Extended Data Figure 6.

Effect of metformin and knockdown of mGPD by siRNA on glucose production from various substrates in primary hepatocytes, and metformin-mediated increase in glycerol-3-phosphate concentrations [G-3-P]. (a) Metformin treatment (100 μM) and siRNA knockdown of mGPD in rat primary hepatocytes inhibited glucose production at higher [lactate]:[pyruvate] ratios, but lower at redox state induced by decreased [lactate]:[pyruvate] abrogated the ability of metformin and mGPD knockdown by siRNA to decrease glucose production. Decreasing redox state itself inhibited glucose production. (b) Metformin only inhibited glucose production from lactate and glycerol, but not from substrates that do not increase cytosolic redox state. (c) mGPD knockdown by siRNA showed a similar substrate-selective inhibition of glucose production. (d) Both metformin and mGPD siRNA treatment increased [G-3-P] levels in hepatocytes, and (e) acute metformin (50 mg/kg, IV) treatment in vivo increased liver [G-3-P] levels without significantly altering [G-3-P] levels in other tissues, suggesting an impasse at the mGPD catalytic step. (f) siRNA treatment did not induce cytotoxicity as determined by trypan blue exclusion, (g) CyQuant proliferation assay and (h) the absence of cytochrome c release into the cytosolic fraction from mitochondria of treated cells. Data are mean ± SEM, (n=5 technical replicates, n=3 for cytotoxicity tests (f–h)). * P<0.05, **P<0.01, ***P<0.001 by unpaired t-test.