THE ROLE OF RENALASE IN BROWN ADIPOSE TISSUE AND OBESITY Monica Chand Supervisor: Dr Harpal S Randeva Warwick Medical School, University of Warwick, Gibbet Hill, Coventry, CV4 7AL INTRODUCTION Obesity is the most visible and most worrying health problem in the UK and world today, hence the term “globesity”. The consequences of obesity and high fat diets result in a range of life-threatening health conditions including diabetes, hypertension, stroke, cancers, cardiovascular diseases and many more life-threatening illnesses. Renalase is a novel flavin adenine dinucleotide-dependent amine oxidase enzyme that regulates cardiac function and systemic blood pressure by metabolising catecholamines. Brown adipose tissue (BAT) is involved in thermoregulatory thermogenesis and energy balance. It is the so-called “good” brown fat as the more we have of it, the less likely we are to lay down excess energy as white fat. It decreases with age, body fat mass and in diabetes. It is also known to increase in cold conditions. Since the prevalence of BAT increases with body fat mass, there may be a link with energy balance and obesity. UCP-1 is an uncoupling protein found in brown adipocytes which is used to generate heat and may be linked to obesity-resistance. No study, conducted in the past, has looked at the link between renalase expression in brown adipose tissue and the relationship with obesity. DISCUSSION CONCLUSION ACKNOWLEDGEMENTS REFERENCES  Homogenisation of mice adipose tissue  RNA extraction  cDNA conversion  Quantitative Reverse Transcriptase-Polymerase Chain Reaction (qRT-PCR)  Western Blotting Acknowledgements to Dr. Harpal Randeva and his Molecular Endocrinology group, Dr. Jing Chen and Dr. Manjunath Ramanjaneya. Xu J, Li G, Wang P, Velazquez H, Yao X, Li Y, et al. Renalase is a novel soluble monoamine oxidase that regulates cardiac function and blood pressure. Journal of Clinical Investigation. 2005;115:1275–1280. Richard D, Monge-Roffarello B, Chechi K, M. Labbé S, E. Turcotte E. Control and physiological determinants of sympathetically mediated brown adipose tissue thermogenesis. Frontiers in Endocrinology The data obtained from the western blot and the PRC product (FIG. 1 & FIG. 2), confirmed that renalase is indeed expressed in BAT. In addition, the results from the qRT-PCR shows that the renalase expression differed between undifferentiated BAT (with a lower expression), and differentiated BAT, with a much higher expression (FIG. 3). These findings may suggest that renalase has a key role in differentiated (9 day) BAT. Another major finding was a significant difference in renalase expression in cold-exposed BAT and room temperature-exposed BAT (FIG. 4A). A much high renalase mRNA expression was found in the cold- exposed BAT. It has been found that the amount of BAT increases in colder environments, which leads us to believe the results found here, suggesting a link between renalase expression in BAT, with decreased temperatures increasing renalase production. In cold temperatures there is an increase in thermogenesis, therefore there may be an increase in the level of catecholamines. This increase in renalase expression in colder environments may act as a counter-regulatory process which acts to reduce this increase in catecholamines. The increase in UCP1 in the cold (FIG. 3B) reflects the increase in thermogenesis. In order to determine the link with obesity and renalase in BAT, mice BAT with a normal diet was compared with that of a high fat diet. However, no significant difference was found in mice given both a 12-week and 20-week diet (FIG. 5). Renalase was also looked at in subcutaneous and omental fat with mice given a 20- week normal/high fat diet, but no significant difference was found (FIG. 6). Future work would involve a more detailed study, maybe using a longer-term diet with high fat and normal BAT or look at a wider range of temperatures to see how levels of renalase changes. In conclusion, our results confirm that there is a clear difference between renalase expression in BAT exposed to different levels of differentiation and also BAT exposed to different temperatures. Since an increase in fat, especially omental fat, is associated with obesity, diabetes and cardiovascular dieases, if renalase was strongly linked with obesity then we would have seen a significant difference between fat from a high fat diet compared to a normal diet. However, no major significant difference was found in this study. FIG. 1. Western blot showing renalase protein expression Western blot showing renalase expression in H295R (H), perirenal tissue (P), omental fat (OM), subcutaneous fat (SC) and brown adipose tissue (BAT). FIG. 6. Renalase mRNA expression in normal diet and high fat diet subcutaneous and omental fat No significant difference was found in the renalase expression in subcutaneous fat (SC) exposed to a high fat diet (HF) and in omental fat (OM) exposed to a normal diet (ND) in a 20-week feeding program (A). A FIG. 4. Renalase and UCP1 mRNA expression in cold and room temperature BAT BAT exposed to the cold temperature (4°c) showed a significant difference in the renalase mRNA expression compared to BAT exposed to room temperature (20°c), (A). UCP1 was used as a positive control (B). (*, P > 0.05; ***, P > 0.001). METHODS RESULTS FIG. 3. Renalase and UCP1 mRNA expression in undifferentiated and differentiated BAT Differentiated t37i cells showed a significant difference in the renalase mRNA expression compared to undifferentiated T37i cells (A). Differentiated BAT was treated with and without Isoproterenol. UCP1 was used as a positive control (B). (***, P > 0.001). FIG. 5. Renalase mRNA expression in normal diet and high fat diet BAT No significant difference was found in the renalase expression in BAT exposed to a high fat diet (HF) and BAT exposed to a normal diet (ND) in both a 20-week feeding program (A) and a 20-week feeding program (B). AIMS To identify the difference of renalase expression in: Differentiated and undifferentiated BAT (0 day, 3 day and 9 day) Cold and room temperature BAT High fat diet and normal diet BAT Subcutaneous and omental fat FIG. 2. Gel electrophoresis images of the PCR products A shows renalase expression (131 bp) in subcutaneous fat (1), omental fat (2) and undifferentiated and differentiated BAT (3 and 4). M is the marker. B shows renalase expression in 0 day, undifferentiated (1) and 9 day, differentiated (2) BAT. B A B A B A B MY URSS EXPERIENCE The URSS has helped me with a range of skills, including fundamental laboratory techniques (PCR and Western blot), communication skills, teamwork and how to work under time pressure. These skills are invaluable for my final year of studying biomedical science. UCP1 expression in BAT exposed to different temperatures Renalase expression in BAT exposed to different temperatures UCP1 expression in undifferentiated and differentiated T37i cells Renalase expression in undifferentiated and differentiated T37i cells Renalase expression in 12-week diet BAT Renalase expression in 20-week diet BAT Renalase expression in 20-week diet SC Renalase expression in 20-week diet OM