Rare Antagonistic Leptin Variants and Severe, Early-Onset Obesity.

Hormone absence or inactivity is common in congenital disease, but hormone antagonism remains controversial. Here, we characterize two novel homozygous leptin variants that yielded antagonistic proteins in two unrelated children with intense hyperphagia, severe obesity, and high circulating levels of leptin. Both variants bind to the leptin receptor but trigger marginal, if any, signaling. In the presence of nonvariant leptin, the variants act as competitive antagonists. Thus, treatment with recombinant leptin was initiated at high doses, which were gradually lowered. Both patients eventually attained near-normal weight. Antidrug antibodies developed in the patients, although they had no apparent effect on efficacy. No severe adverse events were observed. (Funded by the German Research Foundation and others.).

The obesity-linked human lncRNA AATBC stimulates mitochondrial function in adipocytes.

Adipocytes are critical regulators of metabolism and energy balance. While white adipocyte dysfunction is a hallmark of obesity-associated disorders, thermogenic adipocytes are linked to cardiometabolic health. As adipocytes dynamically adapt to environmental cues by functionally switching between white and thermogenic phenotypes, a molecular understanding of this plasticity could help improving metabolism. Here, we show that the lncRNA Apoptosis associated transcript in bladder cancer (AATBC) is a human-specific regulator of adipocyte plasticity. Comparing transcriptional profiles of human adipose tissues and cultured adipocytes we discovered that AATBC was enriched in thermogenic conditions. Using primary and immortalized human adipocytes we found that AATBC enhanced the thermogenic phenotype, which was linked to increased respiration and a more fragmented mitochondrial network. Expression of AATBC in adipose tissue of mice led to lower plasma leptin levels. Interestingly, this association was also present in human subjects, as AATBC in adipose tissue was inversely correlated with plasma leptin levels, BMI, and other measures of metabolic health. In conclusion, AATBC is a novel obesity-linked regulator of adipocyte plasticity and mitochondrial function in humans.

An integrated approach to identify environmental modulators of genetic risk factors for complex traits.

Complex traits and diseases can be influenced by both genetics and environment. However, given the large number of environmental stimuli and power challenges for gene-by-environment testing, it remains a critical challenge to identify and prioritize specific disease-relevant environmental exposures. We propose a framework for leveraging signals from transcriptional responses to environmental perturbations to identify disease-relevant perturbations that can modulate genetic risk for complex traits and inform the functions of genetic variants associated with complex traits. We perturbed human skeletal-muscle-, fat-, and liver-relevant cell lines with 21 perturbations affecting insulin resistance, glucose homeostasis, and metabolic regulation in humans and identified thousands of environmentally responsive genes. By combining these data with GWASs from 31 distinct polygenic traits, we show that the heritability of multiple traits is enriched in regions surrounding genes responsive to specific perturbations and, further, that environmentally responsive genes are enriched for associations with specific diseases and phenotypes from the GWAS Catalog. Overall, we demonstrate the advantages of large-scale characterization of transcriptional changes in diversely stimulated and pathologically relevant cells to identify disease-relevant perturbations.

Metabolic and other morbid complications in congenital generalized lipodystrophy type 4.

Morbidity and mortality rates in patients with autosomal recessive, congenital generalized lipodystrophy type 4 (CGL4), an ultra-rare disorder, remain unclear. We report on 30 females and 16 males from 10 countries with biallelic null variants in CAVIN1 gene (mean age, 12 years; range, 2 months to 41 years). Hypertriglyceridemia was seen in 79% (34/43), hepatic steatosis in 82% (27/33) but diabetes mellitus in only 21% (8/44). Myopathy with elevated serum creatine kinase levels (346-3325 IU/L) affected all of them (38/38). 39% had scoliosis (10/26) and 57% had atlantoaxial instability (8/14). Cardiac arrhythmias were detected in 57% (20/35) and 46% had ventricular tachycardia (16/35). Congenital pyloric stenosis was diagnosed in 39% (18/46), 9 had esophageal dysmotility and 19 had intestinal dysmotility. Four patients suffered from intestinal perforations. Seven patients died at mean age of 17 years (range: 2 months to 39 years). The cause of death in four patients was cardiac arrhythmia and sudden death, while others died of prematurity, gastrointestinal perforation, and infected foot ulcers leading to sepsis. Our study highlights high prevalence of myopathy, metabolic abnormalities, cardiac, and gastrointestinal problems in patients with CGL4. CGL4 patients are at high risk of early death mainly caused by cardiac arrhythmias.

Metabolomics in human SGBS cells as new approach method for studying adipogenic effects: Analysis of the effects of DINCH and MINCH on central carbon metabolism.

Growing evidence suggests that exposure to certain metabolism-disrupting chemicals (MDCs), such as the phthalate plasticizer DEHP, might promote obesity in humans, contributing to the spread of this global health problem. Due to the restriction on the use of phthalates, there has been a shift to safer declared substitutes, including the plasticizer diisononyl-cyclohexane-1,2-dicarboxylate (DINCH). Notwithstanding, recent studies suggest that the primary metabolite monoisononyl-cyclohexane-1,2-dicarboxylic acid ester (MINCH), induces differentiation of human adipocytes and affects enzyme levels of key metabolic pathways. Given the lack of methods for assessing metabolism-disrupting effects of chemicals on adipose tissue, we used metabolomics to analyze human SGSB cells exposed to DINCH or MINCH. Concentration analysis of DINCH and MINCH revealed that uptake of MINCH in preadipocytes was associated with increased lipid accumulation during adipogenesis. Although we also observed intracellular uptake for DINCH, the solubility of DINCH in cell culture medium was limited, hampering the analysis of possible effects in the µM concentration range. Metabolomics revealed that MINCH induces lipid accumulation similar to peroxisome proliferator-activated receptor gamma (PPARG)-agonist rosiglitazone through upregulation of the pyruvate cycle, which was recently identified as a key driver of de novo lipogenesis. Analysis of the metabolome in the presence of the PPARG-inhibitor GW9662 indicated that the effect of MINCH on metabolism was mediated at least partly by a PPARG-independent mechanism. However, all effects of MINCH were only observed at high concentrations of 10 µM, which are three orders of magnitudes higher than the current concentrations of plasticizers in human serum. Overall, the assessment of the effects of DINCH and MINCH on SGBS cells by metabolomics revealed no adipogenic potential at physiologically relevant concentrations. This finding aligns with previous in vivo studies and supports the potential of our method as a New Approach Method (NAM) for the assessment of adipogenic effects of environmental chemicals.

The age of the obesity onset is a very important factor for the development of metabolic complications and cardiovascular risk in children and adolescents with severe obesity.

Severe obesity defined as BMI value corresponding to an adult > 40 kg/m2 affects 1-5% of children and adolescents in Europe. The purpose of this study was to assess the occurrence of cardiovascular risk factors in children and adolescents with severe obesity. The analysis included 140 patients (75 female) at the mean age of 14 ± 2.1 SD (range 10-18) years (all recruited in 4 regional reference centers in Poland). Severe obesity was defined as BMI > 35 kg/m2 (children 6-14 years), and BMI > 40 kg/m2 (> 14 years). Fasting plasma samples have been obtained in all patients, and OGTT was performed in all patients. The metabolic risk factors were defined as high blood pressure (BP > 90 percentile for height, age, and sex), HDL cholesterol < 1.03 mmol/L, TG ≥ 1.7 mmol/L, and hyperglycemic state (fasting blood glucose > 5.6 mmol/L, or blood glucose 120' after oral glucose load > 7.8 mmol/L). Additionally, the MetS z-score was calculated using Metabolic Syndrome Severity Calculator. One hundred twenty-four (89%) participants presented with high BP, 117 (84%) with abnormal lipid profile, and 26 with the hyperglycemic. Only 12 (9%) were free of metabolic complications. More than 60% of patients had more than one cardiovascular risk factor. The high BP was significantly associated with the severity of obesity (F = 9.9, p = 0.002). Patients with at least one metabolic complication presented with significantly younger age of the onset of obesity (the mean age of the patients with no overt obesity complications was 10 years, while the mean age of those who presented at least one was 4.7 ± 3.5 SD years (p = 0.002)). A significant positive association between in the value of the Mets BMI z-score with age was observed (R = 0.2, p < 0.05). There were no differences between girls and boys regarding Mets BMI z-score (1.7 ± 0.8 vs 1.7 ± 0.7, p = 0.8).Conclusions: The most common metabolic risk factor in children and adolescents with severe obesity was high BP. The most important factor determining presence of obesity complications, and thus the total metabolic risk, seems to be younger (< 5 years) age of onset of obesity. What is Known? • It is estimated that 1-5% of children and adolescents in Europe suffer from severe obesity corresponding to an adult BMI > 40 kg/m2, and it is the fastest growing subcategory of childhood obesity. • Children with severe obesity face substantial health risk that may persist into adulthood, encompassing chronic conditions, psychological disorders and premature mortality. What is new: • The most common complication is high BP that is significantly associated with the severity of obesity (BMI z-score), contrary to dyslipidemia and hyperglycemic state, which do not depend on BMI z-score value. • The most important factor determining presence of obesity complications, and thus the total metabolic risk, seems to be younger (< 5 years) age of onset of obesity.

[Improved Care and Treatment Options for Patients with Hyperphagia-Associated Obesity in Bardet-Biedl Syndrome]. / Verbesserte Versorgungs-und Behandlungsoptionen für Patienten mit Hyperphagie-assoziierter Adipositas bei Bardet-Biedl-Syndrom.

Bardet-Biedl syndrome (BBS) is a rare, autosomal recessive multisystem disease. The pathophysiological origin is a dysfunction of the primary cilium. Clinical symptoms are heterogeneous and variable: retinal dystrophy, obesity, polydactyly, kidney abnormalities, hypogenitalism and developmental delays are the most common features. By the approval of the melanocortin 4 receptor agonist setmelanotide, a drug therapy for BBS-associated hyperphagia and obesity can be offered for the first time. Hyperphagia and severe obesity represent a considerable burden and are associated with comorbidity and increased mortality risk. Due to the limited experience with setmelanotide in BBS, a viable comprehensive therapy concept is to be presented. Therapy decision and management should be conducted in expert centers. For best therapeutic effects with setmelanotide adequate information of the patient about the modalities of the therapy (daily subcutaneous injection) and possible adverse drug events are necessary. Furthermore, the involvement of psychologists, nutritionists and nursing services (support for the application) should be considered together with the patient. The assessment of therapy response should be carried out with suitable outcome measurements and centrally reported to an adequate register.

Chromatin accessibility and gene expression during adipocyte differentiation identify context-dependent effects at cardiometabolic GWAS loci.

Chromatin accessibility and gene expression in relevant cell contexts can guide identification of regulatory elements and mechanisms at genome-wide association study (GWAS) loci. To identify regulatory elements that display differential activity across adipocyte differentiation, we performed ATAC-seq and RNA-seq in a human cell model of preadipocytes and adipocytes at days 4 and 14 of differentiation. For comparison, we created a consensus map of ATAC-seq peaks in 11 human subcutaneous adipose tissue samples. We identified 58,387 context-dependent chromatin accessibility peaks and 3,090 context-dependent genes between all timepoint comparisons (log2 fold change>1, FDR<5%) with 15,919 adipocyte- and 18,244 preadipocyte-dependent peaks. Adipocyte-dependent peaks showed increased overlap (60.1%) with Roadmap Epigenomics adipocyte nuclei enhancers compared to preadipocyte-dependent peaks (11.5%). We linked context-dependent peaks to genes based on adipocyte promoter capture Hi-C data, overlap with adipose eQTL variants, and context-dependent gene expression. Of 16,167 context-dependent peaks linked to a gene, 5,145 were linked by two or more strategies to 1,670 genes. Among GWAS loci for cardiometabolic traits, adipocyte-dependent peaks, but not preadipocyte-dependent peaks, showed significant enrichment (LD score regression P<0.005) for waist-to-hip ratio and modest enrichment (P < 0.05) for HDL-cholesterol. We identified 659 peaks linked to 503 genes by two or more approaches and overlapping a GWAS signal, suggesting a regulatory mechanism at these loci. To identify variants that may alter chromatin accessibility between timepoints, we identified 582 variants in 454 context-dependent peaks that demonstrated allelic imbalance in accessibility (FDR<5%), of which 55 peaks also overlapped GWAS variants. At one GWAS locus for palmitoleic acid, rs603424 was located in an adipocyte-dependent peak linked to SCD and exhibited allelic differences in transcriptional activity in adipocytes (P = 0.003) but not preadipocytes (P = 0.09). These results demonstrate that context-dependent peaks and genes can guide discovery of regulatory variants at GWAS loci and aid identification of regulatory mechanisms.

A single-cell CRISPRi platform for characterizing candidate genes relevant to metabolic disorders in human adipocytes.

CROP-Seq combines gene silencing using CRISPR interference with single-cell RNA sequencing. Here, we applied CROP-Seq to study adipogenesis and adipocyte biology. Human preadipocyte SGBS cell line expressing KRAB-dCas9 was transduced with a sgRNA library. Following selection, individual cells were captured using microfluidics at different timepoints during adipogenesis. Bioinformatic analysis of transcriptomic data was used to determine the knockdown effects, the dysregulated pathways, and to predict cellular phenotypes. Single-cell transcriptomes recapitulated adipogenesis states. For all targets, over 400 differentially expressed genes were identified at least at one timepoint. As a validation of our approach, the knockdown of PPARG and CEBPB (which encode key proadipogenic transcription factors) resulted in the inhibition of adipogenesis. Gene set enrichment analysis generated hypotheses regarding the molecular function of novel genes. MAFF knockdown led to downregulation of transcriptional response to proinflammatory cytokine TNF-α in preadipocytes and to decreased CXCL-16 and IL-6 secretion. TIPARP knockdown resulted in increased expression of adipogenesis markers. In summary, this powerful, hypothesis-free tool can identify novel regulators of adipogenesis, preadipocyte, and adipocyte function associated with metabolic disease.NEW & NOTEWORTHY Genomics efforts led to the identification of many genomic loci that are associated with metabolic traits, many of which are tied to adipose tissue function. However, determination of the causal genes, and their mechanism of action in metabolism, is a time-consuming process. Here, we use an approach to determine the transcriptional outcome of candidate gene knockdown for multiple genes at the same time in a human cell model of adipogenesis.

Myricetin attenuates hypoxia-induced inflammation in human adipocytes.

BACKGROUND: Adipose tissue hypoxia plays a crucial role in the development of chronic low-grade systemic inflammation which has been associated with the pathogenesis of obesity-related diseases. Myricetin is a natural compound present in numerous plant-based foods with presumed anti-inflammatory and beneficial health effects. The impact of this flavonoid on hypoxia-induced expression of inflammatory adipokines and hypoxia-regulated pathways is unknown so far and has been addressed in the present study. METHODS: Differentiated human Simpson-Golabi-Behmel syndrome (SGBS) adipocytes were cultured with or without myricetin under normoxic and hypoxic conditions for varying time periods. The effect of hypoxia and myricetin on the expression of the investigated adipokines was measured by real-time RT-PCR. Western blot analysis was used for the detection of transcription factors involved in hypoxia-regulated pathways. RESULTS: Myricetin interfered in the hypoxia-induced regulation of adipokines and the underlying pathways, which are involved in transmitting the inflammatory response. It strongly repressed hypoxia-induced expression of apelin, leptin, chemerin, asprosin, and DPP-4 and HIF-1α accumulation in the nucleus was diminished. Furthermore, the activation of the key regulators in the inflammatory response NF-κB, Akt, and CREB was suppressed by myricetin under hypoxic conditions. Myricetin also decreased hypoxia-induced accumulation of the pro-tumorigenic transcription factors Snail and Slug in the nucleus. CONCLUSION: Taken together, our results indicated that myricetin regulated hypoxia-induced expression of adipokines and hypoxia-regulated pathways in human adipocytes. Our study therefore provided evidence of the anti-inflammatory effects of myricetin in hypoxia-treated human adipocytes.

Pre- and early postpartum psychosocial stress trajectories in mothers and child body mass index at 3 years: a birth cohort study.

BACKGROUND: Child overweight remains a prevalent public health concern, but the impact of maternal psychosocial stress and related constructs, the timing, and possible trajectories on child body mass index (BMI) is controversial. We aimed to investigate the association of maternal stress, depression and anxiety symptoms, and maternal hair cortisol concentrations (HCC) at delivery, 6, and 12 months postpartum with child BMI and age- and sex-standardized BMI (BMI-SDS) at age 3 years. METHODS: Data were derived from the Ulm SPATZ Health Study with a baseline examination between 04/2012 and 05/2013 at the University Medical Centre Ulm, Germany, the only maternity clinic in Ulm, with a good representation of the source population. Adjusted regression analyses based on BMI/BMI-SDS (dependent) and trajectories of stress, depression, and anxiety (independent variables) were investigated in 596 mothers and children. Multiple imputation of missing covariates was performed. RESULTS: Various trajectories in independent variables were identified, trajectories of maternal anxiety symptom differed between child sexes. We did not find an association between trajectories of maternal chronic stress, depression symptoms, or HCC and child BMI/BMI-SDS. However, trajectories of low-increasing maternal anxiety symptoms were linked to higher child BMI compared to a low-stable trajectory group (b = 0.58 kg/m2, 95% Confidence Interval: 0.11; 1.04) in girls. CONCLUSIONS: Trajectories of maternal anxiety symptoms were associated with the child's BMI/BMI-SDS in girls at age 3 years. However, further large scale studies should include variables to determine the causal pathway and enlighten sex-specific differences.

Insulin-inducible THRSP maintains mitochondrial function and regulates sphingolipid metabolism in human adipocytes.

BACKGROUND: Thyroid hormone responsive protein (THRSP) is a lipogenic nuclear protein that is highly expressed in murine adipose tissue, but its role in humans remains unknown. METHODS: We characterized the insulin regulation of THRSP in vivo in human adipose tissue biopsies and in vitro in Simpson-Golabi-Behmel syndrome (SGBS) adipocytes. To this end, we measured whole-body insulin sensitivity using the euglycemic insulin clamp technique in 36 subjects [age 40 ± 9 years, body mass index (BMI) 27.3 ± 5.0 kg/m2]. Adipose tissue biopsies were obtained at baseline and after 180 and 360 min of euglycemic hyperinsulinemia for measurement of THRSP mRNA concentrations. To identify functions affected by THRSP, we performed a transcriptomic analysis of THRSP-silenced SGBS adipocytes. Mitochondrial function was assessed by measuring mitochondrial respiration as well as oxidation and uptake of radiolabeled oleate and glucose. Lipid composition in THRSP silencing was studied by lipidomic analysis. RESULTS: We found insulin to increase THRSP mRNA expression 5- and 8-fold after 180 and 360 min of in vivo euglycemic hyperinsulinemia. This induction was impaired in insulin-resistant subjects, and THRSP expression was closely correlated with whole-body insulin sensitivity. In vitro, insulin increased both THRSP mRNA and protein concentrations in SGBS adipocytes in a phosphoinositide 3-kinase (PI3K)-dependent manner. A transcriptomic analysis of THRSP-silenced adipocytes showed alterations in mitochondrial functions and pathways of lipid metabolism, which were corroborated by significantly impaired mitochondrial respiration and fatty acid oxidation. A lipidomic analysis revealed decreased hexosylceramide concentrations, supported by the transcript concentrations of enzymes regulating sphingolipid metabolism. CONCLUSIONS: THRSP is regulated by insulin both in vivo in human adipose tissue and in vitro in adipocytes, and its expression is downregulated by insulin resistance. As THRSP silencing decreases mitochondrial respiration and fatty acid oxidation, its downregulation in human adipose tissue could contribute to mitochondrial dysfunction. Furthermore, disturbed sphingolipid metabolism could add to metabolic dysfunction in obese adipose tissue.

20 Years with SGBS cells - a versatile in vitro model of human adipocyte biology.

20 years ago, we described a human cell strain derived from subcutaneous adipose tissue of an infant supposed to have Simpson-Golabi-Behmel Syndrome (SGBS), thus called "SGBS cells". Since then, these cells have emerged as the most commonly used cell model for human adipogenesis and human adipocyte biology. Although these adipocyte derived stem cells have not been genetically manipulated for transformation or immortalization, SGBS cells retain their capacity to proliferate and to differentiate into adipocytes for more than 50 population doublings, providing an almost unlimited source of human adipocyte progenitor cells. Original data obtained with SGBS cells led to more than 200 peer reviewed publications comprising investigations on adipogenesis and browning, insulin sensitivity, inflammatory response, adipokine production, as well as co-culture models and cell-cell communication. In this article, we provide an update on the characterization of SGBS cells, present basic methods for their application and summarize results of a systematic literature search on original data obtained with this cell strain.

Functional Screening of Candidate Causal Genes for Insulin Resistance in Human Preadipocytes and Adipocytes.

Rationale: Genome-wide association studies have identified genetic loci associated with insulin resistance (IR) but pinpointing the causal genes of a risk locus has been challenging. Objective: To identify candidate causal genes for IR, we screened regional and biologically plausible genes (16 in total) near the top 10 IR-loci in risk-relevant cell types, namely preadipocytes and adipocytes. Methods and Results: We generated 16 human Simpson-Golabi-Behmel syndrome preadipocyte knockout lines each with a single IR-gene knocked out by lentivirus-mediated CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system. We evaluated each gene knockout by screening IR-relevant phenotypes in the 3 insulin-sensitizing mechanisms, including adipogenesis, lipid metabolism, and insulin signaling. We performed genetic analyses using data on the genotype-tissue expression portal expression quantitative trait loci database and accelerating medicines partnership type 2 diabetes mellitus Knowledge Portal to evaluate whether candidate genes prioritized by our in vitro studies were expression quantitative trait loci genes in human subcutaneous adipose tissue, and whether expression of these genes is associated with risk of IR, type 2 diabetes mellitus, and cardiovascular diseases. We further validated the functions of 3 new adipose IR genes by overexpression-based phenotypic rescue in the Simpson-Golabi-Behmel syndrome preadipocyte knockout lines. Twelve genes, PPARG, IRS-1, FST, PEPD, PDGFC, MAP3K1, GRB14, ARL15, ANKRD55, RSPO3, COBLL1, and LYPLAL1, showed diverse phenotypes in the 3 insulin-sensitizing mechanisms, and the first 7 of these genes could affect all the 3 mechanisms. Five out of 6 expression quantitative trait loci genes are among the top candidate causal genes and the abnormal expression levels of these genes (IRS-1, GRB14, FST, PEPD, and PDGFC) in human subcutaneous adipose tissue could be associated with increased risk of IR, type 2 diabetes mellitus, and cardiovascular disease. Phenotypic rescue by overexpression of the candidate causal genes (FST, PEPD, and PDGFC) in the Simpson-Golabi-Behmel syndrome preadipocyte knockout lines confirmed their function in adipose IR. Conclusions: Twelve genes showed diverse phenotypes indicating differential roles in insulin sensitization, suggesting mechanisms bridging the association of their genomic loci with IR. We prioritized PPARG, IRS-1, GRB14, MAP3K1, FST, PEPD, and PDGFC as top candidate genes. Our work points to novel roles for FST, PEPD, and PDGFC in adipose tissue, with consequences for cardiometabolic diseases.

Development and evaluation of a patient education programme for children, adolescents, and young adults with differences of sex development (DSD) and their parents: study protocol of Empower-DSD.

BACKGROUND: Differences in sexual development (DSD) are rare diseases, which affect the chromosomal, anatomical or gonadal sex differentiation. Although patient education is recommended as essential in a holistic care approach, standardised programmes are still lacking. The present protocol describes the aims, study design and methods of the Empower-DSD project, which developed an age-adapted multidisciplinary education programme to improve the diagnosis-specific knowledge, skills and empowerment of patients and their parents. METHODS: The new patient education programme was developed for children, adolescents and young adults with congenital adrenal hyperplasia, Turner syndrome, Klinefelter syndrome or XX-/or XY-DSD and their parents. The quantitative and qualitative evaluation methods include standardised questionnaires, semi-structured interviews, and participatory observation. The main outcomes (assessed three and six months after the end of the programme) are health-related quality of life, disease burden, coping, and diagnosis-specific knowledge. The qualitative evaluation examines individual expectations and perceptions of the programme. The results of the quantitative and qualitative evaluation will be triangulated. DISCUSSION: The study Empower-DSD was designed to reduce knowledge gaps regarding the feasibility, acceptance and effects of standardised patient education programmes for children and youth with DSD and their parents. A modular structured patient education programme with four generic and three diagnosis-specific modules based on the ModuS concept previously established for other chronic diseases was developed. The topics, learning objectives and recommended teaching methods are summarised in the structured curricula, one for each diagnosis and age group. At five study centres, 56 trainers were qualified for the implementation of the training programmes. A total of 336 subjects have been already enrolled in the study. The recruitment will go on until August 2022, the last follow-up survey is scheduled for February 2023. The results will help improve multidisciplinary and integrated care for children and youth with DSD and their families. TRIAL REGISTRATION: German Clinical Trials Register, DRKS00023096 . Registered 8 October 2020 - Retrospectively registered.

miR-146a regulates insulin sensitivity via NPR3.

The pathogenesis of obesity-related metabolic diseases has been linked to the inflammation of white adipose tissue (WAT), but the molecular interconnections are still not fully understood. MiR-146a controls inflammatory processes by suppressing pro-inflammatory signaling pathways. The aim of this study was to characterize the role of miR-146a in obesity and insulin resistance. MiR-146a-/- mice were subjected to a high-fat diet followed by metabolic tests and WAT transcriptomics. Gain- and loss-of-function studies were performed using human Simpson-Golabi-Behmel syndrome (SGBS) adipocytes. Compared to controls, miR-146a-/- mice gained significantly more body weight on a high-fat diet with increased fat mass and adipocyte hypertrophy. This was accompanied by exacerbated liver steatosis, insulin resistance, and glucose intolerance. Likewise, adipocytes transfected with an inhibitor of miR-146a displayed a decrease in insulin-stimulated glucose uptake, while transfecting miR-146a mimics caused the opposite effect. Natriuretic peptide receptor 3 (NPR3) was identified as a direct target gene of miR-146a in adipocytes and CRISPR/Cas9-mediated knockout of NPR3 increased insulin-stimulated glucose uptake and enhanced de novo lipogenesis. In summary, miR-146a regulates systemic and adipocyte insulin sensitivity via downregulation of NPR3.

Measuring hyperphagia in patients with monogenic and syndromic obesity.

BACKGROUND: Hyperphagia is a key symptom in patients with monogenic obesity, but the assessment is challenging. OBJECTIVES: We aimed to investigate the applicability of Dykens' Hyperphagia Questionnaire in patients with monogenic and syndromic obesity to assess the quality and severity of hyperphagia, and to compare our results with those reported in the literature. METHODS: Patients with biallelic leptin receptor variants (LEPR, n = 8), heterozygous melanocortin-4 receptor variants (MC4R, n = 7) and 16p11.2 deletions, leading to a deletion of the Src homology 2B adaptor protein gene (n = 5) were included in the study. Hyperphagia was assessed by the parent-based, 13-item hyperphagia questionnaire from Dykens et al. (2007). A literature research was performed to identify published hyperphagia scores assessed by Dykens' Hyperphagia Questionnaire. RESULTS: The total hyperphagia scores were similar in patients with biallelic LEPR and monoallelic MC4R variants (32.0 ± 9.3 vs. 31.4 ± 5.4), but significantly lower in patients with 16p11.2 deletions (21.4 ± 5.5, p < 0.05). Compared to patients with syndromic obesity (27.6 ± 9.0) from the literature, patients with LEPR and MC4R variants had higher total hyperphagia scores. Total hyperphagia scores in patients with 16p11.2 deletions were lower than for patients with other syndromic obesity forms (21.4 ± 5.5 vs. 24.6 ± 8.1), but similar to those for individuals with obesity without a genetic cause (22.9 ± 7.2). CONCLUSIONS: Dykens' Hyperphagia Questionnaire seems to be a useful tool to assess hyperphagic behaviour in patients with monogenic and syndromic obesity.

HAND2 is a novel obesity-linked adipogenic transcription factor regulated by glucocorticoid signalling.

AIMS/HYPOTHESIS: Adipocytes are critical cornerstones of energy metabolism. While obesity-induced adipocyte dysfunction is associated with insulin resistance and systemic metabolic disturbances, adipogenesis, the formation of new adipocytes and healthy adipose tissue expansion are associated with metabolic benefits. Understanding the molecular mechanisms governing adipogenesis is of great clinical potential to efficiently restore metabolic health in obesity. Here we investigate the role of heart and neural crest derivatives-expressed 2 (HAND2) in adipogenesis. METHODS: Human white adipose tissue (WAT) was collected from two cross-sectional studies of 318 and 96 individuals. In vitro, for mechanistic experiments we used primary adipocytes from humans and mice as well as human multipotent adipose-derived stem (hMADS) cells. Gene silencing was performed using siRNA or genetic inactivation in primary adipocytes from loxP and or tamoxifen-inducible Cre-ERT2 mouse models with Cre-encoding mRNA or tamoxifen, respectively. Adipogenesis and adipocyte metabolism were measured by Oil Red O staining, quantitative PCR (qPCR), microarray, glucose uptake assay, western blot and lipolysis assay. A combinatorial RNA sequencing (RNAseq) and ChIP qPCR approach was used to identify target genes regulated by HAND2. In vivo, we created a conditional adipocyte Hand2 deletion mouse model using Cre under control of the Adipoq promoter (Hand2AdipoqCre) and performed a large panel of metabolic tests. RESULTS: We found that HAND2 is an obesity-linked white adipocyte transcription factor regulated by glucocorticoids that was necessary but insufficient for adipocyte differentiation in vitro. In a large cohort of humans, WAT HAND2 expression was correlated to BMI. The HAND2 gene was enriched in white adipocytes compared with brown, induced early in differentiation and responded to dexamethasone (DEX), a typical glucocorticoid receptor (GR, encoded by NR3C1) agonist. Silencing of NR3C1 in hMADS cells or deletion of GR in a transgenic conditional mouse model results in diminished HAND2 expression, establishing that adipocyte HAND2 is regulated by glucocorticoids via GR in vitro and in vivo. Furthermore, we identified gene clusters indirectly regulated by the GR-HAND2 pathway. Interestingly, silencing of HAND2 impaired adipocyte differentiation in hMADS and primary mouse adipocytes. However, a conditional adipocyte Hand2 deletion mouse model using Cre under control of the Adipoq promoter did not mirror these effects on adipose tissue differentiation, indicating that HAND2 was required at stages prior to Adipoq expression. CONCLUSIONS/INTERPRETATION: In summary, our study identifies HAND2 as a novel obesity-linked adipocyte transcription factor, highlighting new mechanisms of GR-dependent adipogenesis in humans and mice. DATA AVAILABILITY: Array data have been submitted to the GEO database at NCBI (GSE148699).

IL-20 is involved in obesity by modulation of adipogenesis and macrophage dysregulation.

IL-20 is a proinflammatory cytokine of the IL-10 family and involved in several diseases. However, the regulatory role of IL-20 in obesity is not well understood. We explored the function of IL-20 in the pathogenesis of obesity-induced insulin resistance by ELISA, Western blotting and flow cytometry. The therapeutic potential of IL-20 monoclonal antibody 7E for ameliorating diet-induced obesity was analysed in murine models. Higher serum IL-20 levels were detected in obese patients. It was upregulated in leptin-deficient (ob/ob), leptin-resistant (db/db) and high-fat diet (HFD)-induced murine obesity models. In vitro, IL-20 regulated the adipocyte differentiation and the polarization of bone marrow-derived macrophages into proinflammatory M1 type. It also caused inflammation and macrophage retention in adipose tissues by upregulating TNF-α, monocyte chemotactic protein 1 (MCP-1), netrin 1 and unc5b (netrin receptor) expression in macrophages and netrin 1, leptin and MCP-1 in adipocytes. IL-20 promoted insulin resistance by inhibiting glucose uptake in mature adipocytes through the SOCS-3 pathway. In HFD-induced obesity in mice, 7E treatment reduced body weight and improved glucose tolerance and insulin sensitivity; it also reduced local inflammation and the number of M1-like macrophages in adipose tissues. We have identified a critical role of IL-20 in obesity-induced inflammation and insulin resistance, and we conclude that IL-20 may be a novel target for treating obesity and insulin resistance in patients with metabolic disorders.

Serum IGF1 and linear growth in children with congenital leptin deficiency before and after leptin substitution.

BACKGROUND: Evidence from in vitro and rodent studies suggests that leptin, a key signal of long-term energy reserves, promotes IGF1 synthesis and linear growth. This effect of leptin has not been fully investigated in humans. The aim of our study was to investigate the effect of leptin substitution on growth factors and linear growth in children with congenital leptin deficiency (CLD). METHODS: In this cohort study we included eight pediatric patients (six males), age 0.9-14.8 years, who were diagnosed with CLD and received leptin substitution at our University Medical Center. We calculated standard deviation scores (SDS) for serum levels of IGF1 and IGFBP3, IGF1/IGFBP3 molar ratio, and height at baseline (T0) and 12 months (T12) after the initiation of substitution with metreleptin. RESULTS: All patients had severe obesity (BMI-SDS mean ± SD: 4.14 ± 1.51) at T0 and significant BMI-SDS reduction to 2.47 ± 1.05 at T12. At T0, all patients were taller than the mid-parental median, yet had low IGF1 and IGF1/IGFBP3 molar ratios (IGF1-SDS[Formula: see text]T0: -1.58 ± 0.92, IGF1/IGFBP3 molar ratio-SDS[Formula: see text]T0: -1.58 ± 0.88). At T12, IGF1-SDS increased significantly (∆T0-12: 1.63 ± 1.40, p = 0.01), and IGFBP3-SDS and IGF1/IGFBP3 molar ratio-SDS showed a trend toward an increase. In the three children within the childhood growth period (post-infancy, pre-puberty) height-SDS increased (∆height-SDST0-12: 0.57 ± 0.06, p = 0.003) despite substantial weight loss. CONCLUSIONS: These results in CLD patients are contrary to observations in children with idiopathic obesity who typically have above-mean IGF1 levels that decrease with weight loss, and therefore suggest that leptin increases IGF1 levels and promotes linear growth.