NAFLD
'Non-alcoholic fatty liver disease' (NAFLD) is the most common liver disease worldwide affecting at least a third of individuals eating western diets and being associated closely with the current pandemic of paediatric obesity, insulin resistance and type 2 diabetes, can be considered as the liver manifestation of metabolic syndrome. The term NAFLD is used to describe a range of conditions stating with benign hepatic fat accumulation in the absence of excess alcohol consumption ('fatty liver'; steatosis), progressing through fat accumulation with associated inflammation (non-alcoholic steatohepatitis; NASH) and advanced fibrotic infiltration (cirrhosis), leading eventually to hepatocellular carcinoma (HCC), one of the leading causes of cancer-related death in the western world. Intriguingly, only a proportion of individuals at risk progress from fatty liver to subsequent disease stages, although reasons for progression or the lack of it are currently not well understood, though there is a genetic predisposition to HCC.
The potential worldwide burden of NAFLD, coupled with risks associated with active sampling from diagnosed individuals requires the development of tractable experimental models in which human disease mechanisms can be examined. Small animal models of NAFLD are limited by interspecies differences in plasma and tissue biochemistry, while large animal models such as primates and pigs are favoured by similar physiology, their use is hampered by cost, length of time for experimentation and ethical considerations. The alternative approach of developing in vitro models using primary human hepatocytes (PHHs) or human cell lines derived from hepatocellular carcinomas has been limited by inter-donor variability and loss of liver-specific function in culture, although there has been a move towards cell lines such as the human HepG2-derived C3A cell subline, selected for displaying an inducible hepatic phenotype.
Our laboratory has previously developed an in vitro model of fatty liver, using the C3A cell line that recapitulates the cellular events proposed to occur in the NAFLD disease progression described above, including morphological fat infiltration, enhanced reactive oxygen species (ROS) production, oxidatibe stress and inflammation. Briefly, hepatic C3A cells are cultured for 72 hours in a lactate-pyruvate-octanoate-ammonia (LPON) 'nutrient excess' mix, which promotes lipogenesis and triglyceride accumulation, while accelerating the TCA cycle causing oxidative stress. Subsequently, lipid droplet accumulation and ROS formation can be assessed by established laboratory methods, allowing investigation of the effects of excess triglycerides on mitochondrial function. However, the ability to model the effects of metabolic overload on, e.g. altered drug metabolism in C3A cells is limited by low levels of key members of the cytochrome P450 heme-containing protein super-family which are active in detoxifying drugs & products of endogenous liver metabolism and the extreme interventions required to induce expression of these enzymes.
To circumvent these problems & to explore the potential for more representative NAFLD in vitro models, we have applied our established 'nutrient overload' protocol to the recently-characterised human liver bi-potential (hepatocyte: cholangiocyte) progenitor HepaRG cell line. HepaRG cells maintain differentiated liver-specific functions in long term cell culture, including levels of cytochrome P450 family expression comparable to those found in primary human hepatocytes. Proof-of principle biochemical & morphological characterisation experiments have shown that the nutrient overload model can be adapted to HepaRG cells. Ongoing characterisation includes (i) BODIPY 493/503 staining to confirm lipid droplet accumulation; (ii) mitochondrial trackers to assess mitochondrial membrane potential, ADP/ATP ratios and lactate & superoxide production as measures of mitochondrial function and viability.