Liver p53 is stabilized upon starvation and required for amino acid catabolism and gluconeogenesis

Abstract

The ability to adapt cellular metabolism to nutrient availability is critical for survival. The liver plays a central role in the adaptation to starvation by switching from glucose-consuming processes and lipid synthesis to providing energy substrates like glucose to the organism. Here we report a previously unrecognized role of the tumor suppressor p53 in the physiologic adaptation to food withdrawal. We found that starvation robustly increases p53 protein in mouse liver. This induction was posttranscriptional and mediated by a hepatocyte-autonomous and AMP-activated protein kinase-dependent mechanism. p53 stabilization was required for the adaptive expression of genes involved in amino acid catabolism. Indeed, acute deletion of p53 in livers of adult mice impaired hepatic glycogen storage and induced steatosis. Upon food withdrawal, p53-deleted mice became hypoglycemic and showed defects in the starvation-associated utilization of hepatic amino acids. In summary, we provide novel evidence for a p53-dependent integration of acute changes of cellular energy status and the metabolic adaptation to starvation. Because of its tumor suppressor function, p53 stabilization by starvation could have implications for both metabolic and oncological diseases of the liver.-Prokesch, A., Graef, F. A., Madl, T., Kahlhofer, J., Heidenreich, S., Schumann, A., Moyschewitz, E., Pristoynik, P., Blaschitz, A., Knauer, M., Muenzner, M., Bogner-Strauss, J. G., Dohr, G., Schulz, T. J., Schupp, M. Liver p53 is stabilized upon starvation and required for amino acid catabolism and gluconeogenesis.

Keywords: AMPK; fasting; hepatic steatosis; liver metabolism; nutrient deprivation.

Figure 1.

p53 protein in liver is stabilized by starvation. Four-month-old C57BL/6N mice were either fed ad libitum, starved for 24 h, or refed for 2 h after 24-h starvation. Livers were sectioned and shock frozen for subsequent RNA or protein extraction. A) qPCR of RNA extracted from shock-frozen liver samples to determine expression of p53 target genes. Mean expression in fed group was set to 1. Ddit4, DNA damage inducible transcript 4; Lpin1, lipin 1; Sesn2, sestrin 2. B) Liver lysates were subjected to Western blot analysis to detect p53 protein in fed, starved, and refed mice. Expression of β-actin (ACTB) served as loading control. C) Densitometric quantification of signals (B). Value in fed group was set to 1 (n = 3). D) qPCR of RNA extracted from liver samples to determine expression of p53 and Mdm2 (mouse double minute 2 homolog). Mean expression in fed group was set to 1. All qPCR data above are derived from 5 to 7 mice per group. *P < 0.05 compared to fed group, ^#P < 0.05 compared to starved group.

Figure 2.

p53 is stabilized by starvation in a hepatocyte-autonomous way. A–C) Western blot analysis of p53 protein in primary mouse (A) and human (B) hepatocytes and HepG2 cells (C) treated with indicated compounds and appropriate vehicle controls for 24 h. Concentrations: 5 nM glucagon, 1 µM Dex, 0.5 mM IBMX, 10 µM nutlin-3a, and 20 µM quinacrine. Quinacrine and nutlin-3a were used as positive controls and human β-actin (ACTB) or mouse RAN served as loading controls. D, E) Hepatocytes were starved in HBSS/HEPES for 6 h [primary mouse hepatocytes (D)] or 24 h [primary human hepatocytes (E)]. F) HepG2 cells were starved for indicated times or treated for 24 h with 10 µM nutlin-3a or 20 µg/ml etoposide as positive controls for p53 accumulation. Western blot analysis was performed to determine p53 and p21 protein levels, and ACTB or RAN served as loading controls. G–I) qPCR analysis of p53 mRNA expression after 12 h [primary mouse hepatocytes (G)] or 24 h [primary human hepatocytes (H), HepG2 (I)] of starvation. Expression levels are set to 1 in control.

Figure 3.

Full p53 stabilization under starvation requires AMPK signaling. A) HepG2 cells were starved for indicated times, and Western blot analysis was performed to determine protein levels of AMPK subunits α and β and their respective phosphorylated forms. β-Actin (ACTB) served as loading controls. B) Primary human hepatocytes from 2 donors were treated for 24 h with 0.5 mM AICAR or with DMSO as vehicle control and analyzed for p53 protein expression. Phosphorylated form of ACC1 is shown to validate AMPK activation; ACTB served as loading control. C) HepG2 cells were treated for 24 h with 10 µM nutlin-3a, 0.5 mM AICAR, or respective vehicle controls and analyzed for p53 protein expression. D) qPCR analysis of p53 target gene expression after starvation or treatment with 0.5 mM AICAR or 10 µM nutlin-3a. Expression levels are set to 1 in control. Sesn1, sestrin 1; Tigar, Trp53 induced glycolysis regulatory phosphatase. *P < 0.05 compared to control. E) Western blot analysis validating knockdown of AMPKα and decreased AMPK activity by reduced phosphorylation of downstream target ACC1. F) Two days after transfection with control or AMPKα siRNA, HepG2 cells were starved for 6 h or received fresh medium. Western blot analysis was used to determine p53 protein levels. G) Quantification of p53 protein expression from 3 independent experiments (F). Protein abundance was set to 1 for starved cells transfected with control siRNA. *P < 0.05 compared to siControl.

Figure 4.

Effects of acute and liver-specific p53 deletion on hepatic glucose and lipid metabolism. p53 fl/fl mice were tail-vein injected with adenoviral constructs expressing GFP (control) or CRE (p53 deletion). A) qPCR to detect p53 and p21 transcript levels in liver during fed state. Expression was set to 1 in GFP group. B) Blood glucose measured in GFP and CRE mice fed ad libitum (n = 4–5). C) Glucose concentrations were measured by NMR spectroscopy in methanol-extracted liver lysates of GFP and CRE mice fed ad libitum (n = 4–5). D) Periodic acid–Schiff staining of GFP and CRE liver sections with subsequent segmentation and quantification to determine liver glycogen content (n = 4–5). E) Glycogen content measured by NMR spectroscopy in methanol-extracted liver lysates of GFP and CRE mice fed ad libitum (n = 4–5). F) Oil Red O staining of GFP and CRE liver cryosections with subsequent segmentation and quantification to determine neutral lipids (n = 4–5). G) Liver triglycerides of GFP and CRE mice measured by colorimetric assay (n = 4–5). *P < 0.05 compared to GFP. Scale bars, 100 μm.

Figure 5.

Acute and liver-specific p53 deletion blunts expression of genes related to hepatic amino acid metabolism. Microarray analysis of RNA isolated from livers of ad libitum fed GFP and CRE mice (n = 3 per group). A) DAVID functional annotation using GO terms (biologic processes) and KEGG pathways of genes down-regulated more than 1.5-fold in p53-deleted livers compared to GFP control. Only terms that are significant after correction for multiple testing are shown. P < 0.05 by Benjamini-Hochberg correction. B) Heat map showing differential expression of down-regulated genes comprising DAVID categories (A) after normalization per gene and hierarchical clustering using Genesis software.

Figure 6.

Effects of acute and liver-specific p53 deletion on hepatic starvation response. p53 fl/fl mice were tail-vein injected with adenoviral constructs expressing GFP (control) or CRE (p53 deletion). A) qPCR to detect p53 and p21 transcript levels in liver after 24 h of starvation. Expression is set to 1 in GFP group. B) Blood glucose in GFP and CRE mice starved for 24 h (n = 5–6). C) Glucose concentration were measured by NMR spectroscopy in methanol extracted liver lysates of GFP and CRE mice starved for 24 h (n = 5–6). *P < 0.05 compared to GFP.

Figure 7.

Acute and liver-specific p53 deletion impairs starvation-adapted gene expression. A) GSEA of whole-transcriptome data ranked by differential expression between fed and 24 h starved states of CRE and GFP groups (n = 3 per group). Overlay of normalized enrichment scores of top 20 enriched GO_BP terms and KEGG pathways in GFP (blue bars) and CRE (orange bars) are sorted by decreased normalized enrichment score in GFP group. B) Indicated amino acids in methanol extracts from liver lysates were determined by NMR spectroscopy (n = 4 per group). Mean value in GFP fed group is set to 100%. One-way ANOVA followed by post hoc test was performed. For pairwise comparisons, *P < 0.05 compared to respective fed group, and ^#P < 0.05 and between GFP and CRE group under starvation. C) Validation of siRNA-mediated p53 knockdown in primary hepatocytes isolated from wild-type mice. Representative results of 3 independent experiments are shown. D) OCR in primary hepatocytes was measured 3 d after siRNA application using Seahorse Bioscience XF96 extracellular flux analyzer. Representative results of 3 independent experiments are shown. *P < 0.05 compared to si_Control.

Figure 8.

Schematic summary of p53 action in liver. Starvation-induced stabilization of liver p53 is hepatocyte autonomous and, at least in part, mediated by AMPK activation. While basal p53 activity is necessary for glycogen storage and to prevent hepatic steatosis during feeding, p53 protein stabilization is required for gluconeogenesis and amino acid catabolism in starved state, thus establishing p53 as key mediator of metabolic flexibility.