Metabolic effects of IGF-I and insulin in spontaneously diabetic BB/w rats.

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.

Characterization of a polyethylene glycol conjugate of recombinant human interferon-gamma.

Recombinant human interferon-gamma was conjugated with polyethylene glycol (PEG) using succinimidyl coupling of amino groups in the protein. The PEG conjugated material showed antiviral, growth inhibitory and macrophage activation activities indistinguishable from those of the unmodified protein. The PEG conjugation reduced the receptor binding affinity slightly, but increased the half-life of the protein when measured in rats. Almost no clearance was observed within 6 hr after injection for the PEG conjugated protein, whereas a rapid clearance was seen for the unmodified interferon-gamma. Two possible sites of PEG attachment were identified in the protein: the N-terminal amino group and either lysine 129 or 131.

Differential effect of neurointermediate lobectomy on central oxytocin and vasopressin.

Removal of the neurointermediate pituitary (NIL) affects the secretion of anterior pituitary (AP) hormones. It is not known if these effects are due to changes in central neuropeptide neurons. Two neuropeptides implicated in the neuroendocrine regulation of AP hormone secretion, and which are located in the NIL, are oxytocin and vasopressin. The present study evaluated whether removal of the NIL affected oxytocin and vasopressin concentrations in discrete brain areas containing cell bodies (paraventricular nucleus), fibers (arcuate nucleus), and/or terminals (median eminence) of these central neurons. Adult male rats underwent removal (NIL-X) or visualization (SHAM controls) of the NIL using a parapharyngeal approach. Oxytocin levels increased in the paraventricular nucleus and median eminence following NIL-X, whereas vasopressin concentrations were relatively unaffected by NIL-X. The data suggest that at least part of the influence of the NIL on AP hormone secretion may result from the ability of feedback from the NIL to differentially affect central neuropeptide neurons.

Yeast-derived recombinant human insulin-like growth factor I: production, purification, and structural characterization.

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.

Acute effects of insulin-like growth factor I on glucose and amino acid metabolism in the awake fasted rat. Comparison with insulin.

To elucidate the acute metabolic actions of insulin-like growth factor I (IGF-I), we administered a primed (250 micrograms/kg), continuous (5 micrograms/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 less than 0.005) due to a transient twofold increase (P less than 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 [3-3H]glucose 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 [1-14C]leucine into muscle and liver protein (P less than 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 less than 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.

Antiviral and antitumor effects of a human interferon analog, IFN-alpha Con 1, assessed in hamsters.

An analog of human alpha-and beta-interferons (IFN-alpha and -beta) (generally consisting of the most frequently observed amino acid residue at each position in the molecule) has pronounced antiviral and antiproliferative activity in human and hamster cells. Intraperitoneal administration of this analog (designated IFN-alpha Con 1) to hamsters at 10(6) to 10(8) U/kg resulted in proportional increases in plasma concentrations through 6 h of monitoring. IFN-alpha Con 1 at these doses effectively limited encephalomyocarditis virus (EMCV) infections of hamsters. A natural human IFN-alpha preparation was also active against virus infections in hamsters. The antitumor activity of IFN-alpha Con 1 and natural human IFN-alpha was assessed in hamsters inoculated with lethal TBD932 lymphosarcoma. Various IFN treatment schedules resulted in prolonged survival following tumor challenge. IFN-alpha Con 1 administered at 10(5) to 10(6) U/hamster daily for 9-12 days following tumor challenge was effective in delaying tumor development, as was a natural human IFN-alpha preparation. The efficacies of combined IFN and cyclophosphamide therapies were determined. Unlike the natural human subtype IFN-alpha A, IFN-alpha Con 1 did not diminish the efficacy of cyclophosphamide (2.5 mg/hamster for 3 days) against the lymphosarcoma. However, an ineffective dose of cyclophosphamide (0.05 mg/hamster for 3 days) when combined with IFN-alpha Con 1 treatment showed enhanced antitumor activity. Combinations of cimetidine (16 mg/hamster for 4 days) and IFN-alpha Con 1 treatment did not prolong survival in this model system.

Prolactin release after 5-hydroxytryptophan treatment requires an intact neurointermediate pituitary lobe.

The present study was designed to evaluate the role of the neurointermediate pituitary lobe (NIL) in the 5-hydroxytryptophan (5-HTP)-induced increase in plasma PRL levels. The neurochemical mechanisms involved in this neuroendocrine regulation were also analyzed by examining the concentrations of serotonin (5-HT), norepinephrine, and dopamine as well as their primary metabolites in the median eminence (ME), NIL, and anterior pituitary (AP) after different treatments. Removal of the NIL (NIL-X) did not significantly affect basal plasma PRL concentrations or amine metabolism in the ME or AP. 5-HTP administration resulted in a 5-fold increase in plasma PRL in sham-operated (SHAM) animals, but NIL-X completely abolished this PRL response. 5-HTP injection elicited large increases in 5-HT and 5-hydroxyindole-3-acetic acid concentrations in ME, NIL, and AP in SHAM animals, and similar changes within the ME and AP in NIL-X animals, but did not significantly affect norepinephrine or dopamine metabolism in the ME, NIL, or AP of NIL-X or SHAM animals. Moreover, tissue concentrations of 5-HTP after 5-HTP injection increased similarly in SHAM and NIL-X animals. Inhibition of peripheral decarboxylase activity with MK486 prevented the 5-HTP-induced increase in PRL in SHAM animals and the increase in 5-HT metabolism in both SHAM and NIL-X groups. These data indicate that an intact NIL is required for the 5-HTP-induced increase in plasma PRL, but does not seem to be essential for the regulation of basal PRL release. They also demonstrate that the 5-HTP-induced activation of 5-HT metabolism in both ME and AP is not sufficient, by itself, to enhance PRL release.

ACTH and corticosterone secretion in rats following removal of the neurointermediate lobe of the pituitary gland.

Surgical removal of the neurointermediate lobe of the pituitary gland (NIL-X) in the rat resulted in two abnormalities in ACTH secretion: (1) plasma ACTH and corticosterone levels were elevated in the morning and over a 24-hour period compared with levels in control (NIL-C) rats, and (2) although NIL-X and NIL-C rats had acute increases in plasma ACTH and corticosterone of equal magnitude after interoceptive stimuli (hemorrhage, surgery), NIL-X rats demonstrated markedly smaller elevations in plasma levels of these hormones after neurotropic stimuli (noise, novel environment). This subnormal adrenocortical response of NIL-X rats was not due to an impairment in perception of a neurotropic stimulus; these rats had normal latencies to paw licking and to jumping off a heated surface, yet smaller increases in plasma corticosterone after the stimulus. The impairment in ACTH response was not related to stimulus intensity, as NIL-X and NIL-C rats had equal ACTH and corticosterone secretion during both low and high levels of insulin-induced hypoglycemia. NIL-X rats demonstrated a significant elevation in daily water intake, although hematocrit, plasma Na+, K+, osmolality and protein were normal. Significant diurnal rhythms in plasma corticosterone levels and in water intake were maintained as well. The elevated morning plasma ACTH levels, the blunted hormone increases after noise, and the increase in water intake persisted in NIL-X rats 2 months after surgery. These data indicate that removal of the NIL results in (1) chronic elevations in basal ACTH and corticosterone secretion, and (2) chronic impairment in adrenocortical responses to neurotropic stimuli, but not to interoceptive stimuli. The deficit is not due to impaired perceptual capacity nor to the intensity of the stimulus.

Measurement of hormones and blood gases during hypoxia in conscious cannulated rats.

Simple inexpensive methods are described for implanting chronic arterial cannulas for remote stress-free blood sampling of conscious unrestrained rats and for delivering acute isocapnic hypoxia to these rats in their home cages. The day before experimentation cages were placed in plastic bags with air (21% O2) flowing through at 15 1/min (normoxia). The next morning either normoxia was continued or isocapnic hypoxia (arterial PO2 38 Torr, arterial PCO2 38 Torr) was administered without handling or disturbing the rats. Repeated arterial samples were collected for measurement of blood gases, hematocrit, corticosterone, and ACTH. Blood pressure increased transiently (by 10 mmHg at 10 min) and plasma corticosterone and ACTH levels increased fivefold; hematocrit and heart rate did not change significantly. In rats receiving normoxia, all of these variables remained low. This preparation is useful for studying in conscious rats the regulation of endocrine systems easily stimulated by handling and for studying endocrine and cardiovascular adaptations to environmental stimuli such as hypoxia.

Effects of housing and chronic cannulation on plasma ACTH and corticosterone in the rat.

Effects of housing and cannulation on plasma ACTH and corticosterone levels were evaluated to validate a method for obtaining repeated blood samples for ACTH from conscious rats. Resting morning, evening, or noise-stimulated (4 min of metal rattling) levels of plasma ACTH or corticosterone obtained by decapitation were similar in rats housed alone or in pairs. Individually housed, cannulated rats had slightly elevated morning levels of plasma ACTH and corticosterone, but the evening and noise-stimulated increases in hormone levels were similar to those in unoperated, decapitated rats. A hemorrhage stimulus administered on separate days to cannulated rats evoked highly replicable elevations in plasma ACTH and corticosterone. These results demonstrate that 1) ACTH and corticosterone secretion are similar in rats housed alone versus in pairs, and 2) long-term cannulation permits multiple intra-animal comparisons of basal and acutely stimulated plasma ACTH and corticosterone levels that are useful reflections of those measured in decapitated rats.

Involvement of the neurointermediate lobe of the pituitary gland in the secretion of prolactin and luteinizing hormone in the rat.

The relationship between prolactin (PRL) secretion and the neurointermediate lobe (NIL) of the pituitary gland was investigated. Plasma PRL concentrations in rats bearing anterior pituitaries autografted with or without the NIL to the renal capsule were elevated to equal extents at 1 through 6 weeks after surgery (p greater than 0.10). PRL levels in ovariectomized rats in which the NIl had been removed surgically (NIL-X) or only visualized (NIL-C) were 3-7 ng/ml 4, 7, and 28 days after surgery (p greater than 0.10); however, they were slightly higher in NIL-X vs. NIL-C rats 14 days after surgery (p less than 0.05). Plasma luteinizing hormone (LH) concentrations in NIL-C rats increased by 36% from 2 to 4 weeks after surgery (p less than 0.5); this increase was not detected in NIL-X rats. PRL and LH surges were induced by estradiol implants in ovariectomized NIL-X and NIL-C rats; the profiles of the PRL surges were superimposable, although the magnitude of the LH surge was only 50% that in NIL-C rats (p less than 0.5). These results cast doubt on the importance of the NIL in the regulation of PRL secretion either via secreting hypophysiotropic hormones or via conducting anterior pituitary hormones directly to the median eminence. However, the NIL may have a physiologically important role in the regulation of LH secretion.

The effect of dopamine on thyrotropin-releasing hormone-induced prolactin secretion in vitro.

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.