Metformin suppresses hepatic gluconeogenesis and lowers fasting blood glucose levels through reactive nitrogen species in mice.
OBJECTIVE
Metformin, the major target of which is liver, is commonly used to treat type 2 diabetes. Although metformin activates AMP-activated protein kinase (AMPK) in hepatocytes, the mechanism of activation is still not well known. To investigate AMPK activation by metformin in liver, we examined the role of reactive nitrogen species (RNS) in suppression of hepatic gluconeogenesis.


METHODS
To determine RNS, we performed fluorescence examination and immunocytochemical staining in mouse hepatocytes. Since metformin is a mild mitochondrial complex I inhibitor, we compared its effects on suppression of gluconeogenesis, AMPK activation and generation of the RNS peroxynitrite (ONOO(-)) with those of rotenone, a representative complex I inhibitor. To determine whether endogenous nitric oxide production is required for ONOO(-) generation and metformin action, we used mice lacking endothelial nitric oxide synthase (eNOS).


RESULTS
Metformin and rotenone significantly decreased gluconeogenesis and increased phosphorylation of AMPK in wild-type mouse hepatocytes. However, unlike rotenone, metformin did not increase the AMP/ATP ratio. It did, however, increase ONOO(-) generation, whereas rotenone did not. Exposure of eNOS-deficient hepatocytes to metformin did not suppress gluconeogenesis, activate AMPK or increase ONOO(-) generation. Furthermore, metformin lowered fasting blood glucose levels in wild-type diabetic mice, but not in eNOS-deficient diabetic mice.


CONCLUSIONS
Activation of AMPK by metformin is dependent on ONOO(-). For metformin action in liver, intra-hepatocellular eNOS is required.
Fujita
Y
Y
Department of Diabetes and Clinical Nutrition, Graduate School of Medicine, Kyoto University, 54 Shogoin, Kawahara-cho, Sakyo-ku, Kyoto, 606-8507, Japan.
Hosokawa
M
M
Fujimoto
S
S
Mukai
E
E
Abudukadier
A
A
Obara
A
A
Ogura
M
M
Nakamura
Y
Y
Toyoda
K
K
Nagashima
K
K
Seino
Y
Y
Inagaki
N
N
D000818:Animals; D001786:Blood Glucose / Q000187:drug effects*; D002478:Cells, Cultured; D005943:Gluconeogenesis / Q000187:drug effects*; D022781:Hepatocytes / Q000187:drug effects* / Q000378:metabolism*; D007004:Hypoglycemic Agents / Q000494:pharmacology*; D015151:Immunoblotting; D007150:Immunohistochemistry; D008297:Male; D008687:Metformin / Q000494:pharmacology*; D051379:Mice; D008810:Mice, Inbred C57BL; D026361:Reactive Nitrogen Species / Q000378:metabolism*
D001786:Blood Glucose; D007004:Hypoglycemic Agents; D026361:Reactive Nitrogen Species; D008687:Metformin