AJP - Endo Watch the video to learn how APS reaches out to developing nations.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Endocrinol Metab 295: E356-E367, 2008. First published May 20, 2008; doi:10.1152/ajpendo.90256.2008
0193-1849/08 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Figures and Tables
Right arrow All Versions of this Article:
295/2/E356    most recent
90256.2008v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kus, V.
Right arrow Articles by Kopecky, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kus, V.
Right arrow Articles by Kopecky, J.

Induction of muscle thermogenesis by high-fat diet in mice: association with obesity-resistance

Vladimir Kus,1 Tomas Prazak,1 Petr Brauner,1 Michal Hensler,1 Ondrej Kuda,1 Pavel Flachs,1 Petra Janovska,1 Dasa Medrikova,1 Martin Rossmeisl,1 Zuzana Jilkova,1 Bohumir Stefl,4,{dagger} Eva Pastalkova,3 Zdenek Drahota,2 Josef Houstek,2 and Jan Kopecky1

Departments of 1Adipose Tissue Biology, 2Bioenergetics, and 3Neurophysiology of Memory and Computational Neuroscience, Institute of Physiology, Academy of Sciences of the Czech Republic; and 4Department of Physiology and Developmental Biology, Faculty of Science, Charles University, Prague, Czech Republic

Submitted 27 February 2008 ; accepted in final form 20 May 2008


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The obesogenic effect of a high-fat (HF) diet is counterbalanced by stimulation of energy expenditure and lipid oxidation in response to a meal. The aim of this study was to reveal whether muscle nonshivering thermogenesis could be stimulated by a HF diet, especially in obesity-resistant A/J compared with obesity-prone C57BL/6J (B/6J) mice. Experiments were performed on male mice born and maintained at 30°C. Four-week-old mice were randomly weaned onto a low-fat (LF) or HF diet for 2 wk. In the A/J LF mice, cold exposure (4°C) resulted in hypothermia, whereas the A/J HF, B/6J LF, and B/6J HF mice were cold tolerant. Cold sensitivity of the A/J LF mice was associated with a relatively low whole body energy expenditure under resting conditions, which was normalized by the HF diet. In both strains, the HF diet induced uncoupling protein-1-mediated thermogenesis, with a stronger induction in A/J mice. Only in A/J mice: 1) the HF diet augmented activation of whole body lipid oxidation by cold; and 2) at 30°C, oxygen consumption, total content, and phosphorylation of AMP-activated protein kinase (AMPK), and AICAR-stimulated palmitate oxidation in soleus muscle was increased by the HF diet in parallel with significantly increased leptinemia. Gene expression data in soleus muscle of the A/J HF mice indicated a shift from carbohydrate to fatty acid oxidation. Our results suggest a role for muscle nonshivering thermogenesis and lipid oxidation in the obesity-resistant phenotype of A/J mice and indicate that a HF diet could induce thermogenesis in oxidative muscle, possibly via the leptin-AMPK axis.

nonshivering thermogenesis; leptin; adenosine monophosphate-activated protein kinase

MANY HEALTH PROBLEMS IN AFFLUENT SOCIETIES are linked to the increased incidence of obesity. Especially meals with high content of fat are obesogenic, due to a low energetic cost of nutrient storage and a low potency of fat intake to promote fat oxidation (20). Induction of lipid catabolism is crucial for adaptive thermogenesis, which is regulated by the sympathetic nervous system, thyroid hormones, insulin (reviewed in Refs. 19 and 26), and leptin (see below) and is possibly involved in the regulation of body weight in humans (12, 26, 49). However, the mechanisms and organs contributing to adaptive thermogenesis need to be better characterized.

In small mammalian species, hibernators, and mammalian neonates, adaptive thermogenesis largely depends on uncoupling protein-1 (UCP1), which is located in brown adipose tissue. Thermogenesis in brown fat could be activated in response to both cold exposure and a meal (6), and its capacity is increased by adaptation to cold or intake of high-fat (HF) diets. Diet can even act as a preacclimation to cold (6, 36), and brown fat thermogenesis serves to maintain both body temperature and energy balance. However, the capacity does not exceed 60% of total adaptive nonshivering thermogenesis (reviewed in Ref. 19), suggesting a role for other organs in this process (16, 24, 50). Whether skeletal muscle can mediate adaptive nonshivering thermogenesis in mammals is a matter of a long-lasting controversy. Skeletal muscle is an important site of whole body energy expenditure, which can account for 20–30% of the total oxygen uptake in the resting state (6, 19, 25). Differences in resting muscle metabolism explain part of the variance in resting metabolic rate among adult humans and may play a role in the pathogenesis of obesity (55).

A unique role in the complex control of energy homeostasis and thermogenesis is played by adipocyte hormone leptin, which acts both centrally in the hypothalamus and also directly in the peripheral tissues (reviewed in Ref. 35). The administration of leptin reverts reduced metabolic rate, depression of body temperature, and excessive adiposity in genetically obese ob/ob mice lacking functional leptin (31). Even in normal mice, leptin induces the capacity for UCP1-mediated thermogenesis (38), and it also stimulates lipid oxidation and uptake of glucose in skeletal muscle by activating AMP-activated protein kinase [AMPK (29)]. The direct thermogenic effect of leptin was also demonstrated in murine skeletal muscle, where exogenous leptin stimulated oxygen consumption (41). Leptin's effects occurred primarily in the oxidative but not glycolytic type of muscle (29, 41).

We hypothesized that muscle nonshivering thermogenesis could be complementary to that mediated by UCP1 in brown fat and could be activated under the conditions of a strong thermogenic response to a HF diet, possibly in association with obesity resistance. Therefore, mice of two different inbred strains were studied: 1) obesity-prone C57BL/6J (B/6J) mice and 2) A/J mice, which are relatively resistant to the obesogenic environment (45, 46, 53). The different propensities to obesity have previously been shown to be associated with a stronger activation of thermogenesis mediated by UCP1 and UCP2 in brown (17, 48, 52) and white (8, 13, 48, 52) adipose tissue of A/J mice. Mice of the A/J strain also showed a much stronger induction of leptin by the HF diet especially during the early postweaning period (47, 52). In our experiments, to eliminate the confounding effect of cold-induced thermogenesis, all mice were born and maintained at 30°C, i.e., close to their thermoneutral temperature (1), weaned onto a low-fat (LF) or HF diet, and studied 2 wk after weaning. In contrast to B/6J mice, A/J mice could not maintain their body temperature in cold when fed the LF diet, reflecting their relatively low metabolic rate at 30°C. Our results suggest that HF diet induced nonshivering thermogenesis in oxidative skeletal muscle of A/J mice.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals and study design. All procedures were in accordance with the guidelines for the use and care of laboratory animals of the Institute of Physiology, the directive of the European Communities Council (86/609/EEC), and the Principles of Laboratory Animal Care (NIH publication no. 85-23, revised 1985). B/6J and A/J mice were obtained from the Jackson Laboratory (Bar Harbor, ME) and bred at the Institute of Physiology for several generations. Animals were housed in a controlled environment (22°C; 12:12-h light-dark cycle; light from 6:00 AM) with free access to water and chow, a LF diet (ST-1 diet; Velaz, Prague, Czech Republic). The LF diet contained 25, 9, and 66% calories as protein, fat, and carbohydrate, respectively. To obtain animals for this study, pregnant mice were transferred to 30°C 1 wk before delivery, and newborn mice were maintained at this temperature until the end of the experiment. When indicated, some animals born and reared at 22°C were also analyzed. Only male pups were used. Mice were weaned at 4 wk of age, caged singly in Eurostandard type II mouse plastic cages (~6,000 ml; Techniplast, Milan, Italy), and assigned randomly to either the LF or HF diet. The HF diet, proven to be obesogenic in B/6J mice (22), contained 15, 59, and 26% calories as protein, fat, and carbohydrate, respectively. Fatty acid composition of both diets (LF vs. HF, in mol%) was characterized (29.7 vs. 19.6 for saturated fatty acid, 28.3 vs. 55.2 for monounsaturated fatty acid, and 42.1 vs. 25.2 for polyunsaturated fatty acid), and energy densities of LF and HF diets were 3.4 and 5.3 kcal/g, respectively (37). To eliminate the confounding effect of the large differences in adiposity, all analyses described below were performed 2 wk after weaning, except for 1) 24-h food consumption measurements, which were performed at 2, 4, 9, and 13 days after weaning (Table 1); and 2) a separate experiment at 30°C, in which the effect of the diet on body weight was followed until 4 mo after weaning (Fig. 1). For the collection of plasma and tissues, ad libitum-fed mice were killed by cervical dislocation between 9:00 and 10:00 AM. EDTA-plasma was prepared from truncal blood and stored at –70°C. Subcutaneous (dorsolumbar) and epididymal white adipose tissue, interscapular brown fat, and gastrocnemius and soleus muscles were dissected and stored in liquid nitrogen for analysis of gene expression. For measurements of oxygen consumption and fatty acid oxidation, freshly dissected muscles were used. For Western blot analysis, soleus muscle was dissected under pentobarbital sodium anesthesia and jet-frozen in liquid nitrogen (see below).


View this table:
[in this window]
[in a new window]

  		Table 1. Growth characteristics and plasma parameters

 

Figure 1
View larger version (8K):
[in this window]
[in a new window]

  		Fig. 1. Body weights in B/6J and A/J mice during prolonged feeding by low-fat (LF) and high-fat (HF) diets. Mice were born and maintained at 30°C. At 4 wk of age (week 0), animals were caged singly and weaned either on LF or HF diet. Data are means ± SE (n =7–8). *Significant effect of diet; there was also a significant interaction between diet and genotype (repeated-measures ANOVA).

 
Body temperature and shivering during cold exposure. Two weeks after weaning, singly caged mice fed either the LF or HF diet were transferred to 4°C. Core body temperature was measured just before cold exposure (time 0) and at 1, 2, 3, 24, 48, and 72 h in the cold, using a temperature probe (Thermocouple Thermometer; Cole Parmer, Vernon Hills, IL) inserted into the colon. During the cold exposure, animals had free access to food and water.

Shivering was monitored in a separate group of mice by means of electromyography (EMG). Before the experiment, mice were transferred to 20°C and anesthetized using diethyl ether, and 0.5-cm Ni-Cr wire (150 µm diameter) electrodes were inserted under the skin. Two recording electrodes were placed above the left and right shoulders of the forelimbs, and a reference electrode was implanted above the rear back. Immediately after recovery from anesthesia, mice were caged singly without bedding, water, or food. Action potentials were amplified (5,000 times), filtered (300–6,000 Hz), and digitized (32-bit resolution, 32 kHz), and data were stored for analysis performed using custom-made software (AcX; Andre A. Fenton, Institute of Physiology, Prague, Czech Republic). After a 20-min period of initial recording at 20°C, the cages were transferred to 4°C, and recording was performed for 50 min. After the experiment, mice were killed by cervical dislocation. During offline analysis of the data, moving artifacts were eliminated on the basis of their high amplitude and time intervals. Voltage values were integrated over 10-min periods.

Indirect calorimetry. Energy expenditure was evaluated using the indirect calorimetry system INCA (Somedic, Horby, Sweden) as described before (1). To minimize stress in the animals, measurements were performed in the standard cages, in which animals had been maintained since weaning (see above), and they were transferred into sealed chambers equipped with thermostatically controlled heat exchangers. Calibration of oxygen sensors was performed before each measurement. Measurements proceeded under a constant airflow rate (1,000 ml/min). Oxygen consumption (VO2) and carbon dioxide production (VCO2) were recorded every 2 min. The system allowed for four individually housed mice to be monitored simultaneously. During the measurements, lasting for 46 h and starting at 3:00 PM (12:12-h light-dark cycle; light from 6:00 AM), animals had ad libitum access to food (i.e., LF or HF diet, see above). During the initial 23 h, the temperature was set to 30°C followed by a drop to 15°C during a 30-min interval at the beginning of the second 23-h period and kept at 15°C until the end of the measurement. The level of substrate partitioning was estimated by calculating respiratory exchange ratio (RER; i.e., VCO2/VO2 ratio). To compare subtle differences between subgroups, the percent relative cumulative frequency (PRCF) curves were also drawn (see Fig. 5), based on RER values pooled from all animals within a given subgroup (24, 34).


Figure 5
View larger version (13K):
[in this window]
[in a new window]

  		Fig. 5. Plots of percent relative cumulative frequency (PRCF) of respiratory exchange ratio (RER) values obtained by indirect calorimetry. RER data from the experiment (described in Table 2 and Fig. 4) were used to construct PRCF curves, each of which represents the data pooled from all mice (n = 6–8) within a given subgroup (~4,200–5,600 RER measurements per curve). For statistical analysis, see METHODS. Values of log EC50 (50th %ile values; see also corresponding RER data in Table 2) are significantly different between HF- and LF-fed mice within each genotype and irrespective of experimental temperature (P < 0.001). Only in A/J HF mice, values of log EC50 at 15 and 30°C are also significantly different (P = 0.004). At 15°C, the Hill-slope values are significantly different between HF- and LF-fed mice within each genotype, whereas at 30°C a significant difference between HF- and LF-fed mice was found only in B/6J mice.

 
Norepinephrine-stimulated metabolic rate (NEMR) was measured in anesthetized mice as described before (42) and calculated as a difference between VO2 measured 15–20 min before and after intraperitoneal injection of L-norepinephrine D-bitartrate (Sigma-Aldrich; in 5% D-glucose at a dose of 0.6 mg/kg body wt).

Lipids, metabolites, and hormones in plasma. Triglycerides (TG) were estimated using diagnostic kit no. 320-A (Sigma-Aldrich). The concentration of nonesterified fatty acids (NEFA) was evaluated enzymatically using a NEFA C kit (Wako Chemicals, Richmond, VA). Leptin content was assessed by a Mouse Leptin RIA Kit (Linco Research, St. Charles, MO). Serum triiodothyronine (T3) and thyroxine (T4) levels were determined with total T4 and total T3 RIA kits (Immunotech, Beckman Coulter, Czech Republic).

Measurements of VO2 in skeletal muscles. Dissected soleus or gastrocnemius muscle (10 mg wet wt) was placed in a respiratory chamber of Oroboros Oxygraph [Paar, Graz, Austria (15)] filled with 2 ml of freshly oxygenated (95% O2, 5% CO2) Krebs-Ringer bicarbonate buffer (pH 7.4), containing 10 mM glucose and warmed to 37°C. VO2 was measured continuously and recorded at 1-s intervals between 10 and 35 min after the start of incubation by use of a computer data acquisition system (Datlab; Oroboros, Innsbruck, Austria). Mean values of VO2 were calculated for 5-min intervals. Results were normalized to wet weight of the tissue and also to protein content estimated in the tissue homogenate by using the bicinchoninic acid procedure and BSA as a standard (40), prepared after the respirometry.

Measurements of fatty acid oxidation in muscles. Soleus and gastrocnemius muscle was incubated as described above, except that the final volume was 3 ml, and the Krebs-Ringer bicarbonate buffer contained 5 mM glucose and fatty acid-free bovine serum albumin (5 mg/ml, ICN Biomedicals, High Wycombe, UK) complexed (1:3) with palmitic acid (Sigma-Aldrich) and [U-14C]palmitate (0.1 µCi/ml; LACOMED, Czech Republic); see online APPENDIX for details. Muscles from left and right limb were incubated in the absence or presence of AMPK activator 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR; Sigma-Aldrich) added at a 2 mM final concentration. After 1 h of shaking at 37°C in a closed glass vial (15 ml total volume), the reaction was terminated by injecting 0.3 ml of 5 M H2SO4 into the vial, and liberated CO2 was trapped in 0.3 ml of hyamine hydroxide (PerkinElmer, Waltham, MA) contained in an Eppendorf tube inserted into the incubation vial. After 60 min of CO2 trapping, 14CO2 in hyamine hydroxide was quantified by liquid scintillation counting, and oxidation rate was normalized to tissue protein as above.

Gene expression analysis. Total RNA was isolated (Tri Reagent; MRC, Cincinnati, OH), and levels of different transcripts were evaluated using real-time quantitative PCR (qRT-PCR), a DyNamo Capillary SYBR Green qPCR kit (Finnzymes, Espoo, Finland), and a LightCycler 2.0 instrument (Roche Diagnostics, Mannheim, Germany) as before (11). Lasergene software (DNA Star, Madison, WI) was used to design oligonucleotide primers (see online APPENDIX, Supplemental Table S1). To correct for intersample variations, the level of each transcript was normalized to elongation factor-1{alpha} (EF-1{alpha}), used as an internal standard, and expressed in arbitrary units (AU). Similar results were obtained using cyclophilin-β as an internal standard (not shown).

Evaluation of UCP1 and protein content. UCP1 was quantified by Western blots (39) in crude cell membranes (100,000 g) prepared from homogenates of adipose tissue, using purified UCP1 as a standard (23). Total protein concentration was assessed as above (40).

Quantification of AMPK and acetyl-CoA carboxylase (ACC) in soleus muscle. Muscle lysates were prepared as before (27). Total amount of the {alpha}1 and {alpha}2 catalytic subunits of AMPK (AMPK{alpha}1, AMPK{alpha}2) and the phosphorylated form of AMPK (p-AMPK) were determined by Western blotting using a mixture of primary antibodies: 1) sheep antibodies against AMPK{alpha}1 and -{alpha}2 [1:3,500; kind gift from D. Grahame Hardie, University of Dundee, UK (53)], and 2) phosphospecific rabbit antibodies against the Thr172 of the {alpha}-subunits of AMPK (1:1,000; no. 2535, lot no. 6; Cell Signaling Technology, Danvers, MA). As secondary antibodies, a mixture of infrared dye-labeled antibodies was used: 1) donkey anti-sheep IgG conjugated to infrared dye 680 (1:5,000, no. A-21102, Lot no. 93C2-1; Alexa fluor 680 donkey anti-sheep IgG; Molecullar Probes, Leiden, The Netherlands), and 2) donkey anti-rabbit IgG conjugated to infrared dye 800 (1:5,000, no. 611-732-127, Lot no. 13176; infrared dye 800 anti-rabbit IgG donkey; Rockland, Gilbertsville, PA). Both total and p-AMPK were quantified on the same blot using the Odyssey IR Imaging Systems (Li-Cor Biosciences, Lincoln, NE). Total content of ACC and its phosphorylated form were quantified using Western blotting and specific antibodies as before (3). Prestained protein Mr standards (PageRuler Prestained Protein Ladder; Fermentas, Burlington, ON, Canada) were used to locate positions of AMPK or ACC on the blots. Signals on different blots were compared using a standard prepared from the liver of adult LF diet-fed B/6J mice and expressed in arbitrary units. The value for each mouse represents the mean of values obtained from the left and right soleus muscles.

Statistics. The data were analyzed by a two-way ANOVA, using SigmaStat statistical software. Logarithmic transformation was used to stabilize variance in cells when necessary. Repeated ANOVA measurements were performed to analyze VO2 in muscles. To the analyze statistical significance of differences in PRCF of RER values in the indirect calorimetry (see Indirect calorimetry), data from each mouse were treated separately. The PRCF curves were fitted with sigmoidal dose-response (variable slope) function using GraphPad Prism v. 4.03 for Windows (GraphPad Software, San Diego, CA). Both log EC50 and Hill slope values were compared in a two-way ANOVA (diet x temperature). Tukey's test for all pairwise multiple comparisons was used. All values are presented as means ± SE. Comparisons were judged to be significant at P ≤ 0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Gross phenotypes and plasma parameters. B/6J and A/J mice were born and maintained at 30°C, weaned onto LF or HF diet at 4 wk of age, and killed 2 wk later. At weaning, as well as at the time of euthanasia, mice of both strains had similar body weights independent of the type of diet. Accordingly, body weight gains during the 2-wk postweaning period were similar in all animal subgroups (Table 1). Caloric intake was also similar in all the subgroups. Feeding mice of both strains the HF diet resulted in a decrease of interscapular brown fat (with a significant effect only in B/6J mice). In accord with a previous study (52) performed at 20°C and using a HF diet of a similar composition as in our experiments, the weight of white fat depots was increased by HF diet at 2 wk after weaning, with a stronger effect seen in A/J mice (Table 1). However, when the animals were fed the HF diet for up to 4 mo while still maintained at 30°C, B/6J mice gained more weight than A/J mice, and only in the former mice a strong obesogenic effect of the HF diet on body weight was observed (Fig. 1). Thus, in adult mice, the different propensity to dietary obesity, as detected in previous studies in B/6J and A/J mice maintained at 20–22°C (45, 46, 53), became clearly apparent even close to thermoneutrality, indicating that obesity resistance of A/J mice did not depend on the induction of energy expenditure by cold. On the other hand, the induction of adiposity by HF diet in both strains after weaning may reflect relatively low protein intake for actively growing mice.

At 2 wk after weaning, plasma levels of TG and NEFA were not affected by the diet in either genotype, except for a small induction of NEFA by the HF diet in B/6J mice (Table 1). As expected (47, 52), the HF diet strongly increased (~2.8-fold) leptin levels in A/J mice, whereas no significant induction of leptin could be detected in B/6J mice (Table 1). In contrast, plasma levels of both T4 and T3 were not affected by the HF diet in A/J mice, but they were increased in B/6J mice, indicating the stimulatory effect of the HF diet on thyroid function in B/6J but not in A/J mice (Table 1).

Body temperature and shivering during cold exposure. Assuming that the difference between B/6J and A/J mice in their propensity to HF diet-induced obesity reflected different thermogenic capacity of these two strains, sensitivity of the animals to acute cold exposure might be affected by the diet. To verify this hypothesis, the mice adapted to 30°C were exposed to an ambient temperature of 4°C, and their deep body temperature was measured (Fig. 2). Before the cold exposure (time 0), body temperature was similar in all the subgroups, whereas the temperature tended to be increased by HF diet specifically in A/J mice (legend to Fig. 2). During the first hour in the cold, body temperature decreased in all the subgroups, while the most pronounced decrease (~2.5°C) was observed in the A/J LF mice. However, LF diet-fed mice of both strains showed similar activation of shivering in the cold (Fig. 3). During the next 2 h, body temperature remained stable in all subgroups except for the A/J LF mice, which became hypothermic, and this prompted us to terminate the experiment in this subgroup (Fig. 2). In contrast, all the other mice were able to maintain their body temperature relatively stable up to 3 days in the cold (not shown). The strain-specific response to cold was not apparent in mice born and reared at 22°C, since under these conditions even A/J LF mice were able to maintain their body temperature above 36°C during a 3-day period in the cold (not shown). Thus, the adaptation of the A/J LF mice to the temperature close to thermoneutrality was essential for unmasking the relatively low thermogenic potential of these mice when exposed to cold and for demonstration of the induction of the thermogenic capacity of the A/J mice by HF diet.


Figure 2
View larger version (6K):
[in this window]
[in a new window]

  		Fig. 2. Deep-body temperature in mice during cold exposure. Singly caged mice maintained at 30°C and fed either LF or HF diet for 2 wk after weaning were used. Animals in their cages containing food, water, and bedding were transferred from 30 to 4°C, and their colonic temperature was measured just before (36.24 ± 0.11, 36.31 ± 0.07, 36.05 ± 0.11, and 36.43 ± 0.13°C in B/6J LF, B/6J LH, A/J LF, and A/J HF, respectively) and during cold exposure. In A/J LF mice, cold exposure was finished after 3 h due to the development of severe hypothermia, whereas all the other mice could maintain their body temperature relatively constant for 3 days in the cold (not shown). Data are means ± SE (n = 7–8). *Significant effect of diet.

 

Figure 3
View larger version (10K):
[in this window]
[in a new window]

  		Fig. 3. Shivering during cold exposure. Singly caged B/6J (open symbols) and A/J (filled symbols) mice maintained at 30°C and fed LF diet for 2 wk after weaning were used. After implantation of recording and reference electrodes during anesthesia at 20°C, animals in their cages without food, water, and bedding were transferred to 4°C, and EMG recording was performed before (time 0) and during 50 min of cold exposure. Voltage values were integrated over 10-min periods before the indicated time points. Data are means ± SE (n = 7–8). In mice of both strains, shivering tended to be increased during the first 10 min, but the effect was not statistically significant.

 
Whole body energy expenditure in awake animals. To characterize whole body energy expenditure and its changes in response to diet in mice of both genotypes, indirect calorimetry was used to analyze VO2 and RER in ad libitum-fed mice reared at 30°C. Continuous monitoring in individually caged mice was performed for 23 h at 30°C, followed by a period of 23 h at 15°C (Fig. 4). When measured at 30°C and during the light phase of the day, energy expenditure in the A/J mice was affected by the type of diet, with a significant 38% increase in response to HF diet (Table 2). In fact, this increase was related to a relatively low VO2 during the light phase in the A/J LF mice compared with all other subgroups, and the VO2 values in A/J HF mice were similar to that in B/6J mice (Table 2). No effect of diet on energy expenditure was detected in the B/6J mice. During the dark phase, VO2 was not affected by diet in either strain of mice (Table 2). Upon a decrease of ambient temperature from 30 to 15°C, mean VO2 values increased approximately twofold in all the subgroups and the differences in VO2 between the genotypes and diets disappeared (Table 2).


Figure 4
View larger version (25K):
[in this window]
[in a new window]

  		Fig. 4. Time course of VO2 measurements in mice maintained at 30°C and fed either LF or HF diet for 2 wk after weaning. Indirect calorimetry was performed on singly caged mice with free access to water and diet initially at 30°C for a period of 23 h followed by 23 h at 15°C. VO2 data were sampled at 60 s for every 2 min; however, only mean values of single recordings at 1-h intervals are shown. Arrows indicate beginning of a 30-min period during which the temperature was dropped. Gray areas represent the dark phases of diurnal cycle. A biphasic circadian rhythm of VO2 was observed in B/6J mice, with 2 maxima: 1 in the middle of the day and the other at the end of the dark phase. This pattern was only marginally affected by diet. In contrast, in A/J mice, a monophasic rhythm of VO2 was recorded, with maximum around the beginning of the dark phase of the day and lower VO2 values recorded during the light phase. Data are means ± SE (n = 6–8). For statistical analysis of these data, see Table 2.

 

View this table:
[in this window]
[in a new window]

  		Table 2. Indirect calorimetry

 
The HF diet lowered RER in all animal subgroups, especially during the dark phase (Table 2), suggesting a shift toward lipid oxidation. This shift was also illustrated by the PRCF curves of RER values pooled from all the measurements performed within the same ambient temperature (Fig. 5). Since all the PRCF curves represented normally distributed data (not shown), the mean RER values corresponded to the 50th percentile values of the PRCF curves (34). In A/J but not in B/6J mice, RER values were also decreased in response to the lower ambient temperature. This shift was more significant in mice fed the HF compared with the LF diet (Table 2), resulting also in the steepest PRCF curve in the A/J HF mice when measured at 15°C (Fig. 5). The slope of the PRCF curve is a marker of metabolic flexibility, with a bigger slope suggesting a lower metabolic flexibility between oxidation of lipids and carbohydrates (34). Therefore, the results highlighted modulation of the resting energy expenditure in A/J mice by the dietary treatments and high reliance of A/J mice on fatty acid oxidation during cold exposure.

UCP1-mediated nonshivering thermogenesis. Capacity for UCP1-mediated thermogenesis was assessed as an increment in metabolic rate after the injection of norepinephrine to anesthetized animals (NEMR; Refs. 16, 19, 42). NEMR was similar in LF diet-fed mice of both genotypes, it was stimulated by HF diet, and the stimulation was higher in A/J than in B/6J mice (Fig. 6). UCP1 protein content in interscapular brown fat was also increased by HF diet, and this increase was significantly higher in A/J mice (~10-fold) compared with B/6J mice (~3-fold), resulting in approximately twofold higher UCP1 levels in brown fat in A/J than in B/6J mice. Expression of UCP1 was also detected in subcutaneous white fat (see online APPENDIX, Table S2) and skeletal muscles (Table 3; see also Ref. 2) of mice of both genotypes, but the levels of UCP1 in these tissues were by at least two orders of magnitude lower than in the interscapular fat, indicating a negligible quantitative importance for thermogenesis. In some of the previous studies, UCP2 transcript levels in white fat have also been correlated with obesity resistance in A/J mice (13, 48, 52). However, in our experiments, UCP2 expression was similar in all fat depots studied, including interscapular brown and subcutaneous and epididymal white fat, and no effect of either genotype or diet on UCP2 expression was observed (see online APPENDIX, Table S2).


Figure 6
View larger version (8K):
[in this window]
[in a new window]

  		Fig. 6. Norepinephrine (NE)-induced thermogenesis. Mice maintained at 30°C and fed either LF or HF diet for 2 wk after weaning were anesthetized by pentobarbital sodium, and NE metabolic rate (NEMR) was measured as difference between VO2 before and after ip injection of NE. Before injection, VO2 values were similar in all subgroups of anesthetized mice (28.1 ± 3.1, 29.5 ± 3.0, 26.2 ± 2.7, and 25.6 ± 2.1 ml·kg–1·min–1 in B/6J LF, B/6J HF, A/J LF, and A/J HF mice, respectively). Data are means ± SE (n = 6–9). *Significant effect of diet.

 

View this table:
[in this window]
[in a new window]

  		Table 3. Gene expression in glycolytic and oxidative skeletal muscles

 
Thus, in both genotypes, the stimulation of NEMR by HF diet could be explained by the induction of UCP1 protein in the interscapular brown fat. A relatively strong induction in A/J mice was associated with the development of cold tolerance of these mice. On the other hand, the cold sensitivity of the A/J LF mice could be attributed to their relatively low whole body energy expenditure during the light phase of the day and could not be explained by an insufficient capacity of either UCP1-mediated energy expenditure or shivering thermogenesis, since no differences in this respect were detected between A/J and B/6J mice fed the LF diet.

Gene expression in muscles. A marked increase of VO2 in response to the HF diet in A/J mice during the light phase of the day, when the locomotor activity of animals is minimal (9), suggested involvement of an adaptive mechanism of energy expenditure, compatible with a sustained induction of nonshivering thermogenesis in muscle via the leptin-AMPK axis (see introductory remarks). Therefore, to characterize muscle metabolism, expression of several genes was assessed in gastrocnemius (a predominantly fast, glycolytic fiber type) and soleus (a predominantly slow, oxidative fiber type) muscle (Table 3). Strong effects of both diet and genotype were detected in the case of stearoyl-CoA desaturase-1 (SCD-1), a gene that is specifically repressed by leptin, which also stimulates fatty acid oxidation (4, 7). In both strains and in both types of muscles, the expression of SCD-1 was downregulated by the HF diet. The strongest suppression, approximately fourfold, was observed in the soleus muscle of A/J mice. These results were in agreement with the assumption that leptin was involved in the induction of nonshivering thermogenesis in soleus muscle of A/J mice by HF diet.

There was no effect of genotype or diet on genes engaged in the control of mitochondrial biogenesis and oxidative capacity, as assessed by quantification of the transcripts for nuclear respiratory factor-1 and subunit VIa of mitochondrial cytochrome oxidase (21), respectively. The expression of PPAR{gamma} coactivator-1{alpha} (PGC-1{alpha}), which links nuclear receptors to the transcriptional program of mitochondrial biogenesis and oxidative metabolism (33), could not explain the stimulatory effect of HF diet on energy expenditure in skeletal muscle of A/J mice either (Table 3). These results were in agreement with the lack of any effect of HF diet on the activity of cytochrome oxidase in soleus muscle of A/J mice (not shown).

The HF diet induced expression of mitochondrial acyl-CoA thioesterase, with the most pronounced effect in the soleus muscle of B/6J mice, and only in B/6J mice did it also upregulate UCP3 (Table 3). This was in agreement with the known stimulatory effect of thyroid hormones (30) on the expression of UCP3 and mitochondrial acyl-CoA thioesterase genes and with the B/6J strain-specific stimulation of thyroid status by HF diet (see above). The induction of UCP3 in both muscles of B/6J mice was associated with upregulation of long-chain acyl-CoA synthetase (required for activation and transport of fatty acid into mitochondria), whereas expression of cytosolic acyl-CoA thioesterase was unchanged (Table 3). These results suggest thyroid hormone-dependent induction of a mechanism of lipid handling by HF diet, which may preserve the CoASH pool in mitochondria, specifically in B/6J mice, independently of thermogenesis (18).

The expression of superoxide dismutase-1, a marker of reactive oxygen species formation, was similar in all the subgroups. Except for muscle type-specific differences in gene expression, relatively small variations in mRNA levels of PPAR{alpha}, UCP1 (see above and Ref. 2), acyl-CoA oxidase-1, and the "muscle" isoform of carnitine palmitoyltransferase I (CPT IB) were observed in response to the diet. In contrast, strain-specific differences in the expression of CPT IA ("liver" isoform) were observed both in soleus and in gastrocnemius muscles. In this case, the expression was affected more by the genotype than by the diet, with the most pronounced effect in the soleus muscle, resulting in an approximately twofold higher CPT IA expression in A/J than in B/6J mice. An opposite effect of the genotype on the expression of CPT IA was observed in the gastrocnemius muscle of HF diet-fed mice (Table 3). Furthermore, high levels of mRNA for isoenzyme 4 of pyruvate dehydrogenase kinase (PDK-4) were found in soleus muscles of A/J mice, indicative of sustained lipid delivery and oxidation (Table 3; Ref. 44). Expression of PDK-4 mRNA in A/J mice was not affected in response to the HF diet; however, its expression was stimulated in both the soleus and the gastrocnemius muscles of B/6J mice. All together, the results above suggested a strain-specific loss of regulation of glucose/fatty acid partitioning in the soleus muscle of A/J mice, favoring oxidation of fatty acids.

Oxidative metabolism in muscles and AMPK. To analyze a possible induction of oxidative metabolism by HF diet in skeletal muscles of A/J mice, ex vivo measurements were performed. First, the rate of endogenous VO2 in the gastrocnemius and soleus muscles was assessed during a 35-min period (Fig. 7 and Table 4). Feeding mice the HF diet tended to increase the respiratory rate in gastrocnemius muscle of both B/6J and A/J mice, but this effect was not statistically significant. On the contrary, in the soleus muscle of A/J mice, HF diet increased the respiration ~1.8-fold, while no effect was observed in B/6J mice.


Figure 7
View larger version (14K):
[in this window]
[in a new window]

  		Fig. 7. Ex vivo muscle VO2 during 35-min incubation period at 37°C. Muscle samples were obtained from mice adapted to 30°C and fed either LF or HF diet. Fragments of gastrocnemius or whole soleus muscles were incubated in freshly oxygenated (95% O2-5% CO2) Krebs-Ringer bicarbonate buffer (pH 7.4) containing 10 mM glucose, and VO2 was measured using polarography (see also Table 4). Data are means ± SE for a 5-min interval, beginning 10 min after insertion of tissue fragments into measuring chambers. Results were normalized to tissue protein (n = 7–9). *Significant effect of diet (repeated-measures ANOVA: diet vs. time).

 

View this table:
[in this window]
[in a new window]

  		Table 4. VO2 in skeletal muscles

 
Second, oxidation of exogenous palmitate in the muscles was measured (Table 5). In either genotype, no significant effect of HF diet on palmitate oxidation was observed. However, when palmitate oxidation was evaluated in the presence of AICAR, an AMPK activator, an ~70% increase in the rate of oxidation was observed in the soleus muscle of the A/J HF mice. No stimulatory effect of AICAR was observed in the soleus muscle of either the A/J LF mice or the B/6J mice fed both types of diets. In the gastrocnemius muscle, no stimulation by AICAR or diet was detected in mice of either genotype (not shown).


View this table:
[in this window]
[in a new window]

  		Table 5. Palmitate oxidation in soleus muscle

 
To further investigate a possible involvement of AMPK in the strain-specific effect of HF diet, AMPK content and phosphorylation (p-AMPK) were measured in the soleus muscle (Fig. 8). In B/6J mice, the HF diet had no effect on either total AMPK or p-AMPK muscle content. In contrast, in A/J mice the HF diet caused an ~1.7- and ~1.4-fold increase in the content of AMPK and pAMPK, respectively; however, only the effect on total AMPK content reached statistical significance (two-way ANOVA). The stimulatory effect of HF diet on AMPK in soleus muscle of A/J mice was further confirmed by assessing the level of phosphorylation of ACC, a downstream target of AMPK, which showed an ~1.4-fold increase in response to HF diet, whereas no effect on ACC phosphorylation was found in B/6J mice (not shown). These results indicated a genotypic difference in the effect of HF diet on energy expenditure in soleus muscle, where the inducibility of respiration by HF diet in the A/J mice was associated with a relatively high potency of the AMPK regulatory pathway.


Figure 8
View larger version (29K):
[in this window]
[in a new window]

  		Fig. 8. Content and phosphorylation of AMPK in soleus muscle of B/6J and A/J mice. Mice maintained at 30°C and fed either LF (open bars) or HF (filled bars) diet for 2 wk after weaning were used. Total amount of AMPK (AMPK{alpha}1 + -{alpha}2) was assessed in tissue extracts with a mixture of primary antibodies against AMPK{alpha}1 and -{alpha}2, whereas phosphorylated (p-)AMPK was detected using primary antibodies specific for phosphorylated Thr172. Both forms of AMPK were visualized and quantified on the same blot using two different secondary infrared dye-labeled antibodies and the Odyssey IR Imager. A: picture of representative blot with muscle samples (15 µg protein/lane) from A/J mice, liver standards isolated from adult LF diet-fed B/6J mice (10 and 15 µg protein/lane), and prestained Mr standards. B: quantification of total amount of AMPK. C: quantification of p-AMPK. Data are means ± SE (B/6J, n = 7–8; A/J, n = 10–11). *Significant effect of diet.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The principal finding of this study is the stimulation of muscle thermogenesis and lipid oxidation by HF diet in obesity-resistant A/J mice. Our results indicate that an HF diet could induce nonshivering thermogenesis in oxidative muscle, possibly by the leptin-AMPK axis.

We have demonstrated, for the first time, a profound difference between A/J and B/6J mice in their ability to resist acute cold exposure, providing that the animals were first adapted to the temperature close to thermoneutrality and maintained on an LF diet. Nevertheless, a relatively low whole body energy expenditure of the A/J LF mice, which would predispose these animals to obesity, was enhanced in response to the HF diet. This induction was apparently stronger than in B/6J mice, which exhibited higher energy expenditure than A/J mice when fed the LF diet but are prone to HF diet-induced obesity. That the HF diet induced a larger increase in whole body energy expenditure in A/J than in B/6J mice was also in agreement with the tendency of HF diet to increase body temperature in the former mice in this and in the previous study (47).

Without preacclimation to ambient temperatures around 20–22°C, as used in previous studies (8, 17, 48, 52), the capacity of nonshivering thermogenesis in brown adipose tissue was relatively low, but it was similar in both strains of mice, unless it was stimulated by HF diet. Since the ability to activate shivering was also similar in both B/6J and A/J mice fed the LF diet (Fig. 3), the cold sensitivity of the A/J LF mice could be explained, at least in part, by their relatively low VO2 as measured at 30°C during the light phase of the day. Although locomotor activity was not assessed in this study, it could have contributed to the differences in VO2 as well in cold sensitivity (5, 9, 14).

The stronger obesogenic effect of HF diet in B/6J compared with A/J mice has been previously explained by strain-specific differences in the adrenergic control of thermogenesis (8) and sensitivity to leptin (32), with only A/J mice retaining sensitivity of adipose tissue to both stimuli after several weeks of feeding on a HF diet. The present study confirmed the previous results (8, 17, 48, 52), indicating that the differential response to HF diet could be attributed to the relatively high induction of UCP1 in the interscapular brown fat of A/J mice. In addition, our study demonstrated a HF diet-induced increase of VO2 in soleus muscle, which was specific for A/J mice, and suggested activation of nonshivering thermogenesis in this muscle. The association with increased leptin levels, and the selective involvement of the oxidative muscle, strongly suggested the involvement of the leptin-AMPK axis (29, 41). AMPK plays a major role in the regulation of fatty acid oxidation in skeletal muscle while inhibiting ACC by phosphorylation, reducing malonyl-CoA levels, and, thus, enhancing activity of CPT I and β-oxidation of fatty acids (7, 29). Direct stimulation of muscle thermogenesis ex vivo required both AMPK and phosphatidylinositol 3-kinase and could be prevented by pharmacological inhibition of AMPK, and activation of the AMPK axis was required for leptin-induced substrate cycling between de novo lipogenesis and lipid oxidation (41).

The acute stimulation of AMPK by leptin reflects an increased phosphorylation of Thr172 in the enzyme {alpha}-subunit (29). On the other hand, chronic hyperleptinemia in rats resulted in an increased content of both total AMPK and p-AMPK and increased phosphorylation of ACC (43) similarly to the results of our experiments. Our results have documented the HF diet-induced increase of AICAR-dependent fatty acid oxidation in soleus muscle of A/J but not B/6J mice. These results suggest a much higher inducibility of lipid oxidation by AMPK in the soleus muscle of A/J than in B/6J mice. However, without AICAR, no effect on fatty acid oxidation was noticed. Perhaps, oxidation of the exogenously added fatty acids could be limited by the transport of fatty acids from the medium, unless the AMPK and CPT I activities are maximally stimulated by AICAR. In resting soleus muscle, exogenous fatty acid provide only a very small fraction (~2%) of total lipid metabolized while catabolism of intramuscular lipid represents the bulk [~98% (10)]. Oxidation of intramuscular lipid should be better reflected by the ex vivo measurements of endogenous respiration in the muscle. However, the quantitative interpretation of these measurements, even in a relatively small whole soleus muscle, is limited due, for example, to a diffusion barrier for oxygen or a release of electrolytes (particulary potassium), which can alter the respiration. Nevertheless, the combined results of both approaches strongly suggest induction of energy expenditure by HF diet in the soleus muscle of A/J mice involving AMPK-stimulated fatty acid oxidation.

The strongest suppression of SCD-1 expression, accompanied by the highest levels of PDK-4 transcript in soleus muscle of the A/J HF mice, further supports the preferential use of lipids for adaptive thermogenesis in the muscle. Downregulation of SCD-1 results in increased β-oxidation of fatty acids, enhanced thermogenesis, and resistance to obesity (reviewed in Refs. 4, 7, 25), whereas activation of PDK-4 leads to suppression of glucose oxidation (44). Of note, upregulation of PDK-4 was also detected in soleus muscle of cold-acclimated UCP1 knockout mice (50). A relatively high expression of CPT IA, the liver isoform of the enzyme, in the soleus muscle of the A/J HF mice is also in favor of intense β-oxidation of fatty acids, since it is primarily the muscle isoform that is more sensitive to inhibition by malonyl-CoA (28).

That lipid oxidation was important for the cold defense abilities of the A/J HF mice was further documented by indirect calorimetry. Lowering of ambient temperature during the measurements augmented the activation of lipid oxidation by HF diet in A/J but not in B/6J mice (see the PRCF data in Fig. 5, and RER data in Table 2). This strain-specific enhancement of the whole-body lipid oxidation in cold probably reflected the increased capacity for both UCP1-mediated and muscle thermogenesis by HF diet. On the other hand, the HF diet-induced elevation of VO2 during the light phase in A/J mice maintained at 30°C probably resulted from the sustained increase in muscle nonshivering thermogenesis, induced by leptin and independent of acute stimulation of sympathetic activity due to cold or meal (see also Ref. 6).

The role and mechanism of leptin-mediated adaptive thermogenesis have recently been investigated by Ukropec et al. (51) using ob/ob mice with a targeted inactivation of UCP1 gene (Ucp1–/–.Lep–/– mice). These mice could not adapt to temperatures below 12°C unless they were administered either leptin or T3 (51). Thermogenesis was possibly activated due to the induction of Ca2+ cycling associated with an increased expression of sarcoplasmic reticulum Ca2+ ATPase-2 (SERCA2) in oxidative muscle (51). On the contrary, the stimulation of thermogenesis in the oxidative muscle of the A/J HF mice was not associated either with upregulation of SERCA2 (in fact, in both the experiments on Ucp1–/–.Lep–/– mice and in our study, both SERCA2a and SERCA2b mRNA were quantified together) or with changes of the thyroid hormone status (Tables 1 and 3). The qualitative difference in the thermogenic response between Ucp1–/–.Lep–/– and A/J mice may thus reflect a much higher induction of thermogenic mechanisms required to substitute for defective UCP1-mediated thermogenesis in the transgenic mice. Additional thermogenic mechanisms may function in this transgenic model that do not operate in A/J mice. However, in both Ucp1–/–.Lep–/– and A/J mice, oxidative but not glycolytic muscle was involved in the adaptive thermogenesis, supporting further the importance of lipid catabolism and mitochondria for muscle thermogenesis.

Our results do not answer the question whether the induction of muscle thermogenesis by leptin could result from a direct interaction of leptin with muscle or whether it is mediated centrally via the activation of the adrenergic system (29). With respect to a possible extrapolation to human subjects, where the control and significance of adaptive nonshivering thermogenesis is less defined than in rodent species, an increase of sympathetic activity is known to be critical for resistance to obesity (see other references in Ref. 9).

In summary, our results demonstrate that both the UCP1-mediated thermogenesis in brown fat and muscle thermogenesis are associated with resistance to dietary obesity in mice. Only in the model of obesity-resistant A/J, but not in obesity-prone B/6J mice, stimulation of thermogenesis in oxidative skeletal muscle in response to an HF diet was observed. Our results further suggest the involvement of the leptin-AMPK axis, with a preferential use of lipids as metabolic substrates, in the induction of nonshivering thermogenesis in oxidative muscle by HF diet. Thus, at least in mice, the adaptive stimulation of lipid oxidation and muscle thermogenesis in response to increased fat content in the diet might contribute to cold resistance and obesity resistance.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by Research Project AV0Z50110509 and by grants from the Ministry of Education, Youth and Sports of the Czech Republic (1M6837805002), the Czech Science Foundation (305/08/H037,303/07/0781, and the European Commission (Projects EARNEST: FOOD-CT-2005-007036 and EXGENESIS: LSHM-CT-2004-005272).


   ACKNOWLEDGMENTS
 
We thank Grahame D. Hardie (University of Dundee, Dundee, UK) for the generous gift of the sheep antibodies against AMPK{alpha}1 and -{alpha}2 and Leslie P. Kozak (Pennington Biomedical Research Center, Baton Rouge, LA) and Jan Bures (Institute of Physiology, Academy of Sciences of the Czech Republic, Prague) for critical reading of the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. Kopecky, Dept. of Adipose Tissue Biology, Inst. of Physiology, Academy of Sciences of the Czech Republic, Videnska 1083, 142 20 Prague, Czech Republic (e-mail: kopecky{at}biomed.cas.cz)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

{dagger} Deceased. 1 10, 2008. Back


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Alberts P, Johansson BG, McArthur RA. Measurement and characterization of energy expenditure as a tool in the development of drugs for metabolic diseases, such as obesity and diabetes. In: Current Protocols in Pharmacology, edited by Enna SJ, Williams M, Ferkany JW, Kenakin T, Porsolt RD, Sullivan JP. Wiley and Sons, 2005, p. 1–15.
  2. Almind K, Manieri M, Sivitz WI, Cinti S, Kahn CR. Ectopic brown adipose tissue in muscle provides a mechanism for differences in risk of metabolic syndrome in mice. Proc Natl Acad Sci USA 104: 2366–2371, 2007.[Abstract/Free Full Text]
  3. Andersson U, Filipsson K, Abbott CR, Woods A, Smith K, Bloom SR, Carling D, Small CJ. AMP-activated protein kinase plays a role in the control of food intake. J Biol Chem 279: 12005–12008, 2004.[Abstract/Free Full Text]
  4. Biddinger SB, Miyazaki M, Boucher J, Ntambi JM, Kahn CR. Leptin suppresses stearoyl-CoA desaturase 1 by mechanisms independent of insulin and sterol regulatory element-binding protein-1c. Diabetes 55: 2032–2041, 2006.[Abstract/Free Full Text]
  5. Brownlow BS, Petro A, Feinglos MN, Surwit RS. The role of motor activity in diet-induced obesity in C57BL/6J mice. Physiol Behav 60: 37–41, 1996.[CrossRef][Medline]
  6. Cannon B, Nedergaard J. Brown adipose tissue: function and physiological significance. Physiol Rev 84: 277–359, 2004.[Abstract/Free Full Text]
  7. Cohen P, Friedman JM. Leptin and the control of metabolism: role for stearoyl-CoA desaturase-1 (SCD-1). J Nutr 134: 2455S–2463S, 2004.[Abstract/Free Full Text]
  8. Collins S, Daniel KW, Petro AE, Surwit RS. Strain-specific response to beta3-adrenergic receptor agonist treatment of diet-induced obesity in mice. Endocrinology 138: 405–413, 1997.[Abstract/Free Full Text]
  9. DeRuisseau LR, Parsons AD, Overton JM. Adaptive thermogenesis is intact in B6 and A/J mice studied at thermoneutrality. Metabolism 53: 1417–1423, 2004.[CrossRef][Web of Science][Medline]
  10. Dyck DJ, Bonen A. Muscle contraction increases palmitate esterification and oxidation and triacylglycerol oxidation. Am J Physiol Endocrinol Metab 275: E888–E896, 1998.[Abstract/Free Full Text]
  11. Flachs P, Horakova O, Brauner P, Rossmeisl M, Pecina P, Franssen-van Hal NL, Ruzickova J, Sponarova J, Drahota Z, Vlcek C, Keijer J, Houstek J, Kopecky J. Polyunsaturated fatty acids of marine origin upregulate mitochondrial biogenesis and induce beta-oxidation in white fat. Diabetologia 48: 2365–2375, 2005.[CrossRef][Web of Science][Medline]
  12. Flatt JP. Exaggerated claim about adaptive thermogenesis. Int J Obes (Lond) 31: 1626–1628, 2007.[CrossRef][Medline]
  13. Fleury C, Neverova M, Collins S, Raimbault S, Champigny O, Levi-Meyrueis C, Bouillaud F, Seldin MF, Surwit RS, Ricquier D, Warden CH. Uncoupling protein-2: a novel gene linked to obesity and hyperinsulinemia. Nat Genet 15: 269–272, 1997.[CrossRef][Web of Science][Medline]
  14. Gelegen C, Collier DA, Campbell IC, Oppelaar H, Kas MJ. Behavioral, physiological, and molecular differences in response to dietary restriction in three inbred mouse strains. Am J Physiol Endocrinol Metab 291: E574–E581, 2006.[Abstract/Free Full Text]
  15. Gnaiger E, Steinlechner-Maran R, Mendez G, Eberl T, Margreiter R. Control of mitochondrial and cellular respiration by oxygen. J Bioenerg Biomembr 27: 583–596, 1995.[CrossRef][Web of Science][Medline]
  16. Granneman JG, Burnazi M, Zhu Z, Schwamb LA. White adipose tissue contributes to UCP1-independent thermogenesis. Am J Physiol Endocrinol Metab 285: E1230–E1236, 2003.[Abstract/Free Full Text]
  17. Guerra C, Koza RA, Yamashita H, King KW, Kozak LP. Emergence of brown adipocytes in white fat in mice is under genetic control. Effects on body weight and adiposity J Clin Invest 102: 412–420, 1998.[Medline]
  18. Himms-Hagen J, Harper ME. Physiological role of UCP3 may be export of fatty acids from mitochondria when fatty acid oxidation predominates: an hypothesis. Exp Biol Med (Maywood) 226: 78–84, 2001.[Abstract/Free Full Text]
  19. Jansky I. Humoral thermogenesis and its role in maintaining energy balance. Physiol Rev 75: 237–259, 1995.[Free Full Text]
  20. Jéquier E. Regulation of thermogenesis and nutrient metabolism in the human: relevance for obesity. In: Obesity, edited by Björntorp P, Brodoff BN. Philadelphia, PA: Lippincott, 1992, p. 130–135.
  21. Kadenbach B, Huttemann M, Arnold S, Lee I, Bender E. Mitochondrial energy metabolism is regulated via nuclear-coded subunits of cytochrome c oxidase. Free Radic Biol Med 29: 211–221, 2000.[CrossRef][Web of Science][Medline]
  22. Kopecky J, Hodny Z, Rossmeisl M, Syrovy I, Kozak LP. Reduction of dietary obesity in the aP2-Ucp transgenic mice: physiology and adipose tissue distribution. Am J Physiol Endocrinol Metab 270: E768–E775, 1996.[Abstract/Free Full Text]
  23. Kopecky J, Rossmeisl M, Hodny Z, Syrovy I, Horakova M, Kolarova P. Reduction of dietary obesity in the aP2-Ucp transgenic mice: mechanism and adipose tissue morphology. Am J Physiol Endocrinol Metab 270: E776–E786, 1996.[Abstract/Free Full Text]
  24. Liu X, Rossmeisl M, McClaine J, Kozak LP. Paradoxical resistance to diet-induced obesity in UCP1-deficient mice. J Clin Invest 111: 399–407, 2003.[CrossRef][Web of Science][Medline]
  25. Mainieri D, Summermatter S, Seydoux J, Montani JP, Rusconi S, Russell AP, Boss O, Buchala AJ, Dulloo AG. A role for skeletal muscle stearoyl-CoA desaturase 1 in control of thermogenesis. FASEB J 20: 1751–1753, 2006.[Abstract/Free Full Text]
  26. Major GC, Doucet E, Trayhurn P, Astrup A, Tremblay A. Clinical significance of adaptive thermogenesis. Int J Obes (Lond) 31: 204–212, 2007.[CrossRef][Medline]
  27. Matejkova O, Mustard KJ, Sponarova J, Flachs P, Rossmeisl M, Miksik I, Thomason-Hughes M, Hardie DG, Kopecky J. Possible involvement of AMP-activated protein kinase in obesity resistance induced by respiratory uncoupling in white fat. FEBS Lett 569: 245–248, 2004.[CrossRef][Web of Science][Medline]
  28. McGarry JD, Mills SE, Long CS, Foster DW. Observations on the affinity for carnitine, and malonyl-CoA sensitivity, of carnitine palmitoyltransferase I in animal and human tissues. Demonstration of the presence of malonyl-CoA in non-hepatic tissues of the rat. Biochem J 214: 21–28, 1983.
  29. Minokoshi Y, Kim YB, Peroni OD, Fryer LG, Muller C, Carling D, Kahn BB. Leptin stimulates fatty-acid oxidation by activating AMP-activated protein kinase. Nature 415: 339–343, 2002.[CrossRef][Web of Science][Medline]
  30. Moreno M, Lombardi A, de Lange P, Silvestri E, Ragni M, Lanni A, Goglia F. Fasting, lipid metabolism, and triiodothyronine in rat gastrocnemius muscle: interrelated roles of uncoupling protein 3, mitochondrial thioesterase, and coenzyme Q. FASEB J 17: 1112–1114, 2003.[Abstract/Free Full Text]
  31. Pelleymounter MA, Cullen MJ, Baker MB, Hecht R, Winters D, Boone T, Collins F. Effects of the obese gene product on body weight regulation in ob/ob mice. Science 269: 540–543, 1995.[Abstract/Free Full Text]
  32. Prpic V, Watson PM, Frampton IC, Sabol MA, Jezek GE, Gettys TW. Differential mechanisms and development of leptin resistance in A/J versus C57BL/6J mice during diet-induced obesity. Endocrinology 144: 1155–1163, 2003.[Abstract/Free Full Text]
  33. Puigserver P, Spiegelman BM. Peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1 alpha): transcriptional coactivator and metabolic regulator. Endocr Rev 24: 78–90, 2003.[Abstract/Free Full Text]
  34. Riachi M, Himms-Hagen J, Harper ME. Percent relative cumulative frequency analysis in indirect calorimetry: application to studies of transgenic mice. Can J Physiol Pharmacol 82: 1075–1083, 2004.[CrossRef][Web of Science][Medline]
  35. Rosen ED, Spiegelman BM. Adipocytes as regulators of energy balance and glucose homeostasis. Nature 444: 847–853, 2006.[CrossRef][Web of Science][Medline]
  36. Rothwell NJ, Stock MJ. Similarities between cold- and diet- induced thermogenesis in the rat. Can J Physiol Pharmacol 58: 842–848, 1980.[Web of Science]
  37. Ruzickova J, Rossmeisl M, Prazak T, Flachs P, Sponarova J, Vecka M, Tvrzicka E, Bryhn M, Kopecky J. Omega-3 PUFA of marine origin limit diet-induced obesity in mice by reducing cellularity of adipose tissue. Lipids 39: 1177–1185, 2004.[CrossRef][Web of Science][Medline]
  38. Scarpace PJ, Matheny M. Leptin induction of UCP1 gene expression is dependent on sympathetic innervation. Am J Physiol Endocrinol Metab 275: E259–E264, 1998.[Abstract/Free Full Text]
  39. Skarka L, Bardova K, Brauner P, Flachs P, Jarkovska D, Kopecky J, Ostadal B. Expression of mitochondrial uncoupling protein 3 and adenine nucleotide translocase 1 genes in developing rat heart: putative involvement in control of mitochondrial membrane potential. J Mol Cell Cardiol 35: 321–330, 2003.[CrossRef][Web of Science][Medline]
  40. Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goekke NM, Olson BJ, Klenk BC. Measurement of protein using bicinchoninic acid. Anal Biochem 150: 76–85, 1985.[CrossRef][Web of Science][Medline]
  41. Solinas G, Summermatter S, Mainieri D, Gubler M, Pirola L, Wymann MP, Rusconi S, Montani JP, Seydoux J, Dulloo AG. The direct effect of leptin on skeletal muscle thermogenesis is mediated by substrate cycling between de novo lipogenesis and lipid oxidation. FEBS Lett 577: 539–544, 2004.[CrossRef][Web of Science][Medline]
  42. Stefl B, Janovska A, Hodny Z, Rossmeisl M, Horakova M, Syrovy I, Bemova J, Bendlova B, Kopecky J. Brown fat is essential for cold-induced thermogenesis but not for obesity resistance in aP2-Ucp mice. Am J Physiol Endocrinol Metab 274: E527–E533, 1998.[Abstract/Free Full Text]
  43. Steinberg GR, Rush JW, Dyck DJ. AMPK expression and phosphorylation are increased in rodent muscle after chronic leptin treatment. Am J Physiol Endocrinol Metab 284: E648–E654, 2003.[Abstract/Free Full Text]
  44. Sugden MC, Holness MJ. Recent advances in mechanisms regulating glucose oxidation at the level of the pyruvate dehydrogenase complex by PDKs. Am J Physiol Endocrinol Metab 284: E855–E862, 2003.[Abstract/Free Full Text]
  45. Surwit RS, Feinglos MN, Rodin J, Sutherland A, Petro AE, Opara EC, Kuhn CM, Rebuffe-Scrive M. Differential effects of fat and sucrose on the development of obesity and diabetes in C57BL/6J and A/J mice. Metabolism 44: 645–651, 1995.[CrossRef][Web of Science][Medline]
  46. Surwit RS, Kuhn CM, Cochrane C, McCubbin JA, Feinglos MN. Diet-induced type II diabetes in C57BL/6J mice. Diabetes 37: 1163–1167, 1988.[Abstract]
  47. Surwit RS, Petro AE, Parekh P, Collins S. Low plasma leptin in response to dietary fat in diabetes- and obesity-prone mice. Diabetes 46: 1516–1520, 1997.[Abstract]
  48. Surwit RS, Wang S, Petro AE, Sanchis D, Raimbault S, Ricquier D, Collins S. Diet-induced changes in uncoupling proteins in obesity-prone and obesity-resistant strains of mice. Proc Natl Acad Sci USA 95: 4061–4065, 1998.[Abstract/Free Full Text]
  49. Tremblay A, Pelletier C, Doucet E, Imbeault P. Thermogenesis and weight loss in obese individuals: a primary association with organochlorine pollution. Int J Obes Relat Metab Disord 28: 936–939, 2004.[CrossRef][Web of Science][Medline]
  50. Ukropec J, Anunciado RP, Ravussin Y, Hulver MW, Kozak LP. UCP1-independent thermogenesis in white adipose tissue of cold-acclimated Ucp1–/– mice. J Biol Chem 281: 31894–31908, 2006.[Abstract/Free Full Text]
  51. Ukropec J, Anunciado RV, Ravussin Y, Kozak LP. Leptin is required for uncoupling protein-1-independent thermogenesis during cold stress. Endocrinology 147: 2468–2480, 2006.[Abstract/Free Full Text]
  52. Watson PM, Commins SP, Beiler RJ, Hatcher HC, Gettys TW. Differential regulation of leptin expression and function in A/J vs. C57BL/6J mice during diet-induced obesity. Am J Physiol Endocrinol Metab 279: E356–E365, 2000.[Abstract/Free Full Text]
  53. West DB, Boozer CN, Moody DL, Atkinson RL. Dietary obesity in nine inbred mouse strains. Am J Physiol Regul Integr Comp Physiol 262: R1025–R1032, 1992.[Abstract/Free Full Text]
  54. Woods A, Salt I, Scott J, Hardie DG, Carling D. The alpha1 and alpha2 isoforms of the AMP-activated protein kinase have similar activities in rat liver but exhibit differences in substrate specificity in vitro. FEBS Lett 397: 347–351, 1996.[CrossRef][Web of Science][Medline]
  55. Zurlo F, Larson K, Bogardus C, Ravussin E. Skeletal muscle metabolism is a major determinant of resting energy expenditure. J Clin Invest 86: 1423–1427, 1990.[Web of Science][Medline]



This article has been cited by other articles:


Home page
 Physiol. GenomicsHome page
C. Bourdon, S. Hojna, M. Jordan, J. Berube, V. Kren, M. Pravenec, P. Liu, S. Arab, and Z. Pausova
Genetic locus on rat chromosome 20 regulates diet-induced adipocyte hypertrophy: a microarray gene expression study
Physiol Genomics, June 10, 2009; 38(1): 63 - 72.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Figures and Tables
Right arrow All Versions of this Article:
295/2/E356    most recent
90256.2008v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kus, V.
Right arrow Articles by Kopecky, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kus, V.
Right arrow Articles by Kopecky, J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2008 by the American Physiological Society.