Proton Leak Research Articles

High Salt Induced Alterations in Substrate Metabolism, Respiratory Activity, and Acidification in Isolated Proximal Tubules from Dahl Salt-Sensitive Rats

Rationale: The renal nephron actively reabsorbs nearly 99% of Na+ from the glomerular filtrate to maintain fluid and solute homeostasis, roughly 70% of which occurs in the proximal tubules (PT), where mitochondria consume a great amount of O2 for ATP production to meet the energy demand. However, the effect of a high salt diet upon the PT substrate metabolism and respiratory activity towards energy production and associated cellular acidification is not well-known. Method: To determine alterations in the PT substrate metabolism, respiratory activity, and acidification with a high salt diet, the PT of adult Dahl salt-sensitive (SS) rats were isolated by collagenase digestion and sieving with the rats fed either a 0.4% (LS) or a 4.0% (HS) NaCl diet for 7, 14 and 21 days (age-matched). Responses of the PT O2 consumption rate (OCR) and extracellular acidification rate (ECAR) were determined using a high-throughput Agilent Seahorse XF96 Extracellular Flux Analyzer and a modified mitochondrial stress test protocol, which combined a substrate addition step to the mitochondrial stress reagent addition steps. Basal OCR and ECAR were assessed with only amino acids (basic Seahorse medium) used as fuel for respiration. Subsequently, OCR and ECAR responses were determined by adding different circulating substrates followed by perturbations to the electron transport chain (ETC) in sequential order with oligomycin to inhibit ATP synthase, FCCP to uncouple oxidative phosphorylation, and rotenone + antimycin A to inhibit complexes I and III of the ETC. From these measurements, different OCR and ECAR parameters were derived for each substrate and each salt load condition. Results: The OCR data show that the PT of SS rats preferred to use lactate, glutamine, and palmitate for ATP production under the LS diet. However, the HS diet resulted in significantly reduced OCR when supplied with the same substrates. Interestingly, the basal OCR which occurred with amino acids as metabolic substrates was increased in the PT obtained from HS fed SS rats starting on day 7. These alterations in substrate metabolism were also apparent from the same parametric analysis of the ECAR data. An increased mitochondrial proton leak induced respiration was also deduced form both OCR and ECAR data under the HS diet. Since it is known that uncoupling proteins (UCP) and ADP/ATP translocase (ANT) enhance proton leak, gene expression was quantified from the isolated PT segments. Increased Ucp2 gene expression was found in response to the HS diet (days 7, 14, 21) compared to the LS fed SS rats but no changes were observed in the expression of Slc25A4 (ANT1) or Slc25A5 (ANT2). Summary: HS diet resulted in significant metabolic changes in the PT segments, including substrate metabolism, respiratory activity, and cellular acidification. HS diet also increased PT basal respiration and proton leak, which was associated with an increase of Ucp2 gene expression. Considering the increased ATP demand in the PT segments with a HS diet, these changes would result in increased O2 consumption and increased reactive oxygen species production. Together with the physiological changes on renal hemodynamics that we have observed, we propose that the observed metabolomic changes on the PT upon HS diet contribute to the onset of associated regional ischemia, tissue inflammation, and renal injury observed in hypertensive SS rats. NIH R01-HL151587. 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.

Cell-autonomous Action of the FGF19 Promotes UCP1 Expression and Function in Human Brown Adipocytes

Fibroblast growth factor 19 (FGF19) is an intestine-derived hormone that regulates glucose and energy metabolism. Central and systemic administration of human FGF19 in mice promotes increased energy expenditure and body weight loss through mechanisms that are not fully understood. In this study, we investigated whether FGF19 can activate thermogenesis in brown adipocytes (BA) in a cell-autonomous manner. We differentiated immortalized human brown preadipocytes into mature adipocytes with adipogenic induction media in the presence and absence of FGF19 for 18 days to evaluate the effects of FGF19 on adipogenesis and thermogenesis. Through qPCR and RNA-sequencing analyses, we observed that FGF19 receptors were expressed in both brown preadipocytes and mature BA. However, the obligatory FGF19 co-receptor, β-klotho, was not expressed in preadipocytes, suggesting that the effects of FGF19 must occur preferentially in mature BA. Lipid staining with Oil Red O as well as gene expression of the adipogenic markers fatty acid binding protein (FABP4) and peroxisome proliferator activated receptor-γ (PPAR-γ) showed that adipogenesis was not affected by FGF19. We then treated mature BA with FGF19 (100 ng/mL) acutely and chronically to evaluate FGF19-induced changes in signaling, gene transcription, and thermogenic function. FGF19 signaling was assessed by measuring ERK1/2 phosphorylation levels, which increased within 5 min and remained elevated for at least 30 min after treatment. Using Seahorse assays, we observed that 24-hour FGF19 treatment promoted increased basal respiration, ATP production, and proton leak in human BA, indictive of enhanced mitochondrial function. Since proton leak across the inner membrane through the uncoupling protein 1 (UCP1) generates heat, we assessed the expression of UCP1 and other thermogenic genes. Interestingly, FGF19 treatment specifically increased UCP1 gene expression without affecting other classical adrenergic-mediated thermogenic genes, such as deiodinase 2 (DIO2), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1-α), PR domain containing 16 (PRDM16), and cell death-inducing DNA fragmentation factor A (CIDEA). These findings suggest that FGF19 regulates UCP1 expression through non-canonical pathways. Future work will elucidate the mechanism by which FGF19 promotes UCP1 gene expression in mature BA. Taken together, FGF19 stimulates ERK1/2 phosphorylation and UCP1 expression in human BA, leading to enhanced mitochondrial respiration. These results provide new mechanistic insights into FGF19’s effects on metabolic regulation. Topic subcategories: Adipocyte biology. Funding sources: Sao Paulo Research Foundation (FAPESP): 2022/15148-2 and NIH R01 DK102898. 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.

Characterization of Sex-Dependent Differences in Brain Microvascular Bioenergetics

Background: Women experience increased incidence of stroke-related deaths and lifetime risk of Alzheimer's disease (AD). Microvascular dysfunction has been implicated in the pathogenesis of ischemic stroke and AD. Our objective is to investigate the sex-dependent differences in bioenergetics of young mouse brain microvessels (BMVs). Methods: BMVs were isolated from young male and female mice (3-4 months), using a combination of gradient centrifugation and filtration (300μm and 40μm filters) methods. Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of BMVs were measured utilizing the Agilent Seahorse XFe 24 analyzer. The levels of proteins and mRNAs of mitochondrial respiratory complexes were measured by western blot and RT-PCR, respectively. Citrate synthase (CS) enzyme activity and ATP levels were determined in lysates of BMVs. Results: Real-Time ATP Rate assay demonstrated a decreased total ATP production rate and ATP contribution from both glycolysis and oxidative phosphorylation (OxPhos) in BMVs from females compared to males. Mitochondrial stress test assay revealed significantly lower values of several mitochondrial respiratory parameters, including basal respiration, ATP production, maximal respiration, spare respiratory capacity, and proton leak in BMVs from females compared to males. Glycolytic Rate assay uncovered reduced basal glycolysis, proton efflux rate (PER) originating from basal glycolysis, and the mitoOCR/glycoPER ratio in BMVs from females compared to males. Interestingly, no sex-dependent differences were observed in basal PER and post-2-DG acidification rates in BMVs. Mito Fuel Flex Test assay showed no sex-dependent difference in the utilization of glucose, glutamine, and long-chain fatty acids as fuel substrates in BMVs. ATP measurements confirmed the reduced ATP levels in BMVs from females compared to males. Measurements of CS activity revealed no sex-dependent difference in the BMVs. Lastly, Western blot and RT-PCR experiments indicated no differences in either protein or mRNA levels of various mitochondrial complex protein levels.Conclusions: Female BMVs showed reduced mitochondrial respiratory and glycolytic parameters compared to male BMVs although relative contribution of various substrates for energy production were not different. These sex-dependent differences involving glycolysis and OxPhos make the microvasculature vulnerable to injury in females. Funding sources: NIH grants AG074489 and NS114286 (PVGK and RM). 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.

Effects of heat perturbation to rhinoceros’ cellular metabolism

Climate change is associated with increasing global mean temperatures and extreme heat events. These drastic changes in temperature can have significant deleterious physiological effects, particularly on large strict homeothermic mammals such as the threatened Southern White Rhino (SWR - Ceratotherium simum simum). Developing alternative strategies to understand how climatic events impact species that are logistically challenging to study are crucial. Dermal fibroblasts have proven to be effective in vitro models for studying species resilience to climate since they can reflect an organism's thermosensitivity. These cells can provide an integrative and mechanistic explanation on how cellular function can inform organismal responses to environmental changes. We investigated the effects of heat on mitochondrial function and cellular biology by exposing SWR primary dermal fibroblasts (n = 10 biological replicates) to 24 hours of heat (41°C) or 37°C as a control in culture. Then, we evaluated mitochondrial function using mitochondrial stress test, glycolytic rate test and immunofluorescence (IF) to track nucleus size and mitochondrial distribution in the cytoplasm. Metabolic function was assessed using Agilent Seahorse analyzer and IF images were analyzed on CellProfilerTM software. Our results indicate that compared to the control (37°C), heat exposure decreases mitochondrial respiration (basal: t = 3.7, df = 9, p < 0.01; maximum: t = 3.0, df = 9, p < 0.02), ATP production (t = 5.8, df = 9, p < 0.001), and coupling effciency (t = 7.6, df = 9, p < 0.001). No changes were observed in spare respiratory capacity (t = 0.7, df = 9, p = 0.5), proton leak (t = 0.7, df = 9, p = 0.5) and glycolysis (basal: t = 0.3, df = 4, p = 0.4; compensatory: t = 0.6, df = 4, p = 0.6). Furthermore, heat exposure increases nucleus size and mitochondrial distribution area (Nucleus area: t = 6.8, d.f. = 9, P < 0.001, Mitochondrial coverage area: t = 5.3, d.f. = 9, p < 0.001) and decreases fluorescence intensity of the mitochondrial dye and solidity area covered by mitochondria (Fluorescence: t = 2.3, d.f. = 9, p = 0.04, Mitochondrial area solidity: t = 5.6, d.f. = 9, p < 0.001). These findings show that acute heatwaves trigger a series of complex and interconnected responses in SWR fibroblasts resulting in mitochondrial disfunction, possibly mitophagy and/or mitochondria disintegration, and larger dispersion through the cytoplasm. The thermosensitive phenotype exhibited by SWR fibroblasts provides insights into the likely consequences of global warming on the performance of this species, as cellular and mitochondrial functioning plays a fundamental role in an organism's ability to adjust and thrive under environmental change. Funded by NSF and San Diego Zoo Wildlife Alliance. 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.

Implications of alcohol use for aging-associated skeletal muscle dysfunction

Background: Alcohol use decreases skeletal muscle (SKM) strength and function. Alcohol use is disproportionately on the rise among older individuals, with 25% engaging in heavy drinking and 11% reporting binge drinking. The significant increase in life expectancy in the United States leads us to predict that alcohol use will be an important factor exacerbating SKM function in chronological aging, increasing the risk of poor physical performance and frailty. The objective of the study was to assess the impact of recent alcohol use on measures of physical performance and on muscle differentiation and bioenergetic capacity among people over the age of 60. Methods: We conducted a prospective cross-sectional study. Sedentary people over the age of 60 years were recruited from the Greater New Orleans Area. Participants were assessed for physical performance and vastus lateralis muscle biopsies taken for biochemical analysis. Recent alcohol misuse was defined as phosphatidyl ethanol (PEth), a biomarker of alcohol use, levels >20 ng/ml. Results: Preliminary analysis of data from 13 participants (PEth> 20 ng/ml, n=5) showed no differences in hand grip strength (Means± SD-31.08±9.8 and 33.17±8.4 kg respectively), 30-sec sit to stand test (10.5± 1.8 and 10.6± 3.9 number respectively), and Timed up and go Test (13.5± 2.6 and 11.4± 3.1 seconds respectively) among participants with PEth < 20 ng/ml compared to those with PEth >20ng/ml. Data collected from myotubes derived from myoblasts isolated from SKM biopsies from participants with PEth >20 ng/ml showed increased proton leak (indicator of poor mitochondrial bioenergetic health) and lower myotube density (p=0.06, Cohen’s d = 1.9) compared to myotubes from participants with PEth <20 ng/ml. Conclusions: Preliminary analysis indicates that recent alcohol use dysregulates muscle bioenergetic and differentiation capacity that are not associated with significant differences in physical performance tests. Ongoing analysis will identify confounding factors, and whether cumulative lifetime alcohol use contributes more significantly to physical performance and frailty measures among older people. R21AA030869, Pilot funding Department of Physiology LSUHSC-NO, P60AA009803. 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.

Platelet Bioenergetics and Associations With Delirium and Coma in Patients With Sepsis: A Prospective Cohort Study

BackgroundAcute brain dysfunction during sepsis, which manifests as delirium or coma, is common and associated with multiple adverse outcomes including longer periods of mechanical ventilation, prolonged hospital stays, and increased mortality. Delirium and coma during sepsis may be manifestations of alteration in systemic metabolism. Since access to brain mitochondrial is a limiting factor, measurement of peripheral platelet bioenergetics offers a potential opportunity to understand metabolic changes associated with acute brain dysfunction during sepsis. Research QuestionAre altered platelet mitochondrial bioenergetics associated with acute brain dysfunction during sepsis? Study Design and MethodsWe assessed participants with critical illness in the ICU for the presence of delirium or coma via validated assessment measures. Blood samples were collected and processed to isolate and measure platelet mitochondrial oxygen consumption. We used Seahorse extracellular flux to directly measure baseline, proton leak, maximal oxygen consumption rate, and extracellular acidification rate. We calculated ATP-linked, spare respiratory capacity, and non-mitochondrial oxygen consumption rate from the measured values. ResultsMaximal oxygen consumption was highest in patients with coma, as was spare respiratory capacity and ECAR in unadjusted analysis. After adjusting for age, sedation, modified Sequential Organ Failure Assessment (SOFA) without the neurological component, and pre-existing cognitive function, increased spare respiratory capacity remained associated with coma. Delirium was not associated with any platelet mitochondrial bioenergetics. InterpretationIn this single-center, exploratory, prospective cohort study, we found increased platelet mitochondrial spare respiratory capacity was associated with coma in patients with sepsis. Future studies powered to determine any relationship between delirium and mitochondrial respiration bioenergetics are needed.

Unique features of β-cell metabolism are lost in type 2 diabetes.

Pancreatic β cells play an essential role in the control of systemic glucose homeostasis as they sense blood glucose levels and respond by secreting insulin. Upon stimulating glucose uptake in insulin-sensitive tissues post-prandially, this anabolic hormone restores blood glucose levels to pre-prandial levels. Maintaining physiological glucose levels thus relies on proper β-cell function. To fulfill this highly specialized nutrient sensor role, β cells have evolved a unique genetic program that shapes its distinct cellular metabolism. In this review, the unique genetic and metabolic features of β cells will be outlined, including their alterations in type 2 diabetes (T2D). β cells selectively express a set of genes in a cell type-specific manner; for instance, the glucose activating hexokinase IV enzyme or Glucokinase (GCK), whereas other genes are selectively "disallowed", including lactate dehydrogenase A (LDHA) and monocarboxylate transporter 1 (MCT1). This selective gene program equips β cells with a unique metabolic apparatus to ensure that nutrient metabolism is coupled to appropriate insulin secretion, thereby avoiding hyperglycemia, as well as life-threatening hypoglycemia. Unlike most cell types, β cells exhibit specialized bioenergetic features, including supply-driven rather than demand-driven metabolism and a high basal mitochondrial proton leak respiration. The understanding of these unique genetically programmed metabolic features and their alterations that lead to β-cell dysfunction is crucial for a comprehensive understanding of T2D pathophysiology and the development of innovative therapeutic approaches for T2D patients.

Progression of herpesvirus infection remodels mitochondrial organization and metabolism.

Viruses target mitochondria to promote their replication, and infection-induced stress during the progression of infection leads to the regulation of antiviral defenses and mitochondrial metabolism which are opposed by counteracting viral factors. The precise structural and functional changes that underlie how mitochondria react to the infection remain largely unclear. Here we show extensive transcriptional remodeling of protein-encoding host genes involved in the respiratory chain, apoptosis, and structural organization of mitochondria as herpes simplex virus type 1 lytic infection proceeds from early to late stages of infection. High-resolution microscopy and interaction analyses unveiled infection-induced emergence of rough, thin, and elongated mitochondria relocalized to the perinuclear area, a significant increase in the number and clustering of endoplasmic reticulum-mitochondria contact sites, and thickening and shortening of mitochondrial cristae. Finally, metabolic analyses demonstrated that reactivation of ATP production is accompanied by increased mitochondrial Ca2+ content and proton leakage as the infection proceeds. Overall, the significant structural and functional changes in the mitochondria triggered by the viral invasion are tightly connected to the progression of the virus infection.

The E-liquid flavoring vanillin alters energy and autophagic pathways in human proximal tubule (HK-2) epithelial cells

The use of flavored e-liquids in electronic nicotine delivery systems (ENDS) has become very popular in recent years, but effects of these products have not been well characterized outside the lung. In this study, acute exposure to the popular flavoring vanillin (VAN) was performed on human proximal tubule (HK-2) kidney cells. Cells were exposed to 0–1000 μM VAN for 24 or 48 h and cellular stress responses were determined. Mitochondrial viability using MTT assay showed a significant decrease between the control and 1000 μM group by 48 h. Seahorse XFp analysis showed significantly increased basal respiration, ATP production, and proton leak after 24 h exposure. By 48 h exposure, these parameters remained significantly increased in addition to non-mitochondrial respiration and maximal respiration. Glycolytic activity after 24 h exposure showed significant decreases in glycolysis, glycolytic capacity, glycolytic reserve, and non-glycolytic acidification. The autophagy markers microtubule-associated protein 1A/1B light chain 3 (LC3B–I and LC3B-II) were probed via western blotting. The ratio of LC3B-II/LC3B–I was significantly increased after 24 h exposure to VAN, but by 48 h this ratio significantly decreased. The mitophagy marker PINK1 showed an increasing trend at 24 h, and its downstream target Parkin was significantly increased between the control and 750 μM group only. Finally, the oxidative stress marker 4-HNE was significantly decreased after 48 h exposure to VAN. These results indicate that acute exposure to VAN in the kidney HK-2 model can induce energy and autophagic changes within the cell.

Enhancing Escherichia coli abiotic stress resistance through ornithine lipid formation

Escherichia coli is a common host for biotechnology and synthetic biology applications. During growth and fermentation, the microbes are often exposed to stress conditions, such as variations in pH or solvent concentrations. Bacterial membranes play a key role in response to abiotic stresses. Ornithine lipids (OLs) are a group of membrane lipids whose presence and synthesis have been related to stress resistance in bacteria. We wondered if this stress resistance could be transferred to bacteria not encoding the capacity to form OLs in their genome, such as E. coli. In this study, we engineered different E. coli strains to produce unmodified OLs and hydroxylated OLs by expressing the synthetic operon olsFC. Our results showed that OL formation improved pH resistance and increased biomass under phosphate limitation. Transcriptome analysis revealed that OL-forming strains differentially expressed stress- and membrane-related genes. OL-producing strains also showed better growth in the presence of the ionophore carbonyl cyanide 3-chlorophenylhydrazone (CCCP), suggesting reduced proton leakiness in OL-producing strains. Furthermore, our engineered strains showed improved heterologous violacein production at phosphate limitation and also at low pH. Overall, this study demonstrates the potential of engineering the E. coli membrane composition for constructing robust hosts with an increased abiotic stress resistance for biotechnology and synthetic biology applications.Key points• Ornithine lipid production in E. coli increases biomass yield under phosphate limitation.• Engineered strains show an enhanced production phenotype under low pH stress.• Transcriptome analysis and CCCP experiments revealed reduced proton leakage.

Adipocyte-derived inflammatory molecules induce senescent B cells through metabolic pathways.

The aim of this study was to demonstrate that an adipocyte tissue-derived conditioned medium (ACM) contains inflammatory molecules that induce senescence in B cells. We incubated blood-derived B cells from lean donors with ACM obtained from the adipose tissue of adult female donors with obesity undergoing weight reduction surgery or with medium as control. After 24 h, cells were harvested, and the expression of transcripts for proinflammatory cytokines (TNF/IL-6), chemokines (IL-8), and for markers of the senescence-associated secretory phenotype (SASP) was measured by quantitative polymerase chain reaction. B cells were also stained with the marker of immunosenescence β-galactosidase, and their metabolic status was evaluated in Seahorse using a Mito Stress Test. We show that the incubation of B cells from lean donors with ACM induces the expression of transcripts for inflammatory and SASP transcripts, increases the amount of β-galactosidase staining, and induces a metabolic phenotype characterized by higher basal and maximal oxygen consumption, spare respiratory capacity (difference between maximal and basal respiration), nonmitochondrial oxygen consumption, ATP production, and proton leak. These results demonstrate that B cells from lean individuals, after incubation with ACM, become inflammatory and senescent, and this occurs through metabolic pathways needed to support their secretory phenotype.

Innovative low-energy enrichment of sulfuric acid using PVDF-HFP anion exchange membranes with acid-blocking properties

In recent years, the electrodialysis (ED) technology has gained substantial attention for its role in recovering sulfuric acid from industrial wastewater. However, the typical anion exchange membranes (AEMs) have been known to exhibit notable proton leakage during ED, which can severely hinder the efficiency of acid concentration. To overcome this issue, we utilized a substrate made of poly(vinylidene fluoride-hexafluoropropylene) (PVDF-HFP). This choice capitalizes on the presence of fluorine-containing groups for enhanced acid stability and tertiary amine groups to minimize water content, resulting in a significant improvement in proton-blocking capabilities of the AEMs. It is worth noting that, through a combination of partial cross-linking and quaternization, these AEMs not only maintain their acid concentration performance but also substantially reduce energy consumption. Additionally, we conducted a thorough analysis of the delicate balance between current efficiency and energy consumption in acid recovery systems. In contrast to conventional AEMs and commercially available proton-blocking membranes such as ACM, we highlight the exceptional acid concentration capability (CH+ = 1.28 M) and the low energy consumption (3.3 kWh/kg) of AEM-1.2. This research breakthrough marks a significant advancement in the development of acid-blocking AEMs and strongly advocates for more efficient sulfuric acid recovery from industrial wastewater.

Abstract B044: Elongation control of mRNA translation drives Group 3 medulloblastoma adaptation to nutrient deprivation

Abstract Background: Group 3 affiliation and MYC genetic amplification are associated with poor life expectancy and substantial morbidity in children suffering from medulloblastoma (MB). The high metabolic demand induced by MYC-driven transformation sensitizes MYC-overexpressing MB to cell death under conditions of nutrient deprivation (ND). Additionally, MYC-driven transformation is known to promote mitochondrial oxidative phosphorylation (OXPHOS). We previously reported that eukaryotic Elongation Factor Kinase 2 (eEF2K), the master regulator of mRNA translation elongation, promotes survival of MYC-overexpressing tumors under ND. Interestingly, eEF2K is overexpressed in MYC-driven MB and our preliminary proteomics data highlight large-scale alterations in OXPHOS components affecting eEF2K deficient MB cells. We therefore hypothesized that eEF2K activity is required for the selective translation of mRNAs needed for efficient OXPHOS, and for the progression of MYC-driven MB. METHODS: Multiplexed enhanced Protein Dynamic Mass Spectrometry performed in eEF2K knockdown MYC-overexpressing D425 MB cells to identify mRNAs selectively translated upon eEF2K activation. Time course experiments under ND were conducted in eEF2K knockout (KO) D425 cells to assess the presence of electron transport chain (ETC) complexes I-IV in their native state (via BN-PAGE), as well as transcript expression of individual ETC complex components (by qPCR). The effects of eEF2K inactivation on oxygen consumption, metabolic fluxes and mitochondrial membrane potential were studied with Seahorse technology and JC1/TMRE staining. The viability of eEF2K KO D425 cells was assessed by Incucyte system. Finally, MB orthotopic xenograft mouse models were used to confirm in vitro observations. RESULTS: Multiple (9 out of 10 detected) components of the mitochondrial OXPHOS pathway are selectively translated upon eEF2K activation. Inactivation of eEF2K by genetic KO leads to the disassembly of ETC complexes I-IV without affecting mRNA levels of their respective components. Consistently, eEF2K KO MB cells display decreased mitochondrial membrane potential and ~20% increased proton leak through the mitochondrial membrane. In addition, eEF2K inactivation results in increased D425 cell death under ND and doubles survival of MB bearing mice fed with calorie restricted diets (p<0.05). CONCLUSION: Control of mRNA translation elongation by eEF2K is critical for mitochondrial ETC complex assembly and efficient OXPHOS in MYC-overexpressing MB, likely representing an adaptive response by which MYC-driven MB cells cope with acute metabolic stress. Future therapeutic studies will aim to combine eEF2K inhibition with caloric restriction mimetic drugs as eEF2K activity appears critical under metabolic stress conditions. Citation Format: Alberto Delaidelli, Fares Burwag, Jessica Oliveira de Santis, Betty Yao, Haifeng Zhang, Yue Zhou Huang, Gian Luca Negri, Christopher Hughes, Gabriel Leprivier, Poul Sorensen. Elongation control of mRNA translation drives Group 3 medulloblastoma adaptation to nutrient deprivation [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr B044.

Catalpolaglycone disrupts mitochondrial thermogenesis by specifically binding to a conserved lysine residue of UCP2 on the proton leak tunnel

BackgroundCatalpol (CAT), a naturally occurring iridoid glycoside sourced from the root of Rehmannia glutinosa, affects mitochondrial metabolic functions. However, the mechanism of action of CAT against pyrexia and its plausible targets remain to be fully elucidated. PurposeThis study aimed to identify the specific targets of CAT for blocking mitochondrial thermogenesis and to unveil the unique biological mechanism of action of the orthogonal binding mode between the hemiacetal group and lysine residue on the target protein in vivo. MethodsLipopolysaccharide (LPS)/ carbonyl cyanide 3-chlorophenylhydrazone (CCCP)-induced fever models were established to evaluate the potential antipyretic effects of CAT. An alkenyl-modified CAT probe was designed to identify and capture potential targets. Binding capacity was tested using in-gel imaging and a cellular thermal shift assay. The underlying antipyretic mechanisms were explored using biochemical and molecular biological methods. Catalpolaglycone (CA) was coupled with protein profile identification and molecular docking analysis to evaluate and identify its binding mode to UCP2. ResultsAfter deglycation of CAT in vivo, the hemiacetal group in CA covalently binds to Lys239 of UCP2 in the mitochondria of the liver via an ɛ-amine nucleophilic addition. This irreversible binding affects proton leakage and improves mitochondrial membrane potential and ADP/ATP transformation efficiency, leading to an antipyretic effect. ConclusionOur findings highlight the potential role of CA in modulating UCP2 activity or function within the mitochondria and open new avenues for investigating the therapeutic effects of CA on mitochondrial homeostasis.

Immortalised murine R349P desmin knock-in myotubes exhibit a reduced proton leak and decreased ADP/ATP translocase levels in purified mitochondria

Desmin gene mutations cause myopathies and cardiomyopathies. Our previously characterised R349P desminopathy mice, which carry the ortholog of the common human desmin mutation R350P, showed marked alterations in mitochondrial morphology and function in muscle tissue. By isolating skeletal muscle myoblasts from offspring of R349P desminopathy and p53 knock-out mice, we established an immortalised cellular disease model. Heterozygous and homozygous R349P desmin knock-in and wild-type myoblasts could be well differentiated into multinucleated spontaneously contracting myotubes. The desminopathy myoblasts showed the characteristic disruption of the desmin cytoskeleton and desmin protein aggregation, and the desminopathy myotubes showed the characteristic myofibrillar irregularities. Long-term electrical pulse stimulation promoted myotube differentiation and markedly increased their spontaneous contraction rate. In both heterozygous and homozygous R349P desminopathy myotubes, this treatment restored a regular myofibrillar cross-striation pattern as seen in wild-type myotubes. High-resolution respirometry of mitochondria purified from myotubes by density gradient ultracentrifugation revealed normal oxidative phosphorylation capacity, but a significantly reduced proton leak in mitochondria from the homozygous R349P desmin knock-in cells. Consistent with a reduced proton flux across the inner mitochondrial membrane, our quantitative proteomic analysis of the purified mitochondria revealed significantly reduced levels of ADP/ATP translocases in the homozygous R349P desmin knock-in genotype. As this alteration was also detected in the soleus muscle of R349P desminopathy mice, which, in contrast to the mitochondria purified from cultured cells, showed a variety of other dysregulated mitochondrial proteins, we consider this finding to be an early step in the pathogenesis of secondary mitochondriopathy in desminopathy.

Kaempferol Improves Cardiolipin and ATP in Hepatic Cells: A Cellular Model Perspective in the Context of Metabolic Dysfunction-Associated Steatotic Liver Disease.

Targeting mitochondrial function is a promising approach to prevent metabolic dysfunction-associated steatotic liver disease (MASLD). Cardiolipin (CL) is a unique lipid comprising four fatty acyl chains localized in the mitochondrial inner membrane. CL is a crucial phospholipid in mitochondrial function, and MASLD exhibits CL-related anomalies. Kaempferol (KMP), a natural flavonoid, has hepatoprotective and mitochondrial function-improving effects; however, its influence on CL metabolism in fatty liver conditions is unknown. In this study, we investigated the effects of KMP on mitochondrial function, focusing on CL metabolism in a fatty liver cell model (linoleic-acid-loaded C3A cell). KMP promoted mitochondrial respiratory functions such as ATP production, basal respiration, and proton leak. KMP also increased the gene expression levels of CPT1A and PPARGC1A, which are involved in mitochondrial β-oxidation. Comprehensive quantification of CL species and related molecules via liquid chromatography/mass spectrometry showed that KMP increased not only total CL content but also CL72:8, which strongly favors ATP production. Furthermore, KMP improved the monolysocardiolipin (MLCL)/CL ratio, an indicator of mitochondrial function. Our results suggest that KMP promotes energy production in a fatty liver cell model, associated with improvement in mitochondrial CL profile, and can serve as a potential nutrition factor in preventing MASLD.

Saturated fatty acids differently affect mitochondrial function and the intestinal epithelial barrier depending on their chain length in the in vitro model of IPEC-J2 enterocytes.

Introduction: Maintenance of the intestinal barrier mainly relies on the mitochondrial function of intestinal epithelial cells that provide ATP through oxidative phosphorylation (OXPHOS). Dietary fatty acid overload might induce mitochondrial dysfunction of enterocytes and may increase intestinal permeability as indicated by previous in vitro studies with palmitic acid (C16:0). Yet the impact of other dietary saturated fatty acids remains poorly described. Methods: To address this question, the in vitro model of porcine enterocytes IPEC-J2 was treated for 3days with 250µM of lauric (C12:0), myristic (C14:0), palmitic (C16:0) or stearic (C18:0) acids. Results and discussion: Measurement of the transepithelial electrical resistance, reflecting tight junction integrity, revealed that only C16:0 and C18:0 increased epithelial permeability, without modifying the expression of genes encoding tight junction proteins. Bioenergetic measurements indicated that C16:0 and C18:0 were barely β-oxidized by IPEC-J2. However, they rather induced significant OXPHOS uncoupling and reduced ATP production compared to C12:0 and C14:0. These bioenergetic alterations were associated with elevated mitochondrial reactive oxygen species production and mitochondrial fission. Although C12:0 and C14:0 treatment induced significant lipid storage and enhanced fusion of the mitochondrial network, it only mildly decreased ATP production without altering epithelial barrier. These results point out that the longer chain fatty acids C16:0 and C18:0 increased intestinal permeability, contrary to C12:0 and C14:0. In addition, C16:0 and C18:0 induced an important energy deprivation, notably via increased proton leaks, mitochondrial remodeling, and elevated ROS production in enterocytes compared to C12:0 and C14:0.

Functional promiscuity of small multidrug resistance transporters from Staphylococcus aureus, Pseudomonas aeruginosa, and Francisella tularensis.

Small multidrug resistance transporters efflux toxic compounds from bacteria and are a minimal system to understand multidrug transport. Most previous studies have focused on EmrE, the model SMR from Escherichia coli, finding that EmrE has a broader substrate profile than previously thought and that EmrE may perform multiple types of transport, resulting in substrate-dependent resistance or susceptibility. Here, we performed a broad screen to identify potential substrates of three other SMRs: PAsmr from Pseudomonas aeruginosa; FTsmr from Francisella tularensis; and SAsmr from Staphylococcus aureus. This screen tested metabolic differences in E.coli expressing each transporter versus an inactive mutant, for a clean comparison of sequence and substrate-specific differences in transporter function, and identified many substrates for each transporter. In general, resistance compounds were charged, and susceptibility substrates were uncharged, but hydrophobicity was not correlated with phenotype. Two resistance hits and two susceptibility hits were validated via growth assays and IC50 calculations. Susceptibility is proposed to occur via substrate-gated proton leak, and the addition of bicarbonate antagonizes the susceptibility phenotype, consistent with this hypothesis.

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

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

Significant and Various Effects of ML329-Induced MITF Suppression in the Melanoma Cell Line.

To study the inhibitory effects on microphthalmia-associated transcription factor (MITF)-related biological aspects in malignant melanomas (MMs) in the presence or absence of the low-molecular MITF specific inhibitor ML329, cell viability, cellular metabolic functions, and three-dimensional (3D) spheroid formation efficacy were compared among MM cell lines including SK-mel-24, A375, dabrafenib- and trametinib-resistant A375 (A375DT), and WM266-4. Upon exposure to 2 or 10 μM of ML329, cell viability was significantly decreased in WM266-4, SK-mel-24, and A375DT cells, but not A375 cells, in a dose-dependent manner, and these toxic effects of ML329 were most evident in WM266-4 cells. Extracellular flux assays conducted using a Seahorse bioanalyzer revealed that treatment with ML329 increased basal respiration, ATP-linked respiration, proton leakage, and non-mitochondrial respiration in WM266-4 cells and decreased glycolytic function in SK-mel-24 cells, whereas there were no marked effects of ML329 on A375 and A375DT cells. A glycolytic stress assay under conditions of high glucose concentrations also demonstrated that the inhibitory effect of ML329 on the glycolytic function of WM266-4 cells was dose-dependent. In addition, ML329 significantly decreased 3D-spheroid-forming ability, though the effects of ML329 were variable among the MM cell lines. Furthermore, the mRNA expression levels of selected genes, including STAT3 as a possible regulator of 3D spheroid formation, KRAS and SOX2 as oncogenic-signaling-related factors, PCG1a as the main regulator of mitochondrial biogenesis, and HIF1a as a major hypoxia transcriptional regulator, fluctuated among the MM cell lines, possibly supporting the diverse ML329 effects mentioned above. The findings of diverse ML329 effects on various MM cell lines suggest that MITF-associated biological activities are different among various types of MM.