Hypometabolism during Daily Torpor in Mice is Dominated by Reduction in the Sensitivity of the Thermoregulatory System

Abstract

Some mammals enter a hypometabolic state either daily torpor (minutes to hours in length) or hibernation (days to weeks), when reducing metabolism would benefit survival. Hibernators demonstrate deep torpor by reducing both the sensitivity (H) and the theoretical set-point temperature (T_R) of the thermogenesis system, resulting in extreme hypothermia close to ambient temperature. However, these properties during daily torpor remain poorly understood due to the very short steady state of the hypometabolism and the large variation among species and individuals. To overcome these difficulties in observing and evaluating daily torpor, we developed a novel torpor-detection algorithm based on Bayesian estimation of the basal metabolism of individual mice. Applying this robust method, we evaluated fasting induced torpor in various ambient temperatures (T_As) and found that H decreased 91.5% during daily torpor while T_R only decreased 3.79 °C in mice. These results indicate that thermogenesis during daily torpor shares a common property of sensitivity reduction with hibernation while it is distinct from hibernation by not lowering T_R. Moreover, our findings support that mice are suitable model animals to investigate the regulation of the heat production during active hypometabolism, thus suggesting further study of mice may provide clues to regulating hypometabolism in mammals.

Figure 1. System for recording the metabolism of free-moving mice under controlled ambient temperature.

(a) A block diagram of the thermoregulatory system in mammals when the animal is not moving, therefore assumed to exert no external work. The upper loop and the lower loop represent the heat loss and heat production loop, respectively. The time derivative of body temperature (T_B) is derived from the difference of heat production (Q_in) and heat loss (Q_out) divided by the thermal capacity (C). Q_out is derived from the difference of ambient temperature (T_A) and T_B multiplied by heat conductance (G). Q_in is derived from the difference of theoretically defined set-point temperature (T_R) and T_Bmultiplied by H, which is the open-loop gain of the thermoregulatory feedback system. (b) A system for evaluating the metabolism of free-moving mice. The temperature-controlled animal chamber (left panel) and the inside of the chamber (middle panel), in which four mice can be recorded at once, are shown. Each animal had an intraperitoneally implanted body-temperature transmitter (right panel, top). Each animal was housed in a metabolic chamber and the VO₂ were recorded by gas mass spectrometry. (c) A representative recording of mouse metabolism for three consecutive days. The animal was placed in the chamber, and the T_A was maintained at 16 °C. Once the mouse was placed in the metabolic chamber, there was no physical contact with researchers during the recording period. Note the clear circadian rhythm seen in the T_B, VO₂ and locomotion. Yellow shading shows the light-on period. (d) A representative recording of metabolism during fasting-induced daily torpor. The mouse was placed in the chamber for three days; food was removed on the second day (filled triangle). The T_A was maintained at 16 °C. Daily torpor started during the latter half of the second day. The mouse returned to a euthermic state immediately after the food was returned to the chamber (unfilled triangle). Yellow shading shows the light-on period.

Figure 2. Modelling and predicting metabolism from a single day recording.

(a,b) The distribution of the T_B (a) and VO₂ (b) of four animals (mice 1 to 4) kept at a T_A of 12 °C for two days. The upper panels show the time series; light and dark periods are indicated by yellow and grey bars along the horizontal axis. The distribution for each animal is shown in the lower panels; each colour represents a different animal. (c) The estimated baseline metabolism dynamics of mouse 5. The mouse was kept at a T_A of 16 °C for three days. The baseline dynamics for 24 hours were fitted from the three-day-length data, and the standard deviation of the error (σ₂) for both T_B and VO₂ was estimated. The red and blue lines denote the median of the posterior distribution of the estimated T_B and VO_2, respectively. The data for the remaining three animals (mice 6 to 8) are available in Supplementary Fig. 2a. (d) The probability density of the experimentally obtained and estimated data for T_B and VO₂ for mouse 5. Black histograms represent experimental data; red and blue histograms show the estimated probability density. The data for mice 6 to 8 are available in Supplementary Fig. 2b. (e) The distribution of the estimated σ₂, which is the standard deviation of the error of the secondary trend, for T_B and VO₂. (f) The estimated baseline metabolism dynamics of mouse 9 with credible intervals (CIs). The T_A was kept at 16 °C for three days. The first 24 hours were used for estimation. The red and blue lines denote the median of the posterior distribution of the estimated T_B and VO₂. The red and blue shaded areas denote the CI. The data for mice 10 to12 are available in Supplementary Fig. 2c. (g) CI coverage rate of the metabolism on the second and third days when applying different CI ratios. The estimation was based on the first day. For both T_B and VO₂, 99.9% of the CIs covered more than 99% of the sampling points of the latter two days.

Figure 3. Defining daily torpor as an outlying low metabolism.

(a) The daily torpor-detection pipeline. The first 24-hour data set is used to estimate the baseline metabolism of the individual animal (first panel). The estimated baseline is then applied to the rest of the recordings (second panel). The baseline estimation provides the CI for the prediction from the distribution of the posterior estimates (third panel). Torpor, which is defined as a lower outlier from the CI, is marked in red dots (fourth panel). The filled and unfilled triangles denote food removal and return, respectively. (b) Multiple torpor definitions were compared in four mice (mice 13 to 16). The animals were placed in a constant T_A of 12 °C for three days, and food was restricted during the second day. Results are shown for mouse 13. The two leftmost panels show daily torpor defined by a fixed threshold T_B of 31 °C or 34 °C. The third panel shows daily torpor defined by a lower outlier of the 99.9% CI of the estimated T_B. The fourth panel includes the T_B-based definition further narrowed down by adding the condition of lower outliers from the 99.9% CI of the estimated VO₂. The filled and unfilled triangles denote food removal and return, respectively. The data for the remaining three animals are available in Supplementary Fig. 3a. (c) Boxplots for various torpor statistics according to the different torpor definitions listed in Fig. 3b. The band inside the box, the bottom of the box, and the top of the box represent the median, the first quartile, and the third quartile, respectively. The end of the upper whisker is the highest value that is within 1.5 times the inter-quartile range (IQR). The end of the lower whisker is the lowest value that is within 1.5 times the IQR. All data points are shown as grey dots.

Figure 4. Body-temperature homeostasis is actively controlled during daily torpor.

(a) Protocol of the fasting-induced daily torpor experiment. Animals were placed in the T_A-constant chamber on Day 0; data were recorded for 72 hours from the beginning of Day 1. Food was removed and returned at the beginning of Day 2 and Day 3. Water was freely accessible throughout the experiment. (b) The minimum T_B at various T_As. Including the following panels, red and blue denote normal and torpid status, respectively. For normal status, the minimum T_B of the dark phase of Day 1 was used for analysis. For torpid status, the minimum T_B during torpor was used for analysis. As in the following panel d, the dots with the vertical error bars denote the observed mean and SEM of the minimum variables (T_B in b, VO₂ in d) at each T_A, and the line and the shaded area denote the mean and the 89% HPDI intervals of the estimated minimum variables. (c) The posterior distribution of the slope (a₁) of T_A−T_B relationship. Including the following distribution panels in this figure, the bold and thin lines denote the mean and the 89% HPDI intervals of the estimated values. The bin size is 0.005. (d) The minimum VO₂ at various T_As. Minimum VO₂ was defined as the VO₂ recorded when the T_B was minimum. (e) The posterior distribution of the slope (a₂) of T_A-VO₂ relationship. The bin size is 0.001 ml/g/hr/°C. (f) The rate of successful daily torpor induction at various T_As. When T_A was above 12 °C, all animals entered daily torpor. (g) The averaged torpor duration for each episode. One torpor episode is tended to be shorter when the T_A gets higher. (h, i) The posterior distribution of the estimated mean (n = 6) of T_B (h) and VO₂ (i) at a T_A of 8 °C. Because the observed mean (dashed lines) is larger than the 89% HPDI in both T_B and VO₂, when T_A = 8 °C, the animal exhibited higher metabolism than expected. The bin size is 0.1 °C and 0.01 ml/g/hr for T_B and VO₂, respectively.

Figure 5. The sensitivity of the heat production system is largely reduced during daily torpor while the reduction of set-point temperature was small.

(a) The relationship between the difference of T_B from T_A and VO₂. Including the following panels, red and blue denote normal and torpid status, respectively. The slope of this relationship is the heat conductance, G. As in panel c, the dots represent the observed values, and the lines and shaded areas represent the means and the 89% HPDI intervals of the estimated values. (b) The posterior distribution of the estimated G. During torpor, G is smaller than during normal states. Decrease in G results in heat preservation. However, the T_B decrease seen in daily torpor is indicating the decrease in G is overridden or induced by decrease of heat production. The bin size is 0.001 ml/g/hr/°C. As in panel d and e, the bold and thin lines denote the mean and the 89% HPDI intervals of the estimated values. (c) The relationship between minimum T_B and VO₂ seen during normal and torpid states among various T_As. The brightness of the dots is indicating the T_A. The horizontal intercept of the line indicates the theoretical set-point of T_B, which is T_R (See Fig. 1a). During normal states, T_B is kept relatively constant by employing oxygen and producing heat to fill the gap between T_R and T_A. On the other hand, during daily torpor, the sensitivity against T_R - T_B is weakened which is visualized by less steep slope, which is H, the open-loop negative feedback gain of the heat production loop (See Fig. 1a). (d) The posterior distribution of the estimated T_R. During daily torpor, T_R became smaller than normal states, although the mean difference was 3.79 °C. The bin size is 0.1 °C. (e) The posterior distribution of the estimated H. During daily torpor, H became dramatically smaller than normal states, which the mean difference reached to 4.70 ml/g/hr/°C. This is clearly showing that the open-loop gain of the heat production system reduced to 8.5% during daily torpor from the normal state. The bin size is 0.05 ml/g/hr/°C.