Streszczenie

Wprowadzenie: Receptor CD36 to błonowa glikoproteina uczestnicząca w usuwaniu oxLDL (utlenionych cząstek LDL) z osocza, wiązaniu produktów glikacji białek, patogenezie insulinooporności, cukrzycy typu 2, mikro- i makroangiopatii cukrzycowej. Ekspresja receptora CD36 na makrofagach zwięk­sza się u pacjentów z hiperglikemią i genetycznie uwarunkowaną otyłością. Jego rola w rozwoju cukrzycy typu 2 i insulinooporności jest niejednoznaczna. Celem pracy było stwierdzenie czy u dzieci z otyłością polimorfizm genu CD36 wiąże się z zaburzeniami gospodarki węglowodano­wej oraz zmiennością stężenia CD36 w osoczu.
Materiał/Metody: Badaniami objęto 60 dzieci w wieku 10-15 lat: 30 z masą ciała >97 centyla i 30 z prawidłową masą ciała. Wykonano pomiary hemoglobiny glikowanej, wysokości, masy ciała, obwodu talii i bioder oraz RR. Obliczono BMI, WHR oraz MAP. Wykonano DTTG z pomiarem stężenia in­suliny. Amplikony eksonów 4-6 CD36 z przyległymi intronami analizowano metodą dHPLC. Produkty PCR z wykrytymi zmianami były sekwencjonowane. Osoczowe stężenie CD36 ozna­czono z użyciem testu ELISA.
Wyniki: Zidentyfikowano dwie intronowe zmiany: IVS3-6 T/C (rs3173798) i IVS4-10 G/A (rs3211892), niesynonimiczną substytucję G367A (Glu123Lys, rs183461468) w eksonie 5 oraz dwie synoni­miczne zmiany w eksonie 6: G573A (Pro191Pro, rs5956) i A591T (Thr197Thr, rs141680676). Nie stwierdzono istotnych statystycznie różnic pomiędzy badanymi grupami genotypowymi w żad­nym z morfometrycznych i biochemicznych parametrów.
Dyskusja: Brak jest związku polimorfizmów badanego fragmentu CD36 z zaburzeniami gospodarki węglo­wodanowej oraz zmiennością stężenia CD36 w osoczu dzieci otyłych. Jednakże ze względu na stosunkowo małą liczebność grup oraz brak danych co do funkcjonalnych efektów badanych po­limorfizmów konieczne są dalsze badania.

Słowa kluczowe:CD36 • otyłość • czynniki ryzyka cukrzycy

Summary

Introduction: CD36 may play an important role in removal of oxidized LDLs from plasma, protein glycation, the pathogenesis of insulin resistance, type 2 diabetes, and diabetic micro- and macroangiopathy. Some reports have pointed to decreased expression of macrophages in association with mutations of the CD36 gene in hyperglycemic and obese subjects. The aim of the study was to search for an association between CD36 gene polymorphism and carbohydrate metabolism disturbances or variability of plasma soluble CD36 concentrations in obese children.
Material/Methods: The study included 60 children aged 10 to 15 years: 30 with (study group) and 30 without (control group) obesity. Each patient’s glycated hemoglobin, weight, height, waist and hip circumference, and systolic and diastolic blood pressure were measured, BMI, WHR and MAP were calculated, and oral glucose tolerance test was performed with glucose and insulin concentration measure­ments. Amplicons of exons 4-6 of CD36 were studied using DHPLC technique. The PCR products with alterations were bidirectionally sequenced. Plasma concentrations of human antigen CD36 was measured using a commercially available enzyme-linked immunosorbent assay (ELISA).
Results: We found two intronic alterations: IVS3-6 T/C (rs3173798) and IVS4-10 G/A (rs3211892), one non­synonymous substitution: G367A (Glu123Lys, rs183461468) in exon 5 and two synonymous trans­itions in exon 6: G573A (Pro191Pro, rs5956) and A591T (Thr197Thr, rs141680676). There were no significant differences in any biochemical or morphometric parameters between genotype groups.
Discussion: The polymorphisms of the studied fragment of CD36 are not associated with carbohydrate meta­bolism disturbances or the variability of plasma soluble CD36 concentrations in obese children, but further research is necessary to assess their functional implications.

Key words: CD36 • obesity • diabetes risk factors

Introduction

Diabetes in adults increases the risk of coronary heart di­sease and cardiovascular complications [1]. Also, insulin resistance in patients with normoglycemia or impaired glucose tolerance is a risk factor for atherosclerosis [9]. Recent studies indicate the involvement of CD36 protein in the pathogenesis of insulin resistance and type 2 diabe­tes and diabetic micro-and macrovasculopathy [4,5,10,24]. The CD36 receptor is a membrane glycoprotein present on the surface of many cells. It participates in the removal of oxidized LDL (oxLDL) in plasma and the binding prote­in glycation products [18,20,23]. It was found that glyca­tion of LDL particles more strongly than their oxidation increases the expression of CD36 receptor, oxLDL uptake and accumulation of cholesterol in macrophages. It there­fore appears that glycation of LDL particles in diabetes in­itiates foam cell formation and accelerated atherosclerosis [14]. Some researchers have found that monocytes in sub­jects with hyperglycemia have increased expression of the CD36 receptor in comparison to those with normal gluco­se concentration [22]. In addition, patients with mutations in the gene encoding CD36 are more likely to have type 2 diabetes [6]. CD36 receptor expression in macrophages in patients with diabetes is directly proportional to blood glucose level [8]. Increased expression of the CD36 re­ceptor on macrophages was found in genetically determi­ned obesity, which is associated with insulin resistance. This is probably a consequence of disturbances of insulin signaling in these cells [2,17]. On the other hand, in the Japanese population a CD36-receptor defect was not found to be associated with the occurrence of impaired glucose tolerance, insulin resistance, or diabetes [25]. It is possi­ble that in the future plasma circulating soluble CD36 re­ceptor could be added to non-classical cardiovascular risk factors [11]. However, its role in the development of insu­lin resistance and type 2 diabetes is not clearly understo­od, and needs further investigations. The available litera­ture contains no reports on the importance of the CD36 receptor in obese children. Thus, for a fuller understan­ding of the role of CD36 in glucose metabolism pathoge­nesis, further study is required. The aim of the present stu­dy was to search for an association between CD36 gene polymorphism and carbohydrate metabolism disturbances or the variability of plasma soluble CD36 concentrations in obese children.

Material and Methods

The study included 60 Caucasian children (34 girls and 26 boys) aged 10-15 years: 30 with overweight or obesi­ty (study group) and 30 with normal mass (control gro­up) [12,21]. All patients were treated at the Independent Laboratory of Propedeutics in Pediatrics of Pomeranian Medical University in Szczecin (northwestern Poland) in 2008-2010. The patients were Polish residents. The study complies with the principles outlined in the Declaration of Helsinki and was approved by our institutional Ethics Committee. Informed consent was obtained from each pa­tient. Patients with endocrine or chronic diseases were exc­luded from the study.

There were no significant differences between the study and control groups as regards age (11.9±3.0 and 12.7±2.1 years, p=0.16; 11.9±3.0 and 12.7±2.1 years, respectively, p=0.16) or gender (male 11 and 15, respectively, p=0.20). Each patient’s weight, height, waist and hip circumference, and systolic and diastolic blood pressure were measured. The body mass index (BMI), waist-to-hip ratio (WHR), and mean arterial pressure (MAP) were calculated. A fa­sting blood sample (7 mL) was taken for glycated hemo­globin measurement and DNA extraction. Genomic DNA was isolated as previously described [13]. Moreover, an oral glucose tolerance test (1.75 g glucose/kg body mass, max. dose 75 g) was performed with insulin measurement.

Amplicons of exons 4-6 (region encoding the oxLDL doma­in) including fragments of introns were studied using dena­turing high-performance liquid chromatography (DHPLC) technique as previously described [19]. The PCR products with alterations detected by DHPLC were bidirectionally sequenced using the Applied Biosystems Dye-terminator Cycle Sequencing Ready Reaction kit, according to the manufacturer’s protocol. Semi-automated sequence ana­lysis was performed using a 373A DNA fragment analy­zer (Applied Biosystems, Foster City, CA). Plasma con­centrations of human antigen CD36 (Platelet Membrane Glycoprotein IV) were measured using the commercially available enzyme-linked immunosorbent assay (ELISA) kits (EIAab, Wuhan EIAab Science Co., Ltd., China) ac­cording to the manufacturer’s protocol.

Differences between subgroups of patients classified ac­cording to the intron 3 polymorphism (IVS3-6 T/C) and exon 6 (G573A) were tested with the Mann-Whitney test for quantitative variables and Fisher’s exact test for quali­tative variables.

Results

Changes detected by DHPLC comprised 2 single nucle­otide substitutions in introns (IVS3-6 T/C – rs3173798 and IVS4-10 G/A – rs3211892) and 2 synonymous polymorphi­sms in exon 6 (G573A – rs5956 and A591T – rs141680676). IVS4-10 G/A alteration was detected in one case in both groups and A591T (Thr197Thr) in two cases in the control group. Moreover, a non-synonymous substitution G367A (Glu123Lys – rs183461468) was detected in case in exon 5. Due to low statistical power of the single polymorphi­sms (IVS4-10 G/A, A591T, G367A) we analyzed statisti­cally only the associations with IVS3-6 T/C and G573A genotypes. The IVS3-6C allele frequency in the whole children population (10.8%) was similar to that described earlier in the Caucasian populations (6.2% to 11.2%), ac­cording to the NCBI dbSNP database. The 573A allele fre­quency (5.8%) was slightly higher than that described in Caucasians (4.2-4.5%) according to the dbSNP database. Genotype distributions were consistent with the Hardy- Weinberg equilibrium for all sequence changes (p=1).

There were no significant differences between the study and control groups as regards genotype frequency (p=0.74 for IVS3-6 T/C and p=0.34 for G573A). But we identified si­gnificant differences between these two groups in CD36 pla­sma concentration (p=0.02 for IVS3-6 T/C and p=0.05 for G573G). There were no significant differences between the genotype subgroups in terms of any of the clinical, morpho­metric or biochemical analyzed parameters (Table 1). We fo­und only a tendency (p=0.06) to higher fasting insulin level in IVS3-6 TC heterozygotes than in wild-type homozygotes.

Table 1. Clinical, morphometric and biochemical, parameters of obese and normal-weight children stratified by IVS3-6 T/C and G573A CD36 genotypes

Discussion

No data have been published so far that would suggest an association between variation in the CD36 gene and carbo­hydrate metabolism disturbances or plasma soluble CD36 concentrations in obese children. Available studies on adults did not analyze changes in the sequence presented in this paper. Lepretre et al. [16] observed no association betwe­en the IVS4-10 G/A alteration and type 2 diabetes in the Caucasian population. There was found a low serum adi­ponectin level and the associated insulin resistance accom­panied by A (-178) C alteration in the promoter of CD36 [16]. In addition there was found an association of low plasma concentrations of adiponectin with a rare nonsen­se mutation T1079G in exon 10, leading to premature ter­mination of translation and the formation of nonfunctio­nal CD36 receptor protein in patients with type 2 diabetes, including in the Caucasian population [17]. Other authors have often (44%) found in Caucasian adults with type 2 diabetes the C (-3489) T (rs1527479) alteration in the pro­moter of CD36. The proportion of TT genotype within the study group was 26.5% [3]. By contrast 539AC deletion in exon 6, resulting in a reading frame shift, was indica­ted as the CD36 gene mutation often associated with the presence of type 2 diabetes in the Japanese population [7].

This report is an attempt to draw attention to the possibili­ty of association of CD36 gene polymorphism with impa­ired glucose metabolism and the variability of plasma con­centrations of the protein CD36 in obese children, which, in contrast to adults, has not been studied by researchers. It seems reasonable to continue the research and its exten­sion to a larger group of patients to be able to draw better conclusions for the pediatric population.

Our results suggest no association of the analyzed fragment CD36 polymorphism with impaired glucose metabolism or the variability of plasma concentrations of CD36 pro­tein in obese children. However, due to the relatively small group size and the lack of data regarding the functional ef­fects of polymorphisms studied, further research is needed.

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The authors have no potential conflicts of interest to declare.

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