Animals and Diet

We used C57BL/6 male mice (12–14 weeks of age) obtained from the National Institutes of Health. We determined the sample size with the power that could perform the comparison at a 0.05 level of significance, with a 90% chance of detecting a true difference of all the measurements between the two groups; N = 9–10 per group were used in the study. After arriving at our facilities, each mouse was given its own cage housed in a specific pathogen-free facility to avoid microbiome transfer62. The control regimen (type F1515) consisted of 3.79 kcal/gm pellets composed of 65.2% carbohydrates, 18.1% protein, 5.1% fat, 4.8% fiber, 2.9% ash, and less than 10% moisture. The KD regimen (type F3666), in the form of a 7.24 kcal/gm paste, consisted of 75.1% fat (composed of saturated, monounsaturated, and polyunsaturated fatty acids), 8.6% protein, 4.8% fiber, 3.2% carbohydrates, 3.0% ash, and less than 10% moisture. Both diets were obtained from Bio-Serv. All mice were fed ad libitum for 16 weeks, and body weight was measured once a week. The amount of remaining diet was weighted each week to determine the food intake of the mice. We did not find a significant difference in food intake between the control (46 ± 3 g/mouse/week) and KD mice (47 ± 2 g/mouse/week; p > 0.5). All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Kentucky (UK) and in compliance with the ARRIVE guidelines63.

Cerebral Blood Flow Measurement

We measured CBF using MRI-based arterial spin labeling techniques. Details have been described in a previous study23. Briefly, MRI experiments were performed on a 7T MR scanner (Clinscan, Bruker BioSpin, Germany) at the Magnetic Resonance Imaging & Spectroscopy Center of the University of Kentucky. Mice were anesthetized with 4.0% isoflurane for induction and then maintained in a 1.2% isoflurane and air mixture using a nose cone. Heart rate (90–110 bpm), respiration rate (50–80 breaths/min), and rectal temperature (37 ± 1 °C) was continuously monitored and maintained. A water bath with circulating water at 45–50 °C was used to maintain the body temperature. A whole-body volume coil was used for transmission and a mouse brain surface coil was placed on top of the head for receiving. We measured CBF using MRI-based pseudo-continuous arterial spin labeling (pCASL) techniques23. Paired control and label images were acquired in an interleaved fashion with a train of Hanning window-shaped radiofrequency pulses of duration/spacing = 200/200 μs, flip angle = 25° and slice-selective gradient = 9 mT/m, and a labeling duration = 2100 ms. The images were acquired by 2D multi-slice spin-echo echo planner imaging with FOV = 18 × 18 mm2, matrix = 128 × 128, slice thickness = 1 mm, 10 slices, TR = 4,000 ms, TE = 35 ms, and 120 repetitions. pCASL images were analyzed with in-house written codes in MATLAB (MathWorks, Natick, MA) to obtain quantitative CBF (with units of mL/g per minute). Brain structural T 2 -weighted images were acquired with field of view (FOV) = 18 × 18 mm2, matrix = 256 × 256; slice thickness = 1 mm, 10 slices, repetition time (TR) = 1500 ms, and echo time (TE) = 35 ms. The CBF images were then superimposed to the corresponding structural images using Multi-Image Analysis GUI (Mango) software (http://rii.uthscsa.edu/mango/).

Gut Microbiome Analyses

Fecal DNA Amplification

The experimental protocol has been described in a previous study64. Fecal samples were collected from control and KD mice and frozen at −80 °C until further use. The PowerSoil DNA Isolation Kit (MO BIO Laboratories, Inc.) was used for fecal DNA extraction, according to the manufacturer’s protocol. Genomic DNA was PCR amplified with primers 515 F modified and 926R65 targeting the V4-V5 regions of microbial 16S rRNA genes using a two-stage “targeted amplicon sequencing (TAS)” protocol66. The primers contained 5′ common sequence tags (known as common sequence 1 and 2, CS1 and CS2) as described previously67. First stage amplifications were performed with the following thermocycling conditions: 95 °C for 3 mins, 28 cycles of 95 °C for 45 sec, 50 °C for 45 sec, 72 °C for 1:30 minutes and final elongation at 72 °C for 10 minutes. The PCR master mix was made in a total volume of 25 µl of reaction mixture containing 4 µl (100 ng) of DNA template, 0.5 µl (20 µM) of each forward and reverse primers, 12.5 µl of PCR ready-to-use mixture (MyTaq HS Mix 2x, Bioline, London, UK) and 7.5 µl of distilled water.

Subsequently, a second PCR amplification was performed in 10 microliter reactions in 96-well plates. A mastermix for the entire plate was made using the MyTaq HS 2X mastermix. Each well received a separate primer pair with a unique 10-base barcode, obtained from the Access Array Barcode Library for Illumina (Fluidigm, South San Francisco, CA; Item# 100-4876). These Access Array primers contained the CS1 and CS2 linkers at the 3′ ends of the oligonucleotides. Cycling conditions were as follows: 95 °C for 5 minutes, followed by 8 cycles of 95 °C for 30”, 60 °C for 30” and 72 °C for 30”. A final, 7-minute elongation step was performed at 72 °C. PCR products were purified using SequalPrep plates (Life Technologies) according to the manufacturer’s instructions. Subsequently, these PCR products were quantified using a Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher), implemented on a Genios Pro Fluorescence microplate reader (Tecan). PCR products were then pooled using PicoGreen quantification results, using an epMotion5075 liquid handling workstation (Eppendorf).

The pooled libraries, with a 15% phiX spike-in, were loaded on to a MiSeq v3 flow cell, and sequenced using an Illumina MiSeq sequencer, with paired-end 300 base reads. Fluidigm sequencing primers, targeting the CS1 and CS2 linker regions, were used to initiate sequencing. De-multiplexing of reads was performed on instrument. Second stage PCR amplification and library pooling was performed at the DNA Services (DNAS) facility, Research Resources Center (RRC), University of Illinois at Chicago (UIC). Sequencing was performed at the W.M. Keck Center for Comparative and Functional Genomics at the University of Illinois at Urbana-Champaign (UIUC).

Microbial Analysis

Forward and reverse reads were merged using PEAR68. Primer sequences were identified using Smith-Watermann alignment and trimmed from the sequence. Reads that lacked either primer sequence were discarded. Sequences were then trimmed based on quality scores using a modified Mott algorithm with PHRED quality threshold of p = 0.01, and sequences shorter than 300 bases after trimming were discarded. QIIME v1.8 was used to generate OTU tables and taxonomic summaries24. Briefly, the resulting sequence files were merged with sample information. OTU clusters were generated in a de novo manner using the UCLUST algorithm with a 97% similarity threshold69. Chimeric sequences were identified using the USEARCH61 algorithm with the GreenGenes 13_8 reference sequences70. Taxonomic annotations for each OTU were using the UCLUST algorithm and GreenGenes 13_8 reference with a minimum similarity threshold of 90%69,70. Taxonomic and OTU abundance data were merged into a single OTU table and summaries of absolute abundances of taxa were generated for all phyla, classes, orders, families, genera, and species present in the dataset. The taxonomic summary tables were then rarefied to a depth of 10,000 counts per sample.

Shannon and Bray-Curtis indices were calculated with default parameters in software package Primer771. The rarefied species data, taxonomic level 6, were used to calculate both indices. Significant difference among tested groups was determined using the Kruskal-Wallis one-way analysis of variance. The group significance tests were performed on the rarefied species data using the group_significance.py script within the QIIME v1.8 package. The gene amplicon sequence data generated as part of this study have been submitted to the NCBI BioProject database (PRJNA401034).

P-glycoprotein (P-gp) Transport Determination and Western Blotting

Capillary isolation

Details of the experiments have been described in previous studies23,64. Brain capillaries were isolated from mice according to a previously described protocol23. Briefly, mice were euthanized by CO 2 inhalation and decapitated; brains were immediately harvested and collected in ice-cold DPBS buffer supplemented with 5 mM D-glucose and 1 mM Na-pyruvate, pH 7.4. Brains were dissected by removing meninges, choroid plexus and white matter, and homogenized in DPBS. The brain homogenate was mixed with Ficoll® and centrifuged at 5,800 g for 15 min at 4 °C. The capillary pellet was resuspended in 1% BSA buffer and first passed through a 300 µm nylon mesh followed by filtration through a 27 µm nylon mesh. Capillaries retained by the 27 µm nylon mesh were collected and washed with DPBS buffer, and used for experiments.

P-glycoprotein transport activity

Isolated brain capillaries were incubated for 1 h at room temperature with 2 μM NBD-CSA (custom-synthesized by R. Wenger, Basel, Switzerland) in DPBS buffer. Per treatment group, 10 capillary images were acquired by confocal microscopy (Leica TSP SP5 Confocal Microscope with Environmental Chamber, 63 × D-Water UV objective, numerical aperture 1.2, 488-nm line of an argon laser, Leica Microsystems). Confocal images were analyzed by quantitating luminal NBD-CSA fluorescence with Image J software (v.1.45 s; Wayne Rasband, NIH). Specific, luminal NBD-CSA fluorescence was taken as the difference between total luminal fluorescence and fluorescence in the presence of the P-glycoprotein specific inhibitor PSC833 (5 μM, Novartis, Basel, Switzerland).

Western blotting and quantification

To determine protein expression, isolated brain capillaries were homogenized in tissue lysis buffer containing protease inhibitor cocktail. Homogenized brain capillary samples were centrifuged at 10,000 g for 15 min at 4 °C, followed by centrifugation of the denucleated supernatants at 100,000 g for 90 min at 4 °C. Pellets (crude brain capillary plasma membranes) were resuspended and protein concentrations were determined using the Bradford protein assay. Western blots were performed using the NuPage™ electrophoresis and blotting system from Invitrogen (Carlsbad, CA, USA). Blotting membranes were incubated overnight with antibody to P-gp (C219; MA1-26528, ThermoFisher, 1 μg/ml), mTOR (ab134903, Abcam, 1 μg/ml), GLUT1 (ab652, Abcam, 1 μg/ml), and β-actin (ab8226 from Abcam, 1:1000, 1 μg/ml). Proteins were detected using SuperSignal® West Pico Chemoluminescent substrate (Pierce, Rockford, IL, USA) and protein bands were visualized with a BioRad Gel Doc™ XRS imaging system. Image Lab 5.0 software from Bio-Rad Laboratories was used for densitometric analyses of band intensities and digital molecular weight analyses; the molecular weight marker was RPN800E (GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK). Linear adjustments of contrast and brightness were applied to entire Western blot images. None of the Western blots shown were modified by nonlinear adjustments.

Blood Glucose and Ketone Bodies Measurements

The procedure has been described in a previous study43. When the mice were sacrificed, blood samples were collected in 500 μl lithium heparin 12.5 IU Terumo Capiject Capillary blood collection tubes (Vacutainer K2 EDTA) to avoid blood coagulation. A total of 1–2 μl of blood sample were used to measure blood glucose level using a blood glucose meter and a test strip (Clarity Plus, Boca Raton, FL, USA). Another 10 μl of blood sample was used for ketone bodies level measurement using a STAT-Site M (β-Hydroxybutyrate) meter and a test strip (Standbio Ketosite STAT-Site M-β HB, Boerne, TX, USA).

Statistics

Statistical analyses were performed using GraphPad Prism 7 (GraphPad, San Diego, CA, USA). One-tailed Student’s t-test was performed for determination of differences between the two groups. Values of p < 0.05 were considered statistically significant.