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Recruitment and activation of brown adipocytes as preventive and curative therapy for type 2 diabetes

Final Report Summary - DIABAT (Recruitment and activation of brown adipocytes as preventive and curative therapy for type 2 diabetes)

Executive Summary:
Obesity and its pathological consequences - especially type 2 diabetes - have reached an alarming high prevalence in the European Union and beyond. According to the International Diabetes Foundation (IDF), more than 50 Million individuals in Europe were suffering from type 2 diabetes in 2013. Numbers have rapidly escalated in the recent past, and are expected to further increase in the future, mainly due to the rising numbers of overweight and obese persons. The socioeconomic burden of diabetes is immense, indicated by the fact that approximately 147 Billion Euros have been spent for treatment and management of diabetes and associated complications in 2013 in European countries (IDF atlas). The threat for public health emanating from obesity and its associated metabolic disorders is recognized as highly relevant by the European authorities, highlighted by the publication of the White Paper on “A Strategy for Europe on Nutrition, Overweight and Obesity related health issues” by the European Commission in 2007 (COM 2007 279).

Lifestyle-directed measures to combat obesity have mostly failed due to lacking compliance or re-gain of weight subsequent to temporary reduction. While oral diabetes therapeutics are principally available, they have certain efficacy and safety limitations. They neither affect obesity as the underlying cause, nor are they suited for diabetes prevention. Novel strategies to combat obesity and associated metabolic disorders are therefore still urgently needed.

The discovery of thermogenic, energy dissipating brown adipose tissue (BAT) in adult humans less that a decade ago, revived interest in this tissue among basic researchers and clinicians. As active BAT is able to burn huge amounts of energy from sugar and lipids in order to generate heat, it has the potential to counteract obesity and improve metabolic health, thereby ultimately ameliorating metabolic diseases including type 2 diabetes. The overall aims of the DIABAT project were to gain a better understanding of brown adipocyte cell biology, and to functionally analyse BAT regarding its impact on energy homeostasis in vivo. Exploitation of this new knowledge generated in appropriate model systems will ultimately help to develop novel BAT-directed anti-diabetes therapies.

The DIABAT consortium involved an interdisciplinary team of researchers from 17 academic laboratories, 2 SMEs and 1 biotech company located in 12 European countries. Leading European adipose tissue biologists teamed up with renowned clinical scientists and engineers in order to assess the therapeutic potential of BAT in humans. In the past four years, the team made huge advances in definition of brown adipocyte precursor cells and understanding how they differentiate into mature brown fat cells. Novel pathways activating BAT or inducing “browning” of white adipocytes were identified, which are potential targets for directed treatment of metabolic diseases. Furthermore, an arsenal of nutritional and pharmacological compounds activating thermogenesis was found during the project and validated in mice and men. Improved imaging technology enabled DIABAT researchers to investigate BAT activity in situ in humans.

Project Context and Objectives:
Obesity and type 2 diabetes create a major economic burden through loss of productivity and income, and consume 2–8% of overall health care budgets, equal to estimated health care costs of approximately 147 Billion Euros in the European Community per year. The threat for public health emanating from obesity and its associated metabolic disorders is recognized as highly relevant by the European authorities, highlighted by the publication of the White Paper on “A Strategy for Europe on Nutrition, Overweight and Obesity related health issues” by the European Commission in 2007 (COM 2007 279). In this context, the DIABAT project directly addressed the topic HEALTH-2011-2.4.3-2: “Development of novel treatment strategies based on knowledge of cellular dysfunction”. It recognizes the significant impact of obesity-related type 2 diabetes (“diabesity”) on public health, as well as socio-economic and societal concerns.

The overall objective of the DIABAT project was to recruit and re-activate endogenous energy-dissipating BAT as a preventive and/or remedial measure for weight and blood sugar control in obesity-related type 2 diabetes (“diabesity”), thereby halting or preventing destruction and facilitating recovery of pancreatic beta-cells under diabetic conditions. Accordingly, the specific objectives of DIABAT were:

I. To establish the molecular definition of brown adipocyte progenitor cell populations

− Definition of molecular and structural identities of BAT/BRITE stem cell populations by large-scale lineage tracing and gene knockout experiments in mice to obtain specific genome-wide coding and non-coding gene expression profiles, as well as cell surface fingerprints.
− Study of adipocyte precursors and signalling cues specifying determination and/or differentiation of “BRITE” adipocytes in response to environmental challenges, nutritional and pharmacological interventions, and chronic inflammation in diabetes.

II. To uncover the recruitment and activation of BRITE/brown adipocytes and its impact on the activity of pancreatic beta-cells

− Investigation of endo-/paracrine as well as inflammatory mechanisms determining BAT and BRITE adipocyte activities, triggered - at least partly – by communicating with pancreatic beta-cells and other tissues under conditions of insulin resistance and diabetes in cell and animals models.
− Implementation of pharmacological, nutritional or environmental intervention studies to recruit and activate/expand BRITE/brown adipocytes/BAT in both animal models and obese diabetic patients.
− Development of new transplantation protocols for BAT through targeted ex vivo regeneration studies.

III. To develop clinical targeting and monitoring of BAT depots and progenitor cells in humans

− Correlation of the activity of endogenous BAT depots in adult diabetic and obese pre-diabetic humans via prospective clinical studies using positron emission tomography (PET) scanning.
− Exploration and improvement of alternative imaging technology to estimate BAT depot size in humans, including MRI and PET/CT studies using free fatty acid tracers.
− Assessment of the glucose-consuming capacity of BAT depots in normal, glucose intolerant, as well as type 2 diabetic subjects, to determine the significance of increasing this capacity for diabetic hyperglycemia.

To achieve these goals, 6 Work Packages (WP) have been defined as specified below.


• WP1: BAT and BRITE stem cell definition

The general aim of this WP was to unravel pathways and identify key factors that are involved in differentiation of brown/BRITE adipocytes. To this end, the consortium performed studies to provide better characterization at physiological, cellular and molecular levels of the endogenous and inducible brown adipocyte formation. In total, 11 partners were involved in this WP. The specific objectives of WP1 were:

− Lineage tracing in transgenic mouse models
− Stem cell isolation
− Molecular gene expression profiling
− Cell surface analysis


• WP2: BAT and BRITE stem cell isolation and cultivation

The major aim of this WP was to design standard operating procedures for the isolation and cultivation of BAT progenitor cells from stem and mature cell populations. To this end, the consortium established protocols for the isolation of these cells and the in vitro/ex vivo expansion and cultivation of both mature BAT and BRITE adipocytes and their precursors in mouse and human tissues. In total, 8 partners were involved in this WP. The specific objectives of WP2 were:

− Validation of surface markers/generation of antibodies
− In vitro expansion and testing of functional integrity of experimentally derived BAT and BRITE cells


• WP3: In vitro BAT/BRITE adipocyte recruitment and activation

The general aim of this WP was to obtain efficient and safe recruitment of brown adipocytes in vitro and to determine cellular cross talk with beta cells. To this end, cell culture studies with BRITE and BAT adipocytes were employed. In total, 12 partners were involved in this WP. The specific objectives of WP3 were:

− To achieve pharmacological activation of BAT adipocytes
− To achieve pharmacological recruitment of BRITE adipocytes
− To manipulate mRNA levels in BAT/BRITE adipocytes during differentiation
− To manipulate microRNA levels in BAT/BRITE adipocytes during differentiation
− To co-culture BAT/BRITE adipocytes and beta cells


• WP4: In vivo BAT/BRITE adipocyte recruitment and activation

The general aim of this WP was to reverse diabetic phenotypes upon BAT/BRITE recruitment and/or transplantation. To this end, DIABAT researchers were implementing nutritional, environmental and pharmacological intervention to recruit and activate/expand BRITE/brown adipocytes/BAT in vivo. 16 partners were involved in this WP. The specific objectives of WP4 were:

− Nutritional regulation of BAT/BRITE recruitment and its use to prevent diabetes and obesity in susceptible animal models
− To characterize the cold-dependent switch from WAT to BAT
− To characterize microRNA-mediated regulation of BAT/BRITE recruitment
− To develop transplantation protocols for BAT to prevent/reverse insulin resistance


• WP5: BAT-centered therapies in type 2 diabetes

The general aim of this WP was to evolve BAT-centred therapy options in obesity-related type 2 diabetes. To this end, the consortium developed clinical monitoring and treatment protocols for diabetic patients based on BAT activation and recruitment. 10 partners were involved in this WP. The specific objectives of WP5 were:

− To measure cold-induced activity of endogenous BAT depots (by FDG PET/CT) in adult type 2 diabetic (T2D), obese, and prediabetic humans and correlate the activity with metabolic parameters
− Histology of human BAT and BRITE cells
− To explore alternative imaging technology for measurement of BAT
− To employ dietary and pharmacological cues to activate/expand BAT/BRITE depots in human patient and/or healthy volunteer cohorts
− To develop of an ambulant monitoring device for the measurement of BAT-related metabolic parameters (temperature, heart rate, blood glucose, physical activity) for monitoring purposes in clinical tests


WP6: Coordination, management and dissemination

The scientific WPs 1-5 were supported by a management support team located at the coordinator’s premises. The team consisted of the leading scientist as well as a scientific and an administrative project manager. The specific objectives of WP6 were:

− To perform budgetary and administrative controlling
− To assure the timely delivery of deliverables and reports
− To control the scientific progress of the project
− To assure intra-project communication
− To promote project visibility and exploitation of results


DIABAT employed existing experience and knowledge of the function, dysfunction and physiological regulation of brown adipocytes to develop innovative therapeutic and preventive strategies for type 2 diabetes. By combining a multi-disciplinary approach ranging from molecular cell biology and genomics to in-patient clinical trials, DIABAT research was expected to translate state-of-the-art knowledge on the role of cellular dysfunction in white and brown adipose tissues into novel treatment strategies and pathways in anti-diabesity therapy, thereby focusing on a largely unexplored area of inter-organ communication between WAT and BAT as well as BAT and dysfunctional beta cell activity.
Project Results:
The overall objective of the DIABAT project was to recruit and re-activate endogenous energy-dissipating BAT as a preventive and/or remedial measure for weight and blood sugar control in obesity-related type 2 diabetes (“diabesity”), thereby halting or preventing destruction and facilitating recovery of pancreatic beta-cells under diabetic conditions.
To this end, a set of tools and methods has been generated during the initial phase of the project. These tools and technology include but are not limited to novel cell and animal models as well as common protocols and standard operation procedures. Moreover, huge transcriptomic and proteomic data sets have been generated by the consortium through high- and medium-throughput screening efforts. The data sets are publically available. Upon bioinformatical evaluation, these data have been employed for further in-depth functional characterization of factors involved in BAT/BRITE recruitment and activation. With respect to the clinical focus of this project, novel technologies for brown adipose tissue imaging have been tested and validated in human subjects in the context of metabolic health and disease. Ultimately, the consortium made substantial advances towards pharmacological and nutritional activation of human BAT.
The progress of the individual scientific DIABAT Work Packages is specified below.

Work Package 1: BAT and BRITE stem cell definition
In order to define strategies targeting thermogenically active adipocytes, the identification and molecular characterization of BAT and BRITE stem cells was essential. Consequently, this was the focus of the initial project phase of DIABAT and generated the basis for research performed in other Work Packages.
Initially, high and medium throughput screening approaches were performed in order to define specific molecular signatures of distinct types of adipocytes and their progenitor cells. The data sets were made publically available and can be used by the scientific community (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE51080 and http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE63031). It became clear though, that most of the markers suggested based on –omics approaches did not properly distinguish between BAT, BRITE and WAT, and that even markers that have the best distinctive capacity still left questions regarding the identity of some tissues. This finding clearly indicated the need for additional ways to characterize brown, BRITE and white adipose tissues and adipocytes, which will be achieved by employing the novel animal lines in the future. Nevertheless, the consortium progressed significantly towards molecular characterization of various mouse adipose tissues and cultured human adipocytes based on the expression of a large set of proposed marker genes.

Work Package 2: BAT and BRITE stem cell isolation and cultivation
Despite difficulties in defining an unambiguous marker signature for distinct adipocytes, an isolation procedure that allows for rapid and selective isolation of brown and BRITE progenitor cells from intact tissue has been developed based on combination of selection markers. Technically, the isolation was achieved by a combination of a novel tissue separation methodology with fluorescence-based (FACS) and magnetic cell sorting (MACS). The procedure has been developed and validated in a collaborative effort of an academic and an industry partner and is now ready for commercialisation.
In addition, the consortium agreed to define common standard operating procedures for the cultivation of BAT progenitor cells.

Work Package 3: In vitro BAT/BRITE adipocyte recruitment and activation
A major focus in this WP has been on the in vitro activation and recruitment of brown and BRITE adipocytes, respectively, by pharmacological means in order to increase energy expenditure. To this end, the consortium tested, identified and validated a list of nutritional and pharmacological compounds regulating brown and BRITE adipocyte differentiation and function. Apart from that, a major focus of the consortium was to elucidate the role of specific microRNAs in differentiation as well as thermogenic activation of adipocytes.
In the next step, DIABAT researchers functionally characterised the mechanisms underlying induction of thermogenesis in distinct types of adipocytes. It became evident that brown and BRITE adipocytes represent different cell lineages but at least with respect to energy dissipation do not demonstrate major functional differences. There may, however, be possibilities to selectively activate the cells, which could prove pharmacologically advantageous.
Adipose tissue is considered an endocrine organ. The consortium found that distinct types of adipocytes secrete specific sets of factors. Consequently, co-cultivation assays of adipocytes and cells of other tissues were performed to assess potential remote effects of adipocyte-secreted factors. As preliminary results were quite promising, approaches to identify specific factors with metabolic effects on metabolically active tissues will be followed in the future.

Work Package 4: In vivo BAT/BRITE adipocyte recruitment and activation
This WP had the purpose to translate the results from WP3 in an in vivo setting using appropriate animal models. To this end, various nutritional compounds that were shown to induce activation or recruitment of thermogenically active adipocytes have been validated in rodents and ferrets. Based on the interesting findings from in vitro studies in WP3, the role of particular microRNAs on brown/BRITE activation and recruitment has been analysed in vivo. Detailed analysis of the effects of dietary lipids and proteins on brown/BRITE adipose tissue was performed, and it could be demonstrated that various metabolic intermediates have a specific impact on systemic metabolism. The most promising effects were observed with certain fatty acids. Notably, the work of the consortium showed that particular metabolic effects of certain diets used in mouse studies depended on the strain that has been used.
Another focus of this WP was to understand the mechanisms underlying the cold-induced browning of white adipose tissue. In this context, gene expression analyses of adipose tissues from cold- and warm-exposed animals were performed, which resulted in the identification of several pathways involved in the cold-dependent adipose tissue switch.
As the detailed understanding of in vivo brown/BRITE activation has now notably improved based on DIABAT work, future projects will aim for development of protocols for transplantation of activated thermogenic tissues in order to improve the energy balance on the recipient. To this end, a mouse model has been developed, which express proteins that can be used to visualize thermogenic activity in adipose tissues. Using fat cells derived from these animals, transplants can be followed in vivo.

Work Package 5: BAT-centered therapies in type 2 diabetes
This WP had two main foci: a) improvement of BAT imaging technology in humans, and b) activation of thermogenic tissues in humans. Huge progress has been made regarding BAT imaging, including PET and MRI technology. An animal study used high-resolution laser Doppler imaging to analyse blood flow in brown fat depots. Improvements in imaging technology were highly useful for comparative studies of BAT induction in lean and obese, as well as healthy and diabetic human subjects that were performed in the DIABAT project. Notably, cold-acclimation has been shown to be efficient in combating insulin resistance in type 2 diabetic human patients.
Furthermore, a specific bile acid has been demonstrated to efficiently activate human BAT. Further pilot studies were performed using human adipose tissue derived stem cells, which are a novel model system for analysis of adipocyte differentiation in a human setting.

Potential Impact:
The threat for public health emanating from obesity and its associated metabolic disorders is recognized as highly relevant by the European authorities, highlighted by the publication of the White Paper on “A Strategy for Europe on Nutrition, Overweight and Obesity related health issues” by the European Commission in 2007 (COM 2007 279). In this context, the DIABAT project directly addressed the topic HEALTH-2011-2.4.3-2: “Development of novel treatment strategies based on knowledge of cellular dysfunction”. It recognizes the significant impact of obesity-related type 2 diabetes (“diabesity”) on public health, as well as socio-economic and societal concerns.
DIABAT activities were anticipated to have substantial impact on the improvement of public health. In particular, DIABAT research aimed at the removal of risks associated to common therapies of type 2 diabetes by taking a strict translational approach to exploit the potential of thermogenic adipose tissue for increasing energy expenditure by rebuilding and/or re-activating energy consuming BAT and BRITE depots in the adult diabetic organism. This has been achieved by an extensive molecular characterization of BAT and BRITE precursor cells and the pathways involved in their differentiation towards mature thermogenic adipocytes. Definition of BAT and BRITE progenitor cells at the molecular level as performed in the DIABAT project, substantially promoted advances in the characterization and diagnostic usage of these cells as checkpoints in energy homeostasis and the pathogenesis of insulin resistance and obesity-related type 2 diabetes. Furthermore, the identification of pharmacological and nutritional compounds involved in adipocyte differentiation as well as “browning” of white fat paved the road towards development of BAT/BRITE directed
DIABAT experimental routes were designed to have a high potential for commercialisation. Consequently, we expect the results of this project to have a strong impact on the pharmaceutical, as well as the biotechnology industries in Europe. Industrial and SME partners had central roles in the consortium, demonstrating the high potential that European industrial companies detect in the research concept proposed here. The clinical and screening protocols established by DIABAT will be made available to scientific and commercial partners. The regenerative technologies established within this project will thus help to strengthen the competitiveness and technological possibilities of European companies in this field and will thereby assist in the generation and the securing of highly qualified employment possibilities.
The substantial impact the DIABAT project on the scientific community is highlighted by more than 120 peer-reviewed publications, including articles in Cell, Cell Metabolism, Nature, Diabetes and the Journal of Clinical Investigation. Roughly 100 oral presentations were given by DIABAT researchers at national and international conferences. Visibility of DIABAT research was particularly highlighted by the organisation of the Keystone Symposium on Beige and Brown Fat in Snowbird, UT, USA, in April 2015 by a DIABAT partner. A major number of DIABAT researchers attended the symposium, which is one of the most-recognized international meetings in metabolic research, and now for the first time had a focus on brown fat. Among consortium members attending the Keystone Symposium were 5 invited speakers presenting project-related work.
DIABAT researchers held lectures and seminars on brown fat biology for students at their respective institutions and beyond. 49 PhD students were involved in DIABAT, of which 16 already finished their thesis within the funding period.
Finally, DIABAT work was highly recognized by the general public, which is documented by approximately 50 appearances in newspapers, radio and TV-shows.

List of Websites:
Website and contact

www.diabat.org

Contact:

Dr. Tobias Schafmeier
European Research Administrator

Scientific Coordinator

Institute for Diabetes and Cancer (IDC)
Helmholtz Center Munich
and
Joint Heidelberg-IDC Translational Diabetes Program
Heidelberg University Hospital

Ingolstädter Landstraße 1
85764 Neuherberg
Germany

T: +49 89 3187-1048
M: tobias.schafmeier@helmholtz-muenchen.de
W: www.helmholtz-muenchen.de/IDC