| Background:Ghrelin was discovered in 1999 by Kojima and colleagues as the endogenous ligand of the long known growth hormone secretagogue receptor 1a (GHS-R1a) isoform.Ghrelin positive X/A-like cells distributed throughout the gastric oxyntic mucosa are the main source of circulating ghrelin as demonstrated by the sharp decline of ghrelin levels following gastrectomy. ghrelin has been detected in the central nervous system in the arcuate nucleus of the hypothalamus as well as in neurons adjacent to the third ventricle. The arcuate nucleus is strongly implicated in the regulation of food intake. Ghrelin containing neurons in the arcuate nucleus send projections to neuropeptide Y (NPY) and agouti-related peptide (AgRP) positive neurons. NPY and AgRP are orexigenic neuropeptides and regulated by ghrelin. Peripheral injection of ghrelin selectively activates NPY-containing neurons in the arcuate nucleus in mice.Likewise, intracerebroventricular (i.c.v) administration of ghrelin activates NPY/AgRP-expressing neurons and stimulates the expression of NPY and AgRP mRNA in the arcuate nucleus. Total ghrelin levels inversely correlate with body mass index with increased levels in anorexic and cachectic patients and decreased levels under conditions of obesity. Circulating ghrelin levels increase before and decline after a meal in experimental animals and humans. Relatively speaking, the postprandial suppression of plasma ghrelin has been considerably better studied than the preprandial peak. Although the physiological importance of this effect is not ye clear,an appealing possibility is that the suppression of this orexigenic hormone plays a role in the satiating effect of ingested nutrients. We don't know the brain mechanism of the preprandial peak of plasma ghrelin.Nesfatin-1 was identified by Oh IS et al.in the rat hypothalamus and reported to decrease food intake upon 3rd ventricle injection and named nesfatin for NUCB2 encoded satiety and fat-influencing protein.Post-translational processing results in nesfatin-1,nesfatin-2 and nesfatin-3,however, only nesfatin-1 exhibits a food intake-reducing effect. The nesfatin-1 precursor NUCB2 is more prominently expressed at the mRNA and protein level in the rat gastric oxyntic mucosa than in other viscera such as the heart and even the brain.Moreover, NUCB2 mRNA expression is significantly enriched in a population of gastric oxyntic small endocrine cells. So far, we can't identify the nesfatin-1 receptor. Oh IS et al reported that nesfatin-1 induced anorexia occurs in Zucker rats with a leptin receptor mutation, and an anti-nesfatin-1 antibody does not block leptin-induced anorexia. Central injection of a-melanocyte stimulating hormone elevates NUCB2 gene expression in the paraventricular nucleus. So nesfatin-1 as a satiety molecule that is associated with melanocortin signaling in the hypothalamus.Stengel A et al recently reported that fasting for 24 h decreases NUCB2 mRNA expression in a pool of enriched small gastric endocrine cells and significantly reduces nesfatin-1 plasma levels in rats.the novel satiety factor nesfatin-1 has been shown to decrease food intake and body weight in rodents after i.c.v. injection, they raised the possibility that nesfatin/NUCB2 gene expression may be regulated by nutritional status, suggesting that nesfatin-1 in the stomach might play a role in satiety. However, no further developments regarding the true patho-physiological relevance of nesfatin-1 in obesity and type 1 diabetes mellitus (T1 DM) and type 2 diabetes mellitus (T2 DM) have been reported.Imbalance in energy intake and expenditure underlies the development and reversal of obesity, but the brain mechanisms underlying the control of these processes and the resulting alterations in WAT lipid deposition or mobilization are not precisely known. The preponderance of research on energy intake/energy expenditure has focused on hypothalamic forebrain sites/circuits. An even narrower focus has been placed on the hypothalamic arcuate nucleus (Arc) for numerous reasons, not the least of which is the identification of the largely adipocyte-derived cytokine leptin and the localization of substantial numbers of leptin receptors on Arc neurons. Within the ARC, there are two major cell types that are targets for leptin.One cell type produces neuropeptide Y (NPY) and agouti-related protein (AgRP), two orexigenic peptides, whereas the other cell type produces proopiomelanocortin (POMC) and cocaine-associated related transcripts (CART), two anorexigenic peptides. A classic approach to understanding the role of any brain site for a given physiological response, such as energy balance, is to destroy it. An alternative approach to Arc destruction is offered by neonatal administration of monosodium glutamate (MSG). MSG is able to penetrate the brain via the underdeveloped neonatal blood brain barrier (BBB) in several brain areas, including the area postrema.Object:To investigate the fasting levels of plasma nesfatin-1 in diabetes mellitus patients and the nutrient-related fluctuation of nesfatin-1 level in normal humans,and whether NUCB2/nesfatin is peripherally produced in the human gastric mucosa.To test whether destruction of the Arc by MSG,interferes with the ability of food deprivation to mobilize body fat stores in mice. Whether Arc lesion affect the ghrelin and nesfatin-1 level of plasma and stomach.Methods:In the present study, fasting levels in plasma nesfatin-1,insulin and glucose were measured and analyzed by ELISA in healthy subjects and in patients with T1 DM and T2 DM. Plasma nesfatin-1 levels were measured 6 times before and after oral glucose ingestion in healthy subjects. NUCB2 mRNA and protein expression were analyzed by RT-PCR and Western blot analysis in the human gastric mucosa, NUCB2/nesfatin-1 immunoreactive cells were characterized using immunofluorescent double labeling of mucosal sections.Neonatal mice from our breeding colony, were injected subcutaneously into the dorsal dermis area just below the interscapular region on d 1,3,5,7, and 9 after birth. With 10μl of MSG (Sigma-Aldrich, St. Louis, MO) to deliver 4 mg/g body mass,Control rats were treated with saline. At 4 wk of age, the pups were weaned, sexed, and housed in groups according to treatment. Food intake was assessed by measuring the weight of the food containers (±0.1g) by electronic precision scales (Feeding and Activity Analyser 47552-002, UGO BASILE, ITALY).Food intake was measured weekly after weaning. Body mass and stomach tissues were measured before and after food deprivation. At fasting 48h, before and after fasting, animals were lightly anesthetized with isoflurane and orbital blood was taken for serum measurement of ghrelin. Animals were then anesthetized (pentobarbital sodium, 50 mg/kg ip), stomach tissues were removed, and the right inguinal WAT (1WAT), bilateral retroperitoneal WAT (RWAT), bilateral parametrial WAT (PWAT), and interscapular brown adipose tissue (IBAT) depots were removed and weighed. Ghrelin and nesfatin-1 mRNA of stomach was measured by RT-PCR, ghrelin and nesfatin-1/NUCB2 protein of stomach were measured by western blot. Brains were removed and stored in a 4% paraformaldehyde solution for Arc lesion verification by histological examination.Results:No sex differences in plasma nesfatin-1 were found. The mean fasting plasma nesfatin-1 levels were slightly but not significantly higher in T1 DM patients compared to healthy subjects. However, fasting plasma nesfatin-1 levels were significantly lower in T2 DM patients compared to healthy subjects and T1 DM patients. Plasma nesfatin-1 did not change acutely, although a small rise in circulating nesfatin-1 occurred within 30 min after the beginning of an oral glucose ingestion (from a mean basal value of 0.99±0.23 ng/ml to a maximum of 1.08±0.24 ng/ml). No significant difference in plasma nesfatin-1 before and after an oral glucose was observed. NUCB2 mRNA was detected in the human gastric mucosa. Furthermore, Western blot analysis confirmed nesfatin-1 protein in this tissue. Fluorescent immunoreactivity of nesfatin-1 was detected in endocrine cells of the oxyntic mucosa. Most nesfatin-1 immunoreactivity (67.8%) of the oxyntic mucosa co-localized with ghrelin immunoreactive endocrine cells. The majority of ghrelin positive cells (88.6%) were also positive for nesfatin-1.Compared with saline controls, MSG treatment significantly decreased Nissl staining, NPY-ir fibers, and TH-ir cells of the Arc (P<0.01);MSG treatment significantly reduced TH-ir(P<0.01)of the AP.MSG treatment increased body mass in 3-mo-old mice (P<0.01)compared with the saline controls, but there were no significant different of food intake from 1-mo-old to 3-mo-old in two groups. MSG significantly increased WAT pad mass for all depots assayed (IWAT, RWAT, and gonadal WAT; P<0.01)as well as IBAT. Total dissected WAT also was significantly increased by MSG treatment compared with saline-treated controls (P<0.05). Neonatal MSG injections did not block or diminish the food deprivation-induced lipid mobilization from any of the WAT pads assayed as evidenced by their decreased masses compared with the saline-treated controls. Thus both MSG and saline treated animals had significant food deprivation-induced decreases in IWAT and RWAT mass (P<0.05) compared with their respective ad libitum-fed counterparts, but GWAT mass did not decrease with food deprivation for either group compared to their respective ad libitum-fed controls.Plasma ghrelin levels were significantly increased in MSG treated mice and saline treated mice fasted for 48h compared with their respective before fasting and refeeding counterparts (P<0.01),but there were no significant different of plasma ghrelin level between MSG treated mice and saline treated mice at before, after fasting 48h and refeeding time.Ghrelin mRNA of stomach were significantly increased in MSG treated mice and saline treated mice fasted for 48h compared with their respective before fasting counterparts (P<0.05), but there were no significant different of ghrelin mRNA of stomach between MSG treated mice and saline treated mice before and after fasting.NUCB2 mRNA of stomach were significantly decreased in MSG treated mice and saline treated mice fasted for 48h compared with their respective before fasting counterparts (P<0.05), but there were no significant different of NUCB2 mRNA of stomach between MSG treated mice and saline treated mice before and after fasting.Proghrelin protein of stomach were significantly increased in MSG treated mice and saline treated mice fasted for 48h compared with their respective before fasting counterparts (P<0.01), but there were no significant different of proghrelin protein of stomach between MSG treated mice and saline treated mice before and after fasting.NUCB2/nesfatin-1 protein of stomach were significantly decreased in MSG treated mice and saline treated mice fasted for 48h compared with their respective before fasting counterparts (P<0.01), but there were no significant different of NUCB2/nesfatin-1 protein of stomach between MSG treated mice and saline treated mice before and after fasting.Conclusions:Fasting nesfatin-1 were significantly lower in T2 DM patients compared to healthy subjects and T1 DM patients. The significance of this result is unclear, but the reduction in fasting nesfatin-1 may be one of the appetite-related hormones involved in diabetic hyperphagia. In addition, neither glucose nor saline ingestions affected plasma nesfatin-1, suggesting that gastric chemosensation is not sufficient for the nesfatin-1 response under the present conditions.NUCB2 mRNA and protein were detected in the human gastric mucosa, the majority of gastric oxyntic ghrelin-producing P/Dl cells co-express NUCB2/nesfatin-1The MSG-produced Arc lesions and AP lesions were substantial, and the normal function of these areas likely was compromised, yet food deprivation-induced WAT Hpid mobilization appeared normal. An intact Arc and/or AP are not necessary for food deprivation-induced lipid mobilization from WAT but could play a role in lipid mobilization triggered by other energy-demanding conditions such as exercise or cold exposure.Although Arc lesion, plasma ghrelin levels were significantly increased in MSG treated mice fasted for 48h compared with it's before fasting and refeeding counterparts. Ghrelin positive X/A-like cells distributed throughout the gastric oxyntic mucosa are the main source of circulating ghrelin. Arc lesion did not affect the preprandial peak of plasma ghrelin, so Arc are not necessary for the preprandial peak of plasma ghrelin, Further studies are warranted to elucidate the brain mechanism of preprandial peak of plasma ghrelin.Fasting for 48 h decreases NUCB2 mRNA expression of stomach, there were no significant different of NUCB2/nesfatin-1 of stomach between MSG treated mice and saline treated mice before and after fasting. So Arc lesion didn't affect the synthesis and releasing...
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