Ghrelin对高糖诱导的海马神经细胞tau蛋白磷酸化和糖代谢的影响及机制研究
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摘要
Ghrelin是28个氨基酸残基构成的小分子多肽,主要由胃底粘膜的X／A样细胞分泌。它可与生长素促泌物受体(growth hormone secretagogue receptor 1a, GHSR-1a)结合促进生长素(growth hormone, GH)分泌。体内循环的ghrelin有两种形式：一种是无活性的(unacylated ghrelin, UAG), N端去辛酰基化；另一种是有活性的(acylated ghrelin, AG), N端辛酰基化,该结构是维持ghrelin的生物学活性所必需的。GHSR-1a在外周组织和中枢神经系统均有表达,ghrelin与其结合后在中枢神经系统的神经内分泌调节中起到重要作用,如对饮食和食物摄取的调节,能量和葡萄糖内环境稳定的调节,学习和记忆能力的调节。有研究证实,ghrelin是海马学习记忆过程中重要的调节因子。
糖尿病(diabetes mellitus, DM)是由多种病因引起的胰岛素分泌绝对或相对不足以及靶细胞对胰岛素敏感性降低,继而引起糖、蛋白质、脂肪和水、电解质代谢异常的一种综合症,以慢性高血糖为主要特征。高血糖在2型糖尿病(T2DM)中不仅是次于胰岛素抵抗的重要体征,也是引起靶器官胰岛素抵抗的主要原因。作为一种系统性疾病,糖尿病可以引起多种组织、器官的结构和功能的改变,其中继发于糖尿病的视网膜、肾病及周围神经等病变已经得到人们广泛认识和关注。有研究表明高血糖是神经系统损伤的重要原因,糖尿病患者中痴呆的危险性明显增加,往往伴有认知功能障碍,如学习和记忆等脑功能损害。
流行病学研究显示伴有胰岛素抵抗的糖尿病患者有30-65%可发展为阿尔茨海默病(Alzheimer disease, AD)。最近也有研究发现大脑的胰岛素缺乏和脑内的胰岛素抵抗状态和AD发病有关,有学者提出AD可能是“3型糖尿病”。因此,认为胰岛素抵抗导致代谢紊乱和认知障碍是糖尿病和AD共同的病理机制。Ghrelin可以改善AD模型大鼠的认知动作。有研究显示ghrelin可以调节胰岛素的敏感性,并提出可能是一种潜在的新的治疗糖尿病和AD导致的脑功能异常的药物。但是潜在的机制目前国内外很少有人报道。
葡萄糖是哺乳动物脑内主要的能量来源,对维持正常的大脑功能有重要作用。目前发现葡萄糖转运蛋白(GLUTs)家族有14种亚型。在脑内,葡萄糖转运体1(GLUT1)主要在血脑屏障和胶质细胞中表达,葡萄糖转运体3 (GLUT3)负责神经细胞的葡萄糖转运且不依赖胰岛素。但是,这些葡萄糖转运体不能负责海马区的整个葡萄糖代谢,而且,GLUT3位于神经细胞的突起而不在细胞体,因此有位于细胞体的其它葡萄糖转运蛋白负责神经细胞的葡萄糖摄取和利用。在胰岛素敏感的外周组织,细胞通过GLUT4易位不断的摄取葡萄糖,同样这个过程也发生在大脑的某些区域,包括海马神经细胞。
神经纤维原缠结(neurofibrillary tangles, NFTs)是AD在组织病理学重要标志之一。NFTs由高度磷酸化的tau蛋白组成,其数量和认知障碍相关。在tau蛋白磷酸化的过程中,GSK-3p起到重要作用。磷脂酰肌醇3-激酶／蛋白激酶B(Phosphoinositide 3-kinase/Protein kinaseB, PI3-Kinase/AKT)是胰岛素信号转导的经典途径。胰岛素信号系统受损导致PI3-K/Akt信号通路障碍,引起GSK-3p活性增加,导致tau蛋白高度磷酸化。AS160是Akt底物,一种与Rab蛋白具有特异性作用的GTP酶激活蛋白(GAP),对GLUT4的易位有重要作用,最新的研究表明AS160是胰岛素信号中最接近GLUT4易位的信号分子。Grillol发现神经细胞中胰岛素利用同外周组织一样的信号转导系统刺激葡萄糖转运体4(GLUT4)向细胞膜易位摄取葡萄糖。
Ghrelin能调节胰岛素敏感性,刺激胰岛素诱导的葡糖糖摄取增加,有调节记忆的作用。因此,我们以原代培养的大鼠海马神经细胞为研究对象,探讨ghrelin对高糖诱导的神经细胞tau蛋白磷酸化和葡萄糖代谢的影响。进一步探讨其潜在的作用机制,包括PI3-K/Akt-GSK-3p、AS160及GLUT4表达的变化。进而深入了解ghrelin的功能,为DM是AD的危险因素提供进一步的理论依据,为临床改善糖尿病及AD引起的认知功能障碍提供的新的可能的治疗方向。
材料与方法
一、实验材料
1、原代乳鼠海马神经细胞培养相关试剂
2、MTT相关试剂
3、神经细胞葡萄糖消耗实验相关试剂
4、神经细胞葡萄糖摄取实验相关试剂
5、免疫细胞化学染色相关试剂
6、半定量RT-PCR检测基因的相关试剂
7、Western blotting印迹杂交相关试剂
二、实验方法
1、乳鼠海马神经细胞的提取、培养及鉴定
(1)乳鼠海马神经细胞的提取
取新生(出生24h以内)的SD大鼠,冰浴麻醉约10min,用75%乙醇消毒。断头后放在冰袋上的90mm培养皿中。去除颅骨,取出双侧的海马放入盛有预冷的D-Hanks液的培养皿中,逐一分离海马组织上的软脑膜和毛细血管网。当所有的海马组织分离后,清洗2次,放入培养皿中。将组织剪成1mm3的小块,移入50ml离心管中,加入与组织等体积的0.25%胰蛋白酶(含EDTA)。在37℃,5%CO2条件下消化30min左右,用含有血清的培养液终止反应,用lml移液器吹打混匀组织液。1200rmp,离心10min,弃上清,加入10ml含10%胎牛血清、10%马血清的DMEM培养基,用移液器轻微混悬细胞。用孔径为200目的尼龙网过滤后,血细胞计数板计数细胞。用种植液稀释细胞悬液,以1.5×105、5×105或2.5×106／孔的密度分别接种于事先用多聚赖氨酸浸泡过的96孔培养孔板、24孔培养孔板或6孔培养孔板内,将培养板移入湿润的培养箱,在37℃和5%CO2条件下培养细胞。
(2)乳鼠海马神经细胞的原代培养
将提取到的神经细胞用DMEM培养基(含10%胎牛血清、10%马血清、1%谷氨酰胺和100U／ml青霉素、100μg／ml链霉素)在37℃,5%CO2的条件下湿润的培养箱中培养,于接种24h后全量换液,换成Neurobasal Medium/B27无血清培养基,以后每2天换液1／3—1／2,待7天后,细胞生长成熟可以用于实验。NeurobasalMedium培养液的葡萄糖浓度是25mM,高于其3倍的浓度可以视为高糖(75mM)环境。
(3)乳鼠海马神经细胞免疫细胞化学染色鉴定
用神经元烯醇化酶(NSE)抗体作为神经细胞的识别标志。当细胞生长成熟后,用PBS清洗一次；4%的多聚甲醛溶液在室温固定20min; 3%H2O2阻断内源性过氧化物酶10min；加入非免疫性动物血清封闭液(正常山羊血清),20℃下孵育15min；加入一抗兔抗鼠NSE多克隆抗体(1：250稀释),4℃水盒孵育过夜；滴加二抗(生物素标记的山羊抗兔IgG)20℃下15min,辣根过氧化酶(HRP)标记的链霉菌抗生物素蛋白孵育15min,新鲜配制的DAB显色,苏木精复染,倒置相差显微镜照相记录染色情况。
2、MTT法检测细胞活性和数量
将5mg/ml的MTT原液与无血清的DMEM培养液按体积1：9配成MTT培养液,待孔内原有培养液移出后,每孔加入MTT培养液50μl,37℃继续培养,4h后终止培养,并小心吸弃孔内培养上清夜,每孔加入200μl二甲基亚砜(DMSO),微型震荡器震荡10min使结晶物充分溶解。选择490nm波长,在酶标仪上测定各孔的吸光度值(OD值),以光密度值反映细胞活性和数量的多少。
3、神经细胞葡萄糖消耗实验
神经细胞的葡萄糖消耗量通过葡萄糖氧化酶-过氧化物酶(GOD-POD)法来检测。细胞以1.5×105个／孔接种在96孔板中。在培养第7天,原始的培养基换成不同的处理因素。按各实验组细胞经分组处理后用于实验。将各样本浓度稀释5倍,取2μl放入200μl的检测液中,在室温反应30min达到平衡并保持稳定,在490nm酶标仪中测定各管吸光度值(OD值),根据公式计算葡萄糖浓度,再乘以稀释倍数,与未接种细胞的空白孔的含量糖均值相减,计算葡萄糖的消耗量。
4、[3H]-2-deoxy-D-glucose ([3H]-DG)示踪法检测神经细胞葡萄糖摄取的变化
海马神经细胞培养成熟后按各实验组处理后用于实验。Lock's Buffor缓冲液洗涤细胞3次,用400μl缓冲液37℃孵育细胞30min。弃去缓冲液,各组加或不加胰岛素共同继续孵育30min。加入[3H]-DG至终浓度0.2μCi/ml 30min。用预冷的PBS终止反应,冲洗3次,将细胞裂解于O.1N NaOH中,反复吹打后,在37℃中裂解2h,吹打混匀细胞,将各样本液体滴在微孔滤纸上,经真空滤泵后,室温晾干。将微孔滤膜浸入12ml液闪液中,液体闪烁计数法测定检测各组的每分钟总衰变数(CPM)。测定细胞蛋白浓度。计算葡萄糖摄取量。每组重复5次。
5、RT-PCR法检测GLUT3、4, PPARymRNA的表达
用于实验的细胞按分组分别干预后,弃去培养液,在无菌条件下用D-Hanks液轻微冲洗2次,加入预冷的1ml TRIzol,反复吹打,充分裂解细胞后移入新的经DEPC水处理过的无菌Eppendoff (Ep)管中,采用一步法提取总RNA。RNA纯度及浓度的测定；逆转录合成cDNA;引物序列及反应条件；3%琼脂糖凝胶电泳；紫外透射仪观察,应用凝胶图像处理系统扫描各条带的光密度,以GAPDH产物的光密度值作为内参,计算不同条件下各基因扩增量的比值,进行半定量分析。
6、免疫细胞化学染色检测ptau[Ser199]、GLUT4的表达
细胞接种在盖玻片上,培养7d后,用4%的多聚甲醛溶液在室温固定20min；每张盖玻片加3%H202阻断内源性过氧化物酶10min, PBS洗涤3次,每次5min；加入非免疫性动物血清封闭液(正常山羊血清),20℃下孵育15min；加入一抗为兔抗鼠的ptau[Ser199](1:500稀释)、GLUT4 (1:250稀释)多克隆抗体,4℃水盒孵育过夜；滴加二抗(生物素标记的山羊抗兔IgG)20℃下15min,辣根过氧化酶(HRP)标记的链霉菌抗生物素蛋白孵育15min,新鲜配制的DAB显色,自来水冲洗5min,60℃烘干后中性树脂封片。倒置相差显微镜照相记录染色情况。
7、Western blotting检测pAkt[Ser473]、ptau[Serl99]、pGSK-3p[Ser9]和AS160磷酸化
实验细胞用预冷PBS缓冲液洗2遍,细胞刮收集细胞,根据蛋白提取试剂盒操作分别提取各组细胞总蛋白,采用Bradford法测定蛋白浓度,每组蛋白质样本20μg在95℃下变性5min,进行SDS—PAGE电泳,转膜后3%BSA封闭2h,用TBST洗膜后分别加5%BSA/TBS稀释的多克隆抗体,4℃孵育过夜,再次用TBST洗膜后,加入辣根过氧化物酶标记的二抗在室温下杂交2h,洗膜后ECL超敏发光液进行显色,每组重复3次,结果用Image Analysis Softwave V7.0分析软件对目的条带进行灰度值分析。
8、统计学分析
所有数据用SPSS 13.0软件处理,结果用均数±标准差(x±SD)表示,每组实验重复3次或3次以上。采用单因素方差分析(ANOVA)检验,post-hoc分析,t检验进行均数间的多重比较,以p<0.05为统计学有显著差异。
结果
1、高糖环境对神经细胞的糖代谢有抑制作用；ghrelin可以增加神经细胞的葡萄糖代谢。经过不同浓度的ghrelin (1-100nM)干预,神经细胞在1nM作用下糖代谢有所增加但是没有明显变化,在10nM和100nM作用下糖代谢明显增加,呈剂量依赖性,经100nM胰岛素诱导后增加更明显。
2、免疫细胞化学法检测tau蛋白磷酸化。高糖环境下,tau蛋白磷酸化增加,表现为胞浆染色和正常糖环境相比明显加深,经过ghrelin干预后,胞浆染色变浅,tau蛋白磷酸化减少。
3、Western blotting检测tau蛋白磷酸化的变化。高糖组tau蛋白磷酸化增加,ghrelin可以减弱tau蛋白磷酸化。总蛋白无明显变化。
4、RT-PCR检测GLUT3、GLUT4和PPARy的基因表达。在原代培养的海马神经细胞中除GLUT3表达外,也有GLUT4的表达,但是后者比前者的表达量要少。在高糖环境下,ghrelin和胰岛素干预前后神经细胞中GLUT4和PPARy的基因表达没有发生变化。说明在短时间内ghrelin可能并不是通过PPARy变化增加胰岛素敏感性的；对葡萄糖摄取的改善不是通过GLUT4量的改变实现的,可能和GLUT4易位有关。
5、Western blotting法检测Akt、AS160及GSK-3β磷酸化的变化。Ghrelin可以增加不同葡萄糖浓度的神经细胞Akt、AS 160及GSK-3β磷酸化水平。在高糖组Akt. AS 160和GSK-3β磷酸化水平较正常组降低明显。经过ghrelin干预后,Akt、AS 160和GSK-3β磷酸化增加明显,总蛋白变化不明显。
6、免疫细胞化学法检测GUT4易位的变化。GLUT4在正常对照组的胞浆和胞膜均有表达,为棕色至棕褐色,经高糖作用24h后,易位受到抑制,经ghrelin干预后,易位增加,经胰岛素诱导后这种作用增加更明显。
7、加入PI3-K特异性抑制剂wortmannin(50nM)和ghrelin共同作用,可以削弱ghrelin改善tau蛋白磷酸化和增加葡萄糖摄取的作用。
结论
1、高糖环境能使海马神经细胞tau蛋白磷酸化增加,糖代谢降低,可能存在胰岛素抵抗。
2、Ghrelin能增加高糖诱导的神经细胞的糖代谢,改善胰岛素抵抗。而加入胰岛素后,这种作用更明显。Ghrelin和胰岛素促进神经细胞糖代谢是协同作用。
3、Ghrelin可以降低高糖导致的tau蛋白异常磷酸化。
4、Ghrelin对海马神经细胞的葡萄糖摄取的调节最终可能是通过GLUT4易位实现的。
5、Ghrelin增加高糖诱导的神经细胞的Akt、AS 160和GSK-3p磷酸化水平,这种作用在正常糖浓度下更明显。Ghrelin至少是通过P13-K/Akt-GSK-3β途径改善tau蛋白异常磷酸化和胰岛素抵抗。
Ghrelin is a 28-amino acid peptide secreted predominantly from X/A-like cells of the gastric fundus as the endogenous ligand for the growth hormone (GH) secretagogue-receptor la (GHS-Rla). Circulating hormone has two forms:unacylated ghrelin (UAG) and acylated ghrelin (AG), which is essential for ghrelin bioactivity. Ghrelin has been on its role in neuroendocrine regulation in the central nervous system, for instance modulating appetite and food take, energy and glucose homeostasis, learning and memory performance. The data confirmed that ghrelin is a key mediator of hippocampal-dependent learning processes.
Diabetes mellitus is an endocrine disorder of carbohydrate metabolism resulting from inadequate insulin release or insulin insensitivity, then causing sugar, protein, water abnormal metabolism. Hyperglycemia, as diabetic fundamental biochemistry character, has an important role in diabetes. Hyperglycemia in type 2 diabetes is not only a secondary manifestation of insulin resistance, but could also be responsible for directly inducing insulin resistance in the target tissue. Studies have been shown that hyperglycemia is a primary reason for nervous system impaired. Diabetic demented danger increased significantly, generally accompany with cognition dysfunction, such as learning and memory. Epidemiology studies have shown that diabetic patients with insulin resistant have a 30-65%increased risk of developing AD. Recent data indicate that brain insulin deficiency and the insulin-resistant brain state are related to Alzheimer's disease (AD), de la suggests that AD could be "Type 3 diabetes". Therefore, the common pathomechanism of diabetic encephalopathy and AD was insulin resistance, which led to metabolic disturbance and caused cognitive dysfunction. Ghrelin improved the cognitive action of AD model rats. Recent study suggests that ghrelin may modulate insulin sensitivity and offer a potentially novel therapeutic approach for the cognitive dysfunction seen in Type 2 DM. However, the underlying mechanisms have not been fully elucidated.
Glucose is the primary energy source for mammalian brain and a continuous supply of this substrate is essential to maintain normal cerebral function. Currently,14 members of this sugar/polyol transporter protein family have been described. In the brain, although GLUT1 is expressed at the blood-brain barrier and in astrocytes, whereas GLUT3 is responsible for the neuronal glucose uptake independent insulin, GLUT4 translocation and consequently cellular glucose uptake in peripheral insulin-sensitive tissue as well as in some brain regions, such as in hippocampal neurons.
AD has two pathological hallmarks:P-amyloid (Aβ) deposition and neurofibrillary tangles, which is composed of hyperphosphorylation tau protein and positive relation with cognitive dysfunction. In the process of tau phosphorylation, GSK-3βplays an important role. Schubert demonstrated that insulin signaling system disturbance resulted in Phosphatidylinositol-3 kinase (PI3-K)/Akt signaling pathway dysfunction, which caused GSK-3βactivity increase and led to tau hyperphosphorylation.
PI3-K/Akt is the classical insulin signaling transduction pathway. AS 160 is one of Akt substrate, a GTP enzymes activation protein with Rab protein specificity action, which has an importance to GLUT4 traslocation. The last study showed that AS 160 was an signaling molecule the most closed to GLUT4 translocation. Grillol et al found that neuronal insulin utilized similar signal transduction even as peripheral tissues to stimulate GLUT4 trafficking to the plasma membrane.
Evidences indicate that ghrelin may modulate insulin sensitivity, stimulate insulin-induced glucose uptake, moreover it also modulates memory. Therefore, we hypothesized that ghrelin may promote hippocampal neurons glucose uptake and improving insulin resistance exposure to high glucose, furthermore ghrelin may attenuate tau abnormal phosphorylation. In addition, examined potential mechanisms, including PI3-K/Akt-GSK-3βand GLUT4 expression. Further understand the function of ghrelin, and supply the theory for the relation of DM and AD.
Materials and Methods
Materials
1. The agent of primary rat hippocampal neurons
2. The agent of MTT
3. The agent of GOD-POD of neurons
4; The agent of [3H]-2-deoxy-D-glucose ([3H]-DG) of neurons
5. The agent of immunofluorescence stain
6. The agent of demi-quantitate RT-PCR to detect gene
7. The agent of western blotting cross hybridization
Methods
1. Rat hippocampal neurons isolate, culture and differentiation.
(1) Rat hippocampal neurons isolate
Hippocampal neurons were isolated from Sprague-Dawley neonate rats within 24 h of birth. The rats were decapitated and the heads were put in a 90 mm Petri dish placed on an ice-filled bag. The brains were removed from the skull and the bilateral hippocampi were derived. The hippocampi were carefully separated along with ablated vascellum and meninges, which had an extreme affect on neuronal purity. When all the hippocampi were isolated and washed twice in other Petri dishes using ice-cold D-Hank's, they were then chopped into approximately 1 mm3 pieces. The pieces were collected into a 50 ml conical tube, incubated in an equal volume of 0.25% trypsin/EDTA for approximately 30 min. To counteract the trypsin, an equal volume of a neuronal plating medium was added containing DMEM,10%heat-inactivated horse serum,10%heat-inactivated fetal bovine serum,1%glutamine,100 U/ml penicillin, and 100μg/ml streptomycin. This was centrifuged at 1200rmp for 10 min. The trypsinized hippocampi were triturated slowly and gently using a compatible plastic pipette until the tissue was completely dispersed. Then, neurons were slowly and gently dispersed with a plastic pipette and at 1.5×105, 5×105 or 2.5×106/well on polyethylenimine-coated 96,24 or 6 tissue culture plate and incubated in a humidified 5%CO2 incubator at 37℃.
(2) Rat hippocampal neurons primary culture
Neurons were incubated in DMEM, including 10%heat-inactivated horse serum, 10%heat-inactivated fetal bovine serum,1%glutamine,100 U/ml penicillin, and 100μg/ml streptomycin and incubated in a humidified 5%CO2 incubator at 37℃. A neurobasal medium with B27 was used instead of the culture medium after the first 24h. After 7 days, neurons will be used. Neurobasal medium contains 25mM glucose, therefore,3 times of the concentration (75mM) will be as high glucose concentration.
(3) Rat neurons immunohistochemical identification
Neurons were identified by NSE immunocytochemistry staining. Rat neurons were cleaned by PBS; and fixed by 4%paraformaldehyde for 20 min; 3%H2O2 blocked endogenous peroxydase 10min. Nonimmune animal serum for 15 min; The cells were then incubated overnight at 4℃the polyclonal anti-NSE-antibody (1:250); Immunoreactivity was detected using rabbit anti-rat IgG for 15min. Streptomycete avidin labeled HRP for 15 min. DAB colored and campeachy re-colored. Color status were recorded via inverted phase contrast microscope. Neurons accounted for about 90%of total cells and the experiments were executed on the 7th day of neurons being cultured.
2. Cell viability test
The viability of neuronal cells was determined via colorimetric 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay (MTT).0.5 mg/ml of MTT was added to each well for 4 h at 37℃. Medium with MTT was removed and 200μl of dimethylsulfoxide was added to each well to dissolve the prunosuscolored formazan particles. Absorbance was read at 490 nm with a microplate reader.
3. Neuronal glucose consumption via GOD-POD assay
Glucose consumption was detected with a glucose GOD-POD assay kit according to the manufacturer's protocol. Cells plated in 96-well plates at a concentration of 1.5×105 cells/well. At the cultured the 7 day, preliminary medium was instead of different treating factors. Detected the remnant glucose concentration of medium via GOD-POD assay, the glucose concentration of uncultured cells as basal data, then counting the cells'glucose consumption.
4. [3H]-2-deoxy-D-glucose uptake assay
Cultured neuronal cells were washed twice with Locke's buffer and incubated for 2 h at 37℃After incubation with or without insulin (100nM) for 30 min, [3H]-2-deoxy-D-glucose were added to the culture, and incubation was continued for 10 min. [3H]-2-deoxy-D-glucose was added to each culture well to give a final concentration of 600 nM (0.2μCi/ml). The cells were washed three times with ice-cold 0.1M PBS buffer and digested with 0.1 N NaOH. Cell lysates were used for determining radioactivity by means of a beta liquid scintillation counter and for an assay of total cell.protein. The experiment was carried out in triplicate with 5 wells per condition per replication.
5. Demi-quantitate RT-PCR assay
After the cells were washed twice with asepsis D-Hanks and added pre-cold TRIzol 1ml and to get out cell total RNA. To gather total RNA; To detect the putity and level of RNA; reverse transcription reaction; primer sequence and react condition; to prepare 3%agarose gel; electrophoresis; to detect electrophoresis density; to assay scanning flame. The OD value of GAPDH production as basal data was count each gene ratio, and semiquantitative analysis.
6. Immunocytochemical stain detected ptau[Ser 199] expression
Hippocampal neurons were cultured on coverslips. Rat neurons were cleaned by PBS; and fixed by 4%paraformaldehyde for 20 min; 3%H2O2 blocked endogenous peroxydase 10min. Nonimmune animal serum for 15 min; The cells were then incubated overnight at 4℃the polyclonal ptau[Serl99](1:500), GLUT4(1:250); Immunoreactivity was detected using rabbit anti-rat IgG for 15min. Streptomycete avidin labeled HRP for 15 min. DAB colored. Water flushed 5min, neutro-resin coverd. Color status were recorded via inverted phase contrast microscope.
7. Western blotting analysis pAkt [Ser473], ptau [Serl99], pGSK-3β[Ser9] and AS 160 phosphorylation
The cells were lysed using a RIPA lysis buffer. The protein underwent SDS-PAGE and was transferred to polyvinylidene fluoride membranes. After being washed three times with a 1×TBST (pH 7.5) buffer, the membranes were soaked in 3%BSA for 2 h at room temperature and incubated with the antibodies at 4℃overnight. Then the membranes were washed five times with the 1×TBST (pH 7.5) buffer again. After incubation with the HRP-conjugated secondary antibody for 2 h at room temperature, the immune complexes were visualized by enhanced chemiluminescence methods. The band intensity was measured and half quantitated. The resulting images were analyzed with Image Analysis Software V 7.0.
8. Statistical analysis
All data were expressed as means±SD and analyzed by SPSS 13.0 software. Statistical analysis of the data for multiple comparisons was performed by an analysis of variance (ANOVA) followed by post-hoc analysis. For single comparison, the significance of differences between means was determined by a t-test. p<0.05 was considered significant.
Results
1. Neuronal glucose metabolism decreased significantly in high glucose compared to in normal glucose; in 1-100nM ghrelin administration, there is no great change at lnM ghrelin, however, there is significant change at lOnM and 100nM ghrelin, in a dose-depent fashion. After 100nM insulin stimulated, this change is much more significant.
2. Immunocytochemical method was detected the change of phosphorylation. Tau hyperphosphorylation is colored deeper in high glucose group than in normal group, and treating with ghrelin made the color change light.
3. Western blotting was detected the change of tau phosphorylation. Tau hyperphosphorylation increased in high glucose group, ghrelin made the change much more. However, total tau protein had no change.
4. RT-PCR was detected the gene expression of GLUT3, GLUT4and PPARy. In primary cultured hippocampal neurons have the expression of GLUT4 besides GLUT3, however, the latter is much more than the former. With or without ghrelin and insulin, the expression of GLUT4 and PPARy have no change, which suggested that the effect of ghrelin improvement on insulin resistant was not via PPARy and on glucose uptake was not via the quantity of GLUT4 in short time. The change of glucose uptake may be linked with GLUT4 translocation.
5. Western blotting was detected the change of Akt, AS 160 and GSK-3βphosphorylation. Ghrelin increased the level of Akt, AS 160 and GSK-3p phosphorylation in different glucose concentration.Under high glucose group, the level of Akt, AS 160 and GSK-3βphosphorylation decreased significantly compared to normal group.Treated with ghrelin, Akt, AS 160 and GSK-3βphosphorylation increased greatly, however, total protein had no significant change.
6. Immunocytochemical stain detected GLUT4 translocation. GLUT4 was expressed in plasm and membrane. Under high glucose 24h, translocation was refrained, after insulin and/or ghrelin treatment,the action was improved.
7. Additon PI3-K specific inhibitor wortmannin (50nM) attenuated the effect of ghrelin on increase glucose uptake and improvement tau abnormal phosphorylaiton.
Conclusion
1. Hippocampal neuronal tau hyperphosphorylation and glucose metabolism decreased, existed insulin resistance in high glucose concentration probably.
2. Ghrelin increased hippocampal neuronal glucose metabolism in high glucose concentration and improved insulin resistance. Additon insulin, the effect increased significantly. Ghrelin and insulin is synergistic action on glucose metabolism.
3. Ghrelin decreased tau hyperphosphorylation resulted from high glucose.
4. Ghrelin regulated hippocampal neuronal glucose uptake via GLUT4 translocation probably at last.
5. Ghrelin ameliorated insulin resistance and tau hyperphosphorylation partly via PI3-K/Akt-GSK-3βsignaling pathway.
糖尿病(diabetes mellitus, DM)是由多种病因引起的胰岛素分泌绝对或相对不足以及靶细胞对胰岛素敏感性降低,继而引起糖、蛋白质、脂肪和水、电解质代谢异常的一种综合症,以慢性高血糖为主要特征。高血糖在2型糖尿病(T2DM)中不仅是次于胰岛素抵抗的重要体征,也是引起靶器官胰岛素抵抗的主要原因。作为一种系统性疾病,糖尿病可以引起多种组织、器官的结构和功能的改变,其中继发于糖尿病的视网膜、肾病及周围神经等病变已经得到人们广泛认识和关注。有研究表明高血糖是神经系统损伤的重要原因,糖尿病患者中痴呆的危险性明显增加,往往伴有认知功能障碍,如学习和记忆等脑功能损害。
流行病学研究显示伴有胰岛素抵抗的糖尿病患者有30-65%可发展为阿尔茨海默病(Alzheimer disease, AD)。最近也有研究发现大脑的胰岛素缺乏和脑内的胰岛素抵抗状态和AD发病有关,有学者提出AD可能是“3型糖尿病”。因此,认为胰岛素抵抗导致代谢紊乱和认知障碍是糖尿病和AD共同的病理机制。Ghrelin可以改善AD模型大鼠的认知动作。有研究显示ghrelin可以调节胰岛素的敏感性,并提出可能是一种潜在的新的治疗糖尿病和AD导致的脑功能异常的药物。但是潜在的机制目前国内外很少有人报道。
葡萄糖是哺乳动物脑内主要的能量来源,对维持正常的大脑功能有重要作用。目前发现葡萄糖转运蛋白(GLUTs)家族有14种亚型。在脑内,葡萄糖转运体1(GLUT1)主要在血脑屏障和胶质细胞中表达,葡萄糖转运体3 (GLUT3)负责神经细胞的葡萄糖转运且不依赖胰岛素。但是,这些葡萄糖转运体不能负责海马区的整个葡萄糖代谢,而且,GLUT3位于神经细胞的突起而不在细胞体,因此有位于细胞体的其它葡萄糖转运蛋白负责神经细胞的葡萄糖摄取和利用。在胰岛素敏感的外周组织,细胞通过GLUT4易位不断的摄取葡萄糖,同样这个过程也发生在大脑的某些区域,包括海马神经细胞。
神经纤维原缠结(neurofibrillary tangles, NFTs)是AD在组织病理学重要标志之一。NFTs由高度磷酸化的tau蛋白组成,其数量和认知障碍相关。在tau蛋白磷酸化的过程中,GSK-3p起到重要作用。磷脂酰肌醇3-激酶／蛋白激酶B(Phosphoinositide 3-kinase/Protein kinaseB, PI3-Kinase/AKT)是胰岛素信号转导的经典途径。胰岛素信号系统受损导致PI3-K/Akt信号通路障碍,引起GSK-3p活性增加,导致tau蛋白高度磷酸化。AS160是Akt底物,一种与Rab蛋白具有特异性作用的GTP酶激活蛋白(GAP),对GLUT4的易位有重要作用,最新的研究表明AS160是胰岛素信号中最接近GLUT4易位的信号分子。Grillol发现神经细胞中胰岛素利用同外周组织一样的信号转导系统刺激葡萄糖转运体4(GLUT4)向细胞膜易位摄取葡萄糖。
Ghrelin能调节胰岛素敏感性,刺激胰岛素诱导的葡糖糖摄取增加,有调节记忆的作用。因此,我们以原代培养的大鼠海马神经细胞为研究对象,探讨ghrelin对高糖诱导的神经细胞tau蛋白磷酸化和葡萄糖代谢的影响。进一步探讨其潜在的作用机制,包括PI3-K/Akt-GSK-3p、AS160及GLUT4表达的变化。进而深入了解ghrelin的功能,为DM是AD的危险因素提供进一步的理论依据,为临床改善糖尿病及AD引起的认知功能障碍提供的新的可能的治疗方向。
材料与方法
一、实验材料
1、原代乳鼠海马神经细胞培养相关试剂
2、MTT相关试剂
3、神经细胞葡萄糖消耗实验相关试剂
4、神经细胞葡萄糖摄取实验相关试剂
5、免疫细胞化学染色相关试剂
6、半定量RT-PCR检测基因的相关试剂
7、Western blotting印迹杂交相关试剂
二、实验方法
1、乳鼠海马神经细胞的提取、培养及鉴定
(1)乳鼠海马神经细胞的提取
取新生(出生24h以内)的SD大鼠,冰浴麻醉约10min,用75%乙醇消毒。断头后放在冰袋上的90mm培养皿中。去除颅骨,取出双侧的海马放入盛有预冷的D-Hanks液的培养皿中,逐一分离海马组织上的软脑膜和毛细血管网。当所有的海马组织分离后,清洗2次,放入培养皿中。将组织剪成1mm3的小块,移入50ml离心管中,加入与组织等体积的0.25%胰蛋白酶(含EDTA)。在37℃,5%CO2条件下消化30min左右,用含有血清的培养液终止反应,用lml移液器吹打混匀组织液。1200rmp,离心10min,弃上清,加入10ml含10%胎牛血清、10%马血清的DMEM培养基,用移液器轻微混悬细胞。用孔径为200目的尼龙网过滤后,血细胞计数板计数细胞。用种植液稀释细胞悬液,以1.5×105、5×105或2.5×106／孔的密度分别接种于事先用多聚赖氨酸浸泡过的96孔培养孔板、24孔培养孔板或6孔培养孔板内,将培养板移入湿润的培养箱,在37℃和5%CO2条件下培养细胞。
(2)乳鼠海马神经细胞的原代培养
将提取到的神经细胞用DMEM培养基(含10%胎牛血清、10%马血清、1%谷氨酰胺和100U／ml青霉素、100μg／ml链霉素)在37℃,5%CO2的条件下湿润的培养箱中培养,于接种24h后全量换液,换成Neurobasal Medium/B27无血清培养基,以后每2天换液1／3—1／2,待7天后,细胞生长成熟可以用于实验。NeurobasalMedium培养液的葡萄糖浓度是25mM,高于其3倍的浓度可以视为高糖(75mM)环境。
(3)乳鼠海马神经细胞免疫细胞化学染色鉴定
用神经元烯醇化酶(NSE)抗体作为神经细胞的识别标志。当细胞生长成熟后,用PBS清洗一次；4%的多聚甲醛溶液在室温固定20min; 3%H2O2阻断内源性过氧化物酶10min；加入非免疫性动物血清封闭液(正常山羊血清),20℃下孵育15min；加入一抗兔抗鼠NSE多克隆抗体(1：250稀释),4℃水盒孵育过夜；滴加二抗(生物素标记的山羊抗兔IgG)20℃下15min,辣根过氧化酶(HRP)标记的链霉菌抗生物素蛋白孵育15min,新鲜配制的DAB显色,苏木精复染,倒置相差显微镜照相记录染色情况。
2、MTT法检测细胞活性和数量
将5mg/ml的MTT原液与无血清的DMEM培养液按体积1：9配成MTT培养液,待孔内原有培养液移出后,每孔加入MTT培养液50μl,37℃继续培养,4h后终止培养,并小心吸弃孔内培养上清夜,每孔加入200μl二甲基亚砜(DMSO),微型震荡器震荡10min使结晶物充分溶解。选择490nm波长,在酶标仪上测定各孔的吸光度值(OD值),以光密度值反映细胞活性和数量的多少。
3、神经细胞葡萄糖消耗实验
神经细胞的葡萄糖消耗量通过葡萄糖氧化酶-过氧化物酶(GOD-POD)法来检测。细胞以1.5×105个／孔接种在96孔板中。在培养第7天,原始的培养基换成不同的处理因素。按各实验组细胞经分组处理后用于实验。将各样本浓度稀释5倍,取2μl放入200μl的检测液中,在室温反应30min达到平衡并保持稳定,在490nm酶标仪中测定各管吸光度值(OD值),根据公式计算葡萄糖浓度,再乘以稀释倍数,与未接种细胞的空白孔的含量糖均值相减,计算葡萄糖的消耗量。
4、[3H]-2-deoxy-D-glucose ([3H]-DG)示踪法检测神经细胞葡萄糖摄取的变化
海马神经细胞培养成熟后按各实验组处理后用于实验。Lock's Buffor缓冲液洗涤细胞3次,用400μl缓冲液37℃孵育细胞30min。弃去缓冲液,各组加或不加胰岛素共同继续孵育30min。加入[3H]-DG至终浓度0.2μCi/ml 30min。用预冷的PBS终止反应,冲洗3次,将细胞裂解于O.1N NaOH中,反复吹打后,在37℃中裂解2h,吹打混匀细胞,将各样本液体滴在微孔滤纸上,经真空滤泵后,室温晾干。将微孔滤膜浸入12ml液闪液中,液体闪烁计数法测定检测各组的每分钟总衰变数(CPM)。测定细胞蛋白浓度。计算葡萄糖摄取量。每组重复5次。
5、RT-PCR法检测GLUT3、4, PPARymRNA的表达
用于实验的细胞按分组分别干预后,弃去培养液,在无菌条件下用D-Hanks液轻微冲洗2次,加入预冷的1ml TRIzol,反复吹打,充分裂解细胞后移入新的经DEPC水处理过的无菌Eppendoff (Ep)管中,采用一步法提取总RNA。RNA纯度及浓度的测定；逆转录合成cDNA;引物序列及反应条件；3%琼脂糖凝胶电泳；紫外透射仪观察,应用凝胶图像处理系统扫描各条带的光密度,以GAPDH产物的光密度值作为内参,计算不同条件下各基因扩增量的比值,进行半定量分析。
6、免疫细胞化学染色检测ptau[Ser199]、GLUT4的表达
细胞接种在盖玻片上,培养7d后,用4%的多聚甲醛溶液在室温固定20min；每张盖玻片加3%H202阻断内源性过氧化物酶10min, PBS洗涤3次,每次5min；加入非免疫性动物血清封闭液(正常山羊血清),20℃下孵育15min；加入一抗为兔抗鼠的ptau[Ser199](1:500稀释)、GLUT4 (1:250稀释)多克隆抗体,4℃水盒孵育过夜；滴加二抗(生物素标记的山羊抗兔IgG)20℃下15min,辣根过氧化酶(HRP)标记的链霉菌抗生物素蛋白孵育15min,新鲜配制的DAB显色,自来水冲洗5min,60℃烘干后中性树脂封片。倒置相差显微镜照相记录染色情况。
7、Western blotting检测pAkt[Ser473]、ptau[Serl99]、pGSK-3p[Ser9]和AS160磷酸化
实验细胞用预冷PBS缓冲液洗2遍,细胞刮收集细胞,根据蛋白提取试剂盒操作分别提取各组细胞总蛋白,采用Bradford法测定蛋白浓度,每组蛋白质样本20μg在95℃下变性5min,进行SDS—PAGE电泳,转膜后3%BSA封闭2h,用TBST洗膜后分别加5%BSA/TBS稀释的多克隆抗体,4℃孵育过夜,再次用TBST洗膜后,加入辣根过氧化物酶标记的二抗在室温下杂交2h,洗膜后ECL超敏发光液进行显色,每组重复3次,结果用Image Analysis Softwave V7.0分析软件对目的条带进行灰度值分析。
8、统计学分析
所有数据用SPSS 13.0软件处理,结果用均数±标准差(x±SD)表示,每组实验重复3次或3次以上。采用单因素方差分析(ANOVA)检验,post-hoc分析,t检验进行均数间的多重比较,以p<0.05为统计学有显著差异。
结果
1、高糖环境对神经细胞的糖代谢有抑制作用；ghrelin可以增加神经细胞的葡萄糖代谢。经过不同浓度的ghrelin (1-100nM)干预,神经细胞在1nM作用下糖代谢有所增加但是没有明显变化,在10nM和100nM作用下糖代谢明显增加,呈剂量依赖性,经100nM胰岛素诱导后增加更明显。
2、免疫细胞化学法检测tau蛋白磷酸化。高糖环境下,tau蛋白磷酸化增加,表现为胞浆染色和正常糖环境相比明显加深,经过ghrelin干预后,胞浆染色变浅,tau蛋白磷酸化减少。
3、Western blotting检测tau蛋白磷酸化的变化。高糖组tau蛋白磷酸化增加,ghrelin可以减弱tau蛋白磷酸化。总蛋白无明显变化。
4、RT-PCR检测GLUT3、GLUT4和PPARy的基因表达。在原代培养的海马神经细胞中除GLUT3表达外,也有GLUT4的表达,但是后者比前者的表达量要少。在高糖环境下,ghrelin和胰岛素干预前后神经细胞中GLUT4和PPARy的基因表达没有发生变化。说明在短时间内ghrelin可能并不是通过PPARy变化增加胰岛素敏感性的；对葡萄糖摄取的改善不是通过GLUT4量的改变实现的,可能和GLUT4易位有关。
5、Western blotting法检测Akt、AS160及GSK-3β磷酸化的变化。Ghrelin可以增加不同葡萄糖浓度的神经细胞Akt、AS 160及GSK-3β磷酸化水平。在高糖组Akt. AS 160和GSK-3β磷酸化水平较正常组降低明显。经过ghrelin干预后,Akt、AS 160和GSK-3β磷酸化增加明显,总蛋白变化不明显。
6、免疫细胞化学法检测GUT4易位的变化。GLUT4在正常对照组的胞浆和胞膜均有表达,为棕色至棕褐色,经高糖作用24h后,易位受到抑制,经ghrelin干预后,易位增加,经胰岛素诱导后这种作用增加更明显。
7、加入PI3-K特异性抑制剂wortmannin(50nM)和ghrelin共同作用,可以削弱ghrelin改善tau蛋白磷酸化和增加葡萄糖摄取的作用。
结论
1、高糖环境能使海马神经细胞tau蛋白磷酸化增加,糖代谢降低,可能存在胰岛素抵抗。
2、Ghrelin能增加高糖诱导的神经细胞的糖代谢,改善胰岛素抵抗。而加入胰岛素后,这种作用更明显。Ghrelin和胰岛素促进神经细胞糖代谢是协同作用。
3、Ghrelin可以降低高糖导致的tau蛋白异常磷酸化。
4、Ghrelin对海马神经细胞的葡萄糖摄取的调节最终可能是通过GLUT4易位实现的。
5、Ghrelin增加高糖诱导的神经细胞的Akt、AS 160和GSK-3p磷酸化水平,这种作用在正常糖浓度下更明显。Ghrelin至少是通过P13-K/Akt-GSK-3β途径改善tau蛋白异常磷酸化和胰岛素抵抗。
Ghrelin is a 28-amino acid peptide secreted predominantly from X/A-like cells of the gastric fundus as the endogenous ligand for the growth hormone (GH) secretagogue-receptor la (GHS-Rla). Circulating hormone has two forms:unacylated ghrelin (UAG) and acylated ghrelin (AG), which is essential for ghrelin bioactivity. Ghrelin has been on its role in neuroendocrine regulation in the central nervous system, for instance modulating appetite and food take, energy and glucose homeostasis, learning and memory performance. The data confirmed that ghrelin is a key mediator of hippocampal-dependent learning processes.
Diabetes mellitus is an endocrine disorder of carbohydrate metabolism resulting from inadequate insulin release or insulin insensitivity, then causing sugar, protein, water abnormal metabolism. Hyperglycemia, as diabetic fundamental biochemistry character, has an important role in diabetes. Hyperglycemia in type 2 diabetes is not only a secondary manifestation of insulin resistance, but could also be responsible for directly inducing insulin resistance in the target tissue. Studies have been shown that hyperglycemia is a primary reason for nervous system impaired. Diabetic demented danger increased significantly, generally accompany with cognition dysfunction, such as learning and memory. Epidemiology studies have shown that diabetic patients with insulin resistant have a 30-65%increased risk of developing AD. Recent data indicate that brain insulin deficiency and the insulin-resistant brain state are related to Alzheimer's disease (AD), de la suggests that AD could be "Type 3 diabetes". Therefore, the common pathomechanism of diabetic encephalopathy and AD was insulin resistance, which led to metabolic disturbance and caused cognitive dysfunction. Ghrelin improved the cognitive action of AD model rats. Recent study suggests that ghrelin may modulate insulin sensitivity and offer a potentially novel therapeutic approach for the cognitive dysfunction seen in Type 2 DM. However, the underlying mechanisms have not been fully elucidated.
Glucose is the primary energy source for mammalian brain and a continuous supply of this substrate is essential to maintain normal cerebral function. Currently,14 members of this sugar/polyol transporter protein family have been described. In the brain, although GLUT1 is expressed at the blood-brain barrier and in astrocytes, whereas GLUT3 is responsible for the neuronal glucose uptake independent insulin, GLUT4 translocation and consequently cellular glucose uptake in peripheral insulin-sensitive tissue as well as in some brain regions, such as in hippocampal neurons.
AD has two pathological hallmarks:P-amyloid (Aβ) deposition and neurofibrillary tangles, which is composed of hyperphosphorylation tau protein and positive relation with cognitive dysfunction. In the process of tau phosphorylation, GSK-3βplays an important role. Schubert demonstrated that insulin signaling system disturbance resulted in Phosphatidylinositol-3 kinase (PI3-K)/Akt signaling pathway dysfunction, which caused GSK-3βactivity increase and led to tau hyperphosphorylation.
PI3-K/Akt is the classical insulin signaling transduction pathway. AS 160 is one of Akt substrate, a GTP enzymes activation protein with Rab protein specificity action, which has an importance to GLUT4 traslocation. The last study showed that AS 160 was an signaling molecule the most closed to GLUT4 translocation. Grillol et al found that neuronal insulin utilized similar signal transduction even as peripheral tissues to stimulate GLUT4 trafficking to the plasma membrane.
Evidences indicate that ghrelin may modulate insulin sensitivity, stimulate insulin-induced glucose uptake, moreover it also modulates memory. Therefore, we hypothesized that ghrelin may promote hippocampal neurons glucose uptake and improving insulin resistance exposure to high glucose, furthermore ghrelin may attenuate tau abnormal phosphorylation. In addition, examined potential mechanisms, including PI3-K/Akt-GSK-3βand GLUT4 expression. Further understand the function of ghrelin, and supply the theory for the relation of DM and AD.
Materials and Methods
Materials
1. The agent of primary rat hippocampal neurons
2. The agent of MTT
3. The agent of GOD-POD of neurons
4; The agent of [3H]-2-deoxy-D-glucose ([3H]-DG) of neurons
5. The agent of immunofluorescence stain
6. The agent of demi-quantitate RT-PCR to detect gene
7. The agent of western blotting cross hybridization
Methods
1. Rat hippocampal neurons isolate, culture and differentiation.
(1) Rat hippocampal neurons isolate
Hippocampal neurons were isolated from Sprague-Dawley neonate rats within 24 h of birth. The rats were decapitated and the heads were put in a 90 mm Petri dish placed on an ice-filled bag. The brains were removed from the skull and the bilateral hippocampi were derived. The hippocampi were carefully separated along with ablated vascellum and meninges, which had an extreme affect on neuronal purity. When all the hippocampi were isolated and washed twice in other Petri dishes using ice-cold D-Hank's, they were then chopped into approximately 1 mm3 pieces. The pieces were collected into a 50 ml conical tube, incubated in an equal volume of 0.25% trypsin/EDTA for approximately 30 min. To counteract the trypsin, an equal volume of a neuronal plating medium was added containing DMEM,10%heat-inactivated horse serum,10%heat-inactivated fetal bovine serum,1%glutamine,100 U/ml penicillin, and 100μg/ml streptomycin. This was centrifuged at 1200rmp for 10 min. The trypsinized hippocampi were triturated slowly and gently using a compatible plastic pipette until the tissue was completely dispersed. Then, neurons were slowly and gently dispersed with a plastic pipette and at 1.5×105, 5×105 or 2.5×106/well on polyethylenimine-coated 96,24 or 6 tissue culture plate and incubated in a humidified 5%CO2 incubator at 37℃.
(2) Rat hippocampal neurons primary culture
Neurons were incubated in DMEM, including 10%heat-inactivated horse serum, 10%heat-inactivated fetal bovine serum,1%glutamine,100 U/ml penicillin, and 100μg/ml streptomycin and incubated in a humidified 5%CO2 incubator at 37℃. A neurobasal medium with B27 was used instead of the culture medium after the first 24h. After 7 days, neurons will be used. Neurobasal medium contains 25mM glucose, therefore,3 times of the concentration (75mM) will be as high glucose concentration.
(3) Rat neurons immunohistochemical identification
Neurons were identified by NSE immunocytochemistry staining. Rat neurons were cleaned by PBS; and fixed by 4%paraformaldehyde for 20 min; 3%H2O2 blocked endogenous peroxydase 10min. Nonimmune animal serum for 15 min; The cells were then incubated overnight at 4℃the polyclonal anti-NSE-antibody (1:250); Immunoreactivity was detected using rabbit anti-rat IgG for 15min. Streptomycete avidin labeled HRP for 15 min. DAB colored and campeachy re-colored. Color status were recorded via inverted phase contrast microscope. Neurons accounted for about 90%of total cells and the experiments were executed on the 7th day of neurons being cultured.
2. Cell viability test
The viability of neuronal cells was determined via colorimetric 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay (MTT).0.5 mg/ml of MTT was added to each well for 4 h at 37℃. Medium with MTT was removed and 200μl of dimethylsulfoxide was added to each well to dissolve the prunosuscolored formazan particles. Absorbance was read at 490 nm with a microplate reader.
3. Neuronal glucose consumption via GOD-POD assay
Glucose consumption was detected with a glucose GOD-POD assay kit according to the manufacturer's protocol. Cells plated in 96-well plates at a concentration of 1.5×105 cells/well. At the cultured the 7 day, preliminary medium was instead of different treating factors. Detected the remnant glucose concentration of medium via GOD-POD assay, the glucose concentration of uncultured cells as basal data, then counting the cells'glucose consumption.
4. [3H]-2-deoxy-D-glucose uptake assay
Cultured neuronal cells were washed twice with Locke's buffer and incubated for 2 h at 37℃After incubation with or without insulin (100nM) for 30 min, [3H]-2-deoxy-D-glucose were added to the culture, and incubation was continued for 10 min. [3H]-2-deoxy-D-glucose was added to each culture well to give a final concentration of 600 nM (0.2μCi/ml). The cells were washed three times with ice-cold 0.1M PBS buffer and digested with 0.1 N NaOH. Cell lysates were used for determining radioactivity by means of a beta liquid scintillation counter and for an assay of total cell.protein. The experiment was carried out in triplicate with 5 wells per condition per replication.
5. Demi-quantitate RT-PCR assay
After the cells were washed twice with asepsis D-Hanks and added pre-cold TRIzol 1ml and to get out cell total RNA. To gather total RNA; To detect the putity and level of RNA; reverse transcription reaction; primer sequence and react condition; to prepare 3%agarose gel; electrophoresis; to detect electrophoresis density; to assay scanning flame. The OD value of GAPDH production as basal data was count each gene ratio, and semiquantitative analysis.
6. Immunocytochemical stain detected ptau[Ser 199] expression
Hippocampal neurons were cultured on coverslips. Rat neurons were cleaned by PBS; and fixed by 4%paraformaldehyde for 20 min; 3%H2O2 blocked endogenous peroxydase 10min. Nonimmune animal serum for 15 min; The cells were then incubated overnight at 4℃the polyclonal ptau[Serl99](1:500), GLUT4(1:250); Immunoreactivity was detected using rabbit anti-rat IgG for 15min. Streptomycete avidin labeled HRP for 15 min. DAB colored. Water flushed 5min, neutro-resin coverd. Color status were recorded via inverted phase contrast microscope.
7. Western blotting analysis pAkt [Ser473], ptau [Serl99], pGSK-3β[Ser9] and AS 160 phosphorylation
The cells were lysed using a RIPA lysis buffer. The protein underwent SDS-PAGE and was transferred to polyvinylidene fluoride membranes. After being washed three times with a 1×TBST (pH 7.5) buffer, the membranes were soaked in 3%BSA for 2 h at room temperature and incubated with the antibodies at 4℃overnight. Then the membranes were washed five times with the 1×TBST (pH 7.5) buffer again. After incubation with the HRP-conjugated secondary antibody for 2 h at room temperature, the immune complexes were visualized by enhanced chemiluminescence methods. The band intensity was measured and half quantitated. The resulting images were analyzed with Image Analysis Software V 7.0.
8. Statistical analysis
All data were expressed as means±SD and analyzed by SPSS 13.0 software. Statistical analysis of the data for multiple comparisons was performed by an analysis of variance (ANOVA) followed by post-hoc analysis. For single comparison, the significance of differences between means was determined by a t-test. p<0.05 was considered significant.
Results
1. Neuronal glucose metabolism decreased significantly in high glucose compared to in normal glucose; in 1-100nM ghrelin administration, there is no great change at lnM ghrelin, however, there is significant change at lOnM and 100nM ghrelin, in a dose-depent fashion. After 100nM insulin stimulated, this change is much more significant.
2. Immunocytochemical method was detected the change of phosphorylation. Tau hyperphosphorylation is colored deeper in high glucose group than in normal group, and treating with ghrelin made the color change light.
3. Western blotting was detected the change of tau phosphorylation. Tau hyperphosphorylation increased in high glucose group, ghrelin made the change much more. However, total tau protein had no change.
4. RT-PCR was detected the gene expression of GLUT3, GLUT4and PPARy. In primary cultured hippocampal neurons have the expression of GLUT4 besides GLUT3, however, the latter is much more than the former. With or without ghrelin and insulin, the expression of GLUT4 and PPARy have no change, which suggested that the effect of ghrelin improvement on insulin resistant was not via PPARy and on glucose uptake was not via the quantity of GLUT4 in short time. The change of glucose uptake may be linked with GLUT4 translocation.
5. Western blotting was detected the change of Akt, AS 160 and GSK-3βphosphorylation. Ghrelin increased the level of Akt, AS 160 and GSK-3p phosphorylation in different glucose concentration.Under high glucose group, the level of Akt, AS 160 and GSK-3βphosphorylation decreased significantly compared to normal group.Treated with ghrelin, Akt, AS 160 and GSK-3βphosphorylation increased greatly, however, total protein had no significant change.
6. Immunocytochemical stain detected GLUT4 translocation. GLUT4 was expressed in plasm and membrane. Under high glucose 24h, translocation was refrained, after insulin and/or ghrelin treatment,the action was improved.
7. Additon PI3-K specific inhibitor wortmannin (50nM) attenuated the effect of ghrelin on increase glucose uptake and improvement tau abnormal phosphorylaiton.
Conclusion
1. Hippocampal neuronal tau hyperphosphorylation and glucose metabolism decreased, existed insulin resistance in high glucose concentration probably.
2. Ghrelin increased hippocampal neuronal glucose metabolism in high glucose concentration and improved insulin resistance. Additon insulin, the effect increased significantly. Ghrelin and insulin is synergistic action on glucose metabolism.
3. Ghrelin decreased tau hyperphosphorylation resulted from high glucose.
4. Ghrelin regulated hippocampal neuronal glucose uptake via GLUT4 translocation probably at last.
5. Ghrelin ameliorated insulin resistance and tau hyperphosphorylation partly via PI3-K/Akt-GSK-3βsignaling pathway.
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3 Hebert L E, Scherr PA, Bienias J L, et al. Alzheimer disease in the US population:prevalence estimates using the 2000 census. Arch.Neurol.2003; 60:1119-1122.
4 Arvanitakis Z, Wilson R S, Bienias J L, et al. Diabetes mellitus and risk of Alzheimer disease and decline in cognitive function. Arch.Neurol.2004; 61:661-666.
5 Biessels G J, Bravenboer B, Gispen W H, et al. Glucose, insulin and the brain—modulation of cognition and synaptic plasticity in health and disease:a preface. Eur.J. Pharmacol.2004; 490: 1-4.
6 Qiu W Q, Folstein M F. Insulin,insulin-degrading enzyme and amyloid-beta peptide in Alzheimer's disease:review and hypothesis. Neurobiol Aging.2006; 27:190-198.
7 Wild S, Oglic G, Reen A, et al. Global prevalence of diabetes:estimates for the year 2000 and projections for 2030. Diabetes Care.2004; 27:1047-1053.
8 Schnaider Beeri M, Goldbourt U, Silverman J M, et al. Diabetes mellitus in midlife and the risk of dementia three decades later. Neurology.2004; 63:1902-1907.
9 Haan M N. Therapy Insight:type 2 diabetes mellitus and the risk of late-onset Alzheimer's disease. Nat Clin Pract Neurol.2006; 2:159-166.
10 Rivera E J, Goldin A, Fulmer N, et al. Insulin and insulin-like growth factor expression and function deteriorate with progression of Alzheimer's disease:link to brain reductions in acetylcholine. J.Alzheimer's Dis.2005; 8:247-268.
11 Vanhanen M, Soininen H. Glucose intolerance,cognitive impairment and Alzheimer s disease. Curr Opin Neurol.1998; 11:673-677.
12 Peila R, Rodriguez, B.L, Launer LJ,et al. Type 2 diabetes, APOE gene, and the risk for dementia and related pathologies:The Honolulu-Asia Aging Study. Diabetes.2002; 51: 1256-1262.
13 Hoyer S. Causes and consequences of disturbances of cerebral glucose metabolism in sporadic Alzheimer disease:therapeutic implications. Adv.Exp.Med.Biol.2004; 541:135-152.
14 Liu Y, Liu F, Iqbal K, et al. Decreased glucose transporters correlate to abnormal hyperphosphorylation of tau in Alzheimer disease. FEBS Letters.2008; 582:359-364.
15 Scheepers A, Joost H G, Schumann A. The glucose transporter families SGLT and GLUT: molecular basis of normal and aberrant function. JPEN J.Parenter.Enteral.Nutr.2004; 28: 364-371.
16 McEwen B S, Reagan L P. Glucose transporter expression in the central nervous system: relationship to synaptic function. Eur.J.Pharmacol.2004; 490:13-24.
17 Schubert D. Glucose metabolism and Alzheimer's disease. Ageing Res. Rev.2005; 4:240-257.
18 Qutub A A, Hunt C A. Glucose transport to the brain:a systems model. Brain Res. BrainRes.Rev. 2005; 49:595-617.
19 Dwyer D S, Vannucci S, et al. Expression, regulation, and functional role of glucose transporters (GLUTs) in brain. IntRev.Neurobiol.2002; 51:159-188.
20 Yu S, Zhao T, Guo M, et al. Hypoxic preconditioning up-regulates glucose transport activity and glucose transporter (GLUT1 and GLUT3) gene expression after acute anoxic exposure in the cultured rat hippocampal neurons and astrocytes. Brain research.2008; 1211:22-29.
21 Etsuro Uemura. Heather West Greenlee. Amyloid β-Peptide inhibits neuronal glucose uptake by preventing exocytosis. Experimental Neurology.2001; 170:270-276.
22 Choeiri C, Staines W, Miki T, et al. Glucose transporter plasticity during memory processing. Neuroscience.2005; 130:591-600.
23 Ho, Qin L, Pompl W, et al. Diet-induced insulin resistance promotes amyloidosis in a transgenic mouse model of Alzheimer's disease. FASEB J.2004; 18:902-904.
24 Adam R C, Arlene A, Green C, et al. Molecular connexions between dementia and diabetes. Neuroscience and Biobehavioral Review.2007; 31:1046-1063.
25 Zhao W Q, Chen H, Quon M J, et al. Insulin and insulin receptor in experimental models of learning and memory. Eur J Pharmacol.2004; 490:71-81.
26 Park C R. Cognitive effects of insulin in the central nervous system. Neurosci Biobehav Rev. 2001; 25:311-323.
27 Schulingkamp R J, Pagano T C, Hung D, et al. Insulin rceptors and insulin action in the brain:review and clinical implication. Neurosci Biobehav Rev.2000; 24:855-872.
28 Zhao W Q, Alkon D L. Role of insulin and insulin receptor in learning and memory. Molecular and Cellular Endocrinology.2001; 177:125-134.
29 Hutchinsona D S, Summers R J, Gibbs M E, et al. Energy metabolism and memory processing:Role of glucose transport and glycogen in responses to adrenoceptor activation in the chicken. Brain Research Bulletin.2008; 76:224-234.
30 Cole G M, Frautschy S A. The role of insulin and neurotrophic factor signaling in brain aging and Alzheimer's Disease. Experimental Gerontology.2007; 42:10-21.
31 Pickup J C. Inflammation and activated innate immunity in the pathogenesis of type 2 diabetes. Diabetes Care.2004; 27:813-823.
32 van der Heide L P, Ramakers G M, Smidt M P. Insulin signaling in the central nervous system: Learning to survive. Progress in Neurobiology.2006; 79:205-221.
33 Sui L,Wang J, Li B M. Role of the phosphoinositide 3-kinase-Akt-mammalian target of the rapamycin signaling pathway in long-term potentiation and trace fear conditioning memory in rat medial prefrontal cortex. Lear Mem.2008; 15:762-776.
34 Brazil D P, Hemmings B A. Ten years of protein kinase B signalling:a hard Akt to follow. Trends Biochem Sci.2001; 26:657-664.
35 Martin D, Salinas M, Lopez-Valdaliso R, et al. Effect of the Alzheimer amyloid fragment Abeta(25-35)on Akt/PKB kinaseand survival of PC12 cells. J Neurochem.2001; 78:1000-1008.
36孟艳,张景艳,王蓉,等人神经母细胞瘤株SY5Y细胞P13-K信号转导通路的研究.首都医科大学学报.2008：29：167-170.
37 Takahashi M, Tomizawa K, Ishiguro K. Distribution of tau protein kinaseI/glucogen synthase kinase-3 beta,phosphatases 2A and 2B, and phosphorylated tau in the developing rat brain. Brain Res.2000; 857:193-206.
38 Schubert M, Gautam D, Surjo D, et al. Rolefor neuronal insulin resistance in neurodegenetative diseases. Pro Natl Acad Sci.2004; 101:3100-3105.
39 Li X, Lu F, Tian Q, et al. Activarionofglyeogensynrhasekinase-3 induees Alzheimer like tauhy PerPhos Phory lation in rat hippocampus slices in culture. J Neural. Transm.2006; 113:93-102.
40 Mushii M, Grande L, Hayes M, et al. Congnitive dysfunction is associated with poor diabetes control in older adults. Diabetes Care.2006; 29:1794-1799.
41 Last D, Alsop D C, Abdulhalil A M, et al. Global and regional effects of type 2 diabetes on brain tissue voumes and cerebral vasoreactivity. Diabetes Care.2007; 30:1193-1199.
42 Watson GS, Craf S. Insulin resistance, inflammation, and cognition in Alzheimer's Disease: Lessons for multiple sclerosis. Journal of the Neurological Sciences.2006; 245:21-33.
43 McNay E C. Insulin and ghrelin peripheral hormones modulating memory and hippocampal function. Current Opinion in Pharmacology.2007; 7:628-632.
44 Carlini V P, Varas M M, Cragnolini A B, et al. Differential role of the hippocampus,amygdala,and dorsal raphe nucleus in regulating feeding,memory,and anxiety like behavioral responses to ghrelin. Biochem Biophys Res Commun.2004; 313:635-641.
45 Diano S, Farr S A, Benoit S C, et al. Ghrelin controls hippocampal spine synapse density and memory performance. Nat Neurosci.2006; 9:381-388.
46 Rasgon N L, Kenna H A. Insulin resistance in depressive disorders and Alzheimer's disease: Revisiting the missing link hypothesis. Neurobiology of Aging.2005; 26:103-107.
47 Carlini V P, Varas M M, Cragnolini A B, et al. Differential role of the hippocampus, amygdala, and dorsal raphe nucleus in regulating feeding, memory, and anxiety-like behavioral responses to ghrelin. Biochemical and Biophysical Research Communications.2004; 313:635-641.
48 Katsuya D, Hedeyuki S, Toshihiko Y. Ghrelin is a physiological regulator of insulin release in pancreatic islets and glucose homeostasis. Pharmacology& Therapeutics.2008; 118: 239-249.