Design, synthesis, modeling, biological evaluation and photoaffinity labeling studies of novel series of photoreactive benzamide probes for histone deacetylase 2

The design, modeling, synthesis, biological evaluation of a novel series of photoreactive benzamide probes for class I HDAC isoforms is reported. The probes are potent and selective for HDAC1 and 2 and are efficient in crosslinking to HDAC2 as demonstrated by photolabeling experiments. The probes exhibit a time-dependent inhibition of class I HDACs. The inhibitory activities of the probes were influenced by the positioning of the aryl and alkyl azido groups necessary for photocrosslinking and attachment of the biotin tag. The probes inhibited the deacetylation of H4 in MDA-MB-231 cell line, indicating that they are cell permeable and target the nuclear HDACs.     

Novel histone deacetylase 8 ligands without a zinc chelating group: Exploring an ‘upside-down’ binding pose

A novel series of HDAC8 inhibitors without a zinc-chelating hydroxamic acid moiety is reported. Photoaffinity labeling and molecular modeling studies suggest that these ligands are likely to bind in an ‘upside-down’ fashion in a secondary binding site proximal to the main catalytic site. The most potent ligand in the series exhibits an IC₅₀ of 28 μM for HDAC8 and is found to inhibit the deacetylation of H4 but not α-tubulin in SH-SY5Y cell line.     

The farnesoid X receptor modulates adiposity and peripheral insulin sensitivity in mice

The farnesoid X receptor (FXR) is a bile acid (BA)-activated nuclear receptor that plays a major role in the regulation of BA and lipid metabolism. Recently, several studies have suggested a potential role of FXR in the control of hepatic carbohydrate metabolism, but its contribution to the maintenance of peripheral glucose homeostasis remains to be established. FXR-deficient mice display decreased adipose tissue mass, lower serum leptin concentrations, and elevated plasma free fatty acid levels. Glucose and insulin tolerance tests revealed that FXR deficiency is associated with impaired glucose tolerance and insulin resistance. Moreover, whole-body glucose disposal during a hyperinsulinemic euglycemic clamp is decreased in FXR-deficient mice. In parallel, FXR deficiency alters distal insulin signaling, as reflected by decreased insulin-dependent Akt phosphorylation in both white adipose tissue and skeletal muscle. Whereas FXR is not expressed in skeletal muscle, it was detected at a low level in white adipose tissue in vivo and induced during adipocyte differentiation in vitro. Moreover, mouse embryonic fibroblasts derived from FXR-deficient mice displayed impaired adipocyte differentiation, identifying a direct role for FXR in adipocyte function. Treatment of differentiated 3T3-L1 adipocytes with the FXR-specific synthetic agonist GW4064 enhanced insulin signaling and insulin-stimulated glucose uptake. Finally, treatment with GW4064 improved insulin resistance in genetically obese ob/ob mice in vivo. Although the underlying molecular mechanisms remain to be unraveled, these results clearly identify a novel role of FXR in the regulation of peripheral insulin sensitivity and adipocyte function. This unexpected function of FXR opens new perspectives for the treatment of type 2 diabetes.     

Continuous monitoring of cholesterol oleate hydrolysis by hormone-sensitive lipase and other cholesterol esterases

Hormone-sensitive lipase (HSL) contributes importantly to the hydrolysis of cholesteryl ester in steroidogenic tissues, releasing the cholesterol required for adrenal steroidogenesis. HSL has broad substrate specificity, because it hydrolyzes triacylglycerols (TAGs), diacylglycerols, monoacylglycerols, and cholesteryl esters. In this study, we developed a specific cholesterol esterase assay using cholesterol oleate (CO) dispersed in phosphatidylcholine and gum arabic by sonication. To continuously monitor the hydrolysis of CO by HSL, we used the pH-stat technique. For the sake of comparison, the hydrolysis of CO dispersion was also tested using other cholesteryl ester-hydrolyzing enzymes. The specific activities measured on CO were found to be 18, 100, 27, and 3 micromol/min/mg for HSL, cholesterol esterase from Pseudomonas species, Candida rugosa lipase-3, and cholesterol esterase from bovine pancreas, respectively. The activity of HSL on CO is approximately 4- to 5-fold higher than on long-chain TAGs. In contrast, with all other enzymes tested, the rates of TAG hydrolysis were higher than those of CO hydrolysis. The relatively higher turnover of HSL on CO observed in vitro adds further molecular insight on the physiological importance of HSL in cholesteryl ester catabolism in vivo. Thus, HSL could be considered more as a cholesteryl ester hydrolase than as a TAG lipase.     

Sensitive assay for hormone-sensitive lipase using NBD-labeled monoacylglycerol to detect low activities in rat adipocytes

The recent finding that p-nitrobenzofurazan (NBD)-FA is incorporated into and released from the acylglycerols of isolated rat adipocytes in an insulin-sensitive manner [G. Muller, H. Jordan, C. Jung, H. Kleine, and S. Petry. 2003. Biochimie. 85: 1245-1246] suggests that NBD-FA-labeled acylglycerols are cleaved by rat adipocyte hormone-sensitive lipase (HSL) in vivo. In the present study, we developed a continuous, sensitive in vitro lipase assay using a monoacylglycerol (MAG) containing NBD (NBD-MAG). NBD-MAG was found to provide an efficient substrate for rat adipocyte and human recombinant HSL. Ultrasonic treatment applied in the presence of phospholipids leads to the incorporation of NBD-MAG into the phospholipid liposomes and to a concomitant change of its spectrophotometric properties. The enzymatic release of NBD-FA and its dissociation from the carrier liposomes is accompanied by the recovery of the original spectrophotometric characteristics. The rate of lipolysis was monitored by measuring the increase in optical density at 481 nm, which was found to be linear with time and linearly proportional to the amount of lipase added. To assess the specific activity of recombinant HSL, we determined the molar extinction coefficient of NBD-FA under the assay conditions. This convenient assay procedure based on NBD-MAG should facilitate the search for small molecule HSL inhibitors.     

Hydrous and photosynthetic adaptations of common and durum wheat to saline stress

Seven varieties of bred wheat and seven varieties of durum wheat were cultivated in three different sites from the area of Errachidia (southeastern Morocco). These sites differ by the degree of salinity in the irrigation water. Results obtained showed that the reduction in leaf area is the principal strategy that makes it possible to attenuate the effects of the reduction in the availability of water under saline stress. Bread wheat, which limited the reduction in the leaf area, with the risk to undergo some hydrous problems, seems to better preserve its photosynthetic potentialities and grain productivity.     

Antioxidant status and oxidative stress at rest and in response to acute exercise in judokas and sedentary men

It is well recognized that acute strenuous exercise is accompanied by an increase in free-radical production and subsequent oxidative stress, in addition to changes in blood antioxidant status. Chronic exercise provides protection against exercise-induced oxidative stress by upregulating endogenous antioxidant defense systems. Little is known regarding the protective effect afforded by judo exercise. Therefore, we determined antioxidant and oxidative stress biomarkers at rest and in response to acute exercise in 10 competitive judokas and 10 sedentary subjects after mixed exercise (anaerobic followed by aerobic). The subjects performed a Wingate test, followed by 30 minutes of aerobic exercise performed at 60% of maximal aerobic power. Blood samples were taken, by an intravenous catheter, at rest (R), immediately after the physical exercise (P0), and at 5 (P5), 10 (P10), and 20 (P20) minutes postexercise. The measured parameters included the activity of the antioxidant enzymes superoxide dismutase, glutathione peroxidase, and glutathione reductase, in addition to α-tocopherol, and total antioxidant status. Malondialdehyde was measured as a representation of lipid peroxidation. At rest, the judokas had higher values for all antioxidant and oxidative stress markers as compared to the sedentary subjects (p < 0.05). Plasma concentrations of all parameters except for α-tocopherol increased significantly above resting values for both the judokas and sedentary subjects (p < 0.05) and remained elevated at 20 minutes postexercise. A significant postexercise decrease was observed for α-tocopherol (p < 0.05) at P20 for judokas and at P5 for sedentary subjects. These data indicate that competitive judo athletes have higher endogenous antioxidant protection compared to sedentary subjects. However, both groups of subjects experience an increase in exercise-induced oxidative stress that is not different.