| Leptin receptor (LEPR) is a key regulator of energy hemeostasis. Adipose triglyceride lipase (ATGL) and Lipoprotein lipase (LPL) are enzymes that are essential for lipid metabolism. Toll-like receptor 4 (TLR4) and Interleukin-6 (IL-6), which are critical for innate and adaptive immunity, have been proved to be responsible for fatty-acid related inflammatory activity. In this study, the full-length cDNA sequence of goose LEPR and the full length CDS (coding sequence) of the rest four genes were cloned, sequenced and analyzed. The tissue distribution and expression change after over-feeding treatment of the six genes were examined by using real-time PCR. The effects of LEPR on the expression of adipocyte differentiation, metabolism and immunity related genes were performed by RNA interference (RNAi). Furthermore, the regulation of genes in adipose cells by rosiglitazone and GW-9662 were also studied, indicating the possible role of LEPR in Peroxisome proliferators-activated receptor gamma (PPARy) signaling transduction. The results are as follows:1. cDNA sequences of goose LEPR, ATGL, LPL, TLR4 and IL-6 cloning and analysisThe full-length cDNA sequence goose LEPR is 4,370bp with accession number HQ699480, comprising 628bp 5'UTR,3,468bp coding sequence (CDS) and 239bp 3'UTR. The deduced protein sequence is 1,156 aa in length, which has 90% identity with duck LEPR, more than 75% identity with those of chicken and turkey, while less than 50% identity with that of mammals.The obtained goose ATGL gene is 1,462bp in length with accession number HQ914789, containing 14bp 5'UTR and 1,446bp CDS sequence. Goose ATGL gene encodes a 482-amino-acid peptide, which has the percent identities of 90%,86% with those of duck and chicken, respectively.The obtained goose LPL gene is 1,756bp in length with accession number JF343526, which contains 1.482bp CDS sequence and 283bp 3'UTR. The translated goose LPL protein is 494 aa in length, with 97%,94% and 92% identities to those of duck, chicken and zebra finch, respectively.The obtained goose TLR4 gene is 2,641 bp in length with accession number HQ436371, containing 79bp 5'UTR,2,532bp CDS and 30bp 3'UTR. The deduced protein is 843 aa in length, which has more than 70% identity to that of chicken and zebra finch, while less than 50% identity to that of mammals.The goose IL-6 cDNA sequence obtained in this study is 802bp in length with accession number JF437643, which contains 70bp 5'UTR,705bp CDS and 27bp 3'UTR. Goose IL-6 gene encodes a 234-amino-acid peptide, with 74% and 39% identities to those of chicken and human, respectively.2. Study on the tissue distribution of goose LEPR, ATGL, LPL, TLR4 and IL-6 genes and their expression change after over-feeding treatmentQuantitative real-time analysis reveals that goose LEPR mRNA was abundant in brain, liver, skeletal muscle, lung and abdominal fat, while the expression level in intestine was very low. Goose ATGL mRNA was highly expressed in subcutaneous fat and lowly in stomach and lung. The mRNA level of goose LPL was high in heart, subcutaneous fat and skeletal muscle, while very low in spleen. The expression of TLR4 was detected highly in liver and lowly in subcutaneous fat and lung. Goose IL-6 was predominantly expressed in liver, the expression level in brain and intestine were barely detected.After over-feeding treatment, the mRNA level of goose LEPR was significantly increased in subcutaneous fat, kidney and intestine. The expression of ATGL in subcutaneous fat was lesser than control, while the mRNA level of ATGL in abdominal fat and lung were higher than control. The expression level of goose LPL was increased in abdominal fat, liver and intestine, while decreased in heart. The expression of goose TLR4 gene was increased in subcutaneous fat and intestine after over-feeding treatment. Similar to TLR4, the mRNA level of goose IL-6 was increased in subcutaneous fat and intestine. What's more, after over-feeding, the IL-6 levels in goose stomach and brain were also promoted. The expression change of these six genes after over-feeding treatment may indicate their potential role in goose lipid metabolism.3. Study on the effect of LEPR on differentiation and metabolism related genes in goose adipocytesGoose preadipocytes were successfully cultured in vitro. The effect of LEPR on the expression of differentiation and metabolism related genes were studied by using RNA interference (RNAi). The results showed that the expression of goose LEPR, ATGL, LPL, TLR4 and IL-6 gene were increased as the differentiation progress. The maximal expression level of ATGL was at 6 d of culturing. The maximal level of LEPR, LPL and TLR4 were at 8 d. The expression of IL-6 was a little complicated, the mRNA level was high at 4 d, while decreased at 6 d, then increased gradually and reached a maxima value at 10 d. Similar to previous report on chicken, oleic acid could stimulate the differentiation of goose preadipocyte. Oleic acid improved the mRNA level of LEPR, LPL, FAS and PPARy, while reduced the expression of ATGL A effective siRNA for goose LEPR was screened out, the interference effects reached more 60%. Knockdown of LEPR significantly reduced the expression of FAS, PPARy and ATGL, while improved the mRNA level of adiponectin. Moreover, knockdown of LEPR reduced the sensitivity of genes on oleic acid in goose adipocytes, indicating that LEPR might be required in the differentiation stimulated by oleic acid.4. Study on the effect of LEPR on TLR4 and IL-6 in goose adipocytesThe effect of LEPR on the expression of TLR4 and IL-6 was studied by using RNAi. The results showed that the expression levels of TLR4 and IL-6 could be improved by oleic acid and LPS. Knockdown of LEPR didn't change the mRNA levels of TLR4 and IL-6 significantly. However, knockdown of LEPR seemed to reduce the sensitivity of TLR4 and IL-6 on oleic acid/LPS stimulation. These results indicated that the effect of LEPR on the expression of TLR4 and IL-6 was not obvious, while LEPR might be involved in oleic acid/LPS stimulated inflammation signal transduction.5. Study on the effect of rosiglitazone and GW-9662 on genes in goose adipocytesRosiglitazone and GW-9662 were widely studied as PPARy's agonist and antagonist in mammals. In this study, rosiglitazone was proved to be able to promote the expression of LEPR, ATGL, PPARy, FAS and adiponectin in goose adipocytes, while reduced the mRNA level of TLR4 and IL-6. When treated with GW-9662, all eight genes in this study were inhibited. Compared with the control group, treated with rosiglitazone in the LEPR-knockdown cells reduced the expression of PPRγ,ATGL,FAS and LPL, while improved adiponectin expression. Compared with the control group, treated with GW-9662 in the LEPR-knockdown cells reduced the expression of PPARγand FAS,promoted the mRNA level of LPL, adiponectin and IL-6, while had no significant effect on ATGL and TLR4. These results indicated that rosiglitazone and GW-9662 could regulate gene expression in goose adipocytes, and LEPR might be involved in this process.
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