RIPK3 dampens mitochondrial bioenergetics and lipid droplet dynamics in metabolic liver disease.

BACKGROUND AND AIMS: Receptor-interacting protein kinase 3 (RIPK3) mediates NAFLD progression, but its metabolic function is unclear. Here, we aimed to investigate the role of RIPK3 in modulating mitochondria function, coupled with lipid droplet (LD) architecture in NAFLD. APPROACH AND RESULTS: Functional studies evaluating mitochondria and LD biology were performed in wild-type (WT) and Ripk3-/- mice fed a choline-deficient, amino acid-defined (CDAA) diet for 32 and 66 weeks and in CRISPR-Cas9 Ripk3 -null fat-loaded immortalized hepatocytes. The association between hepatic perilipin (PLIN) 1 and 5, RIPK3, and disease severity was also addressed in a cohort of patients with NAFLD and in PLIN1 -associated familial partial lipodystrophy. Ripk3 deficiency rescued impairment in mitochondrial biogenesis, bioenergetics, and function in CDAA diet-fed mice and fat-loaded hepatocytes. Ripk3 deficiency was accompanied by a strong upregulation of antioxidant systems, leading to diminished oxidative stress upon fat loading both in vivo and in vitro. Strikingly, Ripk3-/- hepatocytes displayed smaller size LD in higher numbers than WT cells after incubation with free fatty acids. Ripk3 deficiency upregulated adipocyte and hepatic levels of LD-associated proteins PLIN1 and PLIN5. PLIN1 upregulation controlled LD structure and diminished mitochondrial stress upon free fatty acid overload in Ripk3-/- hepatocytes and was associated with diminished human NAFLD severity. Conversely, a pathogenic PLIN1 frameshift variant was associated with NAFLD and fibrosis, as well as with increased hepatic RIPK3 levels in familial partial lipodystrophy. CONCLUSIONS: Ripk3 deficiency restores mitochondria bioenergetics and impacts LD dynamics. RIPK3 inhibition is promising in ameliorating NAFLD.

O-GlcNAc transferase acts as a critical nutritional node for the control of liver homeostasis.

Background & Aims: O-GlcNAcylation is a reversible post-translational modification controlled by the activity of two enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). In the liver, O-GlcNAcylation has emerged as an important regulatory mechanism underlying normal liver physiology and metabolic disease. Methods: To address whether OGT acts as a critical hepatic nutritional node, mice with a constitutive hepatocyte-specific deletion of OGT (OGTLKO) were generated and challenged with different carbohydrate- and lipid-containing diets. Results: Analyses of 4-week-old OGTLKO mice revealed significant oxidative and endoplasmic reticulum stress, and DNA damage, together with inflammation and fibrosis, in the liver. Susceptibility to oxidative and endoplasmic reticulum stress-induced apoptosis was also elevated in OGTLKO hepatocytes. Although OGT expression was partially recovered in the liver of 8-week-old OGTLKO mice, hepatic injury and fibrosis were not rescued but rather worsened with time. Interestingly, weaning of OGTLKO mice on a ketogenic diet (low carbohydrate, high fat) fully prevented the hepatic alterations induced by OGT deletion, indicating that reduced carbohydrate intake protects an OGT-deficient liver. Conclusions: These findings pinpoint OGT as a key mediator of hepatocyte homeostasis and survival upon carbohydrate intake and validate OGTLKO mice as a valuable model for assessing therapeutical approaches of advanced liver fibrosis. Impact and Implications: Our study shows that hepatocyte-specific deletion of O-GlcNAc transferase (OGT) leads to severe liver injury, reinforcing the importance of O-GlcNAcylation and OGT for hepatocyte homeostasis and survival. Our study also validates the Ogt liver-deficient mouse as a valuable model for the study of advanced liver fibrosis. Importantly, as the severe hepatic fibrosis of Ogt liver-deficient mice could be fully prevented upon feeding on a ketogenic diet (i.e. very-low-carbohydrate, high-fat diet) this work underlines the potential interest of nutritional intervention as antifibrogenic strategies.

The necroptosis-inducing pseudokinase mixed lineage kinase domain-like regulates the adipogenic differentiation of pre-adipocytes.

Receptor-interacting protein kinase-3 (RIPK3) and mixed lineage kinase domain-like (MLKL) proteins are key regulators of necroptosis, a highly pro-inflammatory mode of cell death, which has been involved in various human diseases. Necroptotic-independent functions of RIPK3 and MLKL also exist, notably in the adipose tissue but remain poorly defined. Using knock-out (KO) cell models, we investigated the role of RIPK3 and MLKL in adipocyte differentiation. Mlkl-KO abolished white adipocyte differentiation via a strong expression of Wnt10b, a ligand of the Wnt/ß-catenin pathway, and a downregulation of genes involved in lipid metabolism. This effect was not recapitulated by the ablation of Ripk3. Conversely, Mlkl and Ripk3 deficiencies did not block beige adipocyte differentiation. These findings indicate that RIPK3 and MLKL have distinct roles in adipogenesis. The absence of MLKL blocks the differentiation of white, but not beige, adipocytes highlighting the therapeutic potential of MLKL inhibition in obesity.

[Cell death mechanisms in non-alcoholic steatohepatitis]. / Les mécanismes de mort cellulaire dans la stéatohépatite non alcoolique.

Continuous cell death associated with inflammation is a key trigger of disease progression notably in chronic liver diseases such as non-alcoholic steatohepatitis (NASH). Apoptosis has been studied as a potential target for reducing cell death in NASH. However, recent studies suggest that caspase inhibition is inefficient to treat NASH patients and may aggravate the disease by redirecting cells to alternative mechanisms of cell death. Alternative forms of lytic cell death have recently been identified and are known to induce strong inflammatory responses due to cell membrane permeabilization. Therefore, controlling lytic cell death modes offers new opportunities for potential therapeutic intervention in NASH. This review summarizes the underlying molecular mechanisms of apoptosis and lytic cell death modes, including necroptosis, pyroptosis and ferroptosis, and discusses their relevance in NASH.

TITLE: Les mécanismes de mort cellulaire dans la stéatohépatite non alcoolique. ABSTRACT: La mort hépatocellulaire chronique et l'inflammation qui en résulte sont des évènements clés dans la progression de la stéatose hépatique non alcoolique (NAFL) vers la stéatohépatite non alcoolique (NASH). La NASH est un état sévère de la maladie qui est associé au développement de la fibrose et qui peut à terme évoluer vers la cirrhose et le cancer du foie. L'apoptose a initialement été étudiée comme cible potentielle pour réduire la mort des hépatocytes dans la NASH. Cependant, des études récentes suggèrent que l'inhibition des caspases est inefficace pour traiter les patients atteints de NASH et pourrait même aggraver la maladie en redirigeant les hépatocytes vers d'autres voies de mort cellulaire. De nouvelles formes de mort cellulaire dites lytiques ont récemment été identifiées et induisent de fortes réponses inflammatoires causées par la perméabilisation des membranes cellulaires. Le contrôle de ces voies de mort lytiques offre par conséquent de nouvelles opportunités thérapeutiques pour traiter la NASH. Cette revue résume les mécanismes moléculaires déclenchant l'apoptose et les voies de mort lytiques, parmi lesquelles la nécroptose, la pyroptose et la ferroptose, et discute de leur pertinence dans la NASH.

In Vitro and In Vivo Models of Non-Alcoholic Fatty Liver Disease: A Critical Appraisal.

Non-alcoholic fatty liver disease (NAFLD), including non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH), represents the hepatic manifestation of obesity and metabolic syndrome. Due to the spread of the obesity epidemic, NAFLD is becoming the most common chronic liver disease and one of the principal indications for liver transplantation. However, no pharmacological treatment is currently approved to prevent the outbreak of NASH, which leads to fibrosis and cirrhosis. Preclinical research is required to improve our knowledge of NAFLD physiopathology and to identify new therapeutic targets. In the present review, we summarize advances in NAFLD preclinical models from cellular models, including new bioengineered platforms, to in vivo models, with a particular focus on genetic and dietary mouse models. We aim to discuss the advantages and limits of these different models.