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© 2008 American Society for Nutrition J. Nutr. 138:674-679, April 2008

Biochemical, Molecular, and Genetic Mechanisms

Serum Amyloid A Protein Regulates the Expression of Porcine Genes Related to Lipid Metabolism1–3,

Chia H. Chen, Pei H. Wang, Bing H. Liu, Hao H. Hsu, Harry J. Mersmann4 and Shih T. Ding*

Department of Animal Science and Technology, Institute of Biotechnology, National Taiwan University, Taipei 106, Taiwan

* To whom correspondence should be addressed. E-mail: sding{at}ntu.edu.tw.


   ABSTRACT
TOP
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
Serum amyloid A protein (SAA) is an apolipoprotein that can replace apolipoprotein A1 (apoA1) as the major apolipoprotein of HDL. Porcine hepatic SAA mRNA is increased by dietary docosahexaenoic acid (DHA) treatment. The purpose of this study was to investigate the role of SAA protein in regulating gene expression related to lipid metabolism in pigs. First, we demonstrated that the 100-µmol/L DHA treatment increased SAA and apoA1 mRNA expression in porcine hepatic cell cultures (P < 0.05). Secondly, we produced porcine SAA recombinant protein and found that the addition of SAA to porcine preadipocytes in culture stimulated interleukin-6 (IL-6) mRNA expression (P < 0.05), indicating a similar biological function of porcine SAA and human SAA. We also found PPAR{alpha} and PPAR{gamma} mRNA were decreased (40 and 60%, respectively) in differentiated adipocytes after treatment with 2 µmol/L SAA. SAA treatment also increased inflammatory cytokine gene expression (IL-6 and tumor necrosis factor {alpha}) and glycerol release (P < 0.05), indicating increased lipolysis. Because the expression of perilipin, a lipid droplet–protective protein, was reduced by the SAA treatment, we hypothesized that SAA increased lipolysis by decreasing the expression of perilipin, which would then allow an increase in hormone sensitive lipase activity. In conclusion, we demonstrated that the DHA-induced SAA gene expression decreased PPAR expression and consequently downregulated the expression of several genes involved in lipid metabolism. Accordingly, SAA may play a critical role in mediating the function of dietary DHA on lipid metabolism and could be a factor in regulating obesity.


   Introduction
TOP
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
Adipose tissue is an important organ for fatty acid (FA)5 synthesis and energy metabolism in pigs. Porcine adipocytes are frequently used to study the effect of PUFA on gene expression (1,2). Both eicosapentaenoic acid and docosahexaenoic acid (DHA) downregulate sterol regulatory element binding protein 1c (SREBP1c) mRNA and protein expression (3); this major transcription factor regulates lipogenesis by increasing expression of FA synthase and acetyl-CoA carboxylase 1 (ACC1) (2,4,5). Although there are differences in the effects of individual (n-3) PUFA on lipolysis, with considerable disparity between eicosapentaenoic acid and DHA (6), we have concentrated on studying the bioactivity of the latter.

In the past, one emphasis of research on the effect of dietary PUFA to reduce lipogenesis and increase lipolysis concerned the molecular mechanisms involved in the function of the SREBP1 and PPAR{alpha} transcription factors in mammals (7–11). Many other genes mediating PUFA effects have also been reported, and the gene list is still growing [reviewed by (12,13)].

Pigs fed DHA supplements have decreased hepatic SREBP1c mRNA and protein, whereas there is no change in adipose tissue SREBP1c (4,14). Dietary DHA also significantly reduced plasma triacylglycerol concentration in pigs (14). Because hepatic SREBP1c is not highly expressed in porcine liver (1), reduction in this protein may only partially explain the reduction in triacylglycerol. These findings suggest that there may be other mechanisms by which DHA reduces lipid deposition in pigs.

We previously demonstrated that dietary DHA treatments increased the expression of porcine hepatic serum amyloid A protein (SAA), a secretive protein that circulates in the blood and may affect overall body metabolism (15). This protein was expressed in the liver but not in the adipose tissue (15). Recent data show that SAA is associated with obesity and lipid metabolism and is highly correlated with BMI (16,17). However, the ability of SAA to regulate the expression of genes involved in lipid metabolism has not been demonstrated. Therefore, we studied the effect of DHA on the expression of SAA in hepatocytes and generated the porcine SAA recombinant protein (pSAA) to study its function in adipocytes.


   Materials and Methods
TOP
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 LITERATURE CITED
 
    Isolation of porcine hepatocytes and cell culture. The animal protocol was approved by the Animal Usage Committee at National Taiwan University. A total of 3 7-d-old small-ear pigs were killed by electrocution and exsanguination for hepatocyte isolation. The liver was perfused with PBS to wash out RBC before tissue was removed, minced, and passed through a 100-µm mesh. This hepatic mince contains hepatocytes but also other hepatic cells, including Kupffer cells, bile duct cells, vascular cells, etc. These hepatic cells were used for hepatocyte culture experiments. Hepatic cells were treated with different concentrations of DHA (0, 25, 50, and 100 µmol/L) in Williams' Medium E for 24 h (37°C, 5% CO2). The TRI reagent (Sigma) was used to extract total RNA. A total of 2 µg RNA was then reverse transcribed to cDNA. The mRNA concentrations of apolipoprotein A1 (apoA1), SAA, and β-actin were determined by real-time PCR. Liver tissue from these pigs was used to clone the full-length cDNA for SAA (15) by RT-PCR using AccuPrime pfx DNA polymerase (Invitrogen). The primer pair for the cloning of porcine SAA was 5' (5'-GCA GCT CAG CTT CAC CAG GA-3') and 3' (5'-CTG CTC ACA GGA GGC TCA CA-3'). Primers were designed from the human sequence and included sequences before the start codon for the sense primer and after the stop codon for the antisense primer (15). The cDNA was used for recombinant protein production.

    Gene construction and recombinant protein production. The full-length cDNA fragment without a signal peptide sequence was subcloned into the QIAexpressionist protein expression vector, pQE30 (Qiagen). Isopropyl β-D-thiogalactoside was used to stimulate the expression of the recombinant protein. The affinity column with Ni2+ (ProBond Purification System from Invitrogen) was used to purify the recombinant protein (pSAA). The histidine tag was used as a binding sequence for Ni2+ during recombinant protein purification.

    Isolation of porcine stromal/vascular cells. Porcine adipose tissue samples were removed and stromal/vascular (S/V) cells were isolated and cultured, as previously described (18). The S/V cells were treated with 0, 40, or 200 nmol/L of pSAA for 24 h to test the bioactivity by detecting the effect of pSAA on the expression of interleukin-6 (IL-6) mRNA. The concentrations were chosen according to those reported in the literature to be within physiological range (16).

    Cell culture and differentiation of porcine adipocytes. The medium for S/V cells was removed and replaced by serum-free, hormone-supplemented differentiation medium (DMEM/F12 containing sodium bicarbonate, 0.5 µmol/L insulin, 10 mg/L transferrin, 2 mmol/L L-glutamine, 33 µmol/L biotin, 17 µmol/L pantothenate, 1 µmol/L dexamethasone, 1 nmol/L triiodothyronine, 0.25 mmol/L 3-isbutyl-methylxanthine, 100 kU/L penicillin, 100 mg/L streptomycin, 1.5 mg/L amphotericin B, and 1 µmol/L rosiglitazone) for 3 d to induce adipogenesis. The medium was then changed to a differentiation medium without rosiglitazone. The medium was replaced every 3 d. After 6 d, the well-differentiated adipocytes (~80% differentiation) were treated with a medium containing 0, 0.2, or 2 µmol/L of pSAA for 24 h to test the effect of pSAA on adipocyte gene expression. The medium from all treatments was collected to determine its glycerol concentration, following the method of Kreutz (19) for estimation of lipolysis activity. Total glycerol was determined (mg/L) and relative concentrations were calculated and presented. The results were the means of 3 independent experiments with S/V cells isolated from 3 different pigs.

    Real-time PCR analysis. The RNA samples were digested with DNase I to remove genomic DNA contamination and were reverse transcribed at 42°C with a High Capacity cDNA Reverse Transcription kit (Applied Biosystems). The mRNA for various genes was quantified with the FastStart SYBR Green real-time PCR kit (Roche). They were quantified by an Opticon 2 Real-Time PCR Detection System (Bio-Rad Laboratories). The PCR was performed under conditions typically consisting of 40 cycles with paired-sense and antisense primers designed from porcine gene sequences. The primer pairs and optimized annealing temperature for the genes are listed in Supplemental Table 1. The conditions for PCR were denaturation at 94°C for 30 s (10 min in cycle 1), annealing at optimized annealing temperature for 30 s, and extension at 72°C for 30 s. The mRNA concentration of each gene was normalized to its β-actin mRNA concentration. Amplification of specific transcripts was further confirmed by melting curve profile analysis and agarose gel electrophoresis. Threshold cycle (Ct) values were obtained and relative gene expression was calculated using the formula (1/2)Ct target genes– Ct β-actin (20). The PCR amplification efficiency was high for all treatments.

    Statistical analyses. For each replicate, the control value for a variable was set to 100, with other variables expressed relative to the control. Routinely, there were 3 replicates, each using preadipocytes isolated from a different pig. Homogeneity of the variance was determined and data were analyzed using ANOVA to determine the effects of DHA or pSAA at different concentrations. The data for tumor necrosis factor (TNF)-{alpha} and IL-6 mRNA (Fig. 5) were log transformed before analysis to establish homogeneity of the variance. Tukey's test was used to evaluate differences among means (SAS Inst.). Differences were considered significant at P ≤ 0.05.


Figure 5
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  		FIGURE 5  The effect of pSAA on the expression of adipocytokines in porcine adipocytes. The well-differentiated adipocytes, as described in Figure 3, were treated with medium containing 0, 0.2, or 2 µmol/L of pSAA for 24 h. The total RNA was extracted to determine the effect of pSAA on the expression of cytokines related to lipid metabolism, i.e., TNF{alpha} (A) and IL-6 (B) by porcine adipocytes. The data were log transformed before statistical analysis. Bars are means ± SE, n = 3 independent measures. Means without a common letter differ, P ≤ 0.05.

 

   Results
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 LITERATURE CITED
 
    Effects of DHA on the expression of genes in porcine hepatic cells. Treatment with 100 µmol/L DHA tended to increase the expression of apoA1 mRNA ~100% (P = 0.09, Fig. 1A) and significantly increased SAA mRNA expression ~3-fold (Fig. 1B). These results suggest that this hepatic cell culture system functions as expected and the hepatic cells are responsive to DHA treatment. The lower concentrations of DHA did not affect the expression of these 2 genes.


Figure 1
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  		FIGURE 1  The effect of DHA treatment on porcine hepatic apoA1 (A) and SAA (B) mRNA levels. Hepatocytes were treated with different concentrations of DHA for 24 h before mRNA concentrations were measured and normalized to β-actin mRNA. Bars are means ± SE, n = 3 independent measures. Means without a common letter differ, P ≤ 0.05.

 
    Effect of pSAA on the expression of IL-6 in porcine preadipocytes. Treatment of porcine preadipocytes (S/V cells) with 40 and 200 nmol/L pSAA for 24 h increased the expression of IL-6 mRNA by 1.3 and 2.2 times compared with the control group, respectively (P < 0.05, data not shown), indicating that the recombinant protein generated in the current study is biologically active.

    Effects of pSAA on porcine adipocytes. Treatment with 2 µmol/L pSAA significantly increased the release of glycerol to adipocyte culture medium 4-fold (Fig. 2A), indicating that pSAA increased lipolysis in porcine adipocytes. We hypothesized that the increase was via an increase in the expression of hormone sensitive lipase (HSL) or a decrease in the expression of a lipid droplet–protective protein, perilipin. The expression of perilipin mRNA was reduced by 85% when cells were treated with 2 µmol/L pSAA (P < 0.05, Fig. 2B).


Figure 2
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  		FIGURE 2  The effect of pSAA on glycerol release (A) and the expression of perilipin mRNA (B) in porcine preadipocytes cultured in serum-free, hormone-supplemented differentiation medium for 3 d to induce adipogenesis. After 6 d, the well-differentiated adipocytes were treated with medium containing 0, 0.2, or 2 µmol/L of pSAA for 24 h. Bars are means ± SE, n = 3 independent measures. Means without a common letter differ, P ≤ 0.05.

 
The expression of the transcription factors, CCAAT/enhancer binding protein {alpha} and retinoid X receptor {alpha}, were not affected by the pSAA treatments, whereas 2 µmol/L reduced the expression of PPAR{alpha} mRNA ~40% and that of PPAR{gamma} mRNA ~60% (Fig. 3). The lower concentration of pSAA (0.2 µmol/L) had no effect. Expression of mRNA for HSL and acyl-CoA oxidase were decreased by 2 µmol/L SAA, suggesting that lipolysis and FA oxidation may be reduced, although carnitine palmitoylCoA transferase I (CPTI) mRNA was increased by 0.2 µmol/L SAA (Fig. 4A). The expression of several lipogenic genes, including ACC1, adipocyte fatty acid binding protein (aP2), and lipoprotein lipase (LPL), were significantly reduced to ~50% of the control group by the treatment with pSAA, indicating that pSAA may downregulate lipogenesis through effects on these genes (Fig. 4B). Concomitantly, the expression of TNF{alpha} and IL-6, 2 lipolytic adipocytokines, were greatly increased by pSAA treatment (Fig. 5).


Figure 3
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  		FIGURE 3  The effect of pSAA on the expression of transcription factors related to lipid metabolism in porcine adipocytes. The well-differentiated adipocytes, as described in Figure 2, were treated with medium containing 0, 0.2, or 2 µmol/L of pSAA for 24 h. The total RNA was extracted to determine the effect of SAA on the expression of transcription factors, CCAAT/enhancer binding protein {alpha} (C/EBP{alpha}), retinoid X receptor {alpha} (RXR{alpha}), PPAR{alpha}, and PPAR{gamma} in porcine adipocytes. Bars are means ± SE, n = 3 independent measures. Labeled means without a common letter differ, P ≤ 0.05.

 

Figure 4
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  		FIGURE 4  The effect of pSAA on the expression of genes related to lipid metabolism in porcine adipocytes. The well-differentiated adipocytes, as described in Figure 3, were treated with medium containing 0, 0.2, or 2 µmol/L of pSAA for 24 h. The total RNA was extracted to determine the effect of SAA on the expression of genes related to lipid metabolism, HSL, ACO, CPTI (A), ACC1, aP2, and LPL (B) in porcine adipocytes. Bars are means ± SE, n = 3 independent measures. Means without a common letter differ, P ≤ 0.05.

 

   Discussion
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
LITERATURE CITED
 
Because apoA1 is one of the PPAR{alpha}-regulated genes (21) and DHA can enhance the activity of PPAR{alpha} (22), we measured the response of porcine hepatic cell apoA1 mRNA to DHA treatment. The DHA treatment tended to increase the expression of apoA1 mRNA. In humans, dietary (n-3) PUFA supplementation increases serum apoA1, indicating that (n-3) PUFA increases the expression of apoA1 in the liver (23). We also found that the mRNA concentration for SAA was increased by the 100-µmol/L DHA treatment, confirming our experiment in vivo, which showed that dietary DHA increased the expression of porcine hepatic SAA (15). Stimulation of the expression of SAA by inflammatory agents, but not by DHA, is well studied in several animal species (17,24,25). Whether the regulation of SAA expression by DHA is direct or mediated by other mechanisms is not known.

The observation that porcine hepatic SAA mRNA is increased by dietary DHA suggests that the SAA protein was increased, secreted into the circulation, and affected physiological functions throughout the body. The expression of SAA mRNA is specific to the liver and not found in the adipose tissues in pigs (15). However, in humans, SAA is expressed in liver and adipose tissue and the SAA expression is associated with body composition and nutritional condition (16,26). In mice, there are 4 types of SAA expressed, with SAA3 mainly expressed in adipocytes and considered an adipocytokine (27). Therefore, there is species specificity for expression of the SAA genes in various tissues.

In human preadipocytes, treatment with SAA increases the expression of IL-6 (17). In this study, we found that the pSAA increased the IL-6 in porcine preadipocytes. Therefore, we demonstrated that this recombinant protein is biologically active.

In adipose tissue, energy is stored primarily as triacylglycerol in lipid droplets. The cellular lipid droplet is covered with perilipin protein to protect it from lipolytic enzyme action. When energy is needed, triacylglycerol is cleaved by HSL, monoacylglycerol lipase, or adipose triglyceride lipase to FFA and glycerol (28). The FFA is released to the blood circulation, transported into the mitochondria for β-oxidation to provide energy, or reesterified to triacylglycerol. Because there is no glycerol kinase in adipocytes, the glycerol is released into the circulation in vivo or into the culture medium in vitro. Therefore, the measurement of culture medium glycerol concentration monitors the lipolytic activity. In this experiment, we found that pSAA induced the release of glycerol into the culture medium (Fig. 2), indicating that pSAA can increase the lipolytic activity in the adipocytes. A similar observation for human SAA treatment in human adipocytes was reported (17). Because we have found that the perilipin mRNA was concomitantly reduced with the increase of medium glycerol, i.e., lipolysis, we speculate that pSAA increased lipolysis through reduction of the perilipin protein. The direct effects of SAA on the modification of HSL activity remain to be demonstrated. In addition to perilipin effects on lipolysis, other mechanisms to modify HSL activity (not evaluated) include cAMP concentration, protein kinase A expression and activity, and the phosphorylation status of HSL. The lipolytic rate was greater when the HSL mRNA was reduced by pSAA treatment. We speculate that even in the presence of reduced HSL protein, the activation of HSL catalytic activity by phosphorylation may be adequate to sustain the increased lipolytic rate or other lipolytic enzymes, such as adipose triacylglycerol lipase (29), or triacylglycerol hydrolase (30) may be functional. This triacylglycerol lipase is responsible for the basal lipolysis in adipose tissues (30).

The major transcription factor to induce the expression of genes related to FA β-oxidation, e.g., ACO and CPTI is PPAR{alpha} (22). We found that the PPAR{alpha} mRNA in adipocytes was reduced by pSAA treatment and the ACO and CPT1 mRNA were reduced and increased, respectively. The reduction in PPAR{gamma} and PPAR{alpha} mRNA can then be expected to reduce the expression of ACC1, as observed. The reduction of ACC1 would decrease the formation of malonyl-CoA, an active inhibitor of the CPTI activity (31). Therefore, even though pSAA treatment decreased the expression of ACO, the increased expression of CPTI coupled with the reduction of the inhibitor, malonyl-CoA, suggests an increase in FA oxidation by pSAA. Although FA oxidation rates have not been measured in porcine adipose tissue, the high expression of PPAR{alpha}, ACO, and CPTI mRNA suggests there is active FA oxidation (32). In humans, the expression of these genes is low in adipose tissue but high in the liver (33).

The reduction in expression of PPAR{gamma} and its target genes, aP2, LPL, and ACC1, after pSAA treatment indicates that the SAA protein reduces the lipogenic activity. This inhibitory effect of SAA on lipogenic genes suggests that SAA may have a function in the depression of obesity.

The expression of SAA is stimulated by cytokines, such as TNF{alpha} and IL-6 (34). Also, pSAA upregulated TNF{alpha} and IL-6 in porcine adipocytes, similar to the observation in human adipose explants (17). There is evidence to show that IL-6 stimulates the oxidation of FA (35) and increases lipolysis in human adipocytes (36); our observations with porcine adipocytes are similar to both findings. Moreover, IL-6 knockout mice have adult-onset obesity that can be treated with IL-6 injection (37). Therefore, we speculate that the lipolytic and β-oxidation enhancing ability of SAA may result, at least partially, through an increase of IL-6.

The TNF{alpha}, secreted by adipocytes, stimulates lipolysis and also inhibits lipogenesis in 3T3-L1 adipocytes, resulting in a high FFA concentration in the culture medium (38). We speculate that TNF{alpha} promotes lipolysis by inhibiting the expression of perilipin through mitogen-activated protein kinases (39). In addition, TNF{alpha} inhibits the expression of HSL and LPL (40). Similarly, we found that pSAA treatment increased the expression of TNF{alpha} and concomitantly reduced the expression of HSL and LPL. It was suggested that the effect of TNF{alpha} on adipocyte dedifferentiation is to increase phosphorylation of perilipin through protein kinase A to reduce the perilipin function of protecting lipid droplets from degradation (16). Such a reduction in perilipin would then allow HSL access to lipid droplets to catalyze hydrolysis of triacylglycerol. We suggest that this mechanism was functional in our experiments with porcine adipocytes.

The expression of perilipin is regulated by PPAR{gamma} (41). The PPAR{gamma} transcription factor increases the expression of perilipin to reduce lipid droplet hydrolysis and increase lipid deposition (42). We observed that pSAA inhibited the expression of PPAR{gamma}, providing a mechanism for the reduction in perilipin expression and the increase in lipolysis.

Taken together, porcine SAA secreted by hepatocytes may be able to increase lipolysis and FA oxidation through the following mechanisms. First, SAA increased TNF{alpha} and reduced PPAR{gamma} expression, both of which reduced the expression of perilipin. Decreased perilipin diminished the stability of the fat droplet to allow increased lipolysis in the adipocyte. Secondly, SAA increased the expression of CPTI to increase FA oxidation. Thirdly, a reduction of PPAR{gamma} by SAA treatment reduced the expression of ACC1, resulting in the decreased production of malonyl-CoA and thus a more active CPTI and greater FA oxidation activity. It is clear that SAA can affect lipid metabolism through regulation of the expression of multiple genes related to lipid metabolism. This protein, not usually considered in schemes describing the regulation of adipocyte lipid metabolism, may be a major player in the control of fat deposition in pigs and other mammals.


   FOOTNOTES
 
1 Supported in part by the National Science Council in Taiwan. Back

2 Author disclosures: C. H. Chen, P. H. Wang, B. H. Liu, H. H. Hsu, H. J. Mersmann, and S. T. Ding, no conflicts of interest. Back

3 Supplemental Table 1 is available with the online posting of this paper at jn.nutrition.org. Back

4 Visiting professor at National Taiwan University. Back

5 Abbreviations used: ACC1, acetyl-CoA carboxylase 1; apoA1, apolipoprotein A1; aP2, adipocyte fatty acid binding protein; CPTI, carnitine palmitoylCoA transferase I; Ct, threshold cycle; DHA, docosahexaenoic acid; FA, fatty acid; HSL, hormone sensitive lipase; IL-6, interleukin-6; LPL, lipoprotein lipase; pSAA, porcine SAA recombinant protein; SAA, serum amyloid A protein; SREBP1c, sterol regulatory element binding protein 1c; S/V, stromal/vascular; TNF{alpha}, tumor necrosis factor {alpha}. Back

Manuscript received 11 September 2007. Initial review completed 30 October 2007. Revision accepted 5 January 2008.


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 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
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