Changes in concentration of serum adiponectin multimeric forms following weight reduction programme in prepubertal obese children*
Zmiana stężeń mulrimetrycznych form adiponektyny w surowicy krwi dzieci otyłych w okresie przedpokwitaniowym po zastosowaniu programu terapeutycznego
Joanna Gajewska1, Halina Weker2, Jadwiga Ambroszkiewicz1, Magdalena Chełchowska1, Małgorzata Więch2, Teresa Laskowska-Klita1
1 Screening Department
Head of Department: dr. M. Ołtarzewski
2 Department of Nutrition
Head of Department: prof. nadzw. dr hab. n. med. H. Weker
Institute of Mother and Child in Warsaw
Director: S. Janus
* This work was supported by grant from Polish Ministry of Science and Higher Education nr NN 407173534.
Introduction: It is widely recognized that lifestyle intervention with modification of dietary habits and physical activity is effective in weight reduction and may improve the biochemical parameters in obese children and adolescents. However, the levels of adiponectin multimeric complexes during lifestyle intervention have not been extensively studied in obese children.
Aim: The aim of this study was to investigate the effect of the 3-months weight-reduction programme on serum adiponectin multimeric complexes in obese prepubertal children.
Material and methods: Changes in clinical, anthropometric and metabolic parameters including total adiponectin and its multimeric forms were assessed in 30 obese children aged 4-10 years, after a 3-months lifestyle intervention programme. This programme consisted of dietary and physical activity modifications and behaviour therapy including individual psychological care for the child and its family. The recommended daily intake from low-energy diet was 1200-1400 kcal/day. The reference group consisted of 35 healthy normal-weight children. Concentration of serum total adiponectin (Total-A) and its multimeric complexes: low molecular weight (LMW) adiponectin, medium molecular weight (MMW) adiponectin and high molecular weight (HMW) adiponectin were measured by ELISA kit.
Results: We observed that the concentrations of total adiponectin and HMW adiponectin were 25% (p<0.01) and 45% (p<0.0001) lower respectively, in obese children compared to controls. HMW/ Total-A ratio was lower in the obese children than in the controls (p<0.001), whereas LMW/Total-A ratio was higher in the obese as compared to the normal-weight children (p<0.001). After 3-months therapy the increases of total adiponectin concentration (by 20%, p<0.001), HMW (by 25%, p<0.001) and MMW adiponectin (by 30%, p<0.05) were recorded in these patients in comparison to baseline values. The increase of HMW/Total-A ratio (p<0.05) and the decrease of LMW/Total-A ratio (p<0.05) were also found in obese patients after weight-reduction programme as compared to the level before therapy. After modification of the diet and physical activity, the BMI of the obese children declined by 10% (p<0.01).
Conclusions: Our results indicate that the weight loss in children after 3-months therapy is associated with the increase of total and HMW, MMW adiponectin concentration. This suggests that the intervention programme is sufficient to detect significant changes in adiponectin multimeric profile, which confirms the efficacy of this therapy in prepubertal obese children
Key words: adiponectin, multimeric complex, obesity, children, weight loss
Wprowadzenie: Zmiana stylu życia obejmująca wprowadzenie odpowiedniej diety i zwiększenie aktywności fizycznej może skutecznie obniżać masę ciała oraz normalizować niektóre parametry biochemiczne u otyłych dzieci i młodzieży. Do tej pory niewiele jest danych dotyczących poziomu multimerycznych kompleksów adiponektyny w surowicy dzieci otyłych oraz poddanych terapii obniżającej masę ciała.
Cel: Celem pracy było zbadanie efektu 3-miesięcznego programu leczniczego na stężenie form multimerycznych adiponektyny u otyłych dzieci w okresie przedpokwitaniowym.
Materiał i metody: Grupę badaną stanowiło 30 dzieci otyłych w wieku 4-10 lat przed i po zastosowaniu 3-miesięcznego programu leczniczego obejmującego wprowadzenie diety ubogoenergetycznej i zwiększenie aktywności fizycznej. Wartość energetyczna całodziennej racji pokarmowej wynosiła 1200-1400 kcal. Równocześnie wprowadzono poradnictwo wychowawcze i indywidualną psychoterapię dla dzieci i ich rodzin. Oceniono zmiany wskaźników antropometrycznych oraz metabolicznych obejmujących stężenie adiponektyny i jej form multimerycznych we krwi. Grupę kontrolną stanowiło 35 zdrowych dzieci o prawidłowej masie ciała w wieku odpowiadającym grupie badanej. Metodą ELISA oznaczono w surowicy krwi stężenie adiponektyny całkowitej i jej form multimerycznych o małej (LMW), średniej (MMW) i dużej masie (HMW) cząsteczkowej.
Wyniki: W grupie dzieci otyłych w porównaniu do kontroli stwierdzono niższe stężenie adiponektyny całkowitej oraz jej formy HMW odpowiednio o 25% (p<0,01) i 45% (p<0,0001). Stosunek HMW/adiponektyna całkowita był niższy (p<0,001) podczas gdy stosunek LMW/adiponektyna całkowita był wyższy (p<0,001) u dzieci otyłych niż w grupie kontrolnej. U pacjentów po 3 miesiącach terapii wykazano wzrost stężenia całkowitej adiponektyny (o 20%, p<0,001), HMW (o 25%, p<0,001) i MMW (o 30%, p<0,05) w porównaniu do wartości uzyskanych przed wprowadzeniem programu leczniczego. U dzieci otyłych po leczeniu wykazano wzrost stosunku HMW/adiponektyna całkowita (p<0,05) oraz obniżenie stosunku LMW/adiponektyna całkowita (p<0,05). Stwierdzono również obniżenie wskaźnika masy ciała średnio o około 10% (p<0,05).
Wnioski: Uzyskane wyniki wskazują, że po 3-miesięcznej terapii odchudzającej u dzieci otyłych wraz z obniżeniem masy ciała wzrasta stężenie całkowitej adiponektyny i jej form HMW i MMW. Tego rodzaju zmiana profilu form multimetrycznych adiponektyny może potwierdzać skuteczność zastosowanego programu leczniczego u dzieci otyłych w okresie przedpokwitaniowym.
Słowa kluczowe: adiponektyna, kompleks multimeryczny, otyłość, dzieci, obniżenie masy ciała
Adipose tissue is a dynamic endocrine organ that secretes various adipokines, among them adiponectin, that play a role in the regulation of adipose tissue and whole-body metabolism. Adiponectin is one of the most abundant plasma protein and is structurally similar to complement 1q, which belongs to a family of proteins that form characteristic multimers (1, 2). Adiponectin monomer comprising 244 amino acids has an aminoterminal collagen-like domain and a carboxy-terminal globular domain. However, monomeric gene product is not found in the circulation, because adiponectin undergoes extensive posttranslational modification and circulates in trimeric (LMW adiponectin, low molecular weight adiponectin), hexameric (MMW adiponectin, medium molecular weight adiponectin) and oligomeric (HMW adiponectin, high molecular weight adiponectin) forms (3). The protein forms homotrimers via the noncovalent interactions of the collagenous domain in a triple helix. Hydrophobic interactions between globular domains may also contribute to trimer stability and help to initiate triple helix formation. For the integrity of adiponectin multimers intertrimer disulfide bonds via a cysteine residue in the variable domains are necessary (4). The three multimeric forms are found in the circulation. Adiponectin production by the adipocytes is a multistep process that can be regulated at the level of gene expression, secretion and/or formation of the multimeric forms of the protein. Reactive oxygen species (ROS) and pro-inflammatory cytokines are potent inhibitors of adiponectin gene expression in cultured adipocytes and could contribute to lowering adiponectin release by adipose tissue (5, 6). Regulation of adiponectin action is complicated and occurs at many levels, including availability of its receptor isoforms. Adipo R1 and AdipoR2 (adiponectin cell-surface receptors) are expressed in muscle, liver and adipose tissue (7). Adipo R1 is ubiquitously expressed, including abundant expression in skeletal muscle, whereas AdipoR2 is most abundantly expressed in liver. In myocytes and hepatocytes adiponectin stimulates phosphorylation and the activation of AMPK (5’-AMP-activated protein kinase), a key regulatory enzyme in glucose and lipid metabolism, inducing glucose uptake and fatty acid oxidation in muscle and reducing hepatic gluconeogenesis (8, 9).
Adiponectin acts through AMPK stimulation, which mediates several cellular processes influenced by adiponectin in vascular endothelial cells and the heart, which may participate in its protective effect against cardiovascular diseases (CVD) (10). Hypoadiponectemia has been demonstrated in patients with obesity, diabetes and coronary artery disease, all of which are linked to insulin resistance. Decreased plasma adiponectin levels have been shown to be closely associated with the clinical phenotype of metabolic syndrome (11).
Paediatric obesity is an important risk factor for adult hypertension and type 2 diabetes, and approximately one third of obese children are hypertensive and/or hyperinsulinemic (12). Additionally, early childhood obesity may play an important role in the development of a metabolic syndrome characterized by abnormalities in several major cardiovascular risk factors. It is widely recognized that lifestyle intervention with modification of dietary habits and physical activity is effective in weight reduction and in improvement of biochemical parameters in obese children and adolescents. Some authors observed that weight reduction is also accompanied by an increase of adiponectin concentrations in the plasma (13-15). Adiponectin levels in obese children were negatively correlated to age, body fat, and insulin resistance and decreased in puberty (13). However, it has been suggested that assessment of total adiponectin may be insufficient and that analysis of the relative levels of the multimeric forms should be analysed (16). To date, the relations of oligomeric, hexameric and trimeric adiponectin during lifestyle intervention have not been extensively studied in obese subjects. Therefore, the aim of this study was to investigate the effect of the 3-months weight-reduction programme on plasma adiponectin multimeric complexes in obese prepubertal children.
Material and methods
The changes in clinical, anthropometric and metabolic parameters including total adiponectin and its multimeric forms in 30 prepubertal patients (mean age±SD 7.8±1.3 years) before and after the 3-months intervention programme, were determined. Additionally, follow-up visits of patients took place after 6 weeks of therapy. Healthy normal-weight children (n=35; mean age±SD 7.2±2.0 years) were the reference group. Physical examination was performed and body mass index (BMI) calculated (Tab. I). Children were classified as obese (z-score BMI≥2) and non-obese (z-score BMI <-1+1>). Percentage body fat was calculated based on a skinfold-thickness equation according to Slaughter at al. (17). Patients with endocrine disorders or genetic syndromes, including syndromic obesity were excluded. Obese and non-obese children who were taking medications that could affect growth, pubertal development, nutritional status or dietary intake were not included. The lifestyle intervention programme consisted of dietary and physical activity modifications and behavior therapy including individual psychological care for the child and its family. The dietary guidelines, recommending the low-energy diet based on a balanced distribution of carbohydrates, proteins and lipids for children and their parents were described in a previous study (18). The recommended daily intake from low-energy diet was 1200-1400 kcal/day (tab. I). Patients had 3-5 meals per day. The dietary intake and physical activity data were collected using randomly selected 3-day records before and after treatment. Average daily food rations and their nutritional value were calculated using nutritional computer programme (Dietetyk2®, National Food and Nutrition Institute, Warsaw). Physical activity of children before and during therapy was evaluated by their parents using a questionnaire (18). Patients received the instruction concerning physical exercises which were described by Oblacińska and Weker (19). The children were advised to reduce sedentary behavior including television and computer games to less than two hours a day. This study had been approved by the Ethics Committee of the Institute of Mother and Child. Informed consent was obtained from the parents of all the examined children.
Venous blood samples were collected between 800 and 1000 in the morning after overnight fasting and configured (1000 g for 10 min at 4° C). Serum levels of total, HMW, MMW and LMW adiponectins were determined using ELISAkits (ALPCO Diagnostics, USA). Adiponectin multimers were selectively measured after sample pretreatment with two proteases that specifially digest5ed the trimeric forms or both the hexameric and trimeric forms. In this assay total adiponectin and HMW levels are determined directly, while the LMW and MMW levels are calculated indirectly. MMW concentration is calculated by substracting the concentration of HMWfrom the combined concentration of MMW + HMW adiponectin. The dynamic range of this kit is 0.075-4.8 ng/ml. Intraassay variations (CV%) were 5.3%, 4.1% and 3.3% for total adiponectin, MMW + HMW and HMW respectively. Interaassay variations (CV%) were 5.0%, 6.0% and 5.7% for total adiponectin, MMW + HMW and HMW respectively. Parameters were determined twice in obese children before and after 3-months therapyand once in the control subjects. To reduce interaassay variance, samples obtained before and after therapy were analyzed in one assay.
The Statistica (version 8.0) computer software was used for statistical analysis. The results were statistically analyzed by applying paired or unpaired Student’s t-test. Paired Student’s t-test was used to compare values before and after therapy within groups. Linear regression analysis with Pearson coefficient was used for correlation. The data are presented as means±SD, with p<0.05 considered statistically significant.
The average daily food rations of studied obese children and healthy normal-weight children are presented in table I. The diet of obese patients before therapy was characterized by the energy intake higher by about 20% (p<0.01) in comparison to diet of the control group. Energy intake in the diet of obese children was lower by 25% (p<0.0001) after 3-months of therapy and similar to that of their normal-weight counterparts. Additionally, average daily food ration energy from protein of the obese children was higher by about 15% (p<0.01) after therapy as compared to the value obtained before. The proportions of fat and carbohydrates in energy intake of these diets were similar to daily intake of diet in the control group.
Table II presents clinical characteristics of prepubertal obese children and normal-weight controls. Obese children before therapy had their BMI higher by 60% on average and their fat mass two times higher than age-matched controls. BMI and fat mass were lower by 10% (p<0.02; p<0.05) in obese children after modification of their diet and physical activity. However, value of BMI as well as fat mass of these patients after therapy were still higher by about 45% and 80%, respectively, than in control group.
Serum concentrations of adiponectin and its multimeric forms in obese children and controls are shown on figure 1. The mean level of total adiponectin and its HMW form in patients before treatment were lower by 25% (p<0.01) and 45% (p<0.0001), respectively, than in controls. Similar values of MWM and LMW adiponectins in both groups were observed. Additionally HMW and LMW forms correlated with total adiponectin in obese children (r=0.902, p<0.0001; r=0.639, p<0.001) as well as in controls (r=0.835, p<0.0001; r=0.524, p<0.001).
After 3-months therapy higher concentrations of total adiponectin (by 20%, p<0.001), HMW (by 25%, p<0.001) and MMW (by 30%, p<0.05) forms were found in obese children in comparison to baseline (fig. 1). The mean value of HMW in patients after the therapy was lower by 30% (p<0.01) than in controls. The concentration of LMW adiponectin remained unchanged in obese children before and after 3-months therapy. HMW and LMW concentrations correlated with total adiponectin in obese children after therapy (r=0.770, p<0.0001; r=0.464, p<0.01). Total adiponectin, HMW and LMW forms correlated before and after therapy (r=0.790, p<0.0001; r=0.892, p<0.0001; r=0.514, p<0.01, respectively). No correlations were found between total adiponectin, multimeric forms and BMI in the studied groups.
The fractional adiponectin levels relative to total adiponectin (Total-A) are shown on figure 2. HMW/ Total-A ratio was lower (p<0.001), whereas LMW/Total A ratio was higher in the obese children as compared to the normal-weight children (p<0.001). In obese patients after the weight-reduction programme we observed the increase of HMW/Total-A ratio (p<0.05) and the decrease of LMW/Total-A ratio (p<0.05) compared to their level before the therapy. MMW/Total-A ratio was similar in obese children before and after therapy as it was in controls.
We investigated the effect of the 3-months weight reduction therapy on total adiponectin concentrations and the distribution of adiponectin isoforms in serum of obese prepubertal children. Additionally, we compared the distribution of each multimer of serum adiponectin in healthy non-obese subjects. In our study, total adiponectin concentrations were significantly lower in obese children before therapy than in normal weight children in contrast to Martos-Moreno et al. (20), who found higher level of this protein in obese than non-obese subjects. However, our results concerning total adiponectin confirmed previous observations of Reinehr et al. (13), Cambuli et al. (14) and Araki et al. (21). Plasma adiponectin levels have also been reported to be reduced in obese adults, particularly those with visceral obesity, and to correlate inversely with insulin resistance (22).
In our study we obtained differences in profile of adiponectin isoforms between obese and normal-weight children. Significantly lower concentrations of HMW form in obese subjects were found, which support the findings of other authors (20, 21, 23). According to Araki et al. (21) HMW adiponectin reflects metabolic abnormalities due to obesity in children better than total adiponectin. Selective decrease in HMW concentration and HMW/ Total-A ratio was shown by authors in the group with metabolic syndrome as was the case in a recent adult study (11). Additionally, Hara et al (11) have reported that HMW form is also more clinically useful to estimate the risk of coronary artery disease and atherosclerosis. Incipient atherosclerosis in obese juveniles and adolescents is associated with specifically altered subfractions of adiponectin. According to Mangge et al. (23) HMW form in this group showed a better correlation with the carotid intima-media thickness as compared with total adiponectin. Based on these observations the HMW form may be more clinically relevant.
In our study we observed similar values of MMW and LMW adiponectins in obese and non-obese groups (5-10 years). The lower level of MMW form and higher level of LMW form in the obese Japanese children (5-15 years) compared to controls were found by Araki et. al (21). Similarly to our results Mangge et al. (23) obtained unchanged concentrations of the MMW and LMW forms as well as lower concentrations of total adiponectin and the HMW form in obese juveniles and adolescents (10-19 years) in comparison to the aged-matched controls. It is known that process of oligomerization of adiponectin monomers to the HMW adiponectin may be very important for the biological activity of this adipokine. In obese individuals the oligomerization may be disturbed due to oxidative stress induced by chronic inflammation (23). The decreased HMW/Total-A ratio and the increased LMW/Total-A ratio showed previously by Mangge et al. (23) in obese juveniles and by us in obese children may be a consequence of a disturbance in oligomerization process from LMW to HMW adiponectin. Furthermore, our observations suggest that the changes in profile of adiponectin isoforms in obese individuals may exist from prepubertal period.
The results concerning changes in total plasma adiponectin concentration after weight loss in obese adults are inconsistent. According to Kovacova 2009 (24) eight weeks of very low-calorie diet promoting changes in body weight and insulin sensitivity did not induce changes in secretion and plasma levels of adiponectin isoforms. No change in total plasma adiponectin following a moderate weight loss was also found in studies (25), whereas an increase in plasma adiponectin following large weight reduction has been described by other authors (26). Studies investigating changes in adiponectin multimeric complexes in plasma following lifestyle interventions have also showed contradictory results. Some showed no changes in their distribution (27, 28), while others found increased quantity of some of the multimeric forms (29, 30) or only increase of HMW form (31).
Reinehr et al. (13), Cambuli et al. (14) and Roth et al. (15) demonstrated the increase in adiponectin levels in obese children as a result of a significant weight loss after 1 year of lifestyle intervention. Martos-Moreno (20) obtained similar results in prepubertal children during longitudinal therapy. In our study, we observed weight loss and the increase of total adiponectin concentrations in obese children after 3-months therapy in comparison to the baseline. In agreement with the previous findings (20) we also recorded the increased level of HMW form and additionally MMW form as well as significant changes in HMW/Total-A and LMW/Total-A ratios after therapy. The LMW/Total-A ratio decreased in children after therapy to values observed in control group. Moreover, HMW/ Total-A ratio was still lower in these patients than in controls but was significantly higher as compared to the value obtained before therapy.
Our results showed significantly changes in profile of multimeric forms after 3-months therapy in obese children. However, it has not yet been clearly established which adiponectin forms are biologically active. According to Polak et al. (30) and Waki et al. (32) both the HMW and MMW forms are able to stimulate AMPK in primary culture hepatocytes and might therefore have similar effects on hepatocytes . However, the specific role and function of the LMW form relative to the other adiponectin multimeric complexes has not yet been elucidated.
Our results suggest that the weight loss in children after 3-months weight reduction therapy is associated with the increase of total and HMW, MMW adiponectin concentration. Moreover, this therapy also improved HMW/Total-A and LMW/Total-A ratio in studied obese subjects. It suggests that the intervention programme is sufficient to detect significant changes in adiponectin multimeric profile, which confirms the efficacy of this therapy in prepubertal obese children.
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