Limits...
SIRT1 inhibits adipogenesis and promotes myogenic differentiation in C3H10T1/2 pluripotent cells by regulating Wnt signaling.

Zhou Y, Zhou Z, Zhang W, Hu X, Wei H, Peng J, Jiang S - Cell Biosci (2015)

Bottom Line: This study aimed to determine the effect of sirtuin 1 (SIRT1) on adipogenesis and myogenic differentiation of C3H10T1/2 cells.The results showed that resveratrol inhibits adipogenic differentiation and improves myogenic differentiation, whereas nicotinamide promotes adipogenic differentiation.Together, our results suggested that SIRT1 inhibits adipogenesis and stimulates myogenic differentiation by activating Wnt signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Nutrition and Feed Science, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan, 430070 People's Republic of China ; State Key Laboratory of Agricultural Microbiology, Division of Animal Infectious Disease, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070 People's Republic of China ; The Cooperative Innovation Center for Sustainable Pig Production, Wuhan, 430070 People's Republic of China.

ABSTRACT

Background: The directed differentiation of mesenchymal stem cells (MSCs) is tightly controlled by a complex network. Wnt signaling pathways have an important function in controlling the fate of MSCs. However, the mechanism through which Wnt/β-catenin signaling is regulated in differentiation of MSCs remains unknown. SIRT1 plays an important role in the regulation of MSCs differentiation.

Results: This study aimed to determine the effect of sirtuin 1 (SIRT1) on adipogenesis and myogenic differentiation of C3H10T1/2 cells. First, the MSC commitment and differentiation model was established by using 5-azacytidine. Using the established model, C3H10T1/2 cells were treated with SIRT1 activator/inhibitor during differentiation. The results showed that resveratrol inhibits adipogenic differentiation and improves myogenic differentiation, whereas nicotinamide promotes adipogenic differentiation. Notably, during commitment, resveratrol blocked adipocyte formation and promoted myotubes differentiation, whereas nicotinamide enhanced adipogenic potential of C3H10T1/2 cells. Furthermore, resveratrol elevated the expression of Cyclin D1 and β-catenin in the early stages. The luciferase assay showed that knockdown SIRT1 inhibits Wnt/β-catenin signaling, while resveratrol treatment or overexpression SIRT1 activates Wnt/β-catenin signaling. SIRT1 suppressed the expression of Wnt signaling antagonists sFRP2 and DACT1. Knockdown SIRT1 promoted adipogenic potential of C3H10T1/2 cells, whereas overexpression SIRT1 inhibited adipogenic differentiation and promoted myogenic differentiation.

Conclusions: Together, our results suggested that SIRT1 inhibits adipogenesis and stimulates myogenic differentiation by activating Wnt signaling.

No MeSH data available.


Related in: MedlinePlus

5-Azacytidine induces C3H10T1/2 cells into differentiated adipocytes and myocytes. C3H10T1/2 cells were treated with 5, 10, 20, and 40 μM 5-azacytidine for 3 days, then changed GM culture treated for next 14 days. a Cells were fixed and stained with DiI on day 14 days after GM Culture. b The relative expression profile of myogenic marker genes: MyoD and MyHc were determined using Real-time PCR at 0, 3, 7, and 14 days after treatment. c Oil Red-O staining of cells on day 14 days after GM culture. d The relative expression profile of adipogenic marker genes: PPARγ and adiponentin were determined using Real-time PCR at 0, 3, 7, and 14 days after treatment. The green arrow points to the myotube. The red arrow points to the lipid droplet. Data represent mean ± SEM from three independent experiments. *P < 0.05; **P < 0.01
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4643498&req=5

Fig1: 5-Azacytidine induces C3H10T1/2 cells into differentiated adipocytes and myocytes. C3H10T1/2 cells were treated with 5, 10, 20, and 40 μM 5-azacytidine for 3 days, then changed GM culture treated for next 14 days. a Cells were fixed and stained with DiI on day 14 days after GM Culture. b The relative expression profile of myogenic marker genes: MyoD and MyHc were determined using Real-time PCR at 0, 3, 7, and 14 days after treatment. c Oil Red-O staining of cells on day 14 days after GM culture. d The relative expression profile of adipogenic marker genes: PPARγ and adiponentin were determined using Real-time PCR at 0, 3, 7, and 14 days after treatment. The green arrow points to the myotube. The red arrow points to the lipid droplet. Data represent mean ± SEM from three independent experiments. *P < 0.05; **P < 0.01

Mentions: To establish a commitment and differentiation model of MSCs, C3H10T1/2 cells were induced with 5, 10, 20 or 40 µM of 5-AZA for 3 days and cultured in GM for another 14 days. Myogenic and adipogenic phenotypes were measured by using DiI staining and Oil Red O staining, respectively. 5-AZA treatment increased myocyte formation (Fig. 1a) and lipid accumulation (Fig. 1c) with the concentration gradient. Treatment with 20 and 40 µM 5-AZA effectively induced myocyte and adipocyte formation. Cells cultured in GM were harvested at 0, 3, 7 and 14 days to determine the mRNA expression profile of marker genes for myogenesis and adipogenesis. The results showed that the myogenic marker gene MyoD was highly expressed at 7 days of inducing differentiation, and the late differentiation marker MyHc was highly expressed at 14 days of differentiation (Fig. 1b). Meanwhile, the adipogenic markers PPARγ and adiponectin were highly expressed at 7 and 14 days of differentiation, respectively (Fig. 1d). The myogenic or adipogenic marker gene expressions of 20 and 40 µM 5-AZA-treated groups were the highest. The values were significantly higher than the 5 and 10 µM recorded for 5-AZA-treated cells. The results indicated that 20 and 40 µM 5-AZA treatment effectively induced MSC commitment to the myocyte and adipocyte lineage. Thus, treatment with 20 µM 5-AZA was chosen to be used for subsequent study.Fig. 1


SIRT1 inhibits adipogenesis and promotes myogenic differentiation in C3H10T1/2 pluripotent cells by regulating Wnt signaling.

Zhou Y, Zhou Z, Zhang W, Hu X, Wei H, Peng J, Jiang S - Cell Biosci (2015)

5-Azacytidine induces C3H10T1/2 cells into differentiated adipocytes and myocytes. C3H10T1/2 cells were treated with 5, 10, 20, and 40 μM 5-azacytidine for 3 days, then changed GM culture treated for next 14 days. a Cells were fixed and stained with DiI on day 14 days after GM Culture. b The relative expression profile of myogenic marker genes: MyoD and MyHc were determined using Real-time PCR at 0, 3, 7, and 14 days after treatment. c Oil Red-O staining of cells on day 14 days after GM culture. d The relative expression profile of adipogenic marker genes: PPARγ and adiponentin were determined using Real-time PCR at 0, 3, 7, and 14 days after treatment. The green arrow points to the myotube. The red arrow points to the lipid droplet. Data represent mean ± SEM from three independent experiments. *P < 0.05; **P < 0.01
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4643498&req=5

Fig1: 5-Azacytidine induces C3H10T1/2 cells into differentiated adipocytes and myocytes. C3H10T1/2 cells were treated with 5, 10, 20, and 40 μM 5-azacytidine for 3 days, then changed GM culture treated for next 14 days. a Cells were fixed and stained with DiI on day 14 days after GM Culture. b The relative expression profile of myogenic marker genes: MyoD and MyHc were determined using Real-time PCR at 0, 3, 7, and 14 days after treatment. c Oil Red-O staining of cells on day 14 days after GM culture. d The relative expression profile of adipogenic marker genes: PPARγ and adiponentin were determined using Real-time PCR at 0, 3, 7, and 14 days after treatment. The green arrow points to the myotube. The red arrow points to the lipid droplet. Data represent mean ± SEM from three independent experiments. *P < 0.05; **P < 0.01
Mentions: To establish a commitment and differentiation model of MSCs, C3H10T1/2 cells were induced with 5, 10, 20 or 40 µM of 5-AZA for 3 days and cultured in GM for another 14 days. Myogenic and adipogenic phenotypes were measured by using DiI staining and Oil Red O staining, respectively. 5-AZA treatment increased myocyte formation (Fig. 1a) and lipid accumulation (Fig. 1c) with the concentration gradient. Treatment with 20 and 40 µM 5-AZA effectively induced myocyte and adipocyte formation. Cells cultured in GM were harvested at 0, 3, 7 and 14 days to determine the mRNA expression profile of marker genes for myogenesis and adipogenesis. The results showed that the myogenic marker gene MyoD was highly expressed at 7 days of inducing differentiation, and the late differentiation marker MyHc was highly expressed at 14 days of differentiation (Fig. 1b). Meanwhile, the adipogenic markers PPARγ and adiponectin were highly expressed at 7 and 14 days of differentiation, respectively (Fig. 1d). The myogenic or adipogenic marker gene expressions of 20 and 40 µM 5-AZA-treated groups were the highest. The values were significantly higher than the 5 and 10 µM recorded for 5-AZA-treated cells. The results indicated that 20 and 40 µM 5-AZA treatment effectively induced MSC commitment to the myocyte and adipocyte lineage. Thus, treatment with 20 µM 5-AZA was chosen to be used for subsequent study.Fig. 1

Bottom Line: This study aimed to determine the effect of sirtuin 1 (SIRT1) on adipogenesis and myogenic differentiation of C3H10T1/2 cells.The results showed that resveratrol inhibits adipogenic differentiation and improves myogenic differentiation, whereas nicotinamide promotes adipogenic differentiation.Together, our results suggested that SIRT1 inhibits adipogenesis and stimulates myogenic differentiation by activating Wnt signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Nutrition and Feed Science, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan, 430070 People's Republic of China ; State Key Laboratory of Agricultural Microbiology, Division of Animal Infectious Disease, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070 People's Republic of China ; The Cooperative Innovation Center for Sustainable Pig Production, Wuhan, 430070 People's Republic of China.

ABSTRACT

Background: The directed differentiation of mesenchymal stem cells (MSCs) is tightly controlled by a complex network. Wnt signaling pathways have an important function in controlling the fate of MSCs. However, the mechanism through which Wnt/β-catenin signaling is regulated in differentiation of MSCs remains unknown. SIRT1 plays an important role in the regulation of MSCs differentiation.

Results: This study aimed to determine the effect of sirtuin 1 (SIRT1) on adipogenesis and myogenic differentiation of C3H10T1/2 cells. First, the MSC commitment and differentiation model was established by using 5-azacytidine. Using the established model, C3H10T1/2 cells were treated with SIRT1 activator/inhibitor during differentiation. The results showed that resveratrol inhibits adipogenic differentiation and improves myogenic differentiation, whereas nicotinamide promotes adipogenic differentiation. Notably, during commitment, resveratrol blocked adipocyte formation and promoted myotubes differentiation, whereas nicotinamide enhanced adipogenic potential of C3H10T1/2 cells. Furthermore, resveratrol elevated the expression of Cyclin D1 and β-catenin in the early stages. The luciferase assay showed that knockdown SIRT1 inhibits Wnt/β-catenin signaling, while resveratrol treatment or overexpression SIRT1 activates Wnt/β-catenin signaling. SIRT1 suppressed the expression of Wnt signaling antagonists sFRP2 and DACT1. Knockdown SIRT1 promoted adipogenic potential of C3H10T1/2 cells, whereas overexpression SIRT1 inhibited adipogenic differentiation and promoted myogenic differentiation.

Conclusions: Together, our results suggested that SIRT1 inhibits adipogenesis and stimulates myogenic differentiation by activating Wnt signaling.

No MeSH data available.


Related in: MedlinePlus