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

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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.