Effects Of Refeeding And β-hydroxybutyrate Infusion On Lipid Metabolism In Starved Yak

Starvation is the main factor to adversely affect the production performance and body health of grazing yaks in the cold season on the Qinghai-Tibet plateau.As the starvation time prolonged,the blood glucose level decreases,then the liver and muscle glycogen,lipids and proteins degrade to meet the basic life activity requirement of animals.Lipid is the most important energy source of starving animals,which provided energy for the extrahepatic tissues through the oxidation of ketones in the liver,such as β-hydroxybutyric acid(BHBA).The BHBA is the main ketone in animals.In recent years,BHBA was confirmed as a signal molecular to regulate the body metabolism in addition to being an energy substance.Therefore,this experiment investigated the effects of refeeding and BHBA intravenous infusion on lipid metabolism of starved yaks through establishing a starving yak animal model and using qPCR,high-throughput sequencing and metabolomics techniques,so as to elucidate the adaptive changes of rumen microorganisms and fat metabolism of yaks in response to starvation stress and reveal the influencing mechanism.Exp.1 Establishment of starved yak models and evaluation of refeeding influencesIn this experiment,12 healthy male yaks with the similar age and weight were selected and randomly divided into the normal feeding group and the starvation group.The yaks in normal feeding group were fed ad libitum.The yaks in the starvation group were starved.Yaks were weighed before starvation and on the day 7 of starvation.blood and rumen fluid samples were collected to evaluate the starved yak model.After the successful establishment of starved yak model,all yaks were fed normally,and the effect of refeeding after starvation was evaluated.The results were shown as following:(1)After starvation,the average weight loss of yaks was 13.95 kg.The daily gain of yaks was significantly reduced.The concentrations of serum glucose(GLU)and triglyceride(TG)were significantly decreased(P < 0.05).Non-esterified fatty acids(NEFA)and β-hydroxybutyrate(BHBA)concentrations significantly increased(P < 0.05).The starved yak model was successfully established.(2)Starvation significantly reduced the index of diversity of rumen microbial(P<0.05),increased the ratio of Firmicutes to Bacteroidetes and decreased the abundance of Prevotella 1,Succiniclasticum and Ruminococcaceae NK4A214 group(P<0.05).The concentration of acetate,propionate,butyrate,total volatile fatty acids,ammonia nitrogen(NH3-N)and microbial protein(MCP)in rumen decreased significantly(P<0.05),suggesting rumen fermentation weakened.(3)After starvation,the activity of fatty acid synthase(FAS)in yak serum decreased significantly(P<0.05),while the activity of acyl coenzyme A oxidase(ACOX)and hormone sensitive esterase(HSL)increased significantly(P<0.05),suggesting the lipid catabolism promoted.(4)After 7 days of refeeding,the daily gain was 376.32 % higher than that of the normal feeding group(P<0.05).The apparent digestibility of crude protein(CP),crude fat(EE)and acid detergent fiber(ADF)in starvation group during the refeeding period was significantly higher than those of the normal feeding group(P<0.05).The ratio of feed to weight(F/G)was 78.00 % lower than that in the normal feeding group(P<0.05),suggesting that the compensatory growth effect was obvious.(5)After refeeding,the rumen microbial diversity index Chao I of yak increased significantly,but it was still significantly lower than that before starvation(P<0.05).The relative abundance of Prevotella 1 and Succinivibrionaceae UCG-002 increased significantly(P<0.05).Euryarchaeota decreased significantly(P<0.05).The concentrations of acetate,total volatile fatty acids and NH3-N and the ratio of acetate to propionate returned to the level of before starvation.The concentration of propionate and MCP after refeeding were significantly higher than before starvation(P<0.05).(6)After refeeding,the activities of FAS,ACOX and HSL in yak serum returned to the levels that were not significantly different from those in the normal feeding group.And the concentration of NEFA and BHBA in serum were significantly lower than those in starvation period(P < 0.05).These results suggested that lipid catabolism was weaker than starvation period.The above results showed that the weight of yaks decreased after starvation,the diversity and abundance of rumen microorganisms decreased and rumen fermentation weakened.The serum GLU concentration decreased significantly,lipid catabolism increased and BHBA increased significantly.After refeeding,the microbial abundance and quantity in rumen increased,rumen fermentation increased,lipid catabolism decreased and the concentration of BHBA in serum decreased significantly.The ADG of yaks in starvation group increased significantly,and compensatory growth of yak was promoted.Exp.2 Effect of starvation and refeeding on serum and urine metabolomics of yakOn the basis of experiment 1,the metabolome of serum and urine of yaks in the experimental group before starvation(normal feeding period,NFP),starvation period(SP)and refeeding period(RFP)were detected by LC-MS.The results were shown as following:(1)Compared with NFP,starvation significantly increased the concentration of serum free saturated fatty acids such as 3-hydroxydecanoic acid,lauric acid and tetradecanoic acid(P<0.05),and significantly decreased the concentration of glycerol 3-phosphate,pyruvate,aconitic acid and citric acid(P<0.05),significantly increased the concentration of branched-chain amino acids.The TCA pathway was inhibited and the catabolism of glycerophospholipids and glycerides was enhanced.(2)Compared with NFP,starvation significantly decreased the concentration of free fatty acids in urine(P<0.05),significantly increased the concentration of BHBA,acetone,oleic amide and choline glycerophosphate in urine(P<0.05),and significantly increased the concentration of isoprenaline and cyclic adenylate(cAMP)in urine(P<0.05).The concentration of lactate,succinic acid and aconitic acid,which are related to the gluconeogenesis and TCA cycle,were reduced.The synthesis pathway of ketone bodies was enhanced and the TCA cycle pathway was weakened.(3)Compared with the starvation period,the concentrations of free saturated fatty acids in yak serum was significantly decreased(P<0.05),and the concentrations of glycerol 3-phosphate,pyruvate,aconitic acid and citric acid significantly increased(P<0.05).The TCA pathway was enhanced and the catabolism of glycerides and glycerophospholipids was inhibited.(4)Compared with starvation period,the concentration of BHBA,acetone,glycerol phosphocholine and Decanoyl carnitine in urine were significantly decreased(P<0.05),the concentration of free fatty acids such as suberic acid,dodecenoic acid,pimelic acid,arachidonic acid and dodecanedioic acid were significantly increased(P<0.05).The concentration of free amino acids in urine was significantly decreased(P<0.05).The concentration of lactate,succinic acid and aconitic acid in urine was increased,but the concentration of 3-phosphoglyceric acid,phenylpyruvic acid and gluconic acid were significantly decreased(P<0.05).The concentration of isoprenaline and cAMP were significantly decreased(P<0.05).The anabolism of ketones was inhibited and the TCA pathway was enhanced.(5)Compared with the normal feeding period,the concentrations of glyceride,linoleic acid,tetradecanoic acid and 3-phosphate glycerol in serum were significantly decreased(P<0.05),and the concentration of citric acid was significantly increased(P<0.05).(6)Compared with the normal feeding period,the concentration of unsaturated fatty acids and their derivatives in urine increased significantly(P<0.05).The concentration of free amino acids such as phenylalanine and proline decreased significantly(P<0.05).The concentration of Phenylpyruvate and gluconic acid decreased significantly(P<0.05).The utilization rate of nitrogen metabolism in the refeeding period was higher than that in the normal feeding period.The above results showed that after starvation,the oxidative decomposition of saturated fatty acids increased,the production of ketone bodies such as BHBA increased,and TCA pathway decreased.After refeeding,the lipid decomposition was weakened,ketone body such as BHBA biosynthesis was inhibited.Exp.3 Effects of intravenous infusion of β-hydroxybutyrate on lipid metabolism of starved yakIn this experiment,18 healthy Jiulong male yaks with similar age and weight were randomly divided into normal feeding group(NG),starvation group(SG)and BHBA infusion group(SBG).The yaks in normal feeding group yaks were fed ad libitum,and the yaks in starvation group and the BHBA infusion group were treated with starvation during the whole period.The 48-hours intravenous perfusion was conducted from the 7th day of the experiment.The NG and the SG were infusion with 0.9 % saline,and the SBG was infusion with BHBA solution of 1.7mmol /L.The results were shown as following:(1)After starvation,the concentration of GLU in serum of yak was significantly decreased on the 3rd day of starvation,while the concentrations of TG and TC were significantly reduced(P < 0.05),and the concentrations of BHBA and NEFA were significantly increased on the 1st and 3rd day of starvation,respectively,to maintain a high level of stability(P < 0.05).BHBA infusion significantly increased the concentration of BHBA in yak serum(P<0.05),significantly decreased the concentration of NEFA(P<0.05),and increased the concentration of serum TG by 25.00 % compared with starvation group.(2)After starvation,the concentrations of insulin(INS),growth hormone(GH)and adiponectin(APN)in serum were significantly decreased(P<0.05),and concentrations of glucagon(GC)and cortisol(Cor)were significantly increased(P<0.05).Infusion of BHBA significantly suppresses the elevated of the GC concentrations induced by starvation(P<0.05),significantly increased the INS and APN concentrations(P<0.05),but Infusion of BHBA had no significant effect on the GH and IGF-Ⅰ(P>0.05).(3)Both starvation and BHBA infusion significantly reduced the proportion of saturated fatty acids(SFA)in yak subcutaneous adipose tissue(P<0.05),and significantly increased the proportion of monounsaturated fatty acids(MUFA)and polyunsaturated fatty acids(PUFA)(P<0.05).Starvation significantly reduced the diameter and area of fat cells in the subcutaneous adipose tissue of yaks(P<0.05).The diameter and area of fat cells after perfusion of BHBA were significantly higher than those in the starvation group(P<0.05),which was still significantly lower than those in the normal feeding group(P <0.05).(4)Starvation significantly reduced the activities of ACC,DGAT1 and FAS in subcutaneous adipose tissue(P<0.05),and significantly increased the activities of ACOX and HSL(P<0.05).BHBA infusion significantly increased the activities of ACC and DGAT1 in starvation yaks(P<0.05),decreased the activities of ACOX and HSL(P<0.05),and the lipid catabolism was weakened.(5)Starvation significantly reduced the expression of m RNA and protein of C/EBP,SREBP1 and PPARγ in the subcutaneous adipose tissue of yaks(P < 0.05),and significantly increased the m RNA and protein expression levels of FOXO1(P < 0.05).BHBA infusion significantly increased the expression of C/EBPa,SREBP 1 and PPARγ in subcutaneous adipose tissue(P<0.05).The expression of FOXO1 was significantly decreased(P<0.05).The lipid anabolism was promoted and lipid catabolism was inhibited.(6)Starvation significantly increased the expression of PKA and CREB in subcutaneous adipose tissue and decreased the expression of PKC and ERK1/2(P<0.05).After BHBA infusion,the expression of GPR109 A was significantly increased(P<0.05),the expression of AC and PKA was inhibited,and the phosphorylation level of CREB was down-regulated,but the expression of MEK,PKC and ERK1/2 was not affected(P>0.05).The above results showed that starvation significantly reduced the concentration of hormones such as INS,GH and APN in yak serum,up-regulated the expression of key factors in the cAMP/PKA/CREB signaling pathway in subcutaneous adipose tissue,down-regulated the expression of key factors in the MEK/PKC/ERK1/2 signaling pathway,promoted lipid catabolism,and significantly increased the concentration of BHBA in serum.Further increasing the concentration of BHBA can promote the expression of the receptor GPR109 A of BHBA,inhibit the cAMP/PKA/CREB signaling pathway,and inhibit lipid decomposition.In conclusion,starvation resulted to the decrease of body weight,rumen microbial diversity and abundance,ability of rumen fermentation,and the serum concentration of GLU.Starvation also significantly increased the concentrations of serum NEFA and BHBA,suggesting a stronger lipid catabolism.With refeeding,the abundance of Prevotella 1 and Succinivibrionaceae UCG 002 were increased,the abundance of Euryarchaeota was decreased,rumen fermentation was enhanced,the concentration of NEFA and BHBA in serum were significantly reduced,and lipid catabolism was weakened.During starvation period,yaks supplied energy by oxidizing saturated fatty acids,increased the catabolism of glycerol phospholipids and glycerides,inhibited the tricarboxylic acid cycle and enhanced ketone synthesis.After refeeding,the catabolism of glycerophospholipids and glycerides were decreased,the activity of TCA pathway was increased.starvation promoted catabolism and increase BHBA concentration of yaks by reducing serum concentrations of INS,GH and APN and increasing concentrations of isoproterenol and other hormones,promoting cAMP/PKA/CREB signaling pathway,inhibiting MEK/PKC/ erk1/2 signaling pathway.The further increasing of serum BHBA concentration could increase serum APN concentration,reduce serum NEFA concentration,up-regulate the m RNA and protein expression of the BHBA receptor GPR109 A in subcutaneous adipose tissue,inhibit the downstream cAMP/PKA/CREB signaling pathway and inhibit lipid decomposition,but it had no effect on the MEK/PKC/ ERK1/2 signaling pathway.