Animals and treatment
In the present study, a mouse model of diet-induced obesity and NAFLD was used. Briefly, 8-week-old male C57/BL6J mice were purchased from Charles River (Japan). All animals were housed under conventional conditions with controlled temperature, humidity, and 12-h light–dark cycle, provided with food and water ad libitum, and housed in transparent polymer X cages (catalog no. CL-0104-2; CLEA Japan, Tokyo, Japan), with a maximum of eight mice housed per cage. The control group (n = 17) were fed a normal chow diet, and the HFD group (n = 17) were fed an HFD (catalog no. D12492; Research Diet, Tokyo, Japan) for eight weeks. In both groups, twelve of the mice were divided into halves and hydrodynamically injected with pLIVE vectors expressing mouse Gdf15 or Fgf21 once a week before the introduction of the specific diet, whereas the remains of the mice were hydrodynamically injected with the control pLIVE vector. At the end of the treatment, liver, epididymal fat, and blood were isolated from each animal for the analyses. The animal study protocol was conducted in accordance with the guidelines for the care and use of laboratory animals established by the Kyoto Prefectural University of Medicine (Kyoto, Japan) and approved by the Institutional Committee on the Ethics of Animal Experiments (permission number of institutional protocol approval M2023-145). All procedures were approved and supervised, and this study was reported in accordance with the ARRIVE guidelines.
Hydrodynamic gene transfer via tail vein injection
Wild-type mouse Gdf15 and Fgf21 cDNA constructs obtained by PCR amplification were subcloned into the pLIVE vector (Mirus Bio, Madison, WI, USA), and the pLIVE vectors expressing mouse Gdf15 or Fgf21 were delivered by hydrodynamic tail vein injection (7). Tail vein injections were performed using 40 µg of the indicated plasmid in the TransIT-EE® solution, according to the manufacturer’s instructions (Mirus Bio).
Analysis of the liver architecture
As previously described, liver sections were stained with hematoxylin/eosin or oil red O using standard techniques (7).
Cell culture and transfection
AML12 cells, a mouse hepatocyte line obtained from the American Type Culture Collection (Manassas, VA), were cultured at 37°C under 5% CO2 in medium including a 1:1 mixture of high-glucose DMEM/Ham’s F-12 supplemented with L-glutamine, 1.0 mM sodium pyruvate, 15 mM HEPES, 10% fetal bovine serum, 1× insulin-transferrin-selenium solution (Invitrogen, Carlsbad, CA), and 40 ng/mL dexamethasone (Sigma Aldrich, St. Louis, MO). The cell cultures were transfected with the pLIVE-control or plive-GDF15 plasmid for 48 h using Lipofectamine 3000 reagent (Invitrogen), according to the manufacturer’s instructions. 42 h after transfection, the cultures were treated with 200 µM palmitic acid and 100 µM oleic acid (Sigma Aldrich) for 24 h. Silencer® Select short interfering RNA (siRNA) (catalog no. s106848; Invitrogen) targeting Cnot6l was transfected at a final concentration of 25 nM using Lipofectamine RNAiMAX transfection agent (Invitrogen), following the manufacturer’s instructions. After transfection, the cultures were incubated for another 48 h before downstream analyses. All transfection assays were repeated at least three times. Intracellular ROS production was measured using the high-sensitive DCFH-DA ROS assay kit (Dojindo, Kumamoto, Japan).
Two-step real-time PCR
PCR was performed as described previously (4, 7). Briefly, total RNA were extracted from whole livers using the RNeasy kit (Qiagen, Valencia, CA) and reverse-transcribed using random primers and Superscript RNase H− reverse transcriptase (Invitrogen). All PCR products were sequenced to confirm the specificity of all primer pairs (Supplemental Table 1). Target gene levels were presented as the ratio of the gene expression in the treated group to the gene expression in the control group. Fold changes were determined using point and interval estimates.
Immunoblotting
Whole-liver protein lysates were separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes, which were probed with primary antibodies to eIF2α, phosphorylated eIF2α (p-eIF2α), spliced X-box binding protein 1 (XBP1s) from Cell Signaling Technology (Beverly, MA); peroxisome proliferator-activated receptor α (PPARα) (GTX101098) from GeneTex (Irvine, CA); unspliced X-box binding protein 1 (XBPu) (clone F-4; sc-8015F-4) from Santa Cruz (Dallas, TX, USA); GDF15 (bs-3818R) from Bioss (Boston, MA) and actin from Sigma Aldrich. Next, the membranes were incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG antibodies (Invitrogen). The proteins were visualized using ECL (GE Healthcare, Chicago, IL). Immunoblots were scanned, and quantitative densitometric analysis of the bands was conducted using ImageJ (NIH, Bethesda, MD).
Tissue and plasma biochemical analyses
Serum levels of aspartate aminotransferase, alanine aminotransferase (ALT), total cholesterol, triglycerides, FFAs, and glucose were measured at SRL (Tokyo, Japan). Serum levels of GDF15, FGF21, and insulin were measured using ELISA kits for mouse GDF15 (ab216947), mouse FGF21 (ab212160), and mouse/rat insulin (Morinaga, Yokohama, Japan). Homeostasis model assessment-insulin resistance (HOMA-IR) was calculated as follows: HOMA-IR = fasting glucose (mM) × fasting insulin (microunits/mL)/22.5.
Statistical analysis
Data were presented as means ± standard error of the mean. Significance was established using two-way repeated ANOVA (Fig. 2A, 2C; Supplemental Fig. S1C, 1E) or Student’s t test and ANOVA, when appropriate. Differences were considered significant at a p level of < 0.05.