Dietary sucrose Defines lifespan anD metabolism in Drosophila

Nutrition affects various life-history traits. We used fruit flies Drosophila melanogaster to determine whether life-history traits, particularly life span and metabolism, are affected by dietary sucrose content. We fed flies by four different diets containing constant yeast concentration and increasing amounts of sugar ranged from 1% to 20%. We found that low sucrose diet increases female lifespan. We also showed, that low dietary sucrose maximized malate dehydrogenase, aspartate aminotransferase activity in males and lactate dehydrogenase activity in females. In addition, dietary carbohydrate has a considerable impact on urea level, suggesting that dietary carbohydrate impacts overall metabolism. Our findings revealed the influence of dietary sugar on metabolic enzymes activities, indicating an existence of optimal nutritional conditions for prolongevity phenotype and confirming an important impact of dietary sugar on life-history traits.

D iet and nutritional factors largely influen ce the biochemical parameters of the body, which results in various physiologi cal changes. Many diseases are associated with nutritional imbalance, according to this a majority of experimental approaches are focused on nutri tional aspects and metabolic regulation. Balance between macronutrients is very important factor affecting fruit fly longevity [1,2]. In drosophila, it was demon strated that protein content in the diet is the main dietary determinant of lifespan, but car bohydrates have little or no effect [3,4]. However, recent studies indicate that carbohydrates also play a central role in determining life expectancy and maintaining health [5]. There are evidences, that dif ferent amount of sucrose in the diet results in distinct changes in the body [2,6]. Moreover, the type of carbohydrate consumed has also important impact on physiological and metabolic processes [7]. Some of these changes are resemble to those complications observed in cardiovascular diseases and diabetes. It is known that high dietary sucrose disrupts the ho abbreviations: LDH, lactate dehydrogenase; MDH, malate dehydrogenase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; AKH, adipokinetic hormone; IS, insulin signaling. understanding the mechanisms underlying the metabolic disease and for developing pharmacologi cal interventions or treatment methods.
Fruit flies have become a promising model for studying the effects of dietary components on various physiological characteristics, including lifespan and metabolism. In studies on drosophila, new important knowledge was gained that the fun damental relationships between lifehistory traits are mediated by macroelements [12]. Using this model organism in the nutrition experiments help us to un derstand the relationship between human nutrition and metabolic disorders.
Here we investigated biochemical parameters and lifespan in Drosophila melanogaster when con suming diets with different concentrations of sucrose ranged from 1 to 20%. We observed, that low carbo hydrate consumption maximized fly lifespan. Ala nine aminotransferase (ALT), aspartate aminotrans ferase (AST), lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) were measured on a series of diets with varying sugar content to ana lyze the course of the main metabolic processes. Our study demonstrated, that the low dietary concentra tion of a particular nutrient in some cases correlates with increased enzymes activities. Interestingly , our study suggested that fly metabolism is driven by a daily caloric intake and our results considered a great impact of macronutrients ratio in aging process.

materials and methods
Insects, maintaining and conditions. We used СantonS (D. melanogaster Meigen) flies which were received from the Bloomington Stock Center (Indiana University, USA). All flies were grown on the medium that contains 4% sucrose, 4% dry yeast, 1.2% agar and 0.18% nipagin as an antifungal agent. Flies were reared at 25°C and relative humidity of 6070% on a 12 h day/night cycle [13]. Flies aged 4 days were separated by sex and kept on the above mentioned medium for one more day to recovery af ter CO 2 anesthesia. Than 5days old flies were placed at standard densities of 200 flies per 1.5 L demo graphic cages. Cages were supplemented with 25 ml plastic vial filled with the 5 ml of experimental medium, containing 4% of dry yeasts and different sucrose concentrations: 1%, 4%, 10%, 20%; 1.2% of agar, 0.18% of nipagin. On the 25th day of the experiment flies were frozen in the liquid nitrogen for subsequent measurements. For measurements preweighted flies were homogenized in 10 mM of icecold sodium phosphate buffer (pH = 7.4) (ratio 1:10) with further centrifugation (16 000 g, 15 min, 4°C). Supernatants were used to measure biochemi cal parameters.
lifespan assay. Flies of both sexes were main tained separately under the conditions, described above. The experimental mediums were changed every second day and dead flies were counted. We conduct two independent lifespan tests, and made survival curves for each group with flies that con sumed different concentration of sucrose; there were approximately 150 flies in each group for each rep licate.
determination of urea. Spectrophotomet ric method was used for urea determination. This method is based on the monitoring of NADH con centration that is used during hydrolysis of urea by urease. Changes in absorbance was recorded on Specol 211 (Jena, Germany) at 340 nm and calcu lation was made using an extinction coefficient of 6220 М 1 ·cm 1 . Urea content was expressed as nano gram per milligram of wet mass (ng/mg wm).
Statistical analysis and graphical representation. Experimental data are presented as mean ± SEM and P < 0.05 is considered as signifi cantly different. Statistical analysis was performed using "Prism" (GraphPad Software, Inc.). Tukey's multiply comparison test has been used to compare activities of LDH, MDH, ALT, AST and urea con centration. Log rank test was used to analyze lifes pan curves. All graph were generated in "Graphpad Prism7".

results and Discussion
Dietary sucrose influence on lifespan of female flies. Longevity is determined by general functional state of the organism as result of its interaction with different environmental factors. Shifts in dietary ma cronutrients ratio lead to changes in functioning of signaling pathways, which, in turn, regulate Dros ophila lifespan [15,16]. Consumption of diet with 1% of sucrose significantly increases lifespan in fly females as compared to those reared on the 4 or 10% (logrank test, P = 0.045; χ 2 = 4.007) ( Fig. 1, B). We observed decreased survival of females fed on 20% sucrose as compared with the 4 and 10% sucrose (log rank, P < 0.001; χ 2 = 12.28) (Fig. 1, B). Our re sults showed, that dietary conditions had no impact on survival of male flies ( Fig. 1, a).
MDH and LDH activities are affected by dietary sucrose. Enzymes which are involved in carbohy drate metabolism including LDH (lactate dehydroge nase) and MDH (malate dehydrogenase) are known to be influenced by nutritional and dietary status. MDH and LDH are two homologous enzymes, which belong to a wide group of 2ketoacid:NAD(P)de pendent dehydrogenases that catalyze the reversible conversion of 2hydroxyacids to the corresponding 2ketoacids and are involved in energy metabolism [17].
LDH activity was dependent on gender (F 3,24 = 3.99, P = 0.193), sucrose concentration in the diet (F 1,24 = 27.73, P < 0.0001), and interaction of both factors (F 3,24 = 7.649, P = 0.0009). The activity of LDH was not affected by dietary sucrose in male flies. The lower activity of LDH was detected in fe males, which consumed medium with 4% and 10% of sucrose as compared to the 1% of sucrose (Fig. 3, a; P < 0.05). Thus, with decreasing sucrose concen tration, LDH activity increases.
alt and aSt activities. Aminotransferases are enzymes that transfer αamino group from the  amino acid to αketoacid. ALT and AST belong to the pyridoxalphosphatedependent aminotrans ferase [17]. In drosophila, changes in ALT and AST activities are mostly associated with cell damage and changes in metabolic processes [18]. However, different concentrations of sucrose in the diet af fect AST activity in male flies. We observed higher AST activity in males, which consumed medium with 1% sucrose compared to groups, which con sumed mediums with 10 and 20% sucrose (Fig 4, B; P < 0.05). AST activity was significantly decreased in the males, which were reared on the medium with 20% sucrose as compared to 4% sucrose (Fig. 5, B; P < 0.004). Consequently, increased AST activity is associated with decreased sucrose concentration in the experimental medium. The activity of ALT was not affected by sucrose concentration in both sexes (Fig. 4, a). Urea content. Urea plays an important role in the metabolism of nitrogencontaining compounds in animals and is the main nitrogencontaining sub stance in the urine of mammals [19]. The content of urea can change due to shifts in proteintocar bohydrate ratio in the diet [20]. Urea concentration in flies significantly depended on sucrose concentra tion in the diet (F 1,24 = 50.44, P < 0.0001), fly gender (F 3,24 = 6.488, P = 0.002) and interaction between sex and diet (F 3,24 = 4.838, P = 0.009). Males that consumed medium with 1% sucrose displayed higher urea content as compared to 10% sucrose (  P < 0.05). Females, which were reared on the me dium with 20% of sucrose had higher urea content compared to those fed on 4% sucrose. Lifespan is critically determined by diet com position mostly by balance between protein and carbohydrate. Numerous studies have shown that changes in dietary concentrations of yeast (a source of protein) and sucrose, as well as their ratio, can have a significant effect on lifespan [2,21]. Under standing the interactions between diet and longevity is critical for protecting against the rising incidence of agerelated metabolic disease.
Our results showed that lower sucrose concen tration in the diet caused lifespan extension in fruit fly. The longevity phenotype in flies, which con sumed low calorie diet is accompanied with higher activities of metabolic enzymes such as LDH, MDH and AST. Hence, we demonstrated, that amount of sucrose in the diet influences metabolism and life span. Low carbohydrate consumption may cause calorie restriction conditions. Reduced food intake without malnutrition has been shown to extend lifes pan in different model organisms [22,23]. Calorie restriction is associated with a decreased glucose level, and as a result insulin signaling (IS) inhibition [24]. Degreased IS affects other signaling pathways involved in lifespan regulation. Under IS inhibition dFOXO (Forkhead box proteins class 'O') remains unphosphorylated and is transported to the nucleus where it activates expression of prolongevity genes [25].
It is known, that organism uses carbohydrates mostly for energy production. AMPK is a key sensor of cellular energy levels and is an upstream regu lator of both the target of rapamycin (TOR) and IS pathways under lownutrient conditions [26]. Acti vated AMPK inhibits energyconsuming processes, such as protein synthesis, through the inhibition of TOR signaling under lowenergy conditions (high AMP:ATP ratio) [27]. Numerous studies demon strated, that IS and TOR signaling inhibition caused lifespan extension in various animal models [28].
Lowering yeast concentrations in the diet has been shown to extend drosophila lifespan [4], sug gesting that protein is the major specific nutrient responsible for modulating lifespan and ageing pro cesses [15,29]. Carbohydrates were not show to have significant effects on lifespan or their impact is too weak [3,15]. However, recent findings have shown that dietary proteintocarbohydrate ratio (P:C) in the food is a key determinant of drosophila lifes pan [2,12,15]. Our study revealed, that low sucrose content in the diet maximized female lifespan. Re striction of glucose and fructose consumption causes mild oxidative stress, which may increase protective potential in drosophila's body [7]. Sucrose is a di saccharide, which consists of glucose and fructose residues. Glucose induces toxicity when circulating in high concentrations, whereas fructose is more prone to induce obesity promoting accumulation of reserve lipids and carbohydrates [7]. In our study, we observed decreased survival of females reared on the medium with high sucrose content that may be asso ciated with hyperglycemia and/or obesity induction.
We also tested how sucrose concentration in the diet influences carbohydrate metabolism. Metabolic changes can be estimated by lactate dehydrogenase (LDH) activity. This activity indicates the predomi nance of anaerobic versus aerobic metabolism in particular tissues [30]. LDH catalyzes the oxidation of lactate to pyruvate when there are high concen trations of lactate and reverse reaction of the reduc tion of pyruvate to lactate occurs in case of oxygen deficiency in the cell. An increase in LDH activity may reflect the hypoxic conditions in the body [31]. MDH activity indicates overall metabolic status. This enzyme plays an important role in the TCA cy cle, and MDH is involved in glucose oxidation. It is known, that decrease in the activity of cytosolic MDH reflects repression of energy metabolism [32]. Our investigation indicates that high sucrose content in diet leads to a decrease in the activity of MDH. Production of oxaloacetic acid in the reaction cata lyzed by MDH may depend on the concentration of energy substrates. So, sufficient carbohydrate sup ply inhibits gluconeogenesis and MDH activity as a result.
Besides carbohydrate metabolism, we suggested , that dietary sucrose may have impact on amino acid metabolism. Aminotransferases are known to play a key role in the intermediary me tabolism of amino acids. ALT and AST take part in maintenance of amino acid pool for protein syn thesis. They supply metabolites for energy metabo lism and provide interactions between protein and carbohydrate metabolism. Elevated AST and ALT activities can be considered as an index of gluco neogenesis [33]. ALT is responsible for reversible transamination between alanine and 2oxoglutarate to generate pyruvate and glutamate, playing a key role in the metabolism of glucose and amino acids. AST catalyzes the interconversion of aspartate and αketoglutarate to oxaloacetate and glutamate. In corporation of αketoglutarate into the TCA cycle is the major step critical for the production of building blocks including nucleotides, lipids and amino acids. It is generally suggested that an increase in the ac tivities of ALT and AST indicates metabolic disorder in various insects [33]. In our investigation we did not observe any changes in ALT activity compared to control in both sexes. We observed a decrease in AST activity in flies when consuming diets with high sucrose content that may be explained by acti vation of gluconeogenesis under well fed state. Our data show that low sucrose content in the diet, on the contrary to high sucrose, increases AST activity with subsequent likely involvement of gluconeogenesis .
Urea derives from nitrogen metabolism, pro tein degradation and amino acids catabolism, and at high concentrations is toxic to organism. The level of urea is markedly affected by both protein content in the diet and protein catabolism rate [19]. Urea is an important scavenger of free radicals in biologi cal systems, including insects [14,34]. Hilliker and colleagues (1992) have found that urate is crucial in antioxidative defense in vivo in D. melanogaster. Lowprotein intake is associated with reduced urea production. Our results show that consumption of the diet with high carbohydrate concentration in crease urea content in females. In addition, low su crose consumption in male flies leads to protein ca tabolism with increased urea production.