Protein kinase A (PKA) plays an important role in cellular life activities. Recently, PKA was found to bind to the inhibitor of nuclear factor-kappaB (IκB), a key protein in the nuclear factor-kappaB (NF-κB) pathway, to form a complex involved in the regulation of inflammatory response. However, the role of PKA in the anti-inflammatory of goose fatty liver is still unclear. A total of 14 healthy 70-d-old male Lander geese were randomly divided into a control group and an overfeeding group. Inflammation level was analyzed by histopathological method in the liver. The mRNA and protein abundance of PKA and tumor necrosis factor-alpha (TNFα), as well as the ubiquitination level of PKA, were detected. Moreover, goose primary hepatocytes were cotreated with glucose, harringtonine, and carbobenzoxy-l-leucyl-l-leucyl-l-leucinal (MG132). Finally, the co-immunoprecipitated samples of PKA from the control and overfeeding group were used for protein mass spectrometry. The results showed that no difference in PKA mRNA expression was observed (P > 0.05), while the PKA protein level in the overfed group was significantly reduced (P < 0.05) when compared with the control group. The ubiquitination level of PKA was higher than that of the control group in fatty liver. The mRNA expression of PKA was elevated but protein abundance was reduced in goose primary hepatocytes with 200 mmol/L glucose treatment (P < 0.05). The PKA protein abundance was dramatically reduced in hepatocytes treated with harringtonine (P < 0.01) when compared with the glucose-supplemented group. Nevertheless, MG132 tended to alleviate the inhibitory effect of harringtonine on PKA protein abundance (P = 0.081). There was no significant difference in TNFα protein level among glucose-treated groups and control (P > 0.05). Protein mass spectrometry analysis showed that 29 and 76 interacting proteins of PKA were screened in goose normal and fatty liver, respectively. Validation showed that PKA interacted with the E3 ubiquitination ligases ring finger protein 135 (RNF135) and potassium channel modulatory factor 1 (KCMF1). In summary, glucose may inhibit the inflammatory response in goose fatty liver by increasing the ubiquitination level of PKA. Additionally, RNF135 and KCMF1 may be involved in the regulation of PKA ubiquitination level as E3 ubiquitination ligases.

中文翻译：

---

葡萄糖通过增加 PKA 的泛素化水平来抑制鹅脂肪肝的炎症反应


蛋白激酶 A （PKA） 在细胞生命活动中起着重要作用。最近，发现 PKA 与核因子-κB （IκB） 抑制剂结合，IκB 是核因子-κB （NF-κB） 通路中的关键蛋白，形成参与炎症反应调节的复合物。然而，PKA 在鹅脂肪肝的抗炎作用仍不清楚。将 14 只健康的 70 d 龄雄性 Lander 鹅随机分为对照组和过量喂养组。通过组织病理学方法分析肝脏炎症水平。检测 PKA 和肿瘤坏死因子-α （TNFα） 的 mRNA 和蛋白丰度，以及 PKA 的泛素化水平。此外，鹅原代肝细胞与葡萄糖、Harringtonine 和碳苯氧基-l-亮氨酰-l-亮氨酰-l-亮氨酸 （MG132） 共处理。最后，将对照组和过量喂养组的 PKA 共免疫沉淀样品用于蛋白质质谱分析。结果显示，与对照组相比，PKA mRNA 表达无差异 （P > 0.05），而过量喂养组 PKA 蛋白水平显著降低 （P < 0.05）。脂肪肝 PKA 泛素化水平高于对照组。200 mmol/L 葡萄糖处理后，鹅原代肝细胞 PKA mRNA 表达升高，但蛋白质丰度降低 （P < 0.05）。与葡萄糖补充组相比，三桔梗果碱处理肝细胞的 PKA 蛋白丰度显著降低 （P < 0.01）。然而，MG132 倾向于减轻 harringtonine 对 PKA 蛋白丰度的抑制作用 （P = 0.081）。 葡萄糖处理组和对照组 TNFα 蛋白水平差异无统计学意义 （P > 0.05）。蛋白质质谱分析显示，在鹅正常肝和脂肪肝中分别筛选出 29 和 76 种 PKA 相互作用蛋白。验证表明 PKA 与 E3 泛素化连接酶无名指蛋白 135 （RNF135） 和钾通道调节因子 1 （KCMF1） 相互作用。综上所述，葡萄糖可能通过增加 PKA 的泛素化水平来抑制鹅脂肪肝的炎症反应。此外，RNF135 和 KCMF1 可能作为 E3 泛素连接酶参与 PKA 泛素化水平的调节。