Regulatory Mechanisms for sOb-R Generation Figure 5. Enhanced release of sOb-R after palmitate incubation is mediated by ADAM10 and ADAM17 and inhibited by oleate. (A) sOb-R levels in the supernatant of Ob-Rfl or Ob-R219.3 transfected cells after incubation with palmitate (0.5–1 mM), oleate (0.5 mM–1 mM or methanol (2%) for 24 and or 48 h. sOb-R levels of palmitate- or oleate-treated cells are displayed relative to those of cells treated with methanol. (B) sOb-R levels in the supernatant of HEK cells transfected with a non-targeting, ADAM10 or ADAM17 specific siRNA (50 nM) and subsequently with Ob- R219.3, after incubation with palmitate (1 mM) or methanol (2%) for 48 h. sOb-R levels of ADAM10 and ADAM17 siRNA transfected cells are shown relative to sOb-R levels of cells transfected with a non-targeting siRNA and treated with methanol. (C) Representative western blot of cleaved PARP after palmitate incubation. Ob-R transfected cells were incubated with palmitate (0.5–1 mM) or Methanol (2%) for 24 and 48 h. Following this incubation cleaved PARP in cell lysates was determined by western blot analysis. (D) sOb-R levels in the supernatant of Ob-Rfl or Ob-R219.3 transfected cells after incubation with palmitate 1 mM and Z-VAD 100 mM or methanol (2%) for 48 h. sOb-R levels of palmitate- and or Z-VAD- stimulated cells are displayed relative to those of cells treated with methanol. (E) Representative western blot of cleaved caspase-3 and cleaved PARP. Ob-R transfected cells were incubated with palmitate (0.5–1 mM), palmitate (0.5–1 mM) and Z-VAD (100 mM) or Methanol (2%) for 48 h. Following this incubation cleaved caspase-3 and cleaved PARP in cell lysates was determined by western blot analysis. (F and G) sOb-R levels in the supernatant of Ob-Rfl or Ob-R219.3 transfected cells after incubation with palmitate (1 mM), oleate (0.1–0.5 mM), palmitate (1 mM) and oleate (0.1–0.5 mM) or methanol (2%) for 48 h. sOb-R levels of palmitate-, oleate– or palmitate- and oleate-treated cells are displayed relative to those of cells treated with methanol. (H) Representative western blot of cleaved PARP after co-incubation with palmitate and oleate. Ob-R transfected cells were incubated with palmitate (0.5–1 mM), palmitate (0.5–1 mM) and oleate (0.1–0.5 mM) or Methanol (2 %) for 48 h. Following this incubation cleaved PARP in cell lysates was determined by western Blot analysis. Data are presented as means 6 SD of n$3 experiments (Fig. 5 D and E, n = 2). doi:10.1371/journal.pone.0034787.g005 sObR generation is regulated by leptin level and ER stress Leptin decreases sOb-R concentrations. In adiposity high leptin levels are correlated with decreased sOb-R levels [27–29]. As such a leptin excess could result in an Ob-R downregulation we asked, whether this process leads to a decline in the constitutive generation of sOb-R. Therefore, we incubated Ob-Rfl or Ob- R219.3 transfected HEK cells for 24 h with 5–100 ng/ml leptin. Fig. 7 demonstrates that 100 ng/ml leptin decreased the amount of sOb-R in the cell supernatant by up to 46%(P,0.001) for Ob- Rfl and up to 39% (P,0.01) for Ob-R219.3. The decrease was concentration-dependent but independent of the length of the transfected Ob-R isoform. ER stress decreases membrane Ob-R expression, impairs leptin signaling and reduces sOb-R generation. Obesity is a state of low grade inflammation associated with ER stress. To investigate whether or not membrane Ob-R expression and leptin signaling are affected by ER stress we applied tunicamycin, an inhibitor of protein N-glycosylation. Ob-Rfl transfected cells were incubated with tunicamycin for 4–24 h and subsequently stimulated with leptin (100 ng/ml) for 30 min. Cells and cell lysates were subjected to 125I-leptin binding assay, P- and total STAT3 ELISA Figure 6. Co-expression of Ob-R219.3 to Ob-Rfl and increased sOb-R levels modulate leptin signaling of Ob-Rfl. (A) Ob-R transfected cells were serum-starved for 16 h and subsequently incubated with leptin (100 ng/ml) for 30 min. P-STAT3 and total STAT3 levels in lysates of HEK cells transfected with equimolar amounts of Ob-Rfl + GFP (equimolar to Ob-R219.3) or Ob-Rfl + Ob-R219.3 were determined by ELISA and western blot. P-STAT3/total STAT3 ratios are displayed relative to P-STAT3/total STAT3 ratio of non-treated cells transfected with Ob-Rfl + GFP. (B) P-STAT3 and total STAT3 levels in lysates of HEK cells transfected with Ob-Rfl. Ob-Rfl-transfected cells were stimulated with media containing leptin (10 ng/ml) and increasing concentrations of sOb-R (0–1000 ng/ml). P-STAT3 and total STAT3 levels are displayed relative to those of leptin-treated control without sOb-R. Data are presented as means 6 SD of n$2 experiments. doi:10.1371/journal.pone.0034787.g006 PLoS ONE | www.plosone.org 9 April 2012 | Volume 7 | Issue 4 | e34787