An Essential Role of IER3IP1 in β-Cell Development and Proinsulin Trafficking

IER3IP1 (immediate early response 3–interacting protein 1) is a small endoplasmic reticulum (ER) membrane protein composed of 82 residues with a central G-patch domain flanked on both sides by two transmembrane domains (1). Patients with recessive mutations in IER3IP1 exhibit a syndrome primarily characterized by microcephaly, epilepsy, and permanent neonatal diabetes (MEDS, also known as microcephaly, epilepsy, and diabetes syndrome 1 [MEDS1]) (2). To date, five IER3IP1 mutations have been reported in 11 patients with MEDS from nine distinct families (2–8). Interestingly, all MEDS-causing mutations are located in the two transmembrane domains, highlighting their critical role in maintaining the normal function of IER3IP1. Since the predominant defects in patients with MEDS occur in brain and pancreatic β-cells, functional studies of IER3IP1 have focused mostly on these two tissues. In brain, MEDS-causing mutations impair neural progenitor differentiation and proliferation, resulting in smaller brain organoids developed from mutant human embryonic stem cells (hESCs) compared with those from wild-type hESCs (9,10). These phenotypes appear to be caused by ER stress and secretion impairment of neuron extracellular matrix proteins, suggesting that IER3IP1 plays an important role in regulating protein trafficking and secretion (9,10). Similarly, in pancreatic β-cells, experimental evidence from knockout/mutant mouse models supports the notion that IER3IP1 is critical for normal ER function and β-cell survival. IER3IP1 deficiency induces ER stress and activates the unfolded protein response (UPR), increases β-cell death, and decreases insulin biosynthesis, leading to elevated blood glucose at the first day after birth and severe insulin-deficient diabetes at 3 weeks of age (11–13). However, to date, there is no direct evidence revealing roles of IER3IP1 in early β-cell development/differentiation and proinsulin intracellular trafficking.

Dynamic transcriptome changes during the early development of pancreatic islets from hESCs indicate that IER3IP1 is highly expressed throughout all stages of islet differentiation, with the highest expression at the final state of functional hESC-derived islets (13,14). From this expression profile of IER3IP1, combined with the fact that insulin-deficient neonatal diabetes is one of the most prominent phenotypes of MEDS, it is reasonable to propose that IER3IP1 may be an essential factor in the early development of pancreatic β-cells, and this important question has not previously been addressed. Reported in this issue of Diabetes, Montaser et al. (13) generated stem cell–derived islets (SC-islets), in which IER3IP1 deficiency was achieved through either CRISPR/Cas9-mediated deletion of the first exon of IER3IP1 or insertion of a MEDS-causing IER3IP1 mutation. The authors demonstrated that IER3IP1 deficiency did not affect the early development of the endodermal and pancreatic progenitors but altered the final stages of β-cell differentiation and maturation, resulting in a decreased β-cell number along with an increased population of α-cells. The methodology using hESCs is thought to mirror the developmental trajectory of human islets, thereby facilitating new insights into the influence of IER3IP1 on the development and functionality of human pancreatic β-cells.

In addition to regulating β-cell early development and maturation, IER3IP1 appears to also be important for β-cells after the neonatal period. Using an inducible β-cell–specific mouse model in which the expression of IER3IP1 in β-cells was downregulated at the age of 8 weeks, we previously demonstrated that IER3IP1 deficiency significantly decreases insulin content and causes diabetes, indicating that IER3IP1 is required for maintaining normal function of β-cells in adulthood (12). Notably, the impaired insulin production is associated with abnormal accumulation of proinsulin in the ER (12). However, the trafficking of proinsulin was not directly examined in previous studies. Montaser et al. (13) have tried to address this important question in IER3IP1-deficient SC-islets using three independent approaches. First, with use of immunofluorescence microscopy and proinsulin ELISA, they find that proinsulin is increasingly colocalized with an ER marker, along with an increased ratio of proinsulin to insulin. Second, with the retention using selective hooks (RUSH) cargo sorting assay in conjunction with time-delayed live cell imaging, they provide direct evidence indicating that IER3IP1 deficiency impairs proinsulin ER-to-Golgi trafficking. Finally, in SC-islets treated with tauroursodeoxycholic acid (TUDCA) (a chemical chaperone shown to facilitate protein folding and alleviate ER stress), no improvement of proinsulin trafficking was observed. These data provide more direct and strong evidence supporting that IER3IP1 plays an important role in regulating proinsulin ER-to-Golgi trafficking (13). These findings are in line with results in HeLa cells, in which IER3IP1 cycles between the ER and early Golgi, and its deletion leads to mistrafficking of various proteins, including the cargo receptors ERGIC53 and KDELR2, as well as some ER-localized chaperone proteins (9).

Despite the above evidence, at least one question remains regarding the primary cause of defective ER-to-Golgi proinsulin trafficking in IER3IP1-deficient β-cells. To date, all studies show that IER3IP1 deletion in β-cells results in markedly distended ER and upregulated UPR, indicating that IER3IP1 deficiency causes ER stress and dysfunction (9), which have been shown to be associated with impaired proinsulin folding (15–17). In fact, misfolded disulfide-linked proinsulin complexes are indeed found in IER3IP1 knockout islets (12). Although TUDCA was used in an attempt to alleviate ER stress and improved proinsulin folding, the study did not show direct evidence of improvement of proinsulin folding and ER stress (13). It therefore remains to be further determined whether the defective proinsulin folding in IER3IP1-deficient cells is caused by a direct effect on proinsulin folding and the ER quality control (causing ER retention of the misfolded proinsulin) or is a secondary effect caused by impaired ER-to-Golgi transport.

Another interesting observation from current and previous studies relates to the role of IER3IP1 in the regulation of UPR. Although IER3IP1 deficiency indeed causes significant ER morphology changes characterized by a severely dilated and distorted ER, the activation of UPR appears to be variable in different studies with different models of β-cells. In most cell types, IER3IP1 deficiency increases the expression of BiP (a master protein regulating UPR) at both transcriptional and translational levels (10–13,18). However, IER3IP1 deletion appears to lead to a dissociation of ER stress from a full activation of UPR in some models. In SC-islets, although transcriptional activation of ATF6 (activating transcription factor 6) and IRE1 (inositol-requiring enzyme 1) was found in IER3IP1-deficient SC-islets, the expression of PERK (protein kinase RNA–like ER kinase) mRNA was not altered (13). In IER3IP1 knockout islets, although the transcriptional activation of Perk was observed, protein phosphorylation of IRE1 and eIF2α was not increased (12). In mouse insulinoma cell line MIN6 cells, downregulation of IER3IP1 surprisingly decreased the expression of all three UPR arms under both basal and ER stress conditions (11). It remains to be determined whether these differences are due to different models/cells that are used in different studies or due to different methodologies used to examine UPR-dependent signaling. Nevertheless, with further research, there is no doubt that deeper insights into the underlying mechanism(s) of IER3IP1 in regulating ER function, and UPR, will be uncovered, demonstrating a critical role in pathophysiology of β-cells and glucose homeostasis.