Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells.

Of paramount importance for the development of cell therapies to treat diabetes is the production of sufficient numbers of pancreatic endocrine cells that function similarly to primary islets. We have developed a differentiation process that converts human embryonic stem (hES) cells to endocrine cells capable of synthesizing the pancreatic hormones insulin, glucagon, somatostatin, pancreatic polypeptide and ghrelin. This process mimics in vivo pancreatic organogenesis by directing cells through stages resembling definitive endoderm, gut-tube endoderm, pancreatic endoderm and endocrine precursor--en route to cells that express endocrine hormones. The hES cell-derived insulin-expressing cells have an insulin content approaching that of adult islets. Similar to fetal beta-cells, they release C-peptide in response to multiple secretory stimuli, but only minimally to glucose. Production of these hES cell-derived endocrine cells may represent a critical step in the development of a renewable source of cells for diabetes cell therapy.

Immunohistochemical identification and effects of atrial natriuretic peptide, pancreastatin, leucine-enkephalin, and galanin in the porcine pancreas.

This study demonstrates the presence and distribution of atrial natriuretic peptide (ANP) pancreastatin (PST), leucineenkephalin (Leu-ENK), galanin (GAL), and insulin in the pig pancreas. The effects of PST, ANP, Leu-ENK, and GAL on protein and amylase secretion were also investigated to determine their functional role in the control of pancreatic secretion. PST-immunoreactive cells were observed in the islet of Langerhans and in the wall of the ducts. Leu-ENK-immunopositive cells were observed in both the endo-and exocrine pancreas. It is colocalized with insulin in the islet of Langerhans. ANP immunoreactivity was discernible in nerve fibers and cells of the exocrine pancreas. GAL-immunopositive cells were observed in close association with insulin-positive cells in the islets of Langerhans and in the exocrine pancreas. Stimulation of isolated pancreatic segments with either ANP or Leu-ENK resulted in increased protein secretion and amylase output. The Leu-ENK-evoked amylase secretion was antagonized by naloxone. Pancreastatin was effective at all concentrations, but low concentration had more marked secretory effects whereas GAL failed to evoke any significant increases in either protein or amylase secretion. The results of the study have demonstrated a close association of peptidergic fibers with the secretory cells of the pancreas. The nerve fibers can release peptides that in turn can stimulate protein and amylase secretion.

Pancreastatin molecular forms in normal human plasma.

Circulating molecular forms with pancreastatin (PST)-like immunoreactivity in plasma from normal subjects were examined. An immunoreactive form corresponding to a human PST-like sequence [human chromogranin-A-(250-301)] (hPST-52) and a larger form (mol wt 15-21 kDa) were detected by gel filtration of plasma from normal subjects. On high performance liquid chromatography, predominant immunoreactive forms coeluted with the three larger forms which were purified from the xenograft of human pancreatic islet cell carcinoma cell line QGP-1N cells and with synthetic hPST-52. The fraction containing larger forms purified from xenograft of QGP-1N cells had biological activity equivalent to that of hPST-52 on the inhibition of pancreatic exocrine secretion. These results suggest that the larger molecular forms as well as hPST-52 may be physiologically important circulating forms of PST in human.

Immunocytochemical localisation of pancreastatin and chromogranin A in porcine neuroendocrine tissues.

Pancreastatin is a 49 amino acid peptide with a C-terminal glycine amide originally isolated from porcine pancreas. In the present study the cellular localisation of pancreastatin in porcine neuroendocrine tissue was examined immunocytochemically using an antiserum raised against porcine pancreastatin (33-49) that does not cross-react with porcine chromogranin A. In order to study the possible precursor-product relationship between chromogranin A and pancreastatin the cellular localisation of both peptides was examined in peripheral tissues using simultaneous double immunostaining. The pancreastatin antiserum immunostained cells and nerve fibers throughout the neuroendocrine system. In most of the examined tissues we found colocalisation of pancreastatin and chromogranin A immunostaining. These results support the precursor-product concept for chromogranin A and pancreastatin. However, in the gastrointestinal tract and the adenohypophysis a minor population of the endocrine cells exhibited immunostaining with only one of the two antibodies. This discrepancy between immunostaining with pancreastatin antiserum and monoclonal chromogranin A antibody could be due to absence of, or extensive, processing of chromogranin A in certain cell populations.

An opioid pancreatic peptide produces ileal muscle inhibition and naloxone-reversible analgesia.

The opioid activity of immunoreactive beta-endorphin-like peptide extracted from pork pancreas duplicates the effects of morphine and synthetic beta-endorphin when measured by inhibition of isolated guinea pig ileal muscle response to electro-stimulation in vitro and by morphine-like analgesia following intravenous injection in the mouse. These responses are reversed by the opiate antagonist naloxone, indicating that a potent opioid mu receptor binding ligand is present in pancreatic extract. These findings imply a pancreatic source of plasma immunoreactive beta-endorphin that may explain a number of physiological and behavioral effects generally attributed to hypophyseal beta-endorphin alone.

Regulation of pancreastatin release from a human pancreatic carcinoid cell line in vitro.

The objective of these experiments was to investigate the influence of activation of three second messenger systems (protein kinase-C, adenylate cyclase-cAMP, and calcium mobilization) on the secretion of pancreastatin (PST) and chromogranin-A (CGA) by a human pancreatic carcinoid cell line (BON) in tissue culture. Stimulation of protein kinase-C by a phorbol ester (0.025-7.5 microM) caused a significant dose-related release of PST (186 +/- 22-4271 +/- 228% over controls). Treatment of BON cells with graded doses of 8-bromo-cAMP (0.14-3.0 mM) and isobutylmethylxanthine (IBMX; 0.01-1.0 mM) also stimulated a dose-related release of PST (107 +/- 22-284 +/- 28 and 16 +/- 12-1076 +/- 100% over controls, respectively). Incubation of BON cells with ionomycin (0.134-13.4 microM) increased the release of PST (102 +/- 15-554 +/- 21% over controls) in a dose-related manner. A combination of IBMX and ionomycin resulted in an additive effect, whereas treatment with a phorbol ester plus IBMX resulted in a synergistic effect on PST release. Pretreatment of BON cells with monensin, an agent that prevents processing of precursors to smaller peptides, significantly decreased PST, but not CGA, secretion in response to phorbol ester or ionomycin. These findings indicate that protein kinase-C, cAMP, and Ca2+ mobilization participate in CGA and PST secretion. Although the observation that secretions of PST and CGA in response to theophylline are quantitatively associated, the absence of a quantitative relationship in the release patterns of PST and CGA in response to phorbol ester and ionomycin do not support a simple precursor-product relationship between CGA and PST. The monensin experiments are consistent with the notion that PST is derived from CGA in BON cells.

Isolation and characterization of a tumor-derived human protein related to chromogranin A and its in vitro conversion to human pancreastatin-48.

A protein with pancreastatin-like immunoreactivity has been isolated and purified from liver metastasis of a patient with insulinoma. NH2-terminal residue analysis, in conjunction with the use of antibodies that are specific for the C-terminal amide peptide of porcine pancreastatin, identified this protein as a 186-amino-acid protein corresponding to human chromogranin A-116-301 (the fragment corresponding to the positions from 116 to 301 of human chromogranin A). Digestion of this protein with trypsin yielded a 48-amino-acid peptide with the retention of full pancreastatin activity. Serum from patient with insulinoma contains a peptide specie(s) that comigrates with the 48-amino-acid pancreastatin, suggesting that this peptide might be a physiologically important circulation form of pancreastatin in humans. A sensitive radioimmunoassay was established using antibody developed against a synthetic 29-amino-acid peptide amide of pancreastatin. Immunocytochemical staining revealed that a major population of human pancreatic islet cells were immunoreactive to the antiserum but with varying intensity of staining. Pancreastatin-like immunoreactivity was not observed in exocrine acinar cells.

Isolation and characterization of a tumor-derived human pancreastatin-related protein.

A protein with pancreastatin-like immunoreactivity has been isolated and purified from liver metastasis of a patient with insulinoma. NH2-terminal sequence analysis in conjunction with the use of antibodies specific for the C-terminal structure of pancreastatin identified this protein as a 186-amino acid residue protein corresponding to human chromogranin A-116-301. Using a sensitive radioimmunoassay it was found that serum from the patient with insulinoma contains two peptide species; one comigrates with the 186-amino acid residue pancreastatin and the other the 48-residue pancreastatin.

Isolation and characterization of bovine pancreastatin.

Bovine pancreastatin, a 47 amino acid residue peptide, was isolated from the pancreas and the pituitary gland using a chemical method which detects its C-terminal glycine amide structure. The complete amino acid sequence of the pancreatic peptide is 74% homologous to that of porcine pancreastatin and is identical to bovine chromogranin A-(248-294), as deduced from its cDNA sequence. The sequence of the first 28 amino-terminal residues of the pituitary peptide was determined to be identical to the corresponding sequence of the pancreatic peptide. Since the pituitary peptide also contains the C-terminal glycine amide, it is therefore likely to be identical in structure to the pancreatic peptide. Thus, we conclude that bovine chromogranin A is the precursor of bovine pancreastatin. Synthetic bovine pancreastatin inhibited pancreatic exocrine secretion in a similar manner to porcine pancreastatin.

Pancreastatin: molecular and immunocytochemical characterization of a novel peptide in porcine and human tissues.

Pancreastatin, a novel 49-amino acid peptide isolated from porcine pancreas, shows over 70% sequence homology to the central part of bovine and human chromogranin-A. Using an N-terminal and C-terminal synthetic peptide, we developed two sensitive and specific RIAs for the detection of pancreastatin-like immunoreactivity (PLI) in porcine and human tissue extracts. PLI was present throughout the gastrointestinal tract and in most endocrine and neuronal tissues. Highest concentrations were measured in the pituitary, adrenal gland, and pancreas (1200-4000 pmol/g), similar to the distribution of chromogranin-A. PLI was also detected in human endocrine tumors, with large quantities in some carcinoids (up to 14 nmol/g). HPLC revealed that extracts from porcine pituitary and pancreas contained small pancreastatin-like peptides, whereas in adrenal medulla large chromogranin-A-like molecular forms predominated. Human endocrine tumors showed a different pattern, with intermediate forms distinct from chromogranin-A and pancreastatin. Biochemical analysis was confirmed by immunocytochemistry localizing PLI in pancreatic islets, adrenal medulla, pituitary, duodenum, and human endocrine tumors. Pancreastatin is present in a variety of gastrointestinal, endocrine, and neuronal tissues and may represent a novel peptide of unknown physiological function, derived from chromogranin-A by proteolytic cleavage.

Phasic release of newly synthesized secretory proteins in the unstimulated rat exocrine pancreas.

Pancreatic lobules from fasted rats secrete pulse-labeled proteins in two phases comprising 15 and 85% of basal output, respectively. The first (0-6.5 h) is initially (less than or equal to 0.5 h) unstimulated by secretagogues, probably represents vesicular traffic of Golgi and post-Golgi origin (including condensing vaculoles/immature granules), and notably contains two groups of polypeptides with distinct release rates: zymogens (t1/2 approximately 2.4 h) and minor nonzymogens plus one unique zymogen (t1/2 approximately 1 h). The second phase (peak at 9-10 h) is stimulable, probably represents basal granule exocytosis (t1/2 approximately 5 h), and contains zymogens exclusively. Newly synthesized proteins released in both phases appear asynchronously, reiterating their asynchronous transport through intracellular compartments. Zymogens in both phases are secreted apically. The sorting of first from second phase zymogen release does not appear to be carrier-mediated, although the sorting of zymogens from other secretory proteins may use this process. Finally, data are presented that suggest that both secretory phases are subject to physiologic regulation.

Pancreastatin, a novel pancreatic peptide that inhibits insulin secretion.

In mammalian tissues the C-terminal amide structure has been found to occur only in neuroactive or hormonally-active peptides. About half known neuropeptide and peptide hormones have this unique chemical feature. Using a chemical detection method, a search for previously unknown peptides that possess the C-terminal amide structure in extracts of brain and intestine was carried out and a number of novel neuropeptides and hormonal peptides, designated neuropeptide Y, PHI, peptide YY, galanin and neuropeptide K were isolated. We recently performed a similar search in porcine pancreas and found a high concentration of a peptide having a glycine amide at its C-terminus. Here we report the isolation, primary structure and biological activity of this novel peptide. The 49-residue peptide strongly inhibits glucose-induced insulin release from the isolated perfused pancreas and was therefore named pancreastatin. It may be important in the regulation of insulin secretion and in the pathogenesis and treatment of diabetes mellitus.

Purification, crystallisation and preliminary X-ray studies on avian pancreatic polypeptide.

A pancreatic polypeptide with some hormonal properties has been purified from chicken and turkey pancreas using acid-ethanol extraction, gel filtration and anion-exchange chromatography. The material has been crystallised. The crystals are monoclinic with space group C2. Preliminary isomorphous replacement experiments have so far provided a single-site derivative with Hg(NO3)2. A low-resolution electron density map phased with this derivative using anomalous scattering considered together with Patterson function calculations suggest that the molecules are partly helical and are arranged as a compact dimer about the crystallographic two-fold axis. The structure and association of these molecules are compared with those of insulin and glucagon, pancreatic protein and polypeptide hormones respectively, which have been studied in great detail.

Immunohistochemical localization of human pancreatic polypeptide (HPP) in the human endocrine pancreas.

In order to clearly identify the cell type responsible for production of human pancreatic polypeptide (HPP), a newly postulated hormone, we have conducted an immunohistochemical detection of this peptide in the human endocrine pancreas at both light and electron microscopy levels. It was shown that HPP containing cells are located both in endocrine and exocrine pancreatic tissue. In the islets, the positive cells are very few in number and are characterized by an elongated shape and the presence of small secretory granules of about 150 nm in diameter. The presence of HPP in an endocrine cell type reinforce the hypothesis that this peptide is a hormone.