Genomewide meta-analysis identifies loci associated with IGF-I and IGFBP-3 levels with impact on age-related traits.

The growth hormone/insulin-like growth factor (IGF) axis can be manipulated in animal models to promote longevity, and IGF-related proteins including IGF-I and IGF-binding protein-3 (IGFBP-3) have also been implicated in risk of human diseases including cardiovascular diseases, diabetes, and cancer. Through genomewide association study of up to 30 884 adults of European ancestry from 21 studies, we confirmed and extended the list of previously identified loci associated with circulating IGF-I and IGFBP-3 concentrations (IGF1, IGFBP3, GCKR, TNS3, GHSR, FOXO3, ASXL2, NUBP2/IGFALS, SORCS2, and CELSR2). Significant sex interactions, which were characterized by different genotype-phenotype associations between men and women, were found only for associations of IGFBP-3 concentrations with SNPs at the loci IGFBP3 and SORCS2. Analyses of SNPs, gene expression, and protein levels suggested that interplay between IGFBP3 and genes within the NUBP2 locus (IGFALS and HAGH) may affect circulating IGF-I and IGFBP-3 concentrations. The IGF-I-decreasing allele of SNP rs934073, which is an eQTL of ASXL2, was associated with lower adiposity and higher likelihood of survival beyond 90 years. The known longevity-associated variant rs2153960 (FOXO3) was observed to be a genomewide significant SNP for IGF-I concentrations. Bioinformatics analysis suggested enrichment of putative regulatory elements among these IGF-I- and IGFBP-3-associated loci, particularly of rs646776 at CELSR2. In conclusion, this study identified several loci associated with circulating IGF-I and IGFBP-3 concentrations and provides clues to the potential role of the IGF axis in mediating effects of known (FOXO3) and novel (ASXL2) longevity-associated loci.

Effects of type I interferons on IGF-mediated autocrine/paracrine growth of human neuroendocrine tumor cells.

We recently demonstrated that interferon (IFN)-beta has a more potent antitumor activity than IFN-alpha in BON cells, a neuroendocrine tumor (NET) cell line. The present study showed the role of type I IFNs in the modulation of the insulin-like growth factor (IGF) system in NETs. BON cells expressed IGF-I, IGF-II, IGF-I receptor, and insulin receptor mRNA. In addition, IGF-I and IGF-II stimulated the proliferation of BON cells and induced an inhibition of DNA fragmentation (apoptosis). As evaluated by quantitative RT-PCR, treatment with IFN-alpha (100 IU/ml) or IFN-beta (100 IU/ml) inhibited the expression of IGF-II mRNA (-42% and -65%, respectively, both P < 0.001), whereas IGF-I receptor mRNA was significantly upregulated by IFN-alpha (+28%, P < 0.001) and downregulated by IFN-beta (-47%, P < 0.001). Immunoreactive IGF-II concentration decreased in the conditioned medium during IFN-alpha (-16%, P < 0.05) and IFN-beta (-69%, P < 0.001) treatment. Additionally, IGF-I receptor bioactivity was reduced (-54%) after IFN-beta treatment. Scatchard analysis of (125)I-labeled IGF-I binding to cell membrane of BON cells revealed a dramatic suppression of maximum binding capacity only in the presence of IFN-beta. Finally, the proapoptotic activity of IFN-beta was partially counteracted by the coadministration of IGF-I and IGF-II (both at 50 nM). In conclusion, these data demonstrate that the IGF system has an important role in autocrine/paracrine growth of BON cells. The more potent antitumor activity of IFN-beta compared with IFN-alpha could be explained by several effects on this system: 1) both IFNs inhibit the transcription of IGF-II, but the suppression is significantly higher after IFN-beta than IFN-alpha and 2) only IFN-beta inhibits the expression of IGF-I receptor.

Insulin-like growth factors, their binding proteins, and prostate cancer risk: analysis of individual patient data from 12 prospective studies.

BACKGROUND: Some, but not all, published results have shown an association between circulating blood levels of some insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) and the subsequent risk for prostate cancer. PURPOSE: To assess the association between levels of IGFs and IGFBPs and the subsequent risk for prostate cancer. DATA SOURCES: Studies identified in PubMed, Web of Science, and CancerLit. STUDY SELECTION: The principal investigators of all studies that published data on circulating concentrations of sex steroids, IGFs, or IGFBPs and prostate cancer risk using prospectively collected blood samples were invited to collaborate. DATA EXTRACTION: Investigators provided individual participant data on circulating concentrations of IGF-I, IGF-II, IGFBP-II, and IGFBP-III and participant characteristics to a central data set in Oxford, United Kingdom. DATA SYNTHESIS: The study included data on 3700 men with prostate cancer and 5200 control participants. On average, case patients were 61.5 years of age at blood collection and received a diagnosis of prostate cancer 5 years after blood collection. The greater the serum IGF-I concentration, the greater the subsequent risk for prostate cancer (odds ratio [OR] in the highest vs. lowest quintile, 1.38 [95% CI, 1.19 to 1.60]; P < 0.001 for trend). Neither IGF-II nor IGFBP-II concentrations were associated with prostate cancer risk, but statistical power was limited. Insulin-like growth factor I and IGFBP-III were correlated (r = 0.58), and although IGFBP-III concentration seemed to be associated with prostate cancer risk, this was secondary to its association with IGF-I levels. Insulin-like growth factor I concentrations seemed to be more positively associated with low-grade than high-grade disease; otherwise, the association between IGFs and IGFBPs and prostate cancer risk had no statistically significant heterogeneity related to stage or grade of disease, time between blood collection and diagnosis, age and year of diagnosis, prostate-specific antigen level at recruitment, body mass index, smoking, or alcohol intake. LIMITATIONS: Insulin-like growth factor concentrations were measured in only 1 sample for each participant, and the laboratory methods to measure IGFs differed in each study. Not all patients had disease stage or grade information, and the diagnosis of prostate cancer may differ among the studies. CONCLUSION: High circulating IGF-I concentrations are associated with a moderately increased risk for prostate cancer.

Long-term efficacy and safety of combined treatment of somatostatin analogs and pegvisomant in acromegaly.

BACKGROUND: We previously reported the efficacy of a combined treatment of active acromegaly with both long-acting somatostatin analogs (SSA) and pegvisomant (PEG-V). OBJECTIVE: Our objective was to assess long-term efficacy and safety in a larger group of acromegalic patients after a period of 138 (35-149) wk [median (range)]. DESIGN: PEG-V was added to high-dose SSA treatment in 32 subjects (13 females) who had not shown a normalization in serum IGF-I concentrations during SSA monotherapy. PEG-V dosage was increased until IGF-I concentration normalized. The maximal dose was 80 mg twice weekly. RESULTS: After dose finding, IGF-I remained within the normal range in all subjects with PEG-V administered once (n = 24) or twice (n = 8) weekly, on a total weekly dose of 60 (40-160) mg. Baseline IGF-I levels were positively correlated with the required dosage of PEG-V (r = 0.48; P = 0.006). PEG-V-dependent liver enzyme disturbances were observed in 11 (6 diabetic) subjects, of which symptomatic gallstones explained two cases. These liver enzyme disturbances were transient in all subjects without discontinuation or dose adaptation of PEG-V. In our series, diabetic patients had a 5.1 times (odds ratio) (confidence interval, 1.02-25.54; P < 0.05) higher risk for developing liver enzyme disturbances. These liver enzyme disturbances seemed to occur earlier. Pituitary adenoma size decreased in four patients. No increase in tumor size was observed in any of the patients. CONCLUSION: Long-term combined treatment with long-acting SSA and (twice) weekly PEG-V for active acromegaly seems to be effective and safe. Patients with acromegaly and diabetes seem to have a higher risk of developing transient liver enzyme disturbances.

Unacylated ghrelin acts as a potent insulin secretagogue in glucose-stimulated conditions.

Acylated and unacylated ghrelin (AG and UAG) are gut hormones that exert pleiotropic actions, including regulation of insulin secretion and glucose metabolism. In this study, we investigated whether AG and UAG differentially regulate portal and systemic insulin levels after a glucose load. We studied the effects of the administration of AG (30 nmol/kg), UAG (3 and 30 nmol/kg), the ghrelin receptor antagonist [D-Lys(3)]GHRP-6 (1 micromol/kg), or various combinations of these compounds on portal and systemic levels of glucose and insulin after an intravenous glucose tolerance test (IVGTT, d-glucose 1 g/kg) in anesthetized fasted Wistar rats. UAG administration potently and dose-dependently enhanced the rise of insulin concentration induced by IVGTT in the portal and, to a lesser extent, the systemic circulation. This UAG-induced effect was completely blocked by the coadministration of exogenous AG at equimolar concentrations. Similarly to UAG, [D-Lys(3)]GHRP-6, alone or in combination with AG and UAG, strongly enhanced the portal insulin response to IVGTT, whereas exogenous AG alone did not exert any further effect. Our data demonstrate that, in glucose-stimulated conditions, exogenous UAG acts as a potent insulin secretagogue, whereas endogenous AG exerts a maximal tonic inhibition on glucose-induced insulin release.

Unacylated ghrelin is active on the INS-1E rat insulinoma cell line independently of the growth hormone secretagogue receptor type 1a and the corticotropin releasing factor 2 receptor.

Both unacylated ghrelin (UAG) and acylated ghrelin (AG) exert metabolic effects. To investigate the interactions between AG and UAG on ghrelin receptors we evaluated the effects of AG and UAG on INS-1E rat insulinoma cells, using insulin secretion after 30min static incubation as a read-out. A possible involvement of the growth hormone secretagogue receptor type 1a (GHS-R1a) or the corticotropin-releasing factor 2 (CRF2) receptor (CRF2R), as a putative receptor for UAG, was also studied determining their mRNA expression and the functional effects of receptor antagonists on insulin release. Both UAG and AG stimulated insulin release dose-dependently in the nanomolar range. The AG-induced insulin output was antagonized by two GHS-R1a antagonists ([d-Lys(3)]GHRP-6 and BIM28163), which did not block UAG actions. These effects occurred in the presence of low levels of GHS-R1a mRNA. Neither CRF2R expression nor effects of the CRF2R antagonist (astressin(2)B) on insulin output were observed. In conclusion, we provide a sensitive and reproducible assay for specific effects of UAG, which in this study is responsible for insulin release by INS-1E cells. Our data support the existence of a specific receptor for UAG, other than the CRF2R and GHS-R1a. The stimulatory effect on insulin secretion by AG in this cell line is mediated by the GHS-R1a.

Limited predictive value of an acute test with subcutaneous octreotide for long-term IGF-I normalization with Sandostatin LAR in acromegaly.

OBJECTIVES: To study whether the growth hormone (GH) response after the subcutaneous administration 50 microg of octreotide (acute octreotide test) has any predictive value for long-term IGF-I normalization with Sandostatin LAR. DESIGN: Twenty four therapy-naive patients with active acromegaly were studied. RESULTS: > 75% GH decrease in the acute octreotide test predicted long-term IGF-I normalization with Sandostatin LAR in 8/11 (73%) of patients. 3/13 (23%) patients with < 75% GH decrease in the acute octreotide test were long-term biochemically controlled with Sandostatin LAR. Using the > 75% GH reduction criterion, the sensitivity and specificity of this test for predicting long-term normalization of serum IGF-I with Sandostatin LAR treatment were 73% and 77%, respectively (positive and negative predictive values: 73% and 77%, respectively). 6/8 (75%) patients with GH suppression to levels < 1.1 microg/l and 9/16 (56%) patients with GH suppression to levels < 2 microg/l in the acute octreotide test showed normalization of serum IGF-I with long-term Sandostatin LAR treatment. The sensitivity and specificity of GH suppression < 1.1 microg/l for predicting of the long-term normalization of serum IGF-I with Sandostatin LAR therapy were 55% and 85%, respectively (positive and negative predictive values: 75% and 69%, respectively). The sensitivity and specificity of GH suppression < 2 microg/l for predicting of the long-term normalization of serum IGF-I with Sandostatin LAR therapy were 82% and 46%, respectively (positive and negative predictive values: 56% and 75%, respectively). CONCLUSION: The acute octreotide is not recommended for clinical decision making with regard to long-term treatment using the long-acting somatostatin analog Sandostatin LAR in acromegaly.

The 'bio-assay' quality of life might be a better marker of disease activity in acromegalic patients than serum total IGF-I concentrations.

OBJECTIVES: To investigate the quality of life (QoL) in acromegalic patients in relation to biochemical parameters. DESIGN AND METHODS: Single-center, open label study in 14 acromegalic patients (eight woman and six men, age 33-77 years), with normal serum IGF-I levels during long-term treatment with monthly injections of 20 mg of long-acting octreotide. We investigated which biochemical parameter might reflect optimal QoL, using the SF-36 questionnaire. RESULTS: We observed that six patients had a low QoL score at baseline in the same range as observed in cancer patients. The other eight patients had a normal QoL. GH, IGF-I nor free IGF-I could discriminate these two subgroups at baseline. After skipping one monthly injection, all six subjects with the low QoL escaped in their free IGF-I concentrations. Also total IGF-I concentrations escaped in four of these six. In the subjects with normal QoL, free IGF-I levels remained normal in all, while total IGF-I levels only escaped in one. CONCLUSIONS: This study tells us that the currently used biochemical criteria for disease control in acromegaly might be sufficient in assessing long-term mortality and morbidity, but they are insufficient in addressing the most important parameter from the patient's perspective--QoL.

Ghrelin stimulates, whereas des-octanoyl ghrelin inhibits, glucose output by primary hepatocytes.

Ghrelin exerts various metabolic activities, including regulation of glucose levels in humans. To verify whether the glucose response to ghrelin reflects a modulation of an insulin-independent hepatic phenomenon, we studied glucose output by primary porcine hepatocytes in suspension culture, after incubation with acylated ghrelin (AG), unacylated ghrelin (UAG), and hexarelin (HEX). AG induced glucose output dose dependently after 20 min of incubation (P < 0.001), whereas HEX, a GH secretagogue receptor type 1a (GHS-R1a) agonist, had no effect. UAG inhibited glucose release also dose dependently and after 20 min (P < 0.001). Moreover, UAG completely reversed AG-induced glucose output (P < 0.01). Using real-time PCR, GHS-R1a gene expression was undetectable in all the hepatocyte preparations studied. The lack of efficacy of HEX, the efficacy of UAG, and the absence of GHS-R1a expression indicate the involvement of a yet uncharacterized ghrelin receptor type. In conclusion, glucose output by primary hepatocytes is time- and dose-dependently stimulated by AG and inhibited by UAG. Moreover, UAG counteracts the stimulatory effect of AG on glucose release. These actions might be mediated by a different receptor than GHS-R1a, and apparently, we must consider AG and UAG as separate hormones that can modify each other's actions on glucose handling, at least in the liver.

Association of the ER22/23EK polymorphism in the glucocorticoid receptor gene with survival and C-reactive protein levels in elderly men.

PURPOSE: We recently demonstrated that a polymorphism in codons 22 and 23 of the glucocorticoid receptor gene is associated with relative glucocorticoid resistance, greater insulin sensitivity, and lower total and low-density lipoprotein cholesterol levels. In the present study, we investigated whether the ER22/23EK polymorphism is associated with survival, cholesterol levels, and two predictors of mortality: serum C-reactive protein and interleukin 6 levels. METHODS: We studied 402 men (mean [+/- SD] age, 77.8 +/- 3.6 years). C-reactive protein was measured by a highly sensitive method using a latex-enhanced immunoephelometric assay. Interleukin 6 was determined by a commercially available immulite assay. RESULTS: After a follow-up of 4 years, 73 (19%) of 381 noncarriers died, while none of the 21 ER22/23EK carriers had died (P = 0.03). C-reactive protein levels were about 50% lower in ER22/23EK carriers (P = 0.01). There were no differences in interleukin 6 levels. CONCLUSION: Carriers of the ER22/23EK polymorphism have better survival than noncarriers, as well as lower C-reactive protein levels.

Central ghrelin production does not substantially contribute to systemic ghrelin concentrations: a study in two subjects with active acromegaly.

INTRODUCTION: In an animal model of acromegaly (PEPCK-hGH transgenic mice), low systemic levels of ghrelin have been observed compared with normal mice. We hypothesized that systemic circulating ghrelin levels are also decreased in humans with active acromegaly and that the contribution of central ghrelin production to systemic ghrelin levels is minimal. OBJECTIVES: The aim of the present study was to investigate, in two subjects with active acromegaly, whether there are differences between systemic ghrelin levels and ghrelin concentrations in the petrosal sinus. DESIGN: We measured systemic and central ghrelin levels in these two acromegalic patients by bilateral simultaneous inferior petrosal sinus sampling. Central and systemic blood samples were drawn before and 1, 5, 10, 15 and 20 min after stimulation with GH-releasing hormone (GHRH). Ghrelin was measured with a commercially available radioimmunoassay. RESULTS: In one acromegalic subject, the baseline systemic and central ghrelin levels were within the same range as in two non-acromegalic obese subjects. No gradient could be observed between central and systemic ghrelin concentrations. Stimulation with GHRH did not change the ghrelin concentrations in this patient. In the other acromegalic subject, the systemic ghrelin levels were also in the same range as in two non-acromegalic obese subjects. However, in this subject, baseline ghrelin concentrations in the right inferior petrosal vein were considerably lower than the systemic ghrelin concentrations, indicating a peripheral over central gradient. Administration of GHRH induced a significant rise in central ghrelin concentrations in the right inferior petrosal vein. Ghrelin levels in the left inferior petrosal vein and systemic ghrelin levels were in the normal range and GHRH stimulation did not change these concentrations. CONCLUSIONS: The absence of a central over peripheral ghrelin gradient in these two acromegalics indicated that circulating ghrelin is mainly produced peripherally. Circulating systemic ghrelin levels were not decreased in these two subjects with active acromegaly.

Ghrelin drives GH secretion during fasting in man.

OBJECTIVES: In humans, fasting leads to elevated serum GH concentrations. Traditionally, changes in hypothalamic GH-releasing hormone and somatostatin release are considered as the main mechanisms that induce this elevated GH secretion during fasting. Ghrelin is an endogenous ligand of the GH secretagogue receptor and is synthesized in the stomach. As ghrelin administration in man stimulates GH release, while serum ghrelin concentrations are elevated during fasting in man, this increase in ghrelin levels might be another mechanism whereby fasting results in stimulation of GH release. DESIGN AND SUBJECTS: In ten healthy non-obese males we performed a double-blind placebo-controlled crossover study comparing fasting with and fasting without GH receptor blockade. GH, ghrelin, insulin, glucose and free fatty acids were assessed. RESULTS: While ghrelin levels do not vary considerably in the fed state, fasting rapidly induced a diurnal rhythm in ghrelin concentrations. These changes in serum ghrelin concentrations during fasting were followed by similar, profound changes in serum GH levels. The rapid development of a diurnal ghrelin rhythm could not be explained by changes in insulin, glucose, or free fatty acid levels. Compared with fasting without pegvisomant, fasting with pegvisomant did not change the ghrelin rhythm. CONCLUSIONS: These data indicate that ghrelin is the main driving force behind the enhanced GH secretion during fasting.