Tsutsumi et al. Nutrition & Metabolism 2014, 11:32
http://www.nutritionandmetabolism.com/content/11/1/32

RESEARCH

Open AccessSudachitin, a polymethoxylated flavone, improves
glucose and lipid metabolism by increasing
mitochondrial biogenesis in skeletal muscle
Rie Tsutsumi1*, Tomomi Yoshida1, Yoshitaka Nii2, Naoki Okahisa2, Shinya Iwata2, Masao Tsukayama2,
Rei Hashimoto1, Yasuko Taniguchi3, Hiroshi Sakaue3, Toshio Hosaka4, Emi Shuto1 and Tohru Sakai1*

Abstract
Background: Obesity is a major risk factor for insulin resistance, type 2 diabetes, and stroke. Flavonoids are effective
antioxidants that protect against these chronic diseases. In this study, we evaluated the effects of sudachitin, a
polymethoxylated flavonoid found in the skin of the Citrus sudachi fruit, on glucose, lipid, and energy metabolism
in mice with high-fat diet-induced obesity and db/db diabetic mice. In our current study, we show that sudachitin
improves metabolism and stimulates mitochondrial biogenesis, thereby increasing energy expenditure and reducing
weight gain.
Methods: C57BL/6 J mice fed a high-fat diet (40% fat) and db/db mice fed a normal diet were treated orally with
5 mg/kg sudachitin or vehicle for 12 weeks. Following treatment, oxygen expenditure was assessed using indirectcalorimetry, while glucose tolerance, insulin sensitivity, and indices of dyslipidemia were assessed by serum biochemistry.
Quantitative polymerase chain reaction was used to determine the effect of sudachitin on the transcription of key
metabolism-regulating genes in the skeletal muscle, liver, and white and brown adipose tissues. Primary myocytes
were also prepared to examine the signaling mechanisms targeted by sudachitin in vitro.
Results: Sudachitin improved dyslipidemia, as evidenced by reduction in triglyceride and free fatty acid levels, andimproved glucose tolerance and insulin resistance. It also enhanced energy expenditure and fatty acid β-oxidation byincreasing mitochondrial biogenesis and function. The in vitro assay results suggest that sudachitin increased Sirt1
and PGC-1α expression in the skeletal muscle.
Conclusions: Sudachitin may improve dyslipidemia and metabolic syndrome by improving energy metabolism.
Furthermore, it also induces mitochondrial biogenesis to protect against metabolic disorders.
Keywords: Sudachitin, Glucose metabolism, Lipid metabolism, Mitochondria

Background
The prevalence of metabolic syndrome is increasing
worldwide, and substantially increases the risk of metabolic and cardiovascular diseases [1]. Metabolic syndrome
is characterized by various metabolic abnormalities, including central obesity, hyperlipidemia, hyperglycemia,
and hypertension. These metabolic abnormalities may becaused by excess accumulation of visceral fat or abnormal* Correspondence: rtsutsumi@tokushima-u.ac.jp; sakai@tokushimau.ac.jp
1
Department of Public Health and Applied and Nutrition, Institute of Health
Bioscience, University of Tokushima, 3-18-15 Kuramoto, Tokushima 770-8503,
Japan
Full list of author information is available at the end of the article

accumulation of fat in non-adipose tissues, particularly
the liver and skeletal muscle [2]. A sedentary lifestyleand high-fat Westernized diets have been implicated in
the increasing prevalence of metabolic syndrome. Asidefrom lifestyle and diet, multiple metabolic pathways are
implicated in the pathogenesis of metabolic disease,including aberrant lipogenesis, increased inflammation,and reduced fatty acid oxidation [3]. Therefore, in
addition to identifying the underlying molecular mechanisms, and designing interventions to improve lifestyleand diet, it is also important to develop new medicines or
identify new phytochemicals, such as flavonoids, that can
improve these disorders.

© 2014 Tsutsumi et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain
Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,
unless otherwise stated.

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Flavonoids are expected to have beneficial effects on
metabolic activities, based on their hypocholesterolemic effects. Resveratrol is known to reduce fat accumulation and improve glucose tolerance and insulin
sensitivity in mice with high-fat diet-induced obesity[4]. In addition to resveratrol, several polymethoxylated flavonoids (PMFs), including tangeretin, nobiletin,
and naringenin, have been shown to exhibit metabolic
effects [5,6]. Tangeretin was reported to decrease diacylglycerol acyltransferase (DGAT) activity, microsomal
triglyceride accumulation, and apolipoprotein (apo) B100
secretion in HepG2 cells [6]. Naringenin was reported toinhibit lipogenesis and the secretion of very low-density
lipoprotein–apoB and improve serum lipid levels in amouse model of dyslipidemia and hyperinsulinemia [5,7].
Meanwhile, a mixture of tangeretin, nobiletin, and other
PMFs was found to improve dyslipidemia and glucose intolerance by regulating adipokine secretion in hamsters
with fructose-induced insulin resistance [8]. Nobiletin increased glucose transporter (GLUT)-4 expression in themuscle and decreased hyperglycemia in ob/ob mice [9].
Naringenin, resveratrol, and other polyphenols also suppressed arteriosclerosis via these metabolic effects in lowdensity lipoprotein receptor knockout and apolipoprotein
E knockout mice [10,11]. Consequently, PMFs and related
compounds were expected to have clinically useful antiarteriosclerotic properties.
Citrus sudachi hort. ex. Shirai (Rutaceae), also known
as sudachi, is an evergreen tree found primarily in theprefecture of Tokushima, Japan. Sudachi is a well-known
citrus fruit in Japan, with approximately 8000 tons offruit grown annually. Approximately half of the fruit issold to be eaten, while the rest is squeezed and processed into juice. The residue from the fruit processing
amounts to approximately 200 tons/year, and there is increasing demand to use it effectively. The flavonoids extracted from the peel of the sudachi fruit have beenshown to exhibit biological activities, including antioxidant,
antimicrobial, and anti-diabetic effects (unpublished data).
The peel can also reduce triglyceride levels and improveobesity or diabetes (data not published). However, it is
not known at this time which component of sudachi is
responsible for these effects, or which signaling pathway
is involved. In this study, we extracted the PMF sudachitin (5,7,4′-trihydroxy-6,8,3′-trimethoxyflavone) fromthe peel of Citrus sudachi and evaluated its anti-obesityand anti-diabetic effects in two distinct models of metabolic dysfunction. C57BL/6 mice fed a high-fat diet exhibit many features of metabolic syndrome including
obesity, hyperinsulinemia, hyperlipidemia, and glucose intolerance [12]. db/db mice, a model of type 2 diabetes, isbased on a leptin receptor mutation and shows hyperphagia, obesity, hyperglycemia, hyperinsulinemia, and hepatic
steatosis [13,14].

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Diabetic changes are generally considered to involve
alterations in the anatomy, endocrine responsiveness,and metabolic function of the white and brown adipose
tissues (WAT and BAT, respectively), liver and skeletal
muscle. In adipose tissue, insulin resistance is manifested
by reduced glucose uptake and increased free fatty acidrelease [15]. Adipokine dysfunction plays an importantrole in the development of obesity and insulin resistance[16,17]. The liver plays an important role in maintaining
glucose homeostasis and controlling lipid metabolism[18], while skeletal muscle is a key tissue involved in glucose uptake. Peroxisome proliferator-activated receptor(PPAR)γ induces adipocyte differentiation and regulates
the expression of several key genes involved in lipid andglucose metabolism [19]. Therefore, we examined theexpression of PPARγ and its target genes.
We hypothesized that sudachitin will exhibit significant anti-obesity and anti-diabetic effects, as it is a tridemethyl form (4′, 5, 7) of nobiletin, and we evaluated its
effect on metabolic parameters in animal models of obesityand diabetes. To elucidate the cellular mechanisms bywhich sudachitin exerts this effect, we evaluated the transcription of genes encoding regulators of glucose and lipid
synthesis, trafficking, and responsiveness.

Methods
Microwave-assisted extraction of Sudachitin

The green peels of Citrus sudachi Hort. ex Shirai werecollected in Tokushima Prefecture, Japan, in September.
The dried peels were ground in a grinder. Microwaveassisted extraction was performed using a micro-reactor(2.45 GHz, 700 W; Shikoku Instrumentation Co., Ltd.,
Kagawa, Japan). A mixture of dried ground sudachi peels(100 g) in MeOH (600 mL) and water (200 mL) was
stirred in a separable three-necked round-bottom flask(1 L) with a condenser, thermo-sensor, and mechanicalstirrer. The mixture was irradiated under microwaves(MW) for 12 min under reflux with stirring. The reaction mixture was extracted with ethyl acetate and separated into the ethyl acetate layer and water-soluble layer.
The ethyl acetate layer was dried with Na2SO4, and ethyl
acetate was subsequently removed under reduced pressure. The resulting dark green residue was chromatographed on silica gel column (elution with t-BuOH/
hexane/ethyl acetate = 0.5 : 5 : 1.2) to yield sudachitin as
yellow needles, which was recrystallized from ethyl acetate
to give sudachitin (0.092 g, melting point 238–240°C). A
mixture of the water-soluble layer (280 mL), MeOH
(280 mL), and concentrated HCl (240 mL) in the roundbottom flask was irradiated by MW for 7 min under reflux
with stirring. The reaction mixture was neutralized with
NaOH, and MeOH was removed under reduced pressure to give a brown residue, which was extracted with
ethyl acetate and dried (Na2SO4). After removal of ethyl

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acetate, the resulting brown residue was chromatographedon silica gel column (elution with t-BuOH/hexane/ethyl
acetate = 0.5 : 5 : 1.2) and further recrystallized from ethyl
acetate to give sudachitin (0.376 g, melting point 238–
240°C) as colorless needles. The purity of sudachitin was
confirmed by HPLC to be above 95%. Sudachitin was
dissolved in 0.2% dimethyl sulfoxide (DMSO).
Animals and experiments

All work involving animals was performed in compliancewith the Guide for the Care and Use of Laboratory
Animals and protocols approved by the Institutional
Animal Care and Use Committee at the University ofTokushima Graduate School (Tokushima, Japan).
C57BL/6 mice at 4 weeks of age were purchased fromJapan SLC, Inc. (Shizuoka, Japan). Mice were housed intemperature- (23 ± 3°C) and humidity-controlled conditions with a 12-h light/dark cycle. Mice were given freeaccess to water and either a control diet (n = 20; 14% ofcalories from fat; Oriental Yeast Co., Ltd., Tokyo, Japan)
or a high-fat diet (n = 20; 40% of calories from fat;
Oriental Yeast Co., Ltd.). Mice were allowed to adapt tothese conditions for 1 week before starting the experimental protocol. During the experimental period, themice were divided into two groups (n = 10) and orally
administered either 5 mg/kg sudachitin dissolved in a
0.2% sodium carbonate solution or a corresponding volume of 0.2% DMSO solution mixed with 0.2% sodium
carbonate daily for 12 weeks. Body weight and food intake of each mouse were measured weekly.
Ten db/db mice at 4 weeks of age were purchasedfrom Charles River Japan, Inc. (Kanagawa, Japan). Mice
were housed under temperature- (23 ± 3°C) and humiditycontrolled conditions with a 12-h light/dark cycle. Mice
were given free access to water and the control diet, as described above, and allowed to adapt to the conditions for
1 week before the beginning of the experimental protocol.
Mice were divided into two groups (n = 5) and orally administered 5 mg/kg sudachitin or 0.2% DMSO, as above,
daily for 12 weeks. The body weights and food intake ofmice were measured once a week.

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Plasma biochemical analyses

Plasma non-esterified fatty acid (NEFA), triglyceride,and total cholesterol levels were measured using enzymatic commercial assay kits (NEFA C-Test, Triglyceride E-Test, and Cholesterol E-Test, respectively; Wako
Pure Chemical Industries, Osaka, Japan). Plasma insulin,
adiponectin, and leptin levels were measured by enzymelinked immunosorbent assays (ELISA; Mouse Insulin
ELISA Kit, Mouse/Rat High Molecular Weight Adiponectin ELISA Kit, and Mouse Leptin ELISA Kit; Shibayagi,
Gunma, Japan).
X-ray CT scan

Total body fat, subcutaneous fat, and visceral fat were
measured in mice under isoflurane anesthesia using
LaTheta X-ray CT scanner LCT-200 (Hitachi-Aloka
Medical, Ltd., Tokyo, Japan). Data was analyzed using
Visualization LaTheta software (Hitachi-Aloka Medical
Ltd., Tokyo, Japan).
Blood glucose, oral glucose tolerance tests (OGTT), andinsulin tolerance tests (ITT)

Blood glucose levels were measured in tail vein bloodusing a glucometer (Arklay, Kyoto, Japan). For OGTT,
blood glucose levels were measured 0, 30, 60, 90, and
120 min after an oral glucose load (1 g/kg) following anovernight fast. For ITT, blood glucose levels were measured 0, 30, 60, and 90 min after an intraperitoneal injection of regular human insulin (Novo Nordisk PharmaInc., Tokyo, Japan). Insulin was administered to C57BL/
6 J mice at 0.75 mU/g body weight and 3 mU/g to db/db
mice, following a 6-h fast.
Total RNA isolation

Total RNA was extracted from WAT and the gastrocnemius muscle using RNeasy Lipid Tissue and RNeasy
Plus Universal Mini Kits (QIAGEN, Valencia, CA, USA)according to manufacturer’s instructions. Total RNA
was extracted from skeletal muscle myocytes using
RNAiso and amplified using CellAmp Whole Transcriptome Amplification Kit Version 2 (Takara Bio Inc.,
Shiga, Japan).

Plasma and tissue collection

Before, during, and at the end of the experimental
period, blood samples were taken from the tails of themice fasted overnight for biochemical analyses. At theend of the study, mice were sacrificed by cervical dislocation after blood collection. Plasma samples were prepared by centrifugation at 3,000 rpm for 10 min at 4°C,
and were stored at −80°C until analysis. Samples of theliver, gastrocnemius muscle, epididymal (visceral) andinguinal (subcutaneous) WAT, and BAT were collected,
rinsed, weighed, frozen in liquid nitrogen, and storedat −80°C until analysis.

Gene expression analyses

Total RNA (1 μg) was reverse-transcribed to cDNA in a
final volume of 20 μL using the Primescript RT Reagent
kit (Takara). Real-time polymerase chain reaction (PCR)
was performed in a final volume of 10 μL containing
50 ng of the cDNA template and primers, using a
StepOnePlus Real-Time PCR System (Life Technologies,
Carlsbad, CA, USA). The primers used for PCR are listed
in Additional file 1: Table S1. We evaluated the RNAlevels of glucose transporters (Glut 1–4), adiponectin,
PPARγ, adipocyte protein 2 (AP2), cluster of differentiation

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36 (CD36, fatty acid translocase), uncoupling protein 1, 2,
and 3 (UCP1, UCP2, and UCP3), and Sirt1 in tissue samples. In cultured skeletal muscle myocytes, we measured
the transcription of genes involved in the synthesis offatty acids (fatty acid synthase (FAS), acetyl-coA carboxylase (ACC1)), triglycerides (diglyceride acetyltransferase (DGAT1 and DGAT2)), or both (SREBP-1), as
well as genes associated with hepatic cholesterol synthesis (farnesyl diphosphate synthase (FDS), squalene
synthase (SS), 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-R), and synthase (HMG-S)), cholesterol sensitivity (microsomal triglyceride transfer protein (MTTP),
low-density lipoprotein receptor (LDLR)), lipid oxidation(UCP2, acyl-coenzyme A oxidase (ACOX), PPARγ coactivator 1α (PGC1α)), lipolysis (hormone-sensitive lipase(HSL), carnitine palmitoyl transferase-α (CPT1α), adipose triglyceride lipase (ATGL)), and glycogenolysis(glucose 6-phosphatase (G6Pase)), phosphoenolypyruvatecarboxykinase (PEPCK)). Evaluation of mitochondrial biogenesis in primary myocytes involved measurement of
nuclear respiratory factor 1 and 2 (NRF1 and NRF2) andmitochondrial transcription factor A (mtTFA).

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Santa Cruz Biotechnology (Santa Cruz, CA, USA). Protein
density were visualized on membranes using horseradish
peroxidase (HRP)-conjugated secondary antibodies (Santa
Cruz Biotechnology) and visualized using the enhanced
chemiluminescence reagent (GE Healthcare; Waukesha,
WI, USA), according to manufacturer’s directions.
Indirect calorimetry

Four-week-old male C57BL/6 J mice were fed high-fat
diet and treated with sudachitin (5 mg/kg) or vehicle for
4 weeks. Oxygen consumption was continuously measured
during the 12-h light–dark cycles using a comprehensive
laboratory animal open-circuit indirect calorimetry monitoring system (Columbus Instruments, Columbus, OH,
USA). Data were corrected for 2 days after 2 days of adaptation to the metabolic cages.
Skeletal muscle enzyme activities

Skeletal muscle samples were homogenized with extraction buffer (0.1 mol/L KH2PO4, 0.1 mol/L NaPHO4, and2 mmol/L EDTA, pH 7.2). Citrate synthase activity wasdetermined using a Citrate Synthase Assay Kit (SigmaAldrich, St. Louis, MO, USA).

Measurement of adipocyte size

Subcutaneous and visceral adipose tissue samples were
fixed in 2% osmium tetroxide and shaken at 37°C for
7 days. The fixed tissues were filtered through a 250-μm
and 25-μm mesh to obtain a single cell suspension. The
diameter and distribution of adipocytes were measured
using a Coulter Multisizer 3 particle counter (Beckman
Coulter, Inc., Brea, CA, USA).
Analysis of visceral adiposity

The volume and distribution of visceral fat at 4 weeks
after starting sudachitin administration were evaluatedby X-ray computed tomography (CT; LaTheta, LCT-200,
Aloka, Tokyo, Japan) of the region between the firstlumbar vertebra and the pubic bone, under isoflurane
anesthesia. Data were analyzed from continuous 2-mm
slice images (for quantitative assessment) using LaTheta
software.
Immunoblotting

Total protein was extracted as described previously [20].
Lysates were separated by SDS-PAGE on 10% polyacrylamide precast gels (Invitrogen, Carlsbad, CA, USA) andtransferred to polyvinylidene difluoride membranes byelectroelution. Membranes were blocked in 20 mmol/Lof TBS solution with 1% Tween containing 5% skim
milk and incubated with primary antibodies overnightat 4°C. Polyclonal antibody sensitive to AMPKα1 andphospho-AMPKα1 (Thr172) was obtained from Cell
Signaling Technology (Danvers, MA, USA) and antibodies against GLUT4 and β-tubulin were obtained fromATP content measurement

ATP content was measured using the AMERIC-ATPkit (Applied Medical Enzyme Research Institute Corporation, Tokushima. Japan) according to manufacturer’s instructions. Briefly, a sample of skeletal muscle(100 mg) tissue was homogenized in 10× volume of extraction buffer (0.25 mol/L sucrose, 10 mmol/L HEPESNaOH, pH 7.4) and centrifuged at 1000 g for 10 min.
The supernatant was diluted 8-fold in phenol-based
extraction buffer and ATP extraction reagent was added.
Following addition of luciferase solution, ATP content
was quantified by measuring the luminescence using aluminometer (Lumat LB9507, Berthold Technologies,
Tokyo, Japan).
Cell culture

Primary myoblasts were isolated from the calf region,
thigh, and pelvic girdle of 8-week-old C57BL/6 J mice.
Satellite cells were isolated by the digestion of muscle
tissue by the type 2 collagenase (Worthington Biochemicals, Lakewood, NJ, USA), followed by pre-plating
to purify cells and remove fibroblasts. Experiments wereconducted after myoblasts had reached 99% purity. Cells
were cultured at 37°C in 5% CO2 in Dulbecco modified
Eagle medium (DMEM), supplanted with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Cells
were differentiated in DMEM/20% horse serum supplanted with basic fibroblast growth factor for 3 days.
The primary cells were treated with vehicle (0.2% DMSO)
or 30 mmol/L sudachitin for 48 h.

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Figure 1 Sudachitin inhibits weight gain in high-fat diet-fed mice. Mice were fed a low-fat control diet or a high-fat diet and weretreated with 5 mg/kg sudachitin or vehicle for 12 weeks (n = 10 per group). Mice were fasted for 16 h at the end of the study. Body weight
was measured weekly (A) and food intake was measured every 2 days (B) for 12 weeks. Values are mean ± standard error of the mean (A) or
mean ± standard deviation (B). *P < 0.05 sudachitin administration vs. control treatment, # P < 0.05 high-fat diet-fed vs. control diet-fed animals.
Cont + Veh: vehicle-treated, control diet group (closed squares in panel A); Cont + Sud: sudachitin-treated, control diet group (open squares);
HFD + Veh: vehicle-treated, high-fat diet group (closed circles); HFD + Sud: sudachitin-treated, high-fat diet group (open circles).

Mitochondrial staining

Skeletal muscle myocytes were grown and differentiated on a glass coverslip coated with Cell-Tak (BDBioscience, Franklin Lakes, NJ, USA). The myocytes
were stained with Mito-tracker dye (Molecular Probes,

Invitrogen, Carlsbad, CA, USA) for 30 min and mitochondrial density was determined using optical microscopy(Eclipse TE 2000-U; Nikon, Tokyo, Japan). The density of
mitochondria was quantified using Image J software (NIH,
Bethesda, MD, USA).

Figure 2 Sudachitin reduces fat in high-fat diet-fed mice. (A) Body composition was assessed by computed tomography, with the relative
body fat content, subcutaneous fat content (g/body weight; B) and visceral fat content (g/body weight; C) quantified. Adipocyte size was measured
after 12 weeks of treatment with sudachitin or vehicle (D). Hematoxylin/eosin staining of WAT after 12 weeks of feeding in vehicle- (upper panel) andsudachitin-treated (lower panel) mice (E). Values are mean ± standard deviation (A-C). *P < 0.05 sudachitin administration vs. control treatment, # P < 0.05
high fat diet vs control diet. Cont + Veh: vehicle-treated, control diet group (closed squares in panel A); Cont + Sud: sudachitin-treated, control diet group(open squares); HFD + Veh: vehicle-treated, high-fat diet group (closed circles); HFD + Sud: sudachitin-treated, high-fat diet group (open circles).

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Statistical analysis

Data were expressed as means ± standard deviation (SD).
Unpaired Student’s t test was used to statistically compare vehicle- and sudachitin-treated groups. Statisticalsignificance was set a priori at P < 0.05. All analyses were
performed using GraphPad Prism version 5.0 (GraphPad
Software, San Diego, CA, USA).

Results
Sudachitin treatment prevents high-fat diet-induced weight
gain, and reduces fat content and adipocyte size

As shown in Figure 1A and B, treatment with 5 mg/kg
body weight sudachitin reduced the weight gain in mice
fed a high-fat diet, without affecting the food intake. By
contrast, treatment with sudachitin did not affect the
body weight in mice fed a low-fat control diet. Treatment of mice fed a high-fat diet with 2 mg/kg sudachitin
did not affect the body weight of the animals (data notshown). The magnitude of weight reduction was similar
between 5 and 10 mg/kg doses of sudachitin (data notshown), which prompted us to use the 5 mg/kg dose forsubsequent investigations.

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A high-fat diet resulted in significantly elevated total
body adipose tissue, increased subcutaneous fat deposits
and elevated visceral fat (enlarged epididymal fat pads)
which were ameliorated by 5 mg/kg sudachitin administration (Figure 2A–C). However, there were no differences in BAT or muscle weight per body weight
among the four groups (data not shown). Sudachitin
treatment also significantly reduced adipocyte size inhigh-fat diet-fed mice, as compared to the vehicletreated mice fed high-fat diet (Figure 2D and E). Since
these changes were independent of food intake andmuscle mass, the results suggest that sudachitin improves energy metabolism.
We next examined the effects of sudachitin on metabolic parameters. As expected, a high-fat diet significantly increased serum triglyceride and NEFA levels by
1.5- and 1.3-fold, respectively, compared with the control diet (Figure 3A and B). Administration of 5 mg/kg
sudachitin prevented these increases in triglycerideand NEFA levels. Total cholesterol levels were notsignificantly different among any of the studied groups(Figure 3C).

Figure 3 Sudachitin reduces serum triglyceride and non-esterified fatty acid. Serum triglyceride (A), non-esterified fatty acid (B), andtotal cholesterol (C levels were evaluated at the end of the 12-week treatment. Values are means ± standard deviation (A-C). *P < 0.05
sudachitin administration vs. control treatment, # P < 0.05 high-fat diet-fed vs control diet-fed animals. Cont + Veh: vehicle-treated, control
diet group (closed squares in panel A); Cont + Sud: sudachitin-treated, control diet group (open squares); HFD + Veh: vehicle-treated, high-fat
diet group (closed circles); HFD + Sud: sudachitin-treated, high-fat diet group (open circles); NEFA: non-esterified fatty acid; T-CHO:
total cholesterol.

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Sudachitin improves glucose tolerance and insulin
sensitivity in high-fat diet-fed mice

As shown in Figure 4A and B, mice fed a high-fat dietexhibited a state of moderate hyperinsulinemia andhyperglycemia, as compared to mice fed the control diet.
Fasting plasma glucose and insulin levels were significantly reduced by sudachitin. Results of the OGTTs revealed that sudachitin improved glucose tolerance inhigh-fat diet-fed mice (Figure 4C). Sudachitin treatment
also re-established normal insulin tolerance in high-fat

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diet-fed mice, normalizing the area under the glucose
plasma concentration curve in these mice (Figure 4D).
Mice fed a high-fat diet also exhibited elevated plasma leptin levels, which were ameliorated by sudachitin (Figure 4E),
consistent with the observed sudachitin-induced decreasein visceral fat (Figure 2). Plasma adiponectin levels were
found to decrease with age and to be significantly lower inmice fed a high-fat diet (Figure 4F). Sudachitin treatment
increased plasma adiponectin levels in mice fed a high-fat
diet but not in mice fed a control diet.

Figure 4 Sudachitin improves glucose and insulin sensitivity in mice fed a high-fat diet. Fasting blood glucose (A) and plasma insulin
concentrations (B) after 12 weeks of sudachitin treatment. Glucose (C) and insulin (D) tolerance tests were performed in high-fat diet-fed mice
after an overnight (16 h) (C) or 6 h (D) fast. Mice received an oral dose of 1 g/kg glucose (C) or 0.75 mU/kg insulin by intraperitoneal injection(D). Blood glucose was measured at the indicated times. Glucose utilization and insulin sensitivity were determined from the area under the curve(AUC; inset). Open symbols: sudachitin-treated group; closed symbols: vehicle-treated groups. Plasma leptin levels were measured after 12 weeks
of treatment (E). Plasma adiponectin levels were measured after 5, 8, and 12 weeks of starting treatment (F). Values are means ± standard
deviation. Closed bars: vehicle-treated, control diet group; dark gray bars: sudachitin-treated, control diet group; light gray bars: vehicle-treated,
high-fat diet group; open bars: sudachitin-treated, high-fat diet group; *P < 0.05 sudachitin administration vs. control treatment, # P < 0.05 high fat
diet vs control diet-fed animals.

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Sudachitin alters the expression of metabolism-related
genes in WAT and liver

As shown in Figure 5A, GLUT4 mRNA levels were significantly increased by the sudachitin treatment, suggesting a
possible molecular mechanism underlying sudachitinmediated improvement in glucose uptake. Transcriptionof the adiponectin gene in high-fat diet-fed mice treatedwith sudachitin showed a trend toward an increase that
did not reach statistical significance, compared to theobservation in vehicle-treated high-fat diet-fed mice.
The mRNA level of PPARγ showed a trend towards anincrease (1.5-fold) which was not statistically significant in the sudachitin-treated group compared with thevehicle-treated group. No significant differences in mRNAtranscripts of adipocyte fatty acid-binding protein andCD36 were observed between the two treatment groups(Figure 5A). mRNA transcripts of uncoupling protein 1
and 3 (UCP1 and UCP3) were significantly increased insudachitin-treated mice, which suggests that WAT gained
a BAT-like phenotype, possibly resulting in increased
thermogenesis.
In the liver, we found that sudachitin decreased thelevels of mRNA transcripts encoding FAS, ACC1 andPage 8 of 14

ACC2, while the expression levels of DGAT1, DGAT2,and SREBP1, were not significantly different between
vehicle- and sudachitin-treated mice fed a high-fat diet
(Figure 5C). In addition, the expression levels of FDS,
SS, HMG-R, and HMG-S were not affected by sudachitin (Figure 5C). The expression levels of MTTP and
LDLR were not significantly different, although MTTP
expression tended to be lower in the sudachitin-treatedmice fed a high-fat diet as compared to the untreatedhigh-fat diet-fed mice, although not significantly. There
were no differences in the transcription of UCP2, ACOX,or PGC-1α between mice treated with sudachitin or vehicle (Figure 5C). However, transcription of the lipolytic
gene HSL was increased in sudachitin-treated mice fed ahigh-fat diet, as was CPT1α, but there was no change in
ATL (Figure 5C). The hepatic transcription of G6Pase and
PEPCK, PPARγ, and adiponectin were not increased bysudachitin.
Sudachitin improved insulin sensitivity in diabetic
db/db mice

As shown in Figure 6A, body weight gain over the first
6 weeks of treatment in db/db mice was not significantly

Figure 5 Effects of sudachitin on mRNA levels in white adipose tissue and liver. Gene transcription was normalized for 36B4 in subcutaneous
white adipose tissue (A-B) or 18S in liver (C). Data are means ± standard deviation. *P < 0.05 vs the indicated groups. Closed bars: vehicle-treated,
high-fat diet group; open bars: sudachitin-treated, high-fat diet group.

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Figure 6 Sudachitin improves fasting glucose, insulin sensitivity, and lipid metabolism in db/db mice. While sudachitin treatment did
not affect weight gain over 9 weeks of treatment (A), it significantly decreased fasting blood glucose levels (B). Insulin tolerance tests were
performed after a 6 h fast (C). Mice received an intraperitoneal injection of 3 mU/kg insulin, and blood glucose levels were measured at indicatedtimes. Serum triglyceride (D), non-esterified fatty acids (E), and total cholesterol levels (F) were measured in db/db mice. Data are presented asmeans ± standard deviation. *P < 0.05 vs the indicated groups. closed circles: vehicle-treated group, open circles: sudachitin-treated group.

suppressed by treatment with 5 mg/kg sudachitin compared with vehicle-treated mice. All db/db mice were
hyperglycemic when the experiment began, as confirmed by the average fasting blood glucose levels of
544 ± 96.0 mg/dL. Six weeks of treatment with sudachitin reduced fasting blood glucose levels comparedwith the untreated db/db mice (Figure 6B). Sudachitin
also improved insulin sensitivity, as determined by the
ITTs performed at this time (Figure 6C). As shown in
Figure 6D–F, sudachitin significantly decreased serum
triglyceride and NEFA, levels, but did not alter the totalcholesterol levels.
Sudachitin promotes energy expenditure by activating
the Sirt1–PGC1α pathway

Because sudachitin increased adiponectin levels and reduced adiposity without reducing food intake, we hypothesized that sudachitin increases the energy expenditure. To
assess whole-body energy expenditure, mice fed a high-fat

diet were treated with sudachitin or vehicle and subjected to indirect calorimetry. Oxygen consumption
during the light and dark cycles was increased by theadministration of sudachitin for 4 weeks, without weightchange compared with control mice, resulting in an increase in total daily energy expenditure by 45% comparedwith vehicle-treated mice (Figure 7A and B). We observed similar increases in energy expenditure in db/db
mice after 4 weeks of administration of 5 mg/kg per dayof sudachitin (Figure 7C-D).
Interestingly, in mice fed a high-fat diet, the respiratory
quotient was not markedly different between mice treated
without or with sudachitin (0.87 ± 0.04 and 0.83 ± 0.03, respectively). Because energy expenditure can be increased
by mitochondrial uncoupling, we examined the expressionof UCP1–3 in the gastrocnemius muscle and in BAT(Figure 8A). There were no significant differences in
UCP3 expression levels in any of the tissues, although its
expression was slightly increased in the skeletal muscle

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Figure 7 Sudachitin increases energy expenditure. Ten-week-old mice treated with vehicle or sudachitin (5 mg/kg) for 4 weeks were placed
into an Oxymax open-circuit indirect calorimetry system for 4 days. Measurements of O2 consumption in control C57/Bl6 mice (A) or db/db mice(C), and energy expenditure were obtained in control C57/Bl6 mice (B) or db/db mice (D).

Sudachitin promotes mitochondrial biogenesis byactivating its signaling pathway in myocytes in vitro

expression was slightly increased in myocytes treatedwith sudachitin for 48 h, whereas UCP1 was not different
between control and sudachitin-treated myocytes. Because GLUT4 expression was increased in sudachitintreated mice, we also determined the expressions of
GLUT1, 3, and 4. As shown in Figure 9A, only the insulin
insensitive transporters GLUT1 and 3 were significantly
increased by sudachitin, whereas GLUT4 expression was
not affected. To examine the effects of sudachitin on
mitochondrial number and activity, we stained the mitochondria of myocytes and present representative images
in Figure 9B and C, with stained mitochondria indicatedwith arrows. Sudachitin treatment increased mitochondrial staining with the fluorophore Mito-Tracker Green
in skeletal muscle myocytes approximately 2.5-fold, incomparison to the cells treated with vehicle.

To determine the direct effects of sudachitin on muscle
metabolism, we assessed the expression of key genes andmitochondrial number in vitro in primary cultured myocytes following incubation with sudachitin. Consistent
with the observed increase in PGC-1α and Sirt1 mRNAin mice (Figure 8), transcription of these genes was significantly increased in differentiated myocytes exposed
to 30 μmol/L sudachitin compared with the vehicletreated cells (Figure 9A). Considering the genes involved
in mitochondrial biogenesis, the expression of NRF1,
NRF2, and mtTFA were increased by 1.5- to 2.5-foldfollowing sudachitin treatment. In addition, the expression of UCP2 was significantly increased and UCP3

Discussion
The present study revealed that sudachitin, a PMF extracted from the peel of the Citrus sudachi fruit, exhibits
positive effects on glucose and energy metabolism in
two animal models of metabolic disorders. Previous
studies have shown that dietary supplementation witha mixture of PMFs nobiletin and tangeretin restores
glucose homeostasis in hamsters with fructose-induced
insulin resistance [21]. Several reports have demonstrated
that citrus flavonoids, including hesperidin, naringin, andnobiletin have hypoglycemic effects [9,22]. In the present
study, we found that plasma glucose and triglycerideof sudachitin-treated mice. By contrast, UCP2 gene expression was significantly increased in skeletal muscle ofsudachitin-treated mice. Sudachitin also induced the expression of PGC-1α and its target gene Sirt1 in skeletal
muscle, which are known to regulate energy metabolism(Figure 8B). As shown in Figures 8C and D, there was astatistically insignificant trend toward increased phosphorylation of AMP protein kinase (AMPK) and Glut4
expression in response to sudachitin treatment. We
also observed a 1.3-fold increase in basal ATP content(Figure 8E), as well as increased citrate synthase activity (Figure 8F) in sudachitin-treated mice, consistent
with enhanced mitochondrial content or activity.

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Figure 8 Sudachitin increases energy metabolism-related gene expressions by activating mitochondrial biogenesis. Relative mRNA
expression of energy metabolism-related genes in the gastrocnemius muscle (A, B) and brown adipose tissue (A). Gene expression was normalized
for GAPDH in both tissues. Phosphorylation of AMPK in sudachitin- or vehicle-treated mice with or without insulin (C). Protein expression of GLUT4
in mice administrated vehicle or sudachitin (D). Skeletal muscle ATP content in mice fed a high-fat diet and treated with sudachitin or vehicle (E).
Citrate synthase activity was measured in mitochondria isolated from high-fat diet-fed mice treated with 5 mg/kg sudachitin or vehicle (F). Data arepresented as mean ± standard deviation. *P < 0.05 vs. the indicated group. Closed bars; vehicle-treated group; open bars: sudachitin-treated group.

levels in C57BL/6 J mice fed a high-fat diet were significantly reduced by treatment with 5 mg/kg sudachitin
within 5 weeks of starting treatment. However, these reductions were not observed in mice treated with 2 mg/kg
body weight sudachitin. Because the reductions in plasma
glucose and triglyceride levels were similar between micetreated with 5 and 10 mg/kg body weight sudachitin, weused the 5 mg/kg dose in this study. This dose seems very
low compared with that required for other flavonoids orother treatments. In experiments using pharmacologicalagents, it is important to use the lowest effective doseto minimize the chances of off target/side effects. We

propose that the observed reduction in weight gain observed in sudachitin-treated mice is a result of alteredglucose and lipid metabolism, caused by the changes inenergy expenditure. This mechanism is supported by thesudachitin-mediated enhanced glucose metabolism observed in db/db mice, even without any weight loss.
How does sudachitin act on skeletal muscle in mice?
So far, the mechanisms involved in the lipid-lowering effects of citrus flavonoids have not yet been fully established. In general, flavonoids are conjugated in blood,
although it is possible that effects in certain tissues are
mediated following local deconjugation. Nevertheless, weTsutsumi et al. Nutrition & Metabolism 2014, 11:32
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Figure 9 Effects of sudachitin on gene expression in mouse primary skeletal muscle myocytes in vitro. Gene transcription in myocytes
treated with 30 μmol/L sudachitin or vehicle for 48 h, normalized for β-actin (A). Data are means ± standard deviation. *P < 0.05 vs the indicatedgroup. Closed bars: untreated cells; open bars: sudachitin-treated cells. The number of mitochondria in myocytes was increased by sudachitin(B, C). Myocytes were incubated with the fluorophore Mito-Tracker Green, which specifically labels mitochondria. Upper panel: control cells; lower
panel: cells treated with 30 μmol/L sudachitin for 48 h. Arrows indicate stained mitochondria (B). Quantitative analysis of mitochondrial staining.
Fluorescence intensity was measured by ImageJ software using the analyze particle function. *P < 0.05.

found that mice treated with sudachitin showed increased skeletal muscle mitochondrial biogenesis andfunction, and possibly increased NAD+ levels in skeletal
muscle. Genetic disorders associated with impaired mitochondrial function are characterized by a rapid onset ofsymptoms that are commonly observed in elderly individuals, including clinical symptoms of type 2 diabetes[23]. There is increasing evidence that a progressive decline in mitochondrial function in asymptomatic, apparently healthy individuals may underlie a number oflate-onset age-related diseases [24,25], and that treatments that stimulate mitochondrial function can delay the
progression or even prevent the manifestation of some ofthese diseases [4,26,27]. Skeletal muscle is one of the primary tissues responsible for insulin-stimulated glucose
uptake, and reduced mitochondrial function plays an important role in the development of insulin resistancecoupled with obesity [23,28]. Therefore, activating upstream pathways that regulate mitochondrial biogenesis
may prove effective in delaying and treating a variety of
metabolic diseases.
PGC-1α is a transcriptional coactivator that regulates
genes involved in energy metabolism and provides a direct link between external physiologic circuits and theregulation of mitochondrial biogenesis [29]. PGC-1α isexpressed at high levels in tissues characterized by abundant mitochondria and active oxidative metabolism,
such as BAT, the heart, and skeletal muscle, whereas its
expression is much lower in the liver and WAT [30]. In
this study, we found an increase in PGC-1α transcription
in skeletal muscle, but not in BAT, following sudachitin
treatment, suggesting that sudachitin, or its metabolite,
act directly on the skeletal muscle. PGC-1α induces thetranscription of NRF1 and NRF2, which in turn stimulate

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the expression of mtTFA. Importantly, PGC-1α has beenshown to affect glucose metabolism. Under fed conditions, skeletal muscle is a major site of glucose disposal,
taking up glucose from the plasma via GLUTs located in
the cell membrane. In our study, the skeletal muscle
tissue of sudachitin-treated mice showed increased transcription of GLUT4, a constitutively expressed gene encoding for an insulin-sensitive transporter protein. On
the other hand, in vitro investigation in cultured myocytes has detected increased expression of GLUT1 andGLUT3, but not GLUT4, following incubation with sudachitin. One explanation for this apparent discrepancy
might be that increased GLUT4 expression was secondary to the improvement in glucose metabolism caused by
the decrease in weight gain in vivo (Figures 1 and 2),
while sudachitin directly influences the expression of
insulin-insensitive GLUTs in myocytes in vitro.
Interestingly, skeletal muscle-specific overexpressionof PGC-1α was shown to be sufficient to delay many
age-related disorders and extend the lifespan of mice[31]. The effects of sudachitin treatment seem to mimic
those of caloric restriction and are similar to the reported effects of resveratrol. However, UCP1 and UCP3
expression were significantly increased in sudachitintreated mice. Based on recent publications, it is possiblethat WAT was transformed into BAT-like tissue via
PGC-1α activity [32], increasing tissue thermogenesis[30]. Clearly, further studies are required to fully understand the effect of sudachitin treatment and to elucidate
the underlying mechanisms in these animal models.
The results of our current study suggest that sudachitin treatment increased serum adiponectin levels and increased Sirt1 expression, which activates PGC-1α in theskeletal muscle. Several reports suggest that AMPK and
Sirt1 act together with PGC-1α, a key regulator of mitochondrial biogenesis, to regulate energy homeostasis
in response to environmental and nutritional stimuli[33,34]. Rasbach et al. reported that overexpression of
PGC-1α caused a shift toward increased oxidative typesof skeletal muscle fibers [35]. Our in vitro data confirm
that treatment with a moderate (30 μmol/L) dose of
sudachitin induces mitochondrial biogenesis, and improves mitochondrial function. This dose also increased
Sirt1 expression, suggesting that AMPK activation might
be dependent upon Sirt1. Recently, Price et al. reported
that moderate doses of resveratrol activate Sirt1, leading
to a deacetylation of liver kinase B1 and AMPK activation, and that Sirt1 and AMPK do not function independently or linearly [36]. The dose of sudachitin used in
our in vivo study was within physiologic levels because itis equivalent to the consumption of the peels of 1.5 small
sudachi fruit per day in humans.
In this study, we examined the Sirt1-dependent activityof sudachitin in skeletal muscle, a tissue with very highPage 13 of 14

energy demands that require highly efficient mitochondrial functioning. As a limitation to the presented work,
it remains unclear whether Sirt1-independent effects observed here result from an accumulation of sudachitinor its metabolites in specific organs, or differences in theactions of sudachitin between tissues and cell types. It isalso unclear how flavonoids, such as sudachitin, modulate Sirt1–PGC1α signaling and regulate specific transcription factors or whether these effects are mediatedindirectly through a separate signaling pathway. Further
studies are needed to gain more insight into the effectsof sudachitin and its mechanisms in other tissues, particularly the liver and brain.

Conclusions
In summary, our study is the first to demonstrate thatsudachitin improves glucose and lipid metabolism by enhancing energy metabolism and mitochondrial biogenesis.
Additionally, our in vivo and in vitro findings suggest thatthese effects are mediated by the Sirt1–AMPK–PGC-1α
pathway. These observations indicate that sudachitin has
potential for use in treating metabolic and age-relateddiseases.
Additional file
Additional file 1: Table S1. Supplemental table Primer sequences.

Competing interests
All authors (Rie Tsutsumi, Tomomi Yoshida, Yoshitaka Nii, Naoki Okahisa,
Shinya Iwata, Masao Tsukayama, Rei Hashimoto, Yasuko Taniguchi, Hiroshi
Sakaue, Toshio Hosaka, Emi Shuto, Tohru Sakai) have no conflicts of interest.
Authors’ contribution
RT designed research, conducted the research, wrote the manuscript, andhas responsibility for the final content; TY, RH and YT conducted research;
HS, TH, ES and TS planed research and edited manuscript. YN, NO, SI and MT
provided essential reagents and analyzed data. All authors have read andapproved the final manuscript.
Acknowledgments
We thank Dr. Katsuya Hirasaka and Dr. Rie Mukai for helpful advice andtechnical support. We also thank Hiromi Sato, Yui Hamasaki, Tomomi Maeno
and Sayaka Sasaga for their technical support.
Funding
Rie Tsutsumi: Funding was provided by the Japan Society for the Promotionof Sciences, Tokyo JSPS KAKENHI 24650489. Tohru Sakai: Funding wasprovided by Grants for the Regional Innovation Cluster.
Author details
1
Department of Public Health and Applied and Nutrition, Institute of Health
Bioscience, University of Tokushima, 3-18-15 Kuramoto, Tokushima 770-8503,
Japan. 2Tokushima Prefectural Industrial Technology Center, Tokushima,
Japan. 3Department of Metabolism and Nutrition, Institute of Health
Bioscience, University of Tokushima, Tokushima, Japan. 4Department ofEndocrinology and Diabetes, Department of General Internal Medicine,
School of Medicine, Saitama Medical University, Saitama, Japan.
Received: 18 April 2014 Accepted: 20 June 2014
Published: 4 July 2014

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doi:10.1186/1743-7075-11-32
Cite this article as: Tsutsumi et al.: Sudachitin, a polymethoxylated
flavone, improves glucose and lipid metabolism by increasing
mitochondrial biogenesis in skeletal muscle. Nutrition & Metabolism
2014 11:32.