Alice S. Green

Consequences of a compromised intrauterine environment on islet function

Low birth weight is an important risk factor for impaired glucose tolerance and diabetes later in life. One hypothesis is that fetal beta-cells inherit a persistent defect as a developmental response to fetal malnutrition, a primary cause of intrauterine growth restriction (IUGR). Our understanding of fetal programing events in the human endocrine pancreas is limited, but several animal models of IUGR extend our knowledge of developmental programing in beta-cells. Pathological outcomes such as beta-cell dysfunction, impaired glucose tolerance, and diabetes are often observed in adult offspring from these animal models, similar to the associations of low birth weight and metabolic diseases in humans. However, the identified mechanisms underlying beta-cell dysfunction across models and species are varied, likely resulting from the different methodologies used to induce experimental IUGR, as well as from intraspecies differences in pancreas development. In this review, we first present the evidence for human beta-cell dysfunction being associated with low birth weight or IUGR. We then evaluate relevant animal models of IUGR, focusing on the strengths of each, in order to define critical periods and types of nutrient deficiencies that can lead to impaired beta-cell function. These findings frame our current knowledge of beta-cell developmental programing and highlight future research directions to clarify the mechanisms of beta-cell dysfunction for human IUGR.

β2-Adrenergic receptor desensitization in perirenal adipose tissue in fetuses and lambs with placental insufficiency-induced intrauterine growth restriction

Placental insufficiency‐induced intrauterine growth restriction (IUGR) fetuses have chronic hypoxaemia and elevated plasma catecholamine concentrations. In this study, we determined whether adrenergic responsiveness becomes desensitized in the perirenal adipose tissue of IUGR fetuses and lambs by measuring adrenergic receptor (AR) mRNA and protein levels. We also tested the ability of adrenaline to mobilize non‐esterified fatty acids (NEFAs) in young lambs. Perirenal adipose tissue was collected from IUGR and control fetuses at 133 days of gestational age (dGA) and lambs at 18 days of age (dA). β2‐AR mRNA concentrations were 59% and 74% lower (P < 0.05) in IUGR fetuses and lambs compared to controls, respectively, which also resulted in lower protein levels (P < 0.05). No treatment differences were detected for α1A‐, α1B‐, α1D‐, α2A‐, α2B‐, α2C‐, β1‐ and β3‐AR expression. mRNA concentrations were also determined for hormone sensitive lipase (HSL), perilipin (lipid droplet‐associated protein), and two adipokines, leptin and adiponectin. Adiponectin and HSL were not different between treatments at either age. Compared to controls, perilipin and leptin mRNA concentrations were lower (P < 0.05) in IUGR fetuses but not in lambs. Because of the β2‐AR results, we challenged a second cohort of lambs with exogenous adrenaline at 21 dA. The ability of adrenaline to mobilize NEFA was 55 ± 15% lower (P < 0.05) in IUGRs than controls. Collectively, our findings indicate that elevated catecholamine exposure in utero causes desensitization of adipose tissue by down‐regulation of β2‐AR, and this persists in lambs. This impairment in adrenergic stimulated lipolysis might partially explain early onset obesity in IUGR offspring.

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.

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.

Chronic Pulsatile Hyperglycemia Reduces Insulin Secretion and Increases Accumulation of Reactive Oxygen Species in Fetal Sheep Islets

Children from diabetic pregnancies have a greater incidence of type 2 diabetes. Our objective was to determine if exposure to mild-moderate hyperglycemia, by modeling managed diabetic pregnancies, affects fetal β-cell function. In sheep fetuses, β-cell responsiveness was examined after 2 weeks of sustained hyperglycemia with 3 pulses/day, mimicking postprandial excursions, and compared to saline-infused controls (n = 10). Two pulsatile hyperglycemia (PHG) treatments were studied: mild (mPHG, n = 5) with +15% sustained and +55% pulse; and moderate (PHG, n = 10) with +20% sustained and +100% pulse. Fetal glucose-stimulated insulin secretion and glucose-potentiated arginine insulin secretion were lower (P < 0.05) in PHG (0.86 ± 0.13 and 2.91 ± 0.39  ng/ml plasma insulin) but not in mPHG fetuses (1.21 ± 0.08 and 4.25 ± 0.56  ng/ml) compared to controls (1.58 ± 0.25 and 4.51 ± 0.56  ng/ml). Islet insulin content was 35% lower in PHG and 35% higher in mPHG vs controls (P < 0.01). Insulin secretion and maximally stimulated insulin release were also reduced (P < 0.05) in PHG islets due to lower islet insulin content. Isolated PHG islets also had 63% greater (P < 0.01) reactive oxygen species (ROS) accumulation at 11.1  mmol/l glucose than controls (P < 0.01), but oxidative damage was not detected in islet proteins. PHG fetuses showed evidence of oxidative damage to skeletal muscle proteins (P < 0.05) but not insulin resistance. Our findings show that PHG induced dysregulation of islet ROS handling and decreased islet insulin content, but these outcomes are independent. The β-cell outcomes were dependent on the severity of hyperglycemia because mPHG fetuses had no distinguishable impairments in ROS handling or insulin secretion but greater insulin content.

Remembering development - epigenetic responses to fetal malnutrition.

Some of the most debilitating diseases affecting global public health that have an extreme monetary impact on health care costs are metabolic diseases such as type 2 diabetes. These complex polymorphic diseases are influenced by multiple genetic loci as well as environmental cues. Fetal malnutrition has been identified as one such cue, causing asymmetric fetal growth as well as being associated with several adult onset diseases (Hales & Barker, 1992). During embryonic and fetal life, cellular expansion and differentiation coordinate the development and growth of tissues and organs to shape an adult. If fetal nutrition is compromised during these important developmental events, adaptations to decreased substrate availability may occur via epigenetic mechanisms (Pinney & Simmons, 2009). These changes can persist postnatally, resulting in inappropriate handling of substrates and likely contributing to the development of chronic metabolic diseases.