| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Obesity: Original Article |
Equipe d’Accueil 3502 and Institut National de la Santé et de la Recherche Médicale (INSERM) "Avenir" and Department de Nutrition (Y.-M.L., A.B., K.C.), Paris VI University, 75004 Paris, France; Unité Mixte de Recherche 7079 (M.M., J.P.), Department de Physiologie et Physiopathologie, Centre National de la Recherche Scientifique, Institut Biomédical des Cordeliers, Paris VI University, 75270 Paris, France; Service de Biochimie et Hormonologie (J.-P.B.), Hôpital Tenon, 75970 Paris, France; Service d’Endocrinologie-Métabolisme (E.B.) and Service de Biochimie (B.H.), La Pitié Salpétrière, 75013 Paris, France; and Unité de Recherches sur les Obésités (N.V., D.L.), INSERM, Institut Louis Bugnard, Centre Hospitalier Universitaire de Toulouse, Université Paul Sabatier, 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.
 |
Abstract |
|---|
 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
, IL-6, resistin, etc.) and nonsecreted (e.g. hormone-sensitive lipase, perilipin, glucose transporter GLUT4, etc.) molecules. It has been documented that adipocyte-produced proteins have a wide range of effects on energy homeostasis, carbohydrate and lipid metabolism, inflammation, and immunity as well as on insulin sensitivity. Dysregulated expression and/or dysfunction of genes encoding adipocyte-synthesized factors have been found in obesity and obesity-related metabolic disorders such as insulin resistance, type 2 diabetes, and dyslipidemia (3, 4, 5, 6, 7). Clinical investigations examining the physiology and pathophysiology of adipocyte-produced proteins are therefore important for the understanding of complex human metabolic diseases such as obesity, diabetes mellitus, hypertension, and related cardiovascular complications (8, 9). A novel nonsecreted adipocyte protein, adiponutrin, was recently discovered in a preadipose cell line and found to be mainly expressed in adipose tissue (10). Identified as a transmembrane protein, adiponutrin is composed of 413 amino acid residues. The corresponding 3.2-kb mRNA appears early and is markedly increased during in vitro 3T3-L1 preadipocyte differentiation (10). In rodents, adiponutrin gene expression is regulated by changes in nutrition and energy balance. Its level decreases upon fasting and rapidly increases with refeeding or feeding with a high-carbohydrate diet (10, 11). Moreover, an increased expression of adiponutrin is observed in brown and white adipose tissues in (fa/fa) obese Zucker rat (10). Although the adiponutrin function is still unknown, these features suggest a possible contribution of adiponutrin to energy homeostasis and adipocyte function.
In humans, there is no available information regarding adiponutrin gene expression in adipose tissue, and its regulation by the variation of energy balance is unknown. Using real time quantitative RT-PCR, we investigated the effects of corpulence and of an acute [2-d very low-calorie diet (VLCD)] change and of a short-term (21-d VLCD) change in energy intake on adiponutrin gene expression in sc adipose tissue of obese women.
|
Subjects and Methods |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
This study included 48 Caucasian women divided into two groups: 1) the lean control group, including 13 unrelated nonobese, nondiabetic women, and 2) the obese group, including 35 unrelated nondiabetic obese women. In this group of obese women, 24 morbidly obese subjects (subgroup 1) were submitted to a 2-d VLCD and a subsequent 2-d refeeding regimen. Subgroup 2, which included 11 obese patients, was subjected to a 21-d VLCD treatment procedure.
Study design
To address the effects of acute changes in energy intake, the 2-d VLCD protocol consisted of a daily energy intake of 573 kcal (31.6% protein, 25.6% lipids, and 42.8% saccharides) for 48 h. This corresponded to a drastic 70% decrease of the usual daily energy intake. After the 2-d VLCD, energy intake was increased to a caloric level corresponding to the resting metabolic rate (RMR) measured at d 1 (baseline).
For the 21-d VLCD protocol, obese subjects reduced their daily energy intake by one third (941 ± 27 kcal/d with 32% protein, 25% lipids, and 43% saccharides).
None of the subjects had a familial or personal history of diabetes or were taking medication. At the time of the study, all obese patients were at their maximal peak weight and were not on a restrictive diet. In our inclusion criteria, patients had to be weight stable for at least 3 months before the VLCD. None of the subjects were involved in an exercise program.
Subcutaneous superficial adipose tissue samples were obtained, after an overnight fast, by needle biopsy from the periumbilical area under local anesthesia (1% Xylocaine, Astra Laboratory, Rueil-Malmaison, France) in the control women (baseline), then at d 1 (baseline), d 3 (2-d VLCD), and d 5 (refeeding) for the subjects involved in the acute-term protocol (subgroup 1). The same type of biopsy was performed at d 1 (baseline) and d 21 for the obese women of subgroup 2. Adipose tissue specimens were immediately frozen in liquid nitrogen and stored at –80 C until analysis. No RNA stabilization reagent was used in this set of experiments. Blood samples were also obtained for biochemical and hormonal evaluation.
Body composition was assessed in fasting condition by dual-energy x-ray absorptiometry (12). We determined body composition at d 1 (baseline) and d 5 (refeeding) for subgroup 1 as well as at d 1 (baseline) and d 21 for subgroup 2. RMR was evaluated after a 1-h resting period in supine position. Oxygen consumption (VO2) and carbon dioxide production (VCO2) were monitored over 30 min by 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 by using indirect calorimetry.
All clinical investigations were performed according to the Declaration of Helsinki. Informed personal consents were obtained, and ethical permission was granted by the local ethics committee (Comité Consultatif de Protection des Personnes dans la Recherche Biomédicale, Hôtel-Dieu and Pitié Salpétrière Paris and Toulouse University Hospitals) in accordance with French laws on bioethics.
Adipose tissue total RNA preparation
Adipose tissue total RNA was extracted by using a modified protocol that combines phenol extraction and silica-based membrane spin columns. Briefly, 0.5 g of adipose tissue was homogenized in 1 ml of guanidine thiocyanate buffer (buffer RLT of RNeasy kit, Qiagen, Coutaboeuf, France) using a rotor-stator homogenizer and followed by phenol and chloroform extraction steps. After chloroform extraction, the upper aqueous phase was subjected to on-column DNase digestion and RNA isolation using RNeasy total RNA kit (Qiagen). RNA concentration was determined by absorbance at 260 nm (A260), and purity was estimated by A260/A280 ratio determination. The integrity, purity, and the 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 Corp., Hopkinton, MA).
Relative-quantitative RT-PCR analysis
Relative quantification of mRNA was performed with quantitative RT-PCR assay. Gene-specific primers were designed using the internet-available software Primer3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) and the sequences accessible in the GenBank database. The gene-specific primers used in the study are shown in Table 1
.
|
Using the TaqMan probe approach, 18S rRNA was quantified for normalization. Briefly, 20 µl PCR mix contains 2.5 ng reverse-transcribed cDNA, 1x LightCycler-FastStart DNA Master Hybridization Probes (Roche Diagnostics), 1x Human 18S primer and probe mix (part no. 4333760F, Applied Biosystems, Courtaboeuf, France), and 3 mM MgCl2. PCR was performed for 36 cycles for 10 sec at 95 C (denaturation) and 40 sec at 60 C (annealing and extension).
A pool of human adipose tissue total RNA was used to make standard curves. After reverse transcription, the cDNA was diluted into 10-fold serial dilution (10–1 to 10–6) and amplified along with sample cDNA. Quantitative data of samples were obtained with the LightCycler software. The standard curve was also used to assess PCR efficiency by examining its slope, which was consistently around –3.3 for each gene in the study. All samples were assayed in duplicates, and the average value of the duplicates was used for quantification. The variation in the two measurements for each sample was generally in a range of 0.1% to approximately 10%. If the variation of a sample exceeded 10%, a novel triplicate assay was carried out for this sample. Because 18S rRNA expression was relatively stable after the 2-d or 21-d VLCD protocols, showing a nonsignificant variation (P = 0.2), we chose 18S rRNA as the endogenous normalizer in the study. The data were expressed as the ratio of the levels of the target gene mRNA on that of 18S rRNA.
Biochemical analysis
Serum leptin in obese subjects was determined using the RIA kits of Linco Research (St. Charles, MO), according to the manufacturer’s recommendation. Serum insulin, blood glucose, lipids, and free fatty acids values were measured using commercially available kits.
Insulin sensitivity calculation
Insulin sensitivity of subjects was evaluated using the quantitative insulin sensitivity check index (QUICKI) method, which is well correlated with the hyperinsulinemic euglycemic clamp method. Calculation was performed from fasting glucose and insulin as described (13).
Statistics analysis
The data are expressed as mean ± SEM and/or as coefficient of variation (CV). Statistical analysis was performed with JMP statistics software (SAS Institute Inc., Cary, NC). One-way ANOVA was used to test the distribution of continuous variables such as age, body mass index (BMI), and biochemical measurements between the three groups in the study. Significant differences of clinical and metabolic characteristics and the mRNA levels of adiponutrin, either between d 1 and 3 (baseline and 2-d VLCD) or between d 3 and 5 (VLCD and refeeding) in the acute-term protocol, and/or between d 1 and d 21 in the protocol of 3 wk, were determined by Wilcoxon nonparametric paired test. Comparison between nonobese and obese subjects was performed using a nonparametric, nonpaired test. The correlations of the adiponutrin mRNA levels with the clinical and metabolic characteristics were examined by the nonparametric Spearman’s rank correlation test. P < 0.05 was the threshold of significance.
|
Results |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
Table 2 shows the characteristics, at baseline, of nonobese and obese subjects at various energy intakes. The subjects assigned to the 2-d VLCD (subgroup 1) were morbidly obese, with high BMI (>40 kg/m2) and fat mass. Those assigned to the 21-d VLCD (subgroup 2) were moderately to severely obese women. In subgroups 1 and 2, all the obese patients had a normal glycemia, with elevated insulin related to their corpulence. The insulin sensitivity assessed by the QUICKI showed, as expected, decreased insulin sensitivity in the obese groups compared with controls. Morbidly obese subjects, assigned to the 2-d VLCD, had a lower QUICKI compared with that of the obese patients involved in the 21-d VLCD (P < 0.01).
|
). After a 2-d refeeding, all of the selected parameters tended to recover their status observed before VLCD (baseline). The 21-d VLCD resulted in a mean reduction of 2.5 kg/m2 in BMI (6.3%; P < 0.001) and a decrease in plasma concentration of glucose (7.3%; P < 0.01), insulin (21.8%; P < 0.001), and leptin (41.3%; P < 0.001) as well as in an improvement of insulin sensitivity (QUICKI increased 5.6%; P < 0.01) (Table 2). Basal levels of adiponutrin mRNA in sc adipose tissue
In a first analysis, we compared the adiponutrin mRNA content in sc adipose tissue of two groups of subjects including lean controls and nondiabetic obese women (Fig. 1). Baseline levels of adiponutrin mRNA in the fasting state displayed a wide distribution for either nondiabetic obese women (range, 0.2–3.8 mRNA/18S rRNA; CV, 65.0%) or lean controls (range, 0.4–2.4 mRNA/18S rRNA; CV, 48.1%). At baseline, the average mRNA levels of adiponutrin were not statistically different between obese subjects (either subgroup 1 or 2) and nonobese subjects (Fig. 1).
|
We found that adiponutrin gene expression in sc adipose tissue acutely responded to energy intake modifications (Fig. 1). The 2-d VLCD reduced the average level of adiponutrin mRNA expression by 36% (P = 0.0016), whereas the 2-d refeeding elevated the expression level of the mRNA by 31% (P = 0.004) (Figs. 1 and 2). Consistently, the 21-d VLCD caused a dramatic 58% fall of the adiponutrin mRNA expression level (P = 0.001) in all the subjects (Fig. 2).
|
Associations of adiponutrin gene expression and biological features
We analyzed the relationship between adiponutrin mRNA expression and several clinical and metabolic parameters related to corpulence and glucose tolerance in the nondiabetic obese subjects. We observed a significant negative association between adiponutrin mRNA levels and plasma glucose concentrations in obese subjects (Rho = –0.62, P < 0.0001, and P = 0.002 adjusted for age and BMI or fat mass). This negative correlation with fasting glycemia was also independently found in both subgroup 1 and subgroup 2 of obese subjects (data not shown). In addition, when we combined the data from obese and nonobese subjects, the negative correlation between fasting glucose and adiponutrin mRNA remains highly significant (Fig. 3).
|
). A correlation between adiponutrin mRNA level and the QUICKI index was mostly found to be significant in the obese women of subgroup 2 (Rho = 0.8, P = 0.002, and P = 0.03 adjusted for age and BMI or fat mass), characterized by a less severe form of obesity and a better insulin resistance index when compared with subgroup 1.
|
|
Discussion |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
Adiponutrin shows a very specific pattern of expression, compared with other important adipose tissue proteins, also modulated by variation of energy balance. Whereas both adiponutrin and leptin expression levels show similar changes after VLCD-induced weight loss (14), acute energy changes only modulate the adipose expression of adiponutrin but not leptin, as previously described (15, 16). In addition, we have described that 2-d VLCD and subsequent refeeding also caused a drop followed by an increase in adiponectin gene expression level (16). However, no association was observed between adiponutrin and adiponectin mRNAs at baseline or after variation in energy balance in the same group of patients. Similarly, no correlation between leptin and adiponutrin expression changes after 21-d VLCD was observed. These observations suggest that adiponutrin gene expression might be regulated by different mechanisms than those that regulate leptin and adiponectin in humans.
The adiponutrin gene pattern of expression during acute or short-term changes of energy balance suggests a substantial interaction between this nonsecreted adipocyte protein and the regulation of energy balance in humans. The fact that acute caloric intake reduction and refeeding leads to transcriptional regulation of the adiponutrin gene suggests that early metabolic events occurring during negative and subsequent positive energy balance may affect the expression of the gene. Two-day drastic variation in energy balance is generally not sufficient to lead to substantial fat loss or regain. Accordingly, in this protocol, body fat mass did not change at d 1 and 5.
Clearly, in vitro adiponutrin gene expression exhibits all the features of lipogenic gene expression such as fatty acid synthase or adipocyte determination and differentiation factor-1/sterol regulatory element binding protein-1c (ADD1/SREBP1) (10). Is adiponutrin implicated in de novo fatty acid synthesis and/or esterification per se? Or is adiponutrin a sensor of substrate availability, regulating the energy balance in adipocytes? This transmembrane nutrient-sensed protein could play a role in membrane trafficking and/or vesicular transport as evoked by in silico analysis. Alternatively, adiponutrin could also participate in a new signaling pathway to adjust adipocyte homeostasis with respect to lipid metabolism.
Whatever the adiponutrin function is, its regulation is associated with energy deprivation. In this regard, its level of expression is associated with markers that rapidly change after variation in the energy balance. In vitro adiponutrin gene transcription is glucose dependent (10), and in vivo adiponutrin mRNA expression is rapidly induced in white adipose tissue of rats fed with a high-sucrose diet (11). In our observation, adiponutrin mRNA level is negatively correlated with fasting plasma level of glucose in obese and nonobese patients. However, this correlation found with fasting glucose levels, in obese and nonobese subjects, does not necessarily reflect causation. To address the possible role of adiponutrin in glucose homeostasis, further studies including dynamic and kinetic protocols after ingestion of glucose are needed. Investigations in adiponutrin gene expression in diabetic obese patients compared with lean controls will also be informative. Otherwise, the present study also shows that adiponutrin mRNA expression level was associated with insulin sensitivity either in the obese and nonobese subjects together or in obese patients alone. This observation suggests that adiponutrin could be involved in the modulation of whole-body insulin sensitivity. To further explore the role of adiponutrin in insulin resistance and energy balance regulation, it will be interesting to pursue longer changes in energy intake.
It is now largely documented that numerous adipocyte proteins, either secreted factors such as adiponectin, TNF-, IL-6, and resistin (17, 18, 19, 20) or nonsecreted molecules such as GLUT4 (21) play a pivotal role in regulation of whole-body insulin action. Adiponutrin could belong to this class of factor. Further studies in groups of subjects with a wider range of insulin sensitivity will be necessary to explore this issue.
To conclude, our study, performed using several groups of obese and nonobese subjects, suggests the contribution of the nonsecreted adipocyte protein, adiponutrin, in the adipocyte regulation of energy balance. In fine, its close relationship to energy status does make it an important gene to consider in the overall energy balance relationship that determines body weight. Further clinical and animal studies will be necessary to dissect the exact physiological function of adiponutrin and its contribution to the regulation of energy balance, adiposity, and related metabolic parameters.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Abbreviations: BMI, Body mass index; CV, coefficient of variation; QUICKI, quantitative insulin sensitivity check index; RMR, resting metabolic rate; VLCD, very low calorie diet.
Received November 14, 2003.
Accepted January 30, 2004.
|
References |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
, 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]
in diabetes and obesity. Curr Diab Rep 2:179–185[Medline]
This article has been cited by other articles:
 |
|
 |
|
  B. Kollerits, S. Coassin, N. D. Beckmann, A. Teumer, S. Kiechl, A. Doring, M. Kavousi, S. C. Hunt, C. Lamina, B. Paulweber, et al. Genetic evidence for a role of adiponutrin in the metabolism of apolipoprotein B-containing lipoproteins Hum. Mol. Genet., December 1, 2009; 18(23): 4669 - 4676. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. E Johansson, U. Lindblad, C. A Larsson, L. Rastam, and M. Ridderstrale Polymorphisms in the adiponutrin gene are associated with increased insulin secretion and obesity Eur. J. Endocrinol., November 1, 2008; 159(5): 577 - 583. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. J. Bujalska, K. N. Hewitt, D. Hauton, G. G. Lavery, J. W. Tomlinson, E. A. Walker, and P. M. Stewart Lack of Hexose-6-Phosphate Dehydrogenase Impairs Lipid Mobilization from Mouse Adipose Tissue Endocrinology, May 1, 2008; 149(5): 2584 - 2591. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. J. Oort, T. A. Knotts, M. Grino, N. Naour, J.-P. Bastard, K. Clement, N. Ninkina, V. L. Buchman, P. A. Permana, X. Luo, et al. {gamma}-Synuclein Is an Adipocyte-Neuron Gene Coordinately Expressed with Leptin and Increased in Human Obesity J. Nutr., May 1, 2008; 138(5): 841 - 848. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. R. Steinberg, B. E. Kemp, and M. J. Watt Adipocyte triglyceride lipase expression in human obesity Am J Physiol Endocrinol Metab, October 1, 2007; 293(4): E958 - E964. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Musaad and E. N. Haynes Biomarkers of Obesity and Subsequent Cardiovascular Events Epidemiol. Rev., May 10, 2007; (2007) mxm005v1. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Faraj, G. Beauregard, E. Loizon, M. Moldes, K. Clement, Y. Tahiri, K. Cianflone, H. Vidal, and R. Rabasa-Lhoret Insulin regulation of gene expression and concentrations of white adipose tissue-derived proteins in vivo in healthy men: relation to adiponutrin. J. Endocrinol., November 1, 2006; 191(2): 427 - 435. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Moldes, G. Beauregard, M. Faraj, N. Peretti, P.-H. Ducluzeau, M. Laville, R. Rabasa-Lhoret, H. Vidal, and K. Clement Adiponutrin gene is regulated by insulin and glucose in human adipose tissue Eur. J. Endocrinol., September 1, 2006; 155(3): 461 - 468. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Y. Kim, K. Tillison, J.-H. Lee, D. A. Rearick, and C. M. Smas The adipose tissue triglyceride lipase ATGL/PNPLA2 is downregulated by insulin and TNF-{alpha} in 3T3-L1 adipocytes and is a target for transactivation by PPAR{gamma} Am J Physiol Endocrinol Metab, July 1, 2006; 291(1): E115 - E127. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. E. Johansson, J. Hoffstedt, H. Parikh, E. Carlsson, M. Wabitsch, A.-G. Bondeson, J. Hedenbro, H. Tornqvist, L. Groop, and M. Ridderstrale Variation in the Adiponutrin Gene Influences Its Expression and Associates With Obesity Diabetes, March 1, 2006; 55(3): 826 - 833. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. C. Lake, Y. Sun, J.-L. Li, J. E. Kim, J. W. Johnson, D. Li, T. Revett, H. H. Shih, W. Liu, J. E. Paulsen, et al. Expression, regulation, and triglyceride hydrolase activity of Adiponutrin family members J. Lipid Res., November 1, 2005; 46(11): 2477 - 2487. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
