The critical role of skeletal muscle in regulating metabolic homeostasis is well-acknowledged. The p21-activated kinase (PAK1), a prominent signaling molecule in skeletal muscle, has garnered attention for its potential modulatory role in glucose and mitochondrial metabolism. By applying conditioned media derived from PAK1-enriched L6 Glut4 myc myotubes or myoblasts to INS-1 832/13 clonal β-cells, we confirmed that a muscle-derived circulating factor(s) could enhance β-cell function. Furthermore, our mass spectrometry analysis in the conditioned media (CM) collected from PAK1 enriched L6.GLUT4myc myotubes revealed elevated levels of one protein in particular, hepatocyte growth factor activator (HGFAC), which is categorized as a secreted myokine. HGFAC is secreted into the circulation, where, upon activation, it can act in a paracrine or endocrine fashion to proteolytically cleave and activate HGF. It has been reported that HGF has a vital role in islet β-cell mass and function and HNFα, a transcription factor that is expressed in pancreatic β-cells regulates β-cell mass or function by modulating HGFAC expression. Thus, we hypothesize that PAK1-enriched skeletal muscle releases HGFAC into the serum-free conditioned medium and the medium carrying HGFAC when applied to islet β-cells enhances insulin secretion. Elevated levels of HGFAC protein were detected in the cells and their associated CM from PAK1-enriched skeletal myotubes, as compared with that of GFP-transduced control myotubes, using sandwich ELISA to quantify HGFAC. Notably, these cells were incubated in a serum-free medium, a means to eliminate serum factors that might impact HGFAC levels or detection capacity. Given this, we tested the effect of this serum-free CM collected from PAK1-enriched L6.GLUT4myc myotubes application to species-matched (rat) clonal β-cells functional capacity. Indeed, CM from PAK1-enriched myotubes significantly enhanced glucose-stimulated insulin secretion (GSIS), as compared that of the control CM. The presence of elevated HGFAC levels in PAK1-enriched cells, and the beneficial impact of this HGFAC-enriched CM upon beta cell function, reveals a new mechanism by which PAK1-enrichment in skeletal muscle imparts improved overall whole-body glucose homeostasis. further hints at a possible intricate relationship between muscle-pancreas crosstalk in the regulation of islet β-cell insulin secretion. Future investigations into possible autocrine functions of PAK1-enriched skeletal muscle are underway. This research was funded by the National Institutes of Health to D.C.T. (DK067912, DK112917, and DK102233), and an American Heart Association Postdoctoral fellowship to R.B. (#905770). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.