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Division of Endocrinology and Diabetes, Department of Internal Medicine, University Hospital Zurich (J.Z., M.G.-P.), 8091 Zurich, Switzerland; Novartis Pharma, Preclinical Research (G.W.), 4002 Basel, Switzerland; M. E. Müller Institute of Biomechanics, University of Bern (E.B.H.), 3010 Bern, Switzerland; and Division of Neuroendocrinology, Institute of Anatomy, University of Zurich (M.R.), 8057 Zurich, Switzerland
Address all correspondence and requests for reprints to: Jürgen Zapf, M.D., Division of Endocrinology and Diabetes, Department of Internal Medicine, University Hospital, 8091 Zurich, Switzerland. E-mail: . ndozaj{at}usz.unizh.ch
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Abstract |
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Introduction |
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The long-acting somatostatin analog octreotide inhibits GH secretion from the pituitary gland. Octreotide is, therefore, used in the treatment of acromegaly. However, some treated acromegalic patients show clinical improvement with normalized serum IGF-I levels, but only a modest or no reduction of GH secretion (7, 8). Similarly, octreotide has been shown to decrease serum IGF-I levels in diabetic patients without significantly suppressing GH levels (9). It has, therefore, been suggested that octreotide, in addition to inhibiting pituitary GH secretion, may act via an alternative mechanism.
It has been reported that octreotide reduces GH-induced IGF-I peptide (10) and mRNA levels (11) in livers of hypophysectomized (hypox) rats as well as GH-induced IGF-I mRNA expression in primary hepatocytes from normal rats (11). We have observed that during a 20-d octreotide infusion in normal rats, GH secretion is significantly inhibited during the first 10 d, but not on d 20 (unpublished results). Serum IGF-I was reduced by 50% after 24 h, but had returned to normal after 5 d and stayed at that level. In contrast, skeletal growth was retarded throughout the entire infusion period. These findings suggested that the growth-inhibiting effect of octreotide might not only be due to the inhibition of GH secretion, but also to interference with local GH action. Thus, octreotide might inhibit local GH-induced IGF-I production and/or reduce the responsiveness of tissues to the action of circulating or locally produced IGF-I, e.g. at the level of IGF-I-mediated receptor phosphorylation or downstream of IGF-I receptor signaling. This reasoning is supported if one considers some of the signaling pathways elicited by interaction of somatostatin with its receptors. These comprise five different subtypes (12). Octreotide binds to three of these five receptors, types 2, 3, and 5, which are expressed in various rat organs (12). Their signaling pathways include inhibition of adenylate cyclase with reduction of intracellular cAMP, lowering of intracellular Ca2+, as well as activation of phosphotyrosine phosphatases. Therefore, it is conceivable that octreotide interferes with IGF-I action, for example by dephosphorylation of the activated (autophosphorylated) type 1 IGF receptor.
To examine these possibilities, we investigated the effects of octreotide on GH- and IGF-I-induced growth of hypox rats. We infused hypox rats with vehicle, IGF-I, or GH, either alone or in combination with octreotide, and compared the growth effects in the presence or absence of this somatostatin analog. Our data show that octreotide reduces liver IGF-I mRNA and peptide, and serum IGF-I levels, and concomitantly inhibits skeletal growth in GH-treated, but not IGF-I-treated, animals. In addition, we observed a significant reduction of IGF-I immunoreactivity at the site of the tibial growth plate cartilage during combined GH/octreotide treatment. Together, these findings demonstrate that octreotide interferes not with IGF-I action, but with local GH-stimulated IGF-I production.
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Materials and Methods |
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Food and water intake (per two cages of three rats each) as well as body weight were measured daily at 0830 h during the infusion period. Two days after starting the infusion, animals were injected sc with 2.5 mg calcein (Fluka, Buchs, Switzerland), dissolved in 0.5 ml 2% NaHCO3. After 6 d of infusion, the rats were anesthetized with Innovar Vet (Pitman Moore, Washington Crossing, NJ; 0.2 ml/100 g body weight) and bled by aortic puncture. Blood glucose was determined immediately with a glucose analyzer (Beckman Coulter, Inc., Fullerton, CA). Excised livers were blotted on filter paper, weighed, immediately frozen in liquid nitrogen, and stored at -80 C until RNA or IGF-I peptide was extracted. Blood was kept on ice for 30 min and then centrifuged for 15 min at 1000 x g at 4 C. Serum was stored in 1-ml aliquots at -20 C for further analysis.
Determination of serum parameters
Serum insulin was measured using an RIA kit from Linco Research, Inc. (St. Charles, MO) according to the protocol provided by the supplier. Serum hGH levels in the hGH-infused animals were determined by RIA using the RIA kit from Schering AG (Baar, Switzerland). Serum octreotide was kindly measured by Novartis Pharma AG, Basel, using an established RIA.
Serum levels of administered rhIGF-I and of endogenous rat IGF-I were determined by two different RIAs as described in detail previously (13). Briefly, 0.15 ml PBS/0.2% human serum albumin (HSA), pH 7.4, was added to 0.1 ml serum, and the mixture was acid-treated and run over Sep-Pak C18 cartridges (Waters Corp., Milford, MA) according to the protocol supplied by Immunonuclear (Stillwater, MN). After reconstitution with 1 ml PBS/0.2% HSA, all samples were assayed at three different dilutions (1:5; 1:10, and 1:20). rhIGF-I was used as a standard for the determination of administered rhIGF-I; rat IGF-I (gift from Dr. M. Kobayashi, Fuji Photo Film Co., Ltd., Tokyo, Japan) served as a standard for the determination of endogenous rat IGF-I. The RIAs were performed with two different antisera at final dilutions of 1:2,000 (14) and 1:20,000 (15), respectively. After preincubation of the antisera with standards or samples for 24 h at 4 C, 25,000–35,000 cpm [125I]IGF-I (Anawa, Wangen, Switzerland; specific activity, 300–400 mCi/mg) were added in a total incubation volume of 0.4 ml. The reaction mixture was then incubated for another 24 h, and the IGF-I/antibody complex was precipitated with the second antibody (goat antirabbit gammaglobulin antiserum) (14).
In the hIGF-I RIA, rat IGF-I does not cross-react at the above dilutions. The values obtained in this RIA therefore reflect only the concentration of the infused rhIGF-I. In the rat IGF-I RIA, hIGF-I cross-reacts 5–6 times better than rat IGF-I, so that endogenous IGF-I concentrations cannot be determined in the animals infused with rhIGF. Total IGF-I levels in these animals can be estimated by adding mean endogenous rat IGF-I levels of control or control/octreotide-treated animals, respectively. As the infusion of rhIGF-II (400–1000 µg/d·rat) does not suppress endogenous IGF-I in hypophysectomized rats (Zapf, J., unpublished observation), it can be assumed that rhIGF-I infusion does not affect endogenous IGF-I either. In animals infused with GH and in the control animals (absence of hIGF-I) the RIA values represent endogenous rat IGF-I concentrations.
RNA isolation and Northern blotting
Total RNA was isolated according to standard procedures (16). Frozen liver tissue (0.5 g) was homogenized in a Polytron homogenizer (Brinkmann Instruments, Inc., Westbury, NY) at 4 C in 3 ml ice-cold 4 M guanidine isothiocyanate containing 5 mM sodium citrate (pH 7.0), 0.1 M ß-mercaptoethanol, and 0.5% sarcosine. The RNA samples were dissolved in diethylpyrocarbonate-treated H2O, and concentrations were determined spectrophotometrically at 280 nm. The integrity of the isolated RNA was checked on a 1% agarose gel containing ethidium bromide. Denatured RNA (20 µg) was electrophoresed on a 1% agarose gel containing 2 M formaldehyde, transferred onto a nylon membrane (Hybond-N, Amersham International, Little Chalfont, UK), and RNA was fixed by UV cross-linking. The filters were prehybridized at 42 C for 2 h in a solution containing 50% formamide, 5x Denhardt’s solution [0.02% (wt/vol) Ficoll, and 0.02% (wt/vol) polyvinyl pyrrolidone), 5x SSPE (20x = 3.6 M NaCl, 0.2 M sodium phosphate, and 0.02 M EDTA, pH 7.7), 0.2% sodium dodecyl sulfate, and 100 µg/ml heat-denatured salmon sperm DNA. The following cDNAs were used for hybridization: rat IGF-I cDNA corresponding to the genomic sequences between nucleotide 2054 of exon 1 and nucleotide 868 of exon 5 (17), PCR-amplified rat GH receptor cDNA corresponding to nucleotides 1239–2161, and yeast 18S cDNA (from Dr. M. Kalousek, University Hospital Zurich, Zurich, Switzerland). The cDNA probes were labeled by random primer extension using a commercial kit (Roche Molecular Biochemicals, Rotkreuz, Switzerland) and [
-32P]deoxy-CTP (3000 Ci/mmol; Amersham International). The radiolabeled cDNA probes had specific activities of 2–4 x 109 cpm/µg DNA. Hybridization was performed in the same solution as that used for prehybridization with 10–15 ng (
2 x 107 cpm) labeled cDNA/filter. After 48 h of incubation at 42 C, the filters were washed twice for 15 min each time at room temperature in 2x SSPE/0.1% sodium dodecyl sulfate and subsequently three times in 0.1x SSC/0.1% sodium dodecyl sulfate for 20 min at 55 C (rat IGF-I and rat GH receptor) or 65 C (18S cDNA probe). mRNA levels were quantitated by scanning densitometry using a video densitometer (Bio-Rad Laboratories, Inc., Richmond, CA). Variations in gel loading were corrected against the corresponding 18S rRNA values.
Liver IGF-I extraction and determination of IGF-I
Liver tissue (0.5 g) was homogenized with a Polytron homogenizer in 2.5 ml ice-cold 0.1 M acetic acid containing 0.2% HSA, 1 µM pepstatin A, 0.3 trypsin inhibitor units/ml aprotinin, 10 µM leupeptin, and 1 mM phenylmethylsulfonylfluoride (all protease inhibitors were from Sigma, St. Louis, MO). The homogenate was centrifuged at 16,000 x g for 10 min at 4 C, and the supernatant was lyophilized. The lyophilized material was dissolved in 2 ml 0.18 M ammonium acetate and centrifuged as described above. The clear supernatant was relyophilized and dissolved in 0.3 ml PBS/0.2% HSA; 0.25 ml was chromatographed on Sep-Pak (protocol of Immunonuclear, Stillwater, MN) and used for IGF-I determination by RIA as described above. The IGF-I values were corrected for the fraction of IGF-I derived from the serum that was entrapped in liver tissue (11 ± 1 µl/g) (18). The recovery for IGF-I by this procedure was 63 ± 4.5%.
Determination of tibial epiphyseal width and accumulated longitudinal bone growth
Tibial epiphyseal width was determined in one tibia according to Greenspan et al. (19). Silver-stained tibiae were photographed under a stereomicroscope (Wild, Heerbrugg, Switzerland) at a x25 magnification. Ten different sections of the tibial epiphyseal width were measured on the photograph, and the mean value was calculated.
The other tibia was used for the determination of accumulated bone growth (growth rate). The proximal tibiae were cut into sagittal slices, fixed in 40% (vol/vol) ethanol for 3 d at ambient temperature, dehydrated in an ascending series of ethanol, and embedded in methacrylate (20, 21). Growth rate was determined in methacrylate-embedded 10-µm tibia sections by incident light fluorescence microscopy as previously described (20, 21).
Immunohistochemical protocol
Methacrylate-embedded slices were cut into 4-µm sections and mounted onto glass slides. The embedding medium was removed with 1-acetoxy-2-methoxyethane (10 min at room temperature). Sections were then treated with 10% H2O2 for 10 min, then washed in PBS (pH 7.4), followed by PBS containing 2% (wt/vol) BSA and 2% (wt/vol) normal goat serum. Sections were incubated with rabbit anti-hIGF-I antiserum diluted 1:200 (22) for 12 h at 4 C. After repeated washing in PBS, the primary antiserum was detected using biotinylated goat antirabbit IgG (diluted 1:100; Bioscience Products, Emmenbrucke, Switzerland) for 30 min at room temperature. Thereafter, sections were washed in PBS and incubated with streptavidin-fluorescein isothiocyanate (diluted 1:100; Bioscience Products) for 30 min at room temperature in the dark.
The specificity of the reaction was tested using the following controls: 1) replacement of the primary antiserum by nonimmune rabbit serum, and 2) preabsorption of the IGF-I antiserum with rhIGF-I, rhIGF-II, or bovine insulin (40 and 400 µg peptide/ml diluted antiserum). Sections of rat pancreas known to contain IGF-I-immunoreactive islet cells (23) were processed in every incubation series and served as positive controls. Preabsorption of the antisera with 40 µg rhIGF-I/ml completely blocked the immunoreaction. IGF-I immunoreactivity was not affected by preabsorption with IGF-II or insulin at concentrations up to 400 µg/ml.
Photomicrographs were taken with am Axiophot (Carl Zeiss, Zurich, Switzerland). The fluorochrome was visualized with a fluorescence module for fluorescein isothiocyanate.
Morphometric evaluation of labeled chondrocytes
Quantitative evaluation of the chondrocytes containing IGF-I immunoreactivity was performed as described previously (22) on 4-µm sections. Five 4-µm sections of each tibia were processed for immunohistochemistry. Growth plate areas were photographed and printed at a final magnification of x720. On these photographs, the total number of chondrocytes in two parallel columns was counted, and the percentage of IGF-I-immunoreactive cells was determined. This procedure was performed twice to obtain a mean value for the two columns of each tibial section. Thus, altogether 60 columns were evaluated for the growth plates of 6 animals/group. The chondrocyte layers were identified using the criteria previously reported (20, 21).
Statistics
Statistical analysis of data was performed using the StatView 4.5 program by ANOVA or Bonferroni/Dunn analysis (for evaluation of IGF-I-labeled cells). All data are expressed as the mean ± SEM.
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Results |
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). GH infusion raised the mean serum IGF-I level to 1208 ± 59 ng/ml, whereas octreotide coinfusion resulted in a 26% decrease to 897 ± 51 ng/ml (P < 0.001). Infusion of rhIGF-I increased rhIGF-I serum levels to 319 ± 21 and 339 ± 9 ng/ml in the absence and presence of octreotide, respectively. Total IGF-I levels in rhIGF-I-infused animals can be estimated by adding mean endogenous rat IGF-I levels of control or control/octreotide-treated animals, respectively (see Materials and Methods). hGH levels in the hGH-infused rats did not differ significantly (21.7 ± 3.10 vs. 19.6 ± 2.6 ng/ml in GH-infused and GH-plus octreotide-infused groups, respectively). Rats infused with GH and octreotide had lower insulin levels than rats treated with GH alone (0.28 ± 0.03 vs. 0.40 ± 0.025 ng/ml; P < 0,05). Octreotide did not significantly decrease serum insulin concentrations in vehicle-treated hypox rats (0.14 ± 0.01 vs. 0.19 ± 0.04 ng/ml). No difference in insulin levels was observed in IGF-I-infused rats treated with or without octreotide. IGF-I further decreased insulin levels compared with those in vehicle-treated controls (0.09 ± 0.02 vs. 0.19 ± 0.04 ng/ml; P < 0.05).
Serum octreotide levels during IGF-I/octreotide and GH/octreotide infusion were identical (191 ± 9 and 179 ± 9 ng/ml).
Food consumption (Fig. 1)
Vehicle-treated rats coinfused with octreotide had consumed slightly less food after 6 d than vehicle-treated control rats, whereas food consumption did not differ between IGF-I-treated and IGF-I/octreotide-treated animals. GH and IGF-I stimulated food intake by approximately 5% and 10%, respectively. With octreotide, the cumulative food intake of GH-treated rats decreased by about 15%.
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Growth parameters of hypox rats infused for 6 d with vehicle, IGF-I, or GH with or without octreotide coinfusion are summarized in Fig. 2. Vehicle-infused control animals with or without octreotide coinfusion did not gain weight during the infusion period. IGF-I and GH infusion increased body weight by 19.8 ± 1.5 and 26.2 ± 1.4 g/6 d, respectively (mean ± SEM). Octreotide coinfusion did not affect IGF-I-stimulated body weight gain, but reduced GH-stimulated body weight gain by 31% to 18.2 ± 1.7 g/6 d (P < 0.001). Tibial epiphyseal width was decreased by 14% in GH/ octreotide-coinfused compared with GH-treated rats (384 ± 9.6 vs. 445 ± 12.7 µm; P < 0.001). Octreotide coadministration did not affect the tibial epiphyseal width of vehicle-infused (185 ± 12 vs. 190 ± 4 µm) and IGF-I-infused (284 ± 15 vs. 285 ± 13 µm) animals (Fig. 2). Accumulated bone growth was reduced by 24% in GH/octreotide-infused compared with GH-infused animals (370 ± 30 vs. 484 ± 28.5 µm/4 d; P < 0.001), whereas it was unaffected by octreotide in IGF-I-infused animals (362 ± 18 vs. 353 ± 14 µm/4 d). In contrast to tibial epiphyseal width, octreotide further reduced accumulated bone growth in vehicle-treated control rats (71 ± 8 vs. 158 ± 20 µm/4 d; P = 0.005; Fig. 2).
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). Octreotide coinfusion resulted in a 27% decrease in GH-stimulated IGF-I mRNA expression (P < 0.001). In parallel, liver IGF-I protein content was decreased by 32% (P < 0.001; Fig. 3).
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IGF-I immunoreactivity in growth plate chondrocytes
IGF-I immunoreactivity was determined in vehicle- and GH-treated animals coinfused with or without octreotide. Because octreotide coinfusion did not affected tibial epiphyseal width or growth rate in IGF-I-infused animals and because IGF-I treatment had no or only minor effects on IGF-I immunostaining in the different growth plate zones (22), no attempt was made to evaluate immunostaining of growth plate chondrocytes in IGF-I- and IGF-I/octreotide-infused animals.
IGF-I immunoreactivity was found in chondrocytes of all zones of tibial growth plate cartilage under all experimental conditions (Fig. 4A). However, the number of labeled cells varied markedly between the different zones and with the experimental condition. Treatment with GH raised the percentage of IGF-I-immunoreactive cells in all zones above the level found in hypox control rats (Fig. 4B). This increase was statistically significant in stem cells and the proliferative and lower hypertrophic zones. In hypox rats treated with GH/octreotide, the percentage of IGF-I-immunoreactive chondrocytes was significantly lower than that in rats treated with GH alone. This decrease in labeling (Fig. 4B) was statistically significant in stem cells and the proliferative and lower hypertrophic zones. As observed previously, the percentage of immunoreactive chondrocytes was not significantly changed in the upper hypertrophic zone after hypophysectomy and remained unchanged during octreotide, GH, and GH/ octreotide treatment.
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Discussion |
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Octreotide coinfusion caused a significant (26%) fall of GH-stimulated serum IGF-I levels, accompanied by 27% and 32% reductions of liver IGF-I mRNA and peptide, respectively. This was not accounted for by a concomitant decrease in hepatic GH receptors, as we, like others (24, 25), found no change in hepatic GH receptor mRNA expression in any of our experimental groups compared with hypox control animals (not shown). Furthermore, we found no effect of octreotide on any of the above indexes in IGF-I-treated or vehicle-treated animals.
The reduction by octreotide of GH-stimulated liver IGF-I mRNA and peptide and of circulating IGF-I might theoretically be caused by the somewhat lower (-15%) food consumption and/or the slightly decreased serum insulin levels. However, Frystyk et al. (26) have shown that even total food deprivation for 1 d does not affect serum IGF-I or liver IGF-I mRNA levels in rats, whereas serum insulin is significantly suppressed. Furthermore, Flyvbjerg et al. (10) reported that 1 wk of pronounced insulinopenia in moderately streptozotocin-diabetic rats did not significantly reduce serum IGF-I levels. Therefore, and in agreement with the study by Ambler et al. (24) and with the finding by Serri et al. that octreotide inhibits GH-induced IGF-I mRNA in primary rat hepatocytes in vitro (11), it appears justified to assume that octreotide directly interferes with GH-stimulated IGF-I mRNA expression and IGF-I peptide synthesis and secretion by the liver, resulting in reduced serum IGF-I levels.
Like Flyvbjerg et al. (10), but in contrast to Serri et al. (11) and Ambler et al. (24), we observed that octreotide caused a significant decrease in GH-stimulated weight gain and tibial epiphyseal width. The latter, when used as the sole parameter to quantitate effects on skeletal growth, may be misleading, because it does not change in proportion to growth rate (20, 27). Therefore, we investigated whether octreotide affected the growth rate of our experimental animals by measuring accumulated bone growth. Octreotide significantly blunted the GH-stimulated growth rate, and this inhibitory effect was greater than that on tibial epiphyseal width, in line with the above statement. Similarly, the residual growth rate of vehicle-treated controls was reduced by octreotide without changes in tibial epiphyseal width, whole body weight, or serum IGF-I.
The inhibitory effect of octreotide on bone growth was obviously overridden by IGF-I in the IGF-I-infused, but not in the GH-infused, animals. This suggested that GH-stimulated local IGF-I production by growth plate chondrocytes (22, 28, 29) might be reduced by octreotide, as observed in the liver (see above) (10, 11). We used a semiquantitative method, described recently (22), to determine IGF-I immunoreactivity in tibial growth plate cartilage. Indeed, we found IGF-I immunoreactivity significantly reduced in stem cells and the proliferative and lower hypertrophic zones of GH/octreotide-treated rats compared with GH-treated animals. Thus, octreotide obviously inhibited GH-stimulated IGF-I production at the site of the growth plate. We cannot definitely exclude that the slightly lower food consumption may have contributed to decrease GH responsiveness, but it is unlikely that it fully accounts for the dramatic changes in IGF-I immunoreactivity.
Although to date somatostatin receptors have not been identified on growth plate chondrocytes themselves, they have been identified on osteoblast precursor cells of the metaphyses of neonatal rats during endochondral ossification in a region adjacent to the hypertrophic cartilage (30). Signaling factors produced by such osteoblast precursor cells might counteract the GH signaling pathway that leads to stimulation of IGF-I. The point at which this interference occurs remains elusive, because the mechanism by which GH induces IGF-I gene expression is still unclear (31). Nevertheless, the present study documents that octreotide does not affect the action of infused IGF-I and thus probably not that of locally produced IGF-I either. Octreotide does, therefore, not appear to interfere with IGF-I receptor signaling in vivo.
That octreotide further inhibited residual growth rate in vehicle-treated hypox control animals appears to be in contrast to the finding that no significant decrease in IGF-I immunoreactivity within the growth plate was detectable, although there was a tendency toward lower levels. As IGF-I immunoreactivity in stem cells and proliferative chondrocytes of hypox rats was only about 4%, it may be difficult to see a further, statistically significant decrease. On the other hand, residual growth of hypox rats may also occur via GH- and thus IGF-I-independent mechanisms, which may be inhibited by octreotide as well.
In the context of octreotide’s inhibition of GH-stimulated bone growth and local IGF-I production, the question arises of whether growth retardation is due to the reduction of the serum IGF-I level or to the suppression of local IGF-I production by growth plate chondrocytes. Although this question cannot be definitely answered, the following arguments favor the latter possibility. 1) Two recent studies in transgenic mice with specific knockout of the liver IGF-I gene have shown that a 75% decrease in the serum IGF-I level does not affect growth of these animals (32, 33). In the face of these studies, it does not appear likely that a much smaller reduction of the serum IGF-I level (26%) in our GH/octreotide-treated animals has essentially contributed to the observed reduction of the bone growth rate. 2) Our previous observation that skeletal growth of normal rats was significantly decreased by octreotide treatment despite normal serum IGF-I levels (unpublished observation) suggests that endogenous circulating IGF-I (in contrast to IGF-I levels raised by IGF-I infusion) is unable to sustain normal growth in the presence of octreotide.
In summary, our findings demonstrate that octreotide does not interfere with IGF-I action but, rather, with local GH-stimulated IGF-I production in the rat tibial growth plate. Thus, octreotide, besides inhibiting pituitary GH secretion, significantly inhibits GH-stimulated longitudinal bone growth.
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Acknowledgments |
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Footnotes |
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Abbreviations: HSA, Human serum albumin; hypox, hypophysectomized; rh, recombinant human.
Received February 13, 2002.
Accepted for publication April 23, 2002.
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| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
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) and control/octreotide (
) and for GH (
) and GH/octreotide (
) treatment on the upper left panel and for IGF-I (
) and IGF-I/octreotide (
) treatment on the upper right panel. Symbols represent means for six animals, and bars show the SEM. Tibial epiphyseal width (lower left) and growth rate (lower right) were determined as described in Materials and Methods. Growth rate is given as increase in microns per 4 d, i.e. following injection of calcein after 2 d of treatment. The height of the columns gives the mean values for six animals, and bars show the SEM. Tibial epiphyseal width: P < 0.001 for GH vs. GH/octreotide, control vs. IGF-I or GH, and IGF-I vs. GH and GH/octreotide; no significant differences between control vs. control/octreotide or between IGF-I vs. IGF-I/ octreotide. Growth rate: P < 0.005 for control vs. control/octreotide; P < 0.001 for control vs. IGF-I and GH, IGF-I vs. GH, and GH vs. GH/octreotide; no significant difference between IGF-I vs. IGF-I/octreotide or GH/octreotide.
