Brown fat in a protoendothermic mammal fuels eutherian evolution

Abstract

Endothermy has facilitated mammalian species radiation, but the sequence of events leading to sustained thermogenesis is debated in multiple evolutionary models. Here we study the Lesser hedgehog tenrec (Echinops telfairi), a phylogenetically ancient, 'protoendothermic' eutherian mammal, in which constantly high body temperatures are reported only during reproduction. Evidence for nonshivering thermogenesis is found in vivo during periodic ectothermic-endothermic transitions. Anatomical studies reveal large brown fat-like structures in the proximity of the reproductive organs, suggesting physiological significance for parental care. Biochemical analysis demonstrates high mitochondrial proton leak catalysed by an uncoupling protein 1 ortholog. Strikingly, bioenergetic profiling of tenrec uncoupling protein 1 reveals similar thermogenic potency as modern mouse uncoupling protein 1, despite the large phylogenetic distance. The discovery of functional brown adipose tissue in this 'protoendothermic' mammal links nonshivering thermogenesis directly to the roots of eutherian evolution, suggesting physiological importance prior to sustained body temperatures and migration to the cold.

Figure 1. Thermoregulation in E. telfairi.

(a,b) Representative Tb traces of a WA (a, Ta =27 °C) and a successively CA (b, 27 °C to 20 °C) tenrec. (c–f) Tb histograms at different Tas (N=6 animals per chart). (g, h) Mean RMR (g) and Tb (h) over a range of Tas, determining the TNZ (WA: N=5–6; CA: N=6; *P=0.001, Student’s t-test, data represent means±s.e.m.).

Figure 2. Heat production (HP) during episodic rewarming.

(a) Temporal-resolved thermogenic profile, including heating events from hypothermia (shaded area: lights off). (b) Resting HP during euthermia and hypometabolism. (c,d) Average HP and average rewarming rates during arousal, calculated over the same temperature range of 25.5–29.75 °C, with or without injection of the β3-adrenergic antagonist SR 59230A (N=5 per group; *P<0.05, paired t-test, data represent means±s.e.m.).

Figure 3. Morphological and molecular and biochemical evidence for functional BAT.

(a) Anatomical location of tenrec BAT. (b–d) Immunological detection of (b) β3-adrenoreceptor protein subunits in tissue homogenates, (c) UCP1 in tissue homogenates and (d) in isolated mitochondria, revealing cold induction. (e,f) COX activity in BAT homogenates and isolated mitochondria, demonstrating increased oxidative capacity in the cold. (g) Mitochondrial content, estimated from the ratio of tissue to mitochondrial COX activity. (*P<0.05, Student’s t-test). (h,i) Mitochondria respiration rates (N=4–5, *P<0.05, Student’s t-test) and proton leak kinetics of isolated BAT mitochondria in response to GDP, FCCP and 5 mM GDP (N=4–5, *P<0.05, paired t-test, data represent means±s.e.m.).

Figure 4. Comparative bioenergetic profiling of tenrec and mouse UCP1 in HEK293 cells.

(a) Representative traces of normalized plate-based respirometry, showing basal respiration, proton leak respiration (after oligomycin treatment), maximal substrate oxidation (FCCP or DNP) and non-mitochondrial respiration (antimycin A/rotenone). (b) BSA-sensitive coupling efficiency (white bars) of mouse UCP1 (mUCP1) and tenrec UCP1 (etUCP1)-containing HEK293 cells (N=5 per group and condition; *P<0.05, Student’s t-test). (c–e) Specific activation of stably expressed mUCP1 and etUCP1, in comparison with wild-type HEK293 (wtHEK293) (N=5–6, P<0.001, one-way ANOVA, lowercase letters indicate statistical difference at P<0.005, data represent means±s.e.m.).

Figure 5. Determination of catalytic centre activities of tenrec and mouse UCP1 in isolated HEK293 cell mitochondria.

(a–c) Proton leak kinetics of wild-type (wt), mouse UCP1 (mUCP1) and tenrec UCP1 (etUCP1) HEK293 cells, under basal conditions after activation with 100 μM palmitate and after inhibition with 1 mM GDP (N=6 per group and condition) (d) Specific UCP1 activity curves reveal similar catalytic power of tenrec and mouse UCP1 (derived from Fig. 4b).

Figure 6. A new model of thermogenic evolution in eutherian mammals.

In this possible evolutionary scenario, potent BAT-derived thermogenesis pre-dated the appearance of systemic homoeothermic endothermy at the roots of eutherian evolution, before migration to the cold. The opportunistic recruitment of thermogenesis, notably including BAT, during periods of parental care, may have been the driving force for sustained eutherian endothermy.