Human intervention protocol

General design. The study was divided into recruitment, prescreening questionnaire, and screening visit and 3 separate study days. Nine healthy, nonsmoking men took part in a randomized, 3-way crossover study. Twenty-four hours before each trial day, the diet and activity of each participant were monitored. On the trial day, participants attended the Medical Research Council — Elsie Widdowson Laboratory (MRC-EWL) for 7 hours and received one of the 3 isoenergetic mixed meals in a randomized order. Nine subjects were randomized into one block. Randomization was generated using a computerized program that generates random permutations. Volunteers were randomized at a 1:1:1 ratio, such that each volunteer consumed all 3 meals within a period of 6 weeks. The isoenergetic meals comprised: control (C; 15% protein, 40% fat, 45% carbohydrate), high-protein (HP; 32% protein, 33% fat, 35% carbohydrate), and high-fat (HF; 14% protein, 62% fat, 24% carbohydrate).

Screening visit. Screening measurements and a blood sample were taken during the screening visit. Participants were also requested to complete a 24-hour food diary. After an overnight 12-hour fast, anthropometric measurements (weight, height, and blood pressure) and a fasting blood sample were taken. The fasting blood sample was taken by venipuncture for analysis of full blood count, liver function, glucose, and insulin and lipid profile, including total cholesterol, HDL, LDL, and triglycerides. These measurements were used to determine whether participants met the inclusion criteria of the study (Table 2).

Table 2 Exclusion criteria of the human study

Study day protocol. Participants arrived in the morning after a 12-hour overnight fast. Participants were cannulated via the antecubital vein of one arm and blood samples were collected, 10 minutes apart. Participants then consumed, within 15 minutes of onset of eating, one of the isoenergetic meals. Subsequently blood samples were collected over a 6-hour period. During the 6-hour period, food consumption was not allowed, and only water was permitted ad libitum after 3 hours had elapsed. Blood samples were collected at 12 time points at various intervals throughout the 6-hour period (T1–T6) to perform mass spectrometry and determine blood lipid changes after an HP meal.

LDL/VLDL purification

Purified LDL/VLDL fractions were obtained using an LDL/VLDL Purification kit (Ultracentrifugation Free; Cell Biolabs Inc.). To 200 μl plasma on ice, 10 μl dextran solution and 100 μl precipitation solution A were added. The samples were incubated for 5 minutes on ice before centrifuging (6000 g) for 10 minutes at 4°C. The remaining pellet (containing LDL/VLDL) was resuspended in 80 μl bicarbonate solution and centrifuged (6000 g) for 10 minutes at 4°C. The supernatant was transferred to a new tube and mixed thoroughly with 1 ml of 1× precipitation solution B and centrifuged (6000 g) for 10 minutes at 4°C. The pellet was resuspended in 40 μl of 5% sodium chloride solution, mixed thoroughly with 1 ml of 1× precipitation solution C, and centrifuged (6000 g) for 10 minutes at 4°C. The above step was repeated and the pellet resuspended in 100 μl sodium chloride solution. To the mixture was added 16 μl of dextran removal solution before incubation for 1 hour at 4°C and then centrifuging (6000 g) for 10 minutes at 4°C. The supernatant (containing purified LDL/VLDL) was recovered and stored at –80°C. After the purified mixture of LDL/VLDL fractions was acquired, metabolites were extracted from these fractions as described below and analyzed by LC-MS of the lipid fraction.

Metabolite extractions

Metabolites were extracted from blood plasma/cells using the modified method of Folch and colleagues (47). Briefly, 15 μl blood plasma or pelleted cells was mixed with chloroform/methanol (2:1, 750 μl), including a mixture of internal standards labeled with deuterium (TAGs 45:0-d29, 48:0-d31, and 54:0-d35; Qmx Laboratories Ltd.). Samples were sonicated for 15 minutes, and water was added (300 μl). Samples were then centrifuged at 13,000 g for 20 minutes. The organic (upper layer) and aqueous phases (lower layer) were separated. The organic samples, containing the lipid extracts, were dried under a stream of nitrogen gas, while the aqueous samples were dried in a CentriVap Centrifugal Concentrator with attached cold trap (78100 series, Labconco).

LC-MS of the organic fractions

The organic fraction was reconstituted in 100 μl chloroform/methanol (1:1) and 10 μl added to 90 μl IPA/acetonitrile/water (2:1:1). Analysis of the fractions was performed using an LTQ Orbitrap Elite Mass Spectrometer (Thermo Scientific). In positive mode, 5 μl sample was injected onto a C18 CSH column, 1.7-μm pore size, 2.1 mm × 50 mm (catalog 186005296, Waters) which was held at 55°C in a Dionex Ultimate 3000 ultra-high performance liquid chromatography system (UHPLC; Thermo Fisher Scientific). A gradient (flow rate, 0.5 ml/min) of mobile phase A (acetonitrile/water 60:40, 10 mmol/l ammonium formate) and B (LC-MS–grade acetonitrile/isopropanol (IPA) 10:90, 10 mmol/l ammonium formate) was used. In negative ion mode, 10 μl of the sample was injected and 10 mmol/l ammonium acetate was used as the additive to aid ionization. In both positive and negative ion mode, the gradient began at 40% B; increased to 43% B at 0.8 minutes, 50% B at 0.9 minutes, 54% B at 4.8 minutes, 70% B at 4.9 minutes, and 81% B at 5.8 minutes; spiked at 99% B at 8 minutes for 0.5 minutes; and subsequently returned to the starting conditions for another 1.5 minutes to reequilibrate the column. The HPLC was coupled to an electrospray ionization (ESI) source before entering the mass spectrometer. The data were collected in both positive and negative ion mode with a mass range of 110–2000 m/z. Default instrument-generated optimization parameters were used. Tandem MS was performed, using normalized collision energy, to fragment the intact lipids listed in Supplemental Table 3 in order to identify the fatty acyl chains contained within.

Picolinyl ester FA derivatization of organic fractions for LC-MS

Picolinyl esters of FAs (PEFAs) were produced using a modification to a method published previously (48) to measure total FA content. To each dried organic extract 200 μl of 10 μmol/l deuterated internal standard mix (containing FAs 13:0, 15:0, 17:0, and 20:0) were added and dried under nitrogen. Each sample was fully resuspended in 200 μl oxalyl chloride (2 mol/l in dichloromethane, catalog 310670, Sigma-Aldrich) and incubated at 65°C in a heating block for 10 minutes, achieving cleavage of FAs from complex lipids and activating the carboxylic group. Samples were then dried under nitrogen, resuspended in 200 μl dichloromethane (catalog 270997, Sigma-Aldrich), and dried again. Each dried residue was then resuspended in 150 μl of 1% 3-hydroxymethylpyridine (catalog P66807, Sigma-Aldrich) in acetonitrile and incubated at room temperature for 5 minutes to produce derivatized FAs. The dichloromethane resuspension was repeated to ensure unreacted 3-hydroxymethylpyridine had evaporated, dried under nitrogen, and stored at –80°C until analysis.

Analysis of total FAs by triple quadrupole mass spectrometry

PEFAs were reconstituted in 100 μl 2:1 methanol/water and sonicated for 15 minutes. Samples were then centrifuged for 15 minutes at 13,000 g to pellet any remaining debris. A 2-μl injection volume of the resulting solution was analyzed on a TSQ Quantiva Triple Quadrupole mass spectrometer attached to a Vanquish UHPLC system. Chromatographic separation was achieved on an Acquity UPLC BEH C18 1.7 μm × 2.1 mm × 50 mm column (catalog 186002350, Waters). Mobile phase A was 100% water with 0.1% formic acid, and mobile phase B was 50:50 acetonitrile/IPA with 0.1% formic acid. The chromatography gradient started at 30% B for 2.33 minutes, increased to 100% B over 1.34 minutes, and decreased to 30% B for 1.23 minutes (to reequilibrate the column) at a flow rate of 0.735 ml/min. Default instrument-generated optimization parameters were used. Xcalibur Software (Thermo Scientific) was used to identify peaks, process mass spectra, and normalize data to the closest-eluting internal standard.

Data processing for open-profiling lipidomics

Samples obtained from human subjects were acquired in 2 analytical batches with the analytical method described above, along with a set of quality controls (QCs, obtained by pooling 15 μl of all samples). The resulting raw data files were converted into mzML format using the tool MSConvert of Proteowizard software (49, 50), and further processed within the R environment (51) with the libraries IPO, XCMS, and CAMERA (52–54) to perform parameter optimization based on the QC samples, peak extraction, grouping, retention time correction, and annotation of adducts and isotopes. The output from these preprocessing steps was exported as a CSV file and imported into an adapted implementation of the KniMet (55) pipeline for postprocessing. The LOESS batch correction utility was used to normalize for differences among the 2 analytical batches (based on QCs for intra-batch correction, and on all samples for inter-batch). Features were filtered first based on their presence in the QC samples on the 2 separate batches, with the QC-based feature filtering functionality (thresholds for missing values and relative standard deviation [RSD]/coefficient of variation [CV] = 50% and 20%, respectively), while median peak area comparison, as previously described by Dunn and coworkers (56), was used on the merged dataset. A summary of the CVs for key lipids is presented in Supplemental Table 3. Metabolites were annotated based on accurate mass using a library built from the LIPID MAPS mass spectrometry combinatorial expansion package (57). Finally, the data matrix to be utilized for multivariate statistical analysis was subjected to missing values imputation with the KniMet MVI-KNN tool.

Growth of AML12 hepatocytes

AML12 cells were purchased from ATCC (CRL-2254) and cultured in 1:1 DMEM and Ham’s F12 medium (Thermo Fisher Scientific), supplemented with 10% FBS, 1% penicillin/streptomycin (100 U/ml and 100 μg/ml, respectively), 1% insulin-transferrin-selenium (ITS; 10 mg/l, 5.5 mg/l, and 6.7 μg/l, respectively), and dexamethasone (100 μmol/l) at 37°C in 5% CO 2 . Cells were removed from liquid nitrogen, thawed rapidly, and initially cultured in a T25 flask (catalog 690175, Greiner). Medium was changed every 2 days, and upon reaching confluence, cells were subcultured in T75 flasks (catalog 7340290, VWR) at a 1:3 ratio. Cells were plated at a density of 50,000 cells/well in collagen 1–coated 12-well plates (catalog 7340295, VWR) and grown to confluence in maintenance medium. Cells were then supplemented with low glucose (7.60 mmol/l) with unlabeled l-glutamate (4 mmol/l) and dialyzed FBS (Thermo Fisher Scientific) for 2 days. After switching medium, cells were serum starved for 20 hours in low-glucose medium supplemented with unlabeled l-glutamate (4 mmol/l).

AML 12 cell 13C 5 -15N-l-glutamate labeling procedure

Cells were supplemented with glucose (7.60 mmol/l, n = 3) with unlabeled-l-glutamate (4 mmol/l) or glucose (7.60 mmol/l, n = 3) with 13C 5 15N-l-glutamate (4 mmol/l, n = 3). Based on previous experiments, the cells were allowed 3 hours so that the 13C 5 -15N-l-glutamate could reach an isotopic steady state in the TCA cycle. Cells and media were harvested at 0, 3, and 24 hours.

Dose response of glutamate, glutamine, leucine and lysine in AML12 hepatocytes

Cells were supplemented with low glucose (7.60 mmol/l, n = 3) without the specific amino acid (0 mmol/l) or with increasing levels of amino acid (2, 4, and 10 mmol/l, n = 3/amino acid). Cells and media were harvested after 24 hours.

Harvesting of AML12 cells for metabolomics

For cells and media undergoing metabolomic analyses, 900 μl medium was taken from each well and frozen at –80°C. Each well was then washed with 1 ml of 0.9% saline, and cells were lifted from the plates by adding 0.5 ml trypsin (10× trypsin-EDTA, catalog 25300045, Invitrogen). Each well was rewashed with 0.5 ml of 0.9% sterile-filtered saline, and the resulting solution was transferred into a 2-ml microcentrifuge tube and centrifuged at 13,000 g for 10 minutes to pellet the cells. The supernatant was removed, and the pellet was resuspended in 750 μl of a 2:1 chloroform/methanol solution to prevent enzymatic degradation of metabolites and frozen at –80°C.

Analysis of aqueous metabolites by triple quadrupole mass spectrometry

Metabolites were extracted as described above, and aqueous extracts were reconstituted in 50 μl of 10 mmol/l ammonium acetate in water before TCA cycle intermediates were separated using reversed-phase liquid chromatography using a Vanquish UHPLC attached to a TSQ Quantiva triple quadrupole mass spectrometer. Multiple reaction monitoring was used in conjunction with positive/negative ion mode switching utilizing the optimized mass transitions. A C18-PFP column (150 mm × 2.1 mm, 2.0 μm; ACE) was utilized at a flow rate of 0.5 ml/min, with a 3.5-μl injection volume. For chromatography on the UHPLC system, mobile phase A was 0.1% formic acid in water, and mobile phase B was 0.1% formic acid in acetonitrile. The gradient started at 30% B, increased to 90% B at 4.5 minutes for 0.5 minutes, and returned to the starting conditions for a further 1.5 minutes to reequilibrate the column. Mass transitions of each species were as follows (precursor > product): d 5 -l-proline 121.2 > 74.2; d 8 -l-valine 126.1 > 80.2; d 10 -l-leucine 142.0 > 96.2; l-glutamate [M] 148.0 > 84.2; l-glutamate [M+1] 149.0 > 85.2; l-glutamate [M+6] 154.1 > 89.1; citrate 191.0 > 111.0; citrate [M+1] 192.0 > 112.0; citrate [M+2] 193.0 > 113.0; citrate [M+3] 194.0 > 114.0; citrate [M+4] 195.0 > 114.0; citrate [M+5] 196.0 > 115.0; citrate [M+6] 197.0 > 116.0. Collision energies and radio frequency (RF) lens voltages were generated for each species using the TSQ Quantiva optimization function. Xcalibur Software was used to identify peaks, process mass spectra, and normalize data to the closest-eluting internal standard.

NAD+/NADH assay

Briefly, cells were lysed using NAD+/NADH extraction buffer, and enzymes that consumed NADH were removed by filtration through a 10 kDa spin column (Abcam, ab93349). NAD+/NADH was measured according to the manufacturer’s protocol (Abcam, ab65348).

RNA extraction and purification from AML12 hepatocytes

Total RNA was extracted and purified from hepatocytes using an RNeasy Mini Kit (QIAGEN) according to the manufacturer’s specifications. Purified RNA concentration was quantified at 260 nm using a NanoDrop 100 (Thermo Fisher Scientific).

cDNA production by reverse transcription

Each purified RNA sample was diluted with RNase-free water to a final concentration of 100 ng/μl. cDNA synthesis and genomic DNA elimination in RNA samples were performed using an RT2 First Strand Synthesis kit (QIAGEN) according to the manufacturer’s specifications. The reactions were stored at –20°C prior to real-time PCR analysis.

Quantitative-PCR

The relative abundance of transcripts of interest was measured by quantitative-PCR (qPCR) in RT2 SYBR Green Mastermix (QIAGEN) with a StepOnePlus detection system (Applied Biosystems). The SYBR Green qPCR Mastermix contained HotStart DNA Taq Polymerase, PCR Buffer, dNTP mix (dATP, dCTP, dGTP, dTTP), and SYBR Green dye. Before adding cDNA to each well of the 96-well plate, cDNA was diluted in RNase-free water to final concentration 8 ng/μl. PCR component mix was prepared by mixing 10 μl SYBR Green qPCR Mastermix with 0.6 μl of 10 μmol/l target primers (forward and reverse; 6 pmol/reaction) and 4.4 μl RNase-free water. To each well of a 96-well plate, 5 μl cDNA (total amount 40 ng) and 15 μl PCR components mix were added. The plate was centrifuged at 1000 g for 30 seconds to ensure that the contents were mixed and to remove any bubbles present in the wells. The plate was placed in the real-time cycler with the following cycling conditions: 10 minutes at 95°C for 1 cycle to activate HotStart DNA Taq Polymerase; 15 seconds at 95°C and 1 minute at 60°C to perform elongation and cooling for 40 cycles. RT2 qPCR Primer Assays for mouse Rn18s, Fasn, Acaca, Acly, Elvol6, Scd1, Dgat2, Mttp, and Apoc3 were purchased from QIAGEN. Expression levels were normalized to the endogenous control, Rn18s, using the ΔΔC t method, and fold changes reported were relative to the control group in the dose response (no extra supplemented amino acid in the media).

Preparation of cell lysates

Cell pellets were lysed in 100 μl cell extraction buffer (10 mmol/l Tris, 100 mmol/l NaCl, 1 mmol/l EDTA, 1 mmol/l EGTA, 10 mmol/l NaF, 20 mmol/l Na 4 P 2 O 7 , 20 mmol/l Na 3 VO 4 , 1% Triton X-100, 10% glycerol, 0.1% sodium dodecyl sulfate, 0.5% deoxycholate, 1 mmol/l phenylmethylsulfonyl fluoride, complete protease inhibitor tablet, and 1% of each phosphatase cocktail inhibitor 2 and 3) for 30 minutes, vortexing at 10-minute intervals. The lysate was centrifuged at 13,000 g for 10 minutes at 4°C, and the supernatant was collected and stored at –80°C.

Cell AKTpS473 and AKT (total) quantification by ELISA

PKB/AKT concentrations were measured using commercial assay kits (Invitrogen). The protocol was followed according to the manufacturer’s specifications. The background absorbance was subtracted from all data points, including standards, and a standard curve was generated. The unknown concentrations were read from the standard curve, and the concentrations were multiplied by the appropriate dilution factor. Values were normalized to protein concentration using a reducing agent–compatible bicinchoninic acid protein assay, and values of AKTpS473 were normalized to AKT (total).

2-Deoxyglucose uptake assay

2-Deoxyglucose uptake was measured using a commercial assay kit (Abcam, ab136955). The protocol was followed according to the manufacturer’s instructions.

Statistics

To calculate the sample size of the study, a paired t test between any 2 comparisons with adjusted multiple comparisons was used. Based on 80% study power, and an adjusted α value of 0.017, 9 subjects were needed for this study.

The distribution of the lipid species did not deviate significantly from a normal distribution (D’Agostino-Pearson omnibus normality test). Multivariate statistical analyses were performed in SIMCA-P software, version 13.0 (Umetrics). All variables were UV scaled and subjected to principal component analysis (PCA) coupled with Hotelling’s T2 test to evaluate the distribution of the observations and identify any possible outliers. Subsequently, samples were classified based on the diet, and a supervised OPLS-DA model was developed to maximize separation between the different classes. The models were validated by a permutation test (n = 100), and their significance (P ≤ 0.05) was assessed by submitting the scores of the models to a CV-ANOVA test. Loadings plots and the VIP were acquired for each model to determine which variables drive the separation between classes (threshold limit >1.0). Once metabolites were annotated in KniMet and analyzed in SIMCA, the most discriminant variables were TAGs. Fragmentation (as described above) was performed on a group of TAGs to confirm their identity (Supplemental Table 4).

These TAGs were subsequently visualized using univariate statistics. The extent to which the model fits and predicts the data is represented by R2X and Q2X, respectively. For univariate statistical analyses, data were visualized using GraphPad (Prism 5.2; GraphPad Software). All data are expressed as mean with SEM. In GraphPad, 1- or 2-way ANOVA was performed where appropriate to determine significant differences between experimental groups. For 1-way ANOVA, Dunnett’s post hoc multiple-comparisons test was performed, while for 2-way ANOVA, Šidák’s post hoc multiple-comparisons test was used. Differences between experimental groups were considered to be statistically significant at P ≤ 0.05.

Spearman’s correlation among the metabolic features annotated as TAGs deriving from open-profiling lipidomics was calculated and visualized within R (51) using the cor function of the stat package and the heatmap.2 function of the gplots library (58), respectively.

Study approval

The protocol of the present human study was approved by both the internal research review board, MRC – EWL, and the Cambridge South Local Research Ethics Committee, Cambridge, United Kingdom. Written informed consent was received from the participants prior to their inclusion in the study.