Structure of O-Polysaccharide and Lipid A of Pantoea Agglomerans 8488.

The Pantoea agglomerans 8488 lipopolysaccharide (LPS) was isolated, purified and characterized by monosaccharide and fatty acid analysis. The O-polysaccharide and lipid A components of the LPS were separated by mild acid degradation. Lipid A was studied by electrospray ionization mass spectrometry (ESI-MS) and found to consist of hexa-, penta-, tetra- and tri-acylated species. Two-dimensional nuclear magnetic resonance (NMR) spectroscopy revealed the following structure of the O-polysaccharide repeating unit →3)-α-L-Rhap-(1→6)-α-D-Manp-(1→3)-α-L-Fucp-(1→3)-ß-D-GlcNAcp-(1→. The LPS showed a low level of toxicity, was not pyrogenic, and reduced the adhesiveness index of microorganisms to 2.12, which was twofold less than the control. LPS modified by complex compounds of germanium (IV) and tin (IV) were obtained. It was found that six LPS samples modified by Sn compounds and two LPS samples modified by Ge compounds lost their toxic activity when administered to mice in a dose of LD50 (105 µg/mice or 5 mg/kg). However, none of the modified LPS samples changed their serological activity in an Ouchterlony double immunodiffusion test in agar.

TOR signaling inhibition in intestinal stem and progenitor cells affects physiology and metabolism in Drosophila.

In all eukaryotic organisms, the control of growth, metabolism, reproduction, and lifespan is realized by interactions of genetic and environmental signals. An important player in the regulatory network is the target of rapamycin (TOR) signaling pathway, which is triggered by nutritional cues. Given the pivotal role of TOR in regulating multiple processes in organisms, we inhibited TOR by inducible expression of specific RNAi in Drosophila intestinal stem and progenitor cells or progenitor cells alone. We found that TOR inhibition in stem and progenitor cells shortened the lifespan on both regular diet and under malnutrition. Moreover, flies became more short-lived under starvation or oxidative stress conditions if TOR was inhibited. TOR-RNAi expression resulted in a decrease in body glycogen and TAG levels. All these physiological and metabolic changes might be partially explained by significant changes in mRNA levels for genes encoding the Drosophila insulin-like peptides (dilp2, dilp3 and dilp5) with subsequent effects on insulin signaling to modulate gene expression in peripheral tissues (e.g. tobi and pepck transcripts). In the gut, a strong increase in transcript levels of cytokines upd2, upd3 and downstream target socs36e of the JAK/STAT signaling pathway in the gut indicate an important role for this signaling pathway when TOR is inhibited.

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.

Exposure to sodium molybdate results in mild oxidative stress in Drosophila melanogaster.

OBJECTIVES: The study was conducted to assess the redox status of Drosophila flies upon oral intake of insulin-mimetic salt, sodium molybdate (Na2MoO4). METHODS: Oxidative stress parameters and activities of antioxidant and associated enzymes were analyzed in two-day-old D. melanogaster insects after exposure of larvae and newly eclosed adults to three molybdate levels (0.025, 0.5, or 10 mM) in the food. RESULTS: Molybdate increased content of low molecular mass thiols and activities of catalase, superoxide dismutase, glutathione-S-transferase, and glucose-6-phosphate dehydrogenase in males. The activities of these enzymes were not affected in females. Males exposed to molybdate demonstrated lower carbonyl protein levels than the control cohort, whereas females at the same conditions had higher carbonyl protein content and catalase activity than ones in the control cohort. The exposure to 10 mM sodium molybdate decreased the content of protein thiols in adult flies of both sexes. Sodium molybdate did not affect the activities of NADP-dependent malate dehydrogenase and thioredoxin reductase in males or NADP-dependent isocitrate dehydrogenase in either sex at any concentration. DISCUSSION: Enhanced antioxidant capacity in upon Drosophila flies low molybdate levels in the food suggests that molybdate can be potentially useful for the treatment of certain pathologies associated with oxidative stress.

Activation of the Tor/Myc signaling axis in intestinal stem and progenitor cells affects longevity, stress resistance and metabolism in drosophila.

The TOR (target of rapamycin) signaling pathway and the transcriptional factor Myc play important roles in growth control. Myc acts, in part, as a downstream target of TOR to regulate the activity and functioning of stem cells. Here we explore the role of TOR-Myc axis in stem and progenitor cells in the regulation of lifespan, stress resistance and metabolism in Drosophila. We found that both overexpression of rheb and myc-rheb in midgut stem and progenitor cells decreased the lifespan and starvation resistance of flies. TOR activation caused higher survival under malnutrition conditions. Furthermore, we demonstrate gut-specific activation of JAK/STAT and insulin signaling pathways to control gut integrity. Both genetic manipulations had an impact on carbohydrate metabolism and transcriptional levels of metabolic genes. Our findings indicate that activation of the TOR-Myc axis in midgut stem and progenitor cells influences a variety of traits in Drosophila.

Systemic corazonin signalling modulates stress responses and metabolism in Drosophila.

Stress triggers cellular and systemic reactions in organisms to restore homeostasis. For instance, metabolic stress, experienced during starvation, elicits a hormonal response that reallocates resources to enable food search and readjustment of physiology. Mammalian gonadotropin-releasing hormone (GnRH) and its insect orthologue, adipokinetic hormone (AKH), are known for their roles in modulating stress-related behaviour. Here we show that corazonin (Crz), a peptide homologous to AKH/GnRH, also alters stress physiology in Drosophila The Crz receptor (CrzR) is expressed in salivary glands and adipocytes of the liver-like fat body, and CrzR knockdown targeted simultaneously to both these tissues increases the fly's resistance to starvation, desiccation and oxidative stress, reduces feeding, alters expression of transcripts of Drosophila insulin-like peptides (DILPs), and affects gene expression in the fat body. Furthermore, in starved flies, CrzR-knockdown increases circulating and stored carbohydrates. Thus, our findings indicate that elevated systemic Crz signalling during stress coordinates increased food intake and diminished energy stores to regain metabolic homeostasis. Our study suggests that an ancient stress-peptide in Urbilateria evolved to give rise to present-day GnRH, AKH and Crz signalling systems.

Anti-aging pharmacology: Promises and pitfalls.

Life expectancy has grown dramatically in modern times. This increase, however, is not accompanied by the same increase in healthspan. Efforts to extend healthspan through pharmacological agents targeting aging-related pathological changes are now in the spotlight of geroscience, the main idea of which is that delaying of aging is far more effective than preventing the particular chronic disorders. Currently, anti-aging pharmacology is a rapidly developing discipline. It is a preventive field of health care, as opposed to conventional medicine which focuses on treating symptoms rather than root causes of illness. A number of pharmacological agents targeting basic aging pathways (i.e., calorie restriction mimetics, autophagy inducers, senolytics etc.) are now under investigation. This review summarizes the literature related to advances, perspectives and challenges in the field of anti-aging pharmacology.

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.

Food odors trigger an endocrine response that affects food ingestion and metabolism.

Food odors stimulate appetite and innate food-seeking behavior in hungry animals. The smell of food also induces salivation and release of gastric acid and insulin. Conversely, sustained odor exposure may induce satiation. We demonstrate novel effects of food odors on food ingestion, metabolism and endocrine signaling in Drosophila melanogaster. Acute exposure to attractive vinegar odor triggers a rapid and transient increase in circulating glucose, and a rapid upregulation of genes encoding the glucagon-like hormone adipokinetic hormone (AKH), four insulin-like peptides (DILPs) and some target genes in peripheral tissues. Sustained exposure to food odors, however, decreases food intake. Hunger-induced strengthening of synaptic signaling from olfactory sensory neurons (OSNs) to brain neurons increases food-seeking behavior, and conversely fed flies display reduced food odor sensitivity and feeding. We show that increasing the strength of OSN signaling chronically by genetic manipulation of local peptide neuromodulation reduces feeding, elevates carbohydrates and diminishes lipids. Furthermore, constitutively strengthened odor sensitivity altered gene transcripts for AKH, DILPs and some of their targets. Thus, we show that food odor can induce a transient anticipatory endocrine response, and that boosted sensitivity to this odor affects food intake, as well as metabolism and hormonal signaling.

High consumption of fructose rather than glucose promotes a diet-induced obese phenotype in Drosophila melanogaster.

During the last 20 years, there has been a considerable scientific debate about the possible mechanisms of induction of metabolic disorders by reducing monosaccharides such as glucose or fructose. In this study, we report the metabolic rearrangement in response to consumption of these monosaccharides at concentrations ranging from 0.25% to 20% in a Drosophila model. Flies raised on high-glucose diet displayed delay in pupation and increased developmental mortality compared with fructose consumers. Both monosaccharides at high concentrations promoted an obese-like phenotype indicated by increased fly body mass, levels of uric acid, and circulating and stored carbohydrates and lipids; and decreased percentage of water in the body. However, flies raised on fructose showed lower levels of circulating glucose and higher concentrations of stored carbohydrates, lipids, and uric acid. The preferential induction of obesity caused by fructose in Drosophila was associated with increased food consumption and reduced mRNA levels of DILP2 and DILP5 in the brain of adult flies. Our data show that glucose and fructose differently affect carbohydrate and lipid metabolism in Drosophila in part by modulation of insulin/insulin-like growth factor signaling. Some reported similarities with effects observed in mammals make Drosophila as a useful model to study carbohydrate influence on metabolism and development of metabolic disorders.

Sodium chromate demonstrates some insulin-mimetic properties in the fruit fly Drosophila melanogaster.

The effects of food supplementation with sodium chromate at concentrations of 1-500 µM on development of Drosophila melanogaster larvae and food intake, carbohydrate and lipid pools in adult fruit flies were investigated. Food supplementation with hexavalent chromium (Na2CrO4) at high concentrations delayed larval development and decreased the percentage of larvae that pupated which indicated a relatively low toxicity. The supplement decreased glucose levels in fly hemolymph, but at concentrations of 5-25 µM increased fly carbohydrate reserves: hemolymph trehalose and whole body trehalose and glycogen. The data on parameters of carbohydrate metabolism show that chromate possesses some insulin-mimetic properties. The changes in metabolism of carbohydrates under chromate exposure were also accompanied by an increase in total lipid levels and in the portion of triacylglycerides among all lipids. Chromate addition to fly food did not affect male or female body mass, but reduced food consumption by females at all concentrations used, whereas in males only 500 µM chromate decreased food consumption. The data show that: (1) Cr(6+) has many of the same effects as Cr(3+) suggesting that it might be just as effective to treat diabetic states, likely as a result of intracellular reduction of Cr(6+) ions, and (2) the Drosophila model can be used to develop new approaches to investigate the molecular mechanisms of chromium as an insulin-mimetic. Although it is usually believed that hexavalent chromium possesses higher toxicity than the trivalent ion, due to its easier penetration into the cell, application of hexavalent chromium may substantially decrease the chromium doses needed to get the desired effects.

Ciona intestinalis NADH dehydrogenase NDX confers stress-resistance and extended lifespan on Drosophila.

An assembled cDNA coding for the putative single-subunit NADH dehydrogenase (NDX) of Ciona intestinalis was introduced into Drosophila melanogaster. The encoded protein was found to localize to mitochondria and to confer rotenone-insensitive substrate oxidation in organello. Transgenic flies exhibited increased resistance to menadione, starvation and temperature stress, and manifested a sex and diet-dependent increase in mean lifespan of 20-50%. However, NDX was able only weakly to complement the phenotypes produced by the knockdown of complex I subunits.

Molybdate partly mimics insulin-promoted metabolic effects in Drosophila melanogaster.

Molybdenum-containing salts have been found to attenuate diabetes complications in mammals by affecting processes normally regulated by insulin and thus were believed to mimic insulin activity. In this study, we used a fruit fly model to test sodium molybdate, Na2MoO4, action in relation to insulin-promoted processes and toxicity. We studied how larval food supplementation with sodium molybdate affected levels of body carbohydrates and lipids in two-day old adult Drosophila melanogaster. Molybdate salt, in the concentrations used (0.025, 0.05, 0.5, 5, and 10mM), showed low toxicity to fly larvae and slightly influenced development and the percentage of pupated animals. Additionally, sodium molybdate decreased the level of hemolymph glucose in males by 30%, and increased the level of hemolymph trehalose in flies of both sexes. These changes were accompanied by an increase in whole body trehalose and glycogen of about 30-90%. Although total lipid levels in flies of both sexes were depleted by 25%, an increased amount of triacylglycerides among total lipids was observed. These effects were not related to changes in food intake. Taken together, the present data let us suggest that sodium molybdate may at least partly mimic insulin-related effects in Drosophila.

Drosophila insulin-producing cells are differentially modulated by serotonin and octopamine receptors and affect social behavior.

A set of 14 insulin-producing cells (IPCs) in the Drosophila brain produces three insulin-like peptides (DILP2, 3 and 5). Activity in IPCs and release of DILPs is nutrient dependent and controlled by multiple factors such as fat body-derived proteins, neurotransmitters, and neuropeptides. Two monoamine receptors, the octopamine receptor OAMB and the serotonin receptor 5-HT1A, are expressed by the IPCs. These receptors may act antagonistically on adenylate cyclase. Here we investigate the action of the two receptors on activity in and output from the IPCs. Knockdown of OAMB by targeted RNAi led to elevated Dilp3 transcript levels in the brain, whereas 5-HT1A knockdown resulted in increases of Dilp2 and 5. OAMB-RNAi in IPCs leads to extended survival of starved flies and increased food intake, whereas 5-HT1A-RNAi produces the opposite phenotypes. However, knockdown of either OAMB or 5-HT1A in IPCs both lead to increased resistance to oxidative stress. In assays of carbohydrate levels we found that 5-HT1A knockdown in IPCs resulted in elevated hemolymph glucose, body glycogen and body trehalose levels, while no effects were seen after OAMB knockdown. We also found that manipulations of the two receptors in IPCs affected male aggressive behavior in different ways and 5-HT1A-RNAi reduced courtship latency. Our observations suggest that activation of 5-HT1A and OAMB signaling in IPCs generates differential effects on Dilp transcription, fly physiology, metabolism and social interactions. However the findings do not support an antagonistic action of the two monoamines and their receptors in this particular system.

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.

Aconitase post-translational modification as a key in linkage between Krebs cycle, iron homeostasis, redox signaling, and metabolism of reactive oxygen species.

Aconitase, an enzyme possessing an iron-sulfur cluster that is sensitive to oxidation, is involved in the regulation of cellular metabolism. There are two isoenzymes of aconitase (Aco)--mitochondrial (mAco) and cytosolic (cAco) ones. The primary role of mAdco is believed to be to control cellular ATP production via regulation of intermediate flux in the Krebs cycle. The cytosolic Aco in its reduced form operates as an enzyme, whereas in the oxidized form it is involved in the control of iron homeostasis as iron regulatory protein 1 (IRP1). Reactive oxygen species (ROS) play a central role in regulation of Aco functions. Catalytic Aco activity is regulated by reversible oxidation of [4Fe-4S]²âº cluster and cysteine residues, so redox-dependent posttranslational modifications (PTMs) have gained increasing consideration as regards possible regulatory effects. These include modifications of cysteine residues by oxidation, nitrosylation and thiolation, as well as Tyr nitration and oxidation of Lys residues to carbonyls. Redox-independent PTMs such as phosphorylation and transamination also have been described. In the presence of a sustained ROS flux, redox-dependent PTMs may lead to enzyme damage and cell stress by impaired energy and iron metabolism. Aconitase has been identified as a protein that undergoes oxidative modification and inactivation in aging and certain oxidative stress-related disorders. Here we describe possible mechanisms of involvement of the two aconitase isoforms, cAco and mAco, in the control of cell metabolism and iron homeostasis, balancing the regulatory, and damaging effects of ROS.

2,4-dinitrophenol partially alleviates ferrocyanide-induced toxicity in Drosophila melanogaster.

The toxicity of potassium ferrocyanide (PFC) and protective effects of 2,4-dinitrophenol (DNP) under PFC treatment were tested on the Drosophila melanogaster model system. Fly larvae were raised on food supplemented with PFC at concentrations of 1.0 mM and mixtures with DNP in concentrations of 0.50 and 1.25 mM, either alone or in combination with 1.0 mM PFC. Food supplementation with PFC decreased larvae viability or pupation height, whereas when larvae were fed by PFC and DNP combination the decrease was less pronounced. Larval exposure to PFC and mixtures of DNP and PFC lowered activities of aconitase. Larval treatment with PFC resulted in higher carbonyl protein, uric acid, and low molecular mass thiols content and higher activity of thioredoxin reductase in adult flies, while DNP in mixtures with PFC relieved these effects. Furthermore, treatment with PFC/DNP mixtures resulted in higher activities of superoxide dismutase and glutathione-S-transferase. It is proposed that PFC toxicity is mainly related to the cyanide and iron ions, released during its decomposition. The potential mechanisms of protective DNP effects against PFC toxicity are discussed.