Activities Of TCA Cycle Enzymes Research Articles

Methylated urolithin A, mitigates cognitive impairment by inhibiting NLRP3 inflammasome and ameliorating mitochondrial dysfunction in aging mice

Effective therapeutic interventions for elderly patients are lacking, despite advances in pharmacotherapy. Methylated urolithin A (mUro A), a modified ellagitannin (ET)-derived metabolite, exhibits anti-inflammatory, antioxidative, and anti-apoptotic effects. Current research has primarily investigated the neuroprotective effects of mUroA in aging mice and explored the underlying mechanisms. Our study used an in vivo aging model induced by d-galactose (D-gal) to show that mUro A notably improved learning and memory, prevented synaptic impairments by enhancing synaptic protein expression and increasing EPSCs, and reduced oxidative damage in aging mice. mUro A alleviated the activation of the NOD-like receptor thermal protein domain-associated protein 3 (NLRP3) inflammasome, leading to reduced glial cell activity and neuroinflammation in both accelerated aging and naturally senescent mouse models. Moreover, mUroA enhanced the activity of TCA cycle enzymes (PDH, CS, and OGDH), decreased 8-OHdG levels, and raised ATP and NAD+ levels within the mitochondria. At the molecular level, mUro A decreased phosphorylated p53 levels and increased the expression of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), thus enhancing mitochondrial function. In conclusion, mUro A alleviates cognitive impairment in aging mice by suppressing neuroinflammation through NLRP3 inflammasome inhibition and restoring mitochondrial function via the p53-PGC-1α pathway. This suggests its potential therapeutic agent for brain aging and aging-related diseases.

Alleviative effects of pinostrobin against cadmium-induced renal toxicity in rats by reducing oxidative stress, apoptosis, inflammation, and mitochondrial dysfunction.

Cadmium (Cd) is a highly toxic heavy metal that can be found everywhere in the environment and can have harmful effects on both human and animal health. Pinostrobin (PSB) is a bioactive natural flavonoid isolated from Boesenbergia rotunda with several pharmacological properties, such as antiinflammatory, anticancer, antioxidant, and antiviral. This investigation was intended to assess the therapeutic potential of PSB against Cd-induced kidney damage in rats. In total, 48 Sprague Dawley rats were divided into four groups: a control, a Cd (5 mg/kg), a Cd + PSB group (5 mg/kg Cd and 10 mg/kg PSB), and a PSB group (10 mg/kg) that received supplementation for 30 days. Exposure to Cd led to a decrease in the activities of catalase (CAT), glutathione reductase (GSR), superoxide dismutase (SOD), and glutathione peroxidase (GSH-PX), whereas levels of reactive oxygen species (ROS) and malondialdehyde (MDA) increased. Cd exposure also caused a substantial increase in urea, kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), and creatinine levels. Moreover, a noticeable decline was noticed in creatinine clearance. Moreover, Cd exposure considerably increased the levels of inflammatory indices, including interleukin-1b (IL-1b), tumor necrosis factor-a (TNF-a), interleukin-6 (IL-6), nuclear factor kappa-B (NF-kB), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) activity. Cd treatment decreased the expression of the antiapoptotic markers (Bcl-2) while increasing the expression of apoptotic markers (Bax and Caspase-3). Furthermore, Cd treatment substantially reduced the TCA cycle enzyme activity, such as alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, and isocitrate dehydrogenase. Moreover, mitochondrial electron transport chain enzymes, succinatedehydrogenase, NADH dehydrogenase, cytochrome c-oxidase, and coenzyme Q-cytochrome reductase activities were also decreased following Cd exposure. PSB administration substantially reduced the mitochondrial membrane potential while inducing significant histological damage. However, PSB treatment significantly reduced Cd-mediated renal damage in rats. Thus, the present investigation discovered that PSB has ameliorative potential against Cd-induced renal dysfunction in rats.

Enzyme a Biosynthesis System on Manifestation of Metabolic Stress and Glutathione System in the CNS under Aluminium Neurotoxicosis

Alzheimer’s-like disease was simulated in female adult Wistar CRL(WI) WUBR rats by 6-week intragastric administration of aluminium chloride at a dose of 200 mg/kg body mass. In the presence of developed oxidative stress (OS), we found a decrease in the activities of tricarboxylic acid cycle (TCA cycle) enzymes and an increase in the activities of pentose phosphate pathway (PPP) dehydrogenases as well as a reduction of SH-and SS-groups in proteins (P) along with the increased SH/SS ratio and glutathionylation with simultaneous decreases of glutathione (GSH) and the GSH/GSSG ratio and its redox potential in the brain hemispheres. The glutathione system enzymes were changed multidirectionally, with glutathione reductase remaining stable. Decreased activities of GSH biosynthesis enzymes and cysteine content were noticed. The intragastric administration of the CoA biosynthesis modulators D-panthenol (PL), D-pantethine or D-homopantothenate (HPA) at a dose of 200 mg/kg since the 5th week of the experiment caused either reduction or leveling of OS manifestations in blood plasma, an increase in acetyl cholinesterase, normalization of the activities of TCA cycle and PPP enzymes, P-SH level (not the SH/SS ratio) and a considerable reduction of S-glutathionylation as well as increases in GSH level, the GSH/GSSG ratio and redox potential in the hemispheres. The effect of CoA system modulators was manifested in activation of glutathione transferase, a decrease of glutathione peroxidase and less evident activation of GSH biosynthesis enzymes (PL) although they contributed to the elevation of cysteine content due to the reduced protein S-cysteinylation. The levels and the ratio of CoA/acetyl-CoA (except for PL) were not changed by toxicosis and the OS modulators. The feasibility of non-conenzyme effects was confirmed by the administration of HPA. The phenomenon of redox activity of the CoA biosynthesis modulators with clearly directional effects on the glutathione system and the TCA cycle and PPP enzymes during alleviation of OS and aluminium neurotoxicosis is discussed.

Intracellular Calcium Localization Mediates the Activity of Venetoclax in Targeting Acute Myeloid Leukemia Stem Cells

Venetoclax (BCL2 inhibitor) based regimens have significantly improved outcomes for elderly patients with acute myeloid leukemia (AML). While most patients initially respond to venetoclax-based regimens, upfront resistance or relapse on therapy occurs in many. Therefore, elucidating the mechanistic link between BCL2 inhibition and AML cell death is crucial to finding novel therapeutic strategies to combat venetoclax resistance and to discover predictors of venetoclax response. Previous studies have shown BCL2 has a non-canonical function in modulating oxidative phosphorylation (OXPHOS) activity of AML stem cells (LSCs). However, the mechanistic link between BCL2 and OXPHOS has not yet been established. BCL2 has been shown to regulate intracellular calcium localization by modulating the activity of calcium channels at the ER and mitochondria. Optimal mitochondrial calcium concentrations are crucial for cell metabolism as calcium overload or decrease can inhibit OXPHOS activity. In preliminary studies, we established venetoclax increases mitochondrial calcium levels (Fig. 1A) leading to a subsequent decrease in OXPHOS activity and changes in the activity of key calcium dependent TCA cycle enzymes in primary human AML LSCs. Genetic inhibition of BCL2 in LSCs phenocopied the effects of venetoclax treatment. Based on these data, we hypothesized that BCL2 exerts its effect on LSC metabolism by modulating calcium localization between the ER and mitochondria. Furthermore, we sought to explore whether dysregulation of calcium localization by venetoclax is a mechanistic component of drug activity that leads to suppression of OXPHOS. To determine how BCL2 may be influencing mitochondrial calcium levels, we assessed the relationship between BCL2 and SERCA3, a key ion channel responsible for calcium uptake into the ER. Venetoclax disrupted the physical interaction between BCL2 and SERCA3. Mechanistically, venetoclax perturbed this interaction by decreasing SERCA3 protein levels. These findings were recapitulated by genetic inhibition of BCL2 in LSCs. Functional analysis of SERCA3 via siRNA-mediated knock-down in primary AML specimens demonstrated increased mitochondrial calcium levels, decreased OXPHOS activity and decreased engraftment of immune deficient NSG-S mice. Based on these data, we propose a model in which venetoclax disrupts SERCA function leading to less calcium uptake at the ER and more calcium sequestered into the mitochondria leading to overload, decreased OXPHOS activity and decreased LSC function. Next, we evaluated how calcium pathways were impacted in venetoclax resistant AML patient specimens. Intriguingly, venetoclax did not affect mitochondrial calcium levels (Fig. 1B) or the relationship between BCL2 and SERCA3. Therefore, we tested whether inhibiting the activity of MCU, the channel responsible for calcium uptake into the mitochondrial matrix, would target venetoclax resistant LSCs as it was overexpressed in this population. MCU inhibition led to decreased mitochondrial calcium levels, decreased OXPHOS activity and decreased LSC engraftment. These data indicate that venetoclax-resistant AML cells are no longer subject to changes in calcium localization induced by drug treatment but do retain an overall reliance on calcium regulation for LSC function/survival. Finally, we assessed whether our findings could be leveraged to predict venetoclax sensitivity. Analysis of single cell RNA-seq data from venetoclax sensitive versus resistant AML patient specimens (n=19) revealed multiple calcium pathway gene sets were enriched in venetoclax resistant LSCs. Additionally, levels of certain genes including SERCA3 and MCU were differentially expressed in primitive versus monocytic clusters between sensitive and resistant specimens. Taken together, our data demonstrate an unexpected and critical link between BCL2 and calcium signaling in human LSCs. A surprising aspect of our work is that venetoclax, a BH3 mimetic, acts to modulate the interaction with SERCA3. Lastly, our findings suggest that targeting intracellular calcium dynamics may represent a novel therapeutic strategy in the context of venetoclax resistant AML. In addition, we show that there are significant differences in calcium gene signatures between venetoclax sensitive vs resistant AML specimens that may be useful in predicting patient response to venetoclax based regimens. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Pseudoislet Aggregation of Pancreatic β-Cells Improves Glucose Stimulated Insulin Secretion by Altering Glucose Metabolism and Increasing ATP Production.

Appropriate glucose-stimulated insulin secretion (GSIS) by pancreatic β-cells is an essential component of blood glucose homeostasis. Configuration of β-cells as 3D pseudoislets (PI) improves the GSIS response compared to 2D monolayer (ML) culture. The aim of this study was to determine the underlying mechanisms. MIN6 β-cells were grown as ML or PI for 5 days. Human islets were isolated from patients without diabetes. Function was assessed by GSIS and metabolic capacity using the Seahorse bioanalyser. Connexin 36 was downregulated using inducible shRNA. Culturing MIN6 as PI improved GSIS. MIN6 PI showed higher glucose-stimulated oxygen consumption (OCR) and extracellular acidification (ECAR) rates. Further analysis showed the higher ECAR was, at least in part, a consequence of increased glycolysis. Intact human islets also showed glucose-stimulated increases in both OCR and ECAR rates, although the latter was smaller in magnitude compared to MIN6 PI. The higher rates of glucose-stimulated ATP production in MIN6 PI were consistent with increased enzyme activity of key glycolytic and TCA cycle enzymes. There was no impact of connexin 36 knockdown on GSIS or ATP production. Configuration of β-cells as PI improves GSIS by increasing the metabolic capacity of the cells, allowing higher ATP production in response to glucose.

Comprehensive investigations of key mitochondrial metabolic changes in senescent human fibroblasts.

There is a paucity of detailed data related to the effect of senescence on the mitochondrial antioxidant capacity and redox state of senescent human cells. Activities of TCA cycle enzymes, respiratory chain complexes, hydrogen peroxide (H2O2), superoxide anions (SA), lipid peroxides (LPO), protein carbonyl content (PCC), thioredoxin reductase 2 (TrxR2), superoxide dismutase 2 (SOD2), glutathione peroxidase 1 (GPx1), glutathione reductase (GR), reduced glutathione (GSH), and oxidized glutathione (GSSG), along with levels of nicotinamide cofactors and ATP content were measured in young and senescent human foreskin fibroblasts. Primary and senescent cultures were biochemically identified by monitoring the augmented cellular activities of key glycolytic enzymes including phosphofructokinase, lactate dehydrogenase, and glycogen phosphorylase, and accumulation of H2O2, SA, LPO, PCC, and GSSG. Citrate synthase, aconitase, α-ketoglutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase, and complex I-III, II-III, and IV activities were significantly diminished in P25 and P35 cells compared to P5 cells. This was accompanied by significant accumulation of mitochondrial H2O2, SA, LPO, and PCC, along with increased transcriptional and enzymatic activities of TrxR2, SOD2, GPx1, and GR. Notably, the GSH/GSSG ratio was significantly reduced whereas NAD+/NADH and NADP+/NADPH ratios were significantly elevated. Metabolic exhaustion was also evident in senescent cells underscored by the severely diminished ATP/ADP ratio. Profound oxidative stress may contribute, at least in part, to senescence pointing at a potential protective role of antioxidants in aging-associated disease.

OLEANOLIC ACID, A PROSPECTIVE PROTECTIVE AGENT AGAINST BRAIN ENERGY METABOLISM AND OXIDATIVE DYSFUNCTIONS FOLLOWING HEXAVALENT CHROMIUM EXPOSURE IN MICE

Objective: Effect of oleanolic acid against hexavalent chromium-induced altered brain energy metabolism associated with oxidative stress was evaluated in the present study. Methods: Swiss albino mice were divided into three groups, Control (n=6), chromium-treated (n=6), and oleanolic acid (OA) supplemented (n=6). The chromium treated group was orally administered with K2Cr2O7 for 30 days at a dose of 10 mg/kg b.w/day. OA supplementation was given at a dose of 5 mg/kg bw/day for the past 14 days of chromium treatment. Control group received the vehicle only. After the treatment, whole brain was removed for examining the parameters such as pyruvic acid, free amino nitrogen, tissue protein, TCA cycle enzyme activities, NADH dehydrogenase function, and oxidative stress markers. Results: Significant decrease in cerebral pyruvic acid content associated with suppressed malate dehydrogenase and succinate dehydrogenase activities were observed. The NADH dehydrogenase activity was inhibited owing to enhanced accumulation of chromium in cerebral tissue. Depletion of proteins and increased free amino acid nitrogen were accompanied with inhibited cathepsin, pronase and trypsin activities, and increased transaminase function. In addition, GSH content was decreased along with increased lipid peroxidation, oxidized GSSG content, TG/GSSG ratio, carbonylated protein content, and tissue free hydroxyl radical formation. Superoxide dismutase, catalase, glutathione reductase, and glutathione peroxidase were also inhibited by hexavalent chromium. Oleanolic acid supplementation was found to have significant protective effect against brain metabolic and oxidative dysfunctions. Conclusion: The present study elucidated therapeutic efficacy of oleanolic acid against hexavalent chromium toxicity in brain tissue of mice.

A study on the curative effect of nobiletin on paraquat induced toxicity in rat

This research aimed to evaluate the curative impact of nobiletin (NOB) on paraquat (PQ) induced mitochondrial impairment in rats' hepatic tissues. Twenty-four male rats were divided into control, PQ, PQ + NOB, and NOB treated groups. The alanine aminotransferase levels, aspartate aminotransferase, and alkaline phosphatase were increased in the experimental animals after exposure with PQ. A significant decrease in the level of catalase, glutathione peroxidase, superoxide dismutase, glutathione, thiobarbituric acid reactive substances, and reactive oxygen species were recorded in rats exposed to PQ. The mitochondrial TCA cycle enzyme activities, including isocitrate-dehydrogenase, succinate-dehydrogenase, alpha-ketoglutarate dehydrogenase, and malate-dehydrogenase, were reduced in PQ exposed rats. The activities of mitochondrial enzymes including NADH dehydrogenase, coenzyme Q-cytochrome reductase, succinic-coenzyme Q and cytochrome c-oxidase were significantly decreased after the PQ-exposure. PQ administration also reduced the mitochondrial membrane potential. However, the administration of mitochondria with NOB can decrease the toxic effect of the PQ in isolated mitochondria. NOB treatment potentially reduced the damaging effects of the PQ in the mitochondria isolated from hepatic tissues. Thus, the current study revealed that the NOB could attenuate PQ-induced mitochondrial damage in rats' hepatic tissues.

Impaired mitochondrial functions and energy metabolism in MPTP-induced Parkinson's disease: comparison of mice strains and dose regimens.

Heterogenous diseases such as Parkinson's disease (PD) needs an efficient animal model to enhance understanding of the underlying mechanisms and to develop therapeutics. MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), a neurotoxin, has been widely used to replicate the pathophysiology of PD in rodents, however, the knowledge about its effects on energy metabolism is limited. Moreover, susceptibility to different dose regimens of MPTP also varies among mice strains. Thus, the present study compares the effect of acute and sub-acute MPTP administration on mitochondrial functions in C57BL/6 and BALB/c mice. In addition, activity of enzymes involved in energy metabolism was also studied along with behavioural alterations. The findings show that acute dose of MPTP in C57BL/6 mice had more profound effect on the activity of electron transport chain complexes. Further, the activity of MAO-B was increased following acute and sub-acute MPTP administration in C57BL/6 mice. However, no significant change was observed in BALB/c mice. Acute MPTP treatment resulted in decreased mitochondrial membrane potential along with increasedswelling of mitochondria in C57BL/6 mice. In addition, perturbations were observed in hexokinase, the rate limiting enzyme of glycolysis and pyruvate dehydrogenase, the enzymes that connects glycolysis and TCA cycle. The activity of TCA cycle enzymes; citrate synthase, aconitase, isocitrate dehydrogenase and fumarase were also altered following MPTP intoxication. Furthermore, acute MPTP administration led to drastic reduction in dopamine levels in striatum of C57BL/6 as compared to BALB/c mice. Behavioral tests such as open field, narrow beam walk and footprint analysis revealed severe impairment in locomotor activity in C57BL/6 mice. These results clearly demonstrate that C57BL/6 strain is more vulnerable to MPTP-induced mitochondrial dysfunctions, perturbations in energy metabolism and motor defects as compared to BALB/c strain. Thus, the findings suggest that the dose and strain of mice need to be considered for pre-clinical studies using MPTP-induced model of Parkinson's disease.

Production of bioactive compounds with bactericidal and antioxidant potential by endophytic fungus Alternaria alternata AE1 isolated from Azadirachta indica A. Juss.

For combating multidrug-resistant microorganisms, exploration of natural compounds from plant endophytes increases the chance of finding novel compounds. An efficient bioactive metabolites producing endophytic fungal strain AE1 was isolated from leaves of Azadirachta indica A. Juss. The metabolites were found to be thermostable, non-proteinacious and produced prominent zones of inhibition against numbers of Gram positive and Gram negative bacteria. Based on 28S rDNA (D1/D2) sequence homology the isolate AE1 was identified as Alternaria alternata. Malt extract broth was found effective for the maximum production of bioactive metabolites by the isolate and was subjected for solvent extraction. The Ethyl acetate (EA) fraction of AE1 showed MIC values of 300–400 μg/ml against Gram positive and Gram negative bacteria tested. The cidal mode of action of EA fraction was detected by treating bacterial cultures at mid log phase. Scanning electron microscopic study supported morphological disintegration of bacterial cells. Release of nucleic acid, protein and potassium ions (K+) also suggested lysis of bacterial cells or leakage of cell membrane upon treatment. In addition, reduction of the activity of EMP pathway, TCA cycle and gluconeogenic enzymes in all bacteria suggested the interference of antibacterial principles with central carbohydrate metabolic pathways. Thin layer chromatographic separation followed by GC-MS analysis of EA fraction suggested numbers of antimicrobial compound production by AE1. In addition, DPPH free radical as well as superoxide radical scavenging assay also suggested strong antioxidant potential of AE1 with an IC50 value of 38.0±1.7 μg/ml and 11.38±1.2 μg/ml respectively. On the basis of above facts it can be concluded that the strain AE1 will be a good source of bioactive compounds having medicinal importance.

Exogenous silicon alters organic acid production and enzymatic activity of TCA cycle in two NaCl stressed indica rice cultivars

The activities of TCA cycle enzymes viz., pyruvate dehydrogenase, citrate synthase, isocitrate dehydrogenase, succinate dehydrogenase and malate dehydrogenase as well as levels of different organic acids viz., pyruvic acid, citric acid, succinic acid and malic acid were studied in two rice cultivars viz. cv. Nonabokra and cv. MTU 1010 differing in salt tolerance grown under 25, 50 and 100 mM NaCl salinity levels. A contrasting response to salt stress on enzyme activities of TCA cycle and accumulation of organic acid was observed between two cultivars during twenty-one days period of study. Salinity caused enhanced organic acid production and increase in all five enzyme activities in cv. Nonabokra whereas in cv. MTU 1010 decrease in both organic acid production and enzymes activities were noted. Joint application of exogenous silicon along with NaCl, altered the organic acids levels and activities of enzymes in both cultivars of rice seedlings conferring tolerance against salt induced stress. Rice cv. MTU 1010 showed better response to exogenous silicon on parameters tested compared to cv. Nonabokra.

The Aspergillus nidulans Pyruvate Dehydrogenase Kinases Are Essential To Integrate Carbon Source Metabolism.

The pyruvate dehydrogenase complex (PDH), that converts pyruvate to acetyl-coA, is regulated by pyruvate dehydrogenase kinases (PDHK) and phosphatases (PDHP) that have been shown to be important for morphology, pathogenicity and carbon source utilization in different fungal species. The aim of this study was to investigate the role played by the three PDHKs PkpA, PkpB and PkpC in carbon source utilization in the reference filamentous fungus Aspergillus nidulans, in order to unravel regulatory mechanisms which could prove useful for fungal biotechnological and biomedical applications. PkpA and PkpB were shown to be mitochondrial whereas PkpC localized to the mitochondria in a carbon source-dependent manner. Only PkpA was shown to regulate PDH activity. In the presence of glucose, deletion of pkpA and pkpC resulted in reduced glucose utilization, which affected carbon catabolite repression (CCR) and hydrolytic enzyme secretion, due to de-regulated glycolysis and TCA cycle enzyme activities. Furthermore, PkpC was shown to be required for the correct metabolic utilization of cellulose and acetate. PkpC negatively regulated the activity of the glyoxylate cycle enzyme isocitrate lyase (ICL), required for acetate metabolism. In summary, this study identified PDHKs important for the regulation of central carbon metabolism in the presence of different carbon sources, with effects on the secretion of biotechnologically important enzymes and carbon source-related growth. This work demonstrates how central carbon metabolism can affect a variety of fungal traits and lays a basis for further investigation into these characteristics with potential interest for different applications.

Levels of Thyroid Hormones and Indices of Energy Metabolism in the Cerebral Cortex of Rats with Experimental Alzheimer’s Disease

The study was carried out on 30 mature WAG rats, 15 intact control animals and 15 rats with modeled Alzheimer’s disease (AD) induced by course injections of scopolamine (27 days, 1 mg/kg, i.p. daily). The contents of ATP, glucose, pyruvate, and lactate were measured spectrophotometrically in homogenates of the brain cortex of the animals. The levels of TSH, T3, and T4 in blood serum and those of T3 and T4 in cortex homogenates were estimated using ELISA. The activities of pyruvate dehydrogenase, creatine phosphokinase, and key enzymes of the tricarboxylic acid cycle (TCA) were measured in the mitochondria using a spectrophotometric technique. It was found that the content of T3 in the brain cortex of rats was much lower than in the controls against the background of higher T4 concentrations, which was indicative of the development of local hypothyroidism in the brain. The increased activity of pyruvate dehydrogenase against the background of low activities of TCA cycle enzymes in brain homogenates was observed. The decreased creatine kinase activity and low ATP contents were also found. Local hypothyroidism in the brain cortex is believed to be one of the significant factors inducing the development of strong energy deficiency and reducing the activity of Krebs cycle enzymes. Consequently, this contributes to the death of neurons in experimental AD in rats.

Combined Therapy with Insulin and Vildagliptin causes Cardiac Dysfunction in Diabetic Rats

There is a strong rationale for using incretin-based therapies with insulin. In particular, hypoglycaemia is the limiting factor in glycemic management of insulin-treated patients of type 2 diabetes mellitus. The present study aimed at gaining greater understanding of the progression of cardiovascular degeneration in diabetes mellitus under combined treatment with vildalgliptin and insulin through various analyses on cardiac tissues such as triphenyl tetrazolium chloride dye test. Experimental diabetic rats exhibited significantly lower TCA cycle enzyme and antioxidant defence enzyme activities with a concomitant increase in lipid peroxidation of the heart tissue. Monotherapy with either vildagliptin or insulin prevented these alteration in the oxidative energy metabolism and restored the TCA cycle enzyme activities to near normal in alloxan diabetic rats whereas the combined therapy altered mitochondrial enzyme levels significantly. These findings suggested that the toxic effects of the combinatorial therapy could be attributed to an increased oxidative damage and altered mitochondrial energy production, which might lead to progression of cardiovascular disease.

Abstract 128: Mitochondrial CaMKII Plays Important Physiological and Pathological Roles

Calcium/calmodulin-dependent protein kinase II (CaMKII) is known for its roles in fight-or-flight responses, where it mobilizes ATP-consuming processes to maximize power output by regulating cellular Ca 2+ . We hypothesized that CaMKII may also influence ATP-generating processes in a feedforward mechanism to match ATP consumption with demand. Most cellular ATP is produced by oxidative phosphorylation (OXPHOS) in mitochondria; therefore, we generated transgenic animals whose mitochondrial CaMKII activity is boosted or inhibited by overexpressing mitochondrial targeted CaMKII (mtCaMKII), or a specific CaMKII inhibitor (mtCaMKII N ). We found that mitochondria from mtCaMKII mice have increased activity of pyruvate dehydrogenase and various TCA cycle enzymes. However, these mitochondria failed to provide sufficient ATP for cardiac function, likely due to detrimental remodeling of electron transport chain complex I. As a result, mtCaMKII mice develop a unique dilated cardiomyopathy soon after birth, which could be rescued by overexpressing the mitochondrial but not the myofibrillar form of creatine kinase. mtCaMKII N mice are protected against lipopolysaccharides (LPS)-induced mortality, ischemia/reperfusion injury, adverse cardiac remodeling after myocardial infarction and asthma. Furthermore, Drosophila melanogaster overexpressing mtCaMKII N have longer lifespan under heat stress (29 o C) and are resistant to paraquat, an ROS-inducing agent. However, in the mice, inhibiting mitochondrial CaMKII reduces metabolic fitness required to sustain the isoproterenol-induced fight-or-flight response, leading to elevated utilization of the cardiac glycogen store and increased lactate production. mtCaMKII N mice have reduced spontaneous activity and a higher tendency to gain fat mass when fed a high-fat diet; mtCaMKII N flies have a shorter lifespan in the absence of thermal stress (25 o C). We are using phosphoproteomics, metabolomics and in silico modeling approaches to determine the mechanisms by which mtCaMKII regulates energy metabolism and stress responses. Our results have provided important insights into the physiological and pathological roles of mitochondrial CaMKII.

Modeling of calcium‐induced swelling in spatially divergent populations of cardiac mitochondria

In addition to ATP synthesis, mitochondria are involved in high cytosolic calcium (Ca2+) waves necessary for the regulation of the cardiac excitation‐contraction coupling, a focal point in the determination of cardio‐pathophysiology. Depending on mitochondrial location within subcellular compartments, mitochondrial functions expands from the buffering of cytosolic Ca2+ necessary for cellular signaling to the initiation of cell death via necrosis or apoptosis depending on ATP availability. The exposure of mitochondria to Ca2+ plays a dual role in determining metabolic activity; the sequestering of low Ca2+ concentration ([Ca2+]) accompanied by a minor matrix swelling enhances ATP production through the upregulation in activity of TCA cycle enzymes, ETC complexes and oxidative phosphorylation. However, excessive matrix swelling at high [Ca2+] can induce the opening of non‐selective permeability transition pores (PTP), sustained (irreversible) opening of which allow for the release Ca2+ to propagate from one mitochondria to the other leading to the wide spread of mitochondrial swelling, thus initiating cell death. Studies have suggested that different mitochondrial sub‐population differ in functional properties such as membrane potential, calcium retention capacity (CRC), and ROS production. In this study, we designed a biophysical model that can explain the kinetics of the transition from a reversible to an irreversible Ca2+‐induced swelling state in heterogenic mitochondrial population including subsarcolemmal (SSM) and intermyofibrillar mitochondria (IFM) of cardiac cells.Model developmentIn this project, a heterogenic population of mitochondria were isolated and purified via differential centrifugation. CRC was determined by the application of numerous Ca2+ pulses until Ca2+ release via PTP opening measured through spectrophotometry. Kinetics model was developed utilizing the 4th order Runge‐Kutta approximation method and fitted to experimental mitochondrial swelling data using the least square method. Reversible and irreversible mitochondrial swelling induced by low and high [Ca2+] in isolated cardiac mitochondria have been measured. Reversibility of mitochondrial swelling was determined in the presence of EGTA, a Ca2+‐chelating agent. Based on experimental data, the development of a kinetic model that simulates the swelling dynamics at low and high [Ca2+] in cardiac mitochondria has been created with a correlation coefficient of r >0.95.ConclusionOur model describes transition from reversible to irreversible swelling in different subcellular populations of mitochondria that is important for understanding the role of mitochondrial heterogeneity in PTP‐induced cell death.Support or Funding InformationSupported by NHLBI NIH (SC1HL118669)

Alterations in Cytosolic and Mitochondrial [U-13C]Glucose Metabolism in a Chronic Epilepsy Mouse Model.

Temporal lobe epilepsy is a common form of adult epilepsy and shows high resistance to treatment. Increasing evidence has suggested that metabolic dysfunction contributes to the development of seizures, with previous studies indicating impairments in brain glucose metabolism. Here we aim to elucidate which pathways involved in glucose metabolism are impaired, by tracing the hippocampal metabolism of injected [U-13C]glucose (i.p.) during the chronic stage of the pilocarpine-status epilepticus mouse model of epilepsy. The enrichment of 13C in the intermediates of glycolysis and the TCA cycle were quantified in hippocampal extracts using liquid chromatography–tandem mass spectroscopy, along with the measurement of the activities of enzymes in each pathway. We show that there is reduced incorporation of 13C in the intermediates of glycolysis, with the percentage enrichment of all downstream intermediates being highly correlated with those of glucose 6-phosphate. Furthermore, the activities of all enzymes in this pathway including hexokinase and phosphofructokinase were unaltered, suggesting that glucose uptake is reduced in this model without further impairments in glycolysis itself. The key findings were 33% and 55% losses in the activities of pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase, respectively, along with reduced 13C enrichment in TCA cycle intermediates. This lower 13C enrichment is best explained in part by the reduced enrichment in glycolytic intermediates, whereas the reduction of key TCA cycle enzyme activity indicates that TCA cycling is also impaired in the hippocampal formation. Together, these data suggest that multitarget approaches may be necessary to restore metabolism in the epileptic brain.

Nephroprotective effect of arjunolic acid on cytosolic lipids and mitochondrial enzymes against cyclosporine induced renal complications in rats

Cyclosporine (CsA) has considerably modified the graft survival in solid organ and bone marrow transplantations. Renal toxicity is the major adverse effect of chronic CsA administration. Deterioration of renal function and renal histopathology are the basic elements of the diagnosis.The present study aimed at investigating the protective effects of Arjunolic acid (10mg/kg body weight) against Cyclosporine (25mg/kg body weight) on the activities of TCA cycle enzymes - SDH (succinate dehydrogenase), MDH (malate dehydrogenase) Acotinase, Citrate synthase and examined the activities of respiratory chain complexes I, II, III, and IV. Mitochondrial enzymes of NADH DH, ICDH and ?KG DH activities in cytosol, Lysosomal enzymes of ?-Cathepsin, ?-Glucuronidase, ?-Galactosidase, ?-D-N-Acetyl Glucosaminidase in cytosol, lipids of Cholesterol, Triglycerides (TG), Free fatty acids (FFA), and phospholipids were estimated. Urea, Uric acid, and Creatinine were also estimated in 24hrs urinary sample. Results showed that CsA caused a marked decrease in the level of SDH, MDH, Acotinase, Citrate synthase, Respiratory chain complexes of I, II, III, IV, NADH DH, ICDH, ?KG DH, in the kidney mitochondrial tissue homogenate and also urea, uric acid and creatinine in urine sample. The lysosomal enzymes of ?-Cathepsin, ?-Glucuronidase, ?-Galactosidase, ?-D-N-Acetyl Glucosaminidase in cytosol, lipids of Cholesterol, Triglycerides (TG), Free fatty acids (FFA), and phospholipids were found to be increased in the cytosol of kidney tissue homogenates. But AA successfully prevented the alterations of these effects in the experimental animals. Our study demonstrated that AA could protect the mitochondrial kidney tissues against CsA induced oxidative stress probably by increasing antioxidative defense activities.

Regulation of leukocyte tricarboxylic acid cycle in drug-naïve Bipolar Disorder

Several lines of evidence suggest a role for mitochondrial dysfunction in the pathophysiology of bipolar disorder (BD). The tricarboxylic acid cycle (TCA cycle) is fundamental for mitochondrial energy production and produces substrates used in oxidative phosphorylation by the mitochondrial electron transport chain. The activity of the key TCA cycle enzymes citrate synthase, malate dehydrogenase, and succinate dehydrogenase has never been evaluated in BD. In the present study, these enzymes were assayed from leukocytes of drug-naïve BD patients in a major depressive episode (n=18) and compared to 24 age-matched healthy controls. Drug-naïve BD patients did not show differences in activities of citrate synthase (p=0.79), malate dehydrogenase (p=0.17), and succinate dehydrogenase (p=0.35) compared with healthy controls. No correlation between any TCA cycle enzyme activity and severity of depressive symptoms was observed. Overall, these data suggest that the activities of the TCA cycle enzymes are not altered in major depressive episodes of recent-onset BD, which may support the concept of illness staging and neuroprogression in BD.

Poly-ß-hydroxybutyrate accumulation by Rhodobacter sphaeroides phase variants

The Rand M variants of a purple photosynthetic bacterium Rhodobacter sphaeroides 2R grown on a medium with acetate accumulate poly- β-hydroxybutyrate (PHB). Accumulation of this polymer occurs in the cells grown either anaerobically on the light or aerobically in the dark. On the medium with C/N imbalance (C/N = 4), PHB content during the stationary growth phase under aerobic conditions in the dark was 40 and 70% of the dry biomass of the R and M variant, respectively. The Rba. sphaeroides M variant is therefore a promising culture for large-scale PHB production. Investigation of activity of the TCA cycle enzymes revealed that decreased activity of citrate synthase, the key enzyme for acetate involvement in the reactions of the tricarboxylic acids cycle, was primarily responsible for enhanced PHB synthesis by Rba. sphaeroides. Moreover, the Rba. sphaeroides M variant grown under aerobic conditions in the dark exhibited considerably lower activity of NADH oxidase, which participates in the oxidation of reduced NADH produced in the TCA cycle during acetate oxidation. The combination of these two factors increases the possibilities for acetate assimilation via an alternative mechanism of PHB synthesis.