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(The FASEB Journal. 1998;12:1739-1745.)
© 1998 FASEB

RESEARCH COMMUNICATION

Overexpression of muscle uncoupling protein 2 content in human obesity associates with reduced skeletal muscle lipid utilization

Jean-Aimé Simoneaua,1, David E. Kelleyb, Maria Neverovac and Craig H. Wardenc

a Division of Kinesiology, Department of Social and Preventive Medicine, Laval University, Ste-Foy, Québec, Canada G1K 7P4
b Department of Endocrinology, University of Pittsburgh, Pennsylvania 15261, USA
c Rowe Program in Genetics and Departments of Pediatrics, Biological Chemistry, and Medicine, UC Davis, California 95616, USA


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Uncoupling proteins (UCP) may influence thermogenesis. Since skeletal muscle plays an important role in energy homeostasis and substrate oxidation, this study was undertaken to test the hypotheses that skeletal muscle UCP2 content is altered in obesity and could be linked to basal energy expenditure, insulin sensitivity, or substrate oxidation within skeletal muscle under postabsorptive (fasting) conditions. To examine these possibilities, limb basal energy expenditure and respiratory quotient (bRQ) were measured in 18 obese nondiabetic (Ob) and lean individuals (L). Total body fat (%) ranged from 11% to 46%. In addition, insulin-stimulated rates of glucose disposal (Rd) were measured under euglycemic hyperinsulinemic conditions. Biopsy of vastus lateralis muscle was used to measure cytochrome c oxidase (COX) enzyme activity and UCP2 content. Whereas low muscle COX activity was found in the Ob compared to L (6.9±1.6 vs. 9.6±1.2 U/g; P<0.001), skeletal muscle UCP2 content in Ob was significantly higher than in L (48±9 vs. 33±12 arbitrary units/g; P<0.05). Moreover, UCP2 content was positively correlated with percent of total body fat (r=0.57; P<0.05) and bRQ (r=0.59; P<0.01), but not with visceral fat (r=0.17; P=0.49), basal energy expenditure (r=0.07; P=0.79) or Rd (r=-0.23; P=0.34). In summary, these results indicate that if development of obesity in humans is mediated by defective expression of UCP2 within skeletal muscle, then this effect is not observed in people with established obesity. The present study also suggests that skeletal muscle UCP2 content is not related to basal energy expenditure or insulin sensitivity in humans. However, the increased content of UCP2 within skeletal muscle in obesity appears to coincide with a reduced postabsorptive lipid utilization by muscle.—Simoneau, J.-A., Kelley, D. E., Neverova, M., Warden, C. H. Overexpression of muscle uncoupling protein 2 content in human obesity associates with reduced skeletal muscle lipid utilization. FASEB J. 12, 1739–1745 (1998)


Key Words: UCP • body fat • substrate utilization • insulin sensitivity • energy expenditure


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
HEAT PRODUCTION in brown adipose tissue is recognized to contribute to whole body energy expenditure in small rodents, and previous studies have shown that a defect in this metabolic process contributes to the development of obesity in these species (1–3). It is unclear whether thermogenesis in brown adipose tissue is relevant to human obesity, since this tissue accounts for about 1% of adult body weight and is localized in small deposits in the neck region and around the heart and kidney in the human adult population exhibiting nonpathologic conditions (4). Estimates of the contribution of brown fat to metabolic rate in human were also low (5). In light of recent investigations, attention is once again focused on the role of mitochondrial uncoupling proteins in human obesity because at least two homologues (UCP2 and UCP3)2 of the protein exclusively expressed in brown adipose tissue were found in different human tissues, including skeletal muscle (6–13). Thus, how these new homologues of UCP can contribute to obesity in humans needs to be demonstrated since skeletal muscle plays an important role in energy homeostasis and substrate oxidation.

There is particular interest in the role of UCP2 since linkages were recently found between genetic markers in the vicinity of the UCP2 gene and obesity phenotypes (14, 15). Such linkages were neither found, however, in families with a propensity to type 2 diabetes (16), in normoglycemic and non-insulin-dependent diabetes mellitus (NIDDM) morbidly obese patients (17) nor in subjects with juvenile and maturity onset forms of obesity and insulin resistance (18). The mRNA expression of muscle UCP2 can also be modulated by environmental (19) or hormonal (20, 21) alterations. Recently, Millet et al. (19) have shown that UCP2 mRNA levels in human white adipose tissue were overexpressed in individuals with a high body mass index (BMI), although they also reported that UCP2 mRNA levels in skeletal muscle were related to neither BMI nor resting metabolic rate.

Considering that the studies referred to above did not examine the protein content of UCP2, but suggest that variation in the content of that protein within skeletal muscle may play a role in obesity, the current study was undertaken to test the hypotheses that the protein content of UCP2 in skeletal muscle is altered in human obesity and could contribute to the interindividual variation in basal energy expenditure, substrate utilization, and insulin sensitivity. Vastus lateralis muscle was obtained by biopsy to characterize the protein content of UCP2 and cytochrome c oxidase activity, an inner mitochondrial membrane marker of oxidative phosphorylation. To examine the role that UCP2 content within skeletal muscle may play in skeletal muscle physiology, basal metabolic rate and substrate utilization were measured using the leg balance method in conjunction with limb indirect calorimetry. Finally, to verify whether UCP2 content is linked to the expression of insulin resistance, glucose utilization during insulin-stimulated conditions was measured. The findings support the hypothesis that UCP2 within skeletal muscle is altered in obesity, but rather than being low, its content is increased. These results also demonstrate that, in skeletal muscle, there is an inverse relationship between fasting rates of lipid oxidation and the protein content of UCP2, though neither basal energy expenditure nor insulin sensitivity are associated with UCP2 content in muscle.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects
Subjects were recruited by advertisement. Potential volunteers had a medical examination prior to participation and those with medical illness were excluded. Obese nondiabetic and lean subjects also had a normal response after an oral glucose tolerance test. The clinical characteristics of lean (5 M/3 F) and obese nondiabetic (5 M/5 F) were as follows: age: 36 ± 3 vs. 32 ± 6 years, weight: 70 ± 16 vs. 96 ± 19 kg, fat-free mass: 53 ± 16 vs. 55 ± 13 kg, percentage body fat: 19 ± 5 vs. 35 ± 7%, fasting blood glucose: 4.6 ± 0.4 vs. 4.7 ± 0.5 mM, and fasting insulin: 54 ± 30 vs. 95 ± 57 pmol/l. Whole body fat mass (FM) and fat-free mass (FFM) were assessed by dual energy X-ray absorptiometry (Lunar model DPX-L, Madison, Wis.), using transverse scans to measure fat and lean tissue mass. Computed tomography (9800 scanner; General Electric, Milwaukee, Wis.) was used to measure intraabdominal adipose tissue using a 10 mm cross-sectional scan through the abdomen centered on the disc space between the fourth and fifth lumbar vertebrae (22). An additional inclusion criteria was that volunteers were sedentary, which was determined by interview at the time of screening. The protocol was approved by the University of Pittsburgh Institutional Review Board and subjects gave written, informed consent prior to their participation.

Postabsorptive measurements of systemic and leg substrate utilization
Subjects were admitted to the University of Pittsburgh General Clinical Research Center on the day before a study. Subjects were instructed to ingest a balanced diet containing at least 200 g of carbohydrate for 3 days preceding a study. On the evening of admission, subjects ingested a standard dinner (10 kcal/kg, 50% carbohydrate, 30% fat, and 20% protein) at 6 PM and then fasted overnight. To measure arteriovenous differences across the leg, catheters were placed in a radial artery and a femoral vein. After an interval of 60 min following cannulations, measurements of postabsorptive metabolism were conducted for 45 min. At 5 min intervals, arterial and femoral venous samples were obtained for measurements of blood O2 and plasma CO2 content to measure leg gas exchange for limb indirect calorimetry, as described previously (23). Leg blood flow was measured in triplicate, at 15 min intervals, using venous occlusion strain gauge plethysmography (Hokanson, Bellevue, Wash.). Systemic indirect calorimetry was performed during the baseline period using an open canopy system (Delta Tract, Anaheim, Calif.). After completion of postabsorptive measurements, a percutaneous needle biopsy of the vastus lateralis muscle was performed, as described previously (24), and muscle was immediately frozen in liquid nitrogen for later analysis.

Measurements of insulin sensitivity
Fifteen minutes after completion of the muscle biopsy, insulin sensitivity was determined using the euglycemic insulin infusion method (25, 26). To measure rates of glucose utilization, a primed (0.20 µCi) continuous (0.20 µCi/min) infusion of 3-[3H]-glucose (New England Nuclear, Boston, Mass.) was started in an antecubital vein approximately 90 min prior to insulin infusion, so that systemic rates of glucose utilization could be determined during the final 30 min (blood being sampled at 10 min intervals) of a 3 h insulin infusion (40 mU/m2-min).

Blood analyses and calculations
Analyses of arterial and vein blood gas and of substrates and hormones, along with calculations, have previously been given in detail in other similar studies (26).

UCP2 content and cytochrome c oxidase enzyme activity in skeletal muscle
Muscle samples (about 15 mg) were homogenized in a glass-glass Duall homogenizer with 40 vol. of ice-cold extracting medium (0.1 M Na-K-phosphate, 2 mM EDTA, pH=7.2). The suspension was magnetically stirred on ice for 15 min and sonicated five times for 5 s at 20 watts, with pauses of 85 s between pulses. The resulting homogenate was used to determine UCP2 content and cytochrome c oxidase (COX) activity level (Vmax). For determination of UCP2 (done in duplicate for each subject), a total volume of 10 µl of Tris buffer (11 mM), ethylenediaminetetraacetic acid (1.1 mM), sodium dodecyl sulfate (SDS; 3.3%), glycerol (11%), and dithiothreitol (40 mM), containing 10 µg of total proteins (BioRad protein assay), was deposited after being heated during 2 min at 95°C, in gel slot (Mini-PROTEAN II electrophoresis cell, BioRad, Mississauga, Canada). A 6% polyacrylamide stacking gel containing Tris buffer (124 mM, pH 6.8), SDS (0.1%), N, N, N', N'-tetra-methyl-ethylenediamine (TEMED; 0.1%) and ammonium persulfate (APS; 0.1%), and a 12% polyacrylamide separating gel containing Tris buffer (0.38 M, pH 8.8), SDS (0.01%), TEMED (0.1%), and APS (0.05%) were used to discern the molecular size of the proteins. The migration of the gel lasted 90 min and was performed at 100 volts in a Tris (25 mM), glycine (192 mM), and SDS (0.1%) electrophoresis buffer (pH 8.0). Separated proteins were electrically transferred (Mini Trans-Blot electrophoretic transfer cell, BioRad, Mississauga, Canada) in a Tris (25 mM), glycine (192 mM), SDS (0.005%), and methanol (20%) buffer (pH 8.0) during 120 min at 100 volts to a polyvinylidene fluoride membrane and immunodecorated with an affinity purified antibody (dilution of 1:6,700) directed against the 15 amino acids at the carboxyl terminus of human UCP2. This region was selected because the last 10 amino acids at the carboxyl-terminal of UCP represent an additional hydrophilic segment not found in ADP/ATP carrier (27). Moreover, almost half (6 out of 15) of the predicted amino acid sequences of human UCP2 and UCP3L are different, whereas UCP1 is even more divergent. The carboxyl-terminal antibody would only recognize human, mouse, and rat UCP2 based on a BLAST search of the nonredundant GeneBank database (http://www.ncbi.nlm.nih.gov). The antibody-antigen complex was visualized with the use of a chemiluminescence-coupled goat-anti-rabbit IgG antibody (dilution of 1:10,000) according to the manufacturer specifications (Western-Star protein detection kit, Tropix, Bedford, Mass.). The reaction product of each blot (as exemplified in Fig. 1) was scanned (Scan Jet 4C, Hewlett Packard, Palo Alto, Calif.) and each band was analyzed twice with the use of the NIH Image analysis software (available on the Internet by anonymous FTP at zippy.nimh.nih.gov). To test for the specificity of the antibody, an immunological analysis was performed in the presence of an excess of the 15 amino acid peptide (50 µg) used to raise the antibody.



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  		Figure 1. Western blot of UCP2 from skeletal muscle of two subjects (one obese; first two left lanes, and one lean; last two right lanes). A Coomassie blue-stained gel containing known molecular mass markers (carbonic anhydrase at 31 kDa and trypsin inhibitor at 21.5 kDa) is also shown.

For an unknown reason, the intensities of at least seven other bands (ranging from about 25 to 75 kDa) were substantially increased (in some cases by as much as 400%) under these experimental conditions whereas the intensity of the 32 kDa band was substantially reduced. It seems that the specificity of the antibody for UCP2 is lost when the peptide is present but increased for other proteins that may have similar and highly conserved amino acid sequences and are most likely close to the last 15 carboxyl terminus amino acids. Standard amounts of human latissimus dorsi muscle (10 µg of proteins deposited in duplicate on each gel) served as internal control on each blot. UCP2 content was expressed in arbitrary units per gram of wet weight tissue (AU/g). COX activity was assayed spectrophotometrically according to a method previously used (28) and its activity was expressed in units of micromoles of substrate per minute, per gram of wet weight tissue (U/g).

Statistics
Data are presented as mean ±SD. Analysis of variance was used to examine for significant differences across groups (lean and obese); Pearson correlations were performed to verify the relationships between muscle UCP2 content, postabsorptive leg indirect calorimetry, and insulin sensitivity measurements using a statistical software (SigmaStat, Jandel Scientific, San Rafael, Calif.).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Range of skeletal muscle UCP2 content and COX activity
Among all subjects, there was a fourfold difference between the lowest (16 AU/g) and highest (64 AU/g) skeletal muscle UCP2 content. The extent of variation between subjects in skeletal muscle UCP2 content was more important than that of COX. There was a twofold difference between the lowest and highest COX activities (from 5 to 11 U/g), and COX activity was significantly (P<0.001) lower in obese (6.9±1.6 U/g) compared to lean (9.6±1.2 U/g) individuals ( Fig. 2) . Contrary to COX, skeletal muscle UCP2 content was significantly higher in obese (48±9 AU/g) compared to lean (33±12 AU/g) individuals ( Fig. 2). To verify whether there was a disproportional expression in UCP2 content and COX enzyme, the ratio of UCP2 to COX activity was determined for each subject. This ratio revealed that the expression of UCP2 compared to COX was twofold higher in obese than in lean individuals (7.1±1.6 vs. 3.6±1.6; P<0.01).



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  		Figure 2. Muscle UCP2 content (AU/g) and cytochrome c oxidase (COX) activity in lean and obese individuals. *Significant (P<0.05) differences between the two groups.

Relation of skeletal muscle UCP2 content to obesity, limb indirect calorimetry, and insulin sensitivity
As shown in Fig. 3, the protein content of skeletal muscle UCP2 was positively and significantly related to percentage of total body fat (r=0.57, P<0.05). However, UCP2 was not significantly correlated with centralized body fat distribution (r=0.17 vs. visceral fat; P=0.49). During postabsorptive conditions, obese individuals did not differ in basal rates of energy expenditure (0.71±0.39 vs. 0.72±0.32 cal/min x 100 ml of leg tissue; P=0.94), but did have higher though nonsignificant basal respiratory quotient (RQ) across the leg (0.92±0.09 vs. 0.85±0.09; P=0.09) compared to lean subjects. As shown in Fig. 4, basal energy expenditure across the leg was not correlated with the content of UCP2 (r=0.07; P=0.79). However, UCP2 content was positively correlated with basal RQ across leg tissue (r=0.59; P<0.01). Based on these RQ values, the relationship observed revealed that the higher the content of muscle UCP2, the lower was the postabsorptive oxidation of lipid by leg tissue. During the 3 h insulin infusion at 40 mU/m2-min, although there were large interindividual differences (from 3.1 to 14.2 µmol/minxkg FFM) and a significant group difference for systemic rates of glucose disposal (Rd: 6.1±2.1 vs. 9.4±3.0 µmol/min x kg FFM; P<0.05), no significant relationship was found between insulin sensitivity (Rd) and UCP2 content within skeletal muscle (r=-0.23; P=0.34).



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  		Figure 3. Relationships between muscle UCP2 content (AU/g) and the percentage of total body fat, and visceral fat.



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  		Figure 4. Relationships between muscle UCP2 content (AU/g) and postabsorptive basal energy expenditure (cal/minx100 ml leg tissue), and basal respiratory quotient across the leg.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Recent studies have suggested that novel members of uncoupling proteins may have weight regulatory and thermogenic properties similar to the well-described UCP1 protein of brown adipose tissue (7–10). Because of these roles, the current study was undertaken to test the hypothesis that skeletal muscle UCP2 content relates to body fat content in humans and can contribute to the interindividual variation in basal energy expenditure, substrate utilization and insulin sensitivity of skeletal muscle. It was recently proposed that identifying obese individuals with decreased expression in UCP2 would help to define whether this protein plays an important role in determining whole body energy expenditure (29). To our knowledge, this is the first study to demonstrate that the protein level of UCP2 in skeletal muscle is up-regulated in human obesity and is positively related to the percentage of body fat. As determined by immunoblots, the amount of UCP2 presents in skeletal muscle of obese subjects is estimated to be about 1.5-fold higher than the level normally detected in skeletal muscle of lean subjects. Moreover, even though UCP2 content overexpression within skeletal muscle coincides with an increased amount of total body fat, the content of that protein was not related to visceral fat content nor to insulin sensitivity, two phenotypes that have been shown to be highly correlated (26).

The absence of significant relationship between insulin sensitivity and UCP2 levels may not be surprising on the basis of recent findings that have shown that neither acute effect of insulin per se (19), a short-term high-fat diet (30), nor diet-induced hyperglycemia and hyperinsulinemia (31) were able to significantly alter the gene expression of UCP2 within skeletal muscle. Moreover, although there was evidence to suggest a linkage between genetic markers located in the vicinity of the uncoupling protein 2 gene and obesity phenotypes (14, 15), such linkages were not found in familial type 2 diabetes (16), in normoglycemic and NIDDM morbidly obese patients (17), or in subjects with juvenile and maturity onset forms of obesity and insulin resistance (18).

Based on experiments undertaken in small rodents, and more recently in humans, other studies, although not all (13), suggest that up-regulation of UCP2 expression in tissues other than skeletal muscle is also found in obesity. Gimeno et al. (9) have reported that in ob/ob and db/db mice, white adipose tissue UCP2 mRNA was up-regulated by approximately fivefold. Fleury et al. (7) also reported that after 18 wk of a high-fat diet, levels of UCP2 mRNA were dramatically increased in epididymal white adipose tissue of obesity-resistant A/J and obesity-prone B6 strains, and that levels of UCP2 were greater in the diet-induced obese B6 mice relative to the leaner A/J strain. These findings were recently confirmed in obesity-prone and obesity-resistant strains of mice fed a high-fat diet for 2 wk (30) and Sprague Dawley rats fed high-fat diet for 4 wk (31). These experiments suggest that an excess of body fat, and more likely its related metabolic perturbations, appears to influence UCP2 expression. Because increased expression of skeletal muscle UCP2 might be expected to decrease rather than to increase body weight (at least from the perspective of postulated thermogenic effects of the uncoupling proteins), the results of the present study indicate that human obesity is unlikely to be maintained by defective expression of skeletal muscle UCP2. However, because UCP2 content in skeletal muscle does differ in obese compared to lean individuals, there may indeed be a connection with the pathogenesis of obesity, but in a manner different than expected. These results do not rule out that UCP2 levels in skeletal muscle contribute to development of obesity. Longitudinal studies of obesity development will be needed to settle this question.

The increase in skeletal muscle UCP2 content in obesity is concomitant with a reduced COX activity. The metabolic implication of an increased UCP2/COX ratio in obesity is that the potential exists for uncoupling between oxidative phosphorylation and respiration, a condition that has been recognized, at least in brown adipose tissue (1), to favor an excess of heat production under stimulating conditions. Although it makes sense to raise this possibility, the results of the present study demonstrate that there is a dissociation between basal energy expenditure and the content of UCP2 in human skeletal muscle. An elegant study recently published (32) led to the conclusion that contrary to UCP-1, UCP-2 has no H+ transport since it does not have the histidine pair H145 and H147, thus reducing the possibility that it plays a major role in the thermogenic process of skeletal muscle. These findings fit nicely with previous investigations that have shown that even though whole body resting metabolic rate, estimated by indirect calorimetry, significantly decreased in lean and obese subjects after 5 days of hypocaloric diet, skeletal muscle UCP2 mRNA expression was found to increase by about twofold (19). These authors also mentioned that there was no relationship between whole body resting metabolic rate and UCP2 mRNA levels in their lean and obese subjects.

It is our hypothesis that the function of UCP2 may not be restricted to thermoregulation. When fatty acids are exposed to mitochondria, they are efficiently taken up by these organelles. The mechanism of this action is most likely due to the transbilayer flip flop movement of undissociated fatty acid from the external leaflet of the mitochondrial membrane to the internal one (33). Because of the dissociation of the fatty acid polar carboxylic ends, absorbed fatty acids are transformed in their anionic form and become impermeable to the membrane bilayer. One hypothesized role of uncoupling proteins, including UCP2, is that these proteins could act as a mitochondrial transmembrane transporter for fatty acid anions (34, 35). A recent structure–activity study of fatty acid interaction with mitochondrial uncoupling protein has supported the existence of a fatty acid cycling mechanism (36). An impairment in the oxidation but not in the uptake of free fatty acids by muscle has been found in obesity during fasting conditions (37), and the present study revealed the presence of an inverse relationship between the content of UCP2 and postabsorptive oxidation of lipid by muscle. The consequence of this metabolic perturbation in obesity could accentuate the availability of free fatty acids within the cytosolic compartment of the muscle cell and thus would favor the transbilayer flip flop movement of undissociated fatty acids (36). An increased content of mitochondrial UCP2 could be seen as a compensatory mechanism that could favor the outwardly translocation of fatty acid anions. In addition to this potential mechanism, fatty acids have been shown, at least in adipose cells, to stimulate UCP2 gene expression (38), although the mechanism is not yet clearly understood. Short-term caloric restriction that increases lipolysis of adipose tissue and levels of circulating fatty acids (39) also causes an increased UCP2 mRNA expression in both white adipose and skeletal muscle in humans (19). This hypothesized mechanism fits nicely with the inverse relationship observed in the present study between postabsorptive basal lipid oxidation by muscle (estimated from basal leg RQ) and the protein content of UCP2 as well as with the recent study showing that the tissue-dependent differential mRNA expression of UCP homologues in rat during food deprivation and refeeding are more consistent with a role for UCP2 in the regulation of lipid as substrate rather than mediators of regulatory thermogenesis (40). It is clear that further work is warranted to elucidate the exact physiological and biochemical functions of skeletal muscle UCP2 in human obesity.

In conclusion, the results of the present study indicate that the protein content of UCP2 in skeletal muscle is about 1.5-fold higher in obese than in lean individuals and that UCP2 is positively correlated with the percentage of body fat in humans. These findings further suggest that although skeletal muscle UCP2 content is not related to basal rates of energy expenditure or to insulin sensitivity in humans, increased content of UCP2 within skeletal muscle in obesity coincides with reduced postabsorptive rates of lipid oxidation by muscle.


   ACKNOWLEDGMENTS
 
The cooperation of our research volunteers is gratefully acknowledged, along with the patient care rendered by the nursing, dietary, and technician staff of the General Clinical Research Center. The authors also wish to acknowledge the contribution of Yves Gélinas for the laboratory work at Laval University. This work was supported by NIH grants DK49200 and DK52581.


   FOOTNOTES
 
1 Correspondence: Jean-Aime.Simoneau@kin.msp. ulaval.ca

2 Abbreviations: AU/g, arbitrary units per grams of wet weight tissue; BMI, body mass index; COX, cytochrome c oxidase; FFM, fat-free mass; FM, fat mass; NIDDM, non-insulin-dependent diabetes mellitus; Rd, rates of glucose disposal; RQ, respiratory quotient; SDS, sodium dodecyl sulfate; TEMED, N, N, N', N'-tetra-methyl-ethylenediamine; UCP, uncoupling proteins; U/g, units of micromoles of subtrate per minute per gram of wet weight tissue.

Received for publication May 29, 1998. Revision received July 10, 1998.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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