CELL BIOLOGY OF TRP64ARG BETA-3-ADRENERGIC RECEPTOR IN OBESITY AND NIDDM

肥胖和非胰岛素依赖型糖尿病 具有很强遗传性的异质性和重叠性疾病 组件。对皮马印第安人的研究表明，静息代谢率 (RMR)是一个家族特征，能量消耗的减少 与肥胖风险增加相关，这反过来又会导致NIDDM。 β-3-肾上腺素能受体(B3AR)是一种跨越G-受体的七层膜。 在脂肪细胞中表达的蛋白连接受体，并参与 脂解作用和生热作用的调节。我们确定了第一个 B3AR基因变异(密码子TGG Trp&Gt；CGG Arg，Trp64Arg)。这 错义突变预测细胞内第一个氨基酸的变化 受体的环，这一区域可能对正常的细胞内 运输和偶联到G蛋白。一些研究，但不是全部，都有 显示出Trp64 B3AR与 NIDDM发病更早，BMI增加和中枢脂肪分布， 高胰岛素血症和舒张压升高。 我们假设Trp64Arg B3AR变体导致 表达异常或信号特性异常导致脂肪分解减少 和能量消耗，进而增加肥胖的易感性和 NIDDM。为了探索这一假设，我们最初计划将 非糖尿病非裔美国人，他们要么是纯合子 Trp64Arg B3AR的正常B3AR、杂合子或纯合子以及 然后，在择期腹内手术期间进行大网膜脂肪活检。 在这三种基因类型中，我们计划比较B3AR基因的水平 和B-激动剂刺激的脂解作为一种定义功能性的手段 Trp64Arg B3AR突变的后果及其在 肥胖与非胰岛素依赖型糖尿病的发展。 在西尔弗博士获得CAP奖的第一年，她开发了 包括特异性核糖核酸酶在内的受体的研究方法 保护法和RT-PCR法检测B3AR mRNA水平， Western Blot定量B3AR蛋白水平，腺苷环化酶分析和 脂解试验。利用RNA模板特异性聚合酶链式反应(RS-PCR)， 证实了Trp64 B3AR mRNA和Arg64的存在 杂合子视网膜脂肪细胞中B3AR基因的表达。此外，通过 使用RS-PCR，她排除了信号是由于 基因组DNA污染一直是其他研究关注的问题。 用SSCP和SSCP技术筛选皮马印第安人和高加索人的B3AR基因 双脱氧测序揭示了第一内含子中新的碱基替换 (G1856t)。鸟嘌呤到胸腺嘧啶内含子的取代引入了一种新的 与公认的剪接供体位点紧密一致的序列。vbl.使用 RT-PCR-ASO，Silver博士无法证明这种情况的存在 这表明异常的G1856T剪接在体内不被使用。 因此，这种替代不太可能对关联起作用 Trp64Arg B3AR与NIDDM发病的关系 肥胖。 在此期间，西尔弗博士还建立了工作关系 与约翰霍普金斯湾景医疗中心的外科医生在一起 霍普金斯医院，马里兰大学医学中心，巴尔的摩 退伍军人管理医院和纽约的圣卢克-罗斯福医院 纽约市需要对大约2000名受试者进行筛查才能找到 本研究所需的25名Trp64Arg变异体纯合子受试者。 由于这种变异体的低流行率和排斥因素， 这项研究的招募工作一直非常糟糕。预计它将 再花很多年的时间来招募足够数量的受试者 学习。因此，西尔弗博士正在改变她的CAP奖项的方向。她 将研究另一种2型候选基因的多态现象 与胰岛素分泌有关的糖尿病，磺脲类 受体(SUR)。通过RPN#AAC92-03-17-01(糖尿病和 肥胖)，我们已经从1700多名受试者那里获得了DNA样本 有兴趣参与临床研究。到目前为止，425名受试者已经 外显子22 SuR多态的基因分型。这种多态具有 等位基因频率为0.03(23例杂合子)。由于受试者 这种多态的纯合子很少见，其他人已经发现 正常和纯合子间胰岛素水平的显著差异 以及杂合子，即SuR外显子22纯合子的受试者 多态不会被纳入这项研究。临床研究将 包括口服和静脉注射葡萄糖耐量试验和胰岛素测量 振荡性。 仅限实验室；没有住院日或门诊 来访。

Obesity and noninsulin dependent diabetes mellitus (NIDDM) are heterogeneous and overlapping disorders both with strong genetic components. Studies in Pima Indians have shown that resting metabolic rate (RMR) is a familial trait and that a reduction in energy expenditure is associated with an increased risk of obesity, which in turn leads to NIDDM. The beta-3-adrenergic receptor (B3AR) is a seven-membrane spanning G- protein-linked receptor that is expressed in adipose cells, and is involved in the regulation of lipolysis and RMR (thermogenesis). We identified the first variant in the B3AR gene (codon 64 TGG Trp > CGG Arg, Trp64Arg). This missenese mutation predicts an amino acid change in the first intracellular loop of the receptor, a region that may be important for proper intracellular trafficking and coupling to G-proteins. Some, but not all, studies have demonstrated significant association or trends of the Trp64 B3AR with an earlier onset of NIDDM, increased BMI and central fat distribution, hyperinsulinemia and increased diastolic blood pressure. We hypothesized that the Trp64Arg B3AR variant results in decreased expression or abnormal signaling properties leading to decreased lipolysis and energy expenditure which in turn increases susceptibility to obesity and NIDDM. To explore this hypothesis, we initially planned to characterize nondiabetic African-American subjects who were either homozygous for the normal B3AR, heterozygous or homozygous for the Trp64Arg B3AR and then during elective intraabdominal surgery obtain omental fat biopsies. Among the three genotypes, we planned to compare B3AR mRNA levels and B-agonist stimulated lipolysis as a means of defining the functional consequences of the Trp64Arg B3AR mutation, and its role in the development of obesity and NIDDM. During the first year of Dr. Silver's CAP award, she developed the methodologies for studying the receptor including specific RNAse protection and RT-PCR assays for measuring levels of B3AR mRNA, Western Blot to quantitate B3AR protein levels, adenyl cyclase assay and lipolysis assay. Using RNA template specific-PCR (RS-PCR), she demonstrated the presence of both the Trp64 B3AR mRNA and the Arg64 B3AR mRNA in omental adipocytes from heterozygotes. Furthermore, by using RS-PCR, she excluded the possibility that the signal was due to genomic DNA contamination which has been a concern with other studies. Screening of the B3AR gene in Pima Indians and Caucasians by SSCP and dideoxy sequencing revealed a new base substitution in the first intron (g1856t). The guanine to thymine intron substitution introduces a new sequence that closely conforms to the consensus splice donor site. Using RT-PCR-ASO, Dr. Silver was unable to demonstrate the presence of this cDNA indicating that the aberrant g1856t splice is not used in vivo. Therefore, it is unlikely that this substitution contributes to the associations observed between the Trp64Arg B3AR and the development of NIDDM or obesity. During this time period, Dr. Silver also established working relationships with surgeons at the Johns Hopkins Bayview Medical Center, Johns Hopkins Hospital,University of Maryland Medical Center, Baltimore Veterans Administration Hospital, and St. Luke's-Roosevelt Hospital in New York City as approximately 2000 subjects would need to be screened to find the 25 subjects homozygous for the Trp64Arg variant needed for this study. Due to the low prevalence of this variant and the exclusion factors, recruitment for this study has been very poor. It is anticipated that it would take many more years to recruit an adequate number of subjects to this study. Therefore, Dr. Silver is changing the direction of her CAP award. She will be studying a polumorphism in another candidate gene for Type 2 diabetes mellitus which is involved in insulin secretion, the sulfonylurea receptor (SuR). Through RPN# AAC92-03-17-01 (Genetics of Diabetes and Obesity), we have obtained DNA samples from over 1700 subjects interested in participating in clinical studies. To date, 425 subjects have been genotyped for the exon 22 SuR polymorphism. The polymorphism has an allele frequency of 0.03 (23 heterozygous subjects). Since subjects homozygous for this polymorphism are rare and others have found significant differences in insulin levels between normal and homozygotes and heterozygotes, subjects who are homozygous for the SuR exon 22 polymorphism will not be entered into this study. Clinical studies will include oral and IV glucose tolerance tests and measurements of insulin oscillation. LABORATORY ONLY; NO INPATIENT DAYS OR OUTPATIENT VISITS.