The Role of Omega-3 Polyunsaturated Fatty Acids from Different Sources in Bone Development
Abstract
:1. Introduction
2. Materials and Methods
2.1. Animals
2.2. Diet Preparation and Composition
2.3. Lipid Extraction and Analysis by Gas Chromatography (GC)
2.4. Histological Staining of Growth-Plate (GP) Sections
2.5. Imaging and Measurement of GPs
2.6. Micro-CT
2.7. Mechanical Testing
2.8. Bone RNA Extraction and RNA-Sequencing
2.8.1. Bioinformatics
2.8.2. Gene-Set Enrichment Analysis
2.9. Statistical Analysis
3. Results
3.1. Dietary n-3 PUFAs Decrease Fat Accumulation and Alter FA Content in Liver and Serum
3.2. Effect of Omega-3 from Different Sources on Growth Pattern and Food Consumption
3.3. The Effect of Diets Rich in n-3 PUFAs on Bone Quality
3.3.1. Diets Rich in n-3 PUFAs Improve Bone Mechanical Properties
3.3.2. Diet Containing Plant Source of n-3 PUFAs (Flaxseed Oil) Improved Trabecular Bone’s Micro-Architecture
3.3.3. Diet Containing n-3 PUFAs from an Animal Source (Fish Oil) Increased BMD
3.4. The Effect of Different Sources of n-3 PUFAs on Bone’s Transcriptional Regulation
IPA Pathway Enrichment Analysis of Genes Related to Dietary n-3 PUFAs
4. Discussion
4.1. Dietary n-3 PUFAs Decrease Hepatic and Serum Fat Levels
4.2. Diet Rich in n-3 PUFAs Affects Postnatal Skeletal Development and Bone Characteristics
4.3. Transcriptional Regulation of Bone by Different Sources n-3 PUFAs
4.4. Dietary n-3 PUFAs Alter Circadian Clock Gene Expression in Bone
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
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Ingredient | g/kg diet | Kcal/kg |
---|---|---|
Cornstarch | 397.486 | 1590 |
Casein (≥85% protein) | 200 | 800 |
Dextrinized cornstarch (90–94% tetrasaccharides) | 132 | 528 |
Sucrose | 100 | 400 |
Corn oil\flaxseed oil\fish oil | 70 | 630 |
Fiber | 50 | |
Mineral mix (AIN-93G-MX) | 35 | |
Vitamin mix (AIN-93-VX) | 10 | |
L-Cystine | 3 | 12 |
Choline bitartrate (41.1% choline) | 2.5 | |
Tert- butylhydroquinone | 0.014 |
Fatty Acids (gr\mouse) | Diet | ||
---|---|---|---|
Control | Flaxseed | Fish | |
SFAs | 1.68 | 1.05 | 3.49 |
MUFAs | 3.21 | 1.88 | 2.65 |
PUFAs | 5.87 | 7.69 | 3.54 |
Total n-6 PUFAs | 5.74 | 1.77 | 0.5 |
Total n-3 PUFAs | 0.12 | 5.89 | 3.01 |
Fatty Acid | Liver | Serum | ||||
---|---|---|---|---|---|---|
Control (mg/100 mg) | Flaxseed (mg/100 mg) | Fish (mg/100 mg) | Control (mg/mL) | Flaxseed (mg/mL) | Fish (mg/mL) | |
Total n-3 | 0.118 (0.02) b | 0.839 (0.15) a | 0.494 (0.10) b | 0.100 (0.03) b | 0.535 (0.07) a | 0.522 (0.09) a |
18:3 ALA (n-3) | 0.011 (0) b | 0.539 (0.12) a | 0.015 (0) b | 0.005 (0.00) b | 0.232 (0.03) a | 0.014 (0.00) b |
20:5 EPA (n-3) | 0.002 (0) b | 0.158 (0.02) a | 0.157 (0.04) a | 0.003 (0.00) c | 0.184 (0.03) b | 0.241 (0.03) a |
22:6 DHA (n-3) | 0.082 (0.02) c | 0.132 (0.02) b | 0.316 (0.05) a | 0.066 (0.03) b | 0.102 (0.01) b | 0.252 (0.05) a |
Total n-6 | 1.710 (0.38) a | 0.449 (0.08) b | 0.274 (0.05) b | 1.381 (0.36) a | 0.587 (0.08) b | 0.430 (0.09) b |
18:2 LA (n-6) | 1.282 (0.34) a | 0.376 (0.07) b | 0.176 (0.04) b | 0.820 (0.31) a | 0.441 (0.07) b | 0.240 (0.06) b |
20:4 AA (n-6) | 0.343 (0.05) a | 0.059 (0.01) b | 0.086 (0) b | 0.480 (0.04) a | 0.101 (0.01) c | 0.147 (0.03) b |
SFA | 1.434 (0.27) a | 0.921 (0.16) b | 1.038 (0.12) b | 0.984 (0.22) a | 0.737 (0.09) b | 0.787 (0.14) ab |
14:0 Mytistic | 0.030 (0.01) a | 0.016 (0) b | 0.025 (0) ab | 0.016 (0) a | 0.013 (0) a | 0.016 (0) a |
16:0 Palmitic | 1.104 (0.24) a | 0.640 (0.11) b | 0.747 (0.10) b | 0.658 (0.18) a | 0.433 (0.06) b | 0.541 (0.10) ab |
18:0 Stearic | 0.294 (0.03) a | 0.261 (0.04) a | 0.261 (0.01) a | 0.300 (0.03) a | 0.270 (0.03) a | 0.220 (0.03) b |
20:0 Arachidic | 0.001 (0) a | 0 (0) b | 0.001 (0) ab | 0.002 (0) a | 0 (0) a | 0 (0) a |
MUFA | 1.619 (0.29) a | 0.815 (0.23) b | 0.615 (0.12)b | 0.582 (0.21) a | 0.372 (0.05) b | 0.389 (0.09) b |
16:1 Palmitoleic | 0.187 (0.04) a | 0.108 (0.03) b | 0.132 (0.04) b | 0.061 (0.01) ab | 0.046 (0.01) b | 0.079 (0.02) a |
18:1 Vaccenic | 0.003 (0) a | 0 (0) b | 0.002 (0) b | 0.008 (0.01) a | 0.004 (0.01) a | 0.002 (0) a |
18:1 Oleic (n-9) | 1.315 (0.24) a | 0.659 (0.18) b | 0.432 (0.06) b | 0.455 (0.18) a | 0.284 (0.03) b | 0.267 (0.06) b |
Total lipids | 4.873 (0.82) a | 3.020 (0.60) b | 2.418 (0.37) b | 3.023 (0.82) a | 2.216 (0.28) b | 2.116 (0.41) b |
Control | ||
18:3 ALA (n-3) | 1.70 | 0.60 |
20:5 EPA (n-3) | 0.05 | 0.11 |
22:6 DHA (n-3) | 0.37 | 4.55 |
18:2 LA (n-6) | 97.45 | 70.47 |
20:4 AA (n-6) | 0.00 | 18.89 |
Flaxseed | ||
18:3 ALA (n-3) | 76.55 | 41.89 |
20:5 EPA (n-3) | 0.04 | 12.32 |
22:6 DHA (n-3) | 0.09 | 10.25 |
18:2 LA (n-6) | 22.76 | 29.20 |
20:4 AA (n-6) | 0.01 | 4.63 |
Fish | ||
18:3 ALA (n-3) | 3.07 | 1.98 |
20:5 EPA (n-3) | 56.44 | 20.65 |
22:6 DHA (n-3) | 25.7 | 41.68 |
18:2 LA (n-6) | 8.61 | 23.16 |
20:4 AA (n-6) | 3.48 | 11.40 |
6 Weeks Old | 9 Weeks Old | |||||
Width (µm) | Control | Flaxseed | Fish | Control | Flaxseed | Fish |
Total | 119.9 (11.8) b | 134.0 (19) a | 128.2 (19.5) a | 89.8 (9.4) b | 101.0(7.4) a | 101.1 (10.6) a |
PZ | 56.3 (9.9) b | 65.1 (13.1) a | 60.1 (13.8) ab | 44.4 (7.9) b | 51.8 (6.6) a | 51.2 (7.7) a |
HZ | 63.6 (9.3) a | 68.9 (14.2) a | 68.0 (15.9) a | 45.4 (7.6) b | 49.2 (5.9) a | 49.9 (8.1) a |
No. of cells | Control | Flaxseed | Fish | Control | Flaxseed | Fish |
Total | 12.2 (0.4) a | 11.9 (0.7) a | 12.2 (1.2) a | 10.4 (0.8) a | 10.3 (0.7) a | 10.4 (0.5) a |
PZ | 8.6 (0.6) a | 7.9 (0.6) a | 8.3 (0.6) a | 7.1 (0.6) a | 6.6 (0.7) a | 6.9 (0.1) a |
HZ | 3.6 (0.2) a | 4.0 (0.5) a | 3.9 (0.8) a | 3.3 (0.3) a | 3.6 (0.2) a | 3.5 (0.5) a |
6 Weeks Old | 9 Weeks Old | |||||
Bone Mechanical Properties | Control | Flaxseed | Fish | Control | Flaxseed | Fish |
Stiffness (N/µ) | 0.031 (0.005) b | 0.038 (0.002) ab | 0.042 (0.009) a | 0.063 (0.013) | 0.062 (0.011) | 0.052 (0.01) |
Max load (N) | 9.025 (0.82) b | 11.264 (1.566) a | 12.022 (1.137) a | 15.474 (1.826) | 15.082 (0.963) | 14.121 (2.905) |
Yield point (N) | 5.07 (0.698) | 6.092 (1.537) | 7.228 (2.426) | 6.628 (1.668) | 7.05 (1.657) | 6.395 (1.037) |
Young’s modulus | 0.405 (0.103) b | 0.522 (0.118) ab | 0.613 (0.178) a | 0.994(0.305) | 0.895(0.304) | 0.72(0.427) |
Trabecular Bone Microarchitecture | Control | Flaxseed | Fish | Control | Flaxseed | Fish |
BV/TV % | 8.04 (1.35) b | 10.28 (0.8) a | 8.81 (1.39) ab | 6.02 (1.06) b | 8.10 (1.32) a | 6.87 (1.82) ab |
Tb.Th (mm) | 0.03 (0) | 0.04 (0) | 0.03 (0) | 0.04 (0) | 0.04 (0) | 0.04 (0) |
Tb.Sp (mm) | 0.22 (0.01) | 0.20 (0.02) | 0.21 (0.03) | 0.27 (0.03) | 0.24 (0.03) | 0.26 (0.03) |
Tb.N (1/mm) | 2.21 (0.33) b | 2.75 (0.41) a | 2.48 (0.46) ab | 1.54 (0.25) b | 2.00 (0.32) a | 1.76 (0.46) ab |
Cortical Bone Microarchitecture | Control | Flaxseed | Fish | Control | Flaxseed | Fish |
B.Ar/T.Ar (%) | 30.22 (1.88) | 31.67 (1.77) | 30.61 (2.79) | 33.18 (1.89) | 35.66 (2.06) | 34.52 (3.19) |
M.Ar (mm2) | 1.39 (0.16) | 1.38 (0.11) | 1.36 (0.09) | 1.36 (0.1) | 1.26 (0.1) | 1.37 (0.11) |
Cs.Th (mm) | 0.11 (0.01) | 0.12 (0) | 0.11 (0.01) | 0.12 (0.01) | 0.13 (0) | 0.13 (0.02) |
BMD (gr/cm3) | 1.17 (0.07) | 1.21 (0.06) | 1.23 (0.08) | 1.33 (0.05) b | 1.30 (0.05) b | 1.41 (0.06) a |
Comparison | Pathway | Molecules |
---|---|---|
Flaxseed vs. control | Circadian Rhythm Signaling | Bmal, bhlhe40, cry2, nr1d1, per2, per3, dpb |
Glutathione Redox Reactions | Gstt1, gstt2/gstt2b | |
Coagulation System | Plat, serpine1 | |
Adipogenesis pathway | Bmal1, nr1d2, per2 | |
Role of Osteoblasts, Osteoclasts and Chondrocytes | Adamts4, adamts5, nfatc2 | |
Fish vs. control | Cell Cycle Control of Replication | Cdc6, cdc7, cdk1, cdk2, cdt1, dbf4, dna2, lig1, mcm2, mcm3, mcm4, mcm5, mcm6, mcm7, orc1, orc2, orc6, pcna, pold1, pole, prim1, top2a |
Role of BRCA1 in DNA Damage Response | Bard1, blm, brca1, brca2, cdkn1a, e2f1, e2f2, e2f4, e2f7, e2f8, fancd2, fancl, gadd45a, mdc1, plk1, rad51, rb1, rbl1, rfc3, rfc4, topbp1 | |
Heme Biosynthesis II | Alad, cpox, fech, hmbs, ppox, urod, uros | |
Circadian Rhythm Signaling | Bmal1, cry2, nr1d1, per2, per3 | |
Hedgehog Signaling | Ccnb1, cdk1, gli2, prkar2b | |
Flaxseed vs. Fish | Heme Biosynthesis II | Alad, cpox, fech, ppox, urod, uros |
Role of BRCA1 in DNA Damage Response | Brca1, brca2, e2f2, e2f4, fancd2, topbp1 | |
Cell Cycle Control of Replication | Mcm3, mcm5, orc1, pole | |
Cytokine Signaling | Blvrb, ccr5, il1rl1, socs3 | |
Hypoxia Signaling | Ube2c, ube2l6, ube2o, ube2t |
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Rozner, R.; Vernikov, J.; Griess-Fishheimer, S.; Travinsky, T.; Penn, S.; Schwartz, B.; Mesilati-Stahy, R.; Argov-Argaman, N.; Shahar, R.; Monsonego-Ornan, E. The Role of Omega-3 Polyunsaturated Fatty Acids from Different Sources in Bone Development. Nutrients 2020, 12, 3494. https://doi.org/10.3390/nu12113494
Rozner R, Vernikov J, Griess-Fishheimer S, Travinsky T, Penn S, Schwartz B, Mesilati-Stahy R, Argov-Argaman N, Shahar R, Monsonego-Ornan E. The Role of Omega-3 Polyunsaturated Fatty Acids from Different Sources in Bone Development. Nutrients. 2020; 12(11):3494. https://doi.org/10.3390/nu12113494
Chicago/Turabian StyleRozner, Reut, Janna Vernikov, Shelley Griess-Fishheimer, Tamar Travinsky, Svetlana Penn, Betty Schwartz, Ronit Mesilati-Stahy, Nurit Argov-Argaman, Ron Shahar, and Efrat Monsonego-Ornan. 2020. "The Role of Omega-3 Polyunsaturated Fatty Acids from Different Sources in Bone Development" Nutrients 12, no. 11: 3494. https://doi.org/10.3390/nu12113494
APA StyleRozner, R., Vernikov, J., Griess-Fishheimer, S., Travinsky, T., Penn, S., Schwartz, B., Mesilati-Stahy, R., Argov-Argaman, N., Shahar, R., & Monsonego-Ornan, E. (2020). The Role of Omega-3 Polyunsaturated Fatty Acids from Different Sources in Bone Development. Nutrients, 12(11), 3494. https://doi.org/10.3390/nu12113494