Neelam Srivastava

Estrogen Up-regulates Apolipoprotein E (ApoE) Gene Expression by Increasing ApoE mRNA in the Translating Pool via the Estrogen Receptor α-Mediated Pathway

The antiatherogenic property of estrogens is mediated via at least two mechanisms: first by affecting plasma lipoprotein profiles, and second by affecting the components of the vessel wall. Raising plasma apolipoprotein E (apoE) in mice protects them against diet-induced atherosclerosis (Shimano, H., Yamada, N., Katsuki, M., Gotoda, T., Harada, K., Murase, T., Fukuzawa, C., Takaku, F., and Yazaka, Y. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 1750–1754). It is possible that estrogen may be antiatherogenic at least in part by increasing plasma apoE levels. Therefore, we studied the regulation of apoE by estrogen. A survey of 15 inbred strains of mice showed that some mouse strains responded to injections or subcutaneously implanted pellets of estradiol by raising their apoB and apoE levels and some did not. We performed detailed studies in two “responder” strains, C57L and C57BL, and two “non-responder” strains, C3H and BALBc. Responders increased their plasma apoE levels 2.5-fold. Non-responders’ levels were altered ±10%. In the responders the distribution of apoE among the plasma lipoproteins shifted from high density lipoprotein toward the apoB-containing lipoprotein fractions. In nonresponders the shift was toward high density lipoprotein. Hepatic apoE mRNA levels and relative rates of apoE mRNA transcription were unchanged in all strains, suggesting that apoE regulation occurred at posttranscriptional loci. Therefore, we measured apoE synthesis in fresh liver slices and on isolated hepatic polysomes. Two-fold increases were noted but only in responders accompanied by selective 1.5-fold increases in polysomal apoE mRNA levels. Similar increases in apoE synthesis were also observed in castrated C57BL mice given either physiological or pharmacological replacement doses of estradiol, but not testosterone, suggesting that the effect of estradiol was specific on the distribution of apoE mRNA in the translationally active polysomal pool. Next, we examined whether the effects of estrogen on apoE translation were mediated by estrogen receptors (ER). ER-α knock-out mice and their wild-type littermates were administered estradiol. As expected, apoE levels and hepatic apoE synthesis increased more than 2-fold in the wild-type littermates, but only 20% increases in the plasma apoE and hepatic synthesis were observed in the ER knock-out mice. Hepatic apoE mRNA levels did not change in either the wild-type or the ER knock-out mice. Thus, estradiol up-regulates apoE gene expression by increasing levels of apoE mRNA in the polysomal translating pool. Furthermore, the increased polysomal recruitment of apoE mRNA is largely mediated by estrogen receptors.

ATP binding cassette transporter A1--key roles in cellular lipid transport and atherosclerosis.

ATP-binding cassette transporter A1 (ABCA1) was recently recognized as the mutant molecule responsible for Tangier disease with low HDL levels, accumulation of cholesteryl esters in tissues, and increased risk of cardiovascular disease. Extensive studies for the past 2 years have recognized the critical role of ABCA1 in cholesterol and phospholipid trafficking. Since the removal of cholesterol from tissues is a key step in the prevention of atherosclerosis, significant attention has been focused on this molecule. Natural ABCA1 mutations in Tangier disease (TD) patients and WHAM chickens together with induced mutation in ABCA1 knock-out mice unequivocally established the important role of ABCA1 in maintaining circulating HDL levels and promoting cholesterol efflux from the arterial wall. Mice lacking ABCA1 showed similar phenotypes observed in Tangier disease patients with low levels of HDL. Further understanding of the roles of ABCA1 in lipid transport and atherosclerosis became clear from studies with ABCA1 transgenic mice. These mice showed enhanced cholesterol efflux from macrophages and reduced atherosclerotic lesion formation. The promoter of the ABCA1 gene has been mapped to a large extent, with the exception of cAMP response element. The present review summarizes recent developments on the role of ABCA1 in cholesterol efflux and prevention of atherosclerosis. Given the antiatherogenic properties of ABCA1, this molecule can serve as an appropriate target for developing drugs to treat individuals with low levels of HDL.

Estrogen increases hepatic lipase levels in inbred strains of mice: a possible mechanism for estrogen-dependent lowering of high density lipoprotein.

We have shown mouse to be an useful animal model for studies on the estrogen-mediated synthesis and secretion of lipoproteins since, unlike in rats, low density lipoprotein receptors are not upregulated in mice [3]. This results into the elevation of plasma levels of apolipoprotein (apo) B and apoE, and lowering of apoA-I-containing particles. The mechanisms of apoB and apoE elevation by estrogen have been elucidated [6], but the mechanism of lowering of plasma levels of HDL is still not known. Among other factors, apoA-I, cholesterol ester transfer protein (CETP), scavenger receptor B1 (SR-B1), and hepatic lipase are potential candidates that modulate plasma levels of HDL. Since estrogen treatment increased hepatic apoA-I mRNA and apoA-I synthesis, and mouse express undetectable levels of CETP, we tested the hypothesis that estradiol-mediated lowering of HDL in mice may occur through modulation of hepatic lipase (HL). Four mouse strains (C57L, C57BL, BALB, C3H) were administered supraphysiological doses of estradiol, and plasma levels of HDL as well as HL mRNA were quantitated. In all 4 strains estradiol decreased plasma levels of HDL by 30%, and increased HL mRNA 2–3 fold. In a separate experiment groups of male C57BL mouse were castrated or sham-operated, and low and high doses of estradiol administered. We found 1.4–2.5 fold elevation of HL mRNA with concomitant lowering of HDL levels. Ten other mouse strains examined also showed estradiol-induced elevation of HL mRNA, but the extent of elevation was found to be strain-specific. Based on these studies, we conclude that hepatic lipase is an important determinant of plasma levels of HDL and that HL mRNA is modulated by estrogen which in turn may participate in the lowering of plasma levels of HDL.

A new apolipoprotein B truncation (apo B-43.7) in familial hypobetalipoproteinemia : Genetic and metabolic studies

We describe a new truncation of apolipoprotein (apo) B in a white kindred with familial hypobetalipoproteinemia (FHBL). Apo B-43.7, found in a daughter and her father, was due to a C --> T change in base position 6162 of the apo B gene converting the arginine (residue 1986) codon CGA to a stop codon TGA. Both subjects were heterozygotes, and both apo B-43.7- and apo B-100-containing particles were present in plasma. On density gradient ultracentrifugation (DGUC), approximately 30% to 40% of apo B-43.7 floated with very-low-density lipoprotein (VLDL)/intermediate-density lipoprotein (IDL)-density particles and 60% to 70% floated with high-density lipoprotein (HDL)-density particles. To assess the metabolism of apo B, 13C-leucine was infused and its rates of appearance in and disappearance from apo B-43.7- and apo B-100-containing particles were quantified by multicompartmental kinetic analysis. Apo B-100 entered plasma via VLDL with a production rate of 30 mg x kg-1 x d-1. Fractional catabolic rates (FCRs) for apo B-100 VLDL, IDL, and low-density lipoprotein (LDL) were 20.0, 16.0, and 0.46 pools x d-1, respectively. The production rate of apo B-43.7 was 9.6 mg x kg-1 x d-1, and FCRs for apo B-43.7 VLDL- and HDL-like particles were 12.0 and 1.8 pools x d-1, respectively. Approximately 30% of apo B-43.7 in HDL-density particles was derived from VLDL apo B-43.7, and about 70% appeared to enter the plasma as HDLs. The relatively low production rate of apo B-43.7 is compatible with previous reports that apo B truncations are produced at lower rates than their apo B-100 counterparts.

Regulation of Creatine Kinase Isoenzymes in Human Placenta During Early, Mid-, and Late Gestation:

Objective: Creative kinase (CK) isoenzymes play an important role in cellular energy transduction. Two isoenzymes of creatine kinase, ubiquitous mitochondrial creatine kinase (uMtCK) and cytosolic brain creatine kinase (BCK), are postulated to form the creatine phosphate (CP) shuttle, in which creatine serves to transport high-energy phosphate from the mitochondria to its site of utilization. Coordinate regulation of these genes is essential for efficient energy transduction. We examined human CK isoenzyme regulation in placentas during all three trimesters of gestation to define the mRNA and protein expression patterns of uMtCK and BCK and to test the CP shuttle hypothesis. Methods: Placental samples were collected from a total of 26 patients from the first, second, and third trimesters. Total RNA and protein were prepared from each sample and quantified. Quantitative RNA analysis was performed by gel electrophoresis and dot blot techniques using isoenzyme-specific human cDNA probes for uMtCK and BCK. Protein expression of uMtCK and BCK was examined by Western blot analysis using isoenzyme-specific antibodies to uMtCK and BCK. Results: Analysis of RNA demonstrated the coordinate expression of uMtCK and BCK mRNAs in human placenta, with peak expression ofboth in the term placentas. Western blot analysis demonstrated coordinate expression of uMtCK and BCK proteins in the first and second trimesters, but not in the term placenta. Expression levels of uMtCK and BCK proteins were not consistent with their respective mRNA levels in the term placenta. Conclusion: Expression of uMtCK and BCK in human placenta is highly regulated, and posttranscriptional regulation of uMtCK and BCK expression occurs in the term placenta. The coordinate regulation of uMtCK and BCK in human placenta supports the CP shuttle hypothesis. This analysis demonstrates that human placenta has high energy needs that can change rapidly; thus, a functioning CP shuttle may be important in the maintenance and termination of pregnancy.

Characterization of the RNA processing enzyme RNase III from wild type and overexpressing Escherichia coli cells in processing natural RNA substrates

1. A precursor to small stable RNA, 10Sa RNA, accumulates in large amounts in a temperature sensitive RNase E mutant at non-permissive temperatures, and somewhat in an rnc (RNase III-) mutant, but not in an RNase P- mutant (rnp) or wild type E. coli cells. 2. Since p10Sa RNA was not processed by purified RNase E and III in customary assay conditions, we purified p10Sa RNA processing activity about 700-fold from wild type E. coli cells. 3. Processing of p10Sa RNA by this enzyme shows an absolute requirement for a divalent cation with a strong preference for Mn2+ over Mg2+. Other divalent cations could not replace Mn2+. 4. Monovalent cations (NH+4, Na+, K+) at a concentration of 20 mM stimulated the processing of p10Sa RNA and a temperature of 37 degrees C and pH range of 6.8-8.2 were found to be optimal. 5. The enzyme retained half of its p10Sa RNA processing activity after 30 min incubation at 50 degrees C. 6. Further characterization of this activity indicated that it is RNase III. 7. To further confirm that the p10Sa RNA processing activity is RNase III, we overexpressed the RNase III gene in an E. coli cells that lacks RNase III activity (rnc mutant) and RNase III was purified using one affinity column, agarose.poly(I).poly(C). 8. This RNase III preparation processed p10Sa RNA in a similar way as observed using the p10Sa RNA processing activity purified from wild type E. coli cells, confirming that the first step of p10Sa RNA processing is carried out by RNase III.

Dietary cholate increases plasma levels of apolipoprotein B in mice by posttranscriptional mechanisms

To induce atherogenesis in mice, a high fat (HF) diet is supplemented with cholic acid (CA), which increases apoB-containing particles and lower apoA-I-containing particles. HF diet without CA increases levels of both HDL and LDL, suggesting that CA may be responsible for the elevation of LDL and lowering of HDL. The mechanism of dietary CA-induced lowering of apoA-I-containing particles has recently been reported. In this study, we examined the mechanism of CA- and HF-induced elevation of apoB-containing lipoproteins in mice. Mice were fed the following four diets: control chow (C), high fat high cholesterol, (HF), control and 0.5% cholate (CA), and HF+CA. Dietary CA increased the plasma levels of apoB-containing particles by approximately 2-fold when compared to control; VLDL levels increased 2-fold, and LDL levels increased 1.3-fold. On HF diet, VLDL increased by 1.4-fold, and LDL by 2-fold, suggesting that CA and HF-induced increases of apoB-containing particles occurred by different mechanisms. We investigated the potential mechanisms regulating plasma levels of apoB in CA- and HF-fed mice. Although hepatic apoB mRNA levels did not change on CA diet, apoB-100 mRNA increased relative to B-48 as a result of decreased editing of apoB mRNA. Measurements of hepatic LDL receptor mRNA suggested that CA diet down-regulated LDL receptor mRNA, possibly by increasing the levels of hepatic cholesterol. Since plasma and hepatic vitamin E levels did not show significant changes on CA-containing diets, it suggests that dietary CA did not act by increasing the absorption of dietary fat. Hepatic lipase, known to modulate plasma levels of apoB-containing particles, did not show changes in CA- or HF-fed mice. Taken together, these results suggest that dietary CA increased apoB-containing particles both in chow-fed and fat-fed mice by enhancing the relative production of apoB-100, and also by reducing LDL receptor-mediated clearance of apoB-containing particles. Thus, dietary cholate modulates plasma levels of apoB primarily by posttranscriptional mechanisms.

The production of 85 kDa N-terminal fragment of apolipoprotein B in mutant HepG2 cells generated by targeted modification of apoB gene occurs by ALLN-inhibitable protease cleavage during translocation.

To study the mechanism of low levels of full length and truncated apoB in individuals heterozygous for apoB truncation, a non-sense mutation was introduced in one of the three alleles of apob gene of HepG2 cells by homologous recombination. Despite very low levels of apoB-82 (1-2%) in the media, a prominent N-terminal apoB protein of 85 kDa (apoB-15) was secreted that fractionated at d>1.065 in density gradient ultracentrifugation. The mechanism of production of this short protein was studied by (35)S-methionine pulse-chase experiment. Oleate prevented presecretory degradation of apoB-100 in the cell and resulted in increased secretion of newly synthesized apoB-100 with decreases in the apoB-15, suggesting that rescue of pre-secretary intracellular degradation of apoB restricted the production and secretion of apoB-15. Further investigation on the degradation of transmembrane forms of apoB, in the presence and absence of a cysteine protease inhibitor, N-acetyl-leucyl-leucyl-norleucinal (ALLN), showed appearance of detectable levels of newly synthesized apoB-82 in the cell and the media together with increased apoB-100 secretion, and reduction in the secretion of apoB-15. Compared to ER membrane, the levels of apoB were higher in the luminal content, and presence of both oleate and ALLN had additive effect on apoB secretion. These results suggest that the presence of improper folding of apoB during translocation led to the cleavage of both apoB-100 and apoB-82 by ALLN-sensitive protease and generation of 85 kDa N-terminal fragment of apoB.

Expression, purification and properties of recombinant E. coli ribonuclease III.

Cloned RNase III gene in a T7 RNA polymerase promoter system was expressed in Escherichia coli cells lacking endogenous RNase III, and the over‐expressed recombinant RNase III was purified to homogeneity using ion exchange, exclusion and affinity column chromatography. The overexpressed RNase III was found to separate with the membrane fraction after sonication, which was solubilized, fractionated with (NH)2SO4 and the active fractions used for further purification. The properties of the purified recombinant RNase III were studied using the synthetic RNA substrate, 3[H]poly[A].poly[U], and the natural substrates, 7S and p10Sa RNAs, and compared with the partially purified RNase III from wild‐type E. coli cells. The recombinant RNase III showed maximal activity at 37°C and at a pH range of 6.9 to 7.4, which was similar to the RNase III purified from the wild‐type cells. Recombinant RNase III efficiently hydrolyzed 3[H].poly[A].poly[U] in the presence of Mg2+. However, the recombinant RNase III cleaved natural RNA substrates efficiently and accurately in the presence of Mn2+. A concentration of Mn2+ ranging from 150 to 300 µM was found to be optimal; concentrations higher than 0.5 mM were inhibitory. Other divalent cations did not support RNase III activity. Monovalent cations, Na+, K+ and NH4+ at 20 mM were equally effective in stimulating RNase III activity although they were not absolutely required for the activity. The thermal stability of the recombinant RNase III was examined at two temperatures, 37° and 50°C. Incubation of RNase III at 37°C for 30 min did not affect activity, but it lost almost 50% of its activity when incubated at 50 °C for 30 min. Thus, the recombinant RNase III prefers Mn2+ for the cleavage of natural substrates and exhibits several properties similar to the wild‐type RNase III.

Lack of Correlation of Plasma HDL With Fecal Cholesterol and Plasma Cholesterol Efflux Capacity Suggests Importance of HDL Functionality in Attenuation of Atherosclerosis

A number of clinical findings suggested HDL-raising as a plausible approach to treat residual risk of CVD. However, lack of CVD risk reduction by elevated HDL cholesterol (HDL-C) through cholesterol ester transfer protein (CETP) inhibition and enhanced risk reduction in apolipoprotein A-I Milano (apoAI-M) individuals with low HDL-C shifted the focus from HDL-C level to HDL function. In the present study, we investigated correlations between HDL-C, HDL function, fecal cholesterol excretion, and ex vivo plasma cholesterol efflux capacity (CEC) in animal models using two HDL modulators, LXR and PPAR-α agonists. In C57Bl mice, LXR agonist, T1317, raised HDL-C by 30%, while PPAR-α agonist, fenofibrate, reduced HDL-C by 30%, but fecal cholesterol showed twofold increase in both cases. CEC showed a 30–40% increase. Combination of LXR and PPAR-α agonists showed no changes in HDL-C, but, interestingly, fecal cholesterol increased by 4.5-fold, and CEC by 40%, suggesting existence of additional pathway for fecal cholesterol excretion. Regression analysis showed a lack of correlation between HDL-C and fecal cholesterol and CEC, while fecal cholesterol showed significant correlation with CEC, a measure of HDL function. ABCA1 and G1, the two important players in RCT showed greater induction with LXR agonist than PPAR-α agonist. HDL-C increased by 40 and 80% in LXR and PPAR-α treated apoA-I transgenic mice, respectively, with 80% increase in fecal cholesterol. A fivefold increase in fecal cholesterol with no correlation with either plasma HDL-C or CEC following co-treatment with LXR and PPAR-α agonists suggested existence of an HDL-independent pathway for body cholesterol elimination. In hyperlipidemic diabetic ob/ob mice also combination of LXR and PPAR-α agonists showed marked increases in fecal cholesterol content (10–20-fold), while HDL-C rise was only 40%, further suggesting HDL-independent elimination of body cholesterol in mice treated with combination of LXR and PPAR-α agonists. Atherosclerosis attenuation by LXR and PPAR-α agonists in LDLr-deficient mice was associated with increased fecal cholesterol, but not HDL-C. However, fecal cholesterol counts showed inverse correlation with aortic cholesteryl ester content. These data suggest: (a) lack of correlation between HDL-C and fecal or aortic cholesterol content; (b) HDL function (CEC) correlated with fecal cholesterol content; (c) association of reduced aortic lipids in LDLr−/− mice with increased fecal cholesterol, but not with HDL-C, and (d) existence of an HDL-independent pathway for fecal cholesterol excretion following co-treatment with LXR and PPAR-α agonists.