ABSTRACT. Uncoupling protein-2 (UCP2) is a mitochondrial protein expressed in a wide range of human tissues. By uncoupling respiration from ATP synthesis ...
0021-972X/98/$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1998 by The Endocrine Society
Vol. 83, No. 7 Printed in U.S.A.
Uncoupling Protein-2 Messenger Ribonucleic Acid Expression During Very-Low-Calorie Diet in Obese Premenopausal Women PIERRE BARBE, LAURENCE MILLET, DOMINIQUE LARROUY, JEAN GALITZKY, MICHEL BERLAN, JEAN-PIERRE LOUVET, AND DOMINIQUE LANGIN INSERM Unit 317 (P.B., L.M., D.L., J.G., M.B., D.L.), Louis Bugnard Institute, Rangueil Hospital, Paul Sabatier University; and Department of Endocrinology and Nutrition (P.B., J.P.L.), Rangueil Hospital, Toulouse, France ABSTRACT Uncoupling protein-2 (UCP2) is a mitochondrial protein expressed in a wide range of human tissues. By uncoupling respiration from ATP synthesis, UCP2 might be involved in the control of energy expenditure. We have investigated UCP2 gene expression in human adipose tissue. In eight subjects, we found a positive correlation (r 5 0.91, P , 0.002) between subcutaneous and visceral fat depots UCP2 messenger RNA (mRNA) levels, suggesting that UCP2 mRNA level in subcutaneous adipose tissue is a good index of UCP2 gene expression in whole body adipose tissues. The effect of a 25-day very-low-calorie diet
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BESITY results from an imbalance between energy intake and energy expenditure. In humans, the resting metabolic rate (RMR), i.e. the obligatory energy expenditure required to maintain physiological tissue function in the resting state is the largest component of daily energy expenditure. A low RMR is a risk factor for body weight gain (1). Comprehension of the molecular mechanisms involved in RMR is essential to understand the regulation of energy expenditure. A substantial part of the RMR results from a leaking of protons across the mitochondrial inner membrane, which results in energy dissipation because of uncoupling of oxygen consumption to ATP synthesis (2, 3). Two recently characterized mitochondrial uncoupling proteins (UCP), designated UCP2 and UCP3, are candidates to explain the proton leak (4 –7). UCP2 and UCP3 expressions in yeast cause a decrease in mitochondrial membrane potential associated with uncoupling of respiration (4, 5, 8). Human UCP2 and UCP3 map to the same region (q13) of chromosome 11 and are apparently organized as a gene cluster (4, 8, 9). This region is co-incident with several quantitative trait loci for obesity (4), and strong evidence of linkage was found between three markers encompassing the UCP2 locus and RMR adjusted for lean body mass (LBM) (10). The tissue distributions of human UCP2 and UCP3 messenger RNAs (mRNAs) are markedly different. UCP2 mRNA is present in
Received March 2, 1998. Revision received March 31, 1998. Accepted April 8, 1998. Address all correspondence and requests for reprints to: Dominique Langin, INSERM U317, Institut Louis Bugnard, Baˆtiment L3, CHU Rangueil, 31403 Toulouse Cedex 4, France. E-mail: langin@rangueil. inserm.fr.
on UCP2 mRNA level and resting metabolic rate was investigated in eight obese premenopausal women. There was no difference in UCP2 mRNA levels before and during the diet. After 25 days of hypocaloric diet, a positive correlation was found between adipose tissue UCP2 mRNA level and resting metabolic rate adjusted for lean body mass (r 5 0.82, P , 0.01). These results show that very-low-calorie diet, unlike short-term fasting, is not associated with an induction in UCP2 mRNA expression, and that adipose tissue UCP2 mRNA levels may be related to variations in resting energy expenditure in humans. (J Clin Endocrinol Metab 83: 2450 –2453, 1998)
many tissues including white adipose tissue, whereas UCP3 mRNA was only detected in skeletal muscle. The factors controlling UCP2 gene expression in humans are currently unknown. We recently reported an unequivocal increase of UCP2 mRNA levels during short-term fasting (11). In the present study, UCP2 mRNA levels were measured in adipose tissue using a sensitive RT-competitive PCR assay. We report here that UCP2 mRNA levels in subcutaneous and visceral adipose tissues are strongly correlated. In obese women following a 25-day very-low-calorie diet (VLCD), no change in subcutaneous adipose tissue UCP2 mRNA expression was observed despite changes in body composition and metabolic parameters. During VLCD, a positive correlation was found between UCP2 mRNA levels and RMR adjusted for LBM. Material and Methods Subjects The patients following VLCD were eight Causasian premenopausal women (mean age 6 sd, 39 6 8 yr; range, 20 – 48 yr). All subjects were obese [body mass index (BMI) range, 30 –37 kg/m2] and had maintained stable body weight for at least 1 month before the beginning of the protocol. All subjects had normal oral glucose tolerance tests (World Health Organization criteria) and plasma lipid profiles. They were not taking drugs except oral contraceptives for one patient. Omental and subcutaneous abdominal adipose tissues were obtained from five female and three male Caucasian subjects (age, 60 6 16 yr; BMI, 29 6 6 kg/m2) undergoing elective open surgery because of eventration or umbilical hernia. All subjects had given written consent, and the protocols were approved by the ethics committee of the Toulouse University Hospitals.
Study procedure The obese women received a 2.5 kJ/day liquid formula diet (Milical, Revel, France) for 25 days. The formula included 45 g protein, 54 g
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UCP-2 mRNA EXPRESSION DURING VLCD carbohydrate, and 24 g fat and the recommended daily allowance of vitamins and minerals. The subjects were outpatients throughout the study. The same investigations were performed before and the 25th day of VLCD. After a 12-h overnight fast, a catheter was inserted at 0800 h into an antecubital vein for blood sampling and kept patent with isotonic saline solution. After a 45-min resting period in supine position, oxygen consumption (VO2) and carbon dioxide production (VCO2) were monitored over 30 min using an open-circuit ventilated-canopy system (Deltatrac II monitor, Datex Instrumentarium Corp., Helsinki, Finland) calibrated with a reference gas. RMR was derived from VO2 and VCO2 using indirect calorimetry (12). Three 10-min interval blood samples were then drawn for determinations of hormonal and metabolic parameters. Following intradermal anesthesia with 50 mL 1% lidocaine (Roger-Bellon, Neuilly-sur-Seine, France), a biopsy of abdominal subcutaneous adipose tissue was performed with a 2-mm-diameter needle. Adipose tissue (200 –300 mg) was drawn by successive suctions into a syringe containing 2 mL saline solution. Samples were frozen immediately in liquid nitrogen and store at 280 C. Body composition was assessed at the end of the session by dual-energy X-ray absiorptiometry performed with a total body scanner (DPX, Software 3.6, Lunar Radiation Corp., Madison, WI) enabling quantification of fat mass (FM), LBM, and total body bone mineral content (13, 14).
Analytical procedures Plasma glucose, nonesterified fatty acid (NEFA) were determined with a glucose oxidase kit (Biotrol, Paris, France) and an enzymatic procedure (Wako, Dardilly, France), respectively. Plasma free T3 and insulin were measured using RIA kits from Institut Pasteur (Paris, France). Serum sex hormone binding globulin (SHBG) concentrations were measured by immunoelectrophoresis (SBP-film, SEBIA, Issy-lesMoulineaux, France). Plasma total cholesterol and triglycerides were analyzed at the hospital routine chemistry laboratory.
Quantifications of mRNAs Total RNA from adipose tissue of patients following VLCD was obtained using the RNeasy kit (QIAGEN, Courtaboeuf, France). Total RNA from visceral and subcutaneous adipose tissues was prepared using guanidinium thiocyanate-phenol-chloroform extraction (15). Human UCP2 mRNA was quantified by RT-competitive PCR using a 235-bp UCP2 competitor DNA as previously described (11, 16). RT was performed on human adipocyte total RNA using 59-ATAGGTGACGAACATCACCACG-39 as primer. The subsequent PCR reaction contained 59-GACCTATGACCTCATCAAGG-39 as sense primer and 59ATAGGTGACGAACATCACCACG-39 as antisense primer. To improve the quantitation of the amplified products, a fluorescent dye-labeled sense oligonucleotide was used. The PCR products were separated and analyzed on an Applied Biosystems 373 DNA sequence analyzer (Perkin Elmer Applied Biosystems, Courtaboeuf, France) using the Genescan software.
Statistical analysis Values are given as means 6 sem. The Wilcoxon nonparametric test for paired values was used for comparisons before and during the diet. Correlations were tested by linear regression analysis. Statistical calculations were performed with a software statistical package (Statview, Abacus Concepts, CA, USA). P , 0.05 was the threshold of significance.
Results
UCP2 mRNA levels were not different in subcutaneous (5.6 6 1.0 amol/mg total RNA) and visceral (6.4 6 1.5 amol/mg total RNA) adipose tissues of the eight subjects tested (P 5 0.29). A strong correlation (r 5 0.91, P , 0.002) was found for UCP2 mRNA levels in the two depots (Fig. 1), suggesting that UCP2 mRNA level in subcutaneous adipose tissue is an index of UCP2 gene expression in body adipose tissues. As expected, there was a strong relationship between RMR
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FIG. 1. Linear relationship between UCP2 mRNA levels in subcutaneous and visceral adipose tissues of five women and three men. TABLE 1. Characteristics of eight obese premenopausal women before and during VLCD
Body weight (kg) FM (kg) LBM (kg) Total body bone mineral content (g) Resting metabolic rate (kJ/day) Resting metabolic rate/kg LBM (kJ/day/kg) Respiratory quotient Glucose (mmol/L) Insulin (pmol/L) NEFA (mmol/L) Total cholesterol (mmol/L) Triglycerides (mmol/L) SHBG (mg/L) (n 5 7) Free-T3 (pmol/L) a b
Before VLCD
During VLCD
88.2 6 2.6 39.7 6 1.1 44.8 6 1.5 2893 6 102 6491 6 465 145 6 2
81.9 6 2.7a 36.7 6 1.7a 41.5 6 1.3a 2911 6 98 5977 6 134a 144 6 3
0.83 6 0.01 4.3 6 0.2 102 6 13 719 6 81 4.81 6 0.23 1.28 6 0.23 2.9 6 0.2 4.5 6 0.1
0.76 6 0.01b 3.8 6 0.1b 68 6 5b 909 6 84b 4.18 6 0.23 1.05 6 0.16 3.8 6 0.5b 4.5 6 0.3
P , 0.01. P , 0.05.
and LBM (r 5 0.85, P , 0.01) in the obese patients of the VLCD protocol. During VLCD, there was a mean decrease of 7% of initial body weight (Table 1). The weight loss was equally accounted for by the reductions in FM and LBM. Fasting plasma glucose and insulin concentrations decreased, and plasma SHBG level increased. Plasma NEFA increased, and the respiratory quotient decreased, indicating increased lipolysis and fat oxidation during the diet. RMR expressed in kilojoules per day decreased significantly, but RMR adjusted for LBM remained unchanged. Individual changes in subcutaneous adipose tissue UCP2 mRNA levels during the diet are presented in Fig. 2. The mean values were not significantly different (11.4 6 3.7 vs. 12.6 6 3.0 amol/mg total RNA before and during VLCD, respectively; P 5 0.78). Next, we studied the relationship between UCP2 gene expression and resting energy expenditure. No relationship was found between UCP2 mRNA level and RMR or LBM. Before the diet, the correlation between UCP2 mRNA level and RMR per kilogram of LBM failed to reach significance (r 5 0.62, P 5 0.09). After 24 days of VLCD (Fig. 3), UCP2
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FIG. 2. Effect of VLCD on UCP2 mRNA levels in subcutaneous adipose tissue of eight obese premenopausal women.
mRNA level was correlated with RMR per kilogram of LBM (r 5 0.82, P , 0.01). Discussion
The potential link between UCP2 and energy balance and the paucity of data on UCP2 gene expression in humans prompted us to investigate UCP2 mRNA expression in human adipose tissue. We studied the effect of VLCD, a routinely prescribed treatment of the obese patients. In a recent work, we reported an increase in subcutaneous adipose tissue UCP2 mRNA levels during a 5-day fasting protocol in obese subjects (11). The induction in UCP2 mRNA expression was not found after 3 weeks of VLCD in obese women of comparable BMI (Fig. 2). The data suggest that the duration of calorie restriction and/or the level of food intake (1 kJ/day during the 5-day hypocaloric diet vs. 2.5 kJ/day during VLCD) influence UCP2 mRNA expression. This situation is reminiscent of the effect of food intake on UCP3 mRNA level in rodent skeletal muscle (17). A 48-h fast markedly increased UCP3 mRNA level, whereas 1 week of 50% food restriction resulted in a decrease of UCP3 mRNA expression. As expected, VLCD induces a decrease in RMR. Because LBM is strongly correlated with RMR and accounts for a large part of interindividual differences in RMR, RMR was expressed per kilogram of LBM. RMR adjusted for LBM was not modified by the diet (Table 1). During VLCD, we found a positive correlation between subcutaneous adipose tissue UCP2 mRNA level and RMR per kilogram of LBM. This result suggests that adipose tissue UCP2 mRNA expression is related to RMR in a population of obese premenopausal women. Factors other than RMR are, however, likely to be associated with UCP2 mRNA levels in adipose tissue, because the correlation failed to reach significance before VLCD. This data may be indicative that variations in diet composition could influence UCP2 gene expression because, in the obese patients, food intake was standardized by the hypocaloric diet. We also found a strong correlation between subcutaneous and visceral adipose tissue UCP2 mRNA levels (Fig. 1). The two fat depots represent most of the adipose tissue mass in humans. Therefore, our data suggest that the level of UCP2 mRNA in subcutaneous adipose tissue reflects the overall level of UCP2 gene expression in total body fat.
FIG. 3. Relationship between UCP2 mRNA level in subcutaneous adipose tissue and RMR per kilogram of LBM before (upper) and during (lower) VLCD.
Hence, the positive correlations found between RMR adjusted for LBM, and subcutaneous UCP2 mRNA level might be extended to UCP2 mRNA level in whole body fat. It remains to be determined whether such relationships are found with UCP2 gene expression in tissues other than adipose tissue. A potential role for adipose tissue UCP2 in the regulation of body weight and energy expenditure is suggested by several lines of evidence. A high-fat diet increases white adipose tissue UCP2 gene expression in the obesity resistant A/J and C57BL/KsJ strains but not in the obesity-prone C57BL/6J mice. Interestingly, the diet does not affect UCP2 and UCP3 mRNA expression in skeletal muscle (4, 18). UCP1-deficient mice do not become obese, and it was proposed that the loss of UCP1, the brown adipose tissue UCP, may be compensated by UCP2 (19). Moreover, the ectopic expression of UCP1 in white adipose tissue results, in transgenic mice, in a decrease of adiposity attributed to an increase of energy dissipation in this tissue (20, 21). A similar role of adipose tissue UCP2 in energy dissipation remains to be demonstrated. To conclude, the present work shows an interesting pattern of UCP2 mRNA expression in human subcutaneous adipose tissue. UCP2 mRNA level in subcutaneous fat
UCP-2 mRNA EXPRESSION DURING VLCD
depot was strongly correlated with UCP2 mRNA level in visceral fat depot. VLCD did not induce the upregulation of UCP2 mRNA expression observed during short-term fasting, suggesting a complex modulation of UCP2 gene expression by food intake. Finally, the positive correlation between UCP2 mRNA level and RMR adjusted for LBM suggests a link between UCP2 expression and energy expenditure.
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