Proton Leak Research Articles

Hypoxia Differently Affects TGF-β2-Induced Epithelial Mesenchymal Transitions in the 2D and 3D Culture of the Human Retinal Pigment Epithelium Cells.

The hypoxia associated with the transforming growth factor-β2 (TGF-β2)-induced epithelial mesenchymal transition (EMT) of human retinal pigment epithelium (HRPE) cells is well recognized as the essential underlying mechanism responsible for the development of proliferative retinal diseases. In vitro, three-dimensional (3D) models associated with spontaneous O2 gradients can be used to recapitulate the pathological levels of hypoxia to study the effect of hypoxia on the TGF-β2-induced EMT of HRPE cells in detail, we used two-dimensional-(2D) and 3D-cultured HRPE cells. TGF-β2 and hypoxia significantly and synergistically increased the barrier function of the 2D HRPE monolayers, as evidenced by TEER measurements, the downsizing and stiffening of the 3D HRPE spheroids and the mRNA expression of most of the ECM proteins. A real-time metabolic analysis indicated that TGF-β2 caused a decrease in the maximal capacity of mitochondrial oxidative phosphorylation in the 2D HRPE cells, whereas, in the case of 3D HRPE spheroids, TGF-β2 increased proton leakage. The findings reported herein indicate that the TGF-β2-induced EMT of both the 2D and 3D cultured HRPE cells were greatly modified by hypoxia, but during these EMT processes, the metabolic plasticity was different between 2D and 3D HRPE cells, suggesting that the mechanisms responsible for the EMT of the HRPE cells may be variable during their spatial spreading.

Genotoxic effects of chlorinated disinfection by-products of 1,3-diphenylguanidine (DPG): Cell-based in-vitro testing and formation potential during water disinfection

1,3-diphenylguanidine (DPG) is a commonly used rubber and polymer additive, that has been found to be one of the main leachate products of tire wear particles and from HDPE pipes. Its introduction to aquatic environments and potentially water supplies lead to further questions regarding the effects of disinfection by-products potentially formed. Using different bioassay approaches and NGS RNA-sequencing, we show that some of the chlorinated by-products of DPG exert significant toxicity. DPG and its chlorinated by-products also can alter cell bioenergetic processes, affecting cellular basal respiration rates and ATP production, moreover, DPG and its two chlorination products, 1,3-bis-(4-chlorophenyl)guanidine (CC04) and 1-(4-chlorophenyl)−3-(2,4-dichlorophenyl)guanidine (CC11), have an impact on mitochondrial proton leak, which is an indicator of mitochondria damage. Evidence of genotoxic effects in the form of DNA double strand breaks (DSBs) was suggested by RNA-sequencing results and further validated by an increased expression of genes associated with DNA damage response (DDR), specifically the canonical non-homologous end joining (c-NHEJ) pathway, as determined by qPCR analysis of different pathway specific genes (XRCC6, PRKDC, LIG4 and XRCC4). Immunofluorescence analysis of phosphorylated histone H2AX, another DSB biomarker, also confirmed the potential genotoxic effects observed for the chlorinated products. In addition, chlorination of DPG leads to the formation of different chlorinated products (CC04, CC05 and CC15), with analysed compounds representing up to 42% of formed products, monochloramine is not able to effectively react with DPG. These findings indicate that DPG reaction with free chlorine doses commonly applied during drinking water treatment or in water distribution networks (0.2–0.5 mg/L) can lead to the formation of toxic and genotoxic chlorinated products.

Regulation of Oxidative Phosphorylation of Liver Mitochondria in Sepsis.

The link between liver dysfunction and decreased mitochondrial oxidative phosphorylation in sepsis has been clearly established in experimental models. Energy transduction is plastic: the efficiency of mitochondrial coupling collapses in the early stage of sepsis but is expected to increase during the recovery phases of sepsis. Among the mechanisms regulating the coupling efficiency of hepatic mitochondria, the slipping reactions at the cytochrome oxidase and ATP synthase seem to be a determining element, whereas other regulatory mechanisms such as those involving proton leakage across the mitochondrial membrane have not yet been formally proven in the context of sepsis. If the dysfunction of hepatic mitochondria is related to impaired cytochrome c oxidase and ATP synthase functions, we need to consider therapeutic avenues to restore their activities for recovery from sepsis. In this review, we discussed previous findings regarding the regulatory mechanism involved in changes in the oxidative phosphorylation of liver mitochondria in sepsis, and propose therapeutic avenues to improve the functions of cytochrome c oxidase and ATP synthase in sepsis.

Insights into the Evolution of Non‐shivering Thermogenesis from UCP1 of the Naked Mole Rat (Heterocephalus glaber)

Recognized as the primary location of non‐shivering thermogenesis (NST) in eutherian mammals, brown adipose tissue (BAT) functions to accelerate rewarming in hibernators and defend body temperature among newborns and small‐bodied species enduring cold stress. Uncoupling protein 1 (UCP1) is highly expressed in BAT and underlies the molecular mechanism of NST. UCP1 resides in the inner mitochondrial membrane where, upon functional induction by free fatty acids, it catalyzes mitochondrial proton leak, collapsing the proton gradient that stores oxidation energy. The ensuing futile cycling of protons increases substrate oxidation and thus heat production. Central questions remain regarding the exact mechanism by which UCP1 facilitates proton leak, stemming from the lack of a crystal structure of the protein. It is also unknown whether UCP1 function has been shaped by evolution to meet differing thermoregulatory demands of various lineages. We adopt a comparative approach, targeting species with extreme ecophysiological characteristics expected to culminate in drastic differences in thermoregulatory demands and thus UCP1 function, to provide insights into protein structure‐function relationships. For instance, the naked mole rat (NMR; Heterocephalus glaber) is well‐suited to survive in hypoxic burrows that exhibit high year‐round temperatures (30‐34°C). Hinting at a reduced NST capacity, NMRs also display poikilothermy, failing to maintain constant body temperatures when artificially cold challenged. Moreover, NMR UCP1 displays several unique amino acid substitutions, one of which, H145Q (histidine to glutamine), occurs at the well‐known “histidine pair motif”. This motif, typically comprised of histidines at positions 145 and 147, has been hypothesized to be a critical final proton acceptor/donor site shuttling protons into the mitochondrial matrix. The exact mutation that occurs in the NMR, H145Q, was previously shown to diminish hamster UCP1‐mediated proton conductance in proteoliposomes by 90%, calling the functional performance of NMR UCP1 into question. We functionally tested wildtype NMR UCP1 as well as histidine pair mutant combinations at positions 145 and 147, not in the highly artificial system of proteoliposomes, which may promote artefactual protein behaviour, but instead in the more native mammalian environment of HEK293 cells. Using plate‐based respirometry techniques, we demonstrate that wildtype NMR UCP1 uncouples, thus NMRs retain this mechanism of NST. While we hypothesized that reversion of the H145Q mutation would increase UCP1‐mediated proton leak, surprisingly both variants function similarly when activated with free fatty acids and inhibited with guanosine diphosphate (GDP). Likewise, mutation of the histidine at position 147, the other half of the “histidine pair motif”, does not drastically alter UCP1 function. Overall, these data suggest that the “histidine pair motif” may not be crucial for protein function, contrary to previous claims. Comparative functional assessments of UCP1 variants provide a deeper understanding of structure‐function relationships of this enigmatic protein and the evolution of mammalian NST in response to ecophysiological constraints.

Pro‐inflammatory Cytokines Increase Glycolysis through Upregulation of Uncoupling Protein and Akt Activation in Colorectal Cancer Cells

Non‐cancerous colonocytes use butyrate, which is a short‐chain fatty acid derived from the fermentation of dietary fiber, as a primary energy source. In contrast, cancerous colonocytes use glucose as their preferred energetic substrate and undergo enhanced glycolysis or the Warburg effect. It has been reported that increased production of pro‐inflammatory cytokines such as interleukin1 beta and tumor necrosis factor alpha (TNFalpha) promote glycolysis. Previously, we have shown that interleukin1 beta increased glycolysis and decreased butyrate oxidation. However, the mechanism underlying how this pro‐inflammatory cytokine increased glycolysis was unclear. Here, we have discovered that Uncoupling Protein 2 (UCP2) appears to mediate the elevation in glycolysis induced by interleukin1 beta. UCP2 is a mitochondrial protein that transports the protons back into the mitochondrial matrix, thus dissipating the proton gradient and reducing ATP production. UCP2 is upregulated in various cancers such as breast, prostate, skin, and colorectal cancers. We hypothesize that interleukin1 beta promotes glycolysis by upregulating UCP2 and activating Akt in colorectal cancer cells. We have found colorectal cancer cells treated with interleukin1 beta showed elevated UCP2 expression and increased Akt activation. Next, we utilized the Seahorse XFe24 analyzer to check whether interleukin1 beta affects proton leak and ATP production in colorectal cancer cells. Preliminary data suggests that interleukin1 beta increases proton leak and decreases ATP production in mitochondria, which is consistent with the upregulation of UCP2 and demonstrates a functional consequence. To study whether UCP2 mediates the interleukin1 beta effect toward increasing glycolysis, we created a stable UCP2 knockdown in colorectal cancer cells and tested whether colorectal cancer cells that had diminished UCP2 from the RNAi knockdown did not show an increase in glycolysis when treated with interleukin1 beta as compared to RNAi mock colorectal cancer cells. Our preliminary data allude to a role for UCP2 in mediating the interleukin1 beta induced increase in glycolysis observed in colorectal cancer cells.

Sex‐dependent Regulation of Mitochondrial Respiratory Function in Mouse Brain Microvessels by Peroxynitrite Decomposition Catalyst

BackgroundPeroxynitrite (PN) is a strong oxidizing and nitrating molecule. PN is a potent inhibitor of mitochondrial respiration and promotes ischemia‐reperfusion injury following stroke. In isolated mouse brain mitochondria, we observed that PN donors inhibit mitochondrial respiratiory function whereas the PN decomposition catalyst, Fe (III) tetrakis (1‐methyl‐4‐pyridyl) porphyrin pentachlorideporphyrin pentachloride (FeTMPyP), enhances mitochondrial state III and state IVo mitochondrial respiration. In addition, we demonstrated that mitochondrial respiration is the primary contributor of cellular energy in brain microvessels (BMVs). The present study tested the hypothesis that FeTMPyP negatively regulates mitochondrial respiration in the mouse BMVs.MethodsBMVs were isolated from male and female mice (C57Bl/6, 2‐4 months) using a combination of filters with pore sizes of 300μm and 40μm followed by gradient centrifugation. BMVs were treated with FeTMPyP at 37oC for 60 minutes. Oxygen consumption rates (OCR) were measured using the Agilent Seahorse XFe24 analyzer and various respiratory parameters were determined following Mitostress test.ResultsIn male BMVs, basal respiration, ATP production, and non‐mitochondrial respiration were not altered by FeTMPyP treatment. Contrary to our hypothesis, in male BMVs, PN decomposition catalyst decreased the mitochondrial maximal respiration by 24.6% (2.1 ± 0.4 vs 2.8 ± 0.3 picomoles of O2/min/µg protein;) whereas the spare respiratory capacity was reduced by 33.3% (1.2 ± 0.3 vs 1.8 ± 0.3 picomoles of O2/min/µg protein; n=17 each, p<0.05). Proton leak was elevated by 70% (0.7 ± 0.1 vs 0.4 ± 0.1 picomoles of O2/min/µg protein) by PN decomposition catalyst in male BMVs. In contrast, in the female BMVs, the PN decomposition catalyst failed to alter mitochondrial respiratory parameters (n=15 each, p=NS). Interestingly, FeTMPyP increased non‐mitochondrial respiration by 63.8% (0.95 ± 0.2 vs 0.58 ± 0.1 picomoles of O2/min/µg protein) in the female BMVs (n=17 each, p<0.05).ConclusionBMVs display sex‐dependent respenses to endogenous PN. Notably, in female mouse BMVs, PN appears to act as an antioxidant as PN inhibited the non‐mitochondrial respiration which mostly contributes to extramitochondrial superoxide generation.

Plate‐Based Respirometry to Assess Zebrafish Embryo Bioenergetics in situ in Changing Environmental Conditions

All species require bioenergetic adaptation to maintain life functions, especially in changing environmental conditions. Global warming and deteriorating water quality may pose threats to aquatic species by interfering with bioenergetic mechanisms. Mitochondrial oxidative metabolism is a key component for bioenergetic responses as it provides the major proportion of cellular energy in the form of adenosine triphosphate (ATP). In our studies, we aim to establish the assessment of zebrafish mitochondrial bioenergetics and investigate the mechanisms, adaptation, and plasticity to temperature, pollutants, and the combination of both to gain insights into the upcoming threats for their energy homeostasis and survival. Using plate‐based respirometry of the Seahorse XF96 analyzer, we measured zebrafish (Danio rerio) embryo oxygen consumption rates in situ. To understand how temperature alters oxidative phosphorylation, we exposed 48 hours post‐fertilization zebrafish embryos to five different water temperatures before measuring oxygen consumption at various assay temperatures. The oxygen consumption rates were challenged using respiratory chain inhibitors and chemical uncouplers to reveal bioenergetic mechanisms and adjustments of mitochondrial efficiency. We show that at 18°C assay temperature, basal oxygen consumption of the embryo is low due to decreased ATP‐linked respiration and not limited by maximum substrate oxidation. Basal oxygen consumption rates increase with assay temperature and remain stable up to 37°C, before rapidly deteriorating at 41°C and above. The pre‐exposure of embryos to 37°C, however, expands the thermal window to higher limits, indicated by stable basal oxygen consumption rates at 41°C. Proton leak respiration, which wastes nutrient energy in parallel to ATP production, increases at assay temperatures above 28°C, thereby reducing the efficiency of ATP synthesis. This is seen as a decrease in coupling efficiency, which is defined as the fraction of oxygen consumption that drives ATP synthesis. Collectively, temperature affects the performance of zebrafish embryos by reducing cellular ATP turnover at lower temperatures and by increasing proton leak at higher temperatures, resulting in an optimum energy conversion temperature that can be shifted by thermal adaptation. In current experiments, we measure how pollutants impact zebrafish bioenergetics and its relation to thermal adaptation.

Mitochondrial Fission is Essential to Maintain Cristae Morphology and Bioenergetics

Mitochondria and peroxisomes are both dynamic signaling organelles that constantly undergo fission. While mitochondrial fission and fusion are known to coordinate cellular metabolism, proliferation, and apoptosis, the physiological relevance of peroxisome dynamics and the implications for cell fate are not fully understood. DRP1 (dynamin‐related protein 1) is an essential GTPase that executes both mitochondrial and peroxisomal fission. Patients with de novo heterozygous missense mutations in the gene that encodes DRP1, DNM1L, present with encephalopathy due to mitochondrial and peroxisomal elongation (EMPF). EMPF is a devastating neurodevelopmental disease with no effective treatment. To interrogate the molecular mechanisms by which DRP1 mutations cause developmental defects, we are using patient‐derived fibroblasts and iPSC‐derived models from patients with mutations in different domains of DRP1 who present with clinically disparate conditions. Using super resolution imaging, we find that patient cells, in addition to displaying elongated mitochondrial and peroxisomal morphology, present with aberrant cristae structure. Given the direct link between cristae morphology and oxidative phosphorylation efficiency, we explored the impact of these mutations on cellular energy production. Patient cells display a lower coupling efficiency of the electron transport chain, increased proton leak, and Complex III deficiency. In addition to these metabolic abnormalities, mitochondrial hyperfusion results in hyperpolarized mitochondrial membrane potential. Intriguingly, human fibroblasts are capable of cellular reprogramming into iPSCs and appear to display peroxisome‐mediated mitochondrial adaptations that could help sustain these cell fate transitions. Understanding the mechanism by which DRP1 mutations cause cellular dysfunction will give insight into the role of mitochondrial and peroxisome dynamics in neurodevelopment.

Altered brown adipose tissue mitochondrial function in newborn fragile X syndrome mice

Brown adipose tissue (BAT) mitochondria generate heat via uncoupled respiration due to excessive proton leak through uncoupling proteins (UCPs). We previously found hyperthermia in a newborn mouse model of fragile X syndrome and excessive leak in Fmr1 KO forebrain mitochondria caused by CoQ deficiency. The inefficient thermogenic nature of Fmr1 mutant forebrain mitochondria was reminiscent of BAT metabolic features. Thus, we aimed to characterize BAT mitochondrial function in these hyperthermic mice using a top-down approach. Although there was no change in steady-state levels of UCP1 expression between strains, BAT weighed significantly less in Fmr1 mutants compared with controls. Fmr1 KO BAT mitochondria demonstrated impaired substrate oxidation, lower mitochondrial membrane potentials and rates of respiration, and CoQ deficiency. The CoQ analog decylubiquinone normalized CoQ-dependent electron flux and unmasked excessive proton leak. Unlike mutant forebrain, where such deficiency resulted in pathological proton leak, CoQ deficiency within BAT mitochondria resulted largely in abnormal substrate oxidation. This suggests that CoQ is important in BAT for uncoupled respiration to produce heat during development. Although our data provide further evidence of a link between fragile X mental retardation protein (FMRP) and CoQ biosynthesis, the results highlight the importance of CoQ in developing tissues and suggest tissue-specific differences from CoQ deficiency. Because BAT mitochondria are primarily responsible for regulating core body temperature, the defects we describe in Fmr1 KOs could manifest as an adaptive downregulated response to hyperthermia or could result from FMRP deficiency directly.

Uncoupling Proteins in Striated Muscle Tissue: Known Facts and Open Questions.

Significance: Uncoupling proteins (UCPs) are a family of proteins that allow proton leakage across the inner mitochondrial membrane. Although UCP1, also known as thermogenin, is well known and important for heat generation in brown adipose tissue, striated muscles express two distinct members of UCP, namely UCP2 and UCP3. Unlike UCP1, the main function of UCP2 and UCP3 does not appear to be heat production. Recent Advances: Interestingly, UCP2 is the main isoform expressed in cardiac tissues, whereas UCP3 is the dominant isoform in skeletal muscles. In the past years, researchers have started to investigate the regulation of UCP2 and UCP3 expression in striated muscles. Furthermore, concepts about the proposed functions of UCP2 and UCP3 in striated muscles are developed but are still a matter of debate. Critical Issues: Potential functions of UCP2 and UCP3 in striated muscles include a role in protection against mitochondria-dependent oxidative stress, as transporter for pyruvate, fatty acids, and protons into and out of the mitochondria, and in metabolic sensing. In this context, the different isoform expression of UCP2 and UCP3 in the skeletal and cardiac muscle may be related to different metabolic requirements of the two organs. Future Directions: The level of expression of UCP2 and UCP3 in striated muscles changes in different disease stages. This suggests that UCPs may become drug targets for therapy in the future. Antioxid. Redox Signal. 37, 324-335.

RMR-Related DNAJC6 Expression Suppresses Adipogenesis in 3T3-L1 Cells.

Obesity causes various complications such as type 2 diabetes, hypertension, fatty liver, cardiovascular diseases, and cancer. In a pilot GWAS study, we screened the DNAJC6 gene which is significantly related to the resting metabolic rate (RMR) in childhood obesity. With DNAJC6-overexpressed 3T3-L1 cells (TgHsp), we investigated the new obesity mechanism caused by an energy imbalance. After differentiation, lipid droplets (Oil red O staining) were not formed in TgHsp cells compared to the control. TgHsp preadipocyte fibroblast morphology was also not clearly observed in the cell morphology assay (DAPI/BODIPY). In TgHsp cells, the expression of PPARγ, C/EBPα, and aP2 (adipogenesis-related biomarkers) decreased 3-, 39-, and 200-fold, respectively. The expression of the adipokines leptin and adiponectin from adipose tissues also decreased 2.4- and 840-fold, respectively. In addition, the levels of pHSL(Ser563) and free glycerol, which are involved in lipolysis, were significantly lower in TgHsp cells than in the control. The reduction in insulin receptor expression in TgHsp cells suppressed insulin signaling systems such as AKT phosphorylation, and GLUT4 expression. Degradation of IRS-1 in 3T3-L1 adipocytes was caused by chronic exposure to insulin, but not TgHsp. Mitochondrial functions such as oxygen consumption and ATP production, as well as proton leak and UCP1 protein expression, decreased in TgHsp cells compared to the control. Moreover, autophagy was observed by increasing autophagosomal proteins, LC3, on Day 8 of differentiation in TgHsp cells. Through our first report on the DNAJC6 gene related to RMR, we found a new mechanism related to energy metabolism in obesity. DNAJC6 expression positively suppressed adipogenesis, leading to the subsequent resistance of lipolysis, adipokine expression, insulin signaling, and mitochondrial functions.

Antidiabetic Sterols from Peniocereus greggii Roots.

The roots of the cactus Peniocereus greggii, which grows in Northern Mexico and in the south of Arizona, are highly valued by the Pima to treat diabetes and other illnesses, such as breast pain and common cold. As part of our chemical and pharmacological investigation on medicinal plants used for treating diabetes, herein we report the hypoglycemic and antihyperglycemic action of a decoction prepared from the roots of the plant. The active compounds were a series of cholestane steroids, namely, peniocerol (2), desoxyviperidone (3), viperidone (4), and viperidinone (5). Also, a new chemical entity was obtained from an alkalinized chloroform extract (CE1), which was characterized as 3,6-dihydroxycholesta-5,8(9),14-trien-7-one (6) by spectroscopic means. Desoxyviperidone (3) showed an antihyperglycemic action during an oral glucose tolerance test. Compound 3 was also able to decrease blood glucose levels during an intraperitoneal insulin tolerance test in hyperglycemic mice only in combination with insulin, thus behaving as an insulin sensitizer agent. Nevertheless, mitochondrial bioenergetic experiments revealed that compounds 3 and 6 increased basal respiration and proton leak, without affecting the respiration associated with ATP production in C2C12 myotubes. Finally, an ultraefficiency liquid chromatographic method for quantifying desoxyviperidone (3) and viperidone (4) in the crude drug was developed and validated. Altogether, our results demonstrate that Peniocereus greggii decoction possesses a hypoglycemic and antihyperglycemic action in vivo, that sterols 2 and 6 promotes insulin secretion in vitro, and that desoxyviperidone (3) physiologically behaves as an insulin sensitizer agent by a mechanism that may involve mitochondrial proton leak.

Multicentric Standardization of Protocols for the Diagnosis of Human Mitochondrial Respiratory Chain Defects.

The quantification of mitochondrial respiratory chain (MRC) enzymatic activities is essential for diagnosis of a wide range of mitochondrial diseases, ranging from inherited defects to secondary dysfunctions. MRC lesion is frequently linked to extended cell damage through the generation of proton leak or oxidative stress, threatening organ viability and patient health. However, the intrinsic challenge of a methodological setup and the high variability in measuring MRC enzymatic activities represents a major obstacle for comparative analysis amongst institutions. To improve experimental and statistical robustness, seven Spanish centers with extensive experience in mitochondrial research and diagnosis joined to standardize common protocols for spectrophotometric MRC enzymatic measurements using minimum amounts of sample. Herein, we present the detailed protocols, reference ranges, tips and troubleshooting methods for experimental and analytical setups in different sample preparations and tissues that will allow an international standardization of common protocols for the diagnosis of MRC defects. Methodological standardization is a crucial step to obtain comparable reference ranges and international standards for laboratory assays to set the path for further diagnosis and research in the field of mitochondrial diseases.

Glycolytic and Oxidative Phosphorylation Defects Precede the Development of Senescence in Primary Human Brain Microvascular Endothelial Cells.

Alterations of mitochondrial and glycolytic energy pathways related to aging could contribute to cerebrovascular dysfunction. We studied the impact of aging on energetics of primary human brain microvascular endothelial cells (HBMECs) by comparing the young (passages 7-9), pre-senescent (passages 13-15), and senescent (passages 20-21) cells. Pre-senescent HBMECs displayed decreased telomere length and undetectable telomerase activity although markers of senescence were unaffected. Bioenergetics in HBMECs were determined by measuring the oxygen consumption (OCR) and extracellular acidification (ECAR) rates. Cellular ATP production in young HBMECs was predominantly dependent on glycolysis with glutamine as the preferred fuel for mitochondrial oxidative phosphorylation (OXPHOS). In contrast, pre-senescent HBMECs displayed equal contribution to ATP production rate from glycolysis and OXPHOS with equal utilization of glutamine, glucose, and fatty acids as mitofuels. Compared to young, pre-senescent HBMECs showed a lower overall ATP production rate that was characterized by diminished contribution from glycolysis. Impairments of glycolysis displayed by pre-senescent cells included reduced basal glycolysis, compensatory glycolysis, and non-glycolytic acidification. Furthermore, impairments of mitochondrial respiration in pre-senescent cells involved the reduction of maximal respiration and spare respiratory capacity but intact basal and ATP production-related OCR. Proton leak and non-mitochondrial respiration, however, were unchanged in the pre-senescent HBMECs. HBMECS at passages 20-21 displayed expression of senescence markers and continued similar defects in glycolysis and worsened OXPHOS. Thus, for the first time, we characterized the bioenergetics of pre-senescent HBMECs comprehensively to identify the alterations of the energy pathways that could contribute to aging.

Proton blockage PVDF-co-HFP-based anion exchange membrane for sulfuric acid recovery in electrodialysis

Recovery of sulfuric acid from industrial wastewater via electrodialysis (ED) has become a hot topic recently. Since the commercially used anion exchange membranes (AEM) generally lead to the severe proton leakage during ED process, the efficiency of the acid recovery was seriously affected. In this work, a series of proton blockage cross-linking AEM has been prepared by modifying poly(vinylidene fluoride-co-hexafluoropropylene) (PVDF-co-HFP) with 1-vinylimidazole, followed by crosslinking with 1,4-dibromobutane. Our investigation demonstrates that the as-prepared PVDF-co-HFP-based AEM has the observable asymmetric structure on the two sides of the AEM with a smooth and dense surface and a rough and porous surface. The asymmetric structure and the hydrophobicity of the fluorinated backbone of AEM render the improved proton blocking performance, and the increased electrical efficiency and reduced energy consumption of acid recovery. For example, the optimized AEM (AEM-0.5) exhibits the acid concentration with the values of cH+ = 2.04 M (initial proton concentration: 1.0 M) in concentrate cell during 12 h-ED process at current density of 20 mA cm−2 and low energy consumption of 1.75 kw·h·kg−1 relative to the commercial NEOSEPTA® AHA and proton blockage ACM AEM. It is believed that this study can provide a guideline for designing advanced proton blocking AEMs.

Beyond appetite regulation: Targeting energy expenditure, fat oxidation, and lean mass preservation for sustainable weight loss.

New appetite‐regulating antiobesity treatments such as semaglutide and agents under investigation such as tirzepatide show promise in achieving weight loss of 15% or more. Energy expenditure, fat oxidation, and lean mass preservation are important determinants of weight loss and weight‐loss maintenance beyond appetite regulation. This review discusses prior failures in clinical development of weight‐loss drugs targeting energy expenditure and explores novel strategies for targeting energy expenditure: mitochondrial proton leak, uncoupling, dynamics, and biogenesis; futile calcium and substrate cycling; leptin for weight maintenance; increased sympathetic nervous system activity; and browning of white fat. Relevant targets for preserving lean mass are also reviewed: growth hormone, activin type II receptor inhibition, and urocortin 2 and 3. We endorse moderate modulation of energy expenditure and preservation of lean mass in combination with efficient appetite reduction as a means of obtaining a significant, safe, and long‐lasting weight loss. Furthermore, we suggest that the regulatory guidelines should be revisited to focus more on the quality of weight loss and its maintenance rather than the absolute weight loss. Commitment to this research focus both from a scientific and from a regulatory point of view could signal the beginning of the next era in obesity therapies.

Comparative study of free respiration in liver mitochondria during oxidation of various electron donors and under conditions of shutdown of complex III of the respiratory chain

The present work shows that the rate of free respiration of liver mitochondria (in the absence of ATP synthesis (state 4) during the oxidation of succinate is 1.7 times higher than during the oxidation of glutamate with malate. In turn, in the case of oxidation of ferrocyanide with ascorbate, this value is 3.1 times greater than in the case of succinate oxidation. A similar pattern is also observed upon stimulation of free respiration by low concentrations (5 and 10 μM) of the protonophore uncoupler 2,4-dinitrophenol (DNP). It is found that the passive leakage rate of protons in state 4 is the same if the H+/O ratios are 10, 6, and 2 upon the oxidation of glutamate with malate, succinate, and ferrocyanide with ascorbate, respectively. At these values of the H+/O ratio, low concentrations of DNP stimulate passive proton leakage equally during the oxidation of these respiration substrates. In the case of succinate oxidation, bypassing complex III by N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) to the maximum degree, as well as switching this complex completely to idle mode by α,ω-hexadecanedioic acid (HDA) cause a 3-fold stimulation of respiration in state 4. We conclude that at mitochondrial free respiration the values of the H+/2e− ratio for complexes I, III, and IV of the respiratory chain are 4, 4, and 2, respectively. It is assumed that the free respiration of mitochondria is carried out by simple diffusion of protons through the inner membrane, and the rate of this diffusion depends on the total number of protons released by the complexes of the electron transport chain into the intermembrane space.

L-Carnitine reduces reactive oxygen species/endoplasmic reticulum stress and maintains mitochondrial function during autophagy-mediated cell apoptosis in perfluorooctanesulfonate-treated renal tubular cells

We previously reported that perfluorooctanesulfonate (PFOS) causes autophagy-induced apoptosis in renal tubular cells (RTCs) through a mechanism dependent on reactive oxygen species (ROS)/extracellular signal-regulated kinase. This study extended our findings and determined the therapeutic potency of l-Carnitine in PFOS-treated RTCs. l-Carnitine (10 mM) reversed the effects of PFOS (100 µM) on autophagy induction and impaired autophagy flux. Furthermore, it downregulated the protein level of p47Phox, which is partly related to PFOS-induced increased cytosolic ROS in RTCs. Moreover, l-Carnitine reduced ROS production in mitochondria and restored PFOS-impeded mitochondrial function, leading to sustained normal adenosine triphosphate synthesis and oxygen consumption and reduced proton leakage in a Seahorse XF stress test. The increased inositol-requiring enzyme 1α expression by PFOS, which indicated endoplasmic reticulum (ER) stress activation, was associated with PFOS-mediated autophagy activation that could be attenuated through 4-phenylbutyrate (5 mM, an ER stress inhibitor) and l-Carnitine pretreatment. Therefore, by reducing the level of IRE1α, l-Carnitine reduced the levels of Beclin and LC3BII, consequently reducing the level of apoptotic biomarkers including Bax and cleaving PARP and caspase 3. Collectively, these results indicate that through the elimination of oxidative stress, extracellular signal–regulated kinase activation, and ER stress, l-Carnitine reduced cell autophagy/apoptosis and concomitantly increased cell viability in RTCs. This study clarified the potential mechanism of PFOS-mediated RTC apoptosis and provided a new strategy for using l-Carnitine to prevent and treat PFOS-induced RTC apoptosis.

Aging related impairment of brain microvascular bioenergetics involves oxidative phosphorylation and glycolytic pathways

Mitochondrial and glycolytic energy pathways regulate the vascular functions. Aging impairs the cerebrovascular function and increases the risk of stroke and cognitive dysfunction. The goal of our study is to characterize the impact of aging on brain microvascular energetics. We measured the oxygen consumption and extracellular acidification rates of freshly isolated brain microvessels (BMVs) from young (2–4 months) and aged (20–22 months) C57Bl/6 male mice. Cellular ATP production in BMVs was predominantly dependent on oxidative phosphorylation (OXPHOS) with glucose as the preferred energy substrate. Aged BMVs exhibit lower ATP production rate with diminished OXPHOS and glycolytic rate accompanied by increased utilization of glutamine. Impairments of glycolysis displayed by aged BMVs included reduced compensatory glycolysis whereas impairments of mitochondrial respiration involved reduction of spare respiratory capacity and proton leak. Aged BMVs showed reduced levels of key glycolysis proteins including glucose transporter 1 and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 but normal lactate dehydrogenase activity. Mitochondrial protein levels were mostly unchanged whereas citrate synthase activity was reduced, and glutamate dehydrogenase was increased in aged BMVs. Thus, for the first time, we identified the dominant role of mitochondria in bioenergetics of BMVs and the alterations of the energy pathways that make the aged BMVs vulnerable to injury.

Effect of Physiological Oxygen on Primary Human Corneal Endothelial Cell Cultures.

PurposePrimary human corneal endothelial cells (HCEnCs) cultured in room air are exposed to significantly higher O2 concentrations [O2] than what is normally present in the eye. We evaluated the growth and metabolism of HCEnCs cultured under physiological [O2] (2.5%; [O2]2.5) and room air ([O2]A).MethodsPrimary cultures of HCEnCs from normal donors and donors with Fuchs dystrophy were grown at [O2]2.5 and [O2]A. Growth and morphology were compared using phase-contrast microscopy, zonula occludens (ZO-1) localization, cell density measurements, and senescence marker staining. CD44 (cell quality) and HIF-1α (hypoxia-inducible factor-1α) levels were evaluated by Western blotting. Cell adaptability to a reversal of [O2] growth conditions was measured with cell viability assays, and cell metabolism was assessed via oxygen consumption and extracellular acidification rates.ResultsHCEnCs grown at [O2]A and [O2]2.5 displayed similar morphologies, ZO-1 localization, CD44 expression, and senescence. Cells from donors with Fuchs dystrophy grew better at [O2]2.5 than at [O2]A. HIF-1α was undetectable. Cells displayed greater viability at [O2]2.5 than at [O2]A. HCEnCs showed significantly greater proton leak (P < 0.01), nonmitochondrial oxygen consumption (P < 0.01), and spare capacity (P < 0.05) for oxygen consumption rates, and greater basal glycolysis (P < 0.05) with a decreased glycolytic reserve capacity (P < 0.05) for extracellular acidification rates.ConclusionsPrimary HCEnCs show unique metabolic characteristics at physiologic [O2]. The effect of [O2] for optimization of HCEnC culture conditions should be considered.Translational RelevanceWith the advance of cell-based therapeutics for corneal endothelial diseases, [O2] should be considered an important variable in the optimization of HCEnC culture conditions.