Department of Experimental Diabetology, German Institute of Human Nutrition Potsdam-Rehbruecke, D-14558 Nuthetal, Germany; German Center for Diabetes Research, D-85764 München-Neuherberg, Germany.
German Center for Diabetes Research, D-85764 München-Neuherberg, Germany; Department of Proteomics and Signal Transduction, Max-Planck Institute of Biochemistry, D-82152 Martinsried, Germany; Institute for Diabetes and Obesity, Helmholtz Zentrum München, D-85764 München-Neuherberg, Germany.
J Hepatol. 2020 Oct;73(4):771-782. doi: 10.1016/j.jhep.2020.04.031. Epub 2020 May 4.
BACKGROUND & AIMS: Currently, only a few genetic variants explain the heritability of fatty liver disease. Quantitative trait loci (QTL) analysis of mouse strains has identified the susceptibility locus Ltg/NZO (liver triglycerides from New Zealand obese [NZO] alleles) on chromosome 18 as associating with increased hepatic triglycerides. Herein, we aimed to identify genomic variants responsible for this association.
Recombinant congenic mice carrying 5.3 Mbp of Ltg/NZO were fed a high-fat diet and characterized for liver fat. Bioinformatic analysis, mRNA profiles and electrophoretic mobility shift assays were performed to identify genes responsible for the Ltg/NZO phenotype. Candidate genes were manipulated in vivo by injecting specific microRNAs into C57BL/6 mice. Pulldown coupled with mass spectrometry-based proteomics and immunoprecipitation were performed to identify interaction partners of IFGGA2.
Through positional cloning, we identified 2 immunity-related GTPases (Ifgga2, Ifgga4) that prevent hepatic lipid storage. Expression of both murine genes and the human orthologue IRGM was significantly lower in fatty livers. Accordingly, liver-specific suppression of either Ifgga2 or Ifgga4 led to a 3-4-fold greater increase in hepatic fat content. In the liver of low-fat diet-fed mice, IFGGA2 localized to endosomes/lysosomes, while on a high-fat diet it associated with lipid droplets. Pulldown experiments and proteomics identified the lipase ATGL as a binding partner of IFGGA2 which was confirmed by co-immunoprecipitation. Both proteins partially co-localized with the autophagic marker LC3B. Ifgga2 suppression in hepatocytes reduced the amount of LC3B-II, whereas overexpression of Ifgga2 increased the association of LC3B with lipid droplets and decreased triglyceride storage.
IFGGA2 interacts with ATGL and protects against hepatic steatosis, most likely by enhancing the binding of LC3B to lipid droplets.
The genetic basis of non-alcoholic fatty liver disease remains incompletely defined. Herein, we identified members of the immunity-related GTPase family in mice and humans that act as regulators of hepatic fat accumulation, with links to autophagy. Overexpression of the gene Ifgga2 was shown to reduce hepatic lipid storage and could be a therapeutic target for the treatment of fatty liver disease.
目前，只有少数遗传变异可以解释脂肪肝疾病的遗传性。对小鼠品系的数量性状基因座（QTL）分析已经确定了染色体 18 上的易感性位点 Ltg/NZO（来自新西兰肥胖 [NZO] 等位基因的肝脏甘油三酯）与肝内甘油三酯增加有关。在此，我们旨在确定导致这种关联的基因组变异。
携带 5.3 Mbp Ltg/NZO 的重组近交系小鼠喂食高脂肪饮食，并对肝脏脂肪进行特征分析。进行生物信息学分析、mRNA 谱分析和电泳迁移率变动分析，以确定导致 Ltg/NZO 表型的基因。通过向 C57BL/6 小鼠注射特定的 microRNA，在体内操纵候选基因。进行下拉实验，结合基于质谱的蛋白质组学和免疫沉淀实验，鉴定 IFGGA2 的相互作用伙伴。
通过定位克隆，我们鉴定出 2 种免疫相关 GTPase（Ifgga2、Ifgga4），它们可以防止肝脏脂质储存。在脂肪肝中，两种鼠基因和人同源物 IRGM 的表达显著降低。相应地，肝脏特异性抑制 Ifgga2 或 Ifgga4 会使肝内脂肪含量增加 3-4 倍。在低脂饮食喂养的小鼠肝脏中，IFGGA2 定位于内体/溶酶体，而在高脂肪饮食中则与脂滴结合。下拉实验和蛋白质组学鉴定出脂肪酶 ATGL 是 IFGGA2 的结合伴侣，这通过共免疫沉淀得到了证实。这两种蛋白质与自噬标志物 LC3B 部分共定位。肝细胞中 Ifgga2 的抑制减少了 LC3B-II 的量，而 Ifgga2 的过表达增加了 LC3B 与脂滴的结合并减少了甘油三酯的储存。
IFGGA2 与 ATGL 相互作用，可防止肝脂肪变性，很可能通过增强 LC3B 与脂滴的结合来实现。
非酒精性脂肪性肝病的遗传基础仍不完全明确。在此，我们在小鼠和人类中鉴定出免疫相关 GTPase 家族成员，它们是调节肝脂肪堆积的调节剂，与自噬有关。Ifgga2 基因的过表达被证明可以减少肝内脂质储存，可能成为治疗脂肪性肝病的治疗靶点。
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