1 Department of Medicine, Karolinska Institute, Huddinge University Hospital, Stockholm, Sweden
2 Obesity Research Unit, Institut National de la Santé et de la Recherche Médicale (INSERM) Unité 586, Institut Louis Bugnard, Centre Hospitalier Universitaire de Toulouse, Université Paul Sabatier, Toulouse, France
3 Institute of Preventive Medicine, Danish Epidemiology Science Centre, Copenhagen University Hospital, Copenhagen, Denmark
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ABSTRACT |
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Adipose tissue secretes a number of proteins with auto-, para-, and endocrine actions, such as tumor necrosis factor- (TNF-
), interleukin (IL)-6 and -8, plasminogen activator inhibitor 1 (PAI-1), leptin, and adiponectin (1,2). Little is known about the regulation of the protein secretory function of human adipose tissue except for leptin (3). The effect of energy restriction is of particular importance for obesity treatment (4). Dietary-induced weight loss normalizes plasma levels and adipose tissue gene expression of several adipocyte-derived proteins (512). Whether the same is true for protein secretion is unknown, except for TNF-
(5,10). Furthermore, the relative roles of loss of body fat, energy restriction per se, and changes in macronutrient supply are not known. It is also unknown which proteins are more or less sensitive to nutritional changes regarding their production by adipose tissue. These questions were investigated by studying the release of leptin, adiponectin, IL-6 and -8, TNF-
, and PAI-1 in subcutaneous adipose tissue of 40 obese women before and after 10 weeks on moderate hypoenergetic diets with either low-fat/high-carbohydrate content or moderate-fat/moderate-carbohydrate content.
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RESEARCH DESIGN AND METHODS |
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Energy expenditure and composition of diets.
The daily energy requirement was estimated as follows: resting energy expenditure was measured by indirect calorimetry (Deltatrac II; Datex-Ohmeda, Helsinki, Finland) and multiplied by 1.3 for physical activity level. The subjects were then prescribed a daily energy intake 600 kcal lower than the estimated energy requirement. The dietary target for fat content was 2025 energy percent (E%) for the low-fat diet and 4045 E% for the moderate-fat diet. Both diets contained 1520 E% from protein and the rest (6065 and 4045 E%, respectively) from carbohydrates. The subjects were given dietary instructions based on an education system consisting of isoenergetic interchangeable units (13). Alcohol was not allowed. The subjects kept food diaries and recorded everything they ate or drank during the study to facilitate compliance. Instructions were given to minimize differences in the amount of fruit and vegetables eaten, type of fat, amount and type of fiber, type of carbohydrate, and meal frequency between the two groups.
Adipose tissue biopsies and dietary intervention.
Before and after the dietary intervention, the subjects were investigated at 8:00 A.M. after an overnight fast. Biopsies (2 g) of subcutaneous abdominal adipose tissue were obtained under local anesthesia (14), venous blood samples were collected, and measurements of weight, circumference of waist and hips, and bioimpedance were taken (QuadScan 4000; Bodystat, Isle of Man, U.K.). Approximately one-half of the tissue was snap frozen for later mRNA analysis.
During the dietary intervention, the subjects visited or had telephone contact with the dietitian every week. The dietitian assessed the compliance of the subjects and checked the content of their diets from the food diaries. The subjects also performed a 3-day weighed food record of 2 weekdays and 1 weekend day before starting the dietary intervention and at the end of the 10-week diet to estimate their habitual diet and compliance with the diet. In addition, 1-day weighed food records were completed in the 2nd, 5th, and 7th week of the intervention. The food records were analyzed using a food-nutrient database. The subjects were weighed when they visited the center (usually every 2nd week).
Adipose tissue.
The adipose tissue was washed in physiological saline and cut into small pieces (10 mg). Then
500 mg were subjected to collagenase isolation (15), and mean fat cell weight and volume were determined (16). The total lipid content in the incubate was measured gravimetrically after organic extraction, and the number of adipocytes was calculated by dividing the total lipid weight by the mean cell weight, as described (16,17). The remaining 400 mg were incubated in 4 ml of Krebs-Ringer phosphate buffer (pH 7.4) supplemented with 40 g/l of BSA and 1 g/l of glucose for 2 h at 37°C in a shaking water bath with air as the gas phase. Then, 2 ml of the medium were removed, frozen in liquid nitrogen, and stored at 70°C to be used for the determination of TNF-
, adiponectin, and IL-6 and -8 as described previously for TNF-
(18). The remaining 2 ml of medium were frozen in liquid nitrogen, freeze dried, and redissolved in 250 µl of distilled water for the determination of leptin and PAI-1 as described (19,20). The incubated adipose tissue was homogenized, and total lipid was extracted as described (21). The secretion of proteins was related to 107 fat cells in the incubated tissue. The methods to determine TNF-
, leptin, and PAI-1 release from human subcutaneous adipose tissue in vitro have been evaluated (5,19,20). For IL-6 and -8 and adiponectin, methodological experiments revealed that 1) recovery from incubation medium was >90% and 2) secretion increased in a linear fashion for at least 3 h.
Protein and metabolite determinations.
Human leptin and adiponectin radioimmunoassay kits from Linco (St. Charles, MO) were used for the determination of leptin and adiponectin concentrations in medium and serum. Quantikine human immunoassays (R&D Systems, Abingdon, U.K.) were used for the quantification of IL-6 and -8 in medium. Quantikine HS (high sensitivity) human immunoassays (R&D systems, Abingdon, U.K.) were used for the determination of IL-6 and TNF- plasma levels and the concentration of TNF-
in medium. TintElize PAI-1 immunoassay (Biopool, Ventura, CA) was used to determine the PAI-1 levels in medium. Plasma enzymatic activity of PAI-1 was determined as described (19). Plasma insulin was determined by radioimmunoassay (Insulin RIA 100; Kabi-Pharmacia, Uppsala, Sweden). The kits were used according to instructions from the manufacturers. The technology needed for the determination of plasma IL-8 levels was not available in the laboratory. Plasma glucose, cholesterol, and triglycerides were determined by the hospitals routine chemistry laboratory. Measures for insulin resistance were obtained using the homeostasis model assessment (HOMA): [fasting plasma insulin (mU/l) x fasting plasma glucose (mmol/l)] ÷ 22.5 (22).
mRNA quantitation.
Total RNA was extracted from subcutaneous adipose tissue biopsies using the RNeasy total RNA Mini Kit (Qiagen, Courtaboeuf, France). Agarose gels were used to check the RNA quality, and the samples from 6 of the 40 subjects (3 from each diet group) were excluded due to inadequate quality. An amount of 1 µg of total RNA was reverse transcribed using random hexamers as primers and Superscript II reverse transcriptase (Invitrogen, Cergy Pontoise, France). Real-time quantitative PCR was performed on a GeneAmp 7000 Sequence Detection System (Applied Biosystems, Foster City, CA). Then, 10 ng of cDNA was used as template for real-time PCR. The thermal cycler parameters for the real-time PCR were 2 min at 50°C, followed by 40 cycles with 10 s at 95°C and 1 min at 60°C. For leptin and adiponectin, a set of primers (Genset-Proligo, Paris, France) was designed using the software Primer Express 1.5 (Applied Biosystems) and used at a final concentration of 300 nmol/l with SYBR Greenbased chemistry. A dissociation curve was generated at the end of the PCR cycles to verify that a single gene product was amplified. For PAI-1, TNF-, and IL-6 and -8, the TaqMan approach was used. Both primers and TaqMan probes were obtained from Applied Biosystems. The probes were labeled with a reporter dye (FAM) on the 5' end. The probe for 18S ribosomal RNA was labeled with the reporter dyes VIC and TAMRA on the 5' end and the 3' end, respectively. For TaqMan assays, because of the very high specificity of the method, checking for nonspecific product formation with dissociation curves is not needed. For each primer pair, a standard curve was obtained using serial dilutions of human adipose tissue cDNA before mRNA quantitation. We used 18S rRNA as control to normalize gene expression using the Ribosomal RNA Control TaqMan assay kit (Applied Biosystems).
Statistical analysis.
Data were compared between and within the two groups by the Mann-Whitney U test, Wilcoxons signed-rank test, Spearmans correlation, and repeated-measures ANOVA using StatView 5.0 (SAS Institute, Cary, NC). The variables in the study were analyzed for normality, and nonnormally distributed variables were log transformed when appropriate. Standard error was used as measure of dispersion. The level of significance was P 0.05. Because of assay failures, the total number of subjects in each analysis was between 34 and 40.
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RESULTS |
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There was a marked difference in the percentage of total calories from carbohydrate and fat between the groups and also in the amount of dietary fiber (P < 0.001 for all of these factors). The percentage of total calories from protein also differed between the groups (P < 0.01). Values for the moderate- and low-fat diets, respectively, were: carbohydrate: 38.9 ± 0.90 vs. 52.4 ± 0.85 E%; dietary fiber: 13.7 ± 0.78 vs. 18.8 ± 0.97 g/day; and protein: 19.6 ± 0.36 vs. 21.1 ± 0.32 E%. The ratio of saturated to monounsaturated to polyunsaturated fatty acids was 2:2:1 in the habitual diet and the two intervention diets. There was no difference in compliance between the groups.
Clinical findings.
There was no statistical difference in age, resting energy expenditure, anthropometric measurements, blood pressure, fat cell volume, or fasting plasma levels of glucose, insulin, triglyceride, and cholesterol between the groups at baseline (data not shown). The average weight reduction for all subjects was 7.7 ± 0.4 kg, i.e., a 7.5% decrease in body weight. There was a continuous loss of weight in both groups (P < 0.0001) (Table 1). The reduction in percent body fat, BMI, and fat cell volume was similar for both groups.
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Adipose tissue mRNA quantitation.
Messenger RNA data are shown in Table 2. At baseline there was no difference between the two diet groups in expression levels for any of the genes. After the diet, leptin mRNA decreased significantly by 25% in the moderate-fat diet group but not in the low-fat diet group, although a tendency was observed (P = 0.076). When both groups were considered together, leptin decreased significantly by
20% (P = 0.0004). IL-6 gene expression decreased significantly in both groups by 1020%. There was no diet effect on TNF-
, IL-8, adiponectin, or PAI-1 mRNA levels when the groups were considered separately or together. No statistically significant interactions between diet, weight change, and changes in mRNA levels were found in ANOVA analysis.
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To test the effect of weight loss on adipose tissue secretion of PAI-1 and adiponectin, we put both groups together and analyzed those 20 who lost the most weight (range 812 kg). In this group adiponectin secretion was 2.4 ± 0.2 µg/107 cells/2 h before and 2.7 ± 0.4 µg/107 cells/2 h after the diet (P = 0.33), and PAI-1 secretion was 17.4 ± 2.3 ng/107 cells/2 h before and 21.0 ± 4.7 µg/107 cells/2 h after the diet (P = 0.77).
The correlation between changes in insulin sensitivity and protein secretion was analyzed in the 20 subjects on either diet with the largest decrease in HOMA index (range 0.54.6). Only the secretion of leptin changed significantly, from 65 ± 7 ng/107 cells/2 h before to 33 ± 4 ng/107 cells/2 h after the diet (P = 0.0013).
Circulating levels.
The serum and plasma levels of leptin, TNF-, IL-6, and adiponectin and the plasma PAI-1 activity are shown in Table 4. There was a 2035% decrease of the serum levels of leptin in the entire group of subjects as well as in both groups after the diet. In Spearmans correlation analysis, this decrease correlated with the change in leptin secretion (P = 0.022). No other correlations were found between changes in mRNA expression, secreted proteins, and circulating levels.
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DISCUSSION |
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The role of dietary fat in the development of obesity is controversial (27,28). Some evidence indicates that moderate-fat diets cause obesity, whereas low-fat diets prevent weight gain (29). In contrast, to succeed in losing weight, it could be of paramount importance to restrict the intake of carbohydrates (30). Very-low-carbohydrate diets (2030 g/day) without energy restriction cause weight loss and improve metabolic profiles (3133) even more efficiently than low-fat, energy-restricted diets (32,33).
The results of this study clearly show that the macronutrient composition, at least the fat and carbohydrate contents, of hypoenergetic diets was not of major importance for the outcome of dietary treatment. The moderate-fat/moderate-carbohydrate and low-fat/high-carbohydrate diets caused a similar weight loss of 7.5% and had similar effects on insulin sensitivity, fat cell volume, circulating and secreted proteins, and mRNA levels. There are differences in circulating levels of leptin and adiponectin between pre- and postmenopausal women (34,35). Five menopausal women were included in this study. We also calculated all results without these women. However, the findings from these calculations did not differ from those obtained with the entire cohort (n = 40).
The effects of the dietary intervention on protein secretion varied, but three clear patterns could be distinguished. First, the weight reduction resulted in a marked reduction in leptin secretion by 40% after both diets. Second, the hypoenergetic diets had a moderate effect on TNF-
and IL-6 and -8 secretion (2030% decrease). Last, PAI-1 and adiponectin were not affected by the diets. It is likely that a more extensive weight reduction is required to cause changes in the secretion of PAI-1 and adiponectin, whereas a rather small weight loss is enough to cause a marked reduction in leptin secretion and a small reduction in the secretion of TNF-
and ILs. Apparently, adiponectin and PAI-1 release is very resistant to weight changes induced by caloric restriction because there was no change in secretion even in the women who had the most marked weight loss on either diet (on average
10% weight loss). It is also possible that a more rapid weight loss during a shorter period of time could give other types of results. Indeed, more rapid or more marked weight reduction than in our study has been shown to induce a change in circulating or adipose PAI-1 and adiponectin (8,9,36,37).
Adipose tissue is the major source of circulating leptin levels (20). Therefore, it is expected that the marked decrease in secretion of leptin after dieting would lead to an almost equally marked decrease in serum leptin levels. It is less clear to what extent human adipose tissue contributes to circulating cytokine levels. TNF- appears above all to be produced and act locally in human fat tissue (18), and there is no in vivo release of TNF-
from subcutaneous adipose tissue to the blood (38). However, human subcutaneous adipose tissue releases IL-6 in vivo (38), which suggests that it acts as an endocrine signal. A diet-induced weight loss of
14 kg in 2 years time caused decreased serum concentrations of IL-6 (37). IL-6 levels in serum and in subcutaneous adipose tissue decreased after a reduction of 2.1 kg/m2 in BMI induced by a 3-week very-low-calorie diet, but TNF-
levels did not change in serum or adipose tissue (6). These results are partly in agreement with the present study: secretion of TNF-
and IL-6 decreased by 2030%, but there was no effect on plasma TNF-
levels and only a small but significant decrease in plasma IL-6 levels. Taken together, the data suggest that adipose tissue has a minor effect on the regulation of circulating TNF-
levels.
A surprising finding is the marked decrease in plasma PAI-1 activity after the energy restriction, despite no change in adipose tissue PAI-1 secretion. This strongly suggests that factors other than adipose tissue regulate the whole-body biological activity of PAI-1, perhaps hepatocytes or endothelial cells (36). In 15 obese subjects after a 3-week very-low-calorie diet causing a weight reduction of 5.8 kg, plasma PAI-1 antigen decreased, but mRNA and protein levels increased (36). However, present and previous studies are not really comparable because Bastard et al. (36) analyzed PAI-1 in homogenized frozen tissue and not PAI-1 secretion from fresh adipose tissue. Although the extent of the weight loss was similar to the weight reduction in the present study, it was induced in a much shorter time. It is quite possible that compensatory effects occur during long-term and slower weight loss, as in our study. In theory, reduced PAI-1 secretion from visceral fat could contribute to the fall in plasma PAI-1 activity. There are regional differences in several adipose-secreted proteins, as reviewed (39), and at least some studies suggest higher secretion from visceral compared with subcutaneous fat (40,41). It is, however, unlikely that visceral fat is responsible for the fall in plasma PAI-1 activity because this depot is relatively small in obese women. For example, the major omentum constitutes <1% of total body fat in very obese subjects (42).
The only major changes in gene expression were decreased leptin and IL-6 mRNA. The expression of IL-6 showed a significant decrease in both diet groups. The mRNA levels of leptin decreased significantly in the moderate-fat diet group and when all subjects were considered together. It also tended to decrease in the low-fat diet group. Thus, leptin and IL-6 are at least in part regulated at the transcriptional level during energy restriction. The mRNA levels of TNF- and IL-8 did not change, although the secretion rates of these proteins decreased in the whole group after diet, suggesting that TNF-
and IL-8 production is subject to posttranscriptional regulation. Recent studies have shown that the expression of TNF-
is limited almost exclusively to macrophages in the stromal-vascular fraction of murine adipose tissue, whereas IL-6 is expressed in both the adipocyte and stromal-vascular fractions (43,44). Whether this is true also for human adipose tissue is not yet clear.
Weight loss improves obesity-associated insulin resistance (45). This was confirmed in the present study and could be a factor behind decreased leptin production (23,46). It is, however, not important for the other secreted proteins because there was no dietary effect on IL-6 or -8, TNF-, adiponectin, or PAI-1 when the subjects who improved their insulin sensitivity the most were analyzed separately (data not shown).
In summary, this study suggests that energy supply per se and not the macronutrient composition is of importance for the regulation of the protein secretory function and gene expression of human adipose tissue, at least during energy restriction. Leptin is most sensitive for energy restriction, followed by cytokines and chemokines, whereas PAI-1 and adiponectin are not affected by a moderate weight decrease/reduction in energy intake. However, other sources for PAI-1 besides subcutaneous adipose tissue are very sensitive to nutritional changes.
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ACKNOWLEDGMENTS |
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The technical assistance of Kerstin Wåhlén, Eva Sjölin, Britt-Marie Leijonhufvud, Katarina Hertel, and Maria Johansson is greatly appreciated.
Address correspondence and reprint requests to Peter Arner, Professor, MD, PhD, Karolinska Institutet, Huddinge University Hospital, M63, SE-141 86 Stockholm, Sweden. E-mail: peter.arner{at}medhs.ki.se
Received for publication January 7, 2004 and accepted in revised form May 4, 2004
HOMA, homeostasis model assessment; IL, interleukin; PAI-1, plasminogen activator inhibitor 1; TNF-, tumor necrosis factor-
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REFERENCES |
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