Jane Y. Edwards

Naringenin prevents dyslipidemia, apolipoprotein B overproduction, and hyperinsulinemia in LDL receptor-null mice with diet-induced insulin resistance.

OBJECTIVE The 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 METHODS LDL 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. RESULTS We 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. CONCLUSIONS Thus, naringenin, through its correction of many of the metabolic disturbances linked to insulin resistance, represents a promising therapeutic approach for metabolic syndrome.

The molecular mechanisms underlying the reduction of LDL apoB-100 by ezetimibe plus simvastatin

The combination of ezetimibe, an inhibitor of Niemann-Pick C1-like 1 protein (NPC1L1), and an HMG-CoA reductase inhibitor decreases cholesterol absorption and synthesis. In clinical trials, ezetimibe plus simvastatin produces greater LDL-cholesterol reductions than does monotherapy. The molecular mechanism for this enhanced efficacy has not been defined. Apolipoprotein B-100 (apoB-100) kinetics were determined in miniature pigs treated with ezetimibe (0.1 mg/kg/day), ezetimibe plus simvastatin (10 mg/kg/day), or placebo (n = 7/group). Ezetimibe decreased cholesterol absorption (−79%) and plasma phytosterols (−91%), which were not affected further by simvastatin. Ezetimibe increased plasma lathosterol (+65%), which was prevented by addition of simvastatin. The combination decreased total cholesterol (−35%) and LDL-cholesterol (−47%). VLDL apoB pool size decreased 26%, due to a 35% decrease in VLDL apoB production. LDL apoB pool size decreased 34% due to an 81% increase in the fractional catabolic rate, both of which were significantly greater than monotherapy. Combination treatment decreased hepatic microsomal cholesterol (−29%) and cholesteryl ester (−65%) and increased LDL receptor (LDLR) expression by 240%. The combination increased NPC1L1 expression in liver and intestine, consistent with increased SREBP2 expression. Ezetimibe plus simvastatin decreases VLDL and LDL apoB-100 concentrations through reduced VLDL production and upregulation of LDLR-mediated LDL clearance.

Selective Up-regulation of LXR-regulated Genes ABCA1, ABCG1, and APOE in Macrophages through Increased Endogenous Synthesis of 24(S),25-Epoxycholesterol

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 SREBP-1c, 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.

Soya phytoestrogens, genistein and daidzein, decrease apolipoprotein B secretion from HepG2 cells through multiple mechanisms

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.

Nobiletin Attenuates VLDL Overproduction, Dyslipidemia, and Atherosclerosis in Mice With Diet-Induced Insulin Resistance

OBJECTIVE Increased 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 METHODS Lipoprotein 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. RESULTS In 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. CONCLUSIONS Nobiletin provides insight into treatments for dyslipidemia and atherosclerosis associated with insulin-resistant states.

Naringenin prevents cholesterol-induced systemic inflammation, metabolic dysregulation, and atherosclerosis in Ldlr−/− mice

Obesity-associated chronic inflammation contributes to metabolic dysfunction and propagates atherosclerosis. Recent evidence suggests that increased dietary cholesterol exacerbates inflammation in adipose tissue and liver, contributing to the proatherogenic milieu. The ability of the citrus flavonoid naringenin to prevent these cholesterol-induced perturbations is unknown. To assess the ability of naringenin to prevent the amplified inflammatory 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 infiltration and inflammation stimulated by dietary cholesterol. Insulin resistance, adipose tissue expansion, and inflammation were alleviated by naringenin. Naringenin attenuated the cholesterol-induced formation of both foam cells and expression of inflammatory markers in peritoneal macrophages. Naringenin significantly decreased atherosclerosis and inhibited the formation of complex lesions, which was associated with normalized aortic lipids and a reversal of aortic inflammation. We demonstrate that in mice fed cholesterol-enriched diets, naringenin attenuates peripheral and systemic inflammation, leading to protection from atherosclerosis. These studies offer a therapeutically relevant alternative for the prevention of cholesterol-induced metabolic dysregulation.

Inhibition of the Apical Sodium-Dependent Bile Acid Transporter Reduces LDL Cholesterol and ApoB by Enhanced Plasma Clearance of LDL ApoB

Objective—Cloning of the ileal apical sodium-dependent bile acid transporter (ASBT) has identified a new pharmacological target for the modulation of plasma lipoproteins. The objective of this study was to determine whether a novel, specific, minimally absorbed ASBT inhibitor (SC-435) decreases LDL cholesterol through the alteration of plasma apoB kinetics. Methods and Results—Miniature pigs were treated for 21 days with 10 mg/kg/day of SC-435 or placebo. SC-435 decreased plasma cholesterol by 9% and LDL cholesterol by 20% with no effect on other lipids. Autologous 131I-VLDL, 125I-LDL, and [3H]-leucine were injected simultaneously to determine apoB kinetics. LDL apoB concentrations decreased significantly by 10% resulting entirely from an increase in LDL–apoB fractional catabolic rate. SC-435 had no effect on either total LDL apoB production or VLDL apoB converted to LDL. SC-435 increased VLDL apoB production by 22%; however, the concentration was unchanged as a result of increased VLDL apoB direct removal. SC-435 increased hepatic mRNA and enzymatic activity for both cholesterol 7&agr;-hydroxylase and HMG-CoA reductase. Hepatic LDL receptor mRNA increased significantly, whereas apoB expression was unaffected. Conclusions—A low dose of the ASBT inhibitor, SC-435, significantly reduces plasma LDL cholesterol through enhanced LDL receptor-mediated LDL apoB clearance, secondary to increased expression of cholesterol 7&agr;-hydroxylase.

Activation of Peroxisome Proliferator-Activated Receptor δ Inhibits Human Macrophage Foam Cell Formation and the Inflammatory Response Induced by Very Low-Density Lipoprotein

Objective—Hypertriglyceridemia is an important risk factor for cardiovascular disease. Elevated plasma very low-density lipoprotein (VLDL) puts insulin-resistant patients at risk for atherosclerosis. VLDL readily induces macrophage lipid accumulation and inflammatory responses, for which targeted therapeutic strategies remain elusive. We examined the ability of VLDL to induce macrophage foam cells and the inflammatory response and sought to define the cell signaling cascades involved. We further examined the potential of peroxisome proliferator-activated receptor (PPAR) &dgr; activation to attenuate both VLDL-stimulated lipid accumulation and cytokine expression. Methods and Results—THP-1 macrophages exposed to VLDL displayed significant triglyceride accumulation, which was attenuated by PPAR&dgr; activation. PPAR&dgr; agonists stimulated a transcriptional program resulting in inhibition of lipoprotein lipase activity, activation of fatty acid uptake, and enhanced &bgr;-oxidation. VLDL-treated macrophages significantly increased the expression of activator protein 1 associated cytokines interleukin-1&bgr;, macrophage inflammatory protein 1&agr;, and intercellular adhesion molecule-1. VLDL treatment significantly increased the phosphorylation of both extracellular signal-related kinase 1 and 2 and p38. VLDL reduced AKT phosphorylation as well as its downstream effector forkhead box protein O1, concomitant with increased nuclear forkhead box protein O1. Cells treated with PPAR&dgr; agonists were completely resistant to VLDL-induced expression of inflammatory cytokines, mediated by normalization of mitogen-activated protein kinase (MAPK)erk and AKT/forkhead box protein O1 signaling. Conclusion—The combined PPAR&dgr;-mediated reductions of lipid accumulation and inflammatory cytokine expression suggest a novel macrophage-targeted therapeutic option in treating atherosclerosis.

Peroxisome Proliferator–Activated Receptor δ Agonist GW1516 Attenuates Diet-Induced Aortic Inflammation, Insulin Resistance, and Atherosclerosis in Low-Density Lipoprotein Receptor Knockout Mice

Objective— The peroxisome proliferator–activated receptor (PPAR) &dgr; regulates systemic lipid homeostasis and inflammation. However, the ability of PPAR&dgr; agonists to improve the pathology of pre-established lesions and whether PPAR&dgr; activation is atheroprotective in the setting of insulin resistance have not been reported. Here, we examine whether intervention with a selective PPAR&dgr; agonist corrects metabolic dysregulation and attenuates aortic inflammation and atherosclerosis. Approach and Results— Low-density lipoprotein receptor knockout mice were fed a chow or a high-fat, high-cholesterol (HFHC) diet (42% fat, 0.2% cholesterol) for 4 weeks. For a further 8 weeks, the HFHC group was fed either HFHC or HFHC plus GW1516 (3 mg/kg per day). GW1516 significantly attenuated pre-established fasting hyperlipidemia, hyperglycemia, and hyperinsulinemia, as well as glucose and insulin intolerance. GW1516 intervention markedly reduced aortic sinus lesions and lesion macrophages, whereas smooth muscle &agr;-actin was unchanged and collagen deposition enhanced. In aortae, GW1516 increased the expression of the PPAR&dgr;-specific gene Adfp but not PPAR&agr;- or &ggr;-specific genes. GW1516 intervention decreased the expression of aortic proinflammatory M1 cytokines, increased the expression of the anti-inflammatory M2 cytokine Arg1, and attenuated the iNos/Arg1 ratio. Enhanced mitogen-activated protein kinase signaling, known to induce inflammatory cytokine expression in vitro, was enhanced in aortae of HFHC-fed mice. Furthermore, the HFHC diet impaired aortic insulin signaling through Akt and forkhead box O1, which was associated with elevated endoplasmic reticulum stress markers CCAAT-enhancer-binding protein homologous protein and 78kDa glucose regulated protein. GW1516 intervention normalized mitogen-activated protein kinase activation, insulin signaling, and endoplasmic reticulum stress. Conclusions— Intervention with a PPAR&dgr; agonist inhibits aortic inflammation and attenuates the progression of pre-established atherosclerosis.

PPARδ activation attenuates hepatic steatosis in Ldlr−/− mice by enhanced fat oxidation, reduced lipogenesis, and improved insulin sensitivity

PPARδ regulates systemic lipid homeostasis and inflammation, but its role in hepatic lipid metabolism remains unclear. Here, we examine whether intervening with a selective PPARδ agonist corrects hepatic steatosis induced by a high-fat, cholesterol-containing (HFHC) diet. Ldlr−/− mice were fed a chow or HFHC diet (42% fat, 0.2% cholesterol) for 4 weeks. For an additional 8 weeks, the HFHC group was fed HFHC or HFHC plus GW1516 (3 mg/kg/day). GW1516-intervention significantly attenuated liver TG accumulation by induction of FA β-oxidation and attenuation of FA synthesis. In primary mouse hepatocytes, GW1516 treatment stimulated AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) phosphorylation in WT hepatocytes, but not AMPKβ1−/− hepatocytes. However, FA oxidation was only partially reduced in AMPKβ1−/− hepatocytes, suggesting an AMPK-independent contribution to the GW1516 effect. Similarly, PPARδ-mediated attenuation of FA synthesis was partially due to AMPK activation, as GW1516 reduced lipogenesis in WT hepatocytes but not AMPKβ1−/− hepatocytes. HFHC-fed animals were hyperinsulinemic and exhibited selective hepatic insulin resistance, which contributed to elevated fasting FA synthesis and hyperglycemia. GW1516 intervention normalized fasting hyperinsulinemia and selective hepatic insulin resistance and attenuated fasting FA synthesis and hyperglycemia. The HFHC diet polarized the liver toward a proinflammatory M1 state, which was reversed by GW1516 intervention. Thus, PPARδ agonist treatment inhibits the progression of preestablished hepatic steatosis.