Wild-type and GM-CSF mice fed the HFD display a similar increase in adipose tissue extract cathepsin D activity and HMGB1
release. Wild-type and GM-CSF–null male mice at 9 weeks of age were fed NCD or HFD for 4 (A) or 8 (B) weeks. Adipose tissue extracts from an entire epididymal adipose tissue fat pad were prepared and assayed for cathepsin
D activity, as described in research design and methods. Cathepsin D activity of the HFD adipose tissue extracts was normalized to their respective NCD adipose tissue extracts.
Data shown are the mean ± standard error of the mean for three to seven mice per group. C and D: 3T3-L1 cells were differentiated for 12 days to generate fully differentiated adipocytes as described in research design and methods. The cells were then treated for 1 h with vehicle (DMSO), 500 μmol/L H2O2, 2 μmol/L ION, or 1 μmol/L STS in serum-free DMEM medium. Then, 30 μL of the culture medium was immunoblotted for HMGB1 (upper panel). The cultured adipocytes were extracted, and 20 μg of protein was immunoblotted for HMBG1 and β-actin as a loading control.
This is a representative immunoblot performed in triplicate. E–H: Wild-type and GM-CSF–null male 9-week-old mice were fed the NCD or HFD for 4 (E, F) or 8 (G and H) weeks. Adipose tissue extracts from an entire epididymal adipose tissue fat pad were prepared and immunoblotted for the
presence of HMGB1 and actin as a loading control. These are representative immunoblots from three independent animals for
each group. The right panels show densitometric quantification of the relative HMBG1/actin levels. For comparison, the ratios from the NCD mice were normalized
to 1.0. Data shown are the mean ± standard error of the mean for three to five mice per group. In panels B, F, and H, the # and * symbols indicate statistical significance (P < 0.05) by Student t test. Data shown in panel D were analyzed as described in statistical analysis. Identical letters indicate values that are not statistically different from each other (P > 0.05).