Endogenous Metabolite
Pyruvate activates c-Jun N-terminal kinase 1 (JNK1) through mitochondrial hydrogen peroxide (H₂O₂) release.
Pyruvate reduces glycogen synthase kinase 3β (GSK-3β) activity via JNK1/RSK3 pathway.

Sodium 2-oxopropionate (acetone) is dephenylized to acetone during the process of scavenging hydrogen peroxide, which ends aerobic metabolism and produces more ROS. ROS concentrations rise in the cytosol and mitochondria when pyruvate activates JNK1 activation. Across a range of pyruvate concentrations, increased JNK1 activity is shown in several cell types [1]. Because sodium 2-oxopropionate (sodium pyruvate) is both H2O2 and O2·-, it shields the top of the lens from oxidation and the ensuing development of white cataracts both internally and externally. Moreover, sodium 2-oxopropionate has been shown to prevent sugar bleaching by binding to protein-NH2 [2].

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In HeLa cells, pyruvate (100 mM) potently activated JNK1 activity, peaking at 1–2 hours after addition. JNK2 and p38 activity were unchanged, while ERK showed a small increase.
Pyruvate (5–100 mM) increased JNK1 activity in various cell types including HeLa, HUVEC, IMR-90 fibroblasts, HEK-293 cells, and 3T3 fibroblasts.
Pyruvate treatment (100 mM) decreased GSK-3β activity in a redox-dependent manner.
Pyruvate increased glycogen synthase activity.
Pyruvate stimulated ribosomal S6 kinase 3 (RSK3) activity, but not p70S6K or AKT1 activity.
Mitochondrial oxidant levels (measured by DHR123 fluorescence) and cytosolic ROS levels (measured by DCFDA fluorescence) increased after pyruvate addition.
Pyruvate-induced JNK1 activation was inhibited by rotenone (mitochondrial complex I inhibitor), extracellular catalase, N-acetylcysteine (NAC), and overexpression of glutathione S-transferase P1 (GSTpi).
Depletion of mitochondria by ethidium bromide treatment abolished pyruvate-induced JNK1 activation.
Expression of dominant negative SEK1 inhibited pyruvate-induced JNK1 activation and GSK-3β activity reduction.

An analysis of serum levels of sodium 2-oxopropionate in neonates revealed a baseline level of pyruvate of 0.30 mM. At the time of sacrifice, serum levels increased approximately six-fold to 1.84 mM after the maximum dose of sodium 2-oxopropionate [1]. The newborns were given Sodium 2-oxopropanoate (acetone acetone; 0.1-10 g/kg) as a bolus injection, and 1 hour later, the JNK1 activity level in the extract was measured.

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In neonatal rats, intraperitoneal injection of pyruvate (0.5–2 g/kg body weight) increased JNK1 activity in liver homogenates in a dose-dependent manner.
Serum pyruvate levels rose approximately sixfold after the highest dose (2 g/kg), from baseline 0.30 mM to 1.84 mM.

Leakage of mitochondrial oxidants contributes to a variety of harmful conditions ranging from neurodegenerative diseases to cellular senescence. We describe here, however, a physiological and heretofore unrecognized role for mitochondrial oxidant release. Mitochondrial metabolism of pyruvate is demonstrated to activate the c-Jun N-terminal kinase (JNK). This metabolite-induced rise in cytosolic JNK1 activity is shown to be triggered by increased release of mitochondrial H(2)O(2). We further demonstrate that in turn, the redox-dependent activation of JNK1 feeds back and inhibits the activity of the metabolic enzymes glycogen synthase kinase 3beta and glycogen synthase. As such, these results demonstrate a novel metabolic regulatory pathway activated by mitochondrial oxidants. In addition, they suggest that although chronic oxidant production may have deleterious effects, mitochondrial oxidants can also function acutely as signaling molecules to provide communication between the mitochondria and the cytosol [1].

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JNK1 activity was measured by immunocomplex kinase assay. Cells were lysed in kinase extraction buffer, JNK1 was immunoprecipitated, and kinase activity was assessed using GST-ATF2Δ as substrate in a reaction containing HEPES, MgCl₂, MnCl₂, DTT, and [γ-³²P]ATP. After incubation at 30°C for 30 min, the reaction was stopped and analyzed by SDS-PAGE and phosphoimaging.
GSK-3β activity was measured by immunoprecipitation followed by kinase assay using a phospho-glycogen synthase peptide substrate in a buffer containing β-glycerophosphate, NaCl, MgCl₂, and [γ-³²P]ATP. Reaction was stopped with phosphoric acid, and phosphate incorporation was measured by liquid scintillation counting.
RSK3, p70S6K, and AKT1 activities were assessed by immune complex kinase assays using specific peptide substrates.
Glycogen synthase activity was assayed by measuring incorporation of ¹⁴C-glucose from UDP-[U-¹⁴C]glucose into glycogen in the presence or absence of glucose-6-phosphate.

Glycation initiated changes in tissue proteins, which are triggered by the Schiff base formation between the sugar carbonyl and the protein -NH2, have been suggested to play an important role in the development of diabetes-related pathological changes such as the formation of cataracts. While the initial reaction takes place by the interaction of >C=O of the parent sugars with the -NH2 of proteins, reactive oxygen species (ROS) dependent generation of more reactive dicarbonyl derivatives from the oxidation of sugars also plays a significant role in these changes, altering the structural as well as functional properties of proteins. The purpose of this study was to examine whether the activities of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), catalase and superoxide dismutase (SOD) could be affected by the high levels of fructose prevalent in diabetic lenses. Incubation of the enzymes with this sugar led to a significant loss of their activities. GAPDH was inactivated within a day. This was followed by the inactivation of catalase (3-4 days) and SOD (6 days). The loss of the activities was prevented significantly by incorporation of pyruvate in the incubation mixture. The protective effect is ascribable to its ability to competitively inhibit glycation as well as to its ROS scavenging activity. Hence, it could play a significant role in the maintenance of lens physiology and cataract prevention [2].

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HeLa cells were treated with pyruvate (100 mM unless specified) in culture medium. For mitochondrial depletion, cells were grown with ethidium bromide (400 ng/mL) for 7 days.
For oxidant measurement, cells were loaded with DHR123 (for mitochondrial H₂O₂) or DCFDA (for cytosolic ROS) and fluorescence was quantified by confocal microscopy or plate reader.
For inhibitor studies, cells were pretreated with rotenone (25–100 µM), TTFA (100 µM), antimycin A (25–100 µM), NAC (2–20 mM), or extracellular catalase (5000 U/mL).
Transient or stable transfection was used for expression of GSTpi, HA-JNK, or dominant negative SEK1.
Intracellular ATP levels were measured using luciferin-luciferase bioluminescence assay.
Intracellular pH was measured using BCECF fluorescent indicator.

Neonatal rats were given intraperitoneal injections of pyruvate at doses of 0.5, 1, or 2 g/kg body weight.
One hour after injection, livers were isolated and homogenized in kinase extraction buffer.
JNK1 activity was measured in liver protein extracts using immunocomplex kinase assay.
Serum pyruvate levels were measured using a lactate dehydrogenase-based diagnostic kit.

[1]. Role for mitochondrial oxidants as regulators of cellular metabolism. Mol Cell Biol. 2000 Oct;20(19):7311-8.

[2]. Fructose induced deactivation of antioxidant enzymes: preventive effect of pyruvate. Free Radic Res. 2000 Jul;33(1):23-30.

Sodium pyruvate is an organic sodium salt containing the pyruvate ion. Pyruvate is a three-carbon metabolite of glucose and can stimulate cellular respiration and the release of mitochondrial oxidants. Mitochondrial-produced H₂O₂ acts as a signaling molecule to activate cytoplasmic JNK1, which in turn inhibits GSK-3β via RSK3, thereby increasing glycogen synthesis and reducing mitochondrial metabolic flux. This pathway forms a feedback loop that regulates cellular metabolism in response to high metabolite supply. This study suggests that mitochondrial oxidants may function as physiological regulators of aerobic metabolism, rather than simply harmful byproducts.

C3H3NAO3

110.0438

109.997

C, 32.74; H, 2.75; Na, 20.89; O, 43.62

113-24-6

Sodium 2-oxopropanoate-13C3;142014-11-7;Pyruvic acid;127-17-3;Sodium 2-oxopropanoate-d3;1316291-18-5;Sodium 2-oxopropanoate-13C;124052-04-6;2-Oxopropanoate-13C5 sodium;89196-78-1; 127-17-3 (free acid)

23662274

Off-white to light yellow solid

1.267g/cm3

165ºC at 760 mmHg

>300 °C(lit.)

54.3ºC

0.968mmHg at 25°C

1.426-1.43

0

3

1

7

88.2

0

[Na+].[O-]C(C(C([H])([H])[H])=O)=O

DAEPDZWVDSPTHF-UHFFFAOYSA-M

InChI=1S/C3H4O3.Na/c1-2(4)3(5)6;/h1H3,(H,5,6);/q;+1/p-1

sodium 2-oxopropanoate

FP-0019 RES100 RES110 FP 0019 FP 0020 RES 001 RES 100 RES 110FP-0020 RES-001 RES-100 RES-110 Pyruvate sodium FP0019 FP0020 RES001

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ~100 mg/mL (~908.76 mM)

Solubility in Formulation 1: 50 mg/mL (454.38 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)