| Food intake has an important impact on animal production performance.Hypothalamus is the central nervous system to regulate appetite by expressing and secreting the appetite neuropeptide Ag RP/NPY and the appetite neuropeptide POMC.Peripheral nutrients such as glucose,fatty acids and amino acids through the blood-brain barrier into the brain,could affect the expression of hypothalamic neuropeptides,participating in the regulation of food intake and the body's energy homeostasis.So far,the effect of fructose on the proliferation and differentiation of the hypothalamic neural stem cells and the function of food intake regulation is unclear.To investigate the effect of fructose on regulating food intake,this research carried out the following four experiments:firstly,the Landrace pigs at 1 day were chosen and hypothalamic neural stem cells were isolated and cultured in the culture medium with different concentrations of fructose,glucose and galactose as control group.Image-Pro Plus 6.0,ELISA,flow cytometry and Western blot were used to analyze the effects of fructose on proliferation and differentiation of porcine hypothalamic neural stem cells,as well as the expression and secretion of Ag RP and POMC.In order to investigatewhether the inflammation signaling pathway induced by fructose was involved in the regulation of hypothalamic neuropeptidessecretion,then TLR4 competitive inhibitor LPS-RS was cotreated with fructose and then detected the activity change of inflammation signaling pathway.Based on this,m Hypo E-N38 and m Hypo A-POMC were selected to explain the response of two cell lines to glucose and fructose,compared with the results of primary cell culture.Finally,verifing the effect of fructose on animal food intake and energy metabolism in vivo,C57 BL / 6J mice were treated with 10% glucose and fructose by drinking water to detect the effect of different hexose treatments energy intake and metabolism.The results are as follows:(1)The addition of 5 mM fructose in the culture medium significantly promoted the proliferation of porcine hypothalamic neural stem cells,but glucose had no significant effect on cell proliferation;meanwhile1 mM galactose significantly inhibited cell proliferation.Besides 5 mM fructose couldsignificantly promote the secretion of Ag RP not POMC.Oppositely 5 mM glucose significantly increased the secretion of POMC,but had no effect on the secretion of Ag RP;addition of galactose could not impact the secretion of Ag RP and POMC in porcine hypothalamic neural stem cells.These results suggested that three differenthexoseshad different effects on theproliferation and neuropeptides secretion of porcine hypothalamic neural stem cells.(2)The effect of glucose and fructose on cell proliferation was further analyzed by flow cytometry and morphological observation.It was found that treatment with 5 mM fructose significantly promoted the growth of neurospheres,increasing the proportion of S phase cells,decreasing the proportion of G0 / G1 phase cells,then significantly promoted the cell proliferation index(PI).(3)5 mM fructose could significantly activate TLR4-mediated JNK / IKK?/?signaling pathway.The effect of fructose and LPS-RS co-treatment on the proliferation and Ag RP secretion of hypothalamic neural stem cells was significantly reversed(P <0.05),but there was no significant effect on TLR4 expression,which indicated that TLR4 signaling pathway involved in the regulation of fructose on the proliferation and neuropeptide secretion of porcine hypothalamic neural stem cells.(4)The following studies showed that 5 mM fructose could significantly promote the expression and secretion of Ag RP in N38 cells.The effect of fructose on the activation of JNK / IKK?/?pathway and the promotion of Ag RP secretion in N38 cells was significantly reversed by LPS-RS(P <0.05).However,the results of m Hypo A-POMC cells showed that both glucose and fructose could significantly promote the expression of POMC(P <0.05),but had on effect on POMC secretion.Treated with LPS-RS and fructose,the secretion of POMC and the expression of My D88/ IKK?/?/IKK?/?,not the expression of TLR4 and p-JNK / JNK,was significantly lower than fructose-treated alone.It was suggested that the response of m Hypo E-N38 and m Hypo A-POMC cell line to glucose and fructose was different,and the result was different fromthe porcine primary cell.(5)In vivo,the resultsshowed that addition of 10% glucose to drinking water significantly increased the water consumptionin mice,suggesting that the degree of preference for glucose was greater than fructose.Meanwhile the total energy intake and body fat mass were significantly higher than that of fructose group,but 10% fructose supplemented with drinking water could significantly increase the body weight and energy conversion rate,especially in cold and fasting-refeeding stress,fructose could significantly improve the basic metabolism level of mice.In summary,we isolated the porcine hypothalamic neural stem cells and found that 5 mM fructose could activate the TLR4-mediated inflammatory signaling pathway,thereby promoting the proliferation and differentiation of porcine hypothalamic neural stem cells,increasing the expression and secretion of Ag RP.In the hypothalamic cell lines of mice,we found that 5 mM fructose could significantly promote the expression and secretion of Ag RP in N38 cells.For primary cells,there were different responses of cell proliferation and neuropeptide secretion to three different hexoses.Fructose and glucose also had different effects on neuropeptide secretion in two different cell lines of mice.In vivo,mice strongly preferred to drinking water added with 10% glucose to fructose,but their body weight gain and energy conversion were significantly lower than those in fructose treatment group.The above results provided a basis for theapplication potential of monosaccharides to regulate animal food intake and energy metabolism. |
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