Activation of the AMPK/Sirt1 pathway by a leucine-metformin combination increases insulin sensitivity in skeletal muscle, and stimulates glucose and lipid metabolism and increases life span in Caenorhabditis elegans

Background. We have previously shown leucine (Leu) to activate Sirt1 by lowering its KM for NAD(+), thereby amplifying the effects of other sirtuin activators and improving insulin sensitivity. Metformin (Met) converges on this pathway both indirectly (via AMPK) and by direct activation of Sirt1, and we recently found Leu to synergize with Met to improve insulin sensitivity and glycemic control while achieving similar to 80% dose-reduction in diet-induced obese mice. Accordingly, we sought here to define the mechanism of this interaction. Methods. Muscle cells C2C12 and liver cells HepG2 were used to test the effect of Met-Leu on Sirt1 activation. Caenorhabditis elegans was used for glucose utilization and life span studies. Results. Leu (0.5 mmol/L) + Met (50-100 gmol/L) synergistically activated Sirt1 (p < 0.001) at low (<= 100 mu mol/L) NAD(+) levels while Met exerted no independent effect. This was associated with an increase in AMPK and ACC, phosphorylation, and increased fatty acid oxidation, which was prevented by AMPK or Sift inhibition or silencing. Met-Leu also increased P-IRS1/IRS1 and P-AKT/AKT and in insulin-independent glucose disposal in myotubes (similar to 50%, p < 0.002) evident within 30 min as well as a 60% reduction in insulin EC50. In addition, in HepG2 liver cells nuclear CREB regulated transcription coactivator 2 (CRTC2) protein expression and phosphorylation of glycogen synthase was decreased, while glycogen synthase kinase phosphorylation was increased indicating decreased gluconeogenesis and glycogen synthesis. We utilized C. elegans to assess the metabolic consequences of this interaction. Exposure to high glucose impaired glucose utilization and shortened life span by similar to 25%, while addition of Leu + Met to high glucose worms increased median and maximal life span by 29 and 15%, respectively (p = 0.023), restored normal glucose utilization and increased fat oxidation-two-fold (p < 0.005), while metformin exerted no independent effect at any concentration (0.1-0.5 mmol/L). Conclusion. Thus, Leu and Met synergize to enable Sirt1 activation at low NAD(+) concentrations (typical of energy replete states). Sirt1 and AMPK activations are required for Met-Leu's full action, which result in improvements in energy metabolism and insulin sensitivity. (C) 2016 Elsevier Inc. All rights reserved.