Edition 75 - September 2019 / Bibliographic Reviews

Bibliographic Reviews – Ed. 75

Marco A. Rivarola y Alicia Belgorosky. Hospital de Pediatria Garrahan, Buenos Aires, Argentina.

For this issue of Endocrinología Pediátrica On Line, we have selected to comment on the following publications:

371
Continuous Free Cortisol Profiles – Circadian Rhythms In Healthy Men. J Clin Endocrinol Metab 2019 [Epub ahead of print]. R Bhake1,2, V Kluckner3, H Stassen3, GM Russell2, J Leendertz2, K Stevens4, ACE Linthorst5, SL Lightman2,6.
1University Hospitals Leicester NHS Trust, Leicester LE1 5WW (Work carried out2); 2Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology; 3Department of Psychiatry, Psychotherapy & Psychosomatics, University of Zurich; 4Medical Statistics, Faculty of Health: Medicine, Dentistry and Human Sciences, University of Plymouth; 5Bristol Medical School, University of Bristol, BS1 3NY; 6University Hospitals Bristol NHS Foundation Trust, Bristol BS2 8HW. 

Abstract.
CONTEX. The pituitary-adrenal axis had historically been considered a representative model for circadian rhythms. A recently developed portable collection device provided the opportunity to evaluate free cortisol profiles using the microdialysis approach in individuals free to conduct their day-to-day activities in their own surroundings. METHODS: Two separate experiments were conducted in healthy male volunteers – ten minutely total and subcutaneous free cortisol were measured for 24-hour period in one and twenty-minutely subcutaneous free cortisol for 72 consecutive hours in free-living individuals in the other experiment. RESULTS: The characteristic circadian rhythm was evident in both serum total and subcutaneous free cortisol with the lowest levels being achieved and maintained in the hours surrounding sleep onset with peak levels occurring in every individual around waking. In all free-living individuals, the circadian rhythm was consistent across 72-hours despite a wide range of activities. All participants also showed increased cortisol following the consumption of lunch. The lowest levels, during all 24-hour, periods were observed during the hours following lights switch-off, at the onset of sleep. CONCLUSIONS: This is the first study to show up to three consecutive 24-hour measurements of subcutaneous free cortisol in healthy individuals. This, authors believe is a landmark study that paves the way for ambulatory monitoring of free cortisol profiles continuously, up to a period of 72 hours, in a free-living individual going about their day to day activities, whether in health or in diseases involving the HPA axis.

Excerpts selected from this article:
Once they reach the circulation, 95% of cortisol is rapidly bound, by either corticosteroid binding globulin (CBG), or by albumin, which bind cortisol in equimolar ratios of 1:1 or a molar ratio of up to 1:10 respectively. It is only the small fraction of free (unbound) cortisol, the one able to pass out of the circulation, and to bind glucocorticoid receptors in the cells, within glucocorticoid-responsive target organs.
A portable microdialysis pump with a sterile pump syringe was connected to a linear microdialysis catheter with a membrane of molecular cut-off of 20 KDa (Microdialysis AB, Stockholm, Sweden). Dialysate samples, were analysed using kits for salivary cortisol (IBL, Hamburg, Germany), which was optimised for use of small dialysate volumes.
Once cortisol reaches the circulation, 95% of the hormone is rapidly bound by either corticosteroid binding globulin (CBG), or by albumin, which bind cortisol in equimolar ratios of 1:1 or a molar ratio of up to 1:10, respectively. Only the small fraction of free unbound cortisol is able to pass out of the circulation, and bind glucocorticoid receptors in the cells within glucocorticoid responsive target organs.
Microdialysis, as a well-established technique for measurement of molecules in extra-cellular fluid, offers three significant advantages. First, it enables the measurement of the active free component of cortisol that is not protein-bound. It is a minimally invasive technique that does not involve venous cannulation or the removal of blood and is relatively free of risk. Repeated blood sampling, the mainstay of human (HPA axis) hormone testing for decades, in contrast, although routine is best carried out at a clinical facility for safety.
As a result, little is known about dynamic functioning of hormonal systems in the most prevalent, and perhaps more relevant, physiological setting of an individual’s own environment. In contrast, by virtue of its safety and lack of need for venous access, microdialysis is feasible in the home/work setting and together with a miniaturized collecting system is suitable for ambulatory sampling.
The term circadian rhythm applies to a periodicity of approximately 24 hours i.e. a day, and rhythms of duration shorter than 24 hours are ascribed the term ultradian. All mammals that have been investigated to date exhibit both circadian and ultradian rhythms, which also operate at the level of other hormonal axes e.g. insulin, but the pituitary-adrenal axis had been historically considered a representative model for these rhythms and a distinct circadian pattern is consistently found across members of an individual species. The characteristic circadian and ultradian rhythm of free corticosterone has been demonstrated in the rat there has been no equivalent study in man.
The use of authors´ recently developed portable collection device (Bhake RC et al., Automated 24-hours sampling of subcutaneous tissue free cortisol in humans. J Med Eng Technol. 2013), provided authors with the opportunity for the prolonged evaluation of free cortisol profiles in individuals going about their day-to-day activities in their own surroundings. This initial study in normal subjects should also open up the feasibility of investigating conditions associated with abnormalities.

STUDY PROTOCOL 1:
Simultaneous determination of the rhythms of total plasma cortisol and subcutaneous free cortisol.
Both serum and subcutaneous (SC) microdialysate samples were collected at ten-minute intervals in experiment one. The dialysate sampling clock-period was 18:55 on day 1 to 19:05 on day 2 (n=8). As blood sampling is episodic but microdialysate sampling continuous, the sample reading of the latter was considered to represent the midpoint of a given sampling duration.

STUDY PROTOCOL 2:
Subcutaneous free cortisol profiles over 3 successive 24-hour periods in subjects at home. A microdialysis catheter was inserted subcutaneously in the lower anterior abdomen. Sampling frequency of dialysate was every twenty minutes and duration was 72 hours, and no blood samples were collected. Participants did not interrupt their normal daily routine and were free to choose their own meal times and meal composition. They kept a sleep diary and voluntarily reported unusual activities such as examinations, consumption of alcohol, and indulgence in leisure activities (poker, sexual activity).
There are many reasons why it will be valuable to measure 24-hour cortisol rhythms in ambulatory subjects – not only to understand normal physiology, but also to diagnose pathology and improve therapy. In terms of normal physiology we need to understand normal changes associated with ageing, aspects of jet-lag and synchronisation to new time zones, and the need to understand what is normal for optimal use in patients. Furthermore, the use of microdialysis allows them to measure free ‘active’ cortisol in the compartment in which it has access to its receptors-so we can have a much clearer view of local regulation of glucocorticoid responsive processes.
The second study deliberately imposed no activity structure on participants other than to avoid contact sport during 72 hours of sampling period. They did not refrain from alcohol or other dietary ingredients, and were encouraged to lead as ‘normal’ a life as possible. A variety of activities including potentially stressful ones e.g. attending examinations, chairing a student body annual meeting, as well as leisure activities like playing poker, watching television/movies, and sexual activity were reported informally during the sampling period. It is striking how similar the day-to-day SC free cortisol profiles are despite such variations in their activities in the awake state as well as their variable duration and pattern of sleep.
In terms of pathology, there are multiple opportunities for use of 24-hour monitoring to improve diagnosis or therapy. Within endocrinology, obvious indications would include the diagnosis of Cushing’s syndrome by a 24-hour or simply an overnight-series of cortisol measurements. Indeed, the study could clarify whether circadian rhythmicity is lost in Cushing’s disease. For cortisol replacement therapy it could get a much clearer view of what is happening at tissue level in response to different replacements regimes. Other indications would include investigations of adrenal incidentalomas and congenital adrenal hyperplasia. In addition to the endocrine indications this technique may help in other conditions associated with disorders of the HPA axis such as depression, sleep disorders and recovery of the HPA axis following glucocorticoid therapy induced HPA suppression. It will also be possible learn more about the physiological deviation from normal rhythm, usually temporary, seen in individuals recovering from major surgery including cardiac surgery and diseases of the cardiovascular system.
The study of free cortisol levels for three successive days was made possible by the use of the novel automated collection device, which is robust enough to allow ambulatory sample collection in individuals who are free to go about their day-today activities. Equally importantly, undisturbed sampling throughout the duration of sleep can now be achieved. This has the dual advantage of prolonged dynamic hormone measurements, and the ability to avoid the use of Clinical Research facilities, which are unnatural environments that not only can affect levels of stress responsive glucocorticoid hormones both in rodents and in human beings, but may also disrupt normal sleep patterns. This second study deliberately imposed no activity structure on participants other than to avoid contact sport during 72 hours of sampling period. They did not refrain from alcohol or other dietary ingredients, and were encouraged to lead as ‘normal’ a life as possible.
Despite their different activities reported, there was a remarkable consistency in each individual’s own profile, with the lowest levels later part of the evening and early sleep hours, and acrophase around awakening time. There are many reasons why it will be valuable to measure 24-hour cortisol rhythms in ambulatory subjects – not only to understand normal physiology, but also to diagnose pathology and improve therapy.
In terms of normal physiology, it is necessary to understand normal changes associated with ageing, aspects of jet-lag and synchronization to new time zones, and the need to understand what is normal for optimal use in patients. Furthermore, the use of microdialysis allows to measure free ‘active’ cortisol in the compartment in which it has access to its receptors-so it is possible to have a much clearer view of local regulation of glucocorticoid responsive processes.
In terms of pathology, there are multiple opportunities for use of 24-hour monitoring to improve diagnosis or therapy. Within endocrinology, obvious indications would include the diagnosis of Cushing’s syndrome by a 24-hour or simply an overnight-series of cortisol measurements. Indeed we could clarify whether circadian rhythmicity is lost in Cushing’s disease. For cortisol replacement therapy, they could get a much clearer view of what is happening at tissue level in response to different replacements regimes. Other indications would include investigations of adrenal incidentalomas and congenital adrenal hyperplasia.
In addition to the endocrine indications, this technique may help in other conditions associated with disorders of the HPA axis such as depression, sleep disorders and recovery of the HPA axis following glucocorticoid therapy induced HPA suppression. They could also learn more about the physiological deviation from normal rhythm, usually temporary, seen in individuals recovering from major surgery including cardiac surgery and diseases of the cardiovascular system.

372
Pharmacology, Physiology, and Mechanisms of Incretin Hormone Action. Cell Metab. 2013 Jun 4;17(6):819-37. Campbell JE1, Drucker DJ.
1Department of Medicine, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, University of Toronto, Toronto, Canada.

Abstract
Incretin peptides, principally GLP-1 and GIP, regulate islet hormone secretion, glucose concentrations, lipid metabolism, gut motility, appetite and body weight, and immune function, providing a scientific basis for utilizing incretin-based therapies in the treatment of type 2 diabetes. Activation of GLP-1 and GIP receptors also leads to nonglycemic effects in multiple tissues, through direct actions on tissues expressing incretin receptors and indirect mechanisms mediated through neuronal and endocrine pathways. Here they contrast the pharmacology and physiology of incretin hormones, and review recent advances in mechanisms coupling incretin receptor signaling to pleiotropic metabolic actions in preclinical studies. They discuss whether mechanisms identified in preclinical studies have potential translational relevance for the treatment of human disease and highlight controversies and uncertainties in incretin biology that require resolution in future studies.

Excerpts selected from this article:
GIP is a 42 amino acid peptide synthesized in and secreted from entero-endocrine K cells located primarily in the duodenum and proximal jejunum, and CNS (central nervous system) production of GIP has also been described. GIP messenger RNA (mRNA) and protein have been localized to the a-cells in mouse and human islets; however, the prohormone is processed by PC2 (rather than PC1/3 as seen in the K cell) to yield a 30 amino acid protein (GIP1–30) (Fujita et al., 2010). GLP-1 increases insulin and inhibits glucagon secretion in a glucose-dependent manner (Drucker, 2006). GLP-1 also increases insulin synthesis, confers glucose sensitivity to glucose-resistant b-cells, stimulates b-cell proliferation and neogenesis, and inhibits b cell apoptosis. The mechanisms through which GLP-1 inhibits glucagon secretion from a-cells are controversial. GLP-1R agonists robustly inhibit glucagon secretion in C-peptide-negative subjects with T1DM (Dupre´ et al., 2004), illustrating that the b-cell is not essential for transducing the glucagonostatic actions of GLP-1. Evidence from experiments employing somatostatin receptor 2 (SSRT2) antagonists and Ssrt2_/_ mice strongly suggests that the inhibitory actions of GLP-1 on a cells are indirect, and mediated through somatostatin-dependent mechanisms (de Heer et al., 2008).

Future of GIP and GLP-1 Research.
The therapeutic potential of manipulating GIP receptor signaling for the treatment of diabetes or obesity remains uncertain. GIPR agonists exhibit potent insulinotropic properties in preclinical studies, and human genome-wide association study data correlate loss-of-function SNPs in the Gipr gene with oral glucose intolerance. However, genetic or pharmacological inhibition of GIPR signaling in preclinical studies prevents or attenuates diet-induced obesity, and the same Gipr SNPs are associated with reduced BMI and waist circumference in human genetic studies. Future experiments should address (1) mechanism(s) that regulate GIPR signaling in the diabetic islet resulting in enhanced glucagon and reduced insulin secretion and (2) the relative importance of GIPR signaling in the b cell versus the adipocyte for adipogenesis. The mechanisms and tissues important for GIPR-dependent control of body weight (b cell versus adipose tissue versus CNS) also require further clarification, as does the complexity of Gipr mRNA splicing and the expression of GIPR isoforms in different tissues (Ahlqvist et al., 2013). The comparative activity of GIP-GLP-1 coagonists, which exhibit promising efficacy in preclinical studies (Tscho¨ p and DiMarchi, 2012), awaits careful assessment in human clinical trials. Although GLP-1 receptors are widely distributed in diverse extra pancreatic tissues and GLP-1R agonists produce favorable pleiotropic effects on many cells and tissues in preclinical  studies, the majority of experiments in vivo do not employ active comparators that provide the same degree of glucose control or weight loss. Hence, the extent to which actions attributed to GLP-1 are direct, or indirect, arising secondary to changes in body weight, glucose, or insulin action, requires more careful elucidation. Furthermore, the majority of ‘‘gain-of-function’’ studies with GLP-1 (and GIP) in rodents and humans achieve circulating levels of these peptides generally far greater than what might be observed even during the postprandial state, requiring consideration of the pharmacological versus the physiological context of the observed action. Similarly, pharmacological levels of GLP-1 and GIP frequently increase insulin levels in acute and chronic preclinical studies, and few experiments employ insulin as an active comparator for analysis of incretin action in vivo.
Of comparable concern is the quality of the current reagents used in studies of incretin action. Ex-9 and most GIPR antagonists are in fact nonselective and function as partial agonists at their respective receptors (Tscho¨ p and DiMarchi, 2012). The majority of available incretin receptor agonists and antagonists do not effectively or selectively penetrate the CNS, rendering uncertain the extent to which CNS incretin action observed after i.c.v. or intracerebral peptide administration may be extrapolated to human biology. Moreover, many studies employing i.c.v. or intracerebral peptide administration fail to assess the extent to which these peptides leak into the systemic circulation. The majority of antisera used to localize mouse and human GLP-1R expression do not in fact recognize the GLP-1R under commonly utilized experimental conditions (Panjwani et al., 2013; Pyke and Knudsen, 2013). These findings mandate a reevaluation of the mechanisms of GLP-1 action in different experimental paradigms as the literature contains dozens of publications using nonspecific antisera to infer GLP-1R expression in many tissues and cell types. Hence, the field requires development of better reagents and much more careful characterization of antisera employed for receptor localization studies.
Ongoing studies employing highly selective incretin antagonists and cell- and tissue-specific inactivation of incretin receptor expression should advance our understanding of how GLP-1 and GIP exert their actions in different cell types. The effective translation of  preclinical data into comparable robust human observations is hampered by the predominant use of young mice and rats in short term preclinical studies. It is now widely recognized that young, healthy animals frequently exhibit profound differences in biological responses in the endocrine pancreas, bone marrow, and immune and cardiovascular systems relative to older subjects with advanced diabetes and atherosclerosis. The considerable body of preclinical data demonstrating induction of insulin gene expression, enhancement of b cell proliferation, reduction of b cell apoptosis, and expansion of b cell mass in young rodents (Drucker, 2003, 2006) has not been matched by robust data from carefully controlled clinical trials attempting to demonstrate preferential preservation of b cell function in human subjects (Drucker, 2011). Hence, more studies of incretin action in older animals with established diabetes, obesity, and cardiovascular disease seem warranted. The increasing use and success of incretinbased therapies to treat T2DM and reduce body weight is based on a robust body of scientific observations, spanning cellular and molecular biology, preclinical animal studies, and human clinical trials.
Future advances in our understanding of incretin biology, and development of coagonists employing two or more incretin-related peptide epitopes within a single molecule (Day et al., 2009; Pocai et al., 2009), may expand the domain of incretin-based therapies into new therapeutic areas and have important implications for the safe and effective use of current incretin-based agents for the treatment of human disease.

 
373
Bile Modulates Secretion of Incretins and Insulin: A Study of Human Extrahepatic Cholestasis. J Clin Endocrinol Metab 104: 2685–2694, 2019. Mezza T1,2, Moffa S1,2, Ferraro PM3,4, Quero G5,6, Capece U1,2, Carfı A 1,2, Chiara MA, Cefalo CMA1,2, Cinti F1,2, Sorice GP1,2, Impronta F1,2 , Mari A7, Pontecorvi A1,2, Alfieri S5,6 , Holst JJ8,and Giaccari A1,2.
1U.O.C., Endocrinologia e Diabetologia, Fondazione Policlinico Universitario Agostino Gemelli IRCCS, 168 Rome, Italy, 2Istituto Patologia Speciale Medica, Universita` Cattolica del Sacro Cuore, Rome, Italy; 3U.O.C., Nefrologia, Fondazione Policlinico Universitario A. Gemelli IRCCS, 00168 Rome, Italy; 4Istituto di Medicina Interna e Geriatria, Universita` Cattolica del Sacro Cuore, 00168 Rome, Italy; 5U.O.C., Chirurgia Digestiva, Fondazione Policlinico Universitario A. Gemelli IRCCS, 00168 Rome, Italy; 6U.O.C., Istituto di Semeiotica Chirurgica, Universita` Cattolica del Sacro Cuore, 00168 Rome, Italy; 7U.O.C., Institute of Neuroscience, National Research Council, Padua, Italy; and 8 NNF Center for Basic Metabolic Research and Department of Biomedical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark.

Abstract
OBJECTIVE: Changes in bile flow after bariatric surgery may beneficially modulate secretion of insulin and incretins, leading to diabetes remission. However, the exact mechanism(s) involved is still unclear. Here, authors proposed an alternative method to investigate the relationship between alterations in physiological bile flow and insulin and incretin secretion by studying changes in gut pancreatic function, in extrahepatic cholestasis, in nondiabetic humans. METHODS: To pursue this aim, 58 nondiabetic patients with recent diagnosis of periampullary tumors underwent an oral glucose tolerance test (OGTT), and a subgroup of 16 patients underwent 4-hour mixed meal tests and hyperinsulinemic-euglycemic clamps. RESULTS: The analysis of the entire cohort revealed a strong inverse correlation between total bilirubin levels and insulinogenic index. When subjects were divided based on bilirubin levels, used as a marker of altered bile flow, subjects with high bilirubin levels displayed inferior glucose control and decreased insulin secretion during the OGTT. Altered bile flow elicited a markedly greater increase in glucagon and glucagon-like peptide 1 (GLP-1) secretion at fasting state, and following the meal, both glucagon and GLP-1 levels remained increased over time. Conversely, glucose-dependent insulinotropic polypeptide (GIP) levels were comparable at the fasting state, whereas the increase following meal ingestion, was significantly blunted with high bilirubin levels. They reveal strong correlations between total bilirubin and glucagon and GLP-1 levels. CONCLUSIONS: These findings suggest that acute extrahepatic cholestasis determines major impairment in enteroendocrine gut-pancreatic secretory function. The altered bile flow may determine a direct deleterious effect on beta-cell function, perhaps mediated by the impairment of incretin hormone function.

Excerpts selected from this article:
Recent clinical evidence has confirmed that bariatric surgery leads to remission of T2D independently of weight loss and that inherent changes in bile flow lead to increased plasma bile acids, fibroblast growth factor (FGF), and incretins. To investigate relationships between altered bile flow and glucose metabolism as well as the secretion of insulin, glucagon, and incretins, authors examined the effects of acute cholestasis in a cohort of subjects with extra-pancreatic obstruction due to periampullary tumors. In the current study, they assessed the relationship between bilirubin levels and glucose metabolism and pancreatic endocrine function and examined changes in levels of glucagon and incretins in nondiabetic individuals with recent cholestasis.
For this, they recruited 58 patients of European ethnicity (35 females, 23 males; mean age, 57 years), who were candidates for pylorus-preserving pancreato-duodenectomy at the Digestive Surgery Unit, and studied at the Center for Endocrine and Metabolic Diseases unit (Agostino Gemelli University Hospital, Rome, Italy).
To characterize the relationship between cholestasis and changes in glucose metabolism and secretion of islet and incretin hormones, they divided subjects according to their levels of total bilirubin, alkaline phosphatase, and g-glutamyl transferase, as well as liver function blood test results. Subjects with cholestasis were studied 2 to 4 days after the clinical occurrence of jaundice. Subjectswith a biochemical and clinical diagnosis of jaundice whose total bilirubin level was above 2.4 mg/dL were classified as “cholestatic,” whereas subjects whose total bilirubin level was below 2.4 mg/dL and whose blood tests were within normal limits were classified as “noncholestatic.” For simplicity, cholestatic subjects were labeled as “high bilirubin” and noncholestatic subjects as “normal bilirubin”.
They observed that acute extrahepatic cholestasis determines substantial impairment in entero-endocrine gut-pancreatic secretory function. The key result was that bile flow alteration was strongly and inversely correlated with insulinogenic index, suggesting a direct effect of altered bile flow on beta-cell function, perhaps mediated by the impairment of incretin hormone function. They observed a greater reduction in insulin secretory capacity and a concomitant increase in glucagon secretion in subjects with bile flow alteration, both in the fasting state and in response to the meal test. This suggests that an imbalance in the insulin secretory machinery due to increasing bilirubin levels also involves a-cell function.
However, this could also be accounted for to an effect of liver dysfunction determining hyper aminoacidemia and increasing glucagon secretion. Moreover, in conditions of altered bile flow with concomitant reduction in bCGS (b-Cell Glucose Sensitivity) ascribable to exhausted b-cell capacity to produce insulin and increase production of GLP-1 amide from the pancreas, the major factor affecting amino acid turnover and determining hyper aminoacidemia, which in turn increases glucagon secretion. Indeed, in line with the interesting new concept of the liver-a axis, they observed increased glucagon secretion in a condition of altered liver function and hepatic insulin resistance, as supported by fasting hyperglycemia. Furthermore, worsening glucose control and decreasing insulin levels may also influence glucagon secretion. Moreover, poor glucose control and comparable insulin clearance in conditions of altered bile flow, along with concomitant reduction in bCGS, could be ascribable mainly to exhausted b-cell capacity to produce insulin for any given increase in glucose level. It canot be excluded that recorded increase in GLP-1 secretion may be partially due to increased production of the inactive form of GLP-1 amide, which could be derived from the pancreas—and could be increased because of the hyper-aminoacidemia.
Thus, in the current study, authors assessed the relationship between bilirubin levels, glucose metabolism and pancreatic endocrine function, and examined changes in levels of glucagon and incretins in nondiabetic individuals with recent cholestasis.
Their findings suggest that acute extrahepatic cholestasis (observed for 2 to 4 days before the investigation) determines substantial impairment in entero-endocrine gut-pancreatic secretory function. The key result is that bile flow alteration is strongly and inversely correlated with insulinogenic index, suggesting a direct effect of altered bile flow on b-cell function, perhaps mediated by the impairment of incretin hormone function. This suggests that an imbalance in the insulin secretory machinery due to increasing bilirubin levels also involves a-cell function. In addition to altered pancreatic endocrine secretion altered bile flow also induces alterations in the secretion of intestinal incretin hormones. Indeed, GLP-1 and GIP displayed opposite trends in altered bile flow conditions: Although GLP-1 levels increased significantly both in the basal state and in response to a meal, no changes in GIP secretion were detected at the basal level, whereas the GIP response was significantly impaired after meals. Furthermore, the increase in GLP-1 basal levels was significantly correlated with worsening hyperbilirubinemia, whereas no correlations were found with basal and total secretion of GIP. GLP-1 and GIP are incretins secreted by the intestine in response to the ingestion of various nutrients. They stimulate insulin secretion from pancreatic b cells independently of glucose, and the impaired secretion and/or action of GLP-1 or GIP contributes to the impairment of b-cell secretion in T2D. Thus, their findings suggest that in a condition of altered bile flow, impaired GIP secretion may contribute to altered insulin secretion and a worsening of glucose metabolism. One possible explanation is that GIP, released from intestinal K cells in response to nutrient ingestion (especially fat), requires bile flux in the duodenum, as demonstrated in patients with obstruction of the common bile duct and impaired GIP secretion, in whom the GIP response to fat loading was restored following bile instillation in the duodenum. On the basis of this observation, they speculated that intestinal fat availability may play a major role in the stimulation of insulin to release GIP in conditions of altered bile flow.
In summary, hyperbilirubinemia induces b-cell dysfunction in nondiabetic humans, and this may be due to impaired b-cell GLP-1 and glucagon sensitivity and the concomitant reduction of GIP production in response to meals. This indicates a deleterious effect of hyperbilirubinemia on b-cell function, which determines the loss of response to incretin stimuli, despite attempts to adapt by increasing levels of glucagon and GLP-1.

374
Genetic Polymorphisms Associated with Idiopathic Short Stature and First-Year Response to Growth Hormone Treatment. Horm Res Paediatr. 2019;91(3):164-174. Quigley CA1, Li YG2, Brown MR3, Pillai SG2, Banerjee P4, Scott RS5, Blum WF6, Parks JS3.
1Endocrinology, Sydney Children’s Hospital, Sydney, New South Wales, Australia, 2Lilly Research Laboratories, Indianapolis, Indiana, USA. 3Pediatric Endocrinology, Emory University, Atlanta, Georgia, USA. 4Regeneron, Tarrytown, New Jersey, USA.5Perinatal Nursing, New Palestine, Indiana, USA. 6University of Giessen, Giessen, Germany.

Abstract
BACKGROUND/AIMS: The term idiopathic short stature (ISS) describes short stature of unknown, but likely polygenic, etiology. This study aimed to identify genetic polymorphisms associated with the ISS phenotype, and with growth response to supplemental GH. METHODS: Using a case-control analysis we compared the prevalence of “tall” versus “short” alleles at 52 polymorphic loci (17 in growth-related candidate genes, 35 identified in prior genome-wide association studies of adult height) in 94 children with ISS followed in the Genetics and Neuroendocrinology of Short Stature International Study, versus 143 controls from the Fels Longitudinal Study.
RESULTS: Four variants were nominally associated with ISS using a genotypic model, confirmed by a simultaneous confident inference approach: compared with controls children with ISS had lower odds of “tall” alleles (odds ratio, 95% CI) for GHR (0.52, 0.29-0.96); rs2234693/ESR1 (0.50, 0.25-0.98); rs967417/BMP2 (0.39, 0.17-0.93), and rs4743034/ZNF462 (0.40, 0.18-0.89). Children with ISS also had lower odds of the “tall” allele (A) at the IGFBP3 -202 promoter polymorphism (rs2855744; 0.40, 0.20-0.80) in the simultaneous confident inference analysis. A significant association with 1st-year height SD score increase during GH treatment was observed with rs11205277, located near 4 known genes: MTMR11, SV2A, HIST2H2AA3, and SF3B4; the latter, in which heterozygous mutations occur in Nager acrofacial dysostosis, appears the most relevant gene.
CONCLUSIONS: In children with ISS authors identified associations with “short” alleles at a number of height-related loci. In addition, a polymorphic variant located near SF3B4 was associated with the GH treatment response in the present cohort. The findings of this small study warrant further investigation.

Excerpts selected from this article:
Recently, it has been shown that cytokines produced by bone (e.g., osteopontin and osteocalcin) might participate in energy metabolism, insulin resistance, and growth. Some newer bone markers such as sclerostin and osteoprotegerin have been associated with insulin resistance and prediabetes in adults.
A number of studies have demonstrated missense mutations in various genes involved in linear growth, from the hypothalamus to the epiphysis. These findings reflect the fact that stature is a polygenic trait, with heritability 60–90% for adult height, depending on the population analyzed. Building on this knowledge, large genome-wide association (GWA) studies have identified hundreds of genetic loci that contribute to the variability of human stature within the normal height range. many children with ISS are treated with supplemental GH under approved indications in various countries. As with all non-GH-deficient causes of short stature, the response to treatment is quite variable, with approximate first-year height gain (i.e.,height increases above expected growth, if untreated) of up to 5 cm, depending on factors such as age at treatment initiation and GH dose [10]. At present there are no reliable markers of GH responsiveness to inform treatment decisions for this patient population. the primary goal of this study was to determine the relative prevalence of “short” versus “tall” alleles of a number of polymorphic loci in children with ISS compared with general population controls, while a secondary aim was to determine whether polymorphisms at any of these loci were associated with the magnitude of the 1st-year response to GH treatment.
GeNeSIS was a prospective, open-label, multinational, observational research program conducted between 1999 and 2015. The Idiopathic Short Stature (ISS) Sub-Study of GeNeSIS ran from 2004 to 2010 as a US-specific addendum to the protocol. Subjects with ISS were diagnosed, enrolled, and treated at the discretion of local investigators based on the individual physicians’diagnostic criteria. Children enrolled in the GeNeSIS ISS sub-study were invited to provide a DNA sample on a voluntary basis at the time of routine clinical blood collection, for an exploratory analysis of genetic polymorphisms associated with ISS.

Patient Analysis Populations:
Three patient populations were defined prospectively for these analyses. The Clinical Population comprised 747 US, non-Hispanic, Caucasian children with the physician-provided diagnosis of ISS. The Case Control Population comprised a subset of 94 children from the Clinical Population who fulfilled the inclusion criteria for polymorphism analysis: pretreatment height more than 2.0 standard deviations (SD) below the mean for age and sex of the reference population; naive to GH treatment at study entry; received GH treatment during study participation, and DNA laboratory results passed quality control criteria. The Treatment Response Population comprised 86 patients from the Case Control Population for whom height measurements were available both at baseline and at approximately 1 year of GH treatment.

Selection of Genetic Loci for Analysis
Twenty-two candidate gene loci were chosen on the basis of published data indicating involvement of the locus in a pathway related to growth or sexual maturation, potential for a direct functional effect on gene expression (e.g., location in an exon, at an mRNA splice site, or in a promoter region) and/or previous reports of an association with stature. The initial targets for investigation included genes encoding proteins in the GH-insulin-like growth factor (IGF) system,factors involved in sex steroid production and response, and factors involved in bone metabolism.
The 52 variants that passed quality control criteria (n = 17 candidate loci; n = 35 GWAS loci) for inclusion in the statistical analyses were grouped into 2 tiers to allow for a 2-step approach to statistical testing, in the effort to mitigate multiplicity issues in the analysis.
Assignment of an individual variant to one or the other tier was based on published statistical evidence, the level of replication and possible relevance to the ISS phenotype, with tier 1 variants being considered those with the strongest prior evidence and the most plausible role in disease biology (tier 1, n = 12 [4 candidate gene loci; 8 GWAS loci]; tier 2, n = 40 [13 candidate gene loci; 27 GWAS loci].
The Clinical Population of US non-Hispanic Caucasian children that formed the pool from which the cohort for genetic analyses was drawn comprised 747 patients with a mean age at the start of GH treatment of 11.6 ± 3.1 years, moderately delayed bone age of 10.4 ± 3.2 years, height SDS –2.4 ± 0.7, BMI SDS –0.7 ± 1.4, and slightly below average mid-parental (target) height (–0.4 ± 0.7 SDS; Table 1). The mean baseline IGF-I SDS was low, at –2.3 ± 1.8 (n = 230) and initial GH dose was 0.3 ± 0.1 mg/kg/week. Baseline data for the Case Control Population (n = 94) and the Treatment Response Population (n = 86) were similar to those of the Clinical Population, They assessed associations between the 52 genetic variants in our analysis set and response to GH treatment. We found an association between tier 2 variant rs11205277 and increase in 1st-year height SDS with all three statistical models: the genotypic model (FDR p = 0.04), the confident inference approach (FDR p = 0.02), and the additive model alone (FDR p = 0.01). For the 75 children with analyzable treatment response data at this locus 1st-year height SDS increase was greatest for those with homozygous “short” (AA) genotype (genotypic model). The height SDS increase was 0.62 ± 0.05 SDS (least squares mean } standard error) for children with AA genotype (n = 29; “short”/major allele), versus 0.31 ± 0.07 for those with GG genotype (n = 15; “tall”/minor allele; minor allele frequency = 0.41), and 0.52 ± 0.05 (n = 31) for those with AG genotype. Using a case-controb approach they determined that, compared with controls who did not havebshort stature, children with ISS had a lower prevalence of “tall” alleles in polymorphisms associated with 4 genes encoding growth-related proteins: GHR, estrogen receptor (ER), bone morphogenetic protein 2 (BMP2), and zinc finger protein 462 (ZF462). Suggestive, but inconclusive, results were observed for variants associated with genes encoding IGFBP-3 and growth-differentiation factor 5 (GDF5). The exon 3-deleted form of the GHR gene (d3GHR; minor allele) is a relatively common polymorphism with homozygosity reported in 8–12% of the general population. This dominant genetic variant, believed to result from an ancestral homologous recombination event between two species-specific retroelements, has been associated with taller stature in some but not other studies. Their finding of 50% greater prevalence of the d3GHR allele in control than ISS subjects (i.e., 21 vs. 14%) is consistent with an association between the d3GHR allele and taller stature. Another hormone receptor, the ERα, also plays a key role in the regulation of linear growth, via stimulation of the pubertal growth spurt and mediation of epiphyseal fusion. The ESR1 rs2234693 polymorphism (PVUII intron 1) has been linked to height during puberty, and adulthood. Furthermore, recessive germline ESR1 mutations causing estrogen resistance and marked pubertal growth delays have been reported in two unrelated patients. The finding of a significantly lower prevalence of the “tall” (C) allele at rs2234693 in our ISS cohort compared with controls (odds ratio 0.50, 95% CI 0.25–0.98) provides independent evidence for an association between ERα and stature, suggesting that variations in estrogen sensitivity may contribute to the impaired growth that characterizes ISS. In addition to the associations with the clinically relevant growth-related loci, GHR and ESR1, they also found associations in their ISS cohort with two loci identified in GWAS of adult height – BMP2 and ZNF462. In conclusion, ISS is a growth disorder of unknown but likely polygenic etiology, representing a phenotype rather than a diagnosis. They found a lower prevalence of “tall” alleles in children with ISS versus controls at 4 polymorphic loci in, or associated with, growth/stature-related genes. They also found an association between the magnitude of 1st-year growth response to exogenous GH and a locus identified in height GWA studies that may represent a functionally important polymorphism. These preliminary data provide intriguing glimpses into facets of the etiology and GH-responsiveness of ISS, but also highlight the challenges of making substantial progress in clarifying the clinical conundrum of this growth disorder. Despite some limitations of this small study, these findings warrant further exploration.

 
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The Bone Markers Sclerostin, Osteoprotegerin, and Bone-Specific Alkaline Phosphatase (B-ALP) Are Related to Insulin Resistance in Children and Adolescents, Independent of Their Association with Growth and Obesity. Horm Res Paediatr2019;91:1–8. Juraj Stanika–c, Jürgen Kratzschd, Kathrin Landgrafa,e, Mandy Vogelf, Joachim Thieryd, Wieland Kiessa,f, Antje Körnera,e, f ,
aCenter for Pediatric Research Leipzig, Hospital forChildren&Adolescents, University of Leipzig, Leipzig, Germany; bDepartment of Pediatrics, Medical Faculty at the Comenius University, Bratislava, Slovakia; cDIABGENE Laboratory, Institute of Experimental Endocrinology, Biomedical Research Center, Slovak Academy of Sciences, Bratislava,Slovakia; dInstitute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University of Leipzig,Leipzig, Germany; eIntegrated Research and Treatment Center (IFB) Adiposity Diseases, University of Leipzig,Leipzig, Germany; f LIFE Leipzig Research Center for Civilization Diseases, University of Leipzig, Leipzig.

Abstract
BACKGROUND/AIMS: Sclerostin, osteoprotegerin, and bone-specific alkaline phosphatase (B-ALP), which are primarily related to bone metabolism, have been linked with insulin resistance in adults. Authors aimed to evaluate the association of these markers with growth, obesity, and parameters of insulin resistance in lean and obese children and adolescents. METHODS: They measured sclerostin, osteoprotegerin, and BALP in fasting and oral glucose tolerance test (oGTT) serum samples from 1,325 children and adolescents, and during 24-h profiles and after exercise and glucose exposure in young adults. RESULTS: In addition to the positive relationship with height standard deviation scores (SDS), sclerostin (r = 0.035, p < 0.001) and B-ALP (r = 0.06, p = 0.028) increased, whereas osteoprotegerin (r = –0.098, p < 0.001) decreased with BMI SDS. Furthermore, B-ALP correlated with fasting and oGTT-derived markers of glucose and insulin metabolism suggestive of insulin resistance. To evaluate potential confounding diurnal variation of bone markers, we performed 24-h profiles. B-ALP and osteo-protegerin had lower night-time levels. Exercise acutely and transiently increased B-ALP and osteoprotegerin levels, but glucose ingestion had no effect. CONCLUSIONS: Besides their association with growth, sclerostin and osteoprotegerin levels are altered in childhood obesity. Particularly B-ALP was related to insulin resistance indices. These findings accent the link between bone, growth, and insulin resistance. Excerpts selected from this article:
Considering that childhood obesity is often accompanied by accelerated growth, and that bone and adipose tissue are embryologically derived from common precursor cells, bone-derived factors come into focus. Recently, it has been shown that cytokines produced by bone (e.g., osteopontin and osteocalcin) might participate in energy metabolism, insulin resistance, and growth. Some newer bone markers such as sclerostin and osteoprotegerin have been associated with insulin resistance and prediabetes in adults.
-Sclerostin is a circulating cytokine produced by the liver, bones, kidneys, and cartilage, and the main target tissue for sclerostin is the bone where it inhibits osteoblast function. Sclerostin serum concentrations are not influenced by age in children and adolescents. The main effects of sclerostin are mediated by the inhibition of the Wnt (Wingless-type mouse mammary tumor virus integration side) signaling pathway. This pathway interacts with insulin secretion and activity, and it has been implicated in various nonskeletal diseases like type 2 diabetes, obesity, early coronary disease, and cancer. Recently, a positive correlation of circulating sclerostin levels with homeostasis model assessment of insulin resistance (HOMA-IR) and a negative association with insulin-mediated total body glucose has been shown in adults with prediabetes, indicating a link to insulin resistance. The role of sclerostin in the early onset of insulin resistance, particularly in children, is unknown so far.
-Osteoprotegerin is a circulating glycoprotein (encoded by the TNFRSF11B gene) and belongs to the TNF receptor superfamily. It is produced by osteoblasts and functions as a negative regulator of bone resorption by decreasing osteoclast development. In several studies, serum osteoprotegerin has been found to increase with liver fat content, and osteoprotegerin levels are higher in adults with diabetes or prediabetes than in those with normal glucose tolerance. Another study found that osteoprotegerin levels increased parallel with the physiological increase of insulin resistance during puberty. Finally, bone-specific alkaline phosphatase (B-ALP) is an enzyme involved in bone mineralization. It is produced by osteoblasts via alternative splicing of the ALPL gene product. B-ALP concentrations are significantly influenced by age in children and adolescents, with the highest concentrations found during adolescence. Higher serum concentrations of both the total and intestinal isoform of ALP have been linked with obesity, but no data is available about children. As several osteokines linked with insulin resistance and bone metabolism are associated with linear growth in children, authors hypothesized that sclerostin, osteoprotegerin, and B-ALP levels are not only associated with linear growth but potentially with the development of insulin resistance in obese children. This study aimed to evaluate the association of sclerostin, osteoprotegerin, and B-ALP levels with growth and parameters of insulin resistance in children and adolescents.
This study shows that the serum levels of the bone markers sclerostin, osteoprotegerin, and B-ALP in children and adolescents are not only related to height SDS, but also to BMI SDS. Independent of these associations with anthropometric parameters, the classical bone marker B-ALP, in particular, was related to levels of fasting and stimulated glucose and insulin and selected insulin resistance indices.
In general, the association of cytokines (e.g., osteocalcin) with bone metabolism and growth is known and is to be expected. Here, data has been added on sclerostin and osteoprotegerin as independent predictors of height SDS. This link could be partially caused by the association of sclerostin and osteoprotegerin with insulin metabolism, and it is also speculated about the influence of skeletal size on sclerostin levels. Moreover, these results support previously published results on the association of B-ALP and growth. Surprisingly, the strongest associations with the majority of the insulin and glucose metabolism parameters found were with the most “classical” bone marker, B-ALP. These data may support a link between B-ALP, glucose and insulin metabolism, independent of growth and the extent of obesity. Finally, besides their association with growth, sclerostin and osteoprotegerin levels are slightly altered in childhood obesity. Independent of BMI SDS, the classical bone marker B-ALP, in particular, was related to insulin resistance indices. These findings accent the link between bone, growth, and insulin resistance. However, further prospective studies will be needed to confirm the associations of sclerostin, osteoprotegerin, and B-ALP with parameters of growth and insulin resistance in children and adolescents.

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Paediatric and Young Adult Manifestations and Outcomes of Multiple Endocrine Neoplasia Type 1. Clin Endocrinol (Oxf). 2019 Jul 26.  Herath M1, Parameswaran V1,2, Thompson M1,2, Williams M2,3, Burgess J1,2.
1Department of Diabetes and Endocrinology, Royal Hobart Hospital. 2School of Medicine, University of Tasmania. 3Department of Paediatrics, Royal Hobart Hospital. Tasmania, Australia. doi: 10.1111/cen.14067.

Abstract
CONTEXT: Multiple Endocrine Neoplasia 1 (MEN 1) is an autosomal dominant disease presenting as hyperplasia and neoplasia of parathyroid, pituitary and enteropancreatic tissues. Over 90% of gene carriers develop phenotypic disease by age 30 years, potentially with onset of asymptomatic disease during childhood and adolescence. OBJECTIVE: To describe the paediatric and young adult manifestations of MEN 1. DESIGN: Descriptive retrospective study of 180 patients with a common MEN1 genotype. The paediatric and young adult (age

Excerpts selected from this article
Multiple Endocrine Neoplasia Type 1 (MEN 1) confers an autosomal dominant predisposition to benign and malignant neoplasia of parathyroid, enteropancreatic, pituitary and adrenal tissues. MEN 1 results from germ-line inactivating mutations of MEN1, a highly penetrant tumour suppressor gene located on Chromosome 11. In addition to the typical endocrine phenotype, a range of non-endocrine manifestations including smooth muscle tumors, cutaneous and subcutaneous neoplasms are also reported. To date, over 400 pathogenic MEN1 mutations have been identified with no overt genotype-phenotype correlation.
Clinically evident disease appears uncommon prior to adolescence, with consensus guidelines currently recommending genetic testing and phenotype screening of confirmed MEN1 mutation carriers commencing by 10 years of age. Recommendations have suggested testing of children as young as age five however, the evidence supporting surveillance screening in early childhood is limited. Pediatric and adolescent case series for patients carrying an MEN1 mutation suggest the likelihood of developing phenotypic disease before 10 years of age is less than 15%, with asymptomatic primary hyperparathyroidism accounting for the majority of events and pituitary and enteropancreatic tumours occurring infrequently. Malignancy and Zollinger-Ellison syndrome appear rare in the first two decades of life, suggesting that the majority of screening effort for pediatric MEN 1 patient should focus on identifying patients with primary hyperparathyroidism, prolactinoma and less commonly, clinically relevant pancreatic neuroendocrine tumours such as insulinoma.
The guidance for therapeutic intervention in pediatric patients with mild and asymptomatic primary hyperparathyroidism (PHPT) and non-functioning pancreatic neuroendocrine tumors is also based on a limited number of case series and case reports. Consequently, there is uncertainty over the merits of early parathyroidectomy versus deferral of surgical intervention until completion of skeletal maturation. For the smaller proportion of patients with prolactinoma, Cushing’s disease and insulinoma, a strong case for intervention exists as the majority of such cases are either symptomatic or at risk of complications if left untreated.

377
Journal of the Endocrine Society, July 2019, 3:1383–1389.
Resistance to GHRH but Not to PTH in a 15-Year-Old Boy With Pseudohypoparathyroidism 1A. Martin Munteanu1, Cordula Kiewert1, Nora Matar1,2, Berthold P. Hauffa1, Nicole Unger3, Olaf Hiort4, Susanne Thiele4, Karin Buiting5, Nuria C. Bramswig2,5, and Corinna Grasemann1,2
1Pediatric Endocrinology and Diabetology, Children’s Hospital, 2Center for Rare Diseases University Hospital Essen, 3Department of Endocrinology, Diabetes and Metabolism; 4Division of Pediatric Endocrinology and Diabetes, 5Institute for Human Genetics, University of Duisburg-Essen Essen, EZSE, Germany.

Abstract
Pseudohypoparathyroidism 1A (PHP1A) consists of signs of Albright hereditary osteodystrophy (AHO) and multiple, variable hormonal resistances. Elevated PTH levels are the biochemical hallmark of the disease. Short stature in PHP1A may be caused by a form of accelerated chondrocyte differentiation leading to premature growth plate closure, possibly in combination with GH deficiency in some patients. Treatment of short stature with recombinant growth hormone (rhGH) in pediatric patients may improve final height if started during childhood. The 10 11/12-year-old boy with clinical signs of AHO presented for evaluation of short stature [height standard deviation score (SDS) -2.72]. Clinically his mother was affected by AHO as well. A heterozygous mutation c.505G.A (p.E169K) in exon 6 of the GNAS gene confirmed a diagnosis of PHP1A in the boy. However, hormonal assessment was unremarkable except for low serum IGF-1 (SDS -2.67). On follow-up, GH deficiency due to GHRH resistance was suspected and confirmed by clonidine and arginine stimulation tests. Treatment with rhGH (0.035 mg/kg) for 2 years resulted in catch-up growth (height SDS -1.52). At age 15 years the PTHlevels and bone age of the patient remain within the normal range. In patients with PHP1A, short stature is caused by the effects of Gs-a deficiency on the growth plate. However, resistance to GHRH and the resulting GH deficiency might also contribute.
Recombinant GH treatment increases growth in these patients. Diagnostic workup for GH deficiency as a factor contributing to short stature is recommended even in the absence of other hormonal resistances.

Excerpts selected from this article:
GNAS (20q13.32) is a complex imprinted gene locus that encodes at least five geneproducts through alternative splicing and promoter activation. One of the known gene products is Gs-α, the α-subunit of the stimulatory G protein that is crucial for signal transduction through mediation of cAMP production after binding of several hormones to their receptors, including CRH, ACTH, GHRH, LH/CG, FSH, TSH, and PTH. Loss-of-function of Gs-α or of genes encoding for proteins downstream of the signaling cascade may cause a variety of signs and symptoms depending on parental origin, type (genomic or epigenetic), and location of the alteration. Hormone resistance with elevated PTH levels, hyperphosphatemia, and hypocalcemia manifests only with mutations located on the maternal allele. Because of the lack of Gs-α derived from the paternal GNAS allele in the renal proximal tubule, thyroid, pituitary, and gonad, maternally derived genomic loss-of-function mutations cannot be compensated for, and consequently may lead to resistance to PTH, TSH, GHRH, LH, and FSH. In most tissues with biallelic (paternal and maternal) GNAS expression a single heterozygous mutation will not interfere with sufficient gene function. However, growth plate chondrocytes seem to require two functional copies of Gs-α for normal development. Accordingly, short stature and shortened metacarpals and metatarsals of AHO are possibly caused by haploinsufficiency of Gs-α in bone tissue independent of parental origin.
Resistance to GHRH is a common finding in PHP1A and may result in decreased IGF-1 levels and pathological response to some GH stimulation tests. Patients presenting with short stature and GH deficiency usually respond with catch-up growth when treated with recombinant growth hormone (rhGH), similar to patients with isolated growth hormone deficiency. However, the pubertal growth spurt seems to be limited because of premature closure of epiphyseal plates, resulting in unsatisfactory final height.

  1. Case Report.

A 10 11/12-year-old boy presented to the pediatric endocrine outpatient clinic for evaluation of short stature. On clinical examination, signs of AHO with a round face and short hands were noted. He attended a school for children with learning disabilities. Medical history revealed mild developmental delay and neurosurgical therapy of craniosynostosis in infancy. The X-ray of the left hand revealed short and broad metacarpals. On hormonal assessment, decreased IGF-I (SDS -2.67) and 25-OH vitamin D, but normal PTH levels were detected. No subcutaneous ossifications were noted. Gs-α activity in erythrocyte membranes was decreased. The Gs- α activity was quantified by measuring the cAMP concentration by a radioimmunoassay according to a method described earlier. Genetic assessment of the GNAS locus revealed a pathogenic heterozygous mutation c.505G.A (p.E169K) in exon 6, which has been previously described in another patient with PHP1A. Methylation status of the GNAS locus investigated by methylation-specific multiplex ligation-dependent probe amplification was normal.
The patient’s mother was also affected by AHO [height 151.9 cm (-2.5 SD), without evidence of hormone resistance.
On follow-up visits an annual growth rate of 3 cm/y (height velocity SDS 22.04), and consistently low IGF-1 levels (SDS 22.54) prompted testing for GH deficiency. Hormonal confirmed GH deficiency. Treatment with rhGH was initiated at age 12 10/12 years and resulted in catch-up growth.  At the most recent visit at age 15 1/12 years, both serum phosphate and PTH were within the normal range.
This case highlights the importance of thorough endocrine evaluation of short stature to differentiate effects of GNAS mutation on the growth plate and on the pituitary. Although GH deficiency has been diagnosed via conventional stimulation tests in the past, the diagnosis of GH deficiency in PHP1A may be difficult because stimulation tests using arginine infusion or insulin-induced hypoglycemia bypass the hypothalamic. GHRH activation by directly acting on the pituitary via somatostatin inhibition. In the case of GH deficiency due to GHRH resistance, these tests may lead to false normal results and may explain the near normal results of the arginine stimulation test in the presented case. Unlike other patients with PHP1A, this patient exhibits a pubertal growth spurt without accelerated bone age. The reason for this finding is unclear
In summary, PTH resistance might develop much later in individual patients with PHP1A than anticipated. Patients with clinical signs of AHO/PHP, short stature, and insufficient height velocity should be tested for GH deficiency, preferably with stimulation tests acting through stimulation of hypothalamic GHRH secretion (clonidine test, L-dopa-test). Because premature closure of epiphyseal plates in patients with PHP is common, timely diagnostic workup and close monitoring of therapy for short stature is recommended.


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