OBJECTIVEThe global epidemic of metabolic syndrome and its complications demands rapid evaluation of new and accessible interventions. Insulin resistance is the central biochemical disturbance in the metabolic syndrome. The citrus-derived flavonoid, naringenin, has lipid-lowering properties and inhibits VLDL secretion from cultured hepatocytes in a manner resembling insulin. We evaluated whether naringenin regulates lipoprotein production and insulin sensitivity in the context of insulin resistance in vivo.RESEARCH DESIGN AND METHODSLDL receptor–null (Ldlr−/−) mice fed a high-fat (Western) diet (42% calories from fat and 0.05% cholesterol) become dyslipidemic, insulin and glucose intolerant, and obese. Four groups of mice (standard diet, Western, and Western plus 1% or 3% wt/wt naringenin) were fed ad libitum for 4 weeks. VLDL production and parameters of insulin and glucose tolerance were determined.RESULTSWe report that naringenin treatment of Ldlr−/− mice fed a Western diet corrected VLDL overproduction, ameliorated hepatic steatosis, and attenuated dyslipidemia without affecting caloric intake or fat absorption. Naringenin 1) increased hepatic fatty acid oxidation through a peroxisome proliferator–activated receptor (PPAR) γ coactivator 1α/PPARα-mediated transcription program; 2) prevented sterol regulatory element–binding protein 1c–mediated lipogenesis in both liver and muscle by reducing fasting hyperinsulinemia; 3) decreased hepatic cholesterol and cholesterol ester synthesis; 4) reduced both VLDL-derived and endogenously synthesized fatty acids, preventing muscle triglyceride accumulation; and 5) improved overall insulin sensitivity and glucose tolerance.CONCLUSIONSThus, naringenin, through its correction of many of the metabolic disturbances linked to insulin resistance, represents a promising therapeutic approach for metabolic syndrome.

OBJECTIVEIncreased plasma concentrations of apolipoprotein B100 often present in patients with insulin resistance and confer increased risk for the development of atherosclerosis. Naturally occurring polyphenolic compounds including flavonoids have antiatherogenic properties. The aim of the current study was to evaluate the effect of the polymethoxylated flavonoid nobiletin on lipoprotein secretion in cultured human hepatoma cells (HepG2) and in a mouse model of insulin resistance and atherosclerosis.RESEARCH DESIGN AND METHODSLipoprotein secretion was determined in HepG2 cells incubated with nobiletin or insulin. mRNA abundance was evaluated by quantitative real-time PCR, and Western blotting was used to demonstrate activation of cell signaling pathways. In LDL receptor–deficient mice (Ldlr−/−) fed a Western diet supplemented with nobiletin, metabolic parameters, gene expression, fatty acid oxidation, glucose homeostasis, and energy expenditure were documented. Atherosclerosis was quantitated by histological analysis.RESULTSIn HepG2 cells, activation of mitogen-activated protein kinase-extracellular signal–related kinase signaling by nobiletin or insulin increased LDLR and decreased MTP and DGAT1/2 mRNA, resulting in marked inhibition of apoB100 secretion. Nobiletin, unlike insulin, did not induce phosphorylation of the insulin receptor or insulin receptor substrate-1 and did not stimulate lipogenesis. In fat-fed Ldlr−/− mice, nobiletin attenuated dyslipidemia through a reduction in VLDL-triglyceride (TG) secretion. Nobiletin prevented hepatic TG accumulation, increased expression of Pgc1α and Cpt1α, and enhanced fatty acid β-oxidation. Nobiletin did not activate any peroxisome proliferator–activated receptor (PPAR), indicating that the metabolic effects were PPAR independent. Nobiletin increased hepatic and peripheral insulin sensitivity and glucose tolerance and dramatically attenuated atherosclerosis in the aortic sinus.CONCLUSIONSNobiletin provides insight into treatments for dyslipidemia and atherosclerosis associated with insulin-resistant states.

Metabolic syndrome is a collection of abnormalities, including obesity, dyslipidemia, hypertension, and insulin resistance, all of which contribute to the development of type 2 diabetes and atherosclerosis. Insulin resistance, dyslipidemia, and atherosclerosis are amplifi ed by the development of a chronic low-grade infl ammatory response ( 1 ). In insulin-resistant states, monocyte-derived macrophages infi ltrate visceral adipose tissue, resulting in proinfl ammatory cytokine synthesis, either from adipocytes or resident macrophages, which impairs insulin sensitivity ( 2, 3 ). Administration of diets rich in saturated fats to Ldlr Ϫ / Ϫ mice represents a model with many characteristics of the metabolic syndrome ( 4-6 ). Recent studies in this Abstract Obesity-associated chronic infl ammation contributes to metabolic dysfunction and propagates atherosclerosis. Recent evidence suggests that increased dietary cholesterol exacerbates infl ammation in adipose tissue and liver, contributing to the proatherogenic milieu. The ability of the citrus fl avonoid naringenin to prevent these cholesterol-induced perturbations is unknown. To assess the ability of naringenin to prevent the amplifi ed infl ammatory response and atherosclerosis induced by dietary cholesterol, male Ldlr ؊ / ؊ mice were fed either a cholesterol-enriched high-fat or low-fat diet supplemented with 3% naringenin for 12 weeks. Naringenin, through induction of hepatic fatty acid (FA) oxidation and attenuation of FA synthesis, prevented hepatic steatosis, hepatic VLDL overproduction, and hyperlipidemia induced by both cholesterol-rich diets. Naringenin attenuated hepatic macrophage infi ltration and infl ammation stimulated by dietary cholesterol. Insulin resistance, adipose tissue expansion, and infl ammation were alleviated by naringenin. Naringenin attenuated the cholesterol-induced formation of both foam cells and expression of infl ammatory markers in peritoneal macrophages. Naringenin signifi cantly decreased atherosclerosis and inhibited the formation of complex lesions, which was associated with normalized aortic lipids and a reversal of aortic infl ammation. We demonstrate that in mice fed cholesterolenriched diets, naringenin attenuates peripheral and systemic infl ammation, leading to protection from atherosclerosis. These studies offer a therapeutically relevant alternative for the prevention of cholesterol-induced metabolic dysregulation. -Assini, J. M., E. E. Mulvihill, B. G. Sutherland, D. E. Telford, C. G. Sawyez, S. L. Felder, S. Chhoker, J. Y. Edwards, R. Gros, and M. W. Huff. Naringenin prevents cholesterol-induced systemic infl ammation, This work was supported by Heart and Stroke Foundation of Ontario (HSFO)Grants , Canadian Foundation for Innovation and Ontario Research Fund (to R.G.), a HSFO Masters Award (to J.M.A.), and a Canadian Institutes of Health Research-Canada Graduate Scholarship Doctoral Award (to E.E.M.). 14 December 2012. Published, JLR Papers in Press, December 19, 2012 DOI 10.1194 Abbreviations: ABCG, ATP-binding...

Microsomal triglyceride transfer protein (MTP) is necessary for hepatocyte assembly and secretion of apolipoprotein (apo)B100-containing lipoproteins. The citrus flavonoid naringenin, like insulin, decreased MTP expression in HepG2 cells, resulting in inhibition of apoB100 secretion; however, the mechanism for naringenin is independent of insulin receptor substrate-1/2. Recently, it was reported that insulin decreased MTP expression in HepG2 cells via the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) (MAPK erk ) pathway. We hypothesized that naringenin acts via a similar mechanism. Inhibition of MAPK kinase (MEK) 1/2 in HepG2 cells significantly attenuated the naringenin-and insulin-induced reduction in MTP expression. Both naringenin and insulin increased ERK1/2 phosphorylation, which was completely inhibited by MEK1/2 inhibition and enhanced by inhibition of MAPK p38 , a negative regulator of MAPK erk activity. Inhibition of MEK1/2 significantly attenuated both the naringenin-and insulin-induced decrease in apoB100 secretion demonstrating a direct link between MAPK erk activation and apoB100 secretion. Furthermore, both compounds increased MAPK p38 activation, and therefore inhibition of MAPK p38 amplified the naringenin-and insulin-induced decrease in apoB100 secretion. We conclude that MAPK erk signaling in hepatocytes is critical for inhibition of apoB100 secretion by naringenin and insulin. Therefore, naringenin may prove useful for activating insulin-signaling pathways important for regulation of hepatocyte lipid homeostasis. Diabetes 54:1676 -1683, 2005 I t is well established that insulin resistance and type 2 diabetes result in overproduction of hepatic apolipoprotein (apo)B-containing lipoproteins, which confers an increased atherosclerotic risk (1,2). Microsomal triglyceride transfer protein (MTP) is rate-limiting for hepatic lipoprotein assembly. MTP catalyzes the transfer of lipids to newly synthesized apoB within the endoplasmic reticulum facilitating secretion of nascent lipoproteins (3). Recent evidence from the fructose-fed hamster model of insulin resistance has shown that an increased activity of MTP contributes to the increased secretion/overproduction of hepatic apoB100 lipoproteins (4). Furthermore, amelioration of hepatic insulin resistance in this model resulted in normalization of MTP expression and reduction of the overproduction of apoB100-containing lipoproteins (5). Under normal culture conditions, insulin negatively regulates the expression of MTP in hepatocytes (6). Therefore, other compounds that activate intracellular insulin-signaling pathways may also decrease MTP expression and potentially be used in patients to treat dyslipidemia associated with insulin resistance.Previous studies in cell culture systems and animal models have shown that the grapefruit flavonoid naringenin dramatically decreases the secretion of hepatic lipoproteins (7-14). We reported that naringenin dosedependently inhibited apoB secretion in HepG2 cells (11) resulting...

Abstract-Oxysterols are key regulators of lipid metabolism and regulate gene expression by activating the liver X receptor (LXR). LXR plays a vital role in macrophage foam cell formation, a central event in atherosclerosis. It is known that addition of exogenous oxysterols to cultured macrophages activates LXR, leading to increased expression of ABCA1 and cholesterol efflux. In this study, we tested the novel hypothesis that stimulation of endogenous oxysterol synthesis would block foam cell formation induced by atherogenic lipoproteins. Macrophage synthesis of 24(S),25-epoxycholesterol, a potent LXR ligand, increased 60-fold by partial inhibition of 2,3-oxidosqualene:lanosterol cyclase (OSC), a microsomal enzyme in both the cholesterol biosynthetic pathway and the alternative oxysterol synthetic pathway. When macrophages were challenged with human hypertriglyceridemic VLDL (HTG-VLDL), cellular cholesteryl ester accumulation increased 12-fold. This was reduced dramatically, by 65%, after preincubation with an OSC inhibitor (OSCi). The HTG-VLDL-induced accumulation of macrophage TG (70-fold) was unaffected by the OSCi or exogenous 24(S),25-epoxycholesterol, an effect associated with suppression of SREBP-1 processing. By contrast, TO901317, a synthetic LXR agonist, increased cellular TG significantly and markedly increased SREBP-1 processing. OSC inhibition decreased HTG-VLDL uptake through downregulation of LDL-receptor expression, despite substantial inhibition of cholesterol synthesis. Furthermore, OSC inhibition significantly upregulated ABCA1 and ABCG1 expression, which led to enhanced macrophage cholesterol efflux, an effect mediated through LXR activation. Therefore, increased macrophage synthesis of endogenous oxysterols represents a new mechanism for the dual regulation of LXR-and SREBP-responsive genes, an approach that inhibits foam cell formation without detrimental effect on TG synthesis. T he uptake of apolipoprotein (apo) B-containing lipoproteins by macrophages within the arterial wall results in cholesteryl ester (CE) deposition and foam cell formation, a hallmark of early and late atherosclerotic lesions. 1 Foam cell formation is the result of an imbalance in processes mediating lipoprotein uptake and cholesterol efflux. 2 The importance of cholesterol efflux in the maintenance of macrophage cellular cholesterol homeostasis arose from elucidation of mutations in the ATP binding cassette protein (ABC) A1 in Tangier disease, which is characterized by macrophage cholesterol accumulation (reviewed by Attie et al 3 ). ABCA1 is a transmembrane protein that controls the transfer of cholesterol and phospholipids to apoA1, the initial step in HDL formation and reverse cholesterol transport (reviewed by Tall et al, 2 Francis et al, 4 and Oram 5 ). The discovery of ABCA1 and its key role in regulating macrophage cholesterol efflux has stimulated development of therapeutic interventions that enhance the removal of cholesterol from arterial cells as a strategy to reduce atherosclerosis. 2 Expression of...

Liver X receptor (LXR) activation represents a mechanism to prevent macrophage foam cell formation. Previously, we demonstrated that partial inhibition of oxidosqualene:lanosterol cyclase (OSC) stimulated synthesis of the LXR agonist 24(S),25-epoxycholesterol (24(S),25-epoxy) and enhanced ABCA1-mediated cholesterol efflux. In contrast to a synthetic, nonsteroidal LXR activator, TO-901317, triglyceride accumulation was not observed. In the present study, we determined whether endogenous 24(S),25-epoxy synthesis selectively enhanced expression of macrophage LXR-regulated cholesterol efflux genes but not genes that regulate fatty acid metabolism. THP-1 human macrophages incubated with the OSC inhibitor (OSCi) RO0714565 (15 nM) significantly reduced cholesterol synthesis and maximized synthesis of 24(S),25-epoxy. Endogenous 24(S),25-epoxy increased ABCA1, ABCG1, and APOE mRNA abundance and consequently increased cholesterol efflux to apoAI. In contrast, OSCi had no effect on LXR-regulated genes LPL (lipoprotein lipase) and FAS (fatty acid synthase). TO-901317 (>10 nM) significantly enhanced expression of all genes examined. OSCi and TO-901317 increased the mRNA and precursor form of SREBP1c, a major regulator of fatty acid and triglyceride synthesis. However, conversion of the precursor to the active form (nSREBP-1c) was blocked by OSCi-induced 24(S),25-epoxy but not by TO-901317 (>10 nM), which instead markedly increased nSREBP-1c. Disruption of nSREBP-1c formation by 24(S),25-epoxy accounted for diminished FAS and LPL expression. In summary, endogenous synthesis of 24(S),25-epoxy selectively up-regulates expression of macrophage LXR-regulated cholesterol efflux genes without stimulating genes linked to fatty acid and triglyceride synthesis.Macrophage-derived cholesteryl ester-rich foam cells develop within the arterial wall as a result of excessive internalization of lipoproteins, which subsequently promote early atherosclerotic plaque formation. In addition, foam cells enhance susceptibility to plaque rupture within advanced stage lesions, leading to further atherosclerosis complications (1, 2). The ligand-activated nuclear receptors known as liver X receptors (LXRs), 5 whose natural ligands are oxysterols, regulate the expression of genes involved in lipid homeostasis through binding to LXR response elements (LXREs) within the promoter of several responsive genes. These include ABCA1 (ATP-binding cassette A1), ABCG1, and APOE (apolipoprotein E), which mediate cellular cholesterol efflux from human and mouse macrophages to extracellular acceptors (3). Additionally, activated LXR increases expression of SREBP-1c (sterol regulatory element-binding protein 1c), FAS (fatty acid synthase), and LPL (lipoprotein lipase), which together act to stimulate cellular free fatty acid synthesis and free fatty acid uptake, respectively, leading to enhanced triglyceride synthesis (4 -6). SREBP-1c is itself a master regulator of genes involved in lipogenesis, such as LPL (7) and FAS (8), and is also self-regulating, since it posit...

The cholesterol biosynthetic pathway produces numerous signaling molecules. Oxysterols through liver X receptor (LXR) activation regulate cholesterol efflux, whereas the non-sterol mevalonate metabolite, geranylgeranyl pyrophosphate (GGPP), was recently demonstrated to inhibit ABCA1 expression directly, through antagonism of LXR and indirectly through enhanced RhoA geranylgeranylation. We used HMG-CoA reductase inhibitors (statins) to test the hypothesis that reduced synthesis of mevalonate metabolites would enhance cholesterol efflux and attenuate foam cell formation. Preincubation of THP-1 macrophages with atorvastatin, dose dependently (1-10 M) stimulated cholesterol efflux to apolipoprotein AI (apoAI, 10 -60%, p < 0.05) and high density lipoprotein (HDL 3 ) (2-50%, p < 0.05), despite a significant decrease in cholesterol synthesis (2-90%). Atorvastatin also increased ABCA1 and ABCG1 mRNA abundance (30 and 35%, p < 0.05). Addition of mevalonate, GGPP or farnesyl pyrophosphate completely blocked the statin-induced increase in ABCA1 expression and apoAI-mediated cholesterol efflux. A role for RhoA was established, because two inhibitors of Rho protein activity, a geranylgeranyl transferase inhibitor and C3 exoenzyme, increased cholesterol efflux to apoAI (20 -35%, p < 0.05), and macrophage expression of dominant-negative RhoA enhanced cholesterol efflux to apoAI (20%, p < 0.05). In addition, atorvastatin increased the RhoA levels in the cytosol fraction and decreased the membrane localization of RhoA. Atorvastatin treatment activated peroxisome proliferator activated receptor ␥ and increased LXR-mediated gene expression suggesting that atorvastatin induces cholesterol efflux through a molecular cascade involving inhibition of RhoA signaling, leading to increased peroxisome proliferator activated receptor ␥ activity, enhanced LXR activation, increased ABCA1 expression, and cholesterol efflux. Finally, statin treatment inhibited cholesteryl ester accumulation in macrophages challenged with atherogenic hypertriglyceridemic very low density lipoproteins indicating that statins can regulate foam cell formation.

Diets containing the soya-derived phytoestrogens, genistein and daidzein, decrease plasma cholesterol in humans and experimental animals. The mechanisms responsible for the hypocholesterolaemic effects of these isoflavones are unknown. The present study was conducted to determine if genistein and daidzein regulate hepatocyte cholesterol metabolism and apolipoprotein (apo) B secretion in cultured human hepatoma (HepG2) cells. ApoB secretion was decreased dose-dependently by up to 63% and 71% by genistein and daidzein (100 microM; P<0.0001) respectively. In contrast, no effect on apoAI secretion was observed. Cellular cholesterol synthesis was inhibited 41% by genistein (100 microM; P<0.005) and 18% by daidzein (100 microM; P<0.05), which was associated with significant increases in 3-hydroxy-3-methylglutaryl-CoA reductase mRNA. Cellular cholesterol esterification was decreased 56% by genistein (100 microM; P<0.04) and 29% by daidzein (100 microM; P<0.04); however, mRNA levels for acyl-CoA:cholesterol acyltransferase (ACAT) 1 and ACAT2 were unaffected. At 100 microM, both isoflavones equally inhibited the activities of both forms of ACAT in cells transfected with either ACAT1 or ACAT2. Genistein (100 microM) and daidzein (100 microM) significantly decreased the activity of microsomal triacylglycerol transfer protein (MTP) by 30% and 24% respectively, and significantly decreased MTP mRNA levels by 35% and 55%. Both isoflavones increased low-density lipoprotein (LDL)-receptor mRNA levels by 3- to 6-fold (100 microM; P<0.03) and significantly increased the binding, uptake and degradation of (125)I-labelled LDL, suggesting that enhanced reuptake of newly secreted apoB-containing lipoproteins contributed to the net decrease in apoB secretion. These results indicate that genistein and daidzein inhibit hepatocyte apoB secretion through several mechanisms, including inhibition of cholesterol synthesis and esterification, inhibition of MTP activity and expression and increased expression of the LDL-receptor.