INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

COLD EXPOSURE IS A STRESSFUL event that elicits different thermogenic adaptive responses in endotherms and exotherms. In mammals, including humans, the physiological responses involve changes in energy expenditure, heat production and dissipation, physical activity, and appetite (31). In rodents, shivering, activation of the sympathetic axis (40, 44), and consequent development of brown adipose tissue (23), with remarkable activity of mitochondrial uncoupling proteins (UCPs) (4, 22) and crucial suppression of white fat leptin production (2, 40), were reported as pivotal mechanisms.

To sustain shivering and uncoupled mitochondrial respiration in brown fat, O₂ uptake and basal metabolic rate (BMR) are increased in animals abruptly exposed to cold (31). During cold acclimation, increased BMR could be sustained for some weeks in rodents (4, 22, 23), but in other species, there was little increase in BMR (13, 32). Furthermore, in some species, cold acclimation is characterized by a marked decline in O₂ uptake in hibernation or torpor (18). Moreover, and depending on leptin activity, conservative mechanisms, such as body insulation due to increased adiposity, may contribute to cold tolerance (39).

It has been recently reported that nitric oxide (NO) regulates mitochondrial O₂ uptake by reversible high-affinity binding to the Cu²⁺ center of cytochrome oxidase (6, 8-10). In the presence of NO, the level of cytochrome oxidase activity depends on the mitochondrial O₂-to-NO concentration ratio (6, 45) and represents an important adaptive response to changes in blood flow (35). Accordingly, activation of endothelial NO synthase (eNOS) by bradykinin induces a prompt decrease in myocardial O₂ uptake (38).

The recent finding of distinct NO synthases (NOS) in mitochondria (mtNOS) by independent groups (19, 21) adds a new perspective to the regulation of mitochondrial O₂ uptake by NO. The mtNOS isoforms exhibit organ- and species-specific characteristics. For instance, after purification, liver mtNOS was immunologically related to inducible NOS (iNOS) (46), as confirmed by proteomic studies (30), whereas a recent study using electrochemical detection identified neuronal NOS (nNOS) in isolated cardiac mitochondria (28). A recent description of mtNOS sequence in different tissues proposes that all mtNOS are nNOS -isoforms with posttranscriptional modifications (14). Likewise, the confinement of NOS in mitochondria ensures NO release vectorially directed to the matrix; therefore, relatively low NO flux and steady-state concentration may exert marked inhibitory effects on the activity of the redox components of the electron transfer chain, particularly on cytochrome oxidase (10, 12, 35).

Hence, we hypothesize that cold modulates mtNOS and, consequently, NO matrix concentration and that this effect could contribute to regulation of mitochondrial and systemic O₂ consumption during exposure to low environmental temperature. The present study was performed in liver and skeletal muscle from the rat, which is basically adaptable to prolonged periods in a cold environment (cold acclimation) but is not a hibernating species. Moreover, liver and skeletal muscle mtNOS may be differentially modulated in other conditions associated with net changes in BMR, such as hypo- and hyperthyroidism (11).

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Chemicals and biochemicals. SDS, glycerol, 2-(-mercaptoethanol), and bromphenol blue were obtained from Bio-Rad (Richmond, CA). L-[³H]arginine was obtained from NEN/DuPont (Boston, MA). The antibody against iNOS was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Western blotting detection system and Hybond-ECL membranes were purchased from Amersham Pharmacia Biotech. Other chemicals and biochemicals were purchased from Sigma Chemical (St. Louis, MO).

Experimental design. Adult female Sprague-Dawley rats (270 ± 30 g body wt) that had been housed for 2 wk at 22°C under standard laboratory conditions (12:12-h light-dark photoperiod) and fed a commercial standard rat diet (1.24 cal/g) with free access to tap water were placed individually in cages. They were assigned to two groups: 1) rats housed at 22°C and 2) rats exposed to cold temperature (4°C) throughout the experiment. The animals were studied at baseline and at days 2, 4, 6, 7, 10, 14, 21, 24, and 28-30. Rat weight and food intake were measured daily at 9 AM with a scale (model CS-2000, Ohaus, Florham Park, NJ). For determinations at the tissue level, the animals were anesthetized with ether and killed by decapitation; blood samples were then obtained by cardiac puncture, and liver and gastrocnemius skeletal muscle were surgically excised. The study followed international ethical guidelines for laboratory animals.

Systemic O₂ uptake. In the experimental conditions, rat movements were restrained and the whole animal O₂ uptake was continuously measured in a nonrecirculating open-flow system for ~30 min after 30 min of equilibration to reach stable conditions. Expired gases were directed to a Douglas bag by means of a constant-flow pump with a flowmeter and then passed through anhydrous CaSO₄ to an O₂ analyzer (model OM-14, Sensor Medics, Beckman Instruments, Anaheim, CA) and a CO₂ analyzer (Katapherometer, Godart, Bilthove, The Netherlands) in series. A standard formula, including inspired and expired O₂ and CO₂ fractions, ambient and chamber temperatures, barometric pressure, and flow, was used to calculate results in STPD conditions (36). Inasmuch as BMR is related to a power function of the body mass (27), O₂ consumption is expressed as ml O₂ · kg body wt^0.75 · min¹ to reflect more accurately the lean mass-specific metabolic rate.

Plasma norepinephrine and nonesterified fatty acids. To estimate sympathetic activity, plasma norepinephrine concentration was determined under anesthesia at baseline and at days 2, 10, and 24 in the cold ambient temperature by HPLC. To analyze fat metabolism, plasma nonesterified fatty acids were studied with an enzymatic commercial assay (NEFA FA 115, Randox Lab, Wako Chemicals) at baseline and at days 2, 4, 7, 10, 14, 21, and 24 in the cold atmosphere.

Isolation and purification of mitochondria. Excised liver (mean weight = 12 g) or gastrocnemius muscle (2 g) was placed in an ice-cold homogenization medium consisting of 0.23 M mannitol, 70 mM sucrose, 10 mM Tris · HCl, 1 mM EDTA, 5 µg of aprotinin, and phenylmethylsulfonyl fluoride (100 µg/ml) with 0.5% BSA (pH 7.4). Mitochondria from gastrocnemius muscle were isolated in Chappell-Perry buffer in the presence of antiproteases. The tissues were finely minced, transferred to a Teflon motorized Potter-Elvejhem homogenizer (Thomas Scientific, Philadelphia, PA), and processed in cold homogenization buffer (8 ml/g tissue). The homogenate was centrifuged at 700 g at 4°C for 10 min. The supernatant was decanted and centrifuged at 7,000 g for 10 min (7). The mitochondrial pellet was further purified in a Percoll gradient to completely remove contaminating organelles and broken mitochondria (26). Briefly, mitochondria were resuspended in 30% Percoll in 225 mM mannitol, 70 mM sucrose, 1 mM EGTA, 25 mM HEPES, and 0.1% BSA (MSHE). The solution was spun at 95,000 g for 30 min, and the ring, with a density of 1.025-1.075 g/ml, was collected and washed twice with MSHE to remove Percoll, twice with 150 mM KCl to remove attached proteins (e.g., arginase), and twice with MSHE without BSA. Mitochondria were finally resuspended to a final concentration of 30 mg protein/ml.

Mitochondrial respiratory activities. O₂ uptake was determined polarographically with a Clark-type electrode placed in a 3-ml chamber at 30°C in a reaction medium consisting of 0.23 M mannitol, 70 mM sucrose, 30 mM Tris · HCl, 4 mM MgCl₂, 5 mM Na₂HPO₄/KH₂PO₄, and 1 mM EDTA (pH 7.4), saturated with room air (225 µM O₂), with 0.5-1 mg mitochondria protein/ml. O₂ uptake was determined with 6 mM malate-glutamate as substrate in the presence (state 3) or absence (state 4) of phosphate acceptor (0.2 mM ADP). Respiratory control ratio was calculated as the ratio of state 3 to state 4 respiration rate. The index was calculated as the ratio of nanomoles of added ADP per nanogram atoms of O₂ utilized ADP/O during state 3 respiration (11).

Western blot analysis. Mitochondrial proteins (50 µg/lane) were separated by electrophoresis on a 7.5% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. Membranes were incubated with a rabbit polyclonal IgG anti-mouse iNOS (1:500) or anti-nNOS (1:1,000) antibody. After membranes were blotted with a donkey anti-rabbit IgG (1:3,000) conjugated to horseradish peroxidase, immunoreactive proteins were detected by a chemiluminescence system. Liver homogenates of lipopolysaccharide-stimulated rats and homogenates of rat brain were used as iNOS and nNOS standard controls, respectively. Cytosolic NOS isoforms were studied as previously described (3).

mtNOS activity. mtNOS activity in purified organelles was determined by conversion of L-[³H]arginine to L-[³H]citrulline (11). The reaction medium consisted of 0.1 µM L-[³H]arginine, 0.1 mM NADPH, 0.3 mM CaCl₂, 0.1 µM calmodulin, 10 µM BH₄, 1 µM FAD, 1 µM FMN, and 50 mM L-valine in 50 mM potassium phosphate buffer (pH 7.5; 0.1 mg mitochondrial protein/ml). The radiolabel in the NOS inhibitor blank [2 mM N-monomethyl-L-arginine (L-NMMA)] was subtracted from that in the tested samples to calculate radiolabel specific for NOS-dependent citrulline formation (11).

Protein determination. Protein concentration was determined by the Lowry assay with BSA as standard.

Statistical analysis. Student's t-test, one-way analysis of variance, and Dunnett's test were used as appropriate. Statistical significance was accepted when P < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

After 30 days of cold exposure, two sequential periods with different thermodynamic responses were delimited. Period A (days 1-10) was characterized by increased systemic O₂ uptake, fat catabolism, and food intake. However, a more prolonged analysis revealed a second phase (period B; days 10-30), which was associated with fat deposition, weight recovery, and reversal of heightened metabolic rate. Rectal temperature remained normal throughout periods A and B (average 36.4-37.2°C).

Effects of cold exposure on eating behavior and body weight. Initially, animals in the cold environment lost ~12% of their body weight (P < 0.001; Fig. 1A). This result agrees with that of Bing et al. (2), who reported 14% weight reduction at 4°C. However, in the present study, after 10 days, there was a marked weight gain in cold-exposed animals with respect to controls (5.8 vs. 0.9 g/day); at the end of the exposure, weight recovered and even increased over the baseline values (Fig. 1A). In the same conditions, the increase in the rate of weight gain at 4°C was maintained at ~2-3 g/day for 4 mo (unpublished observations). The changes in body weight mainly depended on fat metabolism. The variations of plasma free fatty acids indicate increased lipolysis in the initial phase and fat deposition in the later phase (Fig. 1A, inset). In the cold environment, sympathetic outflow is a main determinant of white fat lipolysis and brown fat activity. As a reflection of sympathetic activity, plasma norepinephrine increased from 497 ± 35 at 22°C to 1,283 ± 107 (SE) pg/ml at 4°C on day 10 (P < 0.05) and finally decreased to 663 ± 81 pg/ml at 4°C on day 24.

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Sequential changes of body weight and food intake in a cold environment. Individually housed animals with free access to food and water and exposed to a 4°C (cold) environment () are compared with animals exposed to a 22°C environment (). A: percent body weight variation compared with initial values at day 0 (270 ± 30 g). Inset: variations of plasma free fatty acid (FFA) concentration from the cold-exposed group. B: changes in food intake behavior. Values are means ± SE from 6-19 animals. *P < 0.05 (ANOVA and Dunnett's test).

Course of systemic O₂ uptake during cold acclimation. Systemic O₂ uptake varied significantly during cold acclimation. In period A, rats increased O₂ uptake at a rate of 0.53 ml O₂ · min¹ · kg^0.75 · day¹ to a peak transitional value on day 10 (+5.5 ml O₂ · min¹ · kg^0.75 or 240 µmol O₂ · min¹ · kg^0.75, +32%, P < 0.05; Fig. 2). Conversely, in period B, O₂ uptake progressively declined at a rate of 0.25 ml O₂ · min¹ · kg^0.75 · day¹ to reach the baseline level at the end of the study (Fig. 2). Likewise, different ascendant and descendant slopes of O₂ uptake rate indicated the relative contribution of the concurrent mechanisms for adaptation to cold exposure. Therefore, it is surmised that by day 10 and at constant high energy intake, adapting mechanisms that markedly consume O₂, e.g., facultative thermogenesis and shivering, were in equilibrium with energy-saving mechanisms. Moreover, this transitional plateau anticipated the subsequent redistribution of total energy expenditure during prolonged cold acclimation (Fig. 2).

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Systemic O2 uptake early (period A) and late (period B) in cold acclimation. Resting systemic O2 uptake [basal metabolic rate (BMR)] of controls () and cold-exposed rats () was determined individually over 30 min with O2 and CO2 meters placed in series in an open circuit after 30 min of equilibration. Periods A and B were delimited according to vectorial changes in O2 uptake. Arrow, maximal span of the variation of O2 uptake throughout the experiment. Values were corrected to STPD and referred to body lean mass (27). Values are means ± SE from 6-14 animals. *P < 0.05 vs. baseline, as in Fig. 1.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Differential effects of nitro-L-arginine methyl ester (L-NAME) on O2 uptake in periods A and B. Systemic O2 uptake was determined as described in Fig 2 without L-NAME (open bars) and 24 h after a single dose of L-NAME (30 mg/kg body wt ip; solid bars) administered to rats at 22°C (n = 6) and 4°C [day 5 (n = 5) and day 21 (n = 5)]. *P < 0.05 vs. without L-NAME (Student's t-test); **P < 0.05 vs. 22°C and at 4°C on day 5 (ANOVA and Dunnett's test).

NO-dependent mitochondrial respiration. Mitochondria exhibited acceptable respiratory control (4.0-6.0) and ADP/O (2.8-3.1), indicating a proper coupling of oxidative phosphorylation to electron transfer rates. No significant differences in basal mitochondrial activities were observed at 4°C or 22°C; in the presence of albumin, no fatty acid-induced or UCP-mediated leak was detected in mitochondrial studies. In contrast, supplementation of the organelles with the NOS substrate L-arginine resulted in markedly different responses of mitochondrial O₂ uptake between period A and period B: high BMR in period A and decreasing metabolic rate late in period B. Consequently, in liver mitochondria from controls at 22°C, O₂ uptake was decreased by 15% in the presence of L-arginine. However, in period A, liver mitochondrial O₂ uptake of cold-exposed animals became almost insensitive to L-arginine or the NOS inhibitor L-NMMA. Consequently, in period A, mitochondrial O₂ uptake remained significantly higher than that of controls at 22°C and similar to mitochondrial O₂ uptake at 4°C without L-arginine (Fig. 4). In contrast, in period B, supplementation with L-arginine decreased mitochondrial state 3 O₂ uptake up to 50% compared with the respective controls without the substrate; this decrease was completely reversed by L-NMMA (Fig. 4). Similar results were obtained at resting state 4 O₂ uptake. The effects of L-arginine are consistent with the existence of variations of the mitochondrial NO yield during cold acclimation. On the basis of previously reported NO effects on liver cytochrome oxidase (8, 37), matrix NO steady-state concentration at the different stages could be calculated (Fig. 4, inset).

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Nitric oxide (NO)-dependent modulation of liver mitochondrial O2 uptake during cold adaptation. Mitochondrial state 3 O2 uptake was determined as described in MATERIALS AND METHODS. , Animals at 22°C (day 0) and throughout acclimation at 4°C in the absence of L-arginine; , inhibition of O2 uptake after supplementation of mitochondria with 0.3 mM L-arginine. *P < 0.05 vs. 22°C (Student's t-test); #different from controls at 22°C (by ANOVA). Effects of L-arginine were completely reversed by 3 mM N-monomethyl-L-arginine (L-NMMA). Inset: calculated matrix NO steady-state concentration with respect to NO effects on O2 uptake (37). Values are means ± SE from 5-16 samples.

Activity and expression of liver and skeletal muscle mtNOS. Cold exposure selectively induced changes in the activity or expression of mtNOS, whereas canonical cytosolic NOS isoforms were not modified (data not shown). In agreement with L-arginine-dependent variations of O₂ uptake, activity of liver mtNOS in the cold environment immediately decreased and then increased significantly from day 6 to the end of the study (Fig. 5; P < 0.05). On the basis of previous analyses (35, 37) and with the L-arginine-dependent effects taken into consideration, mtNOS activity (Fig. 5) was consistent with the calculated matrix NO steady-state concentrations in most of the determinations (Fig. 4, inset); however, on day 6, O₂ uptake was unexpectedly high with respect to liver mtNOS activity, which suggests that, at this time, other factors, e.g., increased adrenergic activity, stimulate O₂ uptake and partly counteract the inhibitory mtNOS effects on cytochrome oxidase (20).

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Time course of liver mitochondrial NO synthase (mtNOS) during cold exposure. A: mtNOS protein blots as detected with anti-inducible NOS (iNOS) antibody (50 µg of mitochondrial protein were loaded in each lane) and respective densitometries in arbitrary units (AU) from purified rat liver mitochondria during cold exposure at 4°C. iNOS+, standard positive control (130-kDa liver iNOS from endotoxin-treated rats). B: mtNOS activity from purified rat liver mitochondria under conditions described in MATERIALS AND METHODS. Values are means ± SE from 6 samples determined in triplicate. *P < 0.05 (ANOVA and Dunnett's test).

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View larger version (23K): [in this window] [in a new window]   Fig. 6.   Skeletal muscle mtNOS during cold exposure. A: changes in Western blot from purified rat muscle mitochondria and the respective densitometries throughout cold exposure with respect to controls at 22°C. Skeletal muscle mtNOS (157 kDa) was detected with anti-neuronal NOS (nNOS) antibody (see MATERIALS AND METHODS). nNOS+, standard control (rat brain nNOS). B: mtNOS activity. Values are means ± SE from 6 samples determined in triplicate. *P < 0.05 (ANOVA and Dunnett's test).

Mitochondrial O₂ uptake in skeletal muscle. A transitional shift of mitochondrial O₂ uptake was assessed in skeletal muscle mitochondria during cold exposure (Fig. 7). Similar to liver, organelles from animals at 22°C were able to decrease O₂ uptake by 15% in the presence of L-arginine. Thereafter, at 4°C, muscle mitochondria were not sensitive to L-arginine by day 14, but as a result of the increase in mtNOS, they were significantly inhibited (30%) by the substrate by day 24 (Fig. 7).

View larger version (19K): [in this window] [in a new window]   Fig. 7.   Modulation of skeletal muscle O2 uptake. Skeletal muscle state 3 O2 uptake in the presence of 0.3 mM L-arginine alone (dark gray bars) or L-arginine + 3 mM L-NMMA (light gray bars) on days 14 and 24 of cold exposure is compared with baseline values (22°C, black bars). Values (ng atoms O2 · min1 · mg protein1) are means ± SE from 3-5 samples determined in duplicate. *P < 0.05 vs. respective values in absence of L-arginine or with L-NMMA (ANOVA and Dunnett's test).

Contribution of liver plus muscle mitochondrial O₂ uptake to BMR. At 22°C and with available substrate, the in vivo contribution of liver and muscle to BMR is likely represented by state 3 + state 4 mitochondrial O₂ uptake as inhibited by L-arginine (15%). This value results in ~40% of total BMR (Fig. 8). In period A (up to day 14 in muscle), mitochondrial O₂ uptake was not affected by L-arginine and, thus, was similar to the basal mitochondrial respiratory activities; in these conditions, liver and muscle mitochondrial O₂ uptake increased, and its fractional contribution to increased BMR (39%) was maintained. In period B, the marked effects of L-arginine allowed liver + muscle mitochondrial O₂ uptake to fall to 25% of total BMR at day 24 (Fig. 8).

View larger version (17K): [in this window] [in a new window]   Fig. 8.   Differential liver and muscle fractional contribution to total BMR in cold acclimation. Fractional liver + muscle BMR (black bars) was calculated from the respective measured mitochondrial state 3 and state 4 O2 uptake rates in the presence of 0.3 mM L-arginine and with the assumption of 40-50 mg mitochondrial protein/g tissue (35, 37) and compared with total BMR (resting systemic O2 uptake; gray bars). Proportional contribution of state 3 and state 4 O2 uptake rates to tissue BMR was assumed to be 70% and 30%, respectively, in liver (27) and 50% each in muscle (15, 27). Calculated liver and skeletal muscle O2 uptake rates were corrected by mass-specific exponents of 0.87 and 1, respectively (27); ml O2 was converted to mmol O2 with the assumption that 1 mol O2 = 22.4 liters. Calculation was done at 22°C (basal) and on days 7 and 24 at 4°C. *P < 0.05 vs. period A (ANOVA and Dunnett's test).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

In this study, the putative role of mtNOS in cold acclimation was analyzed in the context of the different responses contributing to preservation of thermoneutrality (Figs. 1 and 2). Initially, exposure to cold was overridden by increasing metabolic rate, which involves sympathetic effects, fat catabolism, and activity of UCPs (4, 31, 41, 44). At persistently high caloric intake, heightened metabolic rate was reversed; accordingly, adaptive thermogenesis with high BMR could be prolonged in mice (22), but the result may be quite variable after some weeks of cold exposure in rats (13), birds (32), rabbits (34), or humans (31).

Liver and skeletal muscle account for ~45% of body mass and represent ~35-40% of total BMR (15); in this study, a similar contribution may be calculated from the basal mitochondrial activities of the tissues. Depending on matrix steady-state concentration, NO tunes up liver and muscle mitochondrial O₂ uptake in vivo (38) and ex vivo (37) through inhibition of cytochrome oxidase; likewise, matrix NO takes part in the modulation and distribution of total BMR (11). The results presented here indicate that this contribution varies from an ambient to a cold environment. Activation or inhibition of liver and muscle mitochondrial O₂ uptake was clearly related to NO levels in early and late phases of cold adaptation. In this context, the variations of matrix NO concentration in mitochondria should account for 25-45% of total BMR variations during acclimation (about +60 and 110 µmol O₂ · min¹ · kg¹ in periods A and B, respectively, or 240 µmol O₂ · min¹ · kg^0.75; Fig. 2); the remaining fraction largely depends on the prevailing relative contribution of other mechanisms, such as brown fat facultative thermogenesis.

Similarly, NO-dependent positive or negative changes in O₂ uptake are a consequence of the amount of NOS operating at the mitochondrial level (11, 20). The mitochondrial response to L-arginine coincided with the modulation of mtNOS; also, liver and muscle mtNOS expression and activity shifted inversely to systemic O₂ uptake (Figs. 2, 5, and 6). As previously reported in the dog (43) and in accord with mitochondrial findings, administration of the NOS inhibitor L-NAME significantly increased in vivo O₂ uptake after 21 days of cold exposure and when mtNOS is fully operative. At this time, this selective response is clearly representative of tissue oxidative metabolism, because it probably includes the less-specific L-NAME effects observed in the rats at 22°C and after 5 days of cold exposure (Fig. 3). Therefore, L-NAME abolished the differences in the time course of O₂ uptake attributed to mitochondrial NO early and late in cold exposure. In addition, although other non-mtNOS isoforms (i.e., eNOS) may be expressed in different tissues, e.g., brown adipose tissue and muscle, the different response to L-NAME suggests that late cold exposure effects mainly depend on mtNOS variations. Moreover, eNOS dysfunction has been reported after prolonged cold exposure (49).

These results suggest the importance of mtNOS activity in the different contribution of tissues to total BMR and energy expenditure throughout acclimation. These findings are consistent with previously reported increased liver and muscle mtNOS correlated to decreased O₂ uptake in experimental hypothyroidism (11); remarkably, hypothyroid animals succumb to sudden cold exposure, whereas cold acclimation in euthyroid animals may be associated with low plasma triiodothyronine concentration (24). Quantitatively, 50 and 30% inhibitions of O₂ uptake probably rely on organ-specific activities of liver and muscle mtNOS, respectively, with mtNOS retaining 30% of the activity of liver NOS (Figs. 5 and 6). It is then surmised that 1) nonshivering thermogenesis may occur in tissues other than brown fat and 2) in cold acclimation, transient inhibition or activation of mtNOS activity contributes to redistribution of the total energy expenditure and balance, favoring liver and muscle nonshivering thermogenesis or body insulation.

In the absence of L-arginine, increase of mitochondrial O₂ uptake could depend on mechanisms other than NO suppression, e.g., adrenergic stimulation and UCP activities. Calorigenic effects of UCP-2 and UCP-3 are controversial (4, 5, 16, 29, 32, 41, 44). However, they could reasonably counteract the NO inhibition on mitochondria; Giulivi et al. (21) reported that mitochondrial uncoupling is associated with a marked decrease of liver mtNOS activity. It is then conceivable that, at particular stages of cold acclimation, NO and UCPs could interact in mitochondria and also that appraisal of UCP activity could require the concurrent activity of mtNOS. Hypothetically, mtNOS expression could be under the influence of UCP genes. It was intriguing that UCP-1-ablated mice are sensitive to cold but not obese (16). In these deficient mice, the lean phenotype is favored by increased proton leak in muscle mitochondria (33); mostly, kinetics of proton leak depend on membrane protonmotive force, a physical parameter easily controlled by the NO-dependent electron transfer rate.

Energy balance between intake and expenditure, as daily distributed among metabolic cost, heat dissipation, and shivering, was likely different throughout cold exposure. From a thermodynamic perspective, averaged caloric availability per single animal increased from 63 cal/day (baseline, neutral balance) to 170 cal/day (caloric intake + fat catabolism in period A, negative balance) and, finally, declined to 125 cal/day (caloric intake in period B, positive balance)¹. Accordingly, the calculated contribution of liver + muscle to the BMR in period B fell from 40% to 25%; in this period, caloric equivalence to ~1.5 mol ATP per animal could be saved.² The fraction of nonoxidized reduction equivalents was likely driven to the synthesis of fat, which is necessary for thermal insulation and energy reserve (Fig. 1). If it is considered that 7 mol of ATP are required per mole of synthesized triglyceride (15), decreased contribution of muscle and liver to BMR should largely account for fat deposition and weight gain in period B (Fig. 1); moreover, an estimated 25% reduction of caloric availability in the transition from period A to period B is almost one-half of the decrease resulting from mtNOS inhibition of mitochondrial oxidative metabolism. In contrast to experimental conditions, where animals are allowed to eat ad libitum, in natural conditions, restricted food availability and intake will directly affect facultative thermogenesis. In these conditions and in consideration of the finding that shivering decreases during prolonged cold exposure (22), fat deposition and body insulation resulting from energy saving may be relevant as a fuel source and in the maintenance of thermal neutrality.

Finally, mitochondrial NO effects could be extended to other conditions associated with changes in BMR; this notion should be examined in hibernating species with markedly diminished metabolic rate. Moreover, regulation of O₂ uptake by NO could be important in those entities associated with an imbalance between energy intake and expenditure, e.g., obesity (44) or other metabolic disorders (48).