Vasu D. Appanna

The Tricarboxylic Acid Cycle, an Ancient Metabolic Network with a Novel Twist

The tricarboxylic acid (TCA) cycle is an essential metabolic network in all oxidative organisms and provides precursors for anabolic processes and reducing factors (NADH and FADH2) that drive the generation of energy. Here, we show that this metabolic network is also an integral part of the oxidative defence machinery in living organisms and α-ketoglutarate (KG) is a key participant in the detoxification of reactive oxygen species (ROS). Its utilization as an anti-oxidant can effectively diminish ROS and curtail the formation of NADH, a situation that further impedes the release of ROS via oxidative phosphorylation. Thus, the increased production of KG mediated by NADP-dependent isocitrate dehydrogenase (NADP-ICDH) and its decreased utilization via the TCA cycle confer a unique strategy to modulate the cellular redox environment. Activities of α-ketoglutarate dehydrogenase (KGDH), NAD-dependent isocitrate dehydrogenase (NAD-ICDH), and succinate dehydrogenase (SDH) were sharply diminished in the cellular systems exposed to conditions conducive to oxidative stress. These findings uncover an intricate link between TCA cycle and ROS homeostasis and may help explain the ineffective TCA cycle that characterizes various pathological conditions and ageing.

Oxidative Stress Evokes a Metabolic Adaptation That Favors Increased NADPH Synthesis and Decreased NADH Production in Pseudomonas fluorescens

The fate of all aerobic organisms is dependent on the varying intracellular concentrations of NADH and NADPH. The former is the primary ingredient that fuels ATP production via oxidative phosphorylation, while the latter helps maintain the reductive environment necessary for this process and other cellular activities. In this study we demonstrate a metabolic network promoting NADPH production and limiting NADH synthesis as a consequence of an oxidative insult. The activity and expression of glucose-6-phosphate dehydrogenase, malic enzyme, and NADP(+)-isocitrate dehydrogenase, the main generators of NADPH, were markedly increased during oxidative challenge. On the other hand, numerous tricarboxylic acid cycle enzymes that supply the bulk of intracellular NADH were significantly downregulated. These metabolic pathways were further modulated by NAD(+) kinase (NADK) and NADP(+) phosphatase (NADPase), enzymes known to regulate the levels of NAD(+) and NADP(+). While in menadione-challenged cells, the former enzyme was upregulated, the phosphatase activity was markedly increased in control cells. Thus, NADK and NADPase play a pivotal role in controlling the cross talk between metabolic networks that produce NADH and NADPH and are integral components of the mechanism involved in fending off oxidative stress.

A Novel Strategy Involved Anti-Oxidative Defense: The Conversion of NADH into NADPH by a Metabolic Network

The reduced nicotinamide adenine dinucleotide phosphate (NADPH) is pivotal to the cellular anti-oxidative defence strategies in most organisms. Although its production mediated by different enzyme systems has been relatively well-studied, metabolic networks dedicated to the biogenesis of NADPH have not been fully characterized. In this report, a metabolic pathway that promotes the conversion of reduced nicotinamide adenine dinucleotide (NADH), a pro-oxidant into NADPH has been uncovered in Pseudomonas fluorescens exposed to oxidative stress. Enzymes such as pyruvate carboxylase (PC), malic enzyme (ME), malate dehydrogenase (MDH), malate synthase (MS), and isocitrate lyase (ICL) that are involved in disparate metabolic modules, converged to create a metabolic network aimed at the transformation of NADH into NADPH. The downregulation of phosphoenol carboxykinase (PEPCK) and the upregulation of pyruvate kinase (PK) ensured that this metabolic cycle fixed NADH into NADPH to combat the oxidative stress triggered by the menadione insult. This is the first demonstration of a metabolic network invoked to generate NADPH from NADH, a process that may be very effective in combating oxidative stress as the increase of an anti-oxidant is coupled to the decrease of a pro-oxidant.

A novel role for calcite in calcium homeostasis

Calcium carbonate (CaCO3) minerals are known to be deposited in a wide array of different organisms, ranging from microbes to vertebrates [(1989) On Biomineralization, Oxford University Press, New York]. Calcite, aragonite and vaterite are the major crystalline structural polymorphs of CaCO3 associated with living systems, and participate in a variety of biological functions [(1989) Biomineralization: Chemical and Biochemical Perspectives. VCH Publishers, Weinham, Germany; (1991) Advances in Inorganic Chemistry 36, 137–200]. Here we report on the ability of a soil bacterium to synthesize calcite in a calcium‐stressed environment. The elaboration of this exocellular crystalline residue enables the organism to regulate its calcium content. The attainment of calcium homeostasis via the exocellular deposition of bacterial calcite with unique crystal habits is a novel biological phenomenon.

Mitochondrial Lactate Dehydrogenase Is Involved in Oxidative-Energy Metabolism in Human Astrocytoma Cells (CCF-STTG1)

Lactate has long been regarded as an end product of anaerobic energy production and its fate in cerebral metabolism has not been precisely delineated. In this report, we demonstrate, for the first time, the ability of a human astrocytic cell line (CCF-STTG1) to consume lactate and to generate ATP via oxidative phosphorylation. 13C-NMR and HPLC analyses aided in the identification of tricarboxylic acid (TCA) cyle metabolites and ATP in the astrocytic mitochondria incubated with lactate. Oxamate, an inhibitor of lactate dehydrogenase (LDH), abolished mitochondrial lactate consumption. Electrophoretic and fluorescence microscopic analyses helped localize LDH in the mitochondria. Taken together, this study implicates lactate as an important contributor to ATP metabolism in the brain, a finding that may significantly change our notion of how this important organ manipulates its energy budget.

α-Ketoglutarate Dehydrogenase and Glutamate Dehydrogenase Work in Tandem To Modulate the Antioxidant α-Ketoglutarate during Oxidative Stress in Pseudomonas fluorescens

Alpha-ketoglutarate (KG) is a crucial metabolite in all living organisms, as it participates in a variety of biochemical processes. We have previously shown that this keto acid is an antioxidant and plays a key role in the detoxification of reactive oxygen species (ROS). In an effort to further confirm this intriguing phenomenon, Pseudomonas fluorescens was exposed to menadione-containing media, with various amino acids as the sources of nitrogen. Here, we demonstrate that KG dehydrogenase (KGDH) and NAD-dependent glutamate dehydrogenase (GDH) work in tandem to modulate KG homeostasis. While KGDH was sharply decreased in cells challenged with menadione, GDH was markedly increased in cultures containing arginine (Arg), glutamate (Glu), and proline (Pro). When ammonium (NH(4)) was utilized as the nitrogen source, both KGDH and GDH levels were diminished. These enzymatic profiles were reversed when control cells were incubated in menadione media. (13)C nuclear magnetic resonance and high-performance liquid chromatography studies revealed how KG was utilized to eliminate ROS with the concomitant formation of succinate. The accumulation of KG in the menadione-treated cells was dependent on the redox status of the lipoic acid residue in KGDH. Indeed, the treatment of cellular extracts from the menadione-exposed cells with dithiothreitol, a reducing agent, partially restored the activity of KGDH. Taken together, these data reveal that KG is pivotal to the antioxidative defense strategy of P. fluorescens and also point to the ROS-sensing role for KGDH.

Metabolic networks to combat oxidative stress in Pseudomonas fluorescens

Oxidative stress is an unavoidable peril that aerobic organisms have to confront. Thus, it is not surprising that intricate strategies are deployed in an effort to fend the dangers associated with living in an O2 environment. In the classical models of anti-oxidative defense mechanisms, a variety of stratagems including the reactive oxygen species (ROS) scavenging systems, the NADPH-generating enzymes and the DNA repair machineries are highlighted. However, it is becoming increasingly clear that metabolism may be intimately involved in anti-oxidative defence. Recent data show that metabolic reprogramming plays a pivotal role in the survival of organisms exposed to oxidative stress. Here, we describe how Pseudomonas fluorescens, the metabolically-versatile soil microbe, manipulates its metabolic networks in an effort to counter oxidative stress. An intricate link between metabolism and anti-oxidative defense is presented. P. fluorescens reconfigures its metabolic processes in an effort to satisfy its need for NADPH during oxidative insult. Seemingly, disparate metabolic modules appear to partner together to concomitantly fine-tune the levels of the anti-oxidant NADPH and the pro-oxidant NADH. Central to this shift in the metabolic production of the pyridine nucleotides is the increase in NAD kinase with the concomitant decrease in NADP phosphatase. The tricarboxylic acid cycle is tweaked in an effort to limit the formation of NADH. This metabolic redox-balancing act appears to afford a potent tool against oxidative challenge and may be a more widespread ROS-combating tactic than hitherto recognized.

Aluminum-Induced Mitochondrial Dysfunction Leads to Lipid Accumulation in Human Hepatocytes: A Link to Obesity

Mitochondrial dysfunction is the cause of a variety of pathologies associated with high energy-requiring tissues like the brain and muscles. Here we show that aluminum (Al) perturbs oxidative-ATP production in human hepatocytes (HepG2 cells). This Al-induced mitochondrial dysfunction promotes enhanced lipogenesis and the accumulation of the very low density lipoprotein (VLDL). Al-stressed HepG2 cells secreted more cholesterol, lipids and proteins than control cells. The level of apolipoprotein B-100 (ApoB-100) was markedly increased in the culture medium of the cells exposed to Al. 13C-NMR and HPLC studies revealed a metabolic profile favouring lipid production and lowered ATP synthesis in Al-treated cells. Electrophoretic and immunoblot analyses pointed to increased activities and expression of lipogenic enzymes such as glycerol 3-phosphate dehydrogenase (G3PDH), acetyl CoA carboxylase (ACC) and ATP-citrate lyase (CL) in the hepatocytes exposed to Al, and a sharp diminution of enzymes mediating oxidative phosphorylation. D-Fructose elicited the maximal secretion of VLDL in the Al-challenged cells. These results suggest that the Al-evoked metabolic shift favours the accumulation of lipids at the expense of oxidative energy production in hepatocytes.

Pseudomonas fluorescens orchestrates a fine metabolic-balancing act to counter aluminium toxicity

Aluminium (Al), an environmental toxin, is known to disrupt cellular functions by perturbing iron (Fe) homeostasis. However, Fe is essential for such metabolic processes as the tricarboxylic acid (TCA) cycle and oxidative phosphorylation, the two pivotal networks that mediate ATP production during aerobiosis. To counter the Fe conundrum induced by Al toxicity, Pseudomonas fluorescens utilizes isocitrate lyase and isocitrate dehydrogenase-NADP dependent to metabolize citrate when confronted with an ineffective aconitase provoked by Al stress. By invoking fumarase C, a hydratase devoid of Fe, this microbe is able to generate essential metabolites. To compensate for the severely diminished enzymes like Complex I, Complex II and Complex IV, the upregulation of a H(2)O-generating NADH oxidase enables the metabolism of citrate, the sole carbon source via a modified TCA cycle. The overexpression of succinyl-CoA synthetase affords an effective route to ATP production by substrate-level phosphorylation in the absence of O(2). This fine metabolic balance enables P. fluorescens to survive the dearth of bioavailable Fe triggered by an Al environment, a feature that may have potential applications in bioremediation technologies.

Aluminum-induced defective mitochondrial metabolism perturbs cytoskeletal dynamics in human astrocytoma cells.

Although aluminum (Al), a known environmental toxin, has been implicated in a variety of neurological disorders, the molecular mechanism responsible for these conditions is not fully understood. In this report, we demonstrate the ability of Al to trigger mitochondrial dysfunction and ineffective adenosine triphosphate (ATP) production. This situation severely affected cytoskeletal dynamics. Whereas the control cells had well‐defined structures, the Al‐exposed astrocytoma cells appeared as globular structures. Creatine kinase (CK) and profilin‐2, two critical modulators of cellular morphology, were markedly diminished in the astrocytoma cells treated with Al. Antioxidants such as α‐ketoglutarate and N‐acetylcysteine mitigated the occurrence of the globular‐shaped cells promoted by Al toxicity. Taken together, these data reveal an intricate link between ATP metabolism and astrocytic dysfunction and provide molecular insights into the pathogenesis of Al‐induced neurological diseases.