Executive summary, entire project

The project “Beta-cell function in Juvenile Diabetes and Obesity” (Beta-JUDO) aimed at investigating the role of the insulin-producing beta-cell in development of obesity and type 2 diabetes mellitus (T2DM) in children. The rationale behind the project was that accentuated insulin response (insulin hypersecretion) is an early phenomenon promoting development of overweight and insulin resistance, since high insulin levels favours storage rather than combustion of energy. Individuals with this secretory phenotype are therefore at risk of developing obesity and T2DM. The project was organised in a translational approach involving studies of obese children and beta-cells from isolated human islets of Langerhans. In Beta-JUDO, paediatric obesity clinics and academic centres with focus on treatment of obese children, beta-cell biology, protein and transcript profiling, genetics, and bioinformatics formed a consortium together with a large drug company and small companies (SMEs) specialized on biomarker discovery and clinical trials.

The overall aim of the project was to identify new pharmacological approaches to normalize the high insulin levels observed in obese children and children of risk to become obese. The aim was addressed by, firstly, defining factors and molecular mechanisms that contribute to insulin hypersecretion, and secondly, to address ways to counteract insulin hypersecretion by investigating pharmacological principles potentially targeting insulin hypersecretion in both isolated islets and in obese children.

Factors contributing to insulin hypersecretion included high circulating levels of the free fatty acid (FFA) palmitate, which were high in obese children with accentuated insulin hypersecretion. Correspondently, in isolated islets such elevated palmitate levels caused accentuated glucose-stimulated insulin secretion. The findings supported a role of elevated levels of palmitate as an underlying factor contributing to insulin hypersecretion. We found that palmitate enhanced islet respiration, which was closely linked with islet insulin hypersecretion. In obese children raised glucagon levels were compatible with a role of altered glucagon response in obese children contributing to hyperinsulinemia. In genetic studies using large obesity cohorts we identified a number of genes that are associated with insulin hypersecretion, such as the gene PCSK1 that is involved in insulin processing. Further underlying factors for islet insulin hypersecretion were found by generating extensive protein and transcript expression patterns.

Strategies to counteract insulin hypersecretion included free fatty acid receptor 1 (FFAR1) antagonism, which normalized insulin hypersecretion from isolated islets exposed to palmitate. FFAR1 may represent a prevention strategy/therapy in subjects with high palmitate levels, but no FFAR1 antagonists are currently available for clinical use. GLP-1 receptor agonism showed promise in normalizing insulin hypersecretion. There are several available clinical intervention options using GLP-1 agonism. Indeed, we have conducted the first extended placebo-controlled, double-blinded intervention trial, where the GLP-1 analogue exenatide was administered to obese adolescents. Significant reduction in weight was observed with no rise in adverse events compared to placebo. The trial will potentially supply this patient group a novel pharmacological alternative.

The Beta-JUDO project has developed a cohort counting to about 700 obese children with detailed phenotype and blood samples collected over time including subjects that develop T2DM. The cohort provides unique possibilities to delineate early events in obesity-related T2DM and identify prognostic markers for subjects at risk. A definition of insulin hypersecretion in obese children has been proposed, which was used when genetic markers for insulin hypersecretion were identified.


 

Project Summary, Final Period 3


Project context and main objectives
 

In Europe more than 20% of children are overweight and 5% even obese. Obesity is a strong risk factor for developing complications including type 2 diabetes mellitus (T2DM), which is now seen in growing numbers in children. These high obesity prevalence numbers in children, which do not decline, pose a major health threat for Europe.

High insulin levels are observed in most of the obese children both at fasting and during oral glucose tolerance test (OGTT). The hyperinsulinemia has been interpreted as a result of decreased insulin sensitivity in insulin target tissue.  Although this is probably correct in many obese adults, the situation may be different in children. In some of the obese children hyperinsulinemia was observed despite essentially normal insulin sensitivity. Insulin is the prime hormone promoting storage of calories. Insulin is secreted from the insulin- producing beta-cells in the pancreas. Evidence is accumulating and supporting that high insulin levels in certain subjects could be a primary event leading to development of overweight and obesity. For these subjects it would be essential to normalize the hyperactive beta-cells so that normal insulin levels were attained.

The project Beta-cell function in juvenile type 2 diabetes and obesity (Beta-JUDO) aimed at reducing childhood obesity and obesity-related complications by normalizing insulin hypersecretion. Beta-JUDO was divided into three parts. In the first part of the project the aim was to define factors that contribute to insulin hyeprsecretion in obese children belonging to childhood obesity cohorts. Special focus was on insulin secretory response during an oral glucose tolerance test (OGTT). In addition to insulin also glucose, free fatty acids (FFAs) and incretins were determined. Whereas glucose levels were generally normal in young obese children, lipid levels including circulating fatty acid palmitate were elevated already in these young subjects. Magnetic resonance (MR) images were obtained from the subjects in order to determine percentages of fat in different body parts. Genetic analyses were conducted, where special focus is on identifying genetic variants correlating with the insulin hypersecreting phenotype.

The second part aimed at defining molecular mechanisms responsible for insulin hypersecretion from islets of Langerhans isolated from human organ-donors. When islets were exposed to elevated levels of palmitate, as observed in obese children, for 2 days, they hypersecreted insulin in response to elevated glucose concentrations. Islets with augmented insulin secretory patterns were analyzed for changes in expression patterns at the transcript (transcriptomics), protein (proteomics) and lipid (lipidomics) levels. These omics data sets were analyzed with the aim of identifying novel molecular players and pathways contributing to insulin hypersecretion.

The third aim was to test novel ways of intervening pharmacologically in the obese pediatric population aiming at normalizing insulin hypersecretion. This aim was addressed by investigating how different pharmacological compounds affect insulin hypersecretion in the isolated islets. These results furthermore founded the basis for an interventional trial performed in obese children.



Work performed since the beginning of the project and the main results achieved



The first aim of the project, where underlying mechanisms of insulin hypersecretion in obese children were to be defined, was addressed within the clinical work. Two childhood obesity cohorts, which contained approximately 4000 subjects, formed the basis for the genetic analysis. Two other cohorts containing approximately 650 subjects had the focus on detailed characterization with special emphasis on insulin secretion. Normal-weight children were also recruited, who were characterized in detail. The genetic work was based on deep exome sequencing and genome-wide association studies (GWAS). These approaches identified new genetic variants that were tested for association with insulin hypersecretion. The cohorts with detailed phenotyping centered around the oral glucose tolerance test (OGTT). From the results a definition of insulin hypersecretion has been proposed. Based on the characterization the oral disposition index was decided to define levels for insulin hypersecretion.


The second aim of the project, where underlying mechanisms of insulin hypersecretion in isolated islets were to be defined, was addressed within the pre-clinical part of the project. Isolated islets were cultured in the presence of palmitate for 2 days. After culture, islets were perifused and glucose-stimulated insulin secretion (GSIS) measured dynamically. GSIS was more than doubled in the palmitate-exposed islets. Also, exendine or metformin was included during culture of islets exposed to palmitate. Both compounds normalized insulin hypersecretion. In order to delineate mechanisms by which hypersecretion occured and how the compounds normalized it, islets exposed to palmitate alone and in the presence of exendine or metformin were used to generate large number of transcripts (transcriptomics), proteins (proteomics) and lipids (lipidomics). These expression data sets together with the related changes in islet phenotype were analyzed and differential pathway activation was suggested based on the analyses.

As the third aim of the project, an interventional study aiming at reducing insulin hypersecretion in obese adolescents was undertaken. The study was a 6-month placebo-controlled double-blind study, where 12-18 years old obese adolescents were given the GLP-1 analogue exenatide once weekly in combination with life-style treatment. The study was conducted in two European countries, which enrolled 44 patients in total. The results showed that the drug caused significant reduction in weight. Furthermore, the drug was well tolerated with no difference in adverse events between the treated and un-treated patients.


Expected final results and their potential impacts and use

 

The translational project Beta-JUDO addressed the role of the insulin-producing beta-cell in obesity-related development of type 2 diabetes mellitus in children. The project has generated results, which will have impact in the following areas:

- Clinical area: The characterization of the obesity cohorts including the genetic work is expected to emphasize the importance of maintaining normal insulin levels and also identify situations and foods that elicit accentuated insulin secretion, which are to be avoided. The successful intervention with exenatide is expected to pave the way for further clinical studies, where the drug will be further tested in larger cohorts for its safety and efficacy in combating childhood obesity. To give this patient group a pharmacological alternative would be very important not only for the patients but also for the European health providers.

- Pre-clinical area: The analyses of transcripts, proteins and lipids, associated with islet insulin hypersecretion gave new information about the molecular mechanisms behind insulin hypersecretion and how different compounds associated with the treatment of obesity and T2DM act at the islet level and affect these mechanisms. Several novel, not yet proposed candidates coming form the Beta-JUDO work and operative in the islets of Langerhans are expected to be validated for a role in insulin hypersecretion. Such candidates may be of interest for further development into intervention strategies.

- Societal and economical impact: It is imperative to meet this challenge and to find 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 results of the project are expected to lead to a reduced number of young individuals with overweight or obesity. 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.

 


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.

 


Summary Report 1-18 Months

Project context and objectives


The number of individuals developing type 2 diabetes mellitus (T2DM) early in life is growing globally. This alarming increase is connected with the rising incidence of childhood obesity. Life-style interventions are not effective in many of these children and additional approaches including pharmacology-based are desperately needed. The project “Beta-cell function in Juvenile Diabetes and Obesity” (Beta-JUDO) aims at investigating the role of the insulin-producing beta-cell in development of obesity and T2DM in children. The hypothesis is that individuals with accentuated insulin responses are at risk of developing obesity and T2DM since high insulin levels favours storage rather than combustion of energy. Thus, high insulin levels would be an early phenomenon preceding obesity development. The hypothesis is addressed in a translational approach involving measuring insulin secretory responses in obese children and from isolated human islets of Langerhans. In Beta-JUDO, paediatric obesity clinics and academic centres with focus on beta-cell biology, brown adipocyte imaging, transcript and protein profiling, genetics, epidemiology and bioinformatics have formed a consortium with a large drug company and with small companies (SMEs) specialized on biomarker discovery and clinical trials.

The main aim of Beta-JUDO is to identify novel pharmacological approaches that reduce weight via modulation of insulin levels. The following questions are addressed:

  1. Are elevated insulin levels in young obese individuals a cause or consequence of the obese state?
  2. What genetic variants, obtained from analysis of the obese children, are associated with yperinsulinemia/insulin hypersecretion?
  3. Under what conditions do isolated islets of Langerhans hypersecrete insulin, and how do drugs used today in adult obesity and T2DM affect insulin secretion from the isolated islet?
  4. What islet cellular mechanisms are connected with insulin hypersecretion and how are these mechanisms affected by drugs modulating insulin hypersecretion?
  5. How does a drug, which modulates islet insulin hypersecretion and is used for adult obesity and T2DM, affect obesity in adolescents?

The following objectives, coupled to the questions above, were formulated:

  1. To characterise insulin secretion in obese children at the childhood obesity clinics by performing oral glucose tolerance tests (OGTTs) and determining levels of glucose, insulin and C-peptide.
  2. To perform genetic analyses on the obese children and associating secretory phenotypes with genotypes.
  3. Characterise insulin secretion from isolated palmitate-treated human islets in the absence and presence of substances used today in the treatment of adult obesity and/or T2DM.
  4. Analyse the islets from a systems biology approach using proteomic, transcriptomic and lipidomic data sets.
  5. Perform a randomized, two-arm, double-blinded, placebo-controlled interventional study with obese adolescents and matched controls with a drug found to affect islet insulin hypersecretion.


Work performed and main results


The main results obtained during the first 18 months period were:

1) Obese and normal weight children have continuously been enrolled and annually followed-up. OGTTs have been performed on 240 patients and 50 controls. Glucose clamps and frequently sampled OGTTs have started. Samples for measurements of free fatty acids have been obtained, and a sensitive method for determination of fatty acids has been developed. Free fatty acid levels in plasma samples from 22 patients and 11 controls have so far been measured. A protocol for MRI analysis has been implemented, and scans of 60 patients and 7 controls have been conducted. DNA samples have been collected from 2250 patient and 240 control children.

2) The aim to define the insulin hypersecreting phenotype has started. First genetic analysis identified genes potentially associated with insulin traits in obese children. In the obese children we found that accentuated insulin levels were more prevalent in the youngest children. When such children were followed longitudinally, we observed a decline in insulin levels. In some of them the decline was to such an extent that glucose levels could no longer be controlled and the children were diagnosed with T2DM. From insulin and C-peptide measurements the contribution of newly secreted insulin was calculated. Based on these calculations of insulin secretion the obese children were stratified. These insulin secretory sub-phenotypes will during the next project period be used in genetic studies..

3) Model of isolated human islets hypersecreting insulin has been established: isolated human islets of Langerhans were used to define mechanisms of insulin hypersecretion in vitro. When islets were cultured in the presence of palmitate for 2 days, glucose-stimulated insulin secretion (GSIS) was accentuated. In contrast, GSIS was lowered in islets cultured for 7 days. This pattern of enhanced GSIS at early time points followed by decline at later time points was reminiscent of the insulin levels/secretion observed in obese children developing T2DM. We hypothesize that the mechanisms responsible for these time-dependent changes in palmitate-exposed hyper- and hypo-secreting islets is of relevance to understanding events occurring in obese children developing T2DM.

4) The first compound normalizing insulin hypersecretion has been selected. Protocols for protein, lipid and transcript profiling of islets have been optimized. Islets exposed to compound normalizing insulin hypersecretion have been subjected to lipidomic and proteomic analyses, data sets curated and statistically analysed. Lipidomics and proteomics data have been bioinfomatically analysed. Second compound normalizing insulin hypersecretion has been identified.

5) A protocol for an interventional study with approximately 50 obese adolescents has been developed. Discussions regarding the proposed active substance, where the substance tested in the islet is considered, are on-going with representatives for ethical review boards and regulatory authorities. Additional compounds will be selected representing novel principles derived from the genotype-phenotype correlation in the obese children and tested for their ability to normalize insulin secretion from the palmitate-treated islets.

A management team was furthermore established with the aim to ensure progression of the project towards its planned objectives and contractual commitments. Dissemination, exploitation and publication strategies were formulated. Project logo and web-portal for communication and data sharing were created. Connections with other FP7-funded diabetes projects were established. Standard operating procedures (SOPs) for clinical and pre-clinical work, data collection and handling were formulated and harmonized between partners. QA/QC work was conducted. Ethical applications and approvals for the clinical and pre-clinical work were collected and scrutinized by an ethical reviewer.



Expected final results, impact and use


The project is expected to generate results about:

  • To what extent insulin hypersecretion is an early phenomenon in the development of childhood obesity.
  • How insulin levels during fasting and OGTT develop in childhood obesity and what specific characteristics are associated with insulin hypersecretion in particular with regard to circulating levels of free fatty acids and white and brown fat characteristics, i.e. visceral/subcutaneous adipose tissue, liver and pancreatic fat.
  • Genetic background to insulin hypersecretion in childhood obesity.
  • Cellular and molecular mechanisms underlying insulin hypersecretion from the pancreatic beta-cell, which will be delineated applying systems biology analysis on data sets generated by multiple omics approaches including transcriptomics, proteomics and lipidomics on human isolated islets.
  • How currently used drugs for the management of adult obesity and T2DM as well as novel principles derived from genetic studies within the project affect islet insulin hypersecretion and cellular and molecular mechanisms underlying insulin hypersecretion.


The project is expected to have impact and use on the following areas:

  • Definition of international standards. OGTT reference values will be generated for obese and normal weight boys and girls of different ages. From these values opportunities to developing standards for insulin secretion and insulin resistance for different age groups will be explored.
  • Pharmacological strategies for intervention in childhood obesity.
  • Basis for the development of new future drugs targeting childhood obesity.
  • Attenuating the rise in in childhood obesity and T2DM in Europe and worldwide.

Childhood obesity has reached levels never seen before. By reducing obesity rates, both major health benefits, and economic benefits can be achieved. This project will raise awareness and knowledge among citizens in Europe, in health care, industry and funding agencies. Scientific benefits will be based on the integrated efforts in elucidating the impact of insulin early in childhood obesity development and developing strategies for the prevention.