Evaluation of GH-IGF-I Axis in Adult Patients with Coeliac Disease

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1 Evaluation of GH-IGF-I Axis in Adult Patients with Coeliac Disease Authors E. Ferrante 1, C. Giavoli 1, L. Elli, A. Redaelli 3, E. Novati 3, A. De Bellis 4, C. L. Ronchi 1, S. Bergamaschi 1, A. Lania 1, M. T. Bardella, G. Bellastella 4, P. Beck-Peccoz 1 Affiliations Affiliation addresses are listed at the end of the article Key words GH d eficiency GH-IGF-I axis coeliac disease Abstract The aim of this study was to evaluate GH / IGF- I axis and other pituitary functions in adult patients with coeliac disease. For this purpose, twenty-eight adult coeliac patients [0M, 8F:19 74 years; body mass index (BMI): kg /m ] were recruited. Basal thyroid, adrenal and gonadal function, serum IGF-I and PRL, and routine parameters were evaluated. Dynamic GH secretion was carried out by GHRH plus arginine test. In 0 patients, antipituitary antibodies (APA) were also evaluated. Seven out of 8 patients, independently from disease onset and the glutenfree diet (GFD), showed an impaired GH secretion (5 % ). All were males, with severe growth hormone deficiency (GHD) and 5 with partial GHD. In patients with GHD, as compared to coeliac patients with normal GH secretion, HOMA (.1 ± 1. vs. 0.9 ± 0.4) and QUICKI (0.35 ± 0.03 vs ± 0.0) levels were significantly higher and lower, respectively, while IGF-I levels were slightly lower (17.7 ± 3.7 vs. 4.7 ± 6.3, p = NS). APA were negative in all 0 patients studied. In conclusion, a significant number of adult coeliac patients show an impaired GH secretion, this alteration being predominant in males and independent from disease onset and diet regimen. Given the absence of APAs, the cause of this pituitary dysfunction remains unclear even if a previous autoimmune involvement in some cases cannot be excluded. Bibliography DOI /s Published online: 009 Horm Metab Res Georg Thieme Verlag KG Stuttgart New York ISSN Correspondence E. Ferrante, MD Endocrinology and Diabetology Unit Department of Medical Sciences Pad. Granelli Via F. Sforza, Milan Italy Tel.: + 39 /0 / Fax: + 39 / 0 / emanuele.ferrante@unimi.it Introduction Coeliac disease (CD) is the most frequent chronic enteropathy in Western countries. Although not completely elucidated, the etiopathology of this disorder is based on an altered immune response triggered by the ingestion of gluten peptides in genetically predisposed subjects carrying the HLA type II DQ / DQ8 aplotypes. The serological presence of IgA antibodies against gliadin and IgA antibodies against tissue transglutaminase, even associated with antibodies responsible for endocrine autoimmune disorders, is the hallmark of this disease. Indeed, type 1 diabetes mellitus, Addison s disease, and Hashimoto s thyroiditis, and other nonendocrine autoimmune diseases, sometimes in the context of polyglandular autoimmunity (PGA) type I or type II, are frequently found in coeliac patients [1, ]. As far as gonadal function is concerned, CD has been found in 4 8 % of women with unexplained infertility [1, 3]. Moreover, a condition of androgen resistance, characterised by relatively high levels of LH, sex hormone-binding globulin (SHBG), and testosterone, has been described in coeliac men following a gluten containing diet. These abnormalities mostly regress during the gluten-free diet [4, 5]. Recent studies strongly support the existence of a relationship between autoimmunity and impaired GH secretion. In particular, high titres of antipituitary antibodies (APA) selectively recognizing somatotroph cells were found in adult and children with apparently idiopathic GH deficiency (GHD), suggesting that the somatotroph may be a target of autoimmune processes [6 8]. Moreover, based on the report of a lack of catch-up growth during gluten-free diet in a significant proportion of children with CD [9], rhgh replacement therapy is recommended in children with coeliac disease and biochemically demonstrated GHD [10, 11]. Accordingly, APA recognizing somatotroph cells have been detected in a subset of CD children with GHD [1]. The aim of this study was to evaluate GH-IGF-I axis and associated metabolic parameters in adult patients with coeliac disease.

2 Patients and Methods Patients For this study, 8 adult coeliac patients [0 M, 8 F; age: 34 ± years, range: years; body mass index (BMI):.6 ±.5, range: ] were recruited. The diagnosis of CD was made on the basis of the presence of serological markers and a Marsh III duodenal histology, as recommended [13]. In 4 patients coeliac disease was newly diagnosed, 5 patients were in gluten-free diet from less than 1 months, and 19 patients were in glutenfree diet from more than 1 months. In particular, 4 out of these 19 patients were diagnosed for CD during childhood. All the patients in GFD treatment showed a good compliance and control of disease. Study protocol In all patients, careful medical history on the presence or absence of symptoms of hypopituitarism was collected. Basal thyroid (TSH, FT4, FT3, antiperoxidase and antithyroglobulin antibodies), adrenal (ACTH, cortisol) and gonadal function (gonadotropins, SHBG, testosterone or estradiol levels), IGF-I, PRL concentrations, and routine parameters (i. e., blood glucose, haemochrome, liver function) were evaluated. In detail, serum prolactin levels were determined in one sample obtained after 30 min of bed rest. Dynamic GH secretion was investigated by GHRH plus arginine test (GHRH1 9; GEREF, Serono, Italy; 1 g / kg i. v. at 0 min; arginine hydrochloride, 0.5 g / kg, up to a maximum of 30 g, i. v. over 30 min from 0 to 30 min). Blood samples for GH evaluation were taken at 0, 15, 30, 45, 60, 90 and 10 min. Severe growth hormone deficiency was defined as a peak response of serum GH less than 11 and 8 μg /l in lean (BMI < 5 kg /m ) and overweight (BMI between 5 and 30 kg /m ) subjects, respectively. Partial growth hormone deficiency was defined as a peak response of serum GH between 16.5 and 11 or 8 μ g / l in lean and overweight subjects, respectively [14 16]. In 0 patients, serum antipituitary antibodies were also evaluated. Insulin resistance and sensitivity were measured using HOMA-IR and QUICKI methods, respectively. These are computer-solved indices based on fasting serum insulin (FI) and glucose concentrations (FG). The formulas are as follows: HOMA-IR = FI (mu /l) FG (mmol /l) /.5 and QUICKI = 1 / [log FG (mg /dl) + log FI (mu /l)] [17, 18]. Assays Serum GH levels were measured by two-site monoclonal immuno fluorimetric assay method (AutoDelfia kit, Wallac, Inc. OY, Turku, Finland). Serum IGF-I levels were measured by a commercial RIA method supplied by Mediagnost, T ü bingen, Germany, obtaining separation of IGF-I from binding proteins by acidification in IGF-II excess. The values were compared with these from an appropriate age- and sex-adjusted range. The sensitivity of the method was 0.0 ng / ml, intra- and interassay coefficient of variation were 3. and 7.4 %, respectively. Serum cortisol, LH, FSH, testosterone, insulin, PRL, TSH, FT4, FT3, AbTg, AbTPO, and SHBG levels were measured by an immunofluorimetric assay (AutoDelfia kit, Wallac, Inc. OY, Turku, Finland). Plasma ACTH levels were measured by chemoluminescence immunometric assay (Nichols Institute Diagnostics, San Juan Capistrano, CA, USA). All the other biochemical and haematological parameters were measured by standard procedures. APA were evaluated in all patients and in normal controls by an indirect immunofluorescence method on cryostat sections of Table 1 Clinical and hormonal parameters of coeliac patients Sex (F/ M) 8 / 0 Age (years) 34.3 ± 11.6 Height (SDS) 0.1 ± 0.8 BMI (kg/ m ).6 ±.5 TSH (mu/ l) [0.6 4.] 1.9 ± 1.0 FT4 (pmol/ l) [9 0] 1.8 ± 1.9 FT3 (pmol/ l) [ ] 6.3 ± 1. ACTH (pg/ ml) [3 60].0 ± 16.8 Cortisol (nmol/ l) [ ] 37.3 ± FSH (U/ l) (M) [1 10.5] 3.6 ± 3.1 LH (U/ l) (M) [1 8.4] 3.3 ± 1.4 Testosterone (nmol/ l) (M) [10 35] 7.4 ± 7.8 PRL (mu/ l) [F: ; M: 70 30] F: 67 ± 86 / M: 30 ± 16 Normal values are shown in square brackets young baboon pituitary gland supplied by BioSystem Italia s. r. l., as previously described [6]. In particular, fluorescein isothiocyanate (FITC) conjugated goat anti-human IgG sera were used to detect the presence of APA and then positive serum samples were tested with FITC-goat anti-human IgG, IgM, and IgA sera, separately. We choose a dilution of 1:8 for titre of APA as cutoff to discriminate the positive sera. Statistical analysis All results are expressed as mean ± SD unless otherwise stated. Unpaired Student s t -test was performed to compare different variables when data were normally distributed. The nonparametric Wilcoxon-Mann-Witney test was used, as appropriate. Fisher s exact test or χ -square test were used to compare different groups of patients, as appropriate. Correlations between different parameters were evaluated by linear regression analysis. Values of p < 0.05 were considered statistically significant. Results Clinical and hormonal parameters of coeliac patients are summarised in Table 1. All the patients had normal height ( 0.1 ± 0.8 SDS). No patients showed a low BMI ( < 18 kg /m ), while 4 subjects were overweight. Basal TSH levels were normal in all but 1 patient, while antithyroid antibodies were detected in 4 patients ( F, M). ACTH and cortisol levels were in the normal range in all patients. Increased prolactin levels were found in 3 men with otherwise normal pituitary function, although values did not exceed 530 mu / l. No clinical signs or symptoms of hypogonadism were observed in any male subject. A slight elevation of FSH was detected in one male patient with new diagnosis of CD. Three male patients, all in gluten-free diet, showed elevated testosterone levels. No significant difference in gonadal function between treated and untreated patients was observed. Among the 8 females, one patient had a history of two spontaneous abortions before diagnosis of coeliac disease, one patient had premature ovarian failure and another reported oligomenorrhea. As far as the GH-IGF-I axis was concerned, 7 out of 8 patients (5 % ) showed an impaired GH secretion after GHRH + arginine test. In particular, two male patients ( / 0, 10 % ) showed a severe GH deficiency, five male patients (5 /0, 5 % ) showed a partial GHD ( Fig. 1 ), while normal GH response was recorded in all female patients. Moreover, HOMA and fasting insulin levels were higher and QUICKI levels lower in patients with GH secretion

3 impairment than in those with normal GH secretion (p < 0.01, Table ). Conversely, IGF-I levels were slightly lower in coeliac GHD patients than in other patients, being significantly lower when considering males patients only (4.7 ± 6.4 vs ± 3.7 nmol /l, p < 0.05). The proportion of patients with GH secretion impairment was independent on diet regimen, GHD being present in 1 out of 4 newly diagnosed patients, out of 5 patients in gluten-free diet from less than 1 year, and in 4 out of 19 patients in gluten-free diet from more than 1 year. Interestingly, 4 patients diagnosed for CD during childhood were found to have normal GH secretion. Moreover, the IGF-I levels in newly diagnosed CD patients and in GFD from less than 1 months were similar to those in GFD from more than 1 months (1.9 ± 4.6 vs. 0.1 ± 7, p = NS). Finally, no significant correlation between GHD or IGF-I levels and time from diagnosis was observed. MRI of pituitary region was performed in two patients with severe GHD, showing a normal gland in one patient and a small anterior pituitary gland with homogenous enhancement in other one. Fasting glucose levels were normal in all but one female patient with impaired fasting glucose, while increased total cholesterol levels were found in 7 cases (3 M and 4 F, mean levels 36 ± 31 mg / dl). All these patients were already in gluten-free diet and 1 male patient was affected by partial GHD. Serum GH peak (µg/l) Lean Patients Overweight Fig. 1 Serum GH peak levels during stimulation test with GHRH plus arginine in lean and overweight coeliac patients. Continuous lines represent cutoff limit for diagnosis of severe GHD in lean and overweight subjects (11 and 8 μ g / l, respectively). Broken line represents cutoff limit for diagnosis of partial GHD (16.5 μ g / l). Finally, APA were negative in all studied patients (n = 0), including subjects with complete or partial GHD. Discussion The main result of the present study is that a consistent proportion of coeliac patients showed an impaired GH secretion. It is well known that coeliac disease is possibly associated with some endocrine autoimmune disorders, such as type 1 diabetes mellitus, Hashimoto s thyroiditis, Graves disease, Addison s disease, and other nonendocrine autoimmune diseases. In the present series of patients, males and females were positive for antithyroid antibodies. No other signs or symptoms of endocrine autoimmune disorders were detected. In previous reports, coeliac disease has been found in 4 8 % of women with unexplained infertility and should be suspected in women with spontaneous abortions [1, 3]. In our patients, one female had a history of two spontaneous abortions before the diagnosis of coeliac disease and another one reported premature ovarian failure of unknown cause. Although in agreement with previous reports, these data are insufficient to support the association between female infertility and coeliac disease. Farthing and co-workers reported an elevation of LH, FSH, testosterone, and SHBG concentrations in untreated coeliac patients [4, 5], thus suggesting the presence of a condition of androgen resistance. Among the male patients studied, one subject showed elevated FSH levels, and 3 other increased testosterone levels. However, no significant difference in testosterone and SHBG levels between treated and untreated patients was detected, and no clinical signs or symptoms of gonadal insufficiency were observed in male subjects. Finally, slightly elevated prolactin levels were detected in 3 out of 0 male patients. However, in these subjects prolactin did not exceed 530 mu / l and hyperprolactinaemia was not associated with biochemical and clinical alteration of pituitary function. As far as the GH-IGF-I axis is concerned, in the last few years an association between coeliac disease and GHD was strongly suspected in coeliac children without catch-up growth after 1 months of gluten-free diet. In these cases, GH secretion should be evaluated and in case of GHD, rhgh replacement therapy is recommended in order to allow a complete growth [9, 10]. Indeed, reports evaluating GH treatment showed that the efficacy in CD children seems to be comparable to that observed in idiopathic GHD [11]. In some children with CD and GHD, similarly to what observed in children with idiopathic GH deficiency or idiopathic short stature, APA recognizing GH-secreting cells Coeliac patients with severe or partial GHD (n = 7) Coeliac patients with normal GH secretion (n = 1) p-value Age (years) 37.3 ± ± 1.5 NS BMI (kg/ m ) 4.1 ± ± 1.8 NS Overweight (BMI 5 30 / BMI < 5 kg / m ) 3 / 4 1 / 0 < 0.05 IGF-I (nmol/ l) 17.7 ± ± 6.8 NS Glucose (mg/ dl) 9.1 ± ± 10.5 NS Insulin (UI/ ml) 9.1 ± ± 1.4 < 0.05 HOMA-IR.1 ± ± 0.4 < 0.05 QUICKI 0.35 ± ± 0.0 < 0.05 Total cholesterol (mg/ dl) 18 ± ± 50 NS HDL (mg/ dl) 53 ± 9 58 ± 1 NS Triglycerides (mg/ dl) 63 ± ± 4 NS Table Hormonal and metabolic parameters of coeliac patients with severe or partial growth hormone deficiency (GHD) and of coeliac patients with normal GH secretion

4 were also detected, thus suggesting the presence of an autoimmune form of hypophysitis involving the somatotrophs cells in these cases [8, 1]. In addition, in adult patients with apparently idiopathic GH deficiency or autoimmune endocrine diseases (i. e., Hashimoto s thyroiditis, Grave s disease, and Addison s disease), high titres of antipituitary antibodies selectively recognizing somatotrophs were detected, suggesting that GH-secreting cells may be a target of the autoimmune processes [6, 7]. In spite of previous report suggesting a possible autoimmune pituitary involvement in children with coeliac disease and the presence of APA [1], none of our adult coeliac patients resulted positive for these antibodies. In addition, MRI study in two patients with severe GHD did not show typical abnormalities of lymphocytic hypophysitis, although a similar finding was reported even in the presence of high titres of APA [6, 7, 1]. These apparently contradictory results could be explained considering that somatotrophs are more active in prepubertal than in adult age, thus more prone to expose antigens to immune system with subsequent appearance of antipituitary antibodies. We speculate that the lack of APA in adult patients can not exclude a previous possible presence of APA in childhood. In fact, during the natural history of several autoimmune diseases a disappearance of respective autoantibodies can be observed over time. Although modifications of IGFs and IGFBPs levels have been demonstrated in adult coeliac patients before and after glutenfree diet [19], little is known about dynamic GH secretion in these subjects. This study demonstrates that the function of GH- IGF-I axis is impaired in a significant proportion of adult coeliac patients, independently to onset of disease. In particular, about one fourth of all our patients showed a partial or severe GHD, this proportion rising when considering male patients only (7 / 0, 35 % ). Indeed, no alteration of GH secretion was demonstrated in female patients. However, given the low prevalence of women in our cohort of patients, this unexpected and interesting observation should be confirmed in a larger number of subjects. GH deficiency in adults is a well-defined clinical syndrome characterised by several metabolic alterations, such as increased body fat (BF), impaired physical performance, altered lipid profile, and insulin resistance. Some authors described metabolic alterations also in patients with partial GHD. In particular, Colao and co-workers [0] observed that total cholesterol and lowdensity lipoprotein cholesterol levels were significantly higher in partial GHD patients than in healthy controls, although no differences in triglycerides and high-density lipoprotein levels were found. In addition, Murray and co-workers [1, ] demonstrated the presence of both abnormal body composition and insulin resistance under conditions of insulin activation, in GHinsufficient patients. The present results showed that coeliac patients with impaired GH secretion (partial or total GHD) present some findings of GHD, such as higher BMI, low IGF-I levels, and insulin resistance in respect to celiac patients with normal GH secretion. However, a larger cohort of subjects as well as the study of typical findings of GHD, such as impaired bone mineral density, body composition and quality of life, are needed in order to confirm these data and to better characterise these patients, before considering a replacement therapy. In conclusion, the study showed an impaired GH secretion in one fourth of adult coeliac patients, this alteration being predominant in males and independent from disease onset and diet regimen. Given the absence of APA, the cause of this pituitary dysfunction is still unclear and requires further elucidations. Acknowledgements The Authors are grateful to all the nursing staff and technical assistants for expert and kind help. The present study was partially supported by Fondazione IRCCS Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena (Milan, Italy). Affiliations 1 Endocrinology and Diabetology Unit, Department of Medical Sciences, University of Milan, Fondazione Ospedale Maggiore Policlinico IRCCS, Milan, Italy Center for the Prevention and Diagnosis of Coeliac Disease, Department of Medical Sciences, University of Milan, Fondazione Ospedale Maggiore Policlinico IRCCS, Milan, Italy 3 Department of Internal Medicine, Digestive Endoscopy and Gastroenterology Unit, Ospedale San Gerardo, Monza, Italy 4 Department of Clinical and Experimental Medicine and Surgery, Chair of Endocrinology, Second University of Naples, Naples, Italy References 1 Collin P, Kaukinen K, Välimäki M, Salmi J. Endocrinological disorders and coeliac disease. Endocr Rev 00 ; 3 : Kumar V, Rajadhyaksha M, Wortsman J. Coeliac disease-associated autoimmune endocrinopathies. Clin Diagn Lab Immunol 001 ; 8 : Collin P, Vilska S, Heinonen PK, H ä llstr ö m O, Pikkarainen P. Infertility and coeliac disease. Gut 1996 ; 39 : Farthing MJ, Rees LH, Dawson AM. 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