Transintestinal transport of the anti-inflammatory drug 4F and the modulation of transintestinal cholesterol efflux.

The site and mechanism of action of the apoA-I mimetic peptide 4F are incompletely understood. Transintestinal cholesterol efflux (TICE) is a process involved in the clearance of excess cholesterol from the body. While TICE is responsible for at least 30% of the clearance of neutral sterols from the circulation into the intestinal lumen, few pharmacological agents have been identified that modulate this pathway. We show first that circulating 4F selectively targets the small intestine (SI) and that it is predominantly transported into the intestinal lumen. This transport of 4F into the SI lumen is transintestinal in nature, and it is modulated by TICE. We also show that circulating 4F increases reverse cholesterol transport from macrophages and cholesterol efflux from lipoproteins via the TICE pathway. We identify the cause of this modulation of TICE either as 4F being a cholesterol acceptor with respect to enterocytes, from which 4F enhances cholesterol efflux, or as 4F being an intestinal chaperone with respect to TICE. Our results assign a novel role for 4F as a modulator of the TICE pathway and suggest that the anti-inflammatory functions of 4F may be a partial consequence of the codependent intestinal transport of both 4F and cholesterol.

Enhancement by LDL of transfer of L-4F and oxidized lipids to HDL in C57BL/6J mice and human plasma.

The apoA-I mimetic peptide L-4F [(Ac-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH2) synthesized from all L-amino acids] has shown potential for the treatment of a variety of diseases. Here, we demonstrate that LDL promotes association between L-4F and HDL. A 2- to 3-fold greater association of L-4F with human HDL was observed in the presence of human LDL as compared with HDL by itself. This association further increased when LDL was supplemented with the oxidized lipid 15S-hydroxy-5Z, 8Z, 11Z, 13E-eicosatetraenoic acid (15HETE). Additionally, L-4F significantly (P = 0.02) promoted the transfer of 15HETE from LDL to HDL. The transfer of L-4F from LDL to HDL was demonstrated both in vitro and in C57BL/6J mice. L-4F, injected into C57BL/6J mice, associated rapidly with HDL and was then cleared quickly from the circulation. Similarly, L-4F loaded onto human HDL and injected into C57BL/6J mice was cleared quickly with T(1/2) = 23.6 min. This was accompanied by a decline in human apoA-I with little or no effect on the mouse apoA-I. Based on these results, we propose that i) LDL promotes the association of L-4F with HDL and ii) in the presence of L-4F, oxidized lipids in LDL are rapidly transferred to HDL allowing these oxidized lipids to be acted upon by HDL-associated enzymes and/or cleared from the circulation.

Structure and function of HDL mimetics.

HDL mimetics have been constructed from a number of peptides and proteins with varying structures, all of which bind lipids found in HDL. HDL mimetics containing a peptide or protein have been constructed with as few as 4 and as many as 243 amino acid residues. Some HDL mimetics have been constructed with lipid but without a peptide or protein component. Some HDL mimetics promote cholesterol efflux, some have been shown to have a remarkable ability to bind oxidized lipids compared to human apolipoprotein A-I (apoA-I). Many of these peptides have been shown to have antiinflammatory properties. Based on studies in a number of animal models and in early human clinical trials, HDL mimetics appear to have promise as diagnostic and therapeutic agents.

Characterization of farnesyl diphosphate farnesyl transferase 1 (FDFT1) expression in cancer.

AIM: To help characterize the FDFT1 gene and protein expression in cancer. Cholesterol represents an important structural component of lipid rafts. These specializations can be involved in pathways stimulating cell growth, survival and other processes active in cancer. This cellular compartment can be expanded by acquisition of cholesterol from the circulation or by its synthesis in a metabolic pathway regulated by the FDFT1 enzyme. Given the critical role this might play in carcinogenesis and in the behavior of cancers, we have examined the level of this enzyme in various types of human cancer. Our demonstration of elevated levels of FDFT1 mRNA and protein in some tumors relative to surrounding normal tissue identifies this as a possible biomarker for disease development and progression, and as a potential new target for the treatment of cancer.

Hepatic farnesyl diphosphate synthase expression is suppressed by polyunsaturated fatty acids.

Dietary vegetable oils and fish oils rich in PUFA (polyunsaturated fatty acids) exert hypocholesterolaemic and hypotriglyceridaemic effects in rodents. The plasma cholesterol-lowering properties of PUFA are due partly to a diminution of cholesterol synthesis and of the activity of the rate-limiting enzyme HMG-CoA reductase (3-hydroxy-3-methylglutaryl-CoA reductase). To better understand the mechanisms involved, we examined how tuna fish oil and individual n-3 and n-6 PUFA affect the expression of hepatic FPP synthase (farnesyl diphosphate synthase), a SREBP (sterol regulatory element-binding protein) target enzyme that is subject to negative-feedback regulation by sterols, in co-ordination with HMG-CoA reductase. Feeding mice on a tuna fish oil diet for 2 weeks decreased serum cholesterol and triacylglycerol levels, by 50% and 60% respectively. Hepatic levels of FPP synthase and HMG-CoA reductase mRNAs were also decreased, by 70% and 40% respectively. Individual n-3 and n-6 PUFA lowered FPP synthase and HMG-CoA reductase mRNA levels in H4IIEC3 rat hepatoma cells to a greater extent than did stearate and oleate, with the largest inhibitory effects occurring with arachidonate, EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid). We observed a similar inhibitory effect on protein levels of FPP synthase. The suppressive effect of PUFA on the FPP synthase mRNA level was not due to a decrease in mRNA stability, but to transcription inhibition. Moreover, a lower nuclear availability of both SREBP-1 and SREBP-2 mature forms was observed in HepG2 human hepatoblastoma cells treated with arachidonate, EPA or DHA. Taken together, these data suggest that PUFA can down-regulate hepatic cholesterol synthesis through inhibition of HMG-CoA reductase and FPP synthase, at least in part through impairment of the SREBP pathway.

Androgens stimulate coordinated lipogenic gene expression in normal target tissues in vivo.

In prostate cancer cell lines in culture androgens cause a marked and coordinated upregulation of the expression of several lipogenic genes. Here, using castrated male Wistar rats as an experimental paradigm, we investigated whether coordinated androgen stimulation of lipogenic gene expression represents a more general physiological regulation in non-cancerous androgen-responsive cells as well. In typical target tissues for androgen action such as the ventral prostate and the lacrimal gland, androgen deprivation resulted in a marked reduction in the mRNA and protein levels of genes involved in fatty acid (fatty acid synthase and acetyl-CoA-carboxylase) and cholesterol synthesis (HMG-CoA-reductase and farnesyl diphosphate synthase). Readministration of testosterone immediately following orchidectomy restored the expression of all four genes. Substitution of testosterone by the non-aromatizable androgen dihydrotestosterone gave rise to comparable changes in the mRNA and protein levels of the lipogenic genes under investigation, confirming the involvement of the androgen receptor in the observed effects. In support of the coordinate nature of this regulation, androgen-induced upregulation of lipogenic gene expression is accompanied by an increase in the nuclear content of SREBP, a key lipogenic transcription factor. Taken together, these findings provide evidence for a coordinate regulation of lipogenic gene expression not only in prostate cancer cell lines in culture but also in non-cancerous androgen-responsive tissues in vivo.

Zwitterionic Sulfobetaine Inhibitors of Squalene Synthase.

A substantial number of sulfobetaines (e.g., 10) have been synthesized and evaluated as inhibitors of squalene synthase (SS) on the basis of the idea that their zwitterionic structure would have properties conducive both to binding in the active site and to passage through cell membranes. When the simple sulfobetaine moiety is incorporated into compounds containing hydrophobic portions like those in farnesyl diphosphate (1) or presqualene diphosphate (2), inhibition of SS in a rat liver microsomal assay was indeed observed. For example, farnesylated sulfobetaine 10 has IC(50) = 10 &mgr;M and aromatic derivative 35 has IC(50) = 2 &mgr;M for SS inhibition. A wide variety of structural modifications, exemplified by compounds 43, 52, 76, 85, 91, 99, 111, and 115, was investigated. Unfortunately, no inhibitors in the submicromolar range were discovered, and exploration of a different type of zwitterion seems necessary if this appealing approach to inhibition of SS is going to provide a potential antihypercholesterolemic agent.

Squalene synthase, a determinant of Raft-associated cholesterol and modulator of cancer cell proliferation.

Several cues for cell proliferation, migration, and survival are transmitted through lipid rafts, membrane microdomains enriched in sphingolipids and cholesterol. Cells obtain cholesterol from the circulation but can also synthesize cholesterol de novo through the mevalonate/isoprenoid pathway. This pathway, however, has several branches and also produces non-sterol isoprenoids. Squalene synthase (SQS) is the enzyme that determines the switch toward sterol biosynthesis. Here we demonstrate that in prostate cancer cells SQS expression is enhanced by androgens, channeling intermediates of the mevalonate/isoprenoid pathway toward cholesterol synthesis. Interestingly, the resulting increase in de novo synthesis of cholesterol mainly affects the cholesterol content of lipid rafts, while leaving non-raft cholesterol levels unaffected. Conversely, RNA interference-mediated SQS inhibition results in a decrease of raft-associated cholesterol. These data show that SQS activity and de novo cholesterol synthesis are determinants of membrane microdomain-associated cholesterol in cancer cells. Remarkably, SQS knock down also attenuates proliferation and induces death of prostate cancer cells. Similar effects are observed when cancer cells are treated with the chemical SQS inhibitor zaragozic acid A. Importantly, although the anti-tumor effect of statins has previously been attributed to inhibition of protein isoprenylation, the present study shows that specific inhibition of the cholesterol biosynthesis branch of the mevalonate/isoprenoid pathway also induces cancer cell death. These findings significantly underscore the importance of de novo cholesterol synthesis for cancer cell biology and suggest that SQS is a potential novel target for antineoplastic intervention.

cDNA cloning and gene expression analysis of human myo-inositol 1-phosphate synthase.

myo-Inositol 1-phosphate synthase (EC 5.5.1.4) (IPS) is a key enzyme in myo-inositol biosynthesis pathway. This study describes the molecular cloning of the full length human myo-inositol 1-phosphate synthase (hIPS) cDNA, tissue distribution of its mRNA and characterizes its gene expression in cultured HepG2 cells. Human testis, ovary, heart, placenta, and pancreas express relatively high level of hIPS mRNA, while blood leukocyte, thymus, skeletal muscle, and colon express low or marginal amount of the mRNA. In the presence of glucose, hIPS mRNA level increases 2- to 4-fold in HepG2 cells. hIPS mRNA is also up-regulated 2- to 3-fold by 2.5 microM lovastain. This up-regulation is prevented by mevalonic acid, farnesol, and geranylgeraniol, suggesting a G-protein mediated signal transduction mechanism in the regulation of hIPS gene expression. hIPS mRNA expression is 50% suppressed by 10mM lithium ion in these cells. Neither 5mM myo-inositol nor the three hormones: estrogen, thyroid hormone, and insulin altered hIPS mRNA expression in these cells.

IDH1 gene transcription is sterol regulated and activated by SREBP-1a and SREBP-2 in human hepatoma HepG2 cells: evidence that IDH1 may regulate lipogenesis in hepatic cells.

The mRNA level for cytosolic NADP-dependent isocitrate dehydrogenase (IDH1) increases 2.3-fold, and enzyme activity of NADP-isocitrate dehydrogenase (IDH) 63%, in sterol-deprived HepG2 cells. The mRNA levels of the NADP- and NAD-dependent mitochondrial enzymes show limited or lack of regulation under the same conditions. Nucleotide sequences that are required, and sufficient, for the sterol regulation of transcription are located within a 67 bp region of an IDH1-secreted alkaline phosphatase promoter-reporter gene. The IDH1 promoter is fully activated by the expression of SREBP-1a in the cells and, to a lesser degree, by that of SREBP-2. A 5'-end truncation of 23 bp containing a CAAT and a GC-Box results in 6.5% residual activity. The promoter region involved in the activation by the sterol regulatory element binding proteins (SREBPs) is located at nucleotides -44 to -25. Mutagenesis analysis identified within this region the IDH1-SRE sequence element GTGGGCTGAG, which binds the SREBPs. Similar to the promoter activation, electrophoretic mobility shifts of probes containing the IDH1-SRE element exhibit preferential binding to SREBP-1a, as compared with SREBP-2. These results indicate that IDH1 activity is coordinately regulated with the cholesterol and fatty acid biosynthetic pathways and suggest that it is the source for the cytosolic NADPH required by these pathways.

Low-temperature effect on the sterol-dependent processing of SREBPs and transcription of related genes in HepG2 cells.

Lowering the growth temperature of HepG2 cells from 37 degrees C to 20 degrees C results in a 73% reduction in human squalene synthase (HSS) protein, a 76% reduction in HSS mRNA, and a 96% reduction in promoter activity of a secreted alkaline phosphatase-HSS reporter gene. A similar decrease in either mRNA or protein levels is observed for 3-hydroxy-3-methylglutaryl CoA reductase, farnesyl diphosphate synthase, the LDL receptor, and fatty acid synthase. All these proteins and mRNAs show either a decrease or a complete loss of sterol-dependent regulation in cells grown at 20 degrees C. In contrast, sterol regulatory element binding proteins (SREBPs)-1 and -2 exhibit a 2- to 3-fold increase in mRNA levels at 20 degrees C. The membrane-bound form of the SREBPs is dramatically increased, but the proteolytic processing to the nuclear (N-SREBP) form is inhibited under these conditions. Overexpression of the N-SREBP or SREBP cleavage-activating protein (SCAP), but not site-1 or site-2 proteases, restores the activation of the HSS promoter at 20 degrees C, most likely by liberating the SCAP-SREBP complex so that it can move to the Golgi for processing. These results indicate that the cholesterol synthesizing machinery is down-regulated at low temperatures, and points to the transport of the SCAP-SREBP complex to the Golgi as the specific down-regulated step.