Figure 8. Maintained adipose tissue function in MC3R^hDM/hDM mice.

Gonadal fat was isolated from fasted female mice fed a high-fat diet for (a) quantitative real-time PCR (MC3R^hWT/hWT n=5; MC3R^hDM/hDM n=8/group), (b–d) western blotting analysis (MC3R^hWT/hWT n=5; MC3R^hDM/hDM n=4), and (e–l) FACS analysis (MC3R^hWT/hWT n=4; MC3R^hDM/hDM n=5) in MC3R^hWT/hWT (open bars) and MC3R^hDM/hDM (closed bars) mice. (a) The expression levels of genes related to macrophage infiltration, inflammation and adipose tissue metabolism were normalized for β-actin expression. Western protein expression results for (b) peroxisome proliferator-activated receptor gamma (PPARγ), (c) adiponectin and (d) phosphospecific 5′-adenosine monophosphate-activated protein kinase (p-AMPK), divided by glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression and normalized relative to average for MC3R^hWT/hWT. The stromal vascular fraction was isolated from gonadal fat (∼2 g) from MC3R^hWT/hWT and MC3R^hDM/hDMmice. For detecting macrophages by flow cytometry, a gating strategy was used to enrich samples from MC3R^hWT/hWT and MC3R^hDM/hDMmice for adipose tissue macrophages by selecting cells in the gate 1 area (e,g). The gate 1 area was stained with a F4/80 antibody (f and h) and the dot plots depict forward scatter (FSC) and side scatter (SSC) (left). For detecting neutrophils, whole cells (non-gated) were stained with CD11b and Gr-1 (i,j). Bar graphs show the average values±s.e.m. for percentage of cells in the stromal vascular fraction that were (k) macrophages and (l) neutrophils. *P<0.05 MC3R^hDM/hDMversus MC3R^hWT/hWT. (m,n) Haematoxylin and eosin stained sections showed no apparent differences in adipocyte morphology between groups. Groups were compared by Student's t-tests (two-tailed) (a–d and k–i). Scale bar, 100 μm. Data are represented as mean±s.e.m. for a-d, k, and l. FACS, fluorescence-activated cell sorting