IT IS WELL known that uncoupling protein-1 (UCP1),

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1 X/99/$03.00/0 Vol. 84, No. 11 The Journal of Clinical Endocrinology & Metabolism Printed in U.S.A. Copyright 1999 by The Endocrine Society Regulation of Uncoupling Protein-2 and -3 by Growth Hormone in Skeletal Muscle and Adipose Tissue in Growth Hormone-Deficient Adults* STEEN B. PEDERSEN, KURT KRISTENSEN, SANNE FISKER, JENS OTTO LUNDE JØRGENSEN, JENS SANDAHL CHRISTIANSEN, AND BJØRN RICHELSEN Department of Endocrinology and Internal Medicine, Aarhus University Hospital, Aarhus Amtssygehus (S.B.P., K.K., B.R.), and Aarhus Kommunehospital (S.F., J.O.L.J., J.S.C.), DK-8000 Aarhus C, Denmark IT IS WELL known that uncoupling protein-1 (UCP1), which is specifically expressed in brown adipose tissue, promotes facultative thermogenesis in rodents (1). In adult humans UCP1 is unlikely to be a major regulator of energy expenditure (EE), because brown adipose tissue is absent or only scarcely present. Recently, two novel members of this protein family, UCP2 and UCP3, have been discovered in several tissues in humans, including white adipose tissue and skeletal muscle (2 4). UCP3 is highly expressed in human skeletal muscle, which is of particular interest as this tissue is the major determinant of resting EE. Thyroid hormones appear to stimulate skeletal muscle UCP3 expression in rodents, and leptin administration is associated with increased expression of both UCP2 and UCP3 in adipose tissue and muscle of rodents (5). Somewhat counterintuitively, severe fasting, which is associated with Received May 13, Revision received July 1, Accepted July 23, Address all correspondence and requests for reprints to: Steen B. Pedersen, M.D., Ph.D., Department of Endocrinology and Internal Medicine, Aarhus University Hospital, Aarhus Amtssygehus, DK-8000 Aarhus C, Denmark. amtssp@aau.dk. * This work was supported by the Novo-Nordisk Foundation, the Institute of Experimental Clinical Research at Aarhus University, the Danish Research Council, the A. P. Møller Foundation, the Løvens Kemiske Fabriks Research Foundation, the Gangsted Foundation, and the Aarhus University-Novo Nordisk Center for Research in Growth and Regeneration. ABSTRACT The newly described uncoupling proteins, UCP2 and UCP3, may play a role in regulating energy expenditure (EE) in humans. GH deficiency (GHD) is associated with decreased lean body mass, increased adiposity, and reduced EE, which are reversed by GH treatment. In the present study we investigated whether GH treatment for 4 months influenced the expression of UCPs in skeletal muscle and adipose tissue in 22 GHD patients who were investigated before and after GH (n 11) or placebo (n 11) treatment. GH treatment increased the amount of lean body mass by 4.5% (P 0.05) and decreased body fat mass by 12% (P 0.05), whereas no changes in these parameters were observed after placebo treatment. The level of UCP3 messenger ribonucleic acid (mrna) increased 3-fold (P 0.005) in skeletal muscle and almost 2-fold (P 0.05) in adipose tissue after GH treatment, with no changes observed after placebo treatment. Skeletal muscle UCP2 mrna was slightly (25%), but significantly (P 0.05), decreased, whereas the level of UCP2 mrna in adipose tissue was unaffected after GH treatment. The T 4 level was positively correlated with skeletal muscle UCP2 and UCP3 expression (r 0.518; P 0.05 and r 0.463; P 0.05, respectively). Furthermore, plasma free fatty acids were positively correlated with the expression of UCP2 (r 0.573; P 0.01) and UCP3 (r 0.518; P 0.05) in skeletal muscle. The marked increase in UCP3 expression after GH treatment indicates that the UCPs might play a role in the effects of GH on EE in GHD patients. Finally, the strong association between thyroid hormone and skeletal muscle UCP and the correlation between plasma free fatty acids and UCP expression in skeletal muscle indicate that these hormones/metabolites might influence UCP expression in humans as previously demonstrated in rodents. (J Clin Endocrinol Metab 84: , 1999) reduced EE, is associated with increased gene expression of UCP2 and UCP3 in adipose tissue and skeletal muscle in rodents (6, 7) and humans (8). It is suggested that this enhanced expression of UCPs is mediated through the accompanying increase in circulating free fatty acids (FFA) during fasting (9). GH is an important anabolic agent that, besides its growthpromoting effects, has important effects on substrate metabolism. The hallmarks include increased protein synthesis, reduced proteolysis, and mobilization and oxidation of lipids. Protein anabolism operates during fed conditions and is partly mediated by insulin-like growth factor I (IGF-I), whereas lipid oxidation occurs in postabsorptive and fasting conditions, presumably through direct effects of GH on adipose tissue. Several lines of evidence suggest that GH modulates EE. Acromegaly is associated with an elevated resting metabolic rate (RMR), which is normalized after successful adenomectomy (10). Short term discontinuation of GH substitution in GH-deficient (GHD) adults induces a decline in the RMR to subnormal levels, which normalize acutely after resuming GH (11). The latter study implies the ability of GH to increase RMR independently of changes in lean body mass. GH administration to hypopituitary patients and to lean and obese subjects is also accompanied by enhanced peripheral conversion of T 4 to T 3 (12, 13), but experimental data suggest that the calorigenic effect of GH is independent of the small elevation in T 3 (14). 4073

2 4074 PEDERSEN ET AL. JCE&M 1999 Vol 84 No 11 These complex effects of GH on body composition, EE, and FFA oxidation provide a rationale for evaluating the impact of GH on UCPs. Therefore, we investigated the effects of 4 months of GH treatment on the expression of UCP2 and UCP3 messenger ribonucleic acid (mrna) in adipose tissue and skeletal muscle in GHD adults. Subjects Subjects and Methods Twenty-two adults with pituitary deficiency, including GH deficiency (GHD), were included in the study. GHD was determined as peak GH less than 5 g/l at two of the following provocative tests: insulininduced hypoglycemia test (blood glucose, 2.2 mmol/l), clonidine test, arginine test, or the heat test. All patients had been adequately substituted with other relevant hormones for at least 1 yr before participation. The study was approved by the national ( ) and the local ( ) ethical committees. The patients were randomized to 4 months of GH treatment (Norditropin, Novo Nordisk A/S, Denmark) in a dose of 2 IU/m 2 (n 11) or placebo treatment for 4 months (n 11). Full GH doses were reached during a 6-week period. Adipose tissue biopsies Adipose tissue biopsies were obtained from the sc abdominal region (periumbilically) after an overnight fast as previously described (15). Each biopsy was performed by needle aspiration (liposuction) after local anesthesia with lidocaine (10 mg/ml). The adipose tissue was washed with isotonic saline to remove blood and then frozen in liquid nitrogen for later RNA extraction. Biopsies were obtained before and after 4 months of GH/placebo treatment. Muscle biopsies Skeletal muscle biopsies were taken from the musculus vastus lateralis with a Bergström needle as previously described (16). After local anesthesia (lidocaine) was applied, a small incision was made through the skin and muscle sheet approximately 15 cm above the knee. The muscle biopsy was quickly cleaned for blood and frozen in liquid nitrogen for later RNA extraction. Biopsies were obtained before and after GH/placebo treatment for 4 months. Isolation of RNA Total RNA was isolated from the biopsies using the TriZol reagent (Life Technologies, Inc., Roskilde, Denmark), RNA was quantitated by measuring absorbency at 260 and 280 nm; the ratio should be 1.8 or higher. Finally, the integrity of the RNA was checked by visual inspection of the two ribosomal RNAs, 18S and 28S, on an agarose gel. Unfortunately, during one of our RNA isolations the RNA was degraded (five adipose tissue samples taken after placebo treatment for 4 months, which were all processed on the same day). RT-PCR assay for detection of UCP2 and UCP3 mrna RTs were performed using random hexamer primers as described by the manufacturer (GeneAmp RNA PCR Kit, Perkin-Elmer Corp./Cetus, Norwalk, CT) at 23 C for 10 min, 42 C for 60 min, and 95 C for 10 min. Then, PCR-Mastermix containing the specific primers and AmpliTaq GOLD DNA polymerase was added. A linear increase in PCR product was observed when using RNA ranging between ng (data not shown); all subsequent PCR reactions were performed using 25 ng RNA. A second set of PCR reactions was designed to determine the appropriate number of cycles to be run, and the protocol was further optimized to coamplify the target and the housekeeping gene by multiplex PCR with primer dropping (17). Semiquantitative multiplex PCR (primer dropping) estimates the relative amount of target RNA to a known housekeeping gene ( -actin) and eliminates the sample to sample variability of the RT-step as well as the PCR step. The lower expression of the target compared to the housekeeping mrna ( -actin) was managed by preamplification of the target complementary DNA by performing a few PCR cycles before the housekeeping primers were added to the PCR tube (primer dropping) (17). The number of initial cycles before dropping the housekeeping primers was 8 for UCP2 and 12 for UCP3 in adipose tissue; 9 for UCP2 and 4 for UCP3 in skeletal muscle. The -actin primers were in all cases coamplified with the target genes for 24 cycles. A similar set-up was used for negative controls, except that the reverse transcriptase was omitted, and no PCR products were detected under these conditions. The following primer pairs were used: UCP2 primers, 5 -TC- CAAGGCCACAGATGTGCC and 5 -TCGGGCAATGGTCTTGTAGGC; UCP3 primers, 5 -GGACTACCACCTGCTCACTG and 5 -CCCGTAA- CATCTGGACTTT; and -actin, 5 -TGTGCCCATCTACGAGGGGTA- TGC-3 and 5 -GGTACATGGTGGTGCCGCCAGACA-3. The PCR protocol was 95 C for 10 min, then cycles of 95 C for 30 s, 57 C for 30 s, and 72 C for 60 s. Finally, the PCR products were extended for 5 min at 72 C. The PCR products were loaded on a 2% agarose gel, stained with ethidium bromide, and analyzed using the Bio-Rad Laboratories, Inc., Gel-Doc 1000 system. The ratio between target PCR product and the -actin PCR product was calculated. The coefficient of variation for the determination of the UCP/ -actin ratio was 12.4% (n 15). Body composition Body composition was determined by dual energy x-ray absorptiometry scan using a QDR-1000W scanner (Hologic, Inc., Waltham, MA) as previously described (18). The amounts of fat (FM), bone mass, and lean body mass (LBM) were determined. LBM comprises both muscle tissue and nonmuscle fat-free soft tissue (including water space). Analysis of blood samples Serum IGF-I was determined using an in-house time-resolved immunofluorometric assay after extraction of serum to remove binding proteins. Serum FFA were measured by an enzymatic colorimetric method (Trichem, Copenhagen, Denmark). Serum leptin was determined by a commercial RIA kit (Linco Research, Inc., St. Charles, MO). Serum total T 4, free T 4, total T 3, free T 3, and rt 3 were measured by RIA as previously described (19). Plasma glucose was determined by the glucose oxidase method with a glucose analyzer (Beckman Coulter, Inc., Fullerton, CA). Insulin was analyzed in an immunoassay (DAKO Corp., Cambridgeshire, UK). Statistical analysis Student s paired t test was used to compare before and after values. Correlation between variables was tested by Pearson s correlation coefficient. Data are given as the mean sem; the level of significance chosen was All analyses were performed with the SPSS statistical package (SPSS, Inc., Chicago, Illinois). Results Effects of GH on body composition and hormones/metabolites GH treatment of GHD patients for 4 months was associated with a significant decrease in total FM (12% decline) and an increase in the amount of LBM, determined by DEXA scan, whereas no change was observed in the placebo-treated group (Table 1). Serum IGF-I, insulin, and blood glucose levels were significantly increased after GH treatment (Table 1), but not after placebo treatment. GH treatment was associated with an increase in the free T 3 to free T 4 ratio (P 0.01) and with a tendency for an increase in free T 3 (0.05 P 0.10). Effects of GH treatment on UCP mrna expression Adipose tissue. Placebo treatment had no influence on UCP2 or UCP3 expression (Fig. 1, A and B), whereas GH treatment

3 EFFECTS OF GH IN GHD ON UCP mrna 4075 TABLE 1. Changes in body composition and hormones after 4 months of GH treatment Placebo-treated group GH-treated group T 0 T 4 T 0 T 4 No. (M/F) 7/4 9/2 Age (yr) BMI (kg/m 2 ) FM (kg) a b LBM (kg) a b IGF-I ( g/l) b FFA (mmol/l) Insulin (mu/l) c Blood glucose (mmol/l) c FT 3 (pmol/l) FT 3 /FT c a P 0.05 between placebo and GH groups before treatment (T 0). b P 0.01 between before (T 0) and after treatment (T 4). c P 0.05 between before (T 0) and after treatment (T 4). TABLE 2. Correlations between UCP expression and body composition and hormones at baseline IGF-I FFA TT 4 FM (%) AT-UCP AT-UCP SM-UCP a a SM-UCP a a b AT-UCP2, UCP2 expression in adipose tissue; AT-UCP3, UCP3 expression in adipose tissue; SM-UCP2, UCP2 expression in skeletal muscle; SM-UCP3, UCP3 expression in skeletal muscle. a P b P for 4 months increased UCP3 expression 1.8-fold (P 0.05) above pretreatment values in adipose tissue (Fig. 1B). UCP2 expression in adipose tissue was not affected by GH treatment (Fig. 1A). Skeletal muscle. Placebo treatment for 4 months did not affect the mrna expression of UCP2 or UCP3 in skeletal muscle (SM-UCP2 or SM-UCP3; Fig. 2, A and B), but GH treatment for 4 months increased SM-UCP3 expression 2.9-fold above initial values (P 0.005; Fig. 2B), whereas the SM-UCP2 mrna level was slightly reduced to 76 8% of the basal value (P 0.05) after GH treatment for 4 months (Fig. 2A). Correlation between UCP mrna expression, hormones, and adiposity UCPs in adipose tissue (AT-UCPs). No significant associations between AT-UCP2 or AT-UCP3 and different hormones or adiposity were detected at baseline (or after GH treatment; data not shown), although a tendency for a negative association between serum IGF-I and AT-UCP3 expression before GH treatment was observed (r ; P 0.1) (Table 2). SM-UCPs. At baseline serum IGF-I levels were negatively correlated with the expression of both SM-UCP2 (r 0.392; P 0.08) and SM-UCP3 (r 0.625; P 0.002). Plasma FFA was positively correlated with SM-UCP2 (r 0.573; P 0.01) and SM-UCP3 expression (r 0.518; P 0.05). T 4 was positively correlated with SM-UCP2 expression (r 0.518; P 0.05) and SM-UCP3 expression (r 0.463; P 0.05) (Table 2). Multiple regression analysis Using SM-UCP3 mrna expression as the dependent variable and serum IGF-I plasma FFA, T 4, and percent FM as independent variables in a backward multiple regression analysis revealed that only FFA (partial correlation coefficient, 0.682; P ), percent FM (partial correlation coefficient, 0.451; P 0.002), and T 4 (partial correlation coefficient, 0.334; P 0.02) remained significantly correlated to SM-UCP3, whereas IGF-I was excluded from the equation. Repeating the analysis using the same independent variables and SM-UCP2 as the dependent demonstrated that only T 4 remained in the equation (r 0.518; P 0.05), whereas all of the other variables were removed (percent FM, IGF-I, and FFA). When analyzing AT-UCP2 and AT-UCP3, none of the independent variables was significantly correlated with the dependent variables, and they were removed from the equations. Discussion GHD patients are characterized by a low EE that can be reversed by GH treatment (20, 21); furthermore, GH can increase EE obese women (13) as well as in healthy subjects (14). As GH treatment is associated with an increase in LBM, EE might be increased secondary to the larger metabolic body size; however, a recent study evaluating the effects of 6 months of GH treatment in GHD patients demonstrated that when adjusting RMR for the differences in FFM, it appeared that other factors also may play a role in the increase in RMR (20). Finally, even short term GH infusion to GHD patients for 24 h was able to increase the EE (11); thus, it seems that GH is able to influence EE independent of any change in LBM. The precise mechanism underlying the GHinduced increase in EE is largely unknown. The newly discovered uncoupling proteins, UCP2 and UCP3, are mitochondrial proteins that are thought to increase EE by uncoupling of oxygen consumption from ATP synthesis, thereby promoting the generation of heat. The precise role for these proteins in the development of obesity and in controlling EE in humans, however, has not been determined. A previous study has shown a positive correlation between AT-UCP2 expression and body mass index

4 4076 PEDERSEN ET AL. JCE&M 1999 Vol 84 No 11 FIG. 1. Effects of GH treatment on UCP mrna expression in adipose tissue. A, The ratio between UCP2 mrna and -actin mrna in adipose tissue before (open boxes) and after (filled boxes) treatment with placebo (left panel) or GH (right panel) for 4 months. B, The ratio between UCP3 mrna and -actin mrna in adipose tissue. *, P 0.05 (before vs. after treatment). (8), whereas other studies found lower UCP2 expression in obese compared to lean individuals (22, 23). In the present study of GHD adults, no correlations between adiposity and UCP expression in adipose tissue or skeletal muscle were found in simple correlation analysis; however, in multiple regression analysis, UCP3 expression in skeletal muscle was negatively correlated with percent FM. supporting a recent study by Schrauwen et al. in Pima Indians demonstrating that body mass index and SM-UCP3 were negatively correlated and that SM-UCP3 was positively correlated with RMR (24). The present study demonstrated that GH treatment of GHD patients, which is known to increase EE, increased skeletal UCP3 expression in adipose tissue and skeletal muscle, indicating that UCP3 might play a role in the increase in EE. Data from animal studies indicate that thyroid hormone (5) and FFA (9) are strong stimulators of UCP expression. Our finding of a positive correlation between thyroid hormone and skeletal muscle UCP expression suggests that thyroid hormones might influence UCP3 expression in skeletal muscle in a similar manner in humans and rodents. On the other hand, we were unable to demonstrate any correlation between the change in thyroid hormones (T 3 and T 4 ) and the

5 EFFECTS OF GH IN GHD ON UCP mrna 4077 FIG. 2. Effects of GH treatment on UCP mrna expression in skeletal muscle. A, The ratio between UCP2 mrna and -actin mrna in skeletal muscle before (open boxes) and after (filled boxes) treatment with placebo (left panel) or GH(right panel) for 4 months. B, The ratio between UCP3 mrna and -actin mrna in skeletal muscle. *, P 0.05; **, P (before vs. after treatment). change in UCP3 expression after GH treatment (data not shown), indicating that the GH effects on UCP3 expression might not be mediated through changes in thyroid hormones, supporting a recent study demonstrating that GH treatment of healthy volunteers increased EE, whereas T 3 administration in a dose selected to mimic the GH-induced increase in peripheral T 3 levels did not increase EE (14). Plasma FFA was positively correlated with UCP2 and UCP3 expression in skeletal muscle, indicating that a high FFA concentration is associated with a high UCP expression in skeletal muscle, thus supporting studies in rodents where FFA stimulates UCP3 expression in skeletal muscle (9). It seems unlikely, however, that the effects of GH on UCP expression were mediated indirectly by changes in plasma FFA, as FFA did not change significantly during GH treatment; furthermore, no association between the changes in FFA and UCP expression were depicted (data not shown). The negative correlation between serum IGF-I and SM-

6 4078 PEDERSEN ET AL. JCE&M 1999 Vol 84 No 11 UCP expression (UCP2 and UCP3) may suggest a role for IGF-I in controlling SM-UCP expression; however, further research is needed to clarify this issue. In conclusion, GH treatment of GHD patients is associated with a marked increase in UCP3 mrna levels in skeletal muscle as well as in adipose tissue. AT-UCP2 expression was unchanged, whereas a slight, but significant, decrease in SM-UCP2 expression was found. These results suggest that UCP3 in both adipose tissue and skeletal muscle might be involved in the increase in EE observed after GH treatment of GHD patients. Furthermore, UCP2 and UCP3 are regulated differentially, indicating that they may have different functions for the intracellular metabolism. Plasma FFA and T 4 were closely correlated with SM-UCP expression, and T 3 was closely correlated to UCP expression in skeletal muscle suggesting that thyroid hormones and FFA might regulate UCPs in humans as already demonstrated in rodents. Acknowledgments We appreciate the expert technical assistance of L. Pedersen and D. Phillip. References 1. Bouillaud F, Ricquier D, Mory G, Thibault J Increased level of mrna for the uncoupling protein in brown adipose tissue of rats during thermogenesis induced by cold exposure or norepinephrine infusion. J Biol Chem. 259: Fleury C, Neverova M, Collins S, et al Uncoupling protein-2: a novel gene linked to obesity and hyperinsulinemia. Nat Genet. 15: Boss O, Samec S, Paoloni-Giacobino A, et al Uncoupling protein-3:a new member of the mitochondrial carrier family with tissue- specific expression. 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