Supporting Information

Supporting Information Materials and Methods Isolation of primary hepatocytes and adenovirus infection. Primary hepatocytes were isolated from male C57BL/6J mice at 8-12 weeks of age according to the procedure described (1). After anesthetization, collagenase perfusion was performed through the portal vein with 50 ml of perfusion buffer (Krebs Ringer buffer with 3.6 mg/ml Glucose, 1 M CaCl 2 and 0.66 mg/ml collagenase I (Worthington) at 37 C. The liver was aseptically removed and cut, and hepatocytes were filtrated and washed with cold Hepatocyte Wash Medium (Gibco) for three times and re-suspended in 15 ml of cold HepatoZYME-SFM (Gibco) medium supplemented with 2 mm L-glutamine, 20 units/ml Penicillin and 20 μg/ml Streptomycin. After the viability was determined by Trypan Blue staining, the hepatocytes were plated at 6 10 5 cells/well in 6-well culture dishes pre-coated with collagen. Cells were cultured for 8 hours before further use. Hepatocytes were infected for 48 or 72 hours with adenoviruses at an MOI of 40 and were subsequently treated with the desired reagents prior to protein extraction for Western immunoblot analysis. Generation of recombinant adenoviruses Recombinant adenoviruses for the overexpression of EGFP and the wild type or mutant forms of IRE1α were generated as previously described (2) with the AdEasy System (Stratagene) according to the manufacturer s instructions. Briefly, DNA fragments encoding the desired proteins were first subcloned into pshuttle-cmv, 1

which were then used to produce recombinant adenoviral plasmids through homologous recombination with padeasy-1 in Escherichia coli BJ5183 cells. Transfection of HEK 293A cells (Invitrogen) was conducted using the linearized recombinant plasmids to produce the recombinant viruses. The short-hairpin (sh) RNA knockdown adenoviruses targeting IRE1α (Ad-shIRE1α- # 1, Ad-shIRE1α- # 2) or PKA (Ad-shPKA- # 1) or expressing a scrambled control shrna (Ad-shCON) were generated with the BLOCK-iT Adenoviral RNAi Expression System (Invitrogen) in HEK 293A cells according to the manufacturer s instructions. DNA fragments encoding shrnas directed against murine IRE1α or PKA, or containing a general scrambled sequence were introduced into pentr/u6 vector under the control of the human U6 promoter. The two shrnas designed for the knockdown of IRE1α had the following IRE1α target sequences: Ad-shIRE1α- # 1, 5 -GCGAGAAGCAGCAGACTTTGT-3, and Ad-shIRE1α- # 2, 5 - GGAATTACTGGCTTCTCATAG. The shrna designed for the knockdown of PKA contained the following PKA target sequence: 5 -GGGTCAATGACATCAAGAACC-3. The control virus, Ad-shCON, had a core scrambled sequence of 5 -GTTCTCCGAACGTGTCACGTTT-3. The shrna for knocking down the expression of XBP1 was as described previously (3). For infection of primary hepatocytes, viruses were used at an MOI (multiplicity of infection) of 40, which was measured according to the manufacturer s instructions. High-titer stocks of amplified recombinant knockdown adenoviruses were purified by two-step ultracentrifugation in cesium chloride gradient. After subsequent dialysis, viral titers 2

were determined by the tissue culture infectious dose 50 (TCID50) method using 293A cells. Viruses were diluted in PBS and administered through tail vein injection, using approximately 4x10 8 pfu/mice. Liver function was assessed by measurement of serum levels of alanine transaminase and aspartate transaminase by Alanine Transaminase/Aspartate Transaminase Determination Kit (ShenSuoYouFu). Glucagon challenge test After a 15-hour fast period, glucagon was administered through i.p. injection (100 μg/kg for WT mice and 150 μg/kg for db/db mice). Tail vein blood was collected at 0, 15, 30, 45, 60 and 75 minutes after glucagon injection. Glucose concentrations were measured by a glucometer (FreeStyle). Glucose and pyruvate tolerance tests After a 6-hour fast, db/db mice infected with the desired adenoviruses were injected i.p. with 1.5 g/kg glucose or 2 g/kg pyuvate. Tail vein blood was taken for glucose measurement at 0, 30, 60 and 120 minutes after glucose or pyruvate injection. Glucose production assay The glucose production assay was conducted according to the protocol described (4). Primary hepatocytes were isolated from mice infected for 5 days with Ad-shCON or Ad-shIRE1α- # 2. After attachment, cells were washed three times with PBS and were then incubated for 4 hours at 37 C with 5% CO 2 in glucose production buffer [DMEM (without glucose, L-glutamine, phenol red, sodium pyruvate or sodium bicarbonate; Sigma) supplemented with 10 mm HEPES (ph 7.4), 0.6% BSA and substrates (5 mm sodium lactate, 5 mm sodium pyruvate)] in the presence of DMSO or 10 μm 3

forskolin(5). The medium was subsequently collected and glucose concentrations were measured with Glucose (GO) Assay Kit (Sigma). Chemical reagents, antibodies, plasmids, and Western immunoblotting Glucagon, thapsigargin, H89, U73122, epinephrine, forskolin, bromo-camp and α-tubulin monoclonal antibody were all purchased from Sigma. IRE1α, CREB, pser 133 -CREB, eif2α and pser 52 -eif2α antibodies were from Cell Signaling, and pser 724 -IRE1α antibody from Novus Biologicals. Antibody against the catalytic α subunit of PKA was from BD Transduction Laboratories, and BiP monoclonal antibody from Stressgen. For the expression of EGFP-IRE1α fusion protein, mouse IRE1α cdna was subcloned in-frame into pegfp-n1 plasmid. For immunoblot analyses, proteins extracted from cells or tissue by RIPA buffer (150 mm NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mm Tris-Cl) were separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membrane filters (Amersham Biosciences). After incubation with the desired antibodies, the blots were developed with Thermo Scientific s SuperSignal West Pico Chemiluminescent Substrate or Millipore s Immobilon Western Chemiluminescent HRP Substrate. To reduce non-specific protein species detected, pser 724 -IRE1α antibody was used after pre-incubation with a PVDF membrane filter that contained transferred mouse cellular proteins. Expression and purification of recombinant PKA, PKC and cyto-ire1α proteins The expression plasmid for the catalytic α subunit of mouse PKA was constructed in pet-15b, which was kindly offered by Susan Taylor (Addgene). Transformation was 4

performed using BL21 DE3 Codon Plus competent cells, and the recombinant PKA protein was subsequently purified by Ni-NTA Agarose (Qiagen) as described (6). The cdna fragment encoding the human full length PKC-ε, a novel isoform of PKC (7), was subcloned into baculovirus expression vector pfastbac-1 (Invitrogen) to produce a GST-PKCε fusion protein. The recombinant virus was generated and amplified according to the manufacturer s instructions. The recombinant PKC-ε protein was expressed in High-Five insect cells and was subsequently purified by GST affinity-chromatography. The enzyme activity of the purified kinase proteins was determined by the Z -LYTE TM Kinase Assay Kit (Invitrogen) using the peptide substrates for PKA and PKC, according to the manufacturer s instructions. His-tagged recombinant protein of the cytoplasmic portion of human IRE1α (cyto-ire1α, spanning amino acid residues 469-977) was bacterially expressed using a PCR-derived cdna fragment which was subcloned into pet-30a(+). Cyto-IRE1α protein was purified with Ni-NTA Agarose (Qiagen) according to the manufacturer s instructions and subsequently used for phosphorylation assays through incubation with purified PKA or PKC proteins. Immunoprecipitation of IRE1α for phosphorylation assays Primary hepatocytes were infected for 48 hours with recombinant adenoviruses expressing Flag-tagged human IRE1α, IRE1α-K599A or IRE1α-S724A proteins. Cells were lysed with the lysis buffer [20 mm Tris-HCl, ph 7.5, 100 mm KCl, 0.1% Nonidet P-40, 1 mm EDTA and 10% Glycerol containing 1% protease inhibitors cocktail (Sigma) and 1% phosphatase inhibitors cocktail I/II (Sigma)] for 20 minutes 5

at 4 o C. After incubation with Flag antibody for 2 hours at 4 o C with gentle rocking, lysates were mixed with a final concentration of 2.5% protein G Sepharose beads (Amersham Biosciences) and incubated for 2 hours at 4 o C on a rotator. After washing three times with the washing buffer [20 mm Tris-HCl ph 7.5, 150 mm KCl, 0.5% Nonidet P-40, 1 mm EDTA, and 10% glycerol supplemented with 1% protease inhibitors cocktail (Sigma) and 1% phosphotase inhibitors cocktail I/II (Sigma)], beads were subsequently incubated with purified PKA or PKCε protein at 30 for 1 hour in the kinase assay buffer (20 mm TrisHCl, ph 7.5, 5 mm MgCl 2, 400 μm ATP, 1 mm DTT, 50 mm NaCl). Reactions were terminated by adding 2 loading buffer and subjected to analysis by immunoblotting. Microarray analysis Primary hepatocytes were infected with the desired adenoviruses or treated with glucagon. Total cellular RNA was isolated with TRIzol (Invitrogen) and subjected to analysis by Affymetrix Mouse Genome 430 2.0 Arrays. Three experiments were independently conducted. The original microarray intensities were log2-transformed and quantile-normalized using 'affy' package in Bioconductor (8). Differences in the expression of genes between each two compared samples were determined by RankProd (9), with proportion of false positive (pfp) < 0.1 considered significant. Parametric Analysis of Gene Set Enrichment (PAGE) (10) was conducted based on KEGG pathways downloaded from the KEGG database (http://www.genome.jp/kegg/) on July 5, 2010. Significance of the enrichment was calculated and false discovery rate (FDR) < 0.01 considered significant. 6

RT-PCR analyses Total RNA was isolated from cells or mouse livers with TRIzol (Invitrogen). After reverse transcription by M-MLV Reverse Transcriptase (Invitrogen), regular PCR was performed with TaKaRa Taq kits (Takara). Quantitative real-time PCR was done with an ABI Prism 7500 sequence detection system, using Power SYBR Green PCR Master Mix (Applied Biosystems) following the manufacturer s recommendations (Applied Biosystems). Actin was used as an internal control for normalization. The oligonucleotide primers for each target gene examined are listed as follows. Mouse Xbp-1: sense 5 -GAACCAGGAGTTAAGGACACG-3, antisense 5 -GGGGATCTCTAAGACTAGAGGCT-3 ; Mouse G6pase: sense 5 -CGACTCGCTATCTCCAAGTGA-3, antisense 5 -GTTGAACCAGTCTCCGACCA-3 ; Mouse Pepck: sense 5 -AAGCATTCAACGCCAGGTTC-3, antisense 5 -GGGCGAGTCTGTCAGTTCAAT-3 ; Mouse Actin: sense 5 -AGTGTGACGTTGACATCCGTA-3, antisense 5 -GCCAGAGCAGTAATCTCCTTCT-3 ; Mouse Bip: sense 5 -ACTTGGGGACCACCTATTCCT-3, antisense 5 -ATCGCCAATCAGACGCTCC-3 ; Mouse Chop: sense 5 -CTGGAAGCCTGGTATGAGGAT-3, antisense 5 -CAGGGTCAAGAGTAGTGAAGGT-3. Mouse Erdj4: sense 5 -ATAAAAGCCCTGATGCTGAAGC-3, antisense 5 - GCCATTGGTAAAAGCACTGTGT-3. 7

Figures and Legends Fig. S1. Glucagon stimulated the expression of gluconeogenic genes but not typical UPR target genes in vivo. Male C57BL/6 mice were treated for the indicated time intervals (n=3/group) by i.p. injection of glucagon (100 μg/kg body weight). The hepatic mrna abundance of G6pase, Pepck, Chop and Bip was analyzed by quantitative real-time RT-PCR, with actin used as an internal control. Results for each gene were normalized to the value at 0 min (set as 1) and shown as the mean ± SEM (n=3 independent experiments). *P < 0.05 versus 0 time point by one-way ANOVA. 8

Fig. S2. Glucagon but not thapsigargin stimulated the expression of gluconeogenic genes in primary hepatocytes. Primary hepatocytes were treated (A) with 100 nm glucagon for 0, 0.5, 1, 2 or 4 hours, or (B) with dimethyl sulfoxide (DMSO) or thapsigargin (Tg, 1 μm) for 1 hour. The mrna abundance of G6pase, Pepck, Chop, and Bip was determined by quantitative real-time RT-PCR, using actin as an internal control. (C) Glucagon or thapsigargin did not affect the expression of actin that was used as the internal control in (A) and (B). The mrna abundance of actin was analyzed by quantitative real-time RT-PCR and shown as relative to 18S rrna. All results were normalized to the value at 0 min for glucagon treatment or to that of DMSO for Tg treatment. Data are shown as the mean ± SEM (n=3 independent 9

experiments). *P < 0.05 versus 0 time point or DMSO control, **P < 0.01 and ***P < 0.001 versus 0 time point by one-way ANOVA. 10

B A GI GI/shIRE1_shCON Pck1 Ppargc1a Igfbp3 Procr Efna1 Afp Nr4a1 Ppp1r3g Cldn4 4931408D14Rik Cldn2 D630004K10Rik Ascl1 2310016C08Rik Thbs1 Thbd Nr4a2 Slc25a25 Slc25a15 Slc16a7 Enc1 Klf15 Got1 Abcg5 Epha4 Socs3 Gadd45g Il33 Rasgef1b Igf1 G6pc Tcp11l2 Spp1 Igfbp4 Grem2 Dppa3 Sgk1 3110043O21Rik Slc7a2 Ctla2b Ccrn4l Akap12 Irs1 Ccng2 Pdgfc Slco2b1 Dnajc12 Irs2 Lnx2 Zfp697 Adh1 Tgfbr1 S100a6 Cldn1 Crim1 Serpine1 Cxcr7 Ypel2 Jarid2 AI452195 Tnfaip6 Steap4 1810011O10Rik Ngf C030033M12Rik Egln3 Tubb2b AA986860 Dusp4 Pah Tnnt2 Nfil3 Klhl24 Abhd2 Npy Aass Raph1 Cd44 Gm4848 98623 Pim3 Esm1 Prei4 2010011I20Rik Cpt1a 4833417J20Rik Hal Maff Fam108c Lgals1 Ppm1k 9430041P20Rik Serpinb6b Igfbp1 Krt20 Uap1l1 Hist1h1c Ctla2a Emp1 Cerk Irx3 Robo1 Cap1 Cpne3 C530047H08Rik F11 5330406M23Rik Gjb4 Akr1b7 Appl2 Plekha6 68124 Ppic Hdac4 Selm Clu Pde4b Gpt2 Stk11 Cp Nnmt Creb3l2 Fst Tbl1xr1 Pmvk Adcy9 Rab20 Nr4a3 Adrb2 Adra2a Mlxip Tle1 B230117O15Rik Adh7 Strm Bckdha Hist3h2a Irf2bp2 Cxcl16 Bcl3 BC024659 Crct1 Tmc6 B230312A22Rik Hspa4 Jub Cd2ap Lpin1 72178 4832420A03Rik Crem Tm4sf4 Rbm47 Arl4a Osbpl3 Fam20a Jhdm1d Arl5b Spty2d1 101755 D7Ertd413e Akr1b10 3110048L19Rik Apoa5 Adam12 Fosl2 Dpysl3 Fbxo32 Shb Mterfd3 4933417E11Rik Grb7 Etnk2 Rnf125 Fbxo31 Slco2a1 Tmem176a Jag1 Pdlim7 Nfkbiz Slc44a3 Gm10452 Lce1f AA408213 Cyp4f18 Id2 Pdgfrl Nid1 Akr1b8 Serinc5 Tat Ank3 Nrk Mtus1 Rora Cobll1 77419 100125272 1200009F10Rik Myo10 Pvrl2 Hps4 Serpina7 Fam117a Amotl2 Egr1 Zfp52 Slc25a4 C730049O14Rik 2900056M20Rik Npdc1 Rab38 Alcam Hk1 Atxn2 Tceal1 Hmga2 103207 Flcn Fnip1 Cdc20 Chrnb1 Fkbp7 Tmem2 Zdhhc14 Fosb 5133401H06Rik Zfp462 Atg2a Gns Lrp1 Dcaf11 Fam102a Ect2 Fam72a Ndrg1 Slc25a22 Fcgrt Slc39a10 Slc38a4 Cpe Baiap2l1 Ccnd2 Eif2c1 D5Wsu152e Wwc2 Itgb1bp1 Tll1 Slc25a33 Plce1 Irx5 BC006779 Nqo2 Rnf217 Mfsd2 Bex2 Cited2 6330564D18Rik Pik3c2a 1110018J18Rik Egfr Csad Zdhhc20 Ppat Skil Fam110a Mapkapk2 Mex3d Cbs 2210403K04Rik Homez Lgr4 9330195I24Rik 68252 Ttc26 9330141E21Rik Paip2 Reep4 Msi2 Prox1 Atf6b 9030411M13Rik Shroom4 Rictor Onecut2 A330041D05Rik Tob1 2610024B07Rik Ccnb1 D18Ertd653e Ece1 Dyrk2 Sh3bgrl3 LOC100134980 Adamts1 Tmem87b Ghr Sik1 Pde10a AA407881 Fgd3 Cul7 Fgd6 Zfhx3 Esrp2 Zrsr1 Oplah Bhmt2 Zfyve26 Ggcx Edem3 2200001I15Rik Tifa Smad7 Bsdc1 Agxt2l1 Sbno1 1700001F09Rik Rbm39 A930001N09Rik Ccdc22 Sh3bgrl Pik3ca Gprc5a Bmp1 Ccnl1 Asah1 Ell2 Fcho2 Hhex Mospd2 Clcf1 Arntl2 1110021L09Rik Tmsb4x B630005N14Rik Abi1 2210402A03Rik Dusp6 Rnf34 Vasp Tnfrsf23 Dusp5 Sdsl Zswim6 B130040O20Rik Nox4 Tmem176b Slc39a4 Scand3 Arg2 Pgpep1 Uggt2 Mtmr10 A630072M18Rik Sec16b Aldh1b1 Rhoc BC022687 Hpn Ube2d1 Tet2 LOC668861 Actr6 Abtb2 G0s2 B130021B11Rik Grpel2 Sdpr Clcn2 Phf17 Pdcd4 Traf3ip1 Mccc1 Ptprk Frat2 D230019N24Rik Hnmt Fam107a C530034L13Rik Krt23 Rcn1 AW987390 Ier3 2810021G02Rik 100039605 Acsl4 7 Mar Rab6 Lipa Plscr2 Ramp3 Anapc1 Ctns Fblim1 Ropn1l Mvp Lpar6 Cetn2 4633401B06Rik Actn1 Pfn2 4833420G17Rik 108805 Agpat9 Atxn3 Stat2 A730017L22Rik 2810416G20Rik Gramd4 Rnf44 Sdc4 Zfp259 Las1l Dexi 93698 Fubp3 Slc4a11 Scfd1 Itm2c D16Ertd472e Pip5k1a Tspyl1 Bpgm Itga3 Rnf11 Tcea3 Gde1 Jmy Sypl Cryab Gas6 Galnt10 Ncald Bmp4 Ankrd16 Gcnt2 Ccdc68 Armcx1 Pxdn Btg3 1810055G02Rik Gm9752 3110062M04Rik Igsf8 Tspan6 Gm10033 Copa Gas2l3 Msn 3930402G23Rik F2r AU040829 B430202K04Rik Cdh1 Wbp2 Gla Plat Ccpg1 Iah1 Nudt4 Fn1 Gm10199 Cnnm2 Sacm1l Picalm Zfp667 9530006C21Rik Tmem140 Pura Wdr92 Tac1 LOC552906 1300001I01Rik Ddah1 Parp4 4933415E08Rik Tap2 Trim25 Fam108a 2010106G01Rik Ube2q1 Supt6h Cbl Ttc30b 4933426M11Rik BB070754 Hmgcs1 Rin2 D230044B12Rik Snx30 Tmem123 Alkbh7 2700078E11Rik Zfp53 Herpud2 Trp53inp2 Rps25 Golt1a Dock4 Dnajc5 Nab1 Cblb Dedd2 Zfp867 2810043O03Rik Tmem50a 2210419D22Rik 4931406P16Rik 1110003O08Rik Pnma1 Rc3h2 Tcf4 Tsc22d2 Dpp9 Col4a3bp Napa D930017J03Rik Pik3r1 Wipi1 Ramp2 5830427D03Rik Bmpr2 Mansc1 Herc4 Crtc2 Gpx7 Map3k2 1500032P08Rik Arfgef1 Gm7993 76579 Reps1 Klhl21 Snrnp25 Rc3h1 Hcfc2 Hrk Aacs Irg1 Zfp418 Aspdh Atp11c Cox19 BB143722 Atp12a 5730508B09Rik Hsd3b7 Fxyd1 Cugbp2 D230046B21Rik Apoh Tcta Fgfr3 Sh3kbp1 Dnmt3a Mup10 St3gal5 Dna2 Sfmbt1 Zyx Afm Synm Insc Serpina6 Hamp Pstpip2 Snai2 Klhl23 BC046404 Efhd2 Klf5 Amy1 V1rb4 1100001G20Rik Pold2 Rbm3 Tmem51 Sult1a1 9530008L14Rik 1700021C14Rik Mapk4 1190002N15Rik Mcm6 Wee1 Pdk1 Elovl7 Pnpla7 Kntc1 Apol7a Fam43a Dhrs13 Casp7 Hsd17b2 AU018778 B130052P14Rik Fas BC037703 Id3 H2 gs10 Nudt11 Rapgef4 Dleu2 Taf9b Acot12 C77370 Ctnnal1 Anubl1 Pigr Cbx2 BC049807 Brip1 BC089597 Asgr1 Phf16 Rchy1 Nrip3 Fam169b Ugt1a9 Cyp4f15 Dut Timd2 Itih1 Hirip3 Tdo2 2810474O19Rik 2310076L09Rik Cpb1 319749 Ttc39a Ugt1a6a Prss29 Stau2 Slco1a1 Slc29a1 Sh3gl3 Ablim1 A030009H04Rik Hesx1 Pdhb Cyp2a12 Mdk Slc9a5 Cml5 Hpgd Eepd1 2210011C24Rik Mcm7 Acaca Kitl Cyp17a1 Samd1 F7 Ttc36 Sult1d1 Donson Gja1 Lrrc1 Vtn Grhl1 2310008H04Rik Aox1 Ccdc67 Mmp19 Tnfsf9 Ugt2b36 Doc2b Dck Tead2 Kctd12 Cdk2 Aim1 Chek1 Prr5l Id4 Slc10a2 Gys2 Klf6 Cyp2j5 Ces6 Mcm5 Cntln 1700040I03Rik Stbd1 Gipc2 Trim47 Slfn9 Asrgl1 Ndufa4l2 Serpinc1 Plod2 Mcm3 Hsd11b1 Arhgef3 Ubtd2 4930422G04Rik Slc37a4 Mdc1 Ankrd37 Cyp2c44 Pmm1 Bnip3 Msh6 Efr3b Serpina1d 2610209A20Rik F13b Zfp367 Tipin C8b Timeless Mid1ip1 Cyp2c70 Ugp2 D330028D13Rik Ccne1 Insm1 0610037M15Rik Slc3a1 Rnf144a Nmral1 6530418L21Rik Nwd1 Ezh2 Fam105b Ankrd34b B3galnt1 Cited1 AI646023 4930528F23Rik Exo1 Arnt2 Gckr Fhl2 Sord Skp2 5033413D22Rik Hopx Khk Shc4 Irf8 73762 2310034O05Rik Tm7sf2 Fam33a Aadac Zcchc3 Rad51ap1 Gjb2 Fam101b Lig1 Hat1 C8g Pbx3 2700038G22Rik Pfkfb3 Tmem209 Hc Leap2 Apcs Alpl F10 Dio1 Gtpbp2 Pzp Slc47a1 9530048O09Rik Apoc4 Cdc7 Dnajc9 Zadh2 Ugt2a3 Gsta3 Chaf1b Hrg Nt5dc2 Ces1 Fkbp5 Psmc3ip Nlrp12 Gm4738 Tmem30b Es1 Trib3 Rpa2 Cdc45l Sult1b1 Plcl2 Stag3 Gck Serpina3n Fam46c Slc27a5 Syce2 Sfn Chac1 Bicc1 Cyp3a25 Cyp4a12a Rad51 5330426P16Rik Ercc6l Pdzrn3 Acsm5 328425 Fbxo5 Chdh Ugt3a2 Ctdspl Igfbp2 Anks4b Mesp2 Car14 Slc6a12 Hmgb2 Ugt2b5 Nrm Parp16 Rdh7 9130221J18Rik Trim59 Plg Arf2 Tshz1 Lce1d Matn2 Garnl4 Aqp9 Rad51c Wdhd1 Mbnl3 Sema6a Selenbp2 Zfp354b Cyp2c68 C8a Pcolce2 Cyp2c54 Gstm1 Rrm1 Gadd45a Angptl6 Mirhg1 Gstm2 Slc2a2 Pon1 Asf1b Tcf19 Ager Lhfpl2 Mlf1 Tex15 Arg1 Hsd3b5 Necab1 Rgn Coro2a 9030208C03Rik Fyn Tk1 Cyp3a11 Adm Car2 Cyp1a2 Fam171b Ccnf Hpd Txnip Gm13889 9130409I23Rik C9 2810417H13Rik Gstm6 Spsb4 AI195470 Adora2b Mug1 Cdt1 Cyp2f2 Cyp2b10 Gm3916 Ccne2 Bhmt Fignl1 Dnmt1 Blnk Srxn1 Defb1 Prim1 Ugt2b35 Cyp2c29 Slc10a1 Gpr120 Rcan1 AI132487 Enpp1 Tacstd2 Selenbp1 Gm15514 Syt9 Otc 2810007J24Rik Hlx Akr1c6 Ttc23 Ugt2b1 Dntt Elovl3 Cdc6 Sdr42e1 Dnase2b Angptl3 Klf2 Plcl1 C730043O17 Gm1381 Arid5a Cyp2e1 Uhrf1 Fabp1 Klf10 Uox Lhx2 Cyp2c50 Vash2 Ugt2b34 Cyp4a10 Vwce Agxt2 Rundc3b Areg Cyp2c37 E2f7 Rrm2 Gjc1 Fam111a Gstm3 Hspa1b Myb 1190002H23Rik Hspa1a C Ascorbate and aldarate metabolism Fructose and mannose metabolism Galactose metabolism Glycolysis / Gluconeogenesis Glycosaminoglycan degradation Insulin signaling pathway Pentose and glucuronate interconversions Pyruvate metabolism Starch and sucrose metabolism Adipocytokine signaling pathway Arachidonic acid metabolism Linoleic acid metabolism Primary bile acid biosynthesis Sphingolipid metabolism Steroid biosynthesis Steroid hormone biosynthesis Alanine aspartate and glutamate metabolism Glutathione metabolism Glycine serine and threonine metabolism Phenylalanine tyrosine and tryptophan biosynthesis Valine leucine and isoleucine degradation Adherens junction Allograft rejection Asthma Autoimmune thyroid disease Caffeine metabolism Cell cycle Complement and coagulation cascades Cytokine cytokine receptor interaction DNA replication Drug metabolism cytochrome P450 Drug metabolism other enzymes Graft versus host disease Hematopoietic cell lineage Homologous recombination Huntington's disease Leukocyte transendothelial migration Lysosome Malaria Metabolism of xenobiotics by cytochrome P450 Mismatch repair mtor signaling pathway Neuroactive ligand receptor interaction Porphyrin and chlorophyll metabolism Primary immunodeficiency Prion diseases Proximal tubule bicarbonate reclamation Pyrimidine metabolism Regulation of actin cytoskeleton Renal cell carcinoma Retinol metabolism Ribosome Spliceosome Tight junction Type I diabetes mellitus Type II diabetes mellitus Enrichment Score Ubiquitin mediated proteolysis Viral myocarditis GI GI/shIRE1_shCON 5 0 5 Annotation Key Carbohydrate metabolism Lipid metabolism Amino acid metabolism Others D Cebpb Cebpa Dgat1 LXRbeta Fasn SREBP1c Ppard LXRalpha Ppara Cebpd Dgat2 Acly ChREBP Pparg Scd1 ACC1 Log2 fold change 2 1 0 1 2 GI GI/shIRE1_shCON 11

Fig. S3. Transcriptomic analysis of the impact of IRE1α knockdown on glucagon-regulated gene expression profiles in liver cells. Primary hepatocytes were infected for 48 hours with Ad-shCON or Ad-shIRE1α- # 2 and then treated with 100 nm glucagon for 1 hour. Hepatocytes infected for 48 hours with EGFP-expressing adenovirus were left untreated. Total cellular RNA was subjected to whole-genome microarray analysis using Affymetrix Mouse Genome 430 2.0 Arrays. (A) Individual heatmaps from three independent experiments showing differentially expressed genes upon glucagon induction (GI), which were aligned with changes of these genes caused by IRE1α knockdown in the presence of glucagon stimulation. Included are genes exhibiting >1.25-fold changes or trends of significant changes as determined by RankProd with proportion of false positive (pfp) < 0.1. Gene labels are also indicated. (B) Percentages of all array-probed genes (All genes) that were downor up-regulated by shire1α as compared with percentages of glucagon-upregulated (GI UP) or -downregulated (GI DOWN) genes that were down- or up-regulated by shire1α relative to shcon control. (C) Heatmaps showing glucagon-regulated cellular pathways or biological processes that were affected by IRE1α knockdown. Parametric Analysis of Gene Set Enrichment (PAGE) was performed using the KEGG database (http://www.genome.jp/kegg/), with false discovery rate (FDR) < 0.01. Pathways related to metabolism of carbohydrates, lipids and amino acids are indicated. (D) Heatmaps showing expression changes of regulatory genes in lipid metabolism and enzymes in triglyceride synthesis as result of IRE1α knockdown in the presence of glucagon stimulation. 12

Fig. S4. Effects of PKA inhibitor on IRE1α phosphorylation during ER stress and stimulation of IRE1α phosphorylation by activation of the camp pathway. (A) H89 had no effect on ER stress-induced phosphorylation of IRE1α. Primary hepatocytes pre-cultured for 30 minutes with DMSO or H89 (at 5 or 10 μm) were treated with 1 μm Tg for 1 hour. (B) camp pathway activators stimulated IRE1α phosphorylation. Primary hepatocytes were treated with DMSO/PBS (Veh), 10 μm forskolin (adenylate cyclase activator), or 100 μm Br-cAMP (camp analog) for 1 hour. Immunoblotting was performed using the indicated antibodies. Results shown are representative of three independent experiments. 13

Fig. S5. Thapsigargin did not induce but overexpressed EGFP-IRE1α caused Ser 724 phosphorylation of the autophosphorylation-defective IRE1α-K599A mutant. (A) Primary hepatocytes were infected for 48 hours with recombinant adenoviruses expressing EGFP, Flag-tagged human wild-type (WT) IRE1α, or IRE1α-K599A and IRE1α-S724A mutants. Cells were then treated with DMSO or 1 μm Tg for 1 hour. (B) HEK293 cells were transfected for 48 hours with plasmids expressing wild-type (WT) IRE1α, IRE1α-K599A or EGFP-IRE1α, or co-transfected with EGFP-IRE1α and IRE1α-K599A. Phosphorylation of IRE1α was analyzed by immunoblotting with IRE1α or phospho-ire1α antibodies. Results shown are representative of three independent experiments. 14

Fig. S6. Enzyme activities of purified PKA and PKC proteins. Recombinant mouse PKA and human PKCε proteins were expressed and purified. Kinase activities were measured with synthetic substrate peptides for PKA (A) and PKC (B) using Z`-LYTE Kinase Assay Kit. 15

A C WT_EGFP S724A_WT Log2 fold change 2 1 0 1 2 D10Bwg1379e Ccnb1 Nucb2 Ube2c Cdc20 Manf Birc5 Fam107a C530034L13Rik Nuf2 Bhlha15 Sec61b Tmed3 Mki67 Selm Gale Ccna2 Ccnb2 Cdca5 Ift20 Sdf2l1 Nme6 Fmo4 Igfbp3 Aurkb 2810482I07Rik Cdca3 Hmmr Pbk Arfgap3 Edem2 Cep55 Gmppb Fgf21 Trabd Uba5 Serp1 Ostc Ica1 Spcs3 Pex11c Lpin3 AI562086 Ficd Ppapdc1b Sec61g Ect2 Gnat2 Magt1 Aurka Herpud1 Esco2 Prc1 Serpina7 Shcbp1 72178 Hspa13 Cdk5rap3 Bet1 2010003K15Rik Slc33a1 A630095E13Rik Ssr2 Spcs2 Sec24d Sec61a1 Wdyhv1 Slc10a2 Nipal1 Creb3l2 Dnajb9 Zfp324 Saa3 Tpx2 Isg20 1110003E01Rik Sec16b Cdca2 74520 Pde9a Kif20a LOC67527 Ssr1 Zfyve21 Gmppa 2810417H13Rik Ikbke Tram1 Igfbp1 Nrg4 Cdc6 Racgap1 Kif18b Tspyl2 Sgk1 Efna1 Cope Pdk2 Mt4 Syvn1 100040799 9130221J18Rik 4931408D14Rik Uso1 Rab4a Pfkfb1 Nans D930003E18Rik 1810074P20Rik Cenpq Mcfd2 Lepre1 Pdia5 Tns1 Lman1 Crip1 Fmo5 Defb1 Rtp3 Kif11 Ncapg Ehhadh Vkorc1 Serpinb6b D630004K10Rik Ccrl2 Plscr2 Cenph Ero1lb P4hb Dad1 Ggcx Melk Cox6b2 4833428C12Rik Dcaf11 Wnt5a Txndc11 Slc35b1 Ank3 623451 Trmt61b Klf15 Edem1 9430088B20Rik Creld2 Ibtk Inhbe Gsg2 Crct1 H47 AI132487 1700001F09Rik Mgat2 Chac1 Ttk Pdk4 Fam114a1 Slc30a7 6030422H21Rik Sec11c Yipf2 Cdkn2d Ncapd2 Pycr2 Paip2b Srprb Apof Calr Aox3 Steap4 Tspyl4 Srp9 Kdelr2 Slc16a7 Depdc1b Id2 Pdia6 Asb13 Slc17a9 Fgb 1810005K13Rik BC043301 Htra2 1200009F10Rik Cdca8 Surf4 2610002M06Rik 4930509E16Rik Anln Pecr Ssr3 Golph3l Fam72a Espl1 Fkbp2 Fmc1 Tnfaip6 Cenpa Hsp90b1 Ccng2 Oat Setdb2 Sgol2 Mfsd2 Guk1 Ifit2 Slc46a3 Nlrp12 Acot2 Cenpp Bub1 Dapp1 Gm447 Gne Cog3 Gm9914 Tasp1 2610039C10Rik 1700029F09Rik Cited2 F3 Nupr1 Wipi1 Zfp697 Ppib Cited1 Myom1 Tmem106b Tnfaip8l1 Tmem107 Snhg11 Pold4 D4Bwg0951e Mterfd3 1700034H14Rik Dnajc12 Acot1 Nde1 Hfe2 7120451J01Rik Serpine1 1500011H22Rik Snrnp25 1110018J18Rik 9030619P08Rik Ppap2b Fam13a Plce1 Pycr1 2900009J06Rik Sf3b5 Srm Slc38a10 Aass Ankrd1 Srp72 9030419F21Rik Sgk2 Hmgcs2 Pebp1 1110020G09Rik Zranb3 Kif4 Hnmt Aldh9a1 Gnai1 BC025446 Arap1 Akap12 Thbs1 Mrps12 Slc10a7 Yipf5 Igsf5 Ankrd37 Spp1 Cxcl12 Ube2t St5 Fam161a Trp53inp2 Cenpi Lysmd3 Wdr92 Snd1 Ppara Dlgap5 Fxc1 Kdelr1 Tmed9 Preb Pxk Zfp810 Kdelr3 Fdx1 Fam122b Dhdpsl Sesn1 Bckdha Nrn1 Tmem125 Phpt1 Rnf5 2610021K21Rik Nusap1 9130016M20Rik Crym Selk Adhfe1 Fbxo5 Vamp7 Fam18b Sdhaf1 1700025K23Rik Zfp9 Sec23b Ckap2l G0s2 Itm2c Ccnd1 Fen1 Porcn 2810021G02Rik Ccdc67 1700007K13Rik Rps25 Lxn Kctd12 Fabp1 Gstt3 Cdr2 Sh2d4a Tmed10 Nudt22 Sgol1 Endog Cenpj Spcs1 Slc31a1 LOC677547 2810408I11Rik Sepx1 Tmprss2 Twf2 4833442J19Rik Olig1 Gen1 Cdk2ap2 Fam81a Idh1 0610007L01Rik Dpagt1 Srp54c Kctd7 Kdelc1 Fam110a Cenpe Mfge8 Hcfc2 Narf Ccbl2 Sorbs2 Akr1c19 Psmb9 Tgds Srp19 Plac8 Hacl1 Rwdd2a Crp Fam165b 3110003A17Rik Cdc2a Gpt2 Necap1 Hnf4a Vamp4 Deb1 Adcy9 Creb3l3 Cks1b S100a6 Trim30 Oas1c Trim14 D4Wsu53e Glt8d1 Dusp19 Gtf2b Slc10a6 Erp29 Srp54b Imp3 Guca1a Mctp2 Spc24 Atl1 Nr0b2 Inpp5b Stt3a Ift80 Actr6 Appl2 Slc6a13 1700040I03Rik Mogs 2810007J24Rik 65104 Zfp473 Fndc4 Gas5 Pgf Sec31a Chek2 Serpina10 Edem3 Zc3h12d 2210021J22Rik Il17rb Dnajc3 Pnpo Ticam1 Birc2 Cul7 Nup37 Trim7 Ttc7b Srp68 Oip5 Orm3 Selenbp2 Ell2 Vasn Chn2 A130022J15Rik 5033406O09Rik Stxbp6 Lgals9 Ddt Phf11 Ccbl1 Ufc1 Aldh3a2 Cyp3a25 Sar1a Cetn2 Spag5 Trip13 Nnt Atp6v0e2 Rint1 Mansc1 Cenpm Polr3k Nln Ttc36 Slc45a3 Bcl2l14 Ccrk 1500035H01Rik 1110002B05Rik AI413582 C130040N14Rik Bhmt2 Gm4848 98623 H2 M3 Tuft1 Xbp1 Acat1 Cln8 Rnf181 Pttg1 Hist1h2ae Dcps Hspa5 Scfd1 Loxl2 Ascl3 Fam83g H2 gs10 Fbxw8 Abcc1 Igsf9 Fignl1 73762 Col18a1 Zfp105 Glce Ank Tcte3 A830021K08Rik Pxmp2 Dsp Gm10129 P4ha2 Spr Ppapdc2 Xrcc1 Cpeb2 Morn2 C8b Tmem48 Nhlrc3 Opn3 Atp13a4 Hn1 Ddah2 Syt11 100039918 Fam134a Commd9 Tusc1 Prr5l ENSMUSG00000074373 Gtf2f1 Grn Gm6484 Snrnp200 C6 Nhlrc1 Phf16 Trim28 Zfp143 Fam40a Ttc13 Tmlhe 2210408I21Rik EG665787 Slc30a1 Ugt3a2 Psap Flot2 Rhob Ccdc158 Plg Tmem98 Lrg1 B3galnt1 Marcks Oaf Klf10 Pigr Paqr7 D030074E01Rik Ift57 Apoa4 Fam171b 73490 Niacr1 Cfh Lpgat1 Gas6 Creb1 Zfp354b Dcbld1 Dera B130052P14Rik Agpat2 Vezt Amfr Nwd1 Zkscan17 F12 BC013529 Tspan17 Leprot Pbxip1 2310033F14Rik Mknk1 Capns1 AW146242 Epdr1 320969 Ankrd50 Pglyrp2 Tm7sf2 Dnmt1 Atp1a1 5330426P16Rik Serpina1a Sharpin Prom1 Atp2b1 C3 Car6 F2 Pcbp2 Clptm1 Nrip3 2310034O05Rik Ttc23 Dpp7 Tmx3 Ccdc122 Gm10840 Cpe Gab1 4930432K21Rik Pon1 C9 Atrnl1 Pzp Igfbp2 Erap1 Baz1a Nup210 Timeless Tex15 Il4ra Galnt2 328425 Unc119b F7 A030009H04Rik Tex2 4933403G14Rik 9130409I23Rik Tpp1 Tprkb Txnrd1 8430419L09Rik Wee1 Orc5l Afm Zdhhc2 Ugt2b35 9130221J17Rik Tapbp Mvp Daglb 2810432L12Rik Serpina1d C77370 Gdpd1 Il17ra Nrip1 Tmprss6 Tug1 Blnk Dnajb2 Klf6 Fam134c Es22 Slc12a7 Smo Dnttip2 Itih1 Ctsb Rnf130 2610029G23Rik Proc Tmem5 Yaf2 Igf2r Inadl Atp13a3 A130034M23Rik Ints9 Tmem209 Cyp2c29 Ces2 Ezr Tpm1 Sidt2 Aim1 Asrgl1 Enpp1 Serping1 9330159M07Rik Man2a2 Pcbp4 Tex264 E130012A19Rik G730046D07Rik Eif3c Spsb4 Ces6 Ndufs2 Rdh5 Slc39a6 Hlx Sema6a Hgsnat C8a Ppp1r15b Pdap1 Pex6 Fam46c Mthfd1l Tmem208 Rif1 Lamp1 Atp6v1h Cyp2f2 Slc27a5 Esd Stag3 Gm4738 Btg1 Tspan3 Zfp644 Ptprf Hectd1 Slc7a11 Smad4 Wnk1 Dntt Gja1 2310003L22Rik Angptl3 Tubb2b Uhrf1 Abcc4 Sema3e Azgp1 Pla1a Blvra Ager Mug1 Ces3 Msn Pmaip1 Dbt Itih4 AU018778 Slc25a17 Vash2 Cyp1a2 Taldo1 Vim Basp1 Furin Bloc1s1 Dgat2 Mettl14 Stim2 2210411K11Rik Nid1 Hc Cxcr7 Ndufa9 Brp44 Plod2 Ces1 2810474O19Rik Klf2 1190002H23Rik Cyp2e1 Hspa1b Es1 Hspa1a Ascorbate and aldarate metabolism Butanoate metabolism Glycolysis / Gluconeogenesis Glycosaminoglycan degradation Insulin signaling pathway N Glycan biosynthesis Other glycan degradation Propanoate metabolism Starch and sucrose metabolism Adipocytokine signaling pathway Arachidonic acid metabolism Linoleic acid metabolism Primary bile acid biosynthesis Sphingolipid metabolism Steroid hormone biosynthesis Glutathione metabolism Glycine serine and threonine metabolism Phenylalanine tyrosine and tryptophan biosynthesis Taurine and hypotaurine metabolism Tryptophan metabolism Adherens junction Aldosterone regulated sodium reabsorption Antigen processing and presentation Apoptosis Bacterial invasion of epithelial cells Base excision repair Calcium signaling pathway Cell adhesion molecules (CAMs) Cell cycle Chronic myeloid leukemia Complement and coagulation cascades DNA replication Drug metabolism cytochrome P450 Drug metabolism other enzymes Endocytosis Focal adhesion Homologous recombination Huntington's disease Jak STAT signaling pathway Lysosome Metabolic pathways Metabolism of xenobiotics by cytochrome P450 Mismatch repair Neuroactive ligand receptor interaction Neurotrophin signaling pathway NOD like receptor signaling pathway Nucleotide excision repair Olfactory transduction Pathways in cancer Prion diseases Protein export Proximal tubule bicarbonate reclamation Purine metabolism Pyrimidine metabolism Regulation of actin cytoskeleton Renal cell carcinoma Retinol metabolism RNA degradation Spliceosome TGF beta signaling pathway Tight junction Ubiquitin mediated proteolysis Vitamin B6 metabolism Wnt signaling pathway B WT_EGFP S724A_WT Enrichment Score 5 0 5 Annotation Key Carbohydrate metabolism Lipid metabolism Amino acid metabolism Others 16

Fig. S7. Transcriptomic analysis of the effect of blocking Ser 724 phosphorylation on IRE1α-evoked gene expression profiles in liver cells. Primary hepatocytes were infected for 48 hours with adenoviruses expressing EGFP, IRE1α or IRE1α-S724A. Total cellular RNA was subjected to whole-genome microarray analysis using Affymetrix Mouse Genome 430 2.0 Arrays. (A) Individual heatmaps from three independent experiments showing differentially expressed genes elicited by overexpressed IRE1α-WT, which were aligned with changes of these genes caused by S724A mutation. Included are genes exhibiting >1.25-fold alterations or trends of significant changes as determined by RankProd with proportion of false positive (pfp) < 0.1. Gene labels are also indicated. (B) Percentage of all arrayed-probed genes (All genes) that were down- or up-regulated by S724A mutant as compared with percentage of IRE1α (WT)-upregulated (WT UP) or -downregulated (WT DOWN) genes which were down- or up-regulated by S724A. (C) Heatmaps showing IRE1α-evoked changes of cellular pathways or biological processes that were influenced by S724A mutation. Parametric Analysis of Gene Set Enrichment (PAGE) was performed using the KEGG database (http://www.genome.jp/kegg/), with false discovery rate (FDR) < 0.01. Pathways related to metabolism of carbohydrates, lipids and amino acids are indicated. 17

18

Fig. S8. Effects of sxbp1 overexpression or XBP1 knockdown on the expression of gluconeogenic genes. (A and B) Adenoviral overexpression of the spliced form of XBP1 suppressed the expression of G6pase and Pepck while inducing the expression of Bip. Primary hepatocytes were infected for 48 hours with recombinant adenoviruses expressing EGFP or Flag-tagged spliced (s) form of XBP1. Infected cells were then treated with PBS or 100 nm glucagons for 1 hour. (A) sxbp1 protein was analyzed by immunoblotting using the Flag antibody. Actin was shown as the loading control. (B) The mrna abundance of G6pase, Pepck and Bip was determined by quantitative real-time RT-PCR using actin as an internal control. **P < 0.01 by two-way ANOVA. (C and D) Adenoviral knockdown of the expression of XBP1 had no effect on glucagon-stimulated expression of G6pase and Pepck. Primary hepatocytes were infected with the control (CON) or XBP1 shrna adenoviruses for 72 hours. (C) The mrna abundance of sxbp1 and txbp1 was analyzed for infected cells without treatment, and the expression of Erdj4, a XBP1 target gene, was assessed for cells upon treatment with 1 μm thapsigargin for 1 hour by quantitative real-time RT-PCR. (D) The mrna abundance of G6pase and Pepck was determined for cells treated with PBS or 100 nm glucagon for 4 hours by quantitative real-time RT-PCR. The mrna expression of actin was used as an internal control. **P < 0.01 and ***P < 0.001 versus shcon by t-test. All data are shown as the mean ± SEM (n=3 independent experiments). 19

Fig. S9. Effect of acute versus chronic exposure to glucagon on XBP1 mrna splicing and the expression of gluconeogenic genes. Primary hepatocytes were treated with 100 nm glucagon for 4 or 16 hours. The medium was changed every 4 hours to keep the glucagon activity during the 16-hour treatment. (A) The ratios of spliced (s) to total (t) XBP1 mrna and (B) the mrna abundance of G6pase and Pepck were determined by real-time RT-PCR analysis, using actin as an internal control. Results were normalized to the value at 0 min. Data are shown as the mean ± SEM (n=3 independent experiments). **P < 0.01 and ***P < 0.001 versus 0 time point by one-way ANOVA. 20

Fig. S10. (A) Elevated blood glucose and glucagon levels in db/db mice. After a 6-hour fast, blood samples were collected from male C57BL/6J db/db mice or their wild-type (+/+) littermates and supplemented with glucagon inhibitor aprotonin. Glucose concentrations were measured by a glucometer and glucagon levels determined by RIA. Data are presented as the mean ± SEM (n=5/genotype). *P < 0.05, **P < 0.01 versus wild-type littermates by t-test. (B) Increased phosphorylation of hepatic IRE1α is PKA-dependent in db/db mice. Male db/db mice were treated for 2 hours with PBS or H89 (5 mg/kg body weight) through i.p. injection. Phosphorylation of liver IRE1α and eif2α was analyzed by immunoblotting as shown in Fig. 4A. Relative p-ire1α/ire1α and p-eif2α/eif2α ratios were determined from densitometric quantifications of the immunoblots and are shown as the mean ± SEM (n=3/group). **P < 0.01 by one-way ANOVA. 21

Fig. S11. Effects of hepatic IRE1α suppression on XBP1 mrna splicing, body weight and the hyperglycemic response to glucagon in db/db mice. Male db/db mice were infected with adenoviruses Ad-shCON or Ad-shIRE1α- # 2 through tail vein injection (n=5/group). (A) Immunoblotting analysis of liver IRE1α from individual mice at 21 days post infection. (B) The spliced (s) and total (t) XBP1 mrna in the liver was determined by real time RT-PCR after a 6-hour fast. (C) Knockdown of hepatic IRE1α expression did not affect body weight. Body weight was determined for db/db mice infected for 21 days. (D) Hepatic IRE1α knockdown reduced glucagon-induced elevations of blood glucose. Glucagon challenge test was performed in mice infected for 11 days through administration i.p. of 150 μg/kg glucagon after a 15-hour fast. Blood glucose was measured at the indicated time points. *P < 0.05 and **P < 0.01 by two-way ANOVA. The bar graph indicates the areas under the curve (AUC) for the glucose levels during the test, shown as the mean ± SEM (n = 5/group). *P < 0.05 by t-test. 22

Fig. S12. Schematic model for the PKA-dependent mechanism that links the IRE1α branch of the UPR with the GPCR signaling pathway. Metabolic signals such as GPCR agonists (glucagon or epinephrine) stimulates PKA, which in turn directly phosphorylates IRE1α at Ser 724, a critical regulatory site within the activation segment of IRE1α. Phosphorylation activation of IRE1α as such plays an important role in glucagon-regulated metabolic programs in the liver, e.g. promoting the expression of gluconeogenic genes in an XBP1-independent fashion. Under obesity-associated metabolic stress, dysregulation of PKA activity, in addition to aberrant ER lipid and calcium metabolism, contributes to increased phosphorylation of hepatic IREα, which constitutes a critical component in perturbation of glucose homeostasis. Thus, IREα also integrates metabolic signals through phosphorylation by PKA of the GPCR pathway in liver cells and is implicated in the control of glucose metabolism. 23

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