Hormonal effects on glucose and ketone metabolism in a perfused liver of an elasmobranch, the North Pacific spiny dogfish, Squalus suckleyi.

Hormonal influence on hepatic function is a critical aspect of whole-body energy balance in vertebrates. Catecholamines and corticosteroids both influence hepatic energy balance via metabolite mobilization through glycogenolysis and gluconeogenesis. Elasmobranchs have a metabolic organization that appears to prioritize the mobilization of hepatic lipid as ketone bodies (e.g. 3-hydroxybutyrate [3-HB]), which adds complexity in determining the hormonal impact on hepatic energy balance in this taxon. Here, a liver perfusion was used to investigate catecholamine (epinephrine [E]) and corticosteroid (corticosterone [B] and 11-deoxycorticosterone [DOC]) effects on the regulation of hepatic glucose and 3-HB balance in the North Pacific Spiny dogfish, Squalus suckleyi. Further, hepatic enzyme activity involved in ketogenesis (3-hydroxybutyrate dehydrogenase), glycogenolysis (glycogen phosphorylase), and gluconeogenesis (phosphoenolpyruvate carboxykinase) were assessed in perfused liver tissue following hormonal application to discern effects on hepatic energy flux. mRNA transcript abundance key transporters of glucose (glut1 and glut4) and ketones (mct1 and mct2) and glucocorticoid function (gr, pepck, fkbp5, and 11ßhsd2) were also measured to investigate putative cellular components involved in hepatic responses. There were no changes in the arterial-venous difference of either metabolite in all hormone perfusions. However, perfusion with DOC increased gr transcript abundance and decreased flow rate of perfusions, suggesting a regulatory role for this corticosteroid. Phosphoenolpyruvate carboxykinase activity increased following all hormone treatments, which may suggest gluconeogenic function; E also increased 3-hydroxybutyrate dehydrogenase activity, suggesting a function in ketogenesis, and decreased pepck and fkbp5 transcript abundance, potentially showing some metabolic regulation. Overall, we demonstrate hormonal control of hepatic energy balance using liver perfusions at various levels of biological organization in an elasmobranch.

Gene knockout of NHX-3 in the soil nematode Caenorhabditis elegans leads to broad-spectrum compensatory regulation of Na+/H+ exchangers, antiporters, and the V-type H+-ATPase.

Na+/H+ exchangers are directly involved in a variety of an animal's essential physiological processes such as ionoregulation, acid-base regulation, nitrogenous waste excretion, and nutrient absorption. While nine NHX isoforms have been identified in Caenorhabditis elegans, the physiological importance of each isoform is not understood. The current study aimed to further our knowledge of NHX-3 which has previously been suggested to be involved in the movement of ammonia and acid-base equivalents across the nematode's hypodermis. Although NHX-3 knockout mutant nematodes exported H+ and imported Na+ at slower rates than wild-type nematodes, attempts to inhibit the NHX activity of mutant nematodes using amiloride and EIPA caused an unexpected increase in hypodermal H+ export and did not impact Na+ fluxes suggesting that the different H+ and Na+ transport profiles of the nematodes are likely due to compensatory changes in the mutants in response to the NHX-3 knockout, rather than the loss of NHX-3's physiological function. Significant changes in the mRNA expression of 7 other NHX isoforms, 2 Na+/H+ antiporter isoforms, and the V-type H+-ATPase were detected between wild-type and mutant nematodes. Furthermore, mutant nematodes possessed significantly reduced rates of cytochrome C oxidase activity and ammonia excretion rates, indicating the knockout of NHX-3 induced fundamental changes in metabolism that could impact the nematode's need to eliminate metabolic end-products like H+ and ammonia that relate to NHX transport. While C. elegans is a popular genetic model with cheap and accessible commercial mutants, our findings suggest caution in interpretation of results in studies using mutants to study physiological traits and the biological significance of specific transporters.

A synthesis of senescence predictions for indeterminate growth, and support from multiple tests in wild lake trout.

Senescence-the deterioration of functionality with age-varies widely across taxa in pattern and rate. Insights into why and how this variation occurs are hindered by the predominance of laboratory-focused research on short-lived model species with determinate growth. We synthesize evolutionary theories of senescence, highlight key information gaps and clarify predictions for species with low mortality and variable degrees of indeterminate growth. Lake trout are an ideal species to evaluate predictions in the wild. We monitored individual males from two populations (1976-2017) longitudinally for changes in adult mortality (actuarial senescence) and body condition (proxy for energy balance). A cross-sectional approach (2017) compared young (ages 4-10 years) and old (18-37 years) adults for (i) phenotypic performance in body condition, and semen quality-which is related to fertility under sperm competition (reproductive senescence)-and (ii) relative telomere length (potential proxy for cellular senescence). Adult growth in these particular populations is constrained by a simplified foodweb, and our data support predictions of negligible senescence when maximum size is only slightly larger than maturation size. Negative senescence (aka reverse senescence) may occur in other lake trout populations where diet shifts allow maximum sizes to greatly exceed maturation size.

NADPH supply and the contribution of NAD(P)+ transhydrogenase (NNT) to H2O2 balance in skeletal muscle mitochondria.

H2O2 is endogenously generated and its removal in the matrix of skeletal muscle mitochondria (SMM) is dependent on NADPH likely provided by NAD(P)+ transhydrogenase (NNT) and isocitrate dehydrogenase (IDH2). Importantly, NNT activity is linked to mitochondrial protonmotive force. Here, we demonstrate the presence of NNT function in detergent-solubilized and intact functional SMM isolated from rats and wild type (Nnt+/+) mice, but not in SMM from congenic mice carrying a mutated NNT gene (Nnt-/-). Further comparisons between SMM from both Nnt mouse genotypes revealed that the NADPH supplied by NNT supports up to 600 pmol/mg/min of H2O2 removal under selected conditions. Surprisingly, SMM from Nnt-/- mice removed exogenous H2O2 at wild-type levels and exhibited a maintained or even decreased net emission of endogenous H2O2 when substrates that support Krebs cycle reactions were present (e.g., pyruvate plus malate or palmitoylcarnitine plus malate). These results may be explained by a compensation for the lack of NNT, since the total activities of concurrent NADP+-reducing enzymes (IDH2, malic enzymes and glutamate dehydrogenase) were ~70% elevated in Nnt-/- mice. Importantly, respiratory rates were similar between SMM from both Nnt genotypes despite differing NNT contributions to H2O2 removal and their implications for an evolving concept in the literature are discussed. We concluded that NNT is capable of meaningfully sustaining NADPH-dependent H2O2 removal in intact SMM. Nonetheless, if the available substrates favor non-NNT sources of NADPH, the H2O2 removal by SMM is maintained in Nnt-/- mice SMM.

Energy and corticosteroid mobilization following an induced stress response in an elasmobranch fish, the North Pacific spiny dogfish (Squalus acanthias suckleyi).

The dominant corticosteroid in elasmobranchs, 1α-hydroxycorticosterone (1α-OHB), has a described role in mineral regulation but a presumptive role in energy balance. Energy demand in vertebrates following exposure to a stressor typically involves an immediate but transient release of glucocorticoids as a means of mobilizing available energy stores, usually in the form of glucose. Although a glucocorticoid role for 1α-OHB would be expected, direct glucocorticoid function of this steroid has yet to be reported in any elasmobranch. In addition, elasmobranchs also utilize the metabolite ß-hydroxybutyrate (ß-HB), which is thought to replace the role fatty acids play in most vertebrates as a predominant fuel source in extrahepatic tissues. To determine the mobilization of metabolites and corticosteroids during a stress event, North Pacific spiny dogfish, Squalus acanthias suckleyi, were cannulated and held in a darkened isolation box to recover (24-48 h) before being subjected to an acute air exposure or corticosterone injection. Dogfish were then serially blood sampled at nine timepoints over 48 h. Glucose, ß-HB, 1α-OHB, corticosterone, as well as lactate, pH, and osmolality were quantified in plasma samples. All measured variables increased in control and treatment groups within 48 h from the start of experimentation, and ß-HB and 1α-OHB remained elevated for the duration of the experiment. There was no linear correlation between glucose and 1α-OHB, but there was a weak (R2 = 0.230) although significant (p = 0.001), positive correlation between ß-HB and 1α-OHB. Interestingly, there were also significant correlations between increasing circulating glucose and corticosterone (R2 = 0.349; p < 0.001), and decreasing ß-HB and corticosterone concentrations (R2 = 0.180; p = 0.008). Our data suggest that following successive stressors of capture, surgery, and confinement, 1α-OHB was not correlated with circulating glucose, only weakly correlated with circulating ß-HB concentrations (R2 = 0.230; p = 0.001), and that corticosterone may also serve a role in energy mobilization in this species.

Life through a wider scope: Brook Trout (Salvelinus fontinalis) exhibit similar aerobic scope across a broad temperature range.

Brook Trout (Salvelinus fontinalis) have been widely introduced throughout the world and are often considered as direct competitors with native salmonid species. Metabolic rate is one metric we can examine to improve our understanding of how well fish perform in different habitats, including across temperature gradients, as metabolism can be directly influenced by environmental temperatures in ectotherms. We estimated the standard metabolic rate, maximum metabolic rate, and aerobic scope of lab-reared juvenile Brook Trout (~1 year) using intermittent-flow respirometry across a range of temperatures (5-23 °C) likely experienced in the wild. We included a diurnal temperature cycle of ±1.5 °C for each treatment temperature to simulate temporal variation observed in natural waterbodies. Standard metabolic rate and maximum metabolic rate both increased with acclimation temperature before appearing to plateau around 20 °C, while mass specific aerobic scope was found to increase from a mean of 287.25 ± 13.03 mg O2·kg-1·h-1 at 5 °C to 384.85 ± 13.31 mg O2·kg-1·h-1 at 15 °C before dropping at higher temperatures. Although a slight peak was found at 15 °C, the generally flat thermal performance curve for aerobic scope suggests Brook Trout are capable of adjusting to a relatively wide range of thermal regimes, appearing to be eurythermal, or a thermal generalist, at least for salmonids. The ability of this population to maintain similar physiological performance across a wide range of temperatures may help explain why Brook Trout succeed in a variety of different thermal habitats.

Sub-lethal temperature thresholds indicate acclimation and physiological limits in brook trout Salvelinus fontinalis.

The upper thermal tolerance of brook trout Salvelinus fontinalis was estimated using critical thermal maxima (CTmax ) experiments on fish acclimated to temperatures that span the species' thermal range (5-25°C). The CTmax increased with acclimation temperature but plateaued in fish acclimated to 20, 23 and 25°C. Plasma lactate was highest, and the hepato-somatic index (IH ) was lowest at 23 and 25°C, which suggests additional metabolic costs at those acclimation temperatures. The results suggest that there is a sub-lethal threshold between 20 and 23°C, beyond which the fish experience reduced physiological performance.

Increased reactive oxygen species production and maintenance of membrane potential in VDAC-less Neurospora crassa mitochondria.

The highly abundant voltage-dependent anion-selective channel (VDAC) allows transit of metabolites across the mitochondrial outer membrane. Previous studies in Neurospora crassa showed that the LoPo strain, expressing 50% of normal VDAC levels, is indistinguishable from wild-type (WT). In contrast, the absence of VDAC (ΔPor-1), or the expression of an N-terminally truncated variant VDAC (ΔN2-12porin), is associated with deficiencies in cytochromes b and aa3 of complexes III and IV and concomitantly increased alternative oxidase (AOX) activity. These observations led us to investigate complex I and complex II activities in these strains, and to explore their mitochondrial bioenergetics. The current study reveals that the total NADH dehydrogenase activity is similar in mitochondria from WT, LoPo, ΔPor-1 and ΔN2-12porin strains; however, in ΔPor-1 most of this activity is the product of rotenone-insensitive alternative NADH dehydrogenases. Unexpectedly, LoPo mitochondria have increased complex II activity. In all mitochondrial types analyzed, oxygen consumption is higher in the presence of the complex II substrate succinate, than with the NADH-linked (complex I) substrates glutamate and malate. When driven by a combination of complex I and II substrates, membrane potentials (Δψ) and oxygen consumption rates (OCR) under non-phosphorylating conditions are similar in all mitochondria. However, as expected, the induction of state 3 (phosphorylating) conditions in ΔPor-1 mitochondria is associated with smaller but significant increases in OCR and smaller decreases in Δψ than those seen in wild-type mitochondria. High ROS production, particularly in the presence of rotenone, was observed under non-phosphorylating conditions in the ΔPor-1 mitochondria. Thus, the absence of VDAC is associated with increased ROS production, in spite of AOX activity and wild-type OCR in ΔPor-1 mitochondria.

Effects of repeated daily acute heat challenge on the growth and metabolism of a cold water stenothermal fish.

Temperature is an important environmental factor influencing fish physiology that varies both spatially and temporally in ecosystems. In small north temperate zone lakes, cold water piscivores rely on nearshore prey; however, this region exceeds the optimal temperature of the foraging species during summer. To cope, piscivores make short excursions into the nearshore to feed and return to cold water to digest their meal, but the physiological impacts of these repeated acute exposures to warm water are not well understood. We exposed juvenile lake trout (Salvelinus namaycush) to treatments where they were held at ∼10°C and exposed to either 17 or 22°C for 5-10 min daily for 53 days mimicking warm-water forays. Control fish, held at an average temperature of ∼10°C but not exposed to thermal variation, consumed more food and grew slightly faster than heat challenged fish, with no clear differences in body condition, hepatosomatic index, ventricle mass, or muscle concentrations of lactate dehydrogenase and cytochrome c oxidase. Aerobic metabolic rates measured at 10°C indicated that standard metabolic rates (SMR) were similar among treatments; however, fish that were repeatedly exposed to 17°C had higher maximum metabolic rates (MMR) and aerobic scopes (AS) than control fish and those repeatedly exposed to 22°C. There were no differences in MMR or AS between fish exposed to 22°C and control fish. These results suggest that although SMR of fish are robust to repeated forays into warmer environments, MMR displays plasticity, allowing fish to be less constrained aerobically in cold water after briefly occupying warmer waters.

Impact of climate change on the American lobster (Homarus americanus): Physiological responses to combined exposure of elevated temperature and pCO2.

The physiological consequences of exposing marine organisms to predicted future ocean scenarios, i.e. simultaneous increase in temperature and pCO2, have only recently begun to be investigated. Adult American lobster (Homarus americanus) were exposed to either current (16 °C, 47 Pa pCO2, pH 8.10) or predicted year 2300 (20 °C, 948 Pa pCO2, pH 7.10) ocean parameters for 14-16 days prior to assessing physiological changes in their hemolymph parameters as well as whole animal ammonia excretion and resting metabolic rate. Acclimation of lobster simultaneously to elevated pCO2 and temperature induced a prolonged respiratory acidosis that was only partially compensated for via accumulation of extracellular HCO3- and ammonia. Furthermore, acclimated animals possessed significantly higher ammonia excretion and oxygen consumption rates suggesting that future ocean scenarios may increase basal energetic demands on H. americanus. Enzyme activity related to protein metabolism (glutamine dehydrogenase, alanine aminotransferase, and aspartate aminotransferase) in hepatopancreas and muscle tissue were unaltered in future ocean scenario exposed animals; however, muscular citrate synthase activity was reduced suggesting that, while protein catabolism may be unchanged, the net energetic output of muscle may be compromised in future scenarios. Overall, H. americanus acclimated to ocean conditions predicted for the year 2300 appear to be incapable of fully compensating against climate change-related acid-base challenges and experience an increase in metabolic waste excretion and oxygen consumption. Combining our study with past literature on H. americanus suggests that the whole lifecycle from larvae to adult stages is at risk of severe growth, survival and reproductive consequences due to climate change.

Ammonia excretion and acid-base regulation in the American horseshoe crab, Limulus polyphemus.

Many studies have investigated ammonia excretion and acid-base regulation in aquatic arthropods, yet current knowledge of marine chelicerates is non-existent. In American horseshoe crabs (Limulus polyphemus), book gills bear physiologically distinct regions: dorsal and ventral half-lamellae, a central mitochondria-rich area (CMRA) and peripheral mitochondria-poor areas (PMPAs). In the present study, the CMRA and ventral half-lamella exhibited characteristics important for ammonia excretion and/or acid-base regulation, as supported by high expression levels of Rhesus-protein 1 (LpRh-1), cytoplasmic carbonic anhydrase (CA-2) and hyperpolarization-activated cyclic nucleotide-gated K+ channel (HCN) compared with the PMPA and dorsal half-lamella. The half-lamellae displayed remarkable differences; the ventral epithelium was ion-leaky whereas the dorsal counterpart possessed an exceptionally tight epithelium. LpRh-1 was more abundant than Rhesus-protein 2 (LpRh-2) in all investigated tissues, but LpRh-2 was more prevalent in the PMPA than in the CMRA. Ammonia influx associated with high ambient ammonia (HAA) treatment was counteracted by intact animals and complemented by upregulation of branchial CA-2, V-type H+-ATPase (HAT), HCN and LpRh-1 mRNA expression. The dorsal epithelium demonstrated characteristics of active ammonia excretion. However, an influx was observed across the ventral epithelium as a result of the tissue's high ion conductance, although the influx rate was not proportionately high considering the ∼3-fold inwardly directed ammonia gradient. These novel findings suggest a role for the coxal gland in excretion and in the maintenance of hemolymph ammonia regulation under HAA. Hypercapnic exposure induced compensatory respiratory acidosis and partial metabolic depression. Functional differences between the two halves of a branchial lamella may be physiologically beneficial in reducing the backflow of waste products into adjacent lamellae, especially in fluctuating environments where ammonia levels can increase.

The physiological stress response of the Atlantic stingray (Hypanus sabinus) to aerial exposure.

Although secondary stress physiology of elasmobranchs is fairly well studied, gaps remain in our understanding of species differences, including stress recovery. We examined the physiological stress response to air exposure in Atlantic stingrays (Hypanus sabinus) using a serial sampling method requiring minimal handling. Many elasmobranch stress studies exclusively quantify glucose, although there is evidence that elasmobranchs are unusually reliant on ketone bodies. Therefore, we also tested the hypothesis that ketone bodies play a significant role in the elasmobranch stress response by examining plasma ß-hydroxybutyrate. Plasma osmolality, urea, trimethylamine-N-oxide, and a suite of ions were also measured to characterize departures from homeostasis due to air exposure. H. sabinus were exposed to air for 30 min and serially sampled at 0, 15, and 30 min, as well as 48 h after the stressor to assess the extent of recovery. Blood lactate and acidosis increased significantly during the stressor and returned to basal levels by 48 h. Glucose values were significantly affected, with the highest values observed at 48 h, suggesting that animals were not fully recovered as initially indicated by other metrics. Average plasma ß-hydroxybutyrate was unaffected by the stressor. This suggests that ketone bodies may not be a major fuel source used during acute stress, at least in the timeframe examined.

A radical shift in perspective: mitochondria as regulators of reactive oxygen species.

Mitochondria are widely recognized as a source of reactive oxygen species (ROS) in animal cells, where it is assumed that over-production of ROS leads to an overwhelmed antioxidant system and oxidative stress. In this Commentary, we describe a more nuanced model of mitochondrial ROS metabolism, where integration of ROS production with consumption by the mitochondrial antioxidant pathways may lead to the regulation of ROS levels. Superoxide and hydrogen peroxide (H2O2) are the main ROS formed by mitochondria. However, superoxide, a free radical, is converted to the non-radical, membrane-permeant H2O2; consequently, ROS may readily cross cellular compartments. By combining measurements of production and consumption of H2O2, it can be shown that isolated mitochondria can intrinsically approach a steady-state concentration of H2O2 in the medium. The central hypothesis here is that mitochondria regulate the concentration of H2O2 to a value set by the balance between production and consumption. In this context, the consumers of ROS are not simply a passive safeguard against oxidative stress; instead, they control the established steady-state concentration of H2O2 By considering the response of rat skeletal muscle mitochondria to high levels of ADP, we demonstrate that H2O2 production by mitochondria is far more sensitive to changes in mitochondrial energetics than is H2O2 consumption; this concept is further extended to evaluate how the muscle mitochondrial H2O2 balance should respond to changes in aerobic work load. We conclude by considering how differences in the ROS consumption pathways may lead to important distinctions amongst tissues, along with briefly examining implications for differing levels of activity, temperature change and metabolic depression.

Does the physiology of chondrichthyan fishes constrain their distribution in the deep sea?

The deep sea is the largest ecosystem on Earth but organisms living there must contend with high pressure, low temperature, darkness and scarce food. Chondrichthyan fishes (sharks and their relatives) are important consumers in most marine ecosystems but are uncommon deeper than 3000 m and exceedingly rare, or quite possibly absent, from the vast abyss (depths >4000 m). By contrast, teleost (bony) fishes are commonly found to depths of ∼ 8400 m. Why chondrichthyans are scarce at abyssal depths is a major biogeographical puzzle. Here, after outlining the depth-related physiological trends among chondrichthyans, we discuss several existing and new hypotheses that implicate unique physiological and biochemical characteristics of chondrichthyans as potential constraints on their depth distribution. We highlight three major, and not mutually exclusive, working hypotheses: (1) the urea-based osmoregulatory strategy of chondrichthyans might conflict with the interactive effects of low temperature and high pressure on protein and membrane function at great depth; (2) the reliance on lipid accumulation for buoyancy in chondrichthyans has a unique energetic cost, which might increasingly limit growth and reproductive output as food availability decreases with depth; (3) their osmoregulatory strategy may make chondrichthyans unusually nitrogen limited, a potential liability in the food-poor abyss. These hypotheses acting in concert could help to explain the scarcity of chondrichthyans at great depths: the mechanisms of the first hypothesis may place an absolute, pressure-related depth limit on physiological function, while the mechanisms of the second and third hypotheses may limit depth distribution by constraining performance in the oligotrophic abyss, in ways that preclude the establishment of viable populations or lead to competitive exclusion by teleosts.

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.

Ammonia excretion in Caenorhabditis elegans: Physiological and molecular characterization of the rhr-2 knock-out mutant.

Previous studies have shown the free living soil nematode Caenorhabditis elegans (N2 strain) to be ammonotelic. Ammonia excretion was suggested to take place partially via the hypodermis, involving the Na(+)/K(+)-ATPase (NKA), V-ATPase (VAT), carbonic anhydrase, NHX-3 and a functional microtubule network and at least one Rh-like ammonia transporter RHR-1. In the current study, we show that a second Rh-protein, RHR-2, is highly expressed in the hypodermis, here also in the apical membrane of that tissue. To further characterize the role of RHR-2 in ammonia excretion, a knock-out mutant rhr-2 (ok403), further referred to as ∆rhr-2, was employed. Compared to wild-type worms (N2), this mutant showed a lower rate of ammonia excretion and a lower hypodermal H(+) excretion rate. At the same time rhr-1, nka, vat, and nhx-3 showed higher mRNA expression levels when compared to N2. Also, in contrast to N2 worms, ∆rhr-2 did not show enhanced ammonia excretion rates when exposed to a low pH environment, suggesting that RHR-2 represents the apical NH3 pathway that allows ammonia trapping via the hypodermis in N2 worms. A hypothetical model for the mechanism of hypodermal ammonia excretion is proposed on the basis of data in this and previous investigations.

Mechanism of ammonia excretion in the freshwater leech Nephelopsis obscura: characterization of a primitive Rh protein and effects of high environmental ammonia.

Remarkably little is known about nitrogenous excretion in freshwater invertebrates. In the current study, the nitrogen excretion mechanism in the carnivorous ribbon leech, Nephelopsis obscura, was investigated. Excretion experiments showed that the ribbon leech is ammonotelic, excreting 166.0 ± 8.6 nmol·grams fresh weight (gFW)(-1)·h(-1) ammonia and 14.7 ± 1.9 nmol·gFW(-1)·h(-1) urea. Exposure to high and low pH hampered and enhanced, respectively, ammonia excretion rates, indicating an acid-linked ammonia trapping mechanism across the skin epithelia. Accordingly, compared with body tissues, the skin exhibited elevated mRNA expression levels of a newly identified Rhesus protein and at least in tendency the Na(+)/K(+)-ATPase. Pharmacological experiments and enzyme assays suggested an ammonia excretion mechanism that involves the V-ATPase, Na(+)/K(+)-ATPase, and carbonic anhydrase, but not necessarily a functional microtubule system. Most importantly, functional expression studies of the identified Rh protein cloned from leech skin tissue revealed an ammonia transport capability of this protein when expressed in yeast. The leech Rh-ammonia transporter (NoRhp) is a member of the primitive Rh protein family, which is a sister group to the common ancestor of vertebrate ammonia-transporting Rh proteins. Exposure to high environmental ammonia (HEA) caused a new adjustment of body ammonia, accompanied with a decrease in NoRhp and Na(+)/K(+)-ATPase mRNA levels, but unaltered ammonia excretion rates. To our knowledge, this is only the second comprehensive study regarding the ammonia excretion mechanisms in a freshwater invertebrate, but our results show that basic processes of ammonia excretion appear to also be comparable to those found in freshwater fish, suggesting an early evolution of ionoregulatory mechanisms in freshwater organisms.

Ammonia excretion in Caenorhabditis elegans: mechanism and evidence of ammonia transport of the Rhesus protein CeRhr-1.

The soil-dwelling nematode Caenorhabditis elegans is a bacteriovorous animal, excreting the vast majority of its nitrogenous waste as ammonia (25.3±1.2 µmol gFW(-1) day(-1)) and very little urea (0.21±0.004 µmol gFW(-1) day(-1)). Although these roundworms have been used for decades as genetic model systems, very little is known about their strategy to eliminate the toxic waste product ammonia from their bodies into the environment. The current study provides evidence that ammonia is at least partially excreted via the hypodermis. Starvation reduced the ammonia excretion rates by more than half, whereas mRNA expression levels of the Rhesus protein CeRhr-2, V-type H(+)-ATPase (subunit A) and Na(+)/K(+)-ATPase (α-subunit) decreased correspondingly. Moreover, ammonia excretion rates were enhanced in media buffered to pH 5 and decreased at pH 9.5. Inhibitor experiments, combined with enzyme activity measurements and mRNA expression analyses, further suggested that the excretion mechanism involves the participation of the V-type H(+)-ATPase, carbonic anhydrase, Na(+)/K(+)-ATPase, and a functional microtubule network. These findings indicate that ammonia is excreted, not only by apical ammonia trapping, but also via vesicular transport and exocytosis. Exposure to 1 mmol l(-1) NH4Cl caused a 10-fold increase in body ammonia and a tripling of ammonia excretion rates. Gene expression levels of CeRhr-1 and CeRhr-2, V-ATPase and Na(+)/K(+)-ATPase also increased significantly in response to 1 mmol l(-1) NH4Cl. Importantly, a functional expression analysis showed, for the first time, ammonia transport capabilities for CeRhr-1 in a phylogenetically ancient invertebrate system, identifying these proteins as potential functional precursors to the vertebrate ammonia-transporting Rh-glycoproteins.

Intertissue differences for the role of glutamate dehydrogenase in metabolism.

The enzyme glutamate dehydrogenase (GDH) plays an important role in integrating mitochondrial metabolism of amino acids and ammonia. Glutamate may function as a respiratory substrate in the oxidative deamination direction of GDH, which also yields α-ketoglutarate. In the reductive amination direction GDH produces glutamate, which can then be used for other cellular needs such as amino acid synthesis via transamination. The production or removal of ammonia by GDH is also an important consequence of flux through this enzyme. However, the abundance and role of GDH in cellular metabolism varies by tissue. Here we discuss the different roles the house-keeping form of GDH has in major organs of the body and how GDH may be important to regulating aspects of intermediary metabolism. The near-equilibrium poise of GDH in liver and controversy over cofactor specificity and regulation is discussed, as well as, the role of GDH in regulation of renal ammoniagenesis, and the possible importance of GDH activity in the release of nitrogen carriers by the small intestine.