Insulin has been shown to be a prominent inhibitor of the IL-6 mediated stimulation of acute phase plasma protein (APP) genes in hepatoma cells. The molecular mechanism of this inhibitory effect has not been established. Recent analysis of the IL-6 receptor (IL-6R) has implicated STAT3 protein to be involved in the IL-6 induction of APPs. We therefore analyzed whether insulin interferes with the STAT3 signaling pathway utilized by the IL-6R.

Rat H-35 hepatoma cells were treated with increasing doses of insulin (0.5, 5.0, 50, 500 and 5000 ng/ml) followed by IL-6. STAT3 gene transcription rate was established by nuclear run-on experiment, STAT3 protein expression was determined by Western blot, and STAT3 protein activation was measured by electrophoretic mobility shift assay(EMSA)using double-stranded SIEm67 oligonucleotide as binding substrate for STAT 3. APP gene transcription rates and mRNA accumulation were also determined.

Relative to cells treated with IL-6 alone, cells exposed to increasing doses of insulin and then to IL-6 exhibited: 1) a 2-fold decrease in STAT3 gene transcription rate, 2) a progressive reduction (9-fold max) in STAT 3 protein expression, 3) a loss of SIF-A complex (STAT3 homodimers) and gradual reduction (3-fold max) in SIF-B complex (STAT1 and STAT3 heterodimers) seen on EMSA, and 4) a progressive reduction in APP mRNA levels (20-fold max). Time course experiments revealed that insulin (500 ng/ml) inhibition of STAT3 protein level and STAT3 activation was apparent by 4h, maximal at 8h, and sustained up to 24h.

These results provide novel evidence linking STAT3 to insulin signaling and suggests that STAT3 serves as a signaling molecule in the insulin inhibition of APPs. STAT3 is therefore one among many biochemical mediators of the complex interaction between cytokines and growth factors. (Funded in part by a research grant from Novo-Nordisk Pharmaceuticals, Inc.)