Skeletal muscle derived CD81+ extracellular vesicles as a potential therapeutic for insulin resistance in adipocytes

Abstract

Obesity and its related metabolic disorders, including insulin resistance (IR), represent a growing public health crisis, with 50% of the U.S. population projected to have obesity by 2030 and an estimated $173 billion cost to the U.S. healthcare system currently. While skeletal muscle and adipose tissue are central to insulin-stimulated glucose disposal, current therapeutic strategies for IR like exercise, pharmacological agents, and bariatric surgery are limited by adherence, accessibility, and a lack of targeted mechanisms. One emerging area of interest in novel therapeutics is the role of extracellular vesicles (EVs), lipid-bound particles released by all cell types that carry bioactive cargo and influence recipient cell behavior. CD81, a tetraspanin protein highly enriched in EVs, plays a structural role in membrane signaling domains and is increasingly implicated as a mediator of insulin sensitivity and adipose tissue homeostasis. Many studies have shown that CD81-expressing EVs are released from skeletal muscle (SkM) and may be capable of modulating metabolic signaling in distal tissues, like adipose tissue. The purpose of the present study was to investigate the functional impact of skeletal muscle-derived CD81+ EVs on adipocyte insulin signaling (Figure 1). Specifically, these studies aimed to determine whether CD81 physically interacts with insulin signaling machinery, whether loss of CD81 impairs insulin sensitivity, and whether delivery of CD81 via SkM-derived EVs could restore insulin action in adipocytes. Using 3T3L1 preadipocytes, we demonstrate that CD81 co-immunoprecipitates with phosphorylated Akt (pAkt), suggesting a physical interaction between CD81 and insulin signaling machinery. While silencing CD81 did not significantly impair insulin signaling, a modest trend toward reduced pAkt suggests a potential role. Notably, treatment with SkM-derived CD81+ EVs successfully restored CD81 content in CD81-silenced cells, confirming that CD81 can be delivered to adipocytes via EVs, but failed to significantly enhance insulin-stimulated pAkt signaling. Taken together, these data provide the first evidence that CD81 physically associates with pAkt in preadipocytes and suggest that CD81+ EVs, isolated by magnetically labelled immunocapture, may modulate membrane protein content without strongly altering downstream signaling under the tested conditions. This work contributes to establishing a framework for exploring skeletal muscle-derived CD81+ EVs as a potential tissue-specific therapy for insulin resistance and metabolic dysfunction.