Sudachitin, a polymethoxylated flavone, improves
glucose and lipid metabolism by increasing
mitochondrial biogenesis in skeletal muscle
Rie Tsutsumi1**
Corresponding author
Email: rtsutsumi@tokushima-u.ac.jp
Tomomi Yoshida1
Email: tomomi.yoshida78@gmail.com
Yoshitaka Nii2
Email: nii@itc.pref.tokushima.jp
Naoki Okahisa2
Email: okahisa@itc.pref.tokushima.jp
Shinya Iwata2
Email: shinya@itc.pref.tokushima.jp
Masao Tsukayama2
Email: tukayama@mc.pikara.ne.jp
Rei Hashimoto1
Email: r2009020286@yahoo.co.jp
Yasuko Taniguchi3
Email: yasukotaniguchi@nutr.med.tokushima-u.ac.jp
Hiroshi Sakaue3
Email: hsakaue@nutr.med.tokushima-u.ac.jp
Toshio Hosaka4
Email: toshio.hosaka@gmail.com
Emi Shuto1
Email: shuto@nutr.med.tokushima-u.ac.jp
Tohru Sakai1
Email: tohrus@nutr.med.tokushima-u.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
2

Tokushima Prefectural Industrial Technology Center, Tokushima, Japan

3

Department of Metabolism and Nutrition, Institute of Health Bioscience,
University of Tokushima, Tokushima, Japan
4

Department of Endocrinology and Diabetes, Department of General Internal
Medicine, School of Medicine, Saitama Medical University, Saitama, Japan

Abstract
BackgroundObesity 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, weevaluated 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 dietinduced 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 treatedorally with 5 mg/kg sudachitin or vehicle for 12 weeks. Following treatment, oxygen
expenditure was assessed using indirect calorimetry, 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 transcriptionof 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, and improved glucose tolerance and insulin resistance. It also enhanced energy
expenditure and fatty acid β-oxidation by increasing 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 byvarious metabolic abnormalities, including central obesity, hyperlipidemia, hyperglycemia,
and hypertension. These metabolic abnormalities may be caused by excess accumulation ofvisceral fat or abnormal accumulation of fat in non-adipose tissues, particularly the liver andskeletal muscle [2]. A sedentary lifestyle and high-fat Westernized diets have been implicatedin the increasing prevalence of metabolic syndrome. Aside from 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 lifestyle and diet, it is also important to develop new medicines or identify new
phytochemicals, such as flavonoids, that can improve these disorders.
Flavonoids are expected to have beneficial effects on metabolic activities, based on their
hypocholesterolemic effects. Resveratrol is known to reduce fat accumulation and improveglucose 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 wasreported to decrease diacylglycerol acyltransferase (DGAT) activity, microsomal triglyceride
accumulation, and apolipoprotein (apo) B100 secretion in HepG2 cells [6]. Naringenin wasreported to inhibit lipogenesis and the secretion of very low-density lipoprotein–apoB and
improve serum lipid levels in a mouse model of dyslipidemia and hyperinsulinemia [5,7].
Meanwhile, a mixture of tangeretin, nobiletin, and other PMFs was found to improvedyslipidemia and glucose intolerance by regulating adipokine secretion in hamsters withfructose-induced insulin resistance [8]. Nobiletin increased glucose transporter (GLUT)-4
expression in the muscle and decreased hyperglycemia in ob/ob mice [9]. Naringenin,
resveratrol, and other polyphenols also suppressed arteriosclerosis via these metabolic effects
in low-density 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 foundprimarily in the prefecture of Tokushima, Japan. Sudachi is a well-known citrus fruit inJapan, with approximately 8000 tons of fruit grown annually. Approximately half of the fruit
is sold to be eaten, while the rest is squeezed and processed into juice. The residue from thefruit processing amounts to approximately 200 tons/year, and there is increasing demand touse 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 improve obesity
or diabetes (data not published). However, it is not known at this time which component ofsudachi 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) from the peelof Citrus sudachi and evaluated its anti-obesity and anti-diabetic effects in two distinct
models of metabolic dysfunction. C57BL/6 mice fed a high-fat diet exhibit many features ofmetabolic syndrome including obesity, hyperinsulinemia, hyperlipidemia, and glucose
intolerance [12]. db/db mice, a model of type 2 diabetes, is based on a leptin receptor
mutation and shows hyperphagia, obesity, hyperglycemia, hyperinsulinemia, and hepatic
steatosis [13,14].

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 ismanifested by reduced glucose uptake and increased free fatty acid release [15]. Adipokine
dysfunction plays an important role 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 andregulates the expression of several key genes involved in lipid and glucose metabolism [19].
Therefore, we examined the expression 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 obesity and 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 were collected in Tokushima Prefecture,
Japan, in September. The dried peels were ground in a grinder. Microwave-assisted
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 (600mL) and water (200 mL) was stirred in a separable three-necked round-bottom flask (1 L)with a condenser, thermo-sensor, and mechanical stirrer. The mixture was irradiated under
microwaves (MW) for 12 min under reflux with stirring. The reaction mixture was extractedwith 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), andconcentrated HCl (240 mL) in the round-bottom flask was irradiated by MW for 7 min under
reflux with stirring. The reaction mixture was neutralized with NaOH, and MeOH wasremoved under reduced pressure to give a brown residue, which was extracted with ethyl
acetate and dried (Na2SO4). After removal of ethyl acetate, the resulting brown residue waschromatographed on 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 compliance with the Guide for the Care andUse of Laboratory Animals and protocols approved by the Institutional Animal Care and Use
Committee at the University of Tokushima Graduate School (Tokushima, Japan).

C57BL/6 mice at 4 weeks of age were purchased from Japan SLC, Inc. (Shizuoka, Japan).
Mice were housed in temperature- (23 ± 3°C) and humidity-controlled conditions with a 12-h
light/dark cycle. Mice were given free access to water and either a control diet (n = 20; 14%of calories from fat; Oriental Yeast Co., Ltd., Tokyo, Japan) or a high-fat diet (n = 20; 40% ofcalories from fat; Oriental Yeast Co., Ltd.). Mice were allowed to adapt to these conditionsfor 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 intakeof each mouse were measured weekly.
Ten db/db mice at 4 weeks of age were purchased from Charles River Japan, Inc. (Kanagawa,Japan). Mice were housed under temperature- (23 ± 3°C) and humidity-controlled 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 foodintake of mice were measured once a week.

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

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, andCholesterol E-Test, respectively; Wako Pure Chemical Industries, Osaka, Japan). Plasma
insulin, adiponectin, and leptin levels were measured by enzyme-linked 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), and insulin tolerance tests(ITT)
Blood glucose levels were measured in tail vein blood using a glucometer (Arklay, Kyoto,
Japan). For OGTT, blood glucose levels were measured 0, 30, 60, 90, and 120 min after anoral glucose load (1 g/kg) following an overnight fast. For ITT, blood glucose levels were
measured 0, 30, 60, and 90 min after an intraperitoneal injection of regular human insulin(Novo Nordisk Pharma Inc., 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).

Gene expression analyses
Total RNA (1 µg) was reverse-transcribed to cDNA in a final volume of 20 µL using thePrimescript 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 RNA levelsof glucose transporters (Glut 1–4), adiponectin, PPARγ, adipocyte protein 2 (AP2), cluster ofdifferentiation 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, wemeasured the transcription of genes involved in the synthesis of fatty acids (fatty acidsynthase (FAS), acetyl-coA carboxylase (ACC1)), triglycerides (diglyceride acetyltransferase(DGAT1 and DGAT2)), or both (SREBP-1), as well as genes associated with hepaticcholesterol synthesis (farnesyl diphosphate synthase (FDS), squalene synthase (SS), 3hydroxy-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 6phosphatase (G6Pase)), phosphoenolypyruvate carboxykinase (PEPCK)). Evaluation of
mitochondrial biogenesis in primary myocytes involved measurement of nuclear respiratory
factor 1 and 2 (NRF1 and NRF2) and mitochondrial transcription factor A (mtTFA).

Measurement of adipocyte size
Subcutaneous and visceral adipose tissue samples were fixed in 2% osmium tetroxide andshaken at 37°C for 7 days. The fixed tissues were filtered through a 250-µm and 25-µm meshto 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 evaluated by X-ray computed tomography (CT; LaTheta, LCT-200, Aloka, Tokyo,
Japan) of the region between the first lumbar 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 SDSPAGE on 10% polyacrylamide precast gels (Invitrogen, Carlsbad, CA, USA) and transferred
to polyvinylidene difluoride membranes by electroelution. Membranes were blocked in 20
mmol/L of TBS solution with 1% Tween containing 5% skim milk and incubated withprimary antibodies overnight at 4°C. Polyclonal antibody sensitive to AMPKα1 and phosphoAMPKα1 (Thr172) was obtained from Cell Signaling Technology (Danvers, MA, USA) andantibodies against GLUT4 and β-tubulin were obtained from Santa Cruz Biotechnology(Santa Cruz, CA, USA). Protein density were visualized on membranes using horseradish
peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz Biotechnology) andvisualized 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 (5mg/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 indirectcalorimetry monitoring system (Columbus Instruments, Columbus, OH, USA). Data werecorrected 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, and 2 mmol/L EDTA, pH 7.2). Citrate synthase activity was determinedusing a Citrate Synthase Assay Kit (Sigma-Aldrich, St. Louis, MO, USA).

ATP content measurement
ATP content was measured using the AMERIC-ATP kit (Applied Medical Enzyme ResearchInstitute Corporation, Tokushima. Japan) according to manufacturer’s instructions. Briefly, asample of skeletal muscle (100 mg) tissue was homogenized in 10× volume of extraction
buffer (0.25 mol/L sucrose, 10 mmol/L HEPES-NaOH, 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 wasquantified by measuring the luminescence using a luminometer (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 topurify cells and remove fibroblasts. Experiments were conducted 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/Lsudachitin for 48 h.

Mitochondrial staining
Skeletal muscle myocytes were grown and differentiated on a glass coverslip coated with
Cell-Tak (BD Bioscience, Franklin Lakes, NJ, USA). The myocytes were stained with Mitotracker 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).

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. Statistical significance 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 theweight 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 weightof the animals (data not shown). The magnitude of weight reduction was similar between 5
and 10 mg/kg doses of sudachitin (data not shown), which prompted us to use the 5 mg/kg
dose for subsequent investigations.
Figure 1 Sudachitin inhibits weight gain in high-fat diet-fed mice. Mice were fed a lowfat control diet or a high-fat diet and were treated 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 inpanel 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).
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 wereameliorated by 5 mg/kg sudachitin administration (Figure 2A–C). However, there were nodifferences in BAT or muscle weight per body weight among the four groups (data notshown). Sudachitin treatment also significantly reduced adipocyte size in high-fat diet-fed
mice, as compared to the vehicle-treated mice fed high-fat diet (Figure 2D and E). Since
these changes were independent of food intake and muscle mass, the results suggest thatsudachitin improves energy metabolism.
Figure 2 Sudachitin reduces fat in high-fat diet-fed mice. (A) Body composition wasassessed 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).
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 triglyceride and NEFA levels. Total cholesterol levels
were not significantly different among any of the studied groups (Figure 3C).
Figure 3 Sudachitin reduces serum triglyceride and non-esterified fatty acid. Serumtriglyceride (A), non-esterified fatty acid (B), and total 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 dietfed 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 (opencircles); NEFA: non-esterified fatty acid; T-CHO: total cholesterol.

Sudachitin improves glucose tolerance and insulin sensitivity in high-fat dietfed mice
As shown in Figure 4A and B, mice fed a high-fat diet exhibited a state of moderate
hyperinsulinemia and hyperglycemia, 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 in high-fat diet-fed mice (Figure4C). Sudachitin treatment also re-established normal insulin tolerance in high-fat 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 wereameliorated by sudachitin (Figure 4E), consistent with the observed sudachitin-induced

decrease in visceral fat (Figure 2). Plasma adiponectin levels were found to decrease with age
and to be significantly lower in mice 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 wasmeasured at the indicated times. Glucose utilization and insulin sensitivity were determinedfrom 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 oftreatment (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: vehicletreated, 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.

Sudachitin alters the expression of metabolism-related genes in WAT andliver
As shown in Figure 5A, GLUT4 mRNA levels were significantly increased by the sudachitin
treatment, suggesting a possible molecular mechanism underlying sudachitin-mediatedimprovement in glucose uptake. Transcription of the adiponectin gene in high-fat diet-fed
mice treated with sudachitin showed a trend toward an increase that did not reach statisticalsignificance, compared to the observation in vehicle-treated high-fat diet-fed mice. The
mRNA level of PPARγ showed a trend towards an increase (1.5-fold) which was notstatistically significant in the sudachitin-treated group compared with the vehicle-treatedgroup. No significant differences in mRNA transcripts of adipocyte fatty acid-binding protein
and CD36 were observed between the two treatment groups (Figure 5A). mRNA transcripts
of uncoupling protein 1 and 3 (UCP1 and UCP3) were significantly increased in sudachitintreated mice, which suggests that WAT gained a BAT-like phenotype, possibly resulting inincreased thermogenesis.
Figure 5 Effects of sudachitin on mRNA levels in white adipose tissue and liver. Genetranscription was normalized for 36B4 in subcutaneous white adipose tissue (A-B) or 18S inliver (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.
In the liver, we found that sudachitin decreased the levels of mRNA transcripts encoding
FAS, ACC1 and 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 notaffected by sudachitin (Figure 5C). The expression levels of MTTP and LDLR were notsignificantly different, although MTTP expression tended to be lower in the sudachitintreated mice fed a high-fat diet as compared to the untreated high-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 a high-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 by sudachitin.

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 suppressed by treatment with 5 mg/kg sudachitin compared withvehicle-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 oftreatment with sudachitin reduced fasting blood glucose levels compared with the untreateddb/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 total cholesterol levels.
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 oftreatment (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 indicated times. Serumtriglyceride (D), non-esterified fatty acids (E), and total cholesterol levels (F) were measured
in db/db mice. Data are presented as means ± standard deviation. *P < 0.05 vs the indicatedgroups. closed circles: vehicle-treated group, open circles: sudachitin-treated group.

Sudachitin promotes energy expenditure by activating the Sirt1–PGC1αpathway
Because sudachitin increased adiponectin levels and reduced adiposity without reducing foodintake, we hypothesized that sudachitin increases the energy expenditure. To assess wholebody energy expenditure, mice fed a high-fat diet were treated with sudachitin or vehicle andsubjected to indirect calorimetry. Oxygen consumption during the light and dark cycles wasincreased by the administration of sudachitin for 4 weeks, without weight change comparedwith 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 day of sudachitin(Figure 7C-D).
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 indirectcalorimetry 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).
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 theexpression of 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 of sudachitin-treated mice. By

contrast, UCP2 gene expression was significantly increased in skeletal muscle of sudachitintreated mice. Sudachitin also induced the expression of PGC-1α and its target gene Sirt1 inskeletal muscle, which are known to regulate energy metabolism (Figure 8B). As shown in
Figures 8C and D, there was a statistically insignificant trend toward increased
phosphorylation of AMP protein kinase (AMPK) and Glut4 expression in response tosudachitin 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.
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 vehicletreated 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 treatedwith 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.

Sudachitin promotes mitochondrial biogenesis by activating its signaling
pathway in myocytes in vitro
To determine the direct effects of sudachitin on muscle metabolism, we assessed theexpression of key genes and mitochondrial number in vitro in primary cultured myocytes
following incubation with sudachitin. Consistent with the observed increase in PGC-1α andSirt1 mRNA in mice (Figure 8), transcription of these genes was significantly increased indifferentiated myocytes exposed to 30 µmol/L sudachitin compared with the vehicle-treatedcells (Figure 9A). Considering the genes involved in mitochondrial biogenesis, the expressionof NRF1, NRF2, and mtTFA were increased by 1.5- to 2.5-fold following sudachitin
treatment. In addition, the expression of UCP2 was significantly increased and UCP3
expression was slightly increased in myocytes treated with sudachitin for 48 h, whereas
UCP1 was not different between control and sudachitin-treated myocytes. Because GLUT4
expression was increased in sudachitin-treated 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 notaffected. 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 indicated with arrows. Sudachitin treatment increased
mitochondrial staining with the fluorophore Mito-Tracker Green in skeletal muscle myocytes
approximately 2.5-fold, in comparison to the cells treated with vehicle.
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 indicated group. Closed bars: untreated cells; open bars: sudachitin-treated cells. The
number of mitochondria in myocytes was increased by sudachitin (B, C). Myocytes wereincubated 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.

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 animalmodels of metabolic disorders. Previous studies have shown that dietary supplementation
with a 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, and nobiletin have hypoglycemic effects [9,22]. In
the present study, we found that plasma glucose and triglyceride 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 treatedwith 2 mg/kg body weight sudachitin. Because the reductions in plasma glucose andtriglyceride levels were similar between mice treated with 5 and 10 mg/kg body weightsudachitin, we used the 5 mg/kg dose in this study. This dose seems very low compared withthat required for other flavonoids or other treatments. In experiments using pharmacologicalagents, it is important to use the lowest effective dose to minimize the chances of off
target/side effects. We propose that the observed reduction in weight gain observed insudachitin-treated mice is a result of altered glucose and lipid metabolism, caused by thechanges in energy expenditure. This mechanism is supported by the sudachitin-mediatedenhanced 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 thelipid-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 found that mice treated withsudachitin showed increased skeletal muscle mitochondrial biogenesis and function, andpossibly increased NAD+ levels in skeletal muscle. Genetic disorders associated with
impaired mitochondrial function are characterized by a rapid onset of symptoms that arecommonly observed in elderly individuals, including clinical symptoms of type 2 diabetes[23]. There is increasing evidence that a progressive decline in mitochondrial function inasymptomatic, apparently healthy individuals may underlie a number of late-onset agerelated diseases [24,25], and that treatments that stimulate mitochondrial function can delay
the progression or even prevent the manifestation of some of these 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 resistance coupled 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 the regulation of
mitochondrial biogenesis [29]. PGC-1α is expressed at high levels in tissues characterized byabundant 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 the transcription of NRF1 and NRF2, which in turn stimulate theexpression of mtTFA. Importantly, PGC-1α has been shown 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, invitro investigation in cultured myocytes has detected increased expression of GLUT1 andGLUT3, but not GLUT4, following incubation with sudachitin. One explanation for thisapparent discrepancy might be that increased GLUT4 expression was secondary to theimprovement 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 overexpression of PGC-1α was shown to be sufficient
to delay many age-related disorders and extend the lifespan of mice [31]. The effects ofsudachitin 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 insudachitin-treated mice. Based on recent publications, it is possible that WAT wastransformed 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 adiponectinlevels and increased Sirt1 expression, which activates PGC-1α in the skeletal 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 andnutritional stimuli [33,34]. Rasbach et al. reported that overexpression of PGC-1α caused ashift toward increased oxidative types of 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 it is equivalent to the consumption of the peels of 1.5 small sudachi fruit per day inhumans.
In this study, we examined the Sirt1-dependent activity of sudachitin in skeletal muscle, atissue with very high 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 sudachitin or its metabolites in specific organs,or differences in the actions of sudachitin between tissues and cell types. It is also unclear
how flavonoids, such as sudachitin, modulate Sirt1–PGC1α signaling and regulate specifictranscription factors or whether these effects are mediated indirectly through a separate
signaling pathway. Further studies are needed to gain more insight into the effects ofsudachitin and its mechanisms in other tissues, particularly the liver and brain.

Conclusions
In summary, our study is the first to demonstrate that sudachitin improves glucose and lipid
metabolism by enhancing energy metabolism and mitochondrial biogenesis. Additionally, ourin vivo and in vitro findings suggest that these effects are mediated by the Sirt1–AMPK–PGC-1α pathway. These observations indicate that sudachitin has potential for use in treating
metabolic and age-related diseases.

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
R.T. designed research, conducted the research, wrote the manuscript, and has responsibility
for the final content; T.Y., R.H. and Y.T. conducted research; H.S., T.H., E.S. and T.S.
planed research and edited manuscript. Y.N., N.O., S.I. and M.T. provided essential reagentsand analyzed data. All authors have read and approved the final manuscript.

Acknowledgments
We thank Dr. Katsuya Hirasaka and Dr. Rie Mukai for helpful advice and technical support.
We also thank Hiromi Sato, Yui Hamasaki, Tomomi Maeno and Sayaka Sasaga for theirtechnical support.

Funding
Rie Tsutsumi: Funding was provided by the Japan Society for the Promotion of Sciences,
Tokyo JSPS KAKENHI 24650489. Tohru Sakai: Funding was provided by Grants for the
Regional Innovation Cluster.

References
1. Kahn SE, Hull RL, Utzschneider KM: Mechanisms linking obesity to insulin resistanceand type 2 diabetes. Nature 2006, 444:840–846.
2. Despres JP, Lemieux I, Bergeron J, Pibarot P, Mathieu P, Larose E, Rodes-Cabau J,
Bertrand OF, Poirier P: Abdominal obesity and the metabolic syndrome: contribution to
global cardiometabolic risk. Arterioscler Thromb Vasc Biol 2008, 28:1039–1049.
3. Glass CK, Olefsky JM: Inflammation and lipid signaling in the etiology of insulin
resistance. Cell Metab 2012, 15:635–645.
4. Baur JA, Pearson KJ, Price NL, Jamieson HA, Lerin C, Kalra A, Prabhu VV, Allard JS,
Lopez-Lluch G, Lewis K, Pistell PJ, Poosala S, Becker KG, Boss O, Gwinn D, Wang M,

Ramaswamy S, Fishbein KW, Spencer RG, Lakatta EG, Le Couteur D, Shaw RJ, Navas P,
Puigserver P, Ingram DK, de Cabo R, Sinclair DA: Resveratrol improves health andsurvival of mice on a high-calorie diet. Nature 2006, 444:337–342.
5. Mulvihill EE, Allister EM, Sutherland BG, Telford DE, Sawyez CG, Edwards JY, Markle
JM, Hegele RA, Huff MW: Naringenin prevents dyslipidemia, apolipoprotein B
overproduction, and hyperinsulinemia in LDL receptor-null mice with diet-induced
insulin resistance. Diabetes 2009, 58:2198–2210.
6. Lin Y, Vermeer MA, Bos W, van Buren L, Schuurbiers E, Miret-Catalan S, Trautwein EA:
Molecular structures of citrus flavonoids determine their effects on lipid metabolism in
HepG2 cells by primarily suppressing apoB secretion. J Agric Food Chem 2011, 59:4496–
4503.
7. Chanet A, Milenkovic D, Deval C, Potier M, Constans J, Mazur A, Bennetau-Pelissero C,
Morand C, Berard AM: Naringin, the major grapefruit flavonoid, specifically affects
atherosclerosis development in diet-induced hypercholesterolemia in mice. J Nutr
Biochem 2012, 23:469–477.
8. Li RW, Theriault AG, Au K, Douglas TD, Casaschi A, Kurowska EM, Mukherjee R:
Citrus polymethoxylated flavones improve lipid and glucose homeostasis and modulate
adipocytokines in fructose-induced insulin resistant hamsters. Life Sci 2006, 79:365–373.
9. Lee YS, Cha BY, Saito K, Yamakawa H, Choi SS, Yamaguchi K, Yonezawa T, Teruya T,
Nagai K, Woo JT: Nobiletin improves hyperglycemia and insulin resistance in obese
diabetic ob/ob mice. Biochem Pharmacol 2010, 79:1674–1683.
10. Mulvihill EE, Assini JM, Sutherland BG, DiMattia AS, Khami M, Koppes JB, Sawyez
CG, Whitman SC, Huff MW: Naringenin decreases progression of atherosclerosis byimproving dyslipidemia in high-fat-fed low-density lipoprotein receptor-null mice.
Arterioscler Thromb Vasc Biol 2010, 30:742–748.
11. Do GM, Kwon EY, Kim HJ, Jeon SM, Ha TY, Park T, Choi MS: Long-term effects ofresveratrol supplementation on suppression of atherogenic lesion formation andcholesterol synthesis in apo E-deficient mice. Biochem Biophys Res Commun 2008,
374:55–59.
12. Surwit RS, Seldin MF, Kuhn CM, Cochrane C, Feinglos MN: Control of expression of
insulin resistance and hyperglycemia by different genetic factors in diabetic C57BL/6 J
mice. Diabetes 1991, 40:82–87.
13. Herberg L, Coleman DL: Laboratory animals exhibiting obesity and diabetes
syndromes. Metabolism 1977, 26:59–99.
14. Yamaguchi K, Yang L, McCall S, Huang J, Yu XX, Pandey SK, Bhanot S, Monia BP, Li
YX, Diehl AM: Inhibiting triglyceride synthesis improves hepatic steatosis but
exacerbates liver damage and fibrosis in obese mice with nonalcoholic steatohepatitis.
Hepatology 2007, 45:1366–1374.

15. Eriksson JW: Metabolic stress in insulin’s target cells leads to ROS accumulation - ahypothetical common pathway causing insulin resistance. FEBS Lett 2007, 581:3734–
3742.
16. Anghel SI, Wahli W: Fat poetry: a kingdom for PPAR gamma. Cell Res 2007,
17:486–511.
17. Fasshauer M, Paschke R: Regulation of adipocytokines and insulin resistance.
Diabetologia 2003, 46:1594–1603.
18. Steiner G, Lewis GF: Hyperinsulinemia and triglyceride-rich lipoproteins. Diabetes
1996, 45(Suppl 3):S24–S26.
19. Rosen ED, Sarraf P, Troy AE, Bradwin G, Moore K, Milstone DS, Spiegelman BM,
Mortensen RM: PPAR gamma is required for the differentiation of adipose tissue in vivo
and in vitro. Mol Cell 1999, 4:611–617.
20. Hirose K, Tsutsumi YM, Tsutsumi R, Shono M, Katayama E, Kinoshita M, Tanaka K,
Oshita S: Role of the O-linked beta-N-acetylglucosamine in the cardioprotection inducedby isoflurane. Anesthesiology 2011, 115:955–962.
21. Nakajima A, Yamakuni T, Haraguchi M, Omae N, Song SY, Kato C, Nakagawasai O,
Tadano T, Yokosuka A, Mimaki Y, Sashida Y, Ohizumi Y: Nobiletin, a citrus flavonoid
that improves memory impairment, rescues bulbectomy-induced cholinergic
neurodegeneration in mice. J Pharmacol Sci 2007, 105:122–126.
22. Jung UJ, Lee MK, Jeong KS, Choi MS: The hypoglycemic effects of hesperidin andnaringin are partly mediated by hepatic glucose-regulating enzymes in C57BL/KsJdb/db mice. J Nutr 2004, 134:2499–2503.
23. Wallace DC: A mitochondrial paradigm of metabolic and degenerative diseases,
aging, and cancer: a dawn for evolutionary medicine. Annu Rev Genet 2005, 39:359–407.
24. de Moura MB, dos Santos LS, Van Houten B: Mitochondrial dysfunction inneurodegenerative diseases and cancer. Environ Mol Mutagen 2010, 51:391–405.
25. Figueiredo PA, Powers SK, Ferreira RM, Appell HJ, Duarte JA: Aging impairs skeletal
muscle mitochondrial bioenergetic function. J Gerontol A Biol Sci Med Sci 2009, 64:21–
33.
26. Fillmore N, Jacobs DL, Mills DB, Winder WW, Hancock CR: Chronic AMP-activatedprotein kinase activation and a high-fat diet have an additive effect on mitochondria in
rat skeletal muscle. J Appl Physiol 2010, 109:511–520.
27. Horvath TL, Erion DM, Elsworth JD, Roth RH, Shulman GI, Andrews ZB: GPA
protects the nigrostriatal dopamine system by enhancing mitochondrial function.
Neurobiol Dis 2011, 43:152–162.

28. Morino K, Petersen KF, Shulman GI: Molecular mechanisms of insulin resistance inhumans and their potential links with mitochondrial dysfunction. Diabetes 2006,
55(Suppl 2):S9–S15.
29. Lin J, Handschin C, Spiegelman BM: Metabolic control through the PGC-1 family oftranscription coactivators. Cell Metab 2005, 1:361–370.
30. Puigserver P, Wu Z, Park CW, Graves R, Wright M, Spiegelman BM: A cold-inducible
coactivator of nuclear receptors linked to adaptive thermogenesis. Cell 1998, 92:829–
839.
31. Wenz T, Rossi SG, Rotundo RL, Spiegelman BM, Moraes CT: Increased muscle PGC1alpha expression protects from sarcopenia and metabolic disease during aging. Proc
Natl Acad Sci U S A 2009, 106:20405–20410.
32. Bostrom P, Wu J, Jedrychowski MP, Korde A, Ye L, Lo JC, Rasbach KA, Bostrom EA,
Choi JH, Long JZ, Kajimura S, Zingaretti MC, Vind BF, Tu H, Cinti S, Højlund K, Gygi SP,
Spiegelman BM: A PGC1-alpha-dependent myokine that drives brown-fat-likedevelopment of white fat and thermogenesis. Nature 2012, 481:463–468.
33. Hardie DG: AMP-activated/SNF1 protein kinases: conserved guardians of cellularenergy. Nat Rev Mol Cell Biol 2007, 8:774–785.
34. Reznick RM, Shulman GI: The role of AMP-activated protein kinase in mitochondrial
biogenesis. J Physiol 2006, 574:33–39.
35. Rasbach KA, Gupta RK, Ruas JL, Wu J, Naseri E, Estall JL, Spiegelman BM: PGC1alpha regulates a HIF2alpha-dependent switch in skeletal muscle fiber types. Proc Natl
Acad Sci U S A 2010, 107:21866–21871.
36. Price NL, Gomes AP, Ling AJ, Duarte FV, Martin-Montalvo A, North BJ, Agarwal B, Ye
L, Ramadori G, Teodoro JS, Hubbard BP, Varela AT, Davis JG, Varamini B, Hafner A,
Moaddel R, Rolo AP, Coppari R, Palmeira CM, de Cabo R, Baur JA, Sinclair DA: SIRT1 isrequired for AMPK activation and the beneficial effects of resveratrol on mitochondrial
function. Cell Metab 2012, 15:675–690.

Additional file
Additional_file_1 as XLSX
Additional file 1: Table S1 Supplemental table Primer sequences.

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Additional files provided with this submission:
Additional file 1: 1847170227127307_add1.xlsx, 11K
http://www.nutritionandmetabolism.com/imedia/4198998351346171/supp1.xlsx