Discussion
In this paper, we investigated the activation energy of metabolismE in rats after injection of a previously developed PITS-composition capable of inducing a daily hypothermia and torpor-like state in rats , as well as in rats after injection of anesthetic xylazine, which can reduce body temperature for a few hours. The use of this anesthetic is based on the fact that it, often in combination with ketamine, can induce hypothermia in animals whereas dexmedetomidine, an analog of xylazine, can be used to initiate hypothermia in humans, and is proposed for protection in the emergency cases or during long-term space travels .
Data on changes in body temperature and oxygen consumption by rats after injection of PITS-composition or xylazine were used to determineE by the linear regression slopes with regard to mass-corrected metabolic rate ln(I/M3/4 ), which was equivalent to heat production, and the inverse body temperature 1/kT . In accordance with the WBE-theory, E should be in the range of -0.6 – -0.7 eV. For example, in hibernating mammals E = -0.69 eV . Our calculations made separately for daily heterotherms and true hibernators revealed some deviations from the above range: in daily heterotherms E = -0.57±0.04 eV, and in true hibernators E= -0.80±0.04 eV. This deviations from a classical viewpoint is not unexpected, especially in the studies of poikilothermic organisms. For example, in the study of fish, E = - 0.5 eV . Even lower values were obtained for the marine copepod , rocky-shore eulittoral-fringe snail (Echinolittorina malaccana ) that experiences fluctuating temperatures , while the teleost fish study revealed excessively large values of E = - 0.79 eV .
The presented above significant deviations in E are inexplicable from the point of view of the WBE-theory , which begins with the observation that temperature controls metabolism through its effect on the rate of biochemical reactions. It is known, that the reaction kinetics depends on temperature according to the Boltzmann factor. In line with Clarke’s criticisms, while statistical thermodynamics provides a very successful description of the behavior of a simple system where temperature is the only variable that changes, organismal metabolism is very different. Organismal metabolism involves a large number of physiological processes, each of which interacts with many others . Although it is generally accepted that changes in temperature should lead to corresponding changes in metabolism, which was demonstrated in model systems or in mitochondrial suspension, the question is how universal the application of this physical principle to processes at the level of multicellular organisms .
We have found that E in rats injected with PITS-composition was close to the corresponding value in natural daily heterotherms, but smaller than that in true hibernators. This suggests that the PITS-composition is able to initiate in homeothermic organisms, like rats, a state close to daily heterotherms, which, however, differ from true hibernation. Indeed, the state of pharmacological torpor that occurs in rats after a single injection of the PITS-composition lasts for about 16 hours, which is characteristic of daily heterotherms rather than of true hibernators, experiencing the state of torpor and hypothermia during many days and even several months .
When rats were injected with the anesthetic xylazine, their body temperature also decreased for several hours (the curve half-width = 3 h), however, E was about three-fold less than that after injection of PITS-composition (E = -0.17±0.071 eV) and did not correspond to the value for a natural state of hibernation . What can be the reason for such a significant difference?
For determination of the resting metabolic rate and correct assessment of E , a stationary metabolic state of animals has to be achieved . In our experiments, the body temperature of animals after injection of the anesthetic xylazine constantly changed: initially decreased, and then increased followed by significant changes in heat production and, accordingly, the animal state could not be regarded as stationary. Therefore, although the anesthetized animals in our experiment were immobile, their metabolism cannot be regarded as the resting metabolism. In homeothermic animals, the body heat production is the source for the elevated body temperature compared to the ambient one. The metabolism plays a leading role in maintaining the body temperature of warm-blooded animals in our understanding of the mechanisms of homeothermy. Thus, changes in body temperature lag behind changes in metabolic rate and, accordingly, the changes in oxygen consumption. Lag compensation by means of an imaginary numerical shift of oxygen consumption by 1 hour allows us to obtain corrected Ec = -0.68 eV, close to the corresponding values for hibernating mammals .
In experiments with PITS-composition, the body temperature initially decreased and then stabilized for a long period of time in a state of hypothermia followed by restoration of the initial temperature level. Thus, in animals under PITS the period of steadily lowered body temperature accounted for a significant part of the time of pharmacological torpor, and can be regarded as a stationary resting state. Oppositely, in anesthetized animals during short-term hypothermia a stationary state is not achieved.
As mentioned above, in the study of poikilothermic organisms, there may be significant variability of E . It can be assumed that in this case, there may also appear a temporary mismatch in temperature and metabolic rate. Since in the cold-blooded organisms the ambient temperature is the main factor determining the body temperature and accordingly the metabolic rate, we suppose that the changes in metabolic rate may occur later than the changes in body temperature for both physiological reasons, including the time necessary for changes of heart rate, respiratory rate, blood vessel conductivity, etc. and even slower metabolic changes in composition of membrane lipids leading to a decrease in phase transition temperature of lipids which is coupled with enhanced cold induction of genes . Thus, if for homeotherms the time lag of temperature behind metabolism was designated as Δt , then for poikilotherms the lag of metabolism behind temperature should be denoted by the opposite sign, as (-)Δt. We assume that as in a case with homeotherms mentioned above, for poikilotherms the correct calculation of E should be made taking into account the time shift mentioned above .