Energetic Macroevolution of Invertebrates

An analysis of research data and published data on comparable standard metabolism in invertebrates is carried out. It is shown that this parameter varies insignificantly within each family and most orders. The mean values of the comparable standard metabolism for orders are grouped around certain values. It is assumed that these values correspond to stationary states, which organisms seek to reach during the evolutionary process. A total of nine levels of stationary states have been identified. The ratio of the values of comparable standard metabolism for neighboring levels is approximately 2.25. The orders occupy ever higher stationary levels in the process of macroevolution of phyla and classes. A possible mechanism for the transition from one stationary level to another is discussed.     

Effect of Fluctuations in Temperature on the Metabolism of Intertidal Invertebrates

SVNOPSIS. Compensatory changes in the level of activity of intertidalorganisms may occur in response to the thermal conditions whichprevail in the habitat. These involve an increase in the activityof those animals subjected to low temperatures and a correspondingsuppression of activity in those animals subjected to high temperaturesso that organisms with a wide geographical range, or livingover a range of shore levels, have comparable rates of activity.However, the rate of activity varies markedly with short-termfluctuations in temperature, as does the rate of respirationof active animals. Recent evidence suggests that the rate ofrespiration of quiescent animals is relatively independent oftemperature over the normal environmental range, and thus themetabolism is well-suited to an environment where rapid fluctuationsin temperature occur. Further, the extent of the thermal rangeover which metabolism is relatively independent of temperatureis modifiable according to season and storage-temperature. Similarchanges occur in the respiration of cell-free homogenates ofcertain intertidal organisms. Acclimation in such organismsinvolves not only a modification in the level of the activeand standard rates of metabolism but also an alteration in theform of the rate/temperature curve such that the range of temperature-independentmetabolism is appropriate to the thermal conditions prevailingin the habitat.     

The importance of arginine in evolution

The special chemistry and metabolism of arginine has been considered in relation to the evolution of metabolic and structural features of animals. Arginine and compounds derived from it act in muscle as a major reserve of ATP and as a regulatory sink for phosphate. The metabolism of arginine in less complex animals has been extended to produce urea, firstly as an osmotic regulator and subsequently as a means for terrestrial animals to excrete surplus nitrogen. The ornithine component of arginine is metabolised to form important polyamines and also proline. It is also argued that the change from phosphoarginine to phosphocreatine was a permissive step in the development of the vertebrates which are rich in connective tissue.     

SENSORY ADAPTATION AND THE STATIONARY STATE

1. Experiments are described which measure the sensitivity of animals exposed to continued illumination to which they have become adapted. It is shown that the amount of outside light energy necessary to stimulate an adapted animal increases with the intensity of the adapting illumination. 2. The data are analyzed quantitatively in terms of the reversible reaction S ⇌ P + A shown previously to account for the photic sensitivity of these animals. This analysis demonstrates that, though the amount of incident energy necessary for a minimal response varies with the adapting intensity, the actual amount of photochemical decomposition required to set off the sensory mechanism is a constant quantity. 3. The ability of these animals to come into sensory equilibrium with any sustained illumination is accounted for quantitatively by the presence of a stationary state in the reversible photochemical reaction S ⇌ P + A during which the concentrations of the three components are constant. 4. It is shown that the concentrations of these substances at the stationary state are automatically controlled by the outside intensity. Therefore, given the sensory mechanism as a basis, the adaptation of the animals to light and the consequent changes in sensitivity, are determined entirely by the light to which the animals are exposed. 5. Because of the properties of the stationary state, and of the constancy of photochemical decomposition for a minimal effect, it is suggested that the sensory system is not only the traditional receptor system, but is also a protecting layer which stabilizes and buffers the relation between the nervous system and the environment.     

Cardiac activity of freshwater crayfish at wakefulness, rest, and “animal hypnosis”

In laboratory experiments, simultaneous continuous recording of the circadian cycle of behavioral reactions and cardiac activity of freshwater crayfish Astacus astacus and Procambarus clarkii was carried out. A non-invasive fiber-optic method of recording of the heart rate (HR) was used. The obtained data were analyzed by the method of variational pulsometry (VP). It was revealed that certain values of HR and characteristics of VP corresponded to the physiological states of active wakefulness and rest. It was found that during long immobilization of crayfish it is possible to identify the states of operative rest and sleep by the animal behavioral reactions and parameters of cardiac activity. Crayfish were studied in the state of artificially evoked immobilization (“animal hypnosis”). During this state, a high HR level, an increase of indexes of tension and autonomic equilibrium, and changes of other VP characteristics were observed. It is suggested that the different level of cardiac activity in different physiological states of crayfish (active wakefulness, operative rest, sleep, and “animal hypnosis”) is regulated by nervous influences analogous to sympathetic and parasympathetic influences in vertebrates. It was concluded that freshwater crayfish as a representative of the highly organized invertebrates can serve an effective model for studying mechanisms of sleep-like states and “animal hypnosis” in animals.     

Metabolic rate depression and biochemical adaptation in anaerobiosis, hibernation and estivation

For many animals, the best defense against harsh environmental conditions is an escape to a hypometabolic or dormant state. Facultative metabolic rate depression is the common adaptive strategy of anaerobiosis, hibernation, and estivation, as well as a number of other arrested states. By reducing metabolic rate by a factor ranging from 5 to 100 fold or more, animals gain a comparable extension of survival time that can support months or even years of dormancy. The present review focuses on the molecular control mechanisms that regulate and coordinate cellular metabolism for the transition into dormancy. These include reversible control over the activity state of enzymes via protein phosphorylation or dephosphorylation reactions, pathway regulation via the association or dissociation of particle-bound enzyme complexes, and fructose-2,6-bisphosphate regulation of the use of carbohydrate reserves for biosynthetic purposes. These mechanisms, their interactions, and the regulatory signals (e.g., second messenger molecules, pH) that coordinate them form a common molecular basis for metabolic depression in anoxia-tolerant vertebrates (goldfish, turtles) and invertebrates (marine molluscs), hibernation in small mammals, and estivation in land snails and terrestrial toads.     

A comparison of the electrophoretic haemoglobin patterns of the vertebrates

A comparative study of the haemoglobins of 648 animals distributed over 300 species of vertebrates and belonging to different classes has been made by horizontal starch gel electrophoresis is tris EDTA-borate pH 8.6. Multiple haemoglobins were found within the different classes. The percentage of multiple haemoglobins varies from class to class. The frequency-distribution of the mobility values of haemoglobins is rather specific for the different classes and in certain instances for the different orders.     

Flux regulation in glycogen-induced oscillatory glycolysis in cell-free extracts of Saccharomyces carlsbergensis

To localise the controlling point of the glycolytic system, the temporal changes of concentrations of glycolytic intermediates have been analysed after addition of glycogen to a substrate-depleted yeast extract. Three sequential metabolic states are clearly observable: a transition state at which there is continuous accumulation of the intermediates before the glyceraldehydephosphate dehydrogenase (GAPDH, EC 1.2.1.12) step; a stationary state with all glycolytic intermediates having concentrations oscillating at nearly stationary mean values; and a depletion state at which the intermediates before the GAPDH step are being depleted due to the exhaustion of glycogen. In all these states, the mean ethanol production rate and the concentration of ATP and the intermediates beyond the GAPDH-step are maintained fairly constant, while the glycogen consumption rate and intermediate concentrations of the upper part of the glycolytic system change considerably: the glycogen consumption rate varies 4-fold and fructose-bis-phosphate concentration more than 10-fold. Doubling of the initial glycogen concentration and the addition of a great excess of fructose-bis-phosphate do not affect the ethanol production rate and the mean glycerate-3-phosphate (3-PGA) and pyruvate levels. By contrast, ethanol production was accelerated by an increase of the net ATP consumption rate resulting from either the addition of apyrase or by substitution of trehalose for glycogen. Neither the mean absolute ATP level nor the adenylate energy charge were measurably affected, however all this data can be interpreted in terms of a very strong stoichiometric regulation and stabilization of the lower part of the glycolytic system.     

Food ingestion and digestive transit time in the euphausiid Meganyctiphanes norvegica as a function of animal size

Laboratory experiments with Meganyctiphanes norvegica of differentsizes (7 to 70 mg dry) fed on Artemia salina show that the dailyspecific ingestion rate varies with animal weight accordingto a power law with a negative exponent of -0.58. This relationis used to calculate the production rate of faeces which ishigher in the smaller individuals when expressed as a percentageof animal weight; a comparison of the M. norvegica data withpublished data for Calanus helgolandicus indicates that thisrate is higher for the smaller species. Under the experimentalconditions used the digestive transit time of food in M. norvegicais appreciably shorter in smaller animals (15 minutes versus30 minutes). The data, combined with other data previously obtainedfor the same ipecies, show that the metabolism of active, well-fedindividuals is about three times higher than the standard metabolism.     

The Use of Gait Transition Speed in Comparative Studies of Fish Locomotion

Physiological and biomechanical inquiries into the principlesof vertebrate locomotion require comparison among animals ofdifferent size, habitat and phyletic association. In designingcomparative studies of locomotion, a major challenge is to isolatethe effects of experimentally imposed variation from the confoundingeffects of variation in animal activity level associated withdifferences in scale and life history. For swimming vertebrates,traditional measures of speed used for comparison, includingsprint speed and critical swimming speed should in theory eachelicit similar efforts from different animals but have practicalshortcomings that can limit their usefulness. This paper presentsan alternative approach, adapted from the work of mammalianphysiologists, which controls for differences in relative activitylevel among swimming animals of different size and habitat throughcomparison at gait transition speeds. The method is illustratedwith examples from study of the teleost fish family Embiotocidae,whose members exhibit a distinct transition from exclusivelypectoral fin oscillation to combined pectoral and caudal finpropulsion with increasing swimming speed. The pectoral-caudalgait transition speed, or any percentage thereof, is shown tobe 'biomechanically equivalent for swimmers of different size.When this performance limit is expressed in terms of body lengthstraveled per unit time, a common normalization of swimming speed,it varies markedly across size and habitat within the family.This finding has the important implication that length specificspeeds may not induce comparable degrees of exercise from differentfishes, and thus kinematic and physiological comparisons atsuch speeds can yield misleading results. The comparative approachdescribed for pectoral fin swimmers, and the limitations oflength-specific speed, should be generally applicable to studiesof other swimming vertebrates.     

The metabolism of 4-androstene-3,17-dione by isolated rat hepatocytes. Maintenance of the sex differences in culture

Hepatocytes from adult rats were isolated and cultivated as primary monolayers for 3 days in a medium containing only 1% homologous serum. The metabolism of 4-androstene-3,17-dione was followed in hepatocytes from male, female and hypophysectomized animals. It was found that immediately after preparation, 5alpha-reductase and 16alpha-hydroxylase activities were present in the cells in amounts comparable to those found in microsomal preparations from liver homogenates. After 3 days in culture, these enzyme activities had decreased to about half the values measured on day 0. During this time the sexual differences in steroid metabolism in the cells were stable, i.e. there was no detectable induction of 16alpha-hydroxylase in cells from female animals, and the relative sex difference in 5alpha-reductase activity persisted.     

Characterization of a microsomal retinol dehydrogenase gene from amphioxus: retinoid metabolism before vertebrates

Amphioxus, a member of the subphylum Cephalochordata, is thought to be the closest living relative to vertebrates. Although these animals have a vertebrate-like response to retinoic acid, the pathway of retinoid metabolism remains unknown. Two different enzyme systems - the short chain dehydrogenase/reductases and the cytosolic medium-chain alcohol dehydrogenases (ADHs) - have been postulated in vertebrates. Nevertheless, recent data show that the vertebrate-ADH1 and ADH4 retinol-active forms originated after the divergence of cephalochordates and vertebrates. Moreover, no data has been gathered in support of medium-chain retinol active forms in amphioxus. Then, if the cytosolic ADH system is absent and these animals use retinol, the microsomal retinol dehydrogenases could be involved in retinol oxidation. We have identified the genomic region and cDNA of an amphioxus Rdh gene as a preliminary step for functional characterization. Besides, phylogenetic analysis supports the ancestral position of amphioxus Rdh in relation to the vertebrate forms.     

Control of oxidative phosphorylation by the extramitochondrial ATP/ADP ratio

The control of mitochondrial ATP synthesis by the extramitochondrial adenine nucleotide pattern was investigated with rat liver mitochondria. It is demonstrated that any stationary state between the two limit states of maximum activity (state 3) and of resting activity (state 4) can be obtained by a hexokinase-glucose trap as an ADP-regenerating system. These intermediate states are characterized by stationary respiratory rates, stationary redox levels of the cytochromes b and c and stationary levels of extramitochondrial ATP and ADP between the rates and levels of the limit states. At a constant concentration of inorganic phosphate the activity of mitochondria between the limit states is controlled by the extramitochondrial ATP/ADP ratio independent of the total concentration of adenine nucleotides present. The control range was found to be between ratios of about 5 and 100 at 10 mM phosphate. At lower ratios the mitochondria are in their maximum phosphorylating state. With succinate + rotenone and glutamate + malate the same control range was observed, indicating that it is independent of the nature of substrate oxidized.The results suggest that in the control range the mitochondrial activity is limited by the competition of ADP and ATP for the adenine nucleotide translocator.     

Vertebrate liver cytokeratins: a comparative study

The structure and composition of intermediate filaments isolated from liver of representatives of different vertebrate classes have been studied by electron microscopy and biochemical and immunochemical methods. It has been shown that the methodological approach for isolation of rat liver intermediate filaments can be efficiently applied to all other classes of vertebrates. The intermediate filaments studied have the same electron microscopic morphology and are species undistinguishable. The molecular weight of intermediate filament proteins varies from 40,000 to 60,000 and their isoelectric point varies from 5.0 to 6.45. Immunological investigations show that in all animals studied the intermediate filaments are built up of cytokeratins belonging to both types of keratins: type I and type II. Only one protein of the type II cytokeratins is present in all vertebrate classes, whereas in lower vertebrates two or even three type I cytokeratins contribute to the structure of liver intermediate filaments. The biochemical and immunochemical results are discussed with regard to the evolution of liver cytokeratins.     

The challenge hypothesis: Where it began and relevance to humans

A contribution to a special issue on Hormones and Human Competition.Over 40 years ago assay methods that allowed the accurate measurement of circulating levels of hormones were developed for the first time enabling us to sample free-living as well as captive animals. This led to a new concept called “field endocrinology”. It quickly became apparent that endocrine profiles of animals under natural conditions were very different from congeners in captivity. Furthermore, hormone data could be organized by functional units (e.g. reproductive states) spaced in time according to natural duration of those states rather than simply by date alone. This approach changed how we interpret data and revealed species-specific patterns of hormone secretion. The “challenge hypothesis”, stating that the temporal patterns of testosterone in blood were determined by a trade-off between the degree of male-male competition that increased testosterone, and the expression of paternal care that required a decrease in testosterone, grew out of a combination of field endocrine investigations that then informed laboratory experimentation. A strong argument can now be made that the challenge hypothesis is highly relevant for understanding social interactions in humans and non-human primates. Investigations on human subjects provide some of the best models for the challenge hypothesis. However, the central mechanisms by which aggressive and other social interactions regulate the hypothalamo-pituitary-gonad axis will depend upon work on not only primates, but also other vertebrates in very different ecological contexts. Research on the challenge hypothesis in humans will play a critical role as new insight on the interrelationships of testosterone and male-male competition comes from new technologies.     

Ameliorating effect of chromium administration on hepatic glucose metabolism in streptozotocin-induced experimental diabetes

Chromium has been recognized as an essential trace element that plays an important role in carbohydrate metabolism. However, the molecular mechanisms involved in its action are not clear. This study was undertaken to understand the mechanism of chromium action in experimental diabetes. Streptozotocin-induced diabetic animals were administered chromium as chromium picolinate (CrP) at a daily dose of 1 mg/kg body weight for a period of 4 weeks. It was observed that chromium complexed with picolinate was effective in lowering plasma glucose levels as well as was able to alleviate polyphagia, polydipsia, and weight loss in diabetic animals. Administration of chromium was also found to normalize glycogen content in liver of diabetic animals to near control levels. The reduction in plasma glucose levels by chromium was accompanied by increase in activity of glycolytic enzymes (e.g., glucokinase, phosphofructokinase, and pyruvate kinase) and by suppression in activity of gluconeogenic enzymes (e.g., glucose-6-phosphatase and phosphoenolpyruvate carboxykinase) in liver. Hepatic glucose uptake was found to be increased by chromium supplementation as demonstrated by decrease in Km and increase in Vmax values in diabetic animals. Chromium levels were lower in the liver of diabetic rats when compared with that of control rats. A negative correlation was observed between plasma glucose and chromium concentration in patients with diabetes. The data suggests that chromium supplementation as CrP is beneficial in correcting hyperglycemia, implying that the modulation of the glucose metabolism by chromium may be therapeutically beneficial in the treatment of diabetes.     

Membranes as possible pacemakers of metabolism.

Basal metabolic rate (BMR) varies dramatically among vertebrate species, both (i) being several fold higher in the endothermic mammals and birds compared to the ectothermic reptiles, amphibians and fish, and (ii) being much greater, on a body mass basis, in small vertebrates compared to large vertebrates. These differences in whole animal BMR are also manifest at the cellular level with substantial contributions to basal metabolic activity from the maintenance of various trans-membrane gradients. The percentage contribution of various processes to basal metabolism is remarkably consistent between different vertebrates and when BMR varies, the components of metabolic activity vary in relative unison. Membrane composition also varies between vertebrates and the degree of polyunsaturation of membrane phospholipids is correlated with cellular metabolic activity. In general, the tissue phospholipids and thus membrane bilayers of endotherms are more polyunsaturated than those from similar-sized ectotherms. In mammals membrane polyunsaturation is allometrically related to body mass. We suggest that membranes can act as pacemakers for overall metabolic activity. We propose that such membrane polyunsaturation increases the molecular activity of many membrane-bound proteins and consequently some specific membrane leak-pump cycles and cellular metabolic activity. We hypothesize a possible mechanistic basis for this effect that is based on a greater transfer of energy during intermolecular collisions of membrane proteins with the unsaturated two carbon units (C=C) of polyunsaturates compared to the single carbon units of saturated acyl chains, as well as the more even distribution of such units throughout the depth of the bilayer when membranes contain polyunsaturated acyl chains compared to monounsaturated ones. The proposed pacemaker role of differences in membrane bilayer composition is briefly discussed with respect to the brain (and sensory cells), evolution of mammalian endothermic metabolism, and its clinical implications for humans.     

Energy metabolism and the direction of evolutionary progress in the class of mammals

The data on dependence of standard metabolism on body weight in 518 mammalian species have been summarized. Standard metabolism differs in representatives of different classes and families. The higher it is, the more complicated is the organization of the animals. Comparison of standard metabolism in different classes of mammals with the time of their paleontological origin demonstrates that evolutionary process in mammals was directed towards the increase in energetic metabolism of animals.     

Prolonged lidocaine metabolizing activity of primary hepatocytes with spheroid culture using polyurethane foam as a culture substratum

Primary rat hepatocytes formed spheroids in the pores of polyurethane foam (PUF) used as a culture substratum. The hepatocytes in monolayer and spheroid stationary culture converted lidocaine to monoethylglycinexylidide (MEGX) which was N-deethylation of lidocaine. The metabolic activity of the hepatocytes/spheroid stationary culture system was 1.5∼2.0-fold higher than that of monolayer culture for 10 days. The activity of albumin production and cell survival of hepatocytes in monolayer and spheroid cultures decrease due to lidocaine treatment dependend on the lidocaine concentration, but the activity and cell survival in PUF/spheroid stationary culture were maintained at a higher level than that in monolayer culture under the lidocaine treatment. We developed a device for an in vitro liver model, drug metabolism simulator (DMS), using a PUF/spheroid packed-bed module including 4.00 ± 0.68 × 10⁷ hepatocytes and analyzed pharmacokinetics of lidocaine in a one-compartment model. Lidocaine clearance and extraction ratio of hepatocytes in the DMS corresponded to 1.354 ± 0.318 ml/min/g-liver and 0.677 ± 0.0159/g-liver, respectively (N=4). These values were comparable with in vivo values, 1.930 ml/min g-liver and 0.965/g-liver reported by Nyberg (1977). Consequently, PUF/spheroid culture maintained high lidocaine metabolizing activity over a long term and seems to provide a promising culture system as a drug metabolism simulator which will be used for drug screening, cytotoxicity tests and prediction of pharmacokinetics. This revised version was published online in July 2006 with corrections to the Cover Date.     

Sequence divergence and diversity suggests ongoing functional diversification of vertebrate NAD metabolism

NAD is not only an important cofactor in redox reactions but has also received attention in recent years because of its physiological importance in metabolic regulation, DNA repair and signaling. In contrast to the redox reactions, these regulatory processes involve degradation of NAD and therefore necessitate a constant replenishment of its cellular pool. NAD biosynthetic enzymes are common to almost all species in all clades, but the number of NAD degrading enzymes varies substantially across taxa. In particular, vertebrates, including humans, have a manifold of NAD degrading enzymes which require a high turnover of NAD. As there is currently a lack of a systematic study of how natural selection has shaped enzymes involved in NAD metabolism we conducted a comprehensive evolutionary analysis based on intraspecific variation and interspecific divergence. We compare NAD biosynthetic and degrading enzymes in four eukaryotic model species and subsequently focus on human NAD metabolic enzymes and their orthologs in other vertebrates. We find that the majority of enzymes involved in NAD metabolism are subject to varying levels of purifying selection. While NAD biosynthetic enzymes appear to experience a rather high level of evolutionary constraint, there is evidence for positive selection among enzymes mediating NAD-dependent signaling. This is particularly evident for members of the PARP family, a diverse protein family involved in DNA damage repair and programmed cell death. Based on haplotype information and substitution rate analysis we pinpoint sites that are potential targets of positive selection. We also link our findings to a three dimensional structure, which suggests that positive selection occurs in domains responsible for DNA binding and polymerization rather than the NAD catalytic domain. Taken together, our results indicate that vertebrate NAD metabolism is still undergoing functional diversification.