All studies were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of North Carolina at Chapel Hill (Approved protocol number: 07-229).
Four week old CONV but specific pathogen free (SPF) C57BL/6 mice were purchased from Jackson Laboratories (Jackson labs, Bar Harbor, ME) and were subjected to dietary intervention in our SPF animal facility. GF C57BL/6 mice were bred in the UNC-CH gnotobiotic facility and maintained in GF conditions throughout the dietary interventions. NF-κBEGFP
knockin mice (C57BL/6) were described previously 
. Briefly, this model contains a single copy of a transgene comprising NF-κB-response elements driving EGFP knocked into the hypoxanthine phosphoribosyltransferase genomic locus. These mice are maintained as hemizygotes and transgenics were identified by examining tail-clips for EGFP expression. Male CONV and GF NF-κBEGFP
mice were used for proposed studies.
CONV mice, CONV NF-κBEGFP
mice and GF mice were randomly selected to receive either HF diet (45% kcal from fat, D01060502, Research Diets, New Brunswick, NJ) or a LF diet (10% kcal from fat, D01060501, Research Diets, New Brunswick, NJ) for 2, 6, or 16 weeks (n
4–8 per group). For experiments in GF mice, diets were subject to irradiation (50 kGy) (Neutron Products, MD). Pilot studies established that CONV mice given irradiated diet gained similar weight as on non-sterile diet.
Body weight and body composition
Body weights were measured at two week intervals. Percentage body fat was assessed by performing Dual Energy x-ray Absorptiometry (DEXA) scan (PIXImus Mouse Densitometer, GE, Piscataway, NJ) at 2, 6, or 16 weeks after beginning the HF or LF diets. DEXA scans were obtained under isoflurane anesthesia to assess fat mass, percent fat and lean body mass.
Animals were euthanized with an overdose of sodium pentobarbital administered intraperitoneally. Cardiac puncture was used to obtain blood, which was placed on ice and centrifuged to collect plasma. Ileum and colon were dissected and carefully stripped of all adherent mesentery or fat. Corresponding segments of tissue from each animal were fixed in 10% formalin or 4% paraformaldehyde (PFA) for histology, or frozen for RNA extraction.
Fasting plasma glucose and insulin measurement
Fasting plasma glucose was measured by a standard biochemical method (#439-90901, Wako Diagnostics, Richmond, VA) and fasting insulin by ELISA (#EZRMI-13K, rat/mouse insulin ELISA kit, Millipore, Billerica, MA). Homeostatic model assessment (HOMA) values were calculated as an estimate of insulin sensitivity, using the formula fasting plasma glucose (mmol/L)×insulin (µU/ml)/22.5. Higher values of HOMA indicate reduced insulin sensitivity.
RNA extraction and Real-time PCR
Total RNA was isolated from ileum and colon using the TRIzol method (Invitrogen, Carlsbad, CA). cDNA was generated using AMV Reverse Transcriptase and Oligo(dT)15 primer in the presence of RNasin Ribonuclease Inhibitor. All reverse transcription reagents were obtained from Promega (Madison, WI). Real-time qPCR was performed on the Light Cycle (Rotor-Gene 3000, Corbett Life Science, San Francisco, CA). Primers and fluorescent reporter probes were obtained from Operon (Huntsville, AL). The sequences were as follows: TNF-α F: 5′-CTGTCTACTGAACTTCGGGGTGAT-3′, R: 5′-CATCAGTTCTATGGCCCAGACC-3′ and FAM-labeled probe: 5′-FAM-ATGAGAAGTTCCCAAATGGCCTCCCTC-TAMRA-3′). The results were normalized to housekeeping gene HMBS F: 5′-TGTGTTGCACGATCCTGAAAC-3′, R: 5′-CTCCTTCCAGGTGCCTCAGAA-3′ and 5′-FAM-TTCGCTGCATTGCTGAAAGGG-TAMRA-3′ probe. Cycle threshold (Ct) values for TNF-α and HMBS were converted to quantification values based on the standard curves. All individual values are expressed as a fold change compared to LF diet at the 2 week time point.
Enhanced GFP (EGFP) imaging
NF-κBEGFP mice given HF or LF diet were anesthetized and euthanized. The intestine was dissected and flushed with ice cold 1×PBS and immediately imaged using a charge-coupled device camera in a light-tight imaging box with a dual-filtered light source and emission filters specific for EGFP (LT-99D2 Illumatools; Lightools Research, Encinitas, CA). Intestines from HF/LF diet fed animals were also photographed using a Leica 16FA stereo dissecting microscope. Identical exposure times were used to capture images within each experiment.
Cellular sites of NF-κB EGFP expression
GFP was visualized directly under confocal microscope on frozen section mounted in gel-mount. Immunofluorescence was performed on frozen sections cut from ileum and colon fixed in 4% PFA, cryoprotected in graded sucrose and embedded in O.C.T. (Tissue-Tek, Torrance, CA). Before the primary antibody incubation, frozen sections were pretreated with 0.05M Tris Triton (TT) epitope retrieval solution at room temperature (RT) for 30 minutes and blocked in 5% normal goat serum (NGS) diluted in TT buffer for 1 hr. The primary antibodies used are listed in . Primary antibodies were diluted in 5% NGS/TT buffer. The sections were incubated with primary antibodies overnight at 4°C in a humidity chamber. After washing 3 times in 0.05M Tris buffer, the sections were incubated with secondary antibodies (see ) for 1 hr at RT. Nucleic acid staining was carried out by labeling with Bisbenzamide for 10 minutes. Following three washes with 0.05M Tris, coverslips were mounted in gel-mount (Electron Microscopy Sciences, Hatfield, PA). Pictures were taken by Leica SP2 Upright Laser Scanning Confocal microscope (Leica) and analyzed with Leica SP2 Upright Laser Scanning Confocal Imaging Software (Leica).
Primary antibodies used in immunofluorescence.
Secondary antibodies used in immunofluorescence.
Conventionalization of GF NF-κB EGFP with fecal slurries from mice given HF or LF diet
Fresh feces were obtained from CONV mice fed HF or LF diet for >16 weeks. Fecal slurries were made by suspending 1.2 g of HF/LF feces into 4 ml sterile 1×PBS (phosphate buffered saline). Whiskers and rectum of each mouse were swabbed with a cotton swab doused in fecal slurry. Colonized GF mice were then fed with sterilized regular chow diet and sterile water for 2 weeks before euthanasia.
Data are expressed as mean ± standard error. Data in CONV or GF mice were compared for an effect of diet by One-Way ANOVA and post-hoc tests used for pair-wise comparisons (Tukey's test). Linear regression using Pearson's correlation coefficients was used to test for correlations between ileal TNF-α and body weight, fat mass, % fat, plasma glucose and insulin levels. All statistical analyses were performed using STATISTICA software version 9 (Statsoft inc. Tulsa, OK). A P value of less than 0.05 was considered statistically significant.