An increased osteoprotegerin serum release characterizes the early onset of diabetes mellitus and may contribute to endothelial cell dysfunction

Abstract

Serum osteoprotegerin (OPG) is significantly increased in diabetic patients, prompting expanded investigation of the correlation between OPG production/release and glycemic levels. Serum levels of OPG, but not of its cognate ligand receptor activator of nuclear factor-kappaB ligand (RANKL), were significantly increased in type 2 diabetes mellitus patients compared with healthy blood donors. Serum OPG was also significantly elevated in a subgroup of recently diagnosed diabetic patients (within 2 years). The relationship between serum OPG and diabetes mellitus onset was next investigated in apoE-null and littermate mice. Serum OPG increased early after diabetes induction in both mouse strains and showed a positive correlation with blood glucose levels and an inverse correlation with the levels of free (OPG-unbound) RANKL. The in vitro addition of tumor necrosis factor-alpha to human vascular endothelial cells, but not human peripheral blood mononuclear cells, markedly enhanced OPG release in culture. In contrast, high glucose concentrations did not modulate OPG release when used alone or in association with tumor necrosis factor-alpha. Moreover, the ability of soluble RANKL to activate the extracellular signal-regulated kinase/mitogen-activated protein kinase and endothelial nitric-oxide synthase pathways in endothelial cells was neutralized by preincubation with recombinant OPG. Altogether, these findings suggest that increased OPG production represents an early event in the natural history of diabetes mellitus, possibly contributing to disease-associated endothelial cell dysfunction.

Figure 1

Serum OPG and RANKL levels in diabetic patients and healthy individuals. Levels of OPG (A), total (B), and free (C) RANKL were determined by ELISA in sera from diabetic patients and from healthy patients. Horizontal bars are median, upper, and lower edges of box are 75th and 25th percentiles; lines extending from box are 10th and 90th percentiles. *P < 0.05.

Figure 2

Relation between serum OPG levels and years from diagnosis in diabetic patients. Serum levels of OPG were analyzed in patient subgroups, based on the indicated years from diagnosis (A) and in relation with patient age (B). Horizontal bars are median, upper, and lower edges of box are 75th and 25th percentiles; lines extending from box are 10th and 90th percentiles. *P < 0.05. Coefficient of correlation is reported in the text.

Figure 3

Atherosclerotic aortic lesions and serum glucose levels in apoE-null mice after induction of diabetes. A: Representative H&E-stained histological cross-sectional sections from aorta of control and diabetic (3 months) apoE-null mice. Arrowhead, a wide atherosclerotic plaque. B: Levels of glucose were determined in serum samples from control and diabetic apoE-null mice at different weeks after diabetes induction by STZ treatment. Horizontal bars are median, upper, and lower edges of box are 75th and 25th percentiles; lines extending from box are 10th and 90th percentiles. *P < 0.05. Original magnifications, ×10.

Figure 4

Serum OPG and RANKL levels in diabetic and control apoE-null mice. Levels of OPG (A) and free RANKL (B) were determined in sera from control and diabetic apoE-null mice at different weeks after induction of diabetes. Horizontal bars are median, upper, and lower edges of box are 75th and 25th percentiles; lines extending from box are 10th and 90th percentiles. *P < 0.05.

Figure 5

Relation between serum levels of OPG and free RANKL or glucose. Relation between serum levels of OPG and free RANKL (A) and between serum levels of OPG and glucose (B) in diabetic apoE-null mice. Coefficients of correlation are indicated.

Figure 6

Serum OPG and RANKL levels in diabetic and control C57 littermate mice. Diabetes was induced by STZ injection in C57Black littermate mice. Levels of glucose (A), OPG (B), and free RANKL (C) were measured in sera from control and diabetic mice at different weeks after STZ treatment. Horizontal bars are median, upper, and lower edges of box are 75th and 25th percentiles; lines extending from box are 10th and 90th percentiles. *P < 0.05.

Figure 7

OPG release in endothelial cells and PBMC cultures. A: HUVECs were either left untreated or stimulated with glucose, insulin, TNF-α, or IL-1β. After 24 hours, the levels of OPG released in culture supernatant were measured by ELISA. B: HUVECs and PBMCs were either left untreated or stimulated with TNF-α ± glucose or STZ. After 72 hours, the levels of OPG released in culture supernatant were measured by ELISA. Results are expressed as means ± SD of three to four independent experiments, each performed in triplicate. *P < 0.05.

Figure 8

Effect of OPG on RANKL-induced intracellular signaling in endothelial cells. Quiescent HUVEC cultures were stimulated with RANKL or RANKL + OPG for the indicated time intervals (0 to 60 minutes). A: Cell lysates were analyzed for ERK1/ERK2 and eNOS activation by Western blot of total and phosphorylated (P) proteins using specific antibodies. Equal loading of protein in each lane was confirmed by staining with the antibody to tubulin. B: Protein bands were quantified by densitometry, and levels of P-ERK1/2 and P-eNOS were calculated for each time point, after normalization to ERK1/2, and eNOS, respectively. Unstimulated basal expression was set as unity. One of three experiments with similar results is shown.