Oxyntomodulin and glicentin: brain-gut peptides in the rat

Glucagon-like materials and glucagon have been identified by immunoassay and immunocytochemistry in the mammalian central nervous system. However, the molecular forms relevant to brain glucagon-like immunoreactivity (GLI) have not been precisely defined. In the rat small intestine, more than 90% of GLI is constituted by two peptides: oxyntomodulin (OXM) and glicentin. This work was initiated to characterize and determine the concentrations of these two peptides and glucagon in the rat central nervous system and to compare their relative proportions with those found in the gut. Different regions from the adult rat brain were analyzed by HPLC in association with RIA, using a central glucagon antiserum and an antibody directed toward the C-terminal end of OXM and glicentin. The elution profiles of hypothalamus extracts were constituted by two main peaks, both detected by the two antibodies used and displaying the same retention times as glicentin and OXM, respectively. A third small peak, which coeluted with glucagon, was constantly recorded with the central glucagon antiserum. The percentages of glicentin, OXM, and glucagon in 10 hypothalami were 37 +/- 1%, 55 +/- 1%, and 8 +/- 2%, respectively (n = 8). This distribution was quite similar to that in small intestinal extracts (38 +/- 1%, 59 +/- 1%, and 1.3 +/- 0.1%, respectively; n = 7); however, the peptide concentrations were almost 50-fold greater in intestine than in hypothalamus. In the medulla oblongata, the same peptide ratio was observed, with 10-fold lower concentrations compared to those in hypothalamus. In olfactory bulb, cerebellum, and cortex the concentrations were close the the detection limit, whereas they could be not detected in the pituitary. The combination of HPLC and specific RIAs allowed us to unambiguously characterize OXM and glicentin as the major components of GLI in the rat hypothalamus and medulla oblongata. The same proportion of these two peptides in the central nervous system and the gut indicates that a similar posttranslational processing exists in these rat tissues, another example of the brain-gut axis.     

Melanin-concentrating hormone: unique peptide neuronal system in the rat brain and pituitary gland.

A unique neuronal system was detected in the rat central nervous system by immunohistochemistry and radioimmunoassay with antibodies to salmon melanin-concentrating hormone (MCH). MCH-like immunoreactive (MCH-LI) cell bodies were confined to the hypothalamus. MCH-LI fibers were found throughout the brain but were most prevalent in hypothalamus, mesencephalon, and pons-medulla regions. High concentrations of MCH-LI were measured in the hypothalamic medial forebrain bundle (MFB), posterior hypothalamic nucleus, and nucleus of the diagonal band. Reversed-phase high-performance liquid chromatography of MFB extracts from rat brain indicate that MCH-like peptide from the rat has a different retention time than that of the salmon MCH. An osmotic stimulus (2% NaCl as drinking water for 120 hr) caused a marked increase in MCH-LI concentrations in the lateral hypothalamus and neurointermediate lobe. The present studies establish the presence of MCH-like peptide in the rat brain. The MCH-LI neuronal system is well situated to coordinate complex functions such as regulation of water intake.     

Human seminal alpha-inhibins: detection in human pituitary, hypothalamus, and serum by immunoreactivity.

An antiserum generated in rabbits against human seminal alpha-inhibin-52 has been used to develop a sensitive radioimmunoassay for the detection of alpha-inhibins. The alpha-inhibin-52 antiserum reacts with alpha-inhibin-92 and alpha-inhibin-31 with equal avidity. These peptides were found to be present in human pituitary, hypothalamus, and serum. In exclusion chromatography on Sephadex G-100, the immunoreactive material eluted in a large molecular size region. Immunoblot analysis of column-derived fractions of these extracts revealed the presence of alpha-inhibin-92. The mean concentrations of immunoreactive alpha-inhibin were found to be 7.2 ng/ml in normal adult male serum, 70.3 ng/g (wet weight) of pituitary, and 12.9 ng/g (wet weight) of hypothalamus. This communication reports on the evidence for the existence of gonadal peptides in the brain.     

Localization of thyrotropin-releasing hormone prohormone messenger ribonucleic acid in rat brain in situ hybridization

We studied the distribution of pro- TRH mRNA in rat brain by in situ hybridization histochemistry using radiolabeled single stranded cRNA probes to confirm the hypothesis that the TRH precursor is distributed beyond regions that contain immunoreactive TRH. All regions of the central nervous system previously recognized to contain TRH showed hybridization. Hypophysiotropic neurons in the medial parvocellular division of the paraventricular nucleus showed more intense hybridization than anterior parvocellular division cells, suggesting regional differences in expression. In addition, regions not previously recognized to contain TRH in neuronal perikarya by immunocytochemistry showed specific hybridization for pro-TRH mRNA. These include cells in the olfactory bulbs, dorsal motor nucleus of the vagus, ventrolateral periaqueductal gray, reticular nucleus of the thalamus, and anterior commissural nucleus. Only a single hybridizing band was observed on Northern blots of RNA extracts of the periaqueductal gray and reticular nucleus, identical to that seen in extracts of the paraventricular nucleus. The appearance of pro-TRH mRNA in neurons not previously recognized to contain TRH but which contain the prohormone suggests that non-TRH peptides within the TRH precursor may be preferentially expressed in certain regions of the brain.     

Glucagon-like peptides GLP-1 and GLP-2, predicted products of the glucagon gene, are secreted separately from pig small intestine but not pancreas

We developed specific antibodies and RIAs for glucagon-like peptides 1 and 2 (GLP-1 and GLP-2), two predicted products of the glucagon gene, and studied the occurrence, nature, and secretion of immunoreactive GLP-1 and GLP-2 in pig pancreas and small intestine. Immunoreactive GLP-1 and GLP-2 were identified in glucagon-producing cells of the pancreatic islets, and in glicentin-producing cells of the small intestine. Immunoreactive GLP-1 and 2 in intestinal extracts corresponded in molecular size to peptides synthesized according to the predicted structure. By reverse phase HPLC, intestinal and synthetic GLP-1 behaved similarly, whereas synthetic and intestinal GLP-2 differed. Pancreatic extracts contained a large peptide with both GLP-1 and GLP-2 immunoreactivity. Secretion was studied using isolated perfused pig pancreas during arginine stimulation, and isolated perfused pig ileum during either luminal glucose stimulation or vascular administration of the neuropeptide, gastrin-releasing peptide (GRP). Immunoreactive GLP-1 and GLP-2 were secreted in parallel with pancreatic glucagon and intestinal glicentin. The molecular forms of secreted immunoreactive GLP-1 and 2 corresponded to those identified in the tissue extracts.     

Vitamin D-dependent calcium-binding protein in rat brain: biochemical and immunocytochemical characterization

Immunoreactive calcium-binding protein (CaBP) has been characterized in rat brain both biochemically and immunocytochemically. In this study antiserum to chick CaBP was used to characterize this protein and to describe its distribution in neurons and fibers of the rat fore- and midbrain. Immunostaining in neuronal elements was judged specific for this protein by the absence of staining in tissue sections after adsorption of the antiserum with either chick intestinal CaBP or the 28,000-dalton fraction from rat brain, but not with other molecular weight fractions with calcium-binding activity. Immunoreactive CaBP was found to have a widespread distribution throughout the central nervous system, and was present in most but not all major neuronal cell groups and fiber tracts. The protein was limited primarily to neuronal elements and some ependymal cells, and was absent in glia and blood vessels. The proportion of immunoreactivity in neuronal perikarya and fibers varied among nuclei and within a given structure at different rostral-caudal levels. Immunoreactivity was prominent in neocortex, hippocampal formation (primarily in CA1 and granular cells of the dentate gyrus), hypothalamus, and amygdala. These areas are responsible for the regulation of a variety of pituitary hormones, and several bind steroids. Immunoreactive CaBP was also a major constituent of nonlimbic system pathways. The widespread distribution of immunoreactive CaBP in the central nervous system suggests that CaBP and the vitamin D endocrine system may play a significant role in the regulation of mammalian brain function.     

Gonadotropin-releasing hormone molecular forms in mammalian hypothalamus

Multiple forms of GnRH have been detected in brain tissue of species from all nonmammalian vertebrate classes, but in mammals it is generally believed a single molecular form of GnRH is present. We have investigated the possibility that additional structural variants of GnRH are present in mammalian (sheep, rat, and human) hypothalamus. Hypothalami were extracted with acetic acid and subjected to gel filtration chromatography and reverse phase HPLC systems specifically designed to separate GnRH analogs. Column fractions were assayed for immunoreactive GnRH using a library of specific antisera raised against the five known vertebrate GnRHs. Biological activity of the fractions was assessed by measuring their ability to release LH and FSH from cultured rat pituitary cells and/or LH release from dispersed chicken pituitary cells. Receptor binding activity was also measured in fractions from the human extract, using rat pituitary membranes. Several immunoreactive and biologically active forms of GnRH were found in sheep, rat, and human hypothalami. The major immunoreactive peptide consistently coeluted with mammalian GnRH. The other forms were not identifiable as any of the other known vertebrate GnRHs. Control experiments suggest these are modified forms of mammalian GnRH, which are artifacts generated during HPLC purification. Chromatographic and immunological studies indicate these forms of GnRH include peptides eluting both earlier and later than mammalian GnRH and which appear to be modified in the middle region and/or at the COOH-terminus of the molecule. Novel immunoreactive forms of GnRH, distinct from modified mammalian GnRH, were not apparent in any of the species. In chicken and rat pituitary cell bioassays and in rat receptor binding studies, the mammalian form of GnRH in HPLC fractions of the sheep and human hypothalamus displayed activity appropriate for this immunoreactive peak being mammalian GnRH. Some of the additional immunoreactive peaks (thought to be modified forms of mammalian GnRH) also displayed LH-releasing activity in the chicken and rat systems. Gonadotropin-releasing activity or receptor binding activity due to a second, novel, GnRH-like substance in HPLC fractions of the sheep and human hypothalamus was not detected. These data provide evidence for a single form of GnRH in sheep, rat, and human hypothalamus, unlike species from other vertebrate classes where two or more GnRHs are present within a single tissue.     

Immunolocalization of the thyrotropin-releasing hormone prohormone in the rat central nervous system

The distribution of immunoreactive TRH prohormone in the rat central nervous system was studied by immunocytochemistry using an antiserum raised against a synthetic decapeptide hypothesized to represent a portion of the mammalian TRH precursor protein. Reaction product was identified in several regions of the brain in a distribution typical of that previously described for the tripeptide. In contrast to TRH, however, immunoreactive pro-TRH was largely confined to neuronal perikarya and only rarely seen in axons or axon terminals. In addition, immunoreactive pro-TRH was present in portions of the telencephalon and brainstem where TRH has not previously been described in neurons by immunocytochemistry. These studies indicate that in most regions of the brain the TRH prohormone is rapidly processed within the cell soma and not during axonal transport, and raise the possibility that in certain regions of the brain processing of the prohormone may be to non-TRH peptides, which may be of biological importance.     

Glucagon-like immunoreactive peptides in a rat ileal epithelial cell line (IEC-18)

The presence of cells containing glucagon-like immunoreactive (GLI) peptides was demonstrated in a rat ileal epithelial cell line (IEC-18) by both immunofluorescence and radioimmunoassay. When cell extracts were subjected to gel filtration chromatography, the cells were found to contain 3.5 Kd glucagon in addition to significant quantities of large molecular weight GLI peptides (apparent molecular weights of 4, 6, 8 and 10 Kd) and a 9 Kd peptide with apparent glucagon immunoreactivity. This was in contrast to extracts of adult rat ileum, which contained only large molecular weight GLI peptides (apparent molecular weights of 6 and 12 Kd). Production of GLI peptides by the IEC-18 cells was stimulated by glucose (p less than 0.02) and inhibited by insulin (p less than 0.01). In conclusion, these results demonstrate that the IEC-18 cells produce both GLI peptides and glucagon, and thus support the notion that proglucagon processing is cell-specific. IEC-18 cells may therefore provide a tool for investigations of some aspects of GLI peptide and glucagon synthesis.     

Localization, quantification, and characterization of pancreatic polypeptide immunoreactivity in the parasitic flatworm Diclidophora merlangi and its fish host (Merlangius merlangus)

Pancreatic polypeptide (PP) immunoreactivity (IR) has been identified, quantified, and subsequently chemically characterised in the parasitic platyhelminth, Diclidophora merlangi, and its specific teleostean host the whiting, Merlangius merlangus. Immunocytochemistry demonstrated PP-IR throughout the central and peripheral nervous systems of the parasite and in open-type endocrine cells of the gastric mucosa of its host. Radioimmunoassay detected PP-IR in alcoholic extracts of whole parasites (39.2 ng/g) and in extracts of gastrointestinal tract (2.1 ng/g), brain (4.6 ng/g), and pancreas (12 ng/g) of the host. Chromatographic analysis of parasite extracts revealed a single immunoreactive species of PP in both high-performance gel permeation and reverse-phase systems. The molecular size of this peptide was similar to bovine PP standard. In contrast, whiting tissues contained two immunoreactive species of PP in both gel permeation and reverse-phase systems. The major species was similar in size to bovine PP standard and the minor species was smaller, with a molecular size comparable to bovine neurotensin. Reverse-phase HPLC revealed that parasite and host peptides were not identical.     

Immunoreactive calcitonin in brain regions and pituitary of sheep

In this study we have investigated the presence of immunoreactive calcitonin in the central nervous system and pituitary of sheep. The calcitonin concentrations were determined radioimmunologically by two different antibodies. We have demonstrated calcitonin in extracts of areas of the central nervous system, whole pituitary, thyroid gland and plasma of 21 sheep. The concentrations were (ng/g wet weight, mean values +/- SE): thyroid 16.0 +/- 4.4, pituitary 2.03 +/- 0.34, reticular formation 1.64 +/- 0.25, substantia nigra 1.53 +/- 0.46, dentate nucleus 1.11 +/- 0.27, putamen 1.05 +/- 0.35, hippocampus 0.97 +/- 0.17, fornix 0.96 +/- 0.15, anterior thalamus 0.92 +/- 0.28, mammillary body 0.88 +/- 0.12, cerebellum 0.86 +/- 0.09, caudate nucleus 0.84 +/- 0.11, posterior hypothalamus 0.83 +/- 0.19, epiphysis 0.75 +/- 0.25, thalamus centralis 0.71 +/- 0.10, almond nucleus 0.69 +/- 0.16, medulla oblongata 0.67 +/- 0.15, anterior hypothalamus 0.66 +/- 0.20, precentral gyrus 0.66 +/- 0.16, globus pallidus 0.63 +/- 0.31, postcentral gyrus 0.36 +/- 0.08 and plasma (ng/ml) 0.058 +/- 0.013. Our results demonstrate that immunoreactive calcitonin is present in the central nervous system (CNS) of sheep, compatible with a neurotransmitter function for this hormone.     

Control of glucagon-like immunoreactive peptide secretion from fetal rat intestinal cultures

Some of the mechanisms underlying intestinal glucagon-like immunoreactive (GLI) peptide secretion from cultured fetal rat intestinal cells were investigated using modulators of the adenylate cyclase pathway [(Bu)2cAMP, theophylline, isobutylmethylxanthine], calcium fluxes (ionomycin, A23187), and protein kinase-C (phorbol ester). All of these agents were found to stimulate GLI peptide release, to 120-230% of paired control values (P less than 0.05-0.001). (Bu)2cAMP, but not the phorbol ester, also increased the total cell content of GLI peptides over the 2-h incubation period (P less than 0.05). No synergism between any of the three pathways was detected. When the mol wt distribution of the stored and secreted GLI peptides was determined in control and (Bu)2 cAMP-stimulated samples, 68 +/- 2% of the peptide corresponded to glicentin, while the remainder eluted with the same distribution coefficient as oxyntomodulin. No 3.5K glucagon was detected in any of the extracts. GLI peptide secretion by the cells was not altered by several pancreatic glucagon secretagogues (cortisol, bombesin, and prostaglandins E1 and D2), but was stimulated by the opioid peptide beta-endorphin (1 microM; P less than 0.02). These studies have indicated that the control of secretion of fetal rat intestinal GLI peptides is complex, involving activation of any one or a combination of the three major second messenger systems. A role for the adenylate cyclase pathway in regulating GLI peptide biosynthesis is also suggested.     

Expression of peroxisome proliferator-activated receptor-gamma in key neuronal subsets regulating glucose metabolism and energy homeostasis

In addition to increasing insulin sensitivity and adipogenesis, peroxisome proliferator-activated receptor (PPAR)-gamma agonists cause weight gain and hyperphagia. Given the central role of the brain in the control of energy homeostasis, we sought to determine whether PPARgamma is expressed in key brain areas involved in metabolic regulation. Using immunohistochemistry, PPARgamma distribution and its colocalization with neuron-specific protein markers were investigated in rat and mouse brain sections spanning the hypothalamus, the ventral tegmental area, and the nucleus tractus solitarius. In several brain areas, nuclear PPARgamma immunoreactivity was detected in cells that costained for neuronal nuclei, a neuronal marker. In the hypothalamus, PPARgamma immunoreactivity was observed in a majority of neurons in the arcuate (including both agouti related protein and alpha-MSH containing cells) and ventromedial hypothalamic nuclei and was also present in the hypothalamic paraventricular nucleus, the lateral hypothalamic area, and tyrosine hydroxylase-containing neurons in the ventral tegmental area but was not expressed in the nucleus tractus solitarius. To validate and extend these histochemical findings, we generated mice with neuron-specific PPARgamma deletion using nestin cre-LoxP technology. Compared with littermate controls, neuron-specific PPARgamma knockout mice exhibited dramatic reductions of both hypothalamic PPARgamma mRNA levels and PPARgamma immunoreactivity but showed no differences in food intake or body weight over a 4-wk study period. We conclude that: 1) PPARgamma mRNA and protein are expressed in the hypothalamus, 2) neurons are the predominant source of PPARgamma in the central nervous system, although it is likely expressed by nonneuronal cell types as well, and 3) arcuate nucleus neurons that control energy homeostasis and glucose metabolism are among those in which PPARgamma is expressed.     

Fetal rat intestinal cells in monolayer culture: a new in vitro system to study the glucagon-like immunoreactive peptides

To establish an in vitro model to investigate the glucagon-related peptides, fetal rat intestinal cells were enzymatically dispersed and placed into culture for up to 7 days. After 1 day in culture, the presence of epithelial-like cells containing glucagon-like immunoreactivity (GLI) was demonstrated using immunocytochemical techniques. The cell peptides were extracted by passage through a cartridge of octadecylsilyl silica and characterized by gel filtration and RIA. Two GLI moieties were detected with apparent mol wts of 11,000-12,000 and 5,000-6,000. The immunoreactive profile obtained for the cells in culture was identical to that of both whole fetal rat intestine and adult rat ileum. The presence of glucagon could not be demonstrated in any of the extracts. The basal levels of GLI and apparent immunoreactive glucagon (IRGa) were 1,457 +/- 381 and 198 +/- 57 pg/dish, respectively, on day 1 of culture. The GLI content of the cells, but not the IRGa, declined with time in culture for up to 5-7 days (P less than 0.03). Addition of insulin to the culture medium (10 or 100 mU/ml) did not influence the decrease in GLI content of the cells, but did inhibit the production of IRGa (P less than 0.05). Addition of 500 mg/dl glucose to the cells in the presence of 20 microU/ml insulin increased the secretion of GLI by 42 +/- 7% over 2 h (P less than 0.05). The stimulation by glucose was not seen in the absence of insulin or with higher insulin concentrations (100 microU/ml), nor did insulin alone (100 microU/ml) have any effect on the release of GLI. Thus, fetal rat intestinal cells in culture produce the GLI peptides, and secrete them in response to glucose. This system may provide a means by which the synthesis and control of secretion of the glucagon-related peptides can be investigated.     

Somatotropin Release-Inhibiting Factor and Galanin Innervation in the Hypothalamus and Pituitary of Seabream (Sparus aurata)

The distribution of galanin (GAL) and somatotropin-release-inhibiting-factor (SRIF) immunoreactivity in the hypothalamus and pituitary of the sea bream (Sparus aurata) was studied by immunocytochemistry. An extensive system of neurons immunoreactive with antisera to the two peptides was identified throughout the brain with staining particularly in the hypothalamus. In the hypothalamus, GAL immunoreactive perikarya were detected principally in the nucleus preopticus and nucleus tuberis. Major nerve tracts were observed to sweep down from the hypothalamic nuclei and reached the pituitary via the preoptico-hypophysial tract. Many of the fibers had varicose swellings indicating they were secretory. SRIF immunoreactivity was distributed similarly to GAL but the network of nerve fibers was less dense; no colocalization of these two peptides was seen. SRIF immunoreactive perikarya were present in the preoptic nucleus, the tuberal nucleus, and the basolateral hypothalamus. These perikarya were large and densely staining and were predominately bipolar, although some multipolar perikarya were observed. In the pituitary GAL and SRIF immunoreactivities were confined principally to the pars distalis where fibers infiltrated between growth hormone, prolactin, and adrenocorticotrophic cells. More of the fibers were immunoreactive for SRIF than for GAL. There was no immunoreaction for GAL or SRIF in any of the pituitary cells. There is thus morphological evidence for a neuroendocrine control of the pars distalis by GAL and SRIF and for a possible functional interaction between these two systems.     

Distribution of active and inactive forms of endorphins in rat pituitary and brain.

The recent isolation and identification of alpha-N-acetyl forms of the C-Fragment of lipotropin (beta-endorphin, residues 61-91) and the C'-Fragment (residues 61-87) [Smyth, D.G., Massey, D.E., Zakarian, S. & Finnie, M. (1979) Nature (London) 279, 252-254] has led to a study of their distribution in the pituitary and brain of the rat. Regions were mapped by the method of immunofluorescent staining and the reactive peptides were determined by immunoassay after extraction, gel filtration, and ion exchange chromatography. The major immunoreactive peptides in both lobes of the pituitary were found to be C'-Fragment and N-acetyl C'-Fragment, which are weakly active or inactive as opiates; the C-Fragment and its N-acetyl derivative represented minor components. This indicates that in the rat the circulating "endorphins" released from pituitary would have little morphinomimetic activity. The same four immunoreactive peptides were observed in rat brain. In the hippocampus the C'-Fragment was the principal component in the midbrain there was more C-Fragment but C'-Fragment predominated; in the hypothalamus the C-Fragment was the major peptide, almost to the exclusion of the other peptides. The results demonstrate that the processing of lipotropin is under differential control in anatomically distinct regions of the central nervous system. The processing of lipotropin in the hypothalamus is directed specifically to the production of lipotropin C-Fragment.     

Immunocytochemical distribution of luteinizing hormone in rat central nervous system

This paper presents the first detailed localization of luteinizing hormone (LH)-containing cells and fibers in the rat central nervous system. These immunoreactive elements were identified by four LH antisera, two directed against the intact LH molecule and two against LHb. Cell bodies, immunoreactive for LH were found throughout the rostral-caudal extent of the hypothalamic arcuate and ventromedial nuclei, the periarcuate area ventral to the ventromedial nucleus, and the retrochiasmatic area. Immunopositive fibers were traced to numerous structures within the brain including discrete regions of the hypothalamus, septal area, nucleus of the diagonal band, bed nucleus of stria terminalis, amygdala, thalamus, periaqueductal gray, raphe nuclei, brainstem reticular nuclei, locus ceruleus, parabrachial nucleus, dorsal motor nucleus of vagus, and the nucleus of the solitary tract, with a few fibers extending into spinal cord central gray. This pattern of fiber distribution corresponds closely with those described for fibers containing several other anterior pituitary hormones. The extensive projection for LH may provide neuroanatomical substrate mediating reproductive events as it does in the pituitary, or it may serve some modulatory function in brain which is independent of its role in reproduction.