Lipids are essential macromolecules required for maintaining various homeostatic, physiologic and cellular processes in the body. Moreover, the dysregulation of lipid and lipoprotein metabolism can contribute to the pathogenesis of a multitude of human diseases such as cardiovascular disease, obesity, diabetes and inflammation. Many of these disorders, including the metabolic syndrome, type 2 diabetes and cardiovascular disease, are reaching epidemic proportions in today's Western societies due primarily to a lack of proper nutrition and physical exercise. Several therapeutic strategies exist to modulate lipid metabolism and prevent metabolic disease, each with their own inherent limitations. Currently, statin drugs, which target the cholesterol synthesis pathway and confer potent cholesterol-lowering effects, are a widely prescribed therapy for preventing mortality associated with atherosclerosis . However, many patients, especially those with the dyslipidemia associated with metabolic syndrome, are unable to reach their lipid treatment goals on statins alone . Also, patients may be statin-intolerant and experience significant side-effects . Fibrates, a class of drugs that reduce elevated plasma triglyceride levels, have little effect on preventing adverse cardiovascular events . Thiazolidinediones are often used in patients with metabolic syndrome to improve insulin sensitivity; however, they have been shown to promote weight gain and are associated with off-target side-effects . Therefore, the development of new therapeutic interventions that modulate lipid metabolism, especially the dyslipidemia of the metabolic syndrome, is imperative. This review focuses on the ability of dietary compounds called citrus flavonoids to regulate aberrant lipid metabolism associated with metabolic dysfunction. Advances in the past 12–18 months will be highlighted.
Flavonoids are a class of polyphenolic molecules, present in foods such as fruits, vegetables and plant-derived juices including tea, coffee and wine . There are several classes of flavonoids including flavanones, flavonols, flavones, flavan-3-ols, anthocyanins and isoflavones, which characterize these molecules based on their carbon structure and level of oxidation. Several studies have demonstrated a relationship between the consumption of flavonoid-rich diets and the prevention of human disease including cancer , type 2 diabetes , neurodegenerative disorders  and osteoporosis [10,11]. A recent prospective study revealed that consumption of citrus flavanones from orange and grapefruit juice was associated with a reduced risk (19%) of ischemic stroke in women, indicating a cardioprotective effect of these molecules [12▪].
Citrus flavonoids encompass several subgroups of flavonoids including flavanones (naringin and hesperidin) and O-polymethoxylated flavones (nobiletin and tangeretin). The bioactivity of these molecules depends on their structure and subsequent metabolism; naringin and hesperidin require hydrolysis to their active aglycone forms naringenin and hesperitin, whereas nobiletin and tangeretin lack a glycoside moiety and are more easily absorbed by the gut [13,14]. Citrus flavonoids can reach significant levels in the plasma from dietary consumption. In humans, following the ingestion of 200 mg of naringenin as grapefruit juice, plasma levels reached 6 μmol/l, whereas plasma concentrations of hesperetin reached 1.3–2.2 μmol/l after ingestion of 130–220 mg of hesperetin as orange juice [15,16]. However, these findings are based on flavonoid intake from food sources, and are therefore affected by the abundance in the food, the amount of flavonoid ingested as well as the inter-individual bioavailability. Therefore, studies of purified flavonoid molecules have provided a more direct assessment of their effectiveness and pharmacological properties.
REGULATION OF DYSLIPIDEMIA AND HEPATIC LIPID METABOLISM BY CITRUS FLAVONOIDS
Dyslipidemia is a major risk factor for atherosclerosis. Citrus flavonoids have been shown to exhibit lipid-lowering properties in humans (reviewed in ). Administration of the purified, soluble hesperidin derivative, glucosyl hesperidin (500 mg/day in capsule form), to hypertriglyceridemic patients for 24 weeks, markedly reduced plasma triglyceride and apolipoprotein B (apoB) concentrations . In a clinical trial involving hypercholesterolemic patients, administration of naringin (400 mg/day, for 8 weeks) reduced plasma LDL-cholesterol and serum apoB by more than 14%, with no change in plasma triglyceride or HDL concentrations . However, in other human studies, capsules of hesperidin (800 mg) or naringin (500 mg) did not affect plasma total cholesterol, LDL-cholesterol or triglyceride after only 4 weeks in moderately hypercholesterolemic individuals . These studies reveal several important factors that impact the effectiveness of citrus flavonoids in clinical studies including the metabolite of flavonoid used, dose, patient population and length of study. It is likely the flavonoid intake is not high enough to achieve a significant metabolic outcome. Therefore careful consideration should be taken when assessing the beneficial lipid-lowering effects of purified citrus flavonoids in humans.
In animal models of the metabolic syndrome and cardiovascular disease, citrus flavonoids have proven to be beneficial in lowering lipids and preventing hepatic steatosis (Fig. 1). In genetically obese db/db mice, the peel of the citrus unshiu fruit (2 g/100 g diet), a rich source of citrus flavonoids, significantly attenuated plasma triglyceride and hepatic steatosis, after 6 weeks of supplementation to a chow diet . Administration of 0.05% naringin to rabbits fed a high-cholesterol diet decreased plasma total and LDL-cholesterol concentrations and reduced hepatic triglyceride and cholesterol levels . Similarly, in C57BL/6 mice fed a high-fat diet, naringin supplementation (0.02%) reduced plasma and liver cholesterol concentrations and improved indices of insulin sensitivity; however, no effect was observed on plasma or hepatic triglyceride . Studies in streptozotocin-treated rats using low doses of naringenin (0.003, 0.006 and 0.012%) or naringin (50–100 mg/kg) demonstrated a reduction in plasma and hepatic triglyceride and cholesterol [24,25], and improved insulin sensitivity .
Studies from our laboratory have shown that in high-fat fed mice lacking the LDL receptor (Ldlr-/-), supplementation of the diet with naringenin (3%) prevented hepatic steatosis leading to decreased hepatic apoB100 and triglyceride production . Moreover, 3% naringenin significantly attenuated the increased sterol response element binding protein 1c (Srebf1c) expression and fatty acid (FA) synthesis observed in high-fat fed mice, and stimulated hepatic FA oxidation thereby contributing to the prevention of dyslipidemia and hepatic lipid accumulation . Addition of nobiletin (0.3%) to a diet high in fat, increased hepatic FA oxidation and suppressed hepatic FA synthesis, which contributed to a marked reduction in hepatic triglyceride, decreased very-low-density (VLDL) apoB secretion and the correction of hepatic and peripheral insulin resistance [27▪▪]. Nobiletin-activated hepatic transcription of peroxisome proliferator-activated receptor (PPAR) gamma coactivator 1-alpha (Pgc1a) and carnitine palmitoyltransferase 1 (Cpt1a) mRNA, in concert with increased mitochondrial DNA and hepatic FA oxidation, and inhibited SREBP1c-stimulated FA synthesis [27▪▪].
The lipid-lowering properties of naringenin and nobiletin in vivo are consistent with in-vitro mechanistic studies whereby microsomal triglyceride transfer protein (MTP) activity in HepG2 cells was inhibited, thereby reducing triglyceride accumulation and decreasing apoB100 secretion by 50–70% [27▪▪,28,29]. Furthermore, naringenin decreased apoB100 secretion from HepG2 cells in the presence of oleate through enhanced apoB degradation . These effects required stimulation of insulin-signaling pathways, although this was independent of the insulin receptor or insulin receptor substrates 1 and 2. These findings highlight the mechanism through which these two flavonoids modulate apoB100 secretion; however, they do not explain the mechanism underlying the enhanced hepatic FA oxidation observed in mice. Although the concentrations of naringenin and nobiletin used to inhibit apoB100 secretion in vitro (100 μM naringenin, 10 μM nobiletin) up-regulated CPT1α mRNA, a PPARα target gene, neither flavonoid directly activated PPARα itself in vitro or in vivo[26,27▪▪]. In contrast, in other studies using different cell lines (U-2OS human osteosarcoma cells and Huh-7 human liver cells), higher concentrations of naringenin (150–160 μM) stimulated PPAR response element (PPRE) reporter activity . Even higher doses of naringenin (240 μM) resulted in activation of the PPARα-GAL4 fusion protein by 24% in HG5LN reporter cells . Studies involving fructose-fed Golden Syrian hamsters supplemented with a mixture of flavonoids (1 : 1; tangeretin:nobiletin, 125 mg/kg/day) and in rats administered streptozotocin or a diabetogenic diet and given naringenin (0.003%, 0.006% and 0.012%) or naringin (50–100 mg/kg), respectively, demonstrated a significant increase in hepatic PPARα protein expression, but not mRNA. However, FA oxidation was not assessed [24,25,31]. Therefore, these studies indicate that several citrus flavonoids may activate PPARα; but highlight that the dose/concentrations and experimental models used for these studies may impact their ability to enhance PPARα transcription.
LIPID-INDUCED INFLAMMATION AND CITRUS FLAVONOIDS
Both obesity and the metabolic syndrome are associated with chronic low-grade inflammation, which is intimately associated with the development of obesity and insulin resistance [32,33]. Adipose tissue plays an important role in storing lipid in the form of triglycerides, as well as secreting a variety of adipokines and cytokines that regulate a number of physiological processes. During obesity, in order to compensate for the excess lipid load, adipose tissue undergoes rapid expansion . Several mouse models have demonstrated obesity-related increases in macrophage infiltration into adipose tissue, leading to inflammatory cytokine production [34,35]. Recently, it has been demonstrated that the addition of cholesterol (0.2%) to a high-fat diet in Ldlr-/- mice significantly increased macrophage infiltration into white adipose tissue, and liver contributing to systemic inflammation and amplified atherogenesis, compared to mice fed a high-fat diet alone [33,36]. Administration of cholesterol-rich diets to other mouse models resulted in increased susceptibility to hepatic steatosis and acute hepatic inflammation, leading to elevated plasma levels of inflammatory markers . Furthermore, cholesterol-fed Ldlr-/- mice display increased circulating serum amyloid A (SAA), an inflammatory mediator that potentiates inflammation in the artery wall . Collectively, these studies indicate that dietary cholesterol promotes tissue and systemic inflammation, thereby contributing to dyslipidemia and atherosclerosis.
Flavonoids have been examined for their anti-inflammatory potential, and several recent studies reveal their ability to attenuate inflammation associated with metabolic disease. In patients with peripheral artery disease, orange juice (500 ml/day) for 28 days reduced circulating high-sensitive C-reactive protein (hsCRP) by 11% . Leukocytes from healthy volunteers following 500 ml/day of orange juice or a control drink and hesperidin for 4 weeks, displayed anti-inflammatory and antiatherogenic gene profile, suggesting a genomic effect of citrus flavonoids . A recent study demonstrated that consumption of red orange juice for 7 days reduced circulating cytokines such as CRP, interleukin (IL)-6 and tumor necrosis factor alpha (TNFα), and improved endothelial function . Oral administration of hesperidin (500 mg/day) for 3 weeks to patients with metabolic syndrome reduced the circulating inflammatory markers hsCRP, SAA and soluble E-selectin [42▪]. Orange juice (300 kcal) added to high-fat, high-cholesterol meal blunted the inflammatory response in peripheral blood monocytes of healthy individuals . In cultured cells, lipoplysaccharide (LPS)-stimulated RAW 264.7 macrophages treated with 10–40 μg/ml 7-O-methylnaringenin dose-dependently down-regulated TNFα, IL-1β and IL-6 expression while preventing LPS-induced phosphorylation of extracellular signal-regulated kinase (ERK1/2), c-Jun N-terminal kinase (JNK) and inhibitor of kappa B alpha (IκBα) . Intracellular lipid accumulation was significantly attenuated in 3T3L1 adipocytes treated with 64 μM nobiletin, whereas there was no effect with the same dose of tangeretin . Nevertheless, monocyte chemotactic protein-1 (MCP-1) secretion into the media was significantly attenuated following treatment of these cells with 128 μM tangeretin or nobiletin .
Several animal studies involving the use of citrus flavonoid compounds have also shown a preventive effect on the inflammation associated with obesity and atherosclerosis (Fig. 1). Obese cats with diet-induced obesity and supplemented with either hesperidin (0.05%) or naringin (0.1%) displayed significantly decreased circulating α1 acid glycoprotein, an acute-phase inflammatory protein secreted from the liver . In streptozotocin-induced diabetic rats, naringin (50 and 100 mg/kg) dose-dependently decreased the elevated circulating TNFα, IL-6 and CRP concentrations . Mice treated with naringin (0.02%) demonstrated decreased serum TNFα . Naringenin treatment (2%) of stretozotocin-induced diabetic mice decreased renal expression of Tnfa, Il1b, Il6 and chemokine (C-C motif) ligand 2 (Ccl2) [47▪]. In db/db mice, citrus unshiu peel (2 g/100 g diet) decreased levels of the proinflammatory markers IL-6, MCP-1, IFNγ and TNFα in plasma or liver . In C57BL6/J mice, supplementation of a high-fat diet with nobiletin (100 mg/kg) decreased adipose tissue expression of Ccl2 and Il6.
Recent studies in our laboratory demonstrated the anti-inflammatory capability of naringenin in Ldlr-/- mice with cholesterol-induced metabolic dysfunction. Addition of naringenin (3%) to a diet high or low in fat and a moderate amount of cholesterol (0.2%) almost completely suppressed liver and adipose tissue expression of Tnfa, Il1b, Ccl2 and macrophage inflammatory protein 1 alpha (Ccl3), concomitant with decreased macrophage infiltration into these tissues. Furthermore, tissue expression and plasma levels of the acute-phase protein Saa1/2 were attenuated demonstrating that naringenin can prevent the chronic low-grade inflammation induced by cholesterol-containing diets (Assini JM and Huff MW, unpublished data). In addition, naringenin completely prevented the expression of Tnfa, Il1b and Ccl2 in peritoneal macrophages and in the aorta, suggesting a direct anti-inflammatory effect of naringenin within cells of the vessel wall that participate in atherogenesis. Although there is evidence that naringenin and naringin suppress the activation of mitogen-activated protein kinase (MAPKerk) and nuclear factor kappa B (NF-κB) [23,47▪], the inflammatory pathways regulated by these flavonoids have not been fully elucidated. Furthermore, it remains to be determined if attenuation of the inflammatory response is a direct transcriptional effect or is secondary to decreased tissue lipid deposition.
ATHEROSCLEROSIS AND CITRUS FLAVONOIDS
In experimental animal models atherosclerosis is attenuated by citrus flavonoids (Fig. 1). Naringenin (0.05%) and naringin (0.1%) reduced aortic fatty streaks in rabbits fed high-cholesterol diets . In Ldlr-/- mice fed a high-fat diet, supplementation with naringenin (3%) markedly suppressed the progression of atherosclerosis in the aortic sinus to more advanced lesions [50▪]. Similar findings were recently observed in mice treated with 0.02% naringin [51▪]. Supplementation of a high-fat diet with nobiletin (0.3%) in Ldlr-/-mice prevented the development of aortic sinus lesions by more than 70% [27▪▪]. In more recent studies in Ldlr-/- mice, the amplified atherosclerosis induced by the addition of cholesterol (0.2%) to a diet high or low in fat markedly suppressed aortic sinus lesion formation by more than 40% (Assini JM and Huff MW, unpublished data). Although flavonoids prevent atherogenesis in these models, from a therapeutic perspective, it will be important to determine whether intervention with flavonoid treatment in animal models of pre-established atherosclerosis will halt the progression or induce regression of lesions.
A number of therapeutically relevant compounds are derived from natural products including foods. Therefore, evaluation of citrus flavonoids as metabolic regulators represents an established avenue for drug discovery. Studies in this review add to the growing body of evidence that citrus flavonoids possess significant lipid and lipoprotein-lowering potential, and demonstrate that these compounds, particularly naringenin and nobiletin, reduce hepatic lipid accumulation and prevent lipoprotein overproduction, normalize insulin sensitivity, blunt tissue inflammation and attenuate the progression of atherosclerosis. These beneficial metabolic effects are mediated, in part, by normalization of hepatic fatty acid metabolism, amplifying insulin signaling and dampening the induction of the inflammatory response. Further studies are essential to fully reveal the interaction of these flavonoids with upstream mediators of these pathways. Clinical studies focussed on flavonoid dose, bioavailability, efficacy and safety are required to expand the limited studies of flavonoid treatment in humans.
Research from the author's laboratory was supported by grants from the Heart and Stroke Foundation of Ontario (HSFO) (T-7707 and PG-5967), Canadian Institutes of Health Research (CHIR) (FRN-8014), Pfizer Canada (NR2580078), a HSFO Masters Award (to J.M.A) and a CIHR Doctoral Award (to E.E.M.).
Conflicts of interest
There are no conflicts of interest.
REFERENCES AND RECOMMENDED READING
Papers of particular interest, published within the annual period of review, have been highlighted as:
▪ of special interest
▪▪ of outstanding interest
Additional references related to this topic can also be found in the Current World Literature section in this issue (pp. 90–91).
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