Crosstalk between CYP2E1 and PPAR α substrates and agonists modulate adipose browning and obesity

Abstract

Although the functions of metabolic enzymes and nuclear receptors in controlling physiological homeostasis have been established, their crosstalk in modulating metabolic disease has not been explored. Genetic ablation of the xenobiotic-metabolizing cytochrome P450 enzyme CYP2E1 in mice markedly induced adipose browning and increased energy expenditure to improve obesity. CYP2E1 deficiency activated the expression of hepatic peroxisome proliferator-activated receptor alpha (PPARα) target genes, including fibroblast growth factor (FGF) 21, that upon release from the liver, enhanced adipose browning and energy expenditure to decrease obesity. Nineteen metabolites were increased in Cyp2e1-null mice as revealed by global untargeted metabolomics, among which four compounds, lysophosphatidylcholine and three polyunsaturated fatty acids were found to be directly metabolized by CYP2E1 and to serve as PPARα agonists, thus explaining how CYP2E1 deficiency causes hepatic PPARα activation through increasing cellular levels of endogenous PPARα agonists. Translationally, a CYP2E1 inhibitor was found to activate the PPARα-FGF21-beige adipose axis and decrease obesity in wild-type mice, but not in liver-specific Ppara-null mice. The present results establish a metabolic crosstalk between PPARα and CYP2E1 that supports the potential for a novel anti-obesity strategy of activating adipose tissue browning by targeting the CYP2E1 to modulate endogenous metabolites beyond its canonical role in xenobiotic-metabolism.

Keywords: CYP2E1; FGF21; Metabolic enzyme; Nuclear receptor; Obesity; PPARα.

Graphical abstract

Figure 1

Cyp2e1 disruption improves high-fat diet (HFD)-induced metabolic syndrome and increases energy expenditure. (A) Fat mass. (B) Adipose tissue/body weight ratios. (C) Circadian rectal temperature on normal condition. (D) Circadian rectal temperature after 14-week HFD feeding. (E) Daily energy expenditure (EE), data are expressed as adjusted means based on body weight to the power 0.75. (F) Respiratory exchange ratio (VCO₂/VO₂), VO₂, oxygen consumption; VCO₂, carbon dioxide production. (G–I), mRNA expression of the thermogenesis genes in brown adipose tissue (BAT), subcutaneous white adipose tissue (SWAT), and epididymal white adipose tissue (EWAT). (J, K) Representative hematoxylin and eosin (H&E) staining (J) and uncoupling protein 1 (UCP1) immunohistochemical staining (K) of SWAT sections from wild-type (WT) and Cyp2e1-null mice, scale bar = 50 μm. All data are presented as mean ± SEM, n = 6 mice/group; ∗P < 0.05, ∗∗P < 0.01 and ∗∗∗P < 0.001, compared with WT mice group, by unpaired two-tailed t test. WT, wild-type. KO, knockout. SWAT, subcutaneous white adipose tissue. BAT, brown adipose tissue. EWAT, epididymal white adipose tissue.

Figure 2

Transcriptome profiles revealed differential genes expression of the wild-type (WT) and Cyp2e1-null (KO) mice. (A) MA plot of differential gene expression levels in the two groups, where expression intensity is on the x-axis and differences in the gene expression levels (fold change, FC) are on the y-axis (log₂ FC), each dot represents one gene, red dots represent genes whose abundance is significantly up-regulated, blue dots represent down-regulated, and black dots represent non-significantly changed genes. (B) Heatmap of differential gene expression in WT and KO mice. (C) Gene ontology annotation of the differentially expressed unigenes. (D) KEGG annotation of the differentially expressed unigenes. (E) Gene–gene interaction network to show the key genes that link with Cyp2e1. WT, wild-type. KO, knockout. FC, fold change. FPKM, fragments per kilobase million.

Figure 3

Cyp2e1 disruption induced white adipose beige via the PPARα–FGF21 axis. (A–F) wild-type (WT) and Cyp2e1-null (KO) mice were fed a HFD for 14 weeks. (A) Hepatic mRNA expression of PPARα target genes under high-fat diet (HFD). (B) Serum FGF21 concentrations. (C) Representative hematoxylin and eosin (H&E) staining of liver sections from WT and KO mice, scale bar = 50 μm. (D, E) Liver triglycerides (TG), total cholesterol (TC). (F) Hepatic expression of mRNAs encoded by hepatic lipogenesis-related genes. All data are presented as mean ± SEM, n = 6; ∗P < 0.05, ∗∗P < 0.01 and ∗∗∗P < 0.001 by unpaired two-tailed t test. WT, wild-type. KO, knockout.

Figure 4

The LysoPC 22:4 was increased in Cyp2e1-null mice and identified as a direct CYP2E1 substrate. (A) Score scatter plot of a PCA model of the hepatic metabolites between wild-type (WT) and Cyp2e1-null (KO) mice, each point represents an individual mouse sample. (B) Heatmap analysis of the different hepatic metabolites in WT and KO mice. (C) Relative response of LysoPC 22:4 in WT and KO mice. (D) Docking pose of LysoPC 22:4 in the human CYP2E1 binding pocket. (E) Synthesis route of LysoPC 22:4. (F) Metabolic sites of LysoPC 22:4 after incubation with recombinant human CYP2E1. Data are presented as mean ± SEM, n = 6 mice/group; ∗∗∗P < 0.001 by unpaired two-tailed t test. WT, wild-type. KO, knockout.

Figure 5

The LysoPC 22:4 was identified as a direct PPARα agonist. (A, B) Surface plasmon resonance (SPR) assay for interaction of Wy-14643 and LysoPC 22:4 with PPARα protein. (C, D) Docking pose of Wy-14643 and LysoPC 22:4 in the human PPARα-AF-2 binding pocket. (E) Luciferase assays for PPARα activation in HEK293 cells after treatment with 10 μmol/L Wy-14643, and 5 and 10 μmol/L LysoPC 22:4, respectively. ∗∗∗P < 0.001, compared with vehicle group, by one-way ANOVA analysis. Data are presented as mean ± SEM; n = 3 per group. RU, response units.

Figure 6

CYP2E1 antagonist alleviates high-fat diet-induced obesity via PPARα–FGF21–beige axis. (A) Body weight gain. (B) Glucose tolerance test. (C) Insulin tolerance test. (D) Representative hematoxylin and eosin (H&E) staining of liver and subcutaneous white adipose tissue (SWAT) sections from phosphate buffered saline (PBS) and diethyldithiocarbamate (DDC)-treated mice, scale bar = 100 μm. (E) Representative uncoupling protein 1 (UCP1) immunohistochemical staining (right) of subcutaneous white adipose tissue (SWAT) sections from PBS and DDC-treated mice, scale bar = 100 μm. (F, G) mRNA expression of the thermogenesis genes in brown adipose tissue (BAT) and SWAT. (H) Rectal temperature. (I) Hepatic mRNA expression of PPARα target genes. (J) Serum FGF21 concentrations. Data are presented as mean ± SEM, n = 6; ∗P < 0.05; ∗∗P < 0.01 and ∗∗∗P < 0.001 by unpaired two-tailed t test.

Figure 7

Anti-obesity effect of Cyp2e1 inhibition is lost in the absence of liver PPARα. (A) Body weight gain. (B) Subcutaneous adipose tissue (SWAT)/body weight ratio. (C) Epididymal adipose tissue (EWAT)/body weight ratio. (D) Glucose tolerance test. (E) Insulin tolerance test. (F) Rectal temperature. (G) Hepatic mRNA expression of PPARα target genes. (H, I) mRNA expression of the thermogenesis genes in brown adipose tissue (BAT) and SWAT. (J) Serum FGF21 concentration. (K) Representative hematoxylin and eosin (H&E) staining of liver and subcutaneous white adipose tissue (SWAT) sections from phosphate buffered saline (PBS) and diethyldithiocarbamate (DDC)-treated mice, scale bar = 50 μm. (L) Representative uncoupling protein 1 (UCP1) staining of SWAT. (M) Liver triglycerides (TG). (N) Liver total cholesterol (TC). (O) Serum TG. (P) Serum TC. (Q) Serum alanine transaminase (ALT) levels. (R) Serum aspartate transaminase (AST) levels. Data are presented as mean ± SEM, n = 6.

Figure 8

Proposed CYP2E1–PPARα–FGF21 axis in inducing white adipose browning for reducing obesity. CYP2E1 deficiency, either by gene disruption or chemical inhibition, leads to hepatic PPARα activation that decreases lipid accumulation as well as increasing the expression and secretion of FGF21 from the liver. Secreted FGF21 then circulates to the peripheral subcutaneous white adipose to enhance beiging, which in turn increases the energy expenditure and decreases obesity.