AGK regulates the progression to NASH by affecting mitochondria complex I function

Abstract

Background: Impaired mitochondrial function contributes to non-alcoholic steatohepatitis (NASH). Acylglycerol kinase (AGK) is a subunit of the translocase of the mitochondrial inner membrane 22 (TIM22) protein import complex. AGK mutation is the leading cause of Sengers syndrome, characterized by congenital cataracts, hypertrophic cardiomyopathy, skeletal myopathy, lactic acidosis, and liver dysfunction. The potential roles and mechanisms of AGK in NASH are not yet elucidated. Methods: Hepatic-specific AGK-deficient mice and AGK G126E mutation (AGK kinase activity arrest) mice were on a choline-deficient and high-fat diet (CDAHFD) and a methionine choline-deficient diet (MCD). The mitochondrial function and the molecular mechanisms underlying AGK were investigated in the pathogenesis of NASH. Results: The levels of AGK were significantly downregulated in human NASH liver samples. AGK deficiency led to severe liver damage and lipid accumulation in mice. Aged mice lacking hepatocyte AGK spontaneously developed NASH. AGK G126E mutation did not affect the structure and function of hepatocytes. AGK deficiency, but not AGK G126E mice, aggravated CDAHFD- and MCD-induced NASH symptoms. AGK deficiency-induced liver damage could be attributed to hepatic mitochondrial dysfunction. The mechanism revealed that AGK interacts with mitochondrial respiratory chain complex I subunits, NDUFS2 and NDUFA10, and regulates mitochondrial fatty acid metabolism. Moreover, the AGK DGK domain might directly interact with NDUFS2 and NDUFA10 to maintain the hepatic mitochondrial respiratory chain complex I function. Conclusions: The current study revealed the critical roles of AGK in NASH. AGK interacts with mitochondrial respiratory chain complex I to maintain mitochondrial integrity via the kinase-independent pathway.

Keywords: NDUFS2; fatty acid metabolism; mitochondrial ROS; mitochondrial respiratory chain.

Figure 1

Liver AGK expression level decreases in NASH patients, and AGK deficiency leads to NASH in mice. (A) Representative liver IHC and positive area analysis of NASH patients (n = 18) and control tissues (n = 4). Black scale bar: 50 µm; White scale bar: 20 µm. ***p < 0.001. (B-E) Serum levels (B-C), Liver H&E, Oil red O staining (D), Sirius red and Masson staining, and immunostaining for αSMA and CD45 (E) in Agk^f/f and Agk^-/- mice at the age of 4 months and 18 months (n = 4-5/group; *p < 0.05, **p < 0.01, ***p < 0.001). ALT, alanine aminotransferase; AST, aspartate aminotransferase; TG, triglyceride; CHOL, cholesterol. Black scale bar: 50 µm. (F) Quantification of Oil red O, Sirius red, and αSMA, CD45 positive areas (n = 3,5/group; *p < 0.05, **p < 0.01, ***p < 0.001). (G) The TBARS levels of plasma in Agk^f/f and Agk^-/- mice at the age of 18 months. TBARS, thiobarbituric acid reactive substances ***p < 0.001. (H) mRNAs levels of fibrosis and inflammation marker genes in Agk^f/f and Agk^-/- mice (n = 4/group; **p < 0.01, ***p < 0.001).

Figure 2

AGK deficiency promotes NASH. (A) Body weight curves and liver weight to body weight (LBW) ratio of Agk^f/f and Agk^-/- mice in the CDAHFD model (n = 5-6/group; *p < 0.05). (B-D) Liver serum levels (B), H&E, Oil red O, Sirius red, Masson staining, and quantification of positive areas (C), immunostaining for αSMA, CD45, and quantification (D) of Agk^f/f and Agk^-/- mice on CDAHFD (n = 3-6; *p < 0.05, ***p < 0.001). Black scale bar: 50 µm. (E) mRNAs levels of fibrosis and inflammation marker genes in Agk^f/f and Agk^-/- mice livers in the CDAHFD group (n = 4; *p < 0.05, **p < 0.01, ***p < 0.001).

Figure 3

AGK G126E mutant has no effect on liver function. (A) Genotyping of AGK-G126E PM mice by sequencing. (B-F) Serum levels (B), liver H&E and Oil red O (C), Masson and Sirius red staining (D), immunostaining for αSMA and CD45 (E), and mRNAs levels of fibrosis and inflammation marker genes (F) in PM and WT mice on chow diet and CDAHFD, respectively (n = 4-5/group; n.s., not significant). Black scale bar: 50 µm.

Figure 4

AGK regulates NASH by affecting mitochondrial function. (A-B) Electron microscopy of mice liver mitochondria. A, Agk^f/f and Agk^-/- mice; B, PM and WT mice. The bars represent 200 nm. (C-D) Extracellular flux analysis of the OCRs of mouse hepatocytes. C, Agk^f/f and Agk^-/- mice; D, PM and WT mice. OCR was normalized to the protein amount (n = 4; *p < 0.05, **p < 0.01, n.s., not significant). (E-F) The membrane potential of mouse liver mitochondria. E, Agk^f/f and Agk^-/- mice; F, PM and WT mice (n = 4; ***p < 0.001, n.s., not significant).

Figure 5

AGK interacts with mitochondrial complex I by anchoring NDUFS2 and NDUFA10 subunits. (A) Coomassie staining of HEK293T cells transfected with AGK-Flag and vector, and the MS/MS spectrum of NDUFS2 (TYLQALPYFDR) and NDUFA10 (YSPGYNTEVGDK). (B-C) Coimmunoprecipitation of the lysates of HEK293T cells transfected with NDUFS2-HA, NDUFA10-HA, and AGK-Flag, respectively. (D) The expression of NDUFS2, NDUFA10, TIMM22, ANT1 and GC-1 in Agk^f/f mice and Agk^-/- mice hepatocytes. (E) The expression of TIMM22, NDUFS2, NDUFA10, and AGK in sgTIM22 cell line. (F-G) Co-immunoprecipitation of the lysates of HEK293T cells transfected with the truncated form of AGK (AGK-1-Flag, 25kd) and full-length AGK (AGK-Flag, 47kd). TM, transmembrane; DGK, diacylglycerol kinase. (H) Sequences of five AGK peptides. (I) The binding levels of AGK peptides with NDUFS2 and NDUFA10. AGK peptide 1 (59Q-92G), AGK peptide 2 (93M-120I), AGK peptide 3 (121V-145S), AGK peptide 4 (146K-177D) and AGK peptide 5 (178A-202P) (n = 6, *p < 0.05, ***p < 0.001; n.s., not significant).

Figure 6

AGK is important for maintaining the stability of NDUFS2 and NDUFA10. (A) Immunofluorescence images of LO₂ cell line transfected with NDUFS2-HA, NDUFA10-HA, and AGK-Flag. The cells were stained with Flag antibodies (Alexa Fluor 488), HA antibodies (rhodamine), DAPI, and mitochondria-targeting dye (Mito-Tracker Deep Red, Alexa Fluor 647). The bars represent 5 µm. (B) mRNAs levels of NDUFS2 and NDUFA10 in Agk^f/f and Agk^-/- mice (n = 4, n.s., not significant). (C) Immunofluorescence images of NDUFS2, NDUFA10 and lysosomes-targeting dye (Lamp1) in the primary hepatocytes of Agk^f/f and Agk^-/- mice. Hepatocytes were stained with NDUFS2 antibodies (rhodamine), NDUFA10 antibodies (rhodamine), DAPI, and Lamp1 (Alexa Fluor 488). The bars represent 10 µm.

Figure 7

AGK-deficiency leads to mitochondrial complex I activity defect and fatty acid accumulation. (A-B) The kinetic activity of mitochondrial complex I in mouse hepatocytes. A, Agk^f/f and Agk^-/- mice; B, PM and WT mice (n = 4; ***p < 0.001, n.s., not significant). (C-D) The ROS levels of mouse liver mitochondria. C. Agk^f/f and Agk^-/- mice; D. PM and WT mice (n = 6; **p < 0.01, n.s., not significant). (E-F) Analysis of long-chain acylcarnitine species by liquid chromatography-mass spectrometry (LC-MS) in mouse livers. E, Agk^f/f and Agk^-/- mice; F, PM and WT mice. (n = 7; *p < 0.05, **p < 0.01, n.s., not significant).