Short communication: Chemical structure, concentration, and pH are key factors influencing antimicrobial activity of conjugated bile acids against lactobacilli.

Effects of chemical structure, concentration, and pH on antimicrobial activity of conjugated bile acids were investigated in 4 strains of lactobacilli. Considerable differences were observed in the antimicrobial activity between the 6 human conjugated bile acids, including glycocholic acid, taurocholic acid, glycodeoxycholic acid, taurodeoxycholic acid, glycochenodeoxycholic acid, and taurochenodeoxycholic acid. Glycodeoxycholic acid and glycochenodeoxycholic acid generally showed significantly higher antimicrobial activity against the lactobacilli, but glycocholic acid and taurocholic acid exhibited the significantly lower antimicrobial activity. Glycochenodeoxycholic acid was selected for further analysis, and the results showed its antimicrobial activity was concentration-dependent, and there was a significantly negative linear correlation (R2 > 0.98) between bile-antimicrobial index and logarithmic concentration of the bile acid for each strain of lactobacilli. Additionally, the antimicrobial activity of glycochenodeoxycholic acid was also observed to be pH-dependent, and it was significantly enhanced with the decreasing pH, with the result that all the strains of lactobacilli were unable to grow at pH 5.0. In conclusion, chemical structure, concentration, and pH are key factors influencing antimicrobial activity of conjugated bile acids against lactobacilli. This study provides theoretical guidance and technology support for developing a scientific method for evaluating the bile tolerance ability of potentially probiotic strains of lactobacilli.

Bile salt hydrolase activity is present in nonintestinal lactic acid bacteria at an intermediate level.

It is generally considered that bile salt hydrolase (BSH) activity is hardly detected in nonintestinal lactic acid bacteria (LAB). The aim of this study was to investigate the distribution and intensity of BSH activity in LAB isolated from naturally fermented vegetables and milk. A total of 624 lactic acid bacterial strains classified into 6 genera and 50 species were isolated from 144 naturally fermented vegetable samples and 103 naturally fermented milk samples, and their BSH activity was screened by gas chromatography with electron capture detection. The BSH-positive strains were further analyzed quantitatively for their deconjugation ability against six human-conjugated bile salts by HPLC based on the disappearance of the conjugated bile salts from the reaction mixture. The results showed that 39% of the strains possessed BSH activity distributed in 24 lactic acid bacterial species. The strains of the fermented vegetable origin showed a 0.5-fold higher incidence of BSH-positive strains than those of the fermented milk origin, and the lactic acid bacilli exhibited 2.5-fold higher incidence of BSH-positive strains than the lactic acid cocci in general. The strains of the fermented vegetable origin generally had greater bile salt deconjugation ability than those of the fermented milk origin. More than 97% and 93% of the BSH-positive strains exhibited a greater substrate preference for glycoconjugated bile salts than tauroconjugated bile salts and for dihydroxy bile salts than trihydroxy bile salts, respectively. This study demonstrated that BSH activity was also present in nonintestinal LAB.

A new method for the in vitro determination of the bile tolerance of potentially probiotic lactobacilli.

A new in vitro method was developed to determine the bile tolerance of potentially probiotic lactobacilli. The overnight culture of various lactobacilli strains was inoculated into sterile, half-strength MRS broth supplemented with and without 0.3% (wt/vol) oxgall, buffered with 0.1 M sodium phosphate buffer at a final pH of 7.3, and incubated at 37 °C for 12 h under anaerobic conditions. The bile tolerance ability of the lactobacilli strains was expressed as the percentage of the propagation generations of the bacterial cells in the presence of oxgall to those in the absence of oxgall. The bile tolerance ability of 11 strains of 8 Lactobacillus species, including 3 bile salt hydrolase (BSH)-negative strains and 8 BSH-positive strains, was analyzed using the newly developed method and two traditional methods. The results showed that bile tolerance ability of the strains was considerably different depending on the analysis method used. The newly developed method mimics the physiological environment of the human small intestine, and avoids changes in pH and bile salt composition during the incubation period, which are drawbacks of the traditional bile tolerance test methods. Therefore, the analysis method developed in this study is more suitable to screen or compare the bile tolerance ability of lactobacilli strains.

[Differentiation and Identification of Alicyclobacillus Strains by Fourier Transform Near-Infrared Spectroscopy].

Fourier transform near-infrared spectroscopy (FT-NIR) can reflect the overall molecular composition of microbial cells to identify different types of microorganisms. To establish an accurate, effective method about the differentiation and identification of Alicyclobacillus strains between different species, the present research performed the following studies by FT-NIR: (1) The FT-NIR spectra about seven type stains was clustered for data analysis. After preprocessing, reduction of data was performed by Principal Component Analysis (PCA) and Linear Discriminant Analysis(LDA), exploring the feasibility of differentiation and identification between different species, the result suggested that the PCA model can cluster the seven species of Alicyclobacillus strains correctly and the LDA model I can predict the unknown species with 100% accuracy. It evidenced that the method could identify different species of Alicyclobacillus strains preliminary. (2)In order to improve the robustness and practicability of the model, a total of 41 Alicyclobacillus strains including type and isolated strains were prepared for LDA model II, using the same methods as mentioned before. The result indicated that the LDA model validated by fifteen sample with 86.67% accuracy. It was more perfect and more comprehensive. As a result, the FT-NIR technology combined with chemometrics method can accurately and effectively identify Alicyclobacillus strains between different microbial species.

[Analysis of changes in minerals contents during cider fermentation process by inductively coupled plasma mass spectrometry].

The changes in mineral elements during cider fermentation process were determined using ICP-MS. The results showed that the main minerals in the fermentation liquor included K, Na, Ca, Mg, Fe, Mn, Zn, Cu, Sr and B. The content of K was the highest in both the apple juice and the cider, being 1 853. 83 and 1 654. 38 mg . L-1 respectively. The content of minerals was in dynamic changes along with the fermentation process. As a whole, during 72-120 h and 144-216 h, most of the minerals contents underwent great fluctuation. Especially when fermented for 192 h, the content of most of the minerals reached peak value or valley value. The content of Fe and Zn achieved their peak value, while the content of K, Na, Ca, Mg, Mn and B achieved valley value. But during the following 24 h, the content of minerals underwent a sharp reversal. After fermentation, the content of K, Mg, Cu, Zn and B decreased significantly, while the content of Na, Ca, Mn, Fe and Sr did not change significantly. The correlational analysis was conducted to evaluate the correlation between the mineral elements, and the result showed that the correlation between Ca and Mn was the most significant, with the correlation index reaching 0. 924. The information of this study will supply sufficient data for the fermentation process control and quality improvement of cider.

[Study on mechanism of inactivated cider yeast adsorbing patulin by Fourier transform infrared spectroscopy].

The mechanism of patulin adsorption by inactivated cider yeast was studied by chemical modification and FTIR The results of patulin removal by various modified yeast biomass showed that the ability of patulin biosorption by acetone-treated yeast and NaOH-treated yeast increased siginificantly, while the methylation of amino group and esterification of carboxylate functionalities of yeast cell surface caused a decrease in patulin binding, which indicated that amino group and carboxyl group presented in the cell walls of yeast might be involved in the binding of patulin to the yeast. The FTIR analysis indicated that the main functional groups were amino group, carboxyl group and hydroxy group which are associated with protein and polysaccharides.

Chondroitin Sulfate Alleviates Diabetic Osteoporosis and Repairs Bone Microstructure via Anti-Oxidation, Anti-Inflammation, and Regulating Bone Metabolism.

Diabetic osteoporosis (DOP) belongs to secondary osteoporosis caused by diabetes; it has the characteristics of high morbidity and high disability. In the present study, we constructed a type 1 diabetic rat model and administered chondroitin sulfate (200 mg/kg) for 10 weeks to observe the preventive effect of chondroitin sulfate on the bone loss of diabetic rats. The results showed that chondroitin sulfate can reduce blood glucose and relieve symptoms of diabetic rats; in addition, it can significantly increase the bone mineral density, improve bone microstructure, and reduce bone marrow adipocyte number in diabetic rats; after 10 weeks of chondroitin sulfate administration, the SOD activity level was upregulated, as well as CAT levels, indicating that chondroitin sulfate can alleviate oxidative stress in diabetic rats. Chondroitin sulfate was also found to reduce the level of serum inflammatory cytokines (TNF-α, IL-1, IL-6, and MCP-1) and alleviate the inflammation in diabetic rats; bone metabolism marker detection results showed that chondroitin sulfate can reduce bone turnover in diabetic rats (decreased RANKL, CTX-1, ALP, and TRACP 5b levels were observed after 10 weeks of chondroitin sulfate administration). At the same time, the bone OPG and RUNX 2 expression levels were higher after chondroitin sulfate treatment, the bone RANKL expression was lowered, and the OPG/RANKL ratio was upregulated. All of the above indicated that chondroitin sulfate could prevent STZ-induced DOP and repair bone microstructure; the main mechanism was through anti-oxidation, anti-inflammatory, and regulating bone metabolism. Chondroitin sulfate could be used to develop anti-DOP functional foods and diet interventions for diabetes.

Optimization of microwave-assisted extraction of polyphenols from apple pomace using response surface methodology and HPLC analysis.

A simple and efficient microwave-assisted extraction of polyphenols from industrial apple pomace was developed and optimized by the maximization of the yield using response surface methodology. A Box-Behnken design was used to monitor the effect of microwave power, extraction time, ethanol concentration and ratio of solvent to raw material (g/mL) on the polyphenols yield. The results showed that the optimal conditions were as follows: microwave power 650.4 W, extraction time 53.7 s, ethanol concentration 62.1% and ratio of solvent to raw material 22.9:1. Validation tests indicated that the actual yield of polyphenols was 62.68±0.35 mg gallic acid equivalents per 100 g dry apple pomace with RSD=0.86% (n=5) under the optimal conditions, which was in good agreement with the predicted yield and higher than those of reflux and ultrasonic-assisted extraction methods. HPLC analysis indicated that the major polyphenols of apple pomace consisted of chlorogenic acid, caffeic acid, syrigin, procyanidin B2, (-)-epicatechin, cinnamic acid, coumaric acid, phlorizin and quercetin, of which procyanidin B2 had the highest content of 219.4 mg/kg.

Bis[4-(2-benzoyl-1-oxidoethen-yl)-3-hy-droxy-phenyl benzoato]diethano-l-cobalt(II).

In the title complex, [Co(C(22)H(15)O(5))(2)(C(2)H(5)OH)(2)], the Co(II) atom (site symmetry ) is coordinated by two O,O'-bidentate 4-(2-benzoyl-1-oxidoethen-yl)-3-hy-droxy-phenyl benzoate anions and two ethanol O atoms, resulting in a slightly distorted CoO(6) octa-hedral coordination. An intra-molecular O-H⋯O hydrogen bond in the ligand generates an S(6) ring. The dihedral angle between the aromatic rings joined to the acetyl-acetonate unit is 6.4 (2)°. The ethanol mol-ecule is disordered over two orientations in a 0.65 (3):0.35 (3) ratio. In the crystal, mol-ecules are linked by O-H⋯O bonds.

[Rapid identification of microorganisms based on Fourier transform near infrared spectroscopy].

Fourier transform-near infrared (FT-NIR) spectra of microorganisms reflect the overall molecular composition of the sample. The spectra were specific and can serve as spectroscopic fingerprints that enable highly accurate identification of microorganisms. Bacterial powders of one yeast and five bacteria strains were prepared to collect FT-NIR spectra. FT-NIR measurements were done using a diffuse reflection-integrating sphere. Reduction of data was performed by principal component analysis (PCA) and two identification models based on linear discriminant analysis (LDA) and artificial neural network (ANN) were established to identify bacterial strains. The reproducibility of the method was proved to be excellent (D(yly2) : 1.61 +/- 1.05-10.97 +/- 6. 65) and high identification accuracy was achieved in both the LDA model (Accuracy rate: 100%) and the ANN model (Average relative error: 5.75%). FT-NIR spectroscopy combined with multivariate statistical analysis (MSA) may provide a novel answer to the fields which need for rapid microbial identification and it will have great prospect in industry.

[Modeling of sugar content based on NIRS during cider-making fermentation].

The sugar content and the matrix always are being changed during cider-making fermentation. In order to measure and monitor sugar content accurately and rapidly, it is necessary for the spectra to be sorted. Calibration models were established at different fermentation stages based on near infrared spectroscopy with artificial neural network. NIR spectral data were collected in the spectral region of 12 000-4 000 cm(-1) for the next analysis. After the different conditions for modeling sugar content were analyzed and discussed, the results indicated that the calibration models developed by the spectral data pretreatment of straight line subtraction(SLS) in the characteristic absorption spectra ranges of 7 502-6 472.1 cm(-1) at stage I and 6 102-5 446.2 cm(-1) at stage II were the best for sugar content. The result of comparison of different data pretreatment methods for establishing calibration model showed that the correlation coefficients of the models (R2) for stage I and II were 98.93% and 99.34% respectively and the root mean square errors of cross validation(RMSECV) for stage I and II were 4.42 and 1.21 g x L(-1) respectively. Then the models were tested and the results showed that the root mean square error of prediction (RMSEP) was 4.07 g x L(-1) and 1.13 g x L(-1) respectively. These demonstrated that the models the authors established are very well and can be applied to quick determination and monitoring of sugar content during cider-making fermentation.

In vivo activation of peroxisome proliferator-activated receptor-delta protects the heart from ischemia/reperfusion injury in Zucker fatty rats.

Peroxisome proliferator-activated receptor (PPAR)-delta is a transcription factor that belongs to the PPAR family. PPAR-delta is abundantly expressed in the heart, and its role in the heart is largely unknown. We tested whether pharmacological activation of PPAR-delta protects the heart from ischemia/reperfusion (I/R) injury in male Zucker fatty rats, a rodent model of obesity and dyslipidemia. A highly selective PPAR-delta agonist, [4-[[[2-[3-fluoro-4-(trifluoromethyl)phenyl]-4-methyl-5-thiazolyl]methyl] thio]-2-methylphenoxy]acetic acid (GW0742), was administered for 7 days at 10 mg/kg/day (p.o., once a day). Ischemic injury was produced by occlusion of the left anterior descending artery for 30 min followed by reperfusion for up to 24 h. Treatment with GW0742 reduced serum levels of cardiac troponin-I and infarct size by 63% (p < 0.01) and 32% (p < 0.01), respectively, and improved left ventricular function. Treatment with GW0742 up-regulated gene expression involved in cardiac fatty acid oxidation, increased fat use in the heart, and reduced serum levels of free fatty acids. The enhanced cardiac expression of interleukin (IL)-6, IL-8, intercellular adhesion molecule-1, and monocyte chemoattractant protein-1 induced by I/R were significantly attenuated by GW0742. Treatment with GW0742 also reduced apoptotic cardiomyocytes by 34% and cardiac caspase-3 activity by 61% (both p < 0.01 versus vehicle). GW0742 differentially regulated Bcl family members, favoring cell survival, and attenuated I/R-induced cardiac mitochondrial damage. In addition, GW0742 treatment augmented the cardiac Akt signaling pathway, as reflected by enhanced phospho-3-phosphoinositide-dependent kinase-1 and p-Akt. The results indicate that activation of PPAR-delta protected the heart from I/R injury in Zucker fatty rats, and multiple mechanisms including amelioration of lipotoxicity, anti-inflammation, and up-regulation of prosurvival signaling contribute together to the cardioprotection.

Systemic activation of the transient receptor potential vanilloid subtype 4 channel causes endothelial failure and circulatory collapse: Part 2.

The transient receptor potential (TRP) vanilloid subtype 4 (V4) is a nonselective cation channel that exhibits polymodal activation and is expressed in the endothelium, where it contributes to intracellular Ca2+ homeostasis and regulation of cell volume. The purpose of the present study was to evaluate the systemic cardiovascular effects of GSK1016790A, a novel TRPV4 activator, and to examine its mechanism of action. In three species (mouse, rat, and dog), the i.v. administration of GSK1016790A induced a dose-dependent reduction in blood pressure, followed by profound circulatory collapse. In contrast, GSK1016790A had no acute cardiovascular effects in the TRPV4-/- null mouse. Hemodynamic analyses in the dog and rat demonstrate a profound reduction in cardiac output. However, GSK1016790A had no effect on rate or contractility in the isolated, buffer-perfused rat heart, and it produced potent endothelial-dependent relaxation of rodent-isolated vascular ring segments that were abolished by nitric-oxide synthase (NOS) inhibition (N-nitro-L-arginine methyl ester; L-NAME), ruthenium red, and endothelial NOS (eNOS) gene deletion. However, the in vivo circulatory collapse was not altered by NOS inhibition (L-NAME) or eNOS gene deletion but was associated with (concentration and time appropriate) profound vascular leakage and tissue hemorrhage in the lung, intestine, and kidney. TRPV4 immunoreactivity was localized in the endothelium and epithelium in the affected organs. GSK1016790A potently induced rapid electrophysiological and morphological changes (retraction/condensation) in cultured endothelial cells. In summary, inappropriate activation of TRPV4 produces acute circulatory collapse associated with endothelial activation/injury and failure of the pulmonary microvascular permeability barrier. It will be important to determine the role of TRPV4 in disorders associated with edema and microvascular congestion.

Bile acid patterns in commercially available oxgall powders used for the evaluation of the bile tolerance ability of potential probiotics.

This study aimed to analyze the bile acid patterns in commercially available oxgall powders used for evaluation of the bile tolerance ability of probiotic bacteria. Qxgall powders purchased from Sigma-Aldrich, Oxoid and BD Difco were dissolved in distilled water, and analyzed. Conjugated bile acids were profiled by ion-pair high-performance liquid chromatography (HPLC), free bile acids were detected as their p-bromophenacyl ester derivatives using reversed-phase HPLC after extraction with acetic ether, and total bile acids were analyzed by enzymatic-colorimetric assay. The results showed that 9 individual bile acids (i.e., taurocholic acid, glycocholic acid, taurodeoxycholic acid, glycodeoxycholic acid, taurochenodeoxycholic acid, glycochenodeoxycholic acid, cholic acid, chenodeoxycholic acid, deoxycholic acid) were present in each of the oxgall powders tested. The content of total bile acid among the three oxgall powders was similar; however, the relative contents of the individual bile acids among these oxgall powders were significantly different (P < 0.001). The oxgall powder from Sigma-Aldrich was closer to human bile in the ratios of glycine-conjugated bile acids to taurine-conjugated bile acids, dihydroxy bile acids to trihydroxy bile acids, and free bile acids to conjugated bile acids than the other powders were. It was concluded that the oxgall powder from Sigma-Aldrich should be used instead of those from Oxoid and BD Difco to evaluate the bile tolerance ability of probiotic bacteria as human bile model.

Hamsters Are a Better Model System than Rats for Evaluating the Hypocholesterolemic Efficacy of Potential Probiotic Strains.

SCOPE: Rats and hamsters are the most commonly used animal models for evaluating the hypocholesterolemic activity of potential probiotic strains, whereas little or no information has been reported on whether the animal models would affect the experimental conclusions regarding the hypocholesterolemic efficacy of the strains. METHODS AND RESULTS: Both high-cholesterol-fed rats and hamsters were intragastrically administered viable cells of bile salt hydrolase-active Lactobacillus acidophilus K16 once daily (1 × 1010 CFU per kg body weight) for 28 d. It was found that the strain did not significantly (p > 0.05) affect the serum and hepatic cholesterol levels in rats, whereas it significantly decreased (p < 0.01 or p < 0.001) the serum total and non-HDL-cholesterol as well as hepatic-free, esterified, and total cholesterol levels in hamsters by 29.6%, 38.8%, 15.8%, 36.2%, and 34.0%, respectively. CONCLUSION: These data suggest that the hypocholesterolemic efficacy of L. acidophilus K16 is substantially different between high-cholesterol-fed hamsters and rats and that hamsters are a better model system than rats for evaluating the hypocholesterolemic efficacy of potential probiotic strains due to their similarity to humans in biliary bile acid composition, including types of bile acids and their conjugation form.

Bile Salt Hydrolase and S-Layer Protein are the Key Factors Affecting the Hypocholesterolemic Activity of Lactobacillus casei-Fermented Milk in Hamsters.

SCOPE: Lactobacillus casei F0822-fermented milk has exhibited significant hypocholesterolemic activity in hamsters in the previous study. Under this premise, the objective of this study is to further explore whether bile salt hydrolase (BSH) and S-layer protein (SLP) of the strain have a significant influence on hypocholesterolemic activity of the fermented milk. METHODS AND RESULTS: Independent and double interposon mutants of BSH and SLP genes are constructed from wild-type L. casei F0822 via chromosomal insertion of chloramphenicol or/and erythromycin resistance genes based on double-crossover homologous recombination. The mutants- and the wild-type strain-fermented milk is prepared (viable counts of approximately 8.0 × 108 colony-forming units mL-1 each) and intragastrically administered to high-cholesterol-fed hamsters once daily at a dose of 1.25 mL d-1 for 28 d, respectively. Both the BSH-deficient mutant- and the SLP-deficient mutant-fermented milk significantly (p < 0.05) increase serum total and LDL-cholesterol levels in hamsters compared with the wild-type strain-fermented milk. However, only the BSH-deficient mutant-fermented milk could significantly (p < 0.05) increase hepatic total and esterified cholesterol levels in hamsters. CONCLUSION: Both BSH and SLP have a significant influence on the hypocholesterolemic activity of L. casei F0822-fermented milk in hamsters. Nevertheless, the BSH is greater than the SLP in this regard.

Rosiglitazone treatment in Zucker diabetic Fatty rats is associated with ameliorated cardiac insulin resistance and protection from ischemia/reperfusion-induced myocardial injury.

The mechanism responsible for the enhanced myocardial susceptibility to ischemic insult in patients with type 2 diabetes is not clear. The present study examines the effect of rosiglitazone treatment on cardiac insulin sensitization and its association with cardioprotection from ischemia/reperfusion injury in an animal model of diabetes. Male Zucker diabetic fatty (ZDF) rats were treated with rosiglitazone (3 mg . kg(-1) . day(-1) orally) or vehicle for 8 days before undergoing 30 min of coronary artery ligation, followed by reperfusion for 4 h (apoptosis) or 24 h (infarction). Rosiglitazone reduced the blood levels of glucose, triglycerides, and free fatty acids; enhanced cardiac glucose oxidation; and increased Akt phosphorylation (Akt-pS473) 2.1-fold and Akt kinase activity 1.8-fold in the ischemic myocardium. The phosphorylation of two downstream targets of Akt, glycogen synthase kinase-3beta and FKHR (forkhead transcription factor), was also enhanced by 2- and 2.9-fold, respectively. In rosiglitazone-treated rats, the number of apoptotic cardiomyocytes and the myocardial infarct size were decreased by 58 and 46%, respectively, and the myocardial contractile dysfunction was improved. Blockade of the insulin-Akt signaling pathway by wortmannin in the 8-day rosiglitazone-treated ZDF rats resulted in a markedly diminished cardioprotective effect of rosiglitazone. In addition, 8-day rosiglitazone treatment in Zucker lean rats or 2-day rosiglitazone treatment in ZDF rats, both of which showed no change in whole-body insulin sensitivity, resulted in a significant reduction in cardiac infarct size, but to a lesser degree when compared with that observed in 8-day rosiglitazone-treated ZDF rats. These results suggest that chronic treatment with rosiglitazone protects the heart against ischemia/reperfusion injury in ZDF rats, and that the enhanced cardiac protection observed after rosiglitazone treatment might be attributable in part to an improvement in cardiac insulin sensitivity.

Nitric oxide mediates the antiapoptotic effect of insulin in myocardial ischemia-reperfusion: the roles of PI3-kinase, Akt, and endothelial nitric oxide synthase phosphorylation.

BACKGROUND: Recent evidence from cultured endothelial cell studies suggests that phosphorylation of endothelial nitric oxide synthase (eNOS) through the PI3-kinase-Akt pathway increases NO production. This study was designed to elucidate the signaling pathway involved in the antiapoptotic effect of insulin in vivo and to test the hypothesis that phosphorylation of eNOS by insulin may participate in the cardioprotective effect of insulin after myocardial ischemia and reperfusion. METHODS AND RESULTS: Male Sprague-Dawley rats were subjected to 30 minutes of myocardial ischemia and 4 hours of reperfusion. Rats were randomized to receive vehicle, insulin, insulin plus wortmannin, or insulin plus L-NAME. Treatment with insulin resulted in 2.6-fold and 4.3-fold increases in Akt and eNOS phosphorylation and a significant increase in NO production in ischemic/reperfused myocardial tissue. Phosphorylation of Akt and eNOS and increase of NO production by insulin were completely blocked by wortmannin, a PI3-kinase inhibitor. Pretreatment with L-NAME, a nonselective NOS inhibitor, had no effect on Akt and eNOS phosphorylation but significantly reduced NO production. Moreover, treatment with insulin markedly reduced myocardial apoptotic death (P<0.01 versus vehicle). Pretreatment with wortmannin abolished the antiapoptotic effect of insulin. Most importantly, pretreatment with L-NAME also significantly reduced the antiapoptotic effect of insulin (P<0.01 versus insulin). CONCLUSIONS: These results demonstrated that in vivo administration of insulin activated Akt through the PI3-kinase-dependent mechanism and reduced postischemic myocardial apoptotic death. Phosphorylation of eNOS and the concurrent increase of NO production contribute significantly to the antiapoptotic effect of insulin.

Antioxidative, antinitrative, and vasculoprotective effects of a peroxisome proliferator-activated receptor-gamma agonist in hypercholesterolemia.

BACKGROUND: Peroxisome proliferator-activated receptor (PPAR) signaling pathways have been reported to exert anti-inflammatory effects and attenuate atherosclerosis formation. However, the mechanisms responsible for their anti-inflammatory and antiatherosclerotic effects remain largely unknown. The present study tested the hypothesis that a PPARgamma agonist may exert significant endothelial protection by antioxidative and antinitrative effects. METHODS AND RESULTS: Male New Zealand White rabbits were randomized to receive a normal (control) or a high-cholesterol diet and treated with vehicle or rosiglitazone (a PPARgamma agonist) 3 mg x kg(-1) x d(-1) for 5 weeks beginning 3 weeks after the high-cholesterol diet. At the end of 8 weeks of a high-cholesterol diet, the rabbits were killed, and the carotid arteries were isolated. Bioactive nitric oxide was determined functionally (endothelium-dependent vasodilatation) and biochemically (the phosphorylation of vasodilator-stimulated phosphoprotein, or P-VASP). Vascular superoxide production, PPARgamma, gp91phox, and inducible nitric oxide synthase (iNOS) expression, and vascular ONOO- formation were determined. Hypercholesterolemia caused severe endothelial dysfunction and reduced P-VASP, despite a marked increase in iNOS expression and total NOx production. Treatment with rosiglitazone enhanced PPARgamma expression, improved endothelium-dependent vasodilatation, preserved P-VASP, suppressed gp91phox and iNOS expression, reduced superoxide and total NOx production, and inhibited nitrotyrosine formation. CONCLUSIONS: The PPARgamma agonist rosiglitazone exerted a significant vascular protective effect in hypercholesterolemic rabbits, most likely by attenuation of oxidative and nitrative stresses. The endothelial protective effects of PPARgamma agonists may reduce leukocyte accumulation in vascular walls and contribute to their antiatherosclerotic effect.

Activation of peroxisome proliferator-activated receptor-alpha protects the heart from ischemia/reperfusion injury.

BACKGROUND: Peroxisome proliferator-activated receptor-alpha (PPAR-alpha) is expressed in the heart and regulates genes involved in myocardial fatty acid oxidation (FAO). The role of PPAR-alpha in acute ischemia/reperfusion myocardial injury remains unclear. METHODS AND RESULTS: The coronary arteries of male mice were ligated for 30 minutes. After reperfusion for 24 hours, ischemic and infarct sizes were determined. A highly selective and potent PPAR-alpha agonist, GW7647, was administered by mouth for 2 days, and the third dose was given 1 hour before ischemia. GW7647 at 1 and 3 mg x kg(-1) x d(-1) reduced infarct size by 28% and 35%, respectively (P<0.01), and myocardial contractile dysfunction was also improved. Cardioprotection by GW7647 was completely abolished in PPAR-alpha-null mice. Ischemia/reperfusion downregulated mRNA expression of cardiac PPAR-alpha and FAO enzyme genes, decreased myocardial FAO enzyme activity and in vivo cardiac fat oxidation, and increased serum levels of free fatty acids. All of these changes were reversed by GW7647. Moreover, GW7647 attenuated ischemia/reperfusion-induced release of multiple proinflammatory cytokines and inhibited neutrophil accumulation and myocardial expression of matrix metalloproteinases-9 and -2. Furthermore, GW7647 inhibited nuclear factor-kappaB activation in the heart, accompanied by enhanced levels of inhibitor-kappaBalpha. CONCLUSIONS: Activation of PPAR-alpha protected the heart from reperfusion injury. This cardioprotection might be mediated through metabolic and antiinflammatory mechanisms. This novel effect of the PPAR-alpha agonist could provide an added benefit to patients treated with PPAR-alpha activators for dyslipidemia.