Nonalcoholic fatty liver disease (NAFLD) has become a common health care burden worldwide. The high heterogeneity of NAFLD remains elusive and impairs outcomes of clinical diagnosis and pharmacotherapy. Several NAFLD classifications have been proposed on the basis of clinical, genetic, alcoholic, or serum metabolic analyses. Yet, accurately predicting the progression of NAFLD to cirrhosis or hepatocellular carcinoma (HCC) in patients remains a challenge. Here, on the basis of a Chinese cohort of patients, we classified NAFLD into three distinct molecular subtypes (NAFLD-mSI, NAFLD-mSII, and NAFLD-mSIII) using integrative multiomics including whole-genome sequencing (WGS), proteomics, phosphoproteomics, lipidomics, and metabolomics across a broad range of liver, blood, and urine specimens. We found that NAFLD-mSI had higher expression of CYP1A2 and CYP3A4, which alleviate hepatic steatosis through mediating free fatty acid/bile acid–mTOR–FXR/PPARα signaling. NAFLD-mSII displayed an elevated risk of liver cirrhosis along with increased hepatic infiltration of M1 and M2 macrophages because of lipid-triggered hepatic CCL2 and CRP production. NAFLD-mSIII exhibited a potential risk for HCC development by increased transcription of CEBPB- and ERCC3-regulated oncogenes because of activation of the EGF-EGFR/CHKA/PI3K-PDK1-AKT cascade. Next, we validated the existence of these three NAFLD molecular subtypes in an external cohort comprising 92 patients with NAFLD across three different Chinese hospitals. These findings may aid in understanding the molecular features underlying NAFLD heterogeneity, thereby facilitating clinical diagnosis and treatment strategies with the aim of preventing the development of liver cirrhosis and HCC.

Semaphorin 7A (SEMA7A) is a membrane‐bound protein that involves axon growth and other biological processes. SEMA7A mutations are associated with vertebral fracture and Kallmann syndrome. Here, we report a case with a mutation in SEMA7A that displays familial cholestasis. WGS reveals a SEMA7A ^R148W homozygous mutation in a female child with elevated levels of serum ALT, AST, and total bile acid (TBA) of unknown etiology. This patient also carried a SLC10A1 ^S267F allele, but Slc10a1 ^S267F homozygous mice exhibited normal liver function. Similar to the child, Sema7a ^R145W homozygous mice displayed elevated levels of serum ALT, AST, and TBA. Remarkably, liver histology and LC‐MS/MS analyses exhibited hepatocyte hydropic degeneration and increased liver bile acid (BA) levels in Sema7a ^R145W homozygous mice. Further mechanistic studies demonstrated that Sema7a ^R145W mutation reduced the expression of canalicular membrane BA transporters, bile salt export pump (Bsep), and multidrug resistance‐associated protein‐2 (Mrp2), causing intrahepatic cholestasis in mice. Administration with ursodeoxycholic acid and a dietary supplement glutathione improved liver function in the child. Therefore, Sema7a ^R145W homozygous mutation causes intrahepatic cholestasis by reducing hepatic Bsep and Mrp2 expression.

Genetic polymorphisms are associated with the development of nonalcoholic fatty liver disease (NAFLD). Semaphorin7a ( Sema7a ) deficiency in mouse peritoneal macrophages reduces fatty acid (FA) oxidation. Here, we identified 17 individuals with SEMA7A heterozygous mutations in 470 patients with biopsy-proven NAFLD. SEMA7A heterozygous mutations increased susceptibility to NAFLD, steatosis severity, and NAFLD activity scores in humans and mice. The Sema7a ^R145W mutation (equivalent to human SEMA7A ^R148W ) significantly induced small lipid droplet accumulation in mouse livers compared with WT mouse livers. Mechanistically, the Sema7a ^R145W mutation increased N-glycosylated Sema7a and its receptor integrin β1 proteins in the cell membranes of hepatocytes. Furthermore, Sema7a ^R145W mutation enhanced its protein interaction with integrin β1 and PKC-α and increased PKC-α phosphorylation, which were both abrogated by integrin β1 silencing. Induction of PKC α _WT, but not PKC α _dominant negative, overexpression induced transcriptional factors Srebp1, Chrebp, and Lxr expression and their downstream Acc1, Fasn, and Cd36 expression in primary mouse hepatocytes. Collectively, our findings demonstrate that the SEMA7A ^R148W mutation is a potentially new strong genetic determinant of NAFLD and promotes intrahepatic lipid accumulation and NAFLD in mice by enhancing PKC-α–stimulated FA and triglyceride synthesis and FA uptake. The inhibition of hepatic PKC-α signaling may lead to novel NAFLD therapies.

Background and Aims: Inflammatory response is crucial for bile acid (BA)-induced cholestatic liver injury, but molecular mechanisms remain to be elucidated. Solute Carrier Family 35 Member C1 (SLC35C1) can transport Guanosine diphosphate-fucose into the Golgi to facilitate protein glycosylation. Its mutation leads to the deficiency of leukocyte adhesion and enhances inflammation in humans. However, little is known about its role in liver diseases. Approach and Results: Hepatic SLC35C1 mRNA transcripts and protein expression were significantly increased in patients with obstructive cholestasis and mouse models of cholestasis. Immunofluorescence revealed that the upregulated SLC35C1 expression mainly occurred in hepatocytes. Liver-specific ablation of Slc35c1 (Slc35c1 cKO) significantly aggravated liver injury in mouse models of cholestasis induced by bile duct ligation and 1% cholic acid–feeding, evidenced by increased liver necrosis, inflammation, fibrosis, and bile ductular proliferation. The Slc35c1 cKO increased hepatic chemokine Ccl2 and Cxcl2 expression and T cell, neutrophil, and F4/80 macrophage infiltration but did not affect the levels of serum and liver BA in mouse models of cholestasis. Liquid chromatography with tandem mass spectrometry analysis revealed that hepatic Slc35c1 deficiency substantially reduced the fucosylation of cell-cell adhesion protein CEACAM1 at N153. Mechanistically, cholestatic levels of conjugated BAs stimulated SLC35C1 expression by activating the STAT3 signaling to facilitate CEACAM1 fucosylation at N153, and deficiency in the fucosylation of CEACAM1 at N135 enhanced the BA-stimulated CCL2 and CXCL2 mRNA expression in primary mouse hepatocytes and Primary Liver Carcinoma/Poliomyelitis Research Foundation/5-ASBT cells. Conclusions: Elevated hepatic SLC35C1 expression attenuates cholestatic liver injury by enhancing CEACAM1 fucosylation to suppress CCL2 and CXCL2 expression and liver inflammation.

Liver fibrosis is a prevalent pathological stage of various chronic liver diseases and has the potential to progress to liver cirrhosis and hepatocellular carcinoma. However, experimental models for in vivo research are limited. Unexpectedly, increased liver inflammation and fibrosis were previously observed in mice treated with aluminum adjuvant (commercial Imject Alum, a mixture of Al(OH)₃ and Mg(OH)₂). Our study aimed to reveal the pathogenesis and pathological features of Imject Alum-induced liver injury and evaluate its potential as an experimental model of fibrotic liver disease. C57BL/6J mice were randomly divided into the following four groups: (i) control group, which received phosphate-buffered saline injections on days 1, 12, 26, 40, and 54; (ii) Imject Alum (Al(OH)₃ 160 mg/kg) D26 group, which was administered with Imject Alum (Al(OH)₃ 160 mg/kg) on days 1, 12, and 26; (iii) Imject Alum (Al(OH)₃ 80 mg/kg) D54; and (iv) Imject Alum (Al(OH)₃ 160 mg/kg) D54 groups, which were treated with 80 mg/kg and 160 mg/kg of Imject Alum (Al(OH)₃), respectively, on days 1, 12, 26, 40, and 54. All reagents were delivered by intraperitoneal injection. Serum biochemical parameters, liver pathology, and expression of genes related to inflammation and fibrogenesis were evaluated. Transcriptome sequencing was performed. The genetic characteristics of the Imject Alum-induced liver lesions in the existing fibrosis model and patients with cirrhosis were determined. Administration of Imject Alum (Al(OH)₃ 160 mg/kg) at certain points for 54 days led to extensive hepatic inflammation and fibrosis, accompanied by disturbed bile acid metabolism in mice. Moreover, Imject Alum aggravated liver inflammation and injury by activating the pyroptosis-related inflammasome pathway. Transcriptome analysis revealed that Imject Alum-induced liver lesions had differentially expressed genes that were significantly enriched in pathways related to inflammation, fibrogenesis, and multiple metabolic processes. Moreover, Imject Alum-induced liver lesions exhibited gene signatures similar to those of existing fibrosis models and patients with cirrhosis. Aluminum adjuvant (Imject Alum; Al(OH)₃ 160 mg/kg) administration at certain points for 54 days resulted in notable liver injury, inflammation, and fibrosis. This model had similar gene expression characteristics with existing fibrosis models and liver samples from patients with cirrhosis. Overall, aluminum adjuvant (Imject Alum)-induced mouse model may be a novel approach for establishing a liver fibrosis animal model.