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1.  Anthropometrics and Body Composition by Dual Energy X-Ray in Children of Obese Women: A Follow-Up of a Randomized Controlled Trial (the Lifestyle in Pregnancy and Offspring [LiPO] Study) 
PLoS ONE  2014;9(2):e89590.
In obese women, 1) to assess whether lower gestational weight gain (GWG) during pregnancy in the lifestyle intervention group of a randomized controlled trial (RCT) resulted in differences in offspring anthropometrics and body composition, and 2) to compare offspring outcomes to a reference group of children born to women with a normal Body Mass Index (BMI).
Research design and methods
The LiPO (Lifestyle in Pregnancy and Offspring) study was an offspring follow-up of a RCT with 360 obese pregnant women with a lifestyle intervention during pregnancy including dietary advice, coaching and exercise. The trial was completed by 301 women who were eligible for follow-up. In addition, to the children from the RCT, a group of children born to women with a normal BMI were included as a reference group. At 2.8 (range 2.5–3.2) years, anthropometrics were measured in 157 children of the RCT mothers and in 97 reference group children with Body Mass Index (BMI) Z-score as a primary outcome. Body composition was estimated by Dual Energy X-ray (DEXA) in 123 successful scans out of 147 (84%).
No differences between randomized groups were seen in mean (95% C.I.) BMI Z-score (intervention group 0.06 [−0.17; 0.29] vs. controls −0.18 [−0.43; 0.05]), in the percentage of overweight or obese children (10.9% vs. 6.7%), in other anthropometrics, or in body composition values by DEXA. Outcomes between children from the RCT and the reference group children were not significantly different.
The RCT with lifestyle intervention in obese pregnant women did not result in any detectable effect on offspring anthropometrics or body composition by DEXA at 2.8 years of age. This may reflect the limited difference in GWG between intervention and control groups. Offspring of obese mothers from the RCT were comparable to offspring of mothers with a normal BMI.
Trial registration NCT00530439, NCT01918319 and NCT01918423. URL: NCT00530439?term = NCT00530439&rank = 1, NCT01918319?term = NCT00530439&rank = 2 and NCT01918423?term = NCT00530439&rank = 3.
PMCID: PMC3933548  PMID: 24586896
2.  A flow cytometric method for characterization of circulating cell-derived microparticles in plasma 
Journal of Extracellular Vesicles  2014;3:10.3402/jev.v3.20795.
Background and aim
Previous studies on circulating microparticles (MPs) indicate that the majority of MPs are of a size below the detection limit of most standard flow cytometers. The objective of the present study was to establish a method to analyze MP subpopulations above the threshold of detection of a new generation BD FACSAria™ III digital flow cytometer.
We analyzed MP subpopulations in plasma from 24 healthy individuals (9 males and 15 females). MPs were identified according to their size (<1.0-µm), by Lactadherin-FITC labelling, and by exposure of cell-specific markers. The sensitivity of the flow cytometer was tested against that of a previous-generation instrument FC500. Reproducibility of the FACSAria and our set-up was investigated, and the percentage of phosphatidylserine (PS) exposing MPs binding Lactadherin was determined.
By using a flow cytometric approach we identified and quantitated MPs derived from platelets, monocytes, erythrocytes and endothelial cells. In addition, levels of tissue factor-positive MPs were determined. The FACSAria demonstrated improved sensitivity and increased MP detection range compared to the FC500 instrument. The reproducibility of PS+PMP and PS+MP measurements was 11.7 and 23.2%, respectively. When expressed as a percentage of total MPs, the PS-positive MP population represented 15.1±5.5%, and PS-positive MPs were significantly increased in men.
We have established a method to measure MPs above the detection limit of a new generation flow cytometer and derived from a number of cell-types in a healthy population of men and women.
PMCID: PMC3916676  PMID: 24511371
extracellular vesicles; flow cytometry; coincidence occurrence; platelet-derived; monocyte-derived; endothelial cell-derived; erythrocyte-derived; tissue-factor; lactadherin
3.  FA1 Induces Pro-Inflammatory and Anti-Adipogenic Pathways/Markers in Human Myotubes Established from Lean, Obese, and Type 2 Diabetic Subjects but Not Insulin Resistance 
Aims: Delta like 1/fetal antigen 1 (Dlk1/FA1) is a protein secreted by hormone producing cells in adult human and mice that is known to inhibit adipogenesis. Recent studies demonstrated the role of Dlk1/FA1 in inducing insulin resistance in mice. To investigate the involvement of circulating Dlk1/FA1 in insulin resistance and type 2 diabetes in human subjects, we studied the effects of chronic FA1 on the intermediary metabolism in myotubes established from lean, obese, and type 2 diabetic (T2D) subjects.
Methods: Myotube cultures were established from lean and obese control subjects, and obese T2D subjects and treated with soluble FA1 for 4 days supplemented with/without palmitate (PA). Lipid- and glucose metabolism were studied with labeled precursors while quantitative expression of genes was analyzed using real-time PCR.
Results: Diabetic myotubes express significantly reduced insulin stimulated glucose metabolism compared to lean myotubes and a significantly decreased basal PA oxidation. Chronic FA1 exposure did not affect the intermediary metabolism in myotubes. Insulin sensitivity of glucose and lipid metabolism was not affected by chronic FA1 exposure in myotubes established from lean, obese, and T2D subjects. Instead, chronic FA1 exposure induced pro-inflammatory cytokines expression (IL-6 and CCL2) in association with reducing adipogenic markers (ADD1, AP2, CD36, and PPARg2) in myotubes. Consistent with this observation, addition of FA1 to cultured myotubes was show to significantly inhibit their differentiation into adipocyte.
Conclusion: Our results exclude direct effects of FA1 on glucose and lipid metabolism in cultured myotubes established from lean, obese, and T2D subjects. Therefore, the pathogenesis of FA1-induced IR might mainly be mediated via the FA1-induced stimulation of pro-inflammatory cytokines, which on turn inhibit adipogenesis in human myotubes.
PMCID: PMC3617402  PMID: 23577002
Dlk1; FA1; Pref-1; insulin resistance; human myotubes; obesity; skeletal muscle; type 2 diabetes
4.  Genome-Wide Analysis of DNA Methylation Differences in Muscle and Fat from Monozygotic Twins Discordant for Type 2 Diabetes 
PLoS ONE  2012;7(12):e51302.
Monozygotic twins discordant for type 2 diabetes constitute an ideal model to study environmental contributions to type 2 diabetic traits. We aimed to examine whether global DNA methylation differences exist in major glucose metabolic tissues from these twins.
Methodology/Principal Findings
Skeletal muscle (n = 11 pairs) and subcutaneous adipose tissue (n = 5 pairs) biopsies were collected from 53–80 year-old monozygotic twin pairs discordant for type 2 diabetes. DNA methylation was measured by microarrays at 26,850 cytosine-guanine dinucleotide (CpG) sites in the promoters of 14,279 genes. Bisulfite sequencing was applied to validate array data and to quantify methylation of intergenic repetitive DNA sequences. The overall intra-pair variation in DNA methylation was large in repetitive (LINE1, D4Z4 and NBL2) regions compared to gene promoters (standard deviation of intra-pair differences: 10% points vs. 4% points, P<0.001). Increased variation of LINE1 sequence methylation was associated with more phenotypic dissimilarity measured as body mass index (r = 0.77, P = 0.007) and 2-hour plasma glucose (r = 0.66, P = 0.03) whereas the variation in promoter methylation did not associate with phenotypic differences. Validated methylation changes were identified in the promoters of known type 2 diabetes-related genes, including PPARGC1A in muscle (13.9±6.2% vs. 9.0±4.5%, P = 0.03) and HNF4A in adipose tissue (75.2±3.8% vs. 70.5±3.7%, P<0.001) which had increased methylation in type 2 diabetic individuals. A hypothesis-free genome-wide exploration of differential methylation without correction for multiple testing identified 789 and 1,458 CpG sites in skeletal muscle and adipose tissue, respectively. These methylation changes only reached some percentage points, and few sites passed correction for multiple testing.
Our study suggests that likely acquired DNA methylation changes in skeletal muscle or adipose tissue gene promoters are quantitatively small between type 2 diabetic and non-diabetic twins. The importance of methylation changes in candidate genes such as PPARGC1A and HNF4A should be examined further by replication in larger samples.
PMCID: PMC3519577  PMID: 23251491
5.  A prospective study of twinning and perinatal mortality in urban Guinea-Bissau 
Despite twinning being common in Africa, few prospective twin studies have been conducted. We studied twinning rate, perinatal mortality and the clinical characteristics of newborn twins in urban Guinea-Bissau.
The study was conducted at the Bandim Health Project (BHP), a health and demographic surveillance site in Bissau, the capital of Guinea-Bissau. The cohort included all newborn twins delivered at the National Hospital Simão Mendes and in the BHP study area during the period September 2009 to August 2011 as well as singleton controls from the BHP study area. Data regarding obstetric history and pregnancy were collected at the hospital. Live children were examined clinically. For a subset of twin pairs zygosity was established by using genetic markers.
Out of the 5262 births from mothers included in the BHP study area, 94 were twin births, i.e. a community twinning rate of 18/1000. The monozygotic rate was 3.4/1000. Perinatal mortality among twins vs. singletons was 218/1000 vs. 80/1000 (RR = 2.71, 95% CI: 1.93-3.80). Among the 13783 hospital births 388 were twin births (28/1000). The hospital perinatal twin mortality was 237/1000.
Birth weight < 2000g (RR = 4.24, CI: 2.39-7.51) and caesarean section (RR = 1.78, CI: 1.06-2.99) were significant risk factors for perinatal twin mortality. Male sex (RR = 1.38, CI: 0.97-1.96), unawareness of twin pregnancy (RR = 1.64, CI: 0.97-2.78) and high blood pressure during pregnancy (RR = 1.77, CI: 0.88-3.57) were borderline non-significant. Sixty-five percent (245/375) of the mothers who delivered at the hospital were unaware of their twin pregnancy.
Twins had a very high perinatal mortality, three-fold higher than singletons. A birth weight < 2000g was the strongest risk factor for perinatal death, and unrecognized twin pregnancy was common. Urgent interventions are needed to lower perinatal twin mortality in Guinea-Bissau.
PMCID: PMC3534574  PMID: 23216795
6.  The LiP (Lifestyle in Pregnancy) Study 
Diabetes Care  2011;34(12):2502-2507.
To study the effects of lifestyle intervention on gestational weight gain (GWG) and obstetric outcomes.
The LiP (Lifestyle in Pregnancy) study was a randomized controlled trial in 360 obese women allocated in early pregnancy to lifestyle intervention or control. The intervention program included dietary guidance, free membership in fitness centers, physical training, and personal coaching.
A total of 360 obese pregnant women were included, and 304 (84%) were followed up until delivery. The intervention group had a significantly lower median (range) GWG compared with the control group of 7.0 (4.7–10.6) vs. 8.6 kg (5.7–11.5; P = 0.01). The Institute of Medicine (IOM) recommendations on GWG were exceeded in 35.4% of women in the intervention group compared with 46.6% in the control group (P = 0.058). Overall, the obstetric outcomes between the two groups were not significantly different.
Lifestyle intervention in pregnancy resulted in limited GWG in obese pregnant women. Overall obstetric outcomes were similar in the two groups. Lifestyle intervention resulted in a higher adherence to the IOM weight gain recommendations; however, a significant number of women still exceeded the upper threshold.
PMCID: PMC3220844  PMID: 21972411
7.  The Danish Centre for Strategic Research in Type 2 Diabetes (DD2) Project: rationale and planned nationwide studies of genetic predictors, physical exercise, and individualized pharmacological treatment 
Clinical Epidemiology  2012;4(Suppl 1):7-13.
Here we provide an overview of the rationale and methods of a series of planned population based studies within the Danish Centre for Strategic Research in Type 2 Diabetes (DD2) Project. The project aims to support and evaluate ongoing political and administrative efforts to implement nationwide guidelines for maintaining metabolic control in newly diagnosed type 2 diabetes (T2D) patients to prevent diabetic complications and improve quality of life. The DD2 is designed as a prospective cohort study (collection of epidemiological data) supplemented by randomized clinical intervention trials (on physical exercise and individualized pharmacological treatment) and the establishment of a biobank comprised of material from a large number of newly diagnosed T2D patients. Inclusion of the majority of newly diagnosed T2D patients as they are diagnosed at their general practitioner or diabetes hospital outpatient clinics and entered into the DD2 cohort will establish a nationwide database comprising a large number of future incident cases of T2D in Denmark. These cases will form the project cohort of the DD2. Within the first 6 months of diagnosis, all patients will be invited to contribute to a biobank of DNA, plasma, urine, and tissue sampling. The DNA biobank will enable future studies of the effect of pharmacological treatment and outcome in subsets of patients with specific genetic risk profiles covering disease etiology and specific drug kinetics and metabolism. We will also perform two clinical intervention trials examining: the effectiveness of physical exercise on diabetes-related outcomes and the impact of trial outcomes on individualized pharmacological treatment. Moreover, the DD2 will serve as a platform for testing and developing new antidiabetic drugs. All together, we expect this study to contribute to substantially improved diabetes care in T2D patients locally and abroad.
PMCID: PMC3469285  PMID: 23071406
type 2 diabetes; prognosis; intervention; physical exercise
8.  The Danish Centre for Strategic Research in Type 2 Diabetes (DD2) study: expected outcome from the DD2 project and two intervention studies 
Clinical Epidemiology  2012;4(Suppl 1):21-26.
The overall aim of the Danish Centre for Strategic Research in Type 2 Diabetes (DD2) is to near-normalize metabolic control in newly diagnosed patients with type 2 diabetes (T2D) using an individualized treatment approach. We hypothesize that this will not only prevent complications and improve quality of life for T2D patients but also result in increased cost efficiency compared with current treatment modalities. This paper provides an overview of the expected outcomes from DD2, focusing on the two main intervention studies. The main data for the DD2 project are collected during patient enrollment and stored using the individual civil registration number. This enables subsequent linking to other national databases where supplemental data can be obtained. All data will be used for designing treatment guidelines and continuously monitoring the development of diabetic complications, thereby obtaining knowledge about predictors for the long-term outcome and identifying targets for new interventions. Further data are being collected from two intervention studies. The aim of the first intervention study is to improve T2D treatment using an individualized treatment modality optimizing medication according to individual metabolic responses and phenotypic characteristics. The aim of the second intervention study is to develop an evidence-based training protocol to be implemented as a treatment modality for T2D and used for initiating lifelong changes in physical activity levels in patients with T2D. An initial pilot study evaluating an interval-based walking protocol is ongoing, and preliminary results indicate that this protocol is an optimal “free-living” training intervention. An initial health-economic analysis will also be performed as a basis for analysis of the data collected during the project. A cost-benefit analysis of the two intervention studies will be conducted. The DD2 project is expected to lead to improved treatment modalities and increased knowledge about existing treatment guidelines, and will also provide a solid base for health-economic decision-making.
PMCID: PMC3469286  PMID: 23071408
type 2 diabetes; epidemiological methods; exercise intervention; clinical intervention; individualized treatment; treatment guidelines
10.  A prospective randomised cross-over study of the effect of insulin analogues and human insulin on the frequency of severe hypoglycaemia in patients with type 1 diabetes and recurrent hypoglycaemia (the HypoAna trial): study rationale and design 
Severe hypoglycaemia still represents a significant problem in insulin-treated diabetes. Most patients do not experience severe hypoglycaemia often. However, 20% of patients with type 1 diabetes experience recurrent severe hypoglycaemia corresponding to at least two episodes per year. The effect of insulin analogues on glycaemic control has been documented in large trials, while their effect on the frequency of severe hypoglycaemia is less clear, especially in patients with recurrent severe hypoglycaemia. The HypoAna Trial is designed to investigate whether short-acting and long-acting insulin analogues in comparison with human insulin are superior in reducing the occurrence of severe hypoglycaemic episodes in patients with recurrent hypoglycaemia. This paper reports the study design of the HypoAna Trial.
The study is a Danish two-year investigator-initiated, prospective, randomised, open, blinded endpoint (PROBE), multicentre, cross-over trial investigating the effect of insulin analogues versus human insulin on the frequency of severe hypoglycaemia in subjects with type 1 diabetes. Patients are randomised to treatment with basal-bolus therapy with insulin detemir / insulin aspart or human NPH insulin / human regular insulin in random order. The major inclusion criterion is history of two or more episodes of severe hypoglycaemia in the preceding year.
In contrast to almost all other studies in this field the HypoAna Trial includes only patients with major problems with hypoglycaemia. The HypoAna Trial will elucidate whether basal-bolus regimen with short-acting and long-acting insulin analogues in comparison with human insulin are superior in reducing occurrence of severe hypoglycaemic episodes in hypoglycaemia prone patients with type 1 diabetes. NCT00346996.
PMCID: PMC3433358  PMID: 22727048
Type 1 diabetes; Severe hypoglycaemia; Human insulin; Insulin analogues; PROBE
11.  Self-Management Support to People with Type 2 Diabetes - A comparative study of Kaiser Permanente and the Danish Healthcare System 
Self-management support is considered to be an essential part of diabetes care. However, the implementation of self-management support within healthcare settings has appeared to be challenging and there is increased interest in “real world” best practice examples to guide policy efforts. In order to explore how different approaches to diabetes care and differences in management structure influence the provision of SMS we selected two healthcare systems that have shown to be comparable in terms of budget, benefits and entitlements. We compared the extent of SMS provided and the self-management behaviors of people living with diabetes in Kaiser Permanente (KP) and the Danish Healthcare System (DHS).
Self-administered questionnaires were used to collect data from a random sample of 2,536 individuals with DM from KP and the DHS in 2006–2007 to compare the level of SMS provided in the two systems and identify disparities associated with educational attainment. The response rates were 75 % in the DHS and 56 % in KP. After adjusting for gender, age, educational level, and HbA1c level, multiple linear regression analyses determined the level of SMS provided and identified disparities associated with educational attainment.
Receipt of SMS varied substantially between the two systems. More people with diabetes in KP reported receiving all types of SMS and use of SMS tools compared to the DHS (p < .0001). Less than half of all respondents reported taking diabetes medication as prescribed and following national guidelines for exercise.
Despite better SMS support in KP compared to the DHS, self-management remains an under-supported area of care for people receiving care for diabetes in the two health systems. Our study thereby suggests opportunity for improvements especially within the Danish healthcare system and systems adopting similar SMS support strategies.
PMCID: PMC3441680  PMID: 22697597
Self-management support; Type 2 diabetes mellitus; Health system; International comparison
12.  Incidence and Prevalence of Unrecognized Myocardial Infarction in People With Diabetes 
Diabetes Care  2011;34(6):1394-1396.
To examine the prevalence and incidence of unrecognized myocardial infarction in a contemporary population with type 2 diabetes.
We performed a retrospective analysis of the electrocardiograms (ECGs) recorded at baseline and after 2 years for the first 1,004 type 2 diabetic individuals to be randomized in the Rosiglitazone Evaluated for Cardiac Outcomes and Regulation of Glycemia in Diabetes (RECORD) study.
ECGs suitable for analysis were obtained from 669 participants. The prevalence of unrecognized Q-wave myocardial infarction at baseline was 1.9% (n = 13). The incidence of unrecognized Q-wave myocardial infarction at the end of 2 years of follow-up was 1.5/1,000-person-years (n = 2). One-third (13 of 39) of prevalent and one-quarter (2 of 8) of incident myocardial infarctions were unrecognized.
Although the prevalence and incidence of myocardial infarction was low, unrecognized Q-wave myocardial infarctions made up a substantial proportion of all events.
PMCID: PMC3114348  PMID: 21562320
13.  Pharmacological Treatment of the Pathogenetic Defects in Type 2 Diabetes 
Diabetes Care  2010;34(1):27-33.
To determine the effect of treatment with insulin aspart compared with NPH insulin, together with metformin/placebo and rosiglitazone/placebo. The hypothesis was that combined correction of major pathogenetic defects in type 2 diabetes would result in optimal glycemic control.
This study was a 2-year investigator-driven randomized partly placebo-controlled multicenter trial in 371 patients with type 2 diabetes on at least oral antiglycemic treatment. Patients were assigned to one of eight treatment groups in a factorial design with insulin aspart at mealtimes versus NPH insulin once daily at bedtime, metformin twice daily versus placebo, and rosiglitazone twice daily versus placebo. The main outcome measurement was change in A1C.
A1C decreased more in patients treated with insulin aspart compared with NPH (−0.41 ± 0.10%, P < 0.001). Metformin decreased A1C compared with placebo (−0.60 ± 0.10%, P < 0.001), as did rosiglitazone (−0.55 ± 0.10%, P < 0.001). Triple therapy (rosiglitazone, metformin, and any insulin) resulted in a greater reduction in A1C than rosiglitazone plus insulin (−0.50 ± 0.14%, P < 0.001) and metformin plus insulin (−0.45 ± 0.14%, P < 0.001). Aspart was associated with a higher increase in body weight (1.6 ± 0.6 kg, P < 0.01) and higher incidence of mild daytime hypoglycemia (4.9 ± 7.5 vs. 1.7 ± 5.4 number/person/year, P < 0.001) compared with NPH.
Insulin treatment of postprandial hyperglycemia results in lower A1C than treatment of fasting hyperglycemia, at the expense of higher body weight and hypoglycemic episodes. However, insulin therapy has to be combined with treatment of both peripheral and liver insulin resistance to normalize blood glucose, and in this case, the insulin regimen is less important.
PMCID: PMC3005476  PMID: 20929990
14.  Plasma osteoprotegerin is related to carotid and peripheral arterial disease, but not to myocardial ischemia in type 2 diabetes mellitus 
Cardiovascular disease (CVD) is frequent in type 2 diabetes mellitus patients due to accelerated atherosclerosis. Plasma osteoprotegerin (OPG) has evolved as a biomarker for CVD. We examined the relationship between plasma OPG levels and different CVD manifestations in type 2 diabetes.
Type 2 diabetes patients without known CVD referred consecutively to a diabetes clinic for the first time (n = 305, aged: 58.6 ± 11.3 years, diabetes duration: 4.5 ± 5.3 years) were screened for carotid arterial disease, peripheral arterial disease, and myocardial ischemia by means of carotid artery ultrasonography, peripheral ankle and toe systolic blood pressure measurements, and myocardial perfusion scintigraphy (MPS). In addition, plasma OPG concentrations and other CVD-related markers were measured.
The prevalence of carotid arterial disease, peripheral arterial disease, and myocardial ischemia was 42%, 15%, and 30%, respectively. Plasma OPG was significantly increased in patients with carotid and peripheral arterial disease compared to patients without (p < 0.001, respectively), however, this was not the case for patients with myocardial ischemia versus those without (p = 0.71). When adjusted for age, HbA1c and U-albumin creatinine ratio in a multivariate logistic regression analysis, plasma OPG remained strongly associated with carotid arterial disease (adjusted OR: 2.12; 95% CI: 1.22-3.67; p = 0.008), but not with peripheral arterial disease or myocardial ischemia.
Increased plasma OPG concentration is associated with carotid and peripheral arterial disease in patients with type 2 diabetes, whereas no relation is observed with respect to myocardial ischemia on MPS. The reason for this discrepancy is unknown.
Trial registration number
at NCT00298844
PMCID: PMC3163516  PMID: 21838881
15.  The SNARE Protein SNAP23 and the SNARE-Interacting Protein Munc18c in Human Skeletal Muscle Are Implicated in Insulin Resistance/Type 2 Diabetes 
Diabetes  2010;59(8):1870-1878.
Our previous studies suggest that the SNARE protein synaptosomal-associated protein of 23 kDa (SNAP23) is involved in the link between increased lipid levels and insulin resistance in cardiomyocytes. The objective was to determine whether SNAP23 may also be involved in the known association between lipid accumulation in skeletal muscle and insulin resistance/type 2 diabetes in humans, as well as to identify a potential regulator of SNAP23.
We analyzed skeletal muscle biopsies from patients with type 2 diabetes and healthy, insulin-sensitive control subjects for expression (mRNA and protein) and intracellular localization (subcellular fractionation and immunohistochemistry) of SNAP23, and for expression of proteins known to interact with SNARE proteins. Insulin resistance was determined by a euglycemic hyperinsulinemic clamp. Potential mechanisms for regulation of SNAP23 were also investigated in the skeletal muscle cell line L6.
We showed increased SNAP23 levels in skeletal muscle from patients with type 2 diabetes compared with that from lean control subjects. Moreover, SNAP23 was redistributed from the plasma membrane to the microsomal/cytosolic compartment in the patients with the type 2 diabetes. Expression of the SNARE-interacting protein Munc18c was higher in skeletal muscle from patients with type 2 diabetes. Studies in L6 cells showed that Munc18c promoted the expression of SNAP23.
We have translated our previous in vitro results into humans by showing that there is a change in the distribution of SNAP23 to the interior of the cell in skeletal muscle from patients with type 2 diabetes. We also showed that Munc18c is a potential regulator of SNAP23.
PMCID: PMC2911056  PMID: 20460426
16.  Microalbuminuria, Preeclampsia, and Preterm Delivery in Pregnant Women With Type 1 Diabetes 
Diabetes Care  2009;33(1):90-94.
To study the association between microalbuminuria and development of preeclampsia and preterm delivery in pregnant women with type 1 diabetes.
This was a population-based prospective study in 846 normoalbuminuric or microalbuminuric women with type 1 diabetes without antihypertensive treatment in early pregnancy. Data were collected prospectively by one to three caregivers in each center and reported to a central registry.
The prevalence of microalbuminuria in the first trimester was 10%, median diabetes duration was 11 years, and third-trimester A1C was 6.6%. The frequencies of preeclampsia and preterm delivery before 34 weeks in the microalbuminuric group were 40 and 13%, both significantly higher than those in the normoalbuminuric group (12 and 6%, respectively, P < 0.001). After adjustments for possible confounders, significant predictors for development of preeclampsia were microalbuminuria (odds ratio 4.0 [95% CI]), nulliparity (3.1 [1.9–5.1]), and third-trimester A1C (1.3 [1.1–1.5] per 1% increase). Delivery before 34 weeks was associated with early microalbuminuria in univariate analyses, but in multivariate analyses A1C was the only significant predictor of this outcome. Preeclampsia was associated with a threefold higher risk of delivery before 34 weeks.
The presence of microalbuminuria in early pregnancy is associated with a fourfold increased risk of developing preeclampsia. A1C values during pregnancy are highly predictive of both preeclampsia and preterm delivery. Future research with antihypertensive treatment in normotensive, microalbuminuric pregnant women to prevent preeclampsia is proposed.
PMCID: PMC2797993  PMID: 19846800
17.  Insulin Resistance Is Not Conserved in Myotubes Established from Women with PCOS 
PLoS ONE  2010;5(12):e14469.
Polycystic ovary syndrome (PCOS) is the most common endocrine disorder among premenopausal women, who often develop insulin resistance. We tested the hypothesis that insulin resistance in skeletal muscle of patients with polycystic ovary syndrome (PCOS) is an intrinsic defect, by investigating the metabolic characteristics and gene expression of in vitro differentiated myotubes established from well characterized PCOS subjects.
Using radiotracer techniques, RT-PCR and enzyme kinetic analysis we examined myotubes established from PCOS subjects with or without pioglitazone treatment, versus healthy control subjects who had been extensively metabolically characterized in vivo. Results Myotubes established from PCOS and matched control subjects comprehensively expressed all insulin-sensitive biomarkers; glucose uptake and oxidation, glycogen synthesis and lipid uptake. There were no significant differences between groups either at baseline or during acute insulin stimulation, although in vivo skeletal muscle was insulin resistant. In particular, we found no evidence for defects in insulin-stimulated glycogen synthase activity between groups. Myotubes established from PCOS patients with or without pioglitazone treatment also showed no significant differences between groups, neither at baseline nor during acute insulin stimulation, although in vivo pioglitazone treatment significantly improved insulin sensitivity. Consistently, the myotube cultures failed to show differences in mRNA levels of genes previously demonstrated to differ in PCOS patients with or without pioglitazone treatment (PLEK, SLC22A16, and TTBK).
These results suggest that the mechanisms governing insulin resistance in skeletal muscle of PCOS patients in vivo are not primary, but rather adaptive.
Trial Registration NCT00145340
PMCID: PMC3012693  PMID: 21209881
18.  Peri-Conceptional A1C and Risk of Serious Adverse Pregnancy Outcome in 933 Women With Type 1 Diabetes 
Diabetes Care  2009;32(6):1046-1048.
To study the association between peri-conceptional A1C and serious adverse pregnancy outcome (congenital malformations and perinatal mortality).
Prospective data were collected in 933 singleton pregnancies complicated by type 1 diabetes.
The risk of serious adverse outcome at different A1C levels was compared with the background population. The risk was significantly higher when peri-conceptional A1C exceeded 6.9%, and the risk tended to increase gradually with increasing A1C. Women with A1C exceeding 10.4% had a very high risk of 16%. Congenital malformation rate increased significantly at A1C above 10.4%, whereas perinatal mortality was increased even at A1C below 6.9%.
These results support recent guidelines of preconceptional A1C levels <7% in women with type 1 diabetes.
PMCID: PMC2681038  PMID: 19265024
19.  Increased Risk of Type 2 Diabetes in Elderly Twins 
Diabetes  2009;58(6):1350-1355.
Genetic susceptibility, low birth weight (LBW), and aging are key etiological factors in the development of type 2 diabetes. LBW is common among twins. It is unknown whether twin status per se is associated with risk of type 2 diabetes, and valid concordance rates of type 2 diabetes in twins on a lifetime perspective are lacking.
A clinical study was done on a population-based cohort of same-sex elderly monozygotic (MZ) and dizygotic (DZ) twins (n = 297) and singleton control subjects (C) (n = 71) including measures of anthropometry and glucose tolerance. In addition, type 2 diabetes incidence cases in twins (n = 626) and singletons (n = 553) were identified through the National Diabetes Register.
Twins were more abdominally obese, insulin resistant, and glucose intolerant, as evidenced by a higher A1C (%) (means ± SD) (MZ: 6.0 ± 1.0, DZ: 5.8 ± 0.7, C: 5.6 ± 0.3, P = 0.004) and 120-min post–oral glucose tolerance test plasma glucose levels (in mmol/l) (MZ: 8.6 ± 4.6, DZ: 8.4 ± 3.9, C: 6.8 ± 2.4, P = 0.003) compared with singletons. Importantly, twins had a higher prevalence of type 2 diabetes (MZ: 17.5% [95% CI 14.4–20.6], DZ: 15.7% [13.1–18.3], C: 5.6% [3.0–8.2], P = 0.03) together with a 60% higher incidence rate of type 2 diabetes compared with singletons. Cumulative concordance rates of type 2 diabetes to the age of 84 years were similar among elderly MZ (0.76 [0.68–0.84]) and DZ (0.71 [0.63–0.78]) twins.
Twin status per se is associated with abdominal obesity, insulin resistance, and increased prevalence of type 2 diabetes in elderly twins. The data support a quantitatively significant impact of the fetal environment as opposed to genetics on risk of type 2 diabetes.
PMCID: PMC2682675  PMID: 19336677
20.  Heart failure events with rosiglitazone in type 2 diabetes: data from the RECORD clinical trial 
European Heart Journal  2010;31(7):824-831.
Thiazolidinediones are insulin sensitizers, and are associated with fluid retention and increased risk of heart failure (HF) in people with type 2 diabetes. We assessed fatal and non-fatal HF events and their outcome, and identified HF predictors in the RECORD (Rosiglitazone Evaluated for Cardiac Outcomes and Regulation of glycaemia in Diabetes) trial population.
Methods and results
In a multicentre, open-label study, we randomized 4447 people with type 2 diabetes on metformin or sulfonylurea monotherapy with a mean HbA1c of 7.9% to add-on rosiglitazone (n = 2220) or to a combination of metformin and sulfonylurea (n = 2227) and followed them over 5.5 years on average. Heart failure hospitalizations and deaths were adjudicated by a Clinical Endpoint Committee using pre-specified criteria. Independent predictors of HF events were identified out of a group of 30 pre-specified clinical, demographic, and biological variables. In the rosiglitazone group, the risk of HF death or hospitalization was doubled: HR = 2.10 (95% CI, 1.35–3.27): the excess HF event rate was 2.6 (1.1–4.1) per 1000 person-years. An excess in HF deaths was observed (10 vs. two), including four HF deaths as first HF events. By contrast, there was no increase in cardiovascular mortality or hospitalization (HR = 0.99, 95% CI, 0.85–1.16) or in cardiovascular deaths (60 vs. 71). Independent predictors of HF were rosiglitazone assignment, age, urinary albumin : creatinine ratio, body mass index, and systolic blood pressure at baseline. A history of previous cardiovascular disease was not predictive of HF. Duration of HF hospitalization and rate of HF re-hospitalization were similar in the two groups.
These findings confirm the increased risk of HF events in people treated with rosiglitazone and support the recommendation that this agent should not continue to be used in people developing symptomatic HF while using the medication. Close follow-up for the risk of HF should be offered to elderly people, people with markedly increased body mass index, people with microalbuminuria/proteinuria, and people with increased systolic blood pressure.
PMCID: PMC2848325  PMID: 20118174
Type 2 diabetes; Thiazolidinediones; Heart failure; Clinical trials
21.  Pioglitazone Enhances Mitochondrial Biogenesis and Ribosomal Protein Biosynthesis in Skeletal Muscle in Polycystic Ovary Syndrome 
PLoS ONE  2008;3(6):e2466.
Insulin resistance is a common metabolic abnormality in women with PCOS and leads to an elevated risk of type 2 diabetes. Studies have shown that thiazolidinediones (TZDs) improve metabolic disturbances in PCOS patients. We hypothesized that the effect of TZDs in PCOS is, in part, mediated by changes in the transcriptional profile of muscle favoring insulin sensitivity. Using Affymetrix microarrays, we examined the effect of pioglitazone (30 mg/day for 16 weeks) on gene expression in skeletal muscle of 10 obese women with PCOS metabolically characterized by a euglycemic-hyperinsulinemic clamp. Moreover, we explored gene expression changes between these PCOS patients before treatment and 13 healthy women. Treatment with pioglitazone improved insulin-stimulated glucose metabolism and plasma adiponectin, and reduced fasting serum insulin (all P<0.05). Global pathway analysis using Gene Map Annotator and Pathway Profiler (GenMAPP 2.1) and Gene Set Enrichment Analysis (GSEA 2.0.1) revealed a significant upregulation of genes representing mitochondrial oxidative phosphorylation (OXPHOS), ribosomal proteins, mRNA processing reactome, translation factors, and proteasome degradation in PCOS after pioglitazone therapy. Quantitative real-time PCR suggested that upregulation of OXPHOS genes was mediated by an increase in PGC-1α expression (P<0.05). Pretreatment expression of genes representing OXPHOS and ribosomal proteins was down-regulated in PCOS patients compared to healthy women. These data indicate that pioglitazone therapy restores insulin sensitivity, in part, by a coordinated upregulation of genes involved in mitochondrial OXPHOS and ribosomal protein biosynthesis in muscle in PCOS. These transcriptional effects of pioglitazone may contribute to prevent the onset of type 2 diabetes in these women.
PMCID: PMC2413008  PMID: 18560589
22.  Effect of the addition of rosiglitazone to metformin or sulfonylureas versus metformin/sulfonylurea combination therapy on ambulatory blood pressure in people with type 2 diabetes: A randomized controlled trial (the RECORD study) 
Hypertension and type 2 diabetes are common co-morbidities. Preliminary studies suggest that thiazolidinediones reduce blood pressure (BP). We therefore used ambulatory BP to quantify BP lowering at 6–12 months with rosiglitazone used in combination with metformin or sulfonylureas compared to metformin and sulfonylureas in people with type 2 diabetes.
Participants (n = 759) in the multicentre RECORD study were studied. Those taking metformin were randomized (open label) to add-on rosiglitazone or sulfonylureas, and those on sulfonylurea to add-on rosiglitazone or metformin.
24-Hour ambulatory BP was measured at baseline, 6 months and 12 months. At 6 and 12 months, reductions in 24-hour ambulatory systolic BP (sBP) were greater with rosiglitazone versus metformin (difference at 6 months 2.7 [95% CI 0.5–4.9] mmHg, p = 0.015; 12 months 2.5 [95% CI 0.2–4.8] mmHg, p = 0.031). Corresponding changes for ambulatory diastolic BP (dBP) were comparable (6 months 2.7 [95% CI 1.4–4.0] mmHg, p < 0.001; 12 months 3.1 [95% CI 1.8–4.5] mmHg, p < 0.001). Similar differences were observed for rosiglitazone versus sulfonylureas at 12 months (sBP 2.7 [95% CI 0.5–4.9] mmHg, p = 0.016; dBP 2.1 [95% CI 0.7–3.4] mmHg, p = 0.003), but differences were smaller and/or not statistically significant at 6 months (sBP 1.5 [95% CI -0.6 to 3.6] mmHg, p = NS; dBP 1.3 [95% CI 0.0–2.5] mmHg, p = 0.049). Changes in BP were not accompanied by compensatory increases in heart rate, did not correlate with basal insulin sensitivity estimates and were not explained by changes in antihypertensive therapy between the various strata.
When added to metformin or a sulfonylurea, 12-month treatment with rosiglitazone reduces ambulatory BP to a greater extent than when metformin and a sulfonylurea are combined.
Trial registration
PMCID: PMC2390513  PMID: 18435852
23.  Counting drugs to understand the disease: The case of measuring the diabetes epidemic 
Diabetes prevalence increases globally with severe consequences for afflicted individuals and societies. Data on diabetes incidence and diabetes related mortality on a population level are, however, scarce. As an alternative to dedicated studies it has been suggested to use pharmacoepidemiological databases that are readily available, at least in the Nordic countries.
For all 470,000 inhabitants in Funen County, Denmark, in the period 1992–2003, data on gender, date of birth, death and migration to and from the county, and any filled prescriptions of an anti-diabetic medication was obtained from the Odense Pharmaco-Epidemiological Database.
Prevalence odds for use of an anti-diabetic medication rose annually 3.5% (95% confidence interval: 3.1%, 3.9%) for females, 4.5% (4.0%, 4.9%) for males. Corresponding incidence rates annually rose 4.8% (3.8%, 5.8%) for females, 4.5% (3.5%, 5.4%) for males. Mortality rates among treated annually declined 2.8% (1.4%, 4.1%) among females, 2.2% (0.9%, 3.5%) among males. The disequilibrium in absolute numbers between incidence and mortality among treated was the main driver for the increasing prevalence, while concurrent trends in incidence and diabetes related mortality only marginally affected prevalence trends. Trend estimates were insensitive to varying the length of the run-in period used for determining treatment status, except when using the naive and methodologically flawed run-in period of variable length.
While pharmacoepidemiological databases provide a useful tool for monitoring pharmacologically treated diabetes, a dedicated diabetes database covering all prevalents and incidents is needed for a more detailed analysis of underlying causes and trends.
PMCID: PMC1805738  PMID: 17313683
24.  Multiple environmental and genetic factors influence skeletal muscle PGC-1α and PGC-1β gene expression in twins 
Journal of Clinical Investigation  2004;114(10):1518-1526.
Genetic and environmental factors contribute to age-dependent susceptibility to type 2 diabetes. Recent studies have reported reduced expression of PPARγ coactivator 1α (PGC-1α) and PGC-1β genes in skeletal muscle from type 2 diabetic patients, but it is not known whether this is an inherited or acquired defect. To address this question we studied expression of these genes in muscle biopsies obtained from young and elderly dizygotic and monozygotic twins without known diabetes before and after insulin stimulation and related the expression to a Gly482Ser variant in the PGC-1α gene. Insulin increased and aging reduced skeletal muscle PGC-1α and PGC-1β mRNA levels. This age-dependent decrease in muscle gene expression was partially heritable and influenced by the PGC-1α Gly482Ser polymorphism. In addition, sex, birth weight, and aerobic capacity influenced expression of PGC-1α in a complex fashion. Whereas expression of PGC-1α in muscle was positively related to insulin-stimulated glucose uptake and oxidation, PGC-1β expression was positively related to fat oxidation and nonoxidative glucose metabolism. We conclude that skeletal muscle PGC-1α and PGC-1β expression are stimulated by insulin and reduced by aging. The data also suggest different regulatory functions for PGC-1α and PGC-1β on glucose and fat oxidation in muscle cells. The finding that the age-dependent decrease in the expression of these key genes regulating oxidative phosphorylation is under genetic control could provide an explanation by which an environmental trigger (age) modifies genetic susceptibility to type 2 diabetes.
PMCID: PMC525741  PMID: 15546003
25.  Randomised controlled trial of structured personal care of type 2 diabetes mellitus 
BMJ : British Medical Journal  2001;323(7319):970.
To assess the effect of a multifaceted intervention directed at general practitioners on six year mortality, morbidity, and risk factors of patients with newly diagnosed type 2 diabetes.
Pragmatic, open, controlled trial with randomisation of practices to structured personal care or routine care; analysis after 6 years.
311 Danish practices with 474 general practitioners (243 in intervention group and 231 in comparison group).
874 (90.1%) of 970 patients aged ⩾40 years who had diabetes diagnosed in 1989-91 and survived until six year follow up.
Regular follow up and individualised goal setting supported by prompting of doctors, clinical guidelines, feedback, and continuing medical education.
Main outcome measures
Predefined clinical non-fatal outcomes, overall mortality, risk factors, and weight.
Predefined non-fatal outcomes and mortality were the same in both groups. The following risk factor levels were lower for intervention patients than for comparison patients (median values): fasting plasma glucose concentration (7.9 v 8.7 mmol/l, P=0.0007), glycated haemoglobin (8.5% v 9.0%, P<0.0001; reference range 5.4-7.4%), systolic blood pressure (145 v 150 mm Hg, P=0.0004), and cholesterol concentration (6.0 v 6.1 mmol/l, P=0.029, adjusted for baseline concentration). Both groups had lost weight since diagnosis (2.6 v 2.0 kg). Metformin was the only drug used more frequently in the intervention group (24% (110/459) v 15% (61/415)).Intervention doctors arranged more follow up consultations, referred fewer patients to diabetes clinics, and set more optimistic goals.
In primary care, individualised goals with educational and surveillance support may for at least six years bring risk factors of patients with type 2 diabetes to a level that has been shown to reduce diabetic complications but without weight gain.
What is already known on this topicEvidence is increasing that control of hyperglycaemia, hypertension, and dyslipidaemia may postpone the development of diabetic complications in patients with type 2 diabetesMaintaining good control over a long period can be difficultWhat this study addsStructured individualised personal care with educational and surveillance support for general practitioners reduced levels of risk factors in type 2 diabetic patients after six yearsRisk factors were reduced to a level that has been shown to have a beneficial effect on diabetic complicationsParticipants also showed modest weight loss
PMCID: PMC59690  PMID: 11679387

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