Homozygosity of the Pro12Ala variant of the peroxisome proliferation-activated receptor-γ2 (PPAR-γ2): 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- γ 2) is associated with juvenile-onset obesity among Danish Caucasianmen and 2) whether the relation of a Pro12Ala polymorphism in PPAR- γ 2 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- γ 2 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] Received: 22 December 1998 and in revised form: 3 March 1999     

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

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] Received: 4 April 2001 and in revised form: 19 July 2001     

Genetic variability of the SUR1 promoter in relation to beta-cell function and Type II diabetes mellitus

Aims/hypothesis: We aimed to examine the promoter of SUR1 for genetic variation and to determine if variants were associated with Type II (non-insulin-dependent) diabetes mellitus or measures of beta-cell function. Methods: We examined 465 bp upstream of the ATG site in 46 Type II diabetic patients and 15 glucose tolerant control subjects by SSCP-heteroduplex analysis. Results: We identified an a → t substitution 437 bp upstream of the ATG site. The allelic frequency was similar in 455 unrelated Type II diabetic patients and in 203 glucose tolerant control subjects matched for age (0.036, [95 % CI 0.019–0.053] vs 0.034 [95 % CI 0.009–0.059]; p = 0.92). Among the glucose tolerant subjects there were no differences between non-carriers (n = 189) and carriers (n = 14) of the variant in fasting values or 30 min values of plasma glucose and serum insulin during an oral glucose tolerance test. In a study of 233 glucose tolerant offspring of and spouses to Danish Caucasian Type II diabetic patients, non-carriers (n = 193) and carriers (n = 37) of the –437 a/t polymorphism did not differ in glucose or tolbutamide stimulated insulin response during an intravenous glucose tolerance test with intravenous tolbutamide injection [AUCs-insulin (0–8) min, 2290 ± 1660 vs 2308 ± 1935 pmol/l · min and AUCs-insulin(20–30 min), 3113 ± 2033 vs. 3393 ± 2830 pmol/l · min, respectively]. Conclusion/interpretation: We have identified a novel a/t polymorphism of the SUR1 gene promoter which is not associated with Type II diabetes mellitus or measures of beta-cell function. Previous reported non-functional variants of SUR1 associated with Type II diabetes mellitus still need to be accounted for. [Diabetologia (2001) 44: 1330–1334] Received: 24 April 2001 and in revised form: 26 June 2001     

Insulin secretion and insulin sensitivity in diabetic subgroups: studies in the prediabetic and diabetic state

Aims/hypothesis. To evaluate insulin sensitivity and insulin secretion in prediabetic and diabetic subjects with mutations in MODY1 (HNF-4α) and MODY3 (HNF-1α) genes, in subjects with GAD antibodies, latent autoimmune diabetes in adults and in subjects with the common form of Type II (non-insulin-dependent) diabetes mellitus. Methods. Insulin secretion was measured as the incremental 30-min insulin (I30) and insulin glucose ratio (I:G30) during OGTT whereas insulin sensitivity was measured as the insulin sensitivity index during OGTT in 131 carriers of MODY mutations [NGT = 38, IFG/IGT = 21, diabetes mellitus (DM) = 72], in 293 subjects with GADA (NGT = 47, IFG/IGT = 29, DM = 217) and in 2961 subjects with a family history of the common form of Type II diabetes but without MODY mutations or GADA (NGT = 1360, IFG/IGT = 857, DM = 744). A subgroup of the subjects underwent a euglycaemic clamp (n = 210) and intravenous glucose tolerance test (n = 337) for the estimation of insulin sensitivity and first-phase insulin secretion. Results. Non-diabetic subjects with MODY mutations had pronounced impaired insulin secretion (I30, I:G30) compared with the two other groups (p = 0.005). Normal or non-diabetic glucose tolerance was maintained by enhanced insulin sensitivity compared with the other two groups (p < 0.05 and p < 0.005). In contrast to patients with Type II diabetes and with adult latent autoimmune diabetes, MODY patients showed only a modest deterioration in insulin sensitivity at onset of diabetes. The 2-h glucose values inversely correlated with insulin sensitivity in subjects with GADA (r = –0.447, p < 0.001) and subjects from Type II diabetic families (r = –0.426, p < 0.001), whereas no such relation was observed in subjects with MODY mutations (r = 0.151, p = NS). There were no statistically significant differences in insulin secretion or insulin sensitivity between subjects with GADA or subjects with a family history of Type II diabetes, either at the NGT or the IFG/IGT stage. Conclusion/interpretation. Glucose-tolerant carriers of MODY mutations are characterised by a severe impairment in insulin secretion. Enhanced insulin sensitivity is the most likely explanation for the normal glucose tolerance. Whereas subjects with positive GADA or Type II diabetes have impaired insulin sensitivity with increasing glucose concentrations, MODY mutation carriers seem to be protected from the effect of glucose toxicity. [Diabetologia (2000) 43: 1476–1483] Received: 23 March 2000 and in revised form: 29 August 2000     

The PPARγ2 amino acid polymorphism Pro 12 Ala is prevalent in offspring of Type II diabetic patients and is associated to increased insulin sensitivity in a subgroup of obese subjects

Aims/hypothesis. Recently a mutation in the coding sequence of the adipocyte specific isoform peroxisome proliferator-activated receptor γ2 (PPARγ2) was described, leading to the substitution of Proline to Alanine at codon 12. Mutations in PPARγ2 could have a role in people who are at increased risk for the development of obesity and Type II (non-insulin-dependent) diabetes mellitus. Methods. Non-diabetic first-degree relatives (n = 108) of subjects with Type II diabetes were characterized by oral glucose tolerance tests and euglycaemic hyperinsulinaemic glucose clamp to determine insulin sensitivity. Results. We found 75 (69 %) probands without the PPARγ ProAla12 substitution, 28 heterozygotes (26 %) and 5 (4 %) homozygotes. When the whole group was analysed for an association between the mutation and insulin sensitivity, no statistical significance could be shown. Only in the group with severe obesity more than 30 kg/m², an association (p = 0.016) of the polymorphism with an increase in insulin sensitivity was found. Conclusion/interpretation. These observations suggest that the mutation in the PPARγ2 molecule may have a role in subgroups prone to the development of obesity and Type II diabetes. [Diabetologia (1999) 42: 758–762] Received: 8 January 1999 and in final revised form: 11 February 1999     

Euglycaemic hyperinsulinaemia does not affect gastric emptying in Type I and Type II diabetes mellitus

Summary Hyperglycaemia slows gastric emptying in both normal subjects and patients with diabetes mellitus. The mechanisms mediating this effect, particularly the potential role of insulin, are uncertain. Hyperinsulinaemia has been reported to slow gastric emptying in normal subjects during euglycaemia. The purpose of this study was to evaluate the effect of euglycaemic hyperinsulinaemia on gastric emptying in Type I (insulin-dependent) and Type II (non-insulin-dependent) diabetes mellitus. In six patients with uncomplicated Type I and eight patients with uncomplicated Type II diabetes mellitus, measurements of gastric emptying were done on 2 separate days. No patients had gastrointestinal symptoms or cardiovascular autonomic neuropathy. The insulin infusion rate was 40 mU · m^–2· min^–1 on one day and 80 mU · m^–2· min^–1 on the other. Gastric emptying and intragastric meal distribution were measured using a scintigraphic technique for 3 h after ingestion of a mixed solid/liquid meal and results compared with a range established in normal volunteers. In both Type I and Type II patients the serum insulin concentration had no effect on gastric emptying or intragastric meal distribution of solids or liquids. When gastric emptying during insulin infusion rates of 40 mU · m^–2· min^–1 and 80 mU · m^–2· min^–1 were compared the solid T₅₀ was 137.8 ± 24.6 min vs 128.7 ± 24.3 min and liquid T₅₀ was 36.7 ± 19.4 min vs 40.4 ± 15.7 min in the Type I patients; the solid T₅₀ was 94.9 ± 19.1 vs 86.1 ± 10.7 min and liquid T₅₀ was 21.8 ± 6.9 min vs 21.8 ± 5.9 min in the Type II patients. We conclude that hyperinsulinaemia during euglycaemia has no notable effect on gastric emptying in patients with uncomplicated Type I and Type II diabetes; any effect of insulin on gastric emptying in patients with diabetes is likely to be minimal. [Diabetologia (1999) 42: 365–372] Received: 3 September 1998 and in revised form: 3 November 1998     

Studies of variability in the PTEN gene among Danish caucasian patients with Type II diabetes mellitus

Abstract Aim/hypothesis. Phosphatase and tensin homologue deleted from chromosome ten (PTEN) has recently been characterized as a novel member in the expanding network of proteins regulating the intracellular effects of insulin. By dephosphorylation of phosphatidyl-inositol-(3, 4, 5)-trisphosphate (PIP₃) the PTEN protein regulates the insulin-dependent phosphoinositide 3-kinase (PI₃K) signalling cassette and accordingly might function as a regulator of insulin sensitivity in skeletal muscle and adipose tissue. In this study we tested PTEN as a candidate gene for insulin resistance and late-onset Type II (non-insulin-dependent) diabetes mellitus in a Danish Caucasian population. Methods. The nine exons of the PTEN, including intronic flanking regions were analysed by PCR-SSCP and heteroduplex analysis in 62 patients with insulin-resistant Type II diabetes. Results. No mutations predicted to influence the expression or biological function of the PTEN protein but four intronic polymorphisms were identified: IVS1–96 A→G (allelic frequency 0.22, 95 % CI: 0.12–0.32), IVS3 + 99 C→T (0.01, CI: 0–0.03), IVS7–3 TT→T (0.10, CI: 0.03–0.18) and IVS8 + 32 G→T (0.35, CI: 0.23–0.47). The IVS8 + 32 G→T polymorphism was used as a bi-allelic marker for the PTEN locus and examined in 379 patients with Type II diabetes and in 224 control subjects with normal glucose tolerance. The IVS8 + 32 G→T polymorphism in the PTEN was not associated with Type II diabetes and it did not have any effect on body-mass index, blood pressure, HOMA insulin resistance index, or concentrations of plasma glucose, serum insulin or serum C peptide obtained during an oral glucose tolerance test (OGTT). Conlusion/interpretation. Variability in the PTEN is not a common cause of Type II diabetes in the Danish Caucasian population. [Diabetologia (2001) 44: 237–240] Received: 31 July 2000 and in revised form: 20 August 2000     

Erythrocyte Na/K ATPase activity and diabetes: relationship with C-peptide level

Summary Erythrocyte Na/K ATPase activity is decreased in Type I diabetic patients; for Type II diabetic patients, literature data are controversial. Therefore, we have compared this enzymatic activity in 81 patients with Type I diabetes mellitus, 87 with Type II diabetes mellitus and 75 control subjects. Mean erythrocyte Na/K ATPase activity was lower in the Type I diabetic patients (285 ± 8 nmol Pi · mg protein^–1· h^–1) than in the control subjects (395 ± 9 nmol Pi · mg protein^–1· h^–1) whereas that of the Type II diabetic patients did not differ from that of control subjects. Sex, age, body mass index, and HbA_{1 c} levels did not influence erythrocyte Na/K ATPase activity. The 25 Type II diabetic patients treated with insulin, however, had lower Na/K ATPase activity than the 62 on oral treatment (264 ± 18 vs 364 ± 16 nmol Pi · mg protein^–1· h^–1, p < 0.001) but similar to that of Type I diabetic patients. Among the Type II diabetic patients, stepwise regression analysis showed that fasting C-peptide level was the only factor independently correlated with Na/K ATPase activity; it explained 23 % of its variance. In fact, in the insulin-treated patients, those with almost total endogenous insulin deficiency (C-peptide < 0.2 nmol · l^–1) had the lower Na/K ATPase activity (181 ± 21 vs 334 ± 17 nmol Pi · mg protein^–1· h^–1, p < 0.0001). The biological effects of treatment with C-peptide have recently led to the suggestion that this peptide could have a physiological role through the same signalling pathway as insulin, involving G-protein and calcium phosphatase and thus restoring Na/K ATPase activity. The relationship we describe between endogenous C-peptide and this activity is a strong argument for this physiological role. [Diabetologia (1998) 41: 1080–1084] Received: 6 November 1997 and in final revised form: 10 April 1998     

The influence of improved glycaemic control with insulin and sulphonylureas on acute phase and endothelial markers in Type II Diabetic subjects

Aims/hypothesis. Improved glycaemic control might reduce both microvascular and macrovascular complications of Type II diabetes (non-insulin-dependent) mellitus. To explore such possible mechanisms, we investigated the effects of intensive treatment on markers of endothelial dysfunction and of acute phase activation, using both sulphonylureas and insulin. Methods. In a randomised cross-over study we gave sulphonylureas or insulin each for a period of 16 weeks to 22 poorly controlled Type II diabetic subjects who were being treated by diet. There was a 4 week washout period between each treatment. Subjects were studied at baseline and at the end of each treatment. Results. Treatment with sulphonylureas and insulin resulted in similar improvements in glycaemic control (glycated haemoglobin, baseline: 11.8 [(SD 2.2)%; after sulphonylureas: 8.6 (1.2)%, p < 0.001; after insulin: 8.6 (1.2)%, p < 0.001] and in insulin sensitivity {metabolic clearance rate of glucose, baseline: median 1.75 [interquartile (IQ) range 1.41, 2.27] ml · kg^–1· min^–1; after sulphonylureas: 2.41 (1.82, 3.01) ml · kg^–1· min^–1, p = 0.001; after insulin: 2.23 (1.92, 2.75) ml · kg^–1· min^–1, p = 0.027}. There were no significant changes in concentrations of endothelial markers von Willebrand factor, cellular fibronectin, thrombomodulin, tissue plasminogen activator, soluble E-selectin or soluble intercellular adhesion molecule-1 or in urinary albumin excretion rate after either treatment period. Concentrations of C-reactive protein were not significantly influenced by sulphonylureas but fell after insulin [baseline: median 4.50 (IQ range 1.37, 6.44) μg · ml^–1; sulphonylureas: 2.69 (0.88, 9.65) μg · ml^–1 (p = 0.53); insulin: 2.07 (1.16, 5.24) μg · ml^–1 (p = 0.017)]. There were, however, no significant effects of either treatment on circulating concentrations of fibrinogen (p = 0.28–0.34) or of the proinflammatory cytokines interleukin-6 or tumour necrosis factor-α (p = 0.65–0.79). Conclusion/interpretation. Markers of endothelial dysfunction and concentrations of proinflammatory cytokines in Type II diabetes are not influenced by improved glycaemic control over 16 weeks. Improved metabolic control with insulin could, however, be associated with reduced concentrations of the acute phase marker C-reactive protein. [Diabetologia (2000) 43: 1099–1106] Received: 11 May 2000 and in revised form: 19 June 2000     

Enlarged subcutaneous abdominal adipocyte size, but not obesity itself, predicts Type II diabetes independent of insulin resistance

Aims/hypothesis. Cross-sectional studies indicate that enlarged subcutaneous abdominal adipocyte size is associated with hyperinsulinaemia, insulin resistance and glucose intolerance. To further explore the pathophysiological significance of these associations, we examined prospectively whether enlarged subcutaneous abdominal adipocyte size predicts Type II (non-insulin-dependent) diabetes mellitus. Methods. Body composition (hydrodensitometry), mean subcutaneous abdominal adipocyte size (fat biopsy), insulin sensitivity (hyperinsulinaemic clamp) and the acute insulin secretory response (25-g i. v. GTT) were assessed in 280 Pima Indians with either normal (NGT), impaired (IGT) or diabetic glucose tolerance (75-g OGTT). Subjects with NGT were then followed prospectively. Results. After adjusting for age, sex and per cent body fat, mean subcutaneous abdominal adipocyte size was 19 % and 11 % higher in subjects with diabetes and IGT, compared with those with NGT (p < 0.001). Insulin sensitivity was inversely correlated with mean subcutaneous abdominal adipocyte size (r = –0.53, p < 0.0001), even after adjusting for per cent body fat (r = –0.31, p < 0.001). In 108 NGT subjects followed over 9.3 ± 4.1 years (33 of whom developed diabetes), enlarged mean subcutaneous abdominal adipocyte size but not high per cent body fat, was an independent predictor of diabetes, in addition to a low insulin sensitivity and acute insulin secretory response [relative hazard 10^th vs 90^th centile (95 % CI): 5.8 (1.7–19.6), p < 0.005]. In 28 NGT subjects with a 9 % weight gain over 2.7 ± 1.3 years, changes in insulin sensitivity were inversely and independently related to changes in mean subcutaneous abdominal adipocyte size and per cent body fat. Conclusion/interpretation. Although enlarged mean subcutaneous abdominal adipocyte size is associated with insulin resistance cross-sectionally, prospectively, both abnormalities are independent and additive predictors of Type II diabetes. [Diabetologia (2000) 43: 1498–1506] Received: 31 July 2000 and in revised form: 6 September 2000     

Human calcium/calmodulin-dependent protein kinase II gamma gene (CAMK2G): cloning,genomic structure and detection of variants in subjects with Type II diabetes

Aims/hypothesis. Ca²⁺/calmodulin-dependent protein kinase II, is expressed in the pancreatic beta cells and is activated by glucose and other secretagogues in a manner correlating with insulin secretion. The activation of Ca²⁺/calmodulin-dependent protein kinase II mediates some of the actions of Ca²⁺ on the exocytosis of insulin. We therefore investigated the gene encoding the gamma isoform (CAMK2G) which has been shown to be expressed in human beta cells as a candidate gene for Type II (non-insulin-dependent) diabetes mellitus. Methods. Human CAMK2G was cloned from a total human P1 artificial chromosome library using a partial Ca²⁺/calmodulin-dependent protein kinase γ _E cDNA probe. Positive PAC clones were localised to chromosome 10q22 by fluorescence in situ hybridisation. To obtain structural information and the sequences of the exon–intron boundaries, the published genomic structures of the rat and mouse genes allowed the putative exon-intron boundaries of human CAMK2G to be amplified by vectorette polymerase chain reaction and sequenced. Sequence variants in each exon were identified using single stranded conformational polymorphism analysis. Results. The human CAMK2G gene comprises 22 exons which range in size between 43 to 230 bp. Screening of the exons and exon–intron boundaries identified two single nucleotide polymorphisms. These did not show association with diabetes in 122 patients and 144 control subjects. Conclusions/interpretation. We have identified the genomic structure of CAMK2G to enable further study of this potential candidate gene. Variation in this gene is not strongly associated with diabetes in Caucasians in the United Kingdom. We have identified two single nucleotide polymorphisms which, with appropriately large case control studies, can be used to assess the role of CAMK2G in the susceptibility to Type II diabetes. [Diabetologia (2002) 45: ▪–▪] Received: 12 October 2001 and in revised form: 9 November 2001     

Studies of the relationship between the ENPP1 K121Q polymorphism and type 2 diabetes, insulin resistance and obesity in 7,333 Danish white subjects

Aims/hypothesis Plasma cell membrane glycoprotein 1 (PC-1) inhibits insulin signalling by direct interaction with the insulin receptor α subunit. This inhibition is enhanced by the minor Q allele of the K121Q polymorphism (rs1044498) in the gene (ENPP1) encoding PC-1. This polymorphism has been studied in relation to insulin resistance, type 2 diabetes and obesity in several populations with conflicting results. We assessed the impact of the ENPP1 K121Q polymorphism on type 2 diabetes, obesity and quantitative metabolic traits in 7,333 Danes.Subjects and methods The K121Q polymorphism was genotyped in the population-based Inter99 study cohort (5,961 subjects) and in a group of 1,386 patients with type 2 diabetes. All subjects were Danish whites.Results No significant associations with type 2 diabetes or related quantitative metabolic traits, including measures of insulin resistance, were detected. However, a meta-analysis of the present and published studies revealed an association with type 2 diabetes (odds ratio per Q allele, 1.17 [95% CI 1.10–1.25], p=1×10⁻⁶). In case–control studies comparing subjects of different BMI strata, we observed a putative association of the codon 121 QQ genotype with being overweight (BMI>25 kg/m²; odds ratio 1.63 [95% CI 1.09–2.46], p=0.015), an association not observed when comparing other levels of BMI or when analysing BMI as a quantitative trait.Conclusions/interpretation In a meta-analysis, the ENPP1 codon 121 Q allele associates with type 2 diabetes. However, a similar association was not found in the present study of Danish white subjects. The effect of this variant on obesity in Danish subjects is contentious and further study is needed.     

Morphometric documentation of abnormal intramyocellular fat storage and reduced glycogen in obese patients with Type II diabetes

Aims/hypothesis. Insulin resistance of skeletal muscle has been associated with increased lipid availability. This study aimed to estimate volume fractions of intramyocellular triglyceride droplets and glycogen granules in skeletal muscle using electron microscopy and furthermore, relate these findings to insulin sensitivity and the level of circulating lipids. Methods. We compared 11 obese patients with Type II (non-insulin-dependent) diabetes mellitus and 11 obese normoglycaemic subjects matched for age and sex. Glucose metabolism was determined using the euglycaemic hyperinsulinaemic clamp technique (40 mU · m^–2· min^–1) coupled with indirect calorimetry and tritiated glucose. On the second day, using an automatic procedure, a fasting muscle biopsy was carried out and processed for electron microscopy. Volume fractions of intramyocellular structures were estimated by pointcounting on photographic pictures in a blinded manner. Results. Insulin-stimulated total glucose disposal rate was lower in the Type II diabetic subjects compared with the obese normoglycaemic subjects (4.96 ± 049 vs 10.35 ± 0.89 mg · min^–1· kg ffm^–1, p < 0.001) as was glucose storage (2.03 ± 0.50 vs 6.59 ± 0.83, p < 0.001). The electron microscopy study revealed that the diabetic subjects had higher intramyocellular amounts of triglyceride (1.43 ± 0.21 vs 0.39 ± 0.07 %, p < 0.001) and lower amounts of glycogen (3.53 ± 0.33 vs 6.94 ± 0.54 %, p < 0.001). Mitochondrial volume was identical indicating equal aerobic capacity. The fractional intramyocellular lipid volume was found to be positively associated with fasting NEFA (r = 0.63, p = < 0.05 and r = 0.79, p = < 0.05) and triglyceride (r = 0.74, p = 0.01 and r = 0.62, p < 0.05) in the obese diabetic and normoglycaemic cohorts respectively. Intramyocellular lipid content was negatively correlated to insulin sensitivity (r = –0.71, p < 0.02) in the obese diabetic group whereas no significant association was found in the obese normoglycaemic group. Conclusion/interpretation. This study shows that fat accumulates intramyocellulary while glycogen stores are simultaneously reduced in obese subjects with Type II (non-insulin-dependent) diabetes mellitus. Quantitatively, a major component of the excessive lipid accumulation could be secondary in origin, related to the diabetic state in itself, although a contribution from the altered insulin action cascade of obesity and diabetes cannot be excluded. In both groups significant positive relations were found between circulating and intramyocellular lipid. [Diabetologia (2001) 44: 824–833] Received: 6 December 2000 and in revised form: 16 March 2001     

Common amino acid substitutions in insulin receptor substrate-4 are not associated with Type II diabetes mellitus or insulin resistance

Summary The family of insulin receptor substrates (IRS1–4) is defined by proteins with an overall similar structure. IRS-1 and IRS-2 have been shown to have key roles in cellular transmission of the action of insulin, insulin-like growth factor-1 and various cytokines. We have previously identified amino acid polymorphisms in the human IRS-1 and IRS-2 proteins. Given the documented importance of IRS-1 and –2 in insulin signalling and the implications of distribution of these genes for the pathogenesis of insulin resistance and diabetes, we decided that the most recently identified member of the IRS family, IRS-4, was a relevant candidate to examine for genetic variability which might be associated with subsets of diabetes or insulin resistance. The gene encoding IRS-4 was analysed by the single strand conformation polymorphism technique in 83 Danish Caucasians with Type II (non-insulin-dependent) diabetes mellitus. Five amino acid polymorphisms were identified: Leu34Phe, Arg411Gly, Gly584Cys, His879Asp and Lys883Thr. In an association study of 324 patients with Type II diabetes and 267 control subjects with normal glucose tolerance the polymorphism at codon 34 was found with allelic frequencies of 3.9 and 2.3 %, respectively, the variant at codon 411 with allelic frequencies of 3.9 and 5.6 %, respectively, and the variant at codon 879 with frequencies of 19.2 and 18.0 %, respectively. Each carrier of the codon 34 polymorphism was also a carrier of the codon 411 and codon 879 variants and similarly, carriers of the variant at codon 411 were also carriers of the polymorphism at codon 879. The variants at codon 584 and 883 were each found in only one Type II diabetic patient. The allelic frequencies of the variants at codon 411 and 879 were also determined in 380 young healthy subjects (4.6 and 18.1 %, respectively). The insulin sensitivity index as estimated by Bergman's minimal model of the young healthy subjects carrying either polymorphism was indistinguishable from the carriers of wild-type IRS-4. Moreover, none of the men were heterozygous for the IRS-4 polymorphisms indicating that the gene is located on the X-chromosome. In conclusion, amino acid polymorphisms in human IRS-4 are common in Caucasians but are not associated with Type II diabetes or with insulin resistance in young healthy subjects. [Diabetologia (1998) 41: 969–974] Received: 25 February 1998 and in revised form: 7 May 1998     

Large-scale studies of the HphI insulin gene variable-number-of-tandem-repeats polymorphism in relation to Type 2 diabetes mellitus and insulin release

Aims/hypothesis The class III allele of the variable-number-of-tandem-repeats polymorphism located 5 of the insulin gene (INS-VNTR) has been associated with Type 2 diabetes and altered birthweight. It has also been suggested, although inconsistently, that the class III allele plays a role in glucose-induced insulin response among NGT individuals.Methods We investigated the impact of the class III allele on Type 2 diabetes susceptibility in a case-control study involving 1462 Type 2 diabetic patients and 4931 NGT subjects. We also examined the potential impact of the class III allele in genotype-quantitative trait studies in three Danish study populations containing (i) 358 young healthy subjects; (ii) 4444 middle-aged NGT subjects, 490 subjects with IFG and 678 subjects with IGT; and (iii) 221 NGT subjects, of whom one parent had Type 2 diabetes.Results There was no difference in frequency of the class III allele or in genotype distribution between the 1462 Type 2 diabetic patients and the 4931 NGT subjects. Among the 358 young subjects the class III/III carriers had significantly reduced post-IVGTT acute serum insulin and C-peptide responses (p=0.04 and 0.03 respectively). However, among the 4444 middle-aged subjects we failed to demonstrate any association between the class III allele and post-OGTT serum insulin and C-peptide levels.Conclusions/interpretation The class III allele of the INS-VNTR does not confer susceptibility to Type 2 diabetes or consistent alterations in glucose-induced insulin release in the examined populations, which consisted of Danish Caucasians.Abbreviations INS insulin gene - HOMA IR HOMA insulin resistance - HOMA IS HOMA insulin secretion - OHA oral hypoglycaemic agents - PCOS polycystic ovary syndrome - S_i insulin sensitivity index - SNP single nucleotide polymorphism - VNTR variable number of tandem repeats     

The common SLC30A8 Arg325Trp variant is associated with reduced first-phase insulin release in 846 non-diabetic offspring of type 2 diabetes patients—the EUGENE2 study

Aims/hypothesis A recent genome-wide association study identified the SLC30A8 rs13266634 polymorphism encoding an Arg325Trp polymorphism in the zinc transporter protein member 8 (ZnT-8) to be associated with type 2 diabetes. Here, we investigate whether the polymorphism is related to altered insulin release in response to intravenous and oral glucose loads in non-diabetic offspring of type 2 diabetic patients. Methods We genotyped SLC30A8 rs13266634 in 846 non-diabetic offspring of type 2 diabetic patients from five different white populations: Danish (n = 271), Finnish (n  = 217), German (n = 149), Italian (n  = 109) and Swedish (n  = 100). Participants were subjected to both IVGTTs and OGTTs, and measurements of insulin sensitivity. Results Homozygous carriers of the major type 2 diabetes C risk-allele showed a 19% decrease in first-phase insulin release (0–10 min) measured during the IVGTT (CC 3,624 ± 3,197; CT 3,763 ± 2,674; TT 4,478 ± 3,032 pmol l⁻¹ min⁻¹, mean ± SD; p = 0.007). We found no significant genotype effect on insulin release measured during the OGTT or on estimates of insulin sensitivity. Conclusions/interpretation Of European non-diabetic offspring of type 2 diabetes patients, 46% are homozygous carriers of the Arg325Trp polymorphism in ZnT-8, which is known to associate with type 2 diabetes. These diabetes-prone offspring are characterised by a 19% decrease in first-phase insulin release following an intravenous glucose load, suggesting a role for this variant in the pathogenesis of pancreatic beta cell dysfunction. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorised users.     

White blood-cell count and the risk of impaired fasting glucose or Type II diabetes in middle-aged Japanese men

Aims/hypothesis: To investigate the association between white blood-cell (WBC) count and the development of diabetes, independent of cigarette smoking. Methods: We examined 2953 Japanese men who were office workers and between 35 and 59 years of age and who did not have impaired fasting glucose (IFG) (a fasting glucose concentration of 6.1–6.9 mmol/l), Type II (non-insulin-dependent) diabetes mellitus (a fasting glucose concentration of ≥ 7.0 mmol/l or more or receipt of hypoglycaemic medication), medication for hypertension, and a history of cardiovascular disease. Fasting glucose concentrations were measured at annual health examinations from May 1994 through May 2000. Results: After controlling for potential predictors of diabetes, the relative risk for IFG or Type II diabetes mellitus compared with a WBC count of less than 5.3 · 10⁹ cells/l was 1.2 (95 %-CI, 0.6–2.3), 1.6 (CI, 0.8–3.1), and 2.5 (CI, 1.2–5.1) among non-smokers (p for trend = 0.009): and 1.0 (CI, 0.4–2.5), 2.3 (CI, 1.0–5.1), and 3.1 (CI, 1.4–7.1) among ex-smokers (p for trend = 0.001) with WBC counts of 5.3–6.1, 6.2–7.2, and 7.3 · 10⁹ cells/l or more, respectively. Among current smokers, the respective multivariate-adjusted relative risks for IFG or Type II diabetes mellitus were 1.1 (CI, 0.6–2.1), 1.4 (CI, 0.8–2.4), and 1.2 (CI, 0.7–2.1) (p for trend = 0.460). Conclusion/hypothesis: Although the selection of a rigorously normoglycaemic cohort might have had an influence on these observations, higher WBC counts seem to predict the development of IFG or Type II diabetes mellitus, primarily in non-smokers. [Diabetologia (2002) 45: 42–48] Received: 16 July 2001 and in revised form: 13 September 2001     

Short-term treatment with GLP-1 increases pulsatile insulin secretion in Type II diabetes with no effect on orderliness

Aims/hypothesis. The enteric incretin hormone, glucagon-like peptide-1 (GLP-1), is a potent insulin secretagogue in healthy humans and patients with Type II (non-insulin-dependent) diabetes mellitus. In this study we assessed the impact of short-term GLP-1 infusion on pulsatile insulin secretion in Type II diabetic patients. Methods. Type II diabetic patients (n = 8) were studied in a randomised cross-over design. Plasma insulin concentration time series were obtained during basal conditions and during infusion with saline or GLP-1 (1.2 pmol/l · kg^–1· min^–1) on 2 separate days. Plasma glucose was clamped at the initial concentration by a variable glucose infusion. Serum insulin concentration time series were evaluated by deconvolution analysis, autocorrelation analysis, spectral analysis and approximate entropy. Results. Serum insulin concentrations increased by approximately 100 % during GLP-1 infusion. Pulsatile insulin secretion was increased as measured by secretory burst mass (19.3 ± 3.8 vs 53.0 ± 10.7 pmol/l/pulse, p = 0.02) and secretory burst amplitude (7.7 ± 1.5 vs 21.1 ± 4.3 pmol/l/min, p = 0.02). A similar increase in basal insulin secretion was observed (3.6 ± 0.9 vs 10.2 ± 2.2 pmol/l/min, p = 0.004) with no changes in the fraction of insulin delivered in pulses (0.50 ± 0.06 vs 0.49 ± 0.02, p = 0.84). Regularity of secretion was unchanged as measured by spectral analysis (normalised spectral power: 5.9 ± 0.6 vs 6.3 ± 0.8, p = 0.86), autocorrelation analysis (autocorrelation coefficient: 0.19 ± 0.04 vs 0.18 ± 0.05, p = 0.66) and the approximate entropy statistic (1.48 ± 0.02 vs 1.51 ± 0.02, p = 0.86). Conclusion/interpretation. Short-term stimulation with GLP-1 jointly increases pulsatile and basal insulin secretion, maintaining but not improving system regularity in Type II diabetic patients. [Diabetologia (2000) 43: 583–588] Received: 11 November 1999 and in revised form: 13 December 1999     

Oral treatment with an antioxidant (raxofelast) reduces oxidative stress and improves endothelial function in men with Type II diabetes

Aims/hypothesis. To determine whether raxofelast, a new water soluble antioxidant decreases oxidative stress and improves endothelial function in men with Type II (non-insulin dependent) diabetes mellitus. Methods. We treated ten normotensive, normocholesterolaemic men with Type II diabetes and as controls ten healthy men matched with them for age with raxofelast (600 mg twice daily) for 1 week. Plasma 8-epi-PGF₂α, a non-enzymic oxidation product of arachidonic acid was measured by gas chromatography/mass spectrometry as an index of oxidative stress. Forearm vasodilator responses to brachial artery infusion of acetylcholine (7.5, 15 and 30 μg min^–1) and of the nitric oxide donor nitroprusside (1, 3 and 10 μg min^–1) were measured by strain gauge plethysmography. Results. Plasma concentrations of 8-epi-PGF₂α were greater in diabetic than in control men (0.99 ± 0.20 vs 0.18 ± 0.01 nmol l^–1, means ± SEM, p < 0.001) and fell after raxofelast (from 0.99 ± 0.20 to 0.47 ± 0.07 nmol l^–1, p < 0.05) in diabetic men but not in control men. Blood flow responses to acetylcholine were lower (p < 0.05) in diabetic than in control men (7.4 ± 1.0 vs 12.9 ± 2.3 ml · min^–1· 100 ml^–1 for the highest dose). In diabetic men, but not in control men, raxofelast increased (p < 0.05) blood flow responses to acetylcholine (from 7.4 ± 1.0 ml · min^–1· 100 ml^–1 to 11.3 ± 2.3 ml · min^–1· 100 ml^–1 at highest dose). Blood flow responses to nitroprusside were similar in control and diabetic men and in both groups were similar before and after raxofelast. Conclusion/interpretation. Oral treatment with raxofelast for 1 week reduces oxidative stress and improves endothelial function in men with Type II diabetes. [Diabetologia (2000) 43: 974–977] Received: 14 October 1999 and in revised form: 28 May 2000     

Visceral adipose tissue is not increased in Pima Indians compared with equally obese Caucasians and is not related to insulin action or secretion

Summary Pima Indians are insulin resistant and hyperinsulinaemic compared with Caucasians. We investigated whether abdominal fat distribution was different between Pimas and Caucasians and whether differences in the amount of visceral fat explained metabolic differences between the groups. Total body fat (absorptiometry) and abdominal fat distribution at L4-L5 (magnetic resonance imaging) were compared in 20 Pima Indians (10 men/10 women) and 20 age-, sex- and BMI-matched Caucasians. Insulin action was measured as glucose disposal during a two-step hyperinsulinaemic-euglycaemic glucose clamp and insulin secretion was assessed in response to oral and intravenous glucose tolerance tests. By design, percent body fat was similar in Pimas and Caucasians. Abdominal visceral and subcutaneous adipose tissue areas were also similar in the two groups (151 ± 16 vs 139 ± 15 cm² and 489 ± 61 vs 441 ± 57 cm² respectively). Plasma insulin concentrations were higher in Pimas than Caucasians in the fasting state (27 ± 6 vs 11 ± 2 mU/ml; p < 0.01) and after a 75-g oral glucose load (area under the curve 19975 ± 2626 vs 9293 ± 1847 mU · l^–1· 180 min^–1; p < 0.005). Glucose disposal was lower in Pimas than Caucasians during both steps of the clamp and negatively correlated (after adjustment for percent body fat and sex) with visceral adipose tissue in Caucasians (partial r = –0.51, p = 0.03), but not in Pima Indians (r = –0.03, p = 0.92). Insulin secretion was not related to visceral fat independently of percent body fat in either group. We conclude that a relative increase in visceral fat does not explain insulin resistance and hyperinsulinaemia in Pima Indians. [Diabetologia (1999) 42: 28–34] Received: 18 May 1998 and in revised form: 28 July 1998