High-Throughput Tissue Bioenergetics Analysis Reveals Identical Metabolic Allometric Scaling for Teleost Hearts and Whole Organisms

Organismal metabolic rate, a fundamental metric in biology, demonstrates an allometric scaling relationship with body size. Fractal-like vascular distribution networks of biological systems are proposed to underlie metabolic rate allometric scaling laws from individual organisms to cells, mitochondria, and enzymes. Tissue-specific metabolic scaling is notably absent from this paradigm. In the current study, metabolic scaling relationships of hearts and brains with body size were examined by improving on a high-throughput whole-organ oxygen consumption rate (OCR) analysis method in five biomedically and environmentally relevant teleost model species. Tissue-specific metabolic scaling was compared with organismal routine metabolism (RMO₂), which was measured using whole organismal respirometry. Basal heart OCR and organismal RMO₂ scaled identically with body mass in a species-specific fashion across all five species tested. However, organismal maximum metabolic rates (MMO₂) and pharmacologically-induced maximum cardiac metabolic rates in zebrafish Danio rerio did not show a similar relationship with body mass. Brain metabolic rates did not scale with body size. The identical allometric scaling of heart and organismal metabolic rates with body size suggests that hearts, the power generator of an organism’s vascular distribution network, might be crucial in determining teleost metabolic rate scaling under routine conditions. Furthermore, these findings indicate the possibility of measuring heart OCR utilizing the high-throughput approach presented here as a proxy for organismal metabolic rate—a useful metric in characterizing organismal fitness. In addition to heart and brain OCR, the current approach was also used to measure whole liver OCR, partition cardiac mitochondrial bioenergetic parameters using pharmacological agents, and estimate heart and brain glycolytic rates. This high-throughput whole-organ bioenergetic analysis method has important applications in toxicology, evolutionary physiology, and biomedical sciences, particularly in the context of investigating pathogenesis of mitochondrial diseases.     

Metabolic level and size scaling of rates of respiration and growth in unicellular organisms

1.  Metabolic rate is conventionally assumed to scale with body mass to the 3/4-power, independently of the metabolic level of the organisms being considered. However, recent analyses in a variety of animals and plants indicate that the power (log–log slope) of this relationship varies significantly with metabolic level, ranging from c . 2/3 to 1.
2.  Here I show that the scaling slopes of rates of respiration and growth are related to the metabolic level of a variety of unicellular organisms, as similarly occurs for respiration rates in multicellular organisms.
3.  The recently proposed 'metabolic-level boundaries hypothesis' provides insight into these effects of metabolic level. As predicted, the scaling slopes for resting (endogenous) respiration rate in prokaryotes, algae and protozoans are negatively related to metabolic level; and in protozoans, the scaling slope increases with starvation. Also as predicted, the scaling slopes of growth rate in algae and protozoans are negatively related to growth level. Unexpectedly, opposite effects of starvation on the metabolic scaling slopes of unicellular prokaryotes (compared to that of eukaryotes) may be a spurious result of respiration measurements that did not adequately consider the effects of rapid cell multiplication in prokaryotes with extremely short generation times.
4.  Analyses of both unicellular and multicellular organisms show that there is no universal metabolic scaling relationship, and that variation in metabolic scaling relationships is systematically and possibly universally related to metabolic level.     

代谢异速生长理论及其在微生物生态学领域的应用

新陈代谢是生物的基本生理过程,影响生物在不同环境中参与物质循环和能量转化的过程.代谢速率作为生物体重要的生命过程指标,几乎影响所有的生物活性速率,且在很多研究中均表现出异速生长现象.所谓代谢异速是指生物体代谢速率与其个体大小(或质量)之间存在的幂函数关系.代谢异速生长理论的提出,从机制模型角度解释了代谢异速关系这一普遍存在的生命现象.该理论利用分形几何学及流体动力学等原理,从生物能量学角度阐释了异速生长规律的机理,证实了3/4权度指数的存在;但同时有研究表明,权度指数因环境因素等影响处于2/3-1范围之间而非定值.随着研究工作的深入,代谢异速生长理论研究从起初的宏观动植物领域拓展到了微生物领域,在研究微生物的代谢异速生长理论时,可将微生物的可操作分类单元(Operational taxonomic unit,OTU)或具有特定功能的功能群视为一个微生物个体,基于其遗传多样性和功能多样性特征进行表征,以便于将微生物群落多样性与其生态功能性联系起来,使该理论在微生物生态学领域得到有效的补充和完善.尽管细菌具有独特的生物学特性,但与宏观生物系统中观测到的现象表现出明显的一致性.有研究表明,3个农田土壤细菌基于遗传多样性的OTU数的平均周转率分别为0.71、0.80和0.84,介于2/3与1之间,可能与生物代谢异速指数有一定关联,为微生物代谢异速指数的研究提出了一个参考解决方案.鉴于微生物个体特征和生物学特性,在分析代谢速率与个体大小关系中,从微生物单位个体的定义、个体大小表征到计量单位的统一,仍需更多的理论支持.分析了代谢异速生长理论在微生物与生态系统功能关系研究中的可能应用,延伸了该理论的应用范围,并对尚待加强的研究问题进行了评述和展望.     

Supply-Side Constraints Are Insufficient to Explain the Ontogenetic Scaling of Metabolic Rate in the Tobacco Hornworm,Manduca sexta

Explanations for the hypoallometric scaling of metabolic rate through ontogeny generally fall into two categories: supply-side constraints on delivery of oxygen, or decreased mass-specific intrinsic demand for oxygen. In many animals, supply and demand increase together as the body grows, thus making it impossible to tease apart the relative contributions of changing supply and demand to the observed scaling of metabolic rate. In larval insects, the large components of the tracheal system are set in size at each molt, but then remain constant in size until the next molt. Larvae of Manduca sexta increase up to ten-fold in mass between molts, leading to increased oxygen need without a concomitant increase in supply. At the molt, the tracheal system is shed and replaced with a new, larger one. Due to this discontinuous growth of the tracheal system, insect larvae present an ideal system in which to examine the relative contributions of supply and demand of oxygen to the ontogenetic scaling of metabolic rate. We observed that the metabolic rate at the beginning of successive instars scales hypoallometrically. This decrease in specific intrinsic demand could be due to a decrease in the proportion of highly metabolically active tissues (the midgut) or to a decrease in mitochondrial activity in individual cells. We found that decreased intrinsic demand, mediated by a decrease in the proportion of highly metabolically active tissues in the fifth instar, along with a decrease in the specific mitochondrial activity, contribute to the hypoallometric scaling of metabolic rate.     

Long-term effects of physiological oxygen concentrations on glycolysis and gluconeogenesis in hepatocyte cultures

Primary cultures of adult rat hepatocytes were kept for 46 h with either insulin ('insulin cells') or glucagon ('glucagon cells') as the dominant hormone under different oxygen concentrations with 13% (v/v) O2 mimicking arterial and 4% hepatovenous levels. Thereafter metabolic rates were measured for a 2 h period under the same ('overall long-term O2 effects') or a different ('short-term O2 effects') oxygen concentration. From the differences of the two effects the 'intrinsic long-term O2 effects' were derived. Glycolysis, as measured in 'insulin-cells', was stimulated by low O2 levels. It was about threefold faster in cells cultured and tested under 4% O2 as compared to cells cultured and tested under 13% O2, indicating the overall long-term effect. Glycolysis was about twofold faster in cells cultured and tested under 4% O2 as compared to cells cultured under 4% O2 but tested under 13% O2, demonstrating the short-term effect. Glycolysis was about 1.5-fold faster in cells cultured and tested under 4% O2 as compared to cells cultured under 13% O2 but tested under 4% O2, showing the intrinsic long-term effect. This difference was roughly parallel to the difference in levels of glucokinase and pyruvate kinase. Gluconeogenesis, as measured in 'glucagon cells', was stimulated by high O2 levels. Similar to glycolysis overall long-term, short-term and intrinsic long-term effects could be distinguished. The intrinsic long-term effects determined under 13% O2 corresponded to a 1.5-fold stimulation and paralleled the difference in phosphoenolpyruvate carboxykinase levels. The present results show that physiological oxygen concentrations also modulate hepatic carbohydrate metabolism by long-term effects and that the O2 gradient over the liver parenchyma thus contributes to the metabolic differences between periportal and perivenous hepatocytes in vivo.     

Body condition helps to explain metabolic rate variation in wolf spiders

1. Metabolism is the fundamental process that powers life. Understanding what drives metabolism is therefore critical to our understanding of the ecology and behaviour of organisms in nature. 2. Metabolic rate generally scales with body size according to a power law. However, considerable unexplained variation in metabolic rate remains after accounting for body mass with scaling functions. 3. We measured resting metabolic rates (oxygen consumption) of 227 field‐caught wolf spiders. Then, we tested for effects of body mass, species, and body condition on metabolic rate. 4. Metabolic rate scales with body mass to the 0.85 power in these wolf spiders, and there are metabolic rate differences between species. After accounting for these factors, residual variation in metabolic rate is related to spider body condition (abdomen:cephalothorax ratio). Spiders with better body condition consume more oxygen. 5. These results indicate that recent foraging history is an important determinant of metabolic rate, suggesting that although body mass and taxonomic identity are important, other factors can provide helpful insights into metabolic rate variation in ecological communities.     

The proton leak across the mitochondrial inner membrane

The proton conductance of the mitochondrial inner membrane increases at high protonmotive force in isolated mitochondria and in mitochondria in situ in rat hepatocytes. Quantitative analysis of its importance shows that about 20-30% of the oxygen consumption by resting hepatocytes is used to drive a heat-producing cycle of proton pumping by the respiratory chain and proton leak back to the matrix. The flux control coefficient of the proton leak pathway over respiration rate varies between 0.9 and zero in mitochondria depending on the rate of respiration, and has a value of about 0.2 in hepatocytes. Changes in the proton leak pathway in situ will therefore change respiration rate. Mitochondria isolated from hypothyroid animals have decreased proton leak pathway, causing slower state 4 respiration rates. Hepatocytes from hypothyroid rats also have decreased proton leak pathway, and this accounts for about 30% of the decrease in hepatocyte respiration rate. Mitochondrial proton leak may be a significant contributor to standard metabolic rate in vivo.     

Model of oxygen transport and metabolism predicts effect of hyperoxia on canine muscle oxygen uptake dynamics.

Previous studies have shown that increased oxygen delivery, via increased convection or arterial oxygen content, does not speed the dynamics of oxygen uptake, Vo(2m), in dog muscle electrically stimulated at a submaximal metabolic rate. However, the dynamics of transport and metabolic processes that occur within working muscle in situ is typically unavailable in this experimental setting. To investigate factors affecting Vo(2m) dynamics at contraction onset, we combined dynamic experimental data across working muscle with a mechanistic model of oxygen transport and metabolism in muscle. The model is based on dynamic mass balances for O(2), ATP, and PCr. Model equations account for changes in cellular ATPase, oxidative phosphorylation, and creatine kinase fluxes in skeletal muscle during exercise, and cellular respiration depends on [ADP] and [O(2)]. Model simulations were conducted at different levels of arterial oxygen content and blood flow to quantify the effects of convection and diffusion of oxygen on the regulation of cellular respiration during step transitions from rest to isometric contraction in dog gastrocnemius muscle. Simulations of arteriovenous O(2) differences and (.)Vo(2m) dynamics were successfully compared with experimental data (Grassi B, Gladden LB, Samaja M, Stary CM, Hogan MC. J Appl Physiol 85: 1394-1403, 1998; and Grassi B, Gladden LB, Stary CM, Wagner PD, Hogan MC. J Appl Physiol 85: 1404-1412, 1998), thus demonstrating the validity of the model, as well as its predictive capability. The main findings of this study are: 1) the estimated dynamic response of oxygen utilization at contraction onset in muscle is faster than that of oxygen uptake; and 2) hyperoxia does not accelerate the dynamics of diffusion and consequently muscle oxygen uptake at contraction onset due to the hyperoxia-induced increase in oxygen stores. These in silico derived results cannot be obtained from experimental observations alone.     

The contribution of the leak of protons across the mitochondrial inner membrane to standard metabolic rate

This paper presents and assesses the hypothesis that the proton leak across the mitochondrial inner membrane is an important contributor to standard metabolic rate, and that increases in the amount of mitochondrial inner membrane may be important in causing changes in proton leak and in the standard metabolic rate. The standard metabolic rate of an animal is known to be a function of body mass, phylogeny and thyroid status, and is largely attributed to the metabolically active internal organs. The total area of mitochondrial inner membrane in these organs correlates well with standard metabolic rate over a wide range of body masses in both ectotherms and endotherms. In hepatocytes isolated from rats, proton leak across the mitochondrial inner membrane accounts for about 30% of the resting oxygen consumption, and the distribution of control over respiration suggests that changes in mitochondrial inner membrane surface area will be accompanied by significant changes in the proton leak. This change in the leak will result in significant changes in resting oxygen consumption, but changes in ATP demand may also have a role to play in determining resting respiration rate. Extrapolation of these results to other tissues and other animals suggests that the hypothesis has the potential to explain a substantial proportion of the variation in standard metabolic rate with body mass, phylogeny and thyroid status. However, in most cases the quantitative contribution of proton leak compared to cellular ATP turnover has yet to be experimentally determined.     

Reversible metabolic depression in hepatocytes of lamprey (Lampetra fluviatilis) during pre-spawning: regulation by substrate availability

The regulation of oxidative metabolism in hepatocytes of lampreys (Lampetra fluviatilis) during the freshwater pre-spawning period of their life cycle was studied. The energy metabolism in these cells is characterized by a simplified scheme, where glycolytic ATP production is insignificant and fatty acids are the major respiratory substrates. Seasonal changes in aerobic cell metabolism include a considerable reversible depression of metabolic rate in lamprey hepatocytes during the winter months of the pre-spawning period. The depression is characterized by a more than twofold decrease in hepatocyte endogenous respiration rate, a reduction of oxidative phosphorylation and drop in cellular ATP content. The addition of fatty acids to the hepatocyte incubation medium prevents the decrease in the metabolic rate. In spring, before spawning, a marked activation of energy metabolism in lamprey hepatocytes is found. These observations support the conclusion that the regulation of lamprey hepatocyte energy metabolism is realized through the availability of fatty acids for oxidation.     

Absence of metabolic rate allometry in an ex vivo model of mammalian skeletal muscle

Within mammalian species, standard metabolic rate (SMR) increases disproportionately with body mass (Mb), such that the mass-specific SMR correlates negatively with Mb. This phenomenon can be explained in part by reduced cellular metabolic rates in larger species. To better understand the cause(s) of this cellular metabolic rate allometry we have used an ex vivo approach to isolate and identify potential contributors. Skeletal myoblasts from mammalian species ranging inMb from 30 g to over 300,000 g were isolated and differentiated into myotubes in vitro. Oxygen consumption rates, citrate synthase (CS) activity, and lactate dehydrogenase (LDH) activity were measured in myotubes under standardized conditions. No correlation of any of these parameters was observedwith speciesMb, suggesting that there is no genetic contribution to between-species differences in cellular metabolic rates. Myotubes were incubated in serum from species ranging from 30 g to 400,000 g to determine whether between-species differences in the levels of metabolically important hormones might produce allometric trends in the cultured cells. However, there was no observed effect of serum donor Mb on any of the metabolic characteristicsmeasured. Thus, there is no evidence for a relationship between skeletal muscle oxidative metabolism and Mb in an ex vivo model.     

Radiosensitivity of parenchymal hepatocytes as a function of oxygen concentration

To investigate the mechanism(s) of hepatocyte radioresistance (D0 2.7 Gy), the radiosensitivities of respiring (37 degrees C) and nonrespiring (0 degrees C) hepatocytes were determined as a function of oxygen concentration. Fischer 344 female rat hepatocytes were isolated by liver perfusion, equilibrated in Leibowitz-15 media with different oxygen tensions, and exposed to 60Co radiation at either 37 or 0 degrees C. Cell survival and DNA single-strand breaks were used as the biological end points of radiosensitivity. The K value for respiring hepatocytes (37 degrees C) was 14.3 +/- 0.5 mm Hg O2 (18.8 +/- 0.7 mumol O2/liter), demonstrating that the K value for freshly isolated parenchymal hepatocytes is significantly greater than those previously obtained for cultured cells. In contrast, the K value for nonrespiring hepatocytes (0 degree C) is 1.4 +/- 0.4 mm Hg O2 (3.7 +/- 1.0 mumol O2/liter) indicating that hepatocyte respiration results in a plasma membrane-to-nucleus oxygen gradient of approximately 12.9 +/- 0.6 mm Hg (15.1 +/- 1.2 microns O2/liter). The hypothesis that the hepatic nucleus typically resides in a hypoxic condition, although the liver is uniformly perfused with well-oxygenated blood, is supported by (1) the nonradom perinuclear distribution of the mitochondria, (2) the high cellular respiration rate, and (3) the large intracellular oxygen diffusion distance in hepatocytes (25 microns diameter).     

Life-history Constraints on the Mechanisms that Control the Rate of ROS Production

The quest to understand why and how we age has led to numerous lines of investigation that have gradually converged to consider mitochondrial metabolism as a major player. During mitochondrial respiration a small and variable amount of the consumed oxygen is converted to reactive species of oxygen (ROS). For many years, these ROS have been perceived as harmful by-products of respiration. However, evidence from recent years indicates that ROS fulfill important roles as cellular messengers. Results obtained using model organisms suggest that ROS-dependent signalling may even activate beneficial cellular stress responses, which eventually may lead to increased lifespan. Nevertheless, when an overload of ROS cannot be properly disposed of, its accumulation generates oxidative stress, which plays a major part in the ageing process. Comparative studies about the rates of ROS production and oxidative damage accumulation, have led to the idea that the lower rate of mitochondrial oxygen radical generation of long-lived animals with respect to that of their short-lived counterpart, could be a primary cause of their slow ageing rate. A hitherto largely under-appreciated alternative view is that such lower rate of ROS production, rather than a cause may be a consequence of the metabolic constraints imposed for the large body sizes that accompany high lifespans. To help understanding the logical underpinning of this rather heterodox view, herein I review the current literature regarding the mechanisms of ROS formation, with particular emphasis on evolutionary aspects.     

The importance of physiological oxygen concentrations in the sandwich cultures of rat hepatocytes on gas‐permeable membranes

Oxygen supply is a critical issue in the optimization of in vitro hepatocyte microenvironments. Although several strategies have been developed to balance complex oxygen requirements, these techniques are not able to accurately meet the cellular oxygen demand. Indeed, neither the actual oxygen concentration encountered by cells nor the cellular oxygen consumption rates (OCR) was assessed. The aim of this study is to define appropriate oxygen conditions at the cell level that could accurately match the OCR and allow hepatocytes to maintain liver specific functions in a normoxic environment. Matrigel overlaid rat hepatocytes were cultured on the polydimethylsiloxane (PDMS) membranes under either atmospheric oxygen concentration [20%‐O₂ (+)] or physiological oxygen concentrations [10%‐O₂ (+), 5%‐O₂ (+)], respectively, to investigate the effects of various oxygen concentrations on the efficient functioning of hepatocytes. In parallel, the gas‐impermeable cultures (polystyrene) with PDMS membrane inserts were used as the control groups [PS‐O₂ (?)]. The results indicated that the hepatocytes under 10%‐O₂ (+) exhibited improved survival and maintenance of metabolic activities and functional polarization. The dramatic elevation of cellular OCR up to the in vivo liver rate proposed a normoxic environment for hepatocytes, especially when comparing with PS‐O₂ (?) cultures, in which the cells generally tolerated hypoxia. Additionally, the expression levels of 84 drug‐metabolism genes were the closest to physiological levels. In conclusion, this study clearly shows the benefit of long‐term culture of hepatocytes at physiological oxygen concentration, and indicates on an oxygen‐permeable membrane system to provide a simple method for in vitro studies. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1401–1410, 2014     

Organ-specific rates of cellular respiration in developing sunflower seedlings and their bearing on metabolic scaling theory

Fifty years ago Max Kleiber described what has become known as the "mouse-to-elephant" curve, i.e., a log-log plot of basal metabolic rate versus body mass. From these data, "Kleiber's 3/4 law" was deduced, which states that metabolic activity scales as the three fourths-power of body mass. However, for reasons unknown so far, no such "universal scaling law" has been discovered for land plants (embryophytes). Here, we report that the metabolic rates of four different organs (cotyledons, cotyledonary hook, hypocotyl, and roots) of developing sunflower (Helianthus annuus L.) seedlings grown in darkness (skotomorphogenesis) and in white light (photomorphogenesis) differ by a factor of 2 to 5 and are largely independent of light treatment. The organ-specific respiration rate (oxygen uptake per minute per gram of fresh mass) of the apical hook, which is composed of cells with densely packaged cytoplasm, is much higher than that of the hypocotyl, an organ that contains vacuolated cells. Data for cell length, cell density, and DNA content reveal that (1) hook opening in white light is caused by a stimulation of cell elongation on the inside of the curved organ, (2) respiration, cell density and DNA content are much higher in the hook than in the stem, and (3) organ-specific respiration rates and the DNA contents of tissues are statistically correlated. We conclude that, due to the heterogeneity of the plant body caused by the vacuolization of the cells, Kleiber's law, which was deduced using mammals as a model system, cannot be applied to embryophytes. In plants, this rule may reflect scaling phenomena at the level of the metabolically active protoplasmic contents of the cells.     

Viability control and oxygen consumption of isolated hepatocytes from thermally acclimated rainbow trout (Salmo gairdnerii)

Hepatocytes were isolated by collagenase perfusion of the liver from rainbow trout acclimated to 10 and 20 degrees C. The suitability of the stimulation of cellular respiration by succinate as criterion of viability was examined and discussed. Endogenous respiration rates of the hepatocytes were a function of cell size to the power of 0.8. Specific oxygen consumption of the hepatocytes and respiratory control ratios of the mitochondria in situ were independent of acclimation temperature.     

Plasticity in metabolic allometry: the role of dietary stoichiometry

Metabolism involves multiple elements. While we know much about the allometry in metabolic response of organisms to energy (carbon, C) availability, little is known about how different-sized organisms respond to the relative availability of elements. I experimentally manipulated availability of phosphorus (P) relative to C, to test whether dietary C : P affects metabolism in four species of Daphnia , spanning an order of magnitude in body mass. Results indicated that the slope of the relationship between individual respiration and body mass was M ^0.83 under a balanced diet (C : P c. 150), and M ^0.67 under an imbalanced diet (C : P c. 800). Increased respiration under dietary imbalance was not due to increased ingestion. The change in the scaling exponent was due to the greater respiratory response of smaller species to altered diets. Diet-induced metabolic plasticity contributes to variation in metabolic allometry, at least at such small scales of body size.     

Meiofauna Metabolism in Suboxic Sediments: Currently Overestimated

Oxygen is recognized as a structuring factor of metazoan communities in marine sediments. The importance of oxygen as a controlling factor on meiofauna (32 µm-1 mm in size) respiration rates is however less clear. Typically, respiration rates are measured under oxic conditions, after which these rates are used in food web studies to quantify the role of meiofauna in sediment carbon turnover. Sediment oxygen concentration ([O₂]) is generally far from saturated, implying that (1) current estimates of the role of meiofauna in carbon cycling may be biased and (2) meiofaunal organisms need strategies to survive in oxygen-stressed environments. Two main survival strategies are often hypothesized: 1) frequent migration to oxic layers and 2) morphological adaptation. To evaluate these hypotheses, we (1) used a model of oxygen turnover in the meiofauna body as a function of ambient [O₂], and (2) performed respiration measurements at a range of [O₂] conditions. The oxygen turnover model predicts a tight coupling between ambient [O₂] and meiofauna body [O₂] with oxygen within the body being consumed in seconds. This fast turnover favors long and slender organisms in sediments with low ambient [O₂] but even then frequent migration between suboxic and oxic layers is for most organisms not a viable strategy to alleviate oxygen limitation. Respiration rates of all measured meiofauna organisms slowed down in response to decreasing ambient [O₂], with Nematoda displaying the highest metabolic sensitivity for declining [O₂] followed by Foraminifera and juvenile Gastropoda. Ostracoda showed a behavioral stress response when ambient [O₂] reached a critical level. Reduced respiration at low ambient [O₂] implies that meiofauna in natural, i.e. suboxic, sediments must have a lower metabolism than inferred from earlier respiration rates conducted under oxic conditions. The implications of these findings are discussed for the contribution of meiofauna to carbon cycling in marine sediments.