Limited proteolysis of serum
insulin-like growth factor (
IGF) binding protein (IGFBP)-3 has been described in various conditions and may increase the bioavailability of IGFs. The physiological regulators of serum
IGFBP-3 protease activity are unknown. To characterize the relationship between
insulin and
IGFBP-3 protease activity, we have examined serum
IGFBP-3 proteolysis in children with untreated
insulin-dependent diabetes mellitus (
IDDM) and have followed the effect of
insulin therapy on serum
IGFBP-3 proteolysis at 1 day, 1 week, and 1 month after the initiation of
insulin therapy.
Ligand blot analysis of sera from untreated children with
IDDM showed that intact
IGFBP-3 was 50 +/- 9% of the age-matched control pool. After the initiation of
insulin treatment,
IGFBP-3 did not change significantly at 1 day
after treatment but increased dramatically at 1 week (90 +/- 13%) and 1 month
after treatment (102 +/- 13%). In contrast, when measured by immunoradiometric assay (which detects both intact and fragments of
IGFBP-3),
IGFBP-3 levels were 70% of the control pool before
insulin therapy and did not increase significantly until 1 month
after treatment. Immunoblot analysis demonstrated that intact
IGFBP-3 doublet was diminished to 41 +/- 7% of controls, whereas the major
IGFBP-3 fragment (30 kDa) was increased in
IDDM sera before
insulin therapy. After
insulin, intact
IGFBP-3 increased and the 30-kDa fragment decreased to values comparable to those observed in controls. In vivo
IGFBP-3 proteolysis, which implies preassay exposure of serum
IGFBP-3 to
proteases, was estimated by immunoblot analysis.
IGFBP-3 proteolysis was increased before
insulin therapy (160 +/- 9%) and decreased to 81 +/- 9% at 1 week and to 71 +/- 11% at 1 month after
insulin treatment. Residual serum
IGFBP-3 protease activity was estimated by a 125I-IGFBP-3 degradation assay. Serum
IGFBP-3 protease activity increased significantly in untreated diabetics, compared with activity in controls (128 +/- 5% vs. 99 +/- 11%). During
insulin therapy, serum
IGFBP-3 protease activity decreased gradually to 91 +/- 5% of control values at 1 month. Molecular sizes of the
IGFBP-3 proteolytic fragments (30 kDa, 24 kDa, and 19 kDa) and inhibition profile of
IGFBP-3 protease were similar in
IDDM and pregnancy sera, indicating that similar
proteases (
cation-dependent
serine proteases) were active in both conditions. These results suggest an important role of
insulin in the regulation of
IGFBP-3 protease activity. Increased
IGFBP-3 proteolysis in the sera of children with
IDDM may serve to counteract the catabolic state induced by
insulin deficiency.