Dietary restriction and the transcription factor clock delay eye aging to extend lifespan in Drosophila Melanogaster.

Many vital processes in the eye are under circadian regulation, and circadian dysfunction has emerged as a potential driver of eye aging. Dietary restriction is one of the most robust lifespan-extending therapies and amplifies circadian rhythms with age. Herein, we demonstrate that dietary restriction extends lifespan in Drosophila melanogaster by promoting circadian homeostatic processes that protect the visual system from age- and light-associated damage. Altering the positive limb core molecular clock transcription factor, CLOCK, or CLOCK-output genes, accelerates visual senescence, induces a systemic immune response, and shortens lifespan. Flies subjected to dietary restriction are protected from the lifespan-shortening effects of photoreceptor activation. Inversely, photoreceptor inactivation, achieved via mutating rhodopsin or housing flies in constant darkness, primarily extends the lifespan of flies reared on a high-nutrient diet. Our findings establish the eye as a diet-sensitive modulator of lifespan and indicates that vision is an antagonistically pleiotropic process that contributes to organismal aging.

Antibody-mediated inhibition of GDF15-GFRAL activity reverses cancer cachexia in mice.

Cancer cachexia is a highly prevalent condition associated with poor quality of life and reduced survival1. Tumor-induced perturbations in the endocrine, immune and nervous systems drive anorexia and catabolic changes in adipose tissue and skeletal muscle, hallmarks of cancer cachexia2-4. However, the molecular mechanisms driving cachexia remain poorly defined, and there are currently no approved drugs for the condition. Elevation in circulating growth differentiation factor 15 (GDF15) correlates with cachexia and reduced survival in patients with cancer5-8, and a GDNF family receptor alpha like (GFRAL)-Ret proto-oncogene (RET) signaling complex in brainstem neurons that mediates GDF15-induced weight loss in mice has recently been described9-12. Here we report a therapeutic antagonistic monoclonal antibody, 3P10, that targets GFRAL and inhibits RET signaling by preventing the GDF15-driven interaction of RET with GFRAL on the cell surface. Treatment with 3P10 reverses excessive lipid oxidation in tumor-bearing mice and prevents cancer cachexia, even under calorie-restricted conditions. Mechanistically, activation of the GFRAL-RET pathway induces expression of genes involved in lipid metabolism in adipose tissues, and both peripheral chemical sympathectomy and loss of adipose triglyceride lipase protect mice from GDF15-induced weight loss. These data uncover a peripheral sympathetic axis by which GDF15 elicits a lipolytic response in adipose tissue independently of anorexia, leading to reduced adipose and muscle mass and function in tumor-bearing mice.

Dietary restriction improves intestinal cellular fitness to enhance gut barrier function and lifespan in D. melanogaster.

Loss of gut integrity is linked to various human diseases including inflammatory bowel disease. However, the mechanisms that lead to loss of barrier function remain poorly understood. Using D. melanogaster, we demonstrate that dietary restriction (DR) slows the age-related decline in intestinal integrity by enhancing enterocyte cellular fitness through up-regulation of dMyc in the intestinal epithelium. Reduction of dMyc in enterocytes induced cell death, which leads to increased gut permeability and reduced lifespan upon DR. Genetic mosaic and epistasis analyses suggest that cell competition, whereby neighboring cells eliminate unfit cells by apoptosis, mediates cell death in enterocytes with reduced levels of dMyc. We observed that enterocyte apoptosis was necessary for the increased gut permeability and shortened lifespan upon loss of dMyc. Furthermore, moderate activation of dMyc in the post-mitotic enteroblasts and enterocytes was sufficient to extend health-span on rich nutrient diets. We propose that dMyc acts as a barometer of enterocyte cell fitness impacting intestinal barrier function in response to changes in diet and age.

Intestinal IRE1 Is Required for Increased Triglyceride Metabolism and Longer Lifespan under Dietary Restriction.

Dietary restriction (DR) is one of the most robust lifespan-extending interventions in animals. The beneficial effects of DR involve a metabolic adaptation toward increased triglyceride usage. The regulatory mechanism and the tissue specificity of this metabolic switch remain unclear. Here, we show that the IRE1/XBP1 endoplasmic reticulum (ER) stress signaling module mediates metabolic adaptation upon DR in flies by promoting triglyceride synthesis and accumulation in enterocytes (ECs) of the Drosophila midgut. Consistently, IRE1/XBP1 function in ECs is required for increased longevity upon DR. We further identify sugarbabe, a Gli-like zinc-finger transcription factor, as a key mediator of the IRE1/XBP1-regulated induction of de novo lipogenesis in ECs. Overexpression of sugarbabe rescues metabolic and lifespan phenotypes of IRE1 loss-of-function conditions. Our study highlights the critical role of metabolic adaptation of the intestinal epithelium for DR-induced lifespan extension and explores the IRE1/XBP1 signaling pathway regulating this adaptation and influencing lifespan.

Peripheral Circadian Clocks Mediate Dietary Restriction-Dependent Changes in Lifespan and Fat Metabolism in Drosophila.

Endogenous circadian clocks orchestrate several metabolic and signaling pathways that are known to modulate lifespan, suggesting clocks as potential targets for manipulation of metabolism and lifespan. We report here that the core circadian clock genes, timeless (tim) and period (per), are required for the metabolic and lifespan responses to DR in Drosophila. Consistent with the involvement of a circadian mechanism, DR enhances the amplitude of cycling of most circadian clock genes, including tim, in peripheral tissues. Mass-spectrometry-based lipidomic analysis suggests a role of tim in cycling of specific medium chain triglycerides under DR. Furthermore, overexpression of tim in peripheral tissues improves its oscillatory amplitude and extends lifespan under ad libitum conditions. Importantly, effects of tim on lifespan appear to be mediated through enhanced fat turnover. These findings identify a critical role for specific clock genes in modulating the effects of nutrient manipulation on fat metabolism and aging.

Control of metabolic adaptation to fasting by dILP6-induced insulin signaling in Drosophila oenocytes.

Metabolic adaptation to changing dietary conditions is critical to maintain homeostasis of the internal milieu. In metazoans, this adaptation is achieved by a combination of tissue-autonomous metabolic adjustments and endocrine signals that coordinate the mobilization, turnover, and storage of nutrients across tissues. To understand metabolic adaptation comprehensively, detailed insight into these tissue interactions is necessary. Here we characterize the tissue-specific response to fasting in adult flies and identify an endocrine interaction between the fat body and liver-like oenocytes that regulates the mobilization of lipid stores. Using tissue-specific expression profiling, we confirm that oenocytes in adult flies play a central role in the metabolic adaptation to fasting. Furthermore, we find that fat body-derived Drosophila insulin-like peptide 6 (dILP6) induces lipid uptake in oenocytes, promoting lipid turnover during fasting and increasing starvation tolerance of the animal. Selective activation of insulin/IGF signaling in oenocytes by a fat body-derived peptide represents a previously unidentified regulatory principle in the control of metabolic adaptation and starvation tolerance.

Mitobolites: the elixir of life.

One of the biggest challenges in biology is to understand how mitochondria influence aging and age-related diseases. Chin et al. (2014) reveal how a mitochondrial metabolite (mitobolite) inhibits mitochondrial ATPase and extends lifespan by mimicking dietary restriction in worms.

Intramyocellular fatty-acid metabolism plays a critical role in mediating responses to dietary restriction in Drosophila melanogaster.

Changes in fat content have been associated with dietary restriction (DR), but whether they play a causal role in mediating various responses to DR remains unknown. We demonstrate that upon DR, Drosophila melanogaster shift their metabolism toward increasing fatty-acid synthesis and breakdown, which is required for various responses to DR. Inhibition of fatty-acid synthesis or oxidation genes specifically in the muscle tissue inhibited life-span extension upon DR. Furthermore, DR enhances spontaneous activity of flies, which was found to be dependent on the enhanced fatty-acid metabolism. This increase in activity was found to be at least partially required for the life-span extension upon DR. Overexpression of adipokinetic hormone (dAKH), the functional ortholog of glucagon, enhances fat metabolism, spontaneous activity, and life span. Together, these results suggest that enhanced fat metabolism in the muscle and physical activity play a key role in the protective effects of DR.

Role of TOR signaling in aging and related biological processes in Drosophila melanogaster.

Extensive studies in model organisms in the last few decades have revealed that aging is subject to profound genetic influence. The conserved nutrient sensing TOR (Target of Rapamycin) pathway is emerging as a key regulator of lifespan and healthspan in various species from yeast to mammals. The TOR signaling pathway plays a critical role in determining how a eukaryotic cell or a cellular system co-ordinates its growth, development and aging in response to constant changes in its surrounding environment? TOR integrates signals originating from changes in growth factors, nutrient availability, energy status and various physiological stresses. Each of these inputs is specialized to sense particular signal(s), and conveys it to the TOR complex which in turn relays the signal to downstream outputs to appropriately respond to the environmental changes. These outputs include mRNA translation, autophagy, transcription, metabolism, cell survival, proliferation and growth amongst a number of other cellular processes, some of which influence organismal lifespan. Here we review the contribution of the model organism Drosophila in the understanding of TOR signaling and the various biological processes it modulates that may impact on aging. Drosophila was the first organism where the nutrient dependent effects of the TOR pathway on lifespan were first uncovered. We also discuss how the nutrient-sensing TOR pathway appears to be critically important for mediating the longevity effects of dietary restriction (DR), a potent environmental method of lifespan extension by nutrient limitation. Identifying the molecular mechanisms that modulate lifespan downstream of TOR is being intensely investigated and there is hope that these are likely to serve as potential targets for amelioration of age-related diseases and enhance healthful lifespan extension in humans.

With TOR, less is more: a key role for the conserved nutrient-sensing TOR pathway in aging.

Target of rapamycin (TOR) is an evolutionarily conserved nutrient-sensing protein kinase that regulates growth and metabolism in all eukaryotic cells. Studies in flies, worms, yeast, and mice support the notion that the TOR signaling network modulates aging. TOR is also emerging as a robust mediator of the protective effects of various forms of dietary restriction (DR), which can extend life span and slow the onset of certain age-related diseases across species. Here we discuss how modulating TOR signaling slows aging through downstream processes including mRNA translation, autophagy, endoplasmic reticulum (ER) stress signaling, stress responses, and metabolism. Identifying the mechanisms by which the TOR signaling network works as a pacemaker of aging is a major challenge and may help identify potential drug targets for age-related diseases, thereby facilitating healthful life span extension in humans.

Dietary restriction and aging, 2009.

Dietary restriction (DR) is a robust nongenetic, nonpharmacological intervention that is known to increase active and healthy lifespan in a variety of species. Despite a variety of differences in the protocols and the way DR is carried out in different species, conserved relationships are emerging among multiple species. 2009 saw the field of DR mature with important mechanistic insights from multiple species. A report of lifespan extension in rapamycin-treated mice suggested that the TOR pathway, a conserved mediator of DR in invertebrates, may also be critical to DR effects in mammals. 2009 also saw exciting discoveries related to DR in various organisms including yeast, worms, flies, mice, monkeys and humans. These studies complement each other and together aim to deliver the promise of postponing aging and age-related diseases by revealing the underlying mechanisms of the protective effects of DR. Here, we summarize a few of the reports published in 2009 that we believe provide novel directions and an improved understanding of dietary restriction.

4E-BP extends lifespan upon dietary restriction by enhancing mitochondrial activity in Drosophila.

Dietary restriction (DR) extends lifespan in multiple species. To examine the mechanisms of lifespan extension upon DR, we assayed genome-wide translational changes in Drosophila. A number of nuclear encoded mitochondrial genes, including those in Complex I and IV of the electron transport chain, showed increased ribosomal loading and enhanced overall activity upon DR. We found that various mitochondrial genes possessed shorter and less structured 5'UTRs, which were important for their enhanced mRNA translation. The translational repressor 4E-BP, the eukaryotic translation initiation factor 4E binding protein, was upregulated upon DR and mediated DR dependent changes in mitochondrial activity and lifespan extension. Inhibition of individual mitochondrial subunits from Complex I and IV diminished the lifespan extension obtained upon DR, reflecting the importance of enhanced mitochondrial function during DR. Our results imply that translational regulation of nuclear-encoded mitochondrial gene expression by 4E-BP plays an important role in lifespan extension upon DR. For a video summary of this article, see the PaperFlick file with the Supplemental Data available online.

A candidate complex approach to study functional mitochondrial DNA changes: sequence variation and quaternary structure modeling of Drosophila simulans cytochrome c oxidase.

A problem with studying evolutionary dynamics of mitochondrial (mt) DNA is that classical population genetic techniques cannot identify selected substitutions because of genetic hitchhiking. We circumvented this problem by employing a candidate complex approach to study sequence variation in cytochrome c oxidase (COX) genes within and among three distinct Drosophila simulans mtDNA haplogroups. First, we determined sequence variation in complete coding regions for all COX mtDNA and nuclear loci and their isoforms. Second, we constructed a quaternary structure model of D. simulans COX. Third, we predicted that six of nine amino acid changes in D. simulans mtDNA are likely to be functionally important. Of these seven, genetic crosses can experimentally determine the functional significance of three. Fourth, we identified two single amino acid changes and a deletion of two consecutive amino acids in nuclear encoded COX loci that are likely to influence cytochrome c oxidase activity. These data show that linking population genetics and quaternary structure modeling can lead to functional predictions of specific mtDNA amino acid mutations and validate the candidate complex approach.

Comparative analysis of mitochondrial genotype and aging.

A common feature across all animals, including humans, is that mitochondrial bioenergetics is linked to oxidative stress, but the nature of these relationships with survival is yet to be properly defined. In this study we included 12 Drosophila simulans isofemale lines: four of each distinct mtDNA haplogroup (siI, -II, and -III). First, we investigated sequence variation in six mtDNA and 13 nuclear encoded genes (nine nuclear-encoded subunits, and the four known isoforms, of complex IV of the electron transport chain). As expected we observed high divergence among the three distinct mitotypes and greatest mtDNA variability in siII-harboring flies. In the nuclear encoded genes, no fixed amino acid differences were observed and levels of polymorphism did not differ significantly among flies harboring distinct mtDNA types. Second, 15,456 flies were included in mortality studies. We observed that mtDNA type influenced survival (siII approximately siIII > siI), flies harboring siII mtDNA had the greatest variation in mortality rates, and in all cases males were longer lived than females. We also assayed maximal rates of hydrogen peroxide (H(2)O(2)) production from complex III of the electron transport chain in mitochondria isolated from 11-day-old flies. Contrary to our prediction, rates of H(2)O(2) production tended to increase with mean survival. This result suggests that higher rates of H(2)O(2) production in younger flies may lead to an upregulation of antioxidants, age-dependent increase in the rate of H(2)O(2) production differ, and/or flies vary in their mitochondrial uncoupling. Alternatively, the whole organism may not regularly, if ever, experience maximal H(2)O(2) production rates.

Sex differences in survival and mitochondrial bioenergetics during aging in Drosophila.

The goal of this study is to test the role of mitochondria and of mitochondrial metabolism in determining the processes that influence aging of female and male Drosophila. We observe that Drosophila simulans females tended to have shorter lifespan, higher levels of hydrogen peroxide production and significantly lower levels of catalase but not superoxide dismutase compared to males. In contrast, mammalian females tend to be longer lived, have lower rates of reactive oxygen species production and higher antioxidant activity. In both Drosophila and mammals, mitochondria extracted from females consume a higher quantity of oxygen when provided with adenosine diphosphate and have a greater mtDNA copy number than males. Combined, these data illustrate important similarities between the parameters that influence aging and mitochondrial metabolism in Drosophila and in mammals but also show surprising differences.

Sympatric Drosophila simulans flies with distinct mtDNA show age related differences in mitochondrial metabolism.

The primary causes of age-related changes in mitochondrial metabolism are not known. The goal of this study is to document the influence of naturally occurring mtDNA variation on age dependent changes in mitochondrial respiration, hydrogen peroxide (H(2)O(2)) generation and antioxidant defenses in the fly Drosophila simulans. Possible changes include an increase in rates of reactive oxygen species production with age and/or an age dependent decrease in antioxidant response. For this study we have used flies harboring distinct siII and siIII mtDNA types. Previously we have shown that males harboring siII mtDNA had higher rates of mitochondrial H(2)O(2) production from complex III at 11d compared to males with the siIII mtDNA type. Here, we corroborate those results and show that Drosophila harboring the siII and siIII mtDNA types exhibit significantly different patterns of pro-oxidant and antioxidant activities as they age. Flies harboring siII mtDNA had higher rates of mitochondrial H(2)O(2) production and manganese superoxide dismutase activity at 11 and 18d of age than siIII mtDNA harboring flies. Copper-zinc superoxide dismutase activity increased from 11 to 25d in siII flies while the accumulation of oxidized glutathione did not change between 11 and 25d. In contrast, siIII harboring flies showed an age dependent increase in H(2)O(2) production, reaching higher production rates on day 25 than that observed in siII flies. Copper-zinc superoxide dismutase activities did not change between 11 and 25d while the oxidized glutathione accumulation increased with age. The results show antioxidant levels correlate with pro-oxidant levels in siII but not siIII flies. These results demonstrate our ability to correlate mtDNA variation with differences in whole mitochondrial physiology and individual complex biochemistry.

Mitochondrial DNA variation is associated with measurable differences in life-history traits and mitochondrial metabolism in Drosophila simulans.

Recent studies have used a variety of theoretical arguments to show that mitochondrial (mt) DNA rarely evolves as a strictly neutral marker and that selection operates on the mtDNA of many species. However, the vast majority of researchers are not convinced by these arguments because data linking mtDNA variation with phenotypic differences are limited. We investigated sequence variation in the three mtDNA and nine nuclear genes (including all isoforms) that encode the 12 subunits of cytochrome c oxidase of the electron transport chain in Drosophila. We then studied cytochrome c oxidase activity as a key aspect of mitochondrial bioenergetics and four life-history traits. In Drosophila simulans, sequence data from the three mtDNA encoded cytochrome c oxidase genes show that there are 76 synonymous and two nonsynonymous fixed differences among flies harboring siII compared with siIII mtDNA. In contrast, 13 nuclear encoded genes show no evidence of genetic subdivision associated with the mtDNA. Flies with siIII mtDNA had higher cytochrome c oxidase activity and were more starvation resistant. Flies harboring siII mtDNA had greater egg size and fecundity, and recovered faster from cold coma. These data are consistent with a causative role for mtDNA variation in these phenotypic differences, but we cannot completely rule out the involvement of nuclear genes. The results of this study have significant implications for the use of mtDNA as an assumed neutral marker and show that evolutionary shifts can involve changes in mtDNA despite the small number of genes encoded in the organelle genome.

Sympatric Drosophila simulans flies with distinct mtDNA show difference in mitochondrial respiration and electron transport.

The role of mitochondrial DNA (mtDNA) in mitochondrial metabolism is understudied yet humans harboring specific mtDNA types age at dissimilar rates, are unequally susceptible to various diseases, and differentially adapt to various environmental conditions. This study compares mitochondrial respiration, proton leak and electron transport of Drosophila simulans males with distinct mtDNA haplogroups (siII and -III) that were collected in sympatry in Kenya. Despite the large divergence among haplogroups there is very low intrahaplogroup variation and no correlated variation in the nuclear genome has been detected. We show that repeatable bioenergetic differences exist between 11d old males harboring siII and siIII mtDNA. Males with siIII mtDNA showed higher (i) state 3 respiration rates from isolated mitochondria for both complex I and complex III based substrates, and (ii) complex IV (cytochrome c oxidase) activity. Males harboring siIII mtDNA had lower (i) hydrogen peroxide formation by both complexes I and III, (ii) proton leak from isolated mitochondria, (iii) mitochondrial ATPase activity, and (iv) mitochondrial cytochrome content. In combination, the results suggest that mitochondria isolated from siIII mtDNA harboring males have more efficient metabolism than siII mtDNA harboring males.

Maternal high-fat diet consumption results in fetal malprogramming predisposing to the onset of metabolic syndrome-like phenotype in adulthood.

Chronic consumption of a high-fat (HF) diet by female rats in their postweaning period resulted in significant increases in body weight and plasma levels of insulin, glucose, and triglycerides during pregnancy compared with female rats consuming a standard rodent laboratory chow (LC). On gestational day 21, plasma insulin levels and the insulin secretory response of islets to various secretogogues were significantly increased in HF fetuses. The HF male progeny weaned onto LC (HF/LC) demonstrated increases in body weight from postnatal day 60 onward. In adulthood, HF/LC male rats were significantly heavier than controls, had increased plasma levels of insulin, glucose, free fatty acids, and triglycerides, and demonstrated glucose intolerance. HF/LC male islets secreted increased amounts of insulin in response to low glucose concentrations, but their response to a high glucose concentration was similar to that of LC/LC islets. In another set of experiments, when the male progeny of HF female rats were weaned onto a high-sucrose diet (HF/HSu), their metabolic profile was further worsened. These results indicate that chronic consumption of a HF diet by female rats malprograms the male progeny for glucose intolerance and development of increased body weight in adulthood. The long-term high-fat feeding to female rats employed in this study bears resemblance to the dietary habits in Western societies. The results of this study implicate dietary practices of women in the etiology of the present epidemic of human obesity and related disorders.

Antimalarials inhibit human erythrocyte membrane acetylcholinesterase.

The current study examined the ability of antimalarials chloroquine (CQ), primaquine (PQ), and quinine (Q) to inhibit human erythrocyte membrane acetylcholinesterase (AChE) and the mechanisms underlying their inhibitory action. CQ was found to be the most effective inhibitor of the enzyme followed by PQ and Q. The concentrations required to obtain 33% inhibition (IC(33)) for CQ and PQ were 22 and 38 microM, respectively, whereas that for Q was 3.2 mM. The concentrations required to obtain 67% inhibition (IC(67)) were about 9 and 7 times higher for CQ and PQ, whereas that for Q was only about 2.5 times higher. Hill plot analysis revealed that CQ shows de-binding above 40 microM. The two kinetic components of AChE were inhibited by the three antimalarials, and the inhibition was of mixed type. Increasing concentrations of antimalarials caused progressive decrease in the V(max) of both components. IC(33) concentrations resulted in 1.6- to 6-fold increase in K(m) of both the components while IC(67) concentration caused 2.8- to 13-fold increases in K(m) with maximum effect being seen with Q. The K(i) values were lowest for CQ suggesting that it was the most potent inhibitor; these values were 3.3 and 60 times higher for PQ and Q. Antimalarials represent the bifunctional compounds that possess anti-inflammatory properties and also inhibit cholinesterases. The results of our studies suggest that 4-aminoquinoline-based antimalarials like CQ and hydroxychloroquine, which are both potent anti-inflammatory agents and inhibitors of cholinesterases, may have potential use as the most effective neuroprotective agents against amyloid-beta-peptide (Abeta) neurotoxicity in Alzheimer's disease.