siRNA reagents and plasmids.
Small interfering RNAs (siRNAs) targeting each individual Forkhead factor or PPARγ, C/EBPα, -β, or -δ and an siRNA control (siGENOMEsiRNA reagents, SMARTpool, and siCONTROL nontargeting siRNA) were purchased from Dharmacon. Foxa3 cDNA was amplified from a mouse liver cDNA library with primers containing a Kozak and a Flag sequence, namely, F (5′-AACAGAATTCGCCACCATGGACTACAAAGACGATGACGATAAACT GGGCTC AGTGAAGAT-3′) and R (5′-CCCGCTCTCTGCTTAATGCATCCTAGGATATCACAA-3′), cloned into pcDNA3.1 (Invitrogen) at the EcoRI and EcoRV sites and subcloned into pMSCV retroviral vector (Clontech) at the EcoRI site. Foxa3-DBD mutant R162P/N165I/M202R/R210P was generated by site-directed mutagenesis (Stratagene) with primers listed in Table S1 in the supplemental material. Plasmids expressing either C/EBPβ or C/EBPδ were purchased from Addgene. The C/EBPα plasmid was a gift of Kai Ge. The mouse PPARγ promoter (−2200 to +1) was amplified from genomic DNA with primers containing NheI and HindIII sites, namely, F (5′-AACAGCTAGCCCCCCACTTTCACCATAGTC-3′) and R (5′-TTGTAAGCTTAACAG CATAAAACAGAGATT-3′, and cloned into pGL3-basic vector (Promega).
To induce adipocyte differentiation, confluent 10T1/2 cells, either transfected with 100 nM siRNA or overexpressing Foxa3, PPARγ, or vector, were cultured in high-glucose Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) supplemented with 5 μg/ml insulin and 10 μM troglitazone, while 3T3-L1 cells were stimulated with DMEM containing 10% FBS and MDI (5 μg/ml insulin, 0.5 mM isobutylmethylxanthine, and 5 mM dexamethasone) for 48 h and subsequently cultured in DMEM containing 10% FBS supplemented with 5 μg/ml insulin (maintenance medium). To generate wild-type (WT) and Foxa3-null stromal-vascular fractions (SVF) of cells from inguinal and visceral depots, epididymal and inguinal adipose tissues were isolated from WT and Foxa3-null mice, minced, and subjected to collagenase digestion (1 mg/ml) for 45 min in a buffer containing 0.123 M NaCl, 5 mM KCl, 1.3 mM CaCl2, 5 mM glucose, 100 mM HEPES, and 4% bovine serum albumin (BSA). Digested tissues were filtered through a 100-μm-pore-size nylon screen and centrifuged for 5 min at 150 × g. Cell pellets were resuspended in DMEM containing 20% FBS, 25 mM glucose, 20 mM HEPES, and 1% penicillin and streptomycin (pen/strep), and the culture medium was changed daily. For differentiation assays, SVF obtained from visceral and subcutaneous fat depots were stimulated for 48 h with medium containing 10% FBS, MDI, and 10 μM troglitazone and subsequently cultured in maintenance medium.
Immunoprecipitations were performed according to protocols described previously (15
). Proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Pierce). Blots were incubated with primary antibodies overnight at 4°C and at room temperature for 1 h with secondary antibodies. Immune complexes were visualized by using ECL Plus (Pierce), following the manufacturer's instructions.
For electromobility shift assays (EMSA), nuclear extracts obtained from U2OS cells transfected with Foxa3-WT or Foxa3-DBD mutant expression plasmids were incubated with a biotin-labeled oligonucleotide (5′-AACTATTCCTTTTTATAGAATTTGGATAGCAGTAACA-3′) corresponding to the putative Foxa binding site identified in the PPARγ promoter. Supershift was obtained by adding 0.4 μg of Foxa3 antibody, and a competition assay was performed using 200-fold unlabeled probe. After incubation, DNA-protein complexes were separated on a 4% nondenaturing polyacrylamide gel followed by transfer to a positively charged nylon membrane (Pierce). After UV cross-linking, detection was performed following the manufacturer's protocol (Pierce).
Chromatin immunoprecipitation (ChIP) assays were performed using a ChIP assay kit (Upstate), according to the manufacturer's instructions. PCR was performed using primers amplifying the C/EBP binding site (5′-GGCCAAATACGTTTATCTGGTG-3′ and 5′-TCACTGTTCTGTGAGGGGC-3′), the Foxa3 binding site (5′-TCACTTAAACATCAACCATTGGA-3′ and 5′-GGTCCAAAATGTTACTGCTATCC-3′), or the TATA box site (5′-GCCCCTCACAGAACAGTGAA-3′ and 5′-AACAGCATAAAACAGAGATTT-3′). PCR products were run on an agarose gel and visualized by ethidium bromide staining. Relative enrichment was quantified by real-time PCR.
Gene expression analysis.
RNA was extracted using TRIzol (Invitrogen), cDNA was generated according to manufacturer's instructions (ABI), and real-time PCR was carried out with SYBR green (Roche). 18S was used for normalization. Mouse Foxa3 mRNA levels were detected with the following primers: F (5′-TGAATCCTGTGCCCACCAT) and R (3′-AGCTGAGTGGGTTCAAGGTCAT). Human FOXA3 was detected with TaqMan primer sets (ABI).
Transfections and luciferase assays.
10T1/2 and 3T3-L1 cells were transfected using a Nucleofector 96-well system (Amaxa) and U2OS cells (ATCC) with FuGENE 6 (Roche), according to the manufacturer's instructions. Luciferase activity was assayed 48 h after transfection, according to the manufacturer protocol (Promega Corporation), using Victor3V (PerkinElmer).
Anti-Flag M2 beads and anti-Flag antibody were purchased from Sigma, and anti-C/EBPα, anti-C/EBPβ, anti-C/EBPδ, and anti-HNF3γ (Foxa3) antibodies were purchased from Santa Cruz. Secondary antibodies were purchased from Amersham and Vector Labs.
All animal experiments were performed according to the guidelines of the National Institute of Diabetes and Digestive and Kidney Disease Animal Care and Use Committee (ACUC). Mice were housed in 12-h light/dark cycles (light on at 6 a.m.) and allowed ad libitum access to food and water. Mice were genotyped by PCR with the following primers: Foxa3-F (forward) (5′-TCCCAAGCTTGGGCACTGGTG GCCA-3′), Foxa3-R (reverse) (5′-GTGGCAGCTGTAGTGGTGGCAG-3′), and lacZ (5′-CGCCATTCGCCATTCAGGCTGC-3′). Mice were sacrificed by ketamine (90 mg/kg of body weight) and xylazine (10 mg/kg) intraperitoneal (i.p.) injection according to NIH ACUC animal study approved procedures. Harvested tissues were snap-frozen in liquid nitrogen or fixed using 4% paraformaldehyde (EMS) and embedded in paraffin for hematoxylin and eosin staining. Adult WT and Foxa3-null mice were fed a high-fat diet (HFD) for 22 weeks (Research Diets, D12492). C57BL/6J mice fed a normal diet or a HFD were purchased from The Jackson Laboratory.
Tissues were dissected and fixed in 10% formalin and embedded in paraffin according to standard procedures. Paraffin-embedded tissues were cut in sections of 5 μm thickness and stained with hematoxylin and eosin (Histoserv) for morphological analysis, following the manufacturer's instructions (Vector Labs). Stained slides were analyzed using a microscope at ×200 magnification (Olympus). Adipocyte sizes were measured using ImageJ software (version 1.45s). At least 200 cells from each animal group were measured.
Human adipose tissue samples.
Fat biopsy specimens from the subcutaneous adipose tissue (SAT) of the midanterior abdominal region and visceral adipose tissue (VAT) of the omental region were obtained from 14 obese women with a body mass index (BMI) of 45.6 kg/m2
(range, 36.1 to 55.6) recruited at the Stanford's Bariatric Surgery Program, with a mean age of 40 years (range, 23 to 59), as previously described (16
). The study was approved by the Stanford University Human Subjects Committee. All subjects gave written informed consent.
ITT, GTT, and serum tests.
For insulin tolerance tests (ITT), mice received an intraperitoneal injection of insulin (1 mU/kg). For glucose tolerance tests (GTT), mice were fasted overnight and injected intraperitoneally with a glucose solution in saline solution (2 g/kg). Plasma glucose levels were measured from tail blood before or at 15, 30, 60, 90, and 120 min after insulin or glucose injections. Blood samples for measurements of serum adiponectin, insulin, and cholesterol levels were collected using a back-eye needle. Serum parameters were measured with kits obtained from Linco Research and Thermo.
All experiments were repeated at least three times. Results are presented as means ± standard errors of the means (SEM). Student's t test and one- or two-way analysis of variance (ANOVA) followed by appropriate posttests were performed with GraphPad software (GraphPad). A P value of less than 0.05 was considered statistically significant.