STUDIES ON ENERGY METABOLISM IN THE HIPPOCAMPUS AFTER ISCHEMIA

The gerbil model of ischemia was used to determine the effect of carotid occlusion on energy metabolites in cellular layers of discrete regions of the hippocampus and dentate gyrus. Levels of glucose, glycogen, ATP and phosphocreatine (PCr) were unchanged after 1 minute of ischemia. However, 3 minutes of ischemia produced a dramatic decrease in net levels of all metabolites. No additional decrease was observed after 15 minutes of ischemia. Re-establishment of the blood flow for 5 minutes after a 15 minute ischemic episode returned all metabolites to pre-ischemia levels. Concentrations of glucose and glycogen were elevated in sham-operated animals as a function of the pentobarbital anesthetic employed. In other studies, elevated GABA levels (produced by inhibiting GABA-transaminase with (gamma)-vinyl-GABA (GVG)) were found to decrease the rate of utilization of the high-energy phosphate metabolites ATP and PCr in the mouse cortex. In addition, glucose and glycogen levels were increased. Thus, tonic inhibition by GABA produced decreased cellular activity. Additional experiments demonstrated the attenuation of ischemia-induced metabolite depletion in cellular layers of regions of the hippocampus, dentate gyrus and cortex after GVG administration. Under ether, 1 minute of bilateral carotid occlusion produced a dramatic decrease in metabolite levels. After GVG treatment, the decrease was blocked completely for glucose, glycogen and ATP, and partially for PCr. Therefore, GABA-transaminase inhibition produced increased levels of GABA which subsequently decreased cellular activity. The protection against ischemia may have been due to (a)decreased metabolic rate; the available energy stores were utilized at a slower rate, and (b)increased levels of energy substrates; additional supplies available to maintain viability. These data suggest that the functional state of neural tissue can determine the response to metabolic stress. ^

Alterations in redox and energy metabolism in Ras-transformed cells: Mechanisms and therapeutic implications

Increasing attention has been given to the connection between metabolism and cancer. Under aerobic conditions, normal cells predominantly use oxidative phosphorylation for ATP generation. In contrast, increase of glycolytic activity has been observed in various tumor cells, which is known as Warburg effect. Cancer cells, compared to normal cells, produce high levels of Reactive Oxygen Species (ROS) and hence are constantly under oxidative stress. Increase of oxidative stress and glycolytic activity in cancer cells represent major biochemical alterations associated with malignant transformation. Despite prevalent upregulation of ROS production and glycolytic activity observed in various cancer cells, underlying mechanisms still remain to be defined. Oncogenic signals including Ras has been linked to regulation of energy metabolism and ROS production. Current study was initiated to investigate the mechanism by which Ras oncogenic signal regulates cellular metabolism and redox status. A doxycycline inducible gene expression system with oncogenic K-ras transfection was constructed to assess the role played by Ras activation in any given studied parameters. Data obtained here reveals that K-ras activation directly caused mitochondrial dysfunction and ROS generation, which appeared to be mechanistically associated with translocation of K-ras to mitochondria and the opening of the mitochondrial permeability transition pore. K-ras induced mitochondrial dysfunction led to upregulation of glycolysis and constitutive activation of ROS-generating NAD(P)H Oxidase (NOX). Increased oxidative stress, upregulation of glycolytic activity, and constitutive activated NOX were also observed in the pancreatic K-ras transformed cancer cells compared to their normal counterparts. Compared to non-transformed cells, the pancreatic K-ras transformed cancer cells with activated NOX exhibited higher sensitivity to capsaicin, a natural compound that appeared to target NOX and cause preferential accumulation of oxidative stress in K-ras transformed cells. Taken together, these findings shed new light on the role played by Ras in the road to cancer in the context of oxidative stress and metabolic alteration. The mechanistic relationship between K-ras oncogenic signals and metabolic alteration in cancer will help to identify potential molecular targets such as NAD(P)H Oxidase and glycolytic pathway for therapeutic intervention of cancer development. ^

A STUDY ON THE FUNCTION OF 14-3-3SIGMA IN REGULATING CANCER ENERGY METABOLISM

Metabolic reprogramming has been shown to be a major cancer hallmark providing tumor cells with significant advantages for survival, proliferation, growth, metastasis and resistance against anti-cancer therapies. Glycolysis, glutaminolysis and mitochondrial biogenesis are among the most essential cancer metabolic alterations because these pathways provide cancer cells with not only energy but also crucial metabolites to support large-scale biosynthesis, rapid proliferation and tumorigenesis. In this study, we find that 14-3-3σ suppresses all these three metabolic processes by promoting the degradation of their main driver, c-Myc. In fact, 14-3-3s significantly enhances c-Myc poly-ubiquitination and subsequent degradation, reduces c-Myc transcriptional activity, and down-regulates c-Myc-induced metabolic target genes expression. Therefore, 14-3-3σ remarkably blocks glycolysis, decreases glutaminolysis and diminishes mitochondrial mass of cancer cells both in vitro and in vivo, thereby severely suppressing cancer bioenergetics and metabolism. As a result, a high level of 14-3-3σ in tumors is strongly associated with increased breast cancer patients’ overall and metastasis-free survival as well as better clinical outcomes. Thus, this study reveals a new role for 14-3-3s as a significant regulator of cancer bioenergetics and a promising target for the development of anti-cancer metabolism therapies.

Energy beverages: content and safety.

Exercise is making a resurgence in many countries, given its benefits for fitness as well as prevention of obesity. This trend has spawned many supplements that purport to aid performance, muscle growth, and recovery. Initially, sports drinks were developed to provide electrolyte and carbohydrate replacement. Subsequently, energy beverages (EBs) containing stimulants and additives have appeared in most gyms and grocery stores and are being used increasingly by "weekend warriors" and those seeking an edge in an endurance event. Long-term exposure to the various components of EBs may result in significant alterations in the cardiovascular system, and the safety of EBs has not been fully established. For this review, we searched the MEDLINE and EMBASE databases from 1976 through May 2010, using the following keywords: energy beverage, energy drink, power drink, exercise, caffeine, red bull, bitter orange, glucose, ginseng, guarana, and taurine. Evidence regarding the effects of EBs is summarized, and practical recommendations are made to help in answering the patient who asks, "Is it safe for me to drink an energy beverage when I exercise?"

Cardiolipin membrane domains in prokaryotes and eukaryotes.

Cardiolipin (CL) plays a key role in dynamic organization of bacterial and mitochondrial membranes. CL forms membrane domains in bacterial cells, and these domains appear to participate in binding and functional regulation of multi-protein complexes involved in diverse cellular functions including cell division, energy metabolism, and membrane transport. Visualization of CL domains in bacterial cells by the fluorescent dye 10-N-nonyl acridine orange is critically reviewed. Possible mechanisms proposed for CL dynamic localization in bacterial cells are discussed. In the mitochondrial membrane CL is involved in organization of multi-subunit oxidative phosphorylation complexes and in their association into higher order supercomplexes. Evidence suggesting a possible role for CL in concert with ATP synthase oligomers in establishing mitochondrial cristae morphology is presented. Hypotheses on CL-dependent dynamic re-organization of the respiratory chain in response to changes in metabolic states and CL dynamic re-localization in mitochondria during the apoptotic response are briefly addressed.

Receptor tyrosine kinase HER2-mediated signaling pathways in metastasis and angiogenesis

Metastasis, the major cause of morbidity and mortality in most cancers, is a highly organized and organ-selective process. The receptor tyrosine kinase HER2 enhances tumor metastasis, however, its role in homing to metastatic organs is poorly understood. The chemokine receptor CXCR4 has recently been shown to mediate the malignant cancer cells to specific organs. Here we show that HER2 enhances the expression of CXCR4 by increasing CXCR4 protein synthesis and inhibiting its degradation. We also observed significant correlation between HER2 and CXCR4 expression in human breast tumor tissues, and an association between CXCR4 expression and a poor overall survival rate in patients with breast cancer. Furthermore, we found that CXCR4 is required for HER2-induced invasion, migration, and adhesion activities in vitro . Finally we established stable transfectants using retroviral RNA interference to inhibit CXCR4 expression and showed that the CXCR4 is required for HER2-mediated lung metastasis in vivo. These results provide a plausible mechanism for HER2-mediated breast tumor metastasis and homing to metastatic organs, and establish a functional link between the receptor tyrosine kinase HER2 and the chemokine receptor CXCR4 signaling pathways. ^ The HER2 overexpression activates PI-3K/Akt pathways and plays an important role in mediating cell survival and tumor development. Hypoxia inducible factors (HIF) are the key regulator for angiogenesis and energy metabolism, and thereby enhance tumor growth and metastasis. HIF activation occurs in the majority of human cancers, including the HER2 overexpressing cancer cells. Previous reports suggested that increased PI-3K/Akt may activate HIF pathway in various tumors, but the detail mechanism is still not completely understood. Here we found that HER2/PI-3K/Akt pathway induces HIF-1α activation, which is independent of hypoxia, but relatively weaker than hypoxic stimulation. This phenomenon was further observed in Akt knock out mouse embryonic fibroblast cells. The PI-3K/Akt pathway does not affect HIF-1α binding with its E3 ligase VHL, but enhances the binding affinity between HIF-1α and β unit. Furthermore, we found Akt phosphorylates HIF-1β at serine 271 and further regulated HIF transcriptional activity. Our findings provided one mechanism that HER2 induce HIF activation via Akt to promote angiogenesis, and this process is independent on hypoxia, which may have implications in the oncogenic activity of HER2 and PI-3K/Akt pathway. ^

Proteomic identification and functional characterization of prohibitin 1 and 2 in T cell activation, expansion, survival and disease

Several immune pathologies are the result of aberrant regulation of T lymphocytes. Pronounced T cell proliferation can result in autoimmunity or hematologic malignancy, whereas loss of T cell activity can manifest as immunodeficiency. Thus, there is a critical need to characterize the signal transduction pathways that mediate T cell activation so that novel and rational strategies to detect and effectively control T cell mediated disease can be achieved. ^ The first objective of this dissertation was to identify and characterize novel T cell regulatory proteins that are differentially expressed upon antigen induced activation. Using a functional proteomics approach, two members of the prohibitin (Phb) family of proteins, Phb1 and Phb2, were determined to be upregulated upon activation of primary human T cells. Furthermore, their regulated expression was dependent upon CD3 and CD28 signaling pathways which synergistically increased their expression. In contrast to previous reports of Phb nuclear localization, both proteins were determined to localize to the mitochondrial inner membrane of human T cells. Additionally, novel Phb phosphorylation sites were identified and characterized using mass spectrometry, phosphospecific antibodies and site directed mutagenesis. ^ Prohibitins have been proposed to play important roles in cancer development however the mechanism of action has not been elucidated. The second objective of this dissertation was to define the functional role of Phbs in T cell activity, survival and disease. Compared to levels in normal human T cells, Phb expression was higher in the human tumor T cell line Kit225 and subcellularly localized to the mitochondrion. Ablation of Phb expression by siRNA treatment of Kit225 cells resulted in disruption of mitochondrial membrane potential and significantly enhanced their sensitivity to cell death, suggesting they serve a protective function in T cells. Furthermore, Q-RT-PCR analysis of human oncology cDNA expression libraries indicated the Phbs may represent hematological cancer biomarkers. Indeed, Phb1 and Phb2 protein levels were 6-10 fold higher in peripheral blood mononuclear cells isolated from malignant lymphoma and multiple myeloma patients compared to healthy individuals. ^ Taken together, Phb1 and Phb2 are novel phosphoproteins upregulated during T cell activation and transformation to function in the maintenance of mitochondrial integrity and perhaps energy metabolism, thus representing previously unrecognized intracellular biomarkers and therapeutic targets for regulating T cell activation and hematologic malignancies. ^

A novel mechanism of 3-bromopyruvate-induced cell death through targeting hexokinase and ubiquitination

Increased dependence on aerobic glycolysis for energy (ATP) supply has been observed in various human cancer cells. It is plausible to exploit this metabolic alteration for therapeutic benefits by inhibiting glycolysis to preferentially abolish cancer energy metabolism and kill the malignant cells. 3-Bromopyruvate has been shown to be a potent inhibitor of glycolysis capable of inducing severe ATP reduction and cell death in various cancer cell lines, especially cancer cells with mitochondrial defects or under hypoxic conditions. However, the detailed mechanisms of this novel anticancer agent still remain unclear. My study demonstrated that 3-Bromopyruvate caused a covalent modification of hexokinase II, a key glycolytic enzyme, and disrupted its association with mitochondria. This led to mitochondrial permeability transition and a substantial release of apoptosis-inducing faction (AIF) prior to cytochrome c release. Dissociation of HK II from mitochondria using a cell permeable specific peptide also induced the release of AIF and cytochrome c, and caused substantial cell death. HK II-targeted peptide did not cause significant change in mitochondria respiration and glycolysis activity, suggesting that dissociation of this molecule from mitochondria alone can also cause cell death, and that this may be a novel mechanism by which 3-Bromopyruvate exerts its potent cytotoxic action, in addition to its inhibition of the enzyme activity. Another significant new discovery was that 3-Bromopyruvate induced rapid reduction of protein ubiquitination in vivo, which occurred within several hours of drug incubation and before ATP reduction and cell death. Further mechanistic studies showed that this was due to the inhibition the ubiquitin activating enzyme E1 and the conjugating enzyme E2. Knocking down ubiquitin protein expression by siRNA did not suppress mitochondria respiration and glycolysis, but caused significant cell death. Taken together, this study demonstrated that induction of HK II dissociation from mitochondria and inhibition of glycolysis are two newly discovered mechanisms that contribute to the potent anticancer activity of 3-Bromopyruvate, and identified this compound as a valuable chemical tool for research in protein ubiquitination. ^

Mitochondrial dysfunction leads to NOX activation: A novel mechanism to maintain high glycolysis in cancer cells

Increased glycolysis and oxidative stress are common features of cancer cells. These metabolic alterations are associated with mitochondrial dysfunction and can be caused by mitochondrial DNA (mtDNA) mutations, oncogenic signals, loss of tumor suppressor, and tumor tissue hypoxia. It is well established that mitochondria play central roles in energy metabolism, maintenance of redox balance, and regulation of apoptosis. However, the biochemical and molecular mechanisms that maintain high glycolysis in cancer cells (the Warburg effect) with mitochondrial dysfunction and oxidative stress remain to be determined. The major goals of this study were to establish a unique experimental system in which the mitochondrial respiratory function can be regulated as desired, and to use this system to investigate the mechanistic link between mitochondrial dysfunction and the Warburg effect along with oxidative stress in cancer cells. To achieve these goals, I have established a tetracycline-inducible system in which a dominant negative form of mitochondrial DNA polymerase y (POLGdn) expression could be regulated by tetracycline; thus controlling mitochondrial respiratory function. Using this cell system, I demonstrated that POLGdn expression resulted in mitochondrial dysfunction through decreasing mtDNA content, depletion of mtDNA encoded mRNA and protein expression. This process was mediated by TFAM proteasome degradation. Mitochondrial dysfunction mediated by POLGdn expression led to a significant increase in cellular glycolysis and oxidative stress. Surprisingly, mitochondrial dysfunction also resulted in increased NAD(P)H oxidase (NOX) enzyme activity, which was shown to be essential for maintaining high glycolysis. Chemical Inhibition of NOX activity by diphenyliodonium (DPI) preferentially impacted the survival of mitochondrial defective cells. The colon cancer HCT116-/- cells that have lost transcriptional regulation of the mitochondrial assembling enzyme SCO2, leading to compromised mitochondrial respiratory function, were found to have increased NOX activity and were highly sensitive to DPI treatment. Ovarian epithelial cells with Ras transformation also exhibited an increase in NOX gene expression and NOX enzyme activity, rendering the cells sensitive to DPI inhibition especially under hypoxic condition. These data together suggest that NOX plays a novel role in maintaining high glycolysis in cancer cells with mitochondrial defects, and that NOX may be a potential target for cancer therapy. ^

Uptake And Metabolism Of 5’-AMP In The Erythrocyte Play Key Roles In The 5’-AMP Induced Model Of Deep Hypometabolism

UPTAKE AND METABOLISM OF 5’-AMP IN THE ERYTHROCYTE PLAY KEY ROLES IN THE 5’-AMP INDUCED MODEL OF DEEP HYPOMETABOLISM Publication No. ________ Isadora Susan Daniels, B.A. Supervisory Professor: Cheng Chi Lee, Ph.D. Mechanisms that initiate and control the natural hypometabolic states of mammals are poorly understood. The laboratory developed a model of deep hypometabolism (DH) initiated by uptake of 5’-adenosine monophosphate (5’-AMP) into erythrocytes. Mice enter DH when given a high dose of 5’-AMP and the body cools readily. Influx of 5’-AMP appears to inhibit thermoregulatory control. In a 15°C environment, mice injected with 5’-AMP (0.5 mg/gw) enter a Phase I response in which oxygen consumption (VO2) drops rapidly to 1/3rd of euthermic levels. The Phase I response appears independent of body temperature (Tb). This is followed by gradual body temperature decline that correlates with VO2 decline, called Phase II response. Within 90 minutes, mouse Tb approaches 15°C, and VO2 is 1/10th of normal. Mice can remain several hours in this state, before gradually and safely recovering. The DH state translates to other mammalian species. Our studies show uptake and metabolism of 5’-AMP in erythrocytes causes biochemical changes that initiate DH. Increased AMP shifts the adenylate equilibrium toward ADP formation, consequently decreasing intracellular ATP. In turn, glycolysis slows, indicated by increased glucose and decreased lactate. 2,3-bisphosphoglycerate levels rise, allosterically reducing oxygen affinity for hemoglobin, and deoxyhemoglobin rises. Less oxygen transport to tissues likely triggers the DH model. The major intracellular pathway for AMP catabolism is catalyzed by AMP deaminase (AMPD). Multiple AMPD isozymes are expressed in various tissues, but erythrocytes only have AMPD3. Mice lacking AMPD3 were created to study control of the DH model, specifically in erythrocytes. Telemetric measurements demonstrate lower Tb and difficulty maintaining Tb under moderate metabolic stress. A more dramatic response to lower dose of 5’-AMP suggests AMPD activity in the erythrocyte plays an important role in control of the DH model. Analysis of adenylates in erythrocyte lysate shows 3-fold higher levels of ATP and ADP but similar AMP levels to wild-type. Taken together, results indicate alterations in energy status of erythrocytes can induce a hypometabolic state. AMPD3 control of AMP catabolism is important in controlling the DH model. Genetically reducing AMP catabolism in erythrocytes causes a phenotype of lower Tb and compromised ability to maintain temperature homeostasis.

XYLITOL METABOLISM IN RAT LIVER AND HEPATOCELLULAR CARCINOMAS

The significance of nutritional factors in cancer research has been strongly emphasized. Such research is concerned not only with epidemiological effects relative to dietary factors on the causation of cancer, but with nutritional effects as an energy source on the prevention of cancer. Many studies speculate that the energy flow between tumor and host can be regulated by dietary intake. However, little knowledge on the comparison of the specific nutritional and energy requirements of different cells and tissues is available. Most popular and essential energy sources for the body are the carbohydrates. Among them, xylitol is known as efficient an energy source as glucose. In carbohydrate metabolism, glycolysis is one of the major energy producing pathways. However, recently the existence of an alternate catabolic pathway in mammals for carbohydrate besides glycolysis, i.e. bypass through triosephosphates to lactate via methylglyoxal has been suggested. This bypass was implicated to regulate glycolysis and also be responsible for the fluctuation in the levels of a regulator of cell growth. Methylglyoxal itself is known as a cancerostatic agent. The alterations of biochemical parameters in xylitol metabolism in animals indicated that xylitol may be metabolized through a methylglyoxal pathway.^ To elucidate the biological effect of xylitol as an energy source and the biological effect of its metabolites as a cancerostatis agent, the mode and extent of metabolism must be understood in tumor-bearing animals. Differential utilization of xylitol and glucose, if any, between tumor and host in such animals may exert tissue selective effects on both in terms of methylglyoxal formation and energy provision. The aim of this work was to assess the extent to which the differential utilization of xylitol might be used to generate different metabolic pathways in tumor and host, and to consider a role of nutrition in cancer.^ The results disclose that the existence of a pathway for biological methylglyoxal formation in normal rat liver has been confirmed in single cell suspension; the metabolic significance of the methylglyoxal pathway in the metabolism of glucose and xylitol has been evaluated quantitatively in normal rat liver and the differential metabolism of glucose and xylitol through overall catabolic pathways of carbohydrates has been studied in normal hepatic cells, AS-30D hepatoma and other several hepatoma lines. ^