Breast milk alkylglycerols sustain beige adipocytes through adipose tissue macrophages

Abstract

Prevalence of obesity among infants and children below 5 years of age is rising dramatically, and early childhood obesity is a forerunner of obesity and obesity-associated diseases in adulthood. Childhood obesity is hence one of the most serious public health challenges today. Here, we have identified a mother-to-child lipid signaling that protects from obesity. We have found that breast milk-specific lipid species, so-called alkylglycerol-type (AKG-type) ether lipids, which are absent from infant formula and adult-type diets, maintain beige adipose tissue (BeAT) in the infant and impede the transformation of BeAT into lipid-storing white adipose tissue (WAT). Breast milk AKGs are metabolized by adipose tissue macrophages (ATMs) to platelet-activating factor (PAF), which ultimately activates IL-6/STAT3 signaling in adipocytes and triggers BeAT development in the infant. Accordingly, lack of AKG intake in infancy leads to a premature loss of BeAT and increases fat accumulation. AKG signaling is specific for infants and is inactivated in adulthood. However, in obese adipose tissue, ATMs regain their ability to metabolize AKGs, which reduces obesity. In summary, AKGs are specific lipid signals of breast milk that are essential for healthy adipose tissue development.

Keywords: Adipose tissue; Immunology; Macrophages; Metabolism; Obesity.

Figure 1. Breast milk AKGs increase beige adipocyte content in infant mice.

(A) Structure of AKGs found in human breast milk. (B) Level of AKGs in mouse breast milk on P3 and P6 normalized to protein content. (C) Levels of AKGs in breast milk of 3 lactating mice and the plasma AKG levels of their respective litters on P3 and P6. One data point represents plasma pooled from 8 to 9 infant mice. Plasma AKG levels on P56. (D) Schematic of AKG treatment of infant mice. (E) iAT weight on P10 of vehicle- or AKG-treated mice. (F–H) Histology of iAT. H&E staining and UCP1 immunohistochemistry (F), semithin sections stained with toluidine blue (G), and transmission electron microscopy of adipocytes (H). lp, lipid vesicle; mt, mitochondria; nc, nucleus. Arrowheads show BeAT; arrow shows fusing lipid vesicles. Scale bars: 25 μm (F and G); 5 μm (H). (I) Mitochondrial content, TG content, adipocyte size, BeAT area in iAT, plasma glycerol levels, and lean weight on P10. (J) Transcription of BeAT hallmark genes in iAT on P10 of vehicle- or AKG-treated mice. *P < 0.05; **P < 0.01; ***P < 0.001, 1-way ANOVA with Dunnett’s post hoc test (C) and Student’s 2-tailed unpaired t test (E, I, J).

Figure 2. Lack of breastfeeding and lack of neonatal AKG intake reduce beige adipocyte content in the iAT.

(A–C) Color-coded CIM showing the relative transcript level of UCP1 in human infant iAT. Also see Supplemental Figure 6A. (A) Breastfed infants, (B) formula-fed infants; age in years and the individual identifier number are indicated for each sample. (C) CIM showing relative UCP1 level in 0.3- to 1.0-yearold infants. UCP1 level was normalized to 3 reference genes (ACTB, TBP, HPRT1), and individual values were compared with the mean value of the 2 study groups. (D) Histology and BeAT content of the iAT in breastfed and formula-fed human infants. Scale bars: 100 μm (H&E); 25 μm (UCP1). (E) Scheme of artificial rearing (AR) experiment. (F) Histology of the iAT at P10 and P34 in breastfed and AR mice. ac, white adipocyte. Scale bars: 80 μm. (G) BeAT content of the iAT at P34. (H) CIM showing relative UCP1 level in iAT on P10 and P34. **P < 0.01; ***P < 0.001, Student’s 2-tailed unpaired t test (D); 1-way ANOVA with Dunnett’s post hoc test (G).

Figure 3. Macrophages are necessary for the effect of AKGs on adipocytes.

(A) Schematic of a Transwell assay with ATMs and 3T3-L1 adipocytes. (B–G) Adipocytes were treated with vehicle or AKGs for 24 hours, without ATMs (–ATMs) or in the presence of ATMs (+ATMs). Each data point represents 1 Transwell assay (B, E–G) or 1 adipocyte (D). (B) MFI of MitoTracker Green in adipocytes after treatment. (C) MitoTracker Red staining and transmission electron microscopy (TEM) images of 3T3-L1 cells treated in the presence of ATMs. Asterisks show lipid droplets; arrowheads show mitochondria. Scale bars: 25 μm (MitoTracker Red); 5 μm (TEM). (D) Mitochondrial content of 3T3-L1 cells, determined with image analysis. (E) Relative NADH-DH activity of adipocytes. (F) Ucp1 transcription in adipocytes. (G) Oxygen consumption of adipocytes treated with conditioned media of ATMs for 18 hours. ATMs were treated with vehicle or AKGs for 4 hours before cell culture media was collected and used for treating adipocytes. (H) CIM representation of BeAT-marker gene transcription in iAT of HFD-fed mice treated with vehicle or AKGs. ATMs were depleted with clodronate liposomes 24 hours before treatment in 1 group of mice (n = 5). (I) AT from human and M. mulatta infants, labeled for CD163 and Iba1. Scale bars: 10 μm (upper panels); 25 μm (lower panels). **P < 0.01; ***P < 0.001, Student’s 2-tailed unpaired t test (B, E–G) 1-way ANOVA with Dunnett’s post hoc test (D).

Figure 4. ATMs release PAF in response to AKGs.

(A) NGS of vehicle- or AKG-treated macrophages. AKGs enriched transcripts related to phosphatidylcholine (PC) and lyso-PC metabolism. Pink bars, relative abundance of GO terms (combined score rankings). (B) AKGs can serve as backbones of the sn-acyl chain of PAF. (C) Top: PAF levels in cell culture media of 3T3-L1 pre-/adipocytes or ATMs treated with vehicle or AKGs for 30 minutes. Bottom: PAF release from 6-day-old neonate (P6) mouse preadipocytes (Pre-ACs), adipocytes (ACs), and ATMs in 30 minutes. (D) AKG-, and PAF-induced transcriptional changes in ATMs. (E) F4/80⁺ ATMs in mouse on P10. Scale bar: 10 μm. (F) TEM image of mouse BeAT on P10. lp, lipid droplet. Scale bar: 4 μm. Arrow shows an ATM-like cell. (G) Transcript levels (left) and FACS analysis (right) of PAF-metabolizing enzymes in mouse ATMs and adipocytes. Iso, isotype control (H) FACS analysis of PAF-metabolizing enzymes in human ATMs and adipocytes. (I) Immunostaining of mouse ATMs and adipocytes cultured in vitro. Scale bars: 10 μm. (J) Effect of PAF synthesis inhibitors on AKG-induced PAF release in macrophages. FRS, farnesol (CHPT1 inhibitor); TSI-01, LPCAT2 inhibitor; LY315920 and 12777, PLA2 inhibitors. **P < 0.01; ***P < 0.001, Student’s 2-tailed unpaired t test. ^#P < 0.05; ^###P < 0.001, 1-way ANOVA with Dunnett’s post hoc test. (K) Schematic of AKG metabolism in adipocytes and ATM.

Figure 5. AKG-induced BeAT development is mediated by PAF/IL-6/STAT3 signaling.

(A) Histology at P7 and P10; BeAT and TG content of AT at P10 in WT and Ptafr^–/– mice. Arrowheads show BeAT. Scale bars: 65 μm. (B) PAF and BeAT content of AT at P10 and AT histology at P7 and P10 in WT and Pla2g2a-Tg (PLA2G2A-overexpressing) mice. Scale bars: 80 μm. (C) Relative transcription of Ucp1 and Ppargc1a in 3T3-L1 adipocytes treated with vehicle, 50 nM PAF, or carbamyl PAF (Carb-PAF) for 18 hours. (D) MitoTracker Red staining and TEM image of 3T3-L1 adipocytes treated with conditioned media of ATMs for 18 hours. ATMS were treated with vehicle or 50 nM PAF. See also Supplemental Figure 7G. Arrowheads show mitochondria. Scale bars: 5 μm. (E) Mitochondrial content and MFI of MitoTracker Green labeling of 3T3-L1 adipocytes treated with vehicle or 50 nM PAF for 18 hours in the presence of ATMs. SR27417, PTAFR blocker. (F) Transcription of BeAT genes in adipocytes treated with 0.2 ng/ml IL-6 or 50 pg/ml TNF-α for 18 hours. (G) Transcription factor–binding site analysis of BeAT genes. (H) STAT3 phosphorylation in adipocytes treated with vehicle or AKGs for 30 minutes in Transwell assay shown in Figure 3A. (I) ATMs were treated with vehicle (Veh) or AKGs for 4 hours, and culture media were added to adipocytes for a further 18 hours. CIM summarizes transcriptional changes evoked in adipocytes. STAT3 was inhibited with 500 nM cucurbitacin I, JAK/STAT3 with 280 nM ruxolitinib. (J) Schematic of the mechanism by which AKGs trigger BeAT gene transcription in adipocytes. **P < 0.01; ***P < 0.001, Student’s 2-tailed unpaired t test.

Figure 6. Effects of dietary AKG supplementation in obesity.

(A) Transcript levels of BeAT genes and genes associated with adipogenesis and lipolysis in iAT of lean, and HFD-fed mice, treated with vehicle or AKGs. (B) Histology and UCP1 immunohistochemistry of iAT of HFD-fed mice treated with vehicle or AKGs. Scale bars: 50 μm. (C) Transcript levels of BeAT genes and genes associated with adipogenesis and lipolysis in iAT of Lepr^db/db mice treated with vehicle or AKGs. (D) Weight gain within 1 week, iAT weight, and TG content of iAT of Lepr^db/db mice treated with vehicle or AKGs. (E) Histology, (F) adipocyte size, and (G) plasma glycerol level in Lepr^db/db mice treated with vehicle or AKGs. Scale bars: 50 μm (H&E); 10 μm (UCP1). Arrowheads show UCP1⁺ adipocyte. (H) NGS analysis of vehicle- and AKG-treated ATMs, showing relative abundance of transcripts. Pink bars, combined score ranking of GO terms. Venn diagram of AKG-suppressed interferon-responsive genes, clustered according to the type of interferon response. (I) Crown-like structures (CLSs) in iAT of Lepr^db/db mice treated with vehicle or AKGs. Scale bars: 50 μm. Quantification of CLSs in iAT of Lepr^db/db mice treated with vehicle or AKGs. *P < 0.05; **P < 0.01; ***P < 0.001, Student’s 2-tailed unpaired t test.

Figure 7. AKG-mediated signaling to adipocytes is inactivated at BeAT/WAT transition.

(A) ATMs were treated with vehicle or AKGs for 30 minutes, and PAF levels were measured in the cell culture media. ATMs were pretreated with vehicle or 10 ng/ml IL-4 for 18 hours. (B) Transcript levels of Agmo in mouse ATMs isolated at P3, P7, P14, and P56. One data point represents pooled ATM samples from 9 to 11 neonate (P3, P7, P14) and 3 to 4 adult (P56) mice. ^##P < 0.01, 1-way ANOVA with Dunnett’s post hoc test. (C) FACS histogram showing ATM expression of AGMO and LPCAT2 at P3 and P56 and following 3-months HFD feeding in mouse. (D) Plasma NPFF levels in C57/BL6 mouse at P3 and P10 and at weaning. **P < 0.01, Student’s 2-tailed unpaired t test. (E) Left: effect of NPFF on Agmo transcription in ATMs and 3T3-L1 adipocytes. Cells were treated with vehicle or 1 nM NPFF for 18 hours in vitro. Another data set of NPFF-induced transcriptional changes in macrophages has been published previously (62) (GEO GSE90658). Right: NGS analysis of 3T3-L1 adipocytes treated with vehicle or NPFF for 18 hours. Ontology of significantly upregulated transcripts (P < 0.001) is shown. Pink bars, combined score ranking; gray bars, rank-based ranking of GO terms. TEM images of 3T3-L1 adipocytes treated with vehicle or 1 nM NPFF for 18 hours. Arrowheads show mitophagosome membranes. mb, multilamellar body. Scale bars: 5 μm; 1 μm (insets). (F) Schematic of AKG-mediated signaling in AT. (a) Breast milk AKGs are metabolized by ATMs into PAF. (b) In an autocrine loop, PAF triggers IL-6 release from ATMs. (c) The secreted IL-6 activates adipocyte JAK/STAT3 signaling. (d) This triggers BeAT gene transcription and prevents a premature BeAT/WAT transition. (e) Under physiological conditions, AKG signaling in AT is active only in infancy and is inactivated in lean adult AT.