All-Trans-Retinoic Acid Enhances Mitochondrial Function in Models of Human Liver

Abstract

All-trans-retinoic acid (atRA) is the active metabolite of vitamin A. The liver is the main storage organ of vitamin A, but activation of the retinoic acid receptors (RARs) in mouse liver and in human liver cell lines has also been shown. Although atRA treatment improves mitochondrial function in skeletal muscle in rodents, its role in modulating mitochondrial function in the liver is controversial, and little data are available regarding the human liver. The aim of this study was to determine whether atRA regulates hepatic mitochondrial activity. atRA treatment increased the mRNA and protein expression of multiple components of mitochondrial b-oxidation, tricarboxylic acid (TCA) cycle, and respiratory chain. Additionally, atRA increased mitochondrial biogenesis in human hepatocytes and in HepG2 cells with and without lipid loading based on peroxisome proliferator activated receptor gamma coactivator 1a and 1b and nuclear respiratory factor 1 mRNA and mitochondrial DNA quantification. atRA also increased b-oxidation and ATP production in HepG2 cells and in human hepatocytes. Knockdown studies of RARa, RARb, and PPARd revealed that the enhancement of mitochondrial biogenesis and b-oxidation by atRA requires peroxisome proliferator activated receptor delta. In vivo in mice, atRA treatment increased mitochondrial biogenesis markers after an overnight fast. Inhibition of atRA metabolism by talarozole, a cytochrome P450 (CYP) 26 specific inhibitor, increased the effects of atRA on mitochondrial biogenesis markers in HepG2 cells and in vivo in mice. These studies show that atRA regulates mitochondrial function and lipid metabolism and that increasing atRA concentrations in human liver via CYP26 inhibition may increase mitochondrial biogenesis and fatty acid b-oxidation and provide therapeutic benefit in diseases associated with mitochondrial dysfunction.

Modest Decreases in Endogenous All-trans-Retinoic Acid Produced by a MouseRdh10Heterozygote Provoke Major Abnormalities in Adipogenesis and Lipid Metabolism

“…Overexpression of the atRAinducible RARb in mouse liver enhances fatty acid oxidation and energy expenditure (31), whereas the RARb2-specific agonist AC261066 reduces liver steatosis in HFD-fed mice (49). Increasing endogenous atRA concentrations by inhibiting the atRA-catabolic Cyp26 isozymes enhances expression of mitochondrial biogenesis markers Nrf1 and Pgc1b 5-to 10-fold (32). These data are consistent with atRA enhancing fatty acid oxidation and mitochondrial biogenesis in vivo and suggest processes contributing to the liver steatosis phenotype.…”

“…We undertook this research to reveal postnatal Rdh10 contributions to atRA function and to assess endogenous atRA actions. Previous studies focusing on specific organs or using vitamin A deficiency models revealed that atRA has widespread impact on metabolism, consistent with organ autonomous effects (15)(16)(17)20,(29)(30)(31)(32). Total Rdh10 ablation impairs the biogenesis of multiple organs, including the pancreas, and causes embryonic lethality between E10.5 and E13, but Rdh10 +/2 mice reportedly were "indistinguishable" from WT (33).…”

In vitro to in vivo extrapolation of the complex drug-drug interaction of bupropion and its metabolites with CYP2D6; simultaneous reversible inhibition and CYP2D6 downregulation

“…At 72 hrs, cells were harvested for mRNA analysis. CYP2D6 and SHP mRNA were quantified using q-RT-PCR (StepOnePlus ™ , Applied Biosystems, Carlsbad, CA) as previously described [27] using GAPDH as a housekeeping gene. Cells were harvested using Tri-reagent (Invitrogen, Grand Island, NY) and mRNA extracted according to the manufacturer’s recommendations.…”

“…At 72 hrs, cells were harvested for protein analysis. Western blot analysis was done to measure CYP2D6 expression with β-actin as aloading control as previously described [27]. In brief, for western blotting, proteins from HepG2 cells were extracted as whole cell extract using whole cell lysis buffer in the presence of protease (Roche Applied Science, Indianapolis, IN) and phosphatase inhibitors (1 mM β-glycerol phosphate, 2.5 mM Na-pyrophosphate, 1 mM Na 3 VO 4 ).…”

“…Cryopreserved human hepatocytes from three donors (3 females, 9, 45 and 59 years of age) were obtained from Xenotech (Lenexa, KS) and cultured as described previously [27]. CYP2D6 activity was measured by formation of dextrorphan from dextromethorphan (1 μM nominal concentration) over a one-hour incubation.…”