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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 vivo by feeding a HFD to mice. A, B: C57BL/6J male mice were fed a HFD or NCD (n = 5 per group) for 16 weeks. A: Homogenate proteins (100 μg) from individual liver samples were resolved by SDS-PAGE. Expression levels of different ACSL isoforms were detected by immunoblotting using isoform-specific antibodies. The protein amount of each ACSL isoform was quantified with the Alpha View software with normalization by signals of GAPDH. Values are mean ± SEM of five samples per group. B: Individual levels of ACSL mRNAs in liver samples of NCD and HFD mice were assessed by real-time qRT-PCR. After normalization with GAPDH mRNA levels, the relative mRNA level in the NCD group is expressed as 1. The results presented are mean ± SEM of five mice per group. C, D: In a second diet study, C57BL/6J male mice were fed a HFD or NCD (n = 3 per group) for 16 weeks. ACSL4 and ACSL3 protein and mRNA levels in the NCD and HFD groups were analyzed as in (A) and (B). The results presented are the mean ± SEM of three mice per groupQ11. * P < 0.05, ** P < 0.01, *** P < 0.001.
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fig1: Downregulation of hepatic ACSL4 expression in vivo by feeding a HFD to mice. A, B: C57BL/6J male mice were fed a HFD or NCD (n = 5 per group) for 16 weeks. A: Homogenate proteins (100 μg) from individual liver samples were resolved by SDS-PAGE. Expression levels of different ACSL isoforms were detected by immunoblotting using isoform-specific antibodies. The protein amount of each ACSL isoform was quantified with the Alpha View software with normalization by signals of GAPDH. Values are mean ± SEM of five samples per group. B: Individual levels of ACSL mRNAs in liver samples of NCD and HFD mice were assessed by real-time qRT-PCR. After normalization with GAPDH mRNA levels, the relative mRNA level in the NCD group is expressed as 1. The results presented are mean ± SEM of five mice per group. C, D: In a second diet study, C57BL/6J male mice were fed a HFD or NCD (n = 3 per group) for 16 weeks. ACSL4 and ACSL3 protein and mRNA levels in the NCD and HFD groups were analyzed as in (A) and (B). The results presented are the mean ± SEM of three mice per groupQ11. * P < 0.05, ** P < 0.01, *** P < 0.001.

Mentions: First, the mRNA and protein levels of ACSL4 in liver tissues from mice that were fed a HFD or a NCD for 16 weeks were measured. Figure 1A shows that HFD feeding markedly reduced the ACSL4 protein levels (∼80%; P < 0.05) in livers of HFD mice as compared with control (NCD) mice. Utilizing a highly specific anti-hamster ACSL3 antibody (17), we showed that in contrast to ACSL4, ACSL3 protein levels remained unchanged in response to HFD feeding. qRT-PCR analyses of four hepatic ACSL isoforms showed that ACSL4 mRNA levels were 40% lower in the HFD group as compared with the NCD group. The mRNA levels of ACSL1 and ACSL5 were unchanged, while ACSL3 mRNA levels were reduced by 50% upon HFD feeding (Fig. 1B) despite the unchanged ACSL3 protein levels. Given a reported association between elevated ACSL4 gene expression and human NAFLD (10), the observed lower levels ACSL4 protein in steatotic liver of HFD mice was unexpected. To confirm this finding, we analyzed liver samples of HFD and NCD mice from another diet experiment. We observed that ACSL4 protein levels in HFD group were 52% lower than those of the NCD group (P < 0.01), while ACSL4 mRNA levels remained unchanged (Fig. 1C, D). Thus, the mean reduction of ACSL4 protein by HFD feeding from the two separate diet studies was approximately 65%.


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 vivo by feeding a HFD to mice. A, B: C57BL/6J male mice were fed a HFD or NCD (n = 5 per group) for 16 weeks. A: Homogenate proteins (100 μg) from individual liver samples were resolved by SDS-PAGE. Expression levels of different ACSL isoforms were detected by immunoblotting using isoform-specific antibodies. The protein amount of each ACSL isoform was quantified with the Alpha View software with normalization by signals of GAPDH. Values are mean ± SEM of five samples per group. B: Individual levels of ACSL mRNAs in liver samples of NCD and HFD mice were assessed by real-time qRT-PCR. After normalization with GAPDH mRNA levels, the relative mRNA level in the NCD group is expressed as 1. The results presented are mean ± SEM of five mice per group. C, D: In a second diet study, C57BL/6J male mice were fed a HFD or NCD (n = 3 per group) for 16 weeks. ACSL4 and ACSL3 protein and mRNA levels in the NCD and HFD groups were analyzed as in (A) and (B). The results presented are the mean ± SEM of three mice per groupQ11. * P < 0.05, ** P < 0.01, *** P < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4109760&req=5

fig1: Downregulation of hepatic ACSL4 expression in vivo by feeding a HFD to mice. A, B: C57BL/6J male mice were fed a HFD or NCD (n = 5 per group) for 16 weeks. A: Homogenate proteins (100 μg) from individual liver samples were resolved by SDS-PAGE. Expression levels of different ACSL isoforms were detected by immunoblotting using isoform-specific antibodies. The protein amount of each ACSL isoform was quantified with the Alpha View software with normalization by signals of GAPDH. Values are mean ± SEM of five samples per group. B: Individual levels of ACSL mRNAs in liver samples of NCD and HFD mice were assessed by real-time qRT-PCR. After normalization with GAPDH mRNA levels, the relative mRNA level in the NCD group is expressed as 1. The results presented are mean ± SEM of five mice per group. C, D: In a second diet study, C57BL/6J male mice were fed a HFD or NCD (n = 3 per group) for 16 weeks. ACSL4 and ACSL3 protein and mRNA levels in the NCD and HFD groups were analyzed as in (A) and (B). The results presented are the mean ± SEM of three mice per groupQ11. * P < 0.05, ** P < 0.01, *** P < 0.001.
Mentions: First, the mRNA and protein levels of ACSL4 in liver tissues from mice that were fed a HFD or a NCD for 16 weeks were measured. Figure 1A shows that HFD feeding markedly reduced the ACSL4 protein levels (∼80%; P < 0.05) in livers of HFD mice as compared with control (NCD) mice. Utilizing a highly specific anti-hamster ACSL3 antibody (17), we showed that in contrast to ACSL4, ACSL3 protein levels remained unchanged in response to HFD feeding. qRT-PCR analyses of four hepatic ACSL isoforms showed that ACSL4 mRNA levels were 40% lower in the HFD group as compared with the NCD group. The mRNA levels of ACSL1 and ACSL5 were unchanged, while ACSL3 mRNA levels were reduced by 50% upon HFD feeding (Fig. 1B) despite the unchanged ACSL3 protein levels. Given a reported association between elevated ACSL4 gene expression and human NAFLD (10), the observed lower levels ACSL4 protein in steatotic liver of HFD mice was unexpected. To confirm this finding, we analyzed liver samples of HFD and NCD mice from another diet experiment. We observed that ACSL4 protein levels in HFD group were 52% lower than those of the NCD group (P < 0.01), while ACSL4 mRNA levels remained unchanged (Fig. 1C, D). Thus, the mean reduction of ACSL4 protein by HFD feeding from the two separate diet studies was approximately 65%.

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