Mitochondrial Oxygen Consumption Rate Research Articles

Abstract 4140181: Mechanisms of SGLT-2 Inhibitor Empagliflozin in Attenuating Intramitochondrial Stress and Restoring Mitochondrial Function in Hyperglycemic Cardiomyocyte

Systemic hyperglycemia causes tissue damage and triggers cardiovascular disease (CVD). Sodium-glucose cotransporter-2 inhibitors (SGLT-2i) are a novel class of glucose-lowering agents that have shown unexpected benefits in clinical trials for the treatment of CVDs. We aim to investigate the underlying mechanisms of how SGLT-2 inhibitors alleviate CVDs associated with elevated glucose stress. iPSC-derived cardiomyocytes (iPSC-CM) were incubated with high glucose concentrations for 72 hours. Mitochondrial function in these cardiomyocytes was assessed by flow cytometry with JC-1 staining and ATP luminescence assay. Intracellular reactive oxygen species (ROS) and intramitochondrial calcium stress were measured using CellROX, MitoSOX, and Rhod-2 AM staining. Patch clamp was employed to determine ion current changes in the cardiomyocytes. Mitochondrial oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were determined using the Seahorse XFe96 analyzer. In addition, qPCR, Western blot, and DM mouse heart histological analysis were performed to assess the regulation of associated molecules. The results indicated that exposure to a high glucose environment caused cardiomyocyte injury and impaired mitochondrial biosynthesis. Empagliflozin exhibited a beneficial effect on mitochondrial function by reducing ROS production and calcium deposition. It also mitigated the reduction in respiratory OCR of cardiomyocytes induced by high glucose incubation. Furthermore, molecular analysis revealed that Empagliflozin attenuated the dysregulation of mitochondrial calcium channels and biosynthesis by reducing associated gene expression, including Bcl2 , Mfn1 , Mx2 , Oas1 , Ant3 , Mcu , Micu1 , Vdac1 , Ryr2 , and Cypd-ppid . Histological analysis of DM mouse hearts demonstrated that reduced MFN2 and ZBP1 were target molecules for hyperglycemia-induced reduction of calcium channel currents in cardiomyocytes and could be restored by Empagliflozin treatment. This study concludes that high glucose stress diminishes mitochondrial calcium channel regulators MFN2 and ZBP1 in cardiomyocyte, which reduces calcium channel currents and leads to sensitization of cardiomyocyte to arrhythmogenesis, resulting in VT/VF. It provides experimental evidence for the clinical efficacy of Empagliflozin in ameliorating CVDs and managing diabetes-related CVDs.

Abstract 4122257: Inhibition of Neutrophil Extracellular Traps Prevents Heart Failure by Preserving Mitochondrial Function of Cardiomyocytes in Mice

Background: We have previously demonstrated that neutrophil extracellular traps (NETs) in myocardial tissue are associated with cardiac dysfunction and adverse outcomes in patients with heart failure with reduced ejection fraction. Here, we aimed to elucidate the underlying mechanisms and to clarify whether inhibiting NETs could rescue the cardiac phenotype using ex vivo and in vivo models. Methods and Results: Given that peptidyl arginine deiminase 4 (PAD4) is essential for the formation of NETs, PAD4 knockout mice were used for this study. First, we assessed the mitochondrial function of cardiomyocytes by conducting real-time measurements of the oxygen consumption rate in ex vivo . Extracellular flux analysis revealed that NETs-containing conditioned medium from wild-type neutrophils or purified NET components led to impaired mitochondrial oxygen consumption of adult mouse cardiomyocytes, while these effects were abolished when PAD4 in neutrophils was genetically ablated (Figure 1). In a murine model of pressure overload-induced heart failure, transverse aortic constriction (TAC) operation was performed on wild-type and PAD4 knockout mice. Immunohistological analysis demonstrated that TAC induced the formation of NETs in the myocardium, with the greatest number of NETs occurring at 1 day, persisting into the failing stage at 4 weeks in wild-type mice, while PAD4-deficient hearts displayed no presence of NETs in the heart tissue (Figure 2). Four weeks after TAC, left ventricular ejection fraction was reduced in wild-type mice, whereas PAD4 knockout mice displayed preserved left ventricular ejection fraction (Figure 2). Finally, we investigated the bioenergetics of cardiomyocytes isolated from PAD4 knockout mice after TAC. Maximal mitochondrial oxygen consumption rate in cardiomyocytes isolated from TAC-operated PAD4 knockout mice was significantly higher than in those from TAC-operated wild-type mice (Figure 3). Conclusion: Our data suggest that NETs directly derived impairment of mitochondrial function in cardiomyocytes. Inhibiting NETs represents a potential therapeutic approach for heart failure, preventing mitochondrial dysfunction of cardiomyocytes.

Targeting ketone body metabolism in mitigating gemcitabine resistance.

Chemotherapy is often combined with surgery for muscle invasive and non-muscle invasive bladder cancer. However, 70% of the patients recur within 5 years. Metabolic reprogramming is an emerging hallmark in cancer chemoresistance. Here, we report a gemcitabine resistance mechanism which promotes cancer reprogramming via the metabolic enzyme, OXCT1. This mitochondrial enzyme, responsible for the rate-limiting step in β-hydroxybutyrate (βHB) catabolism, was elevated in muscle invasive disease and in chemo-resistant bladder cancer patients. Resistant orthotopic tumors presented an OXCT1-dependent rise in mitochondrial oxygen consumption rate, ATP, and nucleotide biosynthesis. In resistant bladder cancer, knocking out OXCT1 restored gemcitabine sensitivity, and administering the non-metabolizable βHB, enantiomer (S-βHB) only partially restored gemcitabine sensitivity. Suggesting an extra-metabolic role for OXCT1, multi-omics analysis of gemcitabine sensitive and resistant cells revealed an OXCT1-dependent signature with the transcriptional repressor, OVOL1, as a master regulator of epithelial differentiation. The elevation of OVOL1 target genes was associated with its cytoplasmic translocation and poor prognosis in a chemotherapy-treated BCa patient cohort. The knockout of OXCT1 restored OVOL1 transcriptional repressive activity by its nuclear translocation. Orthotopic mouse models of bladder cancer supported OXCT1 as a mediator of gemcitabine sensitivity through ketone metabolism and regulating cancer stem cell differentiation.

VLDL induces EZH2/PRC2 complex-mediated cellular senescence in metabolic syndrome to enhance cardiotoxicity

Abstract Introduction In metabolic syndrome, very-low-density lipoprotein (VLDL) carries cardiotoxic lipids that cannot be completely modified by lipid-lowering medications. VLDL isolated from patients with metabolic syndrome (ms-VLDL) induces cardiac remodeling compared with that isolated from non-metabolic syndrome subjects (n-VLDL). Purpose This study focused on identifying dysregulated molecular pathways of ms-VLDL-associated with senescence. Methods RNA-sequencing, accompanied with IPA and KEGG pathway analysis were used to identify differentially expressed genes (DEGs) in cardiomyocytes after two different VLDL incubations, and the identified DEGs were subjected to functional annotation, including enriched molecular pathway and gene ontology analyses. A Seahorse XFe96 Analyzer was used to determine the mitochondrial biosynthesis oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Molecules involved in the mitochondrial pathway were validated using Western blotting, qPCR, and immunofluorescence. Results Compared to the n-VLDL group, the ms-VLDL group had a larger number of significant DEGs involved in several critical biological processes and molecular pathways, such as cardiomyocyte injury (ms-VLDL vs. n-VLDL for significant DEGs numbers, 2,642 vs. 1,725), senescence (259 vs. 184), and mitochondrial dysfunction (137 vs. 68). In terms of mitochondrial biosynthetic function, ms-VLDL induced 8.5 folds OCR reduction compared to n-VLDL, along with intra-mitochondrial calcium overload and excessive ROS. The essential genes involved in these pathways were shown to be differentially regulated, and our analysis identified the Ezh2/PRC2 complex, which is strongly linked to ms-VLDL-induced cardiomyocyte senescence and lipotoxicity. Conclusion This study identified the crucial roles of epigenetic modification enzyme Ezh2/PRC2 complex in induction the senescence pathway, which represents a potential mechanism for ms-VLDL-induced adverse cardiac remodeling in metabolic syndrome. Further functional studies are required to validate their roles in the pathogenesis of lipotoxicity and the connection of cardiomyopahty.RNA-seq of VLDL treated cardiomyocyteSenescence induction of VLDL

Spatheliachromen mitigates methylglyoxal-induced myotube atrophy by activating Nrf2, inhibiting ubiquitin-mediated protein degradation, and restoring mitochondrial function

BackgroundMethylglyoxal (MGO) is a potent precursor of glycative stress that leads to oxidative stress and muscle atrophy in diabetes. Spatheliachromen (FPATM-20), derived from Ficus pumila var. awkeotsang, exhibited potential antioxidant activity. PurposeThis study aimed to evaluate the potential impact and underlying mechanisms of FPATM-20 on MGO-induced myotube atrophy and mitochondrial dysfunction in mouse skeletal C2C12 myotubes. MethodsAtrophic and antioxidant factors were evaluated using immunofluorescence, enzyme-linked immunosorbent assay, and western blotting. Mitochondrial function was assessed using the ATP assay and Seahorse Cell Mito Stress Test. The glycogen content was determined using periodic acid-Schiff staining. Molecular docking was performed to determine the interaction between FPATM-20 and Keap1. ResultsIn myotubes treated with MGO, FPATM-20 activated the Nrf2 pathway, reduced ROS levels, enhanced antioxidant defense, and increased glycogen content. FPATM-20 improved myotube viability and size, upregulated myosin heavy chain (MyHC) expression, modulated ubiquitin-proteasome molecules (nuclear FoxO3a, atrogin-1, MuRF-1, and p62/SQSTM1), and inhibited apoptosis (Bax/Bcl-2 ratio and cleaved caspase 3). Moreover, FPATM-20 restored mitochondrial function, including mitochondrial membrane potential, mitochondrial oxygen consumption rate, and mitochondrial biogenesis pathway (nuclear PGC-1α/TFAM/FNDC5). The inhibition of Nrf2 with ML385 reversed the effects of FPATM-20 on MGO. Furthermore, molecular docking confirmed the binding of FPATM-20 to Keap1, a suppressor of Nrf2, showing the crucial role of Nrf2 in protective effects. ConclusionsFPATM-20 protects myotubes from MGO toxicity by activating the Nrf2 antioxidant defense, reducing protein degradation and apoptosis, and enhancing mitochondrial function. Thus, FPATM-20 may be a novel agent for preventing skeletal muscle atrophy.

Extremely Low-Frequency Electromagnetic Field (ELF-EMF) Increases Mitochondrial Electron Transport Chain Activities and Ameliorates Depressive Behaviors in Mice.

Compromised mitochondrial electron transport chain (ETC) activities are associated with depression in humans and rodents. However, the effects of the enhancement of mitochondrial ETC activities on depression remain elusive. We recently reported that an extremely low-frequency electromagnetic field (ELF-EMF) of as low as 10 μT induced hormetic activation of mitochondrial ETC complexes in human/mouse cultured cells and mouse livers. Chronic social defeat stress (CSDS) for 10 consecutive days caused behavioral defects mimicking depression in mice, and using an ELF-EMF for two to six weeks ameliorated them. CSDS variably decreased the mitochondrial ETC proteins in the prefrontal cortex (PFC) in 10 days, which were increased by an ELF-EMF in six weeks. CSDS had no effect on the mitochondrial oxygen consumption rate in the PFC in 10 days, but using an ELF-EMF for six weeks enhanced it. CSDS inactivated SOD2 by enhancing its acetylation and increased lipid peroxidation in the PFC. In contrast, the ELF-EMF activated the Sirt3-FoxO3a-SOD2 pathway and suppressed lipid peroxidation. Furthermore, CSDS increased markers for mitophagy, which was suppressed by the ELF-EMF in six weeks. The ELF-EMF exerted beneficial hormetic effects on mitochondrial energy production, mitochondrial antioxidation, and mitochondrial dynamics in a mouse model of depression. We envisage that an ELF-EMF is a promising therapeutic option for depression.

Multi-omics analysis reveals phenylalanine enhance mitochondrial function and hypoxic endurance via LKB1/AMPK activation

Many studies have focused on the effects of small molecules, such as amino acids, on metabolism under hypoxia. Recent findings have indicated that phenylalanine levels were markedly elevated in adaptation to chronic hypoxia. This raises the possibility that phenylalanine treatment could markedly improve the hypoxic endurance. However, the importance of hypoxia-regulated phenylalanine is still unclear. This study investigates the role of phenylalanine in hypoxia adaptation using a hypoxic zebrafish model and multi-omics analysis. We found that phenylalanine-related metabolic pathways are significantly up-regulated under hypoxia, contributing to enhanced hypoxic endurance. Phenylalanine treatment reduced ROS levels, improved mitochondrial oxygen consumption rate (OCR), and extracellular acidification rate (ECAR) in hypoxic cells. Western blotting revealed increased phenylalanine uptake via L-type amino transporters (LAT1), activating the LKB1/AMPK signaling pathway. This activation up-regulated peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) and the Bcl-2/Bax ratio, while down-regulating uncoupling protein 2 (UCP2), thereby improving mitochondrial function under hypoxia. This is the first comprehensive multi-omics analysis to demonstrate phenylalanine’s crucial role in hypoxia adaptation, providing insights for the development of anti-hypoxic drugs.Graphical

Germacrone ameliorates acute lung injury induced by intestinal ischemia-reperfusion by regulating macrophage M1 polarization and mitochondrial defects.

Intestinal ischemia-reperfusion (I/R) injury severely affects the lungs. Germacrone (Ger) possesses anti-inflammatory and antioxidant properties. However, it is unclear whether it protects the lungs from I/R injury. In this study, we elucidate the mechanisms by which Ger protects lungs from I/R injury. C57BLKS/J male mice are subjected to I/R injury via complete clamping of the superior mesenteric artery. Ger is administered before intestinal I/R. Mitochondrial morphology is observed via electron microscopy. The histopathology of the lung tissues is monitored via hematoxylin-eosin and immunofluorescence staining. The mitochondrial oxygen consumption rate is measured via an XF96 extracellular flux analyzer. In the I/R mouse model, lung specimens present significant lung damage accompanied by increases in the levels of collagen III, vimentin, and α-SMA in lung tissues. After treatment with Ger, lung impairment and fibrosis in I/R-induced acute lung injury (ALI) model mice are restored, suggesting that Ger improves I/R-ALI. In addition, Ger administration decreases the release of inflammatory factors such as IL-1β, IL-6, and COX2, as well as the expressions of M1 macrophage markers, facilitating cell survival in the I/R-ALI model. Additionally, Ger (EC50: 47.16 μM) ameliorates mitochondrial dysfunction by increasing I/R-ALI-induced apoptosis, increasing the expression of SIRT1, and reducing the levels of HIF1-α, Nrf2, and OGG1 in MLE-12 cells. Ger may affect macrophage polarization and improve subsequent mitochondrial defects through the SIRT1-HIF1α-Nrf2 signaling pathway in MLE-12 cells, which ultimately improves lung function and lung inflammation in the I/R-ALI model.

Real-Time Monitoring of Oxygen-Consumption Rate in Mouse Liver Slices Incubated in Organ-on-a-Chip Devices.

We developed an organ-on-a-chip (OOC) based on precision-cut liver slices to assess liver function in real-time, both in health and disease, in a controlled and noninvasive manner. We achieved this by integrating fiber-optic-based oxygen sensors before and after the microchamber in which a liver slice was incubated under flow, to measure oxygen concentrations in the medium in real time. We first demonstrated that the basal oxygen consumption rate (OCR) of liver slices is a reliable indicator of liver slice viability. By monitoring basal OCR (2.9-5.7 pmol O2/min/μg protein) in incubation medium, we found that it correlated well to cellular adenosine triphosphate (ATP) content (3.0-7.9 pmol/μg protein) (r = 0.82, p < 0.0001). Second, we induced a diseased state in liver slices by targeting the mitochondria, as they play a critical role in liver function and disease. We exposed the liver slices to succinate in abundance (40 mM) for short periods (1 h) to rapidly boost mitochondrial OCR. Two successive treatments of succinate increased the OCR of liver slices by 1.5 pmol/min/μg each time. However, between treatments, the liver slice OCR did not return to its basal OCR, instead decreasing drastically by 60-70%, suggesting succinate toxicity. We confirmed this with ATP analysis (1.0 pmol/μg protein) and hematoxylin and eosin staining, which showed tissue necrosis and apoptosis. Our system could be an advantageous model for future studies assessing liver (patho)physiology in response to potentially toxic drugs or lifestyle-related liver diseases.

Adipose stem cells regulate lipid metabolism by upregulating mitochondrial fatty acid β-oxidation in macrophages to improve the retention rate of transplanted fat

BackgroundAt present, fat transplantation is widely used in the plastic surgery industry, but the long-term preservation rate of transplanted fat decreases because of complications such as oil cysts due to the inability in macrophages to metabolize absorption. In cell-assisted lipotransfer technology, adipose-derived stem cells (ASCs) can influence the inflammatory response of grafts through the immunoregulation in macrophages, and the lipid metabolism in macrophages plays an important role in this process. Therefore, we hypothesized ASCs could improve the retention rate of fat grafts by regulating the progress of lipid metabolism in macrophages.MethodsWe established fat transplantation and ASC-assisted fat transplantation model in C57BL/6 mice in vivo, and bone marrow-derived macrophages cocultured with apoptotic adipocytes were treated with or without ASCs in vitro. Graft retention, tissue structure, fibrosis, macrophage phenotype transformation, lipid deposition, mitochondrial morphology, oxygen consumption rate (OCR), fatty acid β-oxidation (FAO) level, and ATP production were assessed. Additionally, fat transplantation and ASC-assisted fat transplantation model was treated with etomoxir which inhibits mitochondrial FAO. Macrophages pretreated with etomoxir were co-cultured with apoptotic adipocytes and treated with or without ASCs. The method aboved was used for detection and verification.ResultsIn vivo, ASC-assisted fat transplantation improved macrophage mitochondrial expression and FAO level, promoted the early transformation of M2 macrophages, reduced the long-term lipid deposition of macrophages, and improved the retention rate of fat grafts. In vitro, ASCs up-regulated the level of mitochondrial FAO, OCR and ATP production in macrophages, reduced lipid deposition of macrophages and promoted M2 macrophages polarization by paracine function. The ability of ASCs in group pretreated with etomoxir to reduce the foaming of macrophages, promote the transformation to M2 macrophages, and improve the retention rate of fat transplantation was weakened.ConclusionsASCs increased the retention rate of transplanted fat by upregulating mitochondrial FAO to promote M2 polaration in macrophages. In addition, ASCs up-regulate mitochondrial FAO by paracrine effect to reduce foam cells formation and promote M2 transformation in macrophages in vitro.

Protein kinase N1 deficiency results in upregulation of cerebral energy metabolism and is highly protective in in vivo and in vitro stroke models

Background and aimWe recently identified protein kinase N1 (PKN1) as a master regulator of brain development. However, its function in the adult brain has not been clearly established. In this study, we assessed the cerebral energetic phenotype of wildtype (WT) and global Pkn1 knockout (Pkn1−/−) animals under physiological and pathophysiological conditions. MethodsCerebral energy metabolism was analyzed by 13C6-glucose tracing in vivo and real time seahorse analysis of extracellular acidification rates as well as mitochondrial oxygen consumption rates (OCR) of brain slice punches in vitro. Isolated WT and Pkn1−/− brain mitochondria were tested for differences in OCR with different substrates. Metabolite levels were determined by mass spectrometric analysis in brain slices under control and energetic stress conditions, induced by oxygen-glucose deprivation and reperfusion, an in vitro model of ischemic stroke. Differences in enzyme activities were assessed by enzymatic assays, western blotting and bulk RNA sequencing. A middle cerebral artery occlusion stroke model was used to analyze lesion volumes and functional recovery in WT and Pkn1−/− mice. ResultsPkn1 deficiency resulted in a remarkable upregulation of cerebral energy metabolism, in vivo and in vitro. This was due to two separate mechanisms involving an enhanced glycolytic flux and higher pyruvate-induced mitochondrial OCR. Mechanistically we show that Pkn1−/− brain tissue exhibits an increased activity of the glycolysis rate-limiting enzyme phosphofructokinase. Additionally, glucose-1,6-bisphosphate levels, a metabolite that increases mitochondrial pyruvate uptake, were elevated upon Pkn1 deficiency. Consequently, Pkn1−/− brain slices had more ATP and a greater accumulation of ATP degradation metabolites during energetic stress. This translated into increased phosphorylation and activity of adenosine monophosphate (AMP)-activated protein kinase (AMPK) during in vitro stroke. Accordingly, Pkn1−/− brain slices showed a post-ischemic transcriptional upregulation of energy metabolism pathways and Pkn1 deficiency was strongly protective in in vitro and in vivo stroke models. While inhibition of mitochondrial pyruvate uptake only moderately affected the protective phenotype, inhibition of AMPK in Pkn1−/− slices increased post-ischemic cell death in vitro. ConclusionThis is the first study to comprehensively demonstrate an essential and unique role of PKN1 in cerebral energy metabolism, regulating glycolysis and mitochondrial pyruvate-induced respiration. We further uncovered a highly protective phenotype of Pkn1 deficiency in both, in vitro and in vivo stroke models, validating inhibition of PKN1 as a promising new therapeutic target for the development of novel stroke therapies.

Sex differences in respiration and redox homeostasis of heart mitochondria in rats on high-fructose diet

Sex hormones play a leading role in the sexual dimorphism of mitochondrial dysfunction and oxidative stress that are associated with Metabolic Syndrome (MetS) and considered as possible causes of cardiovascular disease. The aim of the work was to determine mitochondrial respiration and redox homeostasis in the heart mitochondria of high-fructose diet-fed (НFD) rats depending on sex. MetS was induced in Wistar rats by 8 weeks intake of fructose (200 g/l) with drinking water. The experiment was performed on 30 rats divided into five groups: control males, control females, HFD-fed males, HFD- fed females with intact ovaries, ovariectomized HFD-fed females. Heart mitochondria were isolated and indicators of redox homeostasis as well as mitochondrial oxygen consumption rate were determined. Heart mitochondria of intact female rats were characterized by a lower intensity of lipid peroxidation, a higher activity of antioxidant defense system and state 3 respiration in comparison with control males. HFD was shown to induce more expressed oxidative stress due to significant inhibition of enzymatic and non-enzymatic components of antioxidant defese and more pronounced dysregulation of mitochondrial respiration in the heart mitochondria of ovariectomized females as compared to males. This data may partially explain the greater cardiovascular risk in women with low estrogen sufficiency and justify the necessity of new sex-specific prevention and treatment of cardiovascular risk approaches. Keywords: antioxidant defense system, heart mitochondria, mitochondrial respiration, oxidative stress, rats, sex differences

Risperidone-induced bioenergetic disruption in the isolated human peripheral blood monocytes

Risperidone (RIS) is a widely used antipsychotic drug with reported alteration in immune response. The current study investigated mitochondrial disruption as the underlying mechanism of RIS-induced immunotoxicity in isolated human peripheral blood monocytes (hPBM). RIS was cytotoxic to hPBM in exposure duration and concentration-dependent patterns. Functionally, RIS was shown to increase the release of IL-6, TNF-α, and IL-8 with a decrease in test particle phagocytosis in concertation and exposure time-based patterns. It was found that RIS decreased ATP production in isolated monocytes' mitochondria, with an estimated EC50 of around 70 μM after 24 h with parallel inhibition of mitochondrial complexes I and III activities and decreased mitochondrial membrane potential and oxygen consumption rates with increased lactate production from by the treated cells in comparison to controls. Structurally, RIS in 100 μM concentration significantly increased the mitochondrial membrane fluidity with significant increase in increased unsaturated/saturated fatty acids ratios of the mitochondrial membranes of the treated cells. Interestingly, water-soluble CoQ10 formulation significantly decreased the cytotoxic effect of RIS and improved the phagocytic activity of RIS-treated cells. To conclude, the current data suggests mitochondrial disruption as the underlying mechanism of RIS-induced immunotoxicity with shown protective effect of water-soluble CoQ10 formulation.

Transcriptomic analysis reveals the mechanism of isorhamnetin in the treatment of diabetes mellitus erectile dysfunction

PurposeExploring the therapeutic effect and mechanism of isorhamnetin in the treatment of DMED. MethodsUsing a high glucose environment to induce endothelial cells damage in the corpus cavernosum, and combining with intervention agents such as ferroptosis inhibitors to observe the process of cell damage and repair, evaluating cell status through CCK-8 and DAPI; To establish the STZ-induced diabetes rat model and detect the erectile function and tissue changes; Perform transcriptomic sequencing on rat models and samples treated with isorhamnetin to analyze differentially expressed genes and their GO functions; Identify critical pathways by combining with the ferroptosis database; Flow cytometry was used to detect ROS and mitochondrial membrane potential, and RT-PCR was used to verify gene expression, Seahorse detects mitochondrial oxygen consumption rate, revealing the mechanism of action of isorhamnetin. ResultsFerroptosis inhibitors and isorhamnetin can effectively reverse the damage of corpus cavernosum endothelial cells induced by high glucose and ferroptosis agonists. Isorhamnetin has the ability to reinstate the erectile function of diabetic rats, while enhancing the quantity of endothelial cells and refining the morphology of collagen fibers. Immunohistochemistry revealed that ferroptosis existed in the penis tissue of diabetes rats. Transcriptomic analysis showed that isorhamnetin improves gene expression in DM rats by regulating genes such as GFER, IGHM, GPX4 and HMOX1, involving multiple pathways and biological processes. Flow cytometry and RT-PCR confirmed that isorhamnetin can reduce reactive oxygen species levels, restore essential gene expression, improve mitochondrial membrane potential, and alleviate oxidative stress and ferroptosis. Seahorse detection found that isorhamnetin can restore mitochondrial oxygen consumption rate. ConclusionIsorhamnetin attenuates high glucose damage to cavernous endothelial cells by inhibiting ferroptosis and oxidative stress, restores erectile function and improves tissue morphology in diabetic rats, and its multi-pathway and multi-targeting regulatory mechanism suggests that it is promising to be an effective drug for the treatment of DMED.

Ginsenoside Rh4 prevents endothelial dysfunction as a novel AMPK activator.

The AMP-activated protein kinase (AMPK) signalling pathway is a desirable target for various cardiovascular diseases (CVD), while the involvement of AMPK-mediated specific downstream pathways and effective interventions in hyperlipidaemia-induced endothelial dysfunction remain largely unknown. Herein, we aim to identify an effective AMPK activator and to explore its efficacy and mechanism against endothelial dysfunction. Molecular docking technique was adopted to screen for the potent AMPK activator among 11 most common rare ginsenosides. In vivo, poloxamer 407 (P407) was used to induce acute hyperlipidaemia in C57BL/6J mice. In vitro, palmitic acid (PA) was used to induce lipid toxicity in HAEC cells. We discovered the strongest binding of ginsenoside Rh4 to AMPKα1 and confirmed the action of Rh4 on AMPK activation. Rh4 effectively attenuated hyperlipidaemia-related endothelial injury and oxidative stress both in vivo and in vitro and restored cell viability, mitochondrial membrane potential and mitochondrial oxygen consumption rate in HAEC cells. Mechanistically, Rh4 bound to AMPKα1 and simultaneously up-regulated AKT/eNOS-mediated NO release, promoted PGC-1α-mediated mitochondrial biogenesis and inhibited P38 MAPK/NFκB-mediated inflammatory responses in both P407-treated mice and PA-treated HAEC cells. The AMPK inhibitor Compound C treatment completely abrogated the regulation of Rh4 on the above pathways and weakened the lowering effect of Rh4 on endothelial impairment markers, suggesting that the beneficial effects of Rh4 are AMPK dependent. Rh4 may serve as a novel AMPK activator to protect against hyperlipidaemia-induced endothelial dysfunction, providing new insights into the prevention and treatment of endothelial injury-associated CVD.

Novel gold-based complex GC7 suppresses cancer cell proliferation via impacting energy metabolism mediated by mitochondria

Due to their pivotal roles in regulating energy metabolism and apoptosis, mitochondria in cancer cells have been considered a vulnerable and feasible target. Many anticancer agents, e.g., metal-based compounds, are found to target and disturb mitochondria primarily, which may lead to the disturbance of energy metabolism and, more importantly, the initiation of apoptosis. In this work, a gold-based complex 7 (GC7) was synthesized and evaluated in a series of different cancer cell lines. The anticancer efficacies of GC7 on cell viability, apoptosis, and colony formation were determined. Cellular thioredoxin reductase (TrxR) activity, oxygen consumption rate (OCR), glucose uptake, and lactate production following GC7 treatment were evaluated and analyzed. The Jeko-1 and A549 xenograft models were used to assess GC7’s tumor-suppressing effects. The results showed that GC7 possessed a broad-spectrum anticancer effect, with IC50 values ranging from 0.43 to 1.2 μM in multiple cancer cell lines, which was more potent than gold-based auranofin (∼2–6 folds). GC7 (0.3 and 1 μM) efficiently induced apoptosis of Jeko-1, A549, and HCT116 cells, and it suppressed the sphere formation of cancer stem cells GSC11 and GSC23 cells at 0.1 μM, and it completely eliminated colony at 0.3 μM. The preliminary mechanistic study showed that GC7 inhibited cellular TrxR activity, suppressed mitochondrial OCR, reduced mitochondrial membrane potential (MMP), decreased glucose uptake, and possibly suppressed glycolysis to reduce lactate production. GC7 was predicted to have a similar yet slightly different pharmacokinetic profile as auranofin. Finally, GC7 (20 mg/kg, oral, 5/week, or 3 mg/kg, IP, 3/week) significantly inhibited tumor growth. In conclusion, GC7 showed great potential in suppressing cancer cell proliferation, probably via inhibiting TrxR and impacting mitochondria-mediated energy metabolism.

Glucose-1,6-bisphosphate: A new gatekeeper of cerebral mitochondrial pyruvate uptake

ObjectiveGlucose-1,6-bisphosphate (G-1,6-BP), a byproduct of glycolysis that is synthesized by phosphoglucomutase 2 like 1 (PGM2L1), is particularly abundant in neurons. G-1,6-BP is sensitive to the glycolytic flux, due to its dependence on 1,3-bisphosphoglycerate as phosphate donor, and the energy state, due to its degradation by inosine monophosphate-activated phosphomannomutase 1. Since the exact role of this metabolite remains unclear, our aim was to elucidate the specific function of G-1,6-BP in the brain. MethodsThe effect of PGM2L1 on neuronal post-ischemic viability was assessed by siRNA-mediated knockdown of PGM2L1 in primary mouse neurons. Acute mouse brain slices were used to correlate the reduction in G-1,6-BP upon ischemia to changes in carbon metabolism by 13C6-glucose tracing. A drug affinity responsive target stability assay was used to test if G-1,6-BP interacts with the mitochondrial pyruvate carrier (MPC) subunits in mouse brain protein extracts. Human embryonic kidney cells expressing a MPC bioluminescence resonance energy transfer sensor were used to analyze how PGM2L1 overexpression affects MPC activity. The effect of G-1,6-BP on mitochondrial pyruvate uptake and oxygen consumption rates was analyzed in isolated mouse brain mitochondria. PGM2L1 and a predicted upstream kinase were overexpressed in a human neuroblastoma cell line and G-1,6-BP levels were measured. ResultsWe found that G-1,6-BP in mouse brain slices was quickly degraded upon ischemia and reperfusion. Knockdown of PGM2L1 in mouse neurons reduced post-ischemic viability, indicating that PGM2L1 plays a neuroprotective role. The reduction in G-1,6-BP upon ischemia was not accompanied by alterations in glycolytic rates but we did see a reduced 13C6-glucose incorporation into citrate, suggesting a potential role in mitochondrial pyruvate uptake or metabolism. Indeed, G-1,6-BP interacted with both MPC subunits and overexpression of PGM2L1 increased MPC activity. G-1,6-BP, at concentrations found in the brain, enhanced mitochondrial pyruvate uptake and pyruvate-induced oxygen consumption rates. Overexpression of a predicted upstream kinase inhibited PGM2L1 activity, showing that besides metabolism, also signaling pathways can regulate G-1,6-BP levels. ConclusionsWe provide evidence that G-1,6-BP positively regulates mitochondrial pyruvate uptake and post-ischemic neuronal viability. These compelling data reveal a novel mechanism by which neurons can couple glycolysis-derived pyruvate to the tricarboxylic acid cycle. This process is sensitive to the glycolytic flux, the cell's energetic state, and upstream signaling cascades, offering many regulatory means to fine-tune this critical metabolic step.

Age-related increase of CD38 directs osteoclastogenic potential of monocytic myeloid-derived suppressor cells through mitochondrial dysfunction in male mice.

An aged immune system undergoes substantial changes where myelopoiesis dominates within the bone marrow. Monocytic-MDSCs (M-MDSCs) have been found to play an important role in osteoclastogenesis and bone resorption. In this study, we sought to provide a more comprehensive understanding of the osteoclastogenic potential of bone marrow M-MDSCs during normal aging through transcriptomic and metabolic changes. Using young mature and aged mice, detailed immunophenotypic analyses of myeloid cells revealed that the M-MDSCs were not increased in bone marrow, however M-MDSCS were significantly expanded in peripheral tissues. Although aged mice exhibited a similar number of M-MDSCs in bone marrow, these M-MDSCs had significantly higher osteoclastogenic potential and greater demineralization activity. Intriguingly, osteoclast progenitors from aged bone marrow M-MDSCs exhibited greater mitochondrial respiration rate and glucose metabolism. Further, transcriptomic analyses revealed the upregulation of mitochondrial oxidative phosphorylation and glucose metabolism genes. Interestingly, there was 8-fold increase in Cd38 mRNA gene expression, consistent with the Mouse Aging Cell Atlas transcriptomic database, and confirmed by qRT-PCR. CD38 regulates NAD+ availability, and 78c, a small molecule inhibitor of CD38, reduced the mitochondrial oxygen consumption rate and glucose metabolism and inhibited the osteoclastogenic potential of aged mice bone marrow-derived M-MDSCs. These results indicate that the age-related increase in Cd38 expression in M-MDSCs bias the transcriptome of M-MDSCs towards osteoclastogenesis. This enhanced understanding of the mechanistic underpinnings of M-MDSCs and their osteoclastogenesis during aging could lead to new therapeutic approaches for age-related bone loss and promote healthy aging.

Mitochondrial control of hypoxia-induced pathological retinal angiogenesis.

Pathological retinal neovascularization is vision-threatening. In mouse oxygen-induced retinopathy (OIR) we sought to define mitochondrial respiration changes longitudinally during hyperoxia-induced vessel loss and hypoxia-induced neovascularization, and to test interventions addressing those changes to prevent neovascularization. OIR was induced in C57BL/6J mice and retinal vasculature was examined at maximum neovessel formation. We assessed total proteome changes and the ratio of mitochondrial to nuclear DNA copy numbers (mtDNA/nDNA) of OIR vs. control retinas, and mitochondrial oxygen consumption rates (OCR) in ex vivo OIR vs. control retinas (BaroFuse). Pyruvate vs. vehicle control was supplemented to OIR mice either prior to or during neovessel formation. In OIR vs. control retinas, global proteomics showed decreased retinal mitochondrial respiration at peak neovascularization. OCR and mtDNA/nDNA were also decreased at peak neovascularization suggesting impaired mitochondrial respiration. In vivo pyruvate administration during but not prior to neovessel formation (in line with mitochondrial activity time course) suppressed NV. Mitochondrial energetics were suppressed during retinal NV in OIR. Appropriately timed supplementation of pyruvate may be a novel approach in neovascular retinal diseases.

Identification of Antioxidant Methyl Derivatives of Ortho-Carbonyl Hydroquinones That Reduce Caco-2 Cell Energetic Metabolism and Alpha-Glucosidase Activity.

α-glucosidase, a pharmacological target for type 2 diabetes mellitus (T2DM), is present in the intestinal brush border membrane and catalyzes the hydrolysis of sugar linkages during carbohydrate digestion. Since α-glucosidase inhibitors (AGIs) modulate intestinal metabolism, they may influence oxidative stress and glycolysis inhibition, potentially addressing intestinal dysfunction associated with T2DM. Herein, we report on a study of an ortho-carbonyl substituted hydroquinone series, whose members differ only in the number and position of methyl groups on a common scaffold, on radical-scavenging activities (ORAC assay) and correlate them with some parameters obtained by density functional theory (DFT) analysis. These compounds' effect on enzymatic activity, their molecular modeling on α-glucosidase, and their impact on the mitochondrial respiration and glycolysis of the intestinal Caco-2 cell line were evaluated. Three groups of compounds, according their effects on the Caco-2 cells metabolism, were characterized: group A (compounds 2, 3, 5, 8, 9, and 10) reduces the glycolysis, group B (compounds 1 and 6) reduces the basal mitochondrial oxygen consumption rate (OCR) and increases the extracellular acidification rate (ECAR), suggesting that it induces a metabolic remodeling toward glycolysis, and group C (compounds 4 and 7) increases the glycolysis lacking effect on OCR. Compounds 5 and 10 were more potent as α-glucosidase inhibitors (AGIs) than acarbose, a well-known AGI with clinical use. Moreover, compound 5 was an OCR/ECAR inhibitor, and compound 10 was a dual agent, increasing the proton leak-driven OCR and inhibiting the maximal electron transport flux. Additionally, menadione-induced ROS production was prevented by compound 5 in Caco-2 cells. These results reveal that slight structural variations in a hydroquinone scaffold led to diverse antioxidant capability, α-glucosidase inhibition, and the regulation of mitochondrial bioenergetics in Caco-2 cells, which may be useful in the design of new drugs for T2DM and metabolic syndrome.