Summary Report 19-36 Months

Project context and objectives

     The prevalence of type 2 diabetes mellitus (T2DM) is increasing in particular among young people, which has been coupled to the dramatic rise in childhood obesity. Higher than normal meal-related insulin secretion?is an early observation in these young obese individuals, also when insulin sensitivity is essentially normal. In isolated islets of Langerhans, which harbor the insulin-producing beta-cells, similar accentuated secretion of insulin can be observed. Based on these observations we propose insulin hypersecretion as an early etiological factor preceding insulin resistance and promoting lipid deposition and T2DM. Reducing insulin hypersecretion would consequently be a strategy to intervene with the development of childhood obesity and obesity-related T2DM. Mechanisms responsible for insulin hypersecretion are not well understood, however. Consequently, knowledge about how therapeutic strategies targeting insulin hypersecretion should be developed is lacking.

     The first aim of the project is to characterize obese children belonging to childhood obesity cohorts and isolated islets of Langerhans with special emphasis on insulin secretion. The obese children are subjected to an oral glucose tolerance test (OGTT). During the test multiple blood samples are obtained in which glucose, islet hormones and incretins are determined. Whereas glucose levels are generally normal in young obese children, lipid levels including circulating free fatty acids (FFAs) are elevated already in these young subjects. FFAs are therefore also measured in the samples obtained during the OGTT. Magnetic resonance (MR) images are obtained from the subjects to determine percentages of fat in different body parts. Questionnaires addressing eating, exercise and sleep patterns are part of the characterization. Lean children are recruited as controls subjects to go through the same procedures as the obese children. Genetic analyses are conducted, where special focus is on identifying genetic variants correlating with the insulin hypersecreting phenotype.

     The second aim is to in vitro characterize isolated human islets of Langerhans obtained from organ-donors with regard to insulin secretion. The knowledge achieved about elevated FFA levels in obese children is utilized when the islets are exposed to the saturated fatty acid palmitate at concentrations measured in the obese children. After different exposure times to palmitate the islets are challenged with an elevated glucose concentration and the release of insulin measured at several time points yielding a dynamic insulin release pattern. Islets showing different insulin secretory patterns will be analyzed for changes in expression patterns at the transcript (transcriptomics), protein (proteomics) and lipid (lipidomics) levels. These omics data sets will be analyzed with the aim of identifying novel molecular players and pathways contributing to insulin hypersecretion.

     The third aim is to identify new ways of intervening pharmacologically in the obese pediatric population aiming at normalizing insulin hypersecretion. The aim is addressed by investigating how different pharmacological compounds affect insulin hypersecretion in the isolated islets. These results together with published results from clinical studies, where pharmacological intervention has been attempted in obese adult subjects, will found the basis for the design of an interventional study in obese children. This study will be to be conducted by two childhood obesity centers during the last year of the Beta-JUDO project.

Work performed since beginning of project and main results

     Mechanisms underlying insulin hypersecretion have been addressed within the clinical work in obese children and pre-clinical work in isolated islets in Beta-JUDO.

     The clinical work includes two, already established childhood obesity cohorts. These cohorts contain approximately 4000 subjects and form the basis for the genetic analysis. In addition two, new childhood obesity cohorts have been initiated. The latter cohorts are focusing on detailed characterization with special emphasis on insulin secretion. These cohorts also recruit normal-weight children, who are also characterized in detail. During the first three years of the Beta-JUDO project 500 subjects were enrolled in the cohorts. The genetic work has been based on deep exome sequencing and GWAS. These approaches have identified new rare genetic variants and SNPs, which will be tested for association with insulin hypersecretion.

     The detailed phenotyping in the new cohorts have centered around the oral glucose tolerance test (OGTT), which has been conducted on close to 90% of the enrolled subjects. Also, annual follow-ups have started with close to 4% already having at least one additional OGTT performed. During the OGTT blood samples are obtained in which glucose and insulin are measured. We have also measured lipids and in particular free fatty acids in the blood samples. Most obese (and all normal-weight) children have normal glucose tolerance (NGT). In contrast however, we have found that lipids and the free fatty acids are elevated in these obese subjects. In both obese and lean controls magnetic resonance imaging (MRI) has been conducted and fat content of different organs determined. Increased fat liver content was observed already in young obese children.

     In the pre-clinical work isolated islets have been cultured in the presence of palmitate for different time periods. After culture, islets were perifused and glucose-stimulated insulin secretion (GSIS) measured dynamically. Difference in GSIS was observed in islets cultured for different time periods. Also, different compounds have been studied to evaluate if they were able to normalize the insulin secretory pattern. Mechanisms for differences in insulin secretory patterns and how compounds affected the secretory patterns were examined by generating expressions of large number of transcripts (transcriptomics), proteins (proteomics) and lipids (lipidomics) from islets cultured for the different time periods. The results from the different omic-approaches together with the related changes in islet phenotype have started to be analyzed in an integrated way.

     The interventional study aiming at reduce insulin hypersecretion in obese adolescents has been planned. From results of experiments with isolated islets exposed to different compounds affecting insulin secretion, and experience in adults, a compound was selected for the interventional trial. A clinical study protocol has been finalized.

Expected final results and their potential impact and use

     The project addresses the role of the insulin-producing beta-cell in obesity-related development of type 2 diabetes mellitus (T2DM) in children. The project is expected to generate results within the following areas:
- Clinical area: the phenotyping of the obesity cohorts is expected to yield information on how insulin secretory changes observed in children that develop T2DM change over time, i.e. give knowledge about early prognostic signs of subjects at risk. Knowledge will be generated how different intervention modes affect the insulin secretory pattern. Importantly, experience with one compound and its efficacy in childhood obesity will be available through the interventional study conducted within the project. The genetic work conducted in obesity childhood cohorts is expected to yield genes associated with insulin hypersecretion.

- Pre-clinical area: the analyses of transcripts, proteins and lipids, associated with the distinct islet secretory phenotypes will define mechanism responsible for difference in islet secretory patterns. Also, detailed molecular knowledge about how different compounds associated with the treatment of obesity and T2DM act at the islet level and affect these mechanisms will be of great value for considering new therapeutic modes of intervention.

     The project spans both clinical and pre-clinical work and its results is expected to have impact at different levels:
- Clinical impact: the project is generating clinical and pre-clinical results that are integrated and used for the design of an interventional study. In the study a drug will be tested for its clinical utility in obese adolescents. Also, information on different therapeutic compounds on their effect on insulin secretion will be of help in attempting new interventional strategies. Another important aspect of the project that is expected to have clinical impact is the knowledge about how insulin secretory patterns change over time. Such knowledge is potentially of great prognostic value. Thus, the impact of the project for the obese subjects is therefore expected to be considerable.
- Scientific impact: the project will supply new knowledge with regard to mechanisms responsible for the T2DM rise in obese children. These mechanisms are focusing on the role of the insulin-producing beta-cell in the etiology of obesity-related T2DM development in adolescents. Results both from the obese children and the isolated islets will be important for this.
- Societal and economical impact: the number of young individuals with obesity is rapidly growing and Europe faces a major challenge with regard to finding ways to reverse this trend. The very limited pharmacology-based alternatives for the treatment of young obese individuals pose a problem not only for the afflicted individuals at risk of developing T2DM and other related diseases but also for European health providers. It is imperative to meet this challenge and finding new ways to attenuate obesity and obesity-related T2DM in the young population and to identify and evaluate new drugs for this rapidly growing patient group. The involvement of SMEs in the project will contribute to that therapeutic strategies proposed by the project will lead to new opportunities for European industry strengthening European health economy.