Study On The Effect And Mechanism Of(-)-Hydroxycitric Acid On Glycolipid Metabolism In Broilers Based On AMPK Signaling

(-)-Hydroxycitric acid[(-)-HCA]is a main natural organic acid in the rind of the fruit Garcinia cambogia,and exhibits many potential biological functions,such as inhibiting fat synthesis,reducing cholesterol content,attenuating inflammation,anti-oxidation and increasing energy consumption in mammals.However,the mechanism of its biological effects is still clear.Previous studies in poultry in our laboratory showed that(-)-HCA inhibited the deposition of abdominal fat by affecting the expression of fat metabolism related factors in poultry;and studies in embryonic broilers illustrated that the mechanism of(-)-HCA inhibiting the deposition of poultry fat may be related to its effect on glucose metabolism in the body,but the specific biochemical mechanism is not clear.Therefore,Ross 308 broiler and primary chicken hepatocytes were chosen as the research object in this study.On the basis of discussing the effect of(-)-HCA on the metabolism of glucose,fat and energy in poultry,labfree technology were used to clarify the key effect factors of(-)-HCA regulating the metabolism of glucose,fat and energy in primary chicken hepatocytes and the main metabolic pathway involved,and the metabolic disorder model of primary chicken hepatocytes induced by oleic acid(OA)was used to further elucidate the exact biochemical mechanism of(-)-HCA regulating the metabolic homeostasis of poultry.The purpose of this study is to provide scientific basis and theoretical basis for the study of physiological function of natural and safe metabolic intermediate(-)-HCA and its application in livestock and poultry production.1 Effects of(-)-hydroxycitric acid on the glucose and fat metabolism in broilers and its biochemical mechanismTo investigate the regulation and mechanism of(-)-HCA on glucose and fat metabolism in Ross 308 broilers.21-day-old Ross 308 broilers were randomly divided into control group and diet supplemented with 1000 mg/kg(low),2000 mg/kg(medium),3000 mg/kg(high)(-)-HCA treatment group(three duplicates of ten broilers per group).After feeding to 49 d,serum and liver samples were taken and tested.The contents of glycogen,triglyceride(TG),blood glucose and the activities of phosphofructokinase-1(PFK-1),pyruvate dehydrogenase(PDH),succinate dehydrogenase(SDH)and malate dehydrogenase(MDH)were measured by colorimetry;the mRNA expression of adiponectin receptor(AdipoR),ATP-citrate lyase(ACLY),acetyl-CoA carboxylase(ACC),fatty acid synthetase(FAS),glycogen synthetase(GS)and glycogen phosphorylase(GP)were detected by quantitative real-time PCR;the protein expression of fatty acid synthesis related factors(ACC?,p-ACC?,FAS),glucose catabolism related factors(PFKL,PDH,SDHA and complex ?),AMP-dependent protein kinase(AMPK?),p-AMPK?,peroxisome proliferator activated receptor y coactivator 1?(PGC-1?),nuclear respiratory factor 1(NRF-1)and mitochondrial transcription factor A(TFAM)were analyzed by Western blot.The current results showed that(-)-HCA decreased the accumulation of lipid droplets and TG content by reducing fatty acid synthase protein level and enhancing phosphorylation of acetyl-CoA carboxylase protein level.(-)-HCA accelerated carbohydrate aerobic metabolisms by increasing the activities of phosphofructokinase-1,pyruvate dehydrogenase,succinate dehydrogenase,and malate dehydrogenase.Furthermore,(-)-HCA increased AdipoR1 mRNA level and enhanced phospho-AMPK?,peroxisome proliferator-activated receptor gamma coactivator-1?,nuclear respiratory factor-1,and mitochondrial transcription factor A protein levels in broiler chickens.These results suggested that(-)-HCA treatment could accelerate energy metabolism by activating the AMPK signal pathway,and ultimately lead to the increasing of glucose decomposition and the decreasing of fat accumulation in broilers.2 Effects of(-)-hydroxycitric acid on the metabolism of glucose,fat and energy in primary chicken hepatocytesBased on the isolation and culture of primary chicken hepatocytes,the present study was designed to investigate the effects of(-)-HCA on glcucose,lipid and energy metabolism related factors and its mechanism in primary chicken hepatocytes.The primary chicken hepatocytes were treated with 0,1,10 and 50 ?mol·L-1(-)-HCA for 1,3,6,12 or 24 h,then the cells or supernatant were collected.The cell viability and death rate were detected by MTT and LDH assay,respectively;the glycogen and TG content,glucose consumption,and the activities of SDH and MDH were measured by colorimetry;the mRNA expression of ACLY,ACC,FAS,sterol regulatory element binding protein 1c(SREBP-1c),carnitine palmitoyl transferase I(CPT-I)and peroxisome proliferator activated receptor ?(PPAR?)were detected by quantitative real-time PCR;the protein contents of glucose kinase(GK),PFK-1,pyruvate kinase(PK),PDH,citrate synthetase(CS),aconitase(ACO),GS,GP,phosphoenolpyruvate carboxykinase(PEPCK),ACLY,NADH dehydrogenase,ATP synthetase and acetyl-CoA were detected by ELISA.In this study,(-)-HCA treatment significantly decreased ACLY,FAS and SREBP-1c mRNA levels and markedly increased PPARa mRNA level,resulting in the inhibition of TG content.Meanwhile,(-)-HCA treatment significantly decreased ACLY activity and acetyl-CoA content in cytosol,but significantly increased glucose consumption.In addition,(-)-HCA treatment promoted the activities/contents of GK,PFK-1,PK,PDH,CS,ACO,SDH,MDH,NADH dehydrogenase and ATP synthase remarkably.These results suggested that(-)-HCA reduced the supply of acetyl-CoA,the precursor of fatty acid synthesis in the cytoplasm,which mainly achieved via inhibiting the ACLY and enhancing oxidative phosphorylation level,and finally inhibited the accumulation of fat in primary chicken hepatocytes.3 Study on the signaling transduction mechanisms of(-)-hydroxycitric acid regulating the metabolism of glucose,fat and energy in primary chicken hepatocytesIn this study,the AMPK pathway key factor inhibitor or RNA interference technology were used to explore the exact signal transduction mechanism of(-)-HCA regulating glucose,fat and energy metabolism in primary chicken hepatocytes.The primary chicken hepatocytes were treated with 0,1,10 and 50 ?mol·L-1(-)-HCA for 24 h or pretreated with the indicated activator,inhibitor or interference vector of AMPK pathway,and then treated with 0 or 10 ?mol-L·1(-)-HCA for 24 h to collect the cells and tested.Triglyceride(TG)content and glucose consumption were measured by colorimetry;the protein expression of fatty acids synthesis metabolism such as ACC?,p-ACC?,FAS and SREBP-1,the expression of glucose and energy metabolism key proteins such as PFKL,PDH,SDH,Complex ?,AMPK?,p-AMPK?,PGC-1a and NRF-1 were detected by Western blot.The results showed that(-)-HCA obviously decreased TG content through inhibiting FAS protein level,and enhancing the protein level of p-ACC?/ACC? in hepatocytes.Meanwhile,(-)-HC A markedly enhanced the protein level of PFK-1,PDH,SDHA and Complex ?,and which led to the enhancing of glucose uptake and catabolism in hepatocytes.In addition,the regulation of(-)-HCA on these key factors associated with lipid and glucose metabolism in hepatocytes was mainly achieved through activation of AMPK-PGC-1?-NRF-1 signaling pathway.These results suggested that(-)-HCA enhanced the oxidative decomposition and oxidative phosphorylation of glucose by activation of AMPK-PGC-1?-NRF-1 signaling pathway,and then accelerated the complete oxidation decomposition of acetyl-CoA to inhibit the synthesis of fatty acids.4 Screening of effector proteins of(-)-hydroxycitric acid regulating the metabolism of glucose,fat and energy in primary chicken hepatocytesIn this study,proteomics was used to screen the expression profile of protein factors after(-)-HCA treatment,and bioinformatics was used to determine the key effect factors and main metabolic pathways of(-)-HCA on the regulation of glucose,fat and energy metabolism in primary chicken hepatocytes.The results showed that core differentially expressed proteins were screened out after 10 ?mol·L-1(-)-HCA treatment in primary chicken hepatocytes.Bioinformatics analysis showed that core differentially expressed proteins were mainly involved in promoting the decomposition and transportion of fat(up-regulated expression of ApoA1,APOA4 and APOB),increasing the uptake of glucose,glycolysis and oxidative phosphorylation of hepatocytes(up-regulated expression of SLC2A2,ATP1A1 and ATP6V0C,down-regulated expression of PDK1,ALDH8A1,etc.),enhancing the immune function of hepatocytes(up-regulated expression of TF,UBB,etc.),regulating the degradation of hepatocyte proteins(up-regulated expression of CUL1,CUL3,CUL4B,CUL5,down-regulated expression of UBE2N,PSMC1,etc.),participating in the main biological processes such as mRNA synthesis and nucleation(up-regulated expression of CDC42,SEH1L,down-regulated expression of UBE2N,COPS5),as well as regulating the protein expression of glycolipid metabolism key signal factor PKA subunit(up-regulated expression of PRKACB and PRKAR2A).These results suggested that(-)-HCA mainly affected the biological effects of glucose,fat and energy metabolism,protein synthesis and degradation,immune function in primary chicken hepatocytes by regulating the mRNA synthesis and protein modification of key effectors,and its regulation of glucose,fat and energy metabolism might be related to the activation of key factors related to PKA.5 Study on the regulation of(-)-hydroxycitric acid on oleic acid-induced metabolism disorder and its signaling transduction mechanisms in primary chicken hepatocytesIn this study,the lipid metabolism disorder model of primary chicken hepatocytes induced by oleic acid(OA)was taken as the research object,in order to further explore the regulation and mechanism of(-)-HCA on the metabolism of homeostasis and inflammation.The primary chicken hepatocytes were treated with 0 or 10 ?mol·L-1(-)-HCA for 4 h and then incubated with 0.6 mmol·L-1 OA for 24 h;meanwhile,the primary chicken hepatocytes were pretreated with the key factor activator,inhibitor or RNA interference vector of AMPK pathway,then treated with 0 or 10 ?mol·L-1(-)-HCA for 4 h and then added with 0.6 mmol·L-1 OA for another 24 h,the cells were collected and tested.Oil red O and Nile red staining were used to detect lipid droplet accumulation;the malondialdehyde(MDA)and TG levels were detected by colorimetry;cellular reactive oxygen species(ROS)content,cell apoptosis rate and mitochondrial membrane potential were detected by flow cytometry;the mRNA expression of inflammation and apoptosis related factors were detected by quantitative real-time PCR;Western blot was used to detect the protein expression of lipid metabolism,apoptosis,inflammation and pathways.The results showed that(-)-HCA reduced FAS and SREBP-1 protein expression,and increased the protein expression of CPT-I,enoyl COA hydratase 1(ECHS1)by activating AMPK signaling,then inhibited OA-induced accumulation of lipid droplets and alleviated lipid metabolism disorder of primary chicken hepatocytes.Meanwhile,(-)-HCA reduced OA-induced excessive accumulation of ROS and alleviated mitochondrial dysfunction caused by fat metabolism disorder through the activation of AMPK-PGC-1?-NRF-1-TFAM signaling pathway in primary chicken hepatocytes.(-)-HCA reduced the expression of apoptogenic factor Bax,caspase 3,caspase-9 and cytochrome c,and increased anti-apoptotic factor Bcl-2 expression and mitochondrial membrane potential,eventually inhibiting OA-induced oxidative damage in primary chicken hepatocytes.In addition,(-)-HCA prevented the nuclear translocation of NF-?B in primary chicken hepatocytes induced by OA,reduced the release of iNOS and COX-2,and finally reduced the inflammatory effect caused by lipid metabolism disorder,which was related to the inhibition of ROS mediated the activation of p38 MAPK signaling pathway.These results suggested that(-)-HCA alleviated lipid metabolism disorder and mitochondrial dysfunction of OA-induced primary hepatocytes by activating AMPK-PGC-1?-NRF-1 pathway,and subsequently achieved anti-oxidation and anti-inflammatory effect by reducing ROS overproduction and inhibiting the activation of p38 MAPK signaling pathway.