Mechanisms of gypenosides in type 2 diabetes mellitus via regulation of m6A methylase METTL3/14 and demethylase FTO.

OBJECTIVES: Type 2 diabetes mellitus (T2DM) is characterized by insufficient insulin secretion and insulin resistance. Gypenosides (GPs) are the major bioactive saponins extracted from Gynostemma pentaphyllum. Previous studies suggest that GPs have beneficial effects on T2DM, but the underlying mechanisms remain unclear. This study aims to investigate whether GPs exert therapeutic effects by influencing RNA N(6)-methyladenosine (m6A) methylation modification, thereby regulating the downstream phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway. METHODS: Expression levels and methylation changes of METTL3, METTL14, and FTO in T2DM were analyzed using public databases, and related pathways were explored via gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. The main active components of GPs were screened from pharmacological databases, followed by compound-target network construction, enrichment analyses, and prediction of potential targets and pathways. A T2DM model was induced in Sprague-Dawley rats using a high-fat/high-sugar diet combined with low-dose streptozotocin. Rats were randomly divided into 4 groups: GPs-treated group (GPG, 400 mg/kg), positive control group (PCG, metformin 100 mg/kg), normal saline control group (CONTROL), and T2DM model group (MODEL). Fasting blood glucose (FBG), oral glucose tolerance test (OGTT)-area under the glucose curve from 0 to 120 min (AUC(0-120)), fasting insulin (FINS), homeostasis model assessment of insulin resistance (HOMA-IR), serum inflammatory factors, and tissue pathology of pancreas and liver hematoxylin and eosin (HE) staining were assessed. Real-time fluorescence quantitative PCR and Western blotting were used to detect RNA and protein expression levels of METTL3, METTL14, FTO, PI3K, and AKT in pancreatic tissues. Molecular docking was applied to evaluate interactions between GPs' main components and METTL3, METTL14, and FTO to infer potential binding modes. RESULTS: Bioinformatic analyses showed downregulation of METTL3/14 and upregulation of FTO in T2DM samples (all P<0.05), with enrichment in pathways related to insulin signaling, PI3K/AKT activation, oxidative stress response, and hormone secretion. Network pharmacology indicated that GPs components may act on targets involved in RNA modification and insulin-related pathways. In diabetic rats, GPs significantly reduced FBG, improved glucose tolerance, decreased HOMA-IR, and decreased the serum tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 levels compared with MODEL (all P<0.05). Pancreatic pathology showed alleviated islet injury and improved cell morphology. GPs treatment up-regulated METTL3/14 mRNA and protein levels, and down-regulated FTO mRNA/protein levels in pancreatic tissue (all P<0.05). PI3K and AKT expression levels increased (both P<0.05), consistent with activation of downstream signals related to glucose uptake and improved insulin sensitivity. Metformin also improved metabolic indices but exhibited a different regulatory pattern on m6A-related enzymes compared with GPs. Molecular docking revealed stable interactions between core GPs saponin structures and methyltransferase-like 3 (METTL3), methyltransferase-like 14 (METTL14), or obesity-associated protein (FTO), suggesting that GPs may directly or indirectly modulate m6A regulatory proteins. CONCLUSIONS: GPs can effectively improve glucose-metabolism disorders, reduce inflammation, and protect pancreatic tissue in T2DM rats. The mechanisms may be associated with METTL3/14 up-regulation and FTO down-regulation, leading to enhanced m6A methylation and subsequent activation of the PI3K/AKT signaling pathway. These findings provide strong evidence for GPs regulation of epigenetic m6A RNA modification and insulin-related downstream pathways, and suggest that natural compounds targeting m6A regulation may be explored in the future for metabolic disease interventions.