Hepatic mitochondrial NAD + transporter SLC25A47 activates AMPKα mediating lipid metabolism and tumorigenesis

Abstract

Background aims: SLC25A47 was initially identified as a mitochondrial HCC-downregulated carrier protein, but its physiological functions and transport substrates are unknown. We aimed to investigate the physiological role of SLC25A47 in hepatic metabolism.

Approach results: In the treatment of hepatocytes with metformin, we found that metformin can transcriptionally activate the expression of Slc25a47 , which is required for AMP-activated protein kinase α (AMPKα) phosphorylation. Slc25a47 -deficient mice had increased hepatic lipid content, triglycerides, and cholesterol levels, and we found that Slc25a47 deficiency suppressed AMPKα phosphorylation and led to an increased accumulation of nuclear SREBPs, with elevated fatty acid and cholesterol biosynthetic activities. Conversely, when Slc25a47 was overexpressed in mouse liver, AMPKα was activated and resulted in the inhibition of lipogenesis. Moreover, using a diethylnitrosamine-induced mouse HCC model, we found that the deletion of Slc25a47 promoted HCC tumorigenesis and development through the activated mammalian target of rapamycin cascade. Employing homology modeling of SLC25A47 and virtual screening of the human metabolome database, we demonstrated that NAD + was an endogenous substrate for SLC25A47, and the activity of NAD + -dependent sirtuin 3 declined in Slc25a47 -deficient mice, followed by inactivation of AMPKα.

Conclusions: Our findings reveal that SLC25A47, a hepatocyte-specific mitochondrial NAD + transporter, is one of the pharmacological targets of metformin and regulates lipid homeostasis through AMPKα, and may serve as a potential drug target for treating NAFLD and HCC.

Graphical abstract

FIGURE 1

Metformin increases the expression of Slc25a47. (A) RNA-sequencing analysis volcano plot of metformin regulation of multiple SLC family proteins. (B) Relative mRNA of Slc25a47 in the primary hepatocytes treated with and without metformin (500 μM) for 6 hours (n=3). (C) Activation of SLC25A47 promoter in HepG2 and Huh7 cells by metformin (HepG2 n=3; Huh7 n=4). (D) Single-cell sequencing analysis of the expression of Slc25a47 in a different type of mouse liver cells. (E) Immunoblots of AMPKα in hepatocytes of WT and Slc25a47-KO mice treated with and without metformin (500 μM) for 6 hours. (F) Inhibitory effect on glucose-6-phosphatase catalytic subunit after metformin treatment in primary hepatocytes of WT and Slc25a47-KO mice (n=3). Ctrl, Control; Met, Metformin. Data are presented as mean±SEM. *p < 0.05, ***p < 0.001,****p < 0.0001, ns, not significant. Abbrevations: KO, knockout; WT, wildtype.

FIGURE 2

Slc25a47-deficiency disrupts liver lipid metabolism homeostasis by increasing fatty acid and cholesterol biosynthesis. (A) The liver appearance and liver index of 12-week-old ob/ob (WT) and ob/ob (Slc25a47-KO) mice (n=6). (B) TAG and CHO contents of serum and liver from 12-week-old ob/ob (WT) and ob/ob (Slc25a47-KO) mice (n=6). (C) Liver H&E staining, ORO staining, and TEM of 12-week-old ob/ob (WT) and ob/ob (Slc25a47-KO) mice. (D) Lipidomic analysis of hepatic lipids from ob/ob (WT) and ob/ob (Slc25a47-KO) mice (n=3). (E) Activation of fatty acid and cholesterol biosynthesis pathways by Slc25a47 knockout (n=3). Immunoblots of fatty acid (F) and cholesterol (G) metabolism pathway in the liver of ob/ob (WT) and ob/ob. (Slc25a47-KO) mice. Immunoblots (H) and its quantitative analysis (I) of full length (fl-) and nuclear (n-) SREBPs expression in the liver of ob/ob (WT) and ob/ob (Slc25a47-KO) mice. IHC staining (J) and its quantitative analysis (K) of SREBPs expression in the liver of ob/ob (WT) and ob/ob (Slc25a47-KO) mice. Data are presented as mean±SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Abbreviations: CHO, cholesterol; IHC, immunohistochemical; KO, knockout; ORO, Oil Red O; SREBPs, sterol regulatory element binding proteins; TAG, triglycerides; transmission electron microscopy; TEM,transmission electron microscopy; WT, wildtype.

FIGURE 3

Slc25a47 deficiency promotes lipid synthesis. (A) The fractional contribution of pyruvate, amino acid, and fatty acid in basal and maximal mitochondrial respiration of primary hepatocytes of WT and Slc25a47-KO mice calculated by the inhibition percentage of UK5099, AOA, and Etomoxir, respectively (n=5). (B) Scheme outlining the path of ¹³C-Glutamine in the TCA cycle of primary hepatocytes. (C) Accumulation kinetics of ¹³C isotope-labeled TCA cycle intermediates during the course of ¹³C-Glutamine tracing (n=4). (D) Malonyl-CoA content in primary hepatocytes of the WT and Slc25a47-KO mice (n=4). (E) Fractional abundance of triglyceride palmitate (C16:0 FA) isotopomers in primary hepatocytes from the WT and Slc25a47-KO mice traced with 10 mM ¹³C-Glutamine (WT n=4; Slc25a47-KO n=3). (F-G) Abundance of the most enriched triglyceride FA (C16:1, C18:0, C18:1) isotopomers in primary hepatocytes from WT and Slc25a47-KO mice traced with 10 mM ¹³C-Glutamine (WT n=4; Slc25a47-KO n=3). Data are presented as mean±SEM. nd means not detected. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant. Abbreviations: AOA, aminooxyacetate; KO, knockout; WT, wildtype.

FIGURE 4

Overexpression of Slc25a47 inhibits lipid accumulation. (A) The liver appearance and liver index of Ad-GFP and Ad-Slc25a47 mice (n=5). (B) Slc25a47 expression in the liver of Ad-GFP and Ad-Slc25a47 mice (n=5). (C) Immunoblots of SLC25A47-His in the liver of Ad-GFP and Ad-Slc25a47 mice. (D) TAG and CHO contents in the serum and liver of Ad-GFP and Ad-Slc25a47 mice (n=5). (E) ORO staining in liver tissues of Ad-GFP and Ad-Slc25a47 mice. (F) Immunoblots of AMPKα and SREBPs in the liver of Ad-GFP and Ad-Slc25a47 mice. (G) Quantitative analysis of immunoblotting for full length (fl-) and nuclear (n-) SREBPs expression in the liver of Ad-GFP and Ad- Slc25a47 mice. (H) Immunoblots of fatty acid and the cholesterol metabolism pathway in the liver of Ad-GFP and Ad-Slc25a47 mice. Data are presented as mean±SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Abbreviations: AMPKα, AMP-activated protein kinase α; CHO, cholesterol; ORO, Oil Red O; SREBPs, sterol regulatory element binding proteins; TAG, triglycerides.

FIGURE 5

SLC25A47 deficiency promotes the pathogenesis of HCC. (A) The expression level of SLC25A47 in HCC samples compared with paracancerous tissues was determined from the GEPIA database (Normal n=110; Tumor n=371). (B) Effect of SLC25A47 expression on the overall survival of HCC patients in the GEPIA database (n=91). (C) IHC staining analysis of the expression of SLC25A47 in HCC tumors and its paracancerous tissues. (D) Survival curve of HCC model induced by DEN treatment in the WT and Slc25a47-KO mice (WT n=10; Slc25a47-KO n=12). Body weight (E), liver appearance, and liver index (F) of DEN-treatment mice at the end of the 10^th month (n=6). (G) H&E, Masson staining, and IHC staining of Ki67, α-SMA, Collagen I, and Fn1 in the liver of WT and Slc25a47-KO mice with DEN-treatment. (H) Immunoblots analysis of fibrosis markers in the liver of WT and Slc25a47-KO mice with DEN treatment. (I) ORO staining of the liver from WT and Slc25a47-KO mice with DEN treatment. (J, K) Immunoblots of AMPKα and its regulated proteins related to fatty acid and cholesterol metabolism pathways in the liver of WT and Slc25a47-KO mice with DEN-treatment. (L) Immunoblots of the mTOR pathway in the liver of WT and Slc25a47-KO mice with DEN treatment. (M) Quantitative analysis of immunoblotting for mTOR pathway-related proteins in the liver of WT and Slc25a47-KO mice with DEN treatment (n=6). Data are presented as mean±SEM. ****p < 0.0001, ns, not significant. Abbreviations: DEN, diethylnitrosamine; GEPIA, Gene Expression Profiling Interactive Analysis; IHC, immunohistochemical; KO, knockout; mTOR, mammalian target of rapamycin; ORO, Oil Red O; WT, wildtype.

FIGURE 6

SLC25A47 is identified as a mitochondrial NAD⁺ transporter. (A) 3D- homology model of human SLC25A47. (B) Uptake of NAD⁺ by liver mitochondria of the WT and Slc25a47-KO mice (n=3). (C, D) Concentration-response and time-response curves of liver mitochondria from the WT and Slc25a47-KO mice to NAD⁺ (n=3). (E) SIRT3 activity in liver mitochondria of the WT and Slc25a47-KO mice (n=3). (F) Immunoblots of SIRT3, p-AMPKα, and AMPKα in hepatocytes of the WT and Slc25a47-KO mice with and without NAD⁺ treatment. (G) mRNA expression of Slc25a47, 51, and 52 in mouse liver (n=2). (H) Close-up view of the binding pocket residues surrounding the docked NAD⁺ molecule. Potential electrostatic/salt-bridge interactions between cationic residues and the phosphate group of NAD⁺ are shown with black lines. (I) Uptake of NAD⁺ by EV, SLC25A47, and mutant SLC25A47 in stably transfected HEK293 cell lines (n=3). (J) A proposed model for the role of SLC25A47 in regulating hepatic lipid metabolism. Data are presented as mean±SEM. nd means not detected. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Abbreviations: AMPKα, AMP-activated protein kinase α; KO, knockout; WT, wildtype.