N-acyl Amino Acids Stimulated UCP1-dependent Or UCP1-independent Effect And Mechanism On Adipocytes Thermogenesis

In livestock production,poor insulation conditions in the pig house will increase the risk of piglet diarrhea and other diseases,resulting in reduced survival rates of newborn piglets.On the other hand,reducing backfat accumulation of finishing pigs to improve lean meat rate is also the pursuit of high quality pork in livestock production.Increasing the thermogenic potential of subcutaneous adiposes tissue in piglets,or reducing the backfat accumulation of pigs by activating thermogenesis,is an effective way to achieve these production goals.Studies in mice and humans have found that uncoupling protein 1(UCP1)plays an important role in the thermogenesis of adipose tissue.However,compared with animals such as humans and mice,pigs lack functional UCP1,which may be the reason for the low heat-producing ability of pig subcutaneous adipose tissue.N-Acyl Amino Acids(NAAs)are a novel type of endogenous fatty acids that exist in adipose tissue.Compared with conventional fatty acids(CFAs),they have one more amino acid structure.It is formed by a conventional fatty acids and an amino acids.Related studies proposed that NAAs can be used as uncouplers to directly target mitochondria to induce thermogenic respiration of adipocytes in both UCP1 positive or negative cells.First,cold exposure,which is a classic method to induce thermogenesis and metabolism of fat in mice,was used to treat piglets to explore the thermogenesis and metabolism cahnges of subcutaneous adipose tissue in pigs.We found that acute cold exposure(4°C,10 h)induced the thermogenesis of porcine subcutaneous white adipose tissue.It showed redness tissue color,smaller size of adipocytes(P<0.05),upregulated m RNA and protein expression of browning marker genes.Meanwhile,UCP1 independent thermogenesis-related genes uncoupling protein 3(UCP3)and creatine kinase1(CKMT1)(P<0.05)were induced,also the catalytic enzyme peptidase M20 domain containing 1(PM20D1)was significantly upregulated(P<0.05).These suggests that the thermogenesis metabolism of porcine subcutaneous adipose may be regulated by UCP1-independent thermogenesis pathways.Among them,the NAAs-mediated pathway attracted our interest.To further study the regulation of NAAs on thermogenic respiration,we constructed cell and mitochondrial models differently expressing UCP1,compared two representative groups of NNAs with its corresponding CFAs: Oleate vs NOLeu,Arachidonate vs NAGly,to study their effects and mechanisms on UCP1-dependent and independent respiration by Oroboros.Our results showed that both CFAs and NAAs could induce UCP1-independent respiration in L6 cells which lacks UCP1 expression.Oleate and NOLeu induced equally respiration in L6 cells.While NOLeu(c<100 ?M)had stronger cytotoxicity than oleate(c<60 ?M).Arachidonate induced higher respiration in L6 cells than NAGly,but they had similar cytotoxicity(both,c<60 ?M).To closer to the physiological state of mice,we further isolated mouse primary brown and white adipocytes,and used rosiglitazone to induce different levels of UCP1 expression in them.In UCP1 negative primary adipocytes,both CFAs and NAAs could induce respiration,while CFAs worked better than NAAs.While in beige adipocytes,CFAs induced respiration significantly,but not NAAs.To further explore the respiratory mechanism of CFAs and NAAs in thermogenesis.Our study extracted brown-fat mitochondria from wild-type mice or global UCP1 knockout mice.We found that both CFAs and NAAs could induce respiration in UCP1 positive and negative mitochondria,but mainly based on a UCP1-dependent way which induced by functionally competitive interaction with GDP.To explore the mechanism of UCP1-independent respiration,mouse liver mitochondria that lack of UCP1 was extracted.It is showed that both CFAs and NAAs could induce respiration in liver mitochondria,and the fatty acids induced respirations could been inhibited by carboxyatractyloside(CATR),which fully indicated that CFAs and NAAs mediated UCP1-independent thermogenic respiration through ANT.This study explored the thermogenic response of porcine adipose tissue to cold exposure,compared studied the effects and mechanisms of NAAs,a novel type of endogenous fatty acid,on adipose thermogenic respiration with different UCP1 expressing cell and mitochondrial models.It is found that both CFAs and NAAs can be used as uncouplers to induce thermogenesis of adipocytes.They both mediate UCP1-dependent respiration through competition with GDP,while mediate UCP1-independent respiration through ANT,but they are mainly based on UCP1-dependent pathway.Moreover,NAAs could not works better than CFAs in thermogenic respiratory,or show a specific induction mechanism.In a nutshell,this study not only enhances current understanding of UCP1(in)-dependent thermogenesis in adipocytes,and provides new ideas for the study of metabolism and heat production in porcine adipose tissue,but also provides theoretical basis for obesity and its-related disease studies.