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Is the Warburg effect an effect of deregulated space occupancy of methylome?

Posted in Anaerobic Glycolysis, Cancer - General, CANCER BIOLOGY & Innovations in Cancer Therapy, Curation, Genomic Expression, Oxidative phosphorylation, Pyruvate Kinase, Warburg effect, tagged anaerobic and aerobic gycolysis, ascorbate, Cancer - General, dihydroascorbate, glutamine, lactate dehydrogenase reaction, Warburg Effect on February 15, 2016| Leave a Comment »

Is the Warburg effect an effect of deregulated space occupancy of methylome?

Larry H. Bernstein and Radoslav Bozov, co-curation

LPBI

 

 

It would appear that pyruvate is directly used by cancer cell machinery for sustaining genome independence, and that CRISP-Cas9 system is essentially a modified CAD protein for making small bases.

¹³C-labeled biochemical probes for the study of cancer metabolism with dynamic nuclear polarization-enhanced magnetic resonance imaging

Lucia Salamanca-Cardona and Kayvan R. Keshari

Cancer & Metabolism 2015; 3:9          http://dx.doi.org:/10.1186/s40170-015-0136-2

In recent years, advances in metabolic imaging have become dependable tools for the diagnosis and treatment assessment in cancer. Dynamic nuclear polarization (DNP) has recently emerged as a promising technology in hyperpolarized (HP) magnetic resonance imaging (MRI) and has reached clinical relevance with the successful visualization of [1-¹³C] pyruvate as a molecular imaging probe in human prostate cancer. This review focuses on introducing representative compounds relevant to metabolism that are characteristic of cancer tissue: aerobic glycolysis and pyruvate metabolism, glutamine addiction and glutamine/glutamate metabolism, and the redox state and ascorbate/dehydroascorbate metabolism. In addition, a brief introduction of probes that can be used to trace necrosis, pH changes, and other pathways relevant to cancer is presented to demonstrate the potential that HP MRI has to revolutionize the use of molecular imaging for diagnosis and assessment of treatments in cancer.

 

Since the hallmark discovery of the Warburg effect in cancer cells in the 1920s, it has been widely accepted that the metabolic properties of cancer cells are vastly different from those of normal cells [1]. Starting from the observation that many cancerous (neoplastic) cells have higher rates of glucose utilization and lactate production, the development of tools and methods to correlate specific cellular metabolic processes to different types of cancer cells has received increased research focus [2, 3]. Several imaging techniques are currently in use for this purpose, including radiography, scintigraphy, positron emission tomography (PET), single-photon emission computed tomography (SPECT), and magnetic resonance (MR) [4, 5].

For more than 30 years, MR has been a revolutionary diagnostic tool, used in a wide range of settings from the central nervous system to cardiomyopathies and cancers. MR imaging (MRI) can outline molecular and cellular processes with high spatial resolution. Typically, MRI of body tissues is achieved via contrast visualization of the protons (¹H) of water, which are present in high abundance in living systems. This can be extended to MR spectroscopy (MRS), which can further differentiate between less abundant, carbon-bearing, biological metabolites in vivo utilizing ¹Hs of these compounds [6, 7]. However, despite its usefulness in imaging whole body tissues, ¹H MRS has low spectral resolution and poor sensitivity for these less abundant metabolites. In addition,¹³C MRS is increasingly difficult, in comparison to ¹H MRS, in that both the gyromagnetic ratio (approximately 25 % of ¹H) and natural abundance (1.1 % of ¹H) are significantly lower, making the detection of carbon-bearing compounds difficult [8, 9]. The low spectral resolution of ¹H MRS for metabolites can be addressed by using ¹³C-enriched compounds, and with this direct ¹³C MRS, metabolic processes can be traced, utilizing enriched tags on specific carbons in a given metabolite [10]. While enrichment of molecules in ¹³C can also moderately address the sensitivity limitation of MRS, recent work in hyperpolarization (HP) provides a means of dramatically increasing sensitivity and enhancing signals, well beyond that of the equilibrium state obtained via MRS. [11, 12]. The focus of this review will be the introduction of this approach in the setting of cancer metabolism, delineating probes of interest, which have been applied to study metabolic processes in vivo.

Obtaining a hyperpolarized probe

In MR, a desired target is placed in a magnetic field where the nuclear spins of molecules are aligned with or against the direction of the magnetic field. The nuclear spins have thus different energies, and an MR signal is detected upon relaxation of nuclear spins of higher energy. At thermal equilibrium, the number of spins aligned with the magnetic field nearly equals the number of spins opposing the direction of the magnetic field. Thus, at thermal equilibrium, spin polarization is in the order of >0.0005 % resulting in a limited signal. Signal increases on the order of 100,000-fold can be achieved by hyperpolarizing the system via the redistribution of the spin population levels found at equilibrium [10, 13]. There are several techniques that have been used to achieve hyperpolarization of various nuclei: spin-exchange optical pumping of ³He and ¹²⁹Xe, parahydrogen-induced polarization (PHIP), and dissolution dynamic nuclear polarization (DNP) [11,14, 15]. Both PHIP and DNP techniques can polarize biologically relevant nuclei like ¹³C and ¹⁵N, although there is a wider range of molecules that can be targeted for hyperpolarization using dissolution DNP [14, 16–18].

The goal of DNP is the transfer of polarization from highly polarized unpaired electron spins to the nuclear spins of a desired target compound. This is achieved by applying an external magnetic field to a free-radical agent in order to polarize electron spins, followed by saturating the electron spin resonance via microwave irradiation in order to obtain polarization transfer. The free-radical agent is generally a stable organic compound that is compatible with aqueous buffers, which are used as solvent in order to obtain a homogeneous distribution of the radical [13]. Nearly 100 % of the electrons on the free-radical agent are polarized when the free-radical/solvent mixture is subjected to high magnetic fields (≥3.3 T) followed by rapid freezing to 1 K using liquid helium in order to obtain a sample frozen to an amorphous state, which is necessary for retention and transfer of polarization [18]. For biological applications, after transfer of electron spin polarization to the nuclei of interest has occurred, the preparation must exist in solution, which can be achieved utilizing a dissolution process in which the solid sample is rapidly melted via injection of a hot solvent, typically a biologically compatible buffer, into the frozen sample [13]. The dissolution process results in a liquid sample at room temperature, while still preserving the enhanced polarization obtained by the microwave irradiation of the frozen sample [8]. Additionally, the use of chelating agents (e.g., EDTA) with the solvent to eliminate trace metals and more recently the use of gadolinium (Gd) chelates with the DNP sample have been used to further enhance and retain polarization in the liquid sample, albeit with caution over potential toxic effects when applied in vivo and the potential for loss of hyperpolarization due to T ₁ shortening [11, 19, 20]. More in-depth exploration of the technical aspects of probe development has been previously reviewed [8, 11].

Considerations in probe selection and current research

The usefulness of a molecule for hyperpolarized MRS is dependent on the polarization lifetime of the nucleus of interest, and this property is determined by the spin-lattice relaxation constant (T₁) [21]. Dipolar coupling, the magnetic field range, and molecular size can also affect the T₁ of a given nucleus. In general, high magnetic fields and large molecular weights decrease the T₁. Dipole-dipole coupling of ¹³C with ¹H is common in biologically relevant molecules, and it shortens relaxation times; therefore, carbon atoms directly bound to ¹H are generally not useful as probes for HP. For example, all carbons present in glucose (an important substrate in cancer cells) have relaxation times shorter than 2 s [22]. On the other hand, carbonyl carbons of biologically relevant molecules generally have T₁’s above 20 s even at high magnetic fields like [1-¹³C] pyruvic acid, which has relaxation times of 67, 48, and 44 s at 3, 11.7, and 14.1 T, respectively [23–25]. Even carbons that are less oxidized than carbonyls, like the hemi-ketal in [2-¹³C] fructose have T₁’s one order of magnitude higher than glucose carbons. Short spin-lattice relaxation times can sometimes be increased by deuterium enrichment of the sample. With this technique, protons that are directly bound to carbons are exchanged for deuterium atoms which results in the reduction of dipole-dipole relaxation, further preserving the hyperpolarized state [26]. This has resulted in increased T₁’s of ¹³C nuclei in molecules such as glucose (T₁ increased from 2 s to 10–14 s), providing the possibility of utilizing them in future metabolic studies [27–29]. Despite the effect of deuterium enrichment, research efforts have largely focused on developing carbonyl-bearing molecules as molecular imaging probes. The focus of this review is to introduce representative compounds relevant to metabolism that are characteristic of cancer tissue and have been applied in the work of multiple groups: aerobic glycolysis, glutamine addiction, and the redox state.

Pyruvate and aerobic glycolysis

Of particular interest to cancer metabolism is the increased conversion of glucose to lactate as a result of modulated aerobic glycolysis. This process, also known as the Warburg effect, is characteristic of many tumors with altered metabolism where pyruvate generated from glucose metabolism via glycolysis is preferentially converted to lactate by lactate dehydrogenase (LDH) as opposed to entering the tricarboxylic acid cycle [1]. With this phenotype, cancer cells show a preference for lactate fermentation even in the presence of oxygen, thus bypassing oxidative respiration for ATP generation. Because of this, pyruvate has been the preferred probe for HP MRS research since it is an intermediate metabolite in pathways characteristic of aberrant metabolism in cancer cells, including increased lactate production as a result of aerobic glycolysis where detection of HP pyruvate-derived lactate can be used as a marker for cancer and response to treatment [30, 31] as well as an intermediate in amino acid metabolism (e.g., interconversion to alanine via transamination with glutamate) (Fig. 1). In addition, as mentioned before, carbonyl carbons in pyruvate have long relaxation times where even the methyl carbon can have T₁’s above 50 s after deuterium enrichment [32]. The interconversion of pyruvate to lactate has been exploited for MRI by using [1-¹³C] pyruvate and detecting the accumulation of increased lactate in cancerous tissue as compared to surrounding benign tissue. Increased conversion of pyruvate to lactate and alanine has been demonstrated to precede MYC-driven tumorigenesis by using HP [1-¹³C] pyruvate in murine models [33]. Furthermore, in the same study, a decrease in the flux of alanine was observed at the tumor stage while a decrease in lactate conversion was indicative of tumor regression [33]. In transgenic adenocarcinoma of mouse prostate (TRAMP) models, in vivo studies using HP [1-¹³C] pyruvate demonstrated that hyperpolarized pyruvate and its metabolic products can be used non-invasively and with high specificity to obtain a profile of the histologic grade of prostate cancers [34]. In vivo imaging following hyperpolarized pyruvate has also been used to evaluate the role of glutaminase and LDH in human lymphoma models [35] as well as to elucidate metabolism of pyruvate in breast cancer [36] and renal cell carcinoma with treatment [30, 37].

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Flux of hyperpolarized [1-¹³C] pyruvate to [1-¹³C] lactate in prostate regions. a MR image from patient with prostate cancer showing regions of cancerous tissue and surrounding normal tissue. b–d Localized dynamic hyperpolarized [1-¹³C]pyruvate and [1-¹³C]lactate spectral from voxels overlapping the contralateral region of prostate (turquoise), a region of prostate cancer (yellow), and a vessel outside the prostate (green). Adapted with permission from ref. [43]

Early work that utilized HP pyruvate to assess the response of tumors to treatment was conducted in mice xenografted with EL-4 lymphoma cells and treated with etoposide, a topoisomerase inhibitor that causes rapid cell death [38, 39]. In this study, tumor necrosis was correlated to a decrease in the flux of hyperpolarized lactate which was suggested to be due to a decrease in NAD+ and NADH in the intracellular pool as well as loss of LDH function. More recently, HP [1-¹³C] pyruvate has been used as a biomarker to evaluate early response to radiation therapy in glioma tumors by observing a decrease in hyperpolarized lactate suggested to be a result of changes in tumor perfusion which can be detected between 24 and 96 h following treatment [40, 41]. HP [1-¹³C] pyruvate has also been used to detect early response to temozolomide (TMZ) treatment on human glioblastoma rat models [42]. The study successfully showed for the first time detection of response to TMZ therapy 1 day after TMZ administration. The continued reports on using HP pyruvate as an imaging probe for assessing treatment response indicate the potential of the compound to become a standard in the field. Moreover, these studies demonstrate the usefulness of HP [1-¹³C] pyruvate as a tool for early assessment of therapy response, which can improve treatment selection at the clinical level. Pyruvate has also been validated as a metabolic imaging marker for use in humans [43]. In a two-phase study, patients with biopsy-proven prostate cancer of various histological grades were injected with HP [1-¹³C] pyruvate. In the first phase, a maximum dose level was determined to establish pharmacological safety of the HP probe while still injecting enough pyruvate to allow visualization. This addressed one of the major challenges faced in translating HP MRI to clinical applications: the potential toxicity of compounds that must be injected into patients. In the second phase, metabolism of pyruvate was visualized in real time and differences in the ratio of [1-¹³C] lactate to [1-¹³C] pyruvate between identified cancerous regions and normal tissue regions were successfully observed (Fig. 1a–d). [1-¹³C] lactate in regions that did not contain tumor was not detected, confirming previous biopsy and preclinical studies that demonstrated low flux of [1-¹³C] pyruvate to lactate and low concentrations of lactate in benign prostate tissues [44, 45]. Preliminary results indicated the possibility of detecting previously unobserved cancerous regions by HP [1-¹³C] pyruvate, later confirmed to be Gleason 4+3 cancer by biopsy, though further investigation into the relationship between grade and metabolism in prostate cancer patients is needed. While there are challenges associated with translation to clinical use for HP [1-¹³C] pyruvate, the first in human study demonstrated the feasibility of hyperpolarization technology as a safe diagnostic tool and provides the potential for utilizing this approach in preclinical models with direct translation to the clinic.

Glutamine metabolism

Glutamine is an amino acid that plays an important cellular role as nitrogen donor in the form of an amide group for purine and pyrimidine biosynthesis, leaving a glutamate molecule in the process although glutamine can also be converted to glutamate by glutaminase in a reaction independent of nucleotide biosynthesis. Glutamate is the primary nitrogen donor for the biosynthesis of non-essential amino acids. Transaminases catalyze the transfer of the amine group from glutamate to α-ketoacids to synthesize alanine, aspartate (precursor for asparagine), serine (precursor for glycine and cysteine), ornithine (precursor for arginine), and proline which is derived from the glutamate carbon backbone. Glutamine is considered a non-essential amino acid as it can be recycled from glutamate and ammonia in a reaction catalyzed by glutamine synthetase; however, some cancer cells show increase consumption of glutamine and are unable to grow in the absence of exogenous glutamine [46, 47]. This metabolic characteristic of cells to require exogenous glutamine for growth has been termed “glutamine addiction” and has generated extensive research interest as an indicator of development of cancerous tissues [48]. In particular to the field of HP MRI, the conversion rate of glutamine to glutamate (Fig. 2) was explored in hepatocellular carcinoma (HCC) using a [5-¹³C] glutamine probe (Fig. 2) [49]. Using the ratio between [5-¹³C] glutamine and [5-¹³C] glutamate, it was demonstrated that HCC cells convert glutamine at a higher rate than normal cells supporting the notion of glutamine addiction. One important aspect of this study was the choice of [5-¹³C] glutamine as a probe as opposed to [1-¹³C] glutamine, which has a longer T₁ (16.1 vs. 24.6 s at 9.4 T) [49, 50]. [5-¹³C] glutamine was selected because the chemical shift change obtained from [1-¹³C] in glutamine and glutamate is far too small, which could prevent proper identification and quantification of the peaks. This highlights the importance of understanding not only the target compound to be hyperpolarized but also the metabolic products to be detected and their resulting spectra in MR. This is further emphasized with studies that demonstrate the usefulness of [1-¹³C] glutamine as a source for [1-¹³C] glutamate in order to follow the metabolism of α-ketoglutarate to observe the metabolic state of the TCA cycle in transformed cells [51]. Furthermore, [1-¹³C] α-ketoglutarate has been hyperpolarized and used to visualize other metabolic events involving [1-¹³C] glutamate such as mutations in IDH1 expression in glioma tumors and pathways dependent on hypoxia-inducible factor (HIF) [51–53]. More recently, [5-¹³C] glutamine has been used to visualize the metabolism of liver cancer in vivo and in vitro, as well as the treatment response of prostate cancer cells in vitro [54]. Based on the promise of glutamine as a biomarker for cancer diagnosis and treatment response, extending the spin-lattice relaxation time of the [5-¹³C] glutamine has been researched and successfully accomplished. The facile synthesis of [5-¹³C-4-²H₂] glutamine has been reported, and its study showed that by relying on the effect of deuterium enrichment to lessen dipolar coupling effects, the T₁ of [5-¹³C] glutamine could be increased from approximately 15 to 30 s [55]. Visualization of real-time conversion of glutamine to glutamate in SF188 cells was achieved using this probe, demonstrating the promise of [5-¹³C-4-²H₂] glutamine as a probe for molecular imaging of metabolic events in real time. Further investigation of this probe applied to in vivo preclinical models will lay the foundation for its clinical translational potential in the future.

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Metabolism of [5-¹³C] glutamine to [5-¹³C] glutamate. a Time-dependent spectral data following conversion of [5-¹³C] glutamine to [5-¹³C] glutamate. The signals are from ¹³C-enriched [5-¹³C]glutamate at 181.5 ppm and [5-¹³C]glutamine at 178.5 ppm and from natural abundance ¹³C label in [1-¹³C]glutamate at 175.2 ppm and [1-¹³C]glutamine at 174.7 ppm. b Plot of the ratio of the signal intensities of [5-¹³C]glutamate/[5-¹³C]glutamine showing the ratio in hepatoma cells (shaded circle), cell lysate (square), and control (triangle). These results demonstrated that hepatoma cancer cells convert glutamine to glutamine at a higher rate than normal cells. Adapted with permission from ref. [49]

Dehydroascorbate as a redox sensor

Reactive oxygen species (ROS) like the hydroxyl radical, superoxide, and hydrogen peroxide have been shown to cause DNA damage and can lead to mutations that transform normal cells into cancerous cells [56]. The reduction/oxidation (redox) state, which is dependent on the balance between oxidizing equivalents like ROS and reducing cofactors, can provide insight into the physiological condition of the cell with respect to potential cancer transformations. Furthermore, the presence of ROS in tissue has been implicated to be a factor in developing resistance to radiation therapies [57]. During oxidative stress (i.e., when there is an increase in ROS), redox homeostasis is maintained by the action of antioxidant compounds, such as ascorbate (or vitamin C, VitC), which can scavenge for ROS and reduce the compounds to rid the cells of damaging agents [58]. In this process, ascorbate that is available to cells in high concentrations can be oxidized to dehydroascorbate (DHA) while reducing ROS. DHA can then be transported into the cell where DHA is reduced back to ascorbate resulting in a process of recycling ascorbate and DHA (Fig. 3) [59]. In this sense, the ratio of DHA to ascorbate can be used as a molecular marker to investigate the redox state and thus the physiological state of tissues. Additionally, conversion of DHA to ascorbate can be enzymatically catalyzed in an NADPH-dependent manner or via oxidation of glutathione (GSH) to glutathione sulfide (GSSG); thus, visualization of ascorbate/DHA metabolism offers a method for probing in vivo metabolism of NADPH as well as determination of GSSG to GSH ratio, both of which have been implicated to be indicators of oxidative stress in the cells, particularly for neurodegenerative, cardiovascular, and cancer diseases [60–62]. Hyperpolarized [1-¹³C] DHA was successfully used in murine models to detect increased reducing capacity in prostate cancer with the purpose of developing a non-invasive, early diagnostic tool for improving selection of treatment therapies [62, 63]. DHA demonstrates a relatively long T₁ at clinically relevant field strengths (>50 s at 3 T) and adequate chemical shift separation between it and its metabolic product ascorbate (δ = 3.8 ppm). Increased reduction of HP [1-¹³C] DHA to ascorbate was observed in tumor tissue compared to normal tissue as well as other metabolic organs (Fig. 3). This was additionally demonstrated in lymphoma cells, further supporting the potential for using DHA as a probe in living systems [64]. A following study validated these results, and the correlation between increased DHA reduction and glutathione was established in vivo, thus showing the utility of [1-¹³C] DHA as a molecular imaging probe to detect events that go beyond the direct metabolism of DHA [63]. Notwithstanding the potential of HP DHA as a diagnostic probe, the toxicity of DHA remains to be validated. Earlier studies on mammalian cells showed DHA toxicity starting at 10 mM, while a study carried on rats demonstrated neurological effects of DHA starting at injections of 50 mg/kg [65, 66]. However, as outlined above, successful use of DHA injections in rats and mice for hyperpolarization has been demonstrated without reported side effects on the animals. More research is needed to determine the parameters regarding the toxicity of DHA in larger animal models using pure formulations to assess its potential for clinical trials. Further work in DHA could demonstrate its applicability for the study of ROS and redox changes in model systems.

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Determination of redox state by imaging of HP [1-¹³C] ascorbate (VitC) and [1-¹³C] dehydroascorbate (DHA). Oxidative stress caused by ROS (1.) can be alleviated by oxidation of ascorbate to DHA (2.), and recycling of DHA to ascorbate can occur indirectly with oxidation of glutathione (3.) or directly with oxidation of NADH (4.). The ratio of [ascorbate] to [DHA] has been successfully used in mice models as a biomarker to determine pH in vivo. Adapted with permission from ref. [62]

Other metabolic imaging probes

While the three probes discussed earlier are the most well studied in metabolic events that are characteristic of cancer cells in general, other molecules have been evaluated in their potential to be used as biomarkers. Hyperpolarized bicarbonate (H¹³CO₃⁻) has been successfully used to determine the pH in extracellular matrix of lymphoma tumors in mice, and a correlation between acidic environments and cancer was established [67]. The relaxation times for bicarbonate compounds at 3 T are between 34 and 50 s, which is enough time to detect the rapid conversion of H¹³CO₃⁻ and ¹³CO₂ catalyzed by carbonic anhydrase [23]. The attractive feature of this probe is based on how ubiquitous acidic extracellular environments are to a wide variety of diseases; thus, HP bicarbonate has the potential for clinical translation beyond cancer research, though extensive work will be necessary to generate a preparation which will result in an adequate dose for the clinic [68, 69]. More recently, the potential of α-ketoisocaproate (KIC) as a molecular probe for in vivo detection of branched chain amino acid transaminase (BCAT) has been explored. BCAT catalyzes the conversion of KIC to leucine, and its expression has been suggested to correlate to genetic characterization of certain tumors. In a pilot study, HP α-keto-[1-¹³C]-isocaproate was shown to have a T₁ of 100 s so its metabolism can be sensitively traced for over a minute after injection [70]. In the same study, metabolism of HP [1-¹³C] KIC to [1-¹³C] leucine by BCAT was observed in murine lymphoma tumor tissue but was absent in rat mammary adenocarcinoma with a correlation between BCAT expression and [1-¹³C] leucine signal detection [70]. Additionally, in the same models, [1-¹³C] pyruvate conversion to [1-¹³C] lactate and [1-¹³C] alanine was detected in both types of tumors. These findings show the promise of using [1-¹³C] KIC as a discriminative probe in addition to pyruvate in order to diagnose different types of cancer [71, 72]. Furthermore, the correlation between BCAT expression and [1-¹³C] leucine detection was also shown in rat brain tissue, confirming the usefulness of HP [1-¹³C] KIC in assessing BCAT activity in vivo [73]. Choline is another compound that has been evaluated as a molecular imaging probe since elevated choline and choline-derived metabolites have been correlated by ¹H-MRS imaging to cancer in the brain, breast, colon, cervix, and prostate [74–76]. Despite its potential as a global marker for cancer because of the long T₁ relaxation times that can be achieved with deuterium and ¹⁵N enrichment [77, 78], HP applications of ¹³C enriched choline are limited due to the small change in chemical shifts of choline and choline-derived metabolites as well as its potential toxicity [16, 79, 80]. It has been shown that choline toxicity occurs at doses of 53 mg/kg in mice, although a recent study successfully detected HP ¹³C choline in vivo without adverse effects in rats at doses of 50 mg/kg by using atropine to prevent cholinergic intoxication [81, 82] though metabolic products have been difficult to visualize in vivo. As mentioned earlier, the usefulness of glucose as a probe is limited due to the short relaxation times of all the carbons present in the molecule and although the T₁’s can be increased through deuterium enrichment, the lifetime of the probe remains a hurdle for clinical applications [27, 28]. Thus, fructose (a pentose analog of glucose) has been successfully used as an alternative to probe glycolytic pathways [83] in TRAMP models where differences in HP [2-¹³C] fructose uptake and metabolism was visualized in tumor regions compared to surrounding normal tissues. Like choline, the limiting factor in the usefulness of [2-¹³C] fructose for in vivo studies is in small chemical shifts between the metabolite and its phosphorylated product. Finally, tumor necrosis can be used as a measure of treatment response, particularly early necrosis. HP [1,4-,¹³C] malate has been visualized in lymphoma mice models after injection of HP [1,4-¹³C] fumarate [84]. In normal cells, fumarate has a slow rate of transport into the mitochondria; however, in necrotic cells where the mitochondrial membrane is degraded, fumarase has access to the HP fumarate and its ubiquitous cofactor, water, thus facilitating rapid conversion to malate. Preliminary studies have shown the potential for its use in animal models though further work is required to determine the necessary density of necrotic cells for detection and the timings required for adequate visualization in patients.

Conclusions

The application of hyperpolarized ¹³C imaging has been extensively investigated in preclinical models, and the successful demonstration of HP [1-¹³C] pyruvate in patients with prostate cancer has validated the potential of HP MRI as a safe diagnostic and treatment assessment tool. Application of other probes beyond pyruvate is still in its infancy, particularly because of the need to further study the currently developed models under conditions that are relevant to a clinical setting (i.e., lower magnetic fields) as well as to study the necessary parameters (probe toxicity dose limits, safety limits for rapid injection) to withstand the necessary hurdles to translation. Nevertheless, these vast research findings are promising and indicate an eventual translation to humans. Furthermore, there is a large variety of biologically relevant molecules that have the potential to be hyperpolarized (Fig. 4), and molecular imaging of metabolic events in real time using not only one single probe but a combination of relevant probes could become an invaluable tool in elucidating so far undiscovered metabolic and proteomic interactions that play a role in cancer development and treatment. This gives HP MRI the great potential to revolutionize current molecular imaging technologies.

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Metabolic pathways with compounds that can be used as molecular imaging probes for HP MRI. A wide variety of metabolic pathways have already been visualized or have the potential to be visualized using hyperpolarization technology that can be applied to different pathological states of the cell including cardiovascular disease and a large variety of cancers. 1. Metabolism of C1 (red dots) in pyruvate. Theasterisks on selected compounds represent enrichment of ¹³C in the second pass of pyruvate in TCA cycle. 2. Metabolism of C1 (brown dots) in DHA using a pool of NADPH derived from the pentose phosphate pathway. 3. Metabolism of C1 (blue dots) and C5 (green dots) of glutamine. 4. Metabolism of C1 and C4 (purple dots) of fumarate unrelated to TCA metabolites. 5. Metabolism of extracellular bicarbonate (gray dots). MTC1 monocarboxylate transporter 1, MTC4 monocarboxylate transporter 4,System ASC amino acid transporter, GLUTs glucose transporters, DCT dicarboxylate transporter, DHARdehydroascorbate reductase, GR glutathione reductase, GSH glutathione, GSSG glutathione disulfide,LDH lactate dehydrogenase, ALT alanine transaminase, CA carbonic anhydrase, PC pyruvate carboxylase,PDH pyruvate dehydrogenase, CS citrate synthase, GLS glutaminase, GLDH glutamate dehydrogenase,IDH isocitrate dehydrogenase, OGDC oxoglutarate dehydrogenase complex, SCS succinyl CoA synthetase, SQR succinate dehydrogenase, FH fumarate hydratase, MDH malate dehydrogenase, FUMfumarase. Cofactors have been omitted for brevity

Abbreviations

ALT:   alanine transaminase;   BCAT:  branched chain amino acid transaminase;   DHA:  dehydroascorbate;   DNP:  dynamic nuclear polarization;   EDTA:  ethylenediaminetetraacetic acid;   GSH:  glutathione;   GSSG:   glutathione sulfide;   HCC:  hepatocellular carcinoma;   HIF:  hypoxia-inducible factor;   HP:  hyperpolarized/hyperpolarization;   IDH:  isocitrate dehydrogenase;   KIC:  ketoisocaproate;   LDH:  lactate dehydrogenase;   MR: magnetic resonance;   MRI:  Magnetic resonance imaging;   MRS:  magnetic resonance spectroscopy;   NAD(H):  nicotinamide adenine dinucleotide;   NADP(H):  nicotinamide adenine dinucleotide phosphate;   PET:  positron emission tomography;   ROS:  reactive oxygen species;   SPECT:  single-photon emission computed tomography;   TRAMP:  transgenic adenocarcinoma of mouse prostate

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sjwilliamspa commented on Is the Warburg effect an effect of deregulated space occupancy of methylome?

Is the Warburg effect an effect of deregulated space occupancy of methylome? Larry H. Bernstein and Radoslav Bozov, …

It would be an interesting figure, although I am not sure anyone has been able to measure it, is the spatial distribution of lactate and pyruvate over the tumor as a function of diffusion distance such as a heat map to see if pyruvate and lactate levels have a gradiant over a solid tumor. I am not sure it would but interesting to see where tumor cells, which undergo Warburg type metabolic phenotype actually exist, if it is a function of angiogenesis or a function of the proliferative capacity of cells in situ.

Response by LHB…

Radoslav Bozov has repeatedly referred to the real problem of space/time in the required experimental view that is intractable, as seen by Erwin Schroedinger.  It is confounded by
the restrictions imposed by research, and to an extent also the dilemma of location and velocity.

I think it is to an extent also inherent in the modern revelations of autophagy and apoptosis that were not part of the view in the mid 20th century.  However, the work of B. Chance led to a substantially better understanding of the hydride transfer in the NAD/NADH.  What is overlooked is the important role cited by NO Kaplan of NADPH/NADP vs NADH/NAD associated with synthetic and, alternatively, catabolic processes in the cell. What role the pyridine nucleotide transhydrogenase would play is anyones guess.   In any case the proliferation of malignant cells is dependent on NADPH.  This would limit the NAD/NADH related reactions. The effect in the cytoplasm is PYR –> LAC, with generation of NAD from NADH.  In addition, the type of isoenzyme favored should be consequential.  For instance, the M-type LDH does not form an abortive ternary complex LDH*NAD+*PYR. In addition, Bernstein, Everse and Grisham showed that in cancer there is an aberrant cytoplasmic MDH.

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Mitochondria: Origin from oxygen free environment, role in aerobic glycolysis, metabolic adaptation

Posted in Biological Networks, Gene Regulation and Evolution, Biomarkers & Medical Diagnostics, CANCER BIOLOGY & Innovations in Cancer Therapy, Cardiovascular Pharmacogenomics, Cell Biology, Signaling & Cell Circuits, Chemical Biology and its relations to Metabolic Disease, Computational Biology/Systems and Bioinformatics, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Genome Biology, Glycobiology: Biopharmaceutical Production, Pharmacodynamics and Pharmacokinetics, International Global Work in Pharmaceutical, Metabolomics, Molecular Genetics & Pharmaceutical, Nutrigenomics, Nutrition, Personalized and Precision Medicine & Genomic Research, Pharmacotherapy of Cardiovascular Disease, Population Health Management, Genetics & Pharmaceutical, Population Health Management, Nutrition and Phytochemistry, Scientist: Career considerations, Systemic Inflammatory Response Related Disorders, Technology Transfer: Biotech and Pharmaceutical, tagged Acetyl-CoA, Adenosine triphosphate, anaerobic and aerobic gycolysis, ATP, ATP demands, Cellular respiration, Citric acid cycle, fermentation, gluconeogenesis, hydrophobic interaction, N:S, overflow at branch points, oxidative stress, Pasteur effect, PFK 2, plant protein diet, protein calorie malnutrition, pyruvate dehydrogenase, Saccharomyces cerevisiae, SIRT3, synthetic small DNA ligands, TCA cycle intermediates, Warburg Effect on September 26, 2012| 26 Comments »

 

English: A diagram of cellular respiration including glycolysis, Krebs cycle, citric acid cycle, and the electron transport chain (Photo credit: Wikipedia)

Reporter and Curator: Larry H Bernstein, MD, FACP

Introduction

Mitochondria are essential for life, and are critical for the generation of ATP. Otto Warburg won the Nobel Prize in 1918 for his studies of respiration and he described a situation of impaired respiration in cancer cells causing them to produce lactic acid, like bacteria. This has been termed facultative anaerobic glycolysis. The metabolic explanation for mitochondrial respiration had to await the Nobel discoveries of the Krebs cycle and high energy ~P in acetyl CoA by Fritz Lippman. The Krebs cycle generates 16 ATPs I respiration compared to 2 ATPs through glycolysis. The discovery of the genetic code with the “Watson-Crick” model and the identification of DNA polymerase opened a window for contuing discovery leading to the human genome project at 20th century end that has now been followed by “ENCODE” in the 21st century. This review opens a rediscovery of the metabolic function of mitochondria and adaptive functions with respect to cancer and other diseases.

Function in aerobic and anaerobic metabolism

Two-carbon compounds – the TCA, the pentose phosphate pathway, together with gluconeogenesis and the glyoxylate cycle are essential for the provision of anabolic precursors. Yeast environmental diversity mostly leads to a vast metabolic complexity driven by carbon and the energy available in environmental habitats. This resulted in much early research on analysis of yeast metabolism associated with glucose catabolism in Saccharomyces cerevisiae, under both aerobic and anaerobic environments. Yeasts may be physiologically classified with respect to the type of energy-generating process involved in sugar metabolism, namely non-, facultative- or obligate fermentative. The nonfermentative yeasts have exclusively a respiratory metabolism and are not capable of alcoholic fermentation from glucose, while the obligate-fermentative yeasts – “natural respiratory mutants” – are only capable of metabolizing glucose through alcoholic fermentation. Most of the yeasts identified are facultative-fermentative ones, and depending on the growth conditions, the type and concentration of sugars and/or oxygen availability, may display either a fully respiratory or a fermentative metabolism or even both in a mixed respiratory-fermentative metabolism (e.g., S. cerevisiae). The sugar composition of the media and oxygen availability are the two main environmental conditions that have a strong impact on yeast metabolic physiology, and three frequently observed effects associated with the type of energy-generating processes involved in sugar metabolism and/or oxygen availability are Pasteur, Crabtree and Custer. In modern terms the Pasteur effect refers to an activation of anaerobic glycolysis in order to meet cellular ATP demands owing to the lower efficiency of ATP production by fermentation compared with respiration. In 1861 Pasteur observed that S. cerevisiae consume much more glucose in the absence of oxygen than in its presence. S. cerevisiae only shows a Pasteur at low growth rates and at resting-cell conditions, where a high contribution of respiration to sugar catabolism occurs owing to the loss of fermentative capacity. The Crabtree effect is defined as the occurrence of alcoholic fermentation under aerobic conditions, explained by a theory involving “limited respiratory capacities” in the branching point of pyruvate metabolism. The Custer effect is known as the inhibition of alcoholic fermentation by the absence of oxygen. It is thought that the Custer effect is caused by reductive stress.

Glycolysis

Once inside the cell, glucose is phosphorylated by kinases to glucose 6-phosphate and then isomerized to fructose 6-phosphate, by phosphoglucose isomerase. The next enzyme is phospho-fructokinase, which is subject to regulation by several metabolites, and further phosphorylates fructose 6-phosphate to fructose 1,6-bisphosphate. These steps of glycolysis require energy in the form of ATP. Glycolysis leads to pyruvate formation associated with a net production of energy and reducing equivalents. Approximately 50% of glucose 6-phosphate is metabolized via glycolysis and 30% via the pentose phosphate pathway in Crabtree negative yeasts. However, about 90% of the carbon going through the pentose phosphate pathway reentered glycolysis at the level of fructose 6-phosphate or glyceraldehyde 3-phosphate. The pentose phosphate pathway in Crabtree positive yeasts (S. cerevisiae) is predominantly used for NADPH production but not for biomass production or catabolic reactions.
Pyruvate branch point. At the pyruvate (the end product of glycolysis) branching point, pyruvate can follow three different metabolic fates depending on the yeast species and the environmental conditions. On the other hand, the carbon flux may be distributed between the respiratory and fermentative pathways. Pyruvate might be directly converted to acetyl–cofactor A (CoA) by the mitochondrial multienzyme complex pyruvate dehydrogenase (PDH) after its transport into the mitochondria by the mitochondrial pyruvate carrier. Alternatively, pyruvate can also be converted to acetyl–CoA in the cytosol via acetaldehyde and to acetate by the so-called PDH-bypass pathway. Compared with cytosolic pyruvate decarboxylase, the mitochondrial PDH complex has a higher affinity for pyruvate and therefore most of the pyruvate will flow through the PDH complex at low glycolytic rates. However, at increasing glucose concentrations, the glycolytic rate will increase and more pyruvate is formed, saturating the PDH bypass and shifting the carbon flux through ethanol production. In the yeast S. cerevisiae, the external glucose level controls the switch between respiration and fermentation.

Rodrigues F, Ludovico P and Leão C. Sugar Metabolism in Yeasts: an Overview of Aerobic and Anaerobic Glucose Catabolism. In Molecular and Structural Biology. Chapter 6. qxd 07/23/05 P117
Eriksson P, Andre L, Ansell R, Blomberg A, Adler L (1995) Cloning and characterization of GPD2, a second gene encoding sn-glycerol 3-phosphate dehydrogenase (NAD+) in Saccharomyces cerevisiae, and its comparison with GPD1. Mol Microbiol 17:95–107.
Flikweert MT, van der Zanden L, Janssen WM, Steensma HY, van Dijken JP, Pronk JT (1996)Pyruvate decarboxylase: an indispensable enzyme for growth of Saccharomyces cerevisiae on glucose. Yeast 12:247–257.

Biogenesis of mitochondrial structures from aerobically grown S. cerevisiae

Under aerobic conditions S. cerevisiae forms mitochondria which are classical in their properties,
but the number, morphology, and enzyme activity of these mitochondria are also affected by catabolite repression, but it cannot respire under anaerobic conditions and lacks cytochromes. These structures were isolated from anaerobically grown yeast cells and contain malate and succinate dehydrogenases, ATPase, and DNA characteristic of yeast mitochondria. These lipid-complete structures consist predominantly of double-membrane vesicles enclosing a dense matrix which contains a folded inner membrane system bordering electron-transparent regions similar to the cristae of mitochondria.

• The morphology of the structures is critically dependent on their lipid composition
• Their unsaturated fatty acid content is similar to that of mitochondria from aerobically grown cells
• The structures from cells grown without lipid supplements have simpler morphology – a dense granular matrix surrounded by a double membrane but have no obvious folded inner membrane system within the matrix
• The lipid-depleted structures are only isolated in intact form from protoplasts
• The synthesis of ergosterol and unsaturated fatty acids is oxygen-dependent and anaerobically grown cells may be depleted of these lipid components
• The cytology of anaerobically grown yeast cells is profoundly affected by both lipid-depletion and catabolite repression
• Lipid-depleted anaerobic cells, membranous mitochondrial profiles were not demonstrable
• The structures from the aerobically and anaerobically grown cells are markedly different in morphology and fatty acid composition, but both contain mitochondrial DNA and a number of mitochondrial enzymes

The phospholipid composition of various strains of Saccharomyces cerevisiae, wild type and petite (cytoplasmic respiratory deficient) yeasts and derived mitochondrial mutants grown under conditions designed to induce variations in the complement of mitochondrial were fractionated into various subcellular fractions and analyzed for cytochrome oxidase (in wild type) and phospholipid composition . 90% or more of the phospholipid, cardiolipin was found in the mitochondrial membranes of wild type and petite yeast . Cardiolipin content differed markedly under various growth conditions .

• Stationary yeast grown in glucose had better developed mitochondria and more cardiolipin than repressed log phase yeast .
• Aerobic yeast contained more cardiolipin than anaerobic yeast .
• Respiration-deficient cytoplasmic mitochondrial mutants, both suppressive and neutral, contained less cardiolipin than corresponding wild types .
• A chromosomal mutant lacking respiratory function had normal cardiolipin content .
• Log phase cells grown in galactose and lactate, which do not readily repress the development of mitochondrial membranes, contained as much cardiolipin as stationary phase cells grown in glucose .
• Cytoplasmic mitochondrial mutants respond to changes in the glucose concentration of the growth medium by variations in their cardiolipin content in the same way as wild type yeast does under similar growth conditions.
• It is of interest that the chromosomal petite, which as far as can be ascertained has qualitatively normal mitochondrial DNA and a normal cardiolipin content when grown under maximally derepressed conditions .

Thus, the genetic defect in this case probably does not diminish the mass of inner mitochondrial membrane under appropriate conditions . This suggests the cardiolipin content of yeast is a good indicator of the state of development of mitochondrial membrane.
Jakovcic S, Getz Gs, Rabinowitz M, Jakob H, Swift H. Cardiolipin Content Of Wild Type and Mutant Yeasts in Relation to Mitochondrial Function and Development. JCB 1971. jcb.rupress.org
Jakovcic S, Haddock J, Getz GS, Rabinowitz M, Swift H. Biochem J. 1971; 121 :341 .
EPHRUSSI, B . 1953 . Nucleocytoplasmic Relations in Microorganisms . Clarendon Press, Oxford.

Mitochondria, hydrogenosomes and mitosomes

Before and after the publication of an unnoticed article in 1905 by Mereschkowsky there were many publications dealing with plant “chimera’s” and cytoplasmic inheritance in plants, which should have favoured the interpretation of plastids as “semi-autonomous” symbiotic entities in the cytoplasm of the eukaryotic plant cell. Twenty years after Mereschkowsky’s plea for an endosymbiotic origin of plastids, Wallin (1925, 1927) postulated the “bacterial nature of mitochondria”. And so it is one of the mysteries of the 20th century that an endosymbiotic origin of plastids had not been generally accepted before the 1970s, primarily because one cannot experience the consequences of mutations in the mitochondrial genome by naked eye.

• Mitochondrial DNA is usually present in multiple copies in one and the same mitochondrion and those in the hundreds to thousands of mitochondria in a single cell are not necessarily identical.
• The random partitioning of the mitochondria in mitosis (and meiosis) frequently results in a more or less biased distribution of the diverent mitochondria in the daughter cells, eventually causing diverent phenotypes in different tissues obscuring the maternal inheritance
• It was not until the 1990s that certain diseases—which had been interpreted as being X-chromosomal with incomplete penetrance—eventually turned out to be

Lastly, the vast majority of mitochondrial proteins are encoded in the nucleus and, consequently, mutations in the corresponding genes exhibit a Mendelian, and not a cytoplasmic, maternal inheritance
In the 1970s and 1980s the unequivocal demonstration of mitochondrial DNA occurred
and mitochondrial mutations at the DNA level provided the final proof for the role of such mutations in a wealth of hereditary diseases in man.

• The genomics era provided the tools to prove the endosymbiont-hypothesis for the origin of the eukaryotic cell

Since DNA does not arise de novo, the genomes of organisms and organelles provide a historical record for the evolution of the eukaryotic cell and its organelles. The DNA sequences of two to three genomes of the eukaryotic cell turned out to be a record of the evolution of the eukaryotic life on earth. The analysis of organelle genomes unequivocally revealed a cyanobacterial origin for plastids and an -proteobacterial origin for mitochondria. Both plastids and mitochondria appear to be monophyletic, i.e. plastids derived from one and the same cyanobacterial ancestor, and mitochondria from one and the same -proteobacterial ancestor.
The evolution of the eukaryotic cell appears to have involved one (in the case of animals) or two (in the case of plants) events that took place 1.5 to 2 billion years ago. However, it appears that symbioses involving one or the other eubacterium arose repeatedly during the billions of years available. For example, photosynthetic algae by phagotrophic eukaryotes, negating the hypothesis of a single eukaryotic event, rather than stringent selection shaping the diversity of present-day life. Recent hypotheses for the origin of the nucleus have postulated that introns, which could be acquired by the uptake of the -proteobacterial endosymbiont, forced the nucleus-cytosol compartmentalization. Lateral gene transfer among eukaryotes is more frequent than was assumed earlier, and “mitochondrial genes” in the nuclear genomes of amitochondrial organisms are not necessarily the consequence of a transient presence of a DNA-containing mitochondrial-like organelle.
To cope with the obvious ubiquity of “mitochondrial” genes and the chimerism of the DNA of present day eukaryotes, the hydrogen hypothesis postulates that an archaeal host took up a eubacterial symbiont that became the ancestor of mitochondria and hydrogenosomes. The hydrogen hypothesis has the potential to explain both the monophyly of the mitochondria, and the existence of “anaerobic” and “aerobic” variants of one and the same original organelle. Based on these observations we have only the terms “mitochondrion”, “hydrogenosome” and “mitosome” to classify the various variants of the mitochondrial family.
Hackstein JHP, Joachim Tjaden J , Huynen M. Mitochondria, hydrogenosomes and mitosomes: products of evolutionary tinkering! Curr Genet (2006) 50:225–245. DOI 10.1007/s00294-006-0088-8.

Lineages

A look at the phylogenetic distribution of characterized anaerobic mitochondria among animal lineages shows that these are not clustered but spread across metazoan phylogeny. The biochemistry and the enzyme equipment used in the facultatively anaerobic mitochondria of metazoans is nearly identical across lineages, strongly indicating a common origin from an archaic metazoan ancestor. The organelles look like hydrogenosomes – anaerobic forms of mitochondria that generate H2 and adenosine triphosphate (ATP) from pyruvateoxidation and which were previously found only in unicellular eukaryotes. The animals harbor structures resembling prokaryotic endosymbionts, reminiscent of the methanogenic endosymbionts found in some hydrogenosome-bearing protists; fluorescence of F420, a typical methanogen cofactor, or lack thereof, will bring more insights as to what these structures are. If we follow the anaerobic lifestyle further back into evolutionary history, beyond the origin of the metazoans, we see that the phylogenetic distribution of eukaryotes with facultative anaerobic mitochondria, eukaryotes with hydrogenosomes and eukaryotes that possess mitosomes (reduced forms of mitochondria with no direct role in ATP synthesis) the picture is similar to that seen for animals. In all six of the major lineages (or supergroups) of eukaryotes that are currently recognized, forms with anaerobic mitochondria have been found. The newest additions to the growing collection of anaerobic mitochondrial metabolisms are the denitrifying foraminiferans. A handful of about a dozen enzymes make the difference between a ‘normal’ O2-respiring mitochondrion found in mammals, and the energy metabolism of eukaryotes with anaerobic mitochondria, hydrogenosomes or mitosomes. Notably, the full complement of those enzymes, once thought to be specific to eukaryotic anaerobes, surprisingly turned up in the green alga Chlamydomonas reinhardtii , which produces O2 in the light, has typical O2-respiring mitochondria but, within about 30 min of exposure to heterotrophic, anoxic and dark conditions, expresses its anaerobic biochemistry to make H2 in the same way as trichomonads, the group in which hydrogenosomes were discovered. Chlamydomonas provides evidence which indicates that the ability to inhabit oxygen-harbouring, as well as anoxic environments, is an ancestral feature of eukaryotes and their mitochondria. The prokaryote inhabitants have existed for well over a billion years, and have reached this new habitat by dispersal, not by adaptive evolution de novo and in situ. Indeed, geochemical evidence has shown that methanogenesis and sulphate reduction, and the niches in which they occur, are truly ancient.
Mentel and Martin. Anaerobic mitochondria: more common all the time. BMC Biology 2010; 8:32. BioMed Central Ltd. http://www.biomedcentral.com/1741-7007/8/32.

Anaerobic mitochondrial enzymes

Mitochondria from the muscle of the parasitic nematode Ascaris lumbricoides var. suum function anaerobically in electron transport-associated phosphorylations under physiological conditions. These helminth organelles have been fractionated into inner and outer membrane, matrix, and inter-membrane space fractions. The distributions of enzyme systems were determined and compared with corresponding distributions reported in mammalian mitochondria. Succinate and pyruvate dehydrogenases as well as NADH oxidase, Mg++-dependent ATPase, adenylate kinase, citrate synthase, and cytochrome c reductases were determined to be distributed as in mammalian mitochondria. In contrast with the mammalian systems, fumarase and NAD-linked “malic” enzyme were isolated primarily from the intermembrane space fraction of the worm mitochondria. These enzymes are required for the anaerobic energy-generating system in Ascaris and would be expected to give rise to NADH in the intermembrane space.
Pyruvate kinase activity is barely detectable in Ascaris muscle. Therefore, rather than giving rise to cytoplasmic pyruvate, CO2 is fixed into phosphoenolpyruvate, resulting in the formation of oxalacetate which, in turn, is reduced by NADH to form malate regenerating glycolytic NAD . Ascaris muscle mitochondria utilize malate anaerobically as their major substrate by means of a dismutation reaction. The “malic” enzyme in the mitochondrion catalyzes theoxidation of malate to form pyruvate, CO2, and NADH. This reaction serves to generate intramitochondrial reducing power in the form of NADH. Concomitantly, fumarase catalyzes thedehydration of an equivalent amount of malate to form fumarate which, in turn, is reduced by an NADH-linked fumarate reductase to succinate. The flavin-linked fumarate reductase reaction results in a site I electron transport-associated phosphorylation of ADP, giving rise to ATP. This identifies a proton translocation system to obtain energy generation.
Rew RS, Saz HJ. Enzyme Localization in the Anaerobic Mitochondria Of Ascaris Lumbricoides. The Journal Of Cell Biology 1974; 63: 125-135. jcb.rupress.org

Mitochondrial redox status

Tumor cells are characterized by accelerated growth usually accompanied by up-regulated pathways that ultimately increase the rate of ATP production. These cells can suffer metabolic reprogramming, resulting in distinct bioenergetic phenotypes, generally enhancing glycolysis channeled to lactate production. These investigators showed metabolic reprogramming by means of inhibitors of histone deacetylase (HDACis), sodium butyrate and trichostatin. This treatment was able to shift energy metabolism by activating mitochondrial systems such as the respiratory chain and oxidative phosphorylation that were largely repressed in the untreated controls.
Amoêdo ND, Rodrigues MF, Pezzuto P, Galina A, et al. Energy Metabolism in H460 Lung Cancer Cells: Effects of Histone Deacetylase Inhibitors. PLoS ONE 2011; 6(7): e22264. doi:10.1371/ journal.pone.0022264
Antioxidant pathways that rely on NADPH are needed for the reduction of glutathione and maintenance of proper redox status. The mitochondrial matrix protein isocitrate dehydrogenase 2 (IDH2) is a major source of NADPH. NAD+-dependent deacetylase SIRT3 is essential for the prevention of age related hearing loss of caloric restricted mice. Oxidative stress resistance by SIRT3 was mediated through IDH2. Inserting SIRT3 Nε-acetyl-lysine into position 413 of IDH2 and has an activity loss by as much as 44-fold. Deacetylation by SIRT3 fully restored maximum IDH2 activity. The ability of SIRT3 to protect cells from oxidative stress was dependent on IDH2, and the deacetylated mimic, IDH2K413R variant was able to protect Sirt3-/- MEFs from oxidative stress through increased reduced glutathione levels. The increased SIRT3 expression protects cells from oxidative stress through IDH2 activation. Together these results uncover a previously unknown mechanism by which SIRT3 regulates IDH2 under dietary restriction. Recent findings demonstrate that IDH2 activities are a major factor in cancer, and as such, these results implicate SIRT3 as a potential regulator of IDH2-dependent functions in cancer cell metabolism.
Wei Yu, Dittenhafer-Reed KE and JM Denu. SIRT3 Deacetylates Isocitrate Dehydrogenase 2 (IDH2) and Regulates Mitochondrial Redox Status. JBC Papers in Press. Published on March 13, 2012 as Manuscript M112.355206. http://www.jbc.org
Computationally designed drug small molecules targeted for metabolic processes: a bridge from the genome to repair of dysmetabolism
New druglike small molecules with possible anticancer applications were computationally designed. The molecules formed stable complexes with antiapoptotic BCL-2, BCL-W, and BFL-1 proteins. These findings are novel because, to the best of the author’s knowledge, molecules that bind all three of these proteins are not known. A drug based on them should be more economical and better tolerated by patients than a combination of drugs, each targeting a single protein. The calculated drug-related properties of the molecules were similar to those found in most commercial drugs. The molecules were designed and evaluated following a simple, yet effective procedure. The procedure can be used efficiently in the early phases of drug discovery to evaluate promising lead compounds in time- and cost-effective ways.
Keywords: small molecule mimetics, antiapoptotic proteins, computational drug design.

Tardigrades

Tardigrades have unique stress-adaptations that allow them to survive extremes of cold, heat, radiation and vacuum. To study this, encoded protein clusters and pathways from an ongoing transcriptome study on the tardigrade Milnesium tardigradum were analyzed using bioinformatics tools and compared to expressed sequence tags (ESTs) from Hypsibius dujardini, revealing major pathways involved in resistance against extreme environmental conditions. ESTs are available on the Tardigrade Workbench along with software and databank updates. Our analysis reveals that RNA stability motifs for M. tardigradum are different from typical motifs known from higher animals. M. tardigradum and H. dujardini protein clusters and conserved domains imply metabolic storage pathways for glycogen, glycolipids and specific secondary metabolism as well as stress response pathways (including heat shock proteins, bmh2, and specific repair pathways). Redox-, DNA-, stress- and protein protection pathways complement specific repair capabilities to achieve the strong robustness of M. tardigradum. These pathways are partly conserved in other animals and their manipulation could boost stress adaptation even in human cells. However, the unique combination of resistance and repair pathways make tardigrades and M. tardigradum in particular so highly stress resistant.
Keywords: RNA, expressed sequence tag, cluster, protein family, adaptation, tardigrada, transcriptome

Epicrisis

This discussion has disparate pieces that are tied together by dysfunctional changes that are

• adaptations from metabolic process in the channeling of energy dependent of mitochondrial enzymes in interaction with three to 6 carbon carbohydrates, high energy phosphate, oxygen and membrane lipid structures, as well as
• proteins rich or poor in sulfur linked with genome specific targets, and semisynthetic modifications, oxidative stress
• leading to a new approach to pharmaceutical targeted drug design.

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• What Causes Aging and How Does Dietary Restriction Increase Life Span? (understandnutrition.com)
• Diaz-Cano SJ. Tumor heterogeneity: mechanisms and bases for a reliable application of molecular marker design. Int J Mol Sci 2012; 13(2):1951-2011 PMID 22408433Bernstein LH. Expanding the Genetic Alphabet and linking the genome to the metabolome.PharmaIntell.wordpress.com. luly 24, 2012.
• Sainani K. Meet the Skeptics: Why Some Doubt Biomedical Models – and What it Takes to Win Them Over.  IMAG Futures meeting.   2009
• Ritusaxena. ENCODE: the key to unlocking the secrets of complex genetic diseases.  PharmaIntell.wordpress.com  2012.
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• Ritusaxena. β Integrin emerges as an important player in mitochondrial dysfunction associated Gastric Cancer. PharmaIntell.wordpress.com 2012

 

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