Elias T. Polymeropoulos

Brown adipose tissue: physiological function and evolutionary significance

In modern eutherian (placental) mammals, brown adipose tissue (BAT) evolved as a specialized thermogenic organ that is responsible for adaptive non-shivering thermogenesis (NST). For NST, energy metabolism of BAT mitochondria is increased by activation of uncoupling protein 1 (UCP1), which dissipates the proton motive force as heat. Despite the presence of UCP1 orthologues prior to the divergence of teleost fish and mammalian lineages, UCP1’s significance for thermogenic adipose tissue emerged at later evolutionary stages. Recent studies on the presence of BAT in metatherians (marsupials) and eutherians of the afrotherian clade provide novel insights into the evolution of adaptive NST in mammals. In particular studies on the ‘protoendothermic’ lesser hedgehog tenrec (Afrotheria) suggest an evolutionary scenario linking BAT to the onset of eutherian endothermy. Here, we review the physiological function and distribution of BAT in an evolutionary context by focusing on the latest research on phylogenetically distinct species.

An ancient look at UCP1

Brown adipose tissue serves as a thermogenic organ in placental mammals to defend body temperature in the cold by nonshivering thermogenesis. The thermogenic function of brown adipose tissue is enabled by several specialised features on the organ as well as on the cellular level, including dense sympathetic innervation and vascularisation, high lipolytic capacity and mitochondrial density and the unique expression of uncoupling protein 1 (UCP1). This mitochondrial carrier protein is inserted into the inner mitochondrial membrane and stimulates maximum mitochondrial respiration by dissipating proton-motive force as heat. Studies in knockout mice have clearly demonstrated that UCP1 is essential for nonshivering thermogenesis in brown adipose tissue. For a long time it had been presumed that brown adipose tissue and UCP1 emerged in placental mammals providing them with a unique advantage to survive in the cold. Our subsequent discoveries of UCP1 orthologues in ectotherm vertebrates and marsupials clearly refute this presumption. We can now initiate comparative studies on the structure-function relationships in UCP1 orthologues from different vertebrates to elucidate when during vertebrate evolution UCP1 gained the biochemical properties required for nonshivering thermogenesis.

Phylogenetic differences of mammalian basal metabolic rate are not explained by mitochondrial basal proton leak.

Metabolic rates of mammals presumably increased during the evolution of endothermy, but molecular and cellular mechanisms underlying basal metabolic rate (BMR) are still not understood. It has been established that mitochondrial basal proton leak contributes significantly to BMR. Comparative studies among a diversity of eutherian mammals showed that BMR correlates with body mass and proton leak. Here, we studied BMR and mitochondrial basal proton leak in liver of various marsupial species. Surprisingly, we found that the mitochondrial proton leak was greater in marsupials than in eutherians, although marsupials have lower BMRs. To verify our finding, we kept similar-sized individuals of a marsupial opossum (Monodelphis domestica) and a eutherian rodent (Mesocricetus auratus) species under identical conditions, and directly compared BMR and basal proton leak. We confirmed an approximately 40 per cent lower mass specific BMR in the opossum although its proton leak was significantly higher (approx. 60%). We demonstrate that the increase in BMR during eutherian evolution is not based on a general increase in the mitochondrial proton leak, although there is a similar allometric relationship of proton leak and BMR within mammalian groups. The difference in proton leak between endothermic groups may assist in elucidating distinct metabolic and habitat requirements that have evolved during mammalian divergence.

Absence of adaptive nonshivering thermogenesis in a marsupial, the fat-tailed dunnart (Sminthopsis crassicaudata)

The presence of nonshivering thermogenesis in marsupials is controversially debated. Survival of small eutherian species in cold environments is crucially dependent on uncoupling protein 1 (UCP1)-mediated, adaptive nonshivering thermogenesis that is executed in brown adipose tissue. In a small dasyurid marsupial species, the fat-tailed dunnart (Sminthopsis crassicaudata), an orthologue of UCP1 has been recently identified which is upregulated during cold exposure resembling adaptive molecular adjustments of eutherian brown adipose tissue. Here, we tested for a thermogenic function of marsupial brown adipose tissue and UCP1 by evaluating the capacity of nonshivering thermogenesis in cold-acclimated dunnarts. In response to an optimal dosage of noradrenaline, cold-acclimated dunnarts (12°C) showed no additional recruitment of noradrenaline-induced maximal thermogenic capacity in comparison to warm-acclimated dunnarts (24°C). While no differences in body temperature were observed between the acclimation groups, basal metabolic rate was significantly elevated after cold acclimation. Therefore, we suggest that adaptive nonshivering thermogenesis does not occur in this marsupial species despite the cold recruitment of oxidative capacity and UCP1 in the interscapular fat deposit. In conclusion, the ancient UCP orthologue in marsupials does not contribute to the classical nonshivering thermogenesis, and may exhibit a different physiological role.

Does hyperthermia constrain flight duration in a short-distance migrant?

While some migratory birds perform non-stop flights of over 11 000 km, many species only spend around 15% of the day in flight during migration, posing a question as to why flight times for many species are so short. Here, we test the idea that hyperthermia might constrain flight duration (FD) in a short-distance migrant using remote biologging technology to measure heart rate, hydrostatic pressure and body temperature in 19 migrating eider ducks (Somateria mollissima), a short-distance migrant. Our results reveal a stop-and-go migration strategy where migratory flights were frequent (14 flights day−1) and short (15.7 min), together with the fact that body temperature increases by 1°C, on average, during such flights, which equates to a rate of heat storage index (HSI) of 4°C h−1. Furthermore, we could not find any evidence that short flights were limited by heart rate, together with the fact that the numerous stops could not be explained by the need to feed, as the frequency of dives and the time spent feeding were comparatively small during the migratory period. We thus conclude that hyperthermia appears to be the predominant determinant of the observed migration strategy, and suggest that such a physiological limitation to FD may also occur in other species. This article is part of the themed issue ‘Moving in a moving medium: new perspectives on flight’.

Growth hormone transgenesis and polyploidy increase metabolic rate, alter the cardiorespiratory response and influence HSP expression in response to acute hypoxia in Atlantic salmon ( Salmo salar ) yolk-sac alevins

Growth hormone (GH)-transgenic Atlantic salmon display accelerated growth rates compared with non-transgenics. GH-transgenic fish also display cardiorespiratory and metabolic modifications that accompany the increased growth rate. An elevated routine metabolic rate has been described for pre- and post-smolt GH-transgenic salmon that also display improvements in oxygen delivery to support the increased aerobic demand. The early ontogenic effects of GH transgenesis on the respiratory and cellular physiology of fish, especially during adverse environmental conditions, and the effect of polyploidy are unclear. Here, we investigated the effects of GH transgenesis and polyploidy on metabolic, heart and ventilation rates and heat shock protein (HSP) levels after exposure to acute hypoxia in post-hatch Atlantic salmon yolk-sac alevins. Metabolic rate decreased with decreasing partial pressures of oxygen in all genotypes. In normoxia, triploid transgenics displayed the highest mass-specific metabolic rates in comparison to diploid transgenics and non-transgenic triploids, which, in contrast, had higher rates than diploid non-transgenics. In hypoxia, we observed a lower mass-specific metabolic rate in diploid non-transgenics compared with all other genotypes. However, no evidence for improved O2 uptake through heart or ventilation rate was found. Heart rate decreased in diploid non-transgenics while ventilation rate decreased in both diploid non-transgenics and triploid transgenics in severe hypoxia. Regardless of genotype or treatment, inducible HSP70 was not expressed in alevins. Following hypoxia, the constitutive isoform of HSP70, HSC70, decreased in transgenics and HSP90 expression decreased in all genotypes. These data suggest that physiological changes through GH transgenesis and polyploidy are manifested during early ontogeny in Atlantic salmon.

Respirometry: Correcting for Diffusion and Validating the Use of Plastic Multiwell Plates with Integrated Optodes

Polystyrene multiwell plates with integrated optodes act as multiple closed-system respirometers that enable the simultaneous measurement of oxygen consumption in small animals. However, the diffusion of oxygen through polystyrene needs to be taken into consideration. Here we provide an equation that accounts for the empirically determined rate of oxygen through a polystyrene well when calculating the instantaneous rate of oxygen consumption. Furthermore, we describe a novel method of calibrating a small respirometer for accuracy using micro-osmotic pumps containing an oxygen scavenger, which is delivered at a constant rate and therefore yields a constant rate of oxygen consumption in an airtight system.

Phylogenetic analysis of the allometry of metabolic rate and mitochondrial basal proton leak

The mitochondrial basal proton leak (MBPL) significantly contributes to high body temperatures (Tb) and basal metabolic rates (BMR) in endotherms. In endotherms at a given body mass (M), liver MBPL is higher than in ectotherms, supporting the notion that MBPL may partly explain the evolutionary increase in metabolic rate (MR), fostering endothermy. Here, we re-addressed this assumption by performing a phylogenetic analysis comparing all available liver MBPL data for ecto- and endotherms. While MBPL within endotherms negatively scales with M and BMR as shown previously, MBPL of ectotherms does not scale allometrically with M. Phylogenetic analysis reveals that this result is confounded by a positive scaling coefficient for MBPL with M for reptiles. Strikingly, the reptilian MBPL reaches endothermic levels above a body mass of 6.6kg. Thus, phylogenetic scaling of MBPL supports previous claims of endotherm-like physiological characteristics in large reptiles. It appears that diversification of ancestral ectothermic tetrapods to a body mass of at least 6kg may have been required to reach a MBPL that is beneficial for sustained high body temperatures. Novel MBPL data for the lesser hedgehog tenrec, a protoendothermic eutherian that displays reptile-like thermoregulatory patterns, fall within the endo- and ectothermic allometric regressions. Finally, we add additional evidence that within endotherms, phylogenetic differences in MR do not correlate with MBPL. Collectively, these data suggest that MBPL does not universally scale with metabolic rate in ecto- or endotherms and that an increasing MBPL with M may have played an important physiological role in the evolutionary history of reptilian thermoregulation.

It Takes Time to Be Cool: On the Relationship between Hyperthermia and Body Cooling in a Migrating Seaduck

The large amount of energy expended during flapping flight is associated with heat generated through the increased work of the flight muscles. This increased muscle work rate can manifest itself in core body temperature (Tb) increase of 1–2°C in birds during flight. Therefore, episodic body cooling may be mandatory in migratory birds. To elucidate the thermoregulatory strategy of a short-distance migrant, common eiders (Somateria mollissima), we implanted data loggers in the body cavity of wild birds for 1 year, and report information on Tb during their entire migration for 19 individuals. We show that the mean body temperature during flight (TbMean) in the eiders was associated with rises in Tb ranging from 0.2 to 1.5°C, largely depending on flight duration. To understand how eiders are dealing with hyperthermia during migration, we first compare, at a daily scale, how Tb differs during migration using a before-after approach. Only a slight difference was found (0.05°C) between the after (40.30°C), the before (40.41°C) and the migration (40.36°C) periods, indicating that hyperthermia during flight had minimal impact at this time scale. Analyses at the scale of a flight cycle (flight plus stops on the water), however, clearly shows that eiders were closely regulating Tb during migration, as the relationship between the storage of heat during flight was highly correlated (slope = 1) with the level of heat dumping during stops, at both inter-individual and intra-individual levels. Because Tb at the start of a flight (TbStart) was significantly and positively related to Tb at the end of a flight (TbEnd), and the maximal attained Tb during a flight (TbMax), we conclude that in absence of sufficient body cooling during stopovers, eiders are likely to become increasingly hyperthermic during migration. Finally, we quantified the time spent cooling down during migration to be 36% of their daily (24 h) time budget, and conclude that behavioral body cooling in relation to hyperthermia represents an important time cost.

Hypoxic acclimation leads to metabolic compensation after reoxygenation in Atlantic salmon yolk-sac alevins

Hypoxia is common in aquatic environments and has substantial effects on development, metabolism and survival of aquatic organisms. To understand the physiological effects of hypoxia and its dependence on temperature, metabolic rate ( [Formula: see text] ) and cardiorespiratory function were studied in response to acute hypoxia (21→5kPa) at different measurement temperatures (Ta; 4, 8 and 12°C) in Salmo salar alevins that were incubated under normoxic conditions (PO2=21kPa) or following hypoxic acclimation (PO2=10kPa) as well as two different temperatures (4°C or 8°C). Hypoxic acclimation lead to a developmental delay manifested through slower yolk absorption. The general response to acute hypoxia was metabolic depression (~60%). Hypoxia acclimated alevins had higher [Formula: see text] s when measured in normoxia than alevins acclimated to normoxia. [Formula: see text] s were elevated to the same degree (~30% per 4°C change) irrespective of Ta. Under severe, acute hypoxia (~5kPa) and irrespective of Ta or acclimation, [Formula: see text] s were similar between most groups. This suggests that despite different acclimation regimes, O2 transport was limited to the same degree. While cardiorespiratory function (heart-, ventilation rate) was unchanged in response to acute hypoxia after normoxic acclimation, hypoxic acclimation led to cardiorespiratory changes predominantly in severe hypoxia, indicating earlier onset and plasticity of cardiorespiratory control mechanisms. Although [Formula: see text] in normoxia was higher after hypoxic acclimation, at the respective acclimation PO2, [Formula: see text] was similar in normoxia and hypoxia acclimated alevins. This is indicative of metabolic compensation to an intrinsic [Formula: see text] at the acclimation condition in hypoxia-acclimated alevins after re-exposure to normoxia.