谷氨酸脱羧酶与Ι型糖尿病发病机制

谷氨酸脱羧酶(glutamic acid decarboxylase,GAD)与I型糖尿病(type 1 diabetes,T1DM)的发病有很大关系,被认为是Ⅰ型糖尿病发病的自身免疫启动靶抗原。谷氨酸脱羧酶对于Ⅰ型糖尿病的预测、诊断、预防和治疗都有很大应用价值。该文阐述了谷氨酸脱羧酶的最新研究进展,以及谷氨酸脱羧酶与I型糖尿病自身免疫发病机制的联系。     

谷氨酸脱羧酶研究进展

谷氨酸脱羧酶(glutamic acid decarboxylase,GAD,EC4.1.1.15)在生物体内广泛存在,其催化产物γ-氨基丁酸(γ-aminobutyric acid,GABA)是哺乳动物体内一种重要的抑制性神经递质。在对自身免疫性疾病以及糖尿病研究中,特别是1型糖尿病,GAD、GABA以及谷氨酸脱羧酶抗体(glutamic acid decarboxylase-antibody,GAD-Ab)等的水平作为病理分析、疾病诊断、免疫治疗的重要参数,历来备受研究者关注。本文就GAD及其催化产物GABA的研究进展进行了综述,为更好地研究自身免疫性疾病的发病机理,探索更加有效安全的治疗方法提供参考。     

重组人谷氨酸脱羧酶65的表达及活性研究

目的：获得有活性的人谷氨酸脱羧酶65（human glutamate decarboxylase 65,hGAD65）用于Ⅰ型糖尿病（type 1 diabetes mellitus,T1DM）检测。方法：将hGAD65基因插入质粒pET32a(+)中,与硫氧还蛋白（thioredox）及六聚组氨酸（hexahistidine）融合,由IPTG诱导融合蛋白（Trx-hGAD65）表达,经两次镍离子亲和层析纯化,通过薄层层析和ELISA研究hGAD65及Trx-hGAD65的活性。结果：在大肠杆菌中实现hGAD65的可溶性表达,经亲和层析得到较纯hGAD65和Trx-hGAD65蛋白,二者具有相同酶学活性,但Trx-hGAD65稳定性更高,并能检测出T1DM患者血清中的hGAD65抗体（hGAD65-Ab）。结论：Trx-hGAD65能在大肠杆菌中可溶性表达,从而为Trx-hGAD65用于T1DM诊断、预防和治疗打下基础。     

谷氨酸脱羧酶若干研究进展

谷氨酸脱羧酶是γ－氨基丁酸的合成酶,主要存在脑和胰岛中。因体内存在多种形成的谷氨酸脱羧酶,现无获得各种均一的谷氨酸脱羧酶的 统一方法。谷氨酸脱羧酶的克隆和表达,既弄清了谷氨酸脱羧酶的基因结构与定位,又为谷氨酸脱酶的大规模应用奠定了基础。目前认为谷氨酸脱羧酶是Ⅰ型糖尿病的始动靶抗原,体内注入谷氨酸脱羧酶可预防或延缓ＮＯＤ（ｎｏｎｏｂｅｓｅ ｄｉａｂｅｔｉｃ）小鼠Ⅰ型糖尿病的发生。     

谷氨酸脱羧酶结构及催化机制的研究概述

谷氨酸脱羧酶(glutamate decarboxylase,GAD)是一种磷酸吡哆醛(pyridoxal-5′-phosphate,PLP)依赖性酶,广泛存在于自然界的动植物和微生物中,在酸性环境下发生结构变化,不可逆地催化L-谷氨酸或谷氨酸盐α-脱羧生成γ-氨基丁酸(γ-aminobutyric acid,GABA)。γ-氨基丁酸在人体中作为一种抑制性神经递质,具有重要的生理功能,可以被广泛应用于食品和制药工业中。本文就谷氨酸脱羧酶结构及催化机制的研究进展进行概述。     

香蕉谷氨酸脱羧酶基因克隆与表达

根据抑制缩减杂交文库获得的香蕉谷氨酸脱羧酶基因片段,利用RACE技术,首次从香蕉果实中克隆了谷氨酸脱羧酶基因的cDNA全长.结果表明,该cDNA的ORF全长1 500 bp,编码499个氨基酸.Blast分析表明,该基因所推导的氨基酸序列与水稻、柑橘、白杨、番茄等具有较高的一致性,分别为82%、81% 、79% 、78%,推测其编码的蛋白质分子量为56.25 kD,等电点5.21,具有与钙调蛋白结合的C端延伸区域和磷酸吡哆醛结合位点.组织特异性和果实采后正常成熟不同阶段表达结果显示,该基因在香蕉根、茎、叶、花、果实中均有表达,果实中的表达量较高,正常成熟表达量的增加可能受内源乙烯诱导并促进乙烯生物合成,推测其可能在不同的生理过程中起作用,并且与果实成熟及乙烯生物合成密切相关.     

L—谷氨酸脱羧酶电极的研制及性能

酶电极是由传感器和酶膜(或其它形式)组成的一种生物传感器,它通过酶催化反应作用,由传感器检测底物到产物转化或其他变化来进行测定,因此具有专一性好,灵敏度高,操作简便等特点。自本世纪六十年代,Updike和Hicks首先提出酶电极这一概念以来,酶电极的研究发展十分迅速,许多成果已被应用于临床、发酵及食品工业、环境保护等领域。氨基酸,可通过氨基酸脱羧酶的作用,由瓦氏呼吸计检测生成的二氧化碳的量而定量测定。1976年,Tong和Rechnitz首先报道了应用赖氨酸脱羧酶电极测定赖氨酸。其后,White和Guilbault及Tran等人分别对之进行了改进;最近,谷氨酸脱羧酶电极的研究也有报道。     

猪脑谷氨酸脱羧酶的分离纯化以及生物学活性鉴定

L-谷氨酸脱羧酶是γ-氨基丁酸合成的关键限速酶,广泛的存在于脊椎动物神经细胞以及β-胰腺细胞,是胰岛素依赖型糖尿病(IDDM)病人以及僵硬综合症(SMS)病人血清的关键抗原。运用sephamryl S-200以及DEAEsepharose可以从猪脑中分离纯化出谷氨酸脱羧酶。纯化的GAD在变性条件下电泳,经考马斯亮蓝R250染色以及Western-Blot鉴定主要有两条带,分子量分别为67kD和44kD。根据L-谷氨酸脱羧酶能够分解谷氨酸产生γ-氨基丁酸和CO2的特性,通过测定产物γ-氨基丁酸推断酶活。以上实验结果表明从猪脑中分离纯化到的是具有生物学活性以及免疫原性的谷氨酸脱羧酶,可进一步改良为IDDM检测试剂盒,用于IDDM的预防和预测。     

Sitagliptin in Glutamic Acid Decarboxylase Antibody-Positive Diabetes Mellitus

ObjectiveTo describe a case illustrating the use of sitagliptin, an inhibitor of dipeptidyl-peptidase-4 (DPP-4), in anti-glutamic acid decarboxylase antibody-positive diabetes mellitus in association with a rare ataxic variant of stiff person syndrome.MethodsWe present our experience with use of the DPP-4 inhibitor sitagliptin for management of autoimmune diabetes in a elderly woman and highlight the association of diabetes with other autoimmune conditions.ResultsA 68-year-old Japanese woman presented with poorly controlled “type 2” diabetes mellitus, cerebral palsy, cerebellar ataxia, and hypothyroidism. She complained of stiffness and spasms, which had resulted in multiple falls and immobility. Antidiabetic medications included gliclazide, rosiglitazone, and acarbose; various insulins had been tried but discontinued because they worsened her stiffness and spasms. Her hemoglobin A_1c values remained above 9% despite maximal doses of the aforementioned orally administered hypoglycemic agents. After sitagliptin therapy was initiated, her hemoglobin A_1c level decreased from 9.3% (78 mmol/mol) to 7.3% (56 mmol/mol) in 5 months. Investigations confirmed the presence of an ataxic variant of stiff person syndrome. On repeated testing 18 months later, her anti-glutamic acid decarboxylase antibody levels had declined by more than 85%.ConclusionApart from the well-known mechanism of an increase in glucagonlike peptide-1, sitagliptin may exert its glucose-lowering effect by other mechanisms in patients with autoimmune diabetes. Further studies should be undertaken to address the effectiveness of DPP-4 inhibitors in non-type 2 diabetes. (Endocr Pract. 2012;18: e65-e68)     

Time Course and Localization of the Effects of Estrogen on Glutamic Acid Decarboxylase Activity1

Abstract: Two approaches were used in an attempt to characterize the effect of estrogen on glutamic acid decarboxylase (GAD) [EC 4.1.1.15] activity in ovariectomized rats. In the first experiment, estradiol-17β (E₂) was unilaterally implanted in one of five different brain areas. After 3 days of estrogen exposure, the animals were sacrificed, and GAD activity in the substantia nigra (SN) and ventral tegmental region (VTR) was measured. Estrogen implanted into the preoptic area and the ventromedial nucleus was ineffective, as were implants of cholesterol, regardless of implant site. However, GAD activity was decreased in the SN when E₂ was implanted into the caudate nucleus or amygdala and in the VTR when implanted into the nucleus accumbens septi. Furthermore, this decrease in GAD activity occurred only in the implanted side. In the second experiment, the time course of changes in GAD activity was measured in ovariectomized rats given a single systemic injection of either 8μg estradiol benzoate (EB) or oil. Rats were sacrificed at 0, 12, 29, or 53 h postinjection. It was found that GAD activity in the SN was maximally suppressed 29 h after EB, whereas decreased GAD activity in the VTR was apparent 12 h after EB but had returned to normal by 29 h. Oil injections had no significant effect on GAD activity. These results suggest that there may be two separate and distinct γ-aminobutyric acid pathways, which are differentially responsive to estrogen.     

Some Characteristics of Glutamic Acid Decarboxylase of Chick Ampullary Cristae

Abstract: In a previous study, it was demonstrated that enzyme-mediated γ-aminobutyric acid (GABA) synthesis occurs in the vestibule of the chick inner ear. As deeper knowledge of the properties of its synthesizing enzyme might contribute to the understanding of the role of GABA in inner ear function, some characteristics of glutamate decarboxylase (GAD) were studied in chick isolated ampullary cristae under conditions in which ¹⁴CO₂ release from [1-¹⁴C]glutamate and [¹⁴C]GABA formation from [U-¹⁴C]glutamate for estimating GAD activity were equal. It was found that K _m for glutamate is 5 m M and that the enzyme pH optimum is 7.3. These values fall within the range described for the corresponding enzyme in nervous tissue of other species. Pyridoxal phosphate (PLP) activates the enzyme and aminooxyacetic acid inhibits it, the same as these agents activate or inhibit GAD from several nervous tissue sources. 2-Mercaptoethanol shows some protection from inactivation of the PLP-de-pendent enzyme and Triton X-100 exerts some inhibition of vestibular GAD activity, as previously shown in other nervous tissue preparations. Although its cellular localization is at present uncertain, these results indicate that GAD of chick vestibular tissue possesses properties resembling those of the brain enzyme and might be controlled in a manner similar to that of GAD in brain, thus possibly participating in the regulation of inner ear function.     

大鼠脑谷氨酸脱羧酶基因的cDNA克隆及序列分析

胰岛β－细胞自身抗原蛋白之一是脑中谷氨酸脱羧酶（Ｇｌｕｔａｍｉｃａｃｉｄｄｅｃａｒｂｏｘｙｌａｓｅ,ＧＡＤ,ＥＣ４．１．１．１５）同源物。以双链ｃＤＮＡ为模板,用ＰＣＲ方法快速克隆了Ｗｉｓｔａｒ大鼠脑ＧＡＤ基因的ｃＤＮＡ,将此包括编码５９３个氨基酸的全长ＤＮＡ片段重组入ｐＵＣ质粒并用双脱氧末端终止法测定了全部序列,证明其全长为１７７９ｂｐ．经比较发现Ｗｉｓｔａｒ大鼠脑与Ｒｕｓｓｅｌｌ报导的大鼠脑ＧＡＤ基因序列,有一处碱基的差别,但并不涉及氨基酸的改变。同时还对用ＰＣＲ扩增长片段ＤＮＡ进行了方法学上的探讨。     

Characterization of Glutamic Acid Decarboxylase Activity in Cerebral Blood Vessels

Abstract: Glutamic acid decarboxylase activity associated with cerebral blood vessels appears to be part of a specific cerebrovascular system involving γ-aminobutyric acid. This activity was characterized kinetically and pharmacologically and compared with that in brain and several nonneuronal tissues. Formation of γ-aminobutyric acid from [¹⁴C]glutamate was measured in a soluble extract of pia-arachnoid blood vessels isolated from bovine brain. The vascular activity was like brain glutamate decarboxylase in that it required pyridoxal phosphate, was completely inhibited by aminooxyacetic acid, and had a similar affinity for glutamate. Cerebrovascular decarboxylase activity differed, however, from brain decarboxylase in that it was less sensitive to sulfhydryl reagents, was stimulated by 3-mercaptopropionic and cysteic acids, and was competitively inhibited by cysteine sulfinic acid. The glutamate decarboxylase activity of the cerebral vessels was similar to that in renal cortex and mesenteric blood vessels in its responses to sulfhydryl reagents and 3-mercaptopropionic acid. These findings are consistent with previous suggestions of a nonneuronal form of the enzyme and offer the possibility that synthesis of γ-aminobutyric acid in cerebral blood vessels can be manipulated independently from that in neuronal tissue.     

Striatal Expression of Glutamic Acid Decarboxylase Gene in Alzheimer's Disease

Abstract: To examine potential alteration of GABAergic striatal neurons in Alzheimer's disease, we used quantitative in situ hybridization to analyze the messenger RNA coding for M_r 67,000 glutamic acid decarboxylase (GAD₆₇ mRNA) in the striatum of five patients with Alzheimer's disease (AD) and nine matched control subjects. We found a 51–57% increase in the optical density of hybridization signal in the caudate nucleus and putamen, corresponding to a 30–42% increase in the number of neurons expressing a detectable amount of GAD₆₇ mRNA. By contrast, no alteration was observed in the ventral striatum. The expression of GAD₆₇ mRNA per neuron was similar in AD and control subjects both in the dorsal and ventral striatum. Taken together, our data indicate that, in AD, GABAergic neurotransmission is increased in the dorsal striatum but not in the ventral striatum. We suggest that this increased GABAergic neurotransmission may explain extrapyramidal signs often observed in AD.     

IGF2BP2 Alternative Variants Associated with Glutamic Acid Decarboxylase Antibodies Negative Diabetes in Malaysian Subjects

Background

The association of Insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) common variants (rs4402960 and rs1470579) with type 2 diabetes (T2D) has been performed in different populations. The aim of this study was to evaluate the association of alternative variants of IGF2BP2; rs6777038, rs16860234 and rs7651090 with glutamic acid decarboxylase antibodies (GADA) negative diabetes in Malaysian Subjects.

Methods/Principal Findings

IGF2BP2; rs6777038, rs16860234 and rs7651090 single nucleotide polymorphisms (SNPs) were genotyped in 1107 GADA negative diabetic patients and 620 control subjects of Asian from Malaysia. The additive genetic model adjusted for age, race, gender and BMI showed that alternative variants; rs6777038, rs16860234 and rs7651090 of IGF2BP2 associated with GADA negative diabetes (OR = 1.21; 1.36; 1.35, P = 0.03; 0.0004; 0.0002, respectively). In addition, the CCG haplotype and diplotype CCG-TCG increased the risk of diabetes (OR = 1.51, P = 0.01; OR = 2.36, P = 0.009, respectively).

Conclusions/Significance

IGF2BP2 alternative variants were associated with GADA negative diabetes. The IGF2BP2 haplotypes and diplotypes increased the risk of diabetes in Malaysian subject.