Zinc finger BED-type containing 3 promotes hepatic steatosis by interacting with polypyrimidine tract-binding protein 1.

AIMS/HYPOTHESIS: The relationship between metabolic dysfunction-associated steatotic liver disease (MASLD) and type 2 diabetes mellitus, insulin resistance and the metabolic syndrome is well established. While zinc finger BED-type containing 3 (ZBED3) has been linked to type 2 diabetes mellitus and the metabolic syndrome, its role in MASLD remains unclear. In this study, we aimed to investigate the function of ZBED3 in the context of MASLD. METHODS: Expression levels of ZBED3 were assessed in individuals with MASLD, as well as in cellular and animal models of MASLD. In vitro and in vivo analyses were conducted using a cellular model of MASLD induced by NEFA and an animal model of MASLD induced by a high-fat diet (HFD), respectively, to investigate the role of ZBED3 in MASLD. ZBED3 expression was increased by lentiviral infection or tail-vein injection of adeno-associated virus. RNA-seq and bioinformatics analysis were employed to examine the pathways through which ZBED3 modulates lipid accumulation. Findings from these next-generation transcriptome sequencing studies indicated that ZBED3 controls SREBP1c (also known as SREBF1; a gene involved in fatty acid de novo synthesis); thus, co-immunoprecipitation and LC-MS/MS were utilised to investigate the molecular mechanisms by which ZBED3 regulates the sterol regulatory element binding protein 1c (SREBP1c). RESULTS: In this study, we found that ZBED3 was significantly upregulated in the liver of individuals with MASLD and in MASLD animal models. ZBED3 overexpression promoted NEFA-induced triglyceride accumulation in hepatocytes in vitro. Furthermore, the hepatocyte-specific overexpression of Zbed3 promoted hepatic steatosis. Conversely, the hepatocyte-specific knockout of Zbed3 resulted in resistance of HFD-induced hepatic steatosis. Mechanistically, ZBED3 interacts directly with polypyrimidine tract-binding protein 1 (PTBP1) and affects its binding to the SREBP1c mRNA precursor to regulate SREBP1c mRNA stability and alternative splicing. CONCLUSIONS/INTERPRETATION: This study indicates that ZBED3 promotes hepatic steatosis and serves as a critical regulator of the progression of MASLD. DATA AVAILABILITY: RNA-seq data have been deposited in the NCBI Gene Expression Omnibus ( www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE231875 ). MS proteomics data have been deposited to the ProteomeXchange Consortium via the iProX partner repository ( https://proteomecentral.proteomexchange.org/cgi/GetDataset?ID=PXD041743 ).

N'-(2-Chloro-benzyl-idene)-2-hydr-oxy-3-methyl-benzohydrazide.

In the title compound, C(15)H(13)ClN(2)O(2), the dihedral angle between the two benzene rings is 3.4 (5)° and the mol-ecule adopts an E configuration with respect to the C=N bond. There is an intra-molecular O-H⋯O hydrogen bond in the mol-ecule, which generates an S(6) loop. In the crystal structure, mol-ecules are linked through inter-molecular N-H⋯O hydrogen bonds, forming C(4) chains running along the a axis.

N'-(2,3-Dimethoxy-benzyl-idene)-2-hydr-oxy-3-methyl-benzohydrazide.

In the title compound, C(17)H(18)N(2)O(4), the dihedral angle between the two benzene rings is 6.0 (2)° and the mol-ecule adopts an E configuration with respect to the C=N bond. There is an intra-molecular O-H⋯O hydrogen bond in the mol-ecule, which generates an S(6) ring. In the crystal, mol-ecules are linked through inter-molecular N-H⋯O hydrogen bonds, forming C(4) chains running along the c axis.

N'-(2,3-Dimethoxy-benzyl-idene)-2,4-dihydroxy-benzohydrazide.

In the title compound, C(16)H(16)N(2)O(5), the dihedral angle between the two benzene rings is 8.5 (3)° and the mol-ecule adopts an E configuration with respect to the C=N bond. There is an intra-molecular N-H⋯O hydrogen bond in the mol-ecule, which generates an S(6) ring. In the crystal, mol-ecules are linked through inter-molecular O-H⋯O hydrogen bonds, forming layers parallel to the bc plane.

2,4-Dihydr-oxy-N'-(2-hydr-oxy-4-methoxy-benzyl-idene)benzohydrazide.

In the title compound, C(15)H(14)N(2)O(5), the dihedral angle between the two benzene rings is 4.3 (3)° and the mol-ecule adopts an E configuration with respect to the C=N bond. Intra-molecular O-H⋯N and N-H⋯O hydrogen bonds are observed. In the crystal structure, the mol-ecules are linked through inter-molecular N-H⋯O and O-H⋯O hydrogen bonds to form layers parallel to the ac plane.

N'-(2,4-Dichloro-benzyl-idene)-2-hydr-oxy-3-methyl-benzohydrazide.

In the title compound, C(15)H(12)Cl(2)N(2)O(2), the dihedral angle between the two benzene rings is 6.3 (2)°. The mol-ecule adopts an E configuration with respect to the C=N bond. An intra-molecular O-H⋯O hydrogen bond is observed. In the crystal structure, the mol-ecules are linked through inter-molecular N-H⋯O and C-H⋯O hydrogen bonds to form chains running along [101].

2-Hydr-oxy-N'-(2-hydr-oxy-3-meth-oxy-5-nitro-benzyl-idene)-3-methyl-benzo-hydrazide.

In the title compound, C(16)H(15)N(3)O(6), the dihedral angle between the two benzene rings is 0.9 (2)°. The mol-ecule adopts an E configuration with respect to the C=N bond. There are intra-molecular O-H⋯N and O-H⋯O hydrogen bonds in the mol-ecule. In the crystal structure, mol-ecules are linked through inter-molecular N-H⋯O hydrogen bonds to form chains running along the c axis.

(E)-2,4-Dihydr-oxy-N'-(2-hydr-oxy-3-meth-oxy-5-nitro-benzyl-idene)benzohydrazide dihydrate.

The asymmetric unit of the title compound, C(15)H(13)N(3)O(7)·2H(2)O, consists of a hydrazone mol-ecule and two solvent water mol-ecules. The mol-ecule adopts an E configuration with respect to the C=N bond. It is relatively planar, with a dihedral angle between the two benzene rings of 2.6 (1)°. There are intra-molecular O-H⋯N and O-H⋯O hydrogen bonds in the hydrazone mol-ecule. In the crystal structure, symmetry-related mol-ecules are linked through inter-molecular N-H⋯O and O-H⋯O hydrogen bonds to form a three-dimensional network.

N'-(2,5-Dihydroxy-benzyl-idene)-2-hydr-oxy-3-methyl-benzohydrazide.

In the title compound, C(15)H(14)N(2)O(4), the dihedral angle between the two benzene rings is 4.1 (2)°. The mol-ecule adopts an E configuration with respect to the C=N bond. There are intra-molecular O-H⋯N and O-H⋯O hydrogen bonds in the mol-ecule. In the crystal structure, mol-ecules are linked through inter-molecular N-H⋯O and O-H⋯O hydrogen bonds, forming chains running along the c axis.

2-Hydr-oxy-N'-(2-hydr-oxy-4-methoxy-benzyl-idene)-3-methyl-benzohydrazide monohydrate.

In the title compound, C(16)H(16)N(2)O(4)·H(2)O, the dihedral angle between the two benzene rings is 12.4 (2)° and the mol-ecule adopts an E configuration with respect to the C=N bond. There are intra-molecular O-H⋯N and O-H⋯O hydrogen bonds in the hydrazone mol-ecule, which both generate S(6) rings. In the crystal structure, mol-ecules are linked by N-H⋯O and O-H⋯O hydrogen bonds, forming layers parallel to the ab plane. The crystal studied was a non-merohedral twin with a domain ratio of 0.887 (3):0.113 (3).