A Sequential Gut–Pancreas–Liver In Vitro Model to Evaluate the Multi-Target Metabolic Effects of a Nutraceutical Formulation in MASLD-Related Conditions

Background/Objectives: Metabolically dysregulated-associated steatotic liver disease (MASLD) is a complex, multifactorial disorder characterised by hepatic lipid accumulation, insulin resistance, oxidative stress, and dysfunction of the gut–liver axis. Given its intricate pathophysiology, multi-target nutritional strategies represent a promising complementary approach. This study aimed to evaluate the biological effects of a multi-component nutraceutical formulation using an integrated in vitro platform replicating intestinal, hepatic, and pancreatic–liver interactions. Methods: The formulation was tested on Caco-2 intestinal cells to assess cell viability, transepithelial electrical resistance (TEER), probiotic functional properties, and glucose absorption. Intestinally processed metabolites were then applied to HepaRG liver cells under hyperglycemic (glucose) or lipotoxic conditions (oleic acid/palmitic acid) to analyse lipid accumulation, cholesterol biomarkers (HMGR, LDL), bile acid production, and cellular damage (ALT, AST). Finally, a pancreas–liver co-culture model (EndoC-βH5 and HepaRG) was employed to investigate insulin secretion and downstream hepatic metabolic signalling (IRS1, GLUT2, glycogen). Results: The formulation preserved intestinal barrier integrity and enhanced probiotic functionality, including aggregation and hydrophobicity. In hepatic models, the treatment significantly reduced intracellular lipid accumulation and triglycerides, while increasing bile acid production and improving cholesterol profiles. Under steatotic stress, it lowered transaminase levels and downregulated lipogenic signalling. In the pancreas–liver axis model, the formulation restored glucose-stimulated insulin secretion and improved hepatic metabolic signalling by increasing IRS1 levels and glycogen synthesis, indicating enhanced insulin sensitivity. Conclusions: These findings support the biological plausibility of a multi-target nutraceutical approach for MASLD. The formulation demonstrates coordinated beneficial effects on intestinal barrier function, hepatic lipid management, and glucose metabolism, providing a strong rationale for further clinical investigation.