Hepatocyte-specific RIG-I loss attenuates metabolic dysfunction-associated steatotic liver disease in mice via changes in mitochondrial respiration and metabolite profiles

Abstract

Pattern recognition receptor (PRR)-mediated inflammation is an important determinant of the initiation and progression of metabolic diseases such as metabolic dysfunction-associated steatotic liver disease (MASLD). In this study, we investigated whether RIG-I is involved in hepatic metabolic reprogramming in a high-fat diet (HFD)-induced MASLD model in hepatocyte-specific RIG-I-KO (RIG-I^∆hep) mice. Our study revealed that hepatic deficiency of RIG-I improved HFD-induced metabolic imbalances, including glucose impairment and insulin resistance. Hepatic steatosis and liver triglyceride levels were reduced in RIG-I-deficient hepatocytes in HFD-induced MASLD mice, and this was accompanied by the reduced expression of lipogenesis genes, such as PPARγ, Dga2, and Pck1. Hepatic RIG-I deficiency alters whole-body metabolic rates in the HFD-induced MASLD model; there is higher energy consumption in RIG-I^∆hep mice. Deletion of RIG-I activated glycolysis and tricarboxylic acid (TCA) cycle-related metabolites in hepatocytes from both HFD-induced MASLD mice and methionine-choline-deficient diet (MCD)-fed mice. RIG-I deficiency enhanced AMPK activation and mitochondrial function in hepatocytes from HFD-induced MASLD mice. These findings indicate that deletion of RIG-I can activate cellular metabolism in hepatocytes by switching on both glycolysis and mitochondrial respiration, resulting in metabolic changes induced by a HFD and stimulation of mitochondrial activity. In summary, RIG-I may be a key regulator of cellular metabolism that influences the development of metabolic diseases such as MASLD.

Supplementary information: The online version contains supplementary material available at 10.1007/s43188-024-00264-x.

Keywords: Metabolic disorder; Metabolic dysfunction-associated steatotic liver disease; Metabolism; Mitochondria; Pattern-recognition receptors.

Fig. 1

Hepatic deficiency of RIG-I alleviates MASLD symptoms in mice fed HFD. Control (fl/fl) and RIG-I^∆hep mice were fed a ND or a HFD for 16 weeks. a Body weight over 16 weeks. b Body weight gain after 16 weeks. c Food intake. d Liver weight. e, f Serum levels of AST and ALT. g, h AUC of the IPGTT and ITT. i Histological analysis of liver sections: H&E staining (upper panel), Oil Red O staining (middle panel), and F4/80 immunostaining (bottom panel). j Steatosis score of liver tissue. k Liver triglyceride levels. l, m, n Expression levels of lipogenesis-related genes PPARγ, Dga2, and Pck1 in liver tissue. Data are presented as mean ± SEM (n = 3–6 mice/group). *p < 0.05

Fig. 2

RIG-I deficiency alters metabolic parameters in a HFD-induced MASLD model. Body composition and metabolic rate were analyzed in mice described in Fig. 1. a Whole-body composition. (B-H) metabolic rates over a 12 h light–dark cycle. Average 24 h values of the metabolic parameters. b Food intake. c Water consumption. d Physical activity levels. e Oxygen consumption rates (VO2). f Carbon dioxide production rates (VCO2). g Respiratory exchange ratio (RER). h Energy expenditure (EE). Data are presented as mean ± SEM (n = 3–5 mice/group, outlier: mean ± 2 SEM). *p < 0.05

Fig. 3

Deletion of hepatic RIG-I activates glycolysis and the TCA cycle in hepatocytes. a–d Seahorse analysis of glycolysis and mitochondrial respiration in hepatocytes from fl/fl and RIG-I^∆hep mice fed an ND, a HFD, and an MCD. The cells were seeded in Seahorse XFe96 cell culture microplates for 24 h. a, c Glycolysis parameters were shown in ECAR values (mpH/min). The cells were cultured 45 min before the experiment in XF assay medium supplemented with 4 mM L-glutamine, and sequentially injected with 10 mM glucose, 1.0 μM oligomycin, and 50 mM 2-DG. b, d Mitochondrial respiration parameters were showed in OCR values (pmol/min). The cells were cultured 45 min before the experiment in XF assay medium supplemented with L-glutamine 4 mM, D-glucose 5.55 mM, and sodium pyruvate 1 mM, and sequentially injected with 1.5 μM oligomycin, 0.5 μM FCCP, and 0.5 μM rotenone/antimycin A. a, b Glycolysis and mitochondrial respiration parameters in hepatocytes of fl/fl and RIG-I^∆hep mice fed an HFD. c, d Glycolysis and mitochondrial respiration parameters in hepatocytes from fl/fl and RIG-I^∆hep mice fed a MCD. e Metabolic profiling of hepatocytes from fl/fl and RIG-I^∆hep mice fed a ND or an HFD for 16 weeks. f The expression levels of the indicated proteins in the livers of fl/fl and RIG-I^∆hep mice fed a ND or an HFD were determined by immunoblotting. g Schematic overview of glycolysis and TCA cycle changes in hepatocytes from HFD RIG-I^∆hep mice. Data are presented as mean ± SEM (n = 3 mice/group). *p < 0.05

Fig. 4

Hepatic RIG-I deficiency enhances AMPK activation and mitochondrial function in hepatocytes. a Immunoblot analysis of AMPK activation in hepatocytes from fl/fl and RIG-I^∆hep mice fed ND or HFD for 16 weeks. b–d Expression of genes related to OXPHOS, mitochondrial dynamics and mitochondrial biogenesis in hepatocytes from fl/fl and RIG-I^∆hep mice fed ND or HFD. The mRNA expression levels were determined by qPCR. e Primary hepatocytes from fl/fl and RIG-I^∆hep mice (8 weeks old) were treated with insulin (100 nM) in the presence or absence of glucose (5.5 or 55 mM). The expression levels of the indicated proteins were determined by immunoblotting. f Schematic showing that liver-specific RIG-I deficiency can alleviate MASLD by restoring cellular metabolism. Data are presented as mean ± SEM (n = 3 mice/group). *p < 0.05