Dietary lipids inhibit mitochondria transfer to macrophages to divert adipocyte-derived mitochondria into the blood

Abstract

Adipocytes transfer mitochondria to macrophages in white and brown adipose tissues to maintain metabolic homeostasis. In obesity, adipocyte-to-macrophage mitochondria transfer is impaired, and instead, adipocytes release mitochondria into the blood to induce a protective antioxidant response in the heart. We found that adipocyte-to-macrophage mitochondria transfer in white adipose tissue is inhibited in murine obesity elicited by a lard-based high-fat diet, but not a hydrogenated-coconut-oil-based high-fat diet, aging, or a corn-starch diet. The long-chain fatty acids enriched in lard suppress mitochondria capture by macrophages, diverting adipocyte-derived mitochondria into the blood for delivery to other organs, such as the heart. The depletion of macrophages rapidly increased the number of adipocyte-derived mitochondria in the blood. These findings suggest that dietary lipids regulate mitochondria uptake by macrophages locally in white adipose tissue to determine whether adipocyte-derived mitochondria are released into systemic circulation to support the metabolic adaptation of distant organs in response to nutrient stress.

Keywords: CD36; EXT1; aging; beige fat; brown adipose tissue; cell-free mitochondria; fatty acids; heparan sulfate; horizontal mitochondria transfer; intercellular mitochondria transfer; lipids; macrophage; mitochondria; obesity; palmitate; white adipose tissue.

Figure 1:. Multidimensional spectral flow cytometry reveals tissue-specific mitochondria transfer axes from adipocytes in white, beige, and brown fat.

(A) Experimental design. (B) UMAP embedding for 384,869 cells randomly sampled, with annotated clustering. (C) Numbers per gram of the indicated cell types in eWAT, iWAT, and BAT from n=8 MitoFat mice. Data are expressed as mean +/− SEM. One-way ANOVA with LSD post-hoc test per tissue. *P<0.05, **P<0.01, ***P<0.001. (D) Relative density of mtD2+ cells. (E) Clock Face Diagram showing the degree of mitochondria transfer from adipocytes to each of the 21 clusters. Dot size reflects relative cluster abundance, and the minute/hour hands are the mean percent mtD2+. (F) Clock Face Diagrams parsed by eWAT, iWAT, and BAT. See also Figure S1 and Tables S1–S2.

Figure 2:. Aging-associated obesity minimally affects mitochondria transfer from adipocytes to other cell types in adipose tissues.

(A) Body weight and (B) eWAT, (C) iWAT, and (D) BAT masses of Young (4–5-months-old, n=5) or Aged (22–24-months-old, n=5) MitoFat mice on a normal chow diet. (E) Clock Face Diagrams comparing mitochondria transfer axes between Young and Aged MitoFat mice, parsed by eWAT, iWAT, and BAT. Mean +/− SEM. Students t-tests. *P<0.05, **P<0.01. See also Figure S2.

Figure 3:. Dietary lipids inhibit mitochondria transfer from adipocytes to macrophages in WAT.

(A) Body weight and (B) eWAT, (C) iWAT, and (D) BAT masses of young MitoFat mice fed a Chow (n=3), Lard-based high fat diet (HFD, n=5), or hydrogenated coconut oil (HCO)-based HFD (n=6) for 9 weeks. (E) Clock Face Diagrams comparing mitochondria transfer axes among the three groups, parsed by eWAT, iWAT, and BAT. Mean +/− SEM. One-way ANOVA with Tukey’s post-hoc test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. See also Figure S3.

Figure 4:. Long-chain fatty acids impair mitochondria uptake in vitro in a heparan sulfate-dependent manner.

(A) Flow cytometry plots of BV2 cells treated with BSA or Palmitate:BSA for 24 hours (h) followed by exposure to mtD2+ mitochondria. (B) Percent mtD2+ and (C) mtD2 MFI (of mtD2+ cells) of BV2 cells treated with BSA for 24h or 4:1 palmitate:BSA for 0.5, 4, or 24h. N=4/group. (D) Mitochondria uptake by BV2 cells treated with BSA or the indicated BSA-conjugated fatty acids (all 11 nM unbound free fatty acids) for 24 hours. N=8/group. (E) Mitochondria uptake in WT (n=4) or CD36 KO (n=4) bone marrow-derived macrophages or (F) WT (n=8) or EXT1 KO (n=8) BV2 cells treated with BSA or 4:1 Palmitate:BSA for 24h. (G) Mitochondria uptake by BV2 cells cultured with low or high glucose or sucrose for 24h. (H) Body weights, (I) fat masses, and (J) mitochondria transfer from adipocytes to macrophages in eWAT, iWAT, and BAT in MitoFat mice fed high sucrose (n=4) or corn starch (n=4) diets for 12 weeks. For B-D, one-way ANOVA with Holm-Sidak post hoc test against BSA control. For E-G, two-way ANOVA with Sidak’s posthoc test. For H-J, Student’s t-tests. Mean +/− SEM. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. See also Table S3.

Figure 5:. Mitochondria transfer is not required for WAT macrophage mitochondrial metabolism but can restore aerobic respiration in metabolically compromised macrophages.

(A) Oxygen consumption rates (OCR), (B) mitochondrial metabolism parameters, and (C) coupling efficiency of F4/80⁺ macrophages isolated from ovarian WAT from Ext1^F/F or Ext1^ΔLyz2 mice fed a chow diet, n=4–5/group with each being a pool of n=2–4 mice. (D) Experimental design for panels E-F. (E) Mitochondria capture by peritoneal macrophages 1 or 2 days after injection, n=5/group. (F) OCR of purified F4/80⁺ peritoneal macrophages on day 1. N=4/group. (G) Experimental design for panels H-J. (H) Basal, (I) maximal, and (J) ATP production-linked respiration (n=6–10/group). For panels E, H-J, two-way ANOVA with Sidak posthoc tests. For panels A and F, two-way ANOVA with Sidak posthoc tests for each condition. For panels B-C, Student’s t-tests. Mean +/− SEM. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Figure 6:. Adipocytes release micron-sized free mitochondria into blood.

(A) Percentages of mtD2⁺ macrophages in eWAT, iWAT, and BAT from mtD2^F/+ (control), MitoFat, and MitoBAT mice (n=3/group). (B) Small particle flow cytometry of plasma with size calibration beads and gating strategy. (C) Representative flow cytometry plots, (D) percentages, and (E) numbers of CD41- CD45− TER-119 mtD2+ adipocyte-derived mitochondria in plasma from control, MitoFat and MitoBAT mice (n=−6/group). (F-H) Percent of adipocyte-derived mitochondria in plasma that are positive or negative for (F) the extracellular vesicle (EV)-associated markers CD63, CD9, or CD81 (n=6/group), (G) one or more of those (EV), (H) the outer mitochondrial membrane protein TOM22 (n=5/group). For A, D, and E, one-way ANOVA with Tukey post-hoc test. F-H, Student’s t-tests. NS, not significant. Mean +/−SEM. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Figure 7:. Macrophages limit the release of adipocyte-derived mitochondria into the blood.

(A) Numbers of adipocyte-derived mitochondria in blood and (B) representative confocal microscopy of native mtD2 signal in hearts from MitoFat mice fed a chow (n=11), Lard-HFD (n=9), or HCO-HFD (n=9) for 9–10 weeks. (C-D) Numbers of adipocyte-derived mitochondria in blood of MitoFat mice (C) fed a high sucrose or corn starch diet for 12 weeks (n=4/group) or (D) that were Young (4-5-months-old, n=4) or Aged (19-months-old, n=3) on a normal chow diet. (E-G) MitoFat mice were treated with control (n=5) or clodronate-loaded liposomes (n=5) twice daily for 2 days. (E) Numbers of macrophages per gram of WAT. (F) Percentage of remaining macrophages that are mtD2+. (G) Numbers of adipocyte-derived mitochondria in blood 0, 1, or 2 days after treatment. For A, one-way ANOVA with Tukey post-hoc test. For C-F, Student’s t-test. For G, two-way ANOVA with repeated measures and Sidak post-hoc testing comparing the two groups each day. Mean +/− SEM. NS, not significant. *P<0.05, **P<0.01, ****P<0.0001.