Elena Bresciani

Obestatin inhibits feeding but does not modulate GH and corticosterone secretion in the rat

Obestatin is a recently discovered 23 amino acids peptide derived from the ghrelin gene. As opposed to ghrelin, obestatin was shown to inhibit food intake in mice. The aims of this research were to study the effects of acute obestatin treatment on feeding behavior in the rat and its effects on GH and corticosterone secretion. Our results demonstrate that in young-adult male rats, obestatin effectively blunts the hunger caused by short-term starvation. Obestatin did not modify GH secretion in 10-day-old rats and did not antagonize the GH-releasing effects of hexarelin. Moreover, obestatin administration had no effects on spontaneous corticosterone secretion. In conclusion, these data demonstrate that in young-adult male rats the newly discovered obestatin can inhibit feeding but does not modify GH and corticosterone release in infant rats.

Acute and late changes in intraarticular cytokine levels following anterior cruciate ligament injury.

Surgical reconstruction of the anterior cruciate ligament (ACL) does not necessarily decrease the risk of developing osteoarthritis (OA). The inflammatory response and relative changes in pro‐ and anti‐inflammatory cytokines could participate in triggering the development of OA. To test this hypothesis we measured the concentrations of IL‐1β, IL‐1ra, IL‐6, IL‐8, IL‐10, and TNF‐α at different times after ACL rupture. The sample population consisted of 48 patients with ACL tear which were assigned to different groups according to the time elapsed from the injury: 22 acute (A), 7 early sub‐acute (ESA), 11 late sub‐acute (LSA), and 8 chronic (C). In group A, there were high levels of IL‐1β, IL‐6, and IL‐8, whereas levels of IL‐1ra and TNF‐α were significantly lower than usually reported. IL‐1β and IL‐8 concentrations returned with time to normal levels in the ESA group. Interestingly, IL‐1ra levels remained always significantly lower than normally reported levels, and TNF‐α levels did not increase after trauma. Our data show increased level of pro‐inflammatory cytokines (IL‐6 and IL‐8) in the acute phase of inflammation which could be responsible for triggering cartilage catabolism and suggest that prompt neutralization of IL‐6 and IL‐8 accumulations in synovial fluid could help prevent development of OA in ACL‐injured knees.

Desacyl-ghrelin and synthetic GH-secretagogues modulate the production of inflammatory cytokines in mouse microglia cells stimulated by β-amyloid fibrils

Data from Alzheimers disease (AD) patients and AD animal models demonstrate the accumulation of inflammatory microglia at sites of insoluble fibrillar β‐amyloid protein (fAβ) deposition. It is known that fAβ binds to CD36, a type B scavenger receptor also involved in internalization of oxidized low‐density lipoprotein (LDL), and initiate a signaling cascade that regulates microglial recruitment, activation, and secretion of inflammatory mediators leading to neuronal dysfunction and death. The recent demonstration of a binding site for the growth hormone secretagogues (GHS) on CD36 prompted us to ascertain whether ghrelin and synthetic GHS could modulate the synthesis of inflammatory cytokines in fAβ‐activated microglia cells. We demonstrate that N9 microglia cells express the CD36 and are a suitable model to study the activation of inflammatory cytokines synthesis. In fact, in N9 cells exposed to fAβ25–35 for 24 hr, the expression of interleukin (IL)‐1β and IL‐6 mRNA significantly increased. Interestingly, 10−7 M desacyl‐ghrelin, hexarelin, and EP80317 in the nanomolar range effectively counteracted fAβ25–35 stimulation of IL‐6 mRNA levels, whereas ghrelin was ineffective. Similarly, the effects of fAβ25–35 on IL‐1β mRNA levels were attenuated by desacyl‐ghrelin, hexarelin, and EP80317, but not ghrelin. Because we have observed that the specific GHS receptor GHS‐R1a is not expressed in N9 cells, the actions of GHS should be mediated by different receptors. Reportedly, hexarelin and EP80317 are capable of binding the CD36 in mouse macrophages and reducing atherosclerotic plaque deposition in mice. We conclude that desacyl‐ghrelin, hexarelin, and EP80317 might interfere with fAβ activation of CD36 in microglia cells.

Characterization of a novel peripheral pro-lipolytic mechanism in mice: role of VGF-derived peptide TLQP-21.

The peptides encoded by the VGF gene are gaining biomedical interest and are increasingly being scrutinized as biomarkers for human disease. An endocrine/neuromodulatory role for VGF peptides has been suggested but never demonstrated. Furthermore, no study has demonstrated so far the existence of a receptor-mediated mechanism for any VGF peptide. In the present study, we provide a comprehensive in vitro, ex vivo and in vivo identification of a novel pro-lipolytic pathway mediated by the TLQP-21 peptide. We show for the first time that VGF-immunoreactivity is present within sympathetic fibres in the WAT (white adipose tissue) but not in the adipocytes. Furthermore, we identified a saturable receptor-binding activity for the TLQP-21 peptide. The maximum binding capacity for TLQP-21 was higher in the WAT as compared with other tissues, and selectively up-regulated in the adipose tissue of obese mice. TLQP-21 increases lipolysis in murine adipocytes via a mechanism encompassing the activation of noradrenaline/β-adrenergic receptors pathways and dose-dependently decreases adipocytes diameters in two models of obesity. In conclusion, we demonstrated a novel and previously uncharacterized peripheral lipolytic pathway encompassing the VGF peptide TLQP-21. Targeting the sympathetic nerve-adipocytes interaction might prove to be a novel approach for the treatment of obesity-associated metabolic complications.

Ontogeny and Tissue-Specific Regulation of Ghrelin mRNA Expression Suggest that Ghrelin Is Primarily Involved in the Control of Extraendocrine Functions in the Rat

Ghrelin is a 28-amino-acid gastric peptide that potently stimulates growth hormone (GH) secretion in vivo and in vitro. Ghrelin-expressing cells have been found in the oxyntic region of the stomach and in the arcuate nucleus of the hypothalamus. The aim of this work was to investigate the regional distribution and developmental changes in ghrelin mRNA levels in the pituitary, hypothalamus and gastrointestinal (GI) tract of the rat using a semiquantitative RT-PCR assay. We also describe the effects of ghrelin immunoneutralization in late gestation and those resulting from induction of an isolated GH deficiency in adult rats. Ghrelin mRNA was already expressed in the fetus by embryonic day 12 (E12), by E17 most of ghrelin mRNA was in the trunk. At E17, in situ hybridization did not reveal a clear expression of ghrelin mRNA in fetal stomach but showed high ghrelin mRNA levels in the placenta. In the pituitary gland, levels of ghrelin mRNA were high after birth but declined significantly with puberty, whereas in the hypothalamus they were barely detectable at birth and remained very low at all subsequent time points tested. In the GI tract, ghrelin mRNA levels were high from birth to 270 days of life. Immunoneutralization of ghrelin at E16 had no effect on survival or development. Rats showed normal somatotropic function, ghrelin expression and onset of puberty. In young adult rats, passive immunization against GHRH did not affect ghrelin mRNA levels in the pituitary, hypothalamus and stomach. Only a 72-hour fasting period induced a significant increase in ghrelin mRNA levels in the stomach, but not in the pituitary and hypothalamus. These results strongly indicate that ghrelin is an important GI hormone expressed early in life and primarily sensitive to nutritional status.

Protective but Not Anticonvulsant Effects of Ghrelin and JMV-1843 in the Pilocarpine Model of Status epilepticus

In models of status epilepticus ghrelin displays neuroprotective effects mediated by the growth hormone secretagogue-receptor 1a (GHS-R1a). This activity may be explained by anticonvulsant properties that, however, are controversial. We further investigated neuroprotection and the effects on seizures by comparing ghrelin with a more effective GHS-R1a agonist, JMV-1843. Rats were treated either with ghrelin, JMV-1843 or saline 10 min before pilocarpine, which was used to induce status epilepticus. Status epilepticus, developed in all rats, was attenuated by diazepam. No differences were observed among the various groups in the characteristics of pilocarpine-induced seizures. In saline group the area of lesion, characterized by lack of glial fibrillary acidic protein immunoreactivity, was of 0.45±0.07 mm2 in the hippocampal stratum lacunosum-moleculare, and was accompanied by upregulation of laminin immunostaining, and by increased endothelin-1 expression. Both ghrelin (P<0.05) and JMV-1843 (P<0.01) were able to reduce the area of loss in glial fibrillary acidic protein immunostaining. In addition, JMV-1843 counteracted (P<0.05) the changes in laminin and endothelin-1 expression, both increased in ghrelin-treated rats. JMV-1843 was able to ameliorate neuronal survival in the hilus of dentate gyrus and medial entorhinal cortex layer III (P<0.05 vs saline and ghrelin groups). These results demonstrate diverse protective effects of growth hormone secretagogues in rats exposed to status epilepticus.

Central Nervous System-Acting Drugs Influencing Hypothalamic-Pituitary-Adrenal Axis Function

The hypothalamic-pituitary-adrenal (HPA) axis is a major integrated system that maintains body homeostasis by regulating the neuroendocrine and sympathetic nervous systems and modulating immune function. It is well established that the central nervous system (CNS) integrates responses to different stimuli secreting a specific corticotropin-releasing hormone (CRH) and vasopressin (AVP). In turn, they stimulate the release of ACTH, which induces steroidogenesis in the adrenal gland. The HPA axis is regulated by diurnal rhythms and negative feedback by corticosteroids. Many neurotransmitters and neuropeptides are responsible for the modulation of CRH and AVP neurons. In addition to synthetic glucocorticoids that inhibit the HPA axis, GABA agonists, opioid peptides and endocannabinoids can inhibit cortisol secretion. On the contrary, serotonin, norepinephrine, dopamine, acetylcholine, ghrelin, angiotensin II and different cytokines can stimulate it. It follows that a number of neuroactive drugs, acting as agonists or antagonists on several brain neurotransmitter or neuropeptide receptors, can influence ACTH/cortisol secretion thereby interfering with clinical testing of the functionality of the HPA axis.

Chronic intracerebroventricular injection of TLQP-21 prevents high fat diet induced weight gain in fast weight-gaining mice

The vgf gene regulates energy homeostasis and the VGF-derived peptide TLQP-21 centrally exerts catabolic effects in mice and hamsters. Here, we investigate the effect of chronic intracerebroventricular (icv) injection of TLQP-21 in mice fed high fat diet (HFD). Fast weight-gaining mice injected with the peptide or cerebrospinal fluid were selected for physiological, endocrine, and molecular analysis. TLQP-21 selectively inhibited the increase in body weight and epididymal white adipose tissue (eWAT) weight induced by HFD in control animals despite both groups having a similar degree of hyperphagia. TLQP-21 normalized the increase in leptin and decrease in ghrelin while increasing epinephrine and epinephrine/norepinephrine ratio when compared to values in controls. Finally, HFD-TLQP-21 mice showed a selective increase of eWAT β3-adrenergic receptor mRNA. Peroxisome-proliferator-activated-receptor-δ and hormone-sensing-lipase mRNA were also upregulated. In conclusion, chronic icv infusion of TLQP-21 prevented the early phase of diet-induced obesity despite overfeeding. These effects were paralleled by activation of catabolic pathways within the eWAT. Our results further support a role for TLQP-21 as a catabolic neuropeptide.

Beneficial effects of desacyl-ghrelin, hexarelin and EP-80317 in models of status epilepticus

It has been reported that ghrelin exerts anticonvulsive effects in models of epilepsy. In this study we aimed to characterize the anticonvulsive activity of ghrelin and other growth hormone secretagogue receptor 1a (GHSR(1a)) ligands in rats exposed to status epilepticus induced by pilocarpine or kainate. Firstly, in three independent experiments, before receiving pilocarpine (380 mg/kg, i.p.), rats were pretreated with one among ghrelin (1.5mg/kg), desacyl-ghrelin (1.5mg/kg), hexarelin (330 μg/kg), EP-80317 (330 μg/kg), JMV-1843 (330 μg/kg), JMV-2959 (330 μg/kg) or saline. Secondly, in the fourth experiment, rats were pretreated with i.p. ghrelin, desacyl-ghrelin, hexarelin, EP-80317 or saline, followed by kainate (15 mg/kg, i.p.). We evaluated: induction of generalized seizures, latency to generalized seizures, status epilepticus, latency to status epilepticus (the time lag between the first tonic-clonic convulsion and the switch to continuous seizures) and mortality. In the pilocarpine model, 60% of rats pretreated with EP-80317 (P<0.05) showed no seizure. Hexarelin and EP-80317 were both able to prevent progression to status epilepticus in pilocarpine-treated rats (P<0.05). When status epilepticus was induced by kainate, seizures developed with few exceptions. However, latency to status epilepticus was significantly (P<0.01) longer in rats pretreated with desacyl-ghrelin, whereas hexarelin and EP-80317 did not display any effect. Almost all GHSR(1a) ligands prevented pilocarpine-induced mortality, which was observed only in rats pretreated with saline or JMV-2959. After kainate administration, all rats survived to status epilepticus. These findings demonstrate that desacyl-ghrelin, hexarelin and EP-80317 but not other GHSR(1a) ligands display relevant anticonvulsive properties in models of limbic seizures.

Central dysregulations in the control of energy homeostasis and endocrine alterations in anorexia and bulimia nervosa

In the last decades we have come to understand that the hypothalamus is a key region in controlling energy homeostasis. A number of control models have been proposed to explain the regulation of feeding behavior in physiological and pathological conditions, but all those based on imbalances of single factors fail to explain the disrupted regulation of energy supply in eating disorders such as anorexia nervosa and bulimia nervosa, as well as other psychiatric disorders. A growing amount of evidence demonstrates that many signaling molecules originated within the brain or coming from the adipose tissue or the gastro-enteric tract are involved in the highly complex process controlling food intake and energy expenditure. The recent discovery of leptin, ghrelin, and other factors have made it possible to penetrate in the still undefined pathophysiology of eating disorders with the hope of finding effective treatments for such diseases.