Search Results (140)

Agonists of the G protein-coupled receptor TGR5 have long been sought-after for their metabolic benefits. Intestinal TGR5 activation induces secretion of the antidiabetic hormone GLP-1, which can systemically improve diabetes phenotypes in multiple organs. However, no TGR5 agonist drug candidate has succeeded in clinical trials due to their low potency and unwanted side effects. A challenge in the field has been the development of TGR5 agonists that are non-toxic, long-acting, and have functional selectivity for G protein-biased agonism. In this study, we propose a systematic pipeline for engineering optimal TGR5 agonists with antidiabetic properties. This pipeline is interdisciplinary, combining in silico, in vitro, and in vivo assays to design and validate drug candidates. We identify 2 lead compounds that outline the necessary beneficial properties for a successful TGR5 agonist against diabetes. We uncover the molecular mechanisms that allow TGR5 agonists to induce the transcription of their target, TGR5, in intestinal enteroendocrine cells. Lastly, we investigate the molecular interactions of our lead candidates in the TGR5 binding pocket to identify optimal parameters for stability and biological activity. Our strategy for TGR5 agonist design and evaluation has the potential to guide the discovery process for targeted TGR5 therapeutics for metabolic diseases. Full article

Background/Objectives: Collagen hydrolysates are widely used as nutritional ingredients for skin and joint health; however, growing evidence indicates that collagen may also exert beneficial effects on cardiometabolic pathways. Short peptides have been shown to modulate angiotensin-converting enzyme (ACE) and dipeptidyl peptidase IV (DPP-IV), key regulators of blood pressure and glucose homeostasis. This study aimed to assess the dual ACE- and DPP-IV inhibitory and GLP-1 stimulation activities, respectively of a tripeptide-enriched formulation (CH). The study was performed using a benchmark collagen hydrolysate (BCH) as reference. Methods: ACE and DPP-IV inhibitory activities were evaluated using in vitro enzymatic assays. Cellular compatibility and in situ DPP-IV inhibition were assessed in Caco-2 intestinal cells, while glucagon-like peptide-1 (GLP-1) secretion was measured in STC-1 enteroendocrine cells. The degree of hydrolysis was determined by OPA assay, and nanoLC–HRMS was used to characterize and compare the proteomic profiles of the samples. Results: Both hydrolysates exhibited dose-dependent ACE and DPP-IV inhibition; however, CH showed significantly higher inhibitory activity at comparable concentrations. CH also reduced cellular DPP-IV activity in Caco-2 cells and stimulated GLP-1 secretion in STC-1 cells, whereas BCH showed limited or non-significant cellular effects. Peptidomic analysis revealed an enrichment of short- and medium-length peptides in CH, while BCH contained a higher proportion of long peptides (>2000 Da). Consistently, CH exhibited a 1.7-fold higher degree of hydrolysis than BCH. Conclusions: The tripeptide-enriched collagen hydrolysate demonstrated superior enzymatic and cellular bioactivity compared with the benchmark formulation, supporting its potential as a multifunctional bioactive ingredient for health applications. Full article

Type 2 diabetes (T2D) is a major global healthcare challenge and burden on the quality of life in affected individuals. While lifestyle management is the mainstay treatment for T2D, the advent of gut-incretin-based therapies with powerful effects on metabolic health, appetite and weight regulation has focussed attention on the role of the gut in the risk, progression and management of T2D. Beyond the tongue, intestinal sweet taste receptors (STRs) are increasingly being identified and functionally characterised. Growing evidence now supports a role for nutrient-activated (e.g., sugars) intestinal STRs in the release of gut hormones from enteroendocrine cells (EECs) and the control of blood glucose and body weight. However, the specific STR pathway and mechanisms linking STRs to these homeostatic controls are poorly understood, with a notable gap existing between evidence from preclinical studies and clinical validation. This review explores intestinal STR-EEC functions and the evidence on how these functions regulate glucose metabolism and energy homeostasis. We further discuss the impact of environmental and dietary factors on these signalling pathways. Full knowledge of the signalling and regulation of intestinal STR-EEC and integrated neural pathways will bridge the current knowledge gap, with a high potential to develop new novel strategies targeting STRs or EECs that preserve hedonic taste rewards and reduce cravings, as well as improve the management of individuals with metabolic diseases. Full article

The aim of our study was to evaluate the effect of dietary cobalt chloride (CoCl₂) supplementation on diarrhea, growth performance, and intestinal development in post-weaning piglets. Twenty-six piglets weaned at 21 days of age (d 21) with similar body weights were randomly assigned to three treatments: a control group (n = 10), a low-dose CoCl₂ group (1 mg/kg of diet; n = 8) and a high dose CoCl₂ group (2 mg/kg of diet, n = 8). Piglets were housed individually and fed the experimental diets for 28 days, with a dietary transition at day 15. During the early post-weaning period (d0 to d14), dietary CoCl₂ supplementation was associated with favorable trends in growth performance parameters, including ADG (average daily gain: linear, 0.05 < p < 0.1) and gain to feed ratio (linear, p < 0.05), as well as reduced fecal scores (Linear, p < 0.05). However, during the later post-weaning period (d15 to d28), increasing dietary CoCl₂ levels were unfavorable trends in feed intake (Linear, p < 0.05) and ADG (Linear, 0.05 < p < 0.1). At the intestinal level, CoCl₂ supplementation was associated with dose-related changes in intestinal morphology, epithelial cell differentiation, and luminal pH. Alterations were observed in duodenal crypt depth (CD) and ileal villus height (VH), and duodenal VH/CD (Linear, p < 0.05), without significant effects on ileal epithelial proliferation and apoptosis (p > 0.1). Changes in the numbers of goblet cells in villi (Quadratic, p < 0.05) and crypt (Linear, p < 0.05), and enteroendocrine cells (Quadratic, p < 0.05) in crypt exhibited dose-dependent trends. In addition, with the increase in the CoCl₂ concentration, the expressions of genes related to nutrient transporters (DMT1, GLUT2, and SGLT1) and metabolism (HIF-1α, FBP1, and FBP2), as well as those related to the NOTCH signaling pathway (LGR5, ATOH1, HES1, and NOTCH2), showed a linear decrease (Liner, p < 0.05). This was the case except for LDHA and DLL4 (Liner, p < 0.05). The expression of the former was the lowest in the high-dose group, while that of the latter was the lowest in the low-dose group. In vitro, CoCl₂ exposure was associated with reduced organoid budding rates (Quadratic, p < 0.01), the budding numbers (Linear, p < 0.05) per organoid, and altered gene expression of SGLT1 and CHGA (Linear, p < 0.05). In summary, dietary supplementation with CoCl₂ exhibited dose- and time-dependent trends in weaned piglets. CoCl₂ supplementation during the early post-weaning period (two weeks after weaning) was associated with favorable trends in growth performance and diarrhea, whereas prolonged supplementation (4 weeks after weaning) or higher dietary level (2 mg/kg of diet) were associated with unfavorable trends in growth performance and intestinal development. These findings suggest that CoCl₂ may have potential as a short-term (two weeks after weaning), low-level (below 2 mg/kg diet) nutritional supplement, while caution is warranted regarding long-term supplementation or higher dietary inclusion levels. Full article

Background: Several studies have attempted to identify the initiating drivers of small intestinal neuroendocrine tumor (SI-NET) development and the molecular mechanisms underlying their progression and metastatic spread. Previous gene expression studies have used bulk microarrays or RNA sequencing to compare tumor tissue with normal intestinal mucosa. However, the intestine comprises multiple distinct cell types, and bulk analyses are limited by this cellular heterogeneity, which can confound tumor-specific signals. Methods: We performed single-cell RNA sequencing on primary SI-NETs and paired normal mucosa from two patients to directly compare tumor cells with their cells of origin, the enterochromaffin (EC) cells. To minimize type I errors, we applied a two-step validation strategy by overlapping differentially expressed genes with an external single-cell dataset and cross-referencing candidate genes for enteroendocrine expression in the Human Protein Atlas. Results: For further distinction and characterization, ECs were subdivided into serotonergic and non-serotonergic clusters. This analysis revealed that the SI-NET cells are transcriptionally more similar to serotonergic ECs, consistent with serum metabolite profiles derived from clinical parameters. Our analyses uncovered a loss-of-expression program characterized by regulators of epithelial differentiation and in parallel, a gain-of-expression program displayed neuronal signaling gene induction, implicating functional reprogramming toward neuronal-like properties. Together, these specific losses and gains suggest that our patient-derived SI-NETs undergo adaptation through both loss of enteroendocrine functions and acquisition of neurobiological-promoting signaling pathways. Conclusions: These findings nominate candidate drivers for further functional validation and highlight potential therapeutic strategies in our patient cohort, including restoring suppressed Notch signaling and targeting aberrant neuronal signaling networks. However, even with a two-step validation procedure, the modest cohort size limits statistical power and generalizability, particularly for the proposed association to a serotonergic phenotype. Larger, multi-patient single-cell studies are required to confirm these mechanisms and establish their clinical relevance. Full article

Objectives: Iron deficiency impairs intestinal mucosal structure and function, yet its impact on intestinal stem cells (ISCs) remains unclear. This study was therefore designed to examine how iron deficiency affects the proliferation and differentiation of ISCs. Methods: Iron-deficient mouse and enteroid models were established. Expression of key cell markers was analyzed using Western blot, qPCR, and immunofluorescence. Results: Iron deficiency led to structural impairment of the intestinal mucosa, characterized by decreased small intestinal villus height. In iron-deficient mice, expression of ChrA (enteroendocrine cell marker), Lyz (Paneth cell marker), and Muc2 (goblet cell marker) was significantly downregulated across duodenum, jejunum and ileum, whereas Vil1 (enterocyte marker) expression increased. Moreover, both Lgr5 (an ISC marker) expression and the number of Ki67-positive proliferating cells were significantly reduced, along with a decrease in Ki67 transcriptional levels under iron-deficient conditions. Similarly, deferoxamine (DFO)-treated enteroids showed fewer Lgr5-positive ISCs, downregulation of Lgr5, Lyz and Muc2, and upregulation of Vil1. RNA-seq further confirmed that iron deficiency skews ISC differentiation toward absorptive lineage. This shift was associated with modulation of the Notch signaling pathway: upregulation of the ligand Dll1, receptors Notch2 and Notch3, and the protease ADAM10, alongside downregulation of the negative regulator Atoh1. These findings indicate that Notch pathway activation promotes enterocyte differentiation under iron deprivation. Conclusions: Iron deficiency suppressed the proliferation of ISCs and induced their differentiation toward enterocytes, which is associated with the modulation of the Notch signaling pathway, providing a mechanistic insights for impaired intestinal repair and the potential for nutrient-targeted therapies. Full article

During gastrointestinal digestion, dietary proteins are hydrolyzed into peptides and free amino acids that modulate enteroendocrine function and satiety-related hormone secretion along the gut–brain axis, thereby contributing to obesity prevention. We investigated whey protein concentrate (WPC) as a source of bioactive peptides and evaluated the effects of its digests on cholecystokinin (CCK) secretion in STC-1 enteroendocrine cells by integrating the standardized INFOGEST in vitro digestion protocol, peptidomics (LC–MS/MS), and in silico bioactivity prediction. In STC-1 cells, the <3 kDa intestinal peptide fraction exhibited the strongest CCK stimulation, positioning these low-molecular-weight peptides as promising bioactive components for satiety modulation and metabolic health applications. Peptidomic analysis of this fraction identified short sequences derived primarily from β-lactoglobulin (β-La) and α-lactalbumin (α-La), enriched in hydrophobic and aromatic residues, including neuropeptide-like sequences containing the Glu–Asn–Ser–Ala–Glu–Pro–Glu (ENSAEPE) motif of β-La f(108–114). In silico bioactivity profiling with MultiPep predicted antihypertensive, angiotensin-converting enzyme (ACE)–inhibitory, antidiabetic, dipeptidyl peptidase-IV (DPP-IV)–inhibitory, antioxidant, antibacterial, and neuropeptide-like activities. Overall, digestion of WPC released low-molecular-weight peptides and amino acids that enhanced CCK secretion in vitro; these findings support their potential use in nutritional strategies to enhance satiety, modulate appetite and energy intake, and improving cardiometabolic health. Full article

Recently, evidence indicating that microplastics (MPs) have a hazardous effect on human health is accumulating. The potential of MPs having a role in carcinogenesis was suggested. Therefore, the aim of this study was to evaluate MPs’ effect on colitis-associated colorectal cancer (CAC) development. The AOM/DSS-induced CAC model was reproduced in two groups of adult male C56BL/6 mice. One of these groups received MPs (5 μm polystyrene microbeads) with drinking water at a dose of 1.48 mg/kg/day throughout the experiment (12 weeks), and the other received water untreated with MPs. In the colons of mice that consumed MPs, there was a higher number of tumor nodules at the macroscopic level, a greater tumor prevalence on histological sections, more pronounced inflammatory infiltration, a higher goblet cell volume fraction, the content of highly sulfated mucins was found in them, and there were more tumors with increased enteroendocrine cell content. We did not find any MP effects on the claudins, mucins, proapoptotic factor Bax, or on the proliferation marker Mki67 gene expression in the medial colon, nor on the serum level of TNFα, IL-1β, IL-6, and IL-10 cytokines. Thus, MPs promote the CAC development in mice by exacerbating intestinal local inflammation and damaging the epithelial barrier, and MPs may represent a potential environmental cofactor contributing to CAC risk. Full article

Colorectal cancer (CRC) is the fourth most common and the third mostly deadly cancer globally. Even with alternative therapies, some patients do not respond to treatment. Identifying modulations in the tumor microenvironment (TME) of CRC is a significant challenge due to the complex and dynamic nature of the TME. The intestinal epithelium comprises different types of secretory lineage cells, including goblet, tuft, Paneth, and enteroendocrine cells (EECs). Yet the relevance of each subtype of secretory intestinal epithelial cell (IEC) within the TME is still debated. This study investigated the involvement of IECs in CRC development through an integrative bioinformatics analysis. We used publicly available datasets from the National Center for Biotechnology Information, the Cancer Genome Atlas Program, and the National Cancer Institute’s Proteomics Tumor Analysis Consortium, encompassing both human and mouse CRC samples. Our findings reveal a CRC microenvironment characterized by elevated expression levels of genes associated with WNT pathway activity. Remarkably, there was increased expression of Paneth cell-associated markers and transcription factors, such as WISP1, LYZ, SOX9, and DEFA1. Conversely, EEC-specific gene markers, such as GCG (encoding glucagon-like peptide-1) and CHGA exhibited significant downregulation in CRC tissue compared with healthy tissue, partially due to Paneth cell activity. Gene ontology analysis showed species-conserved downregulation in hormone/peptide secretion-related pathways in both mouse and human CRC. Of note, lower levels of GCG and CHGA correlated with reduced overall survival and demonstrated a correlation with the cell cycle, apoptosis, and proliferation. These results suggest that the disruption of enteroendocrine cell signaling is a hallmark of CRC development and may hold prognostic and therapeutic value in treating CRC patients. Full article

The mouse enteroendocrine cell line STC-1 has been widely used to investigate the effects of dietary protein-derived peptides on cholecystokinin (CCK) secretion. The studies have also addressed the question of whether specific structural features of a given peptide chain may be related to higher secretagogue activity with respect to others, but a detailed structure–activity relationship in CCK-releasing peptides has not yet been reported. The aim of this study was to list the currently available CCK-releasing peptide sequences; to draw conclusions about the role played by peptide length, peptide amino acid composition and peptide amino acid sequence in differentiating their secretagogue activity; and to highlight the physicochemical properties and sequence motifs shared by the active peptides, and any possible differential feature between CCK-releasing peptides and ineffective peptides. To this end, a method was applied consisting of the fractionation of peptide sets into subsets and the comparison between paired subsets of active and inactive peptides. A few distinctive structural features related to CCK-releasing activity were highlighted for each subset. Actually, minor changes in the primary structure can make the difference between active and inactive peptides, as suggested by previous studies. Hence, the chance of predicting the activity of a peptide that has never been tested in vitro using reference structures must still be considered to be low. Full article

Kisspeptin is a neuropeptide recognised for a pivotal role within the reproductive system, but potentially important endocrine metabolic effects are less well understood. We examined effects of twice-daily intraperitoneal administration of saline vehicle or kisspeptin-10 (25 nmol/kg), for 21 days, on glucose homeostasis, energy balance, circulating hormones as well as the morphology-function of enteroendocrine and islet cells in high-fat diet (HFD) fed female mice, with normal diet (ND) mice as an additional control group. Kisspeptin-10 decreased body weight, blood glucose and energy intake to ND levels. HFD increased circulating follicle-stimulating hormone (FSH) levels, which were further enhanced by kisspeptin-10 along with luteinising hormone (LH) concentrations. Neither HFD nor kisspeptin-10 affected progesterone or corticosterone. In the ileum, kisspeptin-10 decreased crypt depth and restored villi length to ND control levels, as well as increasing the proportion of glucose-dependent insulinotropic polypeptide (GIP) positive cells when compared to HFD mice and glucagon-like peptide-1 (GLP-1) positive cells compared to ND mice. Peptide YY (PYY) immunoreactivity was unaltered by HFD or kisspeptin-10. Plasma GIP was unchanged but circulating GLP-1 and PYY were reduced to ND levels. Within the pancreas, total islet, beta- and alpha-cell areas were similar in all mice, but kisspeptin-10 intervention restored relative insulin area to ND levels. Glucagon radius, an indicator of peripherally located alpha-cells, was reduced in HFD mice but normalised by kisspeptin-10 alongside elevated glucagon-islet area. Notably, beta-cell proliferation was increased by kisspeptin-10 with no alteration in beta-cell apoptosis. Overall, we reveal a previously uncharacterised diverse metabolic role for kisspeptin in directly modulating the gut–pancreatic axis. Full article

WNT/β-catenin signaling is essential for intestinal stem cell development and self-renewal, while its dysregulation can drive tumorigenesis. GSK3, a key negative regulator of β-catenin, in intestinal homeostasis remains incompletely understood. In this study, we investigated the role of GSK3 in intestinal development, niche maintenance, and physiological function. Unlike Apc^Min/+ mice that developed intestinal polyps, neither GSK3α nor GSK3β deficiency disrupted intestinal homeostasis. However, complete GSK3 deletion (DKO) resulted in perinatal lethality, characterized by disturbed crypt–villus architecture, Paneth cell redistribution, and villus elongation. GSK3 deficiency disrupted the intestinal niche, leading to expanded and mislocalized stem cells and Paneth cells, along with reduced tuft and enteroendocrine cells. These alterations impaired nutrient absorption and gut motility. Mechanistically, β-catenin-positive cells were significantly increased following GSK3 deletion. Genetic ablation of β-catenin under GSK3-deficient conditions reduced stem and Paneth cell populations while restoring tuft and enteroendocrine cells, thereby ameliorating niche abnormalities and improving absorptive and peristaltic functions. This study indicates the essential role of GSK3/β-catenin signaling in maintaining intestinal niche integrity and digestive physiology, highlighting potential therapeutic targets for intestinal and digestive disorders. Full article

Intestinal organoids possess self-organizing capacity and recapitulate essential features of intestinal architecture and function, making them powerful models for investigating development, disease mechanisms, pharmacological testing, and host–microbe interactions. Although standardized protocols for chicken intestinal organoids have been established, a defined culture system for ducks has not been available. In this study, we optimized crypt isolation procedures and culture medium composition to establish a reproducible system tailored to duck intestinal stem cells. Among various digestive solutions, ethylene glycol tetraacetic acid (EGTA) achieved the highest crypt isolation efficacy and organoid survival. Suspension culture resulted in better survival, proliferation, and differentiation of intestinal stem cells than air–liquid interface and embedding methods (p < 0.05). Immunofluorescence and real-time PCR indicated the presence of multiple epithelial lineages, including stem cells, Paneth cells, enterocytes, goblet cells, and enteroendocrine cells. Media supplemented with CHIR99021 and LDN193189 (CL) supported growth comparable to that of media with EGF, Noggin, and R-spondin 1 (ENR). Duckling serum and specific factors, such as SB203580 and retinol, further improved organoid formation and promoted differentiation. While long-term passaging and expansion remain technically challenging, this work provides the first duck intestinal organoid model and lays the foundation for future applications in avian intestinal research, including nutrition, disease modeling, and intervention strategies. Full article

Background: Prior research has reported increased expression of duodenal chromogranin-A (CgA), secreted by enteroendocrine cells (EECs), in association with pancreatic fibrosis. However, it remains unknown whether serum CgA levels are also elevated, and whether there is a different distribution pattern of EECs in pancreatic fibrosis and other dyspeptic causes. Aims: This study had three main objectives. First, to compare the serum CgA level between patients with pancreatic fibrosis and those with other dyspeptic conditions. Second, to analyze the distribution pattern of duodenal EECs. Third, to evaluate whether biopsy results varied depending on the specific location within the duodenum. Serum CgA levels were categorized into low and high groups based on a cutoff value of 50 ng/mL. Methods: This cross-sectional prospective case series included 15 patients, with 4 patients in the low CgA group and 11 in the high CgA group. Each participant underwent a serum CgA test, transabdominal ultrasonography, pancreatic elastography, and upper gastrointestinal endoscopy. During endoscopy, a single gastric biopsy and three duodenal biopsies from different locations were obtained. Results: Patients in the high CgA group were generally older (52–68 years) than those in the low CgA group (37–55 years), with a statistically significant difference (p < 0.01). The high CgA group exhibited a clustered and centralized pattern of EECs, whereas the low CgA group showed a more discrete pattern with fewer EECs (p < 0.01). All duodenal ulcer cases were found in the low CgA group, while three cases of pancreatic fibrosis and one case of chronic pancreatitis were identified in the high CgA group. In the high CgA group, five cases of functional dyspepsia showed a band-like EEC distribution pattern, whereas cases with pancreatic fibrosis demonstrated a more uniformly scattered EEC distribution (p < 0.01). Consistency among intra-individual duodenal biopsy results was high across different biopsy sites. Conclusions: Elevated serum CgA (>50 ng/mL) and specific duodenal EEC distribution patterns may serve as potential diagnostic indicators for pancreatic fibrosis or chronic pancreatitis. These characteristics could help differentiate these conditions from functional dyspepsia. Full article

The goal of this review is to expand our understanding of how the cellular organization of the normal colonic crypt is maintained and elucidate how this intricate architecture is disrupted during tumorigenesis. Additionally, it will focus on implications for new therapeutic strategies targeting Epithelial–Mesenchymal Transition (EMT). The colonic crypt is a highly structured epithelial unit that functions in maintaining homeostasis through a complex physiological function of diverse cell types: SCs, transit-amplifying (TA) progenitors, goblet cells, absorptive colonocytes, Paneth-like cells, M cells, tuft cells, and enteroendocrine cells. These cellular subpopulations are spatially organized and regulated by multiple crucial signaling pathways, including WNT, Notch, Bone Morphogenetic Protein (BMP), and Fibroblast Growth Factor (FGF). Specifically, we discuss how these regulatory networks control the precise locations and functions of crypt cell types that are necessary to achieve cellular organization and homeostasis in the normal colon crypt. In addition, we detail how the crypt’s hierarchical structure is profoundly perturbed in colorectal cancer (CRC) development. Tumorigenesis appears to be driven by LGR5+ cancer stem cells (CSCs) and the hyperproliferation of TA cells as colonocytes undergo metabolic reprogramming. Goblet cells lose their secretory phenotype, while REG4+ Paneth-like cells foster SC niches. Tumor microenvironment is also disrupted by upregulation of M cells and by tumor-immune crosstalk that is promoted by tuft cell expansion. Moreover, the presence of enteroendocrine cells in CRC has been implicated in treatment resistance due to its contribution to tumor heterogeneity. These cellular changes are caused by the disruption of homeostasis signaling whereby: overactivation of WNT/β-catenin promotes stemness, dysregulation of Notch inhibits differentiation, suppression of BMP promotes hyperproliferation, and imbalance of FGF/WNT/BMP/NOTCH enhances cellular plasticity and invasion. Further discussion of emerging therapies targeting epithelial markers and regulatory factors, emphasizing current development in novel, precision-based approaches in CRC treatment is also included. Full article