2 型糖尿病家系患者PON2 基因9 Ser311C→G 多态性的研究*

目的:研究黑龙江地区汉族人2型糖尿病家系的PON2基因9 Ser311C→G多态性,探讨其与2型糖尿病发病的关系。方法:应用聚合酶链式反应-限制性内切酶长度多态性(PCR-RFLP)技术,对来自于黑龙江地区120个2型糖尿病家系中的210例2型糖尿病患者及319例正常对照的PON2基因9 Ser311→Cys(C→G)位点进行基因分型。结果:PON2基因9 Ser311C→G三种基因型在病例组和对照组间整体分布没有统计学意义(P=0.610,df=2);各基因型及等位基因在病例组和对照组间分布没有统计学意义(P>0.05)。结论:PON2基因9 Ser311C→G多态性与黑龙江地区汉族人2型糖尿病无关,PON2基因可能不是中国人2型糖尿病发病的相关易感基因。     

PAI-1 Gene 4G/5G polymorphism and risk of type 2 diabetes in a population-based sample

Elevated plasma levels of plasminogen activator inhibitor-1 (PAI-1) increase risk for type 2 diabetes. The PAI-1 4G/5G polymorphism is a major genetic determinant of plasma PAI-1 levels, with 4G/4G homozygotes having elevated PAI-1 levels relative to 5G allele carriers. These observations suggest the hypothesis that the PAI-1 4G/5G polymorphism could be a genetic risk factor for diabetes. We tested this hypothesis among 2169 participants of the Framingham Offspring Study followed for seven examinations over 26 years for 216 cases of type 2 diabetes. PAI-1 4G/4G homozygotes (genotype frequency, 27.4%) were not at significantly (p > 0.05) increased risk of incident diabetes compared with 5G allele carriers and did not have elevated levels of diabetes-related quantitative traits including BMI, fasting plasma glucose, or fasting insulin. In proportional hazards regression models accounting for correlation among siblings, with the 5G/5G genotype as the referent, the hazard ratio for incident diabetes for 4G/5G carriers was 0.93 (95% confidence interval, 0.68 to 1.28) and for 4G/4G carriers was 1.20 (95% confidence interval, 0.83 to 1.92). Results were not altered by further adjustment for sex or levels of BMI, triglycerides, or PAI-1. We conclude that the PAI-1 4G/5G polymorphism is not an important genetic risk factor for type 2 diabetes in this community-based sample. Elevated PAI-1 levels may be associated with an increased risk for diabetes as a marker for underlying endothelial dysfunction rather than by a direct effect of genetically mediated elevated levels.     

Relation between protoplast division,cell-cycle stage and nuclear chromatin structure

Summary Using different sources of protoplasts and two complementary techniques, flow cytometry and image analysis, to study the cell-cycle phases, we sought to define the particular protoplast state associated with the disposition to divide. Both inPetunia and inNicotiana plumbaginifolia, tissues with a higher G2 frequency (from different aged plants) yielded protoplasts capable of increased cell division. InSorghum, the age of the plant does not modify the proportion of G2 nuclei in leaf protoplasts, and we used root protoplasts to increase G2 frequencies. InHelianthus annuus, leaf protoplasts did not divide; however, hypocotyl protoplast preparations with relatively high 4C DNA frequencies do divide. Moreover, image analysis of chromatin structure indicated that leaf nuclei were in the G0 phase, unlike those from hypocotyls which were in G1. A high frequency of protoplasts with G2 nuclei appears to be correlated with the ability of a given preparation to undergo division; conversely, the differentiated G0 state is not conducive to division.     

一个2型糖尿病家系中新发现的线粒体DNA G7444A 突变分析

应用PCR-RFLP和测序对一个2型糖尿病家系的线粒体DNA G7444A的突变进行检测, 并分析其临床资料的特点。结果发现, 27例家系成员中, 11例母系亲属均存在线粒体DNA G7444A突变, 而配偶及父系亲属中未发现该突变。11例突变者中确诊为2型糖尿病患者5例, 糖耐量受损1例, 均表现为乳酸和血糖增高。因此, 线粒体DNA G7444A突变是该家系中糖尿病的遗传易感因素, 是导致2型糖尿病的一个新的突变位点。     

UCP-866G/A和ADIPQ+45T/G基因多态性的交互作用与2型糖尿病 合并冠心病发病风险的关系

目的：探讨UCP2-866G/A 和ADIPOQ+45T/G 基因多态性的交互作用与2型糖尿病合并冠心病发病风险的关系。方法：随机 选取2014 年10 月至2015 年5 月在佳木斯大学附属第一医院就诊的130 例单纯2 型糖尿病患者和128 例2 型糖尿病合并冠心 病患者进行病例对照研究。分别采用聚合酶链反应- 限制性片段长度多态性（PCR-RFLP）方法和聚合酶链反应- 高分辨率溶解曲 线（PCR-HRM）方法检测UCP2-866G/A 和ADIPOQ+45T/G 的基因多态性,并用非条件Logistic 回归分析两基因间的交互作用。 结果：在两组间分别进行UCP2-866G/A 和ADIPOQ+45T/G 基因多态性的单独关联分析,两变异位点的基因型和等位基因的频率 在两组间的分布及遗传模型关联分析均无统计学差异（P>0.05）。两变异位点联合分析发现,UCP2-866 G/A 的GG、GA 分别和 ADIPOQ+45T/G 的TG 在2 型糖尿病合并冠心病中存在正向交互作用（P=0.000,ORI=ORAB/(ORA× ORB)=30.533/(0.549× 0.116) >1;P=0.007,ORI= ORAB/(ORA× ORB)=13.914/(0.525× 0.116)>1。结论：该研究显示： UCP-866G/A 和ADIPOQ+ 45T/G 单一基因的 多态性与2 型糖尿病合并冠心病患病风险无关,而两者之间的交互作用可能增加2 型糖尿病合并冠心病的发病风险。     

Dimorphism in trichome production of <Emphasis Type="Italic">Persicaria lapathifolia</Emphasis> var. <Emphasis Type="Italic">lapathifolia</Emphasis> and Its multiple effects on a leaf beetle

Polymorphisms in plants are main factors that determine the diversity of associated animal communities and their population dynamics. Typically, Persicaria lapathifolia var. lapathifolia (Polygonaceae) has no trichomes on leaf surfaces (glabrous type), but a hairy type does sometimes occur. Based on a cultivation experiment, the presence or absence of trichomes is clarified to be under genetic control. To reveal the defensive function of trichomes against herbivores, laboratory experiments were conducted using a major herbivore, Galerucella grisescens (Coleoptera: Chrysomelidae). In both choice and no-choice feeding tests, the glabrous type was significantly more consumed by G. grisescens adults, while the hairy type was not consumed. In the hairy leaf treatment, larval duration tended to become longer, the adult body weight became significantly lower, and adults laid significantly more eggs than in the glabrous leaf treatment. Hairy leaves contained significantly more total phenolics and condensed tannins than glabrous leaves, suggesting that the hairy type allocates more resources for physical and chemical defence. Because no significant differences in leaf consumption were detected in the feeding experiment using powdered host leaves, G. grisescens seems to have adapted to the chemical defences of P. lapathifolia var. lapathifolia. These results clearly indicate that leaf trichomes of P. lapathifolia var. lapathifolia effectively act as a physical defence against G. grisescens.     

Conformations of Islet Amyloid Polypeptide Monomers in a Membrane Environment: Implications for Fibril Formation

The amyloid fibrils formed by islet amyloid polypeptide (IAPP) are associated with type II diabetes. One of the proposed mechanisms of the toxicity of IAPP is that it causes membrane damage. The fatal mutation of S20G human IAPP was reported to lead to early onset of type II diabetes and high tendency of amyloid formation in vitro. Characterizing the structural features of the S20G mutant in its monomeric state is experimentally difficult because of its unusually fast aggregation rate. Computational work complements experimental studies. We performed a series of molecular dynamics simulations of the monomeric state of human variants in the membrane. Our simulations are validated by extensive comparisons with experimental data. We find that a helical disruption at His18 is common to both human variants. An L-shaped motif of S20G mutant is observed in one of the conformational families. This motif that bends at His18 resembles the overall topology of IAPP fibrils. The conformational preorganization into the fibril-like topology provides a possible explanation for the fast aggregation rate of S20G IAPP.     

Association of the polymorphisms in the 5'-untranslated region of PTEN gene with type 2 diabetes in a Japanese population

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is known to act as a lipid phosphatase hydrolyzing phosphatidylinositol (PI)(3,4,5)P(3) to PI(4,5)P(2). Since the PI3-kinase product, PI(3,4,5)P(3), is an important second messenger leading to the metabolic action of insulin, PTEN functions as a potent negative regulator of insulin signaling and its gene is one of the possible candidates involved in susceptibility to the development of type 2 (non-insulin-dependent) diabetes. In the present study, we investigated the polymorphisms of the PTEN gene in Japanese patients with type 2 diabetes and non-diabetic control subjects. We identified three mutations of the gene in the type 2 diabetes patients. Among these mutations, the frequency of the substitution of C with G at position -9 (-9C-->G) (SNP1), located in the untranslated region of exon 1, was significantly higher in type 2 diabetic patients than in control subjects. In addition, transfection of the PTEN gene with SNP1 resulted in a significantly higher expression level of PTEN protein compared with that of the wild-type PTEN gene in Cos1 and Rat1 cells. Furthermore, insulin-induced phosphorylation of Akt in HIRc cells was decreased more greatly by transfection of SNP1 PTEN gene than that of wild-type PTEN gene. These findings suggest that the change of C to G at position -9 of the PTEN gene is associated with the insulin resistance of type 2 diabetes due possibly to a potentiated hydrolysis of the PI3-kinase product.     

Association of the -112A>C polymorphism of the uncoupling protein 1 gene with insulin resistance in Japanese individuals with type 2 diabetes

The -112A>C polymorphism (rs10011540) of the gene for uncoupling protein 1 (UCP1) has been associated with type 2 diabetes mellitus in Japanese individuals. The aim of the present study was to investigate the effects of this polymorphism, as well as the well-known -3826A>G polymorphism (rs1800592), on clinical characteristics of type 2 diabetes. We determined the genotypes of the two polymorphisms in 93 Japanese patients with type 2 diabetes. Intramyocellular lipid content and hepatic lipid content (HLC) were measured by magnetic resonance spectroscopy. No significant differences in age, sex, BMI, or HbA1c level were detected between type 2 diabetic patients with the -112C allele and those without it. However, homeostasis model assessment for insulin resistance (p=0.0089) and HLC (p=0.012) was significantly greater in patients with the -112C allele. We did not detect an association of the -3826A>G polymorphism (rs1800592) of UCP1 gene with any measured parameters. These results suggest that insulin resistance caused by the -112C allele influences the susceptibility to type 2 diabetes.     

Polymorphism of adiponectin (45T/G) and adiponectin receptor-2 (795G/A) in an Iranian population: relation with insulin resistance and response to treatment with pioglitazone in patients with type 2 diabetes mellitus

Adiponectin, an adipose-derived plasma protein, is reduced in patients with obesity and type 2 diabetes. Thiazolidinediones can increase adiponectin levels and improve insulin sensitivity. This study investigated the associations between type 2 diabetes and two single-nucleotide polymorphisms in the adiponectin (45T/G) and adiponectin receptor-2 gene (795G/A), and investigated whether these genetic variants affect the response to pioglitazone in Iranian patients with type 2 diabetes. We genotyped 128 non-diabetic participants and 101 patients with type 2 diabetes for 45T/G and 795G/A with polymerase chain reaction-restriction fragment length polymorphism assays. Patients were treated with pioglitazone for 12 weeks, after which we compared laboratory parameters in these two groups. Fasting blood sugar differed significantly in individuals with different 795G/A genotypes after pioglitazone treatment (P = 0.009). The mean decrease in insulin/glucose ratio after treatment also differed significantly in individuals with different 45T/G genotypes (P = 0.035). The T allele frequency for 45T/G was 87.11% in controls versus 81.68% in patients (P = 0.071). The TG and GG genotypes were more frequent in patients (P = 0.032). The G allele frequency for 795G/A was 76.17% in controls versus 80.20% in patients (P = 0.179). 795G/A variants were not significantly different between patient and control group. The adiponectin gene 45T/G mutation may be an important determinant of type 2 diabetes in the Iranian population. However, adiponectin 45T/G and adiponectin receptor-2 795G/A polymorphisms were not significantly associated with the response to pioglitazone in our sample.     

Study of <Emphasis Type="Italic">Kir6.2/KCNJ11</Emphasis> gene in a sudden cardiac death pedigree

In clinic, the patients with acute myocardial infarction (AMI) are at high risk to develop ischemia-induced ventricular arrhythmias leading to sudden cardiac death (SCD). Some studies suggest that individual susceptibility to ischemia-induced arrhythmia may be related to the genes encoding ion channels. One of them is the cardiac ATP-sensitive potassium channel (K(ATP)), which is an octamer composed of four pore-forming inwardly rectifying potassium-channel subunits (Kir6.2) and four regulatory sulfonylurea-receptor subunits (SUR2A). They play important roles in the physiology and pathophysiology of cardiovascular system by coupling the metabolic state of the cells to cellular electrical activity. So far, some mutations and polymorphisms of Kir6.2/KCNJ11 gene showed significant correlation with type 2 diabetes. But it was not sure whether it was associated with acute myocardial diseases. Hence a complete mutational analysis of Kir6.2/KCNJ11 gene was performed in a pedigree of sudden cardiac death. The complete coding region and the intron-exon boundaries of KCNJ11 were amplified from genomic DNA using polymerase chain reaction (PCR). Direct sequencing was done to identify any mutations and then further confirmed by restriction site polymorphism (RSP) approach. No mutation was detected in the samples analyzed, a common polymorphism K23E (A>G) was noticed in this pedigree and the proband showed a homozygote genotype (G/G). The result suggests that the Kir6.2/KCNJ11 gene is not related to sudden cardiac death in this family.     

The receptor-bound "empty pocket" state of the heterotrimeric G-protein alpha-subunit is conformationally dynamic

Heterotrimeric G-protein activation by a G-protein-coupled receptor (GPCR) requires the propagation of structural signals from the receptor-interacting surfaces to the guanine nucleotide-binding pocket. To probe conformational changes in the G-protein alpha-subunit (G(alpha)) associated with activated GPCR (R*) interactions and guanine nucleotide exchange, high-resolution solution NMR methods are being applied in studying signaling of the G-protein, transducin, by light-activated rhodopsin. Using these methods, we recently demonstrated that an isotope-labeled G(alpha) reconstituted heterotrimer forms functional complexes under NMR experimental conditions with light-activated, detergent-solubilized rhodopsin and a soluble mimic of R*, both of which trigger guanine nucleotide exchange [Ridge, K. D., et al. (2006) J. Biol. Chem. 281, 7635-7648]. Here, it is shown that both light-activated rhodopsin and the soluble mimic of R form trapped intermediate complexes with a GDP-released "empty pocket" state of the heterotrimer in the absence of GTP (or GTPgammaS). In contrast to guanine nucleotide-bound forms of G(alpha), the NMR spectra of the GDP-released, R-bound empty pocket state of G(alpha) display severe line broadening suggestive of a dynamic intermediate state. Interestingly, the conformation of a GDP-depleted, Mg(2+)-bound state of G(alpha) generated in a manner independent of R* does not exhibit a similar degree of line broadening but rather appears structurally similar to the GDP/Mg(2+)-bound form of the protein. Taken together, these results suggest that R*-mediated changes in the receptor-interacting regions of G(alpha), and not the absence of bound guanine nucleotide, are the predominant factors which dictate G(alpha) conformation and dynamics in this R*-bound state of the heterotrimer.     

Type 2 diabetes in children: clinical aspects and risk factors

In the past, type 2 diabetes mellitus was considered a disease of adults and older individuals, not a paediatric condition. Over the last decade, however, in the USA and the rest of the world there has been a disturbing trend of increasing cases of type 2 diabetes in children, mirroring increasing rates of obesity. The risk factors for paediatric type 2 diabetes are: (1) obesity and increased body mass index; (2) family history of type 2 diabetes; (3) membership of ethnic minority; (4) puberty (mean age of diagnosis is approximately 13.5 years); (5) female gender; and (6) features of 'syndrome X'. The common link among these risk factors is insulin resistance, which plays a pivotal role in the pathophysiology of type 2 diabetes. Both insulin resistance and beta-cell failure are present in the fully established diabetes state. Data will be presented on how these risk factors impact on insulin sensitivity and insulin secretion in childhood, ultimately leading to type 2 diabetes. The clinical presentation of type 2 diabetes in children and its distinction from type 1 diabetes will be discussed.     

A decrease in the sensitivity of adenylyl cyclase and heterotrimeric g proteins to chorionic gonadotrophin and peptide hormones action in the tissues of reproductive system of rats with experimental type 2 diabetes

Patients with different forms of the diabetes, particularly with insulin-independent type 2 diabetes, have a wide spectrum of the disturbances of the functions of reproductive system. It is suggested that the main reason of these disturbances is altered sensitivity of reproductive system tissues to the regulatory action of hormones. The aim of this study was the identification of the changes in functioning of adenylyl cyclase system (ACS) sensitive to human chorionic gonadotrophin (hCG) and the peptide hormones in the ovary, testes and uterus of rats with neonatal streptozotocin (STZ) diabetes that is similar to the type 2 diabetes in humans. The effects of hCG, PACAP-38 and relaxin, realizing their effects via stimulatory G proteins (G_s), and somatostatin, acting via the inhibitory G protein (G_i), on adenylyl cyclase (AC) activity and GTP binding to the G proteins were studied. In rats with STZ type 2 diabetes the regulatory effects of hCG and PACAP-38 decreased in the ovary and testes, while the effects of somatostatin decreased in all investigated tissues (especially in the uterus). This caused attenuation of the hormonal effects, stimulating (hCG and PACAP-38) or inhibiting (somatostatin) AC activity, and in the decrease of their stimulatory effect on the GTP binding. At the same time a significant decrease of ACS sensitivity to relaxin in the tissues of diabetic rats was not found. Data obtained suggest that one of the key reasons for impairments of reproductive functions in experimental type 2 diabetes is the decrease of ACS sensitivity to the hormones, hCG, PACAP-38 and somatostatin, which play an important role in the reproductive system functioning.     

Identification of a novel variant in the phosphoenolpyruvate carboxykinase gene promoter in Japanese patients with type 2 diabetes.

Phospho enolpyruvate carboxykinase (PEPCK) plays an important role in gluconeogenesis and hepatic glucose production. To test the hypothesis that mutations of the PEPCK gene promoter contribute to the increased hepatic glucose production that leads to diabetes, we screened for polymorphisms of the PEPCK promoter region in 252 Japanese type 2 diabetic patients and 188 non-diabetic control subjects. A novel variant at position - 232 (C to G) was found at a similar frequency in type 2 diabetes patients (32 %) and control subjects (35 %) (p = 0.26). However, patients with the - 232 G/G genotype had an earlier age of onset than those with the - 232 C/C or - 232 C/G genotypes (p = 0.028). As the variant might well otherwise influence hormonal action, we transfected PEPCK-luciferase fusion gene constructs with the variant into human hepatoma cells and examined the response to dexamethasone, insulin, and cAMP. The reporter assay showed no significant difference in hormonal responses with the fusion gene containing the variant. Accordingly, the single-base variant at position - 232 of the PEPCK gene promoter is most probably not a major contributor to the pathogenesis of type 2 diabetes. However, this variation may be useful as a genetic marker for other metabolic disorders, especially in Japanese.     

A novel mitochondrial DNA missense mutation at G3421A in a family with maternally inherited diabetes and deafness

OBJECTIVE: Mutations in mtDNA are thought to be responsible for the pathogenesis of maternally inherited diabetes. Here, we report a family with maternally inherited diabetes and deafness whose members did not harbour the mtDNA A3243G mutation, the most frequent point mutation in mitochondrial diabetic patients. This study aimed to investigate a possible other mtDNA mutation and its prevalence in type 2 diabetic patients. METHODS: Height, body weight, waistline, and hip circumference were measured and serum biochemical marks determined in all members of the family. In addition, a 75 g oral glucose tolerance test and electric listening test were conducted in these members. Genomic DNA was prepared from peripheral leukocytes. Direct sequencing of PCR products was used to detect the mtDNA mutation in this family. The prevalence of mtDNA G3421A nucleotide substitutions was investigated by restriction fragment length polymorphism analysis in 1350 unrelated type 2 diabetic patients recruited by random cluster sampling from the central city area of Shanghai, China. RESULTS: (1) A new missense homoplasmic mutation of mtDNA G3421A was found in a maternally inherited diabetic family and existed neither in 1350 unrelated type 2 diabetic patients nor in 50 non-diabetic individuals. (2) The mode of mutation and diabetes transmission was typical maternal inheritance in this family. (3) All diabetic family members were found to have an onset at 35-42 years of age, accompanied by deafness of varying degrees. CONCLUSION: mtDNA G3421A (Val39Ile) found in a family with maternally inherited diabetes and deafness is a novel missense mutation. Whether this is a diabetogenic mutation and its effect on mitochondrial function needs to be further studied.     

新疆维吾尔族人2型糖尿病患者AMPKα2基因多态性的分析研究

目的：研究腺苷酸活化蛋白激酶（AMP-activated protein kinase,AMPK）单核苷酸多态性（SNPs）rs2143754位点与新疆地区维吾尔族人2型糖尿病之间的关系。方法：以聚合酶链式反应-限制性内切酶长度多态性（polymerase chain reaction-restriction flagmerit length polymorphism,PCR-RFLP）技术,对150例2型糖尿病和150例正常对照者AMPK-rs2143754位点进行基因分型。结果：AMPKet2位点T／T、G／T和G／G基因型频率在病例组为（40％,51％和9％）,正常对照组为（28％,49％和29％）,两组基因型和等位基因频率分布差异有显著性（P〈0．05）。结论：AMPKα2多态性与新疆维吾尔族人2型糖尿病有明显相关性。     

Peroxisome proliferator-activated receptor {alpha} is responsible for the up-regulation of hepatic glucose-6-phosphatase gene expression in fasting and db/db Mice

Glucose-6-phosphatase (G6Pase) is a key enzyme that is responsible for the production of glucose in the liver during fasting or in type 2 diabetes mellitus (T2DM). During fasting or in T2DM, peroxisome proliferator-activated receptor α (PPARα) is activated, which may contribute to increased hepatic glucose output. However, the mechanism by which PPARα up-regulates hepatic G6Pase gene expression in these states is not well understood. We evaluated the mechanism by which PPARα up-regulates hepatic G6Pase gene expression in fasting and T2DM states. In PPARα-null mice, both hepatic G6Pase and phosphoenolpyruvate carboxykinase levels were not increased in the fasting state. Moreover, treatment of primary cultured hepatocytes with Wy14,643 or fenofibrate increased the G6Pase mRNA level. In addition, we have localized and characterized a PPAR-responsive element in the promoter region of the G6Pase gene. Chromatin immunoprecipitation (ChIP) assay revealed that PPARα binding to the putative PPAR-responsive element of the G6Pase promoter was increased in fasted wild-type mice and db/db mice. These results indicate that PPARα is responsible for glucose production through the up-regulation of hepatic G6Pase gene expression during fasting or T2DM animal models.     

Association of soluble epoxide hydrolase gene polymorphism with insulin resistance in type 2 diabetic patients

The insulin resistance found in diabetes is influenced by vascular tone and local blood flow. Endothelial-derived hyperpolarizing factor (EDHF) functions as a potent vasodilator to regulate vascular tone, and its production is regulated by soluble epoxide hydrolase (sEH). In this study, we examined the genotype distribution and allele frequency of sEH gene G860A (Arg287Gln) polymorphism in Japanese subjects (n=499) (non-diabetic subjects, n=205; type 2 diabetic patients, n=294). Also, to accurately evaluate insulin resistance, we performed the euglycemic hyperinsulinemic clamp test for each type 2 diabetic patient (n=86) from whom agreement was obtained, and then examined a possible association of sEH gene G860A polymorphism with insulin resistance status. There was no significant difference in genotype distribution and allele frequency between non-diabetic subjects and type 2 diabetic patients. Interestingly, however, there was close association of sEH gene G860A (Arg287Gln) polymorphism with insulin resistance in type 2 diabetic patients, which was not observed in non-diabetic subjects. These results suggest that sEH and EDHF play some important role in the pathogenesis of insulin resistance found in type 2 diabetes.