MC4R variants modulate α-MSH and setmelanotide induced cellular signaling at multiple levels.

CONTEXT: Melanocortin-4 receptor (MC4R) plays an important role in body weight regulation. Pathogenic MC4R variants are the most common cause of monogenic obesity. OBJECTIVE: We have identified 17 MC4R variants in adult and pediatric patients with obesity. Here, we aimed to functionally characterize these variants by analyzing four different aspects of MC4R signaling. In addition, we aimed to analyze the effect of setmelanotide, a potent MC4R agonist, on these MC4R variants. MATERIALS AND METHODS: Cell surface expression and α-MSH- or setmelanotide-induced cAMP response, ß-arrestin-2 recruitment, and ERK activation were measured in cells expressing either wild type (WT) or variant MC4R. RESULTS: We found a large heterogeneity in the function of these variants. We identified variants with a loss of response for all studied MC4R signaling, variants with no cAMP accumulation or ERK activation but normal ß-arrestin-2 recruitment, and variants with normal cAMP accumulation and ERK activation but decreased ß-arrestin-2 recruitment, indicating disrupted desensitization and signaling mechanisms. Setmelanotide displayed a greater potency and similar efficacy as α-MSH, and induced significantly increased maximal cAMP responses of several variants compared to α-MSH. Despite the heterogeneity in functional response, there was no apparent difference in the obesity phenotype in our patients. DISCUSSION: We show that these obesity-associated MC4R variants affect MC4R signaling differently, yet leading to a comparable clinical phenotype. Our results demonstrate the clinical importance of assessing the effect of MC4R variants on a range of molecular signaling mechanisms to determine their association with obesity, which may aid in improving personalized treatment.

Longitudinal changes in acylated versus unacylated ghrelin levels may be involved in the underlying mechanisms of the switch in nutritional phases in Prader-Willi syndrome.

INTRODUCTION: Prader-Willi syndrome (PWS) is characterized by a switch from failure to thrive to excessive weight gain and hyperphagia in early childhood. An elevated, more unfavorable ratio between acylated and unacylated ghrelin (AG/UAG ratio) might play a role in the underlying mechanisms of this switch. We aimed to assess the evolution of the appetite regulating hormones acylated ghrelin (AG) and unacylated ghrelin (UAG) and the AG/UAG ratio and their association with the change in eating behavior in children with PWS, compared to healthy age-matched controls. METHODS: Longitudinal study in 134 children with PWS and 157 healthy controls, from The Netherlands, France and Belgium. Levels of AG and UAG and the AG/UAG ratio were measured and nutritional phases as reported for PWS were scored. RESULTS: The AG/UAG ratio was in the first years of life lower in PWS than in controls and started to increase from the age of 3 years, resulting in a high-normal AG/UAG ratio compared to controls. The AG levels remained stable during the different nutritional phases (p=0.114), while the UAG levels decreased from 290 pg/ml in phase 1a to 137 pg/ml in phase 2b (p<0.001). The AG/UAG ratio increased significantly from 0.81 in phase 2a to 1.24 in phase 2b (p= 0.012). CONCLUSIONS: The change from failure to thrive to excessive weight gain and hyperphagia in infants and children with PWS coincides with an increase in AG/UAG ratio. The increase in AG/UAG ratio occurred during phase 2a, thus before the onset of hyperphagia.

Glucocorticoids, stress and eating: The mediating role of appetite-regulating hormones.

Disrupted hormonal appetite signaling plays a crucial role in obesity as it may lead to uncontrolled reward-related eating. Such disturbances can be induced not only by weight gain itself but also by glucocorticoid overexposure, for example, due to chronic stress, disease, or medication use. However, the exact pathways are just starting to be understood. Here, we present a conceptual framework of how glucocorticoid excess may impair hormonal appetite signaling and, consequently, eating control in the context of obesity. The evidence we present suggests that counteracting glucocorticoid excess can lead to improvements in appetite signaling and may therefore pose a crucial target for obesity prevention and treatment. In turn, targeting hormonal appetite signals may not only improve weight management and eating behavior but may also decrease detrimental effects of glucocorticoid excess on cardio-metabolic outcomes and mood. We conclude that gaining a better understanding of the relationship between glucocorticoid excess and circulating appetite signals will contribute greatly to improvements in personalized obesity prevention and treatment.

Obesity and Hyperphagia With Increased Defective ACTH: A Novel POMC Variant.

OBJECTIVE: Patients with pro-opiomelanocortin (POMC) defects generally present with early-onset obesity, hyperphagia, hypopigmentation and adrenocorticotropin (ACTH) deficiency. Rodent models suggest that adequate cleavage of ACTH to α-melanocortin-stimulating hormone (α-MSH) and desacetyl-α-melanocortin-stimulating hormone (d-α-MSH) by prohormone convertase 2 at the KKRR region is required for regulating food intake and energy balance. METHODS: We present 2 sisters with a novel POMC gene variant, leading to an ACTH defect at the prohormone convertase 2 cleavage site, and performed functional studies of this variant. RESULTS: The patients had obesity, hyperphagia and hypocortisolism, with markerly raised levels of ACTH but unaffected pigmentation. Their ACTH has reduced potency to stimulate the melanocortin (MC) 2 receptor, explaining their hypocortisolism. CONCLUSION: The hyperphagia and obesity support evidence that adequate cleavage of ACTH to α-MSH and d-α-MSH is also required in humans for feeding control.

Ghrelin Levels in Children With Intestinal Failure Receiving Long-Term Parenteral Nutrition.

Background: Children with intestinal failure (IF) require parenteral nutrition (PN). Transition to oral and enteral nutrition (EN) can be difficult also due to abnormal gastrointestinal motility. The gut hormone ghrelin is increased in states of negative energy balance, functioning to preserve euglycemia, and also has appetite stimulating and prokinetic properties. We aimed to evaluate and compare ghrelin levels in children with IF, and to assess the relationship with PN-dependency. Methods: In this exploratory prospective multicenter study, plasma acylated (AG) and unacylated (UAG) ghrelin levels were measured in children with short bowel syndrome (SBS) and with functional IF (pseudo-obstruction or any enteropathy) and compared with healthy control subjects. Spearman's rho (rs) was used to assess correlations of AG and UAG with PN-dependency (%PN) and parenteral glucose intake. Results: Sixty-four samples from 36 IF-patients were analyzed. Median baseline AG and UAG levels were respectively 279.2 and 101.0 pg/mL in children with SBS (n = 16), 126.4 and 84.5 pg/mL in children with functional IF (n = 20) and 82.4 and 157.3 pg/mL in healthy children (n = 39). AG levels were higher in children with SBS and functional IF than in healthy children (p = 0.002 and p = 0.023, respectively). In SBS, AG positively correlated with %PN (rs = 0.5, p = 0.005) and parenteral glucose intake (rs = 0.6, p = 0.003). These correlations were not observed in functional IF. Conclusion: Children with IF had raised AG levels which could be related to starvation of the gut. The positive correlation between AG and glucose infusion rate in SBS suggests an altered glucoregulatory function.

Ghrelin deficiency sex-dependently affects food intake, locomotor activity, and adipose and hepatic gene expression in a binge-eating mouse model.

Binge-eating disorder is the most prevalent eating disorder diagnosed, affecting three times more women than men. Ghrelin stimulates appetite and reward signaling, and loss of its receptor reduces binge-eating behavior in male mice. Here, we examined the influence of ghrelin itself on binge-eating behavior in both male and female mice. Five-wk-old wild-type (WT) and ghrelin-deficient (Ghrl-/-) mice were housed individually in indirect calorimetry cages for 9 wks. Binge-like eating was induced by giving mice ad libitum chow, but time-restricted access to a Western-style diet (WD; 2 h access, 3 days/wk) in the light phase (BE); control groups received ad libitum chow (CO), or ad libitum access to both diets (CW). All groups of BE mice showed binge-eating behavior, eating up to 60% of their 24-h intake during the WD access period. Subsequent dark phase chow intake was decreased in Ghrl-/- mice and remained decreased in Ghrl-/- females on nonbinge days. Also, nonbinge day locomotor activity was lower in Ghrl-/- than in WT BE females. Upon euthanasia, Ghrl-/- BE mice weighed less and had a lower lean body mass percentage than WT BE mice. In BE and CW groups, ghrelin and sex altered the expression of genes involved in lipid processing, thermogenesis, and aging in white adipose tissue and livers. We conclude that, although ghrelin deficiency does not hamper the development of binge-like eating, it sex-dependently alters food intake timing, locomotor activity, and metabolism. These results add to the growing body of evidence that ghrelin signaling is sexually dimorphic.NEW & NOTEWORTHY Ghrelin, a peptide hormone secreted from the gut, is involved in hunger and reward signaling, which are altered in binge-eating disorder. Although sex differences have been described in both binge-eating and ghrelin signaling, this interaction has not been fully elucidated. Here, we show that ghrelin deficiency affects the behavior and metabolism of mice in a binge-like eating paradigm, and that the sex of the mice impacts the magnitude and direction of these effects.

Bariatric Surgery for Hypothalamic Obesity in Craniopharyngioma Patients: A Retrospective, Matched Case-Control Study.

CONTEXT: Craniopharyngioma is a sellar tumor associated with high rates of pituitary deficiencies (~ 98%) and hypothalamic obesity (~ 50%). OBJECTIVE: This work aims to determine the efficacy regarding long-term weight loss after bariatric surgery in obese craniopharyngioma patients with hypothalamic dysfunction. METHODS: This retrospective, case-control, multicenter, international study included obese craniopharyngioma patients (N = 16; of whom 12 are women) with a history of bariatric surgery (12 Roux-en-Y gastric bypass, 4 sleeve gastrectomy; median age 21 years [range, 15-52 years], median follow-up 5.2 years [range, 2.0-11.3 years]) and age/sex/surgery/body mass index-matched obese controls (N = 155). Weight loss and obesity-related comorbidities up to 5 years after bariatric surgery were compared and changes in hormonal replacement therapy evaluated. RESULTS: Mean weight loss at 5-year follow-up was 22.0% (95% CI, 16.1%-27.8%) in patients vs 29.5% (95% CI, 28.0%-30.9%) in controls (P = .02), which was less after Roux-en-Y gastric bypass (22.7% [16.9%-28.5%] vs 32.0% [30.4%-33.6%]; P = .003) but at a similar level after sleeve gastrectomy (21.7% [-1.8% to 45.2%] vs 21.8% [18.2%-25.5%]; P = .96). No major changes in endocrine replacement therapy were observed after surgery. One patient died (unknown cause). One patient had long-term absorptive problems. CONCLUSION: Obese patients with craniopharyngioma had a substantial mean weight loss of 22% at 5-year follow-up after bariatric surgery, independent of type of bariatric surgery procedure. Weight loss was lower than in obese controls after Roux-en-Y gastric bypass. Bariatric surgery appears to be effective and relatively safe in the treatment of obese craniopharyngioma patients.

Overweight and obesity in type 1 diabetes is not associated with higher ghrelin concentrations.

BACKGROUND: Several studies have demonstrated suppressed levels of acylated (AG) and unacylated ghrelin (UAG) in patients with type 2 diabetes. However, the role of these hormones in type 1 diabetes has not been extensively studied. This study assessed the relationship between AG and UAG levels and body composition in patients with type 1 diabetes. METHODS: We selected eighteen patients with type 1 diabetes and divided them into two groups: non-obese (BMI < 25 kg/m2) and overweight (BMI ≥ 25 kg/m2). Demographics, parameters of body composition and serum parameters including AG and UAG, were assessed. RESULTS: The patients with a BMI ≥ 25 kg/m2 were older and had a longer duration of diabetes. AG and UAG levels were not significantly different between non-obese and overweight groups (mean AG non-obese ± SD: 44.5 ± 29.4 pg/ml and mean UAG non-obese 42.4 ± 20.7 pg/ml vs mean AG overweight ± SD: 46.1 ± 29.6 pg/ml and mean UAG overweight 47.2 ± 18.2 pg/ml). AG/UAG ratios did not discriminate between these groups. There was a positive association of insuline dose/kg bodyweight with BMI (r2 = 0.45, p = 0.002). CONCLUSIONS: Surprisingly, unlike non-diabetics and in T2D, we did not observe a difference in plasma levels of AG and UAG between normal weight and overweight adult type 1 diabetics. However, we did observe a positive correlation between BMI and insuline dose/kg bodyweight, suggesting that exogenous insulin is more important than the ghrelin system in the development of obesity in type 1 diabetes.

Unacylated ghrelin binds heparan-sulfate proteoglycans which modulate its function.

Acylated ghrelin (AG) is a gut-derived peptide with growth hormone secretagogue (GHS), orexigenic and other physiological activities mediated by GHS receptor-1a (GHSR). Ghrelin occurs in unacylated form (UAG) with activities opposing AG, although its mechanism of action is unknown. UAG does not antagonize AG at GHSR, and has biological effects on cells that lack this receptor. Because UAG binds to cells, it has been hypothesized that UAG acts via a cell-surface receptor, although this has not been confirmed. This study aimed to identify cell surface proteins to which UAG binds that could modulate or mediate its biological effects. The MCF7 cell-line was used as a model because UAG induces ERK signaling in these cells in the absence of GHSR. Using ligand-receptor capture and LC-MS/MS we identified specific heparan-sulfate proteoglycans (HSPGs) to which UAG interacts on cell surfaces. In line with this, UAG, as well as AG, bind with high affinity to heparin, and heparin and heparinase treatment suppress, whereas HSPG overexpression increases, UAG binding to MCF7 cell surfaces. Moreover, heparin suppresses the ERK response to UAG. However, conversion of the lysines in UAG to alanine, which prevents its binding to heparin and cell surface HSPGs, does not prevent its activation of ERK. Our data show that the interaction of UAG with HSPGs modulates its biological activity in cells. More broadly, the interaction of UAG and AG with HSPGs could be important for the specificity and potency of their biological action in vivo.

Oxytocin in young children with Prader-Willi syndrome: Results of a randomized, double-blind, placebo-controlled, crossover trial investigating 3 months of oxytocin.

CONTEXT: Prader-Willi syndrome (PWS) is characterized by hypothalamic dysfunction, hyperphagia and a typical behavioural phenotype, with characteristics of autism spectrum disorder (ASD) like stubbornness, temper tantrums and compulsivity. It has been suggested that the oxytocin system in patients with PWS is dysfunctional. In ASD, intranasal oxytocin treatment has favourable effects on behaviour. OBJECTIVE: To evaluate the effects of 3 months of twice daily intranasal oxytocin (dose range 16-40 IU/day), compared to placebo, on behaviour and hyperphagia in children with PWS. DESIGN: Randomized, double-blind, placebo-controlled, crossover study in the Dutch PWS Reference Center. PATIENTS: Twenty-six children with PWS aged 3-11 years. MAIN OUTCOME MEASURES: (Change in) behaviour and hyperphagia measured by Oxytocin Questionnaire and Dykens hyperphagia questionnaire. RESULTS: In the total group, no significant effects of oxytocin on social behaviour or hyperphagia were found. However, in boys, the Oxytocin Questionnaire scores improved significantly during oxytocin treatment, compared to a deterioration during placebo (4.5 (-0.8 to 15.3) vs. -4.0 (-11.3 to 0.8), P = .025). The Dykens hyperphagia questionnaire scores remained similar during oxytocin treatment, while there was a deterioration during placebo (0.0 (-0.8 to 4.3) vs. -3.5 (-6.0 to 0.0), P = .046). Patients with a deletion had significant improvements in both questionnaire scores during oxytocin treatment, but deteriorations during placebo. Oxytocin treatment was well tolerated, and there were no serious adverse events. CONCLUSIONS: Intranasal oxytocin treatment has positive effects on social and eating behaviour in 3-11 years aged boys with PWS and in children with a deletion without safety concerns. Intranasal oxytocin in children with PWS might be considered, but individual effects should be carefully evaluated and treatment discontinued if no effects are found.

Efficacy and Safety of switching to Pasireotide in Acromegaly Patients controlled with Pegvisomant and Somatostatin Analogues: PAPE extension study.

OBJECTIVE: to assess the efficacy and safety after 48 weeks of treatment with pasireotide long-acting-release (PAS-LAR) alone or in combination with pegvisomant in patients with acromegaly. In addition, we assessed the relation between insulin secretion and pasireotide-induced hyperglycemia. DESIGN: The PAPE extension study is a prospective follow-up study until 48 weeks after the core study of 24 weeks. METHODS: 59 out of 61 patients entered the extension study. Efficacy was defined as the percentage of patients achieving IGF-I normalization (≤ 1.2 x the Upper Limit of Normal (ULN)) at 48-weeks through protocol-based adjustment of pegvisomant and PAS-LAR doses. At baseline, insulin secretion was assessed by an oral glucose tolerance test (OGTT). RESULTS: At the end of the study median IGF-I was 0.98 x ULN, and 77% of patients achieved normal IGF-I levels with a mean pegvisomant dose of 64 mg/week, and an overall cumulative pegvisomant dose reduction of 52%. Frequency of diabetes mellitus increased from 68% at 24 weeks to 77% at 48 weeks, and 9 patients discontinued PAS-LAR treatment, mainly because of severe hyperglycemia. Pasireotide-induced hyperglycemia was inversely correlated with baseline insulin secretion (r = -0.37, P < 0.005). CONCLUSIONS: PAS-LAR normalizes IGF-I levels in most acromegaly patients, with a fifty percent pegvisomant-sparing effect. However, PAS-LAR treatment coincided with a high incidence of diabetes mellitus. The risk for developing diabetes during PAS-LAR treatment seems inversely related to insulin secretion at baseline.

Efficacy and Safety of Switching to Pasireotide in Patients With Acromegaly Controlled With Pegvisomant and First-Generation Somatostatin Analogues (PAPE Study).

Aim: To assess the efficacy and safety of pasireotide long-acting release (PAS-LAR) alone or in combination with pegvisomant by switching patients with acromegaly who were well controlled with long-acting somatostatin analogues (LA-SSAs) and pegvisomant to PAS-LAR with or without pegvisomant. Methods: Sixty-one patients with acromegaly were enrolled in a prospective open-label study. We included patients with an insulin-like growth factor I (IGF-I) ≤1.2 × upper limit of normal (ULN) during treatment with LA-SSAs and pegvisomant. At baseline, the pegvisomant dose was reduced by 50% up to 12 weeks. When IGF-I remained ≤1.2 × ULN after 12 weeks, patients were switched to PAS-LAR 60 mg monotherapy. When IGF-I was >1.2 × ULN, patients were switched to PAS-LAR 60 mg, and they continued with the 50% reduced pegvisomant dose. Results: At baseline, mean IGF-I was 0.97 × ULN, and the median pegvisomant dose was 80 mg/wk. At 12 weeks, mean IGF-I increased to 1.59 × ULN, and IGF-I levels ≤1.2 ULN were observed in 24.6% of participants. At 24 weeks, IGF-I levels were reduced into the reference range in 73.8% of patients. Between baseline and 24 weeks, the pegvisomant dose was reduced by 66.1%. PAS-LAR was well tolerated, but hyperglycemia was the most frequent adverse event. The frequency of diabetes increased from 32.8% at baseline to 68.9% at 24 weeks. Conclusions: Switching to PAS-LAR, either as monotherapy or combination with pegvisomant, can control IGF-I levels in most patients. PAS-LAR demonstrated a pegvisomant-sparing effect of 66% compared with the combination with LA-SSAs. Hyperglycemia was the most important safety issue.

Unacylated ghrelin modulates circulating angiogenic cell number in insulin-resistant states.

BACKGROUND: Type 2 diabetes (T2D) is associated with reduced numbers and impaired function of circulating angiogenic cells (CAC) which contributes to the progression of atherosclerosis and microvascular disease. Previous studies suggest that short-term infusion of unacylated ghrelin (UAG) normalizes CAC number in patients with T2D. To determine dose-dependent effects of short-term infusion of UAG in T2D patients using a cross-over model, and of long-term infusion of UAG in obese mice, on differentiation of monocyte progenitors into CAC. METHODS: Eight overweight T2D patients were infused overnight with 3 and 10 µg/kg/h of UAG in a double-blind, placebo-controlled cross-over study. To assess the effects of long-term UAG treatment, obese mice were infused with UAG for 4 weeks. Monocyte progenitors were assessed for their ability to differentiate into CAC in vitro. RESULTS: In T2D patients, UAG treatment caused a reduction in differentiation of CAC, dependent on UAG dose and differentiation method. However, mice treated with UAG showed a significant increase in differentiation of bone marrow progenitors into CAC. CONCLUSION: UAG causes a minor suppressive effect on CAC development after short-term treatment in humans, but experiments in mice suggest that long-term treatment has beneficial effects on CAC formation. The Netherlands Trial Register: TC=2487.

The Acylated/Unacylated Ghrelin Ratio Is Similar in Patients With Acromegaly During Different Treatment Regimens.

Background: Data on plasma acylated ghrelin (AG) and unacylated ghrelin (UAG) levels in acromegaly are limited. High AG/UAG ratios are linked with type 2 diabetes, obesity, and hyperphagia (e.g., in Prader-Willi syndrome). Objective: To assess fasting plasma AG and UAG levels, and the AG/UAG ratio in acromegaly patients receiving combination treatment of long-acting somatostatin analogs (LA-SSAs) and pegvisomant (PEGV; n = 60). We used as controls acromegaly patients whose disease was controlled with PEGV monotherapy and medically naïve patients with active acromegaly. Methods: Fasting venous blood samples were collected and directly stabilized to inhibit deacylation of AG. Plasma AG and UAG levels were determined by double-antibody sandwich enzyme immunoassay, and the AG/UAG ratio was calculated. Results: Plasma AG and UAG levels were significantly lower in patients with acromegaly receiving combination treatment [median, interquartile range (IQR): AG: 8.5 pg/mL, 2.9 to 21.1 pg/mL; UAG: 26.9 pg/mL, 11.2 to 42.1 pg/mL] compared with patients using PEGV alone [AG: 60.5 pg/mL (IQR, 58.8 to 77.4 pg/mL); UAG: 153.7 pg/mL (IQR, 127.3 to 196.0 pg/mL)] and medically naïve patients with acromegaly [AG: 24.0 pg/mL (IQR, 12.6 to 49.7 pg/mL); UAG: 56.3 pg/mL (IQR, 43.4 to 61.5 pg/mL)]. However, AG/UAG ratios were similar in all groups. Conclusions: Although plasma AG and UAG are suppressed during combination treatment with LA-SSAs and PEGV, the AG/UAG ratio remained similar. This shows that SSAs decrease both AG and UAG levels, which suggests that they do not alter metabolism significantly in acromegaly patients.

The Effect of the Exon-3-Deleted Growth Hormone Receptor on Pegvisomant-Treated Acromegaly: A Systematic Review and Meta-Analysis.

BACKGROUND: The common exon 3 deletion polymorphism of the growth hormone receptor (d3-GHR) is associated with disease severity in acromegaly patients. The GHR antagonist pegvisomant (PEGV) is highly effective in treating severe acromegaly. Response to PEGV treatment seems to be influenced by d3-GHR and appears to be more responsive to PEGV, although available results remain conflicting. OBJECTIVE: To assess the influence of d3-GHR on the responsiveness of acromegaly patients to PEGV by compiling the evidence derived from the largest available studies. DESIGN: A systematic review of the literature identified three published studies and one conference abstract. Acromegaly patients (n = 324, 49.7% d3-GHR carriers) were treated with either PEGV monotherapy or PEGV combined with long-acting somatostatin analogues and/or cabergoline. A meta-analysis of raw data from these studies was performed. RESULTS: No significant effect of the d3-GHR was observed while bringing insulin-like growth factor I (IGF-I) levels below the upper limit of normal with PEGV, which was defined as the lowest IGF-I level during PEGV treatment (mean difference: -2.3%; 95% CI: -6.5 to 1.8, p = 0.270). The PEGV dose required to achieve the lowest IGF-I levels was also not significantly influenced by individuals carrying d3-GHR (mean difference: 4.1 mg weekly; 95% CI: -5.1 to 13.2, p = 0.385). For both outcomes, separate analysis of PEGV monotherapy and combination treatment gave similar results. CONCLUSION: Our findings suggest that the d3-GHR polymorphism has no effect on biochemical disease control in acromegaly, as it is not of added value for either the prediction of PEGV responsiveness or the determination of the required PEGV dose.

Plasma acylated and plasma unacylated ghrelin: useful new biomarkers in patients with neuroendocrine tumors?

To date, the value of fasting plasma acylated ghrelin (AG) and unacylated ghrelin (UAG) as potential novel biomarkers in patients with neuroendocrine tumors (NETs) is unknown. The aims of this study are to (i) compare fasting AG and UAG levels between nonobese, nondiabetic NET patients (N=28) and age- (±3 years) and sex-matched nonobese, nondiabetic controls (N=28); and (ii) study the relationship between AG, UAG, and AG/UAG ratios and biochemical (chromogranin-A (CgA) and neuron-specific enolase (NSE) levels) and clinical parameters (age at diagnosis, sex, primary tumor location, carcinoid syndrome, ENETS TNM classification, Ki-67 proliferation index, grading, prior incomplete surgery) in NET patients. Fasting venous blood samples (N=56) were collected and directly stabilized with 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride after withdrawal. Plasma AG and UAG levels were determined by ELISA. Expression of ghrelin was examined in tumor tissue by immunohistochemistry. There were no significant differences between NET patients and controls in AG (median: 62.5 pg/mL, IQR: 33.1-112.8 vs median: 57.2pg/mL, IQR: 26.7-128.3, P=0.66) and UAG in levels (median: 76.6pg/mL, IQR: 35.23-121.7 vs median: 64.9, IQR: 27.5-93.1, P=0.44). No significant correlations were found between AG, UAG, and AG/UAG ratios versus biochemical and clinical parameters in NET patients with the exception of age at diagnosis (AG: ρ= -0.47, P=0.012; AG/UAG ratio: ρ= -0.50, P=0.007) and baseline chromogranin-A levels (AG/UAG ratio: ρ= -0.44, P=0.019). In our view, fasting plasma acylated and unacylated ghrelin appear to have no value as diagnostic biomarkers in the clinical follow-up of patients with NETs.

The acylated (AG) to unacylated (UAG) ghrelin ratio in esterase inhibitor-treated blood is higher than previously described.

CONTEXT: The acylated/unacylated ghrelin (AG/UAG) ratio has been reported to range from 0·02 to 0·3, suggesting biologically relevant independent regulation of each ghrelin isoform. However, AG is deacylated to UAG by esterases in blood samples, and esterase inhibition is critical for their accurate measurement. Our hypothesis is that at least part of the variation in reported AG and UAG values is due to inconsistent sample preparation. DESIGN: A non-interventional study. Quantification with two different, commercially available, ELISA formats of AG and UAG in venous plasma stabilized or not with 4-(2-aminoethyl) benzenesulphonyl fluoride (AEBSF) and stored for 0-6 months at -20 or -80 °C. PARTICIPANTS: Healthy, non-obese, adults (n = 8; 4 women), age 26-42 yrs, after an overnight fast. MEASUREMENTS: AG and UAG stability following different methods of sample treatment and storage. RESULTS: Non-AEBSF plasma contained low AG and high UAG (>270 pg/ml) indicating rapid conversion of AG to UAG. However, AEBSF plasma, stored at -80 °C and measured at 0, 1, 3 and 6 months contained AG and UAG ranges of 12-350 and 17-170 pg/ml, respectively. Mean (SEM) AG/UAG ratios were 1·7(0·3), 1·2(0·2), 1·5(0·3) and 1·8(0·5) at each time point with no significant effect of storage period. CONCLUSIONS: AG and UAG levels measured in AEBSF-stabilized plasma indicate that the AG/UAG ratio is markedly higher than previously described and that UAG is a physiological component of the circulation. This highlights the importance of immediately stabilizing blood samples on collection for determination of both AG and UAG concentrations and provides a valuable tool for their measurement in physiological and interventional studies.

Functional characterization of a new human melanocortin-4 receptor homozygous mutation (N72K) that is associated with early-onset obesity.

The melanocortin 4 receptor (MC4R) is expressed in the hypothalamus and is essential for regulation of appetite and energy expenditure. MC4R dysfunction in humans causes hyperphagia, impaired satiety and obesity. We have identified a novel c.216C>A (N72 K) homozygous mutation in MC4R in a girl with severe obesity. The patient presented with early-onset obesity and hyperphagia indicating an effect of the homozygous mutation on her phenotype. In silico analyses indicate a damaging effect on receptor function, and the mutation is unusual in occurring in the first intra-cellular loop of the receptor. Site-directed mutagenesis was used to generate plasmid constructs expressing wild-type and mutant MC4R. These were transfected into HEK293 cells and assessed for cAMP responsiveness to α-MSH. Cells expressing N-terminal HA and C-terminal GFP-tagged MC4R were assessed by immunofluorescence confocal microscopy and flow cytometry for correct cell-surface localization. The maximal response of the mutant MC4R to α-MSH was decreased to 20 ± 1 % of the wild type receptor response, and the EC50 was increased from 16.5 ± 5.4 nM to 37.0 ± 8.3 nM. Localization of N- and C-terminally tagged MC4R by confocal microscopy and flow cytometry showed aberrant retention of the mutant receptor in the cytoplasm. Our data describe a rare homozygous inactivating mutation in the first intra-cellular loop of MC4R that markedly impairs its function and is associated with early-onset obesity and hyperphagia.