Six-week-old male C57BL/6 mice (weight, 18−22 g) were purchased from Shanghai Slack Laboratory Animal Co., Ltd., and raised in specific-pathogen-free conditions at the Laboratory of the Experimental Animal Center of Wenzhou Medical University, with regulated humidity and temperature and a 12/12 h light-dark cycle. During the entire experiment, mice were fed with standard rat chow and tap water. All the experiments followed the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The Institutional Animal Care and Use Committee of Wenzhou Medical University also approved the experiments (document number: wydw2012-0083). We also took steps to minimize the suffering and numbers of mice used.
In the experiment, mice were randomly divided into three groups: control, diabetic, and FGF21-treated diabetic (FGF21) groups. First, T1D was induced using streptozotocin (STZ, i.p., 50 mg/kg), which was freshly prepared in 0.1 M citrate buffer (pH 4.2–4.5), for five consecutive days. The control mice were administered an identical volume of the vehicle. Random blood glucose level in a blood sample drawn from the tail was measured 72 h later using a strip-operated blood glucose monitoring system (One Touch Ultra, Lifescan). Mice with blood glucose levels higher than 11.10 mM were defined as diabetic mice . After inducing hyperglycemia for 9 weeks, the diabetic mice were subdivided into two groups that received either the vehicle or FGF21 (2 mg/kg, i.p.) daily for four consecutive weeks. The control and untreated diabetic groups received sterile saline. The recombinant human FGF21 used in the study was expressed and purified according to previously described procedures .
Morris water maze test
The Morris water maze (MWM) test was performed as previously described [36, 37]. Briefly, after the drug or vehicle administration, the mice in each group were selected to perform a learning task in the MWM. The maze consisted of a white pool (diameter, 200 cm; depth, 50 cm) filled with a mixture of water and a non-toxic white dye (temperature, 26 ± 2°C). The behavior of the mice was traced using a camera connected to a XR-XM101 analysis system (Xinruan Information Technology Co. Ltd, Shanghai). The pool had four quadrants, and each orientation was designated as a starting position (North, South, East, or West). The pool also had a camouflaged escape platform submerged 2 cm below the water surface. The platform was placed in the middle of the second quadrant. The mice were trained for 4 days, and on each training day, the mice were placed in the four quadrants for 20 min intervals. The trial was started by placing a mouse at one of the starting points, with its back facing the platform. The trial was terminated when the mouse reached and stood on the platform, but the mouse was left on the platform for an additional 10 s. However, if a mouse could not find the platform within 60 s, it was guided and placed on the platform for 20 s. During the spatial navigation test, all the mice were trained four times each day. On the 5th day, the probe trial was carried out, wherein the mice were allowed to swim for 60 s, with the platform removed. The swimming distance, latency to find the platform, the number of crossings over the platform, and the time in the target quadrant were measured. After training, each mouse was removed from the pool, dried, and returned to its cage.
Hippocampal samples preparation
Twenty-four hours after the end of the MWM test, the mice were euthanized and their hippocampal tissues were dissected immediately, snap-frozen in liquid nitrogen, and stored at -80°C. The preparation of the brain samples and acquisition of 1H-NMR spectra were performed as previously described [38, 39]. Briefly, the frozen hippocampal tissues were weighed carefully and placed in centrifuge tubes. Methanol (4 mL/g) and water were added into the tube immediately, homogenized at 4°C, and mixed by vortexing. Chloroform (2 mL/g) and distilled water were then added into the tube and mixed again. The tube was kept on ice for 15 min, and then centrifuged for 15 min at 4°C. The obtained aqueous extracts were then dissolved in 600 µL of 99.5% D2O for 1H-NMR spectroscopy.
Multivariate pattern recognition analysis of 1H-NMR spectra
The detailed acquisition protocol of 1H-NMR spectra has been previously described [40, 41]. All NMR spectra were phased and corrected, and then data-reduced to 1,100 integrated regions corresponding to the region of δ 10–0 by using the Topspin 2.1 software. The δ 4.6–5.0 region was removed to eliminate artifacts related to residual water resonance. The spectral segments were normalized to the total of the spectral intensity. The normalized integral values were subjected to multivariate pattern recognition analysis using the SIMCA-P+ V12.0 software package (Umetrics, Umea, Sweden). Supervised partial least squares-discriminant analysis (PLS-DA) was performed for class discrimination and biomarker identification . Metabolites were assigned using Chenomx Profiler, a module of Chenomx NMR Suite version 7.7 evolution edition (Chenomx, Inc., Edmonton, AB, Canada) and the Human Metabolome Database (version 3.6).
Data were visualized using a principal component score plot to provide the most efficient representation of the data, wherein each point represented an individual spectrum. The score plots were used to visualize the separation between the groups. The loading plots, which were colored according to the absolute value of the correlation coefficient, could identify how the metabolites contributed to the separation of the groups. The scores and loading plots complemented each other. A leave-one-out cross-validation and permutation test (200 times) on the first component was also applied to estimate the robustness and credibility of the PLS-DA model. If the plots showed that the Q2 regression line had a negative intercept and that the R2 values on the left were thus lower than those of the original point, the PLS-DA model was considered robust and credible. Meanwhile, three additional parameters were calculated: R2X and R2Y, which explained the variance in the matrix of spectrum data and class membership, respectively, and Q2, which indicated the predictive capability of the model. These parameters are commonly used to determine the quality of a model, with values of R2 and Q2 close to 1.0 representing an excellent model .
LDH activity assessment
LDH activity was determined using a protocol detailed in our previous study. Briefly, to determine the LDH lactate to pyruvate (L→P) activity, we used an LDH kit (Jiancheng Bioengineering Institute, Nanjing) according to the manufacturer’s instructions. LDH L→P activity was detected at 25°C by measuring sample absorbance at 430 nm wavelength. For determining LDH P→L activity, we used an assay designed on the basis of a procedure documented in the literature . Protein lysates were diluted at a concentration of 0.05 mg/mL in 500 mM potassium phosphate buffer (PPB) and were added to the LDH assay reagent containing 100 mM pyruvate and β-nicotinamide adenine dinucleotide (NADH) in 500 mM PPB (5 mg/20 mL; pH 7.5). Changes in NADH absorbance were measured at 25°C, at 1-min intervals for 10 min, at 340 nm wavelength. LDH total activity was calculated in IU/mg protein and expressed as the P:L ratio (LDH P→L/LDH L→P).
ATP levels measurement
The mouse hippocampal tissue/cell samples were homogenized and lysed, and the ATP content was detected using the Enhanced ATP Assay Kit (Beyotime Institute of Biotechnology, Shanghai) according to the manufacturer’s instructions.
NAD+ and NADH contents determination
The mouse hippocampal tissues samples (10–20 mg) were homogenized, and the NAD+/NADH Assay Kit with WST-8 (Beyotime Institute of Biotechnology, Shanghai) was used to detect the NAD+ and NADH contents.
Reverse transcription-polymerase chain reaction analysis
The mouse brain tissue samples (10–20 mg) were homogenized in TRIzol reagent (Invitrogen, Carlsbad, CA) to extract total RNA. The reverse transcription (RT) of RNA into cDNA was performed by using the PrimeScriptTM Real-Time Reagent Kit (Takara, RR037A). The polymerase chain reaction (PCR) analysis was performed using CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA). The primer sequences are provided in supplemental Table S1.
Cells culture and condition medium testing
C6 astrocyte cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (FBS, Gibco) and 1% antibiotics. SH-SY5Y neuron cells were cultured in F12 Medium supplemented with 10% FBS and1% antibiotics. They were incubated in 37°C and 5% of CO2 volume fraction.
C6 cells were cultured with normal glucose, high glucose (HG) for 48 h. Then astrocyte-conditioned medium (ACM) were collected. After filtration, SH-SY5Y cells were respectively treated with ACM and FGF21 for further 48 h. Refer to Figure 6C for the CM testing process [45, 46].
Western blot analysis
We used buffer containing 1% protease and phosphatase inhibitors (Beyotime Biotechnology Institute) to extract the total protein from brain tissues and cell. Protein samples were loaded and electrophoresed, and then transferred to polyvinylidene fluoride membranes. After electrophoresis, the membranes were incubated in 1×TBST containing 5% skimmed milk for 2 h. Thereafter, the membranes were placed in the primary antibody and incubated overnight at 4°C. The primary antibodies used were as follows: MCT1 (1:1000; 20139-1-AP, Proteintech), MCT2 (1:1000; SC-166925, Santa Cruz Biotechnology), MCT4 (1:1000; 22787-1-AP, Proteintech), LDHA (1:1000; 3582S, Cell Signaling Technology), LDH-B (1:1000; 14824-1-AP, Proteintech), c-Fos (1:1000; 2250S, Cell Signaling Technology), EGR1 (1:1000; 97249S, Cell Signaling Technology), SYP (1:1000; ab184176, Abcam), PSD95 (1:1000; 3409S, Cell Signaling Technology), P-PI3K(1:1000; 17366S, Cell Signaling Technology), PI3K(1:1000; 4249S, Cell Signaling Technology), P-Akt (1:1000; 4060S, Cell Signaling Technology), Akt (1:1000; 4691S, Cell Signaling Technology), p-mTOR (1:1000; 5536S, Cell Signaling Technology), mTOR (1:1000; 2983S, Cell Signaling Technology), P-P70 (1:1000; 9250S, Cell Signaling Technology), P70 (1:1000; 2708S, Cell Signaling Technology), GAPDH (1:1000; 60004-1-lg, Beyotime Institute of Biotechnology) and β-Actin (1:1000; A418, Beyotime Institute of Biotechnology). After washing with 1× TBST three times, the membranes were incubated with the secondary antibodies (goat anti-mouse IgG (H+L), 1:5000, SA00001-1, Proteintech; goat anti-rabbit IgG (H+L), 1:5000, SA00001-2, Proteintech) for 1 h at room temperature. We used the ChemiDoc XRS+ Imaging System (Bio-Rad) to detect the bands and Image-Pro Plus 6.0 software to analyze the gray values of the bands.
The brain tissues were isolated and fixed in 4% paraformaldehyde for at least 24 h. Thereafter, they were embedded in paraffin and sectioned using a slicing microtome (Leica, Germany).
For the MCT2 immunohistochemical staining experiments, paraffin sections were placed in an incubator at 60°C for 1 h. The dewaxed and dehydrated paraffin sections were then incubated in 3% H2O2 for 10 min and incubated in boiling citrate buffer for 5–10 min. Thereafter, the sections were blocked with 5% bovine serum albumin for 1 h. The paraffin sections were then incubated with the MCT2 antibody (1:50; SC-166925, Santa Cruz Biotechnology) overnight at 4°C. After washing the sections three times in 1× phosphate-buffered saline (PBS), they were incubated with the secondary antibody (goat anti-mouse IgG, 1:200; SA00001-1, Proteintech) at 37°C for 1 h and stained using a DAB kit (ZLI-9017, ZSGB-BIO). Images of the stained sections were captured using a Nikon ECLIPSE Ti microscope.
For the LDH-B immunofluorescence experiments, most of the protocols were similar to those used in the aforementioned immunohistochemical analysis. The only difference was that the sections were incubated with the fluorescent secondary antibody after washing three times in PBS.
The cells were spread in a 24-hole plate containing the cover glass at a density of 2 × 104/hole, after washing the sections three times in PBS, they were incubated with the 4% Polyformaldehyde for 45 min. Then 0.5% Trition-X-100 punched for 15 min, the plates were blocked with 5% bovine serum albumin. The plates were then incubated with the MCT2 antibody (1:200; SC-166925, Santa Cruz Biotechnology) overnight at 4°C. After washing the plates three times in PBS, they were incubated with the fluorescent secondary antibody for 1 h.
Data are presented as mean ± SEM. All data were analyzed using one-way analyses of variance (ANOVA) followed by Scheffe’s post-hoc test. Values of p < 0.05 were considered statistically significant. SPSS for Windows, Version 13.0 (SPSS Inc., Chicago, IL) was used for all statistical tests. The figures were generated using Prism 5.0 software (GraphPad Software, Inc., San Diego, CA).