Figure 6. RAGE Affects β-Adrenergic-Stimulated Adipocyte Lipolysis and Thermogenic Programming.

Primary adipocytes were prepared from the SVF of 8-week-old WT and Ager null mice fed standard chow.

(A and B) iBAT- (A) and iWAT- (B) derived cells were incubated for 3 h in Krebs-Ringer bicarbonate buffer (KRB) and subjected to vehicle or CL316,243 (CL) (10 μM) for 3 h; glycerol and non-esterified fatty acids (NEFAs) in the media were measured. The means normalized to total protein per well ± SEM are reported from triplicate, independent experiments from cells derived from N = 5 mice/group.

(C) A representative OCR curve for iBAT-derived adipocytes during a mitochondrial stress test is shown under basal condition and following isoproterenol (10 μM). This experiment was performed three times using cells derived from N = 3 mice/group per experiment.

(D) Primary iBAT-derived adipocytes from iBAT of WT or Ager null mice were stimulated with norepinephrine (NE; 5 μM) and T3 (thyroid hormone, 2 nM) for 6 h, and relative Ucp1 mRNA expression was determined by qRT-PCR. The mean ± SEM is reported in N = 4 mice/group.

(E) Western blotting was performed for detection of UCP1/TUBULIN from primary iBAT adipocytes of 8-week-old WT and Ager null mice. The mean ± SEM is reported in N = 6 independent cell lysates per group along with representative blots.

(F and G) Primary adipocytes from (F) iBAT and (G) iWAT of WT mice were incubated with vehicle or the RAGE ligand CML-AGE (300 μg/ml) for 16 h alone or with NE (5 μM) for the final 6 h. qRT-PCR for detection of relative Ucp1 or Ppargc1a mRNA expression was performed. The mean ± SEM is reported from N = 5 mice/group.

(H) Cell lysates from primary adipocytes from iBAT of WT mice treated as in (F) and (G) were used for the detection of UCP1/TUBULIN. The mean ± SEM is reported in cell lysates from N = 3 mice/group.

(I) Schematic of Ucp1 promoter with enhancer elements. Undifferentiated C3H10T1/2 cells were transfected with mouse 3.1 kB Ucp1 promoter luciferase construct (Ucp1-luc) and stimulated for the last 6 h with NE(5 μM) and/or CML-AGE (300 μg/ml) for 16 h. Normalized luciferase activities are shown as fold-change compared to vehicle control.

(J) Ucp1 promoter luciferase constructs were generated and transfected into undifferentiated C3H10T1/2 cells and the cells cultured in medium containing NE (5 μM) for the last 6 h or CML-AGE (300 μg/ml) for 16 h. Normalized luciferase activities are shown relative to vehicle control. The mean ± SEM of three independent experiments is presented.

(K) PKA activity was determined in primary iBAT adipocytes from WT mice treated with CL316,243 (10 μM) for 15 min alone or after pre-incubation with the RAGE ligand CML-AGE (300 μg/ml) for 16 h. The mean ± SEM of N = 8 mice/group is presented.

In (A)–(E), WT (black) and Ager null (green). Data analysis: (A–D, repeated measures) two-way ANOVA followed by post hoc Bonferroni test; (E and H) two-tailed Student’s t test; (F, G, and I–K) one-way ANOVA followed by a post hoc Tukey’s HSD test; *p < 0.05, **p < 0.01, and ***p < 0.001.

See also Figure S6.