| Skeletal muscle and adipose tissue are the principal sites for glucose and fatty acid utilization,playing an important role in regulating lipid metabolism and glucose homeostasis.Cumulative evidences have shown that the development of fetal and postnatal skeletal muscle and adipose tissue is closely associated with maternal nutrition and physiological status.Although maternal energy intake during gestation and lactation plays an essential role in offspring growth and development,the underlying mechanisms remain pooly defined.Resveratrol(RES),a natural polyphenolic compound commonly found in fruits and plants,has been shown to promote myogenesis and inhibit white adipogenesis.However,there is little information available about the effect of maternal RES supplementation on skeletal muscle and adipose tissue development in offspring.In our studies,we firstly investigated the effects of maternal dietary energy intake during gestation on skeletal muscle development characteristics of the embryo and fetuses.Then we evaluated further the effects of resveratrol supplementation of maternal high-energy diet on postnatal offspring skeletal muscle and adipose tissue development,energy metabolism and adaptive thermogenesis.Three experiments are included in this study:Experimental 1.Effects of breed and maternal dietary energy intake on fetal myofiber development in pigs.This experiment aimed to evaluate the breed differences of pig fetal myofiber development and the effects of moderate increase of maternal energy intake from day 1 to 90 of gestation on skeletal muscle development in pig embryo and fetuses.Thirty-six primiparous purebred Large White sows(135.54 ± 0.66 kg)were allocated randomly at mating to a high energy intake group(HE,5.36 Meal NE/d from day 1 to 35 of gestation;6.43 Meal NE/d from day 36 to 90 of gestation)or normal energy intake group following NRC(2012)recommendation for gestating sows(CON,4.65 Meal NE/d from day 1 to 35 of gestation;5.58 Meal NE/d from day 36 to 90 of gestation).36 primiparous purebred Meishan sows(72.84±0.66 kg)were randomly assigned at mating to a high energy intake group(CON,4.65 Meal NE/d from day 1 to 35 of gestation;5.58 Meal NE/d from day 36 to 90 of gestation)or normal energy intake group to mimic the energy levels in traditional diets for the Chinese indigenous pig breed(LE,3.96 Meal NE/d from day 1 to 35 of gestation;4.75 Meal NE/d from day 36 to90 of gestation).After a 12-h overnigh fast on day 35,55 and 90 of gestation,pregnant sows were weighed and four sows per diet group were anaesthetized.Umbilical vein blood was collected from each fetuse on day 55 and 90 of gestation.Four embryo or fetuses with weight close to the average level were selected from each sow at 3 gestational age for longissimus dorsi muscle(LM)sample collection.The results were presented as follows:(1)The mRNA levels of paired box gene 7(Pax7),myogenin,myogenic factor 5(Myf5),muscle regulatory factor 4(MRF4),insulin-like growth factor 1(IGF-1)and IGF-1 receptor(IGF-1R)were increased in LM of Large White pig embryo and fetuses relative to Meishan pig on both days 35 and 90 of gestation(P<0.05).The mRNA levels of peroxisome proliferator activated receptor(PPAR?),embryonic myosin heavy chain(MyHC-embryonic),MyHC-perinatal,MyHC-I,MyHC-?a,MyHC-I? and MyHC-?b were higher in LW embryo than those in MS embryo on day 35 of gestation(P<0.05).Compared to the MS fetuses,the mRNA levels of myogenic differentiation factor 1(MyoDl),myogenin,Desmin,muscle creatine kinase(CKM),growth hormone receptor(GHR),IGF-1,IGF-1R,PPAR? and MyHC-perinatal were lower in LM fetuses only on day 55 of gestation,whereas the opposite were observed for mRNA levels of MyHC-?a and MyHC-?b(P<0.05).The body weight and the mRNA levels of adipocyte fatty-acid-binding protein 4(aP2),MyHC-embryonic,MyHC-perinatal,siruin 1(Sirt1),nuclear respiratory factor 1(NRF-1)and mitochondrial transcription factor A(Tfam)of LW fetuses were higher than those of MS fetuses on day 90 of gestation(P<0.05),whereras the umbilical vein serum T3 concentrations,the activities of lactate dehydrogenase(LDH)and isocitrate dehydrogenase(ICDH)and the concentrations of NAD+and NADH were decreased in Large White pig fetuses(P<0.01).(2)No differences were found for the body weight of Large White pig embryo and fetuses between CON group and HE group.Compared to the CON group,LW sows weight gain was higher in HE group on both days 55 and 90 of gestation(P<0.05).The mRNA levels of MyHC-embryonic,MyHC-perinatal,MyHC-I and MyHC-?a mRNA were decreased in LM of HE embryo relative to NE embryo on day 35 of gestaton(P<0.05).The muscle protein concentrations and CK activity of HE fetuses were lower than those of CON fetuses on both days 55 and 90 of gestation(P<0.05).Compared to CON fetuses,the mRNA levels of Pax7,MyoDl,Myogenin,Myf5,MRF4,CKM,IGF-1 and insulin-like growth factor binding protein 5(IGFBP5)were decreased in HE embryo and fetuses on both days 35 and 90 of gestation,and the mRNA levels of MyoDl and MRF4 and MyoD1 protein expression were decreased only on day 55 of gestation(P<0.05).The umbilical vein serum T3 concentrations,LDH activity,mtDNA content,the ratio of NAD+ to NADH and the mRNA levels of MyHC-?a,MyHC-?x,PPAR y-coactivator a(PGC-1 ?),Sirtl,NRF-1,Tfam and H-ATP synthase catalytic subunit(?-F1-ATPae),as well as the protein expression of MyoD1,myogenin and Fast-MyHC in HE fetuses were lower than those of CON fetuses on day 90 of gestation(P<0.05).No differences were found for the adipogenic genes expression between CON group and HE group.(3)Compared to the LE group,Meishan sows weight gain was higher in high energy intake group on day 90 of gestation(P<0.05).Fetal weights were higher in high energy intake group than in LE group on both days 55 and 90 of gestation(P<0.05).Compared to the LE embryo,the mRNA levels of Pax7,MyoD1,myogenin,Myf5,IGF-1,IGFBP5,MyHC-embryonic,MyHC-perinatal,MyHC-I,MyHC-?a,MyHC-IIx and MyHC-IIb were decreased in embryo of high energy intake group on day 35 of gestation(P<0.05),whereas the opposite was observed for mRNA levels of myogenin,CKM and IGF-1R on day 55 of gestation(P<0.05).On both days 55 and 90 of gestation,the umbilical vein serum T3 concentrations,muscle protein concentrations,CK activity and myogenin protein expression in fetuses of high energy intake group were higher than those of LE fetuses(P<0.05).Compared to the LE fetuses,the umbilical vein serum T4 concentrations,muscle DNA concentrations,activities of ICDH and CS,mtDNA content,ratio of NAD+ to NADH and the mRNA levels of Pax7,MyoDl,Myogenin,Myf5,MRF4,CKM,GHR,IGF-1,PPARy,aP2,MyHC-embryonic,MyHC-perinatal,MyHC-IIa PGC-1?,Sirtl,NRF-1,Tfam,?-F1-ATPae and cytochrome C(Cyt C),as well as fast-MyHC protein expression were increased in fetuses of high energy intake group on day 90 of gestation(P<0.05),whereras the opposite was observed for mRNA levels of MyHC-I.The results indicated that Large White and Meishan pigs exhibit significant differences in prenatal gene expression profiles of skeletal muscle development at different developmentalstages.In comparision of Meishan pigs,development and maturation of fetal skeletal muscle may occur earlier in Large White pigs.Moderate increase of maternal dietary energy intake during gestation delays the differentiation and maturation in skeletal muscle of Large White pig fetuses by adversely affecting the program of myogenic differentiation,fast-MyHC formation and mitochondrial biogenesis and function.Furthermore,moderate increase of maternal dietary energy intake during early gestation inhibits myogenic differentiation in Meishan pig embryo,however,maternal high energy intake during mid-late gestation promotes skeletal muscle growth and development in Meishan pig fetuses.Experimental 2.Effects of maternal dietary energy intake on the proliferation and differentiation of fetal porcine muscle-derived mesenchymal stem cells.The results of experiment 1 showed that moderate increase of maternal dietary energy intake during gestation delayed the differentiation and maturity in skeletal muscle of Large White pig fetuses.In this experiment,mesenchymal stem cells(MSCs)from Large White pig fetal skeletal muscle was used to compare the differences of proliferation and myogenic and adipogenic differentiation between CON group and HE group.The experimental treatment was same as experimental 1.Four sows per diet group were anaesthetized and one fetuse from each sow was collected on day 55 of gestation.MSCs were isolated from longissimus dorsi muscle.Cells were expanded in cultures,and only early passage cells(passages 3-6)were used.The results showed that the positive percentages of CD29 and CD90(MSC markers)were above 98%,and the negative percentages of CD31 and CD34(Endothelial origin markers)were above 99%.The number of muscle-derived cells from HE fetuses was lower than that from CON fetuses after 96 h incubation(P<0.05),and the population doubling time was longer(P<0.05).Compared to the CON group,the mRNA levels of myogenic genes(MyoDl,Myf5,Myogenin and Desmin)in MSCs derived from HE group after 21-day mogenic induction were decreased(P<0.05).No differences were found for mRNA levels of PPAR ?and CCAAT/enhancer-binding proteins(CEBP ?)and lipid accumulation with oil red O staining after 21-day adipogenic induction between CON group and HE group.The results indicated that MSCs of Large White fetuses from HE group exhibit lower ability for proliferation and myogenic differentiation during gestation.Experimental 3.Effects of resveratrol supplementation in high-energy diet fed for pregnant mice on skeletal muscle and adipose tissue development in male offspringHere,we evaluated further the effects of resveratrol supplementation of maternal high-energy and high-fat diet during gestation and lactation on the development of skeletal muscle and adipose tissue and energy metabolism in offspring.Pregnant C57BL/6J mice(3 months old)were housed individually and randomly divided into four dietary groups(n = 10 per group).Two groups were fed with a control diet(CON)or an identical diet supplemented with 0.2%RES(CON+RES).The other two groups were fed with a high-energy diet(HFD)or anidentical diet supplemented with 0.2%RES(HFD+RES).All dams were fed their respective dits from day 0 of gestation until weaning.On postnatal day 21,pups were weaned,and dams and one male pup from each litter were killed by carbon dioxide inhalation and cervical dislocation.Blood samples were collected by cardiac puncture,and the interscapular brown adipose tissue(BAT),inguinal white adipose tissue(IngWAT),epididymal white adipose tissue(EpiWAT),tibialis anterior muscle(TA)and gastrocnemius muscle(GA)were rapidly isolated and weighed.After weaning,the remaining male pups were weaned onto HFD to mimic a postweaning obesogenic environment.After 11 weeks of HFD challenge,glucose tolerance test,acute cold tolerance test and whole-body metabolic rate were determined,then mice were killed and blood was collected by cardiac puncture.The BAT,IngWAT,EpiWAT,TA and GA were harvested and weighed.The results were presented as follows:(1)At weaning,HFD-fed dams had higher body weight,ratio of WAT mass to body weight and the concentrations of serum triglycerides and insulin than CON and CON+RES dams(P<0.05),whereas RES supplementation to HFD dams significantly reduced body weight gain,fat accumulation and the concentratons of serum triglycerides and inuslin(P<0.05).There were significant increases in body weight,ratio of WAT mass to body weight and concentrations of serum triglycerides and insulin in HFD offspring compared to CON and CON+RES groups at weaning and in adulthood,which were reduced by maternal RES treatment(P<0.05).The decreased ratio of muscle mass to body weight in adult HFD offspring was prevented by maternal RES administration(P<0.05).(2)The mRNA levels of Pax7 and MyoD and MHC protein expression in GA muacle of HFD offspring were lower than those of CON and CON+RES offspring at weaning,which were increased by maternal RES treatment(P<0.05).Compared to the CON and CON+RES groups,the mRNA levels of Pax7,MyoD,Myf5 and myogenin and protein expression of myogenin and MHC in GA muscle of adult HFD offspring were decreased,which were reversed by maternal RES supplementation(P<0.05).(3)At weaning and in adulthood,HFD offspring BAT became filled with large lipid droplets,a phenotype similar to white fat,which was recovered following RES supplementation,consistent with changes in UCP1 immunohistochemical staining.In agreement,the UCP1 protein level was reduced by the HFD but recovered by maternal RES supplementation.In addition,the mRNA levels of PR domain-containing 16(PRDM16),PGC-1?,cell death-inducing DFFA-like effector A(Cidea),elongation of very long-chain fatty acids protein 3(Elovl3)and cytochrome c oxidase subunit ?a polypeptide 1(Cox7a1)in BAT of HFD offspring were lower than those of CON and CON+RES offspring at weaning,whereas maternal administration of RES increased the mRNA levels of these thermogenic genes(P<0.05).A similar pattern of changes was observed for protein levels of PRDM16,Sirtl and phosphorylation AMP-activated protein kinase a(p-AMPKa)at weaning.(4)Maternal RES supplementation noticeably increased the abundance of multilocular adipocytes in IngWAT and EpiWAT of HFD offspring at weaning,which is a key characteristic of brown fat-like cells.UCP1 immunostaining showed enhanced UCP1 levels in IngWAT and EpiWAT of HFD+RES offspring compared with HFD offspring.Consistent with the changes in UCP1 immunostaining,UCP1 protein expression was increased in IngWAT and EpiWAT of HFD mice(P<0.05).A similar pattern of changes was observed for UCP1 expression in only IngWAT of adult HFD+RES offspring.In addition,the mRNA levels of UCP1,PRDM16,Elovl3 and CD137 in IngWAT and EpiWAT of HFD offspring were lower than those of CON and CON+RES offspring at weaning,which were recovered by maternal RES supplementation(P<0.05).RES treatment of dams fed HFD increased Cyto C protein levels,accompanied by up-regulated expression of p-AMPKa and Sirtl in IngWAT and EpiWAT at weaning(P<0.05).(5)Maternal RES supplementation improved glucose tolerance in adult HFD offspring.Compared with CON and CON+RES offspring,HFD offspring showed markedly decreased oxygen consumption and heat production during light and dark phases,which was prevented by maternal RES treatment(P<0.05).Moreover,adult HFD+RES offspring had a higher body temperature than HFD offspring during the cold challenge(P<0.05).The results indicated that RES supplementation to mice fed a HFD during gestation and lactation enhances the expression of myogenic genes and MHC,and improves skeletal muscle development in male offspring.Additionally,maternal RES administration promotes a thermogenic programme in BAT and WAT,increases energy expenditure and protects offspring against HFD-induced obesity and insulin resistance in male offspring,which couldbe related to the activation of AMPK/Sirtl signaling pathway.It can be concluded as follows:(1)Large White and Meishan pigs exhibit significant differences in prenatal gene expression profiles of skeletal muscle development at different developmental stages.Compared with Meishan pigs,development and maturation of fetal skeletal muscle may occur earlier in Large White pigs.(2)Moderate increase of maternal dietary energy intake during gestation delays the differentiation and maturation in skeletal muscle of Large White pig fetuses by adversely affecting the program of myogenic differentiation,fast-MyHC formation and mitochondrial biogenesis and function,which was also confirmed in vitro that MSCs of HE fetuses exhibit lower ability for proliferation and myogenic differentiation.(3)Moderate increase of maternal dietary energy intake during early gestation inhibits myogenic differentiation in Meishan pig embryo,which was associated with the expression of myogenic genes.However,maternal high energy intake during mid-late gestation promotes skeletal muscle growth and development in Meishan pig fetuses.(4)RES supplementation of high-energy diet-fed pregnant mice during gestation and lactation enhances the expression of myogenic genes and MHC,and improves skeletal muscle development in male offspring.Additionally,maternal RES administration promotes brown and beige adipocyte development and prevents obesity and insulin resistance in male mice offspring,which could be related to the activation of AMPK/Sirtl signaling pathway. |
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