p53 promotes the expression of gluconeogenesis-related genes and enhances hepatic glucose production
© Goldstein et al.; licensee BioMed Central Ltd. 2013
Received: 20 September 2012
Accepted: 5 December 2012
Published: 4 February 2013
The p53 tumor suppressor protein is a transcription factor that initiates transcriptional programs aimed at inhibiting carcinogenesis. p53 represses metabolic pathways that support tumor development (such as glycolysis and the pentose phosphate pathway (PPP)) and enhances metabolic pathways that are considered counter-tumorigenic such as fatty acid oxidation.
In an attempt to comprehensively define metabolic pathways regulated by p53, we performed two consecutive high-throughput analyses in human liver-derived cells with varying p53 statuses. A gene expression microarray screen followed by constraint-based modeling (CBM) predicting metabolic changes imposed by the transcriptomic changes suggested a role for p53 in enhancing gluconeogenesis (de novo synthesis of glucose). Examining glucogenic gene expression revealed a p53-dependent induction of genes involved in both gluconeogenesis (G6PC, PCK2) and in supplying glucogenic precursors (glycerol kinase (GK), aquaporin 3 (AQP3), aquaporin 9 (AQP9) and glutamic-oxaloacetic transaminase 1 (GOT1)). Accordingly, p53 augmented hepatic glucose production (HGP) in both human liver cells and primary mouse hepatocytes.
These findings portray p53 as a novel regulator of glucose production. By facilitating glucose export, p53 may prevent it from being shunted to pro-cancerous pathways such as glycolysis and the PPP. Thus, our findings suggest a metabolic pathway through which p53 may inhibit tumorigenesis.
Keywordsp53 Gluconeogenesis Hepatic glucose production Diabetes
p53 is a transcription factor that regulates the expression of many genes, thereby eliciting a myriad of cellular responses, most of them culminate in an anti-tumorigenic effect . Recently, the concept of p53 as a regulator of metabolism has emerged with various metabolic pathways found to be regulated by p53 in an anti-tumorigenic effort [2, 3].
Focusing on reactions associated with the gluconeogenesis pathway, we next evaluated the predicted flux rate carried by each of these reactions in each condition. Strikingly, three of the four regulated reactions in the gluconeogenesis pathway (catalyzed by PC, PEPCK and G6Pase) were predicted to carry higher metabolic flux in cells expressing either basal or activated p53 than in any of the other states (Figure 2 and Additional file 1: Table S1). Moreover, the shuttling of glucose-6-phosphate (G6P) from the cytosol to the endoplasmic reticulum (where G6P is dephosphorylated by G6Pase), which is essential for completion of gluconeogenesis, was predicted to carry higher metabolic flux in p53-expressing cells than in HepG2sh-p53 (Figure 2 and Additional file 1: Table S1).
In accordance with our in silico predictions and with the p53-dependent changes in glucogenic gene expression, we found that HepG2 cells exhibit increased hepatic glucose production (HGP) following Nutlin-3a treatment (Figure 3B). Moreover, primary hepatocytes isolated from p53-deficient mice are impaired in their glucogenic capacity compared to wild-type mice (Figure 3C). In order to exclude any effects of cell viability affected by different p53 statuses on total glucose levels, we normalized the glucose level to total protein level yielding similar results (Additional file 3: Figure S1). To summarize, we found that p53 promotes the expression of genes whose encoded proteins are involved in both supplying glucogenic precursors and gluconeogenesis. Accordingly, p53 promotes glucose secretion in both human and mouse hepatocytes.
In combination with gene expression analysis, the iMAT flux-prediction method can infer directionality of reactions and metabolite production capacities. iMAT treats the expression levels of enzymes as cues for the likelihood that their associated reactions carry metabolic flux. It then uses network integration to accumulate these cues into a global, consistent prediction of metabolic behavior. As such, iMAT can reveal levels of post-transcriptional regulation that are not reflected in the gene expression data. Therefore, the proteins associated with reactions predicted to carry higher flux are not necessarily regulated at the expression level and may be either regulated at the level of translation or post-translationally. Moreover, the flux through a certain reaction is calculated not only as a function of gene expression and regulation, but also with respect to the sum of reactions in the cell and is therefore affected by changes in expression of proteins not directly associated with the reaction. Thus, iMAT is a reliable predictor of metabolic fluxes .
By employing iMAT, we were able to find a role for p53 in enhancing gluconeogenesis. Moreover, we found that p53 induces the expression of gluconeogenesis-related genes. As a sequence-specific transcription factor, p53 mostly regulates gene expression by binding to DNA regulatory elements and enhancing transcription. In order to examine the possibility that p53 directly regulates gluconeogenesis-related genes, we utilized published data employing chromatin immunoprecipitation of p53 followed by massive parallel sequencing (ChIP-seq) . The p53 binding events nearest to the genes’ transcription start site (TSS) were several tens of kilobases away from the TSS and in most cases (excluding GOT1) these binding events were closer to other genes. This certainly does not exclude the possibility that p53 regulates these genes by direct DNA binding, since it is becoming evident that a transcription factor binding event (including in the case of p53) is not necessarily responsible for regulating the gene closest to it [17, 18]. Moreover, the ChIP-seq data is from non-hepatic cells which are not glucogenic and the possibility exists that the chromatin around gluconeogenesis-related genes in non-glucogenic cells is inaccessible to transcription factor binding. Another plausible scenario is that p53 regulates gluconeogenesis-related genes in an indirect manner, namely through inducing the transcription or the activity of a glucogenic transcription factor. For example, p53 was found to augment the gene-inducing activity of a known glucogenic transcription factor, glucocorticoid receptor (GR), (although the authors inspected the effect of p53 on properties of GR that are not linked to gluconeogenesis) .
In accordance with the computational analysis and the gene expression data, we found that p53 enhances glucose production in both human and mouse hepatocytes. Our findings are strongly supported by several published data. First, p53 induces the expression of TP53-induced glycolysis and apoptosis regulator (TIGAR), which lowers the levels of fructose-2,6-bisphosphate (F2,6BP), an inhibitor of gluconeogenesis . Second, p53 inhibits glucose-6-phosphate dehydrogenase (G6PD), an enzyme that metabolizes G6P in the pentose phosphate pathway (PPP) . Thus, p53 may enhance gluconeogenesis by both increasing glucogenic flux due to lower F2,6BP (resulting in higher G6P levels) and by blocking the shunting of G6P to the PPP. Third, p53 forms a complex with peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1α), leading to modulation of p53-dependent transcription . The authors also measured HGP as a general indicator of liver function. p53-deficient mice showed reduced glucogenic capacity compared with wild-type mice. PGC1α is an important coactivator of gluconeogenic transcription factors  and the possibility exists that PGC1α directly facilitates p53-dependent gluconeogenesis-related transcription, leading to the observed improved HGP in wild-type mice.
Reduced G6Pase levels are correlated to the development of hepatocellular carcinoma . It has been postulated that blocking the efflux of glucose to the bloodstream leads to its shunting to other pathways which are considered pro-cancerous such as the PPP and glycolysis . Thus, our finding that p53 induces G6PC expression and enhances HGP adds another layer for the metabolic regulation exerted by p53 in an effort to curtail carcinogenesis.
Availability of supporting data
The microarray results were deposited in GEO Accession Number GSE30137.
False discovery rate
Glucose-6-phosphatase, catalytic subunit
Glutamic-oxaloacetic transaminase 1
Non-relevant short hairpin RNA
Short hairpin RNA targeting p53
Hepatic glucose production
Integrative metabolic analysis tool
Phosphoenolpyruvate carboxykinase 2
Peroxisome proliferator-activated receptor gamma coactivator 1 alpha
Pentose phosphate pathway
TP53-induced glycolysis and apoptosis regulator
Transcription start site.
Flight Attendant Medical Research Institute (FAMRI) Center of Excellence grant, ECFP6 grant LSHC-CT-2004-503576; Yad Abraham Center for Cancer Diagnosis and Therapy. VR is the incumbent of the Norman and Helen Asher Professorial Chair Cancer Research at the Weizmann Institute of Science, Israel. Our funding source was not involved in any aspect of this study.
KY is partially supported by a fellowship from the Edmond J. Safra Bioinformatics center at Tel-Aviv University and is grateful to the Azrieli Foundation for the award of an Azrieli Fellowship.
- Vousden KH, Prives C: Blinded by the light: the growing complexity of p53. Cell. 2009, 137: 413-431. 10.1016/j.cell.2009.04.037.View ArticlePubMedGoogle Scholar
- Goldstein I, Rotter V: Regulation of lipid metabolism by p53 - fighting two villains with one sword. Trends Endocrinol Metab. 2012, 23: 567-575. 10.1016/j.tem.2012.06.007.View ArticlePubMedGoogle Scholar
- Maddocks OD, Vousden KH: Metabolic regulation by p53. J Mol Med (Berl). 2011, 89: 237-245. 10.1007/s00109-011-0735-5.View ArticleGoogle Scholar
- Goldstein I, Ezra O, Rivlin N, Molchadsky A, Madar S, Goldfinger N, Rotter V: p53, a novel regulator of lipid metabolism pathways. J Hepatol. 2012, 56: 656-662. 10.1016/j.jhep.2011.08.022.View ArticlePubMedGoogle Scholar
- Oberhardt MA, Palsson BO, Papin JA: Applications of genome-scale metabolic reconstructions. Mol Syst Biol. 2009, 5: 320-PubMed CentralView ArticlePubMedGoogle Scholar
- Duarte NC, Becker SA, Jamshidi N, Thiele I, Mo ML, Vo TD, Srivas R, Palsson BO: Global reconstruction of the human metabolic network based on genomic and bibliomic data. Proc Natl Acad Sci U S A. 2007, 104: 1777-1782. 10.1073/pnas.0610772104.PubMed CentralView ArticlePubMedGoogle Scholar
- Ma H, Sorokin A, Mazein A, Selkov A, Selkov E, Demin O, Goryanin I: The Edinburgh human metabolic network reconstruction and its functional analysis. Mol Syst Biol. 2007, 3: 135-PubMed CentralView ArticlePubMedGoogle Scholar
- Jerby L, Shlomi T, Ruppin E: Computational reconstruction of tissue-specific metabolic models: application to human liver metabolism. Mol Syst Biol. 2010, 6: 401-PubMed CentralView ArticlePubMedGoogle Scholar
- Frezza C, Zheng L, Folger O, Rajagopalan KN, MacKenzie ED, Jerby L, Micaroni M, Chaneton B, Adam J, Hedley A, Kalna G, Tomlinson IP, Pollard PJ, Watson DG, Deberardinis RJ, Shlomi T, Ruppin E, Gottlieb E: Haemoxygenase is synthetically lethal with the tumour suppressor fumaratehydratase. Nature. 2011, 477: 225-228. 10.1038/nature10363.View ArticlePubMedGoogle Scholar
- Shlomi T, Cabili MN, Herrgard MJ, Palsson BO, Ruppin E: Network-based prediction of human tissue-specific metabolism. Nat Biotechnol. 2008, 26: 1003-1010. 10.1038/nbt.1487.View ArticlePubMedGoogle Scholar
- Zur H, Ruppin E, Shlomi T: iMAT: an integrative metabolic analysis tool. Bioinformatics. 2010, 26: 3140-3142. 10.1093/bioinformatics/btq602.View ArticlePubMedGoogle Scholar
- Assaily W, Rubinger DA, Wheaton K, Lin Y, Ma W, Xuan W, Brown-Endres L, Tsuchihara K, Mak TW, Benchimol S: ROS-mediated p53 Induction of Lpin1 regulates fatty acid oxidation in response to nutritional stress. Mol Cell. 2011, 44: 491-501. 10.1016/j.molcel.2011.08.038.View ArticlePubMedGoogle Scholar
- Goldstein I, Rivlin N, Shoshana OY, Ezra O, Madar S, Goldfinger N, Rotter V: Chemotherapeutic agents induce the expression and activity of their clearing enzyme CYP3A4 by activating p53. Carcinogenesis. 2012Google Scholar
- Jitrapakdee S: Transcription factors and coactivators controlling nutrient and hormonal regulation of hepatic gluconeogenesis. Int J Biochem Cell Biol. 2012, 44: 33-45. 10.1016/j.biocel.2011.10.001.View ArticlePubMedGoogle Scholar
- Yoon JC, Puigserver P, Chen G, Donovan J, Wu Z, Rhee J, Adelmant G, Stafford J, Kahn CR, Granner DK, Newgard CB, Spiegelman BM: Control of hepatic gluconeogenesis through the transcriptional coactivator PGC-1. Nature. 2001, 413: 131-138. 10.1038/35093050.View ArticlePubMedGoogle Scholar
- Nikulenkov F, Spinnler C, Li H, Tonelli C, Shi Y, Turunen M, Kivioja T, Ignatiev I, Kel A, Taipale J, Selivanova G: Insights into p53 transcriptional function via genome-wide chromatin occupancy and gene expression analysis. Cell Death Differ. 2012, 19: 1992-2002. 10.1038/cdd.2012.89.PubMed CentralView ArticlePubMedGoogle Scholar
- Bailey ST, Shin H, Westerling T, Liu XS, Brown M: Estrogen receptor prevents p53-dependent apoptosis in breast cancer. Proc Natl Acad Sci U S A. 2012, 109: 18060-18065. 10.1073/pnas.1018858109.PubMed CentralView ArticlePubMedGoogle Scholar
- John S, Sabo PJ, Thurman RE, Sung MH, Biddie SC, Johnson TA, Hager GL, Stamatoyannopoulos JA: Chromatin accessibility pre-determines glucocorticoid receptor binding patterns. Nat Genet. 2011, 43: 264-268. 10.1038/ng.759.View ArticlePubMedGoogle Scholar
- Murphy SH, Suzuki K, Downes M, Welch GL, De Jesus P, Miraglia LJ, Orth AP, Chanda SK, Evans RM, Verma IM: Tumor suppressor protein (p)53, is a regulator of NF-kappaB repression by the glucocorticoid receptor. Proc Natl Acad Sci U S A. 2011, 108: 17117-17122. 10.1073/pnas.1114420108.PubMed CentralView ArticlePubMedGoogle Scholar
- Bensaad K, Tsuruta A, Selak MA, Vidal MN, Nakano K, Bartrons R, Gottlieb E, Vousden KH: TIGAR, a p53-inducible regulator of glycolysis and apoptosis. Cell. 2006, 126: 107-120. 10.1016/j.cell.2006.05.036.View ArticlePubMedGoogle Scholar
- Jiang P, Du W, Wang X, Mancuso A, Gao X, Wu M, Yang X: p53 regulates biosynthesis through direct inactivation of glucose-6-phosphate dehydrogenase. Nat Cell Biol. 2011, 13: 310-316. 10.1038/ncb2172.PubMed CentralView ArticlePubMedGoogle Scholar
- Sen N, Satija YK, Das S: PGC-1alpha, a key modulator of p53, promotes cell survival upon metabolic stress. Mol Cell. 2011, 44: 621-634. 10.1016/j.molcel.2011.08.044.View ArticlePubMedGoogle Scholar
- Puigserver P, Spiegelman BM: Peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1 alpha): transcriptional coactivator and metabolic regulator. Endocr Rev. 2003, 24: 78-90. 10.1210/er.2002-0012.View ArticlePubMedGoogle Scholar
- Wang B, Hsu SH, Frankel W, Ghoshal K, Jacob ST: Stat3-mediated activation of microRNA-23a suppresses gluconeogenesis in hepatocellular carcinoma by down-regulating glucose-6-phosphatase and peroxisome proliferator-activated receptor gamma, coactivator 1 alpha. Hepatology. 2012, 56: 186-197. 10.1002/hep.25632.PubMed CentralView ArticlePubMedGoogle Scholar
- DeBerardinis RJ, Thompson CB: Cellular metabolism and disease: what do metabolic outliers teach us?. Cell. 2012, 148: 1132-1144. 10.1016/j.cell.2012.02.032.PubMed CentralView ArticlePubMedGoogle Scholar