The insulin-like growth factor system- Effects of circulating proteases

The insulin-like growth factor-I and –II (IGF-I/ -II) are peptide hormones important for growth and metabolism. They stimulate cell proliferation and differentiation and inhibit apoptosis. In addition, they have insulin-like effects on glucose and amino acid metabolism. A large quantity of IGFs is synthesized in the liver and is subsequently transported to the circulation to act as endocrine factors. In addition, IGFs produced in extra hepatic tissues may act locally as paracrine/ autocrine factors. In serum most IGF circulate in a ternary complex with IGF-binding protein-3 (IGFBP-3) and the acid labile subunit (ALS). The affinity between IGF and IGFBP-3 exceeds that between IGF and the type 1 IGF receptor demonstrating that most of the circulating IGF is unavailable for the receptor. However, a slight decrease in binding affinity within the ternary complex increases the bioavailability of IGFs. Post-translational modifications of the IGFBPs are common and proteolytic cleavage of IGFBPs represents one example. IGFBP proteolysis results in reduced IGF affinity and subsequently increased IGF bioavailability. Increased IGFBP-3 proteolysis is found in several clinical conditions associated with insulin resistance, e.g. critical illness, postoperatively and type 2 diabetes. IGFBP-3 proteolysis may partly compensate for insulin resistance by increasing IGF availability and thereby also increasing insulin-like effects. In the present licentiate thesis we have studied the mechanisms of IGFBP-3 proteolysis in vivo in humans. We have also attempted to evaluate the molecular consequences of IGFBP-3 proteolysis. We first studied (paper I) whether cannulation and/ or venous stasis increases serum IGFBP-3 proteolysis by activating fibrinolytic and thrombogenic enzymes. These enzymes have the capacity to cleave IGFBP-3 in vitro. We found that limited vascular damage and/ or reduced blood flow in response to cannulation and/ or venous stasis did not affect IGFBP-3 proteolysis. Furthermore, cannulation and/ or venous stasis did not affect the elevated levels of IGFBP-3 proteolysis after surgery. This does not exclude that extensive activation of fibrinolytic and thrombogenic enzymes by myocardial infarction, venous thrombosis or wound healing may result in elevated IGFBP-3 proteolysis. This further demonstrates that the current methods to detect serum IGFBP-3 proteolysis are valid if our sampling recommendations are followed. We then investigated (paper II) whether the cytokine interleukin-6 (IL-6) increases serum IGFBP-3 proteolysis or IGFBP-1 in healthy adults. This was investigated since IL-6 is elevated in several inflammatory diseases associated with increased IGFBP-3 proteolysis. We found that a 3-hour IL-6 infusion did not increase IGFBP-3 proteolysis up to 5 hours post-infusion as compared to saline infusion. This finding is important if we should target the mechanisms resulting in changes in the IGF-system during inflammation and insulin resistance. Based on these results we cannot exclude that higher levels of IL-6 (>0.1 ng/ ml) may immediately stimulate IGFBP-3 proteolysis or that prolonged exposure to IL-6 may affect IGFBP-3. Interestingly, we found that IGFBP-1 was increased after the end of IL-6 infusion by a mechanism independent of insulin. However, the change in IGFBP-1 in response to IL-6 did not affect total or free IGF-I. Finally, we have attempted (project report) to isolate the major IGFBP-3 proteolytic fragment in human postoperative serum in order to 1) obtain amino acid sequence data of the cleavage site to unravel the protease and 2) examine the changes in IGF affinity resulting from proteolytic cleavage. By using IGF-I affinity chromatography, we isolated a 30 kDa IGFBP-3 fragment in post-operative serum. N-terminal amino acid sequencing revealed the sequence GASS. This is identical to the N-terminal of IGFBP-3. The low recovery did not allow us to identify the Cterminal sequence or to obtain enough material for binding studies. Alternative purification strategies were explored including an attempt to raise IGFBP-3 antibodies for affinity chromatography. In summary this licentiate thesis provides human in vivo data demonstrating that IGFBP-3 proteolysis is elevated after abdominal surgery but that it is not affected by venous canulation and/ or stasis or by short-term exposure to IL6. Furthermore, currently established methods to detect IGFBP-3 proteolysis can be used to evaluate potential inductors of IGFBP-3 proteolysis in vivo including fibrinolytic and thrombogenic enzymes. We also demonstrate that IL-6 is a more potent regulator of serum IGFBP-1 compared to IGFBP-3 proteolysis. However, the significance of this is unknown since the level of free IGF-I was unchanged. In addition, we suggest that the development of purification procedures to isolate and characterize IGFBP-3 fragments may be helpful in identifying the proteases that increase serum IGFBP-3 fragmentation in vivo. This may provide useful information about alterations in the IGF-system during inflammation and insulin resistance