Drosophila female precopulatory behavior is modulated by ecdysteroids.

The effect of ecdysteroid signaling on Drosophila female precopulatory behavior was investigated using two types of mutants with either globally reduced ecdysteroid availability or reduced expression of ecdysone receptors in fruitless neurons, known to control sexual behavior. While being courted by males, mutant females performed significantly less full ovipositor extrusion behavior to reject male copulation attempts. Ecdysteroid depleted females (ecdysoneless(1)) performed male-like courtship behaviors, including unilateral wing extension and song production with patterns very similar to male courtship song. These results support the hypothesis that ecdysteroids modulate female sexual behavior, perhaps acting as a regulator of sexual motivation, and as a component affecting the performance of sex specific behavior patterns.

Drosophila male courtship behavior is modulated by ecdysteroids.

Temperature-dependent induction of ecdysteroid deficiency in the ecdysoneless mutant ecd(1) adult Drosophila melanogaster results in altered courtship behavior in males. Ecdysteroid deficiency brings about significantly elevated male-male courtship behavior including song production resembling that directed toward females. Supplementation with dietary 20-hydroxyecdysone reduces male-male attraction, but does not change motor activity, courtship patterns or attraction to females. These observations support the hypothesis that reduced levels of ecdysteroids increase the probability that male fruit flies will display courtship behaviors to male stimuli.

Characterization of aortic root atherosclerosis in ApoE knockout mice: high-resolution in vivo and ex vivo MRM with histological correlation.

In vivo, cardiac-gated, black-blood, and ex vivo magnetic resonance microscopy (MRM) images of the aortic root, and histopathology data were obtained from 12 transgenic and wild-type (WT) mice. MRM was performed using a black-blood imaging spin-echo sequence with upstream and downstream in-flow saturation pulses to obtain aortic root images in three contrast techniques: proton density-weighted (PDW), T(1)- (T(1)W), and T(2)-weighted (T(2)W). Aortic wall thickness and area were measured and correlated with histopathology data (R > 0.90). Ex vivo lesion components (lipid core, fibrous tissue, and cell tissue) were identified and characterized by differing image contrast in PDW, T(1)W, and T(2)W MRM, and by histopathology. The differences between WT and transgenic mice for maximal wall thickness and area were statistically significant (P < 0.05). This study demonstrates the feasibility of in vivo murine aortic root lesion assessment and ex vivo plaque characterization by MRM.

Elevating high-density lipoprotein cholesterol in apolipoprotein E-deficient mice remodels advanced atherosclerotic lesions by decreasing macrophage and increasing smooth muscle cell content.

BACKGROUND: HDL cholesterol levels are inversely correlated with coronary heart disease risk in humans, and in animal studies, HDL elevation decreases formation and progression of foam-cell lesions. The potential for HDL to affect preexisting advanced atherosclerotic lesions is not known. To approach this issue, we used a novel mouse aortic transplantation model. METHODS AND RESULTS: ApoE-deficient (EKO) mice were fed a Western-type diet for 6 months, and thoracic aortic segments containing advanced lesions replaced segments of the abdominal aorta of 4-month-old EKO syngeneic mice not expressing (plasma HDL cholesterol approximately 26 mg/dL) or expressing (HDL approximately 64 mg/dL) a human apoAI (hAI) transgene. Both types of recipients had comparable non-HDL cholesterol levels. Five months after transplantation, mice were killed and grafts analyzed. Compared with lesion area in pretransplant mice (0.14+/-0.04 mm(2), mean+/-SEM), there was progression in the EKO recipients (0.39+/-0.06 mm(2), P<0.01). Compared with EKO recipients, hAI/EKO recipients had retarded progression (0.24+/-0.04 mm(2), P<0.05). Immunostaining for CD68 and other macrophage-associated proteins, monocyte chemoattractant protein-1, acyl coenzyme A:cholesterol acyltransferase, and tissue factor, in lesions of pretransplant and EKO recipient mice showed abundant macrophages. In contrast, compared with any other group, lesional macrophage area in hAI/EKO mice decreased >80% (P<0.003), and smooth muscle cell content (alpha-actin staining) increased >300% (P<0.006). The decrease in macrophages and increase in smooth muscle cells was primarily in the superficial subendothelial layer. CONCLUSIONS: Increasing HDL cholesterol levels in EKO mice retards progression of advanced atherosclerotic lesions and remodels them to a more stable-appearing phenotype.

Dramatic remodeling of advanced atherosclerotic plaques of the apolipoprotein E-deficient mouse in a novel transplantation model.

OBJECTIVE: Regression of atherosclerotic lesions is an important goal. No extensive experimental evidence shows that it can be achieved for advanced lesions. To study this, we developed a model to maintain a long-term change in the plasma lipoprotein environment of advanced arterial lesions of hyperlipidemic (apolipoprotein E [apoE]-deficient) mice. METHODS: The apoE-deficient mice (plasma total cholesterol of 1334 +/- 219 [+/- SEM] mg/dL) on a typical Western diet for 38 weeks had advanced atherosclerotic lesions (ie, beyond the macrophage foam cell stage) throughout the arterial tree. Lesion-containing thoracic aortas were transplanted (replacing a segment of abdominal aorta) into either apoE-deficient or wild-type (WT) (total cholesterol of 86 +/- 10 mg/dL) recipients. Grafts were harvested after 9 weeks. RESULTS: Compared with pretransplant lesions (area = 0.0892 +/- 0.0179 mm(2)), lesion size tended to increase in apoE-deficient to apoE-deficient grafts (0.2411 +/- 0.0636 mm(2); P =.06), whereas a significant reduction was seen in apoE-deficient to WT grafts (0.0214 +/- 0.0049 mm(2); P <.001). Also, foam cells were absent in apoE-deficient to WT grafts, but abundant in pretransplant lesions and apoE-deficient to apoE-deficient grafts. Grafts were evaluated noninvasively in vivo with magnetic resonance imaging, and wall thickening was detected in the apoE-deficient to apoE-deficient group. CONCLUSIONS: Nearly complete regression of advanced atherosclerotic lesions can be achieved with sustained normalization of the plasma lipoprotein profile. Syngeneic arterial transplantation in mice is a novel and valuable model system for atherosclerosis research; and magnetic resonance imaging can detect differences in characteristics in lesions undergoing regression.

Apoprotein B degradation is promoted by the molecular chaperones hsp90 and hsp70.

Apoprotein B (apoB) is the major protein of liver-derived atherogenic lipoproteins. The net production of apoB can be regulated by presecretory degradation mediated by the ubiquitin-proteasome pathway and cytosolic hsp70. To further explore the mechanisms of apoB degradation, we have established a cell-free system in which degradation can be faithfully recapitulated. Human apoB48 synthesized in vitro was translocated into microsomes, glycosylated, and ubiquitinylated. Subsequent incubation with rat hepatic cytosol led to proteasome-mediated degradation. To explore whether hsp90 is required for apoB degradation, geldanamycin (GA) was added during the degradation assay. GA increased the recovery of microsomal apoB48 approximately 3-fold and disrupted the interaction between hsp90 and apoB48. Confirming the hsp90 effect in the cell-free system, we also found that transfection of hsp90 cDNA into rat hepatoma cells enhanced apoB48 degradation. Finally, apoB48 degradation was reconstituted in vitro using cytosol prepared from wild type yeast. Notably, degradation was attenuated when apoB48-containing microsomes were incubated with cytosol supplemented with GA or with cytosol prepared from yeast strains with mutations in the homologues of mammalian hsp70 and hsp90. Overall, our data suggest that hsp90 facilitates the interaction between endoplasmic reticulum-associated apoB and components of the proteasomal pathway, perhaps in cooperation with hsp70.

Acyl-CoA:cholesterol acyltransferase inhibition reduces atherosclerosis in apolipoprotein E-deficient mice.

BACKGROUND: Acyl-COA:cholesterol acyltransferase (ACAT) converts cholesterol to cholesteryl esters. The form of ACAT in macrophages, ACAT1, contributes to foam cell formation in the arterial wall and the development of atherosclerosis. Recent studies in a mouse model of atherosclerosis (the apolipoprotein E [apoE]-deficient mouse), however, have suggested that complete deficiency of ACAT1 activity is not antiatherogenic, in part because of toxicity resulting from adverse effects on tissue cholesterol homeostasis. We have tested whether partial inhibition of ACAT1 and ACAT2 (expressed in liver and intestine) activities reduces atherosclerosis development in apoE-deficient mice and avoids toxicity. METHODS AND RESULTS: ApoE-deficient mice were maintained for 17 weeks on a Western-type diet without (control) or with the ACAT inhibitor F-1394 (effective against ACAT1 and ACAT2) at doses of either 300 (low) or 900 (high) mg/kg. Intimal lesion area at the aortic sinus in controls was 0.69+/-0.06 mm(2). F-1394 treatment significantly decreased lesional area by 39% (low) or 45% (high). F-1394 treatment also reduced lesional immunostaining for macrophages by 61% (low) or 83% (high). En face analysis showed that surface lipid staining in control aortas was 20.0+/-2.8%; F-1394 treatment reduced this by 46% (low) or 62% (high). There were no obvious signs of systemic or vessel wall toxicity associated with F-1394 treatment. CONCLUSIONS: Partial ACAT inhibition by F-1394 had antiatherogenic effects in apoE-deficient mice that were achieved without obvious toxicity. Partial ACAT inhibition may have therapeutic potential in the clinical treatment of atherosclerosis.

The triple threat to nascent apolipoprotein B. Evidence for multiple, distinct degradative pathways.

We previously showed that Omega-3 fatty acids reduce secretion of apolipoprotein B (apoB) from cultured hepatocytes by stimulating post-translational degradation. In this report, we now characterize this process, particularly in regard to the two known processes that degrade newly synthesized apoB, endoplasmic reticulum (ER)-associated degradation and re-uptake from the cell surface. First, we found that Omega-3-induced degradation preferentially reduces the secretion of large, assembled apoB-lipoprotein particles, and apoB polypeptide length is not a determinant. Second, based on several experimental approaches, ER-associated degradation is not involved. Third, re-uptake, the only process known to destroy fully assembled nascent lipoproteins, was clearly active in primary hepatocytes, but Omega-3-induced degradation of apoB continued even when re-uptake was blocked. Cell fractionation showed that Omega-3 fatty acids induced a striking loss of apoB100 from the Golgi, while sparing apoB100 in the ER, indicating a post-ER process. To determine the signaling involved, we used wortmannin, a phosphatidylinositol 3-kinase (PI3K) inhibitor, which blocked most, if not all, of the Omega-3 fatty acid effect. Therefore, nascent apoB is subject to ER-associated degradation, re-uptake, and a third distinct degradative pathway that appears to target lipoproteins after considerable assembly and involves a post-ER compartment and PI3K signaling. Physiologic, pathophysiologic, and pharmacologic regulation of net apoB secretion may involve alterations in any of these three degradative steps.

Co-translational interactions of apoprotein B with the ribosome and translocon during lipoprotein assembly or targeting to the proteasome.

Hepatic lipoprotein assembly and secretion can be regulated by proteasomal degradation of newly synthesized apoB, especially if lipid synthesis or lipid transfer is low. Our previous studies in HepG2 cells showed that, under these conditions, newly synthesized apoB remains stably associated with the endoplasmic reticulum (ER) membrane (Mitchell, D. M., Zhou, M., Pariyarath, R., Wang, H., Aitchison, J. D., Ginsberg, H. N., and Fisher, E. A. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 14733-14738). We now show that independent of lipid synthesis, apoB chains that appear full-length are, in fact, incompletely translated polypeptides still engaged by the ribosome and associated with the ER translocon. In the presence of active lipid synthesis and transfer, translation and lipoprotein assembly are completed, and the complexes exit the ER. Upon omitting fatty acids from, or adding a microsomal triglyceride transfer protein inhibitor to, culture media to reduce lipid synthesis or transfer, respectively, apoB was degraded while it remained associated with the ER and complexed with cytosolic hsp70 and proteasomes. Thus, unlike other ER substrates of the proteasome, such as major histocompatibility complex class I molecules, apoB does not fully retrotranslocate to the cytosol before entering the ubiquitin-proteasome pathway. Although, upon immunofluorescence, apoB in proteasome-inhibited cells accumulated in punctate structures similar in appearance to aggresomes (cytosolic structures containing molecules irreversibly lost from the secretory pathway), these apoB molecules could be secreted when lipid synthesis was stimulated. The results suggest a model in which 1) apoB translation does not complete until lipoprotein assembly terminates, and 2) assembly with lipids or entry into the ubiquitin-proteasome pathway occurs while apoB polypeptides remain associated with the translocon and attached to the ribosome.

The level and species of plasma non-esterified fatty acids are not related to elevated plasma apolipoprotein B levels in familial combined hyperlipidemia.

Familial combined hyperlipidemia (FCHL), the leading cause of familial hyperlipidemia with premature coronary artery disease, has been associated with insulin resistance and elevated plasma levels of apolipoproten B (apoB) and non-esterified fatty acids (NEFA). Becaus dietary fats affect plasma cholesterol levels, and specific saturated fatty acids (FA) are particularly potent stimulators in vitro of apoB secretio from hepatocytes, we hypothesized that FCHL patients would exhibit elevations in plasma levels of total FA or specific saturated species. Five families containing 12 FCHL subjects (5 adults, 7 children and 8 normals (5 adults, 3 children) were assessed by dietary, anthropometric, and plasma measurements (glucose, insulin, lipoproteins, total NEFA, and specific FA types). After adjustment of the data for age, gender, and family affiliation, multivariate ANOVA indicated that FCHL was significantly associated with elevated plasma levels of apoB (p = 0.001) and insulin (p< 0.001) and increased body weight (p=0.043). Nevertheless, dietary intakes of total and saturated fat were comparable in the two groups, as were plasma levels of total NEFA and the major saturated species. In a study population possessing the salient features of FCHL, circulating total NEFA were not elevated, nor were specific saturated NEFA that had been associated with apoB oversecretion in vitro. Despite the speculated link between plasma FA and apoB overproduction in FCHL, our data suggest that other metabolic factors underlie this disease.

Sulindac inhibits neointimal formation after arterial injury in wild-type and apolipoprotein E-deficient mice.

Neointimal hyperplasia is a critical component of restenosis, a major complication of angioplasty and related therapeutic procedures. We studied the effects of hyperlipidemia and the nonsteroidal anti-inflammatory drugs, aspirin (acetyl-salicylic acid; ASA), and sulindac, on neointimal formation in a mouse femoral arterial injury model. At 2 months of age, normolipidemic, wild-type (WT), and hyperlipidemic, apolipoprotein E-deficient (apoE-/-) mice were divided into three treatment groups: Western-type diet (WD), WD + ASA (200 mg/kg food), and WD + sulindac (300 mg/kg food). After 1 week, mice underwent arterial injury and treatments were maintained for 4 weeks. Histomorphometry of the injured arteries showed striking effects of plasma cholesterol levels and drug treatment on neointimal hyperplasia. In the WD or WD + ASA groups, apoE-/- mice had twice the neointimal area than WT mice ( approximately 30,000 vs. 13,000 microm(2) per section; P < 0.0001). Compared with ASA or WD alone, sulindac treatment resulted in approximately 70% (P = 0.0001) and 50% (P = 0.01) reductions in the neointimal area in apoE-/- and WT mice, respectively. ASA, at a dose sufficient to inhibit platelet aggregation, did not affect neointimal formation in mice of either genotype. Evidence of macrophages was noted in the lesions of apoE-/- mice in the WD and WD + ASA groups, but remarkably, none was detectable with sulindac treatment, despite hyperlipidemia, suggesting early steps in the response to injury were abrogated. These results demonstrate sulindac reduces neointimal formation in both normolipidemic and hyperlipidemic settings and raise the possibility that similar benefits may be obtained in patients undergoing angioplasty and related procedures.

The amino-terminal domain of apolipoprotein B does not undergo retrograde translocation from the endoplasmic reticulum to the cytosol. Proteasomal degradation of nascent apolipoprotein B begins at the carboxyl terminus of the protein, while apolipoprotein B is still in its original translocon.

We studied the sequential topology of the NH(2) and COOH termini of apoB during translocation by expressing, in Chinese hamster ovary (CHO) and HepG2 cells, an apoB42 construct with c-Myc and hemagglutinin (HA) tags at 2 and 41% (relative to apoB100) of its amino acid sequence. We conducted similar studies using monoclonal antibodies against the NH(2) and COOH termini of apoB100 in HepG2 cells. After radiolabeling, microsomes were immunoisolated from transfected CHO cells using anti-c-Myc or anti-HA antibodies. Throughout a 60-min chase in the presence of N-acetyl-leucyl-norleucinal, more than 90% of microsomes were isolated by anti-HA antibodies, whereas less than 10% were isolated by anti-c-Myc antibodies. Proteinase K digestion of total microsomes consistently generated two fragments ( approximately 70 and approximately 120 kDa) of apoB42 containing the NH(2) terminus throughout the chase; no fragments containing the COOH terminus were detected. Immunofluorescent studies of transfected CHO cells were consistent with results from the labeling studies. Essentially identical results were obtained from pulse-chase studies in both native and apoB42-transfected HepG2 cells. The present studies support a model in which, in the absence of adequate core lipid synthesis, there is partial translocation of apoB leading to cytosolic exposure, ubiquitination, and proteasomal degradation directly from the original translocation channel.

Release of active tissue factor by human arterial smooth muscle cells.

Tissue factor (TF), the initiator of coagulation, is thought to function predominantly at the cell surface. Recent data have suggested that active TF is present extracellularly in atherosclerotic plaques, the arterial wall, and the blood. This study was conducted to determine whether smooth muscle cells (SMCs), a major source of arterial TF, could generate extracellular TF. Active TF accumulated in the medium of cultured human SMCs, representing approximately 10% of that measured in the underlying cells at 24 hours. Platelet-derived growth factor, phorbol ester, and tumor necrosis factor-alpha caused approximately 3-fold increases in TF activity in the medium. Release of TF into the medium was dependent on the presence of the TF transmembrane domain but not the cytoplasmic domain. Antibodies to TF precipitated most of the activity from the culture medium, whereas antibodies to the beta(1)-integrin subunit precipitated approximately 33% of the activity. Treatment with detergent or phosphatidylserine:phosphatidylcholine did not increase activity, suggesting that all TF released by SMCs was in the appropriate lipid milieu and not encrypted. Western blotting showed that the medium contained full-length TF protein. Fluorescent cytometry showed that extracellular TF was present largely in particles < or =200 nm, which had a density of 1.10 g/mL. We hypothesize that active extracellular TF found in the injured arterial wall and atherosclerotic plaques derives, in part, from SMC microparticles.

Inhibition of translocation of nascent apolipoprotein B across the endoplasmic reticulum membrane is associated with selective inhibition of the synthesis of apolipoprotein B.

In HepG2 cells, inhibition of apolipoprotein B100 (apoB) translocation across the endoplasmic reticulum by an microsomal triglyceride transfer protein (MTP) inhibitor (CP-10447) in the presence of N-acetyl-leucinyl-norleucinal, a proteasomal inhibitor, results in accumulation of newly synthesized apoB in the translocation channel. Here we demonstrated that such accumulation led to a specific reduction of apoB synthesis. ApoB mRNA levels remained unchanged, but we observed reduced rates of elongation of nascent apoB in puromycin-synchronized cells pretreated with MTP inhibitor. This observation was consistent with a longer half-ribosome transit time for the synthesis of apoB in MTP-inhibited cells. Initiation of translation of apoB mRNA was not impaired by MTP inhibition. Overall, these findings suggest that translocation arrest of apoB in the endoplasmic reticulum channel can exert a selective and negative effect on the synthesis of apoB at the stage of elongation.

Hydrolysis of retinyl esters by pancreatic triglyceride lipase.

Previously [van Bennekum, A. M., et al. (1999) Biochemistry 38, 4150-4156] we showed that carboxyl ester lipase (CEL)-deficient (CELKO) mice have normal levels of pancreatic, bile salt-dependent retinyl ester hydrolase (REH) activity. In the present study, we further investigated this non-CEL REH activity in pancreas homogenates of CELKO and wild-type (WT) mice, and rats. REH activity was detected in both the presence and absence of tri- and dihydroxy bile salts in rats, WT mice, and CELKO mice. In contrast, pancreatic cholesteryl ester hydrolase (CEH) activity was only detected in the presence of trihydroxy bile salts and only in rats and WT mice, consistent with CEL-mediated cholesteryl ester hydrolysis. Enzyme assays of pancreatic triglyceride lipase (PTL) showed that there was a colipase-stimulated REH activity in rat and mouse (WT and CELKO) pancreas, consistent with hydrolysis of retinyl ester (RE) by PTL. Pancreatic enzyme activities related to either CEL or PTL were separated using DEAE-chromatography. In both rats and mice (WT and CELKO), REH activity could be attributed mainly to PTL, and to a much smaller extent to CEL. Finally, purified human PTL exhibited similar enzymatic characteristics for triglyceride hydrolysis as well as for retinyl ester hydrolysis, indicating that RE is a substrate for PTL in vivo. Altogether, these studies clearly show that PTL is the major pancreatic REH activity in mice, as well as in rats.

Dietary therapy for children with hypercholesterolemia.

Accumulating evidence clearly shows that atherosclerosis begins in youth. The National Cholesterol Education Program (NCEP) has recommended that children at high risk of developing coronary artery disease as adults be screened so that those with elevated low-density lipoprotein (LDL) cholesterol levels can be treated, primarily by modification of diet. The initial approach to these youthful patients is to use the NCEP step I diet. This diet provides calories and nutrients that support normal growth and development, but limits saturated fat and total fat intake to no more than 10 and 30 percent of total calories, respectively, and cholesterol intake to no more than 100 mg per 1,000 kcal per day, to a maximum of 300 mg. If the goal of reducing the LDL cholesterol level to below 130 mg per dL (3.35 mmol per L) is not achieved, the more restrictive step II diet should be initiated. However, the step II diet may not provide sufficient calories and nutrients to support normal growth and development; therefore, trained nutritionists may be required to effectively manage a child on this diet.

High-density lipoprotein: gene-based approaches to the prevention of atherosclerosis.

Although the atheroprotective role of high-density lipoprotein (HDL) has been well documented in epidemiological and animal studies, highly effective therapeutic approaches for the selective increase of plasma HDL levels or function are not yet available. Several mechanisms by which HDL exerts an atheroprotective effect have been proposed on the basis of experiments in vitro and in vivo. These mechanisms include directing excess cellular cholesterol from the peripheral tissues to the liver in 'reverse cholesterol transport', inhibiting oxidative modification or aggregation of LDL, and modulating inflammatory responses to favour vasoprotection. This review gives an overview of the genes regulating these mechanisms, such as those encoding apolipoprotein AI, lecithin:cholesterol acyltransferase (LCAT), scavenger receptor B1 (SR-BI), and the ATP-binding cassette transporter 1 (ABC1), and the potential to exploit them to develop gene-based therapeutic approaches to increase the level or function of HDL.