Citrus tangeretin improves skeletal muscle mitochondrial biogenesis

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Bioactive Constituents, Metabolites, and Functions

Citrus tangeretin improves skeletal muscle mitochondrial biogenesis via activating the AMPK-PGC1-# pathway in vitro and in vivo: a possible mechanism for its beneficial effect on physical performance Guangning Kou, Zhenqing Li, Chao Wu, Yang Liu, Yan Hu, Liya Guo, Xiaoyu Xu, and Zhiqin Zhou J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b04124 • Publication Date (Web): 28 Oct 2018 Downloaded from http://pubs.acs.org on October 30, 2018

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Journal of Agricultural and Food Chemistry

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Citrus tangeretin improves skeletal muscle mitochondrial biogenesis via activating the

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AMPK-PGC1-α pathway in vitro and in vivo: a possible mechanism for its beneficial

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effect on physical performance

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Guangning Kou†,1, Zhenqing Li†,1, Chao Wu‡, Yang Liu‡, Yan Hu†, Liya Guo§, Xiaoyu Xu‡,*

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and Zhiqin Zhou†,*

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† College of Horticulture and Landscape Architecture, Southwest University, Chongqing

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400716, China;

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‡ College of Pharmaceutical Sciences and Chinese Medicine, Southwest University,

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Chongqing 400716, China;

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§ Key Lab of Physical fitness evaluation and Motor Functional Monitoring, General

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Administration of Sport of China-Southwest University, Chongqing 400715, China;

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1 Co-first authors

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*Corresponding author: Xiaoyu Xu and Zhiqin Zhou

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E-mail address: [email protected] and [email protected]

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ACS Paragon Plus Environment


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Abstract:

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Mitochondrial biogenesis is a key factor which influences the function of skeletal muscle.

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Increasingly, flavonoids are reported to have the potential ability of regulating mitochondrial

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biogenesis. In this study, we investigated the effects of tangeretin, a polymethoxylated

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flavonoid isolated from mandarin fruits, on mitochondrial biogenesis and its underlying

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mechanisms. The tangeretin was obtained from the peel of ‘Dahongpao’ tangerine by

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macroporous

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chromatography. The activity of mitochondrial biogenesis was explored by using mouse

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derived C2C12 myoblasts and Kunming mice. Results showed that the purity of tangeretin

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obtained were 98.64%, and it could effectively activate mitochondrial biogenesis signaling

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pathway both at gene and protein levels in C2C12 myoblasts. Animal experiments showed

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that tangeretin pretreatment could markedly improve exercise performance (the time of

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hanging wire and run to fatigue was obviously increased 1.6 fold and 2.1 fold in the high-dose

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tangeretin group, respectively), and the transmission electron microscopy, western blotting

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and immunohistochemistry further indicated that tangeretin increased mitochondria number

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and activated mitochondrial biogenesis signaling axis. Our findings suggest that tangeretin

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enhance mitochondrial biogenesis via activating the AMPK-PGC1-α pathway, resulting in the

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improvement of exercise performance, and tangeretin may be a potentially novel

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mitochondria regulator in foods.

adsorptive

resins

combined

with preparative-high

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performance

liquid

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Key words: Citrus, Tangeretin, Mitochondria biogenesis, AMPK-PGC1-α pathway.

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Introduction

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Skeletal muscle is one of the largest organs in our body, which requires a large amount

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of energy to maintain muscle function and support physical activity. Mitochondria are the

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major adenosine triphosphate (ATP) synthesizing and energy transducing organelles in the

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skeletal muscle cells. The number of skeletal muscle mitochondria is critical for skeletal

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muscle function, which is also associated with many chronic diseases including obesity, type

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2 diabetes and myasthenia.1,2 The improvement of skeletal muscle mitochondria biogenesis

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(Mt biogenesis) is largely responsible for skeletal muscle’s fatigue delaying, resulting in

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improvement of physical exercise performance.3 In view of the vital function of Mt biogenesis,

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increasing researchers have concentrated on the identification of natural active compounds,

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especially those from foods or herbs, which can activate the Mt biogenesis signaling

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pathway(s) to improve skeletal muscle function.

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AMP-activated protein kinase (AMPK), a serine/threonine heterotrimeric protein complex,

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is considered as an important initiator of muscle Mt biogenesis.4 AMPK stimulates Mt

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biogenesis via peroxisome proliferator-activated receptor gamma coactivator1-alpha

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(PGC1-α), which is the critical regulator to activate nuclear respiratory factors (NRF).5 The

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NRF activation further up-regulates the expression of transcription factor A of the

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mitochondrion (TFAM) which activates Mt DNA duplication and gene transcription, thus

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initiating the mitochondrial proteins coordinated expression.6,7 The mitochondrial biogenesis



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enhancement by activating AMPK/PGC1-α/NRF1/TFAM signaling axis has been well

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reported in the existing literatures.8,9 For examples, curcumin enhances the effect of exercise

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on mitochondrial biogenesis by activating AMPK-PGC1-α pathway. Similarly, sudachitin, a

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polymethoxylated flavone, improves mitochondrial biogenesis in obese mice and diabetic

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mice via activating AMPK-PGC1-α pathway.

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Tangeretin (TG), 5,6,7,8,4’-pentamethoxy flavone, is one of the dominant members of

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polymethoxyfavones (PMFs), which is mainly found in the peel of citrus fruits.10 TG was

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reported to have a broad health benefits including anti-oxidant, anti-inflammatory,

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anti-diabetic and neuroprotective effects.11-14 These findings stimulated the exploration of

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TG’s bioactivities and possible role in human health. Recently, more and more studies have

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reported that the potential ability of plant flavonoids to improve Mt biogenesis both in vivo and

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in vitro.9,15,16 However, the effect of TG on Mt biogenesis, to the best of our knowledge, was

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rarely studied.

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‘Dahongpao’ tangerine (Citrus tangerina Tanaka), one of the most important cultivated

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species in the genus Citrus L., was widely cultivated in southwestern China. The peel of

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‘Dahongpao’ is a rich resource of TG which has been reported in our previous study.17 In this

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study, TG in the peel of ‘Dahongpao’ tangerine was isolated and purified using macroporous

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adsorptive resins (MARs) combined with prep-high performance liquid chromatography

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(prep-HPLC) and its bioactivity on Mt biogenesis were testified in C2C12 myoblasts. Animal

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study was subsequently carried out.

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Materials and methods


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Reagents and plant material. Methanol, ethanol, osmium tetroxide (OT), uranyl acetate

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(UA) and lead citrate (LC) were purchased from Kelong Chemical Reagent Factory, Chengdu,

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China. Gluteraldehyde, paraformaldehyde, Epon 812, paraformaldehyde and citrate buffer

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were obtained from Sangon Biotech, Shanghai, China. Diaminobenzidine and hematoxylin

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were bought from Beyotime Biotech, Shanghai, China. AMPK, p-AMPK (Thr-172) and NRF1

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were purchased from Abcam, Cambridge, UK. PGC1-α, TFAM, COX IV, Histone H3, β-actin,

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anti-mouse lgG and anti-rabbit lgG were bought from Proteintech, Wuhan, China.

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‘Dahongpao’ tangerine (Citrus tangerina Tanaka) was harvested from Xiangping Community

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of TailongTownship in Wanzhou (Chongqing, China), which was numbered as LR0094 in

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Citrus Research Institute of Chinese Academy of Agricultral Science.

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Extraction and isolation. The powdered ‘Dahongpao’ tangerine peels (5.0 kg) were

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extracted with 89% ethanol solution (100 L) for 34 min at 41 °C associated with ultrasound

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(300W). The crude extract was concentrated in vacuo at 45°C and adsorpted by HPD 100

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resin (Baoen Adsorption Material Technology Co., Ltd., Hebei, China) in a concentration of

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8.7 mg/mL. Ethanol (90%) solution was used to desorb the target compounds, and then the

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desorpted solution was freeze-dried as powder and further isolated using MS-Directed

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prep-HPLC (Waters, MA, USA). All the samples were detected by the UPLC-Q-TOF-MS/MS

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method reported in our prior study.18 1H NMR and

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(Sigma, MO, USA) on an AVANCEIII 10600 Bruker NMR (Bruker, Karlsruhe, Germany).

13C

NMR spectra were recorded in CDCl3

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Cell culture and induction of differentiation. The mouse derived C2C12 myoblasts

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(China Center for Type Culture Collection, Beijing, China) were cultured in medium with 89%



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high glucose DMEM (GIBCO, NY, USA) plus 10% FBS (Hyclone, Utah, USA) and 1%

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penicillin/streptomycin antibiotics (Hyclone, Utah, USA), under an incubator controlled

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environment (37 °C, 95% air and 5% CO2). When reached 70-80% confluence, the C2C12

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myoblasts were switched to differentiation medium (DMEM supplemented with 3% HS

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(GIBCO, NY, USA) and 1% P/S antibiotic) for six days to induce differentiation. Experiments

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were carried out on day 7 when C2C12 myotubes differentiation was completed.

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Cell viability assay. For the analysis of cell viability using the CCK-8 (Dojindo,

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Kumamoto, Japan), experiments were performed in different concentrations (50, 100, 200

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μM) of TG for six hours. The ranges of concentration and time of TG were selected after

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performing preliminary experiments based on studies demonstrating the effectiveness of

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similar concentrations.19 Next, the cell added the 10 μl CCK-8 solution was incubated at 37°C

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for 30 min and then measured absorbance using a microplate reader at 450 nm (BioTek,

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Vermont, USA).

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Animal test. Male Kunming mice (6-7 weeks of age, 18-20 g) were purchased from the

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Experimental Animal Center of the Chongqing Medical University (Certification: SCXK

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2017-0001) (Chongqing, China). Mice were maintained in a pathogen-free environment (22 ±

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1 °C, 12 h light/dark cycle, free access to drinking water and standard chow diet). All animals

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were treated in accordance with the guidelines of the NIH for care and use of lab animals.

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And the animal experimental procedures were approved by the Southwest University

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Experimental Animal Ethics Committee to minimize animal suffering throughout the studies.

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After a one-week environmental acclimation, the mice were randomly divided into five


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equal groups as following: normal group (Control), 25 mg/kg tangeretin group (TG25),

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50mg/kg tangeretin group (TG50) and 100 mg/kg tangeretin group (TG100), and each group

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had 8 mice. TG was suspended in 0.25% w/v sodium carboxy-methyl-cellulose (Adamas Co.,

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Ltd, Shanghai, China). The control group was treated with a similar volume of vehicle. TG or

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sodium carboxy-methyl-cellulose was administrated by oral gavage for four consecutive

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weeks. The hanging wire test and exercise tolerance test were carried out in the 29th day and

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the protocols were used as previously reported.20,21 After the final test, mice were sacrificed

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and gastrocnemius muscle were quickly dissected out and washed in ice-cold saline. One

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gastrocnemius muscle was used for transmission electron microscopy test and

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immunohistochemical detection, which immediately fixed in 2.5% gluteraldehyde, and 4%

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paraformaldehyde respectively. The other gastrocnemius muscle was maintained at -80 °C

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for further western blotting analysis.

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Transmission electron microscopy. The transmission electron microscopy (TEM) was

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used to observe the mitochondrial morphology of the gastrocnemius muscle. Sections

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(approximately 1 mm) from control and TG groups of mice were fixed in 2.5% gluteraldehyde

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for 24 hours at 4°C. Then, they were post-fixed in 1% OT for 2 hours at 4°C and dehydrated

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with graded series of ethanol, and subsequently embedded in Epon 812. Finally, the samples

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were cut into 60 nm ultrathin sections and double stained with 2% UA and LC. The thin

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sections were observed by JEOL 1230 (JEOL Ltd., Tokyo, Japan) at 80 kV, and photoed by

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Gatan Orius 830 CCD camera attached to the microscope. To measure the mitochondrial

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number, 10 randomly selected areas per animal were photographed at 10000 × magnification



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and counted.22

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Quantitative real-time PCR. Total RNA was isolated from the C2C12 using Trizol

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reagent (Invitrogen, CA, USA) and mitochondrial DNA was extracted using a kit following the

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manufacturer’s recommendation (Genmed, Boston, USA). RNA purification and cDNA

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synthesis was performed using PrimeScript™ RT reagent Kit with gDNA Eraser (Takara,

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Shiga, Japan). PCR amplifications were carried out in SYBR green mix (Applied Biosystems,

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Carlsbad, USA). The primers of PGC1-α, NRF1, TFAM and mtDNA are listed in supporting

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information 1. β-actin and nuclear DNA were used as the internal control. The data were

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calculated by 2-ΔΔCT value.

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Western blotting. Total and nuclear proteins from cell and gastrocnemius muscle were

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extracted using the total protein extraction kit and nuclear protein extraction kit (Beyotime

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Biotech, Shanghai, China). Samples ran in SDS-PAGE and then electrotransferred 1.5-2

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hours to PVDF membranes (Millipore, Massachusetts, USA). Transferred membranes

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blocked for more than 1.5 hours with 5% bovine serum albumin (for blocking p-AMPK) and

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skimmed milk powder at normal temperature, and then they were bloted with AMPK (1:1000

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dilution), p-AMPK (1:1000 dilution), PGC1-α (1:2500 dilution), NRF1 (1:2500 dilution), TFAM

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(1:1000 dilution), COX IV (1:1000 dilution), Histone H3 (1:2500 dilution) and β-actin (1:2500

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dilution) antibodies overnight at 4°C. The membranes were washed with TBST (10min×3) on

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the second day and incubated with anti-mouse lgG and anti-rabbit lgG (all 1:5000 dilution) for

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1.5-2 h at room temperature. The immunoreactive bands showed in Gel Image Analysis

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System (Tanon-3500R, Shanghai, China) by using enhanced chemiluminescence (Advansta,


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California, USA) and quantified by densitometry. Immunohistochemistry.

The

gastrocnemius

muscles

were

fixed

in

4%

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paraformaldehyde, later, embedded in paraffin to cut into 4-5 μm sections. The antigen

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recovery and endogenous peroxidase quenched were carried out using 0.1 M citrate buffer

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and 3% H2O2. Then, the sample were incubated with 10% goat serum (Vector, CA, USA) for

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1-1.5 h to reduce non-specific binding and bloted with p-AMPK (1:100 dilution), PGC1-α

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(1:200 dilution), NRF1 (1:100 dilution), TFAM (1:200 dilution), COX IV (1:200 dilution)

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overnight at 4°C. The HRP-conjugated secondary antibody was further incubated on the

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second day at room temperature for 1-1.5 h. Chromogenic signal development and counter

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staining were conducted by using diaminobenzidine and hematoxylin after PBS rinsing. The

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sections were captured under light microscope (Carl Zeiss, Oberkochen, Germany) and the

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positive staining of p-AMPK, PGC1-α, NRF1, TFAM, COX IV were the measured by Image

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Pro Plus.

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Statistical analysis. The data were analyzed by using the SPSS 18 software. Results

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were presented as mean ± SD. ANOVA was used to detect differences between groups.

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Tukey’s test was used for multiple comparisons. p < 0.05 was considered to be statistically

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significant.

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Results

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Identification of tangeretin. The results showed that ultrasound-assisted extraction was

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effective in the extraction of total PMFs (yield: 12680 ± 1786 mg/kg DW). The crude extraction

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after treated by HPD 100, most impurities including water-soluble pigments, sugars and



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flavanones were removed (Figure 1). The TG content increased from 5.64 ± 0.23 mg/g to

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67.37 ± 7.10 mg/g, which was an increase of 11.95-folds (Table 1). The obtained compound

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was light yellow powder (5.34 g), and its molecular formula was determined as C20H20O7

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based on the HR-ESI-TOFMS data (m/z: 373.1241 [M+H]+ calcd. 373.1287). 1H NMR (400

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MHz, CDCl3 ) δH 7.88 (1H, d, J=8.92 Hz, H-2’, 6’), 7.03 (H, d, J=8.92 Hz, H-3’, 5’), 6.60 (1H,

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s, H-3), 4.10 (3H, s, OCH3), 4.02 (3H, s, OCH3), 3.95 (6H, s, 2xOCH3).

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CDCl3) δC 177.42 (C-4), 162.42 (C-4’), 161.29 (C-2), 151.48 (C-7), 148.52 (C-5), 147.85

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(C-9), 144.20 (C-6), 138.22 (C-8), 127.82(C-2’, 6’), 124.00(C-1’), 115.04(C-10), 114.64(C-3’,

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5’), 106.83(C-3), 62.36 (C-5, OCH3), 62.13 (C-6, OCH3), 61.93(C-7, OCH3), 61.76 (C-8,

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OCH3), 55.61 (C-4’, OCH3). These data agreed well with those reported in the literature,23 so

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it was identified as TG, and the purity detected by UPLC was 98.64 % (Figure 2 B-E).

13C

NMR (100 MHz,

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Tangeretin up-regulated biogenesis pathway related gene expression in C2C12

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myotubes. The effect of TG on cell viability and Mt biogenesis pathway related gene

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expression were investigated in C2C12 myotubes. As shown in Figure 3 A, C2C12 cell

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viability was not significantly affected in 50, 100, 200 μM TG groups for 6 h, indicating that

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50–200 μM TG had no cytotoxic effects on cells. As shown in Figure 3 B-E, TG significantly

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and dose-dependently enhanced PGC1-α (TG 100 μM 1.95 fold and TG 200 μM 3.01 fold),

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NRF1 (TG100 μM 1.60 fold and TG 200 μM 2.18 fold), TFAM mRNA expression (TG 100 μM

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1.53 fold and TG 200 μM 1.87 fold) and mitochondrial DNA copy number (TG 100 μM 1.46

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fold and TG 200 μM 1.81 fold) compared with control group.

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Tangeretin activated biogenesis pathway related protein expression in C2C12


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myotubes. TG up-regulated mitochondria biogenesis signaling pathway was further

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investigated at protein level. As shown in Figure 4, TG significantly enhanced p-AMPK

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(Thr-172) / AMPK (p

Citrus tangeretin improves skeletal muscle mitochondrial biogenesis

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