Fig. 1

MKRN1 depletion stimulates glucose metabolism by activating AMPK signalling. Analysis of wild-type (WT) or MKRN1-knockout (MK1^−/−) littermate primary mouse embryonic fibroblasts (MEFs) and HepG2 cells transduced with two independent siRNAs targeting MKRN1 (siMK1 #6 and siMK1 #7) or a control siRNA for 48 h. a Glucose consumption in MK1^−/− MEFs (left) or MKRN1-depleted HepG2 cells (right) was measured based on the absorption of 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-d-glucose (2-NBDG) by the cells. b, c Intracellular levels of glycolytic and citric acid cycle intermediates were determined by capillary electrophoresis–time-of-flight mass spectrometry of the MEFs. Each bar represents the relative amount of a metabolite for WT MEFs. G6P glucose-6-phosphate, F6P fructose-6-phosphate, F1,6-BP fructose-1,6-bisphosphate, G3P glucose-3-phosphate, 1,3-BG 1,3-bisphosphoglycerate, 3PG 3-phosphoglycerate, 2PG 2-phosphoglycerate, AcCoA acetyl-CoA, α-KG α-ketoglutarate. d, f To validate the AMPK signalling pathway or mRNA levels of AMPKα subunits, immunoblotting or quantitative real-time PCR analysis was performed using MEFs (d) and HepG2 cells (f). Cell lysates were immunoblotted with antibodies against pAMPKα, AMPKα, pACC, ACC, MKRN1 and actin. e The mRNA levels of glycolytic or lipogenic enzymes in MEFs were analysed by quantitative real-time PCR. g, h FFA-induced steatosis in HepG2 cells. Oil Red O staining (g) and TG levels (scale bar = 100 µm (top), 50 µm (bottom) (h) of the cells treated with FFA/FFA-free bovine serum albumin (BSA), which served as controls. All the experiments with MEFs were conducted in cells within the first 3–6 passages. i Fatty acid oxidation was analysed in MKRN1-depleted HepG2 cells. j The basal oxygen consumption rate (OCR) was measured in WT and MK1^−/− MEFs. k After sequential treatment with oligomycin, FCCP and rotenone/antimycin-A in the presence of BSA or BSA conjugated to palmitate, OCR was measured in WT or MK1^−/− MEFs. The results were normalised against total protein levels using XF-Analyze. All data are presented as the mean ± standard deviation (s.d.) of triplicate samples and are representative of at least three independent experiments. two-tailed Student’s t-test; ^*P ≤ 0.05, ^**P ≤ 0.01, ^***P ≤ 0.001