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
TL;DR
This study shows how introducing alternative mitochondrial enzymes in fruit flies affects their metabolism and oxidative stress levels.
Contribution
The paper demonstrates the metabolic and redox impacts of expressing yeast NDI1 and ascidian AOX in Drosophila.
Findings
NDI1-expressing flies showed increased food intake and oxidative stress markers.
AOX-expressing flies had reduced lipid peroxides and altered glucose metabolism.
NDI1 may increase energy demand, while AOX could reduce superoxide production.
Abstract
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. Female 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 dehydrogenases of isocitrate, lactate, and glucose-6-phosphate. In contrast, AOX-expressing flies had reduced lactate dehydrogenase activity, decreased levels of lipid peroxides, and increased glutathione reductase activity. Lower…
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Taxonomy
TopicsGenetics, Aging, and Longevity in Model Organisms · Invertebrate Immune Response Mechanisms · Coenzyme Q10 studies and effects
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 were 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 the 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 the 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 that 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 the 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 a 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 oxidizing ubiquinol or reducing ubiquinone, respectively, do not produce ROS, owing to a difference in catalytic mechanism, which minimizes the probability of forming the semiquinone radical [18]. Nevertheless, 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 the probability of reverse electron transport through complex I [19]. These peculiarities support a need for phenotyping of the AOX- and ANDH-expressing animals. Fruit flies expressing ANDH were already partially characterized in terms of oxidative stress markers and antioxidants, and related enzymes [20]. However, antioxidant defense and oxidative stress indices were not systematically described for animals expressing AO and ANDH. The present study addresses the following questions: do the alternative oxidase from tunicate Ciona intestinalis (AOX) and the 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-weight 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 they were found to be 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. While AOX and NDI1 have each been studied in Drosophila in the context of mitochondrial function and stress responses [6, 20, 22, 23], a direct comparison of their metabolic and oxidative effects under identical genetic and experimental conditions has not been performed. Here, we systematically investigate AOX and NDI1 expression in the same driver background, focusing on energy storage, feeding behavior, enzyme activities, and late-stage oxidative damage.
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 [23]. 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. [24] earlier. All flies were on w^Dah^ background. Experimental female 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, females expressing yeast NDI1 were of da-GAL4 > UAS-NDI1 genotype (NDI1) 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 on fresh food for an additional 4 days.
Feeding
Food consumption by a single fly was measured by CApillary FEeder (CAFE) assay [25]. Experimental flies were kept in a 1.5 vial supplemented with a 5 µL capillary tube filled with food containing 5% yeast extract, 5% sucrose, 0.1% propionic, and 0.01% phosphoric acid. Ten females per genotype were tested.
Metabolic parameters
Metabolic parameters such as glucose, trehalose, glycogen, triacylglyceride (TAG) and lipids were measured as described earlier by Koliada and coauthors [24]. Briefly, ten frozen flies per sample were weighed 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 13,000 g and 4 °C. The resulting supernatant was transferred to a 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 during 4 h at 37 °C. Measurements were performed using a colorimetric glucose test kit. Samples were then measured at a wavelength of 540 nm.
For TAG determination, reweighted flies were homogenized in 200 mM PBST, boiled for 5 min and centrifuged. Resulting supernatants were used for TAG assay with the Liquick Cor-TG diagnostic. Metabolic parameters were measured in 4–5 independent biological replicates.
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 16,000 g, and 4 °C. Supernatants were collected and used for the determination of enzymatic activities, protein carbonyls, and 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 [26]. The activities of catalase, superoxide dismutase, glutathione-S-transferase, alanine aminotransferase, glutathione reductase, and dehydrogenases of glucose-6-phosphate, isocitrate, and malate were measured by methods described earlier [27, 28]. The levels of protein carbonyls, lipid peroxides, high- and low-molecular-mass thiol-containing compounds were assayed as described by Lozinsky et al. [29]. Activities of enzymes and oxidative stress indices were measured in 4–5 independent biological replicates.
Statistical procedures
Statistical processing of the data was performed using GraphPad Prism 8 software. One-way ANOVA followed by the Tukey test was used to determine a significant difference between groups. Data are shown as mean ± SEM and p value < 0.05 was considered significant.
Results
Alternative respiratory chain enzymes, such as NDI1 and AOX, use the same substrates as proton-pumping electron-transport chain complexes I and III, respectively. Operation of NDI1 and AOX in the fruit fly electron-transport chain might decrease the flow of 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 the accumulation of metabolic stores, such as glycogen and triacylglycerols [30]. 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 control flies (Fig. 1B–F). On the other hand, AOX expression led to increased glycogen while decreased body glucose levels in female flies as compared to both control and NDI1-expressing females (Fig. 1B, C; 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. 1A), indicating that elevated nutrient uptake may help sustain energy-dependent biosynthetic processes.
Fig. 1. Amount of food eaten (A), contents of glucose (B), glycogen (C), trehalose (D), TAG (E), and lipids (F) in flies that express NDI1 and AOX. Control flies were CON (da-GAL4 > w^Dah^), while experimental females NDI1 (da-GAL4 > UAS-NDI1) or AOX (da-GAL4 > UAS-AOX). Values are represented as mean ± SEM for 4–5 independent replicates. Asterisks show significantly different groups with p < 0.05
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 NDI1 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 an increase in protein carbonyls (Fig. 2A; p < 0.05), although AOX-expressing flies showed a two-fold lower level of lipid peroxides compared to the control (Fig. 2B; p = 0.041). There were no differences between the strains studied in the levels of high and low-molecular mass thiol-containing substances (Fig. 2C, D).
Fig. 2. Contents of carbonyl proteins (A), lipid peroxides (B), low- (C) and high- molecular-mass thiol-containing compounds (D) in flies that express NDI1 and AOX. Fly genotypes are as on Fig. 1. Values are represented as mean ± SEM for 4–5 independent replicates. Asterisks show significantly different groups with p < 0.05
Neither superoxide dismutase 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 females as compared to both control and NDI1-expressing flies (Fig. 4A; p < 0.05). Interestingly, NDI1-expressing flies had significantly lower activities of glucose-6-phosphate dehydrogenase by 32% (Fig. 4B; p = 0.027) and NADP-dependent isocitrate dehydrogenase by 24% (Fig. 4C; p = 0.049). Flies of both experimental cohorts had about 1.5-fold lower activity of lactate dehydrogenase compared to that of the control (Fig. 4D; Tukey test: p < 0.02). MDH and ALT activities were not affected either by NDI1 or AOX overexpression (Fig. 4E, F).
Fig. 3. Activities of antioxidant enzymes SOD (A), catalase (B), and glutathione-S-transferase (C) in flies that overexpress yeast NDI1 and AOX. Fly genotypes are as on Fig. 1. Values are represented as mean ± SEM for 4–5 independent replicates. Asterisks show significantly different groups with p < 0.05
Fig. 4. Activities 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 NDI1 and AOX. Fly genotypes are as on Fig. 1. Data are presented as mean ± SEM from 4–5 independent replicates. Asterisks indicate statistically significant differences between groups (p < 0.05)
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 [31]. However, such substrate competition has not been observed for NDI1 [32]. 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 [33]. The current study shows that NDI1 expression did not influence the levels of metabolic stores such as TAG, glycogen, and trehalose. However, the increased food consumption we observe (Fig. 1F) may compensate for a possible biosynthetic shortage if the latter takes place. Moreover, mitochondria may be involved in appetite regulation by affecting the release of adipokinetic hormone as well as several other hormones that promote food consumption [34, 35].
An interesting effect we observe is the decrease in glucose level while the increase in glycogen in AOX-expressing flies (Fig. 1A, 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 the efficiency of oxidative phosphorylation. Nevertheless, we have obtained opposite data, which can be connected with pleiotropic effects of AOX on cell physiology. Specifically, AOX was previously shown to decrease the generation of ROS by ETC complex III [36, 37]. Recent findings imply AOX may directly or indirectly affect cellular calcium homeostasis [38] and apoptosis [38, 39]. Cellular calcium is an important regulator of insulin-like peptide release in insects [40]. Calcium may also affect the downstream insulin signaling pathway, specifically glycogen synthase kinase 3β [41]. Remarkably, 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 [42]. 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 by bypassing 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 the ubiquinone pool, consequently promoting reverse electron transport through ETC complex I [43]. In our current study, both NDI1 and AOX expression in flies lead to an increase in the protein carbonyl levels (Fig. 2A) with a concomitant decrease in LOOH levels (Fig. 2B). The latter suggests a fast breakdown of LOOH that may lead to the formation of the end-products, predominantly aldehydes. Subsequently, aldehydes produced after LOOH breakdown interact with proteins, leading to the formation of additional carbonyl groups [44]. Formation of LOOH and its breakdown are hardly trackable dynamic processes. The breakdown often occurs after the formation of multiple endoperoxides within one fatty acyl chain [45]. Afterward, the end-products can be conjugated with glutathione or other thiols or modified proteins. We saw that NDI1 or AOX expression did not affect thiol levels (Fig. 2C, D). It allows us to assume that in our case, NDI1 and AOX indeed promote ROS production rather than abolish it. However, NDI1 or AOX may 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 dehydrogenases of glucose-6-phosphate, isocitrate, malate, and lactate, can be examples of such proteins. Our results suggest this can be the case for NDI1-expressing flies since the activities of these enzymes were lower compared to those in the control flies [43]. A similar trend can also be seen in AOX-expressing flies [24].
How could it 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 the well-being of mitochondria. An elevation of ROS levels often results in activation of the antioxidant system [46]. It has been shown that reactions occurring at ETC complex III promote the formation of superoxide anion-radical at the mitochondrial matrix side and intermembrane mitochondrial space [17]. The superoxide released in the intermembrane space passes a signal to the cytosol and indirectly activates antioxidant defense [47]. Bypassing complex III by AOX decreases the level of superoxide released to the intermembrane space, thus preventing the activation of the antioxidant system. Indeed, the activity of catalase and superoxide dismutase in AOX-expressing flies is the same as in controls [48]. A higher glutathione reductase activity in these experimental females (Fig. 3) implies a compensatory response to ROS that confers protein carbonylation. Along with interaction between end-products of lipid peroxidation and proteins, this response may account for the respective lower level of LOOH in AOX flies (Fig. 2B).
Lactate dehydrogenase is an enzyme that allows NAD^+^ regeneration for persistent glycolysis. Expression of LDH is regulated by multiple transcription factors, including estrogen-related receptor [49] and hypoxia-inducible factor [50, 51]. Expression of LDH can also be suppressed by 20-hydroxyecdysone (20E) [52]. Interestingly, in insects, several cytochromes of P450 type involved in ecdysone biosynthesis are localized to mitochondria [53, 54]. Cytochromes P450 are heme-containing proteins. Reactions catalyzed by NDI1 and AOX bypass 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 [55]. The latter is activated by NAD^+^ [55]. 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 least, LDH is strongly activated by hypoxia, whereas reoxygenation leads to its rapid downregulation [50, 56]. This regulation is mediated by D. melanogaster homolog of hypoxia-inducible factor 1α (HIF-1α) [50]. In turn, HIF-1α can be activated by ROS [57], whereas NDI1 and AOX operation may decrease ROS levels. Interestingly, type II NADH dehydrogenase NDX from the sea squirt Ciona intestinalis conferred opposite effects on G6PDH and LDH, while not affecting activities of NADP-specific MDH and IDH [22]. Nevertheless, both NDI1 and AOX expression led to an increase in glutathione-S-transferase activity in female 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.
Limitations
A major limitation of this study is that all experiments were conducted exclusively in female flies. Therefore, the results may not be directly generalizable to males, given known sex-specific differences in metabolic and oxidative phenotypes. The absence of UAS-only controls represents an additional limitation. While the driver-only control mitigates concerns about GAL4-related effects, minor contributions from insertion-site or background variation cannot be fully excluded. All conclusions are therefore framed relative to the driver-only genotype, and future work, including additional UAS-only lines, could further confirm these findings.