Abstract

he intestine plays a dual essential role: the digestion and absorption of nutrients, while simultaneously serving as a selective barrier against external threats such as bacteria, toxins, and dietary antigens. To fulfil these functions, it relies on a specialized epithelial barrier that is permeable to nutrients but not to pathogens. This intestinal barrier is maintained through the integrity of tight junctions, a protective mucus layer, and the secretion of antimicrobial peptides. Studies have demonstrated that disruption of this barrier, leading to increased intestinal permeability, is a hallmark of metabolic diseases such as obesity and type 2 diabetes. This compromised barrier allows the translocation of microbial products, particularly lipopolysaccharides (LPS), into the bloodstream, contributing to low-grade systemic inflammation. Several factors have been implicated in this loss of barrier integrity, including high-fat diet (HFD), intestinal dysbiosis, and chronic hyperglycaemia. While the role of hyperglycaemia has been relatively well studied, the direct impact of insulin resistance—another central component of metabolic syndrome—remains poorly understood, partly due to its frequent association with adiposity, which makes it difficult to isolate its specific effects.

To investigate the direct role of intestinal insulin signalling, independently of obesity and hyperglycaemia, we characterized two complementary murine models: (i) a pharmacological model based on systemic inhibition of the insulin receptor using the antagonist S961, and (ii) a genetic model with inducible deletion of the insulin receptor specifically in intestinal epithelial cells (IRΔGUT). Both models are of normal weight, and only the latter is normoglycemic, thereby enabling the dissociation of intestinal insulin resistance from other metabolic syndrome features. Our results show that impaired intestinal insulin signalling leads to a significant increase in colonic permeability, in the absence of obesity, hyperglycaemia, or systemic inflammation. This barrier dysfunction is associated with impaired antimicrobial defenses by Paneth cells, pronounced caecal dysbiosis (including an increase in pro-inflammatory bacteria), closer proximity of the microbiota to the epithelium, and enrichment in metabolites detrimental to barrier integrity. We also identify an intrinsic defect in intestinal stem cells (ISCs), characterized by a metabolic reprogramming toward increased oxidative phosphorylation, reduced expression of stem cell markers, and diminished proliferative capacity. Ex vivo, this manifests as a decrease in the size of ISC-enriched organoids. In vivo, it leads to impaired epithelial regeneration following DSS-induced colitis and heightened susceptibility to enteric infections with Salmonella typhimuriumand Citrobacter rodentium. These defects in permeability, antimicrobial defense, and ISC function are further exacerbated under conditions of S961-induced severe hyperglycaemia, suggesting a synergistic effect of insulin resistance and hyperglycaemia on the intestinal barrier.

In conclusion, our findings demonstrate that intestinal insulin signalling is a key regulator of epithelial barrier integrity, antimicrobial peptide production, and ISC homeostasis. Its disruption, as observed in metabolic syndrome, may actively contribute to dysbiosis, increased intestinal permeability, and ultimately, the chronicity of metabolic inflammation.

Key words: Intestinal barrier – Leaky gut - Metabolic diseases - Obesity – Type 2 diabetes – Paneth cells - Antimicrobial peptides - Intestinal stem cells –Organoids