Neuronostatin Encoded by the Somatostatin Gene Regulates Neuronal, Cardiovascular, and Metabolic Functions

Somatostatin is important in the regulation of diverse neuroendocrine functions. Based on bioinformatic analyses of evolutionarily conserved sequences, we predicted another peptide hormone in pro-somatostatin and named it neuronostatin. Immuno-affinity purification allowed the sequencing of an amidated neuronostatin peptide of 13 residues from porcine tissues. In vivo treatment with neuronostatin induced c-Fos expression in gastrointestinal tissues, anterior pituitary, cerebellum, and hippocampus. In vitro treatment with neuronostatin promoted the migration of cerebellar granule cells and elicited direct depolarizing actions on paraventricular neurons in hypothalamic slices. In a gastric tumor cell line, neuronostatin induced c-Fos expression, stimulated SRE reporter activity, and promoted cell proliferation. Furthermore, intracerebroventricular treatment with neuronostatin increased blood pressure but suppressed food intake and water drinking. Our findings demonstrate diverse neuronal, neuroendocrine, and cardiovascular actions of a somatostatin gene-encoded hormone and provide the basis to investigate the physiological roles of this endogenously produced brain/gut peptide. Somatostatin is important in the regulation of diverse neuroendocrine functions. Based on bioinformatic analyses of evolutionarily conserved sequences, we predicted another peptide hormone in pro-somatostatin and named it neuronostatin. Immuno-affinity purification allowed the sequencing of an amidated neuronostatin peptide of 13 residues from porcine tissues. In vivo treatment with neuronostatin induced c-Fos expression in gastrointestinal tissues, anterior pituitary, cerebellum, and hippocampus. In vitro treatment with neuronostatin promoted the migration of cerebellar granule cells and elicited direct depolarizing actions on paraventricular neurons in hypothalamic slices. In a gastric tumor cell line, neuronostatin induced c-Fos expression, stimulated SRE reporter activity, and promoted cell proliferation. Furthermore, intracerebroventricular treatment with neuronostatin increased blood pressure but suppressed food intake and water drinking. Our findings demonstrate diverse neuronal, neuroendocrine, and cardiovascular actions of a somatostatin gene-encoded hormone and provide the basis to investigate the physiological roles of this endogenously produced brain/gut peptide. Originally discovered in 1972 based on its ability to inhibit pituitary growth hormone release (1Vale W. Brazeau P. Grant G. Nussey A. Burgus R. Rivier J. Ling N. Guillemin R. C. R. Acad. Sci. Hebd. Seances. Acad. Sci. D. 1972; 275: 2913-2916PubMed Google Scholar, 2Brazeau P. Vale W. Burgus R. Ling N. Butcher M. Rivier J. Guillemin R. Science. 1973; 179: 77-79Crossref PubMed Scopus (3045) Google Scholar), somatostatin is one of the most extensively studied peptide hormones (3Reichlin S. N. Engl. J. Med. 1983; 309: 1495-1501Crossref PubMed Scopus (1225) Google Scholar, 4Lamberts S.W. Krenning E.P. Reubi J.C. Endocr. Rev. 1991; 12: 450-482Crossref PubMed Scopus (847) Google Scholar). Somatostatin is widely expressed in neuronal, neuroendocrine, gastrointestinal, inflammatory, immune, and cancer cells and plays important roles in the regulation of neuromodulation, hormone secretion, gastrointestinal functions, immune responses, cell growth, and exocrine secretion (5Low M.J. Best Pract. Res. Clin. Endocrinol. Metab. 2004; 18: 607-622Crossref PubMed Scopus (80) Google Scholar). Two somatostatin isoforms, somatostatin-14 and somatostatin-28, activate five related G protein-coupled receptors with different affinity (6Yamada Y. Post S.R. Wang K. Tager H.S. Bell G.I. Seino S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 251-255Crossref PubMed Scopus (730) Google Scholar, 7Patel Y.C. Front. Neuroendocrinol. 1999; 20: 157-198Crossref PubMed Scopus (1409) Google Scholar, 8Bell G.I. Reisine T. Trends Neurosci. 1993; 16: 34-38Abstract Full Text PDF PubMed Scopus (315) Google Scholar). Somatostatin receptors are also activated by two cortistatin isoforms secreted from different brain regions (9de Lecea L. Criado J.R. Prospero-Garcia O. Gautvik K.M. Schweitzer P. Danielson P.E. Dunlop C.L. Siggins G.R. Henriksen S.J. Sutcliffe J.G. Nature. 1996; 381: 242-245Crossref PubMed Scopus (351) Google Scholar). Based on bioinformatic analysis of evolutionarily conserved sequences in the pro-somatostatin protein, we predicted the existence of another peptide hormone encoded by the somatostatin gene. We generated antibodies against the putative peptide, isolated the endogenous peptide, and found it to be an amidated peptide of 13 residues. This peptide hormone, named neuronostatin, induced c-Fos and c-Jun expression in diverse brain/gut tissues, regulated neuronal functions in vitro, and modulated blood pressure as well as food intake and drinking behavior in vivo. Peptide Hormones—All peptides used were synthesized by Phoenix Pharmaceuticals Inc. (Burlingame, CA) or the PAN facility at Stanford University. Peptide purity was verified by analytical reverse phase HPLC and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) 4The abbreviations used are: MALDI-TOF, matrix-assisted laser desorption/ionization-time of flight; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; RIA, radioimmunoassay; PBS, phosphate-buffered saline; TTX, tetrodotoxin; PVN, paraventricular nucleus; MNC, magnocellular; SRE, serum response element; MAP, mean arterial pressure. mass spectrometry. Unless indicated otherwise, all functional tests utilized human amidated neuronostatin-13. Purification of Endogenous Neuronostatin—To purify endogenous neuronostatin, frozen porcine pancreas and spleen were obtained from Pel-Freez (Rogers, AK) and extracted as described (10Kojima M. Hosoda H. Date Y. Nakazato M. Matsuo H. Kangawa K. Nature. 1999; 402: 656-660Crossref PubMed Scopus (7260) Google Scholar). After acetone precipitation, the supernatant was passed through a Sep-Pak C18 column (Waters, Milford, MA) and lyophilized. Rabbit immunoglobulin G against human neuronostatin-19 was purified using a protein A column and conjugated to glutaraldehyde-activated magnetic beads (BioMag, Polysciences, Warrington, PA). After washing with PBS (phosphate-buffered saline), beads (2 ml at 20 mg/ml) were incubated with reconstituted tissue extracts (58 mg/ml of pancreatic extract and 80 mg/ml of spleen extracts) overnight at 4 °C. After separating beads using a magnet and washing with 6 ml of PBS four times, affinity-captured peptides were eluted by adding 1.5 ml of 60% CH3CN in 0.1% trifluoroacetic acid. Purified peptides and standard peptides were analyzed by a reverse-phase HPLC SCL-10A system (Kratos-Shimadzu, Kyoto, Japan) using a C-18 (5 μm, 250 × 4.6 mm) column. At a flow rate of 1 ml/min, a linear gradient (0-60%) of CH3CN in 0.1% trifluoroacetic acid was passed through the column for 40 min, and the flow-through was monitored by a UV 220-nm detector (30 s/fraction) followed by peptide measurement using an RIA for human/porcine neuronostatin-19. For MALDI-TOF analysis, purified peptides and standard peptides were analyzed using Maxima LNR (Kratos-Shimadzu Co., Kyoto, Japan). Briefly, 2 μl of peptides were mixed with 2 μl of matrix solution of α-cyano-4-hydroxycinnamic acid before mass spectrum determination. Microsequencing was performed from the N terminus by the PAN facility at Stanford University. Immunoassays—Human neuronostatin peptides of 19 residues and rat neuronostatin-13 were synthesized. To increase antigenicity and solubility, these peptides were conjugated to thyroglobulin for immunization in rabbits to raise polyclonal antibodies. RIA for human neuronostatin-19 and rat neuronostatin-13 showed intra-assay variability of 5 and 10%, detection limits of 20 and 14 pg/ml, and EC50 of 65 and 68 pg/ml, respectively. Cross-reactivity of the human neuronostatin-19 RIA to human/porcine neuronostatin-13 and non-amidated human neuronostatin-13 is 10 and 0.2%, respectively. For the determination of tissue content of neuronostatin and somatostatin, different tissues from female rats were minced and boiled for 5 min in 5× volumes of water to inactivate intrinsic proteases. The solution was adjusted to 1 m acetic acid-20 mm HCl before homogenization using a Polytron mixer. After centrifugation for 30 min at 3,000 × g, acetone was added to the supernatant to a final concentration of 66% and precipitated proteins were removed after another centrifugation. The extracted peptide fractions were lyophilized, passed through a Sep-Pak column before RIA measurement of rat neuronostatin-13 and somatostatin-14 content (Phoenix Pharmaceuticals). Animals—All procedures were approved by the institutional animal committees. Adult male BALB/c mice and 7-day-old Sprague-Dawley rats from the Stanford animal facility were used for immunohistochemical staining and c-Fos/c-Jun induction tests. Adult female Sprague-Dawley rats (Charles Rivers Laboratories, Wilmington, MA) were used for peptide extraction. Neonatal Sprague-Dawley rats were used for cerebral growth cone turning assay at Institute of Neuroscience, Shanghai Institutes of Biological Science. Adult Sprague-Dawley rats (225-300 g body weight, Harlan, Indianapolis, IN) were used for pituitary cultures (males and females), blood pressure monitoring (males only), as well as food intake and drinking tests (males only) at St. Louis University. Animals were on with to food and water Sprague-Dawley rats at (Charles were used in the of hypothalamic at University. of were obtained from male mice and with before After in and of were with m at for min followed by 0.1% at for min before using polyclonal antibodies against rat neuronostatin-13 Phoenix human somatostatin MA) or were using the Inc. of were with body of neuronostatin or to c-Fos and c-Jun expression in diverse tissues J. PubMed Scopus Google Scholar). At after were with and the with were in followed by and before staining with antibodies against c-Fos or c-Jun were using the for and were performed using antibodies from For c-Fos induction in brain of neuronostatin body were performed for 1.5 using rats followed by immunohistochemical tissues of rats were incubated in and PBS 0.1% for min at followed by cells were by and on with before in with and serum After for were to a a isolated neurons with were for growth cone turning assay as described Y. Y. K. Wang S. S. Neurosci. PubMed Scopus Google Scholar). Briefly, of neuronostatin and were produced by of a concentration solution from a (2 with a of The of the was from the growth with an of with to the of the granule the concentration the growth cone is to of in the M. Y. J. Neurosci. 1992; 12: PubMed Google Scholar). of were and analyzed using the The turning was by the the of and a the growth cone at the and the of 30 min of and Sprague-Dawley rats (Charles were used in the of hypothalamic slices. were and the brain and solution with and of and A tissue the was obtained and using a were in a water at for at 1 before in of 2 2 and 10 were in a was at with to °C. were using an system on an Japan). cell and were using a CA) and using a and for rate was 5 and were at 10 were with an solution 2 10 2 and 10 with with this of with or a were from was to the cell the were to to neuronostatin was a in was at the of the standard of the mean and or to described J. Neurosci. PubMed Scopus Google Scholar). of c-Fos in gastric tumor cells were obtained from and in For analyses of c-Fos cells were for before was extracted from cells using the and was using before reverse using a were using for c-Fos and were generated by of The used c-Fos was performed using and used for The were performed on a CA) with an of min at followed by of are as expression, to mean of of For staining of c-Fos cells were on a in were with 10 neuronostatin for 1 and in PBS before in in PBS for 20 min at °C. with PBS, cells were incubated with a mm at for 20 After in PBS and in 0.1% in PBS for min at cells were with serum for 1 at °C. This was followed by overnight of cells with the c-Fos antibodies at 4 °C. After cells were in serum in PBS for before treatment with antibodies for 2 at were in serum for 10 min before staining of cell using the for a and were based on the assay monitored a from cells using the cell assay This the of the to T. J. 1983; PubMed Scopus Google Scholar). were on and incubated at °C. After for in were were with or neuronostatin for before adding the for 4 to at using a For the reporter assay in cells were with the reporter CA) for 4 followed by in for to were with different of neuronostatin for 6 before measurement of assay using a For somatostatin of the protein, cells were with a protein P. 1999; PubMed Scopus Google Scholar), the reporter and somatostatin receptors for 4 followed by for were with hormone neuronostatin, or serum for before the and with and St. g body weight, male rats were in a and a mm) the as described J. PubMed Scopus Google Scholar). For cardiovascular a was to a as described Res. 1999; PubMed Scopus Google The was at the of the and with were to a and allowed to for 1 The was with and to a pressure The rats were for an before arterial pressure and rate were and the The of a followed by of 2 μl of or of neuronostatin before monitoring arterial and for an At the of this all an of to in the and in mean arterial pressure of mm For food intake and drinking rats were allowed to from the for at 5 to body rats were to for with to and water J. PubMed Scopus Google Scholar). food and water and body were and were (2 or of neuronostatin at and water were and monitored at and the at and the were and of the was verified by a response to at the of or in a of rats was monitored using an from rats were 0.1% serum and 0.1% before at G. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). cell were × mm and incubated for at in serum and were obtained after centrifugation × g, 10 min, and in 0.1% serum 20 mm and mm with or different of neuronostatin. cells were with neuronostatin in with to or for determination. In the of neuronostatin on and hormone to and hormone to release was In cells from female the of neuronostatin on and hormone hormone release was also After for 30 min, were after centrifugation for the determination of and by RIA using from the and Peptide PubMed Scopus Google Scholar). were by RIA using the rat (Phoenix for tissue peptide are as the mean all are expressed as and were analyzed by analysis of with for the growth cone turning assay was analyzed using the was for with a of 1 or arterial were analyzed by mm from followed by the to the for The were the the and the of the of a of neuronostatin on and Purification of a we for putative with or for in peptide hormones to activate G protein-coupled receptors in the regions were for of and putative peptide regions in diverse This bioinformatic allowed the of a encoded by the pro-somatostatin protein, and we named it neuronostatin. in neuronostatin is after the peptide of secretion in diverse in with a conserved at its and at the of the of residues in diverse neuronostatin isoforms of and 19 residues be all 19 predicted residues are in most rat and peptides a of at We generated antibodies against amidated human neuronostatin-19 and an Endogenous neuronostatin from porcine pancreas and spleen was purified using magnetic beads conjugated to antibodies against human and the purified peptides were monitored using an peptides were a reverse-phase HPLC pressure using a linear gradient of were using a UV 220-nm detector and min, with a at min and a at 30 min This was using HPLC and the purified analyzed using MALDI-TOF mass to a mass of Microsequencing of the purified peptide the residues. Based on mass with non-amidated peptide in the RIA, with amidated peptide in the and the sequencing the endogenous peptide was to be an amidated peptide of 13 residues for the HPLC eluted at 30 min showed a mass to the 30 residues of pro-somatostatin with an residues in the terminus of neuronostatin. For spleen a obtained after MALDI-TOF to amidated neuronostatin-13 was also found and by sequencing In an with a mass to the predicted neuronostatin-19 was in the the of this peptide was for Based on these all tests were performed using amidated human neuronostatin-13. We generated antibodies against rat neuronostatin-13 and an this we found neuronostatin is expressed in diverse neuronal, and gastrointestinal tissues of rats with the in the spleen and followed by and of somatostatin-14 in the indicated in the of neuronostatin and somatostatin in different tissues, of these two peptides from the of in and cells neuronostatin, immunohistochemical staining was In the staining of neuronostatin was found in the cell as somatostatin In the neuronostatin was found in cells of to somatostatin In the staining for neuronostatin and somatostatin was found in the a and of and in investigate cells for neuronostatin, male mice were with neuronostatin. was performed to the expression of c-Fos and response to be induced after diverse J.G. Front. Neuroendocrinol. 1993; PubMed Scopus Google Scholar, K. P. Full Text PDF PubMed Scopus Google Scholar, H. C.L. M.J. S.R. Nature. PubMed Scopus Google Scholar). staining of c-Fos was found in the cells of gastric in mice with neuronostatin but in In the neuronostatin stimulated c-Fos expression in cells of the but staining was found in the of In the anterior pituitary, showed c-Fos staining c-Fos was found in the pancreatic neuronostatin treatment induced c-Jun expression in the of cells and cells of neuronostatin for 1.5 in c-Fos staining was found in granule cells and cells in the in the as well as in cells of the a and In treatment with was and The of neuronostatin treatment on c-Fos expression in granule cells of the this peptide granule cell migration through The of neuronostatin was in a growth cone turning assay using cerebellar granule cells Y. Y. K. Wang S. S. Neurosci. PubMed Scopus Google Scholar). in 5 and a gradient of neuronostatin-13 generated by of or of the peptide from a a turning of the growth cone the 30 A gradient of human neuronostatin-19 growth cone turning at the treatment with somatostatin-14 and also growth cone turning In an peptide with with neuronostatin growth cone turning In hypothalamic we obtained cell from paraventricular neurons and neuroendocrine neurons based on J. Neurosci. PubMed Scopus Google Scholar). of 10 neuronostatin in of mean in A and of these of mean in and the cells showed response to peptide In the of the a of neurons in response to neuronostatin and was in these neurons direct depolarizing actions of neuronostatin through of receptors on these To neuronostatin actions in vitro, human gastric tumor cells were with neuronostatin before c-Fos in analyses indicated a of c-Fos c-Fos staining was 1 after treatment with neuronostatin but in cells We also the ability of neuronostatin to cell based on the assay monitored a from in treatment with human or neuronostatin to a of the of cells with of the of the SRE P.E. H. A. Full Text PDF PubMed Scopus Google Scholar), the ability of neuronostatin to an reporter was also in treatment with human or neuronostatin to a of In treatment with somatostatin-14 was of in performed of neuronostatin in rats and monitored arterial pressure in treatment with neuronostatin to an increase in was in in was the 10 min in all to at the of this and in the a increase in was the A and and the for the were This of neuronostatin on cardiovascular to be to a on as the of neuronostatin elicited the increase in or in a of rats In rat a of and drinking the of by an increase in C. J. PubMed Scopus Google Scholar). was to this endogenous of activity, followed by the monitoring of food and water drinking the In this of and neuronostatin food intake and water drinking in a in 1 of food intake the as with the The of neuronostatin, the phase of a was the of The of neuronostatin food intake the an of the 1 through the of water drinking by neuronostatin was also The of neuronostatin were in food or water were the at or or Somatostatin somatostatin was isolated based on its ability to release (1Vale W. Brazeau P. Grant G. Nussey A. Burgus R. Rivier J. Ling N. Guillemin R. C. R. Acad. Sci. Hebd. Seances. Acad. Sci. D. 1972; 275: 2913-2916PubMed Google Scholar), we the ability of neuronostatin to release from anterior pituitary in hormone or release was by a concentration of neuronostatin. In the anterior pituitary treatment with neuronostatin or or hormone release We neuronostatin with somatostatin a G protein P. 1999; PubMed Scopus Google Scholar), we the ability of somatostatin-14 to the G protein by five different human somatostatin receptors cells were with a protein, the reporter and somatostatin followed by the monitoring of reporter treatment with somatostatin-14 stimulated all five receptors based on treatment with neuronostatin was Based on bioinformatic followed by we the existence of a peptide hormone encoded by the pro-somatostatin induced the expression of response in a of tissues. We the ability of this hormone to neuronal hypothalamic and gastric tumor cell as well as to blood food and water drinking. Somatostatin is expressed in diverse tissues. of neuronostatin also indicated a expression of this peptide but the of neuronostatin-13 and somatostatin-14 are in different tissues. After the of this is to be by one or at residues to somatostatin and neuronostatin somatostatin and neuronostatin is a and is from the the of neuronostatin and somatostatin be of the for the of neuronostatin but somatostatin, expression of in cells somatostatin neuronostatin the neuronostatin the residues at its is conserved from to this amidated hormone plays important roles in diverse In to neuronostatin, we also purified a of pro-somatostatin using the neuronostatin-19 antibodies. peptides with sequences but the neuronostatin peptide, porcine pro-somatostatin H. M. W. PubMed Scopus Google Scholar), porcine M. L. J. Full Text PDF PubMed Google Scholar), and rat pro-somatostatin (1Vale W. Brazeau P. Grant G. Nussey A. Burgus R. Rivier J. Ling N. Guillemin R. C. R. Acad. Sci. Hebd. Seances. Acad. Sci. D. 1972; 275: 2913-2916PubMed Google Scholar, M. Hosoda H. Date Y. Nakazato M. Matsuo H. Kangawa K. Nature. 1999; 402: 656-660Crossref PubMed Scopus (7260) Google R. Ling N. Science. PubMed Scopus Google Scholar). The physiological roles of these peptides are is a brain/gut peptide to its of and its ability to response c-Fos or c-Jun in neuronal, anterior pituitary, and gastrointestinal tissues. the of c-Fos and c-Jun expression in these tissues be to induction by using human tumor cells indicated neuronostatin on gastric Somatostatin is expressed in the and the secretion of pituitary hormones S.W. Endocr. Rev. PubMed Scopus Google Scholar). Somatostatin is also produced in the gastric N. L. Science. PubMed Scopus Google and the secretion of gastrointestinal peptides in a M. Date Y. K. T. K. N. M. Kangawa K. H. Matsuo H. Nakazato M. Res. PubMed Scopus Google Scholar). neuronostatin is expressed in cells and c-Fos in it also as a to somatostatin, neuronostatin is expressed in pancreatic cells and neuronostatin on cells in a somatostatin, discovered based on its ability to inhibit neuronostatin or secretion by anterior pituitary the physiological roles of neuronostatin neuronostatin, somatostatin, is a hormone based on its expression and its ability to c-Fos and c-Jun in different tissues this peptide. showed somatostatin peptides the migration of cerebellar granule cells H. Nature. PubMed Scopus Google Scholar). we found treatment with neuronostatin the migration of the growth cone of granule cell findings neuronostatin, somatostatin, is a in neuronal migration in the indicated neuronostatin and somatostatin are expressed at different regions of the cerebellum, different roles in cerebellar The of neuronostatin as a is by its direct of the of hypothalamic paraventricular treatment with neuronostatin increased in the of was with in of somatostatin also increased arterial by in secretion PubMed Scopus Google Scholar, R. R. U. P. T. J. PubMed Google Scholar). is the of neuronostatin on was to an increase in followed by a to release hormones as in the of neuronostatin depolarizing on to brain and and to the pituitary treatment with neuronostatin also suppressed food intake and water drinking. of neuronostatin to activity, it is the to was to Furthermore, the of direct neuronal of neuronostatin in the hypothalamic a to with the brain Nature. PubMed Scopus Google Scholar). The for neuronostatin is to for peptides D. Neurosci. PubMed Scopus Google Scholar, D. Res. PubMed Scopus Google Scholar). of somatostatin also suppressed food intake in rats R. D. J. PubMed Scopus Google but treatment with a somatostatin induced water drinking Res. PubMed Scopus Google Scholar, J. J. PubMed Google Scholar). findings of the of food intake and drinking by neuronostatin indicated this peptide through a from of human tumor cell functions as by the of c-Fos cell and neuronostatin peptides a of at as with human The ability of human and neuronostatin to the and in cells at by Somatostatin and cortistatin peptides with five somatostatin receptors (6Yamada Y. Post S.R. Wang K. Tager H.S. Bell G.I. Seino S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 251-255Crossref PubMed Scopus (730) Google Scholar, 8Bell G.I. Reisine T. Trends Neurosci. 1993; 16: 34-38Abstract Full Text PDF PubMed Scopus (315) Google to activate the protein T. Bell G.I. Endocr. Rev. 16: Google and one neuronostatin and somatostatin we neuronostatin, somatostatin, by the somatostatin also release by pituitary is neuronostatin with an G protein-coupled or a with on neuronostatin and of the receptors and for this brain/gut hormone with diverse neuroendocrine and physiological functions. We for with

Récupéré en direct depuis OpenAlex et désinversé. Les résumés ne sont pas conservés dans cette base de données : les index inversés représentent 8,6 Go des 9,3 Go de texte de la base, et le serveur dispose de 13 Go libres.