【作者】 李清春；

【导师】 蒋正尧；

【作者基本信息】 青岛大学 ， 生理学， 2010， 博士

【摘要】 背景资料：Ghrelin是1999年由Kojima和他的同事发现,是生长激素促分泌受体la内源性配体(GHS-R1a)。在摄食调节肽中,ghrelin有其独一无二的性能,它的酰基化是在第三位丝氨酸残基,从而增加其亲油性以便能够结合GHS-R1a体。分布在胃泌酸粘膜上的ghrelin阳性X/A样细胞是循环血ghrelin的主要来源,胃切除术后血浆ghrelin有明显的降低。ghrelin也可由位在于下丘脑的弓状核和第三脑室周围的神经元产生。弓状核与摄食调节有很强的关连。弓状核含ghrelin的神经元神经纤维投向神经肽Y(NPY)和刺鼠色蛋白相关蛋白(AgRP)阳性神经元,NPY和AgRP是促进食欲的神经肽,且受ghrelin的调节。外周注射ghrelin选择性激活小鼠弓状核含NPY的神经元。同样,脑室内注射ghrelin激活表达NPY/AgRP的神经元,刺激弓状核NPY和AgRP mRNA的表达。总ghrelin水平与体重指数成反比,在厌食和恶病质病人增加,在肥胖时降低,在实验动物和人血循环中的ghrelin在餐前在餐前有一分泌峰,在餐后降低,呈现餐后抑制。相对而言,进食对血浆ghrelin的餐后抑制研究比较详尽,然而其餐前分泌峰的中枢机制还未明确。nesfatin-1是由Oh IS等在大鼠下丘脑发现的,在第三脑室注射能抑制摄食,命名为nesfatin-1:NUCB2编码的饱感和影响肥胖的肽,翻译后的处理不同生成nesfatin-1、nesfatin、nesfatin-3,只有nesfatin-1具有抑制摄食的效应。nesfatin-1的前体NUCB2无论是mRNA还是蛋白质水平,在胃泌酸粘膜都比其他脏器如心脏、脑表达多。而且,NUCB2 mRNA在胃泌酸的小分泌细胞上有显著的丰富的表达。迄今为止,nesfatin-1的受体尚未得到鉴定。关于nesfatin-1抑制摄食的作用机制,Oh IS等2006年在Nature的首篇报导指出,在leptin受体突变的Zucker大鼠,nesfatin-1仍然可抑制摄食,而促黑激素(a-MSH)受体(MC4-R)阻断剂可阻断nesfatin-1的摄食抑制效应,i.c.v.给予a-MSH引起室旁核NUCB2 mRNA表达增加,由此认为nesfatin-1的摄食抑制效应是通过促黑激素系统(melanocortin signalling)介导的。最近报道大鼠禁食24小时胃内内分泌细胞NUCB2 mRNA表达减少,血浆nesfatin-1水平明显减少。在啮齿类动物,新发现的肽nesfatin-1侧脑室注射能够减少摄食和体重；Stengel等最近研究表明,禁食24h后胃粘膜内分泌细胞NUCB2的mRNA表达减少,他们认为nesfatin/NUCB2基因可能受营养状态的调节,表明外周nesfatin-1在能量稳态中可能有调节作用。然而.关于nesfatin-1与肥胖者、Ⅱ型糖尿病患者的病理生理相关性未见报道。能量摄入和消耗的不平衡成为肥胖发展和逆转的基础,但是对这些过程的控制及白色脂肪组织的脂质沉淀和动员的转换结果的中枢机制还不清楚。对能量摄入和消耗研究的优势主要集中在下丘脑前脑部位及环路,有更多的理由使研究局限的在下丘脑弓状核(arcuate nucleus, Arc),不仅仅是因为大量的脂肪细胞衍生因子瘦素(leptin)的鉴定,且在下丘脑弓状核存在大量的瘦素受体。在弓状核内有两类细胞是瘦素的作用靶点,一类细胞产生神经肽Y(neuropeptide Y,NPY)和刺鼠色蛋白相关蛋白(agouti-related protein,AgRP),这两种肽是促进食欲的肽；另一类是产生阿黑皮素原(proopiomelanocortin,POMC)和可卡因-安非它明调节转录肽(cocaine-associated related transcripts,CART),这两种肽是厌食肽。经典的理解大脑某个部位的生理性反应,如能量平衡,就是损毁它。一种替换传统弓状核损毁的方法是出生后用谷氨酸单钠处理(monosodium glutamate,MSG)来破坏弓状核。主要是因为弓状核血脑屏障不完善,MSG能够渗透到大脑。目的：观察糖尿病患者血浆nesfatin-1的水平及营养素对血浆nesfatin-1水平波动的影响；鉴定人类胃粘膜是否含有NUCB2/nesfatin.检测小鼠弓状核和其它血脑屏障不完善的区域(如最后区)是否被MSG损毁,食物剥夺后动员身体脂肪的贮存的能力是否受到干扰；弓状核损毁后是否影响到胃及血浆的ghrelin、nesfatin-1水平。方法：20例正常人、10例Ⅰ-型糖尿病和47例Ⅱ-型糖尿病患者,空腹采血3ml；16例健康志愿者,禁食12h后,男女各半分为两组,先空腹采血3ml,一组口服300ml生理盐水,另一组口服含75g葡萄糖的300ml液体后30、60、90、150、210min采血。ELISA法检测血浆nesfatin-1和血糖水平的变化。十例胃癌手术后切除的正常胃组织,通过RT-PCR、Western blot、免疫荧光双标实验鉴定人类胃粘膜是否含有NUCB2/nesfatin.新生健康昆明小白鼠,雌雄不限。随机分为正常对照组和MSG处理组,每组16只。MSG处理组小鼠出生后隔日(第1,3,5,7和9天,共5次)于颈部皮下注射MSG(4mg/g体重),而正常对照组只给予同体积的生理盐水。第四周断乳后,放入摄食分析仪测摄食；第十二周后,每组在正常饮食、禁食48h、再进食后,分别内眦取血,测血浆ghrelin水平；此后每组再分为正常饮食组、禁食48h组,分别断头取脑,冰冻切片观察弓状核损毁情况,取胃组织行RT-PCR、Western blot实验,以检测ghrelin、nesfatin-1 mRNA及蛋白质水平；取各部位脂肪称重。结果：血浆nesfatin-1水平无性别变化；Ⅰ型糖尿患者组比正常对照组血浆nesfatin-1水平稍升高,但差别无统计学意义；Ⅱ-型糖尿病患者组血浆nesfatin-1水平明显低于Ⅰ型糖尿患者组和正常对照组。口服葡萄糖30min后,血浆nesfatin-1水平稍高于基础水平(从基础水平0.99±0.23 ng/ml升高1.08±0.24 ng/ml),但观察到各个时间点血浆nesfatin-1水平无明显差别。人类胃粘膜含有NUCB2/nesfatin基因和蛋白质,免疫荧光双标实验显示nesfatin-1阳性细胞,nesfatin-1与ghrelin共表达率为67.8%;ghrelin阳性的细胞,ghrelin与nesfatin-1共表达率为88.6%。与生理盐水组小鼠相比,MSG组小鼠弓状核甲酚紫染色神经元明显减少(P<0.01),TH、NPY免疫阳性神经元也明显减少(P<0.01),最后区TH免疫阳性神经元明显减少(P<0.01)。从第四周断乳后,MSG组小鼠与生理盐水组小鼠比较,每周的摄食量没有明显改变。第十二周后,MSG处理的小鼠比生理盐水处理的小鼠体重明显增加(P<0.01)；MSG组小鼠和生理盐水组小鼠食物剥夺后,与各自自由饮食的小鼠相比体重也明显降低(P<0.05)。与生理盐水处理的小鼠相比,MSG处理的小鼠在所分析的部位(腹膜后、双侧腹股沟、性腺)白色脂肪组织量及肩胛间棕色脂肪组织都明显增加(P<0.01),白色脂肪组织的总量也明显增加(P<0.01)。与生理盐水处理的小鼠相比,新生小鼠MSG处理后不能阻断或减少食物剥夺后引起的白色脂肪组织脂质动员的减少。因此,MSG组小鼠和生理盐水组小鼠食物剥夺后,与各自自由饮食的小鼠相比,腹膜后和双侧腹股沟的白色脂肪量明显降低(P<0.05),而性腺白色脂肪量却没有明显降低。MSG处理的小鼠,在禁食48h后血浆ghrelin水平明显高于禁食前,进食后血浆ghrelin水平又明显降低；生理盐水组小鼠在禁食48h后血浆ghrelin水平明显高于禁食前,进食后血浆ghrelin水平又明显降低(P<0.01)。MSG组小鼠与生理盐水组小鼠比较,无论是在禁食前、禁食48h,还是进食后,血浆ghrelin水平无明显改变。MSG处理的小鼠,在禁食48h后胃粘膜组织ghrelin mRNA表达明显高于禁食前(P<0.01)；生理盐水组小鼠在禁食48h后胃粘膜组织ghrelin mRNA表达也明显高于禁食前(P<0.05)。MSG组小鼠与生理盐水组小鼠比较,无论是在禁食前,还是在禁食48h,胃粘膜组织ghrelin mRNA表达水平无明显改变。MSG处理的小鼠,在禁食48h后胃粘膜组织nesfatin-1前体蛋白NUCB2 mRNA表达明显低于禁食前(P<0.05)；生理盐水组小鼠在禁食48h后胃粘膜组织nesfatin-1前体蛋白NUCB2 mRNA表达明显低于禁食前(P<0.01)。MSG组小鼠与生理盐水组小鼠比较,无论是在禁食前,还是在禁食48h,胃粘膜组织nesfatin-1前体蛋白NUCB2 mRNA表达表达水平无明显改变。MSG处理的小鼠,在禁食48h后胃粘膜组织proghrelin蛋白表达明显高于禁食前(P<0.01)；生理盐水组小鼠在禁食48h后胃粘膜组织proghrelin蛋白表达也明显高于禁食前(P<0.01)。MSG组小鼠与生理盐水组小鼠比较,无论是在禁食前,还是在禁食48h,胃粘膜组织proghrelin蛋白表达水平无明显改变。MSG处理的小鼠,在禁食48h后胃粘膜组织nesfatin-1前体蛋白NUCB2蛋白表达明显低于禁食前(P<0.01)；生理盐水组小鼠在禁食48h后胃粘膜组织nesfatin-1前体蛋白NUCB2蛋白表达明显低于禁食前(P<0.01)。MSG组小鼠与生理盐水组小鼠比较,无论是在禁食前,还是在禁食48h,胃粘膜组织nesfatin-1前体蛋白NUCB2蛋白表达表达水平无明显改变。结论：Ⅱ-型糖尿病患者组血浆nesfatin-1水平明显低于Ⅰ型糖尿患者组和正常对照组,这种结果的意义还不清楚,但血浆nesfatin-1水平降低,可能与糖尿病的多食和胰岛素抵抗有关；另外,不论生理盐水,还是葡萄糖摄入都不能影响血浆nesfatin-1水平,说明目前条件下胃化学状态不足以引起nesfatin-1的反应。人胃粘膜存在NUCB2/nesfatin基因和蛋白质,大多数表达ghrelin的P/D1样细胞也表达NUCB2/nesfatin-1。MSG能够损毁弓状核、最后区。这些部位的正常功能有可能被代偿,食物剥夺后白色脂肪组织的动员也就表现为正常。完整的弓状核、最后区对于食物剥夺引起的白色脂肪组织的脂质动员并不是必需的,但可能在其他能量要求的条件下(如锻炼或寒冷暴露)的脂质动员的发动上起作用。MSG处理的小鼠,尽管损毁了弓状核NPY/AgRP神经元,但禁食48h后血浆ghrelin仍然明显升高,因此弓状核NPY/AgRP神经元损毁并没有影响ghrelin的餐前分泌峰,说明弓状核NPY/AgRP神经元对于ghrelin餐前分泌峰并不是必须的,关于ghrelin餐前分泌峰的中枢机制还需要进一步研究。禁食能引起小鼠胃粘膜NUCB2/nesfatin-1表达减少,MSG损毁小鼠弓状核后,与对照组相比在禁食前后胃粘膜NUCB2/nesfatin-1表达并没有明显变化,说明弓状核损毁并没有影响到胃粘膜NUCB2/nesfatin-1的合成和分泌。更多还原

【Abstract】 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更多还原