Macrophage-Associated Lipin-1 Promotes β-Oxidation in Response to Proresolving Stimuli

Abstract

Macrophages reprogram their metabolism to promote appropriate responses. Proresolving macrophages primarily use fatty acid oxidation as an energy source. Metabolites generated during the catabolism of fatty acids aid in the resolution of inflammation and tissue repair, but the regulatory mechanisms that control lipid metabolism in macrophages are not fully elucidated. Lipin-1, a phosphatidic acid phosphatase that has transcriptional coregulator activity, regulates lipid metabolism in a variety of cells. In this current study, we show that lipin-1 is required for increased oxidative phosphorylation in IL-4 stimulated mouse (Mus musculus) macrophages. We also show that the transcriptional coregulatory function of lipin-1 is required for β-oxidation in response to palmitate (free fatty acid) and apoptotic cell (human) stimulation. Mouse bone marrow-derived macrophages lacking lipin-1 have a reduction in critical TCA cycle metabolites following IL-4 stimulation, suggesting a break in the TCA cycle that is supportive of lipid synthesis rather than lipid catabolism. Together, our data demonstrate that lipin-1 regulates cellular metabolism in macrophages in response to proresolving stimuli and highlights the importance of aligning macrophage metabolism with macrophage phenotype.

FIGURE 1.. Lipin-1 enzymatic activity does not contribute to oxidative phosphorylation.

BMDMs isolated from WT and EKO mice were treated with or without 40 ng/ml IL-4 for 4 h. Oxygen consumption was analyzed via Seahorse extracellular flux analyzer. (A) Untreated WT and EKO macrophages. (B) WT and EKO BMDMs treated with IL-4. (C) Basal and maximal (FCCP) OCR of WT and EKO BMDMs treated with and without IL-4. Graphed data represents mean OCR with SEM. n = 3.

FIGURE 2.. Lipin-1 transcriptional coregulator function contributes to oxidative phosphorylation.

BMDMs isolated from WT and KO mice were treated with or without 40 ng/ml IL-4 for 4 h. Oxygen consumption was analyzed via Seahorse extracellular flux analyzer. (A) Untreated WT and KO macrophages. (B) WT and EKO BMDMs treated with IL-4. (C) Basal and maximal (FCCP) OCR of WT and EKO BMDMs treated with and without IL-4. n = 3. Graphed data represent mean OCR with SEM. *p ≤ 0.5.

FIGURE 3.. Lipin-1 is required for use of free fatty acids.

BMDMs isolated from WT and KO mice were treated with 40 ng/ml for 1.5 h. Immediately before assay, 100 μM palmitate was added to requisite wells, and OCR was analyzed via Seahorse extracellular flux assay. Area under the curve was analyzed via one-way ANOVA. n = 3. Graphed data represent mean OCR with SEM. *p ≤ 0.5.

FIGURE 4.. Lipin-1 does not regulate lipid uptake.

BMDMs isolated from WT and KO mice were treated with 100 μM BODIPY palmitate for 1 h at either 37°C or at 4°C. BMDMs (CD11b⁺, F4/80⁺, FITC⁺, and Ly6G⁻) were analyzed via flow cytometry to determine MFI. (A) A representative histogram of WT and KO BMDMs treated with BODIPY palmitate. (B) Compiled MFI with SEM of WT and KO BMDMs treated with BODIPY palmitate. n = 3. Data were analyzed via a Student t test.

FIGURE 5.. Lipin-1 regulates macrophage metabolism.

Central carbon analysis of WT and KO BMDMs treated with and without 40 ng/ml IL-4 for 4 h (A). Values represent fold change compared with WT control. Quantitative analysis of NADH (B) and NADPH (C) levels in WT and KO BMDMs treated with and without 40 ng/ml IL-4 for 4 h. Graphed data represent mean metabolite concentration with SEM. Student t test was performed as statistical analysis. n = 6. *p ≤ 0.5.

FIGURE 6.. Lipin-1 contributes to efferocytosis.

(A) WT and KO mice subjected to an excisional wound-healing model. Number of macrophages/dead cells was analyzed via flow cytometry. (B) BMDMs from WT and KO mice were subjected to a dual-label in vitro model of continuing efferocytosis, and percentage of macrophages (CD11b⁺ and F4/80⁺) that took up an initial event (primary) and multiple apoptotic bodies (continuing) were analyzed via flow cytometry. WT, EKO (C), and KO (D) mice were subjected to a zymosan model of peritonitis followed by peritoneal injection of PHK26-labeled AC. Percentage of macrophages (CD11b⁺, F4/80⁺, PKH26⁺, and Ly6G⁻) with labeled AC were analyzed via flow cytometry. Graphed data represent mean uptake with SEM. Student t test was used to analyze data. n = 8–12. *p ≤ 0.5.

FIGURE 7.. Lipin-1 enzymatic activity does not contribute to AC-derived lipid use.

OCR of WT (A) and EKO (B) BMDMs pretreated with and without 40 μM etomoxir for 15 min followed by the addition of AC at a 4:1 ratio. Graphed data represent mean OCR with SEM. n = 3. *p ≤ 0.5.

FIGURE 8.. Lipin-1 transcriptional coregulator function is required for degradation of AC-derived lipids.

OCR of WT (A) and KO (B) BMDMs pretreated with and without 40 μM etomoxir for 15 min followed by the addition of AC at a 4:1 ratio. Graphed data represent mean OCR with SEM. n = 3. *p ≤ 0.5.