Novel Regulatory Mechanisms for Generation of the Soluble Leptin Receptor: Implications for Leptin Action Michael Schaab1, Henriette Kausch1, Juergen Klammt2, Marcin Nowicki3, Ulf Anderegg4, Rolf Gebhardt5, Stefan Rose-John6, Juergen Scheller6, Joachim Thiery1, Juergen Kratzsch1* 1 Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University of Leipzig, Leipzig, Germany, 2 Hospital for Children and Adolescents, University of Leipzig, Leipzig, Germany, 3 Institute of Anatomy, Faculty of Medicine, University of Leipzig, Leipzig, Germany, 4 Department of Dermatology, Venerology and Allergology, Faculty of Medicine, University of Leipzig, Leipzig, Germany, 5 Institute of Biochemistry, Faculty of Medicine, University of Leipzig, Leipzig, Germany, 6 Institute of Biochemistry, Christian-Albrechts-University Kiel, Kiel, Germany Abstract Background: The adipokine leptin realizes signal transduction via four different membrane-anchored leptin receptor (Ob-R) isoforms in humans. However, the amount of functionally active Ob-R is affected by constitutive shedding of the extracellular domain via a so far unknown mechanism. The product of the cleavage process the so-called soluble leptin receptor (sOb-R) is the main binding protein for leptin in human blood and modulates its bioavailability. sOb-R levels are differentially regulated in metabolic disorders like type 1 diabetes mellitus or obesity and can, therefore, enhance or reduce leptin sensitivity. Methodology/Principal Findings: To describe mechanisms of Ob-R cleavage and to investigate the functional significance of differential sOb-R levels we established a model of HEK293 cells transiently transfected with different human Ob-R isoforms. Using siRNA knockdown experiments we identified ADAM10 (A Disintegrin And Metalloproteinase 10) as a major protease for constitutive and activated Ob-R cleavage. Additionally, the induction of lipotoxicity and apoptosis led to enhanced shedding shown by increased levels of the soluble leptin receptor (sOb-R) in cell supernatants. Conversely, high leptin concentrations and ER stress reduced sOb-R levels. Decreased amounts of sOb-R due to ER stress were accompanied by impaired leptin signaling and reduced leptin binding. Conclusions: Lipotoxicity and apoptosis increased Ob-R cleavage via ADAM10-dependent mechanisms. In contrast high leptin levels and ER stress led to reduced sOb-R levels. While increased sOb-R concentrations seem to directly block leptin action, reduced amounts of sOb-R may reflect decreased membrane expression of Ob-R. These findings could explain changes of leptin sensitivity which are associated with variations of serum sOb-R levels in metabolic diseases. Citation: Schaab M, Kausch H, Klammt J, Nowicki M, Anderegg U, et al. (2012) Novel Regulatory Mechanisms for Generation of the Soluble Leptin Receptor: Implications for Leptin Action. PLoS ONE 7(4): e34787. doi:10.1371/journal.pone.0034787 Editor: Massimo Federici, University of Tor Vergata, Italy Received May 25, 2011; Accepted March 8, 2012; Published April 24, 2012 Copyright: 2012 Schaab et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Foundation of Pathobiochemistry and Molecular Diagnostics of the German Society of Clinical Chemistry and Laboratory Medicine e.V.(http://www.dgkl.de), the SFB610 TP B7 and the German Science Foundation (http://www.dfg.de). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Juergen.Kratzsch@medizin.uni-leipzig.de Introduction Leptin (Ob), a 16 kDa protein primarily secreted by adipocytes, regulates energy homeostasis via so called central mechanism in the brain. Besides its central effects in the brain, a large number of peripheral actions have been discovered in the last years. Thus, leptin affects the immune system by influencing CD4+ T-cell polarization, B-cell homeostasis as well as renal macrophage infiltration [1–3] and modulates the severity of sepsis [4]. Furthermore, leptin plays a role in the regulation of liver and skeletal muscle lipid oxidation and glucose metabolism [5–9]. Leptin actions are mediated through the leptin receptor (Ob-R) that belongs to the class I cytokine receptor family [10]. So far in humans four different membrane-anchored Ob-R isoforms have been discovered which have identical extracellular, ligand-binding and transmembrane domains but differ in the length of the intracellular domain. Only the long form (Ob-Rfl) has the so called Box 3 motif with Tyr1141 and is therefore considered to possess full signaling capacity via the JAK-STAT (Janus kinase-signal transducer and activator of transcription) pathway [11]. However, latest findings indicate that short isoforms may be involved in leptin signaling as well [12]. In addition, the presence of a soluble receptor form (sOb-R) [13,14] was demonstrated in peripheral blood that represents the main leptin binding activity [15] and reflects the density of membrane Ob-R [16]. Previous findings suggest a predominant inhibitory effect of sOb-R on leptin bioactivity in cell and animal models [17,18]. Thus, high sOb-R concentrations neutralized leptin-mediated STAT3 signaling and anorexic responses in rats [19] and protected mice against experimentally-induced sepsis [4]. Interestingly, transgenic mice with an overexpression of sOb-R showed decreased body weight and increased energy expenditure which would point to a positive correlation between serum sOb-R concentrations and leptin sensitivity [20]. Clinical studies demonstrated both increased and PLoS ONE | www.plosone.org 1 April 2012 | Volume 7 | Issue 4 | e34787