Chromogranin B regulates early-stage insulin granule trafficking from the Golgi in pancreatic islet ß-cells.

Chromogranin B (CgB, also known as CHGB) is abundantly expressed in dense core secretory granules of multiple endocrine tissues and has been suggested to regulate granule biogenesis in some cell types, including the pancreatic islet ß-cell, though the mechanisms are poorly understood. Here, we demonstrate a critical role for CgB in regulating secretory granule trafficking in the ß-cell. Loss of CgB impairs glucose-stimulated insulin secretion, impedes proinsulin processing to yield increased proinsulin content, and alters the density of insulin-containing granules. Using an in situ fluorescent pulse-chase strategy to track nascent proinsulin, we show that loss of CgB impairs Golgi budding of proinsulin-containing secretory granules, resulting in a substantial delay in trafficking of nascent granules to the plasma membrane with an overall decrease in total plasma membrane-associated granules. These studies demonstrate that CgB is necessary for efficient trafficking of secretory proteins into the budding granule, which impacts the availability of insulin-containing secretory granules for exocytic release.This article has an associated First Person interview with the first author of the paper.

Synchronized proinsulin trafficking reveals delayed Golgi export accompanies ß-cell secretory dysfunction in rodent models of hyperglycemia.

The pancreatic islet ß-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 ß-cell function. In this report, we utilized the synchronous protein trafficking system, RUSH, in primary ß-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 ß-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 ß-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 ß-cell secretory defects in the pathogenesis of Type 2 diabetes.

ER Redox Homeostasis Regulates Proinsulin Trafficking and Insulin Granule Formation in the Pancreatic Islet ß-Cell.

Defects in the pancreatic ß-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 ß-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 ß-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.

Mitochondrial Efflux of Citrate and Isocitrate Is Fully Dispensable for Glucose-Stimulated Insulin Secretion and Pancreatic Islet ß-Cell Function.

The defining feature of pancreatic islet ß-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 ß-cell CIC-to-glucose homeostasis has not been established. Here, we generated constitutive and adult CIC ß-cell knockout (KO) mice and demonstrate that 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 ß-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 ß-cell function in ß-cell CIC KO islets. Our data demonstrate that the mitochondrial CIC is not required for glucose-stimulated insulin secretion and that additional complexities exist for the role of Idh1 and NADPH in the regulation of ß-cell function.

RABL6A Promotes Pancreatic Neuroendocrine Tumor Angiogenesis and Progression In Vivo.

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 ß 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.

The Insulinostatic Effect of Ghrelin Requires MRAP2 Expression in δ Cells.

Ghrelin regulates both energy intake and glucose homeostasis. In the endocrine pancreas, ghrelin inhibits insulin release to prevent hypoglycemia during fasting. The mechanism through which this is accomplished is unclear, but recent studies suggest that ghrelin acts on δ cells to stimulate somatostatin release, which in turn inhibits insulin release from ß cells. Recently, the Melanocortin Receptor Accessory Protein 2 (MRAP2) was identified as an essential partner of the ghrelin receptor (GHSR1a) in mediating the central orexigenic action of ghrelin. In this study we show that MRAP2 is expressed in islet δ cells and is required for ghrelin to elicit a calcium response in those cells. Additionally, we show that both global and δ cell targeted deletion of MRAP2 abrogates the insulinostatic effect of ghrelin. Together, these findings establish that ghrelin signaling within δ cells is essential for the inhibition of insulin release and identify MRAP2 as a regulator of insulin secretion.

A High-fat, High-sugar 'Western' Diet Alters Dorsal Striatal Glutamate, Opioid, and Dopamine Transmission in Mice.

Understanding neuroadaptations involved in obesity is critical for developing new approaches to treatment. Diet-induced neuroadaptations within the dorsal striatum have the capacity to drive excessive food seeking and consumption. Five-week-old C57BL/6J mice consumed a high-fat, high-sugar 'western diet' (WD) or a control 'standard diet' (SD) for 16 weeks. Weight gain, glucose tolerance, and insulin tolerance were measured to confirm an obese-like state. Following these 16 weeks, electrophysiological recordings were made from medium spiny neurons (MSNs) in the medial (DMS) and lateral (DLS) portions of dorsal striatum to evaluate diet effects on neuronal excitability and synaptic plasticity. In addition, fast-scan cyclic voltammetry evaluated dopamine transmission in these areas. WD mice gained significantly more weight and consumed more calories than SD mice and demonstrated impaired glucose tolerance. Electrophysiology data revealed that MSNs from WD mice demonstrated increased AMPA-to-NMDA receptor current ratio and prolonged spontaneous glutamate-mediated currents, specifically in the DLS. Evoked dopamine release was also significantly greater and reuptake slower in both subregions of WD striatum. Finally, dorsal striatal MSNs from WD mice were significantly less likely to demonstrate mu-opioid receptor-mediated synaptic plasticity. Neuronal excitability and GABAergic transmission were unaffected by diet in either striatal subregion. Our results demonstrate that a high-fat, high-sugar diet alters facets of glutamate, dopamine, and opioid signaling within the dorsal striatum, with some subregion specificity. These alterations within a brain area known to play a role in food motivation/consumption and habitual behavior are highly relevant for the clinical condition of obesity and its treatment.