Study On Leptin Receptor Gene Regulatory Pathways And Its Physiological Functions In Chicken

Research on Leptin gene has received much more attention in mammals since its sequence has been reported in mice and human, respectively, in1995. It has been demonstrated that Leptin has multiple functions acting as a hormone in the coordination of energy balance, metabolism and neuroendocrine pathways, and playing a role as a cytokine in the regulation of immune responses. However, research on Leptin in birds has suffered from serious limitions, as there is a controversy that whether Leptin exsited in birds. Some researchers claimed that they have cloned Leptin gene in birds successfully, while others debated that they have failed to reproduce the results. In fact, the Leptin gene nucleotide sequence has not been found in the chicken genome until now.On the other hand, a wide range of studies on chicken Leptin recptor (cLEPR) were carried out, simultaneously. It has been confirmed that cLEPR nucleotide sequence exsited in chicken’s genome, which is expressed in at least two isoforms. In mammals, LEPR plays a crucial role in the Leptin function process, which involved in a variety of regulation pathways. In the case of chicken Leptin, if it was not present in the chicken genome, all studies related to chicken Leptin would have no physiological significance except confirming the existence of cLEPR. It is therefore to question that whether cLEPR is an orphan receptor in chicken. If not, what is the function of cLEPR?. Could it activate the corresponding downstream pathway genes as that in mammals? These questions all need to be answered. This study, therefore, will firstly investigate into the regulation pathways of cLEPR, then analyze its physiological functions, and intend to pave the way for further use of cLEPR in genetics and molecular breeding in birds.The main experiments and results are as followings:1. The first experiment was designed to detect whther Leptin gene exsited in chicken genome. In this experiment,42days old Aiweiyin chicken was used as animals. Tissue samples including muscle, fat, brain and liver, as well as blood sample were collected. Primers used in this study including five pairs derived from the literature by which the authors claimed chicken Leptin gene had been successfully cloned, and a pair designed by us.We did not amplify the sequence of chicken Leptin gene, ultimately.2. The second experiment was designed to analyze expression profiles of cLEPR gene. In this experiment, Real-time PCR was used to detect the expression of cLEPR in tissues of muscle, fat, brain and liver. Results showed that the highest expression of cLEPR was detected in the liver, followed by brain and fat, and then the muscle. cLEPR displayed stable expression, but with relatively lower expression abundance, and only a little difference was observed in different individuals.3. The third experiment was designed to investigate the effect of cLEPR knockdown on adipocyte differentiation and expression levels of its related genes in some regulatory processes. In this experiment, RNAi method was used, firstly. Four pairs of chLEPR shRNA were designed according to the published sequence of cLEPR. Results showed that the four pairs of primers displayed good interfere effect, with shRNA-1the best, showing99%interfere efficiency. Then, Real-time PCR method was applied to detect expression levels of LEPR, JAK2, STAT3, SOCS3, AdipoR2, CPT-1and STK11genes. Results showed that STAT3and SOCS3in JAK, STAT signal transduction pathway were activated with the downregulation of cLEPR, both showing significantly higher expression level. However, the expression levels of other genes involved in this study, except STAT3and SOCS3, showed no significantly different.4. The fouth experiment was designed to detect expression of genes involved in fat regulatory pathways after fasting. In this experiment, forty Aiweiyin chicken were assigned randomly to two groups:the test (n=20) and control group (n=20). The test group was fasted for3days before sampling, while the control group fed ad libitum. Animals were killed on17,23,28,33day-old, respectively (n=5for each age and group), and samples of the left abdominal adipose tissue and hypothalamic were removed to extract RNA. Real-time PCR was used to detect LEPR, CPT-1, STAT3, AdipoR2and NPY gene expression levels. Results indicated that there were no significant changes with the fasting days from17-33, in terms of LEPR, CPT-1, STAT3, AdipoR2and NPY expression levels. CPT-1, AdipoR2and NPY genes displayed stable expression level during the experiment, while STAT3and LEPR gene expression level fluctuated significantly.5. The fifth experiment was designed to produce cLEPR gene knock down chicken by transgene technique. In this experiment, we successfully produce3shLEPR transgenic chickens, named as shLEPR-Chicken1, shLEPR-Chicken2, and shLEPR-Chicken3, respectively. They were detected as chimeras, by three methods, including frozen sections, DNA detection and Western-blot. Phenotyping test showed that the weight of transgenic chicken was significantly higher than controls. Gene pathway analysis results showed that LEPR gene in transgenic individuals was significantly lower than the control group. There were no significant differences observed between the two groups in respect of NPY, CPT-1, STAT3, SOCS3gene expression.In summary, in vitro experiments showed that the downregulation of cLEPR could active STAT3/SOCS3path way, which suggested that the LEPR-STAT3-SOCS3signaling pathway is unimpeded. However, in vivo experiments showed that the STAT3-SOCS3signaling pathway might be influenced by multiple factors. Expression levels of NPY, CPT-1and AdipoR2genes changed as fasting, showing a significantly higher trend in fasting group than that in controls. In addtion, study on shRNA transgenic chicken showed that although the weight of transgenic chicken was significantly higher than controls, regular changes of these genes were not observed. It was indicated that cLEPR could have the appropriate physiological functions. In comparision with mammals, genes in this study may not start from Leptin in their pathways. In other words, in food intake, energy metabolism, and fat deposition, cLEPR did not rely on NPY, CPT-1, AdipoR2. Therefore we concluded that cLEPR have biological functions in chicken, but its regulation pathways is different from that in mammals.