Activation of Sirt1 decreases adipocyte formation during osteoblast differentiation of mesenchymal stem cells.
In vitro, mesenchymal stem cells differentiate to osteoblasts when exposed to bone-inducing medium. However, adipocytes are also formed. We showed that activation of the nuclear protein deacetylase Sirt1 reduces adipocyte formation and promotes osteoblast differentiation.


BACKGROUND
Mesenchymal stem cells (MSCs) can differentiate into osteoblasts, adipocytes, chondrocytes, and myoblasts. It has been suggested that a reciprocal relationship exists between the differentiation of MSCs into osteoblasts and adipocytes. Peroxisome proliferator-activated receptor gamma2 (PPARgamma2) is a key element for the differentiation into adipocytes. Activation of Sirt1 has recently been shown to decrease adipocyte development from preadipocytes through inhibition of PPARgamma2.


METHODS
We used the mouse mesenchymal cell line C3H10T1/2 and primary rat bone marrow cells cultured in osteoblast differentiation medium with or without reagents affecting Sirt1 activity. Adipocyte levels were analyzed by light microscopy and flow cytometry (FACS) after staining with Oil red O and Nile red, respectively. Osteoblast and adipocyte markers were studied with quantitative real-time PCR. Mineralization in cultures of primary rat bone marrow stromal cells was studied by von Kossa and alizarin red staining.


RESULTS
We found that Sirt1 is expressed in the mesenchymal cell line C3H10T1/2. Treatment with the plant polyphenol resveratrol as well as isonicotinamide, both of which activate Sirt1, blocked adipocyte development and increased the expression of osteoblast markers. Nicotinamide, which inhibits Sirt1, increased adipocyte number and increased expression of adipocyte markers. Furthermore, activation of Sirt1 prevented the increase in adipocytes caused by the PPARgamma-agonist troglitazone. Finally, activation of Sirt1 in rat primary bone marrow stromal cells increased expression of osteoblast markers and also mineralization.


CONCLUSIONS
In this study, we targeted Sirt1 to control adipocyte development during differentiation of MSCs into osteoblasts. The finding that resveratrol and isonicotinamide markedly inhibited adipocyte and promoted osteoblast differentiation may be relevant in the search for new treatment regimens of osteoporosis but also important for the evolving field of cell-based tissue engineering.
Bäckesjö
Carl-Magnus
CM
Department for Clinical Science, Intervention and Technology (CLINTEC), Division of Orthopedics, Karolinska Institutet, Karolinska University Hospital, Huddinge, Sweden. Magnus.Backesjo@ki.se
Li
Yan
Y
Lindgren
Urban
U
Haldosén
Lars-Arne
LA
D017667:Adipocytes / Q000166:cytology / Q000378:metabolism*; D000818:Animals; D002113:Calcification, Physiologic / Q000187:drug effects / Q000502:physiology; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000494:pharmacology; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D009536:Niacinamide / Q000494:pharmacology; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism*; D010012:Osteogenesis / Q000187:drug effects / Q000502:physiology; D047495:PPAR gamma / Q000037:antagonists & inhibitors / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D000077185:Resveratrol; D056564:Sirtuin 1; D037761:Sirtuins / Q000378:metabolism*; D013267:Stilbenes / Q000494:pharmacology
D004791:Enzyme Inhibitors; D047495:PPAR gamma; D013267:Stilbenes; D009536:Niacinamide; C027681:isonicotinamide; C470545:Sirt1 protein, mouse; C488368:Sirt1 protein, rat; D056564:Sirtuin 1; D037761:Sirtuins; D000077185:Resveratrol