Proteome profiling identifies circulating biomarkers associated with hepatic steatosis in subjects with Prader-Willi syndrome.

Introduction: Prader-Willi syndrome (PWS) is a rare genetic disorder characterized by loss of expression of paternal chromosome 15q11.2-q13 genes. Individuals with PWS exhibit unique physical, endocrine, and metabolic traits associated with severe obesity. Identifying liver steatosis in PWS is challenging, despite its lower prevalence compared to non-syndromic obesity. Reliable biomarkers are crucial for the early detection and management of this condition associated with the complex metabolic profile and cardiovascular risks in PWS. Methods: Circulating proteome profiling was conducted in 29 individuals with PWS (15 with steatosis, 14 without) using the Olink Target 96 metabolism and cardiometabolic panels. Correlation analysis was performed to identify the association between protein biomarkes and clinical variables, while the gene enrichment analysis was conducted to identify pathways linked to deregulated proteins. Receiver operating characteristic (ROC) curves assessed the discriminatory power of circulating protein while a logistic regression model evaluated the potential of a combination of protein biomarkers. Results: CDH2, CTSO, QDPR, CANT1, ALDH1A1, TYMP, ADGRE, KYAT1, MCFD, SEMA3F, THOP1, TXND5, SSC4D, FBP1, and CES1 exhibited a significant differential expression in liver steatosis, with a progressive increase from grade 1 to grade 3. FBP1, CES1, and QDPR showed predominant liver expression. The logistic regression model, -34.19 + 0.85 * QDPR*QDPR + 0.75 * CANT1*TYMP - 0.46 * THOP1*ALDH1A, achieved an AUC of 0.93 (95% CI: 0.63-0.99), with a sensitivity of 93% and specificity of 80% for detecting steatosis in individuals with PWS. These biomarkers showed strong correlations among themselves and were involved in an interconnected network of 62 nodes, related to seven metabolic pathways. They were also significantly associated with cholesterol, LDL, triglycerides, transaminases, HbA1c, FLI, APRI, and HOMA, and showed a negative correlation with HDL levels. Conclusion: The biomarkers identified in this study offer the potential for improved patient stratification and personalized therapeutic protocols.

Fat-Free Mass Is Better Related to Serum Uric Acid Than Metabolic Homeostasis in Prader-Willi Syndrome.

Background: Prader-Willi syndrome (PWS) is conventionally regarded as a model of genetic obesity carrying a metabolically healthier profile and fat compartmentalization than subjects with non-syndromic obesity. Serum uric acid (sUA) is a recognized surrogate marker of metabolic derangement. As no information is currently available on sUA levels in adults with PWS, we aimed to analyze sUA in a large cohort of adult patients with PWS in comparison to a control counterpart; secondly, we aimed to investigate the metabolic and non-metabolic determinants of sUA in PWS. Methods: A cross-sectional study was conducted on 89 consecutive adult patients with genetically confirmed PWS spanning a wide BMI range (17.2-56.7 kg/m2). As controls, 180 age-, sex- and BMI-matched healthy controls were included. sUA levels were analyzed in relation to the PWS status, metabolic variables, hormone status, body composition, and resting energy expenditure (REE). Bivariate correlation and multivariable regression studies were used to test for predictors of sUA in PWS. Results: Despite having similar BMI values, patients with PWS presented with higher FM (p < 0.0001), lower FFM (p < 0.0001) and REE values than controls (p < 0.0001). In PWS, sUA levels were non-significantly different between subjects with and without obesity (5.4 ± 1.3 vs. 4.9 ± 1.1 mg/dL, p = 0.09), and did not vary significantly in relation to genotype, sex steroid or GH replacement, as well as psychiatric treatments. Rates of hyperuricaemia (19.1% vs. 33.7%, p < 0.01) and absolute sUA levels were lower in patients with PWS compared to controls owing to significant differences between subgroups with obesity (5.5 ± 1.4 vs. 6.6 ± 1.6 mg/dL, p < 0.0001). In merged populations, sUA increased in parallel with age, BMI, FM, FFM, REE, glucolipid homeostasis, and inflammatory markers. In a separate analysis in PWS, however, sUA correlations with BMI, FM, and inflammatory markers were null. Stepwise multivariable regression analysis in the PWS group adjusted for karyotype, age, sex, FM, FFM, obesity, triglycerides, and HDL cholesterol, showed that sUA levels were independently associated with FFM (ß = 0.35, p < 0.0001) and, albeit less significantly, with triglycerides (ß = 0.23, p < 0.05). The introduction of height-normalized FFM (FFM index) in the regression model, however, abrogated the predictive role of FFM on sUA. Conclusions: FFM mass is a strong predictor of sUA. PWS is associated to lower sUA levels than controls likely due to genetic predisposition to different body composition and healthier metabolic phenotype. Further studies are warranted to assess purine metabolism and the clinical significance of the FFM index in PWS.

Circulating microRNA Associated to Different Stages of Liver Steatosis in Prader-Willi Syndrome and Non-Syndromic Obesity.

BACKGROUND: Prader-Willi syndrome (PWS) is a rare and poorly characterized disease. Recent genomic and transcriptomic studies contributed to elucidate the molecular bases of the syndrome. In this study, we characterized the expression of circulating miRNAs in patients with PWS compared to those with non-syndromic obesity in association with liver steatosis. METHODS: MiRNAs were studied by qRT-PCR in serum samples from 30 PWS and 30 non-syndromic obese subjects. RESULTS: MiRNA expression was associated with the presence of the syndrome and to the grade of liver steatosis. MiR-122-5p, miR-151a, miR-92a-3p were up-regulated in obese (4.38-fold, p < 0.01; 2.72-fold, p < 0.05; 1.34-fold p < 0.05, respectively) and were able to differentiate obese from PWS (AUC = 0.81, sens/spec 78/71%). When stratifying groups according to the presence of steatosis, the expression of miR-151a-5p, miR-92a-3p, miR-106b-5p, and miR-93-5p were lower in PWS with steatosis grade 1. Within the group with steatosis grade 1, miR-151a-5p was significantly distinguished PWS from obese (AUC = 0.85, sens/spec 80/85%) and the combination of miR-106b-5p and miR-93-5p showed higher performances in discriminating different grades of steatosis in PWS (AUC = 0.84, sens/spec 93/74%). CONCLUSIONS: MiRNAs represent a tool to better classify and characterize PWS, providing new information about the clinical picture and the extent of steatosis.

The relationship between resting energy expenditure and thyroid hormones in response to short-term weight loss in severe obesity.

BACKGROUND: Regulating thermogenesis is a major task of thyroid hormones (THs), and involves TH-responsive energetic processes at the central and peripheral level. In severe obesity, little is known on the relationship between THs and resting energy expenditure (REE) before and after weight loss. METHODS: We enrolled 100 euthyroid subjects with severe obesity who were equally distributed between genders. Each was examined before and after completion of a 4-wk inpatient multidisciplinary dieting program and subjected to measurement of thyroid function, REE, fat-free mass (FFM, kg) and percent fat mass (FM). RESULTS: Baseline REE was lower than predicted in 70 obese patients, and overall associated with BMI, FFM and FM but not thyroid-related parameters. By the study end, both BMI and REE decreased (5.5% and 4.1%, p<0.001 vs. baseline) and their percent changes were significantly associated (p<0.05), while no association related percent changes of REE and FFM or FM. Individually, REE decreased in 66 and increased in 34 patients irrespective of gender, BMI and body composition. Weight loss significantly impacted TSH (-6.3%), FT3 (-3.3%) and FT4 levels (3.9%; p<0.001 for all). By the study end, a significant correlation became evident between REE and FT4 (r = 0.42, p<0.001) as well as FT3 (r = 0.24, p<0.05). In stepwise multivariable regression analysis, however, neither THs nor body composition entered the regression equation for REE response to weight loss. CONCLUSIONS: In severe obesity, short-term weight loss discloses a positive relationship between REE and THs.

Differences in circulating microRNA signature in Prader-Willi syndrome and non-syndromic obesity.

Prader-Willi syndrome (PWS) represents the most common genetic-derived obesity disorder caused by the loss of expression of genes located on the paternal chromosome 15q11.2-q13. The PWS phenotype shows peculiar physical, endocrine and metabolic characteristics compared to those observed in non-syndromic essential obesity. Since miRNAs have now a well-established role in many molecular pathways, including regulatory networks related to obesity, this pilot study was aimed to characterize the expression of circulating miRNAs in PWS compared to essential obesity. The circulating miRNome of 10 PWS and 10 obese subjects, adequately matched for age, BMI and sex, was profiled throughout Genechip miRNA 4.0 microarray analysis. We identified 362 out of 2578 mature miRNAs to be expressed in serum of the studied population. The circulating miRNA signature significantly characterising the two populations include 34 differently expressed RNAs. Among them, miR-24-3p, miR-122 and miR-23a-3p highly differ between the two groups with a FC >10 in obese compared to PWS. In the obese subjects, miR-7107-5p, miR-6880-3p, miR-6793-3p and miR-4258 were associated to the presence of steatosis. A different signature of miRNAs significantly distinguished PWS with steatosis from PWS without steatosis, involving miR-619-5p, miR-4507, miR-4656, miR-7847-3p and miR-6782-5p. The miRNA target GO enrichment analysis showed the different pathway involved in these two different forms of obesity. Although the rarity of PWS actually represents a limitation to the availability of large series, the present study provides novel hints on the molecular pathogenesis of syndromic and non-syndromic obesity.

Long-term echocardiographic and cardioscintigraphic effects of growth hormone treatment in adults with Prader-Willi syndrome.

CONTEXT: In Prader-Willi syndrome (PWS), an altered GH secretion has been related to reduced cardiac mass and systolic function compared to controls. OBJECTIVE: The objective was to evaluate the cardiovascular response to a 4-year GH therapy in adult PWS patients. STUDY PARTICIPANTS: Study participants were nine severely obese PWS adults (three females, six males) and 13 age-, gender-, and body mass index-matched obese controls. METHODS: In an open-label prospective study, assessment of endocrine parameters and metabolic outcome, whole-body and abdominal fat scans, echocardiography, and radionuclide angiography in unstimulated and dobutamine-stimulated conditions were conducted at baseline and after 1 and 4 years of GH treatment. RESULTS: GH treatment increased IGF-1 (P < .0001), decreased C-reactive protein levels (P < .05), improved visceral fat mass (P < .05), and achieved near-significant changes of fat and fat-free body mass in PWS patients. Left ventricle mass indexed by fat mass increased significantly after 1 and 4 years of GH therapy (P < .05) without evident abnormalities of diastolic function, while a trend toward a reduction of the ejection fraction was documented by echocardiography (P = .054). Radionuclide angiography revealed stable values throughout the study of both the left and right ventricle ejection fractions, although this was accompanied by a statistically nonsignificant reduction of the left ventricle filling rate. A positive association between lean body mass and left ventricle ejection fraction was evident during the study (P < .05). CONCLUSIONS: GH therapy increased the cardiac mass of PWS adults without causing overt abnormalities of systolic and diastolic function. Although the association between lean mass and left ventricle ejection fraction during GH therapy corroborates a favorable systemic outcome of long-term GH treatment in adults with PWS, subtle longitudinal modifications of functional parameters advocate appropriate cardiac monitoring in the long-term therapeutic strategy for PWS.

Skeletal muscle characteristics and motor performance after 2-year growth hormone treatment in adults with prader-willi syndrome.

CONTEXT: In adults with Prader-Willi syndrome (PWS), abnormal body composition with decreased lean body mass and skeletal muscle (SM) volume has been related to altered GH secretion and may possibly contribute to greatly reduced motor capacity. OBJECTIVE: The scope of the study was to test the hypothesis that GH treatment has favorable effects on SM characteristics and motor performance in adults with PWS. DESIGN, SETTING, AND PARTICIPANTS: Fifteen obese PWS subjects (nine males and six females; age range, 19-35 y; body mass index, 37.7-59.9 kg/m(2)) were investigated before and after 12 (GH12) and 24 (GH24) months of GH treatment. MAIN OUTCOME MEASURES: SM cross-sectional area and SM attenuation were determined with computed tomography at the lumbar and midthigh levels. Maximal isometric handgrip strength and isokinetic knee extension peak torque were measured. Motor performance was evaluated with different indoor walking tests, whereas exercise endurance was assessed with a treadmill incremental test to exhaustion. RESULTS: A condition of severe GH deficiency was found in six patients (40%). GH treatment significantly increased lean body mass (GH12, P < .05; GH24, P < .05), reduced percentage of body fat (GH12, P < .05; GH24, P < .05), and augmented SM cross-sectional area and SM attenuation of both lumbar (GH12, P < .01; GH24, P < .001) and thigh muscles (GH24, P < .05). Handgrip strength increased by 7% at GH12 (P < .05) and by 13% at GH24 (P < .001). Peak torque of knee extension extrapolated at zero angular velocity was significantly higher at GH24 (P < .01), and exercise endurance rose by 13% (P < .05) and 17% (P < .05) before exhaustion at GH12 and GH24, respectively, whereas no change was detected with walking tests. No significant difference in the response to GH treatment was detected between patients with and without GH deficiency. CONCLUSION: Long-term GH treatment in adult PWS patients improves body composition and muscle size and quality and increases muscle strength and exercise tolerance independently from the GH secretory status.

Lymphocytes and immunoglobulin patterns across the threshold of severe obesity.

The proinflammatory state of metabolic disorders encompasses the alterations in leukocyte counts and acute-phase reactants, and thus, predisposes to acute and chronic cardiovascular events linked to fat accumulation. Leptin is a marker of adiposity and also yields regulatory effects on innate and adaptive immunity; however, its role on the immune function of obese subjects remains to be elucidated. The aim of this study is to determine the influence of obesity and the role of leptin concentrations on lymphocyte counts and immunoglobulin levels as broad markers of immune function. Cross-sectional analysis in 147 obese (64 M, BMI 43 ± 8.1 kg/m(2)) and 111 age- and sex-matched controls (36 M, BMI 22.5 ± 2.6 kg/m(2)) by assessment of peripheral leukocyte counts, immunoglobulin (Ig) A, G, M levels, leptin, glucose and lipid homeostasis, and acute-phase reactants. Compared to controls, all the leukocyte components were significantly increased in obesity (p < 0.0001 for all) except for basophils and eosinophils. While IgA and IgG levels were similar between groups, IgM levels were lower (p < 0.001) in obese individuals. A significant relationship was evident between leptin and leukocyte counts (p < 0.001), with this latter being correlated to insulin resistance, adiposity, and lipid profile. At the stepwise multiple regression analysis, leukocytes were best predicted by leptin (ß = 0.43, p < 0.0001) and male gender (ß = 0.15, p < 0.05), yet when obesity entered the equation, it acted as an independent predictor of leukocytes (ß = 0.51, p < 0.0001). Leptin also acted as a predictor of IgA levels (ß = 0.20, p < 0.01). Current results show that IgM levels are significantly decreased in patients with obesity in association to significant increments in leukocyte counts. These latter are markedly correlated to leptin levels, insulin resistance, lipid profile, and adiposity. This circumstance, and the significant correlation seen between leptin and IgA levels, may suggest an indirect intervention of leptin in the immunologic alterations consequent to obesity and related to its cardiovascular risk.

Investigations of thyroid hormones and antibodies in obesity: leptin levels are associated with thyroid autoimmunity independent of bioanthropometric, hormonal, and weight-related determinants.

OBJECTIVES: Obesity can alter the thyroid hormone status as a result of a dysregulated endocrine loop between the hypothalamo-pituitary unit and adipose tissue. The adipocytokine leptin has been shown to promote autoimmunity; hence, we aimed to clarify whether leptin excess of obesity could increase the susceptibility to develop autoimmune thyroid disease (AITD). STUDY DESIGN: This cross-sectional study was performed in a tertiary care center. METHODS: Free thyroid hormones, TSH, thyroglobulin, and antithyroid antibodies levels were tested in 165 obese and 118 lean subjects. Results were plotted against variables related to body composition, leptin levels, glucose homeostasis, energy expenditure, and pattern of weight accrual. RESULTS: Compared with controls, obese patients had lower free T3 levels and free T4 levels (P<0.01), greater prevalence of hypothyroidism (P<0.05), and higher commonness of antithyroid antibodies (P<0.05). As a marker of AITD, thyroid peroxidase antibodies were more frequent in the obese group (P<0.01). Correlation analysis showed that leptin levels were associated with AITD (P<0.01) independent of bioanthropometric variables. Multiple logistic regression analysis in pooled groups identified female sex and leptin as significant predictors of AITD. CONCLUSIONS: Obesity increases the susceptibility to harbor AITD with an emerging role for leptin as a peripheral determinant, which needs to be confirmed in future investigations.

Conditional cardiovascular response to growth hormone therapy in adult patients with Prader-Willi syndrome.

CONTEXT: In Prader-Willi syndrome (PWS), an altered GH secretion has been related to reduced cardiac mass and systolic function when compared with controls. OBJECTIVES: The objective of the study was to evaluate the cardiovascular response to GH therapy in adult PWS patients. STUDY PARTICIPANTS: Thirteen obese PWS adults (seven males and six females, aged 26.9+/-1.2 yr, body mass index 46.3+/-1.6 kg/m2) participated in the study. METHODS: Determination of IGF-I, metabolic parameters, echocardiography, and cardioscintigraphy with dobutamine stimulation was made during 12 months GH therapy, with results analyzed by repeated-measures ANOVA. RESULTS: GH therapy increased IGF-I (P<0.0001); decreased C-reactive protein levels (P<0.05); and improved lean mass (P<0.001), fat mass (P<0.05), and visceral fat (P<0.001). Echocardiography showed that 6- and 12-month GH therapy increased left ventricle mass in 76 and in 61% of patients, respectively (P<0.05), did not change diastolic function, and slightly decreased the left ventricle ejection fraction (LVEF) (P=0.054). Cardioscintigraphy documented stable values of LVEF throughout the study, whereas right ventricle ejection fraction decreased significantly (P<0.05) being normally responsive to dobutamine infusion. A positive association between IGF-I z-scores and LVEF occurred at the 6- and 12-month follow-up (P<0.05). CONCLUSIONS: In PWS, GH therapy increased cardiac mass devoid of diastolic consequences. The observation of a slight deterioration of right heart function as well as the association between IGF-I and left ventricular function during GH therapy suggest the need for appropriate cardiac and hormonal monitoring in the therapeutic strategy for Prader-Willi syndrome.

The impact of growth hormone/insulin-like growth factor-I axis and nocturnal breathing disorders on cardiovascular features of adult patients with Prader-Willi syndrome.

CONTEXT: Adult patients with Prader-Willi syndrome (PWS) are prone to develop obesity, GH deficiency (GHD), and their related complications, with cardiopulmonary failure explaining more than half of PWS fatalities. OBJECTIVE AND STUDY PARTICIPANTS: This study was undertaken to examine the effect of GHD and sleep breathing disorders on cardiovascular risk factors and heart features of 13 PWS (age 26.9 +/- 1.2 yr) and 13 age-, gender-, and body mass index-matched obese individuals (age 26.2 +/- 0.8 yr). RESULTS: Compared with controls, PWS patients had lower GH response to arginine+GHRH, IGF-I levels, triglycerides, total and LDL-cholesterol, insulin, and insulin resistance measured by a homeostatic model approach. Dual-energy x-ray absorptiometry, abdominal computed tomography scans, and polysomnography revealed a greater fat mass, similar abdominal fat, but greater sleep breathing disorders in PWS than obese subjects. Echocardiography showed no systolic or diastolic alteration, although PWS had lower left ventricle (LV) mass (135.7 +/- 7.7 vs. 163.5 +/- 8.4 g, P < 0.05) and near significantly lower values of LV end-diastole diameter (P = 0.08), compared with obese controls. Baseline radionuclide angiography documented comparable values of systolic and diastolic values between groups. However, adrenergic stimulation with dobutamine caused a lower increase of LV ejection fraction (71.9 +/- 1.9 vs. 76.3 +/- 1.2%, P < 0.05) and heart rate (103 +/- 6.9 vs. 128 +/- 2.8 beats/min, P < 0.05) in PWS than obese individuals. By multivariate analysis, nocturnal oxygen desaturation and IGF-I levels were main significant predictors of LV mass and heart rate in PWS patients. CONCLUSIONS: PWS differs from simple obesity by a healthier metabolic profile, impaired nocturnal breathing, decreased heart geometry, and systolic and chronotropic performance. GHD and the predictive role of IGF-I on structural and functional heart parameters suggest a GH/IGF-I-mediated control of cardiac risk in PWS.

Free and bound plasma leptin in normal weight and obese men and women: relationship with body composition, resting energy expenditure, insulin-sensitivity, lipid profile and macronutrient preference.

OBJECTIVE: The adipose-borne hormone leptin circulates in free and protein-bound forms but little information is available about their biological significance. Free leptin (FL) levels are related to changes in fat mass, whereas bound leptin (BL) appears to be associated with resting energy expenditure (REE). Our aim was to assess FL and BL levels in normal weight and obese subjects and correlate them with metabolic and nutritional variables. DESIGN AND PATIENTS: The partitioning of plasma leptin between FL and BL was evaluated in a population (n = 44) including both genders and different degrees of adiposity [body mass index (BMI) range 18.6-79.6 kg/m2]. MEASUREMENTS: Total leptin and FL and BL concentrations were measured by fast protein liquid chromatography (FPLC) followed by radioimmunoassay (RIA). Body composition, REE, insulin sensitivity, lipid parameters associated with cardiovascular risk and macronutrient preference were also assessed. RESULTS: The BL/FL ratio was significantly reduced in obese subjects due to a major increase in FL compared with BL. Consequently, the gender difference of the %BL/%FL ratio present in lean subjects (35/65 in women; 65/35 in men) was lost in obese subjects. REE was negatively correlated with total leptin (P < 0.0001) and %FL (P < 0.0001), and positively with %BL (P < 0.001). Total leptin and FL were correlated with the diet carbohydrate content in all subjects. CONCLUSIONS: FL increases with the amount of fat mass; the prevalence of FL in normal weight women in comparison to men suggests that this fraction is particularly linked to the amount of subcutaneous fat. Moreover, the correlation of BL with REE and the relationship of FL with food intake favours the view of different biological activities for the two circulating forms of leptin.