Berberine targets the electron transport chain complex I and reveals the landscape of OXPHOS dependency in acute myeloid leukemia with IDH1 mutation / 中国天然药物

Metabolic reprogramming, a newly recognized trait of tumor biology, is an intensively studied prospect for oncology medicines. For numerous tumors and cancer cell subpopulations, oxidative phosphorylation (OXPHOS) is essential for their biosynthetic and bioenergetic functions. Cancer cells with mutations in isocitrate dehydrogenase 1 (IDH1) exhibit differentiation arrest, epigenetic and transcriptional reprogramming, and sensitivity to mitochondrial OXPHOS inhibitors. In this study, we report that berberine, which is widely used in China to treat intestinal infections, acted solely at the mitochondrial electron transport chain (ETC) complex I, and that its association with IDH1 mutant inhibitor (IDH1mi) AG-120 decreased mitochondrial activity and enhanced antileukemic effect in vitro andin vivo. Our study gives a scientific rationale for the therapy of IDH1 mutant acute myeloid leukemia (AML) patients using combinatory mitochondrial targeted medicines, particularly those who are resistant to or relapsing from IDH1mi.

The Structure and Assembly Pathway of MitochondrialRespiratory Chain Complex I / 中国生物化学与分子生物学报

Mitochondria, originated from the last eukaryotic common ancestor by endosymbiosis, are semi-autonomous double-membrane organelles. The oxidative phosphorylation system consists of five complexes that are coordinately encoded by the nuclear and mitochondrial genomes, and establishes the electron transport chain in the mitochondrial inner membrane, concurrently generating ATP using the proton gradient. The proton-pumping Complex I (NADH:ubiquinone oxidoreductase) is the first, large stand most complicated enzyme required in this process. Complex I is an L-shaped multimeric enzyme harbouring over 40 subunits, one FMN molecule and eight Fe-S clusters. In recent years, biochemical, genetic, proteomic and crysta-structure analyses of Complex I in several model systems have provided valuable insights into its function and biogenesis. This review summarizes our current understanding of Complex I structural modules, assembly pathway and factors, discusses their similarities and distinctions between animals and plants, as well as the evolution of Complex I. And we proposes some potentially important but yet unsolved questions of Complex I. Therefore, this review provides a relevant reference for further comprehensively deepening the function and evolution of mitochondrial Complex I.

Complejo I, H2O2 y NO mitocondriales como señales prodrómicas de la disfunción cardíaca en diabetes tipo 1 / Mitochondrial Complex I, H2O2 and NO as Prodromal Signals of Cardiac Dysfunction in Type 1 Diabetes

RESUMEN Introducción: Resultados de nuestro laboratorio sugieren que la disfunción mitocondrial en el corazón precede a la falla miocárdica asociada a la hiperglucemia sostenida. Objetivo: Estudiar los eventos tempranos que ocurren en las mitocondrias de corazón en un modelo de diabetes mellitus tipo 1. Materiales y métodos: Ratas Wistar macho fueron inyectadas con estreptozotocina (STZ; 60 mg/kg, ip) y sacrificadas 10 o 14 días posinyección. Se obtuvo la fracción mitocondrial de corazón. Resultados: El consumo de O2 en estado 3 en presencia de malato-glutamato (21%) o succinato (16%) y las actividades de los complejos I-III (27%), II-III (24%) y IV (22%) fueron menores en los animales diabéticos a los 14 días posinyección. Cuando los animales se sacrificaron al día 10, solo el consumo de O2 en estado 3 en presencia de sustratos del complejo I (23%) y su control respiratorio (30%) fueron menores en las ratas inyectadas con STZ, de acuerdo con una reducción en la actividad del complejo I-III (17%). Estos cambios se acompañaron de un aumento en las velocidades de producción de H2O2 (117%), NO (30%) y ONOO- (∼225%), en la expresión de mtNOS (29%) y en la [O2 -]ss (∼150%) y [NO]ss (∼30%), junto con una disminución de la actividad de la Mn-SOD (15%) y la [GSSG+GSH]mitocondrial (28%), sin cambios en la expresión de PGC-1α. Conclusión: La disfunción del complejo I y el aumento en la generación de H2O2, NO y ONOO- pueden considerarse señales subcelulares prodrómicas del deterioro de la función mitocondrial que precede a la disfunción cardíaca en la diabetes.

ABSTRACT Background: Previous results from our laboratory suggest that heart mitochondrial dysfunction precedes myocardial failure associated with sustained hyperglycemia. Purpose: The aim of this study was to analyze the early events that take place in heart mitochondria in a type 1 diabetes mellitus (DM) model. Methods: Male Wistar rats were injected with streptozotocin (STZ; 60 mg/kg, ip.) to induce DM. They were euthanized 10 or 14 days later and the heart mitochondrial fraction was obtained. Results: State 3 O2 consumption in the presence of malate-glutamate (21%) or succinate (16%), and complex I-III (27%), II-III (24%) and IV (22%) activities were lower in diabetic animals 14 days after STZ injection. When animals were euthanized at day 10, only state 3 O2 consumption sustained by complex I substrates (23%) and its corresponding respiratory control (30%) were lower in rats injected with STZ, in agreement with reduced complex I-III activity (17%). These changes were accompanied by increased H2O2 (117%), NO (30%) and ONOO- (~225%) production rates, mtNOS expression (29%) and O2 - (~150%) and NO (~30%) steady-state concentrations, together with a decrease in Mn-SOD activity (15%) and mitochondrial [GSSG+GSH] (28%), without changes in PGC-1α expression. Conclusion: Complex I dysfunction and increased H2O2, NO and ONOO- production rates can be considered subcellular prodromal signals of the mitochondrial damage that precedes myocardial dysfunction in diabetes.

Mechanism of uptake and retention of 18F-MyoZone in cardiomyocytes: A novel PET myocardial perfusion imaging agent / 解放军医学杂志

Objective To investigate the mechanism of uptake and retention of a novel PET myocardial perfusion imaging agent 18F-MyoZone in cardiomyocytes. Methods 1) Mechanism of inhibition of mitochondrial respiratory chain enzyme activity: With mitochondrial respiratory chain enzyme complex I (MC-I) activity assay kit, a sequence of 19F-MyoZone solution (start at 15 μmol/L, 3 times dilution, 12 points) was interacted with MC-I to detect the half inhibition rate (IC50) of 19F-MyoZone inhibiting mitochondrial respiratory chain activity. 2) Autoradiography experiment of 18F-MyoZone combining with MC-I: Myocardial tissue sections of neonatal rat were hatched with normal saline and 4 known MC-I inhibitors [rotenone (4 μmol/L), 19F-Flurpiridaz (4 μmol/L), 19F-MyoZone (4 μmol/L) and pyridaben (4 μmol/L)], and then 18F-MyoZone was added to autoradiography for detecting whether 18F-MyoZone can specifically bind the cardiomyocytes MC-I; and then the myocardial tissue sections of rat were hatched for 30 min with different concentrations of rotenone or 19F-MyoZone solution (0, 20 μmol/L, 2 μmol/L and 200 nmol/L, 20 nmol/L), then the 18F-MyoZone was added and hatched for 30 min again, developing for 10 min with phosphor storage screen after cleaning the slice, and analyzing and calculating the inhibition rate of each inhibitor concentration. 3) Experiment of rotenone inhibitting the uptake of 18F-MyoZone by cardiomyocytes: Neonate rats' primary cardiomyocytes were cultured for 15 min with different concentrations of 19F-MyoZone or equivalent rotenone (start at 10 μmol/L, 3 times dilution, 12 points), then 50 μl of 18F-MyoZone (about 17 kBq) was added and culturing for 30 min. The lysate was then collected, the radioactivity was counted and the IC50 of rotenone was calculated. 4) Outflow experiment of 18F-MyoZone from cardiomyocytes: Cultured neonate rats' primary cardiomyocytes were interacted with 500 μl of 18F-MyoZone (about 37 kBq) for 30 min, then the cell supernatant and lysate were separated to do photon counts at the time points of 0, 10, 20, 30, 60, 90, 120 and 150 min. The ratio of cardiomyocyte outflow rate was then calculated. Results Enzyme activity studies showed that 19F-MyoZone may effectively inhibit the activity of MC-I(IC50=229.9 nmol/L) in a dose dependent manner. MyoZone could specifically bind to MC-I with binding sites in accordance with the inhibitors rotenone, pyridaben and 19F-Flurpiridaz. Experiment of rotenone inhibitting the uptake of 18F-MyoZone by cardiomyocytes showed that 18F-MyoZone could be absorbed by rat's cardiomyocytes, and with the increase of rotenone concentration, the radio uptake of cardiomyocytes decreased gradually with inhibitor IC50 as 7 nmol/L. Outflow experiment showed that rat's cardiomyocytes could uptake 18F-MyoZone and stably detain over time. The outflow rate increased gradually within 0-30 min, and then maintained constantly from 60 min to 150 min. The amount of retention was about 20% of the entire uptake. Conclusions 18F-MyoZone may specifically bind MC-I and detain for a long time in rat's cardiomyocytes. 18F-MyoZone is a valuable myocardial perfusion imaging agent with great research value.

Leigh Syndrome Due to mtDNA Pathogenic Variants

Abstract Leigh syndrome is a devastating neurodegenerative disease, typically manifesting in infancy or early childhood. Hallmarks of the disease are symmetrical lesions in the basal ganglia or brain stem on MRI, and a clinical course with rapid deterioration of cognitive and motor functions. It is genetically heterogeneous, causative mutations have been disclosed in mitochondrial DNA and nuclear genes involved in the process of energy production in the mitochondria .We investigated the whole mitochondrial DNA in three Brazilian patients with LS, based on their clinical and biochemical data, with the aim to identify the disease-causing mutations. In two of the patients, with complex I deficiency, a novel heteroplasmic variant m.4142G>T (p.R279L) in MT-ND1 and a recurrent homoplasmic mutation m.10197G>A (p.A47T) in MT-ND3 were identified. In the remaining patient, with complex IV deficiency, a de novo heteroplasmic variant in MT-CO1 m.6547T>C (p.L215P) was found. The molecular investigation in mitochondrial diseases have shifted their focus from mitochondrial DNA to nuclear DNA, however, mtDNA protein-coding genes are one of the important genetic causes of mitochondrial disorders for Leigh syndrome. This study expands the molecular and clinical spectrum associated with this disease.

Berberine ameliorates lipid accumulation of mouse primary hepatocytes by decreasing fatty acids uptake / 中国药理学通报

Aim To investigate the role of berberine in mouse primary hepatocytes steatosis and whether aden-osine monophosphate-activated protein kinase(AMPK) is essential in this process in order to explore the mech-anism of non-alcoholic fatty liver disease treatment. Methods Different concentrations of oleic acid(OA) were used in mouse primary hepatocytes to determine the appropriate dose inducing steatosis. Subsequently, hepatocytes were treated with berberine and OA at the same time for 24 h serving metformin as positive con-trol. Lactic dehydrogenase (LDH) release test was performed to investigate cell viability. Lipid level was determined by oil red staining and triglyceride assay. Western blot measured the phosphorylation level of AMPK and Acetyl CoA carboxylase. An AMPK inhibi-tor compound C(CC) pre-treated hepatocytes for 1 h followed by berberine 24 h-treatment. The relationship between free fatty acid(FFA) uptake and mitochondri-al inhibition was evaluated by measuring FFA in the supernatant of OA,berberine and rotenone (mitochon-drial complex I inhibitor) group. Results Berberine could significantly reduce primary hepatocytes steatosis induced by oleic acid and stimulate AMPK and ACC phosphorylation at a non-toxic dose. In addition, CC obviously inhibited AMPK activity,but failed to dimin-ish the lipid dysregulation improvement of berberine. Berberine and rotenone intervention reduced OA up-take by 31.2% and 23.6%,respectively. Conclusion Berberine ameliorates hepatocytes lipid accumulation by suppressing fatty acid uptake,which is probably re-sulted from inhibition of mitochondrial respiratory chain complex I,independently of AMPK activation.

Use of Idebenone for the Treatment of Leber's Hereditary Optic Neuropathy

Abstract Leber's hereditary optic neuropathy (LHON) is one of the most frequent mitochondrial disorders. It is caused by mutations in genes of the mitochondrial DNA coding for subunits of the respiratory chain and leads to severe bilateral vision loss, from which spontaneous recovery is infrequent. Retinal ganglion cells show a selective vulnerability to mitochondrial dysfunction in LHON. Idebenone is the first medication approved for LHON. It is a short-chain benzoquinone, which is an analogue of coenzyme Q10, but with distinct properties and mechanisms of action. Idebenone is a potent antioxidant and inhibitor of lipid peroxidation. Importantly, it facilitates electron flux directly to complex III, bypassing the dysfunctional complex I of the mitochondrial respiratory chain, thereby increasing adenosine triphosphate (ATP) production. In the Rescue of Hereditary Optic Disease Outpatient Study (RHODOS) randomized placebo-controlled clinical trial, 85 patients with LHON were enrolled, in the first 5 years after symptom onset, and randomized to either idebenone 900 mg/d for 6 months or placebo. Idebenone was well tolerated, and although the prespecified primary end point (best recovery in visual acuity [VA]) did not reach statistical significance, all secondary end points (change in best VA, change of VA of best eye at baseline, and change of VA in all eyes) showed a trend toward visual recovery in favor of idebenone. An increasing body of evidence shows that idebenone is effective and safe for the treatment of patients with LHON, including a large retrospective open-label study, several case reports and case series, an expanded access program, and ongoing post-authorization clinical studies. Here, we review the literature on idebenone for the treatment of patients with LHON.

DJ-1 regulates the function of mitochondria in Parkinson’s disease / 中国药理学通报

Mitochondrial dysfunction plays an important role in the process of PD, DJ-1 participates in regulating the function of mitochondria,which has an effect on the protection of mitochon-dria. DJ-1 mutations can lead to the decrease of the activity of mitochondrial complex Ⅰ, the decrease of mitochondrial mem-brane potential and then mitochondrial fragmention and mitoph-agy, and then further damage neurons and trigger PD. This re-view presents the role of DJ-1 in regulating the function of the mitochondria in the pathogenesis of Parkinson's disease(PD).

Effect of inhibiting mitochondrial respiratory chain complex I on migration and invasion capacity of colon cancer cell line Caco2 / 第二军医大学学报

Objective To investigate the effect of inhibiting mitochondrial respiratory chain complex I on the migration and invasion capacity of colon cancer cell line Caco2, and to explore the possible molecular mechanism. Methods Human colon cancer cell line Caco2 was treated with 1 μmol/L rotenone in vitro. Then the relative activity of mitochondrial respiratory chain complex I was examined by chromatometry, the capacity of cell migration and invasion was determined by trans well assay, and the reactive oxygen species (ROS) level in cells was determined using flow cytometry. Results The activity of mitochondrial respiratory chain complex I of Caco2 cells treated with 1 μmol/L rotenone was significantly lower than that of the untreated cells(P<0. 01). In addition, Transwell assay showed that the cell migration rate and invasive rate in Caco2 cells treated with rotenone were significantly higher than those in untreated Caco2 cells after 48 h (migrant rate [30. 4±1. 4]% vs [22. 6 ± 1. 4]%, invasive rate [20. 3 ± 1. 0]% vs [15. 2 ± 1. 3] %, P<0. 01). Furthermore, the ROS level in the rotenone treated cells was significantly higher than that in untreated cells ([5. 68 ± 0. 44] Y vs [3. 46 ± 0. 30]%, P<0. 01). Conclusion Our data suggest that inhibiting the activity of mitochondrial respiratory chain complex I may promote cell migration and invasion by increasing ROS production in colon cancer cells.

Amlodipine Abridged Calcium Associated Complex-I Inhibition in 6-OHDA Lesioned Parkinson’s Rat.

Aims: The present study was carried out to evaluate Amlodipine, a L-type calcium channel blocker for alleviating or reducing the neurodegeneration in 6-OHDA lesioned rat models. Place and Duration of Study: Department of Pharmacology, JSS College of Pharmacy, Rocklands, Ooty, India between October 2011 and may 2012. Methodology: Male adult Wistar rats were given with intra-cerebroventricular injection of 6-hydroxydopamine (6-OHDA) into the median forebrain bundle and treated post 48 hours with Amlodipine (10 mg/kg and 20 mg/kg) per oral for 30 days. Motor coordination, striatal dopamine, mid brain calcium and complex-I activity were estimated. Data were statistically analyzed and p<0.05 was considered significant. Results: Amlodipine regained motor coordination, mid-brain dopamine content, and prevented calcium overload and complex I activity at both dose levels when compared with 6-OHDA control group. Alleviation of calcium overload and complex I inhibition with subsequent increase in dopamine level in Parkinson’s rats were observed at the end of treatment period. Conclusion: The experimental study gave light to a new therapeutic intervention of Amlodipine in preventing neuronal morbidity in Parkinson’s disease (PD). So, further neuro-molecular study with Amlodipine in experimental PD is warranted in future.

Integrative therapy using coenzyme Q10 and minocycline in 1-methyl-4-phenylpyridinium-induced hemiparkinson rats / 中国病理生理杂志

AIM:To investigate the integrative treatment of both coenzyme Q 10 ( CoQ10 ) and minocycline in the rats intranigrally intoxicated with 1-methyl-4-phenylpyridinium ( MPP+) .METHODS:The rat model of Parkinson disease ( PD) was established by intranigral microinjection of MPP +.The degree of microglial activation was measured by immuno-fluorescent density of OX-42 ( a microglia marker ) in the substantia nigra ( SN) .The number of viable dopaminergic neurons was determined by counting the tyrosine hydroxylase ( TH) positive neurons in the SN .The behavioral performances were re-vealed with the number of apomorphine-induced rotations , score of forelimb akinesia and vibrissae-elicited forelimb placing a-symmetry.RESULTS:Pretreatment with CoQ10 or intracerebroventricular (icv) posttreatment with minocycline alone pro-vided partial attenuation against MPP +-induced locomotor defects .Integrative therapy provided enhanced beneficial effects , and resulted in a significant attenuation of locomotor disability than any single therapy (all P<0.01).The results of immu-nohistological analysis showed that the TH positive neurons were maximally protected by integrative therapy compared with minocycline group and CoQ 10 group (P<0.01) .CONCLUSION:The integrative therapy of CoQ 10 combined with minocy-cline may offer additional therapeutic benefit to MPP +-induced hemiparkinson rat model .Such neuroprotective strategy of tar-geting different aspect of the neurodegenerative phenotypes may highlight a new therapeutic strategy for future management of PD.

Effect of monotetrahydrofuran annonaceous acetogenins on mitochondrial complex I of rats / 中草药

Objective: To investigate the activity of mono-tetrahydrofuran (THF) annonaceous acetogenins (ACGs) against mitochondrial complex I of rats. Methods: The inhibitory activity of mono-THF ACGs with six different chemical structures against mitochondrial complex I of rats was investigated to clarify the carbon number and substituted hydroxyl number between THF ring and lactone ring as well as the effect of the core configuration in THF ring on mitochondrial complex I of rats. Results: The results show that mono-THF ACGs can inhibit the mitochondrial complex I of rats. With analysis of the results from the structure-activity relationship between antitumoral activity and their chemical structure of mono-THF ACGs, the less the carbon number between the two rings is, the better their inhibitory activities are; The number of substituted hydroxyl groups is not the decisive factor for influencing its activity in mono-THF ACGs. Conclusion: The inhibitory activity of compound's configurations with th/t/er is better than that of the compound's configurations with th/t/th in mono-THF ACGs.

Niacin metabolism and Parkinson's disease

Epidemiological surveys suggest an important role for niacin in the causes of Parkinson's disease, in that niacin deficiency, the nutritional condition that causes pellagra, appears to protect against Parkinson's disease. Absorbed niacin is used in the synthesis of nicotinamide adenine dinucleotide (NAD) in the body, and in the metabolic process NAD releases nicotinamide by poly(ADP-ribosyl)ation, the activation of which has been reported to mediate 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinson's disease. Recently nicotinamide N-methyltransferase (EC2.1.1.1) activity has been discovered in the human brain, and the released nicotinamide may be methylated to 1-methylnicotinamide (MNA), via this enzyme, in the brain. A deficiency in mitochondrial NADH: ubiquinone oxidoreductase (complex 1) activity is believed to be a critical factor in the development of Parkinson's disease. MNA has been found to destroy several subunits of cerebral complex 1, leading to the suggestion that MNA is concerned in the pathogenesis of Parkinson's disease. Based on these findings, it is hypothesized that niacin is a causal substance in the development of Parkinson's disease through the following processes: NAD produced from niacin releases nicotinamide via poly(ADP-ribosyl)ation, activated by the hydroxyl radical. Released excess nicotinamide is methylated to MNA in the cytoplasm, and superoxides formed by MNA via complex I destroy complex 1 subunits directly, or indirectly via mitochondrial DNA damage. Hereditary or environmental factors may cause acceleration of this cycle, resulting in neuronal death.

Niacin Metabolism and Parkinson\rqs Disease

Epidemiological surveys suggest an important role for niacin in the causes of Parkinson’s disease, in that niacin deficiency, the nutritional condition that causes pellagra, appears to protect against Parkinson’s disease. Absorbed niacin is used in the synthesis of nicotinamide adenine dinucleotide (NAD) in the body, and in the metabolic process NAD releases nicotinamide by poly(ADP-ribosyl)ation, the activation of which has been reported to mediate 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinson’s disease. Recently nicotinamide N-methyltransferase (EC2.1.1.1) activity has been discovered in the human brain, and the released nicotinamide may be methylated to 1-methylnicotinamide (MNA), via this enzyme, in the brain. A deficiency in mitochondrial NADH:ubiquinone oxidoreductase (complex I) activity is believed to be a critical factor in the development of Parkinson’s disease. MNA has been found to destroy several subunits of cerebral complex I, leading to the suggestion that MNA is concerned in the pathogenesis of Parkinson’s disease. Based on these findings, it is hypothesized that niacin is a causal substance in the development of Parkinson’s disease through the following processes: NAD produced from niacin releases nicotinamide via poly(ADP-ribosyl)ation, activated by the hydroxyl radical. Released excess nicotinamide is methylated to MNA in the cytoplasm, and superoxides formed by MNA via complex I destroy complex I subunits directly, or indirectly via mitochondrial DNA damage. Hereditary or environmental factors may cause acceleration of this cycle, resulting in neuronal death.

Molecular Pathway in parkinsons disease: An update on progress / 中国药理学通报

Parkinsons disease(PD) is characterized by a selective lo ss of dopaminergic neurons in the substantia nigra pars compacta. The etiology of PD is still not full y understood. Molecular pathways such as oxidative stress, mitochondrial dysfunc tion and impairment in the ubiquitinproteasomal system, are involved in the proc ess of the dopaminergic neuronal cell death and the progress of PD. The explorat ion and elucidation of these molecular pathways will provide new potential targe ts for the drug therapy of PD.