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J. Anim. Sci. 2005. 83:1516-1525
© 2005 American Society of Animal Science

ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION

Effect of docosahexaenoic acid and arachidonic acid on the expression of adipocyte determination and differentiation-dependent factor 1 in differentiating porcine adipocytes1

B. H. Liu*, C. F. Kuo{dagger}, Y. C. Wang* and S. T. Ding*,2

* Department of Animal Science, National Taiwan University, Taipei 106, Taiwan; and and {dagger} Department of Food Science, Nutrition, and Nutraceutical Biotechnology, Shih Chien University, Taipei 104, Taiwan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Implications
 Literature Cited
 
Adipocyte determination and differentiation-dependent factor 1 (ADD1) drives the expression of several lipogenic genes in mammals. Polyunsaturated fatty acids decrease ADD1 mRNA abundance in differentiating porcine adipocytes. The current study was designed to explore the mechanisms by which PUFA inhibit the expression of ADD1 in porcine adipocytes. Porcine preadipocytes were differentiated for 24 h with 0 or 100 µM of docosahexaenoic acid (DHA) and mixtures of different concentrations of antioxidants to investigate the effect of DHA and antioxidants on the ADD1 mRNA abundance. We found the relative mRNA abundance was decreased by the addition of 100 µM DHA to the medium for porcine differentiating adipocytes, and adding an antioxidant mixture to the medium prevented part of the decrease in ADD1 mRNA abundance. These data suggest that DHA decreased the steady-state transcription factor ADD1 mRNA through a mechanism related to fatty acid peroxidation. Indeed, adding 7.5 µM vitamin E (a natural antioxidant) also restored the concentrations of ADD1 and fatty acid synthase mRNA, which were decreased by DHA treatment; however, the DHA or the antioxidant treatment did not change the expression of antioxidation genes (superoxide dismutase 1 and glutathione peroxidase 1) in porcine stromal vascular cells. When supplemented with the eicosanoid synthesis pathway inhibitors, the inhibition of the expression of ADD1 by arachidonic acid was partially recovered. These results suggest that the mechanism by which PUFA decrease ADD1 mRNA is due to the metabolic product of eicosanoids and peroxidation of these PUFA.

Key Words: Adipocyte Determination and Differentiation-Dependent Factor 1 • Arachidonic Acid • Docosahexaenoic Acid • Fatty Acid Synthase • Polyunsaturated Fatty Acids • Porcine


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Implications
 Literature Cited
 
The polyunsaturated fatty acids, arachidonic acid (ARA; C20:4) and docosahexaenoic acid (DHA; C22:6) are important structural lipid components in biomembranes, and both are necessary for growth and development in young mammals (Mann et al., 1994Go). In addition, dietary PUFA may affect body fat deposition by regulating genes that are involved in upregulating fatty acid oxidation and downregulating lipogenesis (Hsu et al., 2004Go). Adipocyte determination and differentiation-dependent factor 1 (ADD1)/sterol regulatory element binding protein 1 (SREBP1) is a transcription factor that stimulates the expression of genes involved in lipogenesis. Many lipogenic genes contain a sterol regulatory element promoter region (Shimano, 2001Go; Nakatani et al., 2003Go), and transcription is therefore stimulated by ADD1. The ADD1 mRNA concentration is decreased by PUFA in porcine differentiating adipocytes and liver (Hsu and Ding, 2003Go; Hsu et al., 2004Go). The potency of unsaturated fatty acids in inhibiting the expression of lipogenic genes tends to be increased with increasing chain length and degree of unsaturation (Xu et al., 1999Go; Yahagi et al., 1999Go; Hannah et al., 2001Go).

In rodent hepatocytes, the expression of fatty acid synthase (FAS) is downregulated by the generation of PUFA peroxidation products (Foretz et al., 1999Go) and eicosanoid metabolites (Mater et al., 1999Go); however, it is unknown whether PUFA peroxidation products and eicosanoid metabolites have an effect on the expression of ADD1 and FAS mRNA in pigs. Therefore, the current study was designed to study the mechanisms by which PUFA inhibit the expression of ADD1 in porcine adipocytes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Implications
 Literature Cited
 
Animals and Tissue Collection
Crossbred male and female pigs at 7 to 9 d of age were purchased from a commercial pig farm. They were killed by electrical stunning coupled with exsanguination. Adipose tissue was removed under sterile conditions from the dorsal s.c. depot in the neck, shoulder, and back regions. The animal protocol was approved by the Animal Care and Use Committee of the National Taiwan University.

Cell Culture
Under a biohazard hood (<5 min from death of the pigs), 0.66-mm slices of adipose tissue were prepared. The slices were digested with collagenase in sterile Krebs Ringer bicarbonate buffer supplemented with 5.6 mM glucose, 100 U of penicillin/mL, 100 mg of streptomycin/mL (Suryawan and Hu, 1993Go; Ding et al., 1999Go, 2003bGo). The stromal vascular (S/V) cell fraction was isolated by centrifugation at 800 x g, and the pellet was washed with a Dulbecco’s modified Eagle’s medium (DMEM)/F12 medium (a premix produced by Gibco-BRL, catalog No. 12400; Life Technologies, Gaithers-berg, MD) supplemented with NaHCO3, 100 U of penicillin/mL, and 100 mg of streptomycin/mL. After digestion for 90 min at 37°C, the isolated S/V cells were pelleted at 800 x g for 10 min and then washed three times by resuspension coupled with centrifugation using DMEM/F12. The washed S/V cells were resuspended in DMEM/F12 containing 10% fetal bovine serum (vol/vol) and plated at a concentration of 6 x 104 cells/cm2. The S/V cells were then cultured at 37°C in air containing 5% CO2 for 24 h to allow the cells to attach to the plate. After 24 h of incubation, the medium was removed and replaced by serum-free differentiation medium (DMEM/F12 containing 100 nM bovine insulin, 50 ng of hydrocortisone/mL, and 10 µg of transferrin/mL), with or without various treatments. The fatty acid preparation followed the procedures stated by Ding et al. (2000a)Go. In brief, individual fatty acid or treatment reagents were mixed with 1% BSA (final concentration; fatty acid/albumin = 1 mol/1.45 mol) and then dissolved in differentiation medium.

Experiment 1: Effect of Vitamin E, ARA, and DHA on Differentiating Adipocytes
Porcine S/V cells were cultured to confluence in DMEM/F12 containing 10% fetal bovine serum (vol/ vol), and then treated, in a 2 x 3 factorial arrangement, with 0 (Control) or 7.5 µM {alpha}-tocopherol acid succinate (vitamin E), and 0, 100 µM ARA, or 100 µM DHA in differentiation medium for another 24 h. The experimental period of 24 h was chosen because Ding et al. (1999Go, 2002)Go found that the adipocyte differentiation was significantly increased by a 24-h treatment of differentiation medium in porcine S/V cells. Ding et al. (2002)Go and Hsu and Ding (2003)Go also found that the expression of ADD1 mRNA was decreased by a DHA treatment for 24 h. Before treatment media were added, two plates of cells were harvested for mRNA analysis. Cells were obtained from five pigs, and for each pig, there were six wells of cells for each treatment. Total RNA was extracted to determine the concentrations of mRNA for ADD1 and ß-actin, a housekeeping gene. Lactate dehydrogenase activity for medium from each plate was determined according to the procedure of Keiding et al. (1971)Go to monitor the integrity of cells during adipocyte differentiation.

Experiment 2: Effect of DHA and Antioxidants on Gene Expression in Differentiating Adipocytes
Porcine S/V cells were cultured and treated with differentiation medium with 0 (Control), 100 µM DHA, 100 µM DHA + 1x antioxidants, 100 µM DHA + 2x antioxidants, 100 µM DHA + 3x antioxidants, or 100 µM DHA + 4x antioxidants (1x = 100 nM N,N'-diphenyl-1,4-diphenylenediamine-DPPD, 3.75 µM vitamin E, 150 nM butylated hydroxytoluene-BHT, and 40 nM deferoxamine mesylate-DFM; Foretz et al., 1999Go) for 24 h. The DHA initially was used because it had the most double bonds and thereby created the greatest peroxidation pressure. Cells were obtained from six pigs, and for each pig, and there were three wells of cells for each treatment. Cells were used for extraction of total RNA to determine the concentrations of ADD1 mRNA and 18S rRNA.

Experiment 3: Effect of Vitamin E and DHA on ADD1 and FAS mRNA in Differentiating Adipocytes
Porcine S/V cells were cultured and treated with differentiation medium with 0 (Control), 100 µM DHA, 100 µM DHA + 0.75 µM vitamin E, or 100 µM DHA + 7.5 µM vitamin E for 24 h. The concentration of 0.75 µM is close to the normal plasma {alpha}-tocopherol concentration in pigs (Anderson et al., 1995Go). Cells were obtained from four pigs, and for each pig, there were four wells of cells for each treatment. Cells were used for extraction of total RNA to determine the concentrations of ADD1 mRNA, FAS mRNA, and 18S rRNA.

Experiment 4: Effect of ARA Metabolites on ADD1 and FAS mRNA Abundance
Porcine S/V cells were cultured and then treated with 0 (Control), 100 µM ARA, 100 µM ARA + 1, or 10 µM clotrimazole, 100 µM ARA + 1, or 10 µM 5, 8, 11, 14-eicosatetraynoic acid (ETYA), or 100 µM ARA + 1 or 10 µM of indomethacin in differentiation medium for another 24 h (concentrations of the inhibitors followed Qiu and Quilley, 1999Go). Cells were obtained from four pigs, and for each pig, there were four wells of cells for each treatment. Cells were used for extraction of total RNA for determining the mRNA concentration for ADD1 and FAS, as well as the concentration of 18S rRNA.

Transcript Analyses
Total RNA was extracted from the cells by the guanidinium-phenol-chloroform extraction method (Chomczynski and Sacchi, 1987Go), modified by McNeel and Mersmann (1999)Go. The mRNA concentrations of ADD1, FAS, superoxide dismutase1 (SOD-1), and glutathione peroxidase 1 (GPX1) and the concentration of 18S rRNA were quantified by the Northern blot analysis procedure previously described by Ding et al. (1999Go, 2004)Go. The source of the probes for ADD1, FAS, and 18S are stated in Ding et al. (2000Go, 2002Go). The probes for porcine SOD-1 (AF396674), GPX1 (AY743601), and ß-actin were generated from gene fragments cloned by reverse transcription-PCR using mRNA from pig adipose tissue. The transcribed cDNA was amplified by PCR for 36 cycles, using paired sense and antisense primers (5'-AAGGCCGTGTGTGTGCTGAA-3', 5'-CCAATTACAC CACAGGCCAA-3' for SOD1; 5'-TAACCAGTTCGGAC ATCAGG-3', 5'-GTTCCATGCGATGTCATTGC-3' for GPX1, and 5'-GTGGGCCGCTCTAGGCACCA-3, 5'-CG GTTGGCCTTAGGGTTCAGGGGGG-3' for ß-actin). Twenty micrograms of total RNA from each sample was electrophoresed under denaturing conditions, blotted to a nylon membrane, and hybridized with a radiolabeled probe synthesized by PCR. Each RNA sample was represented on two different membranes. All membranes for a given experiment were hybridized at the same time using the same labeled probe to allow for direct comparison within an experiment. The relative density of each mRNA was determined by phosphor-image analysis (BAS-1500, Fujifilm, Kanagawa, Japan) and quantification software program. The density value for each sample was normalized to the density value for 18S rRNA in the same sample to correct for extraction, sampling, gel loading, and membrane transfer variation.

Statistical Analyses
For Experiment 1, the data were analyzed by two-way ANOVA for a factorial arrangement of treatments using GLM procedure of SAS (SAS Inst., Inc., Cary, NC). For the other experiments, the data were analyzed by one-way ANOVA using the GLM procedure of SAS. Significant differences between treatments were tested by Duncan’s Multiple Range Test in SAS. The means and SE for each transcript were presented.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Implications
 Literature Cited
 
The first experiment was designed to study whether vitamin E, ARA, and DHA affect the relative mRNA abundance of ADD1 during porcine adipocyte differentiation. In the current study, the relative mRNA abundance of ADD1 in differentiating adipocytes was increased after 1 d of differentiation induction (P < 0.05; Figure 1Go), suggesting that the expression of lipogenic genes increases after only 1 d of adipocyte differentiation. The addition of ARA and DHA in the medium significantly decreased the ADD1 mRNA abundance in differentiating adipocytes (P < 0.05; Figure 1Go). The addition of 7.5 µM vitamin E allowed for partial recovery of the reduction in ADD1 mRNA abundance. In addition, vitamin E alone did not have a significant effect on the mRNA abundance of ADD1 (Figure 1Go).



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  		Figure 1. The effect of vitamin E, arachidonic acid, and docosahexaenoic acid on adipocyte determination and differentiation-dependent factor 1 (ADD1) mRNA abundance in differentiating adipocytes. Porcine stromal vascular cells were cultured to confluence (d 0) in Dulbecco’s modified Eagle’s medium (DMEM)/F12 containing 10% fetal bovine serum, and then treated with differentiation medium (DMEM/F12 with insulin, hydrocortisone, and transferrin) or differentiation medium containing 7.5 µM vitamin E ({alpha}-tocopherol acid succinate), 100 µM arachidonic acid (ARA), 100 µM ARA + 7.5 µM vitamin E, 100 µM docosahexaenoic acid (DHA), or 100 µM DHA + 7.5 µM vitamin E (E) for 24 h. Total RNA was extracted from cultured cells. The relative mRNA abundance for ADD1 was determined by Northern analysis, and mRNA values were normalized to ß-actin mRNA. After normalization to ß-actin mRNA content, the average of control data was set as 100, and other data were expressed as relative abundance to the control value. The results were presented as means ±SE for cells from five pigs. Means without a common letter differ, P < 0.05.

 
We used a potent combination of antioxidants to inhibit fatty acid oxidation to study whether fatty acid peroxidation products have an effect on the expression of ADD1. The addition of DHA in the medium decreased the ADD1 mRNA abundance in differentiating adipocytes (P < 0.05; Figure 2Go). The addition of a mixture of antioxidants, containing 200 nM DPPD, 7.5 µM vitamin E, 300 nM BHT, and 80 nM DFM, allowed partial recovery of the reduction in ADD1 mRNA abundance.



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  		Figure 2. The effect of docosahexaenoic acid and antioxidants on adipocyte determination and differentiation-dependent factor 1 (ADD1) mRNA abundance in differentiating adipocytes. Porcine stromal vascular cells were cultured to confluence in Dulbecco’s modified Eagle’s medium (DMEM)/F12 containing 10% fetal bovine serum, and then treated with differentiation medium (DMEM/F12 with insulin, hydrocortisone, and transferrin) or differentiation medium containing 100 µM docosahexaenoic acid (DHA), DHA + 1x, DHA + 2x, DHA + 3x, or DHA + 4x antioxidant mixtures. 1x antioxidant mixtures = 100 nM N,N'-diphenyl-1,4-diphenylenediamine, 3.75 µM {alpha}-tocopherol acid succinate, 150 nM butylated hydroxytoluene, and 40 nM deferoxamine mesylate for 24 h. Total RNA was extracted from cultured cells. The ADD1 mRNA abundance was determined by Northern analysis, and mRNA values were normalized to 18S rRNA. After normalization to 18S rRNA content, the average of control data was set as 100, and other data were expressed as relative abundance to the control value. The results were presented as the means ±SE for cells from six pigs. Means without a common letter differ, P < 0.05.

 
Because vitamin E is physiologically present in porcine blood, this experiment was designed to explore whether vitamin E attenuates the effect of DHA on reducing the mRNA concentrations of ADD1 and FAS. An addition of 7.5 µM vitamin E to the adipocyte cell culture reversed the inhibitory effect of DHA on the expression of ADD1 mRNA (P < 0.05; Figure 3Go). The effect was concentration dependent; the addition of 7.5 µM vitamin E created a greater effect than 0.75 µM. Both concentrations of vitamin E had similar effects on the FAS mRNA abundance; however, mRNA abundance for SOD-1 and GPX1, two genes involved in scavenging free radicals, were not affected by the addition of DHA or vitamin E in the medium (Figure 4Go), suggesting that the peroxidation pressure created by the current DHA treatment is not high enough to create a response at the transcript level.



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  		Figure 3. The effect of vitamin E and docosahexaenoic acid on adipocyte determination and differentiation-dependent factor 1 (ADD1) and fatty acid synthase (FAS) mRNA abundance in differentiating adipocytes. Porcine stromal vascular cells were cultured to confluence in Dulbecco’s modified Eagle’s medium (DMEM)/F12 containing 10% fetal bovine serum, and then treated with differentiation medium (DMEM/F12 with insulin, hydrocortisone, and transferrin) or differentiation medium containing 100 µM docosahexaenoic acid (DHA), or DHA + vitamin E (0.75 µM or 7.5 µM {alpha}-tocopherol acid succinate) for 24 h. Total RNA was extracted from cultured cells. The relative mRNA abundance for ADD1 and FAS were determined by Northern analysis, and mRNA values were normalized to 18S rRNA. After normalization to 18S rRNA content, the average of control data was set as 100, and other data were expressed as relative abundance to the control value. The results were presented as means ±SE for cells from four pigs. Means without a common letter differ, P < 0.05.

 


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  		Figure 4. The effect of vitamin E and docosahexaenoic acid on superoxide dismutase 1 (SOD-1) and glutathione peroxidase 1 (GPX1) mRNA abundance in differentiating adipocytes. Porcine stromal vascular cells were cultured to confluence in Dulbecco’s modified Eagle’s medium (DMEM)/F12 containing 10% fetal bovine serum, then treated with differentiation medium (DMEM/F12 with insulin, hydrocortisone, and transferrin) or differentiation medium containing 100 µM docosahexaenoic acid (DHA), or DHA + vitamin E (0.75 µM or 7.5 µM {alpha}-tocopherol acid succinate) for 24 h. Total RNA was extracted from cultured cells. The relative mRNA abundance for SOD-1 and GPX1 were determined by Northern analysis, and mRNA values were normalized to 18S rRNA. After normalization to 18S rRNA content, the average of control data was set as 100, and other data were expressed as relative abundance to the control value. The results were presented as means ±SE for cells from four pigs. There were no significant treatment effects.

 
Inhibitors for cycloogenase (e.g., indomethacin), lipoxygenase (e.g., ETYA), or monooxygenase (e.g., clotrimazole) were added to block the eicosanoid synthesis pathway in order to study the mechanism by which PUFA downregulate the expression of ADD1 and FAS. The addition of 100 µM ARA decreased the steady-state mRNA abundance for ADD1 and FAS (P < 0.05; Figure 5Go). The inhibitory effect was partially reversed by the addition of indomethacin (P < 0.05), suggesting that ARA affects the expression of ADD1 and FAS through an eicosanoid synthesis pathway. The other eicosanoid synthesis pathway inhibitors, ETYA or clotrimazole, were not as effective.



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  		Figure 5. The effect of arachidonic acid metabolites on the adipocyte determination and differentiation-dependent factor 1 (ADD1) and fatty acid synthase (FAS) mRNA abundance in differentiating adipocytes. Porcine stromal vascular cells were cultured to confluence in Dulbecco’s modified Eagle’s medium (DMEM)/F12 containing 10% fetal bovine serum, and then treated with differentiation medium (DMEM/F12 with insulin, hydrocortisone, and transferrin) or differentiation medium containing 100 µM arachidonic acid (ARA), or eicosanoid inhibitors (1C = 1 µM clotrimazole, 10C = 10 µM clotrimazole [Panel A]; 1I = 1 µM indomethacin, 10I = 10 µM indomethacin [Panel B]; 1E = 1 µM 5,8,11,14-eicosatetraynoic acid [ETYA] and 10E = 10 µM ETYA [Panel C]) for 24 h. Total RNA was extracted from cultured cells. The ADD1 and FAS mRNA abundance was determined by Northern analysis, and mRNA values were normalized to 18S rRNA. After normalization to 18S rRNA content, the average of the control data was set as 100, and other data were expressed as relative abundance to the control value. The results were presented as means ±SE for cells from five pigs. Means without a common letter differ, P < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Implications
 Literature Cited
 
The current study confirmed that DHA had an inhibitory effect on the steady-state mRNA concentrations for the transcription factors ADD1 and FAS in differentiating porcine adipocytes (Ding et al., 2002Go; Hsu and Ding, 2003Go). Similar to what was reported for FAS mRNA in rodent hepatocytes (Foretz et al., 1999Go), the addition of 200 nM DPPD, 7.5 µM vitamin E, 300 nM BHT, and 80 nM DFM blocked the inhibitory effect of DHA on ADD1 mRNA. We also have demonstrated that a single dose of 7.5 µM vitamin E can have the same effect as the mixture of antioxidants, whereas a physiological concentration of vitamin E at 0.75 µM did not have a significant effect on reversing the decrease in ADD1 mRNA caused by DHA. The data suggest that a high concentration of vitamin E can partially reverse the peroxidation effect of DHA. It is possible that the inhibitory effect on gene expression by PUFA is a result of its toxicity to the cells. We measured the lactate dehydrogenase activity in the culture medium and found that the enzyme activity was not affected by different treatments (P = 0.31; data not shown), suggesting that the cells were intact under all treatments. Moreover, because we used a housekeeping gene to normalize the transcript data for all the target genes, the effects we observed should have been restricted to the gene of interest.

The expression of genes involved in the free radical-scavenging system, SOD-2 and glutathione transferase, is affected by the addition of dietary PUFA, indicating that these genes are required to scavenge for free radicals created by PUFA (Takahashi et al., 2002Go). In porcine differentiating adipocytes, SOD-1 and GPX1 mRNA abundance were not affected by the addition of DHA, suggesting that the concentration of 100 µM of DHA is not sufficiently high to create a peroxidation pressure that requires additional expression of SOD-1 and GPX1 genes.

Previous studies have found several possible mechanisms by which fatty acids regulate gene functions. Amri et al. (1991)Go found that long-chain fatty acids increase the expression of adipocyte fatty acid binding protein (aP2). This function also can be observed with bromopalmitate, an analog of palmitate that is not metabolized (Grimaldi et al., 1992Go), suggesting that metabolism of the fatty acid is not required to regulate gene expression of aP2. Indeed, Kliewer et al. (1997)Go demonstrated that different fatty acids (including DHA and ARA) have various binding capacities to a family of transcription factors, peroxisomal proliferator-activated receptors (PPAR), which may then regulate the expression of genes, including aP2. Fatty acids activate PPAR{delta} and PPAR{gamma} (Amri et al., 1995Go; Bastie et al., 1999Go; Berger et al., 1999Go) to increase the expression of genes involved in lipid metabolism (e.g., lipoprotein lipase and aP2). Nonetheless, some functions of fatty acid on regulating gene expression may require metabolic transformation (e.g., to eicosanoid molecules; Forman et al., 1995Go; Mater et al., 1998Go; Reginato et al., 1998Go).

To study the involvement of ARA metabolites in regulating gene expression, inhibitors for enzymes involved in the eicosanoid synthesis pathway often are used. The inhibitors for monooxygenase, lipooxygenase, and cyclooxygenase are clotrimazole, ETYA, and indometh-acin, respectively. We used these reagents to study the involvement of eicosanoid metabolism in regulating the expression of porcine ADD1 and FAS and found that indomethacin reversed part of the inhibitory effect of ARA on the concentrations of ADD1 and FAS mRNA. Although the involvement of cyclooxygenase in the expression of ADD1 has not been reported, Long and Pekala (1996)Go demonstrated that cyclooxygenase activity is related to the expression of GLUT4, an ADD1 target gene. The results from the current study suggest that there is a role for cyclooxygenase in the expression of ADD1. In rat hepatocytes, the inhibitory effect of ARA on FAS mRNA is due to the production of PGE2 (Mater et al., 1999Go). Mater et al. (1998)Go found that the inhibition of fatty acid synthase gene expression in 3T3-L1 adipocytes by ARA was blocked by a cyclooxygenase inhibitor, fluriprofen. The current results also indicate that the inhibitory effect of ARA on ADD1 and FAS mRNA abundance is partially related to the metabolites of ARA, especially those produced by cyclooxygenase. Similar to the current observation, Mater et al. (1998)Go demonstrated that inhibitors for lipoxygenase and monooxygenase did not reverse the inhibition of expression of S14 by PUFA. These observations support the assertion that the effect of PUFA on decreasing lipogenic genes is partially through cyclooxygenase-related prostanoids production.

Recently, studies showed that the mechanisms by which PUFA inhibit ADD1 expression are through modifying the proteolytic processing of ADD1 protein (Hannah et al., 2001Go) and mRNA stability in mice (Xu et al., 1999Go, 2001Go). In the current study, we demonstrated that DHA also decreased the ADD1 mRNA abundance through a mechanism that involved fatty acid peroxidation. This effect can be blocked, at least partially, by the addition of antioxidants (vitamin E or a mixture of antioxidants). Therefore, the current experiment demonstrated that in porcine adipocytes, part of the effect of DHA on the expression of ADD1 and FAS is related to peroxidation pressure. In rat hepatocytes, the effect of PUFA to inhibit expression of ADD1 is related to the degree of fatty acid unsaturation (Xu et al., 2001Go), suggesting that the effect of PUFA on ADD1 is related to peroxidation pressure. Results of Xu et al. (2001)Go and Hsu and Ding (2003)Go indicated that the inhibitory effect of PUFA on the ADD1 function is on increasing mRNA degradation, and not on decreasing transcription. Determining whether mRNA degradation is related to PUFA peroxidation pressure will require further study. It also is possible that the effect of the oxidation products from PUFA is an artifact in the cell culture system because the cell in vivo has multiple mechanisms to eliminate oxidation products. Further in vivo studies are needed to test whether such speculation is true.

In pigs, the regulation of dietary PUFA from various sources (fish oil or algal DHA oil) on the expression of lipogenic genes, ADD1 and FAS, in the adipose tissue was not as significant as in the liver (Ding et al., 2003aGo; Hsu et al., 2004Go). However, Allee et al. (1972)Go reported a significant increase of fatty acid synthesis activity by 10% corn oil supplementation (linoleic acid enriched) compared with 10% tallow supplementation (stearic acid enriched). The discrepancy among these reports may be due to the different dietary fatty acid compositions used. In the rat, it was reported by Xu et al. (1999)Go that dietary PUFA significantly decreased ADD1 and FAS mRNA in the liver. Raclot et al. (1997)Go demonstrated that dietary PUFA significantly decreased the expression of FAS and lipoprotein lipase mRNA in the retroperitoneal adipose tissue, but had no effect on the s.c. adipose tissue in the rat. Therefore, the response of gene expression to dietary PUFA is not only species-and tissue-specific, but also site-specific. Although, as described above, the in vitro culture system used in the current experiment may have created artifact conditions that regulate the expression of lipogenic genes, the results form a foundation for exploring possible mechanisms by which PUFA regulate gene expression.


   Implications
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Implications
Literature Cited
 
The current study demonstrated that polyunsaturated fatty acids inhibit the expression of adipocyte determination and differentiation-dependent factor 1 and fatty acid synthase, and that the mechanisms by which polyunsaturated fatty acids regulate gene expression are through peroxidation of polyunsaturated fatty acids and eicosanoid metabolites. Feeding algal docosahexaenoic acid to pigs has been shown to greatly modify liver fatty acid composition but only slightly affect fatty acid composition in adipose tissue. Such a small modification can not decrease the expression of adipocyte determination and differentiation-dependent factor 1 and fatty acid synthase mRNA in the adipose tissue. Inhibition of the expression of these genes can decrease lipogenesis and decrease fat deposition. Understanding the mechanisms by which polyunsaturated fatty acids regulate the expression of lipogenic genes may create novel approaches for decreasing porcine fat deposition.


   Footnotes
 
1 This work was supported by National Science Council in Taiwan (NSC 90-2313-B-002-304). Back

2 Correspondence: 50, Lane 155, Kee-Lung Rd., Sec. 3 (phone: 8862-8732-7350; fax: 8862-2732-4070; e-mail:sding{at}ntu.edu.tw).

Received for publication September 26, 2004. Accepted for publication March 28, 2005.


   Literature Cited
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Implications
 Literature Cited
 


Allee, G. L., D. R. Romsos, G. A. Leveille, and D. H. Baker. 1972. Lipogenesis and enzymatic activity in pig adipose tissue as influenced by source of dietary fat. J. Anim. Sci. 35:41–47.

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Ding, S. T., B. H. Liu, and Y. H. Ko. 2004. Cloning and expression of porcine adiponectin and adiponectin receptor 1 and 2 genes in pigs. J. Anim. Sci. 82:3162–3174.[Abstract/Free Full Text]

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