In the endoplasmic reticulum (ER), the Translocation-Associated Protein complex (TRAP, also called Signal sequence receptor, SSR) includes four integral membrane proteins TRAPα/SSR1, TRAPβ/SSR2 and TRAPδ/SSR4 with the bulk of their extramembranous portions primarily in the ER lumen, whereas the extramembranous portion of TRAPγ/SSR3 is primarily cytosolic. Individually diminished expression of either TRAPα/SSR1, TRAPβ/SSR2 or TRAPδ/SSR4 mRNA is known in each case to lower TRAPα/SSR1 protein levels leading to impaired proinsulin biosynthesis, whereas forced expression of TRAPα/SSR1 at least partially suppresses the proinsulin biosynthetic defect. Here we report that diminished TRAPγ/SSR3 expression in pancreatic β-cells leaves TRAPα/SSR1 levels unaffected while nevertheless inhibiting co-translational and post-translational translocation of preproinsulin into the ER. Crucially, acute exposure to high glucose leads to a rapid upregulation of both TRAPγ/SSR3 and proinsulin protein without change in the respective mRNA levels — observed in cultured rodent β-cell lines and confirmed in human islets. Strikingly, pancreatic β-cells with suppressed TRAPγ/SSR3 expression are blocked in glucose-dependent upregulation of proinsulin (or insulin) biosynthesis. Most remarkable, overexpression of TRAPγ/SSR3 in control β-cells raises proinsulin levels even without boosting extracellular glucose. The data suggest the possibility that TRAPγ/SSR3 may fulfill a rate-limiting function in preproinsulin translocation across the ER membrane for proinsulin biosynthesis.

Throughout evolution, proinsulin has exhibited significant sequence variation in both C-peptide and insulin moieties. As the proinsulin coding sequence evolves, the gene product continues to be under selection pressure both for ultimate insulin bioactivity and for the ability of proinsulin to be folded for export through the secretory pathway of pancreatic β-cells. The substitution proinsulin-R(B22)E is known to yield a bioactive insulin, although R(B22)Q has been reported as a mutation that falls within the spectrum of mutant INS-gene–induced diabetes of youth. Here, we have studied mice expressing heterozygous (or homozygous) proinsulin-R(B22)E knocked into the Ins2 locus. Neither females nor males bearing the heterozygous mutation developed diabetes at any age examined, but subtle evidence of increased proinsulin misfolding in the endoplasmic reticulum is demonstrable in isolated islets from the heterozygotes. Moreover, males have indications of glucose intolerance, and within a few weeks of exposure to a high-fat diet, they developed frank diabetes. Diabetes was more severe in homozygotes, and the development of disease paralleled a progressive heterogeneity of β-cells with increasing fractions of proinsulin-rich/insulin-poor cells as well as glucagon-positive cells. Evidently, subthreshold predisposition to proinsulin misfolding can go undetected but provides genetic susceptibility to diet-induced β-cell failure.

Alterations in endoplasmic reticulum (ER) calcium (Ca²⁺) levels arising from reduced activity of the Sarcoendoplasmic Reticulum Ca²⁺ ATPase (SERCA) pump are associated with impaired insulin secretion and reduce β-cell survival in both major forms of diabetes. However, at present, the mechanisms of how reduced ER Ca²⁺ impairs β-cell function remain incompletely understood. To test this, we analyzed mice with β-cell specific SERCA2 deletion (βS2KO mice) and S2KO INS1 cells. βS2KO mice exhibited age-dependent glucose intolerance, reduced glucose-stimulated insulin secretion, and increased proinsulin secretion without evidence of impaired insulin sensitivity. Immunoblot analysis of βS2KO islets and S2KO INS-1 cells revealed reduced active forms of the proinsulin processing enzymes, PC1/3, PC2, and CPE, while immunofluorescence analysis suggested impaired ER-Golgi trafficking of the convertase enzymes and proinsulin. Because the ER serves as the main site of cellular lipid synthesis and changes in the β-cell lipidome have been linked with diabetes pathophysiology, we hypothesized that ER Ca²⁺ loss may alter protein trafficking in the secretory pathway through changes in ER lipid content. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed on the ER fractions of control and S2KO INS1 cells and revealed increased levels of 16:0, 24:0, 24:1, and total ceramides in S2KO cells. SERCA2 restoration partially rescued convertase enzyme expression, proinsulin processing, and ceramide content. Consistent with these findings, RNA sequencing analysis of βS2KO islets revealed changes in pathways related to vesicle transport, protein processing, and lipid metabolism. We conclude that SERCA2 in the β-cell plays a central role in the maintenance of insulin biosynthesis as well as ceramide metabolism. Disclosure T. Kono: None. H. Iida: None. C. Lee: None. P. Krishnan: None. P. Arvan: None. I. Lindberg: None. C. Evans-molina: Advisory Panel; Self; Provention Bio, Inc., Consultant; Self; Dompe, Other Relationship; Self; Bristol-Myers Squibb Company, Nimbus Pharmaceuticals, Pfizer Inc. Funding National Institutes of Health (R01DK093954)

It has become increasingly appreciated that inflammatory cytokines promote islet dysfunction in both type 1 and type 2 diabetes. Aberrant secretion of the insulin precursor proinsulin is observed in patients with both types of diabetes and may contribute to proinsulin autoimmunity in T1D, yet the mechanisms responsible for proinsulin release remain obscure. To investigate the effects of inflammation on proinsulin trafficking, human islets were treated ex vivo with the diabetes associated cytokines, Interleukin-1-beta and Interferon-gamma, provoking a four-fold release of proinsulin. To identify proinsulin interacting proteins responsible for aberrant trafficking, intracellular proinsulin was affinity purified and its interactome defined by mass spectrometry. Cytokines altered the identify or magnitude of proinsulin’s engagement with binding partners at every stage of the canonical proinsulin biosynthetic pathway that we recently defined in normal human islets. Of particular relevance to potential avenues of proinsulin escape, cytokines promoted proinsulin interactions with: 1) antigen presentation loading complex components TAP2 and calnexin 2) stress granule assembly factors G3BP1, Ataxin-2 and IGF2BP2, that are also found in secreted exosomes, and 3) the immature insulin granule marker and T1D autoantigen ICA1/ICA169. Pathway analysis further revealed that cytokine treatment increased proinsulin binding, even if indirectly, to multiple KIF family microtubule motor proteins. Given that stress granules and immature granules are sensitive to microtubule dynamics we tested the role of microtubules in cytokine induced proinsulin release. Microtubule destabilization with Nocodazole significantly increased proinsulin release above that provoked by cytokines alone. Together, the data quantitatively map the cytokine reshaped proinsulin interactome and provide new insight into how an inflammatory environment can promote proinsulin escape from human islets. Disclosure D. T. Tran: Employee; Self; Encodia Inc. S. Loguercio: None. K. Lee: None. A. Pottekat: None. W. E. Balch: None. P. Arvan: None. R. J. Kaufman: None. P. Itkin-ansari: None. Funding JDRF (2-SRA-2015-47-M-R); National Institutes of Health (R24DK110973)

Analysis of data from clinical cohorts, and more recently from human pancreatic tissue, indicates that reduced prohormone processing is an early and persistent finding in type 1 diabetes. In this article, we review the current state of knowledge regarding alterations in islet prohormone expression and processing in type 1 diabetes and consider the clinical impact of these findings. Lingering questions, including pathologic etiologies and consequences of altered prohormone expression and secretion in type 1 diabetes, and the natural history of circulating prohormone production in health and disease, are considered. Finally, key next steps required to move forward in this area are outlined, including longitudinal testing of relevant clinical populations, studies that probe the genetics of altered prohormone processing, the need for combined functional and histologic testing of human pancreatic tissues, continued interrogation of the intersection between prohormone processing and autoimmunity, and optimal approaches for analysis. Successful resolution of these questions may offer the potential to use altered prohormone processing as a biomarker to inform therapeutic strategies aimed at personalized intervention during the natural history of type 1 diabetes and as a pathogenic anchor for identification of potential disease-specific endotypes.

Background: Peptide hormones are crucial regulators of metabolism. However, maturation of hormone precursors (prohormones) in the endoplasmic reticulum (ER) in vivo remains largely unexplored. Aims: This project aims to study the integrative role of the two major protein quality control pathways in the ER, ER-Associated Degradation (ERAD) and autophagy, in regulating prohormone maturation under physiological conditions. Taking vasopressin precursor (proAVP) as a model, this project will provide novel insights on hormone maturation and the physiological function of ERQC pathways. Methods: We generated AVP neuron specific ERAD and Autophagy deficient mouse models by deleting Sel1L and Atg7, respectively. We also generated Sel1L and Atg7 deficient cell models. We used immunostaining, sucrose sedimentation, and Western blots to dissect the roles of ERAD and autophagy in proAVP folding and maturation. Results: Mice with Atg7 deficiency in AVP neurons maintain normal water homeostasis, while Sel1L deficient mice develop central diabetes insipidus. Further, Atg7 deficient neurons show reduced proAVP protein and altered subcellular distribution, but its ER export remains functional. Mechanically, unlike Sel1L deficiency, Atg7 deficiency alone shows amber accumulation of misfolded proAVP in vitro; however, further blocking autophagy with ERAD deficiency shows increased portion of misfolded proAVP comparing with ERAD deficiency along. Further, combined deletion of Atg7 and Sel1L in AVP neurons leads to earlier development of Diabetes Insipidus comparing with Sel1L deficiency alone, suggesting a critical role of autophagy in maintaining AVP maturation with compromised ERAD. Conclusions: Here we demonstrate that autophagy alone is dispensable for the maturation of proAVP in the ER, but is indispensable in the event of compromised ERAD function both in vivo and in vitro. This study points to the differential roles and crosstalk between ERAD and autophagy in neuroendocrine cells. Disclosure G. Shi: None. X. Pan: None. H. Wang: None. P. Arvan: None. L. Qi: None. Funding National Institutes of Health (R01DK120047); University of Michigan (U064133)

The beta-cell protein synthetic machinery is dedicated to insulin production, and recent reports suggest misfolding of its precursor, proinsulin, is a feature of type 2 diabetes (T2D). Despite the critical role of insulin in organismal homeostasis, the precise network of interactions required for proper proinsulin folding and subsequent trafficking through the Golgi and secretory pathway, remains poorly defined. In the present study we conducted unbiased profiling of the proinsulin interactome in human islets, utilizing a human proinsulin-specific monoclonal antibody for affinity purification and mass spectrometry. Stringent analysis identified a central node of interactions between human proinsulin and sequential secretory pathway proteins that is remarkably conserved across both genders and 3 ethnicities. Among the most prominent proinsulin interactions was with the ER localized oxidoreductase peroxiredoxin-4 (PRDX4). A functional role for PRDX4 was demonstrated by knockdown studies in MIN6 beta-cells that rendered proinsulin susceptible to misfolding, particularly in response to oxidative stress. Conversely, exogenous PRDX4 improved proinsulin folding. Notably, oxidative stress and even high glucose treatment alone induced proinsulin misfolding in human islets and MIN6 cells and this was accompanied by sulfonylation of PRDX4, a modification known to inactivate peroxiredoxins. This finding prompted PRDX4 analysis in a panel of human islet samples that revealed significantly higher levels of sulfonylated (inactive) PRDX4 in islets from patients with T2D compared to that of healthy individuals. Taken together, these data provide a detailed reference map of the human proinsulin interaction network and suggest critical unrecognized areas for study in insulin biosynthesis, beta cell function, and T2D. Disclosure D.T. Tran: None. P. Arvan: None. R.J. Kaufman: None. P. Itkin-Ansari: None. Funding National Institutes of Health (R24DK110973); JDRF (2-SRA-2015-47-M-R)

Decline of proteostasis in the endoplasmic reticulum (ER) is thought to underlie molecular changes in β cell function and drive the pathogenesis of diabetes. ER-associated degradation (ERAD) and macroautophagy (hereafter “autophagy”) are two principal quality-control mechanisms responsible for the clearance of ER-resident proteins or protein aggregates, respectively, in the cell. However, the physiological significance of ERAD-autophagy crosstalk remains unexplored. Previously, using β cell-specific knockout mouse models, we reported that ERAD deficiency leads to β cell dedifferentiation, while autophagy deficiency leads to β cell loss by 4-5 months of age. Interestingly, we observed that loss of ERAD in β cells triggered activation of autophagy pathways, as monitored by LC3 flux. Furthermore, chloroquine-mediated inhibition of autophagy in ERAD-deficient islets led to an increased proinsulin aggregation. In mice, combined deletion of ERAD and autophagy in β cells caused marked hyperglycemia soon after weaning, with extensive loss of β cells and premature death at 2-3 months of age. Thus, our data demonstrate that ERAD and autophagy act synergistically to prevent proinsulin aggregation and promote β cell survival. This study reveals a critical role for crosstalk between the ERAD and autophagy pathways in preventing diabetes and provides the first evidence for synergism between ERAD and autophagy in vivo. Disclosure N. Shrestha: None. R.B. Reinert: None. P. Arvan: None. L. Qi: None. Funding JDRF (2-SRA-2018-539-A-B); National Institutes of Health (1R01DK117639-01, 1R01DK111174)

Impaired proinsulin processing is observed in both type 1 and type 2 diabetes. We have previously shown that reductions in endoplasmic reticulum (ER) calcium (Ca²⁺) in the pancreatic β cell arising from impaired activity of the Sarcoendoplasmic Reticulum Ca²⁺ ATPase (SERCA) pump are associated with increased proinsulin secretion. However, the mechanisms responsible for reduced proinsulin processing in the context of SERCA2 deficiency remain incompletely understood. To test this, we developed mice with β cell specific SERCA2 deletion (βS2KO mice) and S2KO INS1 cells. βS2KO mice exhibited age-dependent glucose intolerance and reduced glucose-stimulated insulin secretion without evidence of impaired insulin sensitivity. ER Ca²⁺ levels in islets from βS2KO mice were significantly reduced, while serum proinsulin/insulin (PI/I) ratios and whole pancreas PI/I content were elevated. Immunoblot analysis of βS2KO islets and S2KO INS-1 cells revealed reduced active forms of the proinsulin processing enzymes, PC1/3, PC2 and CPE. Restoration of SERCA2b via adenoviral transduction in S2KO INS1 cells was sufficient to restore PC1/3 and PC2 maturation and enzyme activity. Brefeldin A treatment in INS1 cells recapitulated the impairments in PC1/3 and PC2 maturation observed in S2KO cells, suggesting a disturbance in protein trafficking between the ER and Golgi. Consistent with this, trafficking assays were performed using a vesicular stomatitis virus G (VSVG) protein construct and revealed a significantly slower rate of VSVG movement from the ER to the Golgi in S2KO INS1 cells. Moreover, pancreas sections from βS2KO mice showed increased co-localization of proinsulin and ProPC2 in the early compartments of the secretory pathway. Taken together, these data suggest that loss of SERCA2 activity and ER Ca²⁺ loss in the pancreatic β cell leads to impaired proinsulin processing via reduced maturation and trafficking of proinsulin processing enzymes. Disclosure T. Kono: None. H. Iida: None. T.S. Jarvela: None. C. Lee: None. X. tong: None. P. Arvan: None. I. Lindberg: None. C. Evans-Molina: Consultant; Self; Bristol-Myers Squibb. Funding National Institutes of Health (R01DK093954); U.S. Department of Veterans Affairs (2-I01BX001733)

The β-cell protein synthetic machinery is dedicated to the production of mature insulin, which requires the proper folding and trafficking of its precursor, proinsulin. The complete network of proteins that mediate proinsulin folding and advancement through the secretory pathway, however, remains poorly defined. Here we used affinity purification and mass spectrometry to identify, for the first time, the proinsulin biosynthetic interaction network in human islets. Stringent analysis established a central node of proinsulin interactions with endoplasmic reticulum (ER) folding factors, including chaperones and oxidoreductases, that is remarkably conserved in both sexes and across three ethnicities. The ER-localized peroxiredoxin PRDX4 was identified as a prominent proinsulin-interacting protein. In β-cells, gene silencing of PRDX4 rendered proinsulin susceptible to misfolding, particularly in response to oxidative stress, while exogenous PRDX4 improved proinsulin folding. Moreover, proinsulin misfolding induced by oxidative stress or high glucose was accompanied by sulfonylation of PRDX4, a modification known to inactivate peroxiredoxins. Notably, islets from patients with type 2 diabetes (T2D) exhibited significantly higher levels of sulfonylated PRDX4 than islets from healthy individuals. In conclusion, we have generated the first reference map of the human proinsulin interactome to identify critical factors controlling insulin biosynthesis, β-cell function, and T2D.

Abnormal interactions between misfolded mutant and wild-type (WT) proinsulin (PI) in the endoplasmic reticulum (ER) drive the molecular pathogenesis of mutant INS gene–induced diabetes of youth (MIDY). How these abnormal interactions are initiated remains unknown. Normally, PI-WT dimerizes in the ER. Here, we suggest that the normal PI-PI contact surface, involving the B-chain, contributes to dominant-negative effects of misfolded MIDY mutants. Specifically, we find that PI B-chain tyrosine-16 (Tyr-B16), which is a key residue in normal PI dimerization, helps confer dominant-negative behavior of MIDY mutant PI-C(A7)Y. Substitutions of Tyr-B16 with either Ala, Asp, or Pro in PI-C(A7)Y decrease the abnormal interactions between the MIDY mutant and PI-WT, rescuing PI-WT export, limiting ER stress, and increasing insulin production in β-cells and human islets. This study reveals the first evidence indicating that noncovalent PI-PI contact initiates dominant-negative behavior of misfolded PI, pointing to a novel therapeutic target to enhance PI-WT export and increase insulin production.

Aim: Risk alleles for type 2 diabetes (T2D) in the STARD10 locus, impair insulin secretion and are associated with decreased proinsulin:insulin ratios. We have shown that the T2D risk associated with variation at this locus is likely to be mediated through lowered STARD10 expression in the β cell. Here, we investigate the mechanisms by which STARD10 may regulate insulin secretion. Materials and Methods: A 3-dimensional model of STARD10 was constructed from the structure of STARD2, using the modelling tools Chimera and Modeller. Islets were isolated from StarD10^fl/fl-Ins1Cre male mice (βStarD10KO). Electron Microscopy (EM) images were obtained from isolated islets after chemical fixation. Total zinc content was measured by inductively coupled plasma mass spectrometry. Pulse-chase analysis of proinsulin processing was performed using ³⁵S-labelled amino acids. RNA-Seq was done on polyadenylated transcripts selected during the preparation of paired-end, directional RNAseq libraries, sequenced on an Illumina HiSeq 4000 machine. Results: Molecular modelling indicated that STARD10 binds either phosphatidylcholine or phosphatidylethanolamine. EM analysis of islets from βStarD10KO mice revealed a markedly altered dense core granule appearance, with a dramatic increase (Fold change = 4.3; p<0.001) in "rod like" dense cores, and a ∼2-fold increase in total islet zinc content. Unexpectedly, pulse-chase studies revealed enhanced basal secretion of newly-synthesised proinsulin from βStarD10KO islets. Whilst RNA sequencing identified several dysregulated genes in βStarD10KO islets, STARD10 deletion did not affect prohormone convertase (Pcsk1, Pcsk2), or ZnT8 (Slc30a8) expression. Conclusion: We identify STARD10 as a critical regulator of insulin granule biogenesis and β cell zinc homeostasis. Our data also suggest that increased β cell Zn²⁺ secretion in risk allele carriers may decrease the clearance of mature insulin to lower plasma proinsulin:insulin ratio. Disclosure G. Carrat: None. E. Haythorne: None. L. Haataja: None. P. Arvan: None. A. Tomas: None. A. Piunti: None. T.J. Pullen: None. E. Georgiadou: None. T. Stylianides: None. V. Salem: None. W. Distaso: None. A. Cakebread: None. D. Hodson: None. A.C. Fung: None. R.B. Sessions: None. F. Alpy: None. A.P. Kong: Advisory Panel; Self; Lilly Diabetes. Research Support; Self; AstraZeneca, Lilly Diabetes. Speaker's Bureau; Self; Abbott. Other Relationship; Self; AstraZeneca, Novartis Pharmaceuticals Corporation, Sanofi. I. Leclerc: None. G.A. Rutter: Consultant; Self; Sun Pharma. Funding Medical Research Council UK; UK Wellcome Trust; Royal Society; Societe Francophone du Diabete

βcell survival and dedifferentiation are two hotly-debated mechanisms underlying βcell loss in type 2 diabetes; however, the underlying molecular mechanism remains largely unclear. Here we compare two principal protein quality control mechanisms, ER-associated protein degradation (ERAD) and autophagy and find that ERAD plays an important role in βcell dedifferentiation. Mice with βcell-specific Sel1L-Hrd1 deletion exhibit early-onset hyperglycemia and glucose intolerance as early as ~5 weeks of age. Surprisingly, in contrast to other tissue specific models of Sel1L-Hrd1 ERAD deficiency in which prohormones accumulate within the ER, Sel1L-Hrd1 ERAD deficiency impairs βcell function by attenuating insulin expression without an overt defect in proinsulin maturation and also triggers marked βcell dedifferentiation. There is, however, no overt activation of unfolded protein response (UPR) or proinsulin aggregation. In striking contrast to ERAD deficiency, loss of macroautophagy, a major mechanism for bulk protein degradation and organelle turnover, in βcells causes massive cell loss and hyperglycemia, but not βcell dedifferentiation. Thus, our data reveal a novel role of Sel1L-Hrd1 ERAD in the maintenance of βcell identify and demonstrate that impairment of ERAD contributes to the progression of type 2 diabetes in a manner distinct from that of autophagy. Disclosure N. Shrestha: None. X. Li: None. Y. Ji: None. R.B. Reinert: None. P. Arvan: None. L. Qi: None. Funding National Institutes of Health

Glucagon-like peptide-1 (GLP-1) is an incretin, secreted in response to a meal that promotes glucose-stimulated insulin secretion in animal models and diabetic and nondiabetic humans. GLP-1 analogues have been successfully used as an effective treatment for both obesity and type 2 diabetes. While the general mechanisms of actions of GLP-1 on β-cell growth and function have been extensively studied, a number of questions regarding the dynamic and heterogeneity of the response of individual islets to GLP-1 in vivo remain. Using a fluorescent reporter mouse model originally created by the Arvan Lab, we have optimized a platform allowing us to monitor the content of individual islets in live animals. In this model, a super-folded GFP is bound to the C-Peptide, allowing the fluorescent visualization and quantification of insulin content within the islets without altering their normal physiology. After an overnight fast, the pancreas is exposed by laparotomy in anesthetized mice and the reporter in individual islets can be imaged repeatedly to quantify their insulin content. Upon glucose challenge, the GFP intensity within the islets progressively decreases in control mice, correlating with the insulin secretion within the bloodstream. In order to study the effects of GLP-1 on individual islet secretion, mice were either treated either with Liraglutide, a stable GLP-1 analog, or subjected to Vertical Sleeve Gastrectomy (VSG), a surgical procedure known to increase GLP-1 levels in obese mice. Surprisingly, with both approaches, in addition to increasing insulin secretion upon a glucose challenge, we could also observe insulin content actually increasing within the islets over the course of the excursion. This finding was later confirmed by measuring total insulin content in liraglutide-treated mice. This data indicates that despite causing enhanced insulin secretion, GLP-1 can also very rapidly increase insulin content within islets. Disclosure C.M.H. Cras-Méneur: None. H. Frikke-Schmidt: None. P. Arvan: None. R.J. Seeley: Consultant; Self; Ironwood Pharmaceuticals, Inc., Novo Nordisk A/S, Scohia Pharma Inc. Research Support; Self; MedImmune, Novo Nordisk A/S, Zafgen, Inc. Stock/Shareholder; Self; Zafgen, Inc.

Although endoplasmic reticulum (ER) chaperone binding to mutant proinsulin has been reported, the role of protein chaperones in the handling of wild-type proinsulin is underinvestigated. Here, we have explored the importance of glucose-regulated protein 94 (GRP94), a prominent ER chaperone known to fold insulin-like growth factors, in proinsulin handling within β-cells. We found that GRP94 coimmunoprecipitated with proinsulin and that inhibition of GRP94 function and/or expression reduced glucose-dependent insulin secretion, shortened proinsulin half-life, and lowered intracellular proinsulin and insulin levels. This phenotype was accompanied by post-ER proinsulin misprocessing and higher numbers of enlarged insulin granules that contained amorphic material with reduced immunogold staining for mature insulin. Insulin granule exocytosis was accelerated twofold, but the secreted insulin had diminished bioactivity. Moreover, GRP94 knockdown or knockout in β-cells selectively activated protein kinase R–like endoplasmic reticulum kinase (PERK), without increasing apoptosis levels. Finally, GRP94 mRNA was overexpressed in islets from patients with type 2 diabetes. We conclude that GRP94 is a chaperone crucial for proinsulin handling and insulin secretion.

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.

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.

Human proinsulin with C-peptide–bearing Superfolder Green Fluorescent Protein (CpepSfGFP) has been expressed in transgenic mice, driven by the Ins1 promoter. The protein, expressed exclusively in β-cells, is processed and stored as CpepSfGFP and human insulin comprising only ∼0.04% of total islet proinsulin plus insulin, exerting no metabolic impact. The kinetics of the release of insulin and CpepSfGFP from isolated islets appear identical. Upon a single acute stimulatory challenge in vitro, fractional release of insulin does not detectably deplete islet fluorescence. In vivo, fluorescence imaging of the pancreatic surface allows, for the first time, visual assessment of pancreatic islet insulin content, and we demonstrate that CpepSfGFP visibly declines upon diabetes progression in live lepR^db/db mice. In anesthetized mice, after intragastric or intravenous saline delivery, pancreatic CpepSfGFP (insulin) content remains undiminished. Remarkably, however, within 20 min after acute intragastric or intravenous glucose delivery (with blood glucose concentrations reaching >15 mmol/L), a small subset of islets shows rapid dispossession of a major fraction of their stored CpepSfGFP (insulin) content, whereas most islets exhibit no demonstrable loss of CpepSfGFP (insulin). These studies strongly suggest that there are “first responder” islets to an in vivo glycemic challenge, which cannot be replicated by islets in vitro.

Pancreatic β-cells normally produce adequate insulin to control glucose homeostasis, but in obesity-related diabetes, there is a presumed deficit in insulin production and secretory capacity. In this study, insulin production was assessed directly in obese diabetic mouse models, and proinsulin biosynthesis was found to be contrastingly increased, coupled with a significant expansion of the rough endoplasmic reticulum (without endoplasmic reticulum stress) and Golgi apparatus, increased vesicular trafficking, and a depletion of mature β-granules. As such, β-cells have a remarkable capacity to produce substantial quantities of insulin in obesity, which are then made available for immediate secretion to meet increased metabolic demand, but this comes at the price of insulin secretory dysfunction. Notwithstanding, it can be restored. Upon exposing isolated pancreatic islets of obese mice to normal glucose concentrations, β-cells revert back to their typical morphology with restoration of regulated insulin secretion. These data demonstrate an unrealized dynamic adaptive plasticity of pancreatic β-cells and underscore the rationale for transient β-cell rest as a treatment strategy for obesity-linked diabetes.