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Currently, over 88 million people are estimated to have adopted a vegan or vegetarian diet. Cysteine is a semi-essential amino acid, which availability is largely dependent on dietary intake of meat, eggs and whole grains. Vegan/vegetarian diets are therefore inherently low in cysteine. Sufficient uptake of cysteine is crucial, as it serves as substrate for protein synthesis and can be converted to taurine and glutathione. We found earlier that intermolecular cystine bridges are essential for the barrier function of the intestinal mucus layer. Therefore, we now investigate the effect of low dietary cystine on the intestine. Mice (8/group) received a high fat diet with a normal or low cystine concentration for 2 weeks. We observed no changes in plasma methionine, cysteine, taurine or glutathione levels or bile acid conjugation after 2 weeks of low cystine feeding. In the colon, dietary cystine restriction results in an increase in goblet cell numbers, and a borderline significant increase mucus layer thickness. Gut microbiome composition and expression of stem cell markers did not change on the low cystine diet. Remarkably, stem cell markers, as well as the proliferation marker Ki67, were increased upon cystine restriction in the small intestine. In line with this, gene set enrichment analysis indicated enrichment of Wnt signaling in the small intestine of mice on the low cystine diet, indicative of increased epithelial proliferation. In conclusion, 2 weeks of cystine restriction did not result in apparent systemic effects, but the low cystine diet increased the proliferative capacity specifically of the small intestine and induced the number of goblet cells in the colon., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 de Jong et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Objective: In vivo studies in humans and mice have implicated the pseudokinase Tribbles 3 (TRIB3) in various aspects of energy metabolism. Whilst cell-based studies indicate a role for TRIB3 in adipocyte differentiation and function, it is unclear if and how these cellular functions may contribute to overall metabolic health., Methods: We investigated the metabolic phenotype of whole-body Trib3 knockout (Trib3 KO ) mice, focusing on adipocyte and adipose tissue functions. In addition, we combined lipidomics, transcriptomics, interactomics and phosphoproteomics analyses to elucidate cell-intrinsic functions of TRIB3 in pre- and mature adipocytes., Results: Trib3 KO mice display increased adiposity, but their insulin sensitivity remains unaltered. Trib3 KO adipocytes are smaller and display higher Proliferating Cell Nuclear Antigen (PCNA) levels, indicating potential alterations in either i) proliferation-differentiation balance, ii) impaired expansion after cell division, or iii) an altered balance between lipid storage and release, or a combination thereof. Lipidome analyses suggest TRIB3 involvement in the latter two processes, as triglyceride storage is reduced and membrane composition, which can restrain cellular expansion, is altered. Integrated interactome, phosphoproteome and transcriptome analyses support a role for TRIB3 in all three cellular processes through multiple cellular pathways, including Mitogen Activated Protein Kinase- (MAPK/ERK), Protein Kinase A (PKA)-mediated signaling and Transcription Factor 7 like 2 (TCF7L2) and Beta Catenin-mediated gene expression., Conclusions: Our findings support TRIB3 playing multiple distinct regulatory roles in the cytoplasm, nucleus and mitochondria, ultimately controlling adipose tissue homeostasis, rather than affecting a single cellular pathway., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Undigested proteins that become available for the microbiota in the hindgut can be used as building blocks for bacterial cells, or can enter various catabolic pathways. Degradation via protein fermentation pathways is least preferred, as several fermentation end-products released can be toxic for the host. Directing microbial protein metabolism towards protein synthesis or degradative pathways that result in less toxic end-products, for example through nutritional interventions, is an interesting strategy for improving health. We studied variation in protein fermentation patterns, resulting from variation in substrate composition. Ileal digesta, obtained from cannulated pigs fed different protein sources, were subjected to fermentation in vitro under different conditions; (1) ileal digesta were fermented as-is, (2) ileal digesta were fermented after standardisation to a constant high C:N ratio, by addition of high fermentable carbohydrates and (3) ileal digesta samples were incubated under limiting N concentrations. Gas production was monitored as an indirect measure of microbial activity, and fermentation end-products at different points in time were analysed by gas chromatography and high resolution mass spectrometry. Using principal component analysis, we identified patterns in protein fermentation end-products and related them to the composition of ileal digesta. Protein-associated fermentation end-product concentrations of e.g. isovaleric-, isobutyric-, phenylacetic acid and p-cresol were negatively affected by the available amount of high fermentable carbohydrates combined with a high C:N ratio. The aforementioned fermentation end-products positively correlated with NH 3 concentrations and negatively with short-chain fatty acid (SCFA) concentrations. Standardisation to a constant high C:N ratio changed their relationship; isovaleric-, isobutyric-, phenylacetic acid and p-cresol lost their correlation with NH 3 concentrations, became positively correlated with SCFA concentrations, and now showed a positive correlation with available amounts of high fermentable carbohydrates. Our observations demonstrate an important role of the C:N ratio in the relationship between fermentation end-products. At constant C:N, protein fermentation end-products correlate with end-products of carbohydrate fermentation and NH 3 , often considered as a proxy for protein fermentation, loses its predictive power., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background and Aims: Recurrent hepatic encephalopathy (HE) is characterized by hyperammonaemia in combination with neuropsychiatric abnormalities and is treated with lactulose and rifaximin. Rifaximin is a pregnane X receptor (PXR) agonist with low systemic and high intestinal bioavailability. The mechanisms by which it alleviates HE are unclear. We used human small intestinal (hSI) organoids to study whether rifaximin, via PXR activation, affects the epithelial biotransformation machinery, and to gain understanding of its low systemic availability., Methods: We generated PXR knockdown hSI organoids via lentiviral delivery of short hairpin RNAs. Organoids were cultured for 24 h with rifaximin or rifampicin. RNA-sequencing and metabolomics were performed to analyse gene expression and amino acid metabolism. Luminal rifaximin was quantified by photospectrometry., Results: Treatment of wild-type hSI organoids with rifaximin resulted in >twofold differential expression of 131 genes compared to DMSO. These effects were largely PXR independent and related to amino acid metabolism. Rifaximin decreased expression of glutaminase-2 and increased expression of asparagine synthetase and solute carrier 7A11, thereby increasing intracellular glutamine and asparagine concentrations, indicating active ammonia detoxification. Rifaximin was apically excreted into the lumen in an ATP binding cassette B1 (ABCB1)-dependent manner., Conclusions: Rifaximin-after uptake into enterocytes-stimulates intracellular nitrogen detoxification by PXR-independent mechanisms. Active apical excretion of rifaximin by ABCB1 into the intestinal lumen explains its low systemic bioavailability. Our study implies that rifaximin, next to modulation of the microbiome, has direct effects on ammonia scavenging in the human small intestinal epithelium., (© 2022 The Authors. Liver International published by John Wiley & Sons Ltd.)

Despite the fact that 5-fluorouracil (5-FU) is the backbone for chemotherapy in colorectal cancer (CRC), the response rates in patients is limited to 50%. The mechanisms underlying 5-FU toxicity are debated, limiting the development of strategies to improve its efficacy. How fundamental aspects of cancer, such as driver mutations and phenotypic heterogeneity, relate to the 5-FU response remains obscure. This largely relies on the limited number of studies performed in pre-clinical models able to recapitulate the key features of CRC. Here, we analyzed the 5-FU response in patient-derived organoids that reproduce the different stages of CRC. We find that 5-FU induces pyrimidine imbalance, which leads to DNA damage and cell death in the actively proliferating cancer cells deficient in p53. Importantly, p53-deficiency leads to cell death due to impaired cell cycle arrest. Moreover, we find that targeting the Warburg effect in KRAS G12D glycolytic tumor organoids enhances 5-FU toxicity by further altering the nucleotide pool and, importantly, without affecting non-transformed WT cells. Thus, p53 emerges as an important factor in determining the 5-FU response, and targeting cancer metabolism in combination with replication stress-inducing chemotherapies emerges as a promising strategy for CRC treatment., (© 2022. The Author(s).)

We report the derivation of 30 patient-derived organoid lines (PDOs) from tumors arising in the pancreas and distal bile duct. PDOs recapitulate tumor histology and contain genetic alterations typical of pancreatic cancer. In vitro testing of a panel of 76 therapeutic agents revealed sensitivities currently not exploited in the clinic, and underscores the importance of personalized approaches for effective cancer treatment. The PRMT5 inhibitor EZP015556, shown to target MTAP (a gene commonly lost in pancreatic cancer)-negative tumors, was validated as such, but also appeared to constitute an effective therapy for a subset of MTAP-positive tumors. Taken together, the work presented here provides a platform to identify novel therapeutics to target pancreatic tumor cells using PDOs.

Purpose: Human leukocyte antigen-B27 (HLA-B27)-positive acute anterior uveitis (AAU) has a higher recurrence rate and shows more anterior chamber cell infiltration compared with HLA-B27-negative patients, suggesting distinct etiologies of these clinically overlapping conditions. To advance our understanding of the biology of AAU, we characterized the metabolic profile of aqueous humor (AqH) of patients with HLA-B27-associated AAU (B27-AAU) and noninfectious idiopathic AAU (idiopathic AAU)., Design: Experimental laboratory study., Methods: AqH samples from 2 independent cohorts totaling 30 patients with B27-AAU, 16 patients with idiopathic AAU, and 20 patients with cataracts underwent 2 individual rounds of direct infusion mass spectrometry. Features predicted by direct infusion mass spectrometry that facilitated maximum separation between the disease groups in regression models were validated by liquid chromatography/tandem mass spectrometry-based quantification with appropriate standards., Results: Partial least square-discriminant analysis revealed metabolite profiles that were able to separate patients with B27-AAU from those with iodiopathic AAU. Pathway enrichment analysis, based on metabolites on which separation of the groups in the partial least square-discriminant analysis model was based, demonstrated the involvement of branched-chain amino acid biosynthesis, ascorbate and aldarate metabolism, the tricarboxylic acid cycle, and glycolysis-diverting pathways (eg, serine biosynthesis) across all investigated cohorts. Notably, the metabolite ketoleucine was elevated in B27-AAU across all 3 runs and moderately-but robustly-correlated with anterior chamber cell count (correlation coefficient range 0.41-0.81)., Conclusions: These results illustrate metabolic heterogeneity between HLA-B27-positive and HLA-B27-negative AAU, including an increase of branched-chain amino acid biosynthesis, that reflects disease activity in AAU., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Genetic lipodystrophies are a group of rare syndromes associated with major metabolic complications - including severe insulin resistance, type 2 diabetes mellitus, and hypertriglyceridemia - which are classified according to the distribution of adipose tissue. Lipodystrophies can be present at birth or develop during life and can range from local to partial and general. With at least 18 different genes implicated so far, definite diagnosis can be challenging due to clinical and genetic heterogeneity. In an adult female patient with clinical and metabolic features of partial lipodystrophy we identified via whole genome sequencing (WGS) a single complex AGPAT2 allele [V67M;V167A], functionally equivalent to heterozygosity. AGPAT2 encodes for an acyltransferase implicated in the biosynthesis of triacylglycerol and glycerophospholipids. So far homozygous and compound heterozygous mutations in AGPAT2 have only been associated with generalized lipodystrophy. A SNP risk score analysis indicated that the index patient is not predisposed to lipodystrophy based on her genetic background. The partial phenotype in our patient is therefore more likely associated to the genetic variants in AGPAT2. To test whether the resulting double-mutant AGPAT2 protein is functional we analyzed its in vitro enzymatic activity via mass spectrometry. The resulting AGPAT2 double mutant is enzymatically inactive. Our data support the view that the current classification of lipodystrophies as strictly local, partial or generalized may have to be re-evaluated and viewed more as a continuum, both in terms of clinical presentation and underlying genetic causes. Better molecular understanding of lipodystrophies may lead to new therapies to treat adipose tissue dysfunction in common and rare diseases.

Background & Aims: Congenital diarrheal disorders are rare inherited intestinal disorders characterized by intractable, sometimes life-threatening, diarrhea and nutrient malabsorption; some have been associated with mutations in diacylglycerol-acyltransferase 1 (DGAT1), which catalyzes formation of triacylglycerol from diacylglycerol and acyl-CoA. We investigated the mechanisms by which DGAT1 deficiency contributes to intestinal failure using patient-derived organoids., Methods: We collected blood samples from 10 patients, from 6 unrelated pedigrees, who presented with early-onset severe diarrhea and/or vomiting, hypoalbuminemia, and/or (fatal) protein-losing enteropathy with intestinal failure; we performed next-generation sequencing analysis of DNA from 8 patients. Organoids were generated from duodenal biopsies from 3 patients and 3 healthy individuals (controls). Caco-2 cells and patient-derived dermal fibroblasts were transfected or transduced with vectors that express full-length or mutant forms of DGAT1 or full-length DGAT2. We performed CRISPR/Cas9-guided disruption of DGAT1 in control intestinal organoids. Cells and organoids were analyzed by immunoblot, immunofluorescence, flow cytometry, chromatography, quantitative real-time polymerase chain reaction, and for the activity of caspases 3 and 7., Results: In the 10 patients, we identified 5 bi-allelic loss-of-function mutations in DGAT1. In patient-derived fibroblasts and organoids, the mutations reduced expression of DGAT1 protein and altered triacylglycerol metabolism, resulting in decreased lipid droplet formation after oleic acid addition. Expression of full-length DGAT2 in patient-derived fibroblasts restored formation of lipid droplets. Organoids derived from patients with DGAT1 mutations were more susceptible to lipid-induced cell death than control organoids., Conclusions: We identified a large cohort of patients with congenital diarrheal disorders with mutations in DGAT1 that reduced expression of its product; dermal fibroblasts and intestinal organoids derived from these patients had altered lipid metabolism and were susceptible to lipid-induced cell death. Expression of full-length wildtype DGAT1 or DGAT2 restored normal lipid metabolism in these cells. These findings indicate the importance of DGAT1 in fat metabolism and lipotoxicity in the intestinal epithelium. A fat-free diet might serve as the first line of therapy for patients with reduced DGAT1 expression. It is important to identify genetic variants associated with congenital diarrheal disorders for proper diagnosis and selection of treatment strategies., (Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.)