To avoid the behavioral influence of estrogen, male rats were selected as experimental animals (Albert and Newhouse, 2019). Sprague-Dawley rats (male, 6–8 weeks old) were purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) and housed in specific pathogen free facilities with controlled temperature (23 ± 2°C) and humidity (50% ± 5%). Rats were given food and water ad libitum in a 12 h light (9:00–21:00)/dark cycle.

After 1-week adaption to environment, animals were assigned to different groups arbitrarily. According to previous studies (Eydipour et al., 2020) and our preliminary study, 21-day continuous corticosterone treatment (40 mg/kg/day) was used to induce animal model of depression. Corticosterone was suspended in saline solution supplied with 1‰ DMSO and 1‰ Tween 80 and given to rats through subcutaneous injection. Rats in control group received the same amount of saline solution (with 1‰ DMSO and 1‰ Tween 80) each day. To study the efficacy and mechanism of fluoxetine, fluoxetine hydrochloride (10 mg/kg/day, with saline as vehicle) was given to corticosterone-stimulated rats by gavage for 21 day. The dosage of fluoxetine hydrochloride (10 mg/kg) was chosen referred to clinical medication guidelines (Avasthi and Grover, 2018) and our previous studies (Pan et al., 2014; Jia et al., 2018).

After 21-day treatment of corticosterone or/and fluoxetine, behavioral tests were conducted within 10:00–15:00 as shown in Figure 1.

Regional brain glucose metabolism was detected by ¹⁸F-FDG PET/CT scans. Animals (n = 4 each group) were randomly selected for this experiment. This work was conducted with the help of MITRO Biotec (Nanjing, China). Concisely, following 12 h fasting, isoflurane (1.5–2.5%, 300–800 ml/min) was applied to anaesthetize rats. Then, 300 μCi ¹⁸F-FDG was administrated to rats via tail vein injection. 1 h post injection of ¹⁸F-FDG, static images were acquired for 10 min using the small animal PET/CT (Inveon, Siemens). A two bed CT scan (80 kV, 500 mA) was obtained for attenuation corrections of PET images. PET images were acquired in a list mode with a coincidence timing window of 1.5 ns. All PET images were reconstructed using OSEM3D/MAP (2 OSEM3D iterations, 16 MAP iterations). PET image dimensions were 128 voxels × 128 voxels × 159 voxels with a voxel size of 0.7764 mm × 0.7764 mm × 0.796 mm (Pan et al., 2021). PMOD software (version3.805, PMOD Technology, Switzerland) was used to analyze PET images. The SUV (standardized uptake value) of different brain regions was obtained.

After 21 day treatment of corticosterone and fluoxetine, prefrontal cortex (PFC) slices were prepared and cultured in oxygen saturated artificial cerebrospinal fluid according to previous report (McNair et al., 2017). 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG, a fluorescent glucose analog) can be taken up by cells through glucose transporters just like glucose. Therefore, 2-NBDG (5 μM) was added into artificial cerebrospinal fluid to evaluate glucose metabolism of PFC slices for 1 h. Sulforhodamine 101 (SR101) was used as a marker of astrocytes because they uptake it selectively (Nimmerjahn et al., 2004; Kafitz et al., 2008). PFC slices were cultured in artificial cerebrospinal fluid with SR101 (0.5 μM) for 20 min. 2-NBDG signal of SR101 + cell in PFC slice was observed by two-photon confocal microscopy. The cell region of astrocytes was manually framed according to SR101 signal. Then, fluorescence intensity of 2-NBDG in this cell region was measured by ImageJ.

After behavioral tests, rats were decapitated. PFCs were quickly separated, cut into pieces, digested by papain with 0.1% DNase. Cell debris was removed and all cells were enriched by Percoll (17-0891-02, GE Healthcare). The isolated cells were fixed by 1% PFA. The fixed cells were incubated with primary antibodies rabbit anti-GLAST1 (1:100, ab416, Abcam) and mouse anti-GLUT1 (1:100, ab40084, Abcam) in permeabilized (0.1% Triton, for total GLUT1 detection) or non-permeabilized (without Triton, for plasma membrane GLUT1 detection) condition for 1 h. Then, cells were incubated with secondary antibody Alexa Flour 488 goat anti-rabbit IgG and Alexa Flour 647 goat anti-mouse IgG for 0.5 h. The fluorescence signal was detected by flow cytometer (Attune NxT, Invitrogen). Astrocytes were identified by GLAST1 expression (Norden et al., 2016).

Three different methods were used to evaluate glucose uptake activity of primary astrocytes.

Primary astrocytes were plated at a density of 2,000 cells/well in XF96 plate. The procedures of the glycolytic rate assay were conducted according to the user guide of Agilent Seahorse XF Glycolytic Rate Assay Kit (103344-100, Agilent Technologies). The day before assay, assay medium was prepared by supplementing base medium with 10 mM glucose, 1 mM pyruvate, 2 mM glutamine and 5.0 mM HEPES (pH 7.4). After 72 h stimulation with corticosterone and fluoxetine, cells were washed with warm assay medium for two times and the XF96 cell culture plate was calibrated in a non-CO₂ incubator at 37°C for 1 h. After calibration, OCR (oxygen consumption rate) and ECAR (extracellular acidification rate) were measured by seahorse analyzer. Rotenone/antimycin A (0.5 μM) and 2-DG (5 mM) were added to detect the compensatory glycolysis and post 2-DG acidification. GlycoPER (glycolytic proton efflux rate) was calculated according to the values of OCR and ECAR. After seahorse glycolytic rate assay, the DNA level per cell was detected by commercial kit (C7026, Invitrogen). The results of glycoPER were normalized by cell content (the ratio of DNA level).

Supernatant lactate level was measured by L-lactate assay kit (ab65330, Abcam). All the operations followed the manufacture’s instruction. The protein contents of primary astrocytes were detected. All the results were normalized by protein ratio.

A human TXNIP promoter (> 1518bp + 50 UTR, Supplementary Table S1) connected with luciferase reporter was cloned to a plasmid. Then, plasmid containing TXNIP promoter, helper plasmids (pGag/Pol, pRev, pVSV-G) and RNAi-mate were added to the medium of HEK293T cells for lentivirus production. Subsequently, TXNIP-luc expressing virus was transduced to U87MG cells. Through puromycin selection, a polyclone of U87MG cells with stable expression of TXNIP-luc was obtained. The stable TXNIP-promote-driven luciferase reporter cell line was developed by Gene Pharm (Shanghai, China). We named the stable cell line “A9433.” A9433 cell was maintained in DMEM medium in the presence of 1 μg/ml puromycin. The activity of firefly luciferase was detected by commercial kit (RG006, Beyotime).

Nuclear, cytoplasmic (CYT) and plasma membrane (PM) protein of PFC tissues or astrocytes were isolated by commercial kits (KGP1100, KeyGEN BioTECH; P0033, Beyotime Biotechnology). Phenylmethanesulfonyl fluoride (PMSF, a protease inhibitor) was used to inhibit protein degradation. The protein concentration of sample was detected by bicinchoninic acid (BCA) protein assay kit (Thermo scientific). Lysates were loaded into 10% sodium dodecyl sulfate-polyacrylamid gel electrophoresis and subsequently transferred to polyvinylidene fluoride membranes. Membranes were blocked in 5% non-fat milk for 1 h. Then, the following first antibodies were used: GLUT1 (1:5000, ab115730, Abcam), TXNIP (1:1000, #14715, CST), Na, K ATPase (1:1000, #3010, CST), GR (1:1000, #3600, CST), Histone H3 (1:500, sc-517576, Santa Cruz) and β-actin (1:2000, #4970, CST). After incubating with first antibody overnight, membranes were incubated goat anti-mouse IgG (1:5000, HAF007, R&D systems) or goat anti-rabbit IgG (1:3000, #7074, CST) for 1.5 h. Protein bands were visualized by ECL (#180–5001, Tanon) and imaged by chemiluminescence imaging instrument (#5200, Tanon). The gray value of images was measured by ImageJ.

Total RNA of PFC tissues or primary astrocytes was extracted by Trizol (15596018, Invitrogen). Isolated RNA was dissolved in diethyl pyrocarbonate (DEPC) H₂O. HiScriptⅡqRT Supermix (Vazyme) was used to reverse RNA to cDNA. Expression of TXNIP was quantified using qRT-PCR. The primers used in this study were listed as follows: TXNIP, sense, TTTCTGCCTCTCTGCTTG and antisense, GAC​TTG​CCT​ACT​GAT​TGC​C; β-Tubulin, sense, CATCACAGGCAAGGAAGA and antisense, GTG​GAA​AAC​CAA​GAA​GCC. SYBR^® Green was combined with DNA during amplification (Bio-Rad CFX96). Relative expression of TXNIP was analyzed by 2^−△△Ct method with β-Tubulin as internal control.

Data were expressed as mean ± SEM and statistical analyzed by Prism 7 (GraphPad Software, San Diego, CA, United States). Data with normal distribution were analyzed by one-way ANOVA followed by Dunnett’s post-hoc test for multiple comparisons. Data without normal distribution were analyzed by Kruskal-Wallis test followed by Dunnett’s post-hoc test for multiple comparisons. p < 0.05 was regarded as statistically significant.

Fluoxetine increased sucrose consumption in SCT (Figure 2A) and locomotor activity in OFT (Figure 2B) as well as reduced despair behavior in FST (Figure 2C) of corticosterone-induced depressive rats. To investigate whether fluoxetine influenced brain glucose metabolism in this rat model of depression, we used ¹⁸F-FDG PET assay and found that fluoxetine increased SUV of ¹⁸F-FDG in frontal association cortex (FAC), medial prefrontal cortex (mPFC), hippocampus and hypothalamus in this animal model, showing the enhancement of brain glucose metabolism, especially in mPFC (Figure 2D; Supplementary Table S2). The obvious recovery of corticosterone-induced rat PFC glucose hypometabolism by fluoxetine was consistent with its antidepressant effects.

Next, fluoxetine was found to increase GLUT1 plasma membrane (PM) translocation in PFC of depressive rats induced by repeated corticosterone injection (Figure 3A). Furthermore, fluoxetine significantly upregulated PM GLUT1 level of GLAST + astrocytes isolated from PFC of this animal model (Figure 3B). Being consistent with the stimulation of GLUT1 PM translocation, fluoxetine increased 2-NBDG uptake of astrocytes (SR101 + cells) in ex vivo cultured PFC slices with corticosterone exposure (Figure 3C). Fluoxetine also promoted glycolytic rate (Figure 3D) and enhanced astrocytic lactate release (Figure 3E) in PFC astrocytes isolated from this animal model.

Subsequently, fluoxetine-treated primary astrocytes isolated from newborn rats were directly used to study the effect on astrocytic glucose metabolism under stress-level corticosterone condition in vitro. Fluoxetine enhanced primary astrocyte PM GLUT1 translocation (Figure 4A), 2-NBDG uptake (Figures 4B,C ), 2DG6P uptake (Figure 4D), glucose uptake (Figure 4E), glycolytic rate (Figure 4F) and lactate release (Figure 4G) compared with corticosterone-treated primary astrocytes. In Figures 3D,E, primary astrocytes were isolated from PFC of depressive rats induced by repeated corticosterone injection after behavioral tests. In Figure 4, primary astrocytes were isolated from PFC of newborn rat and treated with corticosterone in vitro. Because of the difference of cell age and cell density, the glycoPER vale in Figures 3D, 4F was different. But, both the in vivo (Figure 3D) and in vitro (Figure 4F) data showed that fluoxetine increased astrocytic glycolysis. These data indicated that fluoxetine increased astrocytic PM GLUT1 translocation, and subsequent glucose uptake and glycolysis, resulting in recovery of astrocytic glucose hypometabolism in corticosterone-induced depressive condition.

Immunoprecipitation assay showed that TXNIP could interact with GLUT1 in normal primary astrocytes isolated from newborn rats (Supplementary Figure S3). Therefore, we analyzed the effect of fluoxetine on astrocytic TXNIP level in vivo and in vitro. Fluoxetine suppressed TXNIP mRNA (Figure 5A) and protein (Figure 5B) levels in PFC of depressive rats induced by repeated corticosterone injection. Furthermore, it reduced PFC astrocytic (GFAP+) TXNIP levels in this animal model (Figure 5C). Similarly, fluoxetine reduced TXNIP mRNA (Figure 5D) and protein (Figure 5E) levels in cultured primary astrocytes exposed to stress-level corticosterone.

As fluoxetine suppressed astrocytic TXNIP both in mRNA and protein levels, we speculated that fluoxetine reduced corticosterone-induced TXNIP transcription. To confirm this, we first developed a stable TXNIP-promoter-driven luciferase reporter cell line (U87MG), named A9433 cells. In A9433 cells, TXNIP promoter was connected with a luciferase reporter (Figure 5F). The inhibitory potential of fluoxetine on TXNIP transcription was assessed, and its significant decrease of luciferase activity was observed in corticosterone-exposed A9433 cells. Fluoxetine significantly inhibited corticosterone-induced TXNIP transcription in A9433 cells (IC₅₀ = 13.6 μM) (Figure 5G). In this experiment, a reported TXNIP transcriptional inhibitor verapamil (Xu et al., 2012; Harper et al., 2019), presented IC₅₀ value of 125.7 μM (Figure 5H).

Of note, verapamil suppressed TXNIP transcription in A9433 cells with or without corticosterone stimulation, whereas, fluoxetine inhibited TXNIP transcription only in corticosterone stimulated-A9433 cells (Figures 6A,B). Fluoxetine showed no effect on TXNIP mRNA and protein levels of astrocytes isolated from newborn rats without corticosterone stimulation (Figures 6C,D). Thus, fluoxetine may stimulate PFC astrocytic glucose uptake and glycolysis in depressive rats via inhibiting corticosterone-induced TXNIP transcription.

Furthermore, we explored whether fluoxetine suppressed TXNIP transcription via GR. Fluoxetine was found to reduce GR nuclear translocation in corticosterone-exposed primary astrocytes in vitro (Figures 6E,F). Similarly, fluoxetine suppressed astrocytic (GFAP+) GR nuclear translocation in PFC of depressive rats induced by repeated corticosterone injection (Figures 6G,H). In fact, RU486 (a GR antagonist) inhibited corticosterone-induced TXNIP transcription in A9433 cells (Figure 7A), and TXNIP overexpression in primary astrocytes isolated from newborn rats (Figures 7B,C). Consistently, RU486 significantly promoted GLUT1 PM translocation (Figure 7D), 2-NBDG uptake (Figures 7E,F), glucose uptake (Figure 7G), glycolytic rate (Figure 7H) and extracellular lactate level in astrocytes exposed to corticosterone (Figure 7I). These data suggested that inhibition of GR could abolish the effect of corticosterone on TXNIP transcription and glucose metabolism of astrocytes. Thus, fluoxetine suppressed astrocytic TXNIP overexpression possibly via inhibiting GR nuclear translocation in corticosterone-induced depression.