The effect of exercise training and resveratrol supplementation with low fat diet on macrophage infiltration in adipose tissue of obese mice.

2014 한국운동생리학회 동계국제학술대회(샘플)

Hong Gil Dong

exephysio@hanmail.net (교신저자 이메일 꼭 기재 요망)

OO University

Abstract

2012 한국운동생리학회 동계국제학술대회(샘플)

This study was to investigate the effect of exercise training and resveratrol supplement with a low-fat diet(LFD) on macrophage infiltration and inflammatory cytokine in adipose tissue of obese mice. All the mice(C57Bl/6) were divided into five groups; high-fat diet(HD), low-fat diet control(HLC), low-fat diet exercise(HLE), low-fat diet resveratrol(HLR), and low-fat diet exercise plus resveratrol(HLER). All the mice were fed a HFD(45% from fat of total calories) for 12 weeks  and then changed into LFD(10% from fat of total calories) for 7 weeks except HD group. Exercise mice ran on a treadmill at 12-20m/min for 50-60min/day for 7 weeks. Resveratrol supplement mice were orally gavaged with resveratrol(10㎎/㎏ of body weight) dissolved in 50% propylene glycol. Tumor necrosis factor(TNF)-α, interleukin(IL)-6, F4/80, monocyte chemoattractant protein(MCP)-1, inter-cellular adhesion molecule (ICAM)-1, vascular-cellular adhesion molecule(VCAM)-1, CD11c, CD163 and toll-like receptor(TLR)4 mRNA expressions were measured by RT-PCR. A comparison with HD mice, the body weight and epididymal fat mass were reduced in all treatment groups. F4/80, TNF-α, and IL-6 mRNA expression were decreased in HLR, HLE, and HLER mice compared to HD mice. CD11c mRNA expression was reduced by HLE, HLER mice compared to HD mice. In conclusion, exercise training with low fat diet has positive effect to attenuate the obese induced metabolic complication

Key words : obesity, macrophage, inflammation, exercise training, resveratrol

Ⅰ. Purpose

Obesity  and physical inactivity are associated with chronic inflammatory diseases, such as dyslipidemia, Hypertension, type 2 diabetes and  atherosclerosis.  In deed, chronic inflammation has been recognized as a major predictor of metabolic disease(Goodpaster et al., 2005; Mokdad et al., 2001). Previous studies have been conducted to examine the influence of obesity on inflammation(Amar et al., 2007; Chen, 2006; Roth, 2004; Shinkai et al., 1994; Tilg & Moschen, 2006).

Recent study has reported that the inflammation state was associated with a macrophage infiltration in to adipose tissue(Bruun et al., 2006; Permana et al., 2006; Zeyda et al., 2007). However, few study reported that exercise training or resveratrol supplement attenuated inflammation in adipose tissue occurs by suppression of macrophage infiltration in obese mice. Also, none of resveratrol supplementation research showed exercise training effect.

Therefore, the aim of this study is to the effects of exercise training and resveratrol supplementation with low-fat diet on macrophage infiltration in adipose tissue in high-fat diet induced obese mice.

Ⅱ. Method

1. Animals care

30 male C57BL/6 mice (4weeks old) were provided by Orient-Bio. The living conditions were regulated to 22±2°C with 50±5% of humidity, and a night/day circulation of 12-hour intervals (day: 0600 – 1800). All the mice(C57Bl/6) were divided into five groups; high-fat diet(HD), low-fat diet control(HLC), low-fat diet exercise(HLE), low-fat diet resveratrol(HLR), and low-fat diet exercise plus resveratrol(HLER). All the mice were fed a HFD(45% from fat of total calories) for 12 weeks  and then changed into LFD(10% from fat of total calories) for 7 weeks except HD group. All animals had free access to food and water. The whole experiment was approved by the Institutional Animal Care and Use Committee of Chungnam National University.

2. Exercise training and resveratrol supplement

In the early phage of moderate exercise, all the mice of HLE and HLER were run at 8m/min for 10 minutes on the automatically operated rodent treadmill. After that, the speed and time of exercise were gradually increased up to 12 – 20 m/min for 30 –60 minutes. The volume of this protocol represents 60 – 76 % of the maximum oxygen consumption of the rodents as moderate intensity exercise. The exercise groups were subjected to this training for 5 consecutive days within each week, over the 7 weeks period. During training of the exercise group (maximum 60 min day-1), the HD, HLC, and HLR groups were exposed to environmental stress and vibration and noise from the treadmill, and their food and water supply were removed. Resveratrol supplement mice were orally gavaged with resveratrol (10mg/kg of body weight) dissolved in 50% propylene glycol. The other group received the vehicle. Each treatment was administrated once a day, 5 times/week, for 7 weeks before tissue collection.

3. RNA extraction and RT-PCR

Total RNA was isolated from 100 mg of subcutaneus adipose tissue using Trizol  (Qiagen, Hilden, Germany), according to the manufacturer's instructions. RNA was quantified by spectroscopy (NanoDrop, Wilmington, DE). From each sample, 1 μg of total RNA was reverse transcribed to cDNA with primers of random and oligo(dt) using a high-capacity RNA-to-cDNA kit (Roche, Mannheim, Germany) in a total volume of 20 μL, as described in the high-capacity RNA-to-cDNA kit protocol. The amplification was performed in a total volume of 20 μL, which include 2 μL of cDNA, 1 μL of each primer (10 pmol/ μL), and 16 μL of DEPC (Diethyl pyrocarbonate) water. Reactions were carried out in an ABI 7500 system using the following thermal cycling parameters: 95℃ for 2 min, and then 38-40 cycles of 95℃ for 30sec, the appropriate annealing temperature for 30sec, and 72℃ for 2 min. All samples were examined in parallel for β-actin. All primer sequences designed using IDT (Skokie, IL), verify using the National Center for Biotechnology Information (NCBI) BLAST feature, and purchase from MWG Biotech (Huntsville, AL).

5. Statistical analysis

Data on the body weight, adipose tissue fat-pad weights are presented as mean ± SD. RT-PCR data were analyzed using one-way analysis of variance (ANOVA) with LSD post-hoc tests. Statistical significance was defined as p< 0.05.

Ⅲ. Results and Conclusion

Body weight  of all the low fat diet groups was significantly decreased, compared to HD group. Especially, the body weight of exercise groups was more  significantly decreased. The results of epididymal weight were similar to the body weight change.

F4/80, CD11c and CD163 expression were significantly changed in HLE and HLER.

MCP-1, ICAM-1 and VCAM-1 were significantly decreased in HLE and HLER.

IL-6 and TNF-4 were significantly decreased in HLR, HLE and HLER

TLR4 was significantly decreased in HLE.

In conclusion, resveratrol supplementation with low fat diet decreased macrophage infiltration and inflammation expression in adipose tissue partly. However, exercise training with low fat diet has more positive effect to attenuate obese induced metabolic complication.

Reference

Amar, S., Zhou, Q., Shaik-Dasthagirisaheb, Y., and Leeman, S. (2007) Diet-induced obesity in mice causes changes in immune responses and bone loss manifested by bacterial challenge. PNAS, 104,20466-20471

Bruun JM, Helge JW, Richelsen B, Stallknecht B. (2005) Diet and exercise reduce low-grade inflammation and macrophage infiltration in adipose tissue but not in skeletal muscle in severely obese subjects. Am J Physiol Endocrinol Metab 290, E961–E967.

Chen H. Cellular inflammatory responses: novel insights for obesity and insulin resistance. Pharmacol Res. 53,469-477.

Goodpaster BH, Krishnaswami S, Harris TB, et al. (2005) Obesity, regional body fat distribution, and the metabolic syndrome in older men and women. Arch Intren Med. 165,777-783.

Mokdad, A.H.; Bowman, B.A.; Ford, E.S.; Vinicor, F.; Marks, J.S.; Koplan, J.P.(2001) The continuing epidemics of obesity and diabetes in the United States. JAMA 286,1195-1200.

Permana PA, Menge C, Reaven PD. (2006). Macrophage-secreted factors induce adipocyte infalmmation and insulin resistance. Biochem Biophys Res Commun. 341,507-514.

Roth, J. (2004) Obesity research and treatment: where have we been and where are we going? Obes Res. 12:88–101.

Shinkai, S., Watanabe, S., Kurokawa, Y., Toril, J., Asai, H., and Shephard, R. J. (1994)　Effects of 12 weeks of aerobic exercise plus dietary restriction on body composition, resting energy expenditure and aerobic fitness in mildly obese middle aged women. Eur. J. Appl. Physiol., 68,258-265.

Tilg H, Moschen AR.n (2006). Adipocytokines: mediators linking adipose tissue, inflammation and immunity. Nat Rev Immunol. 6,772-783.

Zeyda M, Farmer D, Todoric J, et al. (2007). Human adipose tissue macrophages are of an anti-inflammatory phenotype but capable of excessive pro-inflammatory mediator production. Int J Obes. 31,1420-1428.