Potency of select statin drugs in a new mouse model of hyperlipidemia and atherosclerosis.

Poloxamer-407 (P-407) is a nonionic surfactant that induces atheroma formation in the aortas of C57BL/6 mice with long-term (14 weeks) administration. The objectives of the present study were to determine the mechanism(s) responsible for the induction of hypercholesterolemia as well as to determine whether this animal model may be of potential use in rank ordering the efficacy (lipid lowering) of various statin drugs. The effect of long-term (16 weeks) administration of P-407 on the catalytic activities of rate-limiting enzymes of cholesterol biosynthesis [HMG-CoA reductase (HMGR)] and catabolism [microsomal cholesterol 7alpha-hydroxylase (C7alphaH) and mitochondrial sterol 27 hydroxylase (S27H)] was assessed in C57BL/6 mice. Effects of P-407 on these enzymes were compared in mice fed an atheroma-inducing diet (high-cholesterol, supplemented with cholic acid) and animals maintained on a basal diet and injected with saline (controls) after 16 weeks. The mean value for the activities of C7alphaH in P-407-injected mice was 24.3+/-3.8 pmol min(-1) mg(-1) and was significantly (P<0.05) less than the mean value determined for sham-injected control animals (37.0+/-14.3 pmol min(-1) mg(-1)). In contrast, the mean values for the catalytic activities of S27H and HMGR did not change with P-407 administration. Neither C7alphaH nor S27H activity in mice fed the high-cholesterol diet differed from values for control animals, whereas the mean HMGR activity was drastically reduced (-94%, P<0.05). The hypercholesterolemic effect of P-407 is not due to altered cholesterol biosynthesis, but is mediated by reduced cholesterol catabolism due to decreased activity of the rate limiting enzyme (C7alphaH) in the classic bile acid synthetic pathway. Plasma triglyceride lowering resulting from the oral administration of equal doses of various statin drugs appeared, in general, to be positively correlated with their relative aqueous solubility and paralleled the efficacy of these agents to lower low-density-lipoprotein-associated cholesterol (LDL-C) in humans. The plasma triglyceride lowering effect of the five statin drugs tested produced the following rank order; pravastatin sodium (-44%)>atorvastatin calcium (-36%)>simvastatin (-33%)>lovastatin (-25%)>fluvastatin sodium (-19%). While reductions in plasma total cholesterol following administration of the statin drugs was not as profound as that observed with triglycerides, the relative rank order or trend was preserved. The percent reduction in plasma triglycerides in the present model appears to be a useful parameter with which to predict the relative reduction in plasma LDL-C expected for these agents in humans.

Phosphatidylinositol 3-kinase, not extracellular signal-regulated kinase, regulates activation of the antioxidant-responsive element in IMR-32 human neuroblastoma cells.

The antioxidant-responsive element (ARE) plays an important role in the induction of phase II detoxifying enzymes including NADPH:quinone oxidoreductase (NQO1). We report herein that activation of the human NQO1-ARE (hNQO1-ARE) by tert-butylhydroquinone (tBHQ) is mediated by phosphatidylinositol 3-kinase (PI3-kinase), not extracellular signal-regulated kinase (Erk1/2), in IMR-32 human neuroblastoma cells. Treatment with tBHQ significantly increased NQO1 protein without activation of Erk1/2. In addition, PD 98059 (a selective mitogen-activated kinase/Erk kinase inhibitor) did not inhibit hNQO1-ARE-luciferase expression or NQO1 protein induction by tBHQ. Pretreatment with LY 294002 (a selective PI3-kinase inhibitor), however, inhibited both hNQO1-ARE-luciferase expression and endogenous NQO1 protein induction. In support of a role for PI3-kinase in ARE activation we show that: 1) transfection of IMR-32 cells with constitutively active PI3-kinase selectively activated the ARE in a dose-dependent manner that was completely inhibited by treatment with LY 294002; 2) pretreatment of cells with the PI3-kinase inhibitors, LY 294002 and wortmannin, significantly decreased NF-E2-related factor 2 (Nrf2) nuclear translocation induced by tBHQ; and 3) ARE activation by constitutively active PI3-kinase was blocked completely by dominant negative Nrf2. Taken together, these data clearly show that ARE activation by tBHQ depends on PI3-kinase, which lies upstream of Nrf2.

Lipid emulsions as vehicles for enhanced nasal delivery of insulin.

The objective of this work is to explore lipid emulsion based formulations of insulin as an enhancer of nasal absorption. Insulin was incorporated into the aqueous phases of water-in-oil (w/o) and oil-in-water (o/w) emulsions. The formulations were perfused through the nasal cavity of rats in situ. Enhancement of insulin absorption was observed when insulin was incorporated into the continuous aqueous phase of an o/w emulsion. The presence of a small fraction of oil droplets along with insulin in the aqueous phase appeared to favor insulin absorption. When the oil phase constitutes the external phase, as in w/o emulsion, no insulin absorption was noted. Inhibition of insulin absorption might arise from a rate limiting barrier effect of the membrane completely covered by a stagnant oil layer. The in situ model was validated by in vivo experiments, which also revealed an increase in insulin absorption with o/w emulsions. However at lower insulin doses there was no statistically significant enhancing effect. In situ perfusion experiments across rat nasal pathway appear to be an appropriate model to study the enhancement effect of nasal formulations.

Cadmium-mediated activation of the metal response element in human neuroblastoma cells lacking functional metal response element-binding transcription factor-1.

Metal response element-binding transcription factor-1 (MTF-1) binds specifically to metal response elements (MREs) and transactivates metallothionein (MT) gene expression in response to zinc and cadmium. This investigation contrasts the mechanism of mouse MT gene (mMT-I) promoter activation by cadmium and zinc in IMR-32 human neuroblastoma cells to determine whether MTF-1 binding to the MRE is necessary for activation by these metals. Cadmium activated a mMT-1 promoter (-150 base pairs) luciferase reporter 20-25-fold through a MRE-dependent mechanism. In contrast, zinc had little effect on the mMT-1 luciferase reporter. IMR-32 cells lacked MRE binding activity, and treatment with zinc in vitro or in vivo did not generate a MTF-1. MRE complex, suggesting that IMR-32 cells lack functional MTF-1. Overexpression of mMTF-1 regenerated a zinc-mediated induction of the MRE without affecting cadmium activation. Because no other transition metals tested activated the MRE, this effect appeared to be cadmium-specific. These data demonstrate that in IMR-32 human neuroblastoma cells, zinc and cadmium can use independent mechanisms for activation of the mMT-I promoter and cadmium-mediated MRE activation is independent of MTF-1 and zinc.

Participation of upstream stimulator factor (USF) in cadmium-induction of the mouse metallothionein-I gene.

The roles of the bHLH-Zip protein, upstream stimulatory factor (USF), in mouse metallothionein-I (MT-I) gene expression were examined. The promoter contains a putative USF binding site which overlaps an antioxidant response element (ARE) located at -101 bp relative to the transcription start point. The USF/ARE composite element increases basal expression of the mouse MT-I gene, and partly mediates response to oxidative stress. However, other functions of this composite element and the in vivo roles for USF in MT-I promoter functions have not been examined. We report studies which indicate that USF participates via the USF/ARE element in cadmium responsiveness of the mouse MT-I promoter. During the course of these studies a second, higher affinity USF binding site at -223 bp was identified. Stable and transient transfection assays in mouse hepatoma cells, using the USF/ARE in the context of a minimal promoter and site-directed and truncation mutants of the MT-I promoter, revealed that the USF and the ARE sites contribute to cadmium (2-30 microM) but not zinc responsiveness, and to basal promoter activity. Overexpression of dominant-negative (dn)USF in co-transfection assays significantly attenuated cadmium induction of the USF/ARE in the context of a minimal promoter, and attenuated cadmium, but not zinc, induction of the intact MT-I promoter. A consensus E-box (CACATG) at -223 bp in the MT-I promoter was also found to bind USF in vitro , and to be constitutively footprinted in vivo . The interaction of USF with E-box1 was apparently 10-fold stronger than that with the USF/ARE. However, in contrast, E-box1 was not a strong basal promoter element nor was it metal ions responsive in mouse Hepa cells. In conclusion, these studies demonstrate a role for USF in cadmium-specific induction of the mouse MT-I gene, but bring into question an obligate role for USF in regulating basal activity of this gene. The data further suggest that USF interacts with ARE-binding proteins to influence MT-I gene expression.