Single GH Research Articles

Mean GH profile is more accurate than single fasting GH in the evaluation of acromegaly disease control during somatostatin receptor ligands therapy

PurposeThis study aims to compare the accuracy of mean GH profile (GHP) < 2.5 ng/ml and single fasting GH (SGH) < 1 ng/ml in the evaluation of disease control in acromegaly patients during somatostatin receptor ligands (SRLs) therapy.MethodsWe retrospectively enrolled 100 acromegaly patients, 68 responder, and 32 partial responder to SRLs. Controlled disease has been defined as IGF-I levels within age-related normal limits, while partial response as pathological IGF-I values despite a reduction ≥ 50%. In all patients, GHP, SGH, IGF-I, and IGFBP-3 were evaluated.ResultsMedian GHP levels (1.2 ng/ml, IQR 0.5–2.3 ng/ml) were lower (p = 0.001) than SGH (1.9 ng/ml, IQR 1.0–3.6 ng/ml). Accuracy of GHP was 81%, whereas that of SGH was 55%, with a Kappa index of 0.520 and 0.237, respectively. In multivariable analysis GHP (p = 0.002) and IGFBP-3 (p = 0.004), but not SGH, were independently associated with normal IGF-I levels. At receiver–operator characteristic curve (ROC) analysis GHP cut-off sensitivity and specificity were 94.1% and 50.0%, respectively, while SGH sensitivity and specificity were 35.3% and 93.7%, respectively. Finally, in obese patients the GH cut-off level (both as SGH and GHP) associated to good disease control was significantly different with respect to not obese ones.ConclusionsGHP associates with IGF-I (and therefore with appropriate control of disease) with higher accuracy than SGH. When GH evaluation is needed, the measurement of mean GHP should be preferred and use of BMI-related cut-offs is suggested.

Base and Nucleotide Excision Repair Pathways in DNA Plasmids Harboring Oxidatively Generated Guanine Lesions.

The base and nucleotide excision repair pathways (BER and NER, respectively) are two major mechanisms that remove DNA lesions formed by the reactions of genotoxic intermediates with cellular DNA. We have demonstrated earlier that the oxidatively generated guanine lesions spiroiminodihydantoin (Sp) and 5-guanidinohydantoin (Gh) are excised from double-stranded DNA by competing BER and NER in whole-cell extracts [Shafirovich, V., et al. (2016) J. Biol. Chem. 321, 5309-5319]. In this work we compared the NER and BER yields with single Gh or Sp lesions embedded at the same sites in covalently closed circular pUC19NN plasmid DNA (cccDNA) and in the same but linearized form (linDNA) of this plasmid. The kinetics of the Sp and Gh BER and NER incisions were monitored in HeLa cell extracts. The yield of NER products is ∼5 times greater in covalently closed circular DNA than in the linearized form, while the BER yield is smaller by ∼20-30% depending on the guanine lesion. Control BER experiments with 8-oxo-7,8-dihydroguanine (8-oxoG) show that the BER yield is increased by a factor of only 1.4 ± 0.2 in cccDNA relative to linDNA. These surprising differences in BER and NER activities are discussed in terms of the lack of termini in covalently closed circular DNA and the DNA lesion search dynamics of the NER DNA damage sensor XPC-RAD23B and the BER enzyme OGG1 that recognizes and excises 8-oxoG.

Inhibition of Excision of Oxidatively Generated Hydantoin DNA Lesions by NEIL1 by the Competitive Binding of the Nucleotide Excision Repair Factor XPC-RAD23B.

The interplay between nucleotide excision repair (NER) and base excision repair (BER) of nonbulky, oxidatively generated DNA lesions has long been a subject of significant interest. The hydantoin oxidation products of 8-oxoguanine, spiroiminodihydantoin (Sp) and 5-guanidinohydantoin (Gh), are substrates of both BER and NER in HeLa cell extracts and human cells [Shafirovich, V., et al. (2019) Chem. Res. Toxicol. 32, 753-761]. The primary factor that recognizes DNA lesions is the DNA damage-sensing factor XPC-RAD23B (XPC), while the glycosylase NEIL1 is known to remove Gh and Sp lesions from double-stranded DNA. It is shown here that in aqueous solutions containing nanomolar concentrations of proteins, XPC and NEIL1 compete for binding to 147-mer oligonucleotide duplexes that contain single Gh or Sp lesions under conditions of [protein] ≫ [DNA], thus inhibiting the rate of BER catalyzed by NEIL1. The non-covalently bound NEIL1 molecules can be displaced by XPC at concentration ratios R = [XPC]/[NEIL1] > 0.2, while full displacement of NEIL1 is observed at R ≥ 0.5. In the absence of XPC and under single-turnover conditions, only the burst phase is observable. However, with a progressive increase in the XPC concentration, the amplitude of the burst phase decreases gradually, and a slower time-dependent phase of incision product formation manifests itself with rate constants of 3.0 × 10-3 s-1 (Gh) and 0.90 × 10-3 s-1 (Sp). These slow kinetics are attributed to the dissociation of XPC-DNA complexes that allow for the rebinding of NEIL1 to the temporarily exposed Gh or Sp lesions, and the incisions observed under these steady-state conditions.

Distribution of cytomegalovirus genotypes among ulcerative colitis patients in Okinawa, Japan.

Background/AimsTo determine the prevalence of glycoprotein B (gB), glycoprotein N (gN), and glycoprotein H (gH) genotypes of human cytomegalovirus (HCMV) superimposed on ulcerative colitis (UC) patients in Japan.MethodsFour archived stool samples and 7-archived extracted DNA from stool samples of 11 UC patients with positive multiplex polymerase chain reaction (PCR) results for HCMV were used UL55 gene encoding gB, UL73 gene encoding gN, and UL75 gene encoding gH were identified by PCR. Genotypes of gB and glycoprotein N were determined by sequencing.ResultsAmong 11 samples, 8 samples were amplified through PCR. gB, gN, and gH genotypes were successfully detected in 3 of 8 (37.5%), 4 of 8 (50%), and 8 of 8 (100%), respectively. The distribution of gB and gN genotypes analyzed through phylogenetic analysis were as follows: gB1 (2/3, 66.7%), gB3 (1/3, 33.3%), gN3a (2/4, 50%), and gN3b (2/4, 50%). Other gB genotypes (gB2 and gB4) and gN genotypes (gN1, gN2, and gN4) were not detected in this study. Out of successfully amplified 8 samples of gH genotype, gH1 and gH2 were distributed in 12.5% and 75% samples, respectively. Only 1 sample revealed mixed infection of gH genotype. The distribution of gH1 and gH2 differed significantly (1:6, P<0.05) in UC patients. The distribution of single gH genotype also revealed significant difference in UC patients who were treated with immunosuppressive drug (P<0.05).ConclusionsIn this study, gB1, gN3, and gH2 gene were determined as the most frequently observed genotypes in UC patients, which suggest that there might be an association between these genotypes of HCMV and UC.

Biochemical assessment of disease activity in acromegaly; a comparison of single GH, GH day series mean, OGTT nadir and IGF-1 in 51 patients

Biochemical assessment of disease activity in acromegaly; a comparison of single GH, GH day series mean, OGTT nadir and IGF-1 in 51 patients

GHR and IGF-I gene expression and production characteristics associated with GH gene polymorphism in Nile tilapia

The aim of this study was to investigate the effects of genotype on gene expression and performance characteristics in crossbred GIFT × Chitralada tilapia. We compared the effects of GH single band (GHsb) and GH double band (GHdb) genotypes on performance characteristics, growth hormone receptor (GHR) and insulin-like growth factor I (IGF-I) gene expression in the liver and white muscle. The experiment was conducted in a 2 × 2 factorial design with two categories of weight (20–35 g and 40–70 g) and two genotypes (GHsb and GHdb). Fin fragments were collected for genotyping, while the liver and white muscle samples were collected to analyze gene expression via qRT-PCR. GHdb genotype animals presented greater body weight, empty weight, weight of viscera, and filet weight and length, thereby indicating that GH variation may correlate with performance characteristics. Although some of the phenotypes we measured were correlated with genotype, we cannot say that it is due to the direct effect of the studied polymorphism. However, we recommend further studies be conducted to reveal the genetic nature of this polymorphism. • In this study a new polymorphism in GH gene was observed. • GH double band genotype animals presented the best characteristics of carcass. • GH double band genotype animals presented greater GHR mRNA expression.

GH signaling in skeletal muscle and adipose tissue in healthy human subjects: impact of gender and age.

The mechanisms underlying the impact of age and gender on the GH-IGF1 axis remain unclear. We tested the hypothesis that age and gender have impacts on GH signaling in human subjects in vivo. A total of 20 healthy non-obese adults ('young group'<30 years (5F/5M) and 'old group'>60 years (5F/5M)) were studied after: i) an i.v. GH bolus (0.5 mg) and ii) saline. Muscle and fat biopsies were obtained after 30 and 120 min. Total and phosphorylated STAT5B proteins, gene expression of IGF1, SOCS1, SOCS2, SOCS3 and CISH, body composition, VO2max, and muscle strength were measured. In the GH-unstimulated state, women displayed significantly elevated levels of CISH mRNA in muscle (P=0.002) and fat (P=0.05) and reduced levels of IGF1 mRNA in fat. Phosphorylated STAT5B (pSTAT5b) was maximally increased in all subjects 30 min after GH exposure and more pronounced in women when compared with men (P=0.01). IGF1, SOCS1, SOCS2, SOCS3, and CISH mRNA expression increased significantly in muscle after 120 min in all subjects with no impact of age and gender. GH-induced pSTAT5b correlated inversely with lean body mass (LBM; r=-0.56, P=0.01) and positively with the CISH mRNA response (r=0.533, P=0.05). i) GH signaling in muscle and fat after a single GH bolus in healthy human subjects is age independent, ii) we hypothesize that constitutive overexpression of CISH may contribute to the relative GH resistance in women, and iii) experimental studies on the impact of sex steroid administration and physical training on GH signaling in human subjects in vivo are required.

Age-related changes of GH-IGF1 axis function

Background There is a reduction in the GH-IGF1 axis activity with increasing age, but there is evidence that the pool of GH available for release is not diminished in elderly subjects. We investigated whether there are differences among healthy young-middle aged and old aged subjects in the 24 hour secretory profiles of GH and IGF1. Methods The study was carried out on 15 healthy young-middle aged subjects (age range 36–55 years) and 15 healthy old aged subjects (age range 67–79 years). GH, IGF1, melatonin and cortisol serum levels were measured on blood samples collected every four hours for 24 hours. The fractional variation between single time point GH and IGF1 serum levels, mean photoperiod (06:00h–10:00h–14:00 h) and scotoperiod (18:00h–22:00h–02:00 h) values and the presence of circadian rhythmicity were evaluated. Results A normal circadian rhythmicity was recognizable for GH, melatonin and cortisol secretion in young-middle aged and old aged subjects, MESOR of cortisol was higher in the elderly ( P = 0.002) and the acrophase of cortisol was advanced in the group of old aged subjects ( P = 0.03), whereas the amplitude of IGF1 was higher in the group of young-middle-aged subjects ( P < 0.01). Old aged subjects presented higher mean GH levels in the photoperiod ( P = 0.01), but lack of variability of GH and IGF1 fractional variations, whereas young-middle aged subjects presented significant variability of GH and IGF1 factional variations ( P = 0.05 and P = 0.003, respectively) and increased fractional variation of nocturnal GH and IGF1 serum levels in respect to older subjects ( P = 0.03 and P = 0.05, respectively). There was no statistically significant difference between mean melatonin serum levels in the photoperiod ( P = 0.937) and in the scotoperiod (P = 0.494). There was no statistically significant difference between mean cortisol serum levels in the photoperiod (P = 0.235), whereas mean cortisol serum levels were higher in old aged subjects in the scotoperiod (P = 0.04). Linear regression evidenced a statistically significant negative trend between age and the 24 h mean of GH (P < 0.01) and a statistically significant positive trend between age and the 24 h mean of cortisol (P < 0.001). Conclusions Aging is associated with an altered pattern of GH secretion and IGF1 production.

Distinct Alterations in Chromatin Organization of the Two IGF-I Promoters Precede Growth Hormone-Induced Activation of IGF-I Gene Transcription

Many of the physiological actions of GH are mediated by IGF-I, a secreted 70-residue peptide whose gene expression is induced by GH in the liver and other tissues via mechanisms that remain incompletely characterized but depend on the transcription factor Stat5b. Here we investigate the chromatin landscape of the IGF-I gene in the liver of pituitary-deficient young adult male rats and assess the impact of a single systemic GH injection. Despite minimal ongoing transcription in the absence of GH, both IGF-I promoters appear to reside in open chromatin environments, at least as inferred from relatively high levels of acetylation of core histones H3 and H4 when compared with adjacent intergenic DNA and from enhanced trimethylation of histone H3 at lysine 4. This landscape of open chromatin may reflect maturation of the liver. Surprisingly, in the absence of hormone, IGF-I promoter 1 appears poised to be activated, as evidenced by the presence of the transcriptional coactivator p300 and recruitment of RNA polymerase (Pol) II into a preinitiation complex. By contrast, chromatin surrounding IGF-I promoter 2 is devoid of both p300 and RNA Pol II. Systemic GH treatment causes an approximately 15-fold increase in transcription from each IGF-I promoter within 60 min of hormone administration, leading to a sustained accumulation of IGF-I mRNA. The coordinated induction of both IGF-I promoters by GH is accompanied by hyperacetylation of histones H3 and H4 in promoter-associated chromatin, a decline in monomethylation at lysine 4 of histone H3, and recruitment of RNA Pol II to IGF-I promoter 2. We conclude that GH actions induce rapid and dramatic changes in hepatic chromatin at the IGF-I locus and activate IGF-I gene transcription in the liver by distinct promoter-specific mechanisms: at promoter 1, GH causes RNA Pol II to be released from a previously recruited paused preinitiation complex, whereas at promoter 2, hormone treatment facilitates recruitment and then activation of RNA Pol II to initiate transcription.

Should We Still Use Glucose-Suppressed Growth Hormone Levels for the Evaluation of Acromegaly?

Acromegaly is associated with increased mortality if not adequately treated (1). Currently accepted biochemical criteria for cure of acromegaly include a mean 24-h GH level less than 2.5 g/liter (5 mU/liter) and/or a glucose-suppressed GH level of less than 1 g/liter (2 mU/liter) and a normal IGF-I (2). These criteria wereproposedbyBatesetal. (3)whoshowedthatpatientswithGH levels less than 2.5 g/liter did not show an increase in mortality. Since l993, this GH cutoff has been widely applied based on the relative paucity of data relating normal IGF-I levels to normal life expectancy. More recently, Holdaway et al. (4) demonstrated that IGF-I (expressed as an SD score) was significantly associated with mortality. Specifically, they showed that patients with last follow-up IGF-I of at least 2 SD had a mortality ratio 3.5 times higher than thosewithan SD score less thanzero (4).Theyalso showed that mortality was 1.6 times higher in patients with last follow-up GH levels less than 2 g/liter, whereas mortality was similar to the control population in patients with last follow-up GH levels less than 1 g/liter, suggesting that cure criteria should be modified, at least for levels of GH. No data are available concerning mortality and the GH nadir after glucose, yet it is included in almost all studies examining the efficacy of treatment in acromegaly. There are several areas of debate related to the value of the GH response to glucose. First, the additional diagnostic value over a fasting GH level is limited when the IGF-I level is elevated (5), and additionally, a similar diagnostic accuracy of a GH profile and nadir GH after glucose was demonstrated in a large series of patients (6). Second, the clinical implication of a single unsuppressed GH level in patients studied after surgery and showing normal serum IGF-I levels and GH levels below the threshold of cured patients is still a matter of discussion. In this setting, Freda et al. (7) showed that patients considered cured after surgery (normal IGF-I and postglucose GH nadir 0.14 g/liter) did not demonstrate recurrence of acromegaly in long-term follow-up. These data suggest a very relevant role for assessment of GH after glucose to detect patients with a likelihood for recurrence, which is clearly an important clinical outcome. The threshold GH nadir of no more than 0.14 g/liter is, however, well below that mentioned in the 2000 consensus criteria for cured acromegaly (2); the lower threshold was justified by using a highly sensitive GH assay able to detect levels as low as 0.05 g/liter. Lower nadir GH values of 0.25 and 0.26 g/liter after glucose, also using a highly sensitive GH assay, were reported by Costa et al. (8) and Gullu et al. (9), respectively. Thus, the question as to which GH cutoff value should be used for postglucose GH nadir immediately rises. The assay sensitivity is a crucial aspect of diagnostic accuracy of any biochemical test. It is now known that previously used polyclonal RIAs for GH were insufficiently sensitive to discriminate GH levels around 2 g/liter (5). Modern immunoradiometric assays for GH have a detection limit of 0.05 g/liter, so the ability to discriminate postglucose GH levels in patients with active acromegaly vs. healthy subjects is improved. In this issue of the Journal, Arafat et al. (10) have compared different assays to address further the issue of the appropriate GH nadir after glucose currently used in the treatment evaluation of acromegaly. They measured GH after 75 g glucose in 46 acromegaly patients, 18 with controlled disease and 28 with uncontrolled disease, and in 213 healthy subjects using three commercially available assays (Immulite, Nichols, and DSL) that were calibrated against therecentlyrecommendedGHstandards.Asexpected, their results showed that GH levels measured with all assays were stronglycorrelated(rvalues0.8–0.996)Importantly,however,GH levels assessed with the Immulite assay were, on average, 2.3-fold higher than thoseobtainedwith theNichols assayand6-foldhigher than those obtained with the DSL assay (10). Therefore, in centers where Immulite is thestandardmethod, the likelihoodthatapatient with normal IGF-I will have a discrepant unsuppressed GH after glucose is higher than in centers using other methods. To provide clinical advice, the authors propose that a cutoff GH level of 1 g/liter be used with the Immulite method and 0.5 g/liter with the Nichols method; this allowed the authors to identify 95% of patients with active disease and 78–80% of patients in remission.

Assessment of the Magnitude of Growth Hormone Hypersecretion in Active Acromegaly: Reliability of Different Sampling Models

The pulsatility of GH secretion in acromegaly poses difficulty in ascertaining true daily GH milieu in patients with this disease. Intensive GH sampling [every 10-20 (Q10-20) min for 24 h] is not practical in clinical practice. Our objective was to ascertain reliability of abbreviated sampling protocols to reflect true 24-h mean GH concentrations in patients with acromegaly. An analysis of previously obtained plasma GH profiles was performed. The analysis was performed at the General Clinical Research Center at the University of Michigan. A total of 115 GH profiles obtained in 94 patients with active acromegaly were examined. Frequent blood sampling, i.e. Q10-20 min for 24 h, was performed. Concordance of 24-h mean GH concentrations derived from Q10- to 20-min samplings with abbreviated GH sampling schedules was performed. The study was planned after data collection. All abbreviated schedules of GH sampling correlated well with the true 24-h plasma GH means (i.e. Q10- to 20-min sampling) (R = 0.93-0.98; P < 0.0001 for all). In the GH range more than 20 microg/liter, only 5 and 9-h means had R values more than 0.9. Single GH concentrations less than 1 microg/liter had a positive predictive value of only 0.29, and those with less than 2.5 microg/liter had a positive predictive value of 0.67 vs. their corresponding 24-h mean GH values of the same magnitude. The intensity of GH sampling in patients with acromegaly may vary depending on the nature of the required information. Investigators and clinicians should be aware of the limitations of the abbreviated GH sampling protocols in acromegaly.

Growth hormone deficiency in the adult

Growth hormone deficiency (GHD) in adults may be of either adult or childhood onset and may occur as isolated GHD or as multiple hormone deficiencies. Adult-onset GHD (AoGHD) usually results from damage to the pituitary gland or hypothalamus. GH is frequently undetectable in normal subjects and thus GHD cannot be distinguished from the normal state using a single random GH measurement. In general, a stimulation test is required to recognize GHD. Insulin tolerance test (ITT) has been considered the gold standard by the most important scientific societies, although alternative tests, in particular GHRH plus arginine have been proposed as valuable alternative to ITT. The clinical syndrome associated with AoGHD is characterized by a wide array of symptoms and important chronic complications, such as cardiovascular complications, which may be responsible for an increased mortality. The rationale for GH replacement in adults GHD patients is justified by the beneficial effects on some clinical end-points, such as quality of life (QoL) and cardiovascular risk factors, whereas the effects on mortality risk are still controversial. Over the recent years, guidelines on the use of rhGH as a substitution treatment in adult hypopituitarism have been issued by international (Growth hormone research society-GRS, Endocrine Society) and relevant national (National Institute of Clinical Excellence-UK, NICE) institutions. The aim of the paper is to review and discuss these guidelines.

Effects of gastric bypass on the GH/IGF-I axis in severe obesity – and a comparison with GH deficiency

Overfeeding suppresses GH secretion and makes evaluation of a suspected GH deficiency (GHD) difficult. In normal weight subjects, gender is known to influence GH concentrations, which is most apparent in the ambulatory, morning-fasted state. In this study, we examined the GH/IGF-I axis in obese men and women and the effect of surgically induced weight loss. Sixty-three subjects (body mass index (BMI) 45 +/- 6 kg/m2; 54 women, 9 men) were studied prior to, and 6 and 12 months following Roux-en-Y gastric bypass (RYGBP) surgery. Fifty-four patients with classic GHD (BMI 27 +/- 6 kg/m2; 35 men, 19 women) were included for comparison. Hormones were analysed in fasting morning serum samples. RYGBP resulted in a decreased BMI to 35 +/- kg/m2 at 6 months and 32 +/- 6 kg/m2 at 12 months. GH and IGF-I increased at 6 months in the women and at 12 months in both sexes by > or = 300 and 11% respectively. Prior to RYGBP, GH concentrations were low in the obese men and similar to those of GHD men (mean 0.09 mU/l). Obese women had tenfold higher values than obese men and sevenfold higher than GHD women. IGF-I levels were in the low reference range in the obese and below -2 S.D. for age in 13%. Surgically induced weight loss partially restores GH secretion. Despite a marked suppression of GH values, a gender influence is maintained in severe obesity. In obese women, single morning GH and IGF-I values seem sufficient to exclude a suspicion of classic GHD.

Effect of GH and IGF-I treatment on reproduction, growth, and plasma hormone concentrations in domestic nutria ( Myocastor coypus)

The role of GH and IGF-I in the control of reproduction, growth, and hormone secretion in domestic nutria was examined. In the first series of experiments, we studied the effects of single and multiple (daily for 20 days) injections of recombinant hGH (15 μg/animal) on plasma triiodothyronine (T3), thyroxine (T4), and progesterone (P) concentrations, as well as on the duration of pregnancy (time between start of mating and birth of pups), number of pups born, and body weight of adult females and their newborn pups. In the second series of experiments, the effects of single and multiple (daily for 28 days) injections of recombinant hIGF-I (1 μg/animal) on plasma IGF-I, IGFBP-3, T3, T4 concentrations, the duration of pregnancy, and number of offspring delivered were assessed. It was found that either single or multiple GH treatment resulted in significant increase in plasma T3, T4, but not P concentration. Furthermore, it significantly increased the body weight of adults and newborn pups. No influence of GH on the duration of pregnancy and the number of offspring was observed. IGF-I treatment caused an increase in plasma IGF-I concentration, a reduction in plasma IGFBP-3, T3, and T4 concentrations, and a shorter duration of pregnancy but did not alter the number of pups delivered. Our observations suggest that GH and IGF-I may be involved in the control of hormone secretion, growth, and reproduction in domestic nutria. Reproductive processes are controlled by IGF-I rather than by GH, whilst GH may be involved in the stimulation of prenatal and postnatal growth. The differential effects of these substances on thyroid hormones and reproductive parameters suggest that the actions of GH on these processes are probably not mediated by IGF-I.

Dampening of Cytosolic Ca2+ Oscillations on Propagation to Nucleus

Ca(2+) signals may regulate gene expression. The increase of the cytosolic Ca(2+) concentration ([Ca(2+)](c)) promotes activation and/or nuclear import of some transcription factors, but others require the increase of the nuclear Ca(2+) concentration ([Ca(2+)](N)) for activation. Whether the nuclear envelope may act as a diffusion barrier for propagation of [Ca(2+)](c) signals remains controversial. We have studied the spreading of Ca(2+) from the cytosol to the nucleus by comparing the cytosolic and the nuclear Ca(2+) signals reported by targeted aequorins in adrenal chromaffin, PC12, and GH(3) pituitary cells. Strong stimulation of either Ca(2+) entry (by depolarization with high K(+) or acethylcholine) or Ca(2+) release from the intracellular Ca(2+) stores (by stimulation with caffeine, UTP, bradykinin, or thyrotropin-releasing hormone (TRH)) produced similar Ca(2+) signals in cytosol and nucleus. In contrast, both spontaneous and TRH-stimulated oscillations of cytosolic Ca(2+) in single GH(3) cells were considerably dampened during propagation to the nucleus. These results are consistent with the existence of a kinetic barrier that filters high frequency physiological [Ca(2+)](c) oscillations without disturbing sustained [Ca(2+)](c) increases. Thus, encoding of the Ca(2+) signal may allow differential control of Ca(2+)-dependent mechanisms located at either the cytosol or the nucleus.

Gender and age influence the relationship between serum GH and IGF‐I in patients with acromegaly

In patients with acromegaly serum IGF-I is increasingly used as a marker of disease activity. As a result, the relationship between serum GH and IGF-I is of profound interest. Healthy females secrete three times more GH than males but have broadly similar serum IGF-I levels, and women with GH deficiency require 30-50% more exogenous GH to maintain the same serum IGF-I as GH-deficient men. In a selected cohort of patients with active acromegaly, studied off medical therapy using a single fasting serum GH and IGF-I measurement, we have reported previously that, for a given GH level, women have significantly lower circulating IGF-I. To evaluate the influence of age and gender on the relationship between serum GH and IGF-I in an unselected cohort of patients with acromegaly independent of disease control and medical therapy. Sixty (34 male) unselected patients with acromegaly (median age 51 years (range 24-81 years) attending a colonoscopy screening programme were studied. Forty-five had previously received pituitary radiotherapy. Patients had varying degrees of disease control and received medical therapy where appropriate. Mean serum GH was calculated from an eight-point day profile (n = 45) and values obtained during a 75-g oral glucose tolerance test (n = 15). Serum IGF-I, IGFBP-3 and acid-labile subunit were measured and the dependency of these factors on covariates such as log10 mean serum GH, sex, age and prior radiotherapy was assessed using regression techniques. The median calculated GH value was 4.7 mU/l (range 1-104). A significant linear association was observed between serum IGF-I and log10 mean serum GH for the cohort (R = 0.5, P < 0.0001). After simultaneous adjustment of the above covariates a significant difference in the relationship between mean serum GH and IGF-I was observed for males and females. On average, women had serum IGF-I levels 11.44 nmol/l lower than men with the same mean serum GH (P = 0.03, 95% CI 1.33-21.4 nmol/l). Age significantly influenced the relationship and for a given serum GH, IGF-I was estimated to fall by 0.37 nmol/l per year (P = 0.04, 95% CI 0.015-0.72). In keeping with previous observations of relative GH resistance in normal and GH-deficient females we have observed lower serum IGF-I levels for equivalent mean serum GH levels in females patients with acromegaly. This gender-dependent difference is independent of disease activity and the use of concomitant medical therapy. Additionally, we have demonstrated that for a given serum GH level, age significantly influences IGF-I concentrations in patients with acromegaly. These data have important implications for the use of serum IGF-I and GH as markers of disease activity in acromegaly.

Predictors of the outcome of surgical treatment in acromegaly and the value of the mean growth hormone day curve in assessing postoperative disease activity.

Acromegaly is associated with increased morbidity and mortality unless serum GH levels are persistently less than 5 mU/L ( approximately 2 ng/mL) after treatment. Transsphenoidal surgical resection is the best available treatment for restoring GH to such "safe" levels; however, criteria for the assessment of the response to treatment are not uniform. To determine the clinically most useful method of assessing disease activity postoperatively and identify predictors of a favorable response to surgical treatment, we have analyzed 67 patients with acromegaly who underwent transsphenoidal surgery between 1993 and 1998. We used three different definitions of a satisfactory or safe response: 1) a postoperative mean GH less than 5 mU/L obtained from averaging five serum GH values obtained throughout one day; 2) a random single GH less than 5 mU/L; or 3) a serum insulin-like growth factor I (IGF-I) level within the normal range. Relying on a single GH measurement alone, 9 of the 23 patients with a single postoperative mean GH level less than 5 mU/L obtained at least one GH value of more than 5 mU/L (false positive rate, 28%) and 8 of the patients with a postoperative mean GH value of more than 5 mU/L obtained a single GH value of less than 5 mU/L (false negative rate, 15%). Postoperatively, a significant increase in the fluctuation of random GH values around the mean was observed in patients who were rendered safe (coefficient of variation, from 26 +/- 2% to 53 +/- 6%; P < 0.001) compared with patients with persistence of inadequately controlled disease. However, 13% (3 of 23) of patients with mean postoperative GH levels of less than 5 mU/L had elevated serum IGF-I levels postoperatively, and 17% (8 of 44) of patients with mean serum GH levels more than 5 mU/L had postoperative IGF-I levels within the normal range. There was no difference in the rate of agreement between mean GH less than 5 mU/L and normalization of IGF-I in relation to the interval since operation when IGF-I levels were measured. Preoperative tumor size and pretreatment mean GH levels were the major determinants of the outcome of surgery, as patients who were rendered safe had significantly lower preoperative mean GH levels than patients who were not cured (median, 31 mU/L vs. 78.5 mU/L, P < 0.01). IGF-I levels were weakly correlated with tumor size and could not be used to predict the patients who would be rendered safe. Preoperative PRL levels were higher in patients who failed to achieve a surgical satisfactory outcome [498 mU/L (187-857) vs. 196 mU/L (136-315), P < 0.01]. In summary, although single random GH values and IGF-I values are both significantly correlated with mean GH levels, they should not be used as an alternative to averaging several GH values to assess disease activity, because of the pulsatile nature of GH secretion and the multiple factors that may influence serum IGF-I. Because significant discrepancies occur, particularly postoperatively, mean GH levels remain the more reliable indicator of surgical outcome and disease activity. As there is considerably more evidence relating long-term prognosis to serum GH levels than to IGF-I and discrepancies occur between GH levels and IGF-I, we suggest that mean serum GH levels and single IGF-I levels, measured early in the postoperative period, are currently the best biochemical guide to the adequacy of surgery and, hence, the need for further treatment.

The Human Growth Hormone Gene Cluster Locus Control Region Supports Position-independent Pituitary- and Placenta-specific Expression in the Transgenic Mouse

The human growth hormone (hGH) cluster contains five genes. The hGH-N gene is predominantly expressed in pituitary somatotropes, whereas the remaining four genes, the chorionic somatomammotropin genes (hCS-L, hCS-A, and hCS-B) and hGH-V, are expressed selectively in the placenta. In contrast, the mouse genome contains a single pituitary-specific GH gene and lacks any GH-related CS genes. Activation of the hGH transgene in the mouse is dependent on its linkage to a previously described locus control region (LCR) located -15 to -32 kilobases upstream of the hGH cluster. The sporadic, nonreproducible expression of hCS transgenes lacking the LCR suggests that they may be dependent on hGH LCR activity as well. To determine whether the hCS genes could be expressed with appropriate placental specificity, a series of five transgenic mouse lines carrying an 87-kilobase human genomic insert encompassing the majority of the hGH gene cluster and the entire contiguous LCR was established. All of the hGH cluster genes were appropriately expressed in each of these lines. High level expression of hGH was restricted to the pituitary and hCS to the labyrinthine layer of the placenta. The expression of the GH cluster genes in their respective tissues paralleled transgene copy numbers irrespective of the transgene insertion site in the host mouse genome. These studies have extended the utility of the transgenic mouse model for the analysis of the full spectrum of hGH gene cluster activation. Further, they support a role for the hGH LCR in placental hCS, as well as pituitary hGH gene activation, and expression.

Biochemical tests in the diagnosis of childhood growth hormone deficiency.

GH stimulation tests are widely used in the diagnosis of GH deficiency (GHD), although they are associated with a high false positive rate. We have examined, therefore, the performance of other tests of the GH axis [urinary GH excretion, serum insulin-like growth factor I(IGF-I), and IGF-binding protein-3 (IGFBP-3) levels] compared with GH stimulation tests in identifying children defined clinically as GH deficient. Group I comprised 60 children (mean age, 10.3 +/- 4.8 yr) whose diagnosis of GHD was based on a medical history indicative of pituitary dysfunction (n = 43) or on the typical phenotypic features and appropriate auxological characteristics of isolated GHD (n = 17). Group II comprised 110 short children (mean age, 9.8 +/- 4 yr) in whom GHD was not suspected, but needed exclusion. The best sensitivity for a single GH test was 85% at a peak GH cut-off level of 10 ng/mL, whereas the best specificity was 92% at 5 ng/mL. The sensitivities of IGF-I, IGFBP-3, and urinary GH, using a cut-off of -2 SD score were poor at 34%, 22%, and 25%, respectively, with specificities of 72%, 92%, and 76% respectively. Only 2 of 21 pubertal children in group I and none of the 27 subjects with radiation-induced GHD had an IGFBP-3 SD score less than -1.5. We devised a scoring system based on the positive predictive value of each test, incorporating data from the GH test and the IGF-I and IGFBP-3 levels. A specificity of 94% could be achieved with a score of 10 or more (maximum 17) (sensitivity 34%). The latter could not be improved above 81% with a score of 5 points or more (specificity, 69%). A high score was, therefore, highly indicative of GHD, but was achieved by few patients. A normal IGFBP-3 level, however, did not exclude GHD, particularly in patients with radiation-induced GHD and those in puberty. A GH test with a peak level more than 10 ng/mL was the most useful single investigation to exclude a diagnosis of GHD.

Lithium‐induced decrease in spontaneous Ca2+ oscillations in single GH3 rat pituitary cells

1. Measurement of [Ca2+]i in single rat pituitary GH3 cells by dynamic single cell imaging techniques demonstrated that under basal conditions there is a large variation in the temporal pattern of [Ca2+]i signalling between individual cells ranging from high frequency asynchronous oscillations to quiescence. 2. We have reported previously that treatment of GH3 cells with 1 mM Li+ (a concentration used therapeutically in the treatment of manic depression) for 7 days reduces basal and thyrotrophin-releasing hormone (TRH)-stimulated levels of mass inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]. In the present study, we show that this is associated with a reduction in the number of cells exhibiting basal Ca2+ oscillations over a sampling period of 60 s, whereas the maximum amplitude of oscillations is unaffected. 3. The pattern of [Ca2+]i responses to the agonist TRH varied considerably between individual cells, making quantitation of the responses difficult; however, data obtained from measurements made on a population of cells showed that increases in peak [Ca2+]i induced by high concentrations of TRH were reduced in cells treated with 1 mM Li+ for 7 days relative to control cells. 4. The sensitivity of the phosphoinositide pathway to [Ca2+]i was investigated by loading GH3 cells with BAPTA/AM at a concentration sufficient to lower 'basal' [Ca2+]i in a population of cells and to inhibit agonist-stimulated increases in [Ca2+]i. Under these conditions, basal and TRH-stimulated mass Ins(1,4,5)P3 levels were unaffected. 5. These results demonstrate that a 7-day Li+ treatment leads to an alteration in Ca2+ signalling, in particular by reducing the number of cells exhibiting high frequency Ca2+ oscillations under basal conditions. The significance of these results to the clinical effectiveness of Li+ in the treatment of manic depression is discussed.