GCS in hypothalamic neurons regulates neuronal leptin signaling at the plasma membrane.

<p>(A) Stat3 phosphorylation was markedly decreased in Arc sections of <i>Ugcg</i>^{f/f//CamKCreERT2} mice in response to peripheral leptin (5 mg/kg; 45 min) 6 wk p.i. (<i>n</i> = 16–33). Three independent animal groups were analyzed. (B) Serum leptin levels were unchanged 3 wk p.i. and increased prominently 7 wk p.i. in <i>Ugcg</i>^{f/f//CamKCreERT2} mice, reflecting increased body fat mass (<i>n</i> = 12–14). (C) mRNA expression analysis for suppressor of cytokine signaling 3 (SOCS-3) expression in Arc-enriched hypothalamic tissue was carried out 2, 6, and 9 wk p.i. <i>Socs-3</i> expression normalized to the housekeeping gene tubulin was unaltered (<i>n</i> = 3–5). (D) mRNA expression analysis for the long form of the leptin receptor, <i>Leprb</i>, in mediobasal hypothalamus was carried out 6 wk p.i. <i>Leprb</i> expression normalized to the housekeeping gene tubulin was unaltered at that time point (<i>n</i> = 4–5). (E) Immortalized mouse hypothalamic cells (N-41 cells) were analyzed for cell surface expression of ObR. Non-detergent-treated cells were fixed and simultaneously stained with two ObR antibodies. A proximity ligation assay (PLA) indicated quantifiable and unchanged ObR expression on the surface of controls and cells treated with the specific GCS inhibitor NB-DNJ (<i>n</i> = 41–47 cells). PLA principle is depicted on the right side. (F–G) N-41 cells were incubated with either saline or 100 ng/ml leptin (10 min). Close interactions between GCS-derived neuronal gangliosides GD1a/ObR (F) and GM1/ObR (G) were detected by PLA. Leptin treatment dynamically increased the GD1a/ObR and GM1/ObR PLA spots per cell (<i>n</i> = 48–67 cells). (H) Extracts from saline- and leptin-treated N-41 cells were immunoprecipitated with an ObR antibody, lipids were extracted, and GD1a and GM1 were visualized by immune overlay TLC. GD1a and GM1 co-immunoprecipitated (Co-IP) with ObR, which tended to be stronger in leptin-treated cells. Addition of a blocking peptide almost totally abolished ganglioside signals. Gangliosides GD1b and GT1b, expressed in mouse brain tissue, were not co-precipitated with ObR from hypothalamic tissue of <i>Ugcg</i>^f/f mice (5 mg/kg leptin, 45 min). (I) Co-IP showed significantly decreased leptin-induced complex formation between ObR and Jak in NB-DNJ-treated N-41 cells (<i>n</i> = 4). (J) Sustainable Jak phosphorylation could be induced in N-41 cells after 15 min of leptin treatment (0.5 µg/ml). NB-DNJ-treated cells showed a markedly delayed response to leptin. (K) Thirty minutes after leptin treatment, Jak phosphorylation was decreased in NB-DNJ-treated cells (<i>n</i> = 4). *<i>p</i>≤0.05; ***<i>p</i>≤0.001. Means ± SEM.