Journal of Clinical Endocrinology & Metabolism
, doi:10.1210/jc.2007-1611
The Journal of Clinical Endocrinology & Metabolism Vol. 93, No. 2 634-637
Copyright © 2008 by The Endocrine Society
Association of a Nicotinic Receptor Mutation with Reduced Height and Blunted Physostigmine-Stimulated Growth Hormone Release
Marco Fedi,
Leon A. Bach,
Samuel F. Berkovic,
John O. Willoughby,
Ingrid E. Scheffer and
David C. Reutens
Department of Medicine, Austin Hospital (M.F., S.F.B., I.E.S., D.C.R.), The University of Melbourne, Heidelberg, Victoria 3084 Australia; Department of Medicine, Alfred Hospital (L.A.B.), Monash University, Prahran, Victoria 3181, Australia; Centre for Neuroscience (J.O.W.), Flinders University, Adelaide, South Australia 5001, Australia; and Southern Clinical School, Monash University (D.C.R., M.F.), Clayton, Victoria 3168, Australia
Address all correspondence and requests for reprints to: David C. Reutens, M.D., FRACP, Southern Clinical School, Monash Medical Centre, 246 Clayton Road, Clayton, Victoria 3168, Australia. E-mail: David.Reutens{at}med.monash.edu.au.
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Abstract
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Background: Pulsatile GH secretion from the anterior pituitary is a key mediator of human growth regulation and is affected by a number of genetic and environmental factors. Activation of neuronal nicotinic acetylcholine (nACh) receptors promotes GH release, but the role of these receptors in growth regulation is unknown.
Aim: Our aim was to assess the effect of a mutation in the 
4 subunit of the nACh receptor on cholinergic-mediated GH release.
Methods: Forty-one healthy volunteers (24 male, age 36.2 ± 12.2 yr, mean ± SD) and 13 subjects with the 4-Ser248Phe mutation (four male, age 43.2 ± 16.8 yr) were studied. Serum levels of GH, LH, FSH, prolactin, TSH, free T4, and cortisol were measured at baseline and at regular intervals after infusion of physostigmine. Height and weight were recorded in all participants as well as from additional family members with (n = 11, four male) and without (n = 16, seven male) the mutation.
Results: Subjects with the mutation were shorter (1.62 ± 0.08 vs. 1.72 ± 0.09 m, P < 0.05) and had a greater body mass index (31 ± 6 vs. 24 ± 3 kg/m2, P < 0.05) than healthy volunteers and unaffected members of the pedigree. In controls, physostigmine markedly increased the serum levels of GH (mean increase, +732%). In contrast, the response to physostigmine was markedly blunted in subjects with the mutation (+104%, P > 0.2 vs. control).
Conclusions: These findings suggest a role of the nACh receptor in human growth regulation.
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Introduction
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The study of individuals and families with short stature has provided important insights into the genetic control of human growth. This approach has led to the identification of mutations affecting a key regulator of somatic growth, the GH-IGF-I axis (1). As a result, genetic defects of pituitary GH secretion, the GH receptor, postreceptor mechanisms, and IGF-I have all been identified. Mutations that impair pituitary GH secretion include those that affect the GHRH receptor gene and genes controlling the development of GH-producing cells in the anterior pituitary. Growth is also affected by mutations in the GH gene that give rise to dysfunctional GH variants and by changes in the proximal promoter region of the GH1 gene that affect gene expression (1). Here we provide evidence that the neuronal nicotinic acetylcholine (nACh) receptor affects growth regulation. This report describes alterations in stature and GH secretion associated with a Ser248Phe amino acid exchange in CHRNA4, the 4 subunit of the receptor. This study was part of a neuroimaging project in which we assessed the effect of the 4-Ser248Phe mutation on D1 dopamine receptor binding using positron emission tomography. The mutation is associated with autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE), a syndrome characterized by predominantly nocturnal clusters of brief seizures arising from the prefrontal cortex with no overt endocrinological manifestations (2).
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Subjects and Methods
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Forty-one healthy volunteers (24 male, mean age 36.2 ± 12.2 yr, range 20–67 yr) and 13 subjects with the 4-Ser248Phe mutation from the same large pedigree (3) (four male, mean age 43.2 ± 16.8 yr, range 16–63 yr) underwent biochemical testing of the hypothalamic-pituitary axis before and after cholinergic activation. Height and weight were measured, and the Quetelet index body mass index (BMI) was then calculated. Anthropometric data were obtained from additional family members with (n = 11, four males) and without (n = 16, seven males) the 4-Ser248Phe mutation, yielding anthropometric comparisons between 24 family members with the mutation, 16 family members without the mutation, and 41 controls. All patients had been in seizure remission for several years except for two with ongoing clusters of nocturnal seizures. Mean age at onset and duration of ADNFLE was 8 ± 3 yr (mean ± SD; range 5–14 yr) and 24 ± 16 yr (range 2–54 yr), respectively. Ten patients were on antiepileptic drugs (AEDs) including carbamazepine (seven patients), phenytoin (one), and clonazepam with topiramate (one) or carbamazepine (one). Baseline levels of AEDs at the time of the study were in the therapeutic range (carbamazepine 30.6 ± 6.9 µmol/liter and phenytoin 40 µmol/liter). Five patients and 10 controls were smokers, and their cotinine (the main metabolite of nicotine) levels were not different (controls 49 ± 47 µg/liter, mutation 23 ± 22 µg/liter; P > 0.25).
Indirect cholinergic activation was achieved by infusion of the acetylcholinesterase inhibitor physostigmine salicylate (Sigma Chemical Co., St. Louis, MO) at a dose of 22 µg/kg. Studies were performed between 1000 and 1400 h. All participants fasted for 4 h before the study. The rate of infusion of physostigmine in 500 ml normal saline was similar between the groups (controls 664 ± 26 ml/h, mutation 643 ± 50 ml/h; P > 0.05). The half-life of physostigmine ranges between 16.4 and 21.7 min in humans, and its inhibitory effect on cholinesterase activity lasts up to 240 min (4). To reduce peripheral cholinergic effects, the muscarinic antagonist propantheline bromide (30 mg) was given orally an hour before the study. Physostigmine infusion caused moderate side effects (principally nausea) in all subjects with similar severity, time of onset, and duration. Venous blood samples were collected at baseline and 30 and 60 min after the start of the infusion. Serum was separated and stored at –20 C until assayed for GH, prolactin (PRL), cortisol, TSH, free T4 (FT4), LH, FSH, and IGF-I. Commercially available kits were used to assay hormones by standard RIA. The intra- and interassay coefficients of variation were less than 10% for GH, IGF-I, FT4, LH, and FSH. The protocol was part of a neuroimaging study and was approved by the institutional Human Research Ethics Committee. All subjects gave written consent before the study.
One-way ANOVA was used to assess group differences in the anthropometric measurements and baseline hormone levels. The effect of physostigmine on hormone release was assessed using repeated-measures analysis of covariance with group membership and treatment as factors. Age, BMI, and cotinine and carbamazepine levels were used as covariates. Pearson’s correlation coefficient between carbamazepine levels and change in GH and PRL levels (baseline to 30 min) was calculated. The correlation between BMI and change in GH and PRL levels was also calculated in patients with the mutation. The unpaired t test was used to compare the change in GH and PRL levels between the two groups. Where appropriate, data were log-transformed to satisfy the assumption of normality, and the Green-Geisser correction was used to correct for nonsphericity. SPSS version 7.01 (SPSS Inc., Chicago, IL) was used for the analysis.
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Results
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The anthropometric features are reported in Table 1
. Compared with unrelated and intrafamilial controls, subjects with the 4-Ser248Phe mutation were significantly shorter and had a greater BMI but a similar weight. No overt clinical manifestations of endocrine dysfunction or dysmorphic features were noted. No group differences were seen in the baseline levels of GH, PRL, cortisol, TSH, FSH, FT4, and LH. Of note, IGF-I levels were also not different in patients with ADNFLE (controls 17.2 ± 4.6, mutation 16.3 ± 7.7 nM; P > 0.75).
In subjects with ADNFLE, physostigmine failed to produce changes in GH (mean increase +104%, P >0.2), PRL (+5%, P > 0.8), TSH (P > 0.6), FT4 (P > 0.8), FSH (P > 0.2), and LH (P > 0.6), but it increased cortisol levels (+72%, P < 0.03; Fig. 1). In contrast, in controls, physostigmine increased the levels of GH (mean increase +732%, P < 0.001), PRL (+169%, P < 0.001), and cortisol (+39%, P < 0.02) and slightly reduced FSH levels (–8%, P < 0.002). No changes were seen for LH, TSH, and FT4 (P > 0.1). The response to physostigmine was significantly different between groups for GH (P < 0.04) and PRL (P < 0.01) but not for cortisol (P > 0.2), TSH (P > 0.6), FT4 (P > 0.3), FSH (P > 0.6), and LH (P > 0.3). Covariate analysis did not show any significant effect of gender, age, BMI, or smoking on GH or PRL release in controls or patients. No correlation was found between carbamazepine levels and the change in GH (r= 0.3; P > 0.5) or PRL levels (r = 0.4; P > 0.3) induced by cholinergic activation, or between duration of AEDs and change in GH (r = –0.2; P > 0.3). There was also no correlation between BMI and GH changes induced by physostigmine (controls r = 0.14, P > 0.4; mutation r = 0.2, P > 0.3), and changes in GH and PRL levels did not differ significantly between obese and nonobese subjects with the mutation (P > 0.09).
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FIG. 1. Plasma concentrations (mean ± SE) of GH (top), PRL (center), and cortisol (bottom) in healthy volunteers (•) and patients with ADNFLE ( ) at baseline and 30 and 60 min after physostigmine infusion.
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Discussion
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The major finding reported here is that a mutation in the neuronal nACh receptor CHRNA4 selectively impairs cholinergic-mediated GH and PRL release. This impairment is associated with reduced height and increased frequency of obesity in the presence of normal IGF-I levels.
Growth regulation is affected by several genetic and environmental factors and is predominantly mediated by pulsatile (nocturnal) GH secretion. The finding of reduced height in individuals with the mutations compared with intrafamilial controls (with similar genetic and socioeconomic background) supports an effect of neuronal nACh receptors in growth regulation. Synthesis and secretion of GH and PRL from the anterior pituitary are controlled by factors released from tuberoinfundibular neurons that are in turn regulated by intra- and extrahypothalamic inputs from other functional regions. GH secretion is stimulated by GHRH and ghrelin and inhibited by somatostatin, whereas PRL secretion is controlled by the inhibitory effect of dopamine (5, 6). The cholinergic neural inputs to the hypothalamus play a modulatory role in the regulation of GH and PRL release. First, immunohistochemical studies have demonstrated the presence of a network of cholinergic neurons in the hypothalamus, particularly prominent in the ventromedial, infundibular, and paraventricular nuclei (7), implying a possible influence of cholinergic inputs on tuberoinfundibular neurons. Second, coexpression of cholinergic with GHRHergic, somatostatinergic, and dopaminergic immunoreactivities has been reported in hypothalamic neurons, suggesting an interaction between ACh and the peptides on their own release (8, 9). Third, receptor autoradiography and histochemical studies have documented the presence of nACh receptors containing the 4 subunit in different hypothalamic areas (10). Along these lines, administration of acetylcholinesterase inhibitors or nACh agonists increases GH and PRL release, whereas the nACh receptor antagonist mecamylamine attenuates the release of serum GH induced by ACh precursors (11, 12). A failure of GH secretion in response to physostigmine implies that ACh release or nACh receptor binding is altered in subjects with the 4-Ser248Phe mutation. The presynaptic nACh receptors might be overresponsive and up-regulated (as previously proposed) and so diminish ACh release and its GH-releasing or, more likely, its somatostatin-inhibiting effect (13, 14).
We examined potential confounding factors that may have affected our results. All participants were pretreated with the muscarinic antagonist propantheline to attenuate peripheral cholinergic effects. Given its low lipophilicity, propantheline does not cross the blood-brain barrier. An effect of the mutation on the actions of propantheline in regions with an incomplete blood-brain barrier, such as the median eminence, is possible but unlikely without evidence that the mutation affects muscarinic function. Most of the ADNFLE patients were treated with carbamazepine, a medication that has some cholinergic (15, 16) and neuroendocrine (17) effects. However, ADNFLE patients who were not on carbamazepine showed a similar lack of response to physostigmine, and carbamazepine levels did not correlate with GH changes. It is unlikely that epilepsy itself affected growth regulation (18), because most of the participants had a relatively late onset and infrequent seizures (2). ADNFLE patients had a higher BMI than controls, and obesity reduces cholinergic-mediated GH release (19, 20). The higher BMI in our subjects is highly unlikely to explain our results because we found no correlation between BMI and GH release, and nonobese and obese ADNFLE patients had similar impairment of GH and PRL release. Moreover, the ADNFLE patients were only mildly obese (mean BMI 31 kg/m2). From previous studies of obesity-induced GH hyporesponsiveness, a 30–50% reduction in GH release might have been expected (19), but GH secretion was reduced by 90% in ADNFLE cases. These observations indicate that obesity is not a likely or sole mechanism of impairment of GH secretion in patients with ADNFLE. Taken together, our results suggest a role of the neuronal nACh receptor in growth regulation and extend the spectrum of genetic disorders affecting growth by interfering with the GH-IGF-I axis.
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Footnotes
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Disclosure Statement: The authors have nothing to disclose.
First Published Online November 27, 2007
Abbreviations: ADNFLE, Autosomal dominant nocturnal frontal lobe epilepsy; AED, antiepileptic drug; BMI, body mass index; FT4, free T4; nACh, nicotinic acetylcholine; PRL, prolactin.
Received July 19, 2007.
Accepted November 15, 2007.
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Abstract
Introduction
