Malonylation of Acetyl-CoA Carboxylase 1 Promotes Hepatic Steatosis and Is Attenuated by Ketogenic Diet in NAFLD

Protein post-translational modifications (PTMs) participate in important bioactive regulatory processes and therefore can help elucidate the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Here, we investigate the involvement of PTMs in ketogenic diet (KD)-improved fatty liver by multi-omics and reveal a core target of lysine malonylation, acetyl-CoA carboxylase 1 (ACC1). ACC1 protein levels and Lys1523 malonylation are significantly decreased by KD. It is discovered that a malonylation-mimic mutant in ACC1 increases its enzyme activity and stability to promote hepatic steatosis, whereas the malonylation-null mutant upregulates the ubiquitination degradation of ACC1. A customized Lys1523ACC1 malonylation antibody confirms the increased malonylation of ACC1 in the NAFLD samples. Overall, the lysine malonylation of ACC1 is attenuated by KD in NAFLD and plays an important role in promoting hepatic steatosis. Malonylation is critical for ACC1 activity and stability, highlighting the anti-malonylation effect of ACC1 as a potential strategy for treating NAFLD. Mouse models and dietary intervention Seven-week-old male C57BL/6 mice were purchased from GemPharmatech (Nanjing, China). After an acclimation period of one week, the mice were randomly divided into CD, HFD, iCD, and iKD groups (n = 6/group). Mice were fed a CD (11 % fat [kcal%]; Guangdong Medical Laboratory Animal Center, Guangzhou, China) in the CD group or a HFD (58 % fat [kcal%]; D12331; Research Diets, New Brunswick, NJ, USA) in the HFD group for 16 weeks. Mice in the iCD group were fed a HFD or CD sequentially at 2-week intervals for 16 weeks. Mice in the iKD group were fed a HFD or KD (90.5 % fat [kcal%], TD.160153, Envigo, USA) at 2-week intervals for 16 weeks. Blood and liver samples were collected after an overnight fast. Animal experiments complied with the ARRIVE guidelines and the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All protocols were approved by the Institutional Animal Care and Use Committee of the Sun Yat-sen University (approval number: 2020000276). RNA sequencing Total RNA was isolated using a RNeasy Plus Mini kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. The integrity and concentration of the total RNA were assessed using an Agilent 2100 Bioanalyzer System (Agilent Technologies, Palo Alto, CA, USA) and an Agilent RNA 6000 Nano Kit (Agilent Technologies). Samples with an RNA integrity number >8 were selected for use. RNA sequencing (RNA-Seq) and data analysis were performed by Pan-Guarantee Biotechnology Co. Ltd. (Guangzhou, China). Libraries were constructed using 1 μg of total RNA and prepared using the NEBNext ultra RNA Library Prep Kit (New England Biolabs, Beijing, China). Sequencing was performed using an Illumina HiSeq4000 to generate 150 bp paired-end reads. Quality trimmed reads were mapped onto the mouse genome (mm10) by HISAT233, and gene expression in terms of fragments per kilobase of transcript per million (FPKM) mapped reads of genes overlapping with gene annotations in Ensembl release 96 was calculated using RSEM (v1.1.17) 34. RNAs differential expression analysis was performed using edgeR (v 3.14.0)35 software between two different groups (n= 4). Genes with a false discovery rate < 0.05 and absolute fold change≥ 2 were considered differentially expressed genes.