Transiently restricting individual amino acids protects Drosophila melanogaster against multiple stressors.

Nutrition and resilience are linked, though it is not yet clear how diet confers stress resistance or the breadth of stressors that it can protect against. We have previously shown that transiently restricting an essential amino acid can protect Drosophila melanogaster against nicotine poisoning. Here, we sought to characterize the nature of this dietary-mediated protection and determine whether it was sex, amino acid and/or nicotine specific. When we compared between sexes, we found that isoleucine deprivation increases female, but not male, nicotine resistance. Surprisingly, we found that this protection afforded to females was not replicated by dietary protein restriction and was instead specific to individual amino acid restriction. To understand whether these beneficial effects of diet were specific to nicotine or were generalizable across stressors, we pre-treated flies with amino acid restriction diets and exposed them to other types of stress. We found that some of the diets that protected against nicotine also protected against oxidative and starvation stress, and improved survival following cold shock. Interestingly, we found that a diet lacking isoleucine was the only diet to protect against all these stressors. These data point to isoleucine as a critical determinant of robustness in the face of environmental challenges.

A multi-nutrient array protocol to study disease-diet interactions in Drosophila melanogaster.

The availability of a defined fully synthetic diet for the fruit fly Drosophila melanogaster allows for complete and precise manipulation of its nutritional environment. Here, we present a protocol for performing large-scale multivariate nutrient analysis via the traditional diet preparation approach, or by adding nutrient solutions to a baseline medium. We detail procedures from sample collection to data analysis. This protocol has applications for the study of nutrition-life trait interactions and nutrigenomics, to reveal interactions between genotype and diet composition. For complete details on the use and execution of this protocol, please refer to Martelli et al.1 and Martelli et al.2.

Sex-specific transgenerational effects of diet on offspring life history and physiology.

Dietary variation in males and females can shape the expression of offspring life histories and physiology. However, the relative contributions of maternal and paternal dietary variation to phenotypic expression of latter generations is currently unknown. We provided male and female Drosophila melanogaster grandparents with diets differing in sucrose concentration prior to reproduction, and similarly subjected their grandoffspring to the same treatments. We then investigated the phenotypic consequences of this dietary variation among the grandsons and granddaughters. We observed transgenerational effects of dietary sucrose, mediated through the grandmaternal lineage, which mimic the direct effects of sucrose on lifespan, with opposing patterns across sexes; low sucrose increased female, but decreased male, lifespan. Dietary mismatching of grandoffspring-grandparent diets increased lifespan and reproductive success, and moderated triglyceride levels of grandoffspring, providing insights into the physiological underpinnings of the complex transgenerational effects on life histories.

A defined diet for pre-adult Drosophila melanogaster.

Drosophila melanogaster is unique among animal models because it has a fully defined synthetic diet available to study nutrient-gene interactions. However, use of this diet is limited to adult studies due to impaired larval development and survival. Here, we provide an adjusted formula that reduces the developmental period, restores fat levels, enhances body mass, and fully rescues survivorship without compromise to adult lifespan. To demonstrate an application of this formula, we explored pre-adult diet compositions of therapeutic potential in a model of an inherited metabolic disorder affecting the metabolism of branched-chain amino acids. We reveal rapid, specific, and predictable nutrient effects on the disease state consistent with observations from mouse and patient studies. Together, our diet provides a powerful means with which to examine the interplay between diet and metabolism across all life stages in an animal model.

Short-term fasting of a single amino acid extends lifespan.

Diet and health are strongly linked, though the strict changes in diet required to improve health outcomes are usually difficult to sustain. We sought to understand whether short-term bouts of amino acid-specific modifications to the diet of Drosophila melanogaster could mimic the lifespan and stress resistance benefits of dietary restriction, without the requirement for drastic reductions in food intake. We found that flies that were transiently fed diets lacking the essential amino acid isoleucine, but otherwise nutritionally complete, exhibited enhanced nicotine tolerance, indicating elevated detoxification capacity. The protection from isoleucine deprivation increased with the duration of exposure, up to a maximum at 7-day isoleucine deprivation for flies 2, 3, or 4 weeks of age, and a 5-day deprivation when flies were 5 weeks of age. Because of these beneficial effects on toxin resistance, we intermittently deprived flies of isoleucine during the first 6 weeks of adulthood and monitored the effect on lifespan. Lifespan was significantly extended when flies experienced short-term isoleucine deprivation at 3 and 5 weeks of age, regardless of whether they were also deprived at 1 week. These results indicate that short-term bouts of isoleucine deprivation can extend lifespan and highlight its cumulative and time-dependent benefits. Interestingly, we found that isoleucine-deprived flies lost their protection against nicotine within 3 days of returning to fully fed conditions. Therefore, the mechanisms underlying lifespan extension may involve transient damage clearance during the bouts of isoleucine deprivation rather than sustained enhanced detoxification capacity. These data highlight a new time-restricted, nutritionally precise method to extend life in Drosophila melanogaster and point to a more manageable dietary method to combat ageing.

Identifying potential dietary treatments for inherited metabolic disorders using Drosophila nutrigenomics.

Inherited metabolic disorders are a group of genetic conditions that can cause severe neurological impairment and child mortality. Uniquely, these disorders respond to dietary treatment; however, this option remains largely unexplored because of low disorder prevalence and the lack of a suitable paradigm for testing diets. Here, we screened 35 Drosophila amino acid disorder models for disease-diet interactions and found 26 with diet-altered development and/or survival. Using a targeted multi-nutrient array, we examine the interaction in a model of isolated sulfite oxidase deficiency, an infant-lethal disorder. We show that dietary cysteine depletion normalizes their metabolic profile and rescues development, neurophysiology, behavior, and lifelong fly survival, thus providing a basis for further study into the pathogenic mechanisms involved in this disorder. Our work highlights the diet-sensitive nature of metabolic disorders and establishes Drosophila as a valuable tool for nutrigenomic studies for informing potential dietary therapies.

Exome-informed formulations of food proteins enhance body growth and feed conversion efficiency in ad libitum-fed mice.

Meeting requirements for dietary proteins, especially of essential amino acids (EAAs), is critical for the life-long health of living organisms. However, defining EAA targets for preparing biologically-matched nutrition that satisfies metabolic requirements for protein remains challenging. Previous research has shown the advantages of 'exome matching' in representing the specific requirement of dietary AAs, where the target dietary AA profile was derived from in silico translation of the genome of an organism, specifically responsible for protein expression (the 'exome'). However, past studies have assessed these effects in only one sex, for few parameters (body mass and composition), and have used purified diets in which protein is supplied as a mixture of individual AAs. Here, for the first time, we utilise a computational method to guide the formulation of custom protein blends and test if exome matching can be achieved at the intact protein level, through blending standard protein ingredients, ultimately leading to optimal growth, longevity and reproductive function. Mice were provided ad libitum (ad lib) access to one of the four iso-energetic protein-limited diets, two matched and two mis-matched to the mouse exome target, and fed at a fixed protein energy level of 6.2%. During or following 13-weeks of feeding, the food intake, body growth, composition and reproductive functions were measured. Compared to the two mis-matched diets, male and female animals on the exome-matched diet with protein digestibility correction applied, exhibited significantly improved growth rates and final body mass. The feed conversion efficiency in the same diet was also increased by 62% and 40% over the worst diets for males and females, respectively. Male, not female, exhibited higher accretion of lean body mass with the matched, digestibility-corrected diet. All reproductive function measures in both sexes were comparable among diets, with the exception of testicular daily sperm production in males, which was higher in the two matched diets versus the mis-matched diets. The results collectively demonstrate the pronounced advantages of exome-matching in supporting body growth and improving feed conversion efficiency in both sexes. However, the potential impact of this approach in enhancing fertility needs further investigation.

A Simple Method for Quantifying Larval Locomotion in Drosophila melanogaster.

The fruit fly Drosophila melanogaster is a powerful genetic model that has been used for many decades to study nervous system function, development, and behavior. There are a large number of developmental and behavioral traits that can be measured to provide a broad readout of neurological function. These include patterned motor behaviors, such as larval locomotion, which can be used to assess whether genetic or environmental factors affect nervous system function to provide an entry point for deeper mechanistic studies. Here, we describe a protocol for quantifying larval locomotion using a simple camera setup and a freely available image analysis software. This protocol can be readily applied to human disease models or in toxicology studies, for example, to broadly assess the impact of treatments on neurological function.

Early-adult methionine restriction reduces methionine sulfoxide and extends lifespan in Drosophila.

Methionine restriction (MetR) extends lifespan in various organisms, but its mechanistic understanding remains incomplete. Whether MetR during a specific period of adulthood increases lifespan is not known. In Drosophila, MetR is reported to extend lifespan only when amino acid levels are low. Here, by using an exome-matched holidic medium, we show that decreasing Met levels to 10% extends Drosophila lifespan with or without decreasing total amino acid levels. MetR during the first four weeks of adult life only robustly extends lifespan. MetR in young flies induces the expression of many longevity-related genes, including Methionine sulfoxide reductase A (MsrA), which reduces oxidatively-damaged Met. MsrA induction is foxo-dependent and persists for two weeks after cessation of the MetR diet. Loss of MsrA attenuates lifespan extension by early-adulthood MetR. Our study highlights the age-dependency of the organismal response to specific nutrients and suggests that nutrient restriction during a particular period of life is sufficient for healthspan extension.

GCN2 mediates access to stored amino acids for somatic maintenance during Drosophila ageing.

Many mechanistic theories of ageing argue that a progressive failure of somatic maintenance, the use of energy and resources to prevent and repair damage to the cell, underpins ageing. To sustain somatic maintenance an organism must acquire dozens of essential nutrients from the diet, including essential amino acids (EAAs), which are physiologically limiting for many animals. In Drosophila, adulthood deprivation of each individual EAA yields vastly different lifespan trajectories, and adulthood deprivation of one EAA, phenylalanine (Phe), has no associated lifespan cost; this is despite each EAA being strictly required for growth and reproduction. Moreover, survival under any EAA deprivation depends entirely on the conserved AA sensor GCN2, a component of the integrated stress response (ISR), suggesting that a novel ISR-mediated mechanism sustains lifelong somatic maintenance during EAA deprivation. Here we investigated this mechanism, finding that flies chronically deprived of dietary Phe continue to incorporate Phe into new proteins, and that challenging flies to increase the somatic requirement for Phe shortens lifespan under Phe deprivation. Further, we show that autophagy is required for full lifespan under Phe deprivation, and that activation of the ISR can partially rescue the shortened lifespan of GCN2-nulls under Phe deprivation. We therefore propose a mechanism by which GCN2, via the ISR, activates autophagy during EAA deprivation, breaking down a larvally-acquired store of EAAs to support somatic maintenance. These data refine our understanding of the strategies by which flies sustain lifelong somatic maintenance, which determines length of life in response to changes in the nutritional environment.

Biosynthetic constraints on amino acid synthesis at the base of the food chain may determine their use in higher-order consumer genomes.

Dietary nutrient composition is essential for shaping important fitness traits and behaviours. Many organisms are protein limited, and for Drosophila melanogaster this limitation manifests at the level of the single most limiting essential Amino Acid (AA) in the diet. The identity of this AA and its effects on female fecundity is readily predictable by a procedure called exome matching in which the sum of AAs encoded by a consumer's exome is used to predict the relative proportion of AAs required in its diet. However, the exome matching calculation does not weight AA contributions to the overall profile by protein size or expression. Here, we update the exome matching calculation to include these weightings. Surprisingly, although nearly half of the transcriptome is differentially expressed when comparing male and female flies, we found that creating transcriptome-weighted exome matched diets for each sex did not enhance their fecundity over that supported by exome matching alone. These data indicate that while organisms may require different amounts of dietary protein across conditions, the relative proportion of the constituent AAs remains constant. Interestingly, we also found that exome matched AA profiles are generally conserved across taxa and that the composition of these profiles might be explained by energetic and elemental limitations on microbial AA synthesis. Thus, it appears that ecological constraints amongst autotrophs shape the relative proportion of AAs that are available across trophic levels and that this constrains biomass composition.

Drosophila as a diet discovery tool for treating amino acid disorders.

Amino acid disorders (AADs) are a large group of rare inherited conditions that collectively impact one in 6500 live births, often resulting in rapid neurological decline and death during infancy. For several AADs, including phenylketonuria, dietary modification prevents physiological deterioration and ameliorates symptoms. Despite this remarkable potential for treatment success, dietary therapy for most AADs remains largely unexplored. Although animal models have provided novel insights into AAD mechanisms, few have been used for therapeutic diet discovery. Here, we find that of all the animal models, Drosophila is particularly well suited for nutrigenomic disease modelling, having amino acid pathways conserved with humans, exceptional genetic tractability, and the unique availability of a synthetic customisable diet.

Drosophila melanogaster females prioritise dietary sterols for producing viable eggs.

Limiting calories or specific nutrients without malnutrition, otherwise known as dietary restriction (DR), has been shown to extend lifespan and reduce reproduction across a broad range of taxa. Our recent findings in Drosophila melanogaster show that supplementing flies on macronutrient-rich diets with additional cholesterol can extend lifespan to the same extent as DR, while also sustaining high egg production. Thus, DR may be beneficial for lifespan because it reduces egg production which in turn reduces the mother's demand for sterols, thus supporting longer lifespan. It is also possible that mothers live longer and lay more eggs on high sterol diets because the diet triggers enhanced somatic maintenance and promotes egg production, but at the cost of diminished egg quality. To test this, we measured the viability of eggs and development of offspring from mothers fed either cholesterol-sufficient or cholesterol-limiting diets. We found that even when the mother's diet was completely devoid of cholesterol, viable egg production persisted for ∼10 days. Furthermore, we show that sterol-supplemented flies with long lives lay eggs that have high viability and the same developmental potential as those laid by shorter lived mothers on sterol limiting diets. These findings suggest that offspring viability is not a hidden cost of lifespan extension seen in response to dietary sterol supplementation.

Dietary restriction and lifespan: adaptive reallocation or somatic sacrifice?

Reducing overall food intake, or lowering the proportion of protein relative to other macronutrients, can extend the lifespan of diverse organisms. A number of mechanistic theories have been developed to explain this phenomenon, mostly assuming that the molecules connecting diet to lifespan are evolutionarily conserved. A recent study using Drosophila melanogaster females has pinpointed a single essential micronutrient that can explain how lifespan is changed by dietary restriction. Here, we propose a likely mechanism for this observation, which involves a trade-off between lifespan and reproduction, but in a manner that is conditional on the dietary supply of an essential micronutrient - a sterol. Importantly, these observations argue against previous evolutionary theories that rely on constitutive resource reallocation or damage directly inflicted by reproduction. Instead, they are compatible with a model in which the inverse relationship between lifespan and food level is caused by the consumer suffering from varying degrees of malnutrition when maintained on lab food. The data also indicate that animals on different lab foods may suffer from different nutritional imbalances and that the mechanisms by which dietary restriction benefits the lifespan of different species may vary. This means that translating the mechanistic findings from lab animals to humans will not be simple and should be interpreted in light of the range of challenges that have shaped each organism's lifespan in the wild and the composition of the natural diets upon which they would feed.

Restricting a single amino acid cross-protects Drosophila melanogaster from nicotine poisoning through mTORC1 and GCN2 signalling.

Dietary interventions that restrict protein intake have repeatedly been shown to offer beneficial health outcomes to the consumer. Benefits such as increased stress tolerance can be observed when individual amino acids are restricted, thus mimicking dietary protein restriction. Here, we sought to further understand the relationship between dietary amino acids and stress tolerance using Drosophila melanogaster. Using a chemically defined medium for Drosophila, we found that transiently restricting adult flies of a single essential amino acid generally protects against a lethal dose of the naturally occurring insecticide, nicotine. This protection varied with the identity of the focal amino acid and depended on the duration and intensity of its restriction. To understand the molecular basis of these effects, we modified the signalling of two cellular sensors of amino acids, GCN2 and mTORC1, in combination with amino acid restriction. We found that GCN2 was necessary for diets to protect against nicotine, whereas the suppression of mTORC1 was sufficient to induce nicotine resistance. This finding implies that amino acid restriction acts via amino acid signalling to cross-protect against seemingly unrelated stressors. Altogether, our study offers new insights into the physiological responses to restriction of individual amino acids that confer stress tolerance.

Target of Rapamycin Drives Unequal Responses to Essential Amino Acid Depletion for Egg Laying in Drosophila Melanogaster.

Nutrition shapes a broad range of life-history traits, ultimately impacting animal fitness. A key fitness-related trait, female fecundity is well known to change as a function of diet. In particular, the availability of dietary protein is one of the main drivers of egg production, and in the absence of essential amino acids egg laying declines. However, it is unclear whether all essential amino acids have the same impact on phenotypes like fecundity. Using a holidic diet, we fed adult female Drosophila melanogaster diets that contained all necessary nutrients except one of the 10 essential amino acids and assessed the effects on egg production. For most essential amino acids, depleting a single amino acid induced as rapid a decline in egg production as when there were no amino acids in the diet. However, when either methionine or histidine were excluded from the diet, egg production declined more slowly. Next, we tested whether GCN2 and TOR mediated this difference in response across amino acids. While mutations in GCN2 did not eliminate the differences in the rates of decline in egg laying among amino acid drop-out diets, we found that inhibiting TOR signalling caused egg laying to decline rapidly for all drop-out diets. TOR signalling does this by regulating the yolk-forming stages of egg chamber development. Our results suggest that amino acids differ in their ability to induce signalling via the TOR pathway. This is important because if phenotypes differ in sensitivity to individual amino acids, this generates the potential for mismatches between the output of a pathway and the animal's true nutritional status.

Amino acid quality modifies the quantitative availability of protein for reproduction in Drosophila melanogaster.

Diet composition, especially the relative abundance of key macronutrients, is well known to affect animal wellbeing by changing reproductive output, metabolism and length of life. However, less attention has been paid to the ways the quality of these nutrients modify these macronutrient interactions. Nutritional Geometry can be used to model the effects of multiple dietary components on life-history traits and to compare these responses when diet quality is varied. Previous studies have shown that dietary protein quality can be increased for egg production in Drosophila melanogaster by matching the dietary amino acid proportions to the balance of amino acids used by the sum of proteins in the fly's in silico translated exome. Here, we show that dietary protein quality dramatically alters the effect of protein quantity on female reproduction across a broad range of diets varying in both protein and carbohydrate concentrations. These data show that when sources of ingredients vary, their relative value to the consumer can vastly differ and yield very different physiological outcomes. Such variations could be particularly important for meta analyses that look to draw generalisable conclusions from diverse studies.

A dietary sterol trade-off determines lifespan responses to dietary restriction in Drosophila melanogaster females.

Diet plays a significant role in maintaining lifelong health. In particular, lowering the dietary protein: carbohydrate ratio can improve lifespan. This has been interpreted as a direct effect of these macronutrients on physiology. Using Drosophila melanogaster, we show that the role of protein and carbohydrate on lifespan is indirect, acting by altering the partitioning of limiting amounts of dietary sterols between reproduction and lifespan. Shorter lifespans in flies fed on high protein: carbohydrate diets can be rescued by supplementing their food with cholesterol. Not only does this fundamentally alter the way we interpret the mechanisms of lifespan extension by dietary restriction, these data highlight the important principle that life histories can be affected by nutrient-dependent trade-offs that are indirect and independent of the nutrients (often macronutrients) that are the focus of study. This brings us closer to understanding the mechanistic basis of dietary restriction.

For the past fifteen years, animal studies have consistently shown that a low-protein, high-carbohydrate ('carbs') diet can extend the lifespan of many organisms, but at the cost of the number of offspring an individual can produce. Yet, it is still unclear what the best dietary balance is, and how these effects arise. One potential explanation could be that reproduction damages the body: low levels of proteins would therefore prolong life by lowering the reproductive output. Here, Zanco et al. examined the possibility that protein intake in fruit flies could instead be acting indirectly by changing the levels of a fat-like molecule called cholesterol, which is used to maintain the body and to support reproduction. To test this idea, groups of fruit flies were fed high levels of proteins. This led to increased reproduction rates, in turn depleting the mothers' reserves of cholesterol. Without enough of the molecule in their diet, the insects were less able to maintain their bodies, which reduced their lifespan. When Zanco et al. added cholesterol to a high-protein diet, the flies lived for the normal length of time. Longer lifespan therefore did not require restriction of the diet or any of its components. In fact, the flies that lived the longest ate protein rich diets, and reproduced the most. This study helps to better understand why changes in diet can influence how long an organism lives for, highlighting that the abundance of certain key molecules may be more important than restricting the levels of proteins, carbs or calories actually consumed.

Effects of Short-Term Dietary Protein Restriction on Blood Amino Acid Levels in Young Men.

Pre-clinical studies show that dietary protein restriction (DPR) improves healthspan and retards many age-related diseases such as type 2 diabetes. While mouse studies have shown that restriction of certain essential amino acids is required for this response, less is known about which amino acids are affected by DPR in humans. Here, using a within-subjects diet design, we examined the effects of dietary protein restriction in the fasted state, as well as acutely after meal feeding, on blood plasma amino acid levels. While very few amino acids were affected by DPR in the fasted state, several proteinogenic AAs such as isoleucine, leucine, lysine, phenylalanine, threonine, tyrosine, and valine were lower in the meal-fed state with DPR. In addition, the non-proteinogenic AAs such as 1- and 3-methyl-histidine were also lower with meal feeding during DPR. Lastly, using in silico predictions of the most limiting essential AAs compared with human exome AA usage, we demonstrate that leucine, methionine, and threonine are potentially the most limiting essential AAs with DPR. In summary, acute meal feeding allows more accurate determination of which AAs are affected by dietary interventions, with most essential AAs lowered by DPR.

Restriction of essential amino acids dictates the systemic metabolic response to dietary protein dilution.

Dietary protein dilution (DPD) promotes metabolic-remodelling and -health but the precise nutritional components driving this response remain elusive. Here, by mimicking amino acid (AA) supply from a casein-based diet, we demonstrate that restriction of dietary essential AA (EAA), but not non-EAA, drives the systemic metabolic response to total AA deprivation; independent from dietary carbohydrate supply. Furthermore, systemic deprivation of threonine and tryptophan, independent of total AA supply, are both adequate and necessary to confer the systemic metabolic response to both diet, and genetic AA-transport loss, driven AA restriction. Dietary threonine restriction (DTR) retards the development of obesity-associated metabolic dysfunction. Liver-derived fibroblast growth factor 21 is required for the metabolic remodelling with DTR. Strikingly, hepatocyte-selective establishment of threonine biosynthetic capacity reverses the systemic metabolic response to DTR. Taken together, our studies of mice demonstrate that the restriction of EAA are sufficient and necessary to confer the systemic metabolic effects of DPD.