Endocrine cells are continually regulating the balance between hormone biosynthesis, secretion, and intracellular degradation to ensure that cellular hormone stores are maintained at optimal levels. In pancreatic beta-cells, intracellular insulin stores in beta-granules are mostly upheld by efficiently up-regulating proinsulin biosynthesis at the translational level to rapidly replenish the insulin lost via exocytosis. Under normal circumstances, intracellular degradation of insulin plays a relatively minor janitorial role in retiring aged beta-granules, apparently via crinophagy. However, this mechanism alone is not sufficient to maintain optimal insulin storage in beta-cells when insulin secretion is dysfunctional. Here, we show that despite an abnormal imbalance of glucose/glucagon-like peptide 1 regulated insulin production over secretion in Rab3A(-/-) mice compared with control animals, insulin storage levels were maintained due to increased intracellular beta-granule degradation. Electron microscopy analysis indicated that this was mediated by a significant 12-fold up-regulation of multigranular degradation vacuoles in Rab3A(-/-) mouse islet beta-cells (P <or= 0.001), which by further electron microscopy-tomography analysis was found to be mostly contributed by microautophagic activity. This increased autophagic activity in Rab3A(-/-) mouse islet beta-cells was associated with a specific decrease in islet lysosomal-associated membrane protein 2 gene expression (P <or= 0.05), at both the mRNA and protein expression levels. Lysosomal-associated membrane protein 2 is a documented negative regulator of autophagy. These findings indicate that the up-regulation of degradative pathways provides secretory-deficient endocrine cells with a compensatory mechanism for regulating their intracellular hormone content in vivo. These data may also have implications for the beta-cell's response to diminished insulin secretion during the pathogenesis of type 2 diabetes.