Placenta, a crucial organ linking maternal and fetal for nutrients and waste exchange, undergoes extensive metabolism for its development and function during pregnancy. The interaction between nutrients transport and metabolism within placenta is directly related to the nutrient supply from maternal to fetal to regulate fetal development. To our knowledge, this is the first study providing preliminary data on the influence of maternal nutrition to a broad range of metabolites and global nutrients transporter in placenta in mammals. Important differences in metabolism and transport of lipids, glucose and amino acids were observed in placentas from those sows fed with Leu during late pregnancy.
Amino acids transport and metabolism
The quality and relative composition of amino acids delivered to fetus through placenta is directly related to fetal growth during pregnancy due to the importance of amino acids as basic substance for protein synthesis[15]. In present study, amino acids full spectrum analyses indicates that sow dietary Leu supplementation widely improved amino acids accumulation within placenta, which implying more amino acids supply to fetuses. In porcine placenta, branch chain amino acids (BCAAs), including Leu, Val, Ile, could be degraded to donate their amino groups to synthesize Gln, which is important precursor for protein, purine and pyrimidines synthesis to supply to fetuses[16-18]. In addition, Gln also could be used in the fetus to produce Glu, Asp, and Ala [19, 20]. Thus, Gln accumulated in the placenta either from maternal circulation or placental synthesis, serves as a major nitrogen supply for fetus. Further study found that the amount of Gln in placenta were increased with higher dietary intake of BCAAs during pregnancy [21]. In our present study, we also found that the amount of Gln was significantly increased more than 2 folds in placentas from the sows fed with 0.4% Leu compared with control group. In porcine placenta, those amino acids including Glu, Gln, Ile, Val and Leu showed the highest amount among all amino acids and their accumulation play important role for both placental and fetal development [22]. In the present study, we interestingly found that Leu, Val, Ile, and Glu were enhanced with the addition of Lue and showed the highest VIP scores among all differential amimo acids, which might be growth stimulator for improved fetal development during pregnancy [23].
Amino acids from maternal circulation are accumulated within the placenta by active transport systems for further transportation or metabolism[24, 25]. Amino acids transport cross placenta are also important determinants of the amino acid composition in the fetal circulation [26] and impaired amino acid transport could lead to placental metabolic disorder and restricted fetal growth [27, 28, 29] . In present study, we determined the gene expression of global amino acids transporters in placenta and found that most of transporters were significantly upregulated with the supplementation of Leu. Tau, an essential nutrient in fetal development, need to be mainly supplied by maternal via placenta due to inadequate synthesis from placenta and fetus [30]. Reduced activity of a placental Tau transporter were related with fetal intrauterine growth restriction and impaired placental function [31, 32]. In the present study, the increased level of Tau in placenta from the sows fed with Leu might attribute to elevated transporters expression in those placentas. In addition, Arg could not be metabolized in porcine placenta because of the lack of arginase [33, 34]. The increased Arg within placenta with Lue supplementation might also attribute to the elevated transporters and would be further transported across placenta to promote Arg supply from maternal to fetus.
Metabolotic anysis indicated that amino acid metabolism related to polyamines in placenta were significantly altered by supplementation of Leu in sow diets. Polyamines, which plays essential roles for DNA and protein synthesis in animal cells, are key regulator of placental growth and mammalian embryo early development [29]. Porcine placenta could convert Pro to polyamines by providing the bulk of the carbon skeleton and nitrogen in putrescine, spermidine, and spermine [33]. In the present study, several metobolities related to polyamines including putrescine, spermidine and spermine were up-regulated in the placenta from those sows fed with Leu. In addition, we found the placental glutathione were greatly increased in treatment sow. Glutathione, synthesized in placenta from glutamate, Gly and Cys, are transported to fetus to provide antioxidative protect during development [35]. The enhanced glutathione in placenta has a potential to improve fetal growth during pregnancy.
Fatty acids metabolism and transport
Essential fatty acids and their long-chain polyunsaturated fatty acids play crucial roles for fetal intrauterine development due to their contribution in formation and dynamic properties of biological membranes during pregnancy [36-39]. In placenta, most of fatty acids require facilitated transport by several plasma membrane-located transport/binding proteins such as fatty acid translocase (FAT/CD36), plasma membrane fatty acid binding protein (FABPpm), fatty acid transport protein (FATP 1-6) and intracellular fatty acid binding protein 1-9 (FABP 1-9) [40]. The family of FATPS and FAT/CD36 have been demonstrated to mediate cellular uptake of long-chain and very long chain fatty acids to facilitate fatty acid influx across biological membranes [41]. Previous studied reported that increased expression of CD36 and FATP4 were certainly associated with higher fatty acids uptake in placenta[42, 43]. In present study, the mRNA expression of CD36, FATP1 and FATP2 were significantly enhanced in the placenta from the sows fed with Leu, implying more fatty acid influx from maternal circulation to fetus. Polyunsaturated fatty acids (PUFAs) are essential for fetal development because they are the precursors of eicosanoids and also are essential constituents of the membrane lipids that maintain cellular and organelle integrity [37, 44]. During pregnancy, intrauterine transfer from maternal across placenta is the only supply of PUFAs to the fetus [45]. We found that Leu supplementation resulted in placental higher levels several essential fatty acids (EFA) and their derivatives including arachidonoc, decosahexaenoic acid, and cholesterol in present study, which might be the results of increased gene expression of CD36 and FATP family.
After inside the placenta, fatty acids could be directly transported into the fetal circulation or bound to FABPs to participate in cellular metabolism within placenta [46]. Selective cellular metabolism of certain fatty acids in placenta could contribute to new synthesized lipid supplying from mother to fetus, conversion of certain proportion of arachidonic acid to PGs, incorporation of some fatty acids into phospholipids and triacylglycerols, and the oxidation of fatty acids[47]. Metabolics analysis showed that sow dietary Leu supplementation leads to altered placental lipid metabolism. Placenta has been shown to oxidize fatty acids as an important metabolic fuel to supply energy during the whole gestational process [48, 49] and FABP 3, 4, 5 were upregulated in placenta during fatty acids oxidation [50, 51, 52]. On the other hand, long-chain fatty acid metabolites may exert toxic effects on cellular functions and cause cell injury [53].In present study, gene expression of FABP 3, FABP 5 and FABP 7 were significantly enhanced in placenta from those sows fed with Leu supplementation. But metabolic analysis showed decreased level of several metabolities relalted to lipid oxidation including 9S- hydroxy-octadedienoic acid (HODE) and 13S-HODE, in treatment placenta compared with control. These results indicated that Leu supplementation upregulated fatty acid oxidization to produce more energy to support placental function without boosting cellular transport.
In addition, we interestingly found PGs which is a crucial regulator for pregnancy and has been linked to induction of fetal organ maturation [54, 55], were upregulated with the leu supplementation. During pregnancy, placenta is the major source of PGs within intrauterine tissues. This result might suggest a new regulation effect of leu to PGs synthesis.
Glucose transport and metabolism
Glucose is the main metabolic fuel for fetal intrauterine growth and is one of the major nutrients transported from maternal circulation [56]. In pig placenta, SLC2A family of glucose transporters including 14 members (SLC2A1-SLC2A14), are responsible for transporting glucose in addition to multiple other sugars from maternal to fetus and SLC2A1 and SLC2A3 play the prominent role among these transporters [57, 58]. In present study, we globally determined all transporters belong to SLC2A family and not surprisingly found that SLC2A1 and SLC2A3 were dramatically increased with leu supplementation, suggesting more glucose delivery between maternal and fetus.
Interestingly that although glucose is the major metabolic fuel, but fructose shows the most abundant hexose sugar in porcine endometria and conceptuses to support conceptus development as a subtract in many metabolic pathway [59]. In porcine placenta, glucose could be actively converted to fructose and then enter the hexosamine biosynthesis pathway and stimulate trophectoderm proliferation or transported into fetus to supply energy through glycolytic pathway [60].Because that fructose is undetected in blood of pregnant gilts and sows, placental synthesis from glucose is the main source of fructose for intrauterine fetus during pregnancy [61]. We interestingly found that placenta glucose and fructose amount was both greatly higher in the sows fed with 0.4% Leu compared with control group, but the expression of fructose transporters SLC2A5 and SLC2A8 showed no difference induced by addition of Leu [62]. This result suggested that the promoted fructose synthesis from glucose induced by Leu in placenta just only was metabolized within placenta to provide energy, but no contribution to fetal supply.
Energy metabolism and mTOR signal pathway
Constant and abundant source of energy supply is necessary in placenta for its specialized roles, both nutrient transportation and the synthesis of placental protein hormones, as well as for its nonspecific functions [63]. Previous studies indicated that intensity of energy metabolism in placenta was greatly decreased in intrauterine growth restriction and resulted in disturbances of the placental morphological structure and functional insufficiency. [64]. In present study, the activities of enzymes in placenta involved in energy metabolism including carbohydrate metabolism (hexokinase, glycogen phosphorylase and lactated hydrogenase), tricarboxylic acid cycle enzyme (succinatede hydrogenase) and fatty acid oxidation (hydroxyacyl-CoA-dehydrogenase) were all significantly enhanced with Lue supplementation in sow diet, implying more intensive energy metabolism to provide ATP for placenta development and function.
The mechanisms by which the placenta adjusts signals to meet appropriate intrauterine fetal growth are complex [5, 65]. The mammalian target of rapamycin (mTOR) protein is a Ser/threonine-specific protein kinase that belongs to the PI3K-related kinase (PIKKs) family, which functions in the regulation of cell growth and protein transcription by serving as a nutrient sensor during gestation, and a downstream target of the PI3K and PKA pathways [66, 67]. Previous studies have shown that the inhibition of mTOR activation results in impaired placental function and elevated expression of mTOR as compensation of Intrauterine growth retardation (IUGR) [68]. In current study, we found that mTOR pathway is higher activated in placenta from the sows fed with Leu, demonstrated by elevated levels of p-PI3K, p-Akt, and p-mTOR, implying that PI3K/Akt/mTOR signal pathway was involved in the regulation of leu to placenta nutrient transport and metabolism.