Cholesterol-lowering Drug Lovastatin Research Articles

Estrogen induces LCAT to maintain cholesterol homeostasis and suppress hepatocellular carcinoma development.

Hepatocellular carcinoma (HCC) is an aggressive disease that occurs predominantly in men. Estrogen elicits protective effects against HCC development. Elucidation of the estrogen-regulated biological processes that suppress HCC could lead to improved prevention and treatment strategies. Here, we performed transcriptomic analyses on mouse and human liver cancer and identified LCAT as the most highly estrogen-upregulated gene and a biomarker of favorable prognosis. LCAT upregulation inhibited HCC in vitro and in vivo and mediated estrogen-induced suppression of HCC in an ESR1-dependent manner. LCAT facilitated high-density lipoprotein cholesterol (HDL-C) production and uptake via the LDLR and SCARB1 pathways. Consistently, high HDL-C levels corresponded to a favorable prognosis in HCC patients. The enhanced HDL-C absorption induced by LCAT impaired SREBP2 maturation, which ultimately suppressed cholesterol biosynthesis and dampened HCC cell proliferation. HDL-C alone inhibited HCC growth comparably to the cholesterol-lowering drug lovastatin, and SREBF2 overexpression abolished the inhibitory activity of LCAT. Clinical observations and cross-analyses of multiple databases confirmed the correlation of elevated LCAT and HDL-C levels to reduced cholesterol synthesis and improved HCC patient prognosis. Furthermore, LCAT deficiency mimicked whereas LCAT overexpression abrogated the tumor growth promoting effects of ovariectomy in HCC-bearing female mice. Most importantly, HDL-C and LCAT delayed the development of subcutaneous tumors in nude mice, and HDL-C synergized with lenvatinib to eradicate orthotopic liver tumors. Collectively, this study reveals that estrogen upregulates LCAT to maintain cholesterol homeostasis and dampen hepatocarcinogenesis. LCAT and HDL-C represent potential prognostic and therapeutic biomarkers for targeting cholesterol homeostasis as a strategy for treating HCC.

Biosynthesis of Fungal Natural Products Involving Two Separate Pathway Crosstalk

Fungal natural products (NPs) usually possess complicated structures, exhibit satisfactory bioactivities, and are an outstanding source of drug leads, such as the cholesterol-lowering drug lovastatin and the immunosuppressive drug mycophenolic acid. The fungal NPs biosynthetic genes are always arranged within one single biosynthetic gene cluster (BGC). However, a rare but fascinating phenomenon that a crosstalk between two separate BGCs is indispensable to some fungal dimeric NPs biosynthesis has attracted increasing attention. The hybridization of two separate BGCs not only increases the structural complexity and chemical diversity of fungal NPs, but also expands the scope of bioactivities. More importantly, the underlying mechanism for this hybridization process is poorly understood and needs further exploration, especially the determination of BGCs for each building block construction and the identification of enzyme(s) catalyzing the two biosynthetic precursors coupling processes such as Diels–Alder cycloaddition and Michael addition. In this review, we summarized the fungal NPs produced by functional crosstalk of two discrete BGCs, and highlighted their biosynthetic processes, which might shed new light on genome mining for fungal NPs with unprecedented frameworks, and provide valuable insights into the investigation of mysterious biosynthetic mechanisms of fungal dimeric NPs which are constructed by collaboration of two separate BGCs.

An advanced method for quantitative measurements of cholesterol crystallization

BackgroundCholesterol crystallization within an atherosclerotic plaque significantly contributes to the acceleration of plaque rupture – a problematic event due to the current lack of specific treatments to prevent such formations. Modelling this pathogenic process is also difficult due to the lack of suitable experimental models that enable quantitative analysis of crystal formation and bioactivity screening of potential therapeutic compounds. AimTo develop an in vitro human cell model of cholesterol crystallization combined with an imaging system that incorporates both quantitative analysis and real-time continuous imaging of cholesterol crystal formation. Methods and resultsAn enhanced in vitro model of cholesterol crystallization was developed through the use of acetylated low-density lipoprotein (AcLDL) and 7-ketocholesterol as agents of foam cell induction within a human THP-1 monocytic cell line. Advanced confocal and polarizing microscopies were incorporated into the model so as to allow for quantitation of cholesterol crystallization, with the lipid-loaded group producing significantly greater numbers of cholesterol crystals than the untreated group. The utility of this system was also demonstrated by investigating the effects of the cholesterol-lowering drug lovastatin and therapeutic bile compound ursodeoxycholic acid (UDCA), showing that these drugs influence different aspects of cholesterol crystal formation. ConclusionsThe in vitro human THP-1 monocyte model of cholesterol crystallization provides an effective and efficient means of quantitating cholesterol crystallization in the pre-clinical stage of research. The model also allows for the screening of potentially therapeutic compounds that may be used in attenuating or preventing cholesterol crystallization.

Lovastatin, not Simvastatin, Corrects Core Phenotypes in the Fragile X Mouse Model.

The cholesterol-lowering drug lovastatin corrects neurological phenotypes in animal models of fragile X syndrome (FX), a commonly identified genetic cause of autism and intellectual disability (ID). The therapeutic efficacy of lovastatin is being tested in clinical trials for FX; however, the structurally similar drug simvastatin has been proposed as an alternative due to an increased potency and brain penetrance. Here, we perform a side-by-side comparison of the effects of lovastatin and simvastatin treatment on two core phenotypes in Fmr1-/y mice versus WT littermates: excessive hippocampal protein synthesis and susceptibility to audiogenic seizures (AGSs). We find that simvastatin does not correct excessive hippocampal protein synthesis in the Fmr1-/y hippocampus at any dose tested. In fact, simvastatin significantly increases protein synthesis in both Fmr1-/y and WT. Moreover, injection of simvastatin does not reduce AGS in the Fmr1-/y mouse, while lovastatin significantly reduces AGS incidence and severity versus vehicle-treated animals. These results show that unlike lovastatin, simvastatin does not correct core phenotypes in the Fmr1-/y mouse model.

Cholesterol content in cell membrane maintains surface levels of ErbB2 and confers a therapeutic vulnerability in ErbB2-positive breast cancer

BackgroundErbB2 overexpression identifies a subset of breast cancer as ErbB2-positive and is frequently associated with poor clinical outcomes. As a membrane-embedded receptor tyrosine kinase, cell surface levels of ErbB2 are regulated dynamically by membrane physical properties. The present study aims to investigate the influence of membrane cholesterol contents on ErbB2 status and cellular responses to its tyrosine kinase inhibitors.MethodsThe cholesterol abundance was examined in ErbB2-positive breast cancer cells using filipin staining. Cellular ErbB2 localizations were investigated by immunofluorescence with altered membrane cholesterol contents. The inhibitory effects of the cholesterol-lowering drug lovastatin were assessed using cell proliferation, apoptosis, immunoblotting and immunofluorescence assays. The synergistic effects of lovastatin with the ErbB2 inhibitor lapatinib were evaluated using an ErbB2-positive breast cancer xenograft mouse model.ResultsMembrane cholesterol contents positively correlated with cell surface distribution of ErbB2 through increasing the rigidity and decreasing the fluidity of cell membranes. Reduction in cholesterol abundance assisted the internalization and degradation of ErbB2. The cholesterol-lowering drug lovastatin significantly potentiated the inhibitory effects of ErbB2 kinase inhibitors, accompanied with enhanced ErbB2 endocytosis. Lovastatin also synergized with lapatinib to strongly suppress the in vivo growth of ErbB2-positive breast cancer xenografts.ConclusionThe cell surface distribution of ErbB2 was closely regulated by membrane physical properties governed by cholesterol contents. The cholesterol-lowering medications can hence be exploited for potential combinatorial therapies with ErbB2 kinase inhibitors in the clinical treatment of ErbB2-positive breast cancer.

Impact of high cholesterol in a Parkinson’s disease model: Prevention of lysosomal leakage versus stimulation of α-synuclein aggregation

Parkinson’s disease is characterized by accumulation of intraneuronal cytoplasmic inclusions, Lewy bodies, which mainly consist of aggregated α-synuclein. Controversies exist as to whether high blood cholesterol is a risk factor for the development of the disease and whether statin treatment could have a protective effect. Using a model system of BE(2)-M17 neuroblastoma cells treated with the neurotoxin 1-methyl-4-phenylpyridinium (MPP+), we found that MPP+-induced cell death was accompanied by cholesterol accumulation in a lysosomal-like pattern in pre-apoptotic cells. To study the effects of lysosomal cholesterol accumulation, we increased lysosomal cholesterol through pre-treatment with U18666A and found delayed leakage of lysosomal contents into the cytosol, which reduced cell death. This suggests that increased lysosomal cholesterol is a stress response mechanism to protect lysosomal membrane integrity in response to early apoptotic stress. However, high cholesterol also stimulated the accumulation of α-synuclein. Treatment with the cholesterol-lowering drug lovastatin reduced MPP+-induced cell death by inhibiting the production of reactive oxygen species, but did not prevent lysosomal cholesterol increase nor affect α-synuclein accumulation. Our study indicates a dual role of high cholesterol in Parkinson’s disease, in which it acts both as a protector against lysosomal membrane permeabilization and as a stimulator of α-synuclein accumulation.

Combined treatment with troglitazone and lovastatin inhibited epidermal growth factor-induced migration through the downregulation of cysteine-rich protein 61 in human anaplastic thyroid cancer cells.

Our previous studies have demonstrated that epidermal growth factor (EGF) can induce cell migration through the induction of cysteine-rich protein 61 (Cyr61) in human anaplastic thyroid cancer (ATC) cells. The aim of the present study was to determine the inhibitory effects of combined treatment with the peroxisome proliferator-activated receptor-γ (PPARγ) ligand troglitazone and the cholesterol-lowering drug lovastatin at clinically achievable concentrations on ATC cell migration. Combined treatment with 5 μM troglitazone and 1 μM lovastatin exhibited no cytotoxicity but significantly inhibited EGF-induced migration, as determined using wound healing and Boyden chamber assays. Cotreatment with troglitazone and lovastatin altered the epithelial-to-mesenchymal-transition (EMT) -related marker gene expression of the cells; specifically, E-cadherin expression increased and vimentin expression decreased. In addition, cotreatment reduced the number of filopodia, which are believed to be involved in migration, and significantly inhibited EGF-induced Cyr61 mRNA and protein expression as well as Cyr61 secretion. Moreover, the phosphorylation levels of 2 crucial signal molecules for EGF-induced Cyr61 expression, the cAMP response element-binding protein (CREB) and extracellular signal-regulated kinase (ERK), were decreased in cells cotreated with troglitazone and lovastatin. Performing a transient transfection assay revealed that the combined treatment significantly suppressed Cyr61 promoter activity. These results suggest that combined treatment with low doses of troglitazone and lovastatin effectively inhibits ATC cell migration and may serve as a novel therapeutic strategy for metastatic ATC.

Screening of indigenously isolated fungi for lovastatin production and its in vivo evaluation.

Seven indigenously isolated fungal strains (Aspergillus niger, Aspergillus flavus, Aspergillus terreus, Penicillium citrinum, Penicillium notatum, Pleurotus ostreatus and Trichoderma viradae) were tested for their potential to produce cholesterol lowering drug lovastatin by using different agro-industrial wastes (Corn cobs, corn stover, banana stalk, wheat straw, wheat bran, bagasse) in submerged as well as solid state fermentation. Aspergillus terreus showed maximum production of 18.74 mg/100 mL by wheat bran in solid state fermentation. The fermentation parameters (pH, temperature, Inoculum size, moisture contents and fermentation time) were also optimized for optimum production of lovastatin. It was found that Aspergillus terreus could produce 27.14 mg/100 mL lovastatin under optimum condition of pH (6), temperature (30°C), Inoculum size (2 mL), moisture contents (60%) and fermentation time (120 hrs) in solid state fermentation. The optimized lovastatin was extracted from fermented broth and orally administered to rats. The hypocholesterolemic effect of fermented lovastatin was evaluated on serum ALT, AST, HDL-C, LDL-C, TG and TC level of rats. It was concluded from the study, fermented lovastatin effectively lower the cholesterol level of rats.

Niche-based screening identifies small-molecule inhibitors of leukemia stem cells

Efforts to develop more effective therapies for acute leukemia may benefit from high-throughput screening systems that reflect the complex physiology of the disease, including leukemia stem cells (LSCs) and supportive interactions with the bone marrow microenvironment. The therapeutic targeting of LSCs is challenging because LSCs are highly similar to normal hematopoietic stem and progenitor cells (HSPCs) and are protected by stromal cells in vivo. We screened 14,718 compounds in a leukemia-stroma co-culture system for inhibition of cobblestone formation, a cellular behavior associated with stem-cell function. Among those compounds that inhibited malignant cells but spared HSPCs was the cholesterol-lowering drug lovastatin. Lovastatin showed anti-LSC activity in vitro and in an in vivo bone marrow transplantation model. Mechanistic studies demonstrated that the effect was on target, via inhibition of HMG-CoA reductase. These results illustrate the power of merging physiologically relevant models with high-throughput screening.

Fluvastatin modulates renal water reabsorption in vivo through increased AQP2 availability at the apical plasma membrane of collecting duct cells

X-linked nephrogenic diabetes insipidus (XNDI), a severe pathological condition characterized by greatly impaired urine-concentrating ability of the kidney, is caused by inactivating mutations in the V2 vasopressin receptor (V2R) gene. The lack of functional V2Rs prevents vasopressin-induced shuttling of aquaporin-2 (AQP2) water channels to the apical plasma membrane of kidney collecting duct principal cells, thus promoting water reabsorption from urine to the interstitium. At present, no specific pharmacological therapy exists for the treatment of XNDI. We have previously reported that the cholesterol-lowering drug lovastatin increases AQP2 membrane expression in renal cells in vitro. Here we report the novel finding that fluvastatin, another member of the statins family, greatly increases kidney water reabsorption in vivo in mice in a vasopressin-independent fashion. Consistent with this observation, fluvastatin is able to increase AQP2 membrane expression in the collecting duct of treated mice. Additional in vivo and in vitro experiments indicate that these effects of fluvastatin are most likely caused by fluvastatin-dependent changes in the prenylation status of key proteins regulating AQP2 trafficking in collecting duct cells. We identified members of the Rho and Rab families of proteins as possible candidates whose reduced prenylation might result in the accumulation of AQP2 at the plasma membrane. In conclusion, these results strongly suggest that fluvastatin, or other drugs of the statin family, may prove useful in the therapy of XNDI.

FDA advisers again reject nonprescription lovastatin

For the third time in less than a decade, FDA advisers have recommended that the agency reject an application from Merck & Co. Inc. to market a nonprescription version of the cholesterol-lowering drug lovastatin. “It seemed to me that patients couldn’t figure out who the drug was for and if the drug was for them,” said internal medicine specialist William H. Shrank, of Boston’s Brigham and Women’s Hospital, in describing data presented December 13, 2007, in Silver Spring, Maryland. Shrank, a recently appointed member of FDA’s Nonprescription Drugs Advisory Committee, was 1 of 10 experts who voted against the proposal to make lovastatin 20-mg tablets available without a prescription. The meeting also included participants from FDA’s Endocrinologic and Metabolic Drugs Advisory Committee. Two advisers voted in favor of FDA approval of the application, and one abstained from voting. Shrank’s criticism was shared by many of the advisers and echoed concerns raised nearly three years ago when members of the two committees overwhelmingly voted against Merck’s previous application to market nonprescription lovastatin (see March 1, 2005, AJHP News).

The role of BRCA1 in DNA repair and chemosensitivity

10606 Introduction: The BRCA1 tumor suppressor protein has been implicated in many cellular processes, including several different DNA repair pathways. Inherited mutations in the BRCA1 gene result in an increased risk for developing breast and ovarian cancer. BRCA1 associated tumors are frequently estrogen receptor negative thus rendering them ineffective targets of hormonal manipulations. Therefore chemotherapy is the only treatment option available to patients with metastatic disease. Methods: Global genomic repair activity was detected by an immunoslotblot assay. UV-C and chemosensitivity was determined by the MTT assay. The comet assay was used to measure oxidative DNA damage (ODD). Results: Brca1 deficient cells demonstrate decreased global genomic repair independent of p53 expression. These cells are 3-fold more sensitive to UV-C radiation, 5-fold more sensitive to cisplatin, and 25-fold more sensitive to gemcitabine than Brca1 positive cells. The combination of cisplatin and gemcitabine is synergistic. This synergy is independent of Brca1 protein expression. In addition, the cholesterol lowering drug lovastatin is able mitigate the ODD caused by hydrogen peroxide. Reduced ODD is also observed in breast epithelial cells from high risk patients after 6 months of oral lovastatin use. Discussion: These results confirm our earlier findings that BRCA1 is involved in the nucleotide excision repair pathway. In addition, we demonstrate that this effect is independent of p53 expression. We also find that Brca1 deficient MMECs are more sensitive to UV-C, and the cytotoxic drugs cisplatin and gemcitabine. Cisplatin is known to cause DNA intra- and inter-strand crosslinks whereas gemcitabine is a nucleoside analog. The increased sensitivity of Brca1 deficient MMEC's to these agents supports the role of Brca1 in the different molecular pathways involved in their repair. In addition, we demonstrate that lovastatin can overcome ODD in vitro and in vivo and may be an effective agent to modify breast cancer risk for individuals at increased risk. No significant financial relationships to disclose.

Risks of a nonprescription lovastatin

Recently, Merck, maker of the once blockbuster cholesterol-lowering drug lovastatin (Mevacor), proposed that the product be available without a prescription.[1][1] The company claimed that millions of Americans would benefit and that many lives would be saved by preventing heart disease, the nation

Lovastatin stimulates up-regulation of α7 nicotinic receptors in cultured neurons without cholesterol dependency, a mechanism involving production of the α-form of secreted amyloid precursor protein

The cholesterol-lowering drug lovastatin enhances the secretion of the alpha-secretase cleavage product of amyloid precursor protein (APP). To investigate whether this effect is mediated via activation of alpha7 nicotinic acetylcholine receptors (nAChRs), we treated SH-SY5Y cells and PC12 cells with lovastatin and measured the levels of alpha7 nAChRs, the alpha-form of secreted APP (alphaAPPs), and lovastatin-related lipids, including cholesterol and ubiquinone. The results showed that low concentrations of lovastatin significantly induced up-regulation of alpha7 nAChRs. No effects of lovastatin were observed on alpha3-containing nAChRs, muscarinic receptors, or N-methyl-D-aspartate receptors. alphaAPPs levels increased in the culture medium of cells treated with lovastatin, whereas no change in whole APP was observed. The increase in alphaAPPs was inhibited by prior exposure of these cells to alpha-bungarotoxin, an antagonist of alpha7 nAChRs. The concentrations of lovastatin used in the study did not change the cholesterol content, but high doses can decrease the levels of ubiquinone and cell viability. These results indicate that lovastatin may play a neuronal role that is cholesterol independent. We also show that the up-regulation of alpha7 nAChRs stimulated by lovastatin is involved in a mechanism that enhances production of alphaAPPs during APP processing.

Improved Lymphocyte Function-associated Antigen-1 (LFA-1) Inhibition by Statin Derivatives: MOLECULAR BASIS DETERMINED BY X-RAY ANALYSIS AND MONITORING OF LFA-1 CONFORMATIONAL CHANGES IN VITRO AND EX VIVO

The integrin lymphocyte function-associated antigen-1 (LFA-1) (alphaLbeta2; CD11a/CD18) plays an important role in leukocyte migration and T cell activation. LFA-1 is inhibited by the cholesterol-lowering drug lovastatin, which binds to an allosteric site of the alphaL I domain termed the lovastatin site (L-site). Here we report for the first time the x-ray structures of the LFA-1 I domain complexed with derivatives of lovastatin optimized for LFA-1 inhibition. This analysis identified two new subpockets within the L-site occupied by chemical groups of the statin derivatives but not by lovastatin itself. Occupancy of these L-site subpockets led to distinct conformational changes in LFA-1, which were detectable by an epitope-monitoring assay. We utilized this assay to demonstrate improved LFA-1 inhibition in human blood in vitro and in blood samples from treated animals ex vivo. Moreover, we demonstrate that the novel lovastatin-derived LFA-1 inhibitor LFA878 exhibits potent anti-inflammatory effects in carrageenan-induced rat paw edema. In summary, the findings reported here extend the understanding of LFA-1 inhibition at the molecular level, allow for the identification and design of LFA-1 inhibitors of further enhanced potency, and support the expectation that LFA-1 inhibitors binding to the L-site will be of therapeutic value in treating inflammatory diseases.

Cholesterol regulates ABCD2 gene expression: implications for X-linked adrenoleukodstrophy.

X-linked adrenoleukodystrophy (X-ALD) is a severe neurodegenerative disorder with impaired very long-chain fatty acid (VLCFA) metabolism (Moser et al 2001). The disease-associated ABCD1 (ALD) gene encodes a peroxisomal membrane protein, which belongs to the superfamily of ATP- binding cassette transporters. Currently, no satisfactory therapy is available for X-ALD. The only treatment with some documented benefit for X-ALD patients is bone marrow transplantation when performed at an early stage of the disease (Shapiro et al 2000). Recently, the cholesterol-lowering drug lovastatin, a 3-hydroxy-3-methylglutaryl-coenzyme A (HMGCoA) reductase inhibitor, was proposed as a new therapeutic agent for X-ALD and a cause-and-effect relationship between lovastatin therapy and plasma VLCFA levels was observed when plasma VLCFA and serum cholesterol levels were compared (Singh et al 1998b, Pai et al 2000). However, statin treatment successfully normalized VLCFA levels in the plasma of X-ALD patients in only two out of three studies (Pai et al 2000, Singh et al 1998a, Verrips et al 2000). This investigation aimed to disclose the molecular mechanism of successful reduction of VLCFA accumulation in order to fill in the gap in the understanding how dietary cholesterol lowering affects levels of VLCFA in patients with X-ALD and to allow more efficacious treatment. Overexpression of ABCD2, the closest relative of ABCD1, restores VLCFA accumulation in cultured ABCD1 -deficient cells. Here we show by real-time PCR that the ABCD2 gene is induced in cultured human fibroblasts and monocytes upon sterol-depletion via a mechanism requiring the activation of sterol regulatory element binding proteins (SREBPs), a family of transcription factors that control the metabolism of cholesterol and fatty acids. This is unexpected and the first report that extends the mechanism of transcriptional regulation by SREBPs to a peroxisomal protein, thus providing a closer link between peroxisomes, cholesterol and fatty acid biosynthesis. Using reporter gene studies, site directed mutagenesis and gel shift assays, we identified a functional sterol regulatory element in the proximal promoter region of ABCD2. Finally, we demonstrate that ABCD2 induction by sterol depletion significantly reduced the accumulation of VLCFA in X-ALD fibroblasts. Thus, lowering cholesterol leads to SREBP maturation, increased ABCD2 expression and reduced VLCFA accumulation.KeywordsSterol Regulatory Element Binding ProteinProximal Promoter RegionPeroxisomal ProteinPeroxisomal Membrane ProteinDietary Cholesterol LoweringThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Cholesterol regulates ABCD2 expression: implications for the therapy of X-linked adrenoleukodystrophy.

X-linked adrenoleukodystrophy (X-ALD) is a severe neurodegenerative disorder with impaired very long-chain fatty acid (VLCFA) metabolism. The disease-associated ABCD1 (ALD) gene encodes a peroxisomal membrane protein, which belongs to the superfamily of ATP-binding cassette transporters. Several treatment regimes have been tried without satisfactory clinical benefit. Recently, the cholesterol-lowering drug lovastatin was reported to normalize VLCFA levels in two out of three clinical studies. This investigation aimed to disclose the molecular mechanism of successful reduction of VLCFA accumulation in order to fill in the gap in the understanding how dietary cholesterol lowering affects the levels of VLCFA in patients with X-ALD and to allow more efficacious treatment. Overexpression of ABCD2 (ALDR), the closest relative of ABCD1, restores VLCFA accumulation in cultured ABCD1-deficient cells. Here we show by real-time PCR that the ABCD2 gene is induced in cultured human fibroblasts and monocytes upon sterol depletion via a mechanism requiring the activation of sterol regulatory element-binding proteins (SREBPs), a family of transcription factors that control the metabolism of cholesterol and fatty acids. This is unexpected and the first report that extends the mechanism of transcriptional regulation by SREBPs to a peroxisomal protein, thus providing a closer link between peroxisomes, cholesterol and fatty acid biosynthesis. Using reporter gene studies, site-directed mutagenesis and gel shift assays, we identified a functional sterol regulatory element in the proximal promoter region of ABCD2. Finally, we demonstrated that ABCD2 induction by sterol depletion significantly reduced the accumulation of VLCFA in X-ALD fibroblasts. Thus, lowering cholesterol leads to SREBP maturation, increased ABCD2 expression and reduced VLCFA accumulation.

Inhibition of farnesylation blocks growth but not differentiation in FRTL-5 thyroid cells

The cholesterol lowering drug lovastatin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, blocks DNA synthesis and proliferation of thyrotropin (TSH) primed FRTL-5 rat thyroid cells. The blockade can be completely prevented and/or reversed by mevalonate and largely prevented and/or reversed by farnesol whereas cholesterol and/or other non-sterol mevalonate derivatives such as ubiquinone, dolichol or isopentenyladenosine are ineffective. TSH-dependent augmentation of cyclic-AMP and cAMP dependent differentiated functions, such as iodide uptake, are unaffected by lovastatin. 3H-Thymidine incorporation into DNA is also decreased by α-hydroxyfarnesyl-phosphonic acid, an inhibitor of protein farnesylation which mimicks the effect of lovastatin since it also leaves unaffected TSH stimulated iodide uptake. It is suggested that the HMG-CoA reductase inhibitor lovastatin affects cell proliferation mainly through inhibition of protein farnesylation which results in altered function proteins relevant for proliferation control, notably p21 ras and/or other small GTPases.

Plasma PAF acetylhydrolase in non-insulin dependent diabetes mellitus and obesity: effect of hyperinsulinemia and lovastatin treatment

Insulin resistance is characterized principally by impaired insulin-mediated glucose uptake which provokes a compensatory increase in pancreatic β-cell secretory activity. For a time this may produce well-controlled plasma glucose levels but as the insulin resistance worsens the augmented insulin production becomes inadequate to keep plasma glucose at euglycemia leading to the development of non-insulin dependent diabetes mellitus (NIDDM), accompanied by hyperinsulinemia and hyperglycemia. A number of metabolic defects are associated with NIDDM including obesity, hypercoagulability, cardiovascular disease risk factors such as hypertension and dyslipidemia and these constitute the insulin resistance syndrome. The identity of the biochemical factor that might link all these defects is not yet known. We have hypothsisized that platelet-activating factor (1- O-alkyl-2-acetyl- sn-glycero-3-phosphocholine, PAF) may be such a link. In this study, we measured plasma acetylhydrolase (EC.1.1.48), which degrades PAF to the inactive metabolise lyso-PAF, as a surrogate for PAF activity in three groups of hypercholesterolemic subjects: lean controls ( n=9), non-diabetic obese ( n=6) and NIDDM subjects ( n=6). The ages and body mass indices of the subjects were 46±3.1 and 24.2±2.2 for the lean controls, 52±2.5 and 28.7±0.9 for the NIDDM subjects and 60±2 and 27.6±2.1 for the obese, non-diabetic subjects (mean±S.E.M.). The measurements were made before and after therapy with the cholesterol-lowering drug lovastatin, a 3-hydroxy 3 methylglutaryl (HMG) coenzyme A reductase inhibitor (40 mg/day) for 3 months. Fasting plasma glucose (FPG) levels were 91±11, 96±3 and 146±11 mg/dl, for the lean, obese and NIDDM subjects, respectively, before therapy began. Lovastatin did not affect FPG in any of the three subject groups. Before treatment, the fasting plasma insulin (FPI) levels were 6.1±0.92, 10.83±2.03 and 14.68±3.64 mU/l for the lean, non-diabetic obese and NIDDM subjects, respectively. After lovastatin therapy only the obese group exhibited a significant change in FPI (15.35±2.47 mU/l) ( P<0.05). Total cholesterol levels were similar in all three groups both before and after lovastatin therapy but within each group lovastatin therapy significantly reduced the total cholesterol by 32, 29 and 34% in the lean, obese and NIDDM subject groups respectively ( P<0.0001). Lovastatin therapy reduced LDL-cholesterol levels by 40, 32and 46% in the lean, obese and NIDDM subjects, respectively, but produced no significant effect on HDL or triglyceride levels. Before therapy, the plasma acetylyhydrolase activities were 104±7, 164±7 and 179±7 nmol/ml per min in the lean, obese and NIDDM subjects, respectively. Lovastatin therapy reduced plasma acetylhydrolase levels to 70±7, 87±6 and 86±7 nmol/ml per min in the lean, obese and NIDDM subjects, respectively. Plasma acetylhydrolase activity was predominantly (>80%) associated with LDL cholesterol both before and after lovastatin treatment. Also, plasma acetylhydrolase activity significantly correlated with fasting plasma insulin levels before lovastatin therapy but not after. Taken together, this study clearly implicates PAF metabolism in three defects associated with the insulin resistance syndrome; hypercholesterolemia, obesity and NIDDM. Additionally, we conclude that chronic hyperinsulinemia may play a significant role in the production of plasma acetylhydrolase.

Biotransformation of lovastatin: IV. Identification of cytochrome P450 3A proteins as the major enzymes responsible for the oxidative metabolism of lovastatin in rat and human liver microsomes

Previous studies from our laboratories have shown that the metabolism of the cholesterol-lowering drug lovastatin by rat and human liver microsomes occurs primarily at the 6′-position, giving 6′β-hydroxy- and 6′-exomethylene-lovastatin and that these oxidations are catalyzed by cytochrome P450-dependent monooxygenases. In the present study, the specific cytochrome P450 form involved in lovastatin oxidation was identified through immunoinhibition studies. Among several antibodies prepared against various cytochrome P450s, only anti-rat P450 3A IgG inhibited lovastatin metabolism in liver microsomes from untreated, phenobarbital treated, and pregnenolone-16α-carbonitrile-treated rats. Lovastatin metabolism at the 6′-position was markedly inhibited (6′β-hydroxy, greater than 95%; 6′-exomethylene, 70–80%) by this antibody whereas the effect of anti-rat P450 3A on the 3″-hydroxylation was variable depending on the source of the microsomes. With human liver microsomes, both anti-rat P450 3A and anti-human P450 3A inhibited lovastatin metabolism. Correlation between lovastatin oxidation and the P450 3A content in human liver microsomes (measured by immunoblot analysis) was excellent ( r 2 = 0.97). In addition, preincubation of human liver microsomes with troleandomycin and NADPH inhibited metabolism by 60%. These results clearly indicate that cytochrome P450 3A enzymes are primarily responsible for the metabolism of lovastatin in rat and human liver microsomes.