α-Ketoglutarate prevents hyperlipidemia-induced fatty liver mitochondrial dysfunction and oxidative stress by activating the AMPK-pgc-1α/Nrf2 pathway

Abstract

α-Ketoglutarate (AKG), a crucial intermediate in the tricarboxylic acid cycle, has been demonstrated to mitigate hyperlipidemia-induced dyslipidemia and endothelial damage. While hyperlipidemia stands as a major trigger for non-alcoholic fatty liver disease, the protection of AKG on hyperlipidemia-induced hepatic metabolic disorders remains underexplored. This study aims to investigate the potential protective effects and mechanisms of AKG against hepatic lipid metabolic disorders caused by acute hyperlipidemia. Our observations indicate that AKG effectively alleviates hepatic lipid accumulation, mitochondrial dysfunction, and loss of redox homeostasis in P407-induced hyperlipidemia mice, as well as in palmitate-injured HepG2 cells and primary hepatocytes. Mechanistic insights reveal that the preventive effects are mediated by activating the AMPK-PGC-1α/Nrf2 pathway. In conclusion, our findings shed light on the role and mechanism of AKG in ameliorating abnormal lipid metabolic disorders in hyperlipidemia-induced fatty liver, suggesting that AKG, an endogenous mitochondrial nutrient, holds promising potential for addressing hyperlipidemia-induced fatty liver conditions.

Keywords: Fatty liver; Hyperlipidemia; Mitochondrial dysfunction; Nrf2; Oxidative stress; PGC-1α; α-Ketoglutarate (AKG).

Graphical abstract

Fig. 1

AKG pretreatment attenuates hepatic lipid metabolic disorders in P407-induced hyperlipidemia mice. (A) Schematic representation of animal experiments. Male C57BL/6J mice (8 weeks old) were administered with either ddH₂O or AKG (50 mg/kg/day) for 9 days, followed by intraperitoneal injection with 0.5 g/kg P407 for 24 h. Serum (B) TG content, (C) T-CHO content, (D) FFA content, Relative liver (E) TG content, (F) T-CHO content, (G) FFA content, (H) H&E staining of liver tissue, Serum (I) AST content, (J) ALT content, (K) Western blot image and (L) relative protein content statistical analysis of ACC and FASN. The values are means ± SEM, n = 6. *p < 0.05, **p < 0.01, and ***p < 0.001.

Fig. 2

AKG pretreatment improves hepatic mitochondrial dysfunction in P407-induced hyperlipidemia mice. Liver (A) ATP content, (B) Relative mtDNA copy number, (C) Western blot image and (D) statistical analysis on relative protein content of complexes I–V in mouse liver, (E) Western blot image and (F) statistical analysis on relative protein content of PGC-1α, TFAM, Drp1, MFN1, MFN2, OPA1-L, OPA1-S in mouse liver, (G) Relative activity of mitochondrial complex I–V in hyperlipidemic mouse liver. The values are means ± SEM,c n = 6. *p < 0.05, **p < 0.01, and ***p < 0.001.

Fig. 3

AKG pretreatment inhibits the loss of hepatic redox homeostasis in P407-induced hyperlipidemia mice. Liver (A) ROS, (B) LPO, (C) MDA, (D) protein carbonyl, (E) catalase activity, (F) total SOD activity, (G) SOD2 activity (H) and SOD1 activity. (I) Western blot image and (J) Statistical analysis on the relative protein content of Nrf2, Keap1, NQO1, Catalase, HO-1, SOD2, and SOD1 in the hyperlipidemic mouse liver. (K) Western blot image of SOD2 and AcSOD2, (L) statistical analysis on the relative protein content of AcSOD2/SOD2 in the liver. The values are means ± SEM, n = 6. *p < 0.05, **p < 0.01, and ***p < 0.001.

Fig. 4

AKG pretreatment attenuates PA-induced lipid metabolic disorders, mitochondrial dysfunction, and oxidative stress in HepG2 cells and primary hepatocytes. HepG2 cells were pretreated with AKG (5, 10, 25, and 50 μM) or AKG (25 μM) for 24 h, followed by PA (250 μM) challenge for another 24 h. (A) Cell viability and (B) MMP in HepG2 cells. Primary hepatocytes were pretreated with AKG (25, 50,100 μM) for 6 h, followed by PA (250 μM) challenge for another 24 h. (C) Cell viability in Primary hepatocytes. (D) Schematic representation of follow-up cell experiments. (E) mtDNA copy number, and (F) Relative mitochondria ROS determined by MitoSOX in HepG2 cells. (G) Mitochondrial morphology (stained by Mito-tracker Red) and (H) mean branch length in HepG2 cells. Relative (I) T-CHO, (J) TG, (K) ROS in HepG2 cells. Relative (L) T-CHO, (M) TG, (N) ROS in primary hepatocytes. The values are means ± SEM from at least three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

AKG pretreatment attenuates PA-induced hepatocyte dysfunction through PGC-1α. (A) Western blot image and (B) Statistical analysis on relative protein levels of PGC-1α, MFN1, MFN2, Nrf2, NQO1, HO-1, ACC, and FASN in the HepG2 cells. (C) Western blot image and (D) Statistical analysis on relative protein levels of PGC-1α and Nrf2 in primary hepatocytes. HepG2 cells were treated with the Ctrl siRNA, PGC-1α siRNA, or/and Nrf2 siRNA before adding AKG and PA for 48 h. (E) Relative T-CHO, (F) Relative TG, (G) Relative ROS content. The values are means ± SEM from at least three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001.

Fig. 6

AKG pretreatment inhibits the ubiquitination of endogenous Nrf2 in lipid-overloaded mouse liver. (A) Western blot image and (B) Statistical analysis on the relative protein content of Nrf2 in the cytoplasm and nuclear in the liver. (C) Western blot image and (D) Statistical analysis on the relative protein content of Nrf2 in the cytoplasm and nuclear in the HepG2 cells. (E) Immunoprecipitated Nrf2 in liver tissue then detect the ubiquitination of endogenous Nrf2 by Immunoblot analysis. Add 20 μM MG132 to cells 6 h before collection. (F) Immunoprecipitated Nrf2 in HepG2 cells then detect the ubiquitination of endogenous Nrf2 by Immunoblot analysis. The values are means ± SEM from at least three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001.

Fig. 7

AKG pretreatment activates PGC-1α/Nrf2 by regulating AMPK phosphorylation. (A) Western blot image of AMPK and p-AMPK, statistical analysis on the relative protein content of (B) p-AMPK/AMPK and (C) p-AMPK in mouse liver, (D) Western blot image of AMPK and p-AMPK, statistical analysis on the relative protein levels of (E) p-AMPK/AMPK and (F) p-AMPK in HepG2 cells. HepG2 cells were treated with or without AMPK inhibitor Compound C (10 μM) before adding AKG (25 μM) for 1h. (G) Western blot image and (H) Statistical analysis on PGC-1α, Nrf2, ACC. (I) Relative T-CHO, (J) Relative TG, (K) Relative ROS in HepG2 cells that inhibited AMPK phosphorylation. The values are means ± SEM from at least three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001.