Trimethylamine modulates dauer formation,neurodegeneration, and lifespan through tyra‐3/daf‐11 signaling in Caenorhabditis elegans

In the nematode Caenorhabditis elegans, signals derived from bacteria in the diet, the animal''s major nutrient source, can modulate both behavior and healthspan. Here we describe a dual role for trimethylamine (TMA), a human gut flora metabolite, which acts as a nutrient signal and a neurotoxin. TMA and its associated metabolites are produced by the human gut microbiome and have been suggested to serve as risk biomarkers for diabetes and cardiovascular diseases. We demonstrate that the tyramine receptor TYRA‐3, a conserved G protein‐coupled receptor (GPCR), is required to sense TMA and mediate its responses. TMA activates guanylyl cyclase DAF‐11 signaling through TYRA‐3 in amphid neurons (ASK) and ciliated neurons (BAG) to mediate food‐sensing behavior. Bacterial mutants deficient in TMA production enhance dauer formation, extend lifespan, and are less preferred as a food source. Increased levels of TMA lead to neural damage in models of Parkinson''s disease and shorten lifespan. Our results reveal conserved signaling pathways modulated by TMA in C. elegans that are likely to be relevant for its effects in mammalian systems.     

Metabolic balance in colorectal cancer is maintained by optimal Wnt signaling levels

Wnt pathways are important for the modulation of tissue homeostasis, and their deregulation is linked to cancer development. Canonical Wnt signaling is hyperactivated in many human colorectal cancers due to genetic alterations of the negative Wnt regulator APC. However, the expression levels of Wnt‐dependent targets vary between tumors, and the mechanisms of carcinogenesis concomitant with this Wnt signaling dosage have not been understood. In this study, we integrate whole‐genome CRISPR/Cas9 screens with large‐scale multi‐omic data to delineate functional subtypes of cancer. We engineer APC loss‐of‐function mutations and thereby hyperactivate Wnt signaling in cells with low endogenous Wnt activity and find that the resulting engineered cells have an unfavorable metabolic equilibrium compared with cells which naturally acquired Wnt hyperactivation. We show that the dosage level of oncogenic Wnt hyperactivation impacts the metabolic equilibrium and the mitochondrial phenotype of a given cell type in a context‐dependent manner. These findings illustrate the impact of context‐dependent genetic interactions on cellular phenotypes of a central cancer driver mutation and expand our understanding of quantitative modulation of oncogenic signaling in tumorigenesis.     

Adipose tissue senescence is mediated by increased ATP content after a short‐term high‐fat diet exposure

In the context of obesity, senescent cells accumulate in white adipose tissue (WAT). The cellular underpinnings of WAT senescence leading to insulin resistance are not fully elucidated. The objective of the current study was to evaluate the presence of WAT senescence early after initiation of high‐fat diet (HFD, 1–10 weeks) in 5‐month‐old male C57BL/6J mice and the potential role of energy metabolism. We first showed that WAT senescence occurred 2 weeks after HFD as evidenced in whole WAT by increased senescence‐associated ß‐galactosidase activity and cyclin‐dependent kinase inhibitor 1A and 2A expression. WAT senescence affected various WAT cell populations, including preadipocytes, adipose tissue progenitors, and immune cells, together with adipocytes. WAT senescence was associated with higher glycolytic and mitochondrial activity leading to enhanced ATP content in HFD‐derived preadipocytes, as compared with chow diet‐derived preadipocytes. One‐month daily exercise, introduced 5 weeks after HFD, was an effective senostatic strategy, since it reversed WAT cellular senescence, while reducing glycolysis and production of ATP. Interestingly, the beneficial effect of exercise was independent of body weight and fat mass loss. We demonstrated that WAT cellular senescence is one of the earliest events occurring after HFD initiation and is intimately linked to the metabolic state of the cells. Our data uncover a critical role for HFD‐induced elevated ATP as a local danger signal inducing WAT senescence. Exercise exerts beneficial effects on adipose tissue bioenergetics in obesity, reversing cellular senescence, and metabolic abnormalities.     

Deciphering the physiological response of Escherichia coli under high ATP demand

One long‐standing question in microbiology is how microbes buffer perturbations in energy metabolism. In this study, we systematically analyzed the impact of different levels of ATP demand in Escherichia coli under various conditions (aerobic and anaerobic, with and without cell growth). One key finding is that, under all conditions tested, the glucose uptake increases with rising ATP demand, but only to a critical level beyond which it drops markedly, even below wild‐type levels. Focusing on anaerobic growth and using metabolomics and proteomics data in combination with a kinetic model, we show that this biphasic behavior is induced by the dual dependency of the phosphofructokinase on ATP (substrate) and ADP (allosteric activator). This mechanism buffers increased ATP demands by a higher glycolytic flux but, as shown herein, it collapses under very low ATP concentrations. Model analysis also revealed two major rate‐controlling steps in the glycolysis under high ATP demand, which could be confirmed experimentally. Our results provide new insights on fundamental mechanisms of bacterial energy metabolism and guide the rational engineering of highly productive cell factories.     

Do we still need animals? Surveying the role of animal‐free models in Alzheimer’s and Parkinson’s disease research

The use of animals in neuroscience and biomedical research remains controversial. Policy is built around the “3R” principle of “Refining, Reducing and Replacing” animal experiments, and across the globe, different initiatives stimulate the use of animal‐free methods. Based on an extensive literature screen to map the development and adoption of animal‐free methods in Alzheimer''s and Parkinson''s disease research, we find that at least two in three examined studies rely on animals or on animal‐derived models. Among the animal‐free studies, the relative contribution of innovative models that may replace animal experiments is limited. We argue that the distinction between animal research and alternative models presents a false dichotomy, as the role and scientific value of both animal and animal‐free approaches are intertwined. Calls to halt all animal experiments appear premature, as insufficient non‐animal‐based alternatives are available and their development lags behind. In light of this, we highlight the need for objective, unprejudiced monitoring, and more robust performance indicators of animal‐free approaches.     

IFN‐β rescues neurodegeneration by regulating mitochondrial fission via STAT5, PGAM5, and Drp1

Mitochondrial homeostasis is essential for providing cellular energy, particularly in resource‐demanding neurons, defects in which cause neurodegeneration, but the function of interferons (IFNs) in regulating neuronal mitochondrial homeostasis is unknown. We found that neuronal IFN‐β is indispensable for mitochondrial homeostasis and metabolism, sustaining ATP levels and preventing excessive ROS by controlling mitochondrial fission. IFN‐β induces events that are required for mitochondrial fission, phosphorylating STAT5 and upregulating PGAM5, which phosphorylates serine 622 of Drp1. IFN‐β signaling then recruits Drp1 to mitochondria, oligomerizes it, and engages INF2 to stabilize mitochondria–endoplasmic reticulum (ER) platforms. This process tethers damaged mitochondria to the ER to separate them via fission. Lack of neuronal IFN‐β in the Ifnb ^–/– model of Parkinson disease (PD) disrupts STAT5‐PGAM5‐Drp1 signaling, impairing fission and causing large multibranched, damaged mitochondria with insufficient ATP production and excessive oxidative stress to accumulate. In other PD models, IFN‐β rescues dopaminergic neuronal cell death and pathology, associated with preserved mitochondrial homeostasis. Thus, IFN‐β activates mitochondrial fission in neurons through the pSTAT5/PGAM5/^S622Drp1 pathway to stabilize mitochondria/ER platforms, constituting an essential neuroprotective mechanism.     

Drosophila Sirtuin 6 mediates developmental diet‐dependent programming of adult physiology and survival

Organisms in the wild experience unpredictable and diverse food availability throughout their lifespan. Over‐/under‐nutrition during development and in adulthood is known to dictate organismal survival and fitness. Studies using model systems have also established long‐term effects of developmental dietary alterations on life‐history traits. However, the underlining genetic/molecular factors, which differentially couple nutrient inputs during development with fitness later in life are far less understood. Using Drosophila and loss/gain of function perturbations, our serendipitous findings demonstrate an essential role of Sirtuin 6 in regulating larval developmental kinetics, in a nutrient‐dependent manner. The absence of Sirt6 affected ecdysone and insulin signalling and led to accelerated larval development. Moreover, varying dietary glucose and yeast during larval stages resulted in enhanced susceptibility to metabolic and oxidative stress in adults. We also demonstrate an evolutionarily conserved role for Sirt6 in regulating physiological homeostasis, physical activity and organismal lifespan, known only in mammals until now. Our results highlight gene‐diet interactions that dictate thresholding of nutrient inputs and physiological plasticity, operative across development and adulthood. In summary, besides showing its role in invertebrate ageing, our study also identifies Sirt6 as a key factor that programs macronutrient‐dependent life‐history traits.     

Sodium butyrate inhibits aerobic glycolysis of hepatocellular carcinoma cells via the c‐myc/hexokinase 2 pathway

Aerobic glycolysis is a well‐known hallmark of hepatocellular carcinoma (HCC). Hence, targeting the key enzymes of this pathway is considered a novel approach to HCC treatment. The effects of sodium butyrate (NaBu), a sodium salt of the short‐chain fatty acid butyrate, on aerobic glycolysis in HCC cells and the underlying mechanism are unknown. In the present study, data obtained from cell lines with mouse xenograft model revealed that NaBu inhibited aerobic glycolysis in the HCC cells in vivo and in vitro. NaBu induced apoptosis while inhibiting the proliferation of the HCC cells in vivo and in vitro. Furthermore, the compound inhibited the release of lactate and glucose consumption in the HCC cells in vitro and inhibited the production of lactate in vivo. The modulatory effects of NaBu on glycolysis, proliferation and apoptosis were related to its modulation of hexokinase 2 (HK2). NaBu downregulated HK2 expression via c‐myc signalling. The upregulation of glycolysis in the HCC cells induced by sorafenib was impeded by NaBu, thereby enhancing the anti‐HCC effect of sorafenib in vitro and in vivo. Thus, NaBu inhibits the expression of HK2 to downregulate aerobic glycolysis and the proliferation of HCC cells and induces their apoptosis via the c‐myc pathway.     

IGFBP1 increases β‐cell regeneration by promoting α‐ to β‐cell transdifferentiation

There is great interest in therapeutically harnessing endogenous regenerative mechanisms to increase the number of β cells in people with diabetes. By performing whole‐genome expression profiling of zebrafish islets, we identified 11 secreted proteins that are upregulated during β‐cell regeneration. We then tested the proteins'' ability to potentiate β‐cell regeneration in zebrafish at supraphysiological levels. One protein, insulin‐like growth factor (Igf) binding‐protein 1 (Igfbp1), potently promoted β‐cell regeneration by potentiating α‐ to β‐cell transdifferentiation. Using various inhibitors and activators of the Igf pathway, we show that Igfbp1 exerts its regenerative effect, at least partly, by inhibiting Igf signaling. Igfbp1''s effect on transdifferentiation appears conserved across species: Treating mouse and human islets with recombinant IGFBP1 in vitro increased the number of cells co‐expressing insulin and glucagon threefold. Moreover, a prospective human study showed that having high IGFBP1 levels reduces the risk of developing type‐2 diabetes by more than 85%. Thus, we identify IGFBP1 as an endogenous promoter of β‐cell regeneration and highlight its clinical importance in diabetes.     

Diet and temperature modify the relationship between energy use and ATP production to influence behavior in zebrafish (Danio rerio)

Food availability and temperature influence energetics of animals and can alter behavioral responses such as foraging and spontaneous activity. Food availability, however, is not necessarily a good indicator of energy (ATP) available for cellular processes. The efficiency of energy transduction from food‐derived substrate to ATP in mitochondria can change with environmental context. Our aim was to determine whether the interaction between food availability and temperature affects mitochondrial efficiency and behavior in zebrafish (Danio rerio). We conducted a fully factorial experiment to test the effects of feeding frequency, acclimation temperature (three weeks to 18 or 28°C), and acute test temperature (18 and 28°C) on whole‐animal oxygen consumption, mitochondrial bioenergetics and efficiency (ADP consumed per oxygen atom; P:O ratio), and behavior (boldness and exploration). We show that infrequently fed (once per day on four days per week) zebrafish have greater mitochondrial efficiency than frequently fed (three times per day on five days per week) animals, particularly when warm‐acclimated. The interaction between temperature and feeding frequency influenced exploration of a novel environment, but not boldness. Both resting rate of producing ATP and scope for increasing it were positively correlated with time spent exploring and distance moved in standardized trials. In contrast, behavior was not associated with whole‐animal aerobic (oxygen consumption) scope, but exploration was positively correlated with resting oxygen consumption rates. We highlight the importance of variation in both metabolic (oxygen consumption) rate and efficiency of producing ATP in determining animal performance and behavior. Oxygen consumption represents energy use, and P:O ratio is a variable that determines how much of that energy is allocated to ATP production. Our results emphasize the need to integrate whole‐animal responses with subcellular traits to evaluate the impact of environmental conditions on behavior and movement.     

Structural basis of transport and inhibition of the Plasmodium falciparum transporter PfFNT

The intra‐erythrocyte stage of P. falciparum relies primarily on glycolysis to generate adenosine triphosphate (ATP) and the energy required to support growth and reproduction. Lactic acid, a metabolic byproduct of glycolysis, is potentially toxic as it lowers the pH inside the parasite. Plasmodium falciparum formate–nitrite transporter (PfFNT), a 34‐kDa transmembrane protein, has been identified as a novel drug target as it exports lactate from inside the parasite to the surrounding parasitophorous vacuole within the erythrocyte cytosol. The structure and detailed molecular mechanism of this membrane protein are not yet available. Here we present structures of PfFNT in the absence and presence of the functional inhibitor MMV007839 at resolutions of 2.56 Å and 2.78 Å using single‐particle cryo‐electron microscopy. Genetic analysis and transport assay indicate that PfFNT is able to transfer lactate across the membrane. Combined, our data suggest a stepwise displacement mechanism for substrate transport. The PfFNT membrane protein is capable of picking up lactate ions from the parasite’s cytosol, converting them to lactic acids and then exporting these acids into the extracellular space.     

Differential Involvement of Hedgehog Signaling in Butterfly Wing and Eyespot Development

Butterfly eyespots may have evolved from the recruitment of pre-existent gene circuits or regulatory networks into novel locations on the wing. Gene expression data suggests one such circuit, the Hedgehog (Hh) signaling pathway and its target gene engrailed (en), was recruited from a role in patterning the anterior-posterior insect wing axis to a role patterning butterfly eyespots. However, while Junonia coenia expresses hh and en both in the posterior compartment of the wing and in eyespot centers, Bicyclus anynana lacks hh eyespot-specific expression. This suggests that Hh signaling may not be functioning in eyespot development in either species or that it functions in J. coenia but not in B. anynana. In order to test these hypotheses, we performed functional tests of Hh signaling in these species. We investigated the effects of Hh protein sequestration during the larval stage on en expression levels, and on wing size and eyespot size in adults. Hh sequestration led to significantly reduced en expression and to significantly smaller wings and eyespots in both species. But while eyespot size in B. anynana was reduced proportionately to wing size, in J. coenia, eyespots were reduced disproportionately, indicating an independent role of Hh signaling in eyespot development in J. coenia. We conclude that while Hh signaling retains a conserved role in promoting wing growth across nymphalid butterflies, it plays an additional role in eyespot development in some, but not all, lineages of nymphalid butterflies. We discuss our findings in the context of alternative evolutionary scenarios that led to the differential expression of hh and other Hh pathway signaling members across nymphalid species.     

Glycolysis/gluconeogenesis specialization in microbes is driven by biochemical constraints of flux sensing

Central carbon metabolism is highly conserved across microbial species, but can catalyze very different pathways depending on the organism and their ecological niche. Here, we study the dynamic reorganization of central metabolism after switches between the two major opposing pathway configurations of central carbon metabolism, glycolysis, and gluconeogenesis in Escherichia coli, Pseudomonas aeruginosa, and Pseudomonas putida. We combined growth dynamics and dynamic changes in intracellular metabolite levels with a coarse‐grained model that integrates fluxes, regulation, protein synthesis, and growth and uncovered fundamental limitations of the regulatory network: After nutrient shifts, metabolite concentrations collapse to their equilibrium, rendering the cell unable to sense which direction the flux is supposed to flow through the metabolic network. The cell can partially alleviate this by picking a preferred direction of regulation at the expense of increasing lag times in the opposite direction. Moreover, decreasing both lag times simultaneously comes at the cost of reduced growth rate or higher futile cycling between metabolic enzymes. These three trade‐offs can explain why microorganisms specialize for either glycolytic or gluconeogenic substrates and can help elucidate the complex growth patterns exhibited by different microbial species.     

Drosophila Smad2 Opposes Mad Signaling during Wing Vein Development

In the vertebrates, the BMP/Smad1 and TGF-β/Smad2 signaling pathways execute antagonistic functions in different contexts of development. The differentiation of specific structures results from the balance between these two pathways. For example, the gastrula organizer/node of the vertebrates requires a region of low Smad1 and high Smad2 signaling. In Drosophila, Mad regulates tissue determination and growth in the wing, but the function of dSmad2 in wing patterning is largely unknown. In this study, we used an RNAi loss-of-function approach to investigate dSmad2 signaling during wing development. RNAi-mediated knockdown of dSmad2 caused formation of extra vein tissue, with phenotypes similar to those seen in Dpp/Mad gain-of-function. Clonal analyses revealed that the normal function of dSmad2 is to inhibit the response of wing intervein cells to the extracellular Dpp morphogen gradient that specifies vein formation, as measured by expression of the activated phospho-Mad protein. The effect of dSmad2 depletion in promoting vein differentiation was dependent on Medea, the co-factor shared by Mad and dSmad2. Furthermore, double RNAi experiments showed that Mad is epistatic to dSmad2. In other words, depletion of Smad2 had no effect in Mad-deficient wings. Our results demonstrate a novel role for dSmad2 in opposing Mad-mediated vein formation in the wing. We propose that the main function of dActivin/dSmad2 in Drosophila wing development is to antagonize Dpp/Mad signaling. Possible molecular mechanisms for the opposition between dSmad2 and Mad signaling are discussed.     

Rabl2 GTP hydrolysis licenses BBSome‐mediated export to fine‐tune ciliary signaling

Cilia of higher animals sense various environmental stimuli. Proper ciliary signaling requires appropriate extent of BBSome‐mediated export of membrane receptors across ciliary barrier transition zone (TZ) through retrograde intraflagellar transport (IFT) machinery. How the barrier passage is controlled, however, remains unknown. Here, we show that small GTPase Rabl2 functions as a molecular switch for the outward TZ passage. Rabl2‐GTP enters cilia by binding to IFT‐B complex. Its GTP hydrolysis enables the outward TZ passage of the BBSome and its cargos with retrograde IFT machinery, whereas its persistent association leads to their shedding from IFT‐B during the passing process and consequently ciliary retention. Rabl2 deficiency or expression of a GTP‐locked mutant impairs the ciliary hedgehog signaling without interfering with ciliation and respectively results in different spectrums of mouse developmental disorders. We propose that the switch role of Rabl2 ensures proper turnover of the BBSome and ciliary membrane receptors to fine‐tune cilia‐dependent signaling for normal embryonic development and organismic homeostasis.     

Additive genetic variation,but not temperature,influences warning signal expression in Amata nigriceps moths (Lepidoptera: Arctiinae)

Many aposematic species show variation in their color patterns even though selection by predators is expected to stabilize warning signals toward a common phenotype. Warning signal variability can be explained by trade‐offs with other functions of coloration, such as thermoregulation, that may constrain warning signal expression by favoring darker individuals. Here, we investigated the effect of temperature on warning signal expression in aposematic Amata nigriceps moths that vary in their black and orange wing patterns. We sampled moths from two flight seasons that differed in the environmental temperatures and also reared different families under controlled conditions at three different temperatures. Against our prediction that lower developmental temperatures would reduce the warning signal size of the adult moths, we found no effect of temperature on warning signal expression in either wild or laboratory‐reared moths. Instead, we found sex‐ and population‐level differences in wing patterns. Our rearing experiment indicated that ~70% of the variability in the trait is genetic but understanding what signaling and non‐signaling functions of wing coloration maintain the genetic variation requires further work. Our results emphasize the importance of considering both genetic and plastic components of warning signal expression when studying intraspecific variation in aposematic species.