Supplemental Table 1. Primers used for real-time quantitative RT-PCR assay Name FoxO1 forward FoxO1 reverse GK forward GK reverse INS-1 forward INS-1 reverse INS-2 forward INS-2 reverse Glut2 forward Glut2 reverse MafA forward MafA reverse PDX-1 forward PDX-1 reverse NeuroD forward NeuroD reverse FoxA2 reverse FoxA2 reverse IRS-1 forward IRS-1 reverse IRS-2 forward Nuleotide sequene AAGAGCGTGCCCTACTTCAA CTCTTGCCCAGACTGGAGAG TGGATGACAGAGCCAGGATGG ACTTCTGAGCCTTCTGGGGTG CTTGCCCTCTGGGAGCCCA TGAAGGTCCCCGGGGCTTC CTTCCTCTGGGAGTCCCAC CACCTGCTCCCGGGCCTCCA TCAGAAGACAAGATCACCGG GTGAGCAGATCCTTCAGTCT AGGAGGAGGTCATCCGACTG CTTCTCGCTCTCCAGAATGTG AGCAGTACTACGCGGCCACA GCACTTCGTATGGGGAGATG GATCGTCACTATTCAGAACC CCTCTAGATCCTCATCTTCC CTGGGAGCCGTGAAGATGGA TGAGCCGCTCATGCCCGCCA GCGAGCCCTCCGGATACC GTGTAGAGAGCCACCAGGTGC AGCAGAAGCACGGCCACAA 1
IRS-2 reverse Cnd1 forward Cnd1 reverse Cnd2 forward Cnd2 reverse Cnd3 forward Cnd3 reverse CnA2 forward CnA2 reverse CnH forward CnH reverse Cdk1 forward Cdk1 reverse Cdk2 forward Cdk2 reverse Cdk4 forward Cdk4 reverse Cdk6 forward Cdk6 reverse 2F2 forward 2F2 reverse p16 forward p16 reverse p21 forward TGCTCGTTCTCCGCCGCTA CAAAATGCCAGAGGCGGATG GAAAGTGCGTTGTGCGGTAG CTGCGGAAAAGCTGTGCATT AACTTGAAGTCGGTAGCGCA GCTTCTCCTAGGACTCGCTAAC CATGTGCGGCTTGATCTCCT TCCTTGCTTTTGACTTGGCT ATGACTCAGGCCAGCTCTGT TGCATTTTTGGCCTGCAAAG TCCAACATGGGGTACCGAGT CACCAAGAAGCCGCTTTTCC AAAGTACGGGTGCTTCAGGG GTGTACCCAGCACCATGCTA AAATCTTGCCGAGCCCACTT CTCGCGGCCTGTGTCTATGG TCTGGAGGTGGCCCTTTATCT GCCTATGGGAAGGTGTTCAA GGGCTCTGGAACTTTATCCA ATTTGAAGACCCCACCCGAC CTGTCCGGCACTTCCAATCT GTCGCAGGTTCTTGGTCACT TCTGCACCGTAGTTGAGCAG TCCAGACATTCAGAGCCACA 2
p21 reverse p27 forward p27 reverse p57 forward p57 reverse prb forward prb reverse Cat forward Cat reverse Sod1 forward Sod1 reverse Gpx1 forward Gpx1 reverse 18S RNA forward 18S RNA reverse rsod1 forward rsod1 reverse rgpx1 forward rgpx1 reverse rcat forward rcat reverse rpdx1 forward rpdx1 reverse rbl-2 forward GACCCAGGGCTCAGGTAGA AGATACGAGTGGCAGGAGGT ATGCCGGTCCTCAGAGTTTG CACTCTGTACCATGTGCAAGGAGTA TTTCTCTTTTTGTTTTGCACTGAGA TCAGAAGGTCTGCCAACACC AAGCGCAGCTTTTTCAGTGG GTCCAGTGCGCTGTAGATGT GCGTGTAGGTGTGAATTGCG GTACCAGTGCAGGACCTCAT CGCAATCCCAATCACTCCAC TGCAATCAGTTCGGACACCA AAGGTAAAGAGCGGGTGAGC AAACGGCTACCACATCCAAG CCTCCAATGGATCCTCGTTA ATGAAGGCCGTGTGCGTGCT CATTGCCCAGGTCTCCAACA TGGCACAGTCCACCGTGTAT CATTCTTGCCATTCTCCTGA CAGCCAGCGACCAGATGA CTGGTAATATCGTGGGTGAC GTGAGGAGCAGTACTACGCG AGCTGAGCTGGGAGGTGGT GAGGGGCTACGAGTGGGATA 3
rbl-2 reverse rbax forward rbax reverse rbad forward rbad reverse rcasp3 forward rcasp3 reverse rcasp8 forward rcasp8 reverse rcasp9 forward rcasp9 reverse CGGTAGCGACGAGAGAAGTC AGGGGCCTTTTTGCTACAGG CCAGTTGAAGTTGCCGTCTG AGATCCCAGAGTTTGAGCCG GCGCCTCCATGATGACTGTT ACCCTGAAATGGGCTTGTGT CACAGGTCCGTTCGTTCCAA GAACGTCTGGGCAACGAAGA TCCCGCCGACTGATATGGAA GCGCGACATGATCGAGGATA CCAGGTGGTCTAGGGGTGTA All nuleotide sequenes are in 5ʼ-to-3ʼ orientation and were purhased from Integrated DNA Tehnologies (Coralville, IA). All primers were for mouse mrna speies exept for the ones labeled with the lower ase letter of r for rat mrna speies. 4
Supplemental Figure legends: Supplemental Fig. 1. Central FoxO1 expression and food intake in FoxO1-tg vs. WT mie on regular how: FoxO1-tg mie (male, 12 weeks of age, n=6) and sex/age-mathed WT littermates (n=6) were euthanized and hypothalamus was retrieved for immunoblot analysis using anti- FoxO1 and anti-β-atin antibodies (A) as well as for real-time qrt-pcr analysis for quantifying FoxO1 mrna levels using 18S RNA as ontrol (B). In addition, FoxO1-tg mie (male, 12 weeks of age, n=3) and sex/age-mathed WT littermates (n=3) were euthanized and the whole brain FoxO1 protein levels were determined by anti-foxo1 and anti-β-atin immunoblot analysis (C). FoxO1-tg mie (male, 12 weeks of age, n=1) and sex/age-mathed WT littermates (n=1) were monitored for food intake. The amount of food intake per mouse was determined during a 24-h feeding period (D). NS, not signifiant. Supplemental Fig. 2. Food intake and immunoytohemistry of dispersed islet ells of FoxO1-tg vs. WT mie on high fat diet: (A) Food intake. FoxO1-tg mie (male, 8 weeks old, n=1) and sex/age-mathed WT littermates (n=1) were fed high fat for 8 weeks. Individual mie were monitored for food intake. The amount of food intake per mouse was determined during a 24-h feeding period. In addition, mie were plaed individually in metaboli ages with free aess to food and water in the Oxymax Lab Animal Monitoring System. After alamation for 2 days, oxygen onsumption and respiratory exhange ratio of mie were determined during a 48-h period. (B) The oxygen onsumption rate. (C) The mean oxygen onsumption rate. (D) The respiratory exhange ratio (RR) in a 12-h light/dark yle. 5
() The mean respiratory exhange ratio. To perform immunoytohemistry of dispersed islet ells, FoxO1-tg mie (male, 8 weeks old, n=4) and sex/age-mathed WT littermates (n=4) were fed high fat diet for 8 weeks. Mie were euthanized for isolating islets. Handpiked islets (n=1-15 per mouse) from ontrol and FoxO1-tg mie were dispersed into single ells by DTA-trypsin treatment, followed by anti-insulin immunoytohemistry. Shown are representative pitures of insulin-positive ells dispersed from about 5 islets in WT (F) and FoxO1-tg (G) groups. NS, not signifiant. Bar, 2 µm. Supplemental Fig. 3. Gluose and lipid metabolism in obese C57BL/6J mie: C57BL/6J mie (male, 6 weeks old, n=7 per group) were fed regular how or high fat diet for 1 weeks. Blood gluose and lipid metabolism were determined. (A) Gluose tolerane. Mie were fasted for 16 h, followed by i.p. injetion of gluose (2g/kg). Blood gluose levels were determined before and after gluose administration. (B) Plasma triglyeride levels. (C) Plasma holesterol levels. Plasma triglyeride and holesterol levels were determined in mie after 16-h fasting. *P<.5 and **P<.1 vs. lean mie. Supplemental Fig. 4. ffet of FoxO1 on Pdx1 expression and nulear loalization in β-ells: INS-1 ells were transdued with ontrol and FoxO1 vetor (1 pfu/ell) in ulture. ah ondition was run in tripliate. After 24-h inubation, ells were proessed for real-time qrt-pcr analysis for determining Pdx1 mrna levels (A), as well for immunoblot assay (B) for determining subellular distribution of Pdx1 (C) and FoxO1 (D). Aliquots of INS-1 pre-transdued with ontrol and FoxO1 vetor were subjeted to anti-pdx1 6
immunoytohemistry for visualizing FoxO1 and Pdx1 o-loalization (). Bar, 1 µm. *P<.5 vs. ontrol. NS, not signifiant. Supplemental Fig. 5. Immunohistohemistry of human fetal and mature panreas visualizes o-loalization of FoxO1 and Pdx1 in islet ells: Frozen setions of human fetal panreas (14-week old) were o-immunostained with rabbit anti-foxo1 (A) and guinea pig anti-pdx1 (B) antibodies, and ounter-stained with DAPI (C), followed by immunofluoresent mirosopy. Merged images were shown in (D). Panels (-H) were the same setions examined at high resolution. Similar results were reapitulated in mature human panreas. Frozen setions of human panreas (2.5 years old) were o-immunostained with rabbit anti-foxo1 (I) and guinea pig anti-pdx1 (J), followed by immunofluoresent mirosopy. Merged image was shown in (K). Islets were outlined by dash lines. Islet ells that were positively stained for both FoxO1 and Pdx1 in the nuleus were marked by arrows. Bar, 1 µm. Supplemental Fig. 6. The FoxO1 feedbak loop: FoxO1 ats to transmit insulin signal from ell surfae to downstream targets involved in ell metabolism, survival, proliferation and anti-oxidation. Inreased FoxO1 ativity stimulates the expression of Irs2, whih in turns inhibits FoxO1 ativity via the PI3K- Akt/PKB-dependent mehanism. This forms an autoregulated feedbak loop for regulating FoxO1 ativity in ells. Supplemental Fig. 7. FoxO1 integrates overnutrition to β-ell ompensation: FoxO1 ativity is upregulated in β-ells in response to metaboli stress suh as 7
overnutrition, obesity and/or insulin resistane. This effet primes β-ells to ompensate for obesity and insulin resistane via at least three distint mehanisms by improving β- ell mass and funtion, enhaning β-ell gluose sensing, and augmenting β-ell antioxidative funtion. This model is not exlusive of other potential mehanisms by FoxO1 orhestrates β-ell ompensation for overnutrition and insulin resistane in dietary obesity. 8
Supplemental Figure 1 A B - L. 2. NS a. <( 1.6 2. z >< ::: 1.2 u.. 1.6 (.).8 >< 1.2 o o.4 u.. (.).J:::..8 -. a. o >- I Fox1 o.4.j:::. -atin - a.. >- WT Fox1-tg I WT Fox1-tg NS D 2. 1.6 NS - L. a. 1.2.9 >. C1l ><.8 32 NS u.. -O'l '.6.4 o ;: L. >. ( -o....3 -... O'l Fox1 C1l (.). -atin WT Fox1-tg WT Fox1-tg
Supplemental Figure 2 A.5.4 NS 8 4 -.s:: g, s 35.s.Q a. :::J (/) u 3 25 8 2 >.3.2.1. +----L----1.-...--- WT Fox1-tg -o- WT +- Fox1-tg :2 35 --) :::J - 3 a :::J 25 (/) 8 8 > ro 2 -o-wt +- Fox1 -tg or+. --.-_., Dark -o- WT 1. +- Fox1-tg Light 1. Dark Light -o-wt +- Fox1 -tg (]) C) ro.s:: u X (]).8 a.. (/) :: w ::.9.8.7 o;.... -=============== Dark Light :: w :: ro (]).9.8.7 Dark Light F G WT Fox1-tg
Supplemental Figure 3 A -...J 6 -D-Lean ---Obese "" -> 5 4 C/) 3 () ::J > 2 "" 1 o 3 6 9 12 15 18 21 24 8-18...J "" -a, 1so. 12 "" 9 () 6 >. > 3 I- Lean Time (min) * Obese -2 *...J "" -> 15 - e 1oo... C/) 5 () Lean Obese
Supplemental Figure 4 A B C/) 2. > 1.6 <( 1.2 z a:::.8.4 X '" Cl. NS Nulear Fox1 Cytosoli Fox1 Nulear Pdx1 Cytosoli Pdx1 -a tin Control Fox1 Control Fox1 D Nule u s Cytoplasm C/) N ule u s 1.6 en > 6 > 5 * 1.2 " a; 4 -.8... e... 3 a. a. 2.4...- X '" 1 Cl. X Control Fox1 Control Fox1 LL. Control Fox1 Cytoplasm Control Fox1 Anti-Pdx1 DAPI Merged Control Fox1
Supplemental Figure 5 -en.:::&:...q,... - en u lo... () u a. u +-' - u ::::J I A 8 D F G H FOX1 PDX1 DAP1 Merged en u lo... () u a. lo... ::::J +-' u u ::::J I J FOX1 PDX1 Merged K
Supplemental Figure 6 lnsr PI3K Akt/PK 8! Metabolism Proliferation Anti-oxidation
Supplemental Figure 7 Metaboli Stress (Overnutrition, Obesity or Insulin Resistane) fi-cell Mass/Funtion Gluose Sensing Anti-Oxidation Beta-Cell Compensation