fatty acid oxidation and energy expenditure via suppression of the

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1 Supplemental Material Submission to Genes and Development Dietary obesity-associated Hif1α activation in adipocytes restricts fatty acid oxidation and energy expenditure via suppression of the Sirt2-NAD + system Jaya Krishnan, Carsten Danzer, Tatiana Simka, Josef Ukropec, Katharina Manuela Walter, Susann Kumpf, Peter Mirtschink, Barbara Ukropcova, Daniela Gasperikova, Thierry Pedrazzini, and Wilhelm Krek Supplemental Materials and Methods Mouse physiology ERT2-Cre recombinase activity was induced by intra-peritoneal (ip) injections of tamoxifen citrate (in 40% ethanol/0.9% NaCl) for 5 consecutive days at 20mg/kg body weight. GTT and ITT were performed as previously described (Zehetner et al., 2008). Echocardiography was performed as described (Barrick et al., 2007). Basal body temperature was determined with a microprobe rectal thermometer (Stoelting). Lean body mass was determined by carcass analysis and calculated by subtracting fat mass from the total carcass weight (Brommage, 2003). Mouse and metabolic studies Mice were maintained on a 12-hour:12-hour light dark. Mice were subjected to HFD (D12331, Research Diets Inc. from postnatal week 4. Food and water intake, O 2 consumption, and CO 2 expiration and RER were simultaneously determined for four mice in separate cages per experiment during a 72-hour period in an Oxymax 1

2 metabolic chamber system (Columbus Instruments), as previously described (Silva et al., 2009). REE was calculated using the formula ((3.9x VO 2 ) + (1.1x(VCO 2 )) X 1.44 (van der Kuip et al., 2004). overall food consumption was determined over a period of 3 weeks between weeks post-tamoxifen induction. Metabolic assays Mitochondrial activity was assessed as described (Guzy et al., 2005). β-oxidation capacity was measured with [9,10-3H(N)] palmitate as previously described (Djouadi et al., 2003). Cellular oxygen consumption was measured using a Seahorse bioscience XF24 analyzer in 24 well plates at 37 C, with correction for positional temperature variations adjusted from four empty wells evenly distributed within the plate. 3T3-L1 cells were seeded at cells/well 8-10 days prior to the analysis. Once confluence was reached, differentiation was induced and viral infection performed on day 4 of differentiation. Each experimental condition was performed on 10 biological replicates, and performed as recommended by the manufacturer (Seahorse Bioscience). Adipocyte isolation and 3T3-L1 culture and differentiation. Primary adipocytes were isolated as described (Tozzo et al., 1995). 3T3-L1 preadipocytes (American Type Culture Collection, ATCC) were cultured in DMEM (Sigma) with 10% fetal bovine serum at 37 C and 5% CO 2. Primary preadipocytes were isolated from dissected epididymal and subcutaneous fat pads. Tissues were minced and then incubated with 2mg/ml collagenase (Sigma) at 37 C for one hour while shaking. Cell suspensions were passed through a 70µm cell strainer and then centrifuged at 1000 rpm for 5 minutes. After resuspension and brief incubation in erythrocyte lysis buffer (154mM NH4Cl, 10mM KHCO3, 0.1mM EDTA), cells were centrifuged again, resuspended in DMEM with 10% FCS and plated. Cells were 2

3 grown to confluence and differentiation was induced by adding 100nM insulin, 1 µm dexamethasone and 0.5 mm IBMX (all Sigma) to the serum-containing medium. After 2 days, medium was replaced with fresh serum containing medium including 100nM insulin and the appropriate lentiviruses. After additional 48 hours, medium was replaced with tamoxifen containing medium to induce Cre activation. Adipocytes were ready and used for further assays 7 days after differentiation induction. For 3T3- L1 differentiation, 3T3-L1 cells were grown to confluence. Two days past reaching confluence differentiation was induced by adding 100nM insulin, 1 µm dexamethasone and 0.5 mm IBMX (all Sigma) to the serum-containing medium. After 2 additional days, medium was replaced by DMEM, 10% serum and 100nM insulin, and then media were changed every 2 days until cells were ready for harvesting (6 to 8 days) (Elks and Manganiello, 1985). Transient transfections of differentiated cells were performed on day 4 post-differentiation as described (Shigematsu et al., 2001). All experiments were performed with three biological replicates and repeated three times. Immunofluorescence staining of cryosections Tissue preparation and Immunofluorescence staining was performed as described (Hirschy et al., 2006). Oil Red O staining was performed and quantified as previously described (Krishnan et al., 2009). BODIPY staining was performed with the LipidTox Lipid Stain (Invitrogen) as recommended by the manufacturer. Antibodies, fluorescent reagents and chemicals Cpt1 and Peroxiredoxin 6 antibodies were provided by Rene Lerch (University of Geneva, Switzerland) and Sabine Werner (ETH-Zurich, Switzerland). Sirt1, Sirt2 and acetylated-lysine antibodies were from Cell Signaling. Antibodies against pan- 3

4 cadherin, b-actin and acetylated-tubulin were from Sigma. Hif1α, Hif1β, Pgc1α, tubulin and lamin antibodies were from Santa Cruz Biotechnology. A second Pgc1α antibody from Upstate/Millipore was utilized ti assess the specificity of Pgc1α immunoprecipitation. Phalloidin, and DAPI were used as recommended by the manufacturer (Invitrogen). DMOG (Cayman Chemical) and DFX were used 3mM and 150µM, respectively. All chemical were purchased from Sigma unless indicated otherwise. Lentiviruses and plasmids Lentiviral shrnas were purchased from OpenBioSystems. The Hif1α ΔODD expression construct was generated as described (Huang et al., 1998). SDS-PAGE, immunoblotting and immunoprecipitation Adipose tissue lysates were prepared and immunoblotting performed as described (Hirschy et al., 2006). Immunoprecipitation for Pgc1α was performed as previously described (Rodgers et al., 2005). The in vitro Sirt2 deacetylation assay as previously described with bacterial purified Sirt2 (Ellis et al., 2008). Luciferase assays 1kb upstream of the Sirt2 TSS was amplified from mouse BAC genomic DNA (Imagenes) and cloned into the pgl3 luciferase reporter vector (Stratagene). The Sirt2 ΔHRE-mutant was generated by recombinant PCR (Casonato et al., 2004; Elion et al., 2007). Sequence integrity of the respective wildtype and mutant promoters were verified by sequencing (Microsynth) and BLAST alignment ( The reporter assay was performed by transient cotransfection of the appropriate luciferase reporter, the normalization construct psv- 4

5 β-galactosidase (Promega), in the presence of Hif1α shrna-encoding or of Hif1α overexpressing viruses. The PEPCK-luciferase reporter and the E-box reporter constructs were kindly provided by Pere Puigserver (Dana Farber Cancer Institute, Boston) and Javier León (University of Cantabria, Spain), respectively. Luciferase and β-galactosidase activity was measured using the Luciferase Assay System kit (Promega) as recommended by the manufacturer and analyzed on the MicroLumatPlus LB 96V (luciferase activity: Berthold Technologies) and Spectra MAX 190 plate reader (β-galactosidase activity: Molecular Devices). All experiments were performed with 3-5 biological replicates and repeated three times, unless indicated otherwise. Quantitative RT-PCR and ChIP assays RNA was isolated with Trizol (Invitrogen) and cdna generated using Superscript II (Invitrogen), and performed as recommended by the manufacturer (Roche) and analyzed on the Roche LightCycler 480. The ChIP assay was performed with material from visceral adipose tissue and the assay performed using the ChIP-IT kit (Active Motif) as recommended by the manufacturer. In silico promoter analyses and alignments were performed using MatInspector and DiAlignTF (Genomatix). Sequences of primers used in the various assays are listed in Supplemental Material (Supplemental Tables 2 and 3). Statistical analysis All statistical analyses and tests were carried out using Excel software (Microsoft). Data are given as mean +/- standard error of mean (SEM). Bars in graphs represent standard errors and significance was assessed by two-tailed t-test. 5

6 Supplemental Figure Legends Supplemental Fig. 1 Adipocyte Hif1α inactivation alleviates nutrient overload induced pathologies A-B) Various organs of Hif1α ic(t) and Hif1α icko(t) mice subjected to the NCD/NCD (A) or HFD/HFD (B) protocol were assayed for Hif1α mrna expression at week post-tamoxifen injection. All values were normalized internally to 18S RNA expression and to the Hif1α ic(t) control, respectively. Data are mean +/- SEM of values from 5 mice/group. C) Lean body mass of Hif1α ic(t) and Hif1α icko(t) mice subjected to the NCD/NCD or HFD/HFD protocol was assessed by carcass analysis. D) Cardiac contractile function, expressed as percentage (%) fractional shortening, of Hif1α ic(t) (n=5 NCD/NCD; n=5 HFD/HFD) and Hif1α icko(t) (n=8 NCD/NCD; n=7 HFD/HFD) mice maintained on the HFD/HFD protocol at the indicated timepoints both prior- (week 16), and at 10 and 22 weeks post-tamoxifen-mediated Hif1α excision (*,% p<0.05 vs. Hif1α ic(t) NCD/NCD). Data are mean +/- SEM of values from each group. E) Representative M-mode echocardiographic tracing of Hif1α ic(t) and Hif1α icko(t) mice maintained on the HFD/HFD protocol at week 13 post-tamoxifenmediated Hif1α excision. Closed arrows indicates ventricular lumen internal diameter. F) Representative images depicting the gross morphology of hearts derived from Hif1α ic(t) and Hif1α icko(t) mice maintained on the HFD/HFD protocol. Region corresponding to the lung (lu) and epicardiac fat (epi) are indicated. G) Schematic representation of the NCD/NCD and HFD/NCD protocol. Littermate Hif1α ic and Hif1α icko mice were placed on a NCD or HFD diet from 4 weeks postnatal, for 14 weeks. Thereafter, at week 18, tamoxifen was administered to Hif1α 6

7 ic and Hif1α icko mice of both the NCD and HFD groups. Mice from both groups were maintained for an additional 20 weeks on the NCD diet. GTT measurements were taken prior to Hif1α excision (at week 17), and at 10 and 20 weeks post-hif1α excision (at weeks 28 and 38). H) Body weight measurements of Hif1α ic/hif1α ic(t) (n=9 NCD/NCD; n=10 HFD/NCD) and Hif1α icko/ Hif1α icko(t) (n=8 NCD/NCD; n=12 HFD/NCD) mice subjected to the HFD/NCD protocol were performed at the indicated time points throughout the course of the protocol (* p<0.05 vs. Hif1α icko(t) HFD/HFD, % p<0.05 vs. Hif1α ic HFD/HFD and Hif1α icko HFD/HFD). Data are mean +/- SEM of values from each group. I) GTT and area under curve (AUC) measurements of HFD/NCD maintained Hif1α ic (n=10) and Hif1α icko (n=12) mice prior to Hif1α excision (at week 17). Data are mean +/- SEM of values from each group. J) GTT and AUC measurements of HFD/NCD maintained Hif1α ic(t) (n=10) and Hif1α icko(t) (n=12) mice at 10 weeks post-hif1α excision (* p<0.05). Data are mean +/- SEM of values from each group. K) GTT and AUC measurements of HFD/NCD maintained Hif1α ic(t) (n=10) and Hif1α icko(t) (n=12) mice at 20 weeks post-hif1α excision (* p<0.05). Data are mean +/- SEM of values from each group. L-M) Interscapular BAT (L) and retroperitoneal visceral WAT (M) resected from Hif1α f/f (n=9) and Hif1α BATcKO (n=11) mice were weighed. Data are mean +/- SEM of values from each group. Supplemental Fig. 2 Adipose Hif1α inactivation promotes the expression of fatty acid oxidation genes 7

8 A) Individually housed Hif1α ic(t) (n=10) and Hif1α icko(t) (n=12) mice maintained on a HFD/HFD protocol were assessed for food intake over a period of 1 weeks (between weeks post-tamoxifen administration). Data are mean +/- SEM of values from each group. B) Primary subcutaneous white adipocytes isolated from Hif1α ic(t) and Hif1α icko(t) mice maintained on the HFD/HFD protocol were assessed for palmitate oxidation. Data are means ± SEM of values from 4 mice/group. C) Primary visceral white adipocytes isolated from Hif1α ic(t) and Hif1α icko(t) mice maintained on the NCD/NCD protocol were assessed for palmitate oxidation. Data are means ± SEM of values from 4 mice/group. D) Gene expression profiling of visceral WAT of Hif1α ic(t) and Hif1α icko(t) mice subjected to the HFD/HFD protocol for expression of regulators of fatty acid oxidation and uncoupling protein. All values were normalized internally to 18S RNA expression and to the Hif1α ic(t) control, respectively (* p<0.01 compared to control, set at 1.0). Data are mean +/- SEM of values from 5 mice/group. E-F) Gene expression profiling of subcutaneous WAT of Hif1α ic(t) and Hif1α icko(t) mice subjected to the HFD/HFD protocol for expression of transcriptional (D) and enzymatic (E) regulators of fatty acid oxidation. All values were normalized internally to 18S RNA expression and to the Hif1α ic(t) control, respectively (* p<0.01 compared to control, set at 1.0). Data are mean +/- SEM of values from 5 mice/group. G-H) Preadipocytes isolated from the visceral (G) or subcutaneous (H) fat depots of Hif1α ic(t) and Hif1α icko(t) mice maintained on the HFD/HFD protocol were in vitro differentiated to adipocytes and assessed for oleate-induced oxygen consumption, as a measure of fatty acid β-oxidation, using the Seahorse Bioscience 8

9 24XF extracellular flux analyzer. Normalised oxygen consumption rate (OCR) is shown (* p<0.01 compared to control Hif1α ic(t), set at 1.0). Data are mean +/- SEM of values from 3 mice/group. I) Average body weight of mice maintained on NCD (n=10) or HFD (n=10) for 20 weeks (* p<0.01 vs. NCD fed mice). Data are mean +/- SEM of values from each group. J) Visceral and subcutaneous white adipocytes isolated from wildtype C57/Bl6 mice maintained on NCD or HFD for 20 weeks were assessed for palmitate oxidation (* p < 0.05 vs. WT NCD). Data are means ± SEM of values from 4 mice/group. K-M) Visceral WAT (K), subcutaneous (L) WAT and BAT (M) of mice maintained on NCD or HFD for 20 weeks were assessed for Hif1α protein expression. β-actin serves as a loading control. N) Gene expression profiling of Hif1α-target genes in visceral WAT (vwat), subcutaneous WAT (scwat) and BAT of wildtype C57/Black6 mice maintained on NCD or HFD for 20 weeks. All values were normalized internally to 18S RNA expression and to the NCD control, respectively (* p<0.01 compared to NCD, set at 1.0). Data are mean +/- SEM of values from 5 mice/group. O-P) 3T3-L1-derived adipocytes were subjected to ectopic Hif1α expression (O) or hypoxia (at 3% O 2 ) (P) and assessed for expression of mitochondrial biogenesis and fatty acid oxidation regulators. Black bars indicate mock infectant (O) or normoxia control (20% O 2 ) (P) or, white bars indicate samples subjected to ectopic Hif1α expression (O) or hypoxia (P). All values were normalized internally to 18S RNA expression and to mock infectant (K) or normoxia (L) control, respectively (* p<0.01 compared to control, set at 1.0). Data are mean +/- SEM of values from each group. 9

10 Supplemental Fig. 3 Adipose Hif1α inactivation increases white adipocytes mitochondrial density A) Visceral WAT sections of Hif1α ic(t) and Hif1α icko(t) mice maintained on the HFD/HFD protocol were stained for the mitochondrial marker cytochrome oxidase (COX, red), the neutral lipid marker (BODIPY, green), phalloidin (pink) and DAPI (blue), and analyzed by immunofluorescence confocal microscopy. Supplemental Fig. 4 Hif1α inactivation promotes Sirt2-mediated Pgc1α transcriptional activation A-C) COX3 (A), Cpt1 (B) and PEPCK (C) promoter activity in response to lentiviral shhif1α-mediated Hif1α knockdown, was determined by transient cotransfection of the respective promoters fused to luciferase in 3T3-L1 adipocytes. Adipocytes infected with lentiviral non-silencing shrnas (nsrna) and transfected with the respective reporters serve as controls. All transfections contained equal amounts of a β-galactosidase expression vector for normalization of luciferase activity (* p<0.01 compared to control, set at 1.0). Data are mean +/- SEM of values from each group. D) 3T3-L1-derived adipocytes were subjected to normoxia (20% O 2 ) or hypoxia (3% O 2 ) in the absence or presence of nsrna or shhif1α. Cell lysates were immunoprecipitated for Pgc1α and probed for acetylated lysine. E) Hif1α was stabilized by addition of the prolyl hydroxylase (PHD) inhibitor dimethyloxallyl glycine (DMOG), or by addition of the iron chelator desferrioxamine (DFX) in 293 cells. Lysates were assessed for Hif1α protein levels by immunoblotting (upper panel), and immunoprecipitated with the Pgc1α antibody and probed for lysine acetylation and Pgc1α by immunoblotting (lower panels). F) Visceral WAT of Hif1α ic(t) and Hif1α icko(t) mice subjected to the HFD/HFD 10

11 protocol were assayed for expression of Sirt3, Sirt4, Sirt5, Sirt6 and Sirt7. All values were normalized internally to 18S RNA expression and to the Hif1α ic(t) control, respectively. Data are mean +/- SEM of values from 3 mice/group. G) Visceral WAT sections of HFD/HFD maintained Hif1α ic(t) and Hif1α icko(t) mice were stained for Sirt2 (red), DAPI (blue), the lipid marker BODIPY (green), and phalloidin (pink) and analyzed by immunofluorescence confocal microscopy. Inlay depicts co-localization of Sirt2 with DAPI predominantly in the nucleus. H) Cell fractionation was performed on WAT biopsies of Hif1α icko(t) HFD/HFD mice and probed for Sirt2. Lamin serves as a nuclear marker, while peroxiredoxin 6 was used as a cytoplasmic marker (Kumin et al., 2007). I) 3T3-L1-derived adipocytes were subjected to normoxia (20% O 2 ) or hypoxia (3% O 2 ) in the absence or presence of nsrna or shhif1α, and assessed for Sirt2 expression. All values were normalized internally to 18S RNA expression and to the normoxia control set (* p<0.01 compared to normoxia control, set at 1.0; % p<0.01 compared to hypoxia sample). Data are mean +/- SEM of values from each group. Supplemental Fig. 5 Sirt2 is a Pgc1α deacetylase A-B) 3T3-L1-derived adipocytes were infected with control or Sirt2-encoding (A) lentivirus, or with nsrna or shsirt2 (B), respectively, and assessed for expression of Sirt2 protein. Loading was normalized to β-actin. C) 3T3-L1-derived adipocytes were infected with control or Sirt1-encoding lentivirus and assessed for expression of Sirt1 protein. Loading was normalized to β-actin. D) 3T3-L1-derived adipocytes were infected with nsrna or shpgc1α, and assessed for Pgc1α protein by immunoblotting. Loading was normalized to β-actin. E) Specificity of the Pgc1α antibody used for immunoprecipitation was determined by 11

12 infecting primary neonatal mouse cardiomyocytes (NMC) with nsrna or shpgc1α lentiviruses, and immunoprecipitating and immunoblotting Pgc1α. Lysates derived from visceral WAT of Hif1α ic(t) and Hif1α icko(t) mice maintained on HFD/HFD were run in parallel. NMC express high basal levels of Pgc1α (Ventura- Clapier et al., 2008). Supplemental Fig. 6 Sirt2 is downregulated in a mouse model of dietary-induced obesity A) Sirt2 antibody specificity was verified by lentivirus-mediated knockdown of SIRT2, and immunoblotting for SIRT2 in the human cell lines HeLa and A549. B) Average fasting blood glucose index of mice maintained on NCD (n=10) or HFD (n=10) for 20 weeks (* p<0.01 vs. NCD fed mice). Data are mean +/- SEM of values from each group. C) Visceral WAT biopsies of mice maintained on NCD or HFD for 20 weeks were assessed for Sirt2 protein expression. β-actin serves as a loading control. Supplemental Table Legends Supplemental Table 1 Physiological characteristics of the human lean and obese subjects Adipose tissue area was determined by Magnetic Resonance Imaging (MRI) from a single slice scan of abdomen in the umbilical region (between L4-5 vertebrae). None of the individuals had fasting plasma glucose higher than 5.6 mmol/l. Oral glucose tolerance test (ogtt); M-value, insulin sensitivity index was determined by euglycemic hyperinsulinemic clamp. Data are presented as mean +/- SEM. The two groups were compared by the student t-test and p < 0.05 (compared to lean subjects) 12

13 was considered statistically significant. Supplemental Table 2 List of mouse qpcr and genotyping primers used in the study Supplemental Table 3 List of human qpcr primers used in the study 13

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23 Supplemental References Barrick, C.J., Rojas, M., Schoonhoven, R., Smyth, S.S., and Threadgill, D.W Cardiac response to pressure overload in 129S1/SvImJ and C57BL/6J mice: temporaland background-dependent development of concentric left ventricular hypertrophy. Am J Physiol Heart Circ Physiol 292: H Brommage, R Validation and calibration of DEXA body composition in mice. American journal of physiology 285: E Casonato, A., Cattini, M.G., Soldera, C., Marcato, S., Sartorello, F., Pontara, E., and Pagnan, A A new L1446P mutation is responsible for impaired von Willebrand factor synthesis, structure, and function. J Lab Clin Med 144: Elion, E.A., Marina, P., and Yu, L Constructing recombinant DNA molecules by PCR. Current protocols in molecular biology / edited by Frederick M Ausubel [et al Chapter 3, Unit Elks, M.L., and Manganiello, V.C A role for soluble camp phosphodiesterases in differentiation of 3T3-L1 adipocytes. J Cell Physiol 124: Ellis, D.J., Yuan, Z., and Seto, E Determination of protein lysine deacetylation. Curr Protoc Protein Sci Chapter 14, Unit Hirschy, A., Schatzmann, F., Ehler, E., and Perriard, J.C Establishment of cardiac cytoarchitecture in the developing mouse heart. Developmental biology 289: Huang, L.E., Gu, J., Schau, M., and Bunn, H.F Regulation of hypoxiainducible factor 1alpha is mediated by an O2-dependent degradation domain via the ubiquitin-proteasome pathway. Proceedings of the National Academy of Sciences of the United States of America 95: Kumin, A., Schafer, M., Epp, N., Bugnon, P., Born-Berclaz, C., Oxenius, A., Klippel, A., Bloch, W., and Werner, S Peroxiredoxin 6 is required for blood vessel integrity in wounded skin. J Cell Biol 179: Philp, A., Perez-Schindler, J., Green, C., Hamilton, D.L., and Baar, K Pyruvate suppresses PGC1alpha expression and substrate utilization despite increased respiratory chain content in C2C12 myotubes. Am J Physiol Cell Physiol 299: C

24 Rodgers, J.T., Lerin, C., Haas, W., Gygi, S.P., Spiegelman, B.M., and Puigserver, P Nutrient control of glucose homeostasis through a complex of PGC-1alpha and SIRT1. Nature 434: Shigematsu, S., Miller, S.L., and Pessin, J.E Differentiated 3T3L1 adipocytes are composed of heterogenous cell populations with distinct receptor tyrosine kinase signaling properties. The Journal of biological chemistry 276: Silva, J.P., von Meyenn, F., Howell, J., Thorens, B., Wolfrum, C., and Stoffel, M Regulation of adaptive behaviour during fasting by hypothalamic Foxa2. Nature 462: Tozzo, E., Shepherd, P.R., Gnudi, L., and Kahn, B.B Transgenic GLUT-4 overexpression in fat enhances glucose metabolism: preferential effect on fatty acid synthesis. The American journal of physiology 268: E van der Kuip, M., de Meer, K., Oosterveld, M.J., Lafeber, H.N., and Gemke, R.J Simple and accurate assessment of energy expenditure in ventilated paediatric intensive care patients. Clin Nutr 23: Ventura-Clapier, R., Garnier, A., and Veksler, V Transcriptional control of mitochondrial biogenesis: the central role of PGC-1alpha. Cardiovasc Res 79: Zehetner, J., Danzer, C., Collins, S., Eckhardt, K., Gerber, P.A., Ballschmieter, P., Galvaovskis, J., Shimomura, K., Ashcroft, F.M., Thorens, B., et al PVHL is a regulaor of glucose metabolism and insulin secretion in pancreatic beta cells.genes & development 22: