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Insulin is made from proinsulin, but the extent to which fasting/feeding controls the homeostatically regulated proinsulin pool in pancreatic β-cells remains largely unknown. Here, we first examined β-cell lines (INS1E and Min6, which proliferate slowly and are routinely fed fresh medium every 2-3 days) and found that the proinsulin pool size responds to each feeding within 1 to 2 h, affected both by the quantity of fresh nutrients and the frequency with which they are provided. We observed no effect of nutrient feeding on the overall rate of proinsulin turnover as quantified from cycloheximide-chase experiments. We show that nutrient feeding is primarily linked to rapid dephosphorylation of translation initiation factor eIF2α, presaging increased proinsulin levels (and thereafter, insulin levels), followed by its rephosphorylation during the ensuing hours that correspond to a fall in proinsulin levels. The decline of proinsulin levels is blunted by the integrated stress response inhibitor, ISRIB, or by inhibition of eIF2α rephosphorylation with a general control nonderepressible 2 (not PERK) kinase inhibitor. In addition, we demonstrate that amino acids contribute importantly to the proinsulin pool; mass spectrometry shows that β-cells avidly consume extracellular glutamine, serine, and cysteine. Finally, we show that in both rodent and human pancreatic islets, fresh nutrient availability dynamically increases preproinsulin, which can be quantified without pulse-labeling. Thus, the proinsulin available for insulin biosynthesis is rhythmically controlled by fasting/feeding cycles., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the content of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Preproinsulin entry into the endoplasmic reticulum yields proinsulin, and its subsequent delivery to the distal secretory pathway leads to processing, storage, and secretion of mature insulin. Multiple groups have reported that treatment of pancreatic beta cell lines, rodent pancreatic islets, or human islets with proteasome inhibitors leads to diminished proinsulin and insulin protein levels, diminished glucose-stimulated insulin secretion, and changes in beta-cell gene expression that ultimately lead to beta-cell death. However, these studies have mostly examined treatment times far beyond that needed to achieve acute proteasomal inhibition. Here, we report that although proteasomal inhibition immediately downregulates new proinsulin biosynthesis, it nevertheless acutely increases beta-cell proinsulin levels in pancreatic beta cell lines, rodent pancreatic islets, and human islets, indicating rescue of a pool of recently synthesized WT INS gene product that would otherwise be routed to proteasomal disposal. Our pharmacological evidence suggests that this disposal most likely reflects ongoing endoplasmic reticulum-associated protein degradation. However, we found that within 60 min after proteasomal inhibition, intracellular proinsulin levels begin to fall in conjunction with increased phosphorylation of eukaryotic initiation factor 2 alpha, which can be inhibited by blocking the general control nonderepressible 2 kinase. Together, these data demonstrate that a meaningful subfraction of newly synthesized INS gene product undergoes rapid proteasomal disposal. We propose that free amino acids derived from proteasomal proteolysis may potentially participate in suppressing general control nonderepressible 2 kinase activity to maintain ongoing proinsulin biosynthesis., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

ER-specific autophagy (reticulophagy) has emerged as a critical degradative route for misfolded secretory proteins. Our previous work showed that RTN3 (reticulon 3) drives reticulophagic clearance of disease-causing mutant prohormones. How RTN3, a protein residing on the cytosolic leaflet of the ER bilayer, recruits these lumenally-localized cargos has remained a mystery. To address this question, we used an unbiased proteomics approach to identify RTN3-interacting partners. We discovered that RTN3 recruits misfolded prohormones for lysosomal degradation through the ER transmembrane protein PGRMC1. RTN3 complexes with PGRMC1, which directly binds to misfolded prohormones via its distal ER lumenal domain. Cargos for the RTN3-PGRMC1 degradative axis include mutant POMC (proopiomelanocortin) and proinsulin, each of which oligomerizes in the ER during misfolding, entrapping their wild-type counterparts, leading to secretion defects. Although reticulophagy is thought to degrade large protein aggregates, PGRMC1 instead selectively recruits and promotes degradation of only small oligomers of the mutant prohormones. Of physiological importance, genetic or pharmacological inactivation of PGRMC1 in pancreatic β-cells expressing both wild-type and mutant proinsulin impairs mutant proinsulin turnover and promotes trafficking of wild-type proinsulin. These findings pinpoint PGRMC1 as a possible intervention point for diseases caused by ER protein retention.

The reticulon-3 (RTN3)-driven targeting complex promotes clearance of misfolded prohormones from the endoplasmic reticulum (ER) for lysosomal destruction by ER-phagy. Because RTN3 resides in the cytosolic leaflet of the ER bilayer, the mechanism of selecting misfolded prohormones as ER-phagy cargo on the luminal side of the ER membrane remains unknown. Here we identify the ER transmembrane protein PGRMC1 as an RTN3-binding partner. Via its luminal domain, PGRMC1 captures misfolded prohormones, targeting them for RTN3-dependent ER-phagy. PGRMC1 selects cargos that are smaller than the large size of other reported ER-phagy substrates. Cargos for PGRMC1 include mutant proinsulins that block secretion of wildtype proinsulin through dominant-negative interactions within the ER, causing insulin-deficiency. Chemical perturbation of PGRMC1 partially restores WT insulin storage by preventing ER-phagic degradation of WT and mutant proinsulin. Thus, PGRMC1 acts as a size-selective cargo receptor during RTN3-dependent ER-phagy, and is a potential therapeutic target for diabetes., (© 2021. The Author(s).)

A precondition for efficient proinsulin export from the endoplasmic reticulum (ER) is that proinsulin meets ER quality control folding requirements, including formation of the Cys(B19)-Cys(A20) "interchain" disulfide bond, facilitating formation of the Cys(B7)-Cys(A7) bridge. The third proinsulin disulfide, Cys(A6)-Cys(A11), is not required for anterograde trafficking, i.e., a "lose-A6/A11" mutant [Cys(A6), Cys(A11) both converted to Ser] is well secreted. Nevertheless, an unpaired Cys(A11) can participate in disulfide mispairings, causing ER retention of proinsulin. Among the many missense mutations causing the syndrome of Mutant INS gene-induced Diabetes of Youth (MIDY), all seem to exhibit perturbed proinsulin disulfide bond formation. Here, we have examined a series of seven MIDY mutants [including G(B8)V, Y(B26)C, L(A16)P, H(B5)D, V(B18)A, R(Cpep + 2)C, E(A4)K], six of which are essentially completely blocked in export from the ER in pancreatic β-cells. Three of these mutants, however, must disrupt the Cys(A6)-Cys(A11) pairing to expose a critical unpaired cysteine thiol perturbation of proinsulin folding and ER export, because when introduced into the proinsulin lose-A6/A11 background, these mutants exhibit native-like disulfide bonding and improved trafficking. This maneuver also ameliorates dominant-negative blockade of export of co-expressed wild-type proinsulin. A growing molecular understanding of proinsulin misfolding may permit allele-specific pharmacological targeting for some MIDY mutants., (© 2021. The Author(s).)

Escape of large macromolecular complexes from the endoplasmic reticulum (ER), such as a viral particle or cellular aggregate, likely induces mechanical stress initiated on the luminal side of the ER membrane, which may threaten its integrity. How the ER responds to this threat remains unknown. Here we demonstrate that the cytosolic leaflet ER morphogenic protein reticulon (RTN) protects ER membrane integrity when polyomavirus SV40 escapes the ER to reach the cytosol en route to infection. SV40 coopts an intrinsic RTN function, as we also found that RTN prevents membrane damage during ER escape of a misfolded proinsulin aggregate destined for lysosomal degradation via ER-phagy. Our studies reveal that although ER membrane integrity may be threatened during ER escape of large macromolecular protein complexes, the action of RTN counters this, presumably by deploying its curvature-inducing activity to provide membrane flexibility and stability to limit mechanical stress imposed on the ER membrane., (© 2020 Chen et al.)

Insulin gene coding sequence mutations are known to cause mutant INS-gene-induced diabetes of youth (MIDY), yet the cellular pathways needed to prevent misfolded proinsulin accumulation remain incompletely understood. Here, we report that Akita mutant proinsulin forms detergent-insoluble aggregates that entrap wild-type (WT) proinsulin in the endoplasmic reticulum (ER), thereby blocking insulin production. Two distinct quality-control mechanisms operate together to combat this insult: the ER luminal chaperone Grp170 prevents proinsulin aggregation, while the ER membrane morphogenic protein reticulon-3 (RTN3) disposes of aggregates via ER-coupled autophagy (ER-phagy). We show that enhanced RTN-dependent clearance of aggregated Akita proinsulin helps to restore ER export of WT proinsulin, which can promote WT insulin production, potentially alleviating MIDY. We also find that RTN3 participates in the clearance of other mutant prohormone aggregates. Together, these results identify a series of substrates of RTN3-mediated ER-phagy, highlighting RTN3 in the disposal of pathogenic prohormone aggregates., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Biosynthesis of insulin - critical to metabolic homeostasis - begins with folding of the proinsulin precursor, including formation of three evolutionarily conserved intramolecular disulfide bonds. Remarkably, normal pancreatic islets contain a subset of proinsulin molecules bearing at least one free cysteine thiol. In human (or rodent) islets with a perturbed endoplasmic reticulum folding environment, non-native proinsulin enters intermolecular disulfide-linked complexes. In genetically obese mice with otherwise wild-type islets, disulfide-linked complexes of proinsulin are more abundant, and leptin receptor-deficient mice, the further increase of such complexes tracks with the onset of islet insulin deficiency and diabetes. Proinsulin-Cys(B19) and Cys(A20) are necessary and sufficient for the formation of proinsulin disulfide-linked complexes; indeed, proinsulin Cys(B19)-Cys(B19) covalent homodimers resist reductive dissociation, highlighting a structural basis for aberrant proinsulin complex formation. We conclude that increased proinsulin misfolding via disulfide-linked complexes is an early event associated with prediabetes that worsens with ß-cell dysfunction in type two diabetes., Competing Interests: AA, LH, AP, FP, SK, LZ, AP, JP, BT, PI, RK, ML, PA No competing interests declared, (© 2019, Arunagiri et al.)

The endoplasmic reticulum (ER) is broadly distributed throughout the cytoplasm of pancreatic beta cells, and this is where all proinsulin is initially made. Healthy beta cells can synthesize 6000 proinsulin molecules per second. Ordinarily, nascent proinsulin entering the ER rapidly folds via the formation of three evolutionarily conserved disulfide bonds (B7-A7, B19-A20, and A6-A11). A modest amount of proinsulin misfolding, including both intramolecular disulfide mispairing and intermolecular disulfide-linked protein complexes, is a natural by-product of proinsulin biosynthesis, as is the case for many proteins. The steady-state level of misfolded proinsulin-a potential ER stressor-is linked to (1) production rate, (2) ER environment, (3) presence or absence of naturally occurring (mutational) defects in proinsulin, and (4) clearance of misfolded proinsulin molecules. Accumulation of misfolded proinsulin beyond a certain threshold begins to interfere with the normal intracellular transport of bystander proinsulin, leading to diminished insulin production and hyperglycemia, as well as exacerbating ER stress. This is most obvious in mutant INS gene-induced Diabetes of Youth (MIDY; an autosomal dominant disease) but also likely to occur in type 2 diabetes owing to dysregulation in proinsulin synthesis, ER folding environment, or clearance., (© 2018 New York Academy of Sciences.)

In heterozygous patients with a diabetic syndrome called mutant INS gene-induced diabetes of youth (MIDY), there is decreased insulin secretion when mutant proinsulin expression prevents wild-type (WT) proinsulin from exiting the endoplasmic reticulum (ER), which is essential for insulin production. Our previous results revealed that mutant Akita proinsulin is triaged by ER-associated degradation (ERAD). We now find that the ER chaperone Grp170 participates in the degradation process by shifting Akita proinsulin from high-molecular weight (MW) complexes toward smaller oligomeric species that are competent to undergo ERAD. Strikingly, overexpressing Grp170 also liberates WT proinsulin, which is no longer trapped in these high-MW complexes, enhancing ERAD of Akita proinsulin and restoring WT insulin secretion. Our data reveal that Grp170 participates in preparing mutant proinsulin for degradation while enabling WT proinsulin escape from the ER. In principle, selective destruction of mutant proinsulin offers a rational approach to rectify the insulin secretion problem in MIDY., (© 2017 by the American Diabetes Association.)

Proinsulin folding within the endoplasmic reticulum (ER) remains incompletely understood, but it is clear that in mutant INS gene-induced diabetes of youth (MIDY), progression of the (three) native disulfide bonds of proinsulin becomes derailed, causing insulin deficiency, β-cell ER stress, and onset of diabetes. Herein, we have undertaken a molecular dissection of proinsulin disulfide bond formation, using bioengineered proinsulins that can form only two (or even only one) of the native proinsulin disulfide bonds. In the absence of preexisting proinsulin disulfide pairing, Cys(B19)-Cys(A20) (a major determinant of ER stress response activation and proinsulin stability) preferentially initiates B-A chain disulfide bond formation, whereas Cys(B7)-Cys(A7) can initiate only under oxidizing conditions beyond that existing within the ER of β-cells. Interestingly, formation of these two "interchain" disulfide bonds demonstrates cooperativity, and together, they are sufficient to confer intracellular transport competence to proinsulin. The three most common proinsulin disulfide mispairings in the ER appear to involve Cys(A11)-Cys(A20), Cys(A7)-Cys(A20), and Cys(B19)-Cys(A11), each disrupting the critical Cys(B19)-Cys(A20) pairing. MIDY mutations inhibit Cys(B19)-Cys(A20) formation, but treatment to force oxidation of this disulfide bond improves folding and results in a small but detectable increase of proinsulin export. These data suggest possible therapeutic avenues to ameliorate ER stress and diabetes., (© 2016 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.)

In mutant INS gene-induced diabetes of youth (MIDY), characterized by insulin deficiency, MIDY proinsulin mutants misfold and fail to exit the endoplasmic reticulum (ER). Moreover, these mutants bind and block ER exit of wild-type (WT) proinsulin, inhibiting insulin production. The ultimate fate of ER-entrapped MIDY mutants is unclear, but previous studies implicated ER-associated degradation (ERAD), a pathway that retrotranslocates misfolded ER proteins to the cytosol for proteasomal degradation. Here we establish key ERAD machinery components used to triage the Akita proinsulin mutant, including the Hrd1-Sel1L membrane complex, which conducts Akita proinsulin from the ER lumen to the cytosol, and the p97 ATPase, which couples the cytosolic arrival of proinsulin with its proteasomal degradation. Surprisingly, we find that protein disulfide isomerase (PDI), the major protein oxidase of the ER lumen, engages Akita proinsulin in a novel way, reducing proinsulin disulfide bonds and priming the Akita protein for ERAD. Efficient PDI engagement of Akita proinsulin appears linked to the availability of Hrd1, suggesting that retrotranslocation is coordinated on the lumenal side of the ER membrane. We believe that, in principle, this form of diabetes could be alleviated by enhancing the targeting of MIDY mutants for ERAD to restore WT insulin production., (© 2015 He, Cunningham, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Three principal ER quality-control mechanisms, namely, the unfolded protein response, ER-associated degradation (ERAD), and ER-phagy are each important for the maintenance of ER homeostasis, yet how they are integrated to regulate ER homeostasis and organellar architecture in vivo is largely unclear. Here we report intricate crosstalk among the 3 pathways, centered around the SEL1L-HRD1 protein complex of ERAD, in the regulation of organellar organization in β cells. SEL1L-HRD1 ERAD deficiency in β cells triggers activation of autophagy, at least in part, via IRE1α (an endogenous ERAD substrate). In the absence of functional SEL1L-HRD1 ERAD, proinsulin is retained in the ER as high molecular weight conformers, which are subsequently cleared via ER-phagy. A combined loss of both SEL1L and autophagy in β cells leads to diabetes in mice shortly after weaning, with premature death by approximately 11 weeks of age, associated with marked ER retention of proinsulin and β cell loss. Using focused ion beam scanning electron microscopy powered by deep-learning automated image segmentation and 3D reconstruction, our data demonstrate a profound organellar restructuring with a massive expansion of ER volume and network in β cells lacking both SEL1L and autophagy. These data reveal at an unprecedented detail the intimate crosstalk among the 3 ER quality-control mechanisms in the dynamic regulation of organellar architecture and β cell function.

Insulin is made from proinsulin, but the extent to which fasting/feeding controls the homeostatically regulated proinsulin pool in pancreatic β-cells remains largely unknown. Here, we first examined β-cell lines (INS1E and Min6, which proliferate slowly and are routinely fed fresh medium every 2-3 days) and found that the proinsulin pool size responds to each feeding within 1 to 2 h, affected both by the quantity of fresh nutrients and the frequency with which they are provided. We observed no effect of nutrient feeding on the overall rate of proinsulin turnover as quantified from cycloheximide-chase experiments. We show that nutrient feeding is primarily linked to rapid dephosphorylation of translation initiation factor eIF2a, presaging increased proinsulin levels (and thereafter, insulin levels), followed by its rephosphorylation during the ensuing hours that correspond to a fall in proinsulin levels. The decline of proinsulin levels is blunted by the integrated stress response inhibitor, ISRIB, or by inhibition of eIF2a rephosphorylation with a general control nonderepressible 2 (not PERK) kinase inhibitor. In addition, we demonstrate that amino acids contribute importantly to the proinsulin pool; mass spectrometry shows that β-cells avidly consume extracellular glutamine, serine, and cysteine. Finally, we show that in both rodent and human pancreatic islets, fresh nutrient availability dynamically increases preproinsulin, which can be quantified without pulse-labeling. Thus, the proinsulin available for insulin biosynthesis is rhythmically controlled by fasting/feeding cycles. [ABSTRACT FROM AUTHOR]

Preproinsulin entry into the endoplasmic reticulum yields proinsulin, and its subsequent delivery to the distal secretory pathway leads to processing, storage, and secretion of mature insulin. Multiple groups have reported that treatment of pancreatic beta cell lines, rodent pancreatic islets, or human islets with proteasome inhibitors leads to diminished proinsulin and insulin protein levels, diminished glucose-stimulated insulin secretion, and changes in beta-cell gene expression that ultimately lead to beta-cell death. However, these studies have mostly examined treatment times far beyond that needed to achieve acute proteasomal inhibition. Here, we report that although proteasomal inhibition immediately downregulates new proinsulin biosynthesis, it nevertheless acutely increases beta-cell proinsulin levels in pancreatic beta cell lines, rodent pancreatic islets, and human islets, indicating rescue of a pool of recently synthesized WT INS gene product that would otherwise be routed to proteasomal disposal. Our pharmacological evidence suggests that this disposal most likely reflects ongoing endoplasmic reticulum-associated protein degradation. However, we found that within 60 min after proteasomal inhibition, intracellular proinsulin levels begin to fall in conjunction with increased phosphorylation of eukaryotic initiation factor 2 alpha, which can be inhibited by blocking the general control nonderepressible 2 kinase. Together, these data demonstrate that a meaningful subfraction of newly synthesized INS gene product undergoes rapid proteasomal disposal. We propose that free amino acids derived from proteasomal proteolysis may potentially participate in suppressing general control nonderepressible 2 kinase activity to maintain ongoing proinsulin biosynthesis. [ABSTRACT FROM AUTHOR]