The Secreted Enzyme PM20D1 Regulates Lipidated Amino Acid Uncouplers of Mitochondria - PubMed (original) (raw)

. 2016 Jul 14;166(2):424-435.

doi: 10.1016/j.cell.2016.05.071. Epub 2016 Jun 30.

Katrin J Svensson 1, Leslie A Bateman 2, Hua Lin 3, Theodore Kamenecka 3, Isha A Lokurkar 4, Jesse Lou 4, Rajesh R Rao 1, Mi Ra Chang 3, Mark P Jedrychowski 1, Joao A Paulo 5, Steven P Gygi 5, Patrick R Griffin 3, Daniel K Nomura 6, Bruce M Spiegelman 7

Affiliations

The Secreted Enzyme PM20D1 Regulates Lipidated Amino Acid Uncouplers of Mitochondria

Jonathan Z Long et al. Cell. 2016.

Abstract

Brown and beige adipocytes are specialized cells that express uncoupling protein 1 (UCP1) and dissipate chemical energy as heat. These cells likely possess alternative UCP1-independent thermogenic mechanisms. Here, we identify a secreted enzyme, peptidase M20 domain containing 1 (PM20D1), that is enriched in UCP1(+) versus UCP1(-) adipocytes. We demonstrate that PM20D1 is a bidirectional enzyme in vitro, catalyzing both the condensation of fatty acids and amino acids to generate N-acyl amino acids and also the reverse hydrolytic reaction. N-acyl amino acids directly bind mitochondria and function as endogenous uncouplers of UCP1-independent respiration. Mice with increased circulating PM20D1 have augmented respiration and increased N-acyl amino acids in blood. Lastly, administration of N-acyl amino acids to mice improves glucose homeostasis and increases energy expenditure. These data identify an enzymatic node and a family of metabolites that regulate energy homeostasis. This pathway might be useful for treating obesity and associated disorders.

Copyright © 2016 Elsevier Inc. All rights reserved.

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Conflict of interest statement

CONFLICTS OF INTEREST

BMS is a consultant for Calico, Inc.

Figures

Figure 1

Figure 1. Increased circulating PM20D1 augments whole body energy expenditure

(A) Schematic diagram of search strategy to identify factors expressed by UCP1+ cells. The following publicly available datasets were used for the comparisons: UCP1-TRAP (GSE56248), brown versus white adipose tissues (GSE8044), and inguinal fat following 1 or 5 weeks cold exposure (GSE13432). (B and C) Anti-flag Western blot of plasma 40 days post injection (B) and whole body weight curves (C) from male C57BL/6 mice after tail vein injection of AAV-GFP or AAV-PM20D1 fed high fat diet (HFD) at room temperature. Mice were 7 weeks old at the time of injection, HFD was started 7 days post injection, and mice were maintained at room temperature for the duration of the experiment. For (B), arrow indicates band corresponding to PM20D1-flag. For (C), _n=_8–10/group, mean ± SEM, * p<0.05. (D–F) Whole body weight curves (D), MRI analysis of total body composition (E), and representative images of adipose tissues (F) from male C57BL/6 mice after tail vein injections of AAV-GFP or AAV-PM20D1 fed HFD at thermoneutrality. Mice were placed into thermoneutrality (30°C) at 6 weeks old, injected with virus at 7 weeks old, and HFD was started 7 days post injection. Mice were maintained at 30°C for the duration of the experiment. For (E) and (F), data are from 47 days post injection. _n=_8–10/group, mean ± SEM, * _p_<0.05, ** _p_<0.01. (G and H) VO2 (G) and movement (H) of male C57BL/6 mice over a period of two days. Mice were 7 weeks old at the time of injection, high fat diet (HFD) was started 7 days post injection, and mice were maintained at room temperature for the duration of the experiment. For (G) and (H), measurements were taken at 4 weeks post injection, a time point prior to any sigificant divergence of body weight (body weight means ± SEM: GFP 33.3±1.3 g, PM20D1 31.6±1.3 g, _p_>0.05). _n=_8/group, mean ± SEM, * p<0.05. See also Figure S1, Figure S2, Table S1, and Table S2.

Figure 2

Figure 2. Lack of classical browning and identification of increased _N-_oleoyl phenylalanine in mice injected with AAV-PM20D1

(A and B) mRNA expression of the indicated genes in BAT, iWAT, and eWAT (A) and Western blot of UCP1 and mitochondrial proteins (B) from male C57BL/6 mice at thermoneutrality after tail vein injections of AAV-GFP or AAV-PM20D1. Mice were placed into thermoneutrality (30°C) at 6 weeks old, injected with virus at 7 weeks old, and high fat diet (HFD) was started 7 days post injection. Mice were maintained at 30°C for the duration of the experiment. For (A) and (B), data are from 47 days post injection. For (A), _n=_8/group, mean ± SEM, * p<0.05. For (B), _n=_4–5/group. (C) Chromatogram at m/z = 428 from plasma of male C57BL/6 mice after tail vein injection of AAV-GFP or AAV-PM20D1. For (C), mice were 7 weeks old at the time of injection, high fat diet (HFD) was started 7 days post injection, and mice were maintained at room temperature for the duration of the experiment. The comparative metabolomics was performed on plasma harvested 54 days post injection. _n=_4/group, * p<0.05. (D) Chemical structure of _N-_oleoyl phenylalanine (C18:1-Phe). (E and F) MS/MS spectra (E) and retention time (F) of endogenous (top) or synthetic (bottom) C18:1-Phe. See also Table S3.

Figure 3

Figure 3. PM20D1 regulates the levels of _N-_acyl amino acids in vivo

(A–D) Chromatograms (A and C) and quantitation of fold change (B and D) of various _N-_acyl Phes (A and B) or various C18:1-amino acids (C and D) in plasma of male C57BL/6 mice in thermoneutrality after tail vein injection of AAV-GFP or AAV-PM20D1 by targeted MRM. Mice were placed into 30°C at 6 weeks old, injected with virus at 7 weeks old, and high fat diet (HFD) was started 7 days post injection. Mice were maintained at 30°C for the duration of the experiment. The comparative targeted metabolomics was performed on plasma harvested 47 days post injection. For (A) and (C), chromatograms are from one representative mouse per group. For (B) and (D), the absolute quantitation in AAV-GFP versus AAV-PM20D1 is as follows: C16-Phe, 6 versus 15 nM, respectively; C18:2-Phe, 2 versus 7 nM, respectively; C18:1-Phe, 4 versus 10 nM, respectively; C18:1-Leu, 6 versus 29 nM, respectively. (E) Fold change (cold/room temperature) of the indicated plasma _N-_acyl amino acids following cold exposure for the indicated times by targeted MRM. For (B) and (D–E), _n_=4–5/group, mean ± SEM, * p<0.05, ** p<0.01. See also Figure S3 and Table S4.

Figure 4

Figure 4. Enzymatic activity of PM20D1 in vitro

(A) Schematic of synthase and hydrolase reaction of free fatty acid and free amino acid to _N-_acyl amino acid. (B) Relative levels of C18:1-Phe generated in vitro from Phe (100 μM), oleate (0.03–1.5 mM), and purified mouse PM20D1-flag. (C–E) Relative levels of C18:1-amino acid generated in vitro from the indicated head group (100 μM) and purified mouse PM20D1-flag using either oleate (1.5 mM, C), arachidonate (1.5 mM, D) or oleoyl-coenzyme A (C18:1-CoA, 0.7 mM, E). For (C), EA, ethanolamine. (F) Relative levels of oleate generated in vitro from the indicated _N-_acyl amide substrates (100 μM) and purified mouse PM20D1-flag. C18:1-EA, _N-_oleoyl ethanolamine. (G) Anti-flag Western blot of immunoaffinity purified mouse PM20D1-flag or the indicated point mutants. (H) Relative levels of C18:1-Phe generated in vitro from Phe (100 μM), oleate (1.5 mM), and the indicated wild-type (WT) or mutant PM20D1-flag protein. (I) Relative levels of oleate generated in vitro from C18:1-Phe (100 μM) and the indicated wild-type (WT) or mutant PM20D1-flag protein. (J) Relative levels of C18:1-amino acid generated in vitro from the indicated head group (100 μM), oleate (1.5 mM), and purified human PM20D1-flag. (K) Relative levels of oleate generated in vitro from the indicated _N_-acyl amide substrate (100 μM) and purified human PM20D1-flag. For (B–F) and (H–K), enzymatic assays were carried out in PBS at 37°C for 1.5 hours, _n_=3/group, mean ± SEM, * p<0.05, ** p<0.01, for reaction with PM20D1 versus reaction omitting PM20D1, or reaction with PM20D1 versus reaction with heat-denatured PM20D1. Y-axes indicates relative ion intensity normalized to 1 nmol of a D3,15_N-_serine internal standard that was doped in during the extraction process prior to MS analysis. See also Figure S4.

Figure 5

Figure 5. Effects of _N-_acyl amino acids on respiration in cells

(A–C) Oxygen consumption rates (OCRs) of differentiated primary BAT cells (A), differentiated primary iWAT cells (B), and differentiated primary BAT cells from UCP1-WT or UCP1-KO mice (C), treated with the indicated compounds for the indicated time. For (A–C), adipocytes were differentiated and analyzed on day 5. (D–H) OCRs of C2C12 cells (D–F, and H) or U2OS cells (G) treated with the indicated compounds for the indicated time. For (D–H), cells were seeded and analyzed the following day. For (F–H), data is shown as the maximal increase in OCR as a percentage of the oligomycin basal OCR, which is normalized to 100%. For (A–H), the following concentrations of compounds were used: oligomycin (1 μM), indicated _N-_acyl amino acid or fatty acid (50 μM), FCCP (0.2 μM), or rotenone (3 μM). For (E), the following non-standard abbreviations are used: C18:1-Phe-NH2 (_N-_oleoyl phenylalanine amide), C18:1-Phe-OCH3 (_N-_oleoyl phenylalanine methyl ester). For (H), the following non-standard abbreviations are used: C20:4-NHCH3 (_N_-arachidonoyl _N_-methyl amide), C20:4-NAT (_N_-arachidonoyl taurine), C20:4-GABA (_N_-arachidonoyl gamma-amino butyric acid). For (A–H), _n=_3–6/group, mean ± SEM, * p<0.05, ** p<0.01, *** p<0.001 for treatment versus DMSO at the same time point.

Figure 6

Figure 6. Effects of _N_-acyl amino acids in mitochondria and identification of _N_-acyl amino acid-interacting proteins

(A) Oxygen consumption rates (OCRs) of freshly isolated BAT mitochondria treated with the indicated compounds for the indicated times. Respiration was measured with 10 mM pyruvate and 5 mM malate as substrates, and FCCP and rotenone were used at 2 μM and 3 μM, respectively. _n_=4–5/group, ** p<0.01. (B) Tetramethyl rhodamine methyl ester (TMRM) fluorescence in C2C12 cells following 20 min treatment with oligomycin alone (1 μM), or in combination with C18:1-Phe (10 or 50 μM) or FCCP (0.4 μM). _n=_3/group, mean ± SEM, * _p_<0.05, ** _p_<0.01. (C) Chemical structure of the N-acyl amino acid photocrosslinkable probe (“photo-probe”). (D) OCR of C2C12 cells treated with DMSO or the photo-probe (50 μM). For (D), data is shown as the maximal increase in OCR as a percentage of the oligomycin basal OCR, which is normalized to 100%. _n=_3–4/group, mean ± SEM, ** _p_<0.01 (E) TAMRA in-gel fluorescence of C2C12 cells treated with the photo-probe (50 μM, 20 min), followed by UV irradiation (on ice, 10 min), cell lysis, and click chemistry with TAMRA-N3. For (E), control cells that were not UV irradiated were kept under ambient light (on ice, 10 min). (F) Proteins in C2C12 cells that showed C20:4-Phe competeable photoprobe labeling. For (F), C2C12 cells were incubated with 20 μM photo-probe (“probe only”), or 20 μM photo-probe with 100 μM C20:4-Phe competitor (“probe + competitor”). Cells were then UV irradiated, lysed, subjected to click chemistry with biotin-N3, and analyzed by MS (see Methods). Proteins satisfying the following filtering criteria are shown: >50% reduction in peptide counts with competitor present versus without competitor, and detection of at least one peptide in all three probe only samples. Comparisons in which no peptides were detected in “probe + competitor” samples were assigned a fold-change of 15. See also Table S5.

Figure 7

Figure 7. In vivo effects of chronic C18:1-Leu administration to mice

(A–E) Change in body weight (A), daily and cumulative food intake (B and C), body composition by MRI (D), and GTT (E) of 21 week DIO mice treated daily with vehicle, or C18:1-Leu (25 mg/kg/day, i.p.), or oleate (25 mg/kg/day, i.p.). For (D), MRI measurements were taken on day 7. For (A–E), the initial weights of the mice were not statistically different (means ± SEM: vehicle, 51.9±0.8 g; 25 mg/kg C18:1-Leu, 52.1±1.1 g; 25 mg/kg oleate, 52.9±1.2 g). For (A–E), _n_=9/group, for vehicle and C18:1-Leu, and _n_=5/group for oleate, mean ± SEM, * p<0.05, ** p<0.01, *** p<0.001. For (E), after the last dose on day 7, mice were fasted overnight and the GTT was performed the next morning with glucose at a dose of 1.5 g/kg. (F and G) VO2 (F) and movement (G) of mice treated with vehicle or C18:1-Leu. For (F and G), measurements were recorded for 2 days following 8 days chronic treatment with vehicle or C18:1-Leu (25 mg/kg/day, i.p.); during this time daily administration of the indicated compounds continued. For (F and G), _n_=8/group, mean ± SEM, * p<0.05. See also Figure S5, Figure S6, and Figure S7.

Comment in

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