Inosine

Manufactured by Merck Group

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Inosine is a nucleoside that consists of hypoxanthine attached to a ribose sugar molecule. It functions as a precursor to adenosine, a key component in cellular energy transfer processes.

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Lab products found in correlation

74 protocols using inosine

Quantifying Adipocyte mRNA Expression

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Total RNA was isolated using innuSOLV RNA reagent (Analytik Jena, 845-SB-2090100) and reverse transcribed with the complementary DNA synthesis kit (NEB, ProtoScript II First Strand cDNA Synthesis Kit). Quantitative PCR with reverse transcription was performed with SYBR Green Master Mix (ThermoFisher Scientific, 4309155) using an Applied Biosystems machine (ThermoFisher Scientific). Expression levels were calculated as delta Ct values and normalized to the housekeeping gene Hprt/HPRT. For the lists of murine and humane primer sequences used for real-time PCR, please see Supplementary Tables 1 and 2. Primer pair quality was assessed by analysing the melting curves.
To study the effect of inosine treatment on adipocytes’ mRNA expression, cells were incubated for 16 h with 300 nM inosine (Sigma-Aldrich, I4125), before RNA isolation. To study the effect of secreted factors on adipocytes’ mRNA expression, cells were incubated for 16 h with respective supernatants, before RNA isolation.

Niemann B., Haufs-Brusberg S., Puetz L., Feickert M., Jaeckstein M.Y., Hoffmann A., Zurkovic J., Heine M., Trautmann E.M., Müller C.E., Tönjes A., Schlein C., Jafari A., Eltzschig H.K., Gnad T., Blüher M., Krahmer N., Kovacs P., Heeren J, & Pfeifer A. (2022). Apoptotic brown adipocytes enhance energy expenditure via extracellular inosine. Nature, 609(7926), 361-368.

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3T3-L1 Adipocyte Differentiation Assay

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3T3-L1 murine pre-adipocytes (Cat# SP-L1-F, RRID:CVCL 0123, ZenBio) were maintained following the protocol provided by the manufacturer. The differentiation of 3T3-L1 cells was performed by using 3T3-L1 DIFFERENTIATION KIT (DIF001, Merck). After differentiation, cells were maintained in a complete medium containing 50 μM azelaic acid, inosine, valeric acid, N5-ethylglutamine, Nε-methyl-L-lysine, Nω-methyl-L-arginine, sebacic acid, spermidine, uridine (all purchased from Merck) and vehicle control for 7 days before the harvest. After treatments, cells were subjected to RNA extraction and qRT-PCR analyses.

Chen P.C., Tsai T.P., Liao Y.C., Liao Y.C., Cheng H.W., Weng Y.H., Lin C.M., Kao C.Y., Tai C.C, & Ruan J.W. (2024). Intestinal dual-specificity phosphatase 6 regulates the cold-induced gut microbiota remodeling to promote white adipose browning. NPJ Biofilms and Microbiomes, 10, 22.

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Pharmacological Modulation of Adenosine Signaling

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Compounds (10 μM of AppCH 2 ppA), 10 μM of adenosine, 40 nM of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) (Tocris Bioscience, Bristol, UK), 10 μg/mL of adenosine deaminase (ADA) (Merck, Darmstadt, Germany), 100 μM of suramin hexasodium salt, 10 nM of MRS2500 tetraammonium salt (Tocris Bioscience, Bristol, UK), 100 nM of tertiapin-Q (Tocris Bioscience, Bristol, UK), 100 μM of adenosine 5′-(α,β-methylene) diphosphate (ADP-methylene) (Merck, Darmstadt, Germany) and 500 μM of bupivacaine were added to ACSF-2 and superfused with the same rate as control solution. Inosine (Merck, Darmstadt, Germany) at concentration of 100 μM was added to Kglu intracellular solution.

Pons-Bennaceur A., Tsintsadze V., Bui T.T., Tsintsadze T., Minlebaev M., Milh M., Scavarda D., Giniatullin R., Giniatullina R., Shityakov S., Wright M., Miller A.D., Lozovaya N, & Burnashev N. (2019). Diadenosine-Polyphosphate Analogue AppCH2ppA Suppresses Seizures by Enhancing Adenosine Signaling in the Cortex. Cerebral cortex (New York, N.Y. : 1991), 29(9).

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Synthesis and Purification of Enzyme Inhibitors

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Xanthine oxidase, inosine, ampicillin, IPTG and protease inhibitor cocktail were purchased from Sigma (St. Louis, Mo). Ni-NTA agarose was purchased from Qiagen (Valencia, CA). 5′-methythioinosine was generated from MTA using PfADA as described [13] (link). ImmH and MT-ImmH were synthesized as described previously [6] (link), [17] (link) and were the generous gift of Peter Tyler, Gary B. Evans and Vern Schramm. Crystallography reagents and plates purchased from Hampton Research (HR2-110 and HR3-297) (Aliso Viejo, CA).

Donaldson T.M., Ting L.M., Zhan C., Shi W., Zheng R., Almo S.C, & Kim K. (2014). Structural Determinants of the 5′-Methylthioinosine Specificity of Plasmodium Purine Nucleoside Phosphorylase. PLoS ONE, 9(1), e84384.

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Nutrient Impact on Drosophila Development

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To test the impact of specific nutrients, we prepared 12.5% strength media as above, and selectively added in protein or nucleotides: the precursors and end points of de novo nucleotide biosynthesis. Treatments included casein (for bulk addition of amino acids at 64.1 g/L; Sigma C7078), inosine (0.74 g/L; Sigma I4125), uridine (0.67 g/L; Sigma U3003), or both inosine and uridine at the aforementioned concentrations. The concentrations of additives were calculated as 87.5% of a previously defined holidic diet (25 (link)), to adjust for the 12.5% BDSC media strength. Developmental assays were carried out as described above.

Lindsey A.R., Parish A.J., Newton I.L., Tennessen J.M., Jones M.W, & Stark N. (2023). Wolbachia is a nutritional symbiont in Drosophila melanogaster. bioRxiv.

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Kras-driven Autophagy-deficient Lung Tumor Cell Lines

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Kras-driven TDCLs were generated from p53^−/−;Kras^G12D/+;Atg7^+/+ or p53^−/−;Kras^G12D/+;Atg7^−/− lung tumors and cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, and 1% sodium bicarbonate at 38.5°C with 8.5% CO₂ (Guo et al. 2013 (link)).
For starvation assays, HBSS without glucose was used to maintain pH and osmotic balance as well as provide cells with water and essential inorganic ions.
Glutamine, pyruvate, dimethyl-α-KG, uridine, adenosine, guanosine, inosine, and NAC were purchased from Sigma-Aldrich. [U¹³C₅]-Gln, [U¹³C₆]-Glc, and other ¹³C and ¹⁵N uniformly labeled amino acids were purchased from Cambridge Isotope Laboratories.
pAMPK and AMPK antibodies were purchased from Cell Signaling. β-Actin antibody was purchased from Sigma-Aldrich.

Guo J.Y., Teng X., Laddha S.V., Ma S., Van Nostrand S.C., Yang Y., Khor S., Chan C.S., Rabinowitz J.D, & White E. (2016). Autophagy provides metabolic substrates to maintain energy charge and nucleotide pools in Ras-driven lung cancer cells. Genes & Development, 30(15), 1704-1717.

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Preparation of Gut Microbial Metabolites

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The gut microbial metabolites nisin, inosine, and sodium butyrate and positive standard doxorubicin (DOX) were purchased from Sigma Aldrich, Castle Hill, NSW, Australia. Stock concentrations of 6.5 mg/mL (1.94 mM), 15 mg/mL (55.92 mM), and 500 mg/mL (4.54 M) for nisin, inosine, and sodium butyrate, respectively, were prepared in sterile Milli-Q water based on their solubility. A stock concentration of 1 mM DOX was prepared in sterile DMSO.

Jaye K., Alsherbiny M.A., Chang D., Li C.G, & Bhuyan D.J. (2023). Mechanistic Insights into the Anti-Proliferative Action of Gut Microbial Metabolites against Breast Adenocarcinoma Cells. International Journal of Molecular Sciences, 24(20), 15053.

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Metabolic Regulation by Pharmacological Inhibitors

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¹⁵N-labelled glutamine (Cambridge Isotope Laboratories NLM-557-0.5), D-glucose (Sigma G7528), L-glutamine (Sigma G3126), AZD8330 (Selleckchem, S2134), trametinib (Selleckchem, S2673), GDC0941 (Selleckchem, S1065), leflunomide (Sigma L-5025), brequinar sodium salt (Sigma, SML0113), uridine (Sigma, U3003), inosine (Sigma, I4125), mitochondrial stress kit (Seahorse 101706-100), VECTASTAIN Elite ABC Kit (pk-6100; Vector Labs), DAB (sk-4100; Vector labs).

Santana-Codina N., Roeth A.A., Zhang Y., Yang A., Mashadova O., Asara J.M., Wang X., Bronson R.T., Lyssiotis C.A., Ying H, & Kimmelman A.C. (2018). Oncogenic KRAS supports pancreatic cancer through regulation of nucleotide synthesis. Nature Communications, 9, 4945.

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Quantification of Nucleotide Compounds in Meat Samples

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The nucleotide compounds [adenosine 5’-monophosphate (AMP), inosine
5’-monophosphate (IMP), inosine, and hypoxanthine] were extracted from
the meat samples according to the method of Nakatani et al. (1986) . The extract was filtered through a syringe
filter (0.45 μm) into a glass vial and injected into a high-performance
liquid chromatography (HPLC; LC-10AD, Shimadzu Ltd., Kyoto, Japan). The
analytical conditions were as follows: injection volume, 3 mL; mobile phase,
1% trimethylamine/phosphoric acid (pH 6.5); flow rate, 1.0 mL/min; column
and running temperature, Zorbax Eclipse (150×4.6 mm², 4
μm particles; Agilent Technologies, Palo Alto, USA) at 40°C; and
detector, UV/Vis detector at 254 nm. The peak area was calculated from a
standard curve obtained using a standard AMP (Sigma-Aldrich, St. Louis, USA),
IMP (Sigma-Aldrich), inosine (Sigma-Aldrich), hypoxanthine (Sigma-Aldrich).

Koh K.C., Chung K.Y., Kim H.S., Kang S.J., Choi C.B., Jo C, & Choe J. (2019). Determination of Point of Sale and Consumption for Hanwoo Beef Based on Quality Grade and Aging Time. Food Science of Animal Resources, 39(1), 139-150.

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Nucleoside Binding Affinity to LF-rBmpD

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The binding of nucleosides to LF-rBmpD was monitored with MST (43 (link)). Adenosine, guanosine, inosine, xanthosine (Sigma-Aldrich, Darmstadt, Germany), and ribose (negative-control ligand; Sigma-Aldrich) were mixed with LF-rBmpD (final concentration, 500 nM) in a 24-point serial dilution. The concentration of the ligands ranged from 5 mM to 1.2 nM. Samples were filled into zero-background standard-treated capillaries (product number MO-AZ002; NanoTemper Technologies, Munich, Germany) and were measured with Monolith.NT115 LabelFree equipment (NanoTemper Technologies), using 60% light-emitting diode (LED) power and medium MST power. The data were analyzed by MO.Affinity Analysis software (NanoTemper Technologies) and GraphPad Prism (version 8.0; GraphPad Software, San Diego, CA, USA). No dissociation constants are displayed because results of only one experiment are shown.

Cuellar J., Åstrand M., Elovaara H., Pietikäinen A., Sirén S., Liljeblad A., Guédez G., Salminen T.A, & Hytönen J. (2020). Structural and Biomolecular Analyses of Borrelia burgdorferi BmpD Reveal a Substrate-Binding Protein of an ABC-Type Nucleoside Transporter Family. Infection and Immunity, 88(4), e00962-19.

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