Fig 2: Exposure to plasma from sALS reduces leptin production in human adipocytes. (a) Oil red O coloration demonstrating lipid droplet accumulation (red) in mature adipocytes. (b) Human adipocytes were treated for 12 h with 1:100 diluted mixed plasma samples from healthy controls (12), slow progressors (12) and fast progressors (6). The supernatant was collected, and leptin levels were measured by ELISA (pg/mL) and normalized with plasma sample levels of leptin. (c) Leptin levels (pg/mL) secreted by mature adipocytes after plasma samples treatment. The adipocytes were pretreated with 1 μM of mTOR inhibitor (PP242) or 10 μM of AMPK inhibitor (compound C) for 1h. (d) Proteins from treated adipocytes (c) were extracted using RIPA buffer. Graph exhibits pAMPK/GAPDH fold change observed by immunoblotting with or without 10 μM AMPK inhibitor pretreatment. (e,f) Leptin levels (pg/mL) secreted by mature adipocytes after insulin and sTNF-RII treatment (e) and insulin and CCL16 treatment (f). (g) Immunoblotting showing pAMPK levels in adipocytes treated with increasing concentration of sTNF-RII. (h) Quantification of immunoblotting in (g) showing levels of pAMPK/GAPDH fold change in adipocytes treated with sTNF-RII ((Data are mean ± SEM * p ≤ 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001) n = 3, two-way ANOVA). NS, not significant.