Effect Of Alpha Lipoic Acid Supplementation On Lipid Metabolism Of Broilers Induced By High Fat And Its Mechanism

Alpha lipoic acid(ALA)is a short-chain fatty acid containing 8 carbon atoms,which is easily absorbed by cells due to its lipid solubility and water solubility.As a cofactor of key enzymes in glucose metabolism,it is mainly involved in acyl group transfer in substance metabolism of the body,so it plays a variety of biological functions such as regulating fat metabolism and anti-oxidation in the body.However,the current studies on the biological functions of ALA are mainly focused on rodents,and there are relatively few reports on the regulation of ALA in lipid metabolism of poultry,especially on the regulation of ALA in metabolic disorders induced by high-fat and high-energy diet in broilers.Therefore,in this study,metabolic disorders induced by high fat diet in Ross 308 broilers were studied to clarify the regulatory effects of dietary ALA supplementation on lipid metabolism disorders in the body.Meanwhile,a lipid metabolism disorder model induced by free fatty acids was established to explore the cellular biological mechanism of ALA activating adenosine 5’-monophosphate-activated protein kinase(AMPK)signaling pathway in improving lipid metabolism disorders induced by high fat,focusing on lipid droplet deposition and triglyceride.The aim of this study was to lay a theoretical foundation for the application of ALA as a physiological regulator in promoting healthy broiler breeding.1 Evaluation of dietary α-lipoic acid on safety of broilers induced by high fat dietThis part of the experiment was conducted to investigate the effects of α-lipoic acid(ALA)supplementation on the safety of 21-day-old Ross 308 broilers induced by high fat.Broilers were divided into control group(ND group),high fat diet group(HFD group)and ALA+ high fat diet groups with different doses of ALA,and each treatment group had 9replicates with 3 broilers per replicate.ND group was fed basal diet.HFD group and different doses of ALA+ high fat diet groups were fed with high fat diet supplemented with0 mg/kg,50 mg/kg,100 mg/kg and 150 mg/kg ALA.The experiment lasted for 21 days.Serum and liver samples were collected and tested in the 42 nd day.The number of white and red blood cells were measured by blood cell counting.The cell morphology was observed by blood smear.Serum biochemical indexes related to liver function and kidney function of poultry were detected by colorimetry.Hematoxylin-eosin staining was used to observe the steatosis of liver tissue.The results showed that ALA treatment did not affect the blood routine and blood cell morphology of broilers.At the same time,compared with the control group,ALA treatment did not affect the content of alanine transaminase,aspartate transaminase,total protein,albumin,gamma glutamyltranspeptidase,alkaline phosphatase,total bile acid,total bilirubin,direct bilirubin,lactate dehydrogenase,uric acid,creatinine,urea nitrogen(P > 0.05).ALA treatment of 50-150 mg/kg significantly alleviated liver steatosis induced by high fat in broilers.These results suggested that the ALA dose used in this study was safe for Ross 308 broilers induced by high fat and could improve liver steatosis induced by high fat.2 Effect of α-lipoic acid supplementation on lipid metabolism of broilers induced by high fat dietTo investigate the effect of α-lipoic acid(ALA)treatment on abnormal lipid metabolism of broilers fed high fat diet,21-day-old Ross 308 broilers induced by high fat diet were used as experimental subjects in this study.Treatment and sample collection of broilers were the same as Chapter 1.The contents of triglyceride(TG),total cholesterol(TC),high density lipoprotein(HDL-C),low density lipoprotein(LDL-C),free fatty acid(NEFA)and glucose(GLU)in serum or liver tissues were determined by colorimetry.Real-time q PCR was used to determine the m RNA expression levels of lipid metabolism-related factors in liver tissues.The results showed as follows: high fat diet treatment significantly increased the abdominal fat percentage of broilers(P < 0.05).Adding ALA significantly decreased the abdominal fat weight and abdominal fat percentage of broilers induced by high fat diet(P < 0.01),and significantly decreased the contents of TG,TC and LDL-C in serum(P < 0.05).Meanwhile,high fat diet significantly increased the ACC and ACLY m RNA levels(P < 0.05)while significantly decreased the ATGL,CPT-1,PPARα and LPL m RNA levels in liver(P < 0.05).Different doses of ALA significantly down-regulated the ACC(P < 0.05)and ACLY(P < 0.01)m RNA levels,and up-regulated ATGL m RNA level(P < 0.01)in liver.50 mg/kg ALA significantly increased the expression level of PPARα m RNA(P < 0.05),and extremely significantly increased the expression level of LPL m RNA(P < 0.01)in liver.In addition,compared with the control group,high fat diet significantly decreased LPL and ATGL m RNA levels in adipose tissue(P < 0.05)while different doses of ALA significantly increased the expression level of LPL m RNA in adipose tissue of broilers(P < 0.05).These results suggested that ALA could promote the expression of genes related to fat decomposition and inhibited the expression of genes related to fat synthesis in the liver of broilers induced by high fat,and ultimately reduced the serum lipid content and fat deposition in broilers.3 Effect of α-lipoic acid on lipid metabolism of primary chicken hepatocytes induced by free fatty acids and its mechanismFor further discussion of the effect of alpha lipoic acid(ALA)on fat metabolism of broilers induced by high fat,primary chicken hepatocytes were isolated and cultured.The model of free fatty acids(FFA,oleic acid: palmitic acid = 2:1)inducing hepatocellular fatty degeneration was established,discussing the effect of different doses of ALA treatment on it.Furthermore,the AMPK inhibitor Compound C was used to pretreat chicken primary hepatocytes to reveal the mechanism of ALA regulating hepatocyte steatosis.MTT assay was used to detect cell viability.Nero red staining was used to detect the accumulation of lipid droplets.The contents of triglyceride(TG)and total cholesterol(TC)were determined by colorimetry.Real-time q PCR was used to detect the m RNA expression levels of lipid metabolism-related factors.The results showed that compared with the control group,FFA treatment did not affect the TC content in primary chicken hepatocytes,but significantly increased the TG content(P < 0.01).Compared with FFA group,50-150 μmol/L ALA treatment significantly decreased intracellular TG and TC contents(P < 0.05).At the same time,compared with the control group,FFA treatment significantly up-regulated the ACC,FAS and ACLY m RNA levels of primary chicken hepatocytes(P < 0.05).However,compared with FFA group,ALA treatment did not affect ACC,FAS and ACLY m RNA levels in primary chicken hepatocytes.Compared with the control group,FFA treatment did not affect ATGL and CPT-1 m RNA levels in primary chicken hepatocytes.Compared with FFA group,ALA significantly increased ATGL and CPT-1 m RNA levels in primary chicken hepatocytes(P < 0.05).In addition,compared with FFA group,ALA significantly increased AMPKα1 and AMPKα2 m RNA levels in primary chicken hepatocytes(P < 0.01).Compound C significantly reversed the effect of ALA on AMPKα1,AMPKα2,ATGL and CPT-1 m RNA levels in primary chicken hepatocytes.The results suggested that ALA treatment increased the expression of adiposis-related factors in primary chicken hepatocytes induced by FFA by activating AMPK signaling,and ultimately reduced the abnormal deposition of fat in hepatocytes.