Activation of Naturally Occurring Lecithin:Cholesterol Acyltransferase Mutants by a Novel Activator Compound.

Lecithin:cholesterol acyltransferase (LCAT) is a unique plasma enzyme able to esterify cholesterol, and it plays an important role in HDL maturation and promotion of reverse cholesterol transport. Familial LCAT deficiency (FLD; OMIM number 245900) is a rare recessive disease that results from loss-of-function mutations in the LCAT gene and has no cure. In this study, we assessed the in vitro efficacy of a novel small-molecule LCAT activator. Cholesterol esterification rate (CER) and LCAT activity were tested in plasma from six controls and five FLD homozygous carriers of various LCAT mutations at different doses of the compound (0.1, 1, and 10 µg/ml). In control plasma, the compound significantly increased both CER (P < 0.001) and LCAT activity (P = 0.007) in a dose-dependent manner. Both CER and LCAT activity increased by 4- to 5-fold, reaching maximum activation at the dose of 1 µg/ml. Interestingly, Daiichi Sankyo compound produced an increase in CER in two of the five tested LCAT mutants (Leu372--Arg and Val309--Met), while LCAT activity increased in three LCAT mutants (Arg147--Trp, Thr274--Ile and Leu372--Arg); mutant Pro254--Ser was not activated at any of the tested doses. The present findings form the basis for personalized therapeutic interventions in FLD carriers and support the potential LCAT activation in secondary LCAT defects. SIGNIFICANCE STATEMENT: We characterized the pharmacology of a novel small-molecule LCAT activator in vitro on a subset of naturally occurring LCAT mutants. Our findings form the basis for personalized therapeutic interventions for familial LCAT deficiency carriers, who can face severe complications and for whom no cure exists.

Discovery of novel pyrrole derivatives as potent agonists for the niacin receptor GPR109A.

Novel pyrrole derivatives were discovered as potent agonists of the niacin receptor, GPR109A. During the derivatization, compound 16 was found to be effective both in vitro and in vivo. The compound 16 exhibited a significant reduction of the non-esterified fatty acid in human GPR109A transgenic rats, and the duration of its in vivo efficacy was much longer than niacin.

Treatment of diabetes and atherosclerosis by inhibiting fatty-acid-binding protein aP2.

Adipocyte fatty-acid-binding protein, aP2 (FABP4) is expressed in adipocytes and macrophages, and integrates inflammatory and metabolic responses. Studies in aP2-deficient mice have shown that this lipid chaperone has a significant role in several aspects of metabolic syndrome, including type 2 diabetes and atherosclerosis. Here we demonstrate that an orally active small-molecule inhibitor of aP2 is an effective therapeutic agent against severe atherosclerosis and type 2 diabetes in mouse models. In macrophage and adipocyte cell lines with or without aP2, we also show the target specificity of this chemical intervention and its mechanisms of action on metabolic and inflammatory pathways. Our findings demonstrate that targeting aP2 with small-molecule inhibitors is possible and can lead to a new class of powerful therapeutic agents to prevent and treat metabolic diseases such as type 2 diabetes and atherosclerosis.

A novel inhibitor of stearoyl-CoA desaturase-1 attenuates hepatic lipid accumulation, liver injury and inflammation in model of nonalcoholic steatohepatitis.

Stearoyl-CoA desaturase-1 (SCD-1) catalyzes the biosynthesis of monounsaturated fatty acids, and their abnormality is possibly responsible for obesity, insulin resistance, hepatic steatosis and nonalcoholic steatohepatitis (NASH). A novel SCD-1 inhibitor, N-(2-hydroxy-2-phenylethyl)-6-[4-(2-methylbenzoyl)piperidin-1-yl]pyridazine-3-carboxamide, has been obtained. The compound inhibited liver SCD-1 activity and increased liver triglyceride accumulation in mice fed with non-fat, high-sucrose diets. In order to evaluate the effects of the SCD-1 inhibitor on NASH development, rats were fed with lipogenic methionine and choline-deficient (MCD) diets for 8 weeks. The SCD-1 inhibitor was administered once-daily at a dose of 30 or 100 mg/kg/d by oral gavage. Administration of a high dose of the SCD-1 inhibitor decreased triglyceride accumulation in the liver of NASH rats by 80%. Administration of a high dose of the SCD-1 inhibitor attenuated the increase of aspartate aminotransferase (AST) and alanine transaminase (ALT) by 86% and 78%, respectively. Hepatic steatosis, hepatocellular degeneration and inflammatory cell infiltration were histologically observed in the liver of NASH rats, and administration of the SCD-1 inhibitor ameliorated these crucial observations in NASH. In summary, an SCD-1 inhibitor ameliorated hepatic triglyceride accumulation, liver injury, hepatocellular degeneration and inflammation in experimental NASH models. These results suggest that SCD-1 maybe a promising target for the treatment of NASH.

Adipocyte/macrophage fatty acid binding proteins control integrated metabolic responses in obesity and diabetes.

Fatty acid binding proteins (FABPs) are cytosolic fatty acid chaperones whose biological role and mechanisms of action are not well understood. Here, we developed mice with targeted mutations in two related adipocyte FABPs, aP2 and mal1, to resolve their role in systemic lipid, glucose, and energy metabolism. Mice lacking aP2 and mal1 exhibited a striking phenotype with strong protection from diet-induced obesity, insulin resistance, type 2 diabetes, and fatty liver disease. These mice have altered cellular and systemic lipid transport and composition, leading to enhanced insulin receptor signaling, enhanced muscle AMP-activated kinase (AMP-K) activity, and dramatically reduced liver stearoyl-CoA desaturase-1 (SCD-1) activity underlying their phenotype. Taken together with the previously reported strong protection against atherosclerosis, these results demonstrate that adipocyte/macrophage FABPs have a robust impact on multiple components of metabolic syndrome, integrating metabolic and inflammatory responses in mice and constituting a powerful target for the treatment of these diseases.

Novel spiropiperidine-based stearoyl-CoA desaturase-1 inhibitors: Identification of 1'-{6-[5-(pyridin-3-ylmethyl)-1,3,4-oxadiazol-2-yl]pyridazin-3-yl}-5-(trifluoromethyl)-3,4-dihydrospiro[chromene-2,4'-piperidine].

Cyclization of the benzoylpiperidine in lead compound 2 generated a series of novel and highly potent spiropiperidine-based stearoyl-CoA desaturase (SCD)-1 inhibitors. Among them, 1'-{6-[5-(pyridin-3-ylmethyl)-1,3,4-oxadiazol-2-yl]pyridazin-3-yl}-5-(trifluoromethyl)-3,4-dihydrospiro[chromene-2,4'-piperidine] (19) demonstrated the most powerful inhibitory activity against SCD-1, not only in vitro but also in vivo (C57BL/6J mice). With regard to the pharmacological evaluation, 19 showed powerful reduction of the desaturation index in the plasma of C57BL/6J mice on a non-fat diet after a 7-day oral administration (q.d.) without causing notable abnormalities in the eyes or skin up to the highest dose (3mg/kg) in our preliminary analysis.

Unique profiles of changes in cell membrane fluidity during ethanol-induced yeast-to-pseudohyphal transition in Candida tropicalis.

A dimorphic transition from the yeast form to filamentous one in Candida tropicalis pK233 is triggered by the addition of ethanol into the glucose semi-defined liquid medium and the process of filamentation accompanies temporal depolarization of yeast cells. The transition is completely prevented by further supplementation of myo-inositol at the start of cultivation. The addition of ethanol caused an increase in membrane fluidity during the process of depolarization, and then fluidity was gradually lowered to the level equivalent with that of the stationary-phase yeast cells in accordance with filamentation. The increase in membrane fluidity of ethanol-induced cells appeared parallel with reduction in the content of membrane phosphatidylinositol, which was rich in saturated palmitic acid. Introduction of exogenous myo-inositol or 1 M sorbitol into the ethanol-supplemented culture at the start of cultivation restored yeast growth and the reduction of membrane fluidity occurred, coupled with the recovery of the phosphatidylinositol content.

Novel and potent inhibitors of stearoyl-CoA desaturase-1. Part II: Identification of 4-ethylamino-3-(2-hydroxyethoxy)-N-[5-(3-trifluoromethylbenzyl)thiazol-2-yl]benzamide and its biological evaluation.

The continuing investigation of SAR studies of 3-(2-hydroxyethoxy)-N-(5-benzylthiazol-2-yl)-benzamides as stearoyl-CoA desaturase-1 (SCD-1) inhibitors is reported. Our prior hit-to-lead effort resulted in the identification of 1a as a potent and orally efficacious SCD-1 inhibitor. Further optimization of the structural motif resulted in the identification of 4-ethylamino-3-(2-hydroxyethoxy)-N-[5-(3-trifluoromethylbenzyl)thiazol-2-yl]benzamide (37c) with sub nano molar IC(50) in both murine and human SCD-1 inhibitory assays. This compound demonstrated a dose-dependent decrease in the plasma desaturation index in C57BL/6J mice on a non-fat diet after 7 days of oral administration.

Neuromuscular stimulation ameliorates ischemia-induced walking impairment in the rat claudication model.

Intermittent claudication (IC) is the most common symptom of peripheral arterial disease which significantly deteriorates the quality of life of patients. Exercise training is by far the most effective treatment for IC; however, the underlying mechanisms remain elusive. To determine the local mechanisms by which exercise training improves walking performance in claudicants, we developed an implantable device to locally induce ischemic skeletal muscle contraction mimicking exercise via electrical stimulation (ES). Rats were assigned to four groups, Sham, Ischemia (Isch), Isch + exercise and Isch + ES groups. Following both unilateral femoral and iliac artery occlusion, rats showed sustained impairment of walking performance in the treadmill test. Chronic low-frequency ES of ischemic skeletal muscles for 2 weeks significantly recovered the occlusion-induced walking impairment in the rat claudication model. We further analyzed the ischemic skeletal muscles immunohistochemically following ES or exercise training; both ES and exercise training significantly increased capillaries in the ischemic skeletal muscles and shifted the muscle fibers toward oxidative types. These findings demonstrate that ES takes on common features of exercise in the rat claudication model, which may facilitate investigations on the local mechanisms of exercise-induced functional recovery.

Angiopoietin-like protein3 regulates plasma HDL cholesterol through suppression of endothelial lipase.

OBJECTIVE: A low level of high-density lipoprotein (HDL) in plasma has been recognized as an aspect of metabolic syndrome and as a crucial risk factor of cardiovascular events. However, the physiological regulation of plasma HDL levels has not been completely defined. Current studies aim to reveal the contribution of angiopoietin-like protein3 (angptl3), previously known as a plasma suppressor of lipoprotein lipase, to HDL metabolism. METHODS AND RESULTS: Angptl3-deficient mice showed low plasma HDL cholesterol and HDL phospholipid (PL), and which were increased by ANGPTL3 supplementation via adenovirus. In vitro, ANGPTL3 inhibited the phospholipase activity of endothelial lipase (EL), which hydrolyzes HDL-PL and hence decreases plasma HDL levels, through a putative heparin-binding site in the N-terminal domain of ANGPTL3. Post-heparin plasma in Angptl3-knockout mice had higher phospholipase activity than did that in wild-type mice, suggesting that the activity of endogenous EL is elevated in Angptl3-deficient mice. Furthermore, we established an ELISA system for human ANGPTL3 and found that plasma ANGPTL3 levels significantly correlated with plasma HDL cholesterol and HDL-PL levels in human subjects. CONCLUSIONS: Angptl3 acts as an inhibitor of EL and may be involved in the regulation of plasma HDL cholesterol and HDL-PL levels in humans and rodents.

Novel non-systemic inhibitor of ileal apical Na+-dependent bile acid transporter reduces serum cholesterol levels in hamsters and monkeys.

1-{7-[(1-(3,5-Diethoxyphenyl)-3-{[(3,5-difluorophenyl)(ethyl)amino]carbonyl}-4-oxo-1,4-dihydroquinolin-7-yl)oxy]heptyl}-1-methylpiperidinium bromide, R-146224, is a potent, specific ileum apical sodium-dependent bile acid transporter (ASBT) inhibitor; concentrations required for 50% inhibition of [3H]taurocholate uptake in human ASBT-expressing HEK-293 cells and hamster ileum tissues were 0.023 and 0.73 microM, respectively. In bile-fistula rats, biliary and urinary excretion 48 h after 10 mg/kg [14C]R-146224, were 1.49+/-1.75% and 0.14+/-0.05%, respectively, demonstrating extremely low absorption. In hamsters, R-146224 dose-dependently reduced gallbladder bile [3H]taurocholate uptake (ED50: 2.8 mg/kg). In basal diet-fed hamsters, 14-day 30-100 mg/kg R-146224 dose-dependently reduced serum total cholesterol (approximately 40%), high density lipoprotein (HDL) cholesterol (approximately 37%), non-HDL cholesterols (approximately 20%), and phospholipids (approximately 20%), without affecting serum triglycerides, associated with reduced free and esterified liver cholesterol contents. In normocholesterolemic cynomolgus monkeys, R-146224 specifically reduced non-HDL cholesterol. In human ileum specimens, R-146224 dose-dependently inhibited [3H]taurocholate uptake. Potent non-systemic ASBT inhibitor R-146224 decreases bile acid reabsorption by inhibiting the ileal bile acid active transport system, resulting in hypolipidemic activity.

Ethanol-induced pseudohyphal transition in the cells of Candida tropicalis: participation of phosphoinositide signal transduction.

Ethanol-induced pseudohyphal development in the cells of Candida tropicalis Pk233 was accompanied by the transient accumulation of inositol 1,4,5-trisphosphate (IP3) that occurred at an early growth stage. The concomitant addition of myo-inositol prevented the activation of IP3 accumulation and cancelled pseudohyphal development in the presence of ethanol. The addition of a specific phospholipase C inhibitor, U73 122, inhibited ethanol-induced pseudohyphal transition at the concentrations of subinhibitory levels of cell growth. Pseudohyphal development was also induced by the Ca2+ ionophore, A23 187 in the absence of ethanol. The effect of A23 187 on the development of pseudohyphae was little influenced by myo-inositol, but stimulated by concomitant addition of 12-O-tetradecanoylphorbol 13-acetate. These results suggest that ethanol activated phospholipase C in competition with myo-inositol, and the resulting IP3-Ca2+ and protein kinase C pathways of PI signal transduction may work in pseudohyphal transition.

Ca2+-releasing effect of cerivastatin on the sarcoplasmic reticulum of mouse and rat skeletal muscle fibers.

We analyzed the effect of HMG-CoA reductase inhibitors on Ca(2+) release from the sarcoplasmic reticulum (SR) using chemically skinned skeletal muscle fibers from the mouse and the rat. Cerivastatin (>20 microM) released Ca(2+) from the SR, while pravastatin showed only a little effect. The rates of Ca(2+) release were increased by cerivastatin at all Ca(2+) concentrations tested. Cerivastatin-induced Ca(2+) release in the presence of Ca(2+) was affected by adenosine monophosphate, Mg(2+), and procaine in essentially the same way as for caffeine-induced Ca(2+) release. The Ca(2+)-uptake capacity of the SR was reduced after co-treatment with ryanodine and cerivastatin at pCa 6.0 to a much greater extent than with ryanodine alone. Thus, cerivastatin-induced Ca(2+) release in the presence of Ca(2+) must be a result of the activation of the Ca(2+)-induced Ca(2+) release (CICR) mechanism of the ryanodine receptor. However, even when CICR was maximally inhibited by Mg(2+) and procaine, or in the practical absence of Ca(2+) (pCa >8), cerivastatin still caused Ca(2+) release. These results indicate that cerivastatin causes Ca(2+) release also by activating some other mechanism(s) in addition to the activation of CICR. Either or both of these effects might be related to its adverse effect, rhabdomyolysis.

Ceramide kinase, a novel lipid kinase. Molecular cloning and functional characterization.

Ceramide-1-phosphate is a sphingolipid metabolite that has been implicated in membrane fusion of brain synaptic vesicles and neutrophil phagolysosome formation. Ceramide-1-phosphate can be produced by ATP-dependent ceramide kinase activity, although little is known of this enzyme because it has not yet been highly purified or cloned. Based on sequence homology to sphingosine kinase type 1, we have now cloned a related lipid kinase, human ceramide kinase (hCERK). hCERK encodes a protein of 537 amino acids that has a catalytic region with a high degree of similarity to the diacylglycerol kinase catalytic domain. hCERK also has a putative N-myristoylation site on its NH(2) terminus followed by a pleckstrin homology domain. Membrane but not cytosolic fractions from HEK293 cells transiently transfected with a hCERK expression vector readily phosphorylated ceramide but not sphingosine or other sphingoid bases, diacylglycerol or phosphatidylinositol. This activity was clearly distinguished from those of bacterial or human diacylglycerol kinases. With natural ceramide as a substrate, the enzyme had a pH optimum of 6.0-7.5 and showed Michaelis-Menten kinetics, with K(m) values of 187 and 32 microm for ceramide and ATP, respectively. Northern blot analysis revealed that hCERK mRNA expression was high in the brain, heart, skeletal muscle, kidney, and liver. A BLAST search analysis using the hCERK sequence revealed that putative ceramide kinases (CERKs) exist widely in diverse multicellular organisms including plants, nematodes, insects, and vertebrates. Phylogenetic analysis revealed that CERKs are a new class of lipid kinases that are clearly distinct from sphingosine and diacylglycerol kinases. Cloning of CERK should provide new molecular tools to investigate the physiological functions of ceramide-1-phosphate.

A novel class of apical sodium-dependent bile acid transporter inhibitors: the amphiphilic 4-oxo-1-phenyl-1,4-dihydroquinoline derivatives.

A series of 4-oxo-1-phenyl-1,4-dihydroquinolines possessing a linker and an ammonio moiety were synthesized and found to inhibit the apical sodium-dependent bile acid transporter (ASBT). The potency of ASBT inhibition varied with the position and length of the linking tether. Compound 21e effectively lowered the total serum cholesterol levels in hamsters.