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International Journal of Obesity (2001) 25, 1615–1618 ß 2001 Nature Publishing Group All rights reserved 0307–0565/01 $15.00 www.nature.com/ijo

PAPER Downregulation of uncoupling protein 2 mRNA in women treated with glucocorticoids J Udde´n1, R Folkesson2 and J Hoffstedt1* 1

Centre for Metabolism and Endocrinology, Department of Medicine and Neurotec, Karolinska Institutet at Huddinge University Hospital, Stockholm, Sweden; and 2Department of Experimental Geriatrics, Karolinska Institutet of Huddinge University Hospital, Stockholm, Sweden OBJECTIVE: Glucocorticoids are well-known regulators of energy turnover and adipose tissue metabolism. We investigated the effect of glucocorticoids on the expression of the human uncoupling protein 2 (UCP 2) gene, which has been implicated in energy expenditure. DESIGN: Prednisolone (25 mg) was administered orally daily for 7 days. Subcutaneous adipose tissue UCP 2 mRNA was measured before and after treatment. SUBJECTS: Eight healthy female subjects (age 52 – 63 y; body mass index 25 – 34 kg=m2). RESULTS: No differences in body weight, waist-to-hip ratio or plasma-values of FFA or glucose were found after prednisolone treatment, as compared to pre-treatment values under these conditions. In contrast, plasma insulin levels were significantly increased by glucocorticoid administration, 54 6 before vs 70 12 (mean sem) pmol=l after treatment (P ¼ 0.028). Furthermore, using RT-competitive-PCR, the UCP 2 mRNA level in abdominal subcutaneous adipose tissue was found to be down-regulated by half (6.3 0.4 vs 3.1 0.8 amol=mg RNA, P ¼ 0.012) after glucocorticoid treatment. No difference in expression levels of the reference gene 18SrRNA was observed before, as compared to after prednisolone exposure (249 11 vs 248 30 amol=mg RNA, P ¼ 0.87). CONCLUSION: These data suggest that glucocorticoids may play a role in the regulation of UCP 2 mRNA expression in human adipose tissue in vivo. International Journal of Obesity (2001) 25, 1615 – 1618 Keywords: adipose tissue; cortisol; human; insulin

Introduction Glucocorticoids are potent modulators of adipose tissue function and distribution. Chronic hypercortisolemia, as seen in Cushing’s syndrome, is associated with an enlargement of the abdominal fat mass.1 Also, hyperactivity of the hypothalamic – pituitary – adrenal axis has been demonstrated in subjects with features of the metabolic syndrome, including abdominal obesity and insulin resistance.2 Several mechanisms may be responsible for these observations. In abdominal adipose tissue, glucocorticoids induce the activity of lipoprotein lipase3 and decrease the rate of lipolysis.4 Moreover, abdominal adipocytes show a high density of

*Correspondence: J Hoffstedt, Karolinska Institutet, M61, CME, Huddinge University Hospital, S-141 86 Stockholm, Sweden. E-mail: [email protected] Received 19 January 2000; revised 18 April 2001; accepted 1 May 2001

glucocorticoid receptors.5 In this respect, the function of the recently discovered uncoupling proteins (UCP) 2 and 36,7 could be important. Based on in vitro findings and structural resemblance to the brown adipocyte-specific UCP 1, it has been suggested that UCP 2 and 3 may be involved in regulating thermogenesis and energy expenditure by uncoupling the respiratory chain in muscle or adipose tissue.8,9 Previous animal studies have shown that glucocorticoids may also have a regulatory effect on the expression of uncoupling proteins, although different effects are seen in muscle and brown adipose tissue (BAT). Whereas glucocorticoid administration decreases the expression of UCP 1, 2 and 3 mRNA in BAT,10,11 the level of UCP 3 mRNA in rat muscle is reported to be increased following dexamethasone exposure.12 In the present study, we investigated whether glucocorticoids regulate UCP 2 expression in man. UCP 2 mRNA from abdominal subcutaneous adipose tissue was measured before

UCP 2 regulation of glucocorticoids J Udde´n et al

1616 and after treatment with prednisolone in eight healthy female subjects.

Methods Subjects Eight healthy, postmenopausal women, age 57 y 1.0 (mean s.e.m), range 52 – 63, recruited by an advertisement in a local newspaper, volunteered for the study. The mean body mass index (BMI) of the study group was 28.9 0.8 kg=m2, range 25 – 34. All subjects were investigated before and after daily oral administration of 25 mg of prednisolone for 7 days. All tablets were taken at the same time in the morning. Compliance was controlled by determining serum prednisolone concentrations, which were measured by reversed-phase high performance liquid chromatography. Two women reported mild sleeping disturbances and seven experienced a subjective feeling of increased carbohydrate craving and hunger. No other side effects were reported during or after the period of prednisolone exposure. After an overnight fast, height, weight, BMI and blood pressure were recorded. Body fat percentage was determined by bioimpedance (model Tanita TBF-305, Japan). Venous blood samples were obtained for prednisolone, glucose and insulin measures. Finally, a subcutaneous fat biopsy (0.5 – 1 g) was obtained from the umbilical region under local anesthesia and the sample immediately frozen in liquid nitrogen and stored at 770 C. All biopsies were taken by the same practitioner (JU) using exactly the same procedure, site and depth before and after prednisolone treatment. The study was approved by the Human Ethics Committee of the Karolinska Institute and commenced after oral and written information to obtain informed consent.

Analysis of mRNA levels Total RNA from adipose tissue was prepared using the Rneasy total RNA kit (QIAGEN, Hilden, Germany). The concentrations of RNA were determined spectrophotometrically at 260 nm. The absorption ratios at 260 – 280 nm were between 1.7 and 1.9. The RNA-samples were stored at 770 C. The levels of UCP2-mRNA and 18S ribosomal RNA (18S rRNA) in total RNA were measured using a reverse transcriptase (RT) competitive PCR assay, as described.13,14 In brief, the assay involves the co-amplification of reverse transcribed RNA (cDNA) with known amounts of DNA-competitors. Specific cDNA synthesis from total adipose tissue RNA was performed in duplicate for both the UCP2-mRNA and 18S-rRNA. For the competitive PCR-reaction, 10 ml of the RT medium were added to a PCR-mix (90 ml). Then, four aliquots of 20 ml were transferred to PCR tubes each containing 5 ml of four different concentrations of UCP2 or 18S DNA competitors. The PCR products were resolved and analyzed on a 3% agarose gel. The optical density (OD) of the competitor and target bands was determined and the logarithm (OD competitor band=OD target band) was plotted vs the logarithm of International Journal of Obesity

the initial amount of the competitor added in the PCR. From this curve the amounts of UCP2 mRNA and 18S rRNA were calculated assuming that the initial concentration of the target corresponds to the initial concentration of competitor added at the equivalence point (log ratio ¼ 0), after correction for size differences of the PCR products of the target and competitor. The value obtained at the equivalence point was then multiplied by 2, as the competitor is double-stranded while the target RNA is single-stranded. The expression of the 18S rRNA gene was presumed not to differ before and after glucocorticoid treatment and therefore 18S rRNA was used as a reference.

Statistical analysis Values are given as mean standard error of the mean (s.e.m.). Simple regression analysis and the Wilcoxon signed rank test were used for statistical comparison, the latter since the material was too small to evaluate whether the UCP2 mRNA data were normally distributed or not.

Results Table 1 shows the clinical data before and after 7 days of treatment with prednisolone. The anthropometric data, including BMI, WHR and percentage body fat, were not affected by the treatment. Furthermore, no effect of glucocorticoid administration on either blood pressure levels or plasma values for glucose or FFA was found. In contrast, there was a significant increase in plasma insulin levels after, as compared to before, glucocorticoid treatment. The effect of glucocorticoid treatment on the level of UCP 2 mRNA expression was measured in abdominal subcutaneous adipose tissue. UCP 2 mRNA levels were markedly affected by prednisolone treatment. As illustrated in Figure 1, the level of UCP2 mRNA decreased in each subject following glucocorticoid administration. The mean value for UCP 2 mRNA was about 50% lower after prednisolone treatment, as compared to pre-treatment values (6.3 0.4 vs 3.1 0.8 amol=mg RNA, P ¼ 0.012). Two women showed identical values of UCP 2 mRNA, both before and after treatment. Table 1

Clinical data before and after prednisolone treatment

Weight (kg) BMI (kg=m2) WHR Body fat (%) SBP DBP Glucose (mmol=l) Insulin (pmol=l) FFA (mmol=l) Prednisolone (ng=ml)

Before treatment

After treatment

P

77 3 28.7 0.9 0.86 0.02 41 1 140 6 86 3 5.1 0.3 54.1 5.5 0.8 0.1

77 3 28.3 1.0 0.86 0.02 41 1 136 6 81 6 5.5 0.2 69.6 11.9 0.9 0.1 490 22

NS NS NS NS NS NS NS 0.028 NS

Values are means s.e.m. and were compared using Wilcoxon signed rank test. NS ¼ not significant.


1617 In contrast, no change in 18S rRNA levels before, as compared to after, glucocorticoid treatment was seen (249 11 vs 248 30 amol=mg RNA, P ¼ 0.89). As expected, the ratio of UCP 2 mRNA=18S rRNA was also significantly lower after, as compared to before, treatment (0.026 0.003 vs 0.013 0.002, P ¼ 0.012). We also investigated possible relationships between UCP2 mRNA levels and body weight, BMI, or concentrations of insulin or prednisolone. As shown in Figure 2, a significant correlation was found between UCP 2 mRNA values and insulin concentrations after prednisolone treatment. In contrast, no relations between UCP 2 mRNA and the other parameters analysed were found.

Discussion The present study was designed to evaluate the in vivo effect of glucocorticoids on UCP 2 mRNA expression in man. A marked decrease in the level of UCP 2 mRNA from abdominal subcutaneous adipose tissue was found in eight healthy women after 7 days of prednisolone treatment. The underlying mechanism responsible for the observed downregulation of UCP 2 mRNA expression is unknown. Corticosteroids are known to mediate their effects through binding to specific intracellular receptors, whereafter the

Figure 2 Relationship between adipose tissue UCP2 mRNA levels and plasma insulin concentrations after prednisolone treatment using simple regression analysis.

Figure 1 UCP 2 mRNA levels in abdominal subcutaneous adipose tissue before and after prednisolone treatment.

steroid – receptor complex influences gene transcription.15 The promoter region of the human UCP 2 gene is not reported to have a specific glucocorticoid responsive element,16 which suggests that glucocorticoids may indirectly influence UCP 2 mRNA transcription. Based primarily on animal studies, UCPs have also been implicated in insulin resistance and diabetes.8 For example, the anti-diabetic thiazolidinediones have been shown to induce expression of UCP mRNA in both adipose tissue and skeletal muscle and previous human studies have shown both an increased17 and a decreased18,19 expression of UCPs in subjects with hyperinsulinemia or non-insulin diabetes mellitus. In the present study, the observed downregulation of UCP 2 mRNA upon glucocorticoid treatment was associated with an increased plasma insulin level. This finding was expected, since glucocorticoids are known to induce insulin resistance. However, we also found that the level of UCP 2 mRNA was correlated to plasma insulin after prednisolone treatment, which suggest that hyperinsulinemia, or insulin resistance, may be involved in regulating UCP 2 expression in man. We also measured the levels of FFA, since these substances have been shown to regulate UCP mRNA expression.8 However, we found no effect of glucocorticoid administration on International Journal of Obesity


1618 plasma FFA values. Nevertheless, other intermediate variables affected by prednisolone treatment may be involved in regulating UCP 2 expression in man. Since no control group was included in the present study, it should also be noted that changes in any of the variables are not necessarily attributed to the experimental treatment. No changes in BMI, WHR or percentage body fat were observed before, as compared to after, administration of glucocorticoids. This finding is most probably due to the short duration of treatment, since the development of abdominal obesity is well established in chronic hypercortisolemia.1 Given the putative role of UCPs in energy expenditure and thermogenesis, a glucocorticoid-mediated down-regulation of UCP 2 could be important for abdominal fat accumulation by reducing the metabolic rate in adipose tissue. However, previous studies have presented conflicting results with respect to UCP 2 expression and obesity in man. A decreased UCP 2 mRNA expression in skeletal muscle20 and visceral adipose tissue21 from obese subjects has been demonstrated. In contrast, other studies have reported a positive correlation between adipose tissue UCP 2 mRNA and BMI,14 as well as between UCP 2 protein and body fat.22 Furthermore, it should be emphasised that, with the exception of a single study in mice over-expressing uncoupling protein 3,23 there is as yet no direct evidence for a role of UCPs in regulating metabolic rate. Consequently, the relevance of UCP 2 in normal human physiology as well as in obesity development needs to be further explored. In conclusion, the present study suggests that glucocorticoids may regulate the in vivo expression of UCP 2 in man. A marked 50% reduction of UCP 2 mRNA levels in abdominal subcutaneous adipose tissue from eight healthy women was found following oral prednisolone administration.

Acknowledgements ¨ lin is highly appreThe skilful technical assistance of Eva Sjo ciated. This study was supported by grants from the Foundation of Thuring and the Swedish Society of Medicine.

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