Burning fat: an in vivo and in vitro study of the role of PPAR-delta in regulating fat metabolism in adipose tissue

People in the UK are getting fatter and this has consequences for both the health and wealth of the nation. Obesity increases the risk of a number of diseases including type 2 diabetes, heart disease, stroke and high blood pressure. These diseases will place an increasing burden on the National Health Service and also impair the ability of individuals to work. Central to this problem is energy balance which put basically is the difference between energy coming in as food and energy expenditure of the body. While one obvious solution is to reduce intake of high calorie foods and increase exercise in individuals, national strategies in this area have failed to halt the increase in obesity (or indeed slow the rate of increase). Furthermore, once an individual is obese it may be difficult for that person to exercise and reduce obesity. There are indications that drugs that increase the energy expenditure of the body may be used to reduce obesity and many of the risk factors for other diseases associated with obesity (e.g. insulin resistance, coronary artery disease). Several types of drugs target two proteins found in fat cells referred to as PPAR-gamma and PPAR-delta. These proteins in turn 'switch-on' genes important in either fat metabolism or fat storage. While a large amount of work has been carried out characterising PPAR-gamma, a known target for treating type II diabetes, relatively little work has been performed on PPAR-delta. This proposal sets out to investigate the role that these two receptors play in energy balance in fat cells using a combination of animal studies and in vitro cell culture. For this we will investigate the action of two drugs that target either PPAR-gamma or PPAR-delta in adipose tissue in mice and investigate how they alter the concentration of key metabolites using mass spectrometry and Nuclear Magnetic Resonance (NMR) spectroscopy, gene expression using DNA microarrays and protein content by mass spectrometry based proteomics. The data collected will then be modelled mathematically by statistics to generate hypotheses which can be pursued in cell culture based experiments. The latter approach allows us to manipulate the system more easily and hence probe mechanisms of action. This work will increase our knowledge of the mechanisms controlling energy balance in fat cells and also allow us to develop an experimental approach which could be used to understand other biochemical processes. In addition the information obtained will help better characterise a major potential drug target for obesity and associated complications.

PPAR-delta is a receptor that is highly expressed in adipose tissue, and has been shown to be a potent target for the treatment of obesity. However, to date relatively little work has been carried out on this receptor compared with the other members of the PPAR family, PPAR-alpha and PPAR-gamma, both targets for current treatments of type II diabetes and aspects of the metabolic syndrome. PPAR-delta plays a major role in regulating the transition between fatty acid storage and fatty acid oxidation. Understanding the processes that allow the switch between storage and catabolism of fatty acids in adipose is one of the great challenges in understanding lipid metabolism within the body. This proposal describes a systems biology study of the action of PPAR-delta agonists in adipocytes, making use of a combined in vivo and in vitro approach using a combination of metabolomics, stable isotope analysis, transcriptomics and proteomics. The proposal will examine the metabolic consequences of administrating PPAR-delta, PPAR-gamma and PPARpan (targeting both PPAR-delta and PPAR-gamma) agonists to adipose tissue, examining both acute and chronic (two year) studies in mice using our poly-omic approach. Data fusion will be performed using a variety of multivariate statistics and the use of pathway analysis tools. Key metabolic changes will then be modelled in vitro in cell culture of 3T3-L1 cells using a combination of metabolomics, stable isotope analysis (fluxomics) and molecular interventions such as enzyme inhibitors and RNAi. In addition, to test the validity of mechanistic changes detected in the mouse to human metabolism we will investigate responses in cultured human primary cells. This proposal will both further define a pharmacological system of great relevance to the regulation of human nutrition and obesity, and provide a poly-omic dataset suitable for others to explore and develop tools for systems biology in adipose tissue.

Understanding the roles of the PPARs in regulating energy balance, nutritional status and health has been a highly active area in many pharmaceutical companies both in the UK and across the globe. A number of agonists have been developed to target PPAR-alpha, PPAR-gamma or all three PPARs in order to treat type II diabetes and obesity. To illustrate this, the global sales of three PPAR agonists are listed: The PPAR-gamma agonists Rosiglitazone and pioglitazone have combined 2007 sales of about US$ 6.6 billion. The PPAR alpha agonist fenofibrate from Solvay Pharmaceuticals and Abbott yielded 2007 sales of US$ 1.9 billion. Thus, a better understanding of how PPAR-delta exerts its action may help in the development of new blockbuster drugs. Furthermore, the PPAR agonists are not without unwanted side-effects and safety concerns, at least from animal studies, and by identifying the consequences of chronic stimulation of the receptors one may be able to identify targets downstream of the PPARs which produce the beneficial effects on health without the unwanted side-effects. To ensure our research will be of relevance to the pharmaceutical industry we will continue a close collaboration between Drs Andy Nicholls and John Haselden at GlaxoSmithKline (see letter of support). This collaboration has already produced two project grants and two PhD studentships for the Griffin group. Understanding the regulation of energy balance will have benefit to the public in the UK. As stated in the application the UK is experiencing dramatic increases in obesity and the diseases it causes. This is affecting both the young and old and will have a significant health burden on the National Health Service in the future, as well as the capabilities of the UK's work force. Better understanding of how PPAR-delta upregulates beta-oxidation in adipose tissue will allow the development of drug, and even possibly nutritional interventions to stimulate the receptor. To ensure our work benefits the wider scientific community we will ensure that our results are published as manuscripts, and these, where possible, are open access. In addition Dr. Griffin has been involved in a number of schemes aimed at disseminating the results of his research to the public. This includes the 'Head Start' scheme for 17 year old scientists and the Princes Teaching Institute for school teachers. He has also appeared on BBC Radio 4's Material World discussing his research. The UK has a proud record in the development of mass spectrometry and one aspect of this proposal is the collaboration with Waters to improve structure identification in lipidomics. Although Waters is an international company it has a significant research base in Manchester (formerly Micromass). Our collaboration will help the company develop new solutions for lipidomics, particularly in software tools for interpreting the vast multivariate data produced. The project will also provide training in areas relevant to analytical biochemistry, safety assessment and drug efficacy, skills that are in demand in both academia and industry. This is important for the future of 'UK plc' as the major attraction of pharmaceutical and biotechnology companies to this country is the highly skilled work force found in the UK.