Beta cell chromogranin B is partially segregated in distinct granules and can be released separately from insulin in response to stimulation

Abstract

Aims/hypothesis We investigated, in three beta cell lines (INS-1E, RIN-5AH, betaTC3) and in human and rodent primary beta cells, the storage and release of chromogranin B, a secretory protein expressed in beta cells and postulated to play an autocrine role. We asked whether chromogranin B is stored together with and discharged in constant ratio to insulin upon various stimuli. Methods The intracellular distribution of insulin and chromogranin B was revealed by immunofluorescence followed by three-dimensional image reconstruction and by immunoelectron microscopy; their stimulated discharge was measured by ELISA and immunoblot analysis of homogenates and incubation media. Results Insulin and chromogranin B, co-localised in the Golgi complex/trans-Golgi network, appeared largely segregated from each other in the secretory granule compartment. In INS-1E cells, the percentage of granules positive only for insulin or chromogranin B and of those positive for both was 66, 7 and 27%, respectively. In resting cells, both insulin and chromogranin B were concentrated in the granule cores; upon stimulation, chromogranin B (but not insulin) was largely redistributed to the core periphery and the surrounding halo. Strong stimulation with a secretagogue mixture induced parallel release of insulin and chromogranin B, whereas with 3-isobutyl-1-methylxantine and forskolin ± high glucose release of chromogranin B predominated. Weak, Ca 2+ -dependent stimulation with ionomycin or carbachol induced exclusive release of chromogranin B, suggesting a higher Ca 2+ sensitivity of the specific granules. Conclusions/interpretation The unexpected complexity of the beta cell granule population in terms

SORCS1 is necessary for normal insulin secretory granule biogenesis in metabolically stressed β cells

“…However, 24-hour exposure of Sorcs1 KO lean islets to 16.7 mM glucose with 0.5 mM palmitate led to depletion of islet insulin content as assessed by dithizone staining (Figure mogranin B. In control MIN6 cells, insulin and chromogranin B were partially colocalized throughout the cytoplasm and at the cell surface, consistent with their distribution throughout the secretory pathway and SGs, as previously reported (36). By contrast, in Lum-Sorcs1-expressing cells, a major proportion of the insulin did not colocalize with chromogranin B (Figure 6).…”

Distinguishing signal from autofluorescence in cryogenic correlated light and electron microscopy of mammalian cells

“…Though the secretory machinery of these cells has been extensively studied by both LM and conventional EM (Rubi et al, 2005, Giordano et al, 2008), we sought to advance those earlier efforts by imaging these cells in a near-native, frozen-hydrated state in 3-D via electron cryo-tomography (ECT) (Oikonomou and Jensen, 2016). We were particularly interested in dense core secretory granules (DCSGs), intracellular compartments which are central to the efficient secretion of hormones including insulin (Kim et al, 2001).…”

Excitatory and Inhibitory Neurons in the Hippocampus Exhibit Molecularly Distinct Large Dense Core Vesicles

“…This segregated expression may not be exclusive to the mouse hippocampus as insulin-containing vesicles in pancreatic β-cells mostly contain phogrin with just a small fraction of vesicles with detectable chromogranin B (Giordano et al, 2008). These data indicate that different neuronal populations display distinct LDCVs with non-overlapping molecular markers.…”