Retinoic acid receptors and binding proteins in regulation of gene expression

Abstracts / Chemistry and Physics of Lipids 163S (2010) S1–S18

SO 16 Differential regulation of cardiac GLUT4-mediated glucose and CD36-mediated fatty acid uptake by endosomal pH and actin filaments Laura K.M. Steinbusch 1 , Wino Wijnen 1 , Robert W. Schwenk 1 , Will A. Coumans 1 , Nicole T.H. Hoebers 1 , D. Margriet Ouwens 2 , Michaela Diamant 3 , Arend Bonen 4 , Jan F.C. Glatz 1,∗ , Joost J.F.P. Luiken 1 1

Department of Molecular Genetics, Maastricht University, Maastricht, The Netherlands 2 German Diabetes Center, Düsseldorf, Germany 3 Department of Endocrinology, Diabetes Center, VU University Medical Center, Amsterdam, The Netherlands 4 Department of Human Health and Nutritional Science, University of Guelph, Guelph, Ontario, Canada Insulin and contraction stimulate both cardiac glucose and longchain fatty acid (LCFA) uptake via translocation of the substrate transporters GLUT4 and CD36, respectively, from intracellular compartments to the sarcolemma. Little is known about the role of vesicular trafficking elements in these processes in the heart, especially whether certain trafficking elements are specifically involved in GLUT4 versus CD36 translocation. Therefore, we studied the role of coat proteins, actin/microtubule-filaments and endosomal pH on glucose and LCFA uptake, and GLUT4 and CD36 location, in primary cardiomyocytes under basal conditions and during stimulation with insulin or oligomycin (contraction-like AMP-activated protein kinase activator) by pharmacological inhibition of these vesicular trafficking elements. Inhibition of coat protein targeting to Golgi/endosomes decreased insulin/oligomycin-stimulated glucose (−42%/−51%) and LCFA (−39%/−68%) uptake. Actin disruption decreased insulin/oligomycin-stimulated glucose uptake (−41%/−75%), while not affecting LCFA uptake. Microtubule disruption did not affect substrate uptake under any condition. Endosomal alkalinization increased basal sarcolemmal CD36 (2-fold), but not GLUT4, content, and concomitantly decreased basal intracellular membrane GLUT4 and CD36 content (−60% and −62%, respectively), indicating successful CD36 translocation and incomplete GLUT4 translocation. Additionally, endosomal alkalinization elevated basal LCFA uptake (1.4-fold) in a non-additive manner to insulin/oligomycin, and decreased insulin/oligomycin-stimulated glucose uptake (−32%/−68%). In conclusion: (1) CD36 translocation, just like GLUT4 translocation, is a vesicle-mediated process depending on coat proteins and (2) GLUT4 and CD36 trafficking are differentially dependent on endosomal pH and actin filaments. The latter conclusion suggests novel strategies to alter cardiac substrate preference as part of metabolic modulation therapy. Acknowledgement Supported by the Dutch Diabetes Research Foundation grant 2006.00.044 doi:10.1016/j.chemphyslip.2010.05.035

S11

Session 5: Lipid-binding proteins (A session organized by J. Glatz and J. Storch on behalf of the LBP Society) PL17 Retinoic acid receptors and binding proteins in regulation of gene expression Noa Noy Department of Pharmacology, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA The vitamin A metabolite retinoic acid (RA) plays central roles in embryonic development and in the adult, and is also currently used as a therapeutic agent in pathologies ranging from dermatological disorders to cancer. The pleiotropic biological activities of the hormone are exerted through its ability to regulate gene expression, activities that are mediated by the nuclear receptors RAR and PPAR␤/␦ and by the intracellular lipid-binding proteins CRABP-II and FABP5. The mechanisms by which RA-binding proteins cooperate with RA nuclear receptors to mediate the transcriptional activity of their ligand, and functional consequences of this cooperation will be discussed. doi:10.1016/j.chemphyslip.2010.05.036 PL18 Lipid-regulated sterol transfer between closely apposed membranes by oxysterol-binding protein homologues Sumana Raychaudhuri, Tim Schulz, Mal-Gi Choi, Will Prinz ∗ Laboratory of Cell Biochemistry and Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA Zones of close contact between organelles, known as membrane contacts sites, have been observed in all eukaryotic cells and are thought to play an important role in intracellular communication. They are regions where small molecules, such lipids and calcium ions, or other signals are transferred between organelles. However, in most cases, we have only a relatively poor molecular understanding of how these highly conserved structures are formed and function in cells. Some oxysterol-binding protein-related proteins (ORPs) localize to membrane contact sites. ORPs are a large family of lipid-binding proteins that have been implicated in sterol sensing, vesicular trafficking, and nonvesicular sterol transport. We have found that yeast ORPs use a novel mechanism that allows regulated sterol transfer between closely apposed membranes such as organelle contact sites. We find that the core lipid-binding domain found in all ORPs can simultaneously bind two membranes. Using Osh4p/Kes1p as a representative ORP, we show that ORPs have at least two membrane-binding surfaces; one near the mouth of the sterol-binding pocket and a distal site that can bind a second membrane. The distal site is required for the protein to function in cells and, remarkably, regulates the rate at which Osh4p extracts and delivers sterols in a phosphoinositide-dependent manner. Together, these findings suggest a new model of how ORPs could sense and regulate the lipid composition of adjacent membranes. doi:10.1016/j.chemphyslip.2010.05.037