Spatial Transcriptomic Study Reveals Heterogeneous Metabolic Adaptation and a Role of Pericentral PPARα/CAR/Ces2a Axis During Fasting in Mouse Liver

Abstract

Spatial heterogeneity and plasticity of the mammalian liver are critical for systemic metabolic homeostasis in response to fluctuating nutritional conditions. Here, a spatially resolved transcriptomic landscape of mouse livers across fed, fasted and refed states using spatial transcriptomics is generated. This approach elucidated dynamic temporal-spatial gene cascades and how liver zonation-both expression levels and patterns-adapts to shifts in nutritional status. Importantly, the pericentral nuclear receptor Nr1i3 (CAR) as a pivotal regulator of triglyceride metabolism is pinpointed. It is showed that the activation of CAR in the pericentral region is transcriptionally governed by Pparα. During fasting, CAR activation enhances lipolysis by upregulating carboxylesterase 2a, playing a crucial role in maintaining triglyceride homeostasis. These findings lay the foundation for future mechanistic studies of liver metabolic heterogeneity and plasticity in response to nutritional status changes, offering insights into the zonated pathology that emerge during liver disease progression linked to nutritional imbalances.

Keywords: constitutive androstane receptor (CAR); fasting response; liver zonation; metabolic heterogeneity; peroxisome proliferator‐activated receptor alpha (Pparα); spatial transcriptomics.

Figure 1

Spatial transcriptomic profiling of the murine livers in fed, fasted and refed conditions. A) Left top: H&E staining of liver sections from ctrl, fasted and refed mice. Right: UMAP visualization of 12 identified clusters. Left bottom: Spatial distribution of each cluster in liver tissue sections captured on 10x Visium slides. B) Pseudotime inference by the DPT algorithm. C) Heatmap displaying mRNA expression of selected genes detected by ST from periportal (PN) to centrilobular (CV) regions. D) Projection of selected marker genes in UMAP space. E) Left, Regression analysis defines five zonation patterns, displaying the standard fit model, top five genes by zonation index, and expression values for a representative gene across pseudotime for each pattern. Right, Sankey diagram depicting zonation pattern transitions across nutritional states. F) Gene expression changes along the PN‐CV axis. Orange, red, and green represent ctrl, fasted, and refed samples. Ribbons within the fitted line represent standard error of gene expression.

Figure 2

Downregulated metabolic processes revealed by ST during fasting. A–D) Key anabolic processes downregulated by fasting: A) de novo lipogenesis, B) cholesterol biosynthesis, C) steroidogenesis, and D) xenobiotic biotransformation. Blue indicates downregulation, and red indicates upregulation. E) Quantified profiles of the indicated genes along the PN‐CV axis from ST. F–H) Expression of Thrsp F), Cyp51 G) and Gstm3 H) visualized in tissue spots and detected by IHC in liver sections. The CV and PN are marked.

Figure 3

Enhancement of fatty acid oxidation pathways, including mitochondrial and peroxisomal β‐oxidation and microsomal ω‐oxidation, during fasting. A) Upregulated genes involved in the process of lipid metabolism during fasting are highlighted in red. B) Quantified profiles of the indicated genes along the PN‐CV axis from ST. C,D) Quantified profiles of Cd36 C) and Plin5 D) along the PN‐CV axis from ST, accompanied by corresponding qRT‐PCR analysis, visualization in tissue spots, and IHC staining in liver sections.n = 4 mice for each group.Data are shown in mean ± SEM; ns, not significant, *p < 0.05, **p < 0.01, ****p < 0.0001 by Student's t test.

Figure 4

Fasting‐induced upregulation of CAR is associated with PPARα in the pericentral zone of liver lobules. A) Quantified profiles of Nr1i3 (encoding CAR) along the PN‐CV axis from ST. B) Visualization of Nr1i3 in tissue spots. C) mRNA levels of Nr1i3 in liver samples obtained from the ctrl, fasted and refed mice. n = 4 mice for each group. D) Representative IHC staining of Nr1i3 in the liver sections. E) Heatmap depicting the expression of Nr1i3, and pathway scores determined by ssGSEA for the indicated signaling pathway. F) Scores of PPARα signaling along the PN‐CV axis. G) Sorting strategy for PC and PP hepatocytes, accompanied by qRT‐PCR validation of Cyp2e1 and Cyp2f2. n = 10 mice for each group. H) mRNA levels of Nr1i3 and the genes related to PPARα signaling in PP and PC hepatocytes obtained from ctrl, fasted and refed mice. n = 5 mice for each group. I) Expression of Nr1i3 in livers from WT and Ppara knockout mice upon Wy14643 treatment. n = 3. J) Expression of Nr1i3 in livers from WT and Atgladipo‐/− mice upon fasting. n = 6. K) mRNA levels of the indicated genes in liver samples obtained from the ctrl or fasted mice treated with Vehicle or MK866. n = 5 mice for each group. Data are shown in mean ± SEM; ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001 by Student's t test.

Figure 5

PPARα regulates CAR expression via PPRE binding in its promoter. A) DNase‐seq data illustrating the peak profiles at the Nr1i3 promoter region in the livers of ctrl and fasted mice, with the PPRE motif highlighted. B) Heatmaps displaying the ChIP‐seq signals. C) Profile plots representing the average ChIP signals. D) PPARα‐binding peaks were visualized using IGV software. E) ChIP‐qPCR validation of PPARα binding to the PPRE within the promoters of the indicated genes. n = 3 mice per group. F) Luciferase reporter assays in 293T cells transfected with pGL3 luciferase reporter plasmids the Nr1i3 promoter region (−2000 to ‐1 bp) and a variant lacking the PPRE motif. n = 4. Data are shown in mean ± SEM; ns, not significant, *p < 0.05, **p < 0.01, ****p < 0.0001 by Student's t test.

Figure 6

Ces2a, downstream of CAR, maintains TG homeostasis during fasting. A) Experimental setup for TCPOBOP treatment and analysis of serum β‐BHB, glucose, FFA and liver TG and FFA levels. n = 4 mice per group. B) Left: ChIP‐seq heatmaps showing CAR and IgG signals in mouse livers. Right: Quantification of CAR versus IgG signals. Peaks falling within promoter regions of Ces family genes are indicated. C) Genome browser tracks of CAR and IgG at the indicated Ces family gene promoters, featuring the canonical CAR‐binding site in the Ces2a promoter. D) Schematic of Ces2a promoters (P1/P2: with/without CAR‐binding region) and dual‐luciferase reporter assay. n = 3 per group. E) qRT‐PCR analysis of Ces2a and Ces1e in the liver samples treated with Vehicle or TCPOBOP. n = 6 mice per group. F) mRNA levels of the Ces1e and Ces2a in liver samples obtained from the ctrl, fasted and refed mice. n = 4 mice per group. G) Expression of Ces2a in PP and PC hepatocytes obtained from ctrl, fasted and refed mice. n = 5 mice per group. H) IHC of Ces1 and Ces2 in vehicle‐ or TCPOBOP‐treated livers. I) IHC of Ces2 and Ces1 in fed, fasted and refed livers. J,K) Silencing efficiency of Ces2a‐targeting siRNA assessed by qRT‐PCR J) and immunoblotting K) in mouse livers. n = 5 mice per group. H) Effects of Ces2a knockdown on liver FFA and TG contents and serum levels of TG and β‐HB in mice upon fasting. n = 5 mice per group. Data are shown in mean ± SEM; ns, not significant, **p < 0.01 by Student's t test.