Novel defatting strategies reduce lipid accumulation in primary human culture models of liver steatosis.

Lynda Aoudjehane,Yves Chrétien,Filomena Conti,Wilfried Le Goff,Eric Savier,Philippe Lesnik,Chan Sonavine Nget,Olivier Scatton,Claire Goumard,Chantal Housset,Julia Gilaizeau,Yvon Calmus,Romain Morichon,Muhammad Atif,Jérémie Gautheron

doi:10.1242/dmm.042663

Abstract

ABSTRACTNormothermic perfusion provides a means to rescue steatotic liver grafts, including by pharmacological defatting. In this study, we tested the potential of new drug combinations to trigger defatting in three human culture models, primary hepatocytes with induced steatosis, primary hepatocytes isolated from steatotic liver, and precision-cut liver slices (PCLS) of steatotic liver. Forskolin, L-carnitine and a PPARα agonist were all combined with rapamycin, an immunosuppressant that induces autophagy, in a D-FAT cocktail. D-FAT was tested alone or in combination with necrosulfonamide, an inhibitor of mixed lineage kinase domain like pseudokinase involved in necroptosis. Within 24 h, in all three models, D-FAT induced a decrease in triglyceride content by 30%, attributable to an upregulation of genes involved in free fatty acid β-oxidation and autophagy, and a downregulation of those involved in lipogenesis. Defatting was accompanied by a decrease in endoplasmic reticulum stress and in the production of reactive oxygen species. The addition of necrosulfonamide increased the efficacy of defatting by 8%-12% in PCLS, with a trend towards increased autophagy. In conclusion, culture models, notably PCLS, are insightful to design strategies for liver graft rescue. Defatting can be rapidly achieved by combinations of drugs targeting mitochondrial oxidative metabolism, macro-autophagy and lipogenesis.

Highlights

The ongoing donor-organ supply mismatch is a major barrier to reducing the high mortality rate of patients on the waiting list for a liver transplant
We investigated the beneficial effects of the cocktail and the mechanisms of action using a human hepatocyte primary culture in which steatosis was induced, primary human hepatocytes (PHH) which were isolated from human fatty liver samples, and an ex vivo 3D model: human precision-cut liver slices obtained from fatty liver samples
Model of free fatty acids (FFA)-induced steatosis in PHH Normal hepatocytes in primary culture were incubated with different concentrations of the FFA mixture oleic acid (OA): palmitic acid (PA) in the molar ratio 2:1, for 48 h, to induce steatosis

Summary

Introduction

The ongoing donor-organ supply mismatch is a major barrier to reducing the high mortality rate of patients on the waiting list for a liver transplant. Primarily based on machine perfusion, have been recently developed to rescue marginal liver grafts (Kollmann and Selzner, 2017; Kron et al, 2017). This is interesting from the perspective of steatotic liver grafts, as pharmacological agents can be added to the perfusion circuit during normothermic perfusion to stimulate a defatting process. This strategy has been previously demonstrated in preclinical models (Nagrath et al, 2009). This latter group and others have used different pharmacological modulators of triglyceride (TG) metabolism combined with other drugs to reduce the lipid content in cultured rat fatty hepatocytes, in isolated perfused rat fatty livers (Nagrath et al, 2009; Nativ et al, 2013) and, more recently, in fat-loaded primary human hepatocytes (PHH) (Boteon et al, 2018)

Methods

Results

Conclusion