Conditioned Media from Fructose-treated Hepatocytes Activates Hepatic Stellate cells in Vitro: Implications for Liver Fibrosis
Modern diets comprised of processed foods high in sugar and unhealthy fats contribute to the development of metabolic diseases such as type 2 diabetes obesity and non-alcoholic fatty liver disease (NAFLD). NAFLD is a progressive disease that develops in response to excessive accumulation of fat in hepatocytes and can lead to inflammation, fibrosis, and hepatocellular carcinoma. The molecular mechanisms underlying the development and progression of NAFLD in response to dietary factors remain largely unexplored. The purpose of this study was to determine the effect of fat and sugar on cultured hepatocytes and hepatic stellate cells, which are the primary fibrogenic cells in the liver. We cultured HepG2 cells, a hepatoma cell line, as a model of hepatocytes and treated them with palmitate and different concentrations of fructose. LX-2 cells, an immortalized hepatic stellate cell (HSC) line, were incubated with 10 mM of fructose or conditioned media (CM) from the HepG2 cells treated with fructose. Changes in the expression of genes involved in lipid metabolism, i.e. stearoyl-CoA desaturase (SCD), acetyl-CoA carboxylase alpha (ACC), fatty acid desaturase (FAD), and fatty acid synthase (FAS), in response to different sugars and the combination of fructose with palmitate were measured via RT-qPCR. Oil Red O staining (ORO) was used to visualize accumulation of lipids in treated cells. We determined that cells treated with 10 mM fructose accumulated the greatest amount of fat compared to treatment with 5 mM and 20 mM fructose. We observed that expression of SCD, ACC, and FAD was downregulated by treatment with 5 mM and 20 mM fructose, while FAS expression was upregulated; treatment with 10 mM fructose resulted in downregulation of all genes. Treatment with 1 mM palmitate alone corresponded with upregulated expression of SCD, ACC, FAD and, FAS, and incubation with a combination of 1 mM palmitate and 10 mM fructose resulted in similar expression patterns. Treatment of LX-2 cells with CM from HepG2 cells treated with 1 mM palmitate and 10 mM fructose resulted in increased expression of actin, alpha 2, a marker of HSC activation. These results suggest that communication between hepatocytes and HSCs may propagate the effects of palmitate and fructose in the liver, and may contribute to fibrogenesis attributed to NAFLD.