The Interaction Between Ghrelin And Cannabinoid Type Ⅰ Receptor Signaling In Regulating Food Intake In Acipenser Baerii

In this study,Siberian sturgeon was used to investigate the feeding regulation of CB1 and ghrelin.In this study,the nucleotide sequences of CB1,ghrelin and its receptor GHSR of Siberian sturgeon were firstly obtained through molecular cloning;based on these sequences,the m RNA of the three genes of Siberian sturgeon were detected in 22 tissues by real-time fluorescence quantitative PCR method as well as the expression response to feeding;Finally,the feeding regulation of Siberian sturgeon CB1 and ghrelin were investigated by intraperitoneal injection of CB1 antagonists and intracerebroventricular injection of ghrelin and its receptor antagonists.1.Cloning,tissue distribution and feeding response of CB1,ghrelin and its receptor GHSR m RNA in Siberian sturgeon.In this study,the 1428 bp full-length c DNA of CB1 was obtained by molecular cloning which encoding 475 amino acids;the ghrelin c DNA was 345 bp,encoding 114 amino acids;and the 500 bp GHSR c DNA was partially sequenced.The CB1 m RNA of Siberian sturgeon is widely expressed in 21 tissues(22 detected),among which the expression levels in the forebrain,diencephalon,midbrain,cerebellum,medulla,gonad,eyelid,eyes and skin are relative high.Ghrelin m RNA were expressed in all 22 tissues examined,with high expression in the esophagus,stomach,hernia and eye.The GHSR m RNA expression range were small,involving only 13 tissues.The expression levels in the forebrain,diencephalon,midbrain,cerebellum,medulla,and skin were higher.Telencephalon CB1 m RNA levels presented a significant decrease at 1 h and 3 h in the fed groups compared with the unfed groups(P < 0.01).While in diencephalon,only a slight up-regulation of CB1 m RNA was observed at 0 h where fish started turning their direction to feed pellets.No significant differences in telencephalon and diencephalon were detected across feeding regime within unfed groups.In telencephalon,when comparing to the control(fed),CB1 m RNA expression remained higher in food-deprivation fish for 6 days,which was reversed when fast lasted for 10 and 15 days(P < 0.01).Meanwhile,in telencephalon over the fasting period,a gradual down-regulation of CB1 m RNA abundances was apparent within the unfed fish.As to diencephalon,no significant difference was observed between unfed and control groups.The expression of ghrelin m RNA in the forebrain,diencephalon,and stomach of Siberian sturgeon was significantly decreased(P < 0.05 or P < 0.01).There was no significant difference of ghrelin m RNA in midbrain after feeding and in the control group.After feeding for 1 hour and 3 hours,The GHSR m RNA in the forebrain and diencephalon was significantly reduced(P < 0.01),and there was no significant change in GHSR m RNA in the midbrain.These results suggest that transcriptional regulation may also be one of the pathways involved in the regulation of food intake by CB1 signaling;ghrelin and its receptor GHSR participate in the increase of appetite.2.Orexigenic effects of CB1 and ghrelin signaling in Siberian sturgeonIntraperitoneal injection of 5 μg/g bw AM6545 significantly inhibited the food intake of Siberian sturgeon for 1 hour(P < 0.01).The cumulative food intake was significant within5 hours after intraperitoneal injection of 5 μg/g bw AM6545 and 0.5 μg/g bw Rimonant on the first day.The effect continued until the fifth day(P < 0.01).Furthermore,intraperitoneal injection of 5 μg/g bw AM6545 significantly inhibited the expression of ghrelin m RNA in the forebrain and diencephalon(P < 0.05),significantly increased the expression of CART m RNA in the forebrain(P < 0.01),and significantly increased the intraperitoneal injection of 0.5 μg/g bw Rimonabant.The expression of ghrelin m RNA in the forebrain and diencephalon was inhibited(forebrain P < 0.01,diencephalon P < 0.05),while the expression of NPY m RNA in the diencephalon was also significantly inhibited(P < 0.01),and CART m RNA expression in the midbrain was significantly increased(P < 0.05).Intracerebroventricular administration of ghrelin significantly increased the food intake of Siberian sturgeon(P < 0.01),while intracerebroventricular injection of ghrelin receptor antagonist DLys drastically reduced the food intake of Siberian sturgeon(P < 0.01).DLys can also block the increase effect of ghrelin on food intake.Furthermore,the injection of ghrelin into the lateral ventricle significantly reduced the expression of CART m RNA in the forebrain of the Siberian sturgeon(P < 0.01),and significantly increased the expression of NPY m RNA in the forebrain and midbrain(P <0.01).These results show that both CB1 and ghrelin signal induced feeding in Siberian sturgeon.3.The orexigenic effect of ghrelin depends on CB1 signalIntraperitoneal injection of AM6545(CB1 peripheral restrictive antagonist)with lateral ventricle ghrelin significantly increased dietary intake of Siberian sturgeon(P < 0.01)and NPY m RNA in diencephalon and mesencephalon(P < 0.05 or P < 0.01),while significantly decreased mesencephalon ghrelin expression(P < 0.01).Intraperitoneal injection of Rimonabant(CB1 central nonrestrictive antagonist)+ lateral ventricle injection of ghrelin showed no significant changes in food intake of Siberian sturgeon,while decreased NPY m RNA in the forebrain and ghrelin m RNA in the forebrain(P < 0.01).However,mesencephalon NPY m RNA was significantly elevated(P < 0.01).On the other hand,intracerebroventricular injection of CB1 agonist ACEA significantly increased food intake(P < 0.01)and NPY m RNA(P < 0.01)in the midbrain of Siberian sturgeon.However,intracerebroventricular injection of the ghrelin receptor antagonist DLys did not block the increase of ACEA on food intake in Siberian sturgeon(P < 0.01).Diencephalon ghrelin and NPY m RNA as well as midbrain CART and NPY m RNA were significantly increased by the co-injection of DLys and ACEA(P < 0.01).These results indicate that the function of ghrelin signaling in feeding in the central nervous system of Siberian sturgeon depends on the CB1 signal,which may be related to the telencephalon reward-enhanced neural circuit.Based on the above results,we have obtained the following conclusions:Transcriptional regulation may be one of the pathways in which CB1 signal is involved in the regulation of food intake in Siberian sturgeon;CB1 signal of Siberian sturgeon has the effect of inhibiting feeding,while ghrelin signal has the effect of promoting food intake;The role of ghrelin in promoting food intake depends on the central rather than peripheral CB1 signal.