John K. McDonald

Neuropeptide Y: Direct and indirect action on insulin secretion in the rat

Neuropeptide Y (NPY) was tested for an ability to directly influence the release of insulin using an in vitro isolated rat pancreatic islet system. NPY, at doses ranging from 100 pg/ml to 1 microgram/ml, had no significant effect on the basal release (5.5 mM glucose) of insulin. However, NPY treatment resulted in a significant, dose-dependent (1 ng/ml to 1 microgram/ml) inhibition of glucose-stimulated (11 mM) insulin release. When tested in a perfused rat pancreas preparation in situ, NPY administration led to a marked inhibition of both basal and stimulated insulin release followed by a postinhibitory rebound which exceeded the control insulin levels by 3-fold. In contrast, the intracerebroventricular (ICV) microinjection of NPY (5 micrograms) produced a significant but delayed (30 min) elevation of circulating insulin. It is therefore suggested that the direct action of NPY on insulin release is inhibitory while the central action of NPY indirectly results in an increase in plasma insulin. Thus, NPY may be added to the growing list of peptidergic agents which may affect the endocrine pancreas by acting as neurotransmitters and/or neuromodulators.

High Concentrations of Neuropeptide Y in Pituitary Portal Blood of Rats

Pituitary portal blood was collected from urethane-anesthetized rats and examined for the presence of neuropeptide Y (NPY) using high-performance liquid chromatography and radioimmunoassay. Other rats were perfused with fixative, and coronal sections through the hypothalamus and median eminence were processed for immunohistochemical localization of NPY. Combined high-performance liquid chromatography and radioimmunoassay analysis of pituitary portal plasma and systemic plasma revealed a single peak of NPY immunoreactivity which corresponded in retention time to synthetic porcine NPY. Increasing amounts of portal or systemic plasma produced displacement curves which were parallel to the NPY standard curve. The concentration of NPY immunoreactivity in portal plasma (52.0 +/- 4.0 ng/ml, mean +/- SEM) was three times greater (p less than 0.005) than in systemic plasma (16 +/- 4.5 ng/ml). NPY-labeled fibers were observed in the external zone of the median eminence in the vicinity of hypothalamo-hypophyseal portal vessels. The observation of significantly higher concentrations of NPY immunoreactivity in the portal plasma supports the hypothesis that NPY may be released from the hypothalamus to affect pituitary function.

Role of Neuropeptide Y in Reproductive Function

NPY acts both at the hypothalamus and the anterior pituitary gland to modulate reproductive hormone secretion. Within the hypothalamus, NPY stimulates LHRH secretion in the presence of physiological levels of estrogen and suppresses pulsatile LHRH release following ovariectomy. Intracerebroventricular injection of NPY antiserum blocks or delays the LH surge in steroid-primed ovariectomized rats, thereby adding support for a physiological role of NPY in the neuroendocrine events preceding ovulation. Blockade of alpha 2 adrenergic receptors decreases NPY-stimulated LH release in steroid-primed rats implying a potential noradrenergic mediation of NPY activity. Physiological levels of progesterone do not augment, and may actually suppress NPY-induced LHRH secretion in vitro from median eminences obtained from estrogen-primed ovariectomized rats. The physiological role of progesterone, if any, in modulating NPY effects on LHRH release remains to be determined. Little, if anything, is known about the NPY receptor in the median eminence or the intracellular mechanisms which transduce the NPY signal into activation of LHRH release in estrogen-treated ovariectomized rats although translocation of intracellular calcium is required. Equally puzzling is the mechanism of desensitization of the LHRH-releasing mechanisms of the median eminence of ovariectomized rats or the specific site of NPY suppression of pulsatile LHRH secretion. NPY is released into the hypothalamo-hypophysial portal circulation and this appears correlated with LHRH secretion before the LH surge. NPY affects LH and FSH release from anterior pituitary cells in vitro and enhances LHRH-induced LH secretion. Taken together, the studies described above suggest an important physiological role for NPY as a modulator of neuroendocrine activity which culminates in the preovulatory surge of LH.

Anglerfish islets contain NPY immunoreactive nerves and produce the NPY analog aPY

It has recently been demonstrated that aPY, a peptide which has significant homology with neuropeptide Y (NPY) is present in extracts of anglerfish islets. The purpose of this study was to determine whether cells or nerves which contain NPY-like immunoreactivity could be identified in anglerfish islet tissue and whether aPY is synthesized by this tissue. Antisera against bovine pancreatic polypeptide (BPP), NPY and the 200 kd neurofilament polypeptide were used for immunohistochemical analysis of islets. Identical cells were stained by both the NPY and BPP antisera. The NPY and 200 kd neurofilament antisera also labeled nerve fibers in the tissue which were not stained with the BPP antiserum. The nature of the NPY-like peptide synthesized in islet cells was determined by subjecting differentially radioactively labeled Mr 2,500-8,000 peptides from islet extracts to reverse phase HPLC. Labeled aPY was unequivocally identified in the extracts and was labeled appropriately (as predicted from its sequence) with 13 different radioactive amino acids. These results demonstrate that one form of NPY-like peptide synthesized in anglerfish islets is aPY. The form of NPY-like peptide which was immunolocalized in nerves remains to be determined.

Localization and characterization of neuropeptide Y-like peptides in the brain and islet organ of the anglerfish (Lophius americanus).

SummaryResults from a previous report demonstrate that more than one molecular form of neuropeptide Y-like peptide may be present in the islet organ of the anglerfish (Lophius americanus). Most of the neuropeptide Y-like immunoreactive material was anglerfish peptide YG, which is expressed in a subset of islet cells, whereas an additional neuropeptide Y-like peptide(s) was localized in islet nerves. To learn more about the neuropeptide Y-like peptides in islet nerves, we have employed immunohistochemical and biochemical methods to compare peptides found in anglerfish islets and brain. Using antisera that selectively react with either mammalian forms of neuropeptide Y or with anglerfish peptide YG, subsets of neurons were found in the brain that labelled with only one or the other of the antisera. In separate sections, other neurons that were labelled with either antiserum exhibited similar morphologies. Peptides from brains and islets were subjected to gel filtration and reverse-phase high performance liquid chromatography. Radioimmunoassays employing either the neuropeptide Y or peptide YG antisera were used to examine chromatographic eluates. Immunoreactive peptides having retention times of human neuropeptide Y and porcine neuropeptide Y were identified in extracts of both brain and islets. This indicates that peptides structurally similar to both of these peptides from the neuropeptide Y-pancreatic polypeptide family are expressed in neurons of anglerfish brain and nerve fibers of anglerfish islets. The predominant form of neuropeptide Y-like peptide in islets was anglerfish peptide YG. Neuropeptide Y-immunoreactive peptides from islet extracts that had chromatographic retention times identical to human neuropeptide Y and porcine neuropeptide Y were present in much smaller quantities. These results are consistent with the hypothesis that peptides having significant sequence homology with human neuropeptide Y and porcine neuropeptide Y are present in the nerve fibers that permeate the islet.

Immunocytochemical localization of neuropeptide Y-like immunoreactivity in adrenergic and non-adrenergic neurons of the rat gastrointestinal tract

The immunocytochemical location of neuropeptide Y (NPY)-like immunoreactivity (LI) within the neuronal structures of the rat gastrointestinal (GI) tract was investigated with the indirect immunofluorescence method. NPY immunoreactive neurons were found throughout all regions of the GI tract with the largest number in the duodenum. NPY immunoreactive perikarya were mainly located in the submucosal ganglia. NPY labeled processes were extensively seen in the submucosal and myenteric plexuses, smooth muscles, muscularis mucosa, mucosa and surrounding blood vessels. Following 6-hydroxydopamine (6-OHDA) treatment, NPY immunoreactive nerve fibers around blood vessels disappeared completely and the reactive fibers in other regions were reduced in number. NPY immunoreactive nerve cell bodies in the ganglionic plexuses, however, were not affected by 6-OHDA treatment. Serial sections of the coeliac ganglion showed that NPY-LI was present in cell bodies which also displayed tyrosine hydroxylase (TH) immunoreactivity. Our results suggest that NPY is abundantly contained in both adrenergic and non-adrenergic neurons of the gut and may play an important role in the regulation of the GI tract.

Neuropeptide Y Actions on Reproductive and Endocrine Functions

In this chapter, we have reviewed the current literature concerning the function of neuropeptide Y (NPY) in endocrine and reproductive systems. The reader is referred to other reviews and recent symposia on NPY for consideration of other areas (1–4). The reader is also referred to other chapters in this volume, particularly those by Hendry and by Stanley.

Neuropeptide Y Regulation of LHRH Release in the Median Eminence: Immunocytochemical and Physiological Evidence in Hens

It has been suggested that hypothalamic median eminence (ME) might be a control site for luteinizing hormone-releasing hormone (LHRH) release. Thus, stimulatory and/or inhibitory inputs acting at this site might be involved in regulating LHRH release from the ME and, therefore, luteinizing hormone (LH) release from the anterior pituitary. Since a role for neuropeptide Y (NPY) on LH release has been suggested, we have hypothesized that NPY might act in the ME to control preovulatory LHRH release in hens. To examine this possibility we have determined: (a) the immunocytochemical distribution of LHRH and NPY in the ME of the hen, (b) the basal and NPY-stimulated release of LHRH in vitro from the ME of hens undergoing a natural or a premature preovulatory surge of LH, and (c) the tissue content of LHRH and NPY in microdissected MEs, at various times before and during a natural or a premature preovulatory surge of LH. A potential role for NPY on LHRH release in the ME is suggested for the following reasons. (a) There are opportunities for synaptic interactions between NPY and LHRH-containing axons at this site. LHRH-containing cell bodies localized in the anterior hypothalamus/medial preoptic area project to the ME. NPY-containing perikarya, concentrated in the ventromedial aspect of the arcuate nucleus, might contact LHRH processes going to the ME and/or might themselves send axons to the ME, (b) Addition of NPY to the incubation media increases LHRH release from microdissected ME tissue of hens killed at the time of the natural preovulatory surge of LH, but not in hens killed 7 h before the occurrence of this surge. However, the stimulatory effect of NPY on LHRH release can be induced at this latter time when a premature LH surge is elicited. While the natural preovulatory surge of LH occurs 4 h before the second ovulation in a sequence (C2 ovulation), administration of progesterone (P4) 10-14 h before the expected natural C2 ovulation advances the natural LH surge by 7-8 h. Thus, NPY might act as a physiological stimulus of LHRH release at the ME during the preovulatory surge of LH. This is suggested since in vitro basal LHRH release from denervated ME tissue does not change before and during the natural or the premature LH surge. Therefore, preovulatory release of LHRH in vivo might be under a continuous drive from stimulatory inputs to the LHRH neuron and NPY might be one of these stimulating factors.(ABSTRACT TRUNCATED AT 400 WORDS)

Neuropeptide Y (NPY) and vasopressin (AVP) in the hypothalamo-neurohypophysial axis of salt-loaded or Brattleboro rats.

A close anatomical relationship between nerve terminals containing neuropeptide Y (NPY) and vasopressin (AVP) has been demonstrated in the hypothalamic paraventricular (PVN) and supraoptic nuclei (SON). Furthermore, injections of NPY into the SON increased plasma concentrations of AVP in the rat. These data suggest a potential involvement of hypothalamic NPY in fluid homeostasis in the rat. Therefore, we have studied the effect of elevated plasma osmolality on the concentration of NPY and AVP in the hypothalamus and neurointermediate lobe (NIL) of the pituitary gland. Furthermore, we measured the concentration of NPY in the AVP-deficient Brattleboro rat, which suffers from diabetes insipidus and hyperosmolality. Salt-loading increased plasma osmolality and the concentration of AVP from 2.0 +/- 0.5 to 4.1 +/- 0.6 pg/ml after 7 days. The concentration of NPY in the NIL doubled after 7 days of salt-loading, from 7.9 +/- 0.6 ng/mg protein to 15.2 +/- 1.4 ng/mg protein, whereas AVP concentrations fell from 2285.7 +/- 210.9 ng/mg protein to 187.5 +/- 2.5 ng/mg protein. AVP concentrations in the ME increased transiently after 2 days of salt-loading and returned to control levels after 7 days. In contrast, NPY concentrations in the ME were unchanged at 2 days and were increased 61% after 7 days. NPY concentrations also were significantly elevated after 7 days of salt-loading in the preoptic area (POA) and mediobasal hypothalamus (MBH). The concentration of NPY in the NIL of the homozygous Brattleboro rat was 2-fold greater than in the heterozygous Brattleboro rat and 4-fold greater than in Sprague-Dawley rats used as controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of Hypothalamic Gonadotropin-Releasing Hormone and Neuropeptide Y Concentrations by Progesterone and Corticosteroids in Immature Rats: Correlation with Luteinizing Hormone and Follicle-Stimulating Hormone Release

In a previous study, we demonstrated that progesterone (P4) and the synthetic glucocorticoid triamcinolone acetonide (TA), but not cortisol, could induce LH and FSH release in estrogen-primed ovariectomized immature rats. Therefore, the purpose of this study was to determine if the stimulatory effect of P4 and TA on LH and FSH release were associated with changes in GnRH or NPY concentrations in the medial basal hypothalamus (MBH) or preoptic area (POA). Ovariectomized immature rats primed with estradiol at 27 and 28 days received either vehicle, P4, TA or cortisol (1 mg/kg BW) at 9.00 h on day 29. Animals were killed at 9.30, 10.00, 12.00 and 13.00 h on day 29 for serum LH and FSH measurements, and the MBH and POA were dissected and analyzed for GnRH and NPY concentrations via RIAs. P4- and TA-treated animals showed significantly elevated serum LH and FSH levels from 13.00 h to 15.00 h. Cortisol was without effect. P4 significantly increased MBH GnRH and NPY concentrations at 12.00 h followed by a significant fall at 13.00 h. P4 modulated POA GnRH and NPY concentrations in a fashion similar to that seen in the MBH, except POA NPY concentrations did not fall at 13.00 h after the elevation at 12.00 h. TA had no significant effect on MBH GnRH and NPY levels at 12.00 h compared to the values at 9.30 h and 10.00 h but, as with P4, there was a significant fall in MBH GnRH and NPY levels at 13.00 h. TA had no significant effect on POA GnRH and NPY concentrations at any time point studied.(ABSTRACT TRUNCATED AT 250 WORDS)