Nonalcoholic fatty liver disease (NAFLD) is supposed to manifest its metabolic phenotype in the liver, but it is common to have lean individuals diagnosed with NAFLD, known as lean NAFLD. We conducted a two-stage analysis to identify NAFLD-associated loci in Japanese patients. In stage I, 275 metabolically healthy normal-weight patients with NAFLD were compared with 1,411 non-NAFLD controls adjusted for age, sex, and alcohol consumption by a genomewide association study (GWAS). In stage II, human leukocyte antigen (HLA) in chromosome 6 (chr6) (P = 6.73E-08), microRNA (MIR) MIR548F3 in chr7 (P = 4.25E-07), myosin light chain 2 (MYL2) in chr12 (P = 4.39E-07), and glycoprotein precursor (GPC)6 in chr13 (P = 5.43E-07), as suggested by the GWAS, were assessed by single nucleotide polymorphism (SNP) association analysis of whole NAFLD against non-NAFLD in 9,726 members of the general population. A minor allele of the secondary lead SNP in chr6, rs2076529, was significantly associated (odds ratio [OR], 1.19; 95% confidence interval [CI], 1.11-1.28; P = 2.10E-06) and the lead SNP in chr7 was weakly associated (OR 1.15; 95% CI, 1.04-1.27; P = 6.19E-03) with increased NAFLD risk. Imputation-based typing of HLA showed a significant difference in the distribution of HLA-B, HLA-DR-beta chain 1 (DRB1), and HLA-DQ-beta chain 1 (DQB1) alleles in lean NAFLD GWAS. Next-generation sequence-based typing of HLA in 5,649 members of the general population replicated the significant difference of HLA-B allele distribution and the significant increase of the HLA-B*54:01 allele in whole NAFLD. Fecal metagenomic analysis of 3,420 members of the general population showed significant dissimilarity in beta-diversity analysis of rs2076529 and HLA-B*54:01 allele carriers from noncarriers. Veillonellaceae was increased but Verrucomicrobia was decreased in rs2076529 minor allele and HLA-B*54:01 allele carriers as in NAFLD. Conclusion: HLA was identified as a novel locus associated with NAFLD susceptibility, which might be affected by the alteration of gut microbiota. (Hepatology Communications 2020;4:1124-1135). N onalcoholic fatty liver disease (NAFLD) is defined as liver steatosis without alcoholic liver injury and other liver disease from apparent pathogenic factors, e.g., pharmacological. The global prevalence of NAFLD was estimated at 25.2% by a recent systematic review. (1) The number of patients with NAFLD is increasing annually due to the gradual increment in the prevalence of metabolic syndrome. (2)

N4BP2l1, which is highly expressed in oral squamous cell carcinoma, is associated with poor prognosis. However, N4bp2l1 's role in adipogenesis remains unknown. We aimed to clarify the expression profile and transcriptional regulation of N4bp2l1 to elucidate the functions underlying the role of N4bp2l1 in adipocyte differentiation. Our results revealed that N4bp2l1 mRNA expression increased in 3T3-L1 cells in a differentiation-dependent manner. To investigate the transcriptional regulation of N4bp2l1 , the 2-kb 5′ region upstream of the mouse N4bp2l1 promoter was cloned into a luciferase vector. Luciferase reporter assays indicated that USF1 induces the N4bp2l1 promoter activity. Electrophoretic mobility shift and chromatin immunoprecipitation assays confirmed that USF1 directly binds to the Ebox in the N4bp2l1 promoter. Furthermore, the expressions of adipocyte differentiation markers significantly decreased in N4bp2l1 -knockdown cells compared with those in control cells. Our results demonstrated that N4bp2l1 is a novel USF1 target gene that may be involved in adipogenesis regulation.

The present study aimed to investigate the transcriptional regulation of Kbtbd11 in adipose tissue. To elucidate the physiological role of Kbtbd11 gene expression, adipose Kbtbd11 mRNA expression levels were estimated under various feeding states in wild-type mice. Kbtbd11 expression increased in a time-dependent manner in the adipose tissue in mice fed on chow diet, whereas the promotion of Kbtbd11 mRNA expression by refeeding was attenuated in mice fed on high-fat (HF) diet, suggesting the suppression of Kbtbd11 mRNA expression under HF diets and that changes in mRNA levels were associated with regulation of the transcription activity of Kbtbd11 by some transcription factors. To investigate the transcriptional regulation of Kbtbd11, the fragment upstream of either mouse Kbtbd11 or human KBTBD11 promoter was inserted into a luciferase vector. Luciferase reporter assays revealed that both mouse and human KBTBD11 promoter activity was increased by USF1. Direct USF1 binding to the Ebox in the Kbtbd11 promoter was confirmed by electrophoretic mobility shift and chromatin immunoprecipitation assays. In addition, the adipocyte differentiation marker levels increased instantly in Kbtbd11-overexpressing Usf1 knockdown cells than in Usf1 knockdown cells. These results imply an association of between Kbtbd11 with Usf1 expression and suggest the involvement of Kbtbd11 in a novel adipogenesis pathway.

Our previous genome-wide association study to explore genetic loci associated with lean nonalcoholic fatty liver disease (NAFLD) in Japan suggested four candidate loci, which were mapped to chr6, chr7, chr12 and chr13. The present study aimed to identify the locus involved functionally in NAFLD around the association signal observed in chr13. Chromosome conformation capture assay and a database survey suggested the intermolecular interaction among DNA fragments in association signals with the adjacent four coding gene promoters. The four genes were further screened by knockdown (KD) in mice using shRNA delivered by an adeno-associated virus vector (AAV8), and KD of G protein-coupled receptor 180 (Gpr180) showed amelioration of hepatic lipid storage. Gpr180 knockout (KO) mice also showed ameliorated hepatic and plasma lipid levels without influencing glucose metabolism after high-fat diet intake. Transcriptome analyses showed downregulation of mTORC1 signaling and cholesterol homeostasis, which was confirmed by weakened phosphorylation of mTOR and decreased activated SREBP1 in Gpr180KO mice and a human hepatoma cell line (Huh7). AAV8-mediated hepatic rescue of GPR180 expression in KO mice showed recovery of plasma and hepatic lipid levels. In conclusion, ablation of GPR180 ameliorated plasma and hepatic lipid levels, which was mediated by downregulation of mTORC1 signaling.

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