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Fatty acid synthesis configures the plasma membrane for inflammation in diabetes

Abstract

Dietary fat promotes pathological insulin resistance through chronic inflammation1,2,3. The inactivation of inflammatory proteins produced by macrophages improves diet-induced diabetes4, but how nutrient-dense diets induce diabetes is unknown5. Membrane lipids affect the innate immune response6, which requires domains7 that influence high-fat-diet-induced chronic inflammation8,9 and alter cell function based on phospholipid composition10. Endogenous fatty acid synthesis, mediated by fatty acid synthase (FAS)11, affects membrane composition. Here we show that macrophage FAS is indispensable for diet-induced inflammation. Deleting Fasn in macrophages prevents diet-induced insulin resistance, recruitment of macrophages to adipose tissue and chronic inflammation in mice. We found that FAS deficiency alters membrane order and composition, impairing the retention of plasma membrane cholesterol and disrupting Rho GTPase trafficking—a process required for cell adhesion, migration and activation. Expression of a constitutively active Rho GTPase, however, restored inflammatory signalling. Exogenous palmitate was partitioned to different pools from endogenous lipids and did not rescue inflammatory signalling. However, exogenous cholesterol, as well as other planar sterols, did rescue signalling, with cholesterol restoring FAS-induced perturbations in membrane order. Our results show that the production of endogenous fat in macrophages is necessary for the development of exogenous-fat-induced insulin resistance through the creation of a receptive environment at the plasma membrane for the assembly of cholesterol-dependent signalling networks.

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Figure 1: Macrophage FAS ablation ameliorates diet-induced insulin resistance and inflammation in mice.
Figure 2: Macrophage FAS deficiency attenuates cell-autonomous inflammation and alters detergent-resistant microdomains.
Figure 3: FAS deficiency disrupts Rho GTPases and alters membrane order.
Figure 4: Exogenous palmitate does not rescue inflammation, but exogenous cholesterol restores JNK signalling, membrane order, and Rho GTPase membrane distribution.

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Acknowledgements

This work was supported by NIH grants DK101392, DK076729, DK088083, DK20579, DK56341, RR00954, HL067773 and the Taylor Family Institute for Innovative Psychiatric Research. S. Teitelbaum provided the constitutively active Rac construct, T. Pryse performed confocal microscopy, L. Yang and M. Miller assisted with imaging and L. Mydock-McGrane prepared the cholesterol alkyne.

Author information

Authors and Affiliations

Authors

Contributions

X.W., H.S., L.Y., M.G.R., and R.S. performed the experiments. H.S. performed lipidomic and proteomic analyses. R.S. and D.S.O. designed and performed the analysis for oxysterols. D.F.C. designed experiments using sterol analogues. X.W. and C.F.S. designed the experiments, analysed all data, and wrote the manuscript.

Corresponding author

Correspondence to Clay F. Semenkovich.

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Competing interests

The authors declare no competing financial interests.

Additional information

Reviewer Information Nature thanks A. Chawla, A. Tall and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 FAS is induced by inflammatory stimuli and its deficiency suppresses inflammation.

a, Quantification of subsets of pro-inflammatory macrophages (additionally positive for CD11c, CD18, or ICAM1) in the adipose tissue from HFD-fed control and LysM–FAS mice (n = 4). b, c, Western blotting of FAS protein in bone-marrow-derived macrophages treated with vehicle, palmitate (b; 500 μM) or LPS (c; 100 ng ml−1). d, e, Western blot of FAS protein in RAW 264.7 cells treated with vehicle as control, palmitate (d; 500 μM) or LPS (e; 100 ng ml−1). f, JNK phosphorylation levels after LPS stimulation in the presence of the FAS inhibitors cerulenin or C75. g, Western blotting of FAS protein in RAW 264.7 cells treated with scrambled control or a lentiviral-based shRNA targeting FAS. h, Western blot of pJNK in RAW 264.7 cells after stimulation with palmitate (500 μM). ik, ELISAs for pro-inflammatory cytokines in RAW 264.7 cells stimulated with palmitate (i; 500 μM) or LPS (j, k; 100 ng ml−1), n = 3. *P < 0.05, data are mean ± s.e.m.

Extended Data Figure 2 Tie2–FAS mice are protected from diet-induced insulin resistance.

ac, Glucose tolerance test (a), insulin tolerance test (b) and glucose-stimulated insulin secretion assays (c) in control and Tie2–FAS mice fed with HFD for 3 months (n = 15 control mice and 8 Tie2–FAS). d, e, Glucose infusion rate (GIR) (d), and plasma glucose levels (e) during hyperinsulinaemic–euglycaemic clamping for HFD-fed control (n = 9) and Tie2–FAS mice (n = 6). f, Mean GIR and hepatic glucose production (HGP) suppression in the steady state of the clamp experiment. gi, Western blotting of Akt phosphorylation levels in various indicated mouse tissues during the clamp. j, Western blotting of FAS protein in peritoneal macrophages from control and Tie2–FAS mice. *P < 0.05, data are mean ± s.e.m.

Extended Data Figure 3 Mice with FAS deficiency induced by Tie2–Cre resist diet-induced chronic inflammation.

a, Images of crown-like structures in visceral fat stained for the macrophage marker Mac2. b, Quantification of crown-like structures (n = 4 mice). c, d, Quantification of macrophages (F4/80+ CD11b+; c) and subsets of pro-inflammatory macrophages (those that are additionally positive for CD11c, CD18 and ICAM1; d) in visceral adipose tissue of Tie2–FAS mice by flow cytometry (n = 4 control and 5 Tie2–FAS mice). e, Gene expression in visceral adipose tissue from control (n = 6) and Tie2–FAS mice (n = 9). f, Oil red O staining of liver from Tie2–FAS mice fed HFD. g, Quantification of liver fat content (n = 8 control and 9 Tie2–FAS mice). h, Gene expression in liver from control (n = 6) and Tie2–FAS mice (n = 9). *P < 0.05, data are mean ± s.e.m.

Extended Data Figure 4 Diet effects in Tie2–FAS bone marrow transplant mice.

a, Body weight measurements in mice (n = 15) following bone marrow transplant (BMT) with control or Tie2–FAS marrow on a chow diet. b, Glucose tolerance testing in mice (n = 10) one month after bone marrow transplant on a chow diet. c, d, Food intake (c) and blood counts (d) in mice on HFD following bone marrow transplant (n = 7 control mice and 9 Tie2–FAS bone marrow transplanted). eh, Glucose tolerance testing (e), insulin tolerance testing (f), glucose-stimulated insulin levels (g) and body composition (h) in control (n = 17; grey) and Tie2–FAS bone marrow transplant mice (n = 15; blue) on a HFD. i, Number of inflammatory cells in visceral adipose tissue (n = 4). j, JNK phosphorylation in adipose tissue of bone marrow transplant mice. *P < 0.05, data are mean ± s.e.m.

Extended Data Figure 5 Phospholipid composition of light fractions (DRMs) and heavy fractions (non-DRM) in the presence and absence of FAS in macrophages.

ac, Mass spectroscopic quantification of the relative number of phosphatidylcholine (a; PC), sphingomyelin (b; SM) and phosphatidylethanolamine (c; PE) species of phospholipids in whole membranes (n = 8). d, Quantification of the relative number of phosphatidylcholine, sphingomyelin, and phosphatidylethanolamine species by the detergent method (n = 8 for control, n = 7 for LysM–FAS). e, Relative number of lipid species by the non-detergent, high-pH carbonate buffer method (n = 4). *P < 0.05, data are mean ± s.e.m.

Extended Data Figure 6 FAS is required for compartmentalizing proteins in DRMs.

a, SILAC strategy for DRM proteomic analysis in bone-marrow-derived macrophages (BMDMs). b, Heat map of relative abundance for DRM proteins in both strains (LysM–FAS and Tie2–FAS) of FAS-deficient BMDMs as compared to FAS-replete BMDMs. c, SILAC strategy for whole-membrane proteomic analysis. d, Heat map of relative abundance for membrane-associated proteins in both FAS-deficient models as compared to FAS-replete cells. e, SILAC quantification of DRM (top) and whole-membrane (bottom) proteomes in LysM–FAS and Tie2–FAS BMDMs relative to controls. f, g, DRM proteins affected by FAS deficiency (protein numbers in parentheses) in functional classes (f) and pathway analysis of affected proteins in macrophage inflammation (g). h, Heat map of select proteins from e. i, Western blot confirming heat-map changes of proteins indicated in red in h.

Extended Data Figure 7 Rho family GTPase activity and cell phenotyping in the setting of FAS deficiency.

a, Western blot for activated RhoA (top panel), activated Rac (middle panel) and total RhoA and Rac (bottom panel) in adipose tissue of control and Tie2–FAS BMT mice. b, Western blotting of whole cells (left), or cells with activated Rho GTPases (right with substrate pull-down) in scrambled (control) or FAS-knockdown RAW 264.7 cells. c, Cell spreading images of FAS-replete (control) and FAS-deficient (LysM–FAS) bone-marrow-derived macrophages (left) with corresponding frequency distribution of cell spreading (right). d, Cell area for control (n = 116) and LysM–FAS (n = 103) cells. e, MCP1-induced Transwell migration of Tie2–FAS and LysM–FAS cells (n = 4). f, Cell spreading images of scrambled (control) and FAS-deficient (FAS kd) RAW 264.7 cells (left) with the corresponding frequency distribution of cell spreading (right). g, Cell area for control (n = 108) and FAS knockdown (n = 102) RAW 264.7 cells. h, MCP1-induced Transwell migration of FAS-knockdown RAW 264.7 cells, expressed as a percentage of the value obtained for control cells (n = 4). *P < 0.05, data are mean ± s.e.m.

Extended Data Figure 8 Disruption of Rho GTPase membrane localization with genetic and pharmacologic inhibition of FAS.

a, b, RAW 264.7 cells were transfected with a Rac–EGFP construct then subjected to FAS knockdown (a) or incubated with the nonspecific FAS inhibitor C75 (b), followed by preparation of GPMVs and phase imaging. Liquid-ordered and liquid-disordered domains for quantification are indicated by arrows. Quantification of GFP fluorescence (with domains indicated) is shown in the middle and quantification of Rac partitioning in ordered versus disordered domains is shown on the right. Imaging analyses were repeated in 3 independent experiments and data shown are from representative experiments; n = 6 vesicles per group. *P < 0.05, data are mean ± s.e.m.

Extended Data Figure 9 FAS-dependent macrophage response to chronic palmitate treatment and cholesterol characterization.

a, Changes in pJNK levels in response to acute exposure to high-dose palmitate (500 μM) in control macrophages and in FAS-knockout bone-marrow-derived macrophages chronically pretreated with vehicle or 50 μM palmitate for 24 h. b, Western blots of DRMs of LysM–FAS cells incubated with 50 μM palmitate for 24 h. c, MCP1-induced Transwell migration assays of control and LysM–FAS cells, demonstrating that palmitate incubation does not rescue the defect in macrophage motility with FAS deficiency (n = 4). d, Cell spreading assays for control macrophages, FAS-deficient macrophages, and FAS-deficient macrophages treated with 50 μM palmitate for 24 h. Cell area quantification is shown on the left, with the frequency distribution of cell spreading on the right, and images of macrophage spreading on the bottom. For the frequency distribution of cell spreading, n = 108 for control, n = 101 for LysM–FAS and n = 105 for LysM–FAS with palmitate. e, f, Cholesterol content of fractions from LysM–FAS cells (n = 4). g, Cholesterol release to methyl-β-cyclodextrin in control and FAS-knockdown RAW 264.7 cells (n = 4). h, Cholesterol oxidase activity in control and FAS knockdown RAW 264.7 cells (n = 12). *P < 0.05, except in g where P < 0.001 by nonlinear curve-fit comparison; data are mean ± s.e.m.

Extended Data Figure 10 Schematic rendering of sterols in membranes.

a, Generalized planar and non-planar structures of the sterols used to load macrophages. b, Diagram of FAS-dependent cholesterol retention at the plasma membrane leading to JNK activation. In the FAS-deficient state, the phospholipid environment is characterized by greater levels of unsaturated fatty acids whereas in the FAS-replete state, phospholipids are enriched for saturated fatty acids. Phospholipids are depicted schematically and not intended to represent authentic unsaturated fatty acid structures.

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This file contains Supplementary Tables 1-2 and uncropped blots for Figures 1-4 and Extended Data Figures 1, 2, 6, 7 and 9. (PDF 14367 kb)

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Wei, X., Song, H., Yin, L. et al. Fatty acid synthesis configures the plasma membrane for inflammation in diabetes. Nature 539, 294–298 (2016). https://doi.org/10.1038/nature20117

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