110 Diabetes Volume 65, January 2016 Stefan Norlin,1 Vishal S. Parekh,1 Peter Naredi,2 and Helena Edlund1 Asna1/TRC40 Controls b-Cell Function and Endoplasmic Reticulum Homeostasis by Ensuring Retrograde Transport Diabetes 2016;65:110–119 | DOI: 10.2337/db15-0699 Type 2 diabetes (T2D) is characterized by insulin re- and misfolded proteins accumulate within the ER, and ER sistance and b-cell failure. Insulin resistance per se, stress develops, leading to activation of the unfolded however, does not provoke overt diabetes as long as protein response (UPR). During the development of type 2 compensatory b-cell function is maintained. The in- diabetes (T2D), pancreatic b-cells initially compensate creased demand for insulin stresses the b-cell endo- for insulin resistance successfully by increasing insulin plasmic reticulum (ER) and secretory pathway, and ER biosynthesis and secretion. However, conditions that b stress is associated with -cell failure in T2D. The tail lead to sustained ER stress (i.e., prolonged and persistent recognition complex (TRC) pathway, including Asna1/ insulin resistance and/or failure to reestablish proper ER TRC40, is implicated in the maintenance of endomem- homeostasis) are implicated in the deterioration of b-cell brane trafficking and ER homeostasis. To gain insight function and the development of overt diabetes (1–3). into the role of Asna1/TRC40 in maintaining endomem- Thus, identification of key molecules and factors that en- brane homeostasis and b-cell function, we inactivated b2/2 fi ISLET STUDIES Asna1 b Asna1 sure proper membrane traf cking and ER homeostasis, in -cells of mice. We show that mice b develop hypoinsulinemia, impaired insulin secretion, and thereby -cell function and survival, is important to and glucose intolerance that rapidly progresses to overt gaining insight into the etiology of T2D. diabetes. Loss of Asna1 function leads to perturbed In yeast, the Guided Entry of Tail-anchored proteins plasma membrane-to-trans Golgi network and Golgi- (GET) pathway (i.e., the tail recognition complex [TRC] to-ER retrograde transport as well as to ER stress in pathway equivalent) is associated with a broad range of b-cells. Of note, pharmacological inhibition of retrograde phenotypes (4–9). The GET complex has been suggested transport in isolated islets and insulinoma cells mim- to genetically associate with endomembrane trafficking icked the phenotype of Asna1b2/2 b-cells and resulted pathways (10,11), and inactivation of the GET pathway in reduced insulin content and ER stress. These data results in ER stress and activation of the UPR (12). Mech- support a model where Asna1 ensures retrograde anistic studies, primarily in cell-free systems, have sug- transport and, hence, ER and insulin homeostasis in gested a role for Get3 and the mammalian homolog b-cells. Asna1 (also known as TRC40) in delivering tail-anchored (TA) proteins for posttranslational insertion into the ER through the CAML/WRB receptor complex (13–16). In Secretory proteins (e.g., insulin) are inserted into the agreement with the proposed role for the GET/TRC path- endoplasmic reticulum (ER) where they are posttransla- way in membrane trafficking within the secretory path- tionally modified, folded, and then trafficked further way, key regulators of membrane-mediated transport and through the endomembrane system. If the protein load protein translocation (e.g., soluble NSF attachment pro- exceeds the protein folding capacity of the ER, unfolded tein receptors [SNAREs] such as Sec22b and Sed5 as well 1Umeå Centre for Molecular Medicine, Umeå University, Umeå, Sweden This article contains Supplementary Data online at http://diabetes 2Department of Surgery, Institute of Clinical Sciences, Sahlgrenska Academy, .diabetesjournals.org/lookup/suppl/doi:10.2337/db15-0699/-/DC1. University of Gothenburg, Gothenburg, Sweden © 2016 by the American Diabetes Association. Readers may use this article as Corresponding author: Helena Edlund, [email protected]. long as the work is properly cited, the use is educational and not for profit, and Received 25 May 2015 and accepted 28 September 2015. the work is not altered. diabetes.diabetesjournals.org Norlin and Associates 111 as Sec61b and RAMP4) have been proposed as protein using the DDCq method. TBP was used as an internal ref- clients for this pathway (17). Recently, Get3/Asna1 was erence gene. For detailed information, see Supplementary also shown to function, under oxidative stress conditions, Data. as a molecular chaperone that binds unfolded proteins to Cell Culture, Isolation, In Vitro Culture of Islets prevent their irreversible aggregation (18). In Caenorhab- Islet isolation and insulin secretion were performed ditis elegans, Asna1 function is required for larval growth essentially as previously described (24). For islet insulin and resistance to cisplatin, an oxidative stress–inducing secretion experiments, five equally sized islets were incu- anticancer drug (19,20). bated in CMRL-1066 (#21530; Gibco) supplemented with A mechanistic role for Asna1 in mammalian cells, 10% FBS (#10500; Gibco) at 37°C for 2 h. The islets were however, has not been functionally assessed in vivo equilibrated in ubiquitin (UB) buffer (2.8 mmol/L glucose, because global inactivation of Asna1 in mice results in em- 0.1% BSA) at 37°C for 1 h and then transferred to UB bryonic lethality (21). To explore a potential role for Asna1 buffer containing either 2.8 mmol/L glucose, 16.8 mmol/L in mammalian cells, we generated b-cell–specific Asna1 b2 2 b2 2 glucose, or 30 mmol/L KCl and incubated at 37°C for an mutant mice, denoted Asna1 / mice. Asna1 / mice additional 1 h (Supplementary Data). For Retro-2 treat- displayed pancreatic hypoinsulinemia, impaired insulin se- b2 2 ment experiments, MIN6 cells were passaged 1:3, cultured cretion, and early onset diabetes. b-Cells of Asna1 / for 48 h, and exposed to Retro-2 for 24 h, and islets were mice showed impaired retrograde transport, reduced cultured for 48 h after isolation and exposed to Retro-2 insulin content, and ER stress. Moreover, we show that for an additional 48 h. UB buffer (103) was prepared as Retro-2–mediated pharmacological inhibition of retrograde follows: NaCl 14.6 g, KCl 880 mg, CaCl2 Å; H O 376 mg, transport per se in isolated islets and insulinoma cells leads 2 MgCl Å; 6H O 488 mg, and HEPES 11.9 g was dissolved to decreased insulin content and ER stress. Thus, in addi- 2 2 in 200 mL H O. Upon dilution, pH was set at 7.35 and tion to identifying a role for Asna1 to ensure retrograde 2 0.1% BSA was added (ICN #105033, fatty acid free). transport as well as insulin and ER homeostasis in b-cells, the findings provide independent evidence for a role for Brefeldin A–Induced Retrograde and Anterograde retrograde transport in regulating b-cell function. Transport Assays Isolated islets were first incubated for 1 h in CMRL-1066 RESEARCH DESIGN AND METHODS supplemented with 10% FCS. For the COPI-independent Mouse Strains and Generation of Asna1flox Mice Golgi-to-ER retrograde transport assay, islets were then m A detailed description of the generation and genotyping transferred to media containing brefeldin A (BFA) 0.5 g/mL, fi of the conditional Asna1 allele is described in the Supple- and islets were removed and xed after 0, 2.5, 5, 10, mentary Data. Briefly, two loxP sites flanking exon 2 of 20, and 40 min. For the Golgi anterograde transport as- Asna1 were inserted by a recombination strategy essen- say, islets that had been incubated 1 h in CMRL-1066 tially as previously described (22). CRE recombinase– supplemented with 10% FCS were transferred to media mediated deletion of the intervening exon 2 is predicted with BFA and incubated for an additional 1 h. Islets were to result in translational termination after exon 1. ERAI then washed and incubated in medium without BFA, re- fi mice (23) were provided by the RIKEN BioResource Cen- moved, and xed after 0, 30, 45, 60, and 120 min. ter through the National BioResource Project of the Statistical Analyses MEXT, Japan. The animal studies were approved by the All numerical data are presented as mean 6 SEM. All Institutional Animal Care and Use Committee of Umeå statistical analyses were performed by heteroscedastic University and were conducted in accordance with the two-tailed Student t test. P , 0.05 was considered statis- guidelines for the care and use of laboratory animals. tically significant. Glucose Tolerance and Insulin Secretion Tests RESULTS Glucose tolerance test (GTT) and glucose-stimulated Loss of Asna1 Function in b-Cells Leads to Diabetes insulin secretion (GSIS) were performed on overnight- To elucidate the functional role of Asna1 in vivo, we – fasted (15 17 h) and sedated (Hypnorm and Dormicum) generated b-cell–specific deletion of Asna1 in mice by fl fl mice after intraperitoneal injection of glucose 2 g/kg body breeding Asna1 ox/ ox mice (Supplementary Fig. 1A–H) weight. Area under the curve (AUC) was calculated accord- with Ins1+/Cre mice (i.e., mice where the gene encoding ing to the trapezoidal rule (Supplementary Data). the CRE recombinase was inserted in one of the Ins1 Western Blot Analysis alleles [25,26]). Asna1 gene expression was progressively reduced between 2 and 4 weeks in islets of ;4-week-old Western blot expression data were normalized using b2/2 GAPDH, b-actin, or a-tubulin expression. For detailed Asna1 mice (Supplementary Fig. 2A), and conse- quently, Asna1 protein levels were decreased in information and antibodies, see Supplementary Data. b2 2 Asna1 / islets at 4 weeks (Supplementary Fig. 2B). Quantitative RT-PCR Analyses The residual Asna1 expression at 4 weeks likely predom- All quantitative RT-PCR (qRT-PCR) data are presented as inantly reflects expression in non-b islet cells, although we fold expression relative to the control sample and calculated cannot exclude a potential minor contribution from a few 112 Asna1 Ensures Retrograde Transport in b-Cells Diabetes Volume 65, January 2016 b-cells that had not yet fully deleted both Asna1 alleles at b2 2 this stage.