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The peroxisome proliferator-activated receptor γ coactivator 1 α (PGC-1α) is central to the regulation of cellular and mitochondrial energy homeostasis in mammals, but its role in other vertebrates remains unclear. Indeed, previous work suggests extensive structural and functional divergence of PGC-1α in teleosts but this remains to be directly tested. Here, we describe the initial characterization of heterozygous PGC-1α mutant zebrafish lines created by CRISPR-Cas9 disruptions of an evolutionarily conserved regulatory region of the PGC-1α proximal promoter. Using qPCR, we confirmed the disruption of PGC-1α gene expression in striated muscle, leading to a simultaneous fourfold increase in mixed skeletal muscle PGC-1α mRNA levels and an opposite fourfold downregulation in cardiac muscle. In mixed skeletal muscle, most downstream effector genes were largely unaffected yet two mitochondrial lipid transporters, carnitine palmitoyltransferase-1 and -2, were strongly induced. Conversely, PGC-1α depression in cardiac muscle reduced the expression of several transcriptional regulators (estrogen-related receptor α, nuclear respiratory factor 1, and PGC-1β) without altering metabolic gene expression. Using high-resolution respirometry, we determined that white muscle exhibited increased lipid oxidative capacity with little difference in markers of mitochondrial abundance. Finally, using whole animal intermittent respirometry, we show that mutant fish exhibit a twofold higher basal metabolism than their wild-type counterparts. Altogether, this new model confirms a central but complex role for PGC-1α in mediating energy utilization in zebrafish, and we propose its use as a valuable tool to explore the intricate regulatory pathways of energy homeostasis in a popular biomedical model.

Black queen cell virus (BQCV) is a ubiquitous honeybee virus and a significant pathogen to queen bee (Apis mellifera) larvae. However, many aspects of the virus remain poorly understood, including the transmission dynamics. In this study, we used next-generation sequencing to identify BQCV in Aedes vexans (n = 4,000) collected in 2019 and 2020 from Manitoba, Canada. We assembled de novo the nearly complete (>96%) genome sequence of the virus, which is the first available from North America and the first report of BQCV being harbored by mosquitoes. Phylogenetic tree reconstructions indicated that the genome had 95.5% sequence similarity to a BQCV isolate from Sweden. Sequences of a potential vector (Varroa destructor) and a microsporidian associated with BQCV (Nosema apis) were not identified in the mosquito samples, however, we did detect sequences of plant origin. We, therefore, hypothesize that the virus was indirectly acquired by mosquitoes foraging at the same nectar sources as honeybees.

Larvae of the greater wax moth (

Larvae of the greater wax moth (Galleria mellonella) possess the remarkable ability to consume and rapidly degrade low-density polyethylene. Previous studies have investigated the involvement of the animal's microbiome, but little is known about the host's actual role and if it benefits from biodegradation of this synthetic polymer. We used a combination of RNA sequencing and biochemical approaches to assess caterpillars fed honeycomb, fed polyethylene (PE), or starved for up to 72 h. Sequencing of gut transcripts revealed PE-fed larvae retain an expression profile consistent with normal intestinal function but also show distinct molecular signatures indicative of enhanced fatty acid metabolism (FAM). Further, quantification of total lipid content validated the impact of a PE diet on FAM; in contrast to lipid-depleted starved animals, PE-fed caterpillars maintain lipid reserves similar to honeycomb-fed larvae. Additionally, we found the activity of putative enzymes involved in lipid oxidation (e.g., alcohol dehydrogenase) are considerably higher in PE-fed larvae, indicating that on a functional level, these caterpillars are inducing pathways to effectively metabolize PE. Overall, we put forward a hypothesized model where the similarity in chemical structure between PE and its natural honeycomb diet has endowed larvae of G. mellonella with the extraordinary capability to derive energy from PE as an exclusive food source through pre-existing metabolic pathways.

The caterpillar larvae of the greater wax moth (Galleria mellonella) are avid plastivores, as when provided a diet of low-density polyethylene (LDPE) they actively feed on it. Recent work has highlighted the importance of their microbiome in the putative biodegradation of this plastic polymer, though the impact of plastic metabolism on the insect host is less clear. In the present study, we undertook an integrative approach spanning all levels of biological organization to explore the effects of a plastic diet on the metabolic physiology of this animal model of plastic biodegradation. We demonstrate that an LDPE diet is not sufficient to maintain optimal larval growth and survival. In addition, we confirm that plastic fed waxworms retain their fat body lipid stores in a manner proportional to their individual polyethylene consumption suggesting a direct effect of LDPE biodegradation. At the functional level, the oxidative capacity of the fat body of LDPE-fed larvae is maintained reflecting unaltered metabolic function of the tissue. Finally, metabolomic analyses confirmed fat body lipid stores maintenance in LDPE-fed worms, but uncovered various other nutritional deficiencies. Overall, this work unveils novel insights in the complex interplay between LDPE biodegradation and the metabolic physiology of this model plastivore.

For more than 50 years DEET (N,N-Diethyl-m-toluamide) has been considered the gold standard of repellents. It is applied to the skin or clothing to deter mosquitoes and other blood-sucking invertebrate pests from approaching and/or settling, and ultimately it provides temporary protection from bites. Despite rampant global use, surprisingly little is understood about DEET’s mode of action and the molecular targets of the active ingredient. Furthermore, the theories into its mechanism for repellency are largely based off fruit fly and mosquito research. Since ticks possess a unique sensory structure, the Haller’s organ, the specific genes and pathways associated with DEET avoidance may differ from insects. In these studies, we collected American dog ticks (Dermacentor variabilis) from four natural populations within Manitoba, Canada. We first carried out behavior assays, which showed DEET effectively repelled the ticks. RNA sequencing revealed that DEET caused a rapid and substantial reduction in the abundance of transcripts encoding cytochrome P450 and acetylcholinesterase genes, which gradually recovered over the 24 h time course. Finally, enzymatic kinetics provided functional support for DEET’s role as an effective inhibitor of P450 s. While many facets of its mode of action remain to be worked out, our study provides valuable insights into the molecular underpinnings of DEET’s repellence in ticks.

International audience; The life cycle of gobies of the Sicydiinae subfamily depends on climbing waterfalls. Two sympatric sicydiines species from Reunion Island, Sicyopterus lagocephalus (SIL) and Cotylopus acutipinnis (COA), employ different climbing modes. SIL uses a steady “inching” mode interrupted by short rest periods, whereas COA exhibits short “power-burst” undulatory movements punctuated by longer rest periods. Consequently, we explored the relationship between climbing performance and metabolic activity in these two species. We demonstrated that the two climbing modes are supported by different ecophysiological profiles that promote the interspecific variability of locomotor performance. More specifically, SIL performed better than COA during a climbing experiment because of its inching climbing mode, supported by a generally greater metabolic capacity and a higher potential for oxidative metabolism. Interestingly, we did not detect any difference in metabolic fuel storage and lactate production during climbing in either species, suggesting that these species can maintain fuel reserves and limit lactate accumulation through extensive rest times. Overall, this study provides new insights into the ecophysiology of these two emblematic species and suggests that the better climbing capacity of SIL is supported by its muscular metabolic capacity.

Recently, a few insects, including the caterpillar larva of the greater wax moth Galleria mellonella , have been identified as avid ‘plastivores’. These caterpillars are able to ingest and metabolize polyethylene at unprecedented rates. While it appears that G. mellonella plays an important role in the biodegradation process, the contribution of its intestinal microbiome remains poorly understood and contested. In a series of experiments, we present strong evidence of an intricate relationship between an intact microbiome, low-density polyethylene (LDPE) biodegradation and the production of glycol as a metabolic by-product. First, we biochemically confirmed that G. mellonella larvae consume and metabolize LDPE, as individual caterpillars fed on polyethylene excreted glycol, but those excretions were reduced by antibiotic treatment. Further, while the gut bacterial communities remained relatively stable regardless of diet, we showed that during the early phases of feeding on LDPE (24–72 h), caterpillars exhibited increased microbial abundance relative to those starved or fed on their natural honeycomb diet. Finally, by isolating and growing gut bacteria with polyethylene as their exclusive carbon source for over 1 year, we identified microorganisms in the genus Acinetobacter that appeared to be involved in this biodegradation process. Taken collectively, our study indicates that during short-term exposure, the intestinal microbiome of G. mellonella is intricately associated with polyethylene biodegradation in vivo .

The Gulf toadfish (Opsanus beta) has a fully functional ornithine urea cycle (O-UC) that allows it to excrete nitrogenous waste in the form of urea. Interestingly, urea is excreted in a pulse across the gill that lasts 1-3h and occurs once or twice a day. Both the stress hormone, cortisol, and the neurotransmitter, serotonin (5-HT) are involved in the control of pulsatile urea excretion. This and other evidence suggests that urea pulsing may be linked to toadfish social behavior. The hypothesis of the present study was that toadfish urea pulses can be triggered by waterborne chemical cues from conspecifics. Our findings indicate that exposure to seawater that held a donor conspecific for up to 48h (pre-conditioned seawater; PC-SW) induced a urea pulse within 7h in naive conspecifics compared to a pulse latency of 20h when exposed to seawater alone. Factors such as PC-SW intensity and donor body mass influenced the pulse latency response of naive conspecifics. Fractionation and heat treatment of PC-SW to narrow possible signal candidates revealed that the active chemical was both water-soluble and heat-stable. Fish exposed to urea, cortisol or 5-HT in seawater did not have a pulse latency that was significantly different than seawater alone; however, ammonia, perhaps in the form of NH4Cl, was found to be a factor in the pulse latency response of toadfish to PC-SW and could be one component of a multi-component cue used for chemical communication in toadfish. Further studies are needed to fully identify the chemical cue as well as determine its adaptive significance in this marine teleost fish.

The Colorado potato beetle (Leptinotarsa decemlineata (Say)) is an important pest of the cultivated potato (Solanum tuberosum (L.) [Solanales: Solanaceae]). With its broad resistance toward commonly used insecticides, it is clear that more sophisticated control strategies are needed. Due to their importance in insect development, microRNAs (miRNAs) represent a potential tool to employ in insect control strategies. However, most studies conducted in this area have focused on model species with well-annotated genomes. In this study, next-generation sequencing was used to catalogue the miRNAs produced by L. decemlineata across all eight stages of its development, from eggs to adults. For most stages, the length of miRNAs peaked between 21 and 22 nt, though it was considerably longer for the egg stage (26 nt). Global profiling of miRNAs revealed three distinct developmental clusters: 1) egg stage; 2) early stage (first, second, and third instar); and 3) late stage (fourth instar, prepupae, pupae, and adult). We identified 86 conserved miRNAs and 33 bonafide novel miRNAs, including stage-specific miRNAs and those not previously identified in L. decemlineata. Most of the conserved miRNAs were found in multiple developmental stages, whereas the novel miRNAs were often stage specific with the bulk identified in the egg stage. The identified miRNAs have a myriad of putative functions, including growth, reproduction, and insecticide resistance. We discuss the putative roles of some of the most notable miRNAs in the regulation of L. decemlineata development, as well as the potential applications of this research in Colorado potato beetle management.

The peroxisome proliferator activated receptor γ coactivator-1 (PGC-1) family is composed of three coactivators whose role in regulating mammalian bioenergetics regulation is clear, but is much less certain in other vertebrates. Current evidence suggests that in fish, PGC-1α and PGC-1β may exhibit much less redundancy in the control of fatty acid oxidation and mitochondrial biogenesis compared to mammals. To assess these roles directly, we knocked down PGC-1α and PGC-1β expression with morpholinos in zebrafish embryos, and we investigated the resulting molecular and physiological phenotypes. First, we found no effects of either morpholinos on larval hatching, heart rates and oxygen consumption over the first few days of development. Next, at 3 days post fertilization (dpf), we confirmed by real time PCR a specific knock down of both coactivators, that resulted in a significant reduction in the transcript levels of citrate synthase (CS), 3-hydroxyacyl-CoA dehydrogenase (HOAD), and medium-chain acyl-coenzyme A dehydrogenase (MCAD) in both morphant groups. However, there was no effect on transcription factors' gene expression except for a marked reduction in estrogen related receptor α (ERRα) transcripts in PGC-1α morphants. Finally, we assessed whole embryonic enzyme activity for CS, cytochrome oxidase (COX), HOAD and carnitine palmitoyltransferase I (CPT-1) at 4 dpf. The only significant effect of the knockdown was a reduced CS activity in PGC-1α morphants and a counterintuitive increase of cytochrome oxidase activity in PGC-1β morphants. Overall, our results indicate that in larval zebrafish, PGC-1α and PGC-1β both play a role in regulating expression of important mitochondrial genes potentially through ERRα.

Over the last few decades, plastic waste has become an increasing environmental concern as it accumulates in every environment on our planet. Though traditionally seen as a macroscopic problem (i.e., large plastic debris), plastic pollution is also evident at smaller scales. Indeed, the intentional industrial production of small plastic particles and the physical degradation of larger plastic debris have overtime resulted in an increased environmental prevalence of smaller plastic particles, including microplastics. While the effects of these small polymers on marine biota have been an important research focus, recent global surveys indicate that our freshwater lakes and rivers are also plagued by microplastics. However, despite these discoveries we currently have a limited understanding of the impact these particles may have on freshwater animals, particularly on vertebrate species. Thus, the aim of the present study was to assess the impact of high concentrations of microplastics (5 and 20 mg.L−1) on the early life stages in zebrafish, a model freshwater vertebrate model. To do this, we exposed embryonic and larval zebrafish to fluorescently labelled polyethylene microspheres for up to 14 days and assessed their microplastic content, growth, hatching and oxygen consumption rates. We then explored the molecular underpinnings of the microplastic response by RNA sequencing. Over the course of the exposure, we observed a consistent accumulation of microplastics in the gastrointestinal tract of the fish in a concentration dependent manner, but could not detect any detrimental effects of these particles on larval development, growth or metabolism. However, whole animal transcriptomics revealed that microplastics induced a transient and extensive change in larval gene expression within 48 h exposure, which largely disappeared by 14 days. However, as these transcriptional changes occurred during a critical period of larval development, we suggest that an evaluation of the potential long-term impact of these particles is warranted.

AMP-activated protein kinase is an enzyme that mediates communication between cellular energy status and diverse effector proteins, particularly those that play roles in determining the metabolic phenotype. By phosphorylating metabolic enzymes, transcriptional regulators and proteins involved in cellular structure, it can modify energy metabolism in both the short term and long term. Its basic features are highly conserved, with homologues in all eukaryotes. Gene and/or genome duplications endowed early vertebrates with paralogs of AMPK subunits, though the nature of their subfunctionalization remains uncertain, even in mammals. While most research focuses on the role of the enzyme in human health, a great deal can be learned from comparative studies targeting non-traditional model animals. Fish, in particular, are interesting models because of the diversity in the metabolic properties and complex relationships between metabolism and environmental challenges. In this review, we examine what is known about AMPK structure and function though the lens of comparative physiology, looking for opportunities to better understand how this vital energy sensor has evolved in animals.

Scientific Reports 6: Article number: 34494; published online: 03 October 2016; updated: 19 June 2017 M. Danielle McDonald was omitted from the author list in the original version of this Article. This has been corrected in the PDF and HTML versions of the Article, as well as the Supplementary Information file that accompanies the Article.

In the summer, the plainfin midshipman (Poricththys notatus) migrates to reproduce in the nearshore environment, where oxygen levels are influenced by the tidal cycles. Parental males establish nests under rocks in the intertidal zone, where they reside until the eggs they guard are fully developed. In contrast, females and sneaker males leave the nests shortly after spawning. We examined the physiological resistance and metabolic response of parental male and female adult midshipman to hypoxia to test whether they exhibited sex-specific differences reflecting their reproductive strategies. Further, we assessed whether metabolic enzymes and metabolites were differentially enriched in tissues of parental males and females to explain the differences observed in their hypoxia tolerance. While parental males and females exhibited similar depression of their oxygen consumption in response to graded hypoxia, parental males could withstand significantly longer exposures to severe hypoxic stress. At the biochemical level, parental males showed higher hepatic glycogen reserves and higher glycolytic enzyme capacities in gills and skeletal muscles than females. Although some of these enzymatic variations could be explained by differences in body size, we also observed a significant effect of sex on some of these factors. These results suggest that parental male midshipman may benefit from sexual dimorphism at the whole-organismal (larger body size) and biochemical (enzyme activities) levels, conferring on them a higher glycolytic potential to sustain the extensive hypoxia bouts they experience in nature.

Prior studies of the elasmobranch rectal gland have demonstrated that feeding induces profound and rapid up regulation of the gland's ability to secrete concentrated NaCl solutions and the metabolic capacity to support this highly ATP consuming process. We undertook the current study to attempt to determine the degree to which up regulation of mRNA transcription was involved in the gland's activation. cDNA libraries were created from mRNA isolated from rectal glands of fasted (7days post-feeding) and fed (6h and 22h post-feeding) spiny dogfish sharks (Squalus acanthias), and the libraries were subjected to suppression subtractive hybridization (SSH) analysis. Quantitative real time PCR (qPCR) was also used to ascertain the mRNA expression of several genes revealed by the SSH analysis. In total the treatments changed the abundance of 170 transcripts, with 103 up regulated by feeding, and 67 up regulated by fasting. While many of the changes took place in 'expected' Gene Ontology (GO) categories (e.g., metabolism, transport, structural proteins, DNA and RNA turnover, etc.), KEGG analysis revealed a number of categories which identify oxidative stress as a topic of interest for the gland. GO analysis also revealed that branched chain essential amino acids (e.g., valine, leucine, isoleucine) are potential metabolic fuels for the rectal gland. In addition, up regulation of transcripts for many genes in the anticipated GO categories did not agree (i.e., fasting down regulated in feeding treatments) with previously observed increases in their respective proteins/enzyme activities. These results suggest an 'anticipatory' storage of selected mRNAs which presumably supports the rapid translation of proteins upon feeding activation of the gland.

Bony fish primarily excrete ammonia as adults however the persistence of urea cycle genes may reflect a beneficial role for urea production during embryonic stages in protecting the embryo from toxic effects of ammonia produced from a highly nitrogenous yolk. This study aimed to examine the dynamic scope for changes in rates of urea synthesis and excretion in one such species (zebrafish, Danio rerio) by manipulating the intrinsic developmental rate (by alteration of light:dark cycles), as well as by direct chemical manipulation via ammonia injection (to potentially activate urea production) and nicotinamide exposure (to potentially inhibit urea production). Continuous dark exposure delayed development in embryos as evidenced by delayed appearance of hallmark anatomical features (heartbeat, eye pigmentation, body pigmentation, lateral line, fin buds) at 30 and 48 hr post-fertilization, as well by a lower hatching rate compared to embryos reared in continuous light. Both ammonia and urea excretion were similarly effected and were generally higher in embryos continuously exposed to light. Ammonia injection resulted in significant increases (up to fourfold) of urea N excretion and no changes to ammonia excretion rates along with modest increases in yolk ammonia content during 2-6 hr post-injection. Nicotinamide (an inhibitor of urea synthesis in mammals) reduced the ammonia-induced increase in urea excretion and led to retention of ammonia in the yolk and body of the embryo. Our results indicate that there is a relatively rapid and large scope for increases in urea production/excretion rates in developing embryos. Potential mechanisms for these increases are discussed.

Marine teleost fish produce CaCO3 in their intestine as part of their osmoregulatory strategy. This precipitation is critical for rehydration and survival of the largest vertebrate group on earth, yet the molecular mechanisms that regulate this reaction are unknown. Here, we isolate and characterize an organic matrix associated with the intestinal precipitates produced by Gulf toadfish (Opsanus beta). Toadfish precipitates were purified using two different methods, and the associated organic matrix was extracted. Greater than 150 proteins were identified in the isolated matrix by mass spectrometry and subsequent database searching using an O. beta transcriptomic sequence library produced here. Many of the identified proteins were enriched in the matrix compared to the intestinal fluid, and three showed no substantial homology to any previously characterized protein in the NCBI database. To test the functionality of the isolated matrix, a micro-modified in vitro calcification assay was designed, which revealed that low concentrations of isolated matrix substantially promoted CaCO3 production, where high concentrations showed an inhibitory effect. High concentrations of matrix also decreased the incorporation of magnesium into the forming mineral, potentially providing an explanation for the variability in magnesium content observed in precipitates produced by different fish species.

Elasmobranch diets consist of high quantities of protein and lipids, but very low levels of carbohydrates including glucose. Reflecting this diet, most tissues use lipids and ketone bodies as their main metabolic fuel. However, the rectal gland has been shown to be dependent on glucose as a fuel, so we hypothesized that glucose transporters (GLUTs) would be present and upregulated in the gland during times of activation (e.g. following a meal). In this study, we searched for and identified putative class I GLUTs in three elasmobranchs and a holocephalan using transcriptomes, and used these to reconstruct a Bayesian phylogeny. We determined that each of the four species possessed three of the four class I GLUT sequences, but the identities of the isoforms present in each species differed between the elasmobranchs (GLUT1, 3 and 4) and the holocephalan (GLUT1, 2 and 3). We then used qPCR to measure mRNA levels of these GLUTs in the rectal gland, liver, intestine, and muscle of fed and starved spiny dogfish (Squalus suckleyi). The rectal gland data showed higher mRNA levels of GLUT4 in the starved relative to the fed fish. In the muscle, both GLUT1 and 4 were significantly elevated at 24 h post-feeding, as was the case for GLUT4 in the liver. In the intestine on the other hand, GLUT4 was significantly elevated by 6 h post-feeding, remaining elevated through 48 h. We suggest that GLUT4 has taken on the role of GLUT2 in elasmobranchs as the expression patterns observed in the liver and intestine are representative of GLUT2 in other vertebrates., Summary: Our results indicate the presence of three putative glucose transporters in elasmobranchs (GLUT1, 3, 4) and holocephalans (GLUT1, 2, 3). We determined that GLUT1 and GLUT4 mRNA levels change in various dogfish tissues in response to feeding.

In mammals, the peroxisome proliferator-activated receptor (PPAR) gamma coactivator-1 (PGC-1) family members and their binding partners orchestrate remodelling in response to diverse challenges such as diet, temperature and exercise. In this study, we exposed goldfish to three temperatures (4, 20 and 35 degrees C) and to three dietary regimes (food deprivation, low fat and high fat) and examined the changes in mitochondrial enzyme activities and transcript levels for metabolic enzymes and their genetic regulators in red muscle, white muscle, heart and liver. When all tissues and conditions were pooled, there were significant correlations between the mRNA for the PGC-1 coactivators (both alpha and beta) and mitochondrial transcripts (citrate synthase), metabolic gene regulators including PPARalpha, PPARbeta and nuclear respiratory factor-1 (NRF-1). PGC-1beta was the better predictor of the NRF-1 axis, whereas PGC-1alpha was the better predictor of the PPAR axis (PPARalpha, PPARbeta, medium chain acyl CoA dehydrogenase). In contrast to these intertissue/developmental patterns, the response of individual tissues to physiological stressors displayed no correlations between mRNA for PGC-1 family members and either the NRF-1 or PPAR axes. For example, in skeletal muscles, low temperature decreased PGC-1alpha transcript levels but increased mitochondrial enzyme activities (citrate synthase and cytochrome oxidase) and transcripts for COX IV and NRF-1. These results suggest that in goldfish, as in mammals, there is a regulatory relationship between (i) NRF-1 and mitochondrial gene expression and (ii) PPARs and fatty acid oxidation gene expression. In contrast to mammals, there is a divergence in the roles of the coactivators, with PGC-1alpha linked to fatty acid oxidation through PPARalpha, and PGC-1beta with a more prominent role in mediating NRF-1-dependent control of mitochondrial gene expression, as well as distinctions between their respective roles in development and physiological responsiveness.

In the two decades since the first cloning of the mammalian kidney urea transporter (UT-A), UT genes have been identified in a plethora of organisms, ranging from single-celled bacteria to metazoans. In this review, focusing mainly on vertebrates, we first reiterate the multiple catabolic and anabolic pathways that produce urea, then we reconstruct the phylogenetic history of UTs, and finally we examine the tissue distribution of UTs in selected vertebrate species. Our analysis reveals that from an ancestral UT, three homologues evolved in piscine lineages (UT-A, UT-C and UT-D), followed by a subsequent reduction to a single UT-A in lobe-finned fish and amphibians. A later internal tandem duplication of UT-A occurred in the amniote lineage (UT-A1), followed by a second tandem duplication in mammals to give rise to UT-B. While the expected UT expression is evident in excretory and osmoregulatory tissues in ureotelic taxa, UTs are also expressed ubiquitously in non-ureotelic taxa, and in tissues without a complete ornithine–urea cycle (OUC). We posit that non-OUC production of urea from arginine by arginase, an important pathway to generate ornithine for synthesis of molecules such as polyamines for highly proliferative tissues (e.g. testis, embryos), and neurotransmitters such as glutamate for neural tissues, is an important evolutionary driving force for the expression of UTs in these taxa and tissues.

Aging induces complex changes in myocardium bioenergetic and contractile properties. Using F344BNF1rats, we examined age-dependent changes in myocardial bioenergetic enzymes (catalytic activities and transcript levels) and mRNA levels of putative transcriptional regulators of bioenergetic genes. Very old rats (35 months) showed a 22% increase in ventricular mass with no changes in DNA or RNA per gram. Age-dependent cardiac hypertrophy was accompanied by complex changes in mitochondrial enzymes. Enzymes of the Krebs cycle and electron transport system remained within 15% of the values measured in adult heart, significant decreases occurring in citrate synthase (10%) and aconitase (15%). Transcripts for these enzymes were largely unaffected by aging, although mRNA levels of putative transcriptional regulators of the enzymes (nuclear respiratory factor (NRF) 1 and 2 α subunit) increased by about 30%–50%. In contrast, enzymes of fatty acid oxidation exhibited a more diverse pattern, with a 50% decrease in β-hydroxyacyl-CoA dehydrogenase (HOAD) and no change in long-chain acyl-CoA dehydrogenase or carnitine palmitoyltransferase. Transcript levels for fatty acid oxidizing enzymes covaried with HOAD, which declined significantly by 30%. There were no significant changes in the relative transcript levels of regulators of genes for fatty acid oxidizing enzymes: peroxisome proliferator-activated receptor-α (PPARα), PPARβ, or PPARγ coactivator-1α (PGC-1α). There were no changes in the mRNA levels of Sirt1, a histone-modifying enzyme that interacts with PGC-1α. Collectively, these data suggest that aging causes complex changes in the enzymes of myocardial energy metabolism, triggered in part by NRF-independent pathways as well as post-transcriptional regulation.Key words: PGC-1a, fatty acid oxidation, nuclear respiratory factor (NRF), PPAR, coactivator, transcriptional regulation.

SUMMARY Muscle metabolic properties vary with body size, with larger animals relying relatively less on oxidative metabolism as a result of lower specific activities of mitochondrial enzymes and greater specific activities of glycolytic enzymes. While many have argued reasons why such relationships might be grounded in physical relationships, an explanation for the regulatory basis of the differences in enzyme levels remains unexplored. Focusing on skeletal muscle, we review potential cellular and genetic explanations for the relationship between bioenergetic enzymes and body mass. Differences in myonuclear domain (the ratio of fiber volume to nuclei number) in conjunction with constitutive expression may explain part of the variation in mitochondrial content among fiber types and species. Superimposed on such constitutive determinants are (1) extrinsic signalling pathways that control the muscle contractile and metabolic phenotype and (2) intrinsic signalling pathways that translate changes in cellular milieu (ions, metabolites, oxygen,redox) arising through the contractile phenotype into changes in enzyme synthesis. These signalling pathways work through transcriptional regulation,as well as post-transcriptional, translational and post-translational regulation, acting via synthesis and degradation.

Fuel selection patterns during exercise are thought to be conserved among sea-level native mammals when intensity is expressed relative to maximum aerobic capacity (VO2max). However, this claim is based on data from only a few species larger than rats, and has never been tested statistically. Thus, we investigated fuel use in a small mammal (Mus musculus, CD-1 strain), and combined these with published data on rats, dogs, goats, and humans to evaluate the robustness of the mammalian fuel selection model. We found mice rely less on carbohydrates to power moderate intensity exercise at the same % VO2max than larger mammals. We suggest this difference is due to a decline in aerobic scope (O2 available for exercise above resting metabolism) as body size decreases. We propose a redefined fuel use model that reflects changes in fractional aerobic scope with body size. We find exercise defined as % aerobic scope is a better predictor of fuel use across a wide range of quadruped species from mice to dogs and to running humans.

Although the majority of adult teleosts excrete most of their nitrogenous wastes as ammonia, several fish species are capable of producing urea early in development. In zebrafish, it is unclear whether this results from a functional ornithine-urea cycle (O-UC) and, if so, how it might be regulated. This study examined the spatiotemporal patterns of gene expression of four major O-UC enzymes: carbamoyl phosphate synthase III (CPSIII), ornithine transcarboxylase, arginosuccinate synthetase, and arginosuccinate lyase, using real-time PCR and whole mount in situ hybridization. In addition, we hypothesized that CPSIII gene expression was epigenetically regulated through methylation of its promoter, a widespread mode of differential gene regulation between tissues and life stages in vertebrates. Furthermore, to assess CPSIII functionality, we used morpholinos to silence CPSIII in zebrafish embryos and assessed their nitrogenous waste handling during development, and in response to ammonia injections. Our results suggest that mRNAs of O-UC enzymes are expressed early in zebrafish development and colocalize to the embryonic endoderm. In addition, the methylation status of CPSIII promoter is not consistent with the patterns of expression observed in developing larvae or adult tissues, suggesting other means of transcriptional regulation of this enzyme. Finally, CPSIII morphants exhibited a transient reduction in CPSIII enzyme activity 24 h postfertilization, which was paralleled by reduced urea production during development and in response to an ammonia challenge. Overall, we conclude that the O-UC is functional in zebrafish embryos, providing further evidence that the capacity to produce urea via the O-UC is widespread in developing teleosts.

Bony fish primarily excrete ammonia as adults however the persistence of urea cycle genes may reflect a beneficial role for urea production during embryonic stages in protecting the embryo from toxic effects of ammonia produced from a highly nitrogenous yolk. This study aimed to examine the dynamic scope for changes in rates of urea synthesis and excretion in one such species (zebrafish, Danio rerio) by manipulating the intrinsic developmental rate (by alteration of light:dark cycles), as well as by direct chemical manipulation via ammonia injection (to potentially activate urea production) and nicotinamide exposure (to potentially inhibit urea production). Continuous dark exposure delayed development in embryos as evidenced by delayed appearance of hallmark anatomical features (heartbeat, eye pigmentation, body pigmentation, lateral line, fin buds) at 30 and 48 hr post-fertilization, as well by a lower hatching rate compared to embryos reared in continuous light. Both ammonia and urea excretion were similarly effected and were generally higher in embryos continuously exposed to light. Ammonia injection resulted in significant increases (up to fourfold) of urea N excretion and no changes to ammonia excretion rates along with modest increases in yolk ammonia content during 2-6 hr post-injection. Nicotinamide (an inhibitor of urea synthesis in mammals) reduced the ammonia-induced increase in urea excretion and led to retention of ammonia in the yolk and body of the embryo. Our results indicate that there is a relatively rapid and large scope for increases in urea production/excretion rates in developing embryos. Potential mechanisms for these increases are discussed.

In mammals, the peroxisome proliferator activated receptor (PPAR)gamma coactivator-1alpha (PGC-1alpha) is a central regulator of mitochondrial gene expression, acting in concert with nuclear respiratory factor-1 (NRF-1) and the PPARs. Its role as a "master regulator" of oxidative capacity is clear in mammals, but its role in other vertebrates is ambiguous. In lower vertebrates, although PGC-1alpha seems to play a role in coordinating the PPARalpha axis as in mammals, it does not appear to be involved in NRF-1 regulation of mitochondrial content. To evaluate the evolutionary patterns of this coactivator in fish and mammals, we investigated the evolutionary trajectories of PGC-1alpha homologs in representative vertebrate lineages. A phylogeny of the PGC-1 paralogs suggested that the family diversified through repeated genome duplication events early in vertebrate evolution. Bayesian and maximum likelihood phylogenetic reconstructions of PGC-1alpha in representative vertebrate species revealed divergent evolutionary dynamics across the different functional domains of the protein. Specifically, PGC-1alpha exhibited strong conservation of the activation/PPAR interaction domain across vertebrates, whereas the NRF-1 and MEF2c interaction domains experienced accelerated rates of evolution in actinopterygian (fish lineages) compared to sarcopterygians (tetrapod lineages). Furthermore, analysis of the amino acid sequence of these variable domains revealed successive serine- and glutamine-rich insertions within the teleost lineages, with important ramifications for PGC-1alpha function in these lineages. Collectively, these results suggest modular evolution of the PGC-1alpha protein in vertebrates that could allow for lineage-specific divergences in the coactivating capabilities of this regulator.

In adult zebrafish, 4 weeks of exercise training is known to induce an increase in mitochondrial enzymes such as citrate synthase (CS) when determined in mixed (red and white) muscle. However, this remodeling is not accompanied by changes in PGC-1alpha mRNA, a potent inducer of mitochondrial biogenesis in mammals. To further understand this response, we examined absolute and relative changes in red muscle area by histochemistry after 4 weeks of swim training. We also examined fiber-type specific responses in the expression of metabolic genes and putative regulators in red and white muscle of adult zebrafish at 1 and 8 weeks of training and in recovery from a single bout of exercise. Total red muscle area was unaltered after 4 weeks of training. The mRNA expression of CS was unaffected in red muscle, while it was increased in white muscle after 1 week of training and remained elevated at 8 weeks of training, suggesting an increase in oxidative capacity of this fiber type. In contrast, PGC-1alpha mRNA was elevated in both muscles only after 1 week of training. In both muscles, an acute bout of exercise rapidly (within 0-2 h post-exercise) induced PGC-1alpha mRNA and a delayed (24 h) increase in CS mRNA post-exercise. These results suggest complex temporal and spatial adaptive molecular responses to exercise in the skeletal muscles of zebrafish.

We used expression and reporter gene analysis to understand how changes in transcription factors impinge on mitochondrial gene expression during myogenesis of cultured murine myoblasts (C2C12 and Sol8). The mRNA levels for nuclear respiratory factor-1 (NRF-1) and NRF-2alpha increased 60% by the third day of myogenesis, whereas NRF-1 and NRF-2 reporter gene activity increased by fivefold over the same period. Although peroxisome proliferator activated receptor (PPARalpha) mRNA levels increased almost 10-fold, the activity of a PPAR reporter was unchanged during myogenesis. The PPAR coactivator PPAR-gamma coactivator-1alpha (PGC1alpha), a master controller of mitochondrial biogenesis, was not expressed at detectable levels. However, the mRNA for both PGC1alpha-related coactivator and PGC1beta was abundant, with the latter increasing by 50% over 3 days of differentiation. We also conducted promoter analysis of the gene for citrate synthase (CS), a common mitochondrial marker enzyme. The proximal promoter ( approximately 2,100 bp) of the human CS lacks binding sites for PPAR, NRF-1, or NRF-2. Deletion mutants, a targeted mutation, and an Sp1 site-containing reporter construct suggest that changes in Sp1 regulation also participate in mitochondrial biogenesis during myogenesis. Because most mitochondrial genes are regulated by PPARs, NRF-1, and/or NRF-2, we conducted inhibitor studies to further support the existence of a distinct pathway for CS gene regulation in myogenesis. Although both LY-294002 (a phosphatidylinositol 3-kinase inhibitor) and SB-203580 (a p38-MAPK inhibitor) blocked myogenesis (as indicated by creatine phosphokinase activity), only SB-203580 prevented the myogenic increase in cytochrome oxidase activity, whereas only LY-294002 blocked the increase in CS (enzyme and reporter gene activities). Collectively, these studies help delineate the roles of some transcriptional regulators involved in mitochondrial biogenesis associated with myogenesis and underscore an import role for posttranscriptional regulation of transcription factor activity.