Report of the First ONTOX Stakeholder Network Meeting: Digging Under the Surface of ONTOX Together With the Stakeholders.

The first Stakeholder Network Meeting of the EU Horizon 2020-funded ONTOX project was held on 13-14 March 2023, in Brussels, Belgium. The discussion centred around identifying specific challenges, barriers and drivers in relation to the implementation of non-animal new approach methodologies (NAMs) and probabilistic risk assessment (PRA), in order to help address the issues and rank them according to their associated level of difficulty. ONTOX aims to advance the assessment of chemical risk to humans, without the use of animal testing, by developing non-animal NAMs and PRA in line with 21st century toxicity testing principles. Stakeholder groups (regulatory authorities, companies, academia, non-governmental organisations) were identified and invited to participate in a meeting and a survey, by which their current position in relation to the implementation of NAMs and PRA was ascertained, as well as specific challenges and drivers highlighted. The survey analysis revealed areas of agreement and disagreement among stakeholders on topics such as capacity building, sustainability, regulatory acceptance, validation of adverse outcome pathways, acceptance of artificial intelligence (AI) in risk assessment, and guaranteeing consumer safety. The stakeholder network meeting resulted in the identification of barriers, drivers and specific challenges that need to be addressed. Breakout groups discussed topics such as hazard versus risk assessment, future reliance on AI and machine learning, regulatory requirements for industry and sustainability of the ONTOX Hub platform. The outputs from these discussions provided insights for overcoming barriers and leveraging drivers for implementing NAMs and PRA. It was concluded that there is a continued need for stakeholder engagement, including the organisation of a 'hackathon' to tackle challenges, to ensure the successful implementation of NAMs and PRA in chemical risk assessment.

Intestinal nutrient sensing and blood glucose control.

PURPOSE OF REVIEW: Nutrient-specific sensor systems in enteroendocrine cells detect intestinal contents and cause gut hormone release upon activation. Among these peptide hormones, the incretins glucose-dependent insulinotropic polypeptide and glucagon-like peptide 1 are of particular interest by their role in glucose homeostasis, metabolic control and for proper ß-cell function. This review focuses on intestinal nutrient-sensing processes and their role in health and disease. RECENT FINDINGS: All macronutrients, respectively, their digestion products can cause incretin release by targeting specific sensors. Luminal glucose is the strongest stimulant for incretin release with the Na-dependent glucose transporter as the prime sensor. For peptides, the H-dependent peptide transporter together with calcium-sensing-receptor act as a sensing system. That transporters can function as nutrient-sensing 'transceptors' is conceptually new as G-protein coupled receptors so far were thought to be the sensing entities. This still holds true for GPR40 and GPR120 as sensors for medium/long-chain fatty acids and GPR41 and GPR43 for microbiota-derived short-chain fatty acids. Synthetic agonists for these receptors show impressive effects on glucagon-like peptide 1 output and glycemic control. Moreover, the remarkable and immediate antidiabetic effects of bariatric surgery/gastric bypass put intestinal nutrient sensing into focus of new strategies for metabolic control. SUMMARY: Targeting the intestinal nutrient-sensing machinery by dietary and/or pharmacological means holds promises in particular for treatment of type 2 diabetes. This interest may help to better understand the nutrient-sensing processes and the involvement of the intestine in overall endocrine, neuronal and metabolic control.

Taste and move: glucose and peptide transporters in the gastrointestinal tract.

NEW FINDINGS: What is the topic of this review? Nutrient absorption in the gastrointestinal tract requires membrane proteins embedded in the apical membrane of epithelial cells that allow bulk quantities of nutrients, such as monosaccharides and amino acids, to be moved into epithelial cells. Very recently, a new function of the transporters as nutrient sensors mediating peptide hormone release from enteroendocrine cells has been discovered. What advances does it highlight? The review covers recent advances in membrane transporter functions for the absorption and sensing of dietary peptides and sugars and their putative interplay. Nutrient transporters are integral membrane proteins responsible for uptake into enterocytes and release of nutrients into the circulation. Absorption of food breakdown products, such as fatty acids, monosaccharides or amino acids, requires high-capacity transporters. In the case of glucose, amino acids and peptides, the transporters are electrogenic in nature, coupling substrate flux to ion movement. While glucose absorption is mediated by the Na(+)-dependent SGLT1 protein, uptake of short-chain peptides is mediated by the H(+)-coupled PEPT1 protein. Interestingly, both transporters were recently shown to fulfil an additional role as intestinal 'sensors' in enteroendocrine cells, mediating the release of gastrointestinal peptide hormones into the circulation. Sensing of D-glucose and of di- and tripeptides is particularly relevant for the secretion of the incretins glucose-dependent insulinotrophic polypeptide and glucagon-like peptide 1 that promote insulin output from ß-cells and mediate ß-cell protection. In addition to these sensing pathways, a variety of G-protein-coupled receptors are involved in sensing of intestinal contents. D-Glucose is sensed not only by SGLT1 but also by the sweet taste receptor T1R2/3 expressed in enteroendocrine cells. Activation of T1R2/3 increases SGLT1 levels and intestinal glucose absorption. Although T1R2/3 ligands, such as artificial sweeteners, were shown to elicit incretin secretion from enteroendocrine cell lines or in vitro in tissue preparations, convincing data that this is also relevant in vivo are missing to date. However, there is growing interest in targeting intestinal sensory pathways, involving receptors but also the 'transceptors' PEPT1 and SGLT1, by use of drugs or food constituents to elicit the beneficial effects of incretins on the pancreas and metabolic control.

RANTES (CCL5) reduces glucose-dependent secretion of glucagon-like peptides 1 and 2 and impairs glucose-induced insulin secretion in mice.

Type 2 diabetes is associated with elevated circulating levels of the chemokine RANTES and with decreased plasma levels of the incretin hormone glucagon-like peptide 1 (GLP-1). GLP-1 is a peptide secreted from intestinal L-cells upon nutrient ingestion. It enhances insulin secretion from pancreatic ß-cells and protects from ß-cell loss but also promotes satiety and weight loss. In search of chemokines that may reduce GLP-1 secretion we identified RANTES and show that it reduces glucose-stimulated GLP-1 secretion in the human enteroendocrine cell line NCI-H716, blocked by the antagonist Met-RANTES, and in vivo in mice. RANTES exposure to mouse intestinal tissues lowers transport function of the intestinal glucose transporter SGLT1, and administration in mice reduces plasma GLP-1 and GLP-2 levels after an oral glucose load and thereby impairs insulin secretion. These data show that RANTES is involved in altered secretion of glucagon-like peptide hormones most probably acting through SGLT1, and our study identifies the RANTES-receptor CCR1 as a potential target in diabetes therapy.

Oligopeptides stimulate glucagon-like peptide-1 secretion in mice through proton-coupled uptake and the calcium-sensing receptor.

AIMS/HYPOTHESIS: Ingested protein is a well-recognised stimulus for glucagon-like peptide-1 (GLP-1) release from intestinal L cells. This study aimed to characterise the molecular mechanisms employed by L cells to detect oligopeptides. METHODS: GLP-1 secretion from murine primary colonic cultures and Ca(2+) dynamics in L cells were monitored in response to peptones and dipeptides. L cells were identified and purified based on their cell-specific expression of the fluorescent protein Venus, using GLU-Venus transgenic mice. Pharmacological tools and knockout mice were used to characterise candidate sensory pathways identified by expression analysis. RESULTS: GLP-1 secretion was triggered by peptones and di-/tripeptides, including the non-metabolisable glycine-sarcosine (Gly-Sar). Two sensory mechanisms involving peptide transporter-1 (PEPT1) and the calcium-sensing receptor (CaSR) were distinguishable. Responses to Gly-Sar (10 mmol/l) were abolished in the absence of extracellular Ca(2+) or by the L-type calcium-channel blocker nifedipine (10 µmol/l) and were PEPT1-dependent, as demonstrated by their sensitivity to pH and 4-aminomethylbenzoic acid and the finding of impaired responses in tissue from Pept1 (also known as Slc15a1) knockout mice. Peptone (5 mg/ml)-stimulated Ca(2+) responses were insensitive to nifedipine but were blocked by antagonists of CaSR. Peptone-stimulated GLP-1 secretion was not impaired in mice lacking the putative peptide-responsive receptor lysophosphatidic acid receptor 5 (LPAR5; also known as GPR92/93). CONCLUSIONS/INTERPRETATION: Oligopeptides stimulate GLP-1 secretion through PEPT1-dependent electrogenic uptake and activation of CaSR. Both pathways are highly expressed in native L cells, and likely contribute to the ability of ingested protein to elevate plasma GLP-1 levels. Targeting nutrient-sensing pathways in L cells could be used to mobilise endogenous GLP-1 stores in humans, and could mimic some of the metabolic benefits of bariatric surgery.

Mice lacking the intestinal peptide transporter display reduced energy intake and a subtle maldigestion/malabsorption that protects them from diet-induced obesity.

The intestinal transporter PEPT1 mediates the absorption of di- and tripeptides originating from breakdown of dietary proteins. Whereas mice lacking PEPT1 did not display any obvious changes in phenotype on a high-carbohydrate control diet (HCD), Pept1(-/-) mice fed a high-fat diet (HFD) showed a markedly reduced weight gain and reduced body fat stores. They were additionally protected from hyperglycemia and hyperinsulinemia. Energy balance studies revealed that Pept1(-/-) mice on HFD have a reduced caloric intake, no changes in energy expenditure, but increased energy content in feces. Cecal biomass in Pept1(-/-) mice was as well increased twofold on both diets, suggesting a limited capacity in digesting and/or absorbing the dietary constituents in the small intestine. GC-MS-based metabolite profiling of cecal contents revealed high levels and a broad spectrum of sugars in PEPT1-deficient mice on HCD, whereas animals fed HFD were characterized by high levels of free fatty acids and absence of sugars. In search of the origin of the impaired digestion/absorption, we observed that Pept1(-/-) mice lack the adaptation of the upper small intestinal mucosa to the trophic effects of the diet. Whereas wild-type mice on HFD adapt to diet with increased villus length and surface area, Pept1(-/-) mice failed to show this response. In search for the origin of this, we recorded markedly reduced systemic IL-6 levels in all Pept1(-/-) mice, suggesting that IL-6 could contribute to the lack of adaptation of the mucosal architecture to the diets.

Drug Screening, Oral Bioavailability and Regulatory Aspects: A Need for Human Organoids.

The intestinal epithelium critically contributes to oral bioavailability of drugs by constituting an important site for drug absorption and metabolism. In particular, intestinal epithelial cells (IEC) actively serve as gatekeepers of drug and nutrient availability. IECs' transport processes and metabolism are interrelated to the whole-body metabolic state and represent potential points of origin as well as therapeutic targets for a variety of diseases. Human intestinal organoids represent a superior model of the intestinal epithelium, overcoming limitations of currently used in vitro models. Caco-2 cells or rodent explant models face drawbacks such as their cancer and non-human origin, respectively, but are commonly used to study intestinal nutrient absorption, enterocyte metabolism and oral drug bioavailability, despite poorly correlative data. In contrast, intestinal organoids allow investigating distinct aspects of bioavailability including spatial resolution of transport, inter-individual differences and high-throughput screenings. As several countries have already developed strategic roadmaps to phase out animal experiments for regulatory purposes, intestinal organoid culture and organ-on-a-chip technology in combination with in silico approaches are roads to go in the preclinical and regulatory setup and will aid implementing the 3Rs (reduction, refinement and replacement) principle in basic science.

Intestinal sodium/glucose cotransporter 3 expression is epithelial and downregulated in obesity.

AIM: We aimed to determine whether the sodium/glucose cotransporter family member SGLT3, a proposed glucose sensor, is expressed in the intestine and/or kidney, and if its expression is altered in mouse models of obesity and in humans before and after weight-loss surgery. MAIN METHODS: We used in-situ hybridization and quantitative PCR to determine whether the Sglt3 isoforms 3a and 3b were expressed in the intestine and kidney of C57, leptin-deficient ob/ob, and diabetic BTBR ob/ob mice. Western blotting and immunohistochemistry were also used to assess SGLT3 protein levels in jejunal biopsies from obese patients before and after weight-loss Roux-en-Y gastric bypass surgery (RYGB), and in lean healthy controls. KEY FINDINGS: Sglt3a/3b mRNA was detected in the small intestine (duodenum, jejunum and ileum), but not in the large intestine or kidneys of mice. Both isoforms were detected in epithelial cells (confirmed using intestinal organoids). Expression of Sglt3a/3b mRNA in duodenum and jejunum was significantly lower in ob/ob and BTBR ob/ob mice than in normal-weight littermates. Jejunal SGLT3 protein levels in aged obese patients before RYGB were lower than in lean individuals, but substantially upregulated 6 months post-RYGB. SIGNIFICANCE: Our study shows that Sglt3a/3b is expressed primarily in epithelial cells of the small intestine in mice. Furthermore, we observed an association between intestinal mRNA Sglt3a/3b expression and obesity in mice, and between jejunal SGLT3 protein levels and obesity in humans. Further studies are required to determine the possible role of SGLT3 in obesity.

Organoids to Study Intestinal Nutrient Transport, Drug Uptake and Metabolism - Update to the Human Model and Expansion of Applications.

Intestinal transport and sensing processes and their interconnection to metabolism are relevant to pathologies such as malabsorption syndromes, inflammatory diseases, obesity and type 2 diabetes. Constituting a highly selective barrier, intestinal epithelial cells absorb, metabolize, and release nutrients into the circulation, hence serving as gatekeeper of nutrient availability and metabolic health for the whole organism. Next to nutrient transport and sensing functions, intestinal transporters including peptide transporter 1 (PEPT1) are involved in the absorption of drugs and prodrugs, including certain inhibitors of angiotensin-converting enzyme, protease inhibitors, antivirals, and peptidomimetics like ß-lactam antibiotics. Here, we verify the applicability of 3D organoids for in vitro investigation of intestinal biochemical processes related to transport and metabolism of nutrients and drugs. Establishing a variety of methodologies including illustration of transporter-mediated nutrient and drug uptake and metabolomics approaches, we highlight intestinal organoids as robust and reliable tool in this field of research. Currently used in vitro models to study intestinal nutrient absorption, drug transport and enterocyte metabolism, such as Caco-2 cells or rodent explant models are of limited value due to their cancer and non-human origin, respectively. Particularly species differences result in poorly correlative data and findings obtained in these models cannot be extrapolated reliably to humans, as indicated by high failure rates in drug development pipelines. In contrast, human intestinal organoids represent a superior model of the intestinal epithelium and might help to implement the 3Rs (Reduction, Refinement and Replacement) principle in basic science as well as the preclinical and regulatory setup.

Validation of Arxula Yeast Estrogen Screen assay for detection of estrogenic activity in water samples: Results of an international interlaboratory study.

Endocrine-active substances can adversely impact the aquatic ecosystems. A special emphasis is laid, among others, on the effects of estrogens and estrogen mimicking compounds. Effect-based screening methods like in vitro bioassays are suitable tools to detect and quantify endocrine activities of known and unknown mixtures. This study describes the validation of the Arxula-Yeast Estrogen Screen (A-YES®) assay, an effect-based method for the detection of the estrogenic potential of water and waste water. This reporter gene assay, provided in ready to use format, is based on the activation of the human estrogen receptor alpha. The user-friendly A-YES® enables inexperienced operators to rapidly become competent with the assay. Fourteen laboratories from four countries with different training levels analyzed 17ß-estradiol equivalent concentrations (EEQ) in spiked and unspiked waste water effluent and surface water samples, in waste water influent and spiked salt water samples and in a mixture of three bisphenols. The limit of detection (LOD) for untreated samples was 1.8ng/L 17ß-estradiol (E2). Relative repeatability and reproducibility standard deviation for samples with EEQ above the LOD (mean EEQ values between 6.3 and 20.4ng/L) ranged from 7.5 to 21.4% and 16.6 to 28.0%, respectively. Precision results are comparable to other frequently used analytical methods for estrogens. The A-YES® has been demonstrated to be an accurate, precise and robust bioassay. The results have been included in the ISO draft standard. The assay was shown to be applicable for testing of typical waste water influent, effluent and saline water. Other studies have shown that the assay can be used with enriched samples, which lower the LOD to the pg/L range. The validation of the A-YES® and the development of a corresponding international standard constitute a step further towards harmonized and reliable bioassays for the effect-based analysis of estrogens and estrogen-like compounds in water samples.

Role of Incretin Hormones in Bowel Diseases.

Enteroendocrine cells (EEC) have been studied extensively for their ability to regulate gastrointestinal motility and insulin release by secretion of peptide hormones. In particular, the L cell-derived incretin glucagon-like peptide 1 has gained enormous attention due to its insulinotropic action and relevance in the treatment of type 2 diabetes. Yet, accumulating data indicates a critical role for EEC and incretins in metabolic adaptation and in orchestrating immune responses beyond blood glucose control. EEC actively sense the lamina propria and luminal environment including the microbiota via receptors and transporters, subsequently mediating signals by secreting hormones and cytokines. Data indicate that immune cells and cytokine-mediated signaling impacts EEC numbers and function during infection and chronic inflammation of the gut, suggesting EEC not only to play a role in these pathologies but also being a target of inflammatory processes. This review presents data on the interrelation of incretins and inflammatory signaling. It focuses on the impact of intestinal inflammation, in particular inflammatory bowel disease, on EEC and the potential role of EEC and incretins in these pathologies. Furthermore, it highlights endoplasmic reticulum unfolded protein response, cytokines and the intestinal microbiota as possible targets of inflammatory and EEC signaling.

Inflammation Meets Metabolic Disease: Gut Feeling Mediated by GLP-1.

Chronic diseases, such as obesity and diabetes, cardiovascular, and inflammatory bowel diseases (IBD) share common features in their pathology. Metabolic disorders exhibit strong inflammatory underpinnings and vice versa, inflammation is associated with metabolic alterations. Next to cytokines and cellular stress pathways, such as the unfolded protein response (UPR), alterations in the enteroendocrine system are intersections of various pathologies. Enteroendocrine cells (EEC) have been studied extensively for their ability to regulate gastrointestinal motility, secretion, and insulin release by release of peptide hormones. In particular, the L-cell-derived incretin hormone glucagon-like peptide 1 (GLP-1) has gained enormous attention due to its insulinotropic action and relevance in the treatment of type 2 diabetes (T2D). Yet, accumulating data indicate a critical role for EEC and in particular for GLP-1 in metabolic adaptation and in orchestrating immune responses beyond blood glucose control. EEC sense the lamina propria and luminal environment, including the microbiota via receptors and transporters. Subsequently, mediating signals by secreting hormones and cytokines, EEC can be considered as integrators of metabolic and inflammatory signaling. This review focuses on L cell and GLP-1 functions in the context of metabolic and inflammatory diseases. The effects of incretin-based therapies on metabolism and immune system are discussed and the interrelation and common features of metabolic and immune-mediated disorders are highlighted. Moreover, it presents data on the impact of inflammation, in particular of IBD on EEC and discusses the potential role of the microbiota as link between nutrients, metabolism, immunity, and disease.

Gut barrier impairment by high-fat diet in mice depends on housing conditions.

SCOPE: Diet-induced obesity (DIO) is proposed to cause impairments in intestinal barrier integrity, but contradictory results have been published and it appears that the outcomes depend on other environmental factors. We therefore assessed whether the hygienic status of animal facilities alters the gut barrier in DIO mice. METHODS AND RESULTS: Male C57BL/6N mice were housed in a conventional (CV) or a specific pathogen-free (SPF) animal facility and were fed identical diets represented by a high-fat (60kJ% fat) or control diet (11kJ% fat) for 12 wks. Intestinal barrier function in small and large intestine was evaluated in Ussing chambers by electrical resistance and permeability measurements. Jejunal (p < 0.01) and proximal colonic (p < 0.05) barrier function was altered in CV DIO mice, but not in SPF DIO mice. Moreover, only CV DIO mice were characterized by metabolic endotoxemia and low-grade inflammation. High-throughput 16S rRNA gene sequencing revealed significant differences in fecal bacterial diversity and composition between the two animal facilities, but only in mice fed the HFD. Moreover, cecal DCA concentrations correlated positively with two yet uncultivated Clostridiales species. CONCLUSIONS: We demonstrated that housing conditions and associated changes in gut bacterial colonization are pivotal for maintenance of gut barrier integrity in DIO mice.

Intestinal organoids for assessing nutrient transport, sensing and incretin secretion.

Intestinal nutrient transport and sensing are of emerging interest in research on obesity and diabetes and as drug targets. Appropriate in vitro models are lacking that allow both, studies on transport processes as well as sensing and subsequent incretin hormone secretion including intracellular signaling. We here demonstrate that murine small-intestinal organoids are the first in vitro model system enabling concurrent investigations of nutrient and drug transport, sensing and incretin hormone secretion as well as fluorescent live-cell imaging of intracellular signaling processes. By generating organoid cultures from wild type mice and animals lacking different nutrient transporters, we show that organoids preserve the main phenotypic features and functional characteristics of the intestine. This turns them into the best in vitro model currently available and opens new avenues for basic as well as medical research.

The role of SGLT1 and GLUT2 in intestinal glucose transport and sensing.

Intestinal glucose absorption is mediated by SGLT1 whereas GLUT2 is considered to provide basolateral exit. Recently, it was proposed that GLUT2 can be recruited into the apical membrane after a high luminal glucose bolus allowing bulk absorption of glucose by facilitated diffusion. Moreover, SGLT1 and GLUT2 are suggested to play an important role in intestinal glucose sensing and incretin secretion. In mice that lack either SGLT1 or GLUT2 we re-assessed the role of these transporters in intestinal glucose uptake after radiotracer glucose gavage and performed Western blot analysis for transporter abundance in apical membrane fractions in a comparative approach. Moreover, we examined the contribution of these transporters to glucose-induced changes in plasma GIP, GLP-1 and insulin levels. In mice lacking SGLT1, tissue retention of tracer glucose was drastically reduced throughout the entire small intestine whereas GLUT2-deficient animals exhibited higher tracer contents in tissue samples than wild type animals. Deletion of SGLT1 resulted also in reduced blood glucose elevations and abolished GIP and GLP-1 secretion in response to glucose. In mice lacking GLUT2, glucose-induced insulin but not incretin secretion was impaired. Western blot analysis revealed unchanged protein levels of SGLT1 after glucose gavage. GLUT2 detected in apical membrane fractions mainly resulted from contamination with basolateral membranes but did not change in density after glucose administration. SGLT1 is unequivocally the prime intestinal glucose transporter even at high luminal glucose concentrations. Moreover, SGLT1 mediates glucose-induced incretin secretion. Our studies do not provide evidence for GLUT2 playing any role in either apical glucose influx or incretin secretion.

Live-Cell Calcium Imaging in 3D Intestinal Organoids.

Live-cell Ca2+ imaging is an important tool to detect activation of receptors by a putative ligand/drug and complements studies on transport processes, as intracellular Ca2+ changes provide direct evidence for substrate fluxes. Organoid-based systems offer numerous advantages over other in vitro systems such as cell lines, primary cells, or tissue explants, and in particular, intestinal organoid culture has revolutionized research on functional gastrointestinal processes. Calcium imaging using the fluorescent Ca2+ indicator Fura-2-AM can be applied to 3D intestinal organoids, which show an excellent dye-loading efficiency. Here we describe live-cell Ca2+ imaging in intestinal organoids, an important technique to improve research on malabsorption syndromes, secretory diarrhea, and metabolic disorders.