Hypothermia in mouse is caused by adenosine A1 and A3 receptor agonists and AMP via three distinct mechanisms

Abstract

Small mammals have the ability to enter torpor, a hypothermic, hypometabolic state, allowing impressive energy conservation. Administration of adenosine or adenosine 5'-monophosphate (AMP) can trigger a hypothermic, torpor-like state. We investigated the mechanisms for hypothermia using telemetric monitoring of body temperature in wild type and receptor knock out (Adora1^-/-, Adora3^-/-) mice. Confirming prior data, stimulation of the A₃ adenosine receptor (AR) induced hypothermia via peripheral mast cell degranulation, histamine release, and activation of central histamine H₁ receptors. In contrast, A₁AR agonists and AMP both acted centrally to cause hypothermia. Commonly used, selective A₁AR agonists, including N⁶-cyclopentyladenosine (CPA), N⁶-cyclohexyladenosine (CHA), and MRS5474, caused hypothermia via both A₁AR and A₃AR when given intraperitoneally. Intracerebroventricular dosing, low peripheral doses of Cl-ENBA [(±)-5'-chloro-5'-deoxy-N⁶-endo-norbornyladenosine], or using Adora3^-/- mice allowed selective stimulation of A₁AR. AMP-stimulated hypothermia can occur independently of A₁AR, A₃AR, and mast cells. A₁AR and A₃AR agonists and AMP cause regulated hypothermia that was characterized by a drop in total energy expenditure, physical inactivity, and preference for cooler environmental temperatures, indicating a reduced body temperature set point. Neither A₁AR nor A₃AR was required for fasting-induced torpor. A₁AR and A₃AR agonists and AMP trigger regulated hypothermia via three distinct mechanisms.

Keywords: (1R,2R,3S,5S)-4-(2-chloro-6-((dicyclopropylmethyl)amino)-9H-purin-9-yl)bicyclo[3.1.0]hexane-2,3-diol; (±)-5'-chloro-5'-deoxy-N(6)-endo-norbornyladenosine; 2-Chloro-N(6)-cyclopentyladenosine; A(1)AR; A(3)AR; AMP; Adenosine; Adenosine 5'-monophosphate; CCPA; CHA; CPA; Cl-ENBA; Hypothermia; MRS5474; N(6)-(p-sulfo-phenyl)adenosine; N(6)-R-phenylisopropyladenosine; N(6)-cyclohexyladenosine; N(6)-cyclopentyladenosine; R-PIA; SPA; Torpor.

Fig. 1. Systemic CHA causes hypothermia and decreased physical activity through both A₁AR and A₃AR

(A, B) Tb and physical activity response to the indicated CHA doses injected i.p. into C57BL/6J mice. (C–E) Effect of CHA on average Tb (0–60 min), duration of hypothermia, and physical activity (10–60 min). Data are mean ± SEM, n=5/group; every tenth SEM is shown in A and SEMs were omitted in B for visual clarity. (F,G) Tb response to CHA (0.05 mg/kg, i.p.) or vehicle in C57BL/6J (WT) and Adora1^−/− (KO) mice. (H,I) Tb response to CHA (0.05 mg/kg, i.p.) or vehicle in C57BL/6J (WT) and Adora3^−/− (KO) mice. (J,K) Tb response to CHA (0.05 mg/kg, i.p.) or vehicle in C57BL/6J (WT) and Adora1^−/−;Adora3^−/− (DKO) mice. In F–K, data are mean ± SEM, n=5–10/group in a crossover design; every tenth SEM is shown in F, H, and J; * p<0.05, *** p<0.001.

Fig. 2. Systemic Cl-ENBA acts largely via A₁AR to induce hypothermia

(A,B) Tb response to the indicated Cl-ENBA dose injected i.p. into C57BL/6J mice. (C,D) Tb response to Cl-ENBA (3 mg/kg, i.p.) or vehicle in C57BL/6J (WT) and Adora1^−/− (KO) mice. (E,F) Tb response to Cl-ENBA (3 mg/kg, i.p.) or vehicle in C57BL/6J (WT) and Adora3^−/− (KO) mice. (G,H) Tb response to Cl-ENBA (3 mg/kg, i.p.) or vehicle in C57BL/6J (WT) and Adora1^−/−;Adora3^−/− (KO) mice. Data are mean ± SEM, n=3–6/group in a crossover design; every tenth SEM is shown in C, E, and G; * p<0.05*** p<0.001.

Fig. 3. A₁AR-mediated hypothermia is partially intact and A₃AR-mediated hypothermia is abolished in Kit^W-sh/W-sh mice

(A,B) Tb response to Cl-ENBA (1 mg/kg, i.p.) in Kit^W-sh/W-sh mice. (C,D) Tb response to Cl-ENBA (3 mg/kg, i.p.) in Kit^W-sh/W-sh mice. (E,F) Tb response to MRS5474 (3 mg/kg, i.p.) in Kit^W-sh/W-sh mice. Data are mean ± SEM, n=4–6/group in a crossover design; every tenth SEM is shown in A, C, and E; ** p<0.01.

Fig. 4. Cl-ENBA causes hypothermia via central A₁AR

(A,B) Tb response to Cl-ENBA (3.4 µg/mouse) or vehicle injected i.c.v. into C57BL/6J. (C,D) Tb response to Cl-ENBA (15 µg/mouse) or vehicle injected i.c.v. into C57BL/6J or Adora1^−/− mice. Data are mean ± SEM, n=3–6/group; every tenth SEM is shown in A, C; * p<0.05.

Fig. 5. Systemic Cl-ENBA-induced hypothermia is accompanied by a reduced metabolic rate and preference for a cooler environment

The effect of Cl-ENBA (3 mg/kg, i.p.) or vehicle in Adora3^−/− mice on (A) total energy expenditure (TEE), (B) Tb, and (C) physical activity. The TEE falls before Tb (nadir ~50 min vs ~100 min for Tb). Data are mean ± SEM, n=5/group. Position (D,E) and activity (F,G) of Adora3^−/− mice treated with Cl-ENBA (3 mg/kg, i.p.) or vehicle and placed in a thermal gradient. Mean ± SEM, n=6/group, *p<0.05, ***p<0.001.

Fig. 6. A₁AR or A₃AR is not required for fasting-induced hypothermia

Baseline Tb (A) and activity (B) in WT (n=6) and Adora1^−/− (n=4) littermates during the light or dark phase. Baseline Tb (C) and activity (D) in WT (n=5) and Adora3^−/− (n=4) littermates during the light or dark phase. Baseline Tb (E) and activity (F) in C57BL/6J (n=6) and Adora1^−/−;Adora3^−/− (n=3) mice. during the light or dark phase. Individual traces of Tb in (G) WT and Adora1^−/− littermates, (H) WT and Adora3^−/− littermates, and (I) C57BL/6J and Adora1^−/−;Adora3^−/− mice during a 24 hour fast. White and black bars indicate light and dark phases, respectively. Data are mean ± SEM.

Fig. 7. Systemic AMP causes hypothermia independent of A₁AR, A₃AR, and mast cells

(A,B) Tb response to AMP (100 mg/kg, i.p.) in C57BL/6J (WT) and Adora1^−/− (KO) mice (n=6/group). (C,D) Tb response to AMP (100 mg/kg, i.p.) in C57BL/6J (WT) or Adora3^−/− mice (n=4/group). (E,F) Tb response to AMP (100 mg/kg, i.p.) in C57BL/6J and Adora1^−/−;Adora3^−/− mice (n=5–6/group). (G,H) Tb response to AMP (100 mg/kg, i.p.) in C57BL/6J (WT) or Kit^W-sh/Wsh mice (n=7–8/group). Data are mean ± SEM; every tenth SEM is shown in A, C, E, and G; * p<0.05, ** p<0.01, *** p<0.001.

Fig. 8. Centrally-administered AMP induces hypothermia

(A,B) Tb response to 100 µg (~3.3 mg/kg) AMP injected i.c.v. into C57BL/6J mice or Adora1^−/− (n=4/group). Data are mean ± SEM; every tenth SEM is shown; * p<0.05, ** p<0.01.

Fig. 9. AMP-induced hypothermia is accompanied by a reduced metabolic rate and preference for a cooler environment

The effect of AMP (100 mg/kg, i.p.) or vehicle in C57BL/6J mice on (A) total energy expenditure (TEE), (B) Tb, and (C) physical activity in a calorimetry chamber. The TEE falls before Tb (nadir ~40 min vs ~60 min). Data are mean ± SEM, n=5/group. Position (D,E) and activity (F,G) of C57BL/6J mice treated with AMP (300 mg/kg, i.p.) or vehicle and placed in a thermal gradient. Position (H,I) and activity (J,K) of C57BL/6J mice treated with AMP (100 µg, i.c.v.) or vehicle and placed in a thermal gradient. Mean ± SEM, n=5–6/group, *p<0.05, ***p<0.001