Interplay between Cultured Human Osteoblastic and Skeletal Muscle Cells: Effects of Conditioned Media on Glucose and Fatty Acid Metabolism
Abstract
:1. Introduction
2. Materials and Methods
2.1. Materials
2.2. Ethical Approvals
2.3. Cell Culturing and Harvesting of Conditioned Media
2.4. Total Protein Harvesting and Immunoblotting
2.5. RNA Isolation and qPCR
2.6. Acute Substrate Oxidation Assay
2.7. Lipid Distribution
2.8. Protein Synthesis by Leucine Incorporation
2.9. Proteomic Analysis
2.10. Statistics
3. Results
3.1. Conditioned Medium from Differentiated Osteoblastic Cells Increases Glucose Metabolism in Human Skeletal Muscle Cells
3.2. Conditioned Medium from Differentiated Osteoblastic Cells Reduces Oleic Acid Metabolism in Human Skeletal Muscle Cells
3.3. Conditioned Medium from Differentiated Myotubes Increases Glucose Uptake and Reduces Oleic Acid Uptake in Osteoblastic Cells
3.4. Secretome Analysis of Conditioned Medium from Differentiated Osteoblastic Cells and Myotubes
3.5. SPARC Increases Oleic Acid Oxidation in Human Skeletal Muscle Cells
3.6. Conditioned Medium from Differentiated Osteoblastic Cells Inhibits PGC1α mRNA Expression and Protein Synthesis in Human Skeletal Muscle Cells
3.7. Molecular Proteomic Changes Correlate with Functional Effects of OB-CM on Myotubes Energy Metabolism
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Gene | Acc.no. | Forward Sequence | Reverse Sequence |
---|---|---|---|
CD36 | L06850 | AGTCACTGCGACATGATTAATGGT | CTGCAATACCTGGCTTTTCTCAA |
GLUT1/SLC2A1 | K03195 | CAGCAGCCCTAAGGATCTCTCA | CCGGCTCGGCTGACATC |
MYH2 | C5814 | AAGGTCGGCAATGAGTATGTCA | CAACCATCCACAGGAACATCTTC |
MYH7 | NM_000257.2 | CTCTGCACAGGGAAAATCTGAA | CCCCTGGACTTTGTCTCATT |
PDK4 | BC040239 | TTTCCAGAACCAACCAATTCACA | TGC CCG CAT TGC ATT CTT A |
PGC1α | NM013261.3 | AAAGGATGCGCTCTCGTTCA | TCTACTGCCTGGAGACCTTGATC |
GAPDH | NM002046 | TGCACCACCACCTGCTTAGC | GGCATGGACTGTGGTCAT GAG |
RPLP0 | M17885 | CCATTCTATCATCAACGGGTACAA | AGCAAGTGGGAAGGTGTAATCC |
Uniprot ID | Protein Symbol | Protein Name | Biological Process (Gene Ontology) | ||||||||
---|---|---|---|---|---|---|---|---|---|---|---|
1 | P06733 | ENO1 | Alpha-enolase | ||||||||
2 | P62937 | PPIA | Peptidyl-prolyl cis-trans isomerase A | ||||||||
3 | P63261 | ACTG1 | Actin, cytoplasmic 2 | ||||||||
4 | P08670 | VIM | Vimentin | ||||||||
5 | P09486 | SPARC | Secreted protein acidic and cysteine rich | ||||||||
6 | P02765 | AHSG | Alpha-2-HS-glycoprotein | ||||||||
7 | P23528 | CFL1 | Cofilin-1 | ||||||||
8 | P09382 | LGALS1 | Galectin-1 | ||||||||
9 | P14618 | PKM | Pyruvate kinase | ||||||||
10 | P37802 | TAGLN2 | Transgelin-2 | ||||||||
11 | P68363 | TUBA1B | Tubulin alpha-1B chain | ||||||||
12 | O00299 | CLIC1 | Chloride intracellular channel protein 1 | ||||||||
13 | P26022 | PTX3 | Pentraxin-related protein | ||||||||
14 | P04792 | HSPB1 | Heat shock protein beta-1 | ||||||||
15 | P60709 | ACTB | Actin, cytoplasmic 1 | ||||||||
16 | P00338 | LDHA | L-lactate dehydrogenase A chain | ||||||||
17 | P09211 | GSTP1 | Glutathione S-transferase P | ||||||||
18 | P07437 | TUBB | Tubulin beta chain | ||||||||
19 | P07737 | PFN1 | Profilin-1 | ||||||||
20 | P07355 | ANXA2 | Annexin 2 |
Uniprot ID | Protein Symbol | Protein Name | Biological Process (Gene Ontology) | ||||||||
---|---|---|---|---|---|---|---|---|---|---|---|
1 | P63313 | TMSB10 | Thymosin beta 10 | ||||||||
2 | P62328 | TMSB4X | Thymosin beta 4 X-linked | ||||||||
3 | P63261 | ACTG1 | Actin, cytoplasmic 2 | ||||||||
4 | P07737 | PFN1 | Profilin 1 | ||||||||
5 | P02795 | MT2A | Metallothionein 2A | ||||||||
6 | P08670 | VIM | Vimentin | ||||||||
7 | P09486 | SPARC | Secreted protein acidic and cysteine rich | ||||||||
8 | P63104 | YWHAZ | Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation zeta | ||||||||
9 | P08123 | COL1A2 | Collagen type I alpha 2 chain | ||||||||
10 | P09382 | LGALS1 | Galectin 1 | ||||||||
11 | P02452 | COL1A1 | Collagen type I alpha 1 chain | ||||||||
12 | P0DP25 | CALM3 | Calmodulin 3 | ||||||||
13 | P07951 | TPM2 | Tropomyosin 2 | ||||||||
14 | P62258 | YWHAE | Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation epsilon | ||||||||
15 | Q16270 | IGFBP7 | Insulin-like growth factor binding protein 7 | ||||||||
16 | P17661 | DES | Desmin | ||||||||
17 | P41222 | PTGDS | Prostaglandin D2 synthase | ||||||||
18 | P10599 | TXN | Thioredoxin | ||||||||
19 | P01033 | TIMP1 | TIMP metallopeptidase inhibitor 1 | ||||||||
20 | P06454 | PTMA | Prothymosin alpha |
Uniprot ID | Protein Symbol | Protein Name | Log2 (Fold Change) | p-Value |
---|---|---|---|---|
Q16698 | DECR1 | 2,4-dienoyl-CoA reductase 1 | 0.025562 | 0.01318 |
P35914 | HMGCL | 3-hydroxy-3-methylglutaryl-CoA lyase | 0.0276 | 0.010166 |
Q99798 | ACO2 | Aconitase 2 | 0.010616 | 0.025911 |
O75891 | ALDH1L1 | Aldehyde dehydrogenase 1 family member L1 | 0.029607 | 0.004703 |
P54886 | ALDH18A | Aldehyde dehydrogenase 18 family member A1 | 0.014905 | 0.048835 |
P05091 | ALDH2 | Aldehyde dehydrogenase 2 family member | 0.027491 | 0.006844 |
Q9Y2R0 | COA3 | Cytochrome C oxidase assembly factor 3 | 0.203074 | 0.032505 |
P10515 | DLAT | Dihydrolipoamide S-acetyltransferase | 0.022512 | 0.046349 |
P07954 | FH | Fumarate hydratase | 0.007098 | 0.047717 |
P00367 | GLUD1 | Glutamate dehydrogenase 1 | 0.014062 | 0.037031 |
O94925 | GLS | Glutaminase | 0.048643 | 0.021184 |
Q9Y2Q3 | GSTK1 | Glutathione S-transferase kappa 1 | 0.004066 | 0.031943 |
Q16836 | HADH | Hydroxyacyl-CoA dehydrogenase | 0.02538 | 0.003717 |
P36776 | LONP1 | Lon peptidase 1, mitochondrial | 0.026353 | 0.026783 |
Q8IXM3 | MRPL41 | Mitochondrial ribosomal protein L41 | 0.117037 | 0.021162 |
Q4G0N4 | NADK2 | NAD kinase 2, mitochondrial | 0.08224 | 0.0359 |
P28331 | NDUFS1 | NADH:Ubiquinone oxidoreductase core subunit S1 | 0.015448 | 0.04241 |
O75251 | NDUFS7 | NADH:Ubiquinone oxidoreductase core subunit S7 | 0.033855 | 0.047395 |
Q13423 | NNT | Nicotinamide nucleotide transhydrogenase | 0.012271 | 0.048518 |
Q10713 | PMPCA | Peptidase, mitochondrial processing subunit alpha | 0.029524 | 0.004 |
Q9Y3E5 | PTRH2 | Peptidyl-TRNA hydrolase 2 | 0.019982 | 0.029719 |
P32322 | PYCR1 | Pyrroline-5-carboxylate reductase 1 | 0.034958 | 0.019611 |
P08559 | PDHA1 | Pyruvate dehydrogenase E1 subunit alpha 1 | 0.028673 | 0.012049 |
P11177 | PDHB | Pyruvate dehydrogenase E1 subunit beta | 0.028679 | 0.008729 |
P34897 | SHMT2 | Serine hydroxymethyltransferase 2 | 0.02749 | 0.033103 |
P49411 | TUFM | Tu translation elongation factor, mitochondrial | 0.010111 | 0.035421 |
P07108 | DBI | Acyl-CoA binding protein | −0.04511 | 0.02523 |
Q15046 | KARS1 | Lysyl-TRNA synthetase 1 | −0.01335 | 0.035324 |
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Lunde, N.N.; Osoble, N.M.M.; Fernandez, A.D.; Antobreh, A.S.; Jafari, A.; Singh, S.; Nyman, T.A.; Rustan, A.C.; Solberg, R.; Thoresen, G.H. Interplay between Cultured Human Osteoblastic and Skeletal Muscle Cells: Effects of Conditioned Media on Glucose and Fatty Acid Metabolism. Biomedicines 2023, 11, 2908. https://doi.org/10.3390/biomedicines11112908
Lunde NN, Osoble NMM, Fernandez AD, Antobreh AS, Jafari A, Singh S, Nyman TA, Rustan AC, Solberg R, Thoresen GH. Interplay between Cultured Human Osteoblastic and Skeletal Muscle Cells: Effects of Conditioned Media on Glucose and Fatty Acid Metabolism. Biomedicines. 2023; 11(11):2908. https://doi.org/10.3390/biomedicines11112908
Chicago/Turabian StyleLunde, Ngoc Nguyen, Nimo Mukhtar Mohamud Osoble, Andrea Dalmao Fernandez, Alfreda S. Antobreh, Abbas Jafari, Sachin Singh, Tuula A. Nyman, Arild C. Rustan, Rigmor Solberg, and G. Hege Thoresen. 2023. "Interplay between Cultured Human Osteoblastic and Skeletal Muscle Cells: Effects of Conditioned Media on Glucose and Fatty Acid Metabolism" Biomedicines 11, no. 11: 2908. https://doi.org/10.3390/biomedicines11112908