decreased levels of sOb-R according to the metabolic situation of the patient. A lack of substrates like glucose in the cellular energy stores as shown in newly manifested type 1 diabetes mellitus (T1DM) or anorexia nervosa is associated with an up to a 100-fold molar excess of sOb-R over leptin in blood [21,22]. In newly manifested T1DM, lipotoxic effects by elevated levels of free fatty acid (FFA) may finally cause tissue specific cell death through apoptosis [23–26] and could be of general importance for the shedding process. In contrast, excess of substrates like glucose in the energy stores, as in obesity, was associated with diminished serum levels of sOb-R. In addition, a strong inverse correlation between plasma sOb-R levels and the risk of developing type 2 diabetes mellitus in human patients points to an important role of sOb-R as modulator of leptin action [16,27–29]. Although having increased leptin concentrations many of obese patients are resistant to leptin action. Ob-R downregulation besides other factors like suppressor of cytokine signaling 3 (SOCS3) and protein-tyrosine phosphatase 1B (PTP1B), could be involved in the pathogenesis of leptin resistance [30–33]. However, the molecular mechanisms for receptor downregulation remain completely unclear. Endoplasmic reticulum (ER) stress is a recently identified key mechanism for the development of obesity and leptin resistance [34–36] and therefore could be a trigger for Ob-R downregulation and suppressed sOb-R generation. The aim of this work was to investigate regulatory mechanisms of increased and suppressed generation of sOb-R in a cell model and to elucidate proteases which process the extracellular Ob-R domain. By employing specific inhibitors and siRNA technique we demon- strate the involvement of ADAM10 and even though to a minor degree of ADAM17 in constitutive and activated Ob-R shedding. Moreover we reveal that apoptosis and lipotoxicity induce processing of the extracellular Ob-R domain. In addition, we show that ER stress and high but still physiological leptin concentrations decrease sOb-R concentration in the cell superna- tant. These data present new insights into regulatory mechanisms of sOb-R generation and add new knowledge to the concept of leptin resistance. Materials and Methods Phorbol myristate acetate (PMA), N-[(2R)-2-(hydroxamidocar- bonylmethyl)-4-methylpentano-yl]-L-tryptophan methylamide (GM6001), staurosporine and Z-VAD(OMe)-FMK were pur- chased from Calbiochem (Merck, Darmstadt, Germany). Fetal bovine serum and PBS were purchased from Biochrom (Bio- chrom, Berlin, Germany). DMEM/F12, Opti-MEM, penicillin and streptomycin, glutamine as well as Lipofectamine 2000 and Lipofectamine RNAiMAX were purchased from Invitrogen (Invitrogen by Life Technologies, Darmstadt, Germany). Dimethyl sulfoxide, sodium palmitate, –oleate, bovine serum albumin and fatty acid free bovine serum albumin (BSA) were purchased from Sigma Aldrich (Sigma Aldrich, Munich, Germany). Metallopro- tease inhibitors GI254023X (GI) and GW280264X (GW) were kindly provided by Dr. JD Becherer (GlaxoSmithKline, Research Triangle Park, North Carolina 27709, USA). CCK8-Assay for determination of cell viability was purchased from Dojindo Laboratories (Dojindo Laboratories, Munich, Germany). Plasmid construction cDNA plasmids for the human short Ob-R isoforms were generated by subcloning of the unique parts of the respective isoforms into pcDNA 3.1(+)/ObRfl (kindly provided by Prof. Ross University of Sheffield, UK). Furthermore we subcloned the cDNA for Ob-Rfl and Ob-R219.3 into pcDNA3.1 Zeo-myc to Regulatory Mechanisms for sOb-R Generation obtain Ob-R constructs myc-tagged at the N-terminus. Sequence integrity of the constructs was verified by restriction endonuclease digestion and sequencing. The myc-tagged constructs were used for all experiments except the investigations on the substrates for Ob-R shedding and the influence of leptin on constitutive shedding. Detailed information concerning the cloning of the different constructs is available on request. Cells culture and transient siRNA or plasmid transfection Human embryonic kidney (HEK) 293 cells (American Type Culture Collection (ATCC) Number: CRL-1573) were cultured in DMEM/F12 supplemented with 10% fetal bovine serum, 100 U/ ml penicillin, 100 mg/ml streptomycin and 2 mM glutamine. One day before transient transfection HEK293 cells were seeded into 6- well or 12-well plates in a density of 0.36106 cells or 0.156106 cells/well respectively. For siRNA experiments cells were seeded in a density of 0.26106 cells/6-well. On the day of transfection DMEM/F12 medium was replaced with Opti-MEM and HEK293 cells were transfected with Ob-R plasmids (1 mg) or siRNA (50 nM) using Lipofectamine 2000 or Lipofectamine RNAiMAX, according to the manufacturer’s instructions. 24 hours after siRNA transfection the cells were transfected with the respective Ob-R constructs using Lipofectamine 2000. Stimulation experiments with the indicated substances were carried out in DMEM/F12 medium containing 100 U/ml penicillin, 100 mg/ml streptomycin and 2 mM glutamine, 0,1% BSA or 2% BSA (fatty acid free) for palmitate and oleate incubations. To exclude a possible influence of the solvent of the used substances controls were performed as indicated. RNA Isolation and Reverse Transcription After aspirating the medium, total RNA from transiently transfected cells was isolated using TRIZOL Reagent (Invitrogen) according to the instructions of the manufacturer. Quality of the isolated RNA was confirmed by spectrophotometric measurement at 260 nm and 280 nm (ND 1000, Nano Drop Products, Wilmington, DE) and through gel electrophoresis. 2 mg of RNA were used for first strand DNA-synthesis using SuperScript II (Invitrogen) following the manufacturer’s instruction. cDNA was diluted 1:10 in Tris-EDTA-buffer and kept at 220 C until use. Primer, probes and siRNA Primers (Eurofins MWG Operon, Ebersberg, Germany) were choosen to selectively amplify cDNA and to avoid amplification of genomic DNA traces. ADAM10 (Gen-Bank NM_001110): forward: 59-CAAAGTCTGAGAAGTGTCGGG-39, reverse: 59- CTGCACATTGCCCATTAATG-39, anneal-ing temperature 60uC, ADAM17 (GenBank NM_003183): forward: 59-GAG- GAAAGGAAA GCCCTGTACAGTAG-39, reverse: 59-CA- GCTGGTCAATGAAATCCCAA-39, annealing temperature 60uC, b-actin (GenBank NM_001101): forward: 59-CCT- GGCACCCAGCACAAT-39, reverse: 59-GCCGATCCACACG- GAGTACTT-39, annealing temperature 60uC. The applied fluorescent probes (Eurofins MWG Operon, Ebersberg, Germany) lie across an exon-exon boundary to further improve specificity of the product detection. Sequences were as follows: ADAM17: 59- TTGTGACATGAATGGCAAATGTGAGAAACG-39, b-actin: 59-ATCA AGATCATTGCTCCTCCT GAGCGCA-39. The following siRNAs specific for ADAM10 (Stealth Select RNAi HSS100167) or ADAM17 (Stealth Select RNAi HSS110436) were used in the present investigation and purchased from Invitrogen. The applied non-targeting siRNA (ON-TARGETplus, D-001810- 01-20) was purchased from Dharmacon (Dharmacon, Lafayette, CO). PLoS ONE | www.plosone.org 2 April 2012 | Volume 7 | Issue 4 | e34787