Chronic administration of neuropeptide Y into the lateral ventricle of C57BL/6J male mice produces an obesity syndrome including hyperphagia, hyperleptinemia, insulin resistance, and hypogonadism.

Neuropeptide Y (NPY) is involved in the central regulation of appetite, sexual behavior, and reproductive function. We have previously shown that chronic infusion of NPY into the lateral ventricle of normal rats produced an obesity syndrome characterized by hyperphagia, hyperinsulinism and collapse of reproductive function. We further demonstrated that acute inhibition of LH secretion in castrated rats was preferentially mediated by the NPY receptor subtype 5 (Y(5)). In the present study, the effects of chronic, central infusion of NPY, or the mixed Y2-Y5 agonist PYY(3-36), were evaluated both in normal male C57BL/6J mice and Sprague-Dawley rats. After a 7-day infusion to male mice, both NPY and PYY(3-36) at 5 nmol per day, induced marked hyperphagia leading to significant increases in body and fat pad weights. Furthermore, both compounds markedly reduced several markers of the reproductive axis. In the rat study, PYY(3-36) was more active than NPY to inhibit the pituitary-testicular axis, confirming the importance of the Y5 subtype for such effects. In the mouse, chronic NPY infusion induced a sustained increase in corticosterone and insulin secretion. Plasma leptin levels were also markedly increased possibly explaining the observed reduction in gene expression for hypothalamic NPY. Gene expression for hypothalamic POMC was reduced in the NPY- or PYY(3-36)-infused mice, suggesting that NPY exacerbated food intake by both acting through its own receptor(s), and reducing the satiety signal driven by the POMC-derived alpha-MSH. The present study in the mouse suggests in analogy with available rat data, that constant exposure to elevated NPY in the hypothalamic area unabatedly enhances food intake leading to an obesity syndrome including increased adiposity, insulin resistance, hypercorticism, and hypogonadism, reminiscent of the phenotype of the ob/ob mouse, that displays elevated hypothalamic NPY secondary to lack of leptin negative feedback action.

Two molecular forms of gonadotropin-releasing hormone (GnRH-I and GnRH-II) are expressed by two separate populations of cells in the rhesus macaque hypothalamus.

Gonadotropin-releasing hormone represents the primary neuroendocrine link between the brain and the reproductive axis, and at least two distinct molecular forms of this decapeptide (GnRH-I and GnRH-II) are known to be expressed in the forebrain of rhesus macaques (Macaca mulatta). Although the distribution pattern of the two corresponding mRNAs is largely dissimilar, their expression appears to show some overlap in specific regions of the hypothalamus; this raises the possibility that some cells express both molecular forms of GnRH. To resolve this issue, double-label histochemistry was performed on hypothalamic sections from six male rhesus macaques, using a monoclonal antibody to GnRH-I and a riboprobe to monkey GnRH-II mRNA. In total, more than 2000 GnRH neurons were examined but in no instance were GnRH-I peptide and GnRH-II mRNA found to be coexpressed. This finding emphasizes that GnRH-I and GnRH-II are synthesized by two distinct populations of hypothalamic neurons, and suggests that they may be regulated by different neuroendocrine pathways.

Gonadotropin-releasing hormone genes: phylogeny, structure, and functions.

Gonadotropin-releasing hormone (GnRH, previously called leutinizing hormone-releasing hormone, LHRH) is the final common signaling molecule used by the brain to regulate reproduction in all vertebrates. Recently, genes encoding two other GnRH forms have been discovered. Here we present a phylogenetic analysis that shows that the GnRH genes fall naturally into three distinct branches, each of which shares not only a molecular signature but also characteristic expression sites in the brain. The GnRH genes appear to have arisen through gene duplication from a single ancestral GnRH whose origin predates vertebrates. Several lines of data support this suggestion, including the fact that all three genes share an identical exonic structure. The existence of three distinct GnRH families suggests a new, natural nomenclature for the genes, and in addition, we present a logical proposal for naming the peptide sequences. The two recently discovered GnRH genes are unusual because they encode decapeptides that are identical in all the species in which they have been found. The control of gene expression also differs among the three gene families as might be expected since they have had separate evolutionary trajectories for perhaps 500 million years.

Second form of gonadotropin-releasing hormone in mouse: immunocytochemistry reveals hippocampal and periventricular distribution.

Hypothalamic GnRH (GnRH-I) is known and named for its role in regulating reproductive function in vertebrates by controlling release of gonadotropins from the pituitary. However, another form of GnRH of unknown function (pGlu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly; GnRH-II) is expressed in the mesencephalon of all vertebrate classes except jawless fish. Here we show with immunocytochemical staining that the GnRH-II peptide is localized to the mouse midbrain as in other vertebrates, as well as in cells surrounding the ventricles and in cells adjacent to the hippocampus. Staining of adjacent sections using GnRH-I antibody revealed that the distribution of GnRH-I does not overlap with that of GnRH-II.

Regional expression of mRNA encoding a second form of gonadotropin-releasing hormone in the macaque brain.

In mammals, reproduction is thought to be controlled by a single neuropeptide, gonadotropin-releasing hormone (GnRH-I), which regulates the synthesis and secretion of gonadotropins from the pituitary gland. However, another form of this decapeptide (GnRH-II), of unknown function, also exists in the brain of many vertebrate species, including humans; it is encoded by a different gene and its amino acid sequence is 70% identical to that of GnRH-I. Here we report the cloning of a GnRH-II cDNA from the rhesus macaque (Macaca mulatta), and show for the first time by in situ hybridization that GnRH-II mRNA is expressed in the primate midbrain, hippocampus and discrete nuclei of the hypothalamus, including the supraoptic, paraventricular, suprachiasmatic and arcuate. Because the regional distribution pattern of cells containing GnRH-II mRNA is largely dissimilar to that of cells containing GnRH-I mRNA, it is likely that these two cell populations receive distinct neuroendocrine inputs and thus regulate GnRH synthesis and release differently.

Ontogeny of gonadotropin-releasing hormone (GnRH) gene expression reveals a distinct origin for GnRH-containing neurons in the midbrain.

In the teleost fish, Haplochromis burtoni, three gonadotropin-releasing hormone (GnRH) peptides and their corresponding cDNA sequences and full-length genes have previously been reported. Here we describe the ontogeny of mRNA expression for these three GnRH forms in H. burtoni. Each of the three forms has been shown to have a distinct spatial expression pattern in the adult brain. ¿Ser8¿GnRH (the releasing form) is expressed exclusively in the hypothalamus, ¿His5Trp7Tyr8¿GnRH is expressed in the midbrain mesencephalon, and ¿Trp7Leu8¿GnRH is expressed in the terminal nerve area of the telencephalon. Previous work in other animals has shown that GnRH-containing neurons in the preoptic area arise from the olfactory placode and that these cells migrate into their final positions in the brain during early development. By using molecular probes to identify the cell types expressing distinct GnRH forms, our data are consistent with the migration of both ¿Ser8¿GnRH and ¿Trp7Leu8¿GnRH neurons from the placode to their appropriate adult locations in the brain. In contrast, we show that ¿His5Trp7Tyr8¿GnRH neurons arise from the germinal zone of the third ventricle. By using in situ hybridization with digoxigenin-labeled cRNA probes, ¿His5Trp7Tyr8¿GnRH mRNA was first evident at day 4, ¿Trp7Leu8¿GnRH mRNA at day 8, and ¿Ser8¿GnRH mRNA at day 14. However, by using the reverse-transcriptase polymerase chain reaction (RT-PCR), all three GnRH mRNAs were found in whole embryos at day 4 of the 14 days of embryogenesis. This striking difference may be due to the greater sensitivity of RT-PCR compared with in situ hybridization. Alternatively, it is possible that ¿Ser8¿GnRH and ¿Trp7Leu8¿GnRH are expressed outside the brain during early development and only later inside the brain.

Genomic structure and expression sites of three gonadotropin-releasing hormone genes in one species.

In the teleost fish, Haplochromis burtoni, gonadotropin-releasing hormone (GnRH) peptide has been localized to three distinct regions in the brain. Each GnRH population is associated with expression of a distinct cDNA as previously described. Here we report the complete genomic sequences encoding these three forms and compare their structural organization, putative regulatory elements, and expression patterns in the body. All three genes share a common structure of four exons: the first exon encodes the 5' untranslated region; the second exon encodes the signal sequence, GnRH decapeptide, and the 5' end of the GnRH-associated peptide (GAP); the third exon consists entirely of GAP coding sequence; and the fourth exon encodes the 3' end of GAP and the 3' untranslated region. Each of the three GnRH genes has been shown previously to have a distinct spatial expression pattern in the brain, and here we use reverse transcription and cDNA amplification to demonstrate that each gene is expressed in the body. The gene encoding the releasing form, ¿Ser8¿GnRH, is expressed in the heart, liver, spleen, kidney, and testis, as well as in the preoptic area. The ¿His5Trp7Tyr8¿GnRH gene is expressed in the testis as well as in the midbrain. The ¿Trp7Leu8¿GnRH gene is expressed in the testis and the terminal nerve area. We examined the 500 bp upstream of exon 1 in all three H. burtoni genes and identified putative binding sites for glucocorticoid receptor, androgen receptor, and progesterone receptor, as well as the transcription factors Ap-1 and Sp-1. The genomic sequence encoding the terminal nerve form of GnRH (i.e., ¿Trp7Leu8¿GnRH) in H. burtoni is remarkably similar to that encoding the presumed releasing form of GnRH in salmonids, especially in the 3' intergenic region. Taken together with phylogenetic and mRNA localization data in salmonids, these data suggest that the gene encoding the releasing form of GnRH in salmonids may not yet be described.

Second gene for gonadotropin-releasing hormone in humans.

Gonadotropin-releasing hormone (GnRH) is a decapeptide widely known for its role in regulating reproduction by serving as a signal from the hypothalamus to pituitary gonadotropes. In addition to hypothalamic GnRH (GnRH-I), a second GnRH form (pGln-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly; GnRH-II) with unknown function has been localized to the midbrain of many vertebrates. We show here that a gene encoding GnRH-II is expressed in humans and is located on chromosome 20p13, distinct from the GnRH-I gene that is on 8p21-p11.2. The GnRH-II genomic and mRNA structures parallel those of GnRH-I. However, in contrast to GnRH-I, GnRH-II is expressed at significantly higher levels outside the brain (up to 30x), particularly in the kidney, bone marrow, and prostate. The widespread expression of GnRH-II suggests it may have multiple functions. Molecular phylogenetic analysis shows that this second gene is likely the result of a duplication before the appearance of vertebrates, and predicts the existence of a third GnRH form in humans and other vertebrates.

Catalytic role of cytochrome P4502B6 in the N-demethylation of S-mephenytoin.

In vitro methods were used to identify the cytochrome P450 (CYP) enzyme(s) involved in S-mephenytoin N-demethylation. S-Mephenytoin (200 microM) was incubated with human liver microsomes, and nirvanol formation was quantitated by reversed-phase HPLC. S-Mephenytoin N-demethylase activity in a panel of human liver microsomes ranged 35-fold from 9 to 319 pmol/min/mg protein and correlated strongly with microsomal CYP2B6 activity (r = 0.91). Additional correlations were found with microsomal CYP2A6 and CYP3A4 activity (r = 0.88 and 0.74, respectively). Microsomes prepared from human beta-lymphoblastoid cells transformed with individual P450 cDNAs were assayed for S-mephenytoin N-demethylase activity. Of 11 P450 isoforms (P450s 1A1, 1A2, 2A6, 2B6, 2E1, 2D6, 2C8, 2C9, 2C19, 3A4, and 3A5) tested, only CYP2B6 catalyzed the N-demethylation of S-mephenytoin with an apparent K(m) of 564 microM. Experiments with P450 form-selective chemical inhibitors, competitive substrates, and anti-P450 antibodies were also performed. Troleandomycin, a mechanism-based CYP3A selective inhibitor, and coumarin, a substrate for CYP2A6 and therefore a potential competitive inhibitor, failed to inhibit human liver microsomal S-mephenytoin N-demethylation. In contrast, orphenadrine, an inhibitor of CYP2B forms, produced a 51 +/- 4% decrease in S-mephenytoin N-demethylase activity in human liver microsomes and a 45% decrease in recombinant microsomes expressing CYP2B6. Also, both CYP2B6-marker 7-ethoxytrifluoromethylcoumarin O-deethylase and S-mephenytoin N-demethylase activities were inhibited by approximately 65% by 5 mg anti-CYP2B1 IgG/mg microsomal protein. Finally, polyclonal antibody inhibitory to CYP3A1 failed to inhibit S-mephenytoin N-demethylase activity. Taken together, these studies indicate that the N-demethylation of S-mephenytoin by human liver microsomes is catalyzed primarily by CYP2B6.

Stimulation of in vitro steroidogenesis by pituitary hormones in a turtle (Trachemys scripta) within the temperature-sensitive period for sex determination.

To investigate the possible involvement of pituitary hormones in the regulation of steroidogenesis during reptilian sexual differentiation, we tested the ability of gonadotropin (ovine FSH), adrenocorticotropin (porcine ACTH), and growth hormone (bovine GH) to stimulate in vitro steroidogenesis in embryonic adrenal-kidney-gonad complexes (AKGs) of a turtle, Trachemys scripta, during and after the temperature-sensitive period for sex determination (TSP). Radioimmunoassays were used to measure progesterone, testosterone, estradiol, and corticosterone in incubation media; additionally, immunoreactive ACTH was measured in plasma. Presumptive male and female AKGs were stimulated by both FSH and ACTH at each stage investigated. Secretion of progesterone and corticosterone was usually far greater than that of testosterone or estradiol in both basal and hormone-stimulated incubations. In general, AKGs from presumptive males secreted more progesterone and corticosterone than AKGs from presumptive females. Progesterone and estradiol secretions were stimulated by both FSH and ACTH, but testosterone secretion was stimulated only by ACTH. Corticosterone secretion was strongly stimulated by ACTH. GH failed to significantly stimulate steroid secretion. Plasma ACTH levels were significantly higher in males than in females, and both sexes had significantly higher plasma levels of ACTH after the TSP compared to during the TSP. Our data demonstrate that during the temperature-sensitive period AKGs are responsive to both gonadotropin and ACTH, and that there are significant sex differences in steroidogenesis, sensitivity to gonadotropin and ACTH, and plasma ACTH levels.