Insulin secretion from pancreatic β-cells is a tightly controlled process where hormone synthesis, granule formation and release are regulated in order to maintain whole body glucose homeostasis. Failure to produce or release insulin results in hyperglycemia that may develop into diabetes. Insulin-containing granules exist in different pools that have different propensity for release, yet what determined the fate of a granule after initial formation is not clear. In this study we aimed to identify key steps in the early life of an insulin granule that directs it towards release. Using two different methods for time-dependent labeling, we found that insulin granules shortly after budding from the trans-Golgi network associate with mitochondria. This organelle interaction involves the voltage-dependent anion channel (VDAC) and the vesicular nucleotide transporter (VNUT). Reduced VNUT expression prevented the recruitment of VDAC to insulin granules and redirected granules towards autophagy-dependent lysosomal degradation, resulting in reduced insulin content and impaired insulin secretion. These results show the requirement of granule-mitochondria crosstalk for normal progression through the early stages of the secretory pathway.

Abstract Immediate early response 3 interacting-protein 1 (IER3IP1) is an endoplasmic reticulum resident protein, highly expressed in pancreatic cells and the developing brain cortex. Homozygous mutations in IER3IP1 have been found in individuals with microcephaly and neonatal diabetes, yet the underlying mechanism causing beta cell failure remains unclear. Here, we utilized differentiation of genome edited-stem cells into pancreatic islet cells to elucidate the molecular basis of IER3IP1 neonatal diabetes. Using CRISPR-Cas9, we generated two distinct IER3IP1-mutant human embryonic stem cell lines: a homozygous knock-in model of a patient mutation (IER3IP1V21G), and a knockout model (IER3IP1-/-). While these mutant stem cell lines differentiated normally into definitive endoderm and pancreatic progenitors, we observed that IER3IP1-KO stem cell derived-islets (SC-islets) presented a significant decrease in beta cell numbers and elevated ER stress. Retention Using Selective Hooks (RUSH) assay revealed three-fold reduction in ER-to-Golgi trafficking of proinsulin in IER3IP1 mutant beta cells. Additionally, IER3IP1 mutant SC-islets implanted into immunocompromised mice displayed defective human insulin secretion, indicating the deleterious impact of IER3IP1 mutations on beta cell function. Our study provides valuable insights into the role of IER3IP1 in human beta cell biology and establishes a useful model to investigate ER-to-Golgi trafficking defects within beta cells. Article Highlights: · IER3IP1 mutations are linked to the development of microcephaly, epilepsy, and early-onset diabetes (MEDS1). However, the underlying molecular mechanisms of cell dysfunction are unknown. · Using targeted genome-editing, we generated specific IER3IP1 mutations in hESC lines which were differentiated into pancreatic islet lineages. · Loss of IER3IP1 resulted in a three-fold reduction in ER-to-Golgi trafficking of proinsulin in stem-cell derived beta cells, leading to beta cell dysfunction both in vitro and in vivo. · Loss of IER3IP1 also triggered increased markers of endoplasmic reticulum stress, indicating the pivotal role of the ER-to-Golgi trafficking pathway for beta cell homeostasis and function.

Pancreatic β-cells rely on a delicate balance between the endoplasmic reticulum (ER) and mitochondria to maintain sufficient insulin stores for the regulation of whole animal glucose homeostasis. The ER supports proinsulin maturation through oxidative protein folding, while mitochondria supply the energy and redox buffering that maintain ER proteostasis. In the development of Type 2 diabetes (T2D), the progressive decline of β-cell function is closely linked to disruptions in ER-mitochondrial communication. Mitochondrial dysfunction is a well-established driver of β-cell failure, whereas the downstream consequences for ER redox homeostasis have only recently emerged. This interdependence of ER-mitochondrial functions suggests that an imbalance is both a cause and consequence of metabolic dysfunction. In this review, we discuss the regulatory mechanisms of ER redox control and requirements for mitochondrial function. In addition, we describe how ER redox imbalances may trigger mitochondrial dysfunction in a vicious feed forward cycle that accelerates β-cell dysfunction and T2D onset.

Type 1 diabetes (T1D) is caused by the selective autoimmune ablation of pancreatic β-cells. Emerging evidence reveals β-cell secretory dysfunction arises early in T1D development and may contribute to diseases etiology; however, the underlying mechanisms are not well understood. Our data reveal that proinflammatory cytokines elicit a complex change in the β‑cell’s Golgi structure and function. The structural modifications include Golgi compaction and loss of the inter-connecting ribbon resulting in Golgi fragmentation. We further show that Golgi structural alterations coincide with persistent altered cell-surface glycoprotein composition. Our data demonstrate that iNOS generated nitric oxide (NO) is necessary and sufficient for β-cell Golgi re-structuring. Moreover, the unique sensitivity of the β-cell to NO-dependent mitochondrial inhibition results in β-cell specific Golgi alterations that are absent in other cell types, including α-cells. Examination of human pancreas samples from autoantibody positive and T1D donors with residual β-cells further revealed alterations in β-cell, but not α-cell, Golgi structure that correlate with T1D progression. Collectively, our studies provide critical clues as to how β-cell secretory functions are specifically impacted by cytokines and NO that may contribute to the development of β-cell autoantigens relevant to T1D.

ER stress and proinsulin misfolding are heralded as contributing factors to β cell dysfunction in type 2 diabetes, yet how ER function becomes compromised is not well understood. Recent data identify altered ER redox homeostasis as a critical mechanism that contributes to insulin granule loss in diabetes. Hyperoxidation of the ER delays proinsulin export and limits the proinsulin supply available for insulin granule formation. In this report, we identified glucose metabolism as a critical determinant in the redox homeostasis of the ER. Using multiple β cell models, we showed that loss of mitochondrial function or inhibition of cellular metabolism elicited ER hyperoxidation and delayed ER proinsulin export. Our data further demonstrated that β cell ER redox homeostasis was supported by the metabolic supply of reductive redox donors. We showed that limiting NADPH and thioredoxin flux delayed ER proinsulin export, whereas thioredoxin-interacting protein suppression restored ER redox and proinsulin trafficking. Taken together, we propose that β cell ER redox homeostasis is buffered by cellular redox donor cycles, which are maintained through active glucose metabolism.

A disturbed balance between excitation and inhibition (E/I balance) is increasingly recognized as a key driver of neurodegeneration in multiple sclerosis (MS), a chronic inflammatory disease of the central nervous system. To understand how chronic hyperexcitability contributes to neuronal loss in MS, we transcriptionally profiled neurons from mice lacking inhibitory metabotropic glutamate signaling with shifted E/I balance and increased vulnerability to inflammation-induced neurodegeneration. This revealed a prominent induction of the nuclear receptor NR4A2 in neurons. Mechanistically, NR4A2 increased susceptibility to excitotoxicity by stimulating continuous VGF secretion leading to glycolysis-dependent neuronal cell death. Extending these findings to people with MS (pwMS), we observed increased VGF levels in serum and brain biopsies. Notably, neuron-specific deletion of Vgf in a mouse model of MS ameliorated neurodegeneration. These findings underscore the detrimental effect of a persistent metabolic shift driven by excitatory activity as a fundamental mechanism in inflammation-induced neurodegeneration.

Delayed proinsulin export from the Golgi in diabetic β-cells contributes to decreased insulin granule formation, but the underlying mechanisms are not clear. Here, we explored if dysregulation of Golgi pH can alter Golgi function using β-cell specific knockout (KO) of the Golgi-localized subunit of the v-ATPase, v0a2. We show that partial alkalinization of the Golgi dilates the cisternae, but does not affect proinsulin export, insulin granule formation, insulin secretion, or glucose homeostasis.

OBJECTIVE:Although individual steps have been characterized, there is little understanding of the overall process whereby glucose co-ordinates the biosynthesis of insulin with its export out of the endoplasmic reticulum (ER) and incorporation into insulin secretory granules (ISGs). Here we investigate a role for the transcription factor CREB3L2 in this context. METHODS:MIN6 cells and mouse islets were analysed by immunoblotting after treatment with glucose, fatty acids, thapsigargin and various inhibitors. Knockdown of CREB3L2 was achieved using si or sh constructs by transfection, or viral delivery. In vivo metabolic phenotyping was conducted after deletion of CREB3L2 in β-cells of adult mice using Ins1-CreER+. Islets were isolated for RNAseq and assays of glucose-stimulated insulin secretion (GSIS). Trafficking was monitored in islet monolayers using a GFP-tagged proinsulin construct that allows for synchronised release from the ER. RESULTS:With a Km ≈3.5 mM, glucose rapidly (T1/2 0.9 h) increased full length (FL) CREB3L2 followed by a slower rise (T1/2 2.5 h) in its transcriptionally-active cleavage product, P60 CREB3L2. Glucose stimulation repressed the ER stress marker, CHOP, and this was partially reverted by knockdown of CREB3L2. Activation of CREB3L2 by glucose was not due to ER stress, however, but a combination of O-GlcNAcylation, which impaired proteasomal degradation of FL-CREB3L2, and mTORC1 stimulation, which enhanced its conversion to P60. cAMP generation also activated CREB3L2, but independently of glucose. Deletion of CREB3L2 inhibited GSIS ex vivo and, following a high-fat diet (HFD), impaired glucose tolerance and insulin secretion in vivo. RNAseq revealed that CREB3L2 regulated genes controlling trafficking to-and-from the Golgi, as well as a broader cohort associated with β-cell compensation during a HFD. Although post-Golgi trafficking appeared intact, knockdown of CREB3L2 impaired the generation of both nascent ISGs and proinsulin condensates in the Golgi, implying a defect in ER export of proinsulin and/or its processing in the Golgi. CONCLUSION:The stimulation of CREB3L2 by glucose defines a novel, rapid and direct mechanism for co-ordinating the synthesis, packaging and storage of insulin, thereby minimizing ER overload and optimizing β-cell function under conditions of high secretory demand. Upregulation of CREB3L2 also potentially contributes to the benefits of GLP1 agonism and might in itself constitute a novel means of treating β-cell failure.

IntroductionWith technical advances, confocal and super-resolution microscopy have become powerful tools to dissect cellular pathophysiology. Cell attachment to glass surfaces compatible with advanced imaging is critical prerequisite but remains a considerable challenge for human beta cells. Recently, Phelps et al. reported that human beta cells plated on type IV collagen (Col IV) and cultured in neuronal medium preserve beta cell characteristics. MethodsWe examined human islet cells plated on two commercial sources of Col IV (C6745 and C5533) and type V collagen (Col V) for differences in cell morphology by confocal microscopy and secretory function by glucose-stimulated insulin secretion (GSIS). Collagens were authenticated by mass spectrometry and fluorescent collagen-binding adhesion protein CNA35. ResultsAll three preparations allowed attachment of beta cells with high nuclear localization of NKX6.1, indicating a well-differentiated status. All collagen preparations supported robust GSIS. However, the morphology of islet cells differed between the 3 preparations. C5533 showed preferable features as an imaging platform with the greatest cell spread and limited stacking of cells followed by Col V and C6745. A significant difference in attachment behavior of C6745 was attributed to the low collagen contents of this preparation indicating importance of authentication of coating material. Human islet cells plated on C5533 showed dynamic changes in mitochondria and lipid droplets (LDs) in response to an uncoupling agent 2-[2-[4-(trifluoromethoxy)phenyl]hydrazinylidene]-propanedinitrile (FCCP) or high glucose + oleic acid. DiscussionAn authenticated preparation of Col IV provides a simple platform to apply advanced imaging for studies of human islet cell function and morphology.

The pancreatic islet β-cell's preference for release of newly synthesized insulin requires careful coordination of insulin exocytosis with sufficient insulin granule production to ensure that insulin stores exceed peripheral demands for glucose homeostasis. Thus, the cellular mechanisms regulating insulin granule production are critical to maintaining β-cell function. In this report, we utilized the synchronous protein trafficking system, RUSH, in primary β-cells to evaluate proinsulin transit through the secretory pathway leading to insulin granule formation. We demonstrate that the trafficking, processing, and secretion of the proinsulin RUSH reporter, proCpepRUSH, are consistent with current models of insulin maturation and release. Using both a rodent dietary and genetic model of hyperglycemia and β-cell dysfunction, we show that proinsulin trafficking is impeded at the Golgi and coincides with the decreased appearance of nascent insulin granules at the plasma membrane. Ultrastructural analysis of β-cells from diabetic leptin receptor deficient mice revealed gross morphological changes in Golgi structure, including shortened and swollen cisternae, and partial Golgi vesiculation, which are consistent with defects in secretory protein export. Collectively, this work highlights the utility of the proCpepRUSH reporter in studying proinsulin trafficking dynamics and suggests that altered Golgi export function contributes to β-cell secretory defects in the pathogenesis of Type 2 diabetes.

Insulin is synthesized by pancreatic β-cells and stored into secretory granules (SGs). SGs fuse with the plasma membrane in response to a stimulus and deliver insulin to the bloodstream. The mechanism of how proinsulin and its processing enzymes are sorted and targeted from the trans-Golgi network (TGN) to SGs remains mysterious. No cargo receptor for proinsulin has been identified. Here, we show that chromogranin (CG) proteins undergo liquid–liquid phase separation (LLPS) at a mildly acidic pH in the lumen of the TGN, and recruit clients like proinsulin to the condensates. Client selectivity is sequence-independent but based on the concentration of the client molecules in the TGN. We propose that the TGN provides the milieu for converting CGs into a “cargo sponge” leading to partitioning of client molecules, thus facilitating receptor-independent client sorting. These findings provide a new receptor-independent sorting model in β-cells and many other cell types and therefore represent an innovation in the field of membrane trafficking.

Pancreatic islet β-cells exhibit tremendous plasticity for secretory adaptations that coordinate insulin production and release with nutritional demands. This essential feature of the β-cell can allow for compensatory changes that increase secretory output to overcome insulin resistance early in Type 2 diabetes (T2D). Nutrient-stimulated increases in proinsulin biosynthesis may initiate this β-cell adaptive compensation; however, the molecular regulators of secretory expansion that accommodate the increased biosynthetic burden of packaging and producing additional insulin granules, such as enhanced ER and Golgi functions, remain poorly defined. As these adaptive mechanisms fail and T2D progresses, the β-cell succumbs to metabolic defects resulting in alterations to glucose metabolism and a decline in nutrient-regulated secretory functions, including impaired proinsulin processing and a deficit in mature insulin-containing secretory granules. In this review, we will discuss how the adaptative plasticity of the pancreatic islet β-cell’s secretory program allows insulin production to be carefully matched with nutrient availability and peripheral cues for insulin signaling. Furthermore, we will highlight potential defects in the secretory pathway that limit or delay insulin granule biosynthesis, which may contribute to the decline in β-cell function during the pathogenesis of T2D.

Abstract Defects in the pancreatic β-cell’s secretion system are well-described in type 2 diabetes (T2D) and include impaired proinsulin processing and a deficit in mature insulin-containing secretory granules; however, the cellular mechanisms underlying these defects remain poorly understood. To address this, we used an in situ fluorescent pulse-chase strategy to study proinsulin trafficking. We show that insulin granule formation and the appearance of nascent granules at the plasma membrane are decreased in rodent and cell culture models of prediabetes and hyperglycemia. Moreover, we link the defect in insulin granule formation to an early trafficking delay in endoplasmic reticulum (ER) export of proinsulin, which is independent of overt ER stress. Using a ratiometric redox sensor, we show that the ER becomes hyperoxidized in β-cells from a dietary model of rodent prediabetes and that addition of reducing equivalents restores ER export of proinsulin and insulin granule formation and partially restores β-cell function. Together, these data identify a critical role for the regulation of ER redox homeostasis in proinsulin trafficking and suggest that alterations in ER redox poise directly contribute to the decline in insulin granule production in T2D. This model highlights a critical link between alterations in ER redox and ER function with defects in proinsulin trafficking in T2D. Hyperoxidation of the ER lumen, shown as hydrogen peroxide, impairs proinsulin folding and disulfide bond formation that prevents efficient exit of proinsulin from the ER to the Golgi. This trafficking defect limits available proinsulin for the formation of insulin secretory granules during the development of T2D.

The defining feature of pancreatic islet β-cell function is the precise coordination of changes in blood glucose levels with insulin secretion to regulate systemic glucose homeostasis. While ATP has long been heralded as a critical metabolic coupling factor to trigger insulin release, glucose-derived metabolites have been suggested to further amplify fuel-stimulated insulin secretion. The mitochondrial export of citrate and isocitrate through the citrate-isocitrate carrier (CIC) has been suggested to initiate a key pathway that amplifies glucose-stimulated insulin secretion, though the physiological significance of β-cell CIC to glucose homeostasis has not been established. Here, we generated constitutive and adult CIC β-cell knockout mice and demonstrate these animals have normal glucose tolerance, similar responses to diet-induced obesity, and identical insulin secretion responses to various fuel secretagogues. Glucose-stimulated NADPH production was impaired in β-cell CIC KO islets, whereas glutathione reduction was retained. Furthermore, suppression of the downstream enzyme, cytosolic isocitrate dehydrogenase, Idh1, inhibited insulin secretion in wild type islets, but failed to impact β-cell function in β-cell CIC KO islets. Our data demonstrate that the mitochondrial citrate-isocitrate carrier is not required for glucose-stimulated insulin secretion, and that additional complexities exist for the role of Idh1 and NADPH in the regulation of β-cell function.

1Fraternal Order of Eagles Diabetes Research Center, University of Iowa, Iowa City, IA, USA 8 2Interdisciplinary Graduate Program in Genetics, University of Iowa, Iowa City, IA, USA 9 3Department of Internal Medicine, Division of Endocrinology and Metabolism, University of 10 Iowa, Iowa City, IA, USA 11 4Department of Pharmacology, University of Iowa, Iowa City, IA, USA 12 5Department of Nutrition, Dietetics, and Food Science, Brigham Young University, Provo, Utah, 13 USA 14 6Proteomics Facility, University of Iowa, Iowa City, IA, USA 15 7Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann 16 Arbor, MI, USA 17 8Department of Neurology, School of Medicine, University of Michigan, Ann Arbor, MI, USA 18 19 20 9Corresponding author: 21 Samuel B. Stephens, Ph.D. 22 Fraternal Order of Eagles Diabetes Research Center 23 Department of Internal Medicine, Division of Endocrinology and Metabolism 24 University of Iowa 25 Iowa City, IA 52246 26 Email: samuel-b-stephens@uiowa.edu 27 Tel. 319-335-4843 28 29

Pancreatic neuroendocrine tumors (pNETs) are difficult-to-treat neoplasms whose incidence is rising. Greater understanding of pNET pathogenesis is needed to identify new biomarkers and targets for improved therapy. RABL6A, a novel oncogenic GTPase, is highly expressed in patient pNETs and required for pNET cell proliferation and survival in vitro . Here, we investigated the role of RABL6A in pNET progression in vivo using a well-established model of the disease. RIP-Tag2 (RT2) mice develop functional pNETs (insulinomas) due to SV40 large T-antigen expression in pancreatic islet β cells. RABL6A loss in RT2 mice significantly delayed pancreatic tumor formation, reduced tumor angiogenesis and mitoses, and extended survival. Those effects correlated with upregulation of anti-angiogenic p19ARF and downregulation of proangiogenic c-Myc in RABL6A-deficient islets and tumors. Our findings demonstrate that RABL6A is a bona fide oncogenic driver of pNET angiogenesis and development in vivo .

Perilipin 2 (PLIN2) is the lipid droplet (LD) protein in β cells that increases under nutritional stress. Down-regulation of PLIN2 is often sufficient to reduce LD accumulation. To determine whether PLIN2 positively or negatively affects β cell function under nutritional stress, PLIN2 was down-regulated in mouse β cells, INS1 cells, and human islet cells. β cell specific deletion of PLIN2 in mice on a high fat diet reduced glucose-stimulated insulin secretion (GSIS) in vivo and in vitro. Down-regulation of PLIN2 in INS1 cells blunted GSIS after 24 h incubation with 0.2 mM palmitic acids. Down-regulation of PLIN2 in human pseudoislets cultured at 5.6 mM glucose impaired both phases of GSIS, indicating that PLIN2 is critical for GSIS. Down-regulation of PLIN2 decreased specific OXPHOS proteins in all three models and reduced oxygen consumption rates in INS1 cells and mouse islets. Moreover, we found that PLIN2 deficient INS1 cells increased the distribution of a fluorescent oleic acid analog to mitochondria and showed signs of mitochondrial stress as indicated by susceptibility to fragmentation and alterations of acyl-carnitines and glucose metabolites. Collectively, PLIN2 in β cells have an important role in preserving insulin secretion, β cell metabolism and mitochondrial function under nutritional stress.

Defects in the β-cell’s secretion system are well-described in Type 2 diabetes (T2D), including reduced insulin storage and impaired proinsulin processing; however, the cellular mechanisms underlying these secretory defects and the contribution of chronic hyperglycemia to this process remain poorly understood. In this study, we provide evidence that oxidative protein folding in the endoplasmic reticulum (ER) is perturbed in models of β-cell dysfunction, leading to delays in proinsulin trafficking and insulin granule formation. Using an in situ fluorescent pulse-chase labeling strategy and APEX2-based proximity labeling, we demonstrate that enriched interactions of proinsulin with ER oxidoreductases coincides with a delay in proinsulin ER export. Furthermore, our data show that proinsulin ER export can be regulated by metabolically derived NADPH and reducing equivalent (glutathione) availability. Together, these data highlight an emerging role for nutrient metabolism and mitochondrial dysfunction in the maladaptive remodeling of the β-cell’s secretory pathway during the decline of β-cell function in T2D.

Defects in the pancreatic β-cell’s secretion system are well-described in Type 2 diabetes (T2D) and include impaired proinsulin processing and a deficit in mature insulin-containing secretory granules; however, the cellular mechanisms underlying these defects and the contribution of hyperglycemia to this process remain poorly understood. Here, we used an in situ fluorescent pulse-chase strategy and proximity labeling-based quantitative proteomics analysis to study proinsulin trafficking and demonstrate a direct link to glucose metabolism via the production of redox intermediates that facilitate proinsulin export from the ER. We show that ER export of proinsulin is delayed in T2D models resulting in decreased insulin granule formation and further demonstrate this process can be regulated by NADPH and reducing equivalent availability. Together, these data highlight a critical role for nutrient metabolism and mitochondrial dysfunction in the maladaptive remodeling of the β-cell’s secretory pathway in the decline of β-cell function in T2D.### Competing Interest StatementThe authors have declared no competing interest.