Investigating the Effects of Placental Hormones in an In Vitro Model of Hepatic Steatosis

Non-alcoholic fatty liver disease (NAFLD) represents a range of liver conditions, from lipid accumulation (steatosis) to more serious steatohepatitis and fibrosis. Its global rise is closely linked to increasing obesity rates. Of particular concern, are women of childbearing age and those who are pregnant, who face a rising prevalence of both obesity and NAFLD. During normal pregnancy, maternal metabolic system adapts to support foetal growth, primarily through the development of transient insulin resistance (IR), a process regulated by placental hormones e.g. placental lactogen (PL) and placental growth hormone (pGH). In pregnancies complicated by hepatic steatosis and IR, hepatic metabolism may fail to adapt, leading to pregnancy complications like gestational diabetes mellitus (GDM). The effects of placental hormones on metabolic pathways in the steatotic state remain unclear, particularly if they further impact insulin signalling and contribute to GDM. The project aimed to investigate the effects of PL and pGH on hepatic metabolism and insulin signalling pathways in an in vitro hepatic steatosis model. The model was developed using HepG2 cells incubated in a mixture of oleic and palmitic free fatty acids (FFA) in a 2:1 ratio for 24 hours. Lipid accumulation was confirmed in the model, with triglycerides increasing to 5.3-fold compared to controls. The addition of PL and pGH to steatotic cells, led to a slight decrease and increase in TG content respectively, though not significantly. Metabolomics analysis using 1H NMR-based and multivariate statistical methods revealed that lipid loading of the HepG2 cells affected several pathways associated with energy, glucose and lipid metabolism. For example, FFA-treated cells exhibited increased lactate and glutamate levels, alongside reductions in glucose and creatine. Co-treatment with PL or pGH produced distinct metabolomic profiles compared to lipid-loaded cells, although no clear hormone-specific pattern was observed. Effects of hormones on a major regulatory pathway in lipid metabolism, i.e. AMPK/ACC signalling pathway and on insulin signalling was studied by Western blot of phosphoproteins. FFA treatment increased phosphorylation of AMPK by 4.4-fold and ACC by 2-fold. PL and pGH co-treatments showed opposing trends in AMPK/ACC phosphorylation compared to FFA alone, with PL tending to increase and pGH to reduce phosphorylation levels, though differences were not statistically significant. PL and pGH significantly reduced insulin-stimulated Akt phosphorylation in FFA-loaded HepG2 cells by 66.6% and 76%, respectively, compared to control. While these reductions were not significantly greater than the 44% decrease observed with FFA alone. Overall, FFA loading in HepG2 cells induced significant alterations in lipid accumulation, metabolite levels, AMPK/ACC signalling, and insulin sensitivity. While PL and pGH modulated some pathways, they did not significantly alter the steatotic phenotype.