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Adipose tissue macrophages exert systemic metabolic control by manipulating local iron concentrations

Nature Metabolism volume 4, pages 1474–1494 (2022)Cite this article

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Abstract

Iron is essential to many fundamental biological processes, but its cellular compartmentalization and concentration must be tightly controlled. Although iron overload can contribute to obesity-associated metabolic deterioration, the subcellular localization and accumulation of iron in adipose tissue macrophages is largely unknown. Here, we show that macrophage mitochondrial iron levels control systemic metabolism in male mice by altering adipocyte iron concentrations. Using various transgenic mouse models to manipulate the macrophage mitochondrial matrix iron content in an inducible fashion, we demonstrate that lowering macrophage mitochondrial matrix iron increases numbers of M2-like macrophages in adipose tissue, lowers iron levels in adipocytes, attenuates inflammation and protects from high-fat-diet-induced metabolic deterioration. Conversely, elevating macrophage mitochondrial matrix iron increases M1-like macrophages and iron levels in adipocytes, exacerbates inflammation and worsens high-fat-diet-induced metabolic dysfunction. These phenotypes are robustly reproduced by transplantation of a small amount of fat from transgenic to wild-type mice. Taken together, we identify macrophage mitochondrial iron levels as a crucial determinant of systemic metabolic homeostasis in mice.

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Fig. 1: Obesity leads to mitochondrial iron overload in adipose tissue macrophages.
Fig. 2: Mac-MitoNEETTG mice are protected from HFD-induced obesity and metabolic disorders.
Fig. 3: Mac-FtmtTG mice are more susceptible to high-fat-diet-induced obesity and metabolic disorders.
Fig. 4: Reducing mitochondrial iron content in macrophages after a long-term high-fat-diet feeding reverses obesity and metabolic dysfunction.
Fig. 5: Beneficial systemic effects in Mac-MitoNEETTG mice are due to adipose tissue macrophages.
Fig. 6: Deleterious systemic effects in Mac-FtmtTG mice are equally due to adipose tissue macrophages.
Fig. 7: Beneficial cross-talk between Mac-MitoNEETTG ATMs and adipocytes.
Fig. 8: Deleterious cross-talk between Mac-FtmtTG ATMs and adipocytes.

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All data generated or analysed during this study are available with this paper. Source data are provided with this paper.

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Acknowledgements

We thank the UTSW Animal Resource Center, Histology Core, Metabolic Phenotyping Core, the Live Cell Imaging Core, Transgenic Core and Flow Cytometry Facility for their excellent assistance with experiments performed in this paper. We also thank Shimadzu Scientific Instruments for the collaborative efforts in mass spectrometry technology resources. This study was supported by US National Institute of Health grants RC2-DK118620, R01-DK55758, R01-DK099110, R01-DK127274 and R01-DK131537 to P.E.S.; R01 DK108773 to D.Y.O.; C.C. is supported by K99-DK122019 and R00-DK122019. C.M.G. is supported by F32-DK-122623; V.A.P. was supported by an American Diabetes Association Minority Postdoctoral Fellowship (1-18-PMF-030). J.-B.F. was supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation; grant 414232833).

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Author notes

  1. These authors contributed equally: Nolwenn Joffin, Christy M. Gliniak.

Authors and Affiliations

  1. Touchstone Diabetes Center, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Nolwenn Joffin, Christy M. Gliniak, Jan-Bernd Funcke, Vivian A. Paschoal, Clair Crewe, Shiuhwei Chen, Ruth Gordillo, Christine M. Kusminski, Da Young Oh & Philipp E. Scherer

  2. Department of Cell Biology, Washington University, St. Louis, MO, USA

    Clair Crewe

  3. Department of Pharmaceutical Sciences, West Virginia University, Morgantown, WV, USA

    Werner J. Geldenhuys

  4. Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Philipp E. Scherer

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Contributions

Conceptualization, N.J., C.M.G, V.A.P., D.Y.O. and P.E.S.; methodology, N.J. and C.M.G; formal analysis, N.J., V.A.P. and C.C; investigation, N.J., C.M.G., J.-B.F., V.A.P., C.C., S.C., R.G. and C.M.K.; resources, W.J.G.; original draft writing, N.J. and P.E.S.; manuscript review and editing, N.J., C.M.G., J.-B.F. and P.E.S.; supervision, P.E.S.; funding acquisition, P.E.S.

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Correspondence to Philipp E. Scherer.

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Nature Metabolism thanks Alyssa Hasty and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Christoph Schmitt, in collaboration with the Nature Metabolism team.

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Extended data

Extended Data Fig. 1 Validation of MitoNEET overexpression in macrophages.

(a-j) Six-week-old male control and Mac-MitoNEETTG mice were fed chow dox for 2 weeks. (a) Breeding strategy. (b) Cisd1 mRNA levels relative to control in monocytes (control n = 4, TG n = 3), ATMs (n = 3), and peritoneal macrophages (n = 6). (c) MitoNEET protein expression relative to β-actin in peritoneal macrophages (n = 6). (d) Representative Western blots chosen from 2 independent experiments. (e-f) Mitochondrial respiration in peritoneal macrophages (control n = 3, TG n = 5). (g) Mitochondrial electron transport chain protein expression relative to prohibitin in peritoneal macrophages (n = 6). (h) Representative Western blots. (i-j) Glycolysis stress test in peritoneal macrophages (control n = 6, TG n = 6). Significance in (b-c, f-g, j) between control and Mac-MitoNEETTG was calculated using a two-tailed Student’s t-test. Significance in (e, i) was calculated using a 2-way ANOVA with Sidak’s post-test for multiple comparisons. Error bars represent mean ± S.E.M. * (P < 0.05), ** (p < 0.01), *** (p < 0.0001), **** (p < 0.00001).

Source data

Extended Data Fig. 2 Validation of Ftmt overexpression in macrophages.

(a-j) Six-week-old male control and Mac-FtmtTG mice were fed chow dox for 2 weeks. (a) Breeding strategy. (b) Ftmt mRNA levels relative to control in peritoneal macrophages (control n = 7, TG n = 9). (c) FTMT protein expression relative to β-actin in peritoneal macrophages (control n = 5, TG n = 3). (d) Representative Western blots chosen from 2 independent experiments. (e-f) Mitochondrial respiration in peritoneal macrophages (control n = 6, TG n = 7). (g) Mitochondrial electron transport chain protein expression relative to prohibitin in peritoneal macrophages (control n = 5, TG n = 3). (h) Representative Western blots (control n = 5, TG n = 3). (i-j) Glycolysis stress test in peritoneal macrophages (control n = 4, TG n = 3). Significance in (b-c, f-g, j) between control and Mac-FtmtTG was calculated using a two-tailed Student’s t-test. Significance in (e, i) was calculated using a 2-way ANOVA with Sidak’s post-test for multiple comparisons. Error bars represent mean ± S.E.M. * (P < 0.05), ** (p < 0.01), *** (p < 0.0001), **** (p < 0.00001).

Source data

Extended Data Fig. 3 Macrophages respond to mitoNEET overexpression by up-regulating iron storage.

(a-q) Male control and Mac-MitoNEETTG mice were fed chow dox for 2 weeks. (a) Labile iron pool (LIP) in mitochondrial and cytosolic compartments of peritoneal macrophages (control n = 7, TG n = 6). (b) mRNA levels of iron metabolism genes relative to control in peritoneal macrophages (control n = 10, TG n = 7). (c) Ferritin (control n = 9, TG n = 12), (d) Ferroportin (control n = 3, TG n = 5), (e) CD163 (control n = 3, TG n = 5), (f) CD91 (control n = 3, TG n = 5) and (g) FTMT (control n = 3, TG n = 5) protein expression relative to β-actin in peritoneal macrophages. (h) Representative Western blots chosen from 2 independent experiments (i) mRNA levels of inflammation markers relative to control in peritoneal macrophages (control n = 7, TG n = 10). (j-l) Flow cytometric measurement of total (control n = 6, TG n = 5), M1-like control n = 6, TG n = 5) and M2-like macrophages in eWAT (n = 6). (m) Body weight (control n = 3, TG n = 5). (n-o) Oral glucose tolerance test (control n = 3, TG n = 5). (n) Blood glucose levels. (o) Serum insulin levels. (p) Blood glucose levels during insulin tolerance test (control n = 3, TG n = 5). (q) Serum triglyceride levels during triglyceride clearance (n = 6). Significance in (a-g, i-m) between control and Mac-MitoNEETTG was calculated using a two-tailed Student’s t-test. Significance in (n-q) was calculated using a 2-way ANOVA with Sidak’s post-test for multiple comparisons. Error bars represent mean ± S.E.M. * (P < 0.05), ** (p < 0.01), *** (p < 0.0001), **** (p < 0.00001).

Source data

Extended Data Fig. 4 Macrophages respond to Ftmt overexpression by up-regulating iron uptake.

(a-q) Male control and Mac-FtmtTG mice were fed chow dox for 2 weeks. (a) Labile iron pool (LIP) in mitochondrial and cytosolic compartments of peritoneal macrophages (n = 4). (b) mRNA levels of iron metabolism genes relative to control in peritoneal macrophages (control n = 5, TG n = 7). (c) CD91 (n = 8), (d) CD163 (n = 8), (e) Ferritin (n = 8), (f) Ferroportin (n = 4) and (g) MitoNEET (n = 8) protein expression relative to β-actin in peritoneal macrophages (n = 8). (h) Representative Western blots chosen from 2 independent experiments. (i) mRNA levels of inflammation markers relative to control in peritoneal macrophages (control n = 5; TG n = 7). (j-l) Flow cytometric measurement of total, M1-like and M2-like macrophages in eWAT (n = 6). (m) Body weight (n = 6). (n-o) Oral glucose tolerance test (n = 6). (n) Blood glucose levels (n = 6). (o) Serum insulin levels (control n = 7, TG n = 9). (p) Blood glucose levels during insulin tolerance test (control n = 6, TG n = 5). (q) Serum triglyceride levels during triglyceride clearance (n = 6). Significance in (a-g, i-m) between control and Mac-FtmtTG was calculated using a two-tailed Student’s t-test. Significance in (n-q) was calculated using a 2-way ANOVA with Sidak’s post-test for multiple comparisons. Error bars represent mean ± S.E.M. * (P < 0.05), ** (p < 0.01), *** (p < 0.0001), **** (p < 0.00001).

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Extended Data Fig. 5 Mitochondrial iron depletion in macrophages during HFD increases iron recycling, protecting adipocytes from iron overload.

(a-x) Six-week-old male control and Mac-MitoNEETTG mice were fed HFD dox for 6 weeks. (a) Labile iron pool (LIP) in ATMs from eWAT (control n = 7, TG n = 7) (b) Total iron content measured by ICP-MS in mitochondrial and cytosolic compartments in peritoneal macrophages (control n = 7, TG n = 5). (c-d) LIP in (c) adipocytes and (d) SVF from eWAT (control n = 7, TG n = 5). (e-f) mRNA levels of iron metabolism genes relative to control (e) adipocytes (control n = 5, TG n = 6) and (f) ATMs (control n = 6, TG n = 5) from eWAT. (g) Ferroportin and (h) Ferritin protein expression relative to β-actin in adipocytes from eWAT (n = 6). (i) Representative Western blots chosen from 2 independent experiments. (j) CD163 (n = 9), (k) CD91, (l) Ferroportin, and (m) Ferritin protein expression relative to β-actin in ATMs from eWAT (control n = 9, TG n = 6). (n) Representative Western blots chosen from 2 independent experiments. (o) mRNA levels of mitochondrial iron metabolism genes relative to control in ATMs from eWAT (control n = 5, TG n = 6). (p) MitoNEET and (q) Mitoferritin protein expression relative to β-actin in ATMs from eWAT (n = 3). (r) MitoNEET protein expression relative to β-actin in adipocytes from eWAT (control n = 9, TG n = 6). (s) LIP in mitochondrial and cytosolic compartments of eWAT (control n = 5, TG n = 4). (t-u) mRNA levels of inflammatory marker genes relative to control in (t) ATMs and (u) adipocytes from eWAT (control n = 5, TG n = 6). (v) Representative fluorescence microscopic images of 4-HNE, Perilipin and DAPI staining of eWAT chosen from 3 independent experiments. (w) Quantification of 4-HNE fluorescence in eWAT relative to control (control n = 8, TG n = 7). (x) Protein carbonylation in adipocytes from eWAT (control n = 10, TG n = 11). Significance in (a-h, j-u, w-x) between control and Mac-MitoNEETTG was calculated using a two-tailed Student’s t-test. Error bars represent mean ± S.E.M. * (P < 0.05), ** (p < 0.01), *** (p < 0.0001), **** (p < 0.00001).

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Extended Data Fig. 6 Mitochondrial iron overload in macrophages during HFD promotes adipocyte iron overload and inflammation.

(a-q) Six-week-old male control and Mac-FtmtTG mice were fed HFD dox for 6 weeks. (a) Total iron content measured by ICP-MS in mitochondrial (n = 8) and cytosolic (control n = 4, TG n = 5) compartments in peritoneal macrophages. (b-d) Labile iron pool (LIP) in (b) ATMs (control n = 4, TG n = 5), (c) adipocytes (control n = 9, TG n = 12) and (d) SVF (control n = 7, TG n = 5) from eWAT. (e-f) mRNA levels of iron metabolism genes relative to control in (e) adipocytes (n = 5) and (f) ATMs (n = 6) from eWAT. (g) Ferritin (control n = 7, TG n = 12) and (h) Ferroportin (n = 3) protein expression relative to β-actin in adipocytes from eWAT. (i) Representative Western blots chosen from 3 independent experiments. (j) Ferritin (control n = 7, TG n = 9) and (k) Ferroportin protein expression relative to β-actin in ATMs from eWAT (n = 3). (l) Representative Western blots chosen from 3 independent experiments. (m) mRNA levels of mitochondrial iron metabolism genes relative to control in ATMs from eWAT (n = 6). (i) MitoNEET protein expression relative to β-actin in adipocytes from eWAT (control n = 4, TG n = 10). (o) LIP in mitochondrial and cytosolic compartments of eWAT (n = 4). (p-q) mRNA levels of inflammatory marker genes relative to control in (p) ATMs and (q) adipocytes from eWAT (n = 5). Significance in (a-h, j-k, m-q) between control and Mac-FtmtTG was calculated using a two-tailed Student’s t-test. Error bars represent mean ± S.E.M. * (P < 0.05), ** (p < 0.01), *** (p < 0.0001), **** (p < 0.00001).

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Extended Data Fig. 7 Eliminating MitoNEET in macrophages during HFD promotes insulin resistance and inflammation.

(a-k) Six-week-old male control and Mac-MitoNEETKO mice were fed HFD dox for 7 weeks. (a) Breeding strategy. (b) MitoNEET protein expression relative to β-actin in peritoneal macrophages (n = 3). (c) Representative Western blots (n = 3). (d) Body weight (control n = 9, TG n = 5). (e) Tissue weights (control n = 9, TG n = 5). (f-g) Oral glucose tolerance test. (f) Blood glucose levels (control n = 5, TG n = 3). (g) Serum insulin levels (control n = 9, TG n = 5). (h) Blood glucose levels during insulin tolerance test (control n = 9, TG n = 5). (i-k) Flow cytometric measurement of total, M1-like and M2-like macrophages in eWAT (control n = 5, TG n = 3). Significance in (b, e, i-k) between control and Mac-MitoNEETKO was calculated using a two-tailed Student’s t-test. Significance in (d, f-h) was calculated using a 2-way ANOVA with Sidak’s post-test for multiple comparisons. Error bars represent mean ± S.E.M. * (P < 0.05), ** (p < 0.01), *** (p < 0.0001), **** (p < 0.00001).

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Extended Data Fig. 8 Metabolic analyses of Mac-MitoNEETTG mice.

(a-e) Six-week-old male control and Mac-MitoNEETTG mice were fed HFD for 20 weeks and then switched to HFD dox for 1 week. Metabolic analyses were performed at 3 d before and 7 d after diet change (n = 6). (a) Experimental design. (b) Body weight (n = 5). (c) Hourly food intake (n = 6). (d) Respiratory exchange ratio (n = 6). (e) Energy balance (n = 6). Significance in (b-e) between control and Mac-MitoNEETTG was calculated using a 2-way ANOVA with Sidak’s post-test for multiple comparisons. Error bars represent mean ± S.E.M. * (P < 0.05), ** (p < 0.01), *** (p < 0.0001), **** (p < 0.00001).

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Extended Data Fig. 9 Validation of eWAT transplantation to wild-type mice.

(a-i) Six-week-old male control and Mac-MitoNEETTG mice were injected with PKH67 dye before transplantation of their eWAT to wild-type mice (n = 5). Recipient mice were allowed to recover 3 weeks and then fed HFD dox for 6 weeks. (a-b) Flow cytometric measurement of PKH67 fluorescence in (a) graft and (b) recipient eWAT (n = 5). (c-d) mRNA levels of Cisd1, Ftmt, and Tfam relative to control in (c) graft and (d) recipient eWAT (n = 5). (e-g) Flow cytometric measurement of total, M1-like, and M2-like macrophages in recipient eWAT (n = 5). (h-i) Wild-type mice were transplanted with either control or Mac-FtmtTG eWAT. Transplanted mice were allowed to recover 3 weeks and then fed HFD dox for 6 weeks. (h-i) mRNA levels of Cisd1, Ftmt and Tfam relative to control in (c) graft and (d) recipient eWAT (control n = 7, TG n = 8). Significance in (a-i) between control and transgenic fat-transplanted animals was calculated using a two-tailed Student’s t-test. Error bars represent mean ± S.E.M. * (P < 0.05), ** (p < 0.01), *** (p < 0.0001), **** (p < 0.00001).

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Joffin, N., Gliniak, C.M., Funcke, JB. et al. Adipose tissue macrophages exert systemic metabolic control by manipulating local iron concentrations. Nat Metab 4, 1474–1494 (2022). https://doi.org/10.1038/s42255-022-00664-z

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