Dipeptidyl peptidase IV (DPP IV) and related molecules in type 2 diabetes

Dipeptidyl peptidase IV (DPP IV) is a widely distributed physiological enzyme that can be found solubilized in blood, or membrane-anchored in tissues. DPP IV and related dipeptidase enzymes cleave a wide range of physiological peptides and have been associated with several disease processes including Crohn's disease, chronic liver disease, osteoporosis, multiple sclerosis, eating disorders, rheumatoid arthritis, cancer, and of direct relevance to this review, type 2 diabetes. Here, we place particular emphasis on two peptide substrates of DPP IV with insulin-releasing and antidiabetic actions namely, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). The rationale for inhibiting DPP IV activity in type 2 diabetes is that it decreases peptide cleavage and thereby enhances endogenous incretin hormone activity. A multitude of novel DPP IV inhibitor compounds have now been developed and tested. Here we examine the information available on DPP IV and related enzymes, review recent preclinical and clinical data for DPP IV inhibitors, and assess their clinical significance.

Antimicrobial activity of neuropeptides against a range of micro-organisms from skin, oral, respiratory and gastrointestinal tract sites

Many neuropeptides are similar in size, amino acid composition and charge to antimicrobial peptides. This study aimed to determine whether the neuropeptides substance P (SP), neurokinin A (NKA), calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP), displayed antimicrobial activity against Streptococcus mutans, Lactobacillus acidophilus, Enterococcus faecalis, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans. SP, NPY, VIP and CGRP displayed variable degrees of antimicrobial activity against all the pathogens tested with the exception of S. aureus. These antimicrobial activities add a further dimension to the immunomodulatory roles for neuropeptides in the inflammatory and immune responses. (c) 2008 Elsevier B.V. All rights reserved.

Neuropeptides Regulate Expression of Angiogenic Growth Factors in Human Dental Pulp Fibroblasts

Abstract
INTRODUCTION:
Neuropeptides play an important role in inflammation and repair and have been implicated in mediating angiogenesis. Pulp fibroblasts express neuropeptide receptors, and the aim of this research was to investigate whether neuropeptides could regulate angiogenic growth factor expression in vitro
METHODS:
An angiogenic array was used to determine the levels of 10 angiogenic growth factors expressed by human pulp fibroblasts.
RESULTS:
Pulp fibroblasts were shown to express angiogenin, angiopoietin-2, epidermal growth factor, basic fibroblast growth factor, heparin-binding epidermal growth factor, hepatocyte growth factor, leptin, platelet-derived growth factor, placental growth factor, and vascular endothelial growth factor. Furthermore, the neuropeptides substance P, calcitonin gene-related peptide, vasoactive intestinal polypeptide, and neuropeptide Y altered angiogenic growth factor expression in vitro.
CONCLUSIONS:
The regulation of angiogenic growth factor expression by neuropeptides suggests a novel role for neuropeptides in pulpal inflammation and repair.

Recent Advances in Antidiabetic Drug Therapies Targeting the Enteroinsular Axis

The enteroinsular axis (EIA) constitutes a physiological signalling system whereby intestinal endocrine cells secrete incretin hormones following feeding that potentiate insulin secretion and contribute to the regulation of blood glucose homeostasis. The two key hormones responsible are named glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). Recent years have witnessed sustained development of antidiabetic therapies that exploit the EIA. Current clinical compounds divide neatly into two classes. One concerns analogues or mimetics of GLP-1, such as exenatide (Byetta) or liraglutide (NN2211). The other group comprises the gliptins (e. g. sitagliptin and vildagliptin) which boost endogenous incretin activity by inhibiting the enzyme dipeptidyl peptidase 4 (DPP 4) that degrades both GLP-1 and GIP. Ongoing research indicates that further incretin and gliptin compounds will become available for clinical use in the near future, offering comparable or improved efficacy. For incretin analogues there is the prospect of prolonged duration of action and alternative routes of administration. This review focuses on recent advances in pre-clinical research and their translation into clinical studies to provide future therapies for type 2 diabetes targeting the EIA.

Functional peptidomics of amphibian skin secretion: A novel Kunitz-type chymotrypsin inhibitor from the African hyperoliid frog, Kassina senegalensis

Amphibian skin secretions are, for the most part, complex peptidomes. While many peptide components have been biologically- and structurally-characterised into discrete "families", some of which are analogues of endogenous vertebrate regulatory peptides, a substantial number are of unique structure and unknown function. Among the components of these secretory peptidomes is an array of protease inhibitors. Inhibitors of trypsin are of widespread occurrence in different taxa and are representative of many established structural classes, including Kunitz, Kazal and Bowman-Birk. However, few protease inhibitors with activity against other specific proteases have been described from this source. Here we report for the first time, the isolation and structural characterisation of an inhibitor of chymotrypsin of Kunitz-type from the skin secretion of the African hyperoliid frog, Kassina senegalensis. To this end, we employed a functional peptidomic approach. This scheme involves fractionation of the peptidome, functional end-point screening, structural characterisation of resultant actives followed by molecular cloning of biosynthetic precursor-encoding cDNA(s). The novel mature and active polypeptide identified consisted of 62 amino acid residues (average molecular mass 6776.24 Da), of which 6 were positionally-conserved cysteines. The P(1) position within the active site was occupied by a phenylalanyl residue. Bioinformatic analysis of the sequence using BLAST, revealed a structural similarity to Kunitz-type chymotrypsin inhibitors from other organisms, ranging from silkworms to snakes.

Cellular and subcellular localization of SALMFamide (S1)-like immunoreactivity within the central nervous system of the nematode Ascaris suum (Nematoda, Ascaroidea)

The localization and distribution of SALMFamide (S1)-like immunoreactivity (IR), was determined at both the cellular and subcellular level in the central nervous system (CNS) of the nematode roundworm Ascaris suum. The techniques of indirect immunofluorescence in conjunction with confocal scanning laser microscopy and post-embedding, IgG-conjugated colloidal gold immunostaining were used, respectively. Immunostaining was widespread in the CNS of adult A. suum, with immunoreactivity (IR) being localized in nerve cells and fibres in the ganglia associated with the anterior nerve ring and in the main nerve cords and their commissures. At the subcellular level, gold labeling of peptide was localized exclusively over dense-cored vesicles within nerve cell bodies, nerve axons and nerve terminals of the neuropile of the anterior nerve ring, main ganglia and nerve cords in the CNS. Double-labeling demonstrated an apparent co-localization of S1- and FMRFamide-IR-together IR-together with S1- and pancreatic polypeptide (PP)-IR in the same dense-cored vesicles. Antigen preabsorption experiments indicated little cross-reactivity, if any, between the three antisera; indeed, neither FMRFamide nor PP antigens abolished S1 immunostaining.

SEROTONINERGIC AND PEPTIDERGIC NERVE ELEMENTS IN THE PROTOSCOLEX OF ECHINOCOCCUS-GRANULOSUS (CESTODA, CYCLOPHYLLIDEA)

The localisation and distribution of 5-hydroxytryptamine (5-HT, or serotonin) and neuropeptides in the nervous system of the protoscolex of the hydatid organism Echinococcus granulosus were determined by an indirect immunofluorescence technique. Nerve-cell bodies immunoreactive for 5-HT occurred in the lateral ganglia and in association with the lateral longitudinal nerve cords. 5-HT immunostaining was also evident in the central nerve ring, in the rostellar nerves and in the nerve plexus innervating the suckers. Of the antisera used to screen the protoscolex for neuropeptide immunoreactivity (IR), immunostaining was obtained with those raised against pancreatic polypeptide (PP), peptide YY (PYY), substance P (SP), peptide histidine isoleucine (PI-II) and vasoactive intestinal peptide (VIP). The most extensive pattern of IR occurred with antisera to PP and PYY. Immunoreactive nerve elements were evident in the lateral ganglia, central nerve ring, rostellar nerves, rostellar ganglia, sucker plexus and longitudinal nerve cords. The distribution of SP-, PHI- and VIP-IRs was more restricted: SP-IR occurred in the lateral ganglia and sucker nerves, whilst PHI- and VIP-immunoreactive nerve elements were associated with the lateral longitudinal nerve cords. Protoscoleces cultured in vitro for 29 days were also examined and neuroanatomical changes noted. A greater development of the longitudinal nerve cords and their cross-connectives in the body of the worm was evident, and a group of nerve cells were seen to develop at the posterior end of the main lateral nerve cords.

IMMUNOCYTOCHEMICAL DEMONSTRATION OF PEPTIDERGIC AND SEROTONINERGIC COMPONENTS IN THE ENTERIC NERVOUS-SYSTEM OF THE ROUNDWORM, ASCARIS-SUUM (NEMATODA, ASCAROIDEA)

The localization and distribution of neuropeptides and an indoleamine (serotonin or 5-hydroxytryptamine) in the enteric nervous system (ENS) of the pig roundworm, Ascaris suum, have been determined by the application of an indirect immunofluorescence technique in conjunction with confocal scanning laser microscopy. Whole-mount preparations of pharyngeal, intestinal and rectal regions were screened with antisera to 23 vertebrate peptides, 2 invertebrate peptides and serotonin(= 5-HT). Positive immunoreactivity (IR) was obtained with antisera to pancreatic polypeptide (PP), peptide YY (PYY), FMRFamide, gastrin and serotonin. The only IR observed in the ENS was that evident in the nerve supply to the pharynx and rectal region; no IR was associated with any region of the intestine. The most extensive patterns of IR occurred with antisera to PW, FMRFamide and serotonin. In the pharyngeal component of the ENS, IR was evident in the lateral and dorsal longitudinal pharyngeal nerves, pharyngeal commissures, nerve plexus, and associated nerve cells and fibres. In contrast, the distribution of IR to the PP and gastrin antisera was more restricted and displayed a lower intensity of immunostaining. The other component of the ENS, the rectal enteric system, only yielded immunostaining to FMRFamide. The possible role of neuropeptides and serotonin in the nutritional biology of nematodes is discussed.

IMMUNOCYTOCHEMICAL DEMONSTRATION OF NEUROPEPTIDES IN THE PERIPHERAL NERVOUS-SYSTEM OF THE ROUNDWORM ASCARIS-SUUM (NEMATODA, ASCAROIDEA)

The localisation and distribution of neuropeptides in the peripheral nervous system of the pig roundworm Ascaris suum have been determined by an indirect immunofluorescence technique in conjunction with confocal microscopy. Of the 31 antisera tested, immunostaining was obtained only with antisera to peptide YY (PYY), pancreatic polypeptide (PP) and FMRFamide. Immunostaining for PYY and FMRFamide was evident in the amphidial and papillary ganglia associated with the anterior nerve ring and in the nerves from these ganglia that terminated in sensory receptors within the buccal lips of the parasite. The only peptide immunoreactivity (IR) observed in the reproductive system of either sex was that evident in the nerve supply to the distal region of the vagina in the female worm. It took the form of a well-developed plexus of parallel nerve fibres, cross-connectives and looped commissures. The nerve net diminished in the more proximal region of the vagina. PP-IR was less intense than that for PYY and FMRFamide and was more restricted in distribution, being confined to a small number of nerve fibres in the nerve supply to the vagina; it did not occur in the nerves supplying the anterior sensory receptors. The possible roles of neuropeptides in the sensory and reproductive biology of nematodes are discussed.

IMMUNOELECTRON MICROSCOPIC STUDIES OF REGULATORY PEPTIDES IN THE NERVOUS-SYSTEM OF THE MONOGENEAN PARASITE, DICLIDOPHORA-MERLANGI

Specific antisera, directed against the highly conserved C-terminal hexapeptide amide of mammalian pancreatic polypeptide (PP) and the invertebrate peptide FMRFamide, have been used in conjunction with post-embedding, IgG-conjugated colloidal gold immunostaining to demonstrate peptide immunoreactivity at subcellular level in the nervous system of adult Diclidophora merlangi. Gold labelling revealed that immunoreactivity for PP and FMRFamide was localized exclusively in dense-cored vesicles occupying the majority of axons in the central nervous system. Double-labelling demonstrated an apparent co-localization of PP and FMRFamide in the same dense-cored vesicles. Antigen preabsorption experiments indicated cross-reactivity of the two antisera as unlikely, and that some if not all of the PP/FMRFamide immunostaining in the parasite was due to a neuropeptide F-like peptide.

IMMUNOCYTOCHEMICAL DEMONSTRATION OF NEUROPEPTIDES IN THE CENTRAL-NERVOUS-SYSTEM OF THE ROUNDWORM, ASCARIS-SUUM (NEMATODA, ASCAROIDEA)

The localization and distribution of neuropeptides in the central nervous system of the pig roundworm, Ascaris suum, have been determined by an indirect immunofluorescence technique in conjunction with confocal microscopy. Antisera to 25 vertebrate peptides and two invertebrate peptides were used to screen the worm for immunoreactivity (IR). Immunostaining was obtained with antisera to pancreatic polypeptide (PP), peptide YY (PYY), neuropeptide Y (NPY), gastrin, cholecystokinin (CCK), substance P (SP), atrial natriuretic peptide (ANP), salmon gonadotropin-releasing hormone (SGnRH), mammalian gonadotropin-releasing hormone (MGnRH), chromogranin A (CGA) and FMRFamide. The most extensive patterns of IR occurred with antisera to PYY, FMRFamide and gastrin. IR was evident in nerve cells and fibres in the ganglia associated with the anterior nerve ring and in the main nerve cords and their commissures; IR to FMRFamide also occurred in the posterior nerve ring. Immunostaining for the other peptides was confined to the nerve cords, with the number of immunoreactive nerve fibres varying from peptide to peptide.

IMMUNOCHEMICAL AND CHROMATOGRAPHIC ANALYSES OF A NEUROPEPTIDE FROM THE MONOGENEAN PARASITE, DICLIDOPHORA-MERLANGI - EVOLUTIONARY ASPECTS OF THE NEUROPEPTIDE-Y SUPERFAMILY

1. A neuropeptide exhibiting pancreatic polypeptide-immunoreactivity (PP-IR) has been isolated and characterised from the parasitic platyhelminth, Diclidophora merlangi.

IMMUNOCYTOCHEMICAL AND RADIOIMMUNOMETRICAL DEMONSTRATION OF SEROTONIN-IMMUNOREACTIVITIES AND NEUROPEPTIDE-IMMUNOREACTIVITIES IN THE ADULT-RAT TAPEWORM, HYMENOLEPIS-DIMINUTA (CESTODA, CYCLOPHYLLIDEA)

Standard indirect immunocytochemical techniques have been interfaced with confocal scanning laser microscopy (for whole-mount preparations) and epifluorescence microscopy (for cryosections) to investigate the occurrence and distribution of serotoninergic and peptidergic nerve elements in adult H. diminuta. Serotonin (5-HT)-immunoreactivity (IR) was widespread throughout the worm, occurring in the paired cerebral ganglia, transverse commissure, the 10 longitudinal nerve cords and in a plethora of small nerve fibres of the peripheral nervous system. An abundance of serotoninergic nerve cell bodies was found in association with the lateral nerve cords. The genital atrium and accessory reproductive ducts were richly innervated with serotoninergic nerve fibres. Thirty-five antisera to 20 vertebrate regulatory peptides and 1 invertebrate peptide (FMRFamide) were used to screen the worm for neuropeptide IR. Immunostaining was obtained with antisera raised to pancreatic polypeptide (PP), peptide YY (PYY), neuropeptide Y (NPY), substance P (SP), peptide histidine isoleucine (PHI), xenopsin (XP) and FMRFamide. The most extensive pattern of IR occurred with antisera to PP and PYY, IR being evident in the cerebral ganglia, transverse commissure, longitudinal nerve cords and in small nerve fibres that ramified throughout the parenchyma. A series of bipolar nerve cell bodies between the median nerve cords displayed PP/PYY-IR. The distribution of FMRFamide-IR was reminiscent of the PP/PYY pattern but was less extensive. Comparison of the serotoninergic and peptidergic nervous systems has revealed general similarities and some distinct differences, especially with regard to the distribution of immunoreactive nerve cell bodies. Quantitative data are presented on the levels of PP-, SP-, PHI-, and gastrin-releasing peptide (GRP)-immunoreactivities demonstrable in acid-alcohol extracts of whole worms. The highest level of peptide IR determined was recorded for PP.

NEUROPEPTIDE-F - A NOVEL PARASITIC FLATWORM REGULATORY PEPTIDE FROM MONIEZIA-EXPANSA (CESTODA, CYCLOPHYLLIDEA)

Using a C-terminally directed pancreatic polypeptide (PP) antiserum and immunocytochemical methods, PP-immunoreactivity (IR) was localized throughout the central (CNS) and peripheral nervous systems (PNS) of the cestode, Moniezia expansa. In the CNS, immunostaining was evident in the paired cerebral ganglia (primitive brain), connecting commissure, and the paired longitudinal nerve cords that are cross-linked by numerous regular transverse connectives. The PNS was seen to consist of a fine anastomosing nerve-net of immunoreactive fibres, many of which were closely associated with reproductive structures. Radioimmunoassay of this peptide IR in acid-alcohol extracts of the worm measured 192.8 ng/g of PP-IR. HPLC analyses of the M. expansa PP-IR identified a single molecular form which was purified to homogeneity. Plasma desorption mass spectrometry (PDMS) of purified parasite peptide resolved a single peptide with a molecular mass of 4599 +/- 10 Da. Automated gas-phase Edman degradation identified a 39-amino acid peptide with a C-terminal phenylalaninamide. Examination of its primary structure shows that it displays significant sequence homology with the vertebrate neuropeptide Y superfamily, suggesting that this platyhelminth-derived peptide is the phylogenetic precursor. Neuropeptide F (M. expansa) is the first regulatory peptide to be fully sequenced from the phylum Platyhelminthes and may represent a member of an important new class of invertebrate neuropeptide.

CYTOCHEMICAL DEMONSTRATION OF CHOLINERGIC, SEROTONINERGIC AND PEPTIDERGIC NERVE ELEMENTS IN GORGODERINA-VITELLILOBA (TREMATODA, DIGENEA)

Standard enzyme cytochemical and indirect immunocytochemical techniques have been used in conjunction with light and confocal scanning laser microscopy (CSLM) to visualize cholinergic, serotoninergic and peptidergic nerve elements in whole-mount preparations of the amphibian urinary-bladder fluke, Gorgoderina vitelliloba. Cholinesterase (ChE) activity was localized in paired anterior ganglia, a connecting dorsal commissure and in the origins of the ventral nerve cords. Cholinergic ganglia were also evident in shelled embryos in the uterus. Serotonin-immunoreactivity (IR) was more extensive than ChE activity and was identified in both the central and peripheral nervous systems. Serotoninergic nerve fibres were associated with the somatic musculature and female reproductive ducts. Antisera to nine mammalian peptides and one invertebrate (FMRFamide) peptide have been used to investigate the peptidergic nervous system in the parasite. Immunoreactivity was obtained to five peptides, namely pancreatic polypeptide (PP), peptide YY (PYY), neuropeptide Y (NPY), substance P (SP) and FMRFamide. Peptidergic nerve fibres were found to be more abundant than demonstrable cholinergic or serotoninergic nerve fibres. NPY-IR was identified only in the main components of the central nervous system. However, PP- and PYY-IR occurred in the anterior ganglia, dorsal commissure, main nerve cords and in numerous small varicose fibres that ramified throughout the worm. Additionally, PP-immunoreactive nerve fibres were found to innervate the musculature of the female reproductive tracts. Six sites of IR were found in the acetabulum, using antisera directed towards the C-terminal end of PP and PYY, and these matched with the distribution of six non-ciliated rosette-like papillae observed by scanning electron microscopy. SP- and FMRFamide-IR were identified in the CNS, and FMRFamide-immunopositive nerve fibres were also evident in association with the gonopore/cirrus region and with the terminal excretory pore. Results are discussed with respect to possible roles for each of the neurochemical types.