Characterization of brown adipose tissue thermogenesis in the naked mole-rat (Heterocephalus glaber), a poikilothermic mammal

The naked mole-rat (NMR) is a poikilothermic mammal that forms eusocial colonies consisting of one breeding queen, several breeding kings, and subordinates. Despite their poikilothermic feature, NMRs possess brown adipose tissue (BAT), which in homeothermic mammals induces thermogenesis in cold environments. However, NMR-BAT thermogenic potential is controversial, and its physiological roles are unknown. Here, we show that NMR-BAT has beta-3 adrenergic receptor (ADRB3)-dependent thermogenic potential, which contributes to thermogenesis in the isolated queen in non-cold environments. NMR-BAT expressed several brown adipocyte marker genes and showed noradrenaline-dependent thermogenic activity in vitro and in vivo. Although our ADRB3 inhibition experiments revealed that NMR-BAT thermogenesis slightly delays the decrease in body temperature in a cold environment, it was insufficient to maintain the body temperatures of the NMRs. In a non-cold environment, NMRs are known to increase their body temperature by a heat-sharing behavior. Interestingly, we found that the body temperatures of NMRs isolated from the colony were also significantly higher than the ambient temperature. We also show that queens, but not subordinates, induce BAT thermogenesis in isolated, non-cold conditions. Our research provides novel insights into the role and mechanism of thermoregulation in this unique poikilothermic mammal.

NMR-BAT thermogenic potential is controversial, and its physiological roles are 48 unknown. Here, we show that NMR-BAT has beta-3 adrenergic receptor 49 (ADRB3)-dependent thermogenic potential, which contributes to thermogenesis in the   thermogenically active adipocytes contain smaller lipid droplets than inactive adipocytes 125 24 , we measured the size of the lipid droplets in the HE-stained images, which showed 126 that dBAT contained significantly smaller lipid droplets than the interscapular BAT of ( Fig. S2f). Furthermore, the western blotting showed that the UCP1 protein is highly 144 expressed in dBAT and lBAT (Fig. S3).

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To directly evaluate the thermogenic ability of NMR-BAT, we measured its 146 temperature and the rectum temperature following the administration of noradrenaline by 147 inserting a thermo-probe into the BAT and rectum of anesthetized NMRs. We found that 148 noradrenaline injection caused the BAT temperature to increase by approximately 1.2°C,

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To evaluate whether NMR-BAT thermogenesis depends on the adrenergic 156 beta-3 receptor (ADRB3), which plays a critical role in BAT thermogenesis and has a 157 relatively specific expression in the adipose tissues 1 , we measured 158 noradrenaline-induced oxygen consumption rate of brown adipocytes isolated from a mixture of dBAT and lBAT after a noradrenaline treatment in the presence or absence of the ADRB3 inhibitor SR59230A, using adipocytes isolated from a mixture of dBAT 161 and lBAT. We found that the stimulation with noradrenaline caused the rapid increase in 162 oxygen consumption rate of brown adipocytes, but this increase was inhibited by the 163 pre-treatment with SR59230A (Fig. 1f). Together, these findings indicate that 164 poikilothermic NMRs possess ADRB3-dependent thermogenic BAT.

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Induction of NMR-BAT thermogenesis that slightly delays the decrease in body 167 temperature in a cold environment 168 Next, we investigated the roles of NMR-BAT in physiological conditions. Because NMR 169 skin is almost hairless and quite thin (Fig. 1a), BAT thermogenesis can be monitored by 170 measuring the cervix surface temperature with a thermal camera. We found that the 171 cervix surface temperature was correlated with the BAT temperature, as measured by 172 the thermo-probe (Fig. S4). We measured the cervix temperatures of each NMR in the isolated situation because NMRs are known to enter a behaviorally homeothermic state To evaluate the thermogenic ability of BAT in a cold environment for NMRs 176 (20°C), we monitored the cervix surface temperature and the abdominal core body 177 temperature of free-moving NMR subordinates using a thermal camera and a 178 temperature telemetry system simultaneously. When NMRs were isolated from the 179 colony and transferred to a room at 20 °C, the body and cervix surface temperatures 180 gradually reduced, indicating NMRs were unable to keep their body temperature in cold 181 environment as previously reported 19 . We found that SR59230A initially accelerated the 182 drops in both temperatures; however, SR59230A did not induce further decrease at 183 equilibrium ( Fig. 2a-c). These results suggest that, although BAT thermogenesis 184 contributed to the delay in the decrease in body temperature after cold exposure, the 185 NMRs were unable to maintain their body temperature at 20°C as previously reported 19 186 ( Fig. 2a-c).

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Induction of BAT thermogenesis in naked mole-rat queens under isolated, 189 non-cold conditions that NMR-BAT may also play a role in non-cold environments. Interestingly, we found 193 that NMRs isolated from their colony did not decrease their body temperatures and also 194 showed higher body temperature than the ambient temperature (Fig. 3a). To test 195 whether the thermogenesis of the isolated subordinates depended on BAT, we injected

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NMRs with the ADRB3 inhibitor SR59230A and again measured the body temperature of 197 individuals after isolation. However, no significant change was observed in the 198 subordinates (Fig. 3b, c).

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Notably, we found that the queens showed a tendency toward higher oxygen 200 consumption rates than other members, suggesting that the BAT of queens may be 201 more thermogenic than in other colony members in this situation (Fig. 3d). Although the 202 body weight and age were higher in queen than in the subordinates 27 , we also found that 203 the body weight and age were weakly negatively correlated with the body temperatures 204 of the isolated subordinate NMRs (Fig. S5a, b), suggesting that the higher thermogenic 205 activity of queens was not related to their heavier body weights or older ages. No significant differences in body temperature were found between the sexes (Fig. S5c). To 207 test whether the thermogenesis of the isolated queens depended on BAT, we injected 208 NMRs with the ADRB3 inhibitor SR59230A and again measured the body temperature of 209 individuals after isolation. As a result, we found that the body temperatures of the socially 210 isolated queens significantly decreased following SR59230A injection (Fig. 3e, f). These 211 results indicate that NMR queens, but not subordinates, activate BAT thermogenesis in 212 an isolated, non-cold environment.

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In this study, we provided direct evidence in support of the thermogenic potential of BAT 216 in poikilothermic NMRs, which is ADRB3 dependent. We show that BAT thermogenesis 217 was insufficient to maintain the body temperatures of the NMRs in a cold environment 218 although it can slightly delay the decrease in the body temperatures of NMRs.

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Furthermore, in a non-cold environment, BAT thermogenesis contributes to maintaining 220 the body temperature of the isolated queen. This research provides in vitro and in vivo evidence of NMR-BAT thermogenesis in physiological conditions that have not previously been studied.

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A previous study suggested that the NMR UCP1 gene has a unique sequence 224 that may contribute to the inability of thermogenesis in NMRs 23 ; however, our results 225 clearly show that, NMR-BAT does have thermogenic potential (although we did not 226 compare the levels of thermogenic ability of NMR-BAT with those of other species).

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Whether NMR-BAT has more or less thermogenic potential than the BAT of other

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In conclusion, we revealed that the poikilothermic NMR-BAT is thermogenic,

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The NMRs used in this study were maintained at Kumamoto University, Kumamoto, 281 Japan, where they were housed in four to 10 acrylic chambers that were connected by 282 acrylic tunnels, at 30 ± 0.5°C and 55% ± 5% humidity with a 12 h light/12 h dark cycle 283 (Fig. S1). The body temperatures of NMRs in the colony were assessed using 1-to 284 12-year-old NMRs. The effect of social isolation was assessed using 1-to 13-year-old purchased from CLEA Japan, Inc., and adipose tissues were collected from 1-to 287 2-year-old subordinates and 6-week-old mice for cytological and histological analyses.

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The resulting cDNA was prepared for qPCR using Thunderbird ® qPCR Mix (TOYOBO) 327 in a 384-well plate with the primers listed in Table S1. qPCR was performed on a 328 CFX384 Touch™ Real-Time PCR Detection System (BIO-RAD).

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The floating adipocytes were then collected and diluted with KRBH buffer containing 4% 361 bovine serum albumin and 2.7 mM glucose and then recentrifuged and washed three times. The acquired adipocytes were incubated at room temperature for 1h before 363 measurement.

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The oxygen consumption rate was measured using a Clark-style oxygen

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Expression levels were quantified by quantitative polymerase chain reaction (qPCR) 656 using the primers listed in Table S1 and were normalized to beta-actin (ACTB) (n = 3

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The oblong signal is a microchip for individual identification.