Uncarboxylated osteocalcin inhibits high glucose-induced ROS production and stimulates osteoblastic differentiation by preventing the activation of PI3K/Akt in MC3T3-E1 cells

Liu,Jingli; Yang,Jianhong

doi:10.3892/ijmm.2015.2412

Uncarboxylated osteocalcin inhibits high glucose-induced ROS production and stimulates osteoblastic differentiation by preventing the activation of PI3K/Akt in MC3T3-E1 cells

Authors:
- Jingli Liu
- Jianhong Yang
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Affiliations: College of Life Sciences, University of Chinese Academy of Sciences, Beijing 100049, P.R. China
Published online on: November 17, 2015 https://doi.org/10.3892/ijmm.2015.2412
Pages: 173-181

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Abstract

Uncarboxylated osteocalcin, an osteoblast-derived protein, plays an important role in the regulation of glucose metabolism. It has previously been demonstrated that high glucose levels inhibit osteoblast proliferation and differentiation. However, the mechanisms through which uncarboxylated osteocalcin regulates osteoblast proliferation and differentiation under high glucose conditions remain unclear. Thus, in the present study, we aimed to examine the effects of uncarboxylated osteocalcin on the proliferation and differentiation of MC3T3-E1 cells under high glucose conditions. We demonstrated that high glucose levels induced the production of reactive oxygen species (ROS) in MC3T3-E1 cells, and this production was inhibited by treatment with uncarboxylated osteocalcin and N-acetyl-L-cysteine (NAC), a ROS scavenger. In addition, we found that uncarboxylated osteocalcin reduced high glucose‑induced oxidative stress and increased the mRNA expression of the osteogenic markers, runt-related transcription factor 2 (Runx2), osterix and osteocalcin, as well as the formation of mineralized nodules; it also inhibited adipogenic differentiation, as shown by a decrease in the mRNA expression of the adipogenic markers, peroxisome proliferator‑activated receptor γ (PPARγ), adipocyte fatty acid-binding protein (adipocyte protein 2; aP2) and fatty acid synthase (FAS), and reduced lipid drop accumulation. Furthermore, we found that uncarboxylated osteocalcin inhibited PI3K/Akt signaling which was induced by ROS and facilitated the osteogenic differentiation of MC3T3-E1 cells under high glucose conditions. Taken together and to the best of ou knowledge, our results demonstrate for the first time that uncarboxylated osteocalcin inhibits high glucose-induced ROS production and stimulates osteoblastic differentiation by inhibiting the activation of PI3K/Akt in MC3T3-E1 cells. Therefore, we suggest that uncarboxylated osteocalcin may be a potential therapeutic agent for diabetes-related osteoporosis.

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1	Neve A, Corrado A and Cantatore FP: Osteocalcin: skeletal and extra-skeletal effects. J Cell Physiol. 228:1149–1153. 2013. View Article : Google Scholar
2	Hoang QQ, Sicheri F, Howard AJ and Yang DSC: Bone recognition mechanism of porcine osteocalcin from crystal structure. Nature. 425:977–980. 2003. View Article : Google Scholar : PubMed/NCBI
3	Karsenty G, Kronenberg HM and Settembre C: Genetic control of bone formation. Annu Rev Cell Dev Biol. 25:629–648. 2009. View Article : Google Scholar : PubMed/NCBI
4	Ducy P, Desbois C, Boyce B, Pinero G, Story B, Dunstan C, Smith E, Bonadio J, Goldstein S, Gundberg C, et al: Increased bone formation in osteocalcin-deficient mice. Nature. 382:448–452. 1996. View Article : Google Scholar : PubMed/NCBI
5	Murshed M, Schinke T, McKee MD and Karsenty G: Extracellular matrix mineralization is regulated locally; different roles of two gla-containing proteins. J Cell Biol. 165:625–630. 2004. View Article : Google Scholar : PubMed/NCBI
6	Dubois-Ferrière V, Brennan TC, Dayer R, Rizzoli R and Ammann P: Calcitropic hormones and IGF-I are influenced by dietary protein. Endocrinology. 152:1839–1847. 2011. View Article : Google Scholar : PubMed/NCBI
7	Rutter MM, Markoff E, Clayton L, Akeno N, Zhao G, Clemens TL and Chernausek SD: Osteoblast-specific expression of insulin-like growth factor-1 in bone of transgenic mice induces insulin-like growth factor binding protein-5. Bone. 36:224–231. 2005. View Article : Google Scholar : PubMed/NCBI
8	Garnero P: Biomarkers for osteoporosis management: utility in diagnosis, fracture risk prediction and therapy monitoring. Mol Diagn Ther. 12:157–170. 2008. View Article : Google Scholar : PubMed/NCBI
9	Fukumoto S and Martin TJ: Bone as an endocrine organ. Trends Endocrinol Metab. 20:230–236. 2009. View Article : Google Scholar : PubMed/NCBI
10	Ferron M, McKee MD, Levine RL, Ducy P and Karsenty G: Intermittent injections of osteocalcin improve glucose metabolism and prevent type 2 diabetes in mice. Bone. 50:568–575. 2012. View Article : Google Scholar
11	Lee NK, Sowa H, Hinoi E, Ferron M, Ahn JD, Confavreux C, Dacquin R, Mee PJ, McKee MD, Jung DY, et al: Endocrine regulation of energy metabolism by the skeleton. Cell. 130:456–469. 2007. View Article : Google Scholar : PubMed/NCBI
12	Pi M, Wu Y and Quarles LD: GPRC6A mediates responses to osteocalcin in β-cells in vitro and pancreas in vivo. J Bone Miner Res. 26:1680–1683. 2011. View Article : Google Scholar : PubMed/NCBI
13	Bodine PVN and Komm BS: Evidence that conditionally immortalized human osteoblasts express an osteocalcin receptor. Bone. 25:535–543. 1999. View Article : Google Scholar : PubMed/NCBI
14	Wellendorph P and Bräuner-Osborne H: Molecular cloning, expression, and sequence analysis of GPRC6A, a novel family C G-protein-coupled receptor. Gene. 335:37–46. 2004. View Article : Google Scholar : PubMed/NCBI
15	Schwartz AV: Diabetes mellitus: does it affect bone? Calcif Tissue Int. 73:515–519. 2003. View Article : Google Scholar : PubMed/NCBI
16	McCabe LR: Understanding the pathology and mechanisms of type I diabetic bone loss. J Cell Biochem. 102:1343–1357. 2007. View Article : Google Scholar : PubMed/NCBI
17	Hamann C, Kirschner S, Günther KP and Hofbauer LC: Bone, sweet bone - osteoporotic fractures in diabetes mellitus. Nat Rev Endocrinol. 8:297–305. 2012. View Article : Google Scholar : PubMed/NCBI
18	Hayakawa N and Suzuki A: Diabetes mellitus and osteoporosis. Effect of antidiabetic medicine on osteoporotic fracture. Clin Calcium. 22:1383–1390. 2012.In Japanese. PubMed/NCBI
19	Motyl KJ, McCabe LR and Schwartz AV: Bone and glucose metabolism: a two-way street. Arch Biochem Biophys. 503:2–10. 2010. View Article : Google Scholar : PubMed/NCBI
20	Thrailkill KM, Liu L, Wahl EC, Bunn RC, Perrien DS, Cockrell GE, Skinner RA, Hogue WR, Carver AA, Fowlkes JL, et al: Bone formation is impaired in a model of type 1 diabetes. Diabetes. 54:2875–2881. 2005. View Article : Google Scholar : PubMed/NCBI
21	Hamann C, Goettsch C, Mettelsiefen J, Henkenjohann V, Rauner M, Hempel U, Bernhardt R, Fratzl-Zelman N, Roschger P, Rammelt S, et al: Delayed bone regeneration and low bone mass in a rat model of insulin-resistant type 2 diabetes mellitus is due to impaired osteoblast function. Am J Physiol Endocrinol Metab. 301:E1220–E1228. 2011. View Article : Google Scholar : PubMed/NCBI
22	Zhang Y and Yang JH: Activation of the PI3K/Akt pathway by oxidative stress mediates high glucose-induced increase of adipogenic differentiation in primary rat osteoblasts. J Cell Biochem. 114:2595–2602. 2013. View Article : Google Scholar : PubMed/NCBI
23	Wang W, Zhang X, Zheng J and Yang J: High glucose stimulates adipogenic and inhibits osteogenic differentiation in MG-63 cells through cAMP/protein kinase A/extracellular signal-regulated kinase pathway. Mol Cell Biochem. 338:115–122. 2010. View Article : Google Scholar
24	Movahed A, Larijani B, Nabipour I, Kalantarhormozi M, Asadipooya K, Vahdat K, Akbarzadeh S, Farrokhnia M, Assadi M, Amirinejad R, et al: Reduced serum osteocalcin concentrations are associated with type 2 diabetes mellitus and the metabolic syndrome components in postmenopausal women: The crosstalk between bone and energy metabolism. J Bone Miner Metab. 30:683–691. 2012. View Article : Google Scholar : PubMed/NCBI
25	Zhou M, Ma X, Li H, Pan X, Tang J, Gao Y, Hou X, Lu H, Bao Y and Jia W: Serum osteocalcin concentrations in relation to glucose and lipid metabolism in Chinese individuals. Eur J Endocrinol. 161:723–729. 2009. View Article : Google Scholar : PubMed/NCBI
26	Kim JH, Park S, Kim HW and Jang JH: Recombinant expression of mouse osteocalcin protein in Escherichia coli. Biotechnol Lett. 29:1631–1635. 2007. View Article : Google Scholar : PubMed/NCBI
27	Harada S and Rodan GA: Control of osteoblast function and regulation of bone mass. Nature. 423:349–355. 2003. View Article : Google Scholar : PubMed/NCBI
28	Teitelbaum SL and Ross FP: Genetic regulation of osteoclast development and function. Nat Rev Genet. 4:638–649. 2003. View Article : Google Scholar : PubMed/NCBI
29	Boskey AL, Gadaleta S, Gundberg C, Doty SB, Ducy P and Karsenty G: Fourier transform infrared microspectroscopic analysis of bones of osteocalcin-deficient mice provides insight into the function of osteocalcin. Bone. 23:187–196. 1998. View Article : Google Scholar : PubMed/NCBI
30	Poundarik A, Gundberg C and Vashishth D: Non-collageneous proteins influence bone mineral size, shape and orientation: a SAXS study. J Bone Miner Res. 26(Suppl): S362011.
31	Zhen D, Chen Y and Tang X: Metformin reverses the deleterious effects of high glucose on osteoblast function. J Diabetes Complications. 24:334–344. 2010. View Article : Google Scholar
32	Fujimori S, Osawa M, Iemata M, Hinoi E and Yoneda Y: Increased GABA transport activity in rat calvarial osteoblasts cultured under hyperglycemic conditions. Biol Pharm Bull. 29:297–301. 2006. View Article : Google Scholar : PubMed/NCBI
33	Koh JM, Lee YS, Kim YS, Kim DJ, Kim HH, Park JY, Lee KU and Kim GS: Homocysteine enhances bone resorption by stimulation of osteoclast formation and activity through increased intracellular ROS generation. J Bone Miner Res. 21:1003–1011. 2006. View Article : Google Scholar : PubMed/NCBI
34	Bai XC, Lu D, Liu AL, Zhang ZM, Li XM, Zou ZP, Zeng WS, Cheng BL and Luo SQ: Reactive oxygen species stimulates receptor activator of NF-kappaB ligand expression in osteoblast. J Biol Chem. 280:17497–17506. 2005. View Article : Google Scholar : PubMed/NCBI
35	Lee NK, Choi YG, Baik JY, Han SY, Jeong DW, Bae YS, Kim N and Lee SY: A crucial role for reactive oxygen species in RANKL-induced osteoclast differentiation. Blood. 106:852–859. 2005. View Article : Google Scholar : PubMed/NCBI
36	Wittrant Y, Gorin Y, Woodruff K, Horn D, Abboud HE, Mohan S and Abboud-Werner SL: High d(+)glucose concentration inhibits RANKL-induced osteoclastogenesis. Bone. 42:1122–1130. 2008. View Article : Google Scholar : PubMed/NCBI
37	Wauquier F, Leotoing L, Coxam V, Guicheux J and Wittrant Y: Oxidative stress in bone remodelling and disease. Trends Mol Med. 15:468–477. 2009. View Article : Google Scholar : PubMed/NCBI
38	Liu AL, Zhang ZM, Zhu BF, Liao ZH and Liu Z: Metallothionein protects bone marrow stromal cells against hydrogen peroxide-induced inhibition of osteoblastic differentiation. Cell Biol Int. 28:905–911. 2004. View Article : Google Scholar : PubMed/NCBI
39	Chuang CC, Yang RS, Tsai KS, Ho FM and Liu SH: Hyperglycemia enhances adipogenic induction of lipid accumulation: involvement of extracellular signal-regulated protein kinase 1/2, phosphoinositide 3-kinase/Akt, and peroxisome proliferator-activated receptor gamma signaling. Endocrinology. 148:4267–4275. 2007. View Article : Google Scholar : PubMed/NCBI