Phr1084

Wall-climbing 3rd instar larvae were subjected to an electric shock to induce seizure, with or without previous feeding of drug, as described previously (Marley and Baines, 2011 (link)). For drug feeding studies, eggs were laid on food containing drug and larvae were raised until wall-climbing 3rd instar. Concentration of the drugs used are as follows and the most effective concentration shown in Table 2 is underlined: SB203580 (2.6 and 13 µM, S1076, Selleckchem), Losmapimod (2.6, 13 and 26 µM, S7215, Selleckchem), sodium fluoride (1.2 and 2.4 mM, S7920, Sigma-Aldrich), gemcitabine (3.3, 16.5 and 165 µM, G6423, Sigma-Aldrich), Metformin (1.2, 2.4 and 3.6 mM, PHR1084, Sigma-Aldrich), bestatin (81 and 162 µM, J61106.MC, Alfa Aesar), WP1066 (140 and 281 µM, 573097, Merck), valproic acid (0.6, 1.2 and 2.4 mM, P4543, Sigma-Aldrich) and phenytoin (1.6 mM, D4505, Sigma-Aldrich). In response to electroshock, larvae undergo a transitory paralysis during which they tonically contract and, occasionally, spasm (see (Marley and Baines, 2011 (link)) for details on seizure behavior). Recovery time reported represents the average time for larvae to resume normal crawling behaviour and at least 30 larvae were tested for each chemical inhibitor treatment.

For the mitochondrial morphology assay, WT D1 mothers were treated with 5 μM rotenone (TargetMol, T-2970) and 2 μM CNB dye for 12 h. Offspring were collected for imaging at the embryonic, L2 and L4 stages on rotenone and CNB-free plates. The images of hypodermal mitochondria of L2s were taken with an Axiocam 702 mono camera on a Zeiss LSM800 (Airyscan) confocal microscope using a 63× oil objective. For body length measurement, WT D1 mothers were treated with 5 μM rotenone or 50 mM metformin (Sigma, PHR1084) for 12 h, and embryos were produced on rotenone and metformin-free NGM plates. The maximal adult body lengths of tested animals were measured and quantified.
For the mitochondrial recovery assay, WT OD5 animals were treated with 0.5 mM or 1 mM AICAR (Selleck, S1802) mixed with 2 μM CNB dye from embryos to the L2 stage. The images of hypodermal mitochondria of L2s were taken with an Axiocam 702 mono camera on a Zeiss LSM800 (Airyscan) confocal microscope using a 63× oil objective. For the body length assay, WT OD2 animals were treated with 1 mM AICAR from the embryonic to young adult stages (egg laying start) and then transferred to AICAR-free plates. The maximal adult body lengths of tested animals were measured and quantified.

Eight-week-old female C57BL/6 J mice were purchased from Hunan Stryker Jingda Animal Co., Ltd. Mice were housed in a specific pathogen-free environment under controlled temperature (20–23 °C) and light conditions (12-h dark/light cycle), with a normal chow diet and water provided ad libitum. After one week of adaptation, the 24 individuals were randomly divided into four groups: (1) daily gavage of 10% DMSO as the control group (n = 6); (2) olanzapine-treated mice receiving olanzapine (LY170053, MCE, USA) daily at 6 mg/kg body weight (n = 6); (3) metformin-treated mice receiving metformin (PHR1084, Sigma, USA) daily at 100 mg/kg body weight; and (4) combination therapy with 6 mg/kg body weight olanzapine and 100 mg/kg body weight metformin daily. Olanzapine was prepared as described previously^{18 (link)}, and metformin was dissolved in phosphate-buffered saline (PBS). The weight of mice was monitored weekly.
After 8 weeks of treatment based on groups, mice were fasted overnight, weighed, and euthanized via prompt dislocation of the neck vertebra under anesthesia. The liver was quickly dissected and rinsed with normal saline solution. Liver sections were fixed with 4% paraformaldehyde and embedded in paraffin for histological examination. The rest of the tissue was snap frozen in liquid nitrogen and stored at − 80 °C for analysis of gene/protein expression.

The methods used for the generation of Vav2^Onc/Onc knock-in (C57BL/6 background), Vav2^L332A/L332A knock-in (mixed genetic background), and Vav2^−/− knockout (C57BL/10 genetic background) mice have been described in previous publications^{37 (link),41 (link),76 (link)}. These mice and genetic background-matched WT controls were housed with an artificial 12-h light /12-h dark cycle under controlled temperature (23 °C) and humidity (50%) conditions. They were routinely maintained under ad libitum access to a standard chow global diet (Cat. #2018; Teklad global 18% protein) and tap water. When indicated, 8-week-old mice were shifted to either an HFD (45% fat, 4.73 kcal g⁻¹; Cat. #D12451; Research Diets) or a metformin regimen (2 mg mL⁻¹ in the drinking water; Cat. #PHR1084, Sigma-Aldrich) for 4 months before being euthanized. Endoplasmic reticulum stress was induced in the liver by injecting tunicamycin (2 μg kg⁻¹; Cat. #T7765, Sigma-Aldrich) intraperitoneally to 2-month-old mice. When indicated, 8-week-old mice were shifted to a methionine- and choline-deficient diet (Cat. #A02082002B, Research Diets) for the indicated period of time before being sacrificed. The experiments involving the in vivo infusion of insulin and IGF1 were performed indistinctly in age-matched male and female animals. The rest of the experiments were done using males.

Male C57BL/6 mice were obtained from Chongqing Medical University. To induce hepatic carcinogenesis, 4-week-old C57BL/6 male mice received 75 mg/kg DEN (Sigma-Aldrich, N0258) in two divided doses and CCL4 (mixed with olive oil in a 1:4 ratio, 0.08 ml/10 g) twice a week until 20 weeks. After 8 weeks, fresh liver tissues were obtained through liver biopsy. Immunohistochemical staining was used to detect the level of AMPK phosphorylation. The same criteria with human tissue microarrays were adopted to distinguish the level of p-AMPK expression. In mice with p-AMPK high expression, dorsomorphin (Abcam, ab120843, 0.2 mg/kg d) was used to inhibit p-AMPK, or PBS as a control. In mice with p-AMPK low expression, metformin (Sigma, PHR1084, 250 mg/kg d) or AICAR (Sigma, A9978, 2.5 mg/kg d) were used to activate AMPK. For autophagy inhibition, mice received 50 mg/kg of chloroquine (Santa Cruz Biotechnology, sc-205629) via intraperitoneal injection once every 3 days along with a concurrent application of metformin. All mice were killed at 32 weeks. All animal experiments complied with the ARRIVE guidelines and was carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines.