New insights into proteomic and lipidomic changes during insulin granule aging

Endocrine cells utilize secretory granules for the storage and regulated release of peptide- hormones and neurotransmitters - a process influenced by spatiotemporal factors like the proximity of these organelles to the plasma membrane and their age. While recent progress has been made, a comprehensive view about the molecular composition and aging-related changes of the insulin-containing secretory granules (ISGs) of pancreatic islet beta cells remains elusive. Now, new light was shed on the molecular composition of ISGs, which is relevant to better understand their behavior in healthy conditions and possibly in diabetes, in which insulin release is impaired. A team of researchers from the Paul Langerhans Institute Dresden (PLID) of the German Center for Diabetes Research, the Center of Membrane Biochemistry and Lipid Research at TU Dresden, the Max Planck Institutes for Biochemistry and Medical Research, respectively, as well as the Center for Molecular and Cellular Bioengineering have unveiled a novel immunobased approach for the purification of ISGs. Published in renowned Journal “Cell Reports”, this refined protocol enabled for the first time the accurate proteomic and lipidomic profiling of age-distinct ISG pools.

Exocytosis, a fundamental cellular process responsible for transporting cargoes out of cells, is particularly vital for the maintenance of plasma membrane protein and lipid homeostasis. Regulated exocytosis, triggered by specific signals like elevated intracellular Ca²⁺ levels, is crucial for various cell types, including peptide-hormone-secreting endocrine cells, such as pancreatic islet beta cells. However, up to now the analysis of age-distinct ISG pools has faced challenges due to the lack of reliable purification protocols.

The research team around Dr. Martin Neukam and Prof. Michele Solimena from the PLID, addressed these challenges, by introducing an immunobased approach for purification for ISGs combined with pulse-chase labeling of the ISG-specific bait antigen to restrict its capture after its exit from the Golgi apparatus and sorting into the ISGs. “This innovative technique drastically reduces background interference, enabling the isolation of highly purified ISGs that can be eluted as intact organelles. Importantly, the approach is adaptable for the isolation of age-distinct insulin SG pools”, explains Neukam and continues, "Our protocol takes advantage of the specificity of immunopurification and pulse-chase labeling, providing highly purified ISGs. This method allowed us to conduct a detailed analysis of the proteomics and phospholipidomics profiles of younger and older SGs from INS-1 cells. To our knowledge this is the first-time age-distinct pools of a given cellular organelle could be independently isolated and studied in detail".

The lipidomic and proteomic composition of ISG was scrutinized, revealing intriguing insights. Changes were found about the lipidomic composition of the ISG over time, with a notable shift in the ratio of phosphatidylcholine to phosphatidylethanolamine. This alteration may influence the fluidity and charge of ISG membranes, potentially impacting on their exocytotic properties.

The team also discovered the preferential association of motor protein KIF5b and RAB3a with younger ISGs, hence shedding light on the reasons accounting for the different behavior of age-distinct ISGs. "These molecular data corroborate a model whereby younger ISGs are preferentially released, whereas older ISGs are better poised for degradation by autophagy. The differences in lipids may influence their ability to recruit cytosolic proteins, such as motor proteins involved in microtubule-dependent transport of ISGs, and thus have an impact on their propensity for exocytosis or targeting for intracellular degradation," states Solimena.

Overall, these findings represent a significant step forward in the research field of cell biology, contributing valuable insights into the complex processes governing cellular communication and exocytosis, with potential applications in various biomedical fields, such as diabetes.

Original Publication

Neukam M, Sala P, Brunner AD, Ganß K, Palladini A, Grzybek M, Topcheva O, Vasiljević J, Broichhagen J, Johnsson K, Kurth T, Mann M, Coskun Ü, Solimena M. Purification of time-resolved insulin granules reveals proteomic and lipidomic changes during granule aging. Cell Rep. Volume 43, Issue 3, 26 March 2024, 113836. https://doi.org/10.1016/j.celrep.2024.113836