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. 2023 Aug;55(8):1720-1733.
doi: 10.1038/s12276-023-01047-4. Epub 2023 Aug 1.

Thrap3 promotes nonalcoholic fatty liver disease by suppressing AMPK-mediated autophagy

Hyun-Jun Jang #  1   2 , Yo Han Lee #  1 , Tam Dao  3 , Yunju Jo  3 , Keon Woo Khim  1 , Hye-Jin Eom  1 , Ju Eun Lee  1 , Yi Jin Song  1 , Sun Sil Choi  1 , Kieun Park  1 , Haneul Ji  1 , Young Chan Chae  1 , Kyungjae Myung  4 , Hongtae Kim  1 , Dongryeol Ryu  3 , Neung Hwa Park  5 , Sung Ho Park  6 , Jang Hyun Choi  7
Affiliations

Thrap3 promotes nonalcoholic fatty liver disease by suppressing AMPK-mediated autophagy

Hyun-Jun Jang et al. Exp Mol Med. 2023 Aug.

Abstract

Autophagy functions in cellular quality control and metabolic regulation. Dysregulation of autophagy is one of the major pathogenic factors contributing to the progression of nonalcoholic fatty liver disease (NAFLD). Autophagy is involved in the breakdown of intracellular lipids and the maintenance of healthy mitochondria in NAFLD. However, the mechanisms underlying autophagy dysregulation in NAFLD remain unclear. Here, we demonstrate that the hepatic expression level of Thrap3 was significantly increased in NAFLD conditions. Liver-specific Thrap3 knockout improved lipid accumulation and metabolic properties in a high-fat diet (HFD)-induced NAFLD model. Furthermore, Thrap3 deficiency enhanced autophagy and mitochondrial function. Interestingly, Thrap3 knockout increased the cytosolic translocation of AMPK from the nucleus and enhanced its activation through physical interaction. The translocation of AMPK was regulated by direct binding with AMPK and the C-terminal domain of Thrap3. Our results indicate a role for Thrap3 in NAFLD progression and suggest that Thrap3 is a potential target for NAFLD treatment.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Increased Thrap3 expression level is associated with NAFLD.
a The expression of Thrap3 was measured in HepG2 cells treated with FFA for 24 h. The expression level was normalized to no treatment (NT) (n = 5 per group). Values represent means ± SEMs. *P < 0.05 vs. NT; b Hepatic Thrap3 levels from C57BL/6 J mice fed a high-fat diet (HFD) were normalized to those from C57BL/6 J mice fed a normal chow diet (NCD) (n = 4 per group). Values represent means ± SEMs. **P < 0.01 vs. NCD; c–e Thrap3 expression was analyzed in the livers of obese patients (GSE15653) (Lean, n = 5; Obese, n = 4) and the livers of NAFLD patients (GSE135251) (d, e). Thrap3 expression was compared across NAS scores in NAFL patients by one-way ANOVA (e). ***P < 0.001 vs. Lean (c),**P < 0.01 (d, e).
Fig. 2
Fig. 2. Thrap3 knockout improved the NAFLD phenotype.
a–g Thrap3 F/F and Thrap3 LKO mice were fed a normal chow diet (NCD, n = 5 per group) or a high-fat diet (HFD, n = 6 per group) for 12 weeks. Representative images of liver, H&E staining, Oil Red O staining and Sirius Red staining of liver slides, NAFLD activity score (NAS), fibrosis score, liver weight, hepatic TG, and hepatic cholesterol were analyzed in the indicated mice. Values represent means ± SEMs. *P < 0.05,**P < 0.01 vs. F/F HFD; h–m Thrap3 F/F (n = 6) and Thrap3 LKO (n = 5) mice were fed a methionine- and choline-deficient (HCD) diet for 4 weeks. Representative images of liver, H&E staining, Oil Red O staining and Sirius Red staining of liver slides, NAS, fibrosis score, hepatic TG, and hepatic cholesterol were analyzed in the indicated mice. Values represent means ± SEMs. *P < 0.05,**P < 0.01,***P < 0.001 vs. F/F.
Fig. 3
Fig. 3. Depletion of Thrap3 improved metabolic parameters without expression changes in FA β-oxidation and lipogenesis.
a–m Thrap3 F/F and Thrap3 LKO mice were fed a normal chow diet (NCD, n = 5 per group) or a high-fat diet (HFD, n = 6 per group) for 12 weeks. Representative liver images of mice, body weight, serum ALT, serum AST, serum TG, serum FFA, serum cholesterol, blood glucose, serum insulin, glucose tolerance (GTT), area under the curve (AUC) of GTT, insulin tolerance (ITT), and AUC of ITT were analyzed in the indicated mice. Values represent means ± SEMs. *P < 0.05, **P < 0.01, ***P < 0.001 vs. F/F HFD; n Insulin signals were analyzed in the livers of the indicated mice 20 min after insulin (0.75 U/kg) injection; o Genes related to FA β-oxidation and lipogenesis were determined by quantitative RT‒PCR. Relative values are normalized to Thrap3 F/F NCD (NCD, n = 5 per group; HFD, n = 6 per group). Values represent means ± SEMs. ***P < 0.001.
Fig. 4
Fig. 4. Thrap3 regulated autophagy.
a Liver lysates from HFD-fed mice were subjected to immunoblot analysis using the indicated antibodies; b, c Primary hepatocytes were transfected with GFP-LC3, and GFP-LC3 puncta were analyzed using fluorescence microscopy (F/F, n = 35; LKO, n = 17). Values represent means ± SEMs. ***P < 0.001 vs. F/F; d Lysates from primary hepatocytes treated with BafA1 (10 nM, 24 h) were subjected to immunoblot analysis using the indicated antibodies; e, f Livers from HFD-fed mice were analyzed using a transmission electron microscope (EM). Yellow arrows indicate autophagosomes/autolysosomes (e). The number of autophagic structures was determined from the EM images (F/F, n = 15; LKO, n = 10) (f). Values represent means ± SEMs. ***P < 0.001 vs. F/F.
Fig. 5
Fig. 5. Thrap3 regulated energy expenditure through mitochondrial quality control.
a Primary hepatocytes were transfected with GFP-LC3 and stained with MitoTracker. GFP-LC3 puncta and MitoTracker were analyzed using fluorescence microscopy (F/F, n = 11; LKO, n = 10). Values represent means ± SEMs. ***P < 0.001 vs. F/F; Mitochondria of liver from HFD-fed mice were analyzed using a transmission electron microscope (EM). Representative electron micrographs reveal nuclei (green; N), lipid droplets (red; L) and mitochondria (blue) (b). Cristae volume density (F/F, n = 37; LKO, n = 20) (c), number of mitochondria (d) and % area (e) were determined from the EM images (F/F, n = 15; LKO, n = 10). Values represent means ± SEMs.***P < 0.001, **P < 0.01 vs. F/F; f The mitochondrial DNA copy number was determined by the ratio of the mitochondrial DNA gene Polg to the nuclear DNA gene Actb (n = 3 per group). Values represent means ± SEMs. *P < 0.05 vs. F/F; g The mitochondrial respiratory chain complex proteins and mitophagy markers were determined in HFD-fed mice; h–l Oxygen consumption rate (OCR) (h, i) (n = 5 per group), OCRATP levels (j) (n = 5 per group) and extracellular acidification rate (ECAR) (k, l) (n = 3 per group) were measured in primary hepatocytes from the indicated mice. Values represent means ± SEMs. *P < 0.05, **P < 0.01, ***P < 0.001 vs. F/F; m, n The consumption rate (VO2) of HFD-fed mice was measured using CLAMS (n = 4 per group). Values represent means ± SEMs. *P < 0.05, **P < 0.01 vs. F/F.
Fig. 6
Fig. 6. Thrap3 regulated autophagy through AMPK.
a RNA-seq was performed on samples from the livers of NCD-fed Thrap3 F/F and Thrap3 LKO mice. Volcano plot of the gene expression of Thrap3 LKO (log2-fold change) compared to that of Thrap3 F/F from RNA-seq analysis; b Gene ontology enrichment analysis of differentially expressed genes in Thrap3 LKO compared to Thrap3 F/F; c Genes related to the AMPK signaling pathway were identified in the livers of HFD-fed Thrap3 F/F and Thrap3 LKO mice by quantitative RT‒PCR (n = 5 per group). Relative values are normalized to Thrap3 F/F. Values represent means ± SEMs. *P < 0.05, **P < 0.01, ***P < 0.001 vs. F/F; d Western blot analysis of phospho-AMPK, AMPK, phospho-ACC and ACC levels in the livers of NCD- or HFD-fed mice; e Western blot analysis of phospho-ULK1, ULK1, phospho-AMPK, AMPK, phospho-ACC and ACC levels in primary hepatocytes from indicated mice with nontargeting siRNA (si non) or Ampk-targeting siRNA (si Ampk); f, g Primary hepatocytes were transfected with GFP-LC3 and nontargeting siRNA (si non) or Ampk-targeting siRNA (si Ampk). GFP-LC3 puncta were analyzed using confocal microscopy (n = 8 per group). Values represent means ± SEMs. *P < 0.05 vs. F/F si non. $$P < 0.01 vs. LKO si non; h Western blot analysis of phospho-ULK1, ULK1, phospho-AMPK, AMPK, and LC3 levels in primary hepatocytes from indicated mice incubated with vehicle or 10 μM Compound C for 24 h.
Fig. 7
Fig. 7. Thrap3 regulated cytosolic translocation of AMPK via direct interaction.
a, b HepG2 cell lysates were immunoprecipitated with normal IgG (IgG), an anti-AMPK antibody or an anti-Thrap3 antibody. Precipitates and cell lysates were subjected to immunoblotting for Thrap3 and AMPK; c Schematic of the Thrap3 mutant with functional domain deletion. HepG2 cells were transfected with FLAG-tagged AMPKα and HA-tagged Thrap3 mutant. The cells were then immunoprecipitated with an anti-FLAG antibody. Precipitates and cell lysates were subjected to immunoblotting for FLAG and HA; d Nucleus and cytosolic fractions were isolated from the indicated primary hepatocytes and subjected to immunoblotting for Thrap3, AMPK, Tubulin and Lamin B1; e Nucleus and cytosolic fractions were isolated from the indicated DNA-transfected primary hepatocytes and subjected to immunoblotting for AMPK, Tubulin and Lamin B1; f Primary hepatocytes from Thrap3 F/F and Thrap3 LKO mice were transfected with GFP-Thrap3 WT or GFP-Thrap3 ΔC and immunostained with anti-AMPK antibody. Representative confocal images reveal GFP-Thrap3 WT or GFP-Thrap3 ΔC (green), AMPK (red) and nuclear DNA (blue). The white arrow indicates untransfected Thrap3 LKO hepatocytes. Fluorescence intensity profiles of GFP-Thrap3 WT or GFP-Thrap3 ΔC (green), AMPK (red) and nuclear DNA (blue) signals across nuclei (from a to b) were measured using confocal microscopy; g Lysates from the indicated DNA-transfected primary hepatocytes were subjected to immunoblotting for the indicated proteins; h Representative images of Oil Red O staining from the indicated DNA-transfected primary hepatocytes incubated with FFA for 24 h.
Fig. 8
Fig. 8. Schematic diagram of the mechanism by which Thrap3 affects NAFLD through translocation of AMPK.
Dysregulation of autophagy/mitophagy contributes to the progression of nonalcoholic fatty liver disease (NAFLD). In NAFLD, increased Thrap3 suppresses autophagy/mitophagy by sequestering AMPK in the nucleus. Inhibition of mitophagy does not effectively remove damaged mitochondria and maintain healthy mitochondria, and as a result, it leads to a decrease in energy consumption and exacerbates NAFLD progression. Liver-specific Thrap3 knockout mice show improved lipid accumulation, metabolic parameters, and mitochondrial function and enhanced autophagy/mitophagy in the NAFLD model. Thrap3 may be a potential therapeutic target for preventing and treating NAFLD by regulating the AMPK/autophagy/mitophagy axis.
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