ACE overexpression in myeloid cells increases oxidative metabolism and cellular ATP

Abstract

Angiotensin-converting enzyme (ACE) affects blood pressure. In addition, ACE overexpression in myeloid cells increases their immune function. Using MS and chemical analysis, we identified marked changes of intermediate metabolites in ACE-overexpressing macrophages and neutrophils, with increased cellular ATP (1.7–3.0-fold) and Krebs cycle intermediates, including citrate, isocitrate, succinate, and malate (1.4–3.9-fold). Increased ATP is due to ACE C-domain catalytic activity; it is reversed by an ACE inhibitor but not by an angiotensin II AT1 receptor antagonist. In contrast, macrophages from ACE knockout (null) mice averaged only 28% of the ATP levels found in WT mice. ACE overexpression does not change cell or mitochondrial size or number. However, expression levels of the electron transport chain proteins NDUFB8 (complex I), ATP5A, and ATP5β (complex V) are significantly increased in macrophages and neutrophils, and COX1 and COX2 (complex IV) are increased in macrophages overexpressing ACE. Macrophages overexpressing ACE have increased mitochondrial membrane potential (24% higher), ATP production rates (29% higher), and maximal respiratory rates (37% higher) compared with WT cells. Increased cellular ATP underpins increased myeloid cell superoxide production and phagocytosis associated with increased ACE expression. Myeloid cells overexpressing ACE indicate the existence of a novel pathway in which myeloid cell function can be enhanced, with a key feature being increased cellular ATP. Angiotensin-converting enzyme (ACE) affects blood pressure. In addition, ACE overexpression in myeloid cells increases their immune function. Using MS and chemical analysis, we identified marked changes of intermediate metabolites in ACE-overexpressing macrophages and neutrophils, with increased cellular ATP (1.7–3.0-fold) and Krebs cycle intermediates, including citrate, isocitrate, succinate, and malate (1.4–3.9-fold). Increased ATP is due to ACE C-domain catalytic activity; it is reversed by an ACE inhibitor but not by an angiotensin II AT1 receptor antagonist. In contrast, macrophages from ACE knockout (null) mice averaged only 28% of the ATP levels found in WT mice. ACE overexpression does not change cell or mitochondrial size or number. However, expression levels of the electron transport chain proteins NDUFB8 (complex I), ATP5A, and ATP5β (complex V) are significantly increased in macrophages and neutrophils, and COX1 and COX2 (complex IV) are increased in macrophages overexpressing ACE. Macrophages overexpressing ACE have increased mitochondrial membrane potential (24% higher), ATP production rates (29% higher), and maximal respiratory rates (37% higher) compared with WT cells. Increased cellular ATP underpins increased myeloid cell superoxide production and phagocytosis associated with increased ACE expression. Myeloid cells overexpressing ACE indicate the existence of a novel pathway in which myeloid cell function can be enhanced, with a key feature being increased cellular ATP. Angiotensin-converting enzyme (ACE) 3The abbreviations used are: ACEangiotensin-converting enzymeHBSSHanks' balanced salt solutionMRSAmethicillin-resistant S. aureus2-NBDG2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-d-glucoseFBSfetal bovine serumDAPI4′,6-diamidino-2-phenylindoleVDACvoltage-dependent anion channelMFFmitochondrial fission factorTCAtrichloroacetic acidFCCPcarbonyl cyanide 4-(trifluoromethoxy) phenylhydrazoneTMREtetramethylrhodamine, ethyl esterPMA4-phorbol 12-myristate 13-acetateKOknockoutFDRfalse discovery rateATMatmospheric pressureCVcolumn volumeANOVAanalysis of variance. is a zinc-dependent peptidase that is made by endothelium and many other tissues (1Bernstein K.E. Ong F.S. Blackwell W.L. Shah K.H. Giani J.F. Gonzalez-Villalobos R.A. Shen X.Z. Fuchs S. Touyz R.M. A modern understanding of the traditional and nontraditional biological functions of angiotensin-converting enzyme.Pharmacol. Rev. 2013; 65 (23257181): 1-4610.1124/pr.112.006809Crossref PubMed Scopus (196) Google Scholar). ACE is best known for its effects on blood pressure because it produces the potent vasoconstrictor angiotensin II, and ACE inhibitors are widely used in treating hypertension, heart failure, and other clinical problems (2Mentz R.J. Bakris G.L. Waeber B. McMurray J.J. Gheorghiade M. Ruilope L.M. Maggioni A.P. Swedberg K. Piña I.L. Fiuzat M. O'Connor C.M. Zannad F. Pitt B. The past, present and future of renin-angiotensin aldosterone system inhibition.Int. J. Cardiol. 2013; 167 (23121914): 1677-168710.1016/j.ijcard.2012.10.007Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Less well-recognized is that ACE cleaves many other peptides beside angiotensin II and plays an important function in several other physiologic processes, including immunity, reproduction, and hematopoiesis (3Bernstein K.E. Khan Z. Giani J.F. Cao D.Y. Bernstein E.A. Shen X.Z. Angiotensin-converting enzyme in innate and adaptive immunity.Nat. Rev. Nephrol. 2018; 14 (29578208): 325-33610.1038/nrneph.2018.15Crossref PubMed Scopus (120) Google Scholar). angiotensin-converting enzyme Hanks' balanced salt solution methicillin-resistant S. aureus 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-d-glucose fetal bovine serum 4′,6-diamidino-2-phenylindole voltage-dependent anion channel mitochondrial fission factor trichloroacetic acid carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone tetramethylrhodamine, ethyl ester 4-phorbol 12-myristate 13-acetate knockout false discovery rate atmospheric pressure column volume analysis of variance. In 1975, it was reported that patients with active sarcoidosis have elevated serum ACE levels (4Lieberman J. Elevation of serum angiotensin-converting-enzyme (ACE) level in sarcoidosis.Am. J. Med. 1975; 59 (169692): 365-37210.1016/0002-9343(75)90395-2Abstract Full Text PDF PubMed Scopus (764) Google Scholar). We now know that lesional macrophages and macrophage-derived giant cells in virtually all human granulomatous diseases express abundant ACE, including histoplasmosis, leprosy, miliary tuberculosis, granulomatosis with polyangiitis (Wegener's granulomatosis), and others (5Baudin B. New aspects on angiotensin-converting enzyme: from gene to disease.Clin. Chem. Lab. Med. 2002; 40 (12005216): 256-26510.1515/CCLM.2002.042Crossref PubMed Scopus (121) Google Scholar, 6Brice E.A. Friedlander W. Bateman E.D. Kirsch R.E. Serum angiotensin-converting enzyme activity, concentration, and specific activity in granulomatous interstitial lung disease, tuberculosis, and COPD.Chest. 1995; 107 (7874941): 706-71010.1378/chest.107.3.706Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 7Lieberman J. Rea T.H. Serum angiotensin-converting enzyme in leprosy and coccidioidomycosis.Ann. Intern. Med. 1977; 87 (199098): 423-42510.7326/0003-4819-87-4-422Crossref PubMed Scopus (100) Google Scholar, 8Olle E.W. Deogracias M.P. Messamore J.E. McClintock S.D. Barron A.G. Anderson T.D. Johnson K.J. Screening of serum samples from Wegener's granulomatosis patients using antibody microarrays.Proteomics Clin. Appl. 2007; 1 (21136620): 1212-122010.1002/prca.200600906Crossref PubMed Scopus (9) Google Scholar, 9Weinstock J.V. Production of neuropeptides by inflammatory cells within the granulomas of murine schistosomiasis mansoni.Eur. J. Clin. Invest. 1991; 21 (1905627): 145-15310.1111/j.1365-2362.1991.tb01803.xCrossref PubMed Scopus (33) Google Scholar). Macrophage ACE up-regulation is found in zebra fish granuloma (10Cronan M.R. Beerman R.W. Rosenberg A.F. Saelens J.W. Johnson M.G. Oehlers S.H. Sisk D.M. Jurcic Smith K.L. Medvitz N.A. Miller S.E. Trinh L.A. Fraser S.E. Madden J.F. Turner J. Stout J.E. et al.Macrophage epithelial reprogramming underlies mycobacterial granuloma formation and promotes infection.Immunity. 2016; 45 (27760340): 861-87610.1016/j.immuni.2016.09.014Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Macrophages in Gaucher's disease make abundant ACE, as do the macrophages found in both early and late stages of human atherosclerotic lesions (11Danilov S.M. Tikhomirova V.E. Metzger R. Naperova I.A. Bukina T.M. Goker-Alpan O. Tayebi N. Gayfullin N.M. Schwartz D.E. Samokhodskaya L.M. Kost O.A. Sidransky E. ACE phenotyping in Gaucher disease.Mol. Genet. Metab. 2018; 123 (29478818): 501-51010.1016/j.ymgme.2018.02.007Crossref PubMed Scopus (20) Google Scholar, 12Diet F. Pratt R.E. Berry G.J. Momose N. Gibbons G.H. Dzau V.J. Increased accumulation of tissue ACE in human atherosclerotic coronary artery disease.Circulation. 1996; 94 (8941100): 2756-276710.1161/01.CIR.94.11.2756Crossref PubMed Scopus (399) Google Scholar, 13Ohishi M. Ueda M. Rakugi H. Naruko T. Kojima A. Okamura A. Higaki J. Ogihara T. Enhanced expression of angiotensin-converting enzyme is associated with progression of coronary atherosclerosis in humans.J. Hypertens. 1997; 15 (9383179): 1295-130210.1097/00004872-199715110-00014Crossref PubMed Scopus (98) Google Scholar). ACE increases when the human monocytic cell line THP-1 is differentiated into macrophages (12Diet F. Pratt R.E. Berry G.J. Momose N. Gibbons G.H. Dzau V.J. Increased accumulation of tissue ACE in human atherosclerotic coronary artery disease.Circulation. 1996; 94 (8941100): 2756-276710.1161/01.CIR.94.11.2756Crossref PubMed Scopus (399) Google Scholar). Similarly, when human peripheral blood monocytes are differentiated to macrophages in vitro, cell ACE activity increases 9-fold (14Saijonmaa O. Nyman T. Fyhrquist F. Atorvastatin inhibits angiotensin-converting enzyme induction in differentiating human macrophages.Am. J. Physiol. Heart Circ. Physiol. 2007; 292 (17158648): H1917-H192110.1152/ajpheart.00920.2006Crossref PubMed Scopus (16) Google Scholar). In mice, ACE expression in splenic neutrophils, macrophages, and dendritic cells rapidly increases following Staphylococcus aureus or Listeria monocytogenes infection (15Khan Z. Shen X.Z. Bernstein E.A. Giani J.F. Eriguchi M. Zhao T.V. Gonzalez-Villalobos R.A. Fuchs S. Liu G.Y. Bernstein K.E. Angiotensin-converting enzyme enhances the oxidative response and bactericidal activity of neutrophils.Blood. 2017; 130 (28515091): 328-33910.1182/blood-2016-11-752006Crossref PubMed Scopus (49) Google Scholar, 16Shen X.Z. Billet S. Lin C. Okwan-Duodu D. Chen X. Lukacher A.E. Bernstein K.E. The carboxypeptidase ACE shapes the MHC class I peptide repertoire.Nat. Immunol. 2011; 12 (21964607): 1078-108510.1038/ni.2107Crossref PubMed Scopus (59) Google Scholar). Despite these observations, little is known about the functional role of ACE up-regulation in myeloid cells. Recently, our group investigated two lines of mice in which genetic manipulation was used to selectively increase ACE expression by monocytes and macrophages or by neutrophils (15Khan Z. Shen X.Z. Bernstein E.A. Giani J.F. Eriguchi M. Zhao T.V. Gonzalez-Villalobos R.A. Fuchs S. Liu G.Y. Bernstein K.E. Angiotensin-converting enzyme enhances the oxidative response and bactericidal activity of neutrophils.Blood. 2017; 130 (28515091): 328-33910.1182/blood-2016-11-752006Crossref PubMed Scopus (49) Google Scholar, 17Shen X.Z. Li P. Weiss D. Fuchs S. Xiao H.D. Adams J.A. Williams I.R. Capecchi M.R. Taylor W.R. Bernstein K.E. Mice with enhanced macrophage angiotensin-converting enzyme are resistant to melanoma.Am. J. Pathol. 2007; 170 (17525278): 2122-213410.2353/ajpath.2007.061205Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). In these animals, termed ACE 10/10 and NeuACE mice, the cells overexpressing ACE respond to immune challenge with a consistent enhancement in immune response. For example, ACE 10/10 mice (in which monocytes/macrophages express 16–25-fold WT levels of ACE) resist the growth of B16 melanoma significantly better than WT mice and show increased resistance to infection with Listeria or methicillin-resistant S. aureus (MRSA) (17Shen X.Z. Li P. Weiss D. Fuchs S. Xiao H.D. Adams J.A. Williams I.R. Capecchi M.R. Taylor W.R. Bernstein K.E. Mice with enhanced macrophage angiotensin-converting enzyme are resistant to melanoma.Am. J. Pathol. 2007; 170 (17525278): 2122-213410.2353/ajpath.2007.061205Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 18Okwan-Duodu D. Datta V. Shen X.Z. Goodridge H.S. Bernstein E.A. Fuchs S. Liu G.Y. Bernstein K.E. Angiotensin-converting enzyme overexpression in mouse myelomonocytic cells augments resistance to Listeria and methicillin-resistant Staphylococcus aureus.J. Biol. Chem. 2010; 285 (20937811): 39051-3906010.1074/jbc.M110.163782Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Similarly, NeuACE neutrophils (which express 12–18-fold WT levels of ACE) show increased resistance to MRSA, Klebsiella pneumoniae, or Pseudomonas aeruginosa, as measured both in vivo and in the ability to kill bacteria in vitro (15Khan Z. Shen X.Z. Bernstein E.A. Giani J.F. Eriguchi M. Zhao T.V. Gonzalez-Villalobos R.A. Fuchs S. Liu G.Y. Bernstein K.E. Angiotensin-converting enzyme enhances the oxidative response and bactericidal activity of neutrophils.Blood. 2017; 130 (28515091): 328-33910.1182/blood-2016-11-752006Crossref PubMed Scopus (49) Google Scholar). Both NeuACE neutrophils and ACE 10/10 macrophages make increased levels of NADPH oxidase–dependent superoxide, which contributes to their enhanced killing of bacteria. To investigate the molecular basis of how ACE overexpression affects cells, we studied the metabolism of such cells. Unexpectedly, in both macrophages and neutrophils, ACE overexpression induces a significant increase in cellular ATP, in levels of Krebs cycle intermediates, and in proteins comprising the electron transport chain. These metabolic changes are due to ACE catalytic activity, as they are reverted to WT levels by treating mice with ACE inhibitors. Our studies find that ACE overexpression by both macrophages and neutrophils is associated with a marked change in the metabolism of cells and that this appears to underpin some of the phenotypic differences between these cells and myeloid cells expressing WT levels of ACE. Mass spectrometry was used to measure the intermediate metabolites of ACE 10/10 macrophages and NeuACE neutrophils, as well as equivalent cells from WT mice. Thioglycollate-elicited ACE 10/10 macrophages showed 27 significant differences from WT cells (defined as a false discovery rate–adjusted p value <0.01), with 26 metabolites increased in ACE 10/10 (Fig. S1 and Table S1). Only adenosine was diminished. A similar comparison of freshly isolated NeuACE bone marrow neutrophils with equivalent WT cells showed 15 significant differences, with all being increased in the ACE-overexpressing cells. Perhaps most surprising was the increased amounts of adenosine mono-, di-, and triphosphate in both ACE-overexpressing groups (Fig. 1, A and B). In ACE 10/10 macrophages, the increase in cellular ATP, ADP, and AMP was 3.0-, 2.3-, and 2.6-fold the levels in WT cells. In NeuACE neutrophils, there was an equivalent pattern, although the magnitude of the increase (1.9-, 2.1-, and 1.7-fold) was less than in macrophages. To verify the MS results, we measured levels of cellular ATP chemically in additional samples and found a 2.4- and 1.7-fold increase of ATP in ACE 10/10 macrophages and NeuACE neutrophils (p < 0.001; Fig. 1C). Macrophages from ACE heterozygous mice have roughly 2-fold higher levels of ATP as compared with WT (p < 0.05), whereas, in stark contrast, cells from ACE knockout (null) mice averaged only 28% of the ATP levels found in WT (p < 0.01). For ACE 10/10 macrophages, the ATP/ADP ratio was 2.1 versus 1.4 for WT cells, as determined chemically (Fig. 1D). For NeuACE neutrophils, the ATP/ADP ratio was 3.0 versus 1.9 in WT cells. To examine whether differences in ATP between peritoneal macrophages from ACE 10/10 and WT mice were due to differences in overall cellular protein content, both protein and ATP were measured chemically in another cohort of mice. Whereas ACE 10/10 cells again had significantly more ATP (p < 0.001), the protein content of the cells was equivalent to peritoneal macrophages from WT mice (Fig. S2A). As discussed below, we also evaluated the size of ACE 10/10 and WT macrophages and found no differences (Fig. 2E). Finally, whereas the typical preparation of peritoneal macrophages contained 80–90% macrophages by flow cytometry, ATP was also measured in peritoneal macrophages purified to over 90% using an antibody-based negative cell selection protocol. This too showed more ATP in ACE 10/10 macrophages than equivalent WT cells (see Fig. 7A).Figure 7Decreasing ATP in ACE-overexpressing cells reduces superoxide production and phagocytosis to WT levels. A and B, ACE 10/10 macrophages and WT cells were treated with the ATP synthase inhibitor oligomycin (10 μg/ml) for 20 min. Cellular ATP levels (A) and PMA-induced production of superoxide (B) were measured. C and D, a similar experiment was performed using NeuACE neutrophils and WT cells. For A–D, n ≥ 5 mice, mean ± S.E. (error bars). A and C were studied by one-way ANOVA with the Tukey test. **, p < 0.01; ***, p < 0.001. E and F, peritoneal macrophages from WT, ACE 10/10, and ACE KO mice were cultured with fluorescein-labeled E. coli particles with or without oligomycin (10 μg/ml) for 2 h. Analysis of green fluorescence in CD11b+F4/80+ cells is shown in E, whereas the mean florescence intensity (MFI) is plotted in F. n ≥ 5 mice, mean ± S.E. Analysis was by one-way ANOVA with the Tukey test: *, p < 0.05; **, p < 0.01; NS, no significance.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In the above analysis of ATP, neutrophils were obtained from the bone marrow of mice without any additional stimulation. In contrast, peritoneal macrophages were obtained following stimulation with thioglycollate. When resident peritoneal macrophages were isolated without thioglycollate, ACE 10/10 and WT macrophages showed no difference in ATP levels, as did bone marrow–derived macrophages produced in vitro (Fig. 2, B and C). Thus, macrophage stimulus affects the level of ATP in ACE 10/10 versus WT cells. The increase of cellular ATP is due to the catalytic activity of ACE. Specifically, when animals were continuously treated for 7 days with the ACE inhibitor ramipril, thioglycollate-induced peritoneal macrophages or bone marrow–derived neutrophils showed ATP levels that were now similar to the cells from WT mice also treated with ramipril (Fig. 1, E and F). In contrast, a similar experiment (7-day treatment) performed with the angiotensin II AT1 receptor antagonist losartan showed no significant effects as compared with equivalent animals not treated with the receptor antagonist. WT mice treated with ramipril had less ATP in macrophages and neutrophils than in WT cells from untreated mice, similar to the result in Fig. 1C with ACE knockout (KO) macrophages. Given the differences in intermediate metabolites observed in cells overexpressing ACE, we examined the quantity of cellular glucose transport proteins GLUT1 and GLUT4 by Western blot analysis. No differences were noted between macrophages and neutrophils overexpressing ACE and equivalent WT cells (Fig. 2A and Fig. S3A). Two means of assessing glucose uptake were used: the uptake of the fluorescent glucose analog 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-d-glucose (2-NBDG), as determined by flow cytometry, and an independent chemical analysis of the uptake of 2-deoxyglucose. Neither assay showed increased glucose uptake in ACE-overexpressing cells as compared with equivalent cells with WT ACE expression (Fig. 2B and Fig. S3B). Glycolytic flux ultimately produces lactic acid. We assessed lactic acid levels using two approaches. First, MS determination of cellular lactic acid showed no difference in ACE 10/10 macrophages or NeuACE neutrophils as compared with WT cells (Fig. 2C and Fig. 3C). In addition to this static determination of cellular lactic acid, we also measured the production of lactic acid by cultured macrophages. Thioglycollate-induced ACE 10/10 and WT macrophages were collected and cultured for 12 h in one of three media: medium with 10% fetal bovine serum (FBS), medium without FBS, and medium without FBS and without glucose. We then assayed the lactate concentration in the culture supernatant via the production of NADH by lactate dehydrogenase (Fig. 2D). This showed a small increase of lactate production by ACE 10/10 cells, but these differences were not significant with the number of samples measured. A possible explanation for differences in intermediate metabolites could be due to differences in cell size or mitochondrial number. We investigated this using two assays. First, ACE 10/10 macrophages and NeuACE neutrophils were stained with Mitotracker Green and measured by flow cytometry. This assay of mitochondria showed no significant differences between ACE-overexpressing cells and equivalent cells from mice with WT ACE levels (Fig. S4, A and B). In a more extensive analysis, ACE 10/10 macrophages were studied by confocal microscopy following staining of the nuclei, cytoplasm, and mitochondria with 4′,6-diamidino-2-phenylindole (DAPI), phalloidin green, and Mitotracker Red, respectively (Fig. 2E). Individual images of cells were then quantified for the number of pixels of cytoplasm and of mitochondria. This analysis showed no differences between ACE 10/10 macrophages and WT cells in cell size, mitochondrial number, mitochondrial size, and mitochondrial number per cell size (Fig. S4C). Further, analysis of ACE 10/10 macrophages by EM showed no significant differences in mitochondrial size or cristae structure (Fig. S4D). This conclusion was supported by using Western blotting to measure total cellular dynamin-like 120-kDa protein (OPA1), voltage-dependent anion channel (VDAC), mitochondrial fission factor (MFF), and TOM20 (Fig. 2F). OPA1, VDAC, and MFF were equivalent between ACE 10/10 macrophages and WT cells. Only TOM20 was different; in ACE 10/10 macrophages, it was 1.4-fold the level of WT cells. Thus, it appears that cell size, the number of mitochondria, and mitochondrial size are not major differentiating features between cells overexpressing ACE and equivalent cells with WT ACE levels. As part of the MS analysis of metabolites, we determined the levels of metabolic intermediates found in the TCA cycle. These data showed that ACE 10/10 macrophages have significantly higher levels of citric acid (3.9-fold), isocitrate (5.2-fold), succinate (1.9-fold), and malate (2.0-fold) (Fig. 3 and Table S1). NAD and FAD in ACE 10/10 macrophages were also 1.6- and 1.5-fold higher than in equivalent WT cells. In neutrophils from NeuACE mice, there was a similar trend, although the differences between cells overexpressing ACE and those with WT ACE levels were less pronounced. Specifically, for citrate, isocitrate, succinate, and l-malate, levels were 2.0-, 1.9-, 1.4-, and 1.5-fold higher in NeuACE neutrophils compared with neutrophils from WT mice. The NeuACE neutrophils also had increased NAD (1.3-fold) and FAD (1.4-fold). Given the increased concentration of ATP and TCA cycle intermediates in ACE-overexpressing macrophages, we measured cellular respiration to determine whether there were changes in mitochondrial function. Peritoneal macrophages were obtained after thioglycollate injection and then transferred to culture dishes. Following an overnight incubation, cells were gently detached and acutely adhered to an Agilent Seahorse XF96 plate, and respiratory parameters were measured as described previously (Fig. 4, A and B) (19Divakaruni A.S. Paradyse A. Ferrick D.A. Murphy A.N. Jastroch M. Analysis and interpretation of microplate-based oxygen consumption and pH data.Methods Enzymol. 2014; 547 (25416364): 309-35410.1016/B978-0-12-801415-8.00016-3Crossref PubMed Scopus (264) Google Scholar). The rates of ATP production by glycolysis and oxidative metabolism in ACE 10/10 cells were 31 and 27% higher than WT cells. Total ATP production rates by ACE 10/10 cells were 29% higher than WT (11.2 ± 1.00 pmol of ATP/min/1000 cells (ACE 10/10) versus 8.7 ± 1.37 pmol of ATP/min/1000 cells (WT); p < 0.05, n ≥ 11). To investigate whether the change in oxygen consumption upon ACE overexpression was due to direct mitochondrial changes or simply reflected an increased activation state of the cells, we measured maximal oxygen consumption rates with carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP). After blocking ATP synthase with oligomycin, the addition of FCCP measures maximal respiratory rates when mitochondria are disengaged (uncoupled) from ATP synthesis. Indeed, there was a clear difference in maximal oxygen consumption, which averaged 37% higher in ACE 10/10 cells than in WT macrophages (2.81 ± 0.18 pmol of O2/min/1000 cells (ACE 10/10) versus 2.05 ± 0.24 pmol of O2/min/1000 cells (WT), p < 0.02) (Fig. 4C). To further investigate whether the increase in respiration observed upon ACE overexpression was directly due to mitochondrial changes, we permeabilized the plasma membrane of ACE 10/10 and WT macrophages with recombinant perfringolysin O and offered in situ mitochondria various respiratory substrates (Fig. 4D) (20Divakaruni A.S. Wiley S.E. Rogers G.W. Andreyev A.Y. Petrosyan S. Loviscach M. Wall E.A. Yadava N. Heuck A.P. Ferrick D.A. Henry R.R. McDonald W.G. Colca J.R. Simon M.I. Ciaraldi T.P. Murphy A.N. Thiazolidinediones are acute, specific inhibitors of the mitochondrial pyruvate carrier.Proc. Natl. Acad. Sci. U.S.A. 2013; 110 (23513224): 5422-542710.1073/pnas.1303360110Crossref PubMed Scopus (202) Google Scholar, 21Divakaruni A.S. Rogers G.W. Murphy A.N. Measuring mitochondrial function in permeabilized cells using the Seahorse XF analyzer or a Clark-type oxygen electrode.Curr. Protoc. Toxicol. 2014; 60 (24865646): 25.2.1-25.21610.1002/0471140856.tx2502s60Crossref Scopus (66) Google Scholar). Indeed, ADP-supported respiration was significantly increased in ACE 10/10 in response to pyruvate/malate, glutamate/malate, and succinate/rotenone. Thus, these data indicate a significant increase in respiratory rate in cells overexpressing ACE. The finding of increased cellular ATP, increased concentrations of TCA intermediates, and increased respiration led us to ask whether ACE affects electron transport chain proteins. These were studied using two different approaches. First, analysis of ACE 10/10 and WT macrophages using MS provided information on 28 proteins associated with mitochondria (Fig. 5A and Table S2). Most striking was the increase in proteins from electron transport complex IV (COX2 and COX4) and complex V (ATP5A) found in ACE 10/10. TOM20 was also significantly increased in ACE 10/10 macrophages. As a second means of assessing electron transport chain protein, we used Western blot analysis with six antibodies directed against a protein present in each of the five electron transport chain complexes (NDUFB8, SDHB, UQCRC2, COX1, ATP5A, and ATP5β) of ACE 10/10 macrophages and NeuACE neutrophils (Fig. 5, B and C). An equivalent study used WT cells. In both macrophages and neutrophils overexpressing ACE, there was a significant increase in the proteins from electron transport complex I (NDUFB8) and complex V (ATP5A and ATP5β) as compared with WT data. This is despite the fact that neutrophils predominantly use glycolysis rather than oxidative phosphorylation to generate ATP (22Davies L.C. Rice C.M. McVicar D.W. Weiss J.M. Diversity and environmental adaptation of phagocytic cell metabolism.J. Leukoc. Biol. 2019; 105 (30247792): 37-4810.1002/JLB.4RI0518-195RCrossref PubMed Scopus (32) Google Scholar). ACE 10/10 macrophages also had an increase of COX1 from electron transport complex IV. Thus, these data showing an increase of a subset of electron chain proteins are complementary to the increased respiratory rate found in ACE 10/10 macrophages. Mitochondrial production of ATP is driven by the difference in membrane potential across the inner mitochondrial membrane. This was assessed by flow analysis of macrophages from ACE 10/10, WT, and ACE knockout mice after staining with the mitochondrial dye tetramethylrhodamine, ethyl ester (TMRE) (23Antonenkov V.D. Isomursu A. Mennerich D. Vapola M.H. Weiher H. Kietzmann T. Hiltunen J.K. The human mitochondrial DNA depletion syndrome gene MPV17 encodes a non-selective channel that modulates membrane potential.J. Biol. Chem. 2015; 290 (25861990): 13840-1386110.1074/jbc.M114.608083Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). In this assay, increased membrane potential results in increased fluorescence. We saw that, as compared with WT cells, macrophages from ACE 10/10 mice had a 24% increase of mean fluorescence intensity (increased Δψm), whereas macrophages from ACE knockout mice had a 30% decrease of mean fluorescence intensity (Fig. 5D, p < 0.05). Whereas the NeuACE mice are transgenic, the ACE 10/10 mice were made by substituting the promoter of the endogenous ACE gene. T and B cells from ACE 10/10 do not make increased ACE, but to study whether the genetic manipulation had a generalized effect on all hematopoietic cells, we measured ATP levels and selective electron transport proteins in these cells (Fig. S5, A and B). This showed that ACE 10/10 T and B cells have levels of cellular ATP and mitochondrial proteins equivalent to those of WT cells. Western blotting was also used to assess the level of electron transport proteins in the spleen, heart, lung, liver, kidney, and skeletal muscle of ACE 10 (Fig. 5C). As with lymphocytes, there was no difference between ACE 10/10 and WT mice. ACE is a single polypeptide chain, but it is composed of two homologous catalytic domains termed the N-