Study on the Lipolytic Function of GPR 43 and Its Reduced Expression by DHA

G protein-coupled receptor 43 (GPR43) is a newly-discovered short-chain free fatty acid receptor and its functions remain to be defined. The objective of this study was to investigate the function of GPR43 on lipolysis. We successfully cloned the GPR43 gene from the pig (EU122439), and measured the level of GPR43 mRNA in different tissues and primary pig adipocytes. The expression level of GPR43 mRNA was higher in adipose tissue and increased gradually with adipocyte differentiation. Then we examined GPR43 mRNA level in different types, growth-stages and various regions of adipose tissue of pigs. The results showed that the expression level of GPR43 mRNA was significantly higher in adipose tissue of obese pigs than in lean pigs, and the expression level also gradually increased as age increased. We further found that the abundance of GPR43 mRNA level increased more in subcutaneous fat than visceral fat. Thereafter, we studied the correlation between GPR43 and lipid metabolism-related genes in adipose tissue and primary pig adipocytes. GPR43 gene had significant negative correlation with hormone-sensitive lipase gene (HSL, r = -0.881, p<0.01) and triacylglycerol hydrolase gene (TGH, r = -0.848, p<0.01) in adipose tissue, and had positive correlation with peroxisome proliferator-activated receptor γ gene (PPARγ, r = 0.809, p<0.01) and lipoprotein lipase gene (LPL, r = 0.847, p<0.01) in adipocytes. In addition, we fed different concentrations of docosahexaenoic acid (DHA) to mice, and analyzed expression level changes of GPR43, HSL and TGH in adipose. The results showed that DHA down-regulated GPR43 and up-regulated HSL and TGH mRNA levels; GPR43 also had significant negative correlation with HSL (low: r = -0.762, p<0.01; high: r = -0.838, p<0.01) and TGH (low: r = -0.736, p<0.01; high: r = -0.586, p<0.01). Our results suggested that GPR43 is a potential factor which regulates lipolysis in adipose tissue, and DHA as a receptor of GPR43 might promote lipolysis through down-regulating the expression of GPR43 mRNA. (


INTRODUCTION
Adipose tissue plays an important role in regulating energy metabolism and functioning as an energy reserve organ.The key reaction of energy metabolism is adipose tissue lipolysis in vivo.During lipolysis, triglycerides (TGs) were broken down to energy-rich free fatty acids (FFAs) and glycerol (Gibbons et al., 2000).The effective regulation of lipolysis could decrease body fat and reduce the incidence of obesity and diabetes (Andrea and Philipp, 2005).
G-protein-coupled receptors (GPCRs) are key regulators in several physiological functions.Their roles in cellular signal transduction have made them the target of drug discovery efforts in various therapeutic areas (Howard et al., 2001).GPR43 is one of the GPCRs which coupled to both Gq and Gi/Go proteins.The pathways that were activated by this receptor include release of intracellular Ca 2+ and ERK1/2 activation and inhibition of cAMP accumulation (Le Poul et al., 2003).Pharmacologically, Short chain fatty acids are most potent agonists for GPR43 (Brown et al., 2003;Nilsson et al., 2003).In vivo, GPR43 has been shown to be present in a variety of tissues, particularly evident in fat stores, inflammatory cells and the gastrointestinal tract.Recently, Hong et al. reported that GPR43 expression was detected in adipose tissue and isolated adipocytes.Moreover, propionate and acetate stimulated adipogenesis through GPR43, it was suggested that GPR43 might be involved in lipid metabolism (Hong et al., 2005).
In this study, we examined the expression characteristic of GPR43 in different tissues and primary adipocytes of pig both in vitro and in vivo, and analyzed the correlation between GPR43 and lipid metabolism-related genes.Furthermore, we studied the function of GPR43 on mouse adipose tissue which treated with DHA.Our data could provide some insight into understanding the role of GPR43 on lipolysis.

Materials
Experimental animals : Large White pig is a kind of lean pig.Guanzhong black pig is a kind of obese pig, which belongs to local strain of Chinese pig.Both of them were provided by Shaanxi Guangming pig farm.All animal studies were approved by the Institutional Animal Care and Use Committee of the University of Northwest Agriculture and Forestry.Subcutaneous fat and visceral fat tissue were quickly excised after the animal were slaughtered, then frozen in liquid nitrogen, and stored at -80°C for later use.A range of non-adipose tissues were also collected and stored: muscle, heart, liver, spleen, lung and kidney.
Two-week-old male Kunming mice were obtained from the Fourth Military Medical University (Xi'an, China).They were housed individually in cages at a constant temperature (25°C) and under a 12 h light/12 h dark cycle.The animals had free access to water and standard diet for 1 week of an acclimatization period.Then the mice were divided into two groups, the low concentration group and high concentration group, each group had thirty-five mice.Before operation, mice were fasted for 12 h, then both low and high concentration groups were given 6.25 g/kg and 12.5 g/kg DHA respectively by intragastric administration.At 0, 1, 2, 4, 8, 16, 24 h, five mice of each group were killed by decapitation.Subcutaneous adipose tissues were rapidly separated, immediately frozen in liquid nitrogen, and stored at -80°C until RNA extraction.
Cell culture : Primary pig adipocyte was obtained from new born pig subcutaneous adipose tissue, grown and differentiated as previously described (Hongmei et al., 2007).In brief, tissues were initially washed with PBS containing high concentration of mycillin and then digested with collagenases at 37°C for 1 h.Cells were then filtered through 40 micron nylon membrane to remove tissue debris and concentrated by centrifugation.Isolated cell pellets were resuspended in DMEM/F12.Finally, cells were seeded in culture plates at a density of 5×10 4 cells/cm 2 and cultured at 37°C in a humidified atmosphere containing 5% CO 2 .After 24 h, cells were switched to differentiation medium consisting of DMEM/F12, supplemented with 10% (v/v) FBS, hydrocortisone (50 ng/ml), insulin (10 μg/ml), transferrin (10 μg/ml), and penicillin and streptomycin (MDI).Primary adipocyte was grown for preparation of total RNA at day 0, 2, 4, 6, 8 and 10 respectively.The medium was changed every two days.
Real-time quantitative PCR : GPR43 mRNA from samples of different types, growth-stages and various regions adipose tissue of pigs were determined by real time quantitative PCR.The 20 μl real-time reaction contained 12.5 μl SYBR Premix EX Taq (Takara, Japan), 0.5 μl Forward Primer (10 μmol/L), 0.5 μl Reverse Prime (10 μmol/L), 1 μl cDNA, ddH 2 O 10.5 μl.Reactions were incubated in an TP800 Real-time System (Takara, Japan) for 10 s at 95°C, followed by 40 cycles of 95°C for 5 s and 60°C for30 s, 95°C at 15 s, 60°C at 30 s, 95°C at 15 s.Using the method of 2 -Δ Δ Ct to analysis the expression of gene.The method of 2 -Δ ΔCt always been used to relative quantification of gene expression.The expression level of genes were evaluated by utilizing the comparative critical threshold (Ct).The Ct values for each GPR43 reaction were subtracted from the respective Ct value of the β-actin control, resulting in the ΔCt value.The largest ΔCt value was arbitrarily used as a constant that was subtracted from all other ΔCt values to determine ΔΔCt value.Fold changes were then generated for each GPR43 by calculating 2 -ΔΔCt .
Quantitative PCR : Also, we measured levels of pig and mouse GPR43, PPARγ, LPL, HSL, TGH and β-actin mRNA by use of a simple and similar real-time PCR technique in accordance with the principle of real-time quantitative PCR.The primers and PCR amplification conditions were listed in Table 1.Briefly, for each sample, we prepared 6 tubes for PCR reaction, and the reaction was set as 10, 15, 20, 25, 30 and 35 cycles, respectively.The PCR products were examined by agarose gel electrophoresis, then the expression of GPR43 be corrected by β-actin.In six different cycles, we selected correction value used for statistical analysis while the difference was the biggest between different samples at the same cycle.
Data analysis : Software SPSS 13.0 was used for statistical analysis.The expression of genes was analyzed with one-way ANOVA and LSD multiple comparison.Pearson's correlation coefficients were used to determine statistical linear associations between GPR43 and other genes involved in lipid metabolism.All data from samples was shown as means±standard error (SEM).

Gene clone and bioinformatics analysis
To amplify the pig GPR43 gene, we designed one pair of primers based on human and mice gene sequences as there was no pig GPR43 gene sequences available.Total RNA was prepared from adipose tissue of an adult Large White pig and used for RT-PCR.Agarose gel analysis of PCR product indicated a specific band and the sequencing result showed that the pig GPR43 partial cDNA consists of 488 bp.The pig GPR43 cDNA sequence was submitted to GeneBank (accession no.EU122439).Aligned with other species, pig GPR43 cDNA shares the highest homology with human (89%), followed by mouse (84%) and rat (83%).

Different expression pattern of GPR43 mRNA in pig different tissue
In order to study the expression pattern of GPR43 mRNA in pig different tissues, we redesigned primers of GPR43 gene base on pig gene sequence that we had obtained, and amplified it in pig different tissues.As was shown in Figure 1, GPR43 expressed in multiple tissues including subcutaneous adipose, visceral adipose, muscle, heart, liver, spleen, lung and kidney, and showed its highest expression abundance in lung, which was significantly higher than the other tissues (p<0.01).The expression abundance in subcutaneous adipose was significantly higher than visceral adipose (p<0.01).

Level of GPR43 mRNA during differentiation of pig primary cultured adipocytes
To investigate the involvement of GPR43 in pig primary cultured adipocytes differentiation in vitro, GPR43, LPL and PPARγ mRNA levels were analyzed at different cultured time using quantitative PCR.As showed in Figure 2, the levels of GPR43 mRNA increased after adipocyte differentiation with a peak at day 6, the expression tendency of LPL, PPARγ were same as GPR43.This suggested that GPR43 might be involved in the regulation of pig adipocytes differentiation.

Real-time quantitative PCR determine the expression characteristic of GPR43 mRNA in different types, growth-stages and various regions adipose tissue of pig
The amplification curve and melt curve are necessary for relative quantitation using real-time quantitative PCR.In this experiment, we used β-actin as house keeping gene, to determine the expression level of GPR43 in different types (obese, lean), different growth-stages (5-month-old, 10-month-old) and various regions (Subcutaneous fat and visceral fat).The Figure 3 demonstrated that GPR43 mRNA level was significantly higher in adipose tissue of obese pigs than that of lean pigs (p<0.01), and the expression level also gradually increased as age increased (p<0.01), in addition, the abundance of GPR43 mRNA level increased more in subcutaneous fat than visceral fat (p<0.01).These results suggested that GPR43 might be involved in the regulation of adipose tissue development.

Correlation between GPR43 and some lipid metabolismrelated genes expressed in adipose tissues and primary cells
To support the hypothesis that GPR43 is involved in adipocyte differentiation and adipose tissue development, we next investigated the relationship between GRP43 expression and the expression levels of other lipolysis genes in adipose tissue (Figure 4) by quantitative PCR.HSL and TGH are the main genes that play an important role in lipid metabolism in adipose tissue.Applying Pearson's correlation analysis to the data obtained from the adipose tissue samples, we revealed that the expression level of GPR43 gene had a negative correlation with HSL gene (r = -0.881,p<0.01) and TGH gene (r = -0.848,p<0.01) expression (Table 2).In primary adipocytes, we examined the expression of LPL and PPARγ genes and compared their expression levels with GPR43 gene expression (Figure 2).GPR43 expression showed a postive correlation with PPARγ gene (r = 809, p<0.01) and LPL gene (r = 0.847, p<0.01) gene expression (Table 2).

Effect of GPR43 on DHA treated mice adipose tissue
We next examined the effect of GPR43 on DHA treated mice adipose tissue.As showed in Figure 5, quantitative PCR analysis showed that the level of GPR43 mRNA was down-regulated, on the contrary, the levels of HSL and TGH mRNA were up-regulated at different concentration groups.Applying Pearson's correlation analysis to the data obtained from the adipose tissue samples, we revealed that the expression level of GPR43 still had a negative correlation with HSL gene (low: r = -0.762,p<0.01; high: r = -0.838,p<0.01) and TGH gene (low: r = -0.736,p<0.01; high: r = -0.586,p<0.01) expression (Table 3).The result was same as above.

DISCUSSION
GPR43 gene was originally identified in human by Sawzdargo et al. which located at chromosome 19q13.1 (Sawzdargo et al., 1997).It was initially believed that GPR43 was only expressed in a range of peripheral blood leukocytes, with particularly high levels shown in monocytes and neutrophils (Senga et al., 2003).However, Hong et al. demonstrated that GPR43 was highly expressed in adipose tissue and adipocytes (Hong et al., 2005).In this study, we found that GPR43 also expressed in pig adipose tissue and primary adipocytes.We cloned GPR43 gene from pig adipose tissue and examined its expression pattern in adipose tissues and primary adipocytes.It was found that GPR43 might influence the regulation of lipolysis and adipocyte differentiation.
In this experiment, we examined GPR43 mRNA levels in different types, growth-stage and various regions adipose tissue.The result showed that GPR43 mRNA level was significantly higher in adipose tissue of obese pigs than that of lean pigs.This result was similar to the previous research, which showed that GPR43 mRNA was up-regulated in adipose tissues of mice fed a high-fat chow compared with those on normal chow (Hong et al., 2005).Given the fact that the fat deposition capacity of obese pig significantly higher than lean pig (Gongshe, 1997), so we suggested that GPR43 might be involved in adipose tissue development.Then we found that the expression level of GPR43 in pig adipose gradually increased as age increased, and the abundance of GPR43 mRNA level increased more in subcutaneous fat than visceral fat.Previous research showed that lipolysis gradually decreased as age increased in pig (Mersmann, 1998), and lipolysis capacity of subcutaneous fat better than visceral fat (Bjorntorp, 2000), suggesting that GPR43 might negatively regulate lipolysis in adipose tissue.
PPARγ is a member of the nuclear receptor superfamily of transcription factors and previous studies showed that PPARγ has a important role in adipocyte differentiation and regulates gene expression in many functional pathways (Grimaldi, 2001).In this study, the correlation between GPR43 and PPARγ in pig adipocyte was positive.This result consisted with previous research, which showed that SCFAs stimulated differentiation in 3T3-L1 adipocytes via GPR43, with up-regulation of PPARγ (Hong et al., 2005).LPL hydrolyzes triglycerides (TGs) in chylomicrons and VLDL, thereby generating free fatty acids that enter either storage or oxidative pathways (Guo et al., 2008).Thiazolidinediones affected adipocyte LPL production through activation of PPARγ, promoting the hydrolysis of triglyeride (Schoonjans et al., 1996).Therefore, based on the observation that GPR43 expression was positively correlated with LPL in primary adipocytes, it suggested that GPR43 might be involved in lipid degradation.
Many studies have shown that SCFAs are endogenous ligands for GPR43 (Brown et al., 2003;Kotarsky et al., 2003;Nilsson, 2003) and Isoproterenol-stimulated lipolysis is reduced in a dose dependent manner by acetate or propionate treatment in vitro (Hong et al., 2005), indicated that GPR43 might play a important role in lipolysis.HSL, a key enzyme in fatty acid mobilization, and a rate-limiting enzyme of lipolysis (Holm et al., 1988).TGH is another enzyme that catalyzes the lipolysis of intracellular stored triacylglycerol.during the differentiation of 3T3-L1 adipocytes, lipolysis rate dramatically decreased as TGH mRNA and protein levels increased (Wei et al., 2005).At present, one of the best known mechanisms that activate lipolysis in the adipocyte is the cAMP dependent pathway.cAMP activates PKA which activates HSL by promoting its phosphorylation (Carmen and Victor, 2006).And another group of researchers thought that cAMP acts as a secondary messenger to activate protein kinase which activates HSL by phosphorylation to make it functional in lipid hydrolysis (Zou et al., 2007).We found that GPR43 gene had a negative correlation with HSL gene and TGH gene.It is known that GPR43 can stimulate increasing of IP3, and decreasing of cAMP content when GPR43 expression in recombinant cho cells (Le Poul et al., 2003), suggesting that GPR43 might inhibit lipolysis through cAMP decrease HSL, TGH also be decreased when the expression of GPR43 upregulation.
DHA is one of the polyunsaturated fatty acids which is necessary for the growth and development of the body and have an important role in regulation of fat deposition (Kim et al., 2006).When fed pigs with high DHA may lead to lower body fat deposition (Liu et al., 2005).And if feeding mice diets enriched in for 3 weeks might increase the expression of TGH (Dolinsky et al., 2003), in addition, add polyunsaturated fatty acids to diet of type 2 diabetic patients, HSL activitiy was significantly increased (Rivellese et al., 2007).Those studies are similar to our results.We found that DHA promoted the expression of HSL and TGH in mice adipose tissue.This fully shows DHA may promote lipolysis to regulate fat deposition.Meanwhile, we also detected the expression of GPR43, and found that the expression of GPR43 was significantly decreased with the extension of time.Through the correlation analysis, we also found that GPR43 had a negative correlation with HSL and TGH, this result conformed our above research.Mohamed Soliman et al. reported that the stimulatory effect of SCFAs was abolished by LCFAs (Soliman et al., 2007), GPR43 as a short-chain fatty acid receptor also be inhibited.These results demonstrated that DHA as a long-chain fatty acid may promote adipose lipolysis through inhibiting the expression of GPR43.
In conclusion, in the present study, we found that GPR43 had a negative effect on lipolysis, the role of DHA through down-regulating GPR43 might promote lipolysis.Our data could provide a new insight into the function of GPR43 in lipid metabolism and its possible application to the therapy of obesity.

Figure 2 .Figure 3 .
Figure 2. The expression levels of GPR43, PPARγ and LPL mRNA in the primary cultured adipocytes of pig.A: Electrophoresis results of GPR43, PPARγ and LPL.B: Quantitative PCR Analysis of the expression levels of GPR43, PPARγ and LPL.Values are means±SEM of experiments from four samples, each has three repetition.Means without a common letter differ, p<0.05.

Figure 5 .Figure 4 .
Figure 5.The effect of DHA on GPR43, HSL and TGH expression in mice adipose tissue.A: Electrophoresis results.B: RT-PCR Analysis of the expression level of GPR43.C: RT-PCR Analysis of the expression level of HSL.D: RT-PCR Analysis of the expression level of TGH.Values are means±SEM of experiments from six animals, each has three repetition.Means without a common letter differ, p<0.05.

Table 2 .
Correlation between GPR43 and lipid metabolism related genes in pig Values are means±SEM of experiments, each sample has three repetition.** p<0.01.

Table 3 .
Correlation between GPR43 and other lipolytic-related genes in mouseValues are means±SEM of experiments, each sample has three repetition.** p<0.01.