Cloning of OLR 1 Gene in Pig Adipose Tissue and Preliminary Study on Its Lipid-accumulating Effect

In this study we cloned and characterized a novel lipid-accumulating gene, the oxidized low-density lipoprotein receptor 1 (OLR1), which is associated with lipogenesis. We analyzed the gene structure and detected the mRNA transcriptional expression levels in pig adipose tissues at different months of age (MA) and in different economic types (lean type and obese type) using real-time fluorescence quantitative PCR. OLR1 expression profile in different tissues of pig was analyzed. Finally, we studied the correlation between OLR1 and lipid metabolism related genes including peroxisome proliferator-activated receptorγ2 (PPARγ2), fatty acid synthetase (FAS), triacylglycerol hydrolase (TGH), CAAT/enhancer binding protein α (C/EBPα) and sterol regulatory element binding protein-1c (SREBP-1c). Results indicated that the OLR1 gene of the pig exhibited the highest homology with the cattle (84%), and the lowest with the mouse (27%). The signal peptide located from amino acid 38 to 60 and the domain from amino acid 144 to 256 were shared by the C-type lectin family. The expression level of OLR1 in pig lung was exceedingly higher than other tested tissues (p<0.01). In pig adipose tissue, the expression level of OLR1 mRNA increased significantly with growth (p<0.01). The expression level of OLR1 mRNA in obese-type pigs was significantly higher than that of lean-type pigs of the same monthly age (p<0.05). In adipose tissue, the expression of OLR1 correlated with PPARγ2, FAS and SREBP-1c, but not TGH or C/EBPα. In conclusion, OLR1 was highly associated with fat deposition and its transcription, as suggested by high correlations, was possibly regulated by PPARγ2 and SREBP-1c. (


INTRODUCTION
Obesity has become an important issue in recent years and can be viewed as a state of body lipid-related disorders, including type 2 diabetes and atherosclerosis, that are related to excessive adipose tissue deposition and adipocyte hypertrophy (Larsen et al., 2003;Wang et al., 2008).It is widely accepted that oxidized low-density lipoprotein receptor 1 (OLR1) plays an important role in the process of cardiovascular diseases and shows binding activity to multiple ligands indicating that OLR1 has complicated biological functions (Chen and Du, 2007).In mouse adipose tissue, the overexpression of OLR1 can elevate the cholesterol content and this process is accompanied by an increase uptake of free fatty acid (FFA) and an accumulation of triglyceride (Chui et al., 2005), which demonstrates that OLR1 has a close relationship with the storage of triglyceride and the generation of obesity.
Because of the rapid lipid deposition, the pig is considered as a good model to study lipid metabolism.From this perspective, the local Chinese obesity-type pig is especially good material for many genes in adipocytes.
Besides, there is a high homology among the pig, human and rodent, so the outcomes of our research can give a reference to humans.The current study aimed to clone the OLR1 gene complete CDS in adipose tissue, analyze OLR1 nucleotide and protein structure characteristics, and subsequently study OLR1 expression in different tissues, different months of age (MA) and different economic types of pig adipose tissue.Additionally, investigation of the correlation between OLR1 and other lipid metabolismrelated factors was also carried out to appraise OLR1 lipidaccumulation effects in adipose tissue.The data obtained in this research should be a guide for lean-type pig breeding and could also be a reference for the related diseases caused by lipid metabolism disequilibrium such as obesity and type 2 diabetes.
receptor 1 (OLR1), which is associated with lipogenesis.We analyzed the gene structure and detected the mRNA transcriptional expression levels in pig adipose tissues at different months of age (MA) and in different economic types (lean type and obese type) using real-time fluorescence quantitative PCR.OLR1 expression profile in different tissues of pig was analyzed.Finally, we studied the correlation between OLR1 and lipid metabolism related genes including peroxisome proliferator-activated receptorγ2 (PPARγ2), fatty acid synthetase (FAS), triacylglycerol hydrolase (TGH), CAAT/enhancer binding protein α (C/EBPα) and sterol regulatory element binding protein-1c (SREBP-1c).Results indicated that the OLR1 gene of the pig exhibited the highest homology with the cattle (84%), and the lowest with the mouse (27%).The signal peptide located from amino acid 38 to 60 and the domain from amino acid 144 to 256 were shared by the C-type lectin family.The expression level of OLR1 in pig lung was exceedingly higher than other tested tissues (p<0.01).In pig adipose tissue, the expression level of OLR1 mRNA increased significantly with growth (p<0.01).The expression level of OLR1 mRNA in obese-type pigs was significantly higher than that of lean-type pigs of the same monthly age (p<0.05).In adipose tissue, the expression of OLR1 correlated with PPARγ2, FAS and SREBP-1c, but not TGH or C/EBPα.In conclusion, OLR1 was highly associated with fat deposition and its transcription, as suggested by high correlations, was possibly regulated by PPARγ2 and SREBP-1c.
(Key Words : OLR1, Pig, Lipid Metabolism, Adipose Tissue) Guanzhong Black (GB) pigs at 5 MA were obtained from the animal farm of Northwest A&F University.For each chosen age, 5 pigs were obtained for repeated experiments.After slaughter, samples from visceral adipose tissue, subcutaneous adipose tissue, muscle, heart, liver, spleen, lung and kidney were surgically collected and frozen in liquid nitrogen.

Reagents
TRIpure Reagent was purchased from Beijing BIOTEKE Biotechnology Company (Beijing) and RevertAid TM First Strand cDNA Synthesis Kit was from FERMENTAS (MBI)(Shanghai).Taq enzyme, Premix Ex Taq TM Hot start Version, SYBR Green and pMD18-T vector were from TaKaRa Company (Japan).DL2000Marker and plasmid trace-extraction kits were from Beijing TIANGEN Biochemistry Company (Beijing).E. coli DH 5α was available in our own laboratory.Gel DNA purification kit was from Anhui UGENE Biotechnology Company (Hefei).

Primer design
According to the gene sequences of OLR1 from related species published in GenBank, we obtained relevant gene assemblage sequences by the EST method using DNAstar software, and then the relevant gene primers were designed by Primer5.0 software.The primers for PPARγ2, FAS, TGH, C/EBPα, SREBP-1c and β-actin were directly designed from relevant gene CDS in the pig using Primer5.0 software.Finally, the primer sequences were sent to Shanghai Invitrogen Biotechnology Company for primer synthesis (Table 1).

Cloning of pig ORL1 from adipose tissue
According to the instructions from the normal agarose gel DNA recovery kit, the specific band of GB pig OLR1 gene was recovered from 1.5% normal agarose gel, the recovery product was then connected to pMD-19T vector, and transformed to competent cells for sequencing.

Homology alignment and protein structure analysis for OLR1 in pig adipose tissue
First, the analysis of nucleotide homology alignment was carried out on NCBI web (http://www.ncbi.nlm.nih.gov/) using the Nucleotide BLAST function, then the OLR1 protein differences among related species were analyzed by Clustal X1.8 software, and thirdly, a phylogenetic tree was drawn by the Bootstrap Correction and Position Correction program from Mega2.1 software.Fourthly, the prediction of physical chemistry parameters of OLR1 protein was done with an online tool (http://www.expasy.org/tools/).Lastly, the prediction of OLR1 protein transmembrane structure and signal peptide was carried out using an online tool (http://geome.cbs.dtu.dk/services/) and the analysis of OLR1 protein structure was finished by online tools (http://smart.embl-heidelberg.de/and http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml).

Detection of OLR1 expression abundance in different tissues of pigs
According to the sequencing result of complete OLR1 cDNA, we designed OLR1 gene partial CDS primers and amplified OLR1 and β-actin genes from heart, liver, spleen, lung, kidney, muscle, visceral adipose and subcutaneous adipose of 10 MA LW pigs.Then the amplification products were detected by 1% agarose gel electrophoresis and images were analyzed by the SynGene Bio-image system and the absorbance (A) value of each band was recorded.

Detection of OLR1 expression level in adipose tissues at different MA and in different economic types of pigs
The expression levels of OLR1 and β-actin in subcutaneous adipose of 2, 5 and 10 MA LW and 5 MA GB pigs were detected using real time fluorescence quantitative PCR and the OD value of each band was recorded.

Detection of expression abundances of OLR1 and lipid metabolism related genes in adipose tissue of different economic types of pigs
The expression abundances of OLR1, PPARγ2, FAS, TGH, C/EBPα, SREBP-1c and β-actin were detected in subcutaneous adipose tissue of 5 MA LW and 5 MA GB pigs.After 1% agarose gel electrophoresis, the images were then analyzed by the SynGene Bio-image system and the OD value of each band was recorded.Then correlations between OLR1 and other genes were analyzed.

Data statistics and analysis
The experimental data were processed using SPSS 13.0 software.Analysis of variance was carried out by the function: "Compare Means" → "One-Way ANOVA" → "LSD" method and the results were displayed by means±standard deviation (SD).Correlation analysis for the variances was carried out using the function: "Bivariate Correlation" → "Pearson Correlation" as well as "Graphs" → "Scatter/Dot…" → "Simple scatter".

Cloning and sequencing of OLR1 gene in pig adipose tissue
We extracted total RNA from adipose tissue of GB pigs and amplified OLR1 cDNA complete CDS by RT-PCR, and the total RNA and amplification products were detected by agarose gel electrophoresis (displayed in Figure 1).The three bands of RNA in the image were clearly identified, which confirmed the high quality of extracted RNA and could be used.Further, the size of amplification product nearly matched with the expected fragment size, indicating that OLR1 gene complete CDS was successfully obtained from pig adipose tissue.
Next, we connected the amplification product to the vector for cloning, and then bidirectional sequencing was done.After removal of residues in N and C terminal nontranslating regions by BLAST alignment to the sequencing analysis, we obtained pig adipose tissue OLR1 gene CDS which contained 825 bp and its accession No. in the GenBank is EU122440.

Analysis of OLR1 gene sequence in pig adipose tissue
We aligned the sequence of OLR1 gene CDS in pig adipose tissue with that of related species published in GenBank and found that the OLR1 gene in pig adipose tissue exhibited 99% homology to that in pig aortic endothelial tissue (NM_213805).The homology to cattle (NM_174132), human (NM_002543), rat (NM_133306) and mouse (BC117712) was 84%, 79%, 51% and 27%, respectively.The OLR1 in pig adipose tissue had three basyl transversions compared with that in pig aortic endothelial tissue (NM_213805), which were C308A 、 G402A and C401T, among which G402A was a silent mutation.Therefore, two amino acids were changed eventually: T103K and A137V.
The analysis for physical chemistry parameters of OLR1 protein revealed that OLR1 had a molecular weight of 31,197.8Da and the theoretical PI was 6.60.Instability index was 47.20, which implied an instability characteristic for OLR1 protein, and the aliphatic index was 91.20, which demonstrated that globulin composition was high in OLR1 protein.
We predicted a signal peptide for pig adipose tissue OLR1 protein and the result is displayed in Figure 2. The prediction suggested that amino acid 30 to 64 probably contains a signal peptide area and the other areas were considered to be mature protein.We also predicted transmembrane helices for OLR1 protein and the result demonstrated that the OLR1 protein has one transmembrane helix structure, which belongs to a type 2 membrane protein.
Besides, amino acid 1 to 37 belonged to an intramembrane segment and amino acid 38 to 60 was likely to be a transmembrane helices area, while amino acid 61 to 274 belonged to an extramembrane section.According to the concrete score as well as the prediction result for the signal peptide, we can certify that amino acid 38 to 60 was determined to be a signal peptide area, exactly the same as the transmembrane helices section and the S score shown in Figure 2 had a high trend indicating that OLR1 protein was a secretory protein.The probable cleavage site should be in the middle of amino acid 55 to 56 (VIL-LI).
The analysis for domains and conserved domain of pig OLR1 protein is presented in Figure 3.There were three main functional domains in pig OLR1 protein: a transmembrane domain (38-60), a low complexity region  (118-136), and a C-type lectin domain (or carbohydraterecognition domain), but no glycosylation site was found.To combine the predicted results for domains and conserved domain, we concluded that amino acid 144 to 256 was the conserved domain comprising members of a C-type lectin family.

The expression abundance of OLR1 gene mRNA in different pig tissues
Amplification of a 200 bp fragment of the OLR1 gene in different pig tissues (Figure 4) indicated that the highest relative expression (compared to β-actin) was in the lung and the lowest was in the spleen and visceral adipose tissue, whereas expression in other tissues was at intermediate levels.Expression abundance in subcutaneous adipose tissue was significantly higher than that in visceral adipose tissue (p<0.01).Because subcutaneous adipose has a stronger lipid deposition effect than visceral adipose (Mathieu et al., 2006), we can suppose that OLR1 may play an important role in lipid deposition of subcutaneous adipose.

The expression characteristic of OLR1 in adipose tissue at different MA and in different economic types of pigs
Real time fluorescence quantitative PCR was employed to detect the expression abundance of OLR1 and innerreference β-actin in adipose tissues of 2, 5 and 10 MA LW and 5 MA GB pigs.With increasing age (MA), the expression of OLR1 mRNA in pig adipose tissue increased significantly (p<0.01)(Figure 5), which reflected that the expression abundance of OLR1 had a close relationship with the amount of lipid deposition.At the same MA (5 MA for equality), the expression abundance of OLR1 mRNA in adipose tissue of obese-type pigs (GB) was significantly higher (p<0.05)than in lean-type pigs (LW) (Figure 5), which further illustrated that OLR1 expression abundance was related to a higher accumulation of adipose tissue.

Analysis for expression correlation between OLR1 and lipid metabolism related genes in pig adipose tissue
In order to further explore the role of OLR1 in lipid deposition, we investigated the correlation between the relative expression of OLR1 mRNA and the mRNAs of five lipid metabolism and adipogenesis related genes, PPARγ2, FAS, SREBP-1c, C/EBPα and TGH.Results revealed that, in pig adipose tissue, OLR1 exhibited a significantly positive correlation with PPARγ2 (R = 0.928, p<0.01, Figure 6A) as well as FAS (R = 0.863, p<0.01, Figure 6B) and SREBP-1c (R = 0.746, p<0.05, Figure 6E).However, the correlations with TGH and C/EBPα were not statistically significant (p>0.05, Figure 6C and D).

DISCUSSION
Oxidized low-density lipoprotein receptor 1 (OLR1) was first discovered in cattle aortic endothelial cells in 1997 (Sawamura et al., 1997).Since then, OLR1 has been of interest because a close relationship between OLR1 and atherosclerosis has been acknowledged (Chen and Du, 2007).Here we reported that OLR1 was expressed in adipose tissue and was highly correlated with the expression of PPARγ2, SREBP-1c and FAS, suggesting a potential involvement with lipid deposition.The OLR1 is a type II membrane protein that structurally belongs to the Ctype lectin family with a short intracellular N-terminal hydrophilic and a long extracellular C-terminal hydrophilic domain separated by a hydrophobic domain (Chen et al., 2002).The lectin-like domain of OLR1 protein is highly conserved among different species and is considered to be a ligand-recognition area which plays an essential role for ligand binding to protein (Chen et al., 2001).In our study, we also found this conserved domain, so the lectin-like domain is presumed to structurally influence the regulation of OLR1 in lipid metabolism of different tissues as well as different species.The OLR1 protein collected from pig adipose tissue belongs to a secretory protein and amino acid 38-60 is a signal peptide.
We aligned the OLR1 CDS of pig adipose tissue with those of related species and found that OLR1 of pig adipose tissue has the highest homology with the cattle (84%), and the second highest is with human (79%).This outcome probably results from OLR1 protein of the human possessing more functional domains and more complicated structures than in the pig and the cattle (Yamanaka et al., 1998).We also compared the OLR1 CDS in pig adipose tissue with that in pig aortic endothelial tissue (NM_213805) and found that the OLR1 in pig adipose tissue has three basyl transversions and two amino acids changed eventually: T103K and A137V.Besides, these two amino acids just locate in the transmembrane helices area resulting in an alteration of structure, which may influence function.Whether this change is due to the specificity among tissues and actual difference in function need further investigation.
As no research on the expression characteristics of OLR1 in pig tissues has been reported, we studied the expression abundance changes of OLR1 in various pig tissues.The highest relative expression was in lung, which corresponded to observations on cattle tissues (Yamanaka et al., 1998).The expression levels of OLR1 in liver, kidney, muscle and subcutaneous adipose did not show significant differences.We specially focused on the expression characteristic of OLR1 mRNA in adipose tissue where OLR1 had a significantly higher expression in subcutaneous than in visceral adipose tissue.Subcutaneous adipose tissue is the main depot in pigs (Laplante et al., 2006).
In order to verify the association of the expression of OLR1 with lipid accumulation, we studied the expression of OLR1 in subcutaneous adipose tissue from pigs at different months of age and of different economic types.Results showed that the expression of OLR1 in the 5-month-old LW pig was significantly higher than at 2 months of age, and was significantly lower than in the 10-month-old LW pig (p<0.01).Reiter et al. (2007) elucidated that anabolismrelated genes display their higher expressions in obesitytype than in lean-type pigs, while in the adipose tissue of the lean-type pig the catabolism related genes display their advantages.Chui et al. (2005) reported that in the adipose tissue of an obese mouse model the expression of OLR1 was significantly higher than in the normal controls.We also found that the expression of OLR1 in obese pigs was higher (p<0.05) at the same age (5-month-old) than in lean pigs.These observations indicated that OLR1 may play an important role in lipid metabolism.
The OLR1 has affinity for multiple ligands and exhibits complex biological functions.Experiments in vitro indicate that the OLR1 protein can be induced to express by a variety of factors (Chen and Du, 2007).There is no consensus for the OLR1 signal transduction pathway.Chen et al. (2007) indicated that OLR1 acts to influence atherosclerosis through the P38MAPK, ERK1/2 and PI3K pathway and many factors were involved in this process.However, the exact mechanism still needs to be approached.In mouse adipocytes, Chui et al. (2005) indicated that the OLR1 promoter region contained a peroxisome proliferatoractivated response element (PPRE) suggesting transcriptional regulation by PPARγ.In porcine subcutaneous adipose tissue, expression of OLR1 was highly correlated with expression of PPARγ.The SREBP-1c is another transcription factor closely associated with adipocyte differentiation, fatty acid metabolism and lipid deposition (Roder et al., 2007); in pig adipose tissue, SREBP-1c and OLR1 were highly correlated.The expression level of SREBP-1c is 3.5 times greater in porcine adipose tissue (the primary site of fatty acid biosynthesis in this species) than in liver (Gondret et al., 2001).The expression of OLR1 also was correlated with expression of FAS.A SREBP-1c binding site has been reported in the promoter region of FAS suggesting control of transcription by SREBP-1c (Schweizer et al., 2002).
In our experiment we did not observe a correlation between the expression of OLR1 and that of C/EBPα in adipose tissue.In differentiating porcine adipocytes, the expression of C/EBPα is already elevated before visible lipid accumulation suggesting that C/EBPα is not a limiting transcription factor in this species (Ronald et al., 2000;Hausman, 2003).Although not definitively established, it is widely suggested that TGH plays an important role in adipose tissue catabolism.In adipocytes, Wei et al. (2007) demonstrated that down-regulation of the expression of TGH is accompanied by the accumulation of triglyceride and cholesterol esters.We found no correlation between expression of OLR1 and TGH.
In summary, OLR1 was increased with age and fat accumulation in porcine adipose tissue.This process was accompanied by an increased expression of FAS.The expression of OLR1 in adipose tissue was positively correlated with expression of two key adipocyte transcription factors PPARγ2 and SREBP-1c.However, expression of OLR1 was not correlated with expression of C/EBPα, another important adipocyte transcription factor.The OLR1 protein may be involved in anabolic lipid metabolism and lipid deposition, but at present, definitive conclusions cannot be drawn.

Figure 1 .
Figure 1.Electrophoresis results of total RNA and OLR1 PCR product from pig adipose tissue.The three bands of RNA in the image were clearly identified, which suggested the high quality of extracted RNA.The size of amplification product nearly matched with the expected fragment size.

Figure 2 .
Figure 2. The signal peptide prediction of pig OLR1 protein.The high S score usually appears in the signal peptide area, and the low S score appears in the mature peptide area.The higher the S score is, the more likely it will be the signal peptide area.C score represents the evaluated score for cleavage site.Generally, the highest C score is predicted to be the cleavage site.Y score, which can accurately reflect the position of the cleavage site, is determined by both S score and C score.

Figure 3 .
Figure 3.The domains and conserved domain prediction of pig OLR1 protein.A: C-type lectin domain, B: Low complexity region, C: C-type lectin or carbohydrate-recognition domain.

Figure 5 .
Figure 5.The OLR1 expression rule in pig adipose tissues at different MA and in different economic types.NO.1-3 represent LW pig and NO.4 represents GB pig.1: 2 MA. 2, 4: 5 MA.3: 10 MA.Real-time quantitative PCR was used to detect the expression levels of OLR1.β-actin was used as an internal control.Values on Y-axis were obtained by the comparison of fluorescence intensity of OLR1 and β-actin.Values shown as means±SD of experiments from five animals.Values marked with a different letter differ significantly (capital letters p<0.01; small letters p<0.05).

Figure 6 .
Figure 6.The amplification and correlation result of OLR1 to other lipid metabolism related genes in different varieties of pig adipose tissues."LW" represents LW pig; "GB" represents GB pig; "M" represents Marker; "R" represents Pearson correlation; "P" represents significant level; "N" represents sample number.Values are means±SD of experiments from ten animals, each has two repetitions.Values in the figure represent the correlation of OLR1 and other genes in pig adipose tissue.The "**" indicates p<0.01;The "*" indicates p<0.05.

Table 1 .
PCR parameters for relevant genes F: Forward; R: Reverse.