATGL. However, CaM overexpression upregulated the abundance of LPS-activated p-HSL/HSL, TLR4, IKK, and p-NF-κB p65 and downregulated PLIN1 expression in bovine adipocytes. These data suggest that CaM promotes lipolysis in adipocytes through HSL and PINL1 while activating the TLR4/IKK/NF-κB inflammatory pathway to stimulate an inflammatory response. There is a positive feedback loop between CaM, lipolysis, and inflammation. Inhibiting CaM may act as an adaptive mechanism to alleviate metabolic dysregulation in adipose tissue, thereby relieving lipolysis and inflammatory responses.