Mitochondrial Oxidation Research Articles

Pathophysiology of Carnitine Palmitoyltransferase 2 (CPT2) Deficiency in the Dahl Salt-sensitive R at

Carnitine palmitoyltransferase 2 (CPT2) is an essential enzyme of the carnitine shuttle, which facilitates fatty acid -oxidation in mitochondria. This study aims to characterize the various effects of CPT2 deficiency on the Dahl salt-sensitive (SS) rat metabolome and its concomitant influence on renal homeostasis. CRISPR/Cas9 was used to insert a single base pair into the SS Cpt2 gene, causing a frameshift mutation found to be lethal in homozygotes (SSCpt2−/−). Western blotting and qPCR analysis of renal cortex tissue confirmed a reduced expression of CPT2 in heterozygous (SSCpt2+/-) rats compared to wild-type (WT) littermates; therefore, male and female heterozygotes were used for all experiments. Lipid metabolomics of renal cortex samples collected from SSCpt2+/- animals at 8 weeks old on a baseline 0.4% NaCl diet revealed a significant upregulation in inflammatory lipids such as 11-HETE (p<0.01) and 12-HHT (p<0.05) when compared to WT littermates. Urine analysis from the SSCpt2+/- group demonstrated a severe reduction in acetoacetic acid production (p<0.001), indicating ketogenesis is likely impacted by the loss of CPT2 in -oxidation. Additionally, TCA cycle metabolites fumaric acid (p<0.05) and oxaloacetic acid (p<0.01) are significantly downregulated in urine from SSCpt2+/- animals. Radio telemeters were implanted into the rats at 8 weeks of age to continuously record mean arterial pressure (MAP) and monitor the development of SS hypertension over the course of several dietary treatment protocols. Three weeks of either a 4% NaCl high-salt (HS) diet or a HS + ketogenic (HSK) diet with equivalent caloric content (4:1 caloric ratio, fat: carbohydrate) were administered to the animals. No difference in MAP elevation occurred between groups after the HS challenge alone, but SSCpt2+/- animals on the HSK diet had a significantly attenuated development of hypertension (p=0.03). Further metabolic analysis after the HSK diet illustrated that both groups had similar increases in long-chain fatty acids, liver enzymes, and cholesterol levels with a decrease in short-chain fatty acids. These experiments show that the partial loss of CPT2 in the Dahl SS rat leads to dysregulation across multiple metabolic pathways. Moreover, a ketogenic diet appears to protect heterozygous animals from the development of salt-sensitive hypertension. Funding: NIH R35 HL135749 and VA I01 BX004024. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Exercise Training Mitigates Inhibition of Skeletal Muscle Mitochondrial Respiration with Empagliflozin Treatment in Male and Female Mice

Sodium-glucose cotransporter-2 inhibitors (SGLT2i) are antihyperglycemic drugs designed to lower renal glucose reabsorption, yet recent evidence indicates that SGLT2i have off-target effects on skeletal muscle to inhibit mitochondrial respiration. Such inhibition raises important questions regarding the interaction of SGLT2i and exercise on regulation of skeletal muscle mitochondrial metabolism. The purpose of the current study was to test the hypothesis that SGLT2i treatment (empagliflozin, EMPA) would blunt improvements to skeletal muscle mitochondrial oxidation capacity following aerobic training. Male and female C57BL/6J mice consumed a western diet (40% kcal from fat) for 8 weeks, then performed an additional 8 weeks of either treadmill training (5 times per week progressing up to 23 m/min) or remained sedentary. During the intervention period, all mice consumed either western diet or western diet enriched with EMPA (10mg/kg diet) in a 2x2 design (n=10/group for each sex). We isolated mitochondria from quadriceps muscles collected 72 hours after the final training session (or rest) and measured ex vivo mitochondrial substrate oxidation capacity using high-resolution respirometry. There was an interaction between exercise and EMPA (p=0.037) on complex I-II respiration whereby sedentary mice had lower respiration following EMPA versus untreated (-17% p=0.01) while exercise-trained mice had similar respiration between EMPA and untreated. The interaction was less prominent in complex I-supported respiration (p=0.06) or complex-II alone (p=0.09). Our results show that, contrary to expectations, EMPA treatment did not impair mitochondrial adaptations to exercise training. Instead, exercise training prevented the suppression of mitochondrial respiration during EMPA treatment. These findings indicate that aerobic exercise has positive effects on the off-target inhibition of mitochondrial respiration by SGLT2i. Supported by funding from the Collins Medical Trust (Wilsonville, Oregon) awarded to Matthew Robinson. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Nicotine Exposure Causes Nitrosative and Oxidative Stress in Glomerular Podocytes, Resulting in Renal Damage

Introduction: Nicotine consumption is the leading cause of preventable disease and death. Patients with hypertension or kidney disease are more likely to exhibit renal pathologies associated with nicotine, such as glomerulopathy. In endothelial cells, nicotine was linked to mitochondrial oxidative stress and nitric oxide synthase (NOS) remodeling. However, there is a gap in knowledge regarding the effects of nicotine in glomerular epithelial cells, podocytes. We hypothesized that nicotine promotes nitrosative stress in the podocytes by producing peroxynitrite (ONOO-) and mitochondrial damage, thereby reducing glomerular function. Methods: We performed confocal imaging on human cultured podocytes to detect ONOO− (HPF), Ca2+ (Fluo-8) and nitric oxide NO (DAF-FM) in response to nicotine. Seahorse assay (Agilent XFe24) was used to test mitochondrial respiration. Then, nicotine was chronically infused in Dahl SS rats (0.2 mg/kg/day s.c., 0.4% NaCl, 21 days), and blood pressure was recorded with telemetry. Glomerular damage was evaluated, and electron microscopy was employed to assess mitochondrial ultrastructure. Electron paramagnetic resonance (EPR) spectroscopy was used to access the NO levels in cultured podocytes and in vivo. OriginPro was used for statistical analysis. Results: Acute application of nicotine promoted intracellular Ca2+ and ONOO− transients in podocytes. The ONOO− response was blocked in the presence of SOD, indicating that ONOO− production requires superoxide. The application of specific α7 or α4β2, α2β4, α4β4, and α3β4 nicotinic acetylcholine receptor (nAChR) agonists elicited Ca2+ transients but did not produce ONOO−, suggesting that nitrosative stress occurs independently from Ca2+ influx or nAChR activation. Incubation with nicotine (12 hrs) resulted in a decrease in podocytes’ mitochondrial respiration (two-sample t-test, p<0.05). In vivo, nicotine infusion did not affect blood pressure but promoted mitochondrial damage in podocytes, which exhibited swelling, loss of cristae, and mitophagy (t-test, p<0.05). Histopathological assessment showed higher glomerular damage in nicotine-exposed rats (t-test, p<0.05). Both EPR and confocal approaches demonstrated nicotine-mediated changes in NO bioavailability and an increase in NOS2 activity (t-test, p<0.05) Conclusion: Nicotine elicits superoxide-mediated nitrosative stress and peroxynitrite formation in podocytes, promoting mitochondrial damage and glomerular dysfunction. Revealing the mechanisms of nicotine-mediated damage in the podocytes impacts our progress towards new treatments for smoking and vaping-associated renal pathologies. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 118: Dennd5b Affects Dietary Lipid Absorption And Metabolism In Male And Female Mice

Background: Dietary lipid absorption by the intestine is essential for systemic lipid homeostasis. We previously demonstrated a role for Dennd5b in intestinal lipid absorption. In humans, DENND5B gene variants are correlated with BMI in females, but not males. Female Dennd5b -/- mice have reduced appearance of dietary triacylglyceride (TAG) in plasma and are resistant to diet-induced obesity, however, the metabolic impact of Dennd5b -/- in male mice has not been reported. In this study, we test the hypothesis that the Dennd5b -/- mouse model recapitulates the sex disparity observed in humans with DENND5B polymorphisms. We also investigate the fate of unsecreted intestinal TAG in this model of impaired dietary lipid absorption. Methods and Results: A non-absorbable fatty acid (FA) tracer was used to quantify the impact of Dennd5b -deficiency on lipid absorption in male and female mice (n=7-9 mice/genotype/sex). We observed significant, but relatively modest, reductions in lipid absorption efficiency in Dennd5b -/- mice in both sexes, despite a complete absence of plasma TAG after oil gavage. We hypothesized that this was due to metabolic utilization of TAG by enterocytes. Metabolic cage studies showed that both wildtype and Dennd5b -/- mice shift toward utilization of FAs when fed high-fat diet and Seahorse analysis revealed altered metabolic activity in Dennd5b -/- intestinal tissue. Electron microscopy (EM) revealed large electron dense structures that resemble autophagosomes in Dennd5b -/- enterocytes. Western blots detected altered Lc3 levels in Dennd5b -/- intestine, implicating autophagy. RNA sequencing of intestinal tissue detected changes in expression of genes involved in beta oxidation and EM imaging revealed altered mitochondrial morphology in Dennd5b -/- enterocytes. Conclusions: We conclude that autophagy contributes to degradation of unsecreted TAG in the Dennd5b -/- enterocytes, liberating free FAs which are utilized in mitochondrial oxidation. The sexually dimorphic effect of Dennd5b does not appear to be as prominent in mice as it is in humans. Overall, our findings demonstrate that Dennd5b plays a critical role in TAG secretion by intestinal tissue and that disruption of this process can impact systemic metabolism in both sexes.

Skeletal Muscle Derived Cells from Old Human Donors Show Increased Mitochondrial Lipid Peroxidation and Reduced Mitochondrial Function

Skeletal muscle undergoes atrophy with age progression resulting in decreased mitochondrial function. Similarly, age-related increases in mitochondrial oxidant generation can promote mitochondrial oxidative damage, but the relationship between increased oxidant stress and mitochondrial function is not well understood. Here, we profiled changes in mitochondrial function and mitochondrial lipid peroxidation in intact primary skeletal muscle progenitor cells (myoblasts) derived from the Rectus abdominis muscle of healthy young (19.67 ± 1.70 years) and old (67.0 ± 1.41 years) humans. Using metabolic flux assays, we found that cells derived from young humans showed higher basal and maximal oxygen consumption rates compared to cells derived from old humans (basal: 22.67 ± 6.53 vs. 10.81 ± 5.69; maximal: 41.02 ± 15.20 vs. 17.33 ± 4.84 pmol/min/protein, p < 0.001). Similarly, cells derived from young humans also showed higher ATP production and spare respiratory capacity (ATP: 18.79 ± 5.17 vs. 9.15 ± 4.91; SRC: 18.35 ± 9.62 vs. 6.52 ± 3.42 pmol/min/protein, p < 0.001). In contrast, using live-cell fluorescence microscopy, MitoTracker red and the mitochondrial-target lipid peroxide probe MitoPeDPP, we found higher mitochondrial lipid peroxidation levels in cells derived from old than young humans ( p = 0.0007). We then evaluated whether peroxiredoxin 6 (Prdx6), an enzyme that repairs lipid peroxidation through its phospholipid glutathione peroxidase and phospholipase A2 activities, impacts mitochondrial function using CRISPR/Cas9-mediated knockouts of Prdx6 in C2C12 myoblasts and extracellular flux assays. Spare respiratory capacity was lower in cells with full Prdx6-KO than in cells transfected with non-target gRNAs (NT: 295.10 ± 18.59 vs. Prdx6-KO: 242.20 ± 17.93; p = 0.05). These results show that mitochondrial function decreases with age along with increases in mitochondrial lipid peroxidation in primary muscle cells derived from old humans. Similarly, these results show that the Prdx6 supports mitochondrial function. Overall, our data contributes to our understanding of the mechanistic drivers of age-related skeletal muscle mitochondrial dysfunction. NIGMS-R35GM146951 NIA-R01AG059715. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Mitochondrial function in lungs of rats with different susceptibilities to hyperoxia-induced Acute Respiratory Distress Syndrome (ARDS)

Objective: Rat exposure to hyperoxia is a well-established model of human ARDS. Adult rats exposed to hyperoxia (100% O2) die from respiratory failure within 60-72 hours. However, rats preconditioned by exposure to >95% O2 for 48 hrs followed by a 24-hr “rest period” in room air (H-T) acquire tolerance of the otherwise lethal effects of exposure to 100% O2. In contrast, rats preconditioned by exposure to 60% O2 for 7 days (H-S) become more susceptible to 100% O2. The objective was to evaluate lung tissue mitochondrial bioenergetics in H-T and H-S rats. Methods: Adult rats were exposed to room air (normoxia), >95% O2 for 48 hrs followed by exposure to room air for 24 hrs (H-T), or 60% O2 for 7 days (H-S). Mitochondria were isolated from lung tissue and used to assess mitochondrial bioenergetics. Expressions of electron transport chain complexes were measured in lung tissue homogenate using western blot. Isolated perfused lungs (IPL) were used to determine pulmonary vascular endothelial filtration coeffcient ( Kf) as a measure of pulmonary vascular permeability, and lung tissue mitochondrial membrane potential (ΔΨm). Results: Western blot shows decreased (38%) complex I expression, but increased (70%) complex V expression in H-T lung tissue homogenate compared to normoxia. Complex I expression decreased (43%) in H-S lung tissue homogenate. State 3 oxidative phosphorylation (OxPhos) capacity (Vmax) and respiratory control ratio decreased in mitochondria isolated from H-S lungs. Vmax increased in mitochondria of H-T lungs. Time for ΔΨm repolarization following ADP-stimulated depolarization increased in mitochondria isolated from H-S lungs. IPL studies revealed that tissue ΔΨm is unchanged in H-S and H-T lungs compared to normoxics. Furthermore, complex I plays the dominant role in ΔΨm in H-T and normoxia lungs with no contribution from complex II, whereas complex II has a larger contribution to ΔΨm in H-S lungs than in H-T or normoxia. Kf increased (+178%) in H-S, but not H-T lungs. Discussion: For H-T lungs, decreased complex I expression is countered by increased complex V expression, which could also account for increased Vmax. This along with high tissue glutathione content protects mitochondria from stress such as exposure to 100% O2. For H-S lungs, the effect of decreased complex I expression on lung tissue ΔΨm is countered by a larger contribution from complex II. However, higher dependency of ΔΨm on complex II could lead to higher mitochondrial oxidant production since complex II is the main source of oxidants in rat lungs. This along with decreased Vmax and increased Kf make H-S rats more susceptible to stress such as exposure to 100% O2. These results are clinically relevant since exposure to hyperoxia is a primary therapy for patients with ARDS, and ventilation with 60% O2 is often required for prolonged periods of time, particularly with COVID-19. This study was funded by NHLBI grant 2R15HL129209-03, Department of Veterans Affairs Merit Review Award BX001681, and NSF grant DMS 2153387. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Low Initial Cell Density Promotes the Differentiation and Maturation of Human Pluripotent Stem Cells into Erythrocytes.

Human pluripotent stem cell (hPSC)-derived red blood cells (RBCs) possess great potential for compensating shortages in transfusion medicine. For better RBC generation from hPSCs, we compared the cell seeding density in the embryoid body formation-based hPSC induction protocol. In the selection of low- and high-density inoculation conditions, we found that low-density culture performed better in the final RBC product with more cell output and increased average cellular hemoglobin content. An elaborate study using flow cytometry demonstrated that low inoculation density promoted endothelial-to-hematopoietic transition, followed by improved hematopoietic progenitor formation and erythrocyte generation. The improved transformation from glycolysis to mitochondrial oxidation and reduced apoptosis might be responsible for this effect. Hints from RNA sequencing suggested that molecules involved in microenvironment interaction and metabolic regulation might respond for the different developmental potential. The possible mediators between outer message and intracellular response could be the nutrition sensors FOXO, PRKAA1 (AMPK), and MTOR genes. It is possible that low inoculation density triggered metabolic regulation signals, promoted mitochondrial oxidation, and resulted in enhanced cell amplification and hematopoietic differentiation. The low cell culture density will improve RBC generation from hPSCs.

Interplay between Energy Supply and Glutamate Toxicity in the Primary Cortical Culture.

Limited substrate availability because of the blood-brain barrier (BBB) has made the brain develop specific molecular mechanisms to survive, using lactate synthesized by astrocytes as a source of energy in neurons. To understand if lactate improves cellular viability and susceptibility to glutamate toxicity, primary cortical cells were incubated in glucose- or lactate-containing media and toxic concentrations of glutamate for 24 h. Cell death was determined by immunostaining and lactate dehydrogenase (LDH) release. Mitochondrial membrane potential and nitric oxide (NO) levels were measured using Tetramethylrhodamine, methyl ester (TMRM) and 4-Amino-5-Methylamino-2',7'-Difluorofluorescein Diacetate (DAF-FM) live staining, respectively. LDH activity was quantified in single cells in the presence of lactate (LDH substrate) and oxamate (LDH inhibitor). Nuclei of cells were stained with DAPI and neurons with MAP2. Based on the distance between neurons and glial cells, they were classified as linked (<10 µm) and non-linked (>10 µm) neurons. Lactate increased cell death rate and the mean value of endogenous NO levels compared to glucose incubations. Mitochondrial membrane potential was lower in the cells cultured with lactate, but this effect was reversed when glutamate was added to the lactate medium. LDH activity was higher in linked neurons compared to non-linked neurons, supporting the hypothesis of the existence of the lactate shuttle between astrocytes and at least a portion of neurons. In conclusion, glucose or lactate can equally preserve primary cortical neurons, but those neurons having a low level of LDH activity and incubated with lactate cannot cover high energetic demand solely with lactate and become more susceptible to glutamate toxicity.

Drug-induced impairment of mitochondrial fatty acid oxidation and steatosis: assessment of causal relationship with 45 pharmaceuticals.

Drug-induced liver injury (DILI) represents a major issue for pharmaceutical companies, being a potential cause of black-box warnings on marketed pharmaceuticals, or drug withdrawal from the market. Lipid accumulation in the liver also referred to as steatosis, may be secondary to impaired mitochondrial fatty acid oxidation (mtFAO). However, an overall causal relationship between drug-induced mtFAO inhibition and the occurrence of steatosis in patients has not yet been established with a high number of pharmaceuticals. Hence, 32 steatogenic and 13 nonsteatogenic drugs were tested for their ability to inhibit mtFAO in isolated mouse liver mitochondria. To this end, mitochondrial respiration was measured with palmitoyl-l-carnitine, palmitoyl-CoA + l-carnitine, or octanoyl- l-carnitine. This mtFAO tri-parametric assay was able to predict the occurrence of steatosis in patients with a sensitivity and positive predictive value above 88%. To get further information regarding the mechanism of drug-induced mtFAO impairment, mitochondrial respiration was also measured with malate/glutamate or succinate. Drugs such as diclofenac, methotrexate, and troglitazone could inhibit mtFAO secondary to an impairment of the mitochondrial respiratory chain, whereas dexamethasone, olanzapine, and zidovudine appeared to impair mtFAO directly. Mitochondrial swelling, transmembrane potential, and production of reactive oxygen species were also assessed for all compounds. Only the steatogenic drugs amiodarone, ketoconazole, lovastatin, and toremifene altered all these 3 mitochondrial parameters. In conclusion, our tri-parametric mtFAO assay could be useful in predicting the occurrence of steatosis in patients. The combination of this assay with other mitochondrial parameters could also help to better understand the mechanism of drug-induced mtFAO inhibition.

Exploring novel function of Gpx5 antioxidant activity: Assisting epididymal cells secrete functional extracellular vesicles.

Glutathione peroxisomal-5 (Gpx5) promotes the elimination of H2O2 or organic hydrogen peroxide, and plays an important role in the physiological process of resistance to oxidative stress (OS). To directly and better understand the protection of Gpx5 against OS in epididymal cells and sperm, we studied its mechanism of antioxidant protection from multiple aspects. To more directly investigate the role of Gpx5 in combating oxidative damage, we started with epididymal tissue morphology and Gpx5 expression profiles in combination with the mouse epididymal epithelial cell line PC1 (proximal caput 1) expressing recombinant Gpx5. The Gpx5 is highly expressed in adult male epididymal caput, and its protein signal can be detected in the sperm of the whole epididymis. Gpx5 has been shown to alleviate OS damage induced by 3-Nitropropionic Acid (3-NPA), including enhancing antioxidant activity, reducing mitochondrial damage, and suppressing cell apoptosis. Gpx5 reduces OS damage in PC1 and maintains the well-functioning extracellular vesicles (EVs) secreted by PC1, and the additional epididymal EVs play a role in the response of sperm to OS damage, including reducing plasma membrane oxidation and death, and increasing sperm motility and sperm-egg binding ability. Our study suggests that GPX5 plays an important role as an antioxidant in the antioxidant processes of epididymal cells and sperm, including plasma membrane oxidation, mitochondrial oxidation, apoptosis, sperm motility, and sperm-egg binding ability.

TNF-α signals through ITK-Akt-mTOR to drive CD4+ T cell metabolic reprogramming, which is dysregulated in rheumatoid arthritis.

Upon activation, T cells undergo metabolic reprogramming to meet the bioenergetic demands of clonal expansion and effector function. Because dysregulated T cell cytokine production and metabolic phenotypes coexist in chronic inflammatory disease, including rheumatoid arthritis (RA), we investigated whether inflammatory cytokines released by differentiating T cells amplified their metabolic changes. We found that tumor necrosis factor-α (TNF-α) released by human naïve CD4+ T cells upon activation stimulated the expression of a metabolic transcriptome and increased glycolysis, amino acid uptake, mitochondrial oxidation of glutamine, and mitochondrial biogenesis. The effects of TNF-α were mediated by activation of Akt-mTOR signaling by the kinase ITK and did not require the NF-κB pathway. TNF-α stimulated the differentiation of naïve cells into proinflammatory T helper 1 (TH1) and TH17 cells, but not that of regulatory T cells. CD4+ T cells from patients with RA showed increased TNF-α production and consequent Akt phosphorylation upon activation. These cells also exhibited increased mitochondrial mass, particularly within proinflammatory T cell subsets implicated in disease. Together, these findings suggest that T cell-derived TNF-α drives their metabolic reprogramming by promoting signaling through ITK, Akt, and mTOR, which is dysregulated in autoinflammatory disease.

AICAR confers prophylactic cardioprotection in doxorubicin-induced heart failure in rats

Doxorubicin (DOX) is a widely used chemotherapeutic agent that can cause serious cardiotoxic side effects, leading to heart failure (HF). Impaired mitochondrial function is thought to be key factor driving progression into HF. We have previously shown in a rat model of DOX-HF that heart failure with reduced ejection fraction correlates with mitochondrial loss and dysfunction. Adenosine monophosphate-dependent kinase (AMPK) is a cellular energy sensor, regulating mitochondrial biogenesis and energy metabolism, including fatty acid oxidation. We hypothesised that AMPK activation could restore mitochondrial function and therefore be a novel cardioprotective strategy for the prevention of DOX-HF. Consequently, we set out to assess whether 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR), an activator of AMPK, could prevent cardiac functional decline in this chronic intravenous rat model of DOX-HF. In line with our hypothesis, AICAR improved cardiac systolic function. AICAR furthermore improved cardiac mitochondrial fatty acid oxidation, independent of mitochondrial number, and in the absence of observable AMPK-activation. In addition, we found that AICAR prevented loss of myocardial mass. RNAseq analysis showed that this may be driven by normalisation of pathways associated with ribosome function and protein synthesis, which are impaired in DOX-treated rat hearts. AICAR furthermore prevented dyslipidemia and excessive body-weight loss in DOX-treated rats, which may contribute to preservation of myocardial mass. Though it is unclear whether AICAR exerted its cardioprotective effect through cardiac or extra-cardiac AMPK-activation or via an AMPK-independent effect, these results show promise for the use of AICAR as a cardioprotective agent in DOX-HF to both preserve cardiac function and mass.

Podophyllotoxin via SIRT1/PPAR /NF-κB axis induced cardiac injury in rats based on the toxicological evidence chain (TEC) concept

BackgroundThe study of cardiotoxicity of drugs has become an important part of clinical safety evaluation of drugs. It is commonly known that podophyllotoxin (PPT) and its many derivatives and congeners are broad-spectrum pharmacologically active substances. Clinical cardiotoxicity of PPT and its derivatives has been raised, basic research on the mechanism of cardiotoxicity remains insufficient. PurposeIn present study, our group's innovative concept of toxicological evidence chain (TEC) was applied to reveal the cardiac toxicity mechanism of PPT by targeted metabolomics, TMT-based quantitative proteomics and western blot. MethodsThe injury phenotype evidence (IPE) acquired from the toxicity manifestations, such as weight and behavior observation of Sprague-Dawley rat. The damage to rat hearts were assessed through histopathological examination and myocardial enzymes levels, which were defined as Adverse Outcomes Evidence (AOE). The damage to rat hearts was assessed through histopathological examination and myocardial enzyme levels, which were defined as evidence of adverse outcomes.Overall measurements of targeted metabolomics based on energy metabolism and TMT-based quantitative proteomics were obtained after exposure to PPT to acquire the Toxic Event Evidence (TEE). The mechanism of cardiac toxicity was speculated based on the integrated analysis of targeted metabolomics and TMT-based quantitative proteomics, which was verified by western blot. ResultsThe results indicated that exposure to PPT could result in significant elevation of myocardial enzymes and pathological alterations in rat hearts. In addition, we found that PPT caused disorders in cardiac energy metabolism, characterized by a decrease in energy metabolism fuels. TMT-based quantitative proteomics revealed that the PPAR (Peroxisome proliferators-activated receptor) signaling pathway needs further study. It is worth noting that PPT may suppress the expression of SIRT1, subsequently inhibiting AMPK, decreasing the expression of PGC-1α, PPARα and PPARγ. This results in disorders of glucose oxidation, glycolysis and ketone body metabolism. Additionally, the increase in the expression of p-IKK and p-IκBα, leads to the nuclear translocation of NF-κB p65 from the cytosol, thus triggering inflammation. ConclusionThis study comprehensively evaluated cardiac toxicity of PPT and initially revealed the mechanism of cardiotoxicity,suggesting that PPT induced disorders of energy metabolism and inflammation via SIRT1/PPAR/NF-κB axis, potentially contributing to cardiac injury.

Identification of biomarkers and immune infiltration characterization of lipid metabolism-associated genes in osteoarthritis based on machine learning algorithms.

Osteoarthritis (OA) is a prevalent degenerative condition commonly observed in the elderly, leading to consequential disability. Despite notable advancements made in clinical strategies for OA, its pathogenesis remains uncertain. The intricate association between OA and metabolic processes has yet to receive comprehensive exploration. In our investigation, we leveraged public databases and applied machine learning algorithms, including WGCNA, LASSO, RF, immune infiltration analysis, and pathway enrichment analysis, to scrutinize the role of lipid metabolism-associated genes (LAGs) in the OA. Our findings identified three distinct biomarkers, and evaluated their expression to assess their diagnostic value in the OA patients. The exploration of immune infiltration in these patients revealed an intricate relationship between immune cells and the identified biomarkers. In addition, in vitro experiments, including qRT-PCR, Western blot, chondrocyte lipid droplets detection and mitochondrial fatty acid oxidation measurement, further verified abnormal expressions of selected LAGs in OA cartilage and confirmed the correlation between lipid metabolism and OA.

Functional metabolomics reveals arsenic-induced inhibition of linoleic acid metabolism in mice kidney in drinking water

Arsenic (As) is a heavy metal known for its detrimental effects on the kidneys, but the precise mechanisms underlying its toxicity remain unclear. In this study, we employed an integrated approach combining traditional toxicology methods with functional metabolomics to explore the nephrotoxicity induced by As in mice. Our findings demonstrated that after 28 days of exposure to sodium arsenite, blood urea nitrogen, serum creatinine levels were significantly increased, and pathological examination of the kidneys revealed dilation of renal tubules and glomerular injury. Additionally, uric acid, total cholesterol, and low-density lipoprotein cholesterol levels were significant increased while triglyceride level was decreased, resulting in renal insufficiency and lipid disorders. Subsequently, the kidney metabolomics analysis revealed that As exposure disrupted 24 differential metabolites, including 14 up-regulated and 10 down-regulated differential metabolites. Ten metabolic pathways including linoleic acid and glycerophospholipid metabolism were significantly enriched. Then, 80 metabolic targets and 168 predicted targets were identified using metabolite network pharmacology analysis. Of particular importance, potential toxicity targets, such as glycine amidinotransferase, mitochondrial (GATM), and nitric oxide synthase, and endothelial (NOS3), were prioritized through the “metabolite-target-pathway” network. Receiver operating characteristics curve and molecular docking analyses suggested that 1-palmitoyl-2-myristoyl-sn-glycero-3-PC, linoleic acid, and L-hydroxyarginine might be functional metabolites associated with GATM and NOS3. Moreover, targeted verification result showed that the level of linoleic acid in As group was 0.4951 μg/mL, which was significantly decreased compared with the control group. And in vivo and in vitro protein expression experiments confirmed that As exposure inhibited the expression of GATM and NOS3. In conclusion, these results suggest that As-induced renal injury may be associated with the inhibition of linoleic acid metabolism through the down-regulation of GATM and NOS3, resulting in decreased levels of linoleic acid, 1-palmitoyl-2-myristoyl-sn-glycero-3-PC, and L-hydroxyarginine metabolites.

Triclocarban induces lipid droplet accumulation and oxidative stress responses by inhibiting mitochondrial fatty acid oxidation in HepaRG cells

Mitochondrial fatty acid oxidation (mtFAO) plays an important role in hepatic energy metabolism. Severe mtFAO injury leads to nonalcoholic fatty liver disease (NAFLD) and liver failure. Several drugs have been withdrawn owing to safety issues, such as induction of fatty liver disease through mtFAO disruption. For instance, the antimicrobial triclocarban (TCC), an environmental contaminant that was removed from the market due to its unknown safety in humans, induces NAFLD in rats and promotes hepatic FAO in mice. Therefore, there are no consistent conclusions regarding the effects of TCC on FAO and lipid droplet accumulation. We hypothesized that TCC induces lipid droplet accumulation by inhibiting mtFAO in human hepatocytes. Here, we evaluated mitochondrial respiration in HepaRG cells to investigate the effects of TCC on fatty acid-driven oxidation in cells, electron transport chain parameters, lipid droplet accumulation, and antioxidant genes. The results suggest that TCC increases oxidative stress gene expression (GCLM, p62, HO-1, and NRF2) through lipid droplet accumulation via mtFAO inhibition in HepaRG cells. The results of the present study provide further insights into the effect of TCC on human NAFLD through mtFAO inhibition, and further in vivo studies could be used to validate the mechanisms.

SARS-CoV-2 mitochondrial metabolic and epigenomic reprogramming in COVID-19

To determine the effects of SARS-CoV-2 infection on cellular metabolism, we conducted an exhaustive survey of the cellular metabolic pathways modulated by SARS-CoV-2 infection and confirmed their importance for SARS-CoV-2 propagation by cataloging the effects of specific pathway inhibitors. This revealed that SARS-CoV-2 strongly inhibits mitochondrial oxidative phosphorylation (OXPHOS) resulting in increased mitochondrial reactive oxygen species (mROS) production. The elevated mROS stabilizes HIF-1α which redirects carbon molecules from mitochondrial oxidation through glycolysis and the pentose phosphate pathway (PPP) to provide substrates for viral biogenesis. mROS also induces the release of mitochondrial DNA (mtDNA) which activates innate immunity. The restructuring of cellular energy metabolism is mediated in part by SARS-CoV-2 Orf8 and Orf10 whose expression restructures nuclear DNA (nDNA) and mtDNA OXPHOS gene expression. These viral proteins likely alter the epigenome, either by directly altering histone modifications or by modulating mitochondrial metabolite substrates of epigenome modification enzymes, potentially silencing OXPHOS gene expression and contributing to long-COVID.

SOME FEATURES OF CELLULAR ENERGY SUPPLY OF THE BODY IN PREMATURE INFANTS WITH SEVERE FORMS OF PERINATAL PATHOLOGY

The nature of postnatal adaptation of newborns, their further growth, development, health status and quality of life largelydepend on the well-being of intrauterine development and maturity at birth. Premature birth under hypoxia causes signifi cant metabolic disorders in the body, is accompanied by a high risk of severe organ dysfunction, which requires in-depth research to clarify the features of intracellular metabolism in order to further improve methods of diagnosis and treatment of newborns with various forms of perinatal pathology.Aim of the research. To study the trends of changes in energy metabolism in premature infants with severe forms of perinatal pathology to investigate the possibility of using them as additional criteria of hypoxic damage to the body in conditions of morphological and functional immaturity.Materials and methods. Clinical and laboratory examinations were performed on 68 newborns with severe forms of perinatal pathology at the gestational age of 32 to 34 weeks; the comparison group consisted of 27 conditionally healthy newborns at the gestational age of 34 to 37 weeks. Inclusion criteria for the main study group: gestational age at birth from 32 to 34 weeks, clinical signs of severe perinatal pathology. Exclusion criteria: gestational age at birth &lt; 32 and ≥ 37 weeks, diagnosed congenital malformations and septic conditions. The list of diseases was determined according to the International Classifi cation of Diseases, X Revision. The following blood serum laboratory parameters were studied: lactate level, glycerol-3-phosphate dehydrogenase (GPDH) (EC 1.1.99.5), succinate dehydrogenase (SDH), NADH dehydrogenase (NADH) (EC 1.6.5.3); the electron transport chain coeffi cient (ETC) was calculated. Laboratory studies were performed using micromethods. The study was conducted in accordance with the provisions of GCP (1996), the Convention of the Council of Europe on Human Rights and Biomedicine (April 4, 1997), the Declaration of Helsinki of the World Medical Association (1964-2008), Order of the Ministry of Health of Ukraine No. 690 dated September 23, 2009 (as amended by Order of the Ministry of Health of Ukraine No. 523 dated July 12, 2012). Protocol of scientifi c research of the Biomedical Ethics Committee of BSMU dated September 12, 2015. Informed written consent was obtained from the parents of the patients before the study with an explanationof the purpose, objectives and methods of laboratory testing. Statistical processing of the results was performed using STATISTICA software (StatSoft Inc., USA, version 10). Comparison of quantitative indicators with normal distribution was performed using Student’s t-test, and the probability of diff erences was considered statistically signifi cant at p&lt;0.05. The complex of studies was conducted within the framework of the planned research topics of the Department of Pediatrics, Neonatology and Perinatal Medicine of the Bukovinian State Medical University: «Improvement of the areas of prognosis, diagnostics and treatment of perinatal pathology in newborns and infants, optimization of the schemes of catamenial observation and rehabilitation» (State registration No. 0115U002768, term of execution 01.2015-12.2019) and «Chronobiological and adaptive aspects and features of vegetative regulation in pathological conditions in children of diff erent age groups» (State registration No. 0122U002245, term of execution 01.2020-12.2024).Results. In newborns of the main group, in comparison with the control group, a signifi cant decrease in GFDH and SDHwas found, which indicates a signifi cant impairment of the activity of enzymes of the respiratory chain and explains the lack of oxygen absorption in the body at the cellular and tissue level, causing the severity of the condition of newborns. Determination of the level of GPDH and SDH in the dynamics of observation in children showed some improvement in these indicators. In the severe condition of newborns, a signifi cant increase in the NADPH index was found, with a further probable increase in its level in the dynamics of observation. Calculations of ETC coeffi cient in cord blood showed a signifi cant decrease of this indicator in comparison with the control group. The identifi ed disorders of intracellular energy metabolism under hypoxia indicate the expediency of conducting comprehensive scientifi c research to study the possibilities of appropriate pharmacological correction to improve the eff ectiveness of treatment of severe forms of perinatal pathology in premature infants.Conclusions: 1. The results of the study revealed a signifi cant decrease in GPDH and SDH at elevated levels of NADPH andETC coeffi cient, which confi rms the presence of signifi cant disturbances in intracellular energy metabolism and mitochondrial oxidation processes. 2. Timely detection of metabolic disorders with the determination of mitochondrial oxidation, in particular energy metabolism, is an important area for improving diagnostic measures in severe forms of perinatal pathology in preterm infants with hypoxic damage to the body in the pathogenesis.

Platelet bioenergetic profiling uncovers a metabolic pattern of high dependency on mitochondrial fatty acid oxidation in type 2 diabetic patients who developed in-stent restenosis

Although platelet bioenergetic dysfunction is evident early in the pathogenesis of diabetic macrovascular complications, the bioenergetic characteristics in type 2 diabetic patients who developed coronary in-stent restenosis (ISR) and their effects on platelet function remain unclear. Here, we performed platelet bioenergetic profiling to characterize the bioenergetic alterations in 28 type 2 diabetic patients with ISR compared with 28 type 2 diabetic patients without ISR (non-ISR) and 28 healthy individuals. Generally, platelets from type 2 diabetic patients with ISR exhibited a specific bioenergetic alteration characterized by high dependency on fatty acid (FA) oxidation, which subsequently induced complex III deficiency, causing decreased mitochondrial respiration, increased mitochondrial oxidant production, and low efficiency of mitochondrial ATP generation. This pattern of bioenergetic dysfunction showed close relationships with both α-granule and dense granule secretion as measured by surface P-selectin expression, ATP release, and profiles of granule cargo proteins in platelet releasates. Importantly, ex vivo reproduction of high dependency on FA oxidation by exposing non-ISR platelets to its agonist mimicked the bioenergetic dysfunction observed in ISR platelets and enhanced platelet secretion, whereas pharmaceutical inhibition of FA oxidation normalized the respiratory and redox states of ISR platelets and diminished platelet secretion. Further, causal mediation analyses identified a strong association between high dependency on FA oxidation and increased angiographical severity of ISR, which was significantly mediated by the status of platelet secretion. Our findings, for the first time, uncover a pattern of bioenergetic dysfunction in ISR and enhance current understanding of the mechanistic link of high dependency on FA oxidation to platelet abnormalities in the context of diabetes.

Proanthocyanidins and Phenolic Compounds from the Twigs of Salix chaenomeloides and Their Anti-Lipogenic Effects on 3T3-L1 Preadipocytes.

The present study investigated potential bioactive natural products from the EtOH extract of Salix chaenomeloides twigs using column chromatography, leading to the isolation of six compounds (1-6), which were characterized as two proanthocyanidins, procyanidin B2 (1) and procyanidin B1 (2), and four phenolic compounds, 4-hydroxybenzoic acid β-D-glucosyl ester (3), di-O-methylcrenatin (4), p-coumaric acid glucoside (5), and syringin (6) by the comparison of their NMR spectra with the reported data and high-resolution (HR)-electrospray ionization mass spectroscopy (ESI-MS) analysis. We investigated the potential of six compounds (1-6) to inhibit adipogenesis in 3T3-L1 preadipocytes, which showed that the compounds (1-6) significantly reduced lipid accumulation in 3T3-L1 adipocytes without affecting cell proliferation. Notably, compound 1 demonstrated a remarkable 60% and 90% reduction in lipid levels with 50 and 100 µM treatments, respectively. Oil Red O staining results indicated that compound 1 significantly inhibits the formation of lipid droplets, comparable to the effect of T863, an inhibitor of triglyceride used as a positive control, in adipocytes. Compound 1 had no effect on the regulators PPARγ, C/EBPα, and SREBF1 of adipocyte differentiation in 3T3-L1 preadipocytes, but compound 1 activated the fatty acid oxidation regulator, PPARα, compared to the lipogenic-induced control. It also suppressed fatty acid synthesis by downregulating the expression of fatty acid synthase (FAS). Finally, compound 1 induced the mRNA and protein levels of CPT1A, an initial marker of mitochondrial fatty acid oxidation in 3T3-L1. This finding substantiates the anti-lipogenic and lipolytic effects of procyanidin B2 (1) in 3T3-L1 preadipocytes, emphasizing its pivotal role in modulating obesity-related markers.