Access Type

Open Access Dissertation

Date of Award

January 2016

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Pharmacology

First Advisor

Thomas A. Kocarek

Abstract

Non-alcoholic fatty liver disease is emerging as one of the most common liver disorders worldwide and is characterized by accumulation of triglycerides (TGs) in liver. The endogenous isoprenoid farnesol reduces hepatic TG levels in rodents, and this effect appears to involve at least two nuclear receptors, peroxisome proliferator-activated receptor α and farnesoid X receptor (FXR). However, farnesol’s effects on human hepatic lipid metabolism are currently unknown. The objective of this study is to evaluate how farnesol treatment would affect hepatic lipid accumulation and metabolism in a cellular model of human hepatic steatosis that was created by incubating the hepatocyte-like HepaRG cell line with oleic acid. Farnesol treatment suppressed the OA-induced TGs accumulation in HepaRG cells by 25%. Farnesol upregulated the activity of lipid-sensing nuclear receptors PPARα and constitutive androstane receptor (CAR), which was evident by the increase in the expression of their respective target genes, PLIN2 and CYP2B6; however, farnesol had no effect on FXR activity in HepaRG cells. Gene expression analysis through customized arrays revealed that farnesol modulated mRNA levels of several hepatic lipid- and drug-metabolizing enzymes in both control and OA-overloaded HepaRG cells, and the expression pattern was similar to that achieved through activation of PPARα rather than through CAR or FXR.

In OA-overloaded HepaRG cells, farnesol treatment induced overall fatty acid oxidation rate, which was accompanied by upregulation in the mRNA levels of PPARα target genes involved in hepatic mitochondrial fatty acid oxidation, such as HADHB, ACADS and ACOT12. This effect was lost when cells were co-treated with the PPARα antagonist except for ACOT12. Farnesol had no effect on genes regulating peroxisomal oxidation. OA-induced changes in drug-metabolizing enzymes, such as CYP3A4, CYP2B6, CYP1A2 and CYP2E1 were also attenuated by co-treatment with farnesol. Our findings show for the first time that farnesol regulates human hepatic lipid metabolism and suppresses lipid accumulation in HepaRG cells under steatogenic conditions by upregulating mitochondrial fatty acid oxidation, primarily through PPARα pathway.

Farnesyl pyrophosphate (FPP) is a branch-point intermediate in the mevalonate pathway that is normally converted mainly to squalene by squalene synthase in the first committed step of sterol biosynthesis. Treatment with the squalene synthase inhibitor squalestatin 1 (SQ1) causes accumulation of FPP, its dephosphorylated metabolite farnesol, and several oxidized farnesol-derived metabolites. Also, SQ1 treatment of primary cultured rat hepatocytes increases CYP2B expression through a mechanism that requires FPP synthesis and activation of the constitutive androstane receptor (CAR). Because direct farnesol treatment also increases CYP2B expression, it seems likely that SQ1-mediated CAR activation requires FPP dephosphorylation to farnesol. The lipid phosphatase, phosphatidic acid phosphatase domain containing 2 (PPAPDC2), was recently reported to catalyze FPP dephosphorylation. We therefore determined the effect of overexpressing or knocking down PPAPDC2 on SQ1-mediated CAR activation in primary cultured rat hepatocytes. Co-transfection of rat hepatocytes with a plasmid expressing rat or human PPAPDC2 enhanced SQ1-mediated activation of a CAR-responsive reporter by 1.7- or 2.4-fold over the SQ1-mediated activation that was produced when hepatocytes were co-transfected with empty expression plasmid. Similarly, transduction of rat hepatocytes with a recombinant adenovirus expressing PPAPDC2 enhanced SQ1-mediated CYP2B1 mRNA induction by 1.4-fold over the induction that was seen in hepatocytes transduced with control adenovirus. Co-transfection with a shRNA targeting PPAPDC2 reduced SQ1-mediated CAR activation by ~80% relative to the activation that occurred in hepatocytes transfected with non-targeting shRNA. These results indicate that PPAPDC2 plays an important role in SQ1-mediated CAR activation, most probably by catalyzing the conversion of FPP to farnesol.

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