Biol. Pharm. Bull. 29(5) 1006—1009 (2006)

were first used clinically for diabetes mellitus in the 1950s. Since phenformin was found to have a lethal side effect, lactic acidosis, in the late of 1970s, its usage decreased rapidly in many countries and was finally withdrawn. Then use of other biguanides declined gradually because of their own adverse effects. Lactic acidosis, hypoglycemia, and digestive organ dysfunction have been found to be adverse effects of biguanides. Lactic acidosis caused by biguanides results in lethal metabolic acidosis, so their use was stopped. Cohen and Woods proposed a classification for lactic acidosis that consisted of types A, B1, B2, B3, and B4, and acidosis caused by biguanides belongs to type B2 according to this classification. Type B is lactic acidosis occurring when clinical evidence of poor tissue perfusion or oxygenation is absent and type B2 is due to drugs and toxins. Metformin was recently reported to decrease the risk of diabetes-related endpoints in overweight diabetic patients and, therefore, has begun to be widely used again. The following mechanisms of action of biguanides have been proposed: (i) suppression of glucose release from the liver, (ii) increase of glucose intake in peripheral tissues, (iii) decrease of glucose uptake in an alimentary tract, and (iv) suppression of b-oxidation. Metformin was found to improve resistance to insulin in type II diabetes. Zhou et al. postulated that the suppression of SREBP-1 expression through the activation of AMPactivating protein kinase by metformin eliminated the insulin resistance. The following possible mechanism for the side effect of biguanides has been proposed. The biguanide binds to the mitochondrial membrane and causes the disruption of oxidative phosphorylation in the mitochondria. This results in an increase in the NADH level, a decrease in pyruvate dehydrogenase activity, and deactivation of the TCA cycle. Furthermore, gluconeogenesis is suppressed and pyruvic acid accumulates in the liver. This leads to an increase in production of lactic acid. However, the molecular mechanisms of the side effects of biguanides are still unclear. When we researched which gene expressions underwent a change in order to clarify the mechanism of biguanides induced lactic acidosis, we found that the gene expression of glyceraldehyde 3-phosphate dehydrogenase (GAPD) in HepG2 cells decreases significantly by buformin treatment. Since GAPD is involved in the glucolytic pathway, we believe the decrease in GAPD may have caused the lactic acidosis. In this study, we demonstrate that expression of the gene for GAPD in HepG2 cells was suppressed by buformin treatment in a time-dependent manner. We also examined the effect of the biguanide on the protein expression of GAPD and determined the levels of NAD and ATP in HepG2 cells treated with the biguanide to investigate the mechanism of the lactic acidosis side effect.