RIPK3 acts as a lipid metabolism regulator contributing to inflammation and carcinogenesis in non-alcoholic fatty liver disease

[EN]Objective Receptor-interacting protein kinase 3 (RIPK3) is a key player in necroptosis execution and an emerging metabolic regulator, whose contribution to non-alcoholic fatty liver disease (NAFLD) is controversial. We aimed to clarify the impact of RIPK3 signalling in the pathogenesis of human and experimental NAFLD. Design RIPK3 levels were evaluated in two large independent cohorts of patients with biopsy proven NAFLD diagnosis and correlated with clinical and biochemical parameters. Wild-type (WT) or Ripk3-deficient (Ripk3(-/-)) mice were fed a choline-deficient L-amino acid-defined diet (CDAA) or an isocaloric control diet for 32 and 66 weeks. Results RIPK3 increased in patients with non-alcoholic steatohepatitis (NASH) in both cohorts, correlating with hepatic inflammation and fibrosis. Accordingly, Ripk3 deficiency ameliorated CDAA-induced inflammation and fibrosis in mice at both 32 and 66 weeks. WT mice on the CDAA diet for 66 weeks developed preneoplastic nodules and displayed increased hepatocellular proliferation, which were reduced in Ripk3(-/-) mice. Furthermore, Ripk3 deficiency hampered tumourigenesis. Intriguingly, Ripk3(-/-) mice displayed increased body weight gain, while lipidomics showed that deletion of Ripk3 shifted hepatic lipid profiles. Peroxisome proliferator-activated receptor. (PPAR.) was increased in Ripk3(-/-) mice and negatively correlated with hepatic RIPK3 in patients with NAFLD. Mechanistic studies established a functional link between RIPK3 and PPAR. in controlling fat deposition and fibrosis. Conclusion Hepatic RIPK3 correlates with NAFLD severity in humans and mice, playing a key role in managing liver metabolism, damage, inflammation, fibrosis and carcinogenesis. Targeting RIPK3 and its intricate signalling arises as a novel promising approach to treat NASH and arrest disease progression.Main funding is provided by FEDER funds through the COMPETE programme and by national funds through Fundacao para a Ciencia e a Tecnologia to CMPR (grants SAICTPAC/0019/2015-LISBOA-01-0145--FEDER-016405 and PTDC/MED-FAR/29097/2017 -LISBOA-01-0145-FEDER-029097). Additional funding comes from research grant APEF (Portuguese Association for the Study of Liver)/BAYER 2020 to MBA. JG is funded by the Fondation pour la Recherche Medicale (ARF20170938613), the Institute of Cardiometabolism and Nutrition (PAP17NECJG), the Societe Francophone du Diabete (R19114DD) and the Mairie de Paris (Emergences -R18139DD). MBA, PMR, MMP and ALS were investigators or students funded by Fundacao para a Ciencia e a Tecnologia

Loss of thymidine phosphorylase activity disrupts adipocyte differentiation and induces insulin-resistant lipoatrophic diabetes

Background Thymidine phosphorylase (TP), encoded by the TYMP gene, is a cytosolic enzyme essential for the nucleotide salvage pathway. TP catalyzes the phosphorylation of the deoxyribonucleosides, thymidine and 2 '-deoxyuridine, to thymine and uracil. Biallelic TYMP variants are responsible for Mitochondrial NeuroGastroIntestinal Encephalomyopathy (MNGIE), an autosomal recessive disorder characterized in most patients by gastrointestinal and neurological symptoms, ultimately leading to death. Studies on the impact of TYMP variants in cellular systems with relevance to the organs affected in MNGIE are still scarce and the role of TP in adipose tissue remains unexplored. Methods Deep phenotyping was performed in three patients from two families carrying homozygous TYMP variants and presenting with lipoatrophic diabetes. The impact of the loss of TP expression was evaluated using a CRISPR-Cas9-mediated TP knockout (KO) strategy in human adipose stem cells (ASC), which can be differentiated into adipocytes in vitro. Protein expression profiles and cellular characteristics were investigated in this KO model. Results All patients had TYMP loss-of-function variants and first presented with generalized loss of adipose tissue and insulin-resistant diabetes. CRISPR-Cas9-mediated TP KO in ASC abolished adipocyte differentiation and decreased insulin response, consistent with the patients' phenotype. This KO also induced major oxidative stress, altered mitochondrial functions, and promoted cellular senescence. This translational study identifies a new role of TP by demonstrating its key regulatory functions in adipose tissue. Conclusions The implication of TP variants in atypical forms of monogenic diabetes shows that genetic diagnosis of lipodystrophic syndromes should include TYMP analysis. The fact that TP is crucial for adipocyte differentiation and function through the control of mitochondrial homeostasis highlights the importance of mitochondria in adipose tissue biology.Mairie de Paris grant [R18139DD]; Societe Francophone du Diabete [R19114DD]; Fondation pour la Recherche Medicale [ARF20170938613, EQU202003010517]; Fondation pour la Recherche Medicale grant [EQU201903007868]; Agence nationale de la recherche grant [ANR-21-CE17-0002-01]Mairie de Paris grant R18139DD (JG) Societe Francophone du Diabete grant R19114DD (JG) Fondation pour la Recherche Medicale grants ARF20170938613 & EQU202003010517 (JG) Agence nationale de la recherche grant ANR-21-CE17-0002-01 (JG) Fondation pour la Recherche Medicale grant EQU201903007868 (IJ, CV, BF

EPHX1 mutations cause a lipoatrophic diabetes syndrome due to impaired epoxide hydrolysis and increased cellular senescence

International audienceEpoxide hydrolases (EHs) regulate cellular homeostasis through hydrolysis of epoxides to less-reactive diols. The first discovered EH was EPHX1, also known as mEH. EH functions remain partly unknown, and no pathogenic variants have been reported in humans. We identified two de novo variants located in EPHX1 catalytic site in patients with a lipoatrophic diabetes characterized by loss of adipose tissue, insulin resistance, and multiple organ dysfunction. Functional analyses revealed that these variants led to the protein aggregation within the endoplasmic reticulum and to a loss of its hydrolysis activity. CRISPR-Cas9-mediated EPHX1 knockout (KO) abolished adipocyte differentiation and decreased insulin response. This KO also promoted oxidative stress and cellular senescence, an observation confirmed in patient-derived fibroblasts. Metreleptin therapy had a beneficial effect in one patient. This translational study highlights the importance of epoxide regulation for adipocyte function and provides new insights into the physiological roles of EHs in humans

The Ellagitannin Metabolite Urolithin C is a Glucose‐Dependent Regulator of Insulin Secretion through activation of L‐type Calcium Channels

International audienc

RIPK3 acts as a lipid metabolism regulator contributing to inflammation and carcinogenesis in non-alcoholic fatty liver disease

International audienceObjective Receptor-interacting protein kinase 3 (RIPK3) is a key player in necroptosis execution and an emerging metabolic regulator, whose contribution to non-alcoholic fatty liver disease (NAFLD) is controversial. We aimed to clarify the impact of RIPK3 signalling in the pathogenesis of human and experimental NAFLD.Design RIPK3 levels were evaluated in two large independent cohorts of patients with biopsy proven NAFLD diagnosis and correlated with clinical and biochemical parameters. Wild-type (WT) or Ripk3-deficient (Ripk3 −/−) mice were fed a choline-deficient L-amino acid-defined diet (CDAA) or an isocaloric control diet for 32 and 66 weeks.Results RIPK3 increased in patients with non-alcoholic steatohepatitis (NASH) in both cohorts, correlating with hepatic inflammation and fibrosis. Accordingly, Ripk3 deficiency ameliorated CDAA-induced inflammation and fibrosis in mice at both 32 and 66 weeks. WT mice on the CDAA diet for 66 weeks developed preneoplastic nodules and displayed increased hepatocellular proliferation, which were reduced in Ripk3 −/− mice. Furthermore, Ripk3 deficiency hampered tumourigenesis. Intriguingly, Ripk3 −/− mice displayed increased body weight gain, while lipidomics showed that deletion of Ripk3 shifted hepatic lipid profiles. Peroxisome proliferator-activated receptor γ (PPARγ) was increased in Ripk3 −/− mice and negatively correlated with hepatic RIPK3 in patients with NAFLD. Mechanistic studies established a functional link between RIPK3 and PPARγ in controlling fat deposition and fibrosis.Conclusion Hepatic RIPK3 correlates with NAFLD severity in humans and mice, playing a key role in managing liver metabolism, damage, inflammation, fibrosis and carcinogenesis. Targeting RIPK3 and its intricate signalling arises as a novel promising approach to treat NASH and arrest disease progression

I kappa B Kinase alpha/beta Control Biliary Homeostasis and Hepatocarcinogenesis in Mice by Phosphorylating the Cell-Death Mediator Receptor-Interacting Protein Kinase 1

The I?B-Kinase (IKK) complex-consisting of the catalytic subunits, IKK? and IKK?, as well as the regulatory subunit, NEMO-mediates activation of the nuclear factor ?B (NF-?B) pathway, but previous studies suggested the existence of NF-?B-independent functions of IKK subunits with potential impact on liver physiology and disease. Programmed cell death is a crucial factor in the progression of liver diseases, and receptor-interacting kinases (RIPKs) exerts strategic control over multiple pathways involved in regulating novel programmed cell-death pathways and inflammation. We hypothesized that RIPKs might be unrecognized targets of the catalytic IKK-complex subunits, thereby regulating hepatocarcinogenesis and cholestasis. In this present study, mice with specific genetic inhibition of catalytic IKK activity in liver parenchymal cells (LPCs; IKK?/?(LPC-KO) ) were intercrossed with RIPK1(LPC-KO) or RIPK3(-/-) mice to examine whether RIPK1 or RIPK3 might be downstream targets of IKKs. Moreover, we performed in vivo phospho-proteome analyses and in vitro kinase assays, mass spectrometry, and mutagenesis experiments. These analyses revealed that IKK? and IKK?-in addition to their known function in NF-?B activation-directly phosphorylate RIPK1 at distinct regions of the protein, thereby regulating cell viability. Loss of this IKK?/?-dependent RIPK1 phosphorylation in LPCs inhibits compensatory proliferation of hepatocytes and intrahepatic biliary cells, thus impeding HCC development, but promoting biliary cell paucity and lethal cholestasis.IKK-complex subunits transmit a previously unrecognized signal through RIPK1, which is fundamental for the long-term consequences of chronic hepatic inflammation and might have potential implications for future pharmacological strategies against cholestatic liver disease and cancer. (Hepatology 2016;64:1217-1231).? 2016 by the American Association for the Study of Liver Diseases