Harry W. Chen

Effects of 25-hydroxycholesterol and 7-ketocholesterol, inhibitors of sterol synthesis, administered orally to mice.

When 25-hydroxycholesterol or 7-ketocholesterol was fed to mice with the diet, growth was suppressed and mature mice lost weight. The effect of the 7-ketone upon body weight was effectively counteracted by cholesterol whereas cholestanol and beta-sitosterol were ineffective. Growth repression due to 25-hydroxycholesterol was only partially relieved by cholesterol. The effects of 25-hydroxycholesterol and 7-ketocholesterol upon body weight were related to an apparent effect upon appetite. However the sterols were not unpalatable since diets containing them were not rejected in favor of control diet. Intestinal sterol synthesis was inhibited soon after the administration of dietary 7-ketocholesterol or 25-hydroxycholesterol but inhibition decreased with prolonged feeding. When fed by gavage, the sterols suppressed intestinal sterol synthesis as soon as 2 h after administration. In contrast, cholesterol administered by gavage did not affect intestinal sterol synthesis during a 24 h test period. When fed with the diet 25-hydroxycholesterol and 7-ketocholesterol did not depress hepatic cholesterol synthesis beyond the low levels found in pair-fed controls. Inhibition of intestinal sterol synthesis was accompanied by a decrease in the concentration of cholesterol in the intestinal mucosa and, usually, by a drop in the molar ratio of cholesterol to phospholipids.

Inhibition of protein synthesis blocks the response to 25-hydroxycholesterol by inhibiting degradation of hydroxymethylglutaryl-CoAreductase

The activity of the rate-limiting enzyme of the cholesterol biosynthetic pathway, 3-hydroxy-3-methylglutaryl coenzyme A reductase in Chinese hamster ovary (CHO) cells decreased more rapidly in cells treated with 25-hydroxycholesterol alone (t 1/2 = 1.5 h) than in those incubated with cycloheximide alone (t 1/2 = 5 h). The inhibitory action of 25-hydroxycholesterol on reductase activity was reduced when the sterol and cycloheximide were added together, and was totally abolished when cells were preincubated with cycloheximide for 30 min before the addition of 25-hydroxycholesterol. The effect of puromycin was similar to that of cycloheximide. Treatment of cells with an inhibitor of RNA synthesis, i.e., actinomycin D or cordycepin, had little effect on hydroxymethylglutaryl-CoA reductase activity; however, preincubation of cells with these reagents also decreased the ability of 25-hydroxycholesterol to suppress the reductase activity. These data are consistent with a model which suggests (a) that 25-hydroxycholesterol inhibits the activity of hydroxymethylglutaryl-CoA reductase by repressing its synthesis, (b) that cycloheximide and puromycin affect hydroxymethylglutaryl-CoA reductase activity by blocking the de novo synthesis of the enzyme and by reducing the degradation of the preexisting enzyme, (c) that actinomycin D and cordycepin affect the supply of message for the continuous synthesis of at least one component of a system which degrades hydroxymethylglutaryl-CoA reductase, and (d) that one component of the degradative system has a half-life shorter than 0.5 h.

Effects of various oxygenated sterols on cellular sterol biosynthesis in chinese hamster lung cells resistant to 25-hydroxycholesterol

The effects of a wide variety of oxygenated sterols upon sterol biosynthesis and hydroxymethylglutaryl-CoA reductase (mevalonate: NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34) activity in a wild-type clone and in a 25-hydroxycholesterol-resistant clone of Chinese hamster lung (Dede) cells are described. Derivatives of cholesterol which were oxygenated in the 6, 7 or 15 positions of the sterol nucleus or in the 20, 22, 24 or 25 positions of the sterol side chain were shown to be potent inhibitors of sterol synthesis and reductase activity in the wild-type cells but none of these substitutions had any effect on the 25-hydroxycholesterol-resistant A2 clone. A 32-hydroxylated derivative of lanosterol also suppressed sterol synthesis and reductase activity in wild-type cells but had no significant effect upon the A2 line. It was also appraent that a complete sterol side chain was necessary for inhibitory activity. Studies of a wide range of inhibitory sterols indicated that there was a close correlation between their effects upon sterol synthesis and reductase activity and that their inhibitory action was specific for sterol biosynthesis since little effect was observed upon fatty acid or CO2 synthesis. Previous studies had shown that the uptake of 25-hydroxycholesterol by the resistant A2 line was unimpaired and the present results indicate that metabolism of this oxygenated sterol is also unaltered. These results, in conjunction with previous studies, suggest that the resistant A2 line is defective in feedback regulation of cholesterol synthesis and that all of the oxygenated sterols tested suppress the biosynthetic pathway through at least one common step.

Stimulation of sterol and DNA synthesis in leukemic blood cells by low concentrations of phytohemagglutinin

Abstract The response of leukemic cells from AKR/J mice to phytohemagglutinin (PHA) was compared with that of normal lymphocytes. PHA stimulated first cholesterol synthesis and then DNA synthesis in both lymphocytes and leukemic cells. The neoplastic cells were, however, much more sensitive to PHA, requiring less time and a lower concentration of the lectin for optimum stimulation as compared to lymphocytes. In fact, the amount of PHA which was required to activate lymphocytes to proliferate, as measured by increases in sterol and DNA synthesis, was inhibitory to leukemic cells. The basal level of cholesterol synthesis and the induction of cholesterol synthesis following PHA activation were depressed in lymphocytes and leukemic cells by treatment with 25-hydroxycholesterol and 7-ketocholesterol. These two oxygenated derivatives of cholesterol are known to be potent and specific inhibitors of sterol synthesis. Blockage of sterol synthesis by these reagents also abolished PHA-activated DNA synthesis in lymphocytes and leukemic cells. The results support the hypothesis that the synthesis of cholesterol is an important event leading to cell proliferation.

Stimulation of hepatic RNA synthesis in dwarf mice by ovine prolactin

Dwarf mice (dwdw) have a lower liver RNA to DNA ratio compared to their congenic littermates (+/?). Injection of ovine prolactin into dwarf mice increases the total RNA content of liver. Dwarf mice appear to incorporate less [3H]uridine into total hepatic RNA after pulse injection than normal mice. Prolactin first stimulates the synthesis of guanine-rich then uridine-rich RNA of the whole liver homogenate. In liver nuclei, incorporation of [14C]guanine into RNA after correction for the acid-soluble pool is significantly increased during the first 3 days of treatment, whereas [3H]uridine incorporation rises substantially only after 4–5 days of treatment. In mitochondria, which incorporate labeled precursors into RNA, prolactin stimulates mainly the synthesis of uridine-rich RNA. A single dose of prolactin (10 I.U.7 g body weight) stimulates the assembly of ribosomes into polyribosomes. Total amounts of ribosomes and polyribosomes continuously increase during the 5 days of prolactin treatment.

Cell-substratum and cell-monolayer adhesion are dependent upon cellular cholesterol biosynthesis

Abstract Incubation of Chinese hamster lung (Dede) cells in medium containing 25-hydroxycholesterol resulted in the suppression of cellular cholesterol biosynthesis and a decrease in the cellular concentration of cholesterol so that the cholesterol/phospholipid molar ratio was reduced to approx. 50% of the control value. When these ‘sterol-depleted’ cells were allowed to attach to a serum-coated glass substratum, an inhibition of the rate and extent of attachment relative to the controls was observed. After 1 h incubation, only 10–25% of the 25-hydroxycholesterol-treated cells were attached, compared with virtually 100% of the control cells. Similar results were obtained with Chinese hamster ovary cells and with mouse L cells. Inhibition of adhesion was dependent upon the dose of—and time of exposure to—the oxygenated sterol. This inhibition was prevented when either cholesterol or mevalonic acid (the product of the enzymatic reaction inhibited by 25-hydroxycholesterol) was added with the 25-hydroxycholesterol, indicating that the effect of the sterol was primarily due to suppression of cholesterol synthesis. In addition, the inhibitor had no effect on the growth of a 25-hydroxycholesterol-resistant mutant or on the rate or extent of its adhesion to the substratum. Other oxygenated sterols (e.g. 22-ketocholesterol, 7β-hydroxycholesterol, 20α-hydryxycholesterol and 5α-cholest-8(14)-en-3β-ol-15-one) effected qualitatively similar inhibitions of the rates of sterol synthesis and cell-substratum attachment. The decreased adhesion observed when cultures were treated with inhibitors of cholesterol synthesis was not alleviated at low temperatures, suggesting that the effects of the inhibitor could not be overcome by a temperature-dependent increase in membrane viscosity. The rate and extent of adhesion of either Chinese hamster lung cells or L cell to several cell monolayers was also reduced in response to sterol depletion. These results suggest that membrane lipids, particularly cholesterol, play a significant role in cell-substratum and cell-cell adhesion.

The role of cholesterol and its biosynthesis in lymphocyte proliferation and differentiation.

Most membranes of eucaryotes contain sterols with 27 to 30 carbon atoms. In mammalian cells the major membrane sterol is cholesterol (5-cholesten-3P-01). The physiochemical role of this molecule has been investigated intensively and is today fairly well understood, although some details still require further study. This subject has been reviewed and therefore we restrict ourselves to briefly summarizing the present state of knowledge on the role of membrane sterol in lymphocyte function. The relative distribution of the major membrane constituents in mammals, proteins, phospholipids, and cholesterol, varies considerably according to the types of membranes and cells. Nuclear, mitochondrial, and ergastoplasmic membranes contain relatively small amounts of cholesterol whereas the plasma membrane contains phos-

Effects of cholesterol derivatives on sterol biosynthesis.

Atherosclerosis is a condition marked by the formation of plaques in the endothelium of the arterial system which in the earliest stage are composed largely of cholesterol. The etiology of the disease is not known, although it appears that an elevated serum cholesterol level may contribute to the development and the progression of atherosclerosis in animals and in man. The production of atherosclerotic lesions in experimental animals on a cholesterol-supplemented diet suggested a connection between cholesterol and atherosclerosis.

SYNTHESIS, CELLULAR TURNOVER, AND MASS OF CHOLESTEROL

Publisher Summary The most important of cholesterol functions derives from its interaction with phospholipids to regulate the fluidity of the membranes. Cholesterol in the plasma membrane can derive from endogenous biosynthesis, or from exchange with exogenous cholesterol, usually in the form of serum lipoproteins. The proportion of cellular cholesterol derived from one or the other source is dependent on the composition of the incubation fluid or growth medium. Many methods for measuring cholesterol are available and are in common use for determining blood and tissue cholesterol levels. Nearly all of them involve the generation of a chromophore that is measured with a spectrophotometer. They vary in convenience, sensitivity, and specificity. The amount of esterified cholesterol is obtained by subtracting the value for free cholesterol from that for total cholesterol. Values can be expressed in terms of total cell protein, DNA, or cellular phospholipid.