Jesper O. Clausen

Low Physical Activity Accentuates the Effect of the FTO rs9939609 Polymorphism on Body Fat Accumulation

OBJECTIVE—Three independent studies have shown that variation in the fat mass and obesity-associated (FTO) gene associates with BMI and obesity. In the present study, the effect of FTO variation on metabolic traits including obesity, type 2 diabetes, and related quantitative phenotypes was examined. RESEARCH DESIGN AND METHODS—The FTO rs9939609 polymorphism was genotyped in a total of 17,508 Danes from five different study groups. RESULTS—In studies of 3,856 type 2 diabetic case subjects and 4,861 normal glucose-tolerant control subjects, the minor A-allele of rs9939609 associated with type 2 diabetes (odds ratio 1.13 [95% CI 1.06–1.20], P = 9 × 10−5). This association was abolished when adjusting for BMI (1.06 [0.97–1.16], P = 0.2). Among 17,162 middle-aged Danes, the A-allele associated with overweight (1.19 [1.13–1.24], P = 1 × 10−12) and obesity (1.27 [1.20–1.34], P = 2 × 10−16). Furthermore, obesity-related quantitative traits such as body weight, waist circumference, fat mass, and fasting serum leptin levels were significantly elevated in A-allele carriers. An interaction between the FTO rs9939609 genotype and physical activity (P = 0.007) was found, where physically inactive homozygous risk A-allele carriers had a 1.95 ± 0.3 kg/m2 increase in BMI compared with homozygous T-allele carriers. CONCLUSIONS—We validate that variation in FTO is associated with type 2 diabetes when not adjusted for BMI and with an overall increase in body fat mass. Furthermore, low physical activity seems to accentuate the effect of FTO rs9939609 on body fat accumulation.

Studies of association of variants near the HHEX, CDKN2A/B and IGF2BP2 genes with type 2 diabetes and impaired insulin release in 10,705 Danish subjects – validation and extension of genome-wide association studies

OBJECTIVE— In the present study, we aimed to validate the type 2 diabetes susceptibility alleles identified in six recent genome-wide association studies in the HHEX/KIF11/IDE (rs1111875), CDKN2A/B (rs10811661), and IGF2BP2 (rs4402960) loci, as well as the intergenic rs9300039 variant. Furthermore, we aimed to characterize quantitative metabolic risk phenotypes of the four variants. RESEARCH DESIGN AND METHODS— The variants were genotyped in the population-based Inter99 cohort (n = 5,970), the ADDITION Study (n = 1,626), a population-based sample of young healthy subjects (n = 377), and in additional type 2 diabetic case (n = 2,111) and glucose-tolerant (n = 521) subjects. The case-control studies involved a total of 4,089 type 2 diabetic patients and 5,043 glucose-tolerant control subjects. RESULTS— We validated association of variants near HHEX/KIF11/IDE, CDKN2A/B, and IGF2BP2 with type 2 diabetes. Interestingly, in middle-aged people, the rs1111875 C-allele of HHEX/KIF11/IDE strongly associated with lower acute insulin response during an oral glucose tolerance test (P = 6 × 10−7). In addition, decreased insulin release following intravenous tolbutamide injection was observed in young healthy subjects (P = 0.02). Also, a reduced insulin release was observed for the CDKN2A/B rs10811661 T-allele after both oral and intravenous glucose challenges (P = 0.001 and P = 0.009, respectively). CONCLUSIONS— We validate that variants in the proximity of the HHEX/KIF11/IDE, CDKN2A/B, and IFG2BP2 loci associate with type 2 diabetes. Importantly, variations within the HHEX/KIF11/IDE and CDKN2A/B loci confer impaired glucose- and tolbutamide-induced insulin release in middle-aged and young healthy subjects, suggesting a role for these variants in the pathogenesis of pancreatic β-cell dysfunction.

Insulin Sensitivity and Body Weight Changes in Young White Carriers of the Codon 64 Amino Acid Polymorphism of the β3-Adrenergic Receptor Gene

Recently, a missense mutation replacing tryptophan with arginine at codon 64 of the β3-adrenergic receptor gene was shown to be associated with insulin resistance in nondiabetic subjects and to an earlier onset of NIDDM in Pima Indians. We studied whether the codon 64 amino acid polymorphism of the β3-adrenergic receptor gene in a cohort of young healthy Danes was associated with high birth weight, accelerated weight gain during childhood and adolescence, present obesity, or impaired insulin sensitivity. The protocol included 380 unrelated white subjects in whom insulin sensitivity and secretion were measured during a combined intravenous glucose and tolbutamide tolerance test. A number of biochemical and anthropometric characteristics were determined for each subject. The subjects were genotyped for the codon 64 polymorphism by applying polymerase chain reaction restriction fragment-length polymorphism screening with the use of endonuclease BstNl. The allelic frequency of the mutated allele was 7% (95% CI: 5–10%), and it was similar in obese and nonobese subjects. The β3-adrenergic receptor gene variant was not related to birth weight or weight gain during childhood or adolescence. In its heterozygous form, the gene variant was not associated with an altered insulin sensitivity index (SI) or other features of the insulin resistance syndrome (BMI, blood pressure, fasting serum lipid levels, or fasting serum fibrinolytic variables). Three homozygous carriers of the polymorphism were identified, and each had a significantly higher BMI (27.4 ± 1.3 vs. 23.5 ± 3.7 kg/m2 [mean ± SD]; P = 0.032), lower SI [4.9 ± 2.9 vs. 15.4 ± 9.0 10−5 × (min × pmol/l)−1; P = 0.013], and higher fasting serum C-peptide (730 ± 155 vs. 471 ± 158 pmol/l; P = 0.016) than the wild-type carriers. The homozygous carriers also had significantly higher levels of fasting serum triglyceride (P = 0.042) and serum LDL cholesterol (P = 0.013). When adjustments were made for age, sex, BMI, and VO2max in a multiple regression analysis, a significantly negative association was found between homozygosity for the codon 64 variant and the SI (P = 0.009). We conclude that in young healthy Danes, the homozygous form but not the heterozygous form of the codon 64 amino acid polymorphism of the β3-adrenergic receptor may be associated with obesity and, independent of BMI, with a low SI Since only three homozygous carriers were identified among 380 subjects, the results must be interpreted with caution, and studies of larger population samples are needed.

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.

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.

A Prevalent Amino Acid Polymorphism at Codon 98 in the Hepatocyte Nuclear Factor-1α Gene Is Associated With Reduced Serum C-Peptide and Insulin Responses to an Oral Glucose Challenge

Mutations in the hepatocyte nuclear factor-1α (HNF-1α) gene cause the type 3 form of maturity-onset diabetes of the young (M0DY3), which is characterized by a severe impairment of insulin secretion. In addition to disease-associated mutations, three common amino acid polymorphisms have been identified in the HNF-1α gene: Ile/Leu27, Ala/Val98, and Ser/Asn487. We have addressed the question of whether these variants of the HNF-1α gene are associated with altered glucoseinduced C-peptide and insulin responses or late-onset NIDDM. Among 245 NIDDM patients, the allelic frequency of the Val98 variant was 3.7% (95% CI 2.0–5.4%) vs. 4.4% (2.6–6.2%) among 240 glucose tolerant control subjects (NS). Studies of genotype-phenotype interactions in 240 middle-aged control subjects showed, however, that heterozygous subjects (i.e., genotype Ala/Val98) had an 18% decrease in 30-min serum C-peptide level (P = 0.004) as well as a 23% decrease in 30-min serum insulin level (P = 0.03) during an oral glucose tolerance test. One Val98 homozygote subject had a more severe reduction in stimulated insulin and C-peptide levels. The impact of the homozygous carrier status was similar in a study of 377 healthy young subjects. In contrast, the Ile/Leu27 and Ser/Asn487 polymorphisms were not associated with altered C-peptide and insulin release or NIDDM. In conclusion, 8% of white subjects of Danish ancestry are heterozygous for the Ala/Val98 polymorphism in the HNF-1α gene, which in middle-aged subjects is associated with a ∼20% reduction in serum C-peptide and insulin responses 30 min after an oral glucose challenge. Val98 homozygotes may exhibit a more severe defect in the early glucose-induced insulin response.

Identification of Four Amino Acid Substitutions in Hexokinase II and Studies of Relationships to NIDDM, Glucose Effectiveness, and Insulin Sensitivity

Human hexokinase (HK) II, a glucose phosphorylating enzyme in muscle tissue, plays a central role in glucose metabolism. Since reduced insulin-stimulated glucose uptake and reduced glucose-6-phosphate content in muscle have been demonstrated in pre-non-insulin-dependent diabetes mellitus (pre-NIDDM) and NIDDM subjects, we have examined the coding region of the HKII gene in NIDDM patients to determine whether these patients show genetic polymorphisms that are associated with or contribute to the disease. Single-strand conformational polymorphism analysis and nucleotide sequencing were initially performed on the entire coding region of the HKII gene of 38 insulin-resistant NIDDM patients and 5 healthy control subjects. This analysis revealed four missense mutations at codons 142 (Gln to His), 148 (Leu to Phe), 497 (Arg to Gln), and 844 (Arg to Lys) and an additional six exon polymorphisms that did not predict any change in amino acid composition of the protein. One homozygous and nine heterozygous carriers of the codon 142 mutation were found among the NIDDM patients. The mutations at codons 148, 497, and 844 were each found in one diabetic subject and only on one allele. There were no carriers of compound heterozygous mutations. A subsequent study of 301 patients with NIDDM and 151 healthy control subjects revealed no additional mutations at codons 148, 497, or 844. The total frequency of the mutated allele at codon 142 was 18.9% among the control subjects and 17.0% among the NIDDM patients (X2 = 0.56, P = 0.45). A population-based sample of 383 unrelated young healthy Caucasians was examined during a combined intravenous glucose and tolbutamide test to address whether the codon 142 amino acid polymorphism was associated with alterations in glucose effectiveness or insulin sensitivity. No evidence could, however, be provided for obvious relationships between the two estimates of whole-body insulin action and glucose turnover and the gene variant. In conclusion, four amino acid substitutions have been identified in the human HKII gene. Although the widespread mutation at codon 142 does not seem to be associated with an increased susceptibility to NIDDM or major abnormalities in whole-body glucose effectiveness or insulin sensitivity, further studies are needed to address the potential roles of the HKII mutations in intracellular energy metabolism.

Lewis phenotypes and the insulin resistance syndrome in young healthy white men and women

An increased risk of ischemic heart disease in men with the Lewis blood group phenotype Le(a-b-) has been reported. It has been suggested that the Le(a-b-) phenotype is a genetic marker of the insulin resistance syndrome. To examine whether Le(a-b-) confers the insulin resistance syndrome, we studied a random sample of unrelated healthy young white men and women living in Copenhagen (n = 380, 18 to 32 years). All individuals had their insulin sensitivity estimated using Bergmans minimal model (intravenous glucose in combination with tolbutamide) and systolic blood pressure (SBP) was measured with a London School of Hygiene Sphygmomanometer. A number of anthropometric measurements including body mass index (BMI, kilograms/meters squared) and biochemical characteristics were performed. The Lewis blood group typing was carried out on erythrocytes. Twenty-one men had the Le(a-b-) phenotype. Compared to all other men (N = 165), the Le(a-b-) men had a significantly higher SBP (6 mm Hg, P = .0024). They also had higher values of BMI (8%, P = .016), total body fat mass (25%, P = .015), fasting values of serum insulin (32%, P = .006), serum C-peptide (20%, P = .029), and plasma glucose (8%, P = .003). The fasting values of serum lipids, plasminogen activator inhibitor (PAI-1) activity, tissue plasminogen activator (t-PA) antigen, and insulin sensitivity did not differ between Le(a-b-) men and men with other Lewis phenotypes. Altogether 194 women participated in the study of which 21 women had the Le(a-b-) phenotype. Except for a lower PAI-1 activity (45%, P = .044), no values differed between Le(a-b-) women and women with other Lewis phenotypes. The women were also stratified according to use of oral contraceptives. Le(a-b-) women using oral contraceptives (N = 8) had a significantly lower plasma level of fasting PAI-1 activity (P = .029) and t-PA antigen (P = .004) compared to women using oral contraceptives without the Le(a-b-) phenotype (N = 42). Our data support the hypothesis that Le(a-b-) men exhibit features of the insulin resistance syndrome, including higher levels of BMI, SBP, and fasting levels of serum insulin and plasma glucose. In young women no signs of the insulin resistance syndrome were found in subjects with the Le(a-b-) phenotype.

The g/a Nucleotide Variant at Position −30 in the β-Cell-Specific Glucokinase Gene Promoter Has No Impact on the β-Cell Function in Danish Caucasians

Glucokinase (GCK) is proposed to play a key role in the regulation of insulin secretion in pancreas (1). Mutations in the GCK gene explain the phenotype of MODY 2, a subtype of type 2 diabetes characterized by an early age of onset and an impaired insulin secretion (2). Furthermore, previous studies have reported associations of a microsatellite marker at the GCK gene locus and type 2 diabetes in several populations (3-5). Although mutations in the coding region of the gene are rare in patients with late-onset type 2 diabetes, it has recently been shown that a frequently occurring g/a nucleotide substitution at position -30 in the GCK gene promoter is associated with reduced pancreatic P-cell function, as estimated during an oral glucose tolerance test (OGTT) in Japanese American subjects (6), and with impaired glucose tolerance (IGT) (7) in native Japanese men. In an attempt to replicate these findings in Caucasians, we studied the impact on the 3-cell function of the g/a variant in three different Danish cohorts: i ) 240 unrelated middle-aged glucose-tolerant subjects traced in the Danish central population register. All subjects underwent a standard 75-g OGTT. 2) A population-based sample of 380 healthy subjects randomly recruited from a population of young individuals (8). These participants underwent an intravenous glucose tolerance test (IVGTT). Physiological characteristics of this population sample have been presented previously (8). 3) A total of 267 offspring of type 2 diabetic probands from 62 families were recruited from the Danish family resource bank at the Department of Human Genetics, University of Copenhagen (18 families) and from the outpatient clinic at the Steno Diabetes Center (44 families). All siblings underwent a 4-h OGTT (18 time points) and a frequently sampled IVGTT. Because diabetes may cause secondary changes in insulin secretion, only glucose-tolerant siblings were studied (n = 231). Type 2 diabetes and IGT were diagnosed in accordance with World Health Organization criteria In these families, 65 sib-pairs were discordant for

The importance of adrenaline, insulin and insulin sensitivity as determinants for blood pressure in young Danes

Objective: To study the influence of the adrenergic system, fasting serum insulin level and insulin sensitivity on systolic (SBP) and diastolic blood pressure (DBP) in young individuals. Design and methods: In a population survey we measured SBP and DBP (using the London School of Hygiene sphygmomanometer) and fasting levels of serum catecholamines, serum insulin and insulin sensitivity in 383 randomly recruited subjects (mean age 25.0 years) of both sexes. Insulin sensitivity was estimated from a combined intravenous glucose and tolbutamide tolerance test and calculated using Bergmans minimal model. Confounders were body mass index, waist: hip ratio, maximal aerobic capacity, age, sex, and consumptions of tobacco and alcohol. Results: In a multiple regression analysis including the above factors, the most important determinant of SBP, after sex, was the plasma adrenaline level (partial correlation coefficient, rp=0.23, P<0.01). No significant association was found between plasma noradrenaline level and SBP. A significant association was found between plasma adrenaline level and DBP in females only (rp=0.15, P<0.05). Overall, the plasma adrenaline level was more important than the plasma noradrenaline level. Fasting serum insulin level and insulin sensitivity were each significantly correlated with both SBP and DBP in univariate analyses, but not in a multiple regression analysis. A family history of hypertension was associated with higher SBP level, body mass index and fasting serum insulin level, and with lower insulin sensitivity, but with no difference in circulating plasma adrenaline or noradrenaline compared with individuals without a family history. In a multiple regression analysis with the above confounders, no significant association between SBP and plasma adrenaline level could be found in either sex for subjects with a family history of hypertension. Both male (rp=0.41, P<0.001) and female (rp=0.18, P<0.05) subjects with no history of family hypertension had a significant association between SBP and plasma adrenaline level in a multiple regression analysis. Conclusion: In young healthy Caucasians adrenergic activity is an important determinant for SBP. The importance of fasting serum insulin level and insulin sensitivity on blood pressure level is minor when confounders are considered.