Amino acid deprivation induces TXNIP expression by NRF2 downregulation

Amino acid deprivation induces TXNIP expression by NRF2 downregulation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Amino acid deprivation induces TXNIP expression by NRF2 downregulation Se Hee Ahn, Se-Kyeong Jang, Yu Jin Kim, Gyeongmi Kim, Ki Soo Park, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2915490/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 06 Dec, 2023 Read the published version in IUBMB Life → Version 1 posted You are reading this latest preprint version Abstract TXNIP is an essential regulator of cellular metabolism, including glucose homeostasis, fatty acid synthesis, cholesterol accumulation, and is implicated in metabolic diseases such as obesity, type 2 diabetes, and atherosclerosis. However, how TXNIP expression is regulated in response to amino acid (AA) deprivation is not well understood. In the present study, deprivation of AAs, especially arginine, glutamine, lysine, and methionine, induced TXNIP expression in H460 non-small cell lung cancer cells. Unexpectedly, TXNIP induction by AA deprivation was dependent on NRF2 downregulation, but not ATF4 activation. Furthermore, N-acetyl-L-cysteine, a scavenger of reactive oxygen species (ROS), prevented TXNIP expression in H460 cells deprived of AA. Taken together, TXNIP expression by AA deprivation is mediated by ROS production by NRF2 downregulation. Our findings suggest that TXNIP expression might be associated with the redox homeostasis of AA metabolism. Amino acid ATF4 NRF2 Reactive oxygen species TXNIP Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Thioredoxin-interacting protein (TXNIP, also known as thioredoxin binding protein 2, TBP2 or vitamin D3 upregulated protein 1, VDUP1) is a ubiquitously expressed protein described as a negative regulator of thioredoxin expression and activity, which thereby affects the redox balance 1 . Several studies have revealed the upregulation of TXNIP in diabetes, cardiovascular diseases, and neurodegenerative disease 2–4 . Conversely, other studies have shown that TXNIP expression is decreased in tumors, and that it may act as a tumor suppressor in various cancers 5–7 . TXNIP has emerged as an essential metabolic regulator of lipid and glucose metabolism. In animal models, overexpression of TXNIP has been shown to be associated with metabolic abnormalities, including apoptosis of pancreatic β-cells, reduced insulin sensitivity, and decreased energy expenditure 8–11 . In contrast, TXNIP-deficient animals had normal insulin sensitivity and did not develop diabetes or other metabolic abnormalities 11–13 . TXNIP expression is highly glucose responsive as a result of a carbohydrate response element in the promoter of TXNIP 14,15 . In the presence of increased glucose levels, the transcriptional regulator MondoA accumulates in the nucleus and occupies the promoter of TXNIP to activate TXNIP transcription 16,17 . Glucose-induced TXNIP expression negatively feeds back to the cellular glucose uptake system 12,18 . As such, TXNIP is known to play a critical role in glucose homeostasis, but the regulation of TXNIP expression by amino acids (AAs) is not well understood. In this study, we investigated the regulation of TXNIP expression in response to AA deprivation in H460 non-small cell lung cancer cells. We found that deprivation of AAs, especially arginine, glutamine, lysine, and methionine, strongly induced the expression of TXNIP. Surprisingly, TXNIP expression under AA deprivation was induced by NRF2 downregulation independent of ATF4 activation. A scavenger of reactive oxygen species (ROS), N-acetyl-L-cysteine, blocked TXNIP expression in H460 cells deprived of AA. Taken together, TXNIP expression by AA deprivation is mediated by ROS production by NRF2 downregulation. Our findings suggest that TXNIP expression might be associated with redox homeostasis in AA metabolic processes. Results TXNIP expression under individual amino acid (AA) - deficient conditions First, we investigated the effects of individual AA deprivation on TXNIP expression. H460 non-small cell lung cancer (NSCLC) cells were deprived of each of the 20 AAs for 6 or 12 h, and TXNIP mRNA expression was detected by RT‒PCR. Interestingly, TXNIP mRNA expression was greatly induced in cells deprived of arginine, glutamine, lysine, or methionine (Fig. 1 ). In cells deprived of asparagine, cystine, histidine, hydroxyproline, isoleucine, phenylalanine, proline, threonine, tryptophan, tyrosine, or valine, TXNIP mRNA expression was also slightly induced (Fig. 1 ). There was no change in TXNIP expression in cells deprived of aspartic acid, glutamic acid, glycine, leucine, or serine for 6 or 12 h (Fig. 1 ). These data suggest that there are differences in TXNIP expression in response to individual AA deprivation. AA replenishment reverses the TXNIP expression induced by AA deprivation Next, we investigated TXNIP expression by replenishing AAs in an AA-deprived cell medium. H460 cells were starved of arginine, glutamine, lysine, or methionine for 8 h and then supplemented with each AA. As shown in Fig. 2 , induction of TXNIP in H460 cells deprived of arginine, glutamine, lysine, or methionine was blocked when the medium was replenished with each AA. These data suggest that replenishing AAs reverses the induction of TXNIP in AA-deprived H460 NSCLC cells. TXNIP induction by AA deprivation is independent of ATF4 ATF4 is a critical transcription factor for AA metabolism and is activated by AA deprivation 19 . To investigate whether TXNIP is a downstream gene of ATF4, we examined the expression of TXNIP when ATF4 was overexpressed or knocked down in H460 cells. NUPR1, a target gene of ATF4, was increased in ATF4-overexpressing cells and decreased in ATF4-knockdown cells (Fig. 3 A and 3 B). However, TXNIP expression levels were not affected by increasing or decreasing ATF4 (Fig. 3 A and 3 B). Furthermore, ATF4 knockdown did not affect the TXNIP expression induced by arginine, glutamine, lysine, or methionine deprivation (Fig. 3 C). These data suggest that AA deprivation-induced TXNIP expression is not regulated by ATF4. TXNIP expression by AA deprivation is mediated by reactive oxygen species (ROS) induced by NRF2 downregulation It has been reported that NRF2 negatively regulates TXNIP transcription by binding to the antioxidant response element of the TXNIP promoter 20 . We examined TXNIP expression after NRF2 siRNA treatment. As shown in Fig. 4 A, NRF2 knockdown by siRNA suppressed the expression of NRF2 and the downstream gene GCLC. On the other hand, a significant induction of TXNIP expression at both the mRNA and protein levels was observed upon NRF2 knockdown. Interestingly, the expression levels of NRF2 protein were decreased in H460 cells deprived of arginine, glutamine, lysine, or methionine for 12 h (Fig. 4 B). AA deprivation has been reported to lead to elevated levels of intracellular ROS 21 . To investigate whether ROS induced by AA deprivation is involved in TXNIP expression, we examined TXNIP expression in H460 cells deprived of arginine, glutamine, lysine, or methionine with the ROS scavenger N-acetyl-L-cysteine (NAC). As expected, NAC prevented the elevated TXNIP mRNA expression in cells deprived of each of these AAs (Fig. 4 C). Since the downregulation of the NRF2 pathway resulted in dramatic accumulation of intracellular ROS 22 , we investigated whether TXNIP expression is regulated by NRF2 downregulation-mediated ROS. We treated NAC in H460 cells with NRF2 siRNA. NAC prevented the increased TXNIP mRNA expression in NRF2 knockdown cells (Fig. 4 D). These data suggest that TXNIP expression by AA deprivation is mediated by ROS production by NRF2 downregulation. Discussion TXNIP has attracted considerable attention due to its several aspects of energy metabolism 23 . Overexpression of TXNIP influences metabolic abnormalities such as apoptosis of pancreatic β-cells, reduced insulin sensitivity, and decreased energy expenditure 8–11 . In contrast, genetic ablation of TXNIP promotes normal insulin sensitivity and prevents diabetes or other metabolic abnormalities 11–13 . As such, TXNIP is known to play a critical role in energy homeostasis, but the regulation of TXNIP expression by amino acids (AAs) is not well understood. In this study, we evaluated the effects of single AA deprivation on TXNIP transcription in H460 non-small cell lung cancer (NSCLC) cells. We found that deprivation of glutamine, arginine methionine, or lysine significantly induced the expression of TXNIP mRNA. In addition, deprivation of other AAs slightly increased or did not affect the expression of TXNIP mRNA. These results suggest that TXNIP expression might be different according to the deprivation of each AA. ATF4, which is activated by AA deficiency, has been reported as an important transcription factor for AA metabolism 19 . However, ATF4 knockdown did not affect TXNIP expression by deprivation of arginine, glutamine, lysine, or methionine, suggesting that TXNIP expression by AA deprivation is not regulated by ATF4. Interestingly, we found reduced expression levels of NRF2 protein in H460 cells deprived of arginine, glutamine, lysine, or methionine for 12 h. NRF2 has been reported to negatively regulate TXNIP transcription by binding to the antioxidant response element of the TXNIP promoter 20 . We also observed significant induction of TXNIP in H460 cells with NRF2 knockdown. NRF2 is a key regulator of reactive oxygen species (ROS), and knockdown of NRF2 has been reported to dramatically increase endogenous levels of ROS 22 . The ROS scavenger N-acetyl-L-cysteine, reversed the elevated TXNIP mRNA expression in H460 cells deprived of arginine, glutamine, lysine, or methionine. NAC also reversed the increased TXNIP mRNA expression in NRF2 knockdown H460 cells. These data suggest that TXNIP expression by AA deprivation is mediated by ROS production by NRF2 downregulation. Our findings suggest that TXNIP expression might be associated with redox homeostasis in AA metabolic processes. Further studies are needed to uncover the underlying mechanisms of TXNIP-mediated AA metabolism. Materials and Methods Cell culture and reagents H460 human non-small cell lung cancer cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and were maintained in RPMI 1640 medium (#LM011-01, Welgene, Gyeongsangbuk-do, Republic of Korea) containing 10% fetal bovine serum (#16000-044, Gibco; Thermo Fisher Scientific, Waltham, MA, USA) at 37°C and 5% CO 2 . N-Acetyl-L-cysteine (NAC) was purchased from Sigma-Aldrich (#A7250, Merck KGaA, Darmstadt, Germany). Amino acid (AA) deprivation Individual AA deprivation media were prepared by supplementing the appropriate AA stock solutions (Welgene) for the remaining 19 AAs in AA-free RPMI medium (custom prepared by Welgene) and were supplemented with 10% dialyzed fetal bovine serum (10,000 MW cutoff, #26400-044, Gibco; Thermo Fisher Scientific). Transient transfection Plasmids encoding mouse wild-type and dominant-negative mutant ATF4 (pEF-mATF4 WT-myc and pEF-mATF4ΔRK-myc) were kindly provided by Dr. Jawed Alam 24 . The dominant-negative mutant mATF4 contained a six AA substitution in the DNA binding domain of ATF4 ( 292 RYRQKKR 298 to 292 GYLEAAA 298 ). ATF4 (#sc-35112), NRF2 (sc-37030) and control (#sc-37007) siRNAs were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Transfection with plasmids and siRNAs was performed using Lipofectamine LTX with Plus Reagent (#15338) or Lipofectamine RNAiMAX (#13778), respectively, according to each manufacturer’s instructions (Invitrogen: Thermo Fisher Scientific). RNA extraction and reverse transcription-polymerase chain reaction (RT‒PCR) analysis Total RNA extraction and RT‒PCR analysis were performed as described previously 25 . The following primers were used for PCR: TXNIP (5’-CCTCTGGGAACATCCTTCAA-3’ and 5’-ATTGGCAAGGTAAGTGTGGC-3’; 348-bp product) 26 , ATF4 (5’- AGTCGGGTTTGGGGGCTGAAG-3’ and 5’-TGGGGAAAGGGGAAGAGGTTGTAA-3’; 437-bp product) 27 , NUPR1 (5’-GAGACGGGACTGCGGAGGAAG-3’ and 5’-GTTGCTGCCACCCTGGAGGA-3’, 242-bp product) 28 , GCLC (5’- CCAGTTCCTGCACATCTACC-3’ and 5’- CATGTAACTCCCATACTCTGG-3’ 220-bp product) β-Actin (ACTB) (5’- GGATTCCTATGTGGGCGACAG-3’ and 5’-CGCTCGGTGAGGA TCTTCATG-3’; 438-bp product) 29 . Where indicated, RT‒PCR images were quantified using ImageJ software (version 1.52a; NIH; National Institutes of Health, Bethesda, MD, USA). Western blot analysis Western blotting was performed as described previously 25 . The following antibodies were used: anti-ATF4 antibody (#sc-200)obtained from Santa Cruz Biotechnology; anti-TXNIP (#14715) obtained from Cell Signaling Technology (Beverly, MA, USA); anti-NRF2 (#16396-1-AP) obtained from Proteintech Group (Chicago, IL, USA); and anti-β-actin (#A5316) antibody obtained from Sigma‒Aldrich (Merck KGaA). Declarations Acknowledgments This research was supported by grants from the Korea Institute of Radiological and Medical Sciences (KIRAMS), funded by the Ministry of Science and ICT (MSIT) (50531-2023; 50544-2023) and from the National Research Foundation of Korea (NRF), funded by the MIST (NRF-2022R1F1A1063450; NRF-2023R1A2C1003833), Republic of Korea. Author contributions I.C.P. and H.O.J. developed the concept and designed the study. S.H.A., S.K.J., Y.J.K., G.K., and H.O.J. carried out the experiments. K.S.P. gave technical support and conceptual advice. I.C.P. and H.O.J. wrote the manuscript. All authors have read and agreed to the published version of the manuscript. Data availability statements All data generated or analysed during this study are included in this published article. Competing interests The authors declare no competing interests. References Nishiyama, A. et al. Identification of thioredoxin-binding protein-2/vitamin D(3) up-regulated protein 1 as a negative regulator of thioredoxin function and expression. J Biol Chem 274 , 21645-21650, doi:10.1074/jbc.274.31.21645 (1999). Hu, J. & Yu, Y. The Function of Thioredoxin-Binding Protein-2 (TBP-2) in Different Diseases. Oxid Med Cell Longev 2018 , 4582130, doi:10.1155/2018/4582130 (2018). Tsubaki, H., Tooyama, I. & Walker, D. G. Thioredoxin-Interacting Protein (TXNIP) with Focus on Brain and Neurodegenerative Diseases. Int J Mol Sci 21 , doi:10.3390/ijms21249357 (2020). Masutani, H. Thioredoxin-Interacting Protein in Cancer and Diabetes. Antioxid Redox Signal 36 , 1001-1022, doi:10.1089/ars.2021.0038 (2022). Chen, Y. et al. Research Progress of TXNIP as a Tumor Suppressor Gene Participating in the Metabolic Reprogramming and Oxidative Stress of Cancer Cells in Various Cancers. Front Oncol 10 , 568574, doi:10.3389/fonc.2020.568574 (2020). Xie, M. et al. Thioredoxin interacting protein (TXNIP) acts as a tumor suppressor in human prostate cancer. Cell Biol Int 44 , 2094-2106, doi:10.1002/cbin.11418 (2020). Cadenas, C. et al. Role of thioredoxin reductase 1 and thioredoxin interacting protein in prognosis of breast cancer. Breast Cancer Res 12 , R44, doi:10.1186/bcr2599 (2010). Blouet, C., Liu, S. M., Jo, Y. H., Chua, S. & Schwartz, G. J. TXNIP in Agrp neurons regulates adiposity, energy expenditure, and central leptin sensitivity. J Neurosci 32 , 9870-9877, doi:10.1523/JNEUROSCI.0353-12.2012 (2012). Chen, J., Saxena, G., Mungrue, I. N., Lusis, A. J. & Shalev, A. Thioredoxin-interacting protein: a critical link between glucose toxicity and beta-cell apoptosis. Diabetes 57 , 938-944, doi:10.2337/db07-0715 (2008). Jing, G. et al. Thioredoxin-interacting protein promotes islet amyloid polypeptide expression through miR-124a and FoxA2. J Biol Chem 289 , 11807-11815, doi:10.1074/jbc.M113.525022 (2014). Jo, S. H., Kim, M. Y., Park, J. M., Kim, T. H. & Ahn, Y. H. Txnip contributes to impaired glucose tolerance by upregulating the expression of genes involved in hepatic gluconeogenesis in mice. Diabetologia 56 , 2723-2732, doi:10.1007/s00125-013-3050-6 (2013). Parikh, H. et al. TXNIP regulates peripheral glucose metabolism in humans. PLoS Med 4 , e158, doi:10.1371/journal.pmed.0040158 (2007). Chutkow, W. A. et al. Deletion of the alpha-arrestin protein Txnip in mice promotes adiposity and adipogenesis while preserving insulin sensitivity. Diabetes 59 , 1424-1434, doi:10.2337/db09-1212 (2010). Minn, A. H., Hafele, C. & Shalev, A. Thioredoxin-interacting protein is stimulated by glucose through a carbohydrate response element and induces beta-cell apoptosis. Endocrinology 146 , 2397-2405, doi:10.1210/en.2004-1378 (2005). Cha-Molstad, H., Saxena, G., Chen, J. & Shalev, A. Glucose-stimulated expression of Txnip is mediated by carbohydrate response element-binding protein, p300, and histone H4 acetylation in pancreatic beta cells. J Biol Chem 284 , 16898-16905, doi:10.1074/jbc.M109.010504 (2009). Stoltzman, C. A. et al. Glucose sensing by MondoA:Mlx complexes: a role for hexokinases and direct regulation of thioredoxin-interacting protein expression. Proc Natl Acad Sci U S A 105 , 6912-6917, doi:10.1073/pnas.0712199105 (2008). Kaadige, M. R., Looper, R. E., Kamalanaadhan, S. & Ayer, D. E. Glutamine-dependent anapleurosis dictates glucose uptake and cell growth by regulating MondoA transcriptional activity. Proc Natl Acad Sci U S A 106 , 14878-14883, doi:10.1073/pnas.0901221106 (2009). Chutkow, W. A., Patwari, P., Yoshioka, J. & Lee, R. T. Thioredoxin-interacting protein (Txnip) is a critical regulator of hepatic glucose production. J Biol Chem 283 , 2397-2406, doi:10.1074/jbc.M708169200 (2008). Harding, H. P. et al. An integrated stress response regulates amino acid metabolism and resistance to oxidative stress. Mol Cell 11 , 619-633, doi:10.1016/s1097-2765(03)00105-9 (2003). He, X. & Ma, Q. Redox regulation by nuclear factor erythroid 2-related factor 2: gatekeeping for the basal and diabetes-induced expression of thioredoxin-interacting protein. Mol Pharmacol 82 , 887-897, doi:10.1124/mol.112.081133 (2012). Liao, J. et al. Regulation of stem-like cancer cells by glutamine through beta-catenin pathway mediated by redox signaling. Mol Cancer 16 , 51, doi:10.1186/s12943-017-0623-x (2017). Singh, A. et al. RNAi-mediated silencing of nuclear factor erythroid-2-related factor 2 gene expression in non-small cell lung cancer inhibits tumor growth and increases efficacy of chemotherapy. Cancer Res 68 , 7975-7984, doi:10.1158/0008-5472.CAN-08-1401 (2008). Alhawiti, N. M., Al Mahri, S., Aziz, M. A., Malik, S. S. & Mohammad, S. TXNIP in Metabolic Regulation: Physiological Role and Therapeutic Outlook. Curr Drug Targets 18 , 1095-1103, doi:10.2174/1389450118666170130145514 (2017). He, C. H. et al. Identification of activating transcription factor 4 (ATF4) as an Nrf2-interacting protein. Implication for heme oxygenase-1 gene regulation. J Biol Chem 276 , 20858-20865, doi:10.1074/jbc.M101198200 (2001). Jin, H. O. et al. Induction of HSP27 and HSP70 by constitutive overexpression of Redd1 confers resistance of lung cancer cells to ionizing radiation. Oncol Rep 41 , 3119-3126, doi:10.3892/or.2019.7036 (2019). Jin, H. O. et al. TXNIP potentiates Redd1-induced mTOR suppression through stabilization of Redd1. Oncogene 30 , 3792-3801, doi:10.1038/onc.2011.102 (2011). Carracedo, A. et al. The stress-regulated protein p8 mediates cannabinoid-induced apoptosis of tumor cells. Cancer Cell 9 , 301-312, doi:10.1016/j.ccr.2006.03.005 (2006). Jin, H. O. et al. Nuclear protein 1 induced by ATF4 in response to various stressors acts as a positive regulator on the transcriptional activation of ATF4. IUBMB Life 61 , 1153-1158, doi:10.1002/iub.271 (2009). Jin, H. O. et al. Redd1 inhibits the invasiveness of non-small cell lung cancer cells. Biochem Biophys Res Commun 407 , 507-511, doi:10.1016/j.bbrc.2011.03.047 (2011). Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2915490","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":200563862,"identity":"f1ea29f9-6ebb-445e-bccb-f83dff70afd8","order_by":0,"name":"Se Hee Ahn","email":"","orcid":"","institution":"Korea Institute of Radiological \u0026 Medical Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Se","middleName":"Hee","lastName":"Ahn","suffix":""},{"id":200563863,"identity":"e3103c95-fd6e-4893-972c-3237d25f382c","order_by":1,"name":"Se-Kyeong Jang","email":"","orcid":"","institution":"Korea Institute of Radiological \u0026 Medical Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Se-Kyeong","middleName":"","lastName":"Jang","suffix":""},{"id":200563864,"identity":"f063c5c1-2b52-4ef8-97dc-4464afa50c1b","order_by":2,"name":"Yu Jin Kim","email":"","orcid":"","institution":"Korea Institute of Radiological \u0026 Medical Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yu","middleName":"Jin","lastName":"Kim","suffix":""},{"id":200563865,"identity":"1bccb729-06d9-496b-9393-b1258676cf0c","order_by":3,"name":"Gyeongmi Kim","email":"","orcid":"","institution":"Korea Institute of Radiological \u0026 Medical Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Gyeongmi","middleName":"","lastName":"Kim","suffix":""},{"id":200563866,"identity":"af3fdc82-4c0e-459f-9d98-8b3d036539a9","order_by":4,"name":"Ki Soo Park","email":"","orcid":"","institution":"Konkuk University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ki","middleName":"Soo","lastName":"Park","suffix":""},{"id":200563867,"identity":"7d3aa28f-4d1c-4a73-a331-97aa5e301681","order_by":5,"name":"Hyeon-Ok Jin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4UlEQVRIie3PsQrCMBCA4YPAuSh1DAT0FSIBpSj4KpVAXRJ07CgI9RVaBJ9FKehScC04ieAsDi52UBEUQVLdBPMvIcd9hADYbD8Yvx9ejZLHDD8j4nvSi56zAtKicrEbBn4/nlS2xyyHgQPoH0zEjXwpolTpKSkJpkNw4xEmkYnwTDVZJQz0jCAwPQLO56Wx+S/Z4HQj/RpBclY58G4xUXglymMEkSm8vgKYmEm6F6Kc+o14jNjWIeU0QWkmK7ndlQNZp+sl2ai8w51JKIwEqt7LlQKQ94vPnHnRhs1ms/19F4P1PQ6LvjKjAAAAAElFTkSuQmCC","orcid":"","institution":"Korea Institute of Radiological and Medical Sciences","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Hyeon-Ok","middleName":"","lastName":"Jin","suffix":""},{"id":200563868,"identity":"3ad144a8-deea-47d3-9d9d-98bb52450a2f","order_by":6,"name":"In-Chul Park","email":"","orcid":"","institution":"Korea Institute of Radiological and Medical Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"In-Chul","middleName":"","lastName":"Park","suffix":""}],"badges":[],"createdAt":"2023-05-10 09:59:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2915490/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2915490/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1002/iub.2792","type":"published","date":"2023-12-06T11:09:50+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":37161453,"identity":"136e1ee9-5ce6-4e9e-ad5e-adfd996957e3","added_by":"auto","created_at":"2023-05-17 21:09:44","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":194111,"visible":true,"origin":"","legend":"\u003cp\u003eTXNIP expression in individual\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003eamino acid (AA)\u003c/em\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003cem\u003edeficient conditions\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eH460 cells were deprived of each of the 20 AAs in the medium for 6 or 12 h. The indicated mRNA expression was measured by RT‒PCR analysis. Data are representative of three independent experiments. Arg: arginine; Asn: asparagine; Asp: aspartic acid; Cys: cystine; Gln: glutamine: Glu: glutamic acid; Gly: glycine; His: histidine; Hyp: hydroxyproline; Ile: isoleucine; Leu: leucine; Lys: lysine; Met: methionine; Phe: phenylalanine; Pro: proline; Ser: serine; Thr: threonine; Trp: tryptophan; Tyr: tyrosine; Val: valine.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-2915490/v1/320f808c28dd0f88e2b33c58.png"},{"id":37161454,"identity":"48207186-efa4-493a-afc1-57fc4208a28e","added_by":"auto","created_at":"2023-05-17 21:09:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":118883,"visible":true,"origin":"","legend":"\u003cp\u003eAmino acid (AA) replenishment reverses the TXNIP expression induced by AA deprivation\u003c/p\u003e\n\u003cp\u003e(A) H460 cells deprived of arginine, glutamine, lysine, or methionine for 8 h were supplemented with the indicated concentrations of AAs for 3 h. (B) H460 cells deprived of arginine, glutamine, lysine, or methionine for 8 h were supplemented with 200 mg/L arginine, 300 mg/L glutamine, 40 mg/L lysine, or 15 mg/mL methionine for the indicated times. The indicated mRNA expression was measured by RT‒PCR analysis. Data are representative of three independent experiments. Arg: arginine; Gln: glutamine; Lys: lysine; Met: methionine.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-2915490/v1/a04166d7f9714601f640b38d.png"},{"id":37161184,"identity":"af0533d1-8de6-4b34-865f-c5728ede2299","added_by":"auto","created_at":"2023-05-17 21:01:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":120289,"visible":true,"origin":"","legend":"\u003cp\u003eAmino acid (AA) deprivation induces TXNIP expression independently of the ATF4 pathway\u003c/p\u003e\n\u003cp\u003e(A) H460 cells were transfected with control vector, mATF4 WT, or mATF4ΔRK for 36 h. (B) H460 cells were transfected with control or ATF4 siRNA for 36 h. (C) H460 cells transfected with control or ATF4 siRNA for 24 h were deprived of the indicated AA for 8 h. The protein and mRNA levels were measured by western blot and RT‒PCR analysis, respectively. Data are representative of three independent experiments. Arg: arginine; Gln: glutamine; Lys: lysine; Met: methionine; CTL: control.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-2915490/v1/b50ae6dee1476f5c7401281a.png"},{"id":37161182,"identity":"ae2805b1-eb53-45f7-8985-a9883fc27f17","added_by":"auto","created_at":"2023-05-17 21:01:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":129432,"visible":true,"origin":"","legend":"\u003cp\u003eTXNIP expression by amino acid (AA) deprivation is mediated by reactive oxygen species induced by NRF2 downregulation\u003c/p\u003e\n\u003cp\u003e(A) H460 cells were transfected with control or NRF2 siRNA for 36 h. (B) H460 cells were deprived of the indicated AA for 12 h. (C) H460 cells were incubated in complete medium or medium lacking the indicated AA in combination with 10 mM NAC for 12 h. (D) H460 cells transfected with control or NRF2 siRNA for 24 h were treated with the indicated concentrations of NAC for 12 h. The protein and mRNA levels were measured by western blot and RT‒PCR analysis, respectively. Data are representative of three independent experiments. Arg: arginine; CM: complete medium; CTL: control; Gln: glutamine; Lys: lysine; Met: methionine; NAC: N-Acetyl-L-cysteine.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-2915490/v1/26d81858f3da1ec9fca9d9f6.png"},{"id":52986403,"identity":"e0c5ecab-c2cb-44c8-9fb8-3d80a59a88ea","added_by":"auto","created_at":"2024-03-19 11:09:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":683754,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2915490/v1/d465ec14-80fb-4fe2-b61f-6e1ad9b13228.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Amino acid deprivation induces TXNIP expression by NRF2 downregulation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThioredoxin-interacting protein (TXNIP, also known as thioredoxin binding protein 2, TBP2 or vitamin D3 upregulated protein 1, VDUP1) is a ubiquitously expressed protein described as a negative regulator of thioredoxin expression and activity, which thereby affects the redox balance\u003csup\u003e1\u003c/sup\u003e. Several studies have revealed the upregulation of TXNIP in diabetes, cardiovascular diseases, and neurodegenerative disease \u003csup\u003e2\u0026ndash;4\u003c/sup\u003e. Conversely, other studies have shown that TXNIP expression is decreased in tumors, and that it may act as a tumor suppressor in various cancers\u003csup\u003e5\u0026ndash;7\u003c/sup\u003e. TXNIP has emerged as an essential metabolic regulator of lipid and glucose metabolism. In animal models, overexpression of TXNIP has been shown to be associated with metabolic abnormalities, including apoptosis of pancreatic β-cells, reduced insulin sensitivity, and decreased energy expenditure\u003csup\u003e8\u0026ndash;11\u003c/sup\u003e. In contrast, TXNIP-deficient animals had normal insulin sensitivity and did not develop diabetes or other metabolic abnormalities\u003csup\u003e11\u0026ndash;13\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTXNIP expression is highly glucose responsive as a result of a carbohydrate response element in the promoter of TXNIP\u003csup\u003e14,15\u003c/sup\u003e. In the presence of increased glucose levels, the transcriptional regulator MondoA accumulates in the nucleus and occupies the promoter of TXNIP to activate TXNIP transcription\u003csup\u003e16,17\u003c/sup\u003e. Glucose-induced TXNIP expression negatively feeds back to the cellular glucose uptake system\u003csup\u003e12,18\u003c/sup\u003e. As such, TXNIP is known to play a critical role in glucose homeostasis, but the regulation of TXNIP expression by amino acids (AAs) is not well understood.\u003c/p\u003e \u003cp\u003eIn this study, we investigated the regulation of TXNIP expression in response to AA deprivation in H460 non-small cell lung cancer cells. We found that deprivation of AAs, especially arginine, glutamine, lysine, and methionine, strongly induced the expression of TXNIP. Surprisingly, TXNIP expression under AA deprivation was induced by NRF2 downregulation independent of ATF4 activation. A scavenger of reactive oxygen species (ROS), N-acetyl-L-cysteine, blocked TXNIP expression in H460 cells deprived of AA. Taken together, TXNIP expression by AA deprivation is mediated by ROS production by NRF2 downregulation. Our findings suggest that TXNIP expression might be associated with redox homeostasis in AA metabolic processes.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eTXNIP expression under individual amino acid (AA)\u003cb\u003e-\u003c/b\u003edeficient conditions\u003c/p\u003e \u003cp\u003eFirst, we investigated the effects of individual AA deprivation on TXNIP expression. H460 non-small cell lung cancer (NSCLC) cells were deprived of each of the 20 AAs for 6 or 12 h, and TXNIP mRNA expression was detected by RT‒PCR. Interestingly, TXNIP mRNA expression was greatly induced in cells deprived of arginine, glutamine, lysine, or methionine (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In cells deprived of asparagine, cystine, histidine, hydroxyproline, isoleucine, phenylalanine, proline, threonine, tryptophan, tyrosine, or valine, TXNIP mRNA expression was also slightly induced (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). There was no change in TXNIP expression in cells deprived of aspartic acid, glutamic acid, glycine, leucine, or serine for 6 or 12 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These data suggest that there are differences in TXNIP expression in response to individual AA deprivation.\u003c/p\u003e \u003cp\u003eAA replenishment reverses the TXNIP expression induced by AA deprivation\u003c/p\u003e \u003cp\u003eNext, we investigated TXNIP expression by replenishing AAs in an AA-deprived cell medium. H460 cells were starved of arginine, glutamine, lysine, or methionine for 8 h and then supplemented with each AA. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, induction of TXNIP in H460 cells deprived of arginine, glutamine, lysine, or methionine was blocked when the medium was replenished with each AA. These data suggest that replenishing AAs reverses the induction of TXNIP in AA-deprived H460 NSCLC cells.\u003c/p\u003e \u003cp\u003eTXNIP induction by AA deprivation is independent of ATF4\u003c/p\u003e \u003cp\u003eATF4 is a critical transcription factor for AA metabolism and is activated by AA deprivation\u003csup\u003e19\u003c/sup\u003e. To investigate whether TXNIP is a downstream gene of ATF4, we examined the expression of TXNIP when ATF4 was overexpressed or knocked down in H460 cells. NUPR1, a target gene of ATF4, was increased in ATF4-overexpressing cells and decreased in ATF4-knockdown cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). However, TXNIP expression levels were not affected by increasing or decreasing ATF4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Furthermore, ATF4 knockdown did not affect the TXNIP expression induced by arginine, glutamine, lysine, or methionine deprivation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). These data suggest that AA deprivation-induced TXNIP expression is not regulated by ATF4.\u003c/p\u003e \u003cp\u003eTXNIP expression by AA deprivation is mediated by reactive oxygen species (ROS) induced by NRF2 downregulation\u003c/p\u003e \u003cp\u003eIt has been reported that NRF2 negatively regulates TXNIP transcription by binding to the antioxidant response element of the TXNIP promoter\u003csup\u003e20\u003c/sup\u003e. We examined TXNIP expression after NRF2 siRNA treatment. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, NRF2 knockdown by siRNA suppressed the expression of NRF2 and the downstream gene GCLC. On the other hand, a significant induction of TXNIP expression at both the mRNA and protein levels was observed upon NRF2 knockdown. Interestingly, the expression levels of NRF2 protein were decreased in H460 cells deprived of arginine, glutamine, lysine, or methionine for 12 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eAA deprivation has been reported to lead to elevated levels of intracellular ROS\u003csup\u003e21\u003c/sup\u003e. To investigate whether ROS induced by AA deprivation is involved in TXNIP expression, we examined TXNIP expression in H460 cells deprived of arginine, glutamine, lysine, or methionine with the ROS scavenger N-acetyl-L-cysteine (NAC). As expected, NAC prevented the elevated TXNIP mRNA expression in cells deprived of each of these AAs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Since the downregulation of the NRF2 pathway resulted in dramatic accumulation of intracellular ROS\u003csup\u003e22\u003c/sup\u003e, we investigated whether TXNIP expression is regulated by NRF2 downregulation-mediated ROS. We treated NAC in H460 cells with NRF2 siRNA. NAC prevented the increased TXNIP mRNA expression in NRF2 knockdown cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). These data suggest that TXNIP expression by AA deprivation is mediated by ROS production by NRF2 downregulation.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eTXNIP has attracted considerable attention due to its several aspects of energy metabolism\u003csup\u003e23\u003c/sup\u003e. Overexpression of TXNIP influences metabolic abnormalities such as apoptosis of pancreatic β-cells, reduced insulin sensitivity, and decreased energy expenditure\u003csup\u003e8\u0026ndash;11\u003c/sup\u003e. In contrast, genetic ablation of TXNIP promotes normal insulin sensitivity and prevents diabetes or other metabolic abnormalities\u003csup\u003e11\u0026ndash;13\u003c/sup\u003e. As such, TXNIP is known to play a critical role in energy homeostasis, but the regulation of TXNIP expression by amino acids (AAs) is not well understood.\u003c/p\u003e \u003cp\u003eIn this study, we evaluated the effects of single AA deprivation on TXNIP transcription in H460 non-small cell lung cancer (NSCLC) cells. We found that deprivation of glutamine, arginine methionine, or lysine significantly induced the expression of TXNIP mRNA. In addition, deprivation of other AAs slightly increased or did not affect the expression of TXNIP mRNA. These results suggest that TXNIP expression might be different according to the deprivation of each AA.\u003c/p\u003e \u003cp\u003eATF4, which is activated by AA deficiency, has been reported as an important transcription factor for AA metabolism\u003csup\u003e19\u003c/sup\u003e. However, ATF4 knockdown did not affect TXNIP expression by deprivation of arginine, glutamine, lysine, or methionine, suggesting that TXNIP expression by AA deprivation is not regulated by ATF4.\u003c/p\u003e \u003cp\u003eInterestingly, we found reduced expression levels of NRF2 protein in H460 cells deprived of arginine, glutamine, lysine, or methionine for 12 h. NRF2 has been reported to negatively regulate TXNIP transcription by binding to the antioxidant response element of the TXNIP promoter\u003csup\u003e20\u003c/sup\u003e. We also observed significant induction of TXNIP in H460 cells with NRF2 knockdown. NRF2 is a key regulator of reactive oxygen species (ROS), and knockdown of NRF2 has been reported to dramatically increase endogenous levels of ROS\u003csup\u003e22\u003c/sup\u003e. The ROS scavenger N-acetyl-L-cysteine, reversed the elevated TXNIP mRNA expression in H460 cells deprived of arginine, glutamine, lysine, or methionine. NAC also reversed the increased TXNIP mRNA expression in NRF2 knockdown H460 cells. These data suggest that TXNIP expression by AA deprivation is mediated by ROS production by NRF2 downregulation. Our findings suggest that TXNIP expression might be associated with redox homeostasis in AA metabolic processes. Further studies are needed to uncover the underlying mechanisms of TXNIP-mediated AA metabolism.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eCell culture and reagents\u003c/p\u003e \u003cp\u003eH460 human non-small cell lung cancer cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and were maintained in RPMI 1640 medium (#LM011-01, Welgene, Gyeongsangbuk-do, Republic of Korea) containing 10% fetal bovine serum (#16000-044, Gibco; Thermo Fisher Scientific, Waltham, MA, USA) at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e. N-Acetyl-L-cysteine (NAC) was purchased from Sigma-Aldrich (#A7250, Merck KGaA, Darmstadt, Germany).\u003c/p\u003e \u003cp\u003eAmino acid (AA) deprivation\u003c/p\u003e \u003cp\u003eIndividual AA deprivation media were prepared by supplementing the appropriate AA stock solutions (Welgene) for the remaining 19 AAs in AA-free RPMI medium (custom prepared by Welgene) and were supplemented with 10% dialyzed fetal bovine serum (10,000 MW cutoff, #26400-044, Gibco; Thermo Fisher Scientific).\u003c/p\u003e \u003cp\u003eTransient transfection\u003c/p\u003e \u003cp\u003ePlasmids encoding mouse wild-type and dominant-negative mutant ATF4 (pEF-mATF4 WT-myc and pEF-mATF4ΔRK-myc) were kindly provided by Dr. Jawed Alam \u003csup\u003e24\u003c/sup\u003e. The dominant-negative mutant mATF4 contained a six AA substitution in the DNA binding domain of ATF4 (\u003csup\u003e292\u003c/sup\u003eRYRQKKR\u003csup\u003e298\u003c/sup\u003e to \u003csup\u003e292\u003c/sup\u003eGYLEAAA\u003csup\u003e298\u003c/sup\u003e).\u003c/p\u003e \u003cp\u003eATF4 (#sc-35112), NRF2 (sc-37030) and control (#sc-37007) siRNAs were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Transfection with plasmids and siRNAs was performed using Lipofectamine LTX with Plus Reagent (#15338) or Lipofectamine RNAiMAX (#13778), respectively, according to each manufacturer\u0026rsquo;s instructions (Invitrogen: Thermo Fisher Scientific).\u003c/p\u003e \u003cp\u003eRNA extraction and reverse transcription-polymerase chain reaction (RT‒PCR) analysis\u003c/p\u003e \u003cp\u003eTotal RNA extraction and RT‒PCR analysis were performed as described previously\u003csup\u003e25\u003c/sup\u003e. The following primers were used for PCR: TXNIP (5\u0026rsquo;-CCTCTGGGAACATCCTTCAA-3\u0026rsquo; and 5\u0026rsquo;-ATTGGCAAGGTAAGTGTGGC-3\u0026rsquo;; 348-bp product)\u003csup\u003e26\u003c/sup\u003e, ATF4 (5\u0026rsquo;- AGTCGGGTTTGGGGGCTGAAG-3\u0026rsquo; and 5\u0026rsquo;-TGGGGAAAGGGGAAGAGGTTGTAA-3\u0026rsquo;; 437-bp product)\u003csup\u003e27\u003c/sup\u003e, NUPR1 (5\u0026rsquo;-GAGACGGGACTGCGGAGGAAG-3\u0026rsquo; and 5\u0026rsquo;-GTTGCTGCCACCCTGGAGGA-3\u0026rsquo;, 242-bp product)\u003csup\u003e28\u003c/sup\u003e, GCLC (5\u0026rsquo;- CCAGTTCCTGCACATCTACC-3\u0026rsquo; and 5\u0026rsquo;- CATGTAACTCCCATACTCTGG-3\u0026rsquo; 220-bp product) β-Actin (ACTB) (5\u0026rsquo;- GGATTCCTATGTGGGCGACAG-3\u0026rsquo; and 5\u0026rsquo;-CGCTCGGTGAGGA TCTTCATG-3\u0026rsquo;; 438-bp product)\u003csup\u003e29\u003c/sup\u003e. Where indicated, RT‒PCR images were quantified using ImageJ software (version 1.52a; NIH; National Institutes of Health, Bethesda, MD, USA).\u003c/p\u003e \u003cp\u003eWestern blot analysis\u003c/p\u003e \u003cp\u003eWestern blotting was performed as described previously\u003csup\u003e25\u003c/sup\u003e. The following antibodies were used: anti-ATF4 antibody (#sc-200)obtained from Santa Cruz Biotechnology; anti-TXNIP (#14715) obtained from Cell Signaling Technology (Beverly, MA, USA); anti-NRF2 (#16396-1-AP) obtained from Proteintech Group (Chicago, IL, USA); and anti-β-actin (#A5316) antibody obtained from Sigma‒Aldrich (Merck KGaA).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by grants from the Korea Institute of Radiological and Medical Sciences (KIRAMS), funded by the Ministry of Science and ICT (MSIT) (50531-2023; 50544-2023) and from the National Research Foundation of Korea (NRF), funded by the MIST (NRF-2022R1F1A1063450; NRF-2023R1A2C1003833), Republic of Korea.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eI.C.P. and H.O.J. developed the concept and designed the study. S.H.A., S.K.J., Y.J.K., G.K., and H.O.J. carried out the experiments.\u0026nbsp;K.S.P.\u0026nbsp;gave technical support and conceptual advice. I.C.P. and H.O.J.\u0026nbsp;wrote the manuscript. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eData availability statements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eNishiyama, A.\u003cem\u003e et al.\u003c/em\u003e Identification of thioredoxin-binding protein-2/vitamin D(3) up-regulated protein 1 as a negative regulator of thioredoxin function and expression. \u003cem\u003eJ Biol Chem\u003c/em\u003e \u003cstrong\u003e274\u003c/strong\u003e, 21645-21650, doi:10.1074/jbc.274.31.21645 (1999).\u003c/li\u003e\n\u003cli\u003eHu, J. \u0026amp; Yu, Y. The Function of Thioredoxin-Binding Protein-2 (TBP-2) in Different Diseases. \u003cem\u003eOxid Med Cell Longev\u003c/em\u003e \u003cstrong\u003e2018\u003c/strong\u003e, 4582130, doi:10.1155/2018/4582130 (2018).\u003c/li\u003e\n\u003cli\u003eTsubaki, H., Tooyama, I. \u0026amp; Walker, D. G. Thioredoxin-Interacting Protein (TXNIP) with Focus on Brain and Neurodegenerative Diseases. \u003cem\u003eInt J Mol Sci\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, doi:10.3390/ijms21249357 (2020).\u003c/li\u003e\n\u003cli\u003eMasutani, H. Thioredoxin-Interacting Protein in Cancer and Diabetes. \u003cem\u003eAntioxid Redox Signal\u003c/em\u003e \u003cstrong\u003e36\u003c/strong\u003e, 1001-1022, doi:10.1089/ars.2021.0038 (2022).\u003c/li\u003e\n\u003cli\u003eChen, Y.\u003cem\u003e et al.\u003c/em\u003e Research Progress of TXNIP as a Tumor Suppressor Gene Participating in the Metabolic Reprogramming and Oxidative Stress of Cancer Cells in Various Cancers. \u003cem\u003eFront Oncol\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 568574, doi:10.3389/fonc.2020.568574 (2020).\u003c/li\u003e\n\u003cli\u003eXie, M.\u003cem\u003e et al.\u003c/em\u003e Thioredoxin interacting protein (TXNIP) acts as a tumor suppressor in human prostate cancer. \u003cem\u003eCell Biol Int\u003c/em\u003e \u003cstrong\u003e44\u003c/strong\u003e, 2094-2106, doi:10.1002/cbin.11418 (2020).\u003c/li\u003e\n\u003cli\u003eCadenas, C.\u003cem\u003e et al.\u003c/em\u003e Role of thioredoxin reductase 1 and thioredoxin interacting protein in prognosis of breast cancer. \u003cem\u003eBreast Cancer Res\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, R44, doi:10.1186/bcr2599 (2010).\u003c/li\u003e\n\u003cli\u003eBlouet, C., Liu, S. M., Jo, Y. H., Chua, S. \u0026amp; Schwartz, G. J. TXNIP in Agrp neurons regulates adiposity, energy expenditure, and central leptin sensitivity. \u003cem\u003eJ Neurosci\u003c/em\u003e \u003cstrong\u003e32\u003c/strong\u003e, 9870-9877, doi:10.1523/JNEUROSCI.0353-12.2012 (2012).\u003c/li\u003e\n\u003cli\u003eChen, J., Saxena, G., Mungrue, I. N., Lusis, A. J. \u0026amp; Shalev, A. Thioredoxin-interacting protein: a critical link between glucose toxicity and beta-cell apoptosis. \u003cem\u003eDiabetes\u003c/em\u003e \u003cstrong\u003e57\u003c/strong\u003e, 938-944, doi:10.2337/db07-0715 (2008).\u003c/li\u003e\n\u003cli\u003eJing, G.\u003cem\u003e et al.\u003c/em\u003e Thioredoxin-interacting protein promotes islet amyloid polypeptide expression through miR-124a and FoxA2. \u003cem\u003eJ Biol Chem\u003c/em\u003e \u003cstrong\u003e289\u003c/strong\u003e, 11807-11815, doi:10.1074/jbc.M113.525022 (2014).\u003c/li\u003e\n\u003cli\u003eJo, S. H., Kim, M. Y., Park, J. M., Kim, T. H. \u0026amp; Ahn, Y. H. Txnip contributes to impaired glucose tolerance by upregulating the expression of genes involved in hepatic gluconeogenesis in mice. \u003cem\u003eDiabetologia\u003c/em\u003e \u003cstrong\u003e56\u003c/strong\u003e, 2723-2732, doi:10.1007/s00125-013-3050-6 (2013).\u003c/li\u003e\n\u003cli\u003eParikh, H.\u003cem\u003e et al.\u003c/em\u003e TXNIP regulates peripheral glucose metabolism in humans. \u003cem\u003ePLoS Med\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, e158, doi:10.1371/journal.pmed.0040158 (2007).\u003c/li\u003e\n\u003cli\u003eChutkow, W. A.\u003cem\u003e et al.\u003c/em\u003e Deletion of the alpha-arrestin protein Txnip in mice promotes adiposity and adipogenesis while preserving insulin sensitivity. \u003cem\u003eDiabetes\u003c/em\u003e \u003cstrong\u003e59\u003c/strong\u003e, 1424-1434, doi:10.2337/db09-1212 (2010).\u003c/li\u003e\n\u003cli\u003eMinn, A. H., Hafele, C. \u0026amp; Shalev, A. Thioredoxin-interacting protein is stimulated by glucose through a carbohydrate response element and induces beta-cell apoptosis. \u003cem\u003eEndocrinology\u003c/em\u003e \u003cstrong\u003e146\u003c/strong\u003e, 2397-2405, doi:10.1210/en.2004-1378 (2005).\u003c/li\u003e\n\u003cli\u003eCha-Molstad, H., Saxena, G., Chen, J. \u0026amp; Shalev, A. Glucose-stimulated expression of Txnip is mediated by carbohydrate response element-binding protein, p300, and histone H4 acetylation in pancreatic beta cells. \u003cem\u003eJ Biol Chem\u003c/em\u003e \u003cstrong\u003e284\u003c/strong\u003e, 16898-16905, doi:10.1074/jbc.M109.010504 (2009).\u003c/li\u003e\n\u003cli\u003eStoltzman, C. A.\u003cem\u003e et al.\u003c/em\u003e Glucose sensing by MondoA:Mlx complexes: a role for hexokinases and direct regulation of thioredoxin-interacting protein expression. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e \u003cstrong\u003e105\u003c/strong\u003e, 6912-6917, doi:10.1073/pnas.0712199105 (2008).\u003c/li\u003e\n\u003cli\u003eKaadige, M. R., Looper, R. E., Kamalanaadhan, S. \u0026amp; Ayer, D. E. Glutamine-dependent anapleurosis dictates glucose uptake and cell growth by regulating MondoA transcriptional activity. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e \u003cstrong\u003e106\u003c/strong\u003e, 14878-14883, doi:10.1073/pnas.0901221106 (2009).\u003c/li\u003e\n\u003cli\u003eChutkow, W. A., Patwari, P., Yoshioka, J. \u0026amp; Lee, R. T. Thioredoxin-interacting protein (Txnip) is a critical regulator of hepatic glucose production. \u003cem\u003eJ Biol Chem\u003c/em\u003e \u003cstrong\u003e283\u003c/strong\u003e, 2397-2406, doi:10.1074/jbc.M708169200 (2008).\u003c/li\u003e\n\u003cli\u003eHarding, H. P.\u003cem\u003e et al.\u003c/em\u003e An integrated stress response regulates amino acid metabolism and resistance to oxidative stress. \u003cem\u003eMol Cell\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 619-633, doi:10.1016/s1097-2765(03)00105-9 (2003).\u003c/li\u003e\n\u003cli\u003eHe, X. \u0026amp; Ma, Q. Redox regulation by nuclear factor erythroid 2-related factor 2: gatekeeping for the basal and diabetes-induced expression of thioredoxin-interacting protein. \u003cem\u003eMol Pharmacol\u003c/em\u003e \u003cstrong\u003e82\u003c/strong\u003e, 887-897, doi:10.1124/mol.112.081133 (2012).\u003c/li\u003e\n\u003cli\u003eLiao, J.\u003cem\u003e et al.\u003c/em\u003e Regulation of stem-like cancer cells by glutamine through beta-catenin pathway mediated by redox signaling. \u003cem\u003eMol Cancer\u003c/em\u003e \u003cstrong\u003e16\u003c/strong\u003e, 51, doi:10.1186/s12943-017-0623-x (2017).\u003c/li\u003e\n\u003cli\u003eSingh, A.\u003cem\u003e et al.\u003c/em\u003e RNAi-mediated silencing of nuclear factor erythroid-2-related factor 2 gene expression in non-small cell lung cancer inhibits tumor growth and increases efficacy of chemotherapy. \u003cem\u003eCancer Res\u003c/em\u003e \u003cstrong\u003e68\u003c/strong\u003e, 7975-7984, doi:10.1158/0008-5472.CAN-08-1401 (2008).\u003c/li\u003e\n\u003cli\u003eAlhawiti, N. M., Al Mahri, S., Aziz, M. A., Malik, S. S. \u0026amp; Mohammad, S. TXNIP in Metabolic Regulation: Physiological Role and Therapeutic Outlook. \u003cem\u003eCurr Drug Targets\u003c/em\u003e \u003cstrong\u003e18\u003c/strong\u003e, 1095-1103, doi:10.2174/1389450118666170130145514 (2017).\u003c/li\u003e\n\u003cli\u003eHe, C. H.\u003cem\u003e et al.\u003c/em\u003e Identification of activating transcription factor 4 (ATF4) as an Nrf2-interacting protein. Implication for heme oxygenase-1 gene regulation. \u003cem\u003eJ Biol Chem\u003c/em\u003e \u003cstrong\u003e276\u003c/strong\u003e, 20858-20865, doi:10.1074/jbc.M101198200 (2001).\u003c/li\u003e\n\u003cli\u003eJin, H. O.\u003cem\u003e et al.\u003c/em\u003e Induction of HSP27 and HSP70 by constitutive overexpression of Redd1 confers resistance of lung cancer cells to ionizing radiation. \u003cem\u003eOncol Rep\u003c/em\u003e \u003cstrong\u003e41\u003c/strong\u003e, 3119-3126, doi:10.3892/or.2019.7036 (2019).\u003c/li\u003e\n\u003cli\u003eJin, H. O.\u003cem\u003e et al.\u003c/em\u003e TXNIP potentiates Redd1-induced mTOR suppression through stabilization of Redd1. \u003cem\u003eOncogene\u003c/em\u003e \u003cstrong\u003e30\u003c/strong\u003e, 3792-3801, doi:10.1038/onc.2011.102 (2011).\u003c/li\u003e\n\u003cli\u003eCarracedo, A.\u003cem\u003e et al.\u003c/em\u003e The stress-regulated protein p8 mediates cannabinoid-induced apoptosis of tumor cells. \u003cem\u003eCancer Cell\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 301-312, doi:10.1016/j.ccr.2006.03.005 (2006).\u003c/li\u003e\n\u003cli\u003eJin, H. O.\u003cem\u003e et al.\u003c/em\u003e Nuclear protein 1 induced by ATF4 in response to various stressors acts as a positive regulator on the transcriptional activation of ATF4. \u003cem\u003eIUBMB Life\u003c/em\u003e \u003cstrong\u003e61\u003c/strong\u003e, 1153-1158, doi:10.1002/iub.271 (2009).\u003c/li\u003e\n\u003cli\u003eJin, H. O.\u003cem\u003e et al.\u003c/em\u003e Redd1 inhibits the invasiveness of non-small cell lung cancer cells. \u003cem\u003eBiochem Biophys Res Commun\u003c/em\u003e \u003cstrong\u003e407\u003c/strong\u003e, 507-511, doi:10.1016/j.bbrc.2011.03.047 (2011).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Amino acid, ATF4, NRF2, Reactive oxygen species, TXNIP","lastPublishedDoi":"10.21203/rs.3.rs-2915490/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2915490/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTXNIP is an essential regulator of cellular metabolism, including glucose homeostasis, fatty acid synthesis, cholesterol accumulation, and is implicated in metabolic diseases such as obesity, type 2 diabetes, and atherosclerosis. However, how TXNIP expression is regulated in response to amino acid (AA) deprivation is not well understood. In the present study, deprivation of AAs, especially arginine, glutamine, lysine, and methionine, induced TXNIP expression in H460 non-small cell lung cancer cells. Unexpectedly, TXNIP induction by AA deprivation was dependent on NRF2 downregulation, but not ATF4 activation. Furthermore, N-acetyl-L-cysteine, a scavenger of reactive oxygen species (ROS), prevented TXNIP expression in H460 cells deprived of AA. Taken together, TXNIP expression by AA deprivation is mediated by ROS production by NRF2 downregulation. Our findings suggest that TXNIP expression might be associated with the redox homeostasis of AA metabolism.\u003c/p\u003e","manuscriptTitle":"Amino acid deprivation induces TXNIP expression by NRF2 downregulation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-05-17 21:01:39","doi":"10.21203/rs.3.rs-2915490/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a0c807cb-656a-451f-ab1b-cba2c6887c89","owner":[],"postedDate":"May 17th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-03-19T11:09:50+00:00","versionOfRecord":{"articleIdentity":"rs-2915490","link":"https://doi.org/10.1002/iub.2792","journal":{"identity":"iubmb-life","isVorOnly":true,"title":"IUBMB Life"},"publishedOn":"2023-12-06 11:09:50","publishedOnDateReadable":"December 6th, 2023"},"versionCreatedAt":"2023-05-17 21:01:39","video":"","vorDoi":"10.1002/iub.2792","vorDoiUrl":"https://doi.org/10.1002/iub.2792","workflowStages":[]},"version":"v1","identity":"rs-2915490","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2915490","identity":"rs-2915490","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}