Placentome Nutrient Transporters and Mammalian Target of Rapamycin Signaling Proteins Are Altered by the Methionine Supply during Late Gestation in Dairy Cows and Are Associated with Newborn Birth Weight

Abstract

To our knowledge, most research demonstrating a link between maternal nutrition and both fetal growth and offspring development after birth has been performed with nonruminants. Whether such relationships exist in large ruminants is largely unknown. We aimed to investigate whether increasing the methionine supply during late pregnancy would alter uteroplacental tissue nutrient transporters and mammalian target of rapamycin (mTOR) and their relation with newborn body weight. Multiparous Holstein cows were used in a randomized complete block design experiment. During the last 28 d of pregnancy, cows were fed a control diet or the control diet plus ethylcellulose rumen-protected methionine (0.9 g/kg dry matter intake) (Mepron; Evonik Nutrition & Care GmbH) to achieve a 2.8:1 ratio of lysine to methionine in the metabolizable protein reaching the small intestine. We collected placentome samples at parturition and used them to assess mRNA and protein expression and the phosphorylation status of mTOR pathway proteins. Newborn body weight was greater in the methionine group than in the control group (44.1 kg and 41.8 kg, respectively; ≤ 0.05). Increasing the methionine supply also resulted in greater feed intake (15.8 kg/d and 14.6 kg/d), plasma methionine (11.9 μM and 15.3 μM), and plasma insulin (1.16 μg/L and 0.81 μg/L) in cows during late pregnancy. As a result, mRNA expression of genes involved in neutral amino acid transport [solute carrier (SLC) family members, ,, and ], glucose transport [, , and], and the mTOR pathway [mechanistic target of rapamycin and ribosomal protein S6 kinase B1] were upregulated ( ≤ 0.07) in methionine-supplemented cows. Among 6 proteins in the mTOR pathway, increasing the methionine supply led to greater ( ≤ 0.09) protein expression of α serine-threonine kinase (AKT), phosphorylated (p)-AKT, p-eukaryotic elongation factor 2, and the p-mTOR:mTOR ratio. Supplemental methionine during late gestation increases feed intake and newborn body weight in dairy cows, and this effect may be mediated by alterations in the uteroplacental transport of nondispensable and dispensable amino acids and glucose at least in part through changes in gene transcription and mTOR signaling.

Methionine supply during the periparturient period enhances insulin signaling, amino acid transporters, and mechanistic target of rapamycin pathway proteins in adipose tissue of Holstein cows

“…Under such scenario, conditions that upregulate abundance of total mTOR and p-mTOR in adipose tissue might be the determining factor controlling AA use and the degree of protein synthesis. Although there is no evidence (to our knowledge) that an upregulation of AA transporters results in the activation of mTOR in adipose tissue, nonruminant data indicate that upregulation of placental AA transporters contributes to fetal growth via the activation of mTOR signaling (Jansson et al, 2013;Batistel et al, 2017a). Thus, AA availability and utilization by adipose tissue after calving may not only help in the remodeling process (McNamara et al, 1995) but also may serve ancillary functions such as preventing localized inflammation and oxidative stress (Chimin et al, 2017).…”

“…Total protein was extracted from 100 mg of adipose tissue according to a published procedure from our laboratory (Batistel et al, 2017a).The concentration of total protein was determined using a NanoDrop ND-1000 (Thermo Fisher Scientific). Protein samples were denatured by heating at 100°C for 5 min before loading 75 µg of protein into each lane of a 4 to 20% SDS-PAGE gel (catalog no.…”

N-carbamoylglutamate supplementation in late-gestation dairy cows is associated with increased newborn weight and improved nutrient supply in the placentome

“…Therefore, the increased expression of SLC2A3 observed in NCG cows in this study indicates that the changes in glucose transporters occurred mainly in the microvillus membrane, which is rich in capillaries and may be related to placental angiogenesis. In addition, the AA concentration and supply increased with NCG supplementation in dairy cows [19], contributing to enhanced glucose transport into the placenta [4,33].…”

“…The placentome samples were collected referring to the methods reported by Batistel et al [4]. Brie y, after natural delivery (within 2 h), the placenta was rinsed with physiologic saline, and then, 4-6 placentomes from the central area of the placenta were dissected, rinsed with physiologic saline until clear and then stored at -80°C for later analysis.…”

“…Twenty-three genes of interest were selected based on their key roles in placental functions: (1) genes encoding Arg transporters (SLC6A14, SLC7A1, SLC7A6, SLC7A7, SLC7A9); (2) genes encoding glucose transporters (SLC2A1, SLC2A3, SLC2A4); (3) genes encoding fatty acid transporters (SLC27A1, SLC27A2, SLC27A3); (4) genes associated with angiogenesis (VEGFA, NOS3, GUCY1B3, HIF1A), and (5) genes involved in the mTOR pathway (AKT1, mTOR, RPS6KB1, EIF4BP1, EIF4EBP2, EEF1A1, ELF2, IRS1). In addition, two references genes (GAPDH and ACTB) were included based on published recommendations [4]. Some of the primers targeting these genes were selected based on previous studies, and others were designed using National Center for Biotechnology Information Nucleotide database (http://www.ncbi.nlm.nih.gov/nuccore/), with Bos taurus as the species (Supplementary Table S2).…”