Rai Ajit K. 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.

AMP-activated Protein Kinase: An Emerging Drug Target to Regulate Imbalances in Lipid and Carbohydrate Metabolism to Treat Cardio- Metabolic Diseases

The adenosine monophosphate-activated protein kinase (AMPK) is a metabolic sensor of energy metabolism at the cellular as well as whole-body level. It is activated by low energy status that triggers a switch from ATP-consuming anabolic pathways to ATP-producing catabolic pathways. AMPK is involved in a wide range of biological activities that normalizes lipid, glucose, and energy imbalances. These pathways are dysregulated in patients with metabolic syndrome (MetS), which represents a clustering of major cardiovascular risk factors including diabetes, lipid abnormalities, and energy imbalances. Clearly, there is an unmet medical need to find a molecule to treat alarming number of patients with MetS. AMPK, with multifaceted activities in various tissues, has emerged as an attractive drug target to manage lipid and glucose abnormalities and maintain energy homeostasis. A number of AMPK activators have been tested in preclinical models, but many of them have yet to reach to the clinic. This review focuses on the structure-function and role of AMPK in lipid, carbohydrate, and energy metabolism. The mode of action of AMPK activators, mechanism of anti-inflammatory activities, and preclinical and clinical findings as well as future prospects of AMPK as a drug target in treating cardio-metabolic disease are discussed.

In vivo regulation of low-density lipoprotein receptor and apolipoprotein B gene expressions by dietary fat and cholesterol in inbred strains of mice

Two proteins that may be important in the hypercholesterolemia and atherosclerosis produced by dietary fat and/or cholesterol are apoB and the LDL-receptor. We evaluated the molecular and genetic regulation of these two proteins by two important components of atherogenic diets: dietary fatty acids and dietary cholesterol. The control diet (C) contained 5% corn oil; the high cholesterol (HC) diets, 5% corn oil plus 0.5% or 2% cholesterol; the high fat diet (HF) 1% corn oil and 20% hydrogenated coconut oil; the fat plus cholesterol diets (HF/C) were the same as HF diet plus either 0.5% or 2% cholesterol. Ten strains of inbred mice were fed the C and HF/C (2% cholesterol) diets. Three strains; C3H, C57BL and SWR, were studied in greater detail. In them the effects of dietary fat and cholesterol were assessed separately and together. These three strains were fed all six diets. Lipoprotein profiles of plasma and indexes of lipoprotein composition were obtained by gel filtration chromatography and in selected strains by gradient ultracentrifugation. Relative rates of transcription of LDL-receptor mRNA and apoB mRNA were measured in purified mouse liver nuclei and levels of LDL-receptor mRNA and apoB mRNA in liver and intestine were quantified by RNA excess solution hybridization assays. The HF/C diet produced rises in plasma total-, VLDL- and LDL-cholesterol and apoB concentrations in the ten strains. VLDL and LDL became cholesterol-enriched and the proportion of total cholesterol transported in VLDL and LDL rose at the expense of HDL. This general pattern of HF/C diet-induced changes was similar in all strains, but there were marked quantitative differences between strains with respect to lipid and lipoprotein concentrations, and compositions and the distribution of cholesterol on both the HC and HF/C diets. The strain-related differences were not due to differences in absorption of dietary cholesterol because, for any given diet, hepatic cholesterol levels increased to the same extent in all strains. Nor were the strain-related differences related to alleles of the apoB gene as determined by RFLP analyses. In the three strains, hepatic LDL-receptor mRNA transcription was suppressed by all diets. But, LDL-receptor mRNA levels in both intestine and liver were suppressed only by the HC and HF/C diets and not by the HF diet. Thus, dietary cholesterol decreased LDL-receptor mRNA levels by mechanisms operating at the transcriptional level, while dietary fatty acids, in addition to inhibiting transcription also appeared to enhance mRNA stability.(ABSTRACT TRUNCATED AT 400 WORDS)

AMP-activated protein kinase and ATP-citrate lyase are two distinct molecular targets for ETC-1002, a novel small molecule regulator of lipid and carbohydrate metabolism

ETC-1002 (8-hydroxy-2,2,14,14-tetramethylpentadecanedioic acid) is a novel investigational drug being developed for the treatment of dyslipidemia and other cardio-metabolic risk factors. The hypolipidemic, anti-atherosclerotic, anti-obesity, and glucose-lowering properties of ETC-1002, characterized in preclinical disease models, are believed to be due to dual inhibition of sterol and fatty acid synthesis and enhanced mitochondrial long-chain fatty acid β-oxidation. However, the molecular mechanism(s) mediating these activities remained undefined. Studies described here show that ETC-1002 free acid activates AMP-activated protein kinase in a Ca2+/calmodulin-dependent kinase β-independent and liver kinase β 1-dependent manner, without detectable changes in adenylate energy charge. Furthermore, ETC-1002 is shown to rapidly form a CoA thioester in liver, which directly inhibits ATP-citrate lyase. These distinct molecular mechanisms are complementary in their beneficial effects on lipid and carbohydrate metabolism in vitro and in vivo. Consistent with these mechanisms, ETC-1002 treatment reduced circulating proatherogenic lipoproteins, hepatic lipids, and body weight in a hamster model of hyperlipidemia, and it reduced body weight and improved glycemic control in a mouse model of diet-induced obesity. ETC-1002 offers promise as a novel therapeutic approach to improve multiple risk factors associated with metabolic syndrome and benefit patients with cardiovascular disease.

Scavenger receptor class B type I expression and elemental analysis in cerebellum and parietal cortex regions of the Alzheimer's disease brain ☆

Metal ions play an important role in health and disease by influencing cellular biochemical pathways. The increased concentrations of some metal ions may have cytotoxic effects through their ability to oxidatively modify biomolecules, which may cause oxidative stress-induced brain cell death leading to neurodegenerative disorders observed in Alzheimers disease (AD). We therefore performed elemental analysis of human brain tissues by a sophisticated method of inductively coupled plasma mass spectrometry (ICP-MS) in two regions of the AD brain, the parietal cortex and cerebellum, and compared them with the age-matched control. Our analysis shows the differential distribution of some metal ions in the two regions of the brain. Most importantly, Si, Sn, Al and Mn showed significantly higher levels in the parietal cortex of the AD brain compared to the control. The other metal ions showing moderate increases in the parietal cortex were Na, Te, Cr, Fe and B. Since these metal ions can modify lipoproteins in the brain and modified lipoproteins are taken up by scavenger receptors class B type I (SR-BI), we also determined the presence of SR-BI in the parietal cortex and cerebellum regions of the control and AD brains using a sensitive method, the reverse transcriptase-polymerase chain reaction. Our results suggest that SR-BI are present in the parietal cortex as well as in the cerebellum of the control and AD brains, suggesting that the presence of SR-BI may be involved in the uptake of oxidatively modified lipoproteins and beta-amyloid (Abeta) protein complexed with apoE, suggesting implications in the progression of late onset AD and other neurodegenerative disorders characterized by the deposition of insoluble aggregates observed in the AD brain.

Dietary fatty acids and dietary cholesterol differ in their effect on the in vivo regulation of apolipoprotein A-I and A-II gene expression in inbred strains of mice

Dietary cholesterol and dietary saturated fatty acids affected the plasma concentrations of various HDL components and the hepatic and intestinal expression of the apolipoprotein (apo) A-I gene and the hepatic expression of the A-II gene differently in three inbred strains of female mice. Thus, the HC diet (0.5% cholesterol, no added fatty acids) decreased HDL-cholesterol in C57BL and SWR strains but not in the C3H strain; plasma apo A-I and apo A-II concentrations decreased in all three strains. HDL-C/apo A-I and apo A-I/apo A-II mass ratios increased, suggesting that the HC diet altered both the concentrations and the compositions of HDL particles. In contrast, the HF diet (20% hydrogenated coconut oil, no added cholesterol) increased HDL cholesterol and apo A-I concentrations. The combination diet (HF/C, 20% coconut oil plus 0.5% cholesterol) increased HDL cholesterol and decreased triacylglycerols. Apo A-I concentrations were unaltered except for a significant increase in SWR mice. Apo A-II concentrations decreased in all strains. To examine molecular events that could lead to the changes in plasma apo A-I and apo A-II, we measured transcription rates in hepatic nuclei and steady state mRNA concentrations in liver and intestine and apo A-I synthetic rates in liver. Dietary cholesterol and fatty acids produced differing effects at transcriptional as well as post-transcriptional loci and the changes differed according to mouse strain. The most pronounced strain-related differences for both apo A-I and apo A-II occurred at post-transcriptional loci of apoprotein production. These could represent altered rates of translation in, or secretion from liver and/or intestine, or altered rates of clearance from plasma. In conclusion, the regulation of apo A-I and apo A-II gene expression by diet occurs at several steps of their production and perhaps also in catabolic pathways. This study identifies potential loci of regulation and forms the basis for future studies investigating specific genetic and molecular regulatory mechanisms.

Scavenger receptor class B type I expression in murine brain and regulation by estrogen and dietary cholesterol

The scavenger receptor class B type I (SR-BI), a receptor for high-density lipoproteins (HDL), facilitates cholesterol delivery to steroidogenic tissues, and brings excess body cholesterol to liver for excretion. Scavenger receptors are also involved in the internalization of aggregates of Alzheimers disease (AD) amyloid beta-protein, and selective uptake of HDL-associated vitamin E in the brain. Therefore, modulation of the brain SR-BI may affect these processes. The present study examined the expression of SR-BI receptors in murine brain and their regulation by estradiol administration and cholesterol feeding. Liver and brain appeared to express similar SR-BI transcripts. Expression of SR-BI was highest in the adrenals and lowest in the brain. In rats, estradiol administration decreased SR-BI in liver, but increased in adrenals. In mice, estrogen treatment decreased hepatic SR-BI, but interestingly increased the levels of brain SR-BI mRNA. Cholesterol feeding did not alter mouse hepatic SR-BI mRNA, but increased brain SR-BI levels. ATP-binding cassette transporter A1 (ABCA1), involved in cellular cholesterol transport, increased in cholesterol-fed mouse liver, but did not show changes in the brain. These studies suggest that SR-B1 is expressed in the brain and regulated by hormonal and nutritional stimuli, which may influence the pathophysiology of neurological disorders like AD.

High density lipoprotein, apolipoprotein A-1, and coronary artery disease

High density lipoproteins (HDL), one of the main lipoprotein particles circulating in plasma, is involved in the reverse cholesterol transport. Several lines of evidence suggest that elevated levels of HDL is protective against coronary heart disease. The role of HDL in the removal of body cholesterol and in the regression of atherosclerosis add to the importance of understanding the molecular-cellular processes that determine plasma levels of HDL. Factors modulating plasma levels of HDL may have influence on the predisposition of an individual to premature coronary artery disease. Apolipoprotein (apo) A-I is the main apolipoprotein component of HDL and, to a large extent, sets the plasma levels of HDL. Thus, understanding the regulation of apoA-I gene expression may provide clues to raise plasma levels of HDL. This review discusses the various pathways that alter plasma levels of HDL. Since apoA-I is the main protein component of HDL and determines the plasma levels of HDL, this review also covers the regulation of apoA-I gene expression.

Estrogen-induced regulation of the ATP-binding cassette transporter A1 (ABCA1) in mice: a possible mechanism of atheroprotection by estrogen.

Estrogens are suggested to be antiatherogenic by affecting the vessel wall components. Since ABCA1 was recently shown to be atheroprotective, it was examined if estrogen-induced atheroprotection occurs partly via the regulation of the ABCA1. Since hepatic ABCA1 expression was also suggested to contribute to the bulk HDL levels, regulation of the ABCA1 under conditions of high or low levels of HDL were investigated in mice expressing normal or elevated levels of apoAI. To delineate whether estrogens effect occurs via estrogen receptor-α-mediated pathway, the estrogen receptor-α-deficient (ER-α)−/− mice were also administered either placebo or β-estradiol for 5 consecutive days. Estrogen treatments decreased circulating HDL levels by 30%, but increased hepatic and intestinal ABCA1 mRNA by 2- and 1.5-fold, respectively. Hepatic ABCA1 mRNA also increased in the ER-α−/− mice by 3-fold. These results suggest that estrogen, despite lowering the levels of HDL, it up-regulated the hepatic ABCA1 mRNA, and in the absence of ER-α, ER-β could compensate for ER-α. To study whether HDL levels correlate with the ABCA1 expression, wild-type (WT) and the apoAI transgenic (A1-Tg) mice were fed high fat (HF) diet with or without cholic acid (CA) for 3 weeks. One group of mice was treated with fenofibrate, known to elevate HDL levels. CA without HF decreased HDL levels, while fenofibrate increased HDL levels. However, neither CA nor fenofibrate altered hepatic ABCA1 mRNA levels. HF diet increased the hepatic ABCA1 mRNA 1.8-fold in WT, but lowered ABCA1 mRNA by 2-fold in A1-Tg mice, suggesting that ABCA1 levels did not correlate with circulating HDL levels, while basal levels of HDL influenced ABCA1 expression. These data show for the first time that estrogens antiatherogenic effects may occur via ABCA1-mediated pathway, and circulating HDL levels may influence expression of ABCA1.

Hormonal and nutritional stimuli modulate apolipoprotein B mRNA editing in mouse liver.

Human livers produce apoB-100, a major protein of VLDL, while intestines produce apoB-48, the major protein of chylomicrons. ApoB-48 is translated from apoB-100 mRNAs that are post-transcriptionally edited at codon 2153, converting CAA (glutamine) to TAA, a stop codon. In contrast to humans, mouse and rat livers contain the apoB-100 mRNA editing mechanism. Because hormones and nutrients affect the metabolism of apoB containing lipoproteins, we studied the effects of sex hormones and diets on apoB mRNA editing. Groups of male and female C3H/HeJ mice were castrated and treated with 17 beta-estradiol at 0.16 (E2L) or at 5 micrograms (E2H), or with testosterone propionate at 1 microgram/g body weight/day for 14 days. Plasma apoB levels and ratios of apoB-100/apoB-48 both increased 2-fold, but only in the E2H group. To determine if the increased apoB-100/apoB-48 ratios were associated with altered levels of apoB-100 and apoB-48 mRNA, both forms of apoB mRNA were quantified. We found that indeed ApoB-100 mRNA increased 1.8-fold (p < 0.025) compared to apoB-48 mRNA only in the E2H group. Next, we studied the individual effects of dietary fatty acids and dietary cholesterol on the relative abundance of apoB-100 and apoB-48 mRNA. Contrary to the estrogen effect, the high fat-combination diet increased apoB-48 mRNA relative to apoB-100 mRNA. Total plasma apoB as well as apoB-48 synthesis in liver also increased. Our studies demonstrate that estrogens and high fat diet both modulate apoB editing in mouse liver, but that estrogens and fat diet affected apoB mRNA editing in opposite directions.