To elucidate the acute metabolic actions of insulin-like growth factor I (IGF-I), we administered a primed (250 ,ug/kg), continuous (5 gg/kg. min) infusion of human recombinant (Thr 59) IGF-I or saline to awake, chronically catheterized 24-h fasted rats for 90 min. IGF-I was also infused while maintaining euglycemia (glucose clamp technique) and its effects were compared to those of insulin. IGF-I infusion caused a twofold rise in IGF-I levels and a 75-85% decrease in plasma insulin. When IGF-I alone was given, plasma glucose fell by 30-40 mg/dl (P < 0.005) due to a transient twofold increase (P < 0.05) in glucose uptake; hepatic glucose production and plasma FFA levels remained unchanged. IGF-I infusion with maintenance of euglycemia produced a sustained rise in glucose uptake and a marked stimulation of 13-3Hjglucose incorporation into tissue glycogen, but still failed to suppress glucose production and FFA levels. IGF-I also produced a generalized 30-40% reduction in plasma amino acids, regardless of whether or not hypoglycemia was prevented. This was associated with a decrease in leucine flux and a decline in the incorporation of i1-'4Clleucine into muscle and liver protein (P < 0.05). When insulin was infused in a dosage that mimicked the rise in glucose uptake seen with IGF-I, nearly identical changes in amino acid metabolism occurred. However, insulin suppressed glucose production by 65% and FFA levels by 40% (P < 0.001). Furthermore, insulin was less effective than IGF-I in promoting glycogen synthesis.We conclude that (a) IGF-I produces hypoglycemia by selectively enhancing glucose uptake; (b) IGF-I is relatively ineffective in suppressing hepatic glucose production or FFA levels; and (c) IGF-I, like insulin, lowers circulating amino acids by reducing protein breakdown rather than by stimulating protein synthesis. Thus, IGF-I's metabolic actions in fasted rats are readily distinguished from insulin.

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To examine the influence of insulin-dependent diabetes on the metabolic response to insulin-like growth factor I (IGF-I), awake chronically catheterized diabetic and nondiabetic BB/w rats received IGF-I (5 micrograms.kg-1.min-1) or insulin (2 mU.kg-1.min-1) for 2 h while maintaining euglycemia. In nondiabetic rats, IGF-I and insulin produced similar twofold increases in glucose uptake, but insulin was more effective in reducing hepatic glucose production (90 +/- 15 vs. 5 +/- 11%; P less than 0.001) and beta-hydroxybutyrate levels (94 +/- 1 vs. 19 +/- 6%; P less than 0.001). In diabetic rats, insulin-stimulated glucose uptake was impaired (8.5 +/- 0.9 vs. 11.5 +/- 0.9 mg.kg-1.min-1 in nondiabetics; P less than 0.05). In contrast, IGF-I-stimulated glucose uptake was identical in diabetic and nondiabetic rats. Furthermore, IGF-I suppressed glucose production by 73% (P less than 0.01) and caused a greater lowering of beta-hydroxybutyrate levels (from 2.9 +/- 0.8 to 0.8 +/- 0.3 mumol/l) in diabetic rats. We conclude that 1) the capacity of IGF-I infusion to stimulate glucose uptake is maintained in spontaneously diabetic BB rats that are insulin resistant, and 2) IGF-I infusion suppresses elevated glucose production rates and plasma ketone concentrations in diabetic rats but is relatively ineffective in nondiabetic rats. Thus the metabolic responses to infused IGF-I do not appear to be diminished in diabetic rats with impaired responses to insulin.

Recombinant human insulin-like growth factor I (IGF-I) is efficiently expressed and secreted from Saccharomyces cerevisiae using a yeast alpha-factor leader to direct secretion. However, approximately 10-20% of the IGF-I was in a monomeric form, the remaining materials being disulfide-linked aggregates. When the purified material was subjected to reverse-phase high-performance liquid chromatography (rp-HPLC), it gave two doublet peaks, I and II. Upon reduction, doublet peaks I and II converged to one doublet peak. This suggests that peaks I and II result from different disulfide structures, and the doublet feature of each peak results from other causes. Different disulfide structures between peaks I and II were also suggested from the near UV circular dichroism of these proteins. Only the peak II was biologically active, indicating that peak II has the correct disulfide structure. Concanavalin A affinity chromatography of the purified peak II doublet showed binding of the subpeak with an earlier rp-HPLC retention time, indicating that it was glycosylated. Sequence analysis of tryptic peptides suggested that Thr29 was the site of glycosylation. Site-directed mutagenesis was used to convert Thr29 to Asn29. This substitution reduced, but did not eliminate IGF-I glycosylation, suggesting additional glycosylation sites. The site of carbohydrate addition was consistent with the model that O-glycosylations occur on hydroxyl amino acids near proline residues in beta-turns.

The direct effects on PRL release of acute changes in dopamine (DA) and TRH concentrations were measured in an in vitro perifusion system. Hemisected anterior pituitaries of lactating rats were perifused with medium that received a coinfusion of DA at 20 ng/ml. These tissues released PRL at 35% of the release rate of controls in the absence of DA. Interruption of the DA coinfusion for 9 min caused a 2-fold increase in PRL release, which was resuppressed when the DA treatment was resumed. During continuous Da exposure, TRH administration (10 ng/ml for 12 min) induced a gradual but slight increase in PRL release. However, when this TRH treatment was administered immediately after the end of the DA interruption, it evoked an immediate 2-fold increase in PRL release to 4 times the initial release rate in the presence of DA. This pronounced effect of TRH after the brief DA interruption was also observed when an 18 min interval was imposed between the two manipulations. During continuous coinfusion of DA at 100 ng/ml, TRH was totally ineffective in eliciting PRL release. However, even after this DA treatment had been interrupted briefly and an increase in PRL release had been evoked, TRH still was not an effective stimulus for PRL release. T was imposed between the two manipulations. During continuous coinfusion of DA at 100 ng/ml, TRH was totally ineffective in eliciting PRL release. However, even after this DA treatment had been interrupted briefly and an increase in PRL release had been evoked, TRH still was not an effective stimulus for PRL release. T was imposed between the two manipulations. During continuous coinfusion of DA at 100 ng/ml, TRH was totally ineffective in eliciting PRL release. However, even after this DA treatment had been interrupted briefly and an increase in PRL release had been evoked, TRH still was not an effective stimulus for PRL release. These data indicate that DA not only can serve as a PRL-inhibiting factor for tonic release of PRL but also may determine by its presence or brief absence, and concentration whether acute release occurs in the presence of a PRL-releasing factor. The direct effect of DA on PRL release and its interference with the action of a PRL-releasing factor appear to be independent of each other.