Unacylated ghrelin and obestatin: promising biomarkers of protein energy wasting in children with chronic kidney disease.

BACKGROUND: Impairment in orexigenic/anorexigenic hormone balance may be key in the pathogenesis of protein energy wasting in children with chronic kidney disease (CKD). Measurement of ghrelin and obestatin concentrations in children with CKD would help assess the potential contribution of these hormones to uremic protein energy wasting. METHODS: This was a cross-sectional case-control study. Acylated and unacylated ghrelin and obestatin were measured in 42 children on conservative treatment (CT), 20 children on hemodialysis, 48 pediatric renal transplant (RTx) recipients and 43 controls (CTR) (mean age 11.9, range 5-20 years). Weight, height and bicipital, tricipital, subscapular and suprailiac folds were measured, and the body mass index-standard deviation score (BMI-SDS), percentage of fat mass and fat-free mass were calculated. Urea and creatinine were measured and the glomerular filtration rate (GFR) calculated. RESULTS: Unacylated ghrelin level was higher in patients than controls (p = 0.0001), with the highest levels found in hemodialysis patients (p = 0.001 vs. CKD-CT, p = 0.0001 vs. RTx, p < 0.0001 vs. CTR). Obestatin level was significantly higher in patients on hemodialysis than those on conservative treatment, RTx recipients and controls (p < 0.0001 in each case). Unacylated ghrelin negatively correlated with weight-SDS (p < 0.0001), BMI-SDS (p = 0.0005) and percentage fat mass (p = 0.004) and positively correlated with percentage fat-free mass (p = 0.004). Obestatin concentration negatively correlated with weight-SDS (p = 0.007). Unacylated ghrelin and obestatin concentrations positively correlated with creatinine and urea and inversely with eGFR, even after adjustments for gender, age, puberty and BMI-SDS (p < 0.0001 for each model). CONCLUSIONS: Unacylated ghrelin and obestatin, negatively related to renal function, seem to be promising inverse indicators of nutritional status in children with CKD. Potential therapeutic implications in terms of optimization of their removal in patients on hemodialysis could be hypothesized.

Adiponectin oligomers are similarly distributed in adequate-for-gestational-age obese children irrespective of feeding in their first year.

BACKGROUND: Nutrition and growth in early postnatal life have a role in future diseases. Our aim was to investigate adiponectin oligomers in adequate-for-gestational-age obese children with respect to type and duration of feeding in the first year of life. METHODS: Adiponectin oligomers and cardiometabolic risk factors were measured in 113 adequate-for-gestational-age obese children, divided into group A (prolonged breast feeding, >6 mo), group B (short breast feeding, 1-6 mo), and group C (formula feeding from birth). RESULTS: All the parameters were similar among the groups. Adiponectin oligomers did not correlate with gestational age, months of breast feeding, and time of weaning. Total and high-molecular weight adiponectin were differently distributed across gender and pubertal stages (P < 0.02), being lower in males from the start of puberty. Prepregnancy BMI and at the end of the pregnancy were negatively associated (P < 0.04) with total and medium-molecular weight adiponectin in female and male offspring, respectively. CONCLUSIONS: Adiponectin oligomers and metabolic characteristics are similarly distributed in adequate-for-gestational-age obese children, irrespective of the type and duration of the feeding in the first year of life. Gender and mother's BMI in pregnancy are contributors to adiponectin regulation. Further studies will explain whether breastfeeding protects against metabolic impairment later in life.

Involvement of genes related to inflammation and cell cycle in idiopathic short stature.

Idiopathic Short Stature (ISS) defines a condition in which height is <-2SD compared to the mean of a reference population where systemic, endocrinological, nutritional or chromosomal disorders have not been identified and diagnosis is based on exclusion of any known causes of short stature. JAK/STAT pathway is triggered by GH binding to the GH receptor and promotes cellular growth through transcription of GH-responsive genes. In order to identify "candidate genes" differently expressed in ISS subjects with respect to control ones, we analyzed the expression of 84 genes related to JAK/STAT pathway by RT(2) Profiler PCR array approach in a total of 10 subjects. Then, we validated the observed data by Real Time PCR and ELISA assays in a major number of subjects. We found two genes that were differently expressed in ISS subjects with respect to the control group: CXCL9 and FCGR1A/CD64, both significantly up-regulated (fold change 2.17 and 1.70, respectively) and belonging to family of IFN-γ-inducible factors. Further, ISS subjects showed an increased gene expression of IFN-γ and IFI16, higher serum levels of IFN-γ but similar levels of CXCL9 when compared to healthy subjects. In addition, we showed a pubertal modulation of CXCL9 levels. These data suggest that inflammatory and regulatory factors of the cell cycle may be involved in the ISS condition, introducing a new perspective to its etiology.

Isolated GHD: investigation and implication of JAK/STAT related genes before and after rhGH treatment.

Isolated GH deficiency (IGHD) is a rare disorder that occurs as an idiopathic form in most cases. The pathway JAK/STAT promotes cellular growth and it could be implicated in this condition. In order to characterize IGHD in the pediatric population and identify genes differently expressed before and after GH therapy, we performed a quantitative evaluation of 84 genes related to the JAK/STAT pathway which, by promoting cellular growth. RT(2) Profiler PCR Array and the other/subsequent evaluations were performed in three children with severe IGHD before and after 6 months of GH therapy and in three matched normal children. Gene profiling was modified by the IGHD status and the GH therapy, with a modulation of GHR and some inflammatory genes such as CRP. We found a heterozygous nonsense mutation R43X in the GHR gene in two out of three IGHD subjects, despite a good response to therapy. After therapy cardiovascular markers linked to genes as IL6, IL8 and TNF-α displayed a trend toward reduction. Pre- and post therapy status differently affects gene expression. Mutational screening of GHR may be useful in investigating IGHD's etiology. Genes linked to inflammation suggest to evaluate cardiovascular risks also in pediatric IGHD subjects.

Compound heterozygosity for two GHR missense mutations in a patient affected by Laron Syndrome: a case report.

BACKGROUND: Mutations localized in the Growth Hormone Receptor (GHR) gene are often associated with the pathogenesis of Laron Syndrome, an autosomal recessive hereditary disorder characterized by severe growth retardation. Biochemically, patients present normal to high circulating GH levels, in presence of very low or undetectable IGF-I levels, which do not rise after rhGH treatment. CASE PRESENTATION: We describe the case of a 3.8 years old girl with symmetrical short stature (-3.76 SDS), low IGF-1 and IGFBP-3, in presence of normal GH levels. Parents were not relatives and there was no family history of short stature. During the second day of birth, she developed severe hypoglycaemia that required glucose infusion. She presented frontal bossing and depressed nasal bridge. IGF-1 generation test showed no response, suggesting a GH resistance evidence. In the hypothesis of Laron Syndrome, we decided to perform a molecular analysis of Growth Hormone Receptor (GHR) gene. This analysis demonstrated that the patient was compound heterozygote for two missense mutations. CONCLUSIONS: GHR gene mutations are a well demonstrated cause of GH insensitivity. In heterozygous patients, probably the normal stature may be achieved by a compensatory mechanism of GH secretion or signalling. On the contrary, in homozygous or compound heterozygous patients these compensatory mechanisms are inadequate, and short stature may be the consequence.

Intracoronary Des-Acyl Ghrelin Acutely Increases Cardiac Perfusion Through a Nitric Oxide-Related Mechanism in Female Anesthetized Pigs.

Des-acyl ghrelin (DAG), the most abundant form of ghrelin in humans, has been found to reduce arterial blood pressure and prevent cardiac and endothelial cell apoptosis. Despite this, data regarding its direct effect on cardiac function and coronary blood flow, as well as the related involvement of autonomic nervous system and nitric oxide (NO), are scarce. We therefore examined these issues using both in vivo and in vitro studies. In 20 anesthetized pigs, intracoronary 100 pmol/mL DAG infusion with a constant heart rate and aortic blood pressure, increased coronary blood flow and NO release, whereas reducing coronary vascular resistances (P < .05). Dose responses to DAG were evaluated in five pigs. No effects on cardiac contractility/relaxation or myocardial oxygen consumption were observed. Moreover, whereas the blockade of muscarinic cholinoceptors (n = 5) or α- and ß-adrenoceptors (n = 5 each) did not abolish the observed responses, NO synthase inhibition (n = 5) prevented the effects of DAG on coronary blood flow and NO release. In coronary artery endothelial cells, DAG dose dependently increased NO release through cAMP signaling and ERK1/2, Akt, and p38 MAPK involvement as well as the phosphorylation of endothelial NO synthase. In conclusion, in anesthetized pigs, DAG primarily increased cardiac perfusion through the involvement of NO release. Moreover, the phosphorylation of ERK1/2 and Akt appears to play roles in eliciting the observed NO production in coronary artery endothelial cells.

Interictal ghrelin levels in adult patients with epilepsy.

PURPOSE: In vitro or in animal models of epilepsy, ghrelin showed a clear anticonvulsant action, whose mechanisms are somewhat obscure. In humans however, a controversial relation exists between ghrelin and epilepsy. Yet most studies investigated just total ghrelin levels, without a proper distinction between acylated (AG) or unacylated ghrelin (UAG). We thus evaluated separately AG and UAG interictal levels in adult patients with epilepsy, and their relation to clinical features. METHOD: Cross-sectional study in a tertiary referral centre. Fifty-six patients were recruited: 19 with idiopathic generalized epilepsy, 18 with cryptogenic focal epilepsy and 19 with symptomatic focal epilepsy. Twenty-six healthy subjects of similar age, sex and body mass index (BMI) acted as controls. AG and UAG levels were measured following an overnight fasting and contrasted to the clinical and biometric features. RESULTS: AG and UAG levels were similar between patients and controls. The AG/UAG ratio was higher in patients, also when weighted for covariates (age, BMI, gender, and drugs). Splitting patients according to their epileptic syndrome, drug-resistance or antiepileptic drug number/type resulted in no significant difference in AG, UAG or their ratio. Yet, AG and UAG levels were positively predicted by disease duration, independently by confounders. CONCLUSION: In adult patients with epilepsy, interictal ghrelin levels did not differ from controls, though the AG/UAG ratio was imbalanced. Interpretation of the latter phenomenon is uncertain. Further, levels of AG and UAG were in direct proportion to disease duration, which may represent a long-term compensatory mechanism, antagonistic to the epileptic process.

Pediatric obesity and vitamin D deficiency: a proteomic approach identifies multimeric adiponectin as a key link between these conditions.

Key circulating molecules that link vitamin D (VD) to pediatric obesity and its co-morbidities remain unclear. Using a proteomic approach, our objective was to identify key molecules in obese children dichotomized according to 25OH-vitamin D (25OHD) levels. A total of 42 obese children (M/F = 18/24) were divided according to their 25OHD3 levels into 25OHD3 deficient (VDD; n = 18; 25OHD<15 ng/ml) or normal subjects (NVD; n = 24; >30 ng/ml). Plasma proteomic analyses by two dimensional (2D)-electrophoresis were performed at baseline in all subjects. VDD subjects underwent a 12mo treatment with 3000 IU vitamin D3 once a week to confirm the proteomic analyses. The proteomic analyses identified 53 "spots" that differed between VDD and NVD (p<0.05), amongst which adiponectin was identified. Adiponectin was selected for confirmational studies due to its tight association with obesity and diabetes mellitus. Western Immunoblot (WIB) analyses of 2D-gels demonstrated a downregulation of adiponectin in VDD subjects, which was confirmed in the plasma from VDD with respect to NVD subjects (p<0.035) and increased following 12mo vitamin D3 supplementation in VDD subjects (p<0.02). High molecular weight (HMW) adiponectin, a surrogate indicator of insulin sensitivity, was significantly lower in VDD subjects (p<0.02) and improved with vitamin D3 supplementation (p<0.042). A direct effect in vitro of 1α,25-(OH)2D3 on adipocyte adiponectin synthesis was demonstrated, with adiponectin and its multimeric forms upregulated, even at low pharmacological doses (10(-9) M) of 1α,25-(OH)2D3. This upregulation was paralleled by the adiponectin interactive protein, DsbA-L, suggesting that the VD regulation of adiponectin involves post-transciptional events. Using a proteomic approach, multimeric adiponectin has been identified as a key plasma protein that links VDD to pediatric obesity.

Obestatin levels are associated with C-peptide and antiinsulin antibodies at the onset, whereas unacylated and acylated ghrelin levels are not predictive of long-term metabolic control in children with type 1 diabetes.

CONTEXT AND OBJECTIVE: Ghrelin secretion is altered at the onset and after the start of insulin therapy in children with type 1 diabetes. Contemporary regulation of acylated ghrelin (AG), unacylated ghrelin (UAG), and obestatin (OBST) remains undefined in this disease. It is unknown as to whether they could be good predictors of changes in glucose and metabolic control. DESIGN, SETTING, AND SUBJECTS: This was a longitudinal study conducted in a tertiary care center. AG, UAG, and OBST were measured at baseline and after 2 years of follow-up in 51 children and adolescents with a history of type 1 diabetes extending beyond 1 year. A total of 33 healthy matched subjects were used as controls. RESULTS: Age-, puberty-, and body mass index-adjusted UAG levels were lower (P < .005) and OBST levels were higher (P < .009) in children with type 1 diabetes, with respect to controls. AG levels were similar to controls, but all ratios of the three peptides are altered in diabetic patients. OBST (P < .05) was negatively correlated with C-peptide (P < .05) and insulin antibodies (P < .008) at the onset of diabetes. In diabetic patients, baseline AG and UAG levels were negatively correlated with insulin dosage in the short and long term (P < .001). AG, but not OBST, was positively correlated with C-peptide levels 2 years after diagnosis (P < .05). Overall, the peptides were not predictive of glucose and metabolic control. CONCLUSIONS: UAG, AG, OBST, and their ratios are differently regulated in children with type 1 diabetes, suggesting a role in the metabolic balance of the disease, with insulin a likely regulator of AG and UAG. The peptides do not appear to be good long-term predictors of glucose control, with further investigations needed to explain whether OBST could be a precocious predictor of islet dysfunction.

Endocrine disorders in childhood and adolescence. Natural history of subclinical hypothyroidism in children and adolescents and potential effects of replacement therapy: a review.

OBJECTIVE: Subclinical hypothyroidism (SH) is quite common in children and adolescents. The natural history of this condition and the potential effects of replacement therapy need to be known to properly manage SH. The aim of this review is to analyze: i) the spontaneous evolution of SH, in terms of the rate of reversion to euthyroidism, the persistence of SH, or the progression to over hypothyroidism; and ii) the effects of replacement therapy, with respect to auxological data, thyroid volume, and neuropsychological functions. METHODS: We systematically searched PubMed, Cochrane, and EMBASE (1990-2012) and identified 39 potentially relevant articles of which only 15 articles were suitable to be included. RESULTS AND CONCLUSIONS: SH in children is a remitting process with a low risk of evolution toward overt hypothyroidism. Most of the subjects reverted to euthyroidism or remained SH, with a rate of evolution toward overt hypothyroidism ranging between 0 and 28.8%, being 50% in only one study (nine articles). The initial presence of goiter and elevated thyroglobulin antibodies, the presence of celiac disease, and a progressive increase in thyroperoxidase antibodies and TSH value predict a progression toward overt hypothyroidism. Replacement therapy is not justified in children with SH but with TSH 5-10 mIU/l, no goiter, and negative antithyroid antibodies. An increased growth velocity was observed in children treated with levothyroxine (l-T(4); two articles). l-T(4) reduced thyroid volume in 25-100% of children with SH and autoimmune thyroiditis (two studies). No effects on neuropsychological functions (one study) and posttreatment evolution of SH (one study) were reported.

A novel HESX1 splice mutation causes isolated GH deficiency by interfering with mRNA processing.

OBJECTIVE: Mutations in HESX1 represent a rare cause of GH deficiency (GHD) associated with a broad spectrum of other anomalies. We searched for causative mutations in a cohort of 244 Italian patients affected by combined and isolated GHD (IGHD). METHODS: The HESX1 gene-coding region and exon-intron boundaries were screened by denaturing HPLC scanning. RESULTS: A novel mutation adjacent to the invariant donor splice site of intron 2 (c.357+3G>A) was identified at the heterozygous state in an IGHD patient. The in vitro and in vivo mRNA analysis of the wild-type HESX1 allele revealed the presence of the whole cDNA and two isoforms lacking exon 2 and exons 2-3 respectively. The mutant HESX1 allele yielded only two splicing products, the whole cDNA and the cDNA missing exons 2-3, whereas the mRNA lacking exon 2 was absent. An in vitro assay demonstrated that the exon 2-deleted mRNA, predicting a prematurely truncated protein, is subjected to nonsense-mediated mRNA decay (NMD). CONCLUSIONS: The c.357+3G>A mutation prevents the generation of one of the alternative isoforms normally produced by the wild-type allele, predicting a truncated HESX1 protein. The mutation is likely to cause IGHD in the heterozygous patient by interfering with the downregulation of HESX1 expression mediated by alternative splicing and NMD. Our results open new insight into the mechanism of HESX1 regulation suggesting that the coupling of alternative splicing and NMD might play a fundamental role in directing the HESX1 expression, and that the alteration of this process might lead to severe consequences.

Subclinical hypothyroidism in children and adolescents: a wide range of clinical, biochemical, and genetic factors involved.

OBJECTIVE: The aim of the study was to examine clinical characteristics, biochemical parameters, and TSH-R gene variations in children and adolescents with subclinical hypothyroidism (SH) in order to evaluate their pattern of distribution in SH. PATIENTS: We enrolled 88 patients, each having at least two TSH measurements above the upper limit of the reference range with normal free thyroid hormones and negative thyroid autoantibodies. MAIN OUTCOME MEASURES: Clinical characteristics included height, weight, family history of thyroid diseases, thyroid volume, and echogenicity at ultrasonography. Biochemical parameters included TSH, free thyroid hormones, thyroid autoantibodies, and adjusted daily urinary iodine excretion (UIE). Genetic variations in the TSH-R gene were assessed. RESULTS: The prevalence of overweight/obesity, positive family history of thyroid diseases, and thyroid hypoechogenicity was 28.4, 45.5, and 22.7%, respectively. Median TSH was higher in overweight/obese patients than in normal-weight ones (7.4 vs. 5.7 muIU/ml; P = 0.04) and in overweight/obese patients with hypoechogenicity than in those with normal ultrasound pattern (8.5 vs. 6.8 muIU/ml; P = 0.04). Adjusted daily UIE was lower in subjects without than in those with a positive family history of thyroid diseases (81 vs. 120 mug/d; P = 0.001). The prevalence of a positive family history of thyroid diseases was 1.9-fold higher in patients with nonsynonymous mutations in the TSH-R gene than in patients without any mutation (80 vs. 42%; P = 0.03). A novel mutation at position 1559 in exon 10 (W520X) was detected in one child. CONCLUSIONS: Overweight/obesity, thyroid hypoechogenicity, and nonsynonymous mutations in the TSH-R gene are characterizing features of a large portion of SH children.

Detection of auto-antibodies against cytochrome P4502E1 (CYP2E1) in chronic hepatitis C.

BACKGROUND/AIMS: Chronic hepatitis C (CHC) is often associated with auto-immune reactions. In the light of the role of alcohol in promoting CHC progression, we have investigated the possible presence of auto-reactivity against the ethanol-inducible cytochrome P4502E1 (CYP2E1) in CHC patients with and without alcohol consumption. METHODS: The IgG reactivity against recombinant human CYP2E1 was evaluated by solid-phase immunoassays in 102 CHC patients with different alcohol consumption and 59 HCV-free controls. RESULTS: Auto-antibodies against CYP2E1 were significantly (p<0.0001) increased in CHC patients as compared to controls. Anti-CYP2E1 IgG above the 97th percentile in the controls were evident in 41 (40%) CHC patients. Competition experiments revealed that CYP2E1 recognition was not due to the cross-reactivity with CYP2D6. The detection of anti-CYP2E1 IgG was unrelated to alcohol consumption and no difference in gender, age, aminotransferase levels and virus genotype was evident among the patients with or without anti-CYP2E1 auto-antibodies. However, anti-CYP2E1 auto-reactivity was significantly (p=0.025) associated with the severity of periportal/periseptal interface hepatitis. Moreover, confocal microscopy demonstrated that anti-CYP2E1 IgG associated with CHC recognized CYP2E1 exposed on the outer side of hepatocyte plasma membranes. CONCLUSIONS: HCV infection favours the breaking of self-tolerance against CYP2E1 that might contribute to hepatocyte injury.