Unveiling the molecular legacy of transient insulin resistance: Implications for hepatic metabolic adaptability Alexandre Berthier, Céline Gheeraert, Manjula Vinod, Manuel Johanns, Loïc Guille, Joel T. Haas, Julie Dubois-Chevalier, Jérôme Eeckhoute, Bart Staels, Philippe Lefebvre Journal of Hepatology, 2025 BACKGROUND & AIMS: Insulin plays a central role in coordinating metabolic flexibility (MetF). Insulin resistance (IR) can impair MetF, contributing to type 2 diabetes and obesity. Transient IR episodes, like gestational diabetes or stress-induced hyperglycemia, also heighten the risk of later diabetes development. While the health significance of transient IR is well established, we aimed to better understand the heretofore poorly understood molecular processes that occur after such episodes. METHODS: To do this, we characterized the hepatic response to a high-fat diet challenge in mice previously exposed to a transient IR episode. We integrated transcriptomic, epigenomic, lipidomic, and molecular clock assessments to provide a molecular basis for the observed dysregulations. RESULTS: Our study shows that temporarily blocking the insulin receptor in young mice leads to later-life liver issues by hindering PPARα-mediated adaptation to a high-fat diet. This is linked to decreased histone active marks at PPARα sites and reduced endogenous PPARα ligands. Transient insulin receptor blockade also altered the liver's molecular clock, particularly affecting PPARα transcriptional responsiveness. CONCLUSIONS: Seemingly reversible metabolic challenges in early adulthood may predispose the liver to exacerbated metabolic dysfunctions when confronted with chronic challenges later in life. IMPACT AND IMPLICATIONS: While the health significance of transient insulin resistance is well established, the molecular processes that occur after such episodes are poorly understood. This study thus provides a circadian molecular paradigm for a later-in-life alteration of liver metabolic flexibility following a previous episode of insulin resistance and calls for particular attention to be paid to detecting transient episodes of insulin resistance as they occur in patients with gestational diabetes or stress-induced hyperglycemia. By extension, any transient exposure to compounds altering circadian rhythmicity, such as anti-depressants, might predispose to a compromised metabolic response to an unbalanced diet later in life.
Combined deletion of cytosolic 5′-nucleotidases IA and II lowers glycemia by improving skeletal muscle insulin action and lowering hepatic glucose production Roxane Jacobs, Gaëtan Herinckx, Noémie Galland, Clémence Balty, Didier Vertommen, Mark H. Rider, Manuel Johanns Journal of Biological Chemistry, 2025 Obesity and type 2 diabetes (T2D)-linked hyperglycemia, along with their associated complications, have reached pandemic proportions, constituting a major public health issue. Genetic deletion or pharmacological inhibition of purine nucleotide-metabolizing enzymes has emerged as a potential strategy for treating diseases. We previously showed that cytosolic 5'-nucleotidase II (NT5C2)-deficient mice were protected against high-fat diet (HFD)-induced insulin resistance. This study investigated the effects of dual deletion of cytosolic 5'-nucleotidases IA (NT5C1A) and II (NT5C2) in mice. We found that NT5C1A/NT5C2 double-knockout (NT5C-dKO) mice exhibited mild hypoglycemia, associated with enhanced skeletal muscle insulin action and reduced hepatic glucose production. This phenotype was accompanied by liver and skeletal muscle proteomic alterations notably related to amino acid metabolism, besides the potential involvement of adenosine monophosphate (AMP)-activated protein kinase (AMPK). Our findings support the development of novel anti-diabetic treatments using small-molecule cytosolic 5'-nucleotidase inhibitors.
How mass spectrometry can be exploited to study AMPK Mark H. Rider, Didier Vertommen, Manuel Johanns Essays in Biochemistry, 2024 AMP-activated protein kinase (AMPK) is a key regulator of metabolism and a recognised target for the treatment of metabolic diseases such as Type 2 diabetes (T2D). Here, we review how mass spectrometry (MS) can be used to study short-term control by AMPK via protein phosphorylation and long-term control due to changes in protein expression. We discuss how MS can quantify AMPK subunit levels in tissues from different species. We propose hydrogen-deuterium exchange (HDX)-MS to investigate molecular mechanisms of AMPK activation and thermoproteomic profiling (TPP) to assess off-target effects of pharmacological AMPK activators/inhibitors. Lastly, because large MS data sets are generated, we consider different approaches that can be used for their interpretation.
O-GlcNAcylation controls pro-fibrotic transcriptional regulatory signaling in myofibroblasts Ninon Very, Clémence Boulet, Céline Gheeraert, Alexandre Berthier, Manuel Johanns, Mohamed Bou Saleh, Loïc Guille, Fabrice Bray, Jean-Marc Strub, Marie Bobowski-Gerard, Francesco P. Zummo, Emmanuelle Vallez, Olivier Molendi-Coste, Eloise Woitrain, Sarah Cianférani, David Montaigne, Line Carolle Ntandja-Wandji, Laurent Dubuquoy, Julie Dubois-Chevalier, Bart Staels, Philippe Lefebvre, Jérôme Eeckhoute Cell Death and Disease, 2024 Tissue injury causes activation of mesenchymal lineage cells into wound-repairing myofibroblasts (MFs), whose uncontrolled activity ultimately leads to fibrosis. Although this process is triggered by deep metabolic and transcriptional reprogramming, functional links between these two key events are not yet understood. Here, we report that the metabolic sensor post-translational modification O-linked β-D-N-acetylglucosaminylation (O-GlcNAcylation) is increased and required for myofibroblastic activation. Inhibition of protein O-GlcNAcylation impairs archetypal myofibloblast cellular activities including extracellular matrix gene expression and collagen secretion/deposition as defined in vitro and using ex vivo and in vivo murine liver injury models. Mechanistically, a multi-omics approach combining proteomic, epigenomic, and transcriptomic data mining revealed that O-GlcNAcylation controls the MF transcriptional program by targeting the transcription factors Basonuclin 2 (BNC2) and TEA domain transcription factor 4 (TEAD4) together with the Yes-associated protein 1 (YAP1) co-activator. Indeed, inhibition of protein O-GlcNAcylation impedes their stability leading to decreased functionality of the BNC2/TEAD4/YAP1 complex towards promoting activation of the MF transcriptional regulatory landscape. We found that this involves O-GlcNAcylation of BNC2 at Thr455 and Ser490 and of TEAD4 at Ser69 and Ser99. Altogether, this study unravels protein O-GlcNAcylation as a key determinant of myofibroblastic activation and identifies its inhibition as an avenue to intervene with fibrogenic processes.
The Molecular Circadian Clock Is a Target of Anti-cancer Translation Inhibitors Alexandre Berthier, Céline Gheeraert, Manuel Johanns, Manjula Vinod, Bart Staels, Jérôme Eeckhoute, Philippe Lefebvre Journal of Biological Rhythms, 2024 Circadian-paced biological processes are key to physiology and required for metabolic, immunologic, and cardiovascular homeostasis. Core circadian clock components are transcription factors whose half-life is precisely regulated, thereby controlling the intrinsic cellular circadian clock. Genetic disruption of molecular clock components generally leads to marked pathological events phenotypically affecting behavior and multiple aspects of physiology. Using a transcriptional signature similarity approach, we identified anti-cancer protein synthesis inhibitors as potent modulators of the cardiomyocyte molecular clock. Eukaryotic protein translation inhibitors, ranging from translation initiation (rocaglates, 4-EGI1, etc.) to ribosomal elongation inhibitors (homoharringtonine, puromycin, etc.), were found to potently ablate protein abundance of REV-ERBα, a repressive nuclear receptor and component of the molecular clock. These inhibitory effects were observed both in vitro and in vivo and could be extended to PER2, another component of the molecular clock. Taken together, our observations suggest that the activity spectrum of protein synthesis inhibitors, whose clinical use is contemplated not only in cancers but also in viral infections, must be extended to circadian rhythm disruption, with potential beneficial or iatrogenic effects upon acute or prolonged administration.
Time-of-day-dependent variation of the human liver transcriptome and metabolome is disrupted in MASLD Manuel Johanns, Joel T. Haas, Violetta Raverdy, Jimmy Vandel, Julie Chevalier-Dubois, Loic Guille, Bruno Derudas, Benjamin Legendre, Robert Caiazzo, Helene Verkindt, Viviane Gnemmi, Emmanuelle Leteurtre, Mehdi Derhourhi, Amélie Bonnefond, Philippe Froguel, Jérôme Eeckhoute, Guillaume Lassailly, Philippe Mathurin, François Pattou, Bart Staels, Philippe Lefebvre Jhep Reports, 2024 Background & Aims: Liver homeostasis is ensured in part by time-of-day-dependent processes, many of them being paced by the molecular circadian clock. Liver functions are compromised in metabolic dysfunction-associated steatotic liver disease (MASLD) and metabolic dysfunction-associated steatohepatitis (MASH), and clock disruption increases susceptibility to MASLD progression in rodent models. We therefore investigated whether the time-of-day-dependent transcriptome and metabolome are significantly altered in human steatotic and MASH livers. Methods: Liver biopsies, collected within an 8 h-window from a carefully phenotyped cohort of 290 patients and histologically diagnosed to be either normal, steatotic or MASH hepatic tissues, were analyzed by RNA sequencing and unbiased metabolomic approaches. Time-of-day-dependent gene expression patterns and metabolomes were identified and compared between histologically normal, steatotic and MASH livers. Results: Herein, we provide a first-of-its-kind report of a daytime-resolved human liver transcriptome-metabolome and associated alterations in MASLD. Transcriptomic analysis showed a robustness of core molecular clock components in steatotic and MASH livers. It also revealed stage-specific, time-of-day-dependent alterations of hundreds of transcripts involved in cell-to-cell communication, intracellular signaling and metabolism. Similarly, rhythmic amino acid and lipid metabolomes were affected in pathological livers. Both TNFα and PPARγ signaling were predicted as important contributors to altered rhythmicity. Conclusion: MASLD progression to MASH perturbs time-of-day-dependent processes in human livers, while the differential expression of core molecular clock components is maintained. Impact and implications: This work characterizes the rhythmic patterns of the transcriptome and metabolome in the human liver. Using a cohort of well-phenotyped patients (n = 290) for whom the time-of-day at biopsy collection was known, we show that time-of-day variations observed in histologically normal livers are gradually perturbed in liver steatosis and metabolic dysfunction-associated steatohepatitis. Importantly, these observations, albeit obtained across a restricted time window, provide further support for preclinical studies demonstrating alterations of rhythmic patterns in diseased livers. On a practical note, this study indicates the importance of considering time-of-day as a critical biological variable which may significantly affect data interpretation in animal and human studies of liver diseases.
Specific post-translational modifications of soluble tau protein distinguishes Alzheimer’s disease and primary tauopathies Nathalie Kyalu Ngoie Zola, Clémence Balty, Sébastien Pyr dit Ruys, Axelle A. T. Vanparys, Nicolas D. G. Huyghe, Gaëtan Herinckx, Manuel Johanns, Emilien Boyer, Pascal Kienlen-Campard, Mark H. Rider, Didier Vertommen, Bernard J. Hanseeuw Nature Communications, 2023 Tau protein aggregates in several neurodegenerative disorders, referred to as tauopathies. The tau isoforms observed in post mortem human brain aggregates is used to classify tauopathies. However, distinguishing tauopathies ante mortem remains challenging, potentially due to differences between insoluble tau in aggregates and soluble tau in body fluids. Here, we demonstrated that tau isoforms differ between tauopathies in insoluble aggregates, but not in soluble brain extracts. We therefore characterized post-translational modifications of both the aggregated and the soluble tau protein obtained from post mortem human brain tissue of patients with Alzheimer’s disease, cortico-basal degeneration, Pick’s disease, and frontotemporal lobe degeneration. We found specific soluble signatures for each tauopathy and its specific aggregated tau isoforms: including ubiquitination on Lysine 369 for cortico-basal degeneration and acetylation on Lysine 311 for Pick’s disease. These findings provide potential targets for future development of fluid-based biomarker assays able to distinguish tauopathies in vivo.
Comparison of Different Ex-Vivo Preservation Strategies on Cardiac Metabolism in an Animal Model of Donation after Circulatory Death Stefano Mastrobuoni, Manuel Johanns, Martial Vergauwen, Gwen Beaurin, Mark Rider, Pierre Gianello, Alain Poncelet, Olivier Van Caenegem Journal of Clinical Medicine, 2023 Transplantation of heart following donation after circulatory death (DCD) was recently introduced into clinical practice. Ex vivo reperfusion following DCD and retrieval is deemed necessary in order to evaluate the recovery of cardiac viability after the period of warm ischemia. We tested the effect of four different temperatures (4 °C—18 °C—25 °C—35 °C) on cardiac metabolism during 3-h ex vivo reperfusion in a porcine model of DCD heart. We observed a steep fall in high-energy phosphate (ATP) concentrations in the myocardial tissue at the end of the warm ischemic time and only limited regeneration during reperfusion. Lactate concentration in the perfusate increased rapidly during the first hour of reperfusion and slowly decreased afterward. However, the temperature of the solution does not seem to have an effect on either ATP or lactate concentration. Furthermore, all cardiac allografts showed a significant weight increase due to cardiac edema, regardless of the temperature.
AMPK inhibits liver gluconeogenesis: fact or fiction? Manuel Johanns, Louis Hue, Mark H. Rider Biochemical Journal, 2023 Is there a role for AMPK in the control of hepatic gluconeogenesis and could targeting AMPK in liver be a viable strategy for treating type 2 diabetes? These are frequently asked questions this review tries to answer. After describing properties of AMPK and different small-molecule AMPK activators, we briefly review the various mechanisms for controlling hepatic glucose production, mainly via gluconeogenesis. The different experimental and genetic models that have been used to draw conclusions about the role of AMPK in the control of liver gluconeogenesis are critically discussed. The effects of several anti-diabetic drugs, particularly metformin, on hepatic gluconeogenesis are also considered. We conclude that the main effect of AMPK activation pertinent to the control of hepatic gluconeogenesis is to antagonize glucagon signalling in the short-term and, in the long-term, to improve insulin sensitivity by reducing hepatic lipid content.
PPARs in liver physiology Alexandre Berthier, Manuel Johanns, Francesco Paolo Zummo, Philippe Lefebvre, Bart Staels Biochimica Et Biophysica Acta Molecular Basis of Disease, 2021
TLR9 and beclin 1 crosstalk regulates muscle AMPK activation in exercise Yang Liu, Phong T. Nguyen, Xun Wang, Yuting Zhao, Corbin E. Meacham, Zhongju Zou, Bogdan Bordieanu, Manuel Johanns, Didier Vertommen, Tobias Wijshake, Herman May, Guanghua Xiao, Sanae Shoji-Kawata, Mark H. Rider, Sean J. Morrison, Prashant Mishra, Beth Levine Nature, 2020
