Arrestin-independent constitutive endocytosis of GPR125/ADGRA3.

The orphan receptor GPR125 (ADGRA3) belongs to subgroup III of the adhesion G protein-coupled receptor (aGPCR) family. aGPCRs, also known as class B2 GPCRs, share basic structural and functional properties with other GPCRs. Many of them couple to G proteins and activate G protein-dependent and -independent signaling pathways, but little is known about aGPCR internalization and ß-arrestin recruitment. GPR125 was originally described as a spermatogonial stem cell marker and studied for its role in Wnt signaling and cell polarity. Here, using cell-based assays and confocal microscopy, we show that GPR125 is expressed on the cell surface and undergoes constitutive endocytosis in a ß-arrestin-independent, but clathrin-dependent manner, as indicated by colocalization with transferrin receptor 1, an early endosome marker. These data support that the constitutive internalization of GPR125 contributes to its biological functions by controlling receptor surface expression and accessibility for ligands. Our study sheds light on a new property of aGPCRs, namely internalization; a property described to be important for signal propagation, signal termination, and desensitization of class A (rhodopsin-like) and B1 (VIP/secretin) GPCRs.

Aldosterone to Renin Ratio as Screening Tool in Primary Aldosteronism.

Primary aldosteronism (PA) is a severe and often underdiagnosed form of secondary hypertension. Determining the aldosterone to renin ratio (ARR) in hypertensive patients has been shown to be a valuable screening test for identification of patients suffering from PA. Since the introduction of a more widespread ARR screening the number of PA patients significantly increased worldwide. Interpretation of ARR might be challenging: Several factors from posture to interfering drugs affect the ARR and need to be taken into account when collecting samples. In addition, the wide variety of available assay methods and lack of well-established cut-offs present a challenge to the clinician. This review discusses the usefulness and possible difficulties of ARR screening.

Disruption of the GH Receptor Gene in Adult Mice Increases Maximal Lifespan in Females.

GH and IGF-1 are important for a variety of physiological processes including growth, development, and aging. Mice with reduced levels of GH and IGF-1 have been shown to live longer than wild-type controls. Our laboratory has previously found that mice with a GH receptor gene knockout (GHRKO) from conception exhibit low rates of cancer, resistance to diet-induced diabetes, and extension of lifespan. The GHRKO mouse as well as other mouse lines with reduced GH action display low IGF-1 levels, smaller body size, increased adiposity, and increased longevity. To date, nearly all of these mouse strains carry germline mutations. Importantly, the effect of a long-term suppression of the GH/IGF-1 axis during adulthood, as would be considered for human therapeutic purposes, has not been tested. The goal of this study was to determine whether temporally controlled Ghr gene deletion in adult mice would affect metabolism and longevity. Thus, we produced adult-onset GHRKO (aGHRKO) mice by disrupting the Ghr gene at 6 weeks of age. We found that aGHRKO mice replicate many of the beneficial effects observed in long-lived GHRKO mice. For example, aGHRKO mice, like GHRKO animals, displayed retarded growth and high adiposity with improved insulin sensitivity. Importantly, female aGHRKO animals showed an increase in their maximal lifespan, whereas the lifespan of male aGHRKO mice was not different from controls.

Low-carbohydrate, high-fat diets have sex-specific effects on bone health in rats.

PURPOSE: Studies in humans suggest that consumption of low-carbohydrate, high-fat diets (LC-HF) could be detrimental for growth and bone health. In young male rats, LC-HF diets negatively affect bone health by impairing the growth hormone/insulin-like growth factor axis (GH/IGF axis), while the effects in female rats remain unknown. Therefore, we investigated whether sex-specific effects of LC-HF diets on bone health exist. METHODS: Twelve-week-old male and female Wistar rats were isoenergetically pair-fed either a control diet (CD), "Atkins-style" protein-matched diet (LC-HF-1), or ketogenic low-protein diet (LC-HF-2) for 4 weeks. In females, microcomputed tomography and histomorphometry analyses were performed on the distal femur. Sex hormones were analysed with liquid chromatography-tandem mass spectrometry, and endocrine parameters including GH and IGF-I were measured by immunoassay. RESULTS: Trabecular bone volume, serum IGF-I and the bone formation marker P1NP were lower in male rats fed both LC-HF diets versus CD. LC-HF diets did not impair bone health in female rats, with no change in trabecular or cortical bone volume nor in serum markers of bone turnover between CD versus both LC-HF diet groups. Pituitary GH secretion was lower in female rats fed LC-HF diet, with no difference in circulating IGF-I. Circulating sex hormone concentrations remained unchanged in male and female rats fed LC-HF diets. CONCLUSION: A 4-week consumption of LC-HF diets has sex-specific effects on bone health-with no effects in adult female rats yet negative effects in adult male rats. This response seems to be driven by a sex-specific effect of LC-HF diets on the GH/IGF system.

Clinical validation for the aldosterone-to-renin ratio and aldosterone suppression testing using simultaneous fully automated chemiluminescence immunoassays.

BACKGROUND: As larger numbers of hypertensive patients are screened for primary aldosteronism with the aldosterone-to-renin ratio (ARR), automated analyzers present a practical solution for many laboratories. We report the method-specific ARR cutoff determined with direct, automated chemiluminescence immunoassays allowing the simultaneous measurement of plasma aldosterone concentrations (PACs) and plasma renin concentrations (PRCs). METHODS: Method comparisons to commonly employed assays and tandem mass spectrometry were undertaken. Patients were previously diagnosed based on the local ARR cutoff of 1.2 (ng/dl)/(µIU/ml) in samples collected in upright seated position. Lack of aldosterone suppression in response to salt load to less than 5 ng/dl confirmed primary aldosteronism. For the new assays, the optimal ARR cutoff was established in 152 patients with essential hypertension, 93 with primary aldosteronism and 147 normotensive patients. Aldosterone suppression was assessed in 73 essential hypertensive and 46 primary aldosteronism patients. RESULTS: PAC and PRC were significantly correlated to values determined with currently available methods (P < 0.001). In patients with primary aldosteronism, patients with essential hypertension and controls, mean (95% confidence interval) PAC was 28.4 (25.4-31.8), 6.4 (5.9-6.9) and 6.2 (5.6-6.9) ng/dl, respectively. In the same groups, PRC was 6.6 (5.6-7.7), 12.9 (11.2-14.8) and 26.5 (22.2-31.5) µIU/ml. An ARR cutoff of 1.12 provided 98.9% sensitivity and 78.9% specificity. Employing the new assay aldosterone suppression confirmed the diagnosis of primary aldosteronism and essential hypertension using the cutoff of 5 ng/dl. CONCLUSION: Our data demonstrate that the new assays present a convenient alternative for the measurement of PAC and PRC on a single automated analyzer. Availability of these simultaneous assays should facilitate screening and diagnosis of primary aldosteronism.

Pitfalls of insulin-like growth factor-i and growth hormone assays.

Accurate measurement of growth hormone (GH) and insulin-like growth factor-I (IGF-I) are the key to correct diagnosis of acromegaly and GH deficiency. Unfortunately, there is much variation involved when these hormones are measured at different sites and using different assay methods. There is an ongoing global effort to standardize the measurement process to obtain more comparable results in the future. This review discusses common pitfalls in the measurement of GH and IGF-I and guides laboratories in their analyses of these hormones.

Human metastatic melanoma cell lines express high levels of growth hormone receptor and respond to GH treatment.

Accumulating evidence implicates the growth hormone receptor (GHR) in carcinogenesis. While multiple studies show evidence for expression of growth hormone (GH) and GHR mRNA in human cancer tissue, there is a lack of quantification and only a few cancer types have been investigated. The National Cancer Institute's NCI60 panel includes 60 cancer cell lines from nine types of human cancer: breast, CNS, colon, leukemia, melanoma, non-small cell lung, ovarian, prostate and renal. We utilized this panel to quantify expression of GHR, GH, prolactin receptor (PRLR) and prolactin (PRL) mRNA with real-time RT qPCR. Both GHR and PRLR show a broad range of expression within and among most cancer types. Strikingly, GHR expression is nearly 50-fold higher in melanoma than in the panel as a whole. Analysis of human metastatic melanoma biopsies confirmed GHR gene expression in melanoma tissue. In these human biopsies, the level of GHR mRNA is elevated in advanced stage IV tumor samples compared to stage III. Due to the novel finding of high GHR in melanoma, we examined the effect of GH treatment on three NCI60 melanoma lines (MDA-MB-435, UACC-62 and SK-MEL-5). GH increased proliferation in two out of three cell lines tested. Further analysis revealed GH-induced activation of STAT5 and mTOR in a cell line dependent manner. In conclusion, we have identified cell lines and cancer types that are ideal to study the role of GH and PRL in cancer, yet have been largely overlooked. Furthermore, we found that human metastatic melanoma tumors express GHR and cell lines possess active GHRs that can modulate multiple signaling pathways and alter cell proliferation. Based on this data, GH could be a new therapeutic target in melanoma.

Significance of the disulphide bonds of human growth hormone.

Growth hormone (GH) structure is stabilised by two disulphide bonds, C53-C165 and C182-C189 in human GH. Researchers have investigatedthe role of these structural features since the late 1960s. Early studies implied that the disulphide bonds would not be importantfor biological activity of GH. However, more advanced techniques, as well as clues from patients carrying mutations in their GH1 gene,have demonstrated that the integrity of the disulphide bond between cysteines C53 and C165 is required for biological activity of GH.In contrast, disruption of the C-terminal disulphide bond (C182-C189) has only modest effects on the biological potency of GH, despitedecreased binding affinity to GH receptor and reduced stability as shown by a comprehensive in vitro study.To confirm these results, we generated transgenic mice that express a human GH analogue, C189A, and observed normal growth-promotingand lipolytic activities. In this article, we present new data and review old results concerning the disulphide bonds of GH. We also discussrelevant mutations found in patients with growth disorders.

The GH/IGF-1 axis in ageing and longevity.

Secretion of growth hormone (GH), and consequently that of insulin-like growth factor 1 (IGF-1), declines over time until only low levels can be detected in individuals aged ≥60 years. This phenomenon, which is known as the 'somatopause', has led to recombinant human GH being widely promoted and abused as an antiageing drug, despite lack of evidence of efficacy. By contrast, several mutations that decrease the tone of the GH/IGF-1 axis are associated with extended longevity in mice. In humans, corresponding or similar mutations have been identified, but whether these mutations alter longevity has yet to be established. The powerful effect of reduced GH activity on lifespan extension in mice has generated the hypothesis that pharmaceutically inhibiting, rather than increasing, GH action might delay ageing. Moreover, mice as well as humans with reduced activity of the GH/IGF-1 axis are protected from cancer and diabetes mellitus, two major ageing-related morbidities. Here, we review data on mouse strains with alterations in the GH/IGF-1 axis and their effects on lifespan. The outcome of corresponding or similar mutations in humans is described, as well as the potential mechanisms underlying increased longevity and the therapeutic benefits and risks of medical disruption of the GH/IGF-1 axis in humans.

The role of GH in adipose tissue: lessons from adipose-specific GH receptor gene-disrupted mice.

GH receptor (GHR) gene-disrupted mice (GHR-/-) have provided countless discoveries as to the numerous actions of GH. Many of these discoveries highlight the importance of GH in adipose tissue. For example GHR-/- mice are insulin sensitive yet obese with preferential enlargement of the sc adipose depot. GHR-/- mice also have elevated levels of leptin, resistin, and adiponectin, compared with controls leading some to suggest that GH may negatively regulate certain adipokines. To help clarify the role that GH exerts specifically on adipose tissue in vivo, we selectively disrupted GHR in adipose tissue to produce Fat GHR Knockout (FaGHRKO) mice. Surprisingly, FaGHRKOs shared only a few characteristics with global GHR-/- mice. Like the GHR-/- mice, FaGHRKO mice are obese with increased total body fat and increased adipocyte size. However, FaGHRKO mice have increases in all adipose depots with no improvements in measures of glucose homeostasis. Furthermore, resistin and adiponectin levels in FaGHRKO mice are similar to controls (or slightly decreased) unlike the increased levels found in GHR-/- mice, suggesting that GH does not regulate these adipokines directly in adipose tissue in vivo. Other features of FaGHRKO mice include decreased levels of adipsin, a near-normal GH/IGF-1 axis, and minimal changes to a large assortment of circulating factors that were measured such as IGF-binding proteins. In conclusion, specific removal of GHR in adipose tissue is sufficient to increase adipose tissue and decrease circulating adipsin. However, removal of GHR in adipose tissue alone is not sufficient to increase levels of resistin or adiponectin and does not alter glucose metabolism.

Specific monoclonal antibodies and ultrasensitive immunoassays for 20K and 22K human growth hormone.

OBJECTIVE: Generation of specific monoclonal antibodies (mAbs) against 20K and 22K human growth hormone (hGH) and development of ultrasensitive immunoassays to quantify 20K and 22K hGH. DESIGN: Mice were immunized with recombinant 20K or 22K hGH. Hybridoma cells were screened with biotinylated 20K and 22K hGH simultaneously. The specific mAbs were further characterized and used for construction of isoform specific assays. The ultrasensitive chemiluminescent assays were developed with AMDEX streptavidin-HRP and a sensitive substrate. RESULTS: The 20K hGH specific mAb 1G12 and the 22K hGH specific mAb 5E1 showed less than 0.1% cross-reactivity to 22K or 20K hGH by competitive binding assay, respectively. Western blot analysis also confirmed the specificity of mAb 1G12 and mAb 5E1. Using mAb 1G12 and mAb 5E1, 20K and 22K specific assays with working range of 2-2000 pg/mL were constructed. The 22K hGH concentrations in 103 serum samples from different healthy subjects in the basal GH state were 343.7+/-421.5 pg/mL (18.6-1820 pg/mL). The 20K hGH concentrations were 30.7+/-37.5 pg/mL (2.4-205pg/mL). The ratios of 20K to 20K plus 22K hGH were 9.8+/-4.4% (3.3-28.3%). Both 22K hGH and 20K hGH concentrations in women (465.9+/-476.3 pg/mL and 43.7+/-46.1 pg/mL, n=47) were significantly higher than those (241.1+/-337.0 pg/mL and 20K hGH 19.8+/-23.0 pg/mL, n=56, P<0.01) in men. However, there was no difference in the proportion of 20K to 20K plus 22K between men and women (P>0.05). The strong correlation between 20K and 22K hGH (R=0.914, P<0.01) indicated the constant proportion between 20K and 22K hGH in the basal GH state of healthy subjects. CONCLUSIONS: Specific monoclonal antibodies and ultrasensitive chemiluminescent immunoassays for 20K and 22K hGH were generated. The ultrasensitive immunoassays are essential for the determination of 20K and 22K hGH in the basal GH state. This universal ultrasensitive immunoassay form can be adapted to other immunoassays for broad application.