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Spatial hepatocyte plasticity of gluconeogenesis during the metabolic transitions between fed, fasted and starvation states

Nature Metabolism volume 7, pages 1073–1091 (2025)Cite this article

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Abstract

Hepatocytes are organized along a spatial axis between the portal triad and the central vein to form functionally repetitive units known as lobules. The hepatocytes perform distinct metabolic functions depending on their location within the lobule. Single-cell analysis of hepatocytes across the liver lobule demonstrates that gluconeogenic gene expression is relatively low in the fed state and gradually increases in the periportal hepatocytes during the initial fasting period. As fasting progresses, pericentral hepatocyte gluconeogenic gene expression and gluconeogenic activity also increase and, following entry into a starvation state, the pericentral hepatocytes show similar gluconeogenic gene expression and activity to the periportal hepatocytes. In parallel, starvation suppresses canonical β-catenin signalling and modulates the expression of pericentral and periportal glutamine synthetase and glutaminase, respectively, resulting in enhanced incorporation of glutamine into glucose. Thus, hepatocyte gluconeogenic gene expression and glucose production are spatially and temporally plastic across the liver lobule, underscoring the complexity of defining hepatic insulin resistance and glucose production on a whole-organ level, as well as for a particular fasted or fed condition.

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Fig. 1: Time course of scRNA-seq of hepatocytes in the fed, fasted and starvation states.
Fig. 2: Compass analyses of the temporal changes of hepatocyte scRNA-seq in the fed, fasted and starvation states.
Fig. 3: Quantitative targeted scRNA-seq analyses of hepatocytes in the fed, fasted and starvation states.
Fig. 4: Spatial imaging by smFISH of Pck1 and Fasn mRNA in the fed, fasted and starvation state.
Fig. 5: Bulk RNA-seq analyses of isolated pericentral and periportal hepatocytes in the fed, fasted and starvation states.
Fig. 6: Incorporation of labelled pyruvate carbons into glucose increases in both pericentral and periportal hepatocytes when the liver enters the starvation state.
Fig. 7: Fasting and starvation redistributes expression of glutamine synthetase and glutaminase.

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Data availability

Raw FASTQ files were deposited to the National Center for Biotechnology Information Gene Expression Omnibus database and are available under accession numbers GSE263415, GSE263418 and GSE263419. Proteomics raw files are available in ProteomeXchange (PRIDE) under accession number PXD051593. For KEGG reference pathways, mmu00010, mmu00061 and mmu00062 were used. Free access and query to the complete single-cell dataset is available through the Shiny interactive web application (https://pessinlab.shinyapps.io/liver). Source data are provided with this paper.

Code availability

This study was conducted using only publicly available software; no custom code was used.

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Acknowledgements

This study was supported by grants from the National Institutes of Health (DK110063 (to F.Y., C.E., K.S. and J.E.P.), DK020541 (to V.L.S., I.J.K. and J.E.P.), DK110426 (to K.S.), T32GM007491 (to M.H.), S10OD030286 (to S.S.), P30CA013330 (to D. Reynolds, Genomics Core) and U2CDK135074 (National Mouse Metabolic Phenotyping Center at the University of California Davis). We thank S. Okada for data analysis, the National Mouse Metabolic Phenotyping Center at the University of California Davis, L. Leung for immunofluorescence assistance and D. Reynolds (Genomics Core) for the scRNA-seq processing.

Author information

Author notes

  1. These authors jointly supervised this work: Irwin J. Kurland, Carolina Eliscovich, Kosaku Shinoda, Jeffrey E. Pessin.

Authors and Affiliations

  1. Department of Medicine (Division of Endocrinology), The Albert Einstein College of Medicine, New York, NY, USA

    Junichi Okada, Alus M. Xiaoli, Li Liu, Fajun Yang, Yunping Qiu, Irwin J. Kurland, Kosaku Shinoda & Jeffrey E. Pessin

  2. Fleischer Institute for Diabetes and Metabolism, The Albert Einstein College of Medicine, New York, NY, USA

    Junichi Okada, Austin Landgraf, Alus M. Xiaoli, Li Liu, Maxwell Horton, Victor L. Schuster, Fajun Yang, Simone Sidoli, Yunping Qiu, Irwin J. Kurland, Carolina Eliscovich, Kosaku Shinoda & Jeffrey E. Pessin

  3. Department of Molecular Pharmacology, The Albert Einstein College of Medicine, New York, NY, USA

    Austin Landgraf, Maxwell Horton, Kosaku Shinoda & Jeffrey E. Pessin

  4. Department of Developmental and Molecular Biology, The Albert Einstein College of Medicine, New York, NY, USA

    Alus M. Xiaoli, Fajun Yang & Carolina Eliscovich

  5. Department of Biochemistry, The Albert Einstein College of Medicine, New York, NY, USA

    Maxwell Horton & Simone Sidoli

  6. Department of Medicine (Division of Nephrology), The Albert Einstein College of Medicine, New York, NY, USA

    Victor L. Schuster

  7. Department of Medicine (Division of Hepatology), The Albert Einstein College of Medicine, New York, NY, USA

    Carolina Eliscovich

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Contributions

J.O. performed scRNA-seq, targeted scRNA-seq, smFISH and gluconeogenesis assays as well as drafting and editing the manuscript. A.L. performed periportal and pericentral hepatocyte isolation, bulk RNA-seq, proteomics sample processing, data analyses and manuscript editing. A.M.X. performed AAV-sgRNA preparation, generated the hepatocyte-specific Gls2-KO mice and edited the manuscript. L.L. performed data management and bioinformatic analyses, built the Shiny web application and edited the manuscript. M.H. performed proteomic analyses, bioinformatic data analyses and manuscript editing. V.L.S., F.Y., S.S. and J.E.P. provided experimental advice, data interpretation and manuscript editing. Y.Q. performed the mass spectrometry analyses for gluconeogenesis, experimental advice, data interpretation and manuscript editing. I.J.K. supervised the mass spectrometry, design for stable isotope usage, experimental advice, data interpretation and manuscript editing. C.E. supervised smFISH and provided experimental advice, data interpretation and manuscript editing. K.S. supervised scRNA-seq and provided experimental advice, data interpretation and manuscript editing.

Corresponding author

Correspondence to Junichi Okada.

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The authors declare no competing interests.

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Nature Metabolism thanks Dale S. Edgerton, Jan Tchorz and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Revati Dewal, in collaboration with the Nature Metabolism team.

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Extended data

Extended Data Fig. 1 Day-time and night-time feeding does not significantly affect Pck1/Fasn gene expression or metabolic factors in either male or female mice.

(a, b) Relative mRNA levels of Pck1 (A) and Fasn (B) from total hepatocytes in the feeding time course. N = 3 mice per time point/condition. Ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test was performed to compare -4h with different time points. p values of *p < 0.05, **p < 0.01, and ***p < 0.001 was considered statistically significant when comparing two groups. (N.S.=not statistically significant). (c, d) Relative mRNA levels of lipogenic (Fasn) (C) and gluconeogenic (Pck1) (D) genes from total hepatocytes in the fed (0 h), fasted (8 h) and starvation (24 h) state. N = 3 mice per time point/condition. Ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test was performed to compare 0 h with different time points. p values of *p < 0.05, **p < 0.01, and ***p < 0.001 was considered statistically significant when comparing two groups. (N.S.=not statistically significant). (e-m) Glucagon/insulin ratio, Relative mRNA levels, body weight, blood glucose, and glycogen measurements from male/female mice (day-time fed vs. night-time fed trained) in the fasting time course; fed4h (0 h), fast8h (8 h), fast16h (16 h), fast20h (20 h), and fast24h (24 h). N = 3-4 mice per time point/condition. (E) Glucagon/insulin ratio in plasma from day-time fed vs. night-time fed trained male mice. (f, g) Relative mRNA levels of lipogenic (Pck1) (F) and gluconeogenic (Fasn) (G) genes from day-time fed vs. night-time fed trained male mice. (h) Body weight (g) from day-time fed vs. night-time fed trained male mice. (i) Blood glucose (mg/dl) from day-time fed vs. night-time fed trained male mice. (j, k) Relative mRNA levels of lipogenic (Pck1) (J) and gluconeogenic (Fasn) (K) genes from day-time fed vs. night-time fed trained female mice. (l) Body weight (g) from day-time fed vs. night-time fed trained female mice. (m) Blood glucose (mg/dl) from day-time fed vs. night-time fed trained female mice. (n-q) Glycogen content (mg/g) from total hepatocytes in the fasting time course; day-time fed vs. night-time fed trained male mice (N), day-time fed vs. night-time fed trained female mice (O), day-time fed vs. night-time fed untrained male mice (P), and day-time fed vs. night-time fed untrained female mice (Q). N = 3-4 mice per time point/condition. (r-u) Body weight and blood glucose measurements from male/female mice (day-time fed vs. night-time fed untrained) in the fasting time course; fed4h (0 h), fast8h (8 h), fast16h (16 h), fast20h (20 h), and fast24h (24 h). N = 3-4 mice per time point/condition. (R) Body weight (g) from day-time fed vs. night-time fed untrained male mice. (S) Blood glucose (mg/dl) from day-time fed vs. night-time fed untrained male mice. (T) Body weight (g) from day-time fed vs. night-time fed untrained female mice. (U) Blood glucose (mg/dl) from day-time fed vs. night-time fed untrained female mice.

Source data

Extended Data Fig. 2 Food entrainment effects on the liver local clock and plasma circadian hormone levels.

(A-H) Relative mRNA levels of Bmal1 and Clock from male/female mice in the fasting time course; fed4h (0 h), fast8h (8 h), fast16h (16 h), fast20h (20 h), and fast24h (24 h). N = 3-4 mice per time point/condition. Results aligned to zeitgeber time (ZT); ZT0-12 had the light on, whereas ZT12-0 had the light off. (a, b) Relative mRNA levels of Bmal1 from day-time fed vs. night-time fed trained male (A) and female (B) mice. (c, d) Relative mRNA levels of Clock from day-time fed vs. night-time fed trained male (C) and female (D) mice. (e, f) Relative mRNA levels of Bmal1 from day-time fed vs. night-time fed untrained male (E) and female (F) mice. (g, h) Relative mRNA levels of Clock from day-time fed vs. night-time fed untrained male (G) and female (H) mice. (i-l) Corticosterone and Growth Hormone levels in plasma from male mice in the fasting time course; fed4h (0 h), fast8h (8 h), fast16h (16 h), fast20h (20 h), and fast24h (24 h). N = 3-4 mice per time point/condition. Results aligned to zeitgeber time (ZT); ZT0-12 had the light on, whereas ZT12-0 had the light off. (I, J) Corticosterone and growth hormone levels in plasma from day-time fed vs. night-time fed trained (I) and untrained (J) male mice. (K, L) Growth hormone levels in plasma from day-time fed vs. night-time fed trained (K) and untrained (L) male mice.

Source data

Extended Data Fig. 3 Compass analyses of several gene pathways that are not altered in the fed, fasted or starvation state.

(a, b) Compass-score differential activity analysis of the Vitamin A pathway between 0 h and 8 h pericentral/periportal hepatocytes (A) and between 0 h and 24 h pericentral/periportal hepatocytes (B) based on Cohen’s D analysis. BCDO (BCMO1) is highlighted. (c, d) Compass-score differential activity analysis of the Vitamin B6 pathway between 0 h and 8 h pericentral/periportal hepatocytes (C) and between 0 h and 24 h pericentral/periportal hepatocytes (D) based on Cohen’s D analysis. PNPO is highlighted. (e, f) Compass-score differential activity analysis of the Starch and sucrose (glycogen) pathway between 0 h and 8 h pericentral/periportal hepatocytes (E) and between 0 h and 24 h pericentral/periportal hepatocytes (F) based on Cohen’s D analysis. GLPASE (PYGL) is highlighted. (g) Representative immunoblotting of BCMO1, PNPO, PYGL, CYP2E1, E-Cadherin, and Vinculin from pericentral and periportal isolated hepatocytes.

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Extended Data Fig. 4 Quantitated target scRNA-seq analyses for zonated gene expression.

(a) Sequencing saturation plot of each library. X-axis represents mean reads per cell; Y-axis represents percentage of transcripts sequenced; dotted line is the approximate saturation point. (b) Violin plots of Log2 expression Pck1 and Fasn comparing standard and targeted scRNA-seq in 0 h and 16 h. (c) Ridge plot analysis of gluconeogenic (Pck1, G6pc) and lipogenic (Acly, Fasn) genes from targeted scRNA-seq. X-axis represents log2 expression; Y-axis represents each time point/condition, red dotted line shows the threshold for cells positive of each gene. (d) MFuzz analysis of the targeted scRNA-seq to cluster similar expression profiles. Once percentage of positive cells were defined based on ridge plot analysis (Fig. S4C), each gene expressed in pericentral and periportal hepatocytes were assigned to 6 main clusters; early fasting induced, late fasting induced, cycling, early fasting suppressed, late fasting suppressed, constitutively expressed. X-axis represents time points; Y-axis represents percentage of positive cells.

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Extended Data Fig. 5 Quantification of Pck1 and Fasn transcription sites by spatial smFISH analyses.

(a, b) Representative smFISH images of pericentral (PC) and periportal (PP) areas selected for quantification of TS (Pck1 (A), and Fasn (B)) from 0 h, 4 h, and 24 h in male mice. TS are highlighted with arrows. (c, d) Histogram based on the intensity of each TS (Pck1 (C), and Fasn (D)). X-axis represents the intensity of TS (A.U.); Y-axis represents the number of TS within each intensity. Dotted line shows the mean value of the intensity of TS with SD.

Source data

Extended Data Fig. 6 smFISH spatial analyses of Pck1 and Fasn expression in female mice livers.

(a, b) Representative smFISH images of pericentral (PC) and periportal (PP) areas from 0 h, 4 h, and 24 h in female mice. TS are highlighted with arrows. (c-h) Relative mRNA levels of zonation markers (Cyp2e1 pericentral (C), Cyp2f2 periportal (D)), gluconeogenic genes (Pck1 (E), G6pc (F)), and lipogenic genes (Fasn (G), Acly (H)) from pericentral and periportal isolated hepatocytes in female mice. N = 3 mice for 8hPC/PP and 24hPP, N = 4 mice for 0hPC/PP and 24hPC. Data are presented as mean values +/− SD. Student’s t-test with unpaired two-tailed p values of *p < 0.05, **p < 0.01, and ***p < 0.001 was considered statistically significant when comparing two groups. (N.S.=not statistically significant).

Source data

Extended Data Fig. 7 Identification of nutrition-independent zonation mRNA markers that are unchanged in the fed, fasted and starvation states.

(a) Principal Component Analysis (PCA) of RNA-seq from pericentral and periportal isolated hepatocytes. Each dot represents each time point/condition based on coloring and shape of dot (0 h=red, 8 h=yellow, 24 h=blue; circle=pericentral, triangle=periportal). Green arrow shows how fasting shift the pericentral hepatocytes. (b) The number of differentially expressed genes in pericentral and periportal hepatocytes throughout the fed (0 h), fasted (8 h) and starvation (24 h) state in the RNA-seq from pericentral and periportal isolated hepatocytes. Differentially expressed genes were defined based on log2Fold Change <-1 for pericentral, >1 for periportal, both with a cutoff of p-adj<0.05. (c, e) Venn diagram of differentially expressed genes in pericentral (C) and periportal (E) hepatocytes throughout the fed (0 h), fasted (8 h) and starvation (24 h) state in the RNA-seq from pericentral and periportal isolated hepatocytes. (d, f) Metascape analysis on nutrition-independent upregulated pericentral genes (D) and periportal genes (F). Top 5 pathways in each zone shown (-log10(P)).

Extended Data Fig. 8 The entire mass isotopologue distribution (MID) of glucose from [U-13C] pyruvate or glutamine.

(a) Steady state gluconeogenic time course experiment from [U-13C]-pyruvate using total hepatocytes isolated from 24 h fasted mice. N = 3 mice. Glucose isotopologues with one 13C to six 13C carbons (M1 thru M6 isotopologues) are shown. Data are presented as mean values + SD. (b) Fed, fasting and starvation response of glucose isotopologues with one 13C to six 13C carbons (M1 thru M6 isotopologues) derived from [U-13C]-pyruvate using pericentral and periportal isolated hepatocytes. N = 4 for all conditions except 0hPC, 8hPP, 16hPP, and 24hPC (N = 3). Data are presented as mean values + SD. (c) Fasted 24 hr response of glucose isotopologues with one to three 13C carbons (% enrichment) derived from [U-13C]-glutamine comparing pericentral vs. periportal isolated hepatocytes using Control vs GLS2KO mice 24 h mice. N = 3 mice per condition. Data are presented as mean values + SD.

Source data

Extended Data Fig. 9 Canonical WNT target gene expression decreases in pericentral hepatocytes in the starvations state.

(a, b) RNA-seq data (counts) of pericentral zonation markers (Gulo and Cyp2e1) and periportal zonation markers (Hsd17b13 and Cyp2f2) (A), gluconeogenic genes (Pck1, G6pc) and lipogenic genes (Acly, Fasn) (B) from pericentral and periportal isolated hepatocytes. N = 3 mice per time point/condition. Data are presented as mean values +/− SD. Student’s t-test with unpaired two-tailed p values of *p < 0.05, **p < 0.01, and ***p < 0.001 was considered statistically significant when comparing two groups. (N.S.=not statistically significant). (c) Heatmap of WNT target genes in RNA-seq from pericentral and periportal isolated hepatocytes. Average expression of N = 3 mice from each time point/condition is shown. Coloring based on z-score. 4 main clusters show PC upregulated/fast reduced, fast induced, PP upregulated fast reduced, fast reduced (D) RNA-seq data (counts) of WNT signaling related genes from pericentral and periportal isolated hepatocytes. N = 3 mice per time point/condition. Data are presented as mean values +/− SD. Student’s t-test with unpaired two-tailed p values of *p < 0.05, **p < 0.01, and ***p < 0.001 was considered statistically significant when comparing two groups. (N.S.=not statistically significant).

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Extended Data Fig. 10 HIPPO/YAP and sonic hedgehog zonation gene pathway changes in the fed, fasted and starvation states.

(a) Heatmap of Yap target genes in RNA-seq from pericentral and periportal isolated hepatocytes. Average expression of N = 3 mice from each time point/condition is shown. Coloring based on z-score. (b) RNA-seq data (counts) of Yap related genes from pericentral and periportal isolated hepatocytes. N = 3 mice per time point/condition. Data are presented as mean values +/− SD. Student’s t-test with unpaired two-tailed p values of *p < 0.05, **p < 0.01, and ***p < 0.001 was considered statistically significant when comparing two groups. (N.S.=not statistically significant). (c) Heatmap of Shh target genes in RNA-seq from pericentral and periportal isolated hepatocytes. Average expression of N = 3 mice from each time point/condition is shown. Coloring based on z-score. (d) RNA-seq data (counts) of Shh related genes from pericentral and periportal isolated hepatocytes. N = 3 mice per time point/condition. Data are presented as mean values +/− SD. Student’s t-test with unpaired two-tailed p values of *p < 0.05, **p < 0.01, and ***p < 0.001 was considered statistically significant when comparing two groups. (N.S.=not statistically significant).

Source data

Supplementary information

Source data

Source Data Fig. 2 (download XLSX )

Compass.

Source Data Fig. 4 (download XLSX )

smFISH TS.

Source Data Fig. 5 (download XLSX )

RT–qPCR, western blot quantification.

Source Data Fig. 5 (download PDF )

Unprocessed western blots.

Source Data Fig. 6 (download XLSX )

Stable isotope.

Source Data Fig. 7 (download XLSX )

RNA-seq, IF, proteomics, RT–qPCR, stable isotope.

Source Data Fig. 7 (download PDF )

Unprocessed western blots.

Source Data Extended Data Fig./Table 1 (download XLSX )

RT–qPCR, Insulin/glucagon, BW, BG, glycogen.

Source Data Extended Data Fig./Table 2 (download XLSX )

RT–qPCR, hormones.

Source Data Extended Data Fig./Table 3 (download XLSX )

Compass.

Source Data Extended Data Fig./Table 3 (download PDF )

Unprocessed western blots.

Source Data Extended Data Fig./Table 4 (download XLSX )

MFuzz analysis.

Source Data Extended Data Fig./Table 5 (download XLSX )

smFISH intensities.

Source Data Extended Data Fig./Table 6 (download XLSX )

RT–qPCR.

Source Data Extended Data Fig./Table 8 (download XLSX )

Isotopologue distribution.

Source Data Extended Data Fig./Table 9 (download XLSX )

RNA-seq.

Source Data Extended Data Fig./Table 10 (download XLSX )

RNA-seq.

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Okada, J., Landgraf, A., Xiaoli, A.M. et al. Spatial hepatocyte plasticity of gluconeogenesis during the metabolic transitions between fed, fasted and starvation states. Nat Metab 7, 1073–1091 (2025). https://doi.org/10.1038/s42255-025-01269-y

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