Decreased long-chain acylcarnitine content increases mitochondrial coupling efficiency and prevents ischemia-induced brain damage in rats

Publisher Copyright: © 2023 The AuthorsLong-chain acylcarnitines (LCACs) are intermediates of fatty acid oxidation and are known to exert detrimental effects on mitochondria. This study aimed to test whether lowering LCAC levels with the anti-ischemia compound 4-[ethyl(dimethyl)ammonio]butanoate (methyl-GBB) protects brain mitochondrial function and improves neurological outcomes after transient middle cerebral artery occlusion (MCAO). The effects of 14 days of pretreatment with methyl-GBB (5 mg/kg, p.o.) on brain acylcarnitine (short-, long- and medium-chain) concentrations and brain mitochondrial function were evaluated in Wistar rats. Additionally, the mitochondrial respiration and reactive oxygen species (ROS) production rates were determined using ex vivo high-resolution fluorespirometry under normal conditions, in models of ischemia-reperfusion injury (reverse electron transfer and anoxia-reoxygenation) and 24 h after MCAO. MCAO model rats underwent vibrissae-evoked forelimb-placing and limb-placing tests to assess neurological function. The infarct volume was measured on day 7 after MCAO using 2,3,5-triphenyltetrazolium chloride (TTC) staining. Treatment with methyl-GBB significantly reduced the LCAC content in brain tissue, which decreased the ROS production rate without affecting the respiration rate, indicating an increase in mitochondrial coupling. Furthermore, methyl-GBB treatment protected brain mitochondria against anoxia–reoxygenation injury. In addition, treatment with methyl-GBB significantly reduced the infarct size and improved neurological outcomes after MCAO. Increased mitochondrial coupling efficiency may be the basis for the neuroprotective effects of methyl-GBB. This study provides evidence that maintaining brain energy metabolism by lowering the levels of LCACs protects against ischemia-induced brain damage in experimental stroke models.Peer reviewe

Regulation of Trimethylamine N-oxide in Treatment of Cardiometabolic Diseases. Summary of the Doctoral Thesis

Promocijas darbs izstrādāts Latvijas Organiskās sintēzes institūta Farmaceitiskās farmakoloģijas laboratorijā. Aizstāvēšana: Medicīnas bāzes zinātņu, tai skaitā farmācijas promocijas padomes atklātā sēdē 2023. gada 24. novembrī plkst. 10:00 Hipokrāta auditorijā, Dzirciema ielā 16 un attālināti, tiešsaistes platformā Zoom.Trimetilamīna N-oksīds (TMAO) ir zarnu trakta mikrobioma metabolīts, kas tiek producēts saimniekorganismā no tādām ar uzturu uzņemtām vielām, kā karnitīns, holīns un betaīns. Šie TMAO prekursori ir plaši sastopami dzīvnieku izcelsmes produktos. Pēdējā desmitgadē paaugstināts TMAO līmenis ir izvirzīts kā biomarķieris kardiovaskulāro un metabolo saslimšanu risku novērtēšanai. Turklāt paaugstināta TMAO koncentrācija ir saistīta ar 2. tipa diabētu, sirds mazspēju, būtisku nevēlamu kardiovaskulāro notikumu biežumu un mirstību. Tomēr joprojām nav vienota viedokļa par TMAO lomu sirds un asinsvadu slimību patoģenēzē, jo augsti TMAO līmeņi ir sastopami arī jūras veltēs un zivīs. Šo produktu regulāra lietošana tiek uzskatīta par primāro sirds un asinsvadu slimību profilaksi. Tāpēc šīs disertācijas mērķis bija pētīt TMAO ietekmētos signālceļus kardiometabolo slimību patoģenēzē, kā arī identificēt potenciālās iespējas TMAO līmeņa regulācijai. Lai sasniegtu šo mērķi, tika veikti 3 preklīniskie pētījumi un 1 klīniskais pētījums. Pirmkārt, tika izvērtēti TMAO (līdz 1 mM) akūtie efekti uz sirds un asinsvadu funkcionalitāti ex vivo un in vivo grauzēju eksperimentālajos modeļos. Otrkārt, tika novērtēta ilgtermiņā paaugstināta TMAO līmeņa ietekme (120 mg/kg, 14 nedēļas) uz sirds funkcionalitāti un enerģijas metabolismu monokrotalīna inducētā labā kambara sirds mazspējas modelī žurkām. Treškārt, 2. tipa diabēta eksperimentālajā modelī pelēm tika pārbaudīts TMAO līmeņa samazināšanas potenciāls metformīnam (250 mg/kg dienā), kas ir visplašāk lietotais pretdiabēta medikaments. Papildus tika veikti eksperimenti ar baktēriju monokultūrām, lai noteiktu iespējamos metformīna darbības mehānismus TMAO līmeņu regulācijai. Visbeidzot, tika pārbaudīta piecu dienu badošanos imitējošas diētas kā uztura stratēģijas ietekme uz TMAO līmeni un vispārējo metabolo veselību raksturojošo biomarķieru koncentrācijām veselos brīvprātīgajos. Akūts TMAO pieaugums (2,5 reizes) neietekmēja asinsvadu funkcionalitāti, bet asinsvadu enerģijas metabolisma procesā tika novērots augstāks taukskābju oksidācijas īpatsvars. Sirds audos akūts TMAO līmeņa paaugstinājums neietekmēja sirds funkciju. Taču labā kambara sirds mazspējas patoloģijas apstākļos ilgstoša TMAO lietošana pārsteidzoši pasargāja no traucējumiem sirds enerģijas metabolismā un palīdzēja saglabāt labā kambara funkciju. Izvirzoties kā iespējama farmakoloģiska stratēģija TMAO līmeņa regulācijai, metformīns divkārt samazināja TMAO koncentrāciju plazmā pelēm 2. tipa diabēta modelī; šo efektu var skaidrot ar metformīna ietekmi uz zarnu mikrobioma sastāvu un specifisku baktēriju aktivitāti. Savukārt, uztura intervences pētījumā, kurā tika pētīta badošanos imitējoša diēta, kas ietver atteikšanos no dzīvnieku izcelsmes olbaltumvielu uzņemšanas un kaloriju ierobežojumu, tika parādīts, ka šāda uztura stratēģija ir efektīva, lai veseliem brīvprātīgajiem samazinātu TMAO līmeni un uzlabotu vispārējo metabolo stāvokli. Iegūtie rezultāti liecina, ka īslaicīgs TMAO koncentrācijas pieaugums neaktivizē kaitīgus molekulāros signālceļus sirds un asinsvadu audos, kamēr ilgstoši paaugstināts TMAO līmenis var pat kalpot kā prekondicionējošs faktors, aizsargājot pret labā kambara sirds mazspēju. Šīs disertācijas rezultāti kopumā sniedz pierādījumus par TMAO līmeņu regulācijas iespējām, izmantojot gan farmakoloģisku līdzekli – metformīnu, gan dzīvesstila pieeju.Šis pētījums tika izstrādāts ar Eiropas Sociālo fondu un Latvijas valsts budžeta finansējumu, projekta Nr. 8.2.2.0/20/I/004 “Atbalsts doktorantu iesaistei zinātniski pētnieciskajā un studiju darbā” ietvaros Rīgas Stradiņa universitāt

Trimetilamīna N-oksīda regulācija kardiometabolo slimību ārstēšanā. Promocijas darbs

The Doctoral Thesis was developed at Latvian Institute of Organic Synthesis, Laboratory of Pharmaceutical Pharmacology. Defence: at the public session of the Promotion Council of Basic Medicine on 2023, November 24 at 10.00 in the Hippocrates Lecture Theatre, 16 Dzirciema Street, Rīga Stradiņš University and remotely via online platform Zoom.Trimethylamine N-oxide (TMAO) is a gut microbiota-derived metabolite synthesised in host organisms from specific food constituents, such as choline, carnitine and betaine, that are highly abundant in products of animal origin. During the last decade, elevated TMAO level has been proposed as biomarker to estimate the risk of cardiometabolic diseases. In addition, elevated concentrations of TMAO have been associated with type 2 diabetes, heart failure, incidence of major adverse cardiovascular events and all-cause mortality. However, there is still no consensus on the exact role of TMAO in the pathogenesis of cardiovascular diseases, since regular consumption of TMAO-rich seafood, is considered beneficial for the primary prevention of cardiovascular events. Therefore, the objective of the present Thesis was to study TMAO-mediated signalling pathways in the pathophysiology of cardiometabolic diseases and to identify possible intervention options. To assess this aim, 3 preclinical studies and 1 clinical study were conducted. First, the impact of short-term treatment with high TMAO concentrations (up to 1 mM) on cardiac and vascular functionality was evaluated in ex vivo and in vivo rodent models. Second, the effects of long-term TMAO supplementation (120 mg/kg, 14 weeks) on cardiac functionality and energy metabolism were assessed in a monocrotaline-induced right ventricular heart failure model in rats. Next, the effect of metformin (250 mg/kg daily), the most widely prescribed oral antidiabetic drug worldwide, on TMAO reduction was tested in experimental model of type 2 diabetes in mice. Additional experiments with bacterial monocultures were performed to test the possible TMAO regulatory mechanisms of action of metformin. Lastly, a fasting mimicking diet was tested for 5 days in healthy volunteers to observe the potential changes in plasma TMAO levels and general markers of metabolic health. An acute 2.5-fold increase in TMAO did not affect vascular functionality, but a higher input of fatty acid oxidation was observed in vascular energy metabolism. In cardiac tissue, acute elevation of the TMAO level did not affect cardiac function. However, in detrimental conditions of right ventricular heart failure, long-term administration of TMAO surprisingly prevented impairment of cardiac mitochondrial energy metabolism and preserved right ventricular function. Regarding potential approaches to reduce TMAO levels, metformin was able to decrease plasma concentrations of TMAO in the type 2 diabetes model in mice nearly twofold; these effects can be attributed to the impact of metformin on the composition and activity of intestinal microbiota. In the dietary intervention study, a 5-day cycle of fasting mimicking diet with limited animal-derived protein intake and caloric restriction was effective in reducing TMAO levels and improving overall metabolic health of the volunteers. To summarise, the obtained results indicate that a short-term increase in TMAO concentrations does not activate detrimental signalling pathways in cardiac and vascular tissues. Meanwhile, long-term elevation of TMAO levels can even serve as a preconditioning factor and protect cardiac function in the right ventricular heart failure model. Furthermore, the results of the present thesis provide evidence on the possibility of targeting TMAO levels using pharmacological approaches, namely metformin, and lifestyle approaches.This research has been developed with financing from the European Social Fund and Latvian state budget within the project No. 8.2.2.0/20/I/004 “Support for involving doctoral students in scientific research and studies” at Rīga Stradiņš Universit

Trimetilamīna N-oksīda regulācija kardiometabolo slimību ārstēšanā. Promocijas darba kopsavilkums

The Doctoral Thesis was developed at Latvian Institute of Organic Synthesis, Laboratory of Pharmaceutical Pharmacology. Defence: at the public session of the Promotion Council of Basic Medicine on 2023, November 24 at 10.00 in the Hippocrates Lecture Theatre, 16 Dzirciema Street, Rīga Stradiņš University and remotely via online platform Zoom.Trimethylamine N-oxide (TMAO) is a gut microbiota-derived metabolite synthesised in host organisms from specific food constituents, such as choline, carnitine and betaine, that are highly abundant in products of animal origin. During the last decade, elevated TMAO level has been proposed as biomarker to estimate the risk of cardiometabolic diseases. In addition, elevated concentrations of TMAO have been associated with type 2 diabetes, heart failure, incidence of major adverse cardiovascular events and all-cause mortality. However, there is still no consensus on the exact role of TMAO in the pathogenesis of cardiovascular diseases, since regular consumption of TMAO-rich seafood, is considered beneficial for the primary prevention of cardiovascular events. Therefore, the objective of the present Thesis was to study TMAO-mediated signalling pathways in the pathophysiology of cardiometabolic diseases and to identify possible intervention options. To assess this aim, 3 preclinical studies and 1 clinical study were conducted. First, the impact of short-term treatment with high TMAO concentrations (up to 1 mM) on cardiac and vascular functionality was evaluated in ex vivo and in vivo rodent models. Second, the effects of long-term TMAO supplementation (120 mg/kg, 14 weeks) on cardiac functionality and energy metabolism were assessed in a monocrotaline-induced right ventricular heart failure model in rats. Next, the effect of metformin (250 mg/kg daily), the most widely prescribed oral antidiabetic drug worldwide, on TMAO reduction was tested in experimental model of type 2 diabetes in mice. Additional experiments with bacterial monocultures were performed to test the possible TMAO regulatory mechanisms of action of metformin. Lastly, a fasting mimicking diet was tested for 5 days in healthy volunteers to observe the potential changes in plasma TMAO levels and general markers of metabolic health. An acute 2.5-fold increase in TMAO did not affect vascular functionality, but a higher input of fatty acid oxidation was observed in vascular energy metabolism. In cardiac tissue, acute elevation of the TMAO level did not affect cardiac function. However, in detrimental conditions of right ventricular heart failure, long-term administration of TMAO surprisingly prevented impairment of cardiac mitochondrial energy metabolism and preserved right ventricular function. Regarding potential approaches to reduce TMAO levels, metformin was able to decrease plasma concentrations of TMAO in the type 2 diabetes model in mice nearly twofold; these effects can be attributed to the impact of metformin on the composition and activity of intestinal microbiota. In the dietary intervention study, a 5-day cycle of fasting mimicking diet with limited animal-derived protein intake and caloric restriction was effective in reducing TMAO levels and improving overall metabolic health of the volunteers. To summarise, the obtained results indicate that a short-term increase in TMAO concentrations does not activate detrimental signalling pathways in cardiac and vascular tissues. Meanwhile, long-term elevation of TMAO levels can even serve as a preconditioning factor and protect cardiac function in the right ventricular heart failure model. Furthermore, the results of the present thesis provide evidence on the possibility of targeting TMAO levels using pharmacological approaches, namely metformin, and lifestyle approaches.This research has been developed with financing from the European Social Fund and Latvian state budget within the project No. 8.2.2.0/20/I/004 “Support for involving doctoral students in scientific research and studies” at Rīga Stradiņš Universit

May 1,2-Dithiolane-4-carboxylic Acid and Its Derivatives Serve as a Specific Thioredoxin Reductase 1 Inhibitor?

Thioredoxin reductase is an essential enzyme that plays a crucial role in maintaining cellular redox homeostasis by catalyzing the reduction of thioredoxin, which is involved in several vital cellular processes. The overexpression of TrxR is often associated with cancer development. A series of 1,2-dithiolane-4-carboxylic acid analogs were obtained to verify the selectivity of 1,2-dithiolane moiety toward TrxR. Asparagusic acid analogs and their bioisoters remain inactive toward TrxR, which proves the inability of the 1,2-dithiolane moiety to serve as a pharmacophore during the interaction with TrxR. It was found that the Michael acceptor functionality-containing analogs exhibit higher inhibitory effects against TrxR compared to other compounds of the series. The most potent representatives exhibited micromolar TrxR1 inhibition activity (IC50 varied from 5.3 to 186.0 μM) and were further examined with in vitro cell-based assays to assess the cytotoxic effects on various cancer cell lines and cell death mechanisms

Development of potent microtubule targeting agent by structural simplification of natural diazonamide

74 p.-6 fig.-4 tab.-1 tab. cont.graph.-+255 p.supp.inf.The marine metabolite diazonamide A exerts low nanomolar cytotoxicity against a range of tumor cell lines; however, its highly complex molecular architecture undermines the therapeutic potential of the natural product. We demonstrate that truncation of heteroaromatic macrocycle in natural diazonamide A, combined with the replacement of the challenging-to-synthesize tetracyclic hemiaminal subunit by oxindole moiety leads to considerably less complex analogues with improved drug-like properties and nanomolar antiproliferative potency. The structurally simplified macrocycles are accessible in 12 steps from readily available indolin-2-one and tert-leucine with excellent diastereoselectivity (99:1 dr) in the key macrocyclization step. The most potent macrocycle acts as a tubulin assembly inhibitor and exerts similar effects on A2058 cell cycle progression and induction of apoptosis as does marketed microtubule-targeting agent vinorelbine.This work was supported by ERDF (Grants 1.1.1.1/16/A/281 and KC-PI-2020/16). V.V. is grateful to MikroTik Ltd. and the University of Latvia Foundation for doctoral scholarship. R.A. and N.K. thank the support from Estonian Reaserch Council (Grant PRG399) and J.F.D. acknowledges the support from Ministerio de Ciencia e Innovación (Grant PID2022-136765OB-I00).Peer reviewe