Islet cholesterol accumulation due to loss of ABCA1 leads to impaired exocytosis of insulin granules.

OBJECTIVE: The ATP-binding cassette transporter A1 (ABCA1) is essential for normal insulin secretion from ß-cells. The aim of this study was to elucidate the mechanisms underlying the impaired insulin secretion in islets lacking ß-cell ABCA1. RESEARCH DESIGN AND METHODS: Calcium imaging, patch clamp, and membrane capacitance were used to assess the effect of ABCA1 deficiency on calcium flux, ion channel function, and exocytosis in islet cells. Electron microscopy was used to analyze ß-cell ultrastructure. The quantity and distribution of proteins involved in insulin-granule exocytosis were also investigated. RESULTS: We show that a lack of ß-cell ABCA1 results in impaired depolarization-induced exocytotic fusion of insulin granules. We observed disturbances in membrane microdomain organization and Golgi and insulin granule morphology in ß-cells as well as elevated fasting plasma proinsulin levels in mice in the absence of ß-cell ABCA1. Acute cholesterol depletion rescued the exocytotic defect in ß-cells lacking ABCA1, indicating that elevated islet cholesterol accumulation directly impairs granule fusion and insulin secretion. CONCLUSIONS: Our data highlight a crucial role of ABCA1 and cellular cholesterol in ß-cells that is necessary for regulated insulin granule fusion events. These data suggest that abnormalities of cholesterol metabolism may contribute to the impaired ß-cell function in diabetes.

Expedited approaches to whole cell electron tomography and organelle mark-up in situ in high-pressure frozen pancreatic islets.

We have developed a simplified, efficient approach for the 3D reconstruction and analysis of mammalian cells in toto by electron microscope tomography (ET), to provide quantitative information regarding 'global' cellular organization at approximately 15-20 nm resolution. Two insulin-secreting beta cells-deemed 'functionally equivalent' by virtue of their location at the periphery of the same pancreatic islet-were reconstructed in their entirety in 3D after fast-freezing/freeze-substitution/plastic embedment in situ within a glucose-stimulated islet of Langerhans isolated intact from mouse pancreata. These cellular reconstructions have afforded several unique insights into fundamental structure-function relationships among key organelles involved in the biosynthesis and release of the crucial metabolic hormone, insulin, that could not be provided by other methods. The Golgi ribbon, mitochondria and insulin secretory granules in each cell were segmented for comparative analysis. We propose that relative differences between the two cells in terms of the number, dimensions and spatial distribution (and for mitochondria, also the extent of branching) of these organelles per cubic micron of cellular volume reflects differences in the two cells' individual capacity (and/or readiness) to respond to secretagogue stimulation, reflected by an apparent inverse relationship between the number/size of insulin secretory granules versus the number/size of mitochondria and the Golgi ribbon. We discuss the advantages of this approach for quantitative cellular ET of mammalian cells, briefly discuss its application relevant to other complementary techniques, and summarize future strategies for overcoming some of its current limitations.

Regulated autophagy controls hormone content in secretory-deficient pancreatic endocrine beta-cells.

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