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. 2018 Dec 18;16(12):515.
doi: 10.3390/md16120515.

Reduced Number of Adipose Lineage and Endothelial Cells in Epididymal fat in Response to Omega-3 PUFA in Mice Fed High-Fat Diet

Katerina Adamcova  1 , Olga Horakova  2 , Kristina Bardova  3 , Petra Janovska  4 , Marie Brezinova  5 , Ondrej Kuda  6 , Martin Rossmeisl  7 , Jan Kopecky  8
Affiliations

Reduced Number of Adipose Lineage and Endothelial Cells in Epididymal fat in Response to Omega-3 PUFA in Mice Fed High-Fat Diet

Katerina Adamcova et al. Mar Drugs. .

Abstract

We found previously that white adipose tissue (WAT) hyperplasia in obese mice was limited by dietary omega-3 polyunsaturated fatty acids (omega-3 PUFA). Here we aimed to characterize the underlying mechanism. C57BL/6N mice were fed a high-fat diet supplemented or not with omega-3 PUFA for one week or eight weeks; mice fed a standard chow diet were also used. In epididymal WAT (eWAT), DNA content was quantified, immunohistochemical analysis was used to reveal the size of adipocytes and macrophage content, and lipidomic analysis and a gene expression screen were performed to assess inflammatory status. The stromal-vascular fraction of eWAT, which contained most of the eWAT cells, except for adipocytes, was characterized using flow cytometry. Omega-3 PUFA supplementation limited the high-fat diet-induced increase in eWAT weight, cell number (DNA content), inflammation, and adipocyte growth. eWAT hyperplasia was compromised due to the limited increase in the number of preadipocytes and a decrease in the number of endothelial cells. The number of leukocytes and macrophages was unaffected, but a shift in macrophage polarization towards a less inflammatory phenotype was observed. Our results document that the counteraction of eWAT hyperplasia by omega-3 PUFA in dietary-obese mice reflects an effect on the number of adipose lineage and endothelial cells.

Keywords: adipocyte; cellularity; fat; nutrition; obesity; proliferation; white adipose tissue.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Morphology and immunohistochemistry of eWAT. Representative histological sections of eWAT from mice fed STD (A,E), HFD (B,F,I) or HFF (C,G,J,K) diet at Week 8. Hematoxylin and eosin staining for morphometry of adipocytes (A,B,C), as evaluated in (D). Immunohistochemical staining using macrophage marker MAC2 for quantification of CLS (E,F,G; arrows), as evaluated in (H). Representative sections showing the detection of macrophage proliferation within CLS based on immunofluorescence staining (I,J; nuclei by DAPI, blue; macrophages by anti-F4/80, green; surface of lipid droplets by anti-perilipin 1, white; proliferating nuclei by anti-Ki67, red; proliferating macrophages are indicated with arrows). Representative section showing multinucleated giant cells (K). Data are means ± SD; n = 6–8. * Significant difference from Week 1 between mice on same diets; † significant difference from HFD for the same period of dietary intervention; # significant difference from STD for the same period of dietary intervention. Bar represents 200 μm (A,B,C,E,F,G) or 20 μm (I,J,K). For morphology and immunohistochemistry of eWAT at Week 1, see Supplementary Figure S1.
Figure 2
Figure 2
Principal component analysis of the lipidomic data from eWAT of the HFD and HFF mice. Score plots of the principal components 1 and 2 were generated using the lipid mediator profiles at Week 1 (A) and Week 8 (B). At Week 8 (C), results were expressed as a contribution score plot showing one bar per variable, indicating which species differ most between the groups and in which direction. Lipids derived from AA (red), LA (orange), ALA (purple), DHA (blue), and EPA (green) are discerned by colors. For the source data and the abbreviations, see Supplementary Table S1.
Figure 3
Figure 3
Gene expression of pro- and anti-inflammatory markers in eWAT of mice fed HFD or HFF diet for 1 or 8 weeks (A), or in stromal-vascular fraction (SVF) cells or adipocytes (ADI) isolated from eWAT of mice fed the respective diets for 8 weeks (B). Data were normalized to the geometrical mean of two reference genes, Hprt and EF1α in (A), and EF1α and Rn18s in (B), and expressed relative to the HFD mice at Week 1 for whole eWAT depot (A) or to SVF of HFD group (Week 8) for SVF and ADI (B). Data are means ± SD; n = 6–8. * Significant difference from Week 1 between mice with the same diet; † significant difference for the same period of dietary intervention.
Figure 4
Figure 4
Flow cytometry analysis of immune cell subsets in SVF isolated from eWAT. Numbers of cells are calculated per depot. Arrows indicate the gating strategy used. Cells were first gated on size and singularity for further analysis. Single cells were gated based on the expression of CD45 to identify leukocytes (A). Leukocytes were then gated on the co-expression of CD11b and F4/80 to identify macrophages (B). Finally, macrophages were further subdivided based on the expression of CD206 and CD11c into: M1 macrophages (CD206−/CD11c+; C), double-negative macrophages (CD206−/CD11c−; D), M2 macrophages (CD206+/CD11c−; E) and double-positive macrophages (CD206+/CD11c+; F). Striped columns (black with white stripes, HFD; white with black stripes, HFF) show the amount of proliferating cells per depot, which were detected using antibodies specific for the Ki67 proliferation marker. Illustrative flow cytometry plots and gating strategy are also shown (G). Data are means ± SD.; n = 6–8. * Significant difference compared to Week 1 with the same diets; † significant difference between the diets for the same period of dietary intervention.
Figure 5
Figure 5
Ratio between M2 and M1 macrophages in eWAT of mice fed HFD or HFF diet for 1 week or 8 weeks as determined by flow cytometry. Data from Figure 4C,E were re-plotted. * Significant difference compared to Week 1 within the diets.
Figure 6
Figure 6
Flow cytometry analysis of non-immune cell subsets in SVF isolated from eWAT. The numbers of cells are calculated per depot. Arrows indicate the gating strategy used. Cells were first gated on size and singularity for further analysis. Single cells were gated on the lack of CD45 expression to identify non-lymfoid cells (A). CD45− cells were then gated on CD31 expression to identify endothelial cells (D). CD31− cells were further subdivided based on the expression of Sca1, CD34 and CD24 into progenitors (CD34+/Sca1+/CD24+; B) and preadipocytes (CD34+/Sca1+/CD24−; C). Striped columns (black with white stripes, HFD; white with black stripes, HFF) show the amount of proliferating cells per depot, which were detected using antibodies specific for the Ki67 proliferation marker. Illustrative flow cytometry plots and gating strategy are also shown (E). Data are means ± SD; n = 5–6. * Significant difference compared to Week 1 for mice fed the same diet; † significant difference between the diets for the same period of dietary intervention.
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