Inhibition Of Creatine Kinase Research Articles

A covalent creatine kinase inhibitor ablates glioblastoma migration and sensitizes tumors to oxidative stress

Glioblastoma is a Grade 4 primary brain tumor defined by therapy resistance, diffuse infiltration, and near-uniform lethality. The underlying mechanisms are unknown, and no treatment has been curative. Using a recently developed creatine kinase inhibitor (CKi), we explored the role of this inhibitor on GBM biology in vitro. While CKi minimally impacted GBM cell proliferation and viability, it significantly affected migration. In established GBM cell lines and patient-derived xenografts, CKi ablated both the migration and invasion of GBM cells. CKi also hindered radiation-induced migration. RNA-seq revealed a decrease in invasion-related genes, with an unexpected increase in glutathione metabolism and ferroptosis protection genes post-CKi treatment. The effects of CKi could be reversed by the addition of cell-permeable glutathione. Carbon-13 metabolite tracing indicated heightened glutathione biosynthesis post-CKi treatment. Combinatorial CKi blockade and glutathione inhibition or ferroptosis activation abrogated cell survival. Our data demonstrated that CKi perturbs promigratory and anti-ferroptotic roles in GBM, identifying the creatine kinase axis as a druggable target for GBM treatment.

Structural Basis for Substrate Binding, Catalysis and Inhibition of Breast Cancer Target Mitochondrial Creatine Kinase by Covalent Inhibitor via Cryo-EM.

Mitochondrial creatine kinases are key players in maintaining energy homeostasis in cells by working in conjunction with cytosolic creatine kinases for energy transport from mitochondria to cytoplasm. High levels of MtCK observed in Her2+ breast cancer and inhibition of breast cancer cell growth by substrate analog, cyclocreatine, indicate dependence of cancer cells on the 'energy shuttle' for cell growth and survival. Hence, understanding the key mechanistic features of creatine kinases and their inhibition plays an important role in the development of cancer therapeutics. Herein, we present the mutational and structural investigation on understudied ubiquitous mitochondrial creatine kinase (uMtCK). Our cryo-EM structures and biochemical data on uMtCK showed closure of the loop comprising residue His61 is specific to and relies on creatine binding and the reaction mechanism of phosphoryl transfer depends on electrostatics in the active site. In addition, the previously identified covalent inhibitor CKi showed inhibition in breast cancer BT474 cells, however our biochemical and structural data indicated that CKi is not a potent inhibitor for breast cancer due to strong dependency on the covalent link formation and inability to induce conformational changes upon binding.

Doxycycline-Mediated Inhibition of Snake Venom Phospholipase and Metalloproteinase.

Warfighters are exposed to life-threatening injuries daily and according to the Joint Trauma System Military Clinical Practice Guideline-Global Snake Envenomation Management snakebites are a concerning threat in all theaters of operation. Snake venom is a complex mixture of toxins including phospholipases A2 (PLA2) and snake venom metalloproteinases (SVMP) that produce myotoxic, hemotoxic, and cytotoxic injuries. Antibody-based antivenom is the standard of care but new approaches including small-molecule inhibitors have gained attention in recent years. Doxycycline is an effective inhibitor of human metalloproteinases and PLA2. The enzymatic activities of 3 phylogenetically distinct snakes: Agkistrodon piscivorus, Naja kaouthia, and Daboia russelii were tested under inhibitory conditions using doxycycline. Enzymatic activity of PLA2 and SVMP was measured in N. kaouthia, D. russelii, and A. piscivorus venom alone and with doxycycline using EnzChek Phospholipase A2 and Gelatinase Assay Kits. A 1-way ANOVA with Tukey's post-hoc test was used to conduct comparative analysis. The median lethal dose of the venoms, the effective dose of doxycycline, and creatine kinase (CK) inhibition levels were measured in a murine model with adult Bagg Albino (BALB/c) mice using intramuscular injections. Median lethal and effective doses were determined using Spearman-Karber's method and a 1-way ANOVA with Tukey's post-hoc test was used to compare CK inhibition levels. Phospholipases A2 activity was reduced to 1.5% to 44.0% in all 3 venoms in a dose-dependent manner using 0.32, 0.16, and 0.08 mg/mL doxycycline when compared to venom-only controls (P < .0001) (Fig.1A). Snake venom metalloproteinases activity was reduced to 4% to 62% in all 3 venoms in a dose-dependent manner using 0.32, 0.16, and 0.08 mg/mL doxycycline (P < .0001) (Fig.1B). The lethal dose (LD50) values of the venoms in the murine model were calculated as follows: A. piscivorus = 20.29 mg/kg (Fig.2A), N. kaouthia = 0.38 mg/kg (Fig.2B), and D. russelii = 7.92 mg/kg (Fig.2C). The effective dose (ED50) of doxycycline in A. piscivorus was calculated to be 20.82 mg/kg and 72.07 mg/kg when treating D. russelii venom. No ED50 could be calculated when treating N. kaouthia venom (Fig.3). Creatine kinase activity was significantly decreased in all 3 venoms treated with doxycycline (P < .0001) (Fig.4). Doxycycline reduced PLA2- and SVMP-related lethality, particularly in A. piscivorus envenomings and in a limited capacity with D. russelii revealing its promise as a treatment for snakebites. In addition, CK activity, a common indicator of muscle damage was inhibited in mice that received doxycycline-treated venom. The doxycycline concentrations identified in the ED50 studies correspond to 1,456 to 5,061 mg dosages for a 70 kg human. Factors including venom yield and snake species would affect the actual dosage needed. Studies into high-dose doxycycline safety and its effectiveness against several snake species is needed to fully translate its use into humans. Based on this work, doxycycline could be used as a treatment en route to higher echelons of care, providing protection from muscle damage and reducing lethality in different snake species.

Β-GPA administration activates slow oxidative muscle signaling pathways and protects soleus muscle against the increased fatigue under 7-days of rat hindlimb suspension

Unloading of slow-twitch muscles results in increased muscle fatigue and the mechanisms of this effect are poorly studied. We aimed to analyze the role of high-energy phosphates accumulation during the first week of rat hindlimb suspension plays in a fiber-type phenotype shift towards fast-type fatigable muscle fibers. Male Wistar rats were divided into 3 groups (n = 8): C - vivarium control; 7HS - 7-day hindlimb suspension; 7HB - 7-day hindlimb suspension with intraperitoneal injection of beta-guanidine propionic acid (β-GPA, 400 mg/kg b w). β‐GPA is a competitive inhibitor of creatine kinase and it reduces concentrations of ATP and phosphocreatine. In the 7HB group, β-GPA treatment protected a slow-type signaling network in an unloaded soleus muscle, including MOTS-C, AMPK, PGC1 α and micro-RNA-499. These signaling effects resulted in a preserved soleus muscle fatigue resistance, slow-type muscle fibers percentage and mitochondrial DNA copy number under muscle unloading.

A creatine kinase inhibitor targeting a redox-regulated cysteine.

A creatine kinase inhibitor targeting a redox-regulated cysteine.

Depletion of creatine phosphagen energetics with a covalent creatine kinase inhibitor.

Creatine kinases (CKs) provide local ATP production in periods of elevated energetic demand, such as during rapid anabolism and growth. Thus, creatine energetics has emerged as a major metabolic liability in many rapidly proliferating cancers. Whether CKs can be targeted therapeutically is unknown because no potent or selective CK inhibitors have been developed. Here we leverage an active site cysteine present in all CK isoforms to develop a selective covalent inhibitor of creatine phosphagen energetics, CKi. Using deep chemoproteomics, we discover that CKi selectively engages the active site cysteine of CKs in cells. A co-crystal structure of CKi with creatine kinase B indicates active site inhibition that prevents bidirectional phosphotransfer. In cells, CKi and its analogs rapidly and selectively deplete creatine phosphate, and drive toxicity selectively in CK-dependent acute myeloid leukemia. Finally, we use CKi to uncover an essential role for CKs in the regulation of proinflammatory cytokine production in macrophages.

β-Cyclodextrins alter the energy metabolism-related enzyme activities in rats.

Although widely used in medicine, separation technology, and other fields, the effects of cyclodextrins on the activities of phosphoryl transfer enzymes have not been previously evaluated. In vivo studies evaluated the function of cyclodextrins as active compounds. Despite the use of cyclodextrins as active compounds, the effects of cyclodextrins on hepatic and renal tissues remain to be fully elucidated. The primary objective of this study was to evaluate the effects of β- cyclodextrins, methyl-β-cyclodextrin (M-β- cyclodextrins), and (2-hydroxypropyl)-β-cyclodextrin (HP-β-cyclodextrins) on enzyme activities regulating the maintenance of energy homeostasis in the kidney and liver tissues in relation to toxicity. Serum levels of liver and kidney markers were measured, and oxidative stress parameters were assessed. After 60-day treatments, we observed that the administration of β-cyclodextrins and M-β-cyclodextrins inhibited the hepatic activity of pyruvate kinase, an irreversible enzyme within the glycolytic pathway. Additionally, administration of HP-β-cyclodextrins inhibited creatine kinase activity and increased the total sulfhydryl content in kidneys. Here, we demonstrated for the first time that β-cyclodextrins, M-β-cyclodextrins, and HP-β-cyclodextrins cause bioenergetic dysfunction in renal and hepatic tissues. These findings suggest that understanding the balance between cyclodextrins' efficacy and adverse effects is essential for better accepting their use in medicine.

Ginkgolide B targets and inhibits creatine kinase B to regulate the CCT/TRiC-SK1 axis and exerts pro-angiogenic activity in middle cerebral artery occlusion mice

Promoting angiogenesis in the ischemic penumbra is a well-established method of ischemic stroke treatment. Ginkgolide B (GB) has long been recognized for its neuroprotective properties following stroke. As previously reported, it appears that stroke-induced neurogenesis and angiogenesis interact or are dependent on one another. Although the pharmacodynamic effect of GB on cerebral blood flow (CBF) following ischemic stroke has been reported, the molecular mechanism underlying this effect remains unknown. As such, this study sought to elucidate the pharmacodynamic effects and underlying mechanisms of GB on post-stroke angiogenesis. To begin, GB significantly increased the proliferation, migration, and tube formation capacity of mouse cerebral hemangioendothelioma cells (b.End3) and human umbilical vein endothelial cells (HUVEC). Additionally, GB significantly improved angiogenesis after oxygen-glucose deprivation/reperfusion (OGD/R) in endothelial cells. The dynamics of CBF, brain microvascular neovascularization and reconstruction, and brain endothelial tissue integrity were examined in middle cerebral artery occlusion (MCAO) mice following GB administration. Through label-free target detection techniques, we discovered for the first time that GB can specifically target Creatine Kinase B (CKB) and inhibit its enzymatic activity. Additionally, we demonstrated through network pharmacology and a series of molecular biology experiments that GB inhibited CKB and then promoted angiogenesis via the CCT/TRiC-SK1 axis. These findings shed new light on novel therapeutic strategies for neurological recovery and endothelial repair following ischemic stroke using GB therapy.

HIF-Dependent CKB Expression Promotes Breast Cancer Metastasis, Whereas Cyclocreatine Therapy Impairs Cellular Invasion and Improves Chemotherapy Efficacy.

Simple SummaryTargeting dysregulated cellular metabolism is a promising avenue to treat metastatic disease. The aim of our study was to identify genes downstream of the hypoxia-inducible factor (HIF)-1 transcription factor that are amenable to therapeutic intervention to treat metastatic breast cancer (MBC). We identified creatine kinase, brain isoform (CKB) as an HIF-dependent gene that strongly promotes invasion and metastasis in estrogen-receptor (ER)-negative breast cancer models. Deletion of Ckb also repressed glycolysis and mitochondrial respiration, leading to a reduction in intracellular ATP. Either the deletion of Ckb or inhibition of creatine kinase (CK) activity using the creatine analog cyclocreatine (cCr) repressed cell invasion, the formation of invadopodia and lung metastasis. In addition, when paired with paclitaxel or doxorubicin, cCr enhanced growth inhibition in an additive or synergistic manner. cCr may be an effective anti-metastatic agent in ER-negative, HIF-1α-positive breast cancers, targeting both cellular metabolism and motility, particularly when paired with conventional cytotoxic agents.The oxygen-responsive hypoxia inducible factor (HIF)-1 promotes several steps of the metastatic cascade. A hypoxic gene signature is enriched in triple-negative breast cancers (TNBCs) and is correlated with poor patient survival. Inhibiting the HIF transcription factors with small molecules is challenging; therefore, we sought to identify genes downstream of HIF-1 that could be targeted to block invasion and metastasis. Creatine kinase brain isoform (CKB) was identified as a highly differentially expressed gene in a screen of HIF-1 wild type and knockout mammary tumor cells derived from a transgenic model of metastatic breast cancer. CKB is a cytosolic enzyme that reversibly catalyzes the phosphorylation of creatine, generating phosphocreatine (PCr) in the forward reaction, and regenerating ATP in the reverse reaction. Creatine kinase activity is inhibited by the creatine analog cyclocreatine (cCr). Loss- and gain-of-function genetic approaches were used in combination with cCr therapy to define the contribution of CKB expression or creatine kinase activity to cell proliferation, migration, invasion, and metastasis in ER-negative breast cancers. CKB was necessary for cell invasion in vitro and strongly promoted tumor growth and lung metastasis in vivo. Similarly, cyclocreatine therapy repressed cell migration, cell invasion, the formation of invadopodia and lung metastasis. Moreover, in common TNBC cell line models, the addition of cCr to conventional cytotoxic chemotherapy agents was either additive or synergistic to repress tumor cell growth.

Paradoxical Increase in Body Mass Induced by Beta-Guanidinopropionic Acid in Juvenile Spontaneously Hypertensive Rats.

BackgroundThe adenosine triphosphate (ATP) regenerating enzyme creatine kinase (CK) is intimately involved in blood pressure generation. Consequently, the creatine transporter and CK inhibitor beta-guanidinopropionic acid (GPA) successfully reduced blood pressure in 16-week-old spontaneously hypertensive rats (SHR), but GPA may cause growth retardation in juvenile mammals. This report considers a serendipity observation of paradoxical growth increase after using GPA to prevent hypertension in three-week-old SHR.MethodsImplementing the “Animal Research: Reporting of In Vivo Experiments” (ARRIVE) guideline, male, three-week-old spontaneously hypertensive rats (N=22) were randomly assigned to standard soy-based (creatine-free) chow with GPA 0.1% vs control chow during four weeks (primary, t=4w) or six weeks of treatment (t=6w). Blood pressure measured by the tail-cuff method was the main outcome. Other outcomes included body mass and contractility characteristics of isolated arteries.Results Body mass at baseline was 28.4 (SE 0.71) g (n=22). With similar food intake/100 gram animal in both groups, GPA-treated rats (n=11) developed a strikingly larger body size and mass: t=4w, GPA 110.4 g (3.7) vs controls (n=11) 65.0 g (4.8) (+69.8%; p<0.001); t=6w, GPA 154.3 (4.7) vs controls 68.0 (4.7) g. There were no significant differences in cardiovascular parameters including blood pressure.DiscussionAn unexpected increase in body mass and size without concurrent blood pressure increase was observed in juvenile SHR on GPA vs control soy-based chow. It is speculated that the partial creatine agonist activity of GPA contributed to these effects. Further studies are needed to confirm these findings and better understand the impact of modulating energy metabolism in juvenile hypertension-prone mammals.

Beta-guanidinopropionic acid does not extend Drosophila lifespan

Activation of AMP activated protein kinase (AMPK) signaling has been demonstrated to extend lifespan and improve healthspan across multiple species. This suggests pharmaceutical approaches to increase AMPK hold the potential to modify the aging process and promote healthy aging. Beta-guanidinopropionic acid (GPA) is a naturally occurring metabolite structurally similar to creatine. GPA is capable of activating AMPK signaling in mammalian models via competitive inhibition of cytosolic creatine kinase. A previous report suggested that dietary GPA supplementation increased lifespan in Drosophila through its effect on AMPK signaling and regulation of autophagy. However, studies in Caenorhabditis have found no beneficial effect of this compound on worm lifespan and that GPA may actually diminish lifespan in at least one Caenorhabditis species. To confirm previous reports of increased longevity in Drosophila, we tested a wide range of GPA concentrations on lifespan and healthspan in both male and female W1118 flies. We report here that GPA does not extend lifespan in Drosophila as previously reported. Moreover, high doses of GPA are detrimental to Drosophila lifespan and stress resistance in male flies. These results suggest the lack of a robust effect of GPA on Drosophila lifespan and highlight the importance of replication studies within the field of aging.

Mitochondrial CaMKII causes adverse metabolic reprogramming and dilated cardiomyopathy

Despite the clear association between myocardial injury, heart failure and depressed myocardial energetics, little is known about upstream signals responsible for remodeling myocardial metabolism after pathological stress. Here, we report increased mitochondrial calmodulin kinase II (CaMKII) activation and left ventricular dilation in mice one week after myocardial infarction (MI) surgery. By contrast, mice with genetic mitochondrial CaMKII inhibition are protected from left ventricular dilation and dysfunction after MI. Mice with myocardial and mitochondrial CaMKII overexpression (mtCaMKII) have severe dilated cardiomyopathy and decreased ATP that causes elevated cytoplasmic resting (diastolic) Ca2+ concentration and reduced mechanical performance. We map a metabolic pathway that rescues disease phenotypes in mtCaMKII mice, providing insights into physiological and pathological metabolic consequences of CaMKII signaling in mitochondria. Our findings suggest myocardial dilation, a disease phenotype lacking specific therapies, can be prevented by targeted replacement of mitochondrial creatine kinase or mitochondrial-targeted CaMKII inhibition.

CK (Creatine Kinase) Is Associated With Cardiovascular Hemodynamics: The HELIUS Study.

The ATP-regenerating enzyme CK (creatine kinase) is strongly associated with blood pressure, which lowers upon experimental CK inhibition. The enzyme is thought to affect cardiovascular hemodynamics through enhanced systemic vascular resistance, stroke volume, and cardiac contractility, but data on these parameters are lacking. We hereby report hemodynamics by CK levels in the multiethnic, cross-sectional HELIUS study (Healthy Life in an Urban Setting). Physical examination included sitting brachial blood pressure and noninvasively assessed supine systemic vascular resistance, stroke volume, cardiac output, and cardiac contractility, which we associated with resting plasma CK. Data from 14 937 men and women (mean age, 43.3; SD, 12.9) indicated that per log CK increase, blood pressure increased with 20.2 (18.9-21.4) mm Hg systolic/13.0 (12.2-13.7) diastolic, an odds ratio for hypertension of 6.1 (5.1-7.2). Outcomes were similar by sex, body mass index, and ancestry, although higher blood pressures in men, with overweight/obesity, and West-African ancestry were partially explained by higher CK, with an adjusted increase in systolic/diastolic pressure of 10.5 (10.0-10.9)/6.4 (6.0-6.7) mm Hg per log CK increase. Systemic vascular resistance, stroke volume, cardiac output, and cardiac contractility (n=7876), increased by respectively 20%, 39%, 14%, and 23% SD per log CK increase. This study indicates that the association of CK with blood pressure likely results from an increase in systemic vascular resistance and stroke volume. These data expand the knowledge on the nature of hypertension associated with CK and may inform further experiments on CK inhibition as a means to lower blood pressure.

Creatine is Neuroprotective to Retinal Neurons In Vitro But Not In Vivo.

To investigate the neuroprotective properties of creatine in the retina using in vitro and in vivo models of injury. Two different rat retinal culture systems (one containing retinal ganglion cells [RGC] and one not) were subjected to either metabolic stress, via treatments with the mitochondrial complex IV inhibitor sodium azide, or excitotoxic stress, via treatment with N-methyl-D-aspartate for 24 hours, in the presence or absence of creatine (0.5, 1.0, and 5.0 mM). Neuronal survival was assessed by immunolabeling for cell-specific antigens. Putative mechanisms of creatine action were investigated in vitro. Expression of creatine kinase (CK) isoenzymes in the rat retina was examined using Western blotting and immunohistochemistry. The effect of oral creatine supplementation (2%, wt/wt) on retinal and blood creatine levels was determined as well as RGC survival in rats treated with N-methyl-D-aspartate (NMDA; 10 nmol) or high IOP-induced ischemia reperfusion. Creatine significantly prevented neuronal death induced by sodium azide and NMDA in both culture systems. Creatine administration did not alter cellular adenosine triphosphate (ATP). Inhibition of CK blocked the protective effect of creatine. Retinal neurons, including RGCs, expressed predominantly mitochondrial CK isoforms, while glial cells expressed exclusively cytoplasmic CKs. In vivo, NMDA and ischemia reperfusion caused substantial loss of RGCs. Creatine supplementation led to elevated blood and retinal levels of this compound but did not significantly augment RGC survival in either model. Creatine increased neuronal survival in retinal cultures; however, no significant protection of RGCs was evident in vivo, despite elevated levels of this compound being present in the retina after oral supplementation.

Calmodulin and protein kinases A/G mediate ciliary beat response in the human nasal epithelium.

Mucociliary clearance of the airway epithelium is an essential function for mucosal defense. We recently proposed a hypothetical mechanism of ciliary beat regulation, in which the pannexin-1 (Panx1)-P2X7 unit serves as an oscillator generating a periodic increase in intracellular Ca2+ ([Ca2+ ]i ). In the present study, we examined the localization of Panx1 and P2X7 at the ultrastructural level, and investigated the regulatory pathway subsequent to [Ca2+ ]i increase. The inferior turbinate mucosa was collected from patients with chronic hypertrophic rhinitis during endoscopic sinonasal surgery. The mucosa was examined by transmission immunoelectron microscopy for Panx1 and P2X7. Alternatively, the mucosa was cut into thin strips, and ciliary beat frequency (CBF) was measured under a phase-contrast light microscope with a high-speed digital video camera. In immunoelectron microscopy, immunoreactivities for Panx1 and P2X7 were localized along the plasma membrane of the entire length of the cilia. CBF was significantly increased by stimulation with 100 µM acetylcholine (Ach). The Ach-induced CBF increase was significantly inhibited by calmidazolium (calmodulin antagonist), SQ22536 (adenylate cyclase inhibitor), ODQ (guanylate cyclase inhibitor), KT5720 (protein kinase A inhibitor), and KT5823 (protein kinase G inhibitor). Fluorodinitrobenzene (creatine kinase inhibitor) completely inhibited the ciliary beat in a time- and dose-dependent manner. These results indicate that Panx1 and P2X7 coexist at the cilia of the human nasal epithelial cells and that the ciliary beat is regulated by calmodulin, adenylate/guanylate cyclases and protein kinases A/G, and crucially depends on creatine kinase.

Essential Role of ATP Synthesized by Creatine Kinase in Contraction of -Toxin Permeabilized Preparations of Tonic Type Smooth Muscle

The role of ATP newly synthesized from ADP and phosphocreatine (PC) by creatine kinase (CK) in the contraction of tonic type smooth muscle, rat femoral artery was studied, since its necessity for phasic type smooth muscle was previously shown. In α-toxin-permeabilized preparations obtained from rat femoral artery, Ca2+ induced a tonic type contraction in the presence of ATP and PC. Omission of PC inhibited significantly the contraction. Treatment of the preparations with 2,4-dinitrofluorobenzene, an inhibitor of CK, also inhibited the contraction. In the presence of ADP and PC, Ca2+ also induced the contraction to a level comparable to that in the presence of ATP and PC. The extent of phosphorylated myosin light chain was fairly consistent with that of Ca2+-induced contraction under all experimental conditions planned above. These results suggest that ATP newly synthesized by CK essentially participates in the whole of the contraction in tonic type smooth muscle, although it participates only in a rapid phasic contraction in phasic type muscle as previously shown.

Grape pomace flour ameliorates Pseudomonas aeruginosa-induced bioenergetic dysfunction in gills of grass carp

Grape pomace is a by-product of the winemaking process, rich in bioactive compounds such as resveratrol and has antioxidant and antimicrobial properties. Recent evidence has demonstrated the protective effects of grape against impairment of the phosphoryl transfer network, a system linked to energetic homeostasis of high-energy phosphoryl transfer, to maintain energetic balance. Also, Pseudomonas aeruginosa infection in fish has been related to severe impairment of the branchial phosphoryl transfer network via inhibition of creatine kinase (CK), adenylate kinase (AK) and pyruvate kinase (PK) activities. Thus, the aim of this study was to evaluate whether dietary supplementation with grape pomace flour (GPF) is able to reduce or prevent the impairment of cellular energetic homeostasis in grass carp (Ctenopharyngodon idella) experimentally infected by P. aeruginosa. Branchial CK (cytosolic and mitochondrial), AK, and PK activities were lower in infected animals on day 15 post-infection (PI) compared to uninfected fish, while lactate dehydrogenase (LDH) activity was higher. Branchial reactive oxygen species (ROS), thiobarbituric acid reactive substances (TBARS), lipid oxidation (LOOH) and protein carbonylation levels were higher, while the level of non-proteic (NPSH) and proteic (PSH) thiols was lower in infected fish on day 15 PI, as compared to uninfected fish. The use of 300 mg of GPF/kg of feed was able to ameliorate the activities of CK, AK, PK, and LDH as compared to infected fish, but their activities remained different from the control group. On the other hand, dietary supplementation with 300 mg of GPF/kg of feed was able to prevent all alterations linked to oxidative damage elicited by disease. Based on this evidence, inhibition of the phosphoryl transfer network may contribute to the impairment of intracellular energetic communication between cellular ATP synthesis and consumption and is mediated by oxidative damage in the gills of fish infected by P. aeruginosa. The use of 300 mg of GPF/kg of feed exerted protective effects on branchial energetic metabolism linked to ATP metabolism by reducing the impairment in cellular energetic homeostasis; its effects can be mediated by the prevention of SH group oxidation.

Tissue oxidative damage mediates impairment on phosphotransfer network during thymol intake: Effects on hepatic and renal bioenergetics

Recent evidences demonstrated that ingestion of several monoterpenes cause hepatic and renal damage due to impairment on mitochondrial energy production, eliciting a collapse on adenosine triphosphate (ATP) synthesis and consequently impairment on bioenergetic homeostasis. Thus, the aim of this study was to evaluate whether phosphotransfer network, catalyzed by creatine kinase (CK), adenylate kinase (AK), and pyruvate kinase (PK), can be a pathway to explain hepatic and renal bioenergetics homeostasis impairment due to thymol ingestion. Daily intake of thymol (40 mg/kg) significantly cause a decreased kidney weight and relative kidney weight compared to control group. The same dose of thymol inhibited renal cytosolic and mitochondrial CK activity as well as renal PK activity compared to control group. Finally, thymol (40 mg/kg) elicited a significant increase on renal reactive oxygen species and lipid damage levels, as well as an inhibition on antioxidant capacity against peroxyl radicals and non-protein thiol levels, which did not occur liver. Doses of 10 and 20 mg/kg of thymol administered orally for 30 consecutive days non-changed these variables. Based on these evidence, the data supported that intake of a high dose of thymol severely inhibits cytosolic and mitochondrial CK activity, a crucial enzyme to maintain cellular energy homeostasis. Moreover, high dietary thymol intake impaired communication between CK isoenzymes, which inhibits the attempts to regenerate ATP or to facilitate the CK/PCr shuttle to improve the intracellular ATP utilization and consumption. Moreover, the inhibition of renal CK and PK activities appears to be mediated by the renal oxidation of lipids and thiol groups, as well as by the reduction of the renal antioxidant capacity.

Creatine kinase, energy reserve, and hypertension: from bench to bedside.

We hypothesized that human variation in the activity of the ATP regenerating enzyme creatine kinase (CK) activity affects hypertension and cardiovascular disease risk. CK is tightly bound close to ATP-utilizing enzymes including Ca2+-ATPase, myosin ATPase, and Na+/K+-ATPase, where it rapidly regenerates ATP from ADP, H+, and phosphocreatine. Thus, relatively high CK was thought to enhance ATP-demanding processes including resistance artery contractility and sodium retention, and reduce ADP-dependent functions. In a series of studies of our group and others, CK was linked to hypertension and bleeding risk. Plasma CK after rest, used as a surrogate measure for tissue CK, was associated with high blood pressure and failure of antihypertensive therapy in case-control and population studies. Importantly, high tissue CK preceded hypertension in animal models and in humans, and human vascular tissue CK gene expression was strongly associated with clinical blood pressure. In line with this, CK inhibition substantially reduced the contractility of human resistance arteries ex vivo. We also presented evidence that plasma CK reduced ADP-dependent platelet aggregation. In subsequent intervention studies, the oral competitive CK inhibitor beta-guanidinopropionic acid (GPA) reduced blood pressure in spontaneously hypertensive rats (SHRs), and a 1-week trial of sub-therapeutic dose GPA in healthy men was uneventful. Thus, based on theoretical concepts, evidence was gathered in laboratory, case-control, and population studies that high CK is associated with hypertension and with bleeding risk, potentially leading to a new mode of cardiovascular risk reduction with CK inhibition.

Therapeutic and Protective Potency of Bee Pollen Against Neurotoxic Effects Induced by Prenatal Exposure of Rats to Methyl Mercury.

MeHg is a widely distributed environmental toxicant with harmful effects on the developing and adult nervous system. This study aimed to evaluate the therapeutic and protective efficacy of pollen grain in improving the toxic effects of MeHg, through the measurement of selected biochemical parameters linked to oxidative stress, energy metabolism, and neurotransmission in brain homogenates of male pups' neonates. Forty healthy pregnant female rats were randomly divided into five groups, and after delivery, each group was consisting of 10 male neonates: (1) neonates delivered by control mothers, (2) neonates delivered by bee pollen treated mothers who received bee pollen at the dose of 200-mg/kg body weight from postnatal day 0 for 4weeks, (3) neonates delivered by MeHg-treated mothers who received MeHg at the dose of 0.5mg/kg/day via drinking water from gestational day 7 till postnatal day 7 of delivery, (4) therapeutic group: neonates delivered by MeHg-treated mothers followed by bee pollen treatment who received bee pollen at the dose of 200-mg/kg body weight from postnatal day 0 for 4weeks, and (5) protective group: neonates delivered by MeHg and bee pollen-treated mothers. Mothers continued receiving the bee pollen at the same dose until day 21. Biochemical parameters linked to oxidative stress and energy metabolism and neurotransmission were investigated in brain homogenates of neonates from all the five groups. MeHg treatment showed an increase in oxidative stress markers like lipid peroxidation and catalase activity coupled with a non-significant decrease in glutathione level. Impaired energy metabolism was ascertained via the inhibition of creatine kinase and lactate dehydrogenase activities. Dramatic decrease of Mg2+ and K+ concentrations confirmed the neurotransmission defect. Interestingly, the bee pollen treatment was highly effective in restoring the catalase, lactate dehydrogenase, and creatine kinase activities in addition to normalizing the levels of Mg2+, K+, lipid peroxidation, and glutathione. Overall, the exposure to MeHg during the developing brain stages was highly effective to show signs and symptoms of neuronal toxicity. Furthermore, it has been concluded that bee pollen can be used safely to ameliorate oxidative stress, poor detoxification as well as metal ion defects, and neuronal death as a critical mechanisms involved in the etiology of numerous neurological disorders.