Alpha-ketoglutarate supplementation in long-lived Drosophila melanogaster: Impact on lifespan and metabolic responses.

Studies on antiaging remedies in insect models sometimes show discrepancies in results. These discrepancies could be explained by different responses of short- and long-lived strains on the antiaging remedies. The purpose of the study was to test whether life-prolonging effects of alpha-ketoglutarate (AKG), observed in nematodes and fruit flies, would be reproduced in long-lived Drosophila melanogaster flies. Lifespan was assayed in flies kept in demographic cages. Fecundity, proportion of flies capable of negative geotaxis, starvation resistance, time of heat coma onset, levels of triacyglycerols, body glucose, glycogen, activities of glutamate dehydrogenase, catalase, glutathione-S-transferase, hexokinase, phosphofructokinase, pyruvate kinase, lactate, and glutamate dehydrogenases were assessed. Dietary AKG did not affect fly lifespan on the diet with 5% yeast and 5% sucrose (5Y:5S) and on the diet with 9% yeast and 1% sucrose (9Y:1S), but increased lifespan on the low-protein diet (1Y:9S). Twenty-five-day-old female flies fed a 5Y:5S diet with 10 mM AKG for 3 weeks, did not differ from the control group (without AKG) in climbing activity, resistance to heat stress, and starvation. The levels of glucose and glycogen were unaffected but the levels of triacylglycerols were lower in AKG-fed female flies. No differences in activities of glycolytic enzymes, NADPH-producing enzymes, glutamate dehydrogenase, oxygen consumption, and levels of oxidative stress markers were observed between the control and AKG-fed flies. However, AKG-fed flies had lower activities of catalase and glutathione-S-transferase. These results suggest that potential antiaging remedies, such as AKG, may not extend lifespan in long-living organisms despite influencing several metabolic parameters.

Mutations in genes cnc or dKeap1 modulate stress resistance and metabolic processes in Drosophila melanogaster.

The transcription factor Nrf2 and its negative regulator Keap1 play important roles in the maintenance of redox homeostasis in animal cells. Nrf2 activates defenses against oxidative stress and xenobiotics. Homologs of Nrf2 and Keap1 are present in Drosophila melanogaster (CncC and dKeap1, respectively). The aim of this study was to explore effects of CncC deficiency (due to mutation in the cnc gene) or enhanced activity (due to mutation in the dKeap1 gene) on redox status and energy metabolism of young adult flies in relation to behavioral traits and resistance to a number of stressors. Deficiency in either CncC or dKeap1 delayed pupation and increased climbing activity and heat stress resistance in 2-day-old adult flies. Males and females of the ∆keap1 line shared some similarities such as elevated antioxidant defense as well as lower triacylglyceride and higher glucose levels. Males of the ∆keap1 line also had a higher activity of hexokinase, whereas ∆keap1 females showed higher glycogen levels and lower values of respiratory control and ATP production than flies of the control line. Mutation of cnc gene in allele cncEY08884 caused by insertion of P{EPgy2} transposon in cnc promotor did not affect significantly the levels of metabolites and redox parameters, and even activated some components of antioxidant defense. These data suggest that the mutation can be hypomorphic as well as CncC protein can be dispensable for adult fruit flies under physiological conditions. In females, CncC mutation led to lower mitochondrial respiration, higher hexokinase activity and higher fecundity as compared with the control line. Either CncC activation or its deficiency affected stress resistance of flies.

Protective effects of alpha-ketoglutarate against aluminum toxicity in Drosophila melanogaster.

In recent years, Drosophila melanogaster has emerged as a model for studies on aluminum toxicity. The current study aimed to disclose the mechanisms of aluminum toxicity in D. melanogaster at larval and adult stages and examined the potential protective effects of dietary alpha-ketoglutarate (AKG). Flies were reared on food containing 10 mM AlCl3, 10 mM AKG or both additives. Rearing on an AlCl3-containing diet induced behavioral defects, and decreased fecundity and long-term survival of female flies. The addition of dietary AKG did not ameliorate locomotor and taste behavior defects or the higher sensitivity to oxidative stress, but improved heat stress resistance, egg-laying capability and survival of females treated with AlCl3. Metabolic effects of AlCl3 exposure on flies included an imbalance of metal content, decreased glucose levels, increased free iron and storage triacylglyceride (TAG) levels, mitochondria dysfunction, and the development of oxidative stress. Dietary AKG did not prevent AlCl3 effects on glucose and TAG, but improved metal homeostasis, inhibited the increase in free Fe and restored the functional activity of iron-containing enzymes such as aconitase. In addition, AKG decreased the intensity of oxidative stress seen in AlCl3-reared adult flies, probably due to inhibition of iron mobilization. The results show that AKG is not a full antidote against Al toxicity but is able to relieve multiple metabolic effects of high aluminum. Furthermore, the modulating ability of AKG can clearly be helpful in exploring the molecular mechanisms of Al toxicity.

Dietary L-arginine accelerates pupation and promotes high protein levels but induces oxidative stress and reduces fecundity and life span in Drosophila melanogaster.

L-Arginine, a precursor of many amino acids and of nitric oxide, plays multiple important roles in nutrient metabolism and regulation of physiological functions. In this study, the effects of L-arginine-enriched diets on selected physiological responses and metabolic processes were assessed in Drosophila melanogaster. Dietary L-arginine at concentrations 5-20 mM accelerated larval development and increased body mass, and total protein concentrations in third instar larvae, but did not affect these parameters when diets contained 100 mM arginine. Young (2 days old) adult flies of both sexes reared on food supplemented with 20 and 100 mM L-arginine possessed higher total protein concentrations and lower glucose and triacylglycerol concentrations than controls. Additionally, flies fed 20 mM L-arginine had higher proline and uric acid concentrations. L-Arginine concentration in the diet also affected oxidative stress intensity in adult flies. Food with 20 mM L-arginine promoted lower protein thiol concentrations and higher catalase activity in flies of both sexes and higher concentrations of low molecular mass thiols in males. When flies were fed on a diet with 100 mM L-arginine, lower catalase activities and concentrations of protein thiols were found in both sexes as well as lower low molecular mass thiols in females. L-Arginine-fed males demonstrated higher climbing activity, whereas females showed higher cold tolerance and lower fecundity, compared with controls. Food containing 20 mM L-arginine shortened life span in both males and females. The results suggest that dietary L-arginine shows certain beneficial effects at the larval stage and in young adults. However, the long-term consumption of L-arginine-enriched food had unfavorable effects on D. melanogaster due to decreasing fecundity and life span.

Dietary alpha-ketoglutarate promotes higher protein and lower triacylglyceride levels and induces oxidative stress in larvae and young adults but not in middle-aged Drosophila melanogaster.

Alpha-ketoglutarate (AKG) is involved in multiple metabolic and regulatory pathways. In this work, the effects of AKG-supplemented diets on selected physiological responses and metabolic processes, including metabolism of reactive oxygen species, was assessed in larvae and adult (both 2 and 24days old) Drosophila melanogaster. Dietary supplementation with AKG resulted in dose-dependent effects on larval development, body composition and antioxidant status of third instar larvae. Larvae and young (2days post-eclosion) adult females fed on AKG shared similar metabolic changes such as higher total protein levels, lower triacylglyceride levels and higher values for oxidative stress indices, namely lipid peroxides and low molecular mass thiols. The latter indicated the development of oxidative stress which, in turn, may induce adaptive responses that can explain the higher resistance of AKG-fed young females to heat shock and hydrogen peroxide exposure. In contrast to young flies, middle-aged females (24days) on AKG-containing diet possessed higher total protein, glucose and triacylglyceride levels, whereas oxidative stress parameters were virtually the same as compared with control females of the same age. In parallel, females fed an AKG-supplemented diet showed lower fecundity, higher heat shock resistance but no change in oxidative stress resistance at middle age which in combination with levels of protein, glucose, and triacylglycerides can be considered as potentially beneficial AKG effects for aging organisms. To our best knowledge, this is the first study on age-matched AKG influence on animals' organism which shows that Drosophila may be used as a model for previous quick study in cost-efficient manner age-related AKG effects in mammals and humans.

Alpha-ketoglutarate reduces ethanol toxicity in Drosophila melanogaster by enhancing alcohol dehydrogenase activity and antioxidant capacity.

Ethanol at low concentrations (<4%) can serve as a food source for fruit fly Drosophila melanogaster, whereas at higher concentrations it may be toxic. In this work, protective effects of dietary alpha-ketoglutarate (AKG) against ethanol toxicity were studied. Food supplementation with 10-mM AKG alleviated toxic effects of 8% ethanol added to food, and improved fly development. Two-day-old adult flies, reared on diet containing both AKG and ethanol, possessed higher alcohol dehydrogenase (ADH) activity as compared with those reared on control diet or diet with ethanol only. Native gel electrophoresis data suggested that this combination diet might promote post-translational modifications of ADH protein with the formation of a highly active ADH form. The ethanol-containing diet led to significantly higher levels of triacylglycerides stored in adult flies, and this parameter was not altered by AKG supplement. The influence of diet on antioxidant defenses was also assessed. In ethanol-fed flies, catalase activity was higher in males and the levels of low molecular mass thiols were unchanged in both sexes compared to control values. Feeding on a mixture of AKG and ethanol did not affect catalase activity but caused a higher level of low molecular mass thiols compared to ethanol-fed flies. It can be concluded that both a stimulation of some components of antioxidant defense and the increase in ADH activity may be responsible for the protective effects of AKG diet supplementation in combination with ethanol. The results suggest that AKG might be useful as a treatment option to neutralize toxic effects of excessive ethanol intake and to improve the physiological state of D. melanogaster and other animals, potentially including humans.

Dietary alpha-ketoglutarate increases cold tolerance in Drosophila melanogaster and enhances protein pool and antioxidant defense in sex-specific manner.

Alpha-ketoglutarate (AKG) is an important intermediate in Krebs cycle which bridges the metabolism of amino acids and carbohydrates. Its effects as a dietary supplement on cold tolerance were studied in Drosophila melanogaster Canton S. Two-day-old adult flies fed at larval and adult stages with AKG at moderate concentrations (5-10mM) recovered faster from chill coma (0°C for 15min or 3h) than control ones. The beneficial effect of AKG on chill coma recovery was not found at its higher concentrations, which suggests hormetic like action of this keto acid. Time of 50% observed mortality after 2h recovery from continuous cold exposure (-1°C for 3-31h) (LTi50) was higher for flies reared on 10mM AKG compared with control ones, showing that the diet with AKG enhanced insect cold tolerance. In parallel with enhancement of cold tolerance, dietary AKG improved fly locomotor activity. Metabolic effects of AKG differed partly in males and females. In males fed on AKG, there were no differences in total protein and free amino acid levels, but the total antioxidant capacity, catalase activity and low molecular mass thiol content were higher than in control animals. In females, dietary AKG promoted higher total antioxidant capacity and higher levels of proteins, total amino acids, proline and low molecular mass thiols. The levels of lipid peroxides were lower in both fly sexes reared on AKG as compared with control ones. We conclude that both enhancement of antioxidant system capacity and synthesis of amino acids can be important for AKG-promoted cold tolerance in D. melanogaster. The involvement of AKG in metabolic pathways of Drosophila males and females is discussed.

Alpha-ketoglutarate attenuates toxic effects of sodium nitroprusside and hydrogen peroxide in Drosophila melanogaster.

The protective effects of dietary alpha-ketoglutarate (AKG) are described that aid fruit flies, Drosophila melanogaster, to resist sodium nitroprusside (SNP) and hydrogen peroxide toxicity. Food supplementation with 10mM AKG alleviated toxic effects of 1mM SNP added to food and improved fly development. Dietary AKG also prevented the increase in levels of oxidative stress markers seen in SNP-reared adult flies. In vitro AKG did not affect the rate of SNP decomposition and did not bind iron and nitrite ions released in this process. Alpha-ketoglutarate also displayed high H2O2-scavenging activity in vitro and efficiently protected adult flies against this compound in combined treatments. Based on the observed antioxidant activity of AKG, it may be suggested that the antioxidant mode of AKG action (apart from its cyanide-binding capability) may be used to prevent the toxic effects of SNP and improve general physiological state of D. melanogaster and other animals and humans.