Metformin is an oral anti-diabetic drug of the biguanide class that is commonly 
used to treat type 2 diabetes mellitus. This study examined the molecular 
mechanism for the action of metformin on osteoblast differentiation. 
Metformin-induced mRNA expression of the osteogenic genes and small heterodimer 
partner (SHP) in MC3T3E1 cells were determined by RT-PCR and real-time PCR. 
Metformin increased significantly the expression of the key osteogenic genes, 
such as alkaline phosphatase (ALP), osteocalcin (OC) and bone sialoprotein (BSP) 
as well as SHP. Transient transfection assays were performed in MC3T3E1 cells to 
confirm the effects of metformin on SHP, OC and Runx2 promoter activities. 
Metformin increased the transcription of the SHP and OC genes, and the metformin 
effect was inhibited by dominant negative form of AMPK (DN-AMPK) or compound C 
(an inhibitor of AMPK). The adenoviral overexpression of SHP increased 
significantly the level of ALP staining and OC production. However, metformin 
did not have any significant effect on osteogenic gene expression, ALP staining 
and activity, and OC production in SHP null (SHP-/-) primary calvarial cells. 
Moreover, upstream stimulatory factor-1 (USF-1) specifically mediated 
metformin-induced SHP gene expression. In addition, metformin-induced AMPK 
activation increased the level of Runx2 mRNA and protein. However, USF-1 and SHP 
were not involved in metformin-induced Runx2 expression. Transient transfection 
and chromatin immunoprecipitation assays confirmed that metformin-induced SHP 
interacts physically and forms a complex with Runx2 on the osteocalcin gene 
promoter in MC3T3E1 cells. These results suggest that metformin may stimulate 
osteoblast differentiation through the transactivation of Runx2 via 
AMPK/USF-1/SHP regulatory cascade in mouse calvaria-derived cells.