Alterations of Central Liver Metabolism of Pediatric Patients with Non-Alcoholic Fatty Liver Disease

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in children and is associated with overweight and insulin resistance (IR). Almost nothing is known about in vivo alterations of liver metabolism in NAFLD, especially in the early stages of non-alcoholic steatohepatitis (NASH). Here, we used a complex mathematical model of liver metabolism to quantify the central hepatic metabolic functions of 71 children with biopsy-proven NAFLD. For each patient, a personalized model variant was generated based on enzyme abundances determined by mass spectroscopy. Our analysis revealed statistically significant alterations in the hepatic carbohydrate, lipid, and ammonia metabolism, which increased with the degree of obesity and severity of NAFLD. Histologic features of NASH and IR displayed opposing associations with changes in carbohydrate and lipid metabolism but synergistically decreased urea synthesis in favor of the increased release of glutamine, a driver of liver fibrosis. Taken together, our study reveals already significant alterations in the NASH liver of pediatric patients, which, however, are differently modulated by the simultaneous presence of IR

Transcriptomic and proteomic insight into the effects of a defined European mistletoe extract in Ewing sarcoma cells reveals cellular stress responses

Background The hydrophobic triterpenes, oleanolic and betulinic acid as well as the hydrophilic mistletoe lectins and viscotoxins possess anticancer properties. They do all occur in combination in European mistletoe (Viscum album L.). Commercial Viscum album L. extracts are aqueous, excluding the insoluble triterpenes. We have previously shown that mistletoe lectins and triterpene acids are effective against Ewing sarcoma in vitro, ex vivo and in vivo. Methods We recreated a total mistletoe effect (viscumTT) by combining an aqueous extract (viscum) and a triterpene extract (TT) solubilised with cyclodextrins and analysed the effects of viscumTT and the single extracts on TC-71 Ewing sarcoma cells in vitro by transcriptomic and proteomic profiling. Results Treatment with the extracts strongly impacted Ewing sarcoma cell gene and protein expression. Apoptosis-associated and stress-activated genes were upregulated, proteasomal protein abundance enhanced and ribosomal and spliceosomal proteins downregulated. The mechanism of action of viscum, TT and viscumTT in TC-71 and MHH-ES-1 cells suggests the involvement of the unfolded protein response. While viscum and viscumTT extract treatment indicate response to oxidative stress and activation of stress-mediated MAPK signalling, TT extract treatment suggests the involvement of TLR signalling and autophagy. Conclusions Since the combinatory extract viscumTT exerts highly effective pro-apoptotic effects on Ewing sarcoma cells in vitro, this phytopolychemotherapy could be a promising adjuvant therapeutic option for paediatric patients with Ewing sarcoma

HEPATOKIN1 is a biochemistry-based model of liver metabolism for applications in medicine and pharmacology

The epidemic increase of non-alcoholic fatty liver diseases (NAFLD) requires a deeper understanding of the regulatory circuits controlling the response of liver metabolism to nutritional challenges, medical drugs, and genetic enzyme variants. As in vivo studies of human liver metabolism are encumbered with serious ethical and technical issues, we developed a comprehensive biochemistry-based kinetic model of the central liver metabolism including the regulation of enzyme activities by their reactants, allosteric effectors, and hormone-dependent phosphorylation. The utility of the model for basic research and applications in medicine and pharmacology is illustrated by simulating diurnal variations of the metabolic state of the liver at various perturbations caused by nutritional challenges (alcohol), drugs (valproate), and inherited enzyme disorders (galactosemia). Using proteomics data to scale maximal enzyme activities, the model is used to highlight differences in the metabolic functions of normal hepatocytes and malignant liver cells (adenoma and hepatocellular carcinoma)

Hypoxia induces a transcriptional early primitive streak signature in pluripotent cells enhancing spontaneous elongation and lineage representation in gastruloids

The cellular microenvironment, together with intrinsic regulators, shapes stem cell identity and differentiation capacity. Mammalian early embryos are exposed to hypoxia in vivo and appear to benefit from hypoxic culture in vitro. Yet, how hypoxia influences stem cell transcriptional networks and lineage choices remain poorly understood. Here, we investigated the molecular effects of acute and prolonged hypoxia on embryonic and extra-embryonic stem cells as well as the functional impact on differentiation potential. We find a temporal and cell type-specific transcriptional response including an early primitive streak signature in hypoxic embryonic stem cells mediated by HIF1α. Using a 3D gastruloid differentiation model, we show that hypoxia-induced T expression enables symmetry breaking and axial elongation in the absence of exogenous WNT activation. When combined with exogenous WNT activation, hypoxia enhances lineage representation in gastruloids, as demonstrated by highly enriched signatures of gut endoderm, notochord, neuromesodermal progenitors and somites. Our findings directly link the microenvironment to stem cell function and provide a rationale supportive of applying physiological conditions in models of embryo development

Variants in MARC1 and HSD17B13 reduce severity of NAFLD in children, perturb phospholipid metabolism, and suppress fibrotic pathways

Background & aims: Genome-wide association studies in adults have identified variants in HSD17B13 and MARC1 as protective against NAFLD. It is not known if they are similarly protective in children and, more generally, whether the peri-portal inflammation of pediatric NAFLD and lobular inflammation seen in adults share common genetic influences. Therefore, we aimed to: establish if these variants are associated with NAFLD in children, and to investigate the function of these variants in hepatic metabolism using metabolomics. Methods: 960 children (590 with NAFLD, 394 with liver histology) were genotyped for rs72613567T>TA in HSD17B13, rs2642438G>A in MARC1. Genotype-histology associations were tested using ordinal regression. Untargeted hepatic proteomics and plasma lipidomics were performed in a subset of samples. In silico tools were used to model the effect of rs2642438G>A (p.Ala165Thr) on MARC1. Results: rs72613567T>TA in HSD17B13 was associated with lower odds of NAFLD diagnosis (OR 0.7 (95%CI 0.6-0.9) and lower grade of portal inflammation (PA in MARC1 was associated with lower grade of hepatic steatosis (P=0.02). Proteomics found reduced expression of HSD17B13 in carriers of the protective allele, whereas MARC1 levels were not affected by genotype. Both variants showed downregulation of hepatic fibrotic pathways, upregulation of retinol metabolism and perturbation of phospholipid species. Modelling suggests that p.Ala165Thr would disrupt the stability and metal-binding of MARC1. Conclusions: There are shared genetic mechanisms between pediatric and adult NAFLD, despite their differences in histology. MARC1 and HSD17B13 are involved in phospholipid metabolism and suppress fibrosis in NAFLD

Hijacking of transcriptional condensates by endogenous retroviruses

Most endogenous retroviruses (ERVs) in mammals are incapable of retrotransposition; therefore, why ERV derepression is associated with lethality during early development has been a mystery. Here, we report that rapid and selective degradation of the heterochromatin adapter protein TRIM28 triggers dissociation of transcriptional condensates from loci encoding super-enhancer (SE)-driven pluripotency genes and their association with transcribed ERV loci in murine embryonic stem cells. Knockdown of ERV RNAs or forced expression of SE-enriched transcription factors rescued condensate localization at SEs in TRIM28-degraded cells. In a biochemical reconstitution system, ERV RNA facilitated partitioning of RNA polymerase II and the Mediator coactivator into phase-separated droplets. In TRIM28 knockout mouse embryos, single-cell RNA-seq analysis revealed specific depletion of pluripotent lineages. We propose that coding and noncoding nascent RNAs, including those produced by retrotransposons, may facilitate ‘hijacking’ of transcriptional condensates in various developmental and disease contexts

Androgen receptor condensates as drug targets

Transcription factors are among the most attractive therapeutic targets, but are considered largely undruggable. Here we provide evidence that small molecule-mediated partitioning of the androgen receptor, an oncogenic transcription factor, into phase-separated condensates has therapeutic effect in prostate cancer models. We show that the phase separation capacity of the androgen receptor is driven by aromatic residues and short unstable helices in its intrinsically disordered activation domain. Based on this knowledge, we developed tool compounds that covalently attach aromatic moieties to cysteines in the receptors’ activation domain. The compounds enhanced partitioning of the receptor into condensates, facilitated degradation of the receptor, inhibited androgen receptor-dependent transcriptional programs, and had antitumorigenic effect in models of prostate cancer and castration-resistant prostate cancer in vitro and in vivo. These results establish a generalizable framework to target the phase- separation capacity of intrinsically disordered regions in oncogenic transcription factors and other disease-associated proteins with therapeutic intent