Decision letter: Listeria monocytogenes requires cellular respiration for NAD+ regeneration and pathogenesis

Open AccessCCS ChemistryRESEARCH ARTICLES22 Dec 2022Unexpected Photocatalytic Degeneration of NAD+ for Inducing Apoptosis of Hypoxia Cancer Cells Shengpeng Xia, Haotian Bai, Endong Zhang, Wen Yu, Zhiqiang Gao, Fengting Lv, Yiming Huang, Daoben Zhu and Shu Wang Shengpeng Xia Beijing National Laboratory for Molecular Sciences, Key Laboratory of Organic Solids, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 College of Chemistry, University of Chinese Academy of Sciences, Beijing 100049 , Haotian Bai *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] E-mail Address: [email protected] Beijing National Laboratory for Molecular Sciences, Key Laboratory of Organic Solids, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 , Endong Zhang Beijing National Laboratory for Molecular Sciences, Key Laboratory of Organic Solids, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 , Wen Yu Beijing National Laboratory for Molecular Sciences, Key Laboratory of Organic Solids, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 College of Chemistry, University of Chinese Academy of Sciences, Beijing 100049 , Zhiqiang Gao Beijing National Laboratory for Molecular Sciences, Key Laboratory of Organic Solids, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 College of Chemistry, University of Chinese Academy of Sciences, Beijing 100049 , Fengting Lv Beijing National Laboratory for Molecular Sciences, Key Laboratory of Organic Solids, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 , Yiming Huang *Corresponding authors: E-mail Address: ba[email protected] E-mail Address: [email protected] E-mail Address: [email protected] Beijing National Laboratory for Molecular Sciences, Key Laboratory of Organic Solids, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 , Daoben Zhu Beijing National Laboratory for Molecular Sciences, Key Laboratory of Organic Solids, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 and Shu Wang *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] E-mail Address: [email protected] Beijing National Laboratory for Molecular Sciences, Key Laboratory of Organic Solids, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190 College of Chemistry, University of Chinese Academy of Sciences, Beijing 100049 https://doi.org/10.31635/ccschem.022.202202463 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail Developing customized chemical reactions that could regulate a specific biological process on demand is regarded as an advanced and promising strategy for treating diseases. However, conventional chemical reactions become challenging in complex physiological environments, which demand mild reaction conditions, high efficiency, good biocompatibility, and strong controllability. Moreover, the effects of the achieved reactions on the real biological system are usually further lessened. Herein, we describe an advanced photocatalytic reaction that irreversibly converted nicotinamide adenine dinucleotide (NAD+) to nicotinamide and adenosine diphosphate (ADP)-ribose by the cationic conjugated poly(fluorene-co-phenylene) (PFP). This reaction was introduced to tumor cells and triggered cell apoptosis. Under white-light illumination, the photocatalytic reaction decreased the NAD+ ratio in tumor cells, disrupted the mitochondrial membrane potential, inhibited the synthesis of adenosine triphosphate (ATP), and effectively induced apoptosis. We propose a mechanism of the reaction where PFP is photoexcited to PFP*, and the obtained photoelectrons are transferred from PFP* to NAD+ to produce nicotinamide and another unstable intermediate, ADP-ribosyl radical. ADP-ribosyl radical quickly reacts with triethanolamine to form ADP-ribose. This intracellular utilization of a specific photocatalytic reaction could offer a new approach to affect biological function for efficient cancer treatment. Download figure Download PowerPoint Introduction Life is based on continuous cellular metabolism processes, where many substrates and products are often involved in various chemical reactions.1,2 These synergetic reactions, including intracellular redox, enzyme cascade, and reversible reaction, maintain homeostasis of the biological system in individual bodies.3 However, biological metabolic processes with erroneous chemical reactions can cause metabolic disorders and dysfunction, even injury, cancer, and autoimmune disease.4–7 Customized chemical reactions that could regulate specific biological processes are regarded as an advanced and promising strategy for treating diseases.8 However, the complex physiological environments, including the near-neutral pH, mild temperature, pressure, and hypoxic microenvironment, also put many preconditions on developing these reactions.9 The additional demands of excellent controllability and biocompatibility make these investigations more challenging.10 Moreover, cellular self-healing ability11 would further weaken the effects of these reactions because living cells prefer to maintain a chemical balance in the body. Cancer, with high morbidity and mortality, has significantly threatened human life.12–14 Hypoxic microenvironments often cause worse disease outcomes by protecting cancer cells against apoptosis and enhancing the ability of tumor metastasis.3,15,16 Nicotinamide adenine dinucleotide (NAD) is one of the most important coenzymes involved in intracellular redox reactions including cancer metabolism,17 and it generally exists in two forms including oxidized NAD+ and reduced NADH.18 NAD+/NADH levels can affect cancer cell growth by inhibiting the related energy production pathways,6 such as glycolysis, the tricarboxylic acid cycle, and the mitochondrial electron transport chain (ETC). Hence, regulation of NAD+/NADH levels provides an effective strategy for the inhibition of cancer cells by driving the NAD+ and NADH interconversion.8,19,20 However, the regulated ratio of NAD+/NADH can be gradually restored to rebalance the redox levels through the cell repair process.11 Photocatalytic reactions exhibit great potential in achieving desirable intracellular reactions due to the strong controllability, mild reaction conditions,21 and noninvasive remote regulation.22,23 But the poor biocompatibility of noble metal catalysts24,25 could be harmful to living organisms resulting in limited applications in vivo. In comparison, water-soluble conjugated polymers (WSCPs) possess high biocompatibility26–29 and electron transfer efficiency that are beneficial to their applications in photoinduced processes in living organisms.30–32 The ability of WSCPs to catalyze cell-mediated polymerizations33–36 could be exploited in intracellular photocatalytic reactions, which create NAD+/NADH imbalance and regulate cell activities. In this work, we report an emerging photocatalytic reaction and realize the irreversible decomposition of NAD+ to nicotinamide and adenosine diphosphate (ADP)-ribose by the cationic poly(fluorene-co-phenylene) (PFP). The chemical reaction was induced in hypoxic tumor cells to successfully disrupt the cell activity (Scheme 1). The mechanistic investigation shows that the PFP acts as the photocatalyst in the system, and the photoelectrons transfer from the excited PFP (PFP*) to NAD+ to yield nicotinamide and ADP-ribose instead of the naturally occurring bioactive NADH. Therefore, the photocatalytic reaction mediated by PFP irreversibly regulates NAD+ rather than the common strategy based on a reversible reaction between NAD+ and active NADH.3 Inside these tumor cells, the reaction compromises mitochondrial functions that are mainly manifested as blockage of ETC, decreased mitochondrial membrane potential (MMP), and decreased adenosine triphosphate (ATP) level. The disturbed cellular redox cycle and irreversible damage of mitochondria by PFP under irradiation can cause apoptosis and finally disrupt the tumor cell activity. Scheme 1 | Schematic illustration of the photocatalysis reaction by PFP for irreversibly converting NAD+ to nicotinamide and ADP-ribose and inducing apoptosis of hypoxia cancer cells. Download figure Download PowerPoint Experimental Section General methods and materials Cationic PFP was synthesized according to the reported literature procedure.32 All chemical reagents are commercially available and used without further purification. 5,5-Dimethyl-1-pyrroline N-oxide (DMPO) was purchased from Sigma-Aldrich (St. Louis, Missouri, USA). 4T1 cells were purchased from the cell culture center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (Beijing, China). Roswell Park Memorial Institute 1640 medium (RPMI 1640) was purchased from Hyclone (Beijing, China). Fetal bovine serum was purchased from Sijiqing Biological Engineering Materials (Hangzhou, China). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Xinjingke Biotechnology Co., Ltd. (Beijing, China). MMP assay kit (JC-1), ATP, and BCA Assay Kits were acquired from Beyotime Biotechnology (Shanghai, China). The NAD+/NADH Ratio Kit was purchased from AAT Bioquest (Pleasanton, California, USA). Deionized water was obtained from a Milli-Q system (Millipore, Bedford, MA, United States). 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine (DiD) was provided by Life Technologies (Waltham, Massachusetts, USA). UV–vis absorption spectra were measured by a Thermo Scientific Evolution 201 spectrophotometer (Thermo Fisher, Waltham, Massachusetts, USA), and the fluorescence spectra were taken on a Hitachi F-4500 fluorometer (Hitachi, Tokyo, Japan) equipped with a Xenon lamp excitation source. Ultraviolet photoelectron spectroscopy (UPS) was conducted on an AXIS ULTRA DLD instrument (Kratos, Manchester, UK). Electrochemistry measurements were conducted on an electrochemical workstation (Autolab PGATAT 302N, Metrohm, Herisau, Switzerland). NMR spectra were obtained from a Bruker Avance HD 400 MHz and Bruker AVANCE 600 MHz (Rheinstetten, Germany). The electron paramagnetic resonance (EPR) spectra were measured by Bruker Xenoe nano (Bruker EMXnano, Fällanden, Switzerland). The MTT assay was recorded on a microplate reader (BioTek, Winooski, Vermont, USA). Confocal laser scanning microscopy (CLSM) images were conducted on confocal laser scanning biological microscope (Olympus FV 1200-BX61, Olympus, Hataya, Japan). Intracellular NAD+/NADH ratio assay 4T1 cells were treated in the medium at 37 °C at a density of 5 × 105 cells/well in 6-well plates overnight. Then, the medium with PFP (8 μM) and triethanolamine (TEOA; 4 mM) was used to cultivate 4T1 cells at 37 °C for 12 h. The cells were transferred to a hypoxic environment (1% O2, 5% CO2, 37 °C) for 6 h. After irradiation with 50 mW/cm2 for 20 min, the cells were treated with 250 μL cell lysis buffer. The centrifugal supernatant was collected and used to measure intracellular NAD+/NADH ratio by commercial kit. MMP measurement The 4T1 were added in confocal dishes at a density of 8 × 104 cells/mL and treated in the RPMI medium containing PFP (8 μM) and TEOA (4 mM) at 37 °C for 12 h. Then the cells were incubated in 1% O2 and 5% CO2 at 37 °C for 6 h. After irradiation under white light with 50 mW/cm2 for 30 min, the 4T1 cells were incubated with JC-1 (5×) for 20 min. Then cells were rapidly washed three times by JC-1 (1.25×). The CLSM images were collected at 520–550 nm (λex, 488 nm), and 575–675 nm (λex, 488 nm), respectively. EPR A mixture of 100 μL PFP (1 mM, dimethyl sulfoxide), 20 μL TEOA (1 M), 100 μL DMPO (1 M), 200 μL NAD+ (50 mM), and 580 μL phosphate-buffered saline was irradiated under white light (50 mW/cm2), and the EPR spectra were recorded every 5 min. (Center of Field, 3443.90 G; Sweep Width, 200.0 G; Power, 5 mW; Power Attenuation, 20.00 dB; Modulation Amplitude, 1.0 G; Number of Scans, 6.) Results and Discussion Characteristics of photocatalyst To verify the feasibility of photocatalytic reaction of PFP to catalytically convert NAD+, the photophysical information, energy level, and fluorescence decay kinetics of PFP were collected accordingly. As shown in Figure 1a, PFP exhibited a maximum absorption wavelength of 380 nm, and the peak of fluorescence emission was 429 nm. The band gap of PFP was calculated with a standard method to be 2.97 eV.4 The photoelectric response under a solar simulator demonstrated the possible conversion of absorbed photons into excited electrons ( Supporting Information Figure S1). The highest occupied molecular orbital (HOMO) was determined to be −5.82 eV by UPS (Figure 1b), and the lowest unoccupied molecular orbital (LUMO) was calculated to be −2.85 eV with an optical band gap of 2.97 eV. The Ered and Eox of PFP were +1.32 V and −1.65 V (vs normal hydrogen electrode [NHE]), respectively, obtained by calculations described in the literature.37,38 The reduction potential of NAD+/NADH is −0.54 V versus NHE,39 which is lower than Ered of PFP (+1.32 V vs NHE), and, therefore, suitable to capture the electron from PFP*. The fluorescence decay kinetic curves show that the fluorescence lifetime of PFP decreased from 104 to 23 ns with the addition of NAD+ (Figure 1c). The electron-transfer pathway from PFP to NAD+ under illumination was predicted based on the HOMO/LUMO potential (Figure 1d). The electron captured from the HOMO of PFP could be transferred to NAD+ in the reaction process. All the above characterizations indicate that under illumination PFP generates the holes and electrons, and then the photoelectrons from the PFP HOMO to LUMO transition might reduce NAD+ into the product. The holes are consumed by TEOA whose redox potential (+0.53 V vs NHE) is more negative than PFP (+1.32 V vs NHE). Figure 1 | Material property characterizations of conjugated polymer PFP. (a) Normalized absorption and fluorescence spectrum (in H2O at 25 °C). (b) Ultraviolet photoelectron spectrum of PFP. (c) The fluorescence decay kinetic curves of PFP (10 μM) and PFP (10 μM) + NAD+ (1 mM) with 100 mM TEOA in H2O at 25 °C (λex: 405 nm). (d) The redox potential of the elements involved in the reaction pathway. Download figure Download PowerPoint The photocatalysis reaction to decompose NAD+ According to NMR and mass spectrometry, the photocatalytic reaction of NAD+ generated nicotinamide and ADP-ribose as the products (Figure 2a and Supporting Information Figure S2). Before illumination, 1H NMR peaks corresponding to the nicotinamide unit (a–d), adenine unit (g and h), and the two glycosidic protons (e and f) in NAD+ were identified (black trace), whereas PFP peaks were invisible due to the low concentration. After illumination, the complicated 1H NMR spectral pattern consisted of free nicotinamide (a′–d′, red trace), ADP-ribose (e′–h′), and unreacted NAD+ (not labeled). Major changes of the peaks of nicotinamide and the glycosidic proton e/e′ indicate that the photocatalytic cleavage occurred between nicotinamide and its adjacent ribose. In contrast, minor peak shifts of adenine and its adjacent glycosidic proton f/f′ suggest an intact glycoside on adenine. To further resolve the products, diffusion ordered spectroscopy (DOSY) was performed on the sample after 1 h illumination (Figure 2b). Peaks corresponding to the free nicotinamide exhibited two- to threefold higher diffusion coefficients than other major peaks, and the trend was consistent with the smaller molar mass of nicotinamide (122 g/mol) compared to NAD+ and ADP-ribose. Although the latter two compounds could not be resolved in 1H NMR or DOSY spectra, they were identified on the electrospray ionization-mass spectrometry (ESI-MS) spectrum. The peaks at m/z of 558.1 and 540.1 correspond to ADP-ribose and its dehydrated derivatives, respectively, (Figure 2c), and the peak at 662.1 is unreacted NAD+. To monitor the reaction progress to characterize the kinetics of the photocatalysis, the 1H NMR integrations of H(d) in NAD+ and H(d′) in nicotinamide were calculated (Figure 2d). After 1 h irradiation, 113 μM nicotinamide was obtained, and the products continued to accumulate with extended irradiation time. Figure 2 | The reaction of NAD+ with PFP as the photocatalyst. (a) 1H NMR (D2O) spectra of the reaction mixture before (black) and after illumination (red). (b) DOSY spectrum for further resolving nicotinamide in the illuminated reaction mixture. (c) Negative ion ESI-MS indicates photocatalytic generation of ADP-ribose. (d) Nicotinamide generation under an extended time of illumination. Download figure Download PowerPoint Intracellular catalysis study and therapeutic efficacy The intracellular catalytic effect of PFP photocatalyst on 4T1 cancer cells was investigated. The stability of the cationic conjugated PFP in various physiological solutions was measured ( Supporting Information Figure S3). The similar absorption spectra indicate excellent stability of the conjugated PFP skeleton as a photocatalyst. Meanwhile, the efficiency of this photocatalytic reaction was measured under hypoxic and acidic conditions to simulate the tumor microenvironment. As shown in Supporting Information Figure S4, the efficiency of the photocatalytic reaction was not affected by the low oxygen concentration or acidic pH. It indicates that this photocatalytic reaction mediated by PFP worked in the tumor microenvironment. To exclude the photosensitization effects of PFP, the reactive oxygen species (ROS) production capacity of PFP was measured by 2,7-dichlorodihydrofluorescein (DCFH; Supporting Information Figure S5). In contrast to the efficient photogeneration of ROS under aerobic conditions, negligible ROS were produced by the illumination of PFP under hypoxic conditions. It indicates that the influence of common ROS is not a major contributing factor in this antitumor system. As illustrated in Supporting Information Figure S6, CLSM images of 4T1 cells showed that the blue signal from PFP was surrounded by an entire membrane (labeled with DiD and emitting red signal), which elucidates the successful internalization of PFP. As shown in Supporting Information Figure S7, the PFP, which located in the lysosome, was taken up into cells over the incubation time and retained intracellularly for more than 72 h. This indicates that PFP has the potential to act as a long-term catalyst in cancer cells. To verify that the catalytic reaction could occur in cells, the intracellular NAD+/NADH ratio in hypoxic 4T1 cells treated with PFP under light irradiation was measured. As shown in Figure 3a, the NAD+/NADH ratio of the optical irradiation group treated with PFP decreased from 73% to 65% compared to the dark group. Thus, PFP can catalytically convert NAD+ into nicotinamide and ADP-ribose under white illumination. As an important coenzyme pair in the mitochondrial ETC, and a raw material for photocatalytic reactions, NAD+ can affect the function of mitochondria. The continuous depletion of NAD+ can lead to mitochondrial apoptosis, thereby affecting cell growth. So, the related MMP was measured to further investigate the influence of the explored photocatalytic reaction in hypoxic 4T1 cells. The commercial MMP probe JC-1 was used to visually observe the apoptosis of mitochondria after treatment with PFP and optical illumination. Typically, JC-1 characterizes the state of MMP (ΔΨm) by its monomer or J-aggregate. The J-aggregate initially attaches to the outer membrane of the normal mitochondria and emits red fluorescence. However, the ΔΨm decreases once the NADH-dependent oxidative phosphorylation (OXPHOS) process is inhibited,40 and the JC-1 changes to the monomer and shows green fluorescence, indicating the mitochondria goes into apoptosis (Figure 3b). As displayed in Figure 3c and Supporting Information Figure S8, 4T1 cells exhibited strong green fluorescence after being treated with PFP upon optical illumination in the hypoxic environment, suggesting that the ΔΨm decreased and the mitochondria may be undergoing apoptosis. The variations of the intracellular ATP level were subsequently measured to confirm the mitochondria damage. As shown in Figure 3d, the ATP level of 4T1 cells in the control group had negligible changes no matter with or without white-light illumination. However, the ATP level of the white-irradiation group decreased to 0.11 μg/mg protein (from 0.20 μg/mg protein), which indicates that the process of NAD+ cleavage and product production led to mitochondrial damage, further affecting the normal metabolic process and finally causing cell apoptosis. To visually show the therapeutic effect of the PFP system on 4T1 cancer cells, cell was performed accordingly. As shown in Figure and Supporting Information Figure the cells treated with PFP in the dark strong green of living cells, indicating a high and the promising of PFP, whereas over of the cells treated with PFP under illumination showed the red signal of cells, that PFP had an therapeutic effect on the of tumor cells under white-light illumination. The MTT assay was performed to the cell in the of of PFP. 4T1 cells had high cell even treated with a high concentration of PFP μM) in dark conditions (Figure further its However, upon white-light illumination, the 4T1 cell decreased as the concentration of PFP 2 4T1 cell after being treated with μM PFP, the good therapeutic efficacy of the photocatalytic system due to the catalytic decomposition of NAD+ by this system that in a metabolic process. TEOA and the nicotinamide involved in the system also exhibited negligible ( Supporting Information and As illustrated in Figure and Supporting Information Figure the apoptosis cell ratio of 4T1 cells after being treated with PFP under white-light illumination significantly from to whereas the control or including the white-light group and group. This indicates the good therapeutic efficacy of the photocatalytic system as as the and biocompatibility of optical and PFP respectively. To study the of the described photocatalytic system, two other tumor cell and were by MTT assay ( Supporting Information Figure concentration of PFP 1 the cell of and that the photocatalytic system was effective for various cancer cells, including human cancer cells. to the common strategy of regulation between NAD+ and active the metabolism regulation by PFP is an irreversible reaction, which can the cellular redox cycle and irreversible damage to mitochondria and even cells. The of the photocatalytic reaction to human cells was measured to study the effect of this photocatalytic reaction on normal cells ( Supporting Information Figure The cells high under light and dark conditions. Figure | of the cell apoptosis by PFP photocatalyst upon optical illumination. (a) of the intracellular NAD+ concentration by PFP under optical illumination for 30 min. (b) Schematic illustration of mitochondrial membrane potential changes by (c) Confocal laser scanning microscopy images to show mitochondrial membrane potential (ΔΨm) changes of 4T1 cells after incubation with PFP with or without optical The fluorescence of JC-1 was collected at 575–675 nm (λex, nm), 520–550 nm (λex, 488 nm), respectively. 50 (d) The intracellular ATP levels before and after catalysis by PFP under optical illumination for 30 min. of living and cells by and The fluorescence of and were collected at nm (λex, 488 nm), 575–675 nm (λex, nm), respectively. 200 of 4T1 cells after being treated with PFP μM) with or without white-light illumination (50 for 30 min. Apoptosis and of 4T1 cells after were according to the fluorescence in and Download figure Download PowerPoint The radical and biological characterizations of the photocatalysis reaction EPR assay was conducted to the specific mechanism of the above photocatalytic process. the radical would be by DMPO and to the species the collected radical signal under irradiation was as radical (Figure It that the NAD+ transferred to radical in this photocatalysis process under illumination. the of Figure the electron that NAD+ captured was from PFP*. on the above a mechanism of photocatalytic reaction by PFP is (Figure Under illumination, electrons from PFP* transfer to NAD+, radical and then the holes are captured by After electron ADP-ribose is obtained from the by the of To the of this system in the activity of production was by the catalytic reaction of acid with the conversion of acid to acid with the of and the reaction be inhibited NADH is by photocatalytic The activity of production was by the absorption at nm, which is the absorption of NADH. As shown in Figure the absorption at nm of photocatalytic products exhibited negligible with the addition of the acid and that the photocatalytic product could not be used as a coenzyme of active the absorption decreased significantly after the acid and Thus, photocatalytic products are to in the normal reactions, which that the products obtained in the photocatalytic reaction can not the cycle of the cells. Therefore, the photocatalytic reaction with the function of cancer cells and hypoxic tumor apoptosis. Figure 4 | The radical and biological characterizations of products from NAD+ by PFP under light illumination. (a) EPR spectrum of radical by DMPO under irradiation 50 (b) The mechanism of the photocatalytic reaction by PFP for irreversibly converting NAD+ to nicotinamide and ADP-ribose. (c) The absorption of the PFP + NAD+ group with under dark and compared to the NADH group. upon production under irradiation compared with NADH by Download figure Download PowerPoint In we report a new photocatalytic reaction where irradiated PFP irreversibly NAD+ to nicotinamide and ADP-ribose. The chemical reaction was used to apoptosis in hypoxic tumor cells of The cell demonstrated that the intracellular NAD+ is converted into nicotinamide and ADP-ribose instead of naturally bioactive which is from the pathway. As a reaction of the cellular redox cycle, the continuous depletion of NAD+ disrupted the balance of coenzyme

Overexpression of the Ski oncogene induces oncogenic transformation of chicken embryo fibroblasts (CEFs). However, unlike most other oncogene-transformed cells, Ski-transformed CEFs (Ski-CEFs) do not display the classical Warburg effect. On the contrary, Ski transformation reduced lactate production and glucose utilization in CEFs. Compared with CEFs, Ski-CEFs exhibited enhanced TCA cycle activity, fatty acid catabolism through β-oxidation, glutamate oxidation, oxygen consumption, as well as increased numbers and mass of mitochondria. Interestingly, expression of PPARγ, a key transcription factor that regulates adipogenesis and lipid metabolism, was dramatically elevated at both the mRNA and protein levels in Ski-CEFs. Accordingly, PPARγ target genes that are involved in lipid uptake, transport, and oxidation were also markedly up-regulated by Ski. Knocking down PPARγ in Ski-CEFs by RNA interference reversed the elevated expression of these PPARγ target genes, as well as the shift to oxidative metabolism and the increased mitochondrial biogenesis. Moreover, we found that Ski co-immunoprecipitates with PPARγ and co-activates PPARγ-driven transcription.

NADH Ties One-Carbon Metabolism to Cellular Respiration

Bacterial antibiotic tolerance, a phenomenon first observed in 1944, is known to be responsible for both onset and exacerbation of recurrent and chronic bacterial infections. The development of antibiotic tolerance was previously thought to be due to a switch to physiological dormancy when bacteria encounter adverse growth conditions. Our recent laboratory findings, however, showed that a set of genes related to the maintenance of proton motive force (PMF) are up-regulated under starvation, indicating that the tolerant sub-population, which are commonly known as persisters, can actively maintain their tolerance phenotypes. In this study, we investigated the relative functional roles of proteins involved in the maintenance and active generation of PMF in mediating tolerance formation in bacteria and found that the PspA and RcsB proteins play a key role in PMF maintenance in persisters, as deletion of genes encoding these two proteins resulted in significantly lower tolerance levels. Consistently, expression of the OsmC and Bdm proteins, which is under regulation by RcsB, is required to maintain PMF and the antibiotic tolerance phenotypes. On the other hand, the NuoL, Ndh, AppC, CyoB, and NuoF proteins, which are electron transport chain (ETC) components, were also found to be actively expressed in persisters in order to generate PMF to support functioning of various tolerance mechanisms such as efflux activities. Our data show that active generation of PMF is even more important than the PMF maintenance functions of PspA and RcsB in the expression of antibiotic tolerance phenotypes in persisters. Assessment of double- and triple-gene knockout strains, in which the PMF maintenance genes and those encoding ETC components were simultaneously deleted, confirms that these two groups of genes are both required for the expression of antibiotic tolerance phenotypes and that a lack of these functions would result in complete PMF dissipation and accumulation of antibiotics in the intracellular compartment of persisters and eventually cell death. Products of these genes are, therefore, ideal targets for future development of anti-tolerance agents. IMPORTANCE In this work, bacteria were found to undergo active generation and maintenance of proton motive force (PMF) under adverse conditions, such as starvation so as to support a range of physiological functions in order to survive under such conditions for a prolonged period. The ability to maintain a substantial level of PMF was found to be directly linked to that exhibiting phenotypic antibiotic tolerance under nutrient starvation or other adverse conditions. These findings infer that bacteria do not simply become physiologically dormant when they become antibiotic tolerant, instead they need to produce a wide range of proteins including those which help prevent PMF dissipation, such as PspA and RcsB, and the electron transport chain components, such as NuoL and Ndh, that actively generate PMF even during long-term starvation. As antibiotic tolerant sub-population is known to play a role in eliciting recurrent and chronic infections, especially among patients with a weakened immune system, the PMF maintenance mechanisms identified in this work are potential targets for the development of new strategies to control recurrent and chronic infections.

Overflow metabolism in the form of aerobic acetate excretion by Escherichia coli is an important physiological characteristic of this common industrial microorganism. Although acetate formation occurs under conditions of high glucose consumption, the genetic mechanisms that trigger this phenomenon are not clearly understood. We report on the role of the NADH/NAD ratio (redox ratio) in overflow metabolism. We modulated the redox ratio in E. coli through the expression of Streptococcus pneumoniae (water-forming) NADH oxidase. Using steady-state chemostat cultures, we demonstrated a strong correlation between acetate formation and this redox ratio. We furthermore completed genome-wide transcription analyses of a control E. coli strain and an E. coli strain overexpressing NADH oxidase. The transcription results showed that in the control strain, several genes involved in the tricarboxylic acid (TCA) cycle and respiration were repressed as the glucose consumption rate increased. Moreover, the relative repression of these genes was alleviated by expression of NADH oxidase and the resulting reduced redox ratio. Analysis of a promoter binding site upstream of the genes which correlated with redox ratio revealed a degenerate sequence with strong homology with the binding site for ArcA. Deletion of arcA resulted in acetate reduction and increased the biomass yield due to the increased capacities of the TCA cycle and respiration. Acetate formation was completely eliminated by reducing the redox ratio through expression of NADH oxidase in the arcA mutant, even at a very high glucose consumption rate. The results provide a basis for studying new regulatory mechanisms prevalent at reduced NADH/NAD ratios, as well as for designing more efficient bioprocesses.

Diabetic patients carry the risk of developing a specific form of cardiac dysfunction, termed diabetic cardiomyopathy, which is not accounted for by coronary artery disease or hypertension, but likely stems from hormonal and metabolic derangements associated with diabetes. Several factors contribute to the pathogenesis of diabetic cardiomyopathy, and one important role is the loss of the heart's metabolic flexibility with overreliance on fatty acid (FA) oxidation for adenosine triphosphate (ATP) production. Such disproportionate FA oxidation provokes myocardial accumulation of toxic intermediates, such as ceramide and diacylglycerol, a pathology termed lipotoxicity, which has been observed in various animal models and also patients with diabetes (Brookheart et al. 2009; Bayeva et al. 2013). Furthermore, extensive remodelling of cardiac energy metabolism impacts numerous cellular processes, such as excitation–contraction coupling and the mitochondrial redox state, contributing to contractile dysfunction, but presumably also redox-dependent cell death and signalling events triggering maladaptive cardiac remodelling of the diabetic heart. In this issue of The Journal of Physiology, Cortassa and colleagues (2019) shed new light on the pathophysiology of metabolic remodelling in the diabetic heart. To this end, the authors took advantage of a commonly used mouse model of spontaneous type 2 diabetes induced by global genetic deletion of the leptin receptor (db/db). As a consequence of metabolic inflexibility and insulin resistance, contractile performance of Langendorff-perfused db/db hearts challenged with the β-agonist isoproterenol is blunted when the myocardium is supplied exclusively with high levels of glucose, mimicking peaks of hyperglycaemia, compared to non-diabetic hearts perfused with euglycaemic medium. Under these conditions, the addition of palmitate to the perfusion medium enhances cardiac function in db/db, but not wild-type, hearts. In fact, 95% of cardiac ATP production is sustained by oxidative phosphorylation, which in turn is supported by the continuous supply of reducing equivalents (NADH and FADH2) produced by the terminal oxidation of acetyl-CoA via the Krebs cycle. Although the healthy heart relies predominantly (60–90%) on FAs for ATP production, its metabolic flexibility enables utilization of different substrates as energy sources depending on the metabolic milieu (Bertero & Maack, 2018). In contrast, the metabolically inflexible db/db hearts display limited capacity for glucose oxidation, and might therefore become energy starved in the absence of FAs. The elegant metabolomic and fluxomic approaches applied in this study provide further mechanistic insights into this observation, revealing the consequences of altered substrate utilization for cytosolic and mitochondrial redox balance. It is noteworthy that in db/db hearts, perfusion with palmitate not only reactivated mitochondrial oxidative metabolism, but also markedly decreased intracellular glucose concentrations and shifted glucose flux away from the polyol pathway, which consumes reducing equivalents, towards the pentose phosphate pathway, regenerating reduced NADPH by converting glucose to pentoses (5-carbon sugars). Altogether, these findings suggest that in addition to ATP production, FA metabolism impacts on contractile performance by modulating cellular redox balance of the diabetic heart. Although Cortassa et al. provide precious new insight into altered cardiac metabolism, the metabolomic and fluxomic results require careful interpretation in the light of two methodological limitations. First, the ex vivo conditions of isolated heart preparations do not take into account the effects of physiological (or even pathological) cardiac load. The latter is generally important when estimating increased metabolic flux caused by rampant rise of myofilament workload to match afterload elevations. Another caveat, as mentioned by the authors themselves, is that the reference fluxes used in the metabolomic-fluxome calculation were extrapolated from isolated (unloaded) cardiac myocytes at rest, in which energy demand is lower, and hence the scaling of reference fluxes to ex vivo whole mouse hearts includes some uncertainty. Furthermore, some open questions still remain. One regards the metabolic consequences of long-term exposure to FAs, and in particular to palmitate, for energy reserve and redox equilibrium. FA metabolism is a tight balance of synthesis, uptake and oxidation (Fig. 1). As mentioned above, the diabetic heart displays intracellular accumulation of lipotoxic intermediates because FA oxidation, in spite of its enhanced rate, does not match the abnormally increased FA uptake (Brookheart et al. 2009). Moreover, the role of FAs is not limited to oxidation for ATP production. FAs differ in size (carbon chain length), degree of saturation (number of double bounds) and geometry (cis or trans). Long-chain FAs, such as palmitate, are also key precursors in phospholipid synthesis. In pathological amounts, palmitate induces abnormal phospholipid remodelling by increasing levels of dipalmitoyl phosphatidic acid, a precursor of phospholipid biosynthesis, but a poor substrate for cardiolipin formation (Ostrander et al. 2001). Cardiolipin, however, is a unique phospholipid exclusively found in the inner mitochondrial membrane and responsible for stabilization of the electron transport chain and, presumably, also a number of other proteins in the inner mitochondrial membrane. Palmitate-induced depletion of cellular cardiolipin, under rather extreme pathological conditions, promotes cardiomyocyte loss (Ostrander et al. 2001). Therefore, future studies should address the impact of chronic, but graded (physiological-to-pathological), elevations of palmitate on mitochondrial ATP regeneration and cellular redox state. Inside the cell, glucose can undergo different ‘fates’: oxidation to pyruvate via the glycolytic pathway, oxidation to ribose 5-phosphate (R5P) via the pentose phosphate (PP) pathway, or storage in the form of glycogen. In hyperglycaemic states, elevated intracellular glucose is converted to sorbitol via the polyol pathway, oxidizing nicotinamide adenine dinucleotide phosphate (NADP+). Similarly, fatty acids (FAs) can be oxidized for ATP production, stored in the form of triacylglycerols (TAG), or used as precursors for membrane phospholipids. Although the diabetic heart relies predominantly on FAs for ATP production, excessive FA uptake overcomes rates of β-oxidation, resulting in myocardial accumulation of toxic lipid species. Red arrows indicate changes in diabetes. ATP, adenosine triphosphate; CPT1/2, carnitine palmitoyltransferase 1/2; ETC, electron transport chain; FA-CoA, fatty acyl-coenzyme A ester; FADH2, reduced form of flavin adenine dinucleotide; FAT/CD36, fatty acid translocase; GLUT-1/4, glucose transporters 1/4; G6P, glucose 6-phosphate; G6PD, glucose 6-phosphate dehydrogenase; NADH, reduced form of nicotinamide adenine dinucleotide; PDH, pyruvate dehydrogenase; PP pathway, pentose phosphate pathway; R5P, ribose 5-phospate; TAG, triacylglycerol. In conclusion, this nice and comprehensive study by Cortassa and colleagues tackles the important question of how metabolic remodelling in diabetes impacts on the cellular redox state. The novel approach of this study paves the way for further preclinical work aiming to translate these insights into therapeutic strategies. None declared. All authors have read and approved the final version of this manuscript and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed. The authors received funding from the Bundesministerium für Bildung und Forschung and Deutsche Forschungsgemeinschaft (DFG) Ma 2528/7-1, SFB 894, TRR-219.

Look into any cell today, and you’ll see remnants of ancient bacteria by the thousands. These mitochondria—tiny organelles in the cell that each possess their own DNA—have come under a growing scientific spotlight; scientists increasingly believe they play a central role in many, if not most, human illnesses. Exquisitely sensitive to environmental threats, mitochondria convert dietary sugars into a high-energy molecule—adenosine triphosphate (ATP)—that cells use as fuel. And when mitochondria falter, cells lose power, just as a flashlight dims when its batteries weaken. Now scientists are linking environmental interactions with the mitochondria to an array of metabolic and age-related maladies, including cancer, autism, type 2 diabetes, Alzheimer disease, Parkinson disease, and cardiovascular illness.

The mitochondrial genome: Dynamics, mechanisms of repair, and a target in disease and therapy

Brain cells normally respond adaptively to bioenergetic challenges resulting from ongoing activity in neuronal circuits, and from environmental energetic stressors such as food deprivation and physical exertion. At the cellular level, such adaptive responses include the "strengthening" of existing synapses, the formation of new synapses, and the production of new neurons from stem cells. At the molecular level, bioenergetic challenges result in the activation of transcription factors that induce the expression of proteins that bolster the resistance of neurons to the kinds of metabolic, oxidative, excitotoxic, and proteotoxic stresses involved in the pathogenesis of brain disorders including stroke, and Alzheimer's and Parkinson's diseases. Emerging findings suggest that lifestyles that include intermittent bioenergetic challenges, most notably exercise and dietary energy restriction, can increase the likelihood that the brain will function optimally and in the absence of disease throughout life. Here, we provide an overview of cellular and molecular mechanisms that regulate brain energy metabolism, how such mechanisms are altered during aging and in neurodegenerative disorders, and the potential applications to brain health and disease of interventions that engage pathways involved in neuronal adaptations to metabolic stress.

Mitochondrial ROS Fire Up T Cell Activation

Research Article| March 01 2009 Teaching Cellular Respiration & Alternate Energy Sources with a Laboratory Exercise Developed by a Scientist-Teacher Partnership Brandon Briggs, Brandon Briggs Search for other works by this author on: This Site PubMed Google Scholar Teri Mitton, Teri Mitton Search for other works by this author on: This Site PubMed Google Scholar Rosemary Smith, Rosemary Smith Search for other works by this author on: This Site PubMed Google Scholar Timothy Magnuson Timothy Magnuson Search for other works by this author on: This Site PubMed Google Scholar The American Biology Teacher (2009) 71 (3): 164–167. https://doi.org/10.2307/27669399 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Tools Icon Tools Get Permissions Cite Icon Cite Search Site Citation Brandon Briggs, Teri Mitton, Rosemary Smith, Timothy Magnuson; Teaching Cellular Respiration & Alternate Energy Sources with a Laboratory Exercise Developed by a Scientist-Teacher Partnership. The American Biology Teacher 1 March 2009; 71 (3): 164–167. doi: https://doi.org/10.2307/27669399 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentThe American Biology Teacher Search This content is only available via PDF. Copyright National Association of Biology Teachers Article PDF first page preview Close Modal You do not currently have access to this content.

Mitochondria are being recognized as key factors in many unexpected areas of biomedical science. In addition to their well-known roles in oxidative phosphorylation and metabolism, it is now clear that mitochondria are also central to cell death, neoplasia, cell differentiation, the innate immune system, oxygen and hypoxia sensing, and calcium metabolism. Disruption to these processes contributes to a range of human pathologies, making mitochondria a potentially important, but currently seemingly neglected, therapeutic target. Mitochondrial dysfunction is often associated with oxidative damage, calcium dyshomeostasis, defective ATP synthesis, or induction of the permeability transition pore. Consequently, therapies designed to prevent these types of damage are beneficial and can be used to treat many diverse and apparently unrelated indications. Here we outline the biological properties that make mitochondria important determinants of health and disease, and describe the pharmacological strategies being developed to address mitochondrial dysfunction.

Mitochondrial bioenergetics and neurodegeneration: a paso doble

Staphylococcus epidermidis is a skin-resident bacterium and a major cause of biomaterial-associated infections. The transition from residing on the skin to residing on an implanted biomaterial is accompanied by regulatory changes that facilitate bacterial survival in the new environment. These regulatory changes are dependent upon the ability of bacteria to "sense" environmental changes. In S. epidermidis, disparate environmental signals can affect synthesis of the biofilm matrix polysaccharide intercellular adhesin (PIA). Previously, we demonstrated that PIA biosynthesis is regulated by tricarboxylic acid (TCA) cycle activity. The observations that very different environmental signals result in a common phenotype (i.e. increased PIA synthesis) and that TCA cycle activity regulates PIA biosynthesis led us to hypothesize that S. epidermidis is "sensing" disparate environmental signals through the modulation of TCA cycle activity. In this study, we used NMR metabolomics to demonstrate that divergent environmental signals are transduced into common metabolomic changes that are "sensed" by metabolite-responsive regulators, such as CcpA, to affect PIA biosynthesis. These data clarify one mechanism by which very different environmental signals cause common phenotypic changes. In addition, due to the frequency of the TCA cycle in diverse genera of bacteria and the intrinsic properties of TCA cycle enzymes, it is likely the TCA cycle acts as a signal transduction pathway in many bacteria.

Commentary: Mitochondrial respiration in right heart failure