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.

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.

Commensal bacteria and essential amino acids control food choice behavior and reproduction.

Choosing the right nutrients to consume is essential to health and wellbeing across species. However, the factors that influence these decisions are poorly understood. This is particularly true for dietary proteins, which are important determinants of lifespan and reproduction. We show that in Drosophila melanogaster, essential amino acids (eAAs) and the concerted action of the commensal bacteria Acetobacter pomorum and Lactobacilli are critical modulators of food choice. Using a chemically defined diet, we show that the absence of any single eAA from the diet is sufficient to elicit specific appetites for amino acid (AA)-rich food. Furthermore, commensal bacteria buffer the animal from the lack of dietary eAAs: both increased yeast appetite and decreased reproduction induced by eAA deprivation are rescued by the presence of commensals. Surprisingly, these effects do not seem to be due to changes in AA titers, suggesting that gut bacteria act through a different mechanism to change behavior and reproduction. Thus, eAAs and commensal bacteria are potent modulators of feeding decisions and reproductive output. This demonstrates how the interaction of specific nutrients with the microbiome can shape behavioral decisions and life history traits.

Drosophila as a model for ageing.

Drosophila melanogaster has been a key model in developing our current understanding of the molecular mechanisms of ageing. Of particular note is its role in establishing the evolutionary conservation of reduced insulin and IGF-1-like signaling in promoting healthy ageing. Capitalizing on its many advantages for experimentation, more recent work has revealed how precise nutritional and genetic interventions can improve fly lifespan without obvious detrimental side effects. We give a brief summary of these recent findings as well as examples of how they may modify ageing via actions in the gut and muscle. These discoveries highlight how expanding our understanding of metabolic and signaling interconnections will provide even greater insight into how these benefits may be harnessed for anti-ageing interventions.

Using Mouse and Drosophila Models to Investigate the Mechanistic Links between Diet, Obesity, Type II Diabetes, and Cancer.

Many of the links between diet and cancer are controversial and over simplified. To date, human epidemiological studies consistently reveal that patients who suffer diet-related obesity and/or type II diabetes have an increased risk of cancer, suffer more aggressive cancers, and respond poorly to current therapies. However, the underlying molecular mechanisms that increase cancer risk and decrease the response to cancer therapies in these patients remain largely unknown. Here, we review studies in mouse cancer models in which either dietary or genetic manipulation has been used to model obesity and/or type II diabetes. These studies demonstrate an emerging role for the conserved insulin and insulin-like growth factor signaling pathways as links between diet and cancer progression. However, these models are time consuming to develop and expensive to maintain. As the world faces an epidemic of obesity and type II diabetes we argue that the development of novel animal models is urgently required. We make the case for Drosophila as providing an unparalleled opportunity to combine dietary manipulation with models of human metabolic disease and cancer. Thus, combining diet and cancer models in Drosophila can rapidly and significantly advance our understanding of the conserved molecular mechanisms that link diet and diet-related metabolic disorders to poor cancer patient prognosis.

A holidic medium for Drosophila melanogaster.

A critical requirement for research using model organisms is a well-defined and consistent diet. There is currently no complete chemically defined (holidic) diet available for Drosophila melanogaster. We describe a holidic medium that is equal in performance to an oligidic diet optimized for adult fecundity and lifespan. This holidic diet supports development over multiple generations but at a reduced rate. Over 7 years of experiments, the holidic diet yielded more consistent experimental outcomes than did oligidic food for egg laying by females. Nutrients and drugs were more available to flies in holidic medium and, similar to dietary restriction on oligidic food, amino acid dilution increased fly lifespan. We used this holidic medium to investigate amino acid-specific effects on food-choice behavior and report that folic acid from the microbiota is sufficient for Drosophila development.

Amino-acid imbalance explains extension of lifespan by dietary restriction in Drosophila.

Dietary restriction extends healthy lifespan in diverse organisms and reduces fecundity. It is widely assumed to induce adaptive reallocation of nutrients from reproduction to somatic maintenance, aiding survival of food shortages in nature. If this were the case, long life under dietary restriction and high fecundity under full feeding would be mutually exclusive, through competition for the same limiting nutrients. Here we report a test of this idea in which we identified the nutrients producing the responses of lifespan and fecundity to dietary restriction in Drosophila. Adding essential amino acids to the dietary restriction condition increased fecundity and decreased lifespan, similar to the effects of full feeding, with other nutrients having little or no effect. However, methionine alone was necessary and sufficient to increase fecundity as much as did full feeding, but without reducing lifespan. Reallocation of nutrients therefore does not explain the responses to dietary restriction. Lifespan was decreased by the addition of amino acids, with an interaction between methionine and other essential amino acids having a key role. Hence, an imbalance in dietary amino acids away from the ratio optimal for reproduction shortens lifespan during full feeding and limits fecundity during dietary restriction. Reduced activity of the insulin/insulin-like growth factor signalling pathway extends lifespan in diverse organisms, and we find that it also protects against the shortening of lifespan with full feeding. In other organisms, including mammals, it may be possible to obtain the benefits to lifespan of dietary restriction without incurring a reduction in fecundity, through a suitable balance of nutrients in the diet.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.