Modulation of GCN2/eIF2α/ATF4 Pathway in the Liver and Induction of FGF21 in Young Goats Fed a Protein- and/or Phosphorus-Reduced Diet
Abstract
:1. Introduction
2. Results
2.1. Intake, Body Weight, and Daily Weight Gain
2.2. Plasma Concentration of Urea, Pi, and Calcium (Ca), Concentration of FGF21 in Serum, and Glucose Concentration in Whole Blood
2.3. Concentration of Essential and Non-Essential Amino Acids
2.4. Hepatic Expression of GCN2, ATF4, FGF21-mRNA
2.5. Hepatic Expression of GCN2—Protein
3. Discussion
3.1. Amino Acids and Amino Acid Response Pathway
3.2. Amino Acids and FGF21
3.3. Interaction of Low N and Low P Diet
4. Materials and Methods
4.1. Animals, Feeding Regimen
4.2. Diets
4.3. Blood and Tissue Sampling
4.4. Biochemical Determinations
4.5. RNA Isolation and Reverse Transcription
4.6. Hepatic Expression of GCN2, ATF4 and FGF21-mRNA
4.7. Hepatic Expression of GCN2—Protein
4.8. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Items | N+/P+ | N−/P+ | N+/P− | N−/P− | p-Value | ||
---|---|---|---|---|---|---|---|
N-Reduction | P-Reduction | Interaction | |||||
DM intake (g/d) | 614 ± 10 a | 594 ± 6 a,b | 528 ± 25 b | 534 ± 25 b | 0.707 | 0.001 | 0.485 |
Concentrate intake (g/d) | 555 ± 12 a | 537 ± 7 a,b | 469 ± 29 b | 475 ± 28 a,b | 0.799 | 0.002 | 0.581 |
N intake (g/d) | 13.39 ± 0.27 a | 6.76 ± 0.09 b | 11.64 ± 0.68 c | 5.87 ± 0.33 b | <0.0001 | 0.003 | 0.308 |
P intake (g/d) | 2.40 ± 0.05 a | 2.49 ± 0.03 a | 0.53 ± 0.03 b | 0.54 ± 0.03 b | 0.189 | <0.0001 | 0.282 |
Ca intake (g/d) | 6.50 ± 0.13 a | 6.20 ± 0.08 a,b | 5.42 ± 0.31 b,c | 5.22 ± 0.29 c | 0.274 | 0.0001 | 0.827 |
Items | N+/P+ | N−/P+ | N+/P− | N−/P− | p-Value | ||
---|---|---|---|---|---|---|---|
N-Reduction | P-Reduction | Interaction | |||||
Initial body weight (kg) * | 19.21 ± 0.75 | 18.86 ± 0.57 | 18.93 ± 1.09 | 19.14 ± 1.01 | 0.936 | >0.9999 | 0.749 |
Final body weight (kg) † | 25.29 ± 0.74 a | 24.21 ± 0.32 a,b | 20.21 ± 0.93 c | 21.14 ± 1.22 b,c | 0.935 | <0.0001 | 0.261 |
Body weight gain (kg/d) | 0.14 ± 0.01 a | 0.12 ± 0.02 a | 0.03 ± 0.01 b | 0.05 ± 0.01 b | 0.907 | <0.0001 | 0.091 |
Feed efficiency (kg/kg DM) | 0.23 ± 0.02 a | 0.21 ± 0.02 a | 0.05 ± 0.02 b | 0.09 ± 0.02 b | 0.822 | <0.0001 | 0.118 |
Items | N+/P+ | N−/P+ | N+/P− | N−/P− | p-Value | ||
---|---|---|---|---|---|---|---|
N-Reduction | P-Reduction | Interaction | |||||
Urea (mmol/L) | 6.76 ± 0.46 a | 1.16 ± 0.09 b | 6.85 ± 0.18 a | 2.22 ± 0.66 b,c | <0.0001 | 0.180 | 0.250 |
Pi (mmol/L) | 2.09 ± 0.12 a | 2.00 ± 0.21 a | 0.70 ± 0.04 b | 1.06 ± 0.09 b,c | 0.301 | <0.0001 | 0.099 |
Ca (mmol/L) | 3.16 ± 0.09 a | 3.11 ± 0.09 a | 4.34 ± 0.17 b | 3.55 ± 0.12 a,c | 0.002 | <0.0001 | 0.005 |
Glucose (mg/dL) | 68.00 ± 2.66 | 67.86 ± 1.10 | 67.43 ± 1.67 | 66.57 ± 2.55 | 0.814 | 0.662 | 0.866 |
FGF21 (mmol/L) | 29.14 ± 16.43 a | 195.3 ± 50.51 b | 44.51 ± 25.16 a | 63.53 ± 20.86 a | 0.007 | 0.074 | 0.027 |
Items | N+/P+ | N−/P+ | N+/P− | N−/P− | p-Value | ||
---|---|---|---|---|---|---|---|
N-Reduction | P-Reduction | Interaction | |||||
Threonine (mg/dL) | 1.13 ± 0.08 a | 0.85 ± 0.04 b | 1.12 ± 0.05 a | 1.13 ± 0.03 a | 0.022 | 0.026 | 0.018 |
Valine (mg/dL) | 3.50 ± 0.22 a | 2.63 ± 0.16 b | 3.45 ± 0.26 a | 2.98 ± 0.11 a,b | 0.002 | 0.447 | 0.325 |
Methionine (mg/dL) | 0.66 ± 0.03 | 0.66 ± 0.04 | 0.62 ± 0.03 | 0.59 ± 0.04 | 0.734 | 0.130 | 0.679 |
Isoleucine (mg/dL) | 1.34 ± 0.10 | 1.18 ± 0.09 | 1.37 ± 0.07 | 1.26 ± 0.04 | 0.105 | 0.504 | 0.766 |
Leucine (mg/dL) | 1.49 ± 0.13 a,c | 1.18 ± 0.11 a | 1.78 ± 0.11 c | 1.24 ± 0.06 a | 0.0005 | 0.111 | 0.299 |
Phenylalanine (mg/dL) | 0.71 ± 0.03 a | 0.99 ± 0.08 b | 0.78 ± 0.03 a,c | 0.96 ± 0.06 b,c | 0.0003 | 0.754 | 0.414 |
Lysine (mg/dL) | 3.54 ± 0.36 a | 2.34 ± 0.24 b | 3.42 ± 0.23 a | 2.64 ± 0.19 a,b | 0.0009 | 0.739 | 0.428 |
Histidine (mg/dL) | 0.78 ± 0.04 a,b | 0.79 ± 0.06 a,b | 0.75 ± 0.03 b | 0.93 ± 0.04 a | 0.218 | 0.056 | 0.065 |
Total EAA † (mg/dL) | 1.65 ± 0.16 | 1.33 ± 0.10 | 1.66 ± 0.15 | 1.47 ± 0.11 | 0.058 | 0.566 | 0.650 |
Items | N+/P+ | N−/P+ | N+/P− | N−/P− | p-Value | ||
---|---|---|---|---|---|---|---|
N-Reduction | P-Reduction | Interaction | |||||
Arginine (mg/dL) | 4.50 ± 0.26 | 3.57 ± 0.18 | 4.61 ± 0.39 | 4.53 ± 0.24 | 0.081 | 0.063 | 0.138 |
Serine (mg/dL) | 0.83 ± 0.10 | 0.97 ± 0.03 | 0.78 ± 0.07 | 0.89 ± 0.06 | 0.089 | 0.378 | 0.919 |
Asparagine (mg/dL) | 0.68 ± 0.06 | 0.63 ± 0.06 | 0.58 ± 0.05 | 0.63 ± 0.04 | 0.960 | 0.330 | 0.321 |
Glutamic acid (mg/dL) | 1.53 ± 0.22 a,b | 2.19 ± 0.22 b | 1.25 ± 0.09 a | 1.36 ± 0.12 a | 0.035 | 0.004 | 0.126 |
Glutamine (mg/dL) | 5.29 ± 0.28 a | 4.38 ± 0.35 a,b | 4.31 ± 0.16 b | 4.29 ± 0.14 b | 0.074 | 0.043 | 0.085 |
Glycine (mg/dL) | 7.24 ± 0.46 a | 10.28 ± 0.86 b,c | 6.72 ± 0.47 a | 9.31 ± 0.93 a,c | 0.0006 | 0.303 | 0.754 |
Alanine (mg/dL) | 2.09 ± 0.10 a,c | 4.00 ± 0.30 b | 1.87 ± 0.21 c | 2.94 ± 0.30 a | <0.0001 | 0.014 | 0.097 |
Tyrosine (mg/dL) | 1.09 ± 0.05 a | 1.63 ± 0.09 b | 1.05 ± 0.09 a | 1.51 ± 0.11 b | <0.0001 | 0.352 | 0.675 |
Proline (mg/dL) | 1.50 ± 0.23 a,c | 2.11 ± 0.13 a,b | 1.36 ± 0.18 c | 2.67 ± 0.09 b | <0.0001 | 0.215 | 0.047 |
Total NEAA † (mg/dL) | 2.75 ± 0.29 | 3.31 ± 0.37 | 2.50 ± 0.27 | 3.13 ± 0.34 | 0.066 | 0.504 | 0.914 |
Items | N+/P+ | N−/P+ | N+/P− | N−/P− | p-Value | ||
---|---|---|---|---|---|---|---|
N-Reduction | P-Reduction | Interaction | |||||
GCN2 | 3.86 × 104 ± 0.32 × 104 a | 5.50 × 104 ± 0.64 × 104 b | 3.55 × 104 ± 0.20 × 104 a | 3.89 × 104 ± 0.36 × 104 a,b | 0.025 | 0.029 | 0.129 |
ATF4 | 9.21 × 106 ± 0.48 × 106 a | 15.15 × 106 ± 1.15 × 106 b | 10.15 × 106 ± 1.14 × 106 a | 11.74 × 106 ± 1.30 × 106 a,b | 0.002 | 0.259 | 0.052 |
FGF21 | 4.92 × 104 ± 2.14 × 104 a | 29.03 × 104 ± 4.74 × 104 b | 2.21 × 104 ± 0.91 × 104 a | 6.82 × 104 ± 1.25 × 104 a | <0.0001 | 0.0001 | 0.002 |
Items | N+/P+ | N−/P+ | N+/P− | N−/P− | p-Value | ||
---|---|---|---|---|---|---|---|
N-Reduction | P-Reduction | Interaction | |||||
GCN2 | 3.96 × 10−2 ± 0.32 × 10−2 a | 6.04 × 10−2 ± 0.55 × 10−2 b | 3.23 × 10−2 ± 0.23 × 10−2 a | 4.13 × 10−2 ± 0.21 × 10−2 a,b | 0.025 | 0.029 | 0.129 |
Items | Wheat Straw | Control | N-Reduction | P-Reduction | N- and P-Reduction |
---|---|---|---|---|---|
Components (g/kg) | |||||
Soybean meal | 68.0 | 57.0 | 68.0 | 57.0 | |
Urea | 24.5 | - | 24.5 | - | |
Wheat starch | 378 | 385 | 379 | 390 | |
Beet pulp | 399 | 415 | 399 | 410 | |
Mineral-vitamin premix † | 10.0 | 10.0 | 10.0 | 10.0 | |
MgHPO4·3H2O | 9.3 | 9.3 | - | - | |
MgO | - | - | 2.2 | 2.2 | |
NaH2PO4·2H2O | 9.7 | 10.0 | 0.4 | 0.9 | |
NaCl | 1.4 | 1.2 | 1.4 | 1.2 | |
NaHCO3 | - | - | 5.0 | 5.0 | |
CaCO3 | 14.3 | 14.2 | 14.3 | 14.2 | |
Sipernat 22S ‡ | 41.8 | 58.1 | 52.3 | 69.5 | |
Molasses | 10.0 | 10.0 | 10.0 | 10.0 | |
Soybean oil | 34.0 | 30.0 | 34.0 | 30.0 | |
Composition * | |||||
DM (g/kg) | 904 | 880 | 874 | 884 | 875 |
Nutrients (g/kg DM) | |||||
Crude ash | 39.8 | 100 | 112 | 101 | 115 |
Crude protein | 25.4 | 165 | 83.5 | 169 | 81.1 |
ADFom | 579 | 87.5 | 96.1 | 87.1 | 84.6 |
aNDFom | 855 | 157 | 169 | 179 | 173 |
Crude fat | 24.3 | 43.2 | 49.2 | 47.5 | 43.4 |
Urea | BDL | 27.8 | BDL | 31.1 | BDL |
Ca | 2.8 | 12.6 | 12.5 | 12.3 | 11.8 |
P | BDL | 4.8 | 5.1 | 1.1 | 1.1 |
Vitamin D3 (IU/kg DM) | BDL | 1000 | 1144 | 1075 | 1051 |
ME (MJ/kg DM) | 8.3 | 12.8 | 12.4 | 12.9 | 12.5 |
Genes | Primers and Probes (5′ → 3′) | Accession Number | Reference |
---|---|---|---|
18s | Forward: AAAAATAACAATACAGGACTCTTTCG Reverse: GCTATTGGAGCTGGAATTACCG FAM-TGGAATGAGTCCACTTTAAATCCTTCCGC-BBQ | AM711869.1 | [65] |
Genes | Primers and Probes (5′ → 3′) | Accession Number | Reference |
---|---|---|---|
GCN2 | Forward: GAGACACCATTGACCAGGGG Reverse: TAGATGGTCGGTGGCCAAAC | XM_018054468.1 and XM_018054469.1 | This study |
ATF4 | Forward: GTTCTCCTGCGACAAGGCTA Reverse: TGGCATGGTTTCCAGGTCAT | XM_018048794.1 | This study |
FGF21 | Forward: CCTCTACACGGATGATGCCC Reverse: GCTTTGGGGTCAAAGTGCAG | XM_005692688.3 | [19] |
RPL19 | Forward: AGCCTGTGACTGTCCATTCC Reverse: ACGTTACCTTCTCGGGCATT | XM_005693740.3 | [66] |
B2M | Forward: CCTTGGTCCTTCTCGGGCTG Reverse: TCTGGCGGGTGTCTTGAGTAT | XM_018053818.1 | [66] |
RPS9 | Forward: CGCCTCGACCAAGAGCTGAAG Reverse: CTCCAGACCTCACGTTTGTTCC | XM_018063497.1 | [67] |
TBP | Forward: AGAAGGCCTTGTGCTAACCC Reverse: AGCAGCCATTACGTCGTCTT | XM_018053502.1 and XM_018053503.1 | This study |
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Weber, S.L.; Hustedt, K.; Schnepel, N.; Visscher, C.; Muscher-Banse, A.S. Modulation of GCN2/eIF2α/ATF4 Pathway in the Liver and Induction of FGF21 in Young Goats Fed a Protein- and/or Phosphorus-Reduced Diet. Int. J. Mol. Sci. 2023, 24, 7153. https://doi.org/10.3390/ijms24087153
Weber SL, Hustedt K, Schnepel N, Visscher C, Muscher-Banse AS. Modulation of GCN2/eIF2α/ATF4 Pathway in the Liver and Induction of FGF21 in Young Goats Fed a Protein- and/or Phosphorus-Reduced Diet. International Journal of Molecular Sciences. 2023; 24(8):7153. https://doi.org/10.3390/ijms24087153
Chicago/Turabian StyleWeber, Sarah L., Karin Hustedt, Nadine Schnepel, Christian Visscher, and Alexandra S. Muscher-Banse. 2023. "Modulation of GCN2/eIF2α/ATF4 Pathway in the Liver and Induction of FGF21 in Young Goats Fed a Protein- and/or Phosphorus-Reduced Diet" International Journal of Molecular Sciences 24, no. 8: 7153. https://doi.org/10.3390/ijms24087153