One of insulin's many biological effects is the increased transcription of AP-1-regulated genes. cJun is the principal component of the AP-1 transcription complex, which is regulated by the newly discovered members of the MAPK superfamily referred to as cJun NH2-terminal kinases (JNKs) or stress-activated protein kinases (SAPKs). We show that insulin stimulates a dose- and time-dependent increase in JNK activity in Rat 1 fibroblasts overexpressing human insulin receptors (Rat 1 HIR cells). Using two different polyclonal anti-JNK antibodies, JNK activity was measured after immunoprecipitation from whole cell extracts by phosphorylation of GSTcJun(1-79). Peak activation occurred 15 min after insulin addition, resulting in a 2.5-fold increase in GSTcJun(1-79) phosphorylation over unstimulated controls. Maximal JNK activation correlated with the onset of AP-1 DNA binding activity. Both insulin-stimulated JNK activity and insulin-induced AP-1 transcriptional activity were found to be Ras-dependent. These data suggest that in Rat 1 cells, JNK activation may play a role in insulin-regulated AP-1 transcriptional activity leading to a mitogenic response.
To determine the effects of glucose on insulin-like growth factor I (IGF-I)-induced mesangial cell (MC) proliferation, we have examined the relationships between IGF binding protein 2 (IGFBP-2) secretion and proliferation in murine MCs (MMCs). MMCs incubated in high glucose (HG, 25 mM) exhibited a 25–30% reduction in IGFBP-2 secretion compared with cells in normal glucose (NG, 5.6 mM). This loss was not due to cell surface binding; it correlated with a 3.1-fold decrease in IGFBP-2 mRNA. IGFBP-2 secretion was stimulated by IGF-I in NG but was unaltered in HG. Insulin treatment yielded similar results at 10-fold higher doses, indicating that this response is IGF-I receptor dependent. MMCs in HG displayed increased IGF-I-stimulated insulin receptor substrate-1/2 phosphorylation and activator protein-1 transcriptional activity compared with NG controls. Accordingly, although IGF-I was not proliferative in NG, it increased [3H]thymidine incorporation and cell number in HG to an extent proportional to the decrease in IGFBP-2. Thus hyperglycemia, as seen in diabetes, may increase MC IGF-I sensitivity by reducing IGFBP-2 expression, in turn increasing its proliferative and secretory responses and contributing to the development of diabetic glomerulosclerosis.
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