Søren A. Urhammer

Mutation analysis of peroxisome proliferator-activated receptor-γ coactivator-1 (PGC-1) and relationships of identified amino acid polymorphisms to Type II diabetes mellitus

Abstract.Aim/hypothesis: This study aimed to investigate if variability in the peroxisome proliferator-activated receptor-γ coactivator-1 (PGC-1) gene is associated with Type II (non-insulin-dependent) diabetes mellitus. Methods: The PGC-1 gene was examined in 53 Type II diabetic patients applying single strand conformational polymorphism analysis followed by nucleotide sequencing. Identified variants were genotyped in an association study comprising 483 Type II diabetic patients and 216 glucose-tolerant control subjects. A replication study was done in an additional 201 Type II diabetic patients and 293 glucose-tolerant subjects. Furthermore, a potential interaction between the Pro12Ala polymorphism of PPAR-γ2 and the PGC-1 Gly482Ser variant on risk of Type II diabetes was investigated. Results: A total of seven variants (Ser74Leu, IVS2 + 52C→A, Thr394Thr, Asp475Asp, Gly482Ser, Thr528Thr, and Thr612Met) were identified and investigated in an association study. Six of the variants showed no association with Type II diabetes in the initial study. However, the Gly482Ser polymorphism, was more frequent among Type II diabetic patients (37.0 %) than among glucose-tolerant subjects (30.8 %) (p = 0.032). In a replication study the difference in allele frequencies of the Gly482Ser variant remained significant (p = 0.0135). The combined study yielded an allele frequency of 37.3 % (34.7–39.9) for Type II diabetic patients and 30.5 % (27.7–33.4) for glucose-tolerant subjects (p = 0.0007). No interaction between this variant and the Pro12Ala polymorphism of PPAR-γ2 was observed. Conclusion/interpretation: A widespread Gly482Ser polymorphism of PGC-1 is associated with a 1.34 genotype relative risk of Type II diabetes. [Diabetologia (2001) 44: 2220–2226]

Homozygosity of the Pro12Ala variant of the peroxisome proliferation-activated receptor-gamma2 (PPAR-gamma2): divergent modulating effects on body mass index in obese and lean Caucasian men

Aims/hypothesis. The objectives of the present investigation were to examine: 1) whether a Pro115Gln variant in the peroxisome proliferator-activated receptor-γ2 (PPAR-γ2) is associated with juvenile-onset obesity among Danish Caucasianmen and 2) whether the relation of a Pro12Ala polymorphism in PPAR-γ2 with BMI and long-term weight regulation differ between lean and obese subjects within the same cohort. Methods. The Pro115Gln and Pro112Ala variants were examined using PCR and RFLP in a group of 752 subjects with a Body Mass Index (BMI) of 31.0 kg/m2 or more and in 869 non-obese control subjects. Results. We did not find Pro115Gln in any of the 1621 male subjects we examined. Among the males with juvenile-onset obesity, the allelic frequency of the Pro12Ala polymorphism was 14 % (95 % confidence interval: 12–16 %) compared with 16 % (14–17 %) among the non-obese control subjects (NS). Heterozygosity of the codon 12 variant was not associated with differences in BMI or changes in body weight regulation during follow up in lean or obese subjects. In the group of obese subjects, 21 homozygous Ala12Ala carriers had, however, a higher BMI (38.9 ± 5.4 kg/m2 (means ± SD) vs 35.5 ± 5.5 kg/m2, p = 0.008) and a higher weight gain (0.27 ± 0.24 kg · m–2· year–1 vs 0.10 ± 0.24 kg · m–2· year–1, p = 0.004), compared with wild-type carriers. Moreover, within the control group of 869 men the 14 homozygous carriers of the variant had a lower BMI (24.4 ± 2.7 kg/m2 vs 26.2 ± 3.7 kg/m2, p = 0.005) and a slower increase in BMI (0.11 ± 0.11 kg · m–2· year–1 vs 0.17 ± 0.11 kg · m–2· year–1, p = 0.002) compared with wild-type carriers. Conclusion/interpretation. The codon 12 variant of PPAR-γ2 is not intrinsically associated with juvenile obesity. The variant may in its homozygous form interact, however, with various combinations of genetic and environmental factors in lean and obese subjects to cause divergent modulating effects on BMI and long-term body weight control. [Diabetologia (1999) 42: 892–895]

Studies of the Pro12Ala polymorphism of the peroxisome proliferator-activated receptor-γ2 (PPAR-γ2) gene in relation to insulin sensitivity among glucose tolerant Caucasians

Abstract.Aims/hypothesis: We examined whether the Pro12-Ala polymorphism of the human peroxisome proliferator-activated receptor-γ2 (PPAR-γ2) gene was related to altered insulin sensitivity among glucose-tolerant subjects or a lower accumulated incidence or prevalence of IGT and Type II (non-insulin-dependent) diabetes mellitus among Scandinavian Caucasians. Methods: The Pro12Ala polymorphism was examined using PCR-RFLP. Whole-body insulin sensitivity measured under hyperinsulinaemic-euglycaemic conditions was estimated in a population-based sample of 616 glucose tolerant Swedish Caucasian men at age 70. In addition, insulin sensitivity index was measured using IVGTT and Bergman minimal modelling in a population-based sample of 364 young healthy Danish Caucasians. Finally, we evaluated whether the polymorphism predicted Type II diabetes and IGT in 841 seventy-year-old Swedish men. A case-control study was carried out in 654 unrelated Danish Type II diabetic patients and 742 Danish glucose tolerant subjects matched for age and sex. Results: Whole-body insulin sensitivity was significantly improved in carriers compared with non-carriers of the Ala-allele of the codon 12 polymorphism in Swedish Caucasian men (6.0 ± 2.5 vs 5.6 ± 2.5 mg · kg–1· min–1· [mU/l]–1· 100, p = 0.044). The same tendency, but not significant, was observed in the insulin sensitivity index among the group of young healthy Danish Caucasians. The incidence of Type II diabetes and IGT among the Swedish subjects at the age of 70 was similar in the three genotype-groups of the Pro12Ala variant and the Ala-allele was not related to a lower prevalence of Type II diabetes in Danish Caucasians. Conclusion/interpretation: The Ala-allele of the PPAR-γ2 polymorphism is associated with improved whole body insulin sensitivity among Swedish Caucasians. [Diabetologia (2001) 44: 1170–1176]

Variations of the interleukin-6 promoter are associated with features of the metabolic syndrome in Caucasian Danes.

Aims/hypothesisThe cytokine interleukin 6 (IL-6) is an essential regulator of the acute phase response associated with insulin-resistant states including type 2 diabetes and obesity. Three polymorphisms at positions −597, −572, and −174 of the IL6 promoter have been reported to influence IL6 transcription. The aim of this study was to investigate whether the IL6 promoter polymorphisms were associated with features of the WHO-defined metabolic syndrome and related quantitative traits in 7,553 Caucasian Danes.MethodsUsing analysis of PCR-generated primer extension products by mass spectrometry we examined −597 G/A, −572 G/C, and −174 G/C IL6 variants in the population-based Inter99 study cohort of middle-aged people (n=6,164) and in a group of type 2 diabetic patients (n=1,389).ResultsThe −174 G/C and −597 G/A polymorphisms were in strong linkage disequilibrium (R2=0.95). In the Inter99 cohort the −174 G-allele was associated with insulin resistance (p<0.02) and dyslipidaemia (p<0.007) whereas the C-allele of the −572 polymorphism was associated with increased serum insulin release during an OGTT (p<0.0005). Composite genotype or haplotype analyses of all 3 IL6 promoter variants showed associations with type 2 diabetes (p<0.002), obesity (p<0.02), and the metabolic syndrome (p<0.01).ConclusionsThe present studies suggest that single-nucleotide polymorphisms and composite genotypes or haplotypes of the IL6 promoter may be associated with several features of the metabolic syndrome in Caucasians.

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.

Impact of the v/v 55 polymorphism of the uncoupling protein 2 gene on 24-h energy expenditure and substrate oxidation.

OBJECTIVE: The gene that codes for a novel uncoupling protein, UCP2, has been linked to obesity in animal models. Markers encompassing the UCP2 locus have been linked to energy expenditure in humans. We studied the role of a common amino acid substitution, replacing an alanine (A) with a valine (V) at codon 55, of the coding region of the UCP2 gene for 24‐h energy expenditure and respiratory quotient (RQ) in healthy subjectsMETHODS:24‐h energy expenditure and RQ were measured in calorimeters in 60 healthy subjects. The UCP2 polymorphism was determined by restriction fragment length polymorphism-generating polymerase chain reaction.RESULTS: Age, gender and body fat were not different between groups, the number of subjects in each groups was A/A: 35% (n=21), A/V: 48% (n=29), and V/V: 17% (n=10). Twenty‐four‐hour energy expenditure, adjusted for fat-free mass, fat mass, and spontaneous physical activity, was 311 kJ/d lower (95% confidence interval: 24–598 kJ/d, P=0.03) in the V/V homozygotes than in the A/A and A/V genotypes. The V/V had ∼20% higher 24‐h spontaneous physical activity, particularly higher at night (P<0.005). Energy expenditure due to higher spontaneous physical activity counteracted the V/V group’s lower 24‐h resting energy expenditure for a given body size and composition. 24‐h RQ adjusted for energy balance, age, sex and spontaneous physical activity, was higher in the V/V homozygotes than in the AA and A /V groups (P<0.05).CONCLUSIONS:Subjects with the V/V genotype of the UCP2 gene exhibit an enhanced metabolic efficiency and lower fat oxidation than the A/A and A/V genotypes.

The BIGTT Test A novel test for simultaneous measurement of pancreatic β-cell function, insulin sensitivity, and glucose tolerance

OBJECTIVE—Insulin resistance and impaired β-cell function are key elements in the pathogenesis of type 2 diabetes. We aimed to develop valid algorithms for estimation of the insulin sensitivity index (SI) and acute insulin response (AIR) derived from simple and cheap physiological measurements that could be used in large-scale metabolic, genetic, and epidemiological studies. RESEARCH DESIGN AND METHODS—For our purpose, data from an oral glucose tolerance test (OGTT) (18 samples during 240 min) and a tolbutamide-modified intravenous glucose tolerance test (IVGTT) (33 samples during 180 min) from 258 individuals with fasting plasma glucose <7 mmol/l and 2-h plasma glucose <7.8 mmol/l were used for model development and internal validation. Data from an additional 28 individuals were used for external validation. Bergman’s minimal model was used to calculate SI, and the trapezoidal method was used to calculate AIR0–8 min. Multiple linear regression was applied to derive predictive equations of log(SI) and log(AIR0–8 min) using data on sex, BMI, plasma glucose, and serum insulin levels obtained during the OGTT. RESULTS—We demonstrate that it is possible to obtain estimates of SI (BIGTT-SI) and AIR (BIGTT-AIR) that are highly correlated to IVGTT-derived values of SI (R2 = 0.77) and AIR (R2 = 0.54). In the two validation datasets we obtained similar results. CONCLUSIONS—Data from OGTTs can provide accurate measures of insulin sensitivity and β-cell function, which can be used in large scale metabolic, genetic, and epidemiological studies.

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.

Mutational analysis of the coding region of the uncoupling protein 2 gene in obese NIDDM patients: impact of a common amino acid polymorphism on juvenile and maturity onset forms of obesity and insulin resistance.

Summary Recently, a gene encoding a novel human uncoupling protein, designated UCP2, was discovered. The murine UCP2 was mapped to a region on mouse chromosome 7 which in several models has been shown to be linked to obesity and hyperinsulinaemia. Single strand conformation polymorphism (SSCP) analysis and direct sequencing of the coding region of the UCP2 gene in 35 obese Caucasian NIDDM patients of Danish ancestry revealed one nucleotide substitution, replacing an alanine with a valine at codon 55. The amino acid polymorphism was present in 24 of the 35 (69 %) examined subjects. The allelic frequency of the A/V55 variant was 48.3 % (95 % CI: 42.5–54.1 %) among 144 subjects with juvenile onset obesity, 45.6 % (40.5–50.7 %) among 182 subjects randomly selected at the draft board examination, and 45.5 % (37.1–53.9 %) among lean control subjects selected from the same study cohort. Within these cohorts there were no differences in BMI values at different ages among wild-type carriers and A/V55 carriers. In a population-based sample of 369 young healthy Caucasians the variant showed no association with alterations in BMI, waist-to-hip ratio, fat mass or weight gain during childhood or adolescence. The A/V55 polymorphism was not related to alterations in fasting values of serum insulin and C-peptide or to an impaired insulin sensitivity index. We conclude that genetic variability in the human UCP2 gene is not a common factor contributing to NIDDM in obese Danish Caucasian subjects and the common A/V55 amino acid polymorphism of the gene is not implicated in the pathogenesis of juvenile or maturity onset obesity or insulin resistance in Caucasians. [Diabetologia (1997) 40: 1227–1230]

Studies of the genetic variability of the coding region of the hepatocyte nuclear factor-4α in Caucasians with maturity onset NIDDM

Summary Mutations in the hepatocyte nuclear factor-4α (HNF-4α) gene cause the type 1 form of maturity onset diabetes of the young (MODY1). To address the question of whether genetic variability of HNF-4α is associated with late onset non-insulin-dependent diabetes mellitus (NIDDM) we have sequenced the coding region and intron/exon boundaries of the gene in 36 randomly recruited Danish NIDDM patients. Two nucleotide substitutions that changed the sequence of HNF-4α were identified: Thr/Ile130, which has been reported previously and a novel Val/Met255. The Val/Met 255 mutation was found in 4 of 477 Danish NIDDM patients and in none of 217 glucose tolerant control subjects; thus it cannot be excluded that this mutation may have an impact on NIDDM susceptibility. Among 509 NIDDM patients the allelic frequency of the Thr/Ile130 variant was 4.7 % (95 % confidence interval: 3.4–6.0 %) compared to 1.9 % (0.7–3.1 %) among 239 control subjects (p = 0.008). However, in a population sample of 942 Swedish men with an average age of 70 years the allelic frequency of the variant was similar in 246 men with either impaired glucose tolerance (5.6 % [2.6–8.6 %]) or NIDDM (5.4 % [2.7–8.1 %]) as compared to 666 glucose tolerant men (5.1 % [3.9–6.3 %]). Also in a population sample of 369 young healthy Danes the prevalence of the codon 130 variant (4.7 % [3.2–6.2 %]) was similar to what was found in Swedish Caucasians. Thus, the allelic frequency of the Thr/Ile130 variant among the control subjects in the Danish case-control study deviates from the prevalence in the two other studies which is why we consider the significant association between the codon 130 variant and NIDDM an incidental finding. In glucose tolerant subjects the codon 130 variant in its heterozygous form had no major effect on glucose-induced insulin and C-peptide release although a tendency to a lower insulin secretion during an oral glucose tolerance test was seen in middle-aged subjects. In conclusion, variability in the coding region of the HNF-4α gene is not a common cause of NIDDM among whites of Danish ancestry. However, a Val/Met255 mutation was found exclusively in NIDDM patients (0.8 % of cases) and functional as well as family segregation studies are needed to determine whether this HNF-4α variant is a NIDDM causing mutation. [Diabetologia (1997) 40: 980–983]