Search

Hedychium coronarium Koen., commonly known as ginger lily, is considered an endemic medicinal plant. In the present study, the antidiabetic action of its rhizomes was investigated by α-amylase and α-glucosidase inhibition assay, and the active compounds were identified through bioactivity guided isolation technique. Among the six different extracts, the EA extract has shown highest inhibition, and the subfractions from active EA extract were separated by silica gel column chromatography. The subfraction showing highest inhibition was investigated for its chemical composition by high-resolution liquid chromatography-mass spectroscopy (HRLC-MS/MS). The fatty acids such as suberic acid and terpenes such as triparanol, ginkgolide C, and swietenine were found to be the major compounds in the subfractions. The present work revealed that H. coronarium rhizome extract and its active constituent could be used as a natural inhibitor of these two carbohydrate-metabolizing enzymes and may play a key role in the management of diabetes.

Channels responsible for slowly activating delayed-rectifier potassium current (I(Ks)) are composed of KCNQ1 and KCNE1 subunits, and these channels play a role in the repolarization of cardiac action potentials. Recently, we showed that the antihyperlipidemic drug probucol, which induces QT prolongation, decreases the I(Ks) after 24-h treatment. In the present study, we investigated the effects of three cholesterol-lowering agents (probucol, an enhancer of cholesterol efflux; simvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor; and triparanol, a 3β-hydroxysterol-▵24-reductase inhibitor) on cholesterol synthesis, the KCNQ1 current (I KCNQ1), and the I(Ks) to clarify the differences in the modes of action of these agents on the I(Ks). Probucol did not inhibit cholesterol synthesis and had no effect on I KCNQ1, while I(Ks) decreased after 24-h treatment. Simvastatin inhibited cholesterol synthesis and decreased I KCNQ1 and I(Ks). Additionally, the activation kinetics of I(Ks) became faster, compared with that of control I(Ks). Triparanol inhibited cholesterol synthesis but did not reduce I KCNQ1 and I(Ks). However, the activation kinetics of I(Ks) became faster. Our data indicated that the mechanism by which probucol inhibits I(Ks) was not mediated by the inhibition of cholesterol synthesis but depended on an interaction with the KCNQ1/KCNE1 complex. Meanwhile, the reduction in cholesterol induced by simvastatin and triparanol is one of the mechanisms that affects the kinetics of I(ks).

Desmoid tumor (also called aggressive fibromatosis) is a lesion of mesenchymal origin that can occur as a sporadic tumor or a manifestation of the preneoplastic syndrome, familial adenomatous polyposis caused by a mutation in adenomatous polyposis coli (APC). This tumor type is characterized by the stabilization of β-catenin and activation of Tcf-mediated transcription. Cell transplantation data suggest that desmoid tumors are derived from mesenchymal progenitor cells (MSCs). As such, modulating cell signaling pathways that regulate MSC differentiation or proliferation, such as hedgehog (Hh) signaling, could alter the tumor phenotype. Here, we found that Hh signaling is activated in human and murine desmoid tumors. Inhibiting Hh signaling in human cell cultures decreased cell proliferation and β-catenin protein levels. Apc(+)/Apc(1638N) mice, which develop desmoid tumors, develop smaller and fewer tumors when Hh signaling was inhibited either genetically (by crossing Apc(+)/Apc(1638N) mice with mice lacking one copy of a Hh-activated transcription factor, Gli2 (+/-) mice) or using a pharmacologic inhibitor. Both in mice and in human tumor cell cultures, β-catenin and Hh-mediated signaling positively regulate each other's activity. These data show that targeting a pathway that regulates MSC differentiation influences desmoid tumor behavior, providing functional evidence supporting the notion that these tumors are derived from mesenchymal progenitors. It also suggests Hh blockade as a therapeutic approach for this tumor type.

The cell cycle is a ubiquitous, multi-step process that is essential for growth and proliferation of cells. The role of membrane lipids in cell cycle regulation is not explored well, although a large number of cytoplasmic and nuclear regulators have been identified. We focus in this work on the role of membrane cholesterol in cell cycle regulation. In particular, we have explored the stringency of the requirement of cholesterol in the regulation of cell cycle progression. For this purpose, we utilized distal and proximal inhibitors of cholesterol biosynthesis, and monitored their effect on cell cycle progression. We show that cholesterol content increases in S phase and inhibition of cholesterol biosynthesis results in cell cycle arrest in G1 phase under certain conditions. Interestingly, G1 arrest mediated by cholesterol biosynthesis inhibitors could be reversed upon metabolic replenishment of cholesterol. Importantly, our results show that the requirement of cholesterol for G1 to S transition is absolute, and even immediate biosynthetic precursors of cholesterol, differing with cholesterol merely in a double bond, could not replace cholesterol for reversing the cell cycle arrest. These results are useful in the context of diseases, such as cancer and Alzheimer's disease, that are associated with impaired cholesterol biosynthesis and homeostasis.

Despite the improved contemporary multidisciplinary regimens treating cancer, majority of cancer patients still suffer from adverse effects and relapse, therefore posing a significant challenge to uncover more efficacious molecular therapeutics targeting signaling pathways central to tumorigenesis. Here, our study have demonstrated that Triparanol, a cholesterol synthesis inhibitor, can block proliferation and induce apoptosis in multiple human cancer cells including lung, breast, liver, pancreatic, prostate cancer and melanoma cells, and growth inhibition can be rescued by exogenous addition of cholesterol. Remarkably, we have proved Triparanol can significantly repress Hedgehog pathway signaling in these human cancer cells. Furthermore, study in a mouse xenograft model of human lung cancer has validated that Triparanol can impede tumor growth in vivo. We have therefore uncovered Triparanol as potential new cancer therapeutic in treating multiple types of human cancers with deregulated Hedgehog signaling., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Like many solid tumors, sarcomas are heterogeneous and include a small fraction of the so-called side population (SP) cells with stem-like tumor-initiating potential. Here, we report that SP cells from a soft tissue tumor of enigmatic origin termed undifferentiated pleomorphic sarcoma (also known as malignant fibrous histiocytoma or MFH sarcoma) display activation of both the Hedgehog and Notch pathways. Blockade to these pathways in murine xenograft models, this human cancer decreased the proportion of SP cells present and suppressed tumor self-renewal, as illustrated by the striking inability of xenograft tumors subjected to pathway blockade to be serially transplanted to new hosts. In contrast, conventional chemotherapies increased the proportion of SP cells present in tumor xenografts and did not affect their ability to be serially transplanted. SP cells from these tumors displayed an unexpectedly high proliferation rate which was selectively inhibited by Hedgehog and Notch blockade compared with conventional chemotherapies. Together, our findings deepen the concept that Hedgehog and Notch signaling are fundamental drivers of tumor self-renewal, acting in a small population of tumor-initiating cells present in tumors. Furthermore, our results suggest not only novel treatment strategies for deadly recurrent unresectable forms of this soft tumor subtype, but also potential insights into its etiology which has been historically controversial.

Lipid rafts are plasma membrane microdomains that are highly enriched with cholesterol and sphingolipids and in which various receptors and other proteins involved in signal transduction reside. In the present work, we analyzed the effect of cholesterol biosynthesis inhibition on lipid raft/caveolae composition and functionality and assessed whether sterol precursors of cholesterol could substitute for cholesterol in lipid rafts/caveolae. 3T3-L1 preadipocytes were treated with distal inhibitors of cholesterol biosynthesis or vehicle (control) and then membrane rafts were isolated by sucrose density gradient centrifugation. Inhibition of cholesterol biosynthesis with either SKF 104976, AY 9944, 5,22-cholestadien-3beta-ol or triparanol, which inhibit different enzymes on the pathway, led to a marked reduction in cholesterol content and accumulation of different sterol intermediates in both lipid rafts and non-raft domains. These changes in sterol composition were accompanied by disruption of lipid rafts, with redistribution of caveolin-1 and Fyn, impairment of insulin-Akt signaling and the inhibition of insulin-stimulated glucose transport. Cholesterol repletion abrogated the effects of cholesterol biosynthesis inhibitors, reflecting they were specific. Our results show that cholesterol is required for functional raft-dependent insulin signaling.

Chondrosarcoma is a malignant cartilage tumor that may arise from benign precursor lesions, such as enchondromas. Some cases of multiple enchondromas are caused by a mutation that results in constitutive activation of Hedgehog-mediated signaling. We found that chondrosarcomas expressed high levels of the Hedgehog target genes PTCH1 and GLI1. Treatment with parathyroid hormone-related protein down-regulated Indian Hedgehog (IHH) expression in normal growth plates but not in chondrosarcoma or enchondroma organ cultures. Treatment of the chondrosarcoma organ cultures with Hedgehog protein increased cell proliferation rate, whereas addition of chemical inhibitors of Hedgehog signaling decreased the proliferation rate. Chondrosarcoma xenografts from 12 different human tumors were established in NOD-SCID mice. Treatment with triparanol, an inhibitor of Hedgehog signaling, resulted in a 60% decrease in tumor volume, a 30% decrease in cellularity, and a 20% reduction in proliferation rate. These results show that Hedgehog signaling is active in chondrosarcoma and benign cartilage tumors and regulates tumor cell proliferation. Our data raise the intriguing possibility that Hedgehog blockade could serve as an effective treatment for chondrosarcoma, a tumor for which there are currently no universally effective nonsurgical management options.

Synovial chondromatosis is a condition affecting joints in which metaplastic cartilage nodules arise from the synovium, causing pain, joint dysfunction, and ultimately joint destruction. Because dysregulation of hedgehog signalling is a feature of several benign cartilaginous tumours, expression of the hedgehog target genes PTC1 and GLI1 was examined in this study in samples from human synovial chondromatosis. Significantly higher expression levels were found in synovial chondromatosis than in the synovium, from which it arises. To determine if hedgehog-mediated transcription predisposes to synovial chondromatosis, the extra-toes mutant mouse, which harbours a heterozygous mutation in the hedgehog transcriptional repressor, Gli3, resulting in decreased expression of Gli3 protein, was studied. The extra-toes mutant mouse has a phenotype consistent with overactive hedgehog signalling, suggesting that Gli3 acts as a transcriptional repressor of limb development. Eighty-five per cent of Gli3 mutant mice developed synovial chondromatosis at 18 months of age, compared with 30% of wild-type littermates (p < 0.05). Three of the ten Gli3 mutant mice treated with triparanol, which blocks hedgehog signalling upstream of the Gli transcription factors, developed synovial chondromatosis, compared with eight of ten control mice. These data demonstrate that hedgehog signalling plays an important role in the development of synovial chondromatosis and suggest that blockade of hedgehog signalling may be a potential treatment for this disorder., (Copyright 2005 Pathological Society of Great Britain and Ireland)

Human disorders caused by inborn errors of cholesterol biosynthesis are characterized by dysmorphogenesis of multiple organs. This includes limb malformations that are observed at high frequency in some disorders, such as the Smith-Lemli-Opitz syndrome, indicating a pivotal role of cholesterol in limb morphogenesis. Recently, it has been demonstrated that cholesterol can modulate the activity of the Hedgehog proteins, that act as morphogens to regulate the precise patterning of many embryonic structures, among which the developing limbs. To provide insight in the functions of cholesterol during limb development and in the potential role of Hedgehog signaling in the genesis of limb defects, we developed an in vivo rat model of cholesterol deficiency. We show here that treatment with Triparanol, a distal inhibitor of cholesterol biosynthesis, induced patterning defects of the autopod at high frequency, including pre-axial syndactyly and post-axial polydactyly, thus reproducing limb anomalies frequently observed in humans. Using in situ hybridization, we show that these malformations originate from a modification of Sonic Hedgehog signaling in the limb bud at 13 days post-coitum, leading to a deficiency of the anterior part of the limb. This deficiency results in an imbalance of Indian Hedgehog expression in the forming cartilage, ultimately leading to reduced interdigital apoptosis and syndactyly. Our study thus unravels the molecular mechanisms underlying the genesis of limb defects associated with cholesterol deficiency in rodents, and most probably in humans.

Triparanol (Trp) is known to cause clinical features similar to those seen in myotonic dystrophy, including myotonia, cataract and baldness. To explore the pathophysiological mechanism of myotonic dystrophy, we examined the effect of Trp on intracellular calcium in cultured skeletal myoblasts and myotubes as well as cardiac myocytes by using a fluorescent indicator. Trp preferentially induced increase of intracellular calcium in myotubes of skeletal muscles. Since the increase of calcium was inhibited by thapsigargin pretreatment but not by extracellular calcium elimination, it appears that triparanol might act mostly on intracellular calcium stores. Trp also inhibited the increase of calcium in myotubes induced by acetylcholine. Trp might cause myotonia possibly through the increase of intracellular calcium from intracellular stores.

Veratrum alkaloids and distal inhibitors of cholesterol biosynthesis have been studied for more than 30 years as potent teratogens capable of inducing cyclopia and other birth defects. Here, it is shown that these compounds specifically block the Sonic hedgehog (Shh) signaling pathway. These teratogens did not prevent the sterol modification of Shh during autoprocessing but rather inhibited the response of target tissues to Shh, possibly acting through the sterol sensing domain within the Patched protein regulator of Shh response.

1. The sigma-drug binding site of guinea-pig liver is carried by a protein which shares significant amino acid sequence similarities with the yeast sterol C8-C7 isomerase (ERG2 protein). Pharmacologically-but not structurally-the sigma 1-site is also related to the emopamil binding protein, the mammalian sterol C8-C7 isomerase. We therefore investigated if sterol C8-C7 isomerase inhibitors are high affinity ligands for the (+)-[3H]-pentazocine labelled sigma 1-binding site. 2. Among the compounds which bound with high affinity to native hepatic and cerebral as well as to yeast expressed sigma 1-binding sites were the agricultural fungicide fenpropimorph (Ki 0.005 nM), the antihypocholesterinaemic drugs triparanol (Ki 7.0 nM), AY-9944 (Ki, 0.46 nM) and MDL28,815 (Ki 0.16 nM), the enantiomers of the ovulation inducer clomiphene (Ki 5.5 and 12 nM, respectively) and the antioestrogene tamoxifen (Ki 26 nM). 3. Except for tamoxifen these affinities are essentially identical with those for the [3H]-ifenprodil labelled sterol C8-C7 isomerase of S. cerevisiae. This demonstrates that sigma 1-binding protein and yeast isomerase are not only structurally but also pharmacologically related. Because of its affiliations with yeast and mammalian sterol isomerases we propose that the sigma 1-binding site is localized on a sterol isomerase related protein, involved in postsqualene sterol biosynthesis.

The serum total bile acid concentration was measured in rhesus monkeys fed diets practically free of cholesterol and with added cholesterol at two levels. Also, the effects of inhibiting cholesterol absorption by feeding plant sterols and inhibiting cholesterol synthesis by feeding triparanol upon the serum total bile acid levels were studied. Cholesterol feeding significantly increased the serum bile acid concentration. The serum bile acid level was decreased in the high responders fed plant sterols but only when the diet contained the highest level of cholesterol. In both groups serum bile acid levels were not altered when cholesterol biosynthesis was inhibited by feeding triparanol. It is suggested that cholesterol feeding increases the serum bile acid level probably due to an increase in the intestinal pool of bile acids as a result of increased production of bile acids in the liver and their excretion into the bile.

Zymosterol (5 alpha-cholesta-8(9),24-dien-3 beta-ol) comprised a negligible fraction of the mass of sterol in cultured human fibroblasts but was well labeled biosynthetically with radioactive acetate. Treatment of cells with triparanol, a potent inhibitor of sterol delta 24-reductase, led to a marked increase in labeled zymosterol while its mass rose to 1 mol% of total sterol. All of this sterol could be chased into cholesterol. Furthermore, cell homogenates converted exogenous radiolabeled zymosterol to cholesterol. Three lines of evidence suggested that biosynthetically labeled zymosterol was associated with the plasma membrane. 1) About 80% of radiolabeled zymosterol was oxidized by the impermeant enzyme, cholesterol oxidase, in glutaraldehyde-fixed intact cells. 2) Sucrose density gradient analysis of homogenates showed that the equilibrium buoyant density profile of newly synthesized zymosterol was identical with that of the plasma membrane. 3) Newly synthesized zymosterol was transferred as readily from fixed intact fibroblasts to exogenous acceptors as was cholesterol. Given that cholesterol is synthesized within the cell, it is unclear why most of the zymosterol is in the plasma membrane. The pathway of cholesterol biosynthesis may compel zymosterol to flux through the plasma membrane. Alternatively, plasma membrane zymosterol may represent a separate pool, in equilibrium with the zymosterol in the intracellular biosynthetic pool.

Paramecium requires oleic acid for growth and can grow in media containing no other fatty acids. In the present study, we have shown that this ciliate utilized oleate mainly as a carbon and energy source, even though this fatty acid was the only substrate available for synthesis of polyunsaturated fatty acids. Culture growth was inhibited by the addition of the drug triparanol. Triparanol decreased the formation of polyunsaturated fatty acids from oleate by preventing desaturation to form the dienoic acid, linoleate. Triparanol inhibition resulted in an altered phospholipid fatty acyl composition, an increased fragility and an altered behavioral response of the cells to a depolarizing stimulation solution. Therefore, although most of the dietary oleate was not used by the cells for polyunsaturated fatty acid synthesis, the desaturation of oleic acid was critical for normal culture growth, cell integrity and swimming behavior, all of which are expected to be dependent on normal membrane lipid composition.

The tetracyclic sterol precursors, cyclolaudenol, cycloartenol and lanosterol, inhibit efficiently the tetrahymanol biosynthesis in the ciliate Tetrahymena pyriformis, as reported earlier for cholesterol and other sterols. The prokaryotic bacteriohopanetetrols have little effect, and diplopterol, another hopanoid, as well as the carotenoid, canthaxanthin, have no effect. In the presence of triparanol, a hypocholesterolemic drug inhibiting the squalene cyclase of T. pyriformis and modifying the fatty acid metabolism, the cells do not grow further, but growth can be restored by the addition to the culture medium of suitable polyterpenoids. Thus, growth in presence of triparanol (13 microM) is almost normal after addition of a sterol such as sitosterol and cyclolaudenol, and longer lag times and lower absorbances than those of untreated cultures are observed in presence of cyclartenol, lanosterol, euphenol (a lanosterol isomer), bacteriohopanetetrols and three carotenoids. No growth at all is observed in the presence of tetrahymanol and diplopterol, although these triterpenoids are the normal reinforcers of the ciliate, probably because of a poor bioavailability. Thus, structurally different polyterpenoids are (at least partially) functionally equivalent and capable of replacing tetrahymanol or sterols and might act as membrane reinforcers in T. pyriformis cells.

Cultured cells (chicken embryo liver cells and rat embryo fibroblasts) were treated with triparanol (MER-29) for various lengths of time. Both types of cells have developed numerous membranous whorls-myeloid bodies in the cytoplasm. Various stages in myeloid bodies development are described. Acid phosphatase activity was cytochemically demonstrated within the myeloid bodies, indicating their lysosmal nature. This activity appeared only at a late stage of the myeloid bodies formation.

The absorption of three basic drugs (morphine, butylscopolamine and mecamylamine) and an acidic drug (phenobarbitone) from the rat small intestine in situ was investigated by using a single perfusion technique. The effect of intestinal damage on absorption was studied by treating rats with triparanol 25-50 mg/kg every 24 h for three weeks. Treatment with triparanol decreased the cholesterol concentration in the intestinal wall. The absorption of morphine and mecamylamine was increased by treatment with triparanol, whereas the absorption of butylscopolamine was decreased and that of phenobarbitone remained unaltered. Treatment with triparanol decreased the concentration of mecamylamine in the intestinal wall, but the concentrations of other drugs were unchanged. When comparing the present in situ and previous in vitro results the decreased absorption of butylscopolamine after triparanol in situ was opposite to the finding in vitro. The increased absorption of morphine and unaltered absorption of phenobarbitone were in accordance with the finding in vitro. In situ the absorption of mecamylamine was increased, although in vitro it was unchanged. The structural damage, differences in composition of the intestinal wall and intestinal blood flow are supposed to be the reasons for changes in absorption.

Diethylstilbestrol, clomiphene, ethamoxytriphetol and triparanol were 0.18, 1.0, 0.02 and 0.01 times as potent in the in vitro inhibition of prostaglandin synthetase, respectively as was indomethacin. In the in vivo carrageenan-induced rat paw edema studies, diethylstilbestrol was more potent as an anti-inflammatory agent than was clomiphene, and ethamoxytriphetol and triparanol were only marginally effective. The most potent of the compounds tested was indomethacin. The results reported demonstrate that the nonsteroidal anti-inflammatory agents and the nonsteroidal estrogens and anti-estrogens share the property of inhibition of prostaglandin synthetase.

1. Functional and biochemical studies were performed on the small intestine of control rats, and the results were compared with similar studies on animals given triparanol at a dosage of 0.114 mmol/kg daily for 10 days. The animals given triparanol were fed with either standard rat food or a gluten-free diet. 2. By using a recirculating-perfusion technique in vivo, it was shown that absorption of galactose from an 8 mmol/l solution was impaired in the ileum but not in the jejunum of the triparanol-treated rats. 3. Assays of marker enzymes for the principal subcellular organelles were performed on isolated jejunal and ileal enterocytes. In the ileum there was a striking decrease in lysosomal enzyme activities and a smaller but significant decrease of lactate dehydrogenase, catalase and malate dehydrogenase activities. In the jejunum there was no significant change in the activities of these enzymes. 4. Measurements of lysosomal integrity indicated that ileal lysosomal fragility was markedly increased and that jejunal lysosomes were affected to a much smaller extent. 5. These effects of triparanol could not be ameliorated by feeding with a gluten-free diet.

24(R,S),25-Iminolanosterol (IL) and triparanol added to cultures of rat hepatoma cells, H4-II-C3 (H4), interrupt the conversion of lanosterol to cholesterol and, depending on their concentrations, cause the accumulation in the cells of intermediates in the lanosterol to cholesterol conversion. At 45 microM, both substances cause the accumulation of 5 alpha-cholesta-8(9),24-dien-3 beta-ol (zymosterol), and at the low concentration of 4.5 microM, they cause the accumulation of cholesta-5.24-dien-3 beta-ol (desmosterol). The effect of intermediate concentrations of 9 or 22.5 microM of either substance is to cause the accumulation in the cells of three sterols: cholesta-5,7,24-trien-3 beta-ol, zymosterol, and desmosterol. The synthesis of these intermediary sterols, not found normally in H4 cells, is particularly pronounced in cultures kept in lipid-depleted media that contain the inhibitors and proceeds by the use of endogenous substrates at the expense of cholesterol. The synthesis of cholesterol from [14C]acetate or [2-14C]mevalonate is completely blocked by either inhibitor even at 4.5 microM. IL or triparanol inhibits the growth of H4 cells. Cells seeded into either full growth or lipid-depleted medium containing 22.5 microM IL will not grow unless the media are supplemented with low density lipoproteins (60 micrograms/ml). Supplementation of the media with 4.6 mM mevalonate does not counteract the inhibitory effect of IL on cell growth.

The drug, triparanol, a known inhibitor of cholesterol biosynthesis, was found to interfere with absorption of cholesterol in rhesus monkeys. The percent luminal cholesterol absorbed decreased by 13 and 21%, respectively, in the high- and low-responding monkeys when the drug was fed along with the low-cholesterol, low-plant sterol diet. When the diet contained large amounts of plant sterols, feeding of the drug also reduced the percent cholesterol absorption by the same order of magnitude. The results showed that triparanol decreased the absorption of endogenous cholesterol in rhesus monkeys. The effect of the drug on cholesterol absorption was independent of that of plant sterols. The mechanism(s) of the observed inhibitory effect of triparanol on cholesterol absorption is not clearly understood.

Incubating Hep G2 cells for 18 h with triparanol, buthiobate and low concentrations (less than 0.5 microM) of U18666A, inhibitors of desmosterol delta 24-reductase, of lanosterol 14 alpha-demethylase and of squalene-2,3-epoxide cyclase (EC 5.4.99.7) respectively, resulted in a decrease of the HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase activity. However, U18666A at concentrations higher than 3 microM increased the HMG-CoA reductase activity in a concentration-dependent manner. None of these inhibitors influenced directly the reductase activity in Hep G2 cell homogenates. Analysis by t.l.c. of 14C-labelled non-saponifiable lipids formed from either [14C]acetate or [14C]mevalonate during the cell incubations confirmed the sites of action of the drugs used. Beside the 14C-labelled substrates of the blocked enzymes and 14C-labelled cholesterol, another non-saponifiable lipid fraction was observed, which behaves as polar sterols on t.l.c. This was the case with triparanol and at those concentrations of U18666A that decreased the reductase activity, suggesting that polar sterols may play a role in suppressing the reductase activity. In the presence of 30 microM-U18666A (sterol formation blocked) the increase produced by simultaneously added compactin could be prevented by addition of mevalonate. This indicates the existence of a non-sterol mevalonate-derived effector in addition to a sterol-dependent regulation. LDL (low-density lipoprotein), which was shown to be able to decrease the compactin-induced increase in reductase activity, could not prevent the U18666A-induced increase. On the contrary, LDL enhanced the U18666A effect, showing that the LDL regulation is not merely the result of introducing cholesterol to the cells.

Primary rat hepatocyte culture cells were used to study the acute regulation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity in response to 25-hydroxycholesterol, 3 beta,5 alpha,6 beta-cholestantriol, and mevalonolactone. All three effectors caused a rapid suppression of HMG-CoA reductase activity. 25-Hydroxycholesterol also caused an increase in the ratio of newly synthesized methyl sterols to newly synthesized C27-sterols. Furthermore, in 25-hydroxycholesterol-treated cells, the relative contribution of delta 24-sterol precursors to the nonsaponifiable lipid fraction increased. Di- and trimethyl-diene sterols were the dominant methyl sterols synthesized in the presence of 25-hydroxycholesterol. 3 beta,5 alpha,6 beta-Cholestrantriol (50 microM) also caused a very strong (97%) suppression of sterol demethylation; 4,4-dimethylmonoene sterols were more prominent (23%) in cells treated with 3 beta,5 alpha,6 beta-cholestrantriol, than in cells treated with 25-hydroxycholesterol (2%). The rates of both unesterified and esterified sterol synthesis increased as a function of exogenous mevalonolactone concentration. C27-sterol synthesis was saturated at a concentration of (R)-mevalonolactone which produced only a 33% suppression of HMG-CoA reductase activity. However, there was a direct relationship between the accumulation of methyl sterols and the decrease in HMG-CoA reductase activity. With the aid of triparanol, it was demonstrated that the suppression of HMG-CoA reductase activity by mevalonolactone was linked with the ability of the cells to convert squalene-2,3-epoxide into sterols. The results described in the present article support an important and perhaps necessary relationship between the rate of methyl sterol conversion of C27-sterols and the suppression or inhibition of HMG-Coa reductase in primary hepatocyte culture cells.

The precursor sources of bile acid and bile neutral sterol were evaluated in the rat using Triparanol to inhibit the terminal reduction in the synthesis of cholesterol. During the initial period of Triparanol administration, the accumulation of hepatic desmosterol acts to segregate relatively newly synthetic hepatic sterol from the bulk of the equilibrated sterol mass. Biliary excretion of newly synthetic sterol can then be determined in acute studies, assuming no great differences between desmosterol and cholesterol as precursors of biliary neutral sterol or bile acid. It has been determined in this model that newly synthetic sterol comprises a mean of about 28% of the total biliary neutral sterol output. This fraction fell when hepatic cholesterogenesis was suppressed by prior cholesterol feeding. By using this approach in conjunction with the administration of labeled mevalonate to a renal pedicle-ligated rat, it was possible to calculate the amount of bile acid produced from either newly synthesized sterol or the equilibrated sterol pool. It has been estimated that the bulk of bile acid synthesis arises from this equilibrated source when these determinations were made within two hours of creating the fistula. With more prolonged fistula times, more of the bile acid originated from the newly synthesized sterol.

Administration of hypocholesterolemic agents to developing rats has been found to selectively induce brain hydrolases. Certain regimes also caused an appreciable increase in total brain protein content. The hypocholesterolemic agents AY-9944 and zuclomiphene were tested individually and in combination. A fourth type of treatment utilized the above drugs in combination with Triparanol. Whenever AY-9944 was used, singly or in combination with other compounds, the beta-glucuronidase activity of developing brain was increased. Acid phosphatase and total brain protein were increased in animals treated with AY-9944 plus zuclomiphene or AY-9944 plus zuclomiphene and Triparanol. Neither AY-9944 nor zuclomiphene alone significantly affected brain total protein or acid phosphatase. Electron microscopic examination of tissue specifically reacted for acid phsophatase demonstrated that the increased enzyme activity was localized in cells in the perivascular spaces. Alkaline phosphatase and N-acetyl-beta-glucosaminidase, two other hydrolytic enzymes assayed, seemed to be much less influenced by the drug treatments.

The influence of three potent lipidosis-inducing agents, i.e. iprindole, triparanol and chloroquine, on the rat adrenal cortex was investigated by determination of the corticosterone excretion in the urine, the responsiveness to ACTH and by determination of the corticosterone content of the adrenals and of the corticosterone concentration in plasma. Administration of iprindole and triparanol left the function of the adrenal cortex unaffected, while under chloroquine treatment an activation with time was observed. Morphologically, similar degrees of lipidosis were found in all experiments. The results demonstrate that the functional capacity of the rat adrenal cortex is not directly correlated to lipidotic alterations.

A sensitivity to dietary cereal has been reported previously in niacin-deficient rats by measuring a change in the intestinal absorption of radioactively-labelled cellobiotol and mannitol. The possibility that other stimuli could produce this sensitivity, the range of cereals that could induce the permeability change and the nature of the toxic component in cereal have now all been investigated. Treatment with triparanol induces sensitivity in rats to wheat, rye, barley, oats, and maize but not to rice or soybean. These cereals caused a similar response in niacin-deficient rats. Mucosal damage produced by methotrexate or cetrimide, however, did not sensitise the intestinal mucosa to dietary cereals. Gluten, zein, and pepsin/trypsin digests of gluten all induced the permeability defect in triparanol-treated rats. It is concluded that although gross disruption of the mucosal structure may not sensitise rats to cereals, various causes of mucosal cell damage can produce a susceptibility to gluten toxicity that resembles gluten-sensitivity in man.

Biliary cholesterol secretion was studied in dogs with chronic bile fistulas, using glucagon, an inhibitor of biliary cholesterol secretion, and triparanol, an inhibitor of cholesterol synthesis. Glucagon inhibited neutral sterol secretion before and after triparanol administration. Triparanol caused a significant accumulation in bile of the cholesterol precursor desmosterol which comprised a significant portion of the neutral sterol in bile but not in blood. Glucagon inhibited both biliary desmosterol and cholesterol secretions to a similar degree. These findings suggest that biliary cholesterol is derived from newly synthesized hepatic sterol as well as from equilibrated sources. Furthermore, glucagon suppressed biliary secretion of both equilibrated as well as newly synthesized neutral sterol, suggesting that glucagon inhibits the movement of neutral sterol to or through the canalicular membrane.

Developing rats were injected intraperitoneally twice weekly with a combination of three hypocholesterolemic agents: Zuclomiphene (formerly called trans-clomiphene; dosage, 30 mg/kg body weight), Triparanol (30 mg/kg body weight) and AY-9944 (3 mg/kg body weight). Treatment was initiated at 4 days of age. Biochemical and electron microscopic examination was conducted on animals sacrificed at 20 days of age. Cytoplasmic inclusion bodies were not seen in the CNS. Isolated edematous changes were seen in myelinated axons. Analysis of the sterol content of the brain and spinal cords of drug-treated animals indicated the presence of abnormal concentrations of five sterols, desmosterol, 5alpha-cholesta-7,24-dien-3beta-ol, zymosterol (5alpha-cholesta-8,24-dien-3beta-ol), 7-dehydrocholesterol (cholesta-5,7-dien-3beta-ol) and 7-dehydrodesmosterol (cholesta-5,7,24-trien-3beta-ol). Zymosterol and 5alpha-cholesta 7,24-dien-3beta-ol were minor constituents (5--7% and 1--1.5% of total sterol, respectively). The 7-dehydrosterols represented approximately one-half (44--52%) of the total CNS sterol.

Extended treatment of developing or adult rats with a variety of hypocholesterolemic drugs has shown that both the central nervous system (CNS) and peripheral nervous system (PNS) sterol content could be affected in both age groups by this type of treatment. Treatment of developing animals was begun at 5 days of age and continued for 45 days. Adult rats, 300 g at onset, were treated for 35 days. The influence of these drugs on PNS sterol composition has not been previously examined. Diazacholesterol administration caused an accumulation of desmosterol in the CNS and 7-dehydrodesmosterol and desmosterol in the PNS. Zuclomiphene induced a build-up of desmosterol in either tissue. Both of these drugs had a more pronounced effect on developing CNS and PNS than on adult CNS and PNS. Addition of AY-9944 or Triparanol to the zuclomiphene treatment of the developing animals reduced desmosterol accumulation but brought about a build-up of 7-dehydrocholesterol and 7-dehydrodesmosterol.

The effects of the triparanol analogues chlorotrianisene, clomiphene, tamoxifen, 5-[p-(fluoren-9-ylidenemethyl)phenyl]-2-piperidineethanol (MDL 10393), MDL 8917v, nafoxidine, 2-[p-(6-methoxy-2-phenylinden-3-yl)phenoxy]triethylamine (U-11555A), 2-[p-(3,4-dihydro-6-methoxy-2-phenyl-1-naphthyl)phenoxy]triethylamine (U-10520A), and nitromifene, as well as triparanol itself, were studied in the P388 murine leukemia cell line and in a doxorubicin-resistant subline (P388/ADR). At noninhibitory concentrations, all the analogues increased the sensitivity of P388/ADR cells to doxorubicin but did not have such an effect on the doxorubicin-sensitive cells. Diethylstilbestrol, deacetylated cyclofenil (F6060), hexestrol, and 17 beta-estradiol did not have such an activity. The effects of tamoxifen on doxorubicin sensitivity of P388/ADR cells could not be reversed by 17 beta-estradiol. Estrogen receptors could not be demonstrated in either cell line. It is therefore suggested that the reversal of the doxorubicin-acquired resistance by the triparanol analogues is unrelated to their estrogenic or antiestrogenic activities. The possible clinical implications of these findings are discussed.