Synthetic Access to Fluorocyclopropylidenes.

Herein we report an approach for the straightforward preparation of fluorocyclopropylidene group from aldehydes and ketones via Julia-Kocienski olefination using the newly developed reagent 5-((2-fluorocyclopropyl)sulfonyl)-1-phenyl-1H-tetrazole. Derivatization of monofluorocyclopropylidene compounds includes hydrogenation to deliver fluorocyclopropylmethyl compounds and fluorinated cyclobutanones. The utility of the described method is demonstrated by the synthesis of a fluorocyclopropyl-containing analogue of ibuprofen. Bioisosteric replacement of isobutyl with the fluorocyclopropyl group may be used for tuning biological properties of drug molecules.

Heart-Type Fatty Acid Binding Protein Binds Long-Chain Acylcarnitines and Protects against Lipotoxicity.

Heart-type fatty-acid binding protein (FABP3) is an essential cytosolic lipid transport protein found in cardiomyocytes. FABP3 binds fatty acids (FAs) reversibly and with high affinity. Acylcarnitines (ACs) are an esterified form of FAs that play an important role in cellular energy metabolism. However, an increased concentration of ACs can exert detrimental effects on cardiac mitochondria and lead to severe cardiac damage. In the present study, we evaluated the ability of FABP3 to bind long-chain ACs (LCACs) and protect cells from their harmful effects. We characterized the novel binding mechanism between FABP3 and LCACs by a cytotoxicity assay, nuclear magnetic resonance, and isothermal titration calorimetry. Our data demonstrate that FABP3 is capable of binding both FAs and LCACs as well as decreasing the cytotoxicity of LCACs. Our findings reveal that LCACs and FAs compete for the binding site of FABP3. Thus, the protective mechanism of FABP3 is found to be concentration dependent.

Data on cardiac and vascular functionality in ex vivo and in vivo models following acute administration of trimethylamine N-oxide.

This dataset describes in detail the outcomes of acute trimethylamine N-oxide (TMAO) administration on cardiac, vascular and mitochondrial functionality in ex vivo and in vivo models. The accumulation of TMAO in target tissues was assessed after performing heart perfusion or by incubating aortic tissue in a solution containing TMAO. To evaluate the impact of TMAO on mitochondrial function, the aortic rings and heart homogenates of Wistar rats were incubated in a solution containing [9,10-3H] palmitate (5 µCi/ml) or D-[U-14C] glucose (0.625 µCi/ml) in the presence or absence of TMAO with subsequent measurement of substrate oxidation and uptake. The effects of TMAO on the vascular reactivity of isolated conductance and resistance vessels were tested by measuring their response to acetylcholine and sodium nitroprusside. The impact of elevated TMAO levels on cardiac function and infarct size caused by ischemia-reperfusion injury was evaluated in Langendorff perfused heart model. Normal and forced heart functioning was analyzed by echocardiography in CD-1 mouse acute cardiac stress model induced by isoproterenol (10 µg/mouse) upon single and 7 repeated daily administrations of TMAO (120 mg/kg). The data presented in the manuscript provide valuable information on measurements performed under conditions of acutely elevated TMAO levels in experimental models of cardiac and vascular function and energy metabolism. Furthermore, the data have high reuse potential as they could be applied in the planning of future in vitro, ex vivo, and in vivo studies addressing the molecular mechanisms targeted by elevated levels of TMAO.

Fasting-Mimicking Diet Reduces Trimethylamine N-Oxide Levels and Improves Serum Biochemical Parameters in Healthy Volunteers.

Elevated plasma levels of trimethylamine N-oxide (TMAO) have been proposed as a diet-derived biomarker of cardiometabolic disease risk. Caloric restriction is the most common dietary intervention used to improve cardiometabolic health; however, novel trends suggest a fasting-mimicking diet (FMD) as a more feasible alternative. FMD is a variation of intermittent fasting, based on caloric restriction and limitation of protein sources of animal origin, applied in daily cycles during a 5-day period. As TMAO is intensively produced by gut microbiota after the consumption of animal-derived products, we aim to investigate whether a 5-day FMD affects plasma TMAO levels and markers of metabolic health. To investigate whether an increase in vegetable intake possesses similar effects on TMAO levels and metabolic parameters, healthy volunteers (n = 24) were subjected to a 5-day FMD and 19 volunteers served as a reference group (VEG). This group of volunteers consumed an additional four servings of vegetables per day, but otherwise stayed on their usual diet. FMD resulted in a twofold decrease in plasma TMAO levels, which was not evident in the volunteers from the VEG group. Moreover, FMD led to a weight loss of 2.8 ± 0.2 kg and a subsequent reduction in BMI compared to baseline. The FMD group exhibited a significant elevation in plasma ketone bodies (14-fold compared to baseline) and a decrease in IGF-1 levels by 37 ± 8 ng/mL. Since fasting glucose and C-peptide levels decreased, all volunteers in the FMD group showed improved insulin sensitivity and a decreased HOMA-IR index. In contrast, in the VEG group, only a slight reduction in plasma levels of fasting glucose and triglycerides was noted. In conclusion, we show that FMD is a viable strategy to reduce plasma levels of TMAO by limiting caloric intake and animal-derived protein consumption. The reduction in the level of TMAO could be an additional benefit of FMD, leading to a reduced risk of cardiometabolic diseases.

Long-Chain Acylcarnitines Decrease the Phosphorylation of the Insulin Receptor at Tyr1151 Through a PTP1B-Dependent Mechanism.

The accumulation of lipid intermediates may interfere with energy metabolism pathways and regulate cellular energy supplies. As increased levels of long-chain acylcarnitines have been linked to insulin resistance, we investigated the effects of long-chain acylcarnitines on key components of the insulin signalling pathway. We discovered that palmitoylcarnitine induces dephosphorylation of the insulin receptor (InsR) through increased activity of protein tyrosine phosphatase 1B (PTP1B). Palmitoylcarnitine suppresses protein kinase B (Akt) phosphorylation at Ser473, and this effect is not alleviated by the inhibition of PTP1B by the insulin sensitizer bis-(maltolato)-oxovanadium (IV). This result indicates that palmitoylcarnitine affects Akt activity independently of the InsR phosphorylation level. Inhibition of protein kinase C and protein phosphatase 2A does not affect the palmitoylcarnitine-mediated inhibition of Akt Ser473 phosphorylation. Additionally, palmitoylcarnitine markedly stimulates insulin release by suppressing Akt Ser473 phosphorylation in insulin-secreting RIN5F cells. In conclusion, long-chain acylcarnitines activate PTP1B and decrease InsR Tyr1151 phosphorylation and Akt Ser473 phosphorylation, thus limiting the cellular response to insulin stimulation.

Chemical composition of Prunus padus L. flower extract and its anti-inflammatory activities in primary bone marrow-derived macrophages.

ETHNOPHARMACOLOGICAL RELEVANCE: Prunus padus L. has been traditionally used in European ethnomedicine as a treatment for internal and external purposes and is mainly used to reduce inflammation, pain and fever. The activities of P. padus flower extracts are not well characterized, and additional experimental studies at the molecular level are needed to confirm the ethnobotanical findings. AIM OF THE STUDY: To assess the potential of P. padus flower extract (PPFE) as a source of bioactive compounds through the characterization of its chemical composition and antioxidant, anti-collagenase, and anti-inflammatory activities. MATERIALS AND METHODS: The ethanolic extract (1:10 w/v in ethanol solution) from P. padus flowers was subjected to phytochemical analysis and evaluation of the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity. Anti-collagenase activity was determined using a spectrophotometric method in vitro. The effect of PPFE on inflammation was evaluated by measuring specific markers using flow cytometry and assessing pro-inflammatory cytokine (IL-6) release by bone marrow-derived macrophages (BMDMs) ex vivo. RESULTS: The major components of the ethanolic extract of P. padus flowers were quercetin diglycosides, chlorogenic acid and N',N″-dicaffeoyl,N‴-coumaroyl spermidine. The total phenolic content of PPFE was 85.19 mg GAE/g extract, and the EC50 value in the DPPH assay was 0.55 mg/ml. PPFE exhibited the ability to inhibit collagenase activity in a dose-dependent manner. Preincubation of BMDMs with PPFE reduced the population of M1 (pro-inflammatory) and increased the population of M2 (anti-inflammatory) macrophages. Furthermore, PPFE decreased pro-inflammatory cytokine IL-6 release from BMDMs. CONCLUSIONS: PPFE is a rich source of bioactive compounds and possesses considerable anti-inflammatory properties, supporting its use in ethnomedicine for the reduction of inflammatory processes.

Protective Effects of Meldonium in Experimental Models of Cardiovascular Complications with a Potential Application in COVID-19.

Right ventricular (RV) and left ventricular (LV) dysfunction is common in a significant number of hospitalized coronavirus disease 2019 (COVID-19) patients. This study was conducted to assess whether the improved mitochondrial bioenergetics by cardiometabolic drug meldonium can attenuate the development of ventricular dysfunction in experimental RV and LV dysfunction models, which resemble ventricular dysfunction in COVID-19 patients. Effects of meldonium were assessed in rats with pulmonary hypertension-induced RV failure and in mice with inflammation-induced LV dysfunction. Rats with RV failure showed decreased RV fractional area change (RVFAC) and hypertrophy. Treatment with meldonium attenuated the development of RV hypertrophy and increased RVFAC by 50%. Mice with inflammation-induced LV dysfunction had decreased LV ejection fraction (LVEF) by 30%. Treatment with meldonium prevented the decrease in LVEF. A decrease in the mitochondrial fatty acid oxidation with a concomitant increase in pyruvate metabolism was noted in the cardiac fibers of the rats and mice with RV and LV failure, respectively. Meldonium treatment in both models restored mitochondrial bioenergetics. The results show that meldonium treatment prevents the development of RV and LV systolic dysfunction by enhancing mitochondrial function in experimental models of ventricular dysfunction that resembles cardiovascular complications in COVID-19 patients.

trans-Fluorine Effect in Cyclopropane: Diastereoselective Synthesis of Fluorocyclopropyl Cabozantinib Analogs.

Investigation of the trans-fluorine effect on the hydrolysis rate of diethyl 2-fluorocyclopropane-1,1-dicarboxylate provides synthetic access to both diastereomers of the fluorocyclopropyl analog of cabozantinib, a c-Met and VEGFR-2 inhibitor used as a first-line treatment for thyroid cancer and as a second-line treatment for renal cell carcinoma. Despite some known potent examples, there are only a few drug molecules that contain fluorocyclopropane moieties. Herein, we present a case study in which the monofluoro analog of a known cyclopropane-containing drug molecule displays an improved in vitro profile compared to the parent nonfluorinated structure. The fluorocyclopropane moiety may offer valuable fine-tuning options for lead optimization in drug discovery.

Rats with congenital hydronephrosis show increased susceptibility to renal ischemia-reperfusion injury.

Many drug candidates have shown significant renoprotective effects in preclinical models; however, there is no clinically used effective pharmacotherapy for acute kidney injury. The failure to translate from bench to bedside could be due to misleading results from experimental animals with undetected congenital kidney defects. This study was performed to assess the effects of congenital hydronephrosis on the functional capacity of tubular renal transporters as well as kidney sensitivity to ischemia-reperfusion (I-R)-induced injury in male Wistar rats. Ultrasonography was used to distinguish healthy control rats from rats with hydronephrosis. L-carnitine or furosemide was administered, and serial blood samples were collected and analyzed to assess the effects of hydronephrosis on the pharmacokinetic parameters. Renal injury was induced by clamping the renal pedicles of both kidneys for 30 min with subsequent 24 hr reperfusion. The prevalence of hydronephrosis reached ~30%. The plasma concentrations after administration of L-carnitine or furosemide were similar in both groups. I-R induced more pronounced renal injury in the hydronephrotic rats than the control rats, which was evident by a significantly higher kidney injury molecule-1 concentration and lower creatinine concentration in the urine of the hydronephrotic rats than the control rats. After I-R, the gene expression levels of renal injury markers were significantly higher in the hydronephrotic kidneys than in the kidneys of control group animals. In conclusion, our results demonstrate that hydronephrotic kidneys are more susceptible to I-R-induced damage than healthy kidneys. Unilateral hydronephrosis does not affect the pharmacokinetics of substances secreted or absorbed in the renal tubules.

Inhibition of CPT2 exacerbates cardiac dysfunction and inflammation in experimental endotoxaemia.

The suppression of energy metabolism is one of cornerstones of cardiac dysfunction in sepsis/endotoxaemia. To investigate the role of fatty acid oxidation (FAO) in the progression of inflammation-induced cardiac dysfunction, we compared the effects of FAO-targeting compounds on mitochondrial and cardiac function in an experimental model of lipopolysaccharide (LPS)-induced endotoxaemia. In LPS-treated mice, endotoxaemia-induced inflammation significantly decreased cardiac FAO and increased pyruvate metabolism, while cardiac mechanical function was decreased. AMP-activated protein kinase activation by A769662 improved mitochondrial FAO without affecting cardiac function and inflammation-related gene expression during endotoxaemia. Fatty acid synthase inhibition by C75 restored both cardiac and mitochondrial FAO; however, no effects on inflammation-related gene expression and cardiac function were observed. In addition, the inhibition of carnitine palmitoyltransferase 2 (CPT2)-dependent FAO by aminocarnitine resulted in the accumulation of FAO intermediates, long-chain acylcarnitines, in the heart. As a result, cardiac pyruvate metabolism was inhibited, which further exacerbated inflammation-induced cardiac dysfunction. In conclusion, although inhibition of CPT2-dependent FAO is detrimental to cardiac function during endotoxaemia, present findings show that the restoration of cardiac FAO alone is not sufficient to recover cardiac function. Rescue of cardiac FAO should be combined with anti-inflammatory therapy to ameliorate cardiac dysfunction in endotoxaemia.

Metformin decreases bacterial trimethylamine production and trimethylamine N-oxide levels in db/db mice.

The current study aimed to explore whether metformin, the most widely prescribed oral medication for the treatment of type 2 diabetes, alters plasma levels of cardiometabolic disease-related metabolite trimethylamine N-oxide (TMAO) in db/db mice with type 2 diabetes. TMAO plasma concentration was up to 13.2-fold higher in db/db mice when compared to control mice, while in db/db mice fed choline-enriched diet, that mimics meat and dairy product intake, TMAO plasma level was increased 16.8-times. Metformin (250 mg/kg/day) significantly decreased TMAO concentration by up to twofold in both standard and choline-supplemented diet-fed db/db mice plasma. In vitro, metformin significantly decreased the bacterial production rate of trimethylamine (TMA), the precursor of TMAO, from choline up to 3.25-fold in K. pneumoniae and up to 26-fold in P. Mirabilis, while significantly slowing the growth of P. Mirabilis only. Metformin did not affect the expression of genes encoding subunits of bacterial choline-TMA-lyase microcompartment, the activity of the enzyme itself and choline uptake, suggesting that more complex regulation beyond the choline-TMA-lyase is present. To conclude, the TMAO decreasing effect of metformin could be an additional mechanism behind the clinically observed cardiovascular benefits of the drug.

Low-intensity exercise stimulates bioenergetics and increases fat oxidation in mitochondria of blood mononuclear cells from sedentary adults.

AIM: Exercise training induces adaptations in muscle and other tissue mitochondrial metabolism, dynamics, and oxidative phosphorylation capacity. Mitochondrial fatty acid oxidation was shown to be pivotal for the anti-inflammatory status of immune cells. We hypothesize that exercise training can exert effects influence mitochondrial fatty acid metabolism in peripheral blood mononuclear cells (PBMCs). The aim was to investigate the effect of exercise on the fatty acid oxidation-dependent respiration in PBMCs. DESIGN: Twelve fasted or fed volunteers first performed incremental-load exercise tests to exhaustion on a cycle ergometer to determine the optimal workload ensuring maximal health benefits in volunteers with a sedentary lifestyle. In addition, the same volunteers performed 60 min of low-intensity constant-load exercise. RESULTS: In the incremental-load exercise, the maximal whole-body fat oxidation rate measured by indirect calorimetry was reached at the fasted state already at a 50 W workload. At the 75-175 W workloads, the contribution of fat oxidation significantly decreased to only 11%, the heart rate increased to 185 BPM, and the study participants reached exhaustion. These results show that low-intensity exercise (50W) is optimal for maximal whole-body fat utilization. After low-intensity exercise, the ROUTINE mitochondrial respiration, as well as fatty acid oxidation-dependent respiration in PBMCs at LEAK and OXPHOS states, were significantly increased by 31%, 65%, and 76%, respectively. In addition, during 60 min of low-intensity (50W) exercise, a 2-fold higher lipolysis rate was observed and 13.5 ± 0.9 g of fat was metabolized, which was 57% more than the amount of fat that was metabolized during the incremental-load exercise. CONCLUSIONS: In individuals with a sedentary lifestyle participating in a bicycle ergometry exercise program, maximal lipolysis and whole-body fat oxidation rate is reached in a fasted state during low-intensity exercise. For the first time, it was demonstrated that low-intensity exercise improves bioenergetics and increases fatty acid oxidation in PBMCs and may contribute to the anti-inflammatory phenotype.

Empagliflozin Protects Cardiac Mitochondrial Fatty Acid Metabolism in a Mouse Model of Diet-Induced Lipid Overload.

PURPOSE: Sodium-glucose cotransporter 2 (SGLT2) inhibitors prevent heart failure and decrease cardiovascular mortality in patients with type 2 diabetes. Heart failure is associated with detrimental changes in energy metabolism, and the preservation of cardiac mitochondrial function is crucial for the failing heart. However, to date, there are no data to support the hypothesis that treatment with a SGLT2 inhibitor might alter mitochondrial bioenergetics in diabetic failing hearts. Thus, the aim of this study was to investigate the protective effects of empagliflozin on mitochondrial fatty acid metabolism. METHODS: Mitochondrial dysfunction was induced by 18 weeks of high-fat diet (HFD)-induced lipid overload. Empagliflozin was administered at a dose of 10 mg/kg in a chow for 18 weeks. Palmitate metabolism in vivo, cardiac mitochondrial functionality and biochemical parameters were measured. RESULTS: In HFD-fed mice, palmitate uptake was 1.7, 2.3, and 1.9 times lower in the heart, liver, and kidneys, respectively, compared with that of the normal chow control group. Treatment with empagliflozin increased palmitate uptake and decreased the accumulation of metabolites of incomplete fatty acid oxidation in cardiac tissues, but not other tissues, compared with those of the HFD control group. Moreover, empagliflozin treatment resulted in fully restored fatty acid oxidation pathway-dependent respiration in permeabilized cardiac fibers. Treatment with empagliflozin did not affect the biochemical parameters related to hyperglycemia or hyperlipidemia. CONCLUSION: Empagliflozin treatment preserves mitochondrial fatty acid oxidation in the heart under conditions of chronic lipid overload.

Neuroprotective and anti-inflammatory activity of DAT inhibitor R-phenylpiracetam in experimental models of inflammation in male mice.

R-phenylpiracetam (R-PhP, (4R)-2-(4-phenyl-2-oxopyrrolidin-1-yl)acetamide) is an optical isomer of phenotropil, a clinically-used nootropic drug that improves physical condition and cognition. Recently, R-PhP was shown to bind to the dopamine transporter (DAT). Since growing evidence suggests that dysfunction of the dopaminergic system is associated with persistent neuroinflammation, the aim of this study was to determine whether R-PhP, an inhibitor of DAT, has neuroprotective and anti-inflammatory effects in male mice. The pharmacokinetic profiles of R-PhP in mouse plasma and its bioavailability in brain tissue were assessed. To study possible molecular mechanisms involved in the anti-inflammatory activity of R-PhP, target profiling was performed using radioligand binding and enzymatic activity assays. To clarify the neuroprotective and anti-inflammatory effects of R-PhP, we used a lipopolysaccharide (LPS)-induced endotoxaemia model characterized by reduced body temperature and overexpression of inflammatory genes in the brain. In addition, the antinociceptive and anti-inflammatory effects of R-PhP were tested using carrageenan-induced paw oedema and formalin-induced paw-licking tests. R-PhP (50 mg/kg) reached the brain tissue 15 min after intraperitoneal (ip) and peroral (po) injections. The maximal concentration of R-PhP in the brain tissues was 28 µg/g and 18 µg/g tissue after ip and po administration, respectively. In radioligand binding assays, DAT was the only significant molecular target found for R-PhP. A single ip injection of R-PhP significantly attenuated the LPS-induced body temperature reduction and the overexpression of inflammatory genes, such as tumour necrosis factor-α (TNF-α), interleukin 1 beta (IL-1ß) and inducible nitric oxide synthase (iNOS). Seven-day po pretreatment with R-PhP dose-dependently reduced paw oedema and the antinociceptive response, as shown by the carrageenan-induced paw oedema test. In addition, R-PhP decreased the nociceptive response during the inflammatory phase in the formalin-induced paw-licking test. Our study showed that R-PhP possesses neuroprotective and anti-inflammatory effects, demonstrating the potential of DAT inhibitors as effective therapeutics.

Microbiota-Derived Metabolite Trimethylamine N-Oxide Protects Mitochondrial Energy Metabolism and Cardiac Functionality in a Rat Model of Right Ventricle Heart Failure.

Aim: Trimethylamine N-oxide (TMAO) is a gut microbiota-derived metabolite synthesized in host organisms from specific food constituents, such as choline, carnitine and betaine. During the last decade, elevated TMAO levels have been proposed as biomarkers to estimate the risk of cardiometabolic diseases. However, there is still no consensus about the role of TMAO in the pathogenesis of cardiovascular disease since regular consumption of TMAO-rich seafood (i.e., a Mediterranean diet) is considered to be beneficial for the primary prevention of cardiovascular events. Therefore, the aim of this study was to investigate the effects of long-term TMAO administration on mitochondrial energy metabolism in an experimental model of right ventricle heart failure. Methods: TMAO was administered to rats at a dose of 120 mg/kg in their drinking water for 10 weeks. Then, a single subcutaneous injection of monocrotaline (MCT) (60 mg/kg) was administered to induce right ventricular dysfunction, and treatment with TMAO was continued (experimental groups: Control; TMAO; MCT; TMAO+MCT). After 4 weeks, right ventricle functionality was assessed by echocardiography, mitochondrial function and heart failure-related gene and protein expression was determined. Results: Compared to the control treatment, the administration of TMAO (120 mg/kg) for 14 weeks increased the TMAO concentration in cardiac tissues up to 14 times. MCT treatment led to impaired mitochondrial function and decreased right ventricular functional parameters. Although TMAO treatment itself decreased mitochondrial fatty acid oxidation-dependent respiration, no effect on cardiac functionality was observed. Long-term TMAO administration prevented MCT-impaired mitochondrial energy metabolism by preserving fatty acid oxidation and subsequently decreasing pyruvate metabolism. In the experimental model of right ventricle heart failure, the impact of TMAO on energy metabolism resulted in a tendency to restore right ventricular function, as indicated by echocardiographic parameters and normalized organ-to-body weight indexes. Similarly, the expression of a marker of heart failure severity, brain natriuretic peptide, was substantially increased in the MCT group but tended to be restored to control levels in the TMAO+MCT group. Conclusion: Elevated TMAO levels preserve mitochondrial energy metabolism and cardiac functionality in an experimental model of right ventricular heart failure, suggesting that under specific conditions TMAO promotes metabolic preconditioning-like effects.