Abstract
The development of hepatocyte cell models that represent fatty acid partitioning within the human liver would be beneficial for the study of the development and progression of nonalcoholic fatty liver disease (NAFLD). We sought to develop and characterize a novel human liver cell line (LIV0APOLY) to establish a model of lipid accumulation using a physiological mixture of fatty acids under low- and high-glucose conditions. LIV0APOLY cells were compared with a well-established cell line (HepG2) and, where possible, primary human hepatocytes. LIV0APOLY cells were found to proliferate and express some mature liver markers and were wild type for the PNPLA3 (rs738409) gene, whereas HepG2 cells carried the Ile148Met variant that is positively associated with liver fat content. Intracellular triglyceride content was higher in HepG2 than in LIV0APOLY cells; exposure to high glucose and/or exogenous fatty acids increased intracellular triglyceride in both cell lines. Triglyceride concentrations in media were higher from LIV0APOLY compared with HepG2 cells. Culturing LIV0APOLY cells in high glucose increased a marker of endoplasmic reticulum stress and attenuated insulin-stimulated Akt phosphorylation whereas low glucose and exogenous fatty acids increased AMPK phosphorylation. Although LIV0APOLY cells and primary hepatocytes stored similar amounts of exogenous fatty acids as triglyceride, more exogenous fatty acids were partitioned toward oxidation in the LIV0APOLY cells than in primary hepatocytes. LIV0APOLY cells offer the potential to be a renewable cellular model for studying the effects of exogenous metabolic substrates on fatty acid partitioning; however, their usefulness as a model of lipoprotein metabolism needs to be further explored.
Highlights
NONALCOHOLIC FATTY LIVER DISEASE (NAFLD) is one of the most common liver diseases affecting approximately one-third of adults in the developed world who do not consume significant amounts of alcohol [9, 39]; obesity is a well-documented risk factor [9]
Western blot antibodies against cytokeratin 18 (CK18), ␣-fetoprotein (AFP), caspase-3, diacylglycerol acyltransferase-2 (DGAT2), and -actin were from Abcam (Cambridge, UK), anti-albumin was from Universal Biologicals (Cambridge, UK), anti-AMPK Thr172, anti-Akt Ser473, anti-Akt, and anti-CCAAT/enhancer-binding protein homologous protein (CHOP) were from Cell Signaling (Danvers, MA), and anti-apoB was from Santa Cruz Biotechnology (Santa Cruz, CA)
Human primary hepatocytes would be utilized; these come with limitations, including availability, culturing times, the effect of isolation on function, terminal differentiation, and lack of robust cryopreservation techniques
Summary
NONALCOHOLIC FATTY LIVER DISEASE (NAFLD) is one of the most common liver diseases affecting approximately one-third of adults in the developed world who do not consume significant amounts of alcohol [9, 39]; obesity is a well-documented risk factor [9]. Other factors that may impact on TG accumulation and fatty acid metabolism include culturing conditions [20] Both HepG2 and HepaRG cell lines have been shown to secrete TG-containing lipoproteins in a glucose-dependent manner [45, 52], and under high-glucose conditions immortalized human hepatocytes have been shown to increase lipoprotein secretion and lipogenic gene expression [45]. Available cell lines provide some insight into the development of NAFLD, it would be useful to develop cell models that are characterized in terms of culture conditions and the effect this may have on intracellular fatty acid partitioning. Effects of glucose on LIV0APOLY cells were compared with effects on HepG2 cells and, where possible, fatty acid partitioning was compared with primary human hepatocytes
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