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Supplementary Information FoxK1 and FoxK2 are Components in Insulin Regulation of Cellular and Mitochondrial Metabolism Sakaguchi et al a ProteomicsProteomics screening screening of IR/IGF1Rof IR/IGF1R binding binding proteins proteinsin the in insulinthe insulin dependent DepenDent manner manner Total: 1469proteins 5 quantified peptide: 707 proteins (Including IRS1 and IRS2) (IR+/IR-1.5, IGF1R+/IGF1R-1.5, IR/IGF1R+/ IR/IGF1R-1.5 ) IGF1R/IR+/ IGf1R/IR-1.5 ) Total 11 proteins IR +/- 2 1.5 3 IGF1R +/-1.5 11 Common proteins →protein 5 FoxK1 (Forkhead box protein K1) 3 IR/IGF1R +/- 1.5 IGF1R/IR +/-1.5 b 5000 4000 3000 2000 1000 FoxK1 Expression FoxK1 … 0 … TA SCF EDL PGF liver Skin Lung Heart RETF BATF Testis Uterus Spleen Soleus Kidney Bladder Stomach Pancreas Cerebellum Quadriceps Epidydimus Gall bladder Hippocampus Adrenal gland Hypothalamus Small intestine Raphe nucleus Gastrocnemius Nucleus tractus Substantia nigra Prefrontal cortex Caudate putamen Ventral tegmental c Nucleus accumbens 3000 2000 Expression 1000 FoxK2 FoxK2 … … 0 … TA SCF EDL PGF liver Skin Lung Heart RETF BATF Testis Uterus Spleen Soleus Kidney Bladder Nucleus Stomach Pancreas Cerebellum Quadriceps Epidydimus Gall bladder Hippocampus Adrenal gland Hypothalamus Small intestine Raphe nucleus Gastrocnemius Nucleus tractus Substantia nigra Prefrontal cortex Caudate putamen Ventral tegmental Sakaguchi et al., Supplementary Figure 1 d DifferentiateD brown adipocytes e Astrocytes Cytoplasm Nucleus Cytoplasm Nucleus Insulin 0 5 10 30 0 5 10 30 min Insulin 0 5 10 30 0 5 10 30 min FoxK1 FoxK1 75 75 75 FoxO1 75 FoxO1 75 75 LaminA/C LaminA/C 37 37 GAPDH GAPDH Supplementary Figure 1. FoxK1 is a novel component of IR- and IGFR-mediated signaling complex. (a) FoxK1 is iDentifieD by the proteomics screening of IR/IGF1R binDing proteins. (b) FoxK1 and (c) FoxK2 expression (qPCR) from 34 tissues from 3-month-olD C57BL/6J mice (All Data are representeD as mean ± SEM, n=3). Data were normalizeD to Tbp. Immunoblotting of FoxO1 anD FoxK1 in nuclear anD cytoplasmic fractions extracteD from DifferentiateD brown preaDipocytes (d) or astrocytes (e) before and after 100 nM insulin stimulation for inDicateD times. Sakaguchi et al., Supplementary Figure 1 a b Insulin - - - - + + + + WT (Brown preaDipocyte) 75 Insulin 0 1 5 10 30 60 min FoxO1 Cytoplasm Cytoplasm FoxK1 FoxK1 75 75 75 37 FoxO1 GAPDH 37 GAPDH 75 FoxO1 FoxK1 Nucleus Nucleus 75 75 FoxK1 FoxO1 75 75 75 LaminA/C Lamin A/C 75 FoxO1 Chromatin FoxK1 75 75 LaminA/C exp 1 2 3 4 1 2 3 4 Supplementary Figure 2. Insulin-induced FoxK1 translocation is reciprocal to FoxO1. (a) Subcellular fractions of cytoplasm, nucleus anD chromatin were prepareD from DKO brown preaDipocytes reexpressing the IR without (0 min) anD with 100 nM insulin stimulation for 30 min. FoxO1 anD FoxK1 in each fraction were assesseD by western blotting, as were markers for Different fractions, cytosol (GAPDH), nuclear anD chromatin (Lamin A/C) (n = 4). (b) Immunoblotting of FoxO1 anD FoxK1 in nuclear anD cytoplasmic fractions extracteD from wilD-type brown preaDipocytes before and after 100 nM insulin stimulation for 30 min. Sakaguchi et al., Supplementary Figure 2 a Vehicle MK2206 U0126 Insulin - - - + + + - - - + + + - - - + + + 75 FoxO1 Cytoplasm FoxK1 75 37 GAPDH 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 exp 75 FoxO1 Nucleus FoxK1 75 75 LaminA/C b vehicle LY294002 Insulin IGF1 PDGF EGF Insulin IGF1 PDGF EGF Insulin - + - + - + - + - + - + - + - + FoxK1 75 Nucleus 75 LaminA/C c ★★★ ★★★ ★★ 3.0 ★★★ 2.5 2.0 1.5 FoxK1 Intensity (AU) 1.0 0.5 0.0 Relative Nuclear - + + + + + + + + Insulin IGF1 Sakaguchi et al., Supplementary Figure 3 d e Insulin - + + + + + IGF1 - + + + + + FoxK1 FoxK1 75 75 lysate 37 GAPDH lysate 37 GAPDH Cytoplasm Cytoplasm Cytoplasm Cytoplasm 100 FoxK1 FoxK1 75 lysate lysate 75 75 Nucleus Nucleus Nucleus Nucleus LaminA/C LaminA/C p-Akt p-Akt (S473) 50 (S473) 50 p-ERK1/2 p-ERK1/2 (T202/Y204) (T202/Y204) lysate lysate 37 37 Whole cell cell Whole Whole cell cell Whole T-Akt T-Akt 50 50 f Supplementary Figure 3. Insulin-induced FoxK1 translocation is regulated by PI3K- Akt rather than ERK MAPK pathway. (a) Immunoblotting of FoxO1 anD FoxK1 in nuclear anD cytoplasmic fractions extracteD from IR-expressing brown preaDipocytes before and after 10 nM insulin stimulation for 30 min in the presence or absence of the Akt inhibitor MK2206 (5 μM) or the MEK1/2 inhibitor U0126 (20 μM) (n = 4). (b) Immunoblotting of FoxK1 in nuclear fraction extracteD from WT-brown preaDipocytes before anD after stimulation with insulin, IGF1, PDGF anD EGF at 15 min in the presence or absence of the PI3K inhibitor (LY-294002). (c) Relative FoxK1 fluorescence intensity in nuclear (DAPI+) area quantifieD by Image J in Figure 2D. All Data are representeD as mean ± SEM. (P < 0.05; **, P < 0.01; ***, P < 0.001) (n = 4). (d,e) Immunoblotting of FoxO1 anD FoxK1 in nuclear anD cytoplasmic fractions extracteD from AML12 cells before or after stimulation with 100 nM insulin anD IGF1 for 30 min in the presence or absence of the Akt inhibitor MK2206 (5 μM) or the MEK1/2 inhibitor U0126 (20 μM). (e) Comparison of the phosphorylation clusters between FoxK1 anD FoxK2 after Insulin/IGF1 stimulation. The significant increase after Insulin/IGF1 stimulation shown in reD anD the Decrease shown in blue. Sakaguchi et al., Supplementary Figure 3 a Vehicle Rapamycin CHIR99021 Insulin - - - + + + - - - + + + - - - + + + FoxK1 80 Cytoplasm FoxK2 65 GAPDH 35 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 exp FoxK1 80 Nucleus FoxK2 80 LaminA/C 50 b Control GSK3α KD c Control GSK3β KD exp 1 2 3 4 1 2 3 4 exp 1 2 3 4 1 2 3 4 55 GSK3α 50 GSK3β 37 GAPDH GAPDH 34 Cytoplasm Nucleus d Control GSK3α/β DKD e Insulin - - - - + + + + - - - - + + + + Cytoplasm GSK3β GSK3β - - FoxK1 Control DKD GSK3α/β +HA Control DKD GSK3α/β +HA 75 DKD GSK3α/β DKD GSK3α/β 37 Insulin - + - + - + - + - + - + GAPDH FoxK1 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 exp 75 Nucleus FoxK1 37 75 GAPDH LaminA/C 75 LaminA/C 75 f Insulin, 15 min g Insulin, 15 min IRb IRb 95 95 FoxK1 95 95 FoxK1 p-IRb 95 p-IRb 95 IRb 95 95 FoxK1 FoxK1 43 95 b-Actin 43 b-Actin Sakaguchi et al., Supplementary Figure 4 h Cytoplasm Nucleus Flag-FoxK1 WT Flag-FoxK1 Flag-FoxK1 WT Flag-FoxK1 S402A/S406A/S454A/S458A S402A/S406A/S454A/S458A Insulin - - - + + + - - - + + + - - - + + + - - - + + + 95 Flag 72 Lamin A/C GAPDH 34 Supplementary Figure 4. Insulin-induced FoxK1 translocation is regulated by not only mTOR but also GSK signaling. (a) Nuclear cytoplasmic fractionation anD immunoblotting for FoxK1 anD FoxK2 extracteD from the AML12 cells before anD after 100 nM insulin at 30 min the presence or absence of 100 nM rapamycin (mTOR inhibitor) or/and 10 μM CHIR99201 (GSK3 inhibitor). GAPDH is cytosolic marker anD Lamin A/C is nuclear marker (n = 3). Immunoblotting Control (NS siRNA), GSK3α-knockdown (GSK3α KD) (b) and GSK3β-knockdown (GSK3β KD) (c) cells by siRNAs with anti-GSK3α anD GSK3β antiboDies (n = 4). (d) Nuclear anD cytoplasmic fractionation anD immunoblotting of FoxK1 extracteD from the Control (NS siRNA) or GSK3α/β Double knockdown cells before (0 min) and after 100 nM insulin stimulation for 30 min. (n = 4). (e) Nuclear anD cytoplasmic fractionation anD immunoblotting of FoxK1 extracteD from the Control (NS siRNA) , GSK3α/β DKD cells anD re-expresseD with HA-GSK3β in GSK3α/β DKD cells. before (0 min) and after 100 nM insulin stimulation for 30 min.(f) Immunoblotting with anti- IRβ, p-IRβ anD FoxK1 antiboDy in lysates extracteD from AML12 cells which were immunoprecipitateD with p-Tyr antiboDy following insulin stimulation. (g) Immunoblotting with anti-IRβ, p-IRβ anD FoxK1 antiboDy in lysates extracteD from liver samples which were immunoprecipitateD with p-Tyr antiboDy following insulin stimulation via vena cava injections. (h) Nuclear anD cytoplasmic fractionation anD immunoblotting of Flag extracteD from the overexpresseD 3XFlag-FoxK1 wilD type (WT) cells or overexpresseD 3XFlag- FoxK1 S402A/S406A/S454A/S458A mutant cells before (0 min) anD after 100 nM insulin stimulation for 30 min. Sakaguchi et al., Supplementary Figure 4 a Control FoxK1 OE FoxK2 OE b FoxK1 3.0 1 2 3 1 2 3 1 2 3 exp ★ ★ - + - + - + - + - + - + - + - + - + Insulin 2.5 Flag 2.0 75 1.5 FoxK1 95 1.0 FoxK2 72 0.5 37 GAPDH Control - Control + FoxK1 OE- FoxK1 OE+ FoxK2 OE- FoxK2 OE+ 0.0 - + - + - + d Control FoxK1 OE FoxK2 OE 1 2 3 1 2 3 1 2 3 exp - + - + - + - + - + - + - + - + - + Insulin p-IR/IGF1R c FoxK2 75 10.0 ★★★ IRb 75 8.0 ★ p-IRS-1 (Y612) 150 6.0 IRS-1 150 4.0 p-Akt (S473) 50 2.0 50 Akt p-ERK1/2 0.0 Control - Control + FoxK1 OE- FoxK1 OE+ FoxK2 OE- FoxK2 OE+ 37 (T202/Y204) - + - + - + 37 ERK1/2 37 p-S6 (S235/236) 37 S6 p-IR/ /IRb p-IRS-1 (Y612) p-Akt (S473) p-ERK1/2 (T202/Y204) p-S6 (S235/236) e f g h 1.5 ★ i 1.5 ★★★ 1.5 1.5 1.5 expression Relative Relative expression Relative expression Relative expression Relative Relative expression Relative NS NS 0.05 1.0 1.0 1.0 1.0 1.0 0.5 0.5 0.5 0.5 0.5 Control - Control + Foxk1 KD - Foxk1 KD + Foxk2 KD - Foxk2 KD + Control - Control + Foxk1 KD - Foxk1 KD + Foxk2 KD - Foxk2 KD + Control - Control + Foxk1 KD - Foxk1 KD + Foxk2 KD - Foxk2 KD + Control - Control + Foxk1 KD - Foxk1 KD + Foxk2 KD - Foxk2 KD + 0.0 Control - Control + Foxk1 KD - Foxk1 KD + Foxk2 KD - Foxk2 KD + 0.0 0.0 0.0 0.0 - + - + - + Insulin - + - + - + - + - + - + - + - + - + - + - + - + Supplementary Figure 5.
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  • Foxk1 and Foxk2 in Insulin Regulation of Cellular and Mitochondrial Metabolism

    Foxk1 and Foxk2 in Insulin Regulation of Cellular and Mitochondrial Metabolism

    ARTICLE https://doi.org/10.1038/s41467-019-09418-0 OPEN FoxK1 and FoxK2 in insulin regulation of cellular and mitochondrial metabolism Masaji Sakaguchi1,2,3, Weikang Cai1,2, Chih-Hao Wang1,2, Carly T. Cederquist1,2, Marcos Damasio1,2, Erica P. Homan1,2, Thiago Batista1,2, Alfred K. Ramirez1,2, Manoj K. Gupta1,2, Martin Steger4, Nicolai J. Wewer Albrechtsen4,5,6, Shailendra Kumar Singh7, Eiichi Araki3, Matthias Mann 4, Sven Enerbäck 8 & C. Ronald Kahn1,2 1234567890():,; A major target of insulin signaling is the FoxO family of Forkhead transcription factors, which translocate from the nucleus to the cytoplasm following insulin-stimulated phosphorylation. Here we show that the Forkhead transcription factors FoxK1 and FoxK2 are also downstream targets of insulin action, but that following insulin stimulation, they translocate from the cytoplasm to nucleus, reciprocal to the translocation of FoxO1. FoxK1/FoxK2 translocation to the nucleus is dependent on the Akt-mTOR pathway, while its localization to the cytoplasm in the basal state is dependent on GSK3. Knockdown of FoxK1 and FoxK2 in liver cells results in upregulation of genes related to apoptosis and down-regulation of genes involved in cell cycle and lipid metabolism. This is associated with decreased cell proliferation and altered mito- chondrial fatty acid metabolism. Thus, FoxK1/K2 are reciprocally regulated to FoxO1 following insulin stimulation and play a critical role in the control of apoptosis, metabolism and mitochondrial function. 1 Sections of Integrative Physiology and Metabolism and Islet Cell Biology and Regenerative Medicine, Joslin Diabetes Center, Boston, MA 02215, USA. 2 Department of Medicine, Harvard Medical School, Boston, MA 02215, USA.
  • Target Genes Regulated by Hsa-Mir-21, by Hsa-Mir-203, by Hsa-Mir-21 and by Hsa-Mir-143

    Target Genes Regulated by Hsa-Mir-21, by Hsa-Mir-203, by Hsa-Mir-21 and by Hsa-Mir-143

    Supplemental table 1: Target genes regulated by hsa-miR-205 Index Target gene Index Target gene Index Target gene Index Target gene Index Target gene 1 KCTD20 35 UBE2Z 69 SLC38A1 103 LPCAT1 137 STK38L 2 MAPK14 36 YWHAH 70 ANGPTL7 104 MARCKS 138 C1orf123 3 TXNL1 37 RBBP4 71 CTGF 105 MED13 139 GUCD1 4 SPDL1 38 LRP1 72 CYR61 106 IPO7 140 CDK6 5 TCF20 39 IMPAD1 73 TP73 107 PHC2 141 CDKN2AIPNL 6 RAN 40 GNAS 74 EGLN2 108 PICALM 142 CLIP1 7 RGS6 41 MED1 75 ERBB2 109 PLAGL2 143 CUL5 8 HOXA11 42 INPPL1 76 PRRG4 110 NDUFA4 144 C6orf201 9 PAPPA-AS1 43 DDX5 77 F2RL2 111 NDUFB2 145 VTI1A 10 PRR15 44 E2F1 78 GOT1 112 NIPA2 146 SLC5A12 11 ACTRT3 45 E2F5 79 NUFIP2 113 NOTCH2 147 MAML2 12 YES1 46 ZEB2 80 IL24 114 PANK1 148 MAP3K9 13 SRC 47 ERBB3 81 IL32 115 PARD6B 149 NUDT21 14 NPRL3 48 PRKCE 82 RNF217 116 TMEM66 150 DNAJA1 15 NFAT5 49 SLC41A1 83 ZNF585B 117 EZR 151 CCDC108 16 XPOT 50 SLC7A2 84 SIGMAR1 118 ENPP4 152 SHISA6 17 KCTD16 51 ZEB1 85 VEGFA 119 LRRTM4 153 ACP1 18 TMSB4X 52 PHF8 86 BCL9L 120 KCNJ10 154 BCL2 19 PLCXD2 53 TMEM201 87 CREB1 121 PHLPP2 155 NCAPG 20 TNFSF8 54 PTPRJ 88 SERINC3 122 YEATS2 156 KLHL5 21 SLC25A25 55 ETNK1 89 HMGB3 123 VAMP1 157 ACSL4 22 C11orf74 56 XPR1 90 SRD5A1 124 RTN3 158 BCL6 23 GM2A 57 MRPL44 91 PTEN 125 RFX7 159 ITGA5 24 SMNDC1 58 TM9SF2 92 ESRRG 126 RAP2B 160 ACSL1 25 BAMBI 59 PAIP2B 93 PRLR 127 TRAF3IP1 161 EID2B 26 LCOR 60 NEK9 94 ICK 128 SERTAD2 162 TEX35 27 TMEM239 61 NOX5 95 LOH12CR1 129 TOLLIP 163 YY1 28 AMOT 62 DMXL2 96 SLC39A14 130 TMEM55B 164 SMAD1 29 CDK1 63 ETF1 97 BDP1 131 TMEM123 165 SMAD4 30 SQLE 64