Development and Application of FASA, a Model for Quantifying Fatty Acid Metabolism Using Stable Isotope Labeling

Abstract

It is well understood that fatty acids can be synthesized, imported, and modified to meet requisite demands in cells. However, following the movement of fatty acids through the multiplicity of these metabolic steps has remained difficult. To better address this problem, we developed Fatty Acid Source Analysis (FASA), a model that defines the contribution of synthesis, import, and elongation pathways to fatty acid homeostasis in saturated, monounsaturated, and polyunsaturated fatty acid pools. Application of FASA demonstrated that elongation can be a major contributor to cellular fatty acid content and showed that distinct pro-inflammatory stimuli (e.g., Toll-like receptors 2, 3, or 4) specifically reprogram homeostasis of fatty acids by differential utilization of synthetic and elongation pathways in macrophages. In sum, this modeling approach significantly advances our ability to interrogate cellular fatty acid metabolism and provides insight into how cells dynamically reshape their lipidomes in response to metabolic or inflammatory signals.

Keywords: fatty acid homeostasis; fatty acid modeling; stable isotope labeling.

Figure 1.. ISA Yields Suboptimal Fits for Fatty Acids Containing 20–24 Carbons

H1299 shCON cells were cultured in medium containing 2% FBS, 100% U-¹³C₆-glucose, and 100% U-¹³C₅-glutamine. After 48 hr of labeling, cells were collected, and isotopologue distributions were determined for fatty acids. (A–C) Representative empirical (black) and ISA-derived (red) isotopologue distributions for (A) 16:0, (B) 20:0, and (C) 24:0. The red bracket indicates overestimation of empirical data by ISA modeling, whereas the blue brackets indicate underestimation. (D) ISA sum of squared error (SSE) for the indicated fatty acids normalized to 14:0 ISA. The isotopologue distributions are representative singlets of biological quadruplicates and are corrected for natural abundance of C, H, and O isotopes. SSEs are the average ± SD of biological quadruplicates. See also Figures S1–S5.

Figure 2.. FASA Significantly Improves Fits for Fatty Acids Containing 20–24 Carbons

H1299 shCON cells were cultured in medium containing 2% FBS, 100% U-¹³C₆-glucose, and 100% U-¹³C₅-glutamine. After 48 hr of labeling, cells were collected, and isotopologue distributions were determined for fatty acids. (A) Schematic of possible contributions to the indicated saturated fatty acids used by FASA. All parameters marked in gray are considered elongated (see STAR Methods for additional details). S, synthesized; I, imported; IE_n, imported and elongated n times. (B) Representative empirical (black), ISA-derived (red), and FASA-derived (blue) isotopologue distributions for 16:0. (C) Model-derived contribution parameters for the 16:0 pool. (D) Representative empirical, ISA-derived, and FASA-derived isotopologue distributions for 24:0. The red bracket indicates overestimation of empirical data by ISA modeling, whereas the blue bracket indicates underestimation. (E) Model-derived contribution parameters for the 24:0 pool. (F) SSE for ISA and FASA, normalized to 14:0 ISA. (G) Model-derived D (fractional contribution of ¹³C-labeled metabolites to the lipogenic acetyl-CoA) for the indicated fatty acids using ISA or FASA. The isotopologue distributions shown are representative singlets of biological quadruplicates and are corrected for natural abundance of C, H, and O isotopes. Data shown for pool contribution parameters, SSEs, and D are the average ± SD of biological quadruplicates. See also Figures S1–S6 and S8–S11 and Table S2.

Figure 3.. FASA Provides Good Fits for Polyunsaturated Fatty Acids

H1299 shCON cells were cultured in medium containing 2% FBS, 100% U-¹³C₆-glucose, and 100% U-¹³C₅-glutamine. After 48 hr of labeling, cells were collected, and isotopologue distributions were determined for fatty acids. When applying FASA to polyunsaturated fatty acids (PUFAs), values for D₀, D₁, and D₂ were fixed using results from 16:0 to facilitate modeling. (A) Schematic of possible contributions to the indicated n-6 PUFAs used by FASA. All parameters marked in gray are considered elongated. (B) Representative empirical isotopologue distribution and FASA modeling for 22:4n-6. (C) FASA-derived contribution parameters for the 22:4n-6 pool. (D) Representative empirical isotopologue distribution and FASA modeling for 20:3n-6. (E) FASA-derived contribution parameters for the 20:3n-6 pool. The isotopologue distributions shown are representative singlets of biological quadruplicates and are corrected for natural abundance of C, H, and O isotopes. Data shown for pool contribution parameters are the average ± SD of biological quadruplicates. See also Figure S7.

Figure 4.. Application of FASA to Cells at iSS and mSS Reveals a Significant Role for Elongation

H1299 cells were cultured in a sub-confluent state for 5 days in medium containing 5% FBS and 100% U-¹³C₆-glucose to achieve iSS and mSS. On day 5, cells were collected, and isotopologue distributions were determined for fatty acids. (A) FASA-derived D parameter values from the indicated SFAs and MUFAs. (B) FASA-derived contribution parameters for selected SFA pools. (C) Diagram for determining elongation and import contribution possibilities to the 20:0 pool. (D) Elongation contribution for the indicated SFAs, MUFAs, and PUFAs. When applying FASA to PUFAs (but not SFAs or MUFAs), values for D₀, D₁, and D₂ were fixed using results from 16:0 to facilitate modeling. Data shown are the average ± SD of biological quadruplicates.

Figure 5.. Interpretation of FASA-Derived Parameters from Fatty Acid Pools Not at iSS and/or mSS

H1299 shCON cells were treated with 15 ng/mL doxycycline for 96 hr. In the final 48 hr of doxycycline treatment, they were also labeled in medium containing 5% FBS, 100% U-¹³C₆-glucose, and 100% U-¹³C₅-glutamine. After 48 hr of labeling, cells were counted on-plate, collected, and analyzed for fatty acid content. When applying FASA, D₀, D₁, and D₂ values for all fatty acids were fixed using results from 16:0 to facilitate modeling. (A) Accumulated n-6 PUFA elongation products. (B) Schematic for which SFA and MUFA origins require 18-to-20 elongation; X = 0 or 1 desaturations in fatty acids. (C) Accumulated 20-, 22-, and 24-carbon SFAs and MUFAs that were subject to 18-to-20 elongation during the 48-hr labeling period. (D) Summation of all accumulated 18-to-20 elongation products as described in (C). (E) Accumulated SFA and MUFA elongation products. (F) Accumulated FASN products. For (B)–(D), red, green, and blue indicate contributions from 20-, 22-, and 24-carbon fatty acids, respectively. Values represent average ± SD of biological quadruplicates.

Figure 6.. Application of FASA to Cells with Acute Metabolic Change by Silencing of SREBP

H1299 shCON and H1299 shSCAP cells were treated with 15 ng/mL doxycycline for 96 hr. In the final 48 hr of doxycycline treatment, they were also labeled in medium containing 5% FBS, 100% U-¹³C₆-glucose, and 100% U-₁₃C₅-glutamine. After 48 hr of labeling, cells were collected for gene expression analysis or counted on-plate and collected for fatty acid analysis. When applying FASA, D₀, D₁, and D₂ values for all fatty acids were fixed using results from 16:0 to facilitate modeling. (A) mRNA expression of SCAP and selected SREBP targets with doxycycline. (B) Accumulated SFA and MUFA elongation products. (C) Accumulated FASN products. (D) Accumulated n-6 PUFA elongation products. Values represent average ± SD (n = 3 for RNA, n = 4 for fatty acids). ns (not significant) p ≥ 0.05, *p < 0.05, **p < 0.01, and ***p < 0.001 (two-tailed heteroscedastic Student’s t test).

Figure 7.. Metabolic Reprogramming of Fatty Acid Elongation Programs by TLRs

BMDMs were treated with a TLR2 agonist (Pam3CSK4 [PAM3], 50 ng/mL), a TLR3 agonist (poly(I:C) [PIC], 1,000 ng/mL), or a TLR4 agonist (LPS, 50 ng/mL) or infected with MHV-68 (MOI = 1) in medium containing 5% FBS, 100% U-¹³C₆-glucose, and 100% U-¹³C₅-glutamine for 48 hr. Cells were collected after 24 h of labeling for gene expression analysis or counted on-plate and collected after 48 hr of labeling for fatty acid analysis. When applying FASA, D₀, D₁, and D₂ values for all fatty acids were fixed using results from 16:0 to facilitate modeling. (A) Accumulated SFAs and MUFAs that were subject to the indicated elongation step for non-treated (NT) and TLR-activated macrophages. (B) Accumulated SFAs and MUFAs that were synthesized by FASN for NT and TLR-activated macrophages. (C) Accumulated n-6 PUFAs that were elongated in NT and TLR-activated macrophages. (D) mRNA expression of Fasn and Elovl1-7 for NT and TLR-activated macrophages. (E) Accumulated FASN products for NT, PIC-treated, and MHV-68-infected macrophages. (F) Accumulated n-6 PUFA elongation products for NT, PIC-treated, and MHV-68-infected macrophages. Values represent average ± SD of biological quadruplicates. ns (not significant) p ≥ 0.05, *p < 0.05, **p < 0.01, and ***p < 0.001 (two-tailed heteroscedastic Student’s t test). See also Figure S12 and Table S3.