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J A H Wass

Elevated growth hormone is a cardinal feature of acromegaly from the biological view point. Growth hormone stimulates IGF-I secretion and that of its major binding protein IGFBP-3. In these circumstances, where hyperinsulinaemia is present, IGFBP-1 levels, which are inversely related to insulin, are suppressed.

Failure of suppression of growth hormone after oral glucose (>2 mU/1 (1 μg/l)) is the cardinal biochemical feature of acromegaly. IGF-I values at diagnosis are almost invariably raised. There is some overlap in the value of basal IGFBP-3 between normal subjects and acromegalics.

For monitoring purposes, growth hormone values, either basal or during the day are useful. There is overlap in the values of IGF-I and IGFBP-3 between normal subjects and patients on treatment.

Prognosis in acromegaly is determined by persistent elevation of growth hormone levels above 5 mU/l (2·5 μg/l). More data are required for the prognostic use of IGF-I.

Journal of Endocrinology (1997) 155, S17–S19

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J A H Wass

Radiotherapy most frequently uses an external beam, though interstitial irradiation with yttrium or gold has also been used, and heavy particle irradiation with alpha particles or protons is also practised in a few centres. Radiosurgery is a recent development and data on long-term use and cure rates are lacking.

External beam radiotherapy is administered after careful planning and delineation of the pituitary tumour, including the position of the diaphragm, the slope of the floor and, in particular, the lateral margins. An individual face mask immobilises the patient and irradiation is administered through a 4 or 10 MeV linear accelerator using three portals (two temporal, one frontal) at 180 cGy/fraction in 26 fractions over 35 days, to deliver a total tumour dose of 4500 cGy. This is now the accepted means worldwide for giving radiotherapy. Parallel opposed-field radiotherapy results in the delivery of greater doses of radiation to the temporal lobes

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J. M. P. Holly and J. A. H. Wass


The list of growth factors which have generalized effects on the development and proliferation of many cells throughout the body has grown considerably; however, the insulin-like growth factors (IGFs) are still considered as a class in their own right. They are peptides with a high degree of structural homology to proinsulin with important effects on cell metabolism (insulin-like effects) and on cell growth, differentiation and mitosis. In man, there are two main forms which are known to exist; IGF-I, which is synonymous with somatomedin C, and IGF-II which in the human circulation is the most prevalent form. This review is not intended to cover the entire subject comprehensively but attempts to bring together some of the more recent developments in a cohesive manner to update our concepts on the mode of action of the IGFs. Although there have been many major advancements regarding IGF-II, particularly pertaining to its receptor,

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V. J. Frost, V. M. Macaulay, J. A. H. Wass, and J. M. P. Holly


Proteolytic modification of circulating insulin-like growth factor binding protein-3 (IGFBP-3) has been described in a number of conditions. Using Western ligand blotting and SDS-PAGE analysis of fragmentation patterns of 125I-labelled IGFBP-3 and 125-labelled IGFBP-1, we have examined conditioned media from cultured human cell lines for the presence of proteolytic activity and compared this with the action of circulating proteases and with characterized enzymes including cathepsin D, kallikrein, plasmin and tissue plasminogen activator.

125I-labelled IGFBP-3 was incubated with serum from pregnant women, patients following heart surgery and patients with cancer of the breast, lung or head/neck. Following separation of the preincubated samples by SDS-PAGE, a distinct pattern of degradation fragments was observed which was similar in all cases. This proteolytic activity was inhibited in the presence of EDTA, phenanthroline and 4(-2-aminoethyl)-benzenesulphonylfluoride,HCl. These proteases had no detectable effect on IGFBP-1. Serum-free conditioned medium from a human dermal fibroblast cell line, a rabdomyosarcoma, a cervical, a bladder, a chorio- and two-tongue squamous cell carcinoma cell lines all contained proteolytic activity which fragmented IGFBP-3. The pattern of fragments was similar in all cell lines but different from that produced by the circulating proteases. Six out of nine cell lines produced protease(s) which degraded IGFBP-1 in addition to IGFBP-3. Whilst all the characterized enzymes tested also fragmented IGFBP-3 and plasmin cleaved IGFBP-1, none of these acted in the same way as either circulating or cell line-derived proteolytic activity. The activity associated with the characterized enzymes and cell lines was inhibited in the presence of serum from normal healthy subjects.

These results demonstrate that the serum of pregnant women, post-operative patients and patients with cancer contain circulating proteases which cause fragmentation of IGFBP-3 but have little effect on IGFBP-1. Cell-derived proteases were shown to act on IGFBP-3 and IGFBP-1 in a number of instances but are not active in the presence of circulating inhibitors. These proteases may play an important role in regulating the availability of IGFs to normal and neoplastic tissues.

Journal of Endocrinology (1993) 138, 545–554

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S. C. Cwyfan Hughes, J. A. H. Wass, and J. M. P. Holly


The presence of proteolytic activity in the circulation directed against insulin-like growth factor binding protein-3 (IGFBP-3) was originally described in pregnancy but has subsequently been described in a number of catabolic and other pathological conditions. However, detection of this proteolytically modified IGFBP-3 has been demonstrated, in most cases, following separation using SDS-PAGE and this has led to speculation that its occurrence may be an artefact of the harsh conditions employed. Using two sitespecific antisera, one of which recognizes as its antigenic site a region of IGFBP-3 which is close to that of the IGF-binding domain, we have developed two radioimmunoassays which, when compared, can reveal alterations in the IGF-binding domain of IGFBP-3. The presence of IGFBP-3 modified by the circulating protease is denoted in this system by a divergence in the IGFBP-3 levels observed; this divergence has been shown to coincide with protease activity as determined by Western ligand blotting. The use of these assays has confirmed that the IGF-binding site of IGFBP-3 undergoes proteolytic modification in the circulation during a number of pathologies.

Journal of Endocrinology (1993) 137, 321–328

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M Jevon, A Sabokbar, Y Fujikawa, T Hirayama, SD Neale, J Wass, and NA Athanasou

A number of bone diseases characterised by excessive osteolysis (e.g. osteoporosis and Paget's disease) exhibit a marked gender difference in prevalence and are more common in the elderly population. Bone resorption is carried out by osteoclasts, which are formed by fusion of circulating mononuclear precursor cells of haematopoietic origin. In this study, we have determined whether there are gender- and age-related differences in osteoclast formation from circulating precursors. Peripheral blood mononuclear cells (PBMCs) were co-cultured with UMR106 osteoblast-like cells in the presence of macrophage-colony stimulating factor (M-CSF) and 1,25 dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) or cultured alone in the presence of sRANKL (soluble receptor activator of nuclear factor kappa B ligand) and M-CSF. As assessed by the formation of tartrate resistant acid phosphatase (TRAP)-positive (TRAP(+)) and vitronectin receptor-positive (VNR(+)) multinucleated cells (MNCs), there was no difference in the number of circulating osteoclast precursors in males and females. Lacunar resorption carried out by osteoclasts formed from these precursors was generally increased in males compared with females (P=0.03). An increase in the number of TRAP(+) and VNR(+) MNCs formed from male PBMCs was noted in response to 1,25(OH)(2)D(3) (P<0.005). An increase in lacunar resorption in cultures of PBMCs (10(5) per well) from males was also noted in response to 10(-9) M 1,25(OH)(2)D(3) (P<0.05) and sRANKL (P=0.05), but not M-CSF. The addition of dexamethasone resulted in a marked increase in osteoclast formation and lacunar resorption in both males and females. Post-menopausal females and males of comparable age showed similar levels of osteoclastogenesis. Pre-menopausal women showed similar levels of osteoclastogenesis but less resorption (P=0.01) compared with males of comparable age. These results show that there are specific gender/age-related differences in osteoclast formation and bone resorption and have implications for evaluating osteoclastogenesis in skeletal diseases such as primary osteoporosis and Paget's disease.

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A. M. Cotterill, J. M. P. Holly, S. C. Davies, V. J. Coulson, P. A. Price, and J. A. H. Wass


Non-islet-cell tumours which induce hypoglycaemia are rare. Insulin-like growth factor-II (IGF-II) produced by some tumours is thought to be responsible for the hypoglycaemia and other systemic effects, despite normal or even low serum IGF-II levels. We studied a 44-year-old woman presenting with symptomatic hypoglycaemia associated with a large intraabdominal haemangiopericytoma. The serum IGF-II level was 455 μg/l when measured after acid-ethanol extraction (normal range (NR) 450–750 μg/l) and 1063 μg/l after acid chromatography (normal human serum pool 1068 μg/l). Levels of fasting plasma insulin, C-peptide, glucose and serum IGF-I levels were low before the operation (< 2 mU/l (NR <2-14), 0·23 nmol/l (NR 0-4-1-2), 3-1 mmol/l, (NR 3-7-5-9) and 002 U/ml respectively). After tumour removal, the symptoms resolved rapidly and the patient made a full recovery. Secretion of both insulin and growth hormone was suppressed before the operation in response to a 75 g glucose meal and to an infusion of 100 μg GH-releasing hormone (GHRH) respectively in comparison with studies after the operation. Serum IGF-II levels 6 weeks and 12 weeks after the operation fell to 385 μg/1 (777 μg/1; acid chromatography) and 280 μg/1 (647 μg/1; acid chromatography) and serum IGF-I levels increased to 0-35 U/ml and 0-26 U/ml. Serum before the operation and tumour extract contained chiefly a large molecular weight precursor IGF-II (molecular weight 15 000–20 000) which disappeared from the serum after the operation. The IGF-binding proteins (IGFBP-1, IGFBP-2, IGFBP-3 and IGFBP-4) were examined. The preoperation fasting serum IGFBP-1 level was lower than expected (31 μg/l (NR 20–70 μg/l)) and similar to levels at 6 weeks after the operation (33 μg/l). This was surprising given the differences in plasma insulin levels before and after the operation (< 2 mU/l versus 13 mU/l). Using Western ligand blotting techniques, serum IGFBP-3 levels were found to be low and IGFBP-2 appeared to be the dominant IGFBP before the operation. Serum IGFBP-3 levels after the operation fell further. This further decrease appeared, in part, to be due to the presence of a cation-dependent IGFBP-3-specific protease which has previously only been described in late pregnancy.

We conclude that in this subject, despite normal serum IGF-II levels, the hypoglycaemia and systemic effects on insulin and GH secretion were due to increased bioavailability of a circulating tumour-produced precursor form of IGF-II. This increased bioavailability appears to be due to alterations in the circulating levels and perhaps affinities of the IGFBPs.

Journal of Endocrinology (1991) 131, 303–311

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M. E. Yateman, D. C. Claffey, S. C. Cwyfan Hughes, V. J. Frost, J. A. H. Wass, and J. M. P. Holly


Human dermal fibroblasts produce a number of insulin-like growth factor-binding proteins (IGFBPs) including the main circulating form, IGFBP-3. It has been suggested that the regulation of IGFBP secretion may play a major role in modulating insulin-like growth factor (IGF) bioactivity. We have quantified the effects of two cytokines, transforming growth factor-β1 (TGF-β1) and tumour necrosis factor-α (TNF-α) which have opposing actions on fibroblast IGFBP-3 production, and examined their subsequent role in IGF-I mitogenesis. TGF-β1 caused a dose-dependent increase in IGFBP-3 in serum-free fibroblast-conditioned media. TGF-β1 (1 μg/l) resulted in immunoreactive IGFBP-3 levels reaching 286·5 ± 22·4% of control after 20 h, the increase being confirmed by Western ligand blot. TNF-α caused a dose-dependent decrease in fibroblast IGFBP-3 secretion, 1 μg TNG-α/l reducing IGFBP-3 levels to 32·1 ± 11·% of control. This effect was not due to cytotoxicity and was not cell-density-dependent. Fibroblast proliferation was examined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric cytochemical bioassay. The addition of IGF-I resulted in dose-dependent growth stimulation after 48 h, the effective range being 20–100 μg/l. The IGF-I analogue Long-R3-IGF-I which has little affinity for the IGFBPs was approximately 20-fold more potent in this assay, and was unaffected by exogenous IGFBP-3. While the addition of 1 μg TGF-β1/l to increasing doses of IGF-I resulted in a fourfold decrease in mitogenic sensitivity to the IGF-I, no such effect was seen with Long-R3-IGF-I. Conversely, 1 μg TNF-α/l increased fibroblast IGF-I sensitivity five-fold, an effect not observed with the IGF-I analogue. Such data suggest that endogenous IGFBP-3 inhibits IGF-I bioactivity and that the regulation of IGF mitogenesis by TGF-β1 and TNF-α can occur via local IGFBP modulation. This may represent a mechanism by which complex growth signals are co-ordinated in vivo.

Journal of Endocrinology (1993) 137, 151–159

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J Svensson, M Diez, J Engel, C Wass, Å Tivesten, J-O Jansson, O Isaksson, T Archer, T Hökfelt, and C Ohlsson

IGF-I is a neuroprotective hormone, and neurodegenerative disorders, including Alzheimer’s disease, have been associated with decreased serum IGF-I concentration. In this study, IGF-I production was inactivated in the liver of adult mice (LI-IGF-I−/−), resulting in an approximately 80–85% reduction of circulating IGF-I concentrations. In young (6-month-old) mice there was no difference between the LI-IGF-I−/− and the control mice in spatial learning and memory as measured using the Morris water maze test. In old (aged 15 and 18 months) LI-IGF-I−/− mice, however, the acquisition of the spatial task was slower than in the controls. Furthermore, impaired spatial working as well as reference memory was observed in the old LI-IGF−/− mice. Histochemical analyses revealed an increase in dynorphin and enkephalin immunoreactivities but decreased mRNA levels in the hippocampus of old LI-IGF-I−/− mice. These mice also displayed astrocytosis and increased metabotropic glutamate receptor 7a-immunoreactivity. These neurochemical disturbances suggest synaptic dysfunction and early neurodegeneration in old LI-IGF-I−/− mice. The decline in serum IGF-I with increasing age may therefore be important for the age-related decline in memory function.

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J. M. P. Holly, S. A. Amiel, R. R. Sandhu, L. H. Rees, and J. A. H. Wass


The insulin and growth hormone (GH)/insulin-like growth factor-I (IGF-I) axis are two endocrine systems that are interlinked at many levels. GH is one of the glucose counter-regulatory hormones, rising in response to hypoglycaemia, it has both intrinsic hyperglycaemic actions and causes insulin resistance. Both IGF-I and its receptor have high structural and functional homology to insulin and its receptor. Insulin can regulate IGF-I production, acting on the GH receptor or at a post-receptor site. Conversely IGF-I is thought to have a permissive effect on the pancreatic insulin response to glucose.

Growth is compromised in poorly controlled diabetic children; however, a causal link with altered GH/IGF-I levels has not been proven. Insulin-dependent diabetes clearly causes derangements in the GH/IGF-I axis. In poorly controlled diabetics GH levels are invariably raised whilst normal or low levels of IGF-I are found, indicating a dissociation between the two factors. Altered IGF-binding protein levels are also found, with high levels of small binding protein and low levels of large binding protein. These derangements are probably the result of interactions at many levels although the exact mechanisms are not fully understood.

Raised GH levels could result from altered hypothalamic/pituitary control or reduced feedback inhibition. The latter could, in turn, result from low IGF-I levels, reduced availability of IGF-I to relevant receptors or increased levels of inhibitors (possibly the small binding protein). Low IGF-I levels could be directly due to deficient insulin levels or simply to lack of available circulating binding protein.

Alternative or altered molecular forms of circulating GH in diabetes seem unlikely on present evidence.

That GH has an effect on glycaemic control is most evident from the abnormal glucose tolerance seen in acromegalics, but is also seen with physiological GH variations such as during the pubertal growth spurt. In diabetics the derangements to the GH/IGF-I axis, caused by poor metabolic control, leads to aggravation of the metabolic problems.

Altered GH/IGF-I levels have been implicated in the long-term complications associated with diabetes, and whilst GH/IGF-I are not essential for the early changes involved in these complications they may still play an important role in their development, especially proliferative retinopathy.