Alan Permutt

Polymorphisms of the HDL Receptor Gene Associated with HDL Cholesterol Levels in Diabetic Kindred from Three Populations

Objective: We examined polymorphisms in the HDL receptor, SR-BI, for association with plasma HDL cholesterol levels. Methods: Study subjects, including 847 women and 725 men, were from families originally ascertained for type 2 diabetes from Finland, Sweden and Israel. Four common polymorphisms were examined in linear regression analysis: an exon 1 missense (EX1), exon 8 silent (EX8), intron 5 (IVS5) and intron 10 (IVS10) variants. Results: Genotype combinations for the three polymorphisms in linkage disequilibrium (IVS5, EX8 and IVS10) were found to be associated with HDL-C among women from the Israeli (p = 0.01) and Swedish (p = 0.06) populations. In Finnish women, the association was only apparent after taking into account effect modification by triglyceride levels (p = 0.04). One specific pattern of genotypes, denoted by presence of the IVS5_T and EX8_C alleles, and absence of the IVS10_G allele, was consistently associated with the lowest mean levels of HDL-C in women from all three populations. These same associations were not found in men. Conclusions: Polymorphic variation of the SR-BI gene may influence HDL-C levels and act in a sex-dependent manner.

Detailed investigation of the role of common and low frequency WFS1 variants in type 2 diabetes risk

OBJECTIVE Wolfram syndrome 1 (WFS1) single nucleotide polymorphisms (SNPs) are associated with risk of type 2 diabetes. In this study we aimed to refine this association and investigate the role of low-frequency WFS1 variants in type 2 diabetes risk. RESEARCH DESIGN AND METHODS For fine-mapping, we sequenced WFS1 exons, splice junctions, and conserved noncoding sequences in samples from 24 type 2 diabetic case and 68 control subjects, selected tagging SNPs, and genotyped these in 959 U.K. type 2 diabetic case and 1,386 control subjects. The same genomic regions were sequenced in samples from 1,235 type 2 diabetic case and 1,668 control subjects to compare the frequency of rarer variants between case and control subjects. RESULTS Of 31 tagging SNPs, the strongest associated was the previously untested 3′ untranslated region rs1046320 (P = 0.008); odds ratio 0.84 and P = 6.59 × 10−7 on further replication in 3,753 case and 4,198 control subjects. High correlation between rs1046320 and the original strongest SNP (rs10010131) (r2 = 0.92) meant that we could not differentiate between their effects in our samples. There was no difference in the cumulative frequency of 82 rare (minor allele frequency [MAF] <0.01) nonsynonymous variants between type 2 diabetic case and control subjects (P = 0.79). Two intermediate frequency (MAF 0.01–0.05) nonsynonymous changes also showed no statistical association with type 2 diabetes. CONCLUSIONS We identified six highly correlated SNPs that show strong and comparable associations with risk of type 2 diabetes, but further refinement of these associations will require large sample sizes (>100,000) or studies in ethnically diverse populations. Low frequency variants in WFS1 are unlikely to have a large impact on type 2 diabetes risk in white U.K. populations, highlighting the complexities of undertaking association studies with low-frequency variants identified by resequencing.

The biological activity of catfish pancreatic somatostatin

Catfish pancreatic somatostatin, which contains eight additional amino acids on the amino terminus of a tetradecapeptide with considerable homology to tetradecapeptide somatostatin (SRIF), is a naturally occurring homolog of the hypothalamic peptide. The purpose of these studies was to determine the biological activity of this somatostatin homolog. Inhibition of 125I-labelled tyr1-SRIF binding to bovine pituitary plasma membranes by catfish pancreatic somatostatin was approximately 33% that of SRIF. Pancreatic somatostatin had full biological activity measured by inhibition of growth hormone release from isolated rat pituitary cells, but 0.01-0.1% the potency of SRIF. Pancreatic somatostatin at 100 ng/ml produced a 50-60% inhibition of insulin and glucagon secretion from perfused rat pancreas, while SRIF produced comparable inhibition at 10 ng/ml. This report demonstrates that a larger molecular form and natural homolog of SRIF, isolated from fish pancreas, has the same (but reduced) biological activities in rat assay systems as somatostatin originally isolated from sheep hypothalamus.

Isolation and Characterization of Immunoreactive Somatostatin from Fish Pancreatic Islets

Using a radioimmunoassay with labeled synthetic tetradecapeptide somatostatin, a large amount of immunoreactive somatostatin was found in the principal pancreatic islet of the channel catfish (Ictalurus punctata). The purpose of these experiments was to isolate and characterize the somatostatin-like material. Extracts of islets were chromatographed on a Bio-Gel P-30 column, and over 90% of the immunoreactive somatostatin migrated with proteins at least twice the size of synthetic tetradecapeptide somatostatin. This fraction was further purified by ion-exchange chromatography on carboxymethyl-cellulose and DEAE-cellulose columns. Two peptides were obtained with identical immunoreactivity, which was approximately 25% that of the synthetic somatostatin. Each peptide was judged to be >95% pure by thin-layer electrophoresis, polyacrylamide gel electrophoresis at pH 8.9, and highpressure liquid chromatography. Further criteria of purity included amino-terminal analysis of fraction IV yielding only aspartic acid. A total of 1.3 mg of fraction II, and 3.8 mg of fraction IV somatostatin-like peptides were obtained from 10 g of fresh frozen islets. Characterization of the two peptides revealed both peptides slightly more acidic than synthetic tetradecapeptide somatostatin. Fraction II had an isoelectric point of 8.0-8.3, and fraction IV 8.3-9.0. Molecular weight estimation by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis revealed similar mobility of both peptides, between pancreatic polypeptide (mol wt 4,500) and glucagon (mol wt 3,500). The mobility was not altered by reduction, and was approximately twice the size of synthetic tetradecapeptide somatostatin (mol wt 1,800). This confirmed that the peptides were single polypeptide chains and not aggregates, or somatostatin bound to larger proteins. Molecular weight determination by gel filtration chromatography on Bio-Gel P-6 in 8 M urea gave an estimated mol wt of 3,700. Amino acid analysis of the two immunoreactive somatostatins indicated that they were very similar in composition. Both pancreatic somatostatins (1 muM) had full biological activity relative to synthetic somatostatin measured as inhibition of growth hormone release from rat anterior pituitary cells.

Radioimmunoassay for catfish pancreatic somatostatin-22☆

Abstract An antiserum to purified catfish pancreatic somatostatin-22 (anti-CPS-22) was obtained and a radioimmunoassay developed which was highly specific for CPS-22. Displacement by tetradecapeptide somatostatin (SST-14) was at least 1000-fold less than with homologous peptide. A discrete population of catfish islet cells were stained in sections treated with anti-CPS-22, which appeared to be somatostatin containing D-cells identified by characteristic granule morphology on electron microscopy. Both SST-14 and CPS-22, presumably the products of two non-allelic somatostatin genes, have been identified in catfish pancreatic islets (Andrews, P.C. and Dixon, J.E. (1981) J. Biol. Chem., 256, 8267). Using the anti-CPS-22 and anti-SST-14 assays, it was estimated that CPS-22 comprised approximately 90%, and SST-14 5–10% of total immunoreactive catfish pancreatic somatostatin. The anti-CPS-22 assay was used on other pancreatic extracts to look for non-allelic forms of somatostatin. Anti-CPS-22 reacted poorly with pigeon pancreas extracts, but in rat pancreas extracts there was as much immunoreactive somatostatin measured with the CPS-22 as with the SST-14 assay. Similar immunochemical determinants in fish and rat somatostatins were suggested by parallel displacement of labeled CPS-22 from anti-CPS-22 with pancreatic extracts. Chromatography on Biogel P-60 in 2.5 M propionic acid, although not a vigorously denaturing condition, indicated that the majority of fish and rat immunoreactive pancreatic somatostatin detected with anti-CPS-22 migrated as a 4000 molecular weight peptide, larger than SST-14. These preliminary observations suggest that anti-CPS-22 may be useful in further characterization of somatostatin-like peptides from mammals as well as more primitive vertebrates.