Metabolites from the Dendrobium Endophyte Pseudomonas protegens CM-YJ44 Alleviate Insulin Resistance in HepG2 Cells via the IRS1/PI3K/Akt/GSK3β/GLUT4 Pathway

<b>Background/Objectives:</b> Endophytes can produce bioactive metabolites similar to their host plants. CM-YJ44 (<i>Pseudomonas protegens</i> CHA0, 99.24% similarity), an endophyte from <i>Dendrobium officinale</i>, has not yet validated hypoglycemic potential. This study aimed to evaluate its anti-insulin resistance (IR) activity and metabolite profile. <b>Methods:</b> The fermentation broth of CM-YJ44 was separated into three fractions (CM-YJ44-1, -2, and -3) using semi-preparative high-performance liquid chromatography (pre-HPLC). An IR HepG2 cell model was constructed to evaluate their glucose uptake capacity. CM-YJ44-3 was further tested for oxidative stress, inflammatory, and insulin signaling pathway activation. Metabolites in CM-YJ44-3 were preliminarily identified using the Q Exactive Focus LC-MS system (QE), and the dendrobine content was quantified by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Molecular docking was performed to predict the binding affinities between dendrobine and target proteins. <b>Results:</b> Among the three fractions, CM-YJ44-3 significantly reduced nitric oxide (NO) and reactive oxygen species (ROS) levels in IR cells, enhanced glycogen synthesis, upregulated the activities of pyruvate kinase (PK) and hexokinase (HK), and suppressed the expression of inflammatory factors. Its mechanism of action was mainly through activation of the IRS1/PI3K/Akt/GSK3β/GLUT4 signaling pathway. QE analysis preliminarily identified 24 metabolites in CM-YJ44-3. Quantitative analysis by UPLC-MS/MS showed that the dendrobine content was 78.73 ± 4.29 ng/mL. Molecular docking results indicated that dendrobine exhibited binding energies below -5 kcal/mol with multiple target proteins involved in this signaling pathway, suggesting it may be a key bioactive component responsible for the anti-IR effect. <b>Conclusions:</b> This study provides the first evidence of hypoglycemic bioactive metabolite production by strain CM-YJ44, indicating its potential as a novel microbial candidate for alleviating IR.