Modelling of steatosis and insulin resistance development in human hepatic 3d spheroids : mechanisms and genetic aspects

Abstract: Hepatic steatosis and insulin resistance are common disease manifestations seen in metabolic syndrome, non-alcoholic fatty liver disease (NAFLD) and type 2 diabetes (T2D) patients. Aberrant hepatic lipid accumulation leads to both morphological changes and metabolic alterations in liver function and may cause serious end-stage liver disease. There is currently an unmet need for better and more sophisticated human in vitro models that can interrogate these liver conditions, emulate key disease mechanisms and thereby facilitate the development of novel therapeutic drugs. The overall aim of this thesis was to develop and assess the suitability of a human three-dimensional (3D) hepatic in vitro system consisting of primary human hepatocytes (PHH) to mimic fatty liver disease and associated hepatic disease manifestations, including insulin resistance. Moreover, we sought to explore potential disease modifying mechanisms using this novel in vitro model and study the importance of genetic polymorphism related both to liver disease development and drug response. We demonstrate that PHH cultured as 3D microtissues, hepatic spheroids, can emulate important hallmarks of fatty liver disease using pathophysiological concentrations of nutrients in cell media. Following this treatment, hepatic spheroids develop steatosis over the course of several days, accumulate several types of lipids such as triacylglycerols (TAG) and diacylglycerols (DAG) and subsequently become insulin resistant as judged by increased expression of gluconeogenic markers. Induced steatosis was found to be reversible following nutrient deprivation and this reversibility was accelerated by different types of drugs. Interindividual variability in fat accumulation in hepatic spheroids was attributed to genetic polymorphisms associated with risk for NAFLD, such as the TM6SF2 E167K genetic variant. By utilizing sequencing data and a bioinformatics approach, we further demonstrate that rare genetic variants in nuclear receptors, metabolic enzymes and cellular transporters could potentially affect disease susceptibility and drug efficacy. The majority of all studied gene variants were rare and around 30% of the functional variability could be attributed to these variants, highlighting the influence of rare genetic variants for interindividual variability in drug treatment responses. The influence of rare variants must be considered also for genes of importance for NAFLD development. In the last study of this thesis, we also unraveled the beneficial role of inorganic nitrate and nitrite, found in leafy vegetables, in the modulation of hepatic steatosis and cardio-metabolic functions. Boosting of the levels of nitric oxide by nitrate and nitrite ameliorated aberrant hepatic fat accumulation in both hepatic 3D spheroids, HepG2 cells and obese mice. Mechanistically, inorganic nitrate and nitrite treatment decreased oxidative stress derived from NADPH oxidase and stimulated AMP-activated protein kinase with beneficial effects on hepatic lipid accumulation. In conclusion, the versatile hepatic 3D spheroid fatty liver disease model developed and presented in this thesis has the potential to provide new mechanistic insights into liver disease and associated metabolic syndrome disorders and thereby gain future drug development efforts. Furthermore, the insights into the extensive genetic variability in genes potentially influencing drug response and disease development incentivize the adoption of these findings into clinical practice to enable personalized medicine.

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