b, ii Blots were quantified using image J and the pAMPK to tAMPK ratio relative to β-actin was determined for each experiment. Bars represent mean ± SD, n = 4 biological samples. c Results show expression of click here Osterix and runt related transcription factor 2 (Runx2) normalised to 18s rRNA in femora of saline and metformin groups after 1 month of treatment. Two separate RNA extractions were performed for each treatment group, each time RNA being pooled
from three femora Discussion With the increasing worldwide prevalence of T2DM which predisposes patients to osteoporosis and increased risk of fractures [33, 34], there is an increasing need to evaluate the skeletal actions of anti-diabetic drugs and to examine their effects on healing of osteoporotic fractures. We show in this study that the anti-diabetic drug metformin is not ‘bone unfriendly’ but has no osteogenic action, as previously reported. In contrast, our data indicate that metformin reduces bone formation rate, has no major effect on bone mass in vivo in rodents and does not promote fracture healing. We first used ovariectomised mice to examine the skeletal effect of metformin in conditions of low bone mass that are more representative of the frequent Belnacasan secondary osteoporosis observed in T2DM patients. Our results, which show no effect of metformin on bone architecture, Luminespib contrast with two previous studies performed in ovariectomised
rats [12, 13], demonstrating that metformin inhibits the trabecular bone loss  and the decrease in bone mineral density  induced by OVX. In both studies, metformin was also administered to OVX rats by gavage at an identical concentration with the one used in our work. Although we did not perform a dose–response of metformin in our study, the concentration of metformin given orally has been extensively used in previous rodent studies [35, 36]. Our metformin treatment was efficient since plasma levels of metformin were detected with a value of approximately 0.3 mg/l. In addition, metformin induced a small
decrease in body weight in our study, a known effect of this anti-diabetic drug which promotes satiety, reducing the food intake . It is therefore difficult to reconcile our data with these previous Carteolol HCl rat studies, all the more since Gao’s study  showed similar trabecular bone mass to ours after OVX and we previously showed that same-age OVX mice on this C57BL/6-129Sv background can experience large increases in trabecular bone volume when treated with intermittent PTH . The duration of metformin treatment is unlikely to explain those differences since we treated our mice with metformin for 1 month, but our rats for 2 months, similarly to the previous rat studies. The effects of metformin on bone may however vary depending on the rodent species and strain utilised, as previously demonstrated for the skeletal effect of rosiglitazone [38, 39].