Valeria Sibilia

Whey proteins as source of dipeptidyl dipeptidase IV (dipeptidyl peptidase-4) inhibitors

Preclinical and clinical studies suggest that whey proteins can reduce postprandial glucose levels and stimulate insulin release in healthy subjects and in subjects with type 2 diabetes by reducing dipeptidyl peptidase-4 (DPP-4) activity in the proximal bowel and hence increasing intact incretin levels. Our aim was to identify DPP-4 inhibitors among short peptides occurring in hydrolysates of β-lactoglobulin, the major whey protein found in the milk of ruminants. We proved that the bioactive peptide Ile-Pro-Ala can be regarded as a moderate DPP-4 inhibitor.

Evidence for a Central Inhibitory Role of Growth Hormone Secretagogues and Ghrelin on Gastric Acid Secretion in Conscious Rats

We examined the possible central and peripheral effects of synthetic growth hormone secretagogues (GHS), hexarelin (Hexa) and EP 40737 (D-Thr-D-Trp (2-Me)-Ala- Trp-D-Phe-Lys-NH2), and of their endogenous counterpart, ghrelin, on gastric acid secretion. The compounds were administered intracerebroventricularly (i.c.v.) or subcutaneously (s.c.) in conscious male rats and the volume of gastric secretion and gastric acid output were examined 3 h after pylorus ligation (Shay-test). Central Hexa, EP 40737 and ghrelin administration (from 0.1 pmol to 1 nmol/rat, i.c.v.) significantly inhibited gastric acid secretion. The maximum inhibitory effect on gastric acid output was detected at the dose of 10 pmol/rat, i.c.v. for Hexa (–51.3%), of 100 pmol/rat, i.c.v. for EP 40737 (–70%) and of 1 pmol/rat, i.c.v. for ghrelin (–60%). All peptides were less effective at the highest dose used (1 nmol/rat, i.c.v.). Hexa, EP 40737 and ghrelin injected s.c. did not modify gastric acid secretion. The inhibitory action of Hexa on gastric acid secretion seems to involve brain somatostatinergic system since Hexa (10 pmol/rat, i.c.v.) did not inhibit gastric acid secretion in rats pretreated (4 h before) with cysteamine (300 mg/kg, s.c.), a depletor of endogenous somatostatin. These results show that synthetic GHS and ghrelin exert a central long-lasting inhibitory effect on gastric acid secretion in conscious pylorus-ligated rats. The fact that very low doses of ghrelin and GHS inhibit gastric secretion, provide evidence for a tonic inhibitory role of the peptides in the central control of gastric secretory function.

Protection by amylin of gastric erosions induced by indomethacin or ethanol in rats

1 The effect of amylin on gastric ulcers induced by oral administration of indomethacin (Indo, 20 mg kg−1 at a dosing volume of 5 ml) or ethanol 50% (EtOH, 1 ml/rat) was investigated in conscious rats. 2 Amylin given intracerebroventricularly (0.22, 0.66 and 2.2 μg/rat, i.c.v.) demonstrated a dose‐dependent cytoprotective effect against both Indo and EtOH‐induced ulcers. In contrast, amylin, given subcutaneously at doses effective in inhibiting acid gastric secretion (2.5, 10 and 40 μg kg−1, s.c), did not show any cytoprotective effect. 3 The interaction between amylin and endogenous nitric oxide (NO) in the maintenance of gastric mucosal integrity was investigated by pretreating the rats with a selective inhibitor of NO‐synthesis, NG‐nitro‐L‐arginine methyl ester (l‐NAME, 25 and 70 mg kg−1, s.c). Administration of L‐NAME to rats did not significantly increase the degree of the Indo‐induced ulcer index and was not able to remove the protective effect of amylin on Indo‐induced ulcers, thus excluding a role for endogenous NO in mediating the protective effect of this peptide. 4 To determine whether the cytoprotective effect of amylin was mediated by endogenous prostaglandins, we studied the effect of amylin (2.2 μg/rat, i.c.v.) on EtOH‐induced ulcers in rats pretreated with Indo (10 mg kg−1, s.c.) to inhibit prostanoid biosynthesis; Indo was injected 30 min before amylin and EtOH after a further 30 min. Pretreatment with Indo did not significantly increase the ulcer index induced by EtOH but counteracted the ability of amylin to prevent the ulcer formation. 5 These findings suggest that amylin exerts a gastroprotective activity that is not strictly related to inhibition of acid gastric secretion and can be partly explained through a prostaglandin‐dependent mechanism mediated by receptors for the peptide in the brain. Amylin might be considered as a new brain‐gut peptide.

Ghrelin inhibits inflammatory pain in rats: Involvement of the opioid system

This study examined the effects of intracerebroventricular (i.c.v.), intraperitoneal (i.p.) or intraplantar (i.pl.) administration of ghrelin, the endogenous ligand of the growth hormone secretagogue receptor, in the development of hyperalgesia and edema induced by intraplantar injection of carrageenan in rats. Central ghrelin (4 ng to 4 microg/rat) given 5 min before carrageenan produced a dose-related reversal of carrageenan-induced mechanical hyperalgesia measured by Randall-Selitto test with an ED50 of 81.7 ng/rat. Ghrelin at the dose of 4 microg/rat i.c.v. was also effective in inhibiting edema. When ghrelin (4 microg/rat i.c.v.) was administered 150 min after carrageenan, it failed to modify either hyperalgesia or the paw volume. Given i.p., 30 min before carrageenan, ghrelin (20-160 microg/kg) induced a significant dose-dependent inhibition of hyperalgesia with an ED50 of 77 microg/kg and a slight reduction of edema. Intraplantar ghrelin (40 ng to 12 microg/rat) did not significantly modify both the hyperalgesic and edematous activities of carrageenan. The anti-hyperalgesic and anti-edematous effects of ghrelin (4 microg/rat i.c.v.) were reversed by naloxone (10 microg/rat i.c.v.). Systemic administration of the peripheral selective opioid antagonist, naloxone methiodide (3 mg/kg s.c.), did not antagonize antinociception elicited by i.p. ghrelin. Overall these data indicate that ghrelin exerts an inhibitory role on inflammatory pain through an interaction with the central opioid system.

Central ghrelin gastroprotection involves nitric oxide/prostaglandin cross-talk

Ghrelin, a gut‐brain peptide, is considered a gastroprotective factor in gastric mucosa. We investigated the role of prostaglandins (PG) and the possible interplay between PGs and nitric oxide (NO) in ghrelin gastroprotection against ethanol (EtOH)‐induced gastric lesions.

Intracerebroventricular acute and chronic administration of obestatin minimally affect food intake but not weight gain in the rat.

We studied the effect of the acute central administration of obestatin on food intake and body weight in short-term starved male rats, and those of 28-day continuous intracerebroventricular (icv) infusion of obestatin in free feeding rats. In 16-h starved rats, obestatin induced a trend toward a reduction of food intake that did not reach statistical significance. In fed rats, the icv infusion of obestatin significantly decreased food consumption in the first day of treatment; but the anorexigenic effect of obestatin vanished thereafter. Interestingly, the body weight of rats infused for 28 days with obestatin was superimposable to that of the respective control at all time intervals. In all, our results indicate that the anorexigenic effect of obestatin is of little account and that the peptide does not modify energy metabolism in the long-term administration.

Calcitonin gene-related peptide: antinociceptive activity in rats, comparison with calcitonin

The effects of synthetic human calcitonin gene-related peptide (CGRP) on nociceptive response were evaluated in rats by two behavioral tests (tail-flick and hot-plate) and by electrophysiological recording of the firing of thalamic neurons evoked by peripheral noxious mechanical stimuli. CGRP was administered intracerebroventricularly (i.c.v.) and its effects were compared with that of salmon calcitonin (sCT). In the tail-flick test, CGRP (0.25, 2.5 and 5 micrograms/rat) dose-dependently increased response latencies, whereas sCT (0.125, 2.5, 5 and 10 micrograms/rat) did not. Conversely, in the hot-plate test CGRP was effective in enhancing response latencies only at the highest dose of 10 micrograms/rat, while sCT (0.125, 0.25 and 2.5 micrograms/rat) inhibited the hot-plate response dose-dependently. In electrophysiological studies, CGRP (2.5 micrograms/rat, i.c.v.) completely inhibited the evoked neuronal thalamic firing and the same dose of sCT induced only a partial reduction. Furthermore, the antinociceptive effects of CGRP in the tail-flick test and in the electrophysiological studies were not prevented by naloxone. These results demonstrate that central administration of CGRP is effective in inhibiting nociceptive responses and its action like that of sCT does not involve an opioid mechanism. The differences in the antinociceptive profiles of CGRP and sCT suggest that the inhibitory effects of these peptides may involve different neuronal pathways.

Endocrine disrupters: the new players able to affect the epigenome

Epigenetics represents the way by which the environment is able to program the genome; there are three main levels of epigenetic control on genome: DNA methylation, post-translational histone modification and microRNA expression. The term Epigenetics has been widened by NIH to include “both heritable changes in gene activity and expression but also stable, long-term alterations in the transcriptional potential of a cell that are not necessarily heritable.” These changes might be produced mostly by the early life environment and might affect health influencing the susceptibility to develop diseases, from cancer to mental disorder, during the entire life span. The most studied environmental influences acting on epigenome are diet, infections, wasting, child care, smoking and environmental pollutants, in particular endocrine disrupters (EDs). These are environmental xenobiotics able to interfere with the normal development of the male and female reproductive systems of wildlife, of experimental animals and possibly of humans, disrupting the normal reproductive functions. Data from literature indicate that EDs can act at different levels of epigenetic control, in some cases transgenerationally, in particular when the exposure to these compounds occurs during the prenatal and earliest period of life. Some of the best characterized EDs will be considered in this review. Among the EDs, vinclozolin (VZ), and methoxychlor (MXC) promote epigenetic transgenerational effects. Polychlorinated biphenils (PCBs), the most widespread environmental EDs, affect histone post-translational modifications in a dimorphic way, possibly as the result of an alteration of gene expression of the enzymes involved in histone modification, as the demethylase Jarid1b, an enzyme also involved in regulating the interaction of androgens with their receptor.

Evidence for a Role of the GHS‐R1a Receptors in Ghrelin Inhibition of Gastric Acid Secretion in the Rat

Ghrelin, the endogenous ligand of the GH secretagogue receptor (GHS‐R) has been previously shown to inhibit gastric acid secretion in pylorus‐ligated rats. Two isoforms of GHS‐R have been identified: GHS‐R1a and GHS‐R1b. The present study aimed: (i) to characterise the type of GHS‐R involved in the central gastric inhibitory activity of ghrelin by using des‐octanoyl ghrelin, and synthetic GHS‐R1a agonist (EP1572) and antagonist (D‐Lys3‐GHRP‐6) and (ii) to investigate the relationship between ghrelin and cortistatin (CST) in the control of gastric acid secretion by using the natural neuropeptide CST‐14 and the synthetic octapeptide CST‐8. The specific interactions of all the compounds with GHS‐R1a were determined by comparing their ability to displace labelled ghrelin or somatostatin from its receptors on rat hypothalamic membranes or on rat cardiomyocyte, respectively. Intracerebroventricular administration of 0.01 and 1 nmol/rat des‐octanoyl ghrelin did not affect gastric acid secretion in pylorus‐ligated rats, whereas EP1572 either i.c.v. (0.01–1 nmol/rat) or i.p. (10 and 20 nmol/kg) inhibited acid gastric secretion. Preteatment with D‐Lys3GHRP‐6 (3 nmol/rat, i.c.v.) was able to remove the inhibitory action of ghrelin (0.01 nmol/rat, i.c.v.) on gastric acid volume and acid output, thus indicating that the type 1a GHS‐R likely mediates the gastric inhibitory action of ghrelin. This is supported by binding data showing that D‐Lys3GHRP‐6, but not des‐octanoyl ghrelin, binds to hypothalamic GHS‐R. CST‐14 (1 nmol/rat, i.c.v.) did not affect either basal or ghrelin inhibition of gastric acid secretion. CST‐8 (1 nmol/rat, i.c.v.) was able to counteract the gastric ghrelin response. The observation that CST‐14 binds both GHR‐S and somatostatin receptors, whereas CST‐8 specifically displaces only ghrelin binding, indicates that CST‐8 behaves as a GHS‐R1a antagonist.

Eel calcitonin binding site distribution and antinociceptive activity in rats

The distribution of binding site for [125I]-eel-calcitonin (ECT) to rat central nervous system, studied by an autoradiographic technique, showed concentrations of binding in the diencephalon, the brain stem and the spinal cord. Large accumulations of grains were seen in the hypothalamus, the amygdala, in the fasciculus medialis prosencephali, in the fasciculus longitudinalis medialis, in the ventrolateral part of the periventricular gray matter, in the lemniscus medialis and in the raphe nuclei. The density of grains in the reticular formation and in the nucleus tractus spinalis nervi trigemini was more moderate. In the spinal cord, grains were scattered throughout the dorsal horns. Binding of the ligand was displaced equally by cold ECT and by salmon CT(sCT), indicating that both peptides bind to the same receptors. Human CT was much weaker than sCT in displacing [125I]-ECT binding. The administration of ECT into the brain ventricles of rats dose-dependently induced a significant and long-lasting enhancement of hot-plate latencies comparable with that obtained with sCT. The antinociceptive activity induced by ECT is compatible with the topographical distribution of binding sites for the peptide and is a further indication that fish CTs are active in the mammalian brain.