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.


Blood and tissue collection
Mice were fasted for 6 h before sacrifi ce. Blood and tissue collection was performed as described previously ( 5,15,17 ). Animals were anesthetized with Avertin, and blood was collected via cardiac puncture and stored at Ϫ 20°C. Hearts were perfused with heparin-PBS, and the heart and full-length aorta were dissected together. The top half of the heart was removed, placed in optimum cutting temperature (OCT) medium and frozen on dry ice. The aorta was placed in 10% formalin. Other tissues, including liver, muscle, and adipose, were removed, weighed, snap frozen in liquid nitrogen, and stored at Ϫ 80°C.

Plasma and tissue analysis
Plasma concentrations of insulin, leptin (ALPCO Diagnostics, Windham, NH), and SAA (Invitrogen, Life Technologies, Mississauga, ON) were determined by mouse-specifi c ELISA as per manufacturer's instructions. Plasma concentrations of TG and total cholesterol (TC) were measured enzymatically ( 5 ). The TC and TG within VLDL, LDL, and HDL particles were determined in fresh plasma by fast performance liquid chromatography (FPLC) as described previously ( 5 ). Gall bladder bile acids were measured using an enzymatic assay ( 18 ). Lipids were extracted from whole aorta and 100 mg of liver using the method of Folch et al. ( 19 ) and quantitated as described previously ( 20 ). FA and cholesterol synthesis were measured following intraperitoneal injection of [1-14 C]acetic acid ( 5 ). FA oxidation was determined in tissue homogenates by conversion of [ 3 H]palmitate to 3

Glucose and insulin tolerance tests
Glucose tolerance tests (GTT) were conducted in mice fasted for 6 h and injected intraperitoneally with a 15% D-glucose solution in 0.9% NaCl (1 g/kg body weight). Blood glucose was measured using a glucometer (Acenscia Elite, Bayer Healthcare, Toronto, ON) at regular intervals up to 120 min postinjection. Insulin tolerance tests (ITT) were conducted after a 5 h fast and intraperitoneal injection with 0.6 IU/kg Novolin ge Toronto (Novo Nordisk, Mississauga, ON). Blood glucose was measured as described above every 15 min for 1 h. Glucose utilization and insulin sensitivity were calculated based on the absolute area under the curve (AUC).

Triglyceride and apoB100 secretion; triglyceride and cholesterol absorption
TG and apoB100 secretion into plasma was determined following intraperitoneal injection of tyloxapol and 200 Ci of Tran [ 35 S] label (1,000 Ci/mmol, MP Biomedicals Inc., Irvine, CA) (for model have implicated elevated dietary cholesterol in the induction of macrophage infi ltration and the infl ammatory response in both adipose tissue and liver, leading to the exacerbation of dyslipidemia, insulin resistance, and atherosclerosis ( 6,7 ).
Cholesterol-fed Ldlr Ϫ / Ϫ mice display increased circulating serum amyloid A (SAA), an infl ammatory mediator that potentiates infl ammation in the artery wall ( 8 ). In hamsters, dietary cholesterol functions synergistically with dietary fat and fructose to intensify dyslipidemia, insulin resistance, and hepatic steatosis ( 9 ). Other studies establish a link between hepatic infl ammation and dyslipidemia. Tumor necrosis factor (TNF) ␣ and interleukin (IL)-1 ␤ stimulate hepatic overproduction of apoB100-containing lipoproteins in vivo and in vitro ( 10,11 ). Collectively, these studies indicate that a moderate increase in dietary cholesterol promotes tissue and systemic infl ammation, thereby contributing to dyslipidemia, insulin resistance, and atherosclerosis. Pharmacological therapies for the treatment and prevention of the chronic low-grade infl ammation associated with atherosclerosis remain elusive.
Epidemiological studies revealed an association between increased consumption of dietary fl avonoids and a reduced risk of cardiovascular disease ( 12 ). Flavonoids have been shown to have anti-infl ammatory properties ( 13 ). Naringenin, a citrus fl avonoid, inhibits apoB100 secretion from cultured hepatocytes ( 14 ). Furthermore, in Ldlr Ϫ / Ϫ mice, addition of naringenin to a high-fat diet attenuates obesity and prevents hepatic triglyceride (TG) accumulation through increased hepatic fatty acid (FA) oxidation, leading to prevention of dyslipidemia, insulin resistance, and atherosclerosis ( 5,15 ). These studies were performed in the context of diets low in cholesterol (0.05%). Due to the evidence that high dietary cholesterol augments the infl ammatory state and potentiates atherogenesis ( 1 ), one objective of the present study was to examine the ability of naringenin to prevent these cholesterol-induced perturbations. Diets that are low in fat but high in sucrose and contain moderate amounts of cholesterol (0.2%) induce significant atherosclerosis in Ldlr Ϫ / Ϫ mice ( 16 ). However, the effect of dietary cholesterol in low-fat diets on the local and systemic infl ammatory response has not been determined. Furthermore, the ability of naringenin to prevent metabolic dysfunction and atherosclerosis in the context of a low-fat atherogenic diet remains to be elucidated. Therefore, a second objective of this study was to examine the ability of naringenin to prevent infl ammation, metabolic dysregulation, and atherosclerosis in mice fed a low-fat (LF), cholesterol-containing diet.
In the current study, in Ldlr Ϫ / Ϫ mice fed either a highfat, high-cholesterol (HFHC, 0.2%) diet or a low-fat, highcholesterol (LFHC, 0.2%) diet, naringenin supplementation prevented dyslipidemia and hyperinsulinemia. By enhancing hepatic FA oxidation and attenuating lipogenesis, naringenin prevented hepatic steatosis. The cholesterol-induced infl ammation in liver, adipose tissue, and aorta were alleviated by naringenin. Collectively, the correction of metabolic abnormalities and the infl ammatory response was associated with a marked attenuation of atherosclerosis.

Peritoneal macrophages
In a separate study, male Ldlr Ϫ / Ϫ mice 8-12 weeks of age (n = 16/group) were fed either standard chow or the HFHC diet ± 3% (wt/wt) naringenin. After 12 weeks, mice were injected intraperitoneally with 2.5 ml 4% Brewer's Thioglycollate Medium (Sigma-Aldrich); fi ve days later, mice were sacrifi ced via CO 2 inhalation. Peritoneal cells were plated at a density of 2.76-3.3 × 10 6 per well and were allowed to adhere for 2 h ( 22 ). A subset of peritoneal cells was plated into wells containing cover slips and stained with Oil Red O. Cellular TC, cholesteryl ester (CE), and TG mass in peritoneal cells were determined enzymatically following extraction with 3:2 (vol:vol) hexane:isopropanol ( 23 ). Cellular lipids were normalized to cell protein. Cellular mRNA levels were determined by quantitative real-time PCR ( 5 ).

Statistical analysis
Data is presented as the mean ± SEM. A one-way ANOVA and posthoc Tukey test was performed using Sigma Plot version 14.0 to determine statistical signifi cance. Different letters indicate statistical signifi cance ( P < 0.05) within each separate study (chow, HFHC, and HFHC + naringenin; LF, LFHC, and LFHC + naringenin). A two-sample t -test was also employed to test the prespecifi ed hypothesis that there is a signifi cant difference ( P < 0.05) between chow and LF groups.

Naringenin prevents dyslipidemia and apoB100 overproduction in cholesterol-fed mice
Hypercholesterolemia was induced in Ldlr Ϫ / Ϫ mice fed the HFHC and LFHC diets for 12 weeks, whereas naringenin supplementation reduced plasma cholesterol by more than 40% ( Table 1 ). Plasma TG was signifi cantly increased in mice fed HFHC (6-fold), LF (3-fold) and LFHC (3-fold) compared with chow. Addition of naringenin to the HFHC or LFHC diets attenuated plasma TG concentrations by ‫ف‬ 50% ( Table 1 ). FPLC revealed that both cholesterol-enriched diets markedly increased VLDL-and LDL-cholesterol, and increased VLDL-TG concentrations (2-to 4-fold) ( Fig. 1A , B ). In contrast, naringenin attenuated VLDL-and LDL-cholesterol levels, and substantially reduced VLDL-TG, in the presence of either high or low dietary fat ( Fig. 1A, B ). Decreased plasma lipids by naringenin were independent of any effect on cholesterol or TG absorption ( Table 1 ). The improvement in plasma lipids by naringenin was primarily due to a signifi cant reduction in hepatic TG secretion into plasma (>55%) compared with HFHC-and LFHC-fed animals ( Fig. 1C ). Furthermore, HFHC-and LFHC-fed mice secreted more apoB100 (2.2-and 1.6-fold, respectively) into plasma compared with chow-fed mice. Addition of naringenin to either cholesterolenriched diet attenuated apoB100 secretion by over 80% ( Fig. 1D ).

Improved liver and muscle lipid metabolism by naringenin supplementation to cholesterol-rich diets
Prominent hepatic steatosis , characterized by significantly elevated TC, CE, free cholesterol (FC), and TG apoB100 secretion only) ( 5 ). Mice were sacrifi ced by CO 2 inhalation, and blood was collected via cardiac puncture. A combined VLDL/IDL fraction (density < 1.019 g/ml) was isolated from 250 l of fresh plasma by ultracentrifugation and separated on 4.5% SDS-PAGE gels ( 14 ). Intestinal cholesterol absorption was determined using a modifi ed fecal isotope ratio method ( 5

Tissue sectioning and histology
Pieces of liver were placed in OCT, frozen, and sectioned at 8 m using a cryostat. Liver sections were stained with Oil Red O and Hoechst 33258 (2.5 g/ml, Sigma) and visualized by fl uorescence microscopy at 493 nm excitation. Immunohistochemistry (IHC) was conducted on liver sections (frozen) for the macrophage marker MAC-2 (Cedarlane, Burlington, ON). Briefl y, slides were fi xed in acetone, quenched with 3% H 2 0 2 in methanol, blocked in 10% BSA, and incubated with rat anti-mouse MAC-2 antibody (1:1000), followed by incubation with a secondary antibody (biotinylated goat anti-rat IgG antibody, 1:200, Vector, Burlington, ON). Sections were incubated in ABC reagent (Vector) followed by incubation in DAB substrate (Vector) and counterstained in hematoxylin.
White adipose tissue (WAT) was fi xed in 10% formalin, embedded in paraffi n, sectioned using a microtome, and stained with hematoxylin and eosin. Adipocyte diameter was calculated using Northern Eclipse 7.0 software on 20× magnifi cation photomicrographs.
Morphometric analysis of lesions in the aortic sinus was performed as described previously ( 15 ). Frozen serial sections were stained with Oil Red O for quantitation of lesion area, and collagen was visualized by staining slides with modifi ed Verhoff and Masson's trichrome. Macrophages were identifi ed by IHC staining with monocyte/macrophage antibody-2 (MOMA-2, Accurate Chemical and Scientifi c Corporation, Westbury, NY), and smooth muscle cells were identifi ed by staining with smooth muscle (SM) ␣ -actin antibody (Clone 1AH, Sigma-Aldrich). The relative area of lesions positively stained for MOMA-2, collagen (trichrome), and SM ␣ -actin was determined relative to the total area of the respective plaque. Cleaned, full-length aortas were fi xed in formalin, stained with Sudan IV, prepared en face, and mounted in glycerol gelatin as described previously ( 15 ). Lesion area was calculated by dividing the stained plaque area by the total area of the aortic arch (ascending and descending aorta) and expressed as a percentage.

Gene expression analysis
Tissue mRNA levels were determined by quantitative real-time PCR as published previously ( 5 ). The expression of each gene was normalized to Gapdh expression, and the value of the control group (chow or LF) was set to 1.

Energy expenditure
Energy expenditure, respiratory quotient, and total activity were assessed using the Comprehensive Laboratory Animal Monitoring System (CLAMS) (Columbus Instruments, Columbus, OH). Lean body mass was calculated from whole-body composition analysis conducted by micro-CT imaging using a Locus Ultra micro-CT scanner (GE Healthcare, London, ON) ( 21 ).
To evaluate the increased hepatic FA oxidation by the addition of naringenin to either the HFHC or LFHC diet, energy balance was assessed in an animal metabolic monitoring system. Total activity was similar among all dietary groups ( Table 1 ). Total energy expenditure (EE) in mice fed the HFHC diet was 14% lower than that of chow-fed mice, whereas total EE was signifi cantly increased (16%) by the addition of naringenin to the HFHC diet. Similarly, naringenin supplementation of the LFHC diet signifi cantly increased EE (15%) compared with the LFHC diet alone ( Fig. 2J and supplementary Fig. II-E). There was no signifi cant change in caloric intake by the addition of naringenin to either the HFHC or LFHC diet ( Table 1 ). Respiratory quotient (RQ) profi les, which refl ect the relative contributions of carbohydrate and fat oxidation to total EE, were decreased in HFHC-fed mice relative to chow but were not further affected by the addition of naringenin to the HFHC. Furthermore, RQ was not changed by the addition of naringenin to the LFHC diet ( Table 1 ). Given the signifi cant increase in total EE in naringenin-treated mice ( Fig. 2J ), the lack of change of RQ profi les suggests that both fat and carbohydrate oxidation were increased in the naringenin-treated mice.

Naringenin attenuates adipose tissue expansion and infl ammation and restores glucose homeostasis in mice fed cholesterol-enriched diets
In obesity, adipocytes expand to accommodate increased TG and are associated with increased infl ammation and insulin resistance ( 25 ). Dietary cholesterol may be a stimulus for both macrophage infi ltration into adipose tissue and infl ammatory cytokine production ( 6 ). The HFHC diet increased the adiposity index 3-fold compared with chow-fed mice ( Fig. 4A ). Both adiposity and weight gain were completely normalized by addition of naringenin to the HFHC diet ( Fig. 4A and Table 1 ). Although the increase in adiposity index in LF-fed mice was not increased further in LFHC-fed mice, supplementation with naringenin reduced adipose tissue accumulation to the levels of chow-fed mice ( Fig. 4A and Table 1 ). The reduction in adipose tissue by the addition of naringenin to either cholesterol-enriched diet was independent of any effect on caloric intake or TG absorption ( Table 1 ). of cholesterol to the LF diet did not further impact glucose or plasma insulin; however, naringenin reduced both parameters to levels observed in chow-fed mice ( Table 1 ). Naringenin normalized both the impaired glucose tolerance and insulin sensitivity induced by the HFHC diet ( Fig. 4F, G ). No difference was observed in either the glucose excursion or the response to exogenous insulin between LF-and LFHC-fed mice, whereas naringenin supple mentation signifi cantly improved glucose and insulin tolerance ( Fig. 4F, G ).
HFHC-fed mice were modestly hyperglycemic (1.3-fold) but signifi cantly hyperinsulinemic (3.8-fold) compared with chow-fed animals ( Table 1 ). In contrast, naringenin completely normalized blood glucose and plasma insulin levels. The LF diet increased blood glucose (1.3-fold) and plasma insulin (2.3-fold) compared with chow. The addition aortic lipid accumulation and infl ammatory gene expression were related to a decrease in macrophage foam cell formation. Peritoneal macrophages from HFHC-fed mice accumulated signifi cantly more CE (15-fold) and TG (4-fold) compared with macrophages from chow-fed mice ( Fig. 6D ). Increased lipid mass correlated with enhanced Oil Red O-stained lipid droplets ( Fig. 6E ). In contrast, macrophages isolated from naringenin-treated mice demonstrated a more than 50% reduction in CE and TG, correlating with a marked reduction in neutral lipid droplets. Analysis of infl ammatory cytokine expression in peritoneal macrophages revealed no induction in HFHC-fed mice ( Fig. 6F ). Nevertheless, naringenin signifi cantly attenuated the expression of Tnfa , Ccl2 , and Ccl3 ( Ϫ 30 to Ϫ 50%) compared with either chow-or HFHC-fed mice ( Fig. 6F ). Thus, naringenin's ability to attenuate infl ammation in peritoneal macrophages may, in part, be independent of its lipid-lowering properties.

DISCUSSION
Hypercholesterolemia is a signifi cant risk factor for the development of atherosclerosis ( 26 ). In Ldlr Ϫ / Ϫ mice, cholesterol enrichment of high-fat diets intensifi es dyslipidemia and the infl ammatory response in adipose tissue and liver, which potentiates atherogenesis ( 1, 6, 7 ). Moreover, increasing the cholesterol content of low-fat, highsucrose diets promotes hypercholesterolemia and atherosclerosis in Ldlr Ϫ / Ϫ mice ( 16 ). In the present study, we demonstrate that addition of naringenin to cholesterolrich diets prevents obesity, glucose intolerance, and insulin resistance. Naringenin markedly attenuates dyslipidemia and hepatic lipid accumulation. Furthermore, naringenin alleviates the cholesterol-induced infl ammatory response in liver, adipose tissue, and aorta, collectively resulting in an attenuation of atherogenesis.
Diets enriched in fat and/or cholesterol induce hepatic steatosis ( 7,24 ), primarily a consequence of increased SREBP-1c-induced lipogenesis and reduced FA oxidation ( 5,17 ). Furthermore, diets rich in simple carbohydrates, such as sucrose or fructose, also induce hepatic TG accumulation in rodents ( 9,27 ). In the present study, the HFHC, LF, and LFHC diets increased hepatic FA synthesis without a compensatory increase in hepatic FA oxidation, resulting in marked hepatic steatosis compared with chowfed mice. Addition of cholesterol to the LF diet resulted in a further increase in hepatic TG, even in the absence of excess dietary fat. Hepatic Srebf1c expression was also increased in LFHC-fed mice, refl ecting the impact of dietary cholesterol on liver X receptor (LXR)-induced SREBP-1cstimulated FA synthesis ( 28 ).
In contrast, naringenin reduced hepatic Srebf1c expression and increased Fgf21 , Pgc1a , and Cpt1a mRNA in concert with signifi cantly decreased hepatic FA synthesis and enhanced FA oxidation. We now demonstrate that when added to a cholesterol-enriched high-fat diet, naringenin maintains its ability to shift hepatic gene Naringenin prevents atherosclerosis and alters plaque morphology in the aorta of cholesterol-fed mice We next evaluated whether naringenin's ability to attenuate plasma and hepatic lipids, improve glucose and insulin homeostasis, and prevent the infl ammatory response extended to protection from atherosclerosis. Compared with chow, both the HFHC and LFHC diets markedly increased Oil Red O-stained plaque area in the aortic sinus to a similar extent ( ‫ف‬ 4.5 µm 2 × 10 5 ) ( Fig. 5A , B ). Conversely, naringenin treatment prevented lesion development in the aortic sinus by ‫ف‬ 50% when added to either cholesterol-rich diet. Lesion area within the aortic arch was substantially increased in HFHC-and LFHC-fed mice to 14% of surface area ( Fig. 5C, D ). Addition of naringenin to either diet reduced plaque area by more than 40%, which is depicted in representative photographs of aortae stained with Sudan IV (supplementary Fig. III).
Analysis of lesion morphology revealed that in cholesterol-fed mice, the Oil Red O-stained lesion area was associated with MOMA-2 staining, indicating the presence of lipid-rich macrophage foam cells (supplementary Figs. IV and V). Plaques in both cholesterol-fed groups also stained positively for collagen, which corresponded to the extent of SM ␣ -actin-stained area, suggesting that increased smooth muscle cells within lesions contributed to the secretion of collagen fi brils (supplementary Figs. IV and V). Quantitatively, the cholesterol-rich diets induced lesions in which the percentage of stained lesion area occupied by MOMA-2 was lower than controls, with a concomitant increase in the percentage area occupied by collagen and SM ␣ -actin ( Fig. 5E, F ). These data suggest that cholesterol-fed mice develop complex lesions that are not only rich in lipid but also rich in collagen, representing a more fibrous phenotype. In contrast, in plaques from naringeninfed mice, the percentage of MOMA-2-stained macrophages was elevated. These lesions contained less collagen and SM ␣ -actin, indicative of lesions at an early stage of development ( Fig. 5E, F and supplementary Figs. IV and V). Thus, in addition to reducing plaque area, naringenin attenuates the development of complex lesions.

Naringenin prevents lipid accumulation and infl ammation in the aorta and in peritoneal macrophages
In aortic tissue, TC and TG mass were signifi cantly increased in both HFHC (1.7-and 2-fold, respectively) and LFHC groups (1.5-and 2.2-fold, respectively) compared with controls ( Fig. 6A , B ). In comparison, naringenin completely prevented the cholesterol-induced deposition of aortic cholesterol and TG. The HFHC diet signifi cantly increased expression of the infl ammatory markers Tnfa , Il1b , and Ccl2 ( ‫ف‬ 2-fold) within the aortae ( Fig. 6C ). Conversely, naringenin supplementation of the HFHC diet normalized infl ammatory cytokine expression. Aortae from the low fat-fed groups were not available for analysis of infl ammatory markers.
Elicited peritoneal macrophages from Ldlr Ϫ / Ϫ mice fed a high-fat, 1.25% cholesterol diet have been used as an in vivo model of macrophage foam cell formation ( 22 ). We determined whether the naringenin-induced reduction in Our results are consistent with the concept that increased hepatic FA oxidation as well as decreased hyperinsulinemia-stimulated FA synthesis contributes to the naringenin-induced decrease in hepatic steatosis and VLDL overproduction, reduced adipose tissue accumulation, and normalization of muscle triglyceride levels. These observations are also consistent with studies in which genetically induced increases in hepatic FA oxidation protects mice from metabolic dysregulation. In global Acc2 Ϫ / Ϫ mice, in which the synthesis of malonyl-CoA is blocked thus relieving the inhibition of CPT1a, hepatic FA oxidation was increased ( ‫ف‬ 15%), and mice were protected from HF diet-induced hepatic steatosis, insulin resistance, and adiposity (31)(32)(33)(34). However, this was not observed in another expression to prevent hepatic TG accumulation. Furthermore, naringenin normalizes hyperinsulinemia and liver TG in mice fed a cholesterol-containing low-fat, high-sucrose diet (LFHC), primarily through diminished FA synthesis.
Hepatic apoB100 overproduction characterizes the dyslipidemia associated with insulin resistance, and increased hepatic lipid drives VLDL production ( 29,30 ). The hepatic steatosis in HFHC-and LFHC-fed mice was associated with an overproduction of VLDL apoB100 and TG into plasma. Supplementation of either cholesterol-rich diet with naringenin abolished apoB100 oversecretion and attenuated hyperlipidemia, concomitant with the striking reduction in hepatic TG and cholesterol. both glucose and FA oxidation rather than fuel competition between fat and carbohydrate in naringenin-treated mice ( 34 ).
The cholesterol-induced infl ammatory response in both liver and adipose tissue in mice fed the HFHC diet is consistent with previous reports ( 6,7 ). The current studies demonstrate that the infl ammatory response is also stimulated by addition of cholesterol to a LF diet, a model in which tissue TG accumulation is primarily derived from de novo lipogenesis. However, the magnitude of infl ammatory response induced by the LFHC diet was not as great as in HFHC-fed mice, even though hepatic steatosis and adiposity were similar, suggesting that cholesterol may interact with exogenous FAs to enhance infl ammation. Although both cholesterol-containing diets increased infl ammatory cytokine expression, we did not distinguish which cell type (Kupffer cell, infi ltrating macrophage, or adipocyte) contributed to infl ammation in liver and adipose, respectively, strain of Acc2 Ϫ / Ϫ mice ( 35 ). Furthermore, our results are completely consistent with studies in rats ( 36 ) or mice ( 37 ) in which hepatic-specifi c overexpression of Cpt1a increased hepatic FA oxidation and protected mice from HF dietinduced hepatic steatosis and the infl ammatory response as well as insulin resistance and obesity.
In this study, we assessed energy balance and found that the decrease in ectopic fat deposition in naringenintreated mice was attributed to a ‫ف‬ 15% increase in wholebody EE in mice fed naringenin-supplemented diets. The fact that EE was increased by naringenin while physical activity was similar in naringenin and nonnaringenin-fed mice suggests that substrate oxidation was higher with naringenin treatment. Although fat oxidation and carbohydrate oxidation are thought to be mutually inhibitory ( 38 ), similar RQ measurements were recorded for naringeninsupplemented and nonsupplemented diets, despite higher EE. This suggests that there is a simultaneous increase in and increased dietary cholesterol potentiates the infl ammatory and atherogenic responses ( 1,16 ). In HFHC-and LFHC-fed mice, increased liver and adipose tissue infl ammation, combined with the development of systemic infl ammation, contributed to accelerated atherogenesis compared with chow-and LF-fed mice. These mice developed lesions comprised not only of lipid-laden macrophages but also an abundance of collagen and SM ␣ -actin, consistent with the formation of more complex lesions ( 53 ). In naringenin-treated mice, the reduced dyslipidemia, improved insulin sensitivity, and decreased hepatic, adipose, and systemic infl ammation collectively contributed to a marked reduction in atherosclerosis. Lesions in naringenin-treated mice were less mature and composed mainly of macrophages, indicating that in a model in which atherogenesis is accelerated by dietary cholesterol, naringenin prevents the progression of lesions to more complex phenotypes. Supplementation of cholesterol-rich diets with naringenin not only prevented lipid accumulation but also infl ammatory cytokine expression within the aorta, demonstrating that the anti-infl ammatory properties of naringenin extend to the arterial wall. Therefore, it was important to establish whether naringenin could inhibit the infl ammatory response at the cellular level, specifically in macrophages, a cell that plays an integral role in mediating infl ammation in lesions ( 54 ). Elicited peritoneal macrophages from HFHC-fed mice, an in vivo model of foam cell formation ( 22 ), were signifi cantly enriched in cellular cholesterol and TG. Paradoxically, lipid accumulation was not associated with increased expression of infl ammatory markers compared with chow-fed mice, indicating that macrophage lipid-loading is not necessarily linked to increased infl ammation. Despite this fi nding, naringenin signifi cantly attenuated peritoneal macrophage lipid content and the expression of Tnfa , Ccl2 , and Ccl3 . This implies that naringenin attenuates infl ammation in macrophages not only through its ability to prevent lipid deposition but also via a direct effect on infl ammatory signaling. Naringenin is known to modulate infl ammation in cultured macrophages through inhibition of NF-B and activator protein 1 (AP-1) signaling ( 48,55 ). However, direct experimental evidence is required to elucidate the mechanisms, in addition to lipid lowering, by which naringenin prevents infl ammatory signaling in macrophages, liver, and adipose tissue.
In conclusion, these studies demonstrate that naringenin prevents metabolic dysregulation induced by dietary cholesterol both in the presence and absence of dietary fat. Naringenin's potent lipid-lowering properties contribute to reduced hyperlipidemia and increased insulin sensitivity. Cholesterol-induced infl ammation associated with obesity and atherosclerosis is prevented by naringenin. These studies also highlight the possibility that naringenin has a direct effect on the infl ammatory processes that potentiate atherogenesis. Therefore, the benefi cial effects of naringenin provide insight into possible therapeutic targets for preventing atherosclerosis associated with metabolic dysregulation. as each cell type can participate in the infl ammatory response ( 25,39 ).
The addition of naringenin to either the HFHC or LFHC diet substantially reduced macrophage content of liver and infl ammatory cytokine expression in both liver and adipose tissue. Protection by naringenin may be through its ability to reduce cholesterol-and FA-induced lipotoxicity. Mechanisms by which dietary cholesterol might promote hepatic infl ammation include increased FC, as observed in mice fed either cholesterol-containing diet. Cholesterol feeding has been shown to increase hepatic mitochondrial FC, resulting in sensitization of liver mitochondria to cytokinemediated injury ( 40 ). Furthermore, elevated cellular FC is known to stimulate infl ammatory signaling pathways in vitro, including mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-B ( 41 ). The ability of naringenin to diminish hepatic cholesterol may alleviate this infl ammatory stimulus.
Incubation of adipocytes and macrophages with saturated FAs enhances infl ammatory cytokine secretion, thereby linking saturated FAs to the infl ammatory process ( 42,43 ). Furthermore, diets rich in saturated fat, including the HFHC diet in the present study, induce macrophage infi ltration into adipose tissue and stimulate the infl ammatory response (44)(45)(46). Increased hepatic oxidation of these FAs in HF-fed mice overexpressing hepatic Cpt1a attenuated the expression of Tnfa , Il6 , Il1b , and Ccl3 in liver or adipose tissue ( 37 ). Moreover, in macrophages exposed to excess palmitate, activation of AMPK increased FA oxidation and suppressed infl ammation ( 47 ). Collectively, these results suggest that naringenin's anti-infl ammatory properties in adipose tissue are secondary to decreased exposure to lipoprotein-derived FAs and that naringenin's anti-infl ammatory properties in liver are due to its ability to stimulate FA oxidation and/or inhibit FA synthesis. However, a direct effect of naringenin in these tissues on infl ammatory signaling pathways cannot be ruled out. Naringenin has been shown to directly inhibit MAPK signaling and NF-B activity in epithelial cells ( 48 ).
Infl ammation in adipose tissue associated with obesity is thought to contribute to insulin resistance ( 44,49 ). In ob/ ob mice, macrophage infi ltration into adipose tissue precedes the onset of hyperinsulinemia, suggesting that insulin resistance is initiated, in part, by adipose tissue infl ammation ( 44 ). In vitro, TNF ␣ inhibits insulin signaling in adipocytes, leading to reduced glucose uptake ( 50 ). In the present study, the ability of naringenin to prevent obesity and cholesterol-induced adipose tissue infl ammation may contribute to the improvement in glucose homeostasis and insulin sensitivity. Two other polyphenolic molecules, resveratrol and quercetin, when incubated with human adipocytes, prevented both TNF ␣ -mediated infl ammatory cytokine secretion and inactivation of the insulin receptor ( 51 ). Further mechanistic studies are required to defi ne any direct contribution of naringenin in adipocytes to its protection from obesity, adipose infl ammation, and insulin resistance.
Obesity-associated chronic low-grade infl ammation is considered a risk factor for cardiovascular disease ( 2,52 ),