Arylacetamide deacetylase attenuates fatty-acid-induced triacylglycerol accumulation in rat hepatoma cells

Abstract

Mobilization of hepatic triacylglycerol stores provides substrates for mitochondrial beta-oxidation and assembly of VLDLs; however, the identity of lipolytic enzymes involved in the regulation of this process remains largely unknown. Arylacetamide deacetylase (AADA) shares homology with hormone-sensitive lipase and therefore could potentially participate in hepatic lipid metabolism, including the regulation of hepatic triacylglycerol levels. We have established McArdle-RH7777 (rat hepatoma) cell lines stably expressing mouse AADA cDNA and performed metabolic labeling as well as lipid mass analyses. Expression of AADA cDNA in McArdle-RH7777 cells significantly reduced intracellular triacylglycerol levels and apolipoprotein B secretion and increased fatty acid oxidation. These results suggest that fatty acids released by AADA-mediated hydrolysis of lipids are channeled for -oxidation rather than for the assembly of lipoproteins.

Fig. 1.

Localization and expression of mouse AADA. A: Localization of endogenous AADA in primary mouse hepatocytes by confocal immunofluorescence microscopy. B: Subcellular fractionation of mouse liver homogenate. AADA was detected with anti-mouse AADA polyclonal antibodies as described in Materials and Methods. C: AADA is expressed in rat liver but not in rat-derived McA cells as determined by RT-PCR. CYC, cyclophilin (control); Liv, liver. D: Twenty micrograms of membrane proteins prepared from individual dishes of McA cells stably transfected with an empty pCI-neo vector or pCI-neo vector containing FLAG-tagged mouse AADA cDNA (A13 and A23) were electrophoresed in SDS-PAGE, proteins were transferred to a nitrocellulose membrane and immunoblotted with anti-FLAG and anti-CNX (loading control) antibodies as described in Materials and Methods. E: Twenty micrograms of membrane protein from stably transfected McA cells and from mouse liver homogenate (MLH) were electrophoresed, transferred to a nitrocellulose membrane, and immunoblotted with anti-mouse AADA and anti-CNX antibodies as described in Materials and Methods. F: Localization of AADA in transfected McA cells by confocal immunofluorescence microscopy. G: Cell lysates from AADA-expressing cells were treated with deglycosylation enzymes and changes in AADA molecular mass was assessed by SDS-PAGE.

Fig. 2.

AADA hydrolyzes soluble and insoluble carboxylesters. Twenty micrograms of membrane protein prepared from McA cells stably transfected with an empty pCIneo vector, FLAG-tagged mouse AADA, or human TGH cDNAs were incubated with pNP-acetate (A) or 4-MUH (B). The release of pNP was monitored at 405 nm and that of 4-umbelliferyl at 460 nm. The results are presented as an average from triplicate measurements. C: Hydrolysis of microsomal lipids. Cells were incubated with radiolabeled oleic acid, and microsomes were isolated and incubated for 4 h as described in Materials and Methods. Lipids were extracted and resolved on TLC plates, and radioactivity was determined by scintillation counting. Data (mean ± SD) from three separate experiments performed in triplicate are presented with radioactivity in DG in pNeo microsomes at time 0 set as 100%. Equal amounts of microsomal protein were used for the assay. The actual starting dpm in microsomal lipid fractions at time 0 were 22,000–25,000 for PC, 12,000–18,000 for TG, and 1,500–2,300 for DG. D: Thirty micrograms of total cellular membrane proteins from McA cells stably transfected with pCI-neo, A13, A23, and human TGH were either not treated (pNeo-NT) or treated with FP-biotin, resolved in SDS-PAGE, and transferred to nitrocellulose membrane, and biotinylated proteins were visualized with streptavidin-HRP. Asterisks indicate other potential endogenous serine hydrolases present in McA cells.

Fig. 3.

AADA expression decreases levels of de novo synthesized TG. McA cells stably transfected with an empty pCI-neo plasmid or pCI-neo plasmid containing FLAG-tagged mouse AADA cDNA (A13 and A23) were incubated with 0.4 mM [³H]OA in the presence or absence of a lipase inhibitor E600 and harvested either after pulse or chase as indicated. Lipids were extracted and analyzed by TLC as described in Materials and Methods. The amount of radioactivity incorporated into TG (A), CE (B), and PC (C) was determined by scintillation counting. Due to slight variation in radioactivity incorporation in three independent experiments, the data presented are shown as percentages of the dpm counts per milligram of protein of control pCI-neo at 4 h pulse. The actual dpm/mg cell protein incorporated into cellular lipids for pCI-neo at 4 h pulse from three independent experiments performed in triplicates (mean ± SD) were between 150,000 ± 30,000 and 190,000 ± 20,000 for TG, 50,000 ± 9000 and 71,000 ± 6300 for CE, and 100,000 ± 5500 and 110,000 ± 26,000 for PC. *P < 0.001, **P < 0.01, and ***P < 0.05 compared with pCI-neo (pNeo) cells.

Fig. 4.

AADA expression decreases cellular TG mass levels. McA cells stably transfected with an empty pCI-neo vector or pCI-neo vector containing FLAG-tagged mouse AADA cDNA (A13 and A23) were incubated in serum-free DMEM in the presence or absence of 0.4 mM OA and in the presence or absence of a lipase inhibitor E600 for 4 h. Cells were harvested in 2 ml of ice-cold PBS, lipids were extracted, and the mass of TG (A), CE (B), and phospholipids (PL) (C) was measured by gas chromatography as described in Materials and Methods. The data presented are representative of two independent experiments performed in triplicates. *P < 0.001.

Fig. 5.

AADA expression decreases apoB and TG secretion. A: Media from 100 mm dishes of McA cells stably transfected with an empty pCI-neo vector or pCI-neo vector containing FLAG-tagged mouse AADA cDNA (A13 and A23) were collected after 4 h incubation with 0.4 mM OA. The samples were incubated with anti-apoB antibodies, and then the antigen-antibody complexes were bound to protein A Sepharose. Collected immunoprecipitates were electrophoresed in SDS-PAGE and immunoblotted with anti-apoB antibodies as described in Materials and Methods. B: Densitometry analysis of the amount of apoB100 secretion is expressed as a percentage of apoB secreted by the control cell line (pCI-neo). C: McA cells were incubated with 0.4 mM [³H]OA for 4 h. Cell media were subjected to lipid extraction and analysis. The amount of TG in media was measured by scintillation counting following TLC. The results in A and B are representative of four independent experiments. The results in C represent mean ± SD from three independent experiments performed in triplicates. *P < 0.001.

Fig. 6.

AADA expression increases fatty acid oxidation. McA cells stably transfected with an empty pCI-neo vector and pCI-neo vector containing FLAG-tagged mouse AADA cDNA (A13 and A23) were incubated with 0.4 mM [³H]OA for 4 h in the presence or absence of a lipase inhibitor E600 (pulse), followed by incubation in DMEM with or without a lipase inhibitor E600 (Chase). Media equivalent to 500 µg of cell protein were analyzed for radioactivity in ASMs as described in Materials and Methods. Due to slight variation in radioactivity incorporation in three independent experiments, the data presented are shown as percentages of control pCI-neo at 4 h pulse. The averages of the actual dpm/mg cell protein in ASM for pCI-neo at 4-h pulse from three independent experiments performed in triplicates (mean ± SD) were between 610,000 ± 70,000 and 880,000 ± 100,000. *P < 0.001, **P < 0.01, and ***P < 0.05.

Fig. 7.

Proposed role of AADA in the glycerol-3-phosphate pathway and its effect on hepatic lipid metabolism. TG is synthesized within the ER lipid bilayer by the enzyme of the glycerol-3-phosphate (also termed Kennedy) pathway. AADA hydrolyzes newly synthesized DG and provides fatty acids for β-oxidation in mitochondria (Mito). The result of AADA-mediated DG hydrolysis is the decreased amount of substrate for TG synthesis for cytoplasmic storage droplet formation and for VLDL assembly. AGPAT, 1-acylglycerol-3-phosphate acyltransferase; G-3-P, glycerol-3-phosphate; GPAT, glycerol-3-phosphate acyltransferase; LPA, lysophosphatidic acid; PA, phosphatidic acid; PAPH, phosphatidic acid phosphohydrolase.