Glucagon-like peptide-1 receptor (GLP-1R) agonists are increasingly explored for the treatment of obesity and metabolic disorders, yet the biological consequences of presenting these ligands on the surface of lipid-based nanocarriers remain insufficiently understood. In this study, exendin-4-functionalized nanoemulsions (ex-4 NE) were developed and evaluated in 3T3-L1 pre-adipocytes and adipocytes with respect to cellular uptake and metabolic activity. Both nanoemulsion types were efficiently internalized, however, ex-4 NE displayed higher uptake rates and larger AUC values than non-targeted PEG NE. Uptake was strongly influenced by differentiation stage but only minimally affected by GLP-1R inhibition. Chlorpromazine reduced uptake only in adipocytes differentiated in presence of exendin-4, indicating a shift toward clathrin-mediated routes upon chronic ex-4 treatment, but overall supporting that nanoemulsion uptake is not directly receptor-dependent. Colocalization analysis showed only modestly higher overlap of ex-4 NE with GLP-1R in comparison to the PEG NE. Despite the absence of pronounced receptor-mediated internalization, ex-4 NE exerted substantially stronger GLP-1R-dependent metabolic effects than free exendin-4. When administered early in differentiation, ex-4 enhanced adipogenesis, whereas addition from day 2 markedly reduced lipid accumulation. Notably, 10 nM ex-4 NE produced effects comparable to 1000 nM free ex-4, suggesting enhanced receptor activation via high local ligand density at the nanoemulsion surface, enabling multivalent interactions. At higher NE concentrations, lipid accumulation increased due to lipid substrate supply, independent of GLP-1R signaling. Taken together, these findings indicate that exendin-4 functionalization enhances nanoemulsion activity primarily through amplification of biological responses rather than through pronounced receptor-mediated uptake.
