Intrauterine Growth Restriction Lambs Research Articles

280 AntagomiR-22-3p Injection alters miRNA expression and muscle fiber type of intrauterine growth restricted lambs

Abstract microRNAs (miRNA) are small non-coding RNAs that modulate muscle satellite cell proliferation. Previous research identified miR-22-3p as differentially expressed during ovine muscle development and in vitro experiments showed that antagomir-22-3p treatment enhanced satellite cell proliferation. The objective of this study was to test in vivo vascular injection of antagomir-22-3p in lateral saphenous vein on miRNA and HDAC family mRNA expression in intrauterine growth restricted lambs. Pregnant ewes (n = 18) with twins were either fed at 100% of NRC (CON) or nutrient restricted (60% NRC; NR) from gestational d 85 to parturition. On d 12 of age, NR lambs (n = 8) were randomly selected and given a systemic injection of antagomir-22-3p in the lateral saphenous vein of the right leg (MIR-NR). CON lambs (n = 8) were also randomly selected and given a sham injection of PBS in the lateral saphenous vein of the right leg (SHAM-CON). Antagomir-22-3p was reconstituted in phosphate buffered saline (PBS). Injections were given for 3 consecutive days and lambs harvested 24 d later. Blood samples were collected from each lamb weekly to monitor miR-22-3p expression. Lambs were slaughtered 24 d after the first injection and muscle mass measured. Muscle samples were removed for miR-22-3p and mRNA expression of potential targets. Cell-free circulating RNA was isolated from blood samples and miR-22-3p expression examined over time. Cell-free circulating miR-22-3p expression was downregulated (P < 0.05) for MIR-NR compared with SHAM-CON on d 14 after injection. On d 21 after injection, miR-22-3p expression in plasma tended (P = 0.08) to be down-regulated in MIR-NR compared with SHAM-CON. Lamb body weight and muscle weights at harvest were similar between MIR-NR and SHAM-CON treatments. In heart and semimembranosus (SM) muscles, expression of miR-22-3p was down-regulated (P < 0.05) in MIR-NR compared with SHAM-CON. In the gastrocnemius and semitendinosus muscle, miR-22-3p expression was unchanged (P > 0.05). The number of type I and IIa muscle fibers were greater (P < 0.05) for MIR-NR than SHAM-CON. Muscle fiber area of Type I, IIa or IIx fibers did not differ (P > 0.05) by treatment. The number of type IIx fibers did not differ (P > 0.05) between treatments. MIR-NR treatment appears to shift muscle fibers towards more oxidative metabolism and down-regulate HDAC-1, 3, and 11 and SIRT6 in SM. The systemic injection of antagomir-22-3P down-regulated miR-22-3p expression in circulation and in muscle tissues, which altered expression of HDAC/SIRT genes involved in muscle fiber type conversion and hypertrophy.

Daily Injection of the β2 Adrenergic Agonist Clenbuterol Improved Muscle Glucose Metabolism, Glucose-Stimulated Insulin Secretion, and Hyperlipidemia in Juvenile Lambs Following Heat-Stress-Induced Intrauterine Growth Restriction.

Stress-induced fetal programming diminishes β2 adrenergic tone, which coincides with intrauterine growth restriction (IUGR) and lifelong metabolic dysfunction. We determined if stimulating β2 adrenergic activity in IUGR-born lambs would improve metabolic outcomes. IUGR lambs that received daily injections of saline or the β2 agonist clenbuterol from birth to 60 days were compared with controls from pair-fed thermoneutral pregnancies. As juveniles, IUGR lambs exhibited systemic inflammation and robust metabolic dysfunction, including greater (p < 0.05) circulating TNFα, IL-6, and non-esterified fatty acids, increased (p < 0.05) intramuscular glycogen, reduced (p < 0.05) circulating IGF-1, hindlimb blood flow, glucose-stimulated insulin secretion, and muscle glucose oxidation. Daily clenbuterol fully recovered (p < 0.05) circulating TNFα, IL-6, and non-esterified fatty acids, hindlimb blood flow, muscle glucose oxidation, and intramuscular glycogen. Glucose-stimulated insulin secretion was partially recovered (p < 0.05) in clenbuterol-treated IUGR lambs, but circulating IGF-1 was not improved. Circulating triglycerides and HDL cholesterol were elevated (p < 0.05) in clenbuterol-treated IUGR lambs, despite being normal in untreated IUGR lambs. We conclude that deficient β2 adrenergic regulation is a primary mechanism for several components of metabolic dysfunction in IUGR-born offspring and thus represents a potential therapeutic target for improving metabolic outcomes. Moreover, benefits from the β2 agonist were likely complemented by its suppression of IUGR-associated inflammation.

241 Increased Inflammatory Tone and Impaired Skeletal Muscle Growth in Intrauterine Growth-Restricted Neonatal Lambs Is Improved By Daily Supplementation of ω-3 Polyunsaturated Fatty Acid Ca2+ Salts

Abstract Low birthweight due to fetal intrauterine growth restriction (IUGR) often results from maternofetal heat stress. IUGR fetuses exhibit enhanced inflammatory tone, which persists after birth and impacts body composition, skeletal muscle growth, and metabolic function. Therefore, our objective was to determine whether daily oral supplementation of Ca2+ salts of ω-3 polyunsaturated fatty acids (ω-3 PUFA) to IUGR-born neonatal lambs improved inflammatory tone, body composition, and growth metrics. Pregnant ewes were exposed to heat stress (40°C, 35% humidity, THI = 85) from d 40 to 95 of gestation to induce IUGR. Lambs were weaned at birth and hand-reared on milk replacer. Daily oral boluses of 0.5 mL molasses containing 0 or 0.42 g/kg bodyweight of ω-3 PUFA (Virtus Nutrition) were administered. Femoral catheters were placed at d 24 and lambs were necropsied at d 30. At birth, IUGR lambs (n = 8; 3.12 ± 0.34 kg) and IUGR+ω-3 lambs (n = 7; 3.67 ± 0.32 kg) were lighter (P &amp;lt; 0.05) than controls (n = 12; 4.28 ± 0.15 kg). Plasma TNFα concentrations were greater (P &amp;lt; 0.05) at birth for IUGR (557 ± 121 pg/mL) and IUGR+ω-3 lambs (437 ± 82 pg/mL) than for controls (213 ± 78 pg/mL). Plasma TNFα concentrations remained increased (P &amp;lt; 0.05) for IUGR lambs but not for IUGR+ω-3 compared with controls at 2, 3, and 4 wk of age. Adjusted 30-d bodyweights were lighter (P &amp;lt; 0.05) for IUGR lambs (9.83 ± 0.6 kg) but not for IUGR+ω-3 lambs (11.59 ± 0.4 kg) than for controls (12.35 ± 0.2 kg). Heart and brain weights were 17% and 6% lighter (P &amp;lt; 0.05), respectively, for IUGR lambs than for controls. Brain weight deficits were resolved in IUGR+ω3 lambs, but heart weights did not differ between IUGR and IUGR+ω-3 IUGR lambs. Crown circumference/ bodyweight, abdominal circumference/ bodyweight, and cannon bone length/ bodyweight (indicators of asymmetric body composition) were 11% to 15% greater (P &amp;lt; 0.05) for IUGR lambs than for IUGR+ω-3 lambs or controls. At necropsy, IUGR lambs had lighter (P &amp;lt; 0.05) whole hindlimbs (29%) and flexor digitorum superficialis (24%), soleus (36%), biceps femoris (25%), semitendinosus (24%), gastrocnemius (29%), and longissimus dorsi (26%) muscles compared with controls. Weight deficits were partially or fully resolved in IUGR+ω-3 lambs for whole hindlimbs and for all muscles but the soleus, which did not differ from IUGR lambs. These findings demonstrate that daily oral supplementation of Ca2+ salts of ω-3 PUFA reduced the enhanced inflammatory tone observed in IUGR-born offspring, which coincided with improved skeletal muscle growth and indicators of symmetrical body composition.

PSVIII-30 Impaired Glucose-Stimulated Insulin Secretion in Intrauterine Growth-Restricted Neonatal Lambs Is Improved By Daily Supplementation of ω-3 Polyunsaturated Fatty Acid Ca2+ Salts

Abstract Maternofetal heat stress during placental development induces fetal intrauterine growth restriction (IUGR), which in turn reduces birthweight. IUGR-born livestock exhibit deficits in insulin stimulus-secretion coupling and glucose metabolism that contribute to poor growth efficiency. Thus, our objective was to determine whether daily oral supplementation of Ca2+ salts of ω-3 polyunsaturated fatty acids (ω-3 PUFA) to IUGR-born neonatal lambs improves glucose-stimulated insulin secretion and other indicators of metabolic efficiency. Maternal heat stress (40°C, 35% humidity, THI = 85) from d 40 to 95 of gestation was used to induce IUGR. Lambs were weaned at birth and hand-reared on milk replacer. Beginning at birth, lambs received daily oral boluses of 0.5 mL molasses containing 0 or 0.42 g/kg bodyweight of ω-3 PUFA (Virtus Nutrition). All lambs were fitted with femoral catheters at d 24 and a square-wave hyperglycemic clamp study was performed on d 28 to assess basal (i.e., resting) and glucose-stimulated insulin secretion (GSIS). Blood glucose concentrations did not differ among IUGR lambs (n = 8), IUGR+ω-3 lambs (n = 7), and controls (n = 12), but by study design were increased (P &amp;lt; 0.05) 205% from basal during hyperglycemic periods. Basal plasma insulin concentrations did not differ among groups, but basal glucose-to-insulin ratios were greater (P &amp;lt; 0.05) for IUGR+ω-3 lambs than for controls or IUGR lambs. During hyperglycemia, plasma insulin concentrations were 53% less (P &amp;lt; 0.05) for IUGR lambs but only 30% less (P &amp;lt; 0.05) for IUGR+ω-3 lambs compared with controls. Hyperglycemic glucose-to-insulin ratios were greater (P &amp;lt; 0.05) for IUGR lambs (3.24 ± 0.59) compared with controls (1.10 ± 0.29) and were intermediate for IUGR+ω-3 lambs (2.08 ± 0.86). Blood lactate concentrations did not differ among groups or between periods, but blood pH was was greater (P &amp;lt; 0.05) for IUGR (7.448 ± 0.006) and IUGR+ω-3 lambs (7.439 ± 0.003) compared with controls (7.422 ± 0.004). Blood partial pressure of CO2 was less (P &amp;lt; 0.05) for IUGR lambs (43.0 ± 0.3 mmHg) than for controls (46.7 ± 0.9 mmHg) and was intermediate for IUGR+ω-3 lambs (44.0 ± 0.4 mmHg). Blood HCO3 concentrations and O2-bound hemoglobin were greater (P &amp;lt; 0.05) for IUGR and IUGR+ω-3 lambs than for controls. Blood Na+ tended to be less (P = 0.07) and blood K+ was less (P &amp;lt; 0.05) for IUGR and IUGR+ω-3 lambs than for controls. Blood Ca2+ was less (P &amp;lt; 0.05) for IUGR lambs but not for IUGR+ω-3 lambs than for controls. These findings demonstrate that daily oral supplementation of Ca2+ salts of ω-3 PUFA improved deficits in insulin stimulus-secretion coupling and several other metabolic indicators observed in IUGR-born offspring. However, it did not improve all electrolyte concentrations or acid-base balance of the blood.

252 Use of Agomir and Antagomir Technologies to Alter Mirna Expression and Muscle Hypertrophy in Intrauterine Growth Restricted Lambs

Abstract microRNAs (miRNA) are present in skeletal muscle and play important roles in regulating gene expression. Previous research identified miR-22-3p and miR-127 as differentially regulated during muscle hypertrophy and in vitro experiments found that antagomir-22-3p and mimic-127 enhanced satellite cell proliferation. The objective of this study was to test in vivo intramuscular injection of agomiR-127 or antagomir-22-3p in the longissimus muscle (LM) on miRNA and target mRNA expression in intrauterine growth restricted lambs. Pregnant ewes (n = 18) with twins were nutrient restricted (60%) from gestational d 85 to parturition. On d 2 of age, lambs were randomly selected and assigned to one of two miRNA treatments, 1) agomiR-127 (AGO127, 100 nM, n = 9) to assess gain of function or 2) antagomiR-22-3p (ANT22, 500 nM, n = 9) to assess loss of function. AgomiR and antagomiR technologies are chemically modified miRNA mimics or inhibitors that do not require transfection for in vivo use. AgomiR-127 or antagomir-22-3p were reconstituted in phosphate buffered saline (PBS) and injected into the center of the LM on the left side every 3-d starting at the 10th rib and moving posterior by 1.27 cm at each injection for a total of 7 injections. A sham control treatment (SHAM127 or SHAM22) of PBS was administered following the same protocol in the right LM to serve as the in-animal control. Lambs were harvested 4 d after the last injection and samples were collected from the injection region of each LM for miRNA and mRNA expression, histology, and proteomic analyses. Data were analyzed using paired t-tests to compare each miRNA treatment to its SHAM within animal. AGO127 up-regulated (P = 0.0071) miR-127 expression compared with SHAM127. mRNA expression of PAX7 and MSTN were up-regulated (P &amp;lt; 0.05) and IGFBP2 expression was down-regulated (P &amp;lt; 0.05) for AGO127 compared with SHAM127. Both protein and DNA content of AGO127 were increased (P &amp;lt; 0.05) but the ratio of protein to DNA did not change (P &amp;gt; 0.05). Proteomic analyses found 11 proteins that were differentially expressed for AGO127 compared with SHAM127. ANT22 down-regulated (P = 0.0024) miR-22-3p expression from SHAM22. Expression of predicted mRNA targets of miR-22-3p (ACVR2a, ACVR2b, SIRT1), myosin heavy chain isoforms (MyHCI, MyHCIIa, MyHCIIx), and other myogenic genes (MAPK6, IGF1, MyoG) were also altered (P &amp;lt; 0.05) in the LM compared with SHAM22. DNA content and the protein to DNA ratio was greater (P &amp;lt; 0.05) in ANT22 than SHAM22. Proteomic analyses identified nine proteins that were differentially expressed (P &amp;lt; 0.05) for ANT22. The direct injection of agomiR and antagomir technologies in the LM up- and down-regulated expression, respectively, for the miRNA of interest, which altered mRNA expression of predicted targets, protein abundance and muscle fiber type.

Use of AgomiR and AntagomiR technologies to alter satellite cell proliferation in vitro, miRNA expression, and muscle fiber hypertrophy in intrauterine growth-restricted lambs.

Introduction: microRNAs (miRNAs) are small non-coding RNAs that work at the posttranscriptional level to repress gene expression. Several miRNAs are preferentially expressed in skeletal muscle and participate in myogenesis. This research was conducted to alter endogenous miRNA expression in skeletal muscle to promote muscle hypertrophy. Methods: Two experiments were conducted using mimic/agomiR or antagomir technologies to alter miRNA expression and examine changes in myoblast proliferation in vitro (experiment 1) and muscle hypertrophy in vivo (experiment 2). In vitro experiments found that antagomiR-22-3p and mimic-127 increased myoblast proliferation compared to other miRNA treatments or controls. These miRNA treatments, antagomiR-22-3p (ANT22) and agomiR-127 (AGO127), were then used for intramuscular injections in longissimus muscle. Results and discussion: The use of antagomiR or mimic/agomiR treatments down-regulated or up-regulated, respectively, miRNA expression for that miRNA of interest. Expression of predicted target KIF3B mRNA for miR-127 was up-regulated and ACVR2a mRNA was up-regulated for miR-22-3p. ANT22 injection also up-regulated the major regulator of protein synthesis (mTOR). Proteomic analyses identified 11 proteins for AGO127 and 9 proteins for ANT22 that were differentially expressed. Muscle fiber type and cross-sectional area were altered for ANT22 treatments to transition fibers to a more oxidative state. The use of agomiR and antagomir technologies allows us to alter miRNA expression in vitro and in vivo to enhance myoblast proliferation and alter muscle fiber hypertrophy in IUGR lambs during early postnatal growth.

Daily injection of the β2 adrenergic agonist clenbuterol improved poor muscle growth and body composition in lambs following heat stress-induced intrauterine growth restriction.

Background: Intrauterine growth restriction (IUGR) is associated with reduced β2 adrenergic sensitivity, which contributes to poor postnatal muscle growth. The objective of this study was to determine if stimulating β2 adrenergic activity postnatal would rescue deficits in muscle growth, body composition, and indicators of metabolic homeostasis in IUGR offspring. Methods: Time-mated ewes were housed at 40°C from day 40 to 95 of gestation to produce IUGR lambs. From birth, IUGR lambs received daily IM injections of 0.8μg/kg clenbuterol HCl (IUGR+CLEN; n = 11) or saline placebo (IUGR; n = 12). Placebo-injected controls (n = 13) were born to pair-fed thermoneutral ewes. Biometrics were assessed weekly and body composition was estimated by ultrasound and bioelectrical impedance analysis (BIA). Lambs were necropsied at 60days of age. Results: Bodyweights were lighter (p ≤ 0.05) for IUGR and IUGR+CLEN lambs than for controls at birth, day 30, and day 60. Average daily gain was less (p ≤ 0.05) for IUGR lambs than controls and was intermediate for IUGR+CLEN lambs. At day 58, BIA-estimated whole-body fat-free mass and ultrasound-estimated loin eye area were less (p ≤ 0.05) for IUGR but not IUGR+CLEN lambs than for controls. At necropsy, loin eye area and flexor digitorum superficialis muscles were smaller (p ≤ 0.05) for IUGR but not IUGR+CLEN lambs than for controls. Longissimus dorsi protein content was less (p ≤ 0.05) and fat-to-protein ratio was greater (p ≤ 0.05) for IUGR but not IUGR+CLEN lambs than for controls. Semitendinosus from IUGR lambs had less (p ≤ 0.05) β2 adrenoreceptor content, fewer (p ≤ 0.05) proliferating myoblasts, tended to have fewer (p = 0.08) differentiated myoblasts, and had smaller (p ≤ 0.05) muscle fibers than controls. Proliferating myoblasts and fiber size were recovered (p ≤ 0.05) in IUGR+CLEN lambs compared to IUGR lambs, but β2 adrenoreceptor content and differentiated myoblasts were not recovered. Semitendinosus lipid droplets were smaller (p ≤ 0.05) in size for IUGR lambs than for controls and were further reduced (p ≤ 0.05) in size for IUGR+CLEN lambs. Conclusion: These findings show that clenbuterol improved IUGR deficits in muscle growth and some metabolic parameters even without recovering the deficit in β2 adrenoreceptor content. We conclude that IUGR muscle remained responsive to β2 adrenergic stimulation postnatal, which may be a strategic target for improving muscle growth and body composition in IUGR-born offspring.

PSIII-A-8 Daily Clenbuterol Administration to Lambs Born with low Birthweight due to Intrauterine Growth Restriction Recovered Growth Rates and Improved Body Composition at 60 D of age

Abstract Low birthweight due to intrauterine growth restriction (IUGR) results from unfavorable in utero conditions (i.e., hypoxemia, hypoglycemia) that impairs growth capacity for muscle and other tissues. This programming effect reduces growth efficiency in IUGR offspring below their genetic potential. Specific programming mechanisms are not known, but recent evidence indicates reduced β2 adrenergic regulation of IUGR muscle. Thus, our objective was to determine if early-life growth deficits in IUGR juvenile lambs are improved by manipulating β2 adrenergic activity with daily IM administration of the β2 agonist, clenbuterol (Clen), from birth to 60 d of age. Maternal heat stress-induced IUGR (n=10) and IUGR+Clen (n=9) lambs were smaller (P&amp;lt; 0.05) than controls (n=11) at birth, weighed 18% and 11% less (P&amp;lt; 0.05), respectively, than controls at 30 d of age, and weighed 16% and 13% less (P&amp;lt; 0.05), respectively, at 60 d of age. Average daily gain at 30 and 60 d of age was reduced (P≤0.05) for IUGR but not IUGR+Clen lambs compared to controls. Bioelectrical impedance analysis was used to estimate body composition in live lambs at 58 d of age. Estimated fat-free lean mass and estimated muscle mass were less (P≤0.05) for IUGR but not IUGR+Clen lambs compared to controls. Estimated protein and fat content as well as protein:fat were likewise reduced (P&amp;lt; 0.05) for IUGR but not IUGR+Clen lambs. At necropsy, whole hindlimbs tended to be lighter (P≤0.09) and flexor digitorum superficialis muscle was lighter (P&amp;lt; 0.05) for IUGR but not IUGR+Clen lambs compared to controls. Loin-eye areas measured in chilled carcasses were 15% smaller (P&amp;lt; 0.05) for IUGR lambs and 19% larger (P&amp;lt; 0.05) for IUGR+Clen lambs compared to controls. From this study, we conclude that poor postnatal growth and body composition observed in IUGR juvenile lambs was improved when clenbuterol was used to target programmed deficits in β2 adrenergic regulation of muscle.

Intermittent maternofetal oxygenation during late gestation improved birthweight, neonatal growth, body symmetry, and muscle metabolism in intrauterine growth-restricted lambs.

In humans and animals, intrauterine growth restriction (IUGR) results from fetal programming responses to poor intrauterine conditions. Chronic fetal hypoxemia elevates circulating catecholamines, which reduces skeletal muscle β2 adrenoceptor content and contributes to growth and metabolic pathologies in IUGR-born offspring. Our objective was to determine whether intermittent maternofetal oxygenation during late gestation would improve neonatal growth and glucose metabolism in IUGR-born lambs. Pregnant ewes were housed at 40 °C from the 40th to 95th day of gestational age (dGA) to produce IUGR-born lambs (n = 9). A second group of IUGR-born lambs received prenatal O2 supplementation via maternal O2 insufflation (100% humidified O2, 10 L/min) for 8 h/d from dGA 130 to parturition (IUGR+O2, n = 10). Control lambs (n = 15) were from pair-fed thermoneutral ewes. All lambs were weaned at birth, hand-reared, and fitted with hindlimb catheters at day 25. Glucose-stimulated insulin secretion (GSIS) and hindlimb hyperinsulinemic-euglycemic clamp (HEC) studies were performed at days 28 and 29, respectively. At day 30, lambs were euthanized and ex vivo HEC studies were performed on isolated muscle. Without maternofetal oxygenation, IUGR lambs were 40% lighter (P < 0.05) at birth and maintained slower (P < 0.05) growth rates throughout the neonatal period compared with controls. At 30 d of age, IUGR lambs had lighter (P < 0.05) hindlimbs and flexor digitorum superficialis (FDS) muscles. IUGR+O2 lambs exhibited improved (P < 0.05) birthweight, neonatal growth, hindlimb mass, and FDS mass compared with IUGR lambs. Hindlimb insulin-stimulated glucose utilization and oxidation rates were reduced (P < 0.05) in IUGR but not IUGR+O2 lambs. Ex vivo glucose oxidation rates were less (P < 0.05) in muscle from IUGR but not IUGR+O2 lambs. Surprisingly, β2 adrenoceptor content and insulin responsiveness were reduced (P < 0.05) in muscle from IUGR and IUGR+O2 lambs compared with controls. In addition, GSIS was reduced (P < 0.05) in IUGR lambs and only modestly improved (P < 0.05) in IUGR+O2. Insufflation of O2 also increased (P < 0.05) acidosis and hypercapnia in dams, perhaps due to the use of 100% O2 rather than a gas mixture with a lesser O2 percentage. Nevertheless, these findings show that intermittent maternofetal oxygenation during late gestation improved postnatal growth and metabolic outcomes in IUGR lambs without improving muscle β2 adrenoceptor content.

Effects of birth weight and dietary fat on intake, body composition, and plasma thyroxine in neonatal lambs.

Intrauterine growth restriction (IUGR) is often observed in one of the fetuses carried by well-fed prolific ewes. This condition is the result of an insufficient placental size to cover the nutritional needs of the fetus during the near exponential growth phase of the last trimester. After birth, these IUGR offspring have an elevated appetite and lower maintenance energy requirements, suggesting dysregulation of homeostatic systems governing energy metabolism. It is also unknown whether the consequent increase in fatness occurs similarly in both visceral and carcass fractions. To address these questions, lambs differing in birth size (BS, IUGR vs. Normal, 2.6 ± 0.05 vs. 4.2 ± 0.07 kg, P < 0.001) were offered unlimited amounts of a low fat [LF; 22% of dry matter (DM)] or a high fat (HF; 38% of DM) milk replacer and slaughtered on day 14 of postnatal age (n = 7 to 8 for each BS × Diet); a second group of IUGR lambs (n = 3 for each diet) was slaughtered when they reached 8.5 kg, corresponding to the weight of Normal lambs on day 14. When normalized to body weight (BW), the DM and energy intake of IUGR lambs were higher than those of Normal lambs over the first 14 d of life (BS, P < 0.01), but contrary to expectations, the HF diet did not exacerbate these effects of the IUGR condition. Intrauterine growth restricted lambs had increased viscera fat with both diets (BS and Diet, P < 0.05) but increased carcass fat only with the LF diet (BS × Diet, P = 0.08); the fatness promoting effect of the IUGR condition was increased in both body fractions when lamb groups were compared at the fixed BW of 8.5 kg. A subset of metabolic hormones was analyzed, including the metabolic rate-setting hormone thyroxine (T4) and its possible positive regulator leptin. Plasma T4 was lower in IUGR than in Normal lambs at birth (P < 0.05) but then disappeared by day 7 of postnatal life (BS × Day, P < 0.01). On the other hand, the HF diet had no effect on plasma T4 over the first 3 d but caused an increase, irrespective of BS by day 11 (Diet × Day, P < 0.001). Plasma leptin increased with dietary fat and time (P < 0.06) but bore no relation to the effects of BS or Diet on plasma T4. These data show that IUGR and Normal lambs are similarly unable to adjust caloric intake in early life and that the fatness promoting effects of the IUGR condition are more pronounced in the viscera than in the carcass. These data also reveal dynamic regulation of plasma T4 by BS and Diet in neonatal lambs.

Dietary Supplementation of L-Arginine and N-Carbamylglutamate Attenuated the Hepatic Inflammatory Response and Apoptosis in Suckling Lambs with Intrauterine Growth Retardation.

L-arginine (Arg) is a semiessential amino acid with several physiological functions. N-Carbamylglutamate (NCG) can promote the synthesis of endogenous Arg in mammals. However, the roles of Arg or NCG on hepatic inflammation and apoptosis in suckling lambs suffering from intrauterine growth restriction (IUGR) are still unclear. The current work is aimed at examining the effects of dietary Arg and NCG on inflammatory and hepatocyte apoptosis in IUGR suckling lambs. On day 7 after birth, 48 newborn Hu lambs were selected from a cohort of 432 twin lambs. Normal-birthweight and IUGR Hu lambs were allocated randomly (n = 12/group) to control (CON), IUGR, IUGR+1% Arg, or IUGR+0.1% NCG groups. Lambs were fed for 21 days from 7 to 28 days old. Compared with CON lambs, relative protein 53 (P53), apoptosis antigen 1 (Fas), Bcl-2-associated X protein (Bax), caspase-3, cytochrome C, tumor necrosis factor alpha (TNF-α), nuclear factor kappa-B (NF-κB) p65, and NF-κB pp65 protein levels were higher (P < 0.05) in liver from IUGR lambs, whereas those in liver from IUGR lambs under Arg or NCG treatment were lower than those in IUGR lambs. These findings indicated that supplementing Arg or NCG reduced the contents of proinflammatory cytokines at the same time when the apoptosis-related pathway was being suppressed, thus suppressing the IUGR-induced apoptosis of hepatic cells.

Postnatal β2 adrenergic treatment improves insulin sensitivity in lambs with IUGR but not persistent defects in pancreatic islets or skeletal muscle.

Key points Previous studies in fetuses with intrauterine growth restriction (IUGR) have shown that adrenergic dysregulation was associated with low insulin concentrations and greater insulin sensitivity.Although whole‐body glucose clearance is normal, 1‐month‐old lambs with IUGR at birth have higher rates of hindlimb glucose uptake, which may compensate for myocyte deficiencies in glucose oxidation.Impaired glucose‐stimulated insulin secretion in IUGR lambs is due to lower intra‐islet insulin availability and not from glucose sensing.We investigated adrenergic receptor (ADR) β2 desensitization by administering oral ADRβ modifiers for the first month after birth to activate ADRβ2 and antagonize ADRβ1/3. In IUGR lambs ADRβ2 activation increased whole‐body glucose utilization rates and insulin sensitivity but had no effect on isolated islet or myocyte deficiencies.IUGR establishes risk for developing diabetes. In IUGR lambs we identified disparities in key aspects of glucose‐stimulated insulin secretion and insulin‐stimulated glucose oxidation, providing new insights into potential mechanisms for this risk. Placental insufficiency causes intrauterine growth restriction (IUGR) and disturbances in glucose homeostasis with associated β adrenergic receptor (ADRβ) desensitization. Our objectives were to measure insulin‐sensitive glucose metabolism in neonatal lambs with IUGR and to determine whether daily treatment with ADRβ2 agonist and ADRβ1/β3 antagonists for 1 month normalizes their glucose metabolism. Growth, glucose‐stimulated insulin secretion (GSIS) and glucose utilization rates (GURs) were measured in control lambs, IUGR lambs and IUGR lambs treated with adrenergic receptor modifiers: clenbuterol atenolol and SR59230A (IUGR‐AR). In IUGR lambs, islet insulin content and GSIS were less than in controls; however, insulin sensitivity and whole‐body GUR were not different from controls. Of importance, ADRβ2 stimulation with β1/β3 inhibition increases both insulin sensitivity and whole‐body glucose utilization in IUGR lambs. In IUGR and IUGR‐AR lambs, hindlimb GURs were greater but fractional glucose oxidation rates and ex vivo skeletal muscle glucose oxidation rates were lower than controls. Glucose transporter 4 (GLUT4) was lower in IUGR and IUGR‐AR skeletal muscle than in controls but GLUT1 was greater in IUGR‐AR. ADRβ2, insulin receptor, glycogen content and citrate synthase activity were similar among groups. In IUGR and IUGR‐AR lambs heart rates were greater, which was independent of cardiac ADRβ1 activation. We conclude that targeted ADRβ2 stimulation improved whole‐body insulin sensitivity but minimally affected defects in GSIS and skeletal muscle glucose oxidation. We show that risk factors for developing diabetes are independent of postnatal catch‐up growth in IUGR lambs as early as 1 month of age and are inherent to the islets and myocytes.

N-Carbamylglutamate and l-Arginine Promote Intestinal Absorption of Amino Acids by Regulating the mTOR Signaling Pathway and Amino Acid and Peptide Transporters in Suckling Lambs with Intrauterine Growth Restriction

Previous studies have revealed that dietary N-carbamylglutamate (NCG) and l-arginine (Arg) improve intestinal integrity, oxidative state, and immune function in Hu suckling lambs with intrauterine growth restriction (IUGR). Whether these treatments alter intestinal nutrient absorption is unknown. The aim of this study was to determine the influence of dietary NCG and Arg treatment during the suckling period on intestinal amino acid (AA) absorption, alterations in the mechanistic target of rapamycin (mTOR) signaling pathway, and the abundance of AA and peptide transporters in IUGR lambs. On day 7 after birth, 48 newborn Hu lambs were selected from a cohort of 424 twin lambs. Normal-birth-weight and IUGR Hu lambs were allocated randomly (n=12/group) to a control (4.09±0.12 kg), IUGR (3.52±0.09 kg), IUGR+0.1% NCG (3.49±0.11 kg), or IUGR+1% Arg (3.53±0.10 kg). At day 28, compared with the IUGR group, the IUGR groups receiving NCG and Arg had 7.4% and 7.2% greater (P<0.05) body weight, respectively. Compared with the IUGR group, the serum concentration of insulin was greater (P<0.05) and the cortisol was lower (P<0.05) in the IUGR groups receiving NCG and Arg. Compared with the IUGR group, the IUGR groups receiving NCG and Arg had 13.2%-62.6% greater (P<0.05) serum concentrations of arginine, cysteine, isoleucine, and proline. Dietary NCG or Arg to IUGR lambs resulted in greater protein abundance (P<0.05) of peptide transporter 1 (41.9% or 38.2%) in the ileum compared with the unsupplemented IUGR lambs, respectively. Furthermore, dietary NCG or Arg treatment normalized the IUGR-induced variation (P<0.05) in the ileal ratio of phosphorylated mTOR to total mTOR protein. Both NCG and Arg can help mitigate the negative effect of IUGR on nutrient absorption in neonatal lambs.

Enhanced insulin secretion and insulin sensitivity in young lambs with placental insufficiency-induced intrauterine growth restriction.

Intrauterine growth restriction (IUGR) is associated with persistent metabolic complications, but information is limited for IUGR infants. We determined glucose-stimulated insulin secretion (GSIS) and insulin sensitivity in young lambs with placental insufficiency-induced IUGR. Lambs with hyperthermia-induced IUGR (n = 7) were compared with control lambs (n = 8). GSIS was measured at 8 ± 1 days of age, and at 15 ± 1 days, body weight-specific glucose utilization rates were measured with radiolabeled d-glucose during a hyperinsulinemic-euglycemic clamp (HEC). IUGR lambs weighed 23% less (P < 0.05) than controls at birth. Fasting plasma glucose and insulin concentrations were not different between IUGR and controls for either study. First-phase insulin secretion was enhanced 2.3-fold in IUGR lambs compared with controls. However, second-phase insulin concentrations, glucose-potentiated arginine-stimulated insulin secretion, and β-cell mass were not different, indicating that IUGR β-cells have an intrinsic enhancement in acute GSIS. Compared with controls, IUGR lambs had higher body weight-specific glucose utilization rates and greater insulin sensitivity at fasting (1.6-fold) and hyperinsulinemic periods (2.4-fold). Improved insulin sensitivity for glucose utilization was not due to differences in skeletal muscle insulin receptor and glucose transporters 1 and 4 concentrations. Plasma lactate concentrations during HEC were elevated in IUGR lambs compared with controls, but no differences were found for glycogen content or citrate synthase activity in liver and muscle. Greater insulin sensitivity for glucose utilization and enhanced acute GSIS in young lambs are predicted from fetal studies but may promote conditions that exaggerate glucose disposal and lead to episodes of hypoglycemia in IUGR infants.

Ventilation-induced lung injury is not exacerbated by growth restriction in preterm lambs.

Intrauterine growth restriction (IUGR) and preterm birth are frequent comorbidities and, combined, increase the risk of adverse respiratory outcomes compared with that in appropriately grown (AG) infants. Potential underlying reasons for this increased respiratory morbidity in IUGR infants compared with AG infants include altered fetal lung development, fetal lung inflammation, increased respiratory requirements, and/or increased ventilation-induced lung injury. IUGR was surgically induced in preterm fetal sheep (0.7 gestation) by ligation of a single umbilical artery. Four weeks later, preterm lambs were euthanized at delivery or delivered and ventilated for 2 h before euthanasia. Ventilator requirements, lung inflammation, early markers of lung injury, and morphological changes in lung parenchymal and vascular structure and surfactant composition were analyzed. IUGR preterm lambs weighed 30% less than AG preterm lambs, with increased brain-to-body weight ratio, indicating brain sparing. IUGR did not induce lung inflammation or injury or alter lung parenchymal and vascular structure compared with AG fetuses. IUGR and AG lambs had similar oxygenation and respiratory requirements after birth and had significant, but similar, increases in proinflammatory cytokine expression, lung injury markers, gene expression, and surfactant phosphatidylcholine species compared with unventilated controls. IUGR does not induce pulmonary structural changes in our model. Furthermore, IUGR and AG preterm lambs have similar ventilator requirements in the immediate postnatal period. This study suggests that increased morbidity and mortality in IUGR infants is not due to altered lung tissue or vascular structure, or to an altered response to early ventilation.

Cerebrovascular adaptations to chronic hypoxia in the growth restricted lamb

Chronic moderate hypoxia induces angiogenic adaptation in the brain, reflecting a modulatory role for oxygen in determining cerebrovascular development. Chronic intrauterine fetal hypoxia, such as occurs in intrauterine growth restriction (IUGR) is likely to lead to a reduction in oxygen delivery to the brain and long-term neurological abnormalities. Thus we investigated whether vascular remodeling and vascular abnormalities were evident in the brain of IUGR newborn lambs that were chronically hypoxic in utero. Single uterine artery ligation (SUAL) surgery was performed in fetuses at ∼105 days gestation (term ∼145 days) to induce placental insufficiency and IUGR. Ewes delivered naturally at term and lambs were euthanased 24h later. IUGR brains (n=9) demonstrated a significant reduction in positive staining for the number of blood vessels (laminin immunohistochemistry) compared with control (n=8): from 1650±284 to 416±47cells/mm2 in subcortical white matter (SCWM) 1793±298 to 385±20cells/mm2 in periventricular white matter (PVWM), and 1717±161 to 405±84cells/mm2 in the subventricular zone (SVZ). The decrease in vascular density was associated with a significant decrease in VEGF immunoreactivity. The percentage of blood vessels exhibiting endothelial cell proliferation (Ki67 positive) varied regionally between 14 to 22% in white matter of control lambs, while only 1–3% of blood vessels in IUGR brains showed proliferation. A 66% reduction in pericyte coverage (α-SMA and desmin) of blood vessels was observed in SCWM, 71% in PVWM, and 73% in SVZ of IUGR lambs, compared to controls. A reduction in peri-vascular astrocytes (GFAP and laminin) was also observed throughout the white matter of IUGR lambs, and extravasation of albumin into the brain parenchyma was present, indicative of increased permeability of the blood brain barrier. Chronic hypoxia associated with IUGR results in a reduction in vascular density in the white matter of IUGR newborn brains. Vascular pericyte coverage and peri-vascular astrocytes, both of which are essential for stabilisation of blood vessels and the maintenance of vascular permeability, were also decreased in the white matter of IUGR lambs. In turn, these vascular changes could lead to inadequate oxygen supply and contribute to under-perfusion and increased vulnerability of white matter in IUGR infants.

Influence of birth weight and gender on lipid status and adipose tissue gene expression in lambs.

Intrauterine growth restriction (IUGR) is a risk factor for obesity, particularly when offspring are born into an unrestricted nutritional environment. In this study, we investigated the impact of IUGR and gender on circulating lipids and on expression of adipogenic, lipogenic and adipokine genes in perirenal adipose tissue. Singleton lambs born to overnourished adolescent dams were normal birth weight (N) or IUGR (32% lower birth weight due to placental insufficiency). IUGR lambs exhibited increased fractional growth rates but remained smaller than N lambs at necropsy (d77). At 48 days, fasting plasma triglycerides, non-esterified fatty acids and glycerol were elevated predominantly in IUGR males. Body fat content was independent of prenatal growth but higher in females than in males. In perirenal fat, relative to male lambs, females had larger adipocytes; higher lipoprotein lipase, fatty acid synthase and leptin and lower IGF1, IGF2, IGF1R, IGF2R and hormone-sensitive lipase mRNA expression levels, and all were independent of prenatal growth category; peroxisome proliferator-activated receptor gamma and glycerol-3-phosphate dehydrogenase (G3PDH) mRNA expression were not affected by IUGR or gender. Adiposity indices were inversely related to G3PDH mRNA expression, and for the population as a whole the expression of IGF system genes in perirenal fat was negatively correlated with plasma leptin, fat mass and adipocyte size, and positively correlated with circulating IGF1 levels. Higher plasma lipid levels in IUGR males may predict later adverse metabolic health and obesity, but in early postnatal life gender has the dominant influence on adipose tissue gene expression, reflecting the already established sexual dimorphism in body composition.

Antenatal antioxidant treatment with melatonin to decrease newborn neurodevelopmental deficits and brain injury caused by fetal growth restriction

Fetal intrauterine growth restriction (IUGR) is a serious pregnancy complication associated with increased rates of perinatal morbidity and mortality, and ultimately with long-term neurodevelopmental impairments. No intervention currently exists that can improve the structure and function of the IUGR brain before birth. Here, we investigated whether maternal antenatal melatonin administration reduced brain injury in ovine IUGR. IUGR was induced in pregnant sheep at 0.7 gestation and a subset of ewes received melatonin via intravenous infusion until term. IUGR, IUGR + melatonin (IUGR + MLT) and control lambs were born naturally, neonatal behavioral assessment was used to examine neurological function and at 24 hr after birth the brain was collected for the examination of neuropathology. Compared to control lambs, IUGR lambs took significantly longer to achieve normal neonatal lamb behaviors, such as standing and suckling. IUGR brains showed widespread cellular and axonal lipid peroxidation, and white matter hypomyelination and axonal damage. Maternal melatonin administration ameliorated oxidative stress, normalized myelination and rescued axonopathy within IUGR lamb brains, and IUGR + MLT lambs demonstrated significant functional improvements including a reduced time taken to attach to and suckle at the udder after birth. Based on these observations, we began a pilot clinical trial of oral melatonin administration to women with an IUGR fetus. Maternal melatonin was not associated with adverse maternal or fetal effects and it significantly reduced oxidative stress, as evidenced by reduced malondialdehyde levels, in the IUGR + MLT placenta compared to IUGR alone. Melatonin should be considered for antenatal neuroprotective therapy in human IUGR.

Impact of birth weight and gender on early postnatal hypothalamic energy balance regulatory gene expression in the young lamb

Intra-uterine growth restriction (IUGR) is involved in developmental metabolic programming and here we test the hypothesis that IUGR affects the developing hypothalamic energy balance regulatory pathways in a sex-specific manner. This experiment investigated early postnatal hypothalamic gene expression for six primary leptin- and insulin-sensitive neuropeptides and receptors in male and female IUGR (n=8 and 9, respectively) and normal (N) birth weight lambs (n=8 per gender) gestated and suckled by overnourished mothers. IUGR lambs were smaller at birth, had increased fractional growth rates (FGR), lower final body weight (11 weeks) and similar body fat content compared with N lambs, while males had higher final body weight and insulinemia but lower body fat and leptinemia than females. In situ hybridization revealed greater gene expression in the hypothalamic arcuate nucleus at 11 weeks for anorexigenic genes in females and orexigenic genes in males, with no effect of IUGR. Leptinemia correlated with gene expression for neuropeptide Y (NPY, negatively) in both sexes and pro-opiomelanocortin (POMC, positively) in females but with leptin receptor (negatively) only in males. Current FGR for girth correlated negatively with gene expression for NPY in males and POMC in females. Neither IUGR nor gender affected suckling activity (proxy for appetite) assessed at 3 weeks, but final NPY gene expression correlated with suckling weight gain in males. This study has revealed no effect of IUGR on early postnatal hypothalamic energy balance gene expression but a major effect of gender associated with major sex differences in adiposity and leptinemia.

The effect of placental restriction on insulin signaling and lipogenic pathways in omental adipose tissue in the postnatal lamb.

Intrauterine growth restriction (IUGR) followed by accelerated growth after birth is associated with an increased risk of abdominal (visceral) obesity and insulin resistance in adult life. The aim of the present study was to determine the impact of IUGR on mRNA expression and protein abundance of insulin signaling molecules in one of the major visceral fat depots, the omental adipose depot. IUGR was induced by placental restriction, and samples of omental adipose tissue were collected from IUGR (n = 9, 5 males, 4 females) and Control (n = 14, 8 males, 6 females) neonatal lambs at 21 days of age. The mRNA expression of the insulin signaling molecules, AMP-kinase (AMPK) and adipogenic/lipogenic genes was determined by qRT-PCR, and protein abundance by Western Blotting. AMPKα2 mRNA expression was increased in male IUGR lambs (0.015 ± 0.002 v. 0.0075 ± 0.0009, P < 0.001). The proportion of the AMPK pool that was phosphorylated (%P-AMPK) was lower in IUGR lambs compared with Controls independent of sex (39 ± 9% v. 100 ± 18%, P < 0.001). The mRNA expression and protein abundance of insulin signaling proteins and adipogenic/lipogenic genes was not different between groups. Thus, IUGR is associated with sex-specific alterations in the mRNA expression of AMPKα2 and a reduction in the percentage of the total AMPK pool that is phosphorylated in the omental adipose tissue of neonatal lambs, before the onset of visceral obesity. These molecular changes would be expected to promote lipid accumulation in the omental adipose depot and may therefore contribute to the onset of visceral adiposity in IUGR animals later in life.