Animals and Treatments

For all experiments, male C57BL/6J mice were purchased from The Jackson Laboratory and group housed in static microisolator cages in a specific-pathogen free animal facility. For experiments investigating the composition of the gut microbiome and transcriptional profiling of the liver, mice were purchased at 8 weeks of age, and diet changes occurred at 9 weeks of age. Approximately 4 months later, cecal contents and livers were collected from mice sacrificed in the morning following an overnight, approximately ~16 hr fast. For experiments in which the gut microbiome was ablated with antibiotics, mice were purchased at 5 weeks of age; starting at 6 weeks of age, mice were randomized at the cage level to receive water or water containing antibiotics as described below. Diet changes occurred at 9 weeks of age, and antibiotic treatment was continued for the duration of the experiment. All procedures involving animals were approved by the Institutional Animal Care and Use Committee of the William S. Middleton Memorial Veterans Hospital (Madison, WI), and all experiments were performed in accordance with relevant guidelines and regulations.

Diets

Prior to 9 weeks of age, animals were fed the standard facility chow (Purina 5001; Purina Mills, Richmond, IN, USA). Amino acid defined animal diets (non-irradiated) were obtained from Envigo (formerly Harlan Laboratories). At 9 weeks of age, animals were switched to either a Control amino acid defined diet (TD.140711; 22.0% of calories derived from amino acids; 59.4% from carbohydrate; 18.6% from fat) or a Low Protein amino acid defined diet (TD.140712; 7.1% of calories derived from amino acids; 74.4% from carbohydrates; 18.5% from fat). The complete composition of these diets has been previously described22.

Antibiotic Treatment

The gut microbiome was ablated using an antibiotic treatment protocol previously described to efficiently ablate the gut microbiome of mice38. Briefly, mice were provided with free access to autoclaved water containing 1 g/L ampicillin, 500 mg/L vancomycin, and 1 g/L neomycin; however, in contrast to the protocol followed in38, aspartame was omitted due to its negative effects on glucose homeostasis and body composition in mice39. The mice and the water bottles were weighed and changed biweekly to monitor water intake. Control mice were provided with autoclaved water not containing antibiotics. To verify the efficacy of the antibiotic treatment, fecal pellets were collected and total DNA was extracted using a modification of a previously described protocol40. Briefly, fecal pellets (~30–50 mg) were resuspended in a solution containing 500 µL of extraction buffer [200 mM Tris (pH 8.0), 200 mM NaCl, 20 mM EDTA], 210 µL of 20% SDS, 500 µL phenol:chloroform:isoamyl alcohol (pH 7.9, 25:24:1) and 500 µL of Fisher Scientific 1.4 mm diameter ceramic beads (Cat# 15340159). Following mechanical disruption using a FastPrep 24 (M.P. Biomedicals), the solution was centrifuged at 8,000 rpm at 4 °C for three minutes. The aqueous layer was then sequentially precipitated using sodium acetate/isopropanol and sodium acetate/ethanol. DNA samples were then quantified using a Nanodrop 2000c.

Mouse metabolic phenotyping

Glucose and alanine tolerance tests (GTT and ATT) were performed by fasting the mice overnight for 16 hr and then injecting either glucose (1 g/kg) or alanine (2 g/kg) intraperitoneally as previously described41,42. Glucose measurements were taken using a Bayer Contour blood glucose meter and test strips. Mouse body composition was determined using an EchoMRI 3-in-1 Body Composition Analyzer. For assay of multiple metabolic parameters (O 2 , CO 2 , food consumption) and activity tracking, mice were acclimated to housing in a Columbus Instruments Oxymax/CLAMS metabolic chamber system for ~24 hr, and data from a continuous 24-hr period was then recorded and analyzed.

Gut microbial community DNA preparation

Approximately 20–50 mg of cecal matter was added to an autoclaved Sarstedt 2 m micro screw-cap tube (Ref# 72.693.005) containing approximately 500 μL of BioSpec Zirconia/Silica beads (Cat# 11079101z) and one large Bio Spec bead (Cat# 11079132ss). To this, 500 μL of 200 mM Tris-HCl, pH 8.0/200 mM NaCl/20 mM EDTA was added, as well as 210 μL 20% SDS. 500 μL of Phenol/Chloroform/isoamyl alcohol, pH 7.9, 25:24:1, was added before bead beating using a FastPrep 24 (M.P. Biomedicals) until sample was fully homogenized in solution. Tubes were centrifuged at 8,000 rpm at 4 °C for three minutes. The aqueous layer, approximately 500 µL, was transferred to a new microcentrifuge tube (Axygen). 60 μL of 3 M NaAcetate was added, then 600 μL of isopropanol, then inverted to mix. The samples were placed on ice for one hour before centrifuging at 13,000 rpm at 4 °C for 20 minutes. Samples were decanted, and pellet was rinsed with 200 μL of 100% EtOH, then decanted and briefly dried. The pellet was dissolved in 100–200 μL of TE buffer. 100 μL of DNA was cleaned using the Macherey-Nagel PCR Clean-up kit, using 2 NT3 washes and eluting with 50–100 μL of elution buffer.

Construction and Sequencing of v3-v4 16S Metagenomic libraries

Purified genomic DNA was submitted to the University of Wisconsin-Madison Biotechnology Center. DNA concentration was verified fluorometrically using either the Qubit® dsDNA HS Assay Kit or Quant-iT™ PicoGreen® dsDNA Assay Kit (ThermoFisher Scientific, Waltham, MA, USA). Samples were prepared in a similar process to the one described in Illumina’s 16 S Metagenomic Sequencing Library Preparation Protocol, Part # 15044223 Rev. B (Illumina Inc., San Diego, California, USA) with the following modifications: The 16 S rRNA gene V3/V4 variable region was amplified with fusion primers (forward primer 341 f: 5′-ACACTCTTTCCCTACACGACGCTCTTCCGATCT(N) 3/6 CCTACGGGNGGCWGCAG -3′, reverse primer 805r: 5′-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT(N) 3/6 GACTACHVGGGTATCTAATCC -3′). Region specific primers were previously described (43; underlined sequences above), and were modified to add 3–6 random nucleotides ((N) 3/6 ) and Illumina adapter overhang nucleotide sequences 5′ of the gene‐specific sequences. Following initial amplification, reactions were cleaned using a 0.7x volume of AxyPrep Mag PCR clean-up beads (Axygen Biosciences, Union City, CA). In a subsequent PCR, Illumina dual indexes and Sequencing adapters were added using the following primers (Forward primer: 5′-AATGATACGGCGACCACCGAGATCTACAC[55555555]ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3′, Reverse Primer: 5′-CAAGCAGAAGACGGCATACGAGAT[77777777] G TGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3′, where bracketed sequences are equivalent to the Illumina Dual Index adapters D501-D508 and D701-D712,N716,N718-N724,N726-N729). Following PCR, reactions were cleaned using a 0.7x volume of AxyPrep Mag PCR clean-up beads (Axygen Biosciences). Quality and quantity of the finished libraries were assessed using an Agilent DNA 1000 kit (Agilent Technologies, Santa Clara, CA) and Qubit® dsDNA HS Assay Kit (ThermoFisher Scientific), respectively. Libraries were pooled in an equimolar fashion and appropriately diluted prior to sequencing. Paired end, 300 bp sequencing was performed using the Illumina MiSeq Sequencer and a MiSeq. 600 bp (v3) sequencing cartridge. Images were analyzed using the standard Illumina Pipeline, version 1.8.2. OTU assignments and diversity plots were created using QIIME analysis pipeline44.

Microbiota analysis using QIIME

Microbiota analysis to obtain OTU assignments and diversity plots were performed using Quantitative Insights Into Microbial Ecology (QIIME)44 version 1.9.1. Illumina sequencing reads were adapter and quality trimmed using the Skewer45 trimming program to remove low quality (<Q25) bases and sequencing adapters. Reads shorter than 100 nucleotides after trimming were discarded. Flash46 was used to merge paired end reads into amplicon sequences using a minimum overlap of 10 nucleotides. Amplicons were then PCR primer trimmed and quality filtered. Sequences were then clustered in OTUs using an open-reference OTU picking protocol based on 97% identity using UCLUST46 against the Greengenes reference database47. Representative sequences (most abundant sequence in OTUs) were picked, aligned to GreenGenes47 Core reference alignment using PyNAST48. Taxonomic assignments were associated with OTUs based on the taxonomy associated with the Greengenes reference sequence defining each OTU. UniFrac distances between samples were calculated using the Greengenes reference tree (ftp://greengenes.microbio.me/greengenes_release/gg_13_5/gg_13_8_otus.tar.gz). The resulting biom-formatted OTU table was filtered to remove singletons and OTUs that could not be aligned using PyNAST. Alpha rarefaction curves were calculated for all samples with a rarefaction upper limit of (median depth/sample count). Samples were removed from further characterization if they did not contain sufficient reads at a depth where the Good’s coverage value for most samples was greater than 0.9. Beta diversity was calculated using weighted and unweighted unifrac on OTU data leveled according to the lowest sample depth. An alternative normalization by CSS49 is also provided for additional downstream analysis.

Liver mRNA preparation and transcriptional profiling

Total liver RNA was extracted with Trireagent (Sigma) as previously described19. Concentration and purity of RNA was initially determined using a Nanodrop 2000c, and then submitted to the University of Wisconsin- Madison Biotechnology Center Gene Expression Center & DNA Sequencing Facility. RNA quality was then assayed using an Agilent RNA NanoChip, and stranded mRNA libraries with polyA enrichment were prepared as described in the Illumina TruSeq Stranded mRNA Sample Preparation Guide Rev. E. DNA sequencing was performed using an Illumina HiSeq. 2500 1 × 100 (TruSeq v3) full flowcell. Pathway enrichment was performed using the “edgeR”50 and “org.Mm.eg.db”51 packages in R version 3.4.452.

Gene expression (qPCR)

Total RNA was isolated from liver with Tri-Reagent, and cDNA was generated as previously described24. Oligo dT primers and primers for real-time PCR were obtained from Integrated DNA Technologies (Coralville, IA,USA). Primer sequences used for qPCR were as follows: Cyp8b1: F: GTTTCTGGGTCCTCTTATTCCTG, R: TGGGAGTGAAAGTGAACGAC; Zfp36l1: F: CACACCAGATCCTAGTCCTTG, R: CTGGGAGTGCTGTAGTTGAG; Cyp7a1: F: AACGATACACTCTCCACCTTT, R: CTGCTTTCATTGCTTCAGGG; Ppp2cb: F: ATGGAAGGATATAACTGGTGCC, R: AGGTGCTGGGTCAAACTG; Map2k1: F: CGTACATCGTGGGCTTCTAC, R: CAGAACTTGATCCAAGGACCC; Shp: F: CTACCCTCAAGAACATTCCAGG, R: CACCAGACTCCATTCCACG; Abcb11: F: CCTCATACGGAAACCCAAGATC, R: CTGACTGTTGATAGGCGATGG; Actb: F: ACCTTCTACAATGAGCTGCG, R: CTGGATGGCTACGTACATGG. Reactions were run on an Applied Biosystems StepOnePlus Real-Time PCR System (Thermo Fisher Scientific) with Invitrogen SYBR Green PCR Master Mix (ThermoFisher Scientific). Actin (Actb) was used to normalize the results from gene-specific reactions.

Statistics

Statistics were carried out in Prism 7 (Graphpad Prism). For each measured parameter, we conducted a two‐factor ANOVA which included an effect of drug treatment (vehicle or antibiotics), an effect of diet (Control or Low AA), and an interaction between diet and treatment. A Sidak’s post-test was performed to determine the significance of factors identified as significant in the two-factor ANOVA. PCA analysis was performed using Clustvis53.