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Carbohydrate responsive element-binding protein (ChREBP) has been identified as a primary transcription factor that maintains energy homeostasis through transcriptional regulation of glycolytic, lipogenic, and gluconeogenic enzymes in response to a high-carbohydrate diet. Amino acids are important substrates for gluconeogenesis, but nevertheless, knowledge is lacking about whether this transcription factor regulates genes involved in the transport or use of these metabolites. Here, we demonstrate that ChREBP represses the expression of the amino acid transporter sodium-coupled neutral amino acid transporter 2 (SNAT2) in response to a high-sucrose diet in rats by binding to a carbohydrate response element (ChoRE) site located -160 bp upstream of the transcriptional start site in the SNAT2 promoter region. Additionally, immunoprecipitation assays revealed that ChREBP and silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) interact with each other, as part of the complex that repress SNAT2 expression. The interaction between these proteins was confirmed by an in vivo chromatin immunoprecipitation assay. These findings suggest that glucogenic amino acid uptake by the liver is controlled by ChREBP through the repression of SNAT2 expression in rats consuming a high-carbohydrate diet.

Nutrient ingestion is known to increase the exercise-induced stimulation of muscle protein synthesis following resistance exercise. Less is known about the effect of nutrients on muscle protein synthesis following sprint exercise. At two occasions separated by 1 mo, 12 healthy subjects performed three 30-s sprints with 20-min rest between bouts. In randomized order, they consumed a drink with essential amino acids and maltodextrin (nutrient) or flavored water (placebo). Muscle biopsies were obtained 80 and 200 min after the last sprint, and blood samples were taken repeatedly during the experiment. Fractional synthetic rate (FSR) was measured by continuous infusion of l-[2H5]phenylalanine up to 200 min postexercise. The mRNA expression and protein expression of SNAT2 were both 1.4-fold higher ( P < 0.05) after nutrient intake compared with placebo at 200 min postexercise. Phosphorylated Akt, mammalian target of rapamycin (mTOR), and p70S6k were 1.7- to 3.6-fold higher ( P < 0.01) 80 min after the last sprint with nutrient ingestion as compared with placebo. In addition, FSR was higher ( P < 0.05) with nutrients when plasma phenylalanine (FSRplasma) was used as a precursor but not when intracellular phenylalanine (FSRmuscle) was used. Significant correlations were also found between FSRplasma on the one hand and plasma leucine and serum insulin on the other hand in the nutrient condition. The results show that nutrient ingestion induces the expression of the amino acid transporter SNAT2 stimulates Akt/mTOR signaling and most likely the rate of muscle protein synthesis following sprint exercise. NEW & NOTEWORTHY There is limited knowledge regarding the effect of nutrients on muscle protein synthesis following sprint as compared with resistance exercise. The results demonstrate that nutrient ingestion during repeated 30-s bouts of sprint exercise induces expression of the amino acid transporter SNAT2 and stimulates Akt/mTOR signaling and most likely the rate of muscle protein synthesis. Future studies to explore the chronic effects of nutritional ingestion during sprint exercise sessions on muscle mass accretion are warranted.

Apelin is an insulin-sensitizing hormone increased in abundance with obesity. Apelin and its receptor, APJ, are expressed in the human placenta, but whether apelin regulates placental function in normal body mass index (BMI) and obese pregnant women remains unknown. We hypothesized that apelin stimulates amino acid transport in cultured primary human trophoblast (PHT) cells and that maternal circulating apelin levels are elevated in obese pregnant women delivering large babies. Treating PHT cells with physiological concentrations of the pyroglutamated form [Pyr1]apelin-13 (0.1–10.0 ng/ml) for 24 h dose-dependently increased System A amino acid transport ( P < 0.05) but did not affect System L transport activity. Mechanistic target of rapamycin (mTOR), extracellular signal-regulated kinase-1/2 (ERK1/2), and AMP-activated protein kinase-α (AMPKα) signaling were unaffected by apelin ( P > 0.05). Plasma apelin was not different in obese women (BMI 35.8 ± 0.7, n = 21) with large babies compared with normal-BMI women (23.1 ± 0.5, n = 16) delivering normal birth weight infants. Apelin was highly expressed in placental villous tissue (20-fold higher vs. adipose), and APJ was present in syncytiotrophoblast microvillous membrane, but neither differed in abundance between normal-BMI and obese women. Phosphorylation (Thr172) of placental AMPKα strongly correlated with microvillous membrane APJ expression ( P < 0.01, R = 0.63) but negatively correlated with placental apelin abundance ( P < 0.01, R = −0.62). Neither placental APJ nor apelin abundance correlated with maternal BMI, plasma insulin, birth weight, or mTOR or ERK1/2 signaling ( P > 0.05). Hence, apelin stimulates trophoblast amino acid uptake, establishing a novel mechanism regulating placental function. We found no evidence that apelin constitutes an endocrine link between maternal obesity and fetal overgrowth.

We have previously shown that Na+-coupled neutral amino acid transporter 1 (SNAT1) modulates nitric oxide (NO) production in pulmonary arterial endothelial cells (PAECs) from newborn piglets. Specifically, the ability to increase NO production in response to the l-arginine-NO precursor l-citrulline is dependent on SNAT1 expression. Elucidating factors that regulate SNAT1 expression in PAECs could provide new insights and therapeutic targets relevant to NO production. Our major goals were to determine if reactive oxygen species (ROS) modulate SNAT1 expression in PAECs from newborn piglets and to evaluate the role of NADPH oxidase 1 (NOX1) and uncoupled endothelial NO synthase, enzymatic sources of ROS, in hypoxia-induced increases in SNAT1 expression. Treatment with either H2O2 or xanthine plus xanthine oxidase increased SNAT1 expression in PAECs from newborn piglets cultured under normoxic conditions. Hypoxia-induced increases in SNAT1 expression were inhibited by treatments with the ROS-removing agents catalase and superoxide dismutase, NOX1 siRNA, and the NO synthase inhibitor NG-nitro-l-arginine methyl ester. Both tetrahydropbiopterin (BH4) and l-citrulline, two therapies that decrease ROS by recoupling endothelial NO synthase, reduced the hypoxia-induced increase in SNAT1 expression. BH4 and l-citrulline treatment improved NO production in hypoxic PAECs despite a reduction in SNAT1 expression. In conclusion, SNAT1 expression is modulated by ROS in PAECs from newborn piglets. However, ROS-mediated decreases in SNAT1 expression per se do not implicate a reduction in NO production. Although SNAT1 may be critical to l-citrulline-induced increases in NO production, therapies designed to alter SNAT1 expression may not lead to a concordant change in NO production. NEW & NOTEWORTHY Na+-coupled neutral amino acid transporter 1 (SNAT1) modulates nitric oxide (NO) production in piglet pulmonary arterial endothelial cells. Factors that regulate SNAT1 expression in pulmonary arterial endothelial cells are unclear. Here, we show that ROS-reducing strategies inhibit hypoxia-induced increases in SNAT1 expression. l-Citrulline and tetrahydropbiopterin decrease SNAT1 expression but increase NO production. Although SNAT1 is modulated by ROS, changes in SNAT1 expression may not cause a concordant change in NO production.

Neonatal pigs are used as a model to study and optimize the clinical treatment of infants who are unable to maintain oral feeding. Using this model, we have shown previously that pulsatile administration of leucine during continuous feeding over 24 h via orogastric tube enhanced protein synthesis in skeletal muscle compared with continuous feeding alone. To determine the long-term effects of leucine pulses, neonatal piglets ( n = 11–12/group) were continuously fed formula via orogastric tube for 21 days, with an additional parenteral infusion of either leucine (CON + LEU; 800 μmol·kg−1·h−1) or alanine (CON + ALA) for 1 h every 4 h. The results show that body and muscle weights and lean gain were ∼25% greater, and fat gain was 48% lower in CON + LEU than CON + ALA; weights of other tissues were unaffected by treatment. Fractional protein synthesis rates in longissimus dorsi, gastrocnemius, and soleus muscles were ∼30% higher in CON + LEU compared with CON + ALA and were associated with decreased Deptor abundance and increased mTORC1, mTORC2, 4E-BP1, and S6K1 phosphorylation, SNAT2 abundance, and association of eIF4E with eIF4G and RagC with mTOR. There were no treatment effects on PKB, eIF2α, eEF2, or PRAS40 phosphorylation, Rheb, SLC38A9, v-ATPase, LAMTOR1, LAMTOR2, RagA, RagC, and LAT1 abundance, the proportion of polysomes to nonpolysomes, or the proportion of mRNAs encoding rpS4 or rpS8 associated with polysomes. Our results demonstrate that pulsatile delivery of a leucine supplement during 21 days of continuous enteral feeding enhances lean growth by stimulating the mTORC1-dependent translation initiation pathway, leading to protein synthesis in skeletal muscle of neonates.

Fetal growth restriction (FGR) is the inability of a fetus to reach its genetically predetermined growth potential. In the absence of a genetic anomaly or maternal undernutrition, FGR is attributable to “placental insufficiency”: inappropriate maternal/fetal blood flow, reduced nutrient transport or morphological abnormalities of the placenta (e.g., altered barrier thickness). It is not known whether these diverse factors act singly, or in combination, having additive effects that may lead to greater FGR severity. We suggest that multiplicity of such dysfunction might underlie the diverse FGR phenotypes seen in humans. Pregnant endothelial nitric oxide synthase knockout (eNOS−/−) dams exhibit dysregulated vascular adaptations to pregnancy, and eNOS−/− fetuses of such dams display FGR. We investigated the hypothesis that both altered vascular function and placental nutrient transport contribute to the FGR phenotype. eNOS−/− dams were hypertensive prior to and during pregnancy and at embryonic day (E) 18.5 were proteinuric. Isolated uterine artery constriction was significantly increased, and endothelium-dependent relaxation significantly reduced, compared with wild-type (WT) mice. eNOS−/− fetal weight and abdominal circumference were significantly reduced compared with WT. Unidirectional maternofetal 14C-methylaminoisobutyric acid (MeAIB) clearance and sodium-dependent 14C-MeAIB uptake into mouse placental vesicles were both significantly lower in eNOS−/− fetuses, indicating diminished placental nutrient transport. eNOS−/− mouse placentas demonstrated increased hypoxia at E17.5, with elevated superoxide compared with WT. We propose that aberrant uterine artery reactivity in eNOS−/− mice promotes placental hypoxia with free radical formation, reducing placental nutrient transport capacity and fetal growth. We further postulate that this mouse model demonstrates “uteroplacental hypoxia,” providing a new framework for understanding the etiology of FGR in human pregnancy.

Small neutral amino acid transporter 2 (SNAT2) is the most abundant and ubiquitous transporter for zwitterionic short-chain amino acids. The activity of this amino acid transporter is stimulated in vivo or in vitro by glucagon or cAMP analogs. However, it is not known whether the increase in activity at the protein level is due to an increase in SNAT2 gene transcription. Thus, the aim of the present work was to study whether cAMP was able to stimulate SNAT2 gene expression and to localize and characterize the presence of cAMP response elements (CRE) in the promoter that controls the expression of the rat SNAT2 gene. We found that consumption of a high-protein diet that increased serum glucagon concentration or the administration of glucagon or incubation of hepatocytes with forskolin increased the SNAT2 mRNA level. We then isolated the 5′ regulatory region of the SNAT2 gene and determined that the transcriptional start site was located 970 bp upstream of the translation start codon. We identified two potential CRE sites located at −354 and −48 bp. Our results, using deletion analysis of the 5′ regulatory region of the SNAT2 gene, revealed that the CRE site located at −48 bp was fully responsible for SNAT2 regulation by cAMP. This evidence was strongly supported by mutation of the CRE site and EMSA and ChIP analysis. Alignment of rat, mouse, and human sequences revealed that this CRE site is highly conserved among species, indicating its essential role in the regulation of SNAT2 gene expression.

The results presented here show that STC-1 cells, a model of intestinal endocrine cells, respond to a broad range of amino acids, including l-proline, l-serine, l-alanine, l-methionine, l-glycine, l-histidine, and α-methyl-amino-isobutyric acid (MeAIB) with a rapid increase in the intracellular Ca2+ concentration ([Ca2+]i). We sought to identify the mechanism by which amino acids induce Ca2+ signaling in these cells. Several lines of evidence suggest that amino acid transport through the Na+-coupled neutral amino acid transporter 2 (SNAT2) is a major mechanism by which amino acids induced Ca2+ signaling in STC-1 cells: 1) the amino acid efficacy profile for inducing Ca2+ signaling in STC-1 cells closely matches the amino acid specificity of SNAT2; 2) amino acid-induced Ca2+ signaling in STC-1 cells was suppressed by removing Na+ from the medium; 3) the nonmetabolized synthetic substrate of amino acid transport MeAIB produced a marked increase in [Ca2+]i; 4) transfection of small interfering RNA targeting SNAT2 produced a marked decrease in Ca2+ signaling in response to l-proline in STC-1 cells; 5) amino acid-induced increase in [Ca2+]i was associated with membrane depolarization and mediated by Ca2+ influx, since it depended on extracellular Ca2+; 6) the increase in [Ca2+]i in response to l-proline, l-alanine, or MeAIB was abrogated by either nifedipine (1–10 μM) or nitrendipine (1 μM), which block L-type voltage-sensitive Ca2+ channels. We hypothesize that the inward current of Na+ associated with the function of SNAT2 leads to membrane depolarization and activation of voltage-sensitive Ca2+ channels that mediate Ca2+ influx, thereby leading to an increase in the [Ca2+]i in enteroendocrine STC-1 cells.

Tubular reabsorption of sulfate is achieved by the sodium-dependent sulfate transporter, NaSi-1, located at the apical membrane, and the sulfate-anion exchanger, sat-1, located at the basolateral membrane. To delineate the physiological role of rat sat-1, [35S]sulfate and [14C]oxalate uptake into sat-1-expressing oocytes was determined under various experimental conditions. Influx of [35S]sulfate was inhibited by bicarbonate, thiosulfate, sulfite, and oxalate, but not by sulfamate and sulfide, in a competitive manner with Kivalues of 2.7 ± 1.3 mM, 101.7 ± 9.7 μM, 53.8 ± 10.9 μM, and 63.5 ± 38.7 μM, respectively. Vice versa, [14C]oxalate uptake was inhibited by sulfate with a Kiof 85.9 ± 9.5 μM. The competitive type of inhibition indicates that these compounds are most likely substrates of sat-1. Physiological plasma bicarbonate concentrations (25 mM) reduced sulfate and oxalate uptake by more than 75%. Simultaneous application of sulfate, bicarbonate, and oxalate abolished sulfate as well as oxalate uptake. These data and electrophysiological studies using a two-electrode voltage-clamp device provide evidence that sat-1 preferentially works as an electroneutral sulfate-bicarbonate or oxalate-bicarbonate exchanger. In kidney proximal tubule cells, sat-1 likely completes sulfate reabsorption from the ultrafiltrate across the basolateral membrane in exchange for bicarbonate. In hepatocytes, oxalate extrusion is most probably mediated either by an exchange for sulfate or bicarbonate.

l-Carnitine is absorbed in the intestinal tract via the carnitine transporter OCTN2 and the amino acid transporter ATB0,+. Loss-of-function mutations in OCTN2 may be associated with inflammatory bowel disease (IBD), suggesting a role for carnitine in intestinal/colonic health. In contrast, ATB0,+ is upregulated in bowel inflammation. Butyrate, a bacterial fermentation product, is beneficial for prevention/treatment of ulcerative colitis. Butyryl-l-carnitine (BC), a butyrate ester of carnitine, may have potential for treatment of gut inflammation, since BC would supply both butyrate and carnitine. We examined the transport of BC via ATB0,+ to determine if this transporter could serve as a delivery system for BC. We also examined the transport of BC via OCTN2. Studies were done with cloned ATB0,+ and OCTN2 in heterologous expression systems. BC inhibited ATB0,+-mediated glycine transport in mammalian cells (IC50, 4.6 ± 0.7 mM). In Xenopus laevis oocytes expressing human ATB0,+, BC induced Na+-dependent inward currents under voltage-clamp conditions. The currents were saturable with a K0.5 of 1.4 ± 0.1 mM. Na+ activation kinetics of BC-induced currents suggested involvement of two Na+ per transport cycle. BC also inhibited OCTN2-mediated carnitine uptake (IC50, 1.5 ± 0.3 μM). Transport of BC via OCTN2 is electrogenic, as evidenced from BC-induced inward currents. These currents were Na+ dependent and saturable ( K0.5, 0.40 ± 0.02 μM). We conclude that ATB0,+ is a low-affinity/high-capacity transporter for BC, whereas OCTN2 is a high-affinity/low-capacity transporter. ATB0,+ may mediate intestinal absorption of BC when OCTN2 is defective.

Amino acid transport via system A plays an important role during lactation, promoting the uptake of small neutral amino acids, mainly alanine and glutamine. However, the regulation of gene expression of system A [sodium-coupled neutral amino acid transporter (SNAT)2] in mammary gland has not been studied. The aim of the present work was to understand the possible mechanisms of regulation of SNAT2 in the rat mammary gland. Incubation of gland explants in amino acid-free medium induced the expression of SNAT2, and this response was repressed by the presence of small neutral amino acids or by actinomycin D but not by large neutral or cationic amino acids. The half-life of SNAT2 mRNA was 67 min, indicating a rapid turnover. In addition, SNAT2 expression in the mammary gland was induced by forskolin and PMA, inducers of PKA and PKC signaling pathways, respectively. Inhibitors of PKA and PKC pathways partially prevented the upregulation of SNAT2 mRNA during adaptive regulation. Interestingly, SNAT2 mRNA was induced during pregnancy and to a lesser extent at peak lactation. β-Estradiol stimulated the expression of SNAT2 in mammary gland explants; this stimulation was prevented by the estrogen receptor inhibitor ICI-182780. Our findings clearly demonstrated that the SNAT2 gene is regulated by multiple pathways, indicating that the expression of this amino acid transport system is tightly controlled due to its importance for the mammary gland during pregnancy and lactation to prepare the gland for the transport of amino acids during lactation.