Real-time TIRF imaging of exocytosis in 3T3-L1 adipocytes stably expressing mCherry fused to human adiponectin.

Abstract

Adiponectin is a peptide hormone abundantly released from adipocytes, and reduced circulating levels are associated with obesity-related diseases, such as type 2 diabetes, insulin resistance and cardiovascular disease. Adiponectin is released by regulated exocytosis of secretory vesicles, but traditional molecular biology and imaging techniques lack the specificity and time resolution to adequately quantify exocytosis and trafficking of adiponectin-containing vesicles. Here we generated 3T3-L1 adipocytes that stably express mCherry-tagged human adiponectin, resulting in robust labelling of small adiponectin vesicles with a diameter of 200-300 nm, in live cells. Total internal reflection fluorescence (TIRF) microscopy was used to visualise and quantify exocytosis and adiponectin release in real-time, observed as rapid disappearance of the fluorescence of individual vesicles. Bulk adiponectin secretion measurements confirmed that the labelled adiponectin was secreted to the surrounding solution under these conditions, and expressed in the same vesicle population as endogenous adiponectin. In contrast to previous electrophysiological results, elevation of cytosolic Ca2+ alone was sufficient to induce exocytosis, although at a lower rate compared to elevated cytosolic cAMP. We conclude that the adiponectin-mCherry-labelled cells are useful for studying adiponectin exocytosis at the single vesicle level, and that an intracellular elevation of either cAMP or Ca2+ can trigger adiponectin vesicle release.