Journal of Clinical Medicine Article Growth Hormone (GH) Treatment Decreases Plasma Kisspeptin Levels in GH-Deficient Adults with Prader–Willi Syndrome Olga Giménez-Palop 1,2,3,† , Laia Casamitjana 1,2,3,† , Raquel Corripio 2,3,4 , Susanna Esteba-Castillo 5,6 , Rocío Pareja 1,2 , Néstor Albiñana 2 , Mercedes Rigla 1,2,3 and Assumpta Caixàs 1,2,3, * 1 Endocrinology and Nutrition Department, Hospital Universitari Parc Taulí, 08208 Sabadell, Spain; ogimenez@tauli.cat (O.G.-P.); lcasamitjana@tauli.cat (L.C.); rpareja@tauli.cat (R.P.); mrigla@tauli.cat (M.R.) 2 Institut d’Investigació i Innovació Parc Taulí (I3PT), 08208 Sabadell, Spain; rcorripio@tauli.cat (R.C.); nalbinana@tauli.cat (N.A.) 3 Department of Medicine, Universitat Autònoma de Barcelona, 08208 Sabadell, Spain 4 Pediatrics Department, Hospital Universitari Parc Taulí, 08208 Sabadell, Spain 5 Specialized Service in Mental Health and Intellectual Disability, Institut Assistència Sanitària (IAS), Parc Hospitalari Martí i Julià, 17190 Salt, Spain; susanna.esteba@ias.cat 6 Neurodevelopment Group (Girona Biomedical Research Institute)—IDIBGI, Institute of Health Assistance (IAS), Parc Hospitalari Martí i Julià, 17190 Salt, Spain * Correspondence: acaixas@tauli.cat † Both authors contributed equally. Abstract: Obesity and growth hormone (GH)-deficiency are consistent features of Prader–Willi  syndrome (PWS). Centrally, kisspeptin is involved in regulating reproductive function and can  stimulate hypothalamic hormones such as GH. Peripherally, kisspeptin signaling influences energy Citation: Giménez-Palop, O.; and metabolic status. We evaluated the effect of 12-month GH treatment on plasma kisspeptin levels Casamitjana, L.; Corripio, R.; in 27 GH-deficient adult PWS patients and analyzed its relationship with metabolic and anthropo- Esteba-Castillo, S.; Pareja, R.; metric changes. Twenty-seven matched obese subjects and 22 healthy subjects were also studied. Albiñana, N.; Rigla, M.; Caixàs, A. Before treatment, plasma kisspeptin concentrations in PWS and obese subjects were similar (140.20 Growth Hormone (GH) Treatment Decreases Plasma Kisspeptin Levels (23.5–156.8) pg/mL vs. 141.96 (113.9–165.6) pg/mL, respectively, p = 0.979)) and higher (p = 0.019) in GH-Deficient Adults with than in healthy subjects (124.58 (107.3–139.0) pg/mL); plasma leptin concentrations were similar Prader–Willi Syndrome. J. Clin. Med. in PWS and obese subjects (48.15 (28.80–67.10) ng/mL vs. 33.10 (20.50–67.30) ng/mL, respectively, 2021, 10, 3054. https://doi.org/ p = 0.152) and higher (p < 0.001) than in healthy subjects (14.80 (11.37–67.30) ng/mL). After GH 10.3390/jcm10143054 therapy, lean body mass increased 2.1% (p = 0.03), total fat mass decreased 1.6% (p = 0.005), and plasma kisspeptin decreased to levels observed in normal-weight subjects (125.1(106.2–153.4) pg/mL, Academic Editor: Claudia Giavoli p = 0.027). BMI and leptin levels remained unchanged. In conclusion, 12-month GH therapy im- proved body composition and decreased plasma kisspeptin in GH deficient adults with PWS. All Received: 12 June 2021 data are expressed in median (interquartile range). Accepted: 8 July 2021 Published: 9 July 2021 Keywords: Prader–Willi Syndrome; kisspeptin; leptin; growth hormone deficiency Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- 1. Introduction iations. Kisspeptin is a hormone that promotes the onset of puberty by stimulating the se- cretion of gonadotropin-stimulating hormone (GnRH). Kisspeptin can also stimulate the release of other pituitary hormones such as prolactin, growth hormone (GH), oxytocin, and Copyright: © 2021 by the authors. vasopressin [1]. Licensee MDPI, Basel, Switzerland. Kisspeptin is mainly synthesized by neurons in the arcuate nucleus of the hypothala- This article is an open access article mus. Its synthesis is modulated by energy balance, decreasing in situations of insufficient distributed under the terms and weight (e.g., anorexia nervosa) or excess weight (e.g., obesity) [2]. Low kisspeptin levels conditions of the Creative Commons are considered the cause of the hypogonadotropic hypogonadism seen in some patients Attribution (CC BY) license (https:// with metabolic syndrome and obesity [2,3]. creativecommons.org/licenses/by/ Kisspeptin-producing neurons express receptors for leptin, a hormone produced 4.0/). in adipose tissue that informs these neurons about the status of energy reserves [4,5]. J. Clin. Med. 2021, 10, 3054. https://doi.org/10.3390/jcm10143054 https://www.mdpi.com/journal/jcm J. Clin. Med. 2021, 10, 3054 2 of 11 Kisspeptin is also expressed in peripheral tissues involved in metabolic functions such as the pancreas, liver, and adipose tissue, and emerging data suggests peripheral kisspeptin plays a role in the regulation of insulin secretion [6]. Prader–Willi Syndrome (PWS) is a rare genetic disease characterized by a series of disorders of hypothalamic functionalism: hyperphagia, obesity, hypogonadotropic hypog- onadism, short stature due to GH deficiency [7–9], and cognitive, praxis, and behavioral disorders [10–12]. Up to 60% of adults with PWS have GH deficiency [13], which results in decreased muscle mass and strength and increased fat mass [14]. These changes can be mitigated with GH treatment, which induces metabolically beneficial changes in body composition [15,16]. Kisspeptin concentrations are decreased in patients with idiopathic hypogonadotropic hypogonadism, diabetes, or metabolic syndrome [2,4,5]. To our knowledge, kisspeptin has not been studied in patients with PWS; however, since these patients have impaired hypothalamic function, obesity (often associated with diabetes), central hypogonadism, and GH deficiency, they would be expected to have decreased kisspeptin levels. Moreover, it remains to be determined whether GH treatment could influence kisspeptin synthesis directly through a negative feedback mechanism or indirectly through changes in body composition. We aimed to determine changes in plasma kisspeptin concentrations in 27 adult pa- tients with PWS after 12 months of GH treatment and whether these changes are associated with changes in anthropometric or metabolic parameters. 2. Materials and Methods 2.1. Subjects We included 27 adults with PWS (15 women, median age: 26 years, range: 18–53) treated at our center between 1 January 2016 and 31 January 2019. Cytogenetic analysis revealed 7 had type 1 deletion, 10 had type 2 deletion, 6 had maternal uniparental disomy, 3 had imprinting defects and 1 had an atypical BP2-BP4 microdeletion. All had GH deficiency diagnosed with GHRH-arginine and/or glucagon stimulation tests [17–19]. The cut off for GH deficiency with GHRH-arginine test was: GH < 11 ng/mL if BMI < 25 kg/m2 , <8 ng/mL if BMI 25–30 kg/m2 , and <4 ng/mL if BMI ≥ 30 kg/m2 . The cut off for glucagon test was GH < 3 ng/mL at any time point. Six patients had type 2 diabetes with good glycemic control (HbA1c < 7.5%) and 15 (7 women) were receiving sex steroids. None had precocious puberty. For comparison, we also evaluated 27 obese subjects matched for age, sex, and BMI and 22 healthy subjects. The study complied with all provisions in the Declaration of Helsinki and was ap- proved by the local ethics committee (Comitè d’Ètica d’Investigació amb medicaments del Parc Taulí). All patients with PWS agreed to participate after being informed together with their parents or caregivers; their legal guardians (usually their parents) signed the consent form before enrollment. All control participants provided written informed consent. 2.2. Methods Blood was extracted from all participants at 8 AM after overnight fasting. Plasma samples for kisspeptin and leptin measurements were kept at −80 ◦ C until analysis. We recorded subjects’ height, determined by a stadiometer (Harpenden, Holtain Ltd., Dyfed, UK); body weight, measured to the nearest 0.1 kg with standard equipment; body mass index (BMI); and body composition determined by dual-energy x-ray absorptiometry (Lunar Prodigy−963, Chicago, IL, USA). To determine concentrations of kisspeptin-1 in plasma, we used the Kisspeptin-1 ELISA kit (Cloud-Clone Corp., Houston, TX, USA) (lower limit of detection, 9.27 pg/mL; intraassay coefficient of variation (CV) <10%; interassay CV < 12%). To determine concen- trations of leptin in plasma, we used the Human Leptin ELISA Kit (Biorbyt, Cambridge, UK) (lower limit of detection, 10 pg/mL; intraassay CV < 7.6%; interassay CV < 8.4%). A routine automated analyzer was used for other laboratory tests. The homeostatic model as- nar Prodigy−963, Chicago, IL, USA). recorded subjects’ height, To determine determined concentrations of by a stadiometer kisspeptin-1 (Harpenden, in plasma, we used Holtainthe Ltd., Dyfed, Kisspeptin-1 2.2. Methods UK); J. Clin. Med. 2021, 10, x FOR PEER REVIEW ELISA body weight, measured kit (Cloud-Clone Corp., to Houston, the nearest TX,0.1USA) kg with standard (lower limit of equipment; detection, body 3mass 9.27 pg/ml; of 11 index Blood (BMI); was and extracted body from all determined composition participantsby atdual-energy 8 AM after x-ray overnight fasting. Plasma absorptiometry (Lu- intraassay coefficient of variation (CV) <10%; interassay CV < 12%). To determine concen- samples nar for kisspeptin andIL,leptin Prodigy−963, measurements were kept at −80 °C until analysis. We trations of leptin Chicago,in plasma, we USA). used the Human Leptin ELISA Kit (Biorbyt, Cambridge, J. Clin. Med. 2021, 10, 3054 recordedTo subjects’ height, determine determined concentrations of by a stadiometer kisspeptin-1 (Harpenden, Holtain Ltd., Dyfed, 3 of 11 UK) (lower limit of detection, 10 pg/ml; 2.2. Methods intraassayinCV plasma, < 7.6%;we used interassay theCV Kisspeptin-1 < 8.4%). A UK); body ELISA kit weight, measured (Cloud-Clone Corp., toHouston, the nearest 0.1 kg with standard equipment; body mass routine automated Blood was analyzer extracted from was allused forTX, participants USA) other (lower laboratory at 8 AM after limit tests. ofThe detection, overnight homeostatic 9.27 fasting. Plasma pg/ml; model index intraassay (BMI); and body composition determined by dual-energy x-ray absorptiometry (Lu- assessmentcoefficientfor insulin ofresistance variation (CV) (HOMA-IR)<10%; interassay index wasCV < 12%). To calculated determine as fasting plasma concen- glu- samples nar for kisspeptin Prodigy−963, Chicago, andIL, leptin USA). measurements were kept at −80 °C until analysis. We trations cose of (mmol/L) sessment leptin in plasma, x fasting for insulin insulin resistance we used the (µIU/mL))/22.5 (HOMA-IR) Humanindex Leptin [20]. ELISA was calculated Kit (Biorbyt, as fasting Cambridge, plasma glucose recorded subjects’ To determine height, determined concentrations by a stadiometer of kisspeptin-1 in (Harpenden, Holtain Ltd., Dyfed, UK) (lower Patients (mmol/L) limit × with of PWS fasting detection, were insulin 10 pg/ml; treated (µIU/mL)/22.5 intraassay with recombinant [20]. CVplasma, < 7.6%; GH we used interassay (Genotonorm the CVKisspeptin-1 < 8.4%). A®, Miniquick UK); body ELISA kit weight, measured (Cloud-Clone Corp., toHouston, the nearest TX, 0.1 USA)kg with (lowerstandard limit of equipment; detection, bodypg/ml; 9.27 mass® routine Pfizer, automated New Patients York, with analyzer NY,PWS USA),werewas used with starting treated for other with a dose laboratory of recombinant 0.2mg/day tests. 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PWS were patients received treated with GH treatment. recombinant After GH 12 months’ (Genotonorm GH treatment, Miniquick patients’ , ELISA routine kit (Cloud-Clone automated analyzerCorp., was Houston, used for TX, otherUSA) (lower laboratory limit tests. of Thedetection, homeostatic 9.27 pg/ml; model Pfizer, analytic New Only and York, PWS NY, USA), patients received anthropometric starting parameters with GH treatment. a dose of wereinterassay 0.2mg/day After 12again measured months’ and withGH adjusting treatment, thedetermine the same protocol. dose at patients’ intraassay assessment coefficient insulinof formonths variation resistance (CV) <10%; (HOMA-IR) wasCV < 12%). To concen- 1, 3, 6, andand analytic 12 to anthropometric achieve high-normal parameters wereindex insulin-like measured calculated growth again with as fasting factor-1 the plasma (IGF-1) same glu- levels protocol. trations cose of leptin (mmol/L) x in plasma, fasting insulin we(µIU/mL))/22.5 used the Human [20].Leptin ELISA Kit (Biorbyt, Cambridge, for 2.3.the patient’s Statistical age. Analyses UK) (lower Patients 2.3.OnlyStatistical limit with ofPWS Analysesdetection, were 10 pg/ml; treated withintraassay recombinant CV < 7.6%; interassay CVMiniquick GH (Genotonorm < 8.4%). A ®, PWS Continuous patients variablesreceived are GH treatment. reported as mediansAfterand 12 months’ interquartile GH treatment, ranges (IQR).patients’ Cate- routineNew Pfizer, automatedYork, analyzer NY, USA), was usedwith starting for other a dose laboratory of 0.2mg/day tests.andTheadjusting homeostatic the model dose at analytic gorical and anthropometric Continuous variables variables are reported parameters are reported as frequencies were as measured medians and again with and interquartile percentages. the To compare same ranges protocol. (IQR). all Categor- the contin- assessment 1, 3, 6, and 12 formonths insulin resistance to achieve (HOMA-IR) high-normal index was calculated insulin-like growth as fasting factor-1 plasma (IGF-1) glu- levels ical uous variables are variables xatfasting reported baseline, as frequencies between and the 3 groups, percentages. To compare all we used Kruskal–Wallis test followed the continuous cose for 2.3. (mmol/L) the patient’s Statistical insulin (µIU/mL))/22.5 [20]. age. between the 3 groups, we used Kruskal–Wallis test followed by the byvariables the at Analyses Mann–Whitneybaseline, Patients Only PWS with PWSUreceived patients test with were treatedBonferroni GH treatment. correction. To with recombinant After GHcompare (Genotonorm 12interquartile months’ GHranges all variables treatment, Miniquick before patients’ ®, Mann–Whitney and Continuous after GH U test with variables treatment we are Bonferroni used reported the correction. as medians Wilcoxon To and signed-rankcompare allTo test. variables study before the (IQR). and Cate- relationship after Pfizer, New analytic and York, NY, USA),parameters anthropometric starting with a dose were of 0.2mg/day measured again and the with adjusting same the dose at protocol. GH treatment gorical between variables variables, we areweused theSpearman's reported used Wilcoxon as frequencies signed-rank and rank-order test. To percentages. correlation study To test.the relationship compare Statistical between all significance the contin- 1, 3, 6, and 12 months to achieve high-normal insulin-like growth factor-1 (IGF-1) levels variables, uous was variables fixed we used