Macrophage-associated markers of metaflammation are linked to metabolic dysfunction in pediatric obesity.

BACKGPOUND: Metabolically driven chronic low-grade adipose tissue inflammation, so-called metaflammation, is a central feature in obesity. This inflammatory tone is largely driven by adipose tissue macrophages (ATM), which express pro- and anti-inflammatory markers and cytokines such as, e.g., IL-1 receptor antagonist (IL-1RA), CD163 and osteopontin (OPN). Metaflammation ultimately leads to the development of cardiometabolic diseases. This study aimed to evaluate the association between selected adipose tissue macrophage-associated markers and metabolic comorbidities in pediatric obesity. METHODS: From a pediatric cohort with obesity (n = 108), clinically thoroughly characterized including diverse routine blood parameters, oral glucose tolerance test and liver MRI, plasma IL-1RA, soluble (s)CD163 and OPN were measured by ELISA. RESULTS: We observed significantly higher IL-1RA, sCD163, and OPN levels in the plasma of children with metabolic-dysfunction associated fatty liver disease (MAFLD) and metabolic syndrome. Moreover, IL-1RA and sCD163 correlated with hepatic disease and apoptosis markers alanine aminotransferase and CK-18. IL-1RA concentrations additionally correlated with insulin resistance, while children with disturbed glucose metabolism had significantly higher levels of sCD163. CONCLUSION: MAFLD and other metabolic disorders in pediatric patients with obesity are associated with an elevation of adipose tissue macrophage-related inflammation markers.

Risk Scores for Metabolic Dysfunction-Associated Fatty Liver Disease in Pediatric Obesity.

INTRODUCTION: Early noninvasive detection of incipient liver damage is crucial to prevent long-term adverse health outcomes. A variety of scores to assess liver status have been proposed, mostly for adult populations. Validation of noninvasive hepatic scores to identify children at risk of metabolic dysfunction-associated fatty liver disease (MAFLD) is a gap in research, particularly in youth with severe obesity considering pubertal stage and sex. METHODS: In a well-characterized pediatric population aged 9-19 years (n = 115), 19 published liver scores were analyzed. The area under the receiver operating characteristic curve (AUROC) for determination of MAFLD as assessed by magnetic resonance imaging was calculated. RESULTS: The pediatric indices PNFI, B-AST, and M-APRI and several scores developed in adults significantly differed in children with MAFLD compared to children without, while some established indices did not. Only nonalcoholic fatty liver disease liver fat score (NAFLD-LFS) and the model by Cao et al. [PLoS One. 2013;8(12):e82092] showed acceptable predictive accuracy (AUROC >0.8) independently of pubertal stage and sex. When stratifying for pubertal stage and sex, the GSG-Index was superior in pubertal girls, and NAFLD-LFS performed best in pubertal boys. CONCLUSION: NAFLD-LFS and the model by Cao et al. [PLoS One. 2013;8(12):e82092] were well suited to predict MAFLD in youth with severe obesity. In pubertal children, GSG-Index and NAFLD-LFS performed best in girls and boys, respectively.

Plasma Myostatin Increases with Age in Male Youth and Negatively Correlates with Vitamin D in Severe Pediatric Obesity.

Obesity already causes non-communicable diseases during childhood, but the mechanisms of disease development are insufficiently understood. Myokines such as myostatin and irisin are muscle-derived factors possibly involved in obesity-associated diseases. This explorative study aims to investigate whether myostatin and irisin are associated with metabolic parameters, including the vitamin D status in pediatric patients with severe obesity. Clinical, anthropometric and laboratory data from 108 patients with severe obesity (>97th percentile) aged between 9 and 19 years were assessed. Myostatin, its antagonist follistatin, and irisin, were measured from plasma by ELISA. Myostatin concentrations, particularly in males, positively correlated with age and pubertal stage, as well as metabolic parameters such as insulin resistance. Irisin concentrations correlated positively with HDL and negatively with LDL cholesterol values. For follistatin, the associations with age and pubertal stage were inverse. Strikingly, a negative correlation of myostatin with serum vitamin D levels was observed that remained significant after adjusting for age and pubertal stage. In conclusion, there is an independent association of low vitamin D and elevated myostatin levels. Further research may focus on investigating means to prevent increased myostatin levels in interventional studies, which might open several venues to putative options to treat and prevent obesity-associated diseases.

Tryptophan Metabolism Is Associated with BMI and Adipose Tissue Mass and Linked to Metabolic Disease in Pediatric Obesity.

The obesity epidemic has contributed to an escalating prevalence of metabolic diseases in children. Overnutrition leads to increased tryptophan uptake and availability. An association between the induction of the tryptophan catabolic pathway via indoleamine 2,3-dioxygenase (IDO) activity and obesity-related inflammation has been observed. This study aimed to investigate the impact of pediatric obesity on tryptophan metabolism and the potential relationship with metabolic disease. In this prospective cohort study, plasma kynurenine, tryptophan, and serotonin levels were measured by ELISA, and IDO activity was estimated by calculating the kynurenine/tryptophan ratio in a clinically characterized population with severe obesity (BMI ≥ 97th percentile) aged 9 to 19 (n = 125). IDO activity and its product kynurenine correlated with BMI z-score and body fat mass, whereas concentrations of serotonin, the alternative tryptophan metabolite, negatively correlated with these measures of adiposity. Kynurenine and tryptophan, but not serotonin levels, were associated with disturbed glucose metabolism. Tryptophan concentrations negatively correlated with adiponectin and were significantly higher in prediabetes and metabolically unhealthy obesity. In conclusion, BMI and body fat mass were associated with increased tryptophan catabolism via the kynurenine pathway and decreased serotonin production in children and adolescents with severe obesity. The resulting elevated kynurenine levels may contribute to metabolic disease in obesity.

Circulating microRNAs 34a, 122, and 192 are linked to obesity-associated inflammation and metabolic disease in pediatric patients.

BACKGROUND: Obesity-associated chronic low-grade inflammation leads to dysregulation of central lipid and glucose metabolism pathways leading to metabolic disorders. MicroRNAs (miRNAs) are known to control regulators of metabolic homeostasis. We aimed to assess the relationship of circulating miRNAs with inflammatory modulators and metabolic disorders in pediatric obesity. METHODS: From a pediatric cohort with severe obesity (n = 109), clinically thoroughly characterized including diverse routine blood parameters, oral glucose tolerance test, and liver MRI, a panel of 16 circulating miRNAs was quantified using qRT-PCR. Additionally, markers of inflammation TNFα, IL1 receptor antagonist, procalcitonin, CRP, and IL-6 were measured. RESULTS: Markers of obesity-associated inflammation, TNFα, IL-1Ra, and procalcitonin, all significantly correlated with concentrations of miRNAs 122 and 192. Concentrations of these miRNAs negatively correlated with serum adiponectin and were among those strongly linked to parameters of dyslipidemia and liver function. Moreover, miRNA122 concentrations correlated with HOMA-IR. Several miRNA levels including miRNAs 34a, 93, 122, and 192 were statistically significantly differing between individuals with prediabetes, impaired glucose tolerance, metabolic syndrome, or nonalcoholic fatty liver disease compared to the respective controls. Additionally, miRNA 192 was significantly elevated in metabolically unhealthy obesity. CONCLUSIONS: A miRNA pattern associated with obesity-associated inflammation and comorbidities may be used to distinguish metabolically healthy from unhealthy pediatric patients with obesity. Moreover, these changes in epigenetic regulation could potentially be involved in the etiology of obesity-linked metabolic disease in children and adolescents.

Cystic Fibrosis Newborn Screening in Austria Using PAP and the Numeric Product of PAP and IRT Concentrations as Second-Tier Parameters.

In Austria, newborns have been screened for cystic fibrosis (CF) by analyzing immunoreactive trypsinogen (IRT) from dried blood spots (DBS)s for nearly 20 years. Recently, pancreatitis-associated protein (PAP) analysis was introduced as a second-tier test with the aim of reducing recalls for second DBS cards while keeping sensitivity high. For 28 months, when IRT was elevated (65-130 ng/mL), PAP was measured from the first DBS (n = 198,927) with a two-step cut-off applied. For the last 12 months of the observation period (n = 85,421), an additional IRT×PAP cut-off was introduced. If PAP or IRT×PAP were above cut-off, a second card was analyzed for IRT and in case of elevated values identified as screen-positive. Above 130 ng/mL IRT in the first DBS, newborns were classified as screen-positive. IRT analysis of first DBS resulted in 1961 (1%) tests for PAP. In the first 16 months, 26 of 93 screen-positive were confirmed to have CF. Two false-negatives have been reported (sensitivity = 92.8%). Importantly, less than 30% of families compared to the previous IRT-IRT screening scheme had to be contacted causing distress. Adding IRT×PAP caused a marginally increased number of second cards and sweat tests to be requested during this period (15 and 3, respectively) compared to the initial IRT-PAP scheme. One case of confirmed CF was found due to IRT×PAP, demonstrating an increase in sensitivity. Thus, the relatively simple and economical algorithm presented here performs effectively and may be a useful model for inclusion of CF into NBS panels or modification of existing schemes.

A branched-chain amino acid-based metabolic score can predict liver fat in children and adolescents with severe obesity.

BACKGROUND: Eighty percent of adolescents with severe obesity suffer from non-alcoholic fatty liver disease (NAFLD). Non-invasive prediction models have been tested in adults, however, they performed poorly in paediatric populations. OBJECTIVE: This study aimed to investigate novel biomarkers for NAFLD and to develop a score that predicts liver fat in youth with severe obesity. METHODS: From a population with a BMI >97th percentile aged 9-19 years (n = 68), clinically thoroughly characterized including MRI-derived proton density fat fraction (MRI-PDFF), amino acids and acylcarnitines were measured by HPLC-MS. RESULTS: In children with NAFLD, higher levels of plasma branched-chain amino acids (BCAA) were determined. BCAAs correlated with MRI-PDFF (R = 0.46, p < .01). We identified a linear regression model adjusted for age, sex and pubertal stage consisting of BCAAs, ALT, GGT, ferritin and insulin that predicted MRI-PDFF (R = 0.75, p < .01). ROC analysis of this model revealed AUCs of 0.85, 0.85 and 0.92 for the detection of any, moderate and severe steatosis, respectively, thus markedly outperforming previously published scores. CONCLUSION: BCAAs could be an important link between obesity and other metabolic pathways. A BCAA-based metabolic score can predict steatosis grade in high-risk children and adolescents and may provide a feasible alternative to sophisticated methods like MRI or biopsy in the future.

DNA methylated alleles of the phenylalanine hydroxylase promoter remodeled at elevated phenylalanine levels in newborns with hyperphenylalaninemia.

OBJECTIVES: Although high phenylalanine (phe) exposure has been shown to influence the DNA methylation status of leukocytes in hyperphenylalaninemia (HPA), the potential of DNA methylation changes as a biomarker of pretreatment high phe exposure in diet free newborns with HPA has not been explored. We therefore investigated the DNA methylation pattern of the phenylalanine hydroxylase (PAH) gene promoter at different phe levels, and the possibility of DNA methylation pattern changes being a biomarker of high phe exposure in diet free newborns with HPA. DESIGN AND METHODS: With a combination of methylated PCR, high resolution melting, and sequencing, the cytosine phosphodiester bond guanine (CpG) dinucleotides in the 5' untranslated region of the PAH gene were analysed 2-15days after birth using leukocyte DNA from diet free 16 newborns with HPA and 16 healthy controls. RESULTS: In 2-3days blood cards, GTGTG and GTGC/TG alleles were both detected at similar low mean phe levels in healthy controls (59.39±14.62 and 55.33±13.43µmol/L) and non-phenylketonuria (PKU) HPA (265.00 and 244.25±73.73µmol/L). In HPA with PKU, the GTGTG and GTGC/TG alleles were both detected at dissimilar elevated mean phe levels (380.80±64.62 and 589.00±191.96µmol/L). In ≥7day blood cards, GTGTG and GTGC/TG alleles were both detected at similar excess mean phe levels in HPA with PKU (2297±374.38 and 1562.66±718.23µmol/L). CONCLUSION: The demethylated GTGTG and partial methylated GTGC/TG alleles are not pathogenic alleles. Our results suggest a specific remodeling of the DNA methylated alleles of the PAH promoter at elevated, but not excess phe levels in diet free newborns with PKU.

Demethylation of the promoter region of GPX3 in a newborn with classical phenylketonuria.

OBJECTIVES: Phenylketonuria (PKU) is characterized by a high phenylalanine (phe) in plasma and oxidative stress. However, the monitoring of oxidative stress in newborns with PKU using the activity levels of antioxidant enzymes is not optimal. We investigated the possibility of monitoring an increased reactive oxygen species (ROS) production using DNA methylation changes of an oxidative stress response element in the promoter region of an enzymatic antioxidant gene. DESIGN AND METHODS: Using DNA extracted from blood leukocytes, the cytosine phosphodiester bond guanine positions of an overlapping CCAAT box/metal response element (CGATTGGCTG) of the glutathione peroxidase 3 promoter activated by oxidative stimuli and expressed in plasma were analysed for methylation changes in 20 newborns with hyperphenylalaninemia and 20 healthy controls. RESULTS: A demethylated allele was detected in a PKU patient at a phe level of 465µmol/L on day 2 after birth, but not in other patients (phe<465µmol/L, ≥day 2 after birth; phe>465µmol/L, ≥day 3 after birth) and healthy controls (phe<465µmol/L, ≥day 2 after birth). CONCLUSIONS: The detection of the demethylated allele could be time and phe concentration dependent. The demethylated allele is suggested as an early epigenetic marker for an extracellular monitoring of an increased ROS production in newborns with PKU.

Subcellular localization of ceramide kinase and ceramide kinase-like protein requires interplay of their Pleckstrin Homology domain-containing N-terminal regions together with C-terminal domains.

Ceramide kinase (CERK) and the ceramide kinase-like protein (CERKL), two related members of the diacylglycerol kinase family, are ill-defined at the molecular level. In particular, what determines their distinctive subcellular localization is not well understood. Here we show that the Pleckstrin Homology (PH) domain of CERK, which is required for Golgi complex localization, can substitute for the N-terminal region of CERKL and allow for wild-type CERKL localization, which is typified by nucleolar accumulation. This demonstrates that determinants for localization of these two enzymes do not lie solely in their PH domain-containing N-terminal regions. Moreover, we present evidence for a previously unrecognized participation of CERK distal sequences in structural stability, localization and activity of the full-length protein. Progressive deletion of CERK and CERKL from the C-terminus revealed similar sequential organization in both proteins, with nuclear import signals in their N-terminal part, and nuclear export signals in their C-terminal part. Furthermore, mutagenesis of individual cysteine residues of a CERK-specific CXXXCXXC motif severely compromised both exportation of CERK from the nucleus and its association with the Golgi complex. Altogether, this work identifies conserved domains in CERK and CERKL as well as new determinants for their subcellular localization. It further suggests a nucleocytoplasmic shuttling mechanism for both proteins that may be defective in CERKL mutant proteins responsible for retinal degenerative diseases.

A conserved cysteine motif essential for ceramide kinase function.

Ceramide kinase (CerK) is a sphingolipid metabolizing enzyme very sensitive to oxidation; however, the determinants are unknown. We show here that the thiol-modifying agent N-ethyl-maleimide abrogates CerK activity in vitro and in a cell based assay, implying that important cysteine residues are accessible in purified as well as endogenous CerK. We replaced every 22 residues in human CerK, by an alanine, and measured activity in the resulting mutant proteins. This led to identification of a cluster of cysteines, C(347)XXXC(351)XXC(354), essential for CerK function. These findings are discussed based on homology modeling of the catalytic domain of CerK.

Neutropenia with impaired immune response to Streptococcus pneumoniae in ceramide kinase-deficient mice.

In mammals, ceramide kinase (CerK)-mediated phosphorylation of ceramide is the only known pathway to ceramide-1-phosphate (C1P), a recently identified signaling sphingolipid metabolite. To help delineate the roles of CerK and C1P, we knocked out the gene of CerK in BALB/c mice by homologous recombination. All in vitro as well as cell-based assays indicated that CerK activity is completely abolished in Cerk-/- mice. Labeling with radioactive orthophosphate showed a profound reduction in the levels of de novo C1P formed in Cerk-/- macrophages. Consistently, mass spectrometry analysis revealed a major contribution of CerK to the formation of C16-C1P. However, the significant residual C1P levels in Cerk-/- animals indicate that alternative routes to C1P exist. Furthermore, serum levels of proapoptotic ceramide in these animals were significantly increased while levels of dihydroceramide as the biosynthetic precursor were reduced. Previous literature pointed to a role of CerK or C1P in innate immune cell function. Using a variety of mechanistic and disease models, as well as primary cells, we found that macrophage- and mast cell-dependent readouts are barely affected in the absence of CerK. However, the number of neutrophils was strikingly reduced in blood and spleen of Cerk-/- animals. When tested in a model of fulminant pneumonia, Cerk-/- animals developed a more severe disease, lending support to a defect in neutrophil homeostasis following CerK ablation. These results identify ceramide kinase as a key regulator of C1P, dihydroceramide and ceramide levels, with important implications for neutrophil homeostasis and innate immunity regulation.

Enhanced ceramide-induced apoptosis in ceramide kinase overexpressing cells.

We evaluated how increased levels of ceramide kinase (CerK) would impact the growth of COS-1 fibroblasts and RBL-2H3 basophils. The low CerK activity in these cells was strongly up-regulated upon recombinant expression of CerK. CerK-overexpressing COS-1 cells depended on higher concentrations of serum for their growth and displayed many filipodia. The two CerK-overexpressing cell lines were more sensitive to C2-ceramide-mediated apoptosis, and this correlated with the production of C2-ceramide-1-phosphate by CerK. This study indicates that ceramide kinase may participate in the control of cell growth, and establishes a novel assay that will be valuable for testing ceramide kinase inhibitors.