Serum Level of Soluble Receptor for Advanced Glycation End Products Is Associated with A Disintegrin And Metalloproteinase 10 in Type 1 Diabetes

<div><p>Background</p><p>The receptor for advanced glycation end products (RAGE) is involved in the pathogenesis of diabetic complications, and soluble forms of the receptor (sRAGE) can counteract the detrimental action of the full-length receptor by acting as decoy. Soluble RAGE is produced by alternative splicing [endogenous secretory RAGE (esRAGE)] and/or by proteolytic cleavage of the membrane-bound receptor. We have investigated the role of A Disintegrin And Metalloproteinase 10 (ADAM10) in the ectodomain shedding of RAGE.</p><p>Methods</p><p>Constitutive and insulin-induced shedding of RAGE in THP-1 macrophages by ADAM10 was evaluated using an ADAM10-specific metalloproteinase inhibitor. Serum ADAM10 level was measured in type 1 diabetes and control subjects, and the association with serum soluble RAGE was determined. Serum total sRAGE and esRAGE were assayed by ELISA and the difference between total sRAGE and esRAGE gave an estimated measure of soluble RAGE formed by cleavage (cRAGE).</p><p>Results</p><p>RAGE shedding (constitutive and insulin-induced) was significantly reduced after inhibition of ADAM10 in macrophages, and insulin stimulated ADAM10 expression and activity. Diabetic subjects have higher serum total sRAGE and esRAGE (p<0.01) than controls, and serum ADAM10 was also increased (p<0.01). Serum ADAM10 correlated with serum cRAGE in type 1 diabetes (r = 0.40, p<0.01) and in controls (r = 0.31. p<0.01) but no correlations were seen with esRAGE. The association remained significant after adjusting for age, gender, BMI, smoking status and HbA1c.</p><p>Conclusion</p><p>Our data suggested that ADAM10 contributed to the shedding of RAGE. Serum ADAM10 level was increased in type 1 diabetes and was a significant determinant of circulating cRAGE.</p></div

Effect of inhibition of ADAM10 on constitutive (A) and insulin-induced shedding of RAGE (B) in THP-1 macrophages.

<p>Cell surface receptors were biotinylated and cell-conditioned media was immunoprecipitated with anti-biotin agarose, electrophoresed and immunobloted with anti-RAGE antibody or streptavidin-HRP (STP-HRP) antibody. Biotinylated RAGE in cell-conditioned media (cRAGE) was significantly reduced by addition of GM6001 (*p<0.05) and GI254023X (**p<0.01) vs control cells (A). Cells were incubated with insulin (10mIU/ml) for 24 h in the presence or absence of inhibitors and biotinylated RAGE in cell-conditioned media was quantified. Data represent the mean ± SEM. ^#p< 0.01 vs insulin-treated cells (B).</p

Correlation between ADAM10 and cRAGE in type 1 diabetes (A) and control (B).

<p>Correlation between ADAM10 and cRAGE in type 1 diabetes (A) and control (B).</p

Clinical characteristics and serum levels of ADAM 10 and soluble RAGE isoforms in controls and type 1 diabetic patients.

<p>Values are mean ± SD, or median (interquartile range) or percentage.</p><p>*p<0.05</p><p>** p<0.01 vs controls.</p><p>Clinical characteristics and serum levels of ADAM 10 and soluble RAGE isoforms in controls and type 1 diabetic patients.</p

Effect of insulin on ADAM10 protein expression (A) and activity (B) and shedding of RAGE (C) in THP-1 macrophages.

<p>THP-1 macrophages were incubated with increasing concentrations of insulin (0 to 50 mIU/ml) or blank medium as control for 24 hours. ADAM10 protein in whole cell lysate was then measured by Western blot (A) and cellular ADAM10 activity was measured by fluorimetric assay (B). Data represent the mean ± SEM. *p<0.05, **p<0.01 vs control. Cell-conditioned media was harvested for quantification of cRAGE and experiments were repeated with the addition of specific ADAM10 inhibitor (C). *p<0.05 vs control, ^##p<0.01 vs corresponding insulin-treated cells.</p