Materials and methods Animals and experimental procedures Experimental procedures used 3-month-old female AZD0156 Wistar rats (Charles River Laboratories, Inc., Margate, UK) and 3-month-old female mice that were in a mixed C57BL/6-129Sv genetic background. These mice were bred in our animal facilities and housed in groups of five in polypropylene cages. Wistar rats were allowed to acclimatise for 1 week after transport before the start of experiments and were housed individually. Both rats and mice were subjected to a 12 h light/dark cycle with room temperature maintained at 21 °C. For mice, metformin (Sigma-Aldrich Company Ltd, Dorset, UK) was given by gavage

100 mg/kg/daily. For rats, metformin was

given in the drinking water at a concentration of 2 mg/ml for 8 weeks. On average, water consumption in rats is 10–12 ml per 100 g body weight daily and metformin did not affect the drinking volume. These metformin doses were previously shown to give similar plasma concentrations in rodents than those found therapeutically in humans. The drinking water, along with food, was available ad libitum. The water bottles were replenished twice a week. All animal experimentation procedures were in compliance with Home Office approval and were LY2835219 in vivo performed under the threshold of the UK Animals (Scientific Procedures) Act 1986. Effect of metformin on bone mass in ovariectomised mice about The first experiment was designed to investigate whether metformin could protect against the bone loss induced by ovariectomy. Eighteen female C57BL/6-129Sv mice aged 3 months were all ovariectomised, as previously performed by us [22, 23]. Four weeks after ovariectomy, mice were divided randomly into two groups, one (n = 9) receiving saline while the other one (n = 9) receiving metformin

(100 mg/kg) daily by gavage for 4 weeks. At days 6 and 3 prior to euthanasia, mice were intraperitoneally injected with calcein (Sigma-Aldrich) and alizarin red complexone (Sigma-Aldrich), respectively, to label bone-forming surfaces in trabecular bone. At the end of the experiment, mice were sacrificed, the serum collected for measurement of metformin concentration, the tibia dissected for micro-CT analysis of cortical and trabecular bone parameters and bone histomorphometry while the femora were used for protein isolation and RT–PCR analysis. Since we did not have a SHAM group, the EPZ5676 success of ovariectomy was evaluated by uterine atrophy observations during dissection. Effect of metformin on bone mass and fracture healing in rats The second experiment was designed to investigate the effect of metformin on basal bone mass. For this study, we used the right contra-lateral tibia of non-ovariectomised female rats which underwent a fracture in the left femur.