Dahleh, 2022 PDF

1 Effects of concurrent-type exercise and γ-oryzanol supplementation on aerobic and
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2 anaerobic metabolism in Drosophila melanogaster
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5 Mustafa Munir Mustafa Dahleh,a Stífani Machado Araujo,a Vandreza Cardoso Bortolotto,a
6 Franciane Cabral Pinheiro,a Franciéle Romero Machado,a Luana Barreto Meichtry,a Elize
7 Aparecida Santos Musachio,a Gustavo Petri Guerra,a and Marina Prigola,*
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9 aLaboratory of Pharmacological and Toxicological Evaluations Applied to Bioactive
10 Molecules - LaftamBio - Federal University of Pampa, Itaqui, CEP 97650-000, RS, Brazil.
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19 *Corresponding author:
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20 Marina Prigol
21 Laboratory of Pharmacological and Toxicological Evaluations Applied to Bioactive Molecules

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22 – LaftamBio – Federal University of Pampa
23 Itaqui, CEP 97.650-000, RS, Brazil.
24 E-mail: marinaprigol@unipampa.edu.br
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25 TEL: (55) 99644-7424
This preprint research paper has not been peer reviewed. Electronic copy available at: https://ssrn.com/abstract=4176845
26 ABSTRACT
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27 Concurrent exercise acts as one of the main ways to decrease physical inactivity and the
28 development of several metabolic pathologies. In this sense, this study demonstrated that
29 concurrent-type exercise and γ-oryzanol supplementation (exercise + ORY) can increase
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30 Drosophila melanogaster mobility through the open field test compared to exercise alone. In
31 the same way, exercise + ORY modulate aerobic metabolism through increased citrate synthase
32 activity, decrease triacylglycerol levels, and increase glucose, glycogen, and lactate levels,
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33 compared to exercise groups, which are essential energetic substrates to improve physical
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34 performance. The exercise and exercise + ORY groups, compared to the control and MLF
35 (movement-limited flies) groups, had higher endurance-like tolerance in the forced swim test
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and lactate dehydrogenase activity. This indicates a possible increase in metabolic energy
37 demand, which may be associated with increased food consumption in these groups, allowing
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38 higher protein levels and possibly a higher survival rate regardless of ORY supplementation.
39 Nonetheless, we verified that reducing the locomotion environment in the MLF group
40 decreased mobility and endurance-like tolerance. The lower mobility reduces glycogen storage
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41 capacity, which is associated with higher triglyceride levels and lower total protein levels and
42 survival rates. Finally, we demonstrated that ORY supplementation combined with concurrent-
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43 type exercise could modulate the metabolic responses of this animal, which were associated
44 with higher locomotion capacity through an open field test, indicating the ergogenic potential
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45 of ORY and its synergistic effects with concurrent-type exercise in Drosophila melanogaster.
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47 Keywords: Concurrent-type exercise; γ-Oryzanol; Drosophila melanogaster; Metabolism.
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51 1. INTRODUCTION
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52 Physical exercise is considered one of the fundamental means of inhibiting the
53 development of pathologies, especially those associated with the prophylactic and therapeutic
54 action of complications resulting from metabolic syndrome, including obesity, high
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55 cholesterol, increased blood pressure, and diabetes [1]. Park et al. (2019) reported that the
56 sedentary lifestyle is associated with 75% of all-cause mortality outcomes, revealing a need for
57 further research on different prophylactic and therapeutic proposals for physical inactivity [2].
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58 Concurrent exercise is characterized by the combination of aerobic and anaerobic
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59 exercises and is one of the leading intervention protocols for physical inactivity; it is associated
60 with regulating metabolic parameters such as blood glucose, triglycerides, cholesterol, and
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body weight [1]. Nevertheless, compounds that can act synergistically with concurrent
62 exercise, increase physical performance, and regulate energy metabolism, are still being
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63 sought.
64 Some phytochemical compounds, including γ-oryzanol (ORY), are evidenced by their
65 ability to regulate parameters of exercise metabolism; ORY increase antioxidant defenses by

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66 increasing superoxide dismutase and catalase activity, suppressing inflammatory pathways
67 such as p38 MAPK (mitogen-activated protein kinases), an important marker that, when highly
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68 expressed, can inhibit proteins including Akt (protein kinase B)/phospho-Akt, essential for an
69 adaptive response to physical exercise [3, 4].

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70 γ-Oryzanol is a phytochemical derived from rice bran oil and has ferulic acid as one of
71 its chemical compositions, which can improve the physical performance of concurrent exercise
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72 by suppressing inflammatory mechanisms (e.g., in rodents) [5]. Moreover, ORY increases
73 PPAR-γ (peroxisome proliferator activated receptor gamma) expression, leading to AMPK(5'
74 AMP-activated protein kinase) phosphorylation and higher PGC-1α (peroxisome proliferator-

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75 activated receptor gamma coactivator 1-alpha) expression, increasing mitochondrial
76 biogenesis, fatty acid oxidation capacity, and the oxidative capacity of muscle tissues, which
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77 is associated with improved phosphorylation of glycolytic reserves, leading to greater physical
78 performance [5].
79 Ferulic acids (e.g., ORY) act in the regulation of parameters associated with increased
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80 longevity and survival rate by reducing reactive oxygen species (ROS) formation, leading to
81 lower lipid peroxidation and increased antioxidant enzyme activity, enabling better
82 mitochondrial functioning and, therefore, improving the aerobic metabolism of the exercise
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83 [5–7]. Despite these mechanisms in mammals, there is still a lack of data on how ORY acts in
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84 modulating aerobic and anaerobic metabolism, contributing to higher physical performance,
85 which is associated with metabolic parameter regulation, involving fatty acid oxidation and
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greater glycolytic reserves, thereby setting a precedent for new approaches for this compound
87 and its association with exercise. However, the current use of mammals for this type of study
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88 has been decreasing, bringing the need to establish an alternative animal model to evaluate
89 metabolic pathways derived from ORY with exercise [8].
90 Alternative animal models with fewer ethical and economic issues are an interesting
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91 option for experimental research and require invertebrates such as Drosophila melanogaster, a
92 relevant model for the study to evaluate ORY and its effects on concurrent-type exercise and
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93 its responses on aerobic and anaerobic metabolism. Given this context, this study aimed to
94 evaluate possible anaerobic and aerobic mechanisms of energy metabolism in Drosophila

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95 melanogaster induced by concurrent-type exercise and ORY supplementation.
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97 2. MATERIALS AND METHODS
98 2.1. Chemicals
99 γ-Oryzanol was obtained from Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan), and
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100 the kits and reagents were directly purchased from Labtest®. The Sigma-Aldrich® (São Paulo,
101 SP, Brazil) reagents were provided by ServyLab®. Other reagents were purchased from
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102 Laftambio (UNIPAMPA, Itaqui).
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104 2.2. Materials and fly culture conditions
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105 Wild-type Drosophila melanogaster (Harwich strain) was obtained from LaftamBio
106 Pampa (UNIPAMPA, Itaqui) using flies of both sexes kept (70% females, 30% males) at room
107 temperature (24–25 ºC) and humidity of 30–50% in a 12-h light/dark cycle (12:12). The flies’
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108 diet consisted of cornflour (76.59%), sugar (7.23%), wheat germ (8.51%), powdered milk
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109 (7.23%), salt (0.43%), and Nipagin (methylparaben 0.08%).
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2.3. Experimental design and concurrent-type exercise protocol
112 Two-day-old adults Drosophila melanogaster flies were separated into the following
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113 groups: control [without exercise + vehicle], movement-limited flies (MLF) [without exercise
114 + vehicle + locomotor limitations], EXE [exercise + vehicle], ORY [without exercise + ORY
115 25 μM], and EXE+ORY [exercise + ORY 25 μM]. The ORY was diluted in 1% sucrose
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116 (vehicle) with a final concentration of 25 μM based on an ORY concentration curve at 25, 50,
117 75, and 100 μM, where the lower concentration was found to have synergistic effects with
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118 concurrent-type exercise on Drosophila melanogaster [7-10]. All groups underwent a 7-day
119 protocol. The MLF group was kept in a 5-cm environment in jars (141.37-cm3 volume) to limit
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120 their mobility; this was done as the other experimental groups were kept in 10-cm environments
121 and jars with a 282.74-cm3 volume [7] (Fig. 1). Twenty flies were kept in each jar, considering
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122 that the Drosophila melanogaster were approximately 3 mm in size [7].
123 The induction of continuous movements in the groups submitted to concurrent-type
124 exercise (Table 1) was recorded and registered by the research group (INPI, BR10202000813;
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125 2020); characterized by having a motor with three rotations per minute (RPM). The
126 concurrent-type exercise was induced through the progression of the training volume
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127 performed, aiming progression during the exercise, as well as adaptation to greater locomotion
128 demands, avoiding maximum exhaustion of the animal [7]. The exercised flies were kept in
129 tubes (6-cm high x 1.5-cm wide) attached to metallic claws anchored to the main rod
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130 responsible for rotating the equipment [7]. With the constant rotations of the equipment, the
131 tubes with the flies were continuously directed to the brightest places in the environment,
132 allowing the flies, through their innate behavior of negative geotaxis, to move constantly from
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133 the base to the top of the tube, always aimed at the brightest place in the environment (positive
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134 phototaxis), allowing the flies to exert continuous effort within a defined period [7]. The
135 experimental behavioral and biochemical analysis was carried out at the end of the eighth day
136 (Fig. 1).

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138 2.4. In vivo analysis
139 All flies submitted to in vivo analysis were randomly selected considering that the
140 behavioral tests were carried out at the end of the eighth day and to avoid possible direct
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141 interferences of tiredness or exhaustion by the experimental design.
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143 2.4.1. Survival rate
144 The survival rate was determined by counting the number of live flies until the end of
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145 the experimental period (7 days) using 120 flies per group. The total number of flies is
146 represented by the sum of 6 independent experiments (20 flies per treatment), with the result
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147 expressed as a percentage of the survival rate.
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149 2.4.2. Locomotor behavior evaluation by the open field test and negative geotaxis
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150 The locomotor behavior was assessed by the open field test according to Hirth (2010)
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151 [11]. The flies were previously anesthetized on ice (0–5 °C) for 2 min and placed on an acrylic
152 plate delimited in 1 x 1-cm squares and kept for 10 min until recovery. Afterward, the
153 quantification of squares covered during 1 min was performed in duplicate with a 10-min
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154 interval for each repetition to avoid interference by fatigue. Around 30 flies were used per
155 group, totaling 6 independent experiments.
156 Fly climbing behavior was evaluated by the negative geotaxis induction method [10].
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157 Each fly was briefly anesthetized on ice (0–5 °C) for 2 min and placed in a 6-cm high and 1.5-
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158 cm wide tube. After 10 min and with the fly recovered, it was gently tapped to the base of the
159 tube, and the time spent to reach the top of the tube (6 cm) was counted, with 2 min being the
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maximum time allowed in the validation of the test. Flies that exceeded this threshold were
161 excluded from testing. Five repetitions were performed for each fly, and the final value was
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162 defined from an average of the repetitions.. Each repetition was considered a 2-min interval,
163 and around 30 flies were used per group, totaling 6 independent experiments.
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165 2.4.3. Endurance-like tolerance evaluation by the forced swim test
166 The forced swim test was performed according to [12], with adaptations by [13, 14].
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167 For the test, containers with 4 mL of 0.04% sodium dodecyl sulfate (SDS; L3771, Sigma-
168 Aldrich ®) were used, which is a surfactant capable of decreasing the surface tension in water,
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169 allowing the fly to swim.
170 The forced swim test induces the animal to exert great efforts, given the need to swim
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171 constantly. Therefore, it was used to evaluate the physical effort capacity of the invertebrate by
172 indirectly measuring the endurance capacity through three parameters: latency until the first
173 immobility (when the animal is positioned to constantly swims until the first moment of
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174 motionless), swimming time (the total swimming time regardless of the moments of
175 immobility), and immobility time during the 5 min of the test (the total amount of time the
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176 animal remained motionless during the experiment) [13, 14]. All flies were removed from the
177 containers, dried on paper towels, and their ability to walk and fly was analyzed; no locomotor
178 impairments were observed. The resistance capacity measured by the forced swim test is
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179 already used in research with rodents, and these parameters are used to evaluate the animals’
180 effort capacity [13, 14]. Around 30 flies were used per group, totaling 6 independent
181 experiments. Given the high metabolic activity, the animals employed in this test were not used
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182 for biochemical analysis.
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184 2.5. Sample preparation
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For the ex vivo analysis, twenty flies were anesthetized and euthanized at -5 °C and
186 homogenized in a HEPES buffer (H3375; Sigma-Aldrich ®), pH 7.0, at a ratio of 1:10 (flies/µL
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187 volume). The samples were centrifuged at 1000 × g at 4 ºC, and the supernatant (S1) was used.
188 The S1 was used for all ex vivo analyses except the food consumption analysis, and its sample
189 preparation method was described.

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191 2.5.1. Food consumption

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192 The method of [15] was used for food consumption, with minor modifications. The flies
193 were subjected to a standard diet containing 0.5% Brilliant blue FCF (80717; Sigma-Aldrich®)
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194 right after the last training session (day 7) and were allowed to feed for 30 min, then removed
195 and anesthetized on ice (0–5 °C) for 2 min. The flies were euthanized and homogenized in 200
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196 µL of distilled water, centrifuged at 12,000 × g for 2 min, and the supernatant was removed
197 and measured in a spectrophotometer at 625 nm. In this test, food consumption was performed
198 after the last day of exercise to indirectly measure metabolic demand. Twenty flies were used
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199 for each experimental group, and the mean of each experiment was represented as n = 1 using
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200 twenty flies, and 6 independent experiments were performed (n = 6).
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202 2.5.2. Glucose and triglycerides levels
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203 A Labtest® kit was used to measure glucose (MS 10009010236; Ref.133) and
204 triglyceride (MS 10009010070; Ref. 87) levels according to the manufacturer’s instructions.
205 Briefly, 5 µL of S1 was transferred to 170 µL of glucose or triglyceride reagent (incubated at
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206 37 ºC for 10 min. In glucose and triglyceride measurements, the reaction was measured in
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207 spectrophotometry at 505 nm. A duplicate was performed using the mean value for each
208 sample, and the results were obtained from a standard of glucose (100 mg/dL) and triglyceride
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(0.5 g/L glycerol; sodium azide 1.54 mmol/L); the results were expressed as mg/dL after
210 normalization by the tissue weight in each sample.

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212 2.5.3. Glycogen levels
213 Glycogen levels were analyzed using the method of [16], with minor changes, for
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214 detecting glycogen in small amounts of tissue. The reaction was briefly started by adding 60
215 µL of K(OH) 30% with 200 µL of S1 and pre-incubated at 100 ºC for 10 min. The reaction was
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216 stopped by placing the sample at -5 °C and adding 50 µL of ethanol after the sample cooled,
217 incubating once again at 70 ºC for 10 min. The glycogen was detected by adding 110 µL of
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218 distilled water to the sample with 90 µL of iodine reagent (1.5 M KI and 0.1 M I2). The sample
219 was analyzed by spectrophotometry at 460 nm. A duplicate was performed for each sample
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220 using the mean value, and the results were expressed as % of the control after normalization
221 by the protein concentration in each sample.
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223 2.5.4. Lactate levels
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224 The lactate levels were analyzed using a Sigma-Aldrich® (MAK064) kit and used
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225 according to the manufacturer’s instructions. Briefly, 150 µL of S1 were added to a 96-well
226 plate with 46 uL of Lactate assay buffer (MAK064A); to start the reaction, 2 µL of Lactate
227 Enzyme Mix (MAK064C) and 2 µL of Lactate Probe (MAK064B) were incubated for 30 min
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228 at room temperature and protected from light. The reading was performed by
229 spectrophotometer at 570 nm, and a duplicate was performed for each sample using the mean
230 value. Data were obtained using a standard lactate curve (MAK064D), and the results were
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231 expressed in ng/dL after normalization by the tissue weight of each sample.
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233 2.5.5. Protein levels
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Protein levels were measured by the method of [17] to quantify total protein content
235 and lean body mass; 10 µL of S1 diluted in 90 µL of distilled water was used. Then, 20 µL of
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236 the sample solution was mixed with 180 µL of Coomassie Blue (100 mg/mL) in a 96-well plate
237 and incubated at room temperature for 5 min and protected from light. A duplicate was
238 performed for each sample using the mean value. The quantification was performed using a
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239 spectrophotometer at 595 nm, and the results were calculated from a standard curve of bovine
240 serum albumin (A7906; Sigma-Aldrich®) and expressed as mg/mL.

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241
242 2.5.6. Glucose-6-phosphatase (G-6-Pase) activity

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243 The G-6-Pase activity was determined according to [18]. Briefly, 150 μL of 250 mM
244 sucrose/1 mM EDTA pH 7.0 buffer were added, mixed with 100 μL of S1, and the reaction
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245 was started with 50 μL of glucose-6-phosphate (50 mM, 10127647001; Sigma-Aldrich®) and
246 incubated at 37 ºC for 30 min. Following the incubation, in order to stop the reaction, 1000 µL
247 of trichloroacetic acid (TCA) 10% (w/v) was added, and the samples were centrifuged at 4000
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248 × g at 4 ºC to remove the protein precipitate and the dosage of inorganic phosphate (Pi) from
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249 the supernatant. A duplicate was performed for each sample using the mean value. The G-6-
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250 Pase activity was obtained by subtracting the Pi content of the null-time blanks from each
251 sample; the data were expressed in µmol Pi min/mg protein.
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253 2.5.7. Citrate synthase activity
254 Citrate synthase activity was assessed using the method of [19] and based on the release
255 of sulfhydryl (SH) groups from CoA-SH in the presence of 5,5-dithiobis-2 nitrobenzoic acid
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256 (DTNB), forming the thionitrobenzoic acid. For the reaction, 25 uL of S1 was added, with 20
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257 uL of DTNB (1 mM, D8130; Sigma-Aldrich®), 795 uL of distilled water, and 30 uL of acetyl-
258 CoA (10 mM, A2056; Sigma-Aldrich®), starting the reaction by adding 50 uL of oxaloacetate
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(10 mM, A2056; Sigma-Aldrich®). The reaction was performed every 3 min, and the Δ was
260 calculated by spectrophotometry. A duplicate was performed for each sample using the mean
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261 value, and the results were expressed in µmol/min/mg of protein.
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263 2.5.8. Lactate dehydrogenase activity

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264 A Labtest® kit was used to measure the lactate dehydrogenase (LDH) activity (MS
265 10009010056; Ref.86) based on the method of [20], with minor modifications. Briefly, 1000
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266 µL of a solution containing 250 mmol/L phosphate buffer pH 7.5, 1.2 µmol/L sodium pyruvate,
267 0.095% sodium azide, and 300 mmol/L NAD+ was added to a tube and mixed with 20 µL of
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268 S1 and incubated at 37 °C for 10 min. A duplicate was performed for each sample using the
269 mean value, and the results were expressed in µg/dL after normalization by the tissue weight
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270 in each sample.
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272 2.5.9. Statistical analysis

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273 The normality test was performed using the Shapiro-Wilk test, and homoscedasticity of
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274 variances was performed using the Bartlett and F tests. Then, a one-way analysis of variance
275 (ANOVA) was performed, followed by Bonferroni post-hoc test. The survival rate (%) was
276 determined by the Kaplan-Meier curve and the statistical significance of the log-rank Mantel-
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277 Cox. Differences between groups were considered significant when p < 0.05. Data are
278 represented as the mean ± SEM.
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280 3. RESULTS
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281 3.1. Increased survival rate in concurrent-type exercised flies
282 In order to assess the responses of exercise induction and ORY compared to the MLF
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groups, a survival analysis was performed in fig. 2. There was a significant increase in the
284 survival rate of the exercise group compared to the control (p = 0.0159) and MLF groups (p <
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285 0.0001), in the same way as exercise + ORY compared to the control (p < 0.0159) and MLF
286 groups (p < 0.0001). The MLF group had a significantly lower survival rate than the control (p
287 = 0.0065).
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289 3.2. Concurrent-type exercise and ORY supplementation increases Drosophila

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290 melanogaster locomotion
291 Locomotion and exploration behaviors were assessed using the open field test
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292 demonstrated in fig. 3A. There was a significant increase in the locomotion of the exercise +
293 ORY flies compared to all experimental groups (p < 0.0001). Exercise showed a significant
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294 increase in locomotion compared to control, MLF, and ORY (p < 0.0001). In the climbing
295 activity demonstrated in fig. 3B, no significant differences were observed, indicating no change
296 in the innate mechanisms of negative geotaxis.

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298 3.3. Endurance-like tolerance increased by concurrent-type exercised flies in forced swim
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299 test parameters
300 In order to evaluate the endurance-like tolerance of the flies, different parameters of the
301 forced swim test were evaluated. The latency time for the first bout, demonstrated in fig. 4A,
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302 was evaluated and determined by the time from swimming until the first act of immobility, in
303 which we observed that the exercise + ORY had longer latency times than control (p < 0.0001),
304 MLF (p < 0.0001), exercise (p = 0.0193) and ORY (p < 0.0001). The exercise group had longer
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305 latency times to control (p = 0.0034), MLF (p < 0.0001), and ORY (p = 0.0007). The MLF
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306 group showed decreased latency time for the first bout compared to the control (p = 0.0386).
307 In the analysis of the total swimming time and resistance to fatigue evaluation,
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demonstrated in fig. 4B, the exercise + ORY showed longer swimming times compared to
309 control (p = 0.0038), MLF (p < 0.0001) and ORY flies (p = 0.0263). The exercise group
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310 showed longer swimming times compared to control (p = 0.0089), MLF (p = 0.0002) and ORY
311 flies (p = 0.0486). The MLF flies showed lower swimming times compared to control (p =
312 0.0284), and ORY (p = 0.0488). In the results of immobility time, an indicator of fatigue,
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313 demonstrated in fig. 4C, exercise + ORY had a shorter immobility time in relation to control
314 (p = 0.0038), MLF (p < 0.0001) and ORY (p = 0.0023), while the exercise group had a shorter
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315 immobility time in relation to control (p = 0.0089), MLF (p = 0.0024) and ORY (p = 0.0486).
316 The MLF flies showed significantly higher values of immobility time than the control (p =
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317 0.0284) and ORY(p = 0.0488).
318
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319 3.4. Concurrent-type exercise is associated with high food consumption in Drosophila
320 melanogaster
321 In order to evaluate an indirect measure of energy expenditure after the concurrent-type
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322 exercise, a food consumption analysis was performed, demonstrated in fig. 4C. The exercise +
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323 ORY had an increased food consumption compared to the control (p = 0.0022), in the same
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324 way that exercise compared to the control (p = 0.0009).
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326 3.5. Concurrent-type exercise and ORY supplementation modulates energetic substrates
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327 of Drosophila melanogaster metabolism
328 In order to evaluate possible metabolic responses in the induction of concurrent-type
329 exercise and ORY supplementation, energetic substrate levels in Drosophila melanogaster
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330 were verified. In fig. 6A, the exercise + ORY group showed higher glucose levels compared to
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331 the control (p = 0.0069), MLF (p < 0.0001), and ORY (p = 0.0006), while it decreased in the
332 exercise group (p = 0.0472). The exercise-only group showed higher glucose levels compared
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to the control (p = 0.0006), MLF (p < 0.0001), and ORY (p = 0.0006).
334 In fig. 6B, the glycogen content was increased in the exercise + ORY group compared
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335 to control (p = 0.0009), MLF (p < 0.0001), exercise (p = 0.0285), and ORY (p < 0.0001). The
336 exercise-only flies had higher glycogen levels than the control (p = 0.0282), MLF (p = 0.0011),
337 and ORY flies (p = 0.0284); the MLF flies showed significantly lower glycogen compared to
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338 the control (p = 0.0329).
339 In fig. 6C, there were higher lactate levels in the exercise + ORY group compared to all
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340 experimental groups (p < 0.0001). The exercise-only flies showed significantly higher lactate
341 levels than the control (p = 0.0442), MLF (p = 0.0004), and ORY flies (p = 0.0004). In fig. 6D,
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342 triacylglycerol levels were lower in the exercise + ORY compared to the control (p = 0.0407)
343 and MLF flies (p < 0.0001), whereas the MLF showed increased levels compared to the control
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344 (p < 0.0001).
345 In fig. 6E, the total protein levels was increased in the exercise + ORY flies compared
346 to the MLF (p < 0.0001) and ORY (p = 0.0341), while the exercise-only group showed higher
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347 total protein levels compared to the MLF group (p = 0.0005); additionally, the MLF had lower
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348 total protein levels than the control (p = 0.0029).
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350 3.6. Concurrent-type exercise and ORY-supplemented flies increase responses of energy
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351 metabolism enzymes in Drosophila melanogaster
352 A concurrent-type exercise protocol in biological models was developed by evaluating
353 anaerobic and aerobic metabolism markers. In fig. 7A, the exercise + ORY flies showed higher
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354 citrate synthase activity than control (p < 0.0001), MLF (p < 0.0001), exercise (p = 0.0289),
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355 and ORY (p = 0.0004). The exercise group had an increase in citrate synthase activity compared
356 to the control (p = 0.0175) and MLF flies (p = 0.0069).
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In fig. 7B, the exercise + ORY flies showed higher LDH activity compared to the
358 control (p = 0.0007), MLF (p = 0.0003), and ORY flies (p = 0.0129), while the exercise group
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359 showed significantly higher activity than the the control (p = 0.0491), MLF (p = 0.0199). In
360 fig. 7C, the exercise + ORY flies had higher G-6-Pase activity than the control (p = 0.0464)
361 and MLF flies (p = 0.0065), whereas the exercise-only group showed higher activity than the
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362 MLF group (p = 0.0174).
363
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364 4. DISCUSSION
365 We found highly promising results about the metabolism of Drosophila melanogaster
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366 induced by concurrent-type exercise and ORY supplementation. At the end of the experimental
367 protocol, we found higher survival rate percentages in the exercise and exercise + ORY flies.
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368 This result may indicate that the chronicity of the induction of concurrent-type exercise in
369 Drosophila melanogaster can modulate mechanisms associated with higher survival rates in
370 this animal, regardless of ORY supplementation, as the compound cannot positively alter
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371 survival in this animal [7]. Wen et al. (2019) [21] demonstrated that exercise alone in
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372 Drosophila could increase its survival rate, generating mito-nuclear adaptations resulting from
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373 the signaling of proteins such as dSir2, an important marker associated with longevity.
374 Furthermore, our survival rate data may also be associated with increased endogenous
375 antioxidant defenses, including enzymatic mechanisms involved in free radical degradation
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376 [7]. Together, these two mechanisms may be associated with the delay in the aging of these
377 animals and increased cell regeneration rates [10, 22]. Nevertheless, our findings show that
378 MLF flies had lower survival rates, which according to Rothschild et al. (2020) [23], may
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379 corroborate the sedentary behavior in humans as associated with higher mortality rates and
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380 decreased endogenous antioxidant defenses.
381 In the mobility of the flies, we observed that the exercise and exercise + ORY groups
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had a high number of crossings in the open field test, in which exercise + ORY supplementation
383 had higher performance than the exercise-only flies. This ergogenic potential of ORY is mainly
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384 associated with increased expression of PPAR-γ, leading to the suppression of inflammatory
385 pathways, including p38 MAPK, derived from the increased ROS levels, and concomitant
386 activation of PGC-1α, responsible for increased mitochondrial biogenesis, oxidative capacity
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387 of muscles, and fatty acid oxidation [5]. We hypothesize that possible mechanisms associated
388 with p38 MAPK preserved in Drosophila melanogaster may increase the reserve of energy
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389 substrates [24] (e.g., glycogen), allowing greater catabolism and release of free glucose to
390 generate energy for physical performance, similar to mammals [25]. The forced swim test
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391 results show that the exercise and exercise + ORY groups had longer swimming time and
392 shorter immobility time, and no ergogenic potential of ORY was noted in these parameters.
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393 However, when verifying the latency time for the first immobility, we observed an increase in
394 relation to the exercise-only group, indicating a higher ergogenic potential of ORY in acute
395 measures of induced exercise.

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396 Despite this, it is noteworthy that the exercise + ORY flies had a ~ 34% increase in
ed
397 swimming time compared to the exercise-only group, while it had a ~ 25% decrease in
398 immobility time. Notably, the swimming behavior in Drosophila melanogaster is similar to
399 flight as flies use the same muscles for the “paddle” behavior, which is characteristic of
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400 Drosophila flight [26].
401 Regarding the MLF flies, we found less mobility in the open field test, with a shorter
402 swimming time and latency for the first immobility, causing a longer immobility time in the
v
403 forced swim test; this is possibly involved with decreased energetic substrates such as
re
404 glycogen, thereby having less glucose formation by glycogen phosphorylase, which are
405 important in situations of fast demand for ATP [23, 25]. It is noteworthy that the decreased
406
er
mobility in the MLF group can lead to lower energy expenditure, leading to triglyceride
407 accumulation, as observed in our study, since MLF had a ~ 65% increase in triglycerides, while
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408 it obtained a ~ 44% decrease in mobility in the open field test, helping in the characterization
409 of sedentary-type model in Drosophila melanogaster [5, 22]. In the results of climbing activity,
410 there were no significant differences between the experimental groups, indicating no change in
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411 the innate behavior of negative geotaxis, which could indicate neuronal impairment, an
412 interferent in the behavioral analysis [7, 27].

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413 The higher energy requirement generated by the exercise and exercise + ORY groups
414 may be caused by the flies’ higher glucose uptake after the last day of exercise, mainly
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415 associated with high food consumption [28]. The increased food consumption in the exercise
416 and exercise + ORY groups also helps us understand the higher glucose levels, which may be
ep
417 directly associated with the increased glycogen levels, an important parameter to verify the
418 adaptation performed by physical exercise in mammals [29].
419 Furthermore, our results showed that the exercise and exercise + ORY groups had
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420 higher glycogen stores, with higher G-6-Pase activity after the interval of the last exercise
19
421 session, which is quite different from what was found in mammals, where the increased G-6-
ed
422 Pase activity is primarily restricted during exercise and returning to baseline activity during the
423 recovery period and then restoring glycogen levels [30–32]. In addition, a considerable part of
424 this glycogen restoration may be associated with the higher energy demand of these flies due
iew
425 to their high food consumption [32].
426 In contrast to mammals, Drosophila melanogaster metabolism tends to maintain high
427 glucose levels in situations of exhaustion or high metabolic demand for energy substrates due
v
428 to the constant need for trehalose to circulate, even over long periods, enabling the signaling
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429 of innate mechanisms of survival in this invertebrate [33]. When we visualized the results in
430 the MLF group, we observed a decrease in the glycogen stores in the flies as well as a lower
431
er
G-6-Pase activity, mainly linked to the lower energy requirement and possibly also an increase
432 in triacylglycerol levels [30].

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433 In exercise, the intensity and chronicity of the effort exerted are proportional to the
434 energy requirement and, therefore, to the energy substrate released and taken up by the muscle
435 fibers [34, 35]. In this sense, verifying possible substrates of anaerobic and aerobic metabolism
ot
436 helps us verify how Drosophila melanogaster metabolism reacts to such interventions. The
437 anaerobic metabolism undergoes alterations in Drosophila melanogaster in response to

tn
438 concurrent-type exercise.
439 In the results of lactate levels, the exercise and exercise + ORY flies showed higher
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440 levels of this substrate, with an increase in lactate, especially when ORY supplementation was
441 combined with concurrent-type exercise. Along with the higher lactate levels, increased LDH
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442 activity was also verified in the exercise and exercise + ORY groups, indicating the
443 interconversion of pyruvate to lactate through NADH (Nicotinamide Adenine Dinucleotide)
444 oxidation [36]. It is well known that higher LDH is involved in lactate formation, although the
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445 NADH/NAD+ ratio (one of the parameters used to recognize the direction of the reaction) is
20
446 unknown in exercised Drosophila melanogaster models. Hence, there is a possibility of lactate
ed
447 interconversion in pyruvate [37], given the increased food consumption in the exercised
448 groups. In both cases, high LDH activity may already indicate exertion generation in flies
449 induced by concurrent exercise and increased anaerobic metabolic activity, regardless of the
iew
450 reaction direction [38].
451 The characterization of concurrent-type exercise involves increased anaerobic and
452 aerobic metabolism [39], as verified by the higher citrate synthase activity in the exercise and
v
453 exercise + ORY groups, associated with the cellular and metabolic adaptation capacity [39]. In
re
454 the study by Kılıç et al. (2014) [40], rats submitted to moderate and strenuous training showed
455 higher citrate synthase activity 24–48 h after the last day of training. These are similar findings
456
er
to what we found when we observed that flies induced to concurrent-type exercise showed
457 higher values of this enzyme, thus predicting that the protocol was efficient in the possible
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458 metabolic adaptation of Drosophila melanogaster [7].
459 Furthermore, the potential of the exercise + ORY group in having higher citrate
460 synthase activity compared to the exercise-only group stands out, which is primarily associated
ot
461 with the higher fatty acid oxidation capacity derived from this phytochemical, which increases
462 fatty acid release, serving as a substrate for acetyl-CoA formation, which reacts with
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463 oxaloacetate, and forms citrate through citrate synthase [7, 41]. However, it is important to
464 mention that part of acetyl-CoA formation is derived from glycolysis [41], and therefore, can
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465 be modulated by food consumption, which did not show differences between the exercise and
466 exercise + ORY groups. In addition, our results demonstrate that there were no changes in
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467 citrate synthase activity in the MLF group; in contrast, it obtained higher triglyceride levels
468 without changes in food consumption, which may indicate that the animal remained with low
469 energy expenditure, as expected due to the environment with limited movement [7].
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21
470 In the exercise and exercise + ORY flies, we observed an increase in the total amount
ed
471 of proteins. These results may indicate the efficiency of concurrent exercise in modulating
472 mechanisms associated with increased tissue protein synthesis [41]. Nonetheless, no
473 differences were observed in the supplemented and exercised alone, and this is similar to the
iew
474 study of Ahn [5], in which ORY could not up-regulate higher protein synthesis. Moreover, the
475 ability of the concurrent-type exercise protocol to induce greater protein synthesis is similar in
476 mammals, as chronic induction of physical exercise is linked to long-term hypertrophy and
v
477 increased physical performance [42]. Nevertheless, we found that MLF flies had a lower
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478 amount of total protein, a result similar to that found in mammals, in which sedentary behavior
479 is associated with muscular dystrophy, leading to several diseases, including metabolic
480 syndrome [43].

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481
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482 5. CONCLUSIONS
483 The concurrent exercise protocol and ORY supplementation increased Drosophila
484 melanogaster mobility and short-term endurance-like tolerance associated with stimulating
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485 anaerobic and aerobic metabolism capable of modulating glucose and lipid metabolism.
486 Nonetheless, long-term endurance-like tolerance was not possible. Concurrent-type exercise
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487 increased the survival rate of flies, irrespective of ORY supplementation. As expected, exercise
488 + ORY could not increase protein levels. Despite our promising findings, further studies are
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489 needed to validate other metabolic mechanisms altered by exercise + ORY supplementation.
490
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491 ACKNOWLEDGMENTS
492 The authors are grateful for the financial support received from the Conselho Nacional
493 de Desenvolvimento Científico e Tecnológico (CNPq) (308120/2020-5), the Fundação de

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494 Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS) (PQG 19/2551-0001913-0),
22
495 and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) - Financial
ed
496 Code 001 for the support and research grants provided. We would also like to thank Atlas
497 Assessoria Linguística for language editing.
498
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499 CONFLICT OF INTEREST
500 The authors declare that they have no known competing for financial interests or
501 personal relationships.
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502
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503 AUTHORS’ CONTRIBUTIONS
504 MMMD and MP designed the study. MMMD, SMA, VCB, and FCP performed the
505
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biochemical analysis. MMMD, SMA, VCB, FRM, EASM, and LBM carried out the behavioral
506 tests. MMMD analyzed the data and drafted the manuscript. SMA, GPG, and MP revised the
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507 manuscript. All authors contributed substantially to the study and approved the final version of
508 the manuscript.
509
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659 https://doi.org/10.1002/jcsm.12347
660
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661
662
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663
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665
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666
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667 FIGURE CAPTIONS
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668 Figure 1. Schematic representation of the experimental design. Effects of concurrent-type
669 exercise induction and supplementation and ORY (25 µM) and model of movement limitation
670 (MLF) on behavioral and biochemical analyses in Drosophila melanogaster.
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671
672 Figure 2. Survival rate (%) of flies induced by concurrent-type exercise + ORY
673 supplementation for seven days. The test was performed by comparing curves of the Mantel-
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674 Cox log-rank tests (n = 6 for each group). aDifference compared to the control group (p < 0.05);
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675 bDifference compared to the MLF group (p < 0.05).
676
677
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Figure 3. Analysis of the number of crossings in the open field test (cm) and climbing index
678 (s) through negative geotaxis test in a concurrent-type exercise and ORY supplementation. A)
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679 open field test; B) negative geotaxis test. Values are provided as mean ± SEM (n = 6 for each
680 group). Significance was determined by one-way analysis of variance (ANOVA) followed by
681 the Bonferroni post hoc test. aDifference compared to the control group (p < 0.05); bDifference
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682 compared to the MLF group (p < 0.05); cDifference compared to the EXE group (p < 0.05);
683 dDifference compared to the ORY group (p < 0.05).

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684
685 Figure 4. Evaluation of the forced swim test parameters for endurance-type tolerance analysis
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686 in Drosophila melanogaster submitted to a concurrent-type exercise and ORY
687 supplementation. A) Latency for first bout to immobility (s); B) swimming time(s); C)
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688 immobility time(s). Values are provided as mean ± SEM (n = 6 for each group). Significance
689 was determined by one-way analysis of variance (ANOVA) followed by the Bonferroni post
690 hoc test. aDifference compared to the control group (p < 0.05); bDifference compared to the
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30
691 MLF group (p < 0.05); cDifference compared to the EXE group (p < 0.05); dDifference
ed
692 compared to the ORY group (p < 0.05).
693
694 Figure 5. Effects of concurrent-type exercise and ORY supplementation on food consumption.
iew
695 Values are provided as mean ± SEM (n = 6 for each group). Significance was determined by
696 one-way analysis of variance (ANOVA) followed by the Bonferroni post hoc test. aDifference
697 compared to the control group (p < 0.05); cDifference compared to the EXE group (p < 0.05);
v
698 dDifference compared to the ORY group (p < 0.05); eDifference compared to the EXE + ORY
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699 group (p < 0.05).
700
701
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Figure 6. Analysis of energetic substrates of Drosophila melanogaster metabolism in
702 concurrent-type exercise. A) glucose levels; B) glycogen levels; C) lactate levels; D)

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703 triglyceride levels; E) total protein levels. Values are provided as mean ± SEM (n = 6 for each
704 group). Significance was determined by one-way analysis of variance (ANOVA) followed by
705 the Bonferroni post hoc test. aDifference compared to the control group (p < 0.05); bDifference
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706 compared to the MLF group (p < 0.05); cDifference compared to the EXE group (p < 0.05);
707 dDifference compared to the ORY group (p < 0.05).

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708
709 Figure 7. Evaluation of aerobic and anaerobic enzymes in Drosophila melanogaster

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710 metabolism. A) citrate synthase activity; B) lactate dehydrogenase activity; C) G-6-Pase
711 activity. Values are provided as mean ± SEM (n = 6 for each group). Significance was
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712 determined by one-way analysis of variance (ANOVA) followed by the Bonferroni post hoc
713 test. aDifference compared to the control group (p < 0.05); bDifference compared to the MLF
714 group (p < 0.05); cDifference compared to the EXE group (p < 0.05); dDifference compared to
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715 the ORY group (p < 0.05);
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1 Effects of concurrent-type exercise and γ-oryzanol supplementation on aerobic and

7 Aparecida Santos Musachio,a Gustavo Petri Guerra,a and Marina Prigola,*

21 Laboratory of Pharmacological and Toxicological Evaluations Applied to Bioactive Molecules

22 – LaftamBio – Federal University of Pampa

23 Itaqui, CEP 97.650-000, RS, Brazil.

25 TEL: (55) 99644-7424

29 concurrent-type exercise and γ-oryzanol supplementation (exercise + ORY) can increase

47 Keywords: Concurrent-type exercise; γ-Oryzanol; Drosophila melanogaster; Metabolism.

54 action of complications resulting from metabolic syndrome, including obesity, high

64 Some phytochemical compounds, including γ-oryzanol (ORY), are evidenced by their

65 ability to regulate parameters of exercise metabolism; ORY increase antioxidant defenses by

66 increasing superoxide dismutase and catalase activity, suppressing inflammatory pathways

69 adaptive response to physical exercise [3, 4].

72 by suppressing inflammatory mechanisms (e.g., in rodents) [5]. Moreover, ORY increases

73 PPAR-γ (peroxisome proliferator activated receptor gamma) expression, leading to AMPK(5'

74 AMP-activated protein kinase) phosphorylation and higher PGC-1α (peroxisome proliferator-

75 activated receptor gamma coactivator 1-alpha) expression, increasing mitochondrial

89 metabolic pathways derived from ORY with exercise [8].

94 evaluate possible anaerobic and aerobic mechanisms of energy metabolism in Drosophila

95 melanogaster induced by concurrent-type exercise and ORY supplementation.

97 2. MATERIALS AND METHODS

104 2.2. Materials and fly culture conditions

122 that the Drosophila melanogaster were approximately 3 mm in size [7].

123 The induction of continuous movements in the groups submitted to concurrent-type

136 (Fig. 1).

141 interferences of tiredness or exhaustion by the experimental design.

143 2.4.1. Survival rate

147 expressed as a percentage of the survival rate.

155 group, totaling 6 independent experiments.

165 2.4.3. Endurance-like tolerance evaluation by the forced swim test

169 allowing the fly to swim.

184 2.5. Sample preparation

189 preparation method was described.

191 2.5.1. Food consumption

202 2.5.2. Glucose and triglycerides levels

205 Briefly, 5 µL of S1 was transferred to 170 µL of glucose or triglyceride reagent (incubated at

210 normalization by the tissue weight in each sample.

212 2.5.3. Glycogen levels

221 by the protein concentration in each sample.

223 2.5.4. Lactate levels

233 2.5.5. Protein levels

240 serum albumin (A7906; Sigma-Aldrich®) and expressed as mg/mL.

242 2.5.6. Glucose-6-phosphatase (G-6-Pase) activity

251 sample; the data were expressed in µmol Pi min/mg protein.

263 2.5.8. Lactate dehydrogenase activity

270 in each sample.

272 2.5.9. Statistical analysis

278 represented as the mean ± SEM.

289 3.2. Concurrent-type exercise and ORY supplementation increases Drosophila

290 melanogaster locomotion

296 in the innate mechanisms of negative geotaxis.

317 0.0284) and ORY(p = 0.0488).

328 In order to evaluate possible metabolic responses in the induction of concurrent-type

338 the control (p = 0.0329).

344 (p < 0.0001).

352 A concurrent-type exercise protocol in biological models was developed by evaluating

356 to the control (p = 0.0175) and MLF flies (p = 0.0069).

362 MLF group (p = 0.0174).

395 measures of induced exercise.

412 interferent in the behavioral analysis [7, 27].

418 adaptation performed by physical exercise in mammals [29].

426 In contrast to mammals, Drosophila melanogaster metabolism tends to maintain high

432 in triacylglycerol levels [30].