Loss of Secretory Pathway Ca2+ ATPase (SPCA1) Impairs Insulin Secretion and Reduces Autophagy in the Pancreatic Islet

The β cell Golgi apparatus serves as a significant store of intracellular Ca2+ and an important site of proinsulin maturation. However, the contribution of Golgi Ca2+ to diabetes pathophysiology is unknown. The Golgi primarily utilizes the Secretory Pathway Ca2+ ATPase (SPCA1) to maintain intraluminal Ca2+ stores, and loss of SPCA1 has been linked to impaired Golgi function in other cell types. Here, we demonstrated that SPCA1 expression is decreased in islets from diabetic mice and human organ donors with type 2 diabetes, suggesting SPCA1 may impact diabetes development. INS-1 β cells lacking SPCA1 (SPCA1KO) showed reduced intraluminal Golgi Ca2+ levels, reduced glucose-stimulated insulin secretion (GSIS), and increased insulin content. Islets from SPCA1 haploinsufficient mice (SPCA1+/-) exhibited reduced GSIS, altered glucose-induced Ca2+ oscillations, and altered insulin granule maturation. Autophagy can regulate granule homeostasis, therefore we induced autophagy with Torin1 and found that SPCA1KO cells and SPCA1+/- islets had reduced levels of the autophagosome marker LC3-II. Furthermore, SPCA1KO LC3-II were unchanged after blocking autophagy initiation or autophagolysosome fusion and acidification. Thus, we concluded that β cell SPCA1 plays an important role in the maintenance of Golgi Ca2+ homeostasis and reduced Golgi Ca2+ impairs autophagy initiation and may impact insulin granule homeostasis.

Gut Metabolite Trimethylamine N-oxide Protects β Cell Insulin Secretion by Reducing Oxidative Stress and Maintaining Insulin Granule Formation

Objectives Elevated circulating levels of the dietary metabolite trimethylamine N-oxide (TMAO) is associated with chronic diseases including cardiovascular disease (CVD) and obesity. While TMAO production via the gut microbiome-liver axis and distribution through the circulation is clear, its molecular effects on metabolic tissues are still unclear. Some clinical studies suggest that elevated TMAO levels increase the risk of type 2 diabetes (T2D) where pancreatic β cell insulin secretion is insufficient for blood glucose management. T2D promoting mechanisms limit functional β cell mass by reducing β cell viability and survival, inhibiting proliferation or decreasing insulin secretory function. We hypothesized that TMAO decreases functional β cell mass by one of these mechanisms to aggravate the T2D phenotype. Methods Using the INS-1 832/13 β cell line and primary murine islets, we screened the effect of various TMAO concentrations on cell viability, proliferation, and function. These parameters were measured under standard and glucolipotoxic (GLT) culture conditions to mimic T2D. We investigated TMAO effects, GLT effects and combined effects. Results TMAO minimally affected viability, proliferation or function under standard culture conditions across 96-hours of treatment. Culturing with GLT impaired viability, proliferation and function after 24 hours of treatment, mimicking T2D onset. Interestingly, adding 40–80 μM TMAO protected against GLT mediated functional impairments in cells and islets. Further, GLT increased oxidative stress by 2.5-fold and adding TMAO was significantly protective. Electron microscopy reveals that GLT alters insulin granule density whereas TMAO maintains proper granule structure. Conclusions These results reject our hypothesis. While TMAO has minor effects on β cells in standard culture conditions, TMAO is sufficient to improve GLT mediated β cell damage by decreasing oxidative stress and maintaining insulin granule formation. These results suggest an early compensatory role for TMAO in countering oxidative damage caused by glucolipotoxicity in β cell function during T2D onset. Funding Sources Funding for this study was provided by the Beatson Foundation and the US Department of Agriculture.

In pancreatic β-cells myosin 1b regulates glucose-stimulated insulin secretion by modulating an early step in insulin granule trafficking from the Golgi

Pancreatic β-cells secrete insulin, which controls blood glucose levels, and defects in insulin secretion are responsible for diabetes mellitus. The actin cytoskeleton and some myosins support insulin granule trafficking and release, although a role for the class I myosin Myo1b, an actin- and membrane-associated load-sensitive motor, in insulin biology is unknown. We found by immunohistochemistry that Myo1b is expressed in islet cells of rat pancreas. In cultured rat insulinoma 832/13 cells Myo1b localized near actin patches, the trans-Golgi network (TGN) marker TGN38, and insulin granules in the perinuclear region. Myo1b depletion by siRNA in 832/13 cells reduced intracellular proinsulin and insulin content and glucose-stimulated insulin secretion (GSIS), and led to the accumulation of (pro)insulin SGs at the TGN. Using an in situ fluorescent pulse-chase strategy to track nascent proinsulin (Bearrows et al., 2019), Myo1b depletion in insulinoma cells reduced the number of (pro)insulin-containing secretory granules budding from the TGN. The studies indicate for the first time that in pancreatic β-cells Myo1b controls GSIS at least in part by mediating an early stage in insulin granule trafficking from the TGN.

Structural and functional polarisation of human pancreatic beta cells in islets from organ donors with and without type 2 diabetes

Aims/hypothesis We hypothesised that human beta cells are structurally and functional polarised with respect to the islet capillaries. We set out to test this using confocal microscopy to map the 3D spatial arrangement of key proteins and live-cell imaging to determine the distribution of insulin granule fusion around the cells. Methods Human pancreas samples were rapidly fixed and processed using the pancreatic slice technique, which maintains islet structure and architecture. Slices were stained using immunofluorescence for polarity markers (scribble, discs large [Dlg] and partitioning defective 3 homologue [Par3]) and presynaptic markers (liprin, Rab3-interacting protein [RIM2] and piccolo) and imaged using 3D confocal microscopy. Isolated human islets were dispersed and cultured on laminin-511-coated coverslips. Live 3D two-photon microscopy was used on cultured cells to image exocytic granule fusion events upon glucose stimulation. Results Assessment of the distribution of endocrine cells across human islets found that, despite distinct islet-to-islet complexity and variability, including multi-lobular islets, and intermixing of alpha and beta cells, there is still a striking enrichment of alpha cells at the islet mantle. Measures of cell position demonstrate that most beta cells contact islet capillaries. Subcellularly, beta cells consistently position polar determinants, such as Par3, Dlg and scribble, with a basal domain towards the capillaries and apical domain at the opposite face. The capillary interface/vascular face is enriched in presynaptic scaffold proteins, such as liprin, RIM2 and piccolo. Interestingly, enrichment of presynaptic scaffold proteins also occurs where the beta cells contact peri-islet capillaries, suggesting functional interactions. We also observed the same polarisation of synaptic scaffold proteins in islets from type 2 diabetic patients. Consistent with polarised function, isolated beta cells cultured onto laminin-coated coverslips target insulin granule fusion to the coverslip. Conclusions/interpretation Structural and functional polarisation is a defining feature of human pancreatic beta cells and plays an important role in the control of insulin secretion. Graphical abstract