AMP-activated protein kinase (AMPK) activation regulates in vitro bone formation and bone mass

• Published in: Bone (New York, N.Y.) N.Y.), 2010-08, Vol.47 (2), p.309-319
• Main Authors: Shah, M, Kola, B, Bataveljic, A, Arnett, T.R, Viollet, B, Saxon, L, Korbonits, M, Chenu, C
• Format: Article
• Language: English
• Subjects: Alkaline Phosphatase - metabolism; Aminoimidazole Carboxamide - analogs & derivatives; Aminoimidazole Carboxamide - pharmacology; AMP-Activated Protein Kinases - deficiency; AMP-Activated Protein Kinases - genetics; AMP-Activated Protein Kinases - metabolism; Animals; Biological and medical sciences; Bone and Bones - cytology; Bone and Bones - drug effects; Bone and Bones - enzymology; Cell Differentiation - drug effects; Cell Proliferation - drug effects; Enzyme Activation - drug effects; Enzyme Activators - pharmacology; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation, Enzymologic - drug effects; Metformin - pharmacology; Mice; Mice, Knockout; Neurosecretory Systems - enzymology; Organ Size - drug effects; Orthopedics; Osteoblasts - cytology; Osteoblasts - drug effects; Osteoblasts - enzymology; Osteogenesis - drug effects; Osteogenesis - physiology; Phenotype; Protein Subunits - deficiency; Protein Subunits - genetics; Protein Subunits - metabolism; Rats; Ribonucleotides - pharmacology; RNA, Messenger - genetics; RNA, Messenger - metabolism; Tibia - drug effects; Tibia - enzymology; Vertebrates: anatomy and physiology, studies on body, several organs or systems
• ISSN: 8756-3282; 1873-2763
• DOI: 10.1016/j.bone.2010.04.596

Abstract Adenosine 5′-monophosphate-activated protein kinase (AMPK), a regulator of energy homeostasis, has a central role in mediating the appetite-modulating and metabolic effects of many hormones and antidiabetic drugs metformin and glitazones. The objective of this study was to determine if AMPK can be activated in osteoblasts by known AMPK modulators and if AMPK activity is involved in osteoblast function in vitro and regulation of bone mass in vivo. ROS 17/2.8 rat osteoblast-like cells were cultured in the presence of AMPK activators (AICAR and metformin), AMPK inhibitor (compound C), the gastric peptide hormone ghrelin and the beta-adrenergic blocker propranolol. AMPK activity was measured in cell lysates by a functional kinase assay and AMPK protein phosphorylation was studied by Western Blotting using an antibody recognizing AMPK Thr-172 residue. We demonstrated that treatment of ROS 17/2.8 cells with AICAR and metformin stimulates Thr-172 phosphorylation of AMPK and dose-dependently increases its activity. In contrast, treatment of ROS 17/2.8 cells with compound C inhibited AMPK phosphorylation. Ghrelin and propranolol dose-dependently increased AMPK phosphorylation and activity. Cell proliferation and alkaline phosphatase activity were not affected by metformin treatment while AICAR significantly inhibited ROS 17/2.8 cell proliferation and alkaline phosphatase activity at high concentrations. To study the effect of AMPK activation on bone formation in vitro , primary osteoblasts obtained from rat calvaria were cultured for 14–17 days in the presence of AICAR, metformin and compound C. Formation of ‘trabecular-shaped’ bone nodules was evaluated following alizarin red staining. We demonstrated that both AICAR and metformin dose-dependently increase trabecular bone nodule formation, while compound C inhibits bone formation. When primary osteoblasts were co-treated with AICAR and compound C, compound C suppressed the stimulatory effect of AICAR on bone nodule formation. AMPK is a αβγ heterotrimer, where α is the catalytic subunit. RT-PCR analysis of AMPK subunits in ROS17/2.8 osteoblastic cells and in mouse tibia showed that the AMPKα1 subunit is the dominant isoform expressed in bone. We analysed the bone phenotype of 4 month-old male wild type (WT) and AMPKα1−/− KO mice using micro-CT. Both cortical and trabecular bone compartments were smaller in the AMPK α1-deficient mice compared to the WT mice. Altogether, our data support a role for AMPK sign