Original Paper
HOR MON E RE SE ARCH I N PÆDIATRIC S
Horm Res Paediatr 2013;80:363–370 DOI: 10.1159/000356046
Received: June 6, 2013 Accepted: September 27, 2013 Published online: November 7, 2013
Increased Leptin/Adiponectin Ratio and Free Leptin Index Are Markers of Insulin Resistance in Obese Girls during Pubertal Development M.A. Donoso a M.T. Muñoz-Calvo b, c V. Barrios b, c G. Martínez d F. Hawkins d J. Argente b, c a
Department of Pediatrics, Hospital Ruber Internacional, b Department of Pediatrics and Pediatric Endocrinology, Hospital Infantil Universitario Niño Jesús, Instituto de Investigación Sanitaria La Princesa and Department of Pediatrics, Universidad Autónoma de Madrid, c Centro de Investigación Biomédica en Red de Fisiopatología de la Obesidad y Nutrición (CIBERobn), Instituto de Salud Carlos III, and d Department of Endocrinology, Hospital 12 de Octubre, Universidad Complutense, Madrid, Spain
Abstract Background: Modifications in body fat in obese patients during puberty determine changes in adipokines that affect insulin sensitivity. Aims: We hypothesized that the leptin/ adiponectin (L/A) ratio and free leptin index (FLI) are good markers of insulin resistance (IR) and total body fat (TBF) during pubertal development. Methods: A prospective study of 32 obese girls (OG) and age-matched control girls (CG) was performed. OG were divided into those that maintained a weight loss (WL) of >1 SD of initial body mass index (BMI) (WL group, n = 25) and those without WL (NWL group, n = 7). Oral glucose tolerance tests (OGTT) were performed to evaluate IR. Correlations of adipokines, L/A, and FLI with BMI, waist circumference, percentage of TBF (%TBF) and IR were performed over pubertal development. Results: The L/A ratio and FLI were increased in OG at baseline. Both indexes decreased in the WL group as puberty progressed, with no change in CG or NWL. In the WL group, a correlation between L/A and FLI with OGTT and %TBF, and L/A and ho-
© 2013 S. Karger AG, Basel 1663–2818/13/0805–0363$38.00/0 E-Mail [email protected] www.karger.com/hrp
meostasis model assessment (HOMA) was found throughout the study. Conclusion: The L/A ratio and FLI are good markers to follow changes in IR and %TBF after WL during puberty. Insulin more accurately reflects the changes in IR than HOMA. © 2013 S. Karger AG, Basel
Introduction
Epidemiological data provide evidence that the frequency of obesity and cardiometabolic risk factors show an increasing tendency in children. A transient decrease in insulin sensitivity during puberty is a well-known physiological process. Hormonal changes and body composition may be important for determining cardiometabolic risk factors [1], given that insulin resistance (IR) plays an important role in the pathogenesis of cardiovascular and associated metabolic disturbances of obesity [2]. Adipose tissue secretes adipokines influencing body weight and glucose and lipid metabolism [3] and vice versa. Leptin and adiponectin are amongst the most studied adipokines in obesity and its secondary complications. Jesús Argente, MD, PhD Hospital Infantil Universitario Niño Jesús Avda. Menéndez Pelayo, 65 ES–28009 Madrid (Spain) E-Mail jesus.argente @ uam.es
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Key Words Adiponectin · Insulin resistance · Leptin · Obesity · Puberty · Fat mass · Cardiovascular risk
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Horm Res Paediatr 2013;80:363–370 DOI: 10.1159/000356046
Subjects and Methods Subjects We carried out a prospective follow-up study during 36 months in 32 obese female adolescent patients (OG), with a mean age of 11.3 ± 0.8 years, and 30 healthy girls at different stages of puberty (Tanner stage II, III, and V, with a mean age of 10.1 ± 0.2, 11.9 ± 1.6, and 12.5 ± 1 years, respectively). Obese patients were enrolled from the Endocrinology Department of the University Hospital Niño Jesús in Madrid at Tanner stage II and followed during pubertal development. These patients were classified into three groups according to the evolution of puberty: group 1: Tanner pubertal stage II (beginning of puberty); group 2: Tanner pubertal stage III (mid-puberty), and group 3: Tanner pubertal stage V (late puberty) [22]. The control age-matched group (control girls, CG), consisting of females at different stages of puberty (II, III, and V) with a similar BA, was recruited from a local school. Obese patients were divided into a group that maintained a WL of >1 SD of body mass index (BMI) (WL group, n = 25) and another group without WL (NWL group, n = 7). The age of menarche was taken into consideration. The study protocol was approved by the Institution’s Ethics Committee and informed written consent was obtained from the patients and their parents. Methods Nutritional and Auxological Evaluation and Quantification of TBF Medical and physical examinations were performed monthly in OG. Nutritional status was determined by the following anthropometric measurements: height, measured by using a stadiometer (Holtain Ltd., Crymych, UK); weight, on a SECA scale, and BMI, calculated as body weight (kg) divided by the squared standing height (m2). All anthropometric measurements are expressed as standard deviation score (SDS) for age and sex referred to a normal Spanish population [23]. Growth velocity was determined in cm/ year and expressed in SDS for the chronological age. Bone age (BA) was assessed every year according to the method of Greulich and Pyle [24]. The waist circumference (WC) was expressed in SDS, and results were compared with those obtained by Moreno et al. [25]. Blood pressure was compared to Task Force reference tables [26]. The recommendation in this group was aimed at promoting WL and included an equilibrated normal calorie diet adjusted to the patient’s age and an increase in physical exercise [27]. TBF was evaluated at baseline (Tanner stage II) and Tanner stage III and V by whole-body energy X-ray absorptiometry (DEXA; QDR-4500W, Hologic Waltham, Mass., USA). The in vivo coefficient of variation of this technique was below 1.5%. The results of TBF are expressed in kilograms and the %TBF calculated. In the control group, TBF and %TBF were also analyzed and calculated at the different stages of puberty. Biochemical Measurements Blood samples were collected after overnight fasting at 08: 00 a.m. at each pubertal stage. Serum leptin and sOB-R levels were measured by radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA), respectively, according to the manufacturer’s directions (Millipore, St. Charles, Mo., USA, and BioVendor Laboratory Medicine, Brno, Czech Republic) as previously reported [20]. Adiponectin levels were analyzed by RIA (Millipore) and insulin by RIA (Diagnostic Products Corp., Los Angeles, Calif.,
Donoso /Muñoz-Calvo /Barrios /Martínez / Hawkins /Argente
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Although their association with various diseases remains controversial, many clinical studies have demonstrated that low plasma concentrations of adiponectin are related to obesity-related diseases, including atherosclerotic cardiovascular diseases, type 2 diabetes mellitus, hypertension, and dyslipidemia [4–7]. Leptin and adiponectin levels have been proposed as markers for predicting metabolic syndrome (MS), type 2 diabetes mellitus, and cardiovascular disease [8, 9] in children, with obese patients having reduced plasma levels of adiponectin [5, 6] and increased leptin levels [10]. Recently, ratios between leptin and adiponectin (L/A) and leptin with its receptor (free leptin index, FLI) are suggested to provide information regarding the risk of MS and cardiovascular disorders [11–14]. Several studies show the importance of weight loss (WL) in improving IR associated with obesity [15, 16] and also in reducing cardiovascular risk factors. Unfortunately, there is no numerical definition of IR in children and adolescents that is accepted worldwide. The gold standard to determine insulin sensitivity is the hyperinsulinemic-euglycemic clamp, but it is not applicable for routine evaluation. Other less invasive methods have been developed to evaluate IR, such as the homeostasis model assessment (HOMA) index and the elevation of insulin levels during the oral glucose tolerance test (OGTT) [17]. Modifications in total body fat (TBF) in obese patients during puberty result in changes in serum levels of leptin, its soluble receptor, and adiponectin, which in turn could be involved in the regulation of energy balance [18]. Leptin circulates freely and bound to its soluble receptor (soluble leptin receptor, sOB-R), which affects its bioavailability. The proportion of leptin bound to sOB-R is related to changes in adiposity, with free leptin more accurately reflecting body fat mass [19]. Circulating levels of leptin increase and sOB-R levels decrease during pubertal development, resulting in an increase in free leptin [20]. Puberty is a critical period for the development of obesity, as changes in hormone levels and body fat composition are coupled to behavioral changes [21]. WL during this stage induces changes in adipokine production and a decrease in IR and cardiovascular risk. Our aims were to determine the utility of measuring leptin and adiponectin levels, as well as L/A and FLI, as markers of IR and to analyze the possible relationship of these indexes with changes in the percentage of TBF (%TBF) in obese girls (OG) during pubertal development.
Statistical Analysis Results are expressed as means ± SD. The normal distribution of each parameter in all groups was assessed, and differences between controls and obese patients were analyzed by Student’s t test if a normal distribution was obtained or by Kolmogorov-Smirnov goodness of fit if not. Comparison of baseline measurements and changes over time were made by ANOVA with repeated measures, followed by Bonferroni post hoc test. Pearson’s correlation coefficient was used to investigate the association between the parameters studied with quantitative variables with a normal distribution. When this requirement was not met, we used the nonparametric Spearman test. The level of significance chosen was p < 0.05. Data were analyzed using SPSS (15.0) software for Windows (MapInfo Corporation, Troy, N.Y., USA).
Results
Obese Patients Have Normal Growth and a Slight Advancement in BA and Pubertal Onset The auxological changes are shown in table 1. The mean chronological age of menarche was 11.7 ± 0.3 years, with an age range between 10 and 13.5 years. The mean BA at menarche was 13.3 ± 0.5 years (range 12–14), with a difference between bone and chronological age of 1.1 ± 1 years throughout the study period. The interval between the onset of puberty and menarche was 1.5 ± 0.4 years (range 1–2.4). There was a peak in growth rate during the first 12 months of follow-up with a total gain of 14.9 ± 2.3 cm (range 11.2–19.2). There was an increase in TBF, as well as in %TBF, in OG during follow-up (table 1). In the WL group, there was a decrease (p < 0.05) in %TBF at Tanner stage III (table 1), but this parameter was elevated compared to CG during the progression of puberty. Leptin/Adiponectin Index in Obese Girls
Table 1. Auxological and anthropometric parameters and %TBF
in OG and CG Tanner Groups stage WL CA at puberty onset II
NWL
CG
10.1 ± 0.2*
10.1 ± 0.2*
11.5 ± 1*
BA-CA
II III V
1.1 ± 1* 1.2 ± 1* 1.2 ± 1*
1.1 ± 1* 1.2 ± 1* 1.2 ± 1*
0.2 ± 1* 0.3 ± 1* 0.2 ± 1*
BMI
II III V
3.8 ± 1** 2.7 ± 1**, ## 2.7 ± 1**, ##
4.8 ± 1** 4 ± 1** 4 ± 1**
0.6 ± 1 0.8 ± 1 0.2 ± 1
WC, cm
II III V
79 ± 1** 78 ± 2** 75 ± 1**, +
60 ± 1 84 ± 2** 62 ± 1 85 ± 1** 87 ± 4**, + 62.6 ± 1
%TBF
II III V
40 ± 1** 35 ± 2**, # 37 ± 1**, #
42 ± 2** 40 ± 1** 39 ± 4**
27 ± 1 24 ± 1 25 ± 1
CA = Chronological age. * p < 0.05, ** p < 0.01, compared with the CG; # p < 0.05, ## p < 0.01, comparing the same group at different moments with baseline; + p < 0.05, WL compared with the NWL group.
Relationship of WL with IR, Lipid Metabolism, and MS In the WL group, the prevalence of hyperinsulinemia was 47.8% at baseline, falling to 42.8% at Tanner stage III and 16.6% at stage V. In this group, we also observed a significant decrease in the percentage of girls with IR as determined by HOMA values higher than 2 SD compared with national reference values according to age, sex, and pubertal stage [29], with 47.8, 42.8, and 11.1% at Tanner stage II, III, and V, respectively. However, in the NWL group, no changes in these parameters were found during follow-up. At Tanner stage II, 52.1% of OG presented IR when insulin values during OGTT were used. In the WL group, this percentage decreased more dramatically throughout the study (52.1% at Tanner stage II, 27.0% at Tanner stage III, and no patients with these criteria at Tanner stage V) than when HOMA was used to determine IR. Insulin levels and HOMA index are shown in table 2. We found a positive correlation between basal insulin levels with BMI, WC, and %TBF (r = 0.61, p < 0.01; r = 0.72, p < 0.01, and r = 0.73, p < 0.01, respectively). No correlation between L/A ratio or FLI and lipid profile were found. There was an increase in HDL-C values during Horm Res Paediatr 2013;80:363–370 DOI: 10.1159/000356046
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USA) as previously reported [28]. Intra- and inter-assay coefficients of variation were below 10% for all assays. Fasting insulin and HOMA were compared with reference values according to age, sex, and pubertal stage. Patients were considered to be hyperinsulinemic when fasting insulin levels were higher than 2 SDS and to be IR if HOMA values were higher than 2 SDS of normal values [29]. In obese patients, an OGTT was performed at each stage of puberty [30], with glucose and insulin levels determined every 30 min during 2 h [17]. IR was also defined as an insulin peak higher than 150 μIU/ml and insulin values at 2 h higher than 75 μIU/ml [31]. We also determined high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), triglycerides (TG), and total cholesterol (TC), and the TC/HDL-C, TG/ HDL-C, and HDL-C/LDL-C indexes were calculated. The FLI was determined as the leptin (ng/ml)/sOB-R (ng/ml) ratio and the L/A index as the ratio between leptin (ng/ml) and adiponectin (μg/ml). We evaluated the presence of MS by using the modified Cook criteria for children and adolescents [32] and alterations in glucose metabolism by using the criteria of the American Diabetes Association [30].
follow-up, and at Tanner stage V, a decrease in TC/HDLC and TG/HDL-C, as well as an increase in HDL-C/LDLC, was found in patients with WL. At Tanner stage II, 16.7% of patients had systolic hypertension, while at Tanner stage III, it was 4.8% and at Tanner stage V, no patient was found to be hypertensive. Three patients who had high blood pressure normalized this parameter with WL during follow-up of >1 SD of BMI. The prevalence of MS was 16% at Tanner stage II and 5% at Tanner stage III, with no child meeting the criteria for MS at Tanner stage V [30]. Adiponectin and Leptin Levels and Correlations with Metabolic Parameters Changes throughout puberty in adiponectin and leptin levels, as well as FLI and L/A indexes, are shown in figure 1. We found an increase in leptin values in OG with respect to CG (p < 0.01), with a decrease after WL (p < 0.05). Increases in sOB-R and adiponectin values after WL (p < 0.05 and p < 0.01, respectively; table 2) were also found. At baseline, the WL and NWL group had increased L/A and FLI indexes compared to CG. Both indexes decreased in the WL group as puberty progressed, with no change in the NWL group (table 2). There was a positive correlation between leptin and BMI, WC, and %TBF and a negative correlation with sOB-R throughout the study in the WL group. In the same group, we observed a direct correlation between the L/A ratio and FLI with insulin values in the OGTT, with this correlation being stronger with FLI. Moreover, these indexes also correlated with the %TBF throughout the study period. A direct correlation between L/A and HOMA was also found in all Tanner stage groups (table 3).
Table 2. Adipokine levels, insulin values during fasting and in
OGTT, and HOMA in OG and CG Tanner Groups stage WL
NWL
CG
Leptin, ng/ml
II III V
36 ± 1** 21 ± 1**, # 22 ± 1**, #
44 ± 1** 33 ± 1** 39 ± 1**, +
6±1 7±1 10 ± 1
Adiponectin, μg/ml
II III V
15 ± 1 19 ± 1*, # 19 ± 1*, #
13 ± 1 14 ± 1 14 ± 1
10 ± 1 10 ± 1 12 ± 1
FLI
II III V
2.9 ± 1 1 ± 1##, ++ 1 ± 1#, +
3.7 ± 1* 4 ± 1*, ++ 3.8 ± 1**, +
1±1 0.6 ± 0.1 0.7 ± 1
L/A index
II III V
2.5 ± 1** 1.3 ± 1*, #, + 1.3 ± 1#, +
0.5 ± 1 3.4 ± 1** 2.2 ± 0.2**, + 0.9 ± 1 2.5 ± 0.4**, + 0.9 ± 1
Insulin, μIU/ml
II III V
21 ± 2* 20 ± 2* 17 ± 1*, #
23 ± 1* 25 ± 1 22.5 ± 1
Insulin peak in OGTT, μIU/ml
II III V
151 ± 96 124 ± 75# 74 ± 20##
196 ± 54 188 ± 46 188 ± 56
Insulin at 120 min, II III μIU/ml V
95.2 ± 78 111 ± 59 47 ± 12##
144 ± 83 135 ± 14 108 ± 32
4.1 ± 1 3.3 ± 1# 2.9 ± 1#
5.4 ± 1 5.6 ± 1 5.3 ± 0
HOMA
II III V
9±1 13 ± 1 10 ± 1
0.9 ± 1 1.2 ± 1 1±0
* p < 0.05, ** p < 0.01, compared with the CG; # p < 0.05, ## p < 0.01, comparing the same group at different moments with baseline; + p < 0.05, ++ p < 0.01, WL compared with the NWL group.
Discussion
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Horm Res Paediatr 2013;80:363–370 DOI: 10.1159/000356046
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Our results show that obese adolescent girls have normal growth, but a slight advancement in bone maturation and pubertal onset, as well as an increased incidence of IR. The major finding of this study is that the L/A ratio and FLI are good markers of IR in OG during pubertal development and are useful in monitoring changes in IR and TBF. Another finding is that insulin values during OGTT are better than the HOMA index to determine changes in IR after WL. We found accelerated bone maturation in OG that could be due to different factors such as nutritional status, body fat content, and physical activity [33, 34]. By con-
trast, ballet dancers, with a low BMI, have a delay in skeletal maturation and pubertal progression [35]. Thus, these divergent situations result in different patterns of pubertal evolution in accordance with the nutritional status and body fat content [35]. Hence, the amount of body fat may influence the onset and progression of puberty, as well as the age of menarche, possibly due, at least in part, to changes in the levels of several hormones, including leptin and adiponectin, that are closely related to the amount of fat [36–38]. The observed hyperleptinemia maintained throughout puberty, with decreased levels of leptin receptor, are consistent with previous studies [20, 39–42].
#
2
**
#
**
1 0
a
5
** **
3
CG WL group NWL group
4
6 5
** **
3
+
# + +
* **
2
# ** +
L/A index
4
6
**
**
FLI index
Adiponectin (μg/ml)
5
#
#
6
1 II
III
0
V
*
+
**
4 3 2
## +
# +
III
V
1 0
V
III
II
b
Tanner
++
*
II
c
Tanner
Tanner
Fig. 1. Adiponectin (a), FLI index (b), and L/A index (c) in OG throughout puberty. # p < 0.05, ## p < 0.01, com-
paring the same group at different moments with baseline; * p < 0.05, ** p < 0.01, compared with the CG; < 0.05, ++ p < 0.01, WL compared with the NWL group.
+ p
Table 3. Correlation between L/A and FLI indexes with HOMA, BMI, and %TBF in OG with WL
L/A index, r Tanner stage: HOMA %TBF BMI Insulin peak in OGTT, μIU/ml Insulin at 120 min, μIU/ml
FLI index, r
II
III
V
II
III
V
0.43* 0.58* 0.59** 0.45** 0.59**
0.48* 0.54* 0.70** 0.44* 0.58*
0.83** 0.86** 0.82** 0.81** 0.77**
0.31 0.86** 0.82** 0.79** 0.891**
0.11 0.78** 0.64* 0.70* 0.790*
0.12 0.83* 0.64* 0.83** 0.850*
The FLI can provide precise information about changes in the bioavailability of leptin [21, 43], with the levels of sOB-R possibly serving as an indicator of free leptin levels; thus, the FLI index, defined as the ratio of leptin to sOB-R, may be a more accurate determinant of leptin function. Here, we report that sOB-R levels are inversely correlated with BMI, as previously shown [44], and lower receptor levels increase free leptin concentrations, which could compensate for increased leptin resistance [45]. In addition, the WL group had a decreased FLI during puberty, which may be a compensatory mechanism of energy saving secondary to WL during puberty [46– 49]. Additionally, we observed a strong correlation of FLI with body fat, in line with previous data. It is suggested that leptin circulates predominantly in the bound form in lean subjects, whereas in obese patients, due to low
sOB-R levels, leptin circulates mainly in the free form [50]. Moreover, adolescent girls with anorexia nervosa have higher sOB-R levels and a lower FLI than healthy CG [51]. In contrast to leptin, adiponectin levels are reduced in OG, again in agreement with the role of body fat in the regulation of circulating levels of this adipokine [52]. However, although no correlation between adiponectin and BMI or body fat was found here, similar to that reported in other studies [53], the relationship of BMI and the %TBF with the L/A ratio suggests a greater sensitivity of this index to changes in body fat than either adiponectin or leptin alone. As leptin and adiponectin levels change inversely in relation to BMI, the L/A ratio has been purported to be a potential index relating adiposity to the development of complications of obesity [54].
Leptin/Adiponectin Index in Obese Girls
Horm Res Paediatr 2013;80:363–370 DOI: 10.1159/000356046
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r = Spearman’s ρ correlation (* p < 0.05, ** p < 0.01).
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tin alone [61], and similarly, we found that this index correlated with TBF and HOMA index. One caveat that should be taken into consideration when evaluating these results is that the control subjects were from a cross-sectional population. Although it could be of interest to follow the parallel evolution of both groups, the control population used in this study may present some advantages, especially regarding comparable anthropometric characteristics at a given age. In addition, in our design, we were able to maintain the number of control subjects at each time point. In conclusion, our results show that in OG with WL both the L/A ratio and FLI correlate with IR as measured by insulin values in OGTT. L/A also correlates with HOMA index. In addition, insulin values during OGTT are better than the HOMA index for determining an improvement in insulin sensitivity after WL.
Acknowledgements This work was supported by grants from Fondo de Investigación Sanitaria (PI10/0747) with the help of European FEDER funding, CIBERobn (CB03/06), and Funda-ción Endocrinología y Nutrición.
Disclosure Statement The authors declare that there is no conflict of interest that may be perceived as prejudicing the impartiality of the research reported.
References
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Excess body weight in children is closely related to IR. The deregulation of certain adipokines, such as leptin and adiponectin, due to excess body fat can have deleterious effects on insulin signaling [9, 36]. Our results show that changes in leptin and adiponectin after WL appear to be associated with a decrease in IR. OG have higher insulin values both at baseline and during OGTT, whereas after WL, there is a decrease in the percentage of girls with hyperinsulinemia and high HOMA values. This is in correlation with lower BMI and %TBF, as previously found [14, 55]. This emphasizes the importance of WL in the reduction of IR. The prevalence of IR decreased in OG after WL. This difference may be due to the fact that impaired glucose tolerance is a more restrictive parameter than fasting glucose and insulin to detect changes in glucose homeostasis and insulin sensitivity, indicating that OGTT is a more specific and stringent method to detect changes in IR [56]. The observed improvement in IR is very important, given that during puberty, insulin sensitivity normally decreases [21], and highlights the importance of WL during pubertal development in obese subjects. A combination of leptin and adiponectin as an index of insulin sensitivity was first suggested in 2003 [57]. Subsequently, many researchers have reported that the L/A ratio is a consistent marker of IR in patients with and without diabetes in comparison with other indicators [58] and may be used as an atherogenic index, providing additional information on the risk of MS [59]. We found a correlation between the L/A index and insulin values during OGTT and with HOMA values, suggesting again the possible utility of this ratio to predict the presence of IR, better than adiponectin or leptin alone [54]. In addition, FLI showed a direct relationship with insulin values in OGTT, but not with HOMA, suggesting that the L/A ratio is more related to IR than FLI. Many studies have described the association between higher body fat and increased cardiovascular risk and impaired lipid profiles [14]. These studies report that total fat is negatively correlated with HDL/LDL and positively correlated with TC/HDL, as we found here. During follow-up, despite the decrease in TBF, these associations persist, suggesting a close relationship between lipoproteins and body fat. We have also found that although TBF increases due to changes during puberty, the percentage is decreased due to WL and this could explain the improvement in the lipid profile and insulin sensitivity and lower L/A index and cardiovascular risk factors [60]. In fact, it has been reported that the L/A ratio correlates more strongly with BMI and HDL-C than does adiponec-
Leptin/Adiponectin Index in Obese Girls
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HOR MON E RE SE ARCH I N PÆDIATRIC S
Horm Res Paediatr 2013;80:363–370 DOI: 10.1159/000356046
Received: June 6, 2013 Accepted: September 27, 2013 Published online: November 7, 2013
Increased Leptin/Adiponectin Ratio and Free Leptin Index Are Markers of Insulin Resistance in Obese Girls during Pubertal Development M.A. Donoso a M.T. Muñoz-Calvo b, c V. Barrios b, c G. Martínez d F. Hawkins d J. Argente b, c a
Department of Pediatrics, Hospital Ruber Internacional, b Department of Pediatrics and Pediatric Endocrinology, Hospital Infantil Universitario Niño Jesús, Instituto de Investigación Sanitaria La Princesa and Department of Pediatrics, Universidad Autónoma de Madrid, c Centro de Investigación Biomédica en Red de Fisiopatología de la Obesidad y Nutrición (CIBERobn), Instituto de Salud Carlos III, and d Department of Endocrinology, Hospital 12 de Octubre, Universidad Complutense, Madrid, Spain
Abstract Background: Modifications in body fat in obese patients during puberty determine changes in adipokines that affect insulin sensitivity. Aims: We hypothesized that the leptin/ adiponectin (L/A) ratio and free leptin index (FLI) are good markers of insulin resistance (IR) and total body fat (TBF) during pubertal development. Methods: A prospective study of 32 obese girls (OG) and age-matched control girls (CG) was performed. OG were divided into those that maintained a weight loss (WL) of >1 SD of initial body mass index (BMI) (WL group, n = 25) and those without WL (NWL group, n = 7). Oral glucose tolerance tests (OGTT) were performed to evaluate IR. Correlations of adipokines, L/A, and FLI with BMI, waist circumference, percentage of TBF (%TBF) and IR were performed over pubertal development. Results: The L/A ratio and FLI were increased in OG at baseline. Both indexes decreased in the WL group as puberty progressed, with no change in CG or NWL. In the WL group, a correlation between L/A and FLI with OGTT and %TBF, and L/A and ho-
© 2013 S. Karger AG, Basel 1663–2818/13/0805–0363$38.00/0 E-Mail [email protected] www.karger.com/hrp
meostasis model assessment (HOMA) was found throughout the study. Conclusion: The L/A ratio and FLI are good markers to follow changes in IR and %TBF after WL during puberty. Insulin more accurately reflects the changes in IR than HOMA. © 2013 S. Karger AG, Basel
Introduction
Epidemiological data provide evidence that the frequency of obesity and cardiometabolic risk factors show an increasing tendency in children. A transient decrease in insulin sensitivity during puberty is a well-known physiological process. Hormonal changes and body composition may be important for determining cardiometabolic risk factors [1], given that insulin resistance (IR) plays an important role in the pathogenesis of cardiovascular and associated metabolic disturbances of obesity [2]. Adipose tissue secretes adipokines influencing body weight and glucose and lipid metabolism [3] and vice versa. Leptin and adiponectin are amongst the most studied adipokines in obesity and its secondary complications. Jesús Argente, MD, PhD Hospital Infantil Universitario Niño Jesús Avda. Menéndez Pelayo, 65 ES–28009 Madrid (Spain) E-Mail jesus.argente @ uam.es
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Key Words Adiponectin · Insulin resistance · Leptin · Obesity · Puberty · Fat mass · Cardiovascular risk
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Subjects and Methods Subjects We carried out a prospective follow-up study during 36 months in 32 obese female adolescent patients (OG), with a mean age of 11.3 ± 0.8 years, and 30 healthy girls at different stages of puberty (Tanner stage II, III, and V, with a mean age of 10.1 ± 0.2, 11.9 ± 1.6, and 12.5 ± 1 years, respectively). Obese patients were enrolled from the Endocrinology Department of the University Hospital Niño Jesús in Madrid at Tanner stage II and followed during pubertal development. These patients were classified into three groups according to the evolution of puberty: group 1: Tanner pubertal stage II (beginning of puberty); group 2: Tanner pubertal stage III (mid-puberty), and group 3: Tanner pubertal stage V (late puberty) [22]. The control age-matched group (control girls, CG), consisting of females at different stages of puberty (II, III, and V) with a similar BA, was recruited from a local school. Obese patients were divided into a group that maintained a WL of >1 SD of body mass index (BMI) (WL group, n = 25) and another group without WL (NWL group, n = 7). The age of menarche was taken into consideration. The study protocol was approved by the Institution’s Ethics Committee and informed written consent was obtained from the patients and their parents. Methods Nutritional and Auxological Evaluation and Quantification of TBF Medical and physical examinations were performed monthly in OG. Nutritional status was determined by the following anthropometric measurements: height, measured by using a stadiometer (Holtain Ltd., Crymych, UK); weight, on a SECA scale, and BMI, calculated as body weight (kg) divided by the squared standing height (m2). All anthropometric measurements are expressed as standard deviation score (SDS) for age and sex referred to a normal Spanish population [23]. Growth velocity was determined in cm/ year and expressed in SDS for the chronological age. Bone age (BA) was assessed every year according to the method of Greulich and Pyle [24]. The waist circumference (WC) was expressed in SDS, and results were compared with those obtained by Moreno et al. [25]. Blood pressure was compared to Task Force reference tables [26]. The recommendation in this group was aimed at promoting WL and included an equilibrated normal calorie diet adjusted to the patient’s age and an increase in physical exercise [27]. TBF was evaluated at baseline (Tanner stage II) and Tanner stage III and V by whole-body energy X-ray absorptiometry (DEXA; QDR-4500W, Hologic Waltham, Mass., USA). The in vivo coefficient of variation of this technique was below 1.5%. The results of TBF are expressed in kilograms and the %TBF calculated. In the control group, TBF and %TBF were also analyzed and calculated at the different stages of puberty. Biochemical Measurements Blood samples were collected after overnight fasting at 08: 00 a.m. at each pubertal stage. Serum leptin and sOB-R levels were measured by radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA), respectively, according to the manufacturer’s directions (Millipore, St. Charles, Mo., USA, and BioVendor Laboratory Medicine, Brno, Czech Republic) as previously reported [20]. Adiponectin levels were analyzed by RIA (Millipore) and insulin by RIA (Diagnostic Products Corp., Los Angeles, Calif.,
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Although their association with various diseases remains controversial, many clinical studies have demonstrated that low plasma concentrations of adiponectin are related to obesity-related diseases, including atherosclerotic cardiovascular diseases, type 2 diabetes mellitus, hypertension, and dyslipidemia [4–7]. Leptin and adiponectin levels have been proposed as markers for predicting metabolic syndrome (MS), type 2 diabetes mellitus, and cardiovascular disease [8, 9] in children, with obese patients having reduced plasma levels of adiponectin [5, 6] and increased leptin levels [10]. Recently, ratios between leptin and adiponectin (L/A) and leptin with its receptor (free leptin index, FLI) are suggested to provide information regarding the risk of MS and cardiovascular disorders [11–14]. Several studies show the importance of weight loss (WL) in improving IR associated with obesity [15, 16] and also in reducing cardiovascular risk factors. Unfortunately, there is no numerical definition of IR in children and adolescents that is accepted worldwide. The gold standard to determine insulin sensitivity is the hyperinsulinemic-euglycemic clamp, but it is not applicable for routine evaluation. Other less invasive methods have been developed to evaluate IR, such as the homeostasis model assessment (HOMA) index and the elevation of insulin levels during the oral glucose tolerance test (OGTT) [17]. Modifications in total body fat (TBF) in obese patients during puberty result in changes in serum levels of leptin, its soluble receptor, and adiponectin, which in turn could be involved in the regulation of energy balance [18]. Leptin circulates freely and bound to its soluble receptor (soluble leptin receptor, sOB-R), which affects its bioavailability. The proportion of leptin bound to sOB-R is related to changes in adiposity, with free leptin more accurately reflecting body fat mass [19]. Circulating levels of leptin increase and sOB-R levels decrease during pubertal development, resulting in an increase in free leptin [20]. Puberty is a critical period for the development of obesity, as changes in hormone levels and body fat composition are coupled to behavioral changes [21]. WL during this stage induces changes in adipokine production and a decrease in IR and cardiovascular risk. Our aims were to determine the utility of measuring leptin and adiponectin levels, as well as L/A and FLI, as markers of IR and to analyze the possible relationship of these indexes with changes in the percentage of TBF (%TBF) in obese girls (OG) during pubertal development.
Statistical Analysis Results are expressed as means ± SD. The normal distribution of each parameter in all groups was assessed, and differences between controls and obese patients were analyzed by Student’s t test if a normal distribution was obtained or by Kolmogorov-Smirnov goodness of fit if not. Comparison of baseline measurements and changes over time were made by ANOVA with repeated measures, followed by Bonferroni post hoc test. Pearson’s correlation coefficient was used to investigate the association between the parameters studied with quantitative variables with a normal distribution. When this requirement was not met, we used the nonparametric Spearman test. The level of significance chosen was p < 0.05. Data were analyzed using SPSS (15.0) software for Windows (MapInfo Corporation, Troy, N.Y., USA).
Results
Obese Patients Have Normal Growth and a Slight Advancement in BA and Pubertal Onset The auxological changes are shown in table 1. The mean chronological age of menarche was 11.7 ± 0.3 years, with an age range between 10 and 13.5 years. The mean BA at menarche was 13.3 ± 0.5 years (range 12–14), with a difference between bone and chronological age of 1.1 ± 1 years throughout the study period. The interval between the onset of puberty and menarche was 1.5 ± 0.4 years (range 1–2.4). There was a peak in growth rate during the first 12 months of follow-up with a total gain of 14.9 ± 2.3 cm (range 11.2–19.2). There was an increase in TBF, as well as in %TBF, in OG during follow-up (table 1). In the WL group, there was a decrease (p < 0.05) in %TBF at Tanner stage III (table 1), but this parameter was elevated compared to CG during the progression of puberty. Leptin/Adiponectin Index in Obese Girls
Table 1. Auxological and anthropometric parameters and %TBF
in OG and CG Tanner Groups stage WL CA at puberty onset II
NWL
CG
10.1 ± 0.2*
10.1 ± 0.2*
11.5 ± 1*
BA-CA
II III V
1.1 ± 1* 1.2 ± 1* 1.2 ± 1*
1.1 ± 1* 1.2 ± 1* 1.2 ± 1*
0.2 ± 1* 0.3 ± 1* 0.2 ± 1*
BMI
II III V
3.8 ± 1** 2.7 ± 1**, ## 2.7 ± 1**, ##
4.8 ± 1** 4 ± 1** 4 ± 1**
0.6 ± 1 0.8 ± 1 0.2 ± 1
WC, cm
II III V
79 ± 1** 78 ± 2** 75 ± 1**, +
60 ± 1 84 ± 2** 62 ± 1 85 ± 1** 87 ± 4**, + 62.6 ± 1
%TBF
II III V
40 ± 1** 35 ± 2**, # 37 ± 1**, #
42 ± 2** 40 ± 1** 39 ± 4**
27 ± 1 24 ± 1 25 ± 1
CA = Chronological age. * p < 0.05, ** p < 0.01, compared with the CG; # p < 0.05, ## p < 0.01, comparing the same group at different moments with baseline; + p < 0.05, WL compared with the NWL group.
Relationship of WL with IR, Lipid Metabolism, and MS In the WL group, the prevalence of hyperinsulinemia was 47.8% at baseline, falling to 42.8% at Tanner stage III and 16.6% at stage V. In this group, we also observed a significant decrease in the percentage of girls with IR as determined by HOMA values higher than 2 SD compared with national reference values according to age, sex, and pubertal stage [29], with 47.8, 42.8, and 11.1% at Tanner stage II, III, and V, respectively. However, in the NWL group, no changes in these parameters were found during follow-up. At Tanner stage II, 52.1% of OG presented IR when insulin values during OGTT were used. In the WL group, this percentage decreased more dramatically throughout the study (52.1% at Tanner stage II, 27.0% at Tanner stage III, and no patients with these criteria at Tanner stage V) than when HOMA was used to determine IR. Insulin levels and HOMA index are shown in table 2. We found a positive correlation between basal insulin levels with BMI, WC, and %TBF (r = 0.61, p < 0.01; r = 0.72, p < 0.01, and r = 0.73, p < 0.01, respectively). No correlation between L/A ratio or FLI and lipid profile were found. There was an increase in HDL-C values during Horm Res Paediatr 2013;80:363–370 DOI: 10.1159/000356046
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USA) as previously reported [28]. Intra- and inter-assay coefficients of variation were below 10% for all assays. Fasting insulin and HOMA were compared with reference values according to age, sex, and pubertal stage. Patients were considered to be hyperinsulinemic when fasting insulin levels were higher than 2 SDS and to be IR if HOMA values were higher than 2 SDS of normal values [29]. In obese patients, an OGTT was performed at each stage of puberty [30], with glucose and insulin levels determined every 30 min during 2 h [17]. IR was also defined as an insulin peak higher than 150 μIU/ml and insulin values at 2 h higher than 75 μIU/ml [31]. We also determined high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), triglycerides (TG), and total cholesterol (TC), and the TC/HDL-C, TG/ HDL-C, and HDL-C/LDL-C indexes were calculated. The FLI was determined as the leptin (ng/ml)/sOB-R (ng/ml) ratio and the L/A index as the ratio between leptin (ng/ml) and adiponectin (μg/ml). We evaluated the presence of MS by using the modified Cook criteria for children and adolescents [32] and alterations in glucose metabolism by using the criteria of the American Diabetes Association [30].
follow-up, and at Tanner stage V, a decrease in TC/HDLC and TG/HDL-C, as well as an increase in HDL-C/LDLC, was found in patients with WL. At Tanner stage II, 16.7% of patients had systolic hypertension, while at Tanner stage III, it was 4.8% and at Tanner stage V, no patient was found to be hypertensive. Three patients who had high blood pressure normalized this parameter with WL during follow-up of >1 SD of BMI. The prevalence of MS was 16% at Tanner stage II and 5% at Tanner stage III, with no child meeting the criteria for MS at Tanner stage V [30]. Adiponectin and Leptin Levels and Correlations with Metabolic Parameters Changes throughout puberty in adiponectin and leptin levels, as well as FLI and L/A indexes, are shown in figure 1. We found an increase in leptin values in OG with respect to CG (p < 0.01), with a decrease after WL (p < 0.05). Increases in sOB-R and adiponectin values after WL (p < 0.05 and p < 0.01, respectively; table 2) were also found. At baseline, the WL and NWL group had increased L/A and FLI indexes compared to CG. Both indexes decreased in the WL group as puberty progressed, with no change in the NWL group (table 2). There was a positive correlation between leptin and BMI, WC, and %TBF and a negative correlation with sOB-R throughout the study in the WL group. In the same group, we observed a direct correlation between the L/A ratio and FLI with insulin values in the OGTT, with this correlation being stronger with FLI. Moreover, these indexes also correlated with the %TBF throughout the study period. A direct correlation between L/A and HOMA was also found in all Tanner stage groups (table 3).
Table 2. Adipokine levels, insulin values during fasting and in
OGTT, and HOMA in OG and CG Tanner Groups stage WL
NWL
CG
Leptin, ng/ml
II III V
36 ± 1** 21 ± 1**, # 22 ± 1**, #
44 ± 1** 33 ± 1** 39 ± 1**, +
6±1 7±1 10 ± 1
Adiponectin, μg/ml
II III V
15 ± 1 19 ± 1*, # 19 ± 1*, #
13 ± 1 14 ± 1 14 ± 1
10 ± 1 10 ± 1 12 ± 1
FLI
II III V
2.9 ± 1 1 ± 1##, ++ 1 ± 1#, +
3.7 ± 1* 4 ± 1*, ++ 3.8 ± 1**, +
1±1 0.6 ± 0.1 0.7 ± 1
L/A index
II III V
2.5 ± 1** 1.3 ± 1*, #, + 1.3 ± 1#, +
0.5 ± 1 3.4 ± 1** 2.2 ± 0.2**, + 0.9 ± 1 2.5 ± 0.4**, + 0.9 ± 1
Insulin, μIU/ml
II III V
21 ± 2* 20 ± 2* 17 ± 1*, #
23 ± 1* 25 ± 1 22.5 ± 1
Insulin peak in OGTT, μIU/ml
II III V
151 ± 96 124 ± 75# 74 ± 20##
196 ± 54 188 ± 46 188 ± 56
Insulin at 120 min, II III μIU/ml V
95.2 ± 78 111 ± 59 47 ± 12##
144 ± 83 135 ± 14 108 ± 32
4.1 ± 1 3.3 ± 1# 2.9 ± 1#
5.4 ± 1 5.6 ± 1 5.3 ± 0
HOMA
II III V
9±1 13 ± 1 10 ± 1
0.9 ± 1 1.2 ± 1 1±0
* p < 0.05, ** p < 0.01, compared with the CG; # p < 0.05, ## p < 0.01, comparing the same group at different moments with baseline; + p < 0.05, ++ p < 0.01, WL compared with the NWL group.
Discussion
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Our results show that obese adolescent girls have normal growth, but a slight advancement in bone maturation and pubertal onset, as well as an increased incidence of IR. The major finding of this study is that the L/A ratio and FLI are good markers of IR in OG during pubertal development and are useful in monitoring changes in IR and TBF. Another finding is that insulin values during OGTT are better than the HOMA index to determine changes in IR after WL. We found accelerated bone maturation in OG that could be due to different factors such as nutritional status, body fat content, and physical activity [33, 34]. By con-
trast, ballet dancers, with a low BMI, have a delay in skeletal maturation and pubertal progression [35]. Thus, these divergent situations result in different patterns of pubertal evolution in accordance with the nutritional status and body fat content [35]. Hence, the amount of body fat may influence the onset and progression of puberty, as well as the age of menarche, possibly due, at least in part, to changes in the levels of several hormones, including leptin and adiponectin, that are closely related to the amount of fat [36–38]. The observed hyperleptinemia maintained throughout puberty, with decreased levels of leptin receptor, are consistent with previous studies [20, 39–42].
#
2
**
#
**
1 0
a
5
** **
3
CG WL group NWL group
4
6 5
** **
3
+
# + +
* **
2
# ** +
L/A index
4
6
**
**
FLI index
Adiponectin (μg/ml)
5
#
#
6
1 II
III
0
V
*
+
**
4 3 2
## +
# +
III
V
1 0
V
III
II
b
Tanner
++
*
II
c
Tanner
Tanner
Fig. 1. Adiponectin (a), FLI index (b), and L/A index (c) in OG throughout puberty. # p < 0.05, ## p < 0.01, com-
paring the same group at different moments with baseline; * p < 0.05, ** p < 0.01, compared with the CG; < 0.05, ++ p < 0.01, WL compared with the NWL group.
+ p
Table 3. Correlation between L/A and FLI indexes with HOMA, BMI, and %TBF in OG with WL
L/A index, r Tanner stage: HOMA %TBF BMI Insulin peak in OGTT, μIU/ml Insulin at 120 min, μIU/ml
FLI index, r
II
III
V
II
III
V
0.43* 0.58* 0.59** 0.45** 0.59**
0.48* 0.54* 0.70** 0.44* 0.58*
0.83** 0.86** 0.82** 0.81** 0.77**
0.31 0.86** 0.82** 0.79** 0.891**
0.11 0.78** 0.64* 0.70* 0.790*
0.12 0.83* 0.64* 0.83** 0.850*
The FLI can provide precise information about changes in the bioavailability of leptin [21, 43], with the levels of sOB-R possibly serving as an indicator of free leptin levels; thus, the FLI index, defined as the ratio of leptin to sOB-R, may be a more accurate determinant of leptin function. Here, we report that sOB-R levels are inversely correlated with BMI, as previously shown [44], and lower receptor levels increase free leptin concentrations, which could compensate for increased leptin resistance [45]. In addition, the WL group had a decreased FLI during puberty, which may be a compensatory mechanism of energy saving secondary to WL during puberty [46– 49]. Additionally, we observed a strong correlation of FLI with body fat, in line with previous data. It is suggested that leptin circulates predominantly in the bound form in lean subjects, whereas in obese patients, due to low
sOB-R levels, leptin circulates mainly in the free form [50]. Moreover, adolescent girls with anorexia nervosa have higher sOB-R levels and a lower FLI than healthy CG [51]. In contrast to leptin, adiponectin levels are reduced in OG, again in agreement with the role of body fat in the regulation of circulating levels of this adipokine [52]. However, although no correlation between adiponectin and BMI or body fat was found here, similar to that reported in other studies [53], the relationship of BMI and the %TBF with the L/A ratio suggests a greater sensitivity of this index to changes in body fat than either adiponectin or leptin alone. As leptin and adiponectin levels change inversely in relation to BMI, the L/A ratio has been purported to be a potential index relating adiposity to the development of complications of obesity [54].
Leptin/Adiponectin Index in Obese Girls
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r = Spearman’s ρ correlation (* p < 0.05, ** p < 0.01).
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tin alone [61], and similarly, we found that this index correlated with TBF and HOMA index. One caveat that should be taken into consideration when evaluating these results is that the control subjects were from a cross-sectional population. Although it could be of interest to follow the parallel evolution of both groups, the control population used in this study may present some advantages, especially regarding comparable anthropometric characteristics at a given age. In addition, in our design, we were able to maintain the number of control subjects at each time point. In conclusion, our results show that in OG with WL both the L/A ratio and FLI correlate with IR as measured by insulin values in OGTT. L/A also correlates with HOMA index. In addition, insulin values during OGTT are better than the HOMA index for determining an improvement in insulin sensitivity after WL.
Acknowledgements This work was supported by grants from Fondo de Investigación Sanitaria (PI10/0747) with the help of European FEDER funding, CIBERobn (CB03/06), and Funda-ción Endocrinología y Nutrición.
Disclosure Statement The authors declare that there is no conflict of interest that may be perceived as prejudicing the impartiality of the research reported.
References
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Excess body weight in children is closely related to IR. The deregulation of certain adipokines, such as leptin and adiponectin, due to excess body fat can have deleterious effects on insulin signaling [9, 36]. Our results show that changes in leptin and adiponectin after WL appear to be associated with a decrease in IR. OG have higher insulin values both at baseline and during OGTT, whereas after WL, there is a decrease in the percentage of girls with hyperinsulinemia and high HOMA values. This is in correlation with lower BMI and %TBF, as previously found [14, 55]. This emphasizes the importance of WL in the reduction of IR. The prevalence of IR decreased in OG after WL. This difference may be due to the fact that impaired glucose tolerance is a more restrictive parameter than fasting glucose and insulin to detect changes in glucose homeostasis and insulin sensitivity, indicating that OGTT is a more specific and stringent method to detect changes in IR [56]. The observed improvement in IR is very important, given that during puberty, insulin sensitivity normally decreases [21], and highlights the importance of WL during pubertal development in obese subjects. A combination of leptin and adiponectin as an index of insulin sensitivity was first suggested in 2003 [57]. Subsequently, many researchers have reported that the L/A ratio is a consistent marker of IR in patients with and without diabetes in comparison with other indicators [58] and may be used as an atherogenic index, providing additional information on the risk of MS [59]. We found a correlation between the L/A index and insulin values during OGTT and with HOMA values, suggesting again the possible utility of this ratio to predict the presence of IR, better than adiponectin or leptin alone [54]. In addition, FLI showed a direct relationship with insulin values in OGTT, but not with HOMA, suggesting that the L/A ratio is more related to IR than FLI. Many studies have described the association between higher body fat and increased cardiovascular risk and impaired lipid profiles [14]. These studies report that total fat is negatively correlated with HDL/LDL and positively correlated with TC/HDL, as we found here. During follow-up, despite the decrease in TBF, these associations persist, suggesting a close relationship between lipoproteins and body fat. We have also found that although TBF increases due to changes during puberty, the percentage is decreased due to WL and this could explain the improvement in the lipid profile and insulin sensitivity and lower L/A index and cardiovascular risk factors [60]. In fact, it has been reported that the L/A ratio correlates more strongly with BMI and HDL-C than does adiponec-
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