新型钾通道开放剂对心血管ATP-敏感性钾通道基因表达的调节作用

目的：研究脂肪胺类的新型钾通道开放剂（KCO）埃他卡林（Ipt）和氰胍类的KCO吡那地尔（Pin）对大鼠心血管ATP-敏感性钾通道（KATP）的亚基SUR1、SUR2、Kir6．1和Kir6．2等在mRNA水平的调节作用。方法：SD大鼠给药1周后处死并取组织,提取总RNA,利用反转录-聚合酶链式反应（RT-PCR）研究以上基因在mRNA水平的改变。结果：与正常对照相比,心脏组织中,Ipt和Pin对KATP的4个亚基在mRNA水平均无显著影响;主动脉平滑肌上,Ipt对4个亚基的mRNA表达无显著影响,但Pin可显著上调SUR2的mRNA表达;尾动脉平滑肌上,Ipt对Kit6．1／Kit6．2、Pin对SUR2／Kir6．1均有显著下调的作用。结论：心肌、大动脉平滑肌和小动脉平滑肌KATP基因表达的调控不同,Ipt选择性调节小动脉平滑肌Kit6．1／Kit6．2;Ipt对心血管KATP基因表达的调节作用不同于Pin。     

ATP合成下降在β细胞胰岛素分泌障碍中的作用

胰岛β细胞胰岛素分泌过程是受多种因素协调精确控制的,ATP合成酶在这一调控网络中起着重要作用.高糖、高脂及炎症细胞因子,通过不同的信号通路,引起线粒体膜电位改变及/或ATP合成酶核心亚基表达下降,导致ATP合成速率下降,是β胰岛素分泌障碍发生的共同核心环节,在2型糖尿病病理生理过程中起了关键性作用.糖尿病动物胰岛β细胞内的ATP含量较正常β细胞明显降低,而上调2型糖尿病患者胰岛细胞ATP合成酶β亚基表达能提高ATP合成速率,增加细胞ATP含量并逆转损伤的胰岛素分泌功能.目前的研究提示,亮氨酸、肠抑素(enterostatin)及过氧化物酶体增殖物激活受体γ(PPAR-γ)能通过调控ATP合成酶β亚基表达或活性提高细胞ATP合成速率,这为改善β细胞功能障碍提供了新的思路和信息.     

3T3-L1脂肪细胞对MIN6胰岛素细胞中Kir6.2表达的抑制作用

为明确脂肪细胞对胰岛素细胞中KATP通道表达的直接影响,MIN6胰岛素细胞被分为两组：一组为对照组,一组与分化的3T3-L1脂肪细胞共培养1周。运用半定量RT-PCR方法测定MIN6细胞中KATP通道蛋白Kir6．2的表达变化,Fura-2荧光方法测定MIN6细胞内钙浓度的变化,放射免疫测定方法明确MIN6细胞的胰岛素分泌功能。结果显示,与3T3-L1脂肪细胞共培养1周后,MIN6细胞中Kir6．2的表达明显减少,其表达水平降低为对照组的65．3％。对照组MIN6细胞在0．1mmoi／L甲苯磺丁脲(KATP通道关闭剂)的刺激下,表现为细胞内钙水平显著性升高和胰岛素分泌显著性增加,而共培养组MIN6细胞则失去了甲苯磺丁脲刺激所引起的细胞内钙升高及胰岛素分泌反应。以上实验结果表明,3T3-L1脂肪细胞可以通过分泌一些活性因子直接降低MIN6细胞中KATP通道蛋白的表达和合成,损害MIN6细胞的胰岛素分泌功能。实验结果提示脂肪细胞直接参与2型糖尿病中胰岛β细胞功能障碍的发生。     

氧化应激与2型糖尿病

胰岛素抵抗、胰岛β细胞功能受损是2型糖尿病的主要病因。高血糖、高血脂导致在代谢过程中,线粒体产生大量活性氧,其可损坏线粒体功能,引起氧化应激反应。氧化应激可以激活细胞内的一系列应激信号通路,如JNK／SAPK、p38MAPK、IKKβ／NF-kβ和氨基己醣通路等。这些应激通路的激活可以产生以下结果：（1）阻断胰岛素作用通路,导致胰岛素抵抗;（2）降低胰岛素基因表达水平;（3）抑制胰岛素分泌;（4）促进β细胞凋亡等。本文主要针对活性氧的产生、氧化应激诱导胰岛素抵抗和胰岛β细胞功能受损等机制加以综述,以便进一步阐明2型糖尿病的发病机理。     

线粒体基因突变相关的糖尿病研究进展

线粒体是细胞的能量工厂,通过氧化磷酸化以ATP的形式为细胞的各种生命活动提供能量。线粒体基因的突变会导致多种疾病的发生,包括LHON病、耳聋、Ⅱ型糖尿病等。本综述描述了与Ⅱ型糖尿病相关的67种线粒体基因突变,重点讨论了线粒体基因3243、3310、16189、14709、15059、10003位点相应碱基突变与Ⅱ型糖尿病的相关性关系,总结和讨论了线粒体基因突变导致糖尿病发生的具体机制,即线粒体基因突变导致胰岛β细胞氧化磷酸化功能缺陷,ATP产量降低,ADP+含量升高,胰岛素分泌受阻,胰岛素产量降低,机体正常糖代谢受阻从而出现高血糖等二型糖尿病症状。最后,本综述对Ⅱ型糖尿病的诊断方法和治疗手段进行了阐述。     

氧化应激与2型糖尿病的研究进展

氧化应激与2型糖尿病(T2DM)的发生、发展密切相关.胰岛素抵搞(Insulin Resistance,IR)、胰岛β细胞功能受损是2型糖尿病的主要病因.而氧化应激可以直接及间接激活细胞内的一系列应激信号通路,如核因子κ-B(Nuclear factor-KappaB,NF-κB)、c-Jun氨基端激酶(NH-terminal Jun kinase,JNK)、蛋白激酶C(protein kinase C,PKC)、p38丝裂原活化蛋白激酶(Mitogen-activated protein kinase,MAPK)等.这些应激通路的激活可以产生以下结果:(1)阻断胰岛素作用通路,导致胰岛素抵抗;(2)降低胰岛素基因表达水平,致胰岛素合成和分泌减少;(3)促进胰岛β细胞凋亡等.本文针对氧化应激诱导胰岛素抵抗和胰岛β细胞功能受损等机制加以综述,以便进一步阐明2型糖尿病的发病机制.     

肝细胞核因子在2型糖尿病发生中的作用研究新进展

肝细胞核因子(Hepatocyte nuclear factors,HNFs)是一类分布在肝、胰、肠、肾等多个组织器官,调节肝脏内基因特异性表达的一类转录因子。其主要亚型为HNF1、HNF3、HNF4和HNF6等,这些转录因子相互作用构成的复杂调控网络。2型糖尿病(Type2 diabetes mellitus,T2DM)的基本病理生理机制是胰岛素抵抗及胰岛β细胞损伤,最终导致高血糖。近年来的研究表明,HNFs在胰岛素抵抗及胰岛β细胞损伤中发挥关键的调控作用。本文对HNFs在T2DM发生中的胰岛素抵抗及胰岛β细胞损伤作用研究新进展作以回顾性综述,旨在为认识T2DM发病机制及提出防治策略提供理论基础。     

胰岛β细胞炎症与2型糖尿病

2型糖尿病属于代谢性疾病,它的发生发展受环境因素和多种基因的共同调控.近年来研究认为2型糖尿病属于代谢性炎症,可能是由细胞因子介导的一种慢性炎症反应性疾病.胰岛作为胰岛素的分泌器官,它的异常是2型糖尿病发病进程中的一个重要病理基础.长期的高糖,高脂及巨噬细胞浸润等因素都会刺激细胞因子的大量生成,造成胰岛β细胞的炎症反应,对胰岛β细胞分泌胰岛素的功能和细胞活力产生不同程度的损伤,导致其功能障碍和凋亡,进而促使2型糖尿病的发生发展.本文根据国内外近几年的研究进展,进一步了探讨胰岛β细胞炎症与2型糖尿病的关系.这种代谢性炎症的研究,进一步阐明了炎症的发生,引起胰岛素抵抗、功能障碍的具体机制,革新了对2型糖尿病发病机理的认识,并为2型糖尿病的防治提供了新的方向.     

胰岛移植的应用及新方法

1型糖尿病是由自体免疫系统破坏胰腺胰岛β细胞引起的,可导致胰岛素严重缺乏。当β细胞被大量破坏时,胰岛素分泌不足引起血糖升高甚至出现酮症酸中毒症状。近年来,胰岛细胞移植发展成为一种有效的治疗1型糖尿病的方法。该文对胰岛移植治疗1型糖尿病的发展史、胰岛移植方法以及面临的问题进行讨论,并详细阐述了提高胰岛移植效率新方法的最新研究进展。这些方法包括扩大供体来源、胰岛细胞和间充质干细胞共移植、应用纳米技术包装胰岛、在胰岛细胞中诱导保护性基因表达等方面。这些新方法可以显著提高胰岛细胞移植效率,不仅可以应用在异体胰岛移植中,也可用在同体胰岛移植过程中。     

1型糖尿病治疗方法的回顾与展望

1型糖尿病是由包括病毒感染、药物接触及自身免疫在内的各种原因导致的胰岛β细胞凋亡所引起的,主要表现为由血清中胰岛素的绝对缺乏引起的高血糖。在过去数十年中,外源补充胰岛素一直是1型糖尿病的最主要治疗方法。随着人们对1型糖尿病机制的深入了解及生命科学相关技术的发展,科研工作者及临床医生开始探索治疗1型糖尿病的新方法,其中包括将分泌胰岛素的外源胰岛或干细胞移植入体内。或将胰岛素基因直接导入体内,合成并分泌体内缺乏的胰岛素等。本文对胰岛移植、干细胞和基因疗法用于治疗1型糖尿病的主要方式做一简要回顾与综述,并重点讨论近年来的研究进展及其临床应用的可行性。     

Compound heterozygous mutations in the SUR1 (ABCC 8) subunit of pancreatic K(ATP) channels cause neonatal diabetes by perturbing the coupling between Kir6.2 and SUR1 subunits

KATP channels regulate insulin secretion by coupling β-cell metabolism to membrane excitability. These channels are comprised of a pore-forming Kir6.2 tetramer which is enveloped by four regulatory SUR1 subunits. ATP acts on Kir6.2 to stabilize the channel closed state while ADP (coordinated with Mg(2+)) activates channels via the SUR1 domains. Aberrations in nucleotide-binding or in coupling binding to gating can lead to hyperinsulinism or diabetes. Here, we report a case of diabetes in a 7-mo old child with compound heterozygous mutations in ABCC8 (SUR1[A30V] and SUR1[G296R]). In unison, these mutations lead to a gain of KATP channel function, which will attenuate the β-cell response to increased metabolism and will thereby decrease insulin secretion. (86)Rb(+) flux assays on COSm6 cells coexpressing the mutant subunits (to recapitulate the compound heterozygous state) show a 2-fold increase in basal rate of (86)Rb(+) efflux relative to WT channels. Experiments on excised inside-out patches also reveal a slight increase in activity, manifested as an enhancement in stimulation by MgADP in channels expressing the compound heterozygous mutations or homozygous G296R mutation. In addition, the IC 50 for ATP inhibition of homomeric A30V channels was increased ~6-fold, and was increased ~3-fold for both heteromeric A30V+WT channels or compound heterozygous (A30V +G296R) channels. Thus, each mutation makes a mechanistically distinct contribution to the channel gain-of-function that results in neonatal diabetes, and which we predict may contribute to diabetes in related carrier individuals.     

Sulfonylureas suppress the stimulatory action of Mg-nucleotides on Kir6.2/SUR1 but not Kir6.2/SUR2A KATP channels: A mechanistic study

Sulfonylureas, which stimulate insulin secretion from pancreatic β-cells, are widely used to treat both type 2 diabetes and neonatal diabetes. These drugs mediate their effects by binding to the sulfonylurea receptor subunit (SUR) of the ATP-sensitive K⁺ (K_ATP) channel and inducing channel closure. The mechanism of channel inhibition is unusually complex. First, sulfonylureas act as partial antagonists of channel activity, and second, their effect is modulated by MgADP. We analyzed the molecular basis of the interactions between the sulfonylurea gliclazide and Mg-nucleotides on β-cell and cardiac types of K_ATP channel (Kir6.2/SUR1 and Kir6.2/SUR2A, respectively) heterologously expressed in Xenopus laevis oocytes. The SUR2A-Y1206S mutation was used to confer gliclazide sensitivity on SUR2A. We found that both MgATP and MgADP increased gliclazide inhibition of Kir6.2/SUR1 channels and reduced inhibition of Kir6.2/SUR2A-Y1206S. The latter effect can be attributed to stabilization of the cardiac channel open state by Mg-nucleotides. Using a Kir6.2 mutation that renders the K_ATP channel insensitive to nucleotide inhibition (Kir6.2-G334D), we showed that gliclazide abolishes the stimulatory effects of MgADP and MgATP on β-cell K_ATP channels. Detailed analysis suggests that the drug both reduces nucleotide binding to SUR1 and impairs the efficacy with which nucleotide binding is translated into pore opening. Mutation of one (or both) of the Walker A lysines in the catalytic site of the nucleotide-binding domains of SUR1 may have a similar effect to gliclazide on MgADP binding and transduction, but it does not appear to impair MgATP binding. Our results have implications for the therapeutic use of sulfonylureas.     

A gating mutation at the internal mouth of the Kir6.2 pore is associated with DEND syndrome

Inwardly rectifying potassium (Kir) channels control cell membrane K+ fluxes and electrical signalling in diverse cell types. Heterozygous mutations in the human Kir6.2 gene (KCNJ11), the pore-forming subunit of the ATP-sensitive (K(ATP)) channel, cause permanent neonatal diabetes mellitus. However, the I296L mutation also results in developmental delay, muscle weakness and epilepsy. We investigated the functional effects of the I296L mutation by expressing wild-type or mutant Kir6.2/SUR1 channels in Xenopus oocytes. The mutation caused a marked increase in resting whole-cell K(ATP) currents by reducing channel inhibition by ATP, in both homomeric and simulated heterozygous states. Kinetic analysis showed that the mutation impaired ATP sensitivity indirectly, by stabilizing the open state of the channel and possibly also by means of an allosteric effect on ATP binding and/or transduction. The results implicate a new region in Kir-channel gating and suggest that disease severity is correlated with the extent of reduction in ATP sensitivity.     

Permanent neonatal diabetes caused by dominant, recessive, or compound heterozygous SUR1 mutations with opposite functional effects

Heterozygous activating mutations in the KCNJ11 gene encoding the pore-forming Kir6.2 subunit of the pancreatic beta cell K(ATP) channel are the most common cause of permanent neonatal diabetes (PNDM). Patients with PNDM due to a heterozygous activating mutation in the ABCC8 gene encoding the SUR1 regulatory subunit of the K(ATP) channel have recently been reported. We studied a cohort of 59 patients with permanent diabetes who received a diagnosis before 6 mo of age and who did not have a KCNJ11 mutation. ABCC8 gene mutations were identified in 16 of 59 patients and included 8 patients with heterozygous de novo mutations. A recessive mode of inheritance was observed in eight patients with homozygous, mosaic, or compound heterozygous mutations. Functional studies of selected mutations showed a reduced response to ATP consistent with an activating mutation that results in reduced insulin secretion. A novel mutational mechanism was observed in which a heterozygous activating mutation resulted in PNDM only when a second, loss-of-function mutation was also present.     

Snowflake Vitreoretinal Degeneration (SVD) Mutation R162W Provides New Insights into Kir7.1 Ion Channel Structure and Function

Snowflake Vitreoretinal Degeneration (SVD) is associated with the R162W mutation of the Kir7.1 inwardly-rectifying potassium channel. Kir7.1 is found at the apical membrane of Retinal Pigment Epithelial (RPE) cells, adjacent to the photoreceptor neurons. The SVD phenotype ranges from RPE degeneration to an abnormal b-wave to a liquid vitreous. We sought to determine how this mutation alters the structure and function of the human Kir7.1 channel. In this study, we expressed a Kir7.1 construct with the R162W mutation in CHO cells to evaluate function of the ion channel. Compared to the wild-type protein, the mutant protein exhibited a non-functional Kir channel that resulted in depolarization of the resting membrane potential. Upon co-expression with wild-type Kir7.1, R162W mutant showed a reduction of I_Kir7.1 and positive shift in ‘0’ current potential. Homology modeling based on the structure of a bacterial Kir channel protein suggested that the effect of R162W mutation is a result of loss of hydrogen bonding by the regulatory lipid binding domain of the cytoplasmic structure.     

恒河猴糖尿病动物模型血清及组织中细胞因子的变化

目的探讨恒河猴糖尿模型病细胞因子的水平和变化规律。方法健康恒河猴5只,小剂量(30mg/kg)多次静脉注射链脲佐菌素(STZ),血糖稳定后3、9、12和19个月检测血清中白细胞介素-6(IL-6)、白细胞介素-10(IL-10)、肿瘤坏死因子-α(TNF-α)的含量。安乐濒死状态的动物,取胰腺、肝脏、肾脏制成石蜡切片,用免疫组化染色法显示组织中IL-6、IL-10、TNF-α的表达,并对结果进行图像分析和统计学处理。结果造模后9个月动物血清中IL-10浓度显著降低(P<0.01)。IL-6、TNF-α水平在采样的各个时间点均显著高于造模前(P<0.01)。胰腺组织中IL-6中等强度着色,对照组弱阳性表达(P<0.05)。对照组IL-10强阳性表达,模型组弱阳性(P<0.01)。模型组TNF-α中等强度表达,对照组弱阳性(P<0.05)。模型组肝脏组织TNF-α中等阳性表达,对照组弱阳性(P<0.05)。结论小剂量多次注射STZ后恒河猴细胞因子的含量及变化与临床类似,可作为相关研究的动物模型。     

Characterization and Functional Restoration of a Potassium Channel Kir6.2 Pore Mutation Identified in Congenital Hyperinsulinism

The inwardly rectifying potassium channel Kir6.2 assembles with sulfonylurea receptor 1 to form the ATP-sensitive potassium (K_ATP) channels that regulate insulin secretion in pancreatic β-cells. Mutations in K_ATP channels underlie insulin secretion disease. Here, we report the characterization of a heterozygous missense Kir6.2 mutation, G156R, identified in congenital hyperinsulinism. Homomeric mutant channels reconstituted in COS cells show similar surface expression as wild-type channels but fail to conduct potassium currents. The mutated glycine is in the pore-lining transmembrane helix of Kir6.2; an equivalent glycine in other potassium channels has been proposed to serve as a hinge to allow helix bending during gating. We found that mutation of an adjacent asparagine, Asn-160, to aspartate, which converts the channel from a weak to a strong inward rectifier, on the G156R background restored ion conduction in the mutant channel. Unlike N160D channels, however, G156R/N160D channels are not blocked by intracellular polyamines at positive membrane potential and exhibit wild-type-like nucleotide sensitivities, suggesting the aspartate introduced at position 160 interacts with arginine at 156 to restore ion conduction and gating. Using tandem Kir6.2 tetramers containing G156R and/or N160D in designated positions, we show that one mutant subunit in the tetramer is insufficient to abolish conductance and that G156R and N160D can interact in the same or adjacent subunits to restore conduction. We conclude that the glycine at 156 is not essential for K_ATP channel gating and that the Kir6.2 gating defect caused by the G156R mutation could be rescued by manipulating chemical interactions between pore residues.     

Compound heterozygous mutations in the SUR1 (ABCC 8) subunit of pancreatic KATP channels cause neonatal diabetes by perturbing the coupling between Kir6.2 and SUR1 subunits

K_ATP channels regulate insulin secretion by coupling β-cell metabolism to membrane excitability. These channels are comprised of a pore-forming Kir6.2 tetramer which is enveloped by four regulatory SUR1 subunits. ATP acts on Kir6.2 to stabilize the channel closed state while ADP (coordinated with Mg²⁺) activates channels via the SUR1 domains. Aberrations in nucleotide-binding or in coupling binding to gating can lead to hyperinsulinism or diabetes. Here, we report a case of diabetes in a 7-mo old child with compound heterozygous mutations in ABCC8 (SUR1[A30V] and SUR1[G296R]). In unison, these mutations lead to a gain of K_ATP channel function, which will attenuate the β-cell response to increased metabolism and will thereby decrease insulin secretion. ⁸⁶Rb⁺ flux assays on COSm6 cells coexpressing the mutant subunits (to recapitulate the compound heterozygous state) show a 2-fold increase in basal rate of ⁸⁶Rb⁺ efflux relative to WT channels. Experiments on excised inside-out patches also reveal a slight increase in activity, manifested as an enhancement in stimulation by MgADP in channels expressing the compound heterozygous mutations or homozygous G296R mutation. In addition, the IC₅₀ for ATP inhibition of homomeric A30V channels was increased ~6-fold, and was increased ~3-fold for both heteromeric A30V+WT channels or compound heterozygous (A30V +G296R) channels. Thus, each mutation makes a mechanistically distinct contribution to the channel gain-of-function that results in neonatal diabetes, and which we predict may contribute to diabetes in related carrier individuals.     

A mutation (R826W) in nucleotide-binding domain 1 of ABCC8 reduces ATPase activity and causes transient neonatal diabetes

Activating mutations in the pore-forming Kir6.2 (KCNJ11) and regulatory sulphonylurea receptor SUR1 (ABCC8) subunits of the K(ATP) channel are a common cause of transient neonatal diabetes mellitus (TNDM). We identified a new TNDM mutation (R826W) in the first nucleotide-binding domain (NBD1) of SUR1. The mutation was found in a region that heterodimerizes with NBD2 to form catalytic site 2. Functional analysis showed that this mutation decreases MgATP hydrolysis by purified maltose-binding protein MBP-NBD1 fusion proteins. Inhibition of ATP hydrolysis by MgADP or BeF was not changed. The results indicate that the ATPase cycle lingers in the post-hydrolytic MgADP.P(i)-bound state, which is associated with channel activation. The extent of MgADP-dependent activation of K(ATP) channel activity was unaffected by the R826W mutation, but the time course of deactivation was slowed. Channel inhibition by MgATP was reduced, leading to an increase in resting whole-cell currents. In pancreatic beta cells, this would lead to less insulin secretion and thereby diabetes.     

豚鼠冠状动脉微动脉细胞静息膜电位的分布特性及机制

目的:观察冠状动脉微动脉细胞静息膜电位(RP)的分布特性及形成机制.方法:离体豚鼠冠状动脉微动脉(直径小于100 μm)上,应用细胞内微电极技术记录细胞RP.结果:①成功记录到112个细胞,细胞平均RP为(-65±4.2)mV,应用高斯函数拟合后细胞RP呈双峰状分布,两个峰值分别为-43和-74 mV,分别称为高和低R...