Supplemental Data
Article
Pharmacological stimulation of NADH oxidation ameliorates obesity and related phenotypes in mice
Jung Hwan Hwang*, Dong Wook Kim*, Eun Jin Jo, Yong Kyung Kim, Young Suk Jo, Ji Hoon Park,Sang Ku Yoo, Myung Kyu Park, Tae Hwan Kwak, Young Lim Kho, Jin Han, Hueng-Sik Choi, Sang-Hee Lee, Jin Man Kim, InKyu Lee, Taeyoon Kyung, Cholsoon Jang, Jongkyeong Chung, Gi Ryang Kweon, Minho Shong
*These authors contributed equally to this work.
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Supplemental Materials and Methods
Generation of NQO1 knockout mice. Restriction analysis and sequencing were used to identify suitable regions for subcloning into the NQO1 targeting vector. Left-arm (NCBI; NW_001030904.1, 35399239~35401310) and right-arm (NCBI; NW_001030904.1, 35404763~35411720) of NQO1 genomic DNA were amplified by PCR using the genomic DNA extracted from J1 embryonic stem (ES) cells. A 3425 bp SalI/BamHI fragment including exons 5 and 6 of NQO1 gene was replaced by the PGK-neomycin cassettes of the pPNT vector. The linearized targeting construct was introduced into J1 ES cells (129/SvJ strain) by electroporation and resistant cells were selected in the presence of G418. Selected ES cell lines were injected into C57BL/6 blastocysts, which were subsequently transferred into pseudopregnant females to generate chimeric offspring. Chimeras were bred with C57BL/6 female mice to produce heterozygotes. The genotypes of mutant mice were determined by PCR and confirmed by Southern blotting of genomic DNA from tail biopsies. Briefly, tail samples were incubated at 100°C for 2 hours in lysis buffer (50 mM NaOH, 1 M Tris-HCl pH 8.0), followed by centrifugation to isolate DNA. PCR was performed using genomic DNA with specific primers for the NQO1 wild-type allele against exon 6 of NQO1 genes (forward, 5’-TGTGTACCGTGTGTATGCAA-3’ and reverse, 5’-CTAAGACCTGGAAGCCACAG-3’) and the NQO1 knockout allele against neomycin genes (forward, 5’-GAAGGGACTGGCTGCTATTG-3’ and reverse, 5’-AATATCACGGGTAGCCAACG-3’), respectively. The following amplification protocol was used: 94°C for 5 min, 30 cycles at 94°C for 30 s, 56°C for 30 s, 72°C for 1 min. For southern blotting, genomic DNA was digested with ScaI and hybridized with a probe corresponding to nucleotides 35386413~35386020 (NCBI; NW_001030904.4) of the NQO1 gene, located outside of the targeting construct. The predicted sizes of the hybridizing fragments in the original and disrupted genes are 11 and 8.3 kb, respectively. Disruption of NQO1 was further analyzed by immunoblotting.
Preparation of MEF and cell culture. For preparation of MEF, a 12.5 to 14.5 day pregnant mouse was anesthetized and its skin and hair were sterilized with 70% ethanol.The embryonic sac was thenremoved with a scissors and placed into a dish. After removing the placenta and membranes from embryos, the head, liver, and tail of embryos were eliminated and trypsinized for 30 min in 37°C incubators and then added with 10 ml of complete media (DMEM with 10% FBS) and incubated for 15 min. The cell suspension was maintained with 95% of cell confluence.HepG2, HEK293, and L6 myoblasts were obtained from ATCC and maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS, 100 units/mL penicillin, and 100 µg/mL streptomycin in a humidified atmosphere maintaining 5% CO2 and 95% air at 37°C.
Synthesis and formulation of compounds. β-Lapachone and other related 1,2-naphthoquinones were prepared by two step synthetic processes, resulting in a purity >99.9% as described previously (1). Briefly, the lithium salt of 2-hydroxy-1,2-naphthoquinone was treated with several allyl-halides, such as 3-bromo-1-propene, 1-bromo-3-methyl-2-butene, 3-bromo-2-methyl-1-propene, 1-bromo-2-butene, to give several 2-hydroxy-3-allyl-1,4-naphthoquinone derivatives. Then each 2-hydroxy-3-allyl-1,4-naphthoquinone derivative was treated with H2SO4 and purified by recrystallization to give several pure 1,2-naphthoquinones, including β-lapachone. βL was administered orally as micronized particles coated with calcium silicate.
NADH recycling assays. Assays were performed with hNQO1 (Sigma, EC number 1.6.99.2). The assay using recombinant hNQO1 (0.03U) was mixed with 200 M NADH in 50 mM potassium phosphate buffer, pH7.0. Reactions were initiated by the addition of 10 M of beta-lapachone, alpha-lapachone, tanshinone IIA, crypto-tanshinone, and then the change in absorbance at 340 nm was measured over time for 5 min as described previously (2).
Analysis of NADH fluorescence. Experiments were performed using a microfluorometric system consisting ofa Nikon inverted fluorescence microscope (Eclipse TE300, Japan) with a dry-typefluorescence objective lens (×40, numerical aperture of 0.85),a photomultiplier tube (type R1527, Hamamatsu Photonics, Hamamatsu,Japan), and a Deltascan illuminator (Photon Technology InternationalInc., Lawrenceville, NJ). Fluorescence was excited with a 75 W xenon arc lamp with a 10 Hz chopper wheel to generate monochromatic light (350 nm) with intensity of 405 ± 15 nmand frequency of 460 ± 10 nm. Although auto-fluorescence at 460 nm can arise from unknown intracellular constituents or cytosolic NADH, the majority of light detected by this method is from mitochondrial NADH as reported previously (3; 4).
Glucose tolerance and insulin stimulation test. Glucose tolerance tests were performed in 12 h-fasted DIO mice and OLETF rats as described previously (5). Insulin stimulation tests were performed in 6 h-fasted DIO mice and OLETF rats as described previously (6).Animals were injected i.p. with glucose (1 g per kg body weight) or human insulin [0.5 international unit (IU) per kg body weight] and blood glucose level was measured.
Analysis of cytotoxicity. For the assessment of cytotoxicity, we used a crystal violet assay, which is based on the inability of dead cells to adhereto the cell culture plates (7). After incubation, cells were washed with cold-PBS to remove dead, non-adherent cells. The viable cells were fixed with 4% paraformaldehyde for 20 min and stained with 1% crystal violet solution at room temperature for 5 min. The plates were thoroughly washed with sterile water and crystal violet was dissolved in DMSO. The absorbance of dissolved dye, corresponding to the number of viable cells was measured in an automated microplate reader at 595 nm.
Analysis of mitochondrial DNA contents. Total genomic and mitochondrial DNA were prepared from soleus muscle of mice treated with vehicle or βL using QIAamp®DNA Mini Kit (QIAGEN, Chatworth, CA) according to the manufacturer’s instructions. mtDNA/genomicDNA ratio was analyzed by quantitative real time PCR using specific primers. Primer sequence for mtDNA and genomicDNA; mtDNA (mitochondrial D-loop region), forward (5’-AATCTACCATCCTCCGTGAAACC-3’), reverse (5’-TCAGTTTAGCTACCCCCAAGTTTAT-3’); genomicDNA (telomerase reverse transcriptase region), forward (5’-CTAGCTCATGTGTCAAGACCCTCTT-3’), reverse (5’-GCCAGCACGTTTCTCTCGTT-3’).
Statistical analyses. Results were expressed as the mean ± SD. Differences between groups were examined for statistical significance using Student's t test and analysis of variance (ANOVA). The difference was considered to be significant if P<0.05.
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SupplementalTable 1. Primer sequences used in quantitative RT-PCR
Gene / Direction / Sequence (5’-3’)PGC-1α / Forward / CGATGTGTCGCCTTCTTGCT
Reverse / CGAGAGCGCATCCTTTGG
NRF1 / Forward / TGATGGAGAGGTGGAACAAA
Reverse / GGTTTCCCCAGACAGGACTA
mtTFA / Forward / ATACCTTCGATTTTCCACAGAAC
Reverse / TCATTTCATTGTCGTAACGAATC
LCPT1 / Forward / TTACTTCAAGGTCTGGCTCTACC
Reverse / CTCCTTTACAGTGTCCATCCTCT
UCP1 / Forward / GGGACCTACAATGCTTACAGAGT
Reverse / GTACAATCCACTGTCTGTCTGGA
UCP2 / Forward / AGCCTACAGATGTGGTAAAGGTC
Reverse / GCTCATAGGTGACAAACATCACT
AMPKα1 / Forward / TTCCGAAGTATCTCTTTCCTGAG
Reverse / ACGCAAATAATAGGGGTTTACAA
AMPKα2 / Forward / AGGAAGTGTGTGAGAAATTCGAG
Reverse / CCAGGTAAAGCTGTAAGCTCATT
PPARα / Forward / TGCAGACCTCAAATCTCTGG
Reverse / TAGCCTTGGCAAATTCTGTG
PPARγ / Forward / ATCTTAACTGCCGGATCCAC
Reverse / TGGTGATTTGTCCGTTGTCT
COX4 / Forward / GACTACCCCTTGCCTGATGT
Reverse / GCAGTGAAGCCAATGAAGAA
COX7 / Forward / GCTCTGGTCCGGTCTTTTAG
Reverse / TCACTTCTTGTGGGGGAAG
AOX / Forward / TATGGTGTCGTACTTGAATGACC
Reverse / GGGTCACATCCTTAAAGTCAAAG
GLUT2 / Forward / GCTGTACTGAGTTCCTTCCAGTT
Reverse / GTCCTGAAATTAGCCCACAATAC
GLUT4 / Forward / CAGCGAGTGACTGGAACACT
Reverse / CAGCATAGCCCTTTTCCTTC
SIRT1 / Forward / AGTAAGCGGCTTGAGGGTAA
Reverse / AAATCCAGATCCTCCAGCAC
SIRT2 / Forward / CTTGCCAAGGAGCTCTATCC
Reverse / ACGATATCGGGCTTTACCAC
SIRT3 / Forward / CACTACAGGCCCAATGTCAC
Reverse / TCACAACGCCAGTACAGACA
FAS / Forward / CCTGCTATCATCTGACTTCCTCT
Reverse / AGGGTGGTTGTTAGAAAGATCAA
LPL / Forward / AGGACACTTGTCATCTCATTCCT
Reverse / AGACATCTACAAAATCAGCGTCA
ATGL / Forward / CGCCTTGCTGAGAATCACCAT
Reverse / AGTGAGTGGCTGGTGAAAGGT
SCD1 / Forward / AGTACGTCTGGAGGAACATCATT
Reverse / GCTTGTAGTACCTCCTCTGGAAC
FigureS1. NADH oxidation by various compounds and requirement of NQO1 forβL-inducedNADH oxidation.
(A) NADH oxidation by various compounds. Recombinant human NQO1 (0.03U,Sigma, EC number 1.6.99.2) was mixed with NADH (200 µM) in potassium phosphate buffer (50 mM, pH7.0). Reactions were initiated by the addition of beta-lapachone (βL),alpha-lapachone (L), tanshinone IIA, or cryptotanshinoneand the change in absorbance at 340 nm was measured after 5min. The concentration of oxidized NADH was calculated and divided by the concentration of compounds.
(B)A transient decrease in NADH levels following βL treatment. The auto-fluorescence of NADH after βL (5 µM) treatment was monitored in HepG2 (red), HEK293 (black), and HEK293 transfectants with wild-type NQO1 (orange) or catalytically-inactive NQO1(NQO1 C609T)(gray). An arrow indicates the time of βL treatment.
Figure S2. Requirement for NQO1 in βL-induced Phosphorylation of AMP kinase and ACC.
(A) L6 myoblasts were treated with DMSO or βLat the indicated time points. Phosphorylation of AMPK and ACC was detected by immunoblotting with antibodies against the indicated proteins.
(B) L6 myoblasts were pre-incubated with NQO1 specific inhibitors, dicoumarol and ES936, for 30 min and treated with βL (5 µM)for 30 min. Lysates were immunoblotted with antibodies against the indicated proteins.
FigureS3. Food intake of pair-fed DIO mice
Food intake of three mouse groups, untreated (; n=6), pair-fed (; n=8), and 50 mg/kg/day βL-treated (; n=8) group, were monitored for 8 weeks after oral administration of βL (*P<0.05;**P<0.005).
Figure S4. The effects of βLtreatment on the weights of adipose and other organs.
The weights of adipose tissues and other solid organs were compared between DIO mice treated with vehicle (n=5)and those treated with 50 mg/kg/day βL (n=5) for 4 weeks.
FigureS5. The effect of βL on glucose homeostasis
(A and B) Intraperitoneal glucose tolerance test (IPGTT)(A) and insulin stress test(5)(B) were performed in DIO mice treated with vehicle (; n=5) or 50 mg/kg/day βL (; n=5) for 3 days. (C and D)IPGTT and IST were performed as described in (A) and (B), except using male Otsuka Long Evans Tokushima Fatty(OLETF) rats treated with vesicle (; n=5)or 300 mg/kg/day βL(; n=5) for 3 days. (*P<0.05;**P<0.005).
Figure S6.Comparison ofNQO1 activity in various tissues of lean and DIO mice
NQO1 activities in the indicated organs of lean (white; n=9) and DIO (black; n=9) male mice were compared(*P<0.05;**P<0.005). BAT: Brown adipose tissue.
Figure S7. Comparison of βL-mediated cytotoxicity between HepG2 and MEF
HepG2 (10,000 cells/well; open bars) and MEF (10,000 cells/well; closed bars) were seeded in 96-well plates and cultured for 24 hours. After incubation with βL for the indicated times, cells were fixed with 4% paraformaldehyde for 20 min and stained with crystal violet (1%) for 5 min. After washing with distilled water, each well was added with 100 µl of DMSO and then the number of viable cells was determined by absorbance at 595 nm.
Figure S8. The effect of palmitate on βL-mediated cytotoxicity in MEF
Cells were seeded in 96-well microtiter plates (10,000 cells/well) and incubated for 24 hours. Cytotoxicity of cells treated with DMSO or βL (5 M) together with palmitate (500 M) or dicoumarol (25 M) for 3 hours was measured using crystal violet. All experiments were performed in triplicate.
SupplementalReferences
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