PMC

Epididymal fat from WT and CD36 KO mice after 16 wks on a HFD. RNA was isolated and Q-PCR was performed. A. Expression of genes associated with inflammation. B. Genes associated with cell death. Data was normalized to 36B4 expression. Values shown are mean ± SD (n = 5), *, p<0.05; **, p<0.001.

Adipocytes were differentiated from the SVF in culture wells as described in . A. Lipid accumulation in adipocytes was determined by Oil Red O staining. B. Differentiated (Oil Red O-positive) cells were calculated as a percentage of total cells. C, D. Cytokine levels in culture medium was determined following 4 h treatment of cells with LPS (10 ng/mL). Values shown are mean ± SD (n = 4) from a representative experiment, *, p<0.05; **, p<0.001. Scale bar, 100 µm.

. Representative images of F4/80 and perilipin stained epididymal fat sections from male WT and CD36 KO mice after 16 wks on a HFD. Viable adipocytes are shown as perilipin-positive (red) and macrophages as F4/80-positive (brown). Cell nuclei were stained with DAPI (blue). Stars (*) indicate the cells devoid of perilipin expression. B. Quantification of dead adipocytes (perilipin-negative cells) expressed as a percentage of total adipocytes. For quantitative analysis, cells from 5 random fields, each containing more than 100 cells/field, were examined and counted. Values shown are mean ± SD (n = 3 mice), *, p<0.05. Scale bar, 100 µm.

A–C. WT and CD36 KO male mice were maintained on a HFD for 16 wks. Body weight was measured weekly. After 16 wks on HFD, lean body mass and fat body mass were determined by MRI. Plasma was collected after 6 h of fasting. D. Glucose tolerance test (GTT). Mice were given a bolus of D-glucose (2 g/kg body weight) i.p. after 6 h of fasting. E, F. Fasting (6 h) insulin levels were determined by ELISA kits (BD Bioscience) and plasma lipid levels were determined by commercially available kits (WAKO). Values shown are mean ± SD (n = 7 except GTT, n = 5). *, p<0.05, CD36 KO vs. WT.

. Contact co-culture. Adipocytes were differentiated from the SVF of WT and CD36 KO mice as described in . Peritoneal macrophages isolated from WT and CD36 KO mice were then layered and cultured on top of the differentiated adipocytes and co-cultured for 16 h. LPS (10 ng/mL) was then applied to the co-cultures for 4 h after which medium and cells were collected for cytokine determination. B, C. Non-contact co-culture in transwells. Primary pre-adipocytes were seeded in the bottom chamber and differentiated into mature adipocytes. Primary peritoneal macrophages were then seeded in the transwell inserts and cells were then co-cultured for 16 h. For LPS treatment groups, both adipocytes and macrophages were exposed to LPS (10 ng/mL) for 4 h after which cellular gene expression was measured by Q-PCR (B). Medium was collected and secreted cytokines were measured by ELISA (C). Values shown are mean ± SD (n = 4). *, p<0.05, **, p<0.001.

A–C. Representative images of F4/80 and CD3 stained epididymal fat sections from male WT and CD36 KO mice after 16 wks on a HFD. A, B F4/80-positive macrophages (brown). C. CD3-positive cells (red). D. The number of CLS was calculated as the percentage of adipocytes that are found in crown-like structures. For quantitative analysis, cells from 5 random fields, each containing more than 100 cells/field, were examined and counted. Values shown are mean ± SD (n = 3 mice), *, p<0.05. E. Adipose tissue gene expression. RNA was extracted and gene expression was determined by Q-PCR. Data was normalized to 36B4 expression. Values shown are mean ± SD (n = 5). *, p<0.05. Scale bar, 200 µm (A), 100 µm (B, C).