Age-related flexibility of energetic metabolism in the honey bee Apis mellifera.

The mechanisms that underpin aging are still elusive. In this study, we suggest that the ability of mitochondria to oxidize different substrates, which is known as metabolic flexibility, is involved in this process. To verify our hypothesis, we used honey bees (Apis mellifera carnica) at different ages, to assess mitochondrial oxygen consumption and enzymatic activities of key enzymes of the energetic metabolism as well as ATP5A1 content (subunit of ATP synthase) and adenylic energy charge (AEC). We also measured mRNA abundance of genes involved in mitochondrial functions and the antioxidant system. Our results demonstrated that mitochondrial respiration increased with age and favored respiration through complexes I and II of the electron transport system (ETS) while glycerol-3-phosphate (G3P) oxidation was relatively decreased. In addition, glycolytic, tricarboxylic acid cycle and ETS enzymatic activities increased, which was associated with higher ATP5A1 content and AEC. Furthermore, we detected an early decrease in the mRNA abundance of subunits of NADH ubiquinone oxidoreductase subunit B2 (NDUFB2, complex I), mitochondrial cytochrome b (CYTB, complex III) of the ETS as well as superoxide dismutase 1 and a later decrease for vitellogenin, catalase and mitochondrial cytochrome c oxidase subunit 1 (COX1, complex IV). Thus, our study suggests that the energetic metabolism is optimized with aging in honey bees, mainly through quantitative and qualitative mitochondrial changes, rather than showing signs of senescence. Moreover, aging modulated metabolic flexibility, which might reflect an underpinning mechanism that explains lifespan disparities between the different castes of worker bees.

Environmental variation associated with overwintering elicits marked metabolic plasticity in a temperate salmonid, Salvelinus fontinalis.

Poleward winters commonly expose animals, including fish, to frigid temperatures and low food availability. Fishes that remain active over winter must therefore balance trade-offs between conserving energy and maintaining physiological performance in the cold, yet the extent and underlying mechanisms of these trade-offs are not well understood. We investigated the metabolic plasticity of brook char (Salvelinus fontinalis), a temperate salmonid, from the biochemical to whole-animal level in response to cold and food deprivation. Acute cooling (1°C day-1) from 14°C to 2°C had no effect on food consumption but reduced activity by 77%. We then assessed metabolic performance and demand over 90 days with exposure to warm (8°C) or cold winter (2°C) temperatures while fish were fed or starved. Resting metabolic rate (RMR) decreased substantially during initial cooling from 8°C to 2°C (Q10=4.2-4.5) but brook char exhibited remarkable thermal compensation during acclimation (Q10=1.4-1.6). Conversely, RMR was substantially lower (40-48%) in starved fish, conserving energy. Thus, the absolute magnitude of thermal plasticity may be masked or modified under food restriction. This reduction in RMR was associated with atrophy and decreases in in vivo protein synthesis rates, primarily in non-essential tissues. Remarkably, food deprivation had no effect on maximum oxygen uptake rates and thus aerobic capacity, supporting the notion that metabolic capacity can be decoupled from RMR in certain contexts. Overall, our study highlights the multi-faceted energetic flexibility of Salvelinus spp. that likely contributes to their success in harsh and variable environments and may be emblematic of winter-active fishes more broadly.

The ovine conceptus utilizes extracellular serine, glucose, and fructose to generate formate via the one carbon metabolism pathway.

Highly proliferative cells rely on one carbon (1C) metabolism for production of formate required for synthesis of purines and thymidine for nucleic acid synthesis. This study was to determine if extracellular serine and/or glucose and fructose contribute the production of formate in ovine conceptuses. Suffolk ewes (n = 8) were synchronized to estrus, bred to fertile rams, and conceptuses were collected on Day 17 of gestation. Conceptuses were either snap frozen in liquid nitrogen (n = 3) or placed in culture in medium (n = 5) containing either: 1) 4 mM D-glucose + 2 mM [U-13C]serine; 2) 6 mM glycine + 4 mM D-glucose + 2 mM [U-13C]serine; 3) 4 mM D-fructose + 2 mM [U-13C]serine; 4) 6 mM glycine + 4 mM D-fructose + 2 mM [U-13C]serine; 5) 4 mM D-glucose + 4 mM D-fructose + 2 mM [U-13C]serine; or 6) 6 mM glycine + 4 mM D-glucose + 4 mM D-fructose + 2 mM [U-13C]serine. After 2 h incubation, conceptuses in their respective culture medium were homogenized and the supernatant analyzed for 12C- and 13C-formate by gas chromatography and amino acids by high performance liquid chromatography. Ovine conceptuses produced both 13C- and 12C-formate, indicating that the [U-13C]serine, glucose, and fructose were utilized to generate formate, respectively. Greater amounts of 12C-formate than 13C-formate were produced, indicating that the ovine conceptus utilized more glucose and fructose than serine to produce formate. This study is the first to demonstrate that both 1C metabolism and serinogenesis are active metabolic pathways in ovine conceptuses during the peri-implantation period of pregnancy, and that hexose sugars are the preferred substrate for generating formate required for nucleotide synthesis for proliferating trophectoderm cells.

Social experience influences thermal sensitivity: lessons from an amphibious mangrove fish.

Understanding the factors affecting the capacity of ectothermic fishes to cope with warming temperature is critical given predicted climate change scenarios. We know that a fish's social environment introduces plasticity in how it responds to high temperature. However, the magnitude of this plasticity and the mechanisms underlying socially modulated thermal responses are unknown. Using the amphibious hermaphroditic mangrove rivulus fish Kryptolebias marmoratus as a model, we tested three hypotheses: (1) social stimulation affects physiological and behavioural thermal responses of isogenic lineages of fish; (2) social experience and acute social stimulation result in distinct physiological and behavioural responses; and (3) a desensitization of thermal receptors is responsible for socially modulated thermal responses. To test the first two hypotheses, we measured the temperature at which fish emerged from the water (i.e. pejus temperature) upon acute warming with socially naive isolated fish and with fish that were raised alone and then given a short social experience prior to exposure to increasing temperature (i.e. socially experienced fish). Our results did not support our first hypothesis as fish socially stimulated by mirrors during warming (i.e. acute social stimulation) emerged at similar temperatures to isolated fish. However, in support of our second hypothesis, a short period of prior social experience resulted in fish emerging at a higher temperature than socially naive fish suggesting an increase in pejus temperature with social experience. To test our third hypothesis, we exposed fish that had been allowed a brief social interaction and naive fish to capsaicin, an agonist of TRPV1 thermal receptors. Socially experienced fish emerged at significantly higher capsaicin concentrations than socially naive fish suggesting a desensitization of their TRPV1 thermal receptors. Collectively, our data indicate that past and present social experiences impact the behavioural response of fish to high temperature. We also provide novel data suggesting that brief periods of social experience affect the capacity of fish to perceive warm temperature.

Taurine depletion impairs cardiac function and affects tolerance to hypoxia and high temperatures in brook char (Salvelinus fontinalis).

Physiological and environmental stressors can cause osmotic stress in fish hearts, leading to a reduction in intracellular taurine concentration. Taurine is a ß-amino acid known to regulate cardiac function in other animal models but its role in fish has not been well characterized. We generated a model of cardiac taurine deficiency (TD) by feeding brook char (Salvelinus fontinalis) a diet enriched in ß-alanine, which inhibits cardiomyocyte taurine uptake. Cardiac taurine levels were reduced by 21% and stress-induced changes in normal taurine handling were observed in TD brook char. Responses to exhaustive exercise and acute thermal and hypoxia tolerance were then assessed using a combination of in vivo, in vitro and biochemical approaches. Critical thermal maximum was higher in TD brook char despite significant reductions in maximum heart rate. In vivo, TD brook char exhibited a lower resting heart rate, blunted hypoxic bradycardia and a severe reduction in time to loss of equilibrium under hypoxia. In vitro function was similar between control and TD hearts under oxygenated conditions, but stroke volume and cardiac output were severely compromised in TD hearts under severe hypoxia. Aspects of mitochondrial structure and function were also impacted in TD permeabilized cardiomyocytes, but overall effects were modest. High levels of intracellular taurine are required to achieve maximum cardiac function in brook char and cardiac taurine efflux may be necessary to support heart function under stress. Taurine appears to play a vital, previously unrecognized role in supporting cardiovascular function and stress tolerance in fish.

Reduced Shmt2 Expression Impairs Mitochondrial Folate Accumulation and Respiration, and Leads to Uracil Accumulation in Mouse Mitochondrial DNA.

BACKGROUND: Adequate cellular thymidylate (dTMP) pools are essential for preservation of nuclear and mitochondrial genome stability. Previous studies have indicated that disruption in nuclear dTMP synthesis leads to increased uracil misincorporation into DNA, affecting genome stability. To date, the effects of impaired mitochondrial dTMP synthesis in nontransformed tissues have been understudied. OBJECTIVES: This study aimed to determine the effects of decreased serine hydroxymethyltransferase 2 (Shmt2) expression and dietary folate deficiency on mitochondrial DNA (mtDNA) integrity and mitochondrial function in mouse tissues. METHODS: Liver mtDNA content, and uracil content in liver mtDNA, were measured in Shmt2+/- and Shmt2+/+ mice weaned onto either a folate-sufficient control diet (2 mg/kg folic acid; C) or a modified diet lacking folic acid (0 mg/kg folic acid) for 7 wk. Shmt2+/- and Shmt2+/+ mouse embryonic fibroblast (MEF) cells were cultured in defined culture medium containing either 0 or 25 nM folate (6S-5-formyl-tetrahydrofolate, folinate) to assess proliferative capacity and mitochondrial function. Chi-square tests, linear mixed models, and 2-factor ANOVA with Tukey post hoc analyses were used to analyze data. RESULTS: Shmt2 +/- mice exhibited a 48%-67% reduction in SHMT2 protein concentrations in tissues. Interestingly, Shmt2+/- mice consuming the folate-sufficient C diet exhibited a 25% reduction in total folate in liver mitochondria. There was also a >20-fold increase in uracil in liver mtDNA in Shmt2+/- mice consuming the C diet, and dietary folate deficiency also increased uracil content in mouse liver mtDNA from both Shmt2+/+ and Shmt2+/- mice. Furthermore, decreased Shmt2 expression in MEF cells reduced cell proliferation, mitochondrial membrane potential, and oxygen consumption rate. CONCLUSIONS: This study demonstrates that Shmt2 heterozygosity and dietary folate deficiency impair mitochondrial dTMP synthesis in mice, as evidenced by the increased uracil in mtDNA. In addition, Shmt2 heterozygosity impairs mitochondrial function in MEF cells. These findings suggest that elevated uracil in mtDNA may impair mitochondrial function.

Differences in mitochondrial efficiency explain individual variation in growth performance.

The physiological causes of intraspecific differences in fitness components such as growth rate are currently a source of debate. It has been suggested that differences in energy metabolism may drive variation in growth, but it remains unclear whether covariation between growth rates and energy metabolism is: (i) a result of certain individuals acquiring and consequently allocating more resources to growth, and/or is (ii) determined by variation in the efficiency with which those resources are transformed into growth. Studies of individually housed animals under standardized nutritional conditions can help shed light on this debate. Here we quantify individual variation in metabolic efficiency in terms of the amount of adenosine triphosphate (ATP) generated per molecule of oxygen consumed by liver and muscle mitochondria and examine its effects, both on the rate of protein synthesis within these tissues and on the rate of whole-body growth of individually fed juvenile brown trout (Salmo trutta) receiving either a high or low food ration. As expected, fish on the high ration on average gained more in body mass and protein content than those maintained on the low ration. Yet, growth performance varied more than 10-fold among individuals on the same ration, resulting in some fish on low rations growing faster than others on the high ration. This variation in growth for a given ration was related to individual differences in mitochondrial properties: a high whole-body growth performance was associated with high mitochondrial efficiency of ATP production in the liver. Our results show for the first time, to our knowledge, that among-individual variation in the efficiency with which substrates are converted into ATP can help explain marked variation in growth performance, independent of food intake. This study highlights the existence of inter-individual differences in mitochondrial efficiency and its potential importance in explaining intraspecific variation in whole-animal performance.

Reversion to developmental pathways underlies rapid arm regeneration in juvenile European cuttlefish, Sepia officinalis (Linnaeus 1758).

Coleoid cephalopods, including the European cuttlefish (Sepia officinalis), possess the remarkable ability to fully regenerate an amputated arm with no apparent fibrosis or loss of function. In model organisms, regeneration usually occurs as the induction of proliferation in differentiated cells. In rare circumstances, regeneration can be the product of naïve progenitor cells proliferating and differentiating de novo . In any instance, the immune system is an important factor in the induction of the regenerative response. Although the wound response is well-characterized, little is known about the physiological pathways utilized by cuttlefish to reconstruct a lost arm. In this study, the regenerating arms of juvenile cuttlefish, with or without exposure at the time of injury to sterile bacterial lipopolysaccharide extract to provoke an antipathogenic immune response, were assessed for the transcription of early tissue lineage developmental genes, as well as histological and protein turnover analyses of the resulting regenerative process. The transient upregulation of tissue-specific developmental genes and histological characterization indicated that coleoid arm regeneration is a stepwise process with staged specification of tissues formed de novo, with immune activation potentially affecting the timing but not the result of this process. Together, the data suggest that rather than inducing proliferation of mature cells, developmental pathways are reinstated, and that a pool of naïve progenitors at the blastema site forms the basis for this regeneration.

Activation of oxygen-responsive pathways is associated with altered protein metabolism in Arctic char exposed to hypoxia.

Fish exposed to fluctuating oxygen concentrations often alter their metabolism and/or behaviour to survive. Hypoxia tolerance is typically associated with the ability to reduce energy demand by supressing metabolic processes such as protein synthesis. Arctic char is amongst the most sensitive salmonid to hypoxia, and typically engage in avoidance behaviour when faced with lack of oxygen. We hypothesized that a sensitive species will still have the ability (albeit reduced) to regulate molecular mechanisms during hypoxia. We investigated the tissue-specific response of protein metabolism during hypoxia. Little is known about protein degradation pathways during hypoxia in fish and we predict that protein degradation pathways are differentially regulated and play a role in the hypoxia response. We also studied the regulation of oxygen-responsive cellular signalling pathways [hypoxia inducible factor (HIF), unfolded protein response (UPR) and mTOR pathways] since most of what we know comes from studies on cancerous mammalian cell lines. Arctic char were exposed to cumulative graded hypoxia trials for 3 h at four air saturation levels (100%, 50%, 30% and 15%). The rate of protein synthesis was measured using a flooding dose technique, whereas protein degradation and signalling pathways were assessed by measuring transcripts and phosphorylation of target proteins. Protein synthesis decreased in all tissues measured (liver, muscle, gill, digestive system) except for the heart. Salmonid hearts have preferential access to oxygen through a well-developed coronary artery, therefore the heart is likely to be the last tissue to become hypoxic. Autophagy markers were upregulated in the liver, whereas protein degradation markers were downregulated in the heart during hypoxia. Further work is needed to determine the effects of a decrease in protein degradation on a hypoxic salmonid heart. Our study showed that protein metabolism in Arctic char is altered in a tissue-specific fashion during graded hypoxia, which is in accordance with the responses of the three major hypoxia-sensitive pathways (HIF, UPR and mTOR). The activation pattern of these pathways and the cellular processes that are under their control varies greatly among tissues, sometimes even going in the opposite direction. This study provides new insights on the effects of hypoxia on protein metabolism. Adjustment of these cellular processes is likely to contribute to shifting the fish phenotype into a more hypoxia-tolerant one, if more than one hypoxia event were to occur. Our results warrant studying these adjustments in fish exposed to long-term and diel cycling hypoxia.

Diel cycling hypoxia enhances hypoxia tolerance in rainbow trout (Oncorhynchus mykiss): evidence of physiological and metabolic plasticity.

Many fish naturally encounter a daily cycle of hypoxia, but it is unclear whether this exposure hardens hypoxia-intolerant fish to future hypoxia or leads to accumulated stress and death. The rainbow trout (Oncorhynchus mykiss) is a putatively hypoxia-sensitive species found in rivers and estuaries that may routinely experience hypoxic events. Trout were exposed to one of four 135 h treatments in a swim-tunnel respirometer: (1) air-saturated control (20.7 kPa PO2 ); (2) diel cycling O2 (20.7-4.2 kPa PO2  over 24 h); (3) acute hypoxia (130 h at 20.7 kPa PO2  followed by 5 h at 4.2 kPa PO2 ); and (4) the mean oxygen tension (12.4 kPa PO2 ) experienced by the diel cycled fish. Some responses were similar in diel O2 cycled and mean PO2 -treated fish, but overall, exposure to ecologically representative diel hypoxia cycles improved hypoxia tolerance. Diel hypoxia-induced protective responses included increased inducible HSP70 concentration and mean corpuscular hemoglobin concentration, as well as reduced plasma cortisol. Acclimation to diel hypoxia allowed metabolic rates to decline during hypoxia, reduced oxygen debt following subsequent exposures, and allowed fish to return to an anabolic phenotype. The data demonstrate that acute diel cycling hypoxia improves hypoxia tolerance in previously intolerant fish through the activation of cellular protective mechanisms and a reduction in metabolic O2 requirements.

Transcriptome and proteome analyses to investigate the molecular underpinnings of cold response in the Colorado potato beetle, Leptinotarsa decemlineata.

The Colorado potato beetle (Leptinotarsa decemlineata (Say)) is an insect that can cope with prolonged periods of low temperatures exposure. The molecular changes required to adapt to such conditions have not been thoroughly investigated in this insect. The current work aims at characterizing deregulated transcripts and proteins in adult L. decemlineata exposed to 15 °C and -5 °C using RNA-sequencing-based transcriptomics and liquid chromatography tandem mass spectrometry (LC-MS/MS)-based proteomics approaches, respectively. RNA-sequencing highlighted the differential expression of several transcripts, including ubiquilin-1 and ubiquitin carboxyl-terminal hydrolase 5, in insects submitted to low temperatures when compared with control insects. In addition, proteomics approach detected 2840 proteins in cold-exposed beetles including elevated levels for 409 proteins and reduced levels for 200 proteins. Cuticular proteins CP1, CP4, CP5 and CP7 as well as eukaryotic translation initiation factor 4B were notable proteins with elevated levels in cold insects. Functional analysis of targets modulated at low temperatures using DAVID indicated processes likely affected under cold conditions including select metabolic cascades and RNA-associated processes. Overall, this work presents molecular candidates impacted by low temperatures exposure in L. decemlineata and builds on the current knowledge associated with response to these conditions in this insect.

Effects of fasting and refeeding on protein and glucose metabolism in Arctic charr.

Refeeding, following a period of food deprivation will often lead to compensatory growth. Although many studies have focused on molecular mechanisms behind this accelerated growth response in fish, little is known on the roles of protein and metabolism. We also assessed, for the first time, the potential roles of miRNAs in regulating compensatory growth. Artcic charr, Salvelinus alpinus, a northern freshwater species, was deprived of food for 101 days and then fed to satiety for 126 days. The refeeding period resulted in compensatory growth, with a partial compensation of body mass. The feed deprivation period lead to a decrease in hepatosomatic index (HSI) and intestinal somatic index (ISI). HSI and ISI were then gradually replenished during early refeeding, following a lag phase prior to the compensatory growth response. mRNA transcripts regulating protein degradation via the autophagy pathway (Cathepsin D and Cathepsin L) in muscle were upregulated during feed restriction and downregulated after refeeding, which could allow for greater protein accretion in muscle, facilitating compensatory growth. Transcript levels from the ubiquitin proteasome pathway (Mafbx and Murf1) and the calpain system (Calpain 7 and Calpastatin) suggested that these pathways were not involved in regulating compensatory growth. Furthermore, we've shown that miRNAs (miR-29a and miR-223) could be involved in fish glycogen homeostasis during the early stages of refeeding. These findings provide a deeper understanding of the molecular mechanisms regulating growth in fish.

Riboflavin Deficiency in Rats Decreases de novo Formate Production but Does Not Affect Plasma Formate Concentration.

Background: The one-carbon metabolism pathway is highly dependent on a number of B vitamins in order to provide one-carbon units for purine and thymidylate biosynthesis as well as homocysteine remethylation. Previous studies have examined folate and vitamin B-12 deficiency and their effects on formate metabolism; as of yet, to our knowledge, no studies on the effects of riboflavin deficiency on formate metabolism have been published.Objective: Our objective was to determine the effects of riboflavin deficiency on formate metabolism.Methods: Weanling male rats were randomly assigned either to control, riboflavin-replete (RR) or to experimental, riboflavin-deficient (RD) versions of the AIN-93G diet for 13 d, at which time a constant infusion of [13C]-formate was carried out to ascertain the effects of deficiency on formate production. Gas chromatography-mass spectrometry was used to measure plasma formate concentration and [13C]-formate enrichment. HPLC, LC-mass spectrometry (MS)/MS, and enzymatic assays were used for the measurement of one-carbon precursors and other metabolites.Results: RD rats had significantly lower rates of formate production (15%) as well as significantly reduced hepatic methylenetetrahydrofolate reductase activity (69%) and protein concentration (54%) compared with RR rats. There was no difference in plasma formate concentrations between the groups. Plasma serine, a potential one-carbon precursor, was significantly higher in RD rats (467 ± 73 µM) than in RR rats (368 ± 52 µM).Conclusions: Although deficiencies in folate and vitamin B-12 lead to major changes in plasma formate concentrations, riboflavin deficiency results in no significant difference; this disagrees with the prediction of a published mathematical model. Our observation of a lower rate of formate production is consistent with a role for flavoproteins in this process.

In vivo kinetics of formate metabolism in folate-deficient and folate-replete rats.

It is now established that the mitochondrial production of formate is a major process in the endogenous generation of folate-linked one-carbon groups. We have developed an in vivo approach involving the constant infusion of [(13)C]formate until isotopic steady state is attained to measure the rate of endogenous formate production in rats fed on either a folate-replete or folate-deficient diet. Formate was produced at a rate of 76 µmol·h(-1)·100 g of body weight(-1) in the folate-replete rats, and this was decreased by 44% in folate-deficient rats. This decreased formate production was confirmed in isolated rat liver mitochondria where formate production from serine, the principal precursor of one-carbon groups, was decreased by 85%, although formate production from sarcosine and dimethylglycine (choline metabolites) was significantly increased. We attribute this unexpected result to the demonstrated production of formaldehyde by sarcosine dehydrogenase and dimethylglycine dehydrogenase from their respective substrates in the absence of tetrahydrofolate and subsequent formation of formate by formaldehyde dehydrogenase. Comparison of formate production with the ingestion of dietary formate precursors (serine, glycine, tryptophan, histidine, methionine, and choline) showed that ∼75% of these precursors were converted to formate, indicating that formate is a significant, although underappreciated end product of choline and amino acid oxidation. Ingestion of a high protein diet did not result in increased production of formate, suggesting a regulation of the conversion of these precursors at the mitochondrial level to formate.

Metabolic rate and rates of protein turnover in food-deprived cuttlefish, Sepia officinalis (Linnaeus 1758).

To determine the metabolic response to food deprivation, cuttlefish (Sepia officinalis) juveniles were either fed, fasted (3 to 5 days food deprivation), or starved (12 days food deprivation). Fasting resulted in a decrease in triglyceride levels in the digestive gland, and after 12 days, these lipid reserves were essentially depleted. Oxygen consumption was decreased to 53% and NH4 excretion to 36% of the fed group following 3-5 days of food deprivation. Oxygen consumption remained low in the starved group, but NH4 excretion returned to the level recorded for fed animals during starvation. The fractional rate of protein synthesis of fasting animals decreased to 25% in both mantle and gill compared with fed animals and remained low in the mantle with the onset of starvation. In gill, however, protein synthesis rate increased to a level that was 45% of the fed group during starvation. In mantle, starvation led to an increase in cathepsin A-, B-, H-, and L-like enzyme activity and a 2.3-fold increase in polyubiquitin mRNA that suggested an increase in ubiquitin-proteasome activity. In gill, there was a transient increase in the polyubiquitin transcript levels in the transition from fed through fasted to the starved state and cathepsin A-, B-, H-, and L-like activity was lower in starved compared with fed animals. The response in gill appears more complex, as they better maintain rates of protein synthesis and show no evidence of enhanced protein breakdown through recognized catabolic processes.

Estimates of metabolic rate and major constituents of metabolic demand in fishes under field conditions: Methods, proxies, and new perspectives.

Metabolic costs are central to individual energy budgets, making estimates of metabolic rate vital to understanding how an organism interacts with its environment as well as the role of species in their ecosystem. Despite the ecological and commercial importance of fishes, there are currently no widely adopted means of measuring field metabolic rate in fishes. The lack of recognized methods is in part due to the logistical difficulties of measuring metabolic rates in free swimming fishes. However, further development and refinement of techniques applicable for field-based studies on free swimming animals would greatly enhance the capacity to study fish under environmentally relevant conditions. In an effort to foster discussion in this area, from field ecologists to biochemists alike, we review aspects of energy metabolism and give details on approaches that have been used to estimate energetic parameters in fishes. In some cases, the techniques have been applied to field conditions; while in others, the methods have been primarily used on laboratory held fishes but should be applicable, with validation, to fishes in their natural environment. Limitations, experimental considerations and caveats of these measurements and the study of metabolism in wild fishes in general are also discussed. Potential novel approaches to FMR estimates are also presented for consideration. The innovation of methods for measuring field metabolic rate in free-ranging wild fish would revolutionize the study of physiological ecology.

Nuclear enrichment of folate cofactors and methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) protect de novo thymidylate biosynthesis during folate deficiency.

Folate-mediated one-carbon metabolism is a metabolic network of interconnected pathways that is required for the de novo synthesis of three of the four DNA bases and the remethylation of homocysteine to methionine. Previous studies have indicated that the thymidylate synthesis and homocysteine remethylation pathways compete for a limiting pool of methylenetetrahydrofolate cofactors and that thymidylate biosynthesis is preserved in folate deficiency at the expense of homocysteine remethylation, but the mechanisms are unknown. Recently, it was shown that thymidylate synthesis occurs in the nucleus, whereas homocysteine remethylation occurs in the cytosol. In this study we demonstrate that methylenetetrahydrofolate dehydrogenase 1 (MTHFD1), an enzyme that generates methylenetetrahydrofolate from formate, ATP, and NADPH, functions in the nucleus to support de novo thymidylate biosynthesis. MTHFD1 translocates to the nucleus in S-phase MCF-7 and HeLa cells. During folate deficiency mouse liver MTHFD1 levels are enriched in the nucleus >2-fold at the expense of levels in the cytosol. Furthermore, nuclear folate levels are resistant to folate depletion when total cellular folate levels are reduced by >50% in mouse liver. The enrichment of folate cofactors and MTHFD1 protein in the nucleus during folate deficiency in mouse liver and human cell lines accounts for previous metabolic studies that indicated 5,10-methylenetetrahydrofolate is preferentially directed toward de novo thymidylate biosynthesis at the expense of homocysteine remethylation during folate deficiency.

Betaine supplementation prevents fatty liver induced by a high-fat diet: effects on one-carbon metabolism.

The purpose of this study was to examine the effects of betaine supplementation on the regulation of one-carbon metabolism and liver lipid accumulation induced by a high-fat diet in rats. Rats were fed one of three different liquid diets: control diet, high-fat diet and high-fat diet supplemented with betaine. The control and high-fat liquid diets contained, respectively, 35 and 71 % of energy derived from fat. Betaine supplementation involved the addition of 1 % (g/L) to the diet. After three weeks on the high-fat diet the rats had increased total liver fat concentration, liver triglycerides, liver TBARS and plasma TNF-α. The high-fat diet decreased the hepatic S-adenosylmethionine concentration and the S-adenosylmethionine/S-adenosylhomocysteine ratio compared to the control as well as altering the expression of genes involved in one-carbon metabolism. Betaine supplementation substantially increased the hepatic S-adenosylmethionine concentration (~fourfold) and prevented fatty liver and hepatic injury induced by the high-fat diet. It was accompanied by the normalization of the gene expression of BHMT, GNMT and MGAT, which code for key enzymes of one-carbon metabolism related to liver fat accumulation. In conclusion, the regulation of the expression of MGAT by betaine supplementation provides an additional and novel mechanism by which betaine supplementation regulates lipid metabolism and prevents accumulation of fat in the liver.

An isotope-dilution, GC-MS assay for formate and its application to human and animal metabolism.

Formate, a crucial component of one-carbon metabolism, is increasingly recognized as an important intermediate in production and transport of one-carbon units. Unlike tetrahydrofolate-linked intermediates, it is not restricted to the intracellular milieu so that circulating levels of formate can provide insight into cellular events. We report a novel isotope-dilution, GC-MS assay employing derivatization by 2,3,4,5,6-pentafluorobenzyl bromide for the determination of formate in biological samples. This assay is robust and sensitive; it may be applied to the measurement of formate in serum, plasma and urine. We demonstrate how this method may be applied by providing the first characterization of formate levels in a human population; formate levels were higher in males than in females. We also show how this procedure may be applied for the measurement of in vivo kinetics of endogenous formate production in experimental animals.

Formate: an essential metabolite, a biomarker, or more?

Plasma and urinary formate concentrations were recently found to be elevated during vitamin B12 and folate deficiencies. It was proposed that formate may be a valuable biomarker of impaired one-carbon metabolism. Formate is an essential intermediary metabolite in folate-mediated one-carbon metabolism and, despite its importance, our knowledge of its metabolism is limited. Formate can be produced from several substrates (e.g., methanol, branched chain fatty acids, amino acids), some reactions being folate-dependent while others are not. Formate removal proceeds via two pathways; the major one being folate-dependent. Formate is a potentially toxic molecule and we suggest that formate may play a role in some of the pathologies associated with defective one-carbon metabolism.