Mitochondrial respiration is lower in the intrauterine growth-restricted fetal sheep heart.

Fetuses affected by intrauterine growth restriction have an increased risk of developing heart disease and failure in adulthood. Compared with controls, late gestation intrauterine growth-restricted (IUGR) fetal sheep have fewer binucleated cardiomyocytes, reflecting a more immature heart, which may reduce mitochondrial capacity to oxidize substrates. We hypothesized that the late gestation IUGR fetal heart has a lower capacity for mitochondrial oxidative phosphorylation. Left (LV) and right (RV) ventricles from IUGR and control (CON) fetal sheep at 90% gestation were harvested. Mitochondrial respiration (states 1-3, LeakOmy, and maximal respiration) in response to carbohydrates and lipids, citrate synthase (CS) activity, protein expression levels of mitochondrial oxidative phosphorylation complexes (CI-CV), and mRNA expression levels of mitochondrial biosynthesis regulators were measured. The carbohydrate and lipid state 3 respiration rates were lower in IUGR than CON, and CS activity was lower in IUGR LV than CON LV. However, relative CII and CV protein levels were higher in IUGR than CON; CV expression level was higher in IUGR than CON. Genes involved in lipid metabolism had lower expression in IUGR than CON. In addition, the LV and RV demonstrated distinct differences in oxygen flux and gene expression levels, which were independent from CON and IUGR status. Low mitochondrial respiration and CS activity in the IUGR heart compared with CON are consistent with delayed cardiomyocyte maturation, and CII and CV protein expression levels may be upregulated to support ATP production. These insights will provide a better understanding of fetal heart development in an adverse in utero environment. KEY POINTS: Growth-restricted fetuses have a higher risk of developing and dying from cardiovascular diseases in adulthood. Mitochondria are the main supplier of energy for the heart. As the heart matures, the substrate preference of the mitochondria switches from carbohydrates to lipids. We used a sheep model of intrauterine growth restriction to study the capacity of the mitochondria in the heart to produce energy using either carbohydrate or lipid substrates by measuring how much oxygen was consumed. Our data show that the mitochondria respiration levels in the growth-restricted fetal heart were lower than in the normally growing fetuses, and the expression levels of genes involved in lipid metabolism were also lower. Differences between the right and left ventricles that are independent of the fetal growth restriction condition were identified. These results indicate an impaired metabolic maturation of the growth-restricted fetal heart associated with a decreased capacity to oxidize lipids postnatally.

IGF-1 infusion increases growth in fetal sheep when euinsulinemia is maintained.

Insulin-like growth factor 1 (IGF-1) is a critical fetal anabolic hormone. IGF-1 infusion to the normally growing sheep fetus increases the weight of some organs but does not consistently increase body weight. However, IGF-1 infusion profoundly decreases fetal plasma insulin concentrations, which may limit fetal growth potential. In this study, normally growing late gestation fetal sheep received an intravenous infusion of either: IGF-1 (IGF), IGF-1 with insulin and dextrose to maintain fetal euinsulinemia and euglycemia (IGF+INS), or vehicle control (CON) for one week. The fetus underwent a metabolic study immediately prior to infusion start and after one week of the infusion to measure uterine and umbilical uptake rates of nutrients and oxygen. IGF+INS fetuses were 23% heavier than CON (P=0.0081) and had heavier hearts, livers, and adrenal glands than IGF and CON (P<0.01). By design, final fetal insulin concentrations in IGF were 62% and 65% lower than IGF+INS and CON, respectively. Final glucose concentrations were similar in all groups. IGF+INS had lower final oxygen content than IGF and CON (P<0.0001) and lower final amino acid concentrations than CON (P=0.0002). Final umbilical oxygen uptake was higher in IGF+INS compared to IGF and CON (P<0.05). Final umbilical uptake of several essential amino acids was higher in IGF+INS compared to CON (P<0.05). In summary, maintaining euinsulinemia and euglycemia during fetal IGF-1 infusion is necessary to maximally support body growth. We speculate that IGF-1 and insulin stimulate placental nutrient transport to support fetal growth.

Absence of Metformin in Fetal Circulation Following Maternal Administration in Late Gestation Pregnant Sheep.

In human pregnancy, metformin administered to the mother crosses the placenta resulting in metformin exposure to the fetus. However, the effects of metformin exposure on the fetus are poorly understood and difficult to study in humans. Pregnant sheep are a powerful large animal model for studying fetal physiology. The objective of this study was to determine if maternally administered metformin at human dose-equivalent concentrations crosses the ovine placenta and equilibrates in the fetal circulation. To test this, metformin was administered to the pregnant ewe via continuous intravenous infusion or supplementation in the drinking water. Both administration routes increased maternal metformin concentrations to human dose-equivalent concentrations of ~ 10 µM, yet metformin was negligible in the fetus even after 3-4 days of maternal administration. In cotyledon and caruncle tissue, expression levels of the major metformin uptake transporter organic cation transporter 1 (OCT1) were < 1% of expression levels in the fetal liver, a tissue with abundant expression. Expression of other putative uptake transporters OCT2 and OCT3, and efflux transporters multidrug and toxin extrusion (MATE)1 and MATE2were more abundant. These results demonstrate that the ovine placenta is impermeable to maternal metformin administration. This is likely due to anatomical differences and increased interhaemal distance between the maternal and umbilical circulations in the ovine versus human placenta limiting placental metformin transport.

Increased hepatic glucose production with lower oxidative metabolism in the growth-restricted fetus.

Fetuses with growth restriction (FGR) have an early activation of hepatic glucose production (HGP), a hallmark of type 2 diabetes (T2D). Here we used fetal hepatic catheterization to directly measure HGP and substrate flux in an FGR sheep model. We hypothesized that FGR fetuses would have increased hepatic lactate and amino acid uptake to support increased HGP. Indeed, FGR compared to normal (CON) fetuses had increased HGP and activation of gluconeogenic genes. Unexpectedly, hepatic pyruvate output was increased while hepatic lactate and gluconeogenic amino acid uptake rates were decreased in FGR fetal liver. Hepatic oxygen consumption and total substrate uptake rates were lower. In FGR liver tissue, metabolite abundance, 13C-metabolite labeling, enzyme activity, and gene expression support decreased pyruvate oxidation and increased lactate production. Isolated hepatocytes from FGR fetuses had greater intrinsic capacity for lactate-fueled glucose production. FGR livers also had lower energy (ATP) and redox state (NADH:NAD+). Thus, reduced hepatic oxidative metabolism may make carbons available for increased HGP but also produces nutrient and energetic stress in FGR fetal liver. Intrinsic programming of these pathways regulating HGP in the FGR fetus may underlie increased HGP and T2D risk postnatally.

Targeting optimal protein delivery in parenteral and enteral nutrition for preterm infants: a review of randomized, controlled trials.

Close attention to nutritional management is essential for optimizing growth and neurodevelopment of the preterm infant. Protein intake and the protein to energy ratio are the main determinants of growth and body composition. Yet large, multi-center, randomized controlled trials are lacking to guide protein delivery for the preterm infant. Until these studies are pursued, smaller trials must be used to inform clinical practice. This review summarizes the randomized controlled trials that have been performed to test the impact of higher vs. lower protein delivery to the preterm infant. We consider the trials that varied protein delivery rates during parenteral and enteral phases of nutrition. Considerable heterogeneity exists across study designs. Still, cumulative evidence from these trials provides a framework for current recommendations for protein intake in the preterm infant.

Metformin Disrupts Signaling and Metabolism in Fetal Hepatocytes.

Metformin is used by women during pregnancy to manage diabetes and crosses the placenta, yet its effects on the fetus are unclear. We show that the liver is a site of metformin action in fetal sheep and macaques, given relatively abundant OCT1 transporter expression and hepatic uptake following metformin infusion into fetal sheep. To determine the effects of metformin action, we performed studies in primary hepatocytes from fetal sheep, fetal macaques, and juvenile macaques. Metformin increases AMP-activated protein kinase (AMPK) signaling, decreases mammalian target of rapamycin (mTOR) signaling, and decreases glucose production in fetal and juvenile hepatocytes. Metformin also decreases oxygen consumption in fetal hepatocytes. Unique to fetal hepatocytes, metformin activates stress pathways (e.g., increased PGC1A gene expression, NRF-2 protein abundance, and phosphorylation of eIF2α and CREB proteins) alongside perturbations in hepatokine expression (e.g., increased growth/differentiation factor 15 [GDF15] and fibroblast growth factor 21 [FGF21] expression and decreased insulin-like growth factor 2 [IGF2] expression). Similarly, in liver tissue from sheep fetuses infused with metformin in vivo, AMPK phosphorylation, NRF-2 protein, and PGC1A expression are increased. These results demonstrate disruption of signaling and metabolism, induction of stress, and alterations in hepatokine expression in association with metformin exposure in fetal hepatocytes. ARTICLE HIGHLIGHTS: The major metformin uptake transporter OCT1 is expressed in the fetal liver, and fetal hepatic uptake of metformin is observed in vivo. Metformin activates AMPK, reduces glucose production, and decreases oxygen consumption in fetal hepatocytes, demonstrating similar effects as in juvenile hepatocytes. Unique to fetal hepatocytes, metformin activates metabolic stress pathways and alters the expression of secreted growth factors and hepatokines. Disruption of signaling and metabolism with increased stress pathways and reduced anabolic pathways by metformin in the fetal liver may underlie reduced growth in fetuses exposed to metformin.

Adaptive responses in uteroplacental metabolism and fetoplacental nutrient shuttling and sensing during placental insufficiency.

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.

Attenuated glucose-stimulated insulin secretion during an acute IGF-1 LR3 infusion into fetal sheep does not persist in isolated islets.

Insulin-like growth factor-1 (IGF-1) is a critical fetal growth hormone that has been proposed as a therapy for intrauterine growth restriction. We previously demonstrated that a 1-week IGF-1 LR3 infusion into fetal sheep reduces in vivo and in vitro insulin secretion suggesting an intrinsic islet defect. Our objective herein was to determine whether this intrinsic islet defect was related to chronicity of exposure. We therefore tested the effects of a 90-min IGF-1 LR3 infusion on fetal glucose-stimulated insulin secretion (GSIS) and insulin secretion from isolated fetal islets. We first infused late gestation fetal sheep (n = 10) with either IGF-1 LR3 (IGF-1) or vehicle control (CON) and measured basal insulin secretion and in vivo GSIS utilizing a hyperglycemic clamp. We then isolated fetal islets immediately following a 90-min IGF-1 or CON in vivo infusion and exposed them to glucose or potassium chloride to measure in vitro insulin secretion (IGF-1, n = 6; CON, n = 6). Fetal plasma insulin concentrations decreased with IGF-1 LR3 infusion (P < 0.05), and insulin concentrations during the hyperglycemic clamp were 66% lower with IGF-1 LR3 infusion compared to CON (P < 0.0001). Insulin secretion in isolated fetal islets was not different based on infusion at the time of islet collection. Therefore, we speculate that while acute IGF-1 LR3 infusion may directly suppress insulin secretion, the fetal ß-cell in vitro retains the ability to recover GSIS. This may have important implications when considering the long-term effects of treatment modalities for fetal growth restriction.

Chronic Fetal Leucine Infusion Increases Rate of Leucine Oxidation but Not of Protein Synthesis in Late Gestation Fetal Sheep.

BACKGROUND: Leucine increases protein synthesis rates in postnatal animals and adults. Whether supplemental leucine has similar effects in the fetus has not been determined. OBJECTIVE: To determine the effect of a chronic leucine infusion on whole-body leucine oxidation and protein metabolic rates, muscle mass, and regulators of muscle protein synthesis in late gestation fetal sheep. METHODS: Catheterized fetal sheep at ∼126 d of gestation (term = 147 d) received infusions of saline (CON, n = 11) or leucine (LEU; n = 9) adjusted to increase fetal plasma leucine concentrations by 50%-100% for 9 d. Umbilical substrate net uptake rates and protein metabolic rates were determined using a 1-13C leucine tracer. Myofiber myosin heavy chain (MHC) type and area, expression of amino acid transporters, and abundance of protein synthesis regulators were measured in fetal skeletal muscle. Groups were compared using unpaired t tests. RESULTS: Plasma leucine concentrations were 75% higher in LEU fetuses compared with CON by the end of the infusion period (P < 0.0001). Umbilical blood flow and uptake rates of most amino acids, lactate, and oxygen were similar between groups. Fetal whole-body leucine oxidation was 90% higher in LEU (P < 0.0005) but protein synthesis and breakdown rates were similar. Fetal and muscle weights and myofiber areas were similar between groups, however, there were fewer MHC type IIa fibers (P < 0.05), greater mRNA expression levels of amino acid transporters (P < 0.01), and a higher abundance of signaling proteins that regulate protein synthesis (P < 0.05) in muscle from LEU fetuses. CONCLUSIONS: A direct leucine infusion for 9 d in late gestation fetal sheep does not increase protein synthesis rates but results in higher leucine oxidation rates and fewer glycolytic myofibers. Increasing leucine concentrations in the fetus stimulates its own oxidation but also increases amino acid transporter expression and primes protein synthetic pathways in skeletal muscle.

Differences in body composition and growth persist postnatally in fetuses diagnosed with severe compared to mild fetal growth restriction.

BACKGROUND: Fetal growth restriction (FGR) is most commonly diagnosed in pregnancy if the estimated fetal weight (EFW) is < 10th%. Those with abnormal Doppler velocimetry, indicating placental insufficiency and pathological FGR, demonstrate reduced fat and lean mass compared to both normally growing fetuses and FGR fetuses with normal Dopplers. The aim of this study was to determine how severity of FGR and abnormal Doppler velocimetry impacts neonatal body composition. Among a cohort of fetuses with an EFW < 10th%, we hypothesized that those with abnormal Dopplers and/or EFW < 3rd% would have persistent reductions in lean body mass and fat mass extending into the neonatal period compared to fetuses not meeting those criteria. METHODS: A prospective cohort of FGR fetuses with an estimated fetal weight (EFW) < 10th% was categorized as severe (EFW < 3rd% and/or abnormal Dopplers; FGR-S) versus mild (EFW 3-10th%; FGR-M). Air Displacement Plethysmography and anthropometrics were performed at birth and/or within the first 6-8 weeks of life. RESULTS: FGR-S versus FGR-M were born one week earlier (P = 0.0024), were shorter (P = 0.0033), lighter (P = 0.0001) with smaller weight-for-age Z-scores (P = 0.0004), had smaller head circumference (P = 0.0004) and lower fat mass (P = 0.01) at birth. At approximately 6-8 weeks postmenstrual age, weight, head circumference, and fat mass were similar but FGR-S neonates were shorter (P = 0.0049) with lower lean mass (P = 0.0258). CONCLUSION: Doppler velocimetry abnormalities in fetuses with an EFW < 10th% identified neonates who were smaller at birth and demonstrated catch-up growth by 6-8 weeks of life that favored fat mass accretion over lean mass and linear growth.

3D Fractional Limb Volume Identifies Reduced Subcutaneous and Lean Mass in Fetal Growth Restriction.

OBJECTIVES: Fetal 2D and 3D fractional limb volume (FLV) measurements by ultrasound can detect fetal lean and subcutaneous mass and possibly percent body fat. Our objectives were to 1) compare FLV measurements in fetuses with fetal growth restriction (FGR) versus small for gestational age (SGA) defined by the International Society of Ultrasound in Obstetrics and Gynecology (ISUOG)-supported international Delphi consensus and 2) correlate FLV findings with birth metrics. We hypothesize that FLV measurements will be significantly smaller in FGR versus SGA fetuses and will correlate closer with Ponderal index (PIx) in the neonate than abdominal circumference (AC). METHODS: Patients were categorized as FGR or SGA as defined by ISUOG. Total thigh volume (TTV), volumes of lean mass (LMV), and fat mass volume (FMV) were calculated from 3D acquisitions. Measurements were compared between groups and correlated with birthweight (BW) and PIx (BW/crown-heal length). RESULTS: The FGR group (n = 37) delivered earlier (37/2 versus 38/0; P = .0847), were lighter (2.2 kg versus 2.6 kg; P = .0003) and had lower PIx (0.023 versus 0.025; P = .0013) than SGAs (n = 22). FGRs had reduced TTV (40.6 versus 48.4 cm3 ; P = .0164), FMV (20.8 versus 25.3 cm3 ; P = .0413), and LMV (19.8 versus 23.1 cm3 ; P = .0387). AC had the highest area under the curve (0.69) for FGR. FMV was more strongly associated with PIx than the AC (P = .0032). CONCLUSIONS: The AC and FLV measurements were significantly reduced in FGR fetuses compared to SGAs. While the AC outperformed FLV in predicting FGR, the FLV correlated best with PIx, which holds investigative promise.

Fetal Sex Does Not Impact Placental Blood Flow or Placental Amino Acid Transfer in Late Gestation Pregnant Sheep With or Without Placental Insufficiency.

Pregnant sheep have been used to model complications of human pregnancies including placental insufficiency and intrauterine growth restriction. Some of the hallmarks of placental insufficiency are slower uterine and umbilical blood flow rates, impaired placental transport of oxygen and amino acids, and lower fetal arterial concentrations of anabolic growth factors. An impact of fetal sex on these outcomes has not been identified in either human or sheep pregnancies. This is likely because most studies measuring these outcomes have used small numbers of subjects or animals. We undertook a secondary analysis of previously published data generated by our laboratory in late-gestation (gestational age of 133 ± 0 days gestational age) control sheep (n = 29 male fetuses; n = 26 female fetuses; n = 3 sex not recorded) and sheep exposed to elevated ambient temperatures to cause experimental placental insufficiency (n = 23 male fetuses; n = 17 female fetuses; n = 1 sex not recorded). The primary goal was to determine how fetal sex modifies the effect of the experimental insult on outcomes related to placental blood flow, amino acid and oxygen transport, and fetal hormones. Of the 112 outcomes measured, we only found an interaction between fetal sex and experimental insult for the uterine uptake rates of isoleucine, phenylalanine, and arginine. Additionally, most outcomes measured did not show a difference based on fetal sex when adjusting for the impact of placental insufficiency. Exceptions included fetal norepinephrine and cortisol concentrations, which were higher in female compared to male fetuses. For the parameters measured in the current analysis, the impact of fetal sex was not widespread.

Lower citrate synthase activity, mitochondrial complex expression, and fewer oxidative myofibers characterize skeletal muscle from growth-restricted fetal sheep.

Skeletal muscle from the late gestation sheep fetus with intrauterine growth restriction (IUGR) has evidence of reduced oxidative metabolism. Using a sheep model of placental insufficiency and IUGR, we tested the hypothesis that by late gestation, IUGR fetal skeletal muscle has reduced capacity for oxidative phosphorylation because of intrinsic deficits in mitochondrial respiration. We measured mitochondrial respiration in permeabilized muscle fibers from biceps femoris (BF) and soleus (SOL) from control and IUGR fetal sheep. Using muscles including BF, SOL, tibialis anterior (TA), and flexor digitorum superficialis (FDS), we measured citrate synthase (CS) activity, mitochondrial complex subunit abundance, fiber type distribution, and gene expression of regulators of mitochondrial biosynthesis. Ex vivo mitochondrial respiration was similar in control and IUGR muscle. However, CS activity was lower in IUGR BF and TA, indicating lower mitochondrial content, and protein expression of individual mitochondrial complex subunits was lower in IUGR TA and BF in a muscle-specific pattern. IUGR TA, BF, and FDS also had lower expression of type I oxidative fibers. Fiber-type shifts that support glycolytic instead of oxidative metabolism may be advantageous for the IUGR fetus in a hypoxic and nutrient-deficient environment, whereas these adaptions may be maladaptive in postnatal life.

Tissue-specific responses that constrain glucose oxidation and increase lactate production with the severity of hypoxemia in fetal sheep.

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, ß-cell area, or genes regulating ß-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.

A Two-Week Insulin Infusion in Intrauterine Growth Restricted Fetal Sheep at 75% Gestation Increases Skeletal Myoblast Replication but Did Not Restore Muscle Mass or Increase Fiber Number.

Intrauterine growth restricted (IUGR) fetuses are born with lower skeletal muscle mass, fewer proliferating myoblasts, and fewer myofibers compared to normally growing fetuses. Plasma concentrations of insulin, a myogenic growth factor, are lower in IUGR fetuses. We hypothesized that a two-week insulin infusion at 75% gestation would increase myoblast proliferation and fiber number in IUGR fetal sheep. Catheterized control fetuses received saline (CON-S, n=6), and the IUGR fetuses received either saline (IUGR-S, n=7) or insulin (IUGR-I, 0.014 ± 0.001 units/kg/hr, n=11) for 14 days. Fetal arterial blood gases and plasma amino acid levels were measured. Fetal skeletal muscles (biceps femoris, BF; and flexor digitorum superficialis, FDS) and pancreases were collected at necropsy (126 ± 2 dGA) for immunochemistry analysis, real-time qPCR, or flow cytometry. Insulin concentrations in IUGR-I and IUGR-S were lower vs. CON-S (P ≤ 0.05, group). Fetal arterial PaO2, O2 content, and glucose concentrations were lower in IUGR-I vs. CON-S (P ≤ 0.01) throughout the infusion period. IGF-1 concentrations tended to be higher in IUGR-I vs. IUGR-S (P=0.06), but both were lower vs. CON-S (P ≤ 0.0001, group). More myoblasts were in S/G2 cell cycle stage in IUGR-I vs. both IUGR-S and CON-S (145% and 113%, respectively, P ≤ 0.01). IUGR-I FDS muscle weighed 40% less and had 40% lower fiber number vs. CON-S (P ≤ 0.05) but were not different from IUGR-S. Myonuclear number per fiber and the mRNA expression levels of muscle regulatory factors were not different between groups. While the pancreatic ß-cell mass was lower in both IUGR-I and IUGR-S compared to CON-S, the IUGR groups were not different from each other indicating that feedback inhibition by endogenous insulin did not reduce ß-cell mass. A two-week insulin infusion at 75% gestation promoted myoblast proliferation in the IUGR fetus but did not increase fiber or myonuclear number. Myoblasts in the IUGR fetus retain the capacity to proliferate in response to mitogenic stimuli, but intrinsic defects in the fetal myoblast by 75% gestation may limit the capacity to restore fiber number.

Uteroplacental nutrient flux and evidence for metabolic reprogramming during sustained hypoxemia.

Gestational hypoxemia is often associated with reduced birth weight, yet how hypoxemia controls uteroplacental nutrient metabolism and supply to the fetus is unclear. This study tested the effects of maternal hypoxemia (HOX) between 0.8 and 0.9 gestation on uteroplacental nutrient metabolism and flux to the fetus in pregnant sheep. Despite hypoxemia, uteroplacental and fetal oxygen utilization and net glucose and lactate uptake rates were similar in HOX (n = 11) compared to CON (n = 7) groups. HOX fetuses had increased lactate and pyruvate concentrations and increased net pyruvate output to the utero-placenta. In the HOX group, uteroplacental flux of alanine to the fetus was decreased, as was glutamate flux from the fetus. HOX fetuses had increased alanine and decreased aspartate, serine, and glutamate concentrations. In HOX placental tissue, we identified hypoxic responses that should increase mitochondrial efficiency (decreased SDHB, increased COX4I2) and increase lactate production from pyruvate (increased LDHA protein and LDH activity, decreased LDHB and MPC2), both resembling metabolic reprogramming, but with evidence for decreased (PFK1, PKM2), rather than increased, glycolysis and AMPK phosphorylation. This supports a fetal-uteroplacental shuttle during sustained hypoxemia whereby uteroplacental tissues produce lactate as fuel for the fetus using pyruvate released from the fetus, rather than pyruvate produced from glucose in the placenta, given the absence of increased uteroplacental glucose uptake and glycolytic gene activation. Together, these results provide new mechanisms for how hypoxemia, independent of AMPK activation, regulates uteroplacental metabolism and nutrient allocation to the fetus, which allow the fetus to defend its oxidative metabolism and growth.

Anemic hypoxemia reduces myoblast proliferation and muscle growth in late-gestation fetal sheep.

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 Pax7+ 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.

Reduced glucose-stimulated insulin secretion following a 1-wk IGF-1 infusion in late gestation fetal sheep is due to an intrinsic islet defect.

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 ß-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.

Late gestation fetal hyperglucagonaemia impairs placental function and results in diminished fetal protein accretion and decreased fetal growth.

KEY POINTS: Fetal glucagon concentrations are elevated in the setting of placental insufficiency, hypoxia and elevated stress hormones. Chronically elevated glucagon concentrations in the adult result in profound decreases in amino acid concentrations and lean body mass. Experimental elevation of fetal glucagon concentrations in a late-gestation pregnant sheep results in lower fetal amino acid concentrations, lower protein accretion and lower fetal weight, in addition to decreased placental function. This study demonstrates a negative effect of glucagon on fetal protein accretion and growth, and also provides the first example of a fetal hormone that negatively regulates placental nutrient transport and blood flow. ABSTRACT: Fetal glucagon concentrations are elevated in the setting of placental insufficiency and fetal stress. Postnatal studies have demonstrated the importance of glucagon in amino acid metabolism, and limited fetal studies have suggested that glucagon inhibits umbilical uptake of certain amino acids. We hypothesized that chronic fetal hyperglucagonaemia would decrease amino acid transfer and increase amino acid oxidation by the fetus. Late gestation singleton fetal sheep received a direct intravenous infusion of glucagon (GCG; 5 or 50 ng/kg/min; n = 7 and 5, respectively) or a vehicle control (n = 10) for 8-10 days. Fetal and maternal nutrient concentrations, uterine and umbilical blood flows, fetal leucine flux, nutrient uptake rates, placental secretion of chorionic somatomammotropin (CSH), and targeted placental gene expression were measured. GCG fetuses had 13% lower fetal weight compared to controls (P = 0.0239) and >28% lower concentrations of 16 out of 21 amino acids (P < 0.02). Additionally, protein synthesis was 49% lower (P = 0.0005), and protein accretion was 92% lower in GCG fetuses (P = 0.0006). Uterine blood flow was 33% lower in ewes with GCG fetuses (P = 0.0154), while umbilical blood flow was similar. Fetal hyperglucagonaemia lowered uterine uptake of 10 amino acids by >48% (P < 0.05) and umbilical uptake of seven amino acids by >29% (P < 0.04). Placental secretion of CSH into maternal circulation was reduced by 80% compared to controls (P = 0.0080). This study demonstrates a negative effect of glucagon on fetal protein accretion and growth. It also demonstrates that glucagon, a hormone of fetal origin, negatively regulates maternal placental nutrient transport function, placental CSH production and uterine blood flow.