Evaluation of the components of insulin‐like growth factor (IGF)‐IGF binding protein (IGFBP) system in adolescents with type 1 diabetes and persistent microalbuminuria: relationship with increased urinary excretion of IGFBP‐3 18 kD N‐terminal fragment
Authors: Spagnoli Anna , Chiarelli Francesco , Vorwerk Peter , Boscherini Brunetto , Rosenfeld Ron G. 
Published: May 2003

OBJECTIVE

IGFs and their binding proteins (IGFBPs) have an important role in controlling glucose homeostasis and there is evidence to support their involvement in complications related to type I diabetes. The aim of this study was to evaluate the components of the IGF‐IGFBP system in adolescents with type 1 diabetes that had developed persistent microalbuminuria (MA).

DESIGN AND PATIENTS

A cohort of 49 adolescents with type 1 diabetes were enrolled in the study. Patients were evaluated at baseline and 1 year later (follow‐up). Twenty‐six patients with persistent urinary albumin excretion (UAE) of more than 20 μg/min/1.73 m2 (21.6–109.4 μg/min/1.73 m2) in three different nocturnal urinary collections within 6 months were considered to have MA (baseline mean: 41.9 ± 22.3 μg/min/1.73 m2; follow‐up: 55.9 ± 24.8 μg/min/1.73 m2). Twenty‐three patients with UAE of less than 20 μg/min/1.73 m2 were assigned to the group without MA (baseline mean: 8.6 ± 3.7 μg/min/1.73 m2; follow‐up: 11.8 ± 4.2 μg/min/1.73 m2). Fasting serum levels of IGFBP‐1, IGFBP‐2, IGFBP‐3, IGF‐I and free‐IGF‐I were determined using appropriate immunoenzymatic, radioimmuno‐ or immunoradiometric assays. Overnight 12‐h urinary collections were obtained and assessed for IGFBP‐3 levels, determined by immunoradiometric assay. Urinary and circulating immunoreactive IGFBP‐3 forms were determined by Western‐immunoblotting (WIB) analysis using a specific polyclonal antibody and monoclonal antibodies directed against N‐terminal and C‐terminal epitopes of IGFBP‐3. IGFBP‐3 protease activity was determined using protease assay and by analysis of the intact over the fragmented immunoreactive forms of IGFBP‐3 determined by WIB analysis.

RESULTS

Patients with MA showed higher levels of urinary IGFBP‐3 (649 ± 440 ng/h/m2) than patients without MA (398 ± 229 ng/h/m2; P < 0.05). Urinary levels of IGFBP‐3 were directly correlated to UAE (P < 0.001). WIB analysis, using monoclonal antibodies directed against characterized N‐terminal and C‐terminal IGFBP‐3 epitopes, determined that the immunoreactive form of IGFBP‐3 found in urine from patients with diabetes was an N‐terminal 18 kD fragment. Serum IGFBP‐3 levels were lower in patients with MA (baseline: 3613 ± 598 μg/l; one year follow‐up: 3347 ± 624 μg/l) compared with patients without MA (baseline: 4701 ± 1484 μg/l; follow‐up: 4177 ± 703 μg/l; P < 0.001). In serum from patients with MA, intact IGFBP‐3 was decreased, as indicated by WIB analysis. Conversely, IGFBP‐3 proteolysis was increased in patients with MA (baseline: 131 ± 21% of control; follow‐up: 130 ± 23% of control), compared to patients with normal UAE (baseline: 96 ± 23% of control; follow‐up: 96 ± 14% of control; P < 0.001). Serum IGFBP‐3 protease activity was directly correlated to urinary IGFBP‐3 levels (P < 0.001). Serum IGFBP‐1 levels were increased in patients with MA (baseline: 36 ± 20 μg/l; follow‐up: 36 ± 17 μg/l) compared with patients without MA (baseline: 17 ± 11 μg/l; follow‐up: 18 ± μg/l; P < 0.05). Serum IGFBP‐2 levels were also persistently increased in patients with MA (baseline: 503 ± 134 μg/l; follow‐up: 484 ± 166 μg/l) compared with patients without MA (baseline: 375 ± 83 μg/l; follow‐up: 390 ± 85 μg/l; P < 0.05). On the other hand, free IGF‐I levels were decreased in patients with MA (baseline: 2.3 ± 1.5 μg/l; follow‐up: 2.5 ± 1.4 μg/l) compared with those patients without MA (baseline: 4.1 ± 2.1 μg/l; follow‐up: 4.0 ± 2.2 μg/l; P < 0.05).

CONCLUSIONS

It has been demonstrated that in adolescents with type 1 diabetes and persistent microalbuminuria, the IGF‐IGFBP system is deranged. A urinary 18 kD N‐terminal IGFBP‐3 fragment has been characterized that, in patients with microalbuminuria, correlates with urinary albumin excretion and with serum IGFBP‐3 protease activity. The mechanisms behind these changes remain unclear, but alterations in circulating levels of IGFBPs may alter IGF‐I bioactivity. Determination of urinary levels of IGFBP‐3 might be supportive to the measurement of urinary albumin as an early sign of diabetic nephropathy.