1Department of Physiology, Hangzhou Medical College, Hangzhou, Zhejiang, China 2Department of Pharmacy, Women's Hospital, School of Medicine, Zhejiang University, Zhejiang, China 3Department of Ophthalmology, Sir Run Run Shaw Hospital, Zhejiang University, Hangzhou, China 4Department of Neurobiology, Key Laboratory of Medical Neurobiology of Ministry of Health of China, Zhejiang University School of Medicine, Zhejiang, China 5Department of Biochemistry and Molecular Biology, Hainan Medical University, Haikou, Hainan, China
Depression is one of the most prevalent neuropsychiatric disorders in modern society. However, traditional drugs, such as monoaminergic agents, have defect showing lag response requiring several weeks to months. Additionally, these drugs have limited efficacy and high resistance rates in patients with depression. Thus, there is an urgent need to develop novel drugs or approaches for the treatment of depression. Here, using biochemical, pharmacological, genetic and behavioral methods, we demonstrate that metformin imparts a fast-acting antidepressant-like effect in naïve mice as well as stressed mice subjected to chronic restraint stress model. Moreover, inhibition of AMP-activated protein kinase (AMPK) activity by compound C or knock down of hippocampal AMPKα occluded the antidepressant-like effect induced by metformin. Our results suggest that metformin may be a viable therapeutic drug for the treatment of stress-induced depression via activation of AMPK.
Heng Ai,Weiqing Fang,Hanyi Hu, et al. Antidiabetic Drug Metformin Ameliorates Depressive-Like Behavior in Mice with Chronic Restraint Stress via Activation of AMP-Activated Protein Kinase[J]. Aging and disease,
2020, 11(1): 31-43.
Figure 1. Dose-dependent antidepressant-like effect of metformin in mice. (A) Schematic illustration of the experimental set up. (B-C) Immobility time of mice after acute treatment of various doses of metformin (intraperitoneal injection) in FST (B) and TST (C), respectively. Data are shown as the mean immobility time ± SEM. Independent groups of mice were used for each behavioral test (n = 8 mice per group). (D-E) Mice receiving different doses of metformin were subjected to the open field test (OFT). The mean distance travelled (D) and the velocity (E) were recorded (n = 8 mice per group). (F) Immunoblots of hippocampal lysate from mice treated with various doses of metformin without behavioral test. The relative levels of p-AMPK (pT172) and AMPKα were analyzed, β-actin served as a loading control. (G-H) Quantification of fold changes in pT172 (G) and AMPKα (H) levels in the hippocampus, n = 5 mice per group. One-way analysis of variance (ANOVA) with Bonferroni post-hoc analysis. *P < 0.05, **P < 0.01, #P < 0.001, n.s. represents not significant. Data are presented as mean ± SEM.
Figure 2. Time course of metformin-mediated antidepressant-like behavioral effects. After treatment with metformin (50 mg/kg, intraperitoneal injection), the immobility time of C57BL/6 mice in FST (A) and TST (B) at different time points was analyzed, respectively. Independent groups of mice were used for each behavioral test and each time point to avoid behavioral habituation (n = 7 mice per group). One-way analysis of variance (ANOVA) with Bonferroni post-hoc analysis. *P < 0.05, **P < 0.01, n.s. represents not significant. Data are presented as mean ± SEM.
Figure 3. Metformin produced rapid antidepressant-like effects and diminished the chronic restraint stress (CRS)-induced reduction of p-AMPK in the hippocampus. (A) Timeline of CRS exposure, metformin administration and behavioral test (n = 8 mice per group). (B-C) Mean immobility time ± SEM in non-stressed and stressed mice in FST (B) and TST (C). (D) Metformin prevented the decrease on the sucrose preference test (SPT) in stressed mice. E. Representative western blot of hippocampal proteins. (F-G) Statistical analysis of pT172 (F) and AMPKα (G) levels in the hippocampus, n = 5 mice per group. One-way analysis of variance (ANOVA) with Bonferroni post-hoc analysis. *P < 0.05, **P < 0.01, n.s. represents not significant. Data are presented as mean ± SEM.
Figure 4. Inhibition of AMPK by compound C prevented the metformin-induced antidepressant-like effect in stressed mice. Compound C (CC) attenuated the ability of metformin to reduce the immobility time of mice in FST (A) and TST (B). (C) CC abolished the metformin-induced elevation of sucrose preference in SPT as compared with metformin-treated stressed mice (n = 8 mice per group for all behavioral tests). (D) Representative western blot of hippocampal proteins in non-stressed or stressed mice treated with metformin or CC plus metformin. Quantification of p-AMPK at T172 (E) and AMPK (F), n = 5 mice per group. One-way ANOVA with Bonferroni post-hoc analysis. #P < 0.001, n.s. represents not significant. Data are presented as mean ± SEM.
Figure 5. Adeno-associated virus (AAV)-mediated knock down (KD) of AMPKα in the hippocampus occluded the antidepressant-like effect in stressed mice. (A) Schematic showing the experimental design and targeting strategy for knock down AMPKα. The eGFP (enhanced green fluorescent protein) was exploited to visualize the infection of the virus and an H1 promoter was used to drive the expression of shRNA. (B) Representative images of coronal slice with intensive GFP signal, indicating successful AAV infection in the hippocampus, scale bar = 300μm. (C-D) KD of AMPKα in the hippocampus hampered the metformin-induced elevation in struggling time in FST (C) and TST (D) of stressed mice, n = 7-8 mice per group. (E) KD of AMPKα in the hippocampus occluded the increased sucrose preference in SPT induced by metformin in stressed mice. (F) Representative blots of hippocampal proteins in AAV-NC- and AAV-shRNA-injected mice after CRS without behavior test. (G-H) Statistical analysis of the pT172 (G) and AMPKα (H) levels in the hippocampus, n = 5 mice per group. The results were analyzed using a two-way ANOVA with Bonferroni post hoc analysis. *P < 0.05, **P < 0.01, n.s. represents not significant. Data are presented as mean ± SEM.
Figure 6. Summary of our findings. Mice subjected to chronic restraint stress (CRS) that did or did not receive metformin show distinct behavior. Metformin reversed the reduction of p-AMPK (pT172) level in the hippocampus of mice subjected to CRS, thereby alleviating the depressive-like behavior caused by CRS.
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