Gonadal hormones influence core body temperature during calorie restriction

ABSTRACT

During calorie restriction (CR), endotherms adjust several physiological processes including the decrease of core body temperature (T_b) and reduction of energy expenditure. We recently found that CR-induced hypothermia is regulated in a sex-dependent manner in mice with lowered central insulin-like growth factor receptor signaling. Here, we describe the contribution of sex hormones to CR-induced hypothermia in wild type C57BL6 mice by measuring T_b of female and male mice following bilateral gonadectomy and hormonal replacement. Specifically, we evaluated the effects of progesterone (P4), 17-ß estradiol (E2), a combination of both (P4 + E2) in females and of 5-α dihydrotestosterone (5-α DHT) in males. Gonadectomy resulted in an earlier and stronger CR-induced hypothermia in both sexes. These effects were fully antagonized in females by E2 replacement, but not by P4, which had only minor and partial effects when used alone and did not prevent the action of E2 during CR when both hormones were given in combination. 5-α-DHT had only minor and transient effects on preventing the reduction of T_b during CR on gonadectomized male mice. These findings indicate that gonadal hormones contribute to sex-specific regulation of T_b and energy expenditure when nutrient availability is scarce.

Abbreviations: AL: ad libitum; ANOVA: analysis of variance; CR: calorie restriction; E2: 17-ß estradiol; GNX: gonadectomy or gonadectomized; IGF-1R: insulin-like growth factor 1 receptor; POA: preoptic area; P4: progesterone; RM: repeated measures; SD: standard deviation; SEM: standard error of mean; T_b: core body temperature; WT: wildtype; 5-α DHT: 5-α dihydrotestosterone.

Introduction

Calorie intake and energy expenditure are the main components of energy homeostasis. Their integration is fundamental for the survival of living organisms, allowing them to adjust their physiological function to survive in extreme environmental conditions and contributing to their health and lifespan [1–6]. When energetic resources are limited, such as during controlled calorie restriction (CR), endotherms limit energy expenditure by lowering core body temperature (T_b) [7,8]. This response is believed to be under genetic control and to have evolved as a mechanism to save energy, prolonging survival until more food becomes available [8,9]. The molecules and the signals that are involved in regulating this physiological adaptation are beginning to be identified [10–14].

We recently reported that the insulin-like growth factor receptor 1 (IGF-1R) signaling pathway is one of the factors that regulates T_b during CR and that it does so in a sex- and dose-specific manner [15]. In fact, while full genetic ablation of IGF-1R reduced the hypothermic response to CR equally in male and female mice, its genetic dosage affected T_b only in female mice also displayed a transient and estrus-specific hypothermia when fed ad libitum. Sex hormones are known for their ability to affect T_b especially in females, where their fluctuating levels throughout the days account for subtle but significant differences in T_b during the estrous cycle [16–18]. We previously demonstrated that gonadectomy eliminated the sex-specific differences in the circadian profile of T_b of mice fed ad libitum [19]. Here we measured the effects that gonadectomy and hormonal replacement had on the hypothermic response to CR of female and male mice.

Results

Gonadectomy enhanced the hypothermic response to calorie restriction

In order to test the hypothesis that gonadal hormones contribute to the regulation of T_b during CR, we compared the T_b profile of wildtype (WT) female (Figure 1(a)) and male (Figure 1(b)) mice subjected to progressive CR before and after gonadectomy (GNX). Upon GNX, female mice significantly decreased their T_b starting on day 2 of 75% CR. This profound hypothermic response to CR is consistent throughout the CR regimen, reaching the lowest T_b of ~24.4°C compared to ~31.3°C before gonadal removal. Gonadectomy also affected T_b in male mice (Figure 1(b)), where it enhanced the hypothermic response as early as day 1 of 75% CR. The amount of T_b reduction increased with the progression of the CR regimen, reaching the lowest value of ~26.8°C compared to ~33.9°C before GNX. Taken together, these data indicate that both female and male gonads and potentially the molecules produced by the gonads contribute to the regulation of the hypothermic response to CR.

Figure 1.

Gonads influence core body temperature. T_b profile of WT female (a), male (b) mice before and after bilateral gonadectomy subjected to CR. Horizontal black lines represent lights off (ZT 12–23; active phase of the day) while the yellow lines represent lights on (ZT 0–11; inactive phase of the day). n = 5 animals per group, *p < 0.05 between treatment.

Progesterone partially prevented CR-induced hypothermia in females

Next, we measured whether replacement with the major gonadal hormones could reverse the effects of gonadectomy on T_b during CR. Three-months-old C57BL6 GNX female mice were subcutaneously implanted with progesterone (P4) (10 mg/21 d) or placebo pellets (10 mg/21 d) during AL feeding. P4 treatment increased T_b during certain intervals of the day, reaching a small but significant difference of ~0.5°C between treatments starting at the inactive phase during ad libitum day 1 (F_{1, 11} = 7.385, p = 0.02) (Supplementary Table 1). P4 thermogenic effects were independent of food intake as P4 and the placebo group did not differ in their food intake (t(11) = 0.01502, p = 0.9883; Figure 2(a)). T_b remained elevated throughout the next three days of ad libitum, in which the daily active and inactive average T_b for the P4 group was higher, ~0.3–0.4°C and ~0.3–0.6°C, respectively, than the placebo group (Figure 3(a) and Supplementary Table 1). The thermogenic effects of progesterone have been previously reported for ad libitum feeding but have not been reported during reduced calorie intake [16]. When the mice were subjected to CR, P4 treatment kept promoting its mild thermogenic effects on treated mice (Figure 3(b)). The average T_b of the P4 group was higher than the placebo group during certain days of the CR regimen, specifically for the inactive phase (Supplementary Table 2). However, P4 only partially and transiently attenuated CR-induced hypothermia during 60% CR (Figure3(b)) and by day 1 of 50% CR, P4 treatment was less effective at preventing CR-induced hypothermia; although the P4 group still presented a higher T_b during the hypothermic phase (32.0°C ± 1.4°C vs 28.6°C ± 0.5°C of the placebo group, F_1,11 = 6.264, p = 0.0294; Figure S1). As expected, body weight was reduced similarly across groups during CR. (Figure 2(b))

Figure 2.

Food intake and body weight of GNX treated mice. Average food intake of 3 d of ad libitum feeding for female (a) and male (c) mice. Body weight measurements throughout the calorie restriction protocol for female (b) and male mice (d). *p < 0.05 between treatments.

Figure 3.

Progesterone alone partially prevents CR-induced hypothermia. Temperature profile of GNX female mice hormonally replaced with progesterone (P4 10 mg/21 d) or Placebo (10 mg/21 d) during four days of ad libitum (a) feeding followed by calorie restriction (b). Horizontal black lines represent lights off (ZT 12–23; active phase of the day) while the yellow lines represent lights on (ZT 0–11; inactive phase of the day) and the green lines represent the hypothermic period of the day (ZT23.5–7.5). n = 6–7 animals per group, *p < 0.05 between treatments.

17-ß estradiol severely reduced the hypothermic response to calorie restriction in females

A different group of GNX female mice was implanted with 17-ß estradiol (E2) pellets (0.5 mg/21 d). Similar to what has been previously reported, estrogen replacement caused an immediate mild reduction of T_b in gonadectomized female mice [20]. Compared to placebo, estrogen lowered T_b by 0.3–0.5°C during the active phase of the day and by ~0.5°C during the inactive phase of the day (Figure 4(a) and Supplementary Table 1). This effect was independent of food intake, since both groups were consuming similar ad libitum amount of food (placebo, 4.9 ± 0.2 g vs E2, 4.6 ± 0.3 g, n = 7, 6, t(11) = 0.9658, p = 0.3549; Figure 2(a)). This mild reduction of T_b was also observed following CR until the first day of 75% CR (Figure 4(b)). However, by day 1 of 60% CR, the hypothermic response seen in the placebo group was significantly prevented in the E2 group (Figure 4(b)). Respectively, the placebo and the E2 groups lowered T_b to a minimum of ~26.6 °C and ~31.5 °C (Supplementary Table 2). CR affected body weight of both placebo and E2 groups in a similar manner; ruling out the possibility that the hypothermic prevention observed in the E2 group was due to mass differences between groups instead of the hormone replacement itself [21] (Figure 2(b)).

Figure 4.

17-ß estradiol alone fully prevents CR-induced hypothermia. Temperature profile of GNX female mice hormonally replaced with 17-ß estradiol (E2 0.5 mg/21 d) or Placebo (10 mg/21 d) during four days of ad libitum (a) feeding followed by calorie restriction (b). Horizontal black lines represent lights off (ZT 12–23; active phase of the day) while the yellow lines represent lights on (ZT 0–11; inactive phase of the day) and the green lines represent the hypothermic period of the day (ZT23.5–7.5). n = 6–7 animals per group, *p < 0.05 between treatments.

Progesterone did not alter the effects of estrogen in preventing the hypothermic response to CR in females

A group of GNX female mice received both progesterone and 17-ß estradiol (P4 + E2) or placebo pellets. When fed AL, T_b of the P4 + E2 group was significantly lower, 36.8 ± 0.3°C, in comparison to the placebo group 37.2 ± 0.3°C only during the active phase of day 2 (F_{1, 10} = 8.064, p = 0.0176; Supplementary Table 1 ). Besides this difference, on average, the P4 + E2 group presented a similar T_b as compared to the placebo group, ~37.1°C during the active phase of the day and ~36.1°C during the inactive phase of the day (Figure 5(a) and Supplementary Table 1). When animals were subjected to CR, T_b reduction in the P4 + E2 group was severely attenuated compared to the placebo group. Similar to what is seen in the E2 group, T_b between P4 + E2 and placebo groups significantly differed by day 1 of 60% feeding (Figure 5(b)). On average, the P4 + E2 group presented a T_b during the hypothermic phase of 35.3 ± 0.1°C in comparison to 30.4 ± 0.9 °C of the placebo group (F_1,10 = 19.58, p = 0.0013) (Figure S1). This difference of 4.0°C to 6.5 °C in T_b between groups during the hypothermic phase was consistent from Day 1 of 60% feeding up to Day 1 of 50% feeding. No differences in food intake during the ad libitum regimen and body weight reduction during CR were observed between P4 + E2 and the placebo group (Figure 2(a)–(b)).

Figure 5.

P4 + 17-ß estradiol prevent hypothermia development during CR. Temperature profile of GNX female mice hormonally replaced with both progesterone (P4 10 mg/21 d) and 17-ß estradiol (E2 0.5 mg/21 d) or Placebo (10 mg/21 d) during four days of ad libitum (a) feeding followed by calorie restriction (b). Horizontal black lines represent lights off (ZT 12–23; active phase of the day) while the yellow lines represent lights on (ZT 0–11; inactive phase of the day) and the green lines represent the hypothermic period of the day (ZT23.5–7.5). n = 5–7 animals per group, *p < 0.05 between treatments.

5-α dihydrotestosterone partially and transiently prevented the hypothermic response to CR in gonadectomized male mice

Hormonal replacement was also carried out in male mice by testing the effects of testosterone (5-α DHT), in regulating CR-induced hypothermia in GNX male mice. Two groups of GNX male mice were subcutaneously implanted with either placebo or 5-α DHT (5 mg/21 d) during AL. Upon 5-α DHT treatment, average T_b during the inactive phase of the first two consecutive days of AL was lower in comparison to placebo-treated animals (Supplementary Table 3). 5-α DHT group consumed more food than the placebo group (placebo, 4.4 ± 0.1 g vs 5-α DHT, 5.0 ± 0.2 g, n = 6, 6, t(10) = 2.358, p = 0.0401), ruling out the possibility that the animals were calorie restricting themselves upon 5-α DHT treatment (Figure 2(c)). Besides these differences, T_b during the active phase of the day was similar between groups, ~37.1 °C for the placebo group and ~37.0 °C for 5-α DHT group (Figure 6(a)). Then, animals were subjected to the same CR regimen described for female animals. Starting with a fixed amount of provided food (100% feeding determined from AL measurements), both groups presented an early reduction in T_b by day 2 in 100% feeding. Nevertheless, the T_b reduction presented by 5-α DHT group was more profound than the placebo group, reaching ~28.9°C in comparison to ~32.9°C of the placebo group (Figure 6(b)). During 75% CR, CR-induced hypothermia was mostly similar in both groups, but by the first day of 60% CR, 5-α DHT treatment moderately prevented the development of a profound hypothermia (~29.7°C) in comparison to the placebo group (~24.5–26°C) (Supplementary Table 4) (F_{1, 10} = 7.054, p = 0.0241). By 50% CR, both groups reduced their T_b almost to room temperature levels, ~24.1°C. Both groups reduced their body weight similarly throughout the CR regimen (Figure 2(d)).

Figure 6.

Testosterone did not fully prevent T_b reduction upon CR in gonadectomized male mice. Temperature profile of GNX male mice hormonally replaced with 5-α dihydrotestosterone (5mg/21 d) or Placebo (10mg/21 d) during four days of ad libitum (a) feeding followed by calorie restriction (b). Horizontal black lines represent lights off (ZT 12–23; active phase of the day) while the yellow lines represent lights on (ZT 0–11; inactive phase of the day) and the green lines represent the hypothermic period of the day (ZT23.5–7.5). n = 6 animals per group, *p < 0.05 between treatments.

Discussion

The main finding of the present study is that the gonads through their hormones can influence the regulation of core body temperature during reduced calorie intake. Specifically, gonadectomy resulted in enhanced hypothermic response to calorie restriction in both sexes (Figure 1). This reduction in T_b consistently occurs from the last hours of the active phase of the day (lights off) into the inactive phase of the day (lights on). This is due to the fact we provide food to the animals 1 h before the active phase of the day starts. The food is consumed within 3–4 h period. The next 20 h of the day present a period of fasting until food is provided again the next day. Thus, the hypothermic response occurs due to this fasting period and the presumed circadian dysregulation of molecules modulating daily energy expenditure in the face of reduced calorie intake [22]. If the food would have been provided during the inactive phase of the day, most likely the animals would have experienced the hypothermic response during the active phase of the day. This would have affected their normal circadian T_b profile, in which reduction of T_b under ad libitum conditions occurs in the inactive phase of the day (lights on) [15,19,20]. Despite this qualitative similarity in T_b reduction, females were more sensitive to this manipulation, displaying a deeper hypothermic response reaching ~29.4°C that was evident as early as day 2 of 75% CR compared to male mice with a hypothermic response of ~33.2°C (Figure 1(a-b)). Hormonal replacement provided the first indication that gonadal molecules may influence this action. Among the two hormones tested in females, estrogen was the most effective in antagonizing the effects of CR on T_b reduction, while progesterone had only partial actions. In males, testosterone only had minor and transient effects on preventing CR-induced hypothermia. The mechanisms by which these hormones contribute to the hypothermic response to CR were not investigated here. However, it is likely that they include a direct action on the neuronal circuitry that regulates temperature homeostasis centrally. Sex hormones readily cross the blood-brain barrier and have been previously proposed to impact T_b by acting directly on neurons of the hypothalamic preoptic area (POA). The POA receives information about ambient and local temperature and coordinates the appropriate physiological response for the maintenance of temperature homeostasis (reviewed in [23]).

Progesterone, estrogen, and androgen receptors have all been reported to be expressed in hypothalamic regions including the POA and to be able to influence T_b [24–29]. Application of estradiol to the POA increased the firing rate of warm as well as some temperature-insensitive neurons and may contribute to explain of estradiol to lowers T_b by increasing vasodilation and heat dissipation [20,30,31]; this reduction in T_b is observed in our ad libitum data. Nevertheless, during CR, estradiol administration prevents T_b reduction. The mechanism by which this physiological response differs due to different calorie intake regimens is unknown. Progesterone increased T_b when applied centrally in the POA of ovariectomized rats or systemically by promoting less vasodilation and heat conservation [16,31]. In electrophysiological studies on neurons in preoptic tissue slices, testosterone was able to excite warm-sensitive and insensitive neurons and to inhibit cold-sensitive neurons [28]. While in castrated male mice during ad libitum feeding or castrated mice subjected to heat exposure, testosterone administration contributed to an increase in T_b, that was prevented in castrated mice without testosterone replacement [19,32]. These observations obtained from experiments carried out in animals fed ad libitum demonstrated the ability of gonadal hormones to affect temperature and suggested some of the mechanisms by which this may occur. Our findings in animals subjected to CR indicate that these actions are important regulators of the physiology of the organism that may have evolved to promote survival of the organism as well as of the species when food is scarce.

Calorie restriction does not only lower T_b but also suppresses reproduction, which is unlikely to be successful if nutrients are limited [9,33–36]. This may be achieved simultaneously by reducing estrogen production [36]. Such effects are only opposite to the already known role of progesterone in maintaining both pregnancy and the elevated T_b that characterizes the first period of gestation [31,37–39]. We speculate that progesterone and estrogen act as metabolic signals integrating nutrient and temperature homeostasis and reproduction. Progesterone has the ability to elevate T_b when nutrients are abundant as seen during ad libitum feeding and gestation, while estrogen reduction contributes to T_b reduction during reduced food intake, thus promoting energy conservation.

5-α DHT is the main active male gonadal sex hormone. Given the strong effects that male gonadectomy had on T_b, we hypothesized that these were mediated by 5-α DHT. However, hormonal replacement had only minor effects on T_b and did not rescue the hypothermic response. Although testosterone was reported to have thermogenic effects upon heat exposure [32], this was not observed upon CR. These results indicate that the hypothermic response to CR in males is influenced by gonadal molecules other than 5-α DHT. Such molecule/s remain to be identified but may include testosterone precursors such as androstenedione, which it is also reduced by bilateral gonadectomy [40], or estrogen that can be synthesized in the testis and expresses its receptors in the POA [24,41]. It could be expected that in GNX male mice, administration of E2 has similar results to those obtained in GNX female mice, reduction in T_b during ad libitum feeding and hypothermic prevention during CR. Finally, the POA is the most dimorphic region of the brain. Thus, it is also possible that sex-specific differences exist in the cellular or molecular composition of the neuronal circuitry that regulates T_b [19,42,43].

Altogether, our data showed that the gonads and the gonadal hormone estrogen are important regulators of T_b during calorie restriction. By contributing to reduce T_b during CR, estrogen participates in the integration of nutrient and temperature homeostasis. Lowering T_b is an effective way to decrease energy expenditure when calorie intake is reduced. The role of sex hormones in this important physiological response is not only fundamental for the survival of the organism but may help explain why, especially in females, body weight gain is more common in post-reproductive age and the efficacy of dieting to reduce body weight is limited. In fact, reduced estrogen levels in postmenopausal women might contribute to an enhanced hypothermic response to dieting that would limit energy expenditure and body weight loss [44].

Materials and methods

Mice and husbandry

All procedures were approved by TSRI IACUC. 129/SvPas wild type mice were used in the experiments presented in Figure 1.The rest of the experiments utilized female and male C57BL/6 mice. Animals were singly caged in standard 12:12 h light:dark (lights on 11:00 pm; Zeitgeber time 0 or ZT0) at a controlled ambient temperature of 22 ± 0.5°C. Water was available AL during CR experiments. Food (Lab Diet 5053 Irradiated Pico Lab containing 3.41 kcal/g, 20.0% protein, 52.9% carbohydrates, 10.6% fat, 4.7% crude fiber and 6.1% ash) was provided AL or restricted during the CR experiments as specified.

Radiotelemetry implant

Radiotelemetry for measuring T_b and was performed using a transmitter (TA10TA-F10; Data Sciences, Inc.) surgically implanted into the peritoneal cavity, as described [45]. Animals were allowed a recovery time of 14 d postsurgery and 2 d of habituation to the experimental environment before beginning experiments. Data were recorded by placing a cage containing an animal implanted with a radio transmitter on a receiver plate (RPC-1; DataScience, Inc.). Data collection and offline analysis were performed using the DATAQUEST A.R.T. software (DataScience, Inc.).

Gonadectomy

Data presented in Figure 1, gonadectomy was performed after a month of being back to ad libitum feeding and 2 wk post-surgery recovery, animals were subjected to the CR regimen described below. Gonadectomy in female mice was performed as previously described [19,46,47]. Briefly, the anesthetized animal with isoflurane was placed in ventral recumbency, and the mid-dorsal region was carefully shaved. A small (approx. 0.5 cm) lateral incision was made through the skin of the back of the mouse in the mid-lumbar region. The skin was slid from side-to-side to locate the position of the ovaries beneath the peritoneal wall. While holding the abdominal wall up, a small incision was made in the abdominal wall over the ovarian fat pad and the ovary exteriorized. The ligaments were carefully torn to release the ovary. A clamp was placed across the tip of the uterine horn, and a ligature was placed just underneath the tip of the uterine horn. The ovary/oviduct was removed by cutting above the ligature. The uterine horn was repositioned into the body cavity. The abdominal wall was closed with one or two simple interrupted sutures. This procedure was repeated for removal of the second ovary. The skin incision was closed with vicryl rapide suture. Post-surgery the animal was administered with the analgesic flunixin 0.25mg/ml to reduce pain.

Gonadectomy in males was performed as previously described [19,46,47]. Briefly, the anesthetized animal with isoflurane was placed in dorsal recumbency, and the scrotal area was carefully shaved. Prior to draping the site, sterile gauze was placed over the incision site. A small (approx. 1 cm) midline incision was made in the scrotum and subsequently through the tunica. The testicle was exteriorized, and a clamp was placed across the spermatic vessels, which was ligated and cut across the top, enabling removal of the testicle. The ligated vessels were allowed to pass back through the tunica incision subsequently closed with one or two simple interrupted 5–0 sutures. The procedure was repeated for removal of the second testicle and flunixin as stated above was administered at the completion of the procedure.

Hormonal replacement

Hormonal replacement was performed 14 d post gonadectomy during ad libitum feeding. Commercially available sterile slow-release pellets were purchased from Innovative Research of America (Sarasota, FL, USA): 17β-estradiol (0.5 mg/21 d), 5α-dihydrotestosterone (5 mg/21 d), and progesterone (10 mg/21 d). Pellets dose was determined following our previous study, and they were implanted subcutaneously as previously described [19]. Briefly, an incision was made along the dorsal midline immediately posterior to the scapulae, just large enough to allow passage of the device. The sterile pellet was placed under the skin in the subcutaneous region. Absorbable sutures were used to close the dorsal incision, and the skin was closed with non-absorbable sutures. In order to minimize the incidence of infection, topical antibiotic treatments (Bacitracin Zinc Ointment) were applied after the completion of surgical procedures.

Calorie restriction

CR was carried out by providing a percentage of the average amount of food consumed during the last 3 d of AL diet upon hormone replacement. Animals were given 100% of the AL diet for 2 d to habituate to daily rations of food, followed by 75% of AL for 4 d, 60% of AL for 4 d and 50% of AL for 5 d. Food was provided daily at a 1-h window before lights were turned off (ZT11 to ZT12). All animals ate the entire amount of food provided during CR experiments. In the graphs we present the odd days of the CR protocol, because during some of the even days of the protocol, animals were retrieved and handled for a longer period of time than 5 min for taking other physiological parameters, thus affecting the continuous T_b recordings.

Hypothermic response

Average hypothermic T_b consists of the lowest T_b during 8 h of the day. From 10:30 pm to 6:30 am (ZT23.5 to ZT7.5). This time-window, named as CR-induced hypothermia was determined using the average T_b of the placebo group. When the average T_b of this group goes below 34.0°C (<33.9°C), we count this time point as the beginning of the hypothermic phase, until the average T_b increases and reaches 34.0°C again.

Estrus cycle determination

Estrus cycle was monitored post gonadectomy to ensure the procedure was successful. This was done by histological analysis of vaginal smear by lavage performed in the first 3 h of the dark cycle using the modified version of the procedure [19]. Briefly, the mouse was gently raised holding it by its tail and the vagina was flushed with 5 μL of sterile saline using a smooth and tapered micropipette tip. Collected samples were placed on a slide and were allowed to dry, then stained and fixed for subsequent examination using a standard laboratory light microscope with a × 100 total magnification. Cytology was used to define cycle stage, and stage assignment was based on the classification of cells and relative abundance of each cell type (percentage of parabasal, intermediate, cornified, and neutrophils).

Statistical analysis

GraphPad Prism 7 was used for data analysis. RM two-way ANOVA followed by Bonferroni’s multiple comparison correction was used to analyze the difference between treatments on T_b and body weight on multiple collected time-points during AL, CR regimen. Food intake differences were analyzed by unpaired two-tailed t-test.

Funding Statement

This work was supported by the National Institute of General Medical Sciences [GM113894].

Acknowledgments

Supported by The National Institute of Health, GM113894. RC-C is supported by the Skaggs Graduate School of Chemical and Biological Sciences and ARCS Foundation.

Disclosure statement

No potential conflict of interest was reported by the authors.

Supplementary material

Supplemental data for this article can be accessed here.