| Kisspeptin-10,-14, and-13 are the split products of kisspeptin-54 encoded by Kiss-1 gene, they are the endogenous ligands of G-protein-coupled receptor, GPR54. The amino acids sequence and the function of kisspeptin-10 is highly conserved among different species. Till now, the data about the function of kisspeptin in birds is really scarce. Only two references reported that the positive signal for kisspeptin has been identified in ducks, and have the ability of regulating the reproductive axis (hypothalamus-pituitary-gonads, HPG) hormones secretion. Sexual maturity in birds is also regulated by the hypothalamic hormone in reproductive axis. Generally, the first egg of birds is considered as the signal of complement of puberty and sexual maturity. Estrogen is the key hormone in the downstreem of the reproductive axis, regulating the reproductive central secretory activity through the feedback system. In female birds, estrogen secreted from ovarian follicles stimulates the hepatic lipids synthesis to meet the demands of yolk formation during the onset and maintenance of egg laying. In hens, the process of reproductive initiation is a process of energy enriching. The data showed that lipid content in each egg is more than ten percent of egg weight. Liver is the main organ for lipid metabolism in birds. Therefore, the aim of this study was to investigate the effects of kisspeptin on sexual maturation and lipid metabolism in liver of Japanese female quails, and to elucidate the direct effect of kisspeptin on hepatic lipids metabolism in vitro.1 Effect of kisspeptin-10 on the lipid metabolism in female quails75 female quails (22 days of age) were subjected to 0 (control, con),0.1 nmol (low dosage, L) and 1.0 nmol (high dosage, H) Kp-10 administration by intraperitoneal injection daily. The experimental time has been lasted for 3 weeks. Kp-10 was disolved in saline and the total volume for injection was 300 ?L. The information of egg laying was recorded daily. At 60 days of age, quails were sacrificed and samples including serum and liver were collected for analysis. Serum concentrations of estradiol (E2), triglyceride (TG), total cholesterol (Tch), high density lipoprotein-cholesterol (HLD-C), low density lipoprotein-cholesterol (LDL-C), non-esterified fatty acid (NEFA), glucose, apoprotein B (ApoB) and apoprotein A (ApoA) were measured by biochemical methods. The expression of genes related to lipids metabolism in liver was detected with real-time RT-PCR, including fatty acid synthestase (FAS), acetyl coenzyme A carboxylase a (ACCa), stearyl coenzyme A dehydrogenase 1 (SCD1), malic enzyme (ME), sterol regulatory element-binding proteins 1 (SREBP-1), sterol regulatory element-binding proteins 2 (SREBP-2),3-hydroxyl-3-methyl glutaryl-coenzyme A reductases (HMGCR), cholesterol 7a-hydroxylase (CYP7A1), apoproteins:ApoVLDL-?, ApoB, ApoA1,fatty acid binding protein 2 (FABP2) and vitellogenin ? (VTG-?), estrogen receptor ? (ERa)?estrogen receptor ? (ERp), and microRNAs (miRNAs) of SREBP-1. SREBP-1, SREBP-2 and ERa protein content were measured with western blot. The results showed that Kp-10 injections with low as well as high dosage significantly increased the egg laying rate (P< 0.05 and P < 0.01, respectively) indicating that Kp-10 consecutive injection advanced the onset of egg laying in Japanese quails. Serum E2 concentrations were increased by Kp-10 treatment on dose dependent manner and reached the statistically significance in H group of quails (P< 0.05). Compared to the control group, the concentrations of Tch and HDL-C in serum of quails in H group was significantly decreased (P< 0.05), while the contents of TG and Tch in liver significantly increased in H group (P< 0.05 and P< 0.01, respectively). Real-time PCR results showed that the expression of SREBP-1, FAS, ApoVLDL-II, VTG-II and CYP7A1 genes in liver was significantly up-regulated by high but not low dosage of Kp-10 treatment (P< 0.05). However, others genes such as ME, SREBP-2, ApoA1, FABP mRNAs expression was not significantly changed by Kp-10 injection (P> 0.05). Western blot showed that hepatic SREBP-1 and ERa protein contents significantly increased in H group compared to the control group (P< 0.01 and P< 0.05, respectively), while there was no significant change of SREBP-2 protein expression in liver of quails (P> 0.05). In conclusion, exogenous kisspeptin-10 injection can advance egg laying and E2 secretion, paralleled a higher ability of hepatic lipid synthese and transporting through circulating system, which might be associated with up-regulation in genes expression and proteins translation in liver.2 Effect of kispeptin-10 on lipid metabolism in hepatocytes primary cultured in vitro isolated from chicken embryo14-day-old Hailan chicken embryos were sacrificed and the liver was collected. The hepatocytes were isolated and purificated, cultured in the serum culture-medium for 24 h, and then cultured in serum-free medium (contain 0.1% insulin-transferrin-Se, ITS). After 24 hours stabilization, the hepatocytes were treated with 100 nmol/L and 1000 nmol/L Kp-10, respectively. The supernatant and the cells were collected for analysis. Immunocytochemistry and immunofluorescence were used to identify the expression of kisspeptin in hepatocyte cultured in vitro. The cell viability was measured by MTT method. Hepatic TG, Tch, HDL-C and LDL-C contents were determined by biochemical methods. Real-time PCR were used to measure the expression of genes involved in lipids metabolism. The SREBP-1 protein content was detected by western blot analysis. The results showed that cells viability was not affected by 100 nmol/L Kp-10 treated for 24 h, but showed a mild decrease at 1000 nmol/L concentration. Therefore, the next further analysis was conducted at 100 nmol/L but not 1000 nmol/L concentration. The results showed that total cholesterol (Tch) contents were increased by 51.23% with Kp-10 treatment, but did not reach the statistical significance (P> 0.05), while the level of TG, HDL-C and low density of LDL-C in hepatocytes were significantly increased by Kp-10 treatment (P< 0.05 and P < 0.01, respectively). Real-time PCR results showed that the expression of SREBP-1, ACCa, Carnitine palmitoyltransferase 1 (CPT1), HMGCR and SCD1 mRNA in hepatocytes were significantly down-regulated (P< 0.05), FAS and ApoAl genes expression tended to decrease (P= 0.08 and 0.075, respectively), while the expression of ApoVLDL-II mRNA tended to increase (P= 0.072). On the contrary, western blot showed that the expression of SREBP-1 protein in hepatocytes was significantly increased by 100 nmol/L Kp-10 treatment (P< 0.05). The expressions of miRNAs targeting SREBP-1 gene were measured by real-time PCR. Compared to the control group, among 5 miRNAs gga-miRNA-100 and gga-miRNA-183 were greatly up-regulated in hepatocytes with 100 nmol/L Kp-10 treatment (P< 0.01) whereas no significant changes were observed in other 3 miRNAs (P> 0.05). It indicates that Kp-10 has the direct effect of stimulating lipids synthesis in primary cultured hepatocytes of chickens. |
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