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Arachidonic acid downregulates acyl-CoA synthetase 4 expression by promoting its ubiquitination and proteasomal degradation.

Kan CF, Singh AB, Stafforini DM, Azhar S, Liu J - J. Lipid Res. (2014)

Bottom Line: AA treatment decreased the half-life of ACSL4 protein in HepG2 cells by approximately 4-fold (from 17.3 ± 1.8 h to 4.2 ± 0.4 h) without causing apoptosis.We further show that ACSL4 is intrinsically ubiquitinated and that AA treatment can enhance its ubiquitination.Collectively, our studies have identified a novel substrate-induced posttranslational regulatory mechanism by which AA downregulates ACSL4 protein expression in hepatic cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterans Affairs Palo Alto Health Care System, Palo Alto, CA 94304.

ABSTRACT
ACSL4 is a member of the long-chain acyl-CoA synthetase (ACSL) family with a marked preference for arachidonic acid (AA) as its substrate. Although an association between elevated levels of ACSL4 and hepatosteatosis has been reported, the function of ACSL4 in hepatic FA metabolism and the regulation of its functional expression in the liver remain poorly defined. Here we provide evidence that AA selectively downregulates ACSL4 protein expression in hepatic cells. AA treatment decreased the half-life of ACSL4 protein in HepG2 cells by approximately 4-fold (from 17.3 ± 1.8 h to 4.2 ± 0.4 h) without causing apoptosis. The inhibitory action of AA on ACSL4 protein stability could not be prevented by rosiglitazone or inhibitors that interfere with the cellular pathways involved in AA metabolism to biologically active compounds. In contrast, treatment of cells with inhibitors specific for the proteasomal degradation pathway largely prevented the AA-induced ACSL4 degradation. We further show that ACSL4 is intrinsically ubiquitinated and that AA treatment can enhance its ubiquitination. Collectively, our studies have identified a novel substrate-induced posttranslational regulatory mechanism by which AA downregulates ACSL4 protein expression in hepatic cells.

No MeSH data available.


Related in: MedlinePlus

Downregulation of hepatic ACSL4 expression in hepatic cells by FAs. A–D: HepG2 or Huh7 cells were treated with a mixture of FAs containing 65 μM PA, 65 μM OA, and 20 μM AA. After 24 h of FA treatment, total cell lysates were isolated for Western blotting and total RNA was isolated for qRT-PCR. After Western blotting, the protein amount of each ACSL isoform was quantified with the Alpha View software with normalization by signals of β-actin. The data are the mean ± SEM of three independent experiments. * P < 0.05, *** P < 0.001. C, control.
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fig2: Downregulation of hepatic ACSL4 expression in hepatic cells by FAs. A–D: HepG2 or Huh7 cells were treated with a mixture of FAs containing 65 μM PA, 65 μM OA, and 20 μM AA. After 24 h of FA treatment, total cell lysates were isolated for Western blotting and total RNA was isolated for qRT-PCR. After Western blotting, the protein amount of each ACSL isoform was quantified with the Alpha View software with normalization by signals of β-actin. The data are the mean ± SEM of three independent experiments. * P < 0.05, *** P < 0.001. C, control.

Mentions: To investigate the underlying mechanism involved in the repression of ACSL4 expression in response to HFD feeding of mice, we attempted to mimic the hyperlipidemic conditions in vitro by culturing hepatic cell lines in medium supplemented with a mixture of saturated (PA), monounsaturated (OA), and polyunsaturated (AA) FAs. Exposure of cells to FAs reduced the ACSL4 protein levels to 37% of control (P < 0.001) in HepG2 cells (Fig. 2A, B) and to 72% of control (P < 0.05) in Huh7 cells (Fig. 2D, E). Again, the expression of ACSL1 or ACSL5 protein was not affected by such manipulation, and ACSL3 protein levels were slightly decreased in HepG2 and Huh7 cells. Furthermore, despite the significant decreases in ACSL4 protein levels, ACSL4 mRNA levels were only minimally impacted by FA treatment in HepG2 cells and unaffected in Huh7 cells (Fig. 2C, F). Altogether, these in vivo and in vitro results suggest that exposure of liver cells to excessive amounts of FAs downregulates ACSL4 expression mainly at the protein level.


Arachidonic acid downregulates acyl-CoA synthetase 4 expression by promoting its ubiquitination and proteasomal degradation.

Kan CF, Singh AB, Stafforini DM, Azhar S, Liu J - J. Lipid Res. (2014)

Downregulation of hepatic ACSL4 expression in hepatic cells by FAs. A–D: HepG2 or Huh7 cells were treated with a mixture of FAs containing 65 μM PA, 65 μM OA, and 20 μM AA. After 24 h of FA treatment, total cell lysates were isolated for Western blotting and total RNA was isolated for qRT-PCR. After Western blotting, the protein amount of each ACSL isoform was quantified with the Alpha View software with normalization by signals of β-actin. The data are the mean ± SEM of three independent experiments. * P < 0.05, *** P < 0.001. C, control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4109760&req=5

fig2: Downregulation of hepatic ACSL4 expression in hepatic cells by FAs. A–D: HepG2 or Huh7 cells were treated with a mixture of FAs containing 65 μM PA, 65 μM OA, and 20 μM AA. After 24 h of FA treatment, total cell lysates were isolated for Western blotting and total RNA was isolated for qRT-PCR. After Western blotting, the protein amount of each ACSL isoform was quantified with the Alpha View software with normalization by signals of β-actin. The data are the mean ± SEM of three independent experiments. * P < 0.05, *** P < 0.001. C, control.
Mentions: To investigate the underlying mechanism involved in the repression of ACSL4 expression in response to HFD feeding of mice, we attempted to mimic the hyperlipidemic conditions in vitro by culturing hepatic cell lines in medium supplemented with a mixture of saturated (PA), monounsaturated (OA), and polyunsaturated (AA) FAs. Exposure of cells to FAs reduced the ACSL4 protein levels to 37% of control (P < 0.001) in HepG2 cells (Fig. 2A, B) and to 72% of control (P < 0.05) in Huh7 cells (Fig. 2D, E). Again, the expression of ACSL1 or ACSL5 protein was not affected by such manipulation, and ACSL3 protein levels were slightly decreased in HepG2 and Huh7 cells. Furthermore, despite the significant decreases in ACSL4 protein levels, ACSL4 mRNA levels were only minimally impacted by FA treatment in HepG2 cells and unaffected in Huh7 cells (Fig. 2C, F). Altogether, these in vivo and in vitro results suggest that exposure of liver cells to excessive amounts of FAs downregulates ACSL4 expression mainly at the protein level.

Bottom Line: AA treatment decreased the half-life of ACSL4 protein in HepG2 cells by approximately 4-fold (from 17.3 ± 1.8 h to 4.2 ± 0.4 h) without causing apoptosis.We further show that ACSL4 is intrinsically ubiquitinated and that AA treatment can enhance its ubiquitination.Collectively, our studies have identified a novel substrate-induced posttranslational regulatory mechanism by which AA downregulates ACSL4 protein expression in hepatic cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterans Affairs Palo Alto Health Care System, Palo Alto, CA 94304.

ABSTRACT
ACSL4 is a member of the long-chain acyl-CoA synthetase (ACSL) family with a marked preference for arachidonic acid (AA) as its substrate. Although an association between elevated levels of ACSL4 and hepatosteatosis has been reported, the function of ACSL4 in hepatic FA metabolism and the regulation of its functional expression in the liver remain poorly defined. Here we provide evidence that AA selectively downregulates ACSL4 protein expression in hepatic cells. AA treatment decreased the half-life of ACSL4 protein in HepG2 cells by approximately 4-fold (from 17.3 ± 1.8 h to 4.2 ± 0.4 h) without causing apoptosis. The inhibitory action of AA on ACSL4 protein stability could not be prevented by rosiglitazone or inhibitors that interfere with the cellular pathways involved in AA metabolism to biologically active compounds. In contrast, treatment of cells with inhibitors specific for the proteasomal degradation pathway largely prevented the AA-induced ACSL4 degradation. We further show that ACSL4 is intrinsically ubiquitinated and that AA treatment can enhance its ubiquitination. Collectively, our studies have identified a novel substrate-induced posttranslational regulatory mechanism by which AA downregulates ACSL4 protein expression in hepatic cells.

No MeSH data available.


Related in: MedlinePlus