Miranda J. Anderson

Morphologic evidence that neurokinin B modulates gonadotropin-releasing hormone secretion via neurokinin 3 receptors in the rat median eminence.

Recent studies suggest that arcuate neurokinin B (NKB) neurons play a role in the regulation of gonadotropin secretion, but there is little information on the relationship between these neurons and the hypothalamic reproductive axis. In the present study, dual‐label fluorescent immunohistochemistry was used to visualize the relationship between gonadotropin‐releasing hormone (GnRH) neurons and either proNKB or NK3 receptor (NK3R) immunoreactivity. Immunocytochemistry was also combined with i.p. injections of the fluorescent retrograde tracer aminostilbamidine to determine whether arcuate neuroendocrine neurons expressed either proNKB or NK3R. A dense interweaving and close apposition of GnRH and proNKB‐immunoreactive (ir) fibers was observed within the rat median eminence, where GnRH axons expressed NK3R immunoreactivity. These data provide morphological evidence that NKB neurons could influence GnRH secretion via interaction with NK3R in the rat median eminence. Colocalization of GnRH and NK3R was also identified in fiber tracts converging within the organum vasculosum of the lamina terminalis. In contrast, only a small number (16%) of GnRH‐ir somata exhibited NK3R staining. ProNKB and NK3R‐ir somata were identified within the arcuate nucleus, but none of these neurons were labeled by aminostilbamidine. Thus, we found no evidence that arcuate NKB neurons project to the primary capillary plexus of the portal system. Arcuate neuroendocrine neurons, however, were surrounded and closely apposed by proNKB‐ir puncta and fibers. These data suggest that NKB neurons could indirectly influence anterior pituitary function by inputs to arcuate neuroendocrine neurons, but through a receptor other than NK3R. Our results provide an anatomic framework for putative interactions between NKB neurons and the hypothalamic reproductive axis. J. Comp. Neurol. 489:372–386, 2005.

Forebrain projections of arcuate neurokinin B neurons demonstrated by anterograde tract-tracing and monosodium glutamate lesions in the rat

Neurokinin B (NKB) and kisspeptin receptor signaling are essential components of the reproductive axis. A population of neurons resides within the arcuate nucleus of the rat that expresses NKB, kisspeptin, dynorphin, NK3 receptors and estrogen receptor alpha (ERalpha). Here we investigate the projections of these neurons using NKB-immunocytochemistry as a marker. First, the loss of NKB-immunoreactive (ir) somata and fibers was characterized after ablation of the arcuate nucleus by neonatal injections of monosodium glutamate. Second, biotinylated dextran amine was injected into the arcuate nucleus and anterogradely labeled NKB-ir fibers were identified using dual-labeled immunofluorescence. Four major projection pathways are described: (1) local projections within the arcuate nucleus bilaterally, (2) projections to the median eminence including the lateral palisade zone, (3) projections to a periventricular pathway extending rostrally to multiple hypothalamic nuclei, the septal region and BNST and dorsally to the dorsomedial nucleus and (4) Projections to a ventral hypothalamic tract to the lateral hypothalamus and medial forebrain bundle. The diverse projections provide evidence that NKB/kisspeptin/dynorphin neurons could integrate the reproductive axis with multiple homeostatic, behavioral and neuroendocrine processes. Interestingly, anterograde tract-tracing revealed NKB-ir axons originating from arcuate neurons terminating on other NKB-ir somata within the arcuate nucleus. Combined with previous studies, these experiments reveal a bilateral interconnected network of sex-steroid responsive neurons in the arcuate nucleus of the rat that express NKB, kisspeptin, dynorphin, NK3 receptors and ERalpha and project to GnRH terminals in the median eminence. This circuitry provides a mechanism for bilateral synchronization of arcuate NKB/kisspeptin/dynorphin neurons to modulate the pulsatile secretion of GnRH.

One process for pancreatic β-cell coalescence into islets involves an epithelial–mesenchymal transition

Islet replacement is a promising therapy for treating diabetes mellitus, but the supply of donor tissue for transplantation is limited. To overcome this limitation, endocrine tissue can be expanded, but this requires an understanding of normal developmental processes that regulate islet formation. In this study, we compare pancreas development in sheep and human, and provide evidence that an epithelial-mesenchymal transition (EMT) is involved in beta-cell differentiation and islet formation. Transcription factors know to regulate pancreas formation, pancreatic duodenal homeobox factor 1, neurogenin 3, NKX2-2, and NKX6-1, which were expressed in the appropriate spatial and temporal pattern to coordinate pancreatic bud outgrowth and direct endocrine cell specification in sheep. Immunofluorescence staining of the developing pancreas was used to co-localize insulin and epithelial proteins (cytokeratin, E-cadherin, and beta-catenin) or insulin and a mesenchymal protein (vimentin). In sheep, individual beta-cells become insulin-positive in the progenitor epithelium, then lose epithelial characteristics, and migrate out of the epithelial layer to form islets. As beta-cells exit the epithelial progenitor cell layer, they acquire mesenchymal characteristics, shown by their acquisition of vimentin. In situ hybridization expression analysis of the SNAIL family members of transcriptional repressors (SNAIL1, -2, and -3; listed as SNAI1, -2, -3 in the HUGO Database) showed that each of the SNAIL genes was expressed in the ductal epithelium during development, and SNAIL-1 and -2 were co-expressed with insulin. Our findings provide strong evidence that the movement of beta-cells from the pancreatic ductal epithelium involves an EMT.

Chronic exposure to elevated norepinephrine suppresses insulin secretion in fetal sheep with placental insufficiency and intrauterine growth restriction

In this study, we examined chronic norepinephrine suppression of insulin secretion in sheep fetuses with placental insufficiency-induced intrauterine growth restriction (IUGR). Glucose-stimulated insulin secretion (GSIS) was measured with a square-wave hyperglycemic clamp in the presence or absence of adrenergic receptor antagonists phentolamine (alpha) and propranolol (beta). IUGR fetuses were hypoglycemic and hypoxemic and had lower GSIS responsiveness (P < or = 0.05) than control fetuses. IUGR fetuses also had elevated plasma norepinephrine (3,264 +/- 614 vs. 570 +/- 86 pg/ml; P < or = 0.05) and epinephrine (164 +/- 32 vs. 60 +/- 12 pg/ml; P < or = 0.05) concentrations. In control fetuses, adrenergic inhibition increased baseline plasma insulin concentrations (1.7-fold, P < or = 0.05), whereas during hyperglycemia insulin was not different. A greater (P < or = 0.05) response to adrenergic inhibition was found in IUGR fetuses, and the average plasma insulin concentrations increased 4.9-fold at baseline and 7.1-fold with hyperglycemia. Unlike controls, basal plasma glucose concentrations fell (P < or = 0.05) with adrenergic antagonists. GSIS responsiveness, measured by the change in insulin, was higher (8.9-fold, P < or = 0.05) in IUGR fetuses with adrenergic inhibition than controls (1.8-fold, not significant), showing that norepinephrine suppresses insulin secretion in IUGR fetuses. Strikingly, in IUGR fetuses, adrenergic inhibition resulted in a greater GSIS responsiveness, because beta-cell mass was 56% lower and the maximal stimulatory insulin response tended (P < 0.1) to be higher than controls. This persistent norepinephrine suppression appears to be partially explained by higher mRNA concentrations of adrenergic receptors alpha(1D), alpha(2A), and alpha(2B) in a cohort of fetuses that were naïve to the antagonists. Therefore, norepinephrine suppression of insulin secretion was maintained, in part, by upregulating adrenergic receptor expression, but the beta-cells also appeared to compensate with enhanced GSIS. These findings may begin to explain why IUGR infants have a propensity for increased glucose requirements if norepinephrine is suddenly decreased after birth.

Reductions in insulin concentrations and β-cell mass precede growth restriction in sheep fetuses with placental insufficiency

In pregnancy complicated by placental insufficiency (PI) and intrauterine growth restriction (IUGR), the fetus near term has reduced basal and glucose-stimulated insulin concentrations and reduced β-cell mass. To determine whether suppression of insulin concentrations and β-cell mass precedes reductions in fetal weight, which would implicate insulin deficiency as a cause of subsequent IUGR, we measured basal and glucose-stimulated insulin concentrations and pancreatic histology at 0.7 gestation in PI fetuses. Placental weights in the PI pregnancies were 40% lower than controls (265 ± 26 vs. 442 ± 41 g, P < 0.05), but fetal weights were not different. At basal conditions blood oxygen content, plasma glucose concentrations, and plasma insulin concentrations were lower in PI fetuses compared with controls (2.5 ± 0.3 vs. 3.5 ± 0.3 mmol/l oxygen, P < 0.05; 1.11 ± 0.09 vs. 1.44 ± 0.12 mmol/l glucose; 0.12 ± 0.01 vs. 0.27 ± 0.02 ng/ml insulin; P < 0.05). During a steady-state hyperglycemic clamp (~2.5 ± 0.1 mmol/l), glucose-stimulated insulin concentrations were lower in PI fetuses than controls (0.28 ± 0.02 vs. 0.55 ± 0.04 ng/ml; P < 0.01). Plasma norepinephrine concentrations were 3.3-fold higher (P < 0.05) in PI fetuses (635 ± 104 vs. 191 ± 91 pg/ml). Histological examination revealed less insulin area and lower β-cell mass and rates of mitosis. The pancreatic parenchyma was also less dense (P < 0.01) in PI fetuses, but no differences were found for pancreatic progenitor cells or other endocrine cell types. These findings show that hypoglycemia, hypoxemia, and hypercatecholaminemia are present and potentially contribute to lower insulin concentrations and β-cell mass due to slower proliferation rates in early third-trimester PI fetuses before discernible reductions in fetal weight.

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.

Placental Insufficiency Decreases Pancreatic Vascularity and Disrupts Hepatocyte Growth Factor Signaling in the Pancreatic Islet Endothelial Cell in Fetal Sheep

Hepatocyte growth factor (HGF) and vascular endothelial growth factor A (VEGFA) are paracrine hormones that mediate communication between pancreatic islet endothelial cells (ECs) and β-cells. Our objective was to determine the impact of intrauterine growth restriction (IUGR) on pancreatic vascularity and paracrine signaling between the EC and β-cell. Vessel density was less in IUGR pancreata than in controls. HGF concentrations were also lower in islet EC-conditioned media (ECCM) from IUGR, and islets incubated with control islet ECCM responded by increasing insulin content, which was absent with IUGR ECCM. The effect of ECCM on islet insulin content was blocked with an inhibitory anti-HGF antibody. The HGF receptor was not different between control and IUGR islets, but VEGFA was lower and the high-affinity VEGF receptor was higher in IUGR islets and ECs, respectively. These findings show that paracrine actions from ECs increase islet insulin content, and in IUGR ECs, secretion of HGF was diminished. Given the potential feed-forward regulation of β-cell VEGFA and islet EC HGF, these two growth factors are highly integrated in normal pancreatic islet development, and this regulation is decreased in IUGR fetuses, resulting in lower pancreatic islet insulin concentrations and insulin secretion.

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.

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.