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. 2018 Sep 4;9:3592. doi: 10.1038/s41467-018-05911-0

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© The Author(s) 2018

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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Fig. 5 — Epithelial-derived milk lipid is transferred to adipocytes during involution. a–g MG ultrastructure at involution day 1 examined by TEM. a LDs in alveolar lumen and within AECs (blue arrowheads). Enlarged micrographs of boxed regions in a containing LD b localized in apical AEC membrane and c associated with vacuoles. d Lipid outside of three alveoli, in interstitial space, and within adipocyte (dotted green outline). Boxed region of d enlarged in e. Boxed regions in e further enlarged to show f adipocytes with visible mitochondria (inset, orange arrowheads), small LDs (purple arrowheads), and g extracellular LDs in interstitial space. TEM data are representative micrographs from two mice. h MG cross sections from Adipoq-Cre; mT/mG mice at involution days 1, 3, and 5, immunostained with ADPH antibodies. Arrows indicate examples of ADPH+ LDs, dotted line traces remodeling alveoli. i Distribution of ADPH+ droplet number per μm of alveolar duct perimeter at involution days 1 and 3. Difference between # of ADPH+ droplets in 28–33 total ducts in three mice per time point was calculated using a two-tailed unpaired t-test. j Schematic of lipidomics experiment to evaluate AEC-produced MCFAs and adipocyte-produced LCFAs in adipocytes. k, l Average quantity of specific and total MCFAs from milk (k) and each WAT depot indicated (l). Significance calculated using two-way ANOVA with Tukey’s multiple comparison test. n mice = 6 (sWAT), 5 (dWAT), 3 (milk), 4 (mgWAT at involution day 4), and 3 (mgWAT at other time points). m Timeline to track BODIPY-FA. n–p Representative images of BODIPY-FA localization in Adipoq-Cre; mT/mG mice at involution days 1 (n) and 3 (o), and quantification of the percentage of adipocytes containing BODIPY-FA at involution day 3 (p). Asterisks indicate background. n = 3 biological repeats per time point. Scale bars are 20 μm (a, d, h), 50 μm (n), and 100 μm (o). Data in p is mean ± SD. *P < 0.05, ***, P < 0.001, ****P < 0.0001. TEM transmission electron microscopy, AECs alveolar epithelial cells, LD lipid droplet. Adip. adipocyte, ADPH adipophilin, MCFAs medium chain fatty acids, LCFAs long chain fatty acids, s/d/mgWAT subcutaneous/dermal/mammary gland white adipose tissue, BODIPY-FA boron–dipyrromethene–fatty acid