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. 2018 Jul 17;115(29):E6937-E6945.
doi: 10.1073/pnas.1803389115. Epub 2018 Jul 2.

Ablation of PM20D1 Reveals N-acyl Amino Acid Control of Metabolism and Nociception

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Free PMC article

Ablation of PM20D1 Reveals N-acyl Amino Acid Control of Metabolism and Nociception

Jonathan Z Long et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

N-acyl amino acids (NAAs) are a structurally diverse class of bioactive signaling lipids whose endogenous functions have largely remained uncharacterized. To clarify the physiologic roles of NAAs, we generated mice deficient in the circulating enzyme peptidase M20 domain-containing 1 (PM20D1). Global PM20D1-KO mice have dramatically reduced NAA hydrolase/synthase activities in tissues and blood with concomitant bidirectional dysregulation of endogenous NAAs. Compared with control animals, PM20D1-KO mice exhibit a variety of metabolic and pain phenotypes, including insulin resistance, altered body temperature in cold, and antinociceptive behaviors. Guided by these phenotypes, we identify N-oleoyl-glutamine (C18:1-Gln) as a key PM20D1-regulated NAA. In addition to its mitochondrial uncoupling bioactivity, C18:1-Gln also antagonizes certain members of the transient receptor potential (TRP) calcium channels including TRPV1. Direct administration of C18:1-Gln to mice is sufficient to recapitulate a subset of phenotypes observed in PM20D1-KO animals. These data demonstrate that PM20D1 is a dominant enzymatic regulator of NAA levels in vivo and elucidate physiologic functions for NAA signaling in metabolism and nociception.

Keywords: N-acyl amino acid; PM20D1; knockout; metabolism; pain.

Conflict of interest statement

Conflict of interest statement: B.M.S. is a consultant for Calico Life Sciences, LLC.

Figures

Fig. 1.
Fig. 1.
Generation of the PM20D1-KO mouse and loss of NAA hydrolase/synthase activity in PM20D1-KO tissues. (A) Nucleotide and predicted protein sequence for the Pm20d1 gene from WT or PM20D1-KO animals. The ∆6-bp out-of-frame deletion is highlighted in gray, and the newly generated early stop codon is identified by red text. (B) Sanger sequencing chromatograms of a PCR product amplified from the Pm20d1 gene in PM20D1-KO mice. The new A–T junction is identified by the arrowhead, and the 25-bp region flanking this junction is shown. (C and D) Relative levels of Pm20d1 mRNA from BAT (C) and livers (D) of WT and PM20D1-KO mice using qPCR primers that anneal either downstream of the ∆6-bp deletion (“endogenous”) or directly on the deleted wild-type sequence (“6-bp specific”). (E) Fold change in protein abundance of 216 proteins detected by quantitative shotgun proteomics from livers of WT and PM20D1-KO mice. The red dashed line indicates a fold change = 1 in KO versus WT mice. (F) Percent conversion of the starting material C20:4-Gly into an arachidonic acid product in the indicated tissues from WT or PM20D1-KO mice. (G) Percent conversion of the C18:1-Phe starting material into an oleate product in livers from WT and PM20D1-KO mice. Hydrolase reactions in F and G were performed by incubating 100 µg of whole-tissue lysate in 100 µL PBS with 100 µM of the indicated NAA for 1 h at 37 °C. (H) Percent conversion of the Phe starting materials into a C18:1-Phe product in livers from WT and PM20D1-KO mice. Synthase reactions were performed by incubating 100 µg of whole-liver lysate in 100 µL PBS with 100 µM Phe and 2 mM oleic acid for 1 h at 37 °C. Data are shown as means ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001 for the indicated comparisons. For C, D, and FH, n = 4 per group. For E, n = 2 per group.
Fig. 2.
Fig. 2.
Bidirectional dysregulation of endogenous NAAs in tissues from PM20D1-KO mice. (AC) Relative levels of the indicated NAA metabolites in blood (A), BAT (B), or liver (C) of WT or PM20D1-KO mice. Metabolites were extracted in using acetonitrile/methanol/water and were analyzed in targeted mode by MRM in unit mass mode. NAAs that were not detected are not shown. For NAAs detected in PM20D1-KO mice for which no corresponding peak was detected in WT mice, the fold change was set to 50. Data are shown as means ± SEM; *P < 0.05, **P < 0.01 for the indicated metabolite in WT versus PM20D1-KO mice; n = 4 per group. (D) Representative extracted ion chromatograms (EICs) of the peaks corresponding to C18:1-Gln and C18:1-Lys in blood and liver from WT and PM20D1-KO mice using alternative chromatographic conditions on a high-resolution mass spectrometer (Methods). EICs for the [M-H] ion were extracted with the indicated mass window, and standards were chemically synthesized as described in Methods. The background in gray indicates the elution time of the C18:1-Gln or C18:1-Lys standard ±1 min.
Fig. 3.
Fig. 3.
Glucose intolerance and reduced insulin sensitivity in PM20D1-KO mice. (A) Whole-body energy expenditure of 8- to 10-wk-old male WT and PM20D1-KO mice over 2 d on a chow diet. Gray background indicates nighttime and white background indicates daytime. (BD) Total body weights (B), lean and fat mass (C), and glucose tolerance test (D) of male WT and PM20D1-KO mice on an HFD (60% kcal from fat) starting at 6–10 wk of age. In C and D, body composition (C) was measured at 5 wk on the HFD, and the glucose tolerance test (D) was administered at 17 wk on the HFD. (EH) Total body weights (E), lean and fat mass (F), glucose tolerance (G), and insulin tolerance (H) of female WT and PM20D1-KO mice on a Western diet (40% kcal from fat, 0.2% cholesterol) starting at 12–16 wk of age. For FH, body composition was measured (F) and the insulin tolerance test was administered (H) at 19 wk on the Western diet, and the glucose tolerance test (G) was administered at 17 wk on the Western diet. Data are shown as means ± SEM; *P < 0.05, **P < 0.01 for WT versus PM20D1-KO. n = 8–13 per group in AD, and n = 6 per group in for EH.
Fig. 4.
Fig. 4.
PM20D1 mice maintain a higher body temperature following cold exposure. (A) Representative BAT sections stained with H&E from WT and PM20D1-KO mice. (Magnification: 10×.) (B and C) Core body temperatures at room temperature and after transfer to 4 °C as measured by rectal thermometer (B) or telemetry probe implanted into the i.p. cavity (C) in WT and PM20D1-KO mice. Immediate transition from room temperature to 4 °C is indicated by the dashed gray line (B), and a slow transition over 3 h from room temperature to 4 °C is indicated by the gray bar (C). (D and E) Protein levels of UCP1, mitochondrial proteins, and tubulin in BAT (D) or iWAT (E) of WT and PM20D1-KO mice at room temperature or after 10 h at 4 °C. (F and G) mRNA levels of the indicated genes in WT and PM20D1-KO mice from BAT (F) or iWAT (G) at room temperature. All mice were males, 8–12 wk of age. Data are shown as means ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001. For B and C, n = 6–12 per group; for F and G, n = 5–6 per group.
Fig. 5.
Fig. 5.
Antinociceptive behaviors of PM20D1-KO mice in response to inflammatory and chemical pain. (A and B) Latency to a tail flick in response to a radiant heat stimulus (A) or latency to hind paw lick, flick, or jump in response to a 52 °C hot plate (B) in WT and PM20D1-KO mice. (C) Time spent licking the injected paw after intraplantar administration of 5% formalin (20 µL per paw) in WT and PM20D1-KO mice. The early and late phases were quantified during the 0- to 5-min and the 20- to 40-min periods, respectively, after formalin injection. (D) Number of abdominal constrictions over 30 min following i.p. injection of 0.6% acetic acid (0.3 mL per mouse) in WT and PM20D1-KO mice. (E and F) Total distance traveled during a 10-min open field assay (E) and latency to fall from a rotarod (F) in WT and PM20D1-KO mice. (G and H) Fold change of all plasma NAAs (G) or individual NAAs (H) following i.p. injection of 0.6% acetic acid (0.3 mL/mouse) in WT mice. For G, individual NAAs over time are shown in gray, and the average fold change across all measured NAAs is shown in blue (***P < 0.001 for the indicated time point versus t = 0). All mice were males between 10–15 wk of age. Data are shown as means ± SEM; *P < 0.05, **P < 0.01. For AD, n = 20–26 per group; for E and F, n = 11–15 per group; for G and H, n = 5 per group.
Fig. 6.
Fig. 6.
Antagonism of TRP channels by PM20D1-regulated C18:1-Gln. (A) Change in relative Fluo-4 fluorescence (∆F/F0) over time in cells transfected with TRPV1 (RFP+) or in neighboring untransfected cells (RFP) following treatment with capsaicin. (B) Agonist response (∆F/F0) in TRPV1-transfected cells after treatment with capsaicin or the indicated NAA (50 µM). (CE) Antagonism of capsaicin-induced ∆F/F0 by the indicated compound (50 µM) in TRPV1-transfected cells. (F) Dose response of C18:1-Gln antagonizing capsaicin-induced ∆F/F0 in TRPV1-transfected cells. (GI) Antagonism of agonist-induced ∆F/F0 by the indicated compound (50 µM) in TRPV3- (G), TRPV4- (H), or TRPA1- (I) transfected cells. For AI, transfection was performed in HEK293A cells. For CI, antagonist studies were performed by preincubating the indicated compound for 2 min. Agonist-induced ∆F/F0 was then quantified 1 min after addition of capsaicin (10 µM) or AITC (10 µM) or 12 s after addition of 2-ABP (500 µM) or GSK1016790A (0.2 µM). Data are shown as means ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001 for the indicated comparison, or versus the corresponding DMSO control group. Fifteen to thirty cells were quantified per data point.
Fig. 7.
Fig. 7.
C18:1-Gln exhibits antinociceptive bioactivity in vivo. (A and B) Number of flinches (A) and time spent licking the injected paw (B) after intraplantar administration of capsaicin (1.5 μg per paw). (C) Time spent licking the injected paw after intraplantar administration of 5% formalin (20 µL per paw). (D) Number of abdominal constrictions over 30 min following i.p. injection of 0.6% acetic acid (0.3 mL per mouse). (E) Total number of line crossings during a 5-min open field assay of 8- to 12-wk-old male wild-type mice treated with vehicle or C18:1-Gln (100 mg/kg, i.p.). C18:1-Gln was administered 15 min before the behavioral assays. Data are shown as means ± SEM; **P < 0.01 for the indicated comparison; n = 12 per group.

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