ROLE OF CAVEOLIN - 1 IN BROWN ADIPOSE T I S S U E Charlotte L. Mattsson Role of caveolin-1 in brown adipose tissue Charlotte L. Mattsson ©Charlotte Mattsson, Stockholm 2010 Picture on cover: Cryosectioned brown adipose tissue (10 m) stained with caveolin-1 (red), the lipid marker bodipy (green) and the nuclei marker Hoechst (blue). The image was aquired in a Ziess LSM 510 Confocal Microscope. ISBN 978-91-7447-005-5 Printed in Sweden by Universitetsservice AB, Stockholm 2010 Distributor: Stockholm University Library “Knowing others is wisdom, know- ing yourself is enlightenment” Lao Tzu “No amount of experimentation can ever prove me right; a single expe- riment can prove me wrong” Albert Einstein To all my loved ones ABSTRACT Caveolae are 50-100 nm invaginations in the plasma membrane. Caveolae and the protein caveolin-1 (Cav1) have been shown to be important in many signaling pathways in different cell types; however, in some cell types ca- veolae and Cav1 do not seem to affect the investigated signaling pathways. In my thesis, I have investigated the role of caveolin-1 (Cav1) in metabolism and 3-adrenergic, LPA-, EGF- and PDGF-receptor signaling in brown adi- pocytes. Brown adipose tissue is responsible for nonshivering thermogenesis. Re- cent studies have shown that not only infants but also adult man can have brown adipose tissue and that the presence is negatively correlated with both obesity and age. By understanding how signaling for proliferation and diffe- rentiation in brown adipocytes is regulated, it could be possible in the future to activate brown adipose tissue to combat obesity and the metabolic syn- drome. In brown adipocytes, both epidermal growth factor (EGF) and platelet- derived growth factor (PDGF) were able to induce proliferation, which was dependent on Erk1/2 activation. However, EGF and PDGF utilized different pathways to activate Erk1/2, with EGF signaling partially occurring via a Src-pathway (not involving PI3K/PKC) and PDGF via a PI3K/PKC/Src- pathway. Furthermore, LPA receptors were able to activate Erk1/2 via two pathways, one Gi/PKC/Src-pathway and one PI3K-pathway. For these recep- tors, Cav1-ablation did not affect the agonist-induced Erk1/2 activation. Cav1 was, however, required for proper 3-adrenergic receptor (3-AR) sig- naling to cAMP and for adenylyl cyclase activity. In Cav1-ablated mice, the adrenergic receptors are desensitized. However, this desensitization could be overcome physiologically, and the Cav1-ablated mice were therefore able to survive in prolonged cold by nonshivering ther- mogenesis. In conclusion, ablation of Cav1 affected certain signaling pathways in brown adipocytes, while other pathways were not affected or could be phy- siologically rescued. This thesis is based on the following papers, which are referred to in the text by their Roman numerals, respectively I Mattsson, C.L., Csikasz, R.I., Shabalina, I.G., Nedergaard, J., Can- non, B. Caveolin-1-ablated mice survive cold by nonshivering thermogenesis, despite desensitized adrenergic receptors. Submitted II Holmström, T.E., Mattsson, C.L., Fälting, J.M., Nedergaard, J. Differential signaling pathways for EGF versus PDGF activation of Erk1/2 MAP kinase and cell proliferation in brown pre-adipocytes. Experimental Cell Research, 2008, 314, 3581-3592 III Holmström, T.E., Mattsson, C.L., Wang, Y., Iakovleva, I., Petrovic, N., Nedergaard, J. Non-transactivational, dual pathways for LPA-induced Erk1/2 activa- tion in primary cultures of brown pre-adipocytes. Submitted IV Mattsson, C.L., Andersson, E.R., Nedergaard, J. Differential involvement of caveolin-1 in brown adipocyte signaling: impaired 3-adrenergic but unaffected LPA, PDGF and EGF receptor signaling Submitted Contents 1. Introduction ........................................................................................... 13 2. Caveolae and caveolin ......................................................................... 14 2.1 The caveolin proteins ....................................................................................... 15 2.2 The cavin proteins ............................................................................................ 19 2.3 The formation and internalization of caveolae ............................................ 20 2.4 Methods to study caveolae ............................................................................. 21 2.5 Caveolin-ablated mouse models .................................................................... 23 2.5.1 Caveolin-1-ablated mice ........................................................................ 24 2.5.2 Caveolin-2-ablated mice ........................................................................ 25 2.5.3 Caveolin-3-ablated mice ........................................................................ 26 2.5.4 Caveolin-1/3-ablated mice .................................................................... 26 3. Brown adipose tissue ........................................................................... 27 3.1 Properties of brown adipose tissue ............................................................... 28 3.2 Lipolysis and nonshivering thermogenesis .................................................. 30 4. Caveolin and metabolism in adipose tissue .................................... 33 4.1 Cav1-ablated mice: resistance to diet-induced obesity ............................ 34 4.2 Cav1-ablated mice: lipolysis and cold tolerance ........................................ 38 4.2.1 Lipolysis in white adipocytes: fasting and 3-agonist stimulation .. 38 4.2.2 Effect of acute cold and fasting ............................................................ 39 4.2.3 Effect of prolonged cold .......................................................................... 40 5. Caveolin and signaling ......................................................................... 42 5.1 -adrenergic receptors .................................................................................... 42 5.2 2-adrenergic receptors................................................................................... 51 5.3 1-adrenergic receptors................................................................................... 53 5.4 LPA receptors .................................................................................................... 56 5.5 EGF receptors .................................................................................................... 60 5.6 PDGF receptors ................................................................................................. 69 5.7 Other receptors and mediators relevant for brown adipose tissue ......... 74 5.7.1 Insulin receptor ........................................................................................ 74 5.7.2 G-proteins and downstream signaling ................................................. 74 5.8 Conclusions of caveolin and signaling .......................................................... 77 6. Summary and conclusion .................................................................... 79 7. Acknowledgements .............................................................................. 83 8. References ............................................................................................. 87 Abbreviations AC Adenylyl cyclase AR Adrenergic receptor ATGL Adipose triglyceride lipase BAT Brown adipose tissue Cav Caveolin cAMP Adenosine 3′,5′-cyclic monophosphate CSD Caveolin scaffolding domain DAG Diacylglycerol EGFR Epidermal growth factor receptor Erk Extracellular-regulated protein kinase FFA Free fatty acid Gi Inhibitory G-protein GPCR G-protein coupled receptor Gs Stimulatory G-protein HSL Hormone-sensitive lipase IP3 Inositol triphosphate LPAxR Lysophosphatidic acid receptor x MAPK Mitogen-activated protein kinase mCD Methyl--cyclodextrin MEF Mouse embryonic fibroblast MEK MAP/Erk kinase mRNA Messenger ribonucleic acid MRI Magnetic resonance imaging NE Norepinephrine PDGFR Platelet-derived growth factor receptor PI3K Phosphatidylinositol-3-kinase PKA, PKB, PKC Protein kinase A, B and C PLA, PLC, PLD Phospholipase A, C and D PPAR Peroxisome proliferator-activated receptor PTRF Polymerase I and transcript release factor RTK Receptor tyrosine kinase siRNA Small interfering ribonucleic acid WAT White adipose tissue UCP1 Uncoupling protein 1 1. Introduction For many decades, the fluid mosaic model proposed by Singer and Nicol- son in 1972 was the basis for the understanding of the plasma membrane. In this model, proteins were floating around in the lipid membrane and the plasma membrane was regarded as being homogeneous (Singer and Nicol- son 1972, Thomas and Smart 2008). However, work during the two last dec- ades has changed this view of the plasma membrane. The plasma membrane is now known to contain both so-called liquid-disordered phases (liquid crystalline lc phase) and liquid-ordered phases (lo) (de Laurentiis et al. 2007). These liquid-ordered phases, or microdomains, were named lipid rafts based on the different lipid composition they contain (mixtures of phospholipids, sphingolipids and cholesterol) compared to the rest of the plasma membrane. In addition to lipid rafts, caveolae were also discovered (Thomas and Smart 2008). It has been proposed that lipid rafts instead should be called membrane rafts, with the definition “small (10-200 nm) heterogeneous, highly dynamic, sterol-and sphingolipid-enriched domains that compartmentalize cellular processes. Small rafts can sometimes be stabilized to form larger platforms through