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Abstract
Background In recent years, the prevalence of Nonalcoholic fatty liver disease (NAFLD) has increased and tends to be younger, which poses a significant burden on health services. As one of the important transcription factors, FOXO1 contributes to glucose and lipid metabolism, while its roles in the progression of NAFLD are controversial. This thesis will explore the molecular mechanism by which hepatocyte FOXO1 regulates NAFLD progression in both animal models in vivo and cell experiments in vitro.
Methods We constructed the NAFLD animal model with a high-fat and high-fructose diet (HFHFr) in wild-type (WT) and hepatocyte FOXO1-specific knockout mice. At the end of the experiment, we compared the liver weight, blood glucose, blood lipid, liver function and liver pathological changes between wild-type and hepatocyte FOXO1 knockout mice. Meanwhile, FOXO1 knockout cell line of hepatocytes was constructed and palmitic acid was used to induce the NAFLD cell model in vitro. Furthermore, the target genes of FOXO1 regulating lipid metabolism were predicted by transcriptome sequencing and bioinformatics analysis. Finally, the molecular mechanism of FOXO1 was verified by luciferase reporter gene assay and recovery assay.
Results The results of animal experiments in vivo showed that depletion of hepatocyte-specific FOXO1 aggravated liver injury and lipid deposition in both the normal diet control group or NAFLD modeling group (IDDF2024-ABS-0334 Figure 1. FOXO1 knockdown aggravated NAFLD phenotype in vivo. (A) The levels of serum ALT and AST (B) Representative images of HE and Oil red O staining (C) Pathology scores of HE and semi-quantitative analysis of oil red O staining). However, FOXO1 seemed to have no effect on the level of blood glucose, suggesting that FOXO1 may affect the progression of NAFLD by regulating lipid metabolism (IDDF2024-ABS-0334 Figure 1. FOXO1 knockdown aggravated NAFLD phenotype in vivo. (D)The levels of serum glucose and insulin (E) The Blood glucose levels of glucose tolerance test (GTT) and insulin tolerance test (ITT) (F) The area under curve of GTT and ITT). The transcriptome analysis revealed that knockout of FOXO1 in HepG2 cells decreased the expression of fatty acid oxidation (FAO) associated genes (IDDF2024-ABS-0334 Figure 2. FOXO1 regulated the expression of lipid oxidation-related genes in vitro. (A) Heat map demonstrating fatty acid oxidation-related genes in WT and FOXO1-KO cells treated with PA (B) The protein expression of CPT1a and PPARα (C)The mRNA expression of CPT1a and PPARα). Further studies indicated that FOXO1 could regulate the transcription of aldehyde dehydrogenase 1 family member L2 (ALDH1L2) by directly binding to its promoter (IDDF2024-ABS-0334 Figure 3. FOXO1 Induced Fatty Acid Oxidation by Targeting ALDH1L2 in vitro and in vivo. (A) Dual luciferase reporter assay with the promoter of ALDH1L2 (B and C) The mRNA and protein levels of ALDH1L2). Depletion of FOXO1 significantly reduced the protein levels of ALDH1L2 and creatine carnitine palmitoyl transferase 1a (CPT1α) in vitro and in vivo (IDDF2024-ABS-0334 Figure 3. FOXO1 Induced Fatty Acid Oxidation by Targeting ALDH1L2 in vitro and in vivo. (D) The expression of FOXO1 and ALDH1L2 was detected via immunohistochemical analysis; and that of CPT1a was detected via immunofluorescence analysis).
FOXO1 knockdown aggravated NAFLD phenotype in vivo. (A) The levels of serum ALT and AST (B) Representative images of HE and Oil red O staining (C) Pathology scores of HE and semi-quantitative analysis of oil red O staining (D)The levels of serum glucose and insulin (E) The Blood glucose levels of glucose tolerance test (GTT) and insulin tolerance test (ITT) (F) The area under curve of GTT and ITT
FOXO1 regulated the expression of lipid oxidation-related genes in vitro. (A) Heat map demonstrating fatty acid oxidation-related genes in WT and FOXO1-KO cells treated with PA. (B) The protein expression of CPT1a and PPARα. (C)The mRNA expression of CPT1a and PPARα
FOXO1 Induced Fatty Acid Oxidation by Targeting ALDH1L2 in vitro and in vivo. (A) Dual luciferase reporter assay with the promoter of ALDH1L2 (B and C) The mRNA and protein levels of ALDH1L2. (D) The expression of FOXO1 and ALDH1L2 was detected via immunohistochemical analysis; and that of CPT1a was detected via immunofluorescence analysis
Conclusions This study demonstrated that hepatocyte FOXO1 promoted fatty acid oxidation by directly targeting ALDH1L2, thus ameliorating the development of NAFLD.