Expression of yeast NADH dehydrogenase and ascidian alternative oxidase affects metabolism and free radical processes in Drosophila

Expression of yeast NADH dehydrogenase and ascidian alternative oxidase affects metabolism and free radical processes in Drosophila | 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 Short Report Expression of yeast NADH dehydrogenase and ascidian alternative oxidase affects metabolism and free radical processes in Drosophila Oleh Lushchak, Dmytro Gospodaryov, Ihor Yurkevych, Olha Strilbytska This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7920896/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 31 Jan, 2026 Read the published version in BMC Research Notes → Version 1 posted 8 You are reading this latest preprint version Abstract Objective: The study aimed to investigate the effect of overexpression of alternative mitochondrial enzymes such as yeast NADH dehydrogenase I (NDI1) and alternative oxidase (AOX) on the metabolism, oxidative stress and feeding behavior of the fruit fly Drosophila melanogaster . Experimental flies with expression of NDI1 or AOX were generated using genetic crosses based on the GAL4-UAS system. Results: Flies with NDI1 expression showed increased food consumption, markers of oxidative stress (elevated carbonyl protein content), and increased activity of the detoxification enzyme glutathione- S -transferase, along with decreased activity of key metabolic enzymes including isocitrate dehydrogenase, lactate dehydrogenase, and glucose-6-phosphate dehydrogenase. In contrast, AOX -expressing flies had reduced lactate dehydrogenase (LDH) activity, decreased level of lipid peroxides, increased glutathione reductase activity. Lower free glucose levels with elevated glycogen stores was found in AOX -expressing flies. The results indicate that the alternative electron transport chain radically transforms energy and redox metabolism. In particular, NDI1 expression could lead to increased energy demand and compensatory hyperphagia. In contrast, AOX bypasses the main steps of proton gradient generation, reducing superoxide generation. NADH dehydrogenase alternative oxidase metabolism oxidative stress Figures Figure 1 Figure 2 Figure 3 Figure 4 INTRODUCTION Non-proton pumping NADH dehydrogenase and ubiquinol oxidase (also known as alternative oxidase) are alternative components of the respiratory systems, present in bacteria, fungi, plants, and some animals [ 1 ]. However, these enzymes are absent in the respiratory systems of mammals. From the late 1990s and early 2000s, alternative NADH dehydrogenase and oxidase are studied as potential tools for gene therapy of mitochondrial diseases, respectively [ 2 , 3 ]. It has also been found that being heterologically expressed, these enzymes may indirectly influence metabolism and signaling of a host organism. Of the observed effects, not directly related to respiration, are the ability of alternative NADH dehydrogenase (ANDH) to prolong lifespan and activate xenobiotic defense systems [ 3 ]. In turn, alternative oxidase (AOX) was found to affect developmental signaling [ 4 ], cell migration [ 5 ], reproductive function [ 6 , 7 ], nutrient utilization [ 7 , 8 ], resistance to a number of environmental toxins and cold [ 8 , 9 ]. Both enzymes have been found to modulate oxygen sensing and signaling mediated by reactive oxygen species (ROS) [ 10 – 13 ]. Thus, phenotyping of the organisms that express AOX or ANDH, or both, brings new insights regarding the evolution of respiratory chain, as well as regarding metabolic signaling. Reactions catalyzed by AOX and ANDH consume substrates of conventional proton-pumping respiratory complexes. In some instances, it has been found they compete with those complexes for substrates [ 14 ]. This competition may lead to a decrease in the rates of adenosine triphosphate (ATP) production in mitochondria. Since ATP is required for various biosynthetic processes, one may expect that organisms that express AOX and/or ANDH will accumulate less metabolic stores, such as glycogen or triglycerides. Interestingly, AOX tackles part of ubiquinol oxidation, decreasing ubiquinol flow through ubiquinol-cytochrome c reductase, or complex III of the mitochondrial respiratory chain. In turn, ANDH reduces part of ubiquinone pool, decreasing ubiquinone flow through proton-pumping NADH dehydrogenase, or complex I. Both complex I and complex III are capable of forming semiquinone anion-radical, a free radical quinone form [ 15 , 16 ]. The unpaired electron from the semiquinone anion-radical can be passed to molecular oxygen yielding superoxide anion-radical. The latter is then converted, giving complete lineage of ROS that oxidize lipid acyl chains, proteins, and nucleic acids. These molecules are constantly renewed, so their oxidative modification does not seriously affect cells’ function, until ROS levels exceed a certain threshold [ 16 , 17 ]. It is believed that AOX and ANDH, while oxidize ubiquinol or reduce ubiquinone, respectively, do not produce ROS, owing to difference in catalytic mechanism which minimizes probability of forming the semiquinone radical [ 18 ]. Neverthelss, several studies show that this may not be true at least for ANDH. When ANDH is co-expressed with functional complex I, it may promote ROS production by contributing to the increase in ubiquinol pool and boosting probability of reverse electron transport through complex I [ 19 ]. These peculiarities support a need in phenotyping of the AOX - and ANDH -expressing animals. Fruit flies expressing ANDH were already partially characterized in terms of oxidative stress markers and antioxidant, and related enzymes [ 20 ]. However, antioxidant defense and oxidative stress indices were not systematically described for animals expressing AO and ANDH. Present study addresses the following questions: do the alternative oxidase from tunicate Ciona intestinalis (AOX) and alternative NADH dehydrogenase NDI1 from the budding yeast Saccharomyces cerevisiae heterologously expressed in the fruit fly Drosophila melanogaster 1) affect nutrient utilization – food consumption and metabolic stores? 2) prevent oxidative damage, affect antioxidant defense, and protect sensitive enzymes from oxidative modification? To test this, we measured how much food is consumed by fruit flies that express NDI1 and AOX , as well as glycogen, trehalose, triglyceride and total lipid, and glucose content in the fruit fly body. To assess oxidative damage, we measured the levels of protein carbonyls, lipid peroxides, as well as low and high molecular thiol-containing compounds. Antioxidant defense markers were activities of superoxide dismutase, catalase, glutathione S-transferase, glutathione reductase, and NADP-reducing enzymes, such as glucose 6-phosphate dehydrogenase, NADP-dependent malate and isocitrate dehydrogenases. The three later enzymes are also potential markers of oxidative modification, since were found sensitive to it [ 21 ]. Additional markers of oxidative modification were lactate dehydrogenase and alanine transaminase, enzymes that help recycle anaerobic byproducts such as lactate or alanine into energy-yielding metabolites during metabolic shuttling between tissues. MATERIALS AND METHODS Fly Husbandry and Transgenic Flies Flies were cultured on standard yeast-molasses medium, composed of dry yeast (5%), corn (6.1%), molasses (7.5%), nipagin (0.18%), and propionic acid (0.4%) at 25°C at 25°C under a 12:12 light:dark cycle [ 22 ]. The GAL4-UAS system was used to generate experimental flies. Flies of Da-GAL4 , UAS-NDI1 , UAS-AOX strains were kindly provided by Professor Alberto Sanz (Glasgow, UK). UAS-NDI1 flies were generated by Sanz and coauthors [ 20 ] and UAS-AOX flies by Fernandez-Ayala et al. [ 23 ] earlier. All flies were on w Dah background. Experimental flies were generated by crossing da-GAL4 females with respective males. Cross of da-GAL4 females with w Dah males was used to generate flies of control genotype da-GAL4 > w Dah (CON). Respectively, flies expressing yeast NDI1 were of da-GAL4 > UAS-NDI1 genotype (NDI) and alternative oxidase da-GAL4 > UAS-AOX genotype (AOX). The resulting eggs (100 per vial) were allowed to develop at 25°C. Newly enclosed flies were kept of fresh food for additional 4 days. Feeding Food consumption by a single fly was measured by CApillary FEeder (CAFE) assay [ 24 ]. Experimental flies were kept in 1.5 vial supplemented with 5 µL capillary tube filled with food containing 5% yeast extract, 5% sucrose, 0.1% propionic, and 0.01% phosphoric acid. Capillaries were changed every day and the amount of food consumed was measured over a period of four days. Vials were kept in closed boxes with distilled water on the bottom to maintain high humidity for evaporation prevention. Three non-feeding vials were monitored for changes in volume to control for evaporation. Ten flies per genotype were tested. Metabolic parameters Metabolic parameter such as glucose, trehalose, glycogen, triacylglyceride (TAG) and lipids were measured as described earlier by Koliada and coauthors [ 22 ]. Briefly, ten frozen flies per sample were weighted and homogenized with 50 mM phosphate buffered saline (PBS) supplemented by 0.09% sodium azide (pH 7.4) at a 1:10 w:v ratio, heat denatured as described and centrifuged for 15 min at 13000g and 4°C. Resulted supernatant were transferred to new vial and used for further determinations. Glycogen was converted into glucose by incubation of the supernatants with 5.6 U of amyloglucosidase from Aspergillus niger (Merck KGaA, Cat No. 10115) during 4 h at 37°C. Measurements were performed using a colorimetric glucose test kit (Gliukoza-Mono, «Reagent», Dnipro) that exploits a combined glucose oxidase and peroxidase principle. Samples were then measured at wavelength 540 nm. For TAG determination reweighted flies were homogenized in 200 mM PBST (phosphate buffered saline with 0.05% Triton X100), boiled for 5 min and centrifuged (13000 g, 10 min). Resulting supernatants were used for TAG assay with the Liquick Cor-TG diagnostic kit (Cormay, Poland). Activities of enzymes and oxidative stress indices Flies were homogenized using a Potter-Elvehjem glass/glass homogenizer (1:10 w/v) in 50 mM KPi (pH 7.5) containing 0.5 mM ethylenediaminetetraacetic acid and 1 mM phenylmethylsulfonyl fluoride and centrifuged 15 min at 16000g, and 4°C (Eppendorf 5415R centrifuge). Supernatants were collected and used for the determination of enzymatic activities, protein carbonyls, levels of high- and low-molecular-mass thiol-containing compounds. Protein content was measured by the Bradford method with serum bovine albumin used as the standard [ 25 ]. The activities of catalase, superoxide dismutase (SOD), glucose 6-phosphate dehydrogenase (G6PDH), NADP-dependent isocitrate dehydrogenase (IDH), and glutathione- S -transferase (GST), glutathione reductase (GR), lactate dehydrogenase (LDH), malate dehydrogenase (MDH) were measured by methods described earlier [ 26 , 27 ]. The levels of protein carbonyls, lipid peroxides, high- and low-molecular-mass thiol-containing compounds were assayed as described by Lozinsky et al. [ 28 ]. Statistical Procedures Statistical processing of the data was performed using GraphPad Prism 8 software. One-way ANOVA followed by Tukey test was used to determine a significant difference between groups. Data are shown as mean ± SEM and p value < 0.05 was considered as significance. RESULTS Alternative respiratory chain enzymes, such as NDI1 and AOX, use the same substrates as proton-pumping electron-transport chain (ETC) complexes I and III, respectively. Operation of NDI1 and AOX in the fruit fly electron-transport chain might decrease flow of reduced nicotinamide adenine dinucleotide (NADH) and ubiquinol through these complexes, thus affecting rates of protons pumped and ATP synthesis. Since ATP is spent for biosynthetic processes, one may expect NDI1 and/or AOX expression affect accumulation of metabolic stores, such as glycogen and triacylglycerols [ 29 ]. Nevertheless, NDI1 -expressing flies did not show any significant difference in the levels of glycogen, TAG, total lipids, trehalose, and glucose in the body, compared to the wild type flies (Fig. 1 B-F). On the other hand, AOX expression led to increased glycogen while decreased body glucose levels in flies as compared to both control and NDI1 -expressing flies (Fig. 1 B, C; Tukey test: p < 0.05). To explore potential compensatory mechanisms preserving metabolic stores, we assessed food intake in transgenic lines. NDI1 -expressing flies displayed increased food consumption versus control (Fig. 1 A), indicating that elevated nutrient uptake may help sustain energy-dependent biosynthetic processes. An important outcome of NDI1 and AOX expression is an expected attenuation of ROS generation. Mitochondria contribute to ROS formation through one-electron reduction or oxidation of ubiquinone by complexes I and III of the ETC. The electron from ubiquinone is then passed to dioxygen, yielding superoxide anion-radical. The data on the capability of NDI 1 or AOX to prevent ROS formation are still ambiguous. Re-testing the indices of antioxidant defense and oxidative damage in the current study showed that expression of either NDI1 or AOX led to increase in protein carbonyls (Fig. 2 A; Tukey test: p < 0.05), although AOX -expressing flies showed a two-fold lower level of lipid peroxides compared to the wild type (Fig. 2 B; Tukey test: p = 0.041). There were no differences between the strains studied in the levels of high and low molecular mass thiol-containing substances (Fig. 2 C, D). Neither superoxide dismutase (SOD), nor catalase activities were affected, whereas glutathione-S-transferase activity was elevated by 19% in NDI1 -expressing flies as compared to the control (Fig. 3 ; Tukey test: p = 0.030). On the other hand, glutathione reductase was elevated by ⁓50% in AOX -expressing flies as compared to both control and NDI1 -expressing flies (Fig. 4 A; Tukey test: p < 0.05). Interestingly, NDI1 -expressing flies had significantly lower activities of glucose-6-phosphate dehydrogenase by 32% (Fig. 4 B; Tukey test: p = 0.027) and NADP-dependent isocitrate dehydrogenase by 24% (Fig. 4 C; Tukey test: p = 0.049). Flies of both experimental cohorts had about 1.5-fold lower activity of lactate dehydrogenase compared to that of the wild type (Fig. 4 D; Tukey test: p < 0.02). MDH and AST activities were not affected either by NDI1 or AOX overexpression (Fig. 4 E, F). DISCUSSION As was mentioned above, NDI1 and AOX may deprive substrates of proton-pumping ETC complexes I and III, respectively. Indeed, competition for substrates has been observed between plant alternative NADH dehydrogenases heterologously expressed in human fibroblasts [ 30 ]. However, such substrate competition has not been observed for NDI1 [ 31 ]. The co-expression of alternative NADH dehydrogenase with proton-pumping NADH dehydrogenase may lead to about 30% decrease in the levels of ATP produced [ 3 ]. In turn, ATP is needed for various biosynthetic processes, including protein, lipid, and glycogen synthesis [ 32 ]. The current study shows that NDI1 expression did actually not influence the levels of metabolic stores such as TAG, glycogen, and trehalose. However, increased food consumption we observe (Fig. 1 F) may compensate for a possible biosynthetic shortage if the latter takes place. Moreover, mitochondria contribute to the appetite regulation by affecting release of adipokinetic hormone as well as several other hormones that promote food consumption [ 33 , 34 ]. An interesting effect we observe is the decrease of glucose level while the increase in glycogen in AOX -expressing flies (Fig. 1 A, E). Similar to NDI1, AOX shunts ETC by-passing its proton-pumping complexes. Thus, we expected AOX expression in fruit flies might lead to a decrease in the levels of metabolic stores by lowering efficiency of oxidative phosphorylation. Nevertheless, we have obtained an opposite data, which can be connected with pleiotropic effects of AOX on cell physiology. Specifically, AOX is believed to decrease generation of ROS by ETC complex III [ 35 , 36 ]. Recent findings imply AOX may directly or indirectly affect cellular calcium homeostasis [ 37 ] and apoptosis [ 37 , 38 ]. Cellular calcium is an important regulator of insulin-like peptide release in insects [ 39 ]. Calcium may also affect downstream insulin signaling pathway, specifically glycogen synthase kinase 3β [ 40 ]. It is remarkable, that calcium signaling has not yet been sufficiently studied in AOX -expressing organisms. Nevertheless, observations from different studies, suggest AOX might promote either calcium efflux from or hinder calcium transport into mitochondria [ 41 ]. This can be mediated by the ability of AOX to either lessen ROS generation or membrane potential. In turn, ROS and membrane potential regulate mitochondrial permeability transition and calcium transport. As mentioned above, both NDI1 and AOX may attenuate ROS generation through by-passing ETC complexes I and III that produce ubisemiquinone anion-radical as a byproduct. However, despite many studies that showed such attenuation in isolated mitochondria, this is not a strict rule. Alternative NADH dehydrogenase were shown to increase ROS production by increasing ubiquinone pool, consequently promoting reverse electron transport through ETC complex I [ 42 ]. In our current study, both NDI1 and AOX expression in flies lead to an increase in the protein carbonyl levels (Fig. 2 A) with a concomitant decrease in lipid peroxide (LOOH) levels (Fig. 2 B). The latter suggests a fast breakdown of LOOH that may lead to formation of the end-products, predominantly aldehydes. Subsequently, aldehydes produced after LOOH breakdown interact with proteins leading to formation of additional carbonyl groups [ 43 ]. Formation of LOOH and their breakdown are hardly trackable dynamic processes. The breakdown often occurs after formation of multiple endoperoxide within one fatty acyl chain [ 44 ]. Afterward, the end-products can be conjugated with glutathione or other thiols or modify proteins. We saw that NDI1 or AOX expression did not affect thiol levels (Fig. 2 C, D). It allows to assume that in our case NDI1 and AOX indeed promote ROS production rather than abolish it. However, NDI1 or AOX likely promote intensive formation of short-living ROS, such as hydroxyl radical and/or superoxide anion-radical, that oxidize fatty acyl chains of membrane lipids. The latter may break down leading to the formation of aldehyde-containing end-products that interact with neighboring proteins. The enzymes such as glucose 6-phosphate dehydrogenase (G6PDH), isocitrate dehydrogenase (IDH), malate dehydrogenase (MDH), and lactate dehydrogenase (LDH) can be examples of such proteins. Our results suggest this can be the case for NDI1 -expressing flies since activities of these enzymes was lower compared to those in the wild type flies [ 42 ]. A similar trend can also be seen in AOX -expressing flies [ 23 ]. How it could be possible that expression of NDI1 and AOX leads to an increase in oxidative modifications? Reactive oxygen species are important signaling molecules whose level indicates about well-being of mitochondria. An elevation of ROS levels often results in activation of antioxidant system [ 45 ]. It has been shown that reactions occurred at ETC complex III promote formation of superoxide anion-radical at the mitochondrial matrix side and intermembrane mitochondrial space [ 17 ]. The superoxide released in the intermembrane space passes signal to cytosol and indirectly activates antioxidant defense [ 46 ]. Bypassing complex III by AOX decreases the level superoxide released to the intermembrane space thus preventing the activation of antioxidant system. Indeed, the activity of catalase and superoxide dismutase in AOX -expressing flies is the same as in control [ 47 ]. A higher glutathione reductase activity in these experimental flies (Fig. 3 ) implies a compensatory response to ROS that confer protein carbonylation. Along with interaction between end-products of lipid peroxidation and proteins, this response may account for the respectively lower level of LOOH in AOX flies (Fig. 2 B). Lactate dehydrogenase (LDH) is an enzyme that allows NAD + regeneration for persisting glycolysis. Expression of LDH is regulated by multiple transcription factors, including estrogen-related receptor [ 48 ] and hypoxia-inducible factor [ 49 , 50 ]. Expression of LDH can also be suppressed by 20-hydroxyecdysone (20E) [ 51 ]. Interestingly, in insects, several cytochromes of P450 type involved in ecdysone biosynthesis are localized to mitochondria [ 52 , 53 ]. Cytochromes P450 are heme-containing proteins. Reactions catalyzed by NDI1 and AOX by-pass those catalyzed by iron-containing ETC complexes. Thus, these two alternative respiratory enzymes may favor redirection of heme and iron incorporation from ETC complexes to the mitochondrial cytochromes P450. In turn, this may enable an increase in 20E titers and downregulation of LDH. Another possible mechanism of LDH downregulation in NDI1 - and AOX -expressing flies is activation of dTIS11, a D. melanogaster tristetraprolin homolog [ 54 ]. The latter is activated by NAD + [ 54 ]. Both, NDI1 and AOX may favor NADH depletion since they are not regulated by membrane potential, unlike ETC complexes I and III. Last but not the least, LDH is strongly activated by hypoxia whereas reoxygenation leads to its rapid downregulation [ 49 , 55 ]. This regulation is mediated by D. melanogaster homolog of hypoxia-inducible factor 1α (HIF-1α) [ 49 ]. In turn, HIF-1α can be activated by ROS [ 56 ] whereas NDI1 and AOX operation may decrease ROS levels. Interestingly, type II NADH dehydrogenase NDX from the seq squirt Ciona intestinalis conferred opposite effects on G6PDH and LDH, while not affecting activities of NADP-specific MDH and IDH [ 57 ]. Nevertheless, both NDI1 and AOX expression lead to an increase in glutathione S-transferase activity in flies. These discrepancies may imply the influence of background, sex [ 58 ], and peculiarities of the enzymes from different sources on the pleiotropic effects of alternative oxidase and NADH dehydrogenase. Abbreviations ANDH Alternative NADH dehydrogenase NDI1 NADH dehydrogenase I AOX Alternative oxidase ROS Reactive oxygen species ATP Adenosine triphosphate CAFE assay CApillary FEeder assay TAG Triacylglyceride SOD Superoxide dismutase G6PDH Glucose 6-phosphate dehydrogenase IDH Isocitrate dehydrogenase GST Glutathione-S-transferase GR Glutathione reductase LDH Lactate dehydrogenase MDH Malate dehydrogenase ETC Electron-transport chain LOOH Lipid peroxide HIF-1α Hypoxia-inducible factor 1α Declarations Author contributions O.L. and D.G. developed the idea for the research and edited the manuscript. D.G. and I.Y. performed all the experiments and collected the data. O.S. and D.G. analyzed and interpreted the data and were major contributors to the writing of the manuscript. All authors read and approved the final manuscript. Data availability The data reported in this study are available from the corresponding authors upon request. Ethics approval and consent to participate Not applicable. Funding Declaration This study received no external funding. Declaration of competing interest The authors declare no competing interests. References McDonald AE, Gospodaryov DV. Alternative NAD(P)H dehydrogenase and alternative oxidase: Proposed physiological roles in animals. Mitochondrion. 2019;45:7–17. Viscomi C, Moore AL, Zeviani M, Szibor M. Xenotopic expression of alternative oxidase (AOX) to study mechanisms of mitochondrial disease. Biochim Biophys Acta Bioenerg. 2023;1864(2):148947. Gospodaryov DV. Alternative NADH dehydrogenase: A complex I backup, a drug target, and a tool for mitochondrial gene therapy. 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Noguchi K, Yokozeki K, Tanaka Y, Suzuki Y, Nakajima K, Nishimura T, Goda N. Sima, a Drosophila homolog of HIF-1α, in fat body tissue inhibits larval body growth by inducing Tribbles gene expression. Genes Cells. 2022;27(2):145–51. Tennessen JM, Thummel CS. Coordinating growth and maturation - insights from Drosophila . Curr Biol. 2011;21(18):R750–7. Petryk A, Warren JT, Marqués G, Jarcho MP, Gilbert LI, Kahler J, Parvy JP, Li Y, Dauphin-Villemant C, O'Connor MB. Shade is the Drosophila P450 enzyme that mediates the hydroxylation of ecdysone to the steroid insect molting hormone 20-hydroxyecdysone. Proc Natl Acad Sci U S A. 2003;100(24):13773–8. Schellens S, Lenaerts C, Pérez Baca MDR, Cools D, Peeters P, Marchal E, Vanden Broeck J. Knockdown of the Halloween Genes spook, shadow and shade Influences Oocyte Development, Egg Shape, Oviposition and Hatching in the Desert Locust. Int J Mol Sci. 2022;23(16):9232. Joe Y, Chen Y, Park J, Kim HJ, Rah SY, Ryu J, Cho GJ, Choi HS, Ryter SW, Park JW, Kim UH, Chung HT. Cross-talk between CD38 and TTP Is Essential for Resolution of Inflammation during Microbial Sepsis. Cell Rep. 2020;30(4):1063–e10765. de Toeuf B, Soin R, Nazih A, Dragojevic M, Jurėnas D, Delacourt N, Vo Ngoc L, Garcia-Pino A, Kruys V, Gueydan C. ARE-mediated decay controls gene expression and cellular metabolism upon oxygen variations. Sci Rep. 2018;8(1):5211. Hamanaka RB, Chandel NS. Mitochondrial reactive oxygen species regulate hypoxic signaling. Curr Opin Cell Biol. 2009;21(6):894–9. Gospodaryov DV, Strilbytska OM, Semaniuk UV, Perkhulyn NV, Rovenko BM, Yurkevych IS, Barata AG, Dick TP, Lushchak OV, Jacobs HT. Alternative NADH dehydrogenase extends lifespan and increases resistance to xenobiotics in Drosophila . Biogerontology. 2020;21(2):155–171. Erratum in: Biogerontology. 2020;21(2):173–4. Lushchak O, Strilbytska O, Storey KB. Gender-specific effects of pro-longevity interventions in Drosophila . Mech Ageing Dev. 2023;209:111754. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 31 Jan, 2026 Read the published version in BMC Research Notes → Version 1 posted Editorial decision: Revision requested 22 Dec, 2025 Reviews received at journal 03 Dec, 2025 Reviewers agreed at journal 23 Nov, 2025 Reviewers invited by journal 11 Nov, 2025 Editor invited by journal 27 Oct, 2025 Editor assigned by journal 26 Oct, 2025 Submission checks completed at journal 26 Oct, 2025 First submitted to journal 22 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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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-7920896","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":543576808,"identity":"4163f71e-59f8-472a-b86a-6d2cd7e29696","order_by":0,"name":"Oleh 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09:08:12","extension":"xml","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":135006,"visible":true,"origin":"","legend":"","description":"","filename":"2aa0254e2b0543d3824530bba17efc051structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7920896/v1/90610ebfdf6ad00498f15351.xml"},{"id":96465491,"identity":"b116030a-0cb7-45a7-a0b8-c642a1c2a315","added_by":"auto","created_at":"2025-11-21 11:14:23","extension":"html","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":144885,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7920896/v1/d6965ecb28d822f277a07cc6.html"},{"id":96465476,"identity":"4b3c793b-ea49-4861-8875-1b3cecec2048","added_by":"auto","created_at":"2025-11-21 11:14:23","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":32358,"visible":true,"origin":"","legend":"\u003cp\u003eAmount of food eaten (A), contents of glucose (B), glycogen (C), trehalose (D), TAG (E), and lipids (F) in flies that express \u003cem\u003eNDI1\u003c/em\u003e and \u003cem\u003eAOX\u003c/em\u003e. Values are represented as mean ± SEM for 4-5 independent replicates. Asterisks show significantly different groups with \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7920896/v1/5a1bec6bab802d6e46f80052.png"},{"id":96465477,"identity":"87030bd0-1234-41a7-a59f-aca041eebf15","added_by":"auto","created_at":"2025-11-21 11:14:23","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":20019,"visible":true,"origin":"","legend":"\u003cp\u003eContents of carbonyl proteins (A), lipid peroxides (B), low- (C) and high- molecular-mass thiol-containing compounds (D) in flies that express \u003cem\u003eNDI1\u003c/em\u003e and \u003cem\u003eAOX\u003c/em\u003e. Values are represented as mean ± SEM for 4-5 independent replicates. Asterisks show significantly different groups with \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7920896/v1/389ae00f02044dd307b85fb3.png"},{"id":96465478,"identity":"57a8728f-21c6-4b48-8f54-845e2e14243e","added_by":"auto","created_at":"2025-11-21 11:14:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":16446,"visible":true,"origin":"","legend":"\u003cp\u003eActivities of antioxidant enzymes SOD (A), catalase (B), and glutathione S-transferase (C) in flies that overexpress yeast \u003cem\u003eNDI1\u003c/em\u003e and \u003cem\u003eAOX\u003c/em\u003e. Values are represented as mean ± SEM for 4-5 independent replicates. Asterisks show significantly different groups with \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7920896/v1/2494eda9ee6470c6d4d4a0e2.png"},{"id":96465480,"identity":"83f31219-c1c3-4b34-826c-7274ad624aef","added_by":"auto","created_at":"2025-11-21 11:14:23","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":76790,"visible":true,"origin":"","legend":"\u003cp\u003eActivities of metabolic enzymes glutathion reductase (A), glucose-6-phosphate dehydrogenase (G6PDH) (B), isocitrate dehydrogenase (IDH) (C), lactate dehydrogenase (LDH), malate dehydrogenase (MDH) and alanine aminotransferase (ALT) in flies overexpressing yeast \u003cem\u003eNDI1\u003c/em\u003eand \u003cem\u003eAOX\u003c/em\u003e. Data are presented as mean ± SEM from 4-5 independent replicates. Asterisks indicate statistically significant differences between groups (\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7920896/v1/664c3780aed9a8b76a44559c.png"},{"id":101690412,"identity":"8e65b41a-aa34-4e02-8aa7-c72e512b2183","added_by":"auto","created_at":"2026-02-02 16:01:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":850494,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7920896/v1/f5a47cb4-2eb1-4ad7-a6ea-42408142132e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Expression of yeast NADH dehydrogenase and ascidian alternative oxidase affects metabolism and free radical processes in Drosophila","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eNon-proton pumping NADH dehydrogenase and ubiquinol oxidase (also known as alternative oxidase) are alternative components of the respiratory systems, present in bacteria, fungi, plants, and some animals [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. However, these enzymes are absent in the respiratory systems of mammals. From the late 1990s and early 2000s, alternative NADH dehydrogenase and oxidase are studied as potential tools for gene therapy of mitochondrial diseases, respectively [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. It has also been found that being heterologically expressed, these enzymes may indirectly influence metabolism and signaling of a host organism. Of the observed effects, not directly related to respiration, are the ability of alternative NADH dehydrogenase (ANDH) to prolong lifespan and activate xenobiotic defense systems [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In turn, alternative oxidase (AOX) was found to affect developmental signaling [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], cell migration [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], reproductive function [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], nutrient utilization [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], resistance to a number of environmental toxins and cold [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Both enzymes have been found to modulate oxygen sensing and signaling mediated by reactive oxygen species (ROS) [\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Thus, phenotyping of the organisms that express AOX or ANDH, or both, brings new insights regarding the evolution of respiratory chain, as well as regarding metabolic signaling. Reactions catalyzed by AOX and ANDH consume substrates of conventional proton-pumping respiratory complexes. In some instances, it has been found they compete with those complexes for substrates [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. This competition may lead to a decrease in the rates of adenosine triphosphate (ATP) production in mitochondria. Since ATP is required for various biosynthetic processes, one may expect that organisms that express AOX and/or ANDH will accumulate less metabolic stores, such as glycogen or triglycerides. Interestingly, AOX tackles part of ubiquinol oxidation, decreasing ubiquinol flow through ubiquinol-cytochrome c reductase, or complex III of the mitochondrial respiratory chain. In turn, ANDH reduces part of ubiquinone pool, decreasing ubiquinone flow through proton-pumping NADH dehydrogenase, or complex I. Both complex I and complex III are capable of forming semiquinone anion-radical, a free radical quinone form [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The unpaired electron from the semiquinone anion-radical can be passed to molecular oxygen yielding superoxide anion-radical. The latter is then converted, giving complete lineage of ROS that oxidize lipid acyl chains, proteins, and nucleic acids. These molecules are constantly renewed, so their oxidative modification does not seriously affect cells\u0026rsquo; function, until ROS levels exceed a certain threshold [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. It is believed that AOX and ANDH, while oxidize ubiquinol or reduce ubiquinone, respectively, do not produce ROS, owing to difference in catalytic mechanism which minimizes probability of forming the semiquinone radical [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Neverthelss, several studies show that this may not be true at least for ANDH. When ANDH is co-expressed with functional complex I, it may promote ROS production by contributing to the increase in ubiquinol pool and boosting probability of reverse electron transport through complex I [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. These peculiarities support a need in phenotyping of the \u003cem\u003eAOX\u003c/em\u003e- and \u003cem\u003eANDH\u003c/em\u003e-expressing animals. Fruit flies expressing \u003cem\u003eANDH\u003c/em\u003e were already partially characterized in terms of oxidative stress markers and antioxidant, and related enzymes [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. However, antioxidant defense and oxidative stress indices were not systematically described for animals expressing AO and ANDH. Present study addresses the following questions: do the alternative oxidase from tunicate \u003cem\u003eCiona intestinalis\u003c/em\u003e (AOX) and alternative NADH dehydrogenase NDI1 from the budding yeast \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e heterologously expressed in the fruit fly \u003cem\u003eDrosophila melanogaster\u003c/em\u003e 1) affect nutrient utilization \u0026ndash; food consumption and metabolic stores? 2) prevent oxidative damage, affect antioxidant defense, and protect sensitive enzymes from oxidative modification? To test this, we measured how much food is consumed by fruit flies that express \u003cem\u003eNDI1\u003c/em\u003e and \u003cem\u003eAOX\u003c/em\u003e, as well as glycogen, trehalose, triglyceride and total lipid, and glucose content in the fruit fly body. To assess oxidative damage, we measured the levels of protein carbonyls, lipid peroxides, as well as low and high molecular thiol-containing compounds. Antioxidant defense markers were activities of superoxide dismutase, catalase, glutathione S-transferase, glutathione reductase, and NADP-reducing enzymes, such as glucose 6-phosphate dehydrogenase, NADP-dependent malate and isocitrate dehydrogenases. The three later enzymes are also potential markers of oxidative modification, since were found sensitive to it [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Additional markers of oxidative modification were lactate dehydrogenase and alanine transaminase, enzymes that help recycle anaerobic byproducts such as lactate or alanine into energy-yielding metabolites during metabolic shuttling between tissues.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eFly Husbandry and Transgenic Flies\u003c/h2\u003e\u003cp\u003eFlies were cultured on standard yeast-molasses medium, composed of dry yeast (5%), corn (6.1%), molasses (7.5%), nipagin (0.18%), and propionic acid (0.4%) at 25\u0026deg;C at 25\u0026deg;C under a 12:12 light:dark cycle [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The GAL4-UAS system was used to generate experimental flies. Flies of \u003cem\u003eDa-GAL4\u003c/em\u003e, \u003cem\u003eUAS-NDI1\u003c/em\u003e, \u003cem\u003eUAS-AOX\u003c/em\u003e strains were kindly provided by Professor Alberto Sanz (Glasgow, UK). \u003cem\u003eUAS-NDI1\u003c/em\u003e flies were generated by Sanz and coauthors [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] and \u003cem\u003eUAS-AOX\u003c/em\u003e flies by Fernandez-Ayala et al. [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] earlier. All flies were on \u003cem\u003ew\u003c/em\u003e\u003csup\u003e\u003cem\u003eDah\u003c/em\u003e\u003c/sup\u003e background. Experimental flies were generated by crossing \u003cem\u003eda-GAL4\u003c/em\u003e females with respective males. Cross of \u003cem\u003eda-GAL4\u003c/em\u003e females with \u003cem\u003ew\u003c/em\u003e\u003csup\u003e\u003cem\u003eDah\u003c/em\u003e\u003c/sup\u003e males was used to generate flies of control genotype \u003cem\u003eda-GAL4\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;\u003cem\u003ew\u003c/em\u003e\u003csup\u003e\u003cem\u003eDah\u003c/em\u003e\u003c/sup\u003e (CON). Respectively, flies expressing yeast \u003cem\u003eNDI1\u003c/em\u003e were of \u003cem\u003eda-GAL4\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;\u003cem\u003eUAS-NDI1\u003c/em\u003e genotype (NDI) and alternative oxidase \u003cem\u003eda-GAL4\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;\u003cem\u003eUAS-AOX\u003c/em\u003e genotype (AOX). The resulting eggs (100 per vial) were allowed to develop at 25\u0026deg;C. Newly enclosed flies were kept of fresh food for additional 4 days.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eFeeding\u003c/h3\u003e\n\u003cp\u003eFood consumption by a single fly was measured by CApillary FEeder (CAFE) assay [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Experimental flies were kept in 1.5 vial supplemented with 5 \u0026micro;L capillary tube filled with food containing 5% yeast extract, 5% sucrose, 0.1% propionic, and 0.01% phosphoric acid. Capillaries were changed every day and the amount of food consumed was measured over a period of four days. Vials were kept in closed boxes with distilled water on the bottom to maintain high humidity for evaporation prevention. Three non-feeding vials were monitored for changes in volume to control for evaporation. Ten flies per genotype were tested.\u003c/p\u003e\n\u003ch3\u003eMetabolic parameters\u003c/h3\u003e\n\u003cp\u003eMetabolic parameter such as glucose, trehalose, glycogen, triacylglyceride (TAG) and lipids were measured as described earlier by Koliada and coauthors [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Briefly, ten frozen flies per sample were weighted and homogenized with 50 mM phosphate buffered saline (PBS) supplemented by 0.09% sodium azide (pH 7.4) at a 1:10 w:v ratio, heat denatured as described and centrifuged for 15 min at 13000g and 4\u0026deg;C. Resulted supernatant were transferred to new vial and used for further determinations. Glycogen was converted into glucose by incubation of the supernatants with 5.6 U of amyloglucosidase from \u003cem\u003eAspergillus niger\u003c/em\u003e (Merck KGaA, Cat No. 10115) during 4 h at 37\u0026deg;C. Measurements were performed using a colorimetric glucose test kit (Gliukoza-Mono, \u0026laquo;Reagent\u0026raquo;, Dnipro) that exploits a combined glucose oxidase and peroxidase principle. Samples were then measured at wavelength 540 nm.\u003c/p\u003e\u003cp\u003eFor TAG determination reweighted flies were homogenized in 200 mM PBST (phosphate buffered saline with 0.05% Triton X100), boiled for 5 min and centrifuged (13000 g, 10 min). Resulting supernatants were used for TAG assay with the Liquick Cor-TG diagnostic kit (Cormay, Poland).\u003c/p\u003e\n\u003ch3\u003eActivities of enzymes and oxidative stress indices\u003c/h3\u003e\n\u003cp\u003eFlies were homogenized using a Potter-Elvehjem glass/glass homogenizer (1:10 w/v) in 50 mM KPi (pH 7.5) containing 0.5 mM ethylenediaminetetraacetic acid and 1 mM phenylmethylsulfonyl fluoride and centrifuged 15 min at 16000g, and 4\u0026deg;C (Eppendorf 5415R centrifuge). Supernatants were collected and used for the determination of enzymatic activities, protein carbonyls, levels of high- and low-molecular-mass thiol-containing compounds. Protein content was measured by the Bradford method with serum bovine albumin used as the standard [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The activities of catalase, superoxide dismutase (SOD), glucose 6-phosphate dehydrogenase (G6PDH), NADP-dependent isocitrate dehydrogenase (IDH), and glutathione-\u003cem\u003eS\u003c/em\u003e-transferase (GST), glutathione reductase (GR), lactate dehydrogenase (LDH), malate dehydrogenase (MDH) were measured by methods described earlier [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The levels of protein carbonyls, lipid peroxides, high- and low-molecular-mass thiol-containing compounds were assayed as described by Lozinsky et al. [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eStatistical Procedures\u003c/h3\u003e\n\u003cp\u003eStatistical processing of the data was performed using GraphPad Prism 8 software. One-way ANOVA followed by Tukey test was used to determine a significant difference between groups. Data are shown as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM and \u003cem\u003ep\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered as significance.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003eAlternative respiratory chain enzymes, such as NDI1 and AOX, use the same substrates as proton-pumping electron-transport chain (ETC) complexes I and III, respectively. Operation of NDI1 and AOX in the fruit fly electron-transport chain might decrease flow of reduced nicotinamide adenine dinucleotide (NADH) and ubiquinol through these complexes, thus affecting rates of protons pumped and ATP synthesis. Since ATP is spent for biosynthetic processes, one may expect \u003cem\u003eNDI1\u003c/em\u003e and/or \u003cem\u003eAOX\u003c/em\u003e expression affect accumulation of metabolic stores, such as glycogen and triacylglycerols [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Nevertheless, \u003cem\u003eNDI1\u003c/em\u003e-expressing flies did not show any significant difference in the levels of glycogen, TAG, total lipids, trehalose, and glucose in the body, compared to the wild type flies (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-F). On the other hand, \u003cem\u003eAOX\u003c/em\u003e expression led to increased glycogen while decreased body glucose levels in flies as compared to both control and \u003cem\u003eNDI1\u003c/em\u003e-expressing flies (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, C; Tukey test: \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). To explore potential compensatory mechanisms preserving metabolic stores, we assessed food intake in transgenic lines. \u003cem\u003eNDI1\u003c/em\u003e-expressing flies displayed increased food consumption versus control (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), indicating that elevated nutrient uptake may help sustain energy-dependent biosynthetic processes.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAn important outcome of \u003cem\u003eNDI1\u003c/em\u003e and \u003cem\u003eAOX\u003c/em\u003e expression is an expected attenuation of ROS generation. Mitochondria contribute to ROS formation through one-electron reduction or oxidation of ubiquinone by complexes I and III of the ETC. The electron from ubiquinone is then passed to dioxygen, yielding superoxide anion-radical. The data on the capability of NDI 1 or AOX to prevent ROS formation are still ambiguous. Re-testing the indices of antioxidant defense and oxidative damage in the current study showed that expression of either \u003cem\u003eNDI1\u003c/em\u003e or \u003cem\u003eAOX\u003c/em\u003e led to increase in protein carbonyls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA; Tukey test: \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), although \u003cem\u003eAOX\u003c/em\u003e-expressing flies showed a two-fold lower level of lipid peroxides compared to the wild type (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB; Tukey test: \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.041). There were no differences between the strains studied in the levels of high and low molecular mass thiol-containing substances (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, D).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eNeither superoxide dismutase (SOD), nor catalase activities were affected, whereas glutathione-S-transferase activity was elevated by 19% in \u003cem\u003eNDI1\u003c/em\u003e-expressing flies as compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e; Tukey test: \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.030). On the other hand, glutathione reductase was elevated by ⁓50% in \u003cem\u003eAOX\u003c/em\u003e-expressing flies as compared to both control and \u003cem\u003eNDI1\u003c/em\u003e-expressing flies (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA; Tukey test: \u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05). Interestingly, \u003cem\u003eNDI1\u003c/em\u003e-expressing flies had significantly lower activities of glucose-6-phosphate dehydrogenase by 32% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB; Tukey test: \u003cem\u003ep\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.027) and NADP-dependent isocitrate dehydrogenase by 24% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC; Tukey test: \u003cem\u003ep\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.049). Flies of both experimental cohorts had about 1.5-fold lower activity of lactate dehydrogenase compared to that of the wild type (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD; Tukey test: \u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.02). MDH and AST activities were not affected either by \u003cem\u003eNDI1\u003c/em\u003e or \u003cem\u003eAOX\u003c/em\u003e overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, F).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eAs was mentioned above, \u003cem\u003eNDI1\u003c/em\u003e and \u003cem\u003eAOX\u003c/em\u003e may deprive substrates of proton-pumping ETC complexes I and III, respectively. Indeed, competition for substrates has been observed between plant alternative NADH dehydrogenases heterologously expressed in human fibroblasts [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. However, such substrate competition has not been observed for NDI1 [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The co-expression of alternative NADH dehydrogenase with proton-pumping NADH dehydrogenase may lead to about 30% decrease in the levels of ATP produced [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In turn, ATP is needed for various biosynthetic processes, including protein, lipid, and glycogen synthesis [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The current study shows that \u003cem\u003eNDI1\u003c/em\u003e expression did actually not influence the levels of metabolic stores such as TAG, glycogen, and trehalose. However, increased food consumption we observe (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF) may compensate for a possible biosynthetic shortage if the latter takes place. Moreover, mitochondria contribute to the appetite regulation by affecting release of adipokinetic hormone as well as several other hormones that promote food consumption [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAn interesting effect we observe is the decrease of glucose level while the increase in glycogen in \u003cem\u003eAOX\u003c/em\u003e-expressing flies (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, E). Similar to NDI1, AOX shunts ETC by-passing its proton-pumping complexes. Thus, we expected \u003cem\u003eAOX\u003c/em\u003e expression in fruit flies might lead to a decrease in the levels of metabolic stores by lowering efficiency of oxidative phosphorylation. Nevertheless, we have obtained an opposite data, which can be connected with pleiotropic effects of AOX on cell physiology. Specifically, AOX is believed to decrease generation of ROS by ETC complex III [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Recent findings imply AOX may directly or indirectly affect cellular calcium homeostasis [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] and apoptosis [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Cellular calcium is an important regulator of insulin-like peptide release in insects [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Calcium may also affect downstream insulin signaling pathway, specifically glycogen synthase kinase 3β [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. It is remarkable, that calcium signaling has not yet been sufficiently studied in \u003cem\u003eAOX\u003c/em\u003e-expressing organisms. Nevertheless, observations from different studies, suggest AOX might promote either calcium efflux from or hinder calcium transport into mitochondria [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. This can be mediated by the ability of AOX to either lessen ROS generation or membrane potential. In turn, ROS and membrane potential regulate mitochondrial permeability transition and calcium transport.\u003c/p\u003e\u003cp\u003eAs mentioned above, both NDI1 and AOX may attenuate ROS generation through by-passing ETC complexes I and III that produce ubisemiquinone anion-radical as a byproduct. However, despite many studies that showed such attenuation in isolated mitochondria, this is not a strict rule. Alternative NADH dehydrogenase were shown to increase ROS production by increasing ubiquinone pool, consequently promoting reverse electron transport through ETC complex I [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. In our current study, both \u003cem\u003eNDI1\u003c/em\u003e and \u003cem\u003eAOX\u003c/em\u003e expression in flies lead to an increase in the protein carbonyl levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) with a concomitant decrease in lipid peroxide (LOOH) levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The latter suggests a fast breakdown of LOOH that may lead to formation of the end-products, predominantly aldehydes. Subsequently, aldehydes produced after LOOH breakdown interact with proteins leading to formation of additional carbonyl groups [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Formation of LOOH and their breakdown are hardly trackable dynamic processes. The breakdown often occurs after formation of multiple endoperoxide within one fatty acyl chain [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Afterward, the end-products can be conjugated with glutathione or other thiols or modify proteins. We saw that \u003cem\u003eNDI1\u003c/em\u003e or \u003cem\u003eAOX\u003c/em\u003e expression did not affect thiol levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, D). It allows to assume that in our case NDI1 and AOX indeed promote ROS production rather than abolish it. However, NDI1 or AOX likely promote intensive formation of short-living ROS, such as hydroxyl radical and/or superoxide anion-radical, that oxidize fatty acyl chains of membrane lipids. The latter may break down leading to the formation of aldehyde-containing end-products that interact with neighboring proteins. The enzymes such as glucose 6-phosphate dehydrogenase (G6PDH), isocitrate dehydrogenase (IDH), malate dehydrogenase (MDH), and lactate dehydrogenase (LDH) can be examples of such proteins. Our results suggest this can be the case for \u003cem\u003eNDI1\u003c/em\u003e-expressing flies since activities of these enzymes was lower compared to those in the wild type flies [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. A similar trend can also be seen in \u003cem\u003eAOX\u003c/em\u003e-expressing flies [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eHow it could be possible that expression of \u003cem\u003eNDI1\u003c/em\u003e and \u003cem\u003eAOX\u003c/em\u003e leads to an increase in oxidative modifications? Reactive oxygen species are important signaling molecules whose level indicates about well-being of mitochondria. An elevation of ROS levels often results in activation of antioxidant system [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. It has been shown that reactions occurred at ETC complex III promote formation of superoxide anion-radical at the mitochondrial matrix side and intermembrane mitochondrial space [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The superoxide released in the intermembrane space passes signal to cytosol and indirectly activates antioxidant defense [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Bypassing complex III by AOX decreases the level superoxide released to the intermembrane space thus preventing the activation of antioxidant system. Indeed, the activity of catalase and superoxide dismutase in \u003cem\u003eAOX\u003c/em\u003e-expressing flies is the same as in control [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. A higher glutathione reductase activity in these experimental flies (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) implies a compensatory response to ROS that confer protein carbonylation. Along with interaction between end-products of lipid peroxidation and proteins, this response may account for the respectively lower level of LOOH in AOX flies (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eLactate dehydrogenase (LDH) is an enzyme that allows NAD\u003csup\u003e+\u003c/sup\u003e regeneration for persisting glycolysis. Expression of LDH is regulated by multiple transcription factors, including estrogen-related receptor [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e] and hypoxia-inducible factor [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Expression of LDH can also be suppressed by 20-hydroxyecdysone (20E) [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Interestingly, in insects, several cytochromes of P450 type involved in ecdysone biosynthesis are localized to mitochondria [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Cytochromes P450 are heme-containing proteins. Reactions catalyzed by NDI1 and AOX by-pass those catalyzed by iron-containing ETC complexes. Thus, these two alternative respiratory enzymes may favor redirection of heme and iron incorporation from ETC complexes to the mitochondrial cytochromes P450. In turn, this may enable an increase in 20E titers and downregulation of LDH.\u003c/p\u003e\u003cp\u003eAnother possible mechanism of LDH downregulation in \u003cem\u003eNDI1\u003c/em\u003e- and \u003cem\u003eAOX\u003c/em\u003e-expressing flies is activation of dTIS11, a \u003cem\u003eD. melanogaster\u003c/em\u003e tristetraprolin homolog [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. The latter is activated by NAD\u003csup\u003e+\u003c/sup\u003e [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Both, NDI1 and AOX may favor NADH depletion since they are not regulated by membrane potential, unlike ETC complexes I and III.\u003c/p\u003e\u003cp\u003eLast but not the least, LDH is strongly activated by hypoxia whereas reoxygenation leads to its rapid downregulation [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. This regulation is mediated by \u003cem\u003eD. melanogaster\u003c/em\u003e homolog of hypoxia-inducible factor 1α (HIF-1α) [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. In turn, HIF-1α can be activated by ROS [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e] whereas NDI1 and AOX operation may decrease ROS levels. Interestingly, type II NADH dehydrogenase NDX from the seq squirt \u003cem\u003eCiona intestinalis\u003c/em\u003e conferred opposite effects on G6PDH and LDH, while not affecting activities of NADP-specific MDH and IDH [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Nevertheless, both \u003cem\u003eNDI1\u003c/em\u003e and \u003cem\u003eAOX\u003c/em\u003e expression lead to an increase in glutathione S-transferase activity in flies. These discrepancies may imply the influence of background, sex [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e], and peculiarities of the enzymes from different sources on the pleiotropic effects of alternative oxidase and NADH dehydrogenase.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eANDH \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Alternative NADH dehydrogenase\u003c/p\u003e\n\u003cp\u003eNDI1\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;NADH dehydrogenase I\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAOX \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Alternative oxidase\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eROS\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Reactive oxygen species\u003c/p\u003e\n\u003cp\u003eATP \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Adenosine triphosphate\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCAFE assay \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;CApillary FEeder assay\u003c/p\u003e\n\u003cp\u003eTAG \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Triacylglyceride\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSOD \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Superoxide dismutase\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;G6PDH \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Glucose 6-phosphate dehydrogenase\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIDH\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Isocitrate dehydrogenase\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGST \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Glutathione-S-transferase\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGR \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Glutathione reductase\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLDH \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Lactate dehydrogenase\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMDH \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Malate dehydrogenase\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eETC \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Electron-transport chain\u003c/p\u003e\n\u003cp\u003eLOOH \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Lipid peroxide\u003c/p\u003e\n\u003cp\u003eHIF-1\u0026alpha; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Hypoxia-inducible factor 1\u0026alpha;\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eO.L. and D.G. developed the idea for the research and edited the manuscript. D.G. and I.Y. performed all the experiments and collected the data. O.S. and D.G. analyzed and interpreted the data and were major contributors to the writing of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data reported in this study are available from the corresponding authors upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study received no external funding.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMcDonald AE, Gospodaryov DV. Alternative NAD(P)H dehydrogenase and alternative oxidase: Proposed physiological roles in animals. 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EMBO Mol Med. 2019;11(1):e9456.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSzibor M, Gizatullina Z, Gainutdinov T, Endres T, Debska-Vielhaber G, Kunz M, Karavasili N, Hallmann K, Schreiber F, Bamberger A, Schwarzer M, Doenst T, Heinze HJ, Lessmann V, Vielhaber S, Kunz WS, Gellerich FN. Cytosolic, but not matrix, calcium is essential for adjustment of mitochondrial pyruvate supply. J Biol Chem. 2020;295(14):4383\u0026ndash;97.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eN\u0026auml;ssel DR, Vanden Broeck J. Insulin/IGF signaling in \u003cem\u003eDrosophila\u003c/em\u003e and other insects: factors that regulate production, release and post-release action of the insulin-like peptides. Cell Mol Life Sci. 2016;73(2):271\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLin JL, Lin PL, Gu SH. Phosphorylation of glycogen synthase kinase-3beta in relation to diapause processing in the silkworm, \u003cem\u003eBombyx mori\u003c/em\u003e. J Insect Physiol. 2009;55(6):593\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLi J, Yang S, Wu Y, Wang R, Liu Y, Liu J, Ye Z, Tang R, Whiteway M, Lv Q, Yan L. Alternative Oxidase: From Molecule and Function to Future Inhibitors. ACS Omega. 2024;9(11):12478\u0026ndash;99.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eScialo F, Mallikarjun V, Stefanatos R, Sanz A. Regulation of lifespan by the mitochondrial electron transport chain: reactive oxygen species-dependent and reactive oxygen species-independent mechanisms. Antioxid Redox Signal. 2013;19(16):1953\u0026ndash;69.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMedina-Navarro R, Nieto-Aguilar R, Alvares-Aguilar C. 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Expression of multiple copies of mitochondrially targeted catalase or genomic Mn superoxide dismutase transgenes does not extend the life span of \u003cem\u003eDrosophila melanogaster\u003c/em\u003e. Free Radic Biol Med. 2010;49(12):2028\u0026ndash;31.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTennessen JM, Baker KD, Lam G, Evans J, Thummel CS. The \u003cem\u003eDrosophila\u003c/em\u003e estrogen-related receptor directs a metabolic switch that supports developmental growth. Cell Metab. 2011;13(2):139\u0026ndash;48.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGorr TA, Tomita T, Wappner P, Bunn HF. Regulation of \u003cem\u003eDrosophila\u003c/em\u003e hypoxia-inducible factor (HIF) activity in SL2 cells: identification of a hypoxia-induced variant isoform of the HIFalpha homolog gene similar. J Biol Chem. 2004;279(34):36048\u0026ndash;58.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNoguchi K, Yokozeki K, Tanaka Y, Suzuki Y, Nakajima K, Nishimura T, Goda N. Sima, a \u003cem\u003eDrosophila\u003c/em\u003e homolog of HIF-1α, in fat body tissue inhibits larval body growth by inducing Tribbles gene expression. Genes Cells. 2022;27(2):145\u0026ndash;51.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTennessen JM, Thummel CS. Coordinating growth and maturation - insights from \u003cem\u003eDrosophila\u003c/em\u003e. Curr Biol. 2011;21(18):R750\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePetryk A, Warren JT, Marqu\u0026eacute;s G, Jarcho MP, Gilbert LI, Kahler J, Parvy JP, Li Y, Dauphin-Villemant C, O'Connor MB. Shade is the \u003cem\u003eDrosophila\u003c/em\u003e P450 enzyme that mediates the hydroxylation of ecdysone to the steroid insect molting hormone 20-hydroxyecdysone. Proc Natl Acad Sci U S A. 2003;100(24):13773\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSchellens S, Lenaerts C, P\u0026eacute;rez Baca MDR, Cools D, Peeters P, Marchal E, Vanden Broeck J. Knockdown of the Halloween Genes spook, shadow and shade Influences Oocyte Development, Egg Shape, Oviposition and Hatching in the Desert Locust. Int J Mol Sci. 2022;23(16):9232.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJoe Y, Chen Y, Park J, Kim HJ, Rah SY, Ryu J, Cho GJ, Choi HS, Ryter SW, Park JW, Kim UH, Chung HT. Cross-talk between CD38 and TTP Is Essential for Resolution of Inflammation during Microbial Sepsis. Cell Rep. 2020;30(4):1063\u0026ndash;e10765.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ede Toeuf B, Soin R, Nazih A, Dragojevic M, Jurėnas D, Delacourt N, Vo Ngoc L, Garcia-Pino A, Kruys V, Gueydan C. ARE-mediated decay controls gene expression and cellular metabolism upon oxygen variations. Sci Rep. 2018;8(1):5211.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHamanaka RB, Chandel NS. Mitochondrial reactive oxygen species regulate hypoxic signaling. Curr Opin Cell Biol. 2009;21(6):894\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGospodaryov DV, Strilbytska OM, Semaniuk UV, Perkhulyn NV, Rovenko BM, Yurkevych IS, Barata AG, Dick TP, Lushchak OV, Jacobs HT. Alternative NADH dehydrogenase extends lifespan and increases resistance to xenobiotics in \u003cem\u003eDrosophila\u003c/em\u003e. Biogerontology. 2020;21(2):155\u0026ndash;171. Erratum in: Biogerontology. 2020;21(2):173\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLushchak O, Strilbytska O, Storey KB. Gender-specific effects of pro-longevity interventions in \u003cem\u003eDrosophila\u003c/em\u003e. Mech Ageing Dev. 2023;209:111754.\u003c/span\u003e\u003c/li\u003e\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":"bmc-research-notes","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"resn","sideBox":"Learn more about [BMC Research Notes](http://bmcresnotes.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/resn/default.aspx","title":"BMC Research Notes","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"NADH dehydrogenase, alternative oxidase, metabolism, oxidative stress","lastPublishedDoi":"10.21203/rs.3.rs-7920896/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7920896/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective:\u003c/strong\u003e The study aimed to investigate the effect of overexpression of alternative mitochondrial enzymes such as yeast NADH dehydrogenase I (NDI1) and alternative oxidase (AOX) on the metabolism, oxidative stress and feeding behavior of the fruit fly \u003cem\u003eDrosophila melanogaster\u003c/em\u003e. Experimental flies with expression of \u003cem\u003eNDI1\u003c/em\u003e or \u003cem\u003eAOX\u003c/em\u003e were generated using genetic crosses based on the \u003cem\u003eGAL4-UAS\u003c/em\u003e system.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eFlies with \u003cem\u003eNDI1\u003c/em\u003e expression showed increased food consumption, markers of oxidative stress (elevated carbonyl protein content), and increased activity of the detoxification enzyme glutathione-\u003cem\u003eS\u003c/em\u003e-transferase, along with decreased activity of key metabolic enzymes including isocitrate dehydrogenase, lactate dehydrogenase, and glucose-6-phosphate dehydrogenase. In contrast, \u003cem\u003eAOX\u003c/em\u003e-expressing flies had reduced lactate dehydrogenase (LDH) activity, decreased level of lipid peroxides, increased glutathione reductase activity. Lower free glucose levels with elevated glycogen stores was found in \u003cem\u003eAOX\u003c/em\u003e-expressing flies. The results indicate that the alternative electron transport chain radically transforms energy and redox metabolism. In particular, \u003cem\u003eNDI1\u003c/em\u003eexpression could lead to increased energy demand and compensatory hyperphagia. In contrast, AOX bypasses the main steps of proton gradient generation, reducing superoxide generation.\u003c/p\u003e","manuscriptTitle":"Expression of yeast NADH dehydrogenase and ascidian alternative oxidase affects metabolism and free radical processes in Drosophila","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-21 11:14:18","doi":"10.21203/rs.3.rs-7920896/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-22T11:44:55+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-04T01:19:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"93033331165723571594352650251786822602","date":"2025-11-24T01:57:11+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-11T23:13:51+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-10-27T10:52:32+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-27T00:09:48+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-27T00:09:37+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Research Notes","date":"2025-10-22T07:20:58+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-research-notes","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"resn","sideBox":"Learn more about [BMC Research Notes](http://bmcresnotes.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/resn/default.aspx","title":"BMC Research Notes","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b6510de2-f2f0-46f7-8700-61c377401c48","owner":[],"postedDate":"November 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-02T16:00:08+00:00","versionOfRecord":{"articleIdentity":"rs-7920896","link":"https://doi.org/10.1186/s13104-026-07692-y","journal":{"identity":"bmc-research-notes","isVorOnly":false,"title":"BMC Research Notes"},"publishedOn":"2026-01-31 15:57:58","publishedOnDateReadable":"January 31st, 2026"},"versionCreatedAt":"2025-11-21 11:14:18","video":"","vorDoi":"10.1186/s13104-026-07692-y","vorDoiUrl":"https://doi.org/10.1186/s13104-026-07692-y","workflowStages":[]},"version":"v1","identity":"rs-7920896","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7920896","identity":"rs-7920896","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}