Ilavenil Soundharrajan1, Da Hye Kim2, Srigopalram Srisesharam1, Palaniselvam Kuppusamy1, Ravikumar Sivanesan3, Ki Choon Choi4. 1. Grassland and Forage Division, National Institute of Animal Science, Rural Development Administration, Cheonan 31000, Republic of Korea. 2. Laboratory of Animal Physiology, Graduate School of Agricultural Science, Tohoku University, Aoba, Sendai 980-8577, Japan. 3. Department of Zoology, Rajah Serfoji Government Arts College, 613204, India. 4. Grassland and Forage Division, National Institute of Animal Science, Rural Development Administration, Cheonan 31000, Republic of Korea. Electronic address: choiwh@korea.kr.
Abstract
BACKGROUND: Limonene is a cyclic monoterpene (CTL) found in citrus fruits and many plant kingdoms. It has attracted attention as potential molecule due to its diverse biological activities. However, molecular mechanism involved in the osteogenic induction of CTL in C2C12 skeletal muscle cells remain unclear. PURPOSE: Skeletal development maintains the bone homeostasis through bone remodeling process. It coordinated between the osteoblast and osteoblast process. Osteoporosis is one of the most common bone diseases caused by a systemic reduction in bone mass. Recent osteoporosis treatment is based on the use of anti-resorptive and bone forming drugs. However, long term use of these drugs is associated with serious side effects and strategies on the discovery of lead compounds from natural products for osteoblast differentiation are urgently needed. Therefore, we planned to find out the role of CTL on osteoblast differentiation and glucose uptake in C2C12 cells and its effect on signaling pathways. METHODS: Cell proliferation, alkaline phosphatase (ALP) activity, calcium deposition, genes, and proteins associated with osteoblast activation and glucose utilization were analysed. RESULTS: CTL did not affect the cell viability. CTL significantly increased ALP activity, calcium depositions and the expression of osteogenic specific genes such as Myogenin, Myogenic differentiation 1 (MyoD), ALP, Run-related transcription factor 2(RUNX2), osteocalcin (OCN). In addition, CTL induced the mRNA expression of bone morphogenetic proteins (BMP-2 BMP-4 BMP-6 BMP-7 BMP-9). CTL treatment enhanced 2-Deoxy-d-glucose (2DG) uptake. Moreover, CTL stimulated the activation of p38 mitogen activated protein kinase (p38MAPK), Protein kinase B (Akt), Extracellular signal related kinase (ERKs) by increasing phosphorylation. CTL treatment abolished p38 inhibitor (SB203580) mediated inhibition of osteoblast differentiation, but no effect was noted by ERKs specific inhibitor (PD98059). CONCLUSION: These results suggest that limonene induces osteoblast differentiation and glucose uptake through activating p38MAPK and Akt signaling pathways, confirming the molecular basis of the osteoblast differentiation by limonene in C2C12 skeletal muscle cells.
BACKGROUND:Limonene is a cyclicmonoterpene (CTL) found in citrus fruits and many plant kingdoms. It has attracted attention as potential molecule due to its diverse biological activities. However, molecular mechanism involved in the osteogenic induction of CTL in C2C12 skeletal muscle cells remain unclear. PURPOSE: Skeletal development maintains the bone homeostasis through bone remodeling process. It coordinated between the osteoblast and osteoblast process. Osteoporosis is one of the most common bone diseases caused by a systemic reduction in bone mass. Recent osteoporosis treatment is based on the use of anti-resorptive and bone forming drugs. However, long term use of these drugs is associated with serious side effects and strategies on the discovery of lead compounds from natural products for osteoblast differentiation are urgently needed. Therefore, we planned to find out the role of CTL on osteoblast differentiation and glucose uptake in C2C12 cells and its effect on signaling pathways. METHODS: Cell proliferation, alkaline phosphatase (ALP) activity, calcium deposition, genes, and proteins associated with osteoblast activation and glucose utilization were analysed. RESULTS: CTL did not affect the cell viability. CTL significantly increased ALP activity, calcium depositions and the expression of osteogenic specific genes such as Myogenin, Myogenic differentiation 1 (MyoD), ALP, Run-related transcription factor 2(RUNX2), osteocalcin (OCN). In addition, CTL induced the mRNA expression of bone morphogenetic proteins (BMP-2BMP-4 BMP-6 BMP-7 BMP-9). CTL treatment enhanced 2-Deoxy-d-glucose (2DG) uptake. Moreover, CTL stimulated the activation of p38 mitogen activated protein kinase (p38MAPK), Protein kinase B (Akt), Extracellular signal related kinase (ERKs) by increasing phosphorylation. CTL treatment abolished p38 inhibitor (SB203580) mediated inhibition of osteoblast differentiation, but no effect was noted by ERKs specific inhibitor (PD98059). CONCLUSION: These results suggest that limonene induces osteoblast differentiation and glucose uptake through activating p38MAPK and Akt signaling pathways, confirming the molecular basis of the osteoblast differentiation by limonene in C2C12 skeletal muscle cells.