Teplizumab treatment may improve C-peptide responses in participants with type 1 diabetes after the new-onset period: a randomised controlled trial.

AIMS/HYPOTHESIS: Type 1 diabetes results from a chronic autoimmune process continuing for years after presentation. We tested whether treatment with teplizumab (a Fc receptor non-binding anti-CD3 monoclonal antibody), after the new-onset period, affects the decline in C-peptide production in individuals with type 1 diabetes. METHODS: In a randomised placebo-controlled trial we treated 58 participants with type 1 diabetes for 4-12 months with teplizumab or placebo at four academic centres in the USA. A central randomisation centre used computer generated tables to allocate treatments. Investigators, patients, and caregivers were blinded to group assignment. The primary outcome was a comparison of C-peptide responses to a mixed meal after 1 year. We explored modification of treatment effects in subgroups of patients. RESULTS: Thirty-four and 29 subjects were randomized to the drug and placebo treated groups, respectively. Thirty-one and 27, respectively, were analysed. Although the primary outcome analysis showed a 21.7% higher C-peptide response in the teplizumab-treated group (0.45 vs 0.371; difference, 0.059 [95% CI 0.006, 0.115] nmol/l) (p = 0.03), when corrected for baseline imbalances in HbA(1c) levels, the C-peptide levels in the teplizumab-treated group were 17.7% higher (0.44 vs 0.378; difference, 0.049 [95% CI 0, 0.108] nmol/l, p = 0.09). A greater proportion of placebo-treated participants lost detectable C-peptide responses at 12 months (p = 0.03). The teplizumab group required less exogenous insulin (p < 0.001) but treatment differences in HbA(1c) levels were not observed. Teplizumab was well tolerated. A subgroup analysis showed that treatment benefits were larger in younger individuals and those with HbA(1c) <6.5% at entry. Clinical responders to teplizumab had an increase in circulating CD8 central memory cells 2 months after enrolment compared with non-responders. CONCLUSIONS/INTERPRETATIONS: This study suggests that deterioration in insulin secretion may be affected by immune therapy with teplizumab after the new-onset period but the magnitude of the effect is less than during the new-onset period. Our studies identify characteristics of patients most likely to respond to this immune therapy. TRIAL REGISTRATION: ClinicalTrials.gov NCT00378508 FUNDING: This work was supported by grants 2007-502, 2007-1059 and 2006-351 from the JDRF and grants R01 DK057846, P30 DK20495, UL1 RR024139, UL1RR025780, UL1 RR024131 and UL1 RR024134 from the NIH.

Use of Gonadotropin-Releasing Hormone Analogs in Children: Update by an International Consortium.

This update, written by authors designated by multiple pediatric endocrinology societies (see List of Participating Societies) from around the globe, concisely addresses topics related to changes in GnRHa usage in children and adolescents over the last decade. Topics related to the use of GnRHa in precocious puberty include diagnostic criteria, globally available formulations, considerations of benefit of treatment, monitoring of therapy, adverse events, and long-term outcome data. Additional sections review use in transgender individuals and other pediatric endocrine related conditions. Although there have been many significant changes in GnRHa usage, there is a definite paucity of evidence-based publications to support them. Therefore, this paper is explicitly not intended to evaluate what is recommended in terms of the best use of GnRHa, based on evidence and expert opinion, but rather to describe how these drugs are used, irrespective of any qualitative evaluation. Thus, this paper should be considered a narrative review on GnRHa utilization in precocious puberty and other clinical situations. These changes are reviewed not only to point out deficiencies in the literature but also to stimulate future studies and publications in this area.

Exogenous growth hormone inhibits growth hormone-releasing factor-induced growth hormone secretion in normal men.

Previous studies from this laboratory and by others in rats, monkeys, and humans support the concept that growth hormone (GH) can regulate its own secretion through an autofeedback mechanism. With the availability of human growth hormone-releasing factor (GRF), the possible existence of such a mechanism was reexplored by examining the effect of exogenous GH on the GH response induced by GRF-44-NH2 in six normal men (mean age, 32.4 yr). In all subjects the plasma GH response evoked by GRF-44-NH2 (1 microgram/kg i.v. bolus) was studied before and after 5 d of placebo (1 ml normal saline i.m. every 12 h), and then before and 12 h after 5 d of biosynthetic methionyl human GH (5 U i.m. every 12 h). The GH response to GRF (maximal increment over time 0 value) was significantly inhibited after GH treatment (0-1.3 vs. 2.3-11.2 ng/ml before treatment, P = 0.05), but was not significantly affected by placebo. This impaired pituitary response to GRF persisted for at least 24 h following exogenous GH treatment in two subjects who underwent further study. Serum somatomedin-C concentrations were significantly increased after 5 d of GH treatment (2.66-5.00 vs. 0.92-1.91 U/ml before treatment, P = less than 0.01). The impaired pituitary response to GRF may be mediated indirectly through somatomedin, somatostatin, by a direct effect of GH on the pituitary somatotropes, or by all of these mechanisms. These data suggest that after GH treatment, the blunted GH response to synthetic GRF is not solely a consequence of the inhibition of hypothalamic GRF secretion.

Regulation of insulin-like growth factor (IGF) I receptor expression during muscle cell differentiation. Potential autocrine role of IGF-II.

Muscle is an important target tissue for insulin-like growth factor (IGF) action. The presence of specific, high affinity IGF receptors, as well as the expression of IGF peptides and binding proteins by muscle suggest that a significant component of IGF action in this tissue is mediated through autocrine and/or paracrine mechanisms. To explore autocrine/paracrine action of IGFs in muscle, we studied the regulation of the IGF-I receptor and the expression of IGF peptides during differentiation of the mouse BC3H-1 muscle cell line. Differentiation from myoblasts to myocytes was associated with a 60% decrease in IGF-I receptor sites determined by Scatchard analysis. Analysis of mRNA abundance and protein labeling studies indicated that the decrease in IGF-I receptor sites was associated with similar reductions in IGF-I receptor gene expression and receptor biosynthesis. IGF-II peptide gene expression was detected in myoblasts and increased 15-fold with differentiation; the increase in IGF-II gene expression preceded the decrease in IGF-I receptor gene expression. In contrast, IGF-I peptide gene expression was low in myoblasts and decreased slightly with differentiation. To explore the potential role of endogenous IGF-II in the differentiation-associated decrease in IGF-I receptor expression, we investigated the effects of IGF-II treatment in myoblasts. The addition of IGF-II to undifferentiated myoblasts resulted in downregulation of the IGF-I receptor which was associated with decreased IGF-I receptor biosynthesis and decreased IGF-I receptor mRNA abundance. These studies suggest, therefore, that IGF-I receptor expression during muscle cell differentiation may be regulated, at least in part, through autocrine production of IGF-II.

Fibroblast growth factor inhibits insulin-like growth factor-II (IGF-II) gene expression and increases IGF-I receptor abundance in BC3H-1 muscle cells.

Muscle is an important target tissue for insulin-like growth factor (IGF) action. We have previously reported that muscle cell differentiation is associated with down-regulation of the IGF-I receptor at the level of gene expression that is concomitant with an increase in the expression and secretion of IGF-II. Furthermore, treatment of myoblasts with IGF-II resulted in a similar decrease in IGF-I receptor mRNA abundance, suggesting an autocrine role of IGF-II in IGF-I receptor regulation. To explore further the role of IGF-II in IGF-I receptor regulation, BC3H-1 mouse muscle cells were exposed to differentiation medium in the presence of basic fibroblast growth factor (FGF), a known inhibitor of myogenic differentiation. FGF treatment of cells resulted in a 50% inhibition of IGF-II gene expression compared to that in control myoblasts and markedly inhibited IGF-II secretion. Concomitantly, FGF resulted in a 60-70% increase in IGF-I binding compared to that in control myoblasts. Scatchard analyses and studies of gene expression demonstrated that the increased IGF-I binding induced by FGF reflected parallel increases in IGF-I receptor content and mRNA abundance. These studies indicate that FGF may up-regulate IGF-I receptor expression in muscle cells through inhibition of IGF-II peptide expression and further support the concept of an autocrine role of IGF-II in IGF-I receptor regulation. In addition, these studies suggest that one mechanism by which FGF inhibits muscle cell differentiation is through inhibition of IGF-II expression.

Progestins induce down-regulation of insulin-like growth factor-I (IGF-I) receptors in human breast cancer cells: potential autocrine role of IGF-II.

Insulin-like growth factor-I (IGF-I) receptors are present in breast cancer cells and may play a role in breast cancer cell growth. We have studied the effect of progestins on IGF-I receptors in T47D human breast cancer cells. T47D cells constitutively express high levels of progesterone receptors and are a model for studying the regulation of cellular functions by progestins. Treatment of T47D cells with either progesterone or the synthetic progestin promegestone (R5020) decreased IGF-I receptor content by approximately 50%, as measured by Scatchard analysis and receptor biosynthesis studies. In contrast to progestins, estradiol, dexamethasone, and dihydrotestosterone did not influence IGF-I receptor content. No effect of R5020 was seen after 12 h of incubation, a near-maximal effect was seen after 24 h, and greatest effects were seen after 72 h. R5020 decreased IGF-I receptor mRNA abundance, indicating that progestins acted at the level of gene expression. However, progestins also increased the secretion of IGF-II, a ligand for the IGF-I receptor. In contrast to IGF-II, T47D cells did not express IGF-I. The addition of exogenous IGF-II to T47D cells down-regulated both IGF-I receptor binding and IGF-I receptor mRNA abundance. This study indicates, therefore, that progestins regulate IGF-I receptors in breast cancer cells and suggests that this regulation occurs via an autocrine pathway involving enhanced IGF-II secretion.

An insulin-like growth factor-II (IGF-II) analog with highly selective affinity for IGF-II receptors stimulates differentiation, but not IGF-I receptor down-regulation in muscle cells.

The insulin-like growth factors (IGFs) stimulate the growth and differentiation of muscle cells. IGF-II, the principal IGF peptide expressed by differentiating muscle cells, has been implicated in at least two autocrine/paracrine actions in this tissue: stimulation of differentiation and down-regulation of the IGF-I receptor. To determine which IGF receptor subtypes mediate these effects of IGF-II, we treated mouse BC3H-1 muscle cells with native IGF-II or [Leu27]IGF-II, an analog with high affinity for IGF-II receptors (comparable to that seen with native IGF-II) but markedly reduced affinity for IGF-I and insulin receptors. Muscle cell differentiation was assessed by the expression of myogenin mRNA and by the binding of alpha-bungarotoxin to the nicotinic acetylcholine receptor. IGF-I receptor down-regulation was assessed by receptor binding and mRNA abundance. Although less potent than IGF-II, the [Leu27]IGF-II analog stimulated myogenin gene expression and acetylcholine receptor binding in concentrations at which the analog interacted with IGF-II receptors, but not significantly with IGF-I receptors. In IGF-I receptor down-regulation studies, IGF-II pretreatment significantly decreased binding of IGF-I to the IGF-I receptor and decreased IGF-I receptor mRNA, whereas the IGF-II analog had only minimal effects. Thus, in addition to the IGF-I receptor which has been previously found to signal IGF-induced myogenesis, these results implicate a role for the IGF-II receptor in this process. In contrast, IGF-I receptor down-regulation induced by IGF-II is mediated through IGF-I, but not IGF-II, receptors in muscle cells.

Growth factor-stimulated phosphorylation of Akt and p70(S6K) is differentially inhibited by LY294002 and Wortmannin.

The phosphoinositide 3-kinase (PI3K) inhibitors, LY294002 (LY) and wortmannin (WM), are widely used to examine the role of PI3K in growth factor signaling. These compounds inhibit the kinase action of PI3K, thus preventing the accumulation of PI(3,4,5)P3 and PI(3,4)P2 (PIs) and subsequent phosphorylation and activation of the downstream effectors of PI3K, Akt and p70(S6K). The efficacy of these inhibitors has been demonstrated by measuring cellular levels of PIs or the kinase activity of immunoprecipitated PI3K. However, their effects on activation of Akt and p70(S6K), more widely used markers of PI3K activation, has not been formally tested. We have examined the effects of LY and WM on phosphorylation of Akt and p70(S6K) by insulin-like growth factor-I, insulin, and platelet-derived growth factor in skeletal muscle cells. LY is much less effective in blocking the phosphorylation of Akt than p70(S6K); at concentrations which completely inhibit phosphorylation of p70(S6K), phosphorylation of Akt is only partially inhibited by LY. WM also inhibits IGF-I-stimulated phosphorylation of Akt and p70(S6K) with unequal potency but is equally effective in blocking insulin-stimulated phosphorylation of these peptides. Our data demonstrate that inhibiting PI3K signaling through one of its downstream mediators (p70(S6K)) may not indicate complete blockage of the PI3K pathway which may be signaling through an alternate downstream branch (Akt). These findings indicate that the efficacy of LY and WM in blocking PI3K-activation of Akt and p70(S6K) must be tested within the context of every experiment, and that the results obtained with the use of these inhibitors must be interpreted according to their specific effects on the PI3K/Akt and PI3K/p70(S6K) signaling pathways.

Skeletal muscle cell-derived insulin-like growth factor (IGF) binding proteins inhibit IGF-I-induced myogenesis in rat L6E9 cells.

The insulin-like growth factors (IGFs) stimulate the differentiation of skeletal muscle cells. IGF binding proteins (IGFBPs), which are expressed by skeletal muscle cells, may enhance or inhibit IGF actions. To explore the role of skeletal muscle-derived IGFBPs in IGF-induced myogenesis, we compared the differentiation-inducing effects of IGF-I and des(1-3)IGF-I in rat L6E9 skeletal myoblasts. Des(1-3)IGF-I is a naturally occurring IGF-I analog with markedly reduced affinity for IGFBPs but with an affinity for the IGF-I receptor that is comparable to that for native IGF-I. We find that rat L6E9 cells produce principally IGFBP-4 and BP-6, with a minor component of IGFBP-5. Both IGFBP-4 and BP-6 accumulate during differentiation and increase further in response to IGF-I or des(1-3)IGF-I treatment. We find that an IGF-I analog with reduced affinity for IGFBPs is significantly more potent than native IGF-I in stimulating myogenesis (as assessed by myogenin messenger RNA abundance and muscle creatine kinase activity), indicating that IGFBPs expressed by skeletal muscle cells inhibit differentiation induced by IGF-I. In view of the relative abundance of IGFBP-4, its relatively high affinity for IGF-I and the low affinity of IGFBP-6 for IGF-I, it is likely that the inhibitory effect of rat skeletal muscle-derived IGFBPs on IGF-I-induced myogenesis is mediated principally by IGFBP-4.

A rapid and simple one step method for isolation of poly(A)+ RNA from cells in monolayer.

A simple method was developed to isolate low abundance hormone receptor poly(A)+ RNA from cells in tissue culture. Adherent cells in tissue culture plates were directly released with proteinase K and solubilized in SDS. Oligo(dT) cellulose was directly added to the lysate to obtain poly(A)+ RNA. Yields and purity of the poly(A)+ RNA were comparable to other more lengthy methods. IGF-I receptor and insulin receptor mRNA could be detected on Northern blot without any degradation.

Short term continuous intravenous infusion of growth hormone (GH) inhibits GH-releasing hormone-induced GH secretion: a time-dependent effect.

We previously reported that GH secretion evoked by GHRH is inhibited after 5 days of treatment with im GH. This impaired pituitary response was associated with a significant increase in the serum concentration of insulin-like growth factor I (IGF-I). To dissociate the possible effects of circulating IGF-I from other effects of GH on the pituitary response to GHRH, we carried out the following study in eight normal men. A bolus injection of GHRH (1 microgram/kg, iv) was administered 2 h after the start of a 4-h continuous iv infusion of GH (180-micrograms bolus dose, then 3 micrograms/min in 150 mmol/L NaCl) or placebo (150 mmol/L NaCl). In addition, a similar injection of GHRH was given 4 h after the start of a 6-h continuous iv GH infusion (180-micrograms bolus dose, then 3 micrograms/min). During the GH infusions, plasma GH levels reached steady state concentrations in the 9-13 micrograms/L range. The mean GHRH-induced GH response was not significantly blunted during the 4-h infusions of GH [724 +/- 163 (+/- SE) vs. 1184 +/- 373 micrograms.min/L during placebo; P = 0.29], but was significantly inhibited during the 6-h GH infusions (226 +/- 105 micrograms.min/L; P = 0.04 vs. control). Serum IGF-I or plasma glucose did not change significantly throughout the GH infusions. During the 6-h GH infusions, plasma FFA increased to levels significantly above basal values during the last 3 h of the 6-h infusion. These results indicate that short term GH infusion inhibits the plasma GH response to GHRH in a time-dependent manner. The inhibition is not due to changes in circulating IGF-I and glucose concentrations. Fluctuations in hypothalamic somatostatin secretion, changes in lipid or other GH-dependent metabolites, paracrine effects of IGF-I, or a direct effect of GH itself may cause the impaired pituitary responsiveness during short term iv GH infusion.

Gonadotropin-independent familial sexual precocity with premature Leydig and germinal cell maturation (familial testotoxicosis): effects of a potent luteinizing hormone-releasing factor agonist and medroxyprogesterone acetate therapy in four cases.

Four boys with sexual precocity are described in whom pubertal concentrations of plasma testosterone were associated with premature Leydig and germinal cell maturation without activation of the hypothalamic-pituitary gonadotropin unit. Extensive laboratory evaluation localized the source of testosterone secretion to the testes, and testicular biopsy revealed maturation of Leydig cells and spermatogenic elements. These events appear to be nongonadotropin-dependent in view of the absence of a pubertal pattern of pulsatile LH secretion, persistence of a prepubertal LH response to LRF even after long standing sexual precocity, prepubertal basal levels of LH and undetectable hCG, and the absence of biologically active LH-hCG by bioassay. Indirect immunofluorescence studies failed to demonstrate an immunoglobulin in the patients' sera that bound to Leydig cells or seminiferous tubules of normal adult human testes. The potent LRF analog D-Trp6-Pro9-NEt-LRF did not result in suppression of plasma testosterone or Leydig cell function even after 3 months of daily treatment in two patients which provides additional support of pituitary gonadotropin independence and of the lack of a direct effect of the analog on Leydig cell function. Oral medroxyprogesterone acetate treatment in two patients was associated with a striking decrease in both plasma testosterone concentration and height velocity. In the only patient in whom a complete family history could be obtained (three of four patients were adopted), sexual precocity was noted in the maternal grandfather. As this familial syndrome is characterized by a prepubertal hypothalamic-pituitary gonadotropin unit and apparent gonadotropin-independent maturation of Leydig cells and germinal epithelium, possibly due to an intratesticular inborn error, we propose the term "familial testotoxicosis" to describe this group of sexually precocious boys.

The effect of pulsatile administration, continuous infusion, and diurnal variation on the growth hormone (GH) response to GH-releasing hormone in normal men.

To examine the relative effectiveness of GH-releasing hormone (GHRH) given either as multiple iv pulses or as a continuous iv infusion, we studied the GH response to a nearly equivalent total dose of GHRH-44 administered by both routes in a group of normal men. Further, in view of the pulsatile nature of GH secretion and its augmentation with sleep, we investigated whether a diurnal difference in GH release was present during chronic pulsatile administration of GHRH during day and night. Seven men received six GHRH pulses (1 microgram/kg, iv) at 2-h intervals during both day (0900-2100 h) and night (2100-0900 h), and four underwent nighttime placebo pulsing. Eight men received a daytime continuous GHRH infusion (0.15 microgram/kg X h for 5 h, followed by 0.75 microgram/kg X h for 5 h) and a separate 10-h placebo infusion. The GH response to a bolus dose of GHRH (1 microgram/kg, iv) was determined after both continuous GHRH and placebo infusions. No significant difference was found in the GH area response (mean +/- SEM) during total day and night GHRH pulsing periods (6095 +/- 1192 vs. 6506 +/- 1483 ng/min X ml; P = NS). GH secretion was blunted after the initial daytime GHRH pulse (P = 0.02), and only two of seven men had a GH increase after the second pulse; responsiveness was restored after the fourth pulse. In contrast, all subjects responded to the second nighttime GHRH pulse. During continuous GHRH infusions, GH secretion was unsustained and pulsatile. The incremental GH response to a single GHRH bolus dose was decreased after GHRH infusion compared to that after placebo (4.4 +/- 1.8 vs. 10.3 +/- 3.4 ng/ml; P less than 0.05). No difference was found in the total GH area response to a nearly equivalent dose of GHRH administered as either multiple pulses or continuous infusion followed by a single GHRH bolus dose. The apparent pulsatile nature of GH secretion during continuous GHRH infusion and the lack of a significant difference in the GH response to a nearly equivalent dose of GHRH administered as either multiple pulses or a continuous infusion suggest that GHRH need not be administered in a pulsatile manner to be an effective therapeutic agent for the stimulation of GH secretion in children with hypothalamic GHRH deficiency.

Synthetic human pancreas growth hormone-releasing factor (hpGRF1-44-NH2) stimulates growth hormone secretion in normal men.

A synthetic replicate of human pancreas growth hormone-releasing factor, hpGRF1-44-NH2, (hpGRF44), a 44-amino acid amidated peptide, was administered to seven normal men. In addition to placebo, 4 subjects received 3 doses of hpGRF44 (0.5 microgram/kg, 5 micrograms/kg, 10 micrograms/kg), while one subject received 2 doses (5 micrograms/kg, 10 micrograms/kg) and 2 subjects received one dose (0.5 microgram/kg or 5 micrograms/kg), each as a rapid intravenous injection. A significant increase in circulating growth hormone (GH) was observed at each dose (0.5 microgram/kg, p = 0.05; 5 micrograms/kg, p less than 0.005; 10 micrograms/kg, p = 0.01) when compared to placebo, with equivalent maximal responses. At all doses studied a rise in plasma GH occurred within 5 min, with a peak response in most patients at 30-45 min. There were no associated changes in any of the other anterior pituitary hormones or insulin. Other than transient flushing and minimal changes in vital signs, no side effects were noted. The results of these studies support the potential usefulness of hpGRF44 for the assessment of GH secretion and reserve, and of this peptide or an analog as a therapeutic agent to stimulate GH release.

Effect of growth hormone (GH)-releasing hormone (GRH) on plasma GH in relation to magnitude and duration of GH deficiency in 26 children and adults with isolated GH deficiency or multiple pituitary hormone deficiencies: evidence for hypothalamic GRH deficiency.

Synthetic, amidated, 44 amino acid GH-releasing hormone ( GRH -44) was administered iv at a dose of 5 micrograms/kg to 20 patients with severe GH deficiency (GHD), 6 children and adolescents with partial GHD, and 6 non-GH deficient ( NGHD ) children and adolescents. The 17 patients with severe GHD that responded to GRH -44 had lower peak concentrations of plasma GH than the NGHD individuals (5.0 +/- 1.2 (SEM) vs. 27.2 +/- 3.5 ng/ml; P less than 0.0001). The children and adolescents with severe GHD tended to have higher peak GH responses to GRH -44 than the GHD adults (6.9 +/- 1.7 vs. 2.4 +/- 0.3 ng/ml) although the difference was not significant. The peak GH concentration was attained earlier in the GHD children and adolescents than in the GHD adults (28 +/- 4.7 vs. 69.3 +/- 13 min, P less than 0.004). There was a negative correlation between chronological age and peak plasma GH response to GRH in the children and adolescents with severe GHD (r = -0.758, P less than 0.02). Children and adolescents with partial GHD had a higher mean peak concentration of plasma GH (13. 1 +/- 1.8 ng/ml) than the children, adolescents, and adults with severe GHD (P less than 0.04), but one lower than the NGHD children and adolescents (P less than 0.05). In both severe and partial GHD the GH response to GRH was greater than that elicited by standard pharmacological tests. Serum somatomedin-C did not increase after a single pulse of GRH -44 in the 12 GHD patients studied. PRL increased minimally 30 min after 5 micrograms/kg iv GRH -44 in patients with multiple hypothalamic-pituitary hormone deficiencies but not in patients with isolated GHD or in NGHD individuals. The GH responses to GRH suggest that the majority of patients with isolated GHD as well as those with multiple hypothalamic-pituitary hormone deficiencies have deficiency of hypothalamic GRH . Lack of a GH response to a single pulse of GRH does not exclude GRH deficiency as priming of the somatotrope with multiple pulses of GRH may be necessary to rule out a hypothalamic defect in the nonresponders. The results of this study support the potential usefulness of GRH or its analogs in the diagnosis and treatment of selected patients with disorders of GH secretion.

Adjunctive growth hormone during ovarian hyperstimulation increases levels of insulin-like growth factor binding proteins in follicular fluid: a randomized, placebo-controlled, cross-over study.

GH increases circulating insulin-like growth factor I (IGF-I), which can promote the growth and differentiated function of ovarian granulosa and theca cells. Reported studies of GH as an adjunct to menotropin stimulation in women, largely those with ovarian dysfunction, have not consistently shown a benefit of GH, despite increases in serum and follicular fluid IGF-I. We hypothesized that changes in intrafollicular IGF-binding proteins (IGFBPs), which can antagonize IGF actions on granulosa cells, may underlie the inconsistent effects of GH. In the present study of GH, administered in double-blind, placebo-controlled, cross-over fashion to regularly cycling women undergoing in vitro fertilization, we found that follicular fluid levels of IGFBP-1, -3, and -4 and serum levels of IGFBP-3, as well as follicular fluid and serum IGF-I, were significantly increased in the GH-treated cycles, when compared with the placebo cycle of the same patient. We suggest that the net increase in intrafollicular IGFBPs in GH cycles may mitigate the potential beneficial effect of increased IGF-I.

Long-term outcome in children and adolescents after transsphenoidal surgery for Cushing's disease.

Cushing's disease refers specifically to an ACTH-producing pituitary adenoma that stimulates excess cortisol production. Transsphenoidal surgery is the treatment of choice in children and adolescents, but disparate cure rates have been reported, ranging from 50-98%. The discrepancies in cure rate are due primarily to the technical success of the surgery and the length and method of follow-up. We studied 42 consecutive children and adolescents (age, < or = 18 yr) who underwent transsphenoidal exploration for the primary treatment of Cushing's disease at University of California-San Francisco from 1974-1993. Only 7 patients had persistent disease, defined as evidence of Cushing's disease within 6 months of surgery, yielding an initial remission rate of 83%. We comprehensively evaluated 26 of the 35 patients who experienced an initial remission, including testing of the ACTH-adrenocortical axis. The mean duration of follow-up is 7.2 yr (range, 1.5-13.6 yr). Seven experienced a relapse of Cushing's disease, yielding a net remission rate of 73%. Relapses occurred an average of 4.2 yr postoperatively (range, 0.75-6.2 yr). Five patients experienced relapse within 5 yr of surgery, whereas 2 relapsed more than 5 yr postoperatively. Repeat transsphenoidal surgery was performed in 8 patients with persistent or recurrent disease, and 6 of these remain in remission. Low serum or urinary cortisol measurements within the first post-operative week predicted remission of Cushing's disease, but were not necessarily predictive of long-term cure. Hypercortisolism had significant effects on bone metabolism, as reflected by both diminished bone density in the majority of patients examined and decreased growth rate. Both parameters improved after surgical care, although they did not fully normalize. We conclude that transsphenoidal surgery is a safe and effective treatment for pediatric Cushing's disease, but long-term surveillance is necessary to detect possible recurrences.

Mechanisms of insulin-like growth factor (IGF)-II-induced IGF-I receptor down-regulation in BC3H-1 muscle cells.

Muscle is an important target tissue for insulin-like growth factor (IGF) action. We have previously demonstrated that treatment of myoblasts with IGF-II decreased IGF-I receptor biosynthesis and steady-state mRNA levels. In addition, muscle cell differentiation was associated with a marked increase in the expression and secretion of IGF-II followed by similar down-regulation of the IGF-I receptor, suggesting an autocrine role for IGF-II in this process. To explore further the mechanisms by which IGF-II decreases IGF-I receptor expression in BC3H-1 muscle cells, dose-response studies of IGF-I and -II treatment on the amount of IGF-I receptor mRNA were carried out. In addition, to determine whether IGF-II decreases IGF-I receptor expression by stimulating receptor protein degradation, pulse/chase experiments with [35S]methionine/cysteine were carried out. Both IGF-I and -II induced significant down-regulation of IGF-I receptor mRNA. At low concentrations, IGF-I was more potent than IGF-II in inhibiting IGF-I receptor mRNA accumulation, suggesting that IGF-I receptor down-regulation induced by IGF-II is mediated principally through the IGF-I receptor in these cells. In addition, IGF-II decreased IGF-I receptor expression by stimulating receptor protein degradation as demonstrated by pulse/chase analysis of metabolically labelled receptors. Thus, IGF-II induces IGF-I receptor down-regulation in muscle cells through multiple mechanisms, including decreasing IGF-I receptor mRNA and stimulating IGF-I receptor protein degradation.

Functional inactivation of the IGF-I receptor delays differentiation of skeletal muscle cells.

Skeletal myoblasts are inherently programmed to leave the cell cycle and begin the differentiation process following removal of exogenous growth factors. Serum withdrawal results in a marked induction of IGF production which is essential for skeletal muscle differentiation in vitro. However, the potential role of the tyrosine kinase IGF-I receptor (thought to be the principal mediator of both IGF-I and II signaling in skeletal muscle) in the decision of myoblasts to begin differentiation following serum withdrawal is unknown. To explore the role of the IGF-I receptor in this decision by skeletal myoblasts, we functionally inactivated endogenous IGF-I receptors in mouse C2C12 cells using a dominant negative, kinase-inactive IGF-I receptor in which the ATP-binding site lysine (K) at residue 1003 has been mutated to alanine (A). Cell lines with the greatest degree of mutant IGF-I receptor expression (A/K cells) demonstrated functional inactivation of endogenous IGF-I receptors as determined by their impaired ability to phosphorylate the principal substrate of the IGF-I receptor, IRS-1, in response to treatment with IGF-I. In addition, the proliferative response of myoblasts to IGF-I was completely abolished in A/K cells. Following withdrawal of exogenous growth factors, A/K cells demonstrated a marked delay in the induction of the gene expression of myogenin, a skeletal muscle-specific transcription factor essential for differentiation, and a subsequent delay in the induction of muscle creatine kinase activity. Delayed differentiation in A/K cells was associated with prolonged phosphorylation of the cell cycle regulatory retinoblastoma (Rb) protein; it is the un- (or hypo-) phosphorylated form of Rb which is known to promote differentiation in skeletal myoblasts. Thus, the IGF-I receptor regulates the timing of myoblast differentiation induced by serum withdrawal. The delayed differentiation of skeletal myoblasts with functionally inactive IGF-I receptors may result, at least in part, from delayed induction of myogenin gene expression and prolonged phosphorylation of the Rb protein.

Growth factor receptor regulation in the Minn-1 leprechaun: defects in both insulin receptor and epidermal growth factor receptor gene expression.

Leprechaunism is a disorder characterized by intrauterine growth retardation, distinctive dysmorphology, and extreme insulin resistance due to structural abnormalities of the insulin receptor (IR). In addition to the IR, it has been suggested that abnormalities of the other growth factor receptors may occur in this syndrome. Using fibroblasts from the Minn-1 leprechaun, we have now investigated the expression of three different growth factor receptor genes: the IR, the insulin-like growth factor-I receptor (IGF-IR), and the epidermal growth factor receptor (EGFR). In agreement with previous studies, we found decreased insulin binding to fibroblasts from the Minn-1 leprechaun. In these cells, the IR transcription rate was not decreased, and sequence analysis of the IR promoter region of the patient showed no abnormalities. Both single-stranded conformational polymorphism analysis (SSCP) and DNA sequencing confirmed a previously reported nonsense mutation in one of the patient's two IR alleles at exon 14. mRNA levels for the IR were markedly decreased, suggesting that IR mRNA turnover was enhanced. We then studied the expression of the closely related IGF-IR Ligand binding, mRNA content, and transcription rate were all normal. In contrast to the IGF-IR, when the EGFR was studied, ligand binding and mRNA content were markedly decreased. These studies therefore raise the possibility that the phenotypic expression of leprechaunism results from defects in the expression of both the IR and the EGFR.