Figure 5.

Ceramides have cell autonomous effects on fuel metabolism in primary brown adipocytes. Primary brown adipocytes were treated with (A) myriocin (10 μM) or (B–C) isoproterenol (1 μM) +/− C₂-ceramide as described in Materials and Methods. (A–B) Thermogenic genes were assessed by qPCR and (C) respiration was assessed by respirometry. Data are representative of 3 independent experiments completed with 4 replicates. Primary brown adipocytes were incubated with adenoviruses encoding for either green fluorescent protein (Gfp) or mouse-CerS6 (D–G). Following 48 h of transfection (D) mRNA expression of CerS6 was quantified by qPCR, (E–F) Ceramide and triglyceride content were determined by mass spectrometry and (G–I) respiration was assessed by respirometry. (J) Primary brown adipocytes were treated with vehicle or C₂-ceramide (Cer) for 2 h prior to stimulating with insulin (1 μM, 10 min). Lysates were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blots were performed using antibodies recognizing phosphorylated (P-Akt) or total Akt (N = 3 independent experiments). (K) Western blots and (L) quantification depicting isoproterenol (Iso) induced phosphorylation of pHSL (S563 phosphorylation sites) following treatment with C₂-ceramide (Cer, 50 μM) for 4 h followed by treatment with the PP2A inhibitor microcystin-LR (MLR) for 1 h in primary brown adipocytes (N = 3 independent experiments). (M) FFA uptake in primary brown adipocytes isolated from control and Sptlc2^δUcp1 mouse (N = 5–7 per group). (N) Schematic summarizing the discrete mechanisms linking ceramides to changes in BAT metabolism. Values are expressed as mean ± SEM, ∗p < 0.05. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.