Central activation of the A1 adenosine receptor in fed mice recapitulates only some of the attributes of daily torpor

Abstract

Mice enter bouts of daily torpor, drastically reducing metabolic rate, core body temperature (T _b), and heart rate (HR), in response to reduced caloric intake. Because central adenosine activation has been shown to induce a torpor-like state in the arctic ground squirrel, and blocking the adenosine-1 (A₁) receptor prevents daily torpor, we hypothesized that central activation of the A₁ adenosine receptors would induce a bout of natural torpor in mice. To test the hypothesis, mice were subjected to four different hypothermia bouts: natural torpor, forced hypothermia (FH), isoflurane-anesthesia, and an intracerebroventricular injection of the selective A₁ receptor agonist N^6-cyclohexyladenosine (CHA). All conditions induced profound hypothermia. T _b fell more rapidly in the FH, isoflurane-anesthesia, and CHA conditions compared to torpor, while mice treated with CHA recovered at half the rate of torpid mice. FH, isoflurane-anesthesia, and CHA-treated mice exhibited a diminished drop in HR during entry into hypothermia as compared to torpor. Mice in all conditions except CHA shivered while recovering from hypothermia, and only FH mice shivered substantially while entering hypothermia. Circulating lactate during the hypothermic bouts was not significantly different between the CHA and torpor conditions, both of which had lower than baseline lactate levels. Arrhythmias were largely absent in the FH and isoflurane-anesthesia conditions, while skipped beats were observed in natural torpor and periodic extended (>1 s) HR pauses in the CHA condition. Lastly, the hypothermic bouts showed distinct patterns of gene expression, with torpor characterized by elevated hepatic and cardiac Txnip expression and all other hypothermic states characterized by elevated c-Fos and Egr-1 expression. We conclude that CHA-induced hypothermia and natural torpor are largely different physiological states.

Keywords: Adenosine; Hibernation; Hypothermia; Targeted temperature management; Torpor.

Figure 1. Body temperature (T_b) and heart rate (HR) relationship during bouts of hypothermia

Mice were implanted with an ECG/T_b telemeter and underwent four different bouts of hypothermia. 1) Natural torpor, induced by chronic 70% caloric restriction at an ambient temperature (T_a) of 22°C. 2) Forced hypothermia (FH), where mice were restrained and exposed to a cold temperature (see Methods). 3) Isoflurane-anesthesia, where mice were kept anesthetized with 2.5% isoflurane in an O₂ stream at an T_a of 22°C. 4) ICV injection of the adenosine receptor 1 agonist, cyclohexyladenosine (CHA) at an T_a of 22°C. A. T_b and HR (derived from the ECG tracing) over the bout of hypothermia. B. HR for each condition is plotted as a function of the concurrent T_b. A typical T_b/HR relationship is observed in natural torpor, with time in a clockwise direction, with the beginning of the torpor bout in the top right section of the graph and moving through each of the four steps shown. For the FH condition, the fall in HR during T_b decline closely tracked the rise in HR during T_b rise. Mice treated with CHA also lacked a discernible breadth to their T_b/HR loop with the HR following the same trajectory during body cooling or warming. The T_b/HR relationship in isoflurane-anesthetized mice showed an intermediate breadth. These qualitative assessments were quantified in part C as the difference between HR at a T_b of 30°C while the mouse was recovering from hypothermia and the HR at a T_b of 30°C as the mouse was entering hypothermia. a = p < 0.05 vs. torpor b = p < 0.05 vs. FH c = p < 0.05 vs. isoflurane-anesthesia

Figure 2. The rate of T_b decline into hypothermia was slowest for natural torpor, while the rate of T_b rise from hypothermia was slowest for CHA-treated mice

Maximal rate of T_b change was identified and calculated in 10-minute windows for each mouse and then averaged per condition. The maximum rate of T_b decline showed 3-4 fold higher rates in isoflurane-anesthesia, FH, and CHA treated mice as compared to torpor. During recovery, the maximum rate of T_b rise in CHA-treated mice was significantly slower the recovery in torpor and FH. a = p < 0.05 vs. torpor b = p < 0.05 vs. FH

Figure 3. Shivering profiles of the four hypothermic conditions

Shivering was approximated as the average noise in ECG tracings. A. Shivering profiles (ECG noise, measured in arbitrary units) for each condition. Torpid mice show periodic bouts of shivering while torpid and substantial shivering while arousing. FH mice show substantial shivering throughout the hypothermic bout. Anesthetized mice only shiver as they recover. CHA mice show minimal shivering throughout the hypothermic bout. B. Representative ECG tracings for each condition from which noise was derived

Figure 4. FH and anesthetized mice show signs of anaerobic metabolism, whereas torpid and CHA-treated mice do not

FH and anesthetized mice show higher circulating lactate levels compared to baseline (taken before treatment). CHA-treated mice and torpid mice show lower than baseline lactate levels. a = p<0.05 vs. baseline b = p<0.05 vs. torpor c = p<0.05 vs. FH d = p<0.05 vs. isoflurane-anesthesia

Figure 5. Long HR pauses are only present in torpor and CHA-induced hypothermia

Poincare plots represent the variability in the interbeat intervals (IBI) by plotting adjacent IBIs against each other. A symmetry line is added as a reference. A. Torpor shows a symmetric pattern indicative of skipped beats. B. FH-treated mice and C. Isoflurane-anesthesia treated mice cluster near the symmetry line, indicating a relatively regular heartbeat. D. CHA shows asymmetries (black arrow) that are indicative of pauses that are not skipped beats. The open arrows point to the long pauses seen in the CHA condition. E. The IBIs of a euthermic mouse cluster tightly around the symmetry line (6 hours of data are shown).

Figure 6. Torpor, FH, isoflurane-anesthesia, and CHA exhibit different gene expression patterns

A-D hepatic gene expression. E-H cardiac gene expression (no data for isoflurane-anesthesia). Steady state mRNA levels for the genes c-Fos and Egr1 are upregulated relative to euthermic control mice in all hypothermic states except torpor. Txnip is upregulated in torpor (heart and liver) and FH (liver only) relative to euthermic controls. Hif1a is only modestly induced in the FH liver, and only modestly reduced in the CHA-treated heart. a = p<0.05 vs. control b = p<0.05 vs. torpor c = p<0.05 vs. FH d = p<0.05 vs. isoflurane-anesthesia