Restriction of glucose and fructose causes mild oxidative stress independently of mitochondrial activity and reactive oxygen species in Drosophila melanogaster

Comparative Biochemistry and Physiology, Part A 187 (2015) 27–39

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Restriction of glucose and fructose causes mild oxidative stress independently of mitochondrial activity and reactive oxygen species in Drosophila melanogaster Bohdana M. Rovenko a, Olga I. Kubrak a, Dmytro V. Gospodaryov a, Ihor S. Yurkevych a, Alberto Sanz b,c, Oleh V. Lushchak a,⁎, Volodymyr I. Lushchak a,⁎ a b c

Department of Biochemistry and Biotechnology, Vassyl Stefanyk Precarpathian National University, Ivano-Frankivsk 76018, Ukraine Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle-Upon-Tyne NE4 5PL, UK Newcastle University Institute for Ageing, Newcastle University, Newcastle-Upon-Tyne NE4 5PL, UK

a r t i c l e

i n f o

Article history: Received 20 February 2015 Received in revised form 17 April 2015 Accepted 23 April 2015 Available online 2 May 2015 Keywords: Glucose Fructose Reactive oxygen species (ROS) Oxidative stress Mitochondria Antioxidant defense

a b s t r a c t Our recent study showed different effects of glucose and fructose overconsumption on the development of obese phenotypes in Drosophila. Glucose induced glucose toxicity due to the increase in circulating glucose, whereas fructose was more prone to induce obesity promoting accumulation of reserve lipids and carbohydrates (Rovenko et al., Comp. Biochem. Physiol. A Mol. Integr. Physiol. 2015, 180, 75–85). Searching for mechanisms responsible for these phenotypes in this study, we analyzed mitochondrial activity, mitochondrial density, mtROS production, oxidative stress markers and antioxidant defense in fruit flies fed 0.25%, 4% and 10% glucose or fructose. It is shown that there is a complex interaction between dietary monosaccharide concentrations, mitochondrial activity and oxidative modifications to proteins and lipids. Glucose at high concentration (10%) reduced mitochondrial protein density and consequently respiration in flies, while fructose did not affect these parameters. The production of ROS by mitochondria did not reflect activities of mitochondrial complexes. Moreover, there was no clear connection between mtROS production and antioxidant defense or between antioxidant defense and developmental survival, shown in our previous study (Rovenko et al., Comp. Biochem. Physiol. A Mol. Integr. Physiol. 2015, 180, 75–85). Instead, mtROS and antioxidant machinery cooperated to maintain a redox state that determined survival rates, and paradoxically, pro-oxidant conditions facilitated larva survival independently of the type of carbohydrate. It seems that in this complex system glucose controls the amount of oxidative modification regulating mitochondrial activity, while fructose regulates steady-state mRNA levels of antioxidant enzymes. © 2015 Elsevier Inc. All rights reserved.

1. Introduction Mitochondria generate most of ATP that cells use to fuel their metabolism and other activities (Haslam and Krebs, 1968; Kang and Pervaiz, 2012). Apart from providing cells with energy, mitochondria are involved in various important cellular processes such as betaoxidation of fatty acids and biosynthesis of pyrimidines, amino acids, nucleotides, phospholipids, hemes, and iron–sulphur clusters. Abbreviations: G6PDH, glucose-6-phosphate dehydrogenase; GR activity of TrxR, glutathione reductase activity of thioredoxin reductase; GST, glutathione-S-transferase; IDH, isocitrate dehydrogenase; MD, mitochondrial protein density; mtROS, mitochondrial ROS; PC, protein carbonyls; PT, protein thiols; ROS, reactive oxygen species; SOD, superoxide dismutase. ⁎ Corresponding authors at: Department of Biochemistry and Biotechnology, Vassyl Stefanyk Precarpathian National University, 57 Shevchenko Str., Ivano-Frankivsk 76018, Ukraine. Tel./fax: +380 342 596171. E-mail addresses: [email protected] (O.V. Lushchak), [email protected] (V.I. Lushchak).

http://dx.doi.org/10.1016/j.cbpa.2015.04.012 1095-6433/© 2015 Elsevier Inc. All rights reserved.

Concomitantly, mitochondria are also the main generators of reactive oxygen species (ROS) (Weinberg et al., 2010; Jimenez-Del-Rio and Velez-Pardo, 2012; Kang and Pervaiz, 2012). It was known that increased steady-state ROS concentration may lead to modification of proteins, lipids, and nucleic acids which along with certain physiological consequences reflect development of oxidative stress (Lushchak, 2011, 2014; Scialo et al., 2013). During the last decade, understanding of relationship between intermediary and ROS metabolisms is notably increased. Recent studies showed that ROS are not only damaging factors, but also play regulatory roles in cell fate determination, hypoxia response, apoptosis, and necrosis (Sena and Chandel, 2012; Filomeni et al., 2015). In addition to the “classical ideas” about oxidative damages that postulated an irreversible pattern of biomolecule damages, nowadays substantial piece of evidence has been accumulated clearly indicating that oxidatively modified molecules might be repaired (Clancy and Birdsall, 2013), or removed by the proteasome (Pickering and Davies, 2012) and autophagy (Filomeni et al., in press).

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B.M. Rovenko et al. / Comparative Biochemistry and Physiology, Part A 187 (2015) 27–39

Despite ROS being constantly generated in living organisms, under “normal” conditions organisms are able to tolerate them. In fact, oxidative stress only happens, when the balance between ROS production and elimination is disturbed resulting in increased ROS levels along with certain physiological consequences (Lushchak, 2011; Scialo et al., 2013). Oxidative stress accompanies many disorders including diabetes, atherosclerosis, cancer, and degenerations, which also depend on type of diet, for example, availability of carbohydrates or amino acids (Sanz et al., 2006; Weinberg et al., 2010; Henriksen et al., 2011; Hulsmans et al., 2012; Jimenez-Del-Rio and Velez-Pardo, 2012). Studies in mammalian models indicated that reducing monosaccharides, glucose and fructose, differently affect mitochondrial function and antioxidant defense (Rizkalla, 2010; Kunde et al., 2011; Shah et al., 2013). However, it is unknown if these changes in antioxidant defense are caused by changes in mitochondrial activity and/or ROS levels. Nowadays, Western countries suffer an epidemic of obesity caused to a large extent by overconsumption of carbohydrates. It is believed that part of the problems is related with the increased intake of glucose and fructose (Basciano et al., 2005; Moeller et al., 2009; Tappy and Lê, 2010; Stanhope et al., 2013). Both, glucose and fructose may stimulate ROS generation via non-enzymatic reactions also known as glycation reactions causing oxidative stress (Kanska and Boratynski, 2002; Schalkwijk et al., 2004; Semchyshyn, 2013, 2014; Semchyshyn et al., 2014). However, it remains unclear if there is any difference in their capability to initiate glycation, since some reports call glucose (Kanska and Boratynski, 2002), whereas others call fructose as a more powerful glycation agent (Bunn and Higgins, 1981; Schalkwijk et al., 2004; Semchyshyn et al., 2011; Semchyshyn, 2013). It is also controversial if excessive consumption of monosaccharides causes oxidative damage via glycation (Schalkwijk et al., 2004; Shangari and O'Brien, 2004; Semchyshyn et al., 2011; Semchyshyn, 2013) or through changes in mitochondrial metabolism (Rizkalla, 2010; Shah et al., 2013; Mortensen et al., 2014). The time scales for these processes seem to be different: if glycation is considered to be rather slow process (Semchyshyn, 2013, 2014), mitochondria may quickly respond to alterations in dietary conditions (Barja, 2007; Scialo et al., 2013). Previous studies from our laboratory (Lushchak et al., 2011; Semchyshyn et al., 2011, 2014; Semchyshyn and Lozinska, 2012; Rovenko et al., 2013; Semchyshyn, 2014) indicated the complex relationship between fructose consumption and ROS-related processes. For example, fructose at high concentrations promoted protein oxidation, while at moderate concentrations it protected Saccharomyces cerevisiae cells from oxidative damage inducing a hormetic response (Semchyshyn et al., 2011, 2014; Semchyshyn and Lozinska, 2012). Similar data were obtained in Drosophila melanogaster, where glucose and fructose demonstrated different effects depending on the genetic background and the stage of the life cycle studied (Lushchak et al., 2011, 2014; Rovenko et al., 2013). Carbohydrates are metabolized through similar pathways in flies and mammals (Kunieda et al., 2006; Zera, 2011). In flies, neurosecretory cells from the brain and corpora cardiaca as well as fat body are instrumental to maintain energy homeostasis, playing the same role that pancreas, liver and adipose tissue in vertebrates (Teleman et al., 2012; Owusu-Ansah and Perrimon, 2014). Although during the past decade substantial progress has been made in understanding carbohydrate metabolism regulation, the energy control upon feeding with different carbohydrates, especially fructose, remains unclear. Recent findings showed that brain possesses a specific receptor for fructose (Gr43a), which is able to sense fructose from circulating sugars regulating feeding behavior (Miyamoto et al., 2012). Several independent studies showed strong preferences of Drosophila to fructose-enriched food (Masek and Scott, 2010; Lushchak et al., 2011; Mishra et al., 2013; Rovenko et al., 2015). Our latest study (Rovenko et al., 2015) showed that metabolic response to fructose overfeeding in flies mimics features of the response in mammals (reviewed in details in Basciano et al., 2005; Tappy and Lê, 2010). Thus, fructose rather than glucose promoted a diet-induced obese

phenotype partly by modulating the insulin/insulin-like growth factor signaling. Glucose overfeeding, in turn, induced glucose toxicity and decreased developmental survival due to the increase in circulating glucose (Rovenko et al., 2015). In this study, we expanded our understanding of the role of glucose and fructose in the regulation of mitochondrial function in Drosophila, and described possible implication of mitochondrial function to observed phenotypes. In particular, we were interested to know: (1) how mitochondrial activity is modified in response to variation in dietary monosaccharide composition and concentration; (2) how changes in mitochondrial function affect mitochondrial ROS production; (3) how changes in mitochondrial ROS metabolism are related to ROS-promoted oxidative modifications of lipids and proteins, and (4) how free radical metabolism interfere with glucose- and fructose-derived metabolic phenotypes. For this, we fed Drosophila flies during development with glucose and fructose, in different concentrations, and analyzed mitochondrial activity, mitochondrial density, and ROS production, along with oxidative stress indices and antioxidant response in young adults.

2. Materials and methods 2.1. Reagents All reagents were purchased from Sigma-Aldrich Corporation (USA) unless otherwise stated. RNA stabilization solution was obtained from Ambion (USA). Oligonucleotides for Q-RT-PCR (qPCR) assay were purchased from TAG Copenhagen A.S. (Denmark). SensiFAST SYBR Hi-ROX Kit was obtained from Bioline Reagents Ltd. (United Kingdom). The other reagents for qPCR assay were from Thermo Fisher Scientific (USA). Manufacturer yeasts (type “Extra”, TM “The Lviv yeast”) were bought from “The Enzyme Company” (Ukraine).

2.2. Flies and experimental design Wild type Canton S flies were used in all experiments. The flies were from Bloomington Drosophila Stock Center at Indiana University (USA). Flies were reared on medium containing 6% (w/v) yeasts, 4% (v/v) molasses, 1.25% (w/v) agar and 0.4% (v/v) propionic acid as a mold growth inhibitor. For egg collection, about 300–400 of 3–7 day-old parental flies were transferred into demographic cages with the open side attached to a Petri dish containing egg collection medium (apple juice with 2% agar and yeast paste). After 18 h eggs were washed out from the egg collection plate with distilled water and counted. About 260–280 eggs were placed into 250 ml glass flasks containing 25 ml of experimental food with 4% yeast, 0.25, 4, or 10% glucose or fructose, 1.25% (w/v) agar, and 0.4% (v/v) propionic acid. The caloric values of diets were calculated by counting calories derived from sucrose and yeast. 4 kcal/g was used as sucrose caloric value (Donato, 1987). The caloric value of yeast was taken as 1.17 kcal/g according to manufacturer's annotation. Total caloric content of the diets is presented in Suppl. Table 1. In natural environment the concentrations of glucose and fructose in Drosophila food varies from 2 to 30% with an average around 4–6% in most fruits (Widdowson and McCance, 1935; Li et al., 2002). According to this information and our previous data concerning developmental survival and food consumption with glucose and fructose (Rovenko et al., 2015), the diet with 0.25% carbohydrate was considered as a carbohydrate restricted diet, while diets with 4 and 10% of monosaccharides were suggested to yield relatively moderate and high concentrations of carbohydrates in the Drosophila food, respectively. Newly eclosed flies were transferred onto fresh food of the same composition, where they have been growing during larval stage and held for two days. Two-day old flies were separated by sex and used for biochemical analyses.

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2.3. Mitochondrial parameters 2.3.1. Mitochondrial respiration Mitochondrial respiration rates were measured by polarography using a Clark-type oxygen electrode Oxygraph 2K (Oroboros Instruments, Innsbruck, Austria) (Stefanatos et al., 2012). Forty flies were gently homogenized with a porcelain mortar and pestle in 1 ml of isolation buffer (250 mM sucrose, 5 mM Tris–HCl, 2 mM EGTA, pH 7.4). Then fly homogenate was filtered through a nylon net with a mesh size of 200 μm to remove un-homogenized fly body parts. All procedures were done at 4 °C. Afterwards, 50 μl of filtered homogenate were added to 1.95 ml of respiration assay buffer (120 mM KCl, 5 mM KH2PO4, 3 mM HEPES, 1 mM EGTA, 1 mM MgCl2, 0.2% (w/v) fatty-acid free bovine serum albumin). After stabilization of the signal, 4 μl 0.5 M ADP (1 mM, here and further in parentheses are shown final concentrations of substrates/inhibitors) and different respiratory substrates were added to measure respiratory fluxes of mitochondrial complexes. Complex I + III + IV respiration was assessed by adding 5 μl 2 M pyruvate and proline (5 mM). Complex III + IV respiration was assessed by adding 30 μl 1.3 M glycerol-3-phospate (20 mM). Respiration of complex IV was induced by adding of 15 μl 0.25 M N,N,N′,N′-tetramethyl-pphenylenediamine (TMPD) with 10 μl 0.8 M ascorbate (2 mM and 4 mM are final concentrations, respectively). In order to analyze the respiratory fluxes of specific complexes, 1 μl 10 mM rotenone (5 μM), 1 μl 50 mM antimycin A (10 μM) or 2 μl 0.1 M KCN (100 μM) were added to inhibit complexes I, III and IV respectively. Respiratory fluxes were normalized per amount of protein in homogenates. Additionally, mitochondrial density (MD) was estimated in the same homogenates.

2.3.2. Mitochondrial density It was measured as the ratio (percentage) of citrate synthase activity in whole fly homogenate to the activity of сitrate synthase in isolated mitochondria according to (Magwere et al., 2006). Citrate synthase activity was measured as changes in the absorbance of the conjugate formed between DTNB and CoA-SH at 410 nm. The reaction mixture contained 90 mM Tris–HCl (pH 7.5), 0.5 mM EDTA, 0.3 mM acetyl CoA, 0.1 mM DTNB, 0.5 mM oxaloacetate and 0.6 μg of protein from whole fly homogenate sample or 0.3 μg of protein obtained after isolation of mitochondria from the same samples. The reaction was initiated by the addition of oxaloacetate to the incubation mixture. Blanks without oxaloacetate were used to subtract the non-enzymatic absorbance changes caused by the reaction between DTNB and endogenic thiol compounds.

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2.4. Assay of oxidative stress markers and activities of antioxidant and related enzymes For lipid peroxide assay, flies were homogenized (1:10 w/v) with a Potter–Elvehjem glass homogenizer in a 95% cold ethanol (4 °C), and centrifuged (5000 g, 5 min, 4 °C). The resulting supernatants were immediately used for the measurements. For protein carbonyl and protein thiol assays as well as for measurements of enzyme activities, frozen flies were homogenized (1:10 w/v) with a Potter–Elvehjem glass homogenizer in 50 mM potassium phosphate buffer (pH 7.5) containing 0.5 mM EDTA (ethylenediaminetetraacetic acid) and 1 mM PMSF (phenylmethylsulfonyl fluoride) and centrifuged (16 000 g, 15 min, 4 °C) in Eppendorf 5415R centrifuge (Germany). The supernatants were used for determination of protein carbonyl and thiol content, low molecular mass thiols as well as the enzyme activities. The markers of oxidative modifications to proteins (protein carbonyl and thiol groups) and lipids (lipid peroxides), and activities of antioxidant and associated enzymes were measured in adult flies as described earlier (Lushchak et al., 2011) with minor modifications. Briefly, content of carbonyl groups in proteins (protein carbonyls, PC) was measured detecting the amount of 2,4-dinitrophenylhydrazone formed in the reaction with DNPH at 370 nm using an extinction coefficient of 22 mM−1 cm−1 (Levine et al., 1994). Protein thiol content (PT) was calculated as a difference between total and low molecular mass thiols, measured in protein-containing and deproteinized fraction of supernatants, respectively, by Elman's method (Ellman, 1959). The results were expressed in nanomoles of PC and PT per milligram of protein. Lipid peroxides were measured using the ferrous oxidation of xylenol orange (FOX) method (Hermes-Lima et al., 1995). The amount of lipid peroxides was normalized to the total lipid amount measured as described previously (Rovenko et al., 2015). One unit of SOD activity was defined as the amount of enzyme (per milligram of protein) that inhibits quercetin oxidation reaction by 50% of maximal inhibition. One unit of catalase, glutathione-S-transferase (GST), GR (glutathione reductase) activity of thioredoxin reductase (TrxR), glucose-6phosphate dehydrogenase (G6PDH), and NADPH-dependent isocitrate dehydrogenase (IDH) activity was defined as the amount of enzyme consuming 1 μmol of substrate or generating 1 μmol of product per minute. The activities were expressed as international units (or milliunits) per milligram soluble protein (I.U./mg protein). Protein concentration was measured by the Bradford method with Coomassie Brilliant Blue G-250 (Bradford, 1976) using bovine serum albumin as standard. 2.5. Analysis of specific mRNA levels of antioxidant enzymes by quantitative real time PCR

2.3.3. Mitochondrial ROS production Was assayed fluorometrically on a Plate Chameleon V microplate reader (Hidex, Finland) by resorufin formation from Amplex Red in the presence of horseradish peroxidase and superoxide dismutase at 530 nm wavelength excitation and 595 nm emission. Mitochondria were isolated according to Miwa et al. (2003) with some minor modifications (Fernandez-Ayala et al., 2009). Mitochondria (0.5 mg/ml of mitochondrial protein) were incubated at 25 °C during 30–40 min in respiration assay buffer containing 0.5 mM Amplex Red, 0.1 U/ml peroxidase, 50 U/ml superoxide dismutase and substrates and/or inhibitors of mitochondrial complexes in the same final concentrations as it was used for estimation of respiration. All measurements were carried out in the absence of substrates and background fluorescence was subtracted. The increment in the fluorescence was used for calculation of hydrogen peroxide production rate. A calibration curve was built using the known amounts of hydrogen peroxide produced at glucose oxidation by glucose oxidase. Values are expressed as nanomoles of hydrogen peroxide produced per one minute per milligram of mitochondrial protein.

Total RNA was stabilized using RNA stabilization solution (Ambion) and extracted with Trizol from 2-day old flies (Stefanatos et al., 2012). For cDNA synthesis 2 μg RNA, 1 μl dNTP mix (10 mM), 0.4 μl Random Primers (0.5 μg/μl) and 9.6 μl DEPC-treated water were incubated at 90 °C for 3 min and then transferred to ice, where 4 μl 5 × M-MuLV reaction buffer and 1 μl RNase inhibitor (40 U/μl) were added. The reaction mixture was incubated at 25 °C for 10 min, then, 2 μl M-MuLV reverse transcriptase (20 U/μl) was added to samples kept on ice, and the mixture was incubated for a further 10 min at 25 °C, 1 h at 37 °C and 70 °C for 10 min. Amount of cDNA synthesized was analyzed in triplicates by q-RT-PCR. Primers used for the analysis of expression of Sod1 (CG11793), Sod2 (CG8905), Cat (CG6871), and Gpx (CG12013) genes are listed in Suppl. Table 2. Expression of the target genes was normalized to the expression levels of the reference gene (CG5178, Act88F), coding thin F-actin-like filament. 10-fold dilution series of cDNA mix from all samples was used for the calibration curve. In all cases calculations were performed for cDNA samples diluted 20-fold in 20 μl reaction volume containing 4 μl of the cDNA template, 0.4 μl of

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20 μM forward and reverse primers, and 10 μl of 2 × SensiFAST SYBR Hi-ROX Kit. The PCR conditions were: 2 min pre-incubation at 95 °C, 40 cycles of 5 s denaturation at 95 °C, 10 s annealing at 60 °C and 10 s extension at 72 °C. Data were extracted and analyzed using Applied Biosystems StepOne software version 2.0. Results are shown as a ratio in fluorescence for genes of interest and reference gene. 2.6. Statistical analysis Data are presented as means ± standard errors of the means (S.E.M.) of 3–8 independent replicates with 20–40 flies in each replicate. Statistical analysis was performed using JMP Pro11 software by two-way ANOVA followed by Tukey's HSD test to compare the parameters at different glucose and fructose concentrations, carbohydrate-dependent and sex differences, and their interaction (Table 2). Additionally, Pearson correlation analysis between oxidative stress/antioxidant defense indices and parameters, which characterize mitochondrial metabolism, was done (Table 3). In order to avoid overloading graph material, in the figures only significant changes in relation to type of carbohydrate and its concentration are shown. 3. Results 3.1. Mitochondrial activity and mtROS levels are determined by the amount and type of carbohydrate and fly sex Our previous studies have shown that consumption of glucose and fructose during larval development results in eclosion of metabolically distinct flies (Lushchak et al., 2011; Rovenko et al., 2013). We hypothesized that the different metabolic profiles associated with glucose and fructose consumption are related to utilisation of these monosaccharides through different metabolic pathways, namely glycolysis and fructolysis. Since intermediates of these pathways have different affinity to be metabolized in mitochondria, first, we studied how carbohydrate concentration (0.25, 4 and 10%) and type of carbohydrate (glucose or fructose) in the diet affected mitochondrial respiration. We found that consumption of glucose and fructose differently influenced mitochondrial respiration (F2,79 = 10.3, P = 0.0001). Oxygen consumption changed in response to alterations in glucose concentration (Fig. 1A–F), whereas changes in fructose concentration did not affect this parameter. Glucose excess in the diet (10%) repressed respiration through all mitochondrial complexes in flies of both sexes (Fig. 1A–F). Trying to understand the mechanisms responsible for repressed mitochondrial respiration upon high glucose feeding, we measured mitochondrial protein density (MD). The latter similar to respiration (F1,55 = 6.10, P = 0.0167) was higher in females than in males (F1,55 = 6.10, P = 0.0167). In response to glucose restriction MD was significantly increased in flies of both sexes (F2,55 =

5.00, P = 0.0101). Changes in fructose concentration did not alter this parameter (Fig. 1G, H). Consequently, MD and mitochondrial respiration showed a strong correlation (r = 0.71, P = 0.0099), indicating that changes in respiration could be explained by changes in the amount of mitochondria. Thus, we concluded that glucose and fructose differently affected mitochondrial activity. The restriction of glucose, but not fructose, increased amount of mitochondria and respiration rate in Drosophila flies. Next, we measured mitochondrial ROS (mtROS) production. In general, it is widely believed that ROS generation in mitochondria is strongly affected by oxygen consumption, although some studies showed the trade-off between these parameters dependently on environmental conditions (Barja, 2007; Cortassa et al., 2014). Accordingly, we found that isolated mitochondria from males developed on diets with 0.25% glucose produced 2-fold more hydrogen peroxide, than mitochondria of males developed on diet with 10% glucose (Fig. 2). Nevertheless, a similar trend was observed in males fed fructose (Fig. 2), where no difference in respiration rate was detected (Fig. 1A–F). The differences in mtROS levels of flies fed high and low fructose diets were smaller (about 15%) (Fig. 2). Production of mtROS by complex III in females was significantly higher than that in males (F2,58 = 212, P b 0.0001). In addition, and oppositely to males, mitochondria isolated from females grown on low monosaccharide diet generated less ROS (Fig. 2B, D). The type of carbohydrate also showed impact on mtROS levels (for complex I + III, F2,71 = 8.27, P = 0.0094; for complex III, F2,58 = 25.0, P b 0.0001). Production of mtROS was by 50–60% higher in males and females developed on high fructose compared to glucose, whereas ROS production in flies grown on low glucose was higher than in fructose fed ones (Fig. 2).

3.2. Restriction of monosaccharides causes oxidative stress independently of fly sex and mitochondrial activity In order to estimate how mtROS production and mitochondrial activity were connected with oxidative stress markers, we measured oxidative modifications of proteins and lipids in two-day-old adult flies fed glucose and fructose in different concentrations. Previously, we found that glucose and fructose differently affected oxidation of biomolecules in flies of different strains (Lushchak et al., 2011; Rovenko et al., 2013, 2015). However, in the above mentioned studies total amount of oxidatively modified proteins and lipids was assessed without taking into account the percentage of proteins and lipids in fly bodies. Later, we noticed that variation in dietary content dramatically changed body composition, affecting the percentage of total lipids (Rovenko et al., 2015). Here, the contents of oxidized proteins and lipid peroxides were normalized to the total amount of protein and lipids. Protein oxidation was assessed by the content of protein carbonyl (PC)

Table 1 Activities of glutathione-S-transferase (GST), thioredoxin reductase (GR activity of TrxR), glucose-6-phosphate dehydrogenase (G6PDH) and NADP-dependent isocitrate dehydrogenase (IDH) in two-day-old flies grown on diets with different concentrations of glucose and fructose. Food

GST activity (mI.U./mg protein)

GR activity of TrxR (mI.U./mg protein)

G6PDH activity (mI.U./mg protein)

IDH activity (mI.U./mg protein)

Males

Females

Males

Females

Males

Females

Males

Females

Glucose 0.25 4 10

358 ± 43⁎ 297 ± 21 288 ± 7♣

349 ± 41⁎ 225 ± 24 240 ± 17♣

10.0 ± 0.8⁎ 8.05 ± 0.55 7.64 ± 0.45♣

11.2 ± 0.38⁎ 8.92 ± 0.65 8.34 ± 0.40♣

17.6 ± 2.1⁎ 52.3 ± 1.8 33.5 ± 7.0⁎♣

6.95 ± 0.85⁎ 23.5 ± 4.6 20.9 ± 4.2♣

195 ± 19 235 ± 38 215 ± 38

133 ± 20 102 ± 9 90 ± 10

Fructose 0.25 4 10

439 ± 44⁎ 314 ± 9 293 ± 14♣

317 ± 26⁎ 240 ± 25 217 ± 28♣

9.13 ± 0.86⁎ 6.61 ± 0.37 6.92 ± 0.40♣

10.3 ± 0.60⁎ 8.48 ± 0.90 8.18 ± 0.62♣

27.2 ± 2.6⁎ 50.6 ± 6.2 34.3 ± 3.1⁎♣

10.7 ± 1.3⁎ 17.4 ± 1.9 14.9 ± 2.8

182 ± 28 187 ± 24 201 ± 26

115 ± 12 81.4 ± 2.3 108 ± 17

Enzyme activities are shown as milliunits normalized to the milligram of total protein. Data are presented as means ± S.E.M., n = 3–6 (independent replicates with 20–40 flies in each replicate). ⁎ Significantly different from the group fed the same monosaccharide at concentration of 4%. ♣ Significantly different between the groups fed the same monosaccharide at concentrations of 0.25% and 10% (P b 0.05).

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Table 2 Statistical analysis (two-way ANOVA followed by Tukey's HSD test) of different indices presented in this study from two-day-old Canton S Drosophila melanogaster flies grown on different concentrations of glucose and fructose. Indices studied

Complex I + III + IV oxygen consumption (Error DF = 79) Complex III + IV oxygen consumption (Error DF = 79) Complex IV oxygen consumption (Error DF = 79) MD (Error DF = 55) mtROS, complex I (Error DF = 71) mtROS, complex III (Error DF = 58) Protein carbonyls (Error DF = 38) Protein thiols (Error DF = 42) Lipid peroxides (Error DF = 49) SOD activity (Error DF = 58) Sod1 mRNA (Error DF = 45) Sod2 mRNA (Error DF = 45) Catalase activity (Error DF = 46) Cat mRNA (Error DF = 45) Gpx expression (Error DF = 45) Low molecular mass thiols (Error DF = 54) GR activity of TrxR (Error DF = 58) GST activity (Error DF = 58) G6PDH activity (Error DF = 47) IDH activity (Error DF = 38)

Concentration, DF = 2

Sex, DF = 1

F ratio

F ratio

P

Concentration ∗ Sex, DF = 2

Concentration ∗ Carbohydrate, DF = 2

Sex ∗ Carbohydrate, DF = 1

F ratio

F ratio

F ratio

F ratio

P

P

P

Carbohydrate ∗ Sex, DF = 2 F ratio

P

0.0027⁎

23.3

0.0001⁎

2.43

0.1232

1.17

0.3159

10.3

0.0001⁎

0.01

0.9608

1.73

0.1844

6.79

0.0018⁎

40.1

b0.0001⁎

1.78

0.1855

3.31

0.0412⁎

15.2

b0.0001⁎

1.02

0.3153

2.35

0.1018

4.90

0.0098⁎

3.75

0.73

0.3967

3.70

0.0289⁎

5.00 4.68 5.17 8.81 3.91 50.2 32.0 3.31 0.40 3.99 0.32 5.49 2.10

0.0101⁎ 0.0215⁎ 0.0086⁎ 0.0007⁎ 0.0291⁎ b0.0001⁎ b0.0001⁎ 0.0482⁎

6.10 61.4 212 6.89 2.07 11.5 0.01 0.13 0.31 44.1 1.25 63.6 1.01

0.3367 0.0442⁎ 0.0040⁎ 0.7064 0.1605 0.0979 0.3510 0.0525⁎ 0.0585⁎

16.1 16.0 17.6 0.02

b0.0001⁎ b0.0001⁎ b0.0001⁎

7.04 13.8 72.5 56.7

0.7236 0.3299 0.5574 0.9646

0.9846

P

Concentration ∗

6.37

0.6677 0.0270⁎ 0.7251 0.0083⁎ 0.1342

P

Carbohydrate, DF = 1

0.0562

3.61

0.0608

0.66

0.5199

8.28

b0.0005⁎

0.0167⁎ b0.0001⁎ b0.0001⁎ 0.0124⁎ 0.1595 0.0014⁎ 0.9113 0.7193 0.5856 b0.0001⁎

0.7219 0.0094⁎ b0.0001⁎

0.1994 0.1751 0.4757 0.9598

1.09 7.84 16.3 2.60 0.02 7.35 0.17 2.50 0.84 16.5 4.35 2.01 0.48

0.3450 0.0031⁎ b0.0001⁎ 0.0877 0.9849 0.0016⁎ 0.844 0.0963 0.4411 b0.0001⁎ 0.0203⁎ 0.1483 0.6240

6.07 7.81 5.89 0.06 0.38 7.89 0.17 4.04 1.66 0.95 1.18 4.78 0.42

0.0041⁎ 0.0396⁎ 0.0047⁎ 0.9462 0.6873 0.0011⁎ 0.8443 0.0262 0.2042 0.9346 0.3203 0.0144⁎ 0.6568

0.09 0.11 18.6 1.00 0.90 3.69 0.57 8.05 0.25 6.25 0.01 5.89 2.64

0.7665 0.7458 b0.0001⁎ 0.3247 0.3504 0.0606 0.5690 0.0074 0.6175 0.0170⁎

0.2719 b0.0001⁎ 0.3208

0.13 8.27 25.0 0.34 0.13 6.10 0.87 25.4 11.8 1.71 1.91 0.52 0.01

0.9510 0.0204 0.1111

1.11 3.39 6.07 0.35 1.93 2.44 1.07 3.00 2.89 0.84 6.93 1.45 0.05

0.0112⁎ b0.0005⁎ b0.0001⁎ b0.0001⁎

2.32 0.27 0.44 2.73

0.1354 0.6074 0.5126 0.1068

0.08 0.05 3.03 3.00

0.9253 0.9518 0.0608 0.07

0.17 0.25 0.09 0.35

0.8479 0.7801 0.9151 0.7061

0.97 2.02 1.58 0.50

0.3307 0.1616 0.2162 0.4861

0.33 1.13 0.59 0.04

0.5634 0.7172 0.0170⁎ 0.3544 b0.0001⁎ 0.0015⁎

0.4404 0.0029⁎ 0.2474 0.9489

⁎ Significant difference.

and thiol (PT) groups, while lipid oxidation was evaluated as content of lipid peroxides. The levels of PC in flies fed high monosaccharide diets (10%) were about 3 nmol/mg of protein (Fig. 3A, B). The diets with low concentration of carbohydrates (0.25%) promoted 30–40% higher PC levels in both sexes than diets with higher carbohydrate concentrations (F2,38 = 8.81, P = 0.0007). Accordingly, PT levels were lower in flies grown on diets containing low concentrations of monosaccharides (Fig. 3C, D; F2,42 = 3.91, P = 0.0291). The levels of lipid peroxides correlated with PC being higher in flies under monosaccharide restriction (Fig. 3E, F; F2,49 = 50.2, P = 0.0001). Additionally, the amount of lipid peroxides was higher in flies fed with glucose when compared to those fed with fructose (F2,49 = 6.10, P = 0.0170) and the differences were sex-related (F2,49 = 11.5, P = 0.0014). To test the role of mtROS and mitochondria in phenotype variation of oxidative stress indices, we performed correlation analysis between oxidative stress indices and parameters, which characterized mitochondrial metabolism. Our data clearly showed that upon glucose feeding PC and to a lesser extent lipid peroxide levels correlated with respiration rate and MPD (Table 3). However, this was not true for the same parameters in flies fed fructose. Neither in case of dietary fructose, nor for glucose correlations between oxidative stress indices and mtROS were found (Table 3). Since flies fed both carbohydrates showed oxidative stress development upon carbohydrate restriction, we concluded that this phenotype could not be mediated by mtROS or mitochondrial activity. 3.3. Glucose and fructose restriction induces different antioxidant responses To evaluate the involvement of antioxidant system in protection of fruit flies against ROS, the activities and mRNA levels of main enzymes detoxifying superoxide and hydrogen peroxide were measured. Superoxide dismutase (SOD) protects biomolecules from oxidative damage caused by ROS. The flies developed on 0.25% carbohydrates had the highest SOD activity (Fig. 4A, B). The activity in males and

females developed on 10% carbohydrates was about 2-fold lower in comparison with flies developed on 0.25% carbohydrates (F2,58 = 32.0, P b 0.0001). The ANOVA also showed significant interaction between concentration and type of carbohydrate and fly sex for levels of Sod1 (F2,45 = 3.00, P = 0.0525) and Sod2 (F2,45 = 32.0, P = 0.0585) transcripts. In particular, mRNA levels of Sod1 and Sod2 in males fed fructose changed in opposite direction to the activity of the enzyme (Fig. 4C–F). Although glucose concentration had no impact on Sod1 and Sod2 transcript levels in females, they were lower in flies fed glucose than in those fed fructose (Fig. 4C–F). The concentration (F2,45 = 16.5, P = 0.0001) as well as the type of monosaccharide (F2,45 = 6.25, P = 0.0170) altered both, specific catalase mRNA steady-state levels and catalase enzymatic activity in a sex-related manner (Fig. 5 A–D). Thus, catalase activity in males, developed on 10% carbohydrate diets, was 50–75% higher than in those developed on 0.25% carbohydrate (Fig. 5A). On the other hand, restriction in carbohydrates resulted in higher catalase activity in females (Fig. 5B). Consequently, catalase activity was changed oppositely to levels of hydrogen peroxide in isolated mitochondria in flies of both sexes. The ANOVA also revealed significant interaction between concentration and type of carbohydrate and fly sex for Cat transcripts (F 2,45 = 6.93, P = 0.0029). Similarly to data reporting SOD expression, levels of glucose did not influence the amount of catalase transcripts. However, fructose levels had opposite effects on the transcript levels and activity of catalase. Interestingly, only fructose excess lead to gain in mRNA level of glutathione peroxidase (F2,45 = 4.78, P = 0.0144) — another enzyme detoxifying peroxides in Drosophila (Fig. 4E, F). In particular, flies fed 10% fructose had almost 2-fold higher transcript levels of glutathione peroxidase than those fed 0.25 and 4% fructose (Fig. 4E, F). In summary, dietary restriction of glucose and fructose: (i) resulted in higher SOD, but not catalase activity, whose activity was rather sex-dependent, (ii) fructose consumption, but not glucose one, affected mRNA levels of antioxidants depending on the concentration of carbohydrates, and (iii) fructose differently affected the activity and the expression of different antioxidants.

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Fig. 1. Mitochondrial respiration (A–F) and mitochondrial protein density (G–H) in two-day-old Canton S flies grown on diets with different concentrations of glucose and fructose. For measurement of total respiration (I + III + IV), pyruvate and proline were used. The respiration of complexes III and IV was assayed using sn-glycerol-3-phosphate in the presence of rotenone. Complex IV respiration was measured using TMPD and ascorbate in the presence of rotenone and antimycin A. Respiratory fluxes are shown as nanomoles of O2 per milligram of protein in homogenates. Mitochondrial density (MD) was estimated in the same samples measured as the ratio (%) of citrate synthase activity in whole fly homogenate to activity of the enzyme in isolated mitochondria. Data are presented as means ± S.E.M., n = 5–8 (independent replicates with 40 flies in each replicate). *Significantly different from the group fed the same monosaccharide at concentration of 4%, and #from the group fed glucose at the same carbohydrate concentration with P b 0.05. ♣Significantly different from the groups fed the same monosaccharide at concentrations of 0.25% and 10% (P b 0.05).

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Fig. 2. The level of hydrogen peroxide produced by complexes I and III (A, B) and complex III itself (C, D) in isolated mitochondria of two-day-old flies Canton S grown on diets with different concentrations of glucose and fructose. For measurement of hydrogen peroxide production by complexes I and III, a cocktail of pyruvate and proline was used. The maximal hydrogen peroxide yield was provided adding rotenone and antimycin. For measurement of complex III hydrogen peroxide production (may include also mitochondrial glycerol-3-phosphate dehydrogenase as additional source of ROS), sn-glycerol-3-phosphate was used as a substrate. Values are expressed as nanomoles of hydrogen peroxide produced per minute per milligram of mitochondrial protein. Data are presented as means ± S.E.M., n = 5–8 (independent replicates with 40 flies in each replicate). *Significantly different from the group fed the same monosaccharide at concentration of 4%, and #from the group fed glucose at the same concentration with P b 0.05. ♣Significantly different from the groups fed the same monosaccharide at concentrations of 0.25% and 10% (P b 0.05).

The concentrations of low molecular mass thiols, mainly represented by glutathione, were virtually the same in flies grown at any diet used and consisted of ~ 1.50 nmol/mg wet mass (not shown). In other words, neither variation in concentration, nor in type of carbohydrate used in this study influenced levels of low molecular mass thiols. Nevertheless, the activities of glutathione-related enzymes in adult flies were affected by diet composition (Table 1). Thus, the ANOVA showed statistically significant effect of monosaccharide concentration on the activity of glutathione-S-transferase (F2,58 = 16.0, P b 0.0001) and GR activity of TrxR (F2,58 = 16.1, P b 0.0001). Flies of both sexes fed 0.25% and 4% diets had about 35% higher activities of glutathione-S-transferase and GR activity of TrxR when compared to those fed a 10% diet. Moreover, fly sex also had impact on activities of glutathione-S-transferase (F2,58 = 13.8, P b 0.0005) and GR activity of TrxR (F2,58 = 7.40, P b 0.0112). The activity of glucose-6-phosphate dehydrogenase (G6PDH), an enzyme providing reductive equivalents of NADPH particularly for glutathione reduction, did not correspond to changes in activities of glutathione-related enzymes and was higher in flies developed on diets with high amounts of carbohydrates (Table 1, F2,47 = 17.6, P b 0.0001). The activity of NADPH-dependent isocitrate dehydrogenase (IDH) was not significantly affected by different dietary regimes (Table 1). However, both NADPH-producing enzymes, namely G6PDH and IDH, showed higher activities in male flies in comparison to females (for G6PDH, F2,47 = 72.5, P b 0.0001; for IDH, F2,38 = 56.7, P b 0.0001). Additionally, in order to find interactions between antioxidant defense and mitochondrial metabolism, we did a correlation analysis between parameters characterizing antioxidant defense and mitochondrial performance. However, we only found few correlations (Table 3), including positive correlation between IDH activity and mitochondrial function parameters and strong negative correlation between GPx expression and production of ROS by complex III. In flies fed glucose SOD activity positively correlated with respiration

rate and MPD, while catalase activity showed negative correlation with ROS produced by complex III (Table 3). 4. Discussion Glucose and fructose are vital reducing monosaccharides with multiple biological functions. During the last decade, nutritional studies have provoked hot debates whether or not diets rich in these carbohydrates trigger metabolic disorders in mammals (Basciano et al., 2005; Moeller et al., 2009; Tappy and Lê, 2010; Stanhope et al., 2013). Oxidative stress was found to be among the main players in carbohydrateinduced metabolic pathologies (Basciano et al., 2005; Moeller et al., 2009; Tappy and Lê, 2010; Henriksen et al., 2011; Hulsmans et al., 2012; Jimenez-Del-Rio and Velez-Pardo, 2012; Semchyshyn, 2013; Stanhope et al., 2013; Semchyshyn et al., 2014). Impairment of mitochondrial function, uncontrolled production of mtROS and generation of toxic byproducts from non-enzymatic reactions of monosaccharides with other biomolecules (via glycation reactions) have been shown as contributors to oxidative stress development upon high glucose and fructose feeding. Several years ago we initiated a set of studies aimed to distinguish the physiological effects of these monosaccharides in the budding yeast S. cerevisiae (Semchyshyn et al., 2011, 2014; Semchyshyn and Lozinska, 2012) and in the fruit fly D. melanogaster (Lushchak et al., 2011; Rovenko et al., 2013; Lushchak et al., 2014; Rovenko et al., 2015). Our studies on Drosophila model showed that the effects of dietary carbohydrates are different in adults and larvae and, thereby, are strongly stage-dependent. Thus, aging of flies was not affected by the type of carbohydrate (glucose or fructose) in spite of the fact that they differently alter the antioxidant system (Lushchak et al., 2011; Rovenko et al., 2013; Lushchak et al., 2014). Indeed, the phenotypes induced by high levels of reducing sugars found in mammals were

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Fig. 3. Oxidative stress markers: protein carbonyls (A–B), protein thiols (C–D), and lipid peroxides (E–F) in two-day-old Canton S flies grown on diets with different concentrations of glucose and fructose. Amounts of protein carbonyls and protein thiols are shown as nanomoles per milligram of total protein, amount of lipid peroxides is expressed in nanomoles per milligram of total lipids. Data are presented as means ± S.E.M., n = 5–6 (independent replicates with 20–40 flies in each replicate). *Significantly different from the group fed the same monosaccharide at concentration of 4%, and #from the group fed glucose at the same concentration with P b 0.05. ♣Significantly different from the groups fed the same monosaccharide at concentrations of 0.25% and 10% (P b 0.05).

observed in flies only when different diets were administrated during development (Rovenko et al., 2015). Using the same developmental model (Rovenko et al., 2015), in this study we aimed to disclose the role, which mitochondrial activity and mtROS play in the increase in oxidative damages (to proteins and lipids) and in enhance capacity of antioxidant defense, and more important, how all these parameters contribute to the deleterious phenotypes caused by glucose and fructose overfeeding. Here, we report that glucose and fructose differently affect mitochondrial activity in Drosophila. The effects of these reducing monosaccharides are determined by their concentration in the diet. Restriction of dietary glucose, but not fructose, augmented mitochondrial respiration probably by an increase in the number of mitochondria. We found that ROS production by mitochondria only correlated with mitochondrial respiration in males fed glucose (r = 0.92, P = 0.0548). In general, mtROS production did not correlate with mitochondrial respiration similarly to earlier studies (reviewed in Barja, 2007). Interestingly, mtROS production by complex III was higher in females

than in males, whereas other studies show opposite results using different fly backgrounds and rearing conditions (Sanz et al., 2010; Cocheme et al., 2011). This indicates that mtROS production is a plastic and complex parameter influenced by the environmental conditions. Restriction of both carbohydrates induced mild oxidative stress in flies of both sexes, which was directly associated neither with mitochondrial activity and mtROS, nor with antioxidant system response. Because of this, it can be suggested that mtROS and antioxidant machinery might be regulated in different ways, but overlap to maintain appropriate redox homeostasis at certain nutritional conditions. Several hypotheses may explain differences in mitochondrial function associated with glucose and fructose rich diets. Firstly, glucose and fructose are metabolized through distinct metabolic pathways such as glycolysis and fructolysis (Basciano et al., 2005; Moeller et al., 2009; Tappy and Lê, 2010; Stanhope et al., 2013). Since fructolysis includes fewer reactions than glycolysis to form Krebs cycle precursors (Basciano et al., 2005; Tappy and Lê, 2010), restriction of fructose should demand less mitochondria to provide the same energetic output. Secondly,

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Table 3 Pearson correlation coefficients between indices characterizing mitochondrial metabolism and oxidative stress indices/antioxidant defense. The data are shown as correlation coefficient (r) and P-value. For presented correlation analysis sex-specific differences were neglected. Parameters studied

I + III + IV respiration

MPD

mtROS (complex I)

mtROS (complex III)

Glucose PC PT Lipid peroxides SOD activity Catalase activity GST activity GR activity G6PDH activity IDH activity SOD1 mRNA/Act88F SOD2 mRNA/Act88F Cat mRNA/Act88F Gpx mRNA/Act88F

0.8381; 0.0372⁎ −0.1800; 0.7329 0.3612; 0.4818 0.8293; 0.0412⁎ −0.2816; 0.5888 0.3734; 0.4659 0.7225; 0.1048 −0.5321; 0.5456 −0.3132; 0.5456 −0.5336; 0.2756 0.1348; 0.7990 0.1259; 0.8121 0.4215; 0.4052

0.9110; 0.0115⁎ −0.6152; 0.1937 0.7380; 0.0940 0.8727; 0.0233⁎ 0.5888; 0.3570 0.5163; 0.2943 0.8387; 0.0369⁎ −0.6020; 0.2060 −0.3170; 0.5405 −0.3809; 0.4562 0.5854; 0.2222 0.2589; 0.6203 0.3183; 0.5387

−0.3515; 0.4945 −0.6349; 0.1756 0.4237; 0.4025 −0.1817; 0.7304 0.2332; 0.6565 0.1135; 0.8305 −0.2104; 0.6890 0.6305; 0.1796 0.5018; 0.3104 0.2911; 0.5757 0.1772; 0.7370 −0.0507; 0.9240 −0.6463; 0.1655

0.5615; 0.2463 0.1086; 0.8377 −0.2419; 0.6442 −0.1041; 0.8444 −0.8829; 0.0198⁎ −0.6316; 0.1786 −0.0398; 0.9404 −0.6735; 0.1425 −0.9294; 0.0073⁎ −0.7489; 0.0867 0.3007; 0.5626 −0.7376; 0.0942 0.7877; 0.0628

Fructose PC PT Lipid peroxides SOD activity Catalase activity GST activity GR activity G6PDH activity IDH activity SOD1 mRNA/Act88F SOD2 mRNA/Act88F Cat mRNA/Act88F Gpx mRNA/Act88F

0.1095; 0.8364 0.3364; 0.5144 −0.3589; 0.4848 −0.3711; 0.4689 −0.6305; 0.1796 −0.7382; 0.0939 0.3801; 0.4573 −0.6556; 0.1575 −0.9653; 0.0018⁎ 0.3690; 0.4716 0.0783; 0.8828 −0.0726; 0.8912 0.7363; 0.0951

0.0977; 0.8540 0.3225; 0.5330 0.2861; 0.5826 −0.3024; 0.5602 −0.6630; 0.1512 −0.6848; 0.1334 0.4467; 0.3746 −0.7099; 0.1141 −0.9329; 0.0066⁎ 0.3385; 0.5117 0.0236; 0.9647 −0.1418; 0.7888 0.7539; 0.0834

−0.4941; 0.3192 0.2152; 0.6821 0.2052; 0.6965 0.1223; 0.8174 0.0699; 0.8954 0.4614; 0.3571 0.4963; 0.3167 0.5777; 0.2299 0.5782; 0.2293 −0.5067; 0.3050 −0.5079; 0.3037 0.6948; 0.1255 −0.0602; 0.9098

−0.0021; 0.9968 0.5278; 0.2819 −0.3203; 0.5360 −0.3413; 0.5079 −0.6673; 0.1477 −0.6891; 0.1300 0.3534; 0.4919 −0.6911; 0.1284 −0.8946; 0.0161⁎ 0.2562; 0.6242 0.0675; 0.8989 −0.0086; 0.9871 0.8793; 0.0210⁎

⁎ Significant correlation with P b 0.05.

negligible effect of fructose concentration on mitochondrial respiration could be explained by different consumption of glucose and fructose when they are available at the same concentration. Our previous studies showed that flies consume more fructose than glucose independently of the developmental stage supporting this hypothesis (Lushchak et al., 2014; Rovenko et al., 2015). Thirdly, fructose may be catabolized to oxalate, whereas glucose does not, and calcium salts of oxaloacetate are known to inhibit mitochondrial respiration and induce the opening of the mitochondrial permeability transition pore (Shangari and O'Brien, 2004). These data are in agreement with recent findings reporting the effect of fructose in rats, where high doses of fructose activated UCP5 uncoupling respiration and ATP production (Mortensen et al., 2014). In this case, increased mitochondrial respiration would be an adaptation to compensate the decrease in ATP production. Accordingly, respiration rate was higher in flies of both sexes fed fructose diets. It is possible that inhibition of respiration activates AMP-activated protein kinase increasing mitochondrial biogenesis to respond to a “false state” of cellular starvation (Cha et al., 2008; Tappy and Lê, 2010). Alternatively, it is possible that excess of glucose inhibits mitochondrial respiration. In numerous cell types an inhibition of respiration has been reported in the presence of glucose at high concentrations in culture medium. This phenomenon is known as “Crabtree effect” (Diaz-Ruiz et al., 2008). Crabtree effect works usually as a short term reversible adaptation and is mostly shown for cell culture, tumor and yeast cells (Diaz-Ruiz et al., 2011). However, it is not excluded that it can take place in the living multicellular organisms with negative consequences on metabolism. Recently, we have shown that high glucose rather than fructose was toxic and decreased developmental survival (Rovenko et al., 2015). Inhibition of mitochondrial respiration could be a possible reason for that. Besides of mammalian models, the adverse effects of glucose supplementation on lifespan of Caenorhabditis elegans have been shown (Lee et al., 2009). In another independent study on C. elegans, restriction of glucose extended lifespan inducing mitochondrial respiration and oxidative stress (Schulz et al., 2007). A recent study in Drosophila showed that a mild perturbation of

mitochondrial functioning in muscle tissue increased lifespan due to mechanisms related with oxidative stress and repression of insulin/insulin-like growth factor signaling (Owusu-Ansah et al., 2013). These mechanisms may be involved in our studies, since glucose restriction is associated with induction of oxidative stress, increased developmental survival and repressed expression of Drosophila insulinlike peptide 3 (Rovenko et al., 2015). However, it is clear that the relationship between mitochondrial function, oxidative stress and levels of carbohydrates is very complex. For example, the respiration rate (except males fed glucose) did not correlate with mtROS production. Instead, mtROS levels were related to fly sex. In this aspect, different metabolic rates in males and females seem to be better predictors of ROS production. The role of mtROS in the regulation of cellular functions is quite complex since they act both as damaging agents (Lushchak, 2011) and regulatory messengers (Weinberg et al., 2010; Yadav and Ramana, 2013). The different roles played by ROS may explain the lack of positive correlation between mitochondrial ROS production and oxidative modifications of lipids and proteins (Table 3). Moreover, even negative feedback between both parameters was found. For example, upon high fructose consumption where higher amount of mtROS is produced the amount of oxidized lipids is lower, compared to glucose. At the same time, we observed that restriction of monosaccharides increased oxidative damage in flies of both sexes, and these differences could not be explained by changes in mtROS production or mitochondrial respiration. If one would extrapolate these data to the developmental survival shown in our previous study (Rovenko et al., 2015), it is clear that oxidative damage is a better predictor of developmental survival than mtROS. Neither respiration, nor mtROS explain oxidative damage received in this study (Table 3). Thereby, it is plausible to suggest that the amount of oxidatively modified biomolecules does not depend only on intensity of ROS production, but also on the activity of antioxidant systems. Indeed, activity of SOD was higher upon monosaccharide restriction. This in combination with enhanced levels of markers of oxidative damages to proteins and lipids collectively

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Fig. 4. Superoxide dismutase (SOD) activity (A–B) and relative mRNA levels of Sod1 (C–D) and Sod2 (E–F) in two-day-old flies Canton S grown on diets with different concentrations of glucose and fructose. SOD activities are shown as units normalized to the milligram of total protein; one unit of enzyme is defined as the amount of enzyme (per milligram of protein) that inhibits quercetin oxidation reaction by 50% of maximal inhibition. mRNA levels of the target genes are normalized to the expression levels of the reference gene (CG5178, Act88F), encoding thin F-actin-like filament. The data are shown as means ± S.E.M., n = 4–7 (independent replicates with 20–40 flies in each replicate). *Significantly different from the group fed the same monosaccharide at concentration of 4%, and #from the group fed glucose at the same concentration with P b 0.05. ♣Significantly different from the groups fed the same monosaccharide at concentrations of 0.25% and 10% (P b 0.05).

indicates development of mild oxidative stress (Lushchak, 2014). At the same time, the activity of catalase negatively correlated to mtROS levels (Table 3). Similar sex-specific differences in rate of ROS and its interaction with catalase activity have been shown in Drosophila simulans (Ballard et al., 2007). We also found here that steady-state specific mRNA levels and enzymatic activities of main antioxidant enzymes are differently affected by glucose and fructose levels in the diet. For instance, our data show that fructose rather than glucose regulates transcription of antioxidant enzymes. Glutathione and systems responsible for its maintaining in reduced state seem to be more related to carbohydrate concentration and fly sex than to the type of carbohydrate. Thus, we can suggest that consumption of glucose and fructose differently affected the levels of oxidative damages to lipids and proteins. Glucose may influence oxidative damage via regulation of mitochondrial activity, whether fructose may regulate expression and activities of antioxidant enzymes, affecting the efficiency of ROS detoxification. However, independent on the type

of carbohydrate and the way of regulation of free radical processes, restriction of glucose and fructose causes mild oxidative stress associated with higher survival in Drosophila (Rovenko et al., 2015). 5. Conclusions Excessive intake of glucose and fructose is considered to be a risk factor for development of diet-induced metabolic disorders in humans (Basciano et al., 2005; Moeller et al., 2009; Tappy and Lê, 2010; Henriksen et al., 2011; Hulsmans et al., 2012; Jimenez-Del-Rio and Velez-Pardo, 2012; Semchyshyn, 2013; Stanhope et al., 2013; Semchyshyn et al., 2014). In our recent study, using a Drosophila developmental model, we demonstrated that glucose and fructose at high concentrations impaired metabolism in different ways: while glucose application resulted in glucose toxicity due to the increased level of circulating glucose, fructose was more prone to induce obesity promoting accumulation of reserve lipids and carbohydrates (Rovenko

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Fig. 5. Catalase activity (A–B) and relative mRNA levels of Cat (C–D) and glutathione peroxidase (Gpx) (E, F) in two-day-old Canton S flies grown on diets with different concentrations of glucose and fructose. Catalase activities are shown as international units normalized to the milligram of total protein. Expression of the target genes is normalized to the expression level of the reference gene (CG5178, Act88F), encoding thin F-actin-like filament. The data of enzyme activity are shown as means ± S.E.M., n = 4–7 (independent replicates with 20–40 flies in each replicate). *Significantly different from the group fed the same monosaccharide at concentration of 4%, and #from the group fed glucose at the same concentration with P b 0.05. ♣ Significantly different from the groups fed the same monosaccharide at concentrations of 0.25% and 10% (P b 0.05).

et al., 2015). Excessive intake of both carbohydrates decreased developmental survival in Drosophila flies (Rovenko et al., 2015). Initially, we hypothesized that changes in mitochondrial activity would explain changes in oxidative damage levels and survival; however, the data presented in this paper do not fully support this hypothesis. Although mitochondrial activity and mtROS production depended on the type of monosaccharide in a concentration and sex-related manner, we did not find any clear connection between mtROS and antioxidant defense or between antioxidant defense and developmental survival. Instead, this study shows a complex interaction between the levels of monosaccharides, mitochondrial function and oxidative damage. It seems that the highest developmental survival is associated with the highest levels of oxidative damages to lipids and proteins, similarly as it was suggested in other studies (Schulz et al., 2007; Owusu-Ansah et al., 2013). Because of this, mtROS and antioxidant machinery act as a well-balanced system maintaining certain state of oxidation to biomolecules. In this complex system, glucose is more prone to control amount of oxidative damage through regulation of mitochondrial activity, whereas fructose acts as a regulator of transcription of antioxidant enzymes. Finally, independently

on the type of monosaccharide, restriction of glucose and fructose causes mild oxidative stress associated with highest survival in Drosophila. These findings partially refute oxidative stress as an obligatory factor linked to carbohydrate-induced obese phenotype at least in fruit flies. At the same time, they support a crucial role of mitochondria in the metabolic response to different carbohydrates: overfeeding of glucose, but not fructose represses mitochondrial respiration; this might be a reason for the adverse effects of glucose on development and survival during development. Competing interests The authors declare no competing interests. Acknowledgments The work was partially supported by the FEBS Collaborative Experimental Scholarship for Central and Eastern Europe to B.R. (#261793). A.S. was supported by an ERC Starting Grant (#260632)

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and by the Academy of Finland as a Research Academy Fellow (#252048). We are grateful to A. Glovyak, A. Sriram and V. Mallikarjun for the excellent technical assistance. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.cbpa.2015.04.012. References Ballard, J.W., Melvin, R.G., Miller, J.T., Katewa, S.D., 2007. Sex differences in survival and mitochondrial bioenergetics during aging in Drosophila. Aging Cell 6 (5), 699–708. Barja, G., 2007. Mitochondrial oxygen consumption and reactive oxygen species production are independently modulated: implications for aging studies. Rejuvenation Res. 10 (2), 215–224. Basciano, H., Federico, L., Adeli, K., 2005. Fructose, insulin resistance, and metabolic dyslipidemia. Nutr. Metab. (Lond.) 2 (1), 5. http://dx.doi.org/10.1186/1743-7075-1182-1185. Bradford, M.M., 1976. 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