ATP Production Rate Research Articles

Ceramides as Mediators of Mitochondrial Dysfunction Driving Kidney Injury

Objective. Perturbations in proximal tubule mitochondrial function and lipid metabolism potentiate acute kidney injury and failed repair. Sphingolipids such as ceramides are recognized as lipotoxic drivers of metabolic dysfunction and cell death. We hypothesized that ceramides drive mitochondrial dysfunction and tubular insult and that lowering ceramides would prevent acute kidney injury in a mouse model of bilateral ischemia reperfusion injury (IRI). Methods. We examined acute effects of ceramide treatment on primary mouse proximal tubule epithelial cell (PTEC) mitochondrial oxygen consumption and mitochondrial supercomplex assembly by Seahorse respirometry and blue native PAGE, respectively. In vivo, we profiled accumulation of ceramides via HPLC-MS/MS in kidney cortex tissue of mice challenged with warm bilateral IRI. We lowered whole-body ceramides via Degs1 inducible global knockout ( Degs1iGKO) in male C57Bl/6J mice prior to challenge with IRI. Clinical, transcriptional, and histopathological renal outcomes were compared to sham-operated and Degs1fl/fl controls 24 hours after kidney reperfusion. Results. Treatment of PTECs with 5-50uM C2-ceramide resulted in acute and dose-dependent decreases in mitochondrial ATP production rates (15-65% decrease from baseline, respectively, p<0.05), as well as disruption of electron transport chain supercomplex structures (i.e., III2+IV and I+III2+IV). In mice, IRI led to a 25% increase in total cortex ceramides (sham: 995±110 vs IRI: 1239±185 pmol lipid/mg protein, p<0.05). Lipid species within the de novo ceramide synthesis pathway were elevated as early as 5 minutes after IRI (e.g., 34% increase in Cer(d18:0/16:0), sham: 2.38±0.27 vs 5 minutes: 3.20±0.81 pmol lipid/mg protein, p<0.05) and continued to accumulate throughout the progression of the AKI up to 24 hours after reperfusion (e.g., 122% increase in Cer(d18:0/16:0), sham: 2.38±0.27 vs 24 hours: 5.29±1.71 pmol lipid/mg protein, p<0.001). Cortex ceramides, particularly deleterious long-chain (e.g., Cer(d18:1/16:0)) species, correlated positively with kidney injury biomarkers (plasma creatinine, Pearson r=0.57; BUN, r=0.74; and cortex expression of Lcn2, r=0.60 , and Havcr1, r=0.62 , p<0.05). Degs1 global knockout decreased cortex ceramide levels by 56% (fl/fl: 995±110 vs KO: 435±174 pmol lipid/mg protein, p<0.001). Degs1iGKO animals had improved cortex histopathology, blood urea nitrogen (fl/fl: 81.02±2.13 vs KO: 49.82±6.34 mg/dL, p <0.001), plasma creatinine (fl/fl: 1.28±0.4 vs KO: 0.32±0.11 mg/dL, p<0.001), and cortex Lcn2 expression (fl/fl: 36.36±21.50 vs KO: 5.31±4.61, p<0.01) following IRI. Conclusions. Renal ceramides are implicated as drivers of kidney injury and mitochondrial dysfunction. Global Degs1 knockout prevented AKI induced by ischemia reperfusion injury. Thus, ceramide-lowering interventions may be an effective strategy to prevent AKI. This work was supported by funding from the National Institutes of Health [R01KD130296 (SAS, WLH), R01DK131609 (SAS), U01CA272529 (SAS), R44DK116450 (SAS), R01DK112826 (WLH), T32DK091317 (RJN), F31DK134088 (RJN)]. 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.

Fasting Metabolic Profiles are Cell-Autonomous in Elephant Seal Primary Muscle Cells

Northern elephant seals ( Mirounga angustirostris) undergo prolonged terrestrial fasts during energetically intensive life stages including breeding, molting, and postnatal development. Fasting-tolerant vertebrates generally extend the second phase of fasting, during which body mass declines but homeostasis is preserved through reliance on lipid fuel sources and sparing of body protein stores. In elephant seals, the postnatal fast coincides with development of the extensive body oxygen stores required to support extended breath-hold diving, as well as an increased capacity for apnea-related metabolic suppression. Studies in grizzly bears, another large-bodied fasting-tolerant mammal, have identified both serum-derived and intrinsic cellular regulation of the adipocyte metabolic phenotype. However, it is unknown whether these mechanisms extend across species, tissue, or fasting type (active vs inactive). Thus, we derived proliferative primary skeletal muscle cells (myoblasts) from weaned elephant seal pups; biopsies that yielded cells were collected at both early (~1 week) and late (~8 weeks) points in the postnatal fast. We found that total ATP production rate was greater in myoblasts collected from animals late in the fast (27% increase over early, p=0.08). Throughout the fast, myoblasts relied predominantly on glycolytic rather than oxidative ATP production (p<0.001), and this reliance increased as the fast progressed (p<0.014). Together, these data suggest that metabolic reliance is at least partially cell-autonomous in elephant seal myoblasts. KNA is supported by an NSF Graduate Research Fellowship (DGE 1752814 & 2146752) and a UC Berkeley Fellowship. JPV-M is supported by a NIH/NIGMS grant (R35GM146951). Research was funded by UC Berkeley startup funds and the Winkler Family Foundation. 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.

Photosynthetic control at the cytochrome b6f complex.

Photosynthetic control (PCON) is a protective mechanism that prevents light-induced damage to PSI by ensuring the rate of NADPH and ATP production via linear electron transfer (LET) is balanced by their consumption in the CO2 fixation reactions. Protection of PSI is a priority for plants since they lack a dedicated rapid-repair cycle for this complex, meaning that any damage leads to prolonged photoinhibition and decreased growth. The imbalance between LET and the CO2 fixation reactions is sensed at the level of the transthylakoid ΔpH, which increases when light is in excess. The canonical mechanism of PCON involves feedback control by ΔpH on the plastoquinol oxidation step of LET at cytochrome b6f. PCON thereby maintains the PSI special pair chlorophylls (P700) in an oxidized state, which allows excess electrons unused in the CO2 fixation reactions to be safely quenched via charge recombination. In this review we focus on angiosperms, consider how photo-oxidative damage to PSI comes about, explore the consequences of PSI photoinhibition on photosynthesis and growth, discuss recent progress in understanding PCON regulation, and finally consider the prospects for its future manipulation in crop plants to improve photosynthetic efficiency.

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.

Abstract 1563: Anoikis associated metabolic modulation by UCA1 in CRC

Abstract Colorectal Cancer (CRC) is the second most lethal cancer in estimated deaths for 2023 according to the American Cancer Society. This is partially due to the low survival rate once CRC is diagnosed at a distant location. This warrants early diagnostic markers and an understanding of the mechanisms underlying the metastatic process, anoikis resistance being one of them. Our laboratory analysis of the TCGA database comprising 519 human CRC tissues shows lncRNA UCA1 overexpressed in tumors compared to normal, and higher expression of lncRNA UCA1 is responsible for the progression, metastasis, and poor survival of CRC patients. In our anchorage-independent growth model (anoikis resistant), using isogenic (oncogenic and metastatic) cell lines, we observed a very high expression of lncRNA UCA1 along with GluT1 with increased cell survival. Cancer cells, however, appear to rapidly increase their glucose uptake and directly ferment it into lactate, even in the presence of oxygen and functional mitochondria. UCA1 has been found to play an essential role in the development of metastasis and dysregulation of glycolysis in various cancers. To verify if lncRNA UCA1 modulates anoikis resistance and glucose metabolism, we generated SW480+UCA1 overexpression (OE) and SW620+shUCA1 knockdown (KD) stable cell lines using lentiviral transduction. The OE GFP-expressing cell lines were further sorted, and grown in puromycin. The KD cell lines were selected with puromycin for the generation of stable cell lines. The stable cell lines were characterized through different phenotypic assays. UCA1 OE cell lines have a higher glucose uptake and the UCA1 KD cell lines have a lower glucose uptake. The lactate production is high in the KD and low in OE cell lines. The OE and KD cell lines along with the control cell lines were subjected to an anoikis model, and glucose and lactate analysis were performed accordingly. RT-PCR and western blot analysis were performed for the target genes responsible for cell survival, stemness, and glucose metabolism. We are analyzing the Extracellular Acidification Rate (ECAR), Oxidative Consumption Rate (OCR), ATP production rate the rate and potential max rate of glycolysis, and the mitochondrial stress of the recombinant cell lines using the “Agilent Seahorse XF”. The role of lncRNA UCA1 in anoikis resistance leading to metastasis and modulating glucose metabolism will provide new insights into the pathophysiology of CRC metastasis. This information will be beneficial for developing more unique strategies for the treatment of this metastatic disease. Citation Format: Sophia Leslie, Kyle Doxtater, Samantha Lopez, Bilal Hafeez, Tamer Oraby, Timothy Loy, Manish K. Tripathi. Anoikis associated metabolic modulation by UCA1 in CRC [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 1563.

Abstract 1787: The β3-adrenergic receptor as novel target of metabolic phenotype in Ewing sarcoma

Abstract Purpose of study: Metabolic targeted therapies may represent an innovative strategy in Ewing sarcoma (ES). Introduction: Ewing sarcoma (ES) is highly aggressive round cell mesenchymal neoplasm, most often occurring in children and young adults. Metabolic peculiarities of ES cells are indeed involved in tumor growth, survival and resistance to therapy. We have further investigated apoptotic role of 𝜷;;3-adrenergic receptor (𝜷;;3-AR) SR59230A (SR) antagonist for its ability to reduce cells viability due increased oxidative stress, now we underline the metabolic role of SR. Materials & Methods: The metabolic profile after SR administration in A673 ES cell model was studied using the Seahorse XF Analyzer. Viability was evaluated with MTS. Changes in gene expression was evaluated with RNA sequencing. Western blot analysis and Immunofluorescence was performed for redox signature and metabolic alteration. Ros production and cell death was assessed by flow cytometry. Results: Metabolic analysis reveals glucose and fatty acid dependency in A673 cells using the Fuel Flex Test of Seahorse XFe Analyzer. Quantification of transcript abundance using Salmon in A673 after SR treatment showed decrease in response to reactive oxygen species, increase of Thioredoxin Interacting Protein (TXNIP) inhibits the antioxidative function of thioredoxin, moreover reduced glucose uptake, consequently, decrease NADPH/NADP resulting in the accumulation of ROS coincident with a decrease of GLUT1, SOD2, TrDX an increase of LC3IIA protein expression. SR enhances A673 pyruvate dependency, and decrease glyco and mito ATP production rate, more over observed under glucose deprivation. Furthermore, apoptotic effect was significantly amplified when cells were under nutrient starvation, revealing a 56% apoptotic rate after 24 hours of SR treatment. Conclusion: SR antagonist of 𝜷;;3-AR and combination of SR with BUF could be an innovative therapeutic option to raise awareness ES. Citation Format: Cristina Banella, Francesco Carrozzo, Laura Zocca, Amada Pasha, Gianluca Mattei, Marina Mola, Lara Ballerini, Nicla Lorito, Annalisa Tondo, Claudio Favre, Maura Calvani. The β3-adrenergic receptor as novel target of metabolic phenotype in Ewing sarcoma [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 1787.

Pioglitazone reverses alcohol-induced alterations in alveolar macrophage mitochondrial phenotype.

People with alcohol use disorder (AUD) have an increased risk of developing pneumonia and pulmonary diseases. Alveolar macrophages (AMs) are immune cells of the lower respiratory tract that are necessary for clearance of pathogens. However, alcohol causes AM oxidative stress, mitochondrial damage and dysfunction, and diminished phagocytic capacity, leading to lung injury and immune suppression. AMs were isolated by bronchoalveolar lavage from people with AUD and male and female C57BL/6J mice given chronic ethanol (20% w/v, 12 weeks) in drinking water. The peroxisome proliferator-activated receptor γ ligand, pioglitazone, was used to treat human AMs exvivo (10 μM, 24 h) and mice invivo by oral gavage (10 mg/kg/day). Levels of AM mitochondrial superoxide and hypoxia-inducible factor-1 alpha (HIF-1α) mRNA, a marker of oxidative stress, were measured by fluorescence microscopy and RT-qPCR, respectively. Mouse AM phagocytic ability was determined by internalized Staphylococcus aureus, and mitochondrial capacity, dependency, and flexibility for glucose, long-chain fatty acid, and glutamine oxidation were measured using an extracellular flux analyzer. Invitro studies used a murine AM cell line, MH-S (±0.08% ethanol, 72 h) to investigate mitochondrial fuel oxidation and ATP-linked respiration. Pioglitazone treatment decreased mitochondrial superoxide in AMs from people with AUD and ethanol-fed mice and HIF-1α mRNA in ethanol-fed mouse lungs. Pioglitazone also reversed mouse AM glutamine oxidation and glucose or long-chain fatty acid flexibility to meet basal oxidation needs. Invitro, ethanol decreased the rate of AM mitochondrial and total ATP production, and pioglitazone improved changes in glucose and glutamine oxidation. Pioglitazone reversed chronic alcohol-induced oxidative stress in human AM and mitochondrial substrate oxidation flexibility and superoxide levels in mouse AM. Decreased ethanol-induced AM HIF-1α mRNA with pioglitazone suggests that this pathway may be a focus for metabolic-targeted therapeutics to improve morbidity and mortality in people with AUD.

Genome-scale metabolic modeling of Thraustochytrium sp. RT2316-16: Effects of nutrients on metabolism.

Marine thraustochytrids produce metabolically important lipids such as the long-chain omega-3 polyunsaturated fatty acids, carotenoids, and sterols. The growth and lipid production in thraustochytrids depends on the composition of the culture medium that often contains yeast extract as a source of amino acids. This work discusses the effects of individual amino acids provided in the culture medium as the only source of nitrogen, on the production of biomass and lipids by the thraustochytrid Thraustochytrium sp. RT2316-16. A reconstructed metabolic network based on the annotated genome of RT2316-16 in combination with flux balance analysis was used to explain the observed growth and consumption of the nutrients. The culture kinetic parameters estimated from the experimental data were used to constrain the flux via the nutrient consumption rates and the specific growth rate of the triacylglycerol-free biomass in the genome-scale metabolic model (GEM) to predict the specific rate of ATP production for cell maintenance. A relationship was identified between the specific rate of ATP production for maintenance and the specific rate of glucose consumption. The GEM and the derived relationship for the production of ATP for maintenance were used in linear optimization problems, to successfully predict the specific growth rate of RT2316-16 in different experimental conditions.

An adenosine derivative promotes mitochondrial supercomplexes reorganization and restoration of mitochondria structure and bioenergetics in a diethylnitrosamine-induced hepatocellular carcinoma model

Hepatocellular carcinoma (HCC) progression is associated with dysfunctional mitochondria and bioenergetics impairment. However, no data about the relationship between mitochondrial supercomplexes (hmwSC) formation and ATP production rates in HCC are available. Our group has developed an adenosine derivative, IFC-305, which improves mitochondrial function, and it has been proposed as a therapeutic candidate for HCC. We aimed to determine the role of IFC-305 on both mitochondrial structure and bioenergetics in a sequential cirrhosis-HCC model in rats. Our results showed that IFC-305 administration decreased the number and size of liver tumors, reduced the expression of tumoral markers, and reestablished the typical architecture of the hepatic parenchyma. The livers of treated rats showed a reduction of mitochondria number, recovery of the mtDNA/nDNA ratio, and mitochondrial length. Also, IFC-305 increased cardiolipin and phosphatidylcholine levels and promoted hmwSC reorganization with changes in the expression levels of hmwSC assembly-related genes. IFC-305 in HCC modified the expression of several genes encoding elements of electron transport chain complexes and increased the ATP levels by recovering the complex I, III, and V activity. We propose that IFC-305 restores the mitochondrial bioenergetics in HCC by normalizing the quantity, morphology, and function of mitochondria, possibly as part of its hepatic restorative effect.

Environmentally Relevant Concentrations of Tetrabromobisphenol A Exposure Impends Neurovascular Formation through Perturbing Mitochondrial Metabolism in Zebrafish Embryos and Human Primary Endothelial Cells.

Tetrabromobisphenol A (TBBPA), the most extensively utilized brominated flame retardant, has raised growing concerns regarding its environmental and health risks. Neurovascular formation is essential for metabolically supporting neuronal networks. However, previous studies primarily concerned the neuronal injuries of TBBPA, its impact on the neurovascularture, and molecular mechanism, which are yet to be elucidated. In this study, 5, 30, 100, 300 μg/L of TBBPA were administered to Tg (fli1a: eGFP) zebrafish larvae at 2-72 h postfertilization (hpf). The findings revealed that TBBPA impaired cerebral and ocular angiogenesis in zebrafish. Metabolomics analysis showed that TBBPA-treated neuroendothelial cells exhibited disruption of the TCA cycle and the Warburg effect pathway. TBBPA induced a significant reduction in glycolysis and mitochondrial ATP production rates, accompanied by mitochondrial fragmentation and an increase in mitochondrial reactive oxygen species (mitoROS) production in neuroendothelial cells. The supplementation of alpha-ketoglutaric acid, a key metabolite of the TCA cycle, mitigated TBBPA-induced mitochondrial damage, reduced mitoROS production, and restored angiogenesis in zebrafish larvae. Our results suggested that TBBPA exposure impeded neurovascular injury via mitochondrial metabolic perturbation mediated by mitoROS signaling, providing novel insight into the neurovascular toxicity and mode of action of TBBPA.

Coccidiosis and necrotic enteritis model may have a greater impact than dietary anti-interleukin-10 on broiler chicken systemic immunometabolic responses

Dietary egg yolk-derived anti-interleukin (IL)-10 may preserve broiler chicken performance during coccidiosis due to Eimeria spp. infection while effects on secondary Clostridium perfringens (necrotic enteritis) are unknown. Some necrotic enteritis models implement Salmonella Typhimurium to improve repeatability; however, Salmonella upregulation of IL-10 may be a confounder when evaluating anti-IL-10. The study objective was to investigate anti-IL-10 effects on systemic cytokine concentrations and immunometabolism during E. maxima ± C. perfringens challenge in models ± S. Typhimurium. Three 25 d replicate studies using Ross 308 chicks were conducted in wire-floor cages (32 cages/ replicate) with chicks assigned to diets ± 0.03% anti-IL-10. 640 chicks (20/ cage; replicates 1 and 2) were inoculated with sterile saline ± 1×108 colony forming units (CFU) S. Typhimurium while 480 chicks (15/ cage) were placed in replicate 3. In all replicates, blood samples were collected on d 14 (6 chicks/treatment) before administering 15,000 sporulated E. maxima M6 oocysts to S. Typhimurium-inoculated (replicates 1 and 2) or challenge-designated chicks (replicate 3). Half the E. maxima-challenged chicks received 1×108 CFU C. perfringens on d 18 and 19. Blood samples were collected at 1, 3, 7, and 11 d post-inoculation (dpi) with E. maxima and 1, 3, and 7 dpi with secondary C. perfringens. Plasma cytokines were determined by ELISA while immunometabolic assays evaluated peripheral blood mononuclear cell ATP production and glycolytic rate responses. Data were analyzed with diet and challenge fixed effects plus associated interactions (SAS 9.4; P ≤ 0.05). Replicates 1 and 2 showed few immunometabolic responses within 3 dpi with E. maxima, but 25 to 31% increased ATP production and 32% increased compensatory glycolysis at 1 dpi with C. perfringens in challenged vs. unchallenged chicks (P ≤ 0.04). In replicate 3, total ATP production and compensatory glycolysis were increased 25 and 40%, respectively, by the E. maxima main effect at 1dpi (P ≤ 0.05) with unobserved responsiveness to C. perfringens. These outcomes indicate that model type had greater impacts on systemic immunity than anti-IL-10.

Alcohol Promotes Lipogenesis in Sebocytes-Implications for Acne.

The oral consumption of alcohol (ethanol) has a long tradition in humans and is an integral part of many cultures. The causal relationship between ethanol consumption and numerous diseases is well known. In addition to the well-described harmful effects on the liver and pancreas, there is also evidence that ethanol abuse triggers pathological skin conditions, including acne. In the present study, we addressed this issue by investigating the effect of ethanol on the energy metabolism in human SZ95 sebocytes, with particular focus on qualitative and quantitative lipogenesis. It was found that ethanol is a strong trigger for lipogenesis, with moderate effects on cell proliferation and toxicity. We identified the non-oxidative metabolism of ethanol, which produced fatty acid ethyl esters (FAEEs), as relevant for the lipogenic effect-the oxidative metabolism of ethanol does not contribute to lipogenesis. Correspondingly, using the Seahorse extracellular flux analyzer, we found an inhibition of the mitochondrial oxygen consumption rate as a measure of mitochondrial ATP production by ethanol. The ATP production rate from glycolysis was not affected. These data corroborate that ethanol-induced lipogenesis is independent from oxygen. In sum, our results give a causal explanation for the prevalence of acne in heavy drinkers, confirming that alcoholism should be considered as a systemic disease. Moreover, the identification of key factors driving ethanol-dependent lipogenesis may also be relevant in the treatment of acne vulgaris.

Alcohol and its metabolites dysregulate cellular bioenergetics and induce oxidative and endoplasmic reticulum stress in primary human bronchial epithelial cells.

Chronic alcohol consumption/misuse is a significant risk factor for pneumonia and lung infection leading to the development of chronic pulmonary disorders such as chronic obstructive pulmonary disease (COPD) and lung fibrosis. In this study, we sought to delineate the mechanism of alcohol-associated lung disease. We did so by measuring invitro mitochondrial, endoplasmic reticulum (ER) oxidative stress in human bronchial epithelial cells (hBECs) treated with ethanol and its oxidative (acetaldehyde) and nonoxidative (fatty acid ethyl esters or FAEEs) metabolites. Primary hBECs from a normal subject were treated with relevant concentrations of ethanol and its metabolites and incubated at 37°C for 24 h. Viability and cytotoxicity were determined using cell viability and lactate dehydrogenase (LDH) assay kits, respectively. Oxidized glutathione (GSSG) and reduced glutathione (GSH) were measured by colorimetric reaction, and 4-hydroxynenonal (4HNE) by immunohistochemistry. Endoplasmic reticulum stress and dysregulated cellular bioenergetics were determined by western blot analysis. Mitochondrial stress and real-time ATP production rates were determined using a Seahorse Extracellular Flux analyzer. Amelioration of ethanol-induced oxidative/ER stress and mitochondrial energetics was determined using an AMPKα agonist. Human bronchial epithelial cells treated with ethanol, acetaldehyde, and FAEEs showed a concentration-dependent increase in the secretion of LDH, oxidative/ER stress, deactivation of AMPKα phosphorylation and mitochondrial stress (decreased spare respiratory capacity) with concomitant decreases in mitochondrial and glycolytic ATP production rates. FAEEs caused greater cytotoxicity, ER stress, and dysregulated cellular bioenergetics than those ethanol and its oxidative metabolite. AMPKα agonist-pretreated cells significantly ameliorated ethanol-induced oxidative/ER stress, deactivation of AMPKα, and dysregulated cellular bioenergetics. Findings of this study suggest that ethanol and its metabolites contribute to cytotoxicity, oxidative/ER stress, and dysregulation of cellular bioenergetics in hBECs. The attenuation of ethanol-induced ER/oxidative stress and mitochondrial respiration by an AMPKα agonist may reflect a potential for it to be developed as a therapeutic agent for chronic alcohol-associated lung disease.

OP25 Dual activation of GCGR/GLP1R signaling ameliorates intestinal fibrosis via metabolic regulation of histone H3K9 lactylation in epithelial cells

Abstract Background Intestinal fibrosis is a significant clinical challenge in inflammatory bowel diseases, but no effective anti-fibrotic therapy is currently available. GCGR and GLP1R are both peptide hormone receptors involved in energy metabolism of epithelial cells. However, their role in intestinal fibrosis and the underlying mechanisms remain largely unexplored. Methods Intestinal tissues from patients with Crohn’s Disease (CD) (n=20) were obtained. AAV9-Glp1r-shRNA, AAV9-Gcgr-shRNA or AAV9-NC-shRNA were used to transfect mice. Murine colons were analyzed by single-cell RNA sequencing (seq) and metabonomics. Glycolytic metabolism of epithelial cells was analyzed with bulk RNAseq and seahorse-XF technology, while lactylation levels were verified using antibodies against diverse forms of lactylated histones. Then a range of GCGR/GLP1R dual-target ultralong-acting peptides were synthesized, and their agonistic activities were assessed in HEK293 cells that stably expressed either human GLP1R or GCGR, using a cAMP response element-driven luciferase reporter. The antifibrotic activity of the leading peptide 1907B was functionally evaluated in vitro and in vivo. Results GCGR and GLP1R were reduced in the stenotic ileum of patients with CD as well as in the fibrotic colon of mice with chronic colitis. In the chronic model, Gcgr/Glp1r dual knockdown worsened the degree of fibrosis in mice, and increased glycolysis-related metabolites, with lactate exhibiting the most prominent alterations. In the TGFβ1-induced epithelial cell model, exogenous and endogenous lactate induced fibrotic genes expression and overwhelming histone lactylation in H3K9 (H3K9la). Further, cells overexpressing H3.1 (K9R) exhibited lower levels of fibrotic genes and proteins after treatment with lactate compared with the H3.1 WT group. Then 1907B, the leading GCGR/GLP1R dual-target peptide, normalized the ATP production rate of glycolysis, extracellular lactate, H3K9la levels of promoters related to fibrosis genes, and further ameliorated intestinal fibrosis in vivo. Conclusion We uncovered the role of GCGR/GLP1R in regulating EMT involved in intestinal fibrosis via histone lactylation. Simultaneously activating GCGR/GLP1R with the novel dual agonist 1907B holds promise as a treatment strategy for alleviating intestinal fibrosis.

Radiation-induced changes in energy metabolism result in mitochondrial dysfunction in salivary glands

Salivary glands are indirectly damaged during radiotherapy for head and neck cancer, resulting in acute and chronic hyposalivation. Current treatments for radiation-induced hyposalivation do not permanently restore function to the gland; therefore, more mechanistic understanding of the damage response is needed to identify therapeutic targets for lasting restoration. Energy metabolism reprogramming has been observed in cancer and wound healing models to provide necessary fuel for cell proliferation; however, there is limited understanding of alterations in energy metabolism reprogramming in tissues that fail to heal. We measured extracellular acidification and oxygen consumption rates, assessed mitochondrial DNA copy number, and tested fuel dependency of irradiated primary salivary acinar cells. Radiation treatment leads to increases in glycolytic flux, oxidative phosphorylation, and ATP production rate at acute and intermediate time points. In contrast, at chronic radiation time points there is a significant decrease in glycolytic flux, oxidative phosphorylation, and ATP production rate. Irradiated salivary glands exhibit significant decreases in spare respiratory capacity and increases in mitochondrial DNA copy number at days 5 and 30 post-treatment, suggesting a mitochondrial dysfunction phenotype. These results elucidate kinetic changes in energy metabolism reprogramming of irradiated salivary glands that may underscore the chronic loss of function phenotype.

Glycolysis: An early marker for vancomycin-specific T-cell activation.

Vancomycin, a glycopeptide antibiotic used for Gram-positive bacterial infections, has been linked with drug reaction with eosinophilia and systemic symptoms (DRESS) in HLA-A*32:01-expressing individuals. This is associated with activation of T lymphocytes, for which glycolysis has been isolated as a fuel pathway following antigenic stimulation. However, the metabolic processes that underpin drug-reactive T-cell activation are currently undefined and may shed light on the energetic conditions needed for the elicitation of drug hypersensitivity or tolerogenic pathways. Here, we sought to characterise the immunological and metabolic pathways involved in drug-specific T-cell activation within the context of DRESS pathogenesis using vancomycin as model compound and drug-reactive T-cell clones (TCCs) generated from healthy donors and vancomycin-hypersensitive patients. CD4+ and CD8+ vancomycin-responsive TCCs were generated by serial dilution. The Seahorse XFe96 Analyzer was used to measure the extracellular acidification rate (ECAR) as an indicator of glycolytic function. Additionally, T-cell proliferation and cytokine release (IFN-γ) assay were utilised to correlate the bioenergetic characteristics of T-cell activation with invitro assays. Model T-cell stimulants induced non-specific T-cell activation, characterised by immediate augmentation of ECAR and rate of ATP production (JATPglyc). There was a dose-dependent and drug-specific glycolytic shift when vancomycin-reactive TCCs were exposed to the drug. Vancomycin-reactive TCCs did not exhibit T-cell cross-reactivity with structurally similar compounds within proliferative and cytokine readouts. However, cross-reactivity was observed when analysing energetic responses; TCCs with prior specificity for vancomycin were also found to exhibit glycolytic switching after exposure to teicoplanin. Glycolytic activation of TCC was HLA restricted, as exposure to HLA blockade attenuated the glycolytic induction. These studies describe the glycolytic shift of CD4+ and CD8+ T cells following vancomycin exposure. Since similar glycolytic switching is observed with teicoplanin, which did not activate T cells, it is possible the master switch for T-cell activation is located upstream of metabolic signalling.

Phytosterol organic acid esters: Characterization, anti-inflammatory properties and a delivery strategy to improve mitochondrial function

Phytosterol organic acid esters are important food resources and the components of biomembrane structure. Due to the lack of extraction and synthesis techniques, more research has been focused on phytosterols, and the research on phytosterol acid esters have encountered a bottleneck, but phytosterol acid esters confer substantial benefits to human health. In this study, stigmasteryl vanillate (VAN), stigmasteryl protocatechuate (PRO) and stigmasteryl sinapate (SIN) were prepared through the Steglich reaction. The processes are promotable and the products reach up to 95% purity. In addition, their stability was evaluated by differential scanning calorimetry and thermogravimetric analysis. HPLC analysis revealed an enhancement in water solubility after esterification with phenolic acid. In an in vitro digestion model, the bioaccessibility of stigmasteryl phenolates was significantly higher than that of stigmasterols (STIs). Regarding the anti-inflammatory properties, VAN, PRO, and SIN exhibit superior effects against TNF-α induced pro-inflammatory responses compared to STI. All stigmasteryl phenolates supplementation increased the ATP production, the basal, and maximal oxygen consumption rate in mitochondrial stress test. Overall, we present a synthesis method for stigmasteryl phenolates. It will contribute to the development and research of phytosterol acid ester analysis, functions and utilization in food. Moreover, the nutrient-stigmasterol hybrids tactic we have constructed is practical and can become a targeted mitochondrial delivery strategy with enhanced anti-inflammatory effects.

Low-Se Diet Increased Mitochondrial ROS to Suppress Myoblasts Proliferation and Promote Apoptosis in Broilers via miR-365-3p/SelT Signaling Axis.

microRNA (miRNA) controls the post-transcriptional translation of mRNA to affect the expression of many genes participating in functional interaction pathways. Selenoproteins are characterized by their antioxidant activity, wherein selenoprotein T (SelT) is an essential membrane-bound selenoprotein serving as a guardian of intracellular homeostasis. During muscle development and regeneration, myoblasts enter the cell cycle and rapidly proliferate. However, the role of SelT in muscle development and selenium (Se) deficiency-induced muscle damage remains poorly investigated. This study established Se deficient broiler models, chicken embryos models, and cultured chicken primary myoblasts in vitro. We showed that Se deficiency induced skeletal muscle damage in broilers, promoted miR-365-3p expression, and downregulated the level of SelT, significantly. The absence of SelT led to the accumulation of mitochondrial superoxide and downregulated mitochondrial dynamics gene expression, which, in turn, induced the disruption of mitochondria potential and blocked the oxidative phosphorylation (OXPHOS) process. Limited ATP production rate caused by mitochondrial ROS overproduction went along with cell cycle arrest, cell proliferation slowness, and myocyte apoptosis increase. Using Mito-TEMPO for mitochondrial ROS elimination could effectively mitigate the above adverse reactions and significantly restore the proliferation potential of myoblasts. Moreover, we identified miR-365-3p, a miRNA that targeted SelT mRNA to inhibit myoblast proliferation by disrupting intracellular redox balance. The omics analysis results showed that Se deficiency led to the significant enrichment of "cell cycle", "oxidative stress response", and "oxidative phosphorylation" pathway genes. Finally, we proved that the effect of the miR-365-3p/SelT signaling axis on muscle development did exist in the chicken embryo stage. In summary, our findings revealed that miR-365-3p was involved in broiler skeletal muscle damage in Se deficiency by targeting SelT, and SelT, serving as an intracellular homeostasis guardian, resisted mitochondrial oxidative stress, and protected ATP generation, promoting myoblast proliferation and inhibiting apoptosis. This study provides an attractive target for the cultivated meat industry and regenerative medicine.

Novel Relationship between Mitofusin 2-Mediated Mitochondrial Hyperfusion, Metabolic Remodeling, and Glycolysis in Pulmonary Arterial Endothelial Cells.

The disruption of mitochondrial dynamics has been identified in cardiovascular diseases, including pulmonary hypertension (PH), ischemia-reperfusion injury, heart failure, and cardiomyopathy. Mitofusin 2 (Mfn2) is abundantly expressed in heart and pulmonary vasculature cells at the outer mitochondrial membrane to modulate fusion. Previously, we have reported reduced levels of Mfn2 and fragmented mitochondria in pulmonary arterial endothelial cells (PAECs) isolated from a sheep model of PH induced by pulmonary over-circulation and restoring Mfn2 normalized mitochondrial function. In this study, we assessed the effect of increased expression of Mfn2 on mitochondrial metabolism, bioenergetics, reactive oxygen species production, and mitochondrial membrane potential in control PAECs. Using an adenoviral expression system to overexpress Mfn2 in PAECs and utilizing 13C labeled substrates, we assessed the levels of TCA cycle metabolites. We identified increased pyruvate and lactate production in cells, revealing a glycolytic phenotype (Warburg phenotype). Mfn2 overexpression decreased the mitochondrial ATP production rate, increased the rate of glycolytic ATP production, and disrupted mitochondrial bioenergetics. The increase in glycolysis was linked to increased hypoxia-inducible factor 1α (HIF-1α) protein levels, elevated mitochondrial reactive oxygen species (mt-ROS), and decreased mitochondrial membrane potential. Our data suggest that disrupting the mitochondrial fusion/fission balance to favor hyperfusion leads to a metabolic shift that promotes aerobic glycolysis. Thus, therapies designed to increase mitochondrial fusion should be approached with caution.

Effect of lipotoxicity on mitochondrial function and epigenetic programming during bovine in vitro embryo production

Maternal metabolic disorders may cause lipotoxic effects on the developing oocyte. Understanding the timing at which this might disrupt embryo epigenetic programming and how this is linked with mitochondrial dysfunction is crucial for improving assisted reproductive treatments, but has not been investigated before. Therefore, we used a bovine in vitro model to investigate if pathophysiological palmitic acid (PA) concentrations during in vitro oocyte maturation and in vitro embryo culture alter embryo epigenetic patterns (DNA methylation (5mC) and histone acetylation/methylation (H3K9ac/H3K9me2)) compared to control (CONT) and solvent control (SCONT), at the zygote and morula stage. Secondly, we investigated if these epigenetic alterations are associated with mitochondrial dysfunction and changes in ATP production rate, or altered expression of epigenetic regulatory genes. Compared to SCONT, H3K9ac and H3K9me2 levels were increased in PA-derived zygotes. Also, 5mC and H3K9me2 levels were increased in PA-exposed morulae compared to SCONT. This was associated with complete inhibition of glycolytic ATP production in oocytes, increased mitochondrial membrane potential and complete inhibition of glycolytic ATP production in 4-cell embryos and reduced SOD2 expression in PA-exposed zygotes and morulae. For the first time, epigenetic alterations in metabolically compromised zygotes and morulae have been observed in parallel with mitochondrial dysfunction in the same study.