Abstract
Impairment of hepatic fatty acid metabolism can lead to liver steatosis and injury. Testing drugs for interference with hepatic fatty acid metabolism is therefore important. To find out whether HepG2 cells are suitable for this purpose, we investigated the effect of three established fatty acid metabolism inhibitors and of three test compounds on triglyceride accumulation, palmitate metabolism, the acylcarnitine pool and dicarboxylic acid accumulation in the cell supernatant and on ApoB-100 excretion in HepG2 cells. The three established inhibitors [etomoxir, methylenecyclopropylacetic acid (MCPA), and 4-bromocrotonic acid (4-BCA)] depleted mitochondrial ATP at lower concentrations than cytotoxicity occurred, suggesting mitochondrial toxicity. They inhibited palmitate metabolism at similar or lower concentrations than ATP depletion, and 4-BCA was associated with cellular fat accumulation. They caused specific changes in the acylcarnitine pattern and etomoxir an increase of thapsic (C18 dicarboxylic) acid in the cell supernatant, and did not interfere with ApoB-100 excretion (marker of VLDL export). The three test compounds (amiodarone, tamoxifen, and the cannabinoid WIN 55,212-2) depleted the cellular ATP content at lower concentrations than cytotoxicity occurred. They all caused cellular fat accumulation and inhibited palmitate metabolism at similar or higher concentrations than ATP depletion. They suppressed medium-chain acylcarnitines in the cell supernatant and amiodarone and tamoxifen impaired thapsic acid production. Tamoxifen and WIN 55,212-2 decreased cellular ApoB-100 excretion. In conclusion, the established inhibitors of fatty acid metabolism caused the expected effects in HepG2 cells. HepG cells proved to be useful for the detection of drug-associated toxicities on hepatocellular fatty acid metabolism.
Highlights
Hepatocellular metabolism of fatty acids is a complex function of hepatocytes
The principle aims of the current study were to investigate the effect of different compounds known to be associated with liver steatosis on a human hepatocyte cell line, to find out underlying mechanisms and to assess the suitability of the different assays used to determine fatty acid metabolism
We included six compounds in our study; three established inhibitors of hepatic fatty acid metabolism [etomoxir as an inhibitor of CPT1A (Lilly et al, 1992; Ceccarelli et al, 2011), methylenecyclopropylacetic acid (MCPA) as an inhibitor of medium chain acyl-CoA dehydrogenase (Ikeda and Tanaka, 1990; Tserng et al, 1991) and 4-bromocrotonic acid (4-BCA) as an inhibitor of 3-keto-acyl-CoA thiolase (Olowe and Schulz, 1982)] and three test compounds known to interfere with hepatic fatty acid metabolism and to be associated with liver steatosis (Purohit et al, 2010; Amacher, 2014) (Supplementary Figure 1)
Summary
Hepatocellular metabolism of fatty acids is a complex function of hepatocytes. During starvation, when fatty acids become the most important fuel, activated fatty acids are mainly transported into the mitochondrial matrix for subsequent β-oxidation (Soeters et al, 2012). After ingestion of a meal, the high insulin concentration blocks transport of fatty acids into the mitochondrial matrix by inhibiting carnitine palmitoyltransferase (CPT) 1A, thereby shifting activated fatty acids into triglyceride synthesis (Foster, 2012). Several drugs have been shown to interfere with hepatic metabolism of fatty acids. Well-known examples are for instance valproic acid, tamoxifen, amiodarone and methotrexate (Fromenty and Pessayre, 1995; Amacher, 2014; Ress and Kaser, 2016).
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