SEL1L-HRD1 ER-associated degradation facilitates prohormone convertase 2 maturation and glucagon production in islet α cells

Domain: chemistry
Published: 2026-02-25
Editorial outcome: major_revision

Abstract
Proteolytic cleavage of proglucagon by prohormone convertase 2 (PC2) is required for islet α cells to generate glucagon. However, the regulatory mechanisms underlying this process remain largely unclear. Here, we report that SEL1L-HRD1 endoplasmic reticulum (ER)-associated degradation (ERAD), a highly conserved protein quality control system responsible for clearing misfolded proteins from the ER, plays a key role in glucagon production by regulating turnover of the nascent proform of the PC2 enzyme (proPC2). Using a mouse model with SEL1L deletion in proglucagon-expressing cells, we observe a progressive decline in stimulated glucagon secretion and a reduction in pancreatic glucagon content. Mechanistically, we find that endogenous proPC2 is a substrate of SEL1L-HRD1 ERAD, and that degradation of misfolded proPC2 ensures the maturation of activation-competent proPC2 protein in the ER. Here, we identify ERAD as a regulator of PC2 biology and an essential mechanism for maintaining α cell function.

Introduction
Diabetes mellitus encompasses a spectrum of metabolic diseases with dysregulated secretion of insulin and glucagon from pancreatic islet β and α cells as a central feature. Interest in α cell biology has been reinvigorated in recent years, particularly with the advent of specific tools to manipulate α cell gene expression and glucagon signaling 1,2. Hyperglucagonemia is observed in both type 1 and type 2 diabetes, contributing to hyperglycemia 3,4. Glucagon receptor antagonists are in clinical development to improve hyperglycemia in diabetes 5,6, but may trigger dramatic α cell hyperplasia 7 like that observed in models with genetic deletion of glucagon 8 or its receptor (Gcgr) 9. Conversely, recent studies have demonstrated a more positive role for α cells in directly promoting β cell function and survival through secretion of proglucagon-derived peptides such as glucagon and/or glucagon-like peptide 1 (GLP-1) and their interaction with GCGR and GLP1R receptors on β cells 10-12. The cellular mechanisms that define the ability of α cells to regulate the production and maturation of proglucagon-derived peptides remains an active area of investigation.

In α cells, proglucagon undergoes limited proteolysis primarily by prohormone convertase 2 (PC2), which generates glucagon as the major peptide product 13. PC2 is initially synthesized and folded as a 75 kDa inactive zymogen, proPC2, in the endoplasmic reticulum (ER). The maturation of proPC2 to its ~64 kDa active form, PC2, is a complex, multi-step process that requires folding in the ER, binding to its chaperone protein 7B2, posttranslational modification in the Golgi network, and sorting into acidic secretory granules, where it is then activated by intramolecular autocatalysis 14,15. Understanding the regulatory mechanisms underlying proPC2 maturation and processing is essential for optimizing glucagon production and developing targeted therapeutic strategies for manipulating α cell function.

The synthesis and packaging of islet hormones and their processing enzymes into secretory granules depends on effective protein quality systems, particularly within the ER. Genome-wide association studies (reviewed in 16) and multi-omic analyses of human islets 17 have linked genes involved in ER homeostasis and ER stress responses with both type 1 and type 2 diabetes, but many of these underlying mechanisms have primarily been studied in β cells 18,19. The specific mechanisms defining ER homeostasis in α cells are poorly understood. As the initial location of protein synthesis, the ER must maintain a healthy environment for proteins to fold into their mature structure, as failure to do so risks pathologic aggregation or premature activation. Cells employ multiple, interrelated quality control mechanisms to respond to the ever-changing burden of protein synthesis and manage protein misfolding, including the unfolded protein response (UPR), autophagy, and ER-associated degradation (ERAD) 20.

ERAD is a key quality control mechanism responsible for targeting misfolded proteins in the ER for cytosolic proteasomal degradation 21,22. The SEL1L-HRD1 protein complex represents the most conserved branch of ERAD, where SEL1L serves as a cognate cofactor for the E3 ligase HRD1 23. We and others have shown that SEL1L controls both HRD1 protein stability 24,25 and ERAD complex formation 26,27. SEL1L-HRD1 ERAD promotes the maturation of several misfolding-prone prohormones, such as pro-arginine vasopressin (proAVP) and pro-opiomelanocortin (POMC) in neurons 28,29. In the absence of SEL1L, proAVP and POMC peptides aggregate in the ER, impairing their maturation and limiting production of their functional hormone derivatives. In pancreatic islet β cells, SEL1L-HRD1 ERAD is critical to maintain cell identity 30,31 and insulin production 32,33. The role of SEL1L-HRD1 ERAD in islet α cells and proglucagon maturation has until now been unexplored.

In this study, we generated a mouse model inactivating Sel1L in proglucagon-expressing cells and found that defective ERAD is associated with impaired glucagon production secondary to impaired maturation of proPC2. Mechanistically, we uncovered an unexpected role of SEL1L-HRD1 ERAD in the quality control of proPC2 in the ER – ensuring that misfolded proPC2 is targeted for degradation, thereby maintaining the integrity of proglucagon processing machinery. In the absence of proper ERAD function, misfolded proPC2 accumulates in the ER, undergoes aberrant proteolytic cleavage and forms high molecular weight disulfide-bonded complexes, leading to impaired maturation and reduced levels of active PC2. This, in turn, limits the cleavage of proglucagon, resulting in diminished glucagon production and secretion.

Results
The ER network and SEL1L-HRD1 ERAD expression in islet α cells
Like islet β cells 30,31, neighboring α cells also have an expansive network of ER, as observed by transmission electron microscopy (TEM) (Fig. 1a, Supplementary Fig. 1a). To explore the role of ER homeostasis in α cells, we examined the ER in glucagon-null Gcg-STOP-flox (“Gcg-/-”) mice 34. In this model, there is a dramatic expansion of the α cell population secondary to hyperaminoacidemia, due to the complete absence of hepatic glucagon signaling 35,36. Despite their inability to make proglucagon and its derivative peptides, the ER remained prominent in Gcg-/- α cells (Fig. 1b, Supplementary Fig. 1a-b). Using confocal microscopy, we found that protein levels of BiP, an ER chaperone protein, is relatively low in normal α cells compared to that in β cells in wild-type mice (Fig. 1c-d). However, BiP was increased in α cells in Gcg-/- mice (Fig. 1c-d). This expansion of ER capacity in Gcg-/- α cells was also reflected by increased expression of SEL1L compared to that in normal α cells (Fig. 1e-f, white arrows). SEL1L was expressed in both α and β cells in human islets, from donors with or without diabetes (Fig. 1g). These findings led us to investigate the role of SEL1L-HRD1 ERAD in α cell ER homeostasis and function.

Targeted deletion of SEL1L in islet α cells
To define the role of SEL1L-HRD1 ERAD in α cells, we developed a mouse model in which SEL1L was inactivated specifically in proglucagon-expressing cells by crossing mice expressing an “improved” constitutively active Cre recombinase under the proglucagon promoter (GcgiCre) 37 with Sel1L-floxed mice on a B6 background 24. In these Sel1LΔGcg mice, almost all α cells showed absence of immunolabeling for SEL1L (Fig. 2a). Confirming that ERAD function was impaired in Sel1LΔGcg α cells, we also observed increased expression of the ERAD substrate OS9 (Fig. 2b) and the ER chaperone BiP (Fig. 2c). By contrast, immunofluorescence of glucagon was slightly reduced in Sel1LΔGcg α cells (Fig. 2d), suggesting that ERAD impairs glucagon production. However, this appears to occur through an indirect mechanism, as ERAD dysfunction did not lead to retention of proglucagon in the ER (Fig. 2d), as has been shown with other misfolding-prone prohormones such as proAVP and POMC 28,29. Despite targeting SEL1L deletion in all proglucagon-expressing cells in Sel1LΔGcg mice, we did not observe increased BiP expression in intestinal L-cells (Supplementary Fig. 2a), as detected by an anti-glucagon antibody that shows colocalization with anti-GLP-1 antibody (Supplementary Fig. 2b) and also detects proglucagon by Western blotting (Supplementary Fig. 1c; antibody details provided in Supplementary Table 1). To better understand the extent of Cre recombinase activity in this model, we generated R26R-EYFPΔGcg reporter mice by crossing ROSA26-STOP-flox-EYFP mice 38 with the same GcgiCre line 37. We found that the vast majority of islet α cells expressed the YFP reporter (Supplementary Fig. 2c), while YFP was not detected in intestinal L-cells (Supplementary Fig. 2d). In line with this notion, there was no difference in glucose-stimulated total GLP-1 levels in serum (Supplementary Fig. 2f) or in GLP-1 content of distal colon epithelium (Supplementary Fig. 2g) from Sel1LΔGcg mice. Together, these data suggest that this GcgiCre line predominantly targets islet α cells.

Attenuated glucagon production in Sel1LΔGcg mice
We next asked how inactivation of SEL1L-HRD1 ERAD affected α cell function. Male and female Sel1LΔGcg mice showed normal growth on chow diet through adulthood (Fig. 3a). There was no change in oral glucose tolerance (Fig. 3b) in 4-8 month-old Sel1LΔGcg mice compared to sex-matched littermate controls. To assess stimulated glucagon secretion in vivo, we subjected adult cohorts to insulin-induced hypoglycemia. At 4-8 months of age, male Sel1LΔGcg mice had significantly decreased glucagon secretion following hypoglycemia (Fig. 3c). Female Sel1LΔGcg mice also showed reduced glucagon secretion in vivo, but starting at 8 months of age (Fig. 3c).

To understand the mechanism underlying lower glucagon levels in Sel1LΔGcg mice, we next investigated glucagon production and secretion in α cells. In both adult male and female Sel1LΔGcg mice, pancreatic glucagon content was reduced by half by 4 months of age (Fig. 3d), as detected by an ELISA assay targeted to detect the N- and C-terminal ends of the mature 29-amino acid glucagon peptide. This did not appear to reflect a developmental defect in α cell formation, as stimulated glucagon secretion was not impaired in 11-week-old Sel1LΔGcg mice (Supplementary Fig. 2h) and pancreatic glucagon content was reduced only in male mice at that age (Supplementary Fig. 2i). The defect in circulating glucagon levels was not due to defects in secretory dynamics or response to stimuli, as isolated islets from Sel1LΔGcg mice showed a similar secretory response to low glucose with or without the secretagogue L-arginine, when normalized to total glucagon content (Fig. 3e). Importantly, the expression of Cre in the native Gcg locus did not affect pancreatic glucagon content in R26R-EYFPΔGcg reporter mice (Supplementary Fig. 2e). Altogether, these data suggest that ERAD dysfunction limits glucagon production in α cells, while having no effect on glucagon secretory dynamics.

ER dilation in Sel1LΔGcg α cells
We next performed TEM to examine α cell morphology and secretory granule formation. In contrast to the prominent stacks of ER and abundant electron-dense granules in normal α cells, the α cells in Sel1LΔGcg mice showed significant ER dilation and, in extreme cases, fewer glucagon granules (Fig. 3f and Supplementary Fig. 3a). Nonetheless, the glucagon granules in Sel1LΔGcg α cells appeared largely normal in morphology (Fig. 3f and Supplementary Fig. 3a). Additionally, β cell architecture and pancreatic insulin content were unchanged in Sel1LΔGcg mice (Supplementary Fig. 3b-c). By immunofluorescence detection with antibodies to the mature glucagon sequence, α cell mass was reduced in adult Sel1LΔGcg mice (Supplementary Fig. 3d), but without significant changes in α cell apoptosis (Supplementary Fig. 3e) or proliferation (Supplementary Fig. 3f), as evaluated by TUNEL assay and Ki67+ cells, respectively. Altogether, these data suggest that ERAD deficiency causes ER dilation and limits production of mature glucagon in α cells.

ERAD deficiency causes the accumulation of proPC2 protein in vivo
Given the greater reduction in mature glucagon relative to proglucagon levels in Sel1LΔGcg α cells, we speculated that the defect may lie in the proteolytic cleavage of proglucagon, a process catalyzed by the PC2 enzyme. PC2 is initially synthesized in the ER as the zymogen proPC2. ProPC2 folds in the ER, forming three disulfide bonds (Fig. 4a), binds its chaperone 7B2 to proceed through the secretory pathway 39, and then becomes activated within secretory granules 40, where the Pro domain is cleaved from the proprotein by autocatalysis. AlphaFold structure modeling was used to visualize the possible conformation of each domain (Fig. 4b). This tertiary structure aligns with prior work showing that proPC2 is susceptible to misfolding and aggregation within the ER in the absence of its chaperone protein 7B2 39,41. We thus asked how SEL1L-HRD1 ERAD affects PC2 biology.

To visualize the expression level and distribution of proPC2 in vivo, we performed immunofluorescence labeling using antibodies specific for different regions of proPC2 39 (Fig. 4a). There was a marked increase in proPC2 levels in Sel1LΔGcg α cells (Fig. 4c). In line with this finding, immunolabeling with antibodies to the catalytic domain of PC2 (which detects both mature PC2 and the proPC2 protein) showed that total PC2 levels were also increased in Sel1LΔGcg α cells (Fig. 4d). These data suggest that SEL1L deficiency causes the accumulation of proPC2 protein in vivo.

Altered proPC2 processing in the absence of SEL1L-HRD1 ERAD
To further explore how SEL1L-HRD1 ERAD regulates proPC2 maturation in α cells, we developed a cell culture model of ERAD deficiency using CRISPR-mediated deletion of either SEL1L or HRD1 in the well-established αTC1-6 α cell line (ΔSel1L and ΔHrd1, respectively) (Fig. 5a, Supplementary Fig. 4a-c). Deletion of SEL1L or HRD1 in αTC1-6 cells caused ERAD dysfunction, as demonstrated by increased protein levels of ERAD substrates IRE1α and OS9 (Supplementary Fig. 4d-f). In addition, ERAD deficiency triggered a mild activation of ER stress responses as measured by the phosphorylation of eIF2α and splicing of Xbp1 mRNA (Supplementary Fig. 4g-k). Compared to cells treated with the ER stressor tunicamycin, the level of Xbp1 mRNA splicing in ERAD-deficient αTC1-6 cells was modest (Supplementary Fig. 4k), similar to our previous observations 29,30.

We then examined (pro)PC2 expression using Western blot with three different antibodies targeted to different regions of the protein (Fig. 4a). Using a proPC2-specific antibody, we found that expression of proPC2 (~75 kDa) was significantly increased in both ΔSel1L and ΔHrd1 α cells compared to control cells (Fig. 5b). Intriguingly, we observed a novel ~55 kDa proPC2 fragment (which we termed proPC2*) that was highly enriched in ERAD-deficient cells (Fig. 5b). In addition, using an antibody targeting the PC2 catalytic domain, we confirmed the accumulation of proPC2 and proPC2* in both ΔSel1L and ΔHrd1 α cells compared to control cells, while mature PC2 (~64 kDa) protein levels were reduced by half (Fig. 5c). The combined increase in full-length proPC2 and the proPC2* fragment led to a slight increase in the overall expression of proPC2-derived proteins in ΔSel1L α cells (Supplementary Fig. 5a-b), reflecting the pattern observed in Sel1LΔGcg islets. We confirmed that proPC2* expression was also increased in Sel1LΔGcg islets by Western blot (Supplementary Fig. 5c). In contrast, using an antibody specific for the C-terminal domain of PC2, we failed to observe proPC2* in ΔSel1L or ΔHrd1 α cells (Fig. 5d), suggesting that proPC2* was generated by an aberrant cleavage at the C-terminal end of the protein. We confirmed that proPC2* was derived from proPC2 given its absence in cells with CRISPR-mediated co-deletion of PC2 and Sel1L (Fig. 5e-f; note that PC2 was significantly reduced but incompletely deleted by this method). We also confirmed that proPC2* was an immature protein found in the ER, given its sensitivity to deglycosylation following endoglycosidase H or PNGase F treatments (Supplementary Fig. 5d).

To explore whether the consequences of ERAD deficiency specifically affected proPC2 maturation and glucagon production in α cells, we next examined expression of the PC1/3 enzyme, which is primarily responsible for generation of GLP-1 from proglucagon. By immunofluorescence, Sel1LΔGcg α cells showed slightly reduced expression of both PC1/3 and GLP-1 (Supplementary Fig. 6a-b). Unlike proPC2, we did not observe aggregation of PC1/3 under nonreducing conditions on Western blot (Supplementary Fig. 6c). Instead, the lower PC1/3 content was attributed in part to reduced expression of Pcsk1 mRNA in ΔSel1L α cells (Supplementary Fig. 6d). Pancreatic total GLP-1 content, as measured by immunoassay, was significantly reduced in adult male but not female Sel1LΔGcg mice (Supplementary Fig. 6e). Thus, ERAD deficiency specifically inhibits maturation of proPC2 in α cells and imparts a lesser effect on PC1/3 expression and GLP-1 production.

The accumulation of both proPC2 and proPC2* was specific to ERAD deficiency, and uncoupled from ER stress, as it was not observed in control αTC1-6 cells following induction of ER stress with either tunicamycin or thapsigargin (Fig. 5g). Importantly, ERAD deficiency was associated with a slight decrease, not increase, in expression of Pcsk2 mRNA (Fig. 5h). Therefore, the accumulation of proPC2 protein was attributed to post-transcriptional regulation. Taken together, these data show that SEL1L-HRD1 ERAD is critical for the maturation of proPC2 in α cells by preventing its abnormal proteolytic cleavage.

Nascent proPC2 is degraded by SEL1L-HRD1 ERAD in α cells
We then explored the molecular mechanism underlying the interplay between ERAD and proPC2. Using cycloheximide to block protein synthesis, we found that both proPC2 and proPC2* were stabilized in both ΔSel1L and ΔHrd1 α cells, as validated by two different antibodies (Fig. 6a-b). Moreover, in the absence of ERAD, proPC2 formed high molecular weight (HMW) protein complexes as visualized by non-reducing SDS-PAGE. The formation of these HMW complex were mediated by aberrant disulfide bonds as they were sensitive to the reducing agent β-mercaptoethanol (lanes 5-6, Fig. 6c). The proPC2* fragment contributed to this process, as it co-migrated with HMW aggregates under non-reducing conditions (lanes 5-6 versus lanes 2-3, Fig. 6c). Supporting the direct role of SEL1L-HRD1 ERAD in regulating proPC2 degradation, we found that proPC2 was polyubiquitinated in a HRD1-dependent manner (Fig. 6d). MG132 treatment also increased the polyubiquitination of proPC2 (lanes 4-5, Fig. 6d), pointing to the involvement of proteasome. Interestingly, MG132 treatment also increased the production of proPC2* in control cells (input lane 4 versus input lanes 1-2, Fig. 6d). This suggests that proPC2* is routinely generated in normal α cells but is undetectable due to degradation by SEL1L-HRD1 ERAD. These data demonstrate that in the absence of SEL1L-HRD1 ERAD, proPC2 accumulates in the α cell ER, is abnormally cleaved, and forms HMW complexes via aberrant disulfide bonds.

To better understand the timing of proPC2 processing defects following ERAD dysfunction, we used an siRNA approach to acutely knock down SEL1L or HRD1 protein levels in control αTC cells (Supplementary Fig. 7). Similar to CRISPR-mediated inactivation of ERAD proteins in αTC cells, we observed accumulation of proPC2 and proPC2* 48 hours after siRNA knockdown of these ERAD proteins (Supplementary Fig. 7a). We also observed accumulation of full-length proglucagon in siSel1L and siHrd1 cells (Supplementary Fig. 7b). Notably, we confirmed the profound accumulation of proglucagon and absence of mature glucagon formation in CRISPR-generated ΔPC2 cells, in which complete inactivation of functional PC2 disrupts proglucagon-to-glucagon conversion, as previously shown 42. These data further support the role of SEL1L-HRD1 ERAD in proPC2 maturation and subsequent PC2-mediated conversion of proglucagon into the mature glucagon peptide.

Impaired proPC2 enzymatic activity in ERAD-deficient α cells
One unique feature of PC2 is its requirement for a protein cofactor, 7B2 (also called secretogranin-5), to serve as a chaperone that binds proPC2 in the ER and inhibits PC2 enzyme activation until it reaches later stages in the secretory pathway 39. In the absence of 7B2, proPC2 is prone to aggregation 41, thus limiting generation of glucagon from proglucagon 43. We found that 7B2 protein expression was reduced in both ΔSel1L and ΔHrd1 α cells compared to their respective controls (Fig. 6e), suggesting a possible contribution to proPC2 misprocessing. To better understand the relationship between 7B2 levels and proPC2 maturation in ERAD deficiency, we investigated 7B2 expression in αTC cells following siRNA-mediated ERAD inactivation. In contrast to ΔSel1L and ΔHrd1 αTC cells with chronic ERAD inactivation, both siSel1L and siHrd1 cells instead showed an increase in 7B2 expression (Supplementary Fig. 7a), suggesting that ERAD function influences 7B2 expression, but independently from its effect on proPC2. Importantly, overexpression of 7B2 was unable to rescue the accumulation and aggregation of proPC2(*) in ΔSel1L and ΔHrd1 αTC cells (Supplementary Fig. 8a-b), supporting a limited role for 7B2 in the proPC2 misprocessing observed in ERAD deficiency.

To determine whether these defects in proPC2 maturation affected PC2 enzymatic function, we measured PC2 activity in αTC cells using a substrate-specific aminomethylcoumarin assay as previously described 44. This assay detects activity of mature PC2 in addition to properly folded proPC2 that is spontaneously autoactivated in the low pH of the reaction buffer 15. Compared to control cells, ΔSel1L and ΔHrd1 αTC cells showed significantly impaired PC2 activity (Fig. 6f). Addition of the 7B2 C-terminal peptide, a potent and specific PC2 inhibitor 44, successfully abolished PC2 activity in all cells as a control (Fig. 6f). Together, these data suggest that the aberrant cleavage and aggregation of proPC2 in ERAD-deficient α cells impairs PC2 activity.

Methods
Study approval. All animal procedures were approved by and done in accordance with the IACUC at the University of Michigan Medical School (PRO00008989/PRO00010658/PRO00011495).

Mice. Sel1lfl/fl mice 24 on the C57BL/6J background were crossed with B6;129S-Gcgtm1.1(icre)Gkg/J (GcgiCre) mice 37 to generate mice with SEL1L deletion in proglucagon-expressing cells (denoted Sel1LΔGcg herein), with Cre-negative (Sel1Lfl/fl) or heterozygous (Sel1LΔGcg/+) littermates used as controls. GcgiCre mice were also crossed with B6.129X1-Gt(ROSA)26Sortm1(EYFP)Cos/J reporter mice (Jackson Laboratory 006148). Glucagon-STOP-flox (Gcg-/-) mice, containing GFP and a poly(A) “stop” signal between exons 2 and 3 of the Gcg gene, were previously described 34. Mice were housed in an ambient temperature room with a 12 hr light cycle and fed a normal-chow diet (13% fat, 57% carbohydrate, and 30% protein, LabDiet 5L0D). Males and females were used equally in experiments, except where sexually dimorphic results were observed and reported.

Human islets. Isolated islets from cadaveric donors were obtained from the Human Islet Resource Center at the University of Pennsylvania through the Human Pancreas Analysis Program 77,78 and Integrated Islet Distribution Program (IIDP) 79, following the guidelines of the Clinical Islet Transplantation Consortium protocol (https://www.isletstudy.org). Briefly, the pancreas was digested following an intraductal injection of collagenase & neutral protease in Hanks’ balanced salt solution and then purified on continuous density gradients (Cellgro/Mediatech) using a COBE 2991 centrifuge. Islets were cultured in CIT cultuAre media and maintained in a humidified 5% CO2 incubator. The islets were distributed per the Integrated Islet Distribution Program (IIDP) protocols. After receipt, islets were hand-picked and cultured overnight prior to embedding in low melting point agarose 80 before fixation in 1% paraformaldehyde and mounting in O.C.T. medium in preparation for cryosectioning and immunohistochemistry as previously described 81,82. Gift of Life, along with other organ procurement organizations, obtained consent from the deceased donors’ families for the use of organs in research. All procedures complied with the University of Pennsylvania IRB, Gift of Life leadership team, and UNOS regulations and standards. Donor information is presented in Supplementary Table 2. Only male donors were available for this study.

Glucose tolerance tests and in vivo glucagon and GLP-1 secretion. Mice were fasted for 4-6 hr prior to each experiment. Blood was sampled via tail nick. For glucose tolerance testing, blood glucose was measured at baseline prior to intraperitoneal or oral gavage-mediated administration of D-glucose at 1.5 g/kg body weight. Blood glucose was measured 15, 30, 60, and 90 min after glucose administration using a OneTouch Ultra glucometer. For assessment of glucagon secretion, blood was collected in heparinized tubes before and 30 minutes following intraperitoneal injection of 0.75 units/kg insulin (Novolin R, Fisher Scientific NC0769896). Serum was collected by centrifugation, immediately frozen at -80C, and thawed prior to measurement by ELISA (Mercodia 10-1281-01). For assessment of total serum GLP-1 levels, mice were given an oral gavage of 3 g/kg D-glucose ten minutes before undergoing isoflurane anesthesia and cardiac puncture. DPPIV inhibitor (Millipore Sigma DPP4) was added to whole blood prior to centrifugation, and serum was frozen at -80C prior to measurement by U-PLEX biomarker assay (Meso Scale Diagnostics K1525UK). Note that negligible levels of glucagon and GLP-1 were detected in pancreas extracts or serum from Gcg-/- mice using these assays (Supplementary Fig. 1d).

Pancreatic and gut hormone content. Immediately following sacrifice by cervical dislocation, mouse pancreata were dissected, trimmed of fat, weighed, placed in 3 ml of acid alcohol (1.5% hydrochloric acid in 75% [v/v] ethanol in water) and homogenized for 30 sec. The suspension was rotated for 48 hr at 4°C, centrifuged at 1540 x g for 30 min at 4°C, then the supernatant stored at -80°C until further analysis. For GLP-1 measurement in gut, the most distal 3 cm of colon was dissected, and gut epithelium was scraped with a coverslip and placed in either acid alcohol or T-PER extraction reagent (Thermo Scientific 78510) with DPPIV inhibitor as previously described 34 for hormone extraction. Glucagon and/or GLP-1 content of diluted supernatant was measured by ELISA, as above. Pancreatic insulin content was measured by ELISA (Crystal Chem 90080).

Histology and islet morphometry. Pancreata were isolated, fixed in 10% neutral buffered formalin (VWR 95042-908) or 4% paraformaldehyde (Electron Microscopy Sciences 15710) for 1.5-16 hours at 4C. Samples for paraffin embedding and sectioning were dehydrated in 70% ethanol and processed by the University of Michigan Comprehensive Cancer Center Tissue and Molecular Pathology Shared Resource. Alternatively, some pancreata were processed for cryosectioning as previously described 83. Measurements of α- and β-cell mass were performed by the University of Michigan Islet Core. Briefly, five sections per pancreas, each at least 150 μm apart, were labeled with antibodies to insulin (Agilent, Santa Clara, CA) and glucagon (Immunostar, Hudson, WI), then imaged by tiling with a computer-controlled AZ-100 microscope with motorized stage using the NIS Elements AR software (Nikon, Cambridge, MA) and a Coolsnap EZ Digital Camera (Photometrics, Tucson, AZ). After acquisition, α- and β-cell areas were determined by intensity thresholding with manual curation using FIJI 2.14.0. Total α- and β-cell mass were then calculated using the following calculation: [(signal area/total pancreatic section area) x pancreas weight].

Immunofluorescence staining. Deparaffinized tissue sections and cryosections were immunolabeled as described previously 30. The following primary antibodies were used: SEL1L (homemade, 30,82, 1:200), insulin (Bio-Rad 5330-0104G, 1:500), glucagon (Peninsula Labs/BMA Biomedicals T-5037, 1:200), proglucagon (Cell Signaling Technology 8233S, 1:200), transthyretin (Invitrogen PA580196/PA580197, 1:500), somatostatin (Abcam ab30788, 1:200), BiP (Abcam 21685, 1:100), OS9 (Abcam ab109510, 1:100), GFP (Abcam 13970, 1:2000), PC2 (Cell Signaling Technology 14013s, 1:800), proPC2 (homemade, 14, 1:200), GLP-1 (Peninsula Labs/BMA Biomedicals T-4363, 1:200), E-cadherin (BD Biosciences 610181, 1:100), PC1/3 (homemade RS20 84-86, 1:200). Details on antigens for key antibodies can be found in Supplementary Table 1. Sections were treated with species-specific secondary antibodies conjugated to Alexa Fluor dyes (Jackson ImmunoResearch; 1:500) followed by mounting with VectaShield (Vector Laboratories, H-1500) or ProLong Gold (Invitrogen P36931) mountant with DAPI. Immunolabeled pancreatic sections were imaged using a Leica DM6000 through the University of Michigan Vision Research Center Morphology and Imaging Core, and Nikon A1 or Leica Stellaris confocal microscopes at the University of Michigan Morphology and Image Analysis Core. Images were prepared for publication using ImageJ software (NIH, version 2.14.0) and figure layouts created in Numbers (MacOS, version 14.4). Immunofluorescence quantification was performed using Imaris software (v10.0, Oxford Instruments).

Proliferation and TUNEL assays. Paraffin embedded pancreas sections were immunolabeled with Ki67 (Abcam 15580; 1:100) and glucagon to detect proliferating α cells. TUNEL assay was performed using the In-Situ Cell Death Detection Kit (Roche 11684795910), with glucagon co-labeling by immunofluorescence. Images were acquired using a Leica DM6000 microscope for counting 15-100 islets per mouse to capture a total of 2000-4000 α cells (n = 3-5 per genotype). Cell proliferation and apoptosis rates were calculated as a percentage of Ki67- or TUNEL-positive cells, respectively, per total number of glucagon-positive cells.

Transmission electron microscopy. Whole pancreas was rapidly dissected, placed in fixative (2.5% glutaraldehyde and 4% paraformaldehyde in 0.1 M Na-cacodylate buffer), minced into ~1–2 mm cubes, and fixed overnight at 4°C followed by washes in buffer. Thereafter, samples were submitted to the University of Michigan Microscopy Core for post-fixation in 1% osmium tetroxide, embedding in resin, sectioning, and mounting on grids. Imaging was performed using a JEOL JEM 1400 PLUS microscope with an AMT NanoSprint 12 scientific grade CMOS camera.

Islet isolation and secretion. Pancreatic islets were isolated from mice as previously described 87. Briefly, mice were sacrificed by cervical dislocation and pancreata perfused by intraductal injection of 2.5 mL collagenase (Liberase TL [Roche 5401020001] in serum-free RPMI 1640 [Gibco 11875-085]). Pancreata were incubated at 37°C for ~12 min with an additional 1 mL of digestion solution. Digestion was stopped by adding cold HBSS with 10% FBS. Islets were washed and hand-picked under a dissecting microscope, then allowed to recover overnight in RPMI medium in a humidified incubator (95% air, 5% CO2) at 37°C prior to further analysis. For static secretion experiments, 10 size-matched purified islets were incubated in 200 μl KRBB (Krebs-Ringer Solution, HEPES-buffered, Thermo-Fisher Scientific J67795.K2) containing D-glucose +/- L-arginine hydrochloride (MilliporeSigma A5131) and media collected at the indicated times. Following the experiments, islets were lysed (50 mM Tris HCl pH 7.4, 1 mM EDTA, protease inhibitor [MilliporeSigma P8340], phosphatase inhibitor [MilliporeSigma P5726]), total protein concentrations determined by Pierce BCA assay (Thermo Fisher Scientific), and media and lysate glucagon levels measured by ELISA as above.

Cell culture and Western blotting. Control αTC(1-6) cells were a kind gift from Ernesto Bernal-Mizrachi and also independently obtained from ATCC (CRL-2934). Cells were cultured in αTC complete medium per manufacturer recommendations. CRISPR-targeting sgRNA oligonucleotides designed against mouse Sel1l, Syvn1, or Pcsk2 (sequences listed in Supplementary Table 3) were inserted into the lentiCRISPR, version 2 vector (Addgene, 52961) prior to transfection in αTC (or HEK293T) cells and puromycin selection of modified cells as previously described 76. Cells transfected with the empty lentiCRISPR vector were used as a control. In some experiments, cells were treated with cycloheximide (50 µg/ml; Sigma-Aldrich C7698), endoglycosidase H (New England Biolabs P0702S), or PNGase F (New England Biolabs P0704S) per manufacturer’s instructions. In siRNA treatment experiments, control αTC cells were transfected with 30 pmol of siRNA targeting Sel1l, Syvn1, or a Silencer Select Negative Control (Invitrogen s73510, s92312 and 4390844) using RNAiMAX (Lipofectamine RNAiMAX Transfection Reagent, #13778075). In the 7B2 overexpression experiments, CRISPR-treated αTC cells were seeded in 6-well plates and cultured until reaching 70-90% confluency. For each well, two 125 μl aliquots of Opti-MEM (Gibco, 31985062) were prepared. One aliquot was used to dilute 3.75 μl of Lipofectamine 3000 (Invitrogen, L3000015), while the other was used to dilute 5 μg of the 7B2 plasmid DNA (From Iris Lindberg Lab) combined with 10 μl of P3000 reagent (Invitrogen, L3000015). The DNA-lipid complexes were then mixed and then added to the cells. Upon experiment completion (typically 48 hours after siRNA or 7B2 plasmid), cells were lysed in NP-40 lysis buffer (50 mM Tris HCl at pH 7.5, 150 mM NaCl, 1% NP-40, 1 mM EDTA) with protease inhibitor (MilliporeSigma P8340), DTT (MilliporeSigma, 1 mM), and phosphatase inhibitor (MilliporeSigma P5726) cocktail. Lysates were incubated on ice for 30 min and centrifuged at 16,000 x g for 10 min. Supernatants were collected and protein concentrations determined using Bradford assay (Bio-Rad). For reducing SDS-PAGE analysis, proteins were heat-denatured at 65-90C for 10 min in 5x SDS sample buffer (250 mM Tris-HCl pH 6.8, 10% sodium dodecyl sulfate, 0.05% bromophenol blue, 50% glycerol, and 1.44 M β-mercaptoethanol). For non-reducing conditions, lysates were prepared at 37°C with 5x non-denaturing sample buffer (250 mM Tris-HCl pH 6.8, 1% sodium dodecyl sulfate, 0.05% bromophenol blue, 50% glycerol) for 60 min 27. Samples were resolved by gel electrophoresis, then transferred to PVDF membranes (Bio-Rad). Some membranes were fixed with PBS containing 4% paraformaldehyde and 0.01% glutaraldehyde for 30 min 88. Membranes were blocked with 5% milk in Tris-buffered saline Tween-20 (TBST) for 30 min, then incubated overnight at 4C with antibodies prepared in 2% bovine serum albumin (BSA) in TBST. The following primary antibodies were used: anti-SEL1L (homemade30, 1:10,000), anti-HRD1 (Proteintech 13473-1, 1:2,000), anti-OS9 (Abcam ab109510, 1:5,000), anti-BiP (Abcam ab21685, 1:5,000), anti-IRE1α (Cell Signaling Technology [CST] 3294, 1:2,000), anti-PERK (CST 3192, 1:1000), anti-eIF2α (CST S9722, 1:1000), anti-phospho-eIF2α (CST 9721, 1:1000), anti-PC2 (CST 14013s, 1:500), anti-PC2 C-terminus (homemade39, 1:200), anti-proPC2 (homemade14, 1:200), anti-7B2 (homemade61, 1:200), anti-PC1/3 (Cell Signaling 18030S, 1:200), anti-glucagon (ABclonal A22702, 1:5000), anti-ubiquitin (Santa Cruz, sc-8017), and anti-HSP90 (Abcam ab13492; 1:2000). Details on antigens for key antibodies can be found in Supplementary Table 1. Membranes were washed and then incubated with goat anti-rabbit or anti-mouse IgG-HRP (Bio-Rad 1706515 and 1706516, 1:3000-1:5000) in 5% milk in TBST for 1 hr at room temperature, washed, and labeled with Clarity ECL substrate (Bio-Rad). Blots were imaged with a Bio-Rad ChemiDoc and protein band intensity was quantified using Image Lab software (Bio-Rad, version 6.1).

Immunoprecipitation and ubiquitination assay. Assay was performed as previously described 75. Briefly, control and ΔHrd1 αTC cells were treated with 10 μM MG132 (Enzo, BML-PI102) for 2 hr. Whole cell lysates were prepared in non-reducing lysis buffer as above. The supernatant was denatured with 1% SDS and 5 mM DTT at 95°C for 10 min, then diluted 1:10 with NP-40 lysis buffer and mixed with control IgG antibody (Cell Signaling Technology 2729) or anti-proPC2 antibody (as above). This mixture was incubated with 15 μl anti-protein A agarose (Millipore Sigma, 11719408001) overnight at 4°C with gentle rocking. Agarose beads were washed three times with NP-40 lysis buffer and eluted in the SDS sample buffer at 95°C for 5 min, followed by SDS-PAGE and Western blotting as above.

PC2 enzyme assay. A substrate-specific PC2 assay was performed as previously described 44. Control, ΔSel1L, and ΔHrd1 αTC cells were washed and sonicated in ice-cold lysis buffer (0.1 M sodium acetate, pH 5.5, 1% Triton X-100, 50 µM trans-epoxysuccinyl-L-leucylamido[4-guanidino]butane [E-64], 50 uM pepstatin, 1 mM phenylmethylsulfonyl fluoride [PMSF]) and centrifuged at 10,000 x g at 4°C for 10 minutes. The supernatant was transferred to clean microcentrifuge tubes and protein concentrations determined using a Bradford assay (Bio-Rad). Samples of equal protein concentration (10 µg) were added to a polypropylene microtiter plate with 40 µl reaction buffer mix (2 mM CaCl2, 0.1 M sodium acetate buffer [pH 5.0], 0.1% Triton X-100, 5.6 mM Nα-tosyl-L-lysine chloromethyl ketone hydrochloride [TLCK], 6 mM N-p-tosyl-L-phenylalanine chloromethyl ketone [TPCK], 2 mM E-64, 2 mM pepstatin). Each assay was performed in duplicate, both with and without the synthetic 7B2 C-terminal peptide (7B2-CT). The reaction was preincubated for 15 min at 37°C, followed by addition of 5 µl of substrate, pGlu-Arg-Thr-Lys-Arg-MCA (RTKR-MCA, final concentration 0.2 mM in dimethyl sulfoxide [DMSO]). The reaction mixture was briefly mixed and the fluorescence of the liberated aminomethyl-coumarin (AMC; excitation 380 nm, emission 460 nm) was documented every two minutes using a fluorometer.

RNA extraction, microarray, cDNA synthesis and qPCR analysis. RNA was isolated by phenol extraction and concentration determined using a NanoDrop 2000 UV-Vis Spectrophotometer. RT-PCR for assessment of Xbp1 mRNA splicing and qPCR analyses were performed as previously described 89. All PCR data were normalized to the ribosomal L32 or Rplp0 gene expression level. All qPCR primers were obtained from Integrated DNA Technologies (IDT) and sequences are shown in Supplementary Table 4.

Protein structure analysis. Protein structures were predicted using the AlphaFold 3 server, located at https://alphafoldserver.com/ 90. Images were rendered with PyMOL (version 2.3.2).

Statistical analysis, reproducibility, and figure generation. Results are expressed as mean ± SEM unless otherwise stated. All experiments were repeated at least twice and performed with several independent biological samples as noted in the figure legend and/or Source Data file, with representative data shown. Statistical analyses and data graph generation were performed in GraphPad Prism (GraphPad Software Inc., version 10.6.1). Comparisons between two groups were made by unpaired two-tailed Student's t test. Comparisons between multiple groups were made by one- or two-way ANOVA followed by Šidák or Tukey post-test, as noted. P values less than 0.05 were considered statistically significant. The model in Figure 7 was generated in the MacOS Numbers program (version 14.4).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This study investigates a novel role for the SEL1L-HRD1 ER-associated degradation (ERAD) system in regulating\nprohormone convertase 2 (PC2), which is required for processing proglucagon in pancreatic alpha cells. The authors\nprovide evidence that SEL1L-HRD1 ERAD is essential for clearing excess proPC2. They also demonstrate that\ndysfunctional SEL1L-HRD1 ERAD leads to accumulation of a C-terminally truncated proPC2 form, which the authors\nspeculate is an aberrantly cleaved/misfolded PC2 that is not properly cleared by ERAD. The study observes a progressive\ndecline in stimulated glucagon secretion and a reduction in pancreatic glucagon content in mice lacking Sel1L, likely linked\nto PC2 dysfunction. The experiments are well-performed and appropriately controlled. The presence and rapid clearance of\nthe truncated proPC2 form is an interesting observation, as well as the finding that ERAD mediated clearance of proPC2 is\nseemingly decoupled from a general ER stress response. These findings may provide\nmechanistic insight into the\nconsequences of human disease variants of ERAD components, or potentially an avenue for controlling glucagon output in\ndiabetes by modulating ERAD / PC2. The study’s findings are novel and of potential importance albeit\nperhaps not too\nsurprising given that SEL1L-HRD1 ERAD is a highly conserved system and proPC2 is already known to be prone to\naggregation and accumulation. There are a few issues that should be addressed to improve the manuscript and strengthen\nits conclusions.\nMajor Concerns:\n1. Some of the important conclusions seem to be dependent on immunostaining data and/or use of antibodies with\nspecificity that is not known or at least described. Complementary biochemical assays for identifying proglucagon forms\nsuch as Western blotting, and better characterization of antibody specificity should be provided, as per some specific\nexamples below:\n(a) total PC2 immunoreactivity is stated to be increased in Fig 4D in alpha cell Sel1L KO alpha cells, but this does not seem\nto match with the aTC KO western data in Fig 5 where proPC2 is increased but total PC2 seems similar. It would be helpful\nto perform westerns as well on the alpha cell Sel1L KO primary islets (contribution of beta cells to PC2 should be small) to\nassess proPC2 processing and total PC2 content, and quantification of total PC2 from westerns in Figure 5.\n(b) The proglucagon and glucagon antibodies used in immunostaining are not detailed or their cross-reactivity with other\nantibodies, and the field is known for cross-reactivity of these antibodies with different molecular forms of\nproglucagon/glucagon derived peptides. Western blot of islet lysates from KO and control mice and mutated aTC cells to\nbetter understand the molecular forms of proglucagon peptides would be informative.\n2. PC2-glucagon link: The data indicating that proPC2 is an ERAD substrate is convincing, but the evidence that\naccumulation of proPC2 and reduction in PC2 activity due to ERAD disruption is the direct cause of loss of glucagon in\nthese models would benefit from additional supporting data or at least explanation, for example:\n(a) If ERAD knockout is causing significant PC2 activity loss, an increased accumulation of proglucagon should be evident,\nand demonstrated by western blot.\n(b) The authors could strengthen their conclusions by showing that loss of PC2 activity with PC2 deletion and/or 7B2\ninhibitor alters proglucagon production / processing.\n(c) As detailed in 1 above, demonstration by western in their models that proglucagon processing is impaired, as\nimmunostaining and ELISAs with available antibodies have unknown or questionable specificity.\n(d) The decrease in total pancreatic glucagon and proglucagon shown in Figure 3D does not seem to account for the\nsignificant and near proportional loss of alpha cell mass in Fig S3D. Could the loss of glucagon be simply related to loss of\nalpha cell mass?\n(e) Why does the functional glucagon defect only manifest in older Sel1LΔGcg mice (4 months male; 8 months female)? If a\nproPC2 processing defect is truly the cause of glucagon dysfunction, would the authors not expect the glucagon defect to be\napparent earlier? One interpretation might be due to loss of alpha cells with decreasing with age associated with ERAD\ndisruption. The authors should assess alpha cell population in younger mice. Further, performing cell viability and\nproliferation assays on the aged mice might be influenced by a survivorship bias.\n(f) Measurement (ELISA) of GLP-1 in ERAD deficient alpha cells would be informative. What happens to Pcsk1 / PC1/3 in\nERAD deficient alpha cells? It would be of interest if the defect was specific for PC2 and resulted in increased GLP-1\nproduction, though this is not clear.\n(g) Does Pcsk2 mRNA expression change in the mutants?\n3. Concerns with some figures:\n(a) Fig 1C – inclusion of insulin staining in top panel of 1C (control) would enable clearer comparison with the Gcg KO panel\nbelow.\n(b) representative images in Fig 2 could be more representative: in Fig 2C it is hard to see BiP expression changes when\nthere are so many more alpha cells in the control than in the Sel1L KOs; Fig 2D does not not convincingly show the slight\nbut significant decrease in proglucagon and glucagon levels in Sel1L knockouts reported by the quantification.\n(c) The presence of a proPC2 band in the PC2 knockout condition is puzzling and would benefit from explanation.\nMinor\n1. The implication that SEL1 is not changed in type 2 diabetes should be softened since it is based on immunostaining from\njust one WT and one diabetic donor, or more donors included.\n2. Please state the species of the homemade proPC2 antibody in the methods section.\n3. Quantification of OS9 expression in Figure 2B in a manner similar to BiP in Figure 2C to provide would strengthen\nconclusion that it is upregulated.", "reviewer_2": "This manuscript by Zhu et al investigates the role of the SEL1L-HRD1 ERAD pathway in islet α cells, demonstrating that it is\nessential for the proper folding, maturation, and enzymatic activation of proPC2, a key enzyme in glucagon biogenesis. The\nauthors provide evidence that Sel1L and Hrd1 knockout in proglucagon-expressing cells leads to aberrant proPC2\nprocessing, accumulation of a truncated proPC2* species, decreased PC2 activity, and ultimately impaired proglucagon\ncleavage. These findings reveal a post-translational regulation in α cell biology and open potential therapeutic avenues in\nmetabolic diseases.\nThe paper is clearly structured: the introduction lays out the need for this study, methods are suitable to test their hypothesis,\nthe data support the main conclusions, and discussions are thorough.\nSeveral mechanistic and contextual points should be addressed to solidify the conclusions.\nMajor concerns:\n1.Mechanism of proPC2* generation\nThe accumulation of proPC2* in ERAD-deficient α cells is intriguing and raises important questions about its origin. While\nthe proposed C-terminal cleavage is plausible, the exact mechanism by which proPC2* is generated remains unclear. Since\nproPC2* appears to be a potentially important intermediate with pathological relevance, it would greatly strengthen the study\nto provide further biochemical insight into how this fragment is formed—whether through a specific protease, misfolding, or\nanother pathway.\n2.Role of 7B2 downregulation and feedback loops\nThe observed downregulation of 7B2 in ERAD-deficient α cells suggests a contributing factor to impaired PC2 maturation.\nHowever, this conclusion is largely correlative. The authors are encouraged to assess whether exogenous 7B2 expression\ncan rescue PC2 activity or reduce proPC2 aggregation. This would provide functional support for the 7B2–PC2 axis as a\ndownstream effector of ERAD in α cells.\n3.Rule out the possible off-target effects of the Gcg-Cre model in NTS\nGiven the use of a Gcg-Cre model, the potential for off-target recombination in L-cells and the central nervous system (e.g.,\nNTS) must be considered carefully. While the authors reference prior studies and the absence of metabolic phenotypes,\nadditional lineage tracing or tissue-specific expression data would help rule out confounding effects.\nRecommendation: Major Revision"}, "author_response": "We thank the reviewer for these positive comments and for the feedback below,\nwhich has greatly improved our manuscript.\nMajor Concerns:\n1. Some of the important conclusions seem to be dependent on immunostaining data\nand/or use of antibodies with specificity that is not known or at least described.\nComplementary biochemical assays for identifying proglucagon forms such as Western\nblotting, and better characterization of antibody specificity should be provided, as per\nsome specific examples below:\nWe agree that a better description of antibody specificity is helpful and have added\nSupplementary Table S1 to summarize the key peptide antigen targets and provide\nreferences to representative publications outlining the generation and/or use of these\nantibodies. We have also added clarifying text to the figure legends where necessary,\noutlining the specific antibodies used.\n(a) total PC2 immunoreactivity is stated to be increased in Fig 4D in alpha cell Sel1L KO\nalpha cells, but this does not seem to match with the aTC KO western data in Fig 5\nwhere proPC2 is increased but total PC2 seems similar. It would be helpful to perform\nwesterns as well on the alpha cell Sel1L KO primary islets (contribution of beta cells to\nPC2 should be small) to assess proPC2 processing and total PC2 content, and\nquantification of total PC2 from westerns in Fig. 5.\nWe agree that this would be useful information. We have added quantification for\ncombined isoforms of (pro)PC2 in ΔSel1L and ΔHrd1 α cells in new Fig. S5a-b,\nwhich confirms that “total” PC2 is slightly increased in ΔSel1L cells.\nIn addition, we performed Western blotting on islet lysates from Sel1LΔGcg mice and\nlittermate controls and found a similar increase in expression of both proPC2 and\nproPC2*, reflecting accumulation of these isoforms as also observed in ΔSel1L αTC\ncells. We have added these data to Fig. S5c and added relevant text (pertaining to\nboth sets of new data) to the Results on page 8.\nNEW Figure S5a-c. a-b Quantification of combined proPC2 and proPC2* isoforms (a) and all (pro)PC2 isoforms\n(“Total PC2,” b) in αTC1-6 cells following CRISPR-mediated deletion of Sel1L (ΔSel1L) or Hrd1 (ΔHrd1)\ncompared to vector-treated αTC\ncontrols. c Western blot of isolated\nislet lysates from Sel1LΔGcg mice\nand Sel1Lfl/fl controls, labeled with\nantibodies to proPC2. Quantification\nof the protein bands normalized to\nHSP90 is shown at right. Each data\npoint in the graphs represents an\nindividual replicate. *P < 0.05, **P < 0.01, ***P < 0.001; NS, not\nsignificant (P > 0.05); unpaired two-\ntailed Student’s t-test or one-way\nANOVA with Šidák post-test.\nRevised text, page 8: “The combined increase in full-length proPC2 and the proPC2* fragment led to a slight\nincrease in the overall expression of proPC2-derived proteins in ΔSel1L α cells (Supplementary Fig. 5a-b),\nreflecting the pattern observed in Sel1LΔGcg islets. We confirmed that proPC2* expression was also increased in\nSel1LΔGcg islets by Western blot (Supplementary Fig. 5c).”\n(b) The proglucagon and glucagon antibodies used in immunostaining are not detailed or\ntheir cross-reactivity with other antibodies, and the field is known for cross-reactivity of\nthese antibodies with different molecular forms of proglucagon/glucagon derived\npeptides. Western blot of islet lysates from KO and control mice and mutated aTC cells to\nbetter understand the molecular forms of proglucagon peptides would be informative.\nThe reviewer raises an excellent point; we agree that these considerations for\nantibodies to proglucagon-derived peptides (PDPs) need to be taken into account.\nThe known targets for antibody generation are now listed in Table S1, along with\nreferences to published manuscripts using these antibodies. Although many\nantibodies to glucagon and GLP-1 are targeted to their mature sequences, we\nacknowledge a likely crossreactivity with the lesser-studied PDPs that also contain\nthese sequences (e.g., glicentin, oxyntomodulin, and the major proglucagon\nfragment) as well as the full-length proglucagon sequence.\nAs such, we attempted to detect the specific\nPDPs by Western blotting using the anti-glucagon\nantibodies that successfully labeled alpha cells by\nimmunofluorescence in our study (though noting\nthat none of these commercial antibodies were\npreviously validated for this method). We were\nunable to reliably detect the ~3 kDa mature\nglucagon by Western blotting using the antibodies\nfrom Cell Signaling Technology or BMA\nBiomedicals, though we did detect the full-length\nproglucagon band with the BMA anti-glucagon\nantibody (new Fig. S1c). Thus, we have revised\nall figures with immunofluorescence data to reflect\nthe detection of “(pro)glucagon” by this technique.\nNEW Figure S1c. Western blot of αTC cells with or without\nCRISPR-mediated inactivation of PC2 using the BMA Biomedicals T-5037 anti-glucagon antibody, revealing\nreliable detection of the full-length proglucagon protein but no detectable bands at the expected ~3 kDa location\nfor mature glucagon.\nWe remain confident in the specificity of the anti-proglucagon antibody given its lack\nof immunofluorescence in Gcg-/- (global knockout) mice (Fig. S1b). In support, this\nanti-proglucagon antibody has been used to label alpha cells in mice harboring a\nspecific deletion of the exons encoding mature glucagon, as typical anti-glucagon\nantibodies are nonreactive in this model (Tellez et al., Nat Metab 2020, PMC7739959;\nCoate et al., Mol Metab 2024, PMC11570739; both referenced in new Table S1).\nWhile the quantification of immunofluorescence images supports our primary claim\nthat SEL1L-HRD1 ERAD is essential to maintain glucagon production in alpha cells,\nwe emphasize that our strongest evidence is derived from quantitation of mature\nglucagon in serum and pancreas/islet extracts by ELISA. The Mercodia sandwich\nELISA kit in this work uses a dual antibody system to target both ends of the mature\nglucagon sequence and reports low cross-reactivity with mouse glicentin (7.0%),\noxyntomodulin (2.0%), and GLP-1 (not detected) (as reported by the company at\nhttps://www.mercodia.com/products/glucagon-elisa-10-µl/). Although there have been\nreports for overestimation of glucagon detection with this assay, this has mainly been\nobserved in human patients who have undergone bariatric surgery and have elevated\nsecretion of (gut-derived) PDPs like glicentin (Albrechtsen et al., Scand J Clin Lab\nInvest 2022, DOI: 10.1080/00365513.2021.2016943). We have confirmed that\npancreas extracts from Gcg-/- mice give negligible readings with this assay (new Fig.\nS1d; this point has been added to the Methods section on page 17).\nRevised text, page 17: “Note that negligible levels of glucagon and GLP-1 were detected in pancreas extracts or\nserum from Gcg-/- mice using these assays (Supplementary Fig. 1d).”\n2. PC2-glucagon link: The data indicating that proPC2 is an ERAD substrate is\nconvincing, but the evidence that accumulation of proPC2 and reduction in PC2 activity\ndue to ERAD disruption is the direct cause of loss of glucagon in these models would\nbenefit from additional supporting data or at least explanation, for example:\n(a) If ERAD knockout is causing significant PC2 activity loss, an increased accumulation\nof proglucagon should be evident, and demonstrated by western blot.\nWe first attempted to answer this question using our CRISPR-generated αTC\nknockout cells but unexpectedly found that proglucagon and glucagon are both\nreduced in this condition, for reasons that we are still exploring (data not shown).\nThus, we performed a new experiment using siRNA to acutely knock down SEL1L or\nHRD1 expression. In this experiment, we observed that acute ERAD dysfunction led\nto accumulation of both proPC2 and proPC2* (as observed in the CRISPR knockout\ncells), as well as accumulation of proglucagon. As discussed above, the detection of\nmature glucagon by Western blot proved technically challenging. These data are now\nshown in the new Fig. S7 with accompanying text on pages 9-10.\nNEW Figure S7. Control αTC cells were subjected to siRNA-mediated inactivation of Sel1L or Hrd1 (siSel1L or\nsiHrd1, respectively) and labeled for proteins as shown. a Representative Western blots and quantification of the\nERAD proteins SEL1L and HRD1, proPC2 isoforms (top middle), total PC2 isoforms (bottom middle), and the\nproPC2 chaperone 7B2. b siRNA-treated cells were evaluated for expression of proglucagon-derived peptides\nusing the ABclonal anti-glucagon antibody. As a control, note the lack of proglucagon-to-glucagon processing in\nαTC cells with CRISPR-mediated inactivation of PC2 (ΔPC2). Each protein band was normalized to the HSP90\nloading control, and each data point in the graphs represents an individual replicate. *P < 0.05, **P < 0.01, ***P\n< 0.001; NS, not significant (P > 0.05); Student’s t-test or one-way ANOVA with Šidák post-test.\nRevised text, pages 9-10: “To better understand the timing of proPC2 processing defects following ERAD\ndysfunction, we used an siRNA approach to acutely knock down SEL1L or HRD1 protein levels in control αTC\ncells (Supplementary Fig. 7). Similar to CRISPR-mediated inactivation of ERAD proteins in αTC cells, we\nobserved accumulation of proPC2 and proPC2* 48 hours after siRNA knockdown of these ERAD proteins\n(Supplementary Fig. 7a). We also observed accumulation of full-length proglucagon in siSel1L and siHrd1 cells\n(Supplementary Fig. 7b). Notably, we confirmed the profound accumulation of proglucagon and absence of\nmature glucagon formation in CRISPR-generated ΔPC2 cells, in which complete inactivation of functional PC2\ndisrupts proglucagon-to-glucagon conversion, as previously shown 42. These data further support the role of\nSEL1L-HRD1 ERAD in proPC2 maturation and subsequent PC2-mediated conversion of proglucagon into the\nmature glucagon peptide.”\n(b) The authors could strengthen their conclusions by showing that loss of PC2 activity\nwith PC2 deletion and/or 7B2 inhibitor alters proglucagon production / processing.\nThe inability of PC2-deficient cells to generate mature glucagon from the proglucagon\nprecursor is well known (Furuta et al., J Biol Chem 2001, DOI:\n10.1074/jbc.M103362200). Similarly, siRNA-mediated reduction in 7B2 has been\nshown to disrupt generation of glucagon through disruption of PC2 activity (Helwig et\nal., J Biol Chem 2011, PMC3234932). In our hands, generation of PC2 knockdown\nαTC cells (using CRISPR) also resulted in accumulation of proglucagon and absence\nof glucagon, which can be found in the new Figures S1c and S7b (copied in\nanswers above). We have added relevant text for these points on pages 9-10.\n(c) As detailed in 1 above, demonstration by western in their models that proglucagon\nprocessing is impaired, as immunostaining and ELISAs with available antibodies have\nunknown or questionable specificity.\nWe would kindly refer the reviewer to our discussion above regarding the Mercodia\nassay (Response 1b) and to our new siRNA experiment (outlined in Response 2a).\n(d) The decrease in total pancreatic glucagon and proglucagon shown in Fig. 3D does\nnot seem to account for the significant and near proportional loss of alpha cell mass in\nFig S3D. Could the loss of glucagon be simply related to loss of alpha cell mass?\nThe reviewer raises a great point. We agree that ERAD may have additional effects\non alpha cell biology, similar to what we reported for beta cells – where ERAD directly\ncontrols TGFβ signaling but also affects beta cell identity (Shrestha et al. J Clin Invest\ndecreased production of glucagon in Sel1LΔGcg mice, we believe this is unlikely to be\nsole cause. Our TEM data shows an ongoing presence of alpha cells with abnormal\nmorphology (i.e., dilated endoplasmic reticulum and fewer secretory granules; Fig.\nS3a), so it is possible that our immunofluorescence-based alpha cell mass\nmeasurement slightly underestimates the total alpha cell population. Our data\nsupports the current model of ERAD affecting proPC2 maturation and glucagon\ngeneration, but does not exclude other mechanisms. We have updated the\nDiscussion on page 14 to reflect these points.\nRevised text, page 14: “Although the current work demonstrates a primary role for ERAD in regulating proPC2\nmaturation in α cells, our data hint at other long-term effects of ER dysfunction in α cell biology. Our CRISPR-\ngenerated ERAD-deficient α cell lines unexpectedly showed lower Pcsk2 and Pcsk1 gene expression, which\nwas not observed following short-term inactivation of ERAD using an siRNA approach. These data suggest\nadditional long-term effects on α cell identity that mimic the “dedifferentiation” we observed in ERAD-deficient β\ncells 30. Similarly, we observed decreased α cell mass in older Sel1LΔGcg mice, which may reflect a progressive\ndecline in α cell number, though it is unclear if the immunofluorescence-based measurement underestimated the\nsize of the glucagon-deficient α cell population. It remains challenging to correlate α cell mass with systemic\nglucagon physiology, as it has been previously shown that near-total ablation of α cells induced by diphtheria\ntoxin dramatically reduced pancreatic glucagon content but had a lesser impact on systemic glucagon levels 71.\nThis suggests a robust capacity for normal α cells (e.g., those that escaped Cre-mediated recombination in our\nmodel) to maintain physiologic levels of glucagon secretion. As our female mice started with nearly double the\nglucagon content of male mice, similar to findings from other groups 72-74, we hypothesize that females have a\ngreater capacity to compensate for α cell dysfunction at younger ages, but (like the males) eventually lose the\nability to produce enough glucagon to mount a normal secretory response to hypoglycemia. Thus, there are\nseveral potential contributors to the glucagon production defect in Sel1LΔGcg mice beyond the observed\nmisprocessing of proPC2. The specific mechanisms underlying these additional effects remain to be explored.”\n(e) Why does the functional glucagon defect only manifest in older Sel1LΔGcg mice (4\nmonths male; 8 months female)? If a proPC2 processing defect is truly the cause of\nglucagon dysfunction, would the authors not expect the glucagon defect to be apparent\nearlier? One interpretation might be due to loss of alpha cells with decreasing with age\nassociated with ERAD disruption. The authors should assess alpha cell population in\nyounger mice. Further, performing cell viability and proliferation assays on the aged mice\nmight be influenced by a survivorship bias.\nThe reviewer raises another excellent point. It is known that a low number of alpha\ncells can be sufficient to maintain glucagon blood levels in mice, as near-total\nablation of alpha cells induced by diphtheria toxin dramatically reduced pancreatic\nglucagon content but had a lesser impact on systemic glucagon levels (Thorel et al.,\nDiabetes 2011, PMC3198058). This suggests a robust capacity for normal alpha cells\n(e.g., those that escaped Cre-mediated recombination in our model) to maintain\nglucagon secretory capacity, particularly as typical stimuli only induce exocytosis of a\nsmall portion of an islet’s cells granules stores.\nWe have added data from 11-week-old mice, which show no difference in glucagon\nsecretion in response to insulin-induced hypoglycemia (combined sexes, new Fig.\nS2h) and a statistically significant reduction in pancreatic glucagon content in male\nbut not female Sel1LΔGcg mice (new Fig. S2i). As our female mice started with nearly\ndouble the glucagon content of male mice (Fig. 3d), similar to findings from other\ngroups (Bonnevie-Nielsen, Metabolism 1980, DOI 10.1016/0026-0495(80)90014-1;\nLing et al., Virchows Arch 2001, DOI 10.1007/s004280000374; Jo et al., J Endocr\nSoc 2023, PMC10590649), we hypothesize that females have a greater capacity to\ncompensate for alpha cell dysfunction at younger ages, but (like the males)\neventually lose the ability to produce enough glucagon to mount a normal secretory\nresponse to hypoglycemia. Thus, we emphasize the difficulty in establishing a precise\ncorrelation between alpha cell mass and/or dysfunction with a systemic glucagon\nsecretory defect. Together, our data are most consistent with ERAD deficiency\nlimiting the total production of mature glucagon in affected cells, with the imperfect\nnature of Cre-lox recombination allowing for (temporary) compensation in glucagon\nsecretion in vivo. We have added these points to the Discussion on page 14 (text\ncopied in response above).\nNEW Figure S2h-i. h Serum glucagon values measured in vivo 30 minutes after insulin-induced hypoglycemia\nin combined male and female mice at 11 weeks of age. i Acid ethanol-extracted pancreatic glucagon content in\n11-week-old mice, with sexes as indicated. Each data point represents one mouse and bars showing mean ±\nSEM. * P < 0.05, NS, not significant (P > 0.05), unpaired two-tailed Student's t test.\n(f) Measurement (ELISA) of GLP-1 in ERAD deficient alpha cells would be informative.\nWhat happens to Pcsk1 / PC1/3 in ERAD deficient alpha cells? It would be of interest if\nthe defect was specific for PC2 and resulted in increased GLP-1 production, though this\nis not clear.\nWe agree that this is an important point. We have added new data (presented in\nFig. S6) to reinforce the finding that ERAD deficiency has a specific and direct effect\non (pro)PC2 maturation in alpha cells, and that any effect of ERAD dysfunction on\nPC1/3 protein expression and GLP-1 production is less pronounced. This new data is\ndiscussed on page 8.\nRevised text, page 8: “To explore whether the consequences of ERAD deficiency specifically affected proPC2\nmaturation and glucagon production in α cells, we next examined expression of the PC1/3 enzyme, which is\nprimarily responsible for generation of GLP-1 from proglucagon. By immunofluorescence, Sel1LΔGcg α cells\nshowed slightly reduced expression of both PC1/3 and GLP-1 (Supplementary Fig. 6a-b). Unlike proPC2, we did\nnot observe aggregation of PC1/3 under nonreducing conditions on Western blot (Supplementary Fig. 6c).\nInstead, the lower PC1/3 content was attributed in part to reduced expression of Pcsk1 mRNA in ΔSel1L α cells\n(Supplementary Fig. 6d). Pancreatic total GLP-1 content, as measured by immunoassay, was significantly\nreduced in adult male but not female Sel1LΔGcg mice (Supplementary Fig. 6e). Thus, ERAD deficiency\nspecifically inhibits maturation of proPC2 in α cells and imparts a lesser effect on PC1/3 expression and GLP-1\nproduction.”\nNEW Figure S6. a-b Representative islets from Sel1LΔGcg mice and Sel1Lfl/fl controls, immunolabeled for\nprohormone convertase 1/3 (PC1/3, a; RS20 antibody) or glucagon-like peptide 1 (GLP-1, b). Scale bars, 25\nμm. Violin plots in a’-b’ show the distribution of immunofluorescence intensity of PC1/3 or GLP-1 in glucagon+\ncells from 855-2029 cells with n = 2-3 mice per genotype, with median line shown in white and quartile lines in\nblack. c Western blot of islet lysates from Sel1lLfl/fl and Sel1LΔGcg mice prepared under reducing and\nnonreducing conditions, labeled with antibodies to PC1/3 (Cell Signaling Technology), with demonstration of\nhigh molecular weight (HMW) isoforms. Quantification of each isoform is shown in c’, with each data point\nrepresenting a single replicate, shown as mean ± SEM. d Quantitative RT-PCR of the Pcsk1 gene encoding\nPC1/3 in αTC cells. Each data point represents a single replicate, shown as mean ± SEM. e Acid ethanol-\nextracted pancreatic total GLP-1 content in adult (4-8 month-old) mice. Each data point represents data from\none mouse, with mean ± SEM shown. *P < 0.05, ***P < 0.001; ns, not significant (P > 0.05); Student’s t-test or\none-way ANOVA with Šidák post-test.\n(g) Does Pcsk2 mRNA expression change in the mutants?\nPcsk2 mRNA is not increased in ΔSel1L and ΔHrd1 αTC\ncells. This data can be found in Fig. 5h.\n3. Concerns with some figures:\n(a) Fig 1C – inclusion of insulin staining in top panel of 1C (control) would enable clearer\ncomparison with the Gcg KO panel below.\nAs BiP expression is low in normal islet alpha cells, we felt that this experiment\nshould have direct labeling of the alpha cells to confirm their location. Insulin labeling\nwas used in the Gcg-/- islets solely to demonstrate the peri-islet location of\n(pro)glucagon-negative alpha cells in this model. To better visualize the alpha cells in\nGcg-/- mice, we have added immunofluorescence data using adjacent tissue sections\nlabeled with the alpha cell marker transthyretin in the new Fig. 1c.\nUPDATED Figure 1c. Representative islets from Gcg‑/- mice and littermate controls, with adjacent tissues\nsections immunolabeled for the ER chaperone BiP (left) or the α cell marker transthyretin (right) and either\nglucagon (GCG, top panels; BMA Biomedicals) or insulin (INS, bottom panels).\n(b) representative images in Fig 2 could be more representative: in Fig 2C it is hard to\nsee BiP expression changes when there are so many more alpha cells in the control than\nin the Sel1L KOs; Fig 2D does not not convincingly show the slight but significant\ndecrease in proglucagon and glucagon levels in Sel1L knockouts reported by the\nquantification.\nWe have updated Fig. 2c-d with more representative islets that reflect the average\nintensity of BiP, proglucagon, and (pro)glucagon immunofluorescence:\nUPDATED Figure 2c-d. Representative islets from adult Sel1LΔGcg mice and Sel1Lfl/fl littermate controls,\nimmunolabeled for (pro)glucagon and the ER chaperone BiP (c) or proglucagon (d). Note that the BMA Biomedicals\nantibody targeting the mature glucagon sequence was found to label full-length proglucagon by Western blot (see\nSupplementary Fig. 1c), so images are labeled as detecting “(pro)glucagon” accordingly. Scale bars of left panels, 50\nμm. Violin plots show the distribution of immunofluorescence intensity of BiP, OS9, proglucagon, or glucagon in a\nrange of 277-1451 α cells from n = 3-4 mice per genotype. ***P < 0.001, unpaired two-tailed Student's t test.\n(c) The presence of a proPC2 band in the PC2 knockout condition is puzzling and would\nbenefit from explanation.\nWe thank the reviewer for pointing out a potential source of confusion. We believe\nthis simply reflects an incomplete CRISPR knockout. We have added clarifying text to\npage 8.\nRevised text, page 8: “note that PC2 was significantly reduced but incompletely deleted by this method”\nMinor\n1. The implication that SEL1 is not changed in type 2 diabetes should be softened since it\nis based on immunostaining from just one WT and one diabetic donor, or more donors\nincluded.\nWe apologize for any misunderstanding; our point was simply that SEL1L expression\nwas observed in alpha cells from islet donors with and without diabetes. We agree\nthat there is not sufficient data to make a conclusion on changes in expression in\ndiabetes. Our comment on this data can be found on page 5, at the end of the first\nResults paragraph.\nText, page 5: “SEL1L was expressed in both α and β cells in human islets, from donors with or without diabetes\n(Fig. 1g).”\n2. Please state the species of the homemade proPC2 antibody in the methods section.\nWe have included this information (antibody generated against mouse proPC2, host\nspecies rabbit) in the new Table S1.\n3. Quantification of OS9 expression in Fig. 2B in a manner similar to BiP in Fig. 2C to\nprovide would strengthen conclusion that it is upregulated.\nWe have performed quantification and added this data to Fig. 2 (see updated figure\ncopied in the response to Major Point 3b above).\nReviewer #2 (Remarks to the Author):\nThis manuscript by Zhu et al investigates the role of the SEL1L-HRD1 ERAD pathway in\nislet α cells, demonstrating that it is essential for the proper folding, maturation, and\nenzymatic activation of proPC2, a key enzyme in glucagon biogenesis. The authors\nprovide evidence that Sel1L and Hrd1 knockout in proglucagon-expressing cells leads to\naberrant proPC2 processing, accumulation of a truncated proPC2* species, decreased\nPC2 activity, and ultimately impaired proglucagon cleavage. These findings reveal a post-\ntranslational regulation in α cell biology and open potential therapeutic avenues in\nmetabolic diseases.\nThe paper is clearly structured: the introduction lays out the need for this study, methods\nare suitable to test their hypothesis, the data support the main conclusions, and\ndiscussions are thorough.\nSeveral mechanistic and contextual points should be addressed to solidify the\nconclusions.\nWe also thank this reviewer for their supportive comments and helpful feedback to\nimprove the manuscript.\nMajor concerns:\n1.Mechanism of proPC2* generation\nThe accumulation of proPC2* in ERAD-deficient α cells is intriguing and raises important\nquestions about its origin. While the proposed C-terminal cleavage is plausible, the exact\nmechanism by which proPC2* is generated remains unclear. Since proPC2* appears to\nbe a potentially important intermediate with pathological relevance, it would greatly\nstrengthen the study to provide further biochemical insight into how this fragment is\nformed—whether through a specific protease, misfolding, or another pathway.\nWe agree that the presence of proPC2* is an unexpected and exciting finding. The\nfact that proPC2* is increased in control αTC cells treated with the MG132\nproteasome inhibitor (Fig. 6d, lanes 1 versus 4) and is also observed in low levels in\ncontrol Sel1Lfl/fl islets (new Fig. S6c) suggests that proPC2* is actively generated in\nnormal alpha cells but is cleared by proteasomal degradation. We now show that\nproPC2* is sensitive to deglycosylation by Endo H and PNGase (new Fig. S5d),\nsuggesting that it is an immature protein. Thus, we conclude that proPC2* is an\nabnormal cleavage product generated in the ER of alpha cells (unlike typical proPC2\ncleavage in the secretory granules), and that nascent proPC2* is actively cleared by\nSEL1L-HRD1 ERAD. We are currently exploring the precise nature of this cleavage\nevent as an independent study and have expanded the Discussion on page 13 as\nsuch.\nNEW Figure S5d. Western blot of\nαTC, ΔSel1L, and ΔHrd1 cells before\nand after treatment with\nendoglycosidase H (Endo H) or\nPeptide:N-glycosidase F (PNGase F).\nRevised text, page 13: “Our data show that the effect of ERAD on proPC2 maturation is direct and uncoupled\nfrom ER stress-related protein misfolding, as chemical ER stressors failed to cause the accumulation of proPC2\nor the novel truncated proPC2* protein. Instead, proPC2 and proPC2* are SEL1L-HRD1 ERAD substrates that\nare susceptible to formation of high molecular weight protein aggregates via aberrant disulfide bonds in the\nabsence of ERAD function. The fact that we could detect proPC2* in control α cells after addition of MG132 and\nin isolated islets from Sel1Lfl/fl mice suggests that proPC2* is generated in normal α cells but is quickly degraded\nby the proteasome, through SEL1L-HRD1 ERAD. Based on the estimated molecular weight of proPC2*,\nwe\npredict that cleavage of proPC2 within the P domain leads to the generation of the truncated proPC2* protein;\nhowever, the specific mechanism producing proPC2* in the ER remains unclear. The neuroendocrine protein\n7B2 is necessary for formation of an activation-competent proPC2 complex that can exit the ER, and in its\nabsence proPC2 is susceptible to misfolding and aggregation in CHO cells 41. However, previous 7B2 structure-\nfunction and knockout analyses did not show evidence of proPC2* generation 41,61,62. Our siRNA and 7B2\noverexpression experiments demonstrated that excess 7B2 alone was insufficient to rescue the accumulation of\nproPC2(*) in ERAD-deficient α cells, which appears to be a rapid consequence of ERAD dysfunction. We\nspeculate that the reduction of available 7B2 cofactor in α cells with chronic ERAD deficiency exacerbates\nproPC2 aggregation within the ER, possibly by limiting export of nascent proPC2 41. Future studies will be\nrequired to clearly define the role of ERAD in the regulation of 7B2 expression and its functional consequences,\nas well as identifying the specific mechanism by which the aggregation-prone proPC2* peptide is generated.”\n2.Role of 7B2 downregulation and feedback loops\nThe observed downregulation of 7B2 in ERAD-deficient α cells suggests a contributing\nfactor to impaired PC2 maturation. However, this conclusion is largely correlative. The\nauthors are encouraged to assess whether exogenous 7B2 expression can rescue PC2\nactivity or reduce proPC2 aggregation. This would provide functional support for the 7B2–\nPC2 axis as a downstream effector of ERAD in α cells.\nWe thank the reviewer for encouraging a closer investigation into the specific role of\n7B2 in ERAD deficiency. The essential role for 7B2 in proPC2 maturation has been\nwell documented (summarized in Muller and Lindberg, Prog Nucleic Acid Res Mol\nBiol 1999, PMID: 10506829). Briefly, 7B2 limits aggregation and prevents premature\nactivation of proPC2, and is required to facilitate proPC2 transport through the\nsecretory pathway. Here, our initial data showed downregulation of 7B2 in αTC cells\nfollowing CRISPR-mediated ERAD inactivation (Fig. 6e). To better define the role of\n7B2 in ERAD deficiency without any confounding effects of long-term ER dysfunction,\nwe have now investigated 7B2 expression in cells with siRNA-mediated ERAD\ninactivation. While both siSel1L and siHrd1 cells also demonstrated proPC2\naccumulation, they showed an unexpected increase in 7B2 expression (new Fig.\nS8a). Furthermore, overexpression of 7B2 was unable to rescue the accumulation\nand aggregation of proPC2(*) in ΔSel1L and ΔHrd1 αTC cells (new Fig. S8),\nwhich\nfits with prior data showing that 7B2 facilitates ER export but not the initial folding of\nproPC2 (Muller et al., J Cell Biol 1997, PMC2141705). Thus, we conclude that\nproPC2(*) aggregation is a rapid and direct consequence of ERAD deficiency in\nalpha cells, independent of 7B2 levels. We have added relevant points regarding this\ndata to the Discussion on page 13.\nRevised text, page 13: “The neuroendocrine protein 7B2 is necessary for formation of an activation-competent\nproPC2 complex that can exit the ER, and in its absence proPC2 is susceptible to misfolding and aggregation in\nCHO cells 41. However, previous 7B2 structure-function and knockout analyses did not show evidence of\nproPC2* generation 41,61,62. Our siRNA and 7B2 overexpression experiments demonstrated that excess 7B2\nalone was insufficient to rescue the accumulation of proPC2(*) in ERAD-deficient α cells, which appears to be a\nrapid consequence of ERAD dysfunction. We speculate that the reduction of available 7B2 cofactor in α cells\nwith chronic ERAD deficiency exacerbates proPC2 aggregation within the ER, possibly by limiting export of\nnascent proPC2 41. Future studies will be required to clearly define the role of ERAD in the regulation of 7B2\nexpression and its functional consequences, as well as identifying the specific mechanism by which the\naggregation-prone proPC2* peptide is generated.”\nNEW Figure S8. a Representative Western blots and quantification of 7B2 and proPC2* in control and CRISPR-\ntreated ΔSel1L and ΔHrd1 cells that were transfected with a plasmid expressing rat 7B2. b Cells without and\nwith 7B2 overexpression were evaluated under reducing and non-reducing conditions to evaluate for high\nmolecular weight (HMW) forms of proPC2(*). Each protein band was normalized to HSP90 or GAPDH, and each\ndata point in the graphs represents an individual replicate. *P < 0.05, **P < 0.01, ***P < 0.001; only significant\nresults are shown. One-way ANOVA with Šidák post-test.\n3.Rule out the possible off-target effects of the Gcg-Cre model in NTS\nGiven the use of a Gcg-Cre model, the potential for off-target recombination in L-cells\nand the central nervous system (e.g., NTS) must be considered carefully. While the\nauthors reference prior studies and the absence of metabolic phenotypes,\nadditional\nlineage tracing or tissue-specific expression data would help rule out confounding effects.\nWe agree that these are important considerations, especially with the expectation\nthat these knock-in Gcg-iCre mice theoretically target recombination in all\nproglucagon-expressing cells. As shown in Fig. S2a-d, we were unable to observe\nevidence of Cre-mediated recombination in intestinal L-cells, with no detectable YFP\nreporter expression and no significant changes in BiP (an anticipated consequence of\nERAD deficiency). In addition to the finding that plasma and distal colon tissue levels\nof GLP-1 were unchanged (Fig. S2f-g), we strongly believe that any effect of SEL1L-\nHRD1 ERAD deficiency in L-cells has a minimal impact on systemic glucose\nphysiology.\nWe have since examined brain sections from Sel1LΔGcg mice and littermate (Cre\nnegative) controls. As shown in Response Fig. 1, we observed YFP reporter\nexpression in (pro)glucagon+ cells of the NTS, but without increased expression of\nthe ER marker KDEL. The lack of neurobehavioral or systemic metabolic phenotypes\nstrongly supports that the primary phenotype observed in this mouse model arises\nfrom ERAD deficiency in islet alpha cells. We have added relevant Discussion points\non page 12.\nResponse Figure 1. Immunohistochemistry of brain cryosections highlighting cells within the nucleus of the\nsolitary tract (NTS) labeled with antibodies to GFP (detecting the YFP reporter), (pro)glucagon, and the ER\nmarker KDEL.\nRevised text, page 12: “It is also unlikely that this model has impactful ERAD dysfunction in neurons given the\nlack of body weight changes or systemic glucose dysregulation as observed with manipulation of proglucagon\nexpression in the CNS 59 and the absence of neurologic dysfunction that we have observed in models inducing\nERAD dysfunction in the brain 27,60. Given that the mature glucagon peptide is derived nearly exclusively from\nislet α cells (excluding observations from pancreatectomized mammals), and given our primary finding of ERAD\ndeficiency causing a reduction in pancreatic and circulating glucagon levels, we believe that any impact of Cre-\nmediated recombination in L-cells or neurons in this model is minimal.”"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have done a thorough job of responding to this reviewer's concerns. While I still have minor reservations about\nthe (pro)glucagon antibodies and their specificity for different molecular forms, the authors have done about as much as can\nbe done with available resources and have described it well with new data and text.", "reviewer_2": "The authors have adequately addressed my concerns.I commend the authors for the high quality and scientific rigor of their\nwork. Overall, I find the study to make a valuable contribution to the field and recommend it for publication."}, "author_response": ""}]

Decision letter