Short-term effects of sodium arsenite (AsIII) and sodium arsenate (AsV) on carbohydrate metabolism in the perfused rat liver.

The actions of arsenite and arsenate on carbohydrate metabolism in the once-through perfused rat liver were investigated. The compound inhibited lactate gluconeogenesis with an IC50 of 25 µM. It also increased glycolysis and fructolysis at concentrations between 10 and 100 µM. This effect was paralleled by strong inhibition of pyruvate carboxylation (IC50 = 4.25 µM) and by a relatively moderate diminution in the ATP levels. The inhibitory action of arsenate on pyruvate carboxylation and lactate gluconeogenesis was 103 times less effective than that of arsenite. For realistic doses and concentrations («1 mM), impairment of metabolism by arsenate can be expected to occur solely after its reduction to arsenite. Arsenite, on the other hand, can be regarded as a strong short-term modifier of lactate gluconeogenesis and other pathways. The main cause of the former is inhibition of pyruvate carboxylation, a hitherto unknown effect of arsenic compounds.

Transient Synaptic Enhancement Triggered by Exogenously Supplied Monocarboxylate in Drosophila Motoneuron Synapse.

Current evidence suggests that glial cells provide C3 carbon sources to fuel neuronal activity; however, this notion has become challenged by biosensor studies carried out in acute brain slices or in vivo, showing that neuronal activity does not rely on the import of astrocyte-produced L-lactate. Rather, stimulated neurons become net lactate exporters, as it was also shown in Drosophila neurons, in which astrocyte-provided lactate returns as lipid droplets to be stored in glial cells. In this view, we investigate whether exogenously supplied monocarboxylates can support Drosophila motoneuron neurotransmitter release (NTR). By assessing the excitatory post-synaptic current (EPSC) amplitude under voltage-clamp as NTR indicative, we found that both pyruvate and L-lactate, as the only carbon sources in the synapses bathing-solution, cause a large transient NTR enhancement, which declines to reach a synaptic depression state, from which the synapses do not recover. The FM1-43 pre-synaptic loading ability, however, is maintained under monocarboxylate, suggesting that SV cycling should not contribute to the synaptic depression state. The NTR recovery was reached by supplementing the monocarboxylate medium with sucrose. However, monocarboxylate addition to sucrose medium does not enhance NTR, but it does when the disaccharide concentration becomes too reduced. Thus, when pyruvate concentrations become too reduced, exogenously supplied L-lactate could be converted to pyruvate and metabolized by the neural mitochondria, triggering the NTR enhancement. SIGNIFICANCE STATEMENT: The question of whether monocarboxylic acids can fuel the Drosophila motoneuron NTR was challenged. Our findings show that exogenously supplied monocarboxylates trigger a large transient synaptic enhancement just under extreme glycolysis reduction but fail to maintain NTR under sustained synaptic demand, still at low frequency stimulation, driven to the synapses to a synaptic depression state. Glycolysis activation, by adding sucrose to the monocarboxylate bath solution, restores the motoneuron NTR ability, giving place to a hexoses role in SV recruitment. Moreover these results suggest exogenously supplied C3 carbon sources could have an additional role beyond providing energetic support for neural activity.

The central role of pyruvate metabolism on the epigenetic maturation and transcriptional profile of bovine oocytes.

In brief: Pyruvate metabolism is one of the main metabolic pathways during oocyte maturation. This study demonstrates that pyruvate metabolism also regulates the epigenetic and molecular maturation in bovine oocytes. Abstract: Pyruvate, the final product of glycolysis, undergoes conversion into acetyl-CoA within the mitochondria of oocytes, serving as a primary fuel source for the tricarboxylic acid (TCA) cycle. The citrate generated in the TCA cycle can be transported to the cytoplasm and converted back into acetyl-CoA. This acetyl-CoA can either fuel lipid synthesis or act as a substrate for histone acetylation. This study aimed to investigate how pyruvate metabolism influences lysine 9 histone 3 acetylation (H3K9ac) dynamics and RNA transcription in bovine oocytes during in vitro maturation (IVM). Bovine cumulus-oocyte complexes were cultured in vitro for 24 h, considering three experimental groups: Control (IVM medium only), DCA (IVM supplemented with sodium dichloroacetate, a stimulant of pyruvate oxidation into acetyl-CoA), or IA (IVM supplemented with sodium iodoacetate, a glycolysis inhibitor). The results revealed significant alterations in oocyte metabolism in both treatments, promoting the utilization of lipids as an energy source. These changes during IVM affected the dynamics of H3K9ac, subsequently influencing the oocyte's transcriptional activity. In the DCA and IA groups, a total of 148 and 356 differentially expressed genes were identified, respectively, compared to the control group. These findings suggest that modifications in pyruvate metabolism trigger the activation of metabolic pathways, particularly lipid metabolism, changing acetyl-CoA availability and H3K9ac levels, ultimately impacting the mRNA content of in vitro matured bovine oocytes.

The concentration of glucose in the media influences the susceptibility of stallion spermatozoa to ferroptosis.

In brief: Although common in many commercial extenders, supraphysiological concentrations of glucose in the media may be detrimental to stallion spermatozoa. In this study, we present evidence that these elevated glucose levels may predispose spermatozoa to ferroptosis. Abstract: Stallion spermatozoa depend on oxidative phosphorylation as their major source of ATP; however, the metabolism of these cells is complex and a great degree of metabolic plasticity exists. The composition of the media in which the spermatozoa are extended, or exposed to in the mare's reproductive tract, exerts a profound effect on sperm function and may even accelerate cell demise. Recent research indicates that high concentrations of glucose in the media, although common in commercial extenders, may be detrimental. To determine if supraphysiological glucose concentration may induce or predispose to ferroptosis (a caspase-independent form of programmed cell death, triggered by oxidative stress), stallion spermatozoa were incubated under different concentrations of glucose, 67 mM (HG) or 1 mM plus 10 mM pyruvate (LG-HP), in the presence or absence of known inductors of ferroptosis. Furthermore, we developed a single-cell flow metabolic assay to identify different metabolic phenotypes in spermatozoa. Storage and incubation of spermatozoa under high glucose concentrations led to an increase in the percentage of necrotic spermatozoa (P < 0.0001). Moreover, ferroptosis was induced more intensely in sperm in media with high glucose concentrations (P < 0.0001). Finally, we observed that induction of ferroptosis modified two proteins (oxoglutarate dehydrogenase and superoxide dismutase 2) in spermatozoa incubated in media containing 67 mM glucose but not in media containing 1 mM glucose and 10 mM pyruvate. The composition of the media, especially the concentration of glucose, exerts a major impact on the functionality and life span of the spermatozoa. The results reported here may pave the way for improvements in existing semen extenders.

Pyruvate enhances stallion sperm function in high glucose media improving overall metabolic efficiency.

If a mechanism of more efficient glycolysis depending on pyruvate is present in stallion spermatozoa, detrimental effects of higher glucose concentrations that are common in current commercial extenders could be counteracted. To test this hypothesis, spermatozoa were incubated in a 67 mM Glucose modified Tyrode's media in the presence of 1- or 10-mM pyruvate and in the Tyrode's basal media which contains 5 mM glucose. Spermatozoa incubated for 3 h at 37 °C in 67 mM Tyrode's media with 10 mM pyruvate showed increased motility in comparison with aliquots incubated in Tyrode's 5 mM glucose and Tyrode's 67 mM glucose (57.1 ± 3.5 and 58.1 ± 1.9 to 73.0 ± 1.1 %; P < 0.01). Spermatozoa incubated in Tyrode's with 67 mM glucose 10 mM pyruvate maintained the viability along the incubation (64.03 ± 15.4 vs 61.3 ± 10.2), while spermatozoa incubated in 67 mM Glucose-Tyrode's showed a decrease in viability (38.01 ± 11.2, P < 0.01). 40 mM oxamate, an inhibitor of the lactate dehydrogenase LDH, reduced sperm viability (P < 0.05, from 76 ± 5 in 67 mM Glucose/10 mM pyruvate to 68.0 ± 4.3 %, P < 0.05). Apoptotic markers increased in the presence of oxamate. (P < 0.01). UHPLC/MS/MS showed that 10 mM pyruvate increased pyruvate, lactate, ATP and NAD+ while phosphoenolpyruvate decreased. The mechanisms that explain the improvement of in presence of 10 mM pyruvate involve the conversion of lactate to pyruvate and increased NAD+ enhancing the efficiency of the glycolysis.

Near-Physiological Concentrations of Extracellular Pyruvate Stimulated Glucose Utilization along with Triglyceride Accumulation and Mitochondrial Activity in HepG2 Cells.

Pyruvate, a key intermediate in energy and nutrient metabolism, probably plays important roles in these regulations. In previous reports using cell lines, extracellular pyruvate of supraphysiological concentrations inhibited the glucose uptake by myotubes while being stimulated by adipocytes. As the effect of pyruvate on the glucose utilization is unclear in cultured hepatocytes. We have investigated the effects of extracellular pyruvate on the glucose utilization and the subsequent metabolic changes using the cell line HepG2. In a 24 h culture, pyruvate enhanced the glucose consumption more potently than 1 µM insulin, and this enhancement was detectable at a near-physiological concentrations of ≤1 mM. For metabolic changes following glucose consumption, the conversion ratio of glucose and pyruvate to extracellular lactate was approximately 1.0 without extracellular pyruvate. The addition of pyruvate decreased the conversion ratio to approximately 0.7, indicating that the glycolytic reaction switched from being an anaerobic to a partially aerobic feature. Consistent with this finding, pyruvate increased the accumulation of intracellular triglycerides which are produced through substrate supply from the mitochondria. Furthermore, pyruvate stimulated mitochondria activity as evidenced by increases in ATP content, mitochondrial DNA copy number, enhanced mitochondria-specific functional imaging and oxygen consumption. Interestingly, 1 mM pyruvate increased oxygen consumption immediately after addition. In this study, we found that near-physiological concentrations of extracellular pyruvate exerted various changes in metabolic events, including glucose influx, lactate conversion rations, TG accumulation, and mitochondrial activity in HepG2 cells.

Hepatic pyruvate and alanine metabolism are critical and complementary for maintenance of antioxidant capacity and resistance to oxidative insult.

OBJECTIVE: Mitochondrial pyruvate is a critical intermediary metabolite in gluconeogenesis, lipogenesis, and NADH production. As a result, the mitochondrial pyruvate carrier (MPC) complex has emerged as a promising therapeutic target in metabolic diseases. Clinical trials are currently underway. However, recent in vitro data indicate that MPC inhibition diverts glutamine/glutamate away from glutathione synthesis and toward glutaminolysis to compensate for loss of pyruvate oxidation, possibly sensitizing cells to oxidative insult. Here, we explored this in vivo using the clinically relevant acetaminophen (APAP) overdose model of acute liver injury, which is driven by oxidative stress. METHODS: We used pharmacological and genetic approaches to inhibit MPC2 and alanine aminotransferase 2 (ALT2), individually and concomitantly, in mice and cell culture models and determined the effects on APAP hepatotoxicity. RESULTS: We found that MPC inhibition sensitizes the liver to APAP-induced injury in vivo only with concomitant loss of alanine aminotransferase 2 (ALT2). Pharmacological and genetic manipulation of neither MPC2 nor ALT2 alone affected APAP toxicity, but liver-specific double knockout (DKO) significantly worsened APAP-induced liver damage. Further investigation indicated that DKO impaired glutathione synthesis and increased urea cycle flux, consistent with increased glutaminolysis, and these results were reproducible in vitro. Finally, induction of ALT2 and post-treatment with dichloroacetate both reduced APAP-induced liver injury, suggesting new therapeutic avenues. CONCLUSIONS: Increased susceptibility to APAP toxicity requires loss of both the MPC and ALT2 in vivo, indicating that MPC inhibition alone is insufficient to disrupt redox balance. Furthermore, the results from ALT2 induction and dichloroacetate in the APAP model suggest new metabolic approaches to the treatment of liver damage.

Harmful effects of chlorhexidine on hepatic metabolism.

Chlorhexidine (CHX) is an over-the-counter antiseptic amply used by the population. There are reports that CHX acts in mitochondria as an uncoupler and inhibitor. The purpose of this study was to investigate the short-term effects of CHX on hepatic metabolic pathways linked to energy metabolism in the perfused rat liver. The compound inhibited both glucose synthesis and the urea cycle. Oxygen consumption was raised at low concentrations (up to 10 µM) and diminished at higher ones. A pronounced diminution in the cellular ATP content was observed. Conversely, CHX stimulated glycolysis and enhanced leakage of cellular enzymes (lactate dehydrogenase and fumarase). In isolated mitochondria, this antiseptic inhibited pyruvate carboxylation, oxidases, and oxygen uptake at very low concentrations (2 µM) and promoted uncoupling. The results described herein raise great concerns about the safety of CHX, as the observed effects can induce hypoglycemia, lactic acidosis, ammonemia as well as cell membrane disruption.

PDK inhibition promotes glucose utilization, reduces hepatic lipid deposition, and improves oxidative stress in largemouth bass (Micropterus salmoides) by increasing pyruvate oxidative phosphorylation.

In omnivorous fish, the pyruvate dehydrogenase kinases (PDKs)-pyruvate dehydrogenase E1α subunit (PDHE1α) axis is essential in the regulation of carbohydrate oxidative catabolism. Among the existing research, the role of the PDKs-PDHE1α axis in carnivorous fish with poor glucose utilization is unclear. In the present study, we determined the effects of PDK inhibition on the liver glycolipid metabolism of largemouth bass (Micropterus salmoides). DCA is a PDK-specific inhibitor that inhibits PDK by binding the allosteric sites. A total of 160 juvenile largemouth bass were randomly divided into two groups, with four replicates of 20 fish each, fed a control diet and a control diet supplemented with dichloroacetate (DCA) for 8 weeks. The present results showed that DCA supplementation significantly decreased the hepatosomatic index, triglycerides in liver and serum, and total liver lipids of largemouth bass compared with the control group. In addition, compared with the control group, DCA treatment significantly down-regulated gene expression associated with lipogenesis. Furthermore, DCA supplementation significantly decreased the mRNA expression of pdk3a and increased PDHE1α activity. In addition, DCA supplementation improved glucose oxidative catabolism and pyruvate oxidative phosphorylation (OXPHOS) in the liver, as evidenced by low pyruvate content in the liver and up-regulated expressions of glycolysis-related and TCA cycle/OXPHOS-related genes. Moreover, DCA consumption decreased hepatic malondialdehyde (MDA) content, enhanced the activities of superoxide dismutase (SOD), and increased transforming growth factor beta (tgf-ß), glutathione S-transferase (gst), and superoxide dismutase 1 (sod1) gene expression compared with the control diet. This study demonstrated that inhibition of PDKs by DCA promoted glucose utilization, reduced hepatic lipid deposition, and improved oxidative stress in largemouth bass by increasing pyruvate OXPHOS. Our findings contribute to the understanding of the underlying mechanism of the PDKs-PDHE1α axis in glucose metabolism and improve the utilization of dietary carbohydrates in farmed carnivorous fish.

Pyruvic acid as attenuator of water deficit in cotton plants varying the phenological stage.

The lack of water during crop growth causes damage to any production system, especially when it occurs during the initial establishment or beginning of the reproductive stage. Although cotton can be properly managed in regions with water limitation, its yield is affected at different levels according to the genetics of the cultivar adopted. Exogenous application of some organic components has shown a stress-mitigating effect and can be a valuable procedure to enhance the yield of water stress-sensitive cultivars. The objective of this work was to evaluate the benefits of exogenous application of pyruvic acid (100 µM) in cotton plants under water deficit varying the phenological stage of the crop. The experiment was conducted in a greenhouse, where the plants were grown in pots and subjected to seven days of water suspension, initiated individually in stages V2 and B1. Each pot contained two plants. The treatments adopted were: T1 - control, T2 - water suppression; and T3 - water suppression + pyruvate application. The design was randomized blocks in a factorial scheme (3 × 3) with three replicates. The reductions in gas exchange and growth of the cultivars BRS Seridó, CNPA 7MH and FM 966 were more significant in the reproductive stage, especially for FM 966, which was more sensitive. Pyruvate application reduced the effects of water suppression on boll production by 31% in BRS Seridó and 34% in CNPA 7MH and FM 966.

Pyruvate and lactate based hydrogel film inhibits UV radiation-induced skin inflammation and oxidative stress.

Solar skin damage is one of the most common diseases among outdoor workers. An important cause for the damage is the ultraviolet and infrared rays in sunlight, which are absorbed by the skin in large amounts, leading to severe skin inflammation and oxidative stress. Therefore, physical prevention by shielding the light from harmful wavelengths can be an effective method of skin protection from radiation. However, for existing skin lesions, prompt treatment is essential to avoid the aggravation of the injury and promote repair. Therefore, to improve the therapeutic effect on sun-damaged skin, we attempted to design a system with a dual purpose of eliminating toxic free radicals and modulating tissue inflammatory response. Here, we designed and synthesized a poly-acryloyl lysine (P-Ac-Lys) and polyvinyl alcohol-dihydroxyphenylalanine (PVA-DOPA) composite hydrogel (PAL@PVA-DOPA Hydrogel) loaded with lactate and pyruvate, that exhibites a good free radical scavenging activity and an excellent ability to modulate the inflammatory response. Experimental results showe that this hydrogel film could effectively reduce the UV-induced skin inflammation response, alleviate pathological damage and promote the recovery of the damaged skin.

Exogenous pyruvate and recombinant human basic fibroblast growth factor maintain pluripotency and enhance global metabolic activity of bovine embryonic stem cells grown on low-density feeder layers.

A suitable microenvironment or niche is essential for self-renewal and pluripotency of stem cells cultured in vitro, including bovine embryonic stem cells (bESCs). Feeder cells participate in the construction of stem cell niche by secreting growth factors and extracellular matrix proteins. In this study, metabolomics and transcriptomics analyses were used to investigate the effects of low-density feeder cells on bESCs. The results showed that bESCs co-cultured with low-density feeder cells experienced a decrease in pluripotent gene expression, cell differentiation, and a reduction of central carbon metabolic activity. When cell-permeable pyruvate (Pyr) and recombinant human basic fibroblast growth factor (rhbFGF) were added to the culture system, the pluripotency of bESCs on low-density feeder layers was rescued, and acetyl-coenzyme A (AcCoA) synthesis and fatty acid de novo synthesis increased. In addition, rhbFGF enhances the effects of Pyr and activates the overall metabolic level of bESCs grown on low-density feeder layers. This study explored the rescue effects of exogenous Pyr and rhbFGF on bESCs cultured on low-density feeder layers, which will provide a reference for improvement of the PSC culture system through the supplementation of energy metabolites and growth factors.

Combined Treatment of Dichloroacetic Acid and Pyruvate Increased Neuronal Survival after Seizure.

During seizure activity, glucose and Adenosine triphosphate (ATP) levels are significantly decreased in the brain, which is a contributing factor to seizure-induced neuronal death. Dichloroacetic acid (DCA) has been shown to prevent cell death. DCA is also known to be involved in adenosine triphosphate (ATP) production by activating pyruvate dehydrogenase (PDH), a gatekeeper of glucose oxidation, as a pyruvate dehydrogenase kinase (PDK) inhibitor. To confirm these findings, in this study, rats were given a per oral (P.O.) injection of DCA (100 mg/kg) with pyruvate (50 mg/kg) once per day for 1 week starting 2 h after the onset of seizures induced by pilocarpine administration. Neuronal death and oxidative stress were assessed 1 week after seizure to determine if the combined treatment of pyruvate and DCA increased neuronal survival and reduced oxidative damage in the hippocampus. We found that the combined treatment of pyruvate and DCA showed protective effects against seizure-associated hippocampal neuronal cell death compared to the vehicle-treated group. Treatment with combined pyruvate and DCA after seizure may have a therapeutic effect by increasing the proportion of pyruvate converted to ATP. Thus, the current research demonstrates that the combined treatment of pyruvate and DCA may have therapeutic potential in seizure-induced neuronal death.

Pyruvate prevents the onset of the cachectic features and metabolic alterations in myotubes downregulating STAT3 signaling.

Cachexia is a systemic disease associated with several pathologies, including cancer, that leads to excessive weight loss due to enhanced protein degradation. Previously, we showed that cachectic features in myotubes are provoked by a metabolic shift toward lactic fermentation. Our previous results led us to hyphotesise that increasing pyruvate concentration could impede the metabolic modifications responsible for induction of cachexia in myotubes. Here, we demonstrated that the addition of sodium pyruvate in conditioned media from CT26 colon cancer cells (CM CT26) prevents the onset of either phenotypic and metabolic cachectic features. Myotubes treated with CM CT26 containing sodium pyruvate show a phenotype similar to the healthy counterpart and display lactate production, oxygen consumption, and pyruvate dehydrogenase activity as control myotubes. The use of the Mitochondrial Pyruvate Carrier inhibitor UK5099, highlights the importance of mitochondrial pyruvate amount in the prevention of cachexia. Indeed, UK5099-treated myotubes show cachectic features as those observed in myotubes treated with CM CT26. Finally, we found that sodium pyruvate is able to decrease STAT3 phosphorylation level, a signaling pathway involved in the induction of cachexia in myotubes. Collectively, our results show that cachexia in myotubes could be prevented by the utilization of sodium pyruvate which impedes the metabolic modifications responsible for the acquisition of the cachectic features.

Pyruvate Prevents Dopaminergic Neurodegeneration and Motor Deficits in the 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine Model of Parkinson's Disease.

Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by the selective loss of dopamine(DA)rgic neurons in the substantia nigra of the midbrain, and primarily causes motor symptoms. While the pathological cause of PD remains uncertain, oxidative damage, neuroinflammation, and energy metabolic perturbation have been implicated. Pyruvate has been shown neuroprotective in animal models for many neurological disorders, presumably owing to its potent anti-oxidative, anti-inflammatory, and energy metabolic properties. We therefore investigated whether exogenous pyruvate could also protect nigral DA neurons from degeneration and reverse the associated motor deficits in an animal model of PD using the DA neuron-specific toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MPTP (20 mg/kg) was injected four times every 2 h into the peritoneum of mice, which resulted in a massive loss of DA neurons as well as an increase in neuronal death and cytosolic labile zinc overload. There were rises in inflammatory and oxidative responses, a drop in the striatal DA level, and the emergence of PD-related motor deficits. In comparison, when sodium pyruvate was administered intraperitoneally at a daily dose of 250 mg/kg for 7 days starting 2 h after the final MPTP treatment, significant relief in the MPTP-induced neuropathology, neurodegeneration, DA depletion, and motor symptoms was observed. Equiosmolar dose of NaCl had no neuroprotective effect, and lower doses of sodium pyruvate did not have any statistically significant effects. These findings suggest that pyruvate has therapeutic potential for the treatment of PD and related neurodegenerative diseases.

Time and charge/pH-dependent activation of K+ channel-mediated K+ influx and K+/H+ exchange in guinea pig heart isolated mitochondria; role in bioenergetic stability.

Mitochondria play an important role not only in producing energy for the cell but also for regulating mitochondrial and cell function depending on the cell's needs and environment. Uptake of cations, anions, and substrates requires a stable, polarized transmembrane charge potential (ΔΨm). Chemiosmosis requires ion exchangers to remove Na+, K+, Ca2+, PO43-, and other charged species that enter mitochondria. Knowledge of the kinetics of mitochondrial (m) cation channels and exchangers is important in understanding their roles in regulating mitochondrial chemiosmosis and bioenergetics. The influx/efflux of K+, the most abundant mitochondrial cation, alters mitochondrial volume and shape by bringing in anions and H2O by osmosis. The effects of K+ uptake through ligand-specific mK+ channels stimulated/inhibited by agonists/antagonists on mitochondrial volume (swelling/contraction) are well known. However, a more important role for K+ influx is likely its effects on H+ cycling and bioenergetics facilitated by mitochondrial (m) K+/H+ exchange (mKHE), though the kinetics and consequences of K+ efflux by KHE are not well described. We hypothesized that a major role of K+ influx/efflux is stimulation of respiration via the influx of H+ by KHE. We proposed to modulate KHE activity by energizing guinea pig heart isolated mitochondria and by altering the mK+ cycle to capture changes in mitochondrial volume, pHm, ΔΨm, and respiration that would reflect a role for H+ influx via KHE to regulate bioenergetics. To test this, mitochondria were suspended in a 150 mM K+ buffer at pH 6.9, or in a 140 mM Cs+ buffer at pH 7.6 or 6.9 with added 10 mM K+, minimal Ca2+ and free of Na+. O2 content was measured by a Clark electrode, and pHm, ΔΨm, and volume, were measured by fluorescence spectrophotometry and light-scattering. Adding pyruvic acid (PA) alone caused increases in volume and respiration and a rapid decrease in the transmembrane pH gradient (ΔpHm = pHin-pHext) at pHext 6.9> > 7.6, so that ΔΨm was charged and maintained. BKCa agonist NS1619 and antagonist paxilline modified these effects, and KHE inhibitor quinine and K+ ionophore valinomycin depolarized ΔΨm. We postulate that K+ efflux-induced H+ influx via KHE causes an inward H+ leak that stimulates respiration, but at buffer pH 6.9 also utilizes the energy of ΔpHm, the smaller component of the overall proton motive force, ΔµH+. Thus ΔpHm establishes and maintains the ΔΨm required for utilization of substrates, entry of all cations, and for oxidative phosphorylation. Thus, K+ influx/efflux appears to play a pivotal role in regulating energetics while maintaining mitochondrial ionic balance and volume homeostasis.

Glycolytic potential enhanced by blockade of pyruvate influx into mitochondria sensitizes prostate cancer to detection and radiotherapy.

OBJECTIVE: This study aimed to evaluate the effects of mitochondrial pyruvate carrier (MPC) blockade on the sensitivity of detection and radiotherapy of prostate cancer (PCa). METHODS: We investigated glycolysis reprogramming and MPC changes in patients with PCa by using metabolic profiling, RNA-Seq, and tissue microarrays. Transient blockade of pyruvate influx into mitochondria was observed in cellular studies to detect its different effects on prostate carcinoma cells and benign prostate cells. Xenograft mouse models were injected with an MPC inhibitor to evaluate the sensitivity of 18F-fluorodeoxyglucose positron emission tomography with computed tomography and radiotherapy of PCa. Furthermore, the molecular mechanism of this different effect of transient blockage towards benign prostate cells and prostate cancer cells was studied in vitro. RESULTS: MPC was elevated in PCa tissue compared with benign prostate tissue, but decreased during cancer progression. The transient blockade increased PCa cell proliferation while decreasing benign prostate cell proliferation, thus increasing the sensitivity of PCa cells to 18F-PET/CT (SUVavg, P = 0.016; SUVmax, P = 0.03) and radiotherapy (P < 0.01). This differential effect of MPC on PCa and benign prostate cells was dependent on regulation by a VDAC1-MPC-mitochondrial homeostasis-glycolysis pathway. CONCLUSIONS: Blockade of pyruvate influx into mitochondria increased glycolysis levels in PCa but not in non-carcinoma prostate tissue. This transient blockage sensitized PCa to both detection and radiotherapy, thus indicating that glycolytic potential is a novel mechanism underlying PCa progression. The change in the mitochondrial pyruvate influx caused by transient MPC blockade provides a critical target for PCa diagnosis and treatment.

Glucose Conferred Irinotecan Chemoresistance through Divergent Actions of Pyruvate and ATP in Cell Death and Proliferation of Colorectal Cancer.

BACKGROUND: Altered glucose metabolism is associated with chemoresistance in colorectal cancer (CRC). This study aimed to illustrate the molecular mechanisms of glucose-mediated chemoresistance against irinotecan, a topoisomerase I inhibitor, focusing on the distinct roles of metabolites such as pyruvate and ATP in modulating cell death and proliferation. METHODS: Four human CRC cell lines, tumorspheres, and mouse xenograft models were treated with various doses of irinotecan in the presence of various concentrations of glucose, pyruvate, or ATP-encapsulated liposomes. RESULTS: In this study, human CRC cell lines treated with irinotecan in high glucose displayed increased cell viability and larger xenograft tumor sizes in mouse models compared to those treated in normal glucose concentrations. Irinotecan induced apoptosis and necroptosis, both mitigated by high glucose. Liposomal ATP prevented irinotecan-induced apoptosis, while it did not affect necroptosis. In contrast, pyruvate attenuated the receptor-interacting protein kinase 1/3-dependent necroptosis via free radical scavenging without modulating apoptotic levels. Regarding the cell cycle, liposomal ATP aggravated the irinotecan-induced G0/G1 shift, whereas pyruvate diminished the G0/G1 shift, showing opposite effects on proliferation. Last, tumorsphere structural damage, an index of solid tumor responsiveness to chemotherapy, was determined. Liposomal ATP increased tumorsphere size while pyruvate prevented the deformation of spheroid mass. CONCLUSIONS: Glucose metabolites confer tumor chemoresistance via multiple modes of action. Glycolytic pyruvate attenuated irinotecan-induced necroptosis and potentiated drug insensitivity by shifting cells from a proliferative to a quiescent state. On the other hand, ATP decreased irinotecan-induced apoptosis and promoted active cell proliferation, contributing to tumor recurrence. Our findings challenged the traditional view of ATP as the main factor for irinotecan chemoresistance and provided novel insights of pyruvate acting as an antioxidant responsible for drug insensitivity, which may shed light on the development of new therapies against recalcitrant cancers.

Tumor cells dictate anti-tumor immune responses by altering pyruvate utilization and succinate signaling in CD8+ T cells.

The tumor microenvironment (TME) is a unique metabolic niche that can inhibit T cell metabolism and cytotoxicity. To dissect the metabolic interplay between tumors and T cells, we establish an in vitro system that recapitulates the metabolic niche of the TME and allows us to define cell-specific metabolism. We identify tumor-derived lactate as an inhibitor of CD8+ T cell cytotoxicity, revealing an unexpected metabolic shunt in the TCA cycle. Metabolically fit cytotoxic T cells shunt succinate out of the TCA cycle to promote autocrine signaling via the succinate receptor (SUCNR1). Cytotoxic T cells are reliant on pyruvate carboxylase (PC) to replenish TCA cycle intermediates. By contrast, lactate reduces PC-mediated anaplerosis. The inhibition of pyruvate dehydrogenase (PDH) is sufficient to restore PC activity, succinate secretion, and the activation of SUCNR1. These studies identify PDH as a potential drug target to allow CD8+ T cells to retain cytotoxicity and overcome a lactate-enriched TME.

The Antitumor Effect of Cinnamaldehyde Derivative CB-PIC in Hepatocellular Carcinoma Cells via Inhibition of Pyruvate and STAT3 Signaling.

Though cinnamaldehyde derivative (CB-PIC), a major compound of cinnamon, is known to have anticancer activity, its underlying mechanism is not fully understood. In the present study, the anticancer mechanism of CB-PIC was investigated in human hepatocellular carcinoma cells (HCCs) in association with signal transducer and activator of transcription 3 (STAT3) signaling. CB-PIC exerted cytotoxicity in HepG2 and Huh7 cells. CB-PIC increased the sub G1 population and attenuated the expression of pro-poly (ADP-ribose) polymerase (PARP) and pro-Caspase3 in HepG2 and Huh7 cells. Interestingly, CB-PIC significantly abrogated the expression of a glycolytic enzyme pyruvate kinase M2 (PKM2) in HepG2 cells more than in LNCaP, A549, and HCT-116 cells. Consistently, CB-PIC reduced the expression of hexokinase 2 (HK2) and PKM2, along with a reduced production of lactate in HepG2 and Huh7 cells. Notably, CB-PIC suppressed the phosphorylation of STAT3 in HepG2 and Huh7 cells and conversely STAT3 depletion enhanced the capacity of CB-PIC to suppress the expression of HK2, PKM2, and pro-caspase3 and to reduce the viability in Huh7 cells. Furthermore, CB-PIC activated the phosphorylation of AMPK and ERK and suppressed expression of IL-6 as STAT3-related genes in HepG2 and Huh7 cells. Conversely, pyruvate treatment reversed the inhibitory effect of CB-PIC on p-STAT3, HK2, PKM2, and pro-PARP in Huh7 cells. Overall, there findings suggest that CB-PIC exerts an apoptotic effect via inhibition of the Warburg effect mediated by p-STAT3 and pyruvate signaling.