Loss of the Golgi-localized v-ATPase subunit does not alter insulin granule formation or pancreatic islet ß-cell function.

Delayed Golgi export of proinsulin has recently been identified as an underlying mechanism leading to insulin granule loss and ß-cell secretory defects in type 2 diabetes (T2D). Because acidification of the Golgi lumen is critical for proinsulin sorting and delivery into the budding secretory granule, we reasoned that dysregulation of Golgi pH may contribute to proinsulin trafficking defects. In this report, we examined pH regulation of the Golgi and identified a partial alkalinization of the Golgi lumen in a diabetes model. To further explore this, we generated a ß-cell specific knockout (KO) of the v0a2 subunit of the v-ATPase pump, which anchors the v-ATPase to the Golgi membrane. Although loss of v0a2 partially neutralized Golgi pH and was accompanied by distension of the Golgi cisternae, proinsulin export from the Golgi and insulin granule formation were not affected. Furthermore, ß-cell function was well preserved. ß-cell v0a2 KO mice exhibited normal glucose tolerance in both sexes, no genotypic difference to diet-induced obesity, and normal insulin secretory responses. Collectively, our data demonstrate the v0a2 subunit contributes to ß-cell Golgi pH regulation but suggest that additional disturbances to Golgi structure and function contribute to proinsulin trafficking defects in diabetes.NEW & NOTEWORTHY Delayed proinsulin export from the Golgi in diabetic ß-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 ß-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.

Independent activation of CREB3L2 by glucose fills a regulatory gap in mouse ß-cells by co-ordinating insulin biosynthesis with secretory granule formation.

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 ß-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 ß-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 ß-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 ß-cell failure.

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.

Loss of FoxOs in muscle increases strength and mitochondrial function during aging.

BACKGROUND: Muscle mitochondrial decline is associated with aging-related muscle weakness and insulin resistance. FoxO transcription factors are targets of insulin action and deletion of FoxOs improves mitochondrial function in diabetes. However, disruptions in proteostasis and autophagy are hallmarks of aging and the effect of chronic inhibition of FoxOs in aged muscle is unknown. This study investigated the role of FoxOs in regulating muscle strength and mitochondrial function with age. METHODS: We measured muscle strength, cross-sectional area, muscle fibre-type, markers of protein synthesis/degradation, central nuclei, glucose/insulin tolerance, and mitochondrial bioenergetics in 4.5-month (Young) and 22-24-month-old (Aged) muscle-specific FoxO1/3/4 triple KO (TKO) and littermate control (Ctrl) mice. RESULTS: Lean mass was increased in Aged TKO compared with both Aged Ctrl and younger groups by 26-33% (P < 0.01). Muscle strength, measured by max force of tibialis anterior (TA) contraction, was 20% lower in Aged Ctrl compared with Young Ctrls (P < 0.01) but was not decreased in Aged TKOs. Increased muscle strength in Young and Aged TKO was associated with 18-48% increased muscle weights compared with Ctrls (P < 0.01). Muscle cross-sectional analysis of TA, soleus, and plantaris revealed increases in fibre size distribution and a 2.5-10-fold increase in central nuclei in Young and Aged TKO mice, without histologic signs of muscle damage. Age-dependent increases in Gadd45a and Ube4a expression as well accumulation of K48 polyubiquitinated proteins were observed in quad and TA but were prevented by FoxO deletion. Young and Aged TKO muscle showed minimal changes in autophagy flux and no accumulation of autophagosomes compared with Ctrl groups. Increased strength in Young and Aged TKO was associated with a 10-20% increase in muscle mitochondrial respiration using glutamate/malate/succinate compared with controls (P < 0.05). OXPHOS subunit expression and complex I activity were decreased 16-34% in Aged Ctrl compared with Young Ctrl but were prevented in Aged TKO. Both Aged Ctrl and Aged TKO showed impaired glucose tolerance by 33% compared to young groups (P < 0.05) indicating improved strength and mitochondrial respiration are not due to improved glycemia. CONCLUSIONS: FoxO deletion increases muscle strength even during aging. Deletion of FoxOs maintains muscle strength in part by mild suppression of atrophic pathways, including inhibition of Gadd45a and Ube4a expression, without accumulation of autophagosomes in muscle. Deletion of FoxOs also improved mitochondrial function by maintenance of OXPHOS in both young and aged TKO.

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.

Liquid-liquid phase separation facilitates the biogenesis of secretory storage granules.

Insulin is synthesized by pancreatic ß-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 ß-cells and many other cell types and therefore represent an innovation in the field of membrane trafficking.

Nutrient Regulation of Pancreatic Islet ß-Cell Secretory Capacity and Insulin Production.

Pancreatic islet ß-cells exhibit tremendous plasticity for secretory adaptations that coordinate insulin production and release with nutritional demands. This essential feature of the ß-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 ß-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 ß-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 ß-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 ß-cell function during the pathogenesis 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.