Mice lacking AMP-activated protein kinase α1 catalytic subunit have increased bone remodelling and modified skeletal responses to hormonal challenges induced by ovariectomy and intermittent PTH treatment

Abstract

AMP-activated protein kinase (AMPK) is a key regulator of cellular and body energy homeostasis. We previously demonstrated that AMPK activation in osteoblasts increases in vitro bone formation while deletion of the Ampkα1 (Prkaa1) subunit, the dominant catalytic subunit expressed in bone, leads to decreased bone mass in vivo. To investigate the cause of low bone mass in the Ampkα1(-/-) mice, we analysed bone formation and resorption in the tibia of these mice by dynamic histomorphometry and determined whether bone turnover can be stimulated in the absence of the Ampkα1 subunit. We subjected 12-week-old Ampkα1(+)(/)(+) and Ampkα1(-/-) mice to ovariectomy (OVX), intermittent PTH (iPTH) administration (80 μg/kg per day, 5 days/week) or both OVX and iPTH hormonal challenges. Tibiae were harvested from these mice and bone micro-architecture was determined by micro-computed tomography. We show for the first time that Ampkα1(-/-) mice have a high bone turnover at the basal level in favour of bone resorption. While both Ampkα1(+)(/)(+) and Ampkα1(-/-) mice lost bone mass after OVX, the bone loss in Ampkα1(-/-) mice was lower compared with controls. iPTH increased trabecular and cortical bone indexes in both ovariectomised Ampkα1(+)(/)(+) and Ampkα1(-/-) mice. However, ovariectomised Ampkα1(-/-) mice showed a smaller increase in bone parameters in response to iPTH compared with Ampkα1(+)(/)(+) mice. By contrast, non-ovariectomised Ampkα1(-/-) mice responded better to iPTH treatment than non-ovariectomised Ampkα1(+)(/)(+) mice. Overall, these data demonstrate that Ampkα1(-/-) mice are less affected by changes in bone turnover induced by OVX but respond better to the anabolic challenge induced by iPTH. These results suggest that AMPKα1 activation may play a role in the hormonal regulation of bone remodelling.

Figure 1

Effect of ovariectomy (OVX) on trabecular and cortical bone parameters in tibia of Ampkα1^+/+ and Ampkα1^−/− mice. (A) Trabecular bone parameters in Ampkα1^+/+ and Ampkα1^−/− mice that have undergone OVX or sham operation. (B) Cortical bone parameters in Ampkα1^+/+ and Ampkα1^−/− mice subjected to OVX or sham operation. Values are mean±s.e.m. of n=7 mice/group, *P<0·05, **P<0·01.

Figure 2

Dynamic bone histomorphometry analyses in Ampkα1^+/+ and Ampkα1^−/− mice. (A) BFR, (B) percentage of TRAP surfaces and (C) number of adipocytes per tissue area in 16-week-old Ampkα1^+/+ and Ampkα1^−/− mice. Values are mean±s.e.m. of n=7 mice/group, **P<0·01.

Figure 3

Effect of 4 weeks of iPTH treatment on trabecular and cortical bone parameters in tibia of ovariectomised Ampkα1^+/+ and Ampkα1^−/− mice. (A) Trabecular bone parameters in ovariectomised Ampkα1^+/+ (OVX–Ampkα1^+/+) and Ampkα1^−/− (OVX–Ampkα1^−/−) mice that have been treated with iPTH or saline. (B) Cortical bone parameters in OVX–Ampkα1^+/+ and OVX–Ampkα1^−/− mice subjected to iPTH or saline treatment. Values are mean±s.e.m. of n=8 mice/group, *P<0·05, **P<0·01, ***P<0·001.

Figure 4

Effect of 4 weeks of iPTH treatment on trabecular and cortical bone parameters in tibia of Ampkα1^+/+ and Ampkα1^−/− mice. (A) Trabecular bone parameters in Ampkα1^+/+ and Ampkα1^−/− mice that have been treated with either iPTH or saline for 4 weeks. (B) Cortical bone parameters in Ampkα1^+/+ and Ampkα1^−/− mice treated with iPTH or saline for 4 weeks. Values are mean±s.e.m. of n=10–11 mice/group, *P<0·05, ***P<0·001.

Figure 5

Effect of iPTH treatment on AMPKα1/2 phosphorylation in bone of ovariectomised Ampkα1^+/+ and Ampkα1^−/− mice. (A, i) Western blot analysis of pAMPKα1/2 and tAMPKα1/2. Proteins were extracted from femora of 16-week-old OVX–Ampkα1^+/+ and OVX–Ampkα1^−/− mice from study 2 and probed with polyclonal antibodies directed against pAMPKα1/2, tAMPKα1/2 and β-actin. Representative immunoblots are shown, which were repeated three times with similar results. (ii) Graph showing the ratio of pAMPK α1/2 to tAMPKα1/2 relative to β-actin determined by densitometry analysis of western blot data using Image J Software. Proteins were extracted from femora of two mice per group and western blot analyses were carried out in triplicates. Values are mean±s.e.m., *P<0·05. (B) RT-PCR analysis of RNA extracted from femora of Ampkα1^+/+ and Ampkα1^−/− mice showing differential subunit expression pattern. Expression pattern of AMPK subunits in femoral muscle from Ampkα1^+/+ mice was carried as a control.