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Insulin‐degrading enzyme inhibition increases the unfolded protein response and favours lipid accumulation in the liver

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British Journal of Pharmacology
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Marine Andres
Marine Andres
Marine Andres
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Nathalie Hennuyer
Nathalie Hennuyer
Nathalie Hennuyer
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Khamis Zibar
Khamis Zibar
Khamis Zibar
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Marie Bicharel‐Leconte
Marie Bicharel‐Leconte
Marie Bicharel‐Leconte
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Abstract and Figures

Background and Purpose Nonalcoholic fatty liver disease refers to liver pathologies, ranging from steatosis to steatohepatitis, with fibrosis ultimately leading to cirrhosis and hepatocellular carcinoma. Although several mechanisms have been suggested, including insulin resistance, oxidative stress, and inflammation, its pathophysiology remains imperfectly understood. Over the last decade, a dysfunctional unfolded protein response (UPR) triggered by endoplasmic reticulum (ER) stress emerged as one of the multiple driving factors. In parallel, growing evidence suggests that insulin‐degrading enzyme (IDE), a highly conserved and ubiquitously expressed metallo‐endopeptidase originally discovered for its role in insulin decay, may regulate ER stress and UPR. Experimental Approach We investigated, by genetic and pharmacological approaches, in vitro and in vivo, whether IDE modulates ER stress‐induced UPR and lipid accumulation in the liver. Key Results We found that IDE‐deficient mice display higher hepatic triglyceride content along with higher inositol‐requiring enzyme 1 (IRE1) pathway activation. Upon induction of ER stress by tunicamycin or palmitate in vitro or in vivo, pharmacological inhibition of IDE, using its inhibitor BDM44768, mainly exacerbated ER stress‐induced IRE1 activation and promoted lipid accumulation in hepatocytes, effects that were abolished by the IRE1 inhibitors 4μ8c and KIRA6. Finally, we identified that IDE knockout promotes lipolysis in adipose tissue and increases hepatic CD36 expression, which may contribute to steatosis. Conclusion and Implications These results unravel a novel role for IDE in the regulation of ER stress and development of hepatic steatosis. These findings pave the way to innovative strategies modulating IDE to treat metabolic diseases.
IDE deletion in mice disrupts lipid metabolism and induces the liver IRE1 pathway. (a) Blood glucose, insulin, circulating free fatty acid, and triglyceride levels in IDE KO versus WT mice (12‐week‐old, n = 11–12 WT, n = 11–13 KO) after a 6‐h fasting. (b) Liver weight expressed as percentage of body weight, (c) hepatic triglyceride (TG) content normalized to liver weight. (d) Oil Red O and haematoxylin staining of liver sections observed by light microscopy. Squares indicate regions that are zoomed in and displayed below. Magnifications are ×10 and ×20. Scale bars indicate 100 μm. (e) Relative hepatic mRNA levels of lipid metabolism‐related genes (expressed as fold change vs. WT) determined by real‐time qPCR and normalized to cyclophilin A. (f) Western blots of IDE and ADRP and quantitative analyses of ADRP in total liver lysates, normalized to GAPDH as loading control. (g) Relative hepatic mRNA levels of IRE1 pathway‐related genes (expressed as fold change vs. WT) determined by real‐time qPCR and normalized to cyclophilin A. (h) Western blots and quantitative analyses of p‐IRE1α and t‐IRE1α protein levels in total liver lysates, normalized to Vinculin as loading control. Experiments (b–h) were performed on IDE KO versus WT 12‐week‐old (n = 6–7 WT, n = 7–12 KO) mice. All data are presented as mean ± SEM, *P < 0.05 versus WT by Student's t‐test.
IDE deletion in mice disrupts lipid metabolism and induces the liver IRE1 pathway. (a) Blood glucose, insulin, circulating free fatty acid, and triglyceride levels in IDE KO versus WT mice (12‐week‐old, n = 11–12 WT, n = 11–13 KO) after a 6‐h fasting. (b) Liver weight expressed as percentage of body weight, (c) hepatic triglyceride (TG) content normalized to liver weight. (d) Oil Red O and haematoxylin staining of liver sections observed by light microscopy. Squares indicate regions that are zoomed in and displayed below. Magnifications are ×10 and ×20. Scale bars indicate 100 μm. (e) Relative hepatic mRNA levels of lipid metabolism‐related genes (expressed as fold change vs. WT) determined by real‐time qPCR and normalized to cyclophilin A. (f) Western blots of IDE and ADRP and quantitative analyses of ADRP in total liver lysates, normalized to GAPDH as loading control. (g) Relative hepatic mRNA levels of IRE1 pathway‐related genes (expressed as fold change vs. WT) determined by real‐time qPCR and normalized to cyclophilin A. (h) Western blots and quantitative analyses of p‐IRE1α and t‐IRE1α protein levels in total liver lysates, normalized to Vinculin as loading control. Experiments (b–h) were performed on IDE KO versus WT 12‐week‐old (n = 6–7 WT, n = 7–12 KO) mice. All data are presented as mean ± SEM, *P < 0.05 versus WT by Student's t‐test.
… 
BDM44768 engages IDE in HepG2 cells in ER stress conditions. Binding of BDM44768 to intracellular IDE (target engagement) in HepG2 cells, (a) representative dot blots showing soluble IDE remaining after heat shocks at several temperatures (40°C to 67°C) and 2‐h treatment with control (DMSO), 0.5 μg·ml⁻¹ tunicamycin (TN) and/or 30‐μM BDM44768 (n = 3) and (b) quantification of soluble IDE obtained by three independent experiments (n = 3, mean ± SEM). (c) Soluble IDE remaining after heat shocks at several temperatures (40°C to 67°C) and 2‐h treatment with control (DMSO), 500‐μM palmitate, and/or 30‐μM BDM44768 (n = 3) and (d) quantification of soluble IDE obtained by three independent experiments (n = 3, mean ± SEM). Stabilization of IDE in the presence of compounds is expressed as the ΔTAgg. All data are presented as mean ± SEM. Statistical analyses were performed by one‐way ANOVA followed by Tukey's post hoc test, *P < 0.05 versus control and ¤P < 0.05versus TN or palmitate.
BDM44768 engages IDE in HepG2 cells in ER stress conditions. Binding of BDM44768 to intracellular IDE (target engagement) in HepG2 cells, (a) representative dot blots showing soluble IDE remaining after heat shocks at several temperatures (40°C to 67°C) and 2‐h treatment with control (DMSO), 0.5 μg·ml⁻¹ tunicamycin (TN) and/or 30‐μM BDM44768 (n = 3) and (b) quantification of soluble IDE obtained by three independent experiments (n = 3, mean ± SEM). (c) Soluble IDE remaining after heat shocks at several temperatures (40°C to 67°C) and 2‐h treatment with control (DMSO), 500‐μM palmitate, and/or 30‐μM BDM44768 (n = 3) and (d) quantification of soluble IDE obtained by three independent experiments (n = 3, mean ± SEM). Stabilization of IDE in the presence of compounds is expressed as the ΔTAgg. All data are presented as mean ± SEM. Statistical analyses were performed by one‐way ANOVA followed by Tukey's post hoc test, *P < 0.05 versus control and ¤P < 0.05versus TN or palmitate.
… 
IDE pharmacological inhibition modulates the IRE1 pathway and IRE1‐dependent lipid droplets accumulation in hepatocytes. (a) Relative mRNA levels (expressed as fold change vs. control) of IRE1 pathway‐related genes determined by real‐time qPCR and normalized to cyclophilin A (n = 3) and (b) western blots and quantitative analyses of p‐IRE1α and t‐IRE1α protein levels normalized to Vinculin as loading control (n = 5) in HepG2 cells after overnight and 24‐h treatment, respectively, with or without 0.5 μg·ml⁻¹ tunicamycin and 15‐μM BDM44768. (c) Relative mRNA levels (expressed as fold change vs. control) of IRE1 pathway‐related genes determined by real‐time qPCR and normalized to cyclophilin A (n = 9) and (d) western blots and quantitative analyses of p‐IRE1α and t‐IRE1α protein levels normalized to Vinculin as loading control (n = 4) in HepG2 cells after overnight treatment with or without 500‐μM palmitate (palm) and 15‐μM BDM44768. (e) IDE, FITM1, Fsp27, and ADRP mRNA expression in HepG2 cells treated with DMSO, BDM44768 (15 μM), palm (500 μM), or a combination of palm + BDM44768 (n = 4). (f) Representative fluorescent microscopy images of LD stained by LipidTOX™ Deep Red Neutral Lipid Stain (red spots) and (g) their quantification expressed as number of LD per cell versus control (Ctrl, dashed line) in HepG2 cells treated for 24 h with 500‐μM palmitate with or without 15‐μM BDM44768 in presence or absence of 20‐μM 4μ8c (n = 4–5). Cells treated with DMSO or BDM44768 alone also are displayed. All data are presented as mean ± SEM. The dotted line represents control values. Statistical analyses were performed by one‐way ANOVA followed by Tukey's post hoc test, *P < 0.05 versus TN or palm.
IDE pharmacological inhibition modulates the IRE1 pathway and IRE1‐dependent lipid droplets accumulation in hepatocytes. (a) Relative mRNA levels (expressed as fold change vs. control) of IRE1 pathway‐related genes determined by real‐time qPCR and normalized to cyclophilin A (n = 3) and (b) western blots and quantitative analyses of p‐IRE1α and t‐IRE1α protein levels normalized to Vinculin as loading control (n = 5) in HepG2 cells after overnight and 24‐h treatment, respectively, with or without 0.5 μg·ml⁻¹ tunicamycin and 15‐μM BDM44768. (c) Relative mRNA levels (expressed as fold change vs. control) of IRE1 pathway‐related genes determined by real‐time qPCR and normalized to cyclophilin A (n = 9) and (d) western blots and quantitative analyses of p‐IRE1α and t‐IRE1α protein levels normalized to Vinculin as loading control (n = 4) in HepG2 cells after overnight treatment with or without 500‐μM palmitate (palm) and 15‐μM BDM44768. (e) IDE, FITM1, Fsp27, and ADRP mRNA expression in HepG2 cells treated with DMSO, BDM44768 (15 μM), palm (500 μM), or a combination of palm + BDM44768 (n = 4). (f) Representative fluorescent microscopy images of LD stained by LipidTOX™ Deep Red Neutral Lipid Stain (red spots) and (g) their quantification expressed as number of LD per cell versus control (Ctrl, dashed line) in HepG2 cells treated for 24 h with 500‐μM palmitate with or without 15‐μM BDM44768 in presence or absence of 20‐μM 4μ8c (n = 4–5). Cells treated with DMSO or BDM44768 alone also are displayed. All data are presented as mean ± SEM. The dotted line represents control values. Statistical analyses were performed by one‐way ANOVA followed by Tukey's post hoc test, *P < 0.05 versus TN or palm.
… 
IDE pharmacological inhibition potentiates liver IRE1 pathway and lipid accumulation in an acute mice model of ER stress. Experiments were performed on 6‐h fasted 20‐week‐old WT mice treated for 8 h with 0.5 mg·kg⁻¹ tunicamycin (TN) with or without 50 mg·kg⁻¹ BDM44768 injection (n = 4). (a) Relative hepatic mRNA levels (expressed as fold change compared with vehicle‐treated WT group [dashed line]) of IRE1 pathway‐related genes determined by real‐time qPCR and normalized to cyclophilin A. (b) Western blots and quantitative analyses of p‐IRE1α and t‐IRE1α protein levels in total liver lysates, normalized to Vinculin as loading control, n = 6–7. (c) Hepatic triglyceride (TG) content normalized to liver weight. (d) Oil Red O and haematoxylin staining of liver sections observed by light microscopy (upper panel, scale bar: 100 μm), and liver sections observed by transmission electron microscopy (lower panel, scale bar 10 μm). (e) Relative hepatic mRNA levels of genes involved in lipid metabolism (expressed as fold change compared with vehicle‐treated WT group [dashed line]). All data are presented as mean ± SEM. Statistical analyses were performed by Student's t‐test, *P < 0.05 versus TN.
IDE pharmacological inhibition potentiates liver IRE1 pathway and lipid accumulation in an acute mice model of ER stress. Experiments were performed on 6‐h fasted 20‐week‐old WT mice treated for 8 h with 0.5 mg·kg⁻¹ tunicamycin (TN) with or without 50 mg·kg⁻¹ BDM44768 injection (n = 4). (a) Relative hepatic mRNA levels (expressed as fold change compared with vehicle‐treated WT group [dashed line]) of IRE1 pathway‐related genes determined by real‐time qPCR and normalized to cyclophilin A. (b) Western blots and quantitative analyses of p‐IRE1α and t‐IRE1α protein levels in total liver lysates, normalized to Vinculin as loading control, n = 6–7. (c) Hepatic triglyceride (TG) content normalized to liver weight. (d) Oil Red O and haematoxylin staining of liver sections observed by light microscopy (upper panel, scale bar: 100 μm), and liver sections observed by transmission electron microscopy (lower panel, scale bar 10 μm). (e) Relative hepatic mRNA levels of genes involved in lipid metabolism (expressed as fold change compared with vehicle‐treated WT group [dashed line]). All data are presented as mean ± SEM. Statistical analyses were performed by Student's t‐test, *P < 0.05 versus TN.
… 
IDE deletion cumulated to acute ER stress in vivo potentiates liver IRE1 pathway and lipid accumulation. Experiments were performed on 11‐week‐old IDE KO versus WT mice (n = 5 WT + TN, n = 4 KO + TN) 18 h after 2 mg·kg⁻¹ tunicamycin (TN) treatment. (a) Relative hepatic mRNA levels (expressed as fold change compared with vehicle‐treated WT group [dashed line]) of IRE1 pathway‐related genes determined by real‐time qPCR and normalized to cyclophilin A. (b) Western blots and quantitative analyses of p‐IRE1α and t‐IRE1α protein levels in total liver lysates, normalized to Vinculin as loading control. (c) Hepatic triglyceride (TG) content normalized to liver weight. (d) Oil Red O and haematoxylin staining of liver sections observed by light microscopy. Squares indicate regions that are zoomed in and displayed below. Magnifications are X10 and X20. Scales are indicated in the images. (e) Relative hepatic mRNA levels of lipid metabolism‐related genes (expressed as fold change vs. vehicle‐treated WT group [dashed line]) determined by real‐time qPCR and normalized to cyclophilin A. All data are presented as mean ± SEM. Statistical analyses were performed by Student's t‐test, *P < 0.05 versus WT + TN.
+1
IDE deletion cumulated to acute ER stress in vivo potentiates liver IRE1 pathway and lipid accumulation. Experiments were performed on 11‐week‐old IDE KO versus WT mice (n = 5 WT + TN, n = 4 KO + TN) 18 h after 2 mg·kg⁻¹ tunicamycin (TN) treatment. (a) Relative hepatic mRNA levels (expressed as fold change compared with vehicle‐treated WT group [dashed line]) of IRE1 pathway‐related genes determined by real‐time qPCR and normalized to cyclophilin A. (b) Western blots and quantitative analyses of p‐IRE1α and t‐IRE1α protein levels in total liver lysates, normalized to Vinculin as loading control. (c) Hepatic triglyceride (TG) content normalized to liver weight. (d) Oil Red O and haematoxylin staining of liver sections observed by light microscopy. Squares indicate regions that are zoomed in and displayed below. Magnifications are X10 and X20. Scales are indicated in the images. (e) Relative hepatic mRNA levels of lipid metabolism‐related genes (expressed as fold change vs. vehicle‐treated WT group [dashed line]) determined by real‐time qPCR and normalized to cyclophilin A. All data are presented as mean ± SEM. Statistical analyses were performed by Student's t‐test, *P < 0.05 versus WT + TN.
… 
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RESEARCH ARTICLE
Insulin-degrading enzyme inhibition increases the unfolded
protein response and favours lipid accumulation in the liver
Marine Andres
1,2
| Nathalie Hennuyer
2
| Khamis Zibar
2
|
Marie Bicharel-Leconte
2
| Isabelle Duplan
2
| Emmanuelle Enée
3
|
Emmanuelle Vallez
2
| Adrien Herledan
1
| Anne Loyens
4
| Bart Staels
2
|
Benoit Deprez
1
| Peter van Endert
3,5
| Rebecca Deprez-Poulain
1,6
| Steve Lancel
2,7
1
Univ. Lille, Inserm, Institut Pasteur de Lille,
U1177 - EGID Drugs and Molecules for Living
Systems, Lille, France
2
Univ. Lille, Inserm, CHU Lille, Institut Pasteur
de Lille, U1011-EGID, Lille, France
3
Université Paris Cité, INSERM, CNRS, Institut
Necker Enfants Malades, Paris, France
4
Univ. Lille, UMR-S 1172-JPArc Centre de
Recherche Jean-Pierre Aubert Neurosciences
et Cancer, Lille, France
5
Service immunologie biologique, AP-HP,
Hôpital Universitaire Necker-Enfants Malades,
Paris, France
6
Institut Universitaire de France (IUF), Paris,
France
7
Univ. Lille, Inserm, CHU Lille, Institut Pasteur
de Lille, U1167 - RID-AGE - Facteurs de risque
et déterminants moléculaires des maladies
liées au vieillissement, Lille, France
Correspondence
Rebecca Deprez-Poulain, Univ. Lille, Inserm,
Institut Pasteur de Lille, U1177 - EGID Drugs
and Molecules for Living Systems, F-59000
Lille, France.
Email: rebecca.deprez@univ-lille.fr
Steve Lancel, Univ. Lille, Inserm, CHU Lille,
Institut Pasteur de Lille, U1011- EGID,
F-59000 Lille, France.
Email: steve.lancel@univ-lille.fr
Funding information
Université de Lille; Institut National de la Santé
et de la Recherche Médicale; Institute Pasteur
De Lille; Fondation pour la Recherche
Médicale, Grant/Award Numbers:
DCM20111223046, EQU201903007853,
FDT202001010917; Centre hospitalier
Background and Purpose: Nonalcoholic fatty liver disease refers to liver pathologies,
ranging from steatosis to steatohepatitis, with fibrosis ultimately leading to cirrhosis
and hepatocellular carcinoma. Although several mechanisms have been suggested,
including insulin resistance, oxidative stress, and inflammation, its pathophysiology
remains imperfectly understood. Over the last decade, a dysfunctional unfolded pro-
tein response (UPR) triggered by endoplasmic reticulum (ER) stress emerged as one
of the multiple driving factors. In parallel, growing evidence suggests that insulin-
degrading enzyme (IDE), a highly conserved and ubiquitously expressed metallo-
endopeptidase originally discovered for its role in insulin decay, may regulate ER
stress and UPR.
Experimental Approach: We investigated, by genetic and pharmacological
approaches, in vitro and in vivo, whether IDE modulates ER stress-induced UPR and
lipid accumulation in the liver.
Key Results: We found that IDE-deficient mice display higher hepatic triglyceride
content along with higher inositol-requiring enzyme 1 (IRE1) pathway activation.
Upon induction of ER stress by tunicamycin or palmitate in vitro or in vivo, pharma-
cological inhibition of IDE, using its inhibitor BDM44768, mainly exacerbated ER
stress-induced IRE1 activation and promoted lipid accumulation in hepatocytes,
effects that were abolished by the IRE1 inhibitors 4μ8c and KIRA6. Finally, we identi-
fied that IDE knockout promotes lipolysis in adipose tissue and increases hepatic
CD36 expression, which may contribute to steatosis.
Conclusion and Implications: These results unravel a novel role for IDE in the regula-
tion of ER stress and development of hepatic steatosis. These findings pave the way
to innovative strategies modulating IDE to treat metabolic diseases.
Abbreviations: AC3, adenylate cyclase 3; ADRP, adipose differentiation-related protein; ATF6, activating transcription factor 6; ERDJ4, endoplasmic reticulum DnaJ homologue 4; FITM1, fat
storage-inducing transmembrane protein 1; FSP27, fat-specific protein 27; HSL, hormone-sensitive lipase; IDE, insulin-degrading enzyme; IRE1, inositol-requiring enzyme 1; MASH, metabolic
dysfunction-associated steatohepatitis; MASLD, metabolic dysfunction-associated steatotic liver disease; PERK, protein kinase RNA-like ER kinase; UPR, unfolded protein response; XBP1s, X-
box binding protein 1 spliced; XBP1u, X-box binding protein 1 unspliced.
Marine Andres, Nathalie Hennuyer, Deprez-Poulain, and Steve Lancel contributed equally to this work.
Received: 5 September 2023 Revised: 3 April 2024 Accepted: 25 April 2024
DOI: 10.1111/bph.16436
Br J Pharmacol. 2024;1–17. wileyonlinelibrary.com/journal/bph © 2024 British Pharmacological Society. 1
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  • Jun 2023
  • J HEPATOL
Unlabelled: The principal limitations of the terms nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) are the reliance on exclusionary confounder terms and the use of potentially stigmatising language. This study set out to determine if content experts and patient advocates were in favour of a change in nomenclature and/or definition. Methods: A modified Delphi process was led by three large pan-national liver associations. Consensus was defined a priori as a supermajority (67%) vote. An independent committee of experts external to the nomenclature process made the final recommendation on the acronym and its diagnostic criteria. Results: A total of 236 panellists from 56 countries participated in four online surveys and two hybrid meetings. Response rates across the 4 survey rounds were 87%, 83%, 83% and 78%, respectively. 74% of respondents felt that the current nomenclature was sufficiently flawed to consider a name change. The terms 'non-alcoholic' and 'fatty' were felt to be stigmatising by 61% and 66% of respondents, respectively. Steatotic liver disease (SLD) was chosen as an overarching term to encompass the various aetiologies of steatosis. The term steatohepatitis was felt to be an important pathophysiological concept that should be retained. The name chosen to replace NAFLD was metabolic dysfunction-associated steatotic liver disease (MASLD). There was consensus to change the definition to include the presence of at least one of five cardiometabolic risk factors. Those with no metabolic parameters and no known cause were deemed to have cryptogenic SLD. A new category, outside pure MASLD, termed MetALD was selected to describe those with MASLD who consume greater amounts of alcohol per week (140 to 350 g/week and 210 to 420 g/week for females and males respectively). Conclusions: The new nomenclature and diagnostic criteria are widely supported, non-stigmatising and can improve awareness and patient identification.
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Identification of indole-based activators of insulin degrading enzyme
Article
  • Nov 2021
  • EUR J MED CHEM
Insulin degrading enzyme (IDE) is a zinc metalloprotease that cleaves numerous substrates among which amyloid-β and insulin. It has been linked through genetic studies to the risk of type-2 diabetes (T2D) or Alzheimer's disease (AD). Pharmacological activation of IDE is an attractive therapeutic strategy in AD. While IDE inhibition gave paradoxal activity in glucose homeostasis, recent studies, in particular in the liver suggest that IDE activators could be also of interest in diabetes. Here we describe the discovery of an original series of IDE activators by screening and structure-activity relationships. Early cellular studies show that hit 1 decreases glucose-stimulating insulin secretion. Docking studies revealed it has an unprecedented extended binding to the polyanion-binding site of IDE. These indole-based pharmacological tools are activators of both Aβ and insulin hydrolysis by IDE and could be helpful to explore the multiple roles of IDE.
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