Genetic deletion of catalytic subunits of AMP-activated protein kinase increases osteoclasts and reduces bone mass in young adult mice

Abstract

AMP-activated protein kinase (AMPK) is a key regulator of cellular and systemic energy homeostasis and a potential therapeutic target for the intervention of cancer and metabolic disorders. However, the role of AMPK in bone homeostasis remains incompletely understood. Here we assessed the skeletal phenotype of mice lacking catalytic subunits of AMPK and found that mice lacking AMPKα1 (Prkaa1(-/-)) or AMPKα2 (Prkaa2(-/-)) had reduced bone mass compared with the WT mice, although the reduction was less in Prkaa2(-/-) mice than in Prkaa1(-/-) mice. Static and dynamic bone histomorphometric analyses revealed that Prkaa1(-/-) mice had an elevated rate of bone remodeling because of increases in bone formation and resorption, whereas AMPKα2 KO-induced bone mass reduction was largely attributable to elevated bone resorption. In agreement with our in vivo results, AMPKα deficiency was associated with increased osteoclastogenesis in vitro. Moreover, we found that AMPKα1 inhibited the receptor activator of nuclear factor κB (RANK) signaling, providing an explanation for AMPK-mediated inhibition of osteoclastogenesis. Therefore, our findings further underscore the importance of AMPK in bone homeostasis, in particular osteoclastogenesis, in young adult mammals.

FIGURE 1.

Genetic deletion of catalytic subunit of AMPK reduces bone mineral density and mass. A–C, μCT analysis of femurs from 3-month-old male mice of Prkaa1^−/− and WT. A, representative three-dimensional reconstructed μCT images of the femoral trabecular compartment. Scale bars, 0.5 mm. B, percentage of BV/TV of Prkaa1^−/− and WT. C, apparent (App.) density of Prkaa1^−/− and WT. Hydroxyapatite (HA)-calibrated true volumetric mineral density of trabecular bone was normalized to the tissue volume of selected region of interest. The graphs were plotted with the means ± S.E., n = 7, p < 0.05 versus WT. D–F, μCT analysis of femurs from 3.5-month-old male mice of Prkaa2^−/− and WT. D, representative three-dimensional reconstructed μCT images of the femoral trabecular compartment. Scale bars, 0.5 mm. E, percentage of BV/TV of Prkaa2^−/− and WT. F, apparent density of Prkaa2^−/− and WT. The data in E and F are presented as the means ± S.E., n = 12 in each group of mice. *, p < 0.05 versus WT.

FIGURE 2.

Genetic deletion of catalytic subunit of AMPK reduces cancellous bone mass. A–E, static histomorphometry of the proximal tibiae from 3-month-old male mice of Prkaa1^−/− and WT. A, representative images of toluidine blue-stained histological tibial bone sections. Scale bars, 0.5 mm. B, percentage of trabecular BV/TV. C, trabecular bone thickness (Tb. Th.). D, trabecular number (Tb. N.). E, trabecular separation (Tb. Sp.). F–I, static histomorphometry of the proximal tibiae for 3.5-month-old male mice of Prkaa2^−/− and WT. F, percentage of trabecular BV/TV. G, trabecular bone thickness (Tb. Th.). H, trabecular number (Tb. N.). I, trabecular separation (Tb. Sp.). The data in B–I are presented as the means ± S.E., The sample size was the same as in Fig. 1. *, p < 0.05 versus WT.

FIGURE 3.

Effect of AMPKα1 deficiency on bone formation and resorption. A–C, dynamic histomorphometry of calcein double-labeled tibiae for WT and Prkaa1^−/− mice. A, representative images of calcein double-labeled trabecular bone surfaces of WT and Prkaa1^−/− mice. Scale bars, 100 μm. B, mineralizing surface per bone surface (MS/BS, %) of WT and Prkaa1^−/− mice. C, bone formation rate per bone surface (BFR/BS) of WT and Prkaa1^−/− mice. D, histomorphometric quantification of osteoblast surface per bone surface (Ob.S./BS) of WT and Prkaa1^−/− mice. E, representative microscopic images of TRAP-stained tibial bone sections from WT and Prkaa1^−/− mice. TRAP-positive osteoclasts are stained in purple on trabecular bone surface (black arrow). Scale bars, 100 μm. F, histomorphometric quantification for osteoclast number per bone perimeter (Oc.N/B.Pm) in WT and Prkaa1^−/− mice. Histomorphomeric measurement of osteoclasts was made in the proximal tibial metaphysis starting ∼0.2 mm distal from the growth plate. The horizontal axis of rectangular region of interest was maintained parallel to the growth plate. G, osteoclast surface per bone surface (Oc.S/BS, %) in WT and Prkaa1^−/− mice. *, p < 0.05 as compared with WT by t test.

FIGURE 4.

Effect of AMPKα2 deficiency on bone formation and resorption. A and B, histomorphometric quantification of osteoclasts for WT and Prkaa2^−/− mice. A, osteoclast number per bone perimeter (Oc.N/B.Pm). B, osteoclast surface per bone surface (Oc.S/BS, %). C and D, dynamic histomorphometry of calcein double-labeled WT and Prkaa2^−/− mice. C, bone formation rate per bone surface (BFR/BS) of WT and Prkaa2^−/− mice. D, representative images of calcein double-labeled trabecular bone surfaces of WT and Prkaa2^−/− mice. Scale bars, 100 μm. NS, not significant.

FIGURE 5.

Deletion of AMPK catalytic subunit augments osteoclastogenesis in vitro. A–C, bone marrow cells from WT and Prkaa1^−/− mice were cultured in the presence of M-CSF until subconfluency. Then the BMMs were cultured in the media supplemented with M-CSF and RANKL for 6 days, and multinucleate osteoclasts were stained for TRAP. A, representative images of TRAP stained osteoclasts from WT and Prkaa1^−/−. Scale bars, 100 μm. B, osteoclast number of WT and Prkaa1^−/−. C, osteoclast size of WT and Prkaa1^−/−. D and E, quantitation of osteoclasts derived from bone marrow cells from WT and Prkaa2^−/− mice. D, osteoclast number of WT and Prkaa2^−/−. E, osteoclast size of WT and Prkaa2^−/−.

FIGURE 6.

Deletion of AMPK catalytic subunit increases expression of osteoclastogenesis-associated genes in vitro. A–F, real time quantitative PCR analysis of osteoclastogenesis-associated genes. Bone marrow cells from WT and Prkaa1^−/− mice were cultured in the presence of M-CSF and RANKL. At days 0, 3, and 6, total RNA was isolated and subjected to reverse transcription and subsequent real time PCR. All of the PCRs were carried out in triplicate. The graph for each gene shows the mean values of fold changes compared with the expression level of WT at day 0. A, NFATc1. B, TRAP. C, DC-STAMP. D, Atp6v0d2. E, cathepsin K. F, CA2. G–L, real time quantitative PCR analysis of osteoclastogenesis-associated gene expression with total RNA from WT and Prkaa2^−/− osteoclast cultures. G, NFATc1. H, TRAP. I, DC-STAMP. J, Atp6v0d2. K, cathepsin K. L, CA2. *, statistically significant difference between WT and KO at the indicated time point, p < 0.05.

FIGURE 7.

AMPKα1 deficiency augments the RANK signaling in BMMs. A, BMMs from WT and Prkaa1^−/− mice were starved in serum-free culture media for 3 h before being stimulated with 100 ng/ml of RANKL. The cell lysates were resolved by SDS-PAGE and then immunoblotted for phosphorylated forms of AMPKα1/2, IKKα/β, p38MAPK, and p42/44MAPK. β-Actin served as an internal control for equal loading of proteins on each lane. B, cell lysates of BMMs from WT and Prkaa2^−/− mice were analyzed as in A for phos-IκBα and IκBα.