Rho-kinase Regulates Energy Balance by Targeting Hypothalamic Leptin Receptor Signaling

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1 Rho-kinase Regulates Energy Balane y Targeting Hypothalami Leptin Reeptor Signaling Hu Huang 1, Dong Kong 1, Kyung Hee Byun 2, Chianping Ye 1, Shuihi Koda 1, Dae Ho Lee 1, Byung-Chul Oh 2, Sam W Lee 3, Bonghee Lee 2, Janie M Zaolotny 1, Min Seon Kim 4, Christian Bjoraek 1, Bradford B Lowell 1 and Young-Bum Kim1, 2, # 1 Division of Endorinology, Diaetes and Metaolism, Beth Israel Deaoness Medial Center and Harvard Medial Shool, Boston, MA 2215, USA 2 Gahon University of Mediine & Siene, Graduate Shools of Mediine, Lee Gil Ya Caner & Diaetes Institute, Inheon , Korea 3 Cutaneous Biology Researh Center, Massahusetts General Hospital and Harvard Medial Shool, Charlestown, MA 2129, USA 4 Department of Internal Mediine, Asan Institute for Life Sienes, University of Ulsan College of Mediine, Seoul , Korea # Corresponding author: ykim2@idm.harvard.edu Nature Neurosiene: doi:1.138/nn.327

2 G (t) a No leptin Leptin RO CK1 LepR C ross orrelation Log[time] (s) RO CK1 LepR C ross orrelation Log[time] (s) pstat3 Stat3 -Gal Adenovirus: Lu sirna: Leptin: Leptin DAPI Merged Control d sirna: RPII Lu Leptin: Nuleus Cytosol inhiitor (AG49) e RPII Leptin: Lu AG49 Nuleus Cytosol f Lu Y27632 Rho-kinase inhiitor (Y-27632) RPII Leptin: Nuleus Cytosol Supplementary Figure 1. Effets of on leptin reeptor signaling. (a) Leptin stimulates a physial interation etween and LepR. GT1-7 ells were serum-starved and treated with leptin (1 nm) or vehile and analysed y fluoresene ross-orrelation spetrosopy. X axis: lag time during measurements, Y axis: autoorrelation and ross-orrelation funtion [G(t)]. Cross-orrelation indiates the physial interation strength etween and LepR. () regulates leptin-stimulated Stat3 phosphorylation. GT1-7 ells were infeted with adenovirus enoding -galatosidase (-gal) or WT-, and CHO ells were transfeted with luiferase (Lu) sirna or sirna, and then treated with vehile or leptin (1 nm) for 15 min. pstat3 Tyr, Stat3,, and atin were measured y immunolotting. (-f) or inhiition loks leptin-dependent nulear export. () GT1-7 ells were treated with vehile or leptin for 15 min in the presene of AG49 (3 M, inhiitor) or Y (1 M, ROCK inhiitor). Cells were stained for or with DAPI. Bar = 2.5 m. (d) GT1-7 ells were transfeted with Lu or sirna, and treated with vehile or leptin (1 nm) for 15 min. (e-f) GT1-7 ells were treated as ()., RNA polymerase II (RPII), and atin were measured y immunolotting of nulear and ytosoli ellular frations. Nature Neurosiene: doi:1.138/nn.327

3 a LepR DAPI Merged d pstat3 Merged e Supplementary Figure 2. expression in hypothalami aruate neurons. (a) Distriution of mrna in rain. Data are from the Allen Brain Atlas ( for Proe No: Rok1 - RP_551_4_G3 - sagittal. () protein in hypothalami aruate neurons. Immunohistohemistry (IHC) for protein (green) was performed on oronal rain setions from 12-week old C57BL/6 male mie. Bar = 2 M. () is o-loalized with the LepR in hypothalami aruate neurons. IHC for (green) was performed on oronal rain setion of the LepR-ires-Cre, lox-td-tomato reporter mie, in whih LepR neurons express the td-tomato red fluoresent protein. Nulei were ounterstained with 4',6-diamidino-2-phenylindole (DAPI)(lue). Bar = 5 M. (d) is o-loalized with leptin-responsive pstat3 in hypothalami aruate neurons. Doule IHC for and pstat3 was performed on oronal rain setion from C57BL/6 mie stimulated with leptin (i.p. 3mg/kg for 3 min)(top images). High magnifiation images demonstrate that all hypothalami aruate neurons expressing leptin-responsive pstat3 also express. Bar = 2 M. (e) Representative high magnifiation images of and leptin-responsive pstat3 o-loalization in two hypothalami aruate neurons. Nature Neurosiene: doi:1.138/nn.327

4 a * d 12 *.5 * Daily food intake (g) Leptin (ng/ml) 8 4 Insulin (ng/ml) Gluose (mg/dl) e Cortiosterone (ng/ml) f Nose to anus length (m) g h i j Femur length (m) BMD (g/m2) BMC (g) mrna (AU) flox/flox POMC-Cre, flox/flox ** POMC AgRP NPY Supplementary Figure 3. Daily food intake, lood ompositions, ody length, asi one profiles, and hypothalami neuropeptide gene expression in POMC neuron-speifi -defiient mie. (a) Daily food intake of 1-week-old male flox/flox (ontrol) and POMC-Cre, flox/flox mie (n = 11-12). () Serum leptin, () serum insulin, (d) plasma gluose, (e) serum ortiosterone levels and (f) nose-to-anus length of 24- week-old male mie (n = 6-1). (g) Femur length, (h) one mineral density (BMD) and (i) one mineral ontent (BMC) of 24-week-old male mie (n =1). (j) POMC, AgRP and NPY gene expression in whole hypothalamus of 12-week-old male mie (n = 11-12). Data are presented as means s.e.m. *p <.5 and **p <.5 vs. ontrol mie y unpaired Student s t test, t = (daily food intake), t = (leptin), t = (insulin), t = 3.59 (NPY mrna). Nature Neurosiene: doi:1.138/nn.327

5 a flox/flox POMC-Cre, flox/flox Cell numer per setion Dendrites length (m) Cell ody area (m 2 ) flox/flox POMC-Cre, flox/flox flox/flox POMC-Cre, flox/flox Supplementary Figure 4. POMC neuron morphology and projetions in hypothalamus of POMC neuron-speifi -defiient mie. (a) Immunohistohemistry for POMC was performed on oronal rain setions from 2-week-old flox/flox (ontrol) and POMC-Cre, flox/flox male mie. For eah image, three individual POMC neurons are shown in insets at high magnifiation (2x). Bar =5 M. () POMC neuron numer, dendrite length, and ell ody area were measured using Neuroluida omputer-assisted graphing software (MiroBrightField Biosiene). Data are present as means s.e.m. (n = 5). Bar = 5 M. () Projetions of aruate nuleus POMC neurons to the DMH were similar in ontrol and POMC neuron-speifi -defiient mie. Nature Neurosiene: doi:1.138/nn.327

6 a Gluose (mg/dl) * flox/flox POMC-Cre, flox/flox ** * Gluose (mg/dl) flox/flox POMC-Cre, flox/flox Time after insulin injetion (min) Time after gluose injetion (min) Numer of movements 3, 2,5 2, 1,5 1, 5 Day: flox/flox POMC-Cre, flox/flox Supplementary Figure 5. Insulin tolerane test (ITT), gluose tolerane test (GTT), and loomotor ativity of POMC neuron-speifi defiient mie. (a) An ITT was performed 5h after food removal in 12-week-old male flox/flox (ontrol) and POMC-Cre, flox/flox mie (n = 6-7). Mie were injeted with insulin (i.p.. U/kg of ody weight). Blood gluose was determined y gluometer at indiated times. Data are presented as means s.e.m. *p <.5, and**p <.1 vs. ontrol mie y unpaire d Student s t test, t = -2. (15 min), t = -4.1 (3 min), t = -2.2 (6 min). () A GTT was performed on 14-week-old male flox/flox (ontrol) and POMC-Cre, flox/flox mie (n = 6-7) after overnight fasting. Mie were injeted with gluose (i.p. 1.g/kg of ody weight). Blood gluose was determined at the indiated times y gluometer. Data are presented as means s.e.m. () Loomotor ativity in 18-week-old male mie was determined using infrared eam reak methodology over a 2 week period. Blak ars indiates dark phase periods Data are presented as means (n = 8). Nature Neurosiene: doi:1.138/nn.327

7 a ROCk1flox/flox POMC-Cre, ROCk1flox/flox pstat3 pstat3 flox/flox POMC-Cre, flox/flox pstat3 POMC POMC Stat3 Leptin: Merged Merged LepR Leptin Aruate Neuron 863 PP py py Neuronal Ativity/Firing pser IRS/PI3K py Tyr py 1138 TyrpY 1138 STAT3 Nuleus Energy Balane FoxO P P P sos3 pom Transriptional Control Supplementary Figure 6. Regulation of Stat3 phosphorylation in POMC neurons y. (a)immunohistohemistry of Stat3 phosphorylation and POMC expression in 2-week-old flox/flox and POMC-Cre, flox/flox male mie. Mie were injeted with leptin (i.p. 3mg/kg) or vehile (saline) and sarified 3 min later. : third ventrile. Bar = 5 M. () Immunolotting of Stat3 phosphorylation in hypothalami ARC of 2-week-old male mie. Mie were injeted with with leptin (i.p. 3mg/kg) or vehile (saline) and sarified 3 min later. pstat3 Tyr and Stat3 were measured y immunolotting. () Proposed model for role of in neuronal leptin reeptor signaling in the hypothalamus aruate neurons. Nature Neurosiene: doi:1.138/nn.327

8 a Targeted allele FRT loxp Neo FRT loxp Ex 5 loxp Flp reominase Flp-deleted allele Cre-deleted allele FRT Ex 4 loxp loxp Ex 5 Ex 6 Crereominase loxp Ex 4 Ex 6 G418-resistant ES ell lones itl BA1 ES ells C57BL/6 129/SvEv Targeted allele = 4.9k WT C57BL/6 allele = 4.k WT 129/SvEv allele = 3.k () marker WT loxp = 38p WT = 32p Supplementary Figure 7. Generation of flox/flox mie. (a) Shemati representation of targeted, Flp-deleted, and Cre-deleted alleles. A loxp flanked Neo assette was inserted on the 5 side of exon 5 and a loxp site was inserted on the 3 side of exon 5. The target region is 462p and inludes exon 5. () Southern lot analysis of G418 resistant ES ells. DNA enoding the targeting vetor was linearized y NotI and then transfeted y eletroporation of itl BA1 (C57BL/6 x 129/SvEv) hyrid emryoni stem ells. After seletion in G418 antiioti, surviving lones were expanded for Southern analysis to identify reominant ES lones. DNA was digested with Pst1. itl BA1 ES ells ontain one allele from C57BL/6 mie and the other from 129 mie. ES ell DNA shows the WT 129/SvEv allele yields a and of 3.k, the WT C57BL/6 allele a and of 4 k, and the targeted C57BL/6 allele whih appears as a and shift from 4 to 4.9k. () Genotyping of flox/flox mie. Targeted itl BA1 C57BL/6 x 129/SvEv hyrid emryoni stem ell lone 162 and 211 were miroinjeted into C57BL/6 lastoysts. Resulting himeras with a high perentage agouti oat olor were mated to wild-type C57BL/6 mie to generate F1 heterozygous offspring. DNA from offspring was amplified y PCR using primers for genomi DNA. With the addition of loxp site, the amplified produt is 38p. For wild-type (or without a loxp site), the amplified produt is 32p. The positive ontrol, indiated y (), is a positive ES lone. Nature Neurosiene: doi:1.138/nn.327

9 a POMC Merged flox/flox POMC-Cre, flox/flox Cortex Liver Kidney Musle Con KO Con KO Con KO Con KO NPY-GFP Merged NPY-hrGFP, flox/flox AgRP-ires-Cre, NPY-hrGFP, flox/flox d Liver Musle Heart Panreas Kidney WAT BAT Con KO Con KO Con KO Con KO Con KO Con KO Con KO Supplementary Figure 8. Generation of POMC or AgRP neuron-speifi -defiient mie. (a) is expressed in POMC expressing neurons. Doule IHC for POMC and was performed in 16- week-old flox/flox (ontrol) and POMC-Cre, flox/flox male mie. White arrows indiate protein loalized within POMC expressing neurons. Bar = 2 M. () protein in entral and peripheral tissues of flox/flox (Con) and POMC-Cre, flox/flox (KO) mie. and atin were visualized y immunolotting. protein levels in these tissues were not different etween genotypes. () is expressed in AgRP/NPY expressing neurons. IHC for was performed on oronal rain setions from 12-week-old male NPY-hrGFP, flox/flox and AgRP-ires-Cre, NPY-hrGFP, flox/flox mie. White arrows indiate protein loalized within AgRP expressing neurons. Bar = 1.5 M. (d) expression in peripheral tissues of flox/flox (Con) and AgRP-ires-Cre, flox/flox (KO) mie. and atin were visualized y immunolotting. protein levels in these tissues were not different etween genotypes. Nature Neurosiene: doi:1.138/nn.327

10 sirna: 15 Lu Fig. 1a p 93 DNA: Fig. 1 Mok DN- p 93 Fig. 1 Control Y p 93 Fig. 1d Control Y Origin 25 sirna: Lu 15 sirna: Lu 37 GAPDH DNA: Mok DNA: Mok DN- DN Control Y p p 41 Fig. 1e DNA: Mok 25 DN- Control Y IgG (heavy hain) 25 p Fig. 2a Fig. 8a sirna: IgG (light hain) 15 Lu 15 AAV-GFP AAV-Cre Fig. 1f 15 sirna: Lu ROCK2 pstat3 4 AAV-GFP AAV-Cre IgG (heavy hain) 15 Ad- GFP Ad- Leptin: sirna: Lu Stat Supplementary Figure 9. Unproessed images of the key immunolots in the paper. Memranes were ut at moleular weight markers flanking the protein of interest efore immunolotting. Boxes (solid lines) indiate memrane orders and oxed areas (dashed lines) indiate image areas shown in the indiated main figures. The position of moleular weight markers are shown (kda). Nature Neurosiene: doi:1.138/nn.327