Evaluation of Plasma TRB3 and Sestrin 2 Levels in Obese and Normal-Weight Children

Abstract

Objective:

Obesity in childhood and adolescence is associated with metabolic syndrome and cardiovascular diseases. TRB3 (Tribbles homolog 3) and sestrin 2 are two newly found proteins that have been identified to play an important role in obesity and its complications.

Aim:

The purpose of this study was to evaluate concentrations of TRB3 and sestrin 2 in plasma of obese and normal-weight children and adolescents, and their association with metabolic and anthropometric parameters.

Methods:

Plasma levels of TRB3, sestrin 2, insulin, fasting plasma glucose (FPG), and lipid profile were evaluated in 70 children and adolescents (34 obese and 36 controls). Insulin resistance was calculated using a homeostasis model assessment of insulin resistance. Metabolic syndrome was defined according to IDF criteria.

Results:

Plasma TRB3 levels of the obese subjects were significantly higher than that of normal weight subjects. TRB3 levels were positively correlated with BMI, BMI z-score, waist circumference, and FPG. The concentration of sestrin 2 was significantly lower in obese subjects compared to normal-weight subjects. A statistically significant positive correlation was observed between plasma concentrations of sestrin 2 and high-density lipoprotein cholesterol. Neither TRB3 nor sestrin 2 were correlated with insulin resistance and metabolic syndrome.

Conclusion:

Both TRB3 and sestrin 2 may contribute to the development of obesity and its complications and can be considered interesting therapeutic target for the treatment of obesity.

Introduction

The prevalence of childhood obesity has increased worldwide in recent decades. There is ample evidence suggesting that childhood obesity leads to adult obesity.^1–3 Visceral adiposity and excessive fat accumulation in adipose tissue in obesity is strongly associated with oxidative stress, an imbalance between oxidants and antioxidants. Oxidative stress plays an important role in the development of many metabolic disturbances related to obesity.^4,5

Childhood obesity is associated with complications such as insulin resistance (IR), glucose intolerance, and inflammation. The simultaneous occurrence of these abnormalities, referred to as metabolic syndrome (MetS) increases the risk for the development of cardiovascular disease, type 2 diabetes, and all-cause mortality.⁶

TRB3 (Tribbles homolog 3), also called NIPK (neuronal cell death-inducible protein kinase), is a mammalian homolog of Drosophila Tribbles gene, which is expressed in various tissues, including liver, adipose tissue, heart, and skeletal muscle. The expression of TRB3 in liver is increased under stressful conditions, such as fasting, endoplasmic reticulum (ER) stress, and nutrient starvation.^7,8 On the other hand, elevated hepatic expression of TRB3 results in hyperglycemia and IR in diabetic mice.⁹ Moreover, TRB3 inhibits adipocyte differentiation by suppressing peroxisome proliferator-activated receptor γ (PPARγ), a master regulator of adipocyte differentiation.¹⁰

Sestrins are a family of stress-inducible proteins that regulate metabolic homeostasis and include three members (sestrin 1–3) in mammals.¹¹ Genetic deficiency of sestrin 2 in mice or its homolog in Drosophila leads to obesity-associated pathologies, including triglyceride (TG) accumulation, increased reactive oxygen species (ROS), mitochondrial dysfunction, IR, and cardiac dysfunction.^12,13 Sestrin 2 is considered an antioxidant protein that is induced by oxidative stress and protects cells against reactive oxygen species and is protective against cardiovascular disorders.¹⁴ It also exerts its protective role by regulation of autophagy and mitophagy.¹⁵ In addition, sestrin 2 can increase lipolysis and fatty acid oxidation through the activation of mammalian target of rapamycin (mTOR) and adenosine monophosphate-activated protein kinase (AMPK)-mediated PPARα.^12,16

Recent evidence indicates that the induction of sestrin 2 might be promising strategy for the treatment of many liver diseases, such as hepatitis, metabolic liver diseases, and hepatocellular carcinoma.^13,17,18 It is also indicated that sestrin 2 by regulating oxidative stress plays an important role in tumorigenesis.^19–21 Among the three mammalian sestrins, sestrin 2 has been most strictly characterized in metabolic organs, such as liver and adipose tissue.^12,13,22

Although TRB3 and sestrin 2 have important roles in metabolic homeostasis, the associations of circulating TRB3 and sestrin 2 levels with obesity have not been investigated to date. Therefore, this study was designed to evaluate plasma TRB3 and sesrin 2 levels in obese and normal-weight subjects, and their correlation with metabolic and anthropometric parameters.

Material and Methods

Subjects

Seventy children and adolescents (29 female and 41 male) aged 8–16 years were enrolled in this study. All the subjects went through careful clinical examination and all the systemic illnesses and possible causes of obesity including endocrine dysfunctions such as hypothyroidism and diabetes were ruled out. They were also given a questionnaire including information about their physical activity and diet. None of the subjects were receiving any medication or supplement and were not on any special diet. They had normal physical activity and were neither involved in any professional sports nor had sedentary lifestyle.

Weight and height were measured using a digital electronic weighing scale and a digital stadiometer, respectively, with the subject in the upright position. BMI was calculated by dividing the body weight (kg) by the height (m) squared and BMI z-score was determined for all subjects. Thirty-six subjects whose BMI levels were above the 95th percentile for their age and sex were included as obese subjects and 31 age- and sex-matched lean subjects with BMI between 5th and 84th percentiles were included as the control group. Case and control groups were also matched according to their physical activity.

Waist and hip circumferences (WC and HC, respectively) were measured with a flexible tape measure while the subjects were in the standing position and their waist-to-hip ratio (WHR) was calculated. WC percentiles were determined according to age and gender of each subject.²³ Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were also measured by mercury sphygmomanometer. Pubertal staging was determined by Tanner's classification.²⁴ The study was approved by the Ethics Committee of Endocrinology and Metabolism Research Institute, Tehran University of Medical Sciences. Written informed consent was obtained from all the subjects and/or their parents.

MetS was defined based on the IDF consensus definition of MetS in children and adolescents.²⁵ According to this criteria, children and adolescents with abdominal obesity (WC above 90th percentile for age and sex), and having two or more other clinical features [elevated fasting plasma glucose (FPG), TGs, blood pressure, or low high-density lipoprotein cholesterol (HDL-C)], were categorized as having MetS. Obese subjects were also divided to metabolically healthy and unhealthy based on the presence of one of the metabolic abnormalities mentioned above for MetS (elevated FPG, TGs, BP, or low HDL-C).

IR was defined using homeostatic model assessment of IR (HOMA-IR) that was calculated by the following formula: serum insulin (μIU/mL) × FPG (mg/dL)/405.²⁶ Those who had HOMA-IR values greater than 3.16 were considered insulin resistant.²⁷

Biochemical Measurements

Blood samples were collected early in the morning after an overnight fasting of about 12 hours. To consider for probable circadian alterations, blood specimens were drawn early in the morning (7:30–8:30) for all case and control subjects. For plasma separation, blood samples were immediately kept on ice and separated in a refrigerated centrifuge to prevent any loss in analytes. All serum and plasma samples were kept frozen at −80°C until later analysis.

FPG, TG, total cholesterol (TC), HDL-C, and low-density lipoprotein cholesterol (LDL-C) were measured using calorimetric kits (Pars Azmoon, Iran). Insulin levels were measured using an enzyme-linked immunosorbent assay (ELISA) kit (Monobind) with intra- and inter-assay coefficients of variation (CV) of 8.0% and 6.8%, respectively. Plasma Sestrin 2 (SESN2) and TRB3 (tribbles-related protein 3) levels were determined by ELISA kits (Cusabio, China). The intra- and inter-assay CV of all the ELISA kits were <8% and 10%, respectively.

Statistical Analysis

SPSS software version 16.0 (SPSS, Inc. Chicago, IL) was used for statistical analyses. Data are expressed as the mean ± standard deviation or the median (interquartile range). Mann–Whitney U test (nonparametric) and Student's t test (parametric) were used to analyze the differences between the obese and the control groups and also those with or without IR. Kruskal–Wallis test was used for the comparison of main parameters between various stages of puberty. The correlations between biochemical and anthropometric parameters were evaluated by Pearson's and Spearman's correlation tests for parametric and nonparametric variables, respectively. The statistical significance was defined as p < 0.05.

Results

The anthropometric and biochemical characteristics of the studied subjects are presented in Table 1. There were no significant differences in age and sex between the two studied groups. Obese group had significantly higher BMI, BMI z-score, WC, WHR, SBP, DBP, TG, LDL, and FPG levels than those in the control group. TC was not significantly different between the studied groups; nevertheless, obese children and adolescents had significantly lower levels of HDL-C (Table 1).

Table 1.

Demographic and Biochemical Characteristics of Study Population

	Participants
Characteristics	Obese (n = 34)	Control (n = 36)	p
Age (years)	10.90 ± 2.26	11.11 ± 2.37	0.71
Gender (female)	12 (35.29%)	17 (47.2%)	0.34
Height (cm)	145.86 ± 9.85	140.82 ± 12.15	0.033
Weight (kg)	63.82 ± 17.63	36.13 ± 9.42	0.00
BMI (kg/m²)	29.22 ± 4.52	17.83 ± 1.99	0.00
BMI z-score	2.23 ± 0.30	−0.008 ± 0.76	0.00
WC (cm)	91.33 ± 8.15	65.50 ± 8.68	0.00
Hip circumference(cm)	98.53 ± 9.33	77.77 ± 7.88	0.00
WHR	0.92 ± 0.05	0.83 ± 0.05	0.00
SBP (mmHg)	125.0 (115.0–135.7)	110.0 (31.5–118.0)	0.00
DBP (mmHg)	80.0 (72.75–85.0)	69.5 (21.0–77.5)	0.00
TG (mg/dL)	105.38 ± 42.80	76.30 ± 39.85	0.00
TC (mg/dL)	163.20 ± 24.29	153.63 ± 25.50	0.11
LDL-C (mg/dL)	82.23 ± 16.08	75.11 ± 12.63	0.04
HDL-C (mg/dL)	48.11 ± 8.50	55.02 ± 12.18	0.00
FPG (mg/dL)	93.30 ± 5.39	87.41 ± 5.98	0.00
Insulin (μIU/mL)	12.90 ± 7.46	6.04 ± 3.18	0.00
HOMA-IR	2.98 ± 1.81	1.32 ± 0.77	0.00
TRB3 (pg/mL)	26.35 (14.5–446.5)	15.5 (7–70.7)	0.03
Sestrin 2 (ng/mL)	0.3 (0.03–0.7)	0.6 (0.27–1.4)	0.009

Values are expressed as mean ± standard deviation, numbers (proportions) or median (interquartile range). Comparison of variables means in two groups was performed by Student's t test.

DBP, diastolic blood pressure; FPG, fasting plasma glucose; HDL-C, high-density lipoprotein cholesterol; HOMA-IR, homeostatic model assessment of insulin resistance; LDL-C, low-density lipoprotein cholesterol; SBP, systolic blood pressure; TC, total cholesterol; TG, triglycerides; WC, waist circumference; WHR, waist-to-hip ratio.

Plasma levels of TRB3 in obese subjects were significantly higher than the control subjects (p < 0.05). While the levels of sestrin 2 were significantly lower in obese subjects compared to control subjects (p = 0.009). Neither TRB3 nor sestrin 2 were significantly different between male and female subjects. Additionally, they did not show a significant difference according to various stages of puberty.

The correlations between TRB3, sestrin 2, and other variables are shown in Table 2. Significant positive correlations were found between TRB3 and BMI, BMI z-score, WC, WHR, and FBG. However, when correlations were adjusted for BMI as a confounding factor, the significant correlations disappeared. On the other hand, the plasma levels of sestrin 2 were only significantly and positively correlated with HDL-C levels. Interestingly, partial correlation with BMI as confounding factor still showed significant positive correlation between sestrin 2 and HDL-C (r = 0.458, p < 0.001). Correlation of sestrin 2 with HDL-C was also significant when analyzed only in the control subjects (r = 0.509, p < 0.01), but not in obese subjects.

Table 2.

Correlations of TRB3 and Sestrin 2 with Other Variables

	TRB3		Sestrin 2
Variables	r	p	r	p
BMI (kg/m²)	0.27	0.02	−0.23	0.05
BMI z-score	0.30	0.01	−0.20	0.09
WC (cm)	0.36	0.005	−0.12	0.37
WHR	0.37	0.006	−0.09	0.50
FPG (mg/dL)	0.28	0.01	−0.04	0.73
TG (mg/dL)	0.19	0.11	0.02	0.82
TC (mg/dL)	0.05	0.67	0.09	0.44
LDL-C (mg/dL)	0.08	0.46	0.001	0.99
HDL-C (mg/dL)	−0.016	0.18	0.29	0.01
Insulin (μIU/dL)	0.10	0.37	−0.23	0.85
HOMA-IR	0.13	0.29	−0.01	0.95

Insulin levels and HOMA-IR were significantly higher in obese subjects compared to control subjects. Based on HOMA-IR, 15.5% (n = 11) of obese subjects had IR. None of the control subjects had IR. No significant difference was detected in plasma TRB3 and sestrin 2 levels between the subjects with IR and those without IR.

MetS was diagnosed in 6% of obese children and adolescents. Of the obese subjects, 64.7% were found to be metabolically unhealthy. None of the control subjects had the features of MetS. TRB3 and sestrin 2 levels were not significantly different in subjects with MetS compared with those without MetS or those who were metabolically unhealthy compared to metabolically healthy ones.

Discussion

Overweight and obesity are defined as abnormal or excessive fat accumulation that presents a major risk factor in the development of many chronic diseases, including diabetes, cardiovascular disease, and cancer.⁴ Obesity in childhood and adolescence leads to development of obesity in adulthood and eventually, MetS and type 2 diabetes.⁶

TRB3, which is a newly identified protein, has been suggested to play important roles in glucose and lipid metabolism in vitro and in mouse models.^7–10 Takahashi et al. suggested that TRB3 is active in the abnormally and excessively differentiated adipocytes that are found in obesity and MetS.¹⁰ It has been reported that obesity causes ER stress.²⁸ Ohoka et al. showed that ER stress induces TRB3 gene expression.⁷ Therefore, TRB3 induced by ER stress, might prevent adipocytes from enhancing further excessive differentiation and accumulating more TG.¹⁰ Circulatory TRB3 concentration and its relationship with metabolic parameters has not been previously investigated in obese children and adolescents. In this study, we measured the TRB3 levels in obese children and normal-weight children.

We found that the plasma TRB3 levels were higher in obese children compared to controls. We also observed significant positive correlations between plasma TRB3 and BMI, BMI z-score, and WC confirming the relationship between TRB3 and obesity. These results confirm a previous report showing TRB3 overexpression during adipocyte differentiation.¹⁰ In addition, overexpression of TRB3 in the liver results in hyperglycemia and glucose intolerance and knockdown of TRB3 improves glucose tolerance.⁹ The functional Q84R polymorphism in TRB3 is associated with IR and type 2 diabetes.^29–31

In this study, we observed a positive correlation between the TRB3 levels and plasma fasting glucose. This result is in agreement with previous data showing that overexpression of TRB3 causes hyperglycemia.^9,32 Therefore, it appears that elevated TRB3 exacerbates the glucose homeostasis and overexpression of TRB3 may be involved in increasing plasma levels of glucose. We also observed a significant positive correlation between TRB3 and WHR. WHR is a suitable index for abdominal obesity in adolescents and has been reported an index of IR.^33,34

Although previous studies have shown a close relationship between TRB3 and IR,^32,35–37 we did not find any significant correlation between TRB3 plasma levels and HOMA-IR and TRB3 was not significantly different in obese subjects with or without IR. This discrepancy might be due to the fact that our subjects were very young and had normal glucose levels.

Studies have shown that obesity in children and adolescents is strongly associated with reduced antioxidant capacity and high levels of reactive oxygen species, which are related to hypertension and atherosclerosis.⁵ We had previously reported increased malondialdehyde as a marker of oxidative stress in obese children and adolescents.³⁸ To minimize detrimental consequences of ROS accumulation, cells are equipped with a variety of antioxidant proteins, including superoxide dismutases, catalases, and sestrins.³⁹ Sestrin 2 (Sesn2) is an antioxidant protein that is induced by various stresses, including oxidative and energetic stresses, and protects cells against these situations.⁴⁰ Loss of endogenous sestrins may lead to several metabolic pathologies, including IR, fat accumulation, mitochondrial dysfunction, and oxidative damage.^12,13 In addition, it has been demonstrated that sestrin 2 is induced by oxidized LDL and can be considered as an effective pharmacological target for the treatment of lipid-related cardiovascular diseases.⁴¹

In this study, we measured plasma sestrin 2 levels in obese and normal-weight children and found that the plasma sestrin 2 levels in obese children were lower than that in the controls. Sestrin 2 activates AMPK,⁴² which is a master regulator of energy balance and plays a key role in obesity prevention.⁴³ Therefore, low sestrin 2 levels may contribute to obesity by reducing AMPK levels. We also observed a significant positive correlation between sestrin 2 and HDL-C. An inverse relationship between ROS production and HDL has been previously reported.⁴⁴ Additionally, HDL levels are reduced in inflammatory states.⁴⁵ On the other hand, sestrin 2 prevents inflammation and inhibits TLR-induced inflammatory gene expression in macrophages.¹⁸ Thus, the effect of sestrin 2 on HDL-C levels may be mediated by its anti-inflammatory and antioxidant properties.

Conclusion

In conclusion, in this study we showed dysregulation of TRB3 in obese children and its association with metabolic parameters, which suggests that TRB3 may be an important contributor to obesity and its metabolic disturbances. On the other hand, the relationship between sestrin 2 and HDL-C levels suggests a protective role for sestrin 2 against atherosclerosis. Thus reduced sestrin 2 may be considered a risk factor for artherosclerosis in obesity.

Footnotes

Acknowledgment

This study was financially supported by a grant from Endocrinology and Metabolism Research Institute, Tehran University of Medical Sciences (grant number 1393-02-104-1831).

Author Disclosure Statement

No competing financial interests exist.

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