Whole-body galactose oxidation as a robust functional assay to assess the efficacy of gene-based therapies in a mouse model of Galactosemia.

Despite the implementation of lifesaving newborn screening programs and a galactose-restricted diet, many patients with classic galactosemia develop long-term debilitating neurological deficits and primary ovarian insufficiency. Previously, we showed that the administration of human GALT mRNA predominantly expressed in the GalT gene-trapped mouse liver augmented the expression of hepatic GALT activity, which decreased not only galactose-1 phosphate (gal-1P) in the liver but also peripheral tissues. Since each peripheral tissue requires distinct methods to examine the biomarker and/or GALT effect, this highlights the necessity for alternative strategies to evaluate the overall impact of therapies. In this study, we established that whole-body galactose oxidation (WBGO) as a robust, noninvasive, and specific method to assess the in vivo pharmacokinetic and pharmacodynamic parameters of two experimental gene-based therapies that aimed to restore GALT activity in a mouse model of galactosemia. Although our results illustrated the long-lasting efficacy of AAVrh10-mediated GALT gene transfer, we found that GALT mRNA therapy that targets the liver predominantly is sufficient to sustain WBGO. The latter could have important implications in the design of novel targeted therapy to ensure optimal efficacy and safety.

CO2 scrubbing, zero gases, Keeling plots, and a mathematical approach to ameliorate the deleterious effects of ambient CO2 during 13 C breath testing in humans and animals.

13 C breath testing is increasingly used in physiology and ecology research because of what it reveals about the different fuels that animals oxidize to meet their energetic demands. Here I review the practice of 13 C breath testing in humans and other animals and describe the impact that contamination by ambient/background CO2 in the air can have on the accuracy of 13 C breath measurements. I briefly discuss physical methods to avoid sample contamination as well as the Keeling plot approach that researchers have been using for the past two decades to estimate δ13 C from breath samples mixed with ambient CO2 . Unfortunately, Keeling plots are not suited for 13 C breath testing in common situations where (1) a subject's VCO2 is dynamic, (2) ambient [CO2 ] may change, (3) a subject is sensitive to hypercapnia, or (4) in any flow-through indirect calorimetry system. As such, I present a mathematical solution that addresses these issues by using information about the instantaneous [CO2 ] and the δ13 CO2 of ambient air as well as the diluted breath sample to back-calculate the δ13 CO2 in the CO2 exhaled by the animal. I validate this approach by titrating a sample of 13 C-enriched gas into an air stream and demonstrate its ability to provide accurate values across a wide range of breath and air mixtures. This approach allows researchers to instantaneously calculate the δ13 C of exhaled gas of humans or other animals in real time without having to scrub ambient CO2 or rely on estimated values.

Resting metabolic rates increase with elevation in a mountain-dwelling lizard.

Individuals that inhabit broad elevational ranges may experience unique environmental challenges. Because temperature decreases with increased elevation, the ectotherms living at high elevations have to manage limited activity time and high thermoregulatory effort. The resting metabolic rate (RMR) of a postabsorptive animal is related to its total energy requirements as well as many other fitness traits. Mesquite lizards (Sceloporus grammicus) living on La Malinche Volcano, Mexico, inhabit a wide elevational range with some populations apparently thriving above the tree line. We measured the RMR of lizards from different elevations (i.e., 2,600, 3,200, and 4,100 m) at four ecologically relevant temperatures (i.e., 15, 25, 30, and 35 °C) and found that RMR of mesquite lizards increased with temperature and body mass. More importantly, lizards from the high-elevation population had mass specific RMR that was higher at all temperatures. While the higher RMRs of high-elevation populations imply higher metabolic costs at a given temperature these lizards were also smaller. Both of these traits may allow these high elevation populations to thrive in the face of the thermal challenges imposed by their environment.

Environmental temperature alters the overall digestive energetics and differentially affects dietary protein and lipid use in a lizard.

Processing food (e.g. ingestion, digestion, assimilation) requires energy referred to as specific dynamic action (SDA) and is at least partially fuelled by oxidation of the nutrients (e.g. proteins and lipids) within the recently ingested meal. In ectotherms, environmental temperature can affect the magnitude and/or duration of the SDA, but is likely to also alter the mixture of nutrients that are oxidized to cover these costs. Here, we examined metabolic rate, gut passage time, assimilation efficiency and fuel use in the lizard Agama atra digesting cricket meals at three ecologically relevant temperatures (20, 25 and 32°C). Crickets were isotopically enriched with 13C-leucine or 13C-palmitic-acid tracers to distinguish between protein and lipid oxidation, respectively. Our results show that higher temperatures increased the magnitude of the SDA peak (by 318% between 32 and 20°C) and gut passage rate (63%), and decreased the duration of the SDA response (by 20% for males and 48% for females). Peak rate of dietary protein oxidation occurred sooner than peak lipid oxidation at all temperatures (70, 60 and 31 h earlier for 20, 25 and 32°C, respectively). Assimilation efficiency of proteins, but not lipids, was positively related to temperature. Interestingly, the SDA response exhibited a notable circadian rhythm. These results show that temperature has a pronounced effect on digestive energetics in A.atra, and that this effect differs between nutrient classes. Variation in environmental temperatures may thus alter the energy budget and nutrient reserves of these animals.

Consequences of prey exoskeleton content for predator feeding and digestion: black widow predation on larval versus adult mealworm beetles.

Predators often feed on a wide range of prey that can vary in behavior, morphology, and physiology. The net benefits that predators gain from prey are likely related to both prey nutrient content and prey morphology or defenses. For invertebrates, the exoskeleton is a morphological trait that varies widely among species and during ontogeny and could affect nutrient extraction by predators. The goal of this study was to determine how prey exoskeleton content affected predator nutrient intake, assimilation, and excretion by comparing spiders feeding on either larval or adult mealworms of similar size. We found that the proportion of prey energy invested in digestion was greatest in spiders consuming adult mealworm beetles which had higher amounts of exoskeleton than larvae. Further, spiders extracted a greater proportion of elements, macronutrients, and energy from the larval mealworms, which had lower amounts of exoskeleton. Interestingly, total nitrogen content of prey was not a predictor of nitrogen assimilation as spiders assimilated more nitrogen from the larval mealworms, which had lower total nitrogen content. While adult beetles had higher total nitrogen content, their discarded remains of prey had large amounts of nitrogen that was nutritionally unavailable for spiders (i.e., exoskeleton). These results suggest that prey exoskeleton can affect assimilation efficiency by predators, and that a combination of macronutrient and elemental analyses may be needed to examine the quality of prey for predators and the potential consequences of predation for nutrient flows (e.g., consumer assimilation, egestion, and excretion) in ecosystems.

Progressive behavioural, physiological and transcriptomic shifts over the course of prolonged starvation in ticks.

Ticks are obligatorily hematophagous but spend the majority of their lives off host in an unfed state where they must resist starvation between bouts of blood feeding. Survival during these extended off-host periods is critical to the success of these arthropods as vectors of disease; however, little is known about the underlying physiological and molecular mechanisms of starvation tolerance in ticks. We examined the bioenergetic, transcriptomic and behavioural changes of female American dog ticks, Dermacentor variabilis, throughout starvation (up to nine months post-bloodmeal). As starvation progressed, ticks utilized glycogen and lipid, and later protein as energy reserves with proteolysis and autophagy facilitating the mobilization of endogenous nutrients. The metabolic rate of the ticks was expectedly low, but showed a slight increase as starvation progressed possibly reflecting the upregulation of several energetically costly processes such as transcription/translation and/or increases in host-seeking behaviours. Starved ticks had higher activity levels, increased questing behaviour and augmented expression of genes related to chemosensing, immunity and salivary gland proteins. The shifts in gene expression and associated behavioural and physiological processes are critical to allowing these parasites to exploit their ecological niche as extreme sit-and-wait parasites. The overall responses of ticks to starvation were similar to other blood-feeding arthropods, but we identified unique responses that could have epidemiological and ecological significance for ticks as ectoparasites that must be tolerant of sporadic feeding.

Learning to starve: impacts of food limitation beyond the stress period.

Starvation is common among wild animal populations, and many individuals experience repeated bouts of starvation over the course of their lives. Although much information has been gained through laboratory studies of acute starvation, little is known about how starvation affects an animal once food is again available (i.e. during the refeeding and recovery phases). Many animals exhibit a curious phenomenon - some seem to 'get better' at starving following exposure to one or more starvation events - by this we mean that they exhibit potentially adaptive responses, including reduced rates of mass loss, reduced metabolic rates, and lower costs of digestion. During subsequent refeedings they may also exhibit improved digestive efficiency and more rapid mass gain. Importantly, these responses can last until the next starvation bout or even be inherited and expressed in the subsequent generation. Currently, however, little is known about the molecular regulation and physiological mechanisms underlying these changes. Here, we identify areas of research that can fill in the most pressing knowledge gaps. In particular, we highlight how recently refined techniques (e.g. stable isotope tracers, quantitative magnetic resonance and thermal measurement) as well as next-generation sequencing approaches (e.g. RNA-seq, proteomics and holobiome sequencing) can address specific starvation-focused questions. We also describe outstanding unknowns ripe for future research regarding the timing and severity of starvation, and concerning the persistence of these responses and their interactions with other ecological stressors.

Maintenance of Distal Intestinal Structure in the Face of Prolonged Fasting: A Comparative Examination of Species From Five Vertebrate Classes.

It was recently shown that fasting alters the composition of microbial communities residing in the distal intestinal tract of animals representing five classes of vertebrates [i.e., fishes (tilapia), amphibians (toads), reptiles (leopard geckos), birds (quail), and mammals (mice)]. In this study, we tested the hypothesis that the extent of tissue reorganization in the fasted distal intestine was correlated with the observed changes in enteric microbial diversity. Segments of intestine adjacent to those used for the microbiota study were examined histologically to quantify cross-sectional and mucosal surface areas and thicknesses of mucosa, submucosa, and tunica muscularis. We found no fasting-induced differences in the morphology of distal intestines of the mice (3 days), quail (7 days), or geckos (28 days). The toads, which exhibited a general increase in phylogenetic diversity of their enteric microbiota with fasting, also exhibited reduced mucosal circumference at 14 and 21 days of fasting. Tilapia showed increased phylogenetic diversity of their enteric microbiota, and showed a thickened tunica muscularis at 21 days of fasting; but this morphological change was not related to microbial diversity or absorptive surface area, and thus, is unlikely to functionally match the changes in their microbiome. Given that fasting caused significant increases and reductions in the enteric microbial diversity of mice and quail, respectively, but no detectable changes in distal intestine morphology, we conclude that reorganization is not the primary factor shaping changes in microbial diversity within the fasted colon, and the observed modest structural changes are more related to the fasted state. Anat Rec, 300:2208-2219, 2017. © 2017 Wiley Periodicals, Inc.

Life-extending dietary restriction and ovariectomy each increase leucine oxidation and alter leucine allocation in grasshoppers.

Reduced reproduction and dietary restriction each extend lifespan in many animal models, but possible contributions of nutrient oxidation and allocation are largely unknown. Ovariectomy and eating 70% of ad libitum-feeding each extend lifespan in lubber grasshoppers. When feeding levels between the two groups are matched, ovariectomy increases fat and protein storage while dietary restriction reduces fat storage. Because of these disparities in nutrient investment, metabolism may differ between these two life-extending treatments. Therefore, we examined the allocation and organismal oxidation of one representative of each macronutrient class: leucine, oleic acid, and glucose. Ovariectomy and dietary restriction each increased oxidation of dietary leucine. Dietary leucine may play a special role in aging because amino acids stimulate cellular growth. Speeding oxidation of leucine may attenuate cellular growth. Allocation of leucine to muscle was the clearest difference between ovariectomy and dietary restriction in this study. Ovariectomy reduced allocation of leucine to femur muscle, whereas dietary restriction increased allocation of leucine to femur muscle. This allocation likely corresponds to muscle maintenance for locomotion, suggesting dietary restriction increases support for locomotion, perhaps to search for food. Last, ovariectomy decreased oxidation of dietary oleic acid and glucose, perhaps to save them for storage, but the site of storage is unclear. This study suggests that the oxidation of branched-chain amino acids may be an underappreciated mechanism underlying lifespan extension.

Exogenous stress hormones alter energetic and nutrient costs of development and metamorphosis.

Variation in environmental conditions during larval life stages can shape development during critical windows and have lasting effects on the adult organism. Changes in larval developmental rates in response to environmental conditions, for example, can trade off with growth to determine body size and condition at metamorphosis, which can affect adult survival and fecundity. However, it is unclear how use of energy and nutrients shape trade-offs across life-stage transitions because no studies have quantified these costs of larval development and metamorphosis. We used an experimental approach to manipulate physiological stress in larval amphibians, along with respirometry and 13C-breath testing to quantify the energetic and nutritional costs of development and metamorphosis. Central to larval developmental responses to environmental conditions is the hypothalamic-pituitary-adrenal/interrenal (HPA/I) axis, which regulates development, as well as energy homeostasis and stress responses across many taxa. Given these pleiotropic effects of HPA/I activity, manipulation of the HPA/I axis may provide insight into costs of metamorphosis. We measured the energetic and nutritional costs across the entire larval period and metamorphosis in a larval amphibian exposed to exogenous glucocorticoid (GC) hormones - the primary hormone secreted by the HPA/I axis. We measured metabolic rates and dry mass across larval ontogeny, and quantified lipid stores and nutrient oxidation via 13C-breath testing during metamorphosis, under control and GC-exposed conditions. Changes in dry mass match metamorphic states previously reported in the literature, but dynamics of metabolism were influenced by the transition from aquatic to terrestrial respiration. GC-treated larvae had lower dry mass, decreased fat stores and higher oxygen consumption during stages where controls were conserving energy. GC-treated larvae also oxidized greater amounts of 13C-labelled protein stores. These results provide evidence for a proximate cause of the physiological trade-off between larval growth and development, and provide insight into the energetic and nutrient costs that shape fitness trade-offs across life stages.

Sex differences in the utilization of essential and non-essential amino acids in Lepidoptera.

The different reproductive strategies of males and females underlie differences in behavior that may also lead to differences in nutrient use between the two sexes. We studied sex differences in the utilization of two essential amino acids (EAAs) and one non-essential amino acid (NEAA) by the Carolina sphinx moth (Manduca sexta). On day one post-eclosion from the pupae, adult male moths oxidized greater amounts of larva-derived AAs than females, and more nectar-derived AAs after feeding. After 4 days of starvation, the opposite pattern was observed: adult females oxidized more larva-derived AAs than males. Adult males allocated comparatively small amounts of nectar-derived AAs to their first spermatophore, but this allocation increased substantially in the second and third spermatophores. Males allocated significantly more adult-derived AAs to their flight muscle than females. These outcomes indicate that adult male and female moths employ different strategies for allocation and oxidation of dietary AAs.

Dehydration Causes Increased Reliance on Protein Oxidation in Mice: A Test of the Protein-for-Water Hypothesis in a Mammal.

During fasting, animals rely on a mixture of fats, carbohydrates, and proteins that are derived solely from endogenous sources. The relative contributions of these metabolic fuels chiefly depend on the duration of the fast, but other factors including previous diet, environmental temperature, and activity level can modulate the fuel mixture. It has long been held that endogenous proteins are spared from catabolism until the final stages of prolonged fasting and contribute a significant proportion of energy once the other metabolic fuels have been depleted. However, evidence is mounting that protein is catabolized supplemental to fat metabolism under some circumstances. This has been shown in migratory birds that exhibit dramatic reductions in lean mass during flights. One hypothesis to explain this seemingly maladaptive metabolic strategy is that the catabolism and oxidation of protein in situ yields five times more metabolic water than that generated through fat oxidation alone. Recent support for this hypothesis is that birds benefit from such a strategy due to their uricotelic nature. However, it remains unclear whether ureotelic mammals would also employ this strategy. Here we test the protein-for-water hypothesis in resting laboratory mice subjected to water deprivation during fasting while we tracked rates of protein and lipid catabolism using endogenously incorporated 13C-leucine and 13C-palmitic acid. We found no differences in instantaneous leucine oxidation; however, cumulative differences in instantaneous leucine oxidation ultimately resulted in a higher total leucine oxidation after 72 h of fasting in water-deprived animals. We also found that lipid oxidation was 8% higher in the hydrated mice, but the difference was not significant presumably because of a concomitant reduction in metabolic rates of the water-deprived mice. Our results indicate that mammals do increase rates of protein catabolism during dehydration but to a lesser degree than birds. The ability of mammals to produce highly concentrated urine and their lower inherent rates of protein turnover apparently preclude mammals from taking full advantage of the protein-for-water strategy during fasting under dehydrating conditions.

More than just sugar: allocation of nectar amino acids and fatty acids in a Lepidopteran.

The ability to allocate resources, even when limited, is essential for survival and fitness. We examine how nutrients that occur in minute amounts are allocated among reproductive, somatic, and metabolic demands. In addition to sugar, flower nectars contain two macronutrients-amino acids and fatty acids. We created artificial nectars spiked with 13C-labelled amino acids and fatty acids and fed these to adult moths (Manduca sexta: Sphingidae) to understand how they allocate these nutrients among competing sinks (reproduction, somatic tissue, and metabolic fuel). We found that both essential and non-essential amino acids were allocated to eggs and flight muscles and were still detectable in early-instar larvae. Parental-derived essential amino acids were more conserved in the early-instars than non-essential amino acids. All amino acids were used as metabolic fuel, but the non-essential amino acids were oxidized at higher rates than essential amino acids. Surprisingly, the nectar fatty acids were not vertically transferred to offspring, but were readily used as a metabolic fuel by the moth, minimizing losses of endogenous nutrient stores. We conclude that the non-carbohydrate components of nectar may play important roles in both reproductive success and survival of these nectar-feeding animals.

Previous Repeated Exposure to Food Limitation Enables Rats to Spare Lipid Stores during Prolonged Starvation.

The risk of food limitation and, ultimately, starvation dates back to the dawn of heterotrophy in animals, yet starvation remains a major factor in the regulation of modern animal populations. Researchers studying starvation more than a century ago suggested that animals subjected to sublethal periods of food limitation are somehow more tolerant of subsequent starvation events. This possibility has received little attention over the past decades, yet it is highly relevant to modern science for two reasons. First, animals in natural populations are likely to be exposed to bouts of food limitation once or more before they face prolonged starvation, during which the risk of mortality becomes imminent. Second, our current approach to studying starvation physiology in the laboratory focuses on nourished animals with no previous exposure to nutritional stress. We examined the relationship between previous exposure to food limitation and potentially adaptive physiological responses to starvation in adult rats and found several significant differences. On two occasions, rats were fasted until they lost 20% of their body mass maintained lower body temperatures, and had presumably lower energy requirements when subjected to prolonged starvation than their naive cohort that never experienced food limitation. These rats that were trained in starvation also had lower plasma glucose set -points and reduced their reliance on endogenous lipid oxidation. These findings underscore (1) the need for biologists to revisit the classic hypothesis that animals can become habituated to starvation, using a modern set of research tools; and (2) the need to design controlled experiments of starvation physiology that more closely resemble the dynamic nature of food availability.

Cold adaptation increases rates of nutrient flow and metabolic plasticity during cold exposure in Drosophila melanogaster.

Metabolic flexibility is an important component of adaptation to stressful environments, including thermal stress and latitudinal adaptation. A long history of population genetic studies suggest that selection on core metabolic enzymes may shape life histories by altering metabolic flux. However, the direct relationship between selection on thermal stress hardiness and metabolic flux has not previously been tested. We investigated flexibility of nutrient catabolism during cold stress in Drosophila melanogaster artificially selected for fast or slow recovery from chill coma (i.e. cold-hardy or -susceptible), specifically testing the hypothesis that stress adaptation increases metabolic turnover. Using (13)C-labelled glucose, we first showed that cold-hardy flies more rapidly incorporate ingested carbon into amino acids and newly synthesized glucose, permitting rapid synthesis of proline, a compound shown elsewhere to improve survival of cold stress. Second, using glucose and leucine tracers we showed that cold-hardy flies had higher oxidation rates than cold-susceptible flies before cold exposure, similar oxidation rates during cold exposure, and returned to higher oxidation rates during recovery. Additionally, cold-hardy flies transferred compounds among body pools more rapidly during cold exposure and recovery. Increased metabolic turnover may allow cold-adapted flies to better prepare for, resist and repair/tolerate cold damage. This work illustrates for the first time differences in nutrient fluxes associated with cold adaptation, suggesting that metabolic costs associated with cold hardiness could invoke resource-based trade-offs that shape life histories.

The postabsorptive and postprandial metabolic rates of praying mantises: Comparisons across species, body masses, and meal sizes.

The metabolic rate of an animal affects the amount of energy available for its growth, activity and reproduction and, ultimately, shapes how energy and nutrients flow through ecosystems. Standard metabolic rate (SMR; when animals are post-absorptive and at rest) and specific dynamic action (SDA; the cost of digesting and processing food) are two major components of animal metabolism. SMR has been studied in hundreds of species of insects, but very little is known about the SMR of praying mantises. We measured the rates of CO2 production as a proxy for metabolic rate and tested the prediction that the SMR of mantises more closely resembles the low SMR of spiders - a characteristic generally believed to be related to their sit-and-wait foraging strategy. Although few studies have examined SDA in insects we also tested the prediction that mantises would exhibit comparatively large SDA responses characteristic of other types of predators (e.g., snakes) known to consume enormous, protein-rich meals. The SMR of the mantises was positively correlated with body mass and did not differ among the four species we examined. Their SMR was best described by the equation µW=1526*g0.745 and was not significantly different from that predicted by the standard 'insect-curve'; but it was significantly higher than that of spiders to which mantises are ecologically more similar than other insects. Mantises consumed meals as large as 138% of their body mass and within 6-12h of feeding and their metabolic rates doubled before gradually returning to prefeeding rates over the subsequent four days. We found that the SDA responses were isometrically correlated with meal size and the relative cost of digestion was 38% of the energy in each meal. We conclude that mantises provide a promising model to investigate nutritional physiology of insect predators as well as nutrient cycling within their ecological communities.

The speed and metabolic cost of digesting a blood meal depends on temperature in a major disease vector.

The energetics of processing a meal is crucial for understanding energy budgets of animals in the wild. Given that digestion and its associated costs may be dependent on environmental conditions, it is necessary to obtain a better understanding of these costs under diverse conditions and identify resulting behavioural or physiological trade-offs. This study examines the speed and metabolic costs - in cumulative, absolute and relative energetic terms - of processing a bloodmeal for a major zoonotic disease vector, the tsetse fly Glossina brevipalpis, across a range of ecologically relevant temperatures (25, 30 and 35°C). Respirometry showed that flies used less energy digesting meals faster at higher temperatures but that their starvation tolerance was reduced, supporting the prediction that warmer temperatures are optimal for bloodmeal digestion while cooler temperatures should be preferred for unfed or post-absorptive flies. (13)C-Breath testing revealed that the flies oxidized dietary glucose and amino acids within the first couple of hours of feeding and overall oxidized more dietary nutrients at the cooler temperatures, supporting the premise that warmer digestion temperatures are preferred because they maximize speed and minimize costs. An independent test of these predictions using a thermal gradient confirmed that recently fed flies selected warmer temperatures and then selected cooler temperatures as they became post-absorptive, presumably to maximize starvation resistance. Collectively these results suggest there are at least two thermal optima in a given population at any time and flies switch dynamically between optima throughout feeding cycles.

(13)C-Breath testing in animals: theory, applications, and future directions.

The carbon isotope values in the exhaled breath of an animal mirror the carbon isotope values of the metabolic fuels being oxidized. The measurement of stable carbon isotopes in carbon dioxide is called (13)C-breath testing and offers a minimally invasive method to study substrate oxidation in vivo. (13)C-breath testing has been broadly used to study human exercise, nutrition, and pathologies since the 1970s. Owing to reduced use of radioactive isotopes and the increased convenience and affordability of (13)C-analyzers, the past decade has witnessed a sharp increase in the use of breath testing throughout comparative physiology--especially to answer questions about how and when animals oxidize particular nutrients. Here, we review the practical aspects of (13)C-breath testing and identify the strengths and weaknesses of different methodological approaches including the use of natural abundance versus artificially-enriched (13)C tracers. We critically compare the information that can be obtained using different experimental protocols such as diet-switching versus fuel-switching. We also discuss several factors that should be considered when designing breath testing experiments including extrinsic versus intrinsic (13)C-labelling and different approaches to model nutrient oxidation. We use case studies to highlight the myriad applications of (13)C-breath testing in basic and clinical human studies as well as comparative studies of fuel use, energetics, and carbon turnover in multiple vertebrate and invertebrate groups. Lastly, we call for increased and rigorous use of (13)C-breath testing to explore a variety of new research areas and potentially answer long standing questions related to thermobiology, locomotion, and nutrition.

Carbon stable-isotope tracking in breath for comparative studies of fuel use.

Almost half a century ago, researchers demonstrated that the ratio of stable carbon isotopes in exhaled breath of rats and humans could reveal the oxidation of labeled substrates in vivo, opening a new chapter in the study of fuel use, the fate of ingested substrates, and aerobic metabolism. Until recently, the combined use of respirometry and stable-isotope tracer techniques had not been broadly employed to study fuel use in other animal groups. In this review, we summarize the history of this approach in human and animal research and define best practices that maximize its utility. We also summarize several case studies that use stable-isotope measurements of breath to explore the limits of aerobic metabolism and substrate turnover among several species and various physiological states. We highlight the importance of a comparative approach in revealing the profound effects that phylogeny, ecology, and behavior can have in shaping aerobic metabolism and energetics as well as the fundamental biological principles that underlie fuel use and metabolic function across taxa. New analytical equipment and refinement of methodology make the combined use of respirometry and stable-isotope tracer techniques simpler to perform, less costly, and more field ready than ever before.

How and When Do Insects Rely on Endogenous Protein and Lipid Resources during Lethal Bouts of Starvation? A New Application for 13C-Breath testing.

Most of our understanding about the physiology of fasting and starvation comes from studies of vertebrates; however, for ethical reasons, studies that monitor vertebrates through the lethal endpoint are scant. Insects are convenient models to characterize the comparative strategies used to cope with starvation because they have diverse life histories and have evolved under the omnipresent challenge of food limitation. Moreover, we can study the physiology of starvation through its natural endpoint. In this study we raised populations of five species of insects (adult grasshoppers, crickets, cockroaches, and larval beetles and moths) on diets labeled with either 13C-palmitic acid or 13C-leucine to isotopically enrich the lipids or the proteins in their bodies, respectively. The insects were allowed to become postabsorptive and then starved. We periodically measured the δ13C of the exhaled breath to characterize how each species adjusted their reliance on endogenous lipids and proteins as energy sources. We found that starving insects employ a wide range of strategies for regulating lipid and protein oxidation. All of the insects except for the beetle larvae were capable of sharply reducing reliance on protein oxidation; however, this protein sparing strategy was usually unsustainable during the entire starvation period. All insects increased their reliance on lipid oxidation, but while some species (grasshoppers, cockroaches, and beetle larvae) were still relying extensively on lipids at the time of death, other species (crickets and moth larvae) allowed rates of lipid oxidation to return to prestarvation levels. Although lipids and proteins are critical metabolic fuels for both vertebrates and insects, insects apparently exhibit a much wider range of strategies for rationing these limited resources during starvation.