Regulatory Mechanisms for sOb-R Generation Figure 8. ER stress causes downregulation of membrane Ob-R, impaired leptin signaling via Ob-Rfl and decreased sOb-R concentrations. (A) Representative western blot for protein expression of ER stress markers BiP (GRP78), CHOP (GADD153) and PDI after treatment of Ob-R transfected HEK cells with tunicamycin (3 mg/ml) or DMSO (0.1%) for 4–24 h. (B) Ob-Rfl transfected cells were incubated with tunicamycin (3 mg/ml) or DMSO (0.1%) for 4–24 h and I125-leptin binding assay was performed. I125-leptin binding of tunicamycin-treated samples is displayed relative to that of non-treated controls. (C and D) P-STAT3 (C) and total STAT3 (D) levels in lysates of Ob-Rfl transfected cells after incubation with tunicamycin (3 mg/ml) or DMSO (0.1%) for 4–24 h and subsequent stimulation with leptin (100 ng/ml) for 30 min. P-STAT3 and total STAT3 levels of leptin-stimulated cells are displayed relative to that of non-treated controls. (E) sOb-R levels of Ob-Rfl or Ob-R219.3 transfected cells after incubation with tunicamycin (3–20 mg/ml) or DMSO (0.2%) for 24 and 48 h. sOb-R levels of tunicamycin-treated cells are displayed relative to those of non- treated cells. Data are presented as means 6 SD of n$3 experiments (Fig. 8 A, n = 1; Fig. 8 B, n = 2). doi:10.1371/journal.pone.0034787.g008 depends in part on the activation of caspases. Since the applied caspase inhibitor did not decrease the shedding rate to the basal level, a participation of caspase-independent mechanisms cannot be excluded. This hypothesis is further supported by the finding that staurosporine induces apoptosis via caspase-dependent as well as -independent ways [48]. Our finding that staurosporine- induced shedding of Ob-R mainly depends on the activity of ADAM10 is similar to mechanisms reported for distinct, even unrelated membrane receptors and proteins such as IL-6R, L- selectin and L1 [38,49–52]. Therefore, ADAM-mediated cleavage of membrane proteins as consequence of induced apoptosis appears to be a general shedding mechanism. Because FFAs, particularly saturated, long chain fatty acids like palmitate or stearate, are also known to induce apoptosis [23,24], we speculated that they could be major players for the induction of shedding as well. Fatty acid-induced shedding of both Ob-R isoforms that we observed in our investigations was mediated primarily by ADAM10 with a minor participation of ADAM17 and activated only by palmitate but not oleate. The latter difference might be due to the distinct cellular fate of these fatty acids. Whereas oleate is used to synthesize triglycerides which are stored without affecting cell viability [24], excess amounts of palmitate are only poorly incorporated into triglycerides and cause lipoapoptosis which in turn could lead to increased Ob-R shedding. Both, palmitate as well as staurosporine seem to directly influence Ob-R cleavage since a knockdown of ADAM10– or ADAM17-activity inhibited staurosporine and palmitate effects on sOb-R concen- tration. An also imaginable passive increase of Ob-R in the supernatant through an efflux across leaky membranes or formation of cell debris from dying cells should not be affected by the siRNA knockdown. Recently it was shown that mono- unsaturated fatty acids like oleate could prevent the lipotoxic effects of saturated fatty acids like palmitate [24]. Our results from oleate and palmitate co-incubation experiments not only affirm these findings but also let us speculate that the ratio of saturated to unsaturated fatty acids may influence the amount of shed Ob-R. PLoS ONE | www.plosone.org 11 April 2012 | Volume 7 | Issue 4 | e34787