目的:定义细胞代谢和信号传导的调节因子对于设计肥胖和NAFLD/NASH的新治疗策略至关重要。E3泛素连接酶通过泛素化介导的蛋白质靶标调节来控制多种细胞功能,因此,它们的功能畸变与许多疾病有关。E3连接酶Ube4A与人类肥胖有关,炎症,和癌症。然而,它的体内功能是未知的,并且没有动物模型可用于研究这种新型蛋白质。
方法:制作了一个Ube4A基因敲除(UKO)全身小鼠模型,并比较了高脂饮食(HFD)喂养的WT和UKO小鼠的各种代谢参数,在他们的肝脏里,脂肪组织,和血清。在HFD喂养的WT和UKO小鼠的肝脏样品中进行脂质组学和RNA-Seq研究。进行蛋白质组学研究以鉴定代谢中Ube4A的靶标。此外,鉴定了Ube4A调节代谢的机制。
结果:尽管年轻人的体重和组成,食物喂养的WT和UKO小鼠相似,基因敲除表现为轻度高胰岛素血症和胰岛素抵抗.HFD喂养显著增加肥胖,高胰岛素血症,和UKO小鼠两性的胰岛素抵抗。HFD喂养的UKO小鼠的白色和棕色脂肪组织储库具有增加的胰岛素抵抗和炎症以及减少的能量代谢。此外,Ube4A缺失会加剧肝脏脂肪变性,炎症,和HFD喂养小鼠的肝损伤,肝细胞的脂质摄取和脂肪生成增加。急性胰岛素治疗导致饲喂UKO小鼠的肝脏和脂肪组织中胰岛素效应蛋白激酶Akt的激活受损。我们将Akt激活蛋白APPL1鉴定为Ube4A相互作用物。Akt和APPL1的K63连接的泛素化(K63-Ub),已知可促进胰岛素诱导的Akt激活,在UKO小鼠中受损。此外,Ube4AK63-泛素化Akt体外。
结论:Ube4A是一种新型的肥胖调节因子,胰岛素抵抗,脂肪组织功能障碍和NAFLD,防止其下调可能会改善这些疾病。
Defining the regulators of cell metabolism and signaling is essential to design new therapeutic strategies in obesity and NAFLD/NASH. E3 ubiquitin ligases control diverse cellular functions by ubiquitination-mediated regulation of protein targets, and thus their functional aberration is associated with many diseases. The E3 ligase Ube4A has been implicated in human obesity, inflammation, and cancer. However, its in vivo function is unknown, and no animal models are available to study this novel protein.
A whole-body Ube4A knockout (UKO) mouse model was generated, and various metabolic parameters were compared in chow- and high fat diet (HFD)-fed WT and UKO mice, and in their liver, adipose tissue, and serum. Lipidomics and RNA-Seq studies were performed in the liver samples of HFD-fed WT and UKO mice. Proteomic studies were conducted to identify Ube4A\'s targets in metabolism. Furthermore, a mechanism by which Ube4A regulates metabolism was identified.
Although the body weight and composition of young, chow-fed WT and UKO mice are similar, the knockouts exhibit mild hyperinsulinemia and insulin resistance. HFD feeding substantially augments obesity, hyperinsulinemia, and insulin resistance in both sexes of UKO mice. HFD-fed white and brown adipose tissue depots of UKO mice have increased insulin resistance and inflammation and reduced energy metabolism. Moreover, Ube4A deletion exacerbates hepatic steatosis, inflammation, and liver injury in HFD-fed mice with increased lipid uptake and lipogenesis in hepatocytes. Acute insulin treatment resulted in impaired activation of the insulin effector protein kinase Akt in liver and adipose tissue of chow-fed UKO mice. We identified the Akt activator protein
APPL1 as a Ube4A interactor. The K63-linked ubiquitination (K63-Ub) of Akt and
APPL1, known to facilitate insulin-induced Akt activation, is impaired in UKO mice. Furthermore, Ube4A K63-ubiquitinates Akt in vitro.
Ube4A is a novel regulator of obesity, insulin resistance, adipose tissue dysfunction and NAFLD, and preventing its downregulation may ameliorate these diseases.