Int J Biometeorol (1990) 34:2z~27

meteorology

Peripheral nervous control of cold-induced reduction in the respiratory quotient of the rat

Roberto Refinetti

Department of Psychology, University of Illinois, Champaign, IL 61820, USA

Received July 31, 1989; revised November 21, 1989 Accepted December 6, 1989

Abstract. Cold-exposed rats show a reduction in the re- spiratory quotient which is indicative of a relative shift f rom carbohydrates to lipids as substrates for oxidative metabolism. In the present study, the effects of food deprivation and cold exposure on the respiratory quo- tient were observed. In addition, the involvement of the three main branches of the peripheral nervous system (sympathetic, parasympathetic, and somatic) was inves- tigated by means of synaptic blockade with propranolol , atropine, and quinine, respectively. Both propranolol and quinine blocked the cold-induced decrease in respi- ratory quotient and increase in heat production, whereas atropine had only minor and very brief effects. It is con- cluded that both the sympathetic and somatic branches are involved in the metabolic changes associated with cold-induced thermogenesis and that the increase in met- abolic heat production involves a shift f rom carbohy- drate to lipid utilization irrespective of which of the two branches is activated.

Key words: Cold-induced thermogenesis - Peripheral nervous system - Respiratory quotient - Rat

Introduction

Several investigators have observed that cold exposure causes a reduction in the respiratory quotient (RQ) of endothermic animals (Heldmaier 1974; Kayser 1937; Naka tsuka et al. 1983 ; Pag6 and Ch6nier 1953; Refinetti 1989). Because a reduction in RQ is an indication of a shift in mean nutrient utilization f rom carbohydrates to lipids (Lindmark et al. 1986), it is believed that lipids are the main fuel for oxidative metabolism in cold-in- duced thermogenesis (Doi et al. 1979; Naka t suka et al. 1983; Wilson et al. 1987). An important aspect that has not been experimentally investigated is the nervous con-

Offprint requests to: R. Refinetti

trol of the shift in nutrient utilization. The elevation in heat production in response to cold in non-cold-accli- mated rats is believed to be due to a sympathetically activated component (non-shivering thermogenesis) and a somatically activated component (shivering thermo- genesis). However, no attention was previously directed to the question of which of the three major branches of the peripheral nervous system (i.e., sympathetic, para- sympathetic, and somatic) are involved in the cold-in- duced reduction in RQ (which is not necessarily tied to the increase in heat production). In the present study, cold-induced thermogenesis was investigated before and after selective chemical blockade of each of the three effector systems.

Methods

Subjects. Male adult Sprague-Dawley rats (390-440 g) were ob- tained from Bantin and Kingman (Fremont, Calif.) and maintained in individual metal cages at 25~ with Purina laboratory chow and water available at all times. Test sessions were always con- ducted at the same time of day during the light phase of the 12:12 h light: dark cycle.

Apparatus. Measurements of respiratory gaseous exchange were conducted in a 3-1 plastic chamber through which air was drawn at 3 1/min. The air leaving the chamber was dried in coIumns of anhydrous CaSO 4 desiccant (Hammond Drierite, Xenia, Ohio) and passed to both a carbon dioxide gas analyzer model LB-2 (Beck- man Instruments, Fullerton, Calif.) and an oxygen analyzer type O.A. 137 (Servomex Controls, Crowborough, UK). The output from the oxygen analyzer required pre-amplification by a 741 op amp module, whereas the output from the CO2 analyzer was sent directly to the chart recorder model 2400 (Gould, Cleveland, Ohio). Ambient temperature was varied by immersing the chamber in water baths controlled by a Lauda circulator model MT (Brink- mann Instruments, Westbury, NY) and a static cooler model u-cool (Neslab Instruments, Portsmouth, NH).

All drugs were purchased in the form of salts from Sigma (St. Louis, Mo.) and mixed each day in 0.9% bacteriostatic saline (Abbott Laboratories, North Chicago, Ill.). Quinine hydrochloride was used as a blocker of striated muscles (DiPalma 1971), DL- propranolol hydrochloride as a sympathetic blocker (Maxwell

1971), and atropine sulfate as a parasympathetic blocker (Cullum- bine 1971).

Procedure. Two series of studies were conducted. The first series was designed to replicate previous observations in the specific con- ditions of the present experiment. Each of eight rats was tested once at each of three conditions: control (fed ad libitum and tested at 25 ~ C), cold (fed ad tibitum and tested at 5 ~ C), and fast (food deprived for 20 h and tested at 25 ~ C). In each session, a single rat was placed in the chamber and its 02 consumption and COz production were continuously monitored. After 15-60 min, the gas- eous exchange was low and stable (indicating that the animal was asleep or very quiet) and the next 20 min of recording were used for data analysis.

In the second series of studies, individual animals were placed in the chamber at 25 ~ C for 60 min and then injected intraperitone- ally with one of the drugs and transferred to 5 ~ C for an additional period of 60 min. Each of eight rats was tested four times, once with each drug (saline, quinine 25 mg/kg, propranolol 5 mg/kg, and atropine i mg/kg). The doses were chosen based on previous studies where quinine was used to attenuate shivering (Satinoff and Shah 1971), propranolol to attenuate nonshivering thermogen- esis (Ma et al. 1987), and atropine to block parasympathetic activi- ty (Shiraishi and Mager 1980).

In both series of studies, heat production (in W/kg) was calcu- lated from O2 consumption (standard temperature and pressure, dry) using a caloric equivalent of approximately 20 J/ml adjusted to the particular RQ (Refinetti 1989). The respiratory quotient was calculated by dividing the amount of CO2 produced by the amount of 02 consumed.

Statistics. The main effects of experimental treatments were ana- !yzed by analysis of variance for repeated measures. Pairwise com- parisons of individual means were conducted by Tukey's HSD test (Kirk 1982).

Results

The m e a n resul ts f rom the first series o f s tudies are shown in Fig. 1. H e a t p r o d u c t i o n (HP) was s ignif icant ly affected by the expe r imen ta l t r ea tmen t s [F (2 ,14 )=72 .7 , P < 0 . 0 1 ] . H P was m a r g i n a l l y lower in food depr ived an ima l s c o m p a r e d to an imals fed ad l ib i tum [q(3 ,14)= 3.77, P = 0.05] and s ignif icant ly h igher in an ima l s tes ted at 5 ~ c o m p a r e d to an imals tes ted at 25 ~ [q(3 ,14)= 12.75, P < 0 . 0 1 ] . The expe r imen ta l t r ea tmen t s also had a s t rong effect on the R Q [F(2,14) = 66.9, P < 0 . 0 1 ] . The R Q was s ignif icant ly lower in food depr ived an imals c o m p a r e d to an imals fed ad l ib i tum [q(3 ,14)= 13.36, P < 0.01]. Co ld exposu re resul ted in a much smal ler bu t still s ignif icant r educ t i on in R Q [q(3,14) = 4.09, P < 0.05].

A n excerp t o f a typ ica l session o f a con t ro l (saline) ra t in the second series o f s tudies is shown in Fig. 2. Af te r an ini t ia l s t ress - induced high o f 11 W / k g , H P slow- ly r eached a p l a t eau o f 4-5 W / k g . W h e n the an ima l was injected wi th saline and t rans fe r red to a 5 ~ C env i ron- ment , H P increased to 12 W / k g and r e m a i n e d at this level unt i l the end o f the session. In p i lo t exper iments , an imals in jec ted wi th saline and r e tu rned to 2 5 ~ showed no increase in HP. The R Q o f the ra t shown in Fig. 2 osc i l la ted a r o u n d 0.99 at 25 ~ and decreased to a b o u t 0.90 u p o n t ransfe r o f the an ima l to 5 ~ C.

In jec t ions o f quinine, p r o p r a n o l o l , and a t rop ine b locked to a var iab le ex tent the increase in H P and de-

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Fig. 1. Heat production and respiratory quotient of a group of eight rats tested under three conditions: control (fed ad libitum and tested at 25 ~ C), coid (fed ad Iibitum and tested at 5 ~ C), and fast (food deprived for 20 h and tested at 25 ~ C)

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Fig. 2. Excerpt from a session of a control animal showing the temporal course of heat production and respiratory quotient in blocks of 5 rain. The rat was transferred from 25 ~ to 5 ~ C at 50 rain after the beginning of the session

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Fig. 3. Change in heat production (HP) and respiratory quotient (RQ) of eight rats injected with different drugs and exposed to a 5 ~ C environment. All values are percentages of values obtained at 25 ~ C (and with no drug). Mean HP at 25 ~ was 6.6 W/kg; mean RQ was 0.96

crease in RQ produced by cold exposure. The effects of quinine and propranolol were stronger and lasted 25- 45 rain; the effects of atropine were less marked and lasted only 5-10 min. The mean results from the second series of studies are shown in Fig. 3. In order to increase statistical sensitivity, the data from each rat during cold exposure were expressed as a percentage of the values obtained during the baseline period immediately preced- ing cold exposure. Data from the last 20 min of the base- line period and from the first 20 min of cold exposure were used in the analyses. A significant effect of the drugs on HP can be observed [F(3,21)= 13.9, P<0.01]. Although saline alone did not prevent the doubling of HP upon transfer to 5 ~ C, atropine had a marginal effect (P= 0.05) and propranolol and quinine had significant effects (P<0.01). An overall significant effect of the drugs on the RQ was also found [F(3,21)=6.43, P < 0.01]. Neither saline nor atropine prevented the cold- induced reduction in RQ P > 0.10) but propranolol and quinine did (P< 0.05).

Discussion

Several classical observations were replicated in the first series of studies: (i) the HP of control rats tested in a thermoneutral environment was found to be between 6 and 7 W/kg (Field et al. 1939; Gordon 1988); (ii) the

RQ of rats fed ad libitum was found to be between 0.9 and 1.0 (Atrens et al. 1987; Lindmark et al. 1986); (iii) food deprivation caused a reduction in both HP (Forsum et al. 1981; Hervey and Tobin 1982) and RQ (Atrens etal. 1987; Pag~ and Ch~nier 1953); and (iv) cold exposure caused an increase in HP (Gordon 1987; Herrington 1940) and a decrease in RQ (Nakatsuka et al. 1983; Pag6 and Ch6nier 1953). It should be noticed that, although both food deprivation and cold exposure caused a reduction in the RQ, the former treatment caused a reduction in HP whereas the latter caused an elevation in HP. Therefore, elevations in HP are not always associated with reductions in RQ.

The results from the second series of studies indicated that the metabolic changes due to cold exposure (i.e., elevation in HP and reduction in RQ) can be partially blocked by injections of quinine or propranolol and, to a lesser extent, by atropine. The data suggest that both the somatic and sympathetic nervous systems play an important role in cold-induced thermogenesis, where- as the parasympathetic nervous system plays only a mi- nor role. Thus, both quinine and propranoloi reduced considerably the increase in HP due to cold exposure and abolished the cold-induced reduction in RQ, where- as atropine had a small and brief effect on HP and did not block the cold-induced reduction in RQ.

In recent years, much attention has been directed at brown adipose tissue (BAT) as a major organ in cold- induced thermogenesis (Foster 1986), but muscles are also likely to contribute significantly (Ivanov 1989). If it is assumed that in the rat the major sympathetic effec- tor organ is BAT and the major somatic organs are the striated muscles, then the present results suggest that the cold-induced reduction in RQ is not the consequence of a shift in the organs involved in oxidative metabolism. As blockade of either BAT or striated muscles prevented the reduction in RQ, at least these two classes of organs might be involved in the phenomenon. However, as the action of cholinergic substances on BAT has not been investigated, and the action of noradrenergic substances on muscles is not negligible (Grubb and Folk 1976; Yak- imenko et al. 1971), it would seem safer at this point not to speculate about which organs are affected by which branches of the peripheral nervous system during cold-induced thermogenesis.

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