Cell Metabolism, Volume 15

Supplemental Information

PPARγ agonists Induce a White-to-Brown Fat Conversion through Stabilization of PRDM16 Protein

Haruya Ohno, Kosaku Shinoda, Bruce M. Spiegelman, and Shingo Kajimura

Supplemental Experimental Procedures

Cell Culture

Fat depots were digested in PBS containing collagenase D (1.5U/ml) and dispase II (2.4U/ml)

supplemented with 10mM CaCl2 at 37˚C for 40-45 min. The primary cells were filtered through 70um

cell strainer and centrifuge at 700rcf to collect stromal vascular (SV) fraction. The SV cell pellets were

rinsed and plated on collagen coated plates. Adipocyte differentiation was induced by treating confluent

cells in DMEM/F12 medium (D-glucose 17.51 mM) containing 10% FBS, 0.5 mM

isobutylmethylxanthine, 125 nM indomethacin, 1 mM dexamethasone, 850 nM insulin, 1 nM T3. Two

days after induction, cells were switched to the maintenance medium containing 10% FBS, 850 nM

insulin and 1 nM T3. The cells were cultured in the presence or absence of PPARγ-ligands at 1μM,

including rosiglitazone, MRL24, nTZDpa, Mbx-102, and BVT.13 (Bruning et al., 2007). For cAMP

treatment, cells were incubated with 10 μM forskolin for 6 hours. Collagenase D and dispase II were

purchased from Roche Applied Science. Pioglitazone, troglitazone, SR202, and T0070907 were

purchased from Tocris Bioscience. MRL24, nTZDpa, Mbx-102, and BVT.13 and SR1664 were kindly

provided by Dr. Griffin at the Scripps Institute. Other chemicals for cell culture were obtained from

Sigma.

Animals

Animal experiments were performed according to procedures approved by Institutional Animal Care and

Use Committee at Beth Israel Deaconess Medical Center and at UCSF. 6-8 week- old male C57BL/6J

mice were obtained from the Jackson Laboratory. PRDM16 transgenic mice were generated as described

previously (Seale et al., 2011). Mice were intraperitoneally injected daily with 10 mg/kg rosiglitazone

for 10 days. Adipose tissues were harvested for molecular, biochemical or histological analyses.

Histology

Fat pads were carefully dissected and fixed in 4% paraformaldehyde for histological analysis. For

immonohistochemistry, paraffin-embedded sections were incubated with anti-UCP1 antibody

(Chemicon), followed by detection using the ABC Vectastain-Elite kit (Vector Labs) following the

methods as described previously (Kajimura et al., 2009).

Gene Expression Analysis

Total RNA was isolated from cells or tissues using Trizol (Invitrogen). Reverse transcriptase reactions

were performed using a cDNA reverse transcription kit (Applied Biosystems). Quantitative real-time

PCR (qRT-PCR) was performed with SYBR green fluorescent dye using an ABI9300 PCR machine or

ABI ViiATM7. TATA-binding protein (TBP) served as an internal control. Primer sequences are

provided in Supplementary Table3.

Microarray Analysis

SV cells were isolated from inguinal WAT. The cells were infected with adenoviral vectors expressing

short-hairpin RNA targeting PRDM16 (sh-PRDM16) or a scramble control (sh-scr). The cells were

differentiated to mature adipocytes in the presence or absence of rosiglitazone at 1μM. Total RNA was

isolated and applied for microarray analysis. Array hybridization and scanning were performed by the

Dana-Farber Cancer Institute Core Facility using Affymetrix GeneChip Mouse Genome 430 2.0 arrays

according to established methods (Lockhart et al., 1996). The array data were analyzed using the DNA-

Chip Analyzer (dChip) software (Li and Wong, 2001). The statistical significance of differences in gene

expression was assessed by unpaired t-test (P< 0.05). Microarray data has been deposited in Gene

Expression Omnibus (GEO): GSE35011.

Western Blotting

Total cell lysates or nuclear extracts were isolated and separated by SDS-page. Antibodies for PRDM16

(Seale et al., 2011), ubiquitin (Santa Cruz), UCP1 (Abcam), β-actin (Sigma) or Pol-II (Cell Signaling)

were used for Western blotting. For interaction assay, COS-7 cells expressing flag-tagged PRDM16 or

PPARγ were harvested 24 hours after transfection. Total cell lysates were incubated overnight at 4 ˚C

with PPARγ antibody. The precipitants were analyzed by Western blot using antibodies for M2 flag

antibody (Sigma).

Protein Stability Assay

Inguinal WAT-derived SV cells were differentiated into mature adipocytes in the presence or absence of

1μM rogiglitazone as described above. Subsequently the cells were incubated in medium containing 20

μg/ml cycloheximide and harvested at the indicated time points. Cell lysates were applied for Western

blotting. β-actin was used for loading control. Image J software was used for quantifying the intensity of

signals.

Oxygen Consumption Assay

SV cells isolated from inguinal WAT were infected with adenoviral vectors expressing short-hairpin

RNA targeting PRDM16 or a scramble control. The cells were differentiated to mature adipocytes in the

presence or absence of rosiglitazone at 1μM. At day 7 or 8 of differentiation, oxygen consumption was

measured as described previously (Kajimura et al., 2009). For cAMP-induced respiration assays, fully

differentiated fat cells were incubated with 0.5 mM dibutyryl cyclic AMP for 12 hours prior to

measuring oxygen consumption.

Statistical Analyses

Significant differences between two groups were assessed by two-tailed Student's t test with unequal

variance. Data are expressed as means ± SEM.

Figure S1 Preferential Browning Effects in Subcutaneous WAT Requires Full Agonism of PPARγ, Related to Figure 1 (A) Primary inguinal white preadipocytes were differentiated in the presence of several known full or partial PPARγ agonists, such as rosiglitazone (rosi), MRL24, nTZDpa, Mbx-102, and BVT.13 at 1μM. mRNA levels for brown fat-selective genes (ucp1, cidea, cox8b) were normalized by those of fabp4. * P<0.05, ** P<0.01 relative to control. The actual numerical levels were shown in the graph. (B) Wild type C57BL/6J mice were injected IP with saline or rosiglitazone at 10mg/kg for 10days. mRNA levels for brown fat-selective genes, cox8b and prdm16 were measured by qRT-PCR in interscapular BAT, inguinal WAT (Ing), anterior subcutaneous WAT (AntSC), epidydimal WAT (Epi), retroperitoneal WAT (retroP) and mesenteric WAT (Mesent). *P<0.05, **P<0.01 relative to saline control. (C) mRNA levels for rb were measured by qRT-PCR in the cells from in primary adipocytes isolated from inguinal WAT and from epidydimal WAT. The cells were differentiated to mature adipocytes in the presence or absence of rosiglitazone at 1μM. *P<0.05 relative to control.

 

Figure S2. Regulation of Brown Fat Gene Program by a PRDM16-PPARγ Pathway, Related to Figure 4 (A) Flag-tagged PRDM16 were transiently expressed along with PPARγ in Cw4S-7 cells. Twenty-four hours after the transfection, the cells were treated with rosiglitazone at 0.1 and 1μM for 2 hours. PPARγ was immunoprecipitated using PPARγ antibody, separated by SDS-PAGE, and PRDM16 was detected by Western blotting. The inputs of each assay are shown in bottom panels. (B) Experimental scheme that was used in the experiment in (C). (C) mRNA levels for prdm16, ucp1, and pgc1a were measured by qRT-PCR in the primary inguinal cells from the samples in (B). **P<0.01 relative to control cells. (D) Primary inguinal cells were treated with rosiglitazone at 1μM for 4h, 24h, 3 days or 6 days prior to harvesting cells. PRDM16 protein levels were analyzed by Western blotting. Total cell lysates were used. (E) mRNA levels for ucp1 (left) and fabp4 (right) were measured by qRT-PCR. **P<0.01 relative to control.

Figure S3. Browning of the White Adipocytes is Tightly Linked to the Rosiglitazone-Induced PRDM16 Protein Stablization, Related to Figure 4 (A) Primary inguinal preadipocytes were differentiated in the presence of several known full or partial PPARγ agonists at 1uM. mRNA levels for ucp1 and prdm16 were measured by qRT-PCR. * P<0.05, ** P<0.01 relative to control. (B) Primary inguinal preadipocytes were differentiated in the presence of rosiglitazone at different doses. ucp1 mRNA was measured by qRT-PCR. ** P<0.01 relative to control. (C) Primary inguinal adipocytes were incubated with rosiglitazone together with selective PPARγ antagonists SR202 (400uM) and T0070907 (1uM). PRDM16 protein levels were analyzed by Western blotting. b-actin was used as a loading control. (D) PRDM16 protein levels were analyzed by Western blotting in BAT and inguinal WAT isolated from fabp4-PRDM16 transgenic mice treated with saline or rosiglitazone. (E) prdm16 mRNA levels were measured by qRT-PCR in BAT and inguinal WAT from wild-type mice and PRDM16 transgenic mice treated with saline or rosiglitazone. *P<0.05, **P<0.01. (F) Primary inguinal SV cells were differentiated in the presence of rosiglitazone at 1uM for 8 days (Day8R), or for 4 days with rosiglitazone and further incubated in the absence of rosiglitazone (Day4R-off). MG132 (100 nM) was added for 54 hours prior to harvesting the cells. Total cell lysates were isolated to analyze PRDM16 protein levels by Western blotting.

Figure S4. Developmental Cascades of Pre-Existing Brown Adipocyte and PPARγ-Ligand Inducible Brown Adipocyte in WAT Brown adipocytes from interscapular BAT and rosiglitazone-inducible brown adipocytes (beige/brite cells) in WAT have distinct developmental origins and molecular signatures. PRDM16 is required for the development of both types of brown adipocytes.