Glutamine metabolism modulates chondrocyte inflammatory response
- Department of Orthopedic Surgery, Washington University School of Medicine, United States
- Department of Mechanical Engineering and Material Sciences, Washington University School of Medicine, United States
- Bone and Mineral Division, Department of Medicine, Washington University School of Medicine, United States
- Shriners Hospital for Children, United States
Figures
Figure 1 with 1 supplement
Chondrocytes rely upon glutamine for energy production.
(A–E) Primary murine chondrocytes were treated with IL-1β (10ng/mL) for 24hr. Gene expression of Gls2, Gdh, Gs, Eaat2, and Asct2 was measured by quantitative PCR. Results from n=6-10 independent …
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Figure 1—source data 1
Depicting original raw data related to Figure 1.
- https://cdn.elifesciences.org/articles/80725/elife-80725-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
Chondrocytes rely upon glutamine for energy production.
(A–D) Primary sternal chondrocytes were cultured in media containing glutamine with or without CB-839 (1mM). Cells were then treated with IL-1β (10ng/mL) for 24hr. All values were normalized to cell …
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Figure 1—figure supplement 1—source data 1
Depicting original raw data related to Figure 1—figure supplement 1.
- https://cdn.elifesciences.org/articles/80725/elife-80725-fig1-figsupp1-data1-v2.xlsx
Figure 2 with 1 supplement
Glutamine deprivation causes metabolic reprogramming to inhibit glycolysis.
(A–C) Primary murine chondrocytes were cultured in media containing 4 mM glutamine or 0 mM glutamine for 24 hr. Cells were then treated with IL-1β (10 ng/mL) for 24 hr. Gene expression of G6pd2, Eno1…
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Figure 2—source data 1
Depicting original raw data related to Figure 2.
- https://cdn.elifesciences.org/articles/80725/elife-80725-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
Glutamine deprivation causes metabolic reprogramming to inhibit glycolysis.
(A–E) Primary murine chondrocytes were cultured in media containing 4 mM glutamine or 0 mM glutamine for 24 hr. Cells were then treated with IL-1β (10 ng/mL) for 24 hr. Gene expression of Gls, Got2, …
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Figure 2—figure supplement 1—source data 1
Depicting original raw data related to Figure 2—figure supplement 1.
- https://cdn.elifesciences.org/articles/80725/elife-80725-fig2-figsupp1-data1-v2.xlsx
Figure 3 with 1 supplement
Glutamine deprivation inhibits the inflammatory response.
(A–B) Primary murine chondrocytes were cultured in media containing 4 mM glutamine or 0 mM glutamine for 24 hr. Cells were then treated with IL-1β (10 ng/mL) for 24 hr. Gene expression of Il6 and Mmp…
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Figure 3—source data 1
Depicting original raw data related to Figure 3.
- https://cdn.elifesciences.org/articles/80725/elife-80725-fig3-data1-v2.xlsx
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Figure 3—source data 2
Original raw data for Figure 3D, F.
- https://cdn.elifesciences.org/articles/80725/elife-80725-fig3-data2-v2.zip
Figure 3—figure supplement 1
Glutamine deprivation inhibits the inflammatory response.
(A) Primary murine chondrocytes were treated with IL-1β in the presence or absence of CB-839 (1 mM) for 24 hr. Gene expression of Il6 was measured by quantitative PCR (qPCR). Results are …
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Figure 3—figure supplement 1—source data 1
Depicting original raw data related to Figure 3—figure supplement 1.
- https://cdn.elifesciences.org/articles/80725/elife-80725-fig3-figsupp1-data1-v2.xlsx
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Figure 3—figure supplement 1—source data 2
Original raw data related to Figure 3—figure supplement 1B.
- https://cdn.elifesciences.org/articles/80725/elife-80725-fig3-figsupp1-data2-v2.zip
Figure 4 with 1 supplement
Glutamine deprivation promotes autophagy, and ammonia inhibits autophagy.
(A) Primary murine chondrocytes were cultured in media containing 4 mM glutamine or 0 mM glutamine for 24 hr. Cells were then treated with IL-1β (10 ng/mL) in the presence or absence of chloroquine …
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Figure 4—source data 1
Depicting original raw data related to Figure 4.
- https://cdn.elifesciences.org/articles/80725/elife-80725-fig4-data1-v2.xlsx
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Figure 4—source data 2
Original raw data related to Figure 4A,C,D.
- https://cdn.elifesciences.org/articles/80725/elife-80725-fig4-data2-v2.zip
Figure 4—figure supplement 1
Glutamine deprivation promotes autophagy, and ammonia inhibits autophagy.
(A–B) Quantification of western blot image displayed in Figure 4A. p62 pixel intensity and actin pixel intensity were measured. Each lane is represented as the ratio of p62/actin pixel density. A …
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Figure 4—figure supplement 1—source data 1
Depicting original raw data related to Figure 4—figure supplement 1.
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Figure 4—figure supplement 1—source data 2
Original raw data related to Figure 4—figure supplement 1G,H,I,O.
- https://cdn.elifesciences.org/articles/80725/elife-80725-fig4-figsupp1-data2-v2.zip
Figure 5 with 1 supplement
Glutamine deprivation modulates mTOR activation.
(A) Primary murine chondrocytes were cultured in media containing 4 mM glutamine or 0 mM glutamine for 24 hr. Cells were then treated with IL-1β (10 ng/mL). After 24 hr, lysates were collected, and …
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Figure 5—source data 1
Depicting original raw data related to Figure 5.
- https://cdn.elifesciences.org/articles/80725/elife-80725-fig5-data1-v2.xlsx
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Figure 5—source data 2
Original raw data related to Figure 5A, B.
- https://cdn.elifesciences.org/articles/80725/elife-80725-fig5-data2-v2.zip
Figure 5—figure supplement 1
Glutamine deprivation modulates mTOR activation.
(A) Primary murine chondrocytes were cultured in media containing 4 mM glutamine or 0 mM glutamine for 24 hr. Cells were then treated with IL-1β (10 ng/mL) for 24 hr in the presence or absence of …
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Figure 5—figure supplement 1—source data 1
Depicting original raw data related to Figure 5—figure supplement 1.
- https://cdn.elifesciences.org/articles/80725/elife-80725-fig5-figsupp1-data1-v2.xlsx
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Figure 5—figure supplement 1—source data 2
Original raw data related to Figure 5—figure supplement 1A.
- https://cdn.elifesciences.org/articles/80725/elife-80725-fig5-figsupp1-data2-v2.zip
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (Mus, musculus) | C57BL/6mice | Jackson Labs | RRID:IMSR_JAX:000664 | |
| Genetic reagent (Mus, musculus) | NF-kB luciferase reporter mice | Jackson Labs | RRID:IMSR_JAX:027529 | |
| Biological sample (human) | Human osteoarthritis chondrocytes | Isolated from discarded human tissues, Arra et al., 2020 | ||
| Biological sample (mouse) | Murine chondrocytes | Isolated from sterna of newborn pups of genetic strains indicated above, Arra et al., 2020 | ||
| Antibody | Anti-ATF4 (rabbit polyclonal) | ThermoFisher | 10835–1-AP, RRID:AB_2058600 | 1:1,000 for Western blot |
| Antibody | Biotinylated secondary (horse anti-rabbit polyclonal) | Vector Biolabs | BP-1100 | 1:1,000 for IHC |
| Antibody | Anti-LC3b (rabbit polyclonal) | Cell Signaling Technology | 2775, RRID:AB_915950 | 1:1,000 Western blot; 1:100 for IF |
| Antibody | Anti-p62 (mouse monoclonal) | Abnova | 2C11, RRID:AB_437085 | 1:1,000 Western blot; 1:100 for IF |
| Antibody | Anti-IkB-z (rat monoclonal) | Invitrogen | 14-16801-82, RRID:AB_11218083 | 1:1,000 Western blot |
| Antibody | Anti-p-Akt (rabbit polyclonal) | Cell Signaling Technology | 9271, RRID:AB_329825 | 1:1,000 Western blot |
| Antibody | Anti-Akt (rabbit polyclonal) | Cell Signaling Technology | 9272, RRID:AB_329827 | 1:1,000 Western blot |
| Antibody | Anti-p-S6 (rabbit polyclonal) | Cell Signaling Technology | 2211, RRID:AB_331679 | 1:1,000 Western blot |
| Antibody | Anti-S6 (rabbit polyclonal) | Cell Signaling Technology | 2217, RRID:AB_331355 | 1:1,000 Western blot |
| Antibody | Anti-Actin (mouse monoclonal) | Sigma-Aldrich | A2228, RRID:AB_476697 | 1:10,000 Western blot |
| Sequence-based reagent | quantitative PCR primers | Integrated DNA technologies | N/A | Custom DNA oligos |
| Peptide, recombinant protein | Collagenase D | Roche | COLLD-RO | |
| Peptide, recombinant protein | Pronase | Roche | PRON-RO | |
| Peptide, recombinant protein | IL-1b | Peprotech | 211-11B | 10ng/mL |
| Commercial assay or kit | Diaminobenzidine (DAB) peroxidase kit | Vector Biolabs | SK4100 | |
| Commercial assay or kit | Lactate assay kit | Eton Biosciences | 1.2E+09 | |
| Commercial assay or kit | Purelink RNA Mini Kit | Ambion | 12183025 | |
| Commercial assay or kit | High capacity cDNA Reverse Transcription Kit | Applied Biosystems | 4368814 | |
| Commercial assay or kit | ATP assay kit | Biovision | K255 | |
| Commercial assay or kit | Luminescence assay kit | GoldBio | I-930 | |
| Commercial assay or kit | Glutamate-Glo Assay kit | Promega | J7021 | |
| Chemical compound and drugs | CB-839 | Selleck | S7655 | |
| Chemical compound and drugs | Rapamycin | MedChem Express | HY-10219 | |
| Chemical compound and drugs | Ammonium Chloride | Sigma-Aldrich | A9434 | |
| Chemical compound and drugs | L-glutamic acid | Sigma-Aldrich | G1626 | |
| Chemical compound and drugs | Streptavidin Horseradish peroxidase (HRP) | Vector Biolabs | SA-5004–1 | |
| Chemical compound and drugs | DAPI | Cell Signaling Technology | 9071 | |
| Chemical compound and drugs | Trizol | ThermoFisher | 15596026 | |
| Chemical compound and drugs | DCFDA | Sigma-Aldrich | D6883 | |
| Chemical compound and drugs | MTT | Sigma-Aldrich | M655 | |
| Chemical compound and drugs | Immunocal | Fisher Scientific | NC9044643 | |
| Chemical compound and drugs | iTaq universal SYBR Green | BioRad | 1725120 | |
| Software and algorithm | Gen5 software | Agilent BioTek | BTGENSCPRIM | |
| Software and algorithm | Prism | Graphpad | RRID:SCR_002798 | |
| Software and algorithm | Wave | Agilent BioTek | RRID:SCR_014526 |
Table 1
List of primers.
| Primer | Sequence (5’→3’) |
|---|---|
| m-Il6 | GCTACCAAACTGGATATAATCAGGA |
| CCAGGTAGCTATGGTACTCCAGAA | |
| m-Mmp13 | GCCAGAACTTCCCAACCAT |
| TCAGAGCCCAGAATTTTCTCC | |
| m-Atf4 | TCGATGCTCTGTTTCGAATG |
| AGAATGTAAAGGGGGCAACC | |
| m-Lc3 | TGGGACCAGAAACTTGGTCT |
| GACCAGCACCCCAGTAAGAT | |
| m-p62 | AGAATGTGGGGGAGAGTGTG |
| TCTGGGGTAGTGGGTGTCAG | |
| m-GLS | CTACAGGATTGCGAACATCTGAT |
| ACACCATCTGACGTTGTCTGA | |
| m-GDH | GGCCGATTGACCTTCAAATA |
| TCCTGTCCTGGAACTCTGCT | |
| m-GS | CATTGACAAACTGAGCAAGAGG |
| AAGTCGTTGATGTTGGAGGTT | |
| m-EAAT2 | GGCAATCCCAAACTCAAGAA |
| GTGCTATTGGCCTCCTCAGA | |
| m-ASCT2 | CAACCAAAGAGGTGCTGGAT |
| CCTCCACCTCACAGAGAAGC | |
| m-G6pd2 | CTGAATGAACGCAAAGCTGA |
| CAATCTTGTGCAGCAGTGGT | |
| m-Eno1 | GCCTCCTGCTCAAAGTCAAC |
| AACGATGAGACACCATGACG | |
| m-Ldha | TGGAAGACAAACTCAAGGGCGAGA |
| TGACCAGCTTGGAGTTCGCAGTTA | |
| m-Mdh | GGTGCAGCCTTAGATAAATACGC |
| AGTCAAGCAACTGAAGTTCTCC | |
| m-Sdha | AACACTGGAGGAAGCACACC |
| AGTAGGAGCGGATAGCAGGA | |
| m-Idh2 | AACCGTGACCAGACTGATGAC |
| ATGGTGGCACACTTGACAGC | |
| m-Got2 | GATCCGTCCCCTGTATTCCA |
| CACCTCTTGCAACCATTGCTT | |
| h-GLS2 | TCTCTTCCGAAAGTGTGTGAGC |
| CCGTGAACTCCTCAAAATCAGG | |
| h-GLUD1 | TATCCGGTACAGCACTGACG |
| GCTCCATGGTGAATCTTCGT | |
| h-GS | CCTGCTTGTATGCTGGAGTC |
| GATCTCCCATGCTGATTCCT | |
| h-GOT2 | GTTTGCCTCTGCCAATCATATG |
| GAGGGTTGGAATACATGGGAC | |
| h-NFKBIZ | CCGTTTCCCTGAACACAGTT |
| AGAAAAGACCTGCCCTCCAT | |
| h-MMP3 | CTGGACTCCGACACTCTGGA |
| CAGGAAAGGTTCTGAACTGACC | |
| h-ATF4 | TCTCCAGCGACAAGGCTAA |
| CAATCTGTCCCGGAGAAGG |
