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Comparative Study
. 2020 Sep 18;18(9):472.
doi: 10.3390/md18090472.

Fermented Oyster Extract Promotes Insulin-Like Growth Factor-1-Mediated Osteogenesis and Growth Rate

Ilandarage Menu Neelaka Molagoda  1 , Jayasingha Arachchige Chathuranga Chanaka Jayasingha  1 , Yung Hyun Choi  2 , Eui Kyun Park  3 , You-Jin Jeon  1 , Bae-Jin Lee  4 , Gi-Young Kim  1
Affiliations
Comparative Study

Fermented Oyster Extract Promotes Insulin-Like Growth Factor-1-Mediated Osteogenesis and Growth Rate

Ilandarage Menu Neelaka Molagoda et al. Mar Drugs. .

Abstract

Fermented oyster (Crassostrea gigas) extract (FO) prevents ovariectomy-induced osteoporosis by inhibiting osteoclastogenesis and activating osteogenesis. However, the molecular mechanisms underlying FO-mediated bone formation and growth rate are unclear. In the current study, we found that FO significantly upregulated the expression of growth-promoting genes in zebrafish larvae including insulin-like growth factor 1 (zigf-1), insulin-like growth factor binding protein 3 (zigfbp-3), growth hormone-1 (zgh-1), growth hormone receptor-1 (zghr-1), growth hormone receptor alpha (zghra), glucokinase (zgck), and cholecystokinin (zccka). In addition, zebrafish larvae treated with 100 μg/mL FO increased in total body length (3.89 ± 0.13 mm) at 12 days post fertilization (dpf) compared to untreated larvae (3.69 ± 0.02 mm); this effect was comparable to that of the β-glycerophosphate-treated zebrafish larvae (4.00 ± 0.02 mm). Furthermore, FO time- and dose-dependently increased the extracellular release of IGF-1 from preosteoblast MC3T3-E1 cells, which was accompanied by high expression of IGF-1. Pharmacological inhibition of IGF-1 receptor (IGF-1R) using picropodophyllin (PPP) significantly reduced FO-mediated vertebrae formation (from 9.19 ± 0.31 to 5.53 ± 0.35) and growth performance (from 3.91 ± 0.02 to 3.69 ± 0.01 mm) in zebrafish larvae at 9 dpf. Similarly, PPP significantly decreased FO-induced calcium deposition in MC3T3-E1 cells by inhibiting GSK-3β phosphorylation at Ser9. Additionally, DOI hydrochloride, a potent stabilizer of GSK-3β, reduced FO-induced nuclear translocation of RUNX2. Transient knockdown of IGF-1Rα/β using specific silencing RNA also resulted in a significant decrease in calcium deposition and reduction in GSK-3β phosphorylation at Ser9 in MC3T3-E1 cells. Altogether, these results indicate that FO increased phosphorylated GSK-3β at Ser9 by activating the autocrine IGF-1-mediated IGF-1R signaling pathway, thereby promoting osteogenesis and growth performance. Therefore, FO is a potential nutritional supplement for bone formation and growth.

Keywords: Crassostrea gigas; GSK-3β; IGF-1; RUNX2; growth performance; osteogenesis.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
FO promotes the growth of zebrafish larvae. Zebrafish larvae (n = 30) at 3 days post fertilization (dpf) were treated with the indicated concentrations of FO (0–100 µg/mL). The total body lengths were measured at (A) 6, 9, and 12 dpf using an Olympus microscope (×4). β-glycerophosphate (GP) at 4 mM was used as a positive control. (B) Graphical representation of the total body length at each dpf. Significant differences among the groups were determined using one-way ANOVA followed by Bonferroni correction. All data are presented as mean ± SEM (001 ** p < 0.01 and *** p < 0.001 vs. untreated zebrafish larvae). FO, fermented oyster (C. gigas) extract; UT, untreated.
Figure 2
Figure 2
FO upregulates the expression of growth-promoting genes. Zebrafish larvae (n = 30) at 3 days post fertilization (dpf) were treated with the indicated concentrations of FO (0–100 µg/mL). β-glycerophosphate (GP) at 4 mM was used as a positive control. (A,B) Total mRNA was extracted at 9 (A) and 12 (B) dpf, and reverse transcription polymerase chain reaction was performed to measure the expression of insulin-like growth factor 1 (zigf-1), insulin-like growth factor binding protein 3 (zigfbp-3), growth hormone 1 (zgh-1), growth hormone receptor 1 (zghr-1), growth hormone receptor alpha (zghra), glucokinase (zgck), and cholecystokinin (zccka). β-actin was used as an internal control. The relative density was calculated using ImageJ software. Significant differences among the groups were determined using one-way ANOVA followed by Bonferroni correction. All data are presented as mean ± SEM (* p < 0.05, ** p < 0.01, and *** p < 0.001 vs. untreated zebrafish larvae). FO, fermented oyster (C. gigas) extract; UT, untreated.
Figure 3
Figure 3
FO upregulates IGF-1 expression in preosteoblast MC3T3-E1 cells. (A) MC3T3-E1 cells were seeded at a density of 2000 cells/cm2 and treated with the indicated concentrations of FO (0–100 µg/mL) for 5 days. β-glycerophosphate (GP) at 2 mM was used as a positive control. Total mRNA was extracted at days 1, 3, and 5 after treatment with FO. Reverse transcription polymerase chain reaction was performed to determine the expression level of IGF-1. GAPDH was used as an internal control. The expression of IGF-1 relative to that of GAPDH level was determined (bottom). (B) In a parallel experiment, the cells were treated with FO or GP for 3 days. Cell culture media were collected, and colorimetric ELISA was performed to quantify the levels of extracellular IGF-1. Significant differences among the groups were determined using one-way ANOVA followed by Bonferroni correction. All data are presented as mean ± SEM (* p < 0.05 and *** p < 0.001 vs. untreated MC3T3-E1 cells). FO, fermented oyster (C. gigas) extract.
Figure 4
Figure 4
Pharmacological inhibition of IGF-1R decreases growth rate and bone formation in zebrafish larvae. Zebrafish larvae (n = 30) at 3 days post fertilization (dpf) were pretreated with 10 µM picropodophyllin (PPP) for 2 h prior to stimulation with 100 µg/mL FO or 4 mM β-glycerophosphate (GP). (A) Images of total body length were captured at 9 dpf (left) and the length is shown in the graph (right). (B) In a parallel experiment, the zebrafish larvae were stained with 0.05% calcein (top). Vertebrae number (bottom left) and relative density (bottom right) were calculated and are depicted in the graph. Significant differences among the groups were determined using one-way ANOVA followed by Bonferroni correction. All data are presented as mean ± SEM (* p < 0.05 and *** p < 0.001 vs. untreated zebrafish larvae). FO, fermented oyster (C. gigas) extract.
Figure 5
Figure 5
FO upregulates GSK-3β phosphorylation at Ser9 and thereby promotes the nuclear translocation of RUNX2, leading to calcium deposition. (A) MC3T3-E1 cells were treated with the indicated concentrations of FO (0–100 µg/mL) for 72 h. Total proteins were isolated and Western blotting was performed to measure the protein level of phosphorylated GSK-3β at Ser9. β-glycerophosphate (GP) at 2 mM was used as a positive control. (B and C) MC3T3-E1 cells were seeded on 3% gelatin-coated coverslips and pretreated with 30 µM DOI hydrochloride (DOI) for 2 h prior to treatment with FO or GP. The nuclear translocation of RUNX2 was measured by immunostaining, in the absence (B) and presence (C) of DOI. Scale bar = 100 µm. (D) MC3T3-E1 cells were seeded at a density of 2000 cells/cm2 and pretreated with 1 µM picropodophyllin (PPP) for 2 h prior to stimulation with 100 µg/mL FO and 2 mM GP. After 7 days, the cells were stained with alizarin red to determine the level of calcium deposition. (E) After 3 days in the presence of PPP, total proteins were isolated and Western blotting was performed to measure the level of phosphorylated GSK-3β at Ser9. Total GSK-3β was used as an internal control. The expression of phosphorylated GSK-3β relative to that of total GSK-3β is illustrated in the graphs. Significant differences among the groups were determined using the one-way ANOVA followed by Bonferroni correction. All data are presented as mean ± SEM (* p < 0.05 and *** p < 0.001 vs. untreated MC3T3-E1 cells). FO, fermented oyster (C. gigas) extract.
Figure 6
Figure 6
Transient knockdown of IGF-1Rαβ reduces calcium deposition concomitantly with a decrease in phosphorylated GSK-3β at Ser9. MC3T3-E1 cells were seeded at a density of 2000 cells/cm2 and transfected with silencing RNA for IGF-1Rαβ (siIGF-1Rαβ) 48 h before stimulation with 100 µg/mL FO or 2 mM GP. (A) Total mRNA was extracted, and reverse transcription polymerase chain reaction was performed. GAPDH was used as an internal control. (B) After 7 days, the cells were stained with alizarin red for calcium deposition, and images were captured. (C) Three days after treatment with FO, total proteins were isolated and Western blotting was performed to measure the protein level of phosphorylated GSK-3β at Ser9 (left). Total GSK-3β and β-actin were used as internal controls. The expression of IGF-1Rαβ and phosphorylated GSK-3β at Ser9 relative to GAPDH and total GSK-3β levels is illustrated (right). Significant differences among the groups were determined using one-way ANOVA followed by Bonferroni correction. All data are presented as mean ± SEM (* p < 0.05 and *** p < 0.001 vs. untreated MC3T3-E1 cells). FO, fermented oyster (C. gigas) extract.
Figure 7
Figure 7
FO promotes bone formation and growth by activating the IGF-1/IGF-1R signaling pathway. FO upregulates growth-promoting genes such as insulin-like growth factor 1 (IGF-1), thereby promoting the release of IGF in osteoblast cells. The secreted IGF-1 binds to its specific receptor, IGF-1R, in an autocrine or paracrine manner and subsequently triggers the downstream signaling pathway. Once the IGF-1/IGF-1R complex is formed, it initiates the GSK-3β phosphorylation at Ser9. Phosphorylated GSK-3β subsequently releases RUNX2, which in turn promotes bone formation and growth. FO, fermented oyster (C. gigas) extract; IGF-1R, insulin-like growth factor-1 receptor; GSK-3β, glycogen synthase kinase-3β; RUNX2, runt-related transcription factor 2.
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References

    1. Laron Z. Insulin-like growth factor 1 (IGF-1): A growth hormone. Mol. Pathol. 2001;54:311–316. doi: 10.1136/mp.54.5.311. - DOI - PMC - PubMed
    1. Duan C., Ren H., Gao S. Insulin-like growth factors (IGFs), IGF receptors, and IGF-binding proteins: Roles in skeletal muscle growth and differentiation. Gen. Comp. Endocrinol. 2010;167:344–351. doi: 10.1016/j.ygcen.2010.04.009. - DOI - PubMed
    1. Vijayakumar A., Novosyadlyy R., Wu Y., Yakar S., Gallagher E.J. Biological effects of growth hormone on carbohydrate and lipid metabolism. Growth Horm. IGF Res. 2010;20:1–7. doi: 10.1016/j.ghir.2009.09.002. - DOI - PMC - PubMed
    1. Lee W.J. IGF-I Exerts an anti-inflammatory effect on skeletal muscle cells through down-regulation of TLR4 signaling. Immune Netw. 2011;11:223–226. doi: 10.4110/in.2011.11.4.223. - DOI - PMC - PubMed
    1. Serra C., Bhasin S., Tangherlini F., Barton E.R., Ganno M., Zhang A., Shansky J., VanDenburgh H.H., Travison T.G., Jasuja R., et al. The role of GH and IGF-I in mediating anabolic effects of testosterone on androgen-responsive muscle. Endocrinology. 2011;152:193–206. doi: 10.1210/en.2010-0802. - DOI - PMC - PubMed

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