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The glucose-stimulated biosynthesis of insulin in pancreatic islet beta cells is post-transcriptionally regulated. Several RNA-binding proteins (RBPs) that regulate Insulin mRNA stability and translation also bind mRNAs coding for other insulin secretory granule (ISG) proteins. However, an overview of these interactions and their glucose-induced remodelling is still missing. Here we identify two distinct sets of RBPs which were preferentially pulled down with the 5’-UTRs of mouse Ins1, Ins2, spliced Ins2, Ica512/Ptprn and Pc2/Pcsk2 mRNAs from extracts of either resting or stimulated mouse insulinoma MIN6 cells compared to those recovered with the 5’-UTR of mouse Tubg1 encoding for γ-tubulin. Among RBPs binding in resting conditions to all tested transcripts for ISG components was hnRNP A2/B1. Hnrnpa2b1 KO MIN6 cells contained lower levels of Ins1 mRNA, proinsulin and insulin compared to control cells. In resting cells, both hnRNP A2/B1 and Insulin mRNAs localized to stress granules, which dissolved upon glucose stimulation. Insulin mRNA-positive RNA granules were also found in human pancreatic beta cells in situ. Our results suggest that resting beta cells store mRNAs for insulin secretory granule proteins in stress granules through specific RNA protein interactions. Glucose stimulation remodels these interactions, releasing the transcripts, and another set of RBPs coordinates their translation.

Type 1 diabetes islet cell autoantigen 512 (ICA512/IA-2) is a tyrosine phosphatase–like intrinsic membrane protein involved in the biogenesis and turnover of insulin secretory granules (SGs) in pancreatic islet b-cells. Whereas its membrane-proximal and cytoplasmic domains have been functionally and structurally characterized, the role of the ICA512 N-terminal segment named “regulated endocrine-specific protein 18 homology domain” (RESP18HD), which encompasses residues 35–131, remains largely unknown. Here, we show that ICA512 RESP18HD residues 91–131 encode for an intrinsically disordered region (IDR), which in vitro acts as a condensing factor for the reversible aggregation of insulin and other b-cell proteins in a pH and Zn21-regulated fashion. At variance with what has been shown for other granule cargoes with aggregating properties, the condensing activity of ICA512 RESP18HD is displayed at a pH close to neutral, i.e. in the pH range found in the early secretory pathway, whereas it is resolved at acidic pH and Zn21 concentrations resembling those present in mature SGs. Moreover, we show that ICA512 RESP18HD residues 35–90, preceding the IDR, inhibit insulin fibrillation in vitro. Finally, we found that glucose-stimulated secretion of RESP18HD upon exocytosis of SGs from insulinoma INS-1 cells is associated with cleavage of its IDR, conceivably to prevent its aggregation upon exposure to neutral pH in the extracellular milieu. Taken together, these findings point to ICA512 RESP18HD being a condensing factor for protein sorting and granulogenesis early in the secretory pathway and for prevention of amyloidogenesis., Instituto Multidisciplinario de Biología Celular

Objective Insulin release from pancreatic islet β cells should be tightly controlled to avoid hypoglycemia and insulin resistance. The cortical actin cytoskeleton is a gate for regulated exocytosis of insulin secretory granules (SGs) by restricting their mobility and access to the plasma membrane. Prior studies suggest that SGs interact with F-actin through their transmembrane cargo islet cell autoantigen 512 (Ica512) (also known as islet antigen 2/Ptprn). Here we investigated how Ica512 modulates SG trafficking and exocytosis. Methods Transcriptomic changes in Ica512−/− mouse islets were analyzed. Imaging as well as biophysical and biochemical methods were used to validate if and how the Ica512-regulated gene villin modulates insulin secretion in mouse islets and insulinoma cells. Results The F-actin modifier villin was consistently downregulated in Ica512−/− mouse islets and in Ica512-depleted insulinoma cells. Villin was enriched at the cell cortex of β cells and dispersed villin−/− islet cells were less round and less deformable. Basal mobility of SGs in villin-depleted cells was enhanced. Moreover, in cells depleted either of villin or Ica512 F-actin cages restraining cortical SGs were enlarged, basal secretion was increased while glucose-stimulated insulin release was blunted. The latter changes were reverted by overexpressing villin in Ica512-depleted cells, but not vice versa. Conclusion Our findings show that villin controls the size of the F-actin cages restricting SGs and, thus, regulates their dynamics and availability for exocytosis. Evidence that villin acts downstream of Ica512 also indicates that SGs directly influence the remodeling properties of the cortical actin cytoskeleton for tight control of insulin secretion., Highlights • Ica512-depletion reduces the genetic expression of the F-actin modifier villin. • Villin-depletion enhances basal insulin granule mobility and exocytosis. • Villin regulates the size of actin cages restraining insulin granules. • Villin acts downstream of insulin granule cargo Ica512. • The Ica512-villin genetic link enables granules to control cytoskeleton plasticity.

Aims/hypothesis Pancreatic islet beta cell failure causes type 2 diabetes in humans. To identify transcriptomic changes in type 2 diabetic islets, the Innovative Medicines Initiative for Diabetes: Improving beta-cell function and identification of diagnostic biomarkers for treatment monitoring in Diabetes (IMIDIA) consortium (www.imidia.org) established a comprehensive, unique multicentre biobank of human islets and pancreas tissues from organ donors and metabolically phenotyped pancreatectomised patients (PPP). Methods Affymetrix microarrays were used to assess the islet transcriptome of islets isolated either by enzymatic digestion from 103 organ donors (OD), including 84 non-diabetic and 19 type 2 diabetic individuals, or by laser capture microdissection (LCM) from surgical specimens of 103 PPP, including 32 non-diabetic, 36 with type 2 diabetes, 15 with impaired glucose tolerance (IGT) and 20 with recent-onset diabetes (

Glucose and GLP-1 stimulate not only insulin secretion, but also the post-transcriptional induction of insulin granule biogenesis. This process involves the nucleocytoplasmic translocation of the RNA binding protein PTBP1. Binding of PTBP1 to the 3′-UTRs of mRNAs for insulin and other cargoes of beta cell granules increases their stability. Here we show that glucose enhances also the binding of PTBP1 to the 5′-UTRs of these transcripts, which display IRES activity, and their translation exclusively in a cap-independent fashion. Accordingly, glucose-induced biosynthesis of granule cargoes was unaffected by pharmacological, genetic or Coxsackievirus-mediated inhibition of cap-dependent translation. Infection with Coxsackieviruses, which also depend on PTBP1 for their own cap-independent translation, reduced instead granule stores and insulin release. These findings provide insight into the mechanism for glucose-induction of insulin granule production and on how Coxsackieviruses, which have been implicated in the pathogenesis of type 1 diabetes, can foster beta cell failure.

Insulin secretion is key for glucose homeostasis. Insulin secretory granules (SGs) exist in different functional pools, with young SGs being more mobile and preferentially secreted. However, the principles governing the mobility of age-distinct SGs remain undefined. Using the time-reporter insulin-SNAP to track age-distinct SGs we now show that their dynamics can be classified into three components: highly dynamic, restricted, and nearly immobile. Young SGs display all three components, whereas old SGs are either restricted or nearly immobile. Both glucose stimulation and F-actin depolymerization recruit a fraction of nearly immobile young, but not old, SGs for highly dynamic, microtubule-dependent transport. Moreover, F-actin marks multigranular bodies/lysosomes containing aged SGs. These data demonstrate that SGs lose their responsiveness to glucose stimulation and competence for microtubule-mediated transport over time while changing their relationship with F-actin.

The type 1 diabetes autoantigen ICA512/IA-2/RPTPN is a receptor protein tyrosine phosphatase of the insulin secretory granules (SGs) which regulates the size of granule stores, possibly via cleavage/signaling of its cytosolic tail. The role of its extracellular region remains unknown. Structural studies indicated that β2- or β4-strands in the mature ectodomain (ME ICA512) form dimers in vitro. Here we show that ME ICA512 prompts proICA512 dimerization in the endoplasmic reticulum. Perturbation of ME ICA512 β2-strand N-glycosylation upon S508A replacement allows for proICA512 dimerization, O-glycosylation, targeting to granules, and conversion, which are instead precluded upon G553D replacement in the ME ICA512 β4-strand. S508A/G553D and N506A/G553D double mutants dimerize but remain in the endoplasmic reticulum. Removal of the N-terminal fragment (ICA512-NTF) preceding ME ICA512 allows an ICA512-ΔNTF G553D mutant to exit the endoplasmic reticulum, and ICA512-ΔNTF is constitutively delivered to the cell surface. The signal for SG sorting is located within the NTF RESP18 homology domain (RESP18-HD), whereas soluble NTF is retained in the endoplasmic reticulum. Hence, we propose that the ME ICA512 β2-strand fosters proICA512 dimerization until NTF prevents N506 glycosylation. Removal of this constraint allows for proICA512 β4-strand-induced dimerization, exit from the endoplasmic reticulum, O-glycosylation, and RESP18-HD-mediated targeting to granules., Instituto Multidisciplinario de Biología Celular