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J Rodriguez-Arnao, J Miell, M Thomas, A M McGregor and R J M Ross

Abstract

Changes in thyroid status have a major effect on the GH/insulin-like growth factor (IGF) axis. The majority of IGF in the circulation is bound to specific IGF-binding proteins (IGFBPs) of which six have been cloned and sequenced. We have studied changes in hepatic gene expression of IGFBP-1, -2 and -3, in male Wistar rats rendered hyperthyroid (thyroxine, 200 μg/kg per day) or hypothyroid (propylthiouracil, 0·1% daily). Littermates of the same age were used as controls (n=6 in each group). Thyroxine was measured by radioimmunoassay, and hepatic IGFBP-1, -2 and -3 mRNA levels by Northern blot analysis using specific rat cDNA probes with a 28S ribosomal probe as a loading control. Mean± s.e.m. thyroxine levels were 247·0±44·5 (hyperthyroid group), <9·0 (hypothyroid group) and 76·0 ± 4·5 nmol/l (control group). IGFBP-1 and -2 mRNA levels in the hypothyroid animals compared with the controls were significantly increased, but similar levels of expression were found in thyrotoxic and control rats. IGFBP-3 mRNA levels in hypothyroid animals were decreased, and increased in thyrotoxic animals. Thus, in the adult rat, hypothyroidism is associated with increased hepatic IGFBP-1 and -2 gene expression, but decreased IGFBP-3 gene expression, while in thyrotoxicosis there are normal IGFBP-1 and -2 mRNA levels but increased IGFBP-3 gene expression. These results suggest that there is specific and different transcriptional regulation for IGFBP-1, -2 and -3 in hypoand hyperthyroid rats.

Journal of Endocrinology (1994) 140, 251–255

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J Rodríguez-Arnao, G Yarwood, C Ferguson, J Miell, C J Hinds and R J M Ross

Abstract

Sepsis is characterized by a severe shift in metabolism, characterized by low IGF-I levels. We have studied the influence of caecal ligation and puncture (CLP) on the levels of circulating IGF-I and hepatic IGF-I and IGF-binding protein (IGFBP)-1, -2 and -3 mRNA in adult male Wistar rats (n=12) and compared it with sham-operated rats (n=6). In order to exclude anorexia-induced changes we also studied animals pair-fed to both groups. IGF-I levels were measured by RIA. Steady-state hepatic IGF-I, IGFBP-1, IGFBP-2 and IGFBP-3 mRNA levels were measured by Northern blot analysis using specific rat cDNA probes. Food intake averaged 13·0 ± 2·0 g/day in the sham-operated rats fed ad libitum during the study period, with a sharp decline in food intake in the CLP animals (2·3 ± 1·3 g/day). After CLP, there was a significant reduction in circulating IGF-I levels (467·2 ± 50·9 μg/l) compared with sham-operated animals (924·0 ± 75·3 μg/l; P=0·04) or those pair-fed to the CLP rats (612·5 ± 52·9 μg/l; P=0·04). Total hepatic IGF-I mRNA levels were significantly reduced (2·57 ± 0·05 densitometric units (DU)) after CLP compared with the sham-operated group (2·71 ± 0·04; P=0·04), or their pair-fed controls (2·75 ± 0·08 DU; P<0·05). Hepatic IGFBP-3 mRNA levels were lower after CLP (0·37 ± 0·04 DU) than in the sham-operated animals (0·66 ± 0·09 DU; P=0·04) or their pair-fed controls (0·61 ± 0·05 DU; P=0·04). On the other hand, hepatic IGFBP-2 mRNA levels were increased after CLP (0·91 ± 0·11 DU) compared with sham-operated animals (0·28 ± 0·06 DU; P=0·01) or with their pair-fed controls (0·22 ± 0·22; P=0·01), as were hepatic IGFBP-1 mRNA levels (CLP animals 0·95 ± 0·11 DU; sham-operated 0·30 ± 0·04 DU, P=0·01; pair-fed 0·30 ± 0·02 DU, P=0·01). No significant difference between sham-operated animals and their pair-fed controls was observed in circulating IGF-I levels (888·0 ± 109·3 μg/l; P=not significant (N.S.)), hepatic mRNA levels for IGF-I (2·72 ± 0·06 DU; P=N.S), IGFBP-3 (0·71 ± 0·07 DU; P=N.S.), IGFBP-2 (0·25 ± 0·07 DU; P=N.S.) or IGFBP-1 (0·27 ± 0·06 DU; P=N.S.).

In summary, after CLP there was a reduction in both circulating and hepatic IGF-I mRNA levels associated with a specific and differential regulation of hepatic IGFBP-1, -2 and -3 mRNA levels. Although we cannot eliminate a possible effect of surgical stress combined with malnutrition, our results suggest that these changes are a specific effect of sepsis rather than simply a result of surgical stress or poor nutrition alone.

Journal of Endocrinology (1996) 151, 287–292

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R J M Ross, J Rodriguez-Arnao, A Donaghy, J Bentham, A Clark, J Holly, R Williams and A Gimson

Abstract

Cirrhosis of the liver, a condition characterised by hepatocyte regeneration, is also associated with elevated insulin levels and insulin resistance. In animal models hepatic regeneration is associated with increased IGFBP-1 gene expression. Insulin is known to be an inhibitor of IGFBP-1 gene expression and circulating insulin levels in man demonstrate a negative correlation with IGFBP-1 levels. To further our understanding of the regulation of IGFBP-1 in cirrhosis we have studied steady state levels of IGFBP-1 mRNA in human liver from three groups of patients: Group 1, tissue obtained at the time of harvesting donor liver for orthotopic liver transplantation (n=4); group 2, patients undergoing major liver resection with no histological evidence of chronic liver disease (n=4); and group 3, patients undergoing orthotopic transplantation for chronic liver failure (n=9). Simultaneous samples of serum were taken at the time of surgery in some patients and in these patients IGFBP-1 mRNA levels were related to circulating levels of IGFBP-1 and insulin.

IGFBP-1 mRNA was detectable in all the human liver samples with the greatest levels seen from the normal livers of group 2 patients. Insulin levels were elevated in the cirrhotic group 3 patients compared to a normal range as were IGFBP-1 levels. There was no relationship between circulating levels of IGFBP-1 and IGFBP-1 gene expression.

In conclusion, IGFBP-1 mRNA is present in human adult liver at the time of surgery and also in cirrhotic liver despite high levels of insulin suggesting that there are factors other than insulin regulating IGFBP-1 gene expression.

Journal of Endocrinology (1994) 141, 377–382

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J. R. T. COUTTS, M. C. MACNAUGHTON, P. E. ROSS and J. WALKER

SUMMARY

In a case of hydatidiform mole accompanied by theca lutein cysts there was a greatly increased urinary excretion of pregnanetriol. Incubation of the mole tissue with [4-14C]pregnenolone resulted in the formation of 17α-hydroxypregnenolone, progesterone, 16α-hydroxyprogesterone and 16βhydroxyprogesterone.

Cholesterol, pregnenolone, 17α-hydroxypregnenolone, pregnanediol, pregnanetriol and androstenedione were isolated from both the mole tissue itself and from the ovarian cyst fluid. Progesterone and 17α-hydroxyprogesterone were characterized only from the cyst fluid. Greater quantities of the metabolic intermediates between pregnenolone and pregnanetriol were isolated from the cyst fluid than from the mole tissue.

On the basis of these results it is concluded that although the mole tissue is active in steroid metabolism the increased urinary excretion of pregnanetriol is a function of the polycystic ovaries.

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J. P. Miell, A. M. Taylor, J. Jones, J. M. P. Holly, R. C. Gaillard, F. P. Pralong, R. J. M. Ross and W. F. Blum

ABSTRACT

Glucocorticoids inhibit somatic growth in man and laboratory animals, and have long been regarded as suppressors of both stimulated GH secretion and insulin-like growth factor (IGF) activity. Recent evidence suggests, however, that glucocorticoids can be potent GH secretagogues in their own right with concomitant increases in circulating IGF-I levels. IGFs circulate tightly bound to a group of high-affinity binding proteins (IGFBPs) which modulate their actions. In order to investigate the effects of glucocorticoids on serum levels of IGFs and IGFBPs, normal male volunteers were sampled over 24-h periods before and directly after treatment with dexamethasone (2 mg twice daily) for 96 h. Following dexamethasone administration, all volunteers showed a marked increase in mean± s.e.m. IGF-I levels over the 24-h sampling period (292·2±31·8 before dexamethasone, 425·9 ±37 μg/l after dexamethasone, P<0·005); there was no change in mean IGF-II levels. Integrated mean insulin levels were raised by dexamethasone treatment (50 ±4·6 before dexamethasone, 117±13·4 mU/l after dexamethasone, P= 0·002) and IGFBP-1 was significantly suppressed (42·9±8·2) before dexamethasone, 28·0±7·9 μg/l after dexamethasone, P<0·001). IGFBP-2 levels were similarly suppressed after dexamethasone (319·5±24·5 before dexamethasone, 214·8±8·5 μg/l after dexamethasone, P=0·002), and there was a significant increase in IGFBP-3 levels from 3·24 ±0·25 to 3·67±0·32 mg/l (P=0·0153). Mean IGF bioactivity over the sampling period after dexamethasone was reduced by approximately 60% (0·93 ±0·39 before dexamethasone, 0·39 ±0·05 U/ml after dexamethasone, P <0·0001).

There were strong negative correlations between both insulin and IGF-I levels and those of IGFBP-2, suggesting the presence of a novel regulation mechanism for this binding protein. The established inverse relationship between insulin and IGFBP-1 was maintained after dexamethasone. These results suggest that glucocorticoids alter the bioactivity of IGFs, possibly by induction of inhibitors, but that neither IGFBP-1 nor IGFBP-2 can be implicated as glucocorticoiddependent inhibitors of IGF bioactivity in man. The study also demonstrates for the first time a strong negative correlation between IGF-I, insulin and IGFBP-2.

Journal of Endocrinology (1993) 136, 525–533

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

ABSTRACT

The insulin-like growth factors (IGF-I and IGF-II) are almost completely bound in the circulation to specific binding proteins (IGFBPs). These IGFBPs appear to play a pivotal role in maintaining circulating levels and modulating the delivery of the IGFs to the tissues. A large proportion of the circulating IGFs are bound with high affinity to one of the binding proteins, IGFBP-3. The mechanism by which these IGFs are transferred from the circulatory pool to the tissue receptors is at present unclear. Recent studies in late pregnancy have demonstrated the presence of specific proteases which may modify the IGFBPs such that their affinities for the IGFs are reduced. In this paper, we have demonstrated the presence of a heat-sensitive cation-dependent proteolytic enzyme specific for IGFBP-3 in the serum of five severely ill patients. The activity of this protease was found to vary in these patients, becoming more apparent during fasting than when studied after commencement of parenteral nutrition, indicating that one of the influencing factors in the activity of this protease is the nutritional intake of the patient. Age- and sex-matched healthy adults were also studied in a similar protocol, but no proteolytic modification of any of the IGFBPs was found in any of the samples examined. As the levels of both IGF-I and IGF-II were found to be low in the patients, the presence of a circulatory protease suggests that this may be an adaptive response to increase the bioavailability of the IGFs and possibly to improve the nitrogen retention and counter the catabolic state in severe illness.

Journal of Endocrinology (1991) 130,469–473

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A. M. Wallace, G. H. Beastall, B. Cook, A. J. Currie, A. M. Ross, R. Kennedy and R. W. A. Girdwood

ABSTRACT

We have assessed the feasibility of screening newborn babies for congenital adrenal hyperplasia (CAH) by the direct measurement of 17-hydroxyprogesterone (17-OHP) in blood spots collected on filter paper (Guthrie cards) for the phenylketonuria, hypothyroidism and galactosaemia screening programmes run in Scotland. The procedure described for CAH uses an iodinated 17-OHP tracer and a specific 17-OHP antiserum sheathed within semipermeable nylon microcapsules. The method does not require a solvent extraction step, is inexpensive, precise, efficient and, therefore, practical for large-scale use. With this system the value of a neonatal screening programme was assessed in a retrospective analysis and a prospective trial.

The retrospective study of 15 paediatric cases of CAH illustrated that at least half were not diagnosed within 3 weeks of birth. Analysis of the original Guthrie card samples revealed increased levels of 17-OHP in all cases. The prevalence of CAH as calculated in the retrospective study was 1 in 20 907 with a range (within 95% confidence limits) of from 1 in 12 675 to 1 in 32 604 (n = 301 450). In the prospective trial a total of 92 051 consecutive samples was screened. Five cases of CAH were correctly identified with a current false positive rate of 0·042%. Analysis of urinary steroids confirmed defective adrenal 21-hydroxylase activity in all positive cases. In the prospective trial the prevalence was 1 in 18 401 with a range of from 1 in 7 422 to 1 in 50006.

We conclude that mass screening for CAH is both feasible and desirable.

J. Endocr. (1986) 108, 299–308

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R. Buffenstein, D. C. Skinner, S. Yahav, G. P. Moodley, M. Cavaleros, D. Zachen, F. P. Ross and J. M. Pettifor

ABSTRACT

The damara mole rat, Cryptomys damarensis, is a strictly subterranean dwelling herbivorous rodent that in its natural habitat has no access to any obvious source of cholecalciferol (D3). We examined the effects of D3 supplementation, at physiological and supraphysiological doses, on calcium metabolism, plasma concentrations of calcium and alkaline phosphatase (ALP) and D3 metabolites. Animals not receiving a D3 supplement maintained normal plasma calcium concentrations. In addition, they exhibited a high apparent fractional mineral absorption efficiency (91%) and maintained a positive mineral flux. The serum concentration of 25-(OH)D3 was undetectable (< 5 nmol/l) and that of 1,25-(OH)2D3 was 41±10 pmol/l. Supplementation at a physiological dose of D3 resulted in increased plasma concentrations of D3 metabolites, food intake, apparent fractional absorption efficiency and apparent fractional retention efficiency. Despite the 1·8-fold increase in food intake, body mass remained constant suggesting that the enhanced energy intake was dissipated in catabolic processes. Plasma calcium and ALP concentrations were not significantly altered with physiological doses of D3. The group given supraphysiological doses of D3 exhibited hypercalcaemia, increased creatinine concentrations and markedly increased ALP levels. These data indicate that a pathological response to D3 intoxication occurred and that hepatic and renal excretory functions were impaired. It appears, therefore, that these animals function optimally at the low concentrations of D3 metabolites found naturally. Supplementation at both physiological and supraphysiological doses of D3 may disadvantage the damara mole rat.

Journal of Endocrinology (1991) 131, 197–202

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M Maamra, A Milward, H Zarkesh Esfahani, L P Abbott, L A Metherell, M O Savage, A J L Clark and R J M Ross

Growth hormone insensitivity syndrome (GHIS) has been reported in a family homozygous for a point mutation in the GH receptor (GHR) that activates an intronic pseudoexon. The resultant GHR (GHR1–656) includes a 36 amino-acids insertion after residue 207, in the region known to be important for homodimerization of GHR. We have examined the functional consequences of such an insertion in mammalian cells transfected with the wild type (GHRwt) and mutated GHR (GHR1–656). Radio-ligand binding and flow cytometry analysis showed that GHR1–656 is poorly expressed at the cell surface compared with GHRwt. Total membrane binding and Western blot analysis showed no such difference in the level of total cellular GHR expressed for GHR1–656 vs GHRwt. Immunofluorescence showed GHR1–656 to have different cellular distribution to the wild type receptor (GHRwt), with the mutated GHR being mainly perinuclear and less vesicular than GHRwt. Western blot analysis showed GH-induced phosphorylation of Jak2 and Stat5 for both GHR1–656 and GHRwt, although reduced Stat5 activity was detected with GHR1–656, consistent with lower levels of expression of GHR1–656 than GHRwt at the cell surface. In conclusion, we report that GHIS, due to a 36 amino-acids insertion in the extracellular domain of GHR, is likely to be explained by a trafficking defect rather than by a signalling defect of GHR.

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R J M Ross, S L Chew, L D'Souza Li, M Yateman, J Rodriguez-Arnao, A Gimson, J Holly and C Camacho-Hubner

Abstract

The liver plays a central role in the IGF-I axis producing the majority of circulating hormone and some of its binding proteins (IGFBPs). Cirrhosis of the liver is characterised by changes in IGF-I and IGFBPs associated with liver fibrosis and regeneration. We have studied steady state levels of mRNA for the genes in the IGF-I axis in normal and cirrhotic human liver, localised the most highly expressed gene, IGFBP-1, and measured circulating IGFBP-3 by radioimmunoassay (RIA), IGFBP-2 and IGFBP-3 by Western ligand blot (WLB), and protease activity for IGFBP-3 in cirrhotic patients. Messenger RNA for IGF-I, IGFBP-1, IGFBP-2, and IGFBP-3 was detectable by Northern blotting in normal and cirrhotic liver although there was considerable variation in expression. IGFBP-2 and IGFBP-3 tended to be more highly expressed in cirrhotic liver and IGFBP-1 was more highly expressed in normal liver, although there were no significant differences. In normal liver, in situ hybridisation localised IGFBP-1 to hepatocytes. In cirrhotic liver the regenerating nodules showed expression of IGFBP-1 while there was none in fibrotic tissue. Circulating IGFBP-3 levels were low as measured by RIA and WLB but protease activity was only found in one patient. IGFBP-2 levels, assessed by WLB, were similar to the normal serum pool. Our data show that key mRNAs involved in the IGF-I axis continue to be expressed in cirrhotic liver despite end stage liver disease. The low levels of IGFBP-3 do not appear to be due to reduced gene transcription or increased protease activity.

Journal of Endocrinology (1996) 149, 209–216