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Aims/hypothesis: We aimed to study diurnal variation in glucose regulation by examining the effects of time of day and fasting duration on fasting plasma glucose (FPG), 2 h post-load plasma glucose (2hPG) and HbA(1c) levels., Methods: We analysed data from 5,978 non-diabetic white men and women from the prospective Whitehall II Study. All studied participants fasted for at least 8 h before a clinical examination, which included an OGTT and anthropometric measurements. We fitted mixed-effects models for FPG, 2hPG and HbA(1c) as outcome variables, and time of day and/or fasting duration as explanatory variables. Models were adjusted for age, BMI and study phase., Results: Time of day and fasting duration were associated inversely with FPG and positively with 2hPG. The mean difference between measures at 08:00 and 15:00 hours in men/women was -0.46 (95% CI -0.50, -0.42) mmol/l/-0.39 (95% CI -0.46, -0.31) mmol/l and 1.39 (95% CI 1.25, 1.52) mmol/l/1.19 (95% CI 0.96, 1.42) mmol/l for FPG and 2hPG, respectively. HbA(1c) levels were independent of either time. Time of day and fasting duration were independently associated with 2hPG. In contrast, the effect of fasting duration on FPG was markedly attenuated with adjustment for time of day. Ageing, but not obesity, was associated with increased diurnal variation in glucose tolerance., Conclusions/interpretation: Both time of day and fasting duration should be considered in clinical practice and epidemiological studies, since they have clinically relevant effects on FPG and 2hPG levels. As biochemically expected, HbA(1c) levels are independent of time of blood sampling and fasting duration.

Aims/hypothesis: Human complex metabolic traits are in part regulated by genetic determinants. Here we applied exome sequencing to identify novel associations of coding polymorphisms at minor allele frequencies (MAFs) >1% with common metabolic phenotypes., Methods: The study comprised three stages. We performed medium-depth (8×) whole exome sequencing in 1,000 cases with type 2 diabetes, BMI >27.5 kg/m(2) and hypertension and in 1,000 controls (stage 1). We selected 16,192 polymorphisms nominally associated (p < 0.05) with case-control status, from four selected annotation categories or from loci reported to associate with metabolic traits. These variants were genotyped in 15,989 Danes to search for association with 12 metabolic phenotypes (stage 2). In stage 3, polymorphisms showing potential associations were genotyped in a further 63,896 Europeans., Results: Exome sequencing identified 70,182 polymorphisms with MAF >1%. In stage 2 we identified 51 potential associations with one or more of eight metabolic phenotypes covered by 45 unique polymorphisms. In meta-analyses of stage 2 and stage 3 results, we demonstrated robust associations for coding polymorphisms in CD300LG (fasting HDL-cholesterol: MAF 3.5%, p = 8.5 × 10(-14)), COBLL1 (type 2 diabetes: MAF 12.5%, OR 0.88, p = 1.2 × 10(-11)) and MACF1 (type 2 diabetes: MAF 23.4%, OR 1.10, p = 8.2 × 10(-10))., Conclusions/interpretation: We applied exome sequencing as a basis for finding genetic determinants of metabolic traits and show the existence of low-frequency and common coding polymorphisms with impact on common metabolic traits. Based on our study, coding polymorphisms with MAF above 1% do not seem to have particularly high effect sizes on the measured metabolic traits.

Aims/hypothesis: There is limited evidence on how multifactorial treatment improves outcomes of diabetes when initiated in the lead time between detection by screening and diagnosis in routine clinical practice. Cardiac autonomic neuropathy (CAN) in people with diabetes indicates widespread damage to the autonomic nervous system, which may severely affect health and quality of life. We examined effects of early detection and subsequent intensive treatment of type 2 diabetes in primary care on the prevalence of CAN at the 6-year follow-up examination in a pragmatic cluster-randomised parallel group trial., Methods: One hundred and ninety general practices were randomised to deliver either intensive multifactorial treatment (IT) or routine care (RC) as recommended by national guidelines to patients with type 2 diabetes, identified through a stepwise screening programme in the primary care setting. 1533 people (IT, n = 910; RC, n = 623) were identified and included. At the 6-year follow-up examination, measures of CAN were applied in an unselected subsample of 777 participants using heart rate variability analysis and standard tests of CAN., Results: At the 6-year follow-up examination, the prevalence of early CAN was 15.1% in the RC group and 15.5% in the IT group, while manifest CAN was present in 7.1% and 7.3%, respectively. We found no statistically significant effect of intensive treatment on the prevalence of CAN compared with routine care., Conclusions/interpretation: In the Danish arm of the ADDITION Study, signs of CAN were highly prevalent 6 years after a screening-based diagnosis of type 2 diabetes. Intensive multifactorial treatment did not significantly affect the prevalence of CAN compared with routine care. However, at follow-up the level of medication was also high in the RC group.

Aims/hypothesis: Cancer is more frequent among diabetes patients, but it is unknown how this excess varies with duration of diabetes and insulin use. The aim of this study was to analyse disease data to examine this issue further., Methods: We linked the Danish National Diabetes Register and Cancer Registry and performed a cohort analysis of the entire Danish population by diabetes status, duration of diabetes and insulin use, comparing cancer incidence rates in diabetic patients with the non-diabetic population for the 15 year period 1995-2009, using Poisson regression with natural splines to describe the variation by duration., Results: We found 20,032 cancer cases among patients not using insulin and 2,794 cancer cases among diabetic patients using insulin. The cancer incidence rate ratio among non-insulin users relative to the non-diabetic population decreased from over 2 at diagnosis to 1.15 after 2 years of diabetes duration. The cancer incidence rate ratio was higher among patients using insulin, decreasing from 5 at the start of insulin treatment to about 1.3 [corrected] after 5 years of insulin use. Among non-insulin users, cancers of the stomach, colorectum, liver, pancreas, lung, corpus uteri, kidney and brain, and lymphomas were elevated. Among insulin users the rate ratio of prostate cancer was decreasing by duration whereas we found higher risk of cancer of the stomach, lung, liver, pancreas and kidney. Breast cancer incidence rates were not affected by either diabetes or insulin use., Conclusions: The observed duration effects suggest that both increased surveillance for cancer in the first years after diagnosis of diabetes, and reverse causation, where undiagnosed cancers increase the likelihood of diabetes diagnosis, play a role. For longer durations, a combination of common causes for diabetes and cancer, as well as the effects of diabetes and insulin exposure per se, may play a role in the association between diabetes and some cancers.

Aims/hypothesis: Genome-wide association studies have identified novel WHR and BMI susceptibility loci. The aim of this study was to elucidate if any of these loci had an effect on quantitative measures of glucose homeostasis, including estimates of insulin release and insulin sensitivity in an epidemiological setting., Methods: By applying an additive genetic model, 14 WHR-associated gene variants and 18 BMI-associated variants were investigated for their relationships with glucose-related metabolic traits in treatment-naive individuals from the population-based Inter99 study sample (n = 6,039)., Results: Of the variants associated with BMI, the QPCTL rs2287019 C allele was associated with an increased insulinogenic index of 7.4% per risk allele (p = 4.0 × 10⁻⁷) and increased disposition index of 5.6% (p = 6.4 × 10⁻⁵). The LRP1B rs2890652 C allele was associated with insulin resistance, showing a 3.3% increase (p = 0.0011) using the HOMA-insulin resistance (HOMA-IR) index and a 2.2% reduction (p = 0.0014) with the Matsuda index. Of the variants associated with WHR, LYPLAL1/SLC30A10 rs4846567 G allele carriers showed a 5.2% lower HOMA-IR (p = 0.00086) in women, indicating improved insulin sensitivity. Female carriers of the VEGFA rs6905288 A allele were insulin resistant, with a 3.7% increase in HOMA-IR (p = 0.00036) and 4.0% decrease in Matsuda index (p = 2 × 10⁻⁴)., Conclusions: Our correlative findings from analysing single-locus data suggest that some variation in validated BMI and WHR loci are associated with either increased or decreased insulin sensitivity and thereby potentially with metabolically healthy or metabolically unhealthy subsets of obesity. The results call for testing in larger study samples and for further physiological exploration of the possible metabolic implications of these loci.

Aims/hypothesis: The glucagon gene (GCG) encodes several hormones important for energy metabolism: glucagon, oxyntomodulin and glucagon-like peptide (GLP)-1 and -2. Variants in GCG may associate with type 2 diabetes, obesity and/or related metabolic traits., Methods: GCG was re-sequenced as a candidate gene in 865 European individuals. Twenty-nine variants were identified. Four variants that were considered to have a likelihood for altered functionality: rs4664447, rs7581952, Ile158Val and Trp169Ter, were genotyped in 17,584 Danes., Results: When examined in 5,760 treatment-naive individuals, homozygous carriers of the low frequency (minor allele frequency 2.3%) G allele of rs4664447, predicted to disrupt an essential splice enhancer binding site, had lower levels of fasting plasma glucose (mean ± SD, 4.8 ± 1.2 vs 5.5 ± 0.8 mmol/l, p = 0.004); fasting serum insulin (22 ± 14 vs 42 ± 27 pmol/l, p = 0.04); glucose-stimulated serum insulin (159 ± 83 vs 290 ± 183 pmol/l, p = 0.01) and adult height (165 ± 10 vs 172 ± 9 cm, p = 0.0009) compared with A allele carriers. During oral glucose tolerance and hyperglycaemic arginine stimulation tests, the plasma AUC for GLP-1 (730 ± 69 vs 1,334 ± 288 pmol/l × min, p = 0.0002) and basal and stimulated levels of serum insulin and plasma glucagon were ∼50% decreased (p < 0.001) among three homozygous carriers compared with nine matched wild-type carriers. rs7581952, Ile158Val and Trp169Ter (where 'Ter' indicates 'termination') variants of GCG did not significantly associate or co-segregate with the metabolic traits examined., Conclusions/interpretation: Re-sequencing of GCG revealed a low frequency intronic variant, rs4664447, and follow-up physiological studies suggest that this variant in homozygous form may cause decreased fasting and stimulated levels of insulin, glucagon and GLP-1. Overall, our findings suggest that variation in GCG has no major impact on carbohydrate metabolism in the study populations examined.

Aims/hypothesis: By combining multiple genome-wide association (GWA) studies and comprehensive replication efforts, 12 novel type 2 diabetes associated loci have recently been discovered. Here we evaluate the effect of lead variants of these loci on estimates of insulin release and insulin resistance derived from an oral glucose tolerance test., Methods: We examined 12 lead variants in or near HMGA2, CENTD2 (also known as ARAP1), KLF14, PRC1, TP53INP1, ZBED3, ZFAND6, CHCHD9, DUSP9, KCNQ1, BCL11A and HNF1A in 5,722 middle-aged people from the population-based Inter99 sample., Results: Carriers of the major diabetogenic allele of rs1552224 in CENTD2 had increased 30-min plasma glucose values (2.0%, p = 2 × 10(-5)) as well as 4.2% reduced insulin release 30 min after an oral glucose load (p = 0.001). Risk allele carriers also had decreased BIGTT-acute insulin release (AIR), which is a surrogate measure of insulin release where sex, BMI, plasma glucose and serum insulin are integrated (5.3%, p = 8 × 10(-7)). In addition, a decreased corrected insulin response (CIR; 9.9%, p = 3 × 10(-8)) was observed. For rs5945326 near DUSP9 on the X-chromosome we stratified according to sex. Male carriers of the risk allele showed nominally decreased BIGTT-AIR (2.6%, p = 0.01). No associations with intermediate metabolic traits were found in women. For the remaining ten lead variants no consistent associations were demonstrated., Conclusions/interpretation: Of the lead variants from 12 novel type 2 diabetes associated loci, CENTD2 significantly associated with increased plasma glucose values and decreased glucose-stimulated insulin release, suggesting that the diabetogenic effect of this locus is mediated through an impaired pancreatic beta cell function.

Aims: Increased glucose excursions and postprandial hyperglycaemia have been suggested as unique risk factors for cardiovascular disease (CVD) and mortality in patients with diabetes mellitus. Much of the evidence is based on a single 2 h glucose value after oral glucose tolerance testing in epidemiological studies. We examined the association between various indices of glycaemia measured during everyday activities and metabolic CVD risk factors in the A1C-Derived Average Glucose (ADAG) study., Methods: Participants (268 with type 1 diabetes, 159 with type 2 diabetes) completed 16 weeks of intensive continuous glucose monitoring (CGM) and self-monitoring of blood glucose (SMBG). From these data, common indices of postprandial glycaemia, overall hyperglycaemia, glucose variability and HbA₁(c) were derived. The associations between glycaemic indices and known CVD risk factors (lipids, high-sensitivity C-reactive protein and blood pressure) were explored in linear regression models., Results: For both diabetes types, the overall strongest associations with CVD risk factors were seen for the measures of average glycaemia (mean blood glucose and HbA₁(c)). Associations between self-monitored postprandial and fasting glucose and CVD risk factors were weaker, but significant. Measurements of blood glucose variability showed non-significant associations. Overall, calculations based on CGM were not more informative than those based on frequent SMBG., Conclusions/interpretation: Mean glycaemia and HbA₁(c) show consistent and stronger associations with CVD risk factors than fasting glucose or postprandial glucose levels or measures of glucose variability in patients with diabetes.

Aims/hypothesis: Real-life glycaemic profiles of healthy individuals are poorly studied. Our aim was to analyse to what extent individuals without diabetes exceed OGTT thresholds for impaired glucose tolerance (IGT) and diabetes., Methods: In the A1C-Derived Average Glucose (ADAG) study, 80 participants without diabetes completed an intensive glucose monitoring period of 12 weeks. From these data, we calculated the average 24 h glucose exposure as time spent above different plasma glucose thresholds. We also derived indices of postprandial glucose levels, glucose variability and HbA(1c)., Results: We found that 93% of participants reached glucose concentrations above the IGT threshold of 7.8 mmol/l and spent a median of 26 min/day above this level during continuous glucose monitoring. Eight individuals (10%) spent more than 2 h in the IGT range. They had higher HbA(1c), fasting plasma glucose (FPG), age and BMI than those who did not. Seven participants (9%) reached glucose concentrations above 11.1 mmol/l during monitoring., Conclusions/interpretation: Even though the non-diabetic individuals monitored in the ADAG study were selected on the basis of a very low level of baseline FPG, 10% of these spent a considerable amount of time at glucose levels considered to be 'prediabetic' or indicating IGT. This highlights the fact that exposure to moderately elevated glucose levels remains under-appreciated when individuals are classified on the basis of isolated glucose measurements.

Aims/hypothesis: We aimed to examine whether sex differences in fasting plasma glucose (FPG), 2 h post-OGTT plasma glucose (2hPG) and HbA(1c) could be explained by differences in body size and/or body composition between men and women in a general non-diabetic Danish population. Moreover, we aimed to study to what degree the newly suggested high-risk HbA(1c) criteria overlapped with the current OGTT-based criteria of glucose intolerance., Methods: We used cross-sectional data from 6,006 non-diabetic men and women. HbA(1c) and FPG levels were measured and a 75 g OGTT was performed in all individuals. Height, weight and waist and hip circumferences were measured and BMI was calculated. Data were analysed in age-adjusted linear regression models., Results: Men had higher FPG and HbA(1c) levels than women, and women had higher 2hPG levels than men. Sex differences in 2hPG levels were explained by differences in height and FPG levels, but sex differences in FPG or HbA(1c) levels were not explained by anthropometric measures. Among individuals with HbA(1c) in the high-risk range (6.0-6.5%), 73% had normal glucose tolerance., Conclusions/interpretation: Sex differences in 2hPG levels after an OGTT may to some extent be a consequence of giving the same amount of glucose to individuals with different body size. In contrast, sex differences in FPG and HbA(1c) levels are likely to have a true physiological basis. In clinical practice, the HbA(1c) assay may be more convenient than the OGTT, but it is important to note that different populations are identified by the two methods.

Aims/hypothesis: The aim of the study was to analyse how strongly commuting and leisure-time physical activity affect progression to diabetes and to study whether this relationship is different in individuals with isolated impaired fasting glucose (i-IFG) and isolated impaired glucose tolerance (i-IGT)., Methods: We studied the incidence of diabetes in 4,031 individuals without diabetes at baseline who participated in the baseline and 5 year follow-up examinations of a population-based primary prevention study, the Inter99 Study. Glucose tolerance status at baseline and at follow-up were based on OGTTs. Commuting and leisure-time physical activity at baseline were assessed by questionnaire. We present rate ratios from Poisson regression analyses adjusted for relevant confounders., Results: The progression rate to diabetes was lower among physically active individuals in the total study population and particularly among those with i-IGT. The associations were attenuated and lost statistical significance after further adjustment for BMI. We observed no impact of physical activity on the progression to diabetes in individuals with i-IFG., Conclusions/interpretation: Physical activity was associated with a lower progression to diabetes in the total study population and in individuals with i-IGT, a condition primarily characterised by muscle insulin resistance. Physical activity did not predict progression to diabetes in individuals with i-IFG, a condition primarily characterised by hepatic insulin resistance. Our results suggest that there is a differential relationship between physical activity and progression to diabetes among those with i-IFG and i-IGT. Therefore, clinical trials testing the effect of physical activity on progression from i-IFG to diabetes are needed., Trial Registration: ClinicalTrials.gov ID No.: NCT00289237, Funding: The Danish Medical Research Council, the Danish Center for Evaluation and Health Technology Assessment, Novo Nordisk, Copenhagen County, the Danish Heart Foundation, the Danish Diabetes Association, the Danish Pharmaceutical Association, the Augustinus Foundation, the Ib Henriksen Foundation and the Becket Foundation.

Aims/hypothesis: Cardiac autonomic neuropathy (CAN) is associated with increased morbidity and mortality in type 1 diabetes. Apart from glycaemic control, risk factors for CAN have not been extensively studied., Methods: As part of the EURODIAB Prospective Complications Study, CAN--defined as either a loss of heart rate variability or postural hypotension on standing--was assessed at baseline and follow-up (7.3+/-0.6 years from baseline) in patients with type 1 diabetes., Results: Follow-up measurements were available for 956 participants without CAN at baseline (age at baseline 31.3+/-8.9 years, duration of diabetes 13.5+/-8.3 years). During follow-up, 163 (17%) subjects developed CAN, yielding an incidence of 23.4 per 1,000 person-years. Blood pressure, weight, the presence of cardiovascular disease, albuminuria, distal symmetrical polyneuropathy (DSP) and retinopathy at baseline were associated with the incidence of CAN after adjustment for sex, duration of diabetes and HbA(1)c. In a multivariate regression model, baseline factors associated with an increased risk of developing CAN were age [odds ratio (OR)=1.3 per decade, 95% CI 1.1-1.7], HbA(1)c (OR=1.2 per percentage point, 95% CI 1.1-1.4), systolic blood pressure (OR=1.1 per 10 mmHg, 95% CI 1.0-1.3), feeling faint on standing (OR=2.0, 95% CI 1.2-3.2), DSP (OR=1.9, 95% CI 1.2-3.0) and retinopathy (OR=1.7, 95% CI 1.1-2.6)., Conclusion/interpretation: This study confirms the importance of exposure to hyperglycaemia as a risk factor for CAN. A small set of variables, including HbA(1)c, hypertension, DSP and retinopathy, predict the risk of CAN. Clinical trials are needed to address the impact of intensive antihypertensive treatment on CAN in type 1 diabetes.