Effects Of Dehydroepiandrosterone On Lipid Metabolism Disorder And Insulin Resistance In High Fat Fed Rats And Its Mechanisms

The production performance of livestock and poultry has greatly improved under the current developing tendency of intensive and large-scale farming.However,over-emphasis on the growth rates of livestock and poultry are likely to increase the incidence of metabolic diseases,which lead to a series of problems such as low feed utilization,excessive body fat deposition and meat quality degradation.It has a serious impact on the economic benefits of breeding industry;More importantly,consumers who eating low-quality meat products will increase the risk of obesity and other metabolic diseases.Since many livestock and poultry farming problems are closely related to the metabolic imbalances,solving metabolic disorders are the key to ensure the healthy livestock and poultry farming.Therefore,there is an urgent need for bioactive substance that can be used to alleviate the metabolic disorders to ensure the healthy framing of livestock and poultry.Dehydroepiandrosterone(DHEA)is one of the important metabolic intermediates produced in the cholesterol metabolism process.It has been attracted much attention due to its effects on regulating the glucose and lipid metabolism,improving the immunity capability,enhancing the antioxidant function,and promoting the healthy development of animals,etc.A large number of clinical studies have shown that the decrease of DHEA content with aging is one of the main factors leading to metabolic diseases.Therefore,in this experiment,high fat fed rats were used as research subject to investigate the effects of DHEA on lipid metabolism disorders and insulin resistance.The mechanisms were further clarified in the Palmitic acid(PA)induced BRL-3 A cell.The study aims to reveal the specific signaling pathway involved in DHEA regulating lipid metabolism disorder and insulin resistance;The results will provide theoretical basis for searching bioactive substances,which can be used to relieve metabolic disorders and promote the healthy livestock and poultry framing.Simultaneously,the results also have important guiding significance that using DHEA as a nutritional supplement to treat obesity and prevent other metabolic diseases.1 Effect of DHEA regulating lipid metabolism disorder in High Fat Fed RatsIn this study,high fat fed Sprague-Dawley(SD)rats were used to investigate the effects of DHEA on rats’ body weight gain,lipid deposition and mitochondrial functions,etc.The research aims to reveal the mechanisms of DHEA on lipid metabolism disorders in high fat fed rats.Thirty SD rats were randomly divided into three groups:normal diet group,high fat diet group and high fat diet with DHEA treatment group.Each test group was fed with 0 or 50 mg/kg of DHEA(dissolved in 1%dimethyl sulfoxide)by gavage one time a day for ten weeks.The rats were slaughtered to collect serum,liver,and visceral fat.Oil red O staining was used to observe lipid droplets in the liver;Biochemical indicators of lipid metabolism and mitochondrial function were determined by kits;RT-PCR was used to analyze mitochondrial DNA(mtDNA)copy number;Real-time PCR and Western Blot were used to determine the key factors expression of lipid metabolism and mitochondrial metabolism functions.The results showed that compared with the high fat diet group,DHEA treatment significantly reduced the body weight gain and visceral fat deposition in high fat fed rats(P<0.05).DHEA treatment significantly reduced the contents of triglyceride(TG),total cholesterol(TC),free fatty acid(FFA)in serum and liver(P<0.05),and significantly reduced the number of lipid droplets in the liver(P<0.01).DHEA treatment significantly reduced the mRNA expression levels of fatty acid synthase(FAS)and sterol-regulatory element binding proteins(SREBP-1c),while the mRNA expression levels of peroxidase proliferationactivated receptor alpha(PPARα)and carnitine palmitoyl transferase 1(CPT-1)were significantly increased(P<0.05).In addition,DHEA treatment also alleviate the decrease ofβ-oxidation ability,mitochondrial DNA(mtDNA)copy number and Complex I activity induced by high fat diet(P<0.05).Western Blot results showed that DHEA treatment significantly increased the protein expression level of phospho-AMP-activated protein kinase(p-AMPK),phospho-Acetyl-CoA carboxylase(p-ACC),Sirtuin(SIRT1),nuclear respiration factor 1(NRF-1),mitochondrial transcription factor A(TFAM)and Complex I protein expression levels(P<0.05).Moreover,the phosphorylation and deacetylation levels of peroxisome proliferator-activated receptor gamma coactivator 1α(PGC-1α)were also significantly increased compared with high fat diet group(P<0.05).All results above indicated that DHEA treatment alleviate lipid metabolism disorders induced by high fat diet in rats through activating the AMPK-PGC-1α-NRF-1 signaling pathway.2 Effect of DHEA alleviating insulin resistance in High Fat Fed RatsIn this study,high-fat fed Sprague-Dawley(SD)rats were used to investigate the effects of DHEA on glucose content,insulin level and insulin receptor substrate(IRS)expression,etc.The research aimed to reveal the mechanisms of DHEA on insulin resistance in rats induced by high fat diet.Thirty SD rats were randomly divided into three groups:normal diet group,high fat diet group and high fat diet with DHEA treatment group.Each test group was fed with 0 or 50 mg/kg of DHEA(dissolved in 1%dimethyl sulfoxide)by gavage one time a day for ten weeks.Six rats were randomly selected from each group for glucose resistance test and insulin tolerance test.Rats were slaughtered to collect serum and liver.Glucose and insulin contents were determined by kits and then evaluated the HOMR-IR;Western Blot was used to detect the key protein expression levels of Insulin signaling pathway.Results showed that compared with the normal diet group,the glucose content and insulin level were significantly increased in high fat diet group(P<0.01).The glucose tolerance and insulin tolerance test also showed that the ability to recover blood glucose contents to normal physiological concentration in the high fat diet group were significantly lower than the normal diet group(P<0.01).Compared with the high fat diet group,DHEA treatment reduced the glucose content and insulin level in high fat fed rats(P<0.05).In addition,DHEA also significantly improved the ability of recovering normal blood glucose concentration in high fat fed rats(P<0.01).Moreover,Western Blot results showed that DHEA significantly reduced the protein expression levels of p-IRS1 S307(P<0.05),and significantly increased the protein expression levels of p-IRS1 Y612,p-AKT and GLUT2(P<0.01).All results above indicated that DHEA could alleviate the insulin resistance induced by high fat diet in rats,and these effects might be achieved by the activation of the IRS1AKT-GLUT2 signaling pathway to promote the transport of glucose.3 Mechanism of DHEA regulating lipid metabolism disorder and mitochondria oxidative damage in PA induced BRL-3A cellIn this study,Palmitic acid(PA)induced BRL-3A cells were used to investigate the effects of DHEA on triglyceride(TG)content,reactive oxygen species(ROS)level,mitochondrial membrane potential(MMP)and mitochondrial DNA(mtDNA)copy number,etc.The research aimed to reveal the mechanisms of DHEA on lipid metabolism disorder and mitochondria oxidative damage in PA induced BRL-3A cell.BRL-3A cells were pretreated with different concentrations of DHEA for 4 h,then added 0.2 mM PA to continue incubating cells for 20 h.Oil red O staining was used to observe lipid droplets in the cells;TG content,Complex I activity and ATP content were determined by kits;Reactive oxygen species(ROS)content and mitochondrial membrane potential(MMP)were analyzed by flow cytometry;RT-PCR was used to evaluate mitochondrial DNA(mtDNA)copy number;The crucial protein expression levels of AMPK-PGC-1α-NRF-1 signaling pathway were determined by Western Blot.The results showed that compared with the PA alone treatment group,100 nM and 1000 nM DHEA treatment significantly reduced the lipid deposition and ROS level in PA induced BRL-3A cells(P<0.05).100 nM and 1000 nM DHEA treatment significantly increased mitochondrial membrane potential,mtDNA copy number and ATP content compared with the PA alone treatment group(P<0.05);Moreover,100 nM and 1000 nM DHEA treatment significantly increased the expression levels of phospho-AMP dependent protein kinase(p-AMPK),phospho-Acetyl-CoA carboxylase(p-ACC)and sirtuin(SIRT1)protein expression levels(P<0.05).In addition,the protein expression levels of phosphorylation and deacetylation of peroxisome proliferator-activated receptor y coactivator la(PGC-la)were significantly increased after DHEA treatment compared with PA alone treatment group(P<0.05).However,the effects of DHEA on the up-regulation expressions of p-ACC,SIRT1 and phosphorylation or deacetylation of PGC-1α were completely reversed after pretreating BRL-3A cells with Compound C(AMPK inhibitor)(P>0.05).In addition,PGC-la siRNA blocked the up-regulation of nuclear respiration factor 1(NRF-1)protein expression induced by DHEA(P>0.05).NRF-1 siRNA also blocked the up-regulation of mitochondrial transcription factor A(TFAM)protein expression induced by DHEA while the Complex I protein expression level and activity were no significant difference compared with the PA alone treatment group(P>0.05).All results above showed that DHEA alleviating PA-induced lipid metabolism disorder and mitochondrial oxidative damage in BRL-3 A cells by activating the AMPK-PGC-1α-NRF-1 signaling pathway.4 Mechanism of DHEA alleviating insulin resistance in PA induced BRL-3A cellIn this study,Palmitic acid(PA)induced BRL-3A cells were used to investigate the effects of DHEA on the insulin resistance.The research aimed to reveal the mechanisms of DHEA on insulin resistance in PA induced BRL-3A cell.BRL-3A cells were pretreated with different concentrations of DHEA for 4 h,then added 0.2 mM PA to continue incubating cells for 20 h.The glucose uptake capacity of BRL-3A cells were determined by kits;The crucial protein expression levels of insulin signaling pathway were detected by Western Blot.Results showed that compared with the PA alone treatment group,100 nM and 1000 nM DHEA treatment significantly increased the glucose uptake capacity in PA induced BRL-3 A cells(P<0.05).100 nM and 1000 nM DHEA treatment significantly reduced p-IRS1 S307 protein expression level(P<0.01),while significantly increased p-IRS1 Y612,p-AKT and glucose transporter protein 2(GLUT2)protein expression levels(P<0.01).However,the effects of DHEA on up-regulating p-AKT and GLUT2 expressions were reversed by the pre-treatment of LY294002(PI3K inhibitor)(P>0.05).All results above indicated that DHEA alleviated insulin resistance via promoting glucose uptake in PA induced BRL-3A cells.These effects are realized by the activation of the IRS1-AKT-GLUT2 signaling pathway.