Stefan Norlin,1 Vishal S. Parekh,1 Peter Naredi,2 and Helena Edlund1

Asna1/TRC40 Controls b-Cell Functionand Endoplasmic ReticulumHomeostasis by Ensuring RetrogradeTransportDiabetes 2016;65:110–119 | DOI: 10.2337/db15-0699

Type 2 diabetes (T2D) is characterized by insulin re-sistance and b-cell failure. Insulin resistance per se,however, does not provoke overt diabetes as long ascompensatory b-cell function is maintained. The in-creased demand for insulin stresses the b-cell endo-plasmic reticulum (ER) and secretory pathway, and ERstress is associated with b-cell failure in T2D. The tailrecognition complex (TRC) pathway, including Asna1/TRC40, is implicated in the maintenance of endomem-brane trafficking and ER homeostasis. To gain insightinto the role of Asna1/TRC40 in maintaining endomem-brane homeostasis and b-cell function, we inactivatedAsna1 in b-cells of mice. We show that Asna1b2/2 micedevelop hypoinsulinemia, impaired insulin secretion,and glucose intolerance that rapidly progresses to overtdiabetes. Loss of Asna1 function leads to perturbedplasma membrane-to-trans Golgi network and Golgi-to-ER retrograde transport as well as to ER stress inb-cells. Of note, pharmacological inhibition of retrogradetransport in isolated islets and insulinoma cells mim-icked the phenotype of Asna1b2/2 b-cells and resultedin reduced insulin content and ER stress. These datasupport a model where Asna1 ensures retrogradetransport and, hence, ER and insulin homeostasis inb-cells.

Secretory proteins (e.g., insulin) are inserted into theendoplasmic reticulum (ER) where they are posttransla-tionally modified, folded, and then trafficked furtherthrough the endomembrane system. If the protein loadexceeds the protein folding capacity of the ER, unfolded

and misfolded proteins accumulate within the ER, and ERstress develops, leading to activation of the unfoldedprotein response (UPR). During the development of type 2diabetes (T2D), pancreatic b-cells initially compensatefor insulin resistance successfully by increasing insulinbiosynthesis and secretion. However, conditions thatlead to sustained ER stress (i.e., prolonged and persistentinsulin resistance and/or failure to reestablish proper ERhomeostasis) are implicated in the deterioration of b-cellfunction and the development of overt diabetes (1–3).Thus, identification of key molecules and factors that en-sure proper membrane trafficking and ER homeostasis,and thereby b-cell function and survival, is important togaining insight into the etiology of T2D.

In yeast, the Guided Entry of Tail-anchored proteins(GET) pathway (i.e., the tail recognition complex [TRC]pathway equivalent) is associated with a broad range ofphenotypes (4–9). The GET complex has been suggestedto genetically associate with endomembrane traffickingpathways (10,11), and inactivation of the GET pathwayresults in ER stress and activation of the UPR (12). Mech-anistic studies, primarily in cell-free systems, have sug-gested a role for Get3 and the mammalian homologAsna1 (also known as TRC40) in delivering tail-anchored(TA) proteins for posttranslational insertion into the ERthrough the CAML/WRB receptor complex (13–16). Inagreement with the proposed role for the GET/TRC path-way in membrane trafficking within the secretory path-way, key regulators of membrane-mediated transport andprotein translocation (e.g., soluble NSF attachment pro-tein receptors [SNAREs] such as Sec22b and Sed5 as well

1Umeå Centre for Molecular Medicine, Umeå University, Umeå, Sweden2Department of Surgery, Institute of Clinical Sciences, Sahlgrenska Academy,University of Gothenburg, Gothenburg, Sweden

Corresponding author: Helena Edlund, helena.edlund@umu.se.

Received 25 May 2015 and accepted 28 September 2015.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db15-0699/-/DC1.

© 2016 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, andthe work is not altered.

110 Diabetes Volume 65, January 2016

ISLETSTUDIES

as Sec61b and RAMP4) have been proposed as proteinclients for this pathway (17). Recently, Get3/Asna1 wasalso shown to function, under oxidative stress conditions,as a molecular chaperone that binds unfolded proteins toprevent their irreversible aggregation (18). In Caenorhab-ditis elegans, Asna1 function is required for larval growthand resistance to cisplatin, an oxidative stress–inducinganticancer drug (19,20).

A mechanistic role for Asna1 in mammalian cells,however, has not been functionally assessed in vivobecause global inactivation of Asna1 in mice results in em-bryonic lethality (21). To explore a potential role for Asna1in mammalian cells, we generated b-cell–specific Asna1mutant mice, denoted Asna1b2/2 mice. Asna1b2/2 micedisplayed pancreatic hypoinsulinemia, impaired insulin se-cretion, and early onset diabetes. b-Cells of Asna1b2/2

mice showed impaired retrograde transport, reducedinsulin content, and ER stress. Moreover, we show thatRetro-2–mediated pharmacological inhibition of retrogradetransport per se in isolated islets and insulinoma cells leadsto decreased insulin content and ER stress. Thus, in addi-tion to identifying a role for Asna1 to ensure retrogradetransport as well as insulin and ER homeostasis in b-cells,the findings provide independent evidence for a role forretrograde transport in regulating b-cell function.

RESEARCH DESIGN AND METHODS

Mouse Strains and Generation of Asna1flox MiceA detailed description of the generation and genotypingof the conditional Asna1 allele is described in the Supple-mentary Data. Briefly, two loxP sites flanking exon 2 ofAsna1 were inserted by a recombination strategy essen-tially as previously described (22). CRE recombinase–mediated deletion of the intervening exon 2 is predictedto result in translational termination after exon 1. ERAImice (23) were provided by the RIKEN BioResource Cen-ter through the National BioResource Project of theMEXT, Japan. The animal studies were approved by theInstitutional Animal Care and Use Committee of UmeåUniversity and were conducted in accordance with theguidelines for the care and use of laboratory animals.

Glucose Tolerance and Insulin Secretion TestsGlucose tolerance test (GTT) and glucose-stimulatedinsulin secretion (GSIS) were performed on overnight-fasted (15–17 h) and sedated (Hypnorm and Dormicum)mice after intraperitoneal injection of glucose 2 g/kg bodyweight. Area under the curve (AUC) was calculated accord-ing to the trapezoidal rule (Supplementary Data).

Western Blot AnalysisWestern blot expression data were normalized usingGAPDH, b-actin, or a-tubulin expression. For detailedinformation and antibodies, see Supplementary Data.

Quantitative RT-PCR AnalysesAll quantitative RT-PCR (qRT-PCR) data are presented asfold expression relative to the control sample and calculated

using the DDCq method. TBP was used as an internal ref-erence gene. For detailed information, see SupplementaryData.

Cell Culture, Isolation, In Vitro Culture of IsletsIslet isolation and insulin secretion were performedessentially as previously described (24). For islet insulinsecretion experiments, five equally sized islets were incu-bated in CMRL-1066 (#21530; Gibco) supplemented with10% FBS (#10500; Gibco) at 37°C for 2 h. The islets wereequilibrated in ubiquitin (UB) buffer (2.8 mmol/L glucose,0.1% BSA) at 37°C for 1 h and then transferred to UBbuffer containing either 2.8 mmol/L glucose, 16.8 mmol/Lglucose, or 30 mmol/L KCl and incubated at 37°C for anadditional 1 h (Supplementary Data). For Retro-2 treat-ment experiments, MIN6 cells were passaged 1:3, culturedfor 48 h, and exposed to Retro-2 for 24 h, and islets werecultured for 48 h after isolation and exposed to Retro-2for an additional 48 h. UB buffer (103) was prepared asfollows: NaCl 14.6 g, KCl 880 mg, CaCl2 Å; H2O 376 mg,MgCl2 Å; 6H2O 488 mg, and HEPES 11.9 g was dissolvedin 200 mL H2O. Upon dilution, pH was set at 7.35 and0.1% BSA was added (ICN #105033, fatty acid free).

Brefeldin A–Induced Retrograde and AnterogradeTransport AssaysIsolated islets were first incubated for 1 h in CMRL-1066supplemented with 10% FCS. For the COPI-independentGolgi-to-ER retrograde transport assay, islets were thentransferred to media containing brefeldin A (BFA) 0.5 mg/mL,and islets were removed and fixed after 0, 2.5, 5, 10,20, and 40 min. For the Golgi anterograde transport as-say, islets that had been incubated 1 h in CMRL-1066supplemented with 10% FCS were transferred to mediawith BFA and incubated for an additional 1 h. Islets werethen washed and incubated in medium without BFA, re-moved, and fixed after 0, 30, 45, 60, and 120 min.

Statistical AnalysesAll numerical data are presented as mean 6 SEM. Allstatistical analyses were performed by heteroscedastictwo-tailed Student t test. P , 0.05 was considered statis-tically significant.

RESULTS

Loss of Asna1 Function in b-Cells Leads to DiabetesTo elucidate the functional role of Asna1 in vivo, wegenerated b-cell–specific deletion of Asna1 in mice bybreeding Asna1flox/flox mice (Supplementary Fig. 1A–H)with Ins1+/Cre mice (i.e., mice where the gene encodingthe CRE recombinase was inserted in one of the Ins1alleles [25,26]). Asna1 gene expression was progressivelyreduced between 2 and 4 weeks in islets of ;4-week-oldAsna1b2/2 mice (Supplementary Fig. 2A), and conse-quently, Asna1 protein levels were decreased inAsna1b2/2 islets at 4 weeks (Supplementary Fig. 2B).The residual Asna1 expression at 4 weeks likely predom-inantly reflects expression in non-b islet cells, although wecannot exclude a potential minor contribution from a few

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b-cells that had not yet fully deleted both Asna1 alleles atthis stage. Nonfasted glucose levels of Asna1b2/2 micewere already mildly increased from ;2 weeks of age, andAsna1b2/2 mice progressed to overt diabetes between 6and 10 weeks of age (Fig. 1A). The increase in nonfastinghyperglycemia and development of diabetes was morepronounced in Asna1b2/2 males (Fig. 1A) than inAsna1b2/2 females (Supplementary Fig. 3). These findingsshow a requirement for Asna1 in the maintenance of b-cellfunction and glucose homeostasis. Subsequent experimentswere performed on 3- to 4-week-old mice (i.e., before theonset of overt diabetes) using a mix of males and females.

To assess how Asna1 ensures b-cell function, we ana-lyzed glucose tolerance and GSIS in response to exoge-nous, intraperitoneal administration of glucose (i.e.,GTT) in Asna1b2/2 mice. The targeted insertion of Creinto one of the Ins1 alleles, Ins1+/Cre; global inactivation ofone Asna1 flox allele, Asna1flox/2; or b-cell conditional in-activation of one Asna1 flox allele, Asna1b+/2, affectedneither glucose tolerance nor GSIS (Supplementary Fig.4A–F). Thus, Asna1b+/2, Asna1+/flox, Asna1flox/flox, andAsna1b+/2 mice, collectively denoted Asna1bctrl, wereall used as controls. In contrast to Asna1b+/2 litter-mates, 3-week-old Asna1b2/2 mice exhibited impaired glu-cose tolerance (Fig. 1B) as well as reduced insulinsecretion in response to both glucose (Fig. 1C) and theinsulin secretagogue arginine (Fig. 1D). Of note, glucosetolerance and insulin secretion were affected in bothmales and females at this stage (Supplementary Fig. 5A–F).Together, these data show that loss of Asna1 in b-cellsleads to diabetes due to impaired b-cell function.

Insulin Biogenesis Is Perturbed in Asna1b2/2 b-CellsTo elucidate the mechanism underlying b-cell failure inAsna1b2/2 mice, we next determined pancreatic islet areaand b-cell number. Pancreatic islet area and b-cell numberwere both normal (Fig. 2A and B), whereas pancreaticinsulin content was reduced by ;60% in Asna1b2/2

mice (Fig. 2C), suggesting that Asna1b2/2 mice developdiabetes as a consequence of reduced amounts of insulin.In mice, insulin is encoded by two highly homologousgenes Ins1 and Ins2, and as expected, Ins1 expressionwas nearly significantly (P = 0.055, n = 4) reduced by;50% in islets of mice carrying the Ins1Cre knockin allele(Supplementary Fig. 6A). However, because of the lowexpression of Ins1 relative to Ins2, the reduced Ins1 ex-pression did not in itself affect total insulin (Ins1 + Ins2)expression or insulin protein content in Ins1+/Cre mice(Supplementary Fig. 6A and B). Islet proinsulin and in-sulin content, however, was reduced by ;70% inAsna1b2/2 islets compared with Asna1b+/2 islets (Fig.2D) without a corresponding difference in total insulin(Ins1 + Ins2) mRNA levels (Supplementary Fig. 7), thusproviding evidence for a posttranscriptional reduction ininsulin biogenesis in b-cells of Asna1b2/2 mice. In con-trast, pancreatic content of the hormone islet amyloidpolypeptide, which is stored and cosecreted with insulin,

Figure 1—Asna1b2/2 mice develop diabetes. A: Nonfasted glucoselevels in Asna1bctrl and Asna1b2/2 males (n = 6–26). B and C: Bloodglucose (B) and plasma insulin (C) profiles and AUC (B and C)during GTT of 3-week-old Asna1b+/2 (n = 13) and Asna1b2/2 (n =12) mice. D: Plasma insulin secretion profiles and AUC of 4-week-old Asna1b+/2 (n = 12) and Asna1b2/2 (n = 14) mice during arginine-stimulated insulin secretion. Data are mean 6 SEM. *P < 0.05,**P < 0.01, ***P < 0.001 (Student t test). ns, not significant.

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was unaffected (Supplementary Fig. 7B). No significantdifference in islet hormone content was observed whencomparing males and females (Supplementary Fig. 7C).

The pancreatic proinsulin/insulin ratio was normal inAsna1b2/2 islets (Fig. 2E), and inhibition of protein deg-radation using proteasomal and lysosomal inhibitors hadsimilar effects on proinsulin and insulin content inAsna1b2/2 and Asna1b+/2 islets (Supplementary Fig. 8Aand B). These results argue against an increased rate of(pro)insulin degradation as the underlying mechanism forthe reduced insulin content in Asna1b2/2 b-cells. In agree-ment with the perturbed glucose tolerance and impairedglucose- and arginine-induced insulin secretion observed in

vivo, isolated Asna1b2/2 islets secreted less insulin in re-sponse to both glucose and the membrane depolarizer KCl(Fig. 2F). Normalization of the amount of secreted insulin-to-total insulin content, to adjust for the reduced insulincontent in Asna1b2/2 islets, however, revealed that therelative insulin secretion potential of Asna1b2/2 isletswas similar to that of control islets (Fig. 2G). Of note,plasma levels of insulin and proinsulin showed an increasedproinsulin/insulin ratio in Asna1b2/2 mice (Supplemen-tary Fig. 8C), suggesting that proinsulin secretion is rela-tively greater than insulin secretion in Asna1b2/2 mice.

The expression of genes involved in glucose uptake andmetabolism (i.e., Glut2 and Gck) was reduced by;70% and;30%, respectively (Supplementary Fig. 9A). The expres-sion of both Glut2 and Gck however, has been observed tobe negatively affected by hyperglycemia (27), leaving openthe possibility that the reduced expression of these genesin Asna1b2/2 islets may be secondary to the modesthyperglycemia observed at the stage of islet isolation(i.e., 3–4 weeks of age). The expression of genes involvedin membrane depolarization (Sur1 and Kir6.2) and in-sulin exocytosis (Rab3a, Rab3b, Rab27, Snap25, Syt7,Stx1a, Stx4a, Vamp2, and Vamp3) was essentially normalwith the exception of Rab3b expression, which was re-duced by ;40% (Supplementary Fig. 9B). Taken together,these findings suggest that Asna1b2/2 mice develop insulininsufficiency and diabetes largely as a consequence of im-paired insulin biogenesis, although it is possible that thereduced expression of Glut2, Gck, and Rab3b may contrib-ute to the impaired GSIS observed in vivo.

Loss of Asna1 in b-Cells Leads to ER StressOn a systemic level, Get3/Asna1 has been associated withmaintenance of ER homeostasis (10,12,28), thus consti-tuting a likely intersection point for Asna1 TA-targetingactivity and b-cell function. The expression and localiza-tion of markers for the ER-Golgi intermediate compart-ment (ERGIC53), cis Golgi (Gm130), trans Golgi network(TGN46), proinsulin vesicles, endosome (EEA1), and lyso-some (Lamp1) compartments all appeared normal inAsna1b2/2 islets (Fig. 3A), whereas the expression ofthe ER stress-response chaperones BiP (Grp78) andGrp94, as judged by KDEL immunostaining, appearedmore intense (Fig. 3A). Moreover, strong nuclear ATF4expression was observed (Fig. 3B) and IRE1a activity,monitored using ERAI reporter mice (23) on anAsna1b2/2 background, was enhanced (Fig. 3C). Addition-ally, transmission electron microscopy revealed dilated ERcisterna in a subset of Asna1b2/2 b-cells at 5–6 weeks ofage (Supplementary Fig. 9C). The expression of UPRgenes, including BiP (Hspa5, grp78), Grp94 (Hsp90b1),DnaJc3, Ero1lb, Erp29, Pdia4 (Erp72), Edem2, HRD,Herpud1, Sel1l, Atf3, Atf4, Chop10 (Ddit3), and Trib3, were,with the exception of Ero1lb, all increased in Asna1b2/2

islets (Fig. 3D). Together, these findings, particularly theactivation of all three branches of the UPR, suggest thatloss of Asna1 in b-cells leads to ER stress.

Figure 2—Reduced pancreatic insulin content in Asna1b2/2 mice.A: Quantification of pancreatic islet cell area in 3-week-oldAsna1bctrl (n = 4) and Asna1b2/2 (n = 4) mice. B: Quantificationof b-cell fraction in islets of 3-week-old Asna1bctrl (n = 5) andAsna1b2/2 (n = 5) mice. C: Total pancreatic insulin content in 3-week-old Asna1bctrl (n = 10) and Asna1b2/2 (n = 5) mice. D and E:Proinsulin and insulin content (D) and proinsulin/insulin ratio (E) inislets from 3–4-week-old Asna1+/2 and Asna1b2/2 mice (n = 6–7). Fand G: Insulin secretion from islets isolated from Asna1+/2 andAsna1b2/2 mice incubated at 2.8 and 16.8 mmol/L glucose and30 mmol/L KCl (F ) and insulin secretion normalized to islet insulincontent (G) (n = 3). Data are mean 6 SEM. *P < 0.05, **P < 0.01,***P < 0.001 (Student t test).

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The normal pancreatic proinsulin/insulin ratio (Fig.2E) together with the normal localization of proinsulin tovesicular structures adjacent to the trans Golgi network(TGN) in Asna1b2/2 b- cells (Fig. 3A) suggest that proin-sulin folding, transport, and processing are largely unaf-fected in Asna1b2/2 b-cells. Moreover, treatment of isletswith the chemical chaperones 4-PBA and TUDCA, which areknown to improve ER folding capacity (29,30), failed tonormalize islets proinsulin and insulin content (Supplemen-tary Fig. 10A and B) and to alleviate ER stress (Supplemen-tary Fig. 10C and D) in Asna1b2/2 islets. Taken together,these results argue against impaired folding of proinsulin asthe underlying cause for ER stress in Asna1b2/2 islets.

Loss of Asna1 Leads to Impaired RetrogradeTransport in b-CellsGet3/Asna1 has been suggested to genetically interactwith the retromer complex, the COG complex, and theCOPI coatomer complex, implying a potential role forGet3/Asna1 in retrograde transport (11). Retrogradetransport between the Golgi and the ER are mediatedby both COPI coatomer-coated vesicles and Rab6-dependent membrane tubules (31). To assess retrograde

transport in Asna1b2/2 islets, we first made use of BFA,which 1) inhibits COPI-dependent traffic by blocking theassembly of the COPI vesicle coat and 2) collapses theGolgi into the ER through the alternative Rab6-dependentroute (32). Similar to HeLa cells with impaired Rab6 ac-tivity (32,33), Asna1b2/2 islets showed a delayed and in-complete collapse of the Golgi compartment whenexposed to BFA (Fig. 4A and Supplementary Fig. 11Aand B). These data suggest that the Rab6-dependent ret-rograde transport route between the Golgi and ERis impaired in Asna1b2/2 b-cells. In contrast, rebuildingof the Golgi after BFA washout appeared largely unal-tered, suggesting that anterograde transport was normalin Asna1b2/2 islets (Fig. 4B).

We next assayed plasma membrane (PM)-to-TGN retro-grade transport by monitoring the uptake and transport offluorescently labeled Ricin toxin (594-RiTx), which is trans-ported through endosomes, TGN, and cis Golgi into the ERafter endocytosis (34). In dispersed b-cells from controlislets, 594-RiTx accumulated in the TGN after 90 min(Fig. 4C). In contrast, accumulation of 594-RiTx in theTGN was significantly reduced by 20% in Asna1b2/2 b-cells(Fig. 4C and D), providing evidence that PM-to-TGN

Figure 3—Loss of Asna1 in b-cells results in ER stress. A and B: Immunostaining of pancreatic sections from Asna1b+/2 and Asna1b2/2

mice using KDEL, Gm130 (green), ERGIC53 (red), TGN46 (red), proinsulin (green), EEA1 (green), and Lamp1 (red) antibodies (A) and ATF4antibodies (B). DAPI (blue) indicates nuclei. C: Activation of the Ire1a UPR pathway as monitored by ERAI-GFP reporter activity on anAsna1b+/2 and Asna1b2/2 background. D: qRT-PCR expression analyses of the indicated UPR genes in islets isolated from 3-week-oldAsna1bctrl and Asna1b2/2 mice (n = 6–8). Scale bar = 10 mm (A) and 25 mm (B and C). Data are mean 6 SEM. *P < 0.05, **P < 0.01, ***P <0.001 (Student t test). ERAD, ER-associated degradation; GFP, green fluorescent protein; Proins, proinsulin vesicles.

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retrograde transport is impaired in Asna1b2/2 b-cells. Theseresults demonstrate that Asna1 is required for sustainedretrograde transport to both the TGN and the ER.

Loss of Asna1 Leads to Mislocalization of SyntaxinsInvolved in Retrograde TransportVesicle transport and membrane recycling among the PM,endosome, and TGN critically depends on TA-SNAREs,such as syntaxin (Stx) 5, Stx6, and Vamp3 (35,36). Thus,

we reasoned that Asna1 might regulate retrograde trans-port by ensuring proper localization of Stx5, Stx6, and/orVamp3 to the cis Golgi, TGN, and endosomes, respec-tively. Consistent with this notion, Stx5, Stx6, andVamp3 proteins were barely detectable in Asna1b2/2

b-cells, whereas the localization of the Golgi TA proteingiantin was unaltered (Fig. 5A). The reduced Golgilocalization of Stx5 and Stx6 was already observed in;1-week-old Asna1b2/2 mice (Supplementary Fig. 12),whereas the loss of Vamp3 was observed first after3 weeks of age (Fig. 5A), suggesting that the loss ofVamp3 may be secondary to impaired retrograde trans-port and/or deterioration of b-cell function.

The protein levels of the major 35-kDa Golgi Stx5isoform and Stx6 were largely unaltered in Asna1b2/2

islets (Fig. 5B), although there was a tendency for theminor 42-kDa Stx5 ER isoform (37) to be slightly reduced.Together, these results show that Stx5 and Stx6 are notdegraded but likely become mislocalized or redistributedfrom their respective Golgi compartment in Asna1b2/2

b-cells. Stx5 was successfully coimmunoprecipitated whencoexpressed with Myc-tagged Asna1 in insulinoma celllines (Fig. 5C), leaving open the possibility that analo-gous to Get3/Asna1-Sed5 interaction (28), Asna1 medi-ates membrane insertion of Stx5 in b-cells. Thelocalization of other proposed Asna1 targets, such asthe ER resident proteins Sec61b and Sec22b (Fig. 5D)as well as the post-ER TA-SNAREs Vamp2 and Stx1a,which are implicated in insulin vesicle exocytosis (Fig.5E), revealed a normal localization and expression, arguingagainst a critical role for Asna1 in the biogenesis of theseTA proteins in b-cells. Taken together, these results pro-vide evidence of a requirement for Asna1 function in en-suring Golgi localization of Stx5 and Stx6 in b-cells.

Inhibition of Retrograde Transport in b-Cells Results inReduced Insulin Biogenesis and ER StressTo test whether the activation of the UPR and/or reducedinsulin content observed in Asna1b2/2 b-cells might besecondary to impaired retrograde transport, we used thesmall molecule inhibitor Retro-2 that inhibits retrogradetransport from early endosomes (EEs) to the TGN (38).Of note, analogous to the phenotype of Asna12/2 b-cells,exposure of isolated wild-type islets to Retro-2 resulted innot only reduced Golgi localization of Stx5 but also, albeitless pronounced, reduced Stx6 Golgi localization (Fig. 6A).However, as observed in Asna12/2 b-cells, the levels ofthe 35-kDa isoform of Stx5 were largely unaltered, whereasthe less abundant 42-kDa expression tended to be reducedin Retro-2–treated islets (Supplementary Fig. 13A). Thus,similar to that previously observed in HeLa cells (38), ex-posure of islets to Retro-2 results in a redistribution ofStx5 from the Golgi compartment. Moreover, and likeAsna12/2b-cells, COPI-independent Golgi-to-ER retro-grade transport was impaired in Retro-2–treated islets(Supplementary Fig. 13C–E). Because ex vivo cultivationof isolated islets in itself provokes ER stress, the functional

Figure 4—Retrograde transport is impaired in Asna1b2/2 islets. Aand B: Time course of retrograde collapse of Golgi into the ERfollowing exposure to BFA (A) and rebuilding of cis Golgi followingBFA washout (B) monitored by Gm130 staining (green) in Asna1b+/2

and Asna1b2/2 islets (n = 3). DAPI (blue) indicates nuclei. C and D:Colocalization analyses (C ) and quantification (D) of endocytosed594-Ricin toxin (red) and the TGN marker TGN46 (green) in dis-persed Asna1bctrl and Asna1b2/2 islets following a 90-min chase(n = 4–5). Arrowheads indicate Golgi remnants in A. Scale bar =10 mm (A and B) and 5 mm (C). Data are mean 6 SEM. **P < 0.01(Student t test).

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effects of Retro-2 on UPR were assessed on day 2 after iso-lation (i.e., at a stage when the acute activation of ERstress genes is somewhat dampened) (SupplementaryFig. 14). Retro-2–exposed islets displayed reduced insulincontent by ;30% and enhanced expression of UPR genes,indicating ER stress (Fig. 6B and C). Additionally, insulinexpression was significantly reduced in Retro-2–exposedislets (Fig. 6D), thus likely contributing to the observedreduction in insulin content in these islets. The subcellu-lar location of Stx5 was unaffected by the ER stress/UPRactivators tunicamycin and thapsigargin (SupplementaryFig. 15), providing evidence that the mislocalization ofStx5 is not secondary to ER stress.

To independently assess the effects of Retro-2 oninsulin biogenesis and UPR activation in b-cells, obviatingpotential confounding effects of ER stress induced by iso-lation and ex vivo cultivation of islets, we next exposedMIN6 cells to Retro-2. Exposure of MIN6 cells to Retro-2also resulted in reduced Golgi localization, but not expres-sion, of Stx5 (Fig. 6E and Supplementary Fig. 13B),reduced insulin content, and enhanced expression ofUPR genes (Fig. 6F and G). However, although insulincontent was reduced by ;30% in Retro-2–treated MIN6

cells, insulin expression was not significantly reduced (Fig.6H). Taken together, these data show that inhibition ofretrograde transport in b-cells results in ER stress andimpaired insulin biogenesis. Additionally, these findingsprovide evidence that the reduced insulin content and ERstress observed in Asna1-deficient b-cells are a consequenceof impaired PM/EE-to-TGN retrograde transport.

DISCUSSION

This study shows a critical role for Asna1 in ensuringb-cell function. Loss of Asna1 in b-cells of mice resultsin pancreatic hypoinsulinemia, impaired insulin secretion,and early onset diabetes. Additionally, b-cells ofAsna1b2/2 mice showed impaired PM-to-TGN as wellas Golgi-to-ER retrograde transport, ER stress, and mis-localization of Stx5 and Stx6. Of note, we also showthat inhibition of retrograde transport at the level ofEE-to-TGN in isolated islet and insulinoma cells resultsin impaired Golgi-to-ER retrograde transport, decreasedinsulin content, and ER stress. Thus, the findings provideevidence that Asna1 is required in b-cells to ensure retro-grade transport, which in turn appears to be essential for ERhomeostasis and proinsulin biogenesis. Additionally, the

Figure 5—Stx5 and Stx6 are mislocalized in Asna1b2/2 islets. A: Immunostaining of pancreatic sections from Asna1b+/2 and Asna1b2/2

mice using antibodies against Stx5 (red), giantin (red), Gm130 (green), Stx6 (green), TGN46 (red), insulin (green), and Vamp3 (red). DAPI(blue) indicates nuclei (n = 3). Insets show individual color channels of selected regions (boxes). *Non-b-cells. B: Representative immuno-blots and quantification of Stx5 (35- and 42-kDa isoforms) and Stx6 protein levels in islets isolated from 4–5-week-old Asna1bctrl andAsna1b2/2 mice (n = 3). C: Representative immunoblots showing anti-Myc immunoprecipitation of Myc-tagged Asna1 (top panel: Asna1 [39kDa], Myc-Asna1 [40 kDa] and coprecipitation of Stx5; bottom panel: 35- and 42-kDa Stx5 isoforms) from MIN6 cells transfected with Myc-tagged Asna1 or LacZ and Stx5 constructs as indicated (n = 3). Dashed lines indicate filter cuts. D and E: Immunostaining of pancreaticsections from Asna1b+/2 and Asna1b2/2 mice with Sec61b (red), Sec22b (red) (D), and Gm130 (green) and Stx1a (red), insulin (red), andVamp2 (green) (E ) antibodies. Scale bar = 10 mm. au, arbitrary unit; IB, immunoblot; IP, immunoprecipitation.

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perturbed Golgi-to-ER retrograde transport in Retro-2–treated primary islets suggests that the impairment of thisstep in Asna1b2/2 b-cells likely is secondary to the inhibi-tion of retrograde transport at the level of EE-to-TGN.

The primary cause of diabetes in Asna1b2/2 mice ap-pears to be insufficient production of insulin. The currentdata do not support a role for Asna1 in the posttransla-tional targeting of insulin itself, such as has been de-scribed for other short secretory proteins (39), becausewe did not observe an increase or alteration in pancreaticproinsulin levels. Although the activation of the UPR in-dicates that ER homeostasis is perturbed in the absence ofAsna1 function, we found no evidence of impaired pro-insulin folding or anterograde transport through the secre-tory pathway. Moreover, vesicular proinsulin localizationappears unaltered in Asna1b2/2 b-cells, pancreatic isletamyloid polypeptide content is unaffected, and insulinmaturation as well as exocytosis seem largely unaffected.Thus, activation of the UPR is likely not provoked by

insufficient insulin folding capacity. Of note, we observedan increased plasma proinsulin/insulin ratio, suggestingthat secretion of newly synthesized proinsulin is in-creased and raising the possibility that Asna1-dependentEE-to-TGN retrograde transport normally counteractsleakage or premature secretion of proinsulin throughthe endosomes and the constitutive-like pathway (40).

Instead, we favor the idea that impaired retrogradetransport in Asna1b2/2 mice provokes reduced insulinbiogenesis as a negative consequence of UPR activationon protein translation (41). Additionally, there was atendency, albeit nonsignificant, for insulin expressionlevels to be reduced in Asna1b2/2 mice, which wouldalso be consistent with a negative effect of UPR activa-tion (e.g., through IRE1a/XBP1 and Trib3 induction, oninsulin transcription [42,43], or as a consequence ofIRE1a/XBP1–mediated degradation of insulin mRNA[44,45]). Moreover, Retro-2–mediated impairment ofretrograde transport resulted in robust activation of

Figure 6—Pharmacological inhibition of EE-to-TGN retrograde transport in islets and insulinoma cells mimics Asna1b2/2 phenotypes. A:Immunostaining of islet cells incubated with vehicle (DMSO) and Retro-2 (50 mmol/L) for 48 h using antibodies Stx5 (red), Gm130 (green),Stx6 (green), and TGN46 (red) (n = 4). Insets show individual color channels of selected regions (boxes). B: qRT-PCR analyses of UPR geneexpression in islets incubated with vehicle (DMSO) and Retro-2 (50 mmol/L) for 48 h (n = 4). C and D: Insulin protein content (C) and qRT-PCR analysis of insulin expression (D) of islets incubated with vehicle (DMSO) and Retro-2 (50 mmol/L) for 48 h (n = 5). E: Immunocyto-chemical staining of MIN6 cells incubated with vehicle (DMSO) and Retro-2 (80 mmol/L) for 24 h using antibodies against Stx5 (red) andGm130 (green) (n = 4). F: qRT-PCR analysis of UPR gene expression in MIN6 cells incubated with vehicle (DMSO) and Retro-2 (80 mmol/L)for 24 h (n = 4). G and H: Insulin protein content (G) and qRT-PCR of insulin expression (H) of MIN6 cells incubated with vehicle (DMSO) andRetro-2 (80 mmol/L) for 24 h (n = 4). DAPI (blue) indicates nuclei (A and E ). Scale bar = 10 mm (A) and 50 mm (E). Data are mean6 SEM. *P<0.05, **P < 0.01, ***P < 0.001 (Student t test).

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UPR genes and decreased insulin protein content in bothisolated islets and MIN6 cells and were accompanied,albeit to a different extent, by suppression of insulintranscription. Thus, although we cannot exclude thatRetro-2 has additional UPR-independent effects on in-sulin biogenesis, the reduced insulin content observed inRetro-2–treated islets and MIN6 cells appears to be sec-ondary to UPR-mediated impairment of insulin biogen-esis at both the transcriptional and the translationallevel. These findings also provide strong evidence thatimpairment of retrograde transport in b-cells by Retro-2in vitro and ex vivo and due to loss of Asna1 function invivo leads to activation of the UPR that in turn nega-tively affects insulin biogenesis, which, in vivo, results ininsulin insufficiency and the development of diabetes(Supplementary Fig. 16).

Previous studies, primarily in cell-free systems, haveoutlined a role for Get3/Asna1/Asna1 in the targeting ofTA proteins to the ER membrane receptor Get1/Get2(CAML/WRB in mammals), thus facilitating their in-sertion into the ER membrane (15,16). The current dataindicate that at least in b-cells, Golgi localization of theTA-SNARE proteins Stx5 and Stx6 depend on Asna1 func-tion. Total levels of Stx5 and Stx6 mRNA and proteinwere unaltered in Asna1b2/2 b-cells, suggesting thatStx5 and Stx6 become redistributed and/or mislocalizedin the absence of Asna1 activity. In contrast, the localiza-tion of the TA proteins Sec61b, Sec22b, giantin, Stx1, andVamp2 was unaffected, suggesting that other chaperonesystems, such as the Hsc70/Hsp40 pair (17), compensatefor the targeting of these TA proteins in b-cells lackingAsna1 function. In agreement with such a notion, theyeast TA proteins Sbh1 and Sbh2 (i.e., the homologs ofSec61b) and Scs2 and Ysy6 all retain a certain level of ERlocalization in yeast GET1/GET2 mutants (28). Consistentwith a potential direct role for Asna1 in the targeting ofStx5 to the ER of b-cells, and like Get3/Asna1-Sed5 in-teractions, we found that Asna1 physically interacts withStx5 in insulinoma cells upon coexpression of these pro-teins. We cannot, however, exclude the possibility thatStx5 and Stx6 are appropriately targeted in the absenceof Asna1 activity but become mislocalized or redistributedto other cellular compartments as a consequence of im-paired retrograde transport.

Exposure of MIN6 insulinoma cells and isolated isletsto the small molecule inhibitor Retro-2 closely mimicskey phenotypes of Asna1b2/2 b-cells, including 1) re-duced insulin content, 2) activated UPR, and 3) redis-tributed Stx5 and Stx6. Although the molecular targetsof Retro-2 are unknown and at what point the Retro-2and Asna1 pathways intersect is unclear, the reduction ofStx5 in the cis Golgi and Stx6 in the TGN is likely to affectAsna1-dependent retrograde transport. Small interferingRNA–mediated knockdown of Stx5 and inhibition of Stx6using blocking antibodies have both been shown to neg-atively affect EE-to-TGN recycling (35,36). However, twoobservations argue for a primary role for Stx5 in the

context of retrograde transport and ER homeostasis.First, the effect of Retro-2 on Stx5 localization is acuteand complete, whereas the effect on Stx6 localization isslower and less severe (Fig. 6A) (38), suggesting that Stx6mislocalization may be secondary to the loss of Stx5 fromthe Golgi. Second, in yeast Get3/Asna1 mutants, Sed5 ismislocalized, and the Bip/grp78 ER chaperone homologKar2 is abnormally secreted (10,28), which may reflect animpaired retention or retrograde transport of Kar2 (10).Importantly, overexpression of the Stx5 homolog Sed5 inGet3/Asna1 mutants rescues the Kar2 (Bip) secretion phe-notype (28). Taken together, these data are consistentwith a chain of events where inactivation of Asna1/Get3, likely through mislocalization of Stx5/Sed5, per-turbs retrograde transport and, thereby, ER homeosta-sis. In mammalian b-cells, impairment of retrogradetransport appears to have the additional consequenceof attenuating insulin biogenesis, thus leading to thedevelopment of diabetes.

Recently, an additional function distinct from its TAprotein–targeting activity was described for Get3/Asna1(18). Under oxidative stress conditions and independentof ATP, Get3/Asna1 was shown to function as a molecularchaperone that binds unfolded proteins to prevent theirirreversible aggregation. The proposed dual role for Get3/Asna1 in yeast is intriguing and implies a potential role formaintenance of both ER and redox homeostasis. Futurestudies are required to separate potential oxidative stress–induced chaperone activity of Asna1 from the TA protein–targeting function to fully understand how Asna1 ensuresretrograde transport as well as insulin and ER homeostasis.

In conclusion, we show that Asna1 is critical for b-cellfunction. The results provide evidence that Asna1 plays arole in ensuring retrograde transport along a PM-to-TGNand a Golgi-to-ER route. Impairment of these functions inb-cells leads to ER stress, insulin insufficiency, and devel-opment of diabetes. The findings suggest that maintenanceand/or restoration of retrograde transport in b-cells maybe therapeutically relevant for T2D. The study was per-formed, however, using genetically modified mice and iso-lated mouse islets. Thus, although the data provide strongevidence for a role for Asna1 and retrograde transport inensuring mouse b-cell function, a potential similar role forASNA1 and/or retrograde transport for b-cell function inhumans will require additional analyses.

Acknowledgments. The authors thank Elisabet Pålsson, JurateStraseviciene, Fredrik Backlund, and Lisa Lundberg (Umeå Centre for Mo-lecular Medicine) for technical assistance and members of the authors’laboratory for technical instructions, suggestions, and helpful discussions.Funding. These studies were facilitated by support from the StrategicResearch Program in Diabetes at Umeå University and supported by grants fromthe Swedish Research Council (521-2013-3215) and the Knut and Alice Wallen-berg Foundation (KAW 2010.0033).Duality of Interest. H.E. is a cofounder, shareholder, and consultant of theunlisted biotech company Betagenon AB. No other potential conflicts of interestrelevant to this article were reported.

118 Asna1 Ensures Retrograde Transport in b-Cells Diabetes Volume 65, January 2016

Author Contributions. S.N. and V.S.P. contributed to the design andperformance of experiments, data interpretation, discussion, and writing andediting of the manuscript. P.N. initiated the study, provided advice, and contrib-uted to the discussion and review of the manuscript. H.E. initiated the study,designed and supervised the study, analyzed and interpreted the data, contrib-uted to the discussion, and wrote the manuscript. H.E is the guarantor of thiswork and, as such, had full access to all the data in the study and takesresponsibility for the integrity of the data and the accuracy of the data analysis.

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