Insulin-sensitizing effects of Monascuspilosus ΔMpclr4 fermentation products: a strain- and time-dependent metabolomic and phenotypic study with candidate mwct echanisms

Background Monascus fermentation products have hypoglycemic and lipid-lowering effects, but the influence of strain and fermentation time on metabolite profiles and insulin resistance (IR) improvement remains unclear. Uninoculated substrate showed no activity, confirming that bioactivity depends on microbial transformation, though the key metabolites and pathways are not fully known. Methods Untargeted LC-MS-based metabolomics was employed to profile the metabolite composition of fermentation products from two Monascus strains, Δ Mpclr4 and MS-1, at 7, 14, and 21 days. Key metabolic pathways were identified through multivariate statistical analysis and KEGG pathway enrichment. An insulin resistance model was established in HepG2 cells using 250 μM palmitic acid to evaluate the activity of the fermentation products, and their effects on cell function and protein expression were assessed by CCK-8 assay, 2-NBDG glucose uptake, intracellular TC/TG measurement, and Western blotting. Results Metabolomic data exhibited good quality with clear group separation. Comparisons (MS-1-21d vs. control, Δ Mpclr4 -21d vs. control, and MS-1-21d vs. Δ Mpclr4 -21d) identified 1,516, 1,439, and 333 differential metabolites (VIP>1, FDR<0.05, |log2FC|>0.263), mainly enriched in tryptophan metabolism and steroid hormone biosynthesis. Among the three time points, Δ Mpclr4 -21d showed the most pronounced glucose uptake improvement and lipid reduction, surpassing the 14-day product. At 21 days, Δ Mpclr4 -21d showed more favorable effects than MS-1-21d on cell viability, glucose uptake, and intracellular TC/TG. In the cell assays, Δ Mpclr4 -21d significantly enhanced glucose uptake and lowered both TC and TG, whereas MS-1-21d only reduced TG and did not improve TC. Δ Mpclr4 -21d was associated with upregulation of 5-HT1A and PRLR and downregulation of GANAB. Conclusion Monascus fermentation products were associated with amelioration of insulin resistance and with enrichment of pathways such as tryptophan metabolism and steroid hormone biosynthesis. However, the metabolomic data were obtained from the fermentation products themselves, not from treated HepG2 cells. The differential expression of 5-HT1A, PRLR, and GANAB is therefore proposed as correlates rather than direct evidence of intracellular pathway modulation. The Δ Mpclr4 product fermented for 21 days exhibited the greatest activity among the three time points. These findings provide preliminary evidence for developing Monascus -based functional foods; however, direct mechanistic evidence and pathway intervention experiments are required to establish causality.