Mitochondrial Citrate Synthase Research Articles

Mitochondrial respiration is lower in the intrauterine growth-restricted fetal sheep heart.

Fetuses affected by intrauterine growth restriction have an increased risk of developing heart disease and failure in adulthood. Compared with controls, late gestation intrauterine growth-restricted (IUGR) fetal sheep have fewer binucleated cardiomyocytes, reflecting a more immature heart, which may reduce mitochondrial capacity to oxidize substrates. We hypothesized that the late gestation IUGR fetal heart has a lower capacity for mitochondrial oxidative phosphorylation. Left (LV) and right (RV) ventricles from IUGR and control (CON) fetal sheep at 90% gestation were harvested. Mitochondrial respiration (states 1-3, LeakOmy, and maximal respiration) in response to carbohydrates and lipids, citrate synthase (CS) activity, protein expression levels of mitochondrial oxidative phosphorylation complexes (CI-CV), and mRNA expression levels of mitochondrial biosynthesis regulators were measured. The carbohydrate and lipid state 3 respiration rates were lower in IUGR than CON, and CS activity was lower in IUGR LV than CON LV. However, relative CII and CV protein levels were higher in IUGR than CON; CV expression level was higher in IUGR than CON. Genes involved in lipid metabolism had lower expression in IUGR than CON. In addition, the LV and RV demonstrated distinct differences in oxygen flux and gene expression levels, which were independent from CON and IUGR status. Low mitochondrial respiration and CS activity in the IUGR heart compared with CON are consistent with delayed cardiomyocyte maturation, and CII and CV protein expression levels may be upregulated to support ATP production. These insights will provide a better understanding of fetal heart development in an adverse in utero environment. KEY POINTS: Growth-restricted fetuses have a higher risk of developing and dying from cardiovascular diseases in adulthood. Mitochondria are the main supplier of energy for the heart. As the heart matures, the substrate preference of the mitochondria switches from carbohydrates to lipids. We used a sheep model of intrauterine growth restriction to study the capacity of the mitochondria in the heart to produce energy using either carbohydrate or lipid substrates by measuring how much oxygen was consumed. Our data show that the mitochondria respiration levels in the growth-restricted fetal heart were lower than in the normally growing fetuses, and the expression levels of genes involved in lipid metabolism were also lower. Differences between the right and left ventricles that are independent of the fetal growth restriction condition were identified. These results indicate an impaired metabolic maturation of the growth-restricted fetal heart associated with a decreased capacity to oxidize lipids postnatally.

Prefrontal cortical synaptoproteome profile combined with machine learning predicts resilience towards chronic social isolation in rats

Chronic social isolation (CSIS) of rats serves as an animal model of depression and generates CSIS-resilient and CSIS-susceptible phenotypes. We aimed to investigate the prefrontal cortical synaptoproteome profile of CSIS-resilient, CSIS-susceptible, and control rats to delineate biochemical pathways and predictive biomarker proteins characteristic for the resilient phenotype. A sucrose preference test was performed to distinguish rat phenotypes. Class separation and machine learning (ML) algorithms support vector machine with greedy forward search and random forest were then used for discriminating CSIS-resilient from CSIS-susceptible and control rats. CSIS-resilient compared to CSIS-susceptible rat proteome analysis revealed, among other proteins, downregulated glycolysis intermediate fructose-bisphosphate aldolase C (Aldoc), and upregulated clathrin heavy chain 1 (Cltc), calcium/calmodulin-dependent protein kinase type II (Cam2a), synaptophysin (Syp) and fatty acid synthase (Fasn) that are involved in neuronal transmission, synaptic vesicular trafficking, and fatty acid synthesis. Comparison of CSIS-resilient and control rats identified downregulated mitochondrial proteins ATP synthase subunit beta (Atp5f1b) and citrate synthase (Cs), and upregulated protein kinase C gamma type (Prkcg), vesicular glutamate transporter 1 (Slc17a7), and synaptic vesicle glycoprotein 2 A (Sv2a) involved in signal transduction and synaptic trafficking. The combined protein differences make the rat groups linearly separable, and 100% validation accuracy is achieved by standard ML models. ML algorithms resulted in four panels of discriminative proteins. Proteomics-data-driven class separation and ML algorithms can provide a platform for accessing predictive features and insight into the molecular mechanisms underlying synaptic neurotransmission involved in stress resilience.

Altered Levels of Natural Autoantibodies against Heat Shock Proteins in Pregnant Women with Hashimoto's Thyroiditis.

The function of natural autoantibodies (nAAbs) in maintaining immunological tolerance has been comprehensively explained; however, their function in pregnant patients dealing with autoimmune diseases has not been thoroughly investigated. As Hashimoto's thyroiditis (HT) is the predominant organ-specific autoimmune condition of women of childbearing age, this study's objective was to evaluate IgM and IgG nAAbs targeting mitochondrial citrate synthase (CS) and heat shock proteins (Hsp60 and Hsp70) in women diagnosed with HT who were pregnant (HTP). Serum samples collected from HTP and healthy pregnant (HP) women in the first and third trimesters were tested using in-house-developed enzyme-linked immunosorbent assays (ELISAs). Our findings indicate the stability of nAAbs against CS and Hsps throughout the pregnancies of both healthy women and those with HT. However, during both trimesters, HTP patients displayed elevated levels of IgM isotype nAAbs against Hsp60 and Hsp70 compared to HP women, suggesting a regulatory role of IgM nAAbs during the pregnancies of patients with HT. Nonetheless, levels of IgG isotype nAAbs against Hsps were lower solely in the third trimester among HTP patients, resulting in a higher IgM/IgG ratio, which indicates their importance in alterations of the nAAb network during pregnancy in patients with HT.

Tissue-specific metabolic enzyme levels covary with whole-animal metabolic rates and life-history loci via epistatic effects.

Metabolic rates, including standard (SMR) and maximum (MMR) metabolic rate have often been linked with life-history strategies. Variation in context- and tissue-level metabolism underlying SMR and MMR may thus provide a physiological basis for life-history variation. This raises a hypothesis that tissue-specific metabolism covaries with whole-animal metabolic rates and is genetically linked to life history. In Atlantic salmon (Salmo salar), variation in two loci, vgll3 and six6, affects life history via age-at-maturity as well as MMR. Here, using individuals with known SMR and MMR with different vgll3 and six6 genotype combinations, we measured proxies of mitochondrial density and anaerobic metabolism, i.e. maximal activities of the mitochondrial citrate synthase (CS) and lactate dehydrogenase (LDH) enzymes, in four tissues (heart, intestine, liver, white muscle) across low- and high-food regimes. We found enzymatic activities were related to metabolic rates, mainly SMR, in the intestine and heart. Individual loci were not associated with the enzymatic activities, but we found epistatic effects and genotype-by-environment interactions in CS activity in the heart and epistasis in LDH activity in the intestine. These effects suggest that mitochondrial density and anaerobic capacity in the heart and intestine may partly mediate variation in metabolic rates and life history via age-at-maturity. This article is part of the theme issue 'The evolutionary significance of variation in metabolic rates'.

Pathway crosstalk between the central metabolic and heme biosynthetic pathways in Phanerochaete chrysosporium

A comprehensive analysis to survey heme-binding proteins produced by the white-rot fungus Phanerochaete chrysosporium was achieved using a biotinylated heme–streptavidin beads system. Mitochondrial citrate synthase (PcCS), glyceraldehyde 3-phosphate dehydrogenase (PcGAPDH), and 2-Cys thioredoxin peroxidase (mammalian HBP23 homolog) were identified as putative heme-binding proteins. Among these, PcCS and PcGAPDH were further characterized using heterologously expressed recombinant proteins. Difference spectra of PcCS titrated with hemin exhibited an increase in the Soret absorbance at 414 nm, suggesting that the axial ligand of the heme is a His residue. The activity of PcCS was strongly inhibited by hemin with Ki oxaloacetate of 8.7 μM and Ki acetyl-CoA of 5.8 μM. Since the final step of heme biosynthesis occurred at the mitochondrial inner membrane, the inhibition of PcCS by heme is thought to be a physiological event. The inhibitory mode of the heme was similar to that of CoA analogues, suggesting that heme binds to PcCS at His347 at the AcCoA–CoA binding site, which was supported by the homology model of PcCS. PcGAPDH was also inhibited by heme, with a lower concentration than that for PcCS. This might be caused by the different location of these enzymes. From the integration of these phenomena, it was concluded that metabolic regulations by heme in the central metabolic and heme synthetic pathways occurred in the mitochondria and cytosol. This novel pathway crosstalk between the central metabolic and heme biosynthetic pathways, via a heme molecule, is important in regulating the metabolic balance (heme synthesis, ATP synthesis, flux balance of the tricarboxylic acid (TCA) cycle and cellular redox balance (NADPH production) during fungal aromatic degradation.Key points• A comprehensive survey of heme-binding proteins in P. chrysosporium was achieved.• Several heme-binding proteins including CS and GAPDH were identified.• A novel metabolic regulation by heme in the central metabolic pathways was found.

IRW improves diet-induced non-alcoholic fatty liver disease by reducing steatosis associated with increased capacity for oxidative phosphorylation

Non-alcoholic fatty liver disease (NAFLD), the hepatic manifestation of the metabolic syndrome, remains without approved pharmacological treatment. Food-derived bioactive peptides can aid in the management of metabolic conditions including hypertension, obesity and insulin resistance. IRW (isoleucine-arginine-tryptophan) is a tripeptide produced from egg white with angiotensin converting enzyme-inhibitory properties. IRW supplementation elicits antihypertensive effects, improves skeletal muscle insulin signaling and glucose tolerance while reducing body weight gain. In this study, we hypothesized that IRW supplementation would prevent high-fat diet (HFD)-induced NAFLD by modulating hepatic lipid metabolism and increasing mitochondrial content. We found that IRW prevents diet-induced NAFLD, while rosiglitazone (ROSI) treatment worsens it. IRW decreases hepatic triglyceride content and lipid droplet size compared to HFD and ROSI. IRW increases the hepatic mitochondrial complexes and citrate synthase activity, phosphorylation of 5'-AMP-activated protein kinase and microsomal triglyceride transfer protein abundance compared to HFD. Compared with ROSI, IRW increases phosphorylated acetyl CoA carboxylase and mitochondrial complexes. Overall, the hypothesis is supported by these findings.

Exposure to fluoride exacerbates the cognitive deficit of diabetic patients living in areas with endemic fluorosis, as well as of rats with type 2 diabetes induced by streptozotocin via a mechanism that may involve excessive activation of the poly(ADP ribose) polymerase-1/P53 pathway

Epidemiology has shown that fluoride exposure is associated with the occurrence of diabetes. However, whether fluoride affects diabetic encephalopathy is unclear. Elderly diabetic patients in areas with endemic (n = 169) or no fluorosis (108) and controls (85) underwent Montreal Cognitive Assessment. Sprague-Dawley rats receiving streptozotocin and/or different fluoride doses were examined for spatial learning and memory, brain morphology, blood-brain barrier, fasting blood glucose and insulin. Cultured SH-SY5Y cells were treated with 50 mM glucose and/or low- or high-dose fluoride, and P53-knockdown or poly-ADP-ribose polymerase-1 (PARP-1) inhibition. The levels of PARP-1, P53, poly-ADP-ribose (PAR), apoptosis-inducing factor (AIF), and phosphorylated-histone H2A.X (ser139) were measured by Western blotting. Reactive oxygen species (ROS), 8-hydroxydeguanosine (8-OHdG), PARP-1 activity, acetyl-P53, nicotinamide adenine dinucleotide (NAD+), activities of mitochondrial hexokinase1 (HK1) and citrate synthase (CS), mitochondrial membrane potential and apoptosis were assessed biochemically. Cognition of diabetic patients in endemic fluorosis areas was poorer than in other regions. In diabetic rats, fasting blood glucose, insulin resistance and blood-brain barrier permeability were elevated, while spatial learning and memory and Nissl body numbers in neurons declined. In these animals, expression and activity of P53 and PARP-1 and levels of NAD+, PAR, ROS, 8-OHdG, p-histone H2A.X (ser139), AIF and apoptosis content increased; whereas mitochondrial HK1 and CS activities and membrane potential decreased. SH-SY5Y cells exposed to glucose exhibited changes identical to diabetic rats. The changes in diabetic rats and cells treated with glucose were aggravated by fluoride. P53-knockout or PARP-1 inhibition mitigated the effects of glucose with/without low-dose fluoride. Elevation of diabetic encephalopathy was induced by exposure to fluoride and the underlying mechanism may involve overactivation of the PARP-1/P53 pathway.

Degradation of citrate synthase lacking the mitochondrial targeting sequence is inhibited in cells defective in Hsp70/Hsp40 chaperones under heat stress conditions.

Most nucleus-encoded mitochondrial precursor proteins are synthesized in the cytosol and imported into mitochondria in a post-translational manner. In recent years, the quality control mechanisms of nonimported mitochondrial proteins have been intensively studied. In a previous study, we established that in budding yeast a mutant form of citrate synthase 1 (N∆Cit1) that lacks the N-terminal mitochondrial targeting sequence, and therefore mislocalizes to the cytosol is targeted for proteasomal degradation by the SCFUcc1 ubiquitin ligase complex. Here, we show that Hsp70 and Hsp40 chaperones (Ssa1 and Ydj1 in yeast, respectively) are required for N∆Cit1 degradation under heat stress conditions. In the absence of Hsp70 function, a portion of N∆Cit1-GFP formed insoluble aggregates and cytosolic foci. However, the extent of ubiquitination of N∆Cit1 was unaffected, implying that Hsp70/Hsp40 chaperones are involved in the postubiquitination step of N∆Cit1 degradation. Intriguingly, degradation of cytosolic/peroxisomal gluconeogenic citrate synthase (Cit2), an endogenous substrate for SCFUcc1-mediated proteasomal degradation, was not highly dependent on Hsp70 even under heat stress conditions. These results suggest that mitochondrial citrate synthase is thermally vulnerable in the cytosol, where Hsp70/Hsp40 chaperones are required to facilitate its degradation.

Senescent hearts from male Ts65Dn mice exhibit preserved function but altered size and nicotinamide adenine dinucleotide pathway signaling.

Down syndrome (DS) is associated with congenital heart defects at birth, but cardiac function has not been assessed at older ages. We used the Ts65Dn mouse, a model of DS, to quantify heart structure and function with echocardiography in 18-mo male Ts65Dn and wild-type (WT) mice. Heart weight, nicotinamide adenine dinucleotide (NAD) signaling, and mitochondrial (citrate synthase) activity were investigated, as these pathways may be implicated in the cardiac pathology of DS. The left ventricle was smaller in Ts65Dn versus WT, as well as the anterior wall thickness of the left ventricle during both diastole (LVAW_d; mm) and systole (LVAW_s; mm) as assessed by echocardiography. Other functional metrics were similar between groups including left ventricular area end systole (mm2), left ventricular area end diastole (mm2), left ventricular diameter end systole (mm), left ventricular diameter end diastole (mm), isovolumetric relaxation time (ms), mitral valve atrial peak velocity (mm/s), mitral valve early peak velocity (mm/s), ratio of atrial and early peak velocities (E/A), heart rate (beats/min), ejection fraction (%), and fractional shortening (%). Nicotinamide phosphoribosyltransferase (NAMPT) protein expression, NAD concentration, and tissue weight were lower in the left ventricle of Ts65Dn versus WT mice. Sirtuin 3 (SIRT3) protein expression and citrate synthase activity were not different between groups. Although cardiac function was generally preserved in male Ts65Dn, the altered heart size and bioenergetic disturbances may contribute to differences in aging for DS.

The PNPLA3 I148M variant increases ketogenesis and decreases hepatic de novo lipogenesis and mitochondrial function in humans

The PNPLA3 I148M variant is the major genetic risk factor for all stages of fatty liver disease, but the underlying pathophysiology remains unclear. We studied the effect of this variant on hepatic metabolism in homozygous carriers and non-carriers under multiple physiological conditions with state-of-the-art stable isotope techniques. After an overnight fast, carriers had higher plasma β-hydroxybutyrate concentrations and lower hepatic de novo lipogenesis (DNL) compared to non-carriers. After a mixed meal, fatty acids were channeled toward ketogenesis in carriers, which was associated with an increase in hepatic mitochondrial redox state. During a ketogenic diet, carriers manifested increased rates of intrahepatic lipolysis, increased plasma β-hydroxybutyrate concentrations, and decreased rates of hepatic mitochondrial citrate synthase flux. These studies demonstrate that homozygous PNPLA3 I148M carriers have hepatic mitochondrial dysfunction leading to reduced DNL and channeling of carbons to ketogenesis. These findings have implications for understanding why the PNPLA3 variant predisposes to progressive liver disease.

The role of citrate synthase HbCS4 in latex regeneration of Hevea brasiliensis (para rubber tree)

The rubber tree, Hevea brasiliensis, is a tropical rubber-yielding crop with economic importance and provides over 90% of natural rubber (NR) for the global rubber industry. NR biosynthesis belongs to a typical secondary metabolism in laticifers. Definitely, NR production is a huge energy-consuming metabolism, especially during latex regeneration. The tricarboxylic acid (TCA) cycle, as the primary energy source, strongly supports NR synthesis in laticifers. In this study, a mitochondrial citrate synthase (CS) gene named as HbCS4 was isolated from rubber tree and related changes of expression patterns, enzyme activity and metabolites assays in response to tapping and ethephon (ethylene-releasing agent) treatments were compared. These molecular analyses were also conducted in two typical varieties Reyan 7–33–97 and PR107 as well as in HbCS4-overexpressing lines. As a result, the expression characteristics of HbCS4 or its enzyme activity tallied with the energy requirement of rubber synthesis in both treatments and the yielding traits of varieties Reyan 7–33–97 and PR107. In addition, in HbCS4-overexpressing lines, the activities of three key enzymes in TCA cycle, namely CS, NAD-IDH and α-KGDH, were all increased simultaneously. To conclude, these results suggested that HbCS4 is the initial enzyme required for energy supply in laticifers, which greatly contributes to rubber production through changing its expression or enzyme activity.

The Novel Role of Mitochondrial Citrate Synthase and Citrate in the Pathophysiology of Alzheimer's Disease.

Citrate synthase is a key mitochondrial enzyme that utilizes acetyl-CoA and oxaloacetate to form citrate in the mitochondrial membrane, which participates in energy production in the TCA cycle and linked to the electron transport chain. Citrate transports through a citrate malate pump and synthesizes acetyl-CoA and acetylcholine (ACh) in neuronal cytoplasm. In a mature brain, acetyl-CoA is mainly utilized for ACh synthesis and is responsible for memory and cognition. Studies have shown low citrate synthase in different regions of brain in Alzheimer's disease (AD) patients, which reduces mitochondrial citrate, cellular bioenergetics, neurocytoplasmic citrate, acetyl-CoA, and ACh synthesis. Reduced citrate mediated low energy favors amyloid-β (Aβ) aggregation. Citrate inhibits Aβ25-35 and Aβ1-40 aggregation in vitro. Hence, citrate can be a better therapeutic option for AD by improving cellular energy and ACh synthesis, and inhibiting Aβ aggregation, which prevents tau hyperphosphorylation and glycogen synthase kinase-3 beta. Therefore, we need clinical studies if citrate reverses Aβ deposition by balancing mitochondrial energy pathway and neurocytoplasmic ACh production. Furthermore, in AD's silent phase pathophysiology, when neuronal cells are highly active, they shift ATP utilization from oxidative phosphorylation to glycolysis and prevent excessive generation of hydrogen peroxide and reactive oxygen species (oxidative stress) as neuroprotective action, which upregulates glucose transporter-3 (GLUT3) and pyruvate dehydrogenase kinase-3 (PDK3). PDK3 inhibits pyruvate dehydrogenase, which decreases mitochondrial-acetyl-CoA, citrate, and cellular bioenergetics, and decreases neurocytoplasmic citrate, acetyl-CoA, and ACh formation, thus initiating AD pathophysiology. Therefore, GLUT3 and PDK3 can be biomarkers for silent phase of AD.

Natural and Pathological Autoantibodies Show Age-Related Changes in a Spontaneous Autoimmune Mouse (NZB) Model.

The natural autoantibody (natAAb) network is thought to play a role in immune regulation. These IgM antibodies react with evolutionary conserved antigens; however, they do not lead to pathological tissue destruction as opposed to pathological autoantibodies (pathAAb). The exact relation between the natAAbs and pathAAbs is still not completely understood; therefore, in the present study, we set out to measure nat- and pathAAb levels against three conserved antigens in a spontaneous autoimmune disease model: the NZB mouse strain which develops autoimmune hemolytic anemia (AIHA) from six months of age. There was an age dependent increase in the natAAb levels in the serum against Hsp60, Hsp70, and the mitochondrial citrate synthase until 6-9 months of age, followed by a gradual decrease. The pathological autoantibodies appeared after six months of age, which corresponded with the appearance of the autoimmune disease. The changes in nat/pathAAb levels were coupled with decreasing B1- and increasing plasma cell and memory B cell percentages. Based on this, we propose that there is a switch from natAAbs towards pathAAbs in aged NZB mice.

The cardioprotective effect of human glucagon-like peptide-1 receptor agonist (semaglutide) on cisplatin-induced cardiotoxicity in rats: Targeting mitochondrial functions, dynamics, biogenesis, and redox status pathways.

The cardiotoxic effect of chemotherapeutic agents as cisplatin has become a major issue recently. Interference with mitochondrial dynamics, biogenesis, redox status, and apoptosis are the most possible underlying mechanisms. Semaglutide is a human glucagon-like peptide-1 receptor agonist (GLP-1R), which is used primarily for the treatment of DM. Various recent studies have investigated (GLP-1R) role in cardiovascular diseases due to antiapoptotic and antioxidant effects. The current study aimed to investigate the curative role of semaglutide's against cisplatin- induced cardiotoxicity and its relation to mitochondrial functions, dynamics, biogenesis, apoptosis, and redox status pathways. The study included 30 male rats divided into three groups: control, cisplatin-induced cardiotoxicity, and cisplatin-induced cardiotoxicity treated with semaglutide. At the end of the experiment heart index, serum cardiotoxicity markers, SOD, GPX activities and H2 O2 level were estimated. Mitochondrial transmembrane potential, complex I and citrate synthase enzyme activities, ATP level, Mfn2 in addition to PGC-1 α levels were assessed as biogenesis markers. Mitophagy markers PINK1 and Parkin mRNA gene expression were estimated. Histopathological examination of cardiac muscles of all studied groups and immunoassay of P53 and caspase 3 in cardiac tissue were examined to assess apoptosis. Cisplatin has disturbed mitochondrial function and dynamics, dysregulate redox status and induced mitophagy and apoptosis, in the other hand semaglutide treatment has normalized dysregulated mitochondrial function and dynamics, redox status and suppressed mitophagy and apoptosis. Semaglutide has ameliorative effect against cisplatin- induced cardiotoxicity via modulation of mitochondrial functions, dynamics, biogenesis, apoptosis, and redox status pathways.

Pancreastatin inhibitor PSTi8 balances energy homeostasis by attenuating adipose tissue inflammation in high fat diet fed mice

Pancreastatin (PST) is an endogenous bioactive peptide. PST is generated from chromogranin A (Chga) protein which is released by chromaffin and neuroendocrine cells. PST exhibits diabetogenic effect by antagonizing the action of insulin in adipocytes. The level of PST rises during obesity, resulting in persistent low-grade inflammation in adipocytes. Pancreastatin inhibitor 8 (PSTi8), which is developed by modification of PST sequence which antagonizes the action of PST. In this study, we investigated the immunometabolic effect of PSTi8 in the diet-induced obesity (DIO) model in C57BL/6 mice. Here we found PSTi8 decreased the body weight gain, fat mass and increased the lean mass in (DIO) mice. It also showed reduction of adipocyte hypertrophy in eWAT and lipid accumulation in liver of DIO mice. Immunoprofiling of stromal vascular fraction isolated from eWAT of PTSi8 treated mice showed increased anti-inflammatory M2 macrophages, Eosinophil, T-regulatory cells and reduced pro-inflammatory M1 macrophages, CD4 and CD8 T cell population. Apart from this, PSTi8 also improved the mitochondrial function by decreasing reactive oxygen species and increasing mitochondrial membrane potential, NADPH/NADP ratio and citrate synthase activity in eWAT of DIO mice. It also increased the protein expression of pAMPK, pAKT, Arginase −1 and decreased the expression of MHC-II and iNOS in eWAT of DIO mice. In conclusion, PSTi8 exerted its beneficial effect on restoring energy expenditure by reducing adipose tissue inflammation.

Agomelatine, Ketamine and Vortioxetine Attenuate Energy Cell Metabolism-In Vitro Study.

This determination of the mitochondrial effect of pharmacologically different antidepressants (agomelatine, ketamine and vortioxetine) was evaluated and quantified in vitro in pig brain-isolated mitochondria. We measured the activity of mitochondrial complexes, citrate synthase, malate dehydrogenase and monoamine oxidase, and the mitochondrial respiratory rate. Total hydrogen peroxide production and ATP production were assayed. The most potent inhibitor of all mitochondrial complexes and complex I-linked respiration was vortioxetine. Agomelatine and ketamine inhibited only complex IV activity. None of the drugs affected complex II-linked respiration, citrate synthase or malate dehydrogenase activity. Hydrogen peroxide production was mildly increased by agomelatine, which might contribute to increased oxidative damage and adverse effects at high drug concentrations. Vortioxetine significantly reduced hydrogen peroxide concentrations, which might suggest antioxidant mechanism activation. All tested antidepressants were partial MAO-A inhibitors, which might contribute to their antidepressant effect. We observed vortioxetine-induced MAO-B inhibition, which might be linked to decreased hydrogen peroxide formation and contribute to its procognitive and neuroprotective effects. Mitochondrial dysfunction could be linked to the adverse effects of vortioxetine, as vortioxetine is the most potent inhibitor of mitochondrial complexes and complex I-linked respiration. Clarifying the molecular interaction between drugs and mitochondria is important to fully understand their mechanism of action and the connection between their mechanisms and their therapeutic and/or adverse effects.

Light Dependent Changes in Adenylate Methylation of the Promoter of the Mitochondrial Citrate Synthase Gene in Maize (Zea mays L.) Leaves.

Limited methyl-specific restriction of genomic DNA by endonuclease MAL1 revealed the changes in its methyl status caused by adenine modification in maize (Zea mays L.) leaves under different light conditions (dark, light, irradiation by red and far-red light). Incubation in the light and irradiation by red light exhibited an activating effect on DNA adenine methylase activity, which was reflected in an increase in the number of methylated adenines in GATC sites. Far-red light and darkness exhibited an opposite effect. The use of nitrite conversion of DNA followed by methyladenine-dependent restriction by MboI nuclease revealed a phytochrome B-dependent mechanism of regulation of the methyl status of adenine in the GATC sites in the promoter of the gene encoding the mitochondrial isoform of citrate synthase. Irradiation of plants with red light caused changes in the adenine methyl status of the analyzed amplicon, as evidenced by the presence of restriction products of 290, 254, and 121 nucleotides. Adenine methylation occurred at all three GATC sites in the analyzed DNA sequence. It is concluded that adenylate methylation is controlled by phytochrome B via the transcription factor PIF4 and represents an important mechanism for the tricarboxylic acid cycle regulation by light.

Effects of SGLT2 inhibitor dapagliflozin in patients with type 2 diabetes on skeletal muscle cellular metabolism

ObjectiveSGLT2 inhibitors increase urinary glucose excretion and have beneficial effects on cardiovascular and renal outcomes; the underlying mechanism may be metabolic adaptations due to urinary glucose loss. Here, we investigated the cellular and molecular effects of 5 weeks of dapagliflozin treatment on skeletal muscle metabolism in type 2 diabetes patients. MethodsTwenty-six type 2 diabetes mellitus patients were randomized to a 5-week double-blind, cross-over study with 6-8-week wash-out. Skeletal muscle acetylcarnitine levels, intramyocellular lipid (IMCL) content and phosphocreatine (PCr) recovery rate were measured by magnetic resonance spectroscopy (MRS). Ex vivo mitochondrial respiration was measured in skeletal muscle fibers using high resolution respirometry. Intramyocellular lipid droplet and mitochondrial network dynamics were investigated using confocal microscopy. Skeletal muscle levels of acylcarnitines, amino acids and TCA cycle intermediates were measured. Expression of genes involved in fatty acid metabolism were investigated. ResultsMitochondrial function, mitochondrial network integrity and citrate synthase and carnitine acetyltransferase activities in skeletal muscle were unaltered after dapagliflozin treatment. Dapagliflozin treatment increased intramyocellular lipid content (0.060 (0.011, 0.110) %, p = 0.019). Myocellular lipid droplets increased in size (0.03 μm2 (0.01–0.06), p < 0.05) and number (0.003 μm−2 (−0.001–0.007), p = 0.09) upon dapagliflozin treatment. CPT1A, CPT1B and malonyl CoA-decarboxylase mRNA expression was increased by dapagliflozin. Fasting acylcarnitine species and C4–OH carnitine levels (0.4704 (0.1246, 0.8162) pmoles∗mg tissue−1, p < 0.001) in skeletal muscle were higher after dapagliflozin treatment, while acetylcarnitine levels were lower (−40.0774 (−64.4766, −15.6782) pmoles∗mg tissue−1, p < 0.001). Fasting levels of several amino acids, succinate, alpha-ketoglutarate and lactate in skeletal muscle were significantly lower after dapagliflozin treatment. ConclusionDapagliflozin treatment for 5 weeks leads to adaptive changes in skeletal muscle substrate metabolism favoring metabolism of fatty acid and ketone bodies and reduced glycolytic flux.The trial is registered with ClinicalTrials.gov, number NCT03338855.

Metabolomic fingerprinting of renal disease progression in Bardet-Biedl syndrome reveals mitochondrial dysfunction in kidney tubular cells

SummaryChronic kidney disease (CKD) is a major clinical sign of patients with Bardet-Biedl syndrome (BBS), especially in those carrying BBS10 mutations. Twenty-nine patients with BBS and 30 controls underwent a serum-targeted metabolomic analysis. In vitro studies were conducted in two kidney-derived epithelial cell lines, where Bbs10 was stably deleted (IMCD3-Bbs10−/−cells) and over-expressed. The CKD status affected plasmatic metabolite fingerprinting in both patients with BBS and controls. Specific phosphatidylcholine and acylcarnitines discriminated eGFR decline only in patients with BBS. IMCD3-Bbs10−/ cells displayed intracellular lipidaccumulation, reduced mitochondrial potential membrane and citrate synthase staining. Mass-Spectrometry-based analysis revealed that human BBS10 interacted with six mitochondrial proteins, in vitro. In conclusion, renal dysfunction correlated with abnormal phosphatidylcholine and acylcarnitines plasma levels in patients with BBS; in vitro, Bbs10 depletion caused mitochondrial defects while human BBS10 interacted with several mitochondria-related proteins, suggesting an unexplored role of this protein.

Skeletal muscle mitochondrial function and whole-body metabolic energetics in the +/G610C mouse model of osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is rare heritable connective tissue disorder that most often arises from mutations in the type I collagen genes, COL1A1 and COL1A2, displaying a range of symptoms including skeletal fragility, short stature, blue-gray sclera, and muscle weakness. Recent investigations into the intrinsic muscle weakness have demonstrated reduced contractile generating force in some murine models consistent with patient population studies, as well as alterations in whole body bioenergetics. Muscle weakness is found in approximately 80% of patients and has been equivocal in OI mouse models. Understanding the mechanism responsible for OI muscle weakness is crucial in building our knowledge of muscle bone cross-talk via mechanotransduction and biochemical signaling, and for potential novel therapeutic approaches. In this study we evaluated skeletal muscle mitochondrial function and whole-body bioenergetics in the heterozygous +/G610C (Amish) mouse modeling mild/moderate human type I/VI OI and minimal skeletal muscle weakness. Our analyses revealed several changes in the +/G610C mouse relative to their wildtype littermates including reduced state 3 mitochondrial respiration, increased mitochondrial citrate synthase activity, increased Parkin and p62 protein content, and an increased respiratory quotient. These changes may represent the ability of the +/G610C mouse to compensate for mitochondrial and metabolic changes that may arise due to type I collagen mutations and may also account for the lack of muscle weakness observed in the +/G610C model relative to the more severe OI models.