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. 2019 Sep 10;9:12941. doi: 10.1038/s41598-019-49418-0

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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(a) Body weight gain was continuously monitored during 4 weeks of either chow (white rectangles), HFD (black squares), or 2 weeks of HFD followed by 2 weeks of chow (reversed, grey circles) feeding of C57BL6/J mice. n = 14–20 animals/group. (b) Adipose tissue weight in (g) (Epididymal (Epi), Inguinal (Ing), Retroperitoneal (Retro)), and (c) non-fasting blood glucose level (mmol/l) at termination of the feeding protocol, n = 8–20 animals/group. (d) Cell size distribution using the coulter counter analysis of epididymal adipose tissue collected at the end of the feeding study. Data displayed as average of n = 4–6 samples/group. (e) Same as in (d) (epididymal) also including the previously published⁸ distribution curve after 2 weeks of HFD feeding for comparison. (f) Representative confocal images showing adipose cell size and F-actin (stained with phalloidin) in the three feeding groups (chow, HFD, and reverse). Upper panel: epididymal (EPI), middle panel Inguinal (ING; lower panel Retroperitoneal (RETRO). Correlation between quantified actin filaments (using Image J analysis) and adipose cell size; chow (white rectangles), HFD (black squares), or reversed (grey circles) in adipocytes isolated from respective adipose tissue depot shown in right panel. n = 27–40 ROI/condition. Correlations are shown for each feeding group condition as well as the overall correlation within each depot (p). (g) Representative western blot analysis of β-actin protein level in supernatant (Sup, G-actin fraction) and pellet (Pellet, F-actin fraction) obtained from F/G actin sedimentation assay of adipocyte lysates collected after chow, HFD or reversed feeding. Pretreatment with Jasplakinolide (Jasp) used as positive control (induced polymerization shown as an increase in F/G actin ratio). Quantification of western blot analysis, n = 5–6 biologic replicates/condition, shown in graph above. Data in (b-c) and (h) presented as mean ± SD, **p ≤ 0.01, and ****p ≤ 0.0001.