Supporting Information

Suhara et al. 10.1073/pnas.1515872112 SI Materials and Methods upon Cre-mediated recombination (5). The primer sequences of Mice Breeding. Phd2F/F mice were crossed with the Cre-ER mice Rn18s locus as a control are shown in Table S2. + + or Alb-Cre mice to generate Phd2 /F;Cre-ER or Phd2 /F;Alb-Cre + + mice, respectively. Phd2 /F;Cre-ER or Phd2 /F;Alb-Cre mice were Primary Hepatocytes Isolation and Culture. Portal veins of 10-wk-old then intercrossed to generate Phd2F/F;Cre-ER (Phd2-SKO) or male mice were perfused with HBSS buffer without calcium and Phd2F/F;Alb-Cre (Phd2-LKO) mice, respectively, as well as rele- magnesium (085-09355; Wako) supplemented with 10 mM Hepes vant littermate controls (Phd2F/F). and 0.5 mM EGTA for 10 min, then perfused with low-glucose DMEM (041-29775, Wako) supplemented with 10 mM Hepes + + − − Lactate Efflux Assay. First, 1 × 106 cells of Phd2 / or Phd2 / and 0.4 mg/mL collagenase type 4 (Worthington). After being 2 MEFs (5) were plated on six-well dishes (9.4 cm surface/well) filtered through a 70-μm cell strainer (352350; BD Biosciences), with 1 mL/well of fresh culture media. Lactate concentrations in the isolated hepatocytes were washed using DMEM low glucose the fresh culture media and the culture supernatant (8 h after and centrifuged at 50 × g for 3 min at 4 °C three times. Viable Δ plating) were compared, and then lactate efflux was calculated hepatocytes were counted and then plated onto the collagen type (pmol/mL/hour/cell). 1-coated 100-mm dishes (4020-010; Iwaki) at 6 × 106 cells per ’ Urinary Lactate Measurement. Eight- to 10-wk-old female Control dish in hepatocyte culture media consisting of William sEme- and Phd2-SKO mice were orally administered 1 mg tamoxifen dium (W4128; Sigma-Aldrich) supplemented with 10% FBS – dissolved in 100 μL corn oil twice on day 0 and day 1. On day 2, (172012; Sigma-Aldrich), 1% antibiotic antimycotic (15240-062; 0.5 mg/g body weight of lactate dissolved in 0.9% NaCl was in- Gibco), 1% MEM nonessential amino acids (11140-050; Gibco), jected into mice intraperitoneally, and urine samples were col- 1% ITS (41400-045; Gibco), 2 mM L-glutamine (35050-061; Gib- lected at 0 and 20 min after the lactate injection. Urinary lactate co), 1 mM sodium pyruvate (11360-070; Gibco), 100 nM dexa- levels were measured using Lactate Pro Test Meter and Lactate methasone (872454; MSD Japan), and 10 ng/mL recombinant Pro Test Strip. murine EGF (315-09; PeproTech). Hepatocytes were cultured at 37 °C in 21% O2 and 5% CO2, and the nonadhering cells were RNA Isolation and Real-Time RT-PCR Analysis. Total RNA was iso- removed by changing the media 4 h after plating. Three days lated from the liver, using TRIzol (15596-026, Invitrogen) and later, the culture media were replaced with hepatocyte culture μ RNeasy column (74104, Qiagen). Purified RNA samples (1 g) media without ITS 3 h before the in vitro tracer assay. Then, were reverse transcribed into cDNA using AffinityScript QPCR hepatocytes were labeled for 5 min with 10 mM 13C -lactate in cDNA Synthesis Kit (600559; Agilent Technologies). Real-time 3 hepatocyte culture media without ITS, of which pH was adjusted RT-PCR was performed with THUNDERBIRD SYBR qPCR Mix (QPS-201; TOYOBO), using real-time PCR system (Model to 7.20 with NaOH. Metabolites were extracted as previously 7300; Applied Biosystems). The mRNA levels were normalized reported (32). to β-actin (Actb). The primer sequences are shown in Table S1. Venous Blood Gas Analysis. Before and 20 min after the i.p. injection – Quantitative PCR for Genomic DNA. Genomic DNA was extracted of 0.5 mg/g body weight of L-lactic acid, 8 10-wk-old Control and from various tissues including the liver, kidney, heart, and skeletal Phd2-LKO female mice were anesthetized with isoflurane, and muscle of 8–10-wk-old Phd2-LKO male mice, using DNeasy whole-blood samples were taken from the retro-orbital sinus using heparinized Micro-Hematocrit Capillary Tubes (22-362-566; Blood & Tissue Kit (69504; Qiagen). A total of 50 ng DNA was − analyzed using THUNDERBIRD SYBR qPCR Mix by real-time Thermo Fisher Scientific Inc.). pH, HCO3 ,baseexcess,and PCR system. The primers were designed to amplify intronic se- anion gap were measured using i-STAT 1 analyzer and EC8+ car- quences (intron 2–3) of Phd2 that are expected to be excised out tridge (Abbott).

Suhara et al. www.pnas.org/cgi/content/short/1515872112 1of5 A GLUT1 PFK-M PGK1 p=0.170 p=0.206 p=0.00469 0.12 12 25 0.10 10 20 0.08 8 0.06 6 15 0.04 4 10 0.02 2 5 Pgk1 / Actb Pgk1 / 0 Actb Pfkm / 0 0 Slc2a1 / Actb Slc2a1 /

LDHA PDK1 p=0.000101 p=0.000105 100 0.8 80 0.6 Control (n=3) 60 0.4 40 Phd2-SKO (n=3) 20 0.2 Ldha / Actb Ldha / 0 Actb Pdk1 / 0

B p=0.468 p=0.280 p=0.359 p=0.00142 1.0 0.8 0.6 0.4 intact allele

frequency 0.2 0 Phd2 Control Phd2-LKO Phd2-LKO Phd2-LKO Phd2-LKO DNA Liver Kidney Heart Muscle

C Ad libitum Fasted p=0.544 p=0.728 200 100 150 80 60 100 40 50 20 0 0 Control (n=9) Control (n=12) Phd2-LKO (n=11)

Blood glucose level (mg/dL) Phd2-LKO (n=9) Blood glucose level (mg/dL)

Fig. S1. Systemic or liver-specific inactivation of Phd2.(A) Real-time RT-PCR analysis of transporters or enzymes involved in the skeletal muscles of Control and Phd2-SKO mice: glucose transporter GLUT1 (Slc2a1), phosphofructokinase, muscle (Pfkm), phosphoglycerate kinase 1 (Pgk1), LDHA (Ldha), and PDH kinase 1(Pdk1). Error bars, SEM. (B) Phd2 intact allele frequency was analyzed in the indicated tissues of Phd2-LKO mice (n = 3) by real-time PCR. The copy number of Phd2 locus and Rn18s locus was compared. All of the values were normalized with the control DNA (genomic DNA from livers of Phd2F/F mice carrying no Cre- recombinase transgene; n = 3). The unpaired Student t-test was performed versus control DNA. Note that gene disruption in Phd2-LKO mice is liver-specific. Error bars, SEM. (C) Blood glucose levels under the ad libitum (Left) or fasted (Right) conditions in Control and Phd2-LKO mice. Error bars, SEM.

Suhara et al. www.pnas.org/cgi/content/short/1515872112 2of5 A MCT1 MCT4 LDHB PC p=0.105 p=0.727 p=0.853 p=0.654 0.4 0.003 0.008 2.0 0.3 0.006 1.5 0.002 0.2 0.004 1.0 0.001 0.1 0.002 0.5 Pcx / Actb 0 0 0 0 Slc16a3 / Actb Slc16a1 / Actb MDH2 MDH1 FBP1 G6PD p=0.789 p=0.687 p=0.700 p=0.262 0.6 6 6 0.015

0.4 4 4 0.010

0.2 2 2 0.005 Mdh1 / Actb Fbp1 / Actb Ldhb / Actb Mdh2 / Actb 0 0 0 G6pd / Actb 0 Control (n=8) Phd2-LKO (n=7) B p=0.635 p=0.694 p=0.338 p=0.0606 12 2.0 2.0 2.0 10 1.5 1.5 1.5 8 6 1.0 1.0 1.0 4 0.5 0.5 0.5

-glucose (mM) 2 -glucose (mM) -glucose (mM) -glucose (mM) 6 1 2 3

C 0 C 0 C 0 C 0.0 12 13 13 13 p=0.00398 p=0.0132 p=0.00506 0.5 0.3 0.15 0.4 Control (n=15) 0.2 0.10 0.3 Phd2-LKO (n=15) 0.2 13 0.1 0.05 Natural abundances of Cx-glucose 0.1 -glucose (mM) -glucose (mM) -glucose (mM) 4 5 6

C 0.0 C 0.0 C 0 13 13 13

Fig. S2. Gluconeogenesis in Phd2-LKO liver. (A) Real-time RT-PCR analysis of transporters or enzymes involved in gluconeogenesis in the livers of Control and Phd2-LKO mice: MCT1 (Slc16a1), monocarboxylate transporter 4 (MCT4) (Slc16a3), lactate dehydrogenase B (LDHB) (Ldhb), pyruvate carboxylase (PC) (Pcx), MDH2 (Mdh2), malate dehydrogenase 1 (MDH1) (Mdh1), fructose-1,6-bisphosphatase 1 (FBP1) (Fbp1), and glucose-6-phosphate dehydrogenase (G6PD) (G6pd). 12 13 13 13 Error bars, SEM. (B) LC/MS-based quantification of nonlabeled ( C6) and C-labeled ( C1– C6) glucose. Note that the shaded bars indicate the estimated 13 natural abundances of Cx-glucose. Error bars, SEM.

Suhara et al. www.pnas.org/cgi/content/short/1515872112 3of5 HK2 PFK-L 0.008 p=0.268 0.06 p=0.0578 0.007 0.05 0.006 0.005 0.04 0.004 0.03 0.003 0.02 Pfkl/Actb Hk2/Actb 0.002 0.001 0.01 0 0 Control Phd2-LKO Control Phd2-LKO

PK-LR 0.4 p=0.262 0.3 0.2 Control (n=8)

Pklr/Actb 0.1 Phd2-LKO (n=7) 0 Control Phd2-LKO

Fig. S3. Expression of glycolytic genes in Phd2-LKO liver. The expression levels of Hk2, Pfkl, and Pklr in the livers of Control or Phd2-LKO mice were analyzed by real-time RT-PCR. Error bars, SEM.

LDHA PDK1 p=0.00000221 4 0.5 p=0.00000783 3 0.4 0.3 2 0.2 1 Ldha/Actb Pdk1/Actb 0.1 0 0 Vehicle GSK360A Vehicle GSK360A

PGK1 LAT1 p=0.00000633 p=0.0239 2.0 0.015 1.5 0.010 1.0 0.005 0.5 Pgk1/Actb Slc7a5/Actb 0 0 Vehicle GSK360A Vehicle GSK360A

Fig. S4. GSK360A-induced hypoxic response. Real-time RT-PCR analysis of Ldha, Pdk1, Pgk1, and L-type amino acid transporter 1 (LAT1) (Slc7a5), known as direct HIF-target mRNAs, in the livers of vehicle or GSK360A treated wild-type male mice (n = 3). Total RNA from the liver was isolated 6 h after the oral administration of vehicle (1% methyl cellulose) or GSK360A (30 mg/kg body weight). Error bars, SEM.

Suhara et al. www.pnas.org/cgi/content/short/1515872112 4of5 Table S1. Primer sequences for mRNA expression analysis Gene Oligonucleotide sequence (forward) Oligonucleotide sequence (reverse)

Phd2 5′-AATGGAGATGGAAGATGCG-3′ 5′-TGGGTTCAATGTCAGCAAAC-3′ Slc2a1 5′-CCCCCCAGAAGGTTATTGAG-3′ 5′-CCAACAGGTTCATCATCAGC-3′ Slc2a2 5′-GTCGCCTCATTCTTTGGTG-3′ 5′-CTGATACACTTCGTCCAGC-3′ Hk2 5′-TACCACACACCCTACAGCAG-3′ 5′-ACCCTCTGGAGACCATTGTC-3′ Pfkl 5′-TACCGTGGACCTGGAGAAAC-3′ 5′-CATAGATGAGGAAGACTTTGGC-3′ Pfkm 5′-GCGTGTCTATGATGCTTCAG-3′ 5′-AAAGTGTCAGCCCCAGTGAG-3′ Pgk1 5′-GATGAGGGTGGACTTCAAC-3′ 5′-TAAGGACAACGGACTTGGC-3′ Pklr 5′-TTCTTCCAGCAGCAGCAAC-3′ 5′-TCATCTCCTTGAGGCGGTC-3′ Pdk1 5′-ACCTCGTTTATGTTTCTGCG-3′ 5′-CAACTCCTGAAGGCTTTGG-3′ Ldha 5′-ACAGTTGTTGGGGTTGGTGC-3′ 5′-CGCAGTTACACAGTAGTCTTTG-3′ Ldhb 5′-CTAAGCACCGTGTGATTGG-3′ 5′-ATTCAGTTCCTGGAGGGAG-3′ Pcx 5′-CCACTATGACTCTCTGCTCG-3′ 5′-GGAACTGCTGGTTGTTGAG-3′ Mdh1 5′-ATGATGGGTGTTCTGGACG-3′ 5′-TCACATTGGCTTTCAGTAGG-3′ Mdh2 5′-CCAGAGCAAATGTGAAAGGC-3′ 5′-ATGGTAGCGTTGGTGTTG-3′ Pck1 5′-GGAAGGACAAAGATGGCAAG-3′ 5′-TCAGGTTCAAGGCGTTTTC-3′ Fbp1 5′-TGGATTGTGGTGTCAACTG-3′ 5′-AGTCCTTGGCATAACCCTC-3′ G6pd 5′-ACCGTCTATTCTACCTGGC-3′ 5′-AGAGAGGAGATGTGGTTCG-3′ Slc16a1 5′-TGGTTGTCTGTCTGGTTGC-3′ 5′-CAGTGGTCGCTTCTTGTAG-3′ Slc16a3 5′-AGCCCAGTGTTCCTTTGTG-3′ 5′-ACAGCAGTTGAGCAGTAGG-3′ Slc16a7 5′-TTCAACACCACCTCCAGTC-3′ 5′-CAGCATAATAGTCCTCCCAC-3′ Slc7a5 5′-GCCCTCATCATTTTGCTCG-3′ 5′-TCAGATAGTTCCATCCTCCG-3′ Actb 5′-AACCGTGAAAAGATGACCC-3′ 5′-TACGACCAGAGGCATACAGG-3′

Table S2. Primer sequences for genotyping and quantitative PCR for genomic DNA Gene Oligonucleotide sequence

Phd2 wild type and conditional allele Fwd 5′-AGATGACCTCCCCAACTCTGCTAC-3′ Rev 5′-CAGTGTTCTGCCTCCATTTAT-3′ Cre-ER or Alb-Cre Fwd 5′-CGGGTCAGAAAAAATGGTGTTG-3′ Rev 5′-CGGTATTGAAACTCCAGCG-3′ Rn18s Fwd 5′-CGGCTACCACATCCAAGGAA-3′ Rev 5′-GCTGGAATTACCGCGGCT-3′

Suhara et al. www.pnas.org/cgi/content/short/1515872112 5of5