PPARα and PPARβ/δ Expression is Associated with Proinammatory Markers in an Obese Pediatric Population


 Background

Obesity configures a pathophysiological profile that predisposes the development of metabolic and cardiovascular diseases, critically impacting public health. The chronic dysregulation of immuno-metabolic components triggered by obesity directly affects the metabolism of various tissues. Peroxisome proliferator-activated receptors (PPARs) are a group of nuclear receptors and transcription factors essential for energy homeostasis of different tissues. Despite its wide-ranging expression and function, the functional mismatch of the PPAR subtypes establish biological mechanisms responsible for metabolic diseases from childhood. In addition, the glucagon-like peptide-1 receptor (GLP-1R) activation can influence insulin secretion, but also the cytokine profile possibly mediated through PPARγ activation. However, to date, the role of PPARs and GLP-1R in leukocytes from obese pediatric patients has been unclear. Therefore, we examined the expression of PPARs isotypes and GLP-1R, and its correlation with metabolic, hormonal, inflammatory, and anthropometric markers in an obese pediatric population.
Results

Obese children and adolescents presented a significant increase in anthropometric and body composition parameters, TG, VLDL, TG/HDL, A/G index, and HOMA score when compared to the control group. Obese participants exhibited a pro-inflammatory profile with an augment of IL-8 (p = 0,0081), IL-6 (p = 0,0005), TNF-α (p = 0,0004), IFN-γ (p = 0,0110), MCP-1 (p = 0,0452), and adipsin (p = 0,0397), whereas displayed a reduction of adiponectin (p = 0,0452). The expression of PPARα and GLP-1R was lower in the leukocytes from obese participants than in lean subjects. Furthermore, PPARα correlates negatively with TNF-α (p = 0,0383), while GLP-1R did not show correlation with any inflammatory variable. However, both receptors correlate negatively with the abdominal skinfold. Although PPARβ/δ expression was similar between groups, it was negatively associated with IL-8 levels (p = 0,0085).
Conclusions

PPARα and PPARβ/δ expression are negatively associated with the proinflammatory markers TNF-α and IL-8, respectively, suggesting participation in the regulation of inflammation observed in pediatric obesity. Furthermore, PPARα and GLP-1R are downregulated in leukocytes from obese children and adolescents, and both receptors are associated with the abdominal skinfold suggesting a role in fat distribution that could indirectly affect the function of different immune cells and the secreted cytokines. Thereby, these findings may impact the understanding and implementation of PPARα or GLP-1R agonists in the clinic.


Obese children and adolescents have an impaired metabolic and anthropometric pro le
The anthropometric, body composition, and biochemical variables of the studied individuals strati ed by BMI percentile status are shown in Table 1. Age and gender of the participants were not signi cantly different among the control and obese group, the median age was 11 years old, and 48% were girls. All mean values of skinfold thickness, waist circumference (WC), arm circumference (AC), waist-to-height ratio (WHR), arm muscle circumference, and neck circumference were signi cantly increased in participants with obesity in comparison with healthy weight children and adolescents (Table 1).
The mean of fasting glucose and 2-hours glucose fell within the normal ranges, as recommended by the American Academy of Pediatrics (Table 1) [20]. However, insulin was signi cantly higher in children with obesity than in the standard weight group. Accordingly, the HOMA score was increased in obese children; this score overcomes the 3,4 cut-off indicating IR (Table 1) [21]. TG and VLDL levels were higher in obese children than those of lean subjects, whereas HDL-c levels were signi cantly reduced in obese subjects in contrast with the control group ( Table 1). The levels of total cholesterol were similar among groups (Table 1).
TG/HDL and A/G ratios are frequently used as indicators of cardiometabolic risk, and here, those ratios were signi cantly elevated in the obese group in comparison with the lean patients. Based on this data, obese children and adolescents have an increased risk of developing cardiovascular disease and T2D.
Incretins gastric inhibitory polypeptide (GIP) and GLP-1 did not show signi cant differences between the study groups (Table 1).
PPARα and GLP-1R gene expression is reduced in leukocytes from obese subjects To determine the expression of PPAR isotypes in the leukocytes, the expression of each isotype was analyzed by qPCR. PPAR-α expression showed a signi cant reduction in the leukocytes from obese patients in comparison with the control group, with a 50% reduction in the expression of this gene (p = 0,0484) (Fig. 1a). PPAR-β did not show differential expression between the study groups (Fig. 1b). In contrast, PPAR-γ expression was undetectable in leukocytes from all the samples. To corroborate this nding, we used adipose tissue cDNA as a positive control, since this isotype is mainly present in adipose cells. This sample presented a positive ampli cation and a single peak in the dissociation curve, indicating that the reaction was speci c (data not shown). Likely, PPAR-γ transcripts were not expressed on leukocytes or are expressed at low levels but just in some cell lineages of these samples.
Besides, analysis from mRNA expression levels of incretin receptors was also evaluated in the studied individuals. GLP-1R expression was signi cantly reduced in the obese group as compared to the healthy weight participants (p = 0,1358) (Fig. 2a), whereas no difference was observed in GIPR expression between the two groups ( Fig. 2b).

Obese children and adolescents showed a proin ammatory pro le of adipokines and cytokines
To identify the in ammatory serum pro le in obese children and adolescents, the levels of several adipokines and cytokines were measured by ow cytometry using a multiplex assay. Obese subjects showed a signi cant increase in the levels of IL-8 (p = 0,0081), IL-6 (p = 0,0005), TNF-α (p = 0,0004) IFN-γ (p = 0,0110), and MCP-1 levels (p = 0,0452) in comparison with the normal weight group ( Fig. 3a and b).
Serum levels of IL-10 and IP-10 were detected but did not differ signi cantly between the groups.
The serum adiponectin concentration presented a signi cant reduction in the obese group (p = 0,0452) compared to the control group (Fig. 4). Conversely, adipsin displayed a concentration signi cantly higher (p = 0,0397) in the obese group in comparison with the control group, which had a concentration of 7,688 ± 6,116 pg/mL. (Fig. 4). Resistin showed similar levels between the study groups, with concentrations of 13,102 ± 8,342 pg/mL and 16,792 ± 8,302 pg/mL for the obese and control participants, respectively ( Fig. 4). Leptin was also analyzed; however, it presented considerably high levels in the group with obesity, which exceeded the concentrations determined in the calibration curve, which did not allow to quantify it using the test used. Finally, RBP4 did not present a representative concentration in any of the study groups, indicating that it is not secreted in serum at detectable levels under the evaluated conditions. PPARα and PPARβ/δ correlate negatively with proin ammatory markers Analysis of the relationships between PPAR-α, PPAR-β, and GLP-1R expression and the levels of cytokines, chemokines, adipokines and anthropometric parameters of the obese subjects showed that PPAR-α transcript levels had a signi cant negative correlation with TNF-α levels (r = -0,583, p = 0,03883) ( Table 3), as well as with abdominal skinfold (r = -0,712, p = 0,0016). Besides PPAR-β showed a signi cant negative correlation with IL-8 levels (r = -0,667, p = 0,0085) and arm fat percentage (r = -0,651, p = 0,0132) (Tables 3 and 4).  In contrast, GLP-1R expression did not correlate with any in ammatory parameters (Table 3). However, GLP-1R showed a negative correlation with Abdominal skinfold (r= -0,678, p = 0,009) ( Table 4).

Discussion
Childhood and adolescence are critical periods where fundamental metabolic and hormonal changes start and have an impact on adulthood health. Excess weight at these life stages is a critical factor for the onset of cardiometabolic disease and T2D. Thus, those stages are vital periods for timely intervention. We evaluated the expression of essential metabolic genes, such as PPARs and GLP-1R, in leukocytes and its correlation with metabolic, immune, and anthropometric factors in a Colombian's obese pediatric population.
PPARs participate in the control of the in ammatory process produced by obesity, modulating the expression of proin ammatory cytokines in adipose cells [12]. However, other tissues can contribute to the in ammatory process. Studies of PPARs, particularly of PPARβ/δ and PPARα, on leukocytes from obese children and adolescents, are scarce. However, the assessment of its expression and relation with variables associated with obesity represents an accessible tool for developing therapeutic strategies and understanding the pathophysiology of obesity [22].
In this work, we identify the expression of PPARα and PPARβ/δ, but not PPARγ in leukocytes from obese children and adolescents. The activation of PPARβ/δ has been mainly studied in "endurance-exercise mimetics" particularly focused on the liver, skeletal muscle, and adipose tissue, to reduce in ammation as an obesity treatment [23,24]. Moreover, PPARβ/δ is involved in lipid metabolism, regulation of fatty acid oxidation (FAO), in ammation, and modulation of glucose and cholesterol levels [25]. Direct activation of this isotype improves insulin sensitivity and the disorders of metabolic syndrome in humans [26]. The use of the agonist GW0742 for PPARβ/δ in obese mice in uences the improvement of metabolic parameters positively, increases Foxp3 + CD4 + T cells, induced FAO gene expression in lymph node and increases FAO in CD4 + T cells [27,28]. FAO can also in uence not exclusively the metabolism, but also the T cells lineages and functions [29]. Those effects are not observed only with exercise and suggest that this transcription factor can present additional effects that need to be explored.
In this study, PPARβ/δ did not show signi cant differences between the standard weight and obese groups. However, this gene displayed a signi cant negative correlation with IL-8 levels and arm fat percentage. IL-8 (CXCL8) is a proin ammatory chemokine that mediates the crosstalk between obesity and cardiovascular disease [30,31]. IL-8 is mainly secreted by adipose tissue, broblasts, endothelial cells, monocytes, and macrophages in conditions such as exposure to IL-1β, TNF-α, or LPS [32]. Indeed, we detected a signi cant increase in TNF-α levels from the serum of obese subjects, which could contribute to the IL-8 secretion in those individuals.
Although the direct link of PPARβ/δ to IL-8 is suggestive, some studies have already related the activation of PPARβ/δ with the modulation of expression of IL-8 in endothelial cells and endometrium tissue participating in in ammation [33][34][35]. In line with this evidence, our results suggest that a decrease in the PPARβ/δ expression in leukocytes might consequently augment the IL-8 levels and, thereby, the in ammatory response during obesity. Indeed, experiments in mice have associated the deletion in the expression of PPARβ/δ, with the reduction in the number of hepatic macrophages of the M2 type, which results in alterations in lipid metabolism, IR, and decreased expression of anti-in ammatory cytokines [36]. PPARβ/δ senses the effects that chylomicron-derived FAs have on macrophages inducing overexpression of angiopoietin-related protein 4 (ANGTPL4), inhibiting postprandial uptake of saturated FAs into mesenteric lymph nodes macrophages and preventing macrophage activation in an in ammatory response derived from fat accumulation [37]. Those ndings show the protective role of PPARβ/δ in the control of lipid metabolism and the development of in ammatory processes.
On the other hand, PPARα acts as a lipid sensor actively expressed in tissues with high metabolic rates, such as the liver, heart, muscle, kidney, intestinal mucosa, and brown adipose tissues [38,39]. The use of natural ligands or agonists for this isotype has revealed its participation in glucose homeostasis, IR development, in ammation, atherogenesis, and in the expression of genes related to lipid metabolism [40,41]. In leukocytes, PPARα expression has barely been studied. However, the expression of PPARα has been reported in immune cells such as monocytes/macrophages, peripheral blood mononuclear cells (PBMCs), and CD4 + T cells [12,42,43]. Also, subjects with MS signi cantly decreased PPARα expression in eosinophils by approximately 21% [44].
Our pediatric obese population showed reduced expression of this isotype in leukocytes when compared to the lean group. Likewise, studies on mice with induced obesity exhibited reduced expression of PPARα and carnitine palmitoyltransferase I (CPT-1) in the liver [45]. PPARγ expression is also reduced in PBMC from obese children and adolescents [46]. Indeed, obese mice treated with Beza brate (BZ), an agonist that preferentially activates PPARα and PPARγ, improves biochemical parameters and reduce the WAT proin ammatory state in obese mice [45]. These ndings suggest that obesity in the early stages of life in uences the expression of PPARα in leukocytes, and consequently, its reduction can be involved in the observed altered lipid pro le and reduced insulin sensitivity.
We observed a signi cant increase in TG and a reduction in HDL levels in obese children and adolescents in comparison with the control group. Both parameters overcome the reference values for pediatric individuals. Insulin sensitivity was also affected in this group. We used the HOMA index to evaluate the IR status, which is the most used method for its diagnosis in the pediatric population. We observed that obese subjects displayed a mean value of 4,28, signi cantly higher than lean subjects, which showed a 1,50 score, predicting IR for the obese group.
As a complementary measure to con rm the prevalence of IR, we calculated the TG/HDL ratio, which closely correlated with this parameter in adults at a value ≥ 3 [47]. This ratio is also considered as a predictor of cardiometabolic risk and re ects an atherogenic lipid pro le [48]. In the pediatric population, a high TG/HDL ratio is associated with lower insulin sensitivity and cardiovascular risk [49]. We found a signi cantly increased TG/HDL ratio for the obese group (3,02 p < 0,0001) in comparison with the lean group (1,60). These results con rm a prevalent condition of IR and cardiovascular risk in obese participants that could be associated with their downregulation of PPARα. Regarding this, PPARα has been reported to improve insulin sensitivity and give a protective property against the development of IR [44,50].
Besides, PPARα is recognized by its anti-in ammatory role. In this study, we observed a proin ammatory pro le in obese pediatric subjects with an increase in the serum levels of adipsin, MCP-1, IL-8, IL-6, IFN-γ, and TNF-α, and a decreased levels of the anti-in ammatory adipokine adiponectin. Hence, PPARα downregulation in leukocytes from obese children suggests an association with their proin ammatory pro le. Indeed, PPARα displayed a signi cant negative correlation with TNF-α levels and the abdominal skinfold. TNF-α promotes IR, decreases the uptake of fatty acids by adipocytes, increasing them in the circulation while stimulating lipolysis [51,52].
Furthermore, TNF-α increases the production of in ammatory cytokines such as IL-6 and suppresses the production of adiponectin [52]. TNF-α altogether with IFN-γ and IL-6 leads to an in ltration of immune cells in adipose tissue and dysfunction of in ammatory immune cells [53]. In this work, it was found that the concentration of TNF-α was signi cantly higher in obese patients than in the control group. These ndings are consistent with the high HOMA index, IL-8 and IL-6 levels, and the decreased adiponectin concentration in obese children and adolescents.
The consequences of obesity on the development of IR and activation of a chronic in ammatory response has allowed establishing a relationship between GLP-1 and PPARs. GLP-1 is a hormonal factor that improves insulin synthesis and secretion on the pancreatic β-cells. GLP-1 signaling through GLP-1R is recognized for its cytoprotective role in those cells [16,18]. Increased GLP-1R expression and improved GLP-1 sensitivity into pancreatic islets may be favored by the activation of PPARα [54]. Interestingly, GLP-1R is expressed in some lineages of immune cells that have an immunomodulatory role in the migration and differentiation of T and B lymphocytes [16,55,56]. GLP-1R also displays anti-atherogenic effects during obesity by reducing monocyte/macrophage migration and in ammatory cytokine production [57,58]. In this study, we observed a reduced expression of GLP-1R in obese children and adolescents. Although its expression did not show a signi cant association with in ammatory parameters, GLP-1R and PPARα expression exhibited a negative correlation with abdominal skinfold. These ndings suggest a role of GLP-1R and PPARα in the lipid distribution that could indirectly affect the role of different immune cells and the secreted cytokines. Previous studies show that GLP-1R induce downregulation of proin ammatory cytokines mediated through PPARγ activation and NF-kB inhibition [19], indicating that it is possible that both receptors present crosstalk in leukocytes from obese pediatric population. This association is highly needed to be explored in the future.
In summary, adipose tissue accumulation in obesity is associated with disturbances in the homeostasis of several tissues, disruption of the mechanisms controlling lipid and glucose metabolism, and dysregulation at the secretion pro le for proin ammatory cytokines and adipokines [53]. Accordingly, it was possible to establish a relationship between anthropometric alterations and body fat distribution observed in obesity with associated comorbidities, such as IR, chronic in ammatory pro le, and risk for the development of cardiovascular diseases. Thus, PPARα and GLP-1R are potential gene targets to explore its role in fat distribution and in ammatory pro le as essential aspects in pediatric obesity.

Conclusion
We have shown that PPARα and PPARβ/δ expression are negatively associated with the proin ammatory markers TNF-α and IL-8, respectively, suggesting participation in the regulation of in ammation observed in pediatric obesity. Furthermore, PPARα and GLP-1R are downregulated in leukocytes from obese children and adolescents, and both receptors are associated with the abdominal skinfold suggesting a role in fat distribution that could indirectly affect the function of different immune cells and the secreted cytokines. Thereby, these ndings may impact the understanding and implementation of PPARα or GLP-1R agonists in the clinic.

Materials And Methods
Participants This is a cross-sectional study with primary data collection performed in children and adolescents from February of 2017 to June of 2018 in Bogota-Colombia. Children and adolescents aged 7 to 17 years old from seven public schools were invited to participate voluntarily with parental authorization. The study population included participants from low-to middle-income, which represent most of the city population.
Finally, 37 participants (female, n = 18, and male, n = 19), which ful lled the inclusion criteria, agreed to participate in this study. The inclusion criteria included female and male participants aged 7-17, with a healthy weight (lean control group) and obesity (de ned as BMI, ≥ 85th, and ≥ 95th percentile) [2].
Exclusion criteria included: children and adolescents with diabetes mellitus (both type I and II), secondary causes of obesity, psychiatric illness, mental disorders, with weight equal to or greater than 100 kilograms, as well as those who were receiving medications which promoted weight loss or gain or altered in a way the metabolic pro le including glycemia.
The Institutional Review Board of the Antonio Nariño University and the Medical Ethics Committee at DEXA DIAB (CE-CC-00721) approved the study protocol. Written informed parental consent and child assent from participants were obtained before any research procedures. This study was carried out following the ethics committee code of the American Medical Association endorsed by the National Institutes of Health (NIH) [59].

Anthropometric assessment
Anthropometric measures were obtained using standard methods [60]. Weight was measured to the nearest 0.1 kg on a calibrated digital scale without shoes and wearing light clothing. Height was measured to the nearest 0.1 cm on a stadiometer. To evaluate the nutritional status of children and adolescents, the BMI Z-score was used, according to the criteria proposed by the World Health Organization [2]. Waist circumference (WC) was measured via exible measuring tape with an accuracy of 1 mm. The measurement was made with the subject standing, taken at the midpoint between the right lower costal ridge and the iliac crest, without skin compression from the measuring tape, and at the end of a normal expiration. BMI and waist-height were calculated as ratios. Skinfold thicknesses were assessed using a Harpenden Skinfold Calliper, according to a previous protocol [60]. The measurements were performed in triplicate at the non-dominant side on biceps, triceps, subscapular, and suprailiac to the nearest 0.1 mm, and the mean values were calculated [60].
The pubertal stage was assessed according to Tanner criteria [61]. Brie y, the participants were classi ed as pre-pubertal when they reached a pubertal state between 2-4 and post-pubertal in state 5.

Measurement of body composition by Dual X-ray Absorptiometry
Measurements of total and regional body composition were acquired using a Dual-Energy X-ray Absorptiometry (DEXA) scan (GE Lunar Prodigy advance, GE Healthcare) by a trained technician. Before each acquisition, the scanner was calibrated according to the manufacturer's instructions. The total effective radiation dose during each examination was < 0.05 micro Sievert for a three-minute scan. For analysis, reference data for standard deviation scores were provided from GE Lunar Body composition software (enCORE 2010; version 11.3; GE Healthcare, Madison, WI, USA)

Biochemical analyses
Blood samples for laboratory analysis were collected by venipuncture from all participants after an overnight fast (10-12 hours). Participants underwent a 2-hour oral glucose tolerance test (OGTT) (1.75 g/kg, maximum 75 g) according to the American Diabetes Association criteria [62].

Gene expression analysis
Leukocyte total RNA was extracted using Trizol reagent (Life Technologies, Inc) according to the manufacturer's instructions. RNA was quanti ed with a Nanodrop ND-1000 spectrophotometer (NanoDrop Technologies), and cDNA was synthesized from 1 µg ARN with the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scienti c, Cat. # 4368814). Real-time PCR was performed in duplicate using CFX96 equipment (BioRad) and SYBR® Green PCR Master Mix, according to the manufacturer's protocol. PPAR-α, PPAR-β, PPAR-γ, incretins receptor genes GLP-1R and GIPR, and reference genes PGK1 and YWHAZ were quantitated using the gene-speci c primers (Supplementary  Table 1). cDNAs were ampli ed for 40 cycles consisting of 10 s of denaturation at 95 °C, 15 s of annealing temperature for each primer (Supplementary Table 1), and 10 s of extension at 72 °C. Standard curves for all genes were generated using serial dilutions of pooled cDNAs from all samples. Relative mRNA expression was calculated with the method ΔΔCt. Data are shown as normalized ratios between target gene expression and geometric media of the two reference genes [63]. All expression assays were performed following the MIQE guidelines [64].

Hormone and cytokine levels
Preprandial GIP and GLP1 levels were measured in serum using a speci c enzyme-linked immunosorbent assay (Elabscience, Cat. #E-EL-H2061 and E-EL-H6025) according to the procedures provided by the manufacturer. Levels were expressed in ng/mL and pg/mL. The presence of adiponectin, adipsin, RBP4, MCP-1, IL-1β, IP-10, IL-10, IL-8, leptin, IL-6, IFN-γ, resistin, and TNF-α in serum samples was analyzed using a multiplex immunoassay based on uorescence-encoded beads according to the manufacturer's instructions (No. 40196, BioLegend). The acquisition was performed in an Accuri C6 ow cytometer. Offline analysis was performed with LEGENDplex™ data analysis software (version 8.0), and the data were expressed as the mean reporter uorescence intensity PE (MFI) as a function of concentration (pg/mL). Each assay was performed with two technical replicates.

Statistical analysis
Statistical analysis was performed using GraphPad Prism 6. Descriptive statistics were expressed as mean ± standard deviation (SD) whenever applicable, and variables were tested for normality by Kolmogorov-Smirnov test. Non-parametric tests were used whenever data were not normally distributed or Student t-test when data were normally distributed. We used Wilcoxon signed-rank tests to assess the signi cance of inter-group differences. The correlation of target genes and evaluated parameters were determined using the Spearman rank test. For all analyses, statistical signi cance was considered as p < 0.05.

Declarations
Ethics approval and consent to participate The study protocol was approved by the Institutional Review Board of the Antonio Nariño University and the Medical Ethics Committee at DEXA DIAB (CE-CC-00721). Written informed parental consent, and child assent from participants were obtained before any research procedures were conducted. This study was carried out in accordance with the ethics committee code of the American Medical Association endorsed by the National Institutes of Health (NIH).

Consent for publication
Not applicable.

Availability of data and materials
The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests Funding This study was supported by the Vicerrectoria de investigación (VCTI) from the Universidad Antonio The values are expressed as a normalized ratio between the target gene expression and the geometric median of the PGK and YWHAZ endogenous genes. Statistical differences were analyzed by Student's t test (* p < 0.05).  Serum proin ammatory cytokines and chemokines are increased in children and adolescents with obesity.IL-8, IL-6, TNF-α, IFN-γ, IL-10,MCP-1 and IP-10 serum levels were measured by ow cytometry using a Legendplex multiplex assay. Statistical differences were analyzed by Mann Whitney test (* p < 0.05, ** p < 0.005, *** p < 0.0005).

Figure 4
Serum proin ammatory adipokines are increased in children and adolescents with obesity.Adiponectin, adipsinand resistinserum levels were measured by ow cytometry using a Legendplex multiplex assay.

Supplementary Files
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