Thyroid hormone is a major determinant of energy expenditure and a key regulator of mitochondrial activity. We have previously identified a mitochondrial triiodothyronine receptor (p43) that acts as a mitochondrial transcription factor of the organelle genome, which leads, in vitro and in vivo, to a stimulation of mitochondrial biogenesis. Here we generated mice specifically lacking p43 to address its physiological influence. We found that p43 is required for normal glucose homeostasis. The p43−/− mice had a major defect in insulin secretion both in vivo and in isolated pancreatic islets and a loss of glucose-stimulated insulin secretion. Moreover, a high-fat/high-sucrose diet elicited more severe glucose intolerance than that recorded in normal animals. In addition, we observed in p43~ mice both a decrease in pancreatic islet density and in the activity of complexes of the respiratory chain in isolated pancreatic islets. These dysfunctions were associated with a down-regulation of the expression of the glucose transporter Glut2 and of Kir6.2, a key component of the KATP channel. Our findings establish that p43 is an important regulator of glucose homeostasis and pancreatic β-cell function and provide evidence for the first time of a physiological role for a mitochondrial endocrine receptor.—Blanchet, E., Bertrand, C., Annicotte, J. S., Schlernitzauer, A., Pessemesse, L., Levin, J., Fouret, G., Feillet-Coudray, C., Bonafos, B., Fajas, L., Cabello, G., Wrutniak-Cabello, C., Casas, F. Mitochondrial T3 receptor p43 regulates insulin secretion and glucose homeostasis. FASEB J. 26, 40–50 (2012). www.fasebj.org
Article14 May 2020Open Access Endoplasmic reticulum stress actively suppresses hepatic molecular identity in damaged liver Vanessa Dubois Vanessa Dubois orcid.org/0000-0001-8894-2980 Inserm, CHU Lille, Institut Pasteur de Lille, U1011-EGID, University of Lille, Lille, France Search for more papers by this author Céline Gheeraert Céline Gheeraert Inserm, CHU Lille, Institut Pasteur de Lille, U1011-EGID, University of Lille, Lille, France Search for more papers by this author Wouter Vankrunkelsven Wouter Vankrunkelsven orcid.org/0000-0003-0943-043X Clinical Division and Laboratory of Intensive Care Medicine, Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium Search for more papers by this author Julie Dubois-Chevalier Julie Dubois-Chevalier Inserm, CHU Lille, Institut Pasteur de Lille, U1011-EGID, University of Lille, Lille, France Search for more papers by this author Hélène Dehondt Hélène Dehondt Inserm, CHU Lille, Institut Pasteur de Lille, U1011-EGID, University of Lille, Lille, France Search for more papers by this author Marie Bobowski-Gerard Marie Bobowski-Gerard orcid.org/0000-0002-0915-0799 Inserm, CHU Lille, Institut Pasteur de Lille, U1011-EGID, University of Lille, Lille, France Search for more papers by this author Manjula Vinod Manjula Vinod orcid.org/0000-0001-8699-0372 Inserm, CHU Lille, Institut Pasteur de Lille, U1011-EGID, University of Lille, Lille, France Search for more papers by this author Francesco Paolo Zummo Francesco Paolo Zummo Inserm, CHU Lille, Institut Pasteur de Lille, U1011-EGID, University of Lille, Lille, France Search for more papers by this author Fabian Güiza Fabian Güiza orcid.org/0000-0001-7026-0957 Clinical Division and Laboratory of Intensive Care Medicine, Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium Search for more papers by this author Maheul Ploton Maheul Ploton Inserm, CHU Lille, Institut Pasteur de Lille, U1011-EGID, University of Lille, Lille, France Search for more papers by this author Emilie Dorchies Emilie Dorchies Inserm, CHU Lille, Institut Pasteur de Lille, U1011-EGID, University of Lille, Lille, France Search for more papers by this author Laurent Pineau Laurent Pineau Inserm, CHU Lille, Institut Pasteur de Lille, U1011-EGID, University of Lille, Lille, France Search for more papers by this author Alexis Boulinguiez Alexis Boulinguiez Inserm, CHU Lille, Institut Pasteur de Lille, U1011-EGID, University of Lille, Lille, France Search for more papers by this author Emmanuelle Vallez Emmanuelle Vallez Inserm, CHU Lille, Institut Pasteur de Lille, U1011-EGID, University of Lille, Lille, France Search for more papers by this author Eloise Woitrain Eloise Woitrain Inserm, CHU Lille, Institut Pasteur de Lille, U1011-EGID, University of Lille, Lille, France Search for more papers by this author Eric Baugé Eric Baugé Inserm, CHU Lille, Institut Pasteur de Lille, U1011-EGID, University of Lille, Lille, France Search for more papers by this author Fanny Lalloyer Fanny Lalloyer Inserm, CHU Lille, Institut Pasteur de Lille, U1011-EGID, University of Lille, Lille, France Search for more papers by this author Christian Duhem Christian Duhem Inserm, CHU Lille, Institut Pasteur de Lille, U1011-EGID, University of Lille, Lille, France Search for more papers by this author Nabil Rabhi Nabil Rabhi UMR 8199 - EGID, CNRS, Institut Pasteur de Lille, University of Lille, Lille, France Search for more papers by this author Ronald E van Kesteren Ronald E van Kesteren orcid.org/0000-0002-9592-3777 Center for Neurogenomics and Cognitive Research, Neuroscience Campus Amsterdam, VU University, Amsterdam, The Netherlands Search for more papers by this author Cheng-Ming Chiang Cheng-Ming Chiang Simmons Comprehensive Cancer Center, Departments of Biochemistry and Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Steve Lancel Steve Lancel Inserm, CHU Lille, Institut Pasteur de Lille, U1011-EGID, University of Lille, Lille, France Search for more papers by this author Hélène Duez Hélène Duez Inserm, CHU Lille, Institut Pasteur de Lille, U1011-EGID, University of Lille, Lille, France Search for more papers by this author Jean-Sébastien Annicotte Jean-Sébastien Annicotte UMR 8199 - EGID, CNRS, Institut Pasteur de Lille, University of Lille, Lille, France Search for more papers by this author Réjane Paumelle Réjane Paumelle Inserm, CHU Lille, Institut Pasteur de Lille, U1011-EGID, University of Lille, Lille, France Search for more papers by this author Ilse Vanhorebeek Ilse Vanhorebeek Clinical Division and Laboratory of Intensive Care Medicine, Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium Search for more papers by this author Greet Van den Berghe Greet Van den Berghe orcid.org/0000-0002-5320-1362 Clinical Division and Laboratory of Intensive Care Medicine, Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium Search for more papers by this author Bart Staels Bart Staels orcid.org/0000-0002-3784-1503 Inserm, CHU Lille, Institut Pasteur de Lille, U1011-EGID, University of Lille, Lille, France Search for more papers by this author Philippe Lefebvre Philippe Lefebvre Inserm, CHU Lille, Institut Pasteur de Lille, U1011-EGID, University of Lille, Lille, France Search for more papers by this author Jérôme Eeckhoute Corresponding Author Jérôme Eeckhoute [email protected] orcid.org/0000-0002-7222-9264 Inserm, CHU Lille, Institut Pasteur de Lille, U1011-EGID, University of Lille, Lille, France Search for more papers by this author Vanessa Dubois Vanessa Dubois orcid.org/0000-0001-8894-2980 Inserm, CHU Lille, Institut Pasteur de Lille, U1011-EGID, University of Lille, Lille, France Search for more papers by this author Céline Gheeraert Céline Gheeraert Inserm, CHU Lille, Institut Pasteur de Lille, U1011-EGID, University of Lille, Lille, France Search for more papers by this author Wouter Vankrunkelsven Wouter Vankrunkelsven orcid.org/0000-0003-0943-043X Clinical Division and Laboratory of Intensive Care Medicine, Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium Search for more papers by this author Julie Dubois-Chevalier Julie Dubois-Chevalier Inserm, CHU Lille, Institut Pasteur de Lille, U1011-EGID, University of Lille, Lille, France Search for more papers by this author Hélène Dehondt Hélène Dehondt Inserm, CHU Lille, Institut Pasteur de Lille, U1011-EGID, University of Lille, Lille, France Search for more papers by this author Marie Bobowski-Gerard Marie Bobowski-Gerard orcid.org/0000-0002-0915-0799 Inserm, CHU Lille, Institut Pasteur de Lille, U1011-EGID, University of Lille, Lille, France Search for more papers by this author Manjula Vinod Manjula Vinod orcid.org/0000-0001-8699-0372 Inserm, CHU Lille, Institut Pasteur de Lille, U1011-EGID, University of Lille, Lille, France Search for more papers by this author Francesco Paolo Zummo Francesco Paolo Zummo Inserm, CHU Lille, Institut Pasteur de Lille, U1011-EGID, University of Lille, Lille, France Search for more papers by this author Fabian Güiza Fabian Güiza orcid.org/0000-0001-7026-0957 Clinical Division and Laboratory of Intensive Care Medicine, Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium Search for more papers by this author Maheul Ploton Maheul Ploton Inserm, CHU Lille, Institut Pasteur de Lille, U1011-EGID, University of Lille, Lille, France Search for more papers by this author Emilie Dorchies Emilie Dorchies Inserm, CHU Lille, Institut Pasteur de Lille, U1011-EGID, University of Lille, Lille, France Search for more papers by this author Laurent Pineau Laurent Pineau Inserm, CHU Lille, Institut Pasteur de Lille, U1011-EGID, University of Lille, Lille, France Search for more papers by this author Alexis Boulinguiez Alexis Boulinguiez Inserm, CHU Lille, Institut Pasteur de Lille, U1011-EGID, University of Lille, Lille, France Search for more papers by this author Emmanuelle Vallez Emmanuelle Vallez Inserm, CHU Lille, Institut Pasteur de Lille, U1011-EGID, University of Lille, Lille, France Search for more papers by this author Eloise Woitrain Eloise Woitrain Inserm, CHU Lille, Institut Pasteur de Lille, U1011-EGID, University of Lille, Lille, France Search for more papers by this author Eric Baugé Eric Baugé Inserm, CHU Lille, Institut Pasteur de Lille, U1011-EGID, University of Lille, Lille, France Search for more papers by this author Fanny Lalloyer Fanny Lalloyer Inserm, CHU Lille, Institut Pasteur de Lille, U1011-EGID, University of Lille, Lille, France Search for more papers by this author Christian Duhem Christian Duhem Inserm, CHU Lille, Institut Pasteur de Lille, U1011-EGID, University of Lille, Lille, France Search for more papers by this author Nabil Rabhi Nabil Rabhi UMR 8199 - EGID, CNRS, Institut Pasteur de Lille, University of Lille, Lille, France Search for more papers by this author Ronald E van Kesteren Ronald E van Kesteren orcid.org/0000-0002-9592-3777 Center for Neurogenomics and Cognitive Research, Neuroscience Campus Amsterdam, VU University, Amsterdam, The Netherlands Search for more papers by this author Cheng-Ming Chiang Cheng-Ming Chiang Simmons Comprehensive Cancer Center, Departments of Biochemistry and Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Steve Lancel Steve Lancel Inserm, CHU Lille, Institut Pasteur de Lille, U1011-EGID, University of Lille, Lille, France Search for more papers by this author Hélène Duez Hélène Duez Inserm, CHU Lille, Institut Pasteur de Lille, U1011-EGID, University of Lille, Lille, France Search for more papers by this author Jean-Sébastien Annicotte Jean-Sébastien Annicotte UMR 8199 - EGID, CNRS, Institut Pasteur de Lille, University of Lille, Lille, France Search for more papers by this author Réjane Paumelle Réjane Paumelle Inserm, CHU Lille, Institut Pasteur de Lille, U1011-EGID, University of Lille, Lille, France Search for more papers by this author Ilse Vanhorebeek Ilse Vanhorebeek Clinical Division and Laboratory of Intensive Care Medicine, Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium Search for more papers by this author Greet Van den Berghe Greet Van den Berghe orcid.org/0000-0002-5320-1362 Clinical Division and Laboratory of Intensive Care Medicine, Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium Search for more papers by this author Bart Staels Bart Staels orcid.org/0000-0002-3784-1503 Inserm, CHU Lille, Institut Pasteur de Lille, U1011-EGID, University of Lille, Lille, France Search for more papers by this author Philippe Lefebvre Philippe Lefebvre Inserm, CHU Lille, Institut Pasteur de Lille, U1011-EGID, University of Lille, Lille, France Search for more papers by this author Jérôme Eeckhoute Corresponding Author Jérôme Eeckhoute [email protected] orcid.org/0000-0002-7222-9264 Inserm, CHU Lille, Institut Pasteur de Lille, U1011-EGID, University of Lille, Lille, France Search for more papers by this author Author Information Vanessa Dubois1,6, Céline Gheeraert1,‡, Wouter Vankrunkelsven2,‡, Julie Dubois-Chevalier1,‡, Hélène Dehondt1,‡, Marie Bobowski-Gerard1, Manjula Vinod1, Francesco Paolo Zummo1, Fabian Güiza2, Maheul Ploton1, Emilie Dorchies1, Laurent Pineau1, Alexis Boulinguiez1, Emmanuelle Vallez1, Eloise Woitrain1, Eric Baugé1, Fanny Lalloyer1, Christian Duhem1, Nabil Rabhi3, Ronald E van Kesteren4, Cheng-Ming Chiang5, Steve Lancel1, Hélène Duez1, Jean-Sébastien Annicotte3, Réjane Paumelle1, Ilse Vanhorebeek2, Greet Van den Berghe2, Bart Staels1, Philippe Lefebvre1,‡ and Jérôme Eeckhoute *,1,‡ 1Inserm, CHU Lille, Institut Pasteur de Lille, U1011-EGID, University of Lille, Lille, France 2Clinical Division and Laboratory of Intensive Care Medicine, Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium 3UMR 8199 - EGID, CNRS, Institut Pasteur de Lille, University of Lille, Lille, France 4Center for Neurogenomics and Cognitive Research, Neuroscience Campus Amsterdam, VU University, Amsterdam, The Netherlands 5Simmons Comprehensive Cancer Center, Departments of Biochemistry and Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX, USA 6Present address: Clinical and Experimental Endocrinology, Department of Chronic Diseases, Metabolism and Ageing (CHROMETA), KU Leuven, Leuven, Belgium ‡These authors contributed equally to this work as second authors ‡These authors contributed equally to this work as third authors ‡These authors contributed equally to this work as senior authors *Corresponding author. Tel: +33 3 20 97 42 20; Fax: +33 3 20 97 42 19; E-mail: [email protected] Molecular Systems Biology (2020)16:e9156https://doi.org/10.15252/msb.20199156 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Liver injury triggers adaptive remodeling of the hepatic transcriptome for repair/regeneration. We demonstrate that this involves particularly profound transcriptomic alterations where acute induction of genes involved in handling of endoplasmic reticulum stress (ERS) is accompanied by partial hepatic dedifferentiation. Importantly, widespread hepatic gene downregulation could not simply be ascribed to cofactor squelching secondary to ERS gene induction, but rather involves a combination of active repressive mechanisms. ERS acts through inhibition of the liver-identity (LIVER-ID) transcription factor (TF) network, initiated by rapid LIVER-ID TF protein loss. In addition, induction of the transcriptional repressor NFIL3 further contributes to LIVER-ID gene repression. Alteration to the liver TF repertoire translates into compromised activity of regulatory regions characterized by the densest co-recruitment of LIVER-ID TFs and decommissioning of BRD4 super-enhancers driving hepatic identity. While transient repression of the hepatic molecular identity is an intrinsic part of liver repair, sustained disequilibrium between the ERS and LIVER-ID transcriptional programs is linked to liver dysfunction as shown using mouse models of acute liver injury and livers from deceased human septic patients. Synopsis Functional genomics analyses shows that acute endoplasmic reticulum stress (ERS) in the liver induces a global loss of molecular identity and partial hepatic dedifferentiation, which characterize mouse and human liver upon acute injury. Acute ERS induces loss of hepatic molecular identity by extensively and preferentially downregulating liver identity (LIVER-ID) genes. ERS-mediated transcriptional repression is an active process involving changes in the transcription factor repertoire with inhibition of the LIVER-ID network and induction of the NFIL3 repressor. This translates into a global loss of activity of cis-regulatory modules densely co-bound by LIVER-ID transcription factors and decommissioning of BRD4 at super-enhancers. The hepatic and ERS transcriptional programs are in competitive equilibrium with direct relevance towards the liver's ability to recover from injury. Introduction The liver exerts instrumental homeostatic and detoxifying functions. This organ is also characterized by a unique capacity to regenerate (Abu Rmilah et al, 2019). Studies in mice subjected to liver regeneration subsequent to partial hepatectomy (PHx), a model of liver resection which is a frequent clinical practice to remove liver tumors (Liu et al, 2015a), have identified a role for endoplasmic reticulum stress (ERS) in this process (Liu et al, 2015b; Argemi et al, 2017). ERS, which results from the accumulation of unfolded/misfolded proteins in the ER lumen, triggers the unfolded protein response (UPR), aimed at restoring ER homeostasis. The UPR is controlled by three major ERS sensors, namely endoplasmic reticulum to nucleus signaling 1 (ERN1/IRE1), eukaryotic translation initiation factor 2 alpha kinase 3 (EIF2AK3/PERK), and activating transcription factor 6 (ATF6; Almanza et al, 2019). Signaling triggered by these sensors leads to activation of the Xbox-binding protein 1 (XBP1S), ATF4 and ATF6 transcription factors (TFs), and subsequent collaborative induction of ERS handling genes such as ER chaperones (Vihervaara et al, 2017; Almanza et al, 2019). Additional, non-transcriptional effects of the UPR involved in alleviating ERS also comprise the regulation of protein synthesis (mRNA translation) and degradation (Almanza et al, 2019). However, it has become clear that ERS bears functions beyond proteostasis per se (Hetz, 2012). For instance, liver regeneration upon PHx requires transient ERS to induce genes involved not only in proteostasis but also in acute-phase and DNA damage responses (Liu et al, 2015b; Argemi et al, 2017). Moreover, ERS has been linked to the (patho)physiological control of lipid and glucose metabolism in the liver (Rutkowski, 2019). The molecular mechanisms involved in ERS-mediated control of liver metabolic functions are still poorly defined. TFs activated by the UPR (including XBP1S, ATF4, DDIT3/CHOP, and ATF6) can directly bind to and modulate expression of specific metabolic genes. In addition, a handful of liver-enriched TFs display reduced expression or activity upon ERS through ill-defined mechanisms (Rutkowski, 2019). In general, while gene silencing substantially contributes to ERS-induced transcriptional regulation, the mechanisms accounting for these downregulations are seldom defined (Vihervaara et al, 2018; Almanza et al, 2019). Hence, ERS-induced transcriptional remodeling, especially gene downregulation, remains to be fully understood and its relevance toward liver pathophysiology to be better defined. We and others have reported that hepatic gene transcription relies on networks of highly interconnected TFs (Kyrmizi et al, 2006), which are co-recruited to cis-regulatory modules (CRMs; Dubois-Chevalier et al, 2017), a conclusion corroborated by several studies in other systems reporting that extensive collaboration of TFs at CRMs is essential for their activities (e.g., Levo et al, 2017). These findings point to a requirement for a comprehensive assessment of changes in global TF expression/activity induced by (patho)physiological signals when aiming to define how transcriptional outputs are controlled. Here, we have used a functional genomics approach to characterize the molecular mechanisms responsible for hepatic gene transcriptional alterations triggered by ERS and to define how this relates to liver damage in mouse models of acute liver injury and livers from deceased human septic patients. Results Acute ERS recapitulates the loss of hepatic molecular identity observed following liver PHx through extensive and preferential repression of liver-identity genes The hepatic response to ERS was defined using transcriptomic analysis of mouse primary hepatocytes (MPH) treated with thapsigargin for 4 h (Appendix Fig S1A). In addition to induction of the UPR (hereafter referred to as the ERS UP genes), we found a substantial fraction of regulated genes (~45%) downregulated upon ERS in MPH (ERS DOWN genes; Appendix Fig S1A). This regulatory pattern was conserved when analyzing the mouse liver transcriptome 8 h after a single intraperitoneal injection of tunicamycin, another ERS-inducing drug (Appendix Fig S1B and C; Arensdorf et al, 2013). Moreover, transcriptomic data mining using Short Time-series Expression Miner (STEM; Ernst et al, 2005; Rib et al, 2018), a tool defining the preferential dynamic patterns of gene expression, confirmed that transient ERS occurs upon PHx (Fig 1A and Appendix Fig S2; Reimold et al, 2000; Liu et al, 2015b). Strikingly, unlike ERS UP genes, which are mostly involved in housekeeping functions, ERS DOWN genes are linked to liver-specific functions (e.g., coagulation, xenobiotic drug metabolism; Fig 1B). Concomitant to transient ERS, mouse liver regeneration following PHx has been linked to transient decrease in metabolic gene expression (White et al, 2005; Argemi et al, 2017). We found this was related to ERS DOWN genes being transiently downregulated upon liver PHx (Fig 1A). As the transcriptome of cells has been proposed to be ruled by ecosystem-like equilibriums where resources required to induce novel programs are used at the expense of established ones (Silveira & Bilodeau, 2018), we monitored the extent of transcriptomic alterations triggered by PHx or chemically induced ERS compared with physiological transcriptomic changes unrelated to liver injury, i.e., triggered by fasting to feeding transition in mice (Benegiamo et al, 2018; Kalvisa et al, 2018). Bagplots (bivariate boxplots showing fold changes in expression relative to baseline mouse liver gene expression levels) revealed that downregulation of the hepatic program upon PHx and ERS was associated with more complex and widespread transcriptomic alterations including a greater induction of genes expressed at low/moderate levels in the healthy mouse liver (Fig 1C). Figure 1. Acute ERS triggers massive transcriptomic alterations characterized by repression of LIVER-ID genes and loss of hepatic molecular identity A. Top 2 significantly overrepresented expression patterns for ERS UP and ERS DOWN genes following PHx. Data show changes in the expression at 4, 10, 48 h, and 1 week after PHx (0 h) for genes comprised within each model profile of dynamic expression identified by STEM. The complete set of identified model profiles is provided in Appendix Fig S2. B. Functional enrichment analyses were performed using ERS UP (upper panels) or ERS DOWN (lower panels) genes and the ToppGene Suite. KEGG Pathways with Bonferroni-corrected P < 10−3 were considered, and similar terms were merged. C. Bagplots showing the breadth of transcriptomic changes for the indicated datasets. Genes were positioned based on their basal expression levels in the control conditions and their FC (Log2) in the indicated (patho)physiological context. The dark blue area is the "bag" (50% of the data points around the median, which is indicated by a red cross), while the light blue area delimits the "loop" (see Materials and Methods for details). Red dots are outliers. D, E. Box plots showing normalized expression in liver (D) and liver-specificity index (E) of LIVER-ID genes, UBQ genes, and other genes. Liver-specificity index was calculated as the difference in normalized expression in liver (2 replicates) and mean of normalized expression in control tissues (2 replicates per tissue) using data from BioGPS (Table EV6) and is reported as Log2. Box plots are composed of a box from the 25th to the 75th percentile with the median as a line and min to max as whiskers. One-way ANOVA with Welch's correction and Dunnett's modified Tukey–Kramer pairwise multiple comparison test was used to assess statistical significance, *P < 0.05. F. Similar analyses as in (A) using LIVER-ID genes. G, H. Box plots showing Log2 FC ERS/control in MPH (3 independent experiments) (G) or mouse liver (3 mice per group) (H) for LIVER-ID genes, UBQ genes, and other genes. Box plots are composed of a box from the 25th to the 75th percentile with the median as a line and min to max as whiskers. One-way ANOVA with Welch's correction and Dunnett's modified Tukey–Kramer pairwise multiple comparison test was used to assess statistical significance, *P < 0.05. I. Enrichment plots from gene set enrichment analyses (GSEA) performed using LIVER-ID genes as the gene set and transcriptomic changes induced by acute ERS in MPH (upper panel) or mouse liver (lower panel) as the ranked gene list. NES and FDR (as in all subsequent GSEA panels) are the normalized enrichment score and the false discovery rate provided by the GSEA software, respectively. J, K. Genes repressed by ERS in MPH (3 independent experiments) (J) or mouse liver (3 mice per group) (K) were ranked based on their Log2 FC ERS/control and divided into quartiles (increased repression from Q1 to Q4). The fraction of LIVER-ID genes in the 4 quartiles was defined and is displayed relative to that obtained for Q1 arbitrarily set to 1. Chi-square test with BH correction for multiple testing was used to assess statistical significance, *P < 0.05. L, M. RT–qPCR analyses of selected ERS UP and LIVER-ID genes monitoring expression changes induced by acute ERS in MPH (3–9 independent experiments) (L) or mouse liver (5–7 mice per group) (M). The bar graphs show means ± SD (standard deviations). One-sample t-test with BH correction for multiple testing was used to determine whether the mean Log2 FC ERS/control is statistically different from 0, *P < 0.05. Panel (L) is also displayed in Appendix Fig S3G. N. Heatmaps showing normalized expression of ERS UP and ERS DOWN genes from MPH (upper panel) or mouse liver (lower panel) in mouse liver at the indicated stages of development. O. Average expression of ERS DOWN genes from MPH in single cells from the hepatobiliary lineage. See Materials and Methods together with Appendix Fig S3I for details regarding data processing. The hepatoblast-to-hepatocyte and hepatoblast-to-cholangiocyte differentiation paths are indicated with arrows. Download figure Download PowerPoint To further characterize the impact of PHx and ERS on the hepatic transcriptional program, we defined cell identity genes, i.e., master liver transcriptional regulators and effector genes. Identity genes establish/maintain tissue-specific functions and distinguish themselves by broad H3K4me3 domains encompassing the transcription start site, a feature functionally related to high transcriptional expression and consistency (Benayoun et al, 2014). We defined liver-identity (LIVER-ID) genes as those displaying this epigenetic feature preferentially in liver (Appendix Fig S3A and Table EV1). We verified that LIVER-ID genes displayed expression levels which are higher (Fig 1D), liver-specific (Fig 1E), and linked to hepatic functions when compared to non-LIVER-ID genes, i.e., ubiquitously labeled with broad H3K4me3 (UBQ genes) or lacking liver broad H3K4me3 (Other genes; Appendix Fig S3B). LIVER-ID genes were transiently downregulated upon liver PHx (Fig 1F) as well as upon ERS both in vitro in MPH and in vivo in mouse liver (Fig 1G and H, Appendix Fig S3C and D). LIVER-ID gene repression was specific and not linked to their high expression levels which could make them more prone to repression, since ERS-mediated repression did not correlate with basal hepatic gene expression (Appendix Fig S3E). Note that while microarray-based transcriptomic analyses reliably define fold changes, this technology under-estimates their magnitude (Dallas et al, 2005). This notion should be taken into account when interpreting microarray-based results throughout the study. Remarkably, LIVER-ID genes were enriched among genes which were most strongly repressed by ERS (Fig 1I–K). Reciprocal induction of ERS UP genes and downregulation of LIVER-ID genes was validated using RT–qPCR in MPH (Fig 1L, Appendix Fig S3F and G) and mouse liver (Fig 1M and Appendix Fig S3F). Interestingly, ERS DOWN genes correspond to genes induced during hepatic differentiation, based on whole liver (Fig 1N and Appendix Fig S3H; Li et al, 2009) or hepatobiliary single-cell (Fig 1O and Appendix Fig S3I and J; Yang et al, 2017) transcriptomic data obtained at different developmental stages. Altogether, these data point to loss of hepatic molecular identity upon liver PHx, which can be recapitulated by acute ERS acting as a widespread repressor of the liver transcriptional program. Acute ERS triggers a global loss of activity of the LIVER-ID TF network and its densely co-bound CRMs To define how liver molecular identity loss is induced by ERS at the transcriptional regulatory level, we monitored changes in CRM activities in MPH using alterations to H3K27 acetylation (H3K27ac) levels as a surrogate. H3K27ac ChIP-seq assays identified regions with increased (62%) or decreased (38%) H3K27ac signal intensities (denoted H3K27ac UP or H3K27ac DOWN), respectively (Appendix Fig S4A and Dataset EV1). Genes linked to H3K27ac UP regions were significantly enriched in ERS UP genes, while ERS DOWN genes were more strongly linked to H3K27ac DOWN regions (Fig 2A). In line, LIVER-ID genes were most significantly linked to H3K27ac DOWN regions (Fig 2B). Figure 2. Acute ERS compromises LIVER-TF expression and activities of their densely co-bound hepatic CRMs A. Comparison of transcriptomics (data from three independent experiments) and H3K27ac ChIP-seq (data from three independent experiments) from MPH. Genes were assigned to H3K27ac regions as described in Materials and Methods. The number of ERS DOWN genes is indicated relative to the number of ERS UP genes for the three categories of H3K27ac regions. Fisher's exact test with BH correction for multiple testing was used to assess statistical significance, *P < 0.05, #P < 0.05. B. Similar analyses to (A). The number of LIVER-ID and UBQ genes is indicated relative to the number of other genes for the three categories of H3K27ac regions. The H3K27ac ChIP-seq data were obtained from three independent MPH experiments. Chi-square test with BH correction for multiple testing was used to assess statistical significance, #P < 0.05. C. Multidimensional scaling (MDS) was performed as described in Materials and Methods, and transcriptional regulator co-recruitment was depicted using density plots for regions with increased (a), decreased (b) or unchanged (c) H3K27ac levels in MPH upon acute ER
Genome-wide association studies have reported that DNA polymorphisms at the CDKN2A locus modulate fasting glucose in human and contribute to type 2 diabetes (T2D) risk. Yet the causal relationship between this gene and defective energy homeostasis remains elusive. Here we sought to understand the contribution of Cdkn2a to metabolic homeostasis. We first analyzed glucose and energy homeostasis from Cdkn2a-deficient mice subjected to normal or high fat diets. Subsequently Cdkn2a-deficient primary adipose cells and human-induced pluripotent stem differentiated into adipocytes were further characterized for their capacity to promote browning of adipose tissue. Finally CDKN2A levels were studied in adipocytes from lean and obese patients. We report that Cdkn2a deficiency protects mice against high fat diet-induced obesity, increases energy expenditure and modulates adaptive thermogenesis, in addition to improving insulin sensitivity. Disruption of Cdkn2a associates with increased expression of brown-like/beige fat markers in inguinal adipose tissue and enhances respiration in primary adipose cells. Kinase activity profiling and RNA-sequencing analysis of primary adipose cells further demonstrate that Cdkn2a modulates gene networks involved in energy production and lipid metabolism, through the activation of the Protein Kinase A (PKA), PKG, PPARGC1A and PRDM16 signaling pathways, key regulators of adipocyte beiging. Importantly, CDKN2A expression is increased in adipocytes from obese compared to lean subjects. Moreover silencing CDKN2A expression during human-induced pluripotent stem cells adipogenic differentiation promoted UCP1 expression. Our results offer novel insight into brown/beige adipocyte functions, which has recently emerged as an attractive therapeutic strategy for obesity and T2D. Modulating Cdkn2a-regulated signaling cascades may be of interest for the treatment of metabolic disorders.
In addition to their well-known role in the control of cellular proliferation and cancer, cell cycle regulators are increasingly identified as important metabolic modulators. Several GWAS have identified SNPs near CDKN2A, the locus encoding for p16INK4a (p16), associated with elevated risk for cardiovascular diseases and type-2 diabetes development, two pathologies associated with impaired hepatic lipid metabolism. Although p16 was recently shown to control hepatic glucose homeostasis, it is unknown whether p16 also controls hepatic lipid metabolism. Using a combination of in vivo and in vitro approaches, we found that p16 modulates fasting-induced hepatic fatty acid oxidation (FAO) and lipid droplet accumulation. In primary hepatocytes, p16-deficiency was associated with elevated expression of genes involved in fatty acid catabolism. These transcriptional changes led to increased FAO and were associated with enhanced activation of PPARα through a mechanism requiring the catalytic AMPKα2 subunit and SIRT1, two known activators of PPARα. By contrast, p16 overexpression was associated with triglyceride accumulation and increased lipid droplet numbers in vitro, and decreased ketogenesis and hepatic mitochondrial activity in vivo Finally, gene expression analysis of liver samples from obese patients revealed a negative correlation between CDKN2A expression and PPARA and its target genes. Our findings demonstrate that p16 represses hepatic lipid catabolism during fasting and may thus participate in the preservation of metabolic flexibility.
We characterized the expression pattern of the nuclear receptors liver X receptor (LXR) alpha and beta during mouse embryonic development and in adulthood by in situ hybridization experiments. LXRalpha and LXRbeta are detected in the liver starting at 11.5 days postcoitum. Later, LXRalpha expression remains high in organs involved in lipid homeostasis, such as liver, intestine, and brown adipose tissue, whereas LXRbeta is more ubiquitously expressed and enriched in tissues of neuronal and endocrine origin.
The glucagon-like peptide 1 (Glp-1) has emerged as a hormone with broad pharmacological potential in type 2 diabetes (T2D) treatment, notably by improving β cell functions. The cell-cycle regulator and transcription factor E2f1 is involved in glucose homeostasis by modulating β cell mass and function. Here, we report that β cell-specific genetic ablation of E2f1 (E2f1β−/−) impairs glucose homeostasis associated with decreased expression of the Glp-1 receptor (Glp1r) in E2f1β−/− pancreatic islets. Pharmacological inhibition of E2F1 transcriptional activity in nondiabetic human islets decreases GLP1R levels and blunts the incretin effect of GLP1R agonist exendin-4 (ex-4) on insulin secretion. Overexpressing E2f1 in pancreatic β cells increases Glp1r expression associated with enhanced insulin secretion mediated by ex-4. Interestingly, ex-4 induces retinoblastoma protein (pRb) phosphorylation and E2f1 transcriptional activity. Our findings reveal critical roles for E2f1 in β cell function and suggest molecular crosstalk between the E2F1/pRb and GLP1R signaling pathways.
Elevation of the dietary saturated fatty acid palmitate contributes to the reduction of functional beta cell mass in the pathogenesis of type 2 diabetes. The diabetogenic effect of palmitate is achieved by increasing beta cell death through induction of the endoplasmic reticulum (ER) stress markers including activating transcription factor 3 (Atf3) and CAAT/enhancer-binding protein homologous protein-10 (Chop). In this study, we investigated whether treatment of beta cells with the MS-275, a HDAC1 and HDAC3 activity inhibitor which prevents beta cell death elicited by cytokines, is beneficial for combating beta cell dysfunction caused by palmitate. We show that culture of isolated human islets and MIN6 cells with MS-275 reduced apoptosis evoked by palmitate. The protective effect of MS-275 was associated with the attenuation of the expression of Atf3 and Chop. Silencing of HDAC3, but not of HDAC1, mimicked the effects of MS-275 on the expression of the two ER stress markers and apoptosis. These data point to HDAC3 as a potential drug target for preserving beta cells against lipotoxicity in diabetes.
In Western society, high-caloric diets rich in fats and sugars have fueled the obesity epidemic and its related disorders. Disruption of the body-brain communication, crucial for maintaining glucose and energy homeostasis, arises from both obesogenic and genetic factors, leading to metabolic disorders. Here, we investigate the role of hypothalamic tanycyte shuttles between the pituitary portal blood and the third ventricle cerebrospinal fluid in regulating energy balance.
