AMP-activated kinase (AMPK) is AMP-activated protein kinase (AMPK)1 is a well conserved eukaryotic protein that senses the energy state of the cell (1-6).AMPK is a heterotrimer consisting of three subunits, ␣, ␤, ␥, each of which has at least two isoforms. The ␣ subunit contains the catalytic site; however, all subunits are necessary for full activity (7). AMPK phosphorylates multiple targets both in vivo and in vitro, including several biosynthetic enzymes such as acetyl-CoA carboxylase (ACC), hydroxymethylglutaryl-CoA (HMG-CoA) reductase, glycogen synthase, and endothelial nitric-oxide synthase (eNOS) (8). AMPK is activated in a very sensitive manner by cellular stresses that deplete ATP either by inhibiting ATP production (e.g. hypoxia), or by accelerating ATP consumption (e.g. exercising muscle, Refs. 1-7).Increases in the AMP/ATP ratio activate AMPK by a number of mechanisms, including direct allosteric activation and covalent modification due to activation by an AMPK kinase (AMPKK) that phosphorylates the ␣ subunit on Thr 172 (9, 10). However, a recent study done by Hawley et al. (11) indicates that metformin, an antidiabetic drug, activates AMPK independently of the AMP/ATP ratio. Several studies also suggest the existence of a second AMPKK that is not . However, the upstream events involved in AMPK signaling remain to be elucidated.Our activates c-Src and PI 3-kinase without a change in cellular AMP or ATP content. Furthermore, this novel activation scheme may be implicated during H/R where we found that AMPK activation depends on ONOO Ϫ formation, as well as c-Src and PI 3-kinase. As evidenced by co-immunoprecipitation, both ONOO Ϫ and H/R lead to physical association of c-Src and AMPK suggesting that this enables this signaling pathway for activation of AMPK downstream targets including ACC.

Purpose of this Review Over the past decades, osteocytes have emerged as mechano-sensors of bone and master regulators of bone homeostasis. This article summarizes latest research and progress made in understanding osteocyte mechanobiology and critically reviews tools currently available to study these cells. Recent Findings Whereas increased mechanical forces promote bone formation, decrease loading is always associated with bone loss and skeletal fragility. Recent studies identified cilia, integrins, calcium channels and G-protein coupled receptors as important sensors of mechanical forces and Ca2+ and cAMP signaling as key effectors. Among transcripts regulated by mechanical forces, sclerostin and RANKL have emerged as potential therapeutic targets for disuse-induced bone loss. Summary In this paper we review the mechanisms by which osteocytes perceive and transduce mechanical cues and the models available to study mechano-transduction. Future directions of the field are also discussed.

Cells of the osteoblast lineage are increasingly identified as participants in whole-body metabolism by primarily targeting pancreatic insulin secretion or consuming energy. Osteocytes, the most abundant bone cells, secrete a Wnt-signaling inhibitor called sclerostin. Here we examined three mouse models expressing high sclerostin levels, achieved through constitutive or inducible loss of the stimulatory subunit of G-proteins (Gsα in mature osteoblasts and/or osteocytes). These mice showed progressive loss of white adipose tissue (WAT) with tendency toward increased energy expenditure but no changes in glucose or insulin metabolism. Interestingly beige adipocytes were increased extensively in both gonadal and inguinal WAT and had reduced canonical β-catenin signaling. To determine if sclerostin directly contributes to the increased beige adipogenesis, we engineered an osteocytic cell line lacking Gsα which has high sclerostin secretion. Conditioned media from these cells significantly increased expression of UCP1 in primary adipocytes, and this effect was partially reduced after depletion of sclerostin from the conditioned media. Similarly, treatment of Gsα-deficient animals with sclerostin-neutralizing antibody partially reduced the increased UCP1 expression in WAT. Moreover, direct treatment of sclerostin to wild-type mice significantly increased UCP1 expression in WAT. These results show that osteocytes and/or osteoblasts secrete factors regulating beige adipogenesis, at least in part, through the Wnt-signaling inhibitor sclerostin. Further studies are needed to assess metabolic effects of sclerostin on adipocytes and other metabolic tissues.

Osteocytes are master orchestrators of bone remodeling; they control osteoblast and osteoclast activities both directly cell-to-cell communication and indirectly secreted factors, and they are the main postnatal source of sclerostin and RANKL (receptor activator of NF-kB ligand), two regulators of osteoblast and osteoclast function. Despite progress in understanding osteocyte biology and function, much remains to be elucidated. Recently developed osteocytic cell lines-together with new genome editing tools-has allowed a closer look at the biology and molecular makeup of these cells. By using single-cell cloning, we identified genes that are associated with high Sost/sclerostin expression and analyzed their regulation and function. Unbiased transcriptome analysis of high- low-Sost/sclerostin-expressing cells identified known and novel genes. Dmp1 (dentin matrix protein 1), Dkk1 (Dickkopf WNT signaling pathway inhibitor 1), and Phex were among the most up-regulated known genes, whereas Srpx2, Cd200, and carbonic anhydrase III (CAIII) were identified as novel markers of differentiated osteocytes. Aspn, Enpp2, Robo2, Nov, and Serpina3g were among the transcripts that were most significantly suppressed in high-Sost cells. Considering that CAII was recently identified as being regulated by Sost/sclerostin and capable of controlling mineral homeostasis, we focused our attention on CAIII. Here, we report that CAIII is highly expressed in osteocytes, is regulated by parathyroid hormone both and , and protects osteocytes from oxidative stress.-Shi, C., Uda, Y., Dedic, C., Azab, E., Sun, N., Hussein, A. I., Petty, C. A., Fulzele, K., Mitterberger-Vogt, M. C., Zwerschke, W., Pereira, R., Wang, K., Divieti Pajevic, P. Carbonic anhydrase III protects osteocytes from oxidative stress.