A microscopic investigation on insulin uptake in human hepatocellular carcinoma-derived HepG2 cells.

An in vitro study was conducted to investigate the cellular uptake and compartmentalization of internalized insulin in human hepatocellular carcinoma-derived HepG2 cells using confocal laser scanning microscopy. The cellular nuclei and actin filaments were stained with Hoechst (λ ex = 405 nm, λ em = 415-485 nm) and Alexa Phalloidin 647 (λ ex = 649 nm, λ em = 658-775 nm) dyes, respectively. Besides, recombinant human insulin, labeled with fluorescein isothiocyanate (λ ex = 491 nm, λ em = 502-600 nm), was used for the uptake studies (37 °C) at a concentration of 0.5 mg mL-1, with timepoints at 15 min and 30 min. The optimized tri-staining protocol proved adequate for confocal microscopy, while the 2D and 3D renditions revealed insulin uptake by HepG2 cells. The internalized (fluorescent) insulin was taken up into vesicles (receptor-mediated endocytosis) and spread throughout the cytoplasm, with little or no interaction with the actin network. Some of the internalized insulin accessed the cellular nuclei. Such nuclear interaction might explain insulin's role in influencing translation, cellular proliferation, and (overall) growth. The study developed an optimized tri-staining procedure that enabled in-depth visualization of HepG2 cells (actin network and nuclei) with internalized insulin, providing a better understanding of insulin's cellular uptake and nuclear interaction.