Intrauterine growth restriction (IUGR) increases the risk for bronchopulmonary dysplasia (BPD). Abnormal lung structure has been noted in animal models of IUGR, but whether IUGR adversely impacts fetal pulmonary vascular development and pulmonary artery endothelial cell (PAEC) function is unknown. We hypothesized that IUGR would decrease fetal pulmonary alveolarization, vascular growth, and in vitro PAEC function. Studies were performed in an established model of severe placental insufficiency and IUGR induced by exposing pregnant sheep to elevated temperatures. Alveolarization, quantified by radial alveolar counts, was decreased 20% (P < 0.005) in IUGR fetuses. Pulmonary vessel density was decreased 44% (P < 0.01) in IUGR fetuses. In vitro, insulin increased control PAEC migration, tube formation, and nitric oxide (NO) production. This response was absent in IUGR PAECs. VEGFA stimulated tube formation, and NO production also was absent. In control PAECs, insulin increased cell growth by 68% (P < 0.0001). Cell growth was reduced in IUGR PAECs by 29% at baseline (P < 0.01), and the response to insulin was attenuated (P < 0.005). Despite increased basal and insulin-stimulated Akt phosphorylation in IUGR PAECs, endothelial NO synthase (eNOS) protein expression as well as basal and insulin-stimulated eNOS phosphorylation were decreased in IUGR PAECs. Both VEGFA and VEGFR2 also were decreased in IUGR PAECs. We conclude that fetuses with IUGR are characterized by decreased alveolar and vascular growth and PAEC dysfunction in vitro. This may contribute to the increased risk for adverse respiratory outcomes and BPD in infants with IUGR.

Reduced maternal glucose supply to the fetus and resulting fetal hypoglycemia and hypoinsulinemia activate fetal glucose production as a means to maintain cellular glucose uptake. However, this early activation of fetal glucose production may be accompanied by hepatic insulin resistance. We tested the capacity of a physiological increase in insulin to suppress fetal hepatic gluconeogenic gene activation following sustained hypoglycemia to determine whether hepatic insulin sensitivity is maintained. Control fetuses (CON), hypoglycemic fetuses induced by maternal insulin infusion for 8 wk (HG), and 8 wk HG fetuses that received an isoglycemic insulin infusion for the final 7 days (HG+INS) were studied. Glucose and insulin concentrations were 60% lower in HG compared with CON fetuses. Insulin was 50% higher in HG+INS compared with CON and four-fold higher compared with HG fetuses. Expression of the hepatic gluconeogenic genes, PCK1, G6PC, FBP1, GLUT2, and PGC1A was increased in the HG and reduced in the HG+INS liver. Expression of the insulin-regulated glycolytic and lipogenic genes, PFKL and FAS, was increased in the HG+INS liver. Total FOXO1 protein expression, a gluconeogenic activator, was 60% higher in the HG liver. Despite low glucose, insulin, and IGF1 concentrations, phosphorylation of AKT and ERK was higher in the HG liver. Thus, a physiological increase in fetal insulin is sufficient for suppression of gluconeogenic genes and activation of glycolytic and lipogenic genes in the HG fetal liver. These results demonstrate that fetuses exposed to sustained hypoglycemia have maintained hepatic insulin action in contrast to fetuses exposed to placental insufficiency.

Maliszewski AM, Gadhia MM, O'Meara MC, Thorn SR, Rozance PJ, Brown LD. Prolonged infusion of amino acids increases leucine oxidation in fetal sheep. Am J Physiol Endocrinol Metab 302: E1483-E1492, 2012. First published March 27, 2012 doi:10.1152/ajpendo.00026.2012.-Maternal high-protein supplements designed to increase birth weight have not been successful. We recently showed that maternal amino acid infusion into pregnant sheep resulted in competitive inhibition of amino acid transport across the placenta and did not increase fetal protein accretion rates. To bypass placental transport, singleton fetal sheep were intravenously infused with an amino acid mixture (AA, n ϭ 8) or saline [control (Con), n ϭ 10] for ϳ12 days during late gestation. Fetal leucine oxidation rate increased in the AA group (3.1 Ϯ 0.5 vs. 1.4 Ϯ 0.6 mol·min Ϫ1 ·kg Ϫ1 , P Ͻ 0.05). Fetal protein accretion (2.6 Ϯ 0.5 and 2.2 Ϯ 0.6 mol·min Ϫ1 ·kg Ϫ1 in AA and Con, respectively), synthesis (6.2 Ϯ 0.8 and 7.0 Ϯ 0.9 mol·min Ϫ1 ·kg Ϫ1 in AA and Con, respectively), and degradation (3.6 Ϯ 0.6 and 4.5 Ϯ 1.0 mol·min Ϫ1 ·kg Ϫ1in AA and Con, respectively) rates were similar between groups. Net fetal glucose uptake decreased in the AA group (2.8 Ϯ 0.4 vs. 3.9 Ϯ 0.1 mg·kg Ϫ1 ·min Ϫ1 , P Ͻ 0.05). The glucose-O2 quotient also decreased over time in the AA group (P Ͻ 0.05). Fetal insulin and IGF-I concentrations did not change. Fetal glucagon increased in the AA group (119 Ϯ 24 vs. 59 Ϯ 9 pg/ml, P Ͻ 0.05), and norepinephrine (NE) also tended to increase in the AA group (785 Ϯ 181 vs. 419 Ϯ 76 pg/ml, P ϭ 0.06). Net fetal glucose uptake rates were inversely proportional to fetal glucagon (r 2 ϭ 0.38, P Ͻ 0.05), cortisol (r 2 ϭ 0.31, P Ͻ 0.05), and NE (r 2 ϭ 0.59, P Ͻ 0.05) concentrations. Expressions of components in the mammalian target of rapamycin signaling pathway in fetal skeletal muscle were similar between groups. In summary, prolonged infusion of amino acids directly into normally growing fetal sheep increased leucine oxidation. Amino acid-stimulated increases in fetal glucagon, cortisol, and NE may contribute to a shift in substrate oxidation by the fetus from glucose to amino acids. pregnancy; insulin; fetal protein metabolism; fetal growth PREVIOUS ATTEMPTS TO IMPROVE fetal outcomes with use of maternal high-protein supplementation have actually increased the risk for small-for-gestational-age birth and infant mortality (26,36). Similarly, supplementation of pregnant sheep with intravenous amino acids for several days at the end of gestation demonstrated adverse fetal effects, including hypoxia and respiratory and metabolic acidosis (34). The mechanisms that explain poor fetal outcomes in response to maternal protein supplementation during pregnancy are unknown.Experiments using normal pregnant sheep have led to some testable explanations for how high-protein supplementation during pregnancy might adversely affect fetal outcomes (4). Short-and long-term maternal amino acid infusions using mixtures of amino acids with a higher essential-nonessential ...

Amino acids and glucose acutely stimulate fetal insulin secretion. In isolated adult pancreatic islets, amino acids potentiate glucose-stimulated insulin secretion (GSIS), but whether amino acids have this same effect in the fetus is unknown. Therefore, we tested the effects of increased fetal amino acid supply on GSIS and morphology of the pancreas. We hypothesized that increasing fetal amino acid supply would potentiate GSIS. Singleton fetal sheep received a direct intravenous infusion of an amino acid mixture (AA) or saline (CON) for 10-14 days during late gestation to target a 25-50% increase in fetal branched-chain amino acids (BCAA). Early-phase GSIS increased 150% in the AA group (P < 0.01), and this difference was sustained for the duration of the hyperglycemic clamp (105 min) (P < 0.05). Glucose-potentiated arginine-stimulated insulin secretion (ASIS), pancreatic insulin content, and pancreatic glucagon content were similar between groups. β-Cell mass and area were unchanged between groups. Baseline and arginine-stimulated glucagon concentrations were increased in the AA group (P < 0.05). Pancreatic α-cell mass and area were unchanged. Fetal and pancreatic weights were similar. We conclude that a sustained increase of amino acid supply to the normally growing late-gestation fetus potentiated fetal GSIS but did not affect the morphology or insulin content of the pancreas. We speculate that increased β-cell responsiveness (insulin secretion) following increased amino acid supply may be due to increased generation of secondary messengers in the β-cell. This may be enhanced by the paracrine action of glucagon on the β-cell.

-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 ϭ 7) or saline (C group, n ϭ 4) for 4 days during late gestation. In the AA group, fetal plasma leucine, isoleucine, valine, and lysine concentrations were increased (P Ͻ 0.05), and threonine was decreased (P Ͻ 0.05). In the AA group, fetal arterial pH (7.365 Ϯ 0.007 day 0 vs. 7.336 Ϯ 0.012 day 4, P Ͻ 0.005), hemoglobinoxygen saturation (46.2 Ϯ 2.6 vs. 37.8 Ϯ 3.6%, P Ͻ 0.005), and total oxygen content

Decreased glucose transfer to the fetus is characteristic of pregnancies complicated by maternal under nutrition and placental insufficiency. Chronic experimental restriction of glucose transfer to the sheep fetus for the final 40% of gestation with a maternal insulin infusion (HG fetuses) results in fetal hypoglycemia, hypoinsulinemia, and decreased rates of fetal growth and protein accretion compared to controls (CON). Lower rates of fetal protein accretion are due to increased fetal protein breakdown and not decreased protein synthesis. However, the specific skeletal muscle pathways responsible for increased protein breakdown have not been determined. Nor has it been determined if low fetal glucose or insulin concentrations are more important for regulating these skeletal muscle protein breakdown pathways. We tested whether chronic restriction of glucose transfer to the fetus increased the ubiquitin–proteosome pathway or autophagy‐lysosome pathway in fetal sheep skeletal muscle and found no evidence for an increase in the autophagy‐lysosome pathway. However, HG fetuses had increase mRNA expression of MaFBx1 (twofold, P <0.01) and a trend for increased mRNA expression of MuRF1 (P =0.08) compared to CON. A subset of chronically hypoglycemic fetuses received an isoglycemic insulin infusion for the final 7 days of the maternal insulin infusion (HG + INS fetuses) and had MaFBx1 and MuRF1 mRNA concentrations similar to CON fetuses. These results demonstrate that fetuses exposed to sustained hypoglycemia have decreased protein accretion due to activation of the skeletal muscle ubiquitin–proteosome pathway and that a fetal hyperinsulinemic clamp can suppress this pathway even in the context of continued hypoglycemia.

Maternal undernutrition during pregnancy and placental insufficiency are characterized by impaired development of fetal pancreatic β-cells. Prolonged reduced glucose supply to the fetus is a feature of both. It is unknown if reduced glucose supply, independent of other complications of maternal undernutrition and placental insufficiency, would cause similar β-cell defects. Therefore, we measured fetal insulin secretion and β-cell mass following prolonged reduced fetal glucose supply in sheep. We also tested whether restoring physiological insulin concentrations would correct any β-cell defects. Pregnant sheep received either a direct saline infusion (CON = control, n = 5) or an insulin infusion (HG = hypoglycemic, n = 5) for 8 wk in late gestation (75 to 134 days) to decrease maternal glucose concentrations and reduce fetal glucose supply. A separate group of HG fetuses also received a direct fetal insulin infusion for the final week of the study with a dextrose infusion to prevent a further fall in glucose concentration [hypoglycemic + insulin (HG+I), n = 4]. Maximum glucose-stimulated insulin concentrations were 45% lower in HG fetuses compared with CON fetuses. β-Cell, pancreatic, and fetal mass were 50%, 37%, and 40% lower in HG compared with CON fetuses, respectively (P < 0.05). Insulin secretion and β-cell mass did not improve in the HG+I fetuses. These results indicate that chronically reduced fetal glucose supply is sufficient to reduce pancreatic insulin secretion in response to glucose, primarily due to reduced pancreatic and β-cell mass, and is not correctable with insulin.