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

Lee AC, Lam JK, Shiu SW, Wong Y, Betteridge DJ, Tan KC - PLoS ONE (2015)

Bottom Line: 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).The association remained significant after adjusting for age, gender, BMI, smoking status and HbA1c.Our data suggested that ADAM10 contributed to the shedding of RAGE.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Hong Kong, Hong Kong, China.

ABSTRACT

Background: 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.

Methods: 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).

Results: 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.

Conclusion: 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.

No MeSH data available.


Related in: MedlinePlus

Effect of insulin on ADAM10 protein expression (A) and activity (B) and shedding of RAGE (C) in THP-1 macrophages.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.
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pone.0137330.g002: Effect of insulin on ADAM10 protein expression (A) and activity (B) and shedding of RAGE (C) in THP-1 macrophages.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.

Mentions: To further investigate whether insulin increased shedding of RAGE by stimulating ADAM10 expression and/or activity, THP-1 macrophages were incubated with increasing concentrations of insulin, and ADAM10 protein expression and activity in cell lysate was determined. Western blot analysis showed that insulin increased ADAM10 expression in a dose-dependent manner (Fig 2A). This was paralleled by an increase in ADAM10 activity in the cell lysate (Fig 2B) and shedding of cell surface RAGE into cell-conditioned media which can be blocked by inhibiting ADAM10 (Fig 2C). Taken together, our data would suggest that insulin increases shedding of RAGE by stimulating ADAM10 expression and activity. In addition, we have shown that insulin stimulates ADAM10 expression and shedding of cell surface RAGE not only in THP-1 macrophages but also in human monocyte-derived macrophages (S1 Fig).


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

Lee AC, Lam JK, Shiu SW, Wong Y, Betteridge DJ, Tan KC - PLoS ONE (2015)

Effect of insulin on ADAM10 protein expression (A) and activity (B) and shedding of RAGE (C) in THP-1 macrophages.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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4556489&req=5

pone.0137330.g002: Effect of insulin on ADAM10 protein expression (A) and activity (B) and shedding of RAGE (C) in THP-1 macrophages.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.
Mentions: To further investigate whether insulin increased shedding of RAGE by stimulating ADAM10 expression and/or activity, THP-1 macrophages were incubated with increasing concentrations of insulin, and ADAM10 protein expression and activity in cell lysate was determined. Western blot analysis showed that insulin increased ADAM10 expression in a dose-dependent manner (Fig 2A). This was paralleled by an increase in ADAM10 activity in the cell lysate (Fig 2B) and shedding of cell surface RAGE into cell-conditioned media which can be blocked by inhibiting ADAM10 (Fig 2C). Taken together, our data would suggest that insulin increases shedding of RAGE by stimulating ADAM10 expression and activity. In addition, we have shown that insulin stimulates ADAM10 expression and shedding of cell surface RAGE not only in THP-1 macrophages but also in human monocyte-derived macrophages (S1 Fig).

Bottom Line: 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).The association remained significant after adjusting for age, gender, BMI, smoking status and HbA1c.Our data suggested that ADAM10 contributed to the shedding of RAGE.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Hong Kong, Hong Kong, China.

ABSTRACT

Background: 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.

Methods: 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).

Results: 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.

Conclusion: 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.

No MeSH data available.


Related in: MedlinePlus