INS-1 832/13 insulin-secreting beta cells were obtained from Dr. Christopher Newgard (Duke University). The 832/13 cells were cultured in RPMI-1640 (Mediatech Cellgro) supplemented with 11 mM glucose, 10% fetal bovine serum, 10 mM HEPES, 1 mM sodium pyruvate, 50 μM β-mercaptoethanol and Antibiotic Antimycotic Solution (Sigma) at 37°C in 5% CO2, 95% air. The cells were sub-cultured twice weekly. For determining relative proliferation, BrdU (1 mM) was added to the cell for 1 hr followed by permeabilization and fixation for 10 minutes with 4% formaldehyde, 0.1% Triton x-100 in PBS. The cells were then denatured with 1N HCL for 30 mins, and anti-BrdU (1:50, DAKO) applied for 1 hour. For estimating cell death the DeadEndTM Fluorometric TUNEL System (Promega, Inc.) was used. Cells were fixed in 4% methanol-free formaldehyde solution for 10 min, permeabilized with 0.2% Triton X-100 solution for 5 minutes, and labeled according to instructions of the kit.
Proinsulin content, insulin content and insulin secretion
After 48 hr post-transduction, 832/13 cell pellets were sonicated in 1 mol/l acetic acid containing 0.1% bovine serum albumin. Aliquots of cell extracts were assayed for proinsulin and insulin content using proinsulin EIA (ALPCO) and Mouse Insulin Ultrasensitive EIA (ALPCO), respectively. The data were normalized to DNA content. Insulin secretion was assayed at 3 mmol and 15 mmol/l glucose. For KATP channel dependent secretion assay, 200 μmol/l tolbutamide or 250 μmol/l diazoxide were added.
Plasmids and adenovirus production
The c-myc tagged dominant-negative mouse Perk transgene (PERKΔC) was kindly provided by Dr. David Ron (New York University). PERKΔC was generated by deleting the kinase domain of Perk residing in the carboxy terminal coding sequence, retaining the amino terminal ER luminal domain (Harding, 1999). The PERKΔC transgene was excised by us from the parent vector by digestion with Spe 1 and Xho 1 and then inserted to the Adenovirus vector pShuttle-IERS-hrGFP-1 (Stratagene). The resulting construct, which we denote as AdDNPerk, was transfected into AD-293 cells using the MBS Mammalian Transfection kit (Stratagene) for amplification. The adenovirus was then purified by Adeno-X™ Virus Purification kit (Clontech) and the titer was determined by the QuickTiter™ Adenovirus Titer Immunoasssay Kit (Cell Biolabs, Inc.). The pShuttle-CMV-lacZ transgene, denoted herein as AdLacZ, served as a control. Transduction of 832/13 cells was carried out at a cell confluency of about 70%, at different multiplicities of infection (MOI) from 5 to 20. A MOI of 20 was used in most experiments unless otherwise stated. After 2 hours of incubation with the virus at 37°C, 5% CO2, cells were washed once in RPMI and cultured for an additional 6-48 h before assay.
To assess ATF6 processing the expression plasmid pCMVshort-EYFP-ATF6-alpha, provided by Kazutoshi Mori, was transfected into AD293 cells.
The Perk knockout strain was previously generated by us  and are congenic for C57BL/6J or 129SvEvTac. Both congenic strains exhibited all of the beta cell defects previously described .
The pancreata from E18.5 to p2 mice were inflated in situ by injecting 3 mg/ml Collagenase P (Invitrogen) in HBSS (Sigma) and digestion was carried out at 37°C for about 12 mins at which time 1 ml of ice-cold HBSS was added to stop the enzyme reactions. The dispersed pancreatic tissue was pelleted and washed once with HBSS. The pellets were then suspended in 1 ml Histopaque-1077, overlaid with 0.2 ml RPMI 1640 medium and centrifuged at 890 g for 12 min to separate the islet from the other cell types and residual debris. Islets were manually picked under a dissection microscope and washed once with ice-cold HBSS.
Gene expression levels
Quantification of gene expression was carried out by using qPCR Core Kit for SYBR Green I (Eurogentec) amplifying cDNA with the ABI Prism 7000 Sequence Detection System. The following cycling conditions were used for all primer pairs: 50°C (2 minutes), 95°C (10 minutes), 40 cycles of (95°C [15 seconds], 60°C [1 minute]). Levels of Xbp1-s (spliced form) were normalized to Xbp1-t (total) levels. All other mRNAs were normalized to the levels of actin and/or GAPDH. The mouse primer sequences were as follows: actin 5'-GCCCTGAGGCTCTTTTCC-3', 5'-TGCCACAGGATTCCATACCC-3'; GAPDH 5'-GGAGCGAGACCCCACTAACA-3', 5'-ACATACTCAGCACCGGCCTC-3'; Insulin I 5'-AGCATCTTTGTGGTCCCCAC-3', 5'-CCCCACACACCAGGTAA-3'; Insulin II 5'-CAGAAGCGTGGCATTGTAGA-3', 5'-TTGCAGTAGTTCTCCAGCTGG-3'; MafA, 5'-GCTGGTATCCATGTCCGTGC-3', 5'-GTCGGATGACCTCCTCCTTG-3'; Pdx-1, 5'-GAGCGTTCCAATACGGACCA-3',5'-TCAGCCGTTCTGTTTCTGGG-3'; ERP72, 5'-TTCCACGTGATGGATGTTCAG-3', 5'-AGTCTTACGATGGCCCACCA-3'; ERP57, 5'-GGCGGATGCAACATATCACC-3', 5'-TGTGGTTCGTACTGTCCCCC-3'; ERP58, 5'-CAAGAGGCTTGCCCCTGAG-3', 5'-GGTGTTTGTGTTGGCAGTGC-3'; Hrd1, 5'-TGGCTTTGAGTACGCCATTCT-3', 5'-CCACGGAGTGCAGCACATAC-3'; Ero1 L, 5'-AAACCCTGCCATTCTGATGAA-3', 5'-ACTCATCCACGGCTCCAA GT-3'; Ero1 beta, 5'-TGATTCGCAGGACCACTTTTG-3', 5'-TAGCCAGTGTACCGTTCCGG-3'; Herpud1, 5'-CCCACCTGAGCCGAGTCTAC-3', 5'-CTTGGAGACACTGGTGATCCAA-3'; ATF3, 5'-CCT ATGCAAAGCAGGATCCC-3', 5'-GCGTTGTCAGCCACAGTGG-3'; GRP94, 5'-CTGGGTCAAGCAGAAAGGAG-3' 5'-TCTCTGTTGCTTCCCGACTT-3'; BiP, 5'-GCTTCGTGTCTCCTCCTGAC-3', 5'-TAGGAGTCCAGCAACAGGCT-3'; Chop, 5'-CCAACAGAGGTCACACGCAC-3', 5'-TGACTGGAATCTGGAGAGCGA-3'; Xbp1-spliced, 5'-GAGTCCGCAGCAGGTG-3', 5'-GTGTCAGAGTCCATGGGA-3'; Xbp1-total, 5'-CACCTTCTTGCCTGCTGGAC-3', 5'-GGGAGCCCTCATATCCACAGT-3'. The rat primer sequences were as follows: Actin, 5'-ATC CTG GCC TCA CTG TCC AC-3', 5'-CTA GAA GCA TTT GCG GTG CA-3'; GAPDH, 5'-CACCACCAACTGCTTAGCCC-3', 5'-TGGCATGGACTGTGGTCATG-3'; Insulin I, 5'-CAGCACCTTTGTGGTCCTCA-3', 5'-CCCACACACCAGGTACAGAGC-3'; Insulin II, 5'-CTGCCCAGGCTTTTGTCAAA-3', 5'-CTTCCACCAAGTGAGAACCACA-3'; MafA, 5'-GGCACATTCTGGAGAGCGA-3', 5'-CCCGCCAACTTCTCGTATTTC-3'; Pdx-1, 5'-CCACCAAAGCTCACGCGT-3', 5'-CTGCGTATGCACCTCCTGC-3'; NeuroD, 5'-GCTTGAAGCCATGAATGCAG-3', 5'-TCCTCTCCCCCATTTCTCAGA-3'; ERp72, 5'-TCTAACCAATCACCGGGCTG-3',5'-TCATGGTAAGGTGCCGAGG-3'; BiP, 5'-ACCCTTACTCGGGCCAAATT-3', 5'-AGAGCGGAACAGGTCCATGT-3'; ERp58, 5'-CGAAAACTTCGAGAGTCGCG-3', 5'-GCAAGCCTCTTGCAATGTCC-3'; ERp57,, 5'-GGACTCAAG CGAAGTGACGG-3'; 5'-TCTGCTGCCAGCAAGAACTG-3'; Ero1 L, 5'-TTCACTGAGGAGGGCGGTT-3', 5'-CAGGACGGTCACTGCAATCA-3'; Ero1 beta, 5'-GTGCTTTGTCAAAGGTGG CC3'; 5'-GCAGAAGGGTCTTGGTGTCAG-3'; Crebpp1, 5'-ACGCCGGAGTCAATTCCTATC-3', 5'-GAGCTGATGTTGCGG GAAGA-3'; Psma5, 5'-TCACACCCCTGTCGTACTCG-3', 5'-TTTCAGTCGTGTGGCCTTTG-3'; Sec61a, 5'-GCTTCTGAATTT CCGGCAAG-3', 5'-AGGCAGGGAGTGTAGTCGGAC-3'; Sec63, 5'-TGGGTGAGTGAGACCTTCCC-3', 5'-AACCCCGGATCTTCCCAGTA-3'; Der1, 5'-CACCAGCCATGCTAAGCAGA-3', 5'-TCAGTGTGGGTCAGGTCCAAG-3'; Herpud1, 5'-GTGCTCTGTTGCTGGAGGCT-3', 5'-AGCACATCGTCATCCTGTGG-3'; Xbp-1 spliced, 5'-CTGAGTCCGAATCAGGTGCAG-3', 5'-ATCCATGGGAAGATGTTCTGG-3'; Xbp-1 total, 5'-CCCTTCTCCCTTCAGCGAC-3', 5'-CGTTGGCAAAAGTGTCCTCC-3'; Rat cyclin D2, 5'-TGCTGACCAAGATCACCCAC-3', 5'-CCTGGCAGGCTTTGAGACAA-3'; Rat cyclin B2, 5'-GCCCCTGAGGATGTCTCCAT-3', 5'-AGAGAAAGCTTGGCAGAGGCT-3'; Rat cyclin A2, 5'-AGTGTGAAGATGCCCTGGCT-3', 5'-TGGCTCCGGGTAAAGAGACA-3'; Rat cyclin E, 5'-GTCGCAGGGTTGCTGTTGAT-3', 5'-CATGCTTGCTCACGACCACT-3'
Total 832/13 cellular protein was extracted with RIPA buffer (1% Nonidet P40, 0.5% sodium doxycholate, 0.1% SDS, 1 × PBS, pH 8.0) containing 1× protease and phosphatase inhibitor cocktails (Sigma, Inc.) Protein expression was assayed by Western blots. Primary antibodies used in the analysis were: ERp72 (1:2500, Stressgen, Inc), GRP78/BiP (1:500, Santa Cruz, Inc), ERp57 (1:300, Santa Cruz), and anti-GFP (Sigma, Inc.) to detect EFYP-ATF6. For Western blots performed on islets from single mice, 60 islets from each mouse were dissolved in 2× SDS sample buffer and then loaded onto a 4-15% gradient gel.
After 16 or 24 hr post-transduction, 832/13 cells were deprived of methionine and cysteine (Met/Cys) for 30 minutes at 37°C in Met/Cys-free DMEM, 10% dialyzed FBS. The cells were then labeled with [35S] Met/Cys (500 μCi/ml) at 37°C for 0, 15, 30 minutes, and the reactions were stopped by the addition of concentrated non-radioactive Met/Cys solution (0.1 M each). After two washes with PBS, the cells were lysed in RIPA buffer and then total cellular protein was precipitated with 10% trichloroacetic acid (TCA). The precipitates were washed with 20% ice-cold acetone, air-dried for 20 minutes and then dissolved in 30 mM Tris, 7 M urea, 2 M thiourea, and 4% CHAPS. The resident radioactivity was measured by scintillation counting and normalized to total protein content.
Paraffin-embedded sections or cryo-sections were subjected to immunohistochemistry using the following primary antibodies: ERp72 (1:1000, Stressgen), insulin (1:500, Linco Research); proinsulin (1:1000, Ole D. Madsen, Beta Cell Biology Consortium); GRP78/BiP (1:500, Santa Cruz). Appropriate secondary antibodies conjugated with Alexa Fluor350, 488 or 555 dye (Molecular Probes) were used to visualize the labeled cells. Fluorescence images were captured and analyzed with a Nikon Eclipse E1000 and Image-Pro Plus (Phase 3 Imaging Systems, GE Healthcare, Inc.).
Determination of the oxidative state of ERp72 and ERp57
At 24 hours post-transfection, the medium was removed, and the cells were briefly washed with ice cold PBS and lysed at 0°C for 5 min in 20% formic acid/2% SDS. Cell lysate proteins were precipitated with 10% TCA. Precipitates were washed twice with ice cold 70% acetone. The TCA precipitates were resuspended in 80 mM Tris-HCl pH 6.8, 2% SDS, supplemented with a cocktail of protease inhibitors with or without 10 mM 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid (AMS, Molecular Probes, Inc.). After incubating for 30 min at room temperature and then for 10 min at 37°C, samples were subjected to non-reducing SDS-PAGE followed by western blot analysis to detect ERp72 and ERp57. Non-transfected cells were treated with 10 mM DTT or 5 mM diamide for 10 min at 37°C as reduced and oxidized controls.