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The PI3K pathway preserves metabolic health through MARCO-dependent lipid uptake by adipose tissue macrophages

Abstract

Adipose tissue macrophages (ATMs) display tremendous heterogeneity depending on signals in their local microenvironment and contribute to the pathogenesis of obesity. The phosphoinositide 3-kinase (PI3K) signalling pathway, antagonized by the phosphatase and tensin homologue (PTEN), is important for metabolic responses to obesity. We hypothesized that fluctuations in macrophage-intrinsic PI3K activity via PTEN could alter the trajectory of metabolic disease by driving distinct ATM populations. Using mice harbouring macrophage-specific PTEN deletion or bone marrow chimeras carrying additional PTEN copies, we demonstrate that sustained PI3K activity in macrophages preserves metabolic health in obesity by preventing lipotoxicity. Myeloid PI3K signalling promotes a beneficial ATM population characterized by lipid uptake, catabolism and high expression of the scavenger macrophage receptor with collagenous structure (MARCO). Dual MARCO and myeloid PTEN deficiencies prevent the generation of lipid-buffering ATMs, reversing the beneficial actions of elevated myeloid PI3K activity in metabolic disease. Thus, macrophage-intrinsic PI3K signalling boosts metabolic health by driving ATM programmes associated with MARCO-dependent lipid uptake.

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Fig. 1: Limiting PI3K signalling via PTEN overexpression in hematopoietic/myeloid cells worsens metabolic health in obesity.
Fig. 2: Sustained myeloid-cell-intrinsic PI3K activity via myeloid-specific PTEN deletion improves metabolic health in obesity.
Fig. 3: Resident FBC206 ATMs are induced by lipids and dysregulation in adipose tissue homeostasis and most efficiently buffer lipids in obesity.
Fig. 4: Myeloid-cell-intrinsic PI3K signalling is critical for FBC206 ATM generation.
Fig. 5: MARCO is a marker for lipid-laden FBC206 ATMs in obesity and is regulated by the PI3K–Nrf2 pathway.
Fig. 6: The scavenger receptor MARCO mediates lipid uptake.
Fig. 7: PI3K activity sustains myeloid lipid uptake and catabolism.
Fig. 8: Beneficial effects of myeloid PI3K in diet-induced obesity are dependent on MARCO.

Data availability

The data that support the findings of this study are available from the corresponding authors upon request. The lipidomic datasets generated and analysed during the current study are available at https://github.com/menchelab/Marco. Microarray data of Fig. 5a and Extended Data Fig. 4h that support the findings of this study are available in Gene Expression Omnibus under accession number GSE8831 (ref. 10). Source data are provided with this paper.

Code availability

All code used in the metabolomic differential analyses and subsequent integration is available at https://github.com/menchelab/Marco.

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Acknowledgements

We thank H. Paar, A. Hladik and M. Salzmann for technical assistance. We thank E. Hirsch (Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center, University of Torino) for the Pi3kγKD/KD mice, M. Serrano (IRB Barcelona, Barcelona Institute of Science and Technology) for the PtenTg/+ mice and C. Tsatsanis (Laboratory of Clinical Chemistry, University of Crete Medical School) for the Akt1loxP/loxPAkt2 loxP/loxPLysM-Cre+/− mice. This research was supported by FWF 30026 and 31106 to G.S. and 31568 to O.S. and the Christian Doppler Laboratory for Arginine Metabolism in Rheumatoid Arthritis and Multiple Sclerosis to G.S. J.S.B., A.V. and A.L. were supported by a DOC fellowship of the Austrian Academy of Sciences. A.B. received funding from the European Research Council under the European Union’s Seventh Framework Programme and Horizon 2020 research and innovation programme (grant agreement no. 677006; CMIL). M.A.F. is supported by a Senior Principal Research Fellowship from the National Health and Medical Research Council of Australia (APP1116936).

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Authors

Contributions

J.S.B., A.V., O.S. and G.S. conceived and designed the study. J.S.B., A.V., A.L., A.K., M.P., M.H., A.H., M. Kieler, L.Q.G., M. Kerndl, M. Kuttke, A.E.B., E.E. and C.L.E. performed experiments. M.C. analysed lipidomic data and performed bioinformatic analysis. I.M. analysed liver steatosis. M.W.G., M. Kulik, P.M.D. and M.S. provided intellectual input. K.K. performed lipidomics analysis. J.S.B., A.V. and A.L. analysed the data. F.G., J.M., A.B., T.W. and M.A.F. provided key resources. J.S.B., A.V., O.S. and G.S. wrote the manuscript. All authors read, revised and approved the final manuscript.

Corresponding authors

Correspondence to Omar Sharif or Gernot Schabbauer.

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The authors declare no competing interests.

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Peer review information Primary Handling Editors: Pooja Jha; George Caputa. Nature Metabolism thanks Yi-bin Feng and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Disruptions of adipose homeostasis and metabolic stress ablate myeloid PI3K/AKT signalling.

a, Serum insulin levels of animals subjected to ST-HFD (n = 4 ND, n = 5 ST-HFD animals), 16 weeks HFD (n = 5 ND, n = 4 HFD animals) or acute lipolysis (n = 6 animals per group) (***P = 0.0006; two-tailed student’s t-test). b, Percentage of viable FB (CD45+CD11b+F4/80+) macrophages and in the adipose tissue of mice post 2 or 16 weeks HFD feeding and acute lipolysis (n = 5 animals; **P = 0.0072, ****P < 0.001, **P = 0.0026; two-tailed student’s t-test). c, Corresponding p-AKT MFI of FB-ATMs shown in (b) (*P = 0.027, ***P = 0.0001, **P = 0.0029; two-tailed student’s t-test). d, p-AKT MFI of macrophages post adipocyte conditioned media (ACM) treatment for 3 h (n = 5 samples; ***P = 0.001; two-tailed student’s t-test). Data are mean ± SEM and representative of one (a) or two (b-d) independent experiments.

Source data

Extended Data Fig. 2 Adaptive immune cell PTEN and p-AKT, hyperinsulinemic-euglycemic clamps, pancreatic macrophages and weights of BMT animals.

a, PTEN and p-AKT expression in adaptive immune cells from blood of WT (n = 3) or PtenTg/+ (n = 2) animals. Cells were pre-gated on viable CD45+CD3+B220+. b, Weights, fat and lean mass of Pten+/+>WT (n = 12) or PtenTg/+>WT (n = 10) mice as determined using Echo MRI. c, Blood glucose levels and glucose infusion rate during clamping of Pten+/+>WT (n = 10) or PtenTg/+>WT (n = 8) mice. d, Basal and insulin-stimulated whole-body glucose disposal rates and hepatic glucose production of mice in (c). e, Pancreatic CD68 expression in Pten+/+>WT or PtenTg/+>WT 16 weeks post HFD (n = 9 animals per genotype; ***P = 0.0003; two-tailed student’s t-test). f, Weights of mice shown in Fig. 1f-i. Data are mean ± SEM and representative of one (a-e) or two (f) independent experiments.

Source data

Extended Data Fig. 3 In vivo effects of modulating myeloid PI3K activity are independent of adiposity differences.

a, PTEN and p-AKT expression in adaptive immune cells from blood of PtenWT (n = 7) or PtenΔmyel (n = 6) mice. Cells were gated on viable CD45+CD3+B220+. Isotype control samples depicted are identical to Extended Data Fig. 2a. b, oGTT and AUC of PtenWT (n = 7 ND, n = 16 HFD) or PtenΔmyel (n = 9 ND, n = 16 HFD) mice, 16 weeks post feeding (**P = 0.0057, **P = 0.0031; two-way ANOVA, Bonferroni’s correction, **P for AUC = 0.009; two-tailed student’s t-test). c, Serum insulin levels post glucose bolus (n = 9 PtenWT, n = 7 PtenΔmyel animals). d, Weights of HFD mice shown in Fig. 2b,c stratified for low weight (LW) and high weight (HW) (n = 4 PtenWT LW, n = 10 PtenWT HW, n = 8 PtenΔmyel LW, n = 9 PtenΔmyel HW). e, Insulin tolerance test (ITT) and area under the curve (AUC) post 16 weeks feeding of PtenWT (n = 4 LW, n = 10 HW) or PtenΔmyel (n = 8 LW, n = 9 HW) mice stratified for low weight (LW) and high weight (HW) (*P = 0.0216, *P = 0.0151, **P = 0.0037; two-way ANOVA, Bonferroni’s correction; **P for AUC = 0.0051; two-tailed student’s t-test). f, Serum AST and ALT levels post 16 weeks HFD feeding of PtenWT (n = 12 HFD, n = 4 LW, n = 8 HW) or PtenΔmyel (n = 10 HFD, n = 4 LW, n = 6 HW) mice stratified for low weight (LW) and high weight (HW) (*P = 0.0185, *P = 0.0126, *P = 0.0293; two-tailed student’s t-test). Data are mean ± SEM and representative of one (a, c) or are pooled from two (d-f) or three (b) independent experiments.

Source data

Extended Data Fig. 4 Gating strategies and FBC206-ATMs are a large, lipid laden, lysotracker, TREM-2high population, mostly of resident origin post ST-HFD.

a, Gating strategy of ATMs. b, Gating strategy of monocytes. c, Gating strategy of hepatic myeloid-cells. d, Representative heatmap of BODIPY MFI in relation to cell size (FSC) of cells isolated from eWAT post 16 weeks HFD. e, Representative percentages of FBC and FBC206 populations within FSC low, intermediate and high low cells are depicted. Cells were pre-gated on live CD45+CD11b+F4/80+. f, Cell size quantification according to forward scatter (FSC) of FB-, FBC- and FBC206-ATMs (n = 8 animals; ***P = 0.0001, ****P < 0.0001; one-way ANOVA, Bonferroni’s correction). g, TREM-2 and CD9 MFI of FB-, FBC- and FBC206-ATMs post ST-HFD (n = 5 animals; ****P < 0.0001, *P = 0.013; one-way ANOVA, Bonferroni’s correction). h, Lysosomal gene expression based on KEGG pathway (mmu04142) of microarray data of obese FBs vs FBCs from C57Bl/6 J Lepob/ob mice (GSE8831). Data depicted as fold change relative to FB condition. i, Lysotracker MFI of the FBC206-ATM population post 16 weeks HFD (n = 7 animals; **P = 0.011; one-way ANOVA, Bonferroni’s correction). j, Post ST-HFD, percentages of GFP- FBC- vs GFP+ FBC-ATMs and GFP- FBC206- vs GFP+ FBC206-ATMs were analysed in eWAT of Ccr2GFP/+ animals. Cells were pre-gated on CD45+CD11b+F4/80+ (n = 5 animals; ****P < 0.0001; two-way ANOVA, Bonferroni’s correction). Data are mean ± SEM and representative of one (g, i), two (j) or four (d-f) independent experiments.

Source data

Extended Data Fig. 5 FBC206 generation of naïve macrophages stimulated with the indicated cytokines, adipokines and lipids.

a, FBC206/FBC ratio of macrophages post stimulation with glucose, insulin and ACM for 24 hours (n = 5 samples; ****P < 0.0001; one-way ANOVA, Bonferroni’s correction). b, FBC206/FBC ratio of macrophages post stimulation with various cytokines and ACM for 24 hours (n = 3 samples; ****P < 0.0001; one-way ANOVA, Bonferroni’s correction). c, FBC206/FBC ratio of macrophages post stimulation with various lipids and ACM for 24 hours (n = 4 samples; ****P < 0.0001; one-way ANOVA, Bonferroni’s correction). d, FBC206/FBC ratio of macrophages post stimulation with glucose, insulin and MMe for 24 hours (n = 5 samples). e, FBC206/FBC ratio and BODIPY MFI of naïve macrophages treated with Resatorvid and ACM for 24 hours (n = 4 samples). Data are mean ± SEM and representative of two (a-e) independent experiments.

Source data

Extended Data Fig. 6 MARCO and p-AKT post ST-HFD and acute lipolysis in the different ATM populations, where PTEN is deleted equally.

a, Percentages of Tomato+ and PTEN+ ATMs represented as percentage of their respective parent (FB-, FBC- or FBC206-ATMs) post ST-HFD in PtenWTR26tdTomato (cre-) and PtenΔmyelR26tdTomato (cre+) mice (n = 3 animals). b/c, MARCO MFI in macrophages post ST-HFD (b) or acute lipolysis (c). Cells were pre-gated on viable FBs (CD45+CD11b+F4/80+) (n = 5 animals; ****P < 0.0001, ***P = 0.0003; one-way ANOVA, Bonferroni’s correction). d, p-AKT MFI of FBC- and FBC206-ATMs of mice post ST-HFD or after acute lipolysis (n = 5 ST-HFD, n = 4 CL316,243 animals; **P = 0.0025, ***P = 0.0001; two-tailed student’s t-test). Data are mean ± SEM and representative of one (a) or two (b-d) independent experiments.

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Extended Data Fig. 7 Impaired lipid buffering of Marco-/- mice is not associated with adiposity differences and weights of animals in Fig. 8d,e.

a, Weight of mice in Fig. 6c. b, Fat depot distribution of lean Marco+/+ and Marcon mice presented as percent of bodyweight (n = 4 mice per genotype). RP: retroperitoneal, eWAT: epididymal, VAT: visceral, SAT: subcutaneous, white adipose tissue depots. c, Marco transcript levels measured in macrophages (n = 5 samples) and adipocytes (n = 4 samples). d, NEFA and glycerol levels of adipose tissue isolated from lean Marco+/+ and Marco-/- mice, stimulated with isoproterenol (ISO) for 2 hours (n = 5 animals; **P = 0.0022, **P = 0.0099, ****P < 0.0001; one-way ANOVA, Bonferroni’s correction). e, Weight of mice in Fig. 6d. f, Fat mass and adipose depot distribution of Marco+/+ and Marco-/- mice post 16 weeks HFD. RP: retroperitoneal, eWAT: epididymal, VAT: visceral, SAT: subcutaneous, white adipose tissue depots (n = 11 mice per genotype). g, Weights of mice shown in Fig. 6e. h, Macrophage content and FBC206/FBC ratios of Marco+/+ (n = 5) and Marco-/- (n = 7) mice prior to HFD feeding. i, Corresponding weights of mice in Fig. 8d (***P = 0.0009, ****P < 0.0001; one-way ANOVA, Bonferroni’s correction). j, Corresponding weights of mice in Fig. 8e (*P = 0.0181; one-way ANOVA, Bonferroni’s correction). Data are mean ± SEM and representative of one (b, c, g, h), two (a, d), or are pooled from two (f, j), four (e) or six (i) independent experiments.

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Extended Data Fig. 8 PI3K-dependent differences in lipid uptake and catabolism are independent of mitochondrial numbers and mitochondrial respiratory chain complex expression of Marco-/- macrophages.

a, AcLDL uptake of naïve macrophages from PtenWT (n = 10) and PtenΔmyel (n = 13) mice depicted as fold change (FC) increase (**P = 0.0025; two-tailed student’s t-test). b, Spare respiratory capacity (SRC) of data in Fig. 7e (*P = 0.0184; two-tailed student’s t-test). c, Mitochondrial numbers in naïve PtenWT and PtenΔmyel macrophages as determined using Mitotracker (n = 4 samples per genotype). d, mRNA levels of Pten and selected mitochondrial marker genes in naïve PtenWT (n = 8, for Pparg n = 7 samples) and PtenΔmyel (n = 8 samples) macrophages (****P < 0.0001, **P = 0.0091, *P = 0.443; two-tailed student’s t-test). e, Extracellular acidification rate (ECAR) of data in Fig. 7e (***P = 0.0007; two-tailed student’s t-test). f, SRC and ECAR of data in Fig. 7f (two-tailed student’s t-test). g, SRC of data in Fig. 7g (*P = 0.0487, *P = 0.0173, *P = 0.0111; two-tailed student’s t-test). h, SRC of data in Fig. 7h (**P = 0.0063, *P = 0.0171; two-tailed student’s t-test). i, mRNA levels of selected mitochondrial complex genes in naïve Marco+/+ (n = 8 samples) and Marco-/- (n = 9 samples) macrophages. The mitochondrial complex (C) represented by each gene is indicated. Data are mean ± SEM and representative of two (b-h), or are pooled from two (a, i) independent experiments.

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Brunner, J.S., Vogel, A., Lercher, A. et al. The PI3K pathway preserves metabolic health through MARCO-dependent lipid uptake by adipose tissue macrophages. Nat Metab 2, 1427–1442 (2020). https://doi.org/10.1038/s42255-020-00311-5

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