<8D6291E D A7788CA985F95B D5D975995BD816A2E706466>

Transcription

1

2 Figure 51 Figure TP53 77 Figure

3 97 Figure 115 reference

4 ACTB β-actin ADIPOQ Adiponectin ATG autophagy-related gene AMPK AMP-activated protein kinase ATP adenosine triphosphate BAF bafilomycin A1 BECN1 Beclin 1 BMI body mass index CASP caspase CCL2 C-C motif chemokine ligand 2 CIDEC cell death-inducing DFFA-like effector c CTSB cathepsin B CTSD cathepsin D CTSL cathepsin L Cont control DEX dexamethasone DMEM Dulbecco s modified eagle medium EDTA ethylenediaminetetraacetic acid ER endoplasmic reticulum FABP4 fatty acid binding protein 4 FFA free fatty acid GAPDH glyceraldehyde-3-phosphate dehydrogenase GFP green fluorescent protein HE hematoxylin eosin HFD high fat diet IBMX 3-isobutyl-1-methylxanthine IL interleukin LAMP lysosomal associated membrane protein; LEP leptin LMNB1 Lamin B1 MAP1LC3B/LC3B microtubule associated protein 1 light chain 3 1

5 MTORC1 mechanistic target of rapamycin complex 1 MEF mouse embryonic fibroblast Neo neomycin ND normal diet NRF2 nuclear factor, erythroid 2 like 2 MDM2 murine double minute PGC-1α PPARgamma coactivator-1alpha PDL poly-d-lysine PPAR peroxisome proliferator-activated receptor PPARGC1A PPARG coactivator 1 alpha ROS reactive oxygen species RPS18 ribosomal protein 18 SD standard deviation SA-GLB1/SA β-gal senescence-associated galactosidase beta 1 SPF specific pathogen free TBP tata-binding protein TFAM transcription factor A of mitochondrial TG triglyceride SERPINE1 serpin family E member 1 SQSTM1 sequestosome 1 TFEB transcription factor EB 2

6 TG (triglyceride) ADIPOQ/adiponectin TNF (tumor necrosis factor) SERPINE1/PAI1 (serpin family E member 1) TG ADIPOQ TNF SERPINE1 TP53/p53 QOL quality of life 2016 ATG autophagy-related gene 3

7 ATP adenosine triphosphate PPARGC1A/PGC1α peroxisome proliferator-activated receptor-γ coactivator-1a PPARGC1A TG 1 cathepsin L CTSL B CTSB 3T3L1 CTSL CTSB CTSB CTSB CTSL CTSB 2 CTSL CTSB 4

8 1 CTSB 1 PLIN1 3 TP53 3T3L1 TP53 PPARGC1A TP53 3T3L1 TP53 4 SQSTM1/p62 SQSTM1 KEAP1/NFE2L2 Nrf2 KEAP1/NFE2L2 NFE2L2 Hmox1 heme oxygenase 1 Nqo1 NAD(P)H quinone dehydrogenase 1 ROS reactive oxygen species SQSTM1-KEAP/NFE2L2 L TP53 TP53 PPARGC1A 5

9 L 6

10 7

11 3 1 (Flegal et al., 2012) , ,000 1,010 8, , , ,000 ( ) (Kohara et al., 2014) QOL ( ) (TG; triglyceride) (LD; lipid droplet) TG (FFAs; free fatty acids) β ATP (Lira et al., 2011; Samaras et al., 2010) FFAs (Kolditz and Langin. 2010) (subcutaneous adipose tissue) (visceral adipose tissue) TG TG TG TG FFAs (Lira et al., 2011; Samaras et al., 2010) 8

12 (Cinti, 2005; Ouchi et al., 2011) ADIPOQ/adiponectin ADIPOQ AMPK (Yamauchi et al., 2001; Ouchi et al., 2011) TNF (tumor necrosis factor alpha) IL6 (interleukin-6) CCL2 (C-C motif chemokine ligand 2)/MCP1 (monocyte chemoattractant protein-1) SERPIN1 (serpin family E member 1)/PAI1 (plasminogen activator inhibitor type 1) (Ouchi et al., 2011) TG TNF SERPIN1 (Lira et al., 2011; Samaru et al., 2010) TG FFAs (macroautophagy) (chaperone-mediated autophagy) (microautophagy) (Levine and Kroemer, 2007; Mizushima et al., 2008) ATG (autophagy-related gene) (Tsukada and Ohsumi, 1993) (Inami et al., 2007; Takamura et al., 2011) (Ebato et al., 2008; Jung et al., 2008) (Hara et 9

13 al., 2006; Komatsu et al., 2006; Liang et al., 2010) ATG ATG8 MAP1LC3 (microtubule associated protein 1 light chain 3)/LC3 LC3 prolc3 C ATG4 LC3-I ATG12-ATG5-ATG16L PE (phosphatidylethanolamine) C LC3-II (Kabeya et al., 2000) LC3-II LC3 (Mizushima et al., 2010) (Ezaki et al., 2011) (Hara et al., 2006; Komatsu et al., 2006; Liang et al., 2010) ROS ATP (Scherz-Shouval and Elazar, 2007) (Rabinowitz and White, 2010) β Atg7 (Ebato et al., 2008; Jung et al., 2008) β β TG 10

14 (Singh et al., 2009) Singh in vitro in vivo TG (lipid droplet; LD) TG TG LD ATG5 ATG7 ER (endoplasmic reticulum) (Yang et al., 2010) TG (Singh et al., 2009; Singh et al.,2009) LC3 SQSTM1/p62 LC3-II LC3-II (Mizushima et al., 2010) SQSTM1 LC3-II (Moscat and Diaz-Mecok, 2009; Katsuragi et al., 2015) LC3-II ( ) SQSTM1 ( ) (Tanida et al., 2005; Moscat and Diaz-Meco, 2009) SQSTM1 SQSTM1 mrna SQSTM1 SQSTM1 SQSTM1 (Moscat and Diaz-Meco, 2009; Katsuragi et al., 2015) SQSTM1 GFP RFP LC3 GFP LC3 (GFP-LC3) LC3 GFP GFP GFP (Mizushima et al., 2010; Ni et al., 2011) GFP-LC3 GFP GFP 11

15 (Ni et al., 2011) GFP (Kuma et al., 2007; Tanida et al., 2008) RFP GFP RFP GFP-LC3 (mrfp-gfp-lc3) RFP GFP GFP RFP (Kimura et al., 2007; Ni et al., 2011) RFP-GFP-LC3 RFP (Katayama et al., 2011) in vitro in vivo LC3-II LC3-II GFP-LC3 GFP-LC3 dot (Iwai-Kanai et al., 2009) LC3-II ( ) LC3-II turnover assay LC3-II bafilomycin A1 (BAF) E64d chloroquine LC3-II LC3-II LC3-II LC3-II LC3-II in vivo 12

16 ph5 20 (Appelqvist et al., 2013; Settembre et al., 2013) 1950 Christian de Duve (cathepsin) A Z (Olson and Joyce, 2015) (cathepsin maturation) A G D E B L (Repnik et al., 2012) B (CTSB) L (CTSL) D (CTSD) (Kaminskyy and Zhivotovsky, 2012) (Cox and Cachón-González. 2012; Platt et al., 2012) α NPC (Niemann-Pick disease, type C1) (Liao et al., 2007) (Nixon, 2013; Inami et al., 2012) ph CTSB CTSD (Coffey et al., 2014; Wolfe et al., 2013) ph CTSL CTSB (Inami et al., 2012) 13

17 ATP (Brookes et al., 2004; Mishra and Chan, 2014; Shadel and Horvath, 2015) ATP ROS (reactive oxygen species) (Ott et al., 2007) Ca 2+ (Cali et al., 2012) (Martinou and Youle, 2015) (Moslehi et al., 2012) (Jitrapakdee et al., 2010) (Brandon et al., 2009) (Exner et al., 2012) (Kusminski et al., 2012) (Choo et al., Kusminski et al., 2012) ( ) (Shadel et al., 2015) NRF1 (nuclear respiratory factor 1) NFE2L2 (nuclear factor, erythroid 2 like 2)/NRF2 PPARs (peroxisome proliferator activated receptor) DNA TFAM (transcription factor A, mitochondrial) (Kelly and Scarpulla, 2004; Wenz, 2009) PPARGC1A/PGC-1α (PPARγ coactivator-1 alpha) PPARGC1A DNA NRF1 NFE2L2 PPARs (Scarpulla, 2008) PPARGC1A ( ) 14

18 (Komatsu and Ichimura, 2010) (mitophagy) (Youle and Narendra, 2011) ROS (Komatsu and Ichimura, 2010) PARK2/PINK1 PINK1 E3 PARK2 PARK2 LC3-II SQSTM1 PINK1 PARK2 (Deas et al., 2011) PLIN1 3 TP53/p53 3T3L1 TP53 PPARGC1A TP53 3T3L1 TP

19 SQSTM1/p62 KEAP1/NFE2L2 Nrf2 TP53 TP53 PPARGC1A 16

20 1 Involvement of Lysosomal Dysfunction in Autophagosome Accumulation and Early Pathologies in Adipose Tissue of Obese Mice 17

21 ATG5 ATG7 TG β (Singh et al., 2009; Zhang et al., 2009) (Mouse embryonic fibroblasts; MEFs) (adipogenesis) (Baerga et al., 2009; Zhang et al., 2009) TG Ost rapamycin chloroquine LC3-II turnover assay LC3-II (Ost et al., 2010) Kovsan (Kovsan et al., 2011) 3T3L1 IL1B IL6 CCL2 (Yoshizaki et al., 2012) 3T3L1 (Mikami et al., 2012) ph (Inami et al., 2012) CTSB (Gornicka et al., 2012) 18

22 CTSB CTSL 19

23 12 (8:00~20:00) 23 SPF ( ) 4 C57BL/6JJcl (, Tokyo, Japan) normal diet (ND) 4 high fat diet (HFD) 22 (18 HFD) 34 (30 HFD) ND MR (, Yokohama, Japan) HFD High fat diet 32 ( ) ( ) 4 C57BL/6JJcl ND HFD HFD ( ) 5 ob/ob C57BL/6 (Yokohama, Japan) ob/ob ND HFD HFD

24 Ex vivo (30 ) 2-3mm 3 10% FBS (Sigma, St. Louis, MO, USA) 1% penicillin/streptomycin (Sigma) DMEM (low glucose; Wako, Osaka, Japan) 37 CO2 1 10μM 500nM chloroquine (Wako, Tokyo, Japan) rapamycin (LC laboratories, Woburn, MA, USA) 24 3T3L1 JCRB (Japanese Collection of Research Bioresourses) 3T3L1 10% FBS (Sigma) 1% penicillin/streptomycin (Sigma, P0781) DMEM (low glucose; Wako) 0.4~ cells/cm 2 Luciferase (Luci) Ctsl shrna 5 -GGCAAAACTCGAGTTCGAACGCGTGATCAATTGTTTAAACAAGGCT Forward primer TTTCTCCAAGGGATATTTATAGTC-3 Reverse primer 5 -GTCGACCACTGTGCTGGC-3 RNAi-KPC-1 mouse U6 promoter 5 NotI, 3 PmeI, MunI, FbaI, BstBI, XhoI (NotI-mouse mu6 promoter -PmeI-MunI-FbaI-BstBI-XhoI) PCR PCR NotI XhoI NotI XhoI pmx-neo pmx-mu6-neo mu6 RNAi-KPC-1 pmxs-puro(-3u)-cul2 sidirect 2.0 ( CTSL DNA off-target sense 21

25 (Patzel et al., 2005; Miyagishi et al., 2004) 2 DNA Operon Biotechnologies (Tokyo, Japan) 2 DNA annealing buffer (50mM Tris-HCl ph 7.4, 100mM NaCl, 5mM MgCl2) PmeI 65 3 BstBI pmx-mu6-neo DNA pmx-mu6-shctsl#1-neo pmx-mu6-shctsl#2-neo Table 1. The oligonucleotide sequence for shluciferase (Luc) and shctsl shluc shctsl#1 forward reverse forward reverse 5'-GTACTGAGCCTGTTTGTGGAAttcaagaga TTTCACAAACGGGCTTAGTACttttt-3' 5'-cgaaaaaGTACTAAGCCCGTTTGTGAAAtctcttgaaTTCCACAAACAGGCTCAGTAC-3' 5'-GATCTGATGGGGCTAGTAAGTttcaagagaGCTTATTAGCCCTATCAGATCttttt-3' 5'-cgaaaaaGATCTGATAGGGCTAATAAGCtctcttgaaACTTACTAGCCCCATCAGATC-3' forward 5'-GTTATAAACGGCTTTGTAATGttcaagagaTATTATAAAGCTGTTTATAACttttt-3' shctsl#2 reverse 5'-cgaaaaaGTTATAAACAGCTTTATAATAtctcttgaaCATTACAAAGCCGTTTATAAC-3' Uppercase letters indicate target sequences against Ctsl or luciferase, whereas lowercase letters indicate BstBI or loop structure sequences. B (CTSB) CTSB OE Addgene hctsb vector (Addgene11249) CTSB mrna 5 Xho1 3 Flag Not1 (Xho1-CTSB-Flag-Not1) PrimeSTAR HS DNA polymerase (Takara, Shiga, Japan) Xho1 NotI CTSB-Flag Xho1 NotI pmxs-amnn-puro pmxs- CTSB-Flag-puro forward reverse 5 -GGGCTCGAGCACCATGTGGCAGCTCTGGGCC-3 (Xho1) 5 -GCCGCGGCCGCTTACTTATCGTCGTCATCCTTGTAATCGATCTTTTCCCAGTACTGATCGGTG-3 (Not1) with restriction enzyme sites indicated in italics and antisense of FLAG-tag coding sequences underlined. shrna CTSB 3T3L1 Platinam-E Packaging Cell Line (plat-e) plat-e cells/cm 2 FuGENE 6 Transfection Reagent (Promega, Madison, WI, USA) 22

26 plat-e 16 2 Millex-GV Filter (Millipore, Billerica, MA, USA) 4 PEG (32% PEG (polyethylglycol 6000; Fluka, Tokyo, Japan), 0.4M NaCl, 40mM HEPES (ph7.4)) 1/ rpm rpm 5 OPTI-MEM (Life Technologies Japan Ltd., Tokyo, Japan) 3T3L1 2μg/mL puromyicn 4 0.4μg/mL puromycin plat-e 3T3L1 3T3L cells/cm % Day-2 Day μM 3-isobutyl-1-methylxanthine (Sigma, I5879) 1μM dexamethasone (Sigma, D1756) 2 10μg/mL insulin (Wako) 50 nm tri-iode thyronine (T3; Sigma) bafilomycin A1 (LC laboratories) E64-d + pepstatin A (Peptide insutitute Inc, Osaka, Japan) 10nM 10ug/mL 11 (Day11) 24 CTSL Z-FY-CHO (Santa Cruz Biotechnology, CA, USA) CTSB CA074ME (Peptide insutitute Inc) 100μM 10μM 11 (Day11) 24 Western Blot SDS sample buffer (50mM Tris-HCl ph6.8, 2% SDS, 3M Urea, 6% glycerol) 95 5 (WAT) SDS sample buffer (12000 g, 30 ) 95 5 BSA Protein Assay Kit (Thermo Scientific, Yokohama, Japan) 0.2~2mg/mL BPB/2-Me (0.25% Bromophenol Blue, 50% 2-Mercaptoethanol) 1/ SDS-PAGE 23

27 (PALL, Port Washington, NY) blocking (2.5% skim milk (Wako)), 0.25% BSA, TTBS (25mM Tris-HCl ph 7.4, 140mM NaCl, 2.5mM KCl, 0.1% Tween 20) 1 (Immuno shot reagent 1 (Cosmo Bio Co.,Ltd., Tokyo, Japan) : blocking = 2 : 1) 4 TTBS 4 (Immuno shot reagent 2 (Cosmo Bio Co.,Ltd) : blocking = 2 : 1) 1 TTBS 4 ImmunoStar LD (Wako, ) (LAS 3000, Fujifilm, Tokyo, Japan) Table 2 3 Table 2. rabbit LC3 PM036 MBL rabbit P62 PM045 MBL rabbit Lamin B1 PM064 MBL rabbit Beclin 1 PD017 MBL rabbit Atg 5 M153-3 MBL rat Lamp 2 Ab13524 abcam rabbit CTSL ab abcam mouse CTSB ab58802 abcam rabbit Fsp27/CIDEC ab16760 abcam mouse FLAG M2 F1804 Sigma mouse β-actin A1978 Sigma mouse GAPDH Wako Table 3. HRP-conjugated F (ab ) 2 fragment of goat anti-mouse IgG HRP-conjugated F (ab ) 2 fragment of goat anti-rabbit IgG HRP-conjugated F (ab ) 2 fragment of goat anti-rat IgG Jackson Immunological Research, Jackson Immunological Research, Jackson Immunological Research, lysis buffer (352mM KH2PO4, 48mM Na2HPO4, 4mM EDTA (ph6.0 by 1N KOH), 0.5% TritonX-100) 60 incubation (100 g, 10min) BSA Protein Assay Kit (Thermo Scientific) CTSB 24

28 (1μg) 60uL reaction buffer 240μL (352mM KH2PO4, 48mM Na2HPO4, 4mM EDTA (ph6.0 by 1N KOH), 4mM DTT) sample buffer 100μL sample buffer 100μL CTSB substrate Z-Arg-Arg-MCA (Peptide institute) Infinite M200 Pro (Tecan Group Ltd., Männedorf, Switzerland) (ex: 350nm, em: 440nm) CTSL (30μg) 60uL reaction buffer 240μL sample buffer 100μL sample buffer 100μL 10μM CTSL substrate Z-Arg-Phe-MCA (Peptide institute) + 10μM CTSB inhibitor CA074 (Peptide institute) Infinite M200 Pro (ex: 350nm, em: 440nm) Nile red 3T3L1 3T3L1/shCtsl#1 or #2 Poly-D-Lysine (Sigma, P0899) (, Tokyo, Japan) 6 well dish 8 PBS 4% paraformaldehyde (PFA) 15 10μg/mL Nile red 1μg/mL Hoechest Slowfade Antifade Kit (Invitrogen, Carlsbad, CA) (, S1226, Osaka, Japan) (FV-1000-IX81, Oympus, Tokyo, Japan) Image J software (National Institutes of Health) Real-time RT PCR 3T3L1 ReliaPrep RNA Tissue Miniprep System (Promega, Madison, WI, USA) total RNA mrna NanoDrop 1000 (Thermo Fisher Scientific, Waltham, MA, USA) 1μg RNA PrimeScript RT reagent Kit (TaKaRa, RR037A, Otsu, Japan) cdna SYBR Premix Ex Taq (TaKaRa, RR041A) CFX Connect Real-Time PCR System (Bio-Rad, Hercules, CA, USA) Table 4 25

29 Table 4. primer Ctsl Ctsb Lc3 Sqstm1 Lamp1 Tbp Rps18 Forward 5 - TCG GTG ACA TGA CCA ATG AG -3 Reverse 5 - CAC ACA ACC CTT TTC TCT CCA G -3 Forward 5 - CAT GAC AAG CCT TCC TTC CAC -3 Reverse 5 -TT GTT CCC GTG CAT CAA AG-3 Forward 5 - CCA GTG ATT ATA GAG CGA TAC AAG G -3 Reverse 5 - AAG AAG GCT TGG TTA GCA TTG AG -3 Forward 5 - GAA GCT GAA ACA TGG ACA CTT TG -3 Reverse 5 - CAT TGG GAT CTT CTG GTG GAG -3 Forward 5 - TCA GCA TCT CCA ACC ATT CAC -3 Reverse 5 - CAA GTT TAC AGC CAA GAT TCA CG -3 Forward 5 - CAG TAC AGC AAT CAA CAT CTC AGC -3 Reverse 5 - CAA GTT TAC AGC CAA GAT TCA CG -3 Forward 5 - TGC GAG TAC TCA ACA CCA ACA -3 Reverse 5 - CTT TCC TCA ACA CCA CAT GAG C -3 ± Student t t-test R Tukey Kramer test Bonferroni correction Figure legends 26

30 HFD ND HFD ND (Figure 1-1A and B) HFD MAP1LC3-II/LC3-II SQSTM1/p62 (Figure 1-1C and E) ATG5 BECN1/Beclin1 (Figure 1-1C, F and G) LC3-II HFD SQSTM1 ph chloroquine (chloro) ex vivo LC3-II ND HFD chloro LC3-II (Figure 1-2A, B and C) rapamycin (rapa) SQSTM1 ND rapa SQSTM1 HFD SQSTM1 (Figure 1-2A, D and E) 2 DNA CTSB CTSL D (CTSD) CTSB CTSL CTSD (Kaminskyy et al., 2012) 3T3L1 CTSD CTSE pepstatin A LC3-II SQSTM1 CTSL CTSB E64-d LC3-II SQSTM1 (Figure 27

31 1-3) CTSL CTSB ND HFD CTSL CTSL (Figure 1-4A, B and C) CTSL (Figure 1-4D) CTSB (Figure 1-4A, F, G and H) CTSL CTSB mrna HFD (Figure 1-4 E and I) HFD (ob/ob ) control C57BL/6 (control ) ob/ob+nd ob/ob+hfd control (Figure 1-5) loading control total protein (CBB ) ob/ob GAPDH (Figure 1-6A and B) HFD LC3-II SQSTM1 ob/ob ob/ob+hfd (Figure 1-6C, D and E) CTSL Ctsl mrna (Figure 1-6C, G, H and I) CTSB mrna HFD (Figure 1-6C, J, K and L) HFD CTSL CTSB 8 18 HFD (Figure 1-7A) (Figure 1-7B and C) CTSL 4HFD CTSL (Figure 1-8 A, B and D) 8HFD CTSL (Figure 1-8 A and C) CTSB 4HFD CTSB (Figure 1-8A, F and G) 8HFD (Figure 1-8 H) CTSL CTSB Ctsl Ctsb Sqstm1 mrna 4HFD 8HFD 18HFD 8HFD (Figure 1-8 E, I and M) CTSL CTSL CTSB SQSTM1 Lc3 mrna 8HFD (Figure 1-8K) LC3-II 28

32 3 3T3L1 CTSL CTSB 3T3L1 CTSL Z-FY-CHO CTSB CA074ME CTSL CTSB E64-d Z-FY-CHO 10μM CTSL 100μM (Figure 1-9A) CTSB (Figure 1-9B) LC3-II SQSTM1 (Figure 1-9C, D and E) Ctsl Ctsb mrna (Figure 1-9F and G) LC3-II SQSTM1 E64-d 10μg/mL CTSL Ctsl Ctsb CTSB (Figure 1-9F and G) CTSL CTSL 3T3L1 CIDEC/FSP27 (Figure 1-10A, E and F) SQSTM1 LC3-II (Figure 1-10A, B and C) CTSB (Figure 1-10D) CTSL CTSB CTSB CTSB 3T3L1 CA074ME CTSB CTSB (Figure 1-10G) CTSB CTSL CTSB (Figure 1-10H) CTSB LC3-II SQSTM1 CIDEC (Figure 1-10I, J, K and L) CTSB 29

33 ATG7 ER (Yang et al., 2010) β HFD (Ebato et al., 2008) ex vivo chloroquine LC3-II turnover assay HFD ND LC3-II LC3-II turnover assay rapamycin SQSTM1 HFD SQSTM1 chloroquine E64-d LC3-II SQSTM1 (Li et al., 2013) V-ATPase ATP6V0C bafilomycin A1 (Mangieri et al., 2014) ex vivo LC3-II SQSTM1 SQSTM1 KEAP1/NFE2L2 TRAF6/NFKB1 (Moscat and Diaz-Mecok, 2009; Katsuragi et al., 2015) SQSTM1 - SQSTM1 30

34 CTSL CTSB CTSL CTSB (Reiser et al., 2010) CTSB CTSL (Deussing et al., 1998; Potts et al., 2004) CTSL CTSB 2-4 (Felbor et al., 2002) CTSL (39kDa) (25-30kDa) CTSB (44kDa) 33kDa single-chain form 24-27kDa 5kDa two-chain form CTSB (Gelb et al., 1997; Wolfe et al., 2013) CTSB (Turk et al., 1995) HFD CTSL CTSL mrna CTSL ph CTSD cystatins thyropins serpins (Turk et al., 2012) lysosomal membrane permeabilization (LMP) (trabecular meshwork cell) (Porter et al., 2013) 4 (4HNE) (Figure S1-1) N-acetylcystein (NAC) CTS (Figure S1-2A) HFD+NAC HFD (Figure S1-2 B, C, D and E) HFD CTSL NAC (Figure S1-3A and D) CTSL (Figure S1-3E) LC3-II SQSTM1 (Figure S1-3A, B and C) CTSL maturation CTSL CTSL HFD 31

35 HFD (Figure S1-4A) HFD (Figure S1-4B, C, D and E) CTSL HFD (Figure S1-5A, B, C and F) CTSB Figure 1-8 HFD HFD (Figure S1-5A, D, E and G) CTSL HFD CTSL HFD CTSL ob/ob CTSB CTSL (Inami et al., 2011) CTS cystain C cystatin C (Naour et al., 2009) cystatin C CTSB CTSL (Turk et al., 2001) cystatin C MHC class II p41 invariant chain (CD74 ) CTSL (Turk et al., 2001; Bevec et al., 1996) CTSL MHC class II p41 invariant chain CTSL ph 3T3L1 CTSL CTSB CTSL Sqstm1 1 C (Niemann Pick type C) CTSB CTSD (Liao et al., 2007) CTSB Hannaford CTSB CTSL mrna (Hannaford et al., 2013) 2 CTSL CTSL HFD 1 CTSL ph CTSD (Turk et al., 2001) CTSB CTSL 32

36 ( ) CTSB CTSL (Figure 1-11B) CTSL CTSB CTSL 33

37 34

38 35

39 36

40 37

41 38

42 39

43 40

44 41

45 42

46 43

47 44

48 45

49 46

50 47

51 48

52 49

53 50

54 2 Involvement of Lysosomal Dysfunction in Autophagosome Accumulation and Early Pathologies in Adipose Tissue of Obese Mice 51

55 CTSL CTSB (Levine and Kroemer, 2008; Mizushima et al., 2008) β ATP (Ebato et al., 2008; Jung et al., 2008) ER (Yang et al., 2010) (Kim et al., 2012) mitokine FGF21 (fibroblast growth factor 21) β WAT β WAT positive (Zhang et al., 2009) CTSL (Zheng et al., 2008) (cellular senescence) SAHF (senescence-associated heterochromatic foci) SA-GLB1 CDKN1A/p21 CDKN2A/p16 INK4A (Campisi et al., 2007) CDKN1A CDKN2A CDK4/6 CDK2 RB RB E2F1 (Campisi et al., 2007) OIS 52

56 (oncogene-induced premature senescence) SIS (stress-induced premature senescence) (Rodier and Campisi, 2011) (Minamino et al., 2009) CTSB CASP1/caspase1 (Hornung et al., 2008; Halle et al., 2009) NLRP3 PYCARD/ASC (PYD and CARD domain containing) CASP1 NLRP3 adaptor PYCARD pro-casp1 pro-casp1 CASP1 CASP1 IL1B IL18 IL1B IL18 proinflammatory cytokine (Lamkanfi and Dixit. 2009; Franchi et al., 2009) ( ) NLRP3 (Vandanmagsar et al., 2012; Stienstra et al., 2011) PLIN1/perilipin1 family PLIN2 (adipophilin) PLIN3 (TIP47) PLIN4 (S3-12) PLIN4 (OXPAT/MLDP) (Brasaemle, 2007) PLIN1 PLIN4 PLIN2 PLIN3 PLIN5 (Brasaemle, 2007) PLIN1 (lipolysis) (Brasaemle, 2007; Nielsen et al., 2014) TG hormone-sensitive lipase (HSL) adipose triglyceride lipase (ATGL) FFAs FFAs β ATP PKA (protein kinase A) ATGL HSL PLIN1 HSL ATGL TG PLIN1 S492 S517 (Miyoshi et al., 2007; Miyoshi et al., 2008) PLIN1 53

57 (Gaidhu et al., 2010) PLIN1/perilipin1 (Kovsan et al., 2007; Ogasawara et al., 2012) 54

58 12 (8:00~20:00) 23 SPF ( ) 4 C57BL/6JJcl (, Tokyo, Japan) normal diet (ND) 4 high fat diet (HFD) 22 (18 HFD) 34 (30 HFD) ND MR (, Yokohama, Japan) HFD high fat diet 32 ( ) ( ) 4 C57BL/6JJcl ( ) ND HFD HFD T3L1 JCRB (Japanese Collection of Research Bioresourses) 3T3L1 10% FBS (sigma, St. Louis, MO, USA) 1% penicillin/streptomycin (Sigma, P0781) DMEM (low glucose; Wako, Osaka, Japan) 0.4~ cells/cm2 Luciferase (Luc) Ctsl shrna Forward primer Reverse primer 5 -GGCAAAACTCGAGTTCGAACGCGTGATCAATTGTTTAAACAAGGCT TTTCTCCAAGGGATATTTATAGTC-3 5 -GTCGACCACTGTGCTGGC-3 55

59 RNAi-KPC-1 mouse U6 promoter 5 NotI, 3 PmeI, MunI, FbaI, BstBI, XhoI (NotI-mouse mu6 promoter PmeI-MunI-FbaI-BstBI-XhoI) PCR PCR NotI XhoI NotI XhoI pmx-neo pmx-mu6-neo mu6 RNAi-KPC-1 pmxs-puro (-3U) -Cul2 sidirect 2.0 ( CTSL DNA off-target sense (Patzel et al., 2005; Miyagishi et al., 2004) 2 DNA Operon Biotechnologies (Tokyo, Japan) 2 DNA annealing buffer (50mM Tris-HCl ph 7.4, 100mM NaCl, 5mM MgCl2) PmeI 65 3 BstBI pmx-mu6-puro DNA pmx-mu6-shctsl#1-neo pmx-mu6-shctsl#2-neo Table 1. The oligonucleotide sequence for shluciferase (Luc) and shctsl shluc shctsl#1 forward reverse forward reverse 5'-GTACTGAGCCTGTTTGTGGAAttcaagaga TTTCACAAACGGGCTTAGTACttttt-3' 5'-cgaaaaaGTACTAAGCCCGTTTGTGAAAtctcttgaaTTCCACAAACAGGCTCAGTAC-3' 5'-GATCTGATGGGGCTAGTAAGTttcaagagaGCTTATTAGCCCTATCAGATCttttt-3' 5'-cgaaaaaGATCTGATAGGGCTAATAAGCtctcttgaaACTTACTAGCCCCATCAGATC-3' forward 5'-GTTATAAACGGCTTTGTAATGttcaagagaTATTATAAAGCTGTTTATAACttttt-3' shctsl#2 reverse 5'-cgaaaaaGTTATAAACAGCTTTATAATAtctcttgaaCATTACAAAGCCGTTTATAAC-3' Uppercase letters indicate target sequences against Ctsl or Luciferase, whereas lowercase letters indicate BstBI or loop structure sequences. 56

60 B (CTSB) D (CTSD) CTSB Addgene hctsb vector (Addgene11249) CTSB mrna 5 Xho1 3 Flag Not1 (Xho1-CTSB-Flag-Not1) PrimeSTAR HS DNA polymerase (Takara, Shiga, Japan) Xho1 NotI CTSB-Flag Xho1 NotI pmxs-amnn-puro pmxs- CTSB-Flag-puro forward reverse 5 -GGGCTCGAGCACCATGTGGCAGCTCTGGGCC-3 (Xho1) 5 -GCCGCGGCCGCTTACTTATCGTCGTCATCCTTGTAATCGATCTTTTCCCAGTACTGATCGGTG-3 (Not1) with restriction enzyme sites indicated in italics and antisense of FLAG-tag coding sequences underlined. CTSD Mouse cdna mouse CTSD mrna 5 Xho1 3 myc Not1 (Xho1-mouse CTSD-myc-Not1) PrimeSTAR HS DNA polymerase (Takara, Shiga, Japan) Xho1 NotI mouse CTSD-myc Xho1 NotI pmxs-amnn-puro pmxs-neo pmxs- mouse CTSD-myc -puro / pmxs- mouse CTSD-myc -neon forward reverse 5'- GGGCTCGAGCACCATGCAGCCCTCCAGCCTTCTGCCG-3' (Xho1) 5'-GCCGCGGCCGCCTACAGATCCTCTTCTGAGATGAGTTTTTGTTCGAGGCGGGCAGCCTCGGC-3' (Not1) with restriction enzyme sites indicated in italics and antisense of myc-tag coding sequences underlined. 3T3L1 Platinam-E Packaging Cell Line (plat-e) plat-e cells/cm 2 FuGENE 6 Transfection Reagent (Promega, Tokyo, Japan) plat-e 16 2 Millex-GV Filter (MILLIPORE, Billerica, MA, SLGVJ13SL) 4 PEG (32% PEG (polyethylglycol 6000; Fluka, Tokyo, Japan, 81253), 0.4M NaCl, 40mM HEPES, ph7.4) 1/ rpm rpm 5 OPTI-MEM (Life Technologies Japan Ltd., Tokyo, Japan) 3T3L1 57

61 2μg/mL puromycin 4 0.4μg/mL puromycin plat-e 3T3L1 3T3L cells/cm % Day-2 Day μM 3-isobutyl-1-methylxanthine (Sigma, I5879) 1 μm dexamethasone (Sigma, D1756) 2 10μg/mL insulin (WAKO) 50nM tri-iode thyronine (T3; Sigma) Bafilomycin A1 (LC laboratories, Woburn, MA, USA) E64-d + pepstatin A (Peptide insutitute Inc) 10 nm 10 ug / ml 11 (Day11) 24 CTSL Z-FY-CHO (Peptide insutitute Inc) CTSB CA074ME (Peptide insutitute Inc) 100μM 10μM 11 (Day11) 24 Western Blot SDS sample buffer (50mM Tris-HCl ph 6.8, 2% SDS, 3M Urea, 6% glycerol) 95 5 (WAT) SDS sample buffer (12000 g, 30 ) 95 5 BSA Protein Assay Kit (Thermo Scientific, Yokohama, Japan) 0.2~2mg/mL BPB/2-Me (0.25% Bromophenol Blue, 50% 2-Mercaptoethanol) 1/ SDS-PAGE (PALL, Port Washington, NY) blocking (2.5% skim milk (Wako), 0.25% BSA, TTBS (25mM Tris-HCl ph 7.4, 140mM NaCl, 2.5mM KCl, 0.1% Tween 20) ) 1 (Immuno shot reagent 1 (Cosmo Bio Co.,Ltd., Tokyo, Japan) : blocking = 2 : 1) 4 TTBS 4 (Immuno shot reagent 2 (Cosmo Bio Co.,Ltd) : blocking = 2 : 1) 1 58

62 TTBS 4 ImmunoStar LD (WAKO, ) (LAS 3000, FUJIFILUM, Tokyo, Japan) Table 2 Table 2. 1 rabbit Lamin B1 PM064 MBL rabbit CDKN1A SC-397 Santa cruz Biotechnology mouse CASP1 SC-514 Santa cruz Biotechnology mouse TP53 (Ab-1) OP03 Millipore rabbit Fsp27/CIDEC ab16760 abcam rabbit PLIN1 (D418) #3470 CST rabbit PLIN1 (C-terminal) ab3526 abcam mouse FLAG M2 F1804 Sigma mouse GAPDH Wako Table 3. 2 HRP-conjugated F (ab ) 2 fragment of goat anti-mouse IgG HRP-conjugated F (ab ) 2 fragment of goat anti-rabbit IgG HRP-conjugated F (ab ) 2 fragment of goat anti-rat IgG Jackson Immunological Research, Jackson Immunological Research, Jackson Immunological Research, lysis buffer (352mM KH2PO4, 48mM Na2HPO4, 4mM EDTA (ph6.0 by 1N KOH), 0.5% TritonX-100) 60 incubation (100 g, 10min) BSA Protein Assay Kit (Thermo Scientific, Yokohama, Japan, 23225) CTSB (1μg) 60uL reaction buffer 240μL (352mM KH2PO4,48mM Na2HPO4, 4mM EDTA (ph6.0 by 1N KOH), 4mM DTT) sample buffer 100μL sample buffer 100μL cathepsin B substrate Z-Arg-Arg-MCA (Peptide institute, Osaka, Japan) Infinite M200 Pro (Tecan Group Ltd., Männedorf, Switzerland) (ex: 350nm, em: 440nm) CTSD 59

63 (μg) 60uL reaction buffer 240μL sample buffer 100 μl sample buffer 100μL 10μM cathepsin L substrate Z-Arg-Phe-MCA (Peptide institute) + 10μM CTSB inhibitor CA074 (Peptide institute) Infinite M200 Pro (ex: 350nm, em: 440nm) Poly-D-Lysine (Sigma, P0899) (, Chiba, Japan, ) 6-well dish PBS 4% paraformaldehyde 15 PBS 0.2% Triton-X PBS blocking (1% goat serum (Invitrogen, ), 0.2% BSA (Sigma, A2153) ) 30 blocking TPBS (0.1% Tween in PBS) (1st antibody in blocking ) 4 TPBS (5 4 ) (2nd antibody in blocking ) TBPS (5 4 ) Slowfade Antifade Kit (Invitrogen, Carlsbad, CA) (, Osaka, Japan, S1226) (FV-1000-IX81, Oympus, Tokyo, Japan) Western blot ( ) 10% 24 REM-710 5μm % 95% 2 PBS blocking (1% goat serum, 0.2% BSA) 30 blocking PBS (1st antibody in blocking ) 4 PBS (5 4 ) (2nd antibody in blocking ) PB5 4 ) 2 Hoechst Slowfade Antifade Kit (Invitrogen, Carlsbad, CA) (, Osaka, Japan, S1226) (Leica, Tokyo, Japan) 60

64 ( ) HE 10% (10% formaldehyde in PBS) 5μm Hematoxylin-Eosin (HE) Eclipse 80i DS-FiLc, DS-L2 (Nikon, Tokyo, Japan) 100 Image J 1.4 (Wayne Rasband National Institute of Health, Bethesda, MD) ± Student t t-test R Tukey Kramer test Bonferroni correction Figure legends 61

65 1. CTSL CTSL 3T3L1 CTSL CTSL Z-FY-CHO ROS CM-H2DCFA ROS MitoSOX (Figure 2-1A and B) Z-FY-CHO adiponectin mrna serpine1 CTSB CA074ME (Figure 2-1C and D) CTSL 2. CTSL TP53/p53 CDKN1A/p21 3T3L1 TP53 CTSL CTSB Western blotting CDKN1A CTSL 3T3L1 (Figure 2-2A and B) CTSL CDKN1A (Figure 2-2D and E) CTSB CDKN1A (Figure 2-2G and H) CTSL HFD CTSL TP53 CDKN1A 4HFD 8HFD (Figure 2-3A, B and C) CTSL 3. CTSB CASP1/caspase-1 (cleaved CASP1, p10) 3T3L1 CTSL 3T3L1 cleave CASP1 (Figure 2-2A and C) CTSL cleaved CASP1 (Figure 2-2D and F) CTSB cleaved CASP1 62

66 (Figure 2-2G, H and I) CTSL CTSB CTSB 8HFD 18HFD pro-casp1 (45kDa) cleaved CASP1 (p10) (Figure 2-3A, D and E) CTSL CTSB 8 HFD (F4/80 Cd11c) crown-like structure (Figure 2-3F, G and H) 4 CTSB PLIN1 (Gaidhu et al., 2010) PLIN1 (Xu et al., 2006; Kovsan et al., 2007; Ogasawara et al., 2012) PLIN1 CTSB PLIN1 8HFD mrna 18HFD (Figure 2-4A, B and D) PLIN1 FSP27/CIDEC 18HFD (Figure 2-4A and C) HFD PLIN1 (Figure 2-4E middle panels) CTSB (Figure 2-4E right panels) CTSB PLIN1 CTSB 3T3L1 PLIN1 (D418: PLIN1 418 C-terminal: PLIN1 C ) 2 Western blotting PLIN1 CTSB PLIN1 (62kDa) (Figure 2-5A) D418 50kDa (Figure 2-5A and B) CTSB CA074ME PLIN1 (62kDa) (Figure 2-5D and E) 50kDa PLIN1 CTSB CTSB PLIN1 C 10-12kDa (Figure 2-7C) 2 PLIN1 PLIN1 (D418) CTSB PLIN1 PLIN1 (C-terminal) CTSB CA074ME (Figure 2-5F) 63

67 CTSD 8HFD 18HFD (Figure 2-6A) CTSD (Benes et al., 2008) CTSD CTSB PLIN1 CTSD 3T3L1 CTSD CTSD pepstatin A (Figure 2-6B) PLIN1 (Figure 2-6C, D and E) CTSD PLIN1 CTSB PLIN1 CTSB PLIN1 C 10-12kDa C PKA (S492 S517) PLIN1 HSL ATGL (Lass et al., 2010) β3- CTSB 3T3L1 β3 isoprotenerol Mock isoproterenol CTSB (Figure 2-7A and B) CTSB PLIN1 C 10-12kDa PKA 64

68 TP53 SA-GBL/ SA β-gal (senescence-associated galactosidase beta 1) TP53 (Minamino et al., 2009) CTSL 3T3L1 CDKN1A (Figure 2-2) CTSL CDKN1A (Figure 2-3) TP53/p53- CDKN1A /p21 pathway CDKN2A/p16 INK4A pathway (Campisi et al., 2007) TP53/p53- CDKN1A /p21 pathway (Smogorzewska et al., 2002) CTSL SASP (senescence-associated secretory phenotype) (Yoshimoto et al., 2013) SASP p53 ( ) CTSL CTSB (Figure 2-2) IL1 IL6 CCL2 (Yoshizaki et al., 2012) 3T3L1 bafilomycin A1 TNF ( ) CTSL CTSB CASP1 (Figure 2-2) CTSB 8 HFD CASP1 (Figure 2-3) CTSB 65

69 CTSB lysosomal membrane permeabization (LMP) CTSB CTSB CASP1 (Halle et al., 2008; Bruchard et al., 2013) LMP CTSB apoptosis (Boya and Kroemer, 2008) CTSB apoptosis crown-like structure ( ) PLIN1 B PLIN1 PLIN1 (Gaidhu et al., 2009) PLIN1 adipogenesis PPARG/PPARγ PLIN1 (Arimura et al., 2004) CTSB Mock 8 (Day8) CTSB Mock (Figure S2-1A and B) PPARG 2 PPARG1 PPARG2 Day2 (Figure S2-1 C and G) CTSB adipogenesis CTSB PLIN1 adipogenesis PLIN1 β3 (Martinez-Botas et al., 2000; Tansey et al., 2001) crown-like structure (Cinti et al., 2005; Strissel et al., 2007) Figure 2-4F 18 PLIN1 18 PLIN1 crown-like structure CTSB 66

70 CTSB (Aronson and Barrett, 1978; Illy et al., 1997) CTSB (44kDa) 33kDa single-chain form 24-27kDa 5kDa two-chain form CTSB (Gelb et al., 1997; Wolfe et al., 2013) CTSB (Premzl et al., 2003) CTSB PLIN1 CTSB (Figure 2-5) PLIN1 CTSB PLIN 10-12kDa (S492 S518) (Figure 2-7C) (Tansey et al., 2004) β3 lipolysis (Figure 2-7) PLIN1 PLIN1 central domain (Garcia et al., 2003) CTSB lysosomal membrane permeabilization (LMP) CTSB CTSB PLIN1 HSL ATGL C PLIN1 (Figure 2-7D) PLIN1 CTSB LMP PLIN1 CTSL CTSB CTSL CTSB 67

71 68

72 69

73 70

74 71

75 72

76 73

77 74

78 75

79 76

80 3 Inhibitory effect of TP53 on mitochondrial content and function during adipogenesis TP53 77

81 TP53/p53 30 TP53-30 (Momand et al., 1992; Shieh et al., 1997) DNA TP53 - DNA (el-deiry et al., 1993, Zhan et al., 1999, Hermeking et al., 1997, Miyashita et al, 1995, Oda et al., 2000, Momand et al., 1992; Shieh et al., 1997) DNA TP53 TP53 AKT AMPK (AMP-activated protein kinase) TP53 (Lavin et al., 2006) TP53 TP53 (Saleem et al., 2011; Wang et al., 2012) (mitochondrial biogenesis) PPARGC1A/PGC1α NRF1 NFE2L2/NRF2 mrna (Kelly and Scarpulla, 2004) TP53 PPARGC1A (Saleem et al., 2009; Sahin et al., 2011; Aquilano et al., 2013) (Villeneuve et al., 2013) TP53 mtdna (Lebedeva et al., 2009) TP53 (differentiation) TP53 ips (induced pluripotent stem) TP53 (Yi et al., 2012) (adipogenesis) (myogenesis) TP53 TP53 MEFs MSCs TP53 3T3L1 78

82 (Molchadsky et al., 2008; Armesilla-Diazet al., 2009) C2C12 TP53 TP53 (Molchadsky et al., 2008 Tamir and Bengal, 1998, Porrello et al., 2000) TP53 TP53 2 3T3L1 C2C12 TP53 79

83 12 (8:00~20:00) 23 SPF ( ) 4 C57BL/6JJcl ND HFD HFD ( ) TP53 C57BL/6 TP53 C57BL/6 TP (ND) (HFD) HFD 4 13 ND 18 ( MEFs; Mouse embryonic fibroblasts) TP PBS 0.05% Trypsin-EDTA in PBS (Invitrogen, Carlsbad, CA, 15400) U/mL penicillin G and streptomycin (SIGMA, St. Louis, MO, P0781) 10% FBS (BOVOGEN, Essendon Vic, Australia, SFBS-F) DMEM -high glucose- (Wako, , Tokyo, Japan) Trypsin-EDTA cell strainer (BD, Franklin Lakes, NJ, ) TP53 allele TP53 intron-2 forward primer exon-3 reverse primer neo primer genotyping Table 1 80

84 Table 1. TP53 genotyping primer sequence Forward 5 - AATTGACAAGTTATGCATCCAACAGTACA -3 Reverse 5 - ACTCCTCAACATCCTGGGGCAGCAACAGAT -3 Forward 5 - CAT GAC AAG CCT TCC TTC CAC -3 Neo primer 5 - GAACCTGCGTGCAATCCATCTTGTTCAATG -3 sidirect ( TP53 GFP DNA off-target sense (Patzel et al., 2005; Miyagishi et al., 2004) 2 DNA Operon Biotechnologies (Tokyo, Japan) 2 DNA annealing buffer (50mM Tris-HCl ph 7.4, 100mM NaCl, 5mM MgCl2) PmeI 65 3 BstBI pmx-mu6-puro DNA pmx-mu6-shctsl#1-neo pmx-mu6-shctsl#2-neo RNAi-KPC-1 Table 2. The oligonucleotide sequence for shluciferase (Luc) and shctsl shgfp forward reverse 5'- GGCTATGTCCAGGGGCGCATCttcaagagaGGTGCGCTCCTGGACGTAGCCttttt -3' 5'- cgaaaaaggctacgtccaggagcgcacctctcttgaagatgcgcccctggacatagcc-3' forward 5'- GTACGTGTGTAGTAGCTTCttcaagagaGGAGCTATTACACATGTACttttt -3' shtp53 reverse 5'- cgaaaaagtacatgtgtaatagctcctctcttgaagaagctactacacacgtac -3' Uppercase letters indicate target sequences against CTSL or luciferase, whereas lowercase letters indicate BstBI or loop structure sequences. shrna 3T3L1 Platinam-E Packaging Cell Line (plat-e) plat-e cells/cm 2 FuGENE 6 Transfection Reagent (Promega, Madison, WI, USA) plat-e 16 2 Millex-GV Filter (Millipore, Billerica, MA, USA) 4 PEG (32% PEG (polyethylglycol 6000; Fluka, Tokyo, Japan, 81253), 0.4M NaCl, 40mM HEPES, ph7.4) 81

85 1/4 3000rpm rpm 5 OPTI-MEM (Life Technologies Japan Ltd., Tokyo, Japan) 3T3L μg / ml G μg/mL G418 plat-e 3T3L1 3T3L cells/cm % Day-2 Day μM 3-isobutyl-1-methylxanthine (Sigma, I5879) 1μM dexamethasone (Sigma, D1756) 2 10μg/mL insulin (Wako) 50nM tri-iode thyronine (T3; Sigma) TP53 MEFs 10% FBS, 100U/mL penicillin G and streptomycin 0.1mM 2-Mercaptoethanol (2-Me) DMEM -high glucose- 2.0~ cells/cm 2 MEFs 3 MEFs 0.8~ cells/cm 2 4 Day mM IBMX (SIGMA, I5879) 1μM DEX (SIGMA, D1756) 10μg/mL insulin (Wako, ) 10μM troglitazone (CAYMAN, Ann Arbor, MI, 71750) 2-Me Western blot SDS sample buffer (50mM Tris-HCl ph 6.8, 2% SDS, 3M Urea, 6% glycerol) 95 5 (WAT) SDS sample buffer (12000 g, 30 ) 95 5 BSA Protein Assay Kit (Thermo Scientific, Yokohama, Japan) 0.2~2mg/mL BPB/2-Me (0.25% Bromophenol Blue, 50% 2-mercaptoethanol) 1/ SDS-PAGE (PALL, Port Washington, NY) blocking (2.5% skim milk (Wako), 0.25% BSA, TTBS (25 mm Tris-HCl ph 7.4, 140 mm NaCl, 2.5 mm KCl, 0.1% Tween 20)) 1 (Immuno shot reagent 1 (Cosmo Bio Co.,Ltd., Tokyo, Japan) : blocking = 2 : 1) 4 TTBS 4 82

86 (Immuno shot reagent 1 (Cosmo Bio Co.,Ltd) : blocking = 2 : 1) 1 TTBS 4 ImmunoStar LD (Wako, ) (LAS 3000, FUJIFILUM, Tokyo, Japan) Table 3 4 Table 3. rabbit TP53 (Ab-1) OP03 Calbiochem mouse TUBA/α-tubulin T9026 Sigma mouse PPARG/PPARγ sc-7273 Santa cruz rabbit FABP Cayman mouse COXIV #11967 CST mouse TOM20 WH M1 Sigma mouse ACTB/β-actin A1978 Sigma Table 4. HRP-conjugated F (ab ) 2 fragment of goat anti-mouse IgG HRP-conjugated F (ab ) 2 fragment of goat anti-rabbit IgG Jackson Immunological Research, Jackson Immunological Research, Oil Red O TG Oil Red O (Sigma, O0625) (1.67mg/mL in isopropanol) 60% Millex-GV Filter (MILLIPORE, Billerica, MA, SLGV033RS) Oil Red O PBS 10% Formalin in PBS (Wako, ) 10 PBS 60% isopropanol 1 Oil red O 20 60% isopropanol 1 PBS BZ-9000 (KEYENCE, Osaka, Japan) TG PBS % isopropanol (50μL/cm2) 490nm Microplate Reader Model 550 (BIO-RAD, Hercules, CA) Real-time RT-PCR 3T3L1 ReliaPrep RNA Tissue Miniprep System (Promega, Madison, WI, USA) total RNA mrna 83

87 NanoDrop 1000 (Thermo Fisher Scientific, Waltham, MA) 1 μg RNA PrimeScript RT reagent Kit (TaKaRa, RR037A) cdna SYBR Premix Ex Taq (TaKaRa, RR041A) Real-time PCR ABI PRISM 7300 (Applied Biosystems, Foster City, CA) Table 5 Table 5. primer Actb Fabp4 Gusb Myod Myog cdkn1a TP53 ppargc1a Pparg Tbp Rps18 Forward 5 - TCT TTG CAG CTC CTT CGT TG -3 Reverse 5 - GGC CTC GTC ACC CAC ATA G -3 Forward 5 - TCG ATG ATT ACA TGA AAG AAG TGG -3 Reverse 5 - CGC CAG TTG AGG AAT C -3 Forward 5 - CCA GAG CGA GTA TGG AGC AGA C -3 Reverse 5 - GGT GAC TGG TTC GTC ATG AAG TC -3 Forward 5 - CTG CTC TGA TGG CAT GAT GG -3 Reverse 5 - TAT GCT GGA CAG GCA GTC G -3 Forward 5 -ATC CAG TAC ATT GAG CGC CTA C -3 Reverse 5 - TGT CCA CGA TGG ACG TAA GG-3 Forward 5 - AGT ACT TCC TCT GCC CTG CTG -3 Reverse 5 - GCG CTT GGA GTG ATA GAA ATC TG -3 Forward 5 - TAA AGG ATG CCC ATG CTA CAG -3 Reverse 5 - GAC CGG GAG GAT TGT GTC TC-3 Forward 5 - AGA CGG ATT GCC CTC ATT TG -3 Reverse 5 - CAG GGT TTG TTC TGA TCC TGT G-3 Forward 5 - CAC AAT GCC ATC AGG TTT GG -3 Reverse 5 - GCG GGA AGG ACT TTA TGT ATG AG-3 Forward 5 - CAG TAC AGC AAT CAA CAT CTC AGC -3 Reverse 5 - CAA GTT TAC AGC CAA GAT TCA CG-3 Forward 5 - TGC GAG TAC TCA ACA CCA ACA -3 Reverse 5 - CTT TCC TCA ACA CCA CAT GAG C -3 DNA -80 WAT 200μL DNA Buffer (150mM NaCl 10mM Tris HCl ph8.0 10mM EDTA) Buffer 1/100 10% SDS 1/100 proteinase K (Wako, ) 55 84

88 Buffer Tris 1 PCI (Tris CIA (Chloroform Isoamylalchol=24 1)=1 1) 1 1/50 RNaseA (Wako, Osaka, Japan, ) 37 1 Tris PCI 2 100% ethanol 1/10 3M sodium acetate DNA 180μL 70% ethanol 2 DNA TE Buffer NanoDrop Real-time PCR ABI PRISM 7300 (Applied Biosystems, Foster City, CA) DNA DNA Cox2 DNA Slc16a1 (Table 2) DNA (Cox2)/ DNA (Slc16a1) Table 6. mtdna primer Cox2 Forward 5 - CTT ACA AGA CGC CAC ATC AC -3 Reverse 5 - CTTACAAGACGCCACATCAC -3 Slc16a1 Forward 5 - TAG CTG GAT CCC TGA TGC GA -3 Reverse 5 - GCA TCA GAC TTC CCA GCT TCC -3 ± Student t t-test R Tukey Kramer test Bonferroni correction Figure legends 85

89 3T3L1 C2C12 Ppargc1 TP53 DNA TP53 MDM2 (MDM2 proto-oncogene) E3 nutlin-3a TP53 (Figure 3-1) (Figure 3-1A) (Figure 3-1B) nutlin-3a TP53 Cdkn1a/p21 mrna 3T3L1 Ppargc1a (Figure 3-1A, right panel) C2C12 (Figure 3-1B, right panel) Nutlin-3a side-effects TP53 Ppargc1a Nutlin-3a TP53 Ppargc1a (Figure 3-1C) (Figure 3-1D) TP53 Ppargc1a TP53 (KD) 3T3L1 ( 3T3L1/shTP53) TP53KD C2C12 Ppargc1a (Figure 3-1E and F) Ppargc1a PPARGC1A Western blotting PPARGC1A (Santa Cruz Biotechnology, Cat. No. sc and Merk-Millipore, Cat. No. ST1202-1SET) PPARGC1A ( ) 3T3L1 TP53 Ppargc1a 2 3T3L1 MEFs TP53 Ppargc1a mtdna Figure 3-1 3T3L1 TP53 3T3L1 (adipogenesis) (Wilson-Fritch et al., 2003; Vankoningsloo et al., 2005) adipogenesis TP53 Oil red O TP53 KD (3T3L1/shTP53) (Figure 3-2A) adipogenesis Pparg Fabp4 mrna ( ) (Figure 3-1) TP53 Ppargc1a (Figure 3-2B) 8 TP53KD mtdna 86

90 (Figure 3-2C) PPARG2 (adipogenesis PPARG 1 ) adipogenesis 8 FABP4 adipogenesis 8 3T3L1/shTP53 3T3L1/shGFP (Figure 3-2D) TP53 3T3L1 (Molchadsky et al., 2013) (MEFs) TP53 WT TP53 KO MEFs MEFs oil red O TP53 KO MEFs (Figure 3-2E) MEFs adipogenesis TP53 Ppargc1a mtdna 16 TP53 KO MEFs TP53 WT MEFs Ppargc1a mtdna (Figure 3-2F and G) (mitochondrial cytochrome oxidase complex) 1 COX4 (mitochondrial import receptor) 1 TOM20 TP53 KD (Figure 3-2H) TP53 adipogensis 3 C2C12 TP53 Ppargc1a mtdna TP53 (Saleem et al., 2009; Park et al., 2009) C2C12 TP53 (Figure 3-1) TP53 TP53 C2C12 (Molchadsky et al., 2008; Tamir amd Bengal, 1998; Porrello et al., 2000) TP53 (Figure 3-3A) (myogenesis) Myog Myod ( ) TP53 Ppargc1a mtdna (Figure 3-3B and C) TP53 4 TP53 TP53 3T3L1/shTP53 3T3L1/shGFP TCA CS (citrate synthase) adipogenesis 12 87

91 IV adipogenesis 8 (Figure 3-4) 5 TP53 Ppargc1a TP53 (Yahagi et al., 2003) (TP53 +/+ ND) TP53 KO (TP53 -/- ND) (TP53 +/+ HFD) Ppargc1a mtdna Ppargc1a TP53 -/- ND TP53 +/+ HFD TP53 -/- HFD Ppargc1a mtdna TP53 -/- ND TP53 +/+ HFD (Figure 3-5A and B) Ppargc1a TP53 -/- ND mtdna TP53 +/+ HFD Ppargc1a mtdna (Figure 3-5C and D) 6 Ppargc1a 3 L TP53 CDKN1A/p21 (4 HFD ) Cdkn1a Ppargc1a 4 HFD Cdkn1a Ppargc1a (Figure 3-6A and B) 88

92 C2C12 TP53 Ppargc1a TP53 ( ) Ppargc1a mtdna WT (Saleem et l., 2009; Park et al., 2009) TP53 Ppargc1a TP53 (EDL; Extensor digitorum longus muscle) (SOL; soleus muscle) (Park et al., 2009) C2C12 C2C12 Ppargc1a TP53 PPARGC1A TP53 PPARGC1A (Saleem et al., 2009; Sahin et al., 2011; Aquilano et al., 2013; Villeneuve et al., 2013) ( ) TP53 Ppargc1a negative regulator PPARGC1A PPARGC1A PPARGC1A PPARGC1A (BAT, brown adipose tissue) (Puigserver et al., 1998; Kleiner et al., 2012) Ppargc1a KO Ppargc1a TCA (Minamino et al., 2009) Ppargc1a (adipogenesis) TP53 (Mikami et al., 2012) 1 2 L ( ) TP53 ( ) PPARGC1A 89

93 9 TP53 C2C12 adipogenesis negative TP53 KO (Minamino et al., 2009) (Cheng et al., 2010) TP53 KO in vivo 90

94 91

95 92

96 93

97 94

98 95

99 96

100 4 Trehalose protects against oxidative stress through the regulation of both Keap1-Nrf2 and autophagy pathways 97

101 1 2 CTSL CTSB PLIN1 D 1,1- (Argüelles, 2000) (Benaroud et al., 2001) (Kaushik and Bhat, 2003) (Minutoli et al.,2008) mtor (mechanistic target of rapamycin) (Tanaka et al., 2004; Sarkar et al., 2007) tau α-synuclein (Emanuele, 2014) β(aβ) (Perucho et al., 2012; Du et al., 2013) α-synuclein (Sarkar et al., 2007) (Arai et al., 2010; Arai et al; 2013) 98

102 Hepa1-6 ATG5 Atg5 / MEFs Atg5 +/+ MEFs RIKEN Bioresource Center (Ibaraki, Japan) Sqstm1/p62 Sqstm1 -/- MEFs Sqstm1 +/+ MEFs Hepa1-6 MEFs 10% FBS (sigma, St. Louis, MO, USA) 1% penicillin/streptomycin (Sigma, P0781) DMEM (High glucose; Wako, Osaka, Japan) cells/cm 2 ( ) D(-)- ( ) ( ) cycloheximide (CHX) chloroquine (CQ) N N-acetylcysteine (NAC, ) Wako (Tokyo, Japan) D (+)- ( ) (Osaka, Japan) (I0231) (Tokyo, Japan) (α,β-trehalose, ON12751) Carbosynth (Compton, UK) Paraquat (36541) Sigma Millex-GV Filter (MILLIPORE, Billerica, MA, SLGVJ13SL) dimethylsulfoxide (DMSO) DMSO 0.1% PBS Atg5 sidirect Atg5 DNA off-target sense (Patzel et al., 2005; Miyagishi et al., 2004) 2 DNA Operon Biotechnologies (Tokyo, Japan) 2 DNA annealing buffer (50 mm Tris-HCl ph 7.4, 100 mm NaCl, 5 mm MgCl2) PmeI 65 3 BstBI pmxs-mu6-neo DNA pmxs-mu6-shatg5/luc-neo RNAi-KPC-1 99

103 Table 1. The oligonucleotide sequence for shluciferase (Luc) and shatg5 shluc forward 5'-GTACTGAGCCTGTTTGTGGAAttcaagaga TTTCACAAACGGGCTTAGTACttttt-3' reverse 5'-cgaaaaaGTACTAAGCCCGTTTGTGAAAtctcttgaaTTCCACAAACAGGCTCAGTAC-3' forward 5'- GATAGCTTTCTTTATATTGGCttcaagagaGCTAATATGAAGAAAGTTATCttttt -3' shatg5 reverse 5'-cgaaaaaGATAACTTTCTTCATATTAGCtctcttgaaGCCAATATAAAGAAAGCTATC-3' Uppercase letters indicate target sequences against luciferase or Atg5, whereas lowercase letters indicate BstBI or loop structure sequences. GFP-LC3 mrfp-gfp-lc3 GFP-LC3 (#21073) mrfp-gfp-lc3 (#21074m) Addgene (Cambridge, MA, USA) pegfp-lc3 ptflc3 Nhe EcoR pires-neo3 Nhe EcoR EcoR NotI pmxs-amnn-puro NruI NotI pmxs-amnn-puro-mrfp-egfp-lc3 pmxs-amnn-puro pmxs-puro ApaI MluI NruI NspV TaKaRa (Shiga, Japan) SQSTM1 pires-puro N3 Flag-Msp62 WT pires-puro N3 Flag-Msp62 S351E EcoR1 Xho1 Not1 Xho1 pmxs-amnn-puro Not1 Xho1 pmxs-amnn-puro-sqstm1 (p62) WT/S351A shrna GFP-LC3/mRFP-GFP-LC3 SQSTM1 Hepa1-6 SQSTM1 -/- MEFs Platinam-E Packaging Cell Line (plat-e) plat-e cells/cm 2 FuGENE 6 Transfection Reagent (Promega, Madison, WI, USA) plat-e

104 Millex-GV Filter (MILLIPORE, Billerica, MA, SLGVJ13SL) 4 PEG (32% PEG (polyethylglycol 6000; Fluka, Tokyo, Japan, 81253), 0.4M NaCl, 40mM HEPES, ph7.4) 1/ pm rpm 5 OPTI-MEM (Life Technologies Japan Ltd., Tokyo, Japan) Hepa1-6 MEFs 2μg/mL puromycin μg/mL puromycin plat-e 300μl buffer A (20 mm HEPES [ph 7.9], 3 mm MgCl2, 20 mm KCl, 0.68 M sucrose, 20% glycerol, 1% Triton X-100) g g μl buffer A 1300 g μl buffer A 1300 g 4 total lysis buffer (50 mm Tris HCl ph 6.8, 2% SDS, 5% glycerol) Western blot SDS sample buffer (50mM Tris-HCl ph 6.8, 2% SDS, 3M Urea, 6% glycerol) 95 5 (WAT) SDS sample buffer (12000 g, 30 ) 95 5 BSA Protein Assay Kit (Thermo Scientific, Yokohama, Japan) 0.2~2mg/mL BPB/2-Me (0.25% Bromophenol Blue, 50% 2-Mercaptoethanol) 1/ SDS-PAGE (PALL, Port Washington, NY) blocking (2.5% skim milk (Wako), 0.25% BSA, TTBS (25mM Tris-HCl ph 7.4, 140mM NaCl, 2.5mM KCl, 0.1% Tween 20)) 1 (Immuno shot reagent 1 (Cosmo Bio Co.,Ltd., Tokyo, Japan) : blocking = 2 : 1) 4 TTBS 4 (Immuno shot reagent 1 (Cosmo Bio Co.,Ltd) : blocking = 2 : 1) 1 TTBS 4 ImmunoStar LD (Wako, ) 101

105 (LAS 3000, FUJIFILUM, Tokyo, Japan) Table 2 3 Table 2. rabbit LC3 PM036 MBL rabbit SQSTM1 PM045 MBL rabbit p-sqstm1 (S351) PM074 MBL rabbit LMNB1/LaminB1 PM064 MBL mouse NFE2L2 sc-722 Santa cruz mouse TUBA/α-tubulin T6199 Sigma mouse β-actin A1978 Sigma Table 3. HRP-conjugated F (ab ) 2 fragment of goat anti-mouse IgG HRP-conjugated F (ab ) 2 fragment of goat anti-rabbit IgG HRP-conjugated F (ab ) 2 fragment of goat anti-rat IgG Jackson Immunological Research, Jackson Immunological Research, Jackson Immunological Research, Lysotracker red Hepa1-6/GFP-LC3 Poly-D-Lysine (Sigma, P0899) ( ) 6 well dish 50nM Lysotracker red (Thermo Fisher Scientific, Waltham, MA, L7528) in DMEM 15 PBS 2 4% paraformaldehyde 15 Slowfade Antifade Kit (Invitrogen, Carlsbad, CA) (, Osaka, Japan, S1226) (FV-1000-IX81, Oympus, Tokyo, Japan) RealtimePCR ReliaPrep RNA Tissue Miniprep System (Promega, Madison, WI, USA) total RNA mrna NanoDrop 1000 (Thermo Fisher Scientific, Waltham, MA, USA) 1 μg RNA PrimeScript RT reagent Kit (TaKaRa, RR037A) cdna SYBR Premix Ex Taq (TaKaRa, RR041A) CFX Connect Real-Time PCR System (Bio-Rad, Hercules, CA, USA) Table 3 102

106 Table. 3 primer Hmox1 Nqo1 Sqstm1 Lamp1 Rps18 Forward 5 - GAACTTTCAGAAGGGTCAGGTG- -3 Reverse 5 - AGGGAAGTAGAGTGGGGCATAG -3 Forward 5 - CGAATCTGACCTCTATGCTATGAAC -3 Reverse 5 - GAACTGAAATATCACCAGGTCTGC -3 Forward 5 - TGGTGGGAACTCGCTATAAGTG -3 Reverse 5 - CCAAAGTGTCCATGTTTCAGC -3 Forward 5 - TCA GCA TCT CCA ACC ATT CAC -3 Reverse 5 - CAA GTT TAC AGC CAA GAT TCA CG -3 Forward 5 - TGC GAG TAC TCA ACA CCA ACA -3 Reverse 5 - CTT TCC TCA ACA CCA CAT GAG C -3 CM-H2DCFDA ROS 96 well plate 10μM CM-H2DCFDA (invitrogen, Carlsbad, CA, C6827) 5μM MitoSOXTM Red (invitrogen, M36008) 37 1 incubate 2 EnVision (PerkinElmer) ± Student t t-test R Tukey Kramer test Bonferroni correction Figure legends 103

107 1 SQSTM1 Hepa1-6 LC3-II SQSTM1 LC3-II SQSTM1 (Figure 4-1A) ( ) LC3-II SQSTM1 (Figure 4-1B) LC3-II SQSTM1 (Figure 4-1B and C) SQSTM1 SQSTM1 Atg5 MEFs Sqstm1 +/+ MEFs Hepa1-6 LC3-II SQSTM1 Atg5 -/- MEFs SQSTM1 (Figure 4-1D) Sqstm1 -/- MEFs LC3-II (Figure 4-1E) SQSTM1 2 SQSTM1 SQSTM1 1 LC3-II turnover assay chloroquine LC3-II SQSTM1 (Figure 4-2A, B and C) LC3-II turnover assay basal mrfp-gfp-lc3 Hepa1-6 LC3 RFP GFP tandem GFP RFP GFP RFP merge GFP RFP dot chloroquine dot 104

108 dot (Figure 4-2D) GFP-LC3 Hepa1-6 lysotracker Chloroquine GFP dot Lysotracker merge dot GFP dot Lysotracker dot (Figure 4-2E) cycloheximide (CHX) SQSTM1 Atg5 Hepa1-6 SQSTM1 chloroquine 24 Hepa1-6/shLuc SQSTM1 Hepa1-6/shLuc SQSTM1 (Figure 4-2F and G) Hepa1-6/shAtg5 Hepa1-6 Hepa1-6/shLuc SQSTM1 (Figure 4-2 F and G) SQSTM1 3 KEAP1-NFE2L2 SQSTM1 KEAP1 (Kelch ECH associating protein 1) NFE2L2/Nrf2 (Komatsu et al., 2010) NFE2L2 ARE (antioxidant responsive element) Nqo1 (NAD (P)H dehydrogenase, quinone 1) Hmox1 (heme oxygenase-1) (Kaspar et al., 2009) NFE2L2 E3 KEAP1 - (Kaspar et al., 2009) ROS KEAP1 NFE2L2 NFE2L2 (Itoh, 2003; Taguchi et al., 2010) KEPA1-NFE2L2 SQSTM1 KEPA1 NFE2L2 (Komatsu et al., 2010; Lau et al., 2010) SQSTM1 S351 KEAP1 NFE2L2 (Komatsu et al., 2010, Ichimura et al., 2013) SQSTM1 KEAP1-NFE2L2 SQSTM1 SQSTM1 (p-s351) (Figure 4-3A) NFE2L2 105

109 (Figure 4-3A) NFE2L2 SQSTM1 SQSTM1 (KO) MEFs (SQSTM1 -/- MEFs) SQSTM1 +/+ MEFs NFE2L2 SQSTM1 -/- MEFs (Figure 4-3B) SQSTM1 (p-s351) Sqstm1 mrna (Figure 4-3C) NFE2L2 SQSTM1 Sqstm1 mrna SQSTM1 -/- MEFs SQSTM1 (WT) (S351E) SQSTM1 WT SQSTM1 S351E SQSTM1 -/- MEFs SQSTM1 SQSTM1 (S3651) (Figure 4-3D) SQSTM1 mrna NFE2L2 4 KEAP1-NFE2L2 NFE2L2 Hmox1 Nqo1 Hmox1 mrna Nqo1mRNA (Figure 4-4A) ROS (Cochemé and Murphy, 2008; Drechsel and Patel, 2009) ROS (reactive oxygen species) Hepa1-6 ROS (Figure 4-5A) N-acetlycystein (NAC) 106

110 mtor (Fleming et al., 2011; Sarker, 2013) (Benaroud et al., 2001) SQSTM1 (Ishii et al., 1997; Nagaoka et al., 2004) SQSTM1 KEAP1 KEAP1-NFE2L2 NFE2L2 ( ) KEAP1-NFE2L2 Hepa1-6 3T3L1 ( ) Atg5 -/- MEFs Sqstm1 mrna SQSTM1 Giorgetti SQSTM1 mrna (Giorgetti et al.,2015) MEFs (Higuchi et al., 2015) SQSTM1 NFE2L2 NFE2L2 NFE2L2 ROS ROS (Scherz-Shouval and Elazar, 2011) NEFE2L2 ROS SQSTM1 NFE2L2 SQSTM1 postive feedback loop (Jain et al., 2010) SQSTM1 KEAP1 NFE2L2 (Ichimura et al., 2013) SQSTM1 107

111 NFE2L2 SQSTM1 positive feedback wild-type SQSTM1 Sqstm1 -/- MEFs SQSTM1 NFE2L2 (Figure 4-3) SQSTM1 TFEB (transcription factor EB) SQSTM1 TFEB (Sardiello et al., 2009; Settembre et al., 2011) TFEB (Sardiello et al., 2009) TFEB TFEB Lamp1 V-ATPase ( ) SQSTM1 TFEB SQSTM1 (Saito et al., 2013) NFE2L2 KEAP1-NFE2L2 (Kansanen et al 2013; Marcotte et al., 2013; Saito et al., 2016) SQSTM1 NFE2L2 (Saito et al., 2016) KEAP1 NFE2L2 HFD (Xu et al, 2012) curcumin NFE2L2 (Kou et al., 2013; Hatcher et al., 2008) Curcumin oltipraz sulforaphane CDDO-Im NFE2L2 ROS ROS (Zhang et al 2015) NFE2L2 oltipraz (Yu et al., 2011) curcumin (He et al., 2012) SQSTM1 NFE2L2 (Lau et al 2008; Ichimura et al 2013; Xu et al, 2012) curcumin SQSTM1 NFE2L2 KEAP1-NFE2L2 108

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119 Figure L 1 L ROS 2 L TP53 CDKNIA 2 L CTSB 1 B 1 B 2 B 2 L 1 HFD L L L TP53 CDKN1A TP53 TP53 Ppargc1a 3 116

120 TP53 Ppargc1a 3 Ppargc1a 3 TP53 Ppargc1a vicious cycle Figure 5-2 SQSTM1 NFE2L2 4 Figure

121 Figure 5-1. ࡅ ࢯࢯ ࢪ ᶵ Figure 5-2. ࢯࢯ ࢥ ไᚚ 118

122 Figure

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