SCFAs/GLP-1 Links The Regulation Of Gut Microbiome On Lipid Metabolism In Broiler Chickens

Gut microbiota can produce SCFAs and affect lipid metabolism.However,the role of gut microbiome and SCFAs in lipid metabolism is not clear.In the present study,by changing the composition of gut microbiota with probiotics,the contents of SCFAs and its impact on lipid metabolism were analyzed.Intestinal epithelial cells were cultured and treated with SCFAs,then followed with primary hepatocytes and adipocytes experiments,to explore the regulation mechanism of SCFAs-mediated GLP-1 on lipid metabolism.Relative expression of FFAR2,FFAR3 and GLP-1R in broiler chickensRelative expression of FFAR2,FFAR3 and GLP-1R in intestinal tract,liver and abdominal adipose tissue were analyzed.Results showed that the m RNA expression level of FFA2 and FFAR3 increased from duodenum,jejunum,ileum,cecum to rectum in AA broilers.In addition,the m RNA expression of target genes could also been detected in liver and adipose tissue.The highest expression of GLP-1R was found in adipose tissue.The transcript expression level of GLP-1R is consistent with that of FFARs.Effects of gut microbiome on SCFAs compsotion and lipid metabolism in broiler chickensCombined probiotics(Clostridium butyrate 4 × 105 cfu/g,bifidobacterium 2 × 105 cfu/g,Lactobacillus plantarum 2 × 105 cfu/g,Lactococcus faecalis 2 × 105 cfu/g)and compound antibiotics(ampicillin 1.0 kg/T,neomycin 0.5 kg/T)were used to change to the composition of gut microbiome,broiler chickens were fed with conventional feed.Results showed that both probiotics and antibiotics treatments significantly altered the community compositon of gut microbiota.Both of them significantly increased the abundance of Firmicutes and decreased the abundance of Bacteroides and Proteus at the level of phylum.At the phylum level,both probiotics and antibiotics increased the abundance of Firmicutes and decrease the abundance of Bacteroidetes and Proteobacteria microorganism.Compared with the control group,results showed that probiotics significantly increased the contents of acetate(p<0.001)and butyrate(p<0.001)and decrease the content of propionate(p<0.001)at d 21.The proportion of butyric acid in total SCFAs was significantly increased(p<0.001)while the ratio of propionic acid was decreased(p<0.001)at d 21 in the group of probiotics.Compared with the control group,except the content of acetate had a trend of increasing in caecum of antibiotics group on d 21,the content of SCFAs in the caecum decreased at the days of 14 and 42.The ratio of propionate significantly decreased at d 14 in the antibiotic group(p< 0.01),while the ratio of acetate had an increasing trend at the days of 14,21 and 42.The butyrate ratio had none difference with that of the control group.Results showed that probiotics significantly decreased the abdominal fat index at 21d(p<0.05).The abdominal fat index of antibiotics group was not consistant at d 21 and 42.Probiotics could significantly increase the expression of FFAR2,FFAR3 and GLP-1R at the m RNA level in colonrectum(p<0.001).The expression of FFAR2 and FFAR3 in colon and rectum were decreased in antibiotics group.Results suggest that SCFAs contents and FFARs expresson can be affected by the gut microbiome.Alteration of gut microbiome affects the lipid metabolism of broiler chickens.Compared to antibiotics,probiotics is more suitable to be used to study the impact of gut microbiome on SCFAs and lipid metabolism.Regulation of gut microbiome on lipid metabolism of HFD broilersIn order to further study the effect of gut microbial community composition on lipid metabolism,high fat diet(HFD)was used to feed broiler chickens.The experiment was divided into three groups: control group(fed HFD basal diet),probiotics group(HFD supplemented with Clostridium butyrate 4×105 cfu/g,bifidobacterium 2×105 cfu/g,Lactobacillus plantarum 2×105 cfu/g,Lactococcus faecalis 2×105 cfu/g)and guar gum(HFD supplemented with 0.1% guar gum)group.Results showed that supplementation of probiotics and guar gum significantly decreased body weight gain(p<0.05)at d 42,the abdominal fat index of probiotics and guar gum groups was significantly lower than that of control group(p<0.05).The morphological analysis of liver and abdominal adipose tissue with HE staining showed that both probiotics and guar gum could decrease the fat deposition.Both probiotics and guar gum reduced the vacuolar necrosis and lipid infiltration of liver cells,and decrease the size of adipocytes in adipose tissue.The physiological and biochemical parameters in VII plasma and liver tissue showed that,compared with the control group,probiotics and guar gum significantly decreased plasma TG concentration(p<0.01),liver TG content(p<0.05)and plasma ALT activity(p<0.001).These results further indicated that probiotics and guar gum had obvious protective effect on liver injury induced by HFD.Through analyzing key genes associated with lipid genesis,it showed that both probiotics and guar gum reduced the expression of fatty acid synthase(FAS),acetyl co A carboxylase(ACC),peroxidase proliferative activator receptor-?(PPARG),sterol regulatory element binding protein(SREBP-1c)in liver and adipose tissue at the m RNA expression level.This further indicated the down regulated effect of lipid synthesis in both probiotics and guar gum groups.Analysis of microbiota in cecum showed that both probiotics and guar gum could increase the relative abundance of Actinobacteia microorganism,and increase the abundance of Bifidobacterium,Campylobacter and Lactobacillus in cecum.The microbial abundance of Proteobacteria and Verrucomicrobia in cecum of HFD broiler was decreased.Further analysis of intestinal SCFAs concentration and relative content showed that both probiotics and guar gum could reduce the ratio of propionate and increase the ratio of butyrate.Probiotics significantly decreased the ratio of propionic acid in cecum(p<0.05)and guar gum significantly increased the ratio of butyric acid(p<0.05).The results showed that the effects of probiotics and guar gum on the gut microbiome and SCFAs were consistent.It suggested that the alteration of SCFAs caused by probiotics play an important role in the regulation of lipid metabolism.Further study showed that,with higher SCFAs content in the gut,the expression of FFAR2,FFAR3 and GLP-1R in ileum,cecum and rectum,especially in rectum were increased both in probiotics and guar gum groups.Signal transduction of SCFAs in the intestinal epithelial cellsThe results showed that all three SCFAs could significantly increase the m RNA expression level of FFAR2(p<0.01)and FFAR3(p<0.001)in IECs,increase the secretion of GLP-1 and improved the expression of GLP-1R in IECs.Western blotting results showed that all three SCFAs significantly activated ERK and p38 MAPK signaling pathways in IECs,the activation of JNK pathway was weaker compared with that of ERK and p38 MAPK.Blocked the MAPK signaling pathway with special inhibitors,the secretion of GLP-1 mediated by acetate,propionate and butyrate was completely inhibited under the ERK and p38 MAPK inhibitors(p<0.01).The effect of JNK inhibitor on the release of GLP-1 was relatively weaker.These results suggested that the SCFAs mediated GLP-1 secretion is more related to ERK and p38 MAPK signaling pathways.Effects of GLP-1 and its analogue Liraglutide on lipid metabolism in hepatocytesIn order to investigate the effect of GLP-1 on lipid metabolism of hepatocytes,primary hepatocytes were treated with 100 n M GLP-1 in vitro.It showed that the content of Oil Red O and TG(p<0.01)were reduced under the treatment with GLP-1.Western blotting analysis showed that GLP-1 could significantly increase the phosphorylation level of AMPK(p<0.01),as well as the phosphorylation level of its downstream target gene ACC(p<0.01).GLP-1 analogue Liraglutide also significantly increased the level of p-AMPK/AMPK(p<0.001)and the phosphorylation of ACC(p<0.001),but Liraglutide treatment did not affect the protein expression level of PPARG and CPT1 in hepatocytes.At the transcriptional level,Liraglutide significantly activated GLP-1R(p<0.01),reduced the transcript expression of FABP4(p<0.05)and SREBP-1c(p<0.001).After GLP-1R was blocked with its special inhibitor Exendin(9-39),the TG content in hepatocytes was restored compared with the control and Liraglutide treatment(p<0.01).Take together,it showed that GLP-1 and its analogue,Liraglutide,can activate GLP-1R,and increase the phosphorylation level of AMPK,then induce the phosphorylation of ACC,thus reduce the synthesis of fatty acids and lipid deposition in hepatocytes.Effects of GLP-1 analogue Liraglutide on lipid metabolism in adipocytesTo study the effect of liraglutide on lipid deposition in preadipocytes,cells were treated with 100 n M liraglutide.Results of Oil Red O staining and TG content showed that the treatment with 100 n M Liraglutide did not decrease lipid deposition in adipocytes,but increased lipid deposition.TG content in adipocytes was significantly increased(p<0.05).During the differentiation of preadipocytes,no obvious changes of AMPK and ACC were detected.Therefore,the effect of liraglutide on lipid metabolism in preadipocytes needs further study.In order to study the effect of liraglutide on lipid deposition in differentiated adipocytes,cells were treated with 100 nm liraglutide.The results of Oil Red O staining showed that liraglutide significantly reduced lipid content and lipid droplet size in adipocytes.Compared with the control group,liraglutide significantly decreased the content of TG in differentiated adipocytes(p<0.01).Western blotting analysis showed that the level of intracellular p-AMPK(p<0.05)was significantly increased with liraglutide treatment for 4 hrs,and the level of intracellular p-ACC(p<0.05)was significantly increased at 2 hrs and could sustain to 8hrs under the liraglutide treatment.Take together,the results showed that Liraglutide could reduce lipid deposition in mature adipocytes.In conclusion,the present study showes that the alteration of gut microbiome can affect the contents and ratios of SCFAs in the intestine,SCFAs can mediate the secretion of GLP-1 through MAPK pathway in the intestinal epithelial L-Cells.GLP-1 and its anologue Liraglide can activate AMPK and Inactivate ACC,and then reduce lipid de novo synthesis in hepatocytes and differentiated adipocytes.