Fig 1: HCF1/OGT complex‐mediated O‐GlcNAcylation of LIMA1 Thr662 enhances LIMA1 protein stability by counteracting its ubiquitylation. A, B) Increased LIMA1 stability was determined by cycloheximide chase (CHX) assay. Following 24 h of PBS/OPA or PBS/NAG‐thiazoline (NGT, 0.008 м) treatment, MPH were exposed to CHX (20 µм) for the indicated times. The cells were then harvested and lysed for western blot analysis. C) Cycloheximide chase assay showing that OGT overexpression increases LIMA1 stabilization. HEK293T cells were transfected with Flag‐LIMA1/His‐OGT and then exposed to CHX (50 µм) for the indicated times. The cells were then harvested and lysed for western blot analysis. D) Co‐IP showing the interaction between LIMA1 and ubiquitin was decreased by HCF1 overexpression in HEK293T cells. The cells were co‐transfected with Flag‐LIMA1/HA‐Ubi/MYC‐HCF1 and treated with MG132 for 4 h. After immunoprecipitation with Flag antibodies, immunoprecipitates were analyzed using an anti‐HA antibody. E) Co‐IP showing the interaction between LIMA1 and ubiquitin was decreased by OGT overexpression in HEK293T cells. The cells were co‐transfected with Flag‐LIMA1/HA‐Ubi/His‐OGT and treated with MG132 for 4 h. F, G) Cycloheximide chase assay shows that HCF1 or OGT overexpression does not change LIMA1 stabilization. HEK293T cells were transfected with Flag‐LIMA1∆T662/MCF‐HCF1 or Flag‐LIMA1∆T662/His‐OGT, then exposed to CHX (50 µм) for the indicated times. The cells were then harvested and lysed for western blot analysis. H) Co‐IP showing the interaction between LIMA1 and ubiquitin was not changed by HCF1 overexpression in HEK293T cells. The cells were co‐transfected with Flag‐LIMA1∆T662/HA‐Ubi/MYC‐HCF1 and treated with MG132 for 4 h. I) Co‐IP showing the interaction between LIMA1 and ubiquitin was not changed by OGT overexpression in HEK293T cells. The cells were co‐transfected with Flag‐LIMA1∆T662/HA‐Ubi/His‐OGT and treated with MG132 for 4 h.
Fig 2: Benzyl‐α‐GalNAc (BAGN) inhibits LIMA1 O‐GlcNAcylation, hepatic steatosis, and liver injury in HFD‐fed mice and CDAHFD‐fed mice. A) Western blot analysis of O‐GlcNAc, LIMA1, OGT, OGA, α‐SMA, and Col3A1 in livers from NCD‐fed mice, HFD‐fed mice, and HFD‐fed mice injected with BAGN. B) Immunofluorescence showing LIMA1 (red) and O‐GlcNAc (green) colocalization in livers from NCD‐fed mice, HFD‐fed mice, and HFD‐fed mice injected with BAGN. Scale bars, 50 µm. C‐F) Changes in body weight, EATW/BW (EATW/BW = relative weight of epididymal adipose tissue to body weight), results of IPGTT and ITT assays, and fast glycemia, serum TG, serum AST, serum ALT levels of NCD‐fed mice, HFD‐fed mice, and HFD‐fed mice injected with BAGN (n = 6). G) Tunel (Scale bars, 50 µm) and Oil red O (Scale bars, 50 µm) staining in livers from NCD‐fed mice, HFD‐fed mice, and HFD‐fed mice injected with BAGN. H) Lipometabolic mRNA expression in the liver of NCD‐fed mice, HFD‐fed mice, and HFD‐fed mice injected with BAGN (n = 6). I) Western blot analysis of O‐GlcNAc, LIMA1, OGT, OGA, α‐SMA, and Col3A1 in livers from NCD‐fed mice, CDAHFD‐fed mice, and CDAHFD‐fed mice injected with BAGN. J) Immunofluorescence showing LIMA1 (red) and O‐GlcNAc (green) colocalization in livers from NCD‐fed mice, CDAHFD‐fed mice, and CDAHFD‐fed mice injected with BAGN. Scale bars, 50 µm. K‐M) Changes in body weight, liver index, EATW/BW, and serum TG, serum AST, serum ALT levels of NCD‐fed mice, CDAHFD‐fed mice, and CDAHFD‐fed mice injected with BAGN (n = 6). N) Tunel (Scale bars, 50 µm), Oil red O (Scale bars, 50 µm), F4/80 (Scale bars, 50 µm), and MASSON (Scale bars, 100 µm) staining in livers from NCD‐fed mice, CDAHFD‐fed mice, and CDAHFD‐fed mice injected with BAGN. O) Lipometabolic mRNA expression in the liver of NCD‐fed mice, CDAHFD‐fed mice, and CDAHFD‐fed mice injected with BAGN (n = 6). The data were plotted as Mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 by one‐way ANOVA.
Fig 3: Lipotoxicity induces O‐GlcNAcylation of LIMA1 threonine 662 and upregulates LIMA1 protein levels in hepatocytes. A) Mass spectrometry analysis of proteins undergoing O‐GlcNAcylation in normal human hepatocytes and OPA‐treated human hepatocytes for 24 h. B) Interaction between LIMA1 and O‐GlcNAc by Co‐IP in OPA‐treated MPH at 0 and 24 h. C) Immunofluorescence showing LIMA1 (red) and O‐GlcNAc (green) colocalization in MPH. Scale bars, 10 µm. D) Interaction between LIMA1 and O‐GlcNAc by Co‐IP in OPA‐treated HepG2 cells at 0 and 24 h. E) Immunofluorescence showing LIMA1 (red) and O‐GlcNAc (green) colocalization in HepG2 cells. Scale bars, 10 µm. F, G) Western blot analysis of LIMA1, O‐GlcNAc, OGT, and OGA in livers from NCD‐fed mice, HFD‐fed mice, and CDAHFD‐fed mice (n = 5). H) Mass spectrometry of O‐GlcNAcylated peptides from LIMA1 in OPA‐treated HepG2 cells. I) Interaction between LIMA1 and O‐GlcNAc by Co‐IP in HepG2 cells transfected with Flag‐LIMA1 and Flag‐LIMA1ΔT662. After immunoprecipitation with Flag antibodies, immunoprecipitates were analyzed using an anti‐O‐GlcNAc antibody. The data were plotted as Mean ± SEM. ** p < 0.01 by Student's t‐test.
Fig 4: LC‐MS/MS analysis of N‐sEV and MPH‐sEV and predictive value of serum‐derived small extracellular vesicles (sEV) LIMA1 for MASH patients. A) Western blot analysis of CD9, ALIX, TSG101, and Calnexin in mouse primary hepatocytes (MPH), normal MPH‐derived from small extracellular vesicles (N‐sEV), and steatotic MPH‐derived from sEV (MPH‐sEV). B) Representative result of nanoparticle tracking analyses (NTA) of N‐sEV and MPH‐sEV. C) Representative transmission electron microscope (TEM) image of N‐sEV and MPH‐sEV. Scale bar, 100 nm. D) Proteomic profiling of MPH‐sEV as compared to N‐sEV was analyzed by LC‐MS/MS (n = 3). Heat map of the top 20 differentially expressed proteins in the metabolism signaling pathway. E, F) Western blot analysis of LIMA1 in MPH and HepG2 cells treated with oleic acid and palmitic acid (OPA) for 0, 8, 16, and 24 h (n = 3). G, H) Western blot analysis of LIMA1 in livers from normal chow diet (NCD)‐fed mice, HFD‐fed mice, and CDAHFD‐fed mice at different weeks (n = 3). I) Relative serum LIMA1 levels in patients with MASH and healthy control (n = 50). J) Receiver operating characteristic curve of the performance of serum LIMA1 levels in diagnosing MASH among 50 patients with biopsy‐proven MASLD. The AUROC is shown. K) Correlation of serum LIMA1 levels with serum aspartate transaminase (AST) and alanine aminotransferase (ALT) levels. L) Receiver operating characteristic curve of the performance of serum LIMA1 levels combined with other clinical risk factors in diagnosing MASH among 50 patients with biopsy‐proven MASLD. The AUROC is shown. M) Relative serum‐derived sEV LIMA1 levels in patients with MASH and healthy control (n = 50). N) Receiver operating characteristic curve of the performance of serum‐derived sEV LIMA1 levels and Fib‐4 (Fib‐4 = age×AST/PLT×√ALT) in diagnosing MASH among 50 patients with biopsy‐proven MASLD. The AUROC is shown. O) Correlation of serum‐derived sEV LIMA1 levels with serum AST and ALT levels. P) Receiver operating characteristic curve of the performance of serum‐derived sEV LIMA1 levels combined with other clinical risk factors in diagnosing MASH among 50 patients with biopsy‐proven MASLD. The data were plotted as Mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 by Student's t‐test for I and M; by one‐way ANOVA for E, F, G and H.
Fig 5: LIMA1 increases lipid deposition in hepatocytes by upregulating FASn expression. A) Gene set enrichment analysis (GSEA) revealed the downregulation of fatty acid biosynthesis‐associated signaling in LIMA1 HKO mice fed with HFD at 20 weeks. B‐D) The gene‐level counts of FASn and LIMA1 from the GEO database (GSE130970) were normalized to log2‐counts per million reads (CPM). Correlation of FASn with liver non‐alcoholic fatty liver activity score (NAS) or LIMA1. E) Western blot analysis of LIMA1 and FASn in OPA‐treated HepG2 cells transfected with siCtr or siLIMA1 (n = 3). F) Nile red staining of intracellular lipid droplets in OPA‐treated HepG2 cells transfected with siCtr or siLIMA1 (n = 6). Scale bars, 20 µm. G) Western blot analysis of LIMA1 and FASn in siLIMA1 transfected OPA‐treated HepG2 cells co‐transfected with pcDNA3.1 vector (pCtr) or pcDNA3.1‐FASn (pFASn). siCtr transfected OPA‐treated HepG2 cells served as a control (n = 3). H) Nile red staining of intracellular lipid droplets in siCtr transfected OPA‐treated HepG2 cells, siLIMA1 transfected OPA‐treated HepG2 cells co‐transfected with pCtr or pFASn (n = 6). Scale bars, 20 µm. I) Nile red staining of intracellular lipid deposition in OPA‐treated HepG2 cells transfected with siCtr or FASn siRNA (siFASn) (n = 6). Scale bars, 20 µm. J) Nile red staining of intracellular lipid droplets in pcDNA3.1‐LIMA1 (pLIMA1) transfected HepG2 cells co‐transfected with siCtr or siFASn. pCtr transfected HepG2 cells served as a control (n = 6). Scale bars, 20 µm. The data were plotted as Mean ± SEM. ** p < 0.01, *** p < 0.001 by one‐way ANOVA.
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