Uncoupling Research Articles

Abstract 4687: Primary study on the anticancer role of BAM15 in lung cancer cells

Abstract Background: Lung cancer remains the leading cause of cancer-related death worldwide. Non-small cell lung cancer (NSCLC) accounts for about 80-85% of all lung cancer cases. BAM15, a mitochondrial uncoupler, is a potential therapeutic agent for many diseases, including obesity, diabetes, nonalcoholic fatty liver disease, Sepsis and cancers. However, there is no report on the anti-tumor role of BAM15 in NSCLC. Methods: To investigate the direct anti-cancer effects of BAM15 in NSCLC, we examined its inhibitory role on cell proliferation of NSCLC cells using the Cell Counting Kit-8(CCK8), colony forming assays and EdU staining assays. The impact of BAM15 on the cell cycle and cell apoptosis of NSCLC cells was analyzed by flow cytometric. A xenotransplanted NSCLC model in mice were used to test the anticancer role of BAM15. Result: Using the CCK8 assay, we found that the inhibitory role of BAM15 on the proliferation and colony formation of NSCLC cells is dose-dependent. The half maximal inhibitory concentration (IC50) values for cell proliferation were 2.71, 4.74, and 9.50 μM for A549, PC9, and H1299 NSCLC cells, respectively. The EdU staining assay demonstrated that BAM15 treatment significantly inhibited DNA synthesis in NSCLC cells. Flow cytometric analysis revealed that BAM15 arrested cell cycle at G2 phase in NSCLC cells and caused a striking increase in the percentage of apoptotic cells in NSCLC cells. The xenotransplanted NSCLC experiment indicated that BAM15 could significantly suppressed the growth of NSCLC tumors compared with the control tumors. Conclusions: Our study demonstrates that BAM15 significantly inhibits cell proliferation and promotes apoptosis in NSCLC cells, indicating that BAM15 is a potential therapeutic candidate for the treatment of NSCLC. Citation Format: Mei-yin Zhang, Yue-Ning Wang, Yu-Feng Zhou, Shuo-Cheng Wang, Shi-Juan Mai, Hui-Yun Wang. Primary study on the anticancer role of BAM15 in lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4687.

Abstract 3071: Differential susceptibility of breast cancer cells to mitochondrial dysfunction induced by Wnt inhibitors

Abstract Advances in research have shown that cancer cells have distinctive bioenergetic alterations to meet their energy requirements. It has been established that certain cancers are significantly reliant on mitochondrial respiration, suggesting that modulation of mitochondrial function may have therapeutic value. The Wnt signaling pathway is a potential therapeutic target of cancer, including breast and colorectal cancers. Recent findings suggest that bi-directional crosstalk between Wnt signaling and mitochondrial function plays a crucial role in tumorigenesis, as well as differentiation and self-renewal of stem cells. In this study, 71 agents known to alter Wnt signaling were screened for effects on mitochondrial function in BT474 and Hs578T breast cancer cell lines, which have highly oxidative and highly glycolytic bioenergetic phenotypes, respectively. This screen was performed using the Agilent Seahorse XF Mito Tox assay, which enables sensitive detection and characterization of mitochondrial modulators via a standardized, quantitative parameter, the Mito Tox Index (MTI). The MTI facilitates the comparison of drug efficacy by detecting modes of mitochondrial toxicity, inhibition, and uncoupling. The MTI-based screening of the Wnt modulators identified one inhibitor and two uncouplers as potent agents inducing mitochondrial dysfunction for both BT474 and Hs578T. Despite their distinctive bioenergetic phenotypes, MTI-based dose-response assays revealed no significant differences in the efficacy of these modulators between BT474 and Hs578T cell types. In contrast, significant differential effects on ATP production rates were detected, showing cellular energy depletion induced by these agents. Using the Agilent Seahorse XF Real-Time ATP Rate assay, BT474 cells showed significant metabolic energy crisis in the presence of all 3 compounds, whereas Hs578T cells maintained stable total ATP production. This differential impact on ATP production rate corresponds directly to differences in cell viability, with BT474 showing higher susceptibility to these compounds. These results suggest that the anti-cancer efficacy of mitochondrial modulators can be different depending on the metabolic phenotype of the target cell and should be considered in therapeutic design. Citation Format: Yoonseok Kam, Lisa Winer, George W. Rogers, Natalia Romero. Differential susceptibility of breast cancer cells to mitochondrial dysfunction induced by Wnt inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3071.

Effect of Graphene Oxide and Ammonia-modified Graphene Oxide Particles on ATPase Activity of Rat Liver Mitochondria

Graphene and its derivatives have become promising materials for biomedical applications in the last decade. Before their widespread application, however, evaluating their toxicity and mechanisms underlying interactions with cellular components is imperative. Aims: Assessment of the effect of two graphene derivatives, pristine graphene oxide (GO) and ammonia-modified GO (GO-NH2) particles, on the ATPase activity of rat liver mitochondria and ROS production. Methods: Liver mitochondria were isolated from male albino rats and treated with different concentrations of GO and GO-NH2 particles (4, 10, 25, and 50 μg/ml). ATPase activity of both, intact and uncoupled by freezing/thawing mitochondria was determined by the measurement of inorganic phosphate (Pi) released from ATP. The generation of hydrogen peroxide (H2O2) after exposure of mitochondria to GO and GO-NH2 particles was determined by a DCFH-D assay. Results: GO and GO-NH2 particles applied at concentrations of 4 and 50 μg/ml did not affect the ATPase activity of intact mitochondria. In contrast, in uncoupled mitochondria, they demonstrated a stimulating effect on ATPase activity. The impact of GO-NH2 was more substantial and concentration-dependent. ROS production was also higher in GO-NH2-treated mitochondria. Conclusion: The present study demonstrated that GO and GO-NH2 particles can exert a cytotoxic effect on mitochondria even after a short-time of exposure to both types of particles.

Effect of the mitochondrial uncoupling agent BAM15 against the Toxoplasma gondii RH strain and Prugniaud strain

BackgroundToxoplasmosis is a zoonotic disease caused by the infection of the protozoa Toxoplasma gondii (T. gondii), and safe and effective therapeutic drugs are lacking. Mitochondria, is an important organelle that maintains T. gondii survival, however, drugs targeting mitochondria are lacking.MethodsThe cytotoxicity of BAM15 was detected by CCK-8 and the in vitro effects of BAM15 was detected by qPCR, plaque assay and flow cytometry. Furthermore, the ultrastructural changes of T. gondii after BAM15 treatment were observed by transmission electron microscopy, and further the mitochondrial membrane potential (ΔΨm), ATP level and reactive oxygen species (ROS) of T. gondii after BAM15 treatment were detected. The pharmacokinetic experiments and in vivo infection assays were performed in mice to determine the in vivo effect of BAM15.ResultsBAM15 had excellent anti-T. gondii activity in vitro and in vivo with an EC50 value of 1.25 μM, while the IC50 of BAM15 in Vero cells was 27.07 μM. Notably, BAM15 significantly inhibited proliferation activity of T. gondii RH strain and Prugniaud strain (PRU), caused T. gondii death. Furthermore, BAM15 treatment induced T. gondii mitochondrial vacuolation and autolysis by TEM. Moreover, the decrease in ΔΨm and ATP level, as well as the increase in ROS production further confirmed the changesConclusionsOur study identifies a useful T. gondii mitochondrial inhibitor, which may also serve as a leading molecule to develop therapeutic mitochondrial inhibitors in toxoplasmosis.’Graphical

Synthesis of Triphenylphosphonium-Linked Derivative of 3,5-Ditert-butyl-4-hydroxybenzylidene-malononitrile (SF6847) via Knoevenagel Reaction Yields an Effective Mitochondria-Targeted Protonophoric Uncoupler.

Mitochondrial uncouplers are actively sought as potential therapeutics. Here, we report the first successful synthesis of mitochondria-targeted derivatives of the highly potent uncoupler 3,5-ditert-butyl-4-hydroxybenzylidene-malononitrile (SF6847), bearing a cationic alkyl(triphenyl)phosphonium (TPP) group. As a key step of the synthesis, we used condensation of a ketophenol with malononitrile via the Knoevenagel reaction. SF-C5-TPP with a pentamethylene linker between SF6847 and TPP, stimulating respiration and collapsing membrane potential of rat liver mitochondria at submicromolar concentrations, proved to be the most effective uncoupler of the series. SF-C5-TPP showed pronounced protonophoric activity on a model planar bilayer lipid membrane. Importantly, SF-C5-TPP exhibited rather low toxicity in fibroblast cell culture, causing mitochondrial depolarization in cells at concentrations that only slightly affected cell viability. SF-C5-TPP was more effective in decreasing the mitochondrial membrane potential in the cell culture than SF6847, in contrast to the case of isolated mitochondria. Like other zwitterionic uncouplers, SF-C5-TPP inhibited the growth of Bacillus subtilis in the micromolar concentration range.

Mitochondrial uncoupling proteins protect human airway epithelial ciliated cells from oxidative damage

Apical cilia on epithelial cells defend the lung by propelling pathogens and particulates out of the respiratory airways. Ciliated cells produce ATP that powers cilia beating by densely grouping mitochondria just beneath the apical membrane. However, this efficient localization comes at a cost because electrons leaked during oxidative phosphorylation react with molecular oxygen to form superoxide, and thus, the cluster of mitochondria creates a hotspot for oxidant production. The relatively high oxygen concentration overlying airway epithelia further intensifies the risk of generating superoxide. Thus, airway ciliated cells face a unique challenge of producing harmful levels of oxidants. However, surprisingly, highly ciliated epithelia produce less reactive oxygen species (ROS) than epithelia with few ciliated cells. Compared to other airway cell types, ciliated cells express high levels of mitochondrial uncoupling proteins, UCP2 and UCP5. These proteins decrease mitochondrial protonmotive force and thereby reduce production of ROS. As a result, lipid peroxidation, a marker of oxidant injury, decreases. However, mitochondrial uncoupling proteins exact a price for decreasing oxidant production; they decrease the fraction of mitochondrial respiration that generates ATP. These findings indicate that ciliated cells sacrifice mitochondrial efficiency in exchange for safety from damaging oxidation. Employing uncoupling proteins to prevent oxidant production, instead of relying solely on antioxidants to decrease postproduction oxidant levels, may offer an advantage for targeting a local area of intense ROS generation.

Conjugating uncoupler compounds with hydrophobic hydrocarbon chains to achieve adipose tissue selective drug accumulation

One potential approach for treating obesity is to increase energy expenditure in brown and white adipose tissue. Here we aimed to achieve this outcome by targeting mitochondrial uncoupler compounds selectively to adipose tissue, thus avoiding side effects from uncoupling in other tissues. Selective drug accumulation in adipose tissue has been observed with many lipophilic compounds and dyes. Hence, we explored the feasibility of conjugating uncoupler compounds with a lipophilic C8-hydrocarbon chain via an ether bond. We found that substituting the trifluoromethoxy group in the uncoupler FCCP with a C8-hydrocarbon chain resulted in potent uncoupling activity. Nonetheless, the compound did not elicit therapeutic effects in mice, likely as a consequence of metabolic instability resulting from rapid ether bond cleavage. A lipophilic analog of the uncoupler compound 2,6-dinitrophenol, in which a C8-hydrocarbon chain was conjugated via an ether bond in the para-position (2,6-dinitro-4-(octyloxy)phenol), exhibited increased uncoupling activity compared to the parent compound. However, in vivo pharmacokinetics studies suggested that 2,6-dinitro-4-(octyloxy)phenol was also metabolically unstable. In conclusion, conjugation of a hydrophobic hydrocarbon chain to uncoupler compounds resulted in sustained or improved uncoupling activity. However, an ether bond linkage led to metabolic instability, indicating the need to conjugate lipophilic groups via other chemical bonds.

Divergent Roles of Escherichia Coli Encoded Lon Protease in Imparting Resistance to Uncouplers of Oxidative Phosphorylation: Roles of marA, rob, soxS and acrB.

Uncouplers of oxidative phosphorylation dissipate the proton gradient, causing lower ATP production. Bacteria encounter several non-classical uncouplers in the environment, leading to stress-induced adaptations. Here, we addressed the molecular mechanisms responsible for the effects of uncouplers in Escherichia coli. The expression and functions of genes involved in phenotypic antibiotic resistance were studied using three compounds: two strong uncouplers, i.e., Carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and 2,4-Dinitrophenol (DNP), and one moderate uncoupler, i.e., Sodium salicylate (NaSal). Quantitative expression studies demonstrated induction of transcripts encoding marA, soxS and acrB with NaSal and DNP, but not CCCP. Since MarA and SoxS are degraded by the Lon protease, we investigated the roles of Lon using a lon-deficient strain (Δlon). Compared to the wild-type strain, Δlon shows compromised growth upon exposure to NaSal or 2, 4-DNP. This sensitivity is dependent on marA but not rob and soxS. On the other hand, the Δlon strain shows enhanced growth in the presence of CCCP, which is dependent on acrB. Interestingly, NaSal and 2,4-DNP, but not CCCP, induce resistance to antibiotics, such as ciprofloxacin and tetracycline. This study addresses the effects of uncouplers and the roles of genes involved during bacterial growth and phenotypic antibiotic resistance. Strong uncouplers are often used to treat wastewater, and these results shed light on the possible mechanisms by which bacteria respond to uncouplers. Also, the rampant usage of some uncouplers to treat wastewater may lead to the development of antibiotic resistance.

Beneficial effects of simultaneously targeting calorie intake and calorie efficiency in diet-induced obese mice.

Semaglutide is an anti-diabetes and weight loss drug that decreases food intake, slows gastric emptying, and increases insulin secretion. Patients begin treatment with low-dose semaglutide and increase dosage over time as efficacy plateaus. With increasing dosage, there is also greater incidence of gastrointestinal side effects. One reason for the plateau in semaglutide efficacy despite continued low food intake is due to compensatory actions whereby the body becomes more metabolically efficient to defend against further weight loss. Mitochondrial uncoupler drugs decrease metabolic efficiency, therefore we sought to investigate the combination therapy of semaglutide with the mitochondrial uncoupler BAM15 in diet-induced obese mice. Mice were fed high-fat western diet (WD) and stratified into six treatment groups including WD control, BAM15, low-dose semaglutide without or with BAM15, and high-dose semaglutide without or with BAM15. Combining BAM15 with either semaglutide dose decreased body fat and liver triglycerides, which was not achieved by any monotherapy, while high-dose semaglutide with BAM15 had the greatest effect on glucose homeostasis. This study demonstrates a novel approach to improve weight loss without loss of lean mass and improve glucose control by simultaneously targeting energy intake and energy efficiency. Such a combination may decrease the need for semaglutide dose escalation and hence minimize potential gastrointestinal side effects.

Differential responses to UCP1 ablation in classical brown versus beige fat, despite a parallel increase in sympathetic innervation

In the cold, the absence of the mitochondrial uncoupling protein 1 (UCP1) results in hyper-recruitment of beige fat, but classical brown fat becomes atrophied. Here we examine possible mechanisms underlying this phenomenon. We confirm that in brown fat from UCP1-knockout (UCP1-KO) mice acclimated to the cold, the levels of mitochondrial respiratory chain proteins were diminished; however, in beige fat, the mitochondria seemed to be unaffected. The macrophages that accumulated massively not only in brown fat but also in beige fat of the UCP1-KO mice acclimated to cold did not express tyrosine hydroxylase, the norepinephrine transporter (NET) and monoamine oxidase-A (MAO-A). Consequently, they could not influence the tissues through the synthesis or degradation of norepinephrine. Unexpectedly, in the cold, both brown and beige adipocytes from UCP1-KO mice acquired an ability to express MAO-A. Adipose tissue norepinephrine was exclusively of sympathetic origin, and sympathetic innervation significantly increased in both tissues of UCP1-KO mice. Importantly, the magnitude of sympathetic innervation and the expression levels of genes induced by adrenergic stimulation were much higher in brown fat. Therefore, we conclude that no qualitative differences in innervation or macrophage character could explain the contrasting reactions of brown versus beige adipose tissues to UCP1-ablation. Instead, these contrasting responses may be explained by quantitative differences in sympathetic innervation: the beige adipose depot from the UCP1-KO mice responded to cold acclimation in a canonical manner and displayed enhanced recruitment, while the atrophy of brown fat lacking UCP1 may be seen as a consequence of supraphysiological adrenergic stimulation in this tissue.

The acute and chronic low-temperature stress responses in Porites lutea from a relatively high-latitude coral reef of the South China Sea

Relatively high-latitude coral reefs could be potential “refuges” for corals under climate change. One of the most important aspects limiting their availability as refuges is low-temperature stress. However, the mechanisms underlying the response of coral holobionts to low-temperature stress is unclear. In this study, we aimed to explore the underlying mechanisms by recording the maximum quantum yields of photosystem II (Fv/Fm) and transcriptome responses of Porites lutea under acute (1–2 weeks) and chronic (6–12 weeks) low-temperature stress at 20°C and 14°C. The P. lutea samples were collected from a relatively high-latitude coral reef in the South China Sea (109°00′–109°15′E and 21°00′–21°10′ N). The study suggested that: (1) Under acute low-temperature stress, the Fv/Fm of Symbiodiniaceae dropped by 64%, which was significantly higher than the 49% observed under chronic stress. Low-temperature stress inhibited photosystem II(PSII) functioning, with greater inhibition under acute stress. (2) Downregulation of sugar metabolism-related genes under low-temperature stress implied that the decrease in energy was due to obstruction of PSII. (3) Under low-temperature stress, calcification-related genes were downregulated in coral hosts, possibly because of energy deprivation caused by inhibited photosynthesis, Symbiodiniaceae expulsion, and oxidative phosphorylation uncoupling in mitochondria. (4) Acute low-temperature stress induced the upregulation of genes related to the TNF signaling pathway and endoplasmic reticulum stress, promoting apoptosis and coral bleaching. However, these phenomena were not observed during chronic stress, suggesting acclimation to chronic low-temperature stress and a greater survival pressure of acute low-temperature stress on coral holobionts. In conclusion, low-temperature stress inhibits Symbiodiniaceae PSII functioning, reducing energy production and affecting calcification in coral holobionts. Acute low-temperature stress is more threatening to coral holobionts than chronic stress.

Pyrazinoic acid, the active form of the anti-tuberculosis drug pyrazinamide, and aromatic carboxylic acid analogs are protonophores.

Pyrazinoic acid is the active form of pyrazinamide, a first-line antibiotic used to treat Mycobacterium tuberculosis infections. However, the mechanism of action of pyrazinoic acid remains a subject of debate, and alternatives to pyrazinamide in cases of resistance are not available. The work presented here demonstrates that pyrazinoic acid and known protonophores including salicylic acid, benzoic acid, and carbonyl cyanide m-chlorophenyl hydrazone all exhibit pH-dependent inhibition of mycobacterial growth activity over a physiologically relevant range of pH values. Other anti-tubercular drugs, including rifampin, isoniazid, bedaquiline, and p-aminosalicylic acid, do not exhibit similar pH-dependent growth-inhibitory activities. The growth inhibition curves of pyrazinoic, salicylic, benzoic, and picolinic acids, as well as carbonyl cyanide m-chlorophenyl hydrazone, all fit a quantitative structure-activity relationship (QSAR) derived from acid-base equilibria with R2 values > 0.95. The QSAR model indicates that growth inhibition relies solely on the concentration of the protonated forms of these weak acids (rather than the deprotonated forms). Moreover, pyrazinoic acid, salicylic acid, and carbonyl cyanide m-chlorophenyl hydrazone all caused acidification of the mycobacterial cytoplasm at concentrations that inhibit bacterial growth. Thus, it is concluded that pyrazinoic acid acts as an uncoupler of oxidative phosphorylation and that disruption of proton motive force is the primary mechanism of action of pyrazinoic acid rather than the inhibition of a classic enzyme activity.

Restricting bioenergetic efficiency enhances longevity and mitochondrial redox capacity in Drosophila melanogaster.

Mitochondria are essential for survival and as such, impairments in organelle homeostasis significantly accelerate age-related morbidity and mortality. Here, we determined the contribution of bioenergetic efficiency to life span and health span in Drosophila melanogaster utilizing the mitochondrial uncoupler BAM15. Life span was determined in flies fed a normal diet (ND) or high fat diet (HFD) supplemented with vehicle or BAM15. Locomotor function was determined by negative geotaxis assay in middle-aged flies fed vehicle or BAM15 under ND or HFD conditions. Redox capacity (high-resolution respirometry/fluorometry), citrate synthase (enzyme activity), mtDNA content (qPCR), gene expression (qPCR), and protein expression (western blot) were assessed in flight muscle homogenates of middle-aged flies fed vehicle or BAM15 ND. The molar ratio of H2O2 and O2 (H2O2:O2) in a defined respiratory state was calculated as a measure of redox balance. BAM15 extended life span by 9% on ND and 25% on HFD and improved locomotor activity by 125% on ND and 53% on HFD. Additionally, BAM15 enhanced oxidative phosphorylation capacity supported by pyruvate + malate, proline, and glycerol 3-phosphate. Concurrently, BAM15 enhanced the mitochondrial H2O2 production rate, reverse electron flow from mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH) to Complex I, mGPDH, and Complex I without altering the H2O2:O2 ratio. BAM15 upregulated transcriptional signatures associated with mitochondrial function and fitness as well as antioxidant defense. BAM15-mediated restriction of bioenergetic efficiency prolongs life span and health span in Drosophila fed a ND or HFD. Improvements in life span and health span in ND were supported by synergistic enhancement of muscular redox capacity.

The role of mitochondrial uncoupling in the regulation of mitostasis after traumatic brain injury

Mitostasis, the maintenance of healthy mitochondria, plays a critical role in brain health. The brain's high energy demands and reliance on mitochondria for energy production make mitostasis vital for neuronal function. Traumatic brain injury (TBI) disrupts mitochondrial homeostasis, leading to secondary cellular damage, neuronal degeneration, and cognitive deficits. Mild mitochondrial uncoupling, which dissociates ATP production from oxygen consumption, offers a promising avenue for TBI treatment. Accumulating evidence, from endogenous and exogenous mitochondrial uncoupling, suggests that mitostasis is closely regulating by mitochondrial uncoupling and cellular injury environments may be more sensitive to uncoupling. Mitochondrial uncoupling can mitigate calcium overload, reduce oxidative stress, and induce mitochondrial proteostasis and mitophagy, a process that eliminates damaged mitochondria. The interplay between mitochondrial uncoupling and mitostasis is ripe for further investigation in the context of TBI. These multi-faceted mechanisms of action for mitochondrial uncoupling hold promise for TBI therapy, with the potential to restore mitochondrial health, improve neurological outcomes, and prevent long-term TBI-related pathology.

Exercise-inducible circulating extracellular vesicle irisin promotes browning and the thermogenic program in white adipose tissue.

Exercise can reduce body weight and promote white fat browning, but the underlying mechanisms remain largely unknown. This study investigated the role of fibronectin type III domain-containing protein 5 (FNDC5)/Irisin, a hormone released from exercising muscle, in the browning of white fat in circulating extracellular vesicles (EVs). Mice were subjected to a 4 weeks of running table exercise, and fat browning was analyzed via histology, protein blotting and qPCR. Circulating EVs were extracted by ultrahigh-speed centrifugation, and ELISA was used to measure the irisin concentration in the circulating EVs. Circulating EVs that differentially expressed irisin were applied to adipocytes, and the effect of EV-irisin on adipocyte energy metabolism was analyzed by immunofluorescence, protein blotting, and cellular oxygen consumption rate analysis. During sustained exercise, the mice lost weight and developed fat browning. FNDC5 was induced, cleaved, and secreted into irisin, and irisin levels subsequently increased in the plasma during exercise. Interestingly, irisin was highly expressed in circulating EVs that effectively promoted adipose browning. Mechanistically, the circulating EV-irisin complex is transported intracellularly by the adipocyte membrane receptor integrin αV, which in turn activates the AMPK signaling pathway, which is dependent on mitochondrial uncoupling protein 1 to cause mitochondrial plasmonic leakage and promote heat production. After inhibition of the AMPK signaling pathway, the effects of the EV-irisin on promoting fat browning were minimal. Exercise leads to the accumulation of circulating EV-irisin, which enhances adipose energy metabolism and thermogenesis and promotes white fat browning in mice, leading to weight loss.

The BCKDH kinase inhibitor BT2 promotes BCAA disposal and mitochondrial proton leak in both insulin-sensitive and insulin-resistant C2C12 myotubes.

Elevated circulating branched-chain amino acids (BCAAs) have been correlated with the severity of insulin resistance, leading to recent investigations that stimulate BCAA metabolism for the potential benefit of metabolic diseases. BT2(3,6-dichlorobenzo[b]thiophene-2-carboxylic acid), an inhibitor of branched-chain ketoacid dehydrogenase kinase, promotes BCAA metabolism by enhancing BCKDH complex activity. The purpose of this report was to investigate the effects of BT2 on mitochondrial and glycolytic metabolism, insulin sensitivity, and de novo lipogenesis both with and without insulin resistance. C2C12 myotubes were treated with or without low or moderate levels of BT2 with or without insulin resistance. Western blot and quantitative real-time polymerase chain reactionwere used to assess protein and gene expression, respectively. Mitochondrial, nuclei, and lipid content were measured using fluorescent staining and microscopy. Cell metabolism was assessed via oxygen consumption and extracellular acidification rate. Liquid chromatography-mass spectrometry was used to quantify BCAA media content. BT2 treatment consistently promoted mitochondrial uncoupling following 24-h treatment, which occurred largely independent of changes in expressional profiles associated with mitochondrial biogenesis, mitochondrial dynamics, BCAA catabolism, insulin sensitivity, or lipogenesis. Acute metabolic studies revealed a significant and dose-dependent effect of BT2 on mitochondrial proton leak, suggesting BT2 functions as a small-molecule uncoupler. Additionally, BT2 treatment consistently and dose-dependently reduced extracellular BCAA levels without altering expression of BCAA catabolic enzymes or pBCKDHa activation. BT2 appears to act as a small-molecule mitochondrial uncoupler that promotes BCAA utilization, though the interplay between these two observations requires further investigation.

Oxaloacetic acid induces muscle energy substrate depletion and fatigue by JNK-mediated mitochondrial uncoupling.

Fatigue is a common phenomenon closely related to physical discomfort and numerous diseases, which is severely threatening the life quality and health of people. However, the exact mechanisms underlying fatigue are not fully characterized. Herein, we demonstrate that oxaloacetic acid (OAA), a crucial tricarboxylic acid cycle intermediate, modulates the muscle fatigue. The results showed that serum OAA level was positively correlated with fatigue state of mice. OAA-treated induced muscle fatigue impaired the exercise performance of mice. Mechanistically, OAA increased the c-Jun N-terminal kinase (JNK) phosphorylation and uncoupling protein 2 (UCP2) levels in skeletal muscle, which led to decreased energy substrate and enhanced glycolysis. On the other hand, OAA boosted muscle mitochondrial oxidative phosphorylation uncoupled with energy production. In addition, either UCP2 knockout or JNK inhibition totally reversed the effects of OAA on skeletal muscle. Therein, JNK mediated UCP2 activation with OAA-treated. Our studies reveal a novel role of OAA in skeletal muscle metabolism, which would shed light on the mechanism of muscle fatigue and weakness.

Cell-free ascites from ovarian cancer patients induces Warburg metabolism and cell proliferation through TGFβ-ERK signaling

Ascites plays a key role in supporting the metastatic potential of ovarian cancer cells. Shear stress and carry-over of cancer cells by ascites flow support carcinogenesis and metastasis formation. In addition, soluble factors may participate in the procarcinogenic effects of ascites in ovarian cancer. This study aimed to determine the biological effects of cell-free ascites on carcinogenesis in ovarian cancer cells. Cell-free ascites from ovarian cancer patients (ASC) non-selectively induced cell proliferation in multiple models of ovarian cancer and untransformed primary human dermal fibroblasts. Furthermore, ASC induced a Warburg-type rearrangement of cellular metabolism in A2780 ovarian cancer cells characterized by increases in cellular oxygen consumption and glycolytic flux; increases in glycolytic flux were dominant. ASC induced mitochondrial uncoupling and fundamentally reduced fatty acid oxidation. Ascites-elicited effects were uniform among ascites specimens. ASC-elicited transcriptomic changes in A2780 ovarian cancer cells included induction of the TGFβ-ERK/MEK pathway, which plays a key role in inducing cell proliferation and oncometabolism. ASC-induced gene expression changes, as well as the overexpression of members of the TGFβ signaling system, were associated with poor survival in ovarian cancer patients. We provided evidence that the activation of the autocrine/paracrine of TGFβ signaling system may be present in bladder urothelial carcinoma and stomach adenocarcinoma. Database analysis suggests that the TGFβ system may feed forward bladder urothelial carcinoma and stomach adenocarcinoma. Soluble components of ASC support the progression of ovarian cancer. These results suggest that reducing ascites production may play an essential role in the treatment of ovarian cancer by inhibiting the progression and reducing the severity of the disease.

Differential effects of milk, yogurt, and cheese on energy homeostasis and brown adipose tissue phenotype in high-fat diet-induced obese mice.

Aim: We hypothesized that milk, yogurt, and cheese have differential impacts on energy expenditure (EE) and obesity in mice fed a high-fat diet (HFD). Methods: C57BL/6 mice (n = 16 per group) were fed a HFD or a HFD supplemented with fat-free milk (MILK), fat-free plain yogurt (YOG), or reduced-fat cheddar cheese (CHE; 19 kcal% fat), each provided at 10% of the daily energy intake, for 8 weeks. EE was quantified using a metabolic chamber. Metabolic pathways related to BAT mitochondrial function and uncoupling protein 1 (UCP1) abundance were assessed. Serum lipidomic profiles were analyzed to identify potential mediators of the observed effects. Results: MILK supplementation lowered weight gain and fat accumulation and enhanced EE and BAT thermogenesis, perhaps via the SIRT1-AMPK-PGC1α axis in BAT. This led to elevated UCP1 abundance and enhanced the abundance of hormone-sensitive lipase (HSL). MILK also altered serum lipid species, indicating enhanced energy use, and promoted BAT thermogenesis and mitochondrial function pathways. YOG exhibited a similar pattern but a lower magnitude of effects than MILK on reducing weight gain and fat mass, increasing EE, and BAT thermogenic proteins, including AMPK-PGC1α-UCP1. Both MILK and YOG showed a relative increase in serum PC 15:0_15:0 and LPC 15:0. In contrast, CHE reduced weight gain and increased EE without impacting BAT thermogenesis proteins or serum lipid species. Conclusion: Our study showed that MILK, YOG, and CHE reduced weight gain in mice on a HFD by increasing EE. MILK and YOG also up-regulated BAT thermogenesis, while both additionally altered lipids involved in fat metabolism and inflammation. CHE did not affect BAT thermogenesis and lipid species compared to HFD.

A mathematical modeling of the mitochondrial proton leak via quantum tunneling

<abstract> <p>The mitochondrion is a vital intracellular organelle that is responsible for ATP production. It utilizes both the concentration gradient and the electrical potential of the inner mitochondrial membrane to drive the flow of protons from the intermembrane space to the matrix to generate ATP via ATP-synthase. However, the proton leak flow, which is mediated via the inner mitochondrial membrane and uncoupling proteins, can reduce the efficiency of ATP production. Protons can exhibit a quantum behavior within biological systems. However, the investigation of the quantum behavior of protons within the mitochondria is lacking particularly in the contribution to the proton leak. In the present study, we proposed a mathematical model of protons tunneling through the inner mitochondrial membrane and the mitochondrial carrier superfamily MCF including uncoupling proteins UCPs and the adenine nucleotide translocases ANTs. According to the model and its assumptions, the quantum tunneling of protons may contribute significantly to the proton leak if it is compared with the classical flow of protons. The quantum tunneling proton leak may depolarize the membrane potential, hence it may contribute to the physiological regulation of ATP synthesis and reactive oxygen species ROS production. In addition to that, the mathematical model of proton tunneling suggested that the proton-tunneling leak may depolarize the membrane potential to values beyond the physiological needs which in turn can harm the mitochondria and the cells. Moreover, we argued that the quantum proton leak might be more energetically favorable if it is compared with the classical proton leak. This may give the advantage for quantum tunneling of protons to occur since less energy is required to contribute significantly to the proton leak compared with the classical proton flow.</p> </abstract>