Reagents and chemicals.

HPLC-grade water, methanol, and acetonitrile were commercially available (Fisher Scientific). 3-(Trimethylsilyl)-1-propanesulfonic acid-d₆ sodium salt (DSS-d₆, 98 atom %), 3-(trimethylsilyl) propionic-2,2,3,3-d₄ acid sodium salt (TSP, 98 atom %), methanol-d4, chloroform-d4, and deuterium oxide (99.9% vol/vol) were obtained from Cambridge Isotope Laboratories (Andover, MA). All other chemicals and reagents were obtained from Sigma-Aldrich (St. Louis, MO).

Animals and treatments.

All experiments were performed in 12-wk-old male Sprague-Dawley (SD) rats (250–300 g) obtained from Central Laboratories (Seoul, Korea) and acclimated for at least 7 days. Rats were group-housed under a controlled temperature (24 ± 1°C) and photoperiod (12:12-h light-dark cycle) and allowed ad libitum access to standard rat chow and tap water. All animal experiments were approved by the Animal Care Committee of Keimyung University, Deagu, Korea, in accordance with the institutional guidelines for care and use of laboratory animals.

DJB operations were initiated by midline incision. The duodenum was ligated with 5-0 silk at the distal edge of the pylorus and the jejunum divided 10 cm beyond the ligament of Treitz. The stomach was created on the greater curvature of the antrum, and a side-to-end gastrojejunostomy was performed with a distal segment of jejunum using 7-0 polydioxanone (PDS) sutures. The open end of the proximal jejunum was anastomosed 15 cm from the gastrojejunal anastomosis in the jejunum using 7-0 PDS sutures. For the sham operations, a midline abdominal incision was performed, and the alimentary circuit of food through the stomach was maintained. Operative time was prolonged to produce a similar degree of anesthetic stress to those mice that underwent DJB. Rats were anesthetized by intraperitoneal injection of zoletil (5–10 mg/kg) and xylazine (10 mg/kg) mixture (1:1).

Rats were fasted overnight before and after surgery, and both groups were fed the same standard chow ad libitum after the operation. Body weight and food intake were measured twice a week for each rat. Rats were euthanized 5 wk after surgery, after which blood and liver tissues were harvested and stored at −80°C.

Sample preparation for metabolomics.

For the serum experiment, 270 μl of the serum sample was transferred into the 5-mm outer tube of a two-tube NMR 517 coaxial insert. The inner tube containing DSS-d₆ (98 atom %) was then inserted into the outer tube.

For the liver experiment, 100 mg of liver was transferred to a 1.5-ml tube containing 2.8 mm zirconium oxide beads and homogenized twice at 5,000 rpm with 210 μl of methanol (d₄) and 90 μl of 0.1 M sodium phosphate buffer [in deuterium oxide (D₂O), pH 7.0 ± 0.05] for 20 s using a Precellys 24 tissue grinder (Bertin Technologies, Ampère Montigny-le-Bretonneux, France). After homogenization, 350 μl of methanol (d₄), 150 μl of 0.1 M sodium phosphate buffer (in D₂O, pH 7.0 ± 0.05), and 400 μl of chloroform (d₁) were added to the tube. The mixture was vortexed vigorously for 30 s, allowed to separate for 15 min at 4°C, and centrifuged for 10 min at 13,000 rpm at 4°C. A total of 600 μl of supernatant was removed and mixed with 60 μl of 3-(trimethylsilyl)-propionic-2,2,3,3-d₄ acid sodium salt (TSP, 2.5 mM, 98 atom %) dissolved in 99.9% D₂O. The mixture was then centrifuged for 5 min at 13,000 rpm at 4°C. The supernatants (550 μl) were subsequently transferred into 5-mm NMR tubes (43).

NMR experiments.

¹H NMR spectra of serum and liver extracts were acquired on a VNMRS 600 MHz NMR using a triple-resonance, HCN salt-tolerant cold probe (Agilent Technologies, Santa Clara, CA). The pulse sequence used for the serum samples was a CPMG pulse sequence collected into 32K data points with 128 transients, a spectral width of 6,720.430 Hz, relaxation delay of 2.0 s, and an acquisition time of 2.381 s. For the liver extracts, a NOESYPRESAT (RD-90°-t1-90°-tm-90°-FID acquisition) pulse sequence was applied to suppress the residual water signal. A total of 64 transients were collected into 64K data points using a spectral width of 8,445.9 Hz with a relaxation delay of 2.0 s, an acquisition time of 4.00 s, and a mixing time of 100 ms. A 0.5-Hz line broadening function was applied to all spectra prior to Fourier transformation (FT).

NMR data processing for multivariate statistical analysis.

The ¹H NMR spectra were phased and baseline corrected using Chenomx NMR Suite 7.1 (Chenomx, Edmonton, AB, Canada). The spectral region (δ 0.6–8.5 ppm for serum, δ 0.4–9.8 ppm for liver) of each spectrum was segmented into equal widths (0.005 ppm for serum or liver extracts), and the signal intensities in each spectrum were calculated by integrating these sections. The regions corresponding to water (δ 4.5–5.2 ppm for serum, δ 4.518–5.12 ppm for liver) signal were removed prior to normalization of the spectrum and spectra alignment. The spectra were then normalized to the total spectral area and converted to ASCII format files to perform the alignment using the correlation-optimized warping (COW) method found in MATLAB (R2008a; The Mathworks, Natick, MA).

Multivariate statistical analysis.

The processed NMR data were imported to SIMCA-P version 12.0 (Umetrics, Umea, Sweden) and, prior to multivariate data analysis, scaled using the Pareto scaling method, in which each variable was divided by the square root of the standard deviation of the column values. Initially, principal component analysis (PCA) was used as an unsupervised pattern recognition method to examine intrinsic variation in the data set. Next, a partial least squared discriminant analysis (PLS-DA) was performed to minimize the possible contribution of intergroup variability and to further improve separation between the two groups. The quality of the models is described by R² and Q² values. R² was defined as the proportion of variance in the data explained by the models and indicates goodness of fit, and Q² is defined as the proportion of variance in the predicted data by the model and indicates predictability.

Targeted metabolite profiling.

Targeted profiling of metabolites obtained from ¹H NMR spectral data was accomplished using Chenomx NMR Suite 7.1, and glutathione and IMP (inosine monophosphate) were measured by applying multiple-reaction monitoring (MRM) transition using an AB Sciex 6500 liquid chromatography-quadruple linear ion trap mass spectrometer (LC-Q-LIT; AB Sciex, Forest City, CA). Levels of methionine, serine, dimethylglycine (DMG), S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), homocysteine (Hcy), cystathionine, and cysteine were determined on an Agilent 1290 Infinity LC and an Agilent 6490 Triple Quadrupole MS system equipped with an Agilent Jet Stream ESI source (Agilent Technologies).

Protein extraction for proteomics.

A total of 100 mg of liver tissue from sham or DJB rats was dissolved in 500-μl sample buffer containing 7 M urea, 2 M thiourea, and 4% (wt/vol) CHAPS. After sonication, the samples were centrifuged at 15,000 rpm and 4°C for 1 h, after which the supernatant was collected. For protein precipitation, 100 μl of supernatant was treated with 10% TCA (vol/vol) and incubated at −20°C for 3 h. The samples were then centrifuged at 15,000 rpm and 4°C for 10 min, and the supernatant was discarded. Protein pellets were lyophilized, dissolved in 500 μl of sample buffer, and centrifuged at 15,000 rpm and 4°C for 30 min. The supernatant was transferred to a new tube and stored at −80°C until further use. Protein concentrations were estimated using a Non-Interfering protein assay kit (Biosciences, St. Louis, MO) according to the manufacturer's instructions.

Two-dimensional electrophoresis experiments, imaging, and identification.

In the first dimension, 150 μg of protein extract from each liver sample was applied to an IPG strip (Immobiline DryStrip, pH 3–10 and pH 4–7, 18 cm; GE Healthcare Life Sciences) for isoelectric focusing (IEF), followed by 200 V for 1 h, 500 V for 30 min, 4,000 V for 30 min, 4,000 V for 1 h, 10,000 V for 1 h, 10,000 V for 13 h, and 50 V for 3 h. The liver protein samples were focused for a total of 110.2 kVh. The strips were equilibrated with 10 mg/ml dithiothreitol in an equilibration buffer for 15 min and then with iodoacetamide for 15 min with continuous shaking. In the second dimension, the equilibrated strips were placed onto 12% SDS-PAGE and run at a constant 20 mA until the dye reached the bottom of the gel. The protein spots in the analytical gels were visualized by silver staining (40).

The gel images were acquired using a BIO-RAD GS-800 scanner and imported into Progenesis SameSpots 2D software (v. 4.1; Nonlinear Dynamics, Newcastle, UK) for analysis. Protein spots with a >2.0-fold (P < 0.05) change in abundance or expression were considered differentially expressed. All spots were confirmed visually and edited manually. Differentially expressed proteins were identified using a Mascot-Peptide Mass Fingerprint (www.matrixscience.com) database search.

Western blot, vitamins B₁₂ and B₆, and reactive oxygen species.

Expressions of betaine-homocysteine methyltransferase (BHMT; Santa Cruz, EU), cystathionine β-synthase (Santa Cruz, EU), glutathione S-transferase (Santa Cruz, EU), glucose-6-phosphate dehydrogenase (G6PD; Cell Signaling Technology), phospho-AMPK (Cell Signaling Technology), and β-actin (Sigma) were determined by Western blotting. Rat vitamin B₁₂, B₆, and reactive oxygen species (ROS) levels were measured using ELISA kits (MyBioSource, San Diego, CA) or the OxiSelect ROS/RNS Assay kit (Cell Biolabs, San Diego, CA) according to the manufacturers' instructions.

Statistical analysis.

Two-sample t-tests, Mann-Whitney tests, Welch t-tests, and Spearman's correlation analyses were performed using SAS Statistical Software Package (release 8.02; SAS Institute, Cary, NC) and SPSS 12.0 (SPSS, Chicago, IL). The accepted level of significance was set as P < 0.05. Data are presented as means ± SE.

Body weight, food intake, blood glucose level, insulin level, and liver lipid content.

Food intake (Fig. 1A) and body weight (Fig. 1B) did not differ significantly between the sham and DJB rats. However, the blood glucose levels at the onset of the light phase in the DJB group were significantly lower than those in the sham group (Fig. 1C). The insulin levels of the sham and DJB groups did not differ significantly (Fig. 1D). Additionally, no significant differences in triglyceride (P = 0.093) and total cholesterol (P = 0.078) liver content in the DJB group were observed after DJB surgery.

Metabolic profiles of sera from sham and DJB rats.

The NMR spectra of sera from sham and DJB rats identified 18 sera metabolites (Fig. 2A). PLS-DA score plot (Fig. 2B) of the global metabolic profiles in the serum showed a different pattern of metabolism between sham controls and DJB rats [R²X: 71.7%, R²Y: 92.0%, Q²: 71.5% in serum model (sham controls n = 7; DJB rats n = 8)]; the reliability of the PLS-DA models was validated using a 100-fold repeated permutation test (Fig. 2B). We observed that the levels of formate, glutamine, succinate, and lactate in the serum were significantly altered after DJB surgery (P < 0.05, Fig. 2C).

Metabolic profiles of livers from sham and DJB rats.

NMR spectra of liver tissue from sham and DJB rats contained 33 hepatic metabolites (Fig. 3A). PLS-DA score plot (Fig. 3B) of the global metabolic profiles in livers acquired using NMR spectrometry showed a different pattern of metabolism between sham controls and DJB rats [R²X: 50.8%, R²Y: 94.4%, Q²: 84.0% in liver model (sham controls n = 7; DJB rats n = 9)], and the reliability of the PLS-DA models was validated using a 100-fold repeated permutation test (Fig. 3B). The levels of ADP, AMP, ATP, alanine, choline, formate, glutamine, hypoxanthine, isoleucine, lactate, NADP+, pyruvate, succinate, and valine in the liver samples significantly differed between the groups (P < 0.05; Fig. 3C).

Correlation analyses of serum and liver metabolites.

Spearman's correlation analyses were applied to assess the associations between serum and liver metabolites that were significantly changed between sham and DJB rats (Fig. 4). Serum formate was the highest correlated metabolite with liver metabolites such as ADP, AMP, ATP, alanine, choline, hypoxanthine, isoleucine, lactate, NADP+, pyruvate, and succinate. Serum glutamine was associated with the presence of isoleucine and pyruvate in the liver. Serum lactate was also strongly correlated with a number of liver metabolites, including ADP, alanine, choline, formate, glutamate, hypoxanthine, isoleucine, pyruvate, and succinate. Finally, serum levels of succinate were positively correlated with those of formate, glutamate, hypoxanthine, and pyruvate in liver.

Proteomic analysis of liver samples from sham and DJB rats.

The 2D gel images of proteins extracted from the livers of the sham (n = 3) and DJB groups (n = 3) were analyzed using computational methods (Fig. 5A). The seven identified proteins, namely, aldehyde dehydrogenase, microtubule-actin cross-linking factor 1, ATP-dependent zinc metalloprotease YME1L1, H(+)-transporting ATP synthase, delta-aminolevulinic acid dehydratase, guanine deaminase, and IMP showed over 2.0-fold changes (increase or decrease) in their relative abundance, with a P value of <0.05 in a t-test between the two groups; thus, their expression significantly differed (Fig. 5B).

Trans-methylation and trans-sulfuration pathway.

Our NMR-based metabolic profiling identified a strong correlation between various serum and liver metabolites. In particular, high levels of circulating formate were associated with the liver metabolites ADP, AMP, ATP, and hypoxanthine, which are intermediates in purine metabolism, an output of one-carbon metabolism (45). In addition, previous studies (17, 18) had suggested that elevated levels of formate in plasma could be related to altered cellular one-carbon metabolism. Thus, we measured the concentrations of vitamin B₁₂ and vitamin B₆, which affect the trans-methylation (remethylation process) and trans-sulfuration pathways, respectively. The concentration of vitamin B₁₂ in the serum from the DJB group was significantly lower than that from the sham control group, but the concentrations of vitamin B₆ from the control and DJB groups did not differ significantly (Fig. 6, A and B). However, the plasma total Hcy was decreased in DJB rats (Fig. 6C). Thus, we further investigated one-carbon metabolism in the liver after DJB surgery.

In the liver, the expression level of BHMT, which transfers a methyl group to a Hcy via the demethylation of betaine to generate dimethylglycine, was decreased in the DJB group compared with the sham controls (Fig. 6D). However, one-carbon metabolites affecting trans-methylation, such as choline, betaine, DMG, methionine, S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), and Hcy, did not significantly differ in the groups receiving normal chow (Fig. 6E).

We observed significantly increased levels of serine, cystathionine, cysteine, and glutathione (Fig. 6E), accompanied by an increased expression of cystathionine β-synthase (CBS; Fig. 6D), glutathione S-transferase (GST; Fig. 6D), and aldehyde dehydrogenase in the livers of the DJB group. These results suggest that DJB increased the trans-sulfuration and its consecutive processes such as detoxification and the scavenging activities of ROS. Thus, we measured the level of ROS in the liver using the fluorescent dye 2′,7′-dichlorodihydrofluorescein (DCF) and observed significantly decreased levels of DCF fluorescence in the DJB group (Fig. 6F).

Purine pathway.

In the liver, DJB rats showed increased levels of purine metabolites (ATP, ADP, and AMP) and the first fully formed purine IMP (Fig. 7A).

Purine de novo synthesis started with phosphoribosyl pyrophosphate (PRPP), which is produced primarily through the pentose phosphate pathway. To determine the activity of the pentose phosphate pathway, we observed the levels of phosphoenolpyruvate carboxykinase-1 (PEPCK1), glucose-6-phosphate phosphatase (G6PC), and G6PD. Expression of G6PC and G6PD were decreased and increased, respectively, in DJB rats (Fig. 7, B and C). Levels of NADP+ also increased in DJB rats compared with sham controls. In addition, both a significant increase in lactate, alanine, and BCAAs (isoleucine and valine) and a decrease in ATP/ADP ratio (Fig. 7D) in the liver were observed. These results were indicative of an enhanced pentose phosphate pathway and reduced gluconeogenesis in DJB rats (35).

In contrast to the increased purine synthesis pathway, proteomics analysis of the liver revealed decreased levels of guanine deaminase, an enzyme involved in GTP degradation, and lower levels of hypoxanthine, a metabolite of purine degradation, both of which are associated with reduced purine degradation. In particular, altered purine metabolism affects the energy-regulating system by altering the AMP/ATP ratio, which is used by the cell to sense how much energy is available. An increased AMP/ATP ratio results in enhanced energy production and catabolic activity, such as fatty acid oxidation and glucose transport, by activating AMPK expression. On the basis of our results, DJB rats showed a 3.17-fold higher AMP/ATP ratio than that shown by the sham controls (P < 0.001; Fig. 7E). In addition, AMPK phosphorylation was increased in the DJB rats compared with that in the sham controls (Fig. 7F).