BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) has been associated with the host dysmetabolism of branched-chain amino acids (BCAAs), however, the implications for the role of BCAA metabolism in PDAC development or progression are not clear. The mitochondrial catabolism of valine, leucine, and isoleucine is a multistep process leading to the production of short-chain R-CoA species. They can be subsequently exported from mitochondria as short-chain carnitines (SC-CARs), utilized in anabolic pathways, or released from the cells. METHODS: We examined the specificities of BCAA catabolism and cellular adaptation strategies to BCAA starvation in PDAC cells in vitro. We used metabolomics and lipidomics to quantify major metabolic changes in response to BCAA withdrawal. Using confocal microscopy and flow cytometry we quantified the fluorescence of BODIPY probe and the level of lipid droplets (LDs). We used BODIPY-conjugated palmitate to evaluate transport of fatty acids (FAs) into mitochondria. Also, we have developed a protocol for quantification of SC-CARs, BCAA-derived metabolites. RESULTS: Using metabolic profiling, we found that BCAA starvation leads to massive triglyceride (TG) synthesis and LD accumulation. This was associated with the suppression of activated FA transport into the mitochondrial matrix. The suppression of FA import into mitochondria was rescued with the inhibitor of the acetyl-CoA carboxylase (ACC) and the activator of AMP kinase (AMPK), which both regulate carnitine palmitoyltransferase 1A (CPT1) activation status. CONCLUSIONS: Our data suggest that BCAA catabolism is required for the import of long chain carnitines (LC-CARs) into mitochondria, whereas the disruption of this link results in the redirection of activated FAs into TG synthesis and its deposition into LDs. We propose that this mechanism protects cells against mitochondrial overload with LC-CARs and it might be part of the universal reaction to amino acid perturbations during cancer growth, regulating FA handling and storage.

1st Faculty of Medicine Charles University Prague Czech Republic

3rd Faculty of Medicine Franco Czech Laboratory for Clinical Research on Obesity Prague Czech Republic

Department of Internal Medicine Královské Vinohrady University Hospital and 3rd Faculty of Medicine Prague Czech Republic

Department of Pathophysiology Center for Research on Nutrition Metabolism and Diabetes 3rd Faculty of Medicine Charles University Prague Czech Republic

Institute of Physiology of the Czech Academy of Sciences Laboratory of Mitochondrial Physiology Vídeňská 1083 142 20 Prague 4 Krč Czech Republic

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Rahib L, Smith BD, Aizenberg R, Rosenzweig AB, Fleshman JM, Matrisian LM. Projecting cancer incidence and deaths to 2030: the unexpected burden of thyroid, liver, and pancreas cancers in the United States. Cancer Res. 2014;74(11):2913–21. doi: 10.1158/0008-5472.CAN-14-0155. PubMed DOI

Ilic I, Ilic M. International patterns in incidence and mortality trends of pancreatic cancer in the last three decades: a joinpoint regression analysis. World J Gastroenterol. 2022;28(32):4698–715. doi: 10.3748/wjg.v28.i32.4698. PubMed DOI PMC

Green CR, Wallace M, Divakaruni AS, Phillips SA, Murphy AN, Ciaraldi TP, Metallo CM. Branched-chain amino acid catabolism fuels adipocyte differentiation and lipogenesis. Nat Chem Biol. 2016;12(1):15–21. doi: 10.1038/nchembio.1961. PubMed DOI PMC

Wallace M, Green CR, Roberts LS, Lee YM, McCarville JL, Sanchez-Gurmaches J, Meurs N, Gengatharan JM, Hover JD, Phillips SA, et al. Enzyme promiscuity drives branched-chain fatty acid synthesis in adipose tissues. Nat Chem Biol. 2018;14:1021–1031. doi: 10.1038/s41589-018-0132-2. PubMed DOI PMC

Avruch J, Long X, Ortiz-Vega S, Rapley J, Papageorgiou A, Dai N. Amino acid regulation of TOR complex 1. Am J Physiol Endocrinol Metab. 2009;296(4):E592–602. doi: 10.1152/ajpendo.90645.2008. PubMed DOI PMC

Chen J, Ou Y, Luo R, Wang J, Wang D, Guan J, Li Y, Xia P, Chen PR, Liu Y. SAR1B senses leucine levels to regulate mTORC1 signalling. Nature. 2021;596(7871):281–4. doi: 10.1038/s41586-021-03768-w. PubMed DOI

Violante S, Ijlst L, Ruiter J, Koster J, van Lenthe H, Duran M, de Almeida IT, Wanders RJA, Houten SM, Ventura FV. Substrate specificity of human carnitine acetyltransferase: implications for fatty acid and branched-chain amino acid metabolism. Biochim et Biophys Acta (BBA) - Mol Basis Disease. 2013;1832(6):773–9. doi: 10.1016/j.bbadis.2013.02.012. PubMed DOI

Carrer A, Trefely S, Zhao S, Campbell SL, Norgard RJ, Schultz KC, Sidoli S, Parris JLD, Affronti HC, Sivanand S, et al. Acetyl-CoA metabolism supports multistep pancreatic tumorigenesis. Cancer Discov. 2019;9(3):416–35. doi: 10.1158/2159-8290.CD-18-0567. PubMed DOI PMC

Li JT, Yin M, Wang D, Wang J, Lei MZ, Zhang Y, Liu Y, Zhang L, Zou SW, Hu LP, et al. BCAT2-mediated BCAA catabolism is critical for development of pancreatic ductal adenocarcinoma. Nat Cell Biology 2020. 2020;22(2):167–74. doi: 10.1038/s41556-019-0455-6. PubMed DOI

Zhu Z, Achreja A, Meurs N, Animasahun O, Owen S, Mittal A, Parikh P, Lo T-W, Franco-Barraza J, Shi J, et al. Nature Metabolism 2020 2:8. Nature Publishing Group; 2020. Tumour-reprogrammed stromal BCAT1 fuels branched-chain ketoacid dependency in stromal-rich PDAC tumours; pp. 775–92. PubMed PMC

Lei MZ, Li XX, Zhang Y, Li JT, Zhang F, Wang YP, Yin M, Qu J, Lei QY. Acetylation promotes BCAT2 degradation to suppress BCAA catabolism and pancreatic cancer growth. Signal Transduct Target Therapy. 2020;5(1 2020):1–9. PubMed PMC

Wang K, Zhang Z, Tsai H-i, Liu Y, Gao J, Wang M, Song L, Cao X, Xu Z, Chen H, et al. Branched-chain amino acid aminotransferase 2 regulates ferroptotic cell death in cancer cells. Cell Death Differ 2020. 2020;28(4):1222–36. doi: 10.1038/s41418-020-00644-4. PubMed DOI PMC

Li JT, Li KY, Su Y, Shen Y, Lei MZ, Zhang F, Yin M, Chen ZJ, Wen WY, Hu WG, et al. Diet high in branched-chain amino acid promotes PDAC development by USP1-mediated BCAT2 stabilization. Natl Sci Rev. 2022;9(5):nwab212. doi: 10.1093/nsr/nwab212. PubMed DOI PMC

Smolková K, Špačková J, Gotvaldová K, Dvořák A, Křenková A, Hubálek M, Holendová B, Vítek L, Ježek P. SIRT3 and GCN5L regulation of NADP+- and NADPH-driven reactions of mitochondrial isocitrate dehydrogenase IDH2. Sci Rep. 2020;10(1):8677. doi: 10.1038/s41598-020-65351-z. PubMed DOI PMC

Špačková J, Gotvaldová K, Dvořák A, Urbančoková A, Pospíšilová K, Větvička D, Leguina-Ruzzi A, Tesařová P, Vítek L, Ježek P, et al. Biochemical Background in Mitochondria Affects 2HG Production by IDH2 and ADHFE1 in Breast Carcinoma. Cancers (Basel) 2021;13(7):1709. doi: 10.3390/cancers13071709. PubMed DOI PMC

Cajka T, Smilowitz JT, Fiehn O. Validating quantitative untargeted Lipidomics Across Nine Liquid Chromatography-High-Resolution Mass Spectrometry platforms. Anal Chem. 2017;89(22):12360–8. doi: 10.1021/acs.analchem.7b03404. PubMed DOI

Janovska P, Melenovsky V, Svobodova M, Havlenova T, Kratochvilova H, Haluzik M, Hoskova E, Pelikanova T, Kautzner J, Monzo L, et al. Dysregulation of epicardial adipose tissue in cachexia due to heart failure: the role of natriuretic peptides and cardiolipin. J Cachexia Sarcopenia Muscle. 2020;11(6):1614–27. doi: 10.1002/jcsm.12631. PubMed DOI PMC

Repetto G, del Peso A, Zurita JL. Neutral red uptake assay for the estimation of cell viability/cytotoxicity. Nat Protoc. 2008;3(7):1125–31. doi: 10.1038/nprot.2008.75. PubMed DOI

Smolkova K, Dvorak A, Zelenka J, Vitek L, Jezek P. Reductive carboxylation and 2-hydroxyglutarate formation by wild-type IDH2 in breast carcinoma cells. Int J Biochem Cell Biol. 2015;65:125–33. doi: 10.1016/j.biocel.2015.05.012. PubMed DOI

Giacomoni F, Le Corguillé G, Monsoor M, Landi M, Pericard P, Pétéra M, Duperier C, Tremblay-Franco M, Martin J-F, Jacob D, et al. Workflow4Metabolomics: a collaborative research infrastructure for computational metabolomics. Bioinformatics. 2014;31(9):1493–5. doi: 10.1093/bioinformatics/btu813. PubMed DOI PMC

Lu Y, Pang Z, Xia J. Comprehensive investigation of pathway enrichment methods for functional interpretation of LC–MS global metabolomics data. Brief Bioinform. 2022;24(1):bbac553. doi: 10.1093/bib/bbac553. PubMed DOI PMC

Smolkova K, Bellance N, Scandurra F, Genot E, Gnaiger E, Plecita-Hlavata L, Jezek P, Rossignol R. Mitochondrial bioenergetic adaptations of breast cancer cells to aglycemia and hypoxia. J Bioenerg Biomembr. 2010;42(1):55–67. doi: 10.1007/s10863-009-9267-x. PubMed DOI

Lee JH, Cho YR, Kim JH, Kim J, Nam HY, Kim SW, Son J. Branched-chain amino acids sustain pancreatic cancer growth by regulating lipid metabolism. Exp Mol Med. 2019;51(11):1–11. PubMed PMC

Singh R, Kaushik S, Wang Y, Xiang Y, Novak I, Komatsu M, Tanaka K, Cuervo AM, Czaja MJ. Autophagy regulates lipid metabolism. Nature. 2009;458(7242):1131–5. doi: 10.1038/nature07976. PubMed DOI PMC

Nguyen TB, Louie SM, Daniele JR, Tran Q, Dillin A, Zoncu R, Nomura DK, Olzmann JA. DGAT1-Dependent lipid Droplet Biogenesis protects mitochondrial function during Starvation-Induced Autophagy. Dev Cell. 2017;42(1):9–e2125. doi: 10.1016/j.devcel.2017.06.003. PubMed DOI PMC

Rambold AS, Cohen S, Lippincott-Schwartz J. Fatty acid trafficking in starved cells: regulation by lipid droplet lipolysis, autophagy, and mitochondrial fusion dynamics. Dev Cell. 2015;32(6):678–92. doi: 10.1016/j.devcel.2015.01.029. PubMed DOI PMC

Jusović M, Starič P, Jarc Jovičić E, Petan T. The Combined Inhibition of Autophagy and Diacylglycerol Acyltransferase-Mediated Lipid Droplet Biogenesis Induces Cancer Cell Death during Acute Amino Acid Starvation. Cancers (Basel) 2023;15(19):4857. PubMed PMC

Wang H, Sreenivasan U, Hu H, Saladino A, Polster BM, Lund LM, Gong D-W, Stanley WC, Sztalryd C. Perilipin 5, a lipid droplet-associated protein, provides physical and metabolic linkage to mitochondria. J Lipid Res. 2011;52(12):2159–68. doi: 10.1194/jlr.M017939. PubMed DOI PMC

Kien B, Kolleritsch S, Kunowska N, Heier C, Chalhoub G, Tilp A, Wolinski H, Stelzl U, Haemmerle G. Lipid droplet-mitochondria coupling via perilipin 5 augments respiratory capacity but is dispensable for FA oxidation. J Lipid Res. 2022;63(3):100172. doi: 10.1016/j.jlr.2022.100172. PubMed DOI PMC

Parker CG, Kuttruff CA, Galmozzi A, Jørgensen L, Yeh CH, Hermanson DJ, Wang Y, Artola M, McKerrall SJ, Josyln CM, et al. Chemical Proteomics identifies SLC25A20 as a functional target of the Ingenol Class of Actinic Keratosis drugs. ACS Cent Sci. 2017;3(12):1276–85. doi: 10.1021/acscentsci.7b00420. PubMed DOI PMC

Lee J, Homma T, Kurahashi T, Kang ES, Fujii J. Oxidative stress triggers lipid droplet accumulation in primary cultured hepatocytes by activating fatty acid synthesis. Biochem Biophys Res Commun. 2015;464(1):229–35. doi: 10.1016/j.bbrc.2015.06.121. PubMed DOI

Shibata M, Yoshimura K, Furuya N, Koike M, Ueno T, Komatsu M, Arai H, Tanaka K, Kominami E, Uchiyama Y. The MAP1-LC3 conjugation system is involved in lipid droplet formation. Biochem Biophys Res Commun. 2009;382(2):419–23. doi: 10.1016/j.bbrc.2009.03.039. PubMed DOI

Ren Y, Shen H-M. Critical role of AMPK in redox regulation under glucose starvation. Redox Biol. 2019;25:101154. doi: 10.1016/j.redox.2019.101154. PubMed DOI PMC

Ruderman N, Prentki M. AMP kinase and malonyl-CoA: targets for therapy of the metabolic syndrome. Nat Rev Drug Discovery. 2004;3(4):340–51. doi: 10.1038/nrd1344. PubMed DOI

Herms A, Bosch M, Reddy BJN, Schieber NL, Fajardo A, Rupérez C, Fernández-Vidal A, Ferguson C, Rentero C, Tebar F, et al. AMPK activation promotes lipid droplet dispersion on detyrosinated microtubules to increase mitochondrial fatty acid oxidation. Nat Commun. 2015;6(1):7176. doi: 10.1038/ncomms8176. PubMed DOI PMC

Tong X, Stein R. Lipid droplets protect human β-Cells from Lipotoxicity-Induced stress and cell identity changes. Diabetes. 2021;70(11):2595–607. doi: 10.2337/db21-0261. PubMed DOI PMC

Jarc E, Kump A, Malavašič P, Eichmann TO, Zimmermann R, Petan T. Lipid droplets induced by secreted phospholipase A2 and unsaturated fatty acids protect breast cancer cells from nutrient and lipotoxic stress. Biochim et Biophys Acta (BBA) - Mol Cell Biology Lipids. 2018;1863(3):247–65. PubMed

Petan T, Jarc E, Jusović M. Lipid droplets in Cancer: guardians of Fat in a Stressful World. Molecules. 2018;23(8):1941. doi: 10.3390/molecules23081941. PubMed DOI PMC

Rossmeislová L, Gojda J, Smolková K. Pancreatic cancer: branched-chain amino acids as putative key metabolic regulators? Cancer Metastasis Rev. 2021;40(4):1115–39. doi: 10.1007/s10555-021-10016-0. PubMed DOI

Mayers JR, Torrence ME, Danai LV, Papagiannakopoulos T, Davidson SM, Bauer MR, Lau AN, Ji BW, Dixit PD, Hosios AM, et al. Tissue of origin dictates branched-chain amino acid metabolism in mutant Kras-driven cancers. Sci (New York NY) 2016;353(6304):1161–5. doi: 10.1126/science.aaf5171. PubMed DOI PMC

McGarrah RW, Zhang GF, Christopher BA, Deleye Y, Walejko JM, Page S, Ilkayeva O, White PJ, Newgard CB. Dietary branched-chain amino acid restriction alters fuel selection and reduces triglyceride stores in hearts of Zucker fatty rats. Am J Physiol Endocrinol Metab. 2020;318(2):E216–23. doi: 10.1152/ajpendo.00334.2019. PubMed DOI PMC

Seo JW, Choi J, Lee SY, Sung S, Yoo HJ, Kang MJ, Cheong H, Son J. Autophagy is required for PDAC glutamine metabolism. Sci Rep. 2016;6(1):37594. doi: 10.1038/srep37594. PubMed DOI PMC

Meng Q, Xu J, Liang C, Liu J, Hua J, Zhang Y, Ni Q, Shi S, Yu X. GPx1 is involved in the induction of protective autophagy in pancreatic cancer cells in response to glucose deprivation. Cell Death Dis. 2018;9(12):1187. doi: 10.1038/s41419-018-1244-z. PubMed DOI PMC

Zhang H, Wang Y, Guan L, Chen Y, Chen P, Sun J, Gonzalez FJ, Huang M, Bi H. Lipidomics reveals carnitine palmitoyltransferase 1 C protects cancer cells from lipotoxicity and senescence. J Pharm Anal. 2020;11(3):340–50. doi: 10.1016/j.jpha.2020.04.004. PubMed DOI PMC

Wilcock DJ, Badrock AP, Wong CW, Owen R, Guerin M, Southam AD, Johnston H, Telfer BA, Fullwood P, Watson J, et al. Oxidative stress from DGAT1 oncoprotein inhibition in melanoma suppresses tumor growth when ROS defenses are also breached. Cell Rep. 2022;39(12):110995. doi: 10.1016/j.celrep.2022.110995. PubMed DOI PMC

Cheng X, Geng F, Pan M, Wu X, Zhong Y, Wang C, Tian Z, Cheng C, Zhang R, Puduvalli V, et al. Targeting DGAT1 ameliorates glioblastoma by Increasing Fat Catabolism and oxidative stress. Cell Metab. 2020;32(2):229–e242228. doi: 10.1016/j.cmet.2020.06.002. PubMed DOI PMC

Noland RC, Koves TR, Seiler SE, Lum H, Lust RM, Ilkayeva O, Stevens RD, Hegardt FG, Muoio DM. Carnitine insufficiency caused by aging and overnutrition compromises mitochondrial performance and metabolic control. J Biol Chem. 2009;284(34):22840–52. doi: 10.1074/jbc.M109.032888. PubMed DOI PMC

Long M, Sanchez-Martinez A, Longo M, Suomi F, Stenlund H, Johansson AI, Ehsan H, Salo VT, Montava‐Garriga L, Naddafi S, et al. DGAT1 activity synchronises with mitophagy to protect cells from metabolic rewiring by iron depletion. EMBO J. 2022;41(10):e109390. doi: 10.15252/embj.2021109390. PubMed DOI PMC

Wang S, Song T, Wang S. Mitochondrial DNA 10158T > C mutation in a patient with mitochondrial encephalomyopathy with lactic acidosis, and stroke-like episodes syndrome: A case-report and literature review (CARE-complaint) Medicine. 2020;99(24):e20310. doi: 10.1097/MD.0000000000020310. PubMed DOI PMC

Bose SK, Kim H, Meyer K, Wolins N, Davidson NO, Ray R. Forkhead Box Transcription Factor Regulation and Lipid Accumulation by Hepatitis C Virus. J Virol. 2014;88(8):4195–203. doi: 10.1128/JVI.03327-13. PubMed DOI PMC

Bocca C, Kane MS, Veyrat-Durebex C, Nzoughet JK, Chao de la Barca JM, Chupin S, Alban J, Procaccio V, Bonneau D, Simard G, et al. Lipidomics reveals Triacylglycerol Accumulation due to impaired fatty acid flux in Opa1-Disrupted fibroblasts. J Proteome Res. 2019;18(7):2779–90. doi: 10.1021/acs.jproteome.9b00081. PubMed DOI

Mitochondria to plasma membrane redox signaling is essential for fatty acid β-oxidation-driven insulin secretion