Muhtashan S. Mondal

Identification of ghrelin and its receptor in neurons of the rat arcuate nucleus

Ghrelin, an acylated peptide originally identified in rat stomach as the endogenous ligand for the growth hormone secretagogue receptor (GHS-R), stimulates both food intake and growth hormone (GH) secretion. Ghrelin is predominantly synthesized by a subset of endocrine cells in the oxyntic gland of human and rat stomach. Previous studies using immunohistochemistry have shown that ghrelin is also present in the hypothalamic arcuate nucleus, a region critical for the control of feeding and GH secretion, but its expression pattern in this region and the details of its molecular form has yet to be clarified. In this report, we examined the presence of ghrelin in the arcuate nucleus using reverse-phase liquid chromatography combined with radioimmunoassay (RIA) and immunohistochemistry. Neurons in the arcuate nucleus were observed to react positively to ghrelin antibodies. In addition, we confirmed the existence of ghrelin mRNA expression using the reverse-transcription polymerase chain reaction (RT-PCR). We also observed the colocalization of GHS-R with neuropeptide Y (NPY) and growth-hormone-releasing hormone (GHRH) in the arcuate nucleus. The present study clearly indicates that ghrelin is synthesized in the arcuate nucleus, which will further our understanding of ghrelins actions in the central nervous system, including feeding behavior and GH secretion.

Distribution of orexin/hypocretin in the rat median eminence and pituitary

We determined the distribution of orexin-A and orexin-B (also known as hypocretin-1 and hypocretin-2) and their receptors in the rat median eminence and pituitary using sensitive radioimmunoassays coupled with high-performance liquid chromatography, immunohistochemistry, and in situ hybridization. Orexin-A and -B were present in the median eminence, adenohypophysis, and neurohypophysis. Orexin fibers were abundant in the median eminence, and a few fibers projected to the neurohypophysis. Both the orexin(1)- and orexin(2)-receptor mRNAs were expressed robustly in the pituitary intermediate lobe, whereas in the anterior lobe, the orexin(1) receptor was more markedly expressed than the orexin(2) receptor. These two receptor mRNAs were also found in the posterior lobe. These findings may implicate orexins involvement in additional as yet undefined physiological functions in the hypothalamo-hypophyseal tract.

Somatostatin suppresses ghrelin secretion from the rat stomach.

Ghrelin is an acylated peptide that stimulates food intake and the secretion of growth hormone. While ghrelin is predominantly synthesized in a subset of endocrine cells in the oxyntic gland of the human and rat stomach, the mechanism regulating ghrelin secretion remains unknown. Somatostatin, a peptide produced in the gastric oxyntic mucosa, is known to suppress secretion of several gastrointestinal peptides in a paracrine fashion. By double immunohistochemistry, we demonstrated that somatostatin-immunoreactive cells contact ghrelin-immunoreactive cells. A single intravenous injection of somatostatin reduced the systemic plasma concentration of ghrelin in rats. Continuous infusion of somatostatin into the gastric artery of the vascularly perfused rat stomach suppressed ghrelin secretion in both dose- and time-dependent manner. These findings indicate that ghrelin secretion from the stomach is regulated by gastric somatostatin.

Central administration of ghrelin preferentially enhances fat ingestion

Ghrelin, a brain-gut peptide discovered from the stomach, stimulates growth hormone release, food intake, adiposity, and weight gain. Circulating ghrelin levels are modulated under conditions of positive and negative energy balance, however its effect on macronutrient selection is not known. The present experiment investigates the effect of ghrelin on single and two-diet feeding paradigms in high-carbohydrate (HC) and high-fat (HF) preferring rats. In the macronutrient selection test in which rats were given free access to either high-carbohydrate or high-fat diet, an intracerebroventricular (i.c.v.) administration of ghrelin potently enhanced fat intake over carbohydrate intake in both HC- and HF-preferring rats. In the diet preference test in which rats were given free access to both high-carbohydrate and high-fat diets simultaneously, an i.c.v. administration of ghrelin also preferentially enhanced fat consumption over carbohydrate in both HF- and HC-preferring rats. Intracerebroventricular administrations of galanin and neuropeptide Y enhanced fat and carbohydrate ingestion, respectively. Centrally administered ghrelin enhanced fat ingestion. These results provide further insights for the role of ghrelin in feeding behavior and the development of obesity.

Distribution of orexin-A and orexin-B (hypocretins) in the rat spinal cord.

Orexin-A and orexin-B (also known as hypocretin-1 and hypocretin-2) are hypothalamic peptides that regulate feeding behavior, energy metabolism, and sleep-wake cycle. We determined the distribution of orexin-A and -B in the rat spinal cord, using sensitive radioimmunoassays and an immunohistochemical technique with three antisera specific for these orexins. Orexins were distributed throughout the spinal cord, and their contents were highest in the cervical region. Orexin fibers were concentrated in lamina I of the dorsal horn and area X surrounding the central canal of the spinal cord. Abundant orexin fibers also were present in the preganglionic sympathetic and parasympathetic cell columns. These findings suggest that orexins in the spinal cord may be involved in the modulation of sensory informations and the autonomic system as neurotransmitters or neuromodulators, both.

Impact of Helicobacter pylori infection on gastric and plasma ghrelin dynamics in humans.

OBJECTIVES:There are contradictory reports on the relationship between Helicobacter pylori and circulating ghrelin. We sought to clarify the influence of H. pylori infection on gastric and plasma ghrelin dynamics in humans.METHODS:Using endoscopic biopsies from the corpus of 56 H. pylori-infected patients and 25 uninfected subjects, ghrelin mRNA expression levels and gastric ghrelin peptide contents were measured by real-time polymerase chain reaction and radioimmunoassay, respectively. We also measured plasma ghrelin concentrations and analyzed the numbers of ghrelin immunoreactive cells in the fundic gland area. Fifty-one patients with H. pylori infection were treated with a 7-day triple therapy consisting of lansoprazole, clarithromycin, and amoxicillin.RESULTS:The gastric ghrelin mRNA expression level of H. pylori-positive patients (1.64 ± 1.27 in arbitrary units) was significantly lower than in H. pylori-negative subjects (4.87 ± 4.1, p < 0.0001). A similar trend was noted for ghrelin peptide contents (31.2 ± 27.5 vs 81.2 ± 64.1 ng/mg protein, respectively, p < 0.0001). There was no significant difference in the number of ghrelin immunoreactive cells/mm2 in terms of H. plyori status. Plasma ghrelin concentrations in H. pylori-infected patients (144.6 ± 7.8.8 fmol/ml) were significantly lower than in uninfected subjects (196.1 ± 97.2, p < 0.05) and increased following cure of the infection. Plasma ghrelin levels correlated positively with the expression levels of ghrelin mRNA (r = 0.583, p < 0.0001) and peptide products (r = 0.574, p < 0.0001). There was a significant stepwise decrease in gastric ghrelin mRNA expression (p < 0.05), peptide contents (p < 0.01) and density of ghrelin immunoreactive cells (p < 0.05) with progression of histological severity of glandular atrophy in the corpus. The histological severity of chronic inflammation also negatively influenced the ghrelin mRNA expression (p < 0.001) and peptide production (p < 0.005).CONCLUSIONS:H. pylori infection has a negative impact on gastric and plasma ghrelin dynamics. Chronic inflammatory and atrophic changes associated with the infection may affect gastric ghrelin biosynthesis and contribute to the low circulating levels.

Peptidomic Identification and Biological Validation of Neuroendocrine Regulatory Peptide-1 and -2

Recent advances in peptidomics have enabled the identification of previously uncharacterized peptides. However, sequence information alone does not allow us to identify candidates for bioactive peptides. To increase an opportunity to discover bioactive peptides, we have focused on C-terminal amidation, a post-translational modification shared by many bioactive peptides. We analyzed peptides secreted from human medullary thyroid carcinoma TT cells that produce amidated peptides, and we identified two novel amidated peptides, designated neuroendocrine regulatory peptide (NERP)-1 and NERP-2. NERPs are derived from distinct regions of the neurosecretory protein that was originally identified as a product of a nerve growth factor-responsive gene in PC12 cells. Mass spectrometric analysis of the immunoprecipitate using specific antibodies as well as reversed phase-high performance liquid chromatography coupled with radioimmunoassay analysis of brain extract demonstrated the endogenous presence of NERP-1 and NERP-2 in the rat. NERPs are abundant in the paraventricular and supraoptic nuclei of the rat hypothalamus and colocalized frequently with vasopressin but rarely with oxytocin. NERPs dose-dependently suppressed vasopressin release induced by intracerebroventricular injection of hypertonic NaCl or angiotensin II in vivo. NERPs also suppressed basal and angiotensin II-induced vasopressin secretion from hypothalamic explants in vitro. Bioactivity of NERPs required C-terminal amidation. Anti-NERP IgGs canceled plasma vasopressin reduction in response to water loading, indicating that NERPs could be potent endogenous suppressors of vasopressin release. These findings suggest that NERPs are novel modulators in body fluid homeostasis.

Characterization of orexin-A and orexin-B in the microdissected rat brain nuclei and their contents in two obese rat models

Orexin-A and orexin-B (also known as hypocretins) are newly discovered hypothalamic peptides that stimulate food intake. Using separate radioimmunoassays for these rat orexins, we determined their distributions in microdissected nuclei of the diencephalon and brainstem which have accumulations of orexin fibers. High orexin contents (orexin-A: between 250 and 350 fmol/mg protein and orexin-B: between 650 and 900 fmol/mg protein) were present in the lateral hypothalamus; ventromedial hypothalamic, paraventricular thalamic and dorsal raphe nuclei; periaqueductal central gray and locus coeruleus. Moderate orexin contents (orexin-A: between 100 and 250 fmol/mg protein and orexin-B: between 300 and 500 fmol/mg protein) were found in the median eminence; suprachiasmatic, paraventricular hypothalamic, arcuate and supraoptic nuclei; substantia nigra and the nucleus of the solitary tract. Mature orexin-A and -B peptides were the major endogenous orexin molecules in these nuclei. The orexin-A and -B contents in the brains of obese Zucker rats that have disrupted leptin receptor were significantly higher than in their lean littermates, but in Otsuka Long-Evans Tokushima Fatty rats that have disrupted cholecystokinin type-A receptor the contents were similar to those of the controls. The widespread orexin distributions in the nuclei of diencephalon and brainstem suggest that orexins serve as neuromodulators, neurotransmitters, or both, in a wide variety of neural networks that regulate the autonomic and neuroendocrine systems.

Characterization of obestatin in rat and human stomach and plasma, and its lack of acute effect on feeding behavior in rodents

Obestatin is a 23-amino acid peptide, initially isolated from rat stomach as an endogenous ligand for the orphan G-protein-coupled receptor. Obestatin is derived from proteolytic cleavage of a 117-amino acid precursor, preproghrelin. Ghrelin increases food intake, body weight, and gastric emptying, whereas obestatin has the opposite effects. In this study, we characterized obestatin in both rat and human stomach, and investigated the peptides effect on feeding behavior. Using reversed-phase high-performance liquid chromatography coupled with RIAs specific for rat and human obestatin, we detected a very small amount of obestatin, compared with ghrelin, in the gastric fundi. The ratios of obestatin to ghrelin are 0.0039 and 1.94% respectively in the rat and human gastric fundi. In humans, plasma obestatin accounted for 5.21% of the ghrelin concentration, whereas it was undetectable in rat plasma. Plasma ghrelin concentration decreased after a meal in normal subjects, whereas obestatin concentration did not change. When administered centrally or peripherally, obestatin did not suppress food intake in either free-feeding or fasted rodents. Administration of obestatin did not antagonize ghrelin-induced feeding. These findings indicate that obestatin is present at very low levels compared with ghrelin in both rat and human, and has no acute effect on feeding behavior.

Ghrelin stimulates growth hormone secretion and food intake in aged rats.

Age-related decreases in energy expenditure have been associated with the loss of skeletal muscle and decline of food intake, possibly through a mechanism involving changes of growth hormone (GH) secretion and feeding behavior. Age-related declines of growth hormone secretion and food intake have been termed the somatopause and anorexia of ageing, respectively. Ghrelin, a 28-amino-acid peptide, was isolated from human and rat stomachs as an endogenous ligand of growth hormone secretagogue receptor. Ghrelin stimulates growth hormone release and food intake when peripherally administered to rodents and humans. Here, we investigate the relationship between age-related decline of growth hormone secretion and/or food intake and ghrelin function. Ghrelin (10 nmol/kg body weight) was administered intravenously to male 3-, 12-, 24-and 27-month-old Long-Evans rats, after which growth hormone concentrations and 2 h food intake were measured. An intravenous administration of ghrelin to rats increased food intake in all generations. In addition, to orexigenic effect by ghrelin, intravenous administration of ghrelin elicited a marked increase in plasma GH levels, with the peak occurring 15 min after administration. These findings suggest that the aged rats maintain the reactivity to administered exogenous ghrelin.