Mark R. Benedict

Evidence for insulin-like growth factor-I regulation of chick aortic elastogenesis

The potential role of insulin-like growth factor-I (IGF-I) as a modulator of chick aortic embryogenesis was examined. Studies were designed to investigate the in vivo relationships between embryonic IGF-I serum concentrations, liver and aortic IGF-I mRNA steady-state levels and the inception and perpetuation of aortic elastogenesis. In addition to aortic tissue, elastogenesis was measured in heart and lung tissues in order to compare the responses of functionally unique elastin-containing tissues to the developmental appearance of IGF-I in embryonic serum. Our results demonstrate that the induction of aortic tropoelastin mRNA steady-state levels coincides with a major increase in serum IGF-I concentration. This is not the case with either lung or heart elastogenic responses. All three tissues examined (aorta, lung, and heart) exhibited different developmental patterns of tropoelastin mRNA steady-state levels during the embryonic ages studied (8- through 10-day). Only aortic tropoelastin mRNA levels paralleled the rise and fall of IGF-I serum levels. Steady-state levels of liver IGF-I mRNA peaked one day (9-day) prior to detectable IGF-I serum levels but otherwise mirrored the gradual, but steady decrease in IGF-I serum levels through 16-day. Aortic tissue also expresses IGF-I mRNA beginning at 8-day and continuing throughout the embryonic ages examined (16-day). Although the relative levels of aortic IGF-I mRNA are very low in comparison to corresponding mRNA levels in liver, the fact that IGF-I mRNA is transcribed in the aorta points to the possibility that autocrine and/or paracrine mechanisms of IGF-I action may be operative in aortic elastogenesis.

MATURATIONAL CHANGES OF INSULIN BINDING TO FETAL HEPATOCYTES

To determine if the reported lack of direct insulin netabolic effects in fetal tissues is due to alterations in hormone binding and/or processing, we characterized the binding, internalization, and degradation of insulin by cultured hepatocytes from rat fetuses of 17, 19, and 21 days gestation. When insulin (100 nM) was incubated with fetal hepatocytes, we observed substantial reductions (66%-100%) in immunoreactive insulin. This loss was greatest in cultures prepared from 19 day fetuses. 125I-Insulin binding at 37 C rapidly reached a peak at 30 min. Specific binding was greatest in 19 day cells; 460 fmole/mg protein compared to 150 and 190 fmole/mg protein in 17 and 21 day fetal hepatocytes, respectively. Prior exposure to insulin (100 nM) induced an inhibition of subsequent binding, increasing with gestational age. Only minimal down-regulation was detectable in 17 day hepatocytes. Both internalization and intracellular degradation of 125I-insulin occurred rapidly, following a similar time course for all three ages. Despite the ability of 17 day fetala hepatocytes to bind, internalize, and degrade insulin, we were unable to demonstrate receptor down-regulation. The dissociation of these related processes raises the possibility that these cells have a more rapid rate of receptor turnover than those from 19 and 21 day fetuses.

HORMONAL REGULATION OF SOMATOMEDIN SECRETION BY FETAL RAT HEPATOCYTES IN CULTURE

To determine which hormones might regulate fetal somatomedin (SM) secretion, we measured SM levels in conditioned medium from primary cultures of fetal rat hepatocytes. We employed a bioassay (3H-thymidine incorporation into DNA of chick embryo fibroblasts), a displacement assay (competition for binding of a radiolabeled rat IGF-II to the SM binding protein) for total somatomedin, and the radioimmunoassay for SM-C. Epidermal growth factor (EGF) and dexamethasone were the most active hormones tested, as measured by the displacement assay. Rat growth hormone (rGH) was much less stimulatory. Human placental lactogen (hPL), glucagon and insulin had little or no effect. Stimulation of SM secretion by both EGF and dexamethasone was time- and dose-dependent. The maximal response occurred at 48 hours, at a concentration of about 1 × 10−7 M of either hormone. In the bioassay, the stimulation by EGF, but not dexamethasone, could be detected. The steroid had enhanced the secretion of a heat-labile inhibitor that completely masked the mitogenic activity of the increased SM levels. The fetal SM secreted exhibited immunological cross-reactivity with human SM-C, but the levels were 500-fold less than those measured by our displacement assay. This suggests that the predominant fetal rat SM is not SM-C. We conclude that EGF and dexamethasone, but not rGH or hPL, stimulated the secretion by fetal hepatocytes of a fetal SM which resembled IGF-II.