Severe hypercholesterolemia, hypertriglyceridemia, and atherosclerosis in mice lacking both leptin and the low density lipoprotein receptor.

Leptin-deficient mice (ob/ob) are an excellent murine model for obesity, insulin resistance, and diabetes, all of which are components of a multiple risk factor syndrome that, along with hypercholesterolemia, precipitates a potential high risk for atherosclerosis. In the current study, we show an unexpectedly severe hyperlipidemia in ob/ob mice on a background of low density lipoprotein receptor (LDLR) deficiency (-/-). Doubly mutant mice (LDLR-/-;ob/ob) exhibited striking elevations in both total plasma cholesterol (TC) and triglyceride (TG) levels (1715 +/- 87 and 1016 +/- 172 mg/dl, respectively), at age 3-4 months, resulting in extensive atherosclerotic lesions throughout the aorta by 6 months. Lipoprotein analyses revealed the elevated TC and TG levels to be due to a large increase in an apoB-containing broad-beta remnant lipoprotein fraction. While fasting, diet restriction, and low level leptin treatment significantly lowered TG levels, they caused only slight changes in TC levels. Hepatic cholesterol and triglyceride contents as well as mRNA levels of cholesterologenic and lipogenic enzymes suggest that leptin deficiency increased hepatic triglyceride production but did not change cholesterol production in ob/ob mice regardless of their LDLR genotype. These data provide evidence that the hypertriglyceridemia and hypercholesterolemia in the doubly mutant mice are caused by distinct mechanisms and point to the possibility that leptin might have some impact on plasma cholesterol metabolism, possibly through an LDLR-independent pathway. This model will be an excellent tool for future studies on the relationship between impaired fuel metabolism, increased plasma remnant lipoproteins, diabetes, and atherosclerosis.

Effect of fungal metabolites cytochalasans on lipid droplet formation in mouse macrophages.

Effects of seven cytochalasans including cytochalasins B, D and E and novel phenochalasins A and B were tested on cytosolic lipid droplet formation and neutral lipid synthesis in mouse peritoneal macrophages. Phenochalasin A inhibited lipid droplet formation in a dose-dependent manner at least up to 20 microM without any morphological changes in macrophages. Cytochalasins D and E also inhibited lipid droplet formation only in a narrow range of concentrations, around 1 and 0.1 microM, respectively. At higher concentrations they gave morphological changes in macrophages. The other four cytochalasans only showed severe morphological changes in macrophages. Phenochalasin A and cytochalasins D and E inhibited cholesteryl ester (CE) synthesis specifically with IC50 values of 0.61, 2.4 and 0.20 microM, respectively, while the other cytochalasans inhibited both CE and triacylglycerol syntheses. Thus, among the cytochalasans tested, phenochalasin A showed very specific inhibition of CE synthesis and gave the lowest morphological changes in macrophages, resulting in the best inhibitor of lipid droplet formation in macrophages.

Phenochalasins, inhibitors of lipid droplet formation in mouse macrophages, produced by Phomopsis sp. FT-0211.

Phomopsis sp. FT-0211, a soil isolate, was found to produce inhibitors of lipid droplet formation in mouse peritoneal macrophages. Structurally related new compounds designated phenochalasins A and B were isolated from the fermentation broth of the producing strain by solvent extraction, ODS column chromatography and preparative HPLC. Phenochalasin A caused a dose-dependent reduction in the number and size of lipid droplets in macrophages without any cytotoxic effect at least up to 20 microm. On the other hand, phenochalasin B showed inhibition of lipid droplet formation with a severe cytotoxic effect on macrophages.

Structure elucidation of fungal phenochalasins, novel inhibitors of lipid droplet formation in mouse macrophages.

The structures of phenochalasins A and B were elucidated by spectroscopic studies including various NMR measurements. Phenochalasins A and B have the cytochalasan skeleton of the 21,23-dioxa, 17,22-dione moiety containing unique phenyl and O-methyl phenyl residues at the C-10 position, respectively.

Structure-specific inhibition of cholesteryl ester transfer protein by azaphilones.

The effect of thirteen different fungal azaphilones, which have a common 6-iso-chromane-like ring, was tested on cholesteryl ester transfer protein (CETP) activity in vitro. Chaetoviridin B showed the most potent inhibitory activity with an IC50 value of < 6.2 microM, followed by sclerotiorin with an IC50 value of 19.4 microM. Rotiorin, chaetoviridin A and rubrorotiorin had moderate inhibitory activity (IC50 ; 30 approximately 40 microM), but others showed very weak or no inhibitory activity. The relationship between the structures and their inhibitory activity indicated that the presence of an electrophilic ketone(s) and/or enone(s) at both C-6 and C-8 positions in the isochromane-like ring is essential for eliciting CETP inhibitory activity. The transfer activity of both CE and TG was inhibited by sclerotiorin to approximately the same extent (IC50: 14.4 and 10.3 microM, respectively). A model of the reaction suggested that sclerotiorin reacts with a primary amine of amino acids such as lysine in the protein to form a covalent bond.

Beauveriolides, specific inhibitors of lipid droplet formation in mouse macrophages, produced by Beauveria sp. FO-6979.

Beauveria sp. FO-6979, a soil isolate, was found to produce inhibitors of lipid droplet formation in mouse peritoneal macrophages. A new compound beauveriolide III was isolated along with a known compound beauveriolide I from the fermentation broth of the producing strain by solvent extraction, ODS column chromatography, silica gel column chromatography and preparative HPLC. Beauveriolides I and III caused a reduction in the number and size of cytosolic lipid droplets in macrophages at 10 microM without any cytotoxic effect on macrophages.

Structure elucidation of fungal beauveriolide III, a novel inhibitor of lipid droplet formation in mouse macrophages.

The structure of fungal beauveriolide III, an inhibitor of lipid droplet formation in mouse macrophages, was elucidated to be cyclo-[(3S,4S)-3-hydroxy-4-methyloctanoyl-L-phenylalanyl-L-alanyl- D-allo-isoleucyl] by spectral analyses and chemical degradation.

Complete inhibition of mouse macrophage-derived foam cell formation by triacsin C.

Primary mouse peritoneal macrophages effectively take up and metabolize phosphatidylcholine/cholesterol liposomes containing a small amount of phosphatidylserine, which results in the massive accumulation in the cytoplasm of oil red O positive lipid droplets consisting of cholesteryl ester (CE) and triacylglycerol (TG) [Nishikawa et al. (1990) J. Biol. Chem. 265, 5226-5231]. A number of inhibitors of CE synthesis have been reported, but their effects on the lipid droplet formation have not been fully examined. Furthermore, the contribution of TG synthesis to lipid droplet formation has been poorly investigated. We have investigated the relationship between CE and TG syntheses and cytosolic lipid droplet formation in macrophages cultured in the presence of inhibitors with different modes of action. When macrophages were cultured with liposomes and [14C]oleic acid in the presence of triacsin C, a potent inhibitor of long chain acyl-CoA synthetase, both [14C]CE and [14C]TG syntheses were inhibited to similar extents with IC50 values of 0.19 and 0.10 microM, respectively. On the other hand, pregnenolone, a well-known inhibitor of cellular cholesterol transport, and CL-283,546, a potent inhibitor of acyl-CoA:cholesterol acyltransferase, inhibited [14C]CE synthesis specifically with IC50 values of 5.0 and 0.038 microM, respectively. Microscopic observation revealed that the inhibitors of cholesterol metabolism produce only a partial decrease in cytosolic lipid droplets even at the highest doses which cause almost complete inhibition of [14C]CE synthesis. However, the triacsin C-dose dependent inhibition of lipid droplet formation was almost complete at 0.59 microM, a concentration that inhibits [14C]CE and [14C]TG syntheses by about 90%. These results show that inhibiton of acyl-CoA synthetase by triacsin C causes a decrease in the cellular levels of acyl-CoA, the common substrate for CE and TG syntheses, leading to an inhibiton of neutral lipid synthesis and eventually to the complete disappearance of cytosolic lipid droplets from macrophages. These findings suggest that TG synthesis, as well as CE synthesis, is responsible for macrophage-derived foam cell formation, and is therefore a potential target for new antiatherosclerotic agents.

Pyripyropenes, novel ACAT inhibitors produced by Aspergillus fumigatus. IV. Structure elucidation of pyripyropenes M to R.

Six new pyripyropenes, M to R, were isolated from the ethyl acetate extracts of the jar fermentation broth of Aspergillus fumigatus FO-1289-2501. Structural elucidation indicated that all the pyripyropenes have the same pyridino-alpha-pyrone sesquiterpene core as pyripyropenes A to L. Among them pyripyropene M showed the most potent inhibition against acyl-CoA : cholesterol acyltransferase activity with an IC50 value of 3.80 microM in rat liver microsomes, but pyripyropenes N to R showed moderate inhibitory activity (IC50 11.0 approximately 78.0 microM).