The influence of 5-fluorouracil on the α-ketoglutarate dehydrogenase complex in rat's cardiac muscle - a preliminary study.

5-fluorouracil (5-FU), which is a commonly used chemotherapy agent exerts undesired cardiac toxicity. Mitochondrial dysfunction is thought to be one of potentially important mechanisms of 5-FU- induced cardiotoxicity. α-ketoglutarate dehydrogenase (α-KGDHC) is the key regulatory enzyme of TCA cycle. The complex consists of multiple copies of three catalytic subunits: α-ketoglutarate dehydrogenase (E1), dihydrolipoamide succinyltransferase (E2) and dihydrolipoamide dehydrogenase (E3). α-KGDHC together with branched chain α-ketoacid dehydrogenase (BCKDH) and pyruvate dehydrogenase (PDH), are the members of 2-oxoacid dehydrogenases family that share some structural and functional similarities. Recently, it has been found that 5-FU stimulates BCKDH in rat's cardiac muscle. Therefore, we hypothesize that 5-FU modifies α-KGDHC activity and affects cardiac muscle metabolism. The aim of this study was to determine the effect of 5-FU on α-KGDHC activity and protein levels of E1 and E2 subunits of the complex in rat's cardiac muscle. Wistar male rats were administered with 4 doses of 5-FU, 150 mg/ kg b.wt. each (study group) or 0.3% methylcellulose (control group). α-KGDHC activity was assayed spectrophotometrically. The E1 and E2 proteins levels were quantified by Western blot. 5-FU administration resulted in stimulation of myocardial α-KGDHC activity in rats. In addition, E2 protein level increased in response to 5-FU treatment, while the E1 protein level remained unchanged. Up-regulation of α-KGDHC appears to result from change in E2 subunit protein level. However, the effect of 5-FU on factors modifying α-KGDHC activity at post-translational level cannot be excluded.

Influence of a diet rich in linoleic acid on mRNA levels for enzymes of branched chain amino acids metabolism in rat's adipose tissue: a pilot study.

White adipose tissue plays an important role in the catabolism of branched chain amino acids (BCAAs). Two initial regulatory steps in BCAAs catabolism are catalyzed by branched chain aminotransferase (BCAT) and branched chain α-keto acid dehydrogenase complex (BCKDH complex), respectively. It has been demonstrated that synthetic ligands for PPARγ receptors increased mRNA levels for enzymes involved in BCAAs catabolism. We hypothesized that feeding rats with diet rich in linoleic acid (LA), a natural PPARγ agonist modifies mRNA levels for enzymes catalyzing BCAAs degradation in adipose tissue. The current pilot study was aimed at the investigation of the effect of diet rich in LA on mRNA levels for BCATm, branched chain α-keto acid dehydrogenase (E1 component of the BCKDH), and mRNA levels for the regulatory enzymes of BCKDH complex, a specific kinase (BDK) and a specific phosphatase (PPM1K) in epididymal white adipose tissue (eWAT). Wistar male rats were fed with high unsaturated fat diet containing mainly linoleic acid (study group) or with the high saturated fat diet (control group). The relative mRNA levels were quantified by reverse transcription-PCR. We have found that in rats fed diet rich in LA mRNA level for BCATm decreased, while mRNA amount for BDK increased. There was no difference between mRNA levels for BCKDH E1 and PPM1K. It is conceivable that changes in mRNA levels for enzymes involved in BCAAs metabolism in eWAT may lead to modification of BCAAs catabolic rate. Further studies are required to fully elucidate this issue.

Effect of 5-fluorouracil on branched-chain α-keto acid dehydrogenase (BCKDH) complex in rat's heart.

Undisturbed branched-chain amino acids (BCAA) catabolism is necessary for normal heart function. The key enzyme in BCAA catabolism is a multienzyme branched-chain α-keto acid dehydro- genase complex (BCKDH). BCKDH activity is regulated mainly by reversible dephosphorylation (activa- tion)/phosphorylation (inactivation) cycle catalyzed by regulatory enzymes, a specific phosphatase (PPM1K) and kinase (BDK). 5-fluorouracil (5-FU) is widely used in the treatment of different types of cancer. 5-FU has the potential to cause a wide spectrum of cardiotoxicity, ranging from asymptomatic electrocardiographic changes to cardiomyopathy and subsequent cardiac failure. We hypothesize that 5-FU modifies BCKDH activity and affects cardiac muscle metabolism. The current study was aimed at the investigation of the in vivo effect of 5-FU on BCKDH activity and mRNA levels for E1, PPM1K and BDK. Wistar male rats were administered with 4 doses of 5-FU, 150 mg/kg b.wt. each (study group) or 0.3% methylcellulose (control group). BCKDH activity was assayed spectrophotometrically. The mRNA levels were quantified by real-time PCR. 5-FU treatment caused an increase in BCKDH activity that appears to result mainly from increased dephosphorylation of the complex and is associated with an increase of PPM1K mRNA level and reduction of BDK and E1 mRNA levels. It is conceivable that 5-FU stimulation of BCKDH is an adaptive reaction with the purpose of enhancing the BCAA catabolism and protecting from toxic effect caused by excessive accumulation of these amino acids in heart.

Impact of High-Sucrose Diet on the mRNA Levels for Elongases and Desaturases and Estimated Protein Activity in Rat Adipose Tissue.

Fatty acids (FAs) present in the adipose tissue (AT) can be modified by elongases and desaturases. These enzymes are regulated by different factors including nutrients. The aim of the study was to evaluate the impact of high-sucrose diet (HSD; 68% sucrose) on the levels of mRNAs for elongases (Elovl2, Elovl5, Elovl6) and desaturases (Fads1, Fads2, Scd) and on the activity of the corresponding proteins in the rat AT. Male Wistar rats were randomized into two study groups: fed with an HSD and with a standard diet (ST). The mRNA levels were determined by a semi-quantitative reverse transcription-PCR. FA composition was analyzed by gas chromatography, and FA ratios were used to estimate the activity of the enzymes. In the HSD rats, the levels of Elovl5, Elovl6, Fads1, and Scd mRNAs were higher, while the level of Fads2 mRNA was lower than in the ST group. Higher levels of Elovl5 and Elovl6 mRNAs corresponded to higher relative activities of these enzymes, while downregulation of the Fads2 mRNA was associated with the lower activity of this desaturase. In contrast, an increase in the level of Scd mRNA was accompanied by a decrease in the enzyme activity. Less monounsaturated FAs were detected in the AT of HSD rats than in the ST group. The composition of individual FAs differed between the groups. This study supports the notion that the regulation of mRNA levels and activity of both elongases and desaturases play an important role in managing the AT lipid composition in response to changes in the dietary status.

Plasma BCAA concentrations during exercise of varied intensities in young healthy men-the impact of endurance training.

BACKGROUND: Branched-chain amino acids (BCAA) i.e., leucine (Leu), isoleucine (Ile) and valine (Val) are important amino acids, which metabolism play a role in maintaining system energy homeostasis at rest and during exercise. As recently shown lowering of circulating BCAA level improves insulin sensitivity and cardiac metabolic health. However, little is known concerning the impact of a single bout of incremental exercise and physical training on the changes in blood BCAA. The present study aimed to determine the impact of a gradually increasing exercise intensity-up to maximal oxygen uptake (VO2max) on the changes of the plasma BCAA [∑BCAA]pl, before and after 5-weeks of moderate-intensity endurance training (ET). METHODS: Ten healthy young, untrained men performed an incremental cycling exercise test up to exhaustion to reach VO2max, before and after ET. RESULTS: We have found that exercise of low-to-moderate intensity (up to ∼50% of VO2max lasting about 12 min) had no significant effect on the [∑BCAA]pl, however the exercise of higher intensity (above 70% of VO2max lasting about 10 min) resulted in a pronounced decrease (p < 0.05) in [∑BCAA]pl. The lowering of plasma BCAA when performing exercise of higher intensity was preceded by a significant increase in plasma lactate concentration, showing that a significant attenuation of BCAA during incremental exercise coincides with exercise-induced acceleration of glycogen utilization. In addition, endurance training, which significantly increased power generating capabilities at VO2max (p = 0.004) had no significant impact on the changes of [∑BCAA]pl during this incremental exercise. CONCLUSION: We have concluded that an exercise of moderate intensity of relatively short duration generally has no effect on the [∑BCAA]pl in young, healthy men, whereas significant decrease in [∑BCAA]pl occurs when performing exercise in heavy-intensity domain. The impact of exercise intensity on the plasma BCAA concentration seems to be especially important for patients with cardiometabolic risk undertaken cardiac rehabilitation or recreational activity.

Novel phosphodiesterases inhibitors from the group of purine-2,6-dione derivatives as potent modulators of airway smooth muscle cell remodelling.

Airway remodelling (AR) is an important pathological feature of chronic asthma and chronic obstructive pulmonary disease. The etiology of AR is complex and involves both lung structural and immune cells. One of the main contributors to airway remodelling is the airway smooth muscle (ASM), which is thickened by asthma, becomes more contractile and produces more extracellular matrix. As a second messenger, adenosine 3',5'-cyclic monophosphate (cAMP) has been shown to contribute to ASM cell (ASMC) relaxation as well as to anti-remodelling effects in ASMC. Phosphodiesterase (PDE) inhibitors have drawn attention as an interesting new group of potential anti-inflammatory and anti-remodelling drugs. Recently, new hydrazide and amide purine-2,6-dione derivatives with anti-inflammatory properties have been synthesized by our team (compounds 1 and 2). We expanded our study of their PDE selectivity profile, ability to increase intracellular cAMP levels, metabolic stability and, above all, their capacity to modulate cell responses associated with ASMC remodelling. The results show that both compounds have subtype specificity for several PDE isoforms (including inhibition of PDE1, PDE3, PDE4 and PDE7). Interestingly, such combined PDE subtype inhibition exerts improved anti-remodelling efficacies against several ASMC-induced responses such as proliferation, contractility, extracellular matrix (ECM) protein expression and migration when compared to other non-selective and selective PDE inhibitors. Our findings open novel perspectives in the search for new chemical entities with dual anti-inflammatory and anti-remodelling profiles in the group of purine-2,6-dione derivatives as broad-spectrum PDE inhibitors.

Adaptation of motor unit contractile properties in rat medial gastrocnemius to treadmill endurance training: Relationship to muscle mitochondrial biogenesis.

This study aimed at investigating the effects of 2, 4 and 8 weeks of endurance training on the contractile properties of slow (S), fast fatigue resistant (FR) and fast fatigable (FF) motor units (MUs) in rat medial gastrocnemius (MG) in relation to the changes in muscle mitochondrial biogenesis. The properties of functionally isolated MUs were examined in vivo. Mitochondrial biogenesis was judged based on the changes in mitochondrial DNA copy number (mtDNA), the content of the electron transport chain (ETC) proteins and PGC-1α in the MG. Moreover, the markers of mitochondria remodeling mitofusins (Mfn1, Mfn2) and dynamin-like protein (Opa1) were studied using qPCR. A proportion of FR MUs increased from 37.9% to 50.8% and a proportion of FF units decreased from 44.7% to 26.6% after 8 weeks of training. The increased fatigue resistance, shortened twitch duration, and increased ability to potentiate force were found as early as after 2 weeks of endurance training, predominantly in FR MUs. Moreover, just after 2 weeks of the training an enhancement of the mitochondrial network remodeling was present as judged by an increase in expression of Mfn1, Opa1 and an increase in PGC-1α in the slow part of MG. Interestingly, no signs of intensification of mitochondrial biogenesis assessed by ETC proteins content and mtDNA in slow and fast parts of gastrocnemius were found at this stage of the training. Nevertheless, after 8 weeks of training an increase in the ETC protein content was observed, but mainly in the slow part of gastrocnemius. Concluding, the functional changes in MUs' contractile properties leading to the enhancement of muscle performance accompanied by an activation of signalling that controls the muscle mitochondrial network reorganisation and mitochondrial biogenesis belong to an early muscle adaptive responses that precede an increase in mitochondrial ETC protein content.

Effect of high carbohydrate diet on elongase and desaturase activity and accompanying gene expression in rat's liver.

BACKGROUND: Hepatic fatty acids (FAs) are modified through different metabolic pathways including elongation and desaturation. These processes are catalyzed by elongases and desaturases, respectively. Glucose, by transcription factors, regulates these processes. The aim of the study was to evaluate the influence of high carbohydrate diet (68%) on the expression of elongase (Elovl-2, Elovl-5, and Elovl-6) and desaturase (∆5D, ∆6D, Scd 1, Scd 2) genes and the activity of the enzymes. The changes in serum lipid profile (triglycerides (TG), total cholesterol (TC), HDL cholesterol) and glucose concentration were measured. Male Wistar rats were randomized into two study groups: animals fed with high carbohydrate diet (n = 6; HiCHO) and a control group fed with a standard diet (n = 6; ST). The expression of mRNA was determinate using reverse transcription PCR (RT-PCR). Hepatic FA composition was determined by gas chromatography, and FA ratios were used to estimate the activity of enzymes. Serum lipid profile and glucose concentration were measured using spectrophotometric methods. RESULTS: The mean values of transcript expression of all examined elongases and desaturases in liver HiCHO rats were higher as compared to ST. Higher expression did not always correspond to higher activity (as index). More monounsaturated FAs (MUFAs) were detected in the liver of HiCHO rats as compared to ST. Serum TG level was higher in the HiCHO than in ST. CONCLUSIONS: These studies support the notion that the regulation of both Elovl and desaturase expression may play an important role in managing hepatic lipid composition in response to changes in dietary status.