Transcriptional Activation of Hepatic ACSL3 and ACSL5 by Oncostatin M Reduces Hypertriglyceridemia Through Enhanced β-Oxidation
Objective— In our previous studies that examined in vivo activities of oncostatin M (OM) in upregulation of hepatic LDL receptor (LDLR) expression, we observed reductions of LDL-cholesterol and triglyceride (TG) levels in OM-treated hyperlipidemic hamsters. Interestingly, the OM effect of lowering plasma TG was more pronounced than LDL-cholesterol reduction, suggesting additional LDLR-independent actions. Here, we investigated mechanisms underlying the direct TG-lowering effect of OM.
Methods and Results— We demonstrate that OM activates transcription of long-chain acyl-coenzymeA (CoA) synthetase isoforms 3 and 5 (ACSL3, ACSL5) in HepG2 cells through the extracellular signal-regulated kinase (ERK) signaling pathway. Increased acyl-CoA synthetase activities in OM-stimulated HepG2 cells and in livers of OM-treated hamsters are associated with decreased TG accumulation and increased fatty acid β-oxidation. We further show that overexpression of ACSL3 or ACSL5 alone in the absence of OM led to fatty acid partitioning into β-oxidation. Importantly, we demonstrate that transfection of siRNAs targeted to ACSL3 and ACSL5 abrogated the enhancing effect of OM on fatty acid oxidation in HepG2 cells.
Conclusions— These new findings identify ACSL3 and ACSL5 as OM-regulated genes that function in fatty acid metabolism and suggest a novel cellular mechanism by which OM directly lowers the plasma TG in hyperlipidemic animals through stimulating the transcription of ACSL specific isoforms in the liver.
In addition to elevated plasma LDL-cholesterol (LDL-c), elevated plasma triglyceride (TG) is increasingly recognized as an independent risk factor for developing coronary heart disease.1 In fasting humans, most plasma TG is carried in liver-derived very low density lipoprotein (VLDL). Hepatic VLDL-TG production is directly related to free fatty acid (FFA) flux, with most fatty acids (FAs) derived from release of hydrolyzed TG from adipose tissue. The liberated free long chain FAs are taken up by hepatocytes or myocytes for reesterification or metabolism through β-oxidation occurring in mitochondria or in peroxisomes.
Inside cells FFAs cannot be used in energy production or in other cellular bioprocesses until they are activated by conversion to acyl-CoA. In mammals, long-chain acyl-CoA synthetase (ACSL) catalyzes the initial step in cellular long-chain fatty acid metabolism. ACSL catalyzes the formation of acyl-CoA from FA, ATP, and CoA. This reaction is essential in mammalian fatty acid metabolism.2 Acyl-CoAs are used both in the synthesis of cellular lipids and in degradation via β-oxidation for energy production.3,4 To date, 5 isoforms of ACSLs have been characterized in humans and in rodents.5 These isoforms differ in their tissue and subcellular compartment distributions, substrate preferences, enzyme kinetics, and responses to nutritional and hormonal regulations.6–9 In rat, an extensive analysis of mRNA expressions of ACSL isoforms showed that ACSL1 was highly expressed in adipose tissues, heart, and liver; ACSL3 was primarily expressed in brain and testis with low level expression in liver and other tissues; ACSL5 was abundantly expressed in brain and liver.10
Several studies in different tissues suggested that ACSL isoforms have distinct roles in fatty acid partitioning. Overexpression of ACSL6 in the pheochromocytoma cell line PC12 increased the uptake and incorporation of docosahexaenoate and arachidonate into phospholipids, but not into TG, during neurite outgrowth.11 Overexpression of ACSL5 in rat hepatoma cells primarily activated exogenous FAs destined for TG synthesis.12 ACSL1 was shown to play a pivotal role in TG synthesis. Overexpression of ACSL1 in HepG2 cells increased cellular TG content without affecting β-oxidation, and adenovirus-mediated expression of ACSL1 in mouse liver also increased liver TG accumulation.13
Studies in rodent models have shown that fasting and refeeding differentially affect the expression and activities of ACSL isoforms. Fasting increased ACSL4 and ACSL1 mRNA levels in rat liver, muscle, and some adipose tissues, whereas refeeding decreased these isoform mRNA expressions.10 Other than diet-induced changes, insulin and peroxisome proliferator-activated receptor-α agonists were shown to increase ACSL1 and ACSL6 mRNA expression in rodent heart or cultured cardiac myocytes.14
Despite the important roles of ACSL in several critical cellular pathways, detailed studies addressing the regulation of ACSL isoforms by cell signaling pathways or by cell mediators such as cytokines are scant. Molecular mechanisms by which ACSL isoforms are differentially regulated at transcriptional levels are largely unknown.
Our laboratory has shown that oncostatin M (OM), a member of the interleukin (IL)-6 family of cytokines, is a strong inducer of LDL receptor (LDLR) expression in HepG2 cells.15,16 The level of LDLR mRNA is markedly elevated after 1 hour of OM treatment and is sustained for over 24 hours in HepG2 cells. The upregulation of hepatic LDLR expression by OM resulted in a reduction of plasma LDL-c and total cholesterol (TC) in vivo. Using hyperlipidemic hamsters, we showed that administration of human recombinant OM for 7 days in hamsters fed a high fat and high cholesterol diet significantly reduced plasma levels of TC, LDL-c, and TG in dose- and time-dependent manners.17 This lipid lowering effect was associated with an elevated hepatic LDLR mRNA expression. Unexpectedly, we observed that the potency of OM in TG reduction was far greater than that for LDL-c reduction in hyperlipidemic hamsters. This observation prompted us to undertake further investigations to elucidate the molecular mechanisms by which OM directly influences plasma TG accumulation independent of its effect on upregulation of liver LDLR expression.
In this study, we demonstrate that OM increases hepatic ACSL3 and ACSL5 gene transcription through the extracellular signal-regulated kinases (ERK) signaling pathway. The increased ACSL enzymatic activities in HepG2 cells and in livers of OM-treated hamsters stimulate FAs partitioning into the β-oxidation pathway with a fall in FAs conversion to TG. These findings suggest a novel cellular mechanism for the OM-mediated TG reduction in plasma and in liver. This study, to the best of our knowledge, is the first demonstration of transcriptional regulation of ACSL isoforms by the IL-6 family of cytokines under in vivo and in vitro conditions.
Materials and Methods
ACSL Activity Assay
HepG2 cells or liver tissues were homogenized on ice in a buffer containing 20 mmol/L HEPES, 1 mmol/L EDTA, and 250 mmol/L sucrose, pH 7.4. After a centrifugation at 16 000 rpm, cell lysates were collected and protein concentrations of cell lysates were determined by BCA method (Pierce) and aliquots were stored at −80°C. ACSL activities of cell lysates were measured in the reaction mixture containing 175 mmol/L Tris-HCl, pH 7.4, 8 mmol/L MgCl2, 5 mmol/L dithiothreitol, 1 mmol/L ATP, 0.2 mmol/L CoASH, 0.5 mmol/L Triton X-100, 10 μmol/L EDTA, and 50 μmol/L palmitate mixed with 0.1 μCi of [3H]palmitic acid.18 The reaction was initiated by the addition of 30 μg protein and terminated after 30 minutes at room temperature by adding 1 mL Dole’s reagent (isopropanol: heptane: 1 mol/L H2SO4=40:10:1). After 2 washes, radioactivities in the lower phase containing labeled acyl-CoA were measured by scintillation counting. OM at a dose of 50 ng/mL was used in all in vitro studies.
All other materials and methods are described in online data supplement section, available at http://atvb.ahajournals.org.
In Vivo Reductions of Plasma and Hepatic Levels of TG and FFA by OM
We used hyperlipidemic hamsters as the animal model to examine in vivo effects of OM on plasma and hepatic levels of TC and TG. After a 2-week diet of high cholesterol (HC), human recombinant OM was administered intraperitoneally (i.p.) at daily doses of 100 μg/kg and 200 μg/kg for 8 days and the control group was injected with OM dilution buffer (0.1 mL of 1 mg/mL BSA in PBS) while all hamsters were maintained on the HC diet. OM treatment resulted in moderate decreases in serum TC and LDL-c but strong reductions of serum TG and FFA (supplemental Figure I). At day 3, TG and FFA levels were already reduced to 55.5% and 76% of control with 100 μg/kg OM treatment, whereas levels of TC and LDL-c had not declined (Figure 1A). Similar to the changes seen in plasma levels, hepatic FFA and TG contents in OM-treated hamsters were substantially decreased (Figure 1B), which was further demonstrated by the reduced Oil Red-O staining of lipid droplets in liver sections of OM treated hamsters (supplemental Figure II). We also performed high-performance liquid chromatography (HPLC) analyses to quantitate abundances of VLDL in fasting serum samples of HC-fed hamsters without OM and with OM 8-day treatment at the dose of 200 μg/kg (Figure 1C). The level of VLDL-TG was reduced to 46% of control by OM treatment and the level of VLDL-c was reduced to 60% of control by OM treatment.
Identification of ACSL3 and ACSL5 as Novel OM-Regulated Genes
To search for unknown OM-regulated genes that are involved in cholesterol and FAs metabolism, we performed cDNA microarray analyses to compare gene profiles of HepG2 cells without and with OM treatment. Cells were treated with 50 ng/mL of OM for 8 and 24 hours. Array results showed that expressions of 7 genes that have functions in cellular metabolism were upregulated by OM by at least 2-fold compared with control at both time points (supplemental Table I). Among them are ACSL3 and ACSL5, the critical enzymes for cellular FA partitioning. Next, Northern blot analyses were performed to examine ACSL3 and ACSL5 mRNA levels in control and OM-treated HepG2 cells. Northern blotting with a 32P-labeled human ACSL3 probe detected the previously reported 3 transcripts of 3.0-, 3.3-, and 5.3-kb19 in HepG2 cells, with the transcript of 3.3 kb being the most abundant one. The level of ACSL3 mRNA was increased 1.5-fold at 2 hours of OM treatment; it reached the maximal level of 2.5-fold at 8 hours and remained elevated during the 24-hour treatment (Figure 2A, left panel). Using a 32P-labeled human ACSL5 probe we detected the reported 2 transcripts of 2.5- and 3.7-kb20 in HepG2 cells. OM treatment resulted in a time-dependent increase in ACSL5 mRNA level with kinetics similar to ACSL3 mRNA (Figure 2A, right panel). We further performed real-time RT-PCR assays to independently examine mRNA expressions of ACSL3, ACSL5, ACSL1, ACSL4, and ACSL6 on OM stimulation. OM increased mRNA levels of ACSL3 and ACSL5 and slightly decreased ACSL1 mRNA levels (Figure 2B). The mRNA levels of ACSL4 were not consistently changed by OM but the mRNA levels of ACSL6 were decreased in OM treated HepG2 cells (supplemental Figure IIIA). Because ACSL6 mRNA expression was extremely low as compared with ACSL3 or ACSL4 (supplemental Figure IIIB) we did not further investigate the OM effect on ACSL6 expression.
To determine whether increased mRNA expressions of ACSL3/ACSL5 occurred at the transcriptional level, actinomycin D was added to HepG2 cells 30 minutes before OM treatment. Inhibition of transcription by actinomycin D totally abolished inducing effects of OM on ACSL3/5 expression (supplemental Figure IVA). The effect of OM on ACSL3 transcription was further examined in HepG2 cells transfected with a human ACSL3 promoter luciferase reporter construct.19 OM caused a 3.4-fold increase in the ACSL3 promoter activity at 24 hours (supplemental Figure IVB). Together, these results corroborated findings from microarray analysis and demonstrated specific stimulating effects of OM on transcriptions of ACSL3/5.
Activation of ACSL3/ACSL5 Gene Transcription by OM Leads to Reduced Hepatic TG Storage and Enhanced β-Oxidation
Next, we investigated the functional relevance of OM-mediated transcriptional activation of specific ACSL isoforms. We first examined effects of OM on ACSL enzymatic activities both in vitro and in vivo. Because it is not feasible to separately measure activities of ACSL3/5 from all other ACSLs, we measured total ACSL activities of HepG2 cells without and with OM treatment of 48 hours. Results derived from 3 separate experiments show that OM increased the cellular ACSL activity by 28% (P<0.01) (Figure 3A). We also measured ACSL activities in individual liver samples of untreated (n=6) and hamsters treated with OM (200 μg/kg, n=6). The mean value of ACSL enzymatic activity in the OM group was 27% (P<0.05) higher than that in the control group (Figure 3B), indicating that the effect of OM seen in vitro is operative in vivo. These data demonstrate that activation of ACSL3/ACSL5 gene transcription by OM leads to an enhanced conversion of the inert FFA into the active esterified form ready for usage in cellular bioprocesses.
In addition to the production of acyl-CoA, it has been shown that ACSL also facilitates the cellular uptake of long-chain fatty acids.21 Using 14C-labeled oleic acid and 14C-labeled palmatic acid as substrates, we measured initial fatty acid uptake in control and OM 15 hour–treated HepG2 cells. Fatty acid uptake was not increased at all by OM treatment (supplemental Figure V), suggesting that reduced plasma levels of TG and FFA did not directly result from increased active FFA uptake.
Acyl-CoAs produced by ACSL are mainly used in syntheses of cellular TG and phospholipids and in degradation of FA via β-oxidation for energy production. To determine the fate of acyl-CoAs under the influence of OM, we measured the cellular content of TG. The results derived from 3 independent assays show that OM lowered the TG content of HepG2 cells by 48% after 24 hours and by 50% after 48-hour treatment (Figure 4A), which is consistent with the in vivo effect of OM in lowering liver TG content.
Several recent studies suggest that ACSL isoforms have distinct roles for channeling long chain FAs into nonoxidative and oxidative pathways.12–14 The facts that increased ACSL3/5 activity led to reduced TG contents of HepG2 cells and of livers of OM-treated hamsters imply that transcriptional activation of these specific ACSL isoforms may direct fatty acyl-CoAs into energy expenditure with a fall in conversion to TG. We measured cellular FA oxidation using [14C]palmitate as the substrate. Indeed, oxidation of [14C]palmitate was 50% higher in HepG2 cells treated with OM for 24 hours than in untreated cells (Figure 4B).
We further measured [14C]palmitate oxidation in the presence of siRNAs of ACSL3 and ACSL5. HepG2 cells were transfected with siRNAs directed against ACSL3 and ACSL5 mRNAs individually or combined. A nonspecific siRNA with a scrambled sequence was included in the transfection as a negative control. Two days after transfection, cells were untreated or treated with OM for 24 hours before the assay of β-oxidation. The oxidation of [14C]palmatic acid was increased 65% by OM in cells transfected with the nonspecific siRNA. This enhancement of β-oxidation by OM was partially reduced to 39% and 36% in ACSL3 and ACSL5 siRNA-transfected cells, respectively. The combination of ACSL3/5 siRNAs almost abolished the effect of OM (Figure 4C). The basal level of oxidation was also significantly reduced by diminishing the cellular abundance of these ACSL isoforms, providing additional evidence that supports the function of these isoforms in FA partitioning in liver cells. Knockdown effects of siRNA on endogenous mRNA levels of ACSL3/5 were demonstrated by quantitative real-time RT-PCR assays (supplemental Figure VI).
Stimulation of FA Oxidation by Overexpressions of ACSL3 and ACSL5
To further ensure functions of ACSL3/5 in acyl-CoA partitioning into the cellular oxidative pathway in the absence of OM, we examined effects of overexpression of each of these 2 isoforms in HepG2 cells on TG content and β-oxidation. To increase ACSL3 expression, we transfected HepG2 cells with a human ACSL3-expressing plasmid (phACS3).22 The transfection efficiency was approximately 15% to 20% as determined by parallel transfections of a plasmid encoding the green fluorescent protein (GFP). To increase ACSL5 expression, we performed adenovirus-mediated transduction of ACSL5 by using an adenoviral construct (Ad-ACSL5) that expresses FLAG-tagged rat ACSL5 and GFP.12 Three days after phACS3 or Ad-ACSL5 transfection, total ACSL activities, β-oxidation, and TG content were measured and compared with that of vector-transfected control cells. Results from 3 to 5 separate transfection experiments are summarized in Figure 5A. In phACS3 transfected cells, total cellular ACSL activity was increased by 30.5% (P<0.01), fatty acid oxidation was increased by 32.5% (P<0.01), and the cellular content of TG was reduced to 67% of the control (P<0.01). Similarly, adenovirus-mediated transduction of rat ACSL5 in HepG2 cells resulted in a 16.7% elevation in ACSL enzymatic activity and a substantial increase of 49.5% in β-oxidation. We did observe a trend of TG reduction in Ad-ACSL5 expressing cells, but it was not statistically significant.
We used the siRNA approach to further address the role of ACSL3/5 in cellular TG. HepG2 cells were separately transfected with ACSL5 siRNA, ACSL3 siRNA, or the scrambled control siRNA. Three days after transfection, the TG content was measured. Figure 5B shows that reduction of ACSL5 or ACSL3 expression both resulted in an increase in TG cellular content, which is consistent with their roles in stimulating FA oxidation. Taken together, these results of overexpression and depletion provide additional evidence linking the OM-induced transcriptional activation of ACSL3/5 to the in vivo function of OM in reducing plasma and hepatic levels of TG.
Activation of ERK Signaling Pathway by OM In Vitro and In Vivo
Because activation of MEK/ERK is required for OM to stimulate LDLR transcription,16,23 we investigated the role of ERK activation in OM-stimulated ACSL3/5 transcription. First, Western blot analyses were conducted to detect phosphorylations of ERK in HepG2 cells and in hamster livers without and with OM treatment. Figure 6A shows that the level of phosphorylated ERK in HepG2 cells was highly elevated after 30 minutes of OM addition and remained at the increased level for 2 hours (Figure 6A). To detect ERK activation in the liver of hamsters, liver tissues from 3 control hamsters and 3 OM-treated hamsters (200 μg/kg) were individually homogenized. Cell lysate was examined for ERK phosphorylation. Levels of phosphorylated ERK were significantly higher in all 3 OM-treated liver tissues than that in control. These data clearly demonstrated the activation of ERK by OM under in vitro and in vivo conditions. Second, real-time RT-PCR assays were conducted to examine the mRNA levels of ACSL3/5 in HepG2 cells without and with OM treatment under the condition of blocking ERK activation. Cells were treated with U0126, a specific inhibitor of the ERK upstream kinase MEK1, for 30 minutes before OM treatment of 5 hours. Real-time RT-PCR assays show that U0126 reduced the basal levels of ACSL3 and ACSL5 mRNA. The inducing effect of OM on ACSL3/5 was completely abolished by this MEK1 inhibitor (Figure 6B). The inset of Figure 6B shows the inhibition of OM-induced ERK phosphorylation in HepG2 cells treated with this compound. We also tested specific inhibitors to several other signaling kinases including p38 kinase inhibitor SB239063, c-jun N-terminal kinase inhibitor SP600125, Rho-kinase inhibitor, and PI-3 kinase inhibitor LY294002. These inhibitors, at effective concentrations, had either no effect or only small effects on the OM activity to induce ACSL gene transcription (supplemental Figure VII). Taken together, these results indicate that ERK activation is primarily required for OM to stimulate ACSL transcription.
Our previous studies have elucidated the cellular mechanism by which OM regulates hepatic LDLR transcription.24 The effect of OM on liver LDLR expression translated into a potent lipid reduction in hyperlipidemic hamsters in our previous report17 and the current study. However, different from cholesterol-lowering drug statins that have strong effects on LDL-c reduction and modest effects on TG reduction,25 the TG-lowering effect of OM is greater than its LDL-lowering effect, which cannot be solely explained by the increased LDLR activity. Reduction of elevated levels of plasma TG could be beneficial for dyslipidemia-related cardiovascular disease and the metabolic syndrome.26 Therefore, an in depth investigation into the role of OM in regulating TG metabolism is warranted. In this current study, through a series of in vitro and in vivo experiments, we have uncovered specific roles of hepatic acyl-CoA synthetase isoforms ACSL3/5 in OM-stimulated FA oxidation leading to a fall in cellular TG, which may account in part for the potent effects of OM in lowering plasma TG and FFA in hyperlipidemic hamsters.
By conducting cDNA microarray, Northern blot, and quantitative real-time PCR analyses, we demonstrate that mRNA levels of ACSL3/5 are time-dependently increased in OM-treated HepG2 cells. The increase in mRNA levels of ACSL3 and ACSL5 results in a 28% increase in total cellular ACSL activity after 48 hours of OM treatment. We were not able to examine the effect of OM on ACSL3/5 mRNA levels under in vivo conditions because of the unavailability of hamster ACSL gene sequences; however, the total liver ACSL enzymatic activity of OM-treated hamsters was 27% higher than controls, supporting an effect of OM on ACSL gene transcription.
Several recent studies suggested that ACSL isoforms have distinct roles in fatty acid partitioning. Their functions in channeling long-chain fatty acids into oxidative and nonoxidative pathways are cell type–specific and also depend on their subcellular localizations. By manipulating the expression levels of ACSL3/5 through overexpression, our in vitro studies demonstrate that the increased ACSL3/5 activity in liver cells alone without OM treatment markedly stimulated FA oxidation, which was accompanied by a substantial decrease in cellular TG content in ACSL3 transfected cells. In our experiments, siRNA knockdown of ACSL5 caused a significant increase in cellular TG, whereas overexpression of ACSL5 only produced a small decrease of cellular TG in HepG2 cells. At present, it is not clear what factors cause this apparent discrepancy. Further investigations are needed to fully understand the role of ACSL5 in TG synthesis and assembly in HepG2 cells.
Using a specific inhibitor to ERK upstream kinase MEK1, we demonstrate that the ERK signaling pathway is activated by OM in HepG2 cells and in OM-treated livers. This activation is critically required for OM to induce ACSL3/5 transcription. Studies to identify the cis-acting element and the trans-acting factors that mediate the OM effects on ACSL3/5 transcription are currently undertaken in our laboratory.
In conclusion, we demonstrate a novel cellular mechanism to lower triglyceride through transcriptional activation of specific ACSL isoforms in liver cells. This mechanism could be explored in therapeutics to treat hypertriglyceridemia.
We thank Dr Rosalind A. Coleman (University of North Carolina) for providing the adenovirus vectors of Ad-ACSL5, Dr Hiroyuki Minekura (Research and Molecular Biology research laboratories, Sankyo Co Ltd, Tokyo) for providing the phACS3 and pACS3 (-3600) plasmids, Dr Fang Zhao (Bristol-Myers Squibb Company, Princeton, NJ) for providing human recombinant OM, and Dr Cong Li for her assistance in microarray analysis.
Sources of Funding
This study was supported by the Department of Veterans Affairs (Office of Research and Development, Medical Research Service) and by grant (1RO1 AT002543-01A1) from the National Center for Complementary and Alterative Medicine.
Original received May 21, 2007; final version accepted August 2, 2007.
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