Bruce H. R. Wolffenbuttel

Characterization of the Human Visceral Adipose Tissue Secretome

Adipose tissue is an endocrine organ involved in storage and release of energy but also in regulation of energy metabolism in other organs via secretion of peptide and protein hormones (adipokines). Especially visceral adipose tissue has been implicated in the development of metabolic syndrome and type 2 diabetes. Factors secreted by the stromal-vascular fraction contribute to the secretome and modulate adipokine secretion by adipocytes. Therefore, we aimed at the characterization of the adipose tissue secretome rather than the adipocyte cell secretome. The presence of serum proteins and intracellular proteins from damaged cells, released during culture, may dramatically influence the dynamic range of the sample and thereby identification of secreted proteins. Part of the study was therefore dedicated to the influence of the culture setup on the quality of the final sample. Visceral adipose tissue was cultured in five experimental setups, and the quality of resulting samples was evaluated in terms of protein concentration and protein composition. The best setup involved one wash after the 1st h in culture followed by two or three additional washes within an 8-h period, starting after overnight culture. Thereafter tissue was maintained in culture for an additional 48–114 h to obtain the final sample. For the secretome experiment, explants were cultured in media containing l-[13C6,15N2]lysine to validate the origin of the identified proteins (adipose tissue- or serum-derived). In total, 259 proteins were identified with ≥99% confidence. 108 proteins contained a secretion signal peptide of which 70 incorporated the label and were considered secreted by adipose tissue. These proteins were classified into five categories according to function. This is the first study on the (human) adipose tissue secretome. The results of this study contribute to a better understanding of the role of adipose tissue in whole body energy metabolism and related diseases.

Universal risk factors for multifactorial diseases: LifeLines: a three-generation population-based study.

The risk for multifactorial diseases is determined by risk factors that frequently apply across disorders (universal risk factors). To investigate unresolved issues on etiology of and individual’s susceptibility to multifactorial diseases, research focus should shift from single determinant-outcome relations to effect modification of universal risk factors. We present a model to investigate universal risk factors of multifactorial diseases, based on a single risk factor, a single outcome measure, and several effect modifiers. Outcome measures can be disease overriding, such as clustering of disease, frailty and quality of life. “Life course epidemiology” can be considered as a specific application of the proposed model, since risk factors and effect modifiers of multifactorial diseases typically have a chronic aspect. Risk factors are categorized into genetic, environmental, or complex factors, the latter resulting from interactions between (multiple) genetic and environmental factors (an example of a complex factor is overweight). The proposed research model of multifactorial diseases assumes that determinant-outcome relations differ between individuals because of modifiers, which can be divided into three categories. First, risk-factor modifiers that determine the effect of the determinant (such as factors that modify gene-expression in case of a genetic determinant). Second, outcome modifiers that determine the expression of the studied outcome (such as medication use). Third, generic modifiers that determine the susceptibility for multifactorial diseases (such as age). A study to assess disease risk during life requires phenotype and outcome measurements in multiple generations with a long-term follow up. Multiple generations will also enable to separate genetic and environmental factors. Traditionally, representative individuals (probands) and their first-degree relatives have been included in this type of research. We put forward that a three-generation design is the optimal approach to investigate multifactorial diseases. This design has statistical advantages (precision, multiple-informants, separation of non-genetic and genetic familial transmission, direct haplotype assessment, quantify genetic effects), enables unique possibilities to study social characteristics (socioeconomic mobility, partner preferences, between-generation similarities), and offers practical benefits (efficiency, lower non-response). LifeLines is a study based on these concepts. It will be carried out in a representative sample of 165,000 participants from the northern provinces of the Netherlands. LifeLines will contribute to the understanding of how universal risk factors are modified to influence the individual susceptibility to multifactorial diseases, not only at one stage of life but cumulatively over time: the lifeline.

Racial and Ethnic Differences in Mean Plasma Glucose, Hemoglobin A1c, and 1,5-Anhydroglucitol in Over 2000 Patients with Type 2 Diabetes

CONTENT Recent studies have reported hemoglobin A(1c) (A1c) differences across racial/ethnic groups. Our diverse population allows for further investigation of potential differences in measurements of glycemia. OBJECTIVES Our objectives were to describe and explore baseline racial/ethnic differences in self-monitored plasma glucose profiles, A1c, and 1,5-anhydroglucitol (1,5-AG) in patients with type 2 diabetes enrolled in the Assessing DURAbility of Basal vs. Lispro Mix 75/25 Insulin Efficacy trial. DESIGN, SETTING, PATIENTS The trial enrolled 2094 patients with type 2 diabetes, ages 30-80 yr, from 11 countries. MAIN OUTCOME MEASURES Estimated mean plasma glucose (MPG), A1c, and 1,5-AG were compared among racial/ethnic groups before and after adjusting for factors affecting glycemia: age, sex, duration of diabetes, body mass index, and MPG. RESULTS Baseline estimated MPG +/- sd was 220.0 +/- 82.0 mg/dl, mean A1c was 9.0 +/- 1.3%, and 1,5-AG was 5.0 +/- 4.1microg/ml. Estimated MPG did not differ between Caucasian and non-Caucasian groups. A1c was higher in Hispanics (9.4 +/- 1.4%; P < 0.001), Asians (9.2 +/- 1.4%; P < 0.01), and patients of other racial/ethnic groups (9.7 +/- 1.5%; P < 0.001) compared with Caucasians (8.9 +/- 1.2%). Paradoxically, 1,5-AG was higher for Asian (5.7 +/- 4.6 microg/ml) and African patients (6.2 +/- 5.4 microg/ml) vs. Caucasians (4.9 +/- 3.9 microg/ml) (P < 0.01). After adjusting for factors affecting glycemia, A1c was higher (all P <or= 0.002) in Hispanics, Asians, Africans, and patients of other racial/ethnic groups, and 1,5-AG was higher in Asian and African patients (P < 0.001) vs. Caucasians. CONCLUSIONS There were differences in A1c and 1,5-AG, but not MPG, among racial/ethnic groups. Comparisons of glycemia across racial/ethnic groups using these parameters may be problematic due to inherent biological variability and methodological issues.

Cohort Profile: LifeLines, a three-generation cohort study and biobank

The LifeLines Cohort Study is a large population-based cohort study and biobank that was established as a resource for research on complex interactions between environmental, phenotypic and genomic factors in the development of chronic diseases and healthy ageing. Between 2006 and 2013, inhabitants of the northern part of The Netherlands and their families were invited to participate, thereby contributing to a three-generation design. Participants visited one of the LifeLines research sites for a physical examination, including lung function, ECG and cognition tests, and completed extensive questionnaires. Baseline data were collected for 167 729 participants, aged from 6 months to 93 years. Follow-up visits are scheduled every 5 years, and in between participants receive follow-up questionnaires. Linkage is being established with medical registries and environmental data. LifeLines contains information on biochemistry, medical history, psychosocial characteristics, lifestyle and more. Genomic data are available including genome-wide genetic data of 15 638 participants. Fasting blood and 24-h urine samples are processed on the day of collection and stored at -80 °C in a fully automated storage facility. The aim of LifeLines is to be a resource for the national and international scientific community. Requests for data and biomaterials can be submitted to the LifeLines Research Office [LLscience@umcg.nl].

Glucagon-Like Peptide 1 Receptor Agonist or Bolus Insulin With Optimized Basal Insulin in Type 2 Diabetes

OBJECTIVE Mealtime insulin is commonly added to manage hyperglycemia in type 2 diabetes when basal insulin is insufficient. However, this complex regimen is associated with weight gain and hypoglycemia. This study compared the efficacy and safety of exenatide twice daily or mealtime insulin lispro in patients inadequately controlled by insulin glargine and metformin despite up-titration. RESEARCH DESIGN AND METHODS In this 30-week, open-label, multicenter, randomized, noninferiority trial with 12 weeks prior insulin optimization, 627 patients with insufficient postoptimization glycated hemoglobin A1c (HbA1c) were randomized to exenatide (10–20 µg/day) or thrice-daily mealtime lispro titrated to premeal glucose of 5.6–6.0 mmol/L, both added to insulin glargine (mean 61 units/day at randomization) and metformin (mean 2,000 mg/day). RESULTS Randomization HbA1c and fasting glucose (FG) were 8.3% (67 mmol/mol) and 7.1 mmol/L for exenatide and 8.2% (66 mmol/mol) and 7.1 mmol/L for lispro. At 30 weeks postrandomization, mean HbA1c changes were noninferior for exenatide compared with lispro (–1.13 and –1.10%, respectively); treatment differences were –0.04 (95% CI –0.18, 0.11) in per-protocol (n = 510) and –0.03 (95% CI –0.16, 0.11) in intent-to-treat (n = 627) populations. FG was lower with exenatide than lispro (6.5 vs. 7.2 mmol/L; P = 0.002). Weight decreased with exenatide and increased with lispro (−2.5 vs. +2.1 kg; P < 0.001). More patients reported treatment satisfaction and better quality of life with exenatide than lispro, although a larger proportion of patients with exenatide experienced treatment-emergent adverse events. Exenatide resulted in fewer nonnocturnal hypoglycemic episodes but more gastrointestinal adverse events than lispro. CONCLUSIONS Adding exenatide to titrated glargine with metformin resulted in similar glycemic control as adding lispro and was well tolerated. These findings support exenatide as a noninsulin addition for patients failing basal insulin.

Wnt Signaling and Dupuytren's Disease

BACKGROUND Dupuytrens disease is a benign fibromatosis of the hands and fingers that leads to flexion contractures. We hypothesized that multiple genetic and environmental factors influence susceptibility to this disease and sought to identify susceptibility genes to better understand its pathogenesis. METHODS We conducted a genomewide association study of 960 Dutch persons with Dupuytrens disease and 3117 controls (the discovery set) to test for association between the disease and genetic markers. We tested the 35 single-nucleotide polymorphisms (SNPs) most strongly associated with Dupuytrens disease (P<1×10(-4)) in the discovery set in three additional, independent case series comprising a total of 1365 affected persons and 8445 controls from Germany, the United Kingdom, and The Netherlands. RESULTS Initially, we observed a significant genomewide association between Dupuytrens disease and 8 SNPs at three loci. Tests of replication and joint analysis of all data from 2325 patients with Dupuytrens disease and 11,562 controls yielded an association with 11 SNPs from nine different loci (P<5.0×10(-8)). Six of these loci contain genes known to be involved in the Wnt-signaling pathway: WNT4 (rs7524102) (P=2.8×10(-9); odds ratio, 1.28), SFRP4 (rs16879765) (P=5.6×10(-39); odds ratio, 1.98), WNT2 (rs4730775) (P=3.0×10(-8); odds ratio, 0.83), RSPO2 (rs611744) (P=7.9×10(-15); odds ratio, 0.75), SULF1 (rs2912522) (P=2.0×10(-13); odds ratio, 0.72), and WNT7B (rs6519955) (P=3.2×10(-33); odds ratio, 1.54). CONCLUSIONS This study implicates nine different loci involved in genetic susceptibility to Dupuytrens disease. The fact that six of these nine loci harbor genes encoding proteins in the Wnt-signaling pathway suggests that aberrations in this pathway are key to the process of fibromatosis in Dupuytrens disease.

The Genome of the Netherlands: design, and project goals

Within the Netherlands a national network of biobanks has been established (Biobanking and Biomolecular Research Infrastructure-Netherlands (BBMRI-NL)) as a national node of the European BBMRI. One of the aims of BBMRI-NL is to enrich biobanks with different types of molecular and phenotype data. Here, we describe the Genome of the Netherlands (GoNL), one of the projects within BBMRI-NL. GoNL is a whole-genome-sequencing project in a representative sample consisting of 250 trio-families from all provinces in the Netherlands, which aims to characterize DNA sequence variation in the Dutch population. The parent–offspring trios include adult individuals ranging in age from 19 to 87 years (mean=53 years; SD=16 years) from birth cohorts 1910–1994. Sequencing was done on blood-derived DNA from uncultured cells and accomplished coverage was 14–15x. The family-based design represents a unique resource to assess the frequency of regional variants, accurately reconstruct haplotypes by family-based phasing, characterize short indels and complex structural variants, and establish the rate of de novo mutational events. GoNL will also serve as a reference panel for imputation in the available genome-wide association studies in Dutch and other cohorts to refine association signals and uncover population-specific variants. GoNL will create a catalog of human genetic variation in this sample that is uniquely characterized with respect to micro-geographic location and a wide range of phenotypes. The resource will be made available to the research and medical community to guide the interpretation of sequencing projects. The present paper summarizes the global characteristics of the project.

DURAbility of Basal Versus Lispro Mix 75/25 Insulin Efficacy (DURABLE) Trial 24-Week Results Safety and efficacy of insulin lispro mix 75/25 versus insulin glargine added to oral antihyperglycemic drugs in patients with type 2 diabetes

OBJECTIVE To compare the ability of two starter insulin regimens to achieve glycemic control in a large, ethnically diverse population with type 2 diabetes. RESEARCH DESIGN AND METHODS During the initiation phase of the DURABLE trial, patients were randomized to a twice-daily lispro mix 75/25 (LM75/25; 75% lispro protamine suspension, 25% lispro) (n = 1,045) or daily glargine (GL) (n = 1,046) with continuation of prestudy oral antihyperglycemic drugs. RESULTS Baseline A1C was similar (LM75/25: 9.1 ± 1.3%; GL: 9.0 ± 1.2%; P = 0.414). At 24 weeks, LM75/25 patients had lower A1C than GL patients (7.2 ± 1.1 vs. 7.3 ± 1.1%, P = 0.005), greater A1C reduction (–1.8 ± 1.3 vs. –1.7 ± 1.3%, P = 0.005), and higher percentage reaching A1C target <7.0% (47.5 vs. 40.3%, P < 0.001). LM75/25 was associated with higher insulin dose (0.47 ± 0.23 vs. 0.40 ± 0.23 units · kg−1· day−1, P < 0.001) and more weight gain (3.6 ± 4.0 vs. 2.5 ± 4.0 kg, P < 0.0001). LM75/25 patients had a higher overall hypoglycemia rate than GL patients (28.0 ± 41.6 vs. 23.1 ± 40.7 episodes · pt−1· year−1, P = 0.007) but lower nocturnal hypoglycemia rate (8.9 ± 19.3 vs. 11.4 ± 25.3 episodes · pt−1· year−1, P = 0.009). Severe hypoglycemia rates were low in both groups (LM75/25: 0.10 ± 1.6 vs. GL: 0.03 ± 0.3 episodes · pt−1· year−1, P = 0.167). CONCLUSIONS Compared with GL, LM75/25 resulted in slightly lower A1C at 24 weeks and a moderately higher percentage reaching A1C target <7.0%. Patients receiving LM75/25 experienced more weight gain and higher rates of overall hypoglycemia but lower rates of nocturnal hypoglycemia. Durability of regimens will be evaluated in the following 2-year maintenance phase.

A Meta-Analysis of Thyroid-Related Traits Reveals Novel Loci and Gender-Specific Differences in the Regulation of Thyroid Function

Thyroid hormone is essential for normal metabolism and development, and overt abnormalities in thyroid function lead to common endocrine disorders affecting approximately 10% of individuals over their life span. In addition, even mild alterations in thyroid function are associated with weight changes, atrial fibrillation, osteoporosis, and psychiatric disorders. To identify novel variants underlying thyroid function, we performed a large meta-analysis of genome-wide association studies for serum levels of the highly heritable thyroid function markers TSH and FT4, in up to 26,420 and 17,520 euthyroid subjects, respectively. Here we report 26 independent associations, including several novel loci for TSH (PDE10A, VEGFA, IGFBP5, NFIA, SOX9, PRDM11, FGF7, INSR, ABO, MIR1179, NRG1, MBIP, ITPK1, SASH1, GLIS3) and FT4 (LHX3, FOXE1, AADAT, NETO1/FBXO15, LPCAT2/CAPNS2). Notably, only limited overlap was detected between TSH and FT4 associated signals, in spite of the feedback regulation of their circulating levels by the hypothalamic-pituitary-thyroid axis. Five of the reported loci (PDE8B, PDE10A, MAF/LOC440389, NETO1/FBXO15, and LPCAT2/CAPNS2) show strong gender-specific differences, which offer clues for the known sexual dimorphism in thyroid function and related pathologies. Importantly, the TSH-associated loci contribute not only to variation within the normal range, but also to TSH values outside the reference range, suggesting that they may be involved in thyroid dysfunction. Overall, our findings explain, respectively, 5.64% and 2.30% of total TSH and FT4 trait variance, and they improve the current knowledge of the regulation of hypothalamic-pituitary-thyroid axis function and the consequences of genetic variation for hypo- or hyperthyroidism.

Quality, quantity and harmony: the DataSHaPER approach to integrating data across bioclinical studies

Background Vast sample sizes are often essential in the quest to disentangle the complex interplay of the genetic, lifestyle, environmental and social factors that determine the aetiology and progression of chronic diseases. The pooling of information between studies is therefore of central importance to contemporary bioscience. However, there are many technical, ethico-legal and scientific challenges to be overcome if an effective, valid, pooled analysis is to be achieved. Perhaps most critically, any data that are to be analysed in this way must be adequately ‘harmonized’. This implies that the collection and recording of information and data must be done in a manner that is sufficiently similar in the different studies to allow valid synthesis to take place. Methods This conceptual article describes the origins, purpose and scientific foundations of the DataSHaPER (DataSchema and Harmonization Platform for Epidemiological Research; http://www.datashaper.org), which has been created by a multidisciplinary consortium of experts that was pulled together and coordinated by three international organizations: P3G (Public Population Project in Genomics), PHOEBE (Promoting Harmonization of Epidemiological Biobanks in Europe) and CPT (Canadian Partnership for Tomorrow Project). Results The DataSHaPER provides a flexible, structured approach to the harmonization and pooling of information between studies. Its two primary components, the ‘DataSchema’ and ‘Harmonization Platforms’, together support the preparation of effective data-collection protocols and provide a central reference to facilitate harmonization. The DataSHaPER supports both ‘prospective’ and ‘retrospective’ harmonization. Conclusion It is hoped that this article will encourage readers to investigate the project further: the more the research groups and studies are actively involved, the more effective the DataSHaPER programme will ultimately be.