D. T. Yates

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.

Characterization of glucose-insulin responsiveness and impact of fetal number and sex difference on insulin response in the sheep fetus

GSIS is often measured in the sheep fetus by a square-wave hyperglycemic clamp, but maximal β-cell responsiveness and effects of fetal number and sex difference have not been fully evaluated. We determined the dose-response curve for GSIS in fetal sheep (0.9 of gestation) by increasing plasma glucose from euglycemia in a stepwise fashion. The glucose-insulin response was best fit by curvilinear third-order polynomial equations for singletons (y = 0.018x(3) - 0.26x(2) + 1.2x - 0.64) and twins (y = -0.012x(3) + 0.043x(2) + 0.40x - 0.16). In singles, maximal insulin secretion was achieved at 3.4 ± 0.2 mmol/l glucose but began to plateau after 2.4 ± 0.2 mmol/l glucose (90% of maximum), whereas the maximum for twins was reached at 4.8 ± 0.4 mmol/l glucose. In twin (n = 18) and singleton (n = 49) fetuses, GSIS was determined with a square-wave hyperglycemic clamp >2.4 mmol/l glucose. Twins had a lower basal glucose concentration, and plasma insulin concentrations were 59 (P < 0.01) and 43% (P < 0.05) lower in twins than singletons during the euglycemic and hyperglycemic periods, respectively. The basal glucose/insulin ratio was approximately doubled in twins vs. singles (P < 0.001), indicating greater insulin sensitivity. In a separate cohort of fetuses, twins (n = 8) had lower body weight (P < 0.05) and β-cell mass (P < 0.01) than singleton fetuses (n = 7) as a result of smaller pancreata (P < 0.01) and a positive correlation (P < 0.05) between insulin immunopositive area and fetal weight (P < 0.05). No effects of sex difference on GSIS or β-cell mass were observed. These findings indicate that insulin secretion is less responsive to physiological glucose concentrations in twins, due in part to less β-cell mass.

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.

Myoblasts from intrauterine growth‐restricted sheep fetuses exhibit intrinsic deficiencies in proliferation that contribute to smaller semitendinosus myofibres

To investigate loss of skeletal muscle mass in intrauterine growth‐restricted (IUGR) fetuses near term, which may result from myoblast dysfunction, we examined semitendinosus myofibre and myoblast morphology in placental insufficiency‐induced IUGR sheep fetuses; we also isolated and cultured IUGR fetal myoblasts to determine whether reduced rates of proliferation were due to intrinsic cellular defects or extrinsic factors associated with serum. Using tests for myogenin and pax7 to identify differentiated and undifferentiated fetal myoblasts, respectively, we found that myofibre area and percentage of myogenin‐positive nuclei were less in IUGR fetal semitendinosus muscles than in controls, but myofibre density and percentage of pax7‐positive nuclei were not different. The percentage of pax7‐positive cells that expressed proliferating cellular nuclear antigen was less in IUGR semitendinosus muscles than in controls, while in myoblasts isolated from fetal sheep and replicated and differentiated in culture, IUGR fetal myoblasts proliferated at slower rates than control myoblasts, under identical culture conditions, but the ability to differentiate was similar between treatments. Media supplemented with IUGR serum decreased replication rates in both IUGR and control myoblasts compared to media supplemented with control fetal serum. These findings show that myoblasts proliferate at slower rates in IUGR fetuses due to a combination of intrinsic cellular characteristics and extrinsic serum factors; the intrinsic defects may explain reduced skeletal muscle mass observed in IUGR newborn children and adults.

Hypoxaemia-induced catecholamine secretion from adrenal chromaffin cells inhibits glucose-stimulated hyperinsulinaemia in fetal sheep

•  Hypoxaemia was previously shown to lower fetal plasma insulin at euglycaemia and hyperglycaemia. Lowering insulin redistributes nutrients and spares glucose and oxygen. •  Hypoxaemia also stimulates adrenal medullary secretion of adrenaline and noradrenaline, but the impact of adrenal catecholamines versus local noradrenaline secretion by sympathetic neurons has not been evaluated on insulin concentrations. •  To determine the impact of adrenal medullary catecholamines on plasma insulin, we surgically demedullated the adrenal glands in fetal sheep and challenged them with acute hypoxaemia. •  We found that fetal adrenal chromaffin cells were the source for hypoxia‐induced increases in plasma adrenaline and noradrenaline. •  Adrenal medullary catecholamines were essential for suppression of glucose‐stimulated hyperinsulinaemia but not for reduced basal insulin concentrations. They also contributed to fetal hyperlactacaemia and hypocarbia independently of their effects on insulin. •  This study demonstrates that fetal hypoxaemia reduces basal and glucose‐stimulated insulin concentrations, but by different mechanisms. Glucose‐stimulated hyperinsulinaemia is reduced by elevated plasma catecholamines secreted from the adrenal medulla, which helps to spare glucose and oxygen resources.

Elevated plasma norepinephrine inhibits insulin secretion, but adrenergic blockade reveals enhanced β-cell responsiveness in an ovine model of placental insufficiency at 0.7 of gestation.

In pregnancies complicated by placental insufficiency (PI), fetal hypoglycemia and hypoxemia progressively worsen during the third trimester, which increases circulating norepinephrine (NE). Pharmacological adrenergic blockade (ADR-block) at 0.9 gestation revealed that NE inhibits insulin secretion and enhanced β-cell responsiveness in fetuses with PI-induced intrauterine growth restriction (IUGR). NE concentrations in PI fetuses at 0.7 gestation were threefold greater compared with age-matched controls, but the levels were similar to near-term controls. Therefore, our objective was to determine whether elevations in plasma NE concentrations inhibit insulin secretion and produce compensatory β-cell responsiveness in PI fetuses at 0.7 gestation. Fetal insulin was measured under basal, glucose-stimulated insulin secretion (GSIS) and glucose-potentiated arginine-stimulated insulin secretion (GPAIS) conditions in the absence and presence of an ADR-block. Placental weights were 38% lower (P < 0.05) in PI fetus than in controls, but fetal weights were not different. PI fetuses had lower (P < 0.05) basal blood oxygen content, plasma glucose, insulin-like growth factor-1 and insulin concentrations and greater plasma NE concentrations (891 ± 211 v. 292 ± 65 pg/ml; P < 0.05) than controls. GSIS was lower in PI fetuses than in controls (0.34 ± 0.03 v. 1.08 ± 0.06 ng/ml; P < 0.05). ADR-block increased GSIS in PI fetuses (1.19 ± 0.11 ng/ml; P < 0.05) but decreased GSIS in controls (0.86 ± 0.02 ng/ml; P < 0.05). Similarly, GPAIS was 44% lower (P < 0.05) in PI fetuses than in controls, and ADR-block increased (P < 0.05) GPAIS in PI fetuses but not in controls. Insulin content per islet was not different between treatments. We conclude that elevations in fetal plasma NE suppress insulin concentrations, and that compensatory β-cell stimulus-secretion responsiveness is present before IUGR.

Enhanced insulin secretion responsiveness and islet adrenergic desensitization after chronic norepinephrine suppression is discontinued in fetal sheep.

Intrauterine growth-restricted (IUGR) fetuses experience prolonged hypoxemia, hypoglycemia, and elevated norepinephrine (NE) concentrations, resulting in hypoinsulinemia and β-cell dysfunction. Previously, we showed that acute adrenergic blockade revealed enhanced insulin secretion responsiveness in the IUGR fetus. To determine whether chronic exposure to NE alone enhances β-cell responsiveness afterward, we continuously infused NE into fetal sheep for 7 days and, after terminating the infusion, evaluated glucose-stimulated insulin secretion (GSIS) and glucose-potentiated arginine-induced insulin secretion (GPAIS). During treatment, NE-infused fetuses had greater (P < 0.05) plasma NE concentrations and exhibited hyperglycemia (P < 0.01) and hypoinsulinemia (P < 0.01) compared with controls. GSIS during the NE infusion was also reduced (P < 0.05) compared with pretreatment values. GSIS and GPAIS were approximately fourfold greater (P < 0.01) in NE fetuses 3 h after the 7 days that NE infusion was discontinued compared with age-matched controls or pretreatment GSIS and GPAIS values of NE fetuses. In isolated pancreatic islets from NE fetuses, mRNA concentrations of adrenergic receptor isoforms (α1D, α2A, α2C, and β1), G protein subunit-αi-2, and uncoupling protein 2 were lower (P < 0.05) compared with controls, but β-cell regulatory genes were not different. Our findings indicate that chronic exposure to elevated NE persistently suppresses insulin secretion. After removal, NE fetuses demonstrated a compensatory enhancement in insulin secretion that was associated with adrenergic desensitization and greater stimulus-secretion coupling in pancreatic islets.

Adrenal Demedullation and Oxygen Supplementation Independently Increase Glucose-Stimulated Insulin Concentrations in Fetal Sheep With Intrauterine Growth Restriction

In pregnancies complicated by placental insufficiency and intrauterine growth restriction (IUGR), fetal glucose and oxygen concentrations are reduced, whereas plasma norepinephrine and epinephrine concentrations are elevated throughout the final third of gestation. Here we study the effects of chronic hypoxemia and hypercatecholaminemia on β-cell function in fetal sheep with placental insufficiency-induced IUGR that is produced by maternal hyperthermia. IUGR and control fetuses underwent a sham (intact) or bilateral adrenal demedullation (AD) surgical procedure at 0.65 gestation. As expected, AD-IUGR fetuses had lower norepinephrine concentrations than intact-IUGR fetuses despite being hypoxemic and hypoglycemic. Placental insufficiency reduced fetal weights, but the severity of IUGR was less with AD. Although basal plasma insulin concentrations were lower in intact-IUGR and AD-IUGR fetuses compared with intact-controls, glucose-stimulated insulin concentrations were greater in AD-IUGR fetuses compared with intact-IUGR fetuses. Interestingly, AD-controls had lower glucose- and arginine-stimulated insulin concentrations than intact-controls, but AD-IUGR and AD-control insulin responses were not different. To investigate chronic hypoxemia in the IUGR fetus, arterial oxygen tension was increased to normal levels by increasing the maternal inspired oxygen fraction. Oxygenation of IUGR fetuses enhanced glucose-stimulated insulin concentrations 3.3-fold in intact-IUGR and 1.7-fold in AD-IUGR fetuses but did not lower norepinephrine and epinephrine concentrations. Together these findings show that chronic hypoxemia and hypercatecholaminemia have distinct but complementary roles in the suppression of β-cell responsiveness in IUGR fetuses.

Effects of bacterial lipopolysaccharide injection on white blood cell counts, hematological variables, and serum glucose, insulin, and cortisol concentrations in ewes fed low- or high-protein diets.

Bacterial lipopolysaccharide endotoxins (LPS) elicit inflammatory responses reflective of acute bacterial infection. We determined if feeding ewes high-CP (15.5%) or low-CP (8.5%) diets for 10 d altered inflammatory responses to an intravenous bolus of 0 (control), 0.75 (L75), or 1.50 (L150) μg of LPS/kg of BW in a 2 × 3 factorial arrangement of treatments (n = 5/treatment). Rectal temperatures, heart and respiratory rates, blood leukocyte concentrations, and serum cortisol, insulin, and glucose concentrations were measured for 24 h after an LPS bolus (bolus = 0 h). In general, rectal temperatures were greater (P ≤ 0.05) in control ewes fed high CP, but LPS increased (P ≤ 0.05) rectal temperatures in a dose-dependent manner at most times between 2 and 24 h after the bolus. Peak rectal temperatures in L75 and L150 occurred 4 h after the bolus. A monophasic, dose-independent increase (P ≤ 0.023) in serum cortisol occurred from 0.5 to 24 h after the bolus, with peak cortisol at 4 h. Serum insulin was increased (P ≤ 0.016) by LPS in a dose-dependent manner from 4 to 24 h after the bolus. Insulin did not differ between control ewes fed high- and low-CP diets but was greater (P < 0.001) in L75 ewes fed low CP compared with high CP and in L150 ewes fed high CP compared with low CP. Increased insulin was not preceded by increased serum glucose. Total white blood cell concentrations were not affected (P ≥ 0.135) by LPS, but the neutrophil and monocyte fractions of white blood cells were increased (P ≤ 0.047) by LPS at 12 and 24 h and at 24 h after the bolus, respectively, and the lymphocyte fraction was increased (P = 0.037) at 2 h and decreased (P ≤ 0.006) at 12 and 24 h after the bolus. Red blood cell and hemoglobin concentrations and hematocrit (%) were increased (P ≤ 0.022) by LPS at 2 and 4 h after the bolus. Rectal temperatures and serum glucose were greater (P ≤ 0.033) in ewes fed a high-CP diet before LPS injection, but these effects were lost at and within 2.5 h of the bolus, respectively. Feeding high-CP diets for 10 d did not reduce inflammation in ewes during the first 24 h after LPS exposure but may benefit livestock by preventing acute insulin resistance when endotoxin exposure is mild.

Technical note: comparison of salivary and serum cortisol concentrations after adrenocorticotropic hormone challenge in ewes.

An ACTH challenge was conducted to determine if salivary cortisol concentration reflects serum cortisol concentration in ewes. Twelve yearling ewes (64.0 +/- 1.2 kg) were administered ACTH (100 IU, intravenously) or saline. Serum and salivary samples were collected at 30-min intervals for 2 h before ACTH administration, at 15-min intervals for 2 h after treatment, and at 30-min intervals for an additional 3 h, and cortisol concentration was determined by RIA. Although ewes responded to ACTH and saline, cortisol concentration was greater (P < 0.001) in ACTH-treated ewes from 15 to 120 min and tended to be greater (P = 0.054) at 150 min after challenge in serum. In saliva, cortisol concentration was greater (P < 0.001) in ACTH-treated ewes from 30 to 120 min and tended to be greater (P = 0.092) at 15 min after challenge. No difference was observed between ACTH-treated ewes and controls for time to peak serum cortisol concentration (P = 0.126) and time to peak salivary cortisol concentration (P = 0.109), or between saliva and serum for time to peak cortisol concentration (P = 0.220) and return to baseline cortisol concentration (P = 0.341). The serum (P = 0.009) and salivary (P = 0.050) cortisol areas under the curve between 0 and 150 min were greater for ACTH-treated ewes than controls, and serum (P = 0.002) and salivary (P < 0.001) cortisol return to baseline concentration was longer for ACTH-treated ewes. The correlation coefficient between serum and salivary cortisol concentrations was 0.88 (P < 0.001). These data indicate that salivary cortisol concentration is closely related to serum cortisol concentration and that the former may represent a suitable noninvasive alternative to blood collection for measurement of cortisol in sheep.