Citrus flavonoids and lipid metabolism

Assini, Julia M.a,b; Mulvihill, Erin E.a,b; Huff, Murray W.a,b

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Author Information
a

Department of Biochemistry, Department of Medicine

Robarts Research Institute, The University of Western Ontario, London, Ontario,

Canada
Correspondence to Murray W. Huff, Robarts Research Institute, 4222a, 100 Perth
Drive, London, ON N6A 5K8, Canada. Tel: +1 519 931 5793; fax: +1 519 931 5227;
e-mail: mhuff@uwo.ca
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Abstract
Purpose of review: Citrus flavonoids are polyphenolic compounds with powerful
biological properties. This review aims to summarize recent advances towards
understanding the ability of citrus flavonoids to regulate lipid metabolism and other
metabolic parameters relevant to the metabolic syndrome, type 2 diabetes and
cardiovascular disease.
Recent findings: Citrus flavonoids, including naringenin, hesperidin, nobiletin and
tangeretin, have emerged as promising therapeutic agents for the treatment of
metabolic dysregulation. Epidemiological studies report that intake of citrus
flavonoid-containing foods attenuates cardiovascular diseases. Experimental and a
limited number of clinical studies reveal lipid-lowering, insulin-sensitizing,
antihypertensive and anti-inflammatory properties. In animal models, citrus flavonoid
supplements prevent hepatic steatosis, dyslipidemia and insulin sensitivity primarily
through inhibition of hepatic fatty acid synthesis and increased fatty acid oxidation.
Citrus flavonoids blunt the inflammatory response in metabolically important tissues
including liver, adipose tissue, kidney and the aorta. The mechanisms underlying
flavonoid-induced metabolic regulation have not been completely established. In
mouse models, citrus flavonoids show marked suppression of atherogenesis through
improved metabolic parameters and also through direct impact on the vessel wall.
Summary: These recent studies suggest an important role of citrus flavonoids in the
treatment of dyslipidemia, insulin resistance, hepatic steatosis, obesity and
atherosclerosis. The favorable outcomes are achieved through multiple mechanisms.
Human studies focussed on dose, bioavailability, efficacy and safety are required to
propel the use of these promising therapeutic agents into the clinical arena.

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INTRODUCTION
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 [1].
However, many patients, especially those with the dyslipidemia associated with
metabolic syndrome, are unable to reach their lipid treatment goals on statins alone [2].
Also, patients may be statin-intolerant and experience significant side-effects [3].
Fibrates, a class of drugs that reduce elevated plasma triglyceride levels, have little
effect on preventing adverse cardiovascular events [4]. 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 sideeffects [5]. 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 1218 months will be highlighted.
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CITRUS FLAVONOIDS
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Flavonoids are a class of polyphenolic molecules, present in foods such as fruits,

vegetables and plant-derived juices including tea, coffee and wine [6]. 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 [7], type 2 diabetes [8], neurodegenerative
disorders [9] 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 200mg

of naringenin as grapefruit juice, plasma levels reached 6

mol/l, whereas plasma concentrations of hesperetin reached 1.32.2mol/l

after
[15,16]
ingestion of 130220mg

of hesperetin as orange juice

. 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 interindividual bioavailability. Therefore, studies of purified flavonoid molecules have
provided a more direct assessment of their effectiveness and pharmacological
properties.
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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 [17]). Administration
of the purified, soluble hesperidin derivative, glucosyl hesperidin (500mg/day

in
capsule form), to hypertriglyceridemic patients for 24 weeks, markedly reduced
plasma triglyceride and apolipoprotein B (apoB) concentrations [18]. In a clinical trial
involving hypercholesterolemic patients, administration of naringin (400mg/day,

for 8
weeks) reduced plasma LDL-cholesterol and serum apoB by more than 14%, with no
change in plasma triglyceride or HDL concentrations [19]. However, in other human
studies, capsules of hesperidin (800mg)

or naringin (500mg)

did not affect plasma

total cholesterol, LDL-cholesterol or triglyceride after only 4 weeks in moderately
hypercholesterolemic individuals [20]. 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 lipidlowering 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 obesedb/db mice, the peel of the citrus unshiu fruit (2
g/100g
diet), a rich source of citrus flavonoids, significantly attenuated plasma
triglyceride and hepatic steatosis, after 6 weeks of supplementation to a chow diet[21].
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 [22]. 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 [23]. Studies in streptozotocin-treated rats using low doses of
naringenin (0.003, 0.006 and 0.012%) or naringin (50100mg/kg)

demonstrated a
[24,25]
reduction in plasma and hepatic triglyceride and cholesterol
, and improved
insulin sensitivity [25].
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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 [26].
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 [26].
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 SREBP1cstimulated 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 5070% [27,28,29]. Furthermore, naringenin decreased
apoB100 secretion from HepG2 cells in the presence of oleate through enhanced apoB
degradation [29]. 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 (100M

naringenin, 10M

nobiletin) up-regulated CPT1 mRNA, a PPAR target gene,

neither flavonoid directly activated PPAR itself in vitro or in vivo[26,27]. In contrast,
in other studies using different cell lines (U-2OS human osteosarcoma cells and Huh7 human liver cells), higher concentrations of naringenin (150160M)

stimulated
[30]
PPAR response element (PPRE) reporter activity . Even higher doses of naringenin
(240M)

resulted in activation of the PPAR-GAL4 fusion protein by 24% in HG5LN

reporter cells [30]. Studies involving fructose-fed Golden Syrian hamsters
supplemented with a mixture of flavonoids (1:1;
tangeretin:nobiletin, 125mg/kg/day)

and in rats administered streptozotocin or a diabetogenic diet and given naringenin

(0.003%, 0.006% and 0.012%) or naringin (50100mg/kg),

respectively,
demonstrated a significant increase in hepatic PPAR protein expression, but not
mRNA. However, FA oxidation was not assessed[24,25,31]. Therefore, these studies
indicate that several citrus flavonoids may activate PPAR; but highlight that the
dose/concentrations and experimental models used for these studies may impact their
ability to enhance PPAR transcription.
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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 [32]. 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 [37]. Furthermore, cholesterol-fed Ldlr-/- mice display increased circulating
serum amyloid A (SAA), an inflammatory mediator that potentiates inflammation in
the artery wall [38]. 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 (500ml/day)

for 28
days reduced circulating high-sensitive C-reactive protein (hsCRP) by 11% [39].
Leukocytes from healthy volunteers following 500ml/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 [40]. 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 (TNF),
and improved endothelial function [41]. Oral administration of hesperidin (500mg/day)

for 3 weeks to patients with metabolic syndrome reduced the circulating inflammatory
markers hsCRP, SAA and soluble E-selectin [42]. Orange juice (300kcal)

added to
high-fat, high-cholesterol meal blunted the inflammatory response in peripheral blood
monocytes of healthy individuals [43]. In cultured cells, lipoplysaccharide (LPS)stimulated RAW 264.7 macrophages treated with 1040g/ml

7-O-methylnaringenin
dose-dependently down-regulated TNF, IL-1 and IL-6 expression while preventing
LPS-induced phosphorylation of extracellular signal-regulated kinase (ERK1/2), cJun N-terminal kinase (JNK) and inhibitor of kappa B alpha (IB) [44]. Intracellular
lipid accumulation was significantly attenuated in 3T3L1 adipocytes treated with 64
M nobiletin, whereas there was no effect with the same dose of tangeretin [45].
Nevertheless, monocyte chemotactic protein-1 (MCP-1) secretion into the media was
significantly attenuated following treatment of these cells with 128M

tangeretin or
nobiletin [45].
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 1 acid glycoprotein, an acute-phase inflammatory protein secreted from
the liver [46]. In streptozotocin-induced diabetic rats, naringin (50 and 100mg/kg)

dose-dependently decreased the elevated circulating TNF, IL-6 and CRP

concentrations [25]. Mice treated with naringin (0.02%) demonstrated decreased serum
TNF [23]. Naringenin treatment (2%) of stretozotocin-induced diabetic mice
decreased renal expression ofTnfa, Il1b, Il6 and chemokine (C-C motif) ligand 2
(Ccl2) [47]. In db/dbmice, citrus unshiu peel (2g/100g

diet) decreased levels of the

proinflammatory markers IL-6, MCP-1, IFN and TNF in plasma or liver [21]. In
C57BL6/J mice, supplementation of a high-fat diet with nobiletin (100mg/kg)

decreased adipose tissue expression of Ccl2 andIl6[48].

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 Saa1/2were 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 mitogenactivated protein kinase (MAPKerk) and nuclear factor kappa B (NF-B) [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.
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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 [49]. 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.
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CONCLUSION
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.

Reduction of plasma cholesterol by citrus flavonoids is associated with effects on

specific liver functions related to lipid handling. In previous in vivo studies,
polymethoxylated flavones (PMF) reduced plasma cholesterol levels at lower
doses than required for flavanones. To delineate hepatic mechanisms that
underlie this differential potency, we used HepG2 cells to quantitate effects on
expression of the LDL receptor (LDLR) gene. A dose-response analysis showed
that 200 mol/L hesperetin, a flavanone present as a disaccharide in oranges,
increased LDLR mRNA levels 3.6- to 4.7-fold of the untreated control. In contrast,
nobiletin, a PMF found at the highest concentration in oranges and tangerines,
achieved maximal stimulation of 1.5- to 1.6-fold of control at only 5 mol/L.
Transcriptional regulation of the LDLR gene by citrus flavonoids has been
implicated but, to our knowledge, not directly demonstrated. Here, using
transfection vector constructs containing the upstream region of the LDLR gene,
we show differences in both potency and efficacy in the induction of transcription,
with peak stimulation of 5.3- to 7.5-fold of control at 150160 mol/L hesperetin
and 3- to 3.8-fold of control at 1020 mol/L nobiletin. Hesperetin sustains
induction, whereas nobiletin is inhibitory at high doses, resulting in an inverted-U
dose response. The sterol regulatory element (SRE) in the LDLR gene upstream
region plays a crucial role, because mutation of this site strongly attenuated
induction in response to hesperetin or nobiletin. Thus, citrus flavonoids are likely
to act through the SRE-binding proteins, with PMF initially activating these
mechanisms at considerably lower concentrations than flavanones.