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).
