Cholesterol synthesis inhibitor U18666A and the role of sterol metabolism and trafficking in numerous pathophysiological processes.

The multiple actions of U18666A have enabled major discoveries in lipid research and contributed to understanding the pathophysiology of multiple diseases. This review describes these advances and the utility of U18666A as a tool in lipid research. Harry Rudney's recognition that U18666A inhibited oxidosqualene cyclase led him to discover a pathway for formation of polar sterols that he proved to be important regulators of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase.

Inhibition of fatty acid synthase by Orlistat accelerates gastric tumor cell apoptosis in culture and increases survival rates in gastric tumor bearing mice in vivo.

Orlistat, an anti-obesity drug, is a potent inhibitor of fatty acid synthase (FAS) and tumor cell viability. It can also induce apoptotic cancer cell death. We examined the effects of Orlistat on cultured NUGC-3 gastric cancer cells.

Status of caveolin-1 in various membrane domains of the bovine lens.

Recent studies of the distribution and relative concentration of caveolin-1 in fractions of bovine lens epithelial and fiber cells have led to the novel concept that caveolin-1 may largely exist as a peripheral membrane protein in some cells. Caveolin-1 is typically viewed as a scaffolding protein for caveolae in plasma membrane. In this study, membrane from cultured bovine lens epithelial cells and bovine lens fiber cells were divided into urea soluble and insoluble fractions.

Comparison of effects of U18666A and enantiomeric U18666A on sterol synthesis and induction of apoptosis.

Treatment of animals or cells with the amphipathic tertiary amine U18666A {3beta-[2-(diethylamino) ethoxy]androst-5-en-17-one} provides models for several human diseases (e.g., cataracts, Niemann-Pick disease, and epilepsy).

Multiple forms of 22 kDa caveolin-1 alpha present in bovine lens cells could reflect variable palmitoylation.

Two-dimensional immunoblots of immunoprecipitated caveolin-1 from cultured bovine lens epithelial cells revealed four to five-22 kDa forms of caveolin-1 alpha with isoelectric points of between pH values 5.5 and 6.6.

Concentration and distribution of ubiquinone (coenzyme Q), the endogenous lipid antioxidant, in the rat lens: effect of treatment with simvastatin.

Purpose: Ubiquinone (Ub) is the only known endogenously synthesized lipid soluble antioxidant. It is synthesized from intermediates in the cholesterol metabolic pathway. Our goal was to identify the Ubs and determine the concentration and distribution of Ubs in the rat lens and the effect of treatment with simvastatin, a cholesterol synthesis inhibitor, on lens levels.

Diethylstilbestrol increases intracellular calcium in lens epithelial cells.

The effects of diethylstilbestrol (DES) on steady-state intracellular calcium concentration ([Ca(2+)](i)) and resting Ca(2+) influx were examined in primary cultures of bovine lens epithelial cells using conventional fluorometric techniques (Fura-2). At low concentrations (10 microM), DES usually induced relatively rapid increases in [Ca(2+)](i) that occurred over an interval of 10-50 s and that persisted for several minutes in the continued presence of the drug. In about 10% of the cells, cyclic oscillations in [Ca(2+)](i) were seen after adding 10 microM DES.

Direct perturbation of lens membrane structure may contribute to cataracts caused by U18666A, an oxidosqualene cyclase inhibitor.

Induction of cataracts in experimental animals is a common toxic feature of oxidosqualene cyclase (OSC) inhibitors. U18666A has been shown to produce irreversible lens damage within a few weeks of treatment. Drug actions, besides reducing the availability of cholesterol, could contribute to cataract formation.

Distribution of caveolin-1 in bovine lens and redistribution in cultured bovine lens epithelial cells upon confluence.

The distribution of caveolin-1 in the lens and lens epithelial cells was determined to assess possible roles in cholesterol trafficking, cell to cell communication and signal transduction. Bovine lenses and cultured bovine lens epithelial cells (BLEC) were divided into subcellular fractions and the distribution of proteins recognized by three different caveolin-1 antibodies determined. The immunolocalization of caveolin-1 in the lens epithelium and in subconfluent and confluent cultured BLEC was probed by fluorescence microscopy and laser scanning confocal microscopy.

Discordant expression of the sterol pathway in lens underlies simvastatin-induced cataracts in Chbb: Thom rats.

Simvastatin rapidly induced cataracts in young Chbb:Thom (CT) but not Sprague Dawley (SD) or Hilltop Wistar (HW) rats. Oral treatment for 14 but not 7 days committed CT rat lenses to cataract formation. The cholesterol to phospholipid molar ratio in lenses of treated CT rats was unchanged.

Rapid, non-genomic actions of progesterone and estradiol on steady-state calcium and resting calcium influx in lens epithelial cells.

The effects of steroids on the steady-state intracellular [Ca(2+)] ([Ca(2+)](i)) and resting Ca(2+) influx in Fura-2-loaded bovine lens epithelial cells were examined to identify potential rapid, non-genomic actions. When administered in the presence of 1-2 mM extracellular Ca(2+) ([Ca(2+)](o)), 100 micro M progesterone produced large (up to 12-fold) and transient (5 min) increases in [Ca(2+)](i). These effects were abolished in EGTA-containing solutions, and were associated with large increases in the rate at which extracellularly administered Mn(2+) quenched the intracellular Fura signal.

Immunomagnetic capture of lens membrane fractions containing steroid binding protein.

This study describes the use of magnetic Dynabeads to purify microsomes from a crude microsomal fraction. A 28 kDa membrane-associated protein is proposed to mediate the binding of progesterone and other steroid hormones to ocular lens membranes and the rapid-nongenomic actions of these steroids. The subcellular location of this membrane steroid binding protein (MSBP) was probed by capture of organelles containing MSBP by magnetic beads displaying an antibody to a cytoplasmic domain of the protein.

Specific labeling of lens aldehyde dehydrogenase class 1 from (3)H-cholesterol or its derivatives.

We investigated the possibility that sterols could covalently modify ocular lens cell proteins. Incubation of cultured bovine lens epithelial cells (BLEC) with (3)H-cholesterol led to the labeling of a cytosolic protein of about 52 kD. Two-dimensional electrophoresis of the BLEC soluble proteins and fluorography revealed one labeled protein of 52 kD, pI = 6.

Characterization of membrane steroid binding protein mRNA and protein in lens epithelial cells.

Epithelial cells of the ocular lens contain a 28 kDa membrane protein which is proposed to mediate high affinity binding of steroid hormones and rapid non-genomic actions of steroid hormones. It has been named membrane steroid binding protein (MSBP). Our purpose was to further characterize this protein from cultured bovine lens epithelial cells (BLEC) and compare it to similar forms of the protein present in other species and tissues.

Evidence for distinct cholesterol domains in fiber cell membranes from cataractous human lenses.

Previous studies in our laboratory have provided direct evidence for the existence of distinct cholesterol domains within the plasma membranes of human ocular lens fiber cells. The fiber cell plasma membrane is unique in that it contains unusually high concentrations of cholesterol, with cholesterol to phospholipid (C/P) mole ratios ranging from 1 to 4. Since membrane cholesterol content is disturbed in the development of cataracts, it was hypothesized that perturbation of cholesterol domain structure occurs in cataracts.

Retinal structure and function in an animal model that replicates the biochemical hallmarks of desmosterolosis.

Desmosterolosis is a rare, autosomal recessive, human disease characterized by multiple congenital anomalies in conjunction with grossly elevated levels of desmosterol and markedly reduced levels of cholesterol in all bodily tissues. Herein, we evaluated retinal sterol composition, histology, and electrophysiological function in an animal model that exhibited the biochemical features of desmosterolosis, produced by treating pregnant rats and their progeny with U18666A, an inhibitor of desmosterol reductase. Treated rats had cataracts, were substantially smaller, and had markedly high levels of desmosterol and profoundly low levels of cholesterol in their retinas and other tissues compared to age-matched controls.

Cholesterol synthesis in the vertebrate retina: effects of U18666A on rat retinal structure, photoreceptor membrane assembly, and sterol metabolism and composition.

Treatment of neonatal rats with U18666A, an inhibitor of desmosterol delta24-reductase, results in accumulation of desmosterol (delta5,24) and depletion of cholesterol (delta5) in various bodily tissues and also causes cataracts. We evaluated the effects of U18666A on the sterol composition, de novo sterol synthesis, and histological structure of the retina. Neonatal Sprague-Dawley rats were injected subcutaneously with U18666A (15 mg/kg, in olive oil ) every other day from birth through 3 wk of age; in parallel, control rats received olive oil alone.

Direct evidence for immiscible cholesterol domains in human ocular lens fiber cell plasma membranes.

The molecular structure of human ocular lens fiber cell plasma membranes was examined directly using small angle x-ray diffraction approaches. A distinct biochemical feature of these membranes is their high relative levels of free cholesterol; the mole ratio of cholesterol to phospholipid (C/P) measured in these membranes ranges from 1 to 4. The organization of cholesterol in this membrane system is not well understood, however.

Lens epithelia contain a high-affinity, membrane steroid hormone-binding protein.

Purpose: To describe the serendipitous discovery of a high-affinity, membrane steroid-binding protein (MSBP) in lens epithelial cells and to examine the binding of progesterone to epithelial cell membranes.

Methods: Bovine lens epithelial cells (BLECs) were cultured in media containing 3H-mevalonolactone to examine protein prenylation by mevalonate-derived isoprenes. Cell proteins were divided into insoluble and soluble fractions, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and label detected by fluorography.

Human lens cholesterol concentrations in patients who used lovastatin or simvastatin.

Objective: To determine whether long-term therapeutic use of the hypocholesterolemic drugs lovastatin and simvastatin significantly alters the distribution and concentration of cholesterol in the human lens. Such changes might precede observable alterations in lens structure.

Methods: Pairs of lenses (9-13 pairs) from patients (age range, 46-81 years) who had been taking lovastatin or simvastatin before their death (estimated for the previous 2-4 years) and lenses from similarly aged control subjects were divided into outer cortex and inner cortex plus nucleus by dissolution in a detergent-containing buffer.

Intense myristoylation of a single protein in the ocular lens.

A single protein of the ocular lens was intensely myristoylated following short term incubation of cultured bovine lens epithelial cells and intact rat lenses with 3H-myristic acid. It was acidic (pI <5), about 19 kDa and present exclusively in the cytosol of both cultured epithelial cells and the epithelium of the young rat lens. Fiber cell proteins were not labeled.

Influence of cholesterol on the interaction of alpha-crystallin with phospholipids.

The influence of cholesterol on the binding of alpha-crystallin to pure phospholipid membranes was studied. The rationale of this investigation stems from two unique aspects of human lens cells: an unusually high level of cholesterol in the membranes and the specific binding of alpha-crystallin to membranes. In the absence of cholesterol, binding of alpha-crystallin liposomes composed of either sphingomyelin, disteroyl-phosphatidylcholine or egg-phosphatidylcholine caused a decrease in the fluorescence intensity and anisotropy of the fluorophore NBD-PE.

Prenylation of proteins by the intact lens.

Purpose: To describe and identify proteins prenylated by the intact rat lens.

Methods: Lenses from young rats were incubated for 24 hours in TC199 medium containing 22 microM lovastatin and 110 microCi/ml [3H]mevalonolactone. Proteins of the epithelium and fiber cells were separated by high-performance liquid chromatography (HPLC) and one- and two-dimensional electrophoresis, and the 3H label was detected by fluorography.