Extended Data Fig. 9: CD169⁺ NAM depletion leads to dysregulated lipid metabolism in adipose tissue.

(a) Schematic representation of the experimental protocol used to deplete CD169-macrophages using CD169 diphtheria toxin receptor (CD169-DTR) mice. 9.5-week-old wild-type (WT) and CD169-DTR male mice (n = 3 per genotype) were injected intra-peritoneally (i.p) with 40 ng/gram diphtheria toxin (DT) on day 0 (D0) and bone marrow was collected and isolated from the femur on day 2 (D2) for analysis via flow cytometry. (b) Quantification of neutrophils, macrophages, and monocytes in WT versus CD169-DTR mice in the bone marrow (BM) cells isolated from the femur. Data is represented as a mean +/- SEM. Statistical significance was determined via non-parametric Mann-Whitney U test with p < 0.05. (c) Schematic representation of the experimental protocol used to deplete CD169-macrophages using CD169 diphtheria toxin receptor (CD169-DTR) mice. 9-month-old wild-type (WT) and CD169-DTR female mice (n = 9-11 per genotype) were injected intraperitoneal (i.p) with 40 ng/gram diphtheria toxin (DT) on day 0 (D0) followed by 4 ng/gram DT on D2 and D4. On day 7 (D7) mice either remained ad libitum-fed (n = 4–5 per genotype) or were fasted for 24-hours (n = 5-6 per genotype) and then scarified on D8. (d) Bar plot showing the proportion of CD169⁺CD11c⁻ nerve-associated macrophages (NAMs) in visceral white adipose tissue (VAT) from 9-month-old wild-type (WT) and CD169-DTR female mice treated with diphtheria toxin (DT). Mice were either fed ad libitum or fasted for 24 hours and sacrificed on day 8 (D8). NAMs are shown as a fraction of the total live CD45⁺ cell population. Data are presented as mean ± SEM. Statistical significance was assessed using a two-tailed Student’s t-test (α = 0.05); p < 0.05 was considered significant.(e) Relative protein quantification of Hormone-sensitive Lipase (HSL), HSL phosphorylated at Ser563 (p-HSL_S563), HSL phosphorylated at Ser660 (p-HSL_S660), Adipose triglyceride lipase (ATGL), and Monoamine oxidase A (MAOA) in VAT lysates. Protein was collected from 9-month-old wild-type (WT) and CD169-DTR female mice treated with diphtheria toxin (DT) and sacrificed on day 8 (D8), under ad libitum-fed conditions (n = 4 per genotype). Data are presented as mean ± SEM. Statistical significance was assessed using a two-tailed Student’s t-test (α = 0.05); p < 0.05 was considered significant.(f) Relative protein quantification of Hormone-sensitive Lipase (HSL), HSL phosphorylated at Ser563 (p-HSL_S563), HSL phosphorylated at Ser660 (p-HSL_S660), Adipose triglyceride lipase (ATGL), and Monoamine oxidase A (MAOA) in VAT lysates. Protein was collected from 9-month-old wild-type (WT) and CD169-DTR female mice treated with diphtheria toxin (DT) and sacrificed on day 8 (D8), under 24-hour fasting conditions (n = 4 per genotype). Data are presented as mean ± SEM. Statistical significance was assessed using a two-tailed Student’s t-test (α = 0.05); p < 0.05 was considered significant.(g) Relative protein quantification of oxidative phosphorylation (OXPHOS) complex subunits in VAT lysates from ad libitum-fed 9-month-old wild-type (WT) and CD169-DTR female mice treated with diphtheria toxin (DT) and sacrificed on day 8 (D8) (n = 4 per genotype). The following mitochondrial proteins were assessed: Complex I (CI): NADH:ubiquinone oxidoreductase subunit B8 (NDUFB8), Complex II (CII): succinate dehydrogenase subunit B, iron-sulfur protein (SDHB), Complex III (CIII): ubiquinol-cytochrome c reductase core protein 2 (UQCRC2), Complex IV (CIV): mitochondrially encoded cytochrome c oxidase I (MTCO1), and Complex V (CV): ATP synthase F1 subunit alpha (ATP5A). Data are presented as mean ± SEM. Statistical significance was assessed using a two-tailed Student’s t-test (α = 0.05); p < 0.05 was considered significant.(h) Relative mRNA expression of Adrb3 (top) and Pparγ (bottom) in the VAT of ad libitum-fed 9-month-old WT and CD169-DTR female mice treated with DT and sacrificed on D8 (n = 3–5 per genotype). Data is represented as a mean +/- SEM. Statistical significance was determined via Student’s two-tailed t-test with α = 0.05 and p < 0.05.