Influencing Glucose Homeostasis via a Novel Communication Between Skeletal Muscle-Derived EVs And Pancreatic Islets

Introduction: The critical role of skeletal muscle (skm) in regulating metabolic homeostasis is well recognized. The p21-activated kinase (PAK1) is a key signaling molecule in skm that has garnered attention for its potential role in modulating glucose uptake and mitochondrial function. By applying conditioned media derived from PAK1-enriched L6.Glut4myc myotubes to INS-1 832/13 clonal β-cells, we previously demonstrated that muscle-derived extracellular factors can enhance β-cell insulin release. Therefore, we hypothesize that PAK1-enriched skm releases myokines into the conditioned medium, thereby, when applied to clonal pancreatic β-cells, enhances insulin secretion. Objective: To evaluate the role of skm-PAK1-induced extracellular factors that promote inter-organ communication with pancreatic β-cells to trigger insulin secretion. Methods: Mass spectrometry analysis of secretory components derived from skm was validated through gene expression studies, immunoblotting, and enzyme-linked immunosorbent assay (ELISA). Extracellular vesicles (EVs) were isolated from skm using differential ultracentrifugation and were characterized using Single Extracellular Vesicle Nanoscopy (SEVEN) and transmission electron microscopy (TEM). Furthermore, skm PAK1 mediated insulin secretion and the molecular mechanisms involved were investigated using PAK1-enriched clonal L6.rat skeletal myotubes and INS-1 832/13 rat islet β-cells. In addition, the impact of skm-specific PAK1 ablation on muscle-derived EV secretion was assessed using novel EV-tracking Exomap1 mice crossed with skm-specific inducible PAK1 knockout mice (skmPAK1-iKO). Results: Mass spectrometry analysis, along with biochemical validation in rat skeletal myotubes, demonstrated that PAK1 is crucial for the secretion of hepatocyte growth factor activator (HGFAC) from muscle tissue. In mice with muscle-specific PAK1 ablation, we observed a significant reduction in muscle HGFAC levels. Additionally, the enrichment of muscle-specific PAK1 was associated with an increase in the levels of EVs containing hepatocyte growth factor (HGF), which is known to play a vital role in the mass and function of islet β-cells. Our SEVEN analysis characterized EVs enriched with membrane-associated HGF. Notably, our newly developed skm-Exomap1-PAK1-iKO mice (compared to wild type) exhibited a decrease in the subpopulation of HGF-laden EVs with lower tetraspanin cargo density and higher hollowness. Interestingly, PAK1-enriched skm-secreted EVs enhanced insulin secretion from islet β-cells via the c-MET®Raf1®ERK1/2 signaling pathway. Conclusion: skm PAK1 plays a pivotal role in orchestrating EV-mediated crosstalk between skm and pancreatic β-cells via the HGF signaling axis, engaging c-MET and ERK1/2 pathways. This inter-organ communication markedly amplifies insulin secretion. Therefore, targeting skm PAK1 signaling could help reduce insulin insensitivity in skm and improve insulin secretion from islet β-cells. This approach holds promise for enhancing the health and longevity of patients with T2D. Funding: This research was funded by the National Institutes of Health to DCT (DK067912, DK112917, and DK102233), the American Heart Association’s Postdoctoral fellowship (905770) and Career Development Award (25CDA1454374) to RB, and fellowships from the Ford Foundation and National Institutes of Health (DK102233-05S1) to DE & DCT. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.