Postprandial sRAGE levels in type 1 diabetes mellitus in children and adolescents
sRAGE, postprandial hyperglycaemia, type 1 diabetes mellitus, metabolic memory
Introduction. Hyperglycaemia state increases the accumulation of the advanced glycation end-products (AGEs) in the body, which, in turn, induce the development of micro – and macroangiopathic lesions through binding with their receptor, RAGE (Receptor for Advanced Glycation Endproducts). Soluble isoform of RAGE receptor (sRAGE), circulating in the serum, binds with AGEs, and inhibits their attachment to the surface receptor. In turn, the above mentioned interaction inhibits the activation of the surface receptor. Previous studies did not provide a clear answer to the question about the importance and role of sRAGE in the development and maintaining of inflammation in children and adolescents with type 1 diabetes mellitus (T1DM). Studies assessing the role of the above mentioned eproteins in postprandial period are scarce. Aim to compare sRAGE levels in T1DM children vs. the control group, and to assess sRAGE postprandial levels in T1DM children and adolescents. Material and methods. The study involved 71 patients (35 girls and 36 boys) aged 7–17 years, diagnosed with T1DM, with treatment duration from 6 to 60 months. The control group consisted of 20 children (9 girls and 11 boys) aged 6.5–16 years. Due to a potential impact on study results, patients with other diseases were excluded. sRAGE levels were measured in the morning on fasting (at blood glucose levels within 80–120mg% range), and also at 1, 2 and 4 hours after a standard meal. Results. In the T1DM group, statistically significant higher sRAGE levels were measured at 0’, 1 and 4h of the study compared to the control group. In the T1DM children with HbA1c level <8 % and diabetes mellitus duration <2 years, statistically significant higher sRAGE levels were observed on fasting, and at 1 and 4h of the study compared to the control group. In patients with T1DM, sRAGE fasting levels were statistically significanty higher compared to 1, 2 and 4h measurements. The results show a significant negative correlation between glucose and sRAGE levels measured at the 2nd hour of the study. However, no correlation between sRAGE and HbA1c levels as well as between diabetes mellitus duration were observed. Conclusions. The inflammation is more pronounced in the T1DM group and hyperglycaemia episodes increase inflammatory processes.
Hyperglycaemia is an established factor involved in the pathogenesis of progressive lesions in blood vessels. It has been proven that even a short-term increase of postprandial glycaemia promotes glycation of proteins, and accumulation of advanced glycation end-products (AEGs) responsible for the development of long-term complications . Previous studies demonstrated that AEGs, by binding with their specific receptor, stimulate release of inflammatory cytokines and promote procoagulant activity in blood [2,3]. RAGE was discovered in 1992 . It is a surface receptor located on macrophages, monocytes, smooth muscle cells, endothelial cells, astrocytes and microglial cells. RAGE is a member of the immunoglobulin-like cell surface receptor superfamily encoded by the gene located on chromosome 6, within the major histocompatibility complex (MHC) class III region. RAGE consists of three fragments – N-terminal extracellular fragment (332aa), transmembrane portion (23aa), and C-terminal intracellular fragment (43aa). N-terminal fragment contains 3 domains: two C-set and one V-set. V-domain binds ligand, while transmembrane portion of the RAGE receptor attaches to the C-terminal intracellular fragment, and is responsible for signal transduction into the cellular nucleus. The RAGE receptor recognizes three dimensional structures of the ligand, and not the amino acid sequence, which, in turn, increases the scale of the activated signalling pathways responsible for many inflammatory processes [4,5].
esRAGE (endogenous secretory receptor for AGEs) is a soluble isoform of the RAGE receptor present in blood serum. It is created through an alternative gene transcription process or cleavage of the transmembrane portion of the cRAGE isoform by the matrix metalloproteinases (MMPs) . Both soluble forms create the so-called sRAGE (soluble receptor for AGEs) pool that binds AGEs circulating in blood serum. As a result, this interaction inhibits binding of AGEs to the surface receptor, thus prevents its activation. Therefore, sRAGE exhibits an antagonistic action with regard to RAGE [5,7]. The available literature data do not allow for a clear determination of sRAGE role in development and maintaining of inflammation in children and adolescents with type 1 diabetes mellitus, especially during postprandial period.
Aim of the paper
The study aimed to compare sRAGE levels in children with T1DM and in the control group, and to assess sRAGE postprandial levels in children and adolescents with T1DM.
Material and methods
The study involved 71 patients (35 girls and 36 boys) aged 7–17 years, diagnosed with T1DM, with treatment duration from 6 to 60 months. Children were receiving an intensive insulin therapy with the use of personal insulin pump (PIP) or multiple injections with insulin pens. Due to a potential impact on study results, patients with infectious and chronic diseases were excluded. The control group consisted of 20 children (9 girls and 11 boys) aged 6.5–16 without carbohydrates metabolism disorders or any other diseases. Children and adolescents with T1DM received the possibility to use a 24h continuous glucose monitoring system (CGMS) that was connected on admission. The blood samples were drawn on fasting from the basilic vein in accordance with commonly followed standards. The following parameters were tested: fasting glucose level, HbA1c, and sRAGE levels. On the second day of hospitalization, the trial was initiated only in patients with fasting glucose levels falling within 80–120mg% range, and without nocturnal hypoglycaemia episodes in CGMS measurements. Patients/parents were trained to use the CGMS device. Based on the retrospective data taken from parents/children, each subject received calculations of the amount of insulin needed per carbohydrate exchange, insulin/carbohydrate exchange ratio, and insulin sensitivity. All children were receiving a balanced formula type Nutridrink [Nutricia], in order to obtain a standardized meal. Additional blood samples for sRAGE levels measurement were drawn at 1, 2 and 4h after administration of a standard meal. sRAGE levels were measured with the use of an immunoenzymatic assay, i.e. ready-to-use ELISA kits manufactured by BioVendor Research and Diagnostic Products (Modrice, Czech Republic). The study was approved by the Ethics Committee. Parent/legal representative and individuals over 16 years of age had to provide a signed copy of an informed consent form (ICF) before any study procedure could start.
In groups with heterogeneous variance, a hypothesis concerning equivalence of means for particular samples was verified with the use of the analysis of variance (ANOVA). In case of small size groups, a non-parametric Kruskal-Wallis test was used (homoscedasticity was verified with the use of Bartlett’s test).
With regard to discrete parameters, the prevalence of a given characteristic within groups was analysed with the use of c2 test with Yates’ correction. The correlation analyses were performed for the selected parameters pairs, and allowed for calculation of Pearson’s correlation (r). In case of p value <0.05, the observed difference was statistically significant. Statistical analysis was performed with the use of computer packages of statistical programs EPIINFO version 126.96.36.199 (from 02.07.2013).
Patients’ clinical characteristics are presented in Table I.
In the T1DM group, statistically significant higher sRAGE levels were measured at 0’, 1 and 4h of the study compared to the control group (p <0.05). Tests results are presented in Table II.
The T1DM children were divided in two groups:
Taking into account of\\the level of HbA1c, the patients were divided into two subgroups:
HbA1c ≤8%, N=54 – Subgroup 1; HbA1c >8%, N=17 – Subgroup 2.
Taking into account the T1DM duration, the patients were divided into two subgroups:
≤2 years, N = 35 – Subgroup A; >2 years, N = 36 – Subgroup B.
Statistically significant higher sRAGE levels on fasting and at 1, 4h of the study were observed only in the subgroup 1 compared to the control group (p <0.05). Comparing the results of children in subgroup 2 to the control group and results of children in both groups divided in regard to the HbA1c level, statistically significant differences have not been observed. Results are presented in Table III–V.
Statistically significant higher sRAGE levels on fasting and at 1, 4h of the study were observed in children from subgroup A compared to the control group. The results are presented in Tables VI. Statistically significant higher sRAGE levels at 1 and 4h of the study were observed in children from subgroup B compared to the control group. The results are presented in Tables VII. Comparing the results of children in both groups divided in regard to the T1DM duration, no statistically significant differences was noticed. The results are presented in Tables VIII.
Next, sRAGE levels were measured at particular time points in children with T1DM. Fasting sRAGE levels were statistically significant higher compared to measurements at 1, 2, and 4h of the study (p = 0.000). The results are presented in Table IX and Fig. 1.
This study evaluated the correlation between sRAGE levels and blood glucose at particular time points of the study. The study revealed a significantly negative correlation between glucose and sRAGE levels measured at the 2nd hour of the study. The results are summarized in Table X. However, no correlation between sRAGE and HbA1c levels as well as between diabetes mellitus duration were observed (Table XI).
Hyperglycaemia is the strongest inducer of the protein glycation process [6–8]. Endo- and exogenous AGEs, directly through molecular modification or indirectly via binding with multiple receptors, induce irreversible changes in the endothelium [2,8]. Transmembrane RAGE is the most known receptor for AGEs . The interaction between AGEs and an endothelial cell results in an increase of endothelial permeability, decrease of thrombomodulin expression, increase of tissue factor (TF) synthesis and expression, and an increase of production of inflammatory markers [8,9]. sRAGE prevent the connection between AGEs and the transmembrane receptor, therefore it may play a role of an anti-inflammatory agent . In patients with an early stage diabetic retinopathy, it was demonstrated that the use of sRAGE limits processes of neoangiogenesis and atrophy of pericytes in the retina . In this paper, statistically significant higher sRAGE levels in children with type 1 diabetes mellitus compared to the control group were observed. The statistically significant difference was observed on fasting as well as at 1 and 4h after a standard meal administration (Table II). Other authors also have observed higher sRAGE levels in children with type 1 diabetes mellitus . In authors’ opinion, this condition may provide a temporary protection, and prevent the initiation of the inflammatory cascade by capturing the circulating AGEs . This may indicate the presence of an inflammatory reaction, during which sRAGEs exhibit the protective action. It is because the amount of surface receptors for AGEs increases in places of damaged and inflamed tissues. It was demonstrated that an increased activation of signalling pathways in the long-term treated diabetic patients may result from RAGE receptors activation . In many papers, sRAGE is presented as a factor binding the marked amount of RAGE-specific ligands. Therefore, sRAGE level reduction indicates an increase of the inflammation, progression of diabetic vascular complications, and an increase of cardiovascular (CV) incidents risk [14–16]. Higher sRAGE level may be a strong marker of CV diseases risk, both in type 1 and type 2 DM patients, as well as in subjects without carbohydrates metabolism disorders . Higher sRAGE levels were also observed in patients with advanced micro- and macrovascular complications . Nevertheless, there have been calls that sRAGE is not a AGEs scavenger, and its level may only reflect the tissue RAGE expression . However, a study demonstrating an increase of AGEs level accompanied by an increase of expression and thus the amount of transmembrane RAGE receptor, with concomitant sRAGE level decrease, provides contrary results . On the other hand, Katakami et al. demonstrated that in T1DM patients sRAGE level is higher compared to the healthy controls; however, this difference was insignificant . However, no relationship between the degree of metabolic control in diabetes mellitus, expressed as HbA1c percentage, and sRAGE level was observed . In our studies, sRAGE levels in children with HbAc1 ≤8% were statistically significant higher, both on fasting and at 1 and 4h post meal, compared to the control group (Table III). Therefore, reports on a potential relationship between sRAGE level and metabolic control of diabetes mellitus are divergent [22,23]. Motawi et al. demonstrated that higher HbA1c levels were statistically significant correlated with lower sRAGE levels . In my own study, I have not observed any statistically significant correlation between HbA1c and sRAGE levels. Other authors also did not confirm such a relationship .
In the present study, the highest sRAGE levels were observed in children with DM duration ≤2 and >2 years. In the above mentioned subgroup, fasting as well as 1- and 4-hour postprandial sRAGE levels were statistically significant higher compared to the control group (Table VI,VII). Koutroumani et al. have observed higher sRAGE levels in children with T1DM duration >5 years compared to the children with disease duration below 5 years . According to other authors, sRAGE level increases from the beginning of the disease, and remains stable thereafter . However, the recent studies also demonstrate a strong impact of an exogenous insulin on sRAGE levels . Perhaps, such a type of insulin therapy impacts the increase of the RAGE expression on cell surface, and sRAGE level increase . According to available literature, postprandial hyperglycaemia episodes, regardless of global patient’s metabolic control, may independently impact the development of diabetic complications . However, it was demonstrated that the underlying inflammation responsible for long-term diabetic complications is induced to a greater extent rather by postprandial blood glucose increase than by chronic hyperglycaemia . Moreover, it has been proven that metabolic reactions activated by persisting high blood glucose levels modify inflammatory response, and in part may compensate the toxic effects of chronic hyperglycaemia [28,29]. According to the established principle of the metabolic memory, even short-term hyperglycaemia episodes during and early phases of T1DM, despite good metabolic control in subsequent years, may adversely impact the risk of vascular complications development through epigenetic modifications . We have also found studies that did not observe any relationship between postprandial hyperglycaemia and sRAGE levels . sRAGE stability has been already described by Bower et al. During a 3-year follow-up, the authors concluded that single measurements of sRAGE levels were not significantly different . In our studies, the highest sRAGE levels were observed before Nutridrink administration, and were decreasing thereafter. Differences between measurements performed on fasting and at 1 hour after a meal, and between 2 and 4 hours after a meal were statistically significant lower (Table IX, Fig. 1). This situation indicates high dynamics of metabolic changes during the postprandial period. It is suggested that sRAGE level decrease during the postprandial period probably results from AEGs binding, whose levels increases along with the change of postprandial blood glucose concentrations . The results show a significant negative correlation between glucose and sRAGE 2-hour postprandial levels (Table X). The tests results demonstrate a complex role of sRAGE in the inflammatory reaction. Therefore, further studies enabling explanation of its role in the inflammation observed in T1DM children and adolescents are warranted.
1. Postprandial changes in sRAGE concentrations have a different profile comparing healthy controls to T1DM children.
2. They are not dependent on a long term diabetes control, however for diabetic children there are negative correlations of sRAGE and contemporary glycemia.
1. Ghazarian L, Simoni Y, Pingris K et al. Regulatory role of NKT cells in the prevention of type 1 diabetes. Med Sci 2013, 29(8-9):722-8.
2. Brandt A, Zorena K, Myśliwiec M. Końcowe produkty glikacji – źródło pochodzenia a rozwój powikłań cukrzycowych. Diabetologia Praktyczna 2008, 9,1:12-17.
3. Padgett LE, Broniowska KA, Hansen PA et al. The role of reactive oxygen species and proinflammatory cytokines in type 1 diabetes pathogenesis. Acad Sci. 2013, 4, 1281:16-35.
4. Pietkiewicz J, Seweryn E, Bartyś A. Receptory końcowych produktów zaawansowanej glikacji- znaczenie fizjologiczne i kliniczne. Postępy Hig Med Dosw 2008, 62:511-523.
5. Yamagishi S, Nakamura K, Matsui T et al. Receptor for Advanced Glycation End Products ( RAGE): A Novel Therapeutic Target for Diabetic Vascular Complication. Current Pharmaceutical Design 2008, 14: 487-495.
6. Koyama H, Nishizawa Y. AGEs/RAGE In CKD: irreversible metabolic memory road toward CVD? European Journal of Clinical Investigation 2010, 40:623-635.
7. Obońska K, Grąbczewska Z, Fisz J et al. Cukrzyca i dysfunkcja śródbłonka- krótkie spojrzenie na złożony problem. Folia Cardiologica Excerpta 2011, 6,2:109-116.
8. Yagamishi S, Nakamura K, Imaizumi T. Acvanced Glycation End Products and Diabetic Vascular Complications. Current Diabetes Reviews 2005, 1: 95-106.
9. Yan S, Ramasamy R, Schmidt AM et al. Soluble RAGE: therapy and biomarkers in untraveling the RAGE axis in chronic disease and aging. Biochem Pharmacol 2010, 79 (10): 1379-1386.
10. Bakker SF, Tushuizen ME, Gözütok E et al. Advanced glycation end products (AGEs) and the soluble receptor for AGE (sRAGE) in patients with type 1 diabetes and coeliac disease. Nutrition, Metabolism & Cardiovascular Diseases 2015,25: 230-235.
11. Zorena K, Raczyńska D, Raczyńska K. Biomarkers in diabetic retinopathy and the therapeutic implications. Mediators Inflamm. 2013; 2013:193604.
12. Dettoraki et al.; Challier M, Jacqueminet S, Benabdesselam O, Grimaldi A, Beaudeux JL. Increased serum concentrations of soluble receptor for advanced glycation endproducts in patients with type 1 diabetes. Clin Chem 2005;51(9):1749-1750.
13. Dettoraki A, Gil AP, Spiliotis BE. Association between serum levels of the soluble receptor (sRAGE) for advanced glycation endproducts (AGEs) and their receptor (RAGE) in peripheral blood mononuclear cells of children with type 1 diabetes mellitus. J Pediatr Endocrinol Metab. 2009, 22 (10):895-904.
14. Thomas MC, Soderlund J, Lehto M et al. Soluble receptor for AGE (RAGE) is a novel independent predictor of all-cause and cardiovascular mortality in type 1 diabetes. Diabetologia 2011, 54: 2669-2677.
15. Grossin N, Wautier MP, Meas T et al. Severity of diabetic microvascular complicationns is associated with a low soluble RAGE level. Diabetes & Metabolism 2008, 34: 392-395.
16. Katakami N, Matsuoka TA, Nakatani Y et al. Decreased endogenous secretory advanced glycation end product receptor in type 1-diabetic patients: its possibile association with diabetic vascular complications. Diabetes Care 2005, 28:2716-21.
17. Nin JWM, Jorsal A, Ferreira I et al. Higher Plasma Soluble Receptor for Advanced Glycation End Products (sRAGE) Levels Are Associatef With Incident Cardiovascular Disease and All-Cause Mortality in Type 1 Diabetes. A 12- Year Follow - Up Study. Diabetes 2010, 59: 2027-32.
18. Challier M, Jacqueminet S, Benabdessalam O et al. Increased Serum Concentrations of Soluble Receptor for Advanced Glycation Endproducts in Patients with Type 1 Diabetes. Clin Chem 2005, 51(9):1749-50.
19. Yamagishi S, Matsui T. Soluble form of a receptor for advanced glycation end products (sRAGE) as a biomarker. Front Biosci 2010, 2: 1184-1195.
20. Tam XHL, Shiu WM, Leng L et al. Enhanced expression of receptor for advanced glycation end – products is associated with low circulating soluble isoforms of the receptor in type 2 diabetes. Clinical Science 2011, 120: 81-89.
21. Fujisawa K, Katakami N, Kaneto H et al. Circulating soluble RAGE as a predictive biomarker of cardiovascular event risk in patients with type 2 diabetes. Atherosclerosis 2013, 227(2):425-8.
22. Motawi T, Abou-Seif M, Bader M et al. Effect of glycemic control on soluble RAGE andoxidative stress in type 2 diabetic patients. BMC Endocrine Disorders2013,13:32.
23. Humpert PM, Djuric Z, Kopf S et al. Soluble RAGE but not endogenous secretory RAGE is associated with albuminuria In patients with type 2 diabetes. Cardiovascular Diabetology 2007, 7, 6:9.
24. Koutroumani N, Partsalaki I, Lamari F et al. Protective mechanisms against oxidative stress and angiopathy In Young patients with diabetes type 1. J Pediatr Endocrinol Metab 2013, 26 (3-4): 309-317.
25. Reis JS, Veloso CA, Volpe MO et al. Soluble RAGE and malondialdehyde in type 1 diabetes patients without chronic complications during the course of the disease. Diabetes& Vascular Disease Research 2012, 9 (4): 309-314.
26. Lam JKY, Wang Y, Shiu SW et al. Effect of insulin on the soluble receptor for advanced glycation end products (RAGE). Diabetic Medicine 2013, 30 (6): 702-709.
27. Prasad K. Low Levels of Serum Soluble Receptors for Advanced Glycation End Products, Biomarkers for Disease State: Myth Or Reality. Int J Angiol 2014, 23 (01): 011-016.
28. Monnier , Mas E., Ginet C et al. Activation of oxidative stress by acute glucose fluctuations compared with sustained chronic hyperglycemia in patients with type 2 diabetes. JAMA 2012,295 (14):1681-7.
29. Keating ST, El-osta A. Epigenetic changes in diabetes. Clin Genet 2013, 84(1):1-10.
30. Wentholt IM, Kulik W, Michels RP et al. Glucose fluctuations and activation of oxidative stress in patients with type 1 diabetes. Diabetologia 2008 ,51(1):183-90.
31. Bower JK, Pankow JS, Lazo M et al. Three – year variability in plasma concentrations of the soluble receptor for advanced glycation end products (sRAGE). Clin Biochem 2014 , 47 (1-2): 132-4.
32. Gordin D, Forsblom C, Ronnback M et al. Acute hyperglycaemia induces an inflammatory response in young patients with type 1 diabetes. Ann Med 2008, 40(8):627-33.