Endotherms like mammals and birds display the unique ability to maintain a high body temperature (T_b) over a wide ambient temperature (T_a) range (Crompton et al., 1978). Unlike in ectotherms, where T_b is generally dependent on T_a, this evolutionary strategy has enabled endotherms to expand into highly specialized niches and a wide geographical distribution. Despite the establishment of the gross classification into endo- and ectothermic animals, the distinction is often not clear cut, with several endothermic taxa displaying distinct ectothermic physiological traits and vice versa.

The lesser hedgehog tenrec (Echinops telfairi) as a prime example, displays reptile-like thermoregulatory behavior with markedly high variability in T_b and metabolic rate (Scholl, 1974; Lovegrove and Génin, 2008; Oelkrug et al., 2013). The tenrec displays one of the lowest normothermic T_b (~32°C) compared to all other placental mammals in winter conditions and exhibits regular daily torpor behavior with substantial reductions in T_b (following reductions in T_a) and metabolic rate. Extraordinarily, E. telfairi females only display constantly high T_b during reproduction and parental care (Poppitt et al., 1994). Hence, this afrotherian species is referred to as protoendothermic, representing an intermediate state of thermoregulatory rigor compared to more “modern” homeothermic eutherian species. This thermoregulatory setup of protoendotherms is therefore often considered a representative characteristic of the evolutionary transition from ecto- to endothermy (Lovegrove and Génin, 2008; Mckechnie and Mzilikazi, 2011).

Interestingly, the basal metabolic rate (BMR) of the tenrec is similar to ectothermic metabolic rates of the same body mass, while the basal mitochondrial proton leak in the liver, a significant contributor of metabolic heat production (Brand et al., 1994; Rolfe and Brown, 1997), is at an intermediate level between endo- and ectotherms (Polymeropoulos et al., 2017).

Prolonged cold exposure triggers complex physiological adjustments to ensure cellular homeostasis at a constant T_b in many mammals. There is evidence that E. telfairi is capable of non-shivering thermogenesis mediated by functional brown adipose tissue (BAT) and hence of a mechanism that is unique to “modern” eutherian mammals (Oelkrug et al., 2013, 2015). The bioenergetic setup of tenrec mitochondria in other tissues may also be highly advanced and equipped to adjust energy metabolism in response to environmental stressors such as low ambient temperatures.

While UCP1-mediated non-shivering thermogenesis in BAT is the most prominent mechanism to enable survival in the cold and arousal from torpid or hibernating states of small eutherian mammals (Cannon and Nedergaard, 2004; Oelkrug et al., 2011), mitochondrial adjustments in other tissues of high oxygen uptake have also been described. It appears that another important thermogenic tissue is skeletal muscle which contributes with shivering and possibly other alternative mechanisms of non-shivering thermogenesis using SERCA pathways (Rowland et al., 2015). The liver is commonly studied to assess the relationship between metabolism and mitochondrial bioenergetics as the liver contributes significantly (10–20%) to the metabolic rate of mammals (Field et al., 1939) and mitochondria are easy to isolate as compared to e.g., skeletal muscle.

In general, mitochondria convert nutritional energy into cellular energy by oxidative phosphorylation, a process that consumes about 90% of the cellular oxygen uptake in mammals (Rolfe and Brown, 1997). Mitochondrial substrates are oxidized and their electrons are transported along the respiratory complexes before reducing oxygen to water. The potential energy of the electrons is used to pump protons out of the matrix, generating a proton motive force that drives the ATP synthesis. The energy transduction to ATP is, however, not fully efficient as the proton motive force is also consumed by the mitochondrial proton leak (Jastroch et al., 2010). The management of the energy budget of an animal can be adjusted by alterations in mitochondrial efficiency when exposed to environmental stress, either by a decrease of substrate oxidation or by changes in proton permeability as shown for many ectotherms (Gnaiger et al., 2000; St-Pierre et al., 2000; Trzcionka et al., 2008).

During episodes of energy shortage such as cold seasons or food scarcity, small mammals use energy conserving mechanisms such as torpor or hibernation to decrease energy demand by dramatic reduction of T_b and metabolic rate. During torpor and hibernation, state 3 (phosphorylating) respiration and maximal substrate oxidation are reversibly suppressed in isolated liver mitochondria (Staples and Brown, 2008; Kutschke et al., 2013). The degree of suppression is, not surprisingly, highly dependent on experimental assay temperature. Interestingly, it appears that the suppression of state 3 respiration in hypothermic states is only detectable above a critical assay temperature of 30°C. Above 30°C assay temperature, passive thermal effects rather than active suppression may become more important for metabolic rate (Staples and Brown, 2008). This is further supported by the lack of active suppression in isolated liver mitochondria of the Golden spiny mouse (Acomys russatus), a desert species that displays significant reduction of energy metabolism without pronounced reduction of T_b (Grimpo et al., 2014). The findings at lower assay temperatures may well be related to changes of the biophysical properties of mitochondrial membranes.

In isolated liver mitochondria of the homeothermic rat (Rattus norvegicus), lowering the assay temperature from 37 to 25°C reduces state 3 (phosphorylating) and state 4 (non-phosphorylating) respiration by 50%, (Dufour et al., 1996). In the heterothermic Djungarian hamster (Phodopus sungorus), substrate oxidation of isolated liver mitochondria is suppressed in torpid animals (T_b < 31°C). The suppression of substrate oxidation affects respiration rates the most at normothermic assay temperatures (37°C) but not at torpor-like assay temperatures (15°C). At 15°C, mitochondrial proton leak is increased in mitochondria of torpid but not normothermic animals (Brown et al., 2007). The respiration of liver mitochondria correlates with body temperature but there is no suppression found for kidney, skeletal muscle, and heart mitochondria, suggesting tissue-specificity of mitochondrial suppression (Kutschke et al., 2013).

Given these findings in rodents, it may be expected that frequent body temperature changes of the tenrec affect mitochondrial respiration through passive Q₁₀ driven changes but it is unknown whether mechanistic alterations in response to cold exposure, such as active suppression, contribute as well.

While the general thermoregulatory patterns of the lesser hedgehog tenrec, a representative protoendothermic species, have become more transparent through recent research, underlying function or mechanisms that have co-evolved with thermoregulatory flexibility, remain unclear. Here, we studied the bioenergetics of isolated liver mitochondria of cold vs. warm acclimated, non-torpid tenrecs at different assay temperatures to understand functional adjustments that may assist the animals to manage energy homeostasis during the transition to various body temperatures.

In this study, we report on adjustments of mitochondrial bioenergetics allowing energy homeostasis in the lesser hedgehog tenrec, a protoendothermic eutherian, during its T_b cycles. We find that the mitochondrial system maintains mitochondrial efficiency at lower assay temperatures, partially by decreases in proton leak, counteracting decreases in temperature-dependent substrate oxidation rates.

While these passive thermal effects appear to control mitochondrial energy turnover, cold acclimation of the tenrec had neither effect on the in vivo physiology such as body mass, T_b and metabolic rate, nor on bioenergetics parameters of isolated liver mitochondria.

The patterns of T_b to acute changes in T_a (Figure 1) as described previously (Oelkrug et al., 2013) clearly support the observations of a distinct ectotherm-like nature where T_b closely matches changes in T_a (Scholl, 1974; Lovegrove and Génin, 2008). This was particularly true for cases where animals entered torpid-like minimal metabolic states during the experiments (Figure 1C). Here, the correlation between T_b and T_a was very close to 1. Although the familiar Scholander-Irving model of thermoregulation is typically only applied to euthermic endotherms (McNab, 2002), metabolic heat production was adjusted similarly to euthermic endotherms below the TNZ when measuring RMR in the tenrecs (Figure 1B), clearly demonstrating the ability of classical euthermic, eutherian thermoregulation. Nevertheless, T_b in the tenrecs is amongst the lowest within mammals and similar to monotremes (Nicol, 2017). Interestingly, when undisturbed, tenrecs will drop their metabolic rates to a minimum, while adjusting their T_b to T_a. Even though the reduction in MR_min with changes in T_a that we observed here (Figure 1B) was only marginal in comparison to changes in RMR, the continuous reduction in MR_min appears relevant given the continuous reduction in T_b. It remains to be investigated whether passive heat loss is the major driver in the reduction of T_b as is the case in many species entering torpor (Nicol and Andersen, 2007; Oelkrug et al., 2011), rather than the controlled reduction in MR and hence T_b.

While our understanding of the overarching thermoregulatory patterns of tenrecs has become clearer, the cellular basis supporting this unique pattern is not well-understood. The bioenergetics of liver mitochondria are well-established nominators of (basal) metabolic rates (Porter and Brand, 1995; Polymeropoulos et al., 2012) and actively adjust in response to hypothermic states in some rodent species (Staples and Brown, 2008; Kutschke et al., 2013). Thus, investigating temperature-dependent bioenergetics in the frequently heterothermic tenrec may yield relevant insights into concepts and mechanisms that occurred during early evolution of eutherian metabolism. Therefore, we investigated the flexibility of mitochondrial function in response to acute and chronic temperature changes in isolated liver mitochondria of tenrecs. In contrast to some ectothermic vertebrates such as fish and amphibians, where mitochondrial state 4 respiration and proton leak rate is suppressed in chronic cold (Jastroch et al., 2007; Trzcionka et al., 2008), acclimation temperature had no such impact on liver mitochondrial proton leak in the tenrec (Figure 3). This finding may indicate that there are no functional, long-term changes accompanied by cold acclimatization. In our experiments, we sacrificed the animals at normothermic temperatures for experimental consistency. Under these experimental conditions, putative, rapid molecular changes in response to hypothermic or hypometabolic states in torpor may be missed.

The capacity of ATP production is cold-sensitive with a 50% reduction of respiration rates in state 4 (Figure 2C) and 70% reduction in state 3 over a 10°C temperature decrease (32 vs. 22°C, Figure 3B). Similar findings, but with lesser physiological significance, have been reported for rat liver mitochondria, where a 12°C decrease in assay temperature resulted in a 50% decrease of state 3 and 4 respiration rates (Dufour et al., 1996).

For tenrec mitochondria, the respiratory control ratio at 22°C was 40% lower than that at 27 and 32°C (Figure 2E). However, the correction of ATP-linked state 3 for proton leak respiration at the state 3 mitochondrial membrane potential reveals maintenance of mitochondrial efficiency of about 70–85%, strongly trending to increase at lower temperatures (Figure 4B). This is a striking finding demonstrating an inherent thermal plasticity of tenrec mitochondria that favors a thermoregulatory strategy allowing optimal mitochondrial function in heterothermic states that are naturally experienced by tenrecs. If this may be a trait unique to tenrecs or protoendotherms remains to be elucidated. Clearly, more data are required to phylogenetically generalize this suggestion.

Within vertebrates, the level of the basal mitochondrial proton leak of E. telfairi is at an intermediate level between endo- and ectotherms (Polymeropoulos et al., 2017). However, on a phylogenetically informed basis, the tenrec mitochondrial proton leak scales within other eutherian mammals but not reptiles, corroborating that the molecular, mitochondrial setup is “mammalian” while the behavioral pattern could be classified as “reptilian.” It would therefore be interesting to study whether mitochondrial efficiency at colder temperatures in ectothermic vertebrate species exhibits a similar pattern, thus being a plesiomorphic trait, or whether these mechanistic adjustments were derived in the need to adjust to endothermic metabolism.

In conclusion, the bioenergetic setup of tenrec liver mitochondria resembles mammalian and ectothermic features. Mammalian features are reflected in the amplitude of tenrec liver proton leak within the eutherian range, while increased mitochondrial efficiency may have enabled to cope with energy conversion at low T_b. The latter possibly enables the unique thermoregulation of the tenrec with exceptionally long ectothermic episodes—an assumed prerogative of eutherian survival at the Cretaceous-Paleogene boundary.

EP and RO equally contributed to this manuscript. RO and MJ conceived the ideas, RO conducted the experiments, EP, RO, and MJ prepared the manuscript and figures and analyzed the data, and approved the manuscript.