PPARδ activation induces hepatic long-chain acyl-CoA synthetase 4 expression in vivo and in vitro

Abstract

The arachidonic acid preferred long-chain acyl-CoA synthetase 4 (ACSL4) is a key enzyme for fatty acid metabolism in various metabolic tissues. In this study, we utilized hamsters fed a normal chow diet, a high-fat diet or a high cholesterol and high fat diet (HCHFD) as animal models to explore novel transcriptional regulatory mechanisms for ACSL4 expression under hyperlipidemic conditions. Through cloning hamster ACSL4 homolog and tissue profiling ACSL4 mRNA and protein expressions we observed a selective upregulation of ACSL4 in testis and liver of HCHFD fed animals. Examination of transcriptional activators of the ACSL family revealed an increased hepatic expression of PPARδ but not PPARα in HCHFD fed hamsters. To explore a role of PPARδ in dietary cholesterol-mediated upregulation of ACSL4, we administered a PPARδ specific agonist L165041 to normolipidemic and dyslipidemic hamsters. We observed significant increases of hepatic ACSL4 mRNA and protein levels in all L165041-treated hamsters as compared to control animals. The induction of ACSL4 expression by L165041 in liver tissue in vivo was recapitulated in human primary hepatocytes and hepatocytes isolated from hamster and mouse. Moreover, employing the approach of adenovirus-mediated gene knockdown, we showed that depletion of PPARδ in hamster hepatocytes specifically reduced ACSL4 expression. Finally, utilizing HepG2 as a model system, we demonstrate that PPARδ activation leads to increased ACSL4 promoter activity, mRNA and protein expression, and consequently higher arachidonoyl-CoA synthetase activity. Taken together, we have discovered a novel PPARδ-mediated regulatory mechanism for ACSL4 expression in liver tissue and cultured hepatic cells.

Identification of Hepatic Lysophosphatidylcholine Acyltransferase 3 as a Novel Target Gene Regulated by Peroxisome Proliferator-activated Receptor δ

“…Depletion of PPAR␦ significantly lowered LPCAT3 mRNA levels to ϳ64% of control (p Ͻ 0.001) and also reduced levels of ACSL4 and ACSL3 mRNA, both of which are known target genes of PPAR␦. In contrast, expression of PPAR␦ shRNA in HepG2 cells did not diminish ACSL1 mRNA levels, which was consistent with our previous in vivo observations that PPAR␦ activation increased ACSL4 and ACSL3 expressions without affecting ACSL1 mRNA levels in hamster liver (18,27).…”

“…PPAR␦ activation in liver is known to activate a transcriptional program to increase cellular abundances of multiple enzymes to modulate FA and PL metabolism (31). Our previous studies have identified ACSL4 as a new molecular target of PPAR␦ in liver cells (18). In this study, we explored the possibility that activation of PPAR␦ in liver tissue might not only increase ACSL4 cellular abundance and its enzymatic activity but could also affect the metabolic fates of ACSL4-produced PUFA-CoAs by altering the availability of ACSL4 fatty substrates, its interacting proteins, and downstream effectors.…”

“…LXR agonist GW3965 and anti-␤-actin monoclonal antibody were obtained from Sigma-Aldrich. Rabbit anti-ACSL4 antibody was reported previously (18). Rabbit anti-PPAR␦ antibody (sc-7197X) was obtained from Santa Cruz Biotechnology, Inc.…”

“…Transfections and Luciferase Assay-The luciferase assay was performed as described previously (18). Briefly, HepG2 cells were seeded at a density of 3 ϫ 10 4 cells/well onto 96-well plates.…”

“…Recent in vitro studies further suggested that ACSL4 has specific functions in the AA incorporation into PLs (16,17). Our previous studies conducted in hepatic cells and in liver tissue of hamsters have identified ACSL4 as a direct target gene of PPAR␦ (18). Activation of PPAR␦ in liver cells led to increased ACSL4 protein abundance and higher arachidonyl-CoA synthetase activity, which could potentially provide more substrates to LPCAT3 as LPCAT3 synthesizes arachidonoyl-PC from AA-CoA and lyso-PCs (3,19).…”