Development of fly tolerance to consuming a high-protein diet requires physiological, metabolic and transcriptional changes.

Mortality in insects consuming high-protein-and-low-carbohydrate diets resembles a type III lifespan curve with increased mortality at an early age and few survivors that live a relatively long lifespan. We selected for a Drosophila line able to live for a long time on an imbalanced high-protein-low-carbohydrate diet by carrying out five rounds of breeding to select for the most long-lived survivors. Adaptation to this diet in the selected line was studied at the biochemical, physiological and transcriptomic levels. The selected line of flies consumed less of the imbalanced food but also accumulated more storage metabolites: glycogen, triacylglycerides, and trehalose. Selected flies also had a higher activity of alanine transaminase and a higher urea content. Adaptation of the selected line on the transcriptomic level was characterized by down-regulation of genes encoding serine endopeptidases (Jon25i, Jon25ii, betaTry, and others) but up-regulation of genes encoding proteins related to the immune system, such as antimicrobial peptides, Turandot-family humoral factors, hexamerin isoforms, and vitellogenin. These sets of down- and up-regulated genes were similar to those observed in fruit flies with suppressed juvenile hormone signaling. Our data show that the physiological adaptation of fruit flies to a high-protein-low-carbohydrate diet occurs via intuitive pathways, namely a decrease in food consumption, conversion of amino acids into ketoacids to compensate for the lack of carbohydrate, and accumulation of storage metabolites to eliminate the negative effects of excess amino acids. Nevertheless, transcriptomic adaptation occurs in a counter-intuitive way likely via an influence of gut microbiota on food digestion.

Insulin Signaling in Intestinal Stem and Progenitor Cells as an Important Determinant of Physiological and Metabolic Traits in Drosophila.

The insulin-IGF-1 signaling (IIS) pathway is conserved throughout multicellular organisms and regulates many traits, including aging, reproduction, feeding, metabolism, stress resistance, and growth. Here, we present evidence of a survival-sustaining role for IIS in a subset of gut cells in Drosophila melanogaster, namely the intestinal stem cells (ISCs) and progenitor cells. Using RNAi to knockdown the insulin receptor, we found that inhibition of IIS in ISCs statistically shortened the lifespan of experimental flies compared with non-knockdown controls, and also shortened their survival under starvation or malnutrition conditions. These flies also showed decreased reproduction and feeding, and had lower amounts of glycogen and glucose in the body. In addition, increased expression was observed for the Drosophila transcripts for the insulin-like peptides dilp2, dilp5, and dilp6. This may reflect increased insulin signaling in peripheral tissues supported by up-regulation of the target of the brain insulin gene (tobi). In contrast, activation of IIS (via knockdown of the insulin pathway inhibitor PTEN) in intestinal stem and progenitor cells decreased fly resistance to malnutrition, potentially by affecting adipokinetic hormone signaling. Finally, Pten knockdown to enhance IIS also activated JAK-STAT signaling in gut tissue by up-regulation of upd2, upd3, and soc36 genes, as well as genes encoding the EGF receptor ligands spitz and vein. These results clearly demonstrate that manipulating insulin levels may be used to modulate various fly traits, which are important determinants of organismal survival.

Correction to: Alternative NADH dehydrogenase extends lifespan and increases resistance to xenobiotics in Drosophila.

The article Alternative NADH dehydrogenase extends lifespan and increases resistance to xenobiotics in Drosophila, written by Dmytro V. Gospodaryov. Olha M. Strilbytska. Uliana V. Semaniuk. Natalia V. Perkhulyn. Bohdana M. Rovenko. Ihor S. Yurkevych. Ana G. Barata. Tobias P. Dick. Oleh V. Lushchak and Howard T. Jacobs, was originally published electronically on the publisher's internet portal on 20 November 2019 without open access. With the author(s)' decision to opt for Open Choice the copyright of the article changed on 27 January 2020 to © The Author(s) 2020 and the article is forthwith distributed under a Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The original article has been corrected.

Alternative NADH dehydrogenase extends lifespan and increases resistance to xenobiotics in Drosophila.

Mitochondrial alternative NADH dehydrogenase (aNDH) was found to extend lifespan when expressed in the fruit fly. We have found that fruit flies expressing aNDH from Ciona intestinalis (NDX) had 17-71% lifespan prolongation on media with different protein-tocarbohydrate ratios except NDX-expressing males that had 19% shorter lifespan than controls on a high protein diet. NDX-expressing flies were more resistant to organic xenobiotics, 2,4-dichlorophenoxyacetic acid and alloxan, and inorganic toxicant potassium iodate, and partially to sodium molybdate treatments. On the other hand, NDX-expressing flies were more sensitive to catechol and sodium chromate. Enzymatic analysis showed that NDX-expressing males had higher glucose 6-phosphate dehydrogenase activity, whilst both sexes showed increased glutathione S-transferase activity.

Parental dietary protein-to-carbohydrate ratio affects offspring lifespan and metabolism in drosophila.

Non-genetic inheritance of metabolic state over multiple generations has been widely reported in insects. The present study uses the fruit fly, Drosophila melanogaster, to assess whether lifespan, physiological traits and metabolism are affected by the dietary protein-to-carbohydrate ratio (P:C) of the prior adult generation. Groups of parental flies were fed diets with different P:C ratios. Their progeny groups were raised on the same diet so the only variable in the experiments was the diet fed to the parents. Parental P:C affected the lifespan of female offspring, however had no impact on F1 males survival. Low parental P:C increased feeding rate in progeny. An increase in the P:C ratio from 0.03 to 0.65 decreased the levels of body glucose and trehalose in the offspring and a similar tendency was observed in the levels of circulating hemolymph glucose and trehalose. Offspring also accumulated less triglycerides but more glycogen when parents were fed a low P:C diet. Our study indicates that the parental dietary P:C ration has a strong impact on the lifespan, reproduction, appetite and metabolism in the offspring generation.

Insulin-Like Peptides Regulate Feeding Preference and Metabolism in Drosophila.

Fruit flies have eight identified Drosophila insulin-like peptides (DILPs) that are involved in the regulation of carbohydrate concentrations in hemolymph as well as in accumulation of storage metabolites. In the present study, we investigated diet-dependent roles of DILPs encoded by the genes dilp1-5, and dilp7 in the regulation of insect appetite, food choice, accumulation of triglycerides, glycogen, glucose, and trehalose in fruit fly bodies and carbohydrates in hemolymph. We have found that the wild type and the mutant lines demonstrate compensatory feeding for carbohydrates. However, mutants on dilp2,3, dilp3, dilp5, and dilp7 showed higher consumption of proteins on high yeast diets. To evaluate metabolic differences between studied lines on different diets we applied response surface methodology. High nutrient diets led to a moderate increase in concentration of glucose in hemolymph of the wild type flies. Mutations on dilp genes changed this pattern. We have revealed that the dilp2 mutation led to a drop in glycogen levels independently on diet, lack of dilp3 led to dramatic increase in circulating trehalose and glycogen levels, especially at low protein consumption. Lack of dilp5 led to decreased levels of glycogen and triglycerides on all diets, whereas knockout on dilp7 caused increase in glycogen levels and simultaneous decrease in triglyceride levels at low protein consumption. Fruit fly appetite was influenced by dilp3 and dilp7 genes. Our data contribute to the understanding of Drosophila as a model for further studies of metabolic diseases and may serve as a guide for uncovering the evolution of metabolic regulatory pathways.

OXIDIZED LIPIDS DID NOT REDUCE LIFESPAN IN THE FRUIT FLY, Drosophila melanogaster.

Aging is often associated with accumulation of oxidative damage in proteins and lipids. However, some studies do not support this view, raising the question of whether high levels of oxidative damage are associated with lifespan. In the current investigation, Drosophila melanogaster flies were kept on diets with 2 or 10% of either glucose or fructose. The lifespan, fecundity, and feeding as well as amounts of protein carbonyls (PC) and lipid peroxides (LOOH), activities of superoxide dismutase (SOD), catalase, glutathione-S-transferase (GST), and glutathione reductase activity of thioredoxin reductase (TrxR) were measured in "young" (10-day old) and "aged" (50-day old) flies. Flies maintained on diets with 10% carbohydrate lived longer than those on the 2% diets. However, neither lifespan nor fecundity was affected by the type of carbohydrate. The amount of PC was unaffected by diet and age, whereas flies fed on diets with 10% carbohydrate had about fivefold higher amounts of LOOH compared to flies maintained on the 2% carbohydrate diets. Catalase activity was significantly lower in flies fed on diets with 10% carbohydrates compared to flies on 2% carbohydrate diets. The activities of SOD, GST, and TrxR were not affected by the diet or age of the flies. The higher levels of LOOH in flies maintained on 10% carbohydrate did not reduce their lifespan, from which we infer that oxidative damage to only one class of biomolecules, particularly lipids, is not sufficient to influence lifespan.

High sucrose consumption promotes obesity whereas its low consumption induces oxidative stress in Drosophila melanogaster.

The effects of sucrose in varied concentrations (0.25-20%) with constant amount of yeasts in larval diet on development and metabolic parameters of adult fruit fly Drosophila melanogaster were studied. Larvae consumed more food at low sucrose diet, overeating with yeast. On high sucrose diet, larvae ingested more carbohydrates, despite consuming less food and obtaining less protein derived from yeast. High sucrose diet slowed down pupation and increased pupa mortality, enhanced levels of lipids and glycogen, increased dry body mass, decreased water content, i.e. resulted in obese phenotype. Furthermore, it suppressed reactive oxygen species-induced oxidation of lipids and proteins as well as the activity of superoxide dismutase. The activity of catalase was gender-related. In males, at all sucrose concentrations used catalase activity was higher than at its concentration of 0.25%, whereas in females sucrose concentration virtually did not influence the activity. High sucrose diet increased content of protein thiols and the activity of glucose-6-phosphate dehydrogenase. The increase in sucrose concentration also enhanced uric acid level in females, but caused opposite effects in males. Development on high sucrose diets was accompanied by elevated steady-state insulin-like peptide 3 mRNA level. Finally, carbohydrate starvation at yeast overfeeding on low sucrose diets resulted in oxidative stress reflected by higher levels of oxidized lipids and proteins accompanied by increased superoxide dismutase activity. Potential mechanisms involved in regulation of redox processes by carbohydrates are discussed.

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

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.

Specific dietary carbohydrates differentially influence the life span and fecundity of Drosophila melanogaster.

The fruit fly, Drosophila melanogaster is a broadly used model for gerontological research. Many studies are dedicated to understanding nutritional effects on ageing; however, the influence of dietary carbohydrate type and dosage is still poorly understood. We show that among three carbohydrates tested, fructose, glucose, and sucrose, the latter decreased life span by 13%-27%, being present in concentrations of 2%-20% in the diet. Life-span shortening by sucrose was accompanied by an increase in age-independent mortality. Sucrose also dramatically decreased the fecundity of the flies. The differences in life span and fecundity were determined to be unrelated to differential carbohydrate ingestion. The highest mitochondrial protein density was observed in flies fed sucrose-containing diet. However, this parameter was not affected by carbohydrate amount in the diet. Fly sensitivity to oxidative stress, induced by menadione, was increased in aged flies and was slightly affected by type and concentration of carbohydrate. In general, it has been demonstrated that sucrose, commonly used in recipes of Drosophila laboratory food, may shorten life span and lower egg-laying capability on the diets with very low protein content.

Lifespan extension and delay of age-related functional decline caused by Rhodiola rosea depends on dietary macronutrient balance.

BACKGROUND: This study was conducted to evaluate the effects of rhizome powder from the herb Rhodiola rosea, a traditional Western Ukraine medicinal adaptogen, on lifespan and age-related physiological functions of the fruit fly Drosophila melanogaster. RESULTS: Flies fed food supplemented with 5.0 mg/ml and 10.0 mg/ml of R. rosea rhizome powder had a 14% to 17% higher median lifespan, whereas at 30.0 mg/ml lifespan was decreased by 9% to 12%. The preparation did not decrease fly fecundity.The effect of R. rosea supplement on lifespan was dependent on diet composition. Lifespan extension by 15% to 21% was observed only for diets with protein-to-carbohydrate ratios less than 1. Lifespan extension was also dependent on total concentration of macronutrients. Thus, for the diet with 15% yeast and 15% sucrose there was no lifespan extension, while for the diet with protein-to-carbohydrate ratio 20:1 R. rosea decreased lifespan by about 10%.Flies fed Rhodiola preparation were physically more active, less sensitive to the redox-cycling compound menadione and had a longer time of heat coma onset compared with controls. Positive effects of Rhodiola rhizome on stress resistance and locomotor activity were highest at the 'middle age'. CONCLUSIONS: The present data show that long-term food supplementation with R. rosea rhizome not only increases D. melanogaster lifespan, but also delays age-related decline of physical activity and increases stress resistance, what depends on protein-to-carbohydrate ratio of the diet.

Balance between macronutrients affects life span and functional senescence in fruit fly Drosophila melanogaster.

It has recently been demonstrated that as the ratio of protein to carbohydrate (P:C) in the diet declines, life span increases in Drosophila. Here we explored how extremely low dietary ratios of protein to carbohydrate affected longevity and a selection of variables associated with functional senescence. An increase in P:C ratio from 1:57 to 1:20 shortened life span by increasing age-dependent mortality; whereas a further decline in P:C from 1:57 to 1:95 caused a modest decrease in life span. Female flies consuming the 1:20 and 1:38 diets laid more eggs than those consuming the lower P:C diets. Flies fed diets with higher ratios were more resistant to heat stress. Flies consuming the diets with lowest P:C ratios needed more time to restore activity after paralysis. Our study has therefore extended to very low P:C ratios available data demonstrating that dietary P:C ratio affects life span, fecundity and heat stress resistance, with fecundity and heat stress responses showing the opposite trend to life span.