Catecholamines Mediate Multiple Fetal Adaptations during Placental Insufficiency That Contribute to Intrauterine Growth Restriction: Lessons from Hyperthermic Sheep

Placental insufficiency (PI) prevents adequate delivery of nutrients to the developing fetus and creates a chronic state of hypoxemia and hypoglycemia. In response, the malnourished fetus develops a series of stress hormone-mediated metabolic adaptations to preserve glucose for vital tissues at the expense of somatic growth. Catecholamines suppress insulin secretion to promote glucose sparing for insulin-independent tissues (brain, nerves) over insulin-dependent tissues (skeletal muscle, liver, and adipose). Likewise, premature induction of hepatic gluconeogenesis helps maintain fetal glucose and appears to be stimulated by both norepinephrine and glucagon. Reduced glucose oxidation rate in PI fetuses creates a surplus of glycolysis-derived lactate that serves as substrate for hepatic gluconeogenesis. These adrenergically influenced adaptive responses promote in utero survival but also cause asymmetric intrauterine growth restriction and small-for-gestational-age infants that are at greater risk for serious metabolic disorders throughout postnatal life, including obesity and type II diabetes

Investigating the causes of low birth weight in contrasting ovine paradigms.

Intrauterine growth restriction (IUGR) still accounts for a large incidence of infant mortality and morbidity worldwide. Many of the circulatory and transport properties of the sheep placenta are similar to those of the human placenta and as such, the pregnant sheep offers an excellent model in which to study the development of IUGR. Two natural models of ovine IUGR are those of hyperthermic exposure during pregnancy, and adolescent overfeeding, also during pregnancy. Both models yield significantly reduced placental weights and an asymmetrically growth-restricted fetus, and display altered maternal hormone concentrations, indicative of an impaired trophoblast capacity. Additionally, impaired placental angiogenesis and uteroplacental blood flow appears to be an early defect in both the hyperthermic and adolescent paradigms. The effects of these alterations in placental functional development appear to be irreversible. IUGR fetuses are both hypoxic and hypoglycaemic, and have reduced insulin and insulin-like growth factor-1 (IGF-1), and elevated concentrations of lactate. However, fetal utilization of oxygen and glucose, on a weight basis, remain constant compared with control pregnancies. Maintained utilization of these substrates, in a substrate-deficient environment, suggests increased sensitivities to metabolic signals, which may play a role in the development of metabolic diseases in later adult life

Chronic late-gestation hypoglycemia upregulates hepatic PEPCK associated with increased PGC1alpha mRNA and phosphorylated CREB in fetal sheep.

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-1alpha mRNA and phosphorylation of cAMP response element binding protein at Ser(133) were both increased, with no change in Akt, forkhead transcription factor FoxO1, hepatocyte nuclear factor-4alpha, or CCAAT enhancer binding protein-beta. These results demonstrate that chronic fetal hypoglycemia triggers signals that can activate gluconeogenesis in the fetal liver

Primary myoblasts from intrauterine growth-restricted fetal sheep exhibit intrinsic dysfunction of proliferation and differentiation that coincides with enrichment of inflammatory cytokine signaling pathways

Intrauterine growth restriction (IUGR) is linked to lifelong reductions in muscle mass due to intrinsic functional deficits in myoblasts, but the mechanisms underlying these deficits are not known. Our objective was to determine if the deficits were associated with changes in inflammatory and adrenergic regulation of IUGR myoblasts, as was previously observed in IUGR muscle. Primary myoblasts were isolated from IUGR fetal sheep produced by hyperthermia-induced placental insufficiency (PI-IUGR; n = 9) and their controls (n = 9) and from IUGR fetal sheep produced by maternofetal inflammation (MI-IUGR; n = 6) and their controls (n = 7). Proliferation rates were less (P \u3c 0.05) for PI-IUGR myoblasts than their controls and were not affected by incubation with IL-6, TNF-α, norepinephrine, or insulin. IκB kinase inhibition reduced (P \u3c 0.05) proliferation of control myoblasts modestly in basal media but substantially in TNF-α-added media and reduced (P \u3c 0.05) PI-IUGR myoblast proliferation substantially in basal and TNF-α-added media. Proliferation was greater (P \u3c 0.05) for MI-IUGR myoblasts than their controls and was not affected by incubation with TNF-α. Insulin increased (P \u3c 0.05) proliferation in both MI-IUGR and control myoblasts. After 72-h differentiation, fewer (P \u3c 0.05) PI-IUGR myoblasts were myogenin+ than controls in basal and IL-6 added media but not TNF-α-added media. Fewer (P \u3c 0.05) PI-IUGR myoblasts were desmin+ than controls in basal media only. Incubation with norepinephrine did not affect myogenin+ or desmin+ percentages, but insulin increased (P \u3c 0.05) both markers in control and PI-IUGR myoblasts. After 96-h differentiation, fewer (P \u3c 0.05) MI-IUGR myoblasts were myogenin+ and desmin+ than controls regardless of media, although TNF-α reduced (P \u3c 0.05) desmin+ myoblasts for both groups. Differentiated PI-IUGR myoblasts had greater (P \u3c 0.05) TNFR1, ULK2, and TNF-α-stimulated TLR4 gene expression, and PI-IUGR semitendinosus muscle had greater (P \u3c 0.05) TNFR1 and IL6 gene expression, greater (P \u3c 0.05) c-Fos protein, and less (P \u3c 0.05) IκBα protein. Differentiated MI-IUGR myoblasts had greater (P \u3c 0.05) TNFR1 and IL6R gene expression, tended to have greater (P = 0.07) ULK2 gene expression, and had greater (P \u3c 0.05) β-catenin protein and TNF-α-stimulated phosphorylation of NFκB. We conclude that these enriched components of TNF-α/TNFR1/NFκB and other inflammatory pathways in IUGR myoblasts contribute to their dysfunction and help explain impaired muscle growth in the IUGR fetus. Lay Summary-- Myoblasts are stems cells whose functional capacity can limit muscle growth. However, stressful intrauterine conditions cause these cells to be intrinsically dysfunctional. This restricts muscle growth capacity, leading to intrauterine growth restriction (IUGR) of the fetus, low birth weight, and less muscle mass after birth. Consequently, meat yield is reduced in IUGR-born food animals and glucose homeostasis is impaired in IUGR-born humans, which contributes to metabolic dysfunction. Intrinsic dysfunction of IUGR myoblasts has been previously observed, but the fetal programming changes (i.e., permanent changes in the development of cellular mechanisms that explains different functional outcomes) have not been identified. This study shows that one mechanism is the enhancement of signaling pathways for TNF-α and other inflammatory cytokines. These cytokines have roles in stress responses and regulation of muscle growth. Programmed enhancement of these pathways means that IUGR myoblasts are more responsive to even normal amounts of circulating cytokines. Unfortunately, the primary response of myoblasts to cytokines is slower differentiation (i.e., cellular transformation necessary for muscle growth). Programmed enhancement of this response directly impedes myoblast-dependent muscle growth, and the deficit is lifelong. However, identifying this mechanism is a fundamental step for developing strategies to improve muscle growth in low birth weight offspring

Differential Effects of Chronic Pulsatile versus Chronic Constant Maternal Hyperglycemia on Fetal Pancreatic β-Cells

Constant maternal hyperglycemia limits, while pulsatile maternal hyperglycemia may enhance, fetal glucose-stimulated insulin secretion (GSIS) in sheep. However, the impact of such different patterns of hyperglycemia on the development of the fetal β-cell is unknown. We measured the impact of one week of chronic constant hyperglycemia (CHG, n=6) versus pulsatile hyperglycemia (PHG, n=5) versus controls (n=7) on the percentage of the fetal pancreas staining for insulin (β-cell area), mitotic and apoptotic indices and size of fetal β-cells, and fetal insulin secretion in sheep. Baseline insulin concentrations were higher in CHG fetuses (P<0.05) compared to controls and PHG. GSIS was lower in the CHG group (P<0.005) compared to controls and PHG. PHG β-cell area was increased 50% (P<0.05) compared to controls and CHG. CHG β-cell apoptosis was increased over 400% (P<0.05) compared to controls and PHG. These results indicate that late gestation constant maternal hyperglycemia leads to significant β-cell toxicity (increased apoptosis and decreased GSIS). Furthermore, pulsatile maternal hyperglycemia increases pancreatic β-cell area but did not increase GSIS, indicating decreased β-cell responsiveness. These findings demonstrate differential effects that the pattern of maternal hyperglycemia has on fetal pancreatic β-cell development, which might contribute to later life limitation in insulin secretion

Developmental Programming in Response to Intrauterine Growth Restriction Impairs Myoblast Function and Skeletal Muscle Metabolism

Fetal adaptations to placental insufficiency alter postnatal metabolic homeostasis in skeletal muscle by reducing glucose oxidation rates, impairing insulin action, and lowering the proportion of oxidative fibers. In animal models of intrauterine growth restriction (IUGR), skeletal muscle fibers have less myonuclei at birth. This means that myoblasts, the sole source for myonuclei accumulation in fibers, are compromised. Fetal hypoglycemia and hypoxemia are complications that result from placental insufficiency. Hypoxemia elevates circulating catecholamines, and chronic hypercatecholaminemia has been shown to reduce fetal muscle development and growth. We have found evidence for adaptations in adrenergic receptor expression profiles in myoblasts and skeletal muscle of IUGR sheep fetuses with placental insufficiency. The relationship of β-adrenergic receptors shifts in IUGR fetuses because Adrβ2 expression levels decline and Adrβ1 expression levels are unaffected in myofibers and increased in myoblasts. This adaptive response would suppress insulin signaling, myoblast incorporation, fiber hypertrophy, and glucose oxidation. Furthermore, this β-adrenergic receptor expression profile persists for at least the first month in IUGR lambs and lowers their fatty acid mobilization. Developmental programming of skeletal muscle adrenergic receptors partially explains metabolic and endocrine differences in IUGR offspring, and the impact on metabolism may result in differential nutrient utilization