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Keywords:

  • calorimetry;
  • metabolic rate;
  • oxygen consumption;
  • respiratory quotient;
  • temperature sensitivity

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Objectives: The aims were to compare the temperature dependence of the metabolic rate in young ob/ob mice with that in mature ob/ob and db/db mice and to examine the effect on the metabolic substrate preference of leptin and etomoxir in ob/ob, C57BL/6J (wild-type), and db/db mice.

Research Methods and Procedures: In vivo oxygen consumption and carbon dioxide production were continuously measured by indirect calorimetry, and body temperature and total locomotor activity were measured by an implanted transponder. Leptin, etomoxir, or vehicle was administered intraperitoneally.

Results: The temperature dependence of the metabolic rate of mature ob/ob and db/db mice were similar to that in wild-type mice. In young 6-week-old ob/ob mice, the metabolic rate was almost doubled at 15 °C. Leptin (2 × 3 mg/kg) decreased the respiratory quotient (RQ) and carbon dioxide production but did not alter oxygen consumption, body temperature, or locomotor activity in ob/ob and C57BL/6J mice and had no effect in the db/db mice. Etomoxir (2 × 30 mg/kg) enhanced RQ and decreased oxygen consumption, carbon dioxide production, and body temperature in ob/ob, C57BL/6J, and db/db mice. Total locomotor activity was reduced in ob/ob and C57BL/6J mice.

Discussion: In young ob/ob mice, the temperature sensitivity was enhanced compared with mature mice. Leptin and etomoxir had opposite effects on metabolic substrate preference. Leptin and lowered environmental temperature increased the relative fat oxidation as indicated by decreased RQ, possibly through activation of the sympathetic nervous system.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Obesity is an increasing medical problem for the Western world of today. The morbidities associated with obesity, including type 2 diabetes, cardiovascular disease, osteoarthritis, and several forms of cancer, represent a major health risk to the obese population (1). Physiologically, obesity is a disorder of energy balance when energy intake exceeds energy expenditure (2). The only source of energy intake is food intake, whereas energy expenditure consists of basal metabolism, physical activity, and adaptive thermogenesis.

Leptin administration is well known to reduce food intake and body weight in ob/ob mice. Effects on the metabolic rate, fatty acid oxidation [respiratory quotient (RQ)],1 as well as the expression of uncoupling protein 1, have been suggested (3, 4, 5). It has been hypothesized that the preferential decrease in fat mass is caused by increased energy expenditure (3, 6, 7, 8, 9). The signaling cascades include the janus activating kinase-signal transducer and activator of transcription pathway, which seems to be regulated by protein-tyrosine phosphatase 1B (10). Recently, leptin has been described as stimulating fatty acid oxidation through activation of 5′-adenosine monophosphate—activated protein kinase and subsequent inhibition of acetyl coenzyme A carboxylase in skeletal muscle. In contrast, in the arcuate and paraventricular hypothalamus leptin inhibits 5′-adenosine monophosphate—activated protein kinase, which reduces food intake (11, 12, 13, 14).

There is controversy, however, regarding the effect of leptin on the metabolic rate. Leptin has been found to increase the metabolic rate in ob/ob mice (3, 15, 16), prevent metabolic rate reduction associated with food restriction in Sprague-Dawley rats (17), to decrease metabolic rate in lean (fa/−) and obese (fa/fa) Zucker rats (18), or not to alter metabolic rate in arctic ground squirrels (19).

Leptin decreased the RQ (RQ = Vco2/Vo2) in ob/ob mice (16, 20), lean (fa/−) and obese (fa/fa) Zucker rats (18), Sprague-Dawley rats (17, 21), and diet-induced obese rats (22). A decrease in RQ reflects a change in metabolism from carbohydrate to fat oxidation. This would be a favorable effect for an obesity drug. Therefore, the effect of leptin on metabolic rate was studied in this study.

Metabolism and oxidation of fatty acids occurs mainly through mitochondrial β-oxidation. The rate-limiting step is the transport of long-chain fatty acids into the mitochondria by the enzyme carnitine O-palmitoyl transferase-1 (CPT1) (23). Leptin increases mRNA and stimulates the activity of CPT1 (24, 25). Etomoxir, an oxirane carboxylic acid derivative pro-drug, is converted in vivo to a competitive and irreversible inhibitor of CPT1 (26). Inhibition of fatty acid oxidation increases food intake in mice, rats (27, 28), and humans adapted to a high-fat diet. Etomoxir has also been found to decrease the high blood glucose in diabetic rats (29) and humans (30). In humans, RQ is increased by etomoxir (31, 32). Interestingly, the effect of etomoxir on RQ does not seem to have been reported in animals.

In this study, two mouse models for obesity and diabetes were used, the ob/ob and db/db mice. The ob/ob mouse lacks functional leptin hormone (33) and was chosen as a positive (sensitive) model to test the effect of exogenously administered leptin (21). The db/db mouse lacks a functional leptin receptor (34, 35) and was used as negative control. Because both the ob/ob and db/db mice were of C57BL background, C57BL/6J mice were used as wild-type controls. The in vivo energy expenditure was measured by indirect calorimetry. From these measurements the RQ was calculated, which allows conclusions regarding which substrate was being metabolized.

Research Methods and Procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Drugs, Chemicals, and Administration

Leptin (≥97%, high-performance liquid chromatography, recombinant, expressed in Escherichia coli; Sigma) was dissolved in the vial by adding hydrochloric acid (15 mM). Then, sodium hydroxide (7.5 mM) was added, the solution titrated to about pH 5.2, and diluted with water. In preliminary experiments, a single dose of leptin given immediately before calorimetry had no effect on RQ, nor did two leptin doses, one given in the evening of day 1 and the second dose in the morning of day 2 just before calorimetry. However, when leptin was administered twice on the day before calorimetry an effect on RQ was observed. Thus, in these experiments, leptin was administered twice intraperitoneally, at 10:00 am and 4:30 pm on day 1, and calorimetry was started at 8:30 am on day 2 under thermoneutral conditions at 30 °C.

Etomoxir [sodium (+)-2[6(4-chlorophenoxy) hexyl]-oxirane carboxylate, >99%, high-performance liquid chromatography; Sigma, St. Louis, MO] was dissolved in water. Etomoxir was administered twice intraperitoneally, at 4:30 pm on day 1 and at 8:00 am on day 2, immediately before start of calorimetry at an environmental temperature of 20 °C.

Isotonic, sterile sodium chloride (physiological saline; 154 mM, 9 mg/mL, 0.9%, 290 mosm/kg, pH 6; Pfizer, Inc., New York, NY) was used as vehicle.

Animals

Male C57BL/6J mice (10 to 15 weeks old, 25 to 35 grams), C57BL/6JBOM-ob (ob/ob) mice (12 to 15 weeks old, 45 to 50 grams; for the temperature-dependence study, 6 to 10 weeks old, 25 to 45 grams), and C57BL/KsBOM-db (db/db) mice (10 to 15 weeks old, 35 to 45 grams; Taconic M&B, Ry, Denmark) were given normal diet (R34; Lactamin AB, Nässjavägen, Ullevi, Vadstena) and water ad libitum and housed one animal per cage and at 22 ± 2 °C, 50 ± 20% humidity, and a 12:12-hour light:dark cycle, with lights on between 7:00 am and 7:00 pm. The animals were fasting during the calorimetry.

The procedures involving animals were in conformity with national and international laws for the care and use of laboratory animals and were approved by the local animal ethical committee.

In Vivo Calorimetry

Energy expenditure was measured using a Somedic INCA (Hörby, Sweden) indirect calorimetry apparatus as previously described (36).

The metabolic chamber (volume ∼4 liters) was supplied with synthetic air containing 21% oxygen in nitrogen (AGA, Lidingö, Sweden) at a flow rate of 1 liter/min. The zirconium oxide detector was calibrated daily using two reference gases containing 18% and 25% oxygen in nitrogen. Oxygen consumption was measured in awake, non-fasting animals, placed individually in an empty, sealed metabolic chamber. The air leaving the chamber was dried with Silica gel (Safegel 1–3 mm with yellow moisture indicator; Merck Prolabo; VWR International, West Chester, PA). The oxygen content was measured during every second minute in the air supplying the chamber and every second minute in the air leaving it. Oxygen consumption was calculated continuously as the difference in the oxygen concentration in the two most recent samples. The value was expressed as the weight-compensated metabolic rate (milliliters per minute per kilogram0.75) (36, 37).

For the temperature-dependence study, the environmental temperature in the metabolic chamber was maintained with a thermoelectric thermostat at 15, 20, 25, 27.5, 30, and 32.5 °C. For temperatures 15–27.5 °C, the mice were kept in the chamber for up to 6 hours. To reduce the potential effects of heat stress at 30 and 32.5 °C, the exposure time was reduced to 3.5 hours. Oxygen consumption was calculated as the lowest stable level during a period of 2–15 minutes.

The lower critical temperature (LCT) of the thermoneutral zone, basal metabolic rate (BMR) (38), temperature sensitivity (TS), and the defended body temperature (DBT) were calculated, where the metabolic rate (MR) is the measured oxygen consumption (milliliters per minute per kilogram0.75), and x is the environmental temperature (36):

  • image
  • image

where the bars (|) denote the absolute value. The parameters were calculated using an iterative regression computer program (Fig P for Windows, Version 3.1; Biosoft, Cambridge, United Kingdom).

Carbon dioxide in the air leaving the metabolic chamber was measured using a sensor based on the dual channel infrared absorption principle, Testo model 0633–1240 connected to a Testo 650 Reference instrument (Testo, Lenzkirch, Germany) (36). The detector was calibrated using two reference gases containing carbon dioxide 0.02% and 0.2% in synthetic air. The oxygen consumption and carbon dioxide production were measured and the mean value for all animals calculated. The RQ according to Haldane (36) was calculated for each animal.

The body core temperature and three-dimensional total locomotor activity were measured continuously using a transponder telemetry system (Model PDT-4000 E-Mitter and ER-4000 Energizer Receiver, Mini-Mitter, Bend, OR) (36). For the temperature-dependence experiments and in db/db mice rectal body temperature was measured manually immediately before and after the oxygen consumption measurements with a Physiotemp (Clifton, NJ) thermometer (BAT-12) and rectal probe (RET-3).

Statistics

For RQ and body temperature measurements the area under the curve for vehicle and compound treated groups was compared using Student's t test. For the cumulative locomotor activity, the mean values were compared using Student's t test. Data are expressed as mean ± standard error.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Temperature-Dependent Effects on Metabolic Rate

The temperature dependence of the metabolic rate (oxygen consumption) was determined for young and mature ob/ob mice. In mature animals, the temperature dependence as well as the parameters derived were similar for several mouse strains, including normal C57BL/6J and ob/ob mice (Figure 1). There were no differences in the calculated resting metabolic rate, BMR, LCT, TS, and DBT (Table 1). The young, 6-week-old, ob/ob mice, however, had an almost doubled metabolism at 15 °C compared with the 10-week-old mice. Because the BMR and LCT were similar for all mice, the slope (TS) for the 6-week-old ob/ob mice was much steeper (Figure 1). The body temperature measured immediately before and after the calorimetry was unchanged at all environmental temperatures. In the 6-week-old animals, the body temperature was 35.4 ± 0.5 °C before and 36.0 ± 0.5 °C (n = 8; p > 0.05) after calorimetry at an environmental temperature of 14 °C.

image

Figure 1. The environmental temperature dependence of resting metabolic rate (MR; mL O2/min/kg0.75) in 6- (n = 8 to 10) and 10-week-old (n = 10) male ob/ob mice. The graphs are drawn using the fitted constants from Equations 1 and 2. The intercept on the ordinate gives the BMR, and the intercept on the abscissa gives the lower critical temperature (LCT) and the defended body temperature (DBT). The slope gives the temperature sensitivity (TS) (36).

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Table 1.  Parameters describing the temperature dependence of oxygen consumption
Mouse strain, age (N), ReferenceLCT (°C)BMR (ml/min/kg0.75)TS (ml/min/kg/0.75°C)n1DBT (°C)n2
  • Number of animals (N) and number of data points (n1, n2) collected for oxygen consumption used in Equations 1 and 2 for calculation of the parameters LCT of the thermoneutral zone, BMR, and TS. The DBT was calculated using values in the range of 15°C to 27.5°C. Statistical difference for TS of 6- vs. 10-week-old ob/ob mice (

  • *

    p < 0.001). LCT, lower critical temperature; BMR, basal metabolic rate; TS, temperature sensitivity; DBT, defended body temperature.

ob/ob6 weeks (40)25.5 ± 0.811.0 ± 0.82.1 ± 0.2*56  
ob/ob 10 weeks (40)25.0 ± 1.19.8 ± 0.50.8 ± 0.16037.5 ± 3.430
db/db 10 weeks (39)24.3 ± 2.09.0 ± 0.30.8 ± 0.26036.3 ± 2.730
C57BL/6J29.0 ± 0.79.8 ± 0.30.7 ± 0.0527242.9 ± 1.4181
20 to 22 weeks (100), (36)      

In db/db mice, the temperature dependence of the oxygen consumption was similar to that in the mature ob/ob mice(Figure 2; Table 1). If anything, the oxygen consumption was slightly decreased at the low environmental temperatures tested. Body temperature measurements before and after calorimetry revealed that the db/db mice had a decreased body temperature after exposure to environmental temperatures <25 °C. For example, the body temperature was 35.6 ± 0.2 °C before and 33.6 ± 0.5 °C (n = 10; p > 0.01) after calorimetry at an environmental temperature of 19 °C, and 35.9 ± 0.4 °C before and 33.0 ± 0.8 °C (n = 10; p > 0.01) after calorimetry at an environmental temperature of 14 °C.

image

Figure 2. The environmental temperature dependence of resting metabolic rate (MR; mL O2/min/kg0.75) in male db/db mice (n = 10). The graph is drawn using the fitted constants from Equations 1 and 2.

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Temperature-Dependent Effects on RQ

The RQ was also dependent on the environmental temperature. The RQ increased with increasing temperature in all of the three mouse strains tested, in the C57BL/6J, ob/ob, and db/db mice (Table 2; Figures 3 −7).

Table 2. . RQ values in different mouse strains, at different environmental temperatures, and after treatment with leptin or extomoxir
 RQ
Mouse strain, treatment20°C25°C30°C
  • Values obtained 1 hour after start of measurement. Statistical difference: p vs. vehicle in the same mouse strain at the same temperature is

  • p < 0.01,

  • p < 0.001. p vs. db/db mouse at 20°C is

  • §

    p < 0.01,

  • p < 0.001. p vs. 30°C in the same mouse strain is

  • *

    p < 0.01. RQ, respiratory quotient.

ob/ob0.83 ± 0.021 0.93 ± 0.051
Vehicle(n = 11) (n = 9)
ob/ob  0.77 ± 0.026
Leptin (2 × 3 mg/kg)  (n = 10)
ob/ob0.96 ± 0.028 ‡,  
Etomoxir (2 × 30 mg/kg)(n = 15)  
db/db0.68 ± 0.020* 0.78 ± 0.021
Vehicle(n = 9) (n = 9)
db/db  0.78 ± 0.028
Leptin (2 × 3 mg/kg)  (n = 10)
db/db0.77 ± 0.021  
Etomoxir (2 × 30 mg/kg)(n = 10)  
C57BL/6J0.75 ± 0.0081§0.86 ± 0.0530.85 ± 0.11
Vehicle(n = 11)(n = 4)(n = 4)
C57BL/6J  0.72 ± 0.096
Leptin (2 × 5.4 mg/kg)  (n = 3)
C57BL/6J0.89 ± 0.023 ‡,0.83 ± 0.14 
Etomoxir (2 × 30 mg/kg)(n = 14)(n = 5) 
image

Figure 3. Effect of leptin (2 × 3 mg/kg; ip) in ob/ob mice on (A) RQ (** p < 0.01, 1 to 181 minutes vs. vehicle), (B) body core temperature (°C), and (C) accumulated total locomotor activity (activity counts/min; p > 0.05). Calorimetry was performed at an environmental temperature of 30 °C.

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Effects of Leptin Treatment

The RQ was decreased by leptin in ob/ob mice (2 × 3 mg/kg; 30 °C; by 16%; Figure 3; Table 2), and in a preliminary experiment in C57BL/6J mice (2 × 5.4 mg/kg; 30 °C; n = 2 to 5; 14%) compared with vehicle. Leptin slightly decreased the carbon dioxide production, but there was no significant difference in oxygen consumption, body temperature, or locomotor activity in any of the mouse strains.

In the negative control, the functional leptin receptor-deficient db/db mice, leptin had no effect on RQ (Figure 4), oxygen consumption, carbon dioxide production, or body temperature. Because it is has been found that the db/db mouse has decreased wound healing (39), transponders were not implanted in the db/db mice and activity was not measured.

image

Figure 4. Effect of leptin (2 × 3 mg/kg; ip) in db/db mice on (A) RQ (p > 0.05) and (B) body core temperature (°C) (p > 0.05). Calorimetry was performed at an environmental temperature of 30 °C.

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Effects of Etomoxir Treatment

Because the RQ was found to be lower at 20 than at 30 °C (Table 2), the effect of etomoxir was tested at 20 °C. Etomoxir (2 × 30 mg/kg; 20 °C) enhanced RQ in ob/ob (20%; Figure 5; Table 2), C57BL/6J (14%; Figure 6), and db/db (17%; Figure 7) mice compared with vehicle. Etomoxir decreased both the carbon dioxide production and oxygen consumption in all mouse strains tested.

image

Figure 5. Effect of etomoxir (2 × 30 mg/kg; ip) in ob/ob mice on (A) RQ (*** p < 0.001), (B) body core temperature (°C), and (C) accumulated total locomotor activity (activity counts/min; * p < 0.05). Calorimetry was performed at an environmental temperature of 20 °C.

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image

Figure 6. Effect of etomoxir (2 × 30 mg/kg; ip) in C57BL/6J mice on (A) RQ (*** p < 0.001), (B) body core temperature (°C) (*** p < 0.001), and (C) accumulated total locomotor activity (activity counts/min; ** p < 0.01). Calorimetry was performed at an environmental temperature of 20 °C.

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image

Figure 7. Effect of etomoxir (2 × 30 mg/kg; ip) in db/db mice on (A) RQ (** p < 0.01) and (B) body core temperature (°C) (* p < 0.05). Calorimetry was performed at an environmental temperature of 20 °C.

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The body temperature was reduced in C57BL/6J (7%) and db/db (8.6%) mice, which prompted shortening of the duration of the calorimetry measuring period. There was only a tendency toward a decreased body temperature in ob/ob mice.

The total locomotor activity was reduced in ob/ob (38%) and C57BL/6J (67%) mice by etomoxir.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Temperature-Dependent Effects on Metabolic Rate

The temperature dependence of the metabolic rate measured as oxygen consumption was determined for young and mature ob/ob and mature db/db mice. Comparison of the mature mice with other mouse strains, including normal C57BL/6J, shows that the resting metabolic rate, BMR, LCT, TS, and DBT values were similar (36). Lowering the temperature below the thermoneutral zone increased the metabolism. In the young, 6-week-old ob/ob mice, the metabolism was even further increased, and at 15 °C, their metabolism was almost doubled compared with the mature mice. Because the BMR and LCT were similar for all mice, the TS for the young mice was higher, suggesting “impaired insulation” or “deliberately increased” heat loss (36). Therefore, the DBT cannot be calculated in the 6-week-old ob/ob mice in a meaningful manner using Equation 2, because it would yield a value below (cf. Figure 1) the measured body temperature. In this respect, the young ob/ob mice resemble the protein-tyrosine phosphatase 1B knock-out mice (36).

In contrast, in 5-week-old ob/ob mice, oxygen consumption has been found to be slightly, although significantly, lower than for wild-type C57BL/6J mice (3). The body temperature of those mice was <35 °C, i.e., lower than in our experiments even when performed at 14 °C, although those experiments were probably done at room temperature because the calorimeter used does not appear to be equipped with a thermostat. Lower oxygen consumption has been reported in old, 70-gram, ob/ob, and in 10- to 14-week-old db/db, than in lean mice (40, 41). However, those measurements were made when the mice were “partially restrained,” a procedure “found to be essential if stable O2 measurements were to be obtained” (40). Thus, those early data were obtained under some degree of stress.

Temperature-Dependent Effects on RQ

The RQ is also dependent on the environmental temperature. The RQ increased with the temperature in all mouse strains tested: C57BL/6J, ob/ob, db/db (Table 2), and KKAy mice (36). Thus, oxidation of fat was increased at environmental temperatures below the thermoneutral zone to maintain body temperature, in accordance with previous observations (36, 42).

During calorimetry under the present experimental conditions in the absence of food, fat metabolism increased. The gradual switch of metabolism was instantly reflected in a lowered RQ (cf. Figures 34567). Moreover, the RQ has been shown to decrease when the animals are fed a high-fat diet at temperatures close to the thermoneutral zone (36). Thus, under these conditions, the animals metabolize what they eat (43, 44).

As a consequence, testing of drug effects for a lowering of the RQ is preferably made at the thermoneutral zone, e.g., 30 °C (cf. leptin), and for an increase of the RQ at lower temperatures, e.g., 20 °C (cf. etomoxir).

Effects of Leptin Treatment

Leptin treatment decreased the RQ in ob/ob and C57BL/6J mice as expected of a compound that stimulates oxidation of fat relative to carbohydrates. Administration of leptin did not alter RQ in the db/db mice, suggesting that the effect was specifically mediated through the leptin receptor. Likewise, there was no effect on activity or body temperature (4, 45). In contrast to previous findings, no significant difference in oxygen consumption for any of the mouse strains was observed (3, 9, 16, 46). However, the experimental protocols are different in several ways. First, these leptin experiments were performed at the thermoneutral zone and not at room temperature. Second, the dose and route of administration were different. Third, the age and sex of the animals were different. Finally, diurnal variation in oxygen consumption may influence the amplitude of the leptin effect. Female ob/ob mice, but not their (+/?) littermates or db/db mice, have a diurnal variation in oxygen consumption with a sharp nadir 2 to 3 hours after start of the light period (16). Leptin increases the oxygen consumption selectively during the first hours of the light period (3, 4, 9, 15, 16, 46), and even more so in food-restricted animals (46), thus ablating the diurnal variation. Interestingly, when given by intracerebroventricular administration, leptin increased oxygen consumption during both light and dark periods (20), an effect that was even more pronounced when compared with the pair-fed group. Furthermore, a high dose of leptin (10 mg/kg) increases oxygen consumption in very young, 5-week-old ob/ob mice with no effect in heterozygous (+/?), C57BL/6J, and db/db mice (3).

While the effects on oxygen consumption were observed during a rather limited interval during the first hours of the light period, those on RQ have been reported to be detected immediately after leptin administration (16), although there were only small effects of leptin on carbon dioxide production. The effect of leptin on the preferred metabolic substrate does not seem be secondary to the effect on food intake, because leptin decreased RQ also when compared with the pair-fed group, which did not show a decrease in RQ (17). Moreover, a low leptin dose with no effect on food intake or body weight still caused a decrease in RQ in the rat (22). Consequently, in these experiments, to increase the sensitivity for drug effects of the oxygen and carbon dioxide measurements, the RQ was calculated for each animal, making each its own control.

These results show that both leptin treatment and lowered environmental temperature decreased the RQ, which reflects an increased fat oxidation. Activation of the sympathetic nervous system is known to increase fat oxidation. Thus, the results suggest that both leptin and environmental temperatures below the thermoneutral zone activated the sympathetic nervous system (47, 48).

During thermoneutral conditions, we found no change in the body core temperature by leptin, whereas it has been reported to be slightly increased in the ob/ob mice when tested below thermoneutrality (3, 4).

Effects of Etomoxir Treatment

The CPT1 blocker etomoxir had the opposite effect from leptin. The increased RQ suggests decreased oxidation of fat relative to carbohydrates. Because the effect was similar for all mouse strains tested, it does not correlate with the degree of obesity and diabetes. Etomoxir has previously been shown to increase RQ in humans (32), but not, to our knowledge, in vivo in mice.

Interestingly, in both vehicle and etomoxir-treated db/db mice, RQ was lower than in wild-type and ob/ob mice at 20 °C. The body temperature of db/db mice was decreased after exposure to temperatures <25 °C, indicating insufficient ability to maintain the body temperature, which might contribute to the low RQ in the db/db mouse. This, together with the very high blood glucose levels (49), may be a reflection of the impaired carbohydrate metabolism in db/db mice (50). RQ values <0.7 may be caused by gluconeogenesis from protein and ketogenesis. However, the db/db mice did increase carbohydrate metabolism when β-oxidation was inhibited.

In contrast to leptin, etomoxir caused a significant decrease in activity in both wild-type and ob/ob mice, indicating that the level of activity did not correlate with RQ under these conditions.

It has been shown that inhibition of fatty acid oxidation can lead to increased food intake in mice, rats (27, 28), and humans (51) adapted to a high-fat diet. In contrast, leptin administration is well documented to decrease food intake and reduce body weight in ob/ob mice (6, 7, 8). None of the mice showed any effect on body weight of etomoxir vs. vehicle (data not shown). Because of the short treatment period, food intake was not measured in this study.

In conclusion, in young ob/ob mice, the TS was enhanced compared with mature mice. Leptin and etomoxir had opposite effects on metabolic substrate preference. Leptin and lowered environmental temperature increased the relative fat oxidation as indicated by decreased RQ, possibly through activation of the sympathetic nervous system.

Acknowledgement

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

The authors thank Dr. Bo Johansson, Somedic, Hörby, Sweden, for advice and support with the calorimetry. There was no funding/outside support for this study.

Footnotes
  • 1

    Nonstandard abbreviations: RQ, respiratory quotient; CPT1, carnitine O-palmitoyl transferase-1; LCT, lower critical temperature; BMR, basal metabolic rate; TS, temperature sensitivity; DBT, defended body temperature; MR, metabolic rate.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References
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