Søren M. Echwald

Aminoacid polymorphisms of insulin receptor substrate-1 in non-insulin-dependent diabetes mellitus.

Since relative or absolute insulin deficiency and insulin insensitivity are involved in the aetiology of non-insulin-dependent diabetes mellitus (NIDDM), we examined whether patients with NIDDM exhibit genetic variability in the coding region of insulin receptor substrate-1 (IRS-1), a candidate gene that is ubiquitous in insulin-sensitive and insulin-like growth factor 1 (IGF1) sensitive tissues, including those that determine glucose production and clearance and those with regulatory effects on pancreatic beta-cell function. IRS-1 has a central role as an adaptor molecule that links the insulin-receptor and IGF1-receptor kinases with enzymes that regulate cellular metabolism and growth. Single-stranded conformation polymorphism analysis and direct nucleotide sequencing were applied to genomic DNA from 86 unrelated patients with NIDDM and 76 normoglycaemic controls. 10 of the patients with NIDDM and 3 of the controls were heterozygous at codon 972 for a polymorphism in which glycine was substituted with arginine. Moreover, at codon 513, 6 patients with NIDDM and 2 controls had a heterozygous polymorphism with a transition from alanine to proline. None of the polymorphism carriers had both aminoacid variants and the total allelic frequency of IRS-1 polymorphisms was about three times higher in patients with NIDDM than in controls (p = 0.02). Both aminoacid substitutions were located close to tyrosine phosphorylation motifs that are putative recognition sites for insulin and IGF1 signal transmission proteins. Analysis of the phenotypes showed that patients with NIDDM who had IRS-1 variants did not differ in their degree of insulin resistance compared with patients without known IRS-1 polymorphisms. However, carriers of the codon 972 variant had significantly lower plasma levels of fasting insulin and C-peptide. Our results suggest that aminoacid polymorphisms in IRS-1 may be involved in the aetiology of a subset of late-onset NIDDM.

Amino Acid Polymorphisms in the ATP-Regulatable Inward Rectifier Kir6.2 and Their Relationships to Glucose- and Tolbutamide-Induced Insulin Secretion, the Insulin Sensitivity Index, and NIDDM

Kir6.2 is an inwardly rectifying potassium channel that is expressed in pancreatic (β-cells and cardiac and skeletal muscle. Expressed together with the highaffinity sulphonylurea receptor, it reconstitutes a sulphonylurea- and also ATP-sensitive potassium channel resembling the native (β-cell channel. The objective of this study was to search for mutations in the Kir6.2 gene that might be associated with NIDDM or related to altered insulin secretion, insulin action, or glucose metabolism in healthy subjects. Using polymerase chain reaction–single-strand conformation polymorphism analysis (PCR-SSCP) on genomic DNA from 69 Danish NIDDM patients and 66 matched control subjects, we report the finding of three missense polymorphisms in otherwise conserved codons and three silent polymorphisms in the gene encoding Kir6.2: codon 23 (GAG/AAG), Glu→Lys; codon 190 (GCT/GCC), Ala→Ala; codon 267 (CTC/CTG), Leu→Leu; codon 270 (CTG/GTG), Leu→Val; codon 337 (ATC/GTC), Ile→Val; codon 381 (AAG/AAA), Lys→Lys. The codon 23 and codon 337 amino acid polymorphisms were always coupled. The allelic frequencies of the polymorphisms were similar in NIDDM patients and control subjects. The amino acid polymorphisms were not associated with altered insulin secretion after intravenous glucose or tolbutamide injections or with altered glucose effectiveness in a phenotype study of 346 young healthy subjects. However, carriers of the maximal load of amino acid variants, the compound homozygous codon 23/337 and heterozygous codon 270, had on average a 62% higher insulin sensitivity index (P = 0.006), compared with noncarriers. We conclude that a combination of common Kir6.2 amino acid variants may contribute to the genetic background behind the large variation of the insulin sensitivity index in the general population.

Genetic Polymorphisms and Weight Loss in Obesity: A Randomised Trial of Hypo-Energetic High- versus Low-Fat Diets

Objectives: To study if genes with common single nucleotide polymorphisms (SNPs) associated with obesity-related phenotypes influence weight loss (WL) in obese individuals treated by a hypo-energetic low-fat or high-fat diet. Design: Randomised, parallel, two-arm, open-label multi-centre trial. Setting: Eight clinical centres in seven European countries. Participants: 771 obese adult individuals. Interventions: 10-wk dietary intervention to hypo-energetic (−600 kcal/d) diets with a targeted fat energy of 20%–25% or 40%–45%, completed in 648 participants. Outcome Measures: WL during the 10 wk in relation to genotypes of 42 SNPs in 26 candidate genes, probably associated with hypothalamic regulation of appetite, efficiency of energy expenditure, regulation of adipocyte differentiation and function, lipid and glucose metabolism, or production of adipocytokines, determined in 642 participants. Results: Compared with the noncarriers of each of the SNPs, and after adjusting for gender, age, baseline weight and centre, heterozygotes showed WL differences that ranged from −0.6 to 0.8 kg, and homozygotes, from −0.7 to 3.1 kg. Genotype-dependent additional WL on low-fat diet ranged from 1.9 to −1.6 kg in heterozygotes, and from 3.8 kg to −2.1 kg in homozygotes relative to the noncarriers. Considering the multiple testing conducted, none of the associations was statistically significant. Conclusions: Polymorphisms in a panel of obesity-related candidate genes play a minor role, if any, in modulating weight changes induced by a moderate hypo-energetic low-fat or high-fat diet.

Novel MODY3 Mutations in the Hepatocyte Nuclear Factor-1α Gene: Evidence for a Hyperexcitability of Pancreatic β-cells to Intravenous Secretagogues in a Glucose-Tolerant Carrier of a P447L Mutation

One form of maturity-onset diabetes of the young (MODY3) results from mutations in the hepatocyte nuclear factor (HNF)-1α gene, located on chromosome 12q24.2. The primary objective of the present study was to search for genetic variation in the HNF-1α gene in nine nonrelated Danish Caucasian subjects with MODY. Direct sequencing of the coding region and intron-exon boundaries of the HNF-1α gene revealed 2 novel and 1 previously reported missense mutations and 2 novel frameshift mutations in five of nine MODY subjects. These five mutations were found in neither 84 NIDDM patients nor 84 control subjects. One glucose-tolerant lean male with a P447L missense mutation, which in his relatives caused MODY, underwent an oral glucose tolerance test (OGTT), a tolbutamide modified frequently sampled intravenous glucose tolerance test, and a glucagon test to examine for a possible early β-cell abnormality. He had a low insulin secretion rate during an OGTT, but a twofold increase in pancreatic β-cell response after intravenous glucose and a 2.5- to 4-fold increase in β-cell response after either intravenous tolbutamide or intravenous glucagon loads. In conclusion, 1) mutations in the HNF-1α gene are common in Danish Caucasian MODY patients, and 2) early stages in the pathogenesis of MODY3 caused by the P447L mutation may be characterized by a hyperexcitability of β-cells to intravenous secretagogues.

Identification of a Common Amino Acid Polymorphism in the p85α Regulatory Subunit of Phosphatidylinositol 3-Kinase: Effects on Glucose Disappearance Constant, Glucose Effectiveness, and the Insulin Sensitivity Index

Phosphatidylinositol 3-kinase (PI3-K) may regulate the basal plasma membrane glucose transporter recycling and the organization of the transporter intracellular pool in addition to being an insulin signal for translocation of glucose transporters to the plasma membrane. The objectives of the present study were to examine for genetic variability in the human regulatory p85α subunit of PI3-K, to look for an association between gene variants and NIDDM in a case-control study, and to relate identified variability to potential changes in whole-body insulin sensitivity and glucose turnover in a phenotype study. Single-strand conformational polymorphism and heteroduplex analysis of the coding region of the regulatory p85α subunit in cDNA isolated from human muscle tissue from 70 insulin-resistant NIDDM patients and 12 control subjects revealed three silent polymorphisms and a missense mutation at nucleotide position 1020 (G→A), changing a Met to He at codon 326. Using allele-specific oligohybridization, we found a similar allelic frequency of the codon 326Met→Ile variant in 404 NIDDM patients (0.15 [95% CI 0.13–0.17]) and 224 matched glucose tolerant control subjects (0.16 [0.13–0.19]). In a random sample of 380 unrelated healthy young Caucasians aged 18–32 years, in whom we have performed a tolbutamide modified intravenous glucose tolerance test, we identified 263 wildtype subjects, 109 heterozygous subjects, and 8 subjects homozygous for the codon 326 variant (allelic frequency = 0.16 [0.13–0.19]). No difference in glucose disappearance constant (KG), insulin sensitivity index (SI), and glucose effectiveness (SG) was observed between wildtype and heterozygous subjects. However, compared with the combined values for wildtype and heterozygous carriers, KG was reduced by 40% (P = 0.004) and SG by 23% (P = 0.03) in homozygous carriers of the p85α variant. Moreover, in homozygous carriers, a 32% reduction was found in SI (P = 0.08). In conclusion, a codon 326Met→Ile variant in the gene encoding the PI3-K p85α regulatory subunit is found in 31% of a random sample of young healthy Caucasians. About 2% of the subjects in this population carry the gene variant in its homozygous form, and these carriers are characterized by significant reductions in whole-body glucose effectiveness and intravenous glucose disappearance constant. In itself, the gene variant does not confer an increased risk of diabetes.

Leptin in obesity

Obesity runs in families, mainly due to genetic factors.1 Several different mendelian inherited, monogenic forms of obesity exist in rodents, but they are rare in humans.1 Analysis of the distribution of fatness in families suggests that a few major genes may act on a polygenic and environmental background, but it remains unclear which genes are involved.1 The identification and sequencing of the mouse obese (ob) gene by Friedmans group in 19942 opened important new avenues in obesity research and have already led to overwhelming research activity.3 In homozygous ob/ob mice, the mutation of the ob gene results in increased food intake, reduced energy expenditure, elevated insulin and cortisol levels, and subsequently, in massive obesity and non-insulin dependent diabetes mellitus.2 The ob gene encodes a protein, leptin, which is produced only in fat cells and secreted into the blood. There are two different strains of ob/ob mice: one with a mutation that establishes a stop codon within the ob gene, resulting in the production of a truncated, inactive protein; the other with a mutation that produces no protein at all.2 Administration of leptin corrects the multiple metabolic disturbances.4 The finding of a human homologue of …

Mutational analysis of the proopiomelanocortin gene in Caucasians with early onset obesity.

OBJECTIVE: Mutations in the human gene encoding the polyhormone peptide proopiomelanocortin (POMC) are associated with obesity in rare cases and the gene co-localizes with a reported quantitative trait loci (QTL) for variations in circulating leptin levels and fat mass on human chromosome 2p21. In this study we have used polymerase chain reaction (PCR) and single strand conformation polymorphism (SSCP) analysis, to test whether variations in the human POMC gene are associated with human obesity.DESIGN AND SUBJECTS: Primary mutational analysis was performed on the coding region of the POMC gene and 500 bp of the putative promoter region, by single strand conformational analysis and sequencing, in 56 subjects with juvenile onset obesity (body mass index (BMI) ≥31 kg/m2 at the draft board examination). The prevalence of two polymorphisms were further studied in 156 obese and 205 control subjects, and in a population based cohort of 380 extensively characterized young healthy subjects.RESULTS: We have identified a total of six gene variants, five were silent nucleotide substitutions (No51(promoter) g→c, No670(5′UTR)g→a, No4512(codon6)c→t Cys/Cys, No7726(codon116)c→t Leu/Leu) of which one was prevalent (No8246(3′UTR)c→t) and one variant changed an amino acid (No8086(codon236)g→c Arg/Gln). The amino acid substitution was only seen in one subject. Comparing the prevalence of the frequent No8246 silent polymorphism, in an association study comprising 156 subjects with juvenile onset obesity and 205 randomly sampled control subjects (mean BMI 23.5±4.7 kg/m2), did not show any relationship to obesity. Also, comparing the prevalence of a known 9 bp insertion/deletion variant in the coding region of the gene between obese and lean, showed no association to obesity. Furthermore, analyzing a population based cohort of 380 young healthy Caucasians for the prevalent 3′UTR polymorphism as well as the 9 bp insertion/deletion variant did not show any association to deviations in body fat contents or fasting serum leptin concentrations.CONCLUSION: In conclusion, it is unlikely that variations in the coding region and the putative promoter of the POMC gene are a major cause of juvenile onset human obesity.

Lower-body fat mass as an independent marker of insulin sensitivity--the role of adiponectin.

AIMS:To study the association between lower-body fat and estimates of whole-body insulin sensitivity in middle-aged men with and without a history of juvenile onset obesity, and to determine the possible mediating role of fasting serum adiponectin level as an insulin-sensitizing peptide.METHODS:A total of 401 men aged 39–65 y, body mass index 18–54 kg/m2, participated in the study. The following variables were measured on the study participants: regional body fat distribution as assessed by dual energy X-ray absorptiometry, abdominal sagittal diameter, maximal oxygen uptake (VO2max), physical activity, fasting and post-glucose load levels of plasma glucose, serum insulin, and blood non-esterified fatty acid plus fasting levels of serum adiponectin and HbA1c.RESULTS:Lower-body fat mass was positively associated with insulin sensitivity as estimated by Matsudas index also after adjusting for age, lean tissue mass, trunkal fat mass, weight changes since draft board examination, VO2max and the level of physical activity. In a subgroup of men selected for a large lower-body fat mass, fasting serum insulin concentration was 24% lower (P<0.01) and fasting serum adiponectin 33% higher (P<0.005) compared to a subgroup of men with a small lower-body fat mass but with similar trunkal fat mass.CONCLUSION:Lower-body fat mass is positively associated with an estimate of insulin sensitivity independently of trunkal fat mass in both lean and obese middle-aged men and this effect could partly be statistically explained by variations in serum adiponectin levels.

The Asn363Ser variant of the glucocorticoid receptor gene is not associated with obesity or weight gain in Danish men.

OBJECTIVE: Testing association of the Asn363Ser variant of the glucocorticoid gene with measures of obesity and weight gain.SUBJECTS: 741 obese subjects (BMI≥31 kg/m2 at selection) and 854 random control subjects from the same population, examined at draft board examination and after on average 27.4± y. A lean control group (n=351) was further selected as the fraction from the cohort group having a BMI below 25.0 kg/m2 at the latest examination.METHODS: Using PCR-RFLP subjects were genotyped for the Asn363Ser variant and grouped according to genotype.RESULTS: The prevalence of the Ser363 allele was 4.7% (95% Cl: 3.3–6.2%) among the obese, 4.1% (2.7–5.5%) among the random cohort subjects and 4.3% (2.1–6.5%) among lean control subjects, respectively, showing no significant differences between the groups (P>0.1). Furthermore, no differences in BMI, waist-hip ratio or weight gain were seen within any of the groups when defined according to the glucocorticoid receptor genotype.CONCLUSION: In the examined population this marker is not a relevant predictor of obesity.

Discovery of amino acid variants in the human glucose-dependent insulinotropic polypeptide (GIP) receptor: the impact on the pancreatic beta cell responses and functional expression studies in Chinese hamster fibroblast cells

Summary The two incretins, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1), are insulinotropic factors released from the small intestine to the blood stream in response to oral glucose ingestion. The insulinotropic effect of GLP-1 is maintained in patients with Type II (non-insulin-dependent) diabetes mellitus, whereas, for unknown reasons, the effect of GIP is diminished or lacking. We defined the exon-intron boundaries of the human GIP receptor, made a mutational analysis of the gene and identified two amino acid substitutions, A207 V and E354Q. In an association study of 227 Caucasian Type II diabetic patients and 224 matched glucose tolerant control subjects, the allelic frequency of the A207 V polymorphism was 1.1 % in Type II diabetic patients and 0.7 % in control subjects (p = 0.48), whereas the allelic frequency of the codon 354 polymorphism was 24.9 % in Type II diabetic patients versus 23.2 % in control subjects. Interestingly, the glucose tolerant subjects (6 % of the population) who were homozygous for the codon 354 variant had on average a 14 % decrease in fasting serum C-peptide concentration (p = 0.01) and an 11 % decrease in the same variable 30 min after an oral glucose load (p = 0.03) compared with subjects with the wild-type receptor. Investigation of the function of the two GIP receptor variants in Chinese hamster fibroblasts showed, however, that the GIP-induced cAMP formation and the binding of GIP to cells expressing the variant receptors were not different from the findings in cells expressing the wild-type GIP receptor. In conclusion, amino acid variants in the GIP receptor are not associated with random Type II diabetes in patients of Danish Caucasian origin or with altered GIP binding and GIP-induced cAMP production when stably transfected in Chinese hamster fibroblasts. The finding of an association between homozygosity for the codon 354 variant and reduced fasting and post oral glucose tolerance test (OGTT) serum C-peptide concentrations, however, calls for further investigations and could suggest that GIP even in the fasting state regulates the beta-cell secretory response. [Diabetologia (1998) 41: 1194–1198]