Figure 3. Metformin regulates AMPD2 activity in human hepatocytes.

A) AMPD2 is the main isoform in HepG2 cells. The expression of AMPD1 and AMPD3 isoforms is minimal compred to AMPD2. Skeletal muscle and spleen are positive controls for AMPD1 and AMPD3 expression, respectively. Quantitative PCR analysis (bottom) demonstrates that isoform 2 is the predominant isoform of AMPD2. **p<0.01 versus isoforms 1 and 3. B) Over-expression of AMPD2 down-regulates the activation of AMPK. Transduction of HepG2 cells with lentiviral particles codifying for the isoform 2 of AMPD2 results in significantly higher levels of AMPD2 protein expression as well as AMPD activity. This is paralleled with reduced levels Thr172 pAMPK expression as well as of their target genes ECH1 and Ser79 pACC. *p<0.05 C–D) Reduction in ECH1 expression in over-expressing AMPD2 hepatocytes is accompanied with lower intracellular β-hydroxybutyrate and higher TG levels. *p<0.05, **p<0.01 E) Over-expression of AMPD2 impairs metformin-induced fat oxidation. Metformin 10 µM significantly increased β- hydroxybutyrate levels in cells transducted with scramble RNA. In contrast, no significant change was observed with 10 µM metformin in cells overexpressing AMPD2. Metformin 50 µM significantly increased β- hydroxybutyrate to levels observed in scramble transducted cells *p<0.05, **p<0.01, ***p<0.001.