Search

Protein supplementation during human pregnancy does not improve fetal growth and may increase small-for-gestational-age birth rates and mortality. To define possible mechanisms, sheep with twin pregnancies were infused with amino acids (AA group, n = 7) or saline (C group, n = 4) for 4 days during late gestation. In the AA group, fetal plasma leucine, isoleucine, valine, and lysine concentrations were increased (P < 0.05), and threonine was decreased (P < 0.05). In the AA group, fetal arterial pH (7.365 [+ or -] 0.007 day 0 vs. 7.336 [+ or -] 0.012 day 4, P < 0.005), hemoglobin-oxygen saturation (46.2 [+ or -] 2.6 vs. 37.8 [+ or -] 3.6%, P < 0.005), and total oxygen content (3.17 [+ or -] 0.17 vs. 2.49 [+ or -] 0.20 mmol/l, P < 0.0001) were decreased on day 4 compared with day 0. Fetal leucine disposal did not change (9.22 [+ or -] 0.73 vs. 8.09 [+ or -] 0.63 [micro]mol x [min.sup.-1] x [kg.sup.-1], AA vs. C), but the rate of leucine oxidation increased 43% in the AA group (2.63 [+ or -] 0.16 vs. 1.84 [+ or -] 0.24 [micro]mol x [min.sup.-1] x [kg-.sup.1-], P < 0.05). Fetal oxygen utilization tended to be increased in the AA group (327 [+ or -] 23 vs. 250 [+ or -] 29 [micro]mol x [min.sup.-1] x [kg.sup.-1], P = 0.06). Rates of leucine incorporation into fetal protein (5.19 [+ or -] 0.97 vs. 5.47 [+ or -] 0.89 [micro]mol x [min.sup.-1] x [kg.sup.-1], AA vs. C), release from protein breakdown (4.20 [+ or -] 0.95 vs. 4.62 [+ or -] 0.74 [micro]mol x [min.sup.-1] x [kg.sup.-1]), and protein accretion (1.00 [+ or -] 0.30 vs. 0.85 [+ or -] 0.25 [micro]mol x [min.sup.-1] x [kg.sup.-1]) did not change. Consistent with these data, there was no change in the fetal skeletal muscle ubiquitin ligases MaFBx1 or MuRF1 or in the protein synthesis regulators 4E-BP1, eEF2, eIF2[alpha], and [p70.sup.S6K]. Decreased concentrations of certain essential amino acids, increased amino acid oxidation, fetal acidosis, and fetal hypoxia are possible mechanisms to explain fetal toxicity during maternal amino acid supplementation. metabolism; threonine; leucine; oxygen

In this study, we determined rates of lysine metabolism in fetal sheep during chronic hypoglycemia and following euglycemic recovery and compared results with normal, age-matched euglycemic control fetuses to explain the adaptive response of protein metabolism to low glucose concentrations. Restriction of the maternal glucose supply to the fetus lowered the net rates of fetal (umbilical) glucose (42%) and lactate (36%) uptake, causing compensatory alterations in fetal lysine metabolism. The plasma lysine concentration was 1.9-fold greater in hypoglycemic compared with control fetuses, but the rate of fetal (umbilical) lysine uptake was not different. In the hypoglycemic fetuses, the lysine disposal rate also was higher than in control fetuses due to greater rates of lysine flux back into the placenta and into fetal tissue. The rate of C[O.sub.2] excretion from lysine decarboxylation was 2.4-fold higher in hypoglycemic than control fetuses, indicating greater rates of lysine oxidative metabolism during chronic hypoglycemia. No differences were detected for rates of fetal protein accretion or synthesis between hypoglycemic and control groups, although there was a significant increase in the rate of protein breakdown (P < 0.05) in the hypoglycemic fetuses, indicating small changes in each rate. This was supported by elevated muscle specific ubiquitin ligases and greater concentrations of 4E-BP1. Euglycemic recovery after chronic hypoglycemia normalized all fluxes and actually lowered the rate of lysine decarboxylation compared with control fetuses (P < 0.05). These results indicate that chronic hypoglycemia increases net protein breakdown and lysine oxidative metabolism, both of which contribute to slower rates of fetal growth over time. Furthermore, euglycemic correction for 5 days returns lysine fluxes to normal and causes an overcorrection of lysine oxidation. pregnancy; intrauterine growth restriction, glucose; amino acids

During late gestation, amino acids and insulin promote skeletal muscle protein synthesis. However, the independent effects of amino acids and insulin on the regulation of mRNA translation initiation in the fetus are relatively unknown. The purpose of this study was to determine whether acute amino acid infusion in the late-gestation ovine fetus, with and without a simultaneous increase in fetal insulin concentration, activates translation initiation pathway(s) in skeletal muscle. Fetuses received saline (C), mixed amino acid infusion plus somatostatin infusion to suppress amino acid-stimulated fetal insulin secretion (AA+S), mixed amino acid infusion with concomitant physiological increase in fetal insulin (AA), or high-dose insulin infusion with euglycemia and euaminoacidemia (HI). After a 2-h infusion period, fetal skeletal muscle was harvested under in vivo steady-state conditions and frozen for quantification of proteins both upstream and downstream of mammalian target of rapamycin (mTOR). In the AA group, we found a threefold increase in ribosomal protein S6 kinase (p[70.sup.S6k]) and Erk1/2 phosphorylation; however, blocking the physiological rise in insulin with somatostatin in the AA+S group prevented this increase. In the HI group, Akt, Erk1/2, p[70.sup.S6k], and ribosomal protein S6 were highly phosphorylated and 4E-binding protein 1 (4E-BP1) associated with eukaryotic initiation factor (eIF)4E decreased by 30%. These data show that insulin is a significant regulator of intermediates involved in translation initiation in ovine fetal skeletal muscle. Furthermore, the effect of amino acids is dependent on a concomitant increase in fetal insulin concentrations, because amino acid infusion upregulates p[70.sup.S6k] and Erk only when amino acid-stimulated increase in insulin occurs. translation initiation: mitogen-activated protein kinase pathway; ribosomal protein S6 kinase; protein synthesis

Hepatic glucose production is normally activated at birth but has been observed in response to experimental hypoglycemia in fetal sheep. The cellular basis for this process remains unknown. We determined the impact of 2 wk of fetal hypoglycemia during late gestation on enzymes responsible for hepatic gluconeogenesis, focusing on the insulin-signaling pathway, transcription factors, and coactivators that regulate gluconeogenesis. Hepatic phosphoenolpyruvate carboxykinase and glucose-6-phosphatase mRNA increased 12-fold and 7-fold, respectively, following chronic hypoglycemia with no change in hepatic glycogen. Chronic hypoglycemia decreased fetal plasma insulin with no change in glucagon but increased plasma cortisol 3.5-fold. Peroxisome proliferator-activated receptor-[gamma] coactivator-1[alpha] mRNA and phosphorylation of cAMP response element binding protein at [Ser.sup.133] were both increased, with no change in Akt, forkhead transcription factor FoxO1, hepatocyte nuclear factor-4[alpha], or CCAAT enhancer binding protein-[beta]. These results demonstrate that chronic fetal hypoglycemia triggers signals that can activate gluconeogenesis in the fetal liver. glucose; gluconeogenesis; cortisol; cAMP response element binding protein; peroxisome proliferator-activated receptor-[gamma] coactivator-1[alpha]; phosphoenolpyruvate carboxykinase

In this study we determined body weight-specific fetal (umbilical) glucose uptake (UGU), utilization (GUR), and production rates (GPR) and insulin action in intrauterine growth-restricted (IUGR) fetal sheep. During basal conditions, UGU from the placenta was 33% lower in IUGR fetuses, but GUR was not different between IUGR and control fetuses. The difference between glucose utilization and UGU rates in the IUGR fetuses demonstrated the presence and rate of fetal GPR (41% of GUR). The mRNA concentrations of the gluconeogenic enzymes glucose-6-phophatase and PEPCK were higher in the livers of IUGR fetuses, perhaps in response to CREB activation, as phosphorylated CREB/total CREB was increased 4.2-fold. A hyperglycemic clamp resulted in similar rates of glucose uptake and utilization in IUGR and control fetuses. The nearly identical GURs in IUGR and control fetuses at both basal and high glucose concentrations occurred at mean plasma insulin concentrations in the IUGR fetuses that were ~70% lower than controls, indicating increased insulin sensitivity. Furthermore, under basal conditions, hepatic glycogen content was similar, skeletal muscle glycogen was increased 2,2-fold, the fraction of fetal GUR that was oxidized was 32% lower, and GLUT1 and GLUT4 concentrations in liver and skeletal muscle were the same in IUGR fetuses compared with controls. These results indicate that insulin-responsive fetal tissues (liver and skeletal muscle) adapt to the hypoglycemic-hypoinsulinemic IUGR environment with mechanisms that promote glucose utilization, particularly for glucose storage, including increased insulin action, glucose production, shunting of glucose utilization to glycogen production, and maintenance of glucose transporter concentrations. pregnancy; fetus; glucose oxidation

We measured the impact of chronic late gestation hypoglycemia on pancreatic islet structure and function to determine the cause of decreased insulin secretion in this sheep model of fetal nutrient deprivation. Late gestation hypoglycemia did not decrease pancreas weight, insulin content, [beta]-cell area, [beta]-cell mass, or islet size. The pancreatic islet isolation procedure selected a group of islets that were larger and had an increased proportion of [beta]-cells compared with islets measured in pancreatic sections, but there were no morphologic differences between islets isolated from control and hypoglycemic fetuses. The rates of glucose-stimulated pancreatic islet glucose utilization (126.2 [+ or -] 25.3 pmol glucose x [islet.sup.-1] x [h.sup.-1], hypoglycemic, vs. 93.5 [+ or -] 5.5 pmol glucose x [islet.sup.-1] x [h.sup.-1], control, P = 0.47) and oxidation (10.5 [+ or -] 1.7 pmol glucose x [islet.sup.-1] x [h.sup.-1], hypoglycemic, vs. 10.6 [+ or -] 1.6 pmol glucose x [islet.sup.-1] x [h.sup.-1], control) were not different in hypoglycemic fetuses compared with control fetuses. Chronic late gestation hypoglycemia decreased insulin secretion in isolated pancreatic islets by almost 70% in response to direct nonnutrient membrane depolarization and in response to increased extracellular calcium entry. [beta]-Cell ultrastructure was abnormal with markedly distended rough endoplasmic reticulum in three of the seven hypoglycemic fetuses studied, but in vitro analysis of hypoglycemic control islets showed no evidence that these changes represented endoplasmic reticulum stress, as measured by transcription of glucose regulatory protein-78 and processing of X-box binding protein-1. In conclusion, these studies show that chronic hypoglycemia in late gestation decreases insulin secretion by inhibiting the later steps of stimulus-secretion coupling after glucose metabolism, membrane depolarization, and calcium entry. insulin secretion; pancreatic islet; glucose; metabolism; unfolded protein response doi:10.1152/ajpendo.00265.2006.

We measured in vivo and in vitro nutrient-stimulated insulin secretion in late gestation fetal sheep to determine whether an intrinsic islet defect is responsible for decreased glucose-stimulated insulin secretion (GSIS) in response to chronic hypoglycemia. Control fetuses responded to both leucine and lysine infusions with increased arterial plasma insulin concentrations (average increase: 0.13 [+ or -] 0.05 ng/ml leucine; 0.99 [+ or -] 0.26 ng/ml lysine). In vivo lysine-stimulated insulin secretion was decreased by chronic (0.37 [+ or -] 0.18 ng/ml) and acute (0.27 [+ or -] 0.19 ng/ml) hypoglycemia. Leucine did not stimulate insulin secretion following acute hypoglycemia but was preserved with chronic hypoglycemia (0.12 [+ or -] 0.09 ng/ml). Isolated pancreatic islets from chronically hypoglycemic fetuses had normal insulin and DNA content but decreased fractional insulin release when stimulated with glucose, leucine, arginine, or lysine. Isolated islets from control fetuses responded to all nutrients. Therefore, chronic late gestation hypoglycemia causes defective in vitro nutrient-regulated insulin secretion that is at least partly responsible for diminished in vivo GSIS. Chronic hypoglycemia is a feature of human intrauterine growth restriction (IUGR) and might lead to an islet defect that is responsible for the decreased insulin secretion patterns seen in human IUGR fetuses and low-birth-weight human infants. hypoglycemia; glucose doi:10.1152/ajpendo.00643.2005

Fetal skeletal muscle growth requires myoblast proliferation, differentiation, and fusion into myofibers in addition to protein accretion for fiber hypertrophy. Oxygen is an important regulator of this process. Therefore, we hypothesized that fetal anemic hypoxemia would inhibit skeletal muscle growth. Studies were performed in late-gestation fetal sheep that were bled to anemic and therefore hypoxemic conditions beginning at ~125 days of gestation (term = 148 days) for 9 ± 0 days (n = 19) and compared with control fetuses (n = 16). A metabolic study was performed on gestational day ~134 to measure fetal protein kinetic rates. Myoblast proliferation and myofiber area were determined in biceps femoris (BF), tibialis anterior (TA), and flexor digitorum superficialis (FDS) muscles. mRNA expression of muscle regulatory factors was determined in BF. Fetal arterial hematocrit and oxygen content were 28% and 52% lower, respectively, in anemic fetuses. Fetal weight and whole body protein synthesis, breakdown, and accretion rates were not different between groups. Hindlimb length, however, was 7% shorter in anemic fetuses. TA and FDS muscles weighed less, and FDS myofiber area was smaller in anemic fetuses compared with controls. The percentage of Pax7þ myoblasts that expressed Ki67 was lower in BF and tended to be lower in FDS from anemic fetuses indicating reduced myoblast proliferation. There was less MYOD and MYF6 mRNA expression in anemic versus control BF consistent with reduced myoblast differentiation. These results indicate that fetal anemic hypoxemia reduced muscle growth. We speculate that fetal muscle growth may be improved by strategies that increase oxygen availability. [ABSTRACT FROM AUTHOR]

We measured the impact of chronic late gestation hypoglycemia on pancreatic islet structure and function to determine the cause of decreased insulin secretion in this sheep model of fetal nutrient deprivation. Late gestation hypoglycemia did not decrease pancreas weight, insulin content, β-cell area, β-cell mass, or islet size. The pancreatic islet isolation procedure selected a group of islets that were larger and had an increased proportion of β-cells compared with islets measured in pancreatic sections, but there were no morphologic differences between islets isolated from control and hypoglycemic fetuses. The rates of glucose-stimulated pancreatic islet glucose utilization (126.2 ± 25.3 pmol glucose·islet-1·h-1, hypoglycemic, vs. 93.5 ± 5.5 pmol glucose·islet-1h-1, control, P = 0.47) and oxidation (10.5 ± 1.7 pmol glucose·islet-1·h-1, hypoglycemic, vs. 10.6 ± 1.6 pmol glucose·islet-1·h-1, control) were not different in hypoglycemic fetuses compared with control fetuses. Chronic late gestation hypoglycemia decreased insulin secretion in isolated pancreatic islets by almost 70% in response to direct nonnutrient membrane depolarization and in response to increased extracellular calcium entry. β-Cell ultrastructure was abnormal with markedly distended rough endoplasmic reticulum in three of the seven hypoglycemic fetuses studied, but in vitro analysis of hypoglycemic control islets showed no evidence that these changes represented endoplasmic reticulum stress, as measured by transcription of glucose regulatory protein-78 and processing of X-box binding protein-I. In conclusion, these studies show that chronic hypoglycemia in late gestation decreases insulin secretion by inhibiting the later steps of stimulus-secretion coupling after glucose metabolism, membrane depolarization, and calcium entry. [ABSTRACT FROM AUTHOR]

Insulin-like growth factor-1 (IGF-1) and insulin are important fetal anabolic hormones. Complications of pregnancy, such as placental insufficiency, can lead to fetal growth restriction (FGR) with low-circulating IGF-1 and insulin concentrations and attenuated glucose-stimulated insulin secretion (GSIS), which likely contribute to neonatal glucose dysregulation. We previously demonstrated that a 1-wk infusion of IGF-1 LR3, an IGF-1 analog with low affinity for IGF-binding proteins and high affinity for the IGF-1 receptor, at 6.6 µg·kg -1 ·h -1 into normal fetal sheep increased body weight but lowered insulin concentrations and GSIS. In this study, FGR fetal sheep received either IGF-1 LR3 treatment at 1.17 ± 0.12 μg·kg -1 ·h -1 (LR3; n = 7) or vehicle (VEH; n = 7) for 1 wk. Plasma insulin, glucose, oxygen, and amino acids were measured before starting treatment and at the end of the treatment period. GSIS was measured on the final treatment day. Fetal body weights, insulin, glucose, oxygen, and GSIS were not different between groups. Amino acid concentrations decreased in LR3 (baseline vs. final individual means comparison P = 0.0232) but not in VEH ( P = 0.3866). In summary, a 1-wk IGF-1 LR3 treatment did not improve growth in FGR fetuses. Insulin concentrations and GSIS were not attenuated by IGF-1 LR3, yet circulating amino acids decreased, which could reflect increased amino acid utilization. We speculate that maintaining amino acid concentrations or raising insulin, glucose, and/or oxygen concentrations to values consistent with normally growing fetuses during IGF-1 LR3 treatment may be necessary to increase fetal growth in the setting of placental insufficiency and FGR. NEW & NOTEWORTHY IGF-1 LR3 treatment administered directly into growth-restricted fetal sheep circulation did not improve fetal growth or attenuate circulating insulin or fetal GSIS. Importantly, IGF-1 LR3 treatment reduced circulating amino acids, notably branched-chain amino acids, which have been shown to potentiate GSIS and protein accretion supporting fetal growth.

We previously demonstrated impaired placental nutrient transfer in chorionic somatomammotropin (CSH) RNA interference (RNAi) pregnancies, with glucose transfer being the most impacted. Thus, we hypothesized that despite experimentally elevating maternal glucose, diminished umbilical glucose uptake would persist in CSH RNAi pregnancies, demonstrating the necessity of CSH for adequate placental glucose transfer. Trophectoderm of sheep blastocysts (9 days of gestational age; dGA) were infected with a lentivirus expressing either nontargeting control (CON RNAi; n = 5) or CSH-specific shRNA (CSH RNAi; n = 7) before transfer into recipient sheep. At 126 dGA, pregnancies were fitted with vascular catheters and underwent steady-state metabolic studies ( 3 H 2 O transplacental diffusion) at 137 ± 0 dGA, before and during a maternal hyperglycemic clamp. Umbilical glucose and oxygen uptakes, as well as insulin and IGF1 concentrations, were impaired ( P ≤ 0.01) in CSH RNAi fetuses and were not rescued by elevated maternal glucose. This is partially due to impaired uterine and umbilical blood flow ( P ≤ 0.01). However, uteroplacental oxygen utilization was greater ( P ≤ 0.05) during the maternal hyperglycemic clamp, consistent with greater placental oxidation of substrates. The relationship between umbilical glucose uptake and the maternal-fetal glucose gradient was analyzed, and while the slope (CON RNAi, Y = 29.54X +74.15; CSH RNAi, Y = 19.05X + 52.40) was not different, the y-intercepts and elevation were ( P = 0.003), indicating reduced maximal glucose transport during maternal hyperglycemia. Together, these data suggested that CSH plays a key role in modulating placental metabolism that ultimately promotes maximal placental glucose transfer. NEW & NOTEWORTHY The current study demonstrated a novel, critical autocrine role for chorionic somatomammotropin in augmenting placental glucose transfer and maintaining placental oxidative metabolism. In pregnancies with CSH deficiency, excess glucose in maternal circulation is insufficient to overcome fetal hypoglycemia due to impaired placental glucose transfer and elevated placental metabolic demands. This suggests that perturbations in glucose transfer in CSH RNAi pregnancies are due to compromised metabolic efficiency along with reduced placental mass.

Glucose, lactate, and amino acids are major fetal nutrients. During placental insufficiency-induced intrauterine growth restriction (PI-IUGR), uteroplacental weight-specific oxygen consumption rates are maintained, yet fetal glucose and amino acid supply is decreased and fetal lactate concentrations are increased. We hypothesized that uteroplacental metabolism adapts to PI-IUGR by altering nutrient allocation to maintain oxidative metabolism. Here, we measured nutrient flux rates, with a focus on nutrients shuttled between the placenta and fetus (lactate-pyruvate, glutamine-glutamate, and glycine-serine) in a sheep model of PI-IUGR. PI-IUGR fetuses weighed 40% less and had decreased oxygen, glucose, and amino acid concentrations and increased lactate and pyruvate versus control (CON) fetuses. Uteroplacental weight-specific rates of oxygen, glucose, lactate, and pyruvate uptake were similar. In PI-IUGR, fetal glucose uptake was decreased and pyruvate output was increased. In PI-IUGR placental tissue, pyruvate dehydrogenase (PDH) phosphorylation was decreased and PDH activity was increased. Uteroplacental glutamine output to the fetus and expression of genes regulating glutamine-glutamate metabolism were lower in PI-IUGR. Fetal glycine uptake was lower in PI-IUGR, with no differences in uteroplacental glycine or serine flux. These results suggest increased placental utilization of pyruvate from the fetus, without higher maternal glucose utilization, and lower fetoplacental amino acid shuttling during PI-IUGR. Mechanistically, AMP-activated protein kinase (AMPK) activation was higher and associated with thiobarbituric acid-reactive substances (TBARS) content, a marker of oxidative stress, and PDH activity in the PI-IUGR placenta, supporting a potential link between oxidative stress, AMPK, and pyruvate utilization. These differences in fetoplacental nutrient sensing and shuttling may represent adaptive strategies enabling the placenta to maintain oxidative metabolism. NEW & NOTEWORTHY These results suggest increased placental utilization of pyruvate from the fetus, without higher maternal glucose uptake, and lower amino acid shuttling in the placental insufficiency-induced intrauterine growth restriction (PI-IUGR) placenta. AMPK activation was associated with oxidative stress and PDH activity, supporting a putative link between oxidative stress, AMPK, and pyruvate utilization. These differences in fetoplacental nutrient sensing and shuttling may represent adaptive strategies enabling the placenta to maintain oxidative metabolism at the expense of fetal growth.

Fetal hypoxemia decreases insulin and increases cortisol and norepinephrine concentrations and may restrict growth by decreasing glucose utilization and altering substrate oxidation. Specifically, we hypothesized that hypoxemia would decrease fetal glucose oxidation and increase lactate and pyruvate production. We tested this by measuring whole body glucose oxidation and lactate production, and molecular pathways in liver, muscle, adipose, and pancreas tissues of fetuses exposed to maternal hypoxemia for 9 days (HOX) compared with control fetal sheep (CON) in late gestation. Fetuses with more severe hypoxemia had lower whole body glucose oxidation rates, and HOX fetuses had increased lactate production from glucose. In muscle and adipose tissue, expression of the glucose transporter GLUT4 was decreased. In muscle, pyruvate kinase ( PKM ) and lactate dehydrogenase B ( LDHB ) expression was decreased. In adipose tissue, LDHA and lactate transporter ( MCT1 ) expression was increased. In liver, there was decreased gene expression of PKLR and MPC2 and phosphorylation of PDH, and increased LDHA gene and LDH protein abundance. LDH activity, however, was decreased only in HOX skeletal muscle. There were no differences in basal insulin signaling across tissues, nor differences in pancreatic tissue insulin content, β-cell area, or genes regulating β-cell function. Collectively, these results demonstrate coordinated metabolic responses across tissues in the hypoxemic fetus that limit glucose oxidation and increase lactate and pyruvate production. These responses may be mediated by hypoxemia-induced endocrine responses including increased norepinephrine and cortisol, which inhibit pancreatic insulin secretion resulting in lower insulin concentrations and decreased stimulation of glucose utilization. NEW & NOTEWORTHY Hypoxemia lowered fetal glucose oxidation rates, based on severity of hypoxemia, and increased lactate production. This was supported by tissue-specific metabolic responses that may result from increased norepinephrine and cortisol concentrations, which decrease pancreatic insulin secretion and insulin concentrations and decrease glucose utilization. This highlights the vulnerability of metabolic pathways in the fetus and demonstrates that constrained glucose oxidation may represent an early event in response to sustained hypoxemia and fetal growth restriction.

Insulin and insulin-like growth factor-1 (IGF-1) are fetal hormones critical to establishing normal fetal growth. Experimentally elevated IGF-1 concentrations during late gestation increase fetal weight but lower fetal plasma insulin concentrations. We therefore hypothesized that infusion of an IGF-1 analog for 1 wk into late gestation fetal sheep would attenuate fetal glucose-stimulated insulin secretion (GSIS) and insulin secretion in islets isolated from these fetuses. Late gestation fetal sheep received infusions with IGF-1 LR3 (IGF-1, n = 8), an analog of IGF-1 with low affinity for the IGF binding proteins and high affinity for the IGF-1 receptor, or vehicle control (CON, n = 9). Fetal GSIS was measured with a hyperglycemic clamp (IGF-1, n = 8; CON, n = 7). Fetal islets were isolated, and insulin secretion was assayed in static incubations (IGF-1, n = 8; CON, n = 7). Plasma insulin and glucose concentrations in IGF-1 fetuses were lower compared with CON ( P = 0.0135 and P = 0.0012, respectively). During the GSIS study, IGF-1 fetuses had lower insulin secretion compared with CON ( P = 0.0453). In vitro, glucose-stimulated insulin secretion remained lower in islets isolated from IGF-1 fetuses ( P = 0.0447). In summary, IGF-1 LR3 infusion for 1 wk into fetal sheep lowers insulin concentrations and reduces fetal GSIS. Impaired insulin secretion persists in isolated fetal islets indicating an intrinsic islet defect in insulin release when exposed to IGF-1 LR3 infusion for 1 wk. We speculate this alteration in the insulin/IGF-1 axis contributes to the long-term reduction in β-cell function in neonates born with elevated IGF-1 concentrations following pregnancies complicated by diabetes or other conditions associated with fetal overgrowth. NEW & NOTEWORTHY After a 1-wk infusion of IGF-1 LR3, late gestation fetal sheep had lower plasma insulin and glucose concentrations, reduced fetal glucose-stimulated insulin secretion, and decreased fractional insulin secretion from isolated fetal islets without differences in pancreatic insulin content.

Insulin-like growth factor-1 (IGF-1) is an important fetal growth factor. However, the role of fetal IGF-1 in increasing placental blood flow, nutrient transfer, and nutrient availability to support fetal growth and protein accretion is not well understood. Catheterized fetuses from late gestation pregnant sheep received an intravenous infusion of LR3 IGF-1 (LR3 IGF-1; n = 8) or saline (SAL; n = 8) for 1 wk. Sheep then underwent a metabolic study to measure uterine and umbilical blood flow, nutrient uptake rates, and fetal protein kinetic rates. By the end of the infusion, fetal weights were not statistically different between groups (SAL: 3.260 ± 0.211 kg, LR3 IGF-1: 3.682 ± 0.183; P = 0.15). Fetal heart, adrenal gland, and spleen weights were higher ( P < 0.05), and insulin was lower in LR3 IGF-1 ( P < 0.05). Uterine and umbilical blood flow and umbilical uptake rates of glucose, lactate, and oxygen were similar between groups. Umbilical amino acid uptake rates were lower in LR3 IGF-1 ( P < 0.05) as were fetal concentrations of multiple amino acids. Fetal protein kinetic rates were similar. LR3 IGF-1 skeletal muscle had higher myoblast proliferation ( P < 0.05). In summary, LR3 IGF-1 infusion for 1 wk into late gestation fetal sheep increased the weight of some fetal organs. However, because umbilical amino acid uptake rates and fetal plasma amino acid concentrations were lower in the LR3 IGF-1 group, we speculate that animals treated with LR3 IGF-1 can efficiently utilize available nutrients to support organ-specific growth in the fetus rather than by stimulating placental blood flow or nutrient transfer to the fetus. NEW & NOTEWORTHY After a 1-wk infusion of LR3 IGF-1, late gestation fetal sheep had lower umbilical uptake rates of amino acids, lower fetal arterial amino acid and insulin concentrations, and lower fetal oxygen content; however, LR-3 IGF-1-treated fetuses were still able to effectively utilize the available nutrients and oxygen to support organ growth and myoblast proliferation.

Chorionic somatomammotropin (CSH) is one of the most abundantly produced placental hormones, yet its exact function remains elusive. Near-term [135 days of gestational age (dGA)], CSH RNA interference (RNAi) results in two distinct phenotypes: 1 ) pregnancies with intrauterine growth restriction (IUGR), and 2 ) pregnancies with normal fetal and placental weights. Here, we report the physiological changes in CSH RNAi pregnancies without IUGR. The trophectoderm of hatched blastocysts (9 dGA) were infected with lentiviral-constructs expressing either a scrambled control (Control RNAi) or CSH-specific shRNA (CSH RNAi), prior to transfer into synchronized recipient ewes. At 126 dGA, Control RNAi ( n = 6) and CSH RNAi ( n = 6) pregnancies were fitted with maternal and fetal catheters. Uterine and umbilical blood flows were measured at 132 dGA and nutrient uptakes were calculated by the Fick's principle. Control RNAi and CSH RNAi pregnancies were compared by analysis of variance, and significance was set at P ≤ 0.05. Absolute (mL/min) and relative (mL/min/kg fetus) uterine blood flows were reduced ( P ≤ 0.05) in CSH RNAi pregnancies, but umbilical flows were not impacted. The uterine artery-to-vein glucose gradient (mmol/L) was significantly ( P ≤ 0.05) increased. The uteroplacental glucose uptake (μmoL/min/kg placenta) was increased ( P ≤ 0.05), whereas umbilical glucose uptake (μmoL/min/kg fetus) was reduced. Our results demonstrate that CSH RNAi has significant physiological ramifications, even in the absence of IUGR, and comparing CSH RNAi pregnancies exhibiting both IUGR and non-IUGR phenotypes may help determine the direct effects of CSH and its potential impact on fetal development.

The effect of chronic of hyperinsulinemia in the fetal liver is poorly understood. Here, we produced hyperinsulinemia with euglycemia for ∼8 days in fetal sheep [hyperinsulinemic (INS)] at 0.9 gestation. INS fetuses had increased insulin and decreased oxygen and amino acid (AA) concentrations compared with saline-infused fetuses [control (CON)]. Glucose (whole body) utilization rates were increased, as expected, in INS fetuses. In the liver, however, there were few differences in genes and metabolites related to glucose and lipid metabolism and no activation of insulin signaling proteins (Akt and mTOR). There was increased p-AMPK activation and decreased mitochondrial mass ( PGC1A expression, mitochondrial DNA content) in INS livers. Using an unbiased multivariate analysis with 162 metabolites, we identified effects on AA and one-carbon metabolism in the INS liver. Expression of the transaminase BCAT2 and glutaminase genes GLS1 and GLS2 was decreased, supporting decreased AA utilization. We further evaluated the roles of hyperinsulinemia and hypoxemia, both present in INS fetuses, on outcomes in the liver. Expression of PGC1A correlated only with hyperinsulinemia, p-AMPK correlated only with hypoxemia, and other genes and metabolites correlated with both hyperinsulinemia and hypoxemia. In fetal hepatocytes, acute treatment with insulin activated p-Akt and decreased PGC1A, whereas hypoxia activated p-AMPK. Overall, chronic hyperinsulinemia produced greater effects on amino acid metabolism compared with glucose and lipid metabolism and a novel effect on one-carbon metabolism in the fetal liver. These hepatic metabolic responses may result from the downregulation of insulin signaling and antagonistic effects of hypoxemia-induced AMPK activation that develop with chronic hyperinsulinemia.

In a sheep model of intrauterine growth restriction (IUGR) produced from placental insufficiency, late gestation fetuses had smaller skeletal muscle mass, myofiber area, and slower muscle protein accretion rates compared with normally growing fetuses. We hypothesized that IUGR fetal muscle develops adaptations that divert amino acids (AAs) from protein accretion and activate pathways that conserve substrates for other organs. We placed hindlimb arterial and venous catheters into late gestation IUGR ( n = 10) and control (CON, n = 8) fetal sheep and included an external iliac artery flow probe to measure hindlimb AA uptake rates. Arterial and venous plasma samples and biceps femoris muscle were analyzed by mass spectrometry-based metabolomics. IUGR fetuses had greater abundance of metabolites enriched within the alanine, aspartate, and glutamate metabolism pathway compared with CON. Net uptake rates of branched-chain AA (BCAA) were lower by 42%-73%, and muscle ammoniagenic AAs (alanine, glycine, and glutamine) were lower by 107%-158% in IUGR hindlimbs versus CON. AA uptake rates correlated with hindlimb weight; the smallest hindlimbs showed net release of ammoniagenic AAs. Gene expression levels indicated a decrease in BCAA catabolism in IUGR muscle. Plasma purines were lower and plasma uric acid was higher in IUGR versus CON, possibly a reflection of ATP conservation. We conclude that IUGR skeletal muscle has lower BCAA uptake and develops adaptations that divert AAs away from protein accretion into alternative pathways that sustain global energy production and nitrogen disposal in the form of ammoniagenic AAs for metabolism in other organs.

Fetal hypoxemia is associated with pregnancy conditions that cause an early activation of fetal glucose production. However, the independent role of hypoxemia to activate this pathway is not well understood. We hypothesized that fetal hypoxemia would activate fetal glucose production by decreasing umbilical glucose uptake and increasing counter-regulatory hormone concentrations. We induced hypoxemia for 9 days with maternal tracheal N 2 gas insufflation to reduce maternal and fetal arterial Po 2 by ~20% (HOX) compared with fetuses from ewes receiving intratracheal compressed air (CON). At 0.9 of gestation, fetal metabolic studies were performed ( n = 7 CON, 11 HOX). Umbilical blood flow rates, net fetal oxygen and glucose uptake rates, and fetal arterial plasma glucose concentrations were not different between the two groups. Fetal glucose utilization rates were lower in HOX versus CON fetuses but not different from umbilical glucose uptake rates, demonstrating the absence of endogenous glucose production. In liver tissue, mRNA expression of gluconeogenic genes G6PC (P < 0.01) and PCK1 ( P = 0.06) were six- and threefold greater in HOX fetuses versus CON fetuses. Increased fetal norepinephrine and cortisol concentrations and hepatic G6PC and PCK1 expression were inversely related to fetal arterial Po 2 . These findings support a role for fetal hypoxemia to act with counter-regulatory hormones to potentiate fetal hepatic gluconeogenic gene expression. However, in the absence of decreased net fetal glucose uptake rates and plasma glucose concentrations, hypoxemia-induced gluconeogenic gene activation is not sufficient to activate fetal glucose production.

Intrauterine growth-restricted (IUGR) fetal sheep have increased hepatic glucose production (HGP) that is resistant to suppression during a hyperinsulinemic-isoglycemic clamp (insulin clamp). We hypothesized that the IUGR fetal liver would have activation of metabolic and signaling pathways that support HGP and inhibition of insulin-signaling pathways. To test this, we used transcriptomic profiling with liver samples from control (CON) and IUGR fetuses receiving saline or an insulin clamp. The IUGR liver had upregulation of genes associated with gluconeogenesis/glycolysis, transcription factor regulation, and cytokine responses and downregulation of genes associated with cholesterol synthesis, amino acid degradation, and detoxification pathways. During the insulin clamp, genes associated with cholesterol synthesis and innate immune response were upregulated in CON and IUGR. There were 20-fold more genes differentially expressed during the insulin clamp in IUGR versus CON. These genes were associated with proteasome activation and decreased amino acid and lipid catabolism. We found increased TRB3, JUN , MYC , and SGK1 expression and decreased PTPRD expression as molecular targets for increased HGP in IUGR. As candidate genes for resistance to insulin's suppression of HGP, expression of JUN , MYC , and SGK1 increased more during the insulin clamp in CON compared with IUGR. Metabolites were measured with 1 H-nuclear magnetic resonance and support increased amino acid concentrations, decreased mitochondria activity and energy state, and increased cell stress in the IUGR liver. These results demonstrate a robust response, beyond suppression of HGP, during the insulin clamp and coordinate responses in glucose, amino acid, and lipid metabolism in the IUGR fetus.

Overcoming impaired growth in an intrauterine growth-restricted (IUGR) fetus has potential to improve neonatal morbidity, long-term growth, and metabolic health outcomes. The extent to which fetal anabolic capacity persists as the IUGR condition progresses is not known. We subjected fetal sheep to chronic placental insufficiency and tested whether prolonged amino acid infusion would increase protein accretion in these IUGR fetuses. IUGR fetal sheep were infused for 10 days with either mixed amino acids providing ~2 g·kg -1 ·day -1 (IUGR-AA) or saline (IUGR-Sal) during late gestation. At the end of the infusion, fetal plasma leucine, isoleucine, lysine, methionine, and arginine concentrations were higher in the IUGR-AA than IUGR-Sal group ( P < 0.05). Fetal plasma glucose, oxygen, insulin, IGF-1, cortisol, and norepinephrine concentrations were similar between IUGR groups, but glucagon concentrations were fourfold higher in the IUGR-AA group ( P < 0.05). Net umbilical amino acid uptake rate did not differ between IUGR groups; thus the total amino acid delivery rate (net umbilical amino acid uptake + infusion rate) was higher in the IUGR-AA than IUGR-Sal group (30 ± 4 vs. 19 ± 1 μmol·kg -1 ·min -1 , P < 0.05). Net umbilical glucose, lactate, and oxygen uptake rates were similar between IUGR groups. Fetal leucine oxidation rate, measured using a leucine tracer, was higher in the IUGR-AA than IUGR-Sal group (2.5 ± 0.3 vs. 1.7 ± 0.3 μmol·kg -1 ·min -1 , P < 0.05). Fetal protein accretion rate was not statistically different between the IUGR groups (1.6 ± 0.4 and 0.8 ± 0.3 μmol·kg -1 ·min -1 in IUGR-AA and IUGR-Sal, respectively) due to variability in response to amino acids. Prolonged amino acid infusion into IUGR fetal sheep increased leucine oxidation rates with variable anabolic response.

Intrauterine growth restriction (IUGR) in premature newborns increases the risk for bronchopulmonary dysplasia, a chronic lung disease characterized by disrupted pulmonary angiogenesis and alveolarization. We previously showed that experimental IUGR impairs angiogenesis; however, mechanisms that impair pulmonary artery endothelial cell (PAEC) function are uncertain. The NF-κB pathway promotes vascular growth in the developing mouse lung, and we hypothesized that IUGR disrupts NF-κB-regulated proangiogenic targets in fetal PAEC. PAECs were isolated from the lungs of control fetal sheep and sheep with experimental IUGR from an established model of chronic placental insufficiency. Microarray analysis identified suppression of NF-κB signaling and significant alterations in extracellular matrix (ECM) pathways in IUGR PAEC, including decreases in collagen 4α1 and laminin α4, components of the basement membrane and putative NF-κB targets. In comparison with controls, immunostaining of active NF-κB complexes, NF-κB-DNA binding, baseline expression of NF-κB subunits p65 and p50, and LPS-mediated inducible activation of NF-κB signaling were decreased in IUGR PAEC. Although pharmacological NF-κB inhibition did not affect angiogenic function in IUGR PAEC, angiogenic function of control PAEC was reduced to a similar degree as that observed in IUGR PAEC. These data identify reductions in endothelial NF-κB signaling as central to the disrupted angiogenesis observed in IUGR, likely by impairing both intrinsic PAEC angiogenic function and NF-κB-mediated regulation of ECM components necessary for vascular development. These data further suggest that strategies that preserve endothelial NF-κB activation may be useful in lung diseases marked by disrupted angiogenesis such as IUGR.

Acute amino acid (AA) infusion increases AA oxidation rates in normal late gestation fetal sheep. Because the fetal oxygen consumption rate does not change with increased AA oxidation, we hypothesized that AA infusion would suppress glucose oxidation pathways and that the additional carbon supply from AA would activate hepatic glucose production. To test this, late gestation fetal sheep were infused intravenously for 3 h with saline or exogenous AA (AA). Glucose tracer metabolic studies were performed and skeletal muscle and liver tissues samples were collected. AA infusion increased fetal arterial plasma branched chain AA, cortisol, and glucagon concentrations. Fetal glucose utilization rates were similar between basal and AA periods, yet the fraction of glucose oxidized and the glucose oxidation rate were decreased by 40% in the AA period. AA infusion increased expression of PDK4 , an inhibitor of glucose oxidation, nearly twofold in muscle and liver. In liver, AA infusion tended to increase PCK1 gluconeogenic gene and PCK1 correlated with plasma cortisol concentrations. AA infusion also increased liver mRNA expression of the lactate transporter gene ( MCT1) , protein expression of GLUT2 and LDHA, and phosphorylation of AMPK, 4EBP1, and S6 proteins. In isolated fetal hepatocytes, AA supplementation increased glucose production and PCK1 , LDHA , and MCT1 gene expression. These results demonstrate that AA infusion into fetal sheep competitively suppresses glucose oxidation and potentiates hepatic glucose production. These metabolic patterns support flexibility in fetal metabolism in response to increased nutrient substrate supply while maintaining a relatively stable rate of oxidative metabolism., (Copyright © 2017 the American Physiological Society.)