| 5-Hydroxytryptamine(5-HT)is a highly conserved biogenic amine that functions as an important vasoactive substance and as a vital neurotransmitter in the body.5-HT is widely involved in the regulation of biological functions and pathological processes in mammals,such as appetite regulation,lipid metabolism,glucose metabolism,bile acid metabolism,immune regulation,intestinal motility and gut microbiota.However,there are wide descrepancies regarding reports from reseachers on its role in lipid metabolism among poultry and mammals.In this study,animal models and cell models were established to elucidate the role of 5-HT in the mechanism of lipid metabolism in chickens.(1)The gene expression of 5-HT receptor in chickens.The mRNA expression of5-HT2A in different organs(abdominal fat,hypothalamus,liver,breast,thigh,spleen,thymus,bursa of fabricius,duodenum,jejunum and ileum)and at different physiological stages(21 d and 35 d)were studied in broiler chickens.The results showed that the highest mRNA expression of 5-HT2A was found in the abdominal fat,followed by the hypothalamus,thigh,spleen,liver and breast,and the lowest expression was found in the jejunum,duodenum,ileum,thymus and bursa of fabricius.Results at different physiological stages were consistent.These results indicated the possible involvement of 5-HT in lipid metabolism and its important functional role in broiler chickens.(2)The effect of dietary tryptophan(Trp)supplementation on immune factors and5-HT synthesis in broiler chickens.Two groups of 7-day-old chicks were randomly subjected to the following treatments for 3 weeks:1%Trp(a substrate of 5-HT)supplementation and controls.The results showed that Trp supplementation significantly reduced the body weight,feed intake and abdominal fat weight of broilers,up-regulated the gene level of tryptophan hydroxylase(TPH1)(5-HT synthesis rate-limiting enzyme)in the liver,duodenum,jejunum and ileum,and down-regulated the expression of interleukin(IL)-10 in the jejunum and IL-1?and tumor necrosis factor(TNF)-?in the ileum(p<0.05).Therefore,1%Trp supplementation could increase 5-HT synthesis and inhibit the expression of intestinal immune factors in broiler chickens.(3)The effect of dietary 5-hydroxytryptophane(5-HTP)supplementation on lipid metabolism and immune factors in broiler chickens.7-day-old AA broilers were administrated 0.2%5-HTP to study the influence of 5-HT on lipid metabolism and intestinal immune factors after 3 weeks.The results showed that 5-HTP treatment could significantly reduce the abdominal fat deposition of broilers.The genes related to lipid metabolism in the liver and abdominal fat were detected,and only adiponectin(ADP)1R and ADP2R genes expression levels were significantly different(p<0.05).Compared with the control group,5-HTP treatment up-regulated TPH1 gene levels in the liver,duodenum and ileum,down-regulated the mRNA expression of IL-1?,IL-10 and TNF-?in the duodenum,the level of IL-10 in the jejunum and the transcriptional expression of TNF-?in the ileum(p<0.05).The results demonstrated that 5-HTP treatment could increase 5-HT synthesis and inhibit the expression of intestinal immune factors in broiler chickens,suggesting its participation in lipid metabolism,however,the specific mechanism behind its regulation still needed further elucidation.(4)The effect of 5-HT on lipid metabolism in hepatocytes.We establish the model of hepatocytes culture in vitro with the E 18 SPF chicken embryos,which were treated with 10?M 5-HT for 3 h to observe the effects on the lipid metabolism.Similar to adipocytes and myoblasts,5-HT treated hepatocytes had significantly lowered intracellular triglyceride(TG)deposition(p<0.05),and the oil red O staining results were consistent with it.By testing the signal pathway,it was found that the protein level of AMP-activated protein kinase(AMPK)/acetyl-CoA carboxylase(ACC)/carnitine palmitoyl transterase(CPT)1/sterol regulatory element-bingding protein(SREBP)-1 were not affected.Then we detected gene levels,5-HT treatment down-regulated the expression of fatty acid synthetase(FAS),ACC,SREBP-1,CPT1,fatty acid-binding protein 4(FABP4),adipose triacylglyceride lipase(ATGL)and transcription factor CCAAT enhancer binding protein(C/EBP)?,but up-regulated the transcription levels of lipoproteinlipase(LPL)and 5-HT 2A(p<0.05).At the same time,we found that compared with the control group,5-HT treatment significantly reduced FAS and malic enzyme(ME)activity in hepatocytes,increased hepatic lipase(HL)activity,and inhibited the uptake of glucose and fatty acid(p<0.05).It is suggested that the inhibition of TG deposition by 5-HT in hepatocytes is due to reduced fat synthesis and increased lipolysis,thus,not related with oxidative utilization.(5)The effect of 5-HT on lipid metabolism in adipocytes.Unlike in mammals where the liver is the main organ for lipid synthesis,the major site for lipid metabolism in poultry is the adipose tissue which majorly acts as fat storage organs with the skeletal muscle serving as the body's main organ for energy utilization.We established an in vitro model of adipocytes culture using E 16 SPF chicken embryos,which were treated with 10?M 5-HT for 3 h to observe the effects on the lipid metabolism.According to the results,we found that 5-HT treatment significantly reduced TG content after 3 h(p<0.05),and oil red O staining results were consistent with this findings.Upon detection of protein level,it was found that 5-HT stimulated adipocytes can activate AMPK/ACC/CPT1 and extracellular signal-regulated kinase(ERK)signaling pathways while down-regulating ATGL gene level(p<0.05),but had no effect on fat synthase.At the same time,the uptake of glucose and fatty acid were reduced in the 5-HT treated group compared to the control group(p<0.05).After administration of Compound C,an AMPK inhibitor,it showed an inhibitory effect on 5-HT in terms of TG content,gene expression and protein expression.It was observed that 5-HT reduces TG deposition in adipocytes mainly by increasing oxidative utilization and activating the AMPK signaling pathway,which is not related to fat synthesis and lipolysis.(6)The effect of 5-HT on lipid metabolism in myoblasts.We established an in vitro model of myoblasts culture with the E 15 SPF chicken embryos,which were treated with 100?M 5-HT for 3 h to observe the effects on the lipid metabolism.5-HT(100?M)treatment did not affect cell viability.The TG content and oil red O staining results indicated that 5-HT treatment could reduce fat deposition.Compared with the control group,100?M 5-HT treatment activated the AMPK/ACC/CPT1 signaling pathway,up-regulated the transcription levels of AMPK,ACC,CPT1,FAS,LPL,peroxisome proliferator-activated receptors(PPAR)-?,ADP,and 5-HT 2A,and down-regulated ATGL expression(p<0.05).Similarly,5-HT treatment reduced the uptake of glucose(p<0.05)but had no effect on the uptake of fatty acid.After treatment with Compound C,an AMPK inhibitor,the effects of5-HT were alleviated at both the gene and protein levels.This shows that 5-HT reduces the TG content in myoblasts mainly through oxidative utilization,which activated the AMPK signaling pathway and then when we inhibited the AMPK signaling pathway,the 5-HT effect was offset.(7)The effects of gut microbiota on 5-HT synthesis and lipid metabolism in broiler chickens.Four groups of 28-week-old chicks were randomly subjected to the following treatments for 5 weeks:4%L-arabimose(L-Ara),10~8 CUF/kg C.butyricum,antibiotics(500mg/kg salin,500 mg/kg flavomycin,100 mg/kg colistin)and Control,to study the influence of L-Ara,C.butyricum and antibiotics on the changesin gut microbiota,5-HT synthesis and lipid metabolism.The results showed that L-Ara,C.butyricum and antibiotics could alter the intestinal microbiota of broilers,especially the L-Ara group,reducing the abundance of the microbiota by ausing an increase in the abundance of Megamonas and Prevotellaceae UCG-001,while reducing the abundance of Faecalibacterium(p<0.05).By detecting the content of 5-HT in blood,we found that changed intestinal microbiota could influence 5-HT synthesis in broilers(p<0.05),but the results were different in the liver and abdominal fat.The content of 5-HT was reduced in the blood whereas 5-HT expressions were increased in the liver and abdominal fat.According to the results of production performance,it was found that although the antibiotics group had lower body weights,significant steatosis occurred in the liver,which was mainly due to increased liver FAS and ME enzyme activities,up-regulated ACC,ME,LPL,and FABP4 gene levels and down-regulated ATGL transcription levels leading to increased fat synthesis and TG deposition in the liver(p<0.05).Changes at gene levels in abdominal fat were consistent with the liver.In order to determine the localization of enterochromaffin cells(EC cell)in broilers,we examined the expression of its specific protein TPH1 in the intestine.Immunohistochemistry and Western Blot results showed that TPH1 was mainly expressed in the duodenum and cecum,with lower expressions in the jejunum and ileum(p<0.05).Also,treatment with C.butyricum significantly reduced the protein expression of TPH1(p<0.05).With the liver as a site for bile acid synthesis,it was found that the L-Ara group and the antibiotics group significantly reduced the gene expressions of cholesterol 7-alpha hydroxylase(CYP7A1),bile salt export pump(BSEP)and Na+/taurocholate cotransporting polypeptide(NTCP)in the liver,and the transcription level of apical sodium-dependent bile acid transporter(ASBT)in the duodenum and ileum(p<0.05).This results indicate that L-Ara,C.butyricum and antibiotics could alter the microbiota in the intestine,thereby changing the 5-HT content,and further affecting lipid metabolism and bile acid metabolism in broiler chickens.In summary,5-HT is involved in lipid metabolism in chickens,with various tissues having different regulatory mechanisms.This action is carried out within the abdominal fat and muscle tissues mainly via increase in oxidative utilization pathway(AMPK signaling pathway),whereas in the liver it is mainly through the reduction in de novo synthesis of lipid(FAS and ME).Alterations in in intestinal microbiota could regulate 5-HT synthesis,possibly by changing the expression of TPH1. |
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