Final Report Summary - TOBI (Targeting OBesity-driven Inflammation)
Obesity has become an epidemic in industrialised countries. In Europe, about 15 % are affected, with a rapidly increasing number of adolescents. Obesity has detrimental effects by promoting diabetes and cardiovascular diseases. Overweight patients suffer from an obesity-driven inflammatory shift originating from chronic low-grade inflammation primarily in fat tissue, which has been implicated to cause insulin resistance and cardiovascular disease. Researchers hope to interfere with common health complications in overweight individuals by targeting inflammatory processes in adipose tissue. Signalling molecules termed adipokines, mediate the interaction between adipocytes and inflammatory cells with detrimental effects on other organs. TOBI addresses pathophysiological mechanisms related to excess fat which promote secondary health problems through adipokine-mediated crosstalk and an inflammatory drift. The release of metabolic intermediates during the breakdown of fat like fatty acids could release adipokines.
The remit of this project is to identify novel strategies to reduce or reverse adipokine-mediated adverse interactions. Special attention has been placed on the study of novel fat-derived adipokines with the goal to identify novel targets for drug development. Adipocyte ultrastructural analyses and studies of how adipose tissue acts on the adjacent vasculature added to the understanding of the relevant pathophysiology. Within 42 months the consortium has made significant progress in all objectives. Critical components of stress and nuclear receptor signalling in inflammatory processes in obesity were identified and its impact on adipocyte structure analysed. The impact of lipolysis and fat-derived adipokines on inflammatory alterations in obese adipose tissue proved the breakdown of fat and fat-derived mediator promotes obesity-related complications. TOBI has led to the identification of a potential drug target, which is already exploited. Important tools have been developed including standards of experimental and clinical protocols. The TOBI bio bank is an important resource of biological specimens related to obesity from more than 250 well-characterised subjects, which will continue to facilitate material exchange between TOBI members. These activities have provided a sustainable basis for a lasting collaboration. Professional project management and dissemination of results significantly contributed to successful collaboration of the beneficiaries. The coordinator has encouraged all beneficiaries to actively disseminate their project aims and results to colleagues, stakeholders and the wider public. Public awareness has been addressed with a website, press releases and other activities, including an event (see http://www.tobi-project.eu online).
TOBI was highly successfully and has met essentially all objectives within schedule. The project was a timely initiative, which contributed significantly to our improved understanding of adipose tissue inflammation and its harmful clinical consequences, which will be corroborated by further publications. The tools established have fostered obesity research in Europe. If the drug target identified will turn out to be valuable for clinical application, TOBI could bring enormous benefit to obese patients, contribute to sustainable health systems in Europe and support the competitiveness of the European health industry.
Project context and objectives
The prevalence of obesity has escalated to epidemic proportions in the past decades in industrialised countries. The problem is the detrimental effects obesity has on health. It is invariably accompanied by metabolic disorders such as diabetes and metabolic syndrome. Reduced insulin action in the body, (insulin resistance) is the basis for the metabolic syndrome and type 2 diabetes and promotes atherosclerotic alterations leading to vascular disease. Enlargement of fat depots by excess food consumption is the basis of obesity but weight reduction turned out to be difficult to achieve and keep in long-term. It is only recently that chronic systemic low-grade inflammation, originating primarily from the fat tissue in obese individuals, has been identified as an important cause of insulin resistance leading to diabetes and vascular disease.
Reversing the obesity-driven inflammatory shift should improve insulin sensitivity and prevent diabetes and cardiovascular disease in obese patients. Adipose tissue produces a large number of adipokines; soluble messenger molecules that mediate the interaction between adipose tissue and inflammation and immunity. Adipokines may be inflammatory or anti-inflammatory in action and are produced by fat and other cells present in fat tissue as well as infiltrating inflammatory cells. In obesity, a large number of inflammatory adipokines affect the function of other organs by different mechanisms. Primary triggering events within adipose tissue must be targeted to curb all adverse adipokine-mediated crosstalk.
Most adipokines are proteins by nature. Adipose tissue secretes considerable amounts of potent messenger molecules derived from fat molecules (lipids). Few of these active lipid molecules have been identified so far. By virtue of their signalling capacity and production by adipose tissue, such lipid-derived adipokines (LDA) effectively qualify as adipokines and are considered to be crucial in the study of obesity-associated adipokine crosstalk. Research has made clear that chronic inflammation is triggered by obesity and that this activates cells in a way that leads to insulin resistance and atherosclerosis. It has become evident that adipokines, produced by adipocytes and infiltrating inflammatory cells are key players in mediating inflammatory signal transduction. Significant study has been carried out on protein adipokines but lipid-derived molecules have not been addressed properly. Mechanisms triggering the inflammatory cascade leading to inflammatory adipokine production and subsequent impact on peripheral tissues remained largely unresolved. Cells stress occurring in the endoplasmic reticulum (ER), an organelle implicated in metabolic actions within the cell, has recently been suggested as a mechanism triggering inflammatory cell activation and insulin resistance in obesity. Factors inducing ER stress such as altered nutrient fluxes in fat cells, e.g. by disorders in the release of fatty acids by lipolysis are still elusive. Structural alterations of the ER could occur in stress situations but those have not yet been defined on an ultrastructural level.
The presence of inflammatory cells (macrophages) in adipose tissue of obese subjects puts another piece into the puzzle of adipose tissue inflammatory alterations. Different inflammatory cell activation pathways could have different impact on adipokine production and crosstalk when activated in fat cells and adipose tissue macrophages (ATM). Inflammatory activation of ATM or fat cells could be significantly modulated by nutrient-sensing nuclear receptors that directly regulate genes in cells. Nuclear receptors represent potential drug targets by nature, but their involvement in fat inflammation has not been elucidated. Crosstalk of adipokines with other organs through the systemic blood circulation promotes the development of insulin resistance leading to development of type 2 diabetes. Adipokine-mediated crosstalk with tissues other than adipose tissue does not necessarily require systemic action. Particularly important tissues are embedded in adipose tissue and could be directly affected by adjacent adipokine production. Research addressing multiple factors that promote inflammatory adipokine crosstalk in obesity is urgently required in order to identify crucial mechanisms and potentially targetable molecules.
Objectives
The overall aim was to integrate research on the interplay between the cellular stress, the inflammatory response involving macrophage infiltration and changes in lipolysis and lipid-derived mediator substances within fat tissue and how they feed into altered adipokine expression leading to the development of insulin resistance and vascular dysfunction. TOBI aimed succeeded in achieving a knowledge base by standardising the analytical approach via the TOBI toolbox, using defined clinical data recording of individuals from which biological materials have been collected, sophisticated biochemical and histological analyses, undertaking genome-wide expression analyses, investigating cells at an ultrastructural level, exploiting newly generated and existing animal models and creating a pipeline for drug development. The objective was to analyse mechanisms that investigate altered adipokine-mediated crosstalk including the involvement of LDA associated with obesity. This approach led to the identification of a novel drug target with the potential to interfere with the obesity-associated inflammatory shift. Several other molecules have been suggested with more work being required for defining their potential as drug targets.
This overall TOBI aim encompassed the following objectives: Identification of cellular triggering and activation events pivotal for the initiation of inflammatory drift through adipokine crosstalk, identification of novel LDA and their contribution to the inflammatory drift and validation of the involved pathways and molecules as potential targets for therapeutic intervention, and in best case assay development for a selected therapeutic target. To this end, TOBI implemented a collaborative research project using a wide variety of methods including genetic approaches in mice, measures of gene regulation, biochemical investigations, ultrastructural analyses up to analyses of clinical specimen. Collectively, the consortium combined all the necessary expertise to investigate the basis of obesity-associated inflammatory drift.
For objective 1, TOBI addressed a variety of pathophysiological mechanisms related to excess fat that are important for initiating and promoting inflammatory adipokine-mediated crosstalk with other tissues. Emphasis has been placed on studying ER stress signalling, the dissection of inflammatory activation pathways and the activation of nuclear receptors as well as lipolysis. The release of fatty acids and metabolic intermediates during lipolysis is linked to objective 1 as a potential triggering mechanism, but also to objective 2, since the release of fatty acids in most instances is a prerequisite for LDA production. Whereas objectives 1 and 2 aimed to elucidate pathophysiological mechanisms, objective 3 aimed to exploit the resulting knowledge by transferring it to drug development, in close collaboration with the respective TOBI partners. In order to address these objectives in a manner that shares resources and allows data comparison between the partners of the consortium, the TOBI Toolbox has been established. The toolbox consists of TOBI standards, i.e. SOP. The TOBI Standards have provided a valuable basis for European research on obesity and promote collaboration among European scientists and enterprises. The TOBI toolbox includes a TOBI bio bank for sharing human material. The TOBI bio bank is accompanied by a database integrating parameters for detailed characterisation of patients. TOBI generated novel analytical tools for LDA and generate novel animal models that were exploited by the consortium or shared with other researchers to strengthen the ERA.
The objectives included identification of novel adipokines, particularly LDA. TOBI has added to the detection of obesity-associated adipokine-mediated adverse interactions. Future research will show to which extent these results could lead to the development of individualised pharmacological strategies in order to optimise the delivery of healthcare to European citizens by tailored medicine. In order to implement this comprehensive collaborative research programme, a separate WP has structured management activities. In addition, another WP was included to realise a variety of dissemination activities directed to the scientific community including envisioning of patent applications. WP2 included numerous dissemination activities to reach the general public and increase the awareness for the TOBI project and more general for obesity as an important health issue.
Project results
WP4 ER stress-induced signalling for inflammatory drift and insulin resistance
Obesity damages human cells by damaging specific subcellular structures called organelles. One such organelle affected by obesity is the ER. In cells, the ER specialises in synthesising protein destined for excretion by the cell and proteins residing in cellular membranes. This WP addressed the question how a malfunctioning ER in obese patients contributes to the development of type 2 diabetes in these patients. Obesity is closely associated with two cellular signalling events; insulin resistance and inflammation. Insulin resistance is the inability of human cells to appropriately respond to the protein hormone insulin. Insulin is released by the pancreas after having had a meal and stimulates the uptake and utilisation of nutrients by cells. An inability to respond to insulin, termed insulin resistance, leads to chronically elevated blood glucose levels, which damages blood vessels and leads to type 2 diabetes and its complications. Inflammation is the response of cells and organisms to damage with the view to repair this damage and to remove damaged cells from the organism.
This WP has characterised molecular mechanisms through which a malfunctioning ER causes insulin resistance. We have evaluated the role of activation of a transmembrane protein of the ER, called IRE1 that reports on correct functioning of the ER, on the ability of insulin to stimulate glucose utilisation by human cells. We have focused on culture models for the main tissues and organs that contribute to blood glucose homeostasis, muscle cells, liver cells and fat cells. We have mimicked the damage caused by obesity to the ER using pharmacological agents that interfere with protein synthesis by this organelle. Using biochemical assays and ultrastructural methods, we confirmed that these drugs damage the ER. We characterised the consequences of this damage on the ability of cells to respond to insulin.
To characterise the effects of damage to the ER we have used biochemical assays to detect the activated, i.e. phosphorylated forms of several signalling molecules activated by insulin. We have observed a biphasic role of damage to the ER on the ability of cells to respond to insulin. Short periods of damage to the ER do not affect the ability of cells to respond to insulin, while prolonged damage to the ER leads to profound insulin resistance. Biochemical characterisation of signalling molecules involved in transducing the insulin signal to promote glucose / nutrient uptake and utilisation by cells has revealed novel interactions between ER damage and the insulin signalling pathway and has identified novel molecules involved in the insulin signalling pathway that are inactivated/inhibited in cells with damage to the ER. We have observed that short periods of ER stress induce a subset of inflammatory signalling events leading to activation of the protein kinase JNK in cells. Activation of these inflammatory signalling pathways was transient. While we find that activation of these inflammatory signalling pathways requires IRE1 and an adaptor protein, called TRAF2, in genetic experiments using gene knock-out cell lines and gene silencing technologies we have not found any evidence for these inflammatory events to contribute to the development of insulin resistance. Detailed molecular genetic characterisation of one molecule activated by damage to the ER has identified the enzymatic activities of this molecule that activate inflammatory signalling in response to ER damage.
WP5 Crosstalk between lipolysis and adipokines
Triacylglycerol are neutral fats that are efficient energy depots. Moreover, they provide essential building blocks for membranes and organelles as well as hormones. These triacylglycerols look somewhat like the letter 'E'. There is one glycerol molecule as backbone with three fatty acids attached. These fatty acids act as signalling molecules, which send signals throughout the body to accelerate or stop biological processes. It is conceivable that a person who is obese sends different fatty acid signals than a lean person. The exact mechanisms of lipid signalling are still poorly understood. The enzymes required to cleave these fatty acids off storage fat molecules are called lipases. Lipases are most active in times of increased energy demand. The first fatty acid is cleaved off by a lipase called adipose triglyceride lipase (ATGL). This enzyme was discovered by the principle investigator of B10, Rudolf Zechner, in 2004. It is the rate-limiting enzyme in the breakdown of storage fat. Working with lipase-deficient mice is the only way to find out the exact roles of the individual enzymes. Mice that lack this enzyme have massive fat accumulation in all organs, especially the heart and die prematurely because of heart failure.
There are human patients that have a defect in their ATGL-gene and suffer from a similar condition like the ATGL-deficient mice. They accumulate excess fat in skeletal and cardiac muscle as well as in the heart and require heart transplantation at an early age. Their condition is called neutral lipid storage disease with myopathy. Despite having more body fat ATGL-mice seem to be protected from developing diabetes. Obese humans have a much higher incidence of diabetes than lean people. There is the hypothesis that fat does not do much harm as long as is stays within the designated fat tissues. When it is broken down and released into the blood it seems to act toxic. Since ATGL-deficient mice cannot mobilise the fat, but retain it in their fat depots, there are only low fatty acid levels observed in their blood. The second step in the breakdown of storage fat is performed by the enzyme hormone-sensitive lipase (HSL) and the last step by monoacylglycerol lipase (MGL). Mice missing either of these two lipases are relatively lean and quite healthy compared to ATGL-deficient mice. HSL-deficient male mice are infertile, underlining the importance of fat as a precursor for hormones and other signalling molecules. All these processes are complex and involve a large number of additional players that fine-tune the rate of fat breakdown. We still do not understand many mechanisms, but try to set up hypotheses that match our experimental data.
Obesity is often associated with inflammation. Within TOBI we raised the question whether mice lacking a certain lipase show more inflammation or stress of the protein synthesising unit of the cell (ER) than normal mice. We wanted to find out if certain signalling molecules that are known or proposed to be involved in the breakdown of fat (adipokines) can influence the activity of lipases. Many approaches have been used to investigate these questions in cooperation with TOBI partners, including biochemical and molecular biology techniques as well as microscopic analysis. We need to continue our work after the end of the project to be able to correctly interpret all of the obtained data.
As a first result we can state that lipase-deficiency with or without elevated fat composition do not always mean that there is increased inflammation in mice. It is difficult to make direct comparison between lipase-deficient mice and obese humans. The scientific data obtained during the project enables us to shed light onto the connection between lipases and adipokines, even though we cannot give an explanation for all experimental data yet. In a particular area of metabolic signalling we have made considerable progress within TOBI, which will most likely result in a high-level publication within the next few months. We suggest a therapy for patients suffering from neutral lipid storage disease with myopathy based on your experimental data with mice. The revision of the manuscript has been sent in to Nature Medicine. Unfortunately, we are not allowed to publically disclose any results which are part of this manuscript prior to the date of publication. As soon as the content of this publication can be made public it will be posted on the TOBI website.
WP6 Identification of LDA and their role in adipokine crosstalk
Adipokines are not necessarily proteins by nature. Due to the release of fatty acids from fats stored in fat (adipose) tissue, lipid-derived molecules are synthesised locally. Many molecules derived from polyunsaturated fatty acids (PUFA) are highly potent mediators to promote or reduce inflammatory reactions. Recently identified molecules derived from omega-3 PUFA which are predominantly found in marine fish oils are of high interest because they actively repress inflammatory reactions by promoting their resolution. A lack of resolving lipid mediators in adipose tissue, LDA from obese patients could contribute to the ongoing inflammatory response that leads to insulin resistance and type 2 diabetes. Within this WP the coordinator has set up a tight collaboration with a small and medium-sized enterprise (SME) partner with top level expertise in small molecule analytics in order to establish methods for the detection of potent PUFA-derived mediators in adipose tissue. A number of substances could be found in adipose tissue from humans and mice. The steric structure of many substances could be resolved. With this tool we analysed LDA from obese and lean humans and different animal models of obesity.
We found that human obesity altered the concentration of a number of LDA and their precursors in adipose tissue. These alterations comprised molecules derived from omega-3 PUFA and omega-6 PUFA, the latter being in general of more pro-inflammatory nature. We found alterations in LDA concentrations in two models of murine obesity. Regional analysis revealed similar obesity-related changes in different adipose depots. Studies from the coordinator's laboratory have revealed that a diet rich in omega-3 PUFA reduces adipose tissue inflammation in mice, whereas omega-6 PUFA were virtually ineffective. We fed obese mice with a diet enriched in omega-3 PUFA and analysed LDA in adipose tissue and their metabolic changes. As expected, measures of adipose tissue inflammation were decreased in obese mice following omega-3 PUFA treatment. We found a number of resolving lipid mediators increased in adipose tissue of omega-3 PUFA-treated animals. Changes in LDA were detectable after short periods of treatment. We studied gene expression of molecules relevant to LDA production and action and found interesting changes. These results indicate that changes in LDA could contribute to the anti-inflammatory action of omega-3 PUFA in obese adipose tissue and their beneficial metabolic action. Based on the promising results in mice we conducted a clinical trial looking for effects of omega-3 PUFA on LDA production in obese patients. Omega-3 PUFA-derived LDA were increased in adipose tissue along with reduced inflammatory changes. These data emphasise potential relevance of omega-3 PUFA-derived LDA in terminating obesity-driven adipose tissue inflammation. Selected LDA and their precursor were tested in mice in order to see whether application of these substances could reduce adipose tissue inflammation in obesity. A selected LDA was particularly potent in reducing adipose tissue inflammation and improving metabolic function. In addition to the importance of adipokines, LDA play important roles in sustained inflammatory response in fat tissue of obese subjects. LDA could be of potential use in reducing obesity-driven inflammation, thereby preventing type 2 diabetes and cardiovascular disease in obese patients.
WP7 Inflammatory signalling pathways that elicit inflammatory adipokine crosstalk
Research from the last dozen of years has demonstrated that low-grade inflammation in the adipose tissue is likely an alarm signal from lipid-filled adipocytes, which are in danger of cell death due to huge amounts of lipid they have to take up. These signals attract immune cells, which try to engulf dying adipocytes. Due to the massive amount of lipids, the immune cells send alarm signals to attract more immune cells. These signals can affect not only the functionality of adipocytes and immune cells locally, but can also impact other tissues. This low-grade inflammation is able to impair the actions of the key hormone insulin, which in healthy people keeps blood glucose and lipid levels in check. When the ability of insulin to normalise blood glucose and lipids decreases the body is able to produce increased levels of insulin. After years of increased production of insulin, the insulin-producing cells stop working properly, giving rise to uncontrolled blood glucose levels (overt diabetes mellitus) from which around 9 % of Europe's population suffers. When inflammatory signals reach cells, they induce the activation of enzymes, termed kinases that can affect basically all intracellular processes. Research from the last 10 years has implicated that these inflammatory enzymes are able to reduce the efficacy of insulin, i.e. can induce insulin resistance. Experiments could demonstrate that one enzyme (c-Jun kinase) is intimately involved in the induction of insulin resistance, and is commonly activated in tissues from obese and diabetic patients. The research efforts during TOBI were aimed to define the tissues in which activation of this kinase affects glucose and lipid metabolism. We generated mouse models in which this kinase was missing only from one tissue.
Mice which lacked this enzyme only in the liver were not protected from insulin resistance and developed diabetes-like symptoms as much as control animals when fed a high-fat diet simulating an unhealthy diet. Mice lacking this kinase in the muscle were not protected from these symptoms. These findings in combination with results from other laboratories indicate that activation of the enzyme in classical tissues involved in metabolism does not play a major role during the development of obesity-associated pathologies. Obesity is a consequence of energy intake exceeding energy consumption. Food intake and utilisation of energy are under control of neuronal circuit. In the last 20 years, it has become obvious that hormones do not only act directly on classical tissues, but also on the brain, to regulate feeding, energy expenditure, glucose and lipid metabolism. The possibility of inflammation in specific brain regions involved in regulating glucose and lipid metabolism in obesity had not yet been addressed. We found that inflammation can be detected in these brain areas of obese animals, similar to what can be detected in liver or adipose tissue. The c-Jun kinase enzyme was activated. We generated mice which lack this enzyme in the brain. These mice were protected from inflammation in the adipose tissue, demonstrated lower blood glucose levels and did not suffer from a fatty liver when challenged with a high-fat diet. These mice were shown to be highly insulin-sensitive both in the brain and in classical tissues. These results demonstrate that inflammatory enzymes may not only act in peripheral tissues, but also in the central tissue involved in regulating body weight, obesity and glucose levels, the brain, and activation of these enzymes in the brain may participate in the development of obesity and diabetes mellitus.
WP8 Nuclear receptor LXR in adipose tissue macrophages
Obesity is a low-grade chronic inflammatory disease characterised by enlarged fat cells associated with a pro-inflammatory state in adipose tissue due to the infiltration of macrophages (ATM), which aggregate in crown-like structures usually surrounding dead adipocytes. Within adipose tissue, ATM are a major source of inflammatory cytokines which may be involved in obesity-related inflammatory disorders and the recruitment of additional immune-inflammatory cells. Adipose tissue macrophage infiltration and ensuing inflammation precedes the development of insulin resistance, at least in animal models. ATM are surrounded by pre-adipocytes and adipocytes and it is likely that paracrine loops exist via the production of adipokines, free fatty acids and derived mediators, leading to inflammatory changes. Pre-adipocytes produce higher levels of pro-inflammatory cytokines compared to adipocytes, suggesting that they play an important role in the induction and maintenance of inflammation and may operate as potent ATM activators.
To characterise the phenotype and function of human ATM, a global gene expression analysis comparing ATM and macrophages (monocyte-derived macrophages or MDM) derived from blood from the same morbidly obese patients undergoing bariatric surgery was performed. Several genes over-expressed in ATM were identified to belong to cytokine-cytokine receptor interaction pathways yielding a specific ATM pattern. In vitro experiments indicated that the phenotype of ATM is related to factors released by the pre-adipocytes, whereas lipids released from mature adipocytes had no effect. ATM displayed a gene expression profile sharing similarities with tumour-associated macrophages (TAM) isolated from human tumours. ATM were found to release a repertoire of growth factors, cytokines, chemokines and enzymes involved in the regulation of tumour growth, generation of blood vessels, tumour spreading to adjacent and remote tissues and organs similar to that observed in TAM. These data indicate that the human ATM phenotype sharing similarity with those of human TAM, is directed by environmental cell types and suggest that factors released by ATM may be involved in cancer initiation and progression in obese patients. ATM-conditioned medium increases the expression of genes leading to lipid accumulation in breast cancer cells. Over-expressing tumours display aggressive behaviour compared to those with normal lipid gene expression levels, indicating a pivotal role in cancer cell survival and in the maintenance or enhancement of the malignant phenotype.
These results indicate that factors released by ATM could affect tumour aggressiveness. Many tumours have increased levels of obesity-related factors in their microenvironment, rendering the tumours more aggressive. Macrophages in peri-tumoural adipose tissue which release obesity-related factors could be locally involved in carcinogenesis. Our data indicate that ATM have a gene expression profile comparable to TAM raising the possibility that ATM may be one of the potential links between obesity and cancer. LXR (liver-X-receptor) is a ligand activated transcription factor playing important role in the control of macrophages functions. Its expression and activity in human ATM were unknown. Human ATM have been isolated from human visceral adipose tissue and cultured in presence or absence of highly specific synthetic LXR agonists. Circulating MDMs have been prepared from the same donors. Gene expression profiling analysis has been performed to potentially identify novel LXR target genes specifically regulated in ATM. Results indicate that expression of LXRa is significantly lower in ATM compared to the MDM isolated from the same obese donors. Results show that only few genes are differentially regulated by LXR in ATM and MDM. Among these LXR target genes, we have identified two chemokines (molecules that attract inflammatory cells) which showed a regulation in ATM. These two genes are strongly inhibited by LXR activation in ATM and not regulated or regulated in an opposite manner by LXR agonists in MDM. These results point to the relevance of gene regulation by LXR for the specific type and function of ATM in obese adipose tissue.
WP9 Ultrastructural analysis of obese adipocytes by electron microscopy
Adipose tissue is an important organ producing hormones able to influence our behaviour and molecules that could change the metabolism of individuals. It was discovered that adipose tissue of obese animals and humans is infiltrated by macrophages producing a mild chronic inflammation. This inflammation seems to be important because interfering with the activity of insulin receptor induce a state of insulin resistance that induce diabetes. Insulin is the hormone produced by pancreas that act as a key that open the door of cells allowing the entrance of glucose, a fundamental fuel for most cells. Insulin receptor are the lock for the key insulin. When the lock is not functioning the key does not open and glucose remains in the blood producing hyperglycaemia (type 2 diabetes). We discovered an important cause of this inflammation; the death of adipocytes. Adipocytes are cells that represent main constituents of adipose tissue. In obese individuals the energy to be stored is high and adipocytes have to expand their volume, thus fat cells become six to seven times bigger. Studies have shown that this increase has several consequences on fat cells. The thickness of the wall of the adipocytes gets reduced and the number and size of mitochondria is reduced.
These alterations were studied by electron microscopy. Two animal models of obesity were investigated, which develop a form of diabetes that is similar to what happens in humans. The alterations of fat cells were not identical in all fat depots. It is known that fat in all mammals is located in two distinct anatomic districts; subcutaneous and visceral. Accumulation of abdominal fat is more dangerous than accumulation of subcutaneous fat. Abdominal fat is dangerous because it is often associated with all metabolic disorders associated with obesity. Visceral fat behaves differently than subcutaneous fat. Studies showed that visceral fat cells are more fragile than subcutaneous fat cells. When they are required to expand, subcutaneous fat cells are able to expand better and reach larger sizes that are not reached by visceral fat cells. Visceral adipocytes die at a size in which subcutaneous fat cells survive. Inflammation of visceral fat is more pronounced than inflammation in subcutaneous fat supporting the importance of inflammation for the metabolic disorders associated with abdominal obesity. Studies highlighted that brown adipocytes surround the area where heart and aorta are contained. In obese animals these cells transform into normal fat cells and die quickly, inducing inflammation with possible immediate cardiovascular consequences. These studies are important to understand the mechanisms that are rapidly expanding plague, affecting millions of citizens. The understanding of basic cell biology underlying this disease is the prerequisite for pharmaceutical research to create new drugs to combat and curb obesity and type 2 diabetes.
WP10 Effects of obesity on adipokine-mediated crosstalk
Research has explored the differences between accumulations of adipose tissue in different sites. Accumulation of fat in the intra-abdominal depot is associated with greater risk of metabolic and cardiovascular complications than accumulation in depots under the skin. Differences in function between the visceral and subcutaneous adipose depots and their crosstalk with other organs have been described. Until now, the importance of the perivascular adipose depot has been neglected. Abnormalities of blood vessels are important in obesity; increased contraction may contribute to high blood pressure. Changes in the vessel's structure are important for maintaining the normal shape of the vessel, for allowing blood flow and regulating forming new vessels. Abnormal remodelling contributes to narrowing of blood vessels which causes cardiovascular disease and is important in adipose tissue in obesity, where lack of oxygen supply exacerbates inflammation within the adipose tissue. Blood vessels are surrounded by perivascular adipose, a layer of fatty tissue. The extent to which this accumulates in obesity may predict cardiovascular disease and perivascular adipose might influence the contraction of the blood vessels. This WP aimed to characterise perivascular adipose tissue in lean and obese mice, with a focus on the crosstalk between the perivascular adipose and the blood vessel wall. Perivascular adipose tissue was collected from mice with or without diet-induced or genetic obesity. The microscopic appearances and pattern of genes active in the perivascular adipose tissue were compared with visceral and subcutaneous adipose tissue. Healthy perivascular adipose has a pattern of gene activity that is distinct from other depots, with a low level of inflammation.
We corroborated some of our findings in tissue biopsies of human perivascular adipose tissue obtained at surgery. In obesity, perivascular adipose accumulates and changes its characteristics to become similar to visceral adipose, producing potentially damageing inflammatory substances. We studied vessels from mice using a device called a myograph in which their responses to various drugs are tested. This showed that obese mice have abnormal vessel contraction. Perivascular adipose has a powerful effect to block vessel contraction by a variety of drugs. We tested which cells in the blood vessel wall are targets for the effect of perivascular adipose tissue. This confirmed a physiological interaction between perivascular adipose and blood vessel function. Its importance in the vascular dysfunction of obesity is less clear; we did not find that the differences in blood vessel function in obesity could be attributed to differences in the substances released from perivascular adipose. To test whether perivascular adipose affects blood vessel remodelling, we set up a laboratory method in which we allowed mouse blood vessels to form new vessel sprouts in a plastic dish. We added material produced by perivascular and other adipose tissue, and showed it had dramatic effects on the number of new vessels formed. This suggests that adipose tissue can directly influence the shaping and reshaping of blood vessels.
Perivascular adipose tissue from obese animals has altered effects on vessel remodelling. To identify factors that might mediate the effect of perivascular adipose tissue on vessel remodelling, we used protein arrays to measure a host of potential factors in the conditioned medium. This identified several candidates that can be potential drug targets. We tested whether release of steroid hormones from the perivascular adipose tissue is important, using adipose tissue from mice lacking an enzyme that makes steroids in the adipose tissue. This showed substantial effects of the enzyme on remodelling activity of adipose tissue. We were able to replicate this finding using an inhibitor of the enzyme. By collaborating with other consortium partners we explored mechanisms that might be responsible for the effects of perivascular adipose on vessel remodelling. This included testing whether LDA influence vessel remodelling. These studies illustrate the utility of the screening assay we developed for dissecting control of vessel remodelling. The data highlight that the effects of perivascular adipose on vessel remodelling are specific to certain mechanisms involved in obesity. This supports our notion that these pathways could be selectively targeted to interfere with the crosstalk between adipose tissue and the vessel wall and to prevent adverse vessel remodelling in obesity.
WP11 and WP12 Validation of a drug target to interfere with adipokine crosstalk
Recent data indicates that inflammatory alterations in white adipose tissue (WAT) play a major pathophysiological role for the development of insulin resistance, predisposing to type 2 diabetes mellitus and cardiovascular diseases in obese patients. Little is known about the sequence of events that lead to chronic inflammation in this organ. In obesity, macrophage and T-lymphocyte infiltration in WAT has been described in both mouse and human. Whereas macrophages are part of the innate immune system and act in an unspecific manner, T-lymphocytes are directed against foreign structures called antigens by the T-cell receptors (TCR). As compared to splenic T-cells, adipose tissue resident T-cells exhibit markedly restricted TCR diversity, which indicates they are a result of a specific selection. The expression of the cytokine interferon gamma increased in enlarged adipose tissue, elicits the production of numerous inflammatory mediators and increases antigen-presenting capacity of diverse cell types in the adipose tissue. The identification of relevant drug targets with the potential to interfere with obesity-associated inflammatory shift is a major goal of the consortium, in addition to research on molecular and physiological mechanisms that contribute to the pathology.
Genfit has accomplished the deorphanisation and pharmacological validation studies for a drug target that is potentially relevant for development of the chronic inflammatory state in the adipose tissue. The expression of the target (TGT) was confirmed in diverse inflammatory cell populations and its functional role in restraining the inflammatory response was confirmed in vitro. Preliminary functional validation experiments showed that increased TGT expression results in a decreased proinflammatory cytokine secretion by cells that participate in both innate and adaptive immune response. An exacerbated pro-inflammatory response was observed in inflammatory cells under TGT-deficient conditions. TGT biology is being pursued by renowned academic groups. Work that describes the metabolic phenotype of TGT-deficient mice was recently published and confirms the importance of that target in regulating glucose and lipid homeostasis in vivo. No data exist that relates to the selective pharmacological modulation of TGT in vivo. The identification of synthetic, drug-like TGT modulators was a major deliverable of Genfit within TOBI. A drug discovery programme was initiated within the project. As a result, small molecule compounds that bind to the target and provide anti-inflammatory action in diverse model systems in vitro were identified in a high throughput screening campaign. Improved, more active, drug-like and structurally novel chemical entities (NCEs) were derived from original hits through functional selection and medicinal chemistry efforts.
Potential impact
Obesity is rapidly increasing has become one of the most important global health issues. Obesity is associated with severe metabolic and cardiovascular sequelae, particularly type 2 diabetes, hypertension, coronary heart disease and stroke. Obesity and associated adipokine-mediated disorders counteract European initiatives to promote healthy ageing. Insulin resistance mediated by inflammatory adipokines probably plays a predominant role in the pathophysiology of excess fat. Interference with adipokine-mediated crosstalk by novel drugs is the ultimate goal. TOBI has used a multidisciplinary basic biomedical approach to achieve a strong translational aspect. TOBI has addressed issues starting from subcellular aspects. All research aimed at identifying potential drug targets or pathophysiological mechanisms to interfere with the adipokine-mediated inflammatory drift with emphasis on adverse interactions that lead to insulin resistance and diabetes as well as cardiovascular disease. TOBI has contributed to the need for novel pharmacological strategies to restrain the epidemic of obesity-associated morbidity in Europe and the whole first world.
Specific impact
The development of TOBI standards and the TOBI bio bank strengthens the European research on obesity. The TOBI standard protocols are available for all researchers upon request. The TOBI bio bank is available for researchers beyond the project. These tools equip European research to more effectively address the obesity epidemic thereby promoting more general impact of obesity research in Europe. Identification of the enzymatic activity in a signalling molecule activated by damage to the ER allows design of drugs to inhibit this activity in order to inactivate the inflammatory response to damage to the ER. This is applicable to the inflammation associated with obesity, but also applicable to inflammation linked to other diseases associated with ER damage, most notably neurodegenerative diseases such as Alzheimer or Parkinson. Identification of novel molecules of the insulin-signalling pathway allows design of drugs that restore function of these molecules. Substances such as chemical chaperones have been approved by the FDA and could be easily available and relatively inexpensive drugs to combat obesity-related complications. Identification of novel detrimental responses of the ER within the TOBI project has enabled molecular characterisation in future work, which may contribute to a better understanding of how cells respond to ER damage. This is important to understand the role of these responses in diseases that are associated with ER stress and to biotechnological production of therapeutic proteins in eukaryotic cells. The importance of lipases for obesity and its complications was emphasised by the discovery of the ATGL by B10. An impact of ATGL and other lipases was found for the inflammatory crosstalk.
The new results on lipases obtained provide novel insight into the biology of lipid breakdown that is highly required for the design of drugs that interfere with lipid synthesis and breakdown in order to combat the obesity epidemic. TOBI was one of the first projects to shed light on lipid-derived mediator molecules and their role in obesity. LDA turned out to be of potential relevance for the obesity epidemic. Certain LDA and their precursors and derivatives could be developed as potential drugs to interfere with inflammatory processes involved in obesity. Long-chain omega-3 polyunsaturated fatty acids are found in marine fish oils and turned out to be useful for the suppression of inflammatory reactions related to obesity. These data complement results from large clinical trials emphasising the preventive impact of long-chain omega-3 polyunsaturated fatty acids on cardiovascular health. The requirement for fish oils to appropriately supplement the European and the world population largely exceeds the availability of fish. Substances derived from omega-3 polyunsaturated fatty acids that mimic their effects at lower concentration could be used as fish oil substitutes and enable sufficient availability. Certain LDA derived from omega-3 polyunsaturated fatty acids inhibit adipose tissue inflammation at lower doses. These are important contributions of TOBI to appropriate supplementation with effective fish-oil-derived material. Inflammation-activated enzymes play a crucial role in the brain in the impairment of body-wide, systemic metabolism.
These results open up the possibility that inflammatory signals in combination with elevated serum fatty acids may reach specific brain parts and thus activate inflammatory enzymes to affect the functionality of insulin and other hormones, which regulate body weight in healthy patients. Chronic activation of these enzymes would induce neuronal resistance against these hormones, which plays a major role in the development of obesity and subsequently diabetes mellitus. These results have been reported in international meetings and have been published in international top journals. Similar results have been reported by other laboratories worldwide. These results have implications for future research and pharmaceutical drug design. If the brain is a key tissue in which inflammation occurs during obesity, potential drugs preventing chronic inflammation may be chemically designed to be targeted to the brain, but sparing peripheral tissues. This may prevent or dampen side effects. We have detected inflammation in the hypothalamus, the key brain region involved in regulating metabolism and body weight. In neurodegenerative diseases such as Alzheimer or Parkinson, activation of inflammatory enzymes has been detected as well. Potentially linking these two findings, obesity increases the risk to suffer from neurodegenerative diseases.
Current research aims to define if obesity-induced inflammation is found in all or specific brain areas, and if specific, which mechanisms protect the non-affected brain areas. Chronic inflammation can lead to cell dysfunction and cell death. One report indicated that in the hypothalamus, neuronal death occurs upon chronic obesity. Chronic activation of inflammatory enzymes may be involved in these phenomena as well. Future studies will be aimed to define the survival of key neurons involved in metabolism in long-term obesity. TOBI results on inflammatory signalling in the brain exceed the field of obesity and could be relevant for the whole field of neurodegenerative disorders.
TOBI results on ATM revealed considerable parallels to the phenotype of tumour-associated macrophages. Obese patient are more prone to develop cancer than lean subjects. It may be hypothesised that ATM could be a crucial link between obesity and cancer. Many tumours have increased levels of obesity-related factors in their microenvironment, rendering, in some cases, the tumours more aggressive. Macrophages in peri-tumoural adipose tissue which release obesity-related factors could be locally involved in carcinogenesis. We found that some genes expressed by the ATM that play a role in cancer development can be controlled by the pharmacological activation of the nuclear receptor LXR. Our results described a novel phenotype of ATM that can be associated to cancer and can be corrected by the activation of LXR with synthetic ligands. These observations can be the basis for novel pharmacological strategies acting at the level of ATM.
The understanding of the link between morphological alterations of adipose tissue occurring in obesity and the development of type 2 diabetes is becoming clearer and could be fundamental in developing pharmacological strategies aimed to curb the epidemic in western society and in developing countries. Within the consortium, fruitful collaborations have been established with laboratories with several expertises to comprehensively study adipocyte ultrastructure in obesity. Many interactions with all partners have been made both from an intellectual and technical point of view in order to implement these investigations. These data showed correlations that will promote our understanding of the pathophysiology of obesity. The expertise of B3 in microscopy was made more broadly available by generating TOBI standards on the collection, fixation and inclusion of adipose tissue to perform light and electron microscopy. An expert-based immunohistochemical protocol for the evaluation of macrophages presence in the adipose tissue has been established as part of the TOBI toolbox.
Discoveries on adipokine crosstalk between perivascular adipose tissue and the blood vessel wall can be exploited for benefits in health and wealth. In the medium term, these data will inform the potentially diverse therapeutic indications for 11beta-HSD1 inhibitors in obesity and diabetes. The concept of 11beta-HSD1 inhibition is protected by patents owned by the University of Edinburgh. There is a large number of new chemical entity patents published from the University of Edinburgh and from numerous large pharmaceutical companies. Several compounds have reached clinical development but some have stalled because of uncertainty about the future regulatory requirements for evidence of cardiovascular protection for drugs in development for type 2 diabetes. The additional evidence provided for potentially beneficial effects on vessel remodelling may enable this field by further assuageing concerns about cardiovascular safety in the development of these agents. Given some of these agents have already completed phase 2 studies, the opportunities for medium term health and wealth benefits are substantial. A potential drug target has been identified and followed up during the project.
As judged from literature and from our preliminary animal model data, modulators of the target should be able to improve glucose and lipid homeostasis and alleviate chronic adipose tissue inflammation. Target modulators should act through non-redundant mechanisms as compared to existing antidiabetic and hypolipidemic drugs, which suggests that their association with existing, first line treatments is theoretically possible and bring additional benefits to patients. These compounds will address unmet medical needs in a large population of patients who suffer from type 2 diabetes and from associated cardiovascular disease. The aim is to position a first in class and proprietary target modulator as a drug candidate ready to enter in full preclinical development. This compound will comply with all early development requirements. Genfit has the ability to provide clinical proof of concept up to the end of phase 2, but for these large therapeutic areas Genfit will partner via pharmaceutical company alliances to complete development and bring the drug to market in the future. In the longer term there may be additional drug targets validated among the pathways identified in the project. Patenting may follow if data are extended to fully exemplify the utility of these targets in improving obesity-associated vascular remodelling and intra-adipose inflammation.
Ultimate benefit for patients will stem not only from the potential therapeutic approaches identified and supported by TOBI, but also from the provision of laboratory measurement protocols and mechanistic knowledge that will help to refine our understanding of inter-individual differences in susceptibility to complications of obesity and hence potentially facilitate patient stratification for risk assessment and therapy. There are numerous direct beneficiaries from the knowledge generated by this research project. These include the investigators working on the project and their staff and students, who have benefited from training and experience and are likely to continue investigating the novel theme of the role of inflammation in obesity research under additional peer-reviewed external funding. Additional beneficiaries are the TOBI partners whose awareness of the role of specific issues involved in obesity-driven inflammation, e.g. perivascular adipose, has been substantially enhanced by regular discussion in project meetings, and the global scientific community, who will benefit from access to data when these are published in a peer-review journal.
Impact on health care issues
TOBI provided progress to effectively interfere with adipokine-mediated crosstalk for prevention of obesity-associated morbidity will help to stabilise the currently escalating costs for obesity-associated disorders. TOBI has ameliorated societal issues related to obesity. Results could help improve the health of European citizens by having addressed an epidemic health problem with an effective approach by investigating its pathophysiological roots in a translational approach. Translation discoveries into clinical application has been taken seriously by linking world-class academic researchers directly to drug developing and analytical companies included in the consortium. The consortium has contributed to the development of new therapies for obesity-related disorders and has started to validate a potential drug target. Novel methods for health promotion and prevention of obesity-associated disorders could be future effects of TOBI, contributing to healthy ageing of the European population. Novel strategies based on TOBI results could include treatment options that potentiate beneficial effects of healthy life style on adipokine-mediated inflammation, e.g. by developing omega-3 fatty acid-derived substances to be used as drugs or included in functional foods. TOBI addressed juvenile obesity, a rapidly increasing health issue with long-term consequences. TOBI has characterised a considerable number of obese adolescents and collected clinical and biochemical data. Further studies on juvenile obesity have been facilitated for a broader scientific community by including these samples in the TOBI bio bank.
Cross-thematic impact
Investigating products of dietary lipids has been part of the research, particularly omega-3 polyunsaturated fatty acids (PUFA). TOBI is cross-thematically related to FP7 topics on food quality. Many LDA are derived from omega-3 PUFA are found in marine fish oils. TOBI perfectly synergises and provides complementarity with themes on 'Food, Agriculture and Fisheries, and Biotechnology'. TOBI has achieved knowledge contributing to the impact of food and nutrition on health in order to fight diet-related disorders. TOBI has contributed to the understanding of beneficial and harmful dietary factors in order to control the development and reduction of diet-related diseases. TOBI could have an impact on European food-related industries. Results could contribute to the development of functional foods and preventive options. The limited availability of fish oils due to ecologic considerations and reduced acceptance of high-dose marine n-3 PUFA ingestion due to gastrointestinal side effects prohibit their population-wide consumption even though omega-3 PUFA prevent the obesity-related inflammatory drift and adipose tissue in mice. TOBI investigations on LDA have identified omega-3 PUFA-derived substances that resolve inflammation and are effective in adipose tissue. Results could provide the knowledge required for developing functional foods or preventive medicines for inflammatory disorders not confined to obesity.
Competitiveness and added value
TOBI has improved the competitiveness of European universities and research-based SMEs by collaboration on methodological and thematic aspects that will bring forth added value to all participants. TOBI has increased the innovative capacity of the European health-related sector by facilitating tight interactions with world-class European. TOBI collaborations have strengthened the international competitiveness of European academies and SMEs that will generate added value for SMEs and academic research. By bringing together internationally renowned experts in closely interacting WPs, TOBI has achieved world-class collaborative research on obesity. The complementary expertise of partners' fields of research has been successfully organised. TOBI facilitated sharing of methodologies between a critical mass of partners, potentiating their scientific impact. TOBI has made technically sophisticated methodologies available for its partners and has brought about breakthroughs in obesity research. TOBI has produced knowledge that will significantly contribute to the development of pharmacologic strategies to prevent and treat obesity and associated complications. TOBI adds to the detection and monitoring of obesity-associated adipokine-mediated adverse interactions. These results could lead to the development of individualised pharmacological strategies in order to optimise the delivery of healthcare to European citizens by tailored medicine.
Dissemination
The coordinator has encouraged all participants to actively disseminate their project aims and results to colleagues, stakeholders and the wider public. The TOBI dissemination activities aimed at linking project topics with topics with almost everyone is affected: overweight, obesity and nutrition. A logo and an overall project design have been created in order to identify the project. Public awareness of TOBI has been addressed by the website http://www.tobi-project.eu The content is regularly checked and updated. The website is available in four languages and will be continued after the project ends. Emphasis has been placed on media-rich presentation of the content (see http://www.youtube.com/user/TheTOBIproject and http://www.vimeo.com/26270197 online). The news-section provides visitors with the latest publications and dissemination activities. TOBI was presented to the public at large during the European Researchers' Night 2009 in Vienna. TOBI organised a scientific cooking event which main goal was to help people understand that a healthier life begins in the kitchen and in their minds.
The consortium takes measures to provide TOBI exposure to the public through conventional PR media work, which will complement the public access website, particularly for those that do not have Internet access. Press kits sent out to journalists and other stakeholders provide them with all project related information. Posters and folders about the impact of overweight and obesity were created to be distributed by partners. The coordinator published several articles in the general press. A success story about TOBI was published by the Austrian National Contact Point (NCP). The scientific community has a vested interest in keeping up with TOBI progress and results to build on the body of knowledge that currently exists. This will be achieved through conventional scientific dissemination means, i.e. peer-reviewed publications and presentations at international conferences. Some results of the project have not yet been published but are submitted to or under revision at scientific journals or manuscripts for scientific journals are written at the time of the report.
Project website: http://www.tobi-project.eu/
Contact details
Project coordinator:
Medizinische Universität Wien
Prof. Thomas Stulnig
Clinical Division of Endocrinology and Metabolism
Department of Medicine III
Wahringer Gurtel 18-20, 1090 Vienna, Austria
E-mail: thomas.stulnig@meduniwien.ac.at
Phone: +43-140-4004368
Fax: +43-140-4007790
Biolution - TOBI office
Karl-Farkas-Gasse 22
1030 Vienna, Austria
E-mail: office@tobi-project.eu
Phone: +43-178-6959512
Fax: +43-178-6959520
List of partners
Medical University of Vienna (Coordinator), Thomas Stulnig, Austria
biolution GmbH (SME), Iris Grunert, Austria
Universitat zu Koln, Jens Bruning, Germany
Universita politecnica delle marche, Saverio Cinti, Italy
Genfit SA (SME), Matthieu Dubruque, France
Pharm-analyt (SME), Hermann Mascher, Austria
University of Durham, Martin Schroeder, United Kingdom
Institut Pasteur de Lille, Bart Staels, France
The University of Edinburgh, Brian Walker, United Kingdom
Universität Graz, Rudolf Zechner, Austria.
The remit of this project is to identify novel strategies to reduce or reverse adipokine-mediated adverse interactions. Special attention has been placed on the study of novel fat-derived adipokines with the goal to identify novel targets for drug development. Adipocyte ultrastructural analyses and studies of how adipose tissue acts on the adjacent vasculature added to the understanding of the relevant pathophysiology. Within 42 months the consortium has made significant progress in all objectives. Critical components of stress and nuclear receptor signalling in inflammatory processes in obesity were identified and its impact on adipocyte structure analysed. The impact of lipolysis and fat-derived adipokines on inflammatory alterations in obese adipose tissue proved the breakdown of fat and fat-derived mediator promotes obesity-related complications. TOBI has led to the identification of a potential drug target, which is already exploited. Important tools have been developed including standards of experimental and clinical protocols. The TOBI bio bank is an important resource of biological specimens related to obesity from more than 250 well-characterised subjects, which will continue to facilitate material exchange between TOBI members. These activities have provided a sustainable basis for a lasting collaboration. Professional project management and dissemination of results significantly contributed to successful collaboration of the beneficiaries. The coordinator has encouraged all beneficiaries to actively disseminate their project aims and results to colleagues, stakeholders and the wider public. Public awareness has been addressed with a website, press releases and other activities, including an event (see http://www.tobi-project.eu online).
TOBI was highly successfully and has met essentially all objectives within schedule. The project was a timely initiative, which contributed significantly to our improved understanding of adipose tissue inflammation and its harmful clinical consequences, which will be corroborated by further publications. The tools established have fostered obesity research in Europe. If the drug target identified will turn out to be valuable for clinical application, TOBI could bring enormous benefit to obese patients, contribute to sustainable health systems in Europe and support the competitiveness of the European health industry.
Project context and objectives
The prevalence of obesity has escalated to epidemic proportions in the past decades in industrialised countries. The problem is the detrimental effects obesity has on health. It is invariably accompanied by metabolic disorders such as diabetes and metabolic syndrome. Reduced insulin action in the body, (insulin resistance) is the basis for the metabolic syndrome and type 2 diabetes and promotes atherosclerotic alterations leading to vascular disease. Enlargement of fat depots by excess food consumption is the basis of obesity but weight reduction turned out to be difficult to achieve and keep in long-term. It is only recently that chronic systemic low-grade inflammation, originating primarily from the fat tissue in obese individuals, has been identified as an important cause of insulin resistance leading to diabetes and vascular disease.
Reversing the obesity-driven inflammatory shift should improve insulin sensitivity and prevent diabetes and cardiovascular disease in obese patients. Adipose tissue produces a large number of adipokines; soluble messenger molecules that mediate the interaction between adipose tissue and inflammation and immunity. Adipokines may be inflammatory or anti-inflammatory in action and are produced by fat and other cells present in fat tissue as well as infiltrating inflammatory cells. In obesity, a large number of inflammatory adipokines affect the function of other organs by different mechanisms. Primary triggering events within adipose tissue must be targeted to curb all adverse adipokine-mediated crosstalk.
Most adipokines are proteins by nature. Adipose tissue secretes considerable amounts of potent messenger molecules derived from fat molecules (lipids). Few of these active lipid molecules have been identified so far. By virtue of their signalling capacity and production by adipose tissue, such lipid-derived adipokines (LDA) effectively qualify as adipokines and are considered to be crucial in the study of obesity-associated adipokine crosstalk. Research has made clear that chronic inflammation is triggered by obesity and that this activates cells in a way that leads to insulin resistance and atherosclerosis. It has become evident that adipokines, produced by adipocytes and infiltrating inflammatory cells are key players in mediating inflammatory signal transduction. Significant study has been carried out on protein adipokines but lipid-derived molecules have not been addressed properly. Mechanisms triggering the inflammatory cascade leading to inflammatory adipokine production and subsequent impact on peripheral tissues remained largely unresolved. Cells stress occurring in the endoplasmic reticulum (ER), an organelle implicated in metabolic actions within the cell, has recently been suggested as a mechanism triggering inflammatory cell activation and insulin resistance in obesity. Factors inducing ER stress such as altered nutrient fluxes in fat cells, e.g. by disorders in the release of fatty acids by lipolysis are still elusive. Structural alterations of the ER could occur in stress situations but those have not yet been defined on an ultrastructural level.
The presence of inflammatory cells (macrophages) in adipose tissue of obese subjects puts another piece into the puzzle of adipose tissue inflammatory alterations. Different inflammatory cell activation pathways could have different impact on adipokine production and crosstalk when activated in fat cells and adipose tissue macrophages (ATM). Inflammatory activation of ATM or fat cells could be significantly modulated by nutrient-sensing nuclear receptors that directly regulate genes in cells. Nuclear receptors represent potential drug targets by nature, but their involvement in fat inflammation has not been elucidated. Crosstalk of adipokines with other organs through the systemic blood circulation promotes the development of insulin resistance leading to development of type 2 diabetes. Adipokine-mediated crosstalk with tissues other than adipose tissue does not necessarily require systemic action. Particularly important tissues are embedded in adipose tissue and could be directly affected by adjacent adipokine production. Research addressing multiple factors that promote inflammatory adipokine crosstalk in obesity is urgently required in order to identify crucial mechanisms and potentially targetable molecules.
Objectives
The overall aim was to integrate research on the interplay between the cellular stress, the inflammatory response involving macrophage infiltration and changes in lipolysis and lipid-derived mediator substances within fat tissue and how they feed into altered adipokine expression leading to the development of insulin resistance and vascular dysfunction. TOBI aimed succeeded in achieving a knowledge base by standardising the analytical approach via the TOBI toolbox, using defined clinical data recording of individuals from which biological materials have been collected, sophisticated biochemical and histological analyses, undertaking genome-wide expression analyses, investigating cells at an ultrastructural level, exploiting newly generated and existing animal models and creating a pipeline for drug development. The objective was to analyse mechanisms that investigate altered adipokine-mediated crosstalk including the involvement of LDA associated with obesity. This approach led to the identification of a novel drug target with the potential to interfere with the obesity-associated inflammatory shift. Several other molecules have been suggested with more work being required for defining their potential as drug targets.
This overall TOBI aim encompassed the following objectives: Identification of cellular triggering and activation events pivotal for the initiation of inflammatory drift through adipokine crosstalk, identification of novel LDA and their contribution to the inflammatory drift and validation of the involved pathways and molecules as potential targets for therapeutic intervention, and in best case assay development for a selected therapeutic target. To this end, TOBI implemented a collaborative research project using a wide variety of methods including genetic approaches in mice, measures of gene regulation, biochemical investigations, ultrastructural analyses up to analyses of clinical specimen. Collectively, the consortium combined all the necessary expertise to investigate the basis of obesity-associated inflammatory drift.
For objective 1, TOBI addressed a variety of pathophysiological mechanisms related to excess fat that are important for initiating and promoting inflammatory adipokine-mediated crosstalk with other tissues. Emphasis has been placed on studying ER stress signalling, the dissection of inflammatory activation pathways and the activation of nuclear receptors as well as lipolysis. The release of fatty acids and metabolic intermediates during lipolysis is linked to objective 1 as a potential triggering mechanism, but also to objective 2, since the release of fatty acids in most instances is a prerequisite for LDA production. Whereas objectives 1 and 2 aimed to elucidate pathophysiological mechanisms, objective 3 aimed to exploit the resulting knowledge by transferring it to drug development, in close collaboration with the respective TOBI partners. In order to address these objectives in a manner that shares resources and allows data comparison between the partners of the consortium, the TOBI Toolbox has been established. The toolbox consists of TOBI standards, i.e. SOP. The TOBI Standards have provided a valuable basis for European research on obesity and promote collaboration among European scientists and enterprises. The TOBI toolbox includes a TOBI bio bank for sharing human material. The TOBI bio bank is accompanied by a database integrating parameters for detailed characterisation of patients. TOBI generated novel analytical tools for LDA and generate novel animal models that were exploited by the consortium or shared with other researchers to strengthen the ERA.
The objectives included identification of novel adipokines, particularly LDA. TOBI has added to the detection of obesity-associated adipokine-mediated adverse interactions. Future research will show to which extent these results could lead to the development of individualised pharmacological strategies in order to optimise the delivery of healthcare to European citizens by tailored medicine. In order to implement this comprehensive collaborative research programme, a separate WP has structured management activities. In addition, another WP was included to realise a variety of dissemination activities directed to the scientific community including envisioning of patent applications. WP2 included numerous dissemination activities to reach the general public and increase the awareness for the TOBI project and more general for obesity as an important health issue.
Project results
WP4 ER stress-induced signalling for inflammatory drift and insulin resistance
Obesity damages human cells by damaging specific subcellular structures called organelles. One such organelle affected by obesity is the ER. In cells, the ER specialises in synthesising protein destined for excretion by the cell and proteins residing in cellular membranes. This WP addressed the question how a malfunctioning ER in obese patients contributes to the development of type 2 diabetes in these patients. Obesity is closely associated with two cellular signalling events; insulin resistance and inflammation. Insulin resistance is the inability of human cells to appropriately respond to the protein hormone insulin. Insulin is released by the pancreas after having had a meal and stimulates the uptake and utilisation of nutrients by cells. An inability to respond to insulin, termed insulin resistance, leads to chronically elevated blood glucose levels, which damages blood vessels and leads to type 2 diabetes and its complications. Inflammation is the response of cells and organisms to damage with the view to repair this damage and to remove damaged cells from the organism.
This WP has characterised molecular mechanisms through which a malfunctioning ER causes insulin resistance. We have evaluated the role of activation of a transmembrane protein of the ER, called IRE1 that reports on correct functioning of the ER, on the ability of insulin to stimulate glucose utilisation by human cells. We have focused on culture models for the main tissues and organs that contribute to blood glucose homeostasis, muscle cells, liver cells and fat cells. We have mimicked the damage caused by obesity to the ER using pharmacological agents that interfere with protein synthesis by this organelle. Using biochemical assays and ultrastructural methods, we confirmed that these drugs damage the ER. We characterised the consequences of this damage on the ability of cells to respond to insulin.
To characterise the effects of damage to the ER we have used biochemical assays to detect the activated, i.e. phosphorylated forms of several signalling molecules activated by insulin. We have observed a biphasic role of damage to the ER on the ability of cells to respond to insulin. Short periods of damage to the ER do not affect the ability of cells to respond to insulin, while prolonged damage to the ER leads to profound insulin resistance. Biochemical characterisation of signalling molecules involved in transducing the insulin signal to promote glucose / nutrient uptake and utilisation by cells has revealed novel interactions between ER damage and the insulin signalling pathway and has identified novel molecules involved in the insulin signalling pathway that are inactivated/inhibited in cells with damage to the ER. We have observed that short periods of ER stress induce a subset of inflammatory signalling events leading to activation of the protein kinase JNK in cells. Activation of these inflammatory signalling pathways was transient. While we find that activation of these inflammatory signalling pathways requires IRE1 and an adaptor protein, called TRAF2, in genetic experiments using gene knock-out cell lines and gene silencing technologies we have not found any evidence for these inflammatory events to contribute to the development of insulin resistance. Detailed molecular genetic characterisation of one molecule activated by damage to the ER has identified the enzymatic activities of this molecule that activate inflammatory signalling in response to ER damage.
WP5 Crosstalk between lipolysis and adipokines
Triacylglycerol are neutral fats that are efficient energy depots. Moreover, they provide essential building blocks for membranes and organelles as well as hormones. These triacylglycerols look somewhat like the letter 'E'. There is one glycerol molecule as backbone with three fatty acids attached. These fatty acids act as signalling molecules, which send signals throughout the body to accelerate or stop biological processes. It is conceivable that a person who is obese sends different fatty acid signals than a lean person. The exact mechanisms of lipid signalling are still poorly understood. The enzymes required to cleave these fatty acids off storage fat molecules are called lipases. Lipases are most active in times of increased energy demand. The first fatty acid is cleaved off by a lipase called adipose triglyceride lipase (ATGL). This enzyme was discovered by the principle investigator of B10, Rudolf Zechner, in 2004. It is the rate-limiting enzyme in the breakdown of storage fat. Working with lipase-deficient mice is the only way to find out the exact roles of the individual enzymes. Mice that lack this enzyme have massive fat accumulation in all organs, especially the heart and die prematurely because of heart failure.
There are human patients that have a defect in their ATGL-gene and suffer from a similar condition like the ATGL-deficient mice. They accumulate excess fat in skeletal and cardiac muscle as well as in the heart and require heart transplantation at an early age. Their condition is called neutral lipid storage disease with myopathy. Despite having more body fat ATGL-mice seem to be protected from developing diabetes. Obese humans have a much higher incidence of diabetes than lean people. There is the hypothesis that fat does not do much harm as long as is stays within the designated fat tissues. When it is broken down and released into the blood it seems to act toxic. Since ATGL-deficient mice cannot mobilise the fat, but retain it in their fat depots, there are only low fatty acid levels observed in their blood. The second step in the breakdown of storage fat is performed by the enzyme hormone-sensitive lipase (HSL) and the last step by monoacylglycerol lipase (MGL). Mice missing either of these two lipases are relatively lean and quite healthy compared to ATGL-deficient mice. HSL-deficient male mice are infertile, underlining the importance of fat as a precursor for hormones and other signalling molecules. All these processes are complex and involve a large number of additional players that fine-tune the rate of fat breakdown. We still do not understand many mechanisms, but try to set up hypotheses that match our experimental data.
Obesity is often associated with inflammation. Within TOBI we raised the question whether mice lacking a certain lipase show more inflammation or stress of the protein synthesising unit of the cell (ER) than normal mice. We wanted to find out if certain signalling molecules that are known or proposed to be involved in the breakdown of fat (adipokines) can influence the activity of lipases. Many approaches have been used to investigate these questions in cooperation with TOBI partners, including biochemical and molecular biology techniques as well as microscopic analysis. We need to continue our work after the end of the project to be able to correctly interpret all of the obtained data.
As a first result we can state that lipase-deficiency with or without elevated fat composition do not always mean that there is increased inflammation in mice. It is difficult to make direct comparison between lipase-deficient mice and obese humans. The scientific data obtained during the project enables us to shed light onto the connection between lipases and adipokines, even though we cannot give an explanation for all experimental data yet. In a particular area of metabolic signalling we have made considerable progress within TOBI, which will most likely result in a high-level publication within the next few months. We suggest a therapy for patients suffering from neutral lipid storage disease with myopathy based on your experimental data with mice. The revision of the manuscript has been sent in to Nature Medicine. Unfortunately, we are not allowed to publically disclose any results which are part of this manuscript prior to the date of publication. As soon as the content of this publication can be made public it will be posted on the TOBI website.
WP6 Identification of LDA and their role in adipokine crosstalk
Adipokines are not necessarily proteins by nature. Due to the release of fatty acids from fats stored in fat (adipose) tissue, lipid-derived molecules are synthesised locally. Many molecules derived from polyunsaturated fatty acids (PUFA) are highly potent mediators to promote or reduce inflammatory reactions. Recently identified molecules derived from omega-3 PUFA which are predominantly found in marine fish oils are of high interest because they actively repress inflammatory reactions by promoting their resolution. A lack of resolving lipid mediators in adipose tissue, LDA from obese patients could contribute to the ongoing inflammatory response that leads to insulin resistance and type 2 diabetes. Within this WP the coordinator has set up a tight collaboration with a small and medium-sized enterprise (SME) partner with top level expertise in small molecule analytics in order to establish methods for the detection of potent PUFA-derived mediators in adipose tissue. A number of substances could be found in adipose tissue from humans and mice. The steric structure of many substances could be resolved. With this tool we analysed LDA from obese and lean humans and different animal models of obesity.
We found that human obesity altered the concentration of a number of LDA and their precursors in adipose tissue. These alterations comprised molecules derived from omega-3 PUFA and omega-6 PUFA, the latter being in general of more pro-inflammatory nature. We found alterations in LDA concentrations in two models of murine obesity. Regional analysis revealed similar obesity-related changes in different adipose depots. Studies from the coordinator's laboratory have revealed that a diet rich in omega-3 PUFA reduces adipose tissue inflammation in mice, whereas omega-6 PUFA were virtually ineffective. We fed obese mice with a diet enriched in omega-3 PUFA and analysed LDA in adipose tissue and their metabolic changes. As expected, measures of adipose tissue inflammation were decreased in obese mice following omega-3 PUFA treatment. We found a number of resolving lipid mediators increased in adipose tissue of omega-3 PUFA-treated animals. Changes in LDA were detectable after short periods of treatment. We studied gene expression of molecules relevant to LDA production and action and found interesting changes. These results indicate that changes in LDA could contribute to the anti-inflammatory action of omega-3 PUFA in obese adipose tissue and their beneficial metabolic action. Based on the promising results in mice we conducted a clinical trial looking for effects of omega-3 PUFA on LDA production in obese patients. Omega-3 PUFA-derived LDA were increased in adipose tissue along with reduced inflammatory changes. These data emphasise potential relevance of omega-3 PUFA-derived LDA in terminating obesity-driven adipose tissue inflammation. Selected LDA and their precursor were tested in mice in order to see whether application of these substances could reduce adipose tissue inflammation in obesity. A selected LDA was particularly potent in reducing adipose tissue inflammation and improving metabolic function. In addition to the importance of adipokines, LDA play important roles in sustained inflammatory response in fat tissue of obese subjects. LDA could be of potential use in reducing obesity-driven inflammation, thereby preventing type 2 diabetes and cardiovascular disease in obese patients.
WP7 Inflammatory signalling pathways that elicit inflammatory adipokine crosstalk
Research from the last dozen of years has demonstrated that low-grade inflammation in the adipose tissue is likely an alarm signal from lipid-filled adipocytes, which are in danger of cell death due to huge amounts of lipid they have to take up. These signals attract immune cells, which try to engulf dying adipocytes. Due to the massive amount of lipids, the immune cells send alarm signals to attract more immune cells. These signals can affect not only the functionality of adipocytes and immune cells locally, but can also impact other tissues. This low-grade inflammation is able to impair the actions of the key hormone insulin, which in healthy people keeps blood glucose and lipid levels in check. When the ability of insulin to normalise blood glucose and lipids decreases the body is able to produce increased levels of insulin. After years of increased production of insulin, the insulin-producing cells stop working properly, giving rise to uncontrolled blood glucose levels (overt diabetes mellitus) from which around 9 % of Europe's population suffers. When inflammatory signals reach cells, they induce the activation of enzymes, termed kinases that can affect basically all intracellular processes. Research from the last 10 years has implicated that these inflammatory enzymes are able to reduce the efficacy of insulin, i.e. can induce insulin resistance. Experiments could demonstrate that one enzyme (c-Jun kinase) is intimately involved in the induction of insulin resistance, and is commonly activated in tissues from obese and diabetic patients. The research efforts during TOBI were aimed to define the tissues in which activation of this kinase affects glucose and lipid metabolism. We generated mouse models in which this kinase was missing only from one tissue.
Mice which lacked this enzyme only in the liver were not protected from insulin resistance and developed diabetes-like symptoms as much as control animals when fed a high-fat diet simulating an unhealthy diet. Mice lacking this kinase in the muscle were not protected from these symptoms. These findings in combination with results from other laboratories indicate that activation of the enzyme in classical tissues involved in metabolism does not play a major role during the development of obesity-associated pathologies. Obesity is a consequence of energy intake exceeding energy consumption. Food intake and utilisation of energy are under control of neuronal circuit. In the last 20 years, it has become obvious that hormones do not only act directly on classical tissues, but also on the brain, to regulate feeding, energy expenditure, glucose and lipid metabolism. The possibility of inflammation in specific brain regions involved in regulating glucose and lipid metabolism in obesity had not yet been addressed. We found that inflammation can be detected in these brain areas of obese animals, similar to what can be detected in liver or adipose tissue. The c-Jun kinase enzyme was activated. We generated mice which lack this enzyme in the brain. These mice were protected from inflammation in the adipose tissue, demonstrated lower blood glucose levels and did not suffer from a fatty liver when challenged with a high-fat diet. These mice were shown to be highly insulin-sensitive both in the brain and in classical tissues. These results demonstrate that inflammatory enzymes may not only act in peripheral tissues, but also in the central tissue involved in regulating body weight, obesity and glucose levels, the brain, and activation of these enzymes in the brain may participate in the development of obesity and diabetes mellitus.
WP8 Nuclear receptor LXR in adipose tissue macrophages
Obesity is a low-grade chronic inflammatory disease characterised by enlarged fat cells associated with a pro-inflammatory state in adipose tissue due to the infiltration of macrophages (ATM), which aggregate in crown-like structures usually surrounding dead adipocytes. Within adipose tissue, ATM are a major source of inflammatory cytokines which may be involved in obesity-related inflammatory disorders and the recruitment of additional immune-inflammatory cells. Adipose tissue macrophage infiltration and ensuing inflammation precedes the development of insulin resistance, at least in animal models. ATM are surrounded by pre-adipocytes and adipocytes and it is likely that paracrine loops exist via the production of adipokines, free fatty acids and derived mediators, leading to inflammatory changes. Pre-adipocytes produce higher levels of pro-inflammatory cytokines compared to adipocytes, suggesting that they play an important role in the induction and maintenance of inflammation and may operate as potent ATM activators.
To characterise the phenotype and function of human ATM, a global gene expression analysis comparing ATM and macrophages (monocyte-derived macrophages or MDM) derived from blood from the same morbidly obese patients undergoing bariatric surgery was performed. Several genes over-expressed in ATM were identified to belong to cytokine-cytokine receptor interaction pathways yielding a specific ATM pattern. In vitro experiments indicated that the phenotype of ATM is related to factors released by the pre-adipocytes, whereas lipids released from mature adipocytes had no effect. ATM displayed a gene expression profile sharing similarities with tumour-associated macrophages (TAM) isolated from human tumours. ATM were found to release a repertoire of growth factors, cytokines, chemokines and enzymes involved in the regulation of tumour growth, generation of blood vessels, tumour spreading to adjacent and remote tissues and organs similar to that observed in TAM. These data indicate that the human ATM phenotype sharing similarity with those of human TAM, is directed by environmental cell types and suggest that factors released by ATM may be involved in cancer initiation and progression in obese patients. ATM-conditioned medium increases the expression of genes leading to lipid accumulation in breast cancer cells. Over-expressing tumours display aggressive behaviour compared to those with normal lipid gene expression levels, indicating a pivotal role in cancer cell survival and in the maintenance or enhancement of the malignant phenotype.
These results indicate that factors released by ATM could affect tumour aggressiveness. Many tumours have increased levels of obesity-related factors in their microenvironment, rendering the tumours more aggressive. Macrophages in peri-tumoural adipose tissue which release obesity-related factors could be locally involved in carcinogenesis. Our data indicate that ATM have a gene expression profile comparable to TAM raising the possibility that ATM may be one of the potential links between obesity and cancer. LXR (liver-X-receptor) is a ligand activated transcription factor playing important role in the control of macrophages functions. Its expression and activity in human ATM were unknown. Human ATM have been isolated from human visceral adipose tissue and cultured in presence or absence of highly specific synthetic LXR agonists. Circulating MDMs have been prepared from the same donors. Gene expression profiling analysis has been performed to potentially identify novel LXR target genes specifically regulated in ATM. Results indicate that expression of LXRa is significantly lower in ATM compared to the MDM isolated from the same obese donors. Results show that only few genes are differentially regulated by LXR in ATM and MDM. Among these LXR target genes, we have identified two chemokines (molecules that attract inflammatory cells) which showed a regulation in ATM. These two genes are strongly inhibited by LXR activation in ATM and not regulated or regulated in an opposite manner by LXR agonists in MDM. These results point to the relevance of gene regulation by LXR for the specific type and function of ATM in obese adipose tissue.
WP9 Ultrastructural analysis of obese adipocytes by electron microscopy
Adipose tissue is an important organ producing hormones able to influence our behaviour and molecules that could change the metabolism of individuals. It was discovered that adipose tissue of obese animals and humans is infiltrated by macrophages producing a mild chronic inflammation. This inflammation seems to be important because interfering with the activity of insulin receptor induce a state of insulin resistance that induce diabetes. Insulin is the hormone produced by pancreas that act as a key that open the door of cells allowing the entrance of glucose, a fundamental fuel for most cells. Insulin receptor are the lock for the key insulin. When the lock is not functioning the key does not open and glucose remains in the blood producing hyperglycaemia (type 2 diabetes). We discovered an important cause of this inflammation; the death of adipocytes. Adipocytes are cells that represent main constituents of adipose tissue. In obese individuals the energy to be stored is high and adipocytes have to expand their volume, thus fat cells become six to seven times bigger. Studies have shown that this increase has several consequences on fat cells. The thickness of the wall of the adipocytes gets reduced and the number and size of mitochondria is reduced.
These alterations were studied by electron microscopy. Two animal models of obesity were investigated, which develop a form of diabetes that is similar to what happens in humans. The alterations of fat cells were not identical in all fat depots. It is known that fat in all mammals is located in two distinct anatomic districts; subcutaneous and visceral. Accumulation of abdominal fat is more dangerous than accumulation of subcutaneous fat. Abdominal fat is dangerous because it is often associated with all metabolic disorders associated with obesity. Visceral fat behaves differently than subcutaneous fat. Studies showed that visceral fat cells are more fragile than subcutaneous fat cells. When they are required to expand, subcutaneous fat cells are able to expand better and reach larger sizes that are not reached by visceral fat cells. Visceral adipocytes die at a size in which subcutaneous fat cells survive. Inflammation of visceral fat is more pronounced than inflammation in subcutaneous fat supporting the importance of inflammation for the metabolic disorders associated with abdominal obesity. Studies highlighted that brown adipocytes surround the area where heart and aorta are contained. In obese animals these cells transform into normal fat cells and die quickly, inducing inflammation with possible immediate cardiovascular consequences. These studies are important to understand the mechanisms that are rapidly expanding plague, affecting millions of citizens. The understanding of basic cell biology underlying this disease is the prerequisite for pharmaceutical research to create new drugs to combat and curb obesity and type 2 diabetes.
WP10 Effects of obesity on adipokine-mediated crosstalk
Research has explored the differences between accumulations of adipose tissue in different sites. Accumulation of fat in the intra-abdominal depot is associated with greater risk of metabolic and cardiovascular complications than accumulation in depots under the skin. Differences in function between the visceral and subcutaneous adipose depots and their crosstalk with other organs have been described. Until now, the importance of the perivascular adipose depot has been neglected. Abnormalities of blood vessels are important in obesity; increased contraction may contribute to high blood pressure. Changes in the vessel's structure are important for maintaining the normal shape of the vessel, for allowing blood flow and regulating forming new vessels. Abnormal remodelling contributes to narrowing of blood vessels which causes cardiovascular disease and is important in adipose tissue in obesity, where lack of oxygen supply exacerbates inflammation within the adipose tissue. Blood vessels are surrounded by perivascular adipose, a layer of fatty tissue. The extent to which this accumulates in obesity may predict cardiovascular disease and perivascular adipose might influence the contraction of the blood vessels. This WP aimed to characterise perivascular adipose tissue in lean and obese mice, with a focus on the crosstalk between the perivascular adipose and the blood vessel wall. Perivascular adipose tissue was collected from mice with or without diet-induced or genetic obesity. The microscopic appearances and pattern of genes active in the perivascular adipose tissue were compared with visceral and subcutaneous adipose tissue. Healthy perivascular adipose has a pattern of gene activity that is distinct from other depots, with a low level of inflammation.
We corroborated some of our findings in tissue biopsies of human perivascular adipose tissue obtained at surgery. In obesity, perivascular adipose accumulates and changes its characteristics to become similar to visceral adipose, producing potentially damageing inflammatory substances. We studied vessels from mice using a device called a myograph in which their responses to various drugs are tested. This showed that obese mice have abnormal vessel contraction. Perivascular adipose has a powerful effect to block vessel contraction by a variety of drugs. We tested which cells in the blood vessel wall are targets for the effect of perivascular adipose tissue. This confirmed a physiological interaction between perivascular adipose and blood vessel function. Its importance in the vascular dysfunction of obesity is less clear; we did not find that the differences in blood vessel function in obesity could be attributed to differences in the substances released from perivascular adipose. To test whether perivascular adipose affects blood vessel remodelling, we set up a laboratory method in which we allowed mouse blood vessels to form new vessel sprouts in a plastic dish. We added material produced by perivascular and other adipose tissue, and showed it had dramatic effects on the number of new vessels formed. This suggests that adipose tissue can directly influence the shaping and reshaping of blood vessels.
Perivascular adipose tissue from obese animals has altered effects on vessel remodelling. To identify factors that might mediate the effect of perivascular adipose tissue on vessel remodelling, we used protein arrays to measure a host of potential factors in the conditioned medium. This identified several candidates that can be potential drug targets. We tested whether release of steroid hormones from the perivascular adipose tissue is important, using adipose tissue from mice lacking an enzyme that makes steroids in the adipose tissue. This showed substantial effects of the enzyme on remodelling activity of adipose tissue. We were able to replicate this finding using an inhibitor of the enzyme. By collaborating with other consortium partners we explored mechanisms that might be responsible for the effects of perivascular adipose on vessel remodelling. This included testing whether LDA influence vessel remodelling. These studies illustrate the utility of the screening assay we developed for dissecting control of vessel remodelling. The data highlight that the effects of perivascular adipose on vessel remodelling are specific to certain mechanisms involved in obesity. This supports our notion that these pathways could be selectively targeted to interfere with the crosstalk between adipose tissue and the vessel wall and to prevent adverse vessel remodelling in obesity.
WP11 and WP12 Validation of a drug target to interfere with adipokine crosstalk
Recent data indicates that inflammatory alterations in white adipose tissue (WAT) play a major pathophysiological role for the development of insulin resistance, predisposing to type 2 diabetes mellitus and cardiovascular diseases in obese patients. Little is known about the sequence of events that lead to chronic inflammation in this organ. In obesity, macrophage and T-lymphocyte infiltration in WAT has been described in both mouse and human. Whereas macrophages are part of the innate immune system and act in an unspecific manner, T-lymphocytes are directed against foreign structures called antigens by the T-cell receptors (TCR). As compared to splenic T-cells, adipose tissue resident T-cells exhibit markedly restricted TCR diversity, which indicates they are a result of a specific selection. The expression of the cytokine interferon gamma increased in enlarged adipose tissue, elicits the production of numerous inflammatory mediators and increases antigen-presenting capacity of diverse cell types in the adipose tissue. The identification of relevant drug targets with the potential to interfere with obesity-associated inflammatory shift is a major goal of the consortium, in addition to research on molecular and physiological mechanisms that contribute to the pathology.
Genfit has accomplished the deorphanisation and pharmacological validation studies for a drug target that is potentially relevant for development of the chronic inflammatory state in the adipose tissue. The expression of the target (TGT) was confirmed in diverse inflammatory cell populations and its functional role in restraining the inflammatory response was confirmed in vitro. Preliminary functional validation experiments showed that increased TGT expression results in a decreased proinflammatory cytokine secretion by cells that participate in both innate and adaptive immune response. An exacerbated pro-inflammatory response was observed in inflammatory cells under TGT-deficient conditions. TGT biology is being pursued by renowned academic groups. Work that describes the metabolic phenotype of TGT-deficient mice was recently published and confirms the importance of that target in regulating glucose and lipid homeostasis in vivo. No data exist that relates to the selective pharmacological modulation of TGT in vivo. The identification of synthetic, drug-like TGT modulators was a major deliverable of Genfit within TOBI. A drug discovery programme was initiated within the project. As a result, small molecule compounds that bind to the target and provide anti-inflammatory action in diverse model systems in vitro were identified in a high throughput screening campaign. Improved, more active, drug-like and structurally novel chemical entities (NCEs) were derived from original hits through functional selection and medicinal chemistry efforts.
Potential impact
Obesity is rapidly increasing has become one of the most important global health issues. Obesity is associated with severe metabolic and cardiovascular sequelae, particularly type 2 diabetes, hypertension, coronary heart disease and stroke. Obesity and associated adipokine-mediated disorders counteract European initiatives to promote healthy ageing. Insulin resistance mediated by inflammatory adipokines probably plays a predominant role in the pathophysiology of excess fat. Interference with adipokine-mediated crosstalk by novel drugs is the ultimate goal. TOBI has used a multidisciplinary basic biomedical approach to achieve a strong translational aspect. TOBI has addressed issues starting from subcellular aspects. All research aimed at identifying potential drug targets or pathophysiological mechanisms to interfere with the adipokine-mediated inflammatory drift with emphasis on adverse interactions that lead to insulin resistance and diabetes as well as cardiovascular disease. TOBI has contributed to the need for novel pharmacological strategies to restrain the epidemic of obesity-associated morbidity in Europe and the whole first world.
Specific impact
The development of TOBI standards and the TOBI bio bank strengthens the European research on obesity. The TOBI standard protocols are available for all researchers upon request. The TOBI bio bank is available for researchers beyond the project. These tools equip European research to more effectively address the obesity epidemic thereby promoting more general impact of obesity research in Europe. Identification of the enzymatic activity in a signalling molecule activated by damage to the ER allows design of drugs to inhibit this activity in order to inactivate the inflammatory response to damage to the ER. This is applicable to the inflammation associated with obesity, but also applicable to inflammation linked to other diseases associated with ER damage, most notably neurodegenerative diseases such as Alzheimer or Parkinson. Identification of novel molecules of the insulin-signalling pathway allows design of drugs that restore function of these molecules. Substances such as chemical chaperones have been approved by the FDA and could be easily available and relatively inexpensive drugs to combat obesity-related complications. Identification of novel detrimental responses of the ER within the TOBI project has enabled molecular characterisation in future work, which may contribute to a better understanding of how cells respond to ER damage. This is important to understand the role of these responses in diseases that are associated with ER stress and to biotechnological production of therapeutic proteins in eukaryotic cells. The importance of lipases for obesity and its complications was emphasised by the discovery of the ATGL by B10. An impact of ATGL and other lipases was found for the inflammatory crosstalk.
The new results on lipases obtained provide novel insight into the biology of lipid breakdown that is highly required for the design of drugs that interfere with lipid synthesis and breakdown in order to combat the obesity epidemic. TOBI was one of the first projects to shed light on lipid-derived mediator molecules and their role in obesity. LDA turned out to be of potential relevance for the obesity epidemic. Certain LDA and their precursors and derivatives could be developed as potential drugs to interfere with inflammatory processes involved in obesity. Long-chain omega-3 polyunsaturated fatty acids are found in marine fish oils and turned out to be useful for the suppression of inflammatory reactions related to obesity. These data complement results from large clinical trials emphasising the preventive impact of long-chain omega-3 polyunsaturated fatty acids on cardiovascular health. The requirement for fish oils to appropriately supplement the European and the world population largely exceeds the availability of fish. Substances derived from omega-3 polyunsaturated fatty acids that mimic their effects at lower concentration could be used as fish oil substitutes and enable sufficient availability. Certain LDA derived from omega-3 polyunsaturated fatty acids inhibit adipose tissue inflammation at lower doses. These are important contributions of TOBI to appropriate supplementation with effective fish-oil-derived material. Inflammation-activated enzymes play a crucial role in the brain in the impairment of body-wide, systemic metabolism.
These results open up the possibility that inflammatory signals in combination with elevated serum fatty acids may reach specific brain parts and thus activate inflammatory enzymes to affect the functionality of insulin and other hormones, which regulate body weight in healthy patients. Chronic activation of these enzymes would induce neuronal resistance against these hormones, which plays a major role in the development of obesity and subsequently diabetes mellitus. These results have been reported in international meetings and have been published in international top journals. Similar results have been reported by other laboratories worldwide. These results have implications for future research and pharmaceutical drug design. If the brain is a key tissue in which inflammation occurs during obesity, potential drugs preventing chronic inflammation may be chemically designed to be targeted to the brain, but sparing peripheral tissues. This may prevent or dampen side effects. We have detected inflammation in the hypothalamus, the key brain region involved in regulating metabolism and body weight. In neurodegenerative diseases such as Alzheimer or Parkinson, activation of inflammatory enzymes has been detected as well. Potentially linking these two findings, obesity increases the risk to suffer from neurodegenerative diseases.
Current research aims to define if obesity-induced inflammation is found in all or specific brain areas, and if specific, which mechanisms protect the non-affected brain areas. Chronic inflammation can lead to cell dysfunction and cell death. One report indicated that in the hypothalamus, neuronal death occurs upon chronic obesity. Chronic activation of inflammatory enzymes may be involved in these phenomena as well. Future studies will be aimed to define the survival of key neurons involved in metabolism in long-term obesity. TOBI results on inflammatory signalling in the brain exceed the field of obesity and could be relevant for the whole field of neurodegenerative disorders.
TOBI results on ATM revealed considerable parallels to the phenotype of tumour-associated macrophages. Obese patient are more prone to develop cancer than lean subjects. It may be hypothesised that ATM could be a crucial link between obesity and cancer. Many tumours have increased levels of obesity-related factors in their microenvironment, rendering, in some cases, the tumours more aggressive. Macrophages in peri-tumoural adipose tissue which release obesity-related factors could be locally involved in carcinogenesis. We found that some genes expressed by the ATM that play a role in cancer development can be controlled by the pharmacological activation of the nuclear receptor LXR. Our results described a novel phenotype of ATM that can be associated to cancer and can be corrected by the activation of LXR with synthetic ligands. These observations can be the basis for novel pharmacological strategies acting at the level of ATM.
The understanding of the link between morphological alterations of adipose tissue occurring in obesity and the development of type 2 diabetes is becoming clearer and could be fundamental in developing pharmacological strategies aimed to curb the epidemic in western society and in developing countries. Within the consortium, fruitful collaborations have been established with laboratories with several expertises to comprehensively study adipocyte ultrastructure in obesity. Many interactions with all partners have been made both from an intellectual and technical point of view in order to implement these investigations. These data showed correlations that will promote our understanding of the pathophysiology of obesity. The expertise of B3 in microscopy was made more broadly available by generating TOBI standards on the collection, fixation and inclusion of adipose tissue to perform light and electron microscopy. An expert-based immunohistochemical protocol for the evaluation of macrophages presence in the adipose tissue has been established as part of the TOBI toolbox.
Discoveries on adipokine crosstalk between perivascular adipose tissue and the blood vessel wall can be exploited for benefits in health and wealth. In the medium term, these data will inform the potentially diverse therapeutic indications for 11beta-HSD1 inhibitors in obesity and diabetes. The concept of 11beta-HSD1 inhibition is protected by patents owned by the University of Edinburgh. There is a large number of new chemical entity patents published from the University of Edinburgh and from numerous large pharmaceutical companies. Several compounds have reached clinical development but some have stalled because of uncertainty about the future regulatory requirements for evidence of cardiovascular protection for drugs in development for type 2 diabetes. The additional evidence provided for potentially beneficial effects on vessel remodelling may enable this field by further assuageing concerns about cardiovascular safety in the development of these agents. Given some of these agents have already completed phase 2 studies, the opportunities for medium term health and wealth benefits are substantial. A potential drug target has been identified and followed up during the project.
As judged from literature and from our preliminary animal model data, modulators of the target should be able to improve glucose and lipid homeostasis and alleviate chronic adipose tissue inflammation. Target modulators should act through non-redundant mechanisms as compared to existing antidiabetic and hypolipidemic drugs, which suggests that their association with existing, first line treatments is theoretically possible and bring additional benefits to patients. These compounds will address unmet medical needs in a large population of patients who suffer from type 2 diabetes and from associated cardiovascular disease. The aim is to position a first in class and proprietary target modulator as a drug candidate ready to enter in full preclinical development. This compound will comply with all early development requirements. Genfit has the ability to provide clinical proof of concept up to the end of phase 2, but for these large therapeutic areas Genfit will partner via pharmaceutical company alliances to complete development and bring the drug to market in the future. In the longer term there may be additional drug targets validated among the pathways identified in the project. Patenting may follow if data are extended to fully exemplify the utility of these targets in improving obesity-associated vascular remodelling and intra-adipose inflammation.
Ultimate benefit for patients will stem not only from the potential therapeutic approaches identified and supported by TOBI, but also from the provision of laboratory measurement protocols and mechanistic knowledge that will help to refine our understanding of inter-individual differences in susceptibility to complications of obesity and hence potentially facilitate patient stratification for risk assessment and therapy. There are numerous direct beneficiaries from the knowledge generated by this research project. These include the investigators working on the project and their staff and students, who have benefited from training and experience and are likely to continue investigating the novel theme of the role of inflammation in obesity research under additional peer-reviewed external funding. Additional beneficiaries are the TOBI partners whose awareness of the role of specific issues involved in obesity-driven inflammation, e.g. perivascular adipose, has been substantially enhanced by regular discussion in project meetings, and the global scientific community, who will benefit from access to data when these are published in a peer-review journal.
Impact on health care issues
TOBI provided progress to effectively interfere with adipokine-mediated crosstalk for prevention of obesity-associated morbidity will help to stabilise the currently escalating costs for obesity-associated disorders. TOBI has ameliorated societal issues related to obesity. Results could help improve the health of European citizens by having addressed an epidemic health problem with an effective approach by investigating its pathophysiological roots in a translational approach. Translation discoveries into clinical application has been taken seriously by linking world-class academic researchers directly to drug developing and analytical companies included in the consortium. The consortium has contributed to the development of new therapies for obesity-related disorders and has started to validate a potential drug target. Novel methods for health promotion and prevention of obesity-associated disorders could be future effects of TOBI, contributing to healthy ageing of the European population. Novel strategies based on TOBI results could include treatment options that potentiate beneficial effects of healthy life style on adipokine-mediated inflammation, e.g. by developing omega-3 fatty acid-derived substances to be used as drugs or included in functional foods. TOBI addressed juvenile obesity, a rapidly increasing health issue with long-term consequences. TOBI has characterised a considerable number of obese adolescents and collected clinical and biochemical data. Further studies on juvenile obesity have been facilitated for a broader scientific community by including these samples in the TOBI bio bank.
Cross-thematic impact
Investigating products of dietary lipids has been part of the research, particularly omega-3 polyunsaturated fatty acids (PUFA). TOBI is cross-thematically related to FP7 topics on food quality. Many LDA are derived from omega-3 PUFA are found in marine fish oils. TOBI perfectly synergises and provides complementarity with themes on 'Food, Agriculture and Fisheries, and Biotechnology'. TOBI has achieved knowledge contributing to the impact of food and nutrition on health in order to fight diet-related disorders. TOBI has contributed to the understanding of beneficial and harmful dietary factors in order to control the development and reduction of diet-related diseases. TOBI could have an impact on European food-related industries. Results could contribute to the development of functional foods and preventive options. The limited availability of fish oils due to ecologic considerations and reduced acceptance of high-dose marine n-3 PUFA ingestion due to gastrointestinal side effects prohibit their population-wide consumption even though omega-3 PUFA prevent the obesity-related inflammatory drift and adipose tissue in mice. TOBI investigations on LDA have identified omega-3 PUFA-derived substances that resolve inflammation and are effective in adipose tissue. Results could provide the knowledge required for developing functional foods or preventive medicines for inflammatory disorders not confined to obesity.
Competitiveness and added value
TOBI has improved the competitiveness of European universities and research-based SMEs by collaboration on methodological and thematic aspects that will bring forth added value to all participants. TOBI has increased the innovative capacity of the European health-related sector by facilitating tight interactions with world-class European. TOBI collaborations have strengthened the international competitiveness of European academies and SMEs that will generate added value for SMEs and academic research. By bringing together internationally renowned experts in closely interacting WPs, TOBI has achieved world-class collaborative research on obesity. The complementary expertise of partners' fields of research has been successfully organised. TOBI facilitated sharing of methodologies between a critical mass of partners, potentiating their scientific impact. TOBI has made technically sophisticated methodologies available for its partners and has brought about breakthroughs in obesity research. TOBI has produced knowledge that will significantly contribute to the development of pharmacologic strategies to prevent and treat obesity and associated complications. TOBI adds to the detection and monitoring of obesity-associated adipokine-mediated adverse interactions. These results could lead to the development of individualised pharmacological strategies in order to optimise the delivery of healthcare to European citizens by tailored medicine.
Dissemination
The coordinator has encouraged all participants to actively disseminate their project aims and results to colleagues, stakeholders and the wider public. The TOBI dissemination activities aimed at linking project topics with topics with almost everyone is affected: overweight, obesity and nutrition. A logo and an overall project design have been created in order to identify the project. Public awareness of TOBI has been addressed by the website http://www.tobi-project.eu The content is regularly checked and updated. The website is available in four languages and will be continued after the project ends. Emphasis has been placed on media-rich presentation of the content (see http://www.youtube.com/user/TheTOBIproject and http://www.vimeo.com/26270197 online). The news-section provides visitors with the latest publications and dissemination activities. TOBI was presented to the public at large during the European Researchers' Night 2009 in Vienna. TOBI organised a scientific cooking event which main goal was to help people understand that a healthier life begins in the kitchen and in their minds.
The consortium takes measures to provide TOBI exposure to the public through conventional PR media work, which will complement the public access website, particularly for those that do not have Internet access. Press kits sent out to journalists and other stakeholders provide them with all project related information. Posters and folders about the impact of overweight and obesity were created to be distributed by partners. The coordinator published several articles in the general press. A success story about TOBI was published by the Austrian National Contact Point (NCP). The scientific community has a vested interest in keeping up with TOBI progress and results to build on the body of knowledge that currently exists. This will be achieved through conventional scientific dissemination means, i.e. peer-reviewed publications and presentations at international conferences. Some results of the project have not yet been published but are submitted to or under revision at scientific journals or manuscripts for scientific journals are written at the time of the report.
Project website: http://www.tobi-project.eu/
Contact details
Project coordinator:
Medizinische Universität Wien
Prof. Thomas Stulnig
Clinical Division of Endocrinology and Metabolism
Department of Medicine III
Wahringer Gurtel 18-20, 1090 Vienna, Austria
E-mail: thomas.stulnig@meduniwien.ac.at
Phone: +43-140-4004368
Fax: +43-140-4007790
Biolution - TOBI office
Karl-Farkas-Gasse 22
1030 Vienna, Austria
E-mail: office@tobi-project.eu
Phone: +43-178-6959512
Fax: +43-178-6959520
List of partners
Medical University of Vienna (Coordinator), Thomas Stulnig, Austria
biolution GmbH (SME), Iris Grunert, Austria
Universitat zu Koln, Jens Bruning, Germany
Universita politecnica delle marche, Saverio Cinti, Italy
Genfit SA (SME), Matthieu Dubruque, France
Pharm-analyt (SME), Hermann Mascher, Austria
University of Durham, Martin Schroeder, United Kingdom
Institut Pasteur de Lille, Bart Staels, France
The University of Edinburgh, Brian Walker, United Kingdom
Universität Graz, Rudolf Zechner, Austria.