Nutrient-dependent regulation of ß-cell proinsulin content.

Insulin is made from proinsulin, but the extent to which fasting/feeding controls the homeostatically regulated proinsulin pool in pancreatic ß-cells remains largely unknown. Here, we first examined ß-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 ß-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.

The Role of TRAPγ/SSR3 in Preproinsulin Translocation Into the Endoplasmic Reticulum.

In the endoplasmic reticulum (ER), the translocation-associated protein complex (TRAP), also called signal sequence receptor (SSR), includes four integral membrane proteins TRAPα/SSR1, TRAPß/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, TRAPß/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 ß-cells leaves TRAPα/SSR1 levels unaffected while nevertheless inhibiting cotranslational and posttranslational 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, as observed in cultured rodent ß-cell lines and confirmed in human islets. Strikingly, pancreatic ß-cells with suppressed TRAPγ/SSR3 expression are blocked in glucose-dependent upregulation of proinsulin (or insulin) biosynthesis. Most remarkably, overexpression of TRAPγ/SSR3 in control ß-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.

Deficient endoplasmic reticulum translocon-associated protein complex limits the biosynthesis of proinsulin and insulin.

The conserved endoplasmic reticulum (ER) membrane protein TRAPα (translocon-associated protein, also known as signal sequence receptor 1, SSR1) has been reported to play a critical but unclear role in insulin biosynthesis. TRAPα/SSR1 is one component of a four-protein complex including TRAPß/SSR2, TRAPγ/SSR3, and TRAPδ/SSR4. The TRAP complex topologically has a small exposure on the cytosolic side of the ER via its TRAPγ/SSR3 subunit, whereas TRAPß/SSR2 and TRAPδ/SSR4 function along with TRAPα/SSR1 largely on the luminal side of the ER membrane. Here, we have examined pancreatic ß-cells with deficient expression of either TRAPß/SSR2 or TRAPδ/SSR4, which does not perturb mRNA expression levels of other TRAP subunits, or insulin mRNA. However, deficient protein expression of TRAPß/SSR2 and, to a lesser degree, TRAPδ/SSR4, diminishes the protein levels of other TRAP subunits, concomitant with deficient steady-state levels of proinsulin and insulin. Deficient TRAPß/SSR2 or TRAPδ/SSR4 is not associated with any apparent defect of exocytotic mechanism but rather by a decreased abundance of the proinsulin and insulin that accompanies glucose-stimulated secretion. Amino acid pulse labeling directly establishes that much of the steady-state deficiency of intracellular proinsulin can be accounted for by diminished proinsulin biosynthesis, observed in a pulse-labeling as short as 5 minutes. The proinsulin and insulin levels in TRAPß/SSR2 or TRAPδ/SSR4 null mutant ß-cells are notably recovered upon re-expression of the missing TRAP subunit, accompanying a rebound of proinsulin biosynthesis. Remarkably, overexpression of TRAPα/SSR1 can also suppress defects in ß-cells with diminished expression of TRAPß/SSR2, strongly suggesting that TRAPß/SSR2 is needed to support TRAPα/SSR1 function.

[Pancreatic proinsulinoma]. / Proinsulinoma podzheludochnoi zhelezy.

OBJECTIVE: To report own experience in the treatment of patients with proinsulinoma. MATERIAL AND METHODS: There were 10 patients with increased proinsulin production and normal insulin level since 2017. Most of them were young women. RESULTS: Fasting hypoglycemia in all patients was severe (up to 0.7 mmol/l). Clinical picture consisted of typical symptoms similar to those in insulinoma. The main difference in the course of proinsulinoma was the absence of weight gain in 7 patients and rapid weight loss (from 210 to 90 kg within 9 months) in 1 patient. All patients with proinsulinoma underwent surgery. In most cases, minimally aggressive surgery was performed. CONCLUSION: Proinsulinoma is an extremely rare endocrine-active neuroendocrine pancreatic tumor. Differential features of proinsulinoma are the absence of weight gain and normal insulin levels in the presence of hypoglycemia. Surgery is the only radical method of treatment.

Human Islets Contain a Beta Cell Type That Expresses Proinsulin But Not the Enzyme That Converts the Precursor to Insulin.

In pancreatic beta cells, proinsulin (ProIN) undergoes folding in endoplasmic reticulum/Golgi system and is translocated to secretory vesicles for processing into insulin and C-peptide by the proprotein convertases (PC)1/3 and PC2, and carboxypeptidase E. Human beta cells show significant variation in the level of expression of PC1/3, the critical proconvertase involved in proinsulin processing. To ascertain whether this heterogeneity is correlated with the level of expression of the prohormone and mature hormone, the expression of proinsulin, insulin, and PC1/3 in human beta cells was examined. This analysis identified a human beta cell type that expressed proinsulin but lacked PC1/3 (ProIN+PC1/3-). This beta cell type is absent in rodent islets and is abundant in human islets of adults but scarce in islets from postnatal donors. Human islets also contained a beta cell type that expressed both proinsulin and variable levels of PC1/3 (ProIN+PC1/3+) and a less abundant cell type that lacked proinsulin but expressed the convertase (ProIN-PC1/3+). These cell phenotypes were altered by type 2 diabetes. These data suggest that these three cell types represent different stages of a dynamic process with proinsulin folding in ProIN+PC1/3- cells, proinsulin conversion into insulin in ProIN+PC1/3+cells, and replenishment of the proinsulin content in ProIN-PC1/3+ cells.

The making of insulin in health and disease.

The discovery of insulin in 1921 has been one of greatest scientific achievements of the 20th century. Since then, the availability of insulin has shifted the focus of diabetes treatment from trying to keep patients alive to saving and improving the life of millions. Throughout this time, basic and clinical research has advanced our understanding of insulin synthesis and action, both in healthy and pathological conditions. Yet, multiple aspects of insulin production remain unknown. In this review, we focus on the most recent findings on insulin synthesis, highlighting their relevance in diabetes. Graphical abstract.

Presumption of innocence for beta cells: why are they vulnerable autoimmune targets in type 1 diabetes?

It is increasingly appreciated that the pathogenic mechanisms of type 1 diabetes involve both the autoimmune aggressors and their beta cell targets, which engage in a conflicting dialogue within and possibly outside the pancreas. Indeed, autoimmune CD8+ T cells, which are the final mediators of beta cell destruction, circulate at similar frequencies in type 1 diabetic and healthy individuals. Hence a universal state of 'benign' islet autoimmunity exists, and we hypothesise that its progression to type 1 diabetes may at least partially rely on a higher vulnerability of beta cells, which play a key, active role in disease development and/or amplification. We posit that this autoimmune vulnerability is rooted in some features of beta cell biology: the stress imposed by the high rate of production of insulin and other granule proteins, their dense vascularisation and the secretion of their products directly into the bloodstream. Gene variants that may predispose individuals to this vulnerability have been identified, e.g. MDA5, TYK2, PTPN2. They interact with environmental cues, such as viral infections, that may drive this genetic potential towards exacerbated local inflammation and progressive beta cell loss. On top of this, beta cells set up compensatory responses, such as the unfolded protein response, that become deleterious in the long term. The relative contribution of immune and beta cell drivers may vary and phenotypic subtypes (endotypes) are likely to exist. This dual view argues for the use of circulating biomarkers of both autoimmunity and beta cell stress for disease staging, and for the implementation of both immunomodulatory and beta cell-protective therapeutic strategies. Graphical abstract.

Characterization of Signaling Pathways Associated with Pancreatic ß-cell Adaptive Flexibility in Compensation of Obesity-linked Diabetes in db/db Mice.

The onset of obesity-linked type 2 diabetes (T2D) is marked by an eventual failure in pancreatic ß-cell function and mass that is no longer able to compensate for the inherent insulin resistance and increased metabolic load intrinsic to obesity. However, in a commonly used model of T2D, the db/db mouse, ß-cells have an inbuilt adaptive flexibility enabling them to effectively adjust insulin production rates relative to the metabolic demand. Pancreatic ß-cells from these animals have markedly reduced intracellular insulin stores, yet high rates of (pro)insulin secretion, together with a substantial increase in proinsulin biosynthesis highlighted by expanded rough endoplasmic reticulum and Golgi apparatus. However, when the metabolic overload and/or hyperglycemia is normalized, ß-cells from db/db mice quickly restore their insulin stores and normalize secretory function. This demonstrates the ß-cell's adaptive flexibility and indicates that therapeutic approaches applied to encourage ß-cell rest are capable of restoring endogenous ß-cell function. However, mechanisms that regulate ß-cell adaptive flexibility are essentially unknown. To gain deeper mechanistic insight into the molecular events underlying ß-cell adaptive flexibility in db/db ß-cells, we conducted a combined proteomic and post-translational modification specific proteomic (PTMomics) approach on islets from db/db mice and wild-type controls (WT) with or without prior exposure to normal glucose levels. We identified differential modifications of proteins involved in redox homeostasis, protein refolding, K48-linked deubiquitination, mRNA/protein export, focal adhesion, ERK1/2 signaling, and renin-angiotensin-aldosterone signaling, as well as sialyltransferase activity, associated with ß-cell adaptive flexibility. These proteins are all related to proinsulin biosynthesis and processing, maturation of insulin secretory granules, and vesicular trafficking-core pathways involved in the adaptation of insulin production to meet metabolic demand. Collectively, this study outlines a novel and comprehensive global PTMome signaling map that highlights important molecular mechanisms related to the adaptive flexibility of ß-cell function, providing improved insight into disease pathogenesis of T2D.

The intermediate proteasome is constitutively expressed in pancreatic beta cells and upregulated by stimulatory, low concentrations of interleukin 1 ß.

A central and still open question regarding the pathogenesis of autoimmune diseases, such as type 1 diabetes, concerns the processes that underlie the generation of MHC-presented autoantigenic epitopes that become targets of autoimmune attack. Proteasomal degradation is a key step in processing of proteins for MHC class I presentation. Different types of proteasomes can be expressed in cells dictating the repertoire of peptides presented by the MHC class I complex. Of particular interest for type 1 diabetes is the proteasomal configuration of pancreatic ß cells, as this might facilitate autoantigen presentation by ß cells and thereby their T-cell mediated destruction. Here we investigated whether so-called inducible subunits of the proteasome are constitutively expressed in ß cells, regulated by inflammatory signals and participate in the formation of active intermediate or immuno-proteasomes. We show that inducible proteasomal subunits are constitutively expressed in human and rodent islets and an insulin-secreting cell-line. Moreover, the ß5i subunit is incorporated into active intermediate proteasomes that are bound to 19S or 11S regulatory particles. Finally, inducible subunit expression along with increase in total proteasome activities are further upregulated by low concentrations of IL-1ß stimulating proinsulin biosynthesis. These findings suggest that the ß cell proteasomal repertoire is more diverse than assumed previously and may be highly responsive to a local inflammatory islet environment.

Cells Deploy a Two-Pronged Strategy to Rectify Misfolded Proinsulin Aggregates.

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.

Proinsulin Expressing Neuroendocrine Tumors of the Pancreas: An Underrecognized Entity.

OBJECTIVES: Rare cases of pancreatic neuroendocrine tumors (PNETs) that produce only proinsulin (PI) and manifest with hypoglycemia have been reported. Proinsulin expression in PNET has not been systematically studied, and the clinicopathologic features of such tumors remain unknown. METHODS: We studied expression of PI by immunohistochemistry (IHC) in 136 PNETs from 2 high-volume surgical oncology centers and assessed all available clinicopathologic data. RESULTS: Thirty-six (26%) of PNETs were positive for PI by IHC, most (89%) of which coexpressed insulin IHC. Nine PI-positive tumors represented functional insulinomas. Patients with PI IHC-positive tumors demonstrated significantly lower mean preoperative serum glucose compared with PI-negative PNET patients, even when insulinomas were excluded. No differences in survival between PI IHC-positive and PI IHC-negative tumors were observed. We identified 2 PI-positive PNETs from hypoglycemic patients, which were not insulinomas or other functional variants and in which serum PI was never tested. These may have been undetected proinsulinomas. CONCLUSIONS: Proinsulin-expressing PNETs (functional or non) are not uncommon. Patients who present with hypoglycemia and normal insulin levels should be screened for proinsulinoma. Proinsulin IHC could also be used to screen for proinsulinoma. To further elucidate the clinical significance of PI expressing PNETs, prospective studies are required.

Biosynthesis, structure, and folding of the insulin precursor protein.

Insulin synthesis in pancreatic ß-cells is initiated as preproinsulin. Prevailing glucose concentrations, which oscillate pre- and postprandially, exert major dynamic variation in preproinsulin biosynthesis. Accompanying upregulated translation of the insulin precursor includes elements of the endoplasmic reticulum (ER) translocation apparatus linked to successful orientation of the signal peptide, translocation and signal peptide cleavage of preproinsulin-all of which are necessary to initiate the pathway of proper proinsulin folding. Evolutionary pressures on the primary structure of proinsulin itself have preserved the efficiency of folding ("foldability"), and remarkably, these evolutionary pressures are distinct from those protecting the ultimate biological activity of insulin. Proinsulin foldability is manifest in the ER, in which the local environment is designed to assist in the overall load of proinsulin folding and to favour its disulphide bond formation (while limiting misfolding), all of which is closely tuned to ER stress response pathways that have complex (beneficial, as well as potentially damaging) effects on pancreatic ß-cells. Proinsulin misfolding may occur as a consequence of exuberant proinsulin biosynthetic load in the ER, proinsulin coding sequence mutations, or genetic predispositions that lead to an altered ER folding environment. Proinsulin misfolding is a phenotype that is very much linked to deficient insulin production and diabetes, as is seen in a variety of contexts: rodent models bearing proinsulin-misfolding mutants, human patients with Mutant INS-gene-induced Diabetes of Youth (MIDY), animal models and human patients bearing mutations in critical ER resident proteins, and, quite possibly, in more common variety type 2 diabetes.

Estrogens Promote Misfolded Proinsulin Degradation to Protect Insulin Production and Delay Diabetes.

Conjugated estrogens (CE) delay the onset of type 2 diabetes (T2D) in postmenopausal women, but the mechanism is unclear. In T2D, the endoplasmic reticulum (ER) fails to promote proinsulin folding and, in failing to do so, promotes ER stress and ß cell dysfunction. We show that CE prevent insulin-deficient diabetes in male and in female Akita mice using a model of misfolded proinsulin. CE stabilize the ER-associated protein degradation (ERAD) system and promote misfolded proinsulin proteasomal degradation. This involves activation of nuclear and membrane estrogen receptor-α (ERα), promoting transcriptional repression and proteasomal degradation of the ubiquitin-conjugating enzyme and ERAD degrader, UBC6e. The selective ERα modulator bazedoxifene mimics CE protection of ß cells in females but not in males.

Age-related changes of proinsulin processing in diabetic and non-diabetic Japanese individuals.

AIM: The present study was carried out to examine whether the insulin secretory mechanism deteriorates during the aging process using the new intact proinsulin assay system in non-diabetic and diabetic individuals. METHODS: A total of 172 participants were separated into four groups according to their age (<64 years and >65 years) and an association of type 2 diabetes; that is, 46 older diabetics (mean age 74.5 ± 6.2 years, glycated hemoglobin [National Glycohemoglobin Standardization Program] 7.5 ± 1.3%), 27 older non-diabetics (mean age 76.9 ± 7.5 years), 48 middle-aged diabetics (mean age 50.8 ± 10.4, glycated hemoglobin 7.8 ± 1.5%) and 51 middle aged non-diabetics (mean age 46.6 ± 13.0 years) participants were enrolled. RESULTS: The proinsulin/insulin (PI/I) ratio of the diabetic group was higher than that of the non-diabetic group in the older group (0.19 ± 0.12 vs 0.11 ± 0.06, P = 0.002). In the middle-aged groups, the PI/I ratio of the diabetic group was higher than that of the non-diabetic group (0.16 ± 0.15 vs 0.09 ± 0.09, P = 0.003). Simple regression analysis showed that male sex (95% CI 0.02-0.01, P = 0.004), age (95% CI 0.00-0.002, P = 0.03), lower body mass index (95% CI -0.06 to 0.00, P = 0.02) and the presence of diabetes mellitus (95% CI 0.04-0.012, P < 0.0001) were significantly associated with the increase in the PI/I ratio. Multivariate regression analysis showed that male sex and age were the independent factors determining the increase in the PI/I ratio in the non-diabetic group. After adjusted for body mass index, the PI/I ratio correlated significantly with age only in the non-diabetic group (r = 0.5, P = 0.004). CONCLUSIONS: The proinsulin processing system might deteriorate not only in diabetics, but also in non-diabetic Japanese individuals with age. Also, sex-related hormones can be protective for the proinsulin processing system. Geriatr Gerontol Int 2018; 18: 1046-1050.

Misfolded proinsulin in the endoplasmic reticulum during development of beta cell failure in diabetes.

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.

cTAGE5 deletion in pancreatic ß cells impairs proinsulin trafficking and insulin biogenesis in mice.

Proinsulin is synthesized in the endoplasmic reticulum (ER) in pancreatic ß cells and transported to the Golgi apparatus for proper processing and secretion into plasma. Defects in insulin biogenesis may cause diabetes. However, the underlying mechanisms for proinsulin transport are still not fully understood. We show that ß cell-specific deletion of cTAGE5, also known as Mea6, leads to increased ER stress, reduced insulin biogenesis in the pancreas, and severe glucose intolerance in mice. We reveal that cTAGE5/MEA6 interacts with vesicle membrane soluble N-ethyl-maleimide sensitive factor attachment protein receptor Sec22b. Sec22b and its interaction with cTAGE5/MEA6 are essential for proinsulin processing. cTAGE5/MEA6 may coordinate with Sec22b to control the release of COPII vesicles from the ER, and thereby the ER-to-Golgi trafficking of proinsulin. Importantly, transgenic expression of human cTAGE5/MEA6 in ß cells can rescue not only the defect in islet structure, but also dysfunctional insulin biogenesis and glucose intolerance on cTAGE5/Mea6 conditional knockout background. Together our data provide more insight into the underlying mechanism of the proinsulin trafficking pathway.

[Synthesis of soluble proinsulin in a cell-free protein synthesis system].

Proinsulin (Pins) is the precursor of insulin. The expression of proinsulin in Escherichia coli forms inclusion body, so that the recombinant protein should be processed with multiple steps to form active insulin. With the development in biotechnology, cell-free protein synthesis (CFPS) system is becoming a valuable tool in protein expression by decoupling the cell growth with protein production, which allows it to express proteins that would interfere with cell physiology. In this study, we synthesized soluble proinsulin in CFPS system in order to establish a new approach for both insulin expression and delivery. The soluble proinsulin was successfully expressed in CFPS system by fusing proinsulin with two types of fluorescent protein. The expression of Pins-mCherry was confirmed by Western blotting analysis, and the Pins-eGFP titer was (12.28±3.45) µg/mL in CFPS system. These results implicated that the proinsulin was expressed partially in soluble form. Here, for the first time, we successfully expressed soluble proinsulin in CFPS system by fluorescent protein fusion. These results provide useful information in developing new insulin expression and delivery method.

Proinsulin Promotes Self-Renewal of a Hematopoietic Progenitor Cell Line In Vitro.

The objective of this study was to assess the effects of exogenously expressed proinsulin on the biological characters of a hematopoietic stem cell line (HSC) and erythroid myeloid lymphoid (EML) cells and explore new strategies for cell therapy for type I diabetes. EML cells were transduced with lentivirus particles carrying the human proinsulin (proINS) gene. The positive transduced cells were selected based on green fluorescence protein (GFP) positivity and puromycin resistance. Overexpression of proINS was confirmed via real-time PCR and Western blotting. The functional activity of the human proINS secreted by EML cells was elucidated by analyzing the activation of insulin receptor and its downstream signaling. Pro-INS + EML cells were able to prime the phosphorylation of insulin receptor as well as induce the expression of downstream genes of insulin receptor. Furthermore, Wnt3a can significantly promote self-renewal of Pro-INS + EML cells. However, we did not observe significant changes in the proliferation and differentiation of INS + EML cells, compared to the control EML cells. Our results might be useful for developing a new therapy for diabetes mellitus.

Jagn1 Is Induced in Response to ER Stress and Regulates Proinsulin Biosynthesis.

The Jagn1 protein was indentified in a SILAC proteomic screen of proteins that are increased in insulinoma cells expressing a folding-deficient proinsulin. Jagn1 mRNA was detected in primary rodent islets and in insulinoma cell lines and the levels were increased in response to ER stress. The function of Jagn1 was assessed in insulinoma cells by both knock-down and overexpression approaches. Knock-down of Jagn1 caused an increase in glucose-stimulated insulin secretion resulting from an increase in proinsulin biosynthesis. In contrast, overexpression of Jagn1 in insulinoma cells resulted in reduced cellular proinsulin and insulin levels. Our results identify a novel role for Jagn1 in regulating proinsulin biosynthesis in pancreatic ß-cells. Under ER stress conditions Jagn1 is induced which might contribute to reducing proinsulin biosynthesis, in part by helping to relieve the protein folding load in the ER in an effort to restore ER homeostasis.

Chimeric Vaccine Stimulation of Human Dendritic Cell Indoleamine 2, 3-Dioxygenase Occurs via the Non-Canonical NF-κB Pathway.

A chimeric protein vaccine composed of the cholera toxin B subunit fused to proinsulin (CTB-INS) was shown to suppress type 1 diabetes onset in NOD mice and upregulate biosynthesis of the tryptophan catabolic enzyme indoleamine 2, 3-dioxygenase (IDO1) in human dendritic cells (DCs). Here we demonstrate siRNA inhibition of the NF-κB-inducing kinase (NIK) suppresses vaccine-induced IDO1 biosynthesis as well as IKKα phosphorylation. Chromatin immunoprecipitation (ChIP) analysis of CTB-INS inoculated DCs showed that RelB bound to NF-κB consensus sequences in the IDO1 promoter, suggesting vaccine stimulation of the non-canonical NF-κB pathway activates IDO1 expression in vivo. The addition of Tumor Necrosis Factor Associated Factors (TRAF) TRAF 2, 3 and TRAF6 blocking peptides to vaccine inoculated DCs was shown to inhibit IDO1 biosynthesis. This experimental outcome suggests vaccine activation of the TNFR super-family receptor pathway leads to upregulation of IDO1 biosynthesis in CTB-INS inoculated dendritic cells. Together, our experimental data suggest the CTB-INS vaccine uses a TNFR-dependent signaling pathway of the non-canonical NF-κB signaling pathway resulting in suppression of dendritic cell mediated type 1 diabetes autoimmunity.