Important Regulators Of Cholesterol Metabolism Research Articles

Abstract 675: Time Restricted Feeding Regulates Cholesterol Efflux And Atherosclerosis In Macrophage-specific Bmal1 Deficient Mice

Background: Macrophages play a critical role in atherosclerosis. We have previously shown that Clock mutant and global or tissue-specific Bmal1 deficient mice show enhanced atherosclerosis. Yet, the role of time restricted feeding (TRF) in cholesterol transport and atherosclerosis in macrophage Bmal1 deficient mice remains unknown. Objectives: To establish a novel regulatory link between TRF, circadian clock, and atherosclerosis. Methods: We studied the effect of day or nighttime TRF on macrophage-specific Bmal1 deficient (M- Bmal1 –/– Apoe –/– ) or control mice on cholesterol efflux to clarify the role of Bmal1 in macrophage function and in atherosclerosis. Results: We found that daytime-TRF increased atherosclerosis. Biochemical studies showed that macrophages from daytime TRF M- Bmal1 –/– Apoe –/– mice exhibit increased expression of Lox-1 and Cd36, and take up more oxLDL compared to nighttime TRF mice. Furthermore, daytime TRF mice showed decreased expression of Abca1 and Abcg1, reduced cellular cholesterol efflux and in vivo reverse cholesterol transport compared to nighttime TRF mice. Thus, TRF regulates macrophage cholesterol metabolism by regulating uptake of oxidized lipoproteins and cholesterol efflux pathways. Molecular studies showed that TRF regulates HDAC3 interactions with p300 to regulate Znf202 gene expression. Thus, TRF regulates Bmal1 and Znf202 to regulate uptake of oxidized lipoproteins and cholesterol efflux. Conclusions: TRF is an important regulator of cholesterol metabolism in macrophages and affects atherosclerosis. It regulates macrophage function by modulating the expression of different transcription factors. It is likely that nighttime-TRF may prevent atherosclerosis.

LXR agonist inhibits inflammation through regulating MyD88 mRNA alternative splicing.

Liver X receptors (LXRs) are important regulators of cholesterol metabolism and inflammatory responses. LXR agonists exhibit potently anti-inflammatory effects in macrophages, which make them beneficial to anti-atherogenic therapy. In addition to transrepressive regulation by SUMOylation, LXRs can inhibit inflammation by various mechanisms through affecting multiple targets. In this study, we found that the classic LXR agonist T0901317 mediated numerous genes containing alternative splice sites, including myeloid differentiation factor 88 (MyD88), that contribute to inflammatory inhibition in RAW264.7 macrophages. Furthermore, T0901317 increased level of alternative splice short form of MyD88 mRNA by down-regulating expression of splicing factor SF3A1, leading to nuclear factor κB-mediated inhibition of inflammation. In conclusion, our results suggest for the first time that the LXR agonist T0901317 inhibits lipopolysaccharide-induced inflammation through regulating MyD88 mRNA alternative splicing involved in TLR4 signaling pathway.

Pathophysiological role of 27-hydroxycholesterol in human diseases.

Oxysterols are oxygenated cholesterol derivatives and important regulators of cholesterol metabolism, lipid homeostasis, the immune system, and membrane fluidity regulation. Although the detailed mechanism of action of oxysterols remains unclear, activation of some nuclear receptors, such as liver X receptor α (LXRα) and RAR-related orphan receptors, have been believed to be critical for the regulation of various physiological processes in multiple tissues. 27-Hydroxycholesterol (27-OHC) is an endogenous oxysterol, which has an intermediate function in cholesterol catabolism to bile acid synthesis. According to previous studies, however, there are opposing opinions on whether 27-OHC activates human LXR. Recently, several studies have shown that 27-OHC can activate or inhibit the function of estrogen receptors ERα and ERβ in a tissue-specific manner, indicating that the understanding of 27-OHC-mediated biological output is very complicated. This review summarizes the pathophysiological relevance of 27-OHC in various tissues, with a special discussion on their functions in human diseases.

Fumonisin B1 regulates LDL receptor and ABCA1 expression in an LXR dependent mechanism in liver (HepG2) cells

The metabolic toxicity of Fumonisin B1 (FB1) converges at the accumulation of sphingoid bases and reduced ceramide levels. Several studies have alluded to a hypercholesterolemic endpoint after FB1 exposure, yet the molecular mechanisms remain elusive. Cell surface receptors are important regulators of cholesterol metabolism by regulating influx of lipids and efflux of cholesterol. Western blot analysis showed that FB1 elevates the expression of ABCA1 (a cholesterol efflux promoter) in an LXR dependent mechanism. We further highlight the potential role of PCSK9 in the degradation of LDL receptor. These data provide important evidence for the mechanism underlying hypercholesterolemia in FB1 treated models. The disruption of lipid homeostasis by FB1 is beginning to shift away from canonical ceramide synthase inhibition, and this changed perspective may shed light on diseases caused by dysregulated cholesterol metabolism such as cancer initiation and promotion.

Maternal fructose induces gender-dependent changes in both LXRα promoter methylation and cholesterol metabolism in progeny

Fructose consumption from added sugars correlates with the epidemic rise in obesity, metabolic syndrome and cardiovascular diseases. However, consumption of beverages containing fructose is allowed during gestation. We have investigated whether maternal fructose intake produces subsequent changes in cholesterol metabolism of progeny. Carbohydrates were supplied to pregnant rats in drinking water (10% w/v solution) throughout gestation. Adult male and female descendants from fructose-fed, control or glucose-fed mothers were studied. Male offspring from fructose-fed mothers had elevated plasma HDL-cholesterol levels, whereas female progeny from fructose-fed mothers presented lower levels of non-HDL cholesterol vs. the other two groups. Liver X-receptor (LXR), an important regulator of cholesterol metabolism, and its target genes such as scavenger receptor B1, ATP-binding cassette (ABC)G5 and cholesterol 7-alpha hydroxylase showed decreased gene expression in males from fructose-fed mothers and the opposite in the female progeny. Moreover, the expression of a number of LXRα target genes related to lipogenesis paralleled to that for LXRα expression. In accordance with this, LXRα gene promoter methylation was increased in males from fructose-fed mothers and decreased in the corresponding group of females. Surprisingly, plasma folic acid levels, an important methyl-group donor, were augmented in males from fructose-fed mothers and diminished in female offspring. Maternal fructose intake produces a fetal programming that influences, in a gender-dependent manner, the transcription factor LXRα epigenetically, and both hepatic mRNA gene expression and plasma parameters of cholesterol metabolism in adult progeny. Changes in the LXRα promoter methylation might be related to the availability of the methyl donor folate.

SREBF2 POLYMORPHISMS AND EXPRESSION LEVELS CORRELATE WITH ALZHEIMER’S DISEASE NEUROPATHOLOGY

To improve understanding of dysregulated cholesterol metabolism in late-onset Alzheimer's disease (LOAD), we used a candidate-gene approach with available genomic, transcriptomic, proteomic, imaging and histopathological data from three independent cohorts: the United Kingdom Brain Expression Consortium (UKBEC), the Alzheimer's Disease Neuroimaging Initiative (ADNI) and the Quebec Founder Population (QFP). We conducted region-specific association studies to identify SREBF2 polymorphisms associated with gene expression levels (from the UKBEC) and with relevant LOAD neuropathological markers (from the ADNI and the QFP). Next, we measured SREBF2 mRNA levels by real time quantitative polymerase chain reaction in controls and LOAD brains (from the QFP). Polymorphisms were found in strong correlation with SREBF2 gene expression levels in all non-affected brain regions assayed in the UKBEC (4.6E-4 ≥ p ≥ 3.4E-15). Four of these SNPs were significantly associated with several LOAD biomarkers from the ADNI and the QFP (4.7E-2 ≥ p ≥ 2.7E-5). In the QFP, brain SREBF2 mRNA levels were significantly increased in LOAD compared with controls (1.3E-2 ≥ p ≥ 7.0E-5), except in the frontal cortex where a stronger genotype effect was observed (p = 7.4E-3). In this specific brain region, SREBF2 mRNA levels correlated negatively with age at death (p = 5.5E-4), suggesting an association with LOAD progression or severity. SREBF2 gene expression levels appear to be influenced by both disease status and the presence of specific polymorphisms. As an important regulator of cholesterol metabolism, SREBF2 may affect the progression or extent of pathology in Alzheimer's disease.

Abstract 334: The LXRβ Selective Agonist, VTP-38443, Significantly Decreases Plaque Cholesterol Ester Content and Inflammation in a Murine Model of Accelerated Atherosclerosis

The ligand-activated transcription factors LXRα and LXRβ are important regulators of cholesterol metabolism and inflammation. Activation of LXR was previously shown to inhibit atherosclerosis in animal models through activation of reverse cholesterol transport (RCT) and suppression of vascular inflammation. VTP-38443 is a potent selective and orally bioavailable modulator of LXRβ that is being pursued for the prevention of subsequent vascular events following an acute coronary syndrome (ACS) episode. VTP-38443 is a potent binder (Ki=12 nM) and activator (EC50=17 nM) of LXRβ. It is ~20x selective for LXRβ versus LXRβ in both binding and activation. In primary human cells, it induces the expression of the cholesterol efflux pumps ABCA1 and ABCG1, markers of RCT, and promotes cholesterol efflux from human fibroblasts at low concentrations (EC50 = 14 nM). Orally dosed VTP-38443 demonstrates robust induction of ABCA1 and ABCG1 in mice (ED50 < 0.3 mg/kg) and primates (ED50 < 0.1 mg/kg). Based on this robust activity, VTP-38443 was tested in the apoE -/- carotid artery ligated mouse model, an accelerated atherosclerosis model. ApoE-/- mice (9 week) were fed a Western diet for 2 weeks at which point the left common carotid artery was ligated. Mice remained on the Western diet for 2 weeks post-surgery and were dosed BID with VTP-38443 (0.05, 0.2 and 1 mg/kg/dose) or a non-selective LXR agonist, TO90137 (20 mg/kg/day) during this time. Plasma cholesterol and TGs were measured as were cholesterol esters and FDG-6-phosphate, a biomarker of plaque inflammation, in the carotid plaques. VTP-38443 gave a dose-dependent reduction in plasma cholesterol (35% at 1 mg/kg) and a modest dose-dependent increase in plasma TGs ranging from 5% to 45% of the TG increase seen with TO90137. Plaque analysis showed a highly significant decrease in carotid cholesterol ester content at all doses, achieving >90% reduction at the highest dose. In addition, there was a significant reduction (~40-50%) in FDG-6 phosphate at all doses of VTP-38443, indicating a decrease in vascular inflammation. These data indicate that LXRβ activation may decrease plaque cholesterol content and reduce plaque inflammation in ACS.

Discovery of New Liver X Receptor Agonists by Pharmacophore Modeling and Shape-Based Virtual Screening

Agonists of liver X receptors (LXR) α and β are important regulators of cholesterol metabolism, but agonism of the LXRα subtype appears to cause hepatic lipogenesis, suggesting LXRβ-selective activators are attractive new lipid lowering drugs. In this work, pharmacophore modeling and shape-based virtual screening were combined to predict new LXRβ-selective ligands. Out of the 10 predicted compounds, three displayed significant LXR activity. Two activated both LXR subtypes. The third compound activated LXRβ 1.8-fold over LXRα.

On the formation and possible biological role of 25-hydroxycholesterol

The oxysterol 25-hydroxycholesterol is a widely used compound displaying an array of pharmacological actions in invitro systems and cell based experimental systems. In spite of the frequent use of this compound over the last few decades and a large number of studies invitro and invivo, its mechanism of formation invivo is still not well understood. Cholesterol autoxidation does not seem to be an important contributor to invivo formation of 25-hydroxycholesterol. A number of different cytochrome P450 enzymes such as CYP27A1 and CYP3A4 have been reported to catalyze the conversion of cholesterol to 25-hydroxycholesterol invitro, but the importance of these reactions invivo remains unclear. The dioxygenase enzyme cholesterol 25-hydroxylase has been shown to generate 25-hydroxycholesterol, but in cholesterol 25-hydroxylase knockout mice there are still significant levels of 25-hydroxycholesterol in several tissues. This suggests that cholesterol 25-hydroxylase is not the sole producer of 25-hydroxycholesterol. The relative importance of different mechanisms of formation of 25-hydroxycholesterol invivo have still to be elucidated. The maintenance of cholesterol homeostasis is of great importance to supply tissues with the appropriate amount of cholesterol and prevent accumulation that may affect health. Numerous articles mention 25-hydroxycholesterol as an important regulator of cholesterol metabolism. However, mice with a disruption of the cholesterol 25-hydroxylase gene regulate cholesterol metabolism normally and patients with highly elevated levels of 25-hydroxycholesterol also display normal cholesterol and bile acid levels. These reports challenge the hypothesis that 25-hydroxycholesterol is an important regulator of cholesterol metabolism. Recent reports suggest that 25-hydroxycholesterol and one of its metabolites may have functions in regulation of humoral immunity. Thus, 25-hydroxycholesterol may be more important as a regulator of immunity than as a regulator of cholesterol metabolism.

Activation of Liver X Receptor (LXR) Inhibits Receptor Activator of Nuclear Factor κB Ligand (RANKL)-induced Osteoclast Differentiation in an LXRβ-dependent Mechanism

Bone destruction is the major pathological process in many bone metabolic diseases and is a result of increased osteoclast formation and bone resorption. The liver X receptors (α,β), important regulators of cholesterol metabolism and inflammatory signaling, have recently been observed to play a role in both physiological and pathological bone turnover. However, the relationship between liver X receptors (LXR) and osteoclast differentiation/formation remains unknown. Here, we report that the LXR ligand GW3965 is able to clearly and potently inhibit the formation of mature osteoclasts from receptor activator of nuclear factor κB ligand (RANKL)-stimulated human and murine osteoclast precursors. This results in a significant inhibition of bone resorption. We observed that GW3965 significantly inhibited expression of the osteoclast markers tartrate-resistant acid phosphatase, cathepsin K, osteoclast-associated receptor (OSCAR), and calcitonin receptor, appearing to act in an NFATc1/p38/microphthalmia-associated transcription factor (MITF)-dependent mechanism, independently of receptor activator of nuclear factor κB or c-Fos and not directly involving the NFκB pathways. GW3965 was less effective in RAW264.7 monocyte/macrophage cells, which are more committed into the osteoclast lineage. Also, GW3965 seemed to act differently depending on the source of the progenitor cells as it had no effect on calvarial osteoclasts, compared with marrow or blood-derived monocytes. As these effects were abolished in osteoclast precursors derived from LXRβ(-/-) mice, we suggest that GW3965 acts via an LXRβ-dependent mechanism. Taken together, our results suggest that the LXR can act as an important inhibitor of RANKL-mediated osteoclast differentiation.

Identification of macrophage liver X receptors as inhibitors of atherosclerosis.

Recent studies have identified the liver X receptors (LXR alpha and LXR beta) as important regulators of cholesterol metabolism and transport. LXRs control transcription of genes critical to a range of biological functions including regulation of high density lipoprotein cholesterol metabolism, hepatic cholesterol catabolism, and intestinal sterol absorption. Although LXR activity has been proposed to be critical for physiologic lipid metabolism and transport, direct evidence linking LXR signaling pathways to the pathogenesis of cardiovascular disease has yet to be established. In this study bone marrow transplantations were used to selectively eliminate macrophage LXR expression in the context of murine models of atherosclerosis. Our results demonstrate that LXRs are endogenous inhibitors of atherogenesis. Additionally, elimination of LXR activity in bone marrow-derived cells mimics many aspects of Tangier disease, a human high density lipoprotein deficiency, including aberrant regulation of cholesterol transporter expression, lipid accumulation in macrophages, splenomegaly, and increased atherosclerosis. These results identify LXRs as targets for intervention in cardiovascular disease.