Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

Hydrogen sulfide stimulates lipid biogenesis from glutamine that is dependent on the mitochondrial NAD(P)H pool

2021; Elsevier BV; Volume: 297; Issue: 2 Linguagem: Inglês

10.1016/j.jbc.2021.100950

ISSN

1083-351X

Autores

Sebastián Carballal, Victor Vitvitsky, Roshan Kumar, David A. Hanna, Marouane Libiad, Aditi Gupta, Jace W. Jones, Ruma Banerjee,

Tópico(s)

Adipose Tissue and Metabolism

Resumo

Mammalian cells synthesize H2S from sulfur-containing amino acids and are also exposed to exogenous sources of this signaling molecule, notably from gut microbes. As an inhibitor of complex IV in the electron transport chain, H2S can have a profound impact on metabolism, suggesting the hypothesis that metabolic reprogramming is a primary mechanism by which H2S signals. In this study, we report that H2S increases lipogenesis in many cell types, using carbon derived from glutamine rather than from glucose. H2S-stimulated lipid synthesis is sensitive to the mitochondrial NAD(P)H pools and is enabled by reductive carboxylation of α-ketoglutarate. Lipidomics analysis revealed that H2S elicits time-dependent changes across several lipid classes, e.g., upregulating triglycerides while downregulating phosphatidylcholine. Direct analysis of triglyceride concentration revealed that H2S induces a net increase in the size of this lipid pool. These results provide a mechanistic framework for understanding the effects of H2S on increasing lipid droplets in adipocytes and population studies that have pointed to a positive correlation between cysteine (a substrate for H2S synthesis) and fat mass. Mammalian cells synthesize H2S from sulfur-containing amino acids and are also exposed to exogenous sources of this signaling molecule, notably from gut microbes. As an inhibitor of complex IV in the electron transport chain, H2S can have a profound impact on metabolism, suggesting the hypothesis that metabolic reprogramming is a primary mechanism by which H2S signals. In this study, we report that H2S increases lipogenesis in many cell types, using carbon derived from glutamine rather than from glucose. H2S-stimulated lipid synthesis is sensitive to the mitochondrial NAD(P)H pools and is enabled by reductive carboxylation of α-ketoglutarate. Lipidomics analysis revealed that H2S elicits time-dependent changes across several lipid classes, e.g., upregulating triglycerides while downregulating phosphatidylcholine. Direct analysis of triglyceride concentration revealed that H2S induces a net increase in the size of this lipid pool. These results provide a mechanistic framework for understanding the effects of H2S on increasing lipid droplets in adipocytes and population studies that have pointed to a positive correlation between cysteine (a substrate for H2S synthesis) and fat mass. Perturbations in the electron transport chain (ETC), which converts energy captured as reducing equivalents from oxidative metabolism into the currencies of ATP and membrane potential, have widespread effects on metabolism. Endogenous modulators of the ETC are therefore of interest as potential regulators of cellular metabolism, rendering it responsive to intrinsic as well as extrinsic cues. H2S is one such modulator and is derived from metabolism of the sulfur amino acids, cysteine and homocysteine (1Singh S. Banerjee R. PLP-dependent H2S biogenesis.Biochim. Biophys. Acta. 2011; 1814: 1518-1527Crossref PubMed Scopus (123) Google Scholar). Despite a growing literature reporting varied cellular and physiological effects of H2S (2Filipovic M.R. Zivanovic J. Alvarez B. Banerjee R. Chemical biology of H2S signaling through persulfidation.Chem. Rev. 2018; 118: 1253-1337Crossref PubMed Scopus (324) Google Scholar, 3Kabil O. Vitvitsky V. Banerjee R. Sulfur as a signaling nutrient through hydrogen sulfide.Ann. Rev. Nutr. 2014; 34: 171-205Crossref PubMed Scopus (75) Google Scholar), mechanistic insights into how H2S signals are limited (4Kumar R. Banerjee R. Regulation of the redox metabolome and thiol proteome by hydrogen sulfide.Crit. Rev. Biochem. Mol. Biol. 2021; 56: 221-235Crossref PubMed Scopus (6) Google Scholar). Complex IV is a bona fide cellular target of H2S, explaining its long-known toxicity as an environmental poison (5Nicholls P. Kim J.K. Sulphide as an inhibitor and electron donor for the cytochrome c oxidase system.Can. J. Biochem. 1982; 60: 613-623Crossref PubMed Scopus (148) Google Scholar). Steady-state concentrations of H2S are very low in most cell types and tissues (6Furne J. Saeed A. Levitt M.D. Whole tissue hydrogen sulfide concentrations are orders of magnitude lower than presently accepted values.Am. J. Physiol. Regul. Integr. Comp. Physiol. 2008; 295: R1479-R1485Crossref PubMed Scopus (503) Google Scholar, 7Vitvitsky V. Kabil O. Banerjee R. High turnover rates for hydrogen sulfide allow for rapid regulation of its tissue concentrations.Antioxid. Redox Signal. 2012; 17: 22-31Crossref PubMed Scopus (128) Google Scholar) and influenced by the kinetics of its synthesis and oxidation. Three enzymes, cystathionine β-synthase (8Singh S. Padovani D. Leslie R.A. Chiku T. Banerjee R. Relative contributions of cystathionine beta-synthase and gamma-cystathionase to H2S biogenesis via alternative trans-sulfuration reactions.J. Biol. Chem. 2009; 284: 22457-22466Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar), γ-cystathionase (9Chiku T. Padovani D. Zhu W. Singh S. Vitvitsky V. Banerjee R. H2S biogenesis by human cystathionine γ-lyase leads to the novel sulfur metabolites lanthionine and homolanthionine and is responsive to the grade of hyperhomocysteinemia.J. Biol. Chem. 2009; 284: 11601-11612Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar), and mercaptopyruvate sulfur transferase (10Yadav P.K. Vitvitsky V. Carballal S. Seravalli J. Banerjee R. Thioredoxin regulates human mercaptopyruvate sulfurtransferase at physiologically-relevant concentrations.J. Biol. Chem. 2020; 295: 6299-6311Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar) synthesize H2S, whereas enzymes in a mitochondrial resident pathway catalyze its oxidation to thiosulfate and sulfate (11Hildebrandt T.M. Grieshaber M.K. Three enzymatic activities catalyze the oxidation of sulfide to thiosulfate in mammalian and invertebrate mitochondria.FEBS J. 2008; 275: 3352-3361Crossref PubMed Scopus (367) Google Scholar). Cells can also be exposed to exogenous H2S particularly at the host–microbiota interface; gut microbial metabolism is estimated to expose colon epithelial cells to 0.2 to 2.4 mM H2S (12Macfarlane G.T. Gibson G.R. Cummings J.H. Comparison of fermentation reactions in different regions of the human colon.J. Appl. Bacteriol. 1992; 72: 57-64PubMed Google Scholar, 13Deplancke B. Finster K. Graham W.V. Collier C.T. Thurmond J.E. Gaskins H.R. Gastrointestinal and microbial responses to sulfate-supplemented drinking water in mice.Exp. Biol. Med. (Maywood). 2003; 228: 424-433Crossref PubMed Scopus (56) Google Scholar). The reversibility of complex IV inhibition by H2S underlies its potential to modulate metabolism by perturbing mitochondrial bioenergetics (4Kumar R. Banerjee R. Regulation of the redox metabolome and thiol proteome by hydrogen sulfide.Crit. Rev. Biochem. Mol. Biol. 2021; 56: 221-235Crossref PubMed Scopus (6) Google Scholar). Sulfide quinone oxidoreductase (SQOR) catalyzes the first step in the H2S oxidation pathway, forming glutathione persulfide (14Libiad M. Yadav P.K. Vitvitsky V. Martinov M. Banerjee R. Organization of the human mitochondrial sulfide oxidation pathway.J. Biol. Chem. 2014; 289: 30901-30910Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 15Landry A.P. Ballou D.P. Banerjee R. H2S oxidation by nanodisc-embedded human sulfide quinone oxidoreductase.J. Biol. Chem. 2017; 292: 11641-11649Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 16Mishanina T.V. Yadav P.K. Ballou D.P. Banerjee R. Transient kinetic analysis of hydrogen sulfide oxidation catalyzed by human sulfide quinone oxidoreductase.J. Biol. Chem. 2015; 290: 25072-25080Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). The latter is oxidized by ETHE1 to sulfite, releasing GSH (17Kabil O. Motl N. Strack M. Seravalli J. Metzler-Nolte N. Banerjee R. Mechanism-based inhibition of human persulfide dioxygenase by gamma-glutamyl-homocysteinyl-glycine.J. Biol. Chem. 2018; 293: 12429-12439Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar). SQOR is a mitochondrial inner membrane protein that transfers electrons released during H2S oxidation to coenzyme Q and connects to the ETC at the level of complex III (18Landry A.P. Ballou D.P. Banerjee R. Hydrogen sulfide oxidation by sulfide quinone oxidoreductase.Chembiochem. 2021; 22: 949-960Crossref PubMed Scopus (7) Google Scholar). Hence, H2S can both provide electrons to and inhibit the ETC, and SQOR plays a critical role as a respiratory shield, reducing exposure of complex IV to H2S (19Libiad M. Vitvitsky V. Bostelaar T. Bak D.W. Lee H.J. Sakamoto N. Fearon E. Lyssiotis C.A. Weerapana E. Banerjee R. Hydrogen sulfide perturbs mitochondrial bioenergetics and triggers metabolic reprogramming in colon cells.J. Biol. Chem. 2019; 294: 12077-12090Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). As SQOR is the committing enzyme in the sulfide oxidation pathway, regulation of SQOR expression levels and/or activity could be instrumental for transiently building up intracellular H2S levels. SQOR deficiency leads to increased sensitivity to H2S poisoning at a cellular level (19Libiad M. Vitvitsky V. Bostelaar T. Bak D.W. Lee H.J. Sakamoto N. Fearon E. Lyssiotis C.A. Weerapana E. Banerjee R. Hydrogen sulfide perturbs mitochondrial bioenergetics and triggers metabolic reprogramming in colon cells.J. Biol. Chem. 2019; 294: 12077-12090Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) and to Leigh's disease in man (20Friederich M.W. Elias A.F. Kuster A. Laugwitz L. Larson A.A. Landry A.P. Ellwood-Digel L. Mirsky D.M. Dimmock D. Haven J. Jiang H. MacLean K.N. Styren K. Schoof J. Goujon L. et al.Pathogenic variants in SQOR encoding sulfide:quinone oxidoreductase are a potentially treatable cause of Leigh disease.J. Inherit. Metab. Dis. 2020; 43: 1024-1036Crossref PubMed Scopus (14) Google Scholar). Population and animal model studies have pointed to a role for cysteine and H2S in regulating lipid metabolism (21Carter R.N. Morton N.M. Cysteine and hydrogen sulphide in the regulation of metabolism: Insights from genetics and pharmacology.J. Pathol. 2016; 238: 321-332Crossref PubMed Scopus (55) Google Scholar). Plasma total cysteine is positively correlated with obesity, specifically with fat mass (22Elshorbagy A.K. Nurk E. Gjesdal C.G. Tell G.S. Ueland P.M. Nygard O. Tverdal A. Vollset S.E. Refsum H. Homocysteine, cysteine, and body composition in the Hordaland Homocysteine Study: Does cysteine link amino acid and lipid metabolism?.Am. J. Clin. Nutr. 2008; 88: 738-746Crossref PubMed Scopus (106) Google Scholar). Of importance, this correlation is not general to amino acids including the other sulfur amino acids: methionine, homocysteine, and cystathionine (23Aasheim E.T. Elshorbagy A.K. Diep L.M. Sovik T.T. Mala T. Valdivia-Garcia M. Olbers T. Bohmer T. Birkeland K.I. Refsum H. Effect of bariatric surgery on sulphur amino acids and glutamate.Br. J. Nutr. 2011; 106: 432-440Crossref PubMed Scopus (18) Google Scholar). Although the underlying mechanism for this correlation is unknown, it has been speculated that cysteine regulates energy expenditure. Correlations between plasma H2S and adiposity have also been reported (24Jain S.K. Micinski D. Lieblong B.J. Stapleton T. Relationship between hydrogen sulfide levels and HDL-cholesterol, adiponectin, and potassium levels in the blood of healthy subjects.Atherosclerosis. 2012; 225: 242-245Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar) but should be viewed with caution, owing to the technical difficulties with and lack of standardization of H2S measurements (reviewed in (21Carter R.N. Morton N.M. Cysteine and hydrogen sulphide in the regulation of metabolism: Insights from genetics and pharmacology.J. Pathol. 2016; 238: 321-332Crossref PubMed Scopus (55) Google Scholar)). γ-Cystathionase knockout mice exhibit lower plasma total cysteine and reduced body weight and white adipose tissue (25Mani S. Yang G. Wang R. A critical life-supporting role for cystathionine gamma-lyase in the absence of dietary cysteine supply.Free Radic. Biol. Med. 2011; 50: 1280-1287Crossref PubMed Scopus (57) Google Scholar). γ-Cystathionase is the second enzyme in the transsulfuration pathway and generates H2S from cysteine and/or homocysteine (9Chiku T. Padovani D. Zhu W. Singh S. Vitvitsky V. Banerjee R. H2S biogenesis by human cystathionine γ-lyase leads to the novel sulfur metabolites lanthionine and homolanthionine and is responsive to the grade of hyperhomocysteinemia.J. Biol. Chem. 2009; 284: 11601-11612Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar). In a study on differentiated adipocytes, H2S was shown to increase the size and number of lipid droplets and to decrease lipolysis (26Tsai C.Y. Peh M.T. Feng W. Dymock B.W. Moore P.K. Hydrogen sulfide promotes adipogenesis in 3T3L1 cells.PLoS One. 2015; 10e0119511PubMed Google Scholar). The molecular mechanism by which H2S influences lipid metabolism is, however, not known. Oxidative metabolism of glucose and glutamine furnish citrate-derived acetyl-CoA for lipid biogenesis. Studies in our laboratory have demonstrated that H2S affects the metabolism of both glucose and glutamine in a manner that predicts opposite effects of these carbon sources on lipid synthesis. Thus, H2S stimulates aerobic glycolysis and leads to the stoichiometric conversion of glucose to two equivalents of lactate (27Vitvitsky V. Kumar R. Libiad M. Maebius A. Landry A. Banerjee R. The mitochondrial NADH pool is involved in hydrogen sulfide signaling and stimulation of aerobic glycolysis.J. Biol. Chem. 2021; 296: 100736Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar). On the other hand, by inducing a reductive shift in the NAD+/NADH ratio, H2S stimulates reductive carboxylation, i.e., the conversion of glutamine-derived α-ketoglutarate to isocitrate (Fig. 1A) (19Libiad M. Vitvitsky V. Bostelaar T. Bak D.W. Lee H.J. Sakamoto N. Fearon E. Lyssiotis C.A. Weerapana E. Banerjee R. Hydrogen sulfide perturbs mitochondrial bioenergetics and triggers metabolic reprogramming in colon cells.J. Biol. Chem. 2019; 294: 12077-12090Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). These observations suggest the hypothesis that H2S reprograms energy metabolism and supports lipid biosynthesis using the glutamine-derived pathway for acetyl-CoA while simultaneously inhibiting β-oxidation by targeting complex IV. In this study, we report that H2S stimulates lipid synthesis from glutamine but not glucose, and that this response is seen across various malignant and nonmalignant cell lines. Of interest, metabolic flux from glutamine to lipids is sensitive to mitochondrial but not cytoplasmic NAD(P)H and is correlated with this pool affecting sulfide-stimulated oxygen consumption kinetics. Lipidomics analysis reveals that H2S elicits time-dependent changes across various classes of lipids. Collectively, these data reveal the ability of H2S to reprogram energy metabolism and impact lipid homeostasis. We examined the effect of sulfide on lipid biogenesis from [U-14C]-glucose or [U-14C]-glutamine in nonmalignant human colonic epithelial cell (HCEC) and malignant HT29 colorectal carcinoma cells (Fig. 1). We have previously demonstrated that, under cell culture conditions, H2S is lost from the growth medium in ∼30 min (27Vitvitsky V. Kumar R. Libiad M. Maebius A. Landry A. Banerjee R. The mitochondrial NADH pool is involved in hydrogen sulfide signaling and stimulation of aerobic glycolysis.J. Biol. Chem. 2021; 296: 100736Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar). To observe sufficient radiolabel incorporation into the lipid pool, exogenous sulfide (100 μM) was added every 3 h over a period of 12 h and samples were collected at t = 13 h (Fig. 1B). Sulfide resulted in a small (≤18%) but statistically significant increase in radiolabel incorporation from [U-14C]-glucose into lipids in HCEC cells but had no effect in HT29 cells (Fig. 1C). Since H2S stimulates reductive carboxylation in malignant colorectal cancer cells (19Libiad M. Vitvitsky V. Bostelaar T. Bak D.W. Lee H.J. Sakamoto N. Fearon E. Lyssiotis C.A. Weerapana E. Banerjee R. Hydrogen sulfide perturbs mitochondrial bioenergetics and triggers metabolic reprogramming in colon cells.J. Biol. Chem. 2019; 294: 12077-12090Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar), lipids are predicted to be labeled by [U-14C]-glutamine (Fig. 1A). Indeed, sulfide elicited a significant increase in radiolabel incorporation from glutamine into lipids in control HT29scr cells (transfected with a scrambled sequence), which was dependent on the concentration of sulfide added (Fig. 1D). Next, we examined the effect of the sulfide oxidation enzymes SQOR and ETHE1 on the ability of sulfide to stimulate glutamine-dependent lipid synthesis. For this, we used HT29 cells in which SQOR or ETHE1 was knocked down using shRNA as previously described (19Libiad M. Vitvitsky V. Bostelaar T. Bak D.W. Lee H.J. Sakamoto N. Fearon E. Lyssiotis C.A. Weerapana E. Banerjee R. Hydrogen sulfide perturbs mitochondrial bioenergetics and triggers metabolic reprogramming in colon cells.J. Biol. Chem. 2019; 294: 12077-12090Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 27Vitvitsky V. Kumar R. Libiad M. Maebius A. Landry A. Banerjee R. The mitochondrial NADH pool is involved in hydrogen sulfide signaling and stimulation of aerobic glycolysis.J. Biol. Chem. 2021; 296: 100736Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar). In comparison with HT29scr control, the sulfide effect was significantly higher in SQOR knockdown cells (Fig. 1E). Basal radiolabel incorporation into lipids was significantly higher in the ETHE1 knockdown cells, whereas the magnitude of sulfide-induced stimulation was comparable with the HT29scr controls. To assess whether sulfide-stimulated labeling of lipids by glutamine is a general metabolic response, seven other cell lines were examined under normoxic (20% O2) versus hypoxic (2% O2) conditions (Fig. 1, F and G). With the exception of HepG2 and J774 cells, hypoxic conditions tended to increase radiolabeling of lipids compared with cells grown under normoxic conditions (Fig. 1F), which is consistent with the reported stimulation of reductive activation by hypoxia (28Wise D.R. Ward P.S. Shay J.E. Cross J.R. Gruber J.J. Sachdeva U.M. Platt J.M. DeMatteo R.G. Simon M.C. Thompson C.B. Hypoxia promotes isocitrate dehydrogenase-dependent carboxylation of alpha-ketoglutarate to citrate to support cell growth and viability.Proc. Natl. Acad. Sci. U. S. A. 2011; 108: 19611-19616Crossref PubMed Scopus (646) Google Scholar). Although the human hepatocellular carcinoma cells HepG2 were unresponsive to hypoxia, a decrease in lipid labeling was observed in J774 murine macrophage cells. Like HT29scr cells, sulfide increased lipid labeling in HCEC, HCT116, J774, 143Bwt, and 143BCytB cells (Fig. 1G). In contrast, sulfide decreased radiolabel incorporation in HepG2 cells. The wild-type osteosarcoma cybrid 143BWT exhibited the highest (10- to 15-fold) sulfide-stimulated increase in radiolabel incorporation into lipids, whereas the 143BCytB cybrids lacking an intact ETC exhibited only a small (10%), but statistically significant, increase. These results suggest an interplay between hypoxia and sulfide-based metabolic reprogramming, which leads to increased glutamine incorporation into lipids via activation of reductive carboxylation (Fig. 1A). Targeted dissipation of the cytoplasmic and mitochondrial NADH pools can be achieved by ectopic expression of the water-forming NADH oxidase, LbNOX and mito-LbNOX (29Titov D.V. Cracan V. Goodman R.P. Peng J. Grabarek Z. Mootha V.K. Complementation of mitochondrial electron transport chain by manipulation of the NAD+/NADH ratio.Science. 2016; 352: 231-235Crossref PubMed Scopus (154) Google Scholar), respectively. Sulfide stimulated similar levels of lipid labeling from glutamine in HT29 cells expressing the empty vector or cytoplasmic LbNOX (Fig. 2A). In contrast, expression of mito-LbNOX significantly decreased sulfide-stimulated radiolabeling of lipids by glutamine. Next, we examined the effect of dissipating the cytoplasmic versus mitochondrial NADPH pool by ectopic expression of the water-forming NADPH oxidase (TPNOX, Fig. S1) (30Cracan V. Titov D.V. Shen H. Grabarek Z. Mootha V.K. A genetically encoded tool for manipulation of NADP(+)/NADPH in living cells.Nat. Chem. Biol. 2017; 13: 1088-1095Crossref PubMed Scopus (39) Google Scholar). In contrast to expression of the empty vector or the cytoplasmic TPNOX, expression of mito-TPNOX significantly decreased sulfide activation of radiolabel incorporation from [U-14C]-glutamine to lipids (Fig. 2B). To understand how the mitochondrial expression of LbNOX affects sulfide metabolism, we examined the kinetics of oxygen consumption. At a relatively low concentration of sulfide (10 μM), HT29 cells expressing the empty vector, the cytoplasmic or mitochondrial form of LbNOX, elicited similar responses, i.e., an increase in the oxygen consumption rate (OCR), which returned to basal levels within ∼2 min (Fig. 3, A–C). At higher concentrations (≥20 μM), differences in the cellular response to sulfide were observed. In response to 20 μM sulfide (Fig. 3, D–F), cells expressing the empty vector or cytoplasmic LbNOX showed similar responses with an increase in OCR following the first injection but signs of inhibition after the second. In contrast, mito-LbNOX-expressing cells were more resistant to inhibition. At 30 μM sulfide, the differences were even more pronounced (Fig. 3, G–I). These data demonstrate that the mitochondrial NADH pool modulates sulfide-dependent OCR and are consistent with enhanced H2S clearance by cells expressing mitochondrial versus cytoplasmic LbNOX (27Vitvitsky V. Kumar R. Libiad M. Maebius A. Landry A. Banerjee R. The mitochondrial NADH pool is involved in hydrogen sulfide signaling and stimulation of aerobic glycolysis.J. Biol. Chem. 2021; 296: 100736Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar). The sulfide-stimulated glutamate-dependent lipid labeling was ∼2-fold at 2% versus 3- to 4-fold at 20% oxygen in HT29scr cells (Fig. 1G). Since colonocytes are exposed to an atmosphere with low oxygen tension, lipidomics analysis was conducted on HT29scr cells cultured in an atmosphere of 2% oxygen. Multivariate analysis revealed significant perturbations in the distribution of lipids in HT29scr cells 1 h after exposure to sulfide (Fig. 4A), which returned to control values after 3 h. Exposure to additional 100 μM doses of sulfide at 3 h intervals resulted in distinct patterns of changes (Fig. 4, A and B). After 1 h, lower levels of ceramides, sphingomyelin, and phosphatidylcholines were observed (Fig. 4C and Table S1). Hexosylceramides, phosphatidylethanolamine, and triglycerides on the other hand, showed a mixed response, with some species being up- and others, downregulated. At 13 h, the time point at which metabolic labeling studies were conducted (Fig. 1), ceramides (70%), hexosylceramides (75%), and triglycerides (140%) were the major lipid groups that were present at higher levels compared with untreated controls, whereas phosphatidylcholine (30%) was downregulated (Fig. 4C). At 13 h, we observed a 70% overall increase in the levels of those lipids that were differentially expressed between H2S treated versus untreated cells. Glutamine-derived acetyl-CoA can support fatty acid and cholesterol synthesis (Fig. 5A). Since cholesterol is not picked up in our lipidomics analysis, we examined whether inhibition of cholesterol synthesis by fluvastatin, a 3-hydroxy-3-methyl-glutaryl CoA reductase inhibitor (31Warita K. Warita T. Beckwitt C.H. Schurdak M.E. Vazquez A. Wells A. Oltvai Z.N. Statin-induced mevalonate pathway inhibition attenuates the growth of mesenchymal-like cancer cells that lack functional E-cadherin mediated cell cohesion.Sci. Rep. 2014; 4: 7593Crossref PubMed Scopus (77) Google Scholar), decreases radiolabel incorporation from glutamine. However, no change in lipid labeling was observed in the presence of fluvastatin (Fig. 5B), whereas the fatty acid synthase inhibitor cerulenin (32Pizer E.S. Wood F.D. Heine H.S. Romantsev F.E. Pasternack G.R. Kuhajda F.P. Inhibition of fatty acid synthesis delays disease progression in a xenograft model of ovarian cancer.Cancer Res. 1996; 56: 1189-1193PubMed Google Scholar) and the acetyl-CoA carboxylase inhibitors, TOFA (33Wang C. Xu C. Sun M. Luo D. Liao D.F. Cao D. Acetyl-CoA carboxylase-alpha inhibitor TOFA induces human cancer cell apoptosis.Biochem. Biophys. Res. Commun. 2009; 385: 302-306Crossref PubMed Scopus (75) Google Scholar) and ND-646 (34Svensson R.U. Parker S.J. Eichner L.J. Kolar M.J. Wallace M. Brun S.N. Lombardo P.S. Van Nostrand J.L. Hutchins A. Vera L. Gerken L. Greenwood J. Bhat S. Harriman G. Westlin W.F. et al.Inhibition of acetyl-CoA carboxylase suppresses fatty acid synthesis and tumor growth of non-small-cell lung cancer in preclinical models.Nat. Med. 2016; 22: 1108-1119Crossref PubMed Scopus (217) Google Scholar), inhibited sulfide-activated lipid labeling as expected (Fig. 5C). To assess whether the increase in metabolic labeling from glutamine to lipids was accompanied by a net increase in lipid biogenesis, we measured triglycerides levels directly, owing to the relative ease with which this lipid group can be detected. Sulfide treatment resulted in a 30 ± 23% (n = 9, p < 0.003) increase in the triglyceride pool in HT29scr cells, which was attenuated by pharmacological inhibition of fatty acid synthase with cerulenin (Fig. 5D). In comparison with the hundreds of protein targets of persulfidation that have been identified and ascribed to H2S signaling (35Gao X.H. Krokowski D. Guan B.J. Bederman I. Majumder M. Parisien M. Diatchenko L. Kabil O. Willard B. Banerjee R. Wang B. Bebek G. Evans C.R. Fox P.L. Gerson S.L. et al.Quantitative H2S-mediated protein sulfhydration reveals metabolic reprogramming during the integrated stress response.Elife. 2015; 4e10067Crossref PubMed Scopus (100) Google Scholar, 36Doka E. Pader I. Biro A. Johansson K. Cheng Q. Ballago K. Prigge J.R. Pastor-Flores D. Dick T.P. Schmidt E.E. Arner E.S. Nagy P. A novel persulfide detection method reveals protein persulfide- and polysulfide-reducing functions of thioredoxin and glutathione systems.Sci. Adv. 2016; 2e1500968Crossref PubMed Scopus (153) Google Scholar), little is known about how H2S influences metabolism (4Kumar R. Banerjee R. Regulation of the redox metabolome and thiol proteome by hydrogen sulfide.Crit. Rev. Biochem. Mol. Biol. 2021; 56: 221-235Crossref PubMed Scopus (6) Google Scholar). Intriguing connections between H2S and lipid synthesis have been described in the literature ranging from the H2S-induced increase in lipid accumulation in Nanochloropsis oceanica for microalgal biodiesel production (37Cheng J. Wang Z. Lu H. Yang W. Fan Z. Hydrogen sulfide improves lipid accumulation in nannochloropsis oceanica through metabolic regulation of carbon allocation and energy supply.ACS Sustainable Chem. Eng. 2020; 8: 2481-2489Crossref Scopus (4) Google Scholar) to cysteine/H2S being pro-obesogenic (reviewed in (21Carter R.N. Morton N.M. Cysteine and hydrogen sulphide in the regulation of metabolism: Insights from genetics and pharmacology.J. Pathol. 2016; 238: 321-332Crossref PubMed Scopus (55) Google Scholar)). In this study, we have elucidated the carbon source used for lipid synthesis by cultured cells treated with H2S and demonstrate that it is linked to the inhibitory effect of H2S on the ETC. The consequent pleiotropic effects on cell metabolism appear to prioritize glycolysis for ATP synthesis as described previously (27Vitvitsky V. Kumar R. Libiad M. Maebius A. Landry A. Banerjee R. The mitochondrial NADH pool is involved in hydrogen sulfide signaling and stimulation of aerobic glycolysis.J. Biol. Chem. 2021; 296: 100736Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar) and shifts operation of the tricarboxylic acid cycle (TCA) cycle in the reductive direction for redox recycling and macromolecular synthesis (19Libiad M. Vitvitsky V. Bostelaar T. Bak D.W. Lee H.J. Sakamoto N. Fearon E. Lyssiotis C.A. Weerapana E. Banerjee R. Hydrogen sulfide perturbs mitochondrial bioenergetics and triggers metabolic reprogramming in colon cells.J. Biol. Chem. 2019; 294: 12077-12090Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). We have recently demonstrated that H2S stimulates aerobic glycolysis and enhances lactate production (Fig. 6) (27Vitvitsky V. Kumar R. Libiad M. Maebius A. Landry A. Banerjee R. The mitochondrial NADH pool is involved in hydrogen sulfide signaling and stimulation of aerobic glycolysis.J. Biol. Chem. 2021; 296: 100736Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar). Under these conditions, glycolysis presumably functions primarily to support cellular ATP needs, with a redox neutral cycle being established between NAD+ reduction by glyceraldehyde 3-phosphate dehydrogenase and NADH oxidation by lactate dehydrogenase. By inhibiting the ETC, H2S decreases the NAD+/NADH ratio (19Libiad M. Vitvitsky V. Bostelaar T. Bak D.W. Lee H.J. Sakamoto N. Fearon E. Lyssiotis C.A. Weerapana E. Banerjee R. Hydrogen sulfide perturbs mitochondrial bioenergetics and triggers metabolic reprogramming in colon cells.J. Biol. Chem. 2019; 294: 12077-12090Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar), disfavoring the oxidative TCA cycle. The anabolic needs of the cell are presumably met by glutamine metabolism under these conditions. Consistent with this model, reductive carboxylation is stimulated by H2S as evidenced by

Referência(s)