University of Cambridge

1. University of Cambridge Overview of our programme of research Lipotoxicity and the metabolic syndrome Lipotoxicity and the metabolic syndrome. Toni Vidal-Puig Nugo 2010

2. Ingestion – Energy expenditure = Fat Deposition Overnutrition/Excess of energy = Obesity Increased demands to adipose tissue expandability ENERGY BALANCE The development of obesity requires a state of positive energy balance What is not that clear is why expansion of the adipose tissue causes metabolic problems.

3. Hypothesis: Lipid induced toxicity, “Lipotoxicity”, as the main cause obesity induced of insulin resistance Lipotoxicity Overnutrition Lipids Activated Blockage

4. Overview of our Programme LIPOTOXICITY: Inappropriate lipid storage in tissues other than adipose is the major underlying factor linking obesity and insulin resistance Hypothesis 1: Improving the capacity for lipid storage in adipose will protect against insulin resistance and diabetes Hypothesis 2: In the advent of a failure to store lipid appropriately in adipose tissue then mitochondrial oxidation of lipids will protect against diabetes Hypothesis 3: When adipose storage and oxidation fail to prevent inappropriate deposition of lipid in other tissues, the type of lipid deposited is more important than the amount of lipid stored.

5. Hypothesis 1: Improving the capacity for lipid storage in adipose will protect against insulin resistance and diabetes (Sethi, J. K., and Vidal-Puig, A. J. (2007) J Lipid Res48(6), 1253-1262) • PPARg Modelling networks related to adipogenesis and lipid metabolism in the context of lipotoxicity and insulin resistance Studies on PPARg2KO and PPARgdn mice Medina-Gomez. (2007) PLoS Genet 3(4), e64 Gray, S. L.,(2006) Diabetes 55(10), 2669-267 Gray, S. L.(2006) Endocrinology 147(12), 5708-5714 Medina-Gomez, G.(2005) Diabetes 54(6), 1706-1716 b) Wnt signalling Predominantly cell biology studies Christodoulides, C (2006) Diabetologia 49(4), 678-684 Christodoulides, C. (2006) J Cell Sci 119(Pt 12), 2613-2620 c) Prostaglandin Metabolism and adipogenesis Prostaglandin synthase related mouse models

6. Hypothesis 2: In the advent of a failure to store lipid appropriately in adipose tissue then mitochondrial oxidation of lipids will protect against diabetes • Mechanism promoting increased fat oxidation through • mitochondria biogenesis or mitochondrial uncoupling. a) PGC1b PGC1 related mouse models Lelliott, C. J., Medina-Gomez, G.(2006) PLoS Biol 4(11), e369 b) Uncoupling proteins UCP related mouse models • Mechanism controlling brown fat differentiation as a strategy • to increase energy dissipation. c) Bone Morphogenic proteins BMP KO and transgenic models

7. Hypothesis 3:When adipose storage and oxidation fail to prevent inappropriate deposition of lipid in other tissues, the type of lipid deposited is more important than the amount of lipid stored. • Identification of organ specific lipid networks may provide key information for specific therapeutic interventions. 2) Specific lipotoxic patterns or species may provide an earlier and more accurate metabolic Biomarker of cardiometabolic risk.

8. Hypothesis 3: When adipose storage and oxidation fail to prevent inappropriate deposition of lipid in other tissues, the type of lipid deposited is more important than the amount of lipid stored. a) Plasmalogens and metabolism Gnpat KO mice Identification of plasmalogens, a type of phospholipids, relevant for lipid membrane homeostasis, siganlling and antioxidant activity. b) Role of elongases on insulin resistance Elov6 and Elov3 KOmice Enzymes responsible of elongation of fatty acids leading to specific Long chain fatty acid species of relevance in metabolism. c) Role of SREBP1 controlling specific lipid pathways SREBP1c KO mice

9. Our research strategies are influenced by an allostatic perspective of lipotoxicity which suggests that in a pathogenic state an organism attempts to maintain homeostasis through changes in parallel systems. We believe that considering diseases from an allostatic perspective will enable the design of treatments that will provide greater health benefits and greater long term efficacy.

10. Mouse models Day 3 Day 4 Day 6 WT KO Circumstantial evidence for robust compensatory mechanisms occurring in vivo Genetic modifications supposed to cause important adipose tissue disturbances paradoxically reveal mild phenotypes. Adipose tissue in the PPARg2KO mouse Lack of differentiation of PPARg2 deficient white preadipocytes in vitro Epididymal WAT WT KO

11. PGS Expression in PPARy2 KO Brown adipose tissue PGS *** Relative Expression (WT=1) WT KO * 1.4 1.2 1 0.8 Relative Expression (WT=1) 0.6 0.4 0.2 0 Cont TZD Allostatic upregulation of PGS in BAT in PPARy2 KO mice PGS x PPARy2 double knock-out mice have worse glucose tolerance when compared to either single mutant 25 $ ** *** $$$ 20 *** WT DKO *** PGS PPARy2 15 Glucose (mMol/dl) ### DKO $ 10 5 0 0 50 100 150 200 Time (min) Sam Virtue

12. Allostasis is a concept from 1980s referring to the mechanisms controlling homeostasis through change initially applied to stress. Some definitions Homeostasis is the maintenance of biological systems to maintain life Allostasis is maintenance of stability through change. Allostatic load is the level of demand on systems for correction Allostatic overload refers to a state of prolonged demand on a given allostatic system that will ultimately cause the system to fail if not corrected

13. Biological Phenotyping Basic phenotyping: 1) targeted to areas of interest OR 2) targeted to a hypothesis predicted phenotype Differences identified and described

14. Identify molecular mechanism to understand the effect of an intervention (KO, TG, drug, diet etc) This involves looking at the: Genome Transcriptome Proteome Metabolome

15. Our experience to date • Samples with large differences: • Able to discriminate, however • In samples with larger biological variability (eg humans); discriminatory strength of lipidomics can be poor despite clear biochemical differences. • Sample size optimisation required • Correction for inter-individual differences required • Able to detect the expected differences • Can identified novel findings, but • Follow-up targeted analysis requires time to set up • Evidence from other modalities do not always fit; an explanation for the discrepancy is not always forthcoming.

16. Our experience to date • Samples with no obvious differences (Differences are either absent or subtle): • Some differences are detected, but • Not easy to decide whether it is worth pursuing and going through the process of validating the findings. (At present this decision is dependent upon data outside of metabolomics) • If the decision is to pursue the metabolomic changes, such subtle findings are difficult interpret. We have learnt a lot about lipidomics, and it is making us rethink how we should set up of our biological experiments, particularly the controls

17. An obese but insulin sensitive mouse model Based on the adipose tissue expandability hypothesis it should be possible to become massively obese without metabolic complications, so long as new adipocytes can be made. • 50% greater body weight than obob • Increased insulin sensitivity • No ectopic deposition in fat • Preferential deposition in ScWAT Mouse over expressing adiponectin in adipose tissue of an obese ob/ob background (Ja-Young Kim, et al 2007 JCI) First example of a mouse with apparently limitless adipose tissue expandability

18. Some adipose tissue data ■ AdTG ■ ob/ob ■ WT fasted ■ WT fed ob/ob Fasted Component2 Separates Fast&Ob/Ob vs Fed&AdTG ob/ob Adiponectin Transgenic Fed Component1 separates ObOb from WT

19. ClinicalParadigms – Clinical research Morbid Obesity with and without Metabolic Syndrome Lipotoxicity markers as predictors of disease Candidate approach Lipotoxicity Non candidate approach Wide approach Adipose tissue Blood Muscle Liver

20. Chong Yew Tan Crystal Mok Daniel Hart Agnes Lukasik Alberto Camacho Andy Whittle Iris Schmitt Ko Ishikawa Gema Medina Helen Westby Janice Carter Margaret Blount Martin Dale Rachel Hagen Mark Campbell Jackie Higgins Sam Virtue Sarah Grocott Sophie Gough Sergio Rodriguez Stefania Carobbio

21. Hope this makes sense Thanks