The GH/IGF-I axis has a clearly established role in somatic growth regulation and there is much evidence suggesting that it can play a contributing role in neoplastic tissue growth; a number of recent epidemiological reports indicate that it may also be an important determinant of cancer incidence. Whilst there have been previous reports of changes to the axis in patients with established cancers, these new studies are distinct in being prospective and the inferences that can be made from this are outlined in this review. The recent studies are considered within the context of other indirect epidemiological evidence, and together indicate that the GH/IGF-I axis may establish the level of predisposition to a number of common cancers and indeed that such risk may be programmed from early life. There is considerable evidence for a number of possible mechanisms, both direct and indirect, which could account for the associations between GH/IGF-I levels and cancer incidence; these mechanisms are briefly summarised. The implications of the new findings are then discussed in relation to the increasing clinical usage of chronic GH administration and the need for further studies to establish any consequent increase in cancer risk. Finally the opportunities for further work to optimise cancer risk assessment and risk reduction strategies are highlighted.
JM Holly, DJ Gunnell and G Davey Smith
AL Crown, XL He, JM Holly, SL Lightman and CE Stewart
In an attempt to address the complex and clinically challenging question of the causes of muscle wasting in patients with cachexia, we have developed a primary adult human skeletal muscle cell model. The cultured cells were characterised by immunocytochemistry using antibodies to the myofibrillar protein constituents desmin and titin. Myotube formation was confirmed biochemically by a fourfold increase in the activity of the muscle-specific enzyme creatinine kinase, and myoblast withdrawal from the cell cycle, which is essential for terminal differentiation, was associated with progressive retinoblastoma protein dephosphorylation. Having successfully confirmed the phenotype of these adult human muscle cells, we assessed their interaction with the insulin-like growth factor (IGF) system. IGF-I is known to stimulate myoblast survival, proliferation and differentiation in cell lines, and, like insulin, is a potent anabolic agent in the regulation of protein metabolism. We have shown that IGF-I stimulated both replication and differentiation of myoblasts, whilst fibroblast growth factor-2 stimulated replication but inhibited differentiation. Examining the IGF system during the process of terminal differentiation, we found that both myoblasts and myotubes expressed insulin, IGF-I and insulin-IGF-I hybrid receptors, with the levels of all three receptor types increasing on differentiation. The cells also produced a wide range of IGF binding proteins (IGFBPs) including IGFBP-2, IGFBP-4 and abundant IGFBP-3, which has not been shown to be produced by any other skeletal muscle cell line examined to date. Both insulin and IGF-I had anabolic effects on myotube protein metabolism at physiological concentrations. Insulin was more potent than IGF-I: use of the IGF analogue long R(3)IGF-I demonstrated that the effects of exogenous IGF-I on protein metabolism were not affected by the high levels of endogenous IGFBP production. In summary, we have developed and characterised a clinically relevant in vitro model with which to address the aetiology of muscle wasting associated with chronic catabolic conditions, and we anticipate that future work will enable the development of novel, effective therapeutic interventions.
EJ Whellams, LA Maile, JK Fernihough, ME Billingham and JM Holly
Increased concentrations of insulin-like growth factor (IGF) system components have previously been observed in rheumatoid arthritis (RA) and osteoarthritis (OA); however, disruption of the IGF axis and the implications for the disease process remain largely unaddressed. This study was undertaken to characterise the IGF binding protein (IGFBP)-3 proteolysis and complex formation systems in synovial fluid and to investigate changes in these systems in arthritic disease, and their impact on the availability of IGF. Western blotting or autoradiography of SDS gels was used to visualise IGFBP-3 or its proteolysis. IGF-I and IGFBP-3 concentrations were determined by radioimmunoassays and acid-labile subunit (ALS) was measured by ELISA. A shift in distribution of IGFBP-3 and IGF-I in RA and OA synovial fluids (RASynF, OASynF) and an associated increase in ALS suggested the presence of 150 kDa ternary complexes. IGFBP-3 proteolysis was decreased in RASynF and OASynF, but was apparent in size-fractionated fluid and resembled serum activity. The presence of serum-like inhibitors of IGFBP-3 proteolysis in RASynF was also demonstrated by the ability of this fluid, and 150 kDa fractions from its size fractionation, to inhibit IGFBP-3 proteolysis in the other synovial fluid. A marked disruption in the IGF system was observed, as considerably more IGF-I was retained in ternary complexes. We also classified the IGFBP-3 proteolysis system in synovial fluid and found it to be disturbed in RASynF and OASynF. These changes may be caused by an increased flux of circulatory proteins into synovial fluid, resulting from an inflammation-induced increase in vascular permeability. The net result in RA and OA would be a decrease in IGF availability in arthritic joints, and therefore loss of a potential anabolic stimulus. This disruption to the IGF axis would influence disease progression in RA and OA.
SC Hughes, HD Mason, S Franks and JM Holly
The presence of IGFs and their associated binding proteins (IGFBPs) in human follicular fluid is well documented. Furthermore, most of the constituents of the IGF system in follicular fluid have been found to vary, either in total amount or by proteolytic cleavage, depending on the health status of the follicle. In this study we have examined the acid-labile subunit (ALS) and found that levels in follicular fluid (mean 146 nmol/l) were almost 50% of those in the circulation. This amount of ALS was considerably greater than that found in other extracirculatory fluids (20.9 for synovial fluid and 31.4 nmol/l for skin blister fluid). As in the circulation, ALS levels were in molar excess and did not vary between atretic and dominant follicles. Although the source of ALS is probably from blood (conditioned medium from ovarian cell cultures had no measurable ALS) it would appear that this glycoprotein is not merely diffusing from the circulation as the capillary endothelium becomes more permeable in dominant follicles and this is not reflected in the level of ALS. Analysis of the distribution of IGF-I, IGF-II and IGFBP-3 in fluid from healthy and atretic follicles revealed that the majority of these growth factors (> 80% of total IGF-II) were in the 150KDa complex, indicating that the ALS present was functional, in that it formed the ternary complex with a molecule of IGFBP-3 and IGF. No free IGF-II was found in any of the follicular fluids analysed nor was there any increase in the amount of unsaturated IGFBP-3 in atretic follicles. In summary, we have shown that the majority of IGF measured in follicular fluid, whether from healthy or atretic follicles, is bound in the ternary complex.
TD Cheetham, JM Holly, RC Baxter, K Meadows, J Jones, AM Taylor and DB Dunger
The long term therapeutic potential of recombinant human (rh) IGF-I administration in insulin-dependent diabetes mellitus (IDDM) may be determined by changes in the IGF binding proteins (IGFBPs) and thus the bioavailability of IGF-I. We have therefore studied the effects of a single subcutaneous dose of rhIGF-I (40 micrograms/kg at 1800 h), when compared with an untreated control night, in 17 subjects with IDDM, on serum concentrations of IGF-I, IGF-II, IGFBP-3, acid labile subunit (ALS), and IGFBP-3 proteolysis. Mean (+/- S.E.M.) IGF-I levels increased from 242 +/- 30 ng/ml to 399 +/- 26 ng/ml (P = 0.01) after rhIGF-I whereas IGF-II levels declined from 600 +/- 45 ng/ml to 533 +/- 30 ng/ml. There was a small overnight reduction in baseline ALS levels from 48 +/- 2.8 to 44.5 +/- 3.2 micrograms/ml (P = 0.04) after rhIGF-I administration. An early fall in IGFBP-3 concentrations on the control night was not seen after rhIGF-I and overall mean levels were increased (5.2 +/- 0.2 micrograms/ml vs 4.9 +/- 0.2 micrograms/ml, P = 0.04, on the control night). On the baseline night, IGFBP-3 levels correlated with the sum of IGF-I and IGF-II (r = 0.73, P = 0.02) and with levels of the ALS (r = 0.7, P = 0.002). However after rhIGF-I, the sum of IGF-I and IGF-II no longer correlated with IGFBP-3, whereas the relationship with ALS was maintained. Immunoblot studies in six subjects indicated that 60%-70% of the IGFBP-3 was detected as a low molecular weight fragment at 1900 h on both study nights, but the amount of fragment declined to approximately 50% at 0100 h and 45% at 0700 h. In conclusion, despite a slight but significant fall in ALS, IGFBP-3 levels rise after rhIGF-I administration in IDDM. This cannot be explained by alterations in IGFBP-3 proteolysis, and may relate to the relative stability of ALS/IGFBP-3 when complexed principally with IGF-I rather than IGF-II.
I Ibanez De Caceres, JM Holly, T Priego, AI Martin, A Lopez-Calderon and MA Villanua
Adjuvant-induced arthritis is a chronic inflammatory illness that induces a catabolic state, with a decrease in pituitary GH and hepatic IGF-I synthesis. We have previously observed an increase in serum IGF-binding protein-3 (IGFBP-3) in arthritic rats, and found that GH administration prevents the increase in circulating IGFBP-3 in arthritic rats. The aim of this work was therefore to study IGFBP-3 synthesis in the liver as well as its proteolysis in serum as the two possible causes of the increased circulating IGFBP-3 in arthritic rats. The effect of recombinant human GH (rhGH) administration was also analysed. Adult male Wistar rats were injected with complete Freund's adjuvant or vehicle, and 14 days later they were injected s.c. daily until day 22 after adjuvant injection with rhGH (3 IU/kg) or saline. Three hours after the last GH injection, all rats were killed by decapitation. Arthritis increased serum IGFBP-3 levels (P<0.01). The increase in serum IGFBP-3 levels in arthritic rats seems to be due to decreased proteolysis (P<0.01) rather than to an increased synthesis, since liver IGFBP-3 mRNA content was not modified by arthritis. GH administration to control rats resulted in an increase in both hepatic IGFBP-3 mRNA content and in serum IGFBP-3 levels in spite of the increase in IGFBP-3 proteolysis in serum. In arthritic rats, GH treatment did not modify liver IGFBP-3 synthesis, but it increased serum proteolysis of IGFBP-3, leading to a serum concentration of IGFBP-3 similar to that of control rats. Furthermore, there was a negative correlation between circulating IGFBP-3 and its proteolytic activity in the serum of adjuvant-induced arthritic rats. These data suggest that in chronic arthritis the increase in IGFBP-3 serum concentration is secondary to a decrease in proteolytic activity, rather than to an increase in hepatic IGFBP-3 gene expression.