CART对2型糖尿病大鼠下丘脑及胰岛β细胞的作用及机制研究
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摘要
1.背景与目的:
2型糖尿病是一种主要由于胰岛素抵抗伴随相对胰岛素不足,或胰岛素分泌缺陷伴有或不伴有胰岛素抵抗而导致慢性高血糖的代谢疾病,占糖尿病总数的90%-95%。2型糖尿病的重要原因之一是由于激素调节紊乱所致的饮食不当而产生的,因此可卡因-苯丙胺调节转录肽(cocaine- and amphetamine-related transcript peptide, CART)在调控进食行为和体内能量平衡方面的作用受到广泛关注。CART分布在许多与进食等相关的脑区,尤其是在下丘脑背内侧核(dorsomedial nucleus,DMH)、腹内侧(ventromedial nucleus,VMH)、弓状核(areuate nucleus,ARC)、外侧下丘脑(1ateral hypothalamus,LH)和室旁核(paraventricular nucleus,PVN)中分布较多。下丘脑不同核团之间构成复杂的调控网络,通过分泌神经递质以及神经元间的投射通路在机体摄食活动和能量平衡调节活动中发挥重要作用。研究表明CART与某些神经递质共存于某些神经元,提示CART参与调节进食行为。CART除了在中枢和外周神经系统中分布外,外周组织也有分布如交感节前神经纤维、胃窦G细胞、胰岛生长抑素细胞(islet somatostatin cells)、肠肌丛等,表明CART是一种脑肠肽,在胃肠道及中枢、外周神经系统中均能发挥作用。上述研究表明CART作为内源性神经递质,在奖赏与强化、食欲、应激、感知、内分泌等生理过程中发挥重要的作用。
因此在本课题中,我们将从整体动物和和细胞水平探讨CART在2型糖尿病大鼠下丘脑及胰岛β细胞中的表达及其作用机制。期望为深入认识CART的生物学功能及其调控机制提供新的实验依据,对CART的深入研究将使CART可能成为基因治疗2型糖尿病的新药物靶点。
2.方法:
2.1 2型糖尿病大鼠模型的建立及血中FBG、TC、TG、FFA和FINS的检测大鼠高脂饲料喂养4周,禁食12h后腹腔注射链脲佐菌素(Streptozotocin,STZ) 35mg/kg,用于建立2型糖尿病模型,正常对照组大鼠腹腔注射等体积的柠檬酸缓冲液。1周后,挑选空腹血糖值≥16.7 mmol/L作为糖尿病模型大鼠。建模前、后,按相应方法进行血中空腹血糖(fasting blood glucose, FBG)、三酰甘油(Triglyceride, TG)、总胆固醇(Total cholesterol, TC)、游离脂肪酸(Free fatty acid, FFA)、血清胰岛素(fasting insulin levels, FINS)等指标的检测。
2.2观察CART对2型糖尿病大鼠下丘脑定位核团进食量的影响及饮食干预对CART mRNA的表达
使用脑立体定位仪结合大鼠脑图谱,按照各核团三维坐标,进行准确定位。术后给予大鼠抗感染措施,恢复7d后进行实验,并在实验结束后,对导管植入位置进行尼森染色确定定位结果的准确性。使用微量注射器将各浓度CART注射入禁食各实验组的ARC、DMH及PVN核团中,或不禁食各实验组的ARC核团中,药物注射后1,2,4,8,24h测定进食量。采用荧光实时定量PCR观察饮食干预后糖尿病大鼠ARC中CART mRNA的水平变化。
2.3探讨神经肽Y(NPY)与CART对进食行为的调节及CART对其他神经递质释放的影响
使用微量注射器将CART或NPY注射入禁食各实验组的ARC核团中,药物注射后1,2h测定进食量。采用免疫荧光双标分析糖尿病大鼠PVN核团中NPY、CART的表达。通过下丘脑脑片孵育,放免法测定CART对下丘脑重要神经递质NPY、AGRP、CRH和α-MSH的释放。
2.4探讨PKA/CREB信号通路在糖尿病大鼠ARC中的作用采用原位杂交和Western blotting技术,在2型糖尿病大鼠ARC内探讨PKA/CREB参与CART mRNA和蛋白的转录调节中的作用。
2.5探讨钙离子内流激活的ERK信号通路在CART调节下丘脑神经元磷酸化中的作用
首先对大鼠下丘脑神经元进行培养,通过Western blotting分析ERK1/2和磷酸化ERK1/2的蛋白表达,用Fluo-3/AM钙荧光探针标记细胞内游离Ca~(2+),激光共聚焦测定神经元[Ca~(2+)]i的变化研究CART对ERK1/2信号通路的调节。
2.6探讨CART在体外细胞模型和2型糖尿病大鼠胰岛β细胞中的表达及作用机制
培养大鼠胰岛β细胞瘤细胞INS-1,观察CART对INS-1细胞胰岛素分泌的影响。研究CART对分离、纯化后的糖尿病大鼠胰岛细胞胰岛素分泌的作用。通过免疫荧光、超薄切片结合胶体金标记蛋白A免疫电镜技术(PAg)研究CART在2型糖尿病大鼠胰岛β细胞中的作用。通过大鼠口服葡萄糖耐量实验(OGTT)研究磷酸西他列汀对糖尿病大鼠血糖、胰高血糖素样肽(GLP-1)、胰岛素的影响,以及CART在磷酸西他列汀治疗糖尿病发生发展中的作用。
3.结果:
3.1 2型糖尿病大鼠模型的建立及血中FBG、TC、TG、FFA和FINS的检测糖尿病模型大鼠血中FBG、TG、TC、FFA均较正常对照大鼠明显增加,而FINS含量较正常对照大鼠明显降低,表明本研究建立的2型糖尿病大鼠模型符合要求。
3.2 CART对2型糖尿病大鼠下丘脑定位核团进食量的影响及饮食干预对CART mRNA的表达
在禁食糖尿病大鼠PVN中,仅注射高浓度CART可以促进糖尿病大鼠2-4h的进食;在禁食糖尿病大鼠ARC和DMH中,注射CART可以促进糖尿病大鼠的进食(0-1,1-2h);在4-8h出现进食量下降(i-ARC);在不禁食糖尿病大鼠ARC中,与禁食大鼠整体比较,进食量明显增高,且高脂饮食可以促进糖尿病大鼠进食量增高。QRT-PCR结果表明,高脂饮食糖尿病大鼠ARC中CART mRNA表达增高,进一步说明CART应对高脂饮食时可能有特殊的方式。
3.3 NPY与CART对进食行为的调节及CART对其他神经递质释放的影响
ARC内微量注射NPY对进食普通和高脂饲料的糖尿病大鼠进食量并无明显区别。ARC内微量注射CART对进食普通和高脂饲料的糖尿病大鼠进食量明显区别。碳水化合物在普通饲料和高脂饲料中的比例为3:1,在NPY引起的摄食增加中,增加碳水化合物的摄入是最主要的。糖尿病大鼠进食高脂饲料2周后,先注射0.2 nmol CART 10min后注射0.2 nmol NPY,与单独注射NPY及普通饲料喂养2周后的CART+NPY其进食量明显增高,表明CART对高脂饮食有反应而NPY对碳水化合物有反应,再次说明了CART在应对高脂饮食时可能有特殊的方式。免疫荧光结果显示NPY和CART蛋白在2型糖尿病大鼠下丘脑PVN核团中广泛分布,且大量表达,具有一定的共表达,糖尿病大鼠PVN内可能存在CART→NPY联系。下丘脑脑片释放实验结果表明,0.4,4,40 nmol/L CART灌流脑片后,NPY-IR升高,4nmol/L CART灌流后明显促进AGRP-IR和CRH-IR的释放(p<0.05),α-MSH-IR各组间无明显差异,CART可能刺激下丘脑促食欲神经递质的释放。
3.4 PKA/CREB信号通路在糖尿病大鼠ARC中的作用
通过原位杂交检测到PKA激动剂forskolin增加ARC核团CART mRNA表达,呈剂量依赖性;PKA抑制剂Rp-cAMPs减少ARC核团CART mRNA表达,两者联合注射可减弱由forskolin引起的CART mRNA释放,这些结果表明PKA信号通路在CART基因表达的调节中具有重要作用。Western blotting显示ARC注射forskolin后,CART蛋白表达增高,含量明显高于对照组,Rp-cAMPs减弱由forskolin介导的CART蛋白表达,此结果与基因转录结果一致(P<0.05)。ARC注射forskolin后,增加磷酸化CREB(p-CREB)蛋白表达,含量明显高于对照组,注射Rp-cAMPs后降低p-CREB蛋白表达,含量明显低于forskolin组,Rp-cAMPs减弱由forskolin介导的CREB磷酸化。
3.5钙离子内流激活的ERK信号通路在CART调节下丘脑神经元磷酸化中的作用
3.5.1下丘脑神经元鉴定
培养的神经元生长迅速,胶质细胞少,4-6d神经元形态饱满,胞体直径,突起长度,均达高峰,且无过于广泛的突触连接和胶质细胞生长。经NF-200染色后计算培养神经元的纯度达96.17±0.06%。
3.5.2 CART对下丘脑神经元ERK1/2磷酸化的调节
Western blotting显示,CART能特异性引起下丘脑神经元快速和持续的ERK1/2磷酸化反应,并呈剂量(1-100nmol/L)、时间(5-90min)依赖关系。MEK的拮抗剂U0126抑制正常的ERK1/2磷酸化并阻断由CART诱导的ERK1/2磷酸化。
3.5.3 CART促进下丘脑神经元[Ca~(2+)]i升高
D-hank’s液和DMSO作为阴性对照不能影响下丘脑神经元内游离钙的水平,100nmol/L CART刺激培养的下丘脑神经元,会导致绝大多数神经元内[Ca~(2+)]i迅速上升并稳定在一个较高的水平,或以较高的浓度水平为中心,反复轻微振荡,MEK阻断剂U0126预处理神经元后,100nmol/L CART不再能够引起下丘脑神经元内[Ca~(2+)]i下降,说明ERK1/2信号途径参与了CART激活细胞内[Ca~(2+)]i的过程。
3.6 CART在体外细胞模型和2型糖尿病大鼠胰岛β细胞中的表达及作用
3.6.1 CART对INS-1细胞胰岛素分泌的影响
培养的INS-1细胞密度在l×106/ml以上,呈扁平不规则多角形。GLP-1能促进INS-1细胞胰岛素和cAMP的释放,在GLP-1的高糖溶液中加入CART后,能增强GLP-1对INS-1细胞胰岛素和cAMP的释放。
3.6.2胰岛细胞灌注后CART对GSIS的影响
每只大鼠分离纯化后获得的胰岛数量为303±44个,DTZ染色显示胰岛纯度>85%,AO-PI染色显示胰岛存活率>95%。在含有GLP-1或GLP-1+CART的高糖中加入PKA抑制剂H89后,与对照组、GLP-1组和GLP-1+CART组比较,INS的释放降低。
3.6.3磷酸西他列汀对糖尿病大鼠OGTT期间血糖、GLP-1、血清胰岛素的影响
糖尿病模型大鼠糖耐量严重受损,口服灌胃磷酸西他列汀能显著提高大鼠糖耐量水平,并呈剂量依赖性。磷酸西他列汀低、中、高剂量组AUC与正常组和模型对照组AUC比较能显著增加血浆GLP-1水平,并呈剂量依赖性。糖尿病模型组大鼠血清中胰岛素水平与正常对照组大鼠比较明显降低;磷酸西他列汀各组大鼠血清中胰岛素水平较糖尿病模型组和正常大鼠组明显升高,并呈剂量依赖性。
3.6.4磷酸西他列汀对2型糖尿病大鼠胰岛病理结构的影响
HE结果显示,和正常对照组大鼠比较,糖尿病模型组大鼠胰腺中胰岛面积明显减小和胰岛β细胞数量明显减少。中、高剂量组大鼠的胰岛面积和胰岛β细胞数量较糖尿病模型组大鼠明显增加和增多,有趋向与正常大鼠水平。
3.6.5 CART-IR、Insulin-IR、Glucagon-IR的表达
免疫荧光结果显示,正常大鼠CART-IR主要在胰岛δ细胞中表达,胰岛β细胞表达很少,糖尿病模型大鼠CART-IR除了在δ细胞表达外,也在胰岛β细胞中大量表达,中、高剂量组能逐步降低CART-IR在胰岛β细胞中的表达,并呈剂量依赖性。正常大鼠Insulin-IR主要在胰岛β细胞中表达,Glucagon-IR在胰岛α细胞中表达,Insulin-IR和Glucagon-IR几乎没有共表达;糖尿病大鼠Insulin-IR在胰岛β细胞中表达减少,Glucagon-IR在胰岛α细胞中表达增加,有一部分共表达;中、高剂量组能逐步增加Insulin -IR在胰岛β细胞中的表达,减少Glucagon-IR在胰岛α细胞中表达,并呈剂量依赖性。
3.6.6 CART在2型糖尿病大鼠胰岛β细胞中的表达
应用超薄切片技术的PAg免疫电镜结果显示,糖尿病大鼠胰岛β细胞中CART阳性呈黑色点状颗粒,分布在β细胞细胞核和界膜之间,细胞核内无阳性,阴性对照无CART标记。胰岛δ细胞中CART阳性呈黑色点状颗粒,分布在细胞核内,细胞核和界膜之间无阳性。
4.结论:
4.1本研究采用高脂饲料喂养加腹腔注射小剂量STZ诱导大鼠的空腹血糖和脂质代谢紊乱,成功复制2型糖尿病大鼠模型。ARC内注射CART1-2h和2-4h后糖尿病大鼠进食量增加,表明CART可能是一种促食欲神经肽。高脂饮食糖尿病大鼠ARC中CART mRNA表达增高,进一步说明CART应对高脂饮食时可能有特殊的方式。
4.2 CART与NPY共同完成对进食行为的调节,CART可促进糖尿病大鼠PVN内NPY-IR阳性表达,可能存在CART→NPY联系。CART可能刺激下丘脑促食欲神经递质的释放。
4.3 p-CREB可以与CART启动子CRE结合位点结合,PKA/CREB通路可能参与CART mRNA和蛋白的转录调节。
4.4 CART能特异地引起下丘脑神经元快速而持续的ERK1/2磷酸化反应,并呈时间和剂量依赖性。CART可能通过胞内Ca~(2+)内流激活ERK1/2,在CART调控下丘脑神经元磷酸化过程中发挥重要作用。
4.5离体实验表明CART在INS-1细胞和分离的胰岛细胞中可能通过PKA信号通路产生作用。磷酸西他列汀显著改善糖尿病大鼠糖耐量水平,提高GLP-1、胰岛素水平,并呈剂量依赖性。免疫荧光和免疫电镜结果表明CART在糖尿病大鼠β和δ细胞中表达,进一步证明了CART是一种脑肠肽,在胃肠道及神经系统中均能发挥重要作用。
1. Background and Objective:
Type 2 diabetes is the most common form of diabetes (accounts for 90-95%) and is primarily characterized by insulin resistance or reduced insulin sensitivity, combined with reduced insulin secretion and hyperglycemia. This metabolic disease is a growing public health problem. Improper diet which is caused by hormone adjustment disorder is the important reason for type 2 diabetes. In recent years, the cocaine- and amphetamine-related transcript peptide (CART) plays an important role on eating behavior and in vivo energy-regulating and draws more and more attention. It was suggested that CART was involved in food intake; this suggestion was based on the distribution of CART peptides in the brain, namely in regions that included the arcuate nucleus, the dorsomedial hypothalamus nucleus, the paraventricular nucleus and the nucleus accumbens, all of which have a role in the regulation of food intake. The discovery of numerous novel neuropeptides with distinct hypothalamic distribution patterns has certainly added to the complexity of the hypothalamus they have also been the key to a better understanding of how the hypothalamus integrates neural and hormonal inputs/signals into coordinated neuroendocrine, autonomic and behavioral responses. Recent studies display that CART peptide containing cell bodies in the hypothalamic nucleus were found to be surrounded by other transmitter nerve terminals, suggesting that CART plays a role in the regulation of feeding. CART peptides and their effects have been found in feeding-relevant parts of the brain, they were also demonstrated to be present in the gut and in vagal nerves (Intestinal muscle plexus, islet cells, somatostatin gastric antrum G cells, beta cells, etc), suggesting that CART peptides have emerged as important islet regulators. CART peptides have emerged as major neurotansmitters and hormones, are widely distributed in the CNS and are involved in regulating many processes, including food intake, the maintenance of body weight, reproduction, stress, pain, reward and endocrine functions. In this study, we investigated the effects and mechanism of CART on STZ-diabetic rats’hypothalamus and isletβcells. Our findings provide a novel insight into understanding of biological function and mechanism of CART. A role for CART in type 2 diabetes pathophysiology could become new drug targets for gene therapy in type 2 diabetes.
2. Methods:
2.1 To induce type 2 diabetic rats and measurement of FBG、TG、TC、FFA and FINS in blood
After 4 weeks feeding of the high-fat diet (to induce hyperlipidemia), fasted rats were rendered diabetic by the intraperitoneal injection of 35 mg/kg streptozotocin dissolved in citrate-phosphate buffer and control rats were injected with the same volume citrate-phosphate buffer. One week later, rats with fasting blood glucose level of above 16.7 mmol/L were considered diabetic and were used in further studies. Total cholesterol (TC), triglyceride (TG), free fatty acid (FFA), serum insulin levels in blood were measured by commercial kit.
2.2 Effects of hypothalamic injection of CART on food intake and dietary intervention on CART mRNA expression
Kopf sterotaxic frame and the rat brain atlas of Paxions and Watson were used to identify the nucleus. Rats were allowed to recover for 7 days after surgery. The placement of the hypothalamic cannulas was verified by histological examination of the brain at the end of the study. In fasted rats, each dose CART were injected in ARC, PVN and DMH, in satiated rats, high dose CART was injected in ARC, after injection, rats were returned to cages and the food remaining 1, 2, 4, 8, 24h after injection was weighed. Quantitative real-time reverse transcription-polymerase chain reaction (QRT-PCR) was used to measure CART mRNA expression in the hypothalamus of rats after dietary intervention.
2.3 Effects of hypothalamic injection of neuropeptide Y (NPY) and CART on food intake and effects of CART on hypothalamic neuropeptide release
In fasted rats, each concentration of NPY or CART or the combined were injected in ARC, after injection, rats were returned to cages and the food remaining 1, 2h after injection was weighed. Double fluorescence immunohistochemistry was used to observe NPY and CART expression in PVN. Static hypothalamic explant culture was used to detect NPY, AGRP,α-MSH and CRH immunoreactivity by radio immunoassay.
2.4 Effects of PKA/CREB-mediated signaling
Using in situ hybridization analysis for CART mRNA expression and Western blotting analysis for CART peptide and CREB/p-CREB expression were carried out. To find CART expression in vivo in the rat ARC is regulated by PKA-mediating signaling and likely through the activation of CREB.
2.5 Effects of ERK signaling pathways activated by calcium influx in the hypothalamus neurons phosphorylation by CART
Firstly, the hypothalamic neurons from 2-3 days old SD neonatal rats were primarily cultured, and then Western blotting analysis for ERK1/2/p-ERK1/2 expression was carried out. The intracellular Ca~(2+) in these cells was marked with calcium fluorescent probe of Fluo-3/AM. The fluorescent values were recorded continuously under confocal laser scanning microscope.
2.6 CART regulates islet hormone secretion and is expressed in theβcells of type 2 diabetic rats
We have examined the role of CART as a regulator of islet hormone secretion using INS-1 cells and isolated rat islets. Fluorescence immunohistochemistry, ultrathin sections and protein A-gold technique were used to detect the CART labeling in theβcells of type 2 diabetic rats. The last part was designed to assess the chornic effects of dipeptidyl peptidase-4 inhibitor Sitagliptin treatment on oral glucose tolerance test, hormone profiles, glycemic control and pancreatic insulin content in type 2 diabetic rats. These data favor a role of CART in islet function and in the pathophysiology of type 2 diabetes.
3. Results:
3.1 To induce type 2 diabetic rats and measurement of FBG、TG、TC、FFA and FINS in blood
Fasting blood glucose, TG, TC, FFA levels in diabetic rats were all significantly higher than that of the control ones, while serum insulin level was significantly lower. These results indicated that type 2 diabetic rats with hyperlipidemia were successfully induced.
3.2 Effects of hypothalamic injection of CART on food intake and dietary intervention on CART mRNA expression
In fasted rats, injection of high dose CART into PVN of diabetic rats significantly increased feeding 2-4h after injection compared with that in the control, vehicle and low dose CART groups, other time points did not differ significantly from control levels. Injection of low and high dose CART into DMH and ARC of diabetic rats significantly increased feeding 1-2h and 2-4h after injection compared with that in the control, vehicle groups. A subsequent decrease in feeding relative to control levels was observed in the 4-8h period after injection of CART into ARC. The groups fed the high fat diet exhibited a clear increase in food intake following injection of CART into non-fasted diabetic rats’ARC. QRT-PCR demonstrated that diabetic rats fed the high fat diet exhibited a clear increase in the CART mRNA/β-actin mRNA ratio, further enhanced that CATR may have special way to high fat diet.
3.3 Effects of hypothalamic injection of NPY and CART on food intake and effects of CART on hypothalamic neuropeptide release
In fasted rats, injection of NPY into ARC of diabetic rats significantly increased feeding in 1-2h and 2-4h after injection compared with that in the control, vehicle groups. However, the difference between the regular chow and high fat diet were not obvious. NPY stimulated feeding was through carbohydrate intake which is most important. A ratio of carbohydrate in regular chow was 3 times higher than the high fat diet, thus, NPY may be the reflection of carbohydrate storage. Group was fed with high fat diet for 2 weeks, combination injection of CART and NPY, the food intake was much higher than the NPY group or the group was fed with regular chow for 2 weeks, combination injection of CART and NPY, further enhanced that CATR may have special way to high fat diet. Double fluorescence immunohistochemistry in the PVN of the type 2 diabetic rats showed that immunoreactivity for NPY and CART in overexpressed neurons in PVN, demonstrating colocalization of two molecules in the PVN neurons. There may be having the connection between NPY and CART. Exposure of hypothalamic explants to 0.4, 4 and 40 nmol/L CART significantly increased NPY-IR. There was a significantly increase in AGRP-IR and CRH-IR following exposure of the explants to 4 nmol/L CART. The release ofα-MSH from hypothalamic explants was not altered after exposure of the exlants to 0.4, 4 and 40 nmol/L compared with basal release. It’s possible that CART could stimulate the release of the hypothalamic orexigenic neuropeptides.
3.4 Effects of PKA/CREB-mediated signaling
Various stimulators and inhibitors of the PKA pathway were administered, and CART mRNA and peptide, as well as p-CREB, levels in the diabetic rat ARC. In situ hybridization demonstrated that PKA stimulators forskolin increased CART mRNA levels, showed dose dependent manner, an effect attenuated by the inhibition of PKA (Rp-cAMPs). Rp-cAMPs alone reduced mRNA levels compared with controls, indicating a tonic regulation by PKA on CART expression. Western blotting indicated that forskolin significantly increased CART peptide levels in a manner consistent with the observed increase in CART mRNA levels (P<0.05). Intra-ARC forskolin administration significantly increased p-CREB levels while simultaneously decreasing the amount of CREB protein in the diabetic raGt ARC. Inhibition of PKA with Rp-cAMPs attenuated p-CREB expression.
3.5 Effects of ERK signaling pathways activated by calcium influx in the hypothalamus neurons phosphorylation by CART
3.5.1 Identification of hypothalamus neurons
The hypothalamus neurons were grown very fast, there were little Glial cells. During the course of neuron growth we measured the axis length and the cell body area of the cultured neurons. 4-6 days, the cell body area and process length of nerve cells reached their highest levels. We used NF-200 to identify the cultured neurons, then calculated the percentage of the neuron that is 96.17±0.06%。
3.5.2 Effects of CART on the phosphorylation of the hypothalamus neurons
Western blotting showed that CART induced the activation of ERK 1/2 in a time- (5-90min) and dose-(1-100nmol/L) dependent manner in hypothalamus neurons. The CART effect was blocked by MEK inhibitor U0126, indicating the involvement of the upstream kinases, MEK 1/2.
3.5.3 CART enhanced the [Ca~(2+)]i of the hypothalamus neurons
D-hank’s and DMSO were as negative control could not affect the change in [Ca~(2+)]i. In Ca~(2+)-free external circumstance, exposure of the hypothalamic neurons to 100 nmol/L CART produced a rapid and significant increase of [Ca~(2+)]i or unregulated fluctuation. Pretreatment of the hypothalamus neurons with MEK inhibitor U0126 30min, 100 nmol/L CART on [Ca~(2+)]i was abolished, there was no statistical difference, indicated that ERK1/2 pathway processed in stimulating the hypothalamus neurons [Ca~(2+)]i by CART.
3.6 CART regulates islet hormone secretion and is expressed in theβcells of type 2 diabetic rats
3.6.1 Effects of CART on insulin secretion from INS-1 cell
The cell density of the cultured INS-1 cell was above l×106/ml, most of the cells were showed in compressed irregular polygons. At 16.7 mmol/L glucose, 100 nmol/L CART evoked significantly augmentation of GLP-1 stimulated GSIS. The cAMP levels in cells treated with GLP-1 together with CART were higher than in cells treated with GLP-1 alone. CART potentiated cAMP-enhanced GSIS.
3.6.2 Effects of CART on islet hormone secretion from isolated islets
Every rat purification for the islet number was 303±44, DTZ staining demonstrated that the islet purity>85%, and AO-PI staining demonstrated that the islet survival rate>95%. To explore whether PKA-dependent or -independent mechanisms account for the observed effects, we used the PKA inhibitor H89. H89 completely abolished the potentiating effect of CART on insulin release.
3.6.3 Effects of Sitagliptin treatment on oral glucose tolerance test, blood glucose, GLP-1 and insulin levels
A single dose of Sitagliptin (a dose-related manner) has been demonstrated to improve glucose tolerance and increase insulin secretion after an oral glucose challenge in STZ induced diabetic rats. Sitagliptin could increase active GLP-1 compared with the control and vehicle group in dose dependent manner. For the 1, 3, 10mg/kg doses, were significantly increased the plasma insulin levels compared with the control and vehicle groups.
3.6.4 Effects of Sitagliptin on histopathology in type 2 diabetic rats islet
Hematoxylin-eosin (HE) staining was used to observe pancreatic histopathology ofβcells. Compared with the normal rats, type 2 diabetic rats’islet area significantly reduced andβcell number were obviously reduced. After Sitagliptin treatment, the islet area andβcell number were obviously increased, in a dose dependent manner.
3.6.5 Expression of CART-IR、Insulin-IR、Glucagon-IR
Fluorescence immunohistochemistry for CART in control rats showed that CART immunoreactivity was largely restricted to theδcells and pancreatic neurons. In type 2 diabetic rats, in addition toδcells and neurons, a great portion of theβcells was CART immunoreactive. Middle and high dose of Sitagliptin treatment, the number of CART- immunoreactiveβcells were reduced compared with the vehicle. High dose of Sitagliptin treatment, CART- immunoreactivity was reduced to a level comparable with the controls. Double fluorescence immunohistochemistry for Insulin and Glugacon in control rats showed that Insulin immunoreactivity was largely restricted to theβcells and Glugacon immunoreactivity was largely restricted to theαcells. Vehicle group demonstratied that colocalization of two molecules in theβcells.
3.6.6 Effects of CART on type 2 diabetic rats’βcell
TEM examination revealed that labeling for CART was present in the secretory granules ofβcells andδcells. In theβcells the gold particles were mainly localized to the electro-lucent halos of the granules, between the dense core and the limiting membrane. Negative control displayed no CART labeling. In theδcells the gold particles were mainly localized to the electro-lucent dense core, not between the halos and the limiting membrane.
4. Conclusions:
4.1 Rats with hyperlipidemia were successfully induced with the high fat diet; type 2 diabetic rats were developed by injection low dose streptozotocin due to parts ofβcells injury. The groups fed the high fat diet exhibited a clear increase in food intake following injection of CART into ARC. These data provided evidence that hypothalamic CART has an orexigenic effect. Diabetic rats fed the high fat diet exhibited a clear increase in the CART mRNA, further enhanced that CATR may have special way to high fat diet.
4.2 CART and NPY are the mediators of the regulation of food intake. CART stimulated the NPY-IR in PVN of the diabetic rats, indicated that the connection between CART and NPY. CART could stimulate the release of the hypothalamic orexigenic neuropeptides.
4.3 Inactive CREB which is associated with the CRE site of the CART promoter, to form transcriptionally active pCREB. PKA/CREB pathway may be involved in regulating CART mRNA and peptide levels.
4.4 CART induced the activation of ERK 1/2 in a time- and dose- dependent manner in hypothalamus neurons. ERK1/2 pathway processed in stimulating the hypothalamus neurons [Ca~(2+)]i by CART.
4.5 The results of INS-1 cells and isolated islet cells demonstrated that it could be explained possibly a stronger ensuring activation of the PKA-dependent pathway. Sitagliptin (a dose-related manner) has been demonstrated to improve glucose tolerance and increase GLP-1 and insulin secretion. Fluorescence immunohistochemistry and TEM examination revealed that labeling for CART was present in the secretory granules ofβcells andδcells in type 2 diabetic rats. CART peptides not only found in the brain but also found in the gut, they are referred to as‘brain-gut’peptides.
2型糖尿病是一种主要由于胰岛素抵抗伴随相对胰岛素不足,或胰岛素分泌缺陷伴有或不伴有胰岛素抵抗而导致慢性高血糖的代谢疾病,占糖尿病总数的90%-95%。2型糖尿病的重要原因之一是由于激素调节紊乱所致的饮食不当而产生的,因此可卡因-苯丙胺调节转录肽(cocaine- and amphetamine-related transcript peptide, CART)在调控进食行为和体内能量平衡方面的作用受到广泛关注。CART分布在许多与进食等相关的脑区,尤其是在下丘脑背内侧核(dorsomedial nucleus,DMH)、腹内侧(ventromedial nucleus,VMH)、弓状核(areuate nucleus,ARC)、外侧下丘脑(1ateral hypothalamus,LH)和室旁核(paraventricular nucleus,PVN)中分布较多。下丘脑不同核团之间构成复杂的调控网络,通过分泌神经递质以及神经元间的投射通路在机体摄食活动和能量平衡调节活动中发挥重要作用。研究表明CART与某些神经递质共存于某些神经元,提示CART参与调节进食行为。CART除了在中枢和外周神经系统中分布外,外周组织也有分布如交感节前神经纤维、胃窦G细胞、胰岛生长抑素细胞(islet somatostatin cells)、肠肌丛等,表明CART是一种脑肠肽,在胃肠道及中枢、外周神经系统中均能发挥作用。上述研究表明CART作为内源性神经递质,在奖赏与强化、食欲、应激、感知、内分泌等生理过程中发挥重要的作用。
因此在本课题中,我们将从整体动物和和细胞水平探讨CART在2型糖尿病大鼠下丘脑及胰岛β细胞中的表达及其作用机制。期望为深入认识CART的生物学功能及其调控机制提供新的实验依据,对CART的深入研究将使CART可能成为基因治疗2型糖尿病的新药物靶点。
2.方法:
2.1 2型糖尿病大鼠模型的建立及血中FBG、TC、TG、FFA和FINS的检测大鼠高脂饲料喂养4周,禁食12h后腹腔注射链脲佐菌素(Streptozotocin,STZ) 35mg/kg,用于建立2型糖尿病模型,正常对照组大鼠腹腔注射等体积的柠檬酸缓冲液。1周后,挑选空腹血糖值≥16.7 mmol/L作为糖尿病模型大鼠。建模前、后,按相应方法进行血中空腹血糖(fasting blood glucose, FBG)、三酰甘油(Triglyceride, TG)、总胆固醇(Total cholesterol, TC)、游离脂肪酸(Free fatty acid, FFA)、血清胰岛素(fasting insulin levels, FINS)等指标的检测。
2.2观察CART对2型糖尿病大鼠下丘脑定位核团进食量的影响及饮食干预对CART mRNA的表达
使用脑立体定位仪结合大鼠脑图谱,按照各核团三维坐标,进行准确定位。术后给予大鼠抗感染措施,恢复7d后进行实验,并在实验结束后,对导管植入位置进行尼森染色确定定位结果的准确性。使用微量注射器将各浓度CART注射入禁食各实验组的ARC、DMH及PVN核团中,或不禁食各实验组的ARC核团中,药物注射后1,2,4,8,24h测定进食量。采用荧光实时定量PCR观察饮食干预后糖尿病大鼠ARC中CART mRNA的水平变化。
2.3探讨神经肽Y(NPY)与CART对进食行为的调节及CART对其他神经递质释放的影响
使用微量注射器将CART或NPY注射入禁食各实验组的ARC核团中,药物注射后1,2h测定进食量。采用免疫荧光双标分析糖尿病大鼠PVN核团中NPY、CART的表达。通过下丘脑脑片孵育,放免法测定CART对下丘脑重要神经递质NPY、AGRP、CRH和α-MSH的释放。
2.4探讨PKA/CREB信号通路在糖尿病大鼠ARC中的作用采用原位杂交和Western blotting技术,在2型糖尿病大鼠ARC内探讨PKA/CREB参与CART mRNA和蛋白的转录调节中的作用。
2.5探讨钙离子内流激活的ERK信号通路在CART调节下丘脑神经元磷酸化中的作用
首先对大鼠下丘脑神经元进行培养,通过Western blotting分析ERK1/2和磷酸化ERK1/2的蛋白表达,用Fluo-3/AM钙荧光探针标记细胞内游离Ca~(2+),激光共聚焦测定神经元[Ca~(2+)]i的变化研究CART对ERK1/2信号通路的调节。
2.6探讨CART在体外细胞模型和2型糖尿病大鼠胰岛β细胞中的表达及作用机制
培养大鼠胰岛β细胞瘤细胞INS-1,观察CART对INS-1细胞胰岛素分泌的影响。研究CART对分离、纯化后的糖尿病大鼠胰岛细胞胰岛素分泌的作用。通过免疫荧光、超薄切片结合胶体金标记蛋白A免疫电镜技术(PAg)研究CART在2型糖尿病大鼠胰岛β细胞中的作用。通过大鼠口服葡萄糖耐量实验(OGTT)研究磷酸西他列汀对糖尿病大鼠血糖、胰高血糖素样肽(GLP-1)、胰岛素的影响,以及CART在磷酸西他列汀治疗糖尿病发生发展中的作用。
3.结果:
3.1 2型糖尿病大鼠模型的建立及血中FBG、TC、TG、FFA和FINS的检测糖尿病模型大鼠血中FBG、TG、TC、FFA均较正常对照大鼠明显增加,而FINS含量较正常对照大鼠明显降低,表明本研究建立的2型糖尿病大鼠模型符合要求。
3.2 CART对2型糖尿病大鼠下丘脑定位核团进食量的影响及饮食干预对CART mRNA的表达
在禁食糖尿病大鼠PVN中,仅注射高浓度CART可以促进糖尿病大鼠2-4h的进食;在禁食糖尿病大鼠ARC和DMH中,注射CART可以促进糖尿病大鼠的进食(0-1,1-2h);在4-8h出现进食量下降(i-ARC);在不禁食糖尿病大鼠ARC中,与禁食大鼠整体比较,进食量明显增高,且高脂饮食可以促进糖尿病大鼠进食量增高。QRT-PCR结果表明,高脂饮食糖尿病大鼠ARC中CART mRNA表达增高,进一步说明CART应对高脂饮食时可能有特殊的方式。
3.3 NPY与CART对进食行为的调节及CART对其他神经递质释放的影响
ARC内微量注射NPY对进食普通和高脂饲料的糖尿病大鼠进食量并无明显区别。ARC内微量注射CART对进食普通和高脂饲料的糖尿病大鼠进食量明显区别。碳水化合物在普通饲料和高脂饲料中的比例为3:1,在NPY引起的摄食增加中,增加碳水化合物的摄入是最主要的。糖尿病大鼠进食高脂饲料2周后,先注射0.2 nmol CART 10min后注射0.2 nmol NPY,与单独注射NPY及普通饲料喂养2周后的CART+NPY其进食量明显增高,表明CART对高脂饮食有反应而NPY对碳水化合物有反应,再次说明了CART在应对高脂饮食时可能有特殊的方式。免疫荧光结果显示NPY和CART蛋白在2型糖尿病大鼠下丘脑PVN核团中广泛分布,且大量表达,具有一定的共表达,糖尿病大鼠PVN内可能存在CART→NPY联系。下丘脑脑片释放实验结果表明,0.4,4,40 nmol/L CART灌流脑片后,NPY-IR升高,4nmol/L CART灌流后明显促进AGRP-IR和CRH-IR的释放(p<0.05),α-MSH-IR各组间无明显差异,CART可能刺激下丘脑促食欲神经递质的释放。
3.4 PKA/CREB信号通路在糖尿病大鼠ARC中的作用
通过原位杂交检测到PKA激动剂forskolin增加ARC核团CART mRNA表达,呈剂量依赖性;PKA抑制剂Rp-cAMPs减少ARC核团CART mRNA表达,两者联合注射可减弱由forskolin引起的CART mRNA释放,这些结果表明PKA信号通路在CART基因表达的调节中具有重要作用。Western blotting显示ARC注射forskolin后,CART蛋白表达增高,含量明显高于对照组,Rp-cAMPs减弱由forskolin介导的CART蛋白表达,此结果与基因转录结果一致(P<0.05)。ARC注射forskolin后,增加磷酸化CREB(p-CREB)蛋白表达,含量明显高于对照组,注射Rp-cAMPs后降低p-CREB蛋白表达,含量明显低于forskolin组,Rp-cAMPs减弱由forskolin介导的CREB磷酸化。
3.5钙离子内流激活的ERK信号通路在CART调节下丘脑神经元磷酸化中的作用
3.5.1下丘脑神经元鉴定
培养的神经元生长迅速,胶质细胞少,4-6d神经元形态饱满,胞体直径,突起长度,均达高峰,且无过于广泛的突触连接和胶质细胞生长。经NF-200染色后计算培养神经元的纯度达96.17±0.06%。
3.5.2 CART对下丘脑神经元ERK1/2磷酸化的调节
Western blotting显示,CART能特异性引起下丘脑神经元快速和持续的ERK1/2磷酸化反应,并呈剂量(1-100nmol/L)、时间(5-90min)依赖关系。MEK的拮抗剂U0126抑制正常的ERK1/2磷酸化并阻断由CART诱导的ERK1/2磷酸化。
3.5.3 CART促进下丘脑神经元[Ca~(2+)]i升高
D-hank’s液和DMSO作为阴性对照不能影响下丘脑神经元内游离钙的水平,100nmol/L CART刺激培养的下丘脑神经元,会导致绝大多数神经元内[Ca~(2+)]i迅速上升并稳定在一个较高的水平,或以较高的浓度水平为中心,反复轻微振荡,MEK阻断剂U0126预处理神经元后,100nmol/L CART不再能够引起下丘脑神经元内[Ca~(2+)]i下降,说明ERK1/2信号途径参与了CART激活细胞内[Ca~(2+)]i的过程。
3.6 CART在体外细胞模型和2型糖尿病大鼠胰岛β细胞中的表达及作用
3.6.1 CART对INS-1细胞胰岛素分泌的影响
培养的INS-1细胞密度在l×106/ml以上,呈扁平不规则多角形。GLP-1能促进INS-1细胞胰岛素和cAMP的释放,在GLP-1的高糖溶液中加入CART后,能增强GLP-1对INS-1细胞胰岛素和cAMP的释放。
3.6.2胰岛细胞灌注后CART对GSIS的影响
每只大鼠分离纯化后获得的胰岛数量为303±44个,DTZ染色显示胰岛纯度>85%,AO-PI染色显示胰岛存活率>95%。在含有GLP-1或GLP-1+CART的高糖中加入PKA抑制剂H89后,与对照组、GLP-1组和GLP-1+CART组比较,INS的释放降低。
3.6.3磷酸西他列汀对糖尿病大鼠OGTT期间血糖、GLP-1、血清胰岛素的影响
糖尿病模型大鼠糖耐量严重受损,口服灌胃磷酸西他列汀能显著提高大鼠糖耐量水平,并呈剂量依赖性。磷酸西他列汀低、中、高剂量组AUC与正常组和模型对照组AUC比较能显著增加血浆GLP-1水平,并呈剂量依赖性。糖尿病模型组大鼠血清中胰岛素水平与正常对照组大鼠比较明显降低;磷酸西他列汀各组大鼠血清中胰岛素水平较糖尿病模型组和正常大鼠组明显升高,并呈剂量依赖性。
3.6.4磷酸西他列汀对2型糖尿病大鼠胰岛病理结构的影响
HE结果显示,和正常对照组大鼠比较,糖尿病模型组大鼠胰腺中胰岛面积明显减小和胰岛β细胞数量明显减少。中、高剂量组大鼠的胰岛面积和胰岛β细胞数量较糖尿病模型组大鼠明显增加和增多,有趋向与正常大鼠水平。
3.6.5 CART-IR、Insulin-IR、Glucagon-IR的表达
免疫荧光结果显示,正常大鼠CART-IR主要在胰岛δ细胞中表达,胰岛β细胞表达很少,糖尿病模型大鼠CART-IR除了在δ细胞表达外,也在胰岛β细胞中大量表达,中、高剂量组能逐步降低CART-IR在胰岛β细胞中的表达,并呈剂量依赖性。正常大鼠Insulin-IR主要在胰岛β细胞中表达,Glucagon-IR在胰岛α细胞中表达,Insulin-IR和Glucagon-IR几乎没有共表达;糖尿病大鼠Insulin-IR在胰岛β细胞中表达减少,Glucagon-IR在胰岛α细胞中表达增加,有一部分共表达;中、高剂量组能逐步增加Insulin -IR在胰岛β细胞中的表达,减少Glucagon-IR在胰岛α细胞中表达,并呈剂量依赖性。
3.6.6 CART在2型糖尿病大鼠胰岛β细胞中的表达
应用超薄切片技术的PAg免疫电镜结果显示,糖尿病大鼠胰岛β细胞中CART阳性呈黑色点状颗粒,分布在β细胞细胞核和界膜之间,细胞核内无阳性,阴性对照无CART标记。胰岛δ细胞中CART阳性呈黑色点状颗粒,分布在细胞核内,细胞核和界膜之间无阳性。
4.结论:
4.1本研究采用高脂饲料喂养加腹腔注射小剂量STZ诱导大鼠的空腹血糖和脂质代谢紊乱,成功复制2型糖尿病大鼠模型。ARC内注射CART1-2h和2-4h后糖尿病大鼠进食量增加,表明CART可能是一种促食欲神经肽。高脂饮食糖尿病大鼠ARC中CART mRNA表达增高,进一步说明CART应对高脂饮食时可能有特殊的方式。
4.2 CART与NPY共同完成对进食行为的调节,CART可促进糖尿病大鼠PVN内NPY-IR阳性表达,可能存在CART→NPY联系。CART可能刺激下丘脑促食欲神经递质的释放。
4.3 p-CREB可以与CART启动子CRE结合位点结合,PKA/CREB通路可能参与CART mRNA和蛋白的转录调节。
4.4 CART能特异地引起下丘脑神经元快速而持续的ERK1/2磷酸化反应,并呈时间和剂量依赖性。CART可能通过胞内Ca~(2+)内流激活ERK1/2,在CART调控下丘脑神经元磷酸化过程中发挥重要作用。
4.5离体实验表明CART在INS-1细胞和分离的胰岛细胞中可能通过PKA信号通路产生作用。磷酸西他列汀显著改善糖尿病大鼠糖耐量水平,提高GLP-1、胰岛素水平,并呈剂量依赖性。免疫荧光和免疫电镜结果表明CART在糖尿病大鼠β和δ细胞中表达,进一步证明了CART是一种脑肠肽,在胃肠道及神经系统中均能发挥重要作用。
1. Background and Objective:
Type 2 diabetes is the most common form of diabetes (accounts for 90-95%) and is primarily characterized by insulin resistance or reduced insulin sensitivity, combined with reduced insulin secretion and hyperglycemia. This metabolic disease is a growing public health problem. Improper diet which is caused by hormone adjustment disorder is the important reason for type 2 diabetes. In recent years, the cocaine- and amphetamine-related transcript peptide (CART) plays an important role on eating behavior and in vivo energy-regulating and draws more and more attention. It was suggested that CART was involved in food intake; this suggestion was based on the distribution of CART peptides in the brain, namely in regions that included the arcuate nucleus, the dorsomedial hypothalamus nucleus, the paraventricular nucleus and the nucleus accumbens, all of which have a role in the regulation of food intake. The discovery of numerous novel neuropeptides with distinct hypothalamic distribution patterns has certainly added to the complexity of the hypothalamus they have also been the key to a better understanding of how the hypothalamus integrates neural and hormonal inputs/signals into coordinated neuroendocrine, autonomic and behavioral responses. Recent studies display that CART peptide containing cell bodies in the hypothalamic nucleus were found to be surrounded by other transmitter nerve terminals, suggesting that CART plays a role in the regulation of feeding. CART peptides and their effects have been found in feeding-relevant parts of the brain, they were also demonstrated to be present in the gut and in vagal nerves (Intestinal muscle plexus, islet cells, somatostatin gastric antrum G cells, beta cells, etc), suggesting that CART peptides have emerged as important islet regulators. CART peptides have emerged as major neurotansmitters and hormones, are widely distributed in the CNS and are involved in regulating many processes, including food intake, the maintenance of body weight, reproduction, stress, pain, reward and endocrine functions. In this study, we investigated the effects and mechanism of CART on STZ-diabetic rats’hypothalamus and isletβcells. Our findings provide a novel insight into understanding of biological function and mechanism of CART. A role for CART in type 2 diabetes pathophysiology could become new drug targets for gene therapy in type 2 diabetes.
2. Methods:
2.1 To induce type 2 diabetic rats and measurement of FBG、TG、TC、FFA and FINS in blood
After 4 weeks feeding of the high-fat diet (to induce hyperlipidemia), fasted rats were rendered diabetic by the intraperitoneal injection of 35 mg/kg streptozotocin dissolved in citrate-phosphate buffer and control rats were injected with the same volume citrate-phosphate buffer. One week later, rats with fasting blood glucose level of above 16.7 mmol/L were considered diabetic and were used in further studies. Total cholesterol (TC), triglyceride (TG), free fatty acid (FFA), serum insulin levels in blood were measured by commercial kit.
2.2 Effects of hypothalamic injection of CART on food intake and dietary intervention on CART mRNA expression
Kopf sterotaxic frame and the rat brain atlas of Paxions and Watson were used to identify the nucleus. Rats were allowed to recover for 7 days after surgery. The placement of the hypothalamic cannulas was verified by histological examination of the brain at the end of the study. In fasted rats, each dose CART were injected in ARC, PVN and DMH, in satiated rats, high dose CART was injected in ARC, after injection, rats were returned to cages and the food remaining 1, 2, 4, 8, 24h after injection was weighed. Quantitative real-time reverse transcription-polymerase chain reaction (QRT-PCR) was used to measure CART mRNA expression in the hypothalamus of rats after dietary intervention.
2.3 Effects of hypothalamic injection of neuropeptide Y (NPY) and CART on food intake and effects of CART on hypothalamic neuropeptide release
In fasted rats, each concentration of NPY or CART or the combined were injected in ARC, after injection, rats were returned to cages and the food remaining 1, 2h after injection was weighed. Double fluorescence immunohistochemistry was used to observe NPY and CART expression in PVN. Static hypothalamic explant culture was used to detect NPY, AGRP,α-MSH and CRH immunoreactivity by radio immunoassay.
2.4 Effects of PKA/CREB-mediated signaling
Using in situ hybridization analysis for CART mRNA expression and Western blotting analysis for CART peptide and CREB/p-CREB expression were carried out. To find CART expression in vivo in the rat ARC is regulated by PKA-mediating signaling and likely through the activation of CREB.
2.5 Effects of ERK signaling pathways activated by calcium influx in the hypothalamus neurons phosphorylation by CART
Firstly, the hypothalamic neurons from 2-3 days old SD neonatal rats were primarily cultured, and then Western blotting analysis for ERK1/2/p-ERK1/2 expression was carried out. The intracellular Ca~(2+) in these cells was marked with calcium fluorescent probe of Fluo-3/AM. The fluorescent values were recorded continuously under confocal laser scanning microscope.
2.6 CART regulates islet hormone secretion and is expressed in theβcells of type 2 diabetic rats
We have examined the role of CART as a regulator of islet hormone secretion using INS-1 cells and isolated rat islets. Fluorescence immunohistochemistry, ultrathin sections and protein A-gold technique were used to detect the CART labeling in theβcells of type 2 diabetic rats. The last part was designed to assess the chornic effects of dipeptidyl peptidase-4 inhibitor Sitagliptin treatment on oral glucose tolerance test, hormone profiles, glycemic control and pancreatic insulin content in type 2 diabetic rats. These data favor a role of CART in islet function and in the pathophysiology of type 2 diabetes.
3. Results:
3.1 To induce type 2 diabetic rats and measurement of FBG、TG、TC、FFA and FINS in blood
Fasting blood glucose, TG, TC, FFA levels in diabetic rats were all significantly higher than that of the control ones, while serum insulin level was significantly lower. These results indicated that type 2 diabetic rats with hyperlipidemia were successfully induced.
3.2 Effects of hypothalamic injection of CART on food intake and dietary intervention on CART mRNA expression
In fasted rats, injection of high dose CART into PVN of diabetic rats significantly increased feeding 2-4h after injection compared with that in the control, vehicle and low dose CART groups, other time points did not differ significantly from control levels. Injection of low and high dose CART into DMH and ARC of diabetic rats significantly increased feeding 1-2h and 2-4h after injection compared with that in the control, vehicle groups. A subsequent decrease in feeding relative to control levels was observed in the 4-8h period after injection of CART into ARC. The groups fed the high fat diet exhibited a clear increase in food intake following injection of CART into non-fasted diabetic rats’ARC. QRT-PCR demonstrated that diabetic rats fed the high fat diet exhibited a clear increase in the CART mRNA/β-actin mRNA ratio, further enhanced that CATR may have special way to high fat diet.
3.3 Effects of hypothalamic injection of NPY and CART on food intake and effects of CART on hypothalamic neuropeptide release
In fasted rats, injection of NPY into ARC of diabetic rats significantly increased feeding in 1-2h and 2-4h after injection compared with that in the control, vehicle groups. However, the difference between the regular chow and high fat diet were not obvious. NPY stimulated feeding was through carbohydrate intake which is most important. A ratio of carbohydrate in regular chow was 3 times higher than the high fat diet, thus, NPY may be the reflection of carbohydrate storage. Group was fed with high fat diet for 2 weeks, combination injection of CART and NPY, the food intake was much higher than the NPY group or the group was fed with regular chow for 2 weeks, combination injection of CART and NPY, further enhanced that CATR may have special way to high fat diet. Double fluorescence immunohistochemistry in the PVN of the type 2 diabetic rats showed that immunoreactivity for NPY and CART in overexpressed neurons in PVN, demonstrating colocalization of two molecules in the PVN neurons. There may be having the connection between NPY and CART. Exposure of hypothalamic explants to 0.4, 4 and 40 nmol/L CART significantly increased NPY-IR. There was a significantly increase in AGRP-IR and CRH-IR following exposure of the explants to 4 nmol/L CART. The release ofα-MSH from hypothalamic explants was not altered after exposure of the exlants to 0.4, 4 and 40 nmol/L compared with basal release. It’s possible that CART could stimulate the release of the hypothalamic orexigenic neuropeptides.
3.4 Effects of PKA/CREB-mediated signaling
Various stimulators and inhibitors of the PKA pathway were administered, and CART mRNA and peptide, as well as p-CREB, levels in the diabetic rat ARC. In situ hybridization demonstrated that PKA stimulators forskolin increased CART mRNA levels, showed dose dependent manner, an effect attenuated by the inhibition of PKA (Rp-cAMPs). Rp-cAMPs alone reduced mRNA levels compared with controls, indicating a tonic regulation by PKA on CART expression. Western blotting indicated that forskolin significantly increased CART peptide levels in a manner consistent with the observed increase in CART mRNA levels (P<0.05). Intra-ARC forskolin administration significantly increased p-CREB levels while simultaneously decreasing the amount of CREB protein in the diabetic raGt ARC. Inhibition of PKA with Rp-cAMPs attenuated p-CREB expression.
3.5 Effects of ERK signaling pathways activated by calcium influx in the hypothalamus neurons phosphorylation by CART
3.5.1 Identification of hypothalamus neurons
The hypothalamus neurons were grown very fast, there were little Glial cells. During the course of neuron growth we measured the axis length and the cell body area of the cultured neurons. 4-6 days, the cell body area and process length of nerve cells reached their highest levels. We used NF-200 to identify the cultured neurons, then calculated the percentage of the neuron that is 96.17±0.06%。
3.5.2 Effects of CART on the phosphorylation of the hypothalamus neurons
Western blotting showed that CART induced the activation of ERK 1/2 in a time- (5-90min) and dose-(1-100nmol/L) dependent manner in hypothalamus neurons. The CART effect was blocked by MEK inhibitor U0126, indicating the involvement of the upstream kinases, MEK 1/2.
3.5.3 CART enhanced the [Ca~(2+)]i of the hypothalamus neurons
D-hank’s and DMSO were as negative control could not affect the change in [Ca~(2+)]i. In Ca~(2+)-free external circumstance, exposure of the hypothalamic neurons to 100 nmol/L CART produced a rapid and significant increase of [Ca~(2+)]i or unregulated fluctuation. Pretreatment of the hypothalamus neurons with MEK inhibitor U0126 30min, 100 nmol/L CART on [Ca~(2+)]i was abolished, there was no statistical difference, indicated that ERK1/2 pathway processed in stimulating the hypothalamus neurons [Ca~(2+)]i by CART.
3.6 CART regulates islet hormone secretion and is expressed in theβcells of type 2 diabetic rats
3.6.1 Effects of CART on insulin secretion from INS-1 cell
The cell density of the cultured INS-1 cell was above l×106/ml, most of the cells were showed in compressed irregular polygons. At 16.7 mmol/L glucose, 100 nmol/L CART evoked significantly augmentation of GLP-1 stimulated GSIS. The cAMP levels in cells treated with GLP-1 together with CART were higher than in cells treated with GLP-1 alone. CART potentiated cAMP-enhanced GSIS.
3.6.2 Effects of CART on islet hormone secretion from isolated islets
Every rat purification for the islet number was 303±44, DTZ staining demonstrated that the islet purity>85%, and AO-PI staining demonstrated that the islet survival rate>95%. To explore whether PKA-dependent or -independent mechanisms account for the observed effects, we used the PKA inhibitor H89. H89 completely abolished the potentiating effect of CART on insulin release.
3.6.3 Effects of Sitagliptin treatment on oral glucose tolerance test, blood glucose, GLP-1 and insulin levels
A single dose of Sitagliptin (a dose-related manner) has been demonstrated to improve glucose tolerance and increase insulin secretion after an oral glucose challenge in STZ induced diabetic rats. Sitagliptin could increase active GLP-1 compared with the control and vehicle group in dose dependent manner. For the 1, 3, 10mg/kg doses, were significantly increased the plasma insulin levels compared with the control and vehicle groups.
3.6.4 Effects of Sitagliptin on histopathology in type 2 diabetic rats islet
Hematoxylin-eosin (HE) staining was used to observe pancreatic histopathology ofβcells. Compared with the normal rats, type 2 diabetic rats’islet area significantly reduced andβcell number were obviously reduced. After Sitagliptin treatment, the islet area andβcell number were obviously increased, in a dose dependent manner.
3.6.5 Expression of CART-IR、Insulin-IR、Glucagon-IR
Fluorescence immunohistochemistry for CART in control rats showed that CART immunoreactivity was largely restricted to theδcells and pancreatic neurons. In type 2 diabetic rats, in addition toδcells and neurons, a great portion of theβcells was CART immunoreactive. Middle and high dose of Sitagliptin treatment, the number of CART- immunoreactiveβcells were reduced compared with the vehicle. High dose of Sitagliptin treatment, CART- immunoreactivity was reduced to a level comparable with the controls. Double fluorescence immunohistochemistry for Insulin and Glugacon in control rats showed that Insulin immunoreactivity was largely restricted to theβcells and Glugacon immunoreactivity was largely restricted to theαcells. Vehicle group demonstratied that colocalization of two molecules in theβcells.
3.6.6 Effects of CART on type 2 diabetic rats’βcell
TEM examination revealed that labeling for CART was present in the secretory granules ofβcells andδcells. In theβcells the gold particles were mainly localized to the electro-lucent halos of the granules, between the dense core and the limiting membrane. Negative control displayed no CART labeling. In theδcells the gold particles were mainly localized to the electro-lucent dense core, not between the halos and the limiting membrane.
4. Conclusions:
4.1 Rats with hyperlipidemia were successfully induced with the high fat diet; type 2 diabetic rats were developed by injection low dose streptozotocin due to parts ofβcells injury. The groups fed the high fat diet exhibited a clear increase in food intake following injection of CART into ARC. These data provided evidence that hypothalamic CART has an orexigenic effect. Diabetic rats fed the high fat diet exhibited a clear increase in the CART mRNA, further enhanced that CATR may have special way to high fat diet.
4.2 CART and NPY are the mediators of the regulation of food intake. CART stimulated the NPY-IR in PVN of the diabetic rats, indicated that the connection between CART and NPY. CART could stimulate the release of the hypothalamic orexigenic neuropeptides.
4.3 Inactive CREB which is associated with the CRE site of the CART promoter, to form transcriptionally active pCREB. PKA/CREB pathway may be involved in regulating CART mRNA and peptide levels.
4.4 CART induced the activation of ERK 1/2 in a time- and dose- dependent manner in hypothalamus neurons. ERK1/2 pathway processed in stimulating the hypothalamus neurons [Ca~(2+)]i by CART.
4.5 The results of INS-1 cells and isolated islet cells demonstrated that it could be explained possibly a stronger ensuring activation of the PKA-dependent pathway. Sitagliptin (a dose-related manner) has been demonstrated to improve glucose tolerance and increase GLP-1 and insulin secretion. Fluorescence immunohistochemistry and TEM examination revealed that labeling for CART was present in the secretory granules ofβcells andδcells in type 2 diabetic rats. CART peptides not only found in the brain but also found in the gut, they are referred to as‘brain-gut’peptides.
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