Characterization of induction of protooncogene c-myc and cellular growth in human vascular smooth muscle cells by insulin and IGF-I.

Insulin and insulin-like growth factor I (IGF-I) are structurally related polypeptides that stimulate DNA synthesis and cellular proliferation, probably through a common pathway. Human arterial smooth muscle cells in culture demonstrated the presence of high-affinity receptors for both these hormones. Insulin and IGF-I both exhibited cross-reactivity to each other's receptors but with an affinity that is 100-fold less than for the homologous receptor. To examine more closely the receptor responsible for producing the growth effects, we used the polyclonal antibody against the insulin receptor, B2, and a monoclonal antibody to the IGF-I receptor, alpha IR3. We studied the growth effects of insulin and IGF-I as measured by stimulation of c-myc, DNA synthesis, and cellular proliferation in the presence and absence of these antibodies. F(ab') fragments of the anti-insulin-receptor antibody at a concentration of 10 micrograms/ml were capable of displacing greater than 90% of the bound insulin, thus establishing an effective insulin-receptor blockade. Under such blockade, insulin and IGF-I were both capable of doubling the amount of DNA synthesis and cell number in cultured human arterial smooth muscle cells. However, in the presence of a 1:2500 dilution of the monoclonal antibody alpha IR3, which caused a 90% displacement of IGF-I bound to its receptor, both the insulin and IGF-I effects on stimulating DNA synthesis or cellular proliferation were inhibited by greater than 90%. These findings demonstrate that the IGF-I receptor is the common pathway for the growth effects of both insulin and IGF-I.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin, insulin-like growth factor I and platelet-derived growth factor interact additively in the induction of the protooncogene c-myc and cellular proliferation in cultured bovine aortic smooth muscle cells.

Vascular smooth muscle cell (SMC) growth is under the influence of various growth factors. We demonstrate that platelet-derived growth factor (PDGF) stimulates DNA synthesis of cultured bovine aortic SMCs by 2.5- to 3.5-fold. PDGF also exhibits additivity with insulin and insulin-like growth factor I (IGF-I) for DNA synthesis and cellular proliferation. Insulin (2 x 10(-6) M), IGF-I (1 x 10(-8) M), and PDGF (1 x 10(-9) M) cause a 60-80% increase in cell numbers over basal, but PDGF with insulin or IGF causes a 40-150% increase over basal. No additivity between insulin and IGF-I is evident. PDGF also induces commitment to DNA synthesis earlier than insulin or IGF-I. After exposure to PDGF for 4 h, SMCs incorporate 3H-thymidine to 60% of maximum (with PDGF alone) levels (achieved after exposure of 12 h or longer). Insulin and IGF-I exposure for 4 h, on the other hand, achieves 3H-thymidine incorporation that is only a 20-30% of maximum (with insulin or IGF-I alone). Insulin, IGF-I, and PDGF increase mRNA levels of the protooncogene c-myc. This induction begins within 30 min of exposure to these growth factors which causes a 4- to 6-fold increase in c-myc mRNA levels. Additivity is also observed between PDGF with insulin or IGF-I, but not between insulin or IGF-I, in c-myc induction. C-myc mRNA levels remain elevated as long as the hormones are present, although there's a tendency for the mRNA levels to fall off with insulin and IGF-I.(ABSTRACT TRUNCATED AT 250 WORDS)

Processing and release of insulin and insulin-like growth factor I by macro- and microvascular endothelial cells.

Insulin and insulin-like growth factor I (IGF-I) processing by macro- and microvascular endothelial cells was investigated. Specific binding of insulin and IGF-I on the capillary endothelial cells derived from rat fat pads was 4 +/- 0.5% (+/- SE) and 4.3 +/- 0.3%/mg protein, respectively, in contrast to bovine aortic endothelial cells, which bound 9.3 +/- 0.3% IGF-I/mg protein. Both binding and processing of insulin and IGF-I were time and temperature dependent in macro- and microvascular endothelial cells. After 30 min at 37 C, between 40-50% of the bound IGF-I and insulin were internalized in both capillary and aortic endothelial cells, whereas 20-25% insulin and 15-20% IGF-I internalization were observed at 15 C. Less than 20% internalization was observed for both insulin and IGF-I at 4 C. Cellular inhibitors of hormone processing, such as chloroquine and monensin, enhanced cell-associated insulin at 37 C on the bovine aortic endothelial cells from 4.7% to 10.4 +/- 1% and 9.9 +/- 2% mg protein, respectively, at 60 min. Similarly, chloroquine and monensin increased the amount of [125I]IGF-I associated with aortic endothelial cells from 4.3 +/- 0.2% to 5.5 +/- 0.3% and 6.2 +/- 0.7%/mg protein, respectively. Chloroquine and monensin increased [125I]insulin associated with rat capillary endothelial cells from a control of 2.9 +/- 0.1% to 4.0 +/- 0.2% and 3.8% +/- 0.37%, respectively. No effect of chloroquine and monensin was observed on [125I]IGF-I binding to rat capillary endothelial cells. Leupeptin, a lysosomal protease inhibitor, did not affect insulin or IGF-I binding in either cell type. The internalized insulin and IGF-I were both rapidly released, with 70-80% of both hormones being detected in the medium by 120 min. The released hormones were mostly intact (greater than 80-90%), as assessed by trichloroacetic acid precipitability, gel filtration, and immunoprecipitation. Both insulin and IGF-I induced corresponding down-regulation of their receptors, as shown by a 66 +/- 7% decrease in insulin binding in the capillary endothelial cells and a 72 +/- 1% and 58 +/- 1% decrease in IGF-I binding in the aortic and capillary endothelial cells, respectively. Thus, macro- and microvascular endothelial cells bind and process insulin and IGF-I by degradative and nondegradative pathways. The predominance of the nondegradative pathway for the processing of insulin and IGF-I and the modulation of their receptors by physiological hormone concentrations suggested that endothelial cells may regulate the access of insulin and IGF-I to their target cells.