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EA 3502 and Institut National de la Santé et de la Recherche Médicale (INSERM) Avenir and Paris VI University, Department of Nutrition (Y.-M.L., C.P., V.P., B.G.-G., A.B., K.C.), Department of Medical Biochemistry (J.-M.L., C.C.), Hôtel-Dieu, 75004 Paris, France; and Obesity Research Unit 586 (N.V., D.L.), INSERM, Louis Bugnard Institute, Paul Sabatier University, 31403 Toulouse, France
Address all correspondence and requests for reprints to: Karine Clément, M.D., Ph.D., Service de Nutrition, Hôtel-Dieu, place du Parvis Notre-Dame, 75004 Paris, France. E-mail: karine.clement{at}htd ap-hop-paris.fr.
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Abstract |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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, IL-6, and eventually resistin, may participate in the relationship between obesity and the development of type 2 diabetes via their effects on the regulation of insulin action (4, 5, 6, 7).
Adiponectin is a collagen-like circulating protein exclusively secreted by mature adipose cells (8, 9, 10, 11). The level of adiponectin is correlated to the level of adiposity (10, 11, 12, 13), but in contrast to other adipokines (such as leptin or TNF), circulating concentrations decrease in obese humans (12) and increase with long-term weight loss (13). Recent studies have suggested that adiponectin is involved in lipid and glucose metabolism (14, 15). Treatment of mice with the globular segment of adiponectin exhibited numerous biological actions, including small induction of weight loss in animals consuming a high fat diet, prevention of the elevation of plasma fatty acid levels caused by administration of lipid, and promotion of fatty acid oxidation in muscle (14). Adiponectin is also a potent insulin enhancer in mouse model of obesity, lipoatrophy, and diabetes (16, 17). Hypoadiponectinemia has been linked to insulin resistance in humans (18).
In humans, little is known regarding the consequences of acute changes in energy balance on adipose tissue secreted proteins and notably adiponectin mRNA expression and its circulating level. The relationship between adipokine gene expression and acute changes in whole body insulin sensitivity remains to be addressed.
In the current study we examined, using a real-time, relative-quantitative RT-PCR assay, the effects of a 2-d very low calorie diet (VLCD) and 2-d refeeding on the gene expression of adiponectin in sc adipose tissue of weight-stable, morbidly obese women.
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Subjects and Methods |
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Fourteen unrelated Caucasian morbidly obese women (mean age, 39.0 ± 2.8 yr; range, 20–53 yr) participated in the clinical investigation. At the time of the study all obese subjects were at their maximal peak weight and were not on a restrictive diet. None of the obese women was involved in an exercise program. Eight unrelated weight-stable lean women with a mean age of 35.3 ± 3.3 yr (range, 26–48 yr) were also included as controls in the study. None of the subjects had a familial or personal history of diabetes or was taking medication likely to affect adipocyte metabolism. Most of the obese and nonobese women were in the follicular phase of their cycles.
Study design
The 14 obese women were submitted to a VLCD of 573-kcal daily energy intake (31.6% protein, 25.6% lipids, and 42.8% saccharides) for 2 d. This corresponded to a drastic 70% decrease in their usual daily energy intake. After the 2-d VLCD, their energy intake was increased to a level corresponding to the resting metabolic rate measured on d 1 (baseline). The sc superficial adipose tissue samples of the obese women were obtained on d 1, d 3 (VLCD), and d 5 (refeeding) by needle biopsy from the periumbilical area under local anesthesia (1% xylocaine). The superficial adipose tissue samples of lean controls were taken from a comparable site of abdominal region. Adipose tissue specimens were immediately frozen in liquid nitrogen and stored at -80 C until analysis. The resting metabolic rate was evaluated by indirect calorimetry. Body fat mass was measured by biphotonic absorptiometry (DXA, Hologic, Inc., Waltham, MA) (19) on d 1 and 5. Informed personal consents were obtained, and ethical permission was granted by the local ethics committee (CCPPRB, Hôtel-Dieu, Paris, France) in accordance with French laws on bioethics.
Total RNA preparation
Total RNA was extracted from adipose tissue samples using a modified protocol that combines phenol extraction and silica-based membrane spin columns. Briefly, 0.5 g adipose tissue was homogenized in 1 ml guanidine thiocyanate buffer (buffer RLT of the RNeasy kit, Qiagen, Courtaboeuf, France) using a rotor-stator homogenizer, followed by phenol and chloroform extraction steps. After chloroform extraction, the upper aqueous phase was subjected to the procedure of on-column deoxyribonuclease digestion and RNA isolation using the RNeasy total RNA kit (Qiagen, Chatsworth, CA). RNA concentrations were determined by absorbance at 260 nm (A260), and purity was estimated by A260/A280 ratio determination. Furthermore, the integrity, purity, and amount of RNA were confirmed by visualization of rRNAs after electrophoresis on normal agarose gels or by analysis using Agilent 2100 Bioanalyzer (Agilent Technologies, Massy, France) and RNA 6000 LabChip kit (Caliper Technologies, Waldems, Germany).
Relative-quantitative RT-PCR analysis
A real-time, two-step RT-PCR assay was developed for mRNA relative quantification. Gene-specific primers were designed using the internet-available software Primer3 and the sequences accessible from the GenBank database. The gene-specific primers for leptin, adiponectin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA quantification are shown in Table 1
. Leptin mRNA was quantified to compare changes in its expression to those in adiponectin. The quantification of GAPDH mRNA and 18S rRNA was performed for sample normalization.
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18S rRNA was quantified using TaqMan probe approach. Briefly, 20 µl PCR mix contain 2.5 ng reverse transcribed RNA, 1x LightCycler-FastStart DNA Master Hybridization Probes (Roche), 1x Human 18S primer and probe mix (Part 4333760F, PE Applied Biosystems, Foster City, CA), and 1 mM MgCl2. PCR was performed in 36 cycles with 10 sec at 95 C (denaturation) and 40 sec at 60 C (annealing and extension).
A pooled human adipose tissue total RNA was used to make standard curves. In summary, after RT, the cDNA was diluted into 10-fold serial dilution (10-1–10-6) and amplified along with samples. By plotting, in arbitrary units, the 10-fold serial dilutions, quantitative data of samples were obtained with LightCycler software. The standard curve was also used to assess PCR efficiency by examining its slope that was consistently around -3.3 for each gene in the study. All total RNA samples were reverse transcribed twice. Each cDNA was quantified in duplicate. The average value of each sample was used for quantification. The variation in measurements for a target gene in each sample generally ranged from 1–10% in the study.
It is noteworthy that GAPDH mRNA expression in sc adipose tissue of the obese women underwent significant changes during the short-term energy intake change procedure. GAPDH mRNA abundance decreased by 66.1% (P < 0.0001) upon VLCD, whereas 18S rRNA expression was relatively stable, showing a nonsignificant variation (P = 0.2). We thus chose 18S rRNA as the endogenous normalizer in the study. The relative-quantitative data were expressed as the ratio of the level of adiponectin or leptin mRNA to that of 18S rRNA in arbitrary units.
Biochemical analysis
Serum adiponectin and leptin were determined using RIA kits (Linco Research, Inc., St. Charles, MO) according to the manufacturer’s recommendation. A human adiponectin standard was provided by the manufacturer as two quality control samples. Serum insulin, blood glucose, lipids, free fatty acids, and estradiol were also measured using commercially available kits.
Insulin sensitivity calculation
Estimation of insulin sensitivity (HOMA-S% index) of obese and nonobese subjects were performed from plasma glucose and insulin values after an overnight fast, using homeostasis model assessment (HOMA) software (HOMA-CIGMA Calculator program version 2.00) as previously described (20).
Statistical analysis
The data are expressed as the mean ± SEM and/or the coefficient of variation (CV). Statistical analysis was performed with JMP statistics software (SAS Institute, Inc., Cary, NC). One-way ANOVA was used to assess the distributions of continuous variables such as age, body mass index (BMI), and biochemical measurements between obese subjects and lean controls. Comparisons of clinical and metabolic characteristics and adiponectin mRNA levels, either between d 1 and 3 (baseline and VLCD) or between d 3 and 5 (VLCD and refeeding), were made with the Wilcoxon nonparametric paired test. The significance of correlations was examined using nonparametric Spearman’s rank correlation test. The Mann-Whitney U test was used to assess differences in the levels of adiponectin mRNA as well as metabolic factors between the subgroups of obese women. P < 0.05 was the threshold of significance.
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Results |
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Table 2 summarizes the 14 obese women’s clinical and biological evaluations throughout the drastic energy variation as well as basal data for 8 lean controls. In the obese group, the VLCD protocol led to a 1.6% fall in body weight (P < 0.001) and a 3% decrease in resting metabolic rate (P = 0.065). Simultaneous decreased plasma concentrations of glucose (6%; P < 0.01), insulin (33%; P < 0.001), and leptin (9%; P = 0.02) were observed, whereas the plasma free fatty acid concentration and insulin sensitivity (HOMA-S%) increased by 33 ± 9% (P = 0.039) and 57% (P = 0.002), respectively, with VLCD. With refeeding, all of the selected parameters tended to recover to their baseline status (Table 2). Circulating adiponectin concentrations remained unchanged upon VLCD (d 1–3; P = 0.65) and refeeding (d 3–5; P = 0.77).
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Baseline adiponectin mRNA abundance in sc adipose tissue of the obese women as well as the nonobese subjects displayed a wide distribution (obese: range, 2.6–14.3 mRNA/18S rRNA; CV, 51.2%; lean: 7.4–15.4 mRNA/18S rRNA; CV, 23.6%), but mean values were significantly lower in the morbidly obese group compared with lean controls (6.2 ± 0. 9 vs. 11.9 ± 1.0; P < 0.001; Fig. 1). The mRNA expression level in sc adipose tissue was correlated with the plasma adiponectin concentration (
= 0.45; P = 0.03) in the whole group of subjects. In addition, negative correlations were observed between the mRNA level and BMI ( = -0.71; P = 0.0002; Fig. 2A) as well as with body weight ( = -0.67; P = 0.0007), fat mass ( = -0.61; P = 0.003), and fasting insulinemia ( = -0.68; P = 0.0005). The adiponectin mRNA level was positively correlated with the HOMA-S% index ( = 0.64; P = 0.0015; Fig. 2B). Consistently, the plasma protein level of adiponectin showed similar negative correlations with BMI ( = -0.51; P = 0.015), body weight ( = -0.49; P = 0.021), and insulinemia ( = -0.62; P = 0.0019) and a similar positive correlation with the HOMA-S% index ( = 0.69; P = 0.0004).
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Effects of short-term changes in energy intake on the levels of adiponectin mRNA in sc adipose tissue
Mean adiponectin mRNA in sc adipose tissue acutely responded to short-term changes in energy intake (Fig. 1
). Two-day VLCD caused a 33% rise (P < 0.01) in average adiponectin mRNA expression, whereas 2-d refeeding caused a 32.8% fall (P < 0.05) in mRNA levels. In contrast to adiponectin, no significant changes in leptin mRNA expression upon either VLCD or refeeding were observed (data not shown).
When examining individual values of adiponectin mRNA, we found that the obese women could be separated into two subgroups, a responder subgroup (n = 8) and a nonresponder subgroup (n = 6), based on the variation in response of adiponectin mRNA to short-term VLCD and subsequent refeeding (Fig. 3). The adiponectin mRNA expression level was significantly higher in the responder subgroup in the basal and VLCD states compared with the nonresponder subgroup (P = 0.02 and P = 0.003, respectively; Fig. 3). With refeeding, the average adiponectin mRNA values of the two subgroups showed no significant difference (P > 0.05). We analyzed whether the increase in the adiponectin mRNA expression in the responder subgroup resulted in an increased circulating protein level, and observed that only four of the eight responders displayed an increased circulating adiponectin concentration (range, 3–28%).
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Although the weight variation (1.6%) was relatively small, the change in adiponectin mRNA expression was negatively correlated to the change in weight ( = -0.62; P = 0.01) upon VLCD in all obese women. Interestingly, we observed substantial differences in insulin sensitivity between the responders and the nonresponders. Although the insulin sensitivity of the responder subgroup increased significantly by 89% (P = 0.008) with VLCD, that of the nonresponder subgroup increased by only 24% (P = 1.56). In addition, the mean body weight and BMI of the responder subgroup were 18 kg and 6 points, respectively, lower than those of the nonresponder subgroup, showing a borderline significance (P = 0.1). No other remarkable change was seen between the two subgroups for other clinical and metabolic parameters.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Our data showed that the basal adiponectin mRNA level in sc adipose tissue of weight-stable, morbidly obese women displayed a wide distribution and was correlated to its plasma protein concentration. In agreement with other studies performed in a less obese population (10, 12), both mRNA levels and plasma adiponectin concentrations were significantly lower than those in the lean subjects. In addition, the circulating concentration of adiponectin was lower in obese patients with higher levels of adiposity and was positively correlated with insulin sensitivity in the morbidly obese women as well as in the whole group of obese and nonobese subjects. An important role for this adipokine in the regulation of insulin action has previously been proposed (15). Lower circulating adiponectin levels have been linked to the onset of insulin resistance and the development of type 2 diabetes in recent studies (18, 21, 22). Our data support the relationship between adiponectin and insulin resistance associated with extreme forms of obesity.
This study showed for the first time that adiponectin mRNA responded to acute changes in energy intake in some obese subjects. This finding led us to question whether the degree of obesity may modulate adiponectin gene expression in response to energy balance variations. By analyzing individual data, we observed that the subjects who showed an acute adiponectin mRNA response to the changes in energy intake had higher basal adiponectin mRNA levels and tended to have a lower BMI than the subjects who showed no or weak adiponectin mRNA response. This observation suggests that a high degree of adiposity may be a factor blunting the regulation of adiponectin mRNA expression by caloric restriction. The possible contributing effect of genetic variations in adiponectin gene on the basal level of adiponectin mRNA merits further consideration. It was recently described that several single nucleotide polymorphisms and combined haplotypes of the adiponectin gene were associated with modified circulating levels (23). Whether these single nucleotide polymorphisms might modulate the variation in adiponectin gene expression and the differential response of the gene to caloric restriction is still to be determined.
The study also showed that the post-VLCD insulin sensitivity of the responder subgroup was significantly higher than that of the nonresponder subgroup. The fact that acute caloric intake reduction leads to transcriptional regulation of the adiponectin gene suggests that metabolic events occurring during negative energy balance may impact on the expression of the adipokine gene. In vitro studies (24) have shown that metabolic factors that usually vary during changes in energy balance control adiponectin gene expression. Insulin and IGF-I are both positive regulators of adiponectin gene expression, whereas glucocorticoids are negative regulators of gene expression (24). Together these data suggest that the modulation in insulin signaling may contribute to the in vivo control of adiponectin gene expression in human sc fat, or, in another words, the blunted response of adiponectin gene to energy change in some subjects may be related to the modified regulation of insulin action.
Albeit the mean adiponectin mRNA in sc adipose tissue of the obese women significantly varied under short-term energy modification, it is unlikely that adiponectin is an acute signal of energy balance, because its circulating concentration showed no or weak variations with VLCD and refeeding in either the whole group or the two subgroups of the obese women. This observation is in contrast to leptin, which showed significant changes in the plasma concentration upon the short-term VLCD and after refeeding, whereas its mRNA levels were similar in the two conditions. Dissociation between leptin mRNA and the circulating protein has been widely reported (25, 26, 27), and several hypotheses regarding posttranscriptional regulation occurring during change in energy balance have been made (26, 28, 29). Dissociation of adiponectin mRNA with its plasma protein level was also recently reported in first degree relatives of type 2 diabetics (30). A recent in vitro study by Motoshima et al. (31) revealed that the secretion rate of adiponectin from visceral adipose tissue is significantly higher than that from sc adipose tissue, suggesting that the main proportion of circulating adiponectin comes from the omental region. Regional differences in the responses of adipose-specific genes to cellular or metabolic stimuli have been largely reported (32, 33, 34). However, until now the effect of energy intake alteration on adiponectin gene expression in visceral adipose tissue remains unknown. Thus, further studies dealing with both visceral and sc adipose tissues will be needed to evaluate the role of this important adipokine in regulation of energy balance.
In conclusion, this study shows that adiponectin mRNA in sc adipose tissue, but not circulating concentrations, can acutely respond to drastic short-term energy changes in obese subjects. Our data mostly suggest that both the levels of adiposity and insulin sensitivity may contribute to the modulation of adiponectin mRNA levels.
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Acknowledgments |
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Footnotes |
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Abbreviations: BMI, Body mass index; CV, coefficient of variation; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HOMA, homeostasis model assessment; VLCD, very low calorie diet.
Received May 21, 2003.
Accepted August 30, 2003.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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expression. J Lipid Res 39:724–730
, leptin and tumor necrosis factor- mRNA expression during very low calorie diet in subcutaneous adipose tissue in obese women. Diabetes Metab Res Rev 15:92–98[CrossRef][Medline]
and RXR agonists. Diabetes 51:718–723This article has been cited by other articles:
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, P < 0.01 compared with values at baseline (d 1) and 2-d VLCD (d 3) in obese women; 
, P < 0.05 compared with values of 2-d VLCD (d 3) and 2-d refeeding (d 5) of obese women.
