Clive W. Coen

Maternal Obesity Induced by Diet in Rats Permanently Influences Central Processes Regulating Food Intake in Offspring

Hypothalamic systems which regulate appetite may be permanently modified during early development. We have previously reported hyperphagia and increased adiposity in the adult offspring of rodents fed an obesogenic diet prior to and throughout pregnancy and lactation. We now report that offspring of obese (OffOb) rats display an amplified and prolonged neonatal leptin surge, which is accompanied by elevated leptin mRNA expression in their abdominal white adipose tissue. At postnatal Day 30, before the onset of hyperphagia in these animals, serum leptin is normal, but leptin-induced appetite suppression and phosphorylation of STAT3 in the arcuate nucleus (ARC) are attenuated; the level of AgRP-immunoreactivity in the hypothalamic paraventricular nucleus (PVH), which derives from neurones in the ARC and is developmentally dependent on leptin, is also diminished. We hypothesise that prolonged release of abnormally high levels of leptin by neonatal OffOb rats leads to leptin resistance and permanently affects hypothalamic functions involving the ARC and PVH. Such effects may underlie the developmental programming of hyperphagia and obesity in these rats.

Oestrogen Receptor β-Immunoreactivity in Gonadotropin Releasing Hormone-Expressing Neurones: Regulation by Oestrogen

Double‐label immunohistochemistry was employed to establish whether immunoreactivity for the β subtype of the oestrogen receptor (ERβ‐IR) is present in gonadotropin releasing hormone (GnRH)‐containing cells. In the immortalized GnRH cell line, GT1‐7, almost all nuclei were immunoreactive for ERβ. In the preoptic area of ovariectomized rats, more than one‐half of the GnRH neurones (52.0–63.5%) contained ERβ‐IR within the nucleus; a smaller proportion of these neurones (5–10%) displayed a particularly intense nuclear signal for ERβ. The presence of ERβ‐IR in the nuclei of GT1‐7 cells and GnRH neurones is consistent with recent reports of ERβ mRNA in these cells. Oestrogen treatment reduced the percentage of GnRH neurones with detectable ERβ‐IR. The range of signal intensity for ERβ and the incidence of the ERβ signal in GnRH neurones were comparable following double‐label immunohistochemistry involving either bright field or fluorescent techniques. These findings raise the possibility that ERβ receptors mediate direct effects of oestrogen on GnRH neurones.

The isoflavone equol mediates rapid vascular relaxation - Ca2+-independent activation of endothelial nitric-oxide synthase/Hsp90 involving ERK1/2 and Akt phosphorylation in human endothelial cells

We recently reported that soy isoflavones increase gene expression of endothelial nitric-oxide synthase (eNOS) and antioxidant defense enzymes, resulting in improved endothelial function and lower blood pressure in vivo. In this study, we establish that equol (1-100 nm) causes acute endothelium- and nitric oxide (NO)-dependent relaxation of aortic rings and rapidly (2 min) activates eNOS in human aortic and umbilical vein endothelial cells. Intracellular Ca2+ and cyclic AMP levels were unaffected by treatment (100 nm, 2 min) with equol, daidzein, or genistein. Rapid phosphorylation of ERK1/2, protein kinase B/Akt, and eNOS serine 1177 by equol was paralleled by association of eNOS with heat shock protein 90 (Hsp90) and NO synthesis in human umbilical vein endothelial cells, expressing estrogen receptors (ER)α and ERβ. Inhibition of phosphatidylinositol 3-kinase and ERK1/2 inhibited eNOS activity, whereas pertussis toxin and the ER antagonists ICI 182,750 and tamoxifen had negligible effects. Our findings provide the first evidence that nutritionally relevant plasma concentrations of equol (and other soy protein isoflavones) rapidly stimulate phosphorylation of ERK1/2 and phosphatidylinositol 3-kinase/Akt, leading to the activation of NOS and increased NO production at resting cytosolic Ca2+ levels. Identification of the nongenomic mechanisms by which equol mediates vascular relaxation provides a basis for evaluating potential benefits of equol in the treatment of postmenopausal women and patients at risk of cardiovascular disease.

Vasoactive intestinal polypeptide (VIP) phase-shifts the rat suprachiasmatic nucleus clock in vitro.

In mammals, the principal circadian pacemaker is housed in the hypothalamic suprachiasmatic nuclei (SCN). The SCN exhibit high levels of vasoactive intestinal polypeptide (VIP) immunoreactivity and two of the three VIP receptors, VPAC2 and PAC1, are found in the rat SCN. However, the role of VIP in the SCN remains unclear. In this study, we examined the phase‐resetting actions of VIP and selective VIP receptor agonists on the electrical activity rhythm of rat SCN neurons in vitro. Application of VIP during the subjective day did not shift the peak in the firing rate rhythm. However, VIP treatment during the early or late subjective night evoked a small phase delay or a large phase advance, respectively. The phase‐advancing effect of VIP was reproduced by the novel VPAC2 receptor agonist RO 25‐1553, but not by pituitary adenylate cyclase‐activating peptide (a potent PAC1 receptor agonist), or by [K15,R16,L27]VIP(1‐7)/GRF(8‐27), a novel, selective VPAC1 receptor agonist. These data show that VIP phase‐dependently phase‐resets the rodent SCN pacemaker in vitro, presumably via the VPAC2 receptor. As the pattern of phase‐shifting evoked by VIP and RO 25‐1553 resembles the phase‐resetting actions of light on rodent behavioural rhythms, these data support a role for VIP and the VPAC2 receptor in photic entrainment of the rodent circadian pacemaker.

Evidence for Sympathetic Origins of Hypertension in Juvenile Offspring of Obese Rats

Maternal obesity in rodents is associated with increased adiposity, impaired glucose tolerance, and hypertension in adult offspring. In this study we investigated the influence of maternal obesity in the rat on blood pressure and blood pressure regulatory pathways in juvenile and adult offspring. Obesity was induced before pregnancy in female Sprague-Dawley rats by feeding a highly palatable energy-dense diet. In juvenile animals (30 days of age), before the onset of obesity and hyperleptinemia, basal nighttime mean arterial pressure was significantly raised in the offspring of obese dams (OffOb) relative to offspring of controls (OffCon; mean arterial pressure, males: OffOb, 121.8±0.6 mm Hg versus OffCon, 115.0±0.5 mm Hg, n=6, P<0.01; females: OffOb, 125.4±0.4 mm Hg versus OffCon, 114.4±0.5 mm Hg, n=6, P<0.001), as was the mean arterial pressure response to restraint stress (P<0.01). The pressor response to a leptin challenge was enhanced in OffOb rats (&Dgr;mean arterial pressure: OffOb, 9.7±0.8 mm Hg versus OffCon, 5.3±1.3 mm Hg; n=8; P<0.05). Renal tissue norepinephrine content (P<0.001) and renin expression (P<0.05) were markedly raised. Analysis of heart rate variability revealed an increased low:high frequency ratio in OffOb versus OffCon rats (P<0.05). At 90 days, hypertension in OffOb rats persisted and was abolished by &agr;1- and &bgr;-adrenergic blockade, and cardiovascular responses to phenylephrine or sodium nitroprusside indicated altered baroreceptor function. The exaggerated pressor response to leptin in OffOb rats was maintained. Hypertension in the offspring of obese rats may arise from persistent sympathoexcitatory hyperresponsiveness acquired in early stages of development.

Evidence for Suprachiasmatic Vasopressin Neurones Innervating Kisspeptin Neurones in the Rostral Periventricular Area of the Mouse Brain: Regulation by Oestrogen

In rodents, a circadian signal from the suprachiasmatic nucleus (SCN) is essential for the pro‐oestrous surge of gonadotrophin‐releasing hormone (GnRH), which, in turn, induces luteinising hormone (LH) surge and ovulation. We hypothesised that kisspeptin (KP) neurones in the anteroventral periventricular and periventricular preoptic nuclei (AVPV/PeN) form part of the communication pathway between the SCN and GnRH neurones. In anterograde track tracing studies, we first identified vasopressin (VP)‐containing axons of SCN origin in apposition to KP‐immunoreactive (IR) neurones. Studies to quantify this input relied on the observation that VP‐synthesising neurones in the SCN differ from other VP systems in their lack of galanin expression. In ovariectomised mice, 30.79 ± 1.63% of KP‐IR perikarya and proximal dendrites within the AVPV/PeN received galanin‐negative VP‐IR varicosities. Oestrogen‐treatment significantly increased the number of KP‐IR neurones, with their percentage apposed by galanin‐negative VP‐IR varicosities (46.95 ± 1.88%) and the number of VP‐IR appositions on individual KP‐IR neurones. At the ultrastructural level, the VP‐IR terminals formed symmetric synapses with KP‐IR neurones, which was in accordance with the morphology of inhibitory synapses established by SCN neurones. By contrast to VP, vasoactive intestinal polypeptide (VIP), which is synthesised by a distinct subset of SCN neurones, occurred only rarely in axons apposed to KP‐IR neurones. Altogether, our results are consistent with the hypothesis that KP neurones located in the mouse AVPV/PeN receive circadian information from the SCN via a vasopressinergic monosynaptic pathway, which is enhanced by oestrogen.

Estrogen receptor-beta in oxytocin and vasopressin neurons of the rat and human hypothalamus: Immunocytochemical and in situ hybridization studies

Topographical distribution of estrogen receptor‐β (ER‐β)‐synthesizing oxytocin (OT) and vasopressin (VP) neurons was studied in the hypothalamic paraventricular and supraoptic nuclei (PVH; SO) of ovariectomized rats. In distinct subregions, 45–98% of OT neurons and 88–99% of VP neurons exhibited ER‐β immunoreactivity that was confined to cell nuclei. Neuronal populations differed markedly with respect to the intensity of the ER‐β signal. Magnocellular OT neurons in the PVH, SO, and accessory cell groups typically contained low levels of the ER‐β signal; in contrast, robust receptor labeling was displayed by OT cells in the ventral subdivision of medial parvicellular subnucleus and in the caudal PVH (dorsal subdivision of medial parvicellular subnucleus and lateral parvicellular subnucleus). Estrogen receptor‐β signal was generally more intense and present in higher proportions of magnocellular and parvicellular VP vs. OT neurons of similar topography. Immunocytochemical observations were confirmed via triple‐label in situ hybridization, an approach combining use of digoxigenin‐, fluorescein‐, and 35S‐labeled cRNA hybridization probes. Further, ER‐β mRNA was also detectable in corticotropin‐releasing hormone neurons in the parvicellular PVH. Finally, double‐label immunocytochemical analysis of human autopsy samples showed that subsets of OT and VP neurons also express ER‐β in the human. These neuroanatomical studies provide detailed information about the topographical distribution and cellular abundance of ER‐β within subsets of hypothalamic OT and VP neurons in the rat. The variable receptor content may indicate the differential responsiveness to estrogen in distinct OT and VP neuronal populations. In addition, a relevance of these findings to the human hypothalamus is suggested. J. Comp. Neurol. 473:315–333, 2004.

Fasting Reduces KiSS-1 Expression in the Anteroventral Periventricular Nucleus (AVPV): Effects of Fasting on the Expression of KiSS-1 and Neuropeptide Y in the AVPV or Arcuate Nucleus of Female Rats

Changes in metabolic state, such as those induced by fasting, have profound effects on reproduction. In rats, the time‐course over which fasting inhibits luteinising hormone (LH) release is reduced to 48 h by the presence of oestradiol‐17β (E2). Hypothalamic kisspeptin plays a key role in mediating the actions of E2 on gonadotrophin‐releasing hormone (GnRH) neurones, and thereby promotes LH release. KiSS‐1‐expressing neurones are found in the anteroventral periventricular nucleus (AVPV) and arcuate nucleus (ARC). Extensive evidence implicates the AVPV in GnRH release and the ARC in energy balance. The latter nucleus also contains neurones that express neuropeptide Y (NPY), an orexigenic peptide implicated in GnRH control. To elucidate the involvement of kisspeptin and/or NPY in hypothalamic responses to fasting, their expression was quantified by in situ hybridisation histochemistry in ovariectomised rats, with or without E2 replacement, before and after 48 h of fasting. In the presence of E2, but not in its absence, the fasting suppressed plasma LH. In the AVPV, the low level of KiSS‐1 expression found in the absence of E2 was unaffected by fasting. By contrast, the elevated level found in the presence of E2 was suppressed by fasting. Independent of E2, fasting had no effect on KiSS‐1 expression in the ARC, but increased NPY expression at that site. The present study has identified the AVPV as a site at which KiSS‐1 expression can be influenced by fasting. The results suggest that inhibition of KiSS‐1 expression in the AVPV may be a significant factor in restraining the gonadotrophic axis in response to negative energy balance in the presence of oestrogen. The extent to which the concurrent rise in NPY expression in the ARC may contribute to the suppression of LH release by influencing AVPV kisspeptin neurones, directly or indirectly, or by actions independent of kisspeptin, remains to be established.

Histamine H3 Receptors Inhibit Serotonin Release in Substantia Nigra Pars Reticulata

The substantia nigra pars reticulata (SNr) plays a key role in basal ganglia function. Projections from multiple basal ganglia nuclei converge at the SNr to regulate nigrothalamic output. The SNr is also characterized by abundant aminergic input, including dopaminergic dendrites and axons containing 5-hydroxytryptamine (5-HT) or histamine (HA). The functions of HA in the SNr include motor control via HA H3 receptors (H3Rs), although the mechanism remains far from elucidated. In Parkinsons disease, there is an increase in H3Rs and the density of HA-immunoreactive axons in the SN. We explored the role of H3Rs in the regulation of 5-HT release in SNr using fast-scan cyclic voltammetry at carbon-fiber microelectrodes in rat midbrain slices. Immunohistochemistry identified a similar distribution for histaminergic and serotonergic processes in the SNr: immunoreactive varicosities were observed in the vicinity of dopaminergic dendrites. Electrically evoked 5-HT release was dependent on extracellular Ca2+ and prevented by NaV+-channel blockade. Extracellular 5-HT concentration was enhanced by inhibition of uptake transporters for 5-HT but not dopamine. Selective H3R agonists (R)-(-)-α-methyl-histamine or immepip inhibited evoked 5-HT release by up to 60%. This inhibition was prevented by the H3R antagonist thioperamide but not by the 5-HT1B receptor antagonist isamoltane. H3R inhibition of 5-HT release prevailed in the presence of GABA or glutamate receptor antagonists (ionotropic and metabotropic), suggesting minimal involvement of GABA or glutamate synapses. The potent regulation of 5-HT by H3Rs reported here not only elucidates HA function in the SNr but also raises the possibility of novel targets for basal ganglia therapies.

Oestrogen Receptor α and β Immunoreactive Cells in the Suprachiasmatic Nucleus of Mice: Distribution, Sex Differences and Regulation by Gonadal Hormones

Oestrogen regulates various aspects of circadian rhythm physiology. The presence of oestrogen receptors within the suprachiasmatic nucleus (SCN), the principal circadian oscillator, indicates that some actions of oestrogen on circadian functions may be exerted at that site. The present study analysed sex differences, topographic distribution, and neurochemical phenotype of neurones expressing the α and β subtypes of oestrogen receptors (ERα and ERβ) in the mouse SCN. We found that relatively few neurones in the SCN are immunoreactive (IR) for ERα (approximately 4.5% in females and 3% in males), but five‐ to six‐fold more SCN neurones express ERβ. ER‐IR neurones are primarily in the shell subdivision of the nucleus and show differences between the sexes, significantly greater numbers being found in females. Treatment of male or female gonadectomised mice with oestradiol benzoate for 24 h substantially reduced the number of ERβ‐IR neurones, but not ERα‐IR neurones. Double‐labelling immunocytochemical experiments to characterise the phenotype of the oestrogen‐receptive neurones showed the presence of the calcium‐binding proteins calretinin or calbindin D28K in approximately 12% and 10%, respectively, of ERα‐IR neurones. A higher proportion (approximately 38%) of ERβ‐IR neurones contains calbindin D28K; a few (approximately 2%) express calretinin or vasopressin. These double‐labelled cells appear primarily in the shell subdivision of the SCN. Neither vasoactive intestinal polypeptide‐ nor gastrin releasing peptide‐immunoreactivity was observed in ER‐IR neurones. These data indicate that the primary target cells for oestrogen are in the shell subdivision of the nucleus. The sexually differentiated expression and distribution of ERα and ERβ in various cell populations of the SCN suggest multiple modes of oestrogen signalling within this nucleus, which may modulate circadian functions.