Since only co-incubation with oleate but not the broad spectrum caspase inhibitor Z-VAD(OMe)-FMK reduced sOb-R to basal level and restored cell viability we suggest that similar to staurosporine caspase– or even apoptosis-independent mecha- nisms are of importance for effects seen after palmitate incubation. Lipotoxicity caused by elevated free fatty acids in tissues other than the adipose tissue [25,26] and apoptosis for example of pancreatic beta cells occur during the manifestation of T1DM. Accordingly, our data suggest that strongly increased sOb-R levels observed in newly manifested T1DM [21,22] could be induced by high levels of saturated FFAs like palmitate. Recently, it was demonstrated that high doses of leptin even without insulin administration normalize glucose and lipid metabolism in type I diabetic mice [53–55]. The high doses of leptin applied in these studies might have been necessary to overcome the inhibitory effect of high sOb- R levels in the type I diabetic mice. This hypothesis is supported by our findings that co-incubation of leptin with increasing amounts of sOb-R resulted in reduced leptin-mediated STAT3 phosphorylation in Ob-Rfl transfected HEK293 cells. Our data suggest that sOb-R levels which exceed a molar ratio of sOb-R to leptin of 2:1 exhibit a predominant inhibitory effect on leptin signaling, a ratio that is frequently observed in catabolic diseases and early childhood [21,22,56]. As we could show that sOb-R mainly originates from short Ob-R isoforms, the signal transduc- ing long Ob-R isoform in the mouse model described before might still occurred at the cell surface at sufficient amounts to mediate beneficial effects of the ligand. Therefore, decreasing Ob-R shedding by use of specific ADAM10 inhibitors perhaps could further improve the effects of a leptin mono therapy. Moreover, we demonstrated that co-expression of the short isoform Ob- R219.3 with Ob-Rfl impaired leptin signalling compared to cells transfected with Ob-Rfl only. These findings are in contrast to Bacart et al. [44,57] who demonstrated that increasing amounts of the co-expressed short isoform Ob-Ra did not alter leptin signaling of the long receptor variant. However, our findings are supported by results of White et al. who demonstrated a modest repression on signaling of wildtype Ob-Rfl when they co-transfected increasing amounts of a truncated Ob-Rfl isoform [58]. In addition to modulation of shedding enzyme activity, regulation of Ob-R supply is a determining factor for the extent of sOb-R generation. Ligand-receptor interactions as well as receptor trafficking from ER to membrane could affect the amount of membrane-anchored Ob-R. Accordingly, we investigated to which extent high leptin levels and low grade inflammation- associated ER stress – two markers linked to obesity – take effect on leptin receptor availability for sOb-R generation. The exposure of Ob-R transfected cells to leptin concentrations in the physiological range led to decreased sOb-R levels. Bjorbaek et al. showed that leptin/ leptin-receptor-complexes were internalized and transported to lysosomes for degradation [59]. This ligand-induced receptor internalization process probably accounts for the temporarily decrease of the plasma membrane Ob-R pool, resulting in decreased sOb-R levels as observed in the present study. The nucleoside antibiotic tunicamycin inhibits N-glycosylation of proteins [60] and increases the protein load of the ER finally leading to ER stress induction. We found a reduced leptin binding and an impaired up to blunted leptin signaling in Ob-Rfl transfected cells in parallel to reduced sOb-R levels after tunicamycin-induced ER stress. These findings support the hypothesis that increased ER stress may decelerate the turnover of Ob-R to the plasma membrane. Concomitantly, constitutive shedding could further decrease the amount of membrane Ob-R which might foster deterioration of leptin signaling. These hypotheses could complement recent findings describing a Regulatory Mechanisms for sOb-R Generation participation of the NF-kB (nuclear factor ‘kappa-light-chain- enhancer’ of activated B-cells) as well as the JNK (c-Jun N- terminal kinase) signaling pathway in the development of ER stress- and obesity-associated leptin resistance [34–36]. An increase in PTP1B and SOCS3 protein levels that were reported previously to be inducers of leptin resistance [11,30–35,61] could accelerate the process of becoming insensitive to leptin on cellular level. We suggest that chronic ER stress in obese patients decreases the amount of sOb-R due to diminished membrane Ob-R expression. Accordingly, the determination of serum sOb-R concentrations may serve as a tool to monitor the development of a leptin resistant state. This suggestion was already supported by clinical data [16]. Future studies are necessary to clarify the exact mechanisms for reduced sOb-R concentrations in response to ER stress and high leptin levels. Such investigations should include the use of receptor endocytosis inhibitors like cytochalasin and the determination of ER stress inducing agent-linked effects on ADAM10 and ADAM17 activity. Since tunicamycin is a synthetic and not a physiological stimulus of ER stress, continuing investigations should be extended to animal models of obesity in order to uncover to which extent ER stress- associated effects on the leptin – leptin receptor axis are of importance in-vivo. Our results base upon transient overexpression of Ob-R isoforms in HEK cells but not upon investigations in primary cells or non-transfected cell lines. This restriction is hardly to overcome as for example endogenous Ob-R expression of primary human hepatocytes or non-transfected HEK cells is to low for the analytical differentiation. Otherwise the contribution of individual isoforms to the overall amount of shed Ob-R and isoform-specific regulation can be only proven by a cell model with receptor transfection. In summary, we provide novel insights into the regulatory mechanisms for sOb-R generation and its importance as a main player in the regulation and for the monitoring of leptin action: high sOb-R concentrations seem to directly modulate leptin effects thereby acting predominantly inhibitory. In contrast, low sOb-R levels, although having no direct influence on leptin action, could serve as one potential indicator for the development of a leptin resistant state in obesity. Supporting Information Figure S1 Normalisation of Ob-R shedding to Ob-R protein expression in whole cell lysates. sOb-R levels in the supernatant of Ob-R transfected HEK cells were normalized to Ob-R-isoform specific protein expression in whole cell lysates. (DOC) Figure S2 Metalloproteases are involved in Ob-R shed- ding. sOb-R levels in the supernatant of Ob-R219.3 transfected HEK cells after incubation with GM6001 (25–100 mM) for 24 h and 48 h. sOb-R levels of GM6001-treated cells are displayed relative to those of non-treated cells. Data are presented as means 6 SD of n$3 experiments. (DOC) Figure S3 Metalloproteases ADAM10 and ADAM17 are involved in PKC activated Ob-R shedding. sOb-R levels in the supernatant of Ob-R219.3 transfected HEK cells that were pre-treated for 30 min with GI/GW (3 mM) and further incubated for 24 h without or with PMA (10 ng/ml). sOb-R levels of PMA- treated cells are displayed relative to those of non-treated cells. Data are presented as means 6 SD of n$3 experiments. (DOC) PLoS ONE | www.plosone.org 12 April 2012 | Volume 7 | Issue 4 | e34787