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Effect of lignin-derived lignophenols on hepatic lipid metabolism in rats fed a high-fat diet
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 4 ( 2 0 1 2 ) 228–234
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/etap
Effect of lignin-derived lignophenols on hepatic lipid metabolism in rats fed a high-fat diet Shin Sato a,∗ , Yuuka Mukai a , Yukari Tokuoka a , Keigo Mikame b , Masamitsu Funaoka b , Shuzo Fujita a a
Department of Nutrition, Faculty of Health Sciences, Aomori University of Health and Welfare, Mase 58-1, Hamadate, Aomori 030-0841, Japan b Department of Environmental Science and Technology, Faculty of Bioresources, Mie University, Tsu 514-8507, Japan
a r t i c l e
i n f o
a b s t r a c t
Article history:
The effect of lignin-derived lignophenols on lipid metabolism in the livers of rats fed a high-
Received 28 December 2011
fat diet was investigated. Rats fed a diet providing 45% of energy from fat were divided into
Received in revised form
2 groups, namely 0% and 0.5% lignophenols-containing diets. The controls were fed a diet
5 April 2012
providing 10% of energy from fat. Plasma blood parameters, protein expression of acetyl-CoA
Accepted 6 April 2012
carboxylase (ACC) and sterol regulatory element-binding protein (SREBP)-1, and SREBP-1c
Available online 13 April 2012
mRNA expression in the livers were examined. The plasma triglyceride levels in the rats fed lignophenols-containing diets were decreased. SREBP-1c mRNA expression in the rats fed
Keywords:
lignophenols-containing diets was significantly reduced compared with the rats fed high-
Lignophenols
fat diets, and phosphorylated ACC protein in the rats fed lignophenols-containing diets was
Obesity
significantly increased. Our results suggested that lignophenols suppress the expression of
Sterol regulatory element-binding
SREBP-1c mRNA and the phosphorylation of ACC in the liver, and may lead to a decrease in
protein-1c
plasma triglyceride levels.
Acetyl-CoA carboxylase
© 2012 Elsevier B.V. All rights reserved.
High-fat diet
1.
Introduction
Obesity has become one of the most prevalent health problems around the world, and a risk factor for a number of chronic diseases such as insulin resistance, type 2 diabetes mellitus, and cardiovascular disease (Browning and Horton, 2004). In general, it is accepted that obesity results from an imbalance between energy intake and expenditure, and is characterized by increased fat accumulation in adipose
tissue and elevated lipid concentrations in the blood. Dietary fat is one of the main environmental factors associated with the incidence of obesity. Thus, it is important to establish strategies for the prevention of obesity. Sterol regulatory element-binding protein (SREBP)-1c is an important regulator of lipogenic enzymes such as acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) (Horton et al., 2002; Jump et al., 2005). Overexpression of SREBP-1c increased triglyceride accumulation and fatty acid synthesis (You and Crabb, 2004). In contrast, polyunsaturated fatty acids were
Abbreviations: LP, lignophenols; HFLP, LP-containing high-fat diet; ACC, acetyl-CoA carboxylase; SREBP, sterol regulatory elementbinding protein; FAS, fatty acid synthase; EGCG, (−)-epigallocatechin-3-gallate; Glc, glucose; Tg, triglyceride; T-Cho, total cholesterol; AST, aspartate aminotransferase; ALT, alanine aminotransferase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. ∗ Corresponding author at: Department of Nutrition, Faculty of Health Sciences, Aomori University of Health and Welfare, Mase 58-1, Hamadate, Aomori 030-8505, Japan, Tel.: +81 17 765 4184; fax: +81 17 765 4184. E-mail address: s [email protected] (S. Sato). 1382-6689/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.etap.2012.04.005
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reported to decrease lipogenic gene transcription via suppression of SREBP-1c (Howell et al., 2009; Kim et al., 1999). Previous studies analyzed the physiological effects of plant polyphenolic compounds such as soy isoflavones, (−)epigallocatechin-3-gallate (EGCG), and quercetin on obesity progression (Ørgaard and Jensen, 2008; Rains et al., 2011; Rivera et al., 2008). Therefore, plant polyphenolic compounds, including tea and coffee polyphenols, are believed to have beneficial effects on the prevention of obesity (Grove and Lambert, 2010; Murase et al., 2011). Lignin, which is one of the most abundant organic substances in the plant kingdom, is seldom utilized because of its resistance to chemical and biological degradation. Lignophenols (LP), which are derivatives of lignin, have been isolated using a phase-separation system (Funaoka et al., 1995; Funaoka and Fukatsu, 1996), and their chemical structure is similar to that of natural lignin. Although LP is known to be highly phenolic and highly stable, and to exhibit antioxidant properties in vitro (Fujita et al., 2003), the physiological role of LP remains unclear. LP derived from bamboo was reported to prevent hydrogen peroxide-induced cell death in vitro (Akao et al., 2004). We also showed that LP reduced oleate-induced apolipoprotein-B secretion and sterol regulatory elementbinding protein (SREBP)-2 in HepG2 cells (Norikura et al., 2010). Moreover, LP attenuated the excess oxidative stress, infiltration and activation of macrophages, and glomerular expansion in the kidneys of streptozotocin-induced diabetic rats (Sato et al., 2009). However, there is limited information about the potential beneficial effects of an LP-rich diet on dietinduced obesity. The present study was designed to evaluate whether lignin-derived LP had an effect on the expression of the SREBP1c gene in the liver of rats fed a high-fat diet. In addition, the effect of LP treatment on lipid metabolism is discussed.
2.
Materials and methods
2.1.
Preparation of LP
LP is composed of a phenol and a concentrated acid, which converts the native lignin into highly phenolic polymers (Fujita et al., 2003; Funaoka et al., 1995). LP was prepared by the 2-step method established by Funaoka and Fukatsu (1996). In brief, 3 mol/C9 (phenyl propane unit of lignin) of p-cresol dissolved in acetone was added to defatted wood meal (beech sugi), and the acetone was evaporated by stirring. Thereafter, sulfuric acid (72%, 5 mL/g wood) was added to the mixture with vigorous stirring at 30 ◦ C for 60 min. The reaction mixture was rapidly poured into distilled water. The insoluble fraction was collected by centrifugation, washed with distilled water until a neutral pH was achieved, and lyophilized. The dried insoluble fraction was extracted with acetone. The acetone solution was concentrated under reduced pressure, added dropwise to an excess amount of a mixture of benzene/h-hexane (= 2/1) with stirring, and washed by diethyl ether. The LP was decomposed into lower molecular weight components by second functional control and heating after alkali treatment. The LP dissolved in 1.0 M NaOH solution was heated at 170 ◦ C for 1 h. After cooling, the reaction mixture was acidified with 1.0 M HCl. The
Table 1 – Composition of the diets. High-fat diets
Fat (% energy) Carbohydrate (% energy) Protein (% energy) Digestible energy (kcal/g) Casein (g/kg) l-Cystine (g/kg) Cornstarch (g/kg) Maltodextrin 10 (g/kg) Sucrose (g/kg) Cellulose (g/kg) Soybean oil (g/kg) Lard (g/kg) Mineral mix S10026 (g/kg) Dicalcium phosphate (g/kg) Calcium carbonate (g/kg) Potassium citrate monohydrate (g/kg) Vitamin mix V10001 (g/kg) Choline bitartrate (g/kg) Lignophenol (g/kg)
Con
HF
HFLP
10.0 70.0 20.0 3.85 189.6 2.8 298.6 33.2 331.7 47.4 23.7 19.0 9.5 12.3 5.2 15.6
44.9 35.1 20.0 4.73 233.1 3.5 84.8 116.5 201.4 58.3 29.1 206.8 11.7 15.1 6.4 19.2
44.9 35.1 20.0 4.73 233.1 3.5 84.8 116.5 201.4 53.3 29.1 206.8 11.7 15.1 6.4 19.2
9.5 1.9 –
11.7 2.3 –
11.7 2.3 5.0
Con: control; HF: high-fat diet; HFLP: high-fat diet containing lignophenols.
precipitant was washed with deionized water and then dried. The molecular weight distribution of the alkalinedepolymerized lignocresol was analyzed by size exclusion chromatography. The chemical structure is illustrated in Fig. 1. The molecular weight of the LP from beech sugi was determined to be approximately 1500.
2.2.
Animals
This study was approved by the Animal Research Committee, Aomori University of Health and Welfare, and all procedures were performed in accordance with the Guidelines for Animal Experimentation of the Aomori University of Health and Welfare. Five-week-old male Sprague-Dawley rats (Charles River Laboratories Japan, Inc., Yokohama, Japan) weighing 260–321 g were used. They were maintained at a temperature of 23 ± 1 ◦ C under a 12 h light-dark cycle starting at 08:00 h. After this period, the animals that received a diet providing 45% of energy from fat were divided into 2 groups comprising 0% and 0.5% LP-containing diets (HFLP). The control animals were fed a diet providing 10% of energy from fat (control). The rats were fed the corresponding diets and tap water ad libitum. The composition of the diets is described in Table 1. As streptozotocin-induced diabetic rats treated with 1.0% LP exhibited significantly lower concentrations of plasma triglyceride compared with untreated diabetic rats in our previous study (Mukai et al., 2011), we used a 0.5% LP diet in the present study. Body weights and food intakes were measured during the LP treatments. Before they were killed at week 8, all animals were fasted overnight and then weighed, and blood samples were collected under ether anesthesia. The livers, kidneys, and hearts were quickly removed, rapidly rinsed, and weighed. Portions of the livers were immediately frozen in liquid nitrogen and stored at −80 ◦ C until used in experiments.
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-
O H3 C
OH
O
O
OH
H3 C OCH 3
OCH 3 O(H)
1.0N NaOH 170ºC
OCH 3
+O
OCH 3
OH
Fig. 1 – Structural features of the LP.
2.3.
Blood chemistry
Plasma samples were separated by centrifugation at 800 × g for 10 min at 4 ◦ C and tested for glucose (Glc), triglyceride (Tg), and total cholesterol (T-Cho) using a commercially available kit (Wako Pure Chemical Industries Ltd., Osaka, Japan). Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the plasma were determined using a blood chemistry autoanalyzer (Fuji Dry-Chem 3500V; Fuji Film, Tokyo, Japan).
2.4.
Western blotting
To separate the nuclear and cytoplasmic fractions from the livers, the CelLyticTM NuCLEARTM Extraction kit (Sigma–Aldrich, St. Louis, MO, USA) was used according to the manufacturer’s instructions. All samples and tubes were handled and chilled on ice, and western blot analyses were carried out on the nuclear and cytoplasmic extracts. The homogenates were centrifuged at 5000 × g for 45 min at 4 ◦ C. Supernatants were collected and the protein concentration was determined using the BCATM Protein Assay Kit (Pierce, Rockford, IL, USA). Proteins were resolved on 10% SDS-PAGE and electrotransferred onto PVDF membranes (GE Healthcare UK Ltd., Buckinghamshire, UK). The membranes were probed with mouse SREBP-1 antibodies (1:500, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and rabbit ACC (1:500; Cell Signaling Technology, Beverly, MA, USA), and phospho-ACC-Ser79 (1:500; Millipore Corp., Billerica, MA, USA). The membranes were then incubated with the appropriate secondary horseradish peroxidase-conjugated antibodies for 1 h at room temperature and enhanced with ECL Western Blotting Detection Reagents (GE Healthcare UK Ltd.) on Hyperfilm (GE Healthcare UK Ltd.). Quantitative analysis of the specific band density was performed using ATTO densitometry software (ATTO Corp., Tokyo, Japan). Protein levels were normalized to the -actin expression from the same sample and reported as fold of the levels obtained for the control rats.
2.5.
Real-time PCR
Total RNA in the liver was extracted using the SV Total RNA Isolation System (Promega Corp., WI, USA) according to the manufacturer’s instructions, and cDNA was synthesized using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). The mRNA levels of SREBP-1c were analyzed using the following
primers and probe: forward, 5 -GGAGCCATGGATTGCACATT3 ; reverse, 5 -AGGAAGGCTTCCAGAGAGGA-3 , probe; 5 -ACATGCTTCAGCTCATCAACAACCAA-3 . Glyceraldehyde3-phosphate dehydrogenase (GAPDH) mRNA was analyzed using the TaqMan Rodent GAPDH Control Reagent Kit (Applied Biosystems) as an endogenous control. Real-time PCR was performed using the Universal PCR Master Mix (Applied Biosystems) on an Applied Biosystems 7000 Sequence Detection System according to the manufacturer’s instructions. Gene expression levels were expressed relative to the GAPDH intensity of each co-amplified sample.
2.6.
Statistical analysis
Statistical analyses were performed by one-way analysis of variance (ANOVA), followed by the Tukey test. Each value was expressed as mean ± SEM. In all cases, P < 0.05 was considered statistically significant.
3.
Results
3.1. Effect of LP treatment on body weights and food intakes Although there was no significant difference between the high-fat diet fed rats and HFLP rats, the latter showed a tendency for decreased weight during the treatments (Fig. 2). At week 2, the food intake in the HFLP group was significantly lower than that in the high-fat diet-fed group. However, no differences in food intake were observed between the groups during treatments.
3.2.
Tissue weights and plasma parameters
The absolute and relative weights of the epididymal fat tissues in the HF group were significantly higher than those in the control group were (Table 2). Conversely, the absolute and relative weights of the epididymal fat tissues in the HFLP group were significantly lower than that in the high-fat dietfed group. Although a statistically significant difference was not achieved, a tendency toward decreased absolute and relative weights of the perirenal fat tissues was observed in the HFLP group compared with the high-fat diet-fed group. The relative weight of the liver in the HFLP group was significantly higher than that in the high-fat diet-fed group (Table 2), and similar to that in the control group.
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700
Body weight (g)
600 500
a
400
a
a
a
a
a
Con
200
HF HFLP
100
30 25
a
300
B Food intake (g)
A
20
b
15 Con 10
HF HFLP
5 0
0
0
1
2
3
4
5
6
7
1
2
3
4
5
6
7
LP treatment periods (weeks) Fig. 2 – Effects of LP treatment on body weight (A) and food intake (B) in rats fed high-fat diet. Values are mean ± SEM (n = 8). a P < 0.05 compared with the control group. b P < 0.05 compared with the high-fat diet-fed group.
Table 2 – Morphological characteristics. High-fat diet
c
Con
HF
± ± ± ± ± ± ± ± ± ± ±
± ± ± ± ± ± ± ± ± ± ±
504 11.90 2.96 1.27 22.85 8.73 23.63 5.88 2.52 28.83 17.28
BW (g) Liver (g) Kidney (g) Heart (g) Perirenal fat tissue Epididymal fat tissue Liver/BW (g/kg) Kidney/BW (g/kg) Heart/BW (g/kg) Perirenal fat tissue/BW (g/kg) Epididymal fat tissue/BW (g/kg)
11 0.35 0.08 0.04 1.57 0.73 0.61 0.11 0.05 2.86 1.29
585 12.97 3.00 1.29 25.49 16.07 22.15 5.14 2.21 43.17 27.24
HFLP a
12 0.42 0.05 0.02 2.22 1.28a 0.50 0.09 0.05 2.98a 1.66a
563 13.75 2.99 1.29 19.80 11.81 24.32 5.31 2.29 34.86 20.88
± ± ± ± ± ± ± ± ± ± ±
23a 0.84 0.14 0.06 1.80 0.87a,b 0.61b 0.14 0.03 2.16 1.08b
Con: control; HF: high-fat diet; HFLP: high-fat diet containing lignophenols; BW: body weight. Values are mean ± SEM (n = 8). a P < 0.05 compared with Con group. b P < 0.05 compared with HF group. c At sacrifice after fasting overnight.
A significant difference in the plasma Tg levels was found between the high-fat diet-fed and HFLP groups (Table 3). No difference in plasma AST was observed between the groups. There was a significant increase of ALT in the HFLP group compared with the high-fat diet-fed group, but no significant difference in AST/ALT ratio was detected between the high-fat diet-fed and HFLP groups.
3.3. Effect of LP treatment on the expression of SREBP-1c mRNA and protein SREBP-1c mRNA expression was significantly higher in the livers of high-fat diet-fed rats than in the control group rats (Fig. 3A). Conversely, the levels of SREBP-1c mRNA in HFLP rats were decreased significantly compared with those in the
Table 3 – Plasma parameters. High-fat diet Con Triglyceride (mg/dL) Total cholesterol (mg/dL) Glucose (mg/dL) Insulin (ng/mL) AST (U/L) ALT (U/L) AST/ALT ratio
67.9 66.4 133.2 1.96 100.0 25.6 3.96
± ± ± ± ± ± ±
10.2 4.5 4.7 0.28 7.3 1.4 0.35
HF 84.4 72.0 133.0 2.02 113.8 32.1 3.65
± ± ± ± ± ± ±
HFLP 8.8 6.6 4.4 0.31 6.6 2.0 0.26
35.9 74.4 131.8 2.54 116.4 41.3 2.83
± ± ± ± ± ± ±
6.9b 7.2 3.3 0.41 11.3 4.0b 0.21a
Con: control; HF: high-fat diet; HFLP: high-fat diet containing lignophenols; AST: aspartate aminotransferase; ALT: alanine aminotransferase. Values are mean ± SEM (n = 7–8). a P < 0.05 compared with Con group. b P < 0.05 compared with HF group.
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Fig. 3 – Effects of LP treatment on the expression of SREBP-1c mRNA (A) and SREBP-1 protein in cytoplasmic extracts (B) of the livers of rats fed a high-fat diet. Values are mean ± SEM (n = 7–8). a P < 0.05 compared with the control group. b P < 0.05 compared with the high-fat diet-fed group.
high-fat diet-fed group, indicating that LP suppressed the mRNA expression of SREBP-1c in the liver. We performed western blot analysis to demonstrate the effect of LP treatment on protein expression in the liver of rats fed high-fat diets. A tendency toward decreased SREBP-1 protein levels in the hepatic cytoplasmic extracts in the HFLP group was observed, as compared with the high-fat diet-fed group (Fig. 3B). However, a statistically significant difference was not achieved. There was no significant difference from the SREBP-1 protein levels in the hepatic nuclear extracts among three groups (data not shown).
3.4. Effect of LP treatment on the expression of ACC protein
In this study, we found that epididymal fat tissue weights and plasma Tg levels were reduced in the HFLP group compared with the high-fat diet-fed group without a change in food intake, suggesting that LP treatment may exert a hypolipidemic effect in rats fed a high-fat diet. Previous studies reported the decrease of plasma parameters such as Tg and T-Cho by plant polyphenolic compounds in animal models of high-fat diet-induced obesity (Lee et al., 2009; Torre-Villalvazo et al., 2008; Wang et al., 2011). For instance, treatment with coffee polyphenols significantly reduced body weight gain, and abdominal and liver fat accumulation in high-fat dietfed mice (Murase et al., 2011). In obese Zucker rats, chronic treatment with quercetin, which is a plant polyphenolic compound, reduced the plasma concentrations of Tg, T-Cho, and free fatty acids (Rivera et al., 2008). Moreover, treatment with
As ACC is one of the target enzymes of SREBP-1c, we performed western blot analysis to measure the expression of ACC protein. Phosphorylated ACC levels in the high-fat dietfed rats were significantly lower than that in the Con group, whereas a significant increase in the abundance of phosphorylated ACC was detected in the HFLP group compared with the high-fat diet-fed group (Fig. 4), indicating that LP treatment was associated with the inactivation of ACC in the liver.
4.
Discussion
LP is a derivative of lignin, which is a component of plant cell walls. LP was produced experimentally using a phaseseparation system, and is composed of a phenol and a concentrated acid, which converts the native lignin into a highly phenolic polymer (Funaoka et al., 1995; Funaoka and Fukatsu, 1996). The chemical structure is similar to that of natural lignin (Fig. 1). Although LP is known to have polyphenolic and antioxidant properties (Fujita et al., 2003), the physiological roles of LP associated with its beneficial effect on diet-induced obesity have not yet been clarified. The major findings of the present study are as follows: (i) the weights of adipose tissues and plasma Tg levels were reduced, (ii) the expression of SREBP-1c mRNA was down-regulated, and (iii) ACC, a target enzyme of SREBP-1c, was inactivated in the livers of rats fed a high-fat diet when lignin-derived LP was provided.
Fig. 4 – Effects of LP treatment on the protein expression of acetyl-CoA carboxylase in the livers of rats fed a high-fat diet. Values are mean ± SEM (n = 7–8). a P < 0.05 compared with the control group. b P < 0.05 compared with the high-fat diet-fed group.
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 4 ( 2 0 1 2 ) 228–234
EGCG decreased liver weight and liver Tg levels in high-fat diet-fed mice (Bose et al., 2008). Recently, we demonstrated that 1.0% LP treatment lowered the plasma levels of Tg and T-Cho in streptozotocin-induced diabetic rats (Mukai et al., 2011). Therefore, because LP is also a polyphenolic compound, it appears likely that LP treatment suppresses the abdominal fat accumulation in obesity. Although the plasma ALT levels were increased in the HFLP group in this study, there was no significant difference in plasma AST between the groups. In addition, the elevated plasma aminotransferase levels in the HFLP group were not associated with a high AST/ALT ratio. These results suggested that LP treatment does not increase liver damage. Interestingly, LP significantly reduced the expression of SREBP-1c mRNA, and a tendency toward decreased SREBP-1 protein levels was observed in the HFLP group. SREBPs are transcription factors that regulate lipid homeostasis, and are encoded by 2 genes, SREBP-1a and SREBP-1c (Horton et al., 2002). SREBP-1c binds the sterol regulator element in the gene promoters of enzymes involved in lipogenesis, such as ACC and FAS (Jump et al., 2005). When fed an EGCG-containing diet, the mRNA levels of SREBP-1c decreased significantly in the epididymal white adipose tissue of high-fat diet-fed mice (Lee et al., 2009). Furthermore, polyphenol extracted from the bark of the black wattle tree (Acacia meansii) reduced the mRNA expressions of the fatty acid synthesis-related genes SREBP1c, ACC, and FAS in the liver of mice with high-fat diet-induced obesity (Ikarashi et al., 2011). On the other hand, several studies reported that EGCG activated AMP-activated protein kinase (AMPK) in cultured cells (Huang et al., 2009; Murase et al., 2009). AMPK is known to phosphorylate a number of metabolic enzymes associated with lipid metabolism in the liver, such as ACC, which is phosphorylated and inactivated by AMPK (Hardie and Pan, 2002; Saha and Ruderman, 2003). Based on the present findings and previous studies, we hypothesized that LP, which is a polyphenolic compound, may reduce fatty acid synthesis, at least in part by suppressing the expression of SREBP-1c and/or by increasing the phosphorylation of ACC in the liver, and may lead to the decrease of plasma Tg levels. In conclusion, we demonstrated that LP treatment suppresses the weight of adipose tissues, plasma Tg levels, and hepatic expression of SREBP-1c mRNA in rats fed a high-fat diet. Although the mechanism underlying the effects of LP treatment on lipid metabolism in diet-induced obesity should be investigated in future studies, our results may facilitate the formulation of preventive care strategies for obesity.
Conflict of interest statement The authors declare that there are no conflicts of interest.
Role of the funding source This study was supported by Adaptable and Seamless Technology transfer Program through target-driven R&D (AS221Z02004G), Japan Science and Technology Agency research grant.
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Acknowledgement The authors thank Keiko Tamakuma for her technical assistance.
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Available online at www.sciencedirect.com
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Effect of lignin-derived lignophenols on hepatic lipid metabolism in rats fed a high-fat diet Shin Sato a,∗ , Yuuka Mukai a , Yukari Tokuoka a , Keigo Mikame b , Masamitsu Funaoka b , Shuzo Fujita a a
Department of Nutrition, Faculty of Health Sciences, Aomori University of Health and Welfare, Mase 58-1, Hamadate, Aomori 030-0841, Japan b Department of Environmental Science and Technology, Faculty of Bioresources, Mie University, Tsu 514-8507, Japan
a r t i c l e
i n f o
a b s t r a c t
Article history:
The effect of lignin-derived lignophenols on lipid metabolism in the livers of rats fed a high-
Received 28 December 2011
fat diet was investigated. Rats fed a diet providing 45% of energy from fat were divided into
Received in revised form
2 groups, namely 0% and 0.5% lignophenols-containing diets. The controls were fed a diet
5 April 2012
providing 10% of energy from fat. Plasma blood parameters, protein expression of acetyl-CoA
Accepted 6 April 2012
carboxylase (ACC) and sterol regulatory element-binding protein (SREBP)-1, and SREBP-1c
Available online 13 April 2012
mRNA expression in the livers were examined. The plasma triglyceride levels in the rats fed lignophenols-containing diets were decreased. SREBP-1c mRNA expression in the rats fed
Keywords:
lignophenols-containing diets was significantly reduced compared with the rats fed high-
Lignophenols
fat diets, and phosphorylated ACC protein in the rats fed lignophenols-containing diets was
Obesity
significantly increased. Our results suggested that lignophenols suppress the expression of
Sterol regulatory element-binding
SREBP-1c mRNA and the phosphorylation of ACC in the liver, and may lead to a decrease in
protein-1c
plasma triglyceride levels.
Acetyl-CoA carboxylase
© 2012 Elsevier B.V. All rights reserved.
High-fat diet
1.
Introduction
Obesity has become one of the most prevalent health problems around the world, and a risk factor for a number of chronic diseases such as insulin resistance, type 2 diabetes mellitus, and cardiovascular disease (Browning and Horton, 2004). In general, it is accepted that obesity results from an imbalance between energy intake and expenditure, and is characterized by increased fat accumulation in adipose
tissue and elevated lipid concentrations in the blood. Dietary fat is one of the main environmental factors associated with the incidence of obesity. Thus, it is important to establish strategies for the prevention of obesity. Sterol regulatory element-binding protein (SREBP)-1c is an important regulator of lipogenic enzymes such as acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) (Horton et al., 2002; Jump et al., 2005). Overexpression of SREBP-1c increased triglyceride accumulation and fatty acid synthesis (You and Crabb, 2004). In contrast, polyunsaturated fatty acids were
Abbreviations: LP, lignophenols; HFLP, LP-containing high-fat diet; ACC, acetyl-CoA carboxylase; SREBP, sterol regulatory elementbinding protein; FAS, fatty acid synthase; EGCG, (−)-epigallocatechin-3-gallate; Glc, glucose; Tg, triglyceride; T-Cho, total cholesterol; AST, aspartate aminotransferase; ALT, alanine aminotransferase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. ∗ Corresponding author at: Department of Nutrition, Faculty of Health Sciences, Aomori University of Health and Welfare, Mase 58-1, Hamadate, Aomori 030-8505, Japan, Tel.: +81 17 765 4184; fax: +81 17 765 4184. E-mail address: s [email protected] (S. Sato). 1382-6689/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.etap.2012.04.005
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reported to decrease lipogenic gene transcription via suppression of SREBP-1c (Howell et al., 2009; Kim et al., 1999). Previous studies analyzed the physiological effects of plant polyphenolic compounds such as soy isoflavones, (−)epigallocatechin-3-gallate (EGCG), and quercetin on obesity progression (Ørgaard and Jensen, 2008; Rains et al., 2011; Rivera et al., 2008). Therefore, plant polyphenolic compounds, including tea and coffee polyphenols, are believed to have beneficial effects on the prevention of obesity (Grove and Lambert, 2010; Murase et al., 2011). Lignin, which is one of the most abundant organic substances in the plant kingdom, is seldom utilized because of its resistance to chemical and biological degradation. Lignophenols (LP), which are derivatives of lignin, have been isolated using a phase-separation system (Funaoka et al., 1995; Funaoka and Fukatsu, 1996), and their chemical structure is similar to that of natural lignin. Although LP is known to be highly phenolic and highly stable, and to exhibit antioxidant properties in vitro (Fujita et al., 2003), the physiological role of LP remains unclear. LP derived from bamboo was reported to prevent hydrogen peroxide-induced cell death in vitro (Akao et al., 2004). We also showed that LP reduced oleate-induced apolipoprotein-B secretion and sterol regulatory elementbinding protein (SREBP)-2 in HepG2 cells (Norikura et al., 2010). Moreover, LP attenuated the excess oxidative stress, infiltration and activation of macrophages, and glomerular expansion in the kidneys of streptozotocin-induced diabetic rats (Sato et al., 2009). However, there is limited information about the potential beneficial effects of an LP-rich diet on dietinduced obesity. The present study was designed to evaluate whether lignin-derived LP had an effect on the expression of the SREBP1c gene in the liver of rats fed a high-fat diet. In addition, the effect of LP treatment on lipid metabolism is discussed.
2.
Materials and methods
2.1.
Preparation of LP
LP is composed of a phenol and a concentrated acid, which converts the native lignin into highly phenolic polymers (Fujita et al., 2003; Funaoka et al., 1995). LP was prepared by the 2-step method established by Funaoka and Fukatsu (1996). In brief, 3 mol/C9 (phenyl propane unit of lignin) of p-cresol dissolved in acetone was added to defatted wood meal (beech sugi), and the acetone was evaporated by stirring. Thereafter, sulfuric acid (72%, 5 mL/g wood) was added to the mixture with vigorous stirring at 30 ◦ C for 60 min. The reaction mixture was rapidly poured into distilled water. The insoluble fraction was collected by centrifugation, washed with distilled water until a neutral pH was achieved, and lyophilized. The dried insoluble fraction was extracted with acetone. The acetone solution was concentrated under reduced pressure, added dropwise to an excess amount of a mixture of benzene/h-hexane (= 2/1) with stirring, and washed by diethyl ether. The LP was decomposed into lower molecular weight components by second functional control and heating after alkali treatment. The LP dissolved in 1.0 M NaOH solution was heated at 170 ◦ C for 1 h. After cooling, the reaction mixture was acidified with 1.0 M HCl. The
Table 1 – Composition of the diets. High-fat diets
Fat (% energy) Carbohydrate (% energy) Protein (% energy) Digestible energy (kcal/g) Casein (g/kg) l-Cystine (g/kg) Cornstarch (g/kg) Maltodextrin 10 (g/kg) Sucrose (g/kg) Cellulose (g/kg) Soybean oil (g/kg) Lard (g/kg) Mineral mix S10026 (g/kg) Dicalcium phosphate (g/kg) Calcium carbonate (g/kg) Potassium citrate monohydrate (g/kg) Vitamin mix V10001 (g/kg) Choline bitartrate (g/kg) Lignophenol (g/kg)
Con
HF
HFLP
10.0 70.0 20.0 3.85 189.6 2.8 298.6 33.2 331.7 47.4 23.7 19.0 9.5 12.3 5.2 15.6
44.9 35.1 20.0 4.73 233.1 3.5 84.8 116.5 201.4 58.3 29.1 206.8 11.7 15.1 6.4 19.2
44.9 35.1 20.0 4.73 233.1 3.5 84.8 116.5 201.4 53.3 29.1 206.8 11.7 15.1 6.4 19.2
9.5 1.9 –
11.7 2.3 –
11.7 2.3 5.0
Con: control; HF: high-fat diet; HFLP: high-fat diet containing lignophenols.
precipitant was washed with deionized water and then dried. The molecular weight distribution of the alkalinedepolymerized lignocresol was analyzed by size exclusion chromatography. The chemical structure is illustrated in Fig. 1. The molecular weight of the LP from beech sugi was determined to be approximately 1500.
2.2.
Animals
This study was approved by the Animal Research Committee, Aomori University of Health and Welfare, and all procedures were performed in accordance with the Guidelines for Animal Experimentation of the Aomori University of Health and Welfare. Five-week-old male Sprague-Dawley rats (Charles River Laboratories Japan, Inc., Yokohama, Japan) weighing 260–321 g were used. They were maintained at a temperature of 23 ± 1 ◦ C under a 12 h light-dark cycle starting at 08:00 h. After this period, the animals that received a diet providing 45% of energy from fat were divided into 2 groups comprising 0% and 0.5% LP-containing diets (HFLP). The control animals were fed a diet providing 10% of energy from fat (control). The rats were fed the corresponding diets and tap water ad libitum. The composition of the diets is described in Table 1. As streptozotocin-induced diabetic rats treated with 1.0% LP exhibited significantly lower concentrations of plasma triglyceride compared with untreated diabetic rats in our previous study (Mukai et al., 2011), we used a 0.5% LP diet in the present study. Body weights and food intakes were measured during the LP treatments. Before they were killed at week 8, all animals were fasted overnight and then weighed, and blood samples were collected under ether anesthesia. The livers, kidneys, and hearts were quickly removed, rapidly rinsed, and weighed. Portions of the livers were immediately frozen in liquid nitrogen and stored at −80 ◦ C until used in experiments.
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-
O H3 C
OH
O
O
OH
H3 C OCH 3
OCH 3 O(H)
1.0N NaOH 170ºC
OCH 3
+O
OCH 3
OH
Fig. 1 – Structural features of the LP.
2.3.
Blood chemistry
Plasma samples were separated by centrifugation at 800 × g for 10 min at 4 ◦ C and tested for glucose (Glc), triglyceride (Tg), and total cholesterol (T-Cho) using a commercially available kit (Wako Pure Chemical Industries Ltd., Osaka, Japan). Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the plasma were determined using a blood chemistry autoanalyzer (Fuji Dry-Chem 3500V; Fuji Film, Tokyo, Japan).
2.4.
Western blotting
To separate the nuclear and cytoplasmic fractions from the livers, the CelLyticTM NuCLEARTM Extraction kit (Sigma–Aldrich, St. Louis, MO, USA) was used according to the manufacturer’s instructions. All samples and tubes were handled and chilled on ice, and western blot analyses were carried out on the nuclear and cytoplasmic extracts. The homogenates were centrifuged at 5000 × g for 45 min at 4 ◦ C. Supernatants were collected and the protein concentration was determined using the BCATM Protein Assay Kit (Pierce, Rockford, IL, USA). Proteins were resolved on 10% SDS-PAGE and electrotransferred onto PVDF membranes (GE Healthcare UK Ltd., Buckinghamshire, UK). The membranes were probed with mouse SREBP-1 antibodies (1:500, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and rabbit ACC (1:500; Cell Signaling Technology, Beverly, MA, USA), and phospho-ACC-Ser79 (1:500; Millipore Corp., Billerica, MA, USA). The membranes were then incubated with the appropriate secondary horseradish peroxidase-conjugated antibodies for 1 h at room temperature and enhanced with ECL Western Blotting Detection Reagents (GE Healthcare UK Ltd.) on Hyperfilm (GE Healthcare UK Ltd.). Quantitative analysis of the specific band density was performed using ATTO densitometry software (ATTO Corp., Tokyo, Japan). Protein levels were normalized to the -actin expression from the same sample and reported as fold of the levels obtained for the control rats.
2.5.
Real-time PCR
Total RNA in the liver was extracted using the SV Total RNA Isolation System (Promega Corp., WI, USA) according to the manufacturer’s instructions, and cDNA was synthesized using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). The mRNA levels of SREBP-1c were analyzed using the following
primers and probe: forward, 5 -GGAGCCATGGATTGCACATT3 ; reverse, 5 -AGGAAGGCTTCCAGAGAGGA-3 , probe; 5 -ACATGCTTCAGCTCATCAACAACCAA-3 . Glyceraldehyde3-phosphate dehydrogenase (GAPDH) mRNA was analyzed using the TaqMan Rodent GAPDH Control Reagent Kit (Applied Biosystems) as an endogenous control. Real-time PCR was performed using the Universal PCR Master Mix (Applied Biosystems) on an Applied Biosystems 7000 Sequence Detection System according to the manufacturer’s instructions. Gene expression levels were expressed relative to the GAPDH intensity of each co-amplified sample.
2.6.
Statistical analysis
Statistical analyses were performed by one-way analysis of variance (ANOVA), followed by the Tukey test. Each value was expressed as mean ± SEM. In all cases, P < 0.05 was considered statistically significant.
3.
Results
3.1. Effect of LP treatment on body weights and food intakes Although there was no significant difference between the high-fat diet fed rats and HFLP rats, the latter showed a tendency for decreased weight during the treatments (Fig. 2). At week 2, the food intake in the HFLP group was significantly lower than that in the high-fat diet-fed group. However, no differences in food intake were observed between the groups during treatments.
3.2.
Tissue weights and plasma parameters
The absolute and relative weights of the epididymal fat tissues in the HF group were significantly higher than those in the control group were (Table 2). Conversely, the absolute and relative weights of the epididymal fat tissues in the HFLP group were significantly lower than that in the high-fat dietfed group. Although a statistically significant difference was not achieved, a tendency toward decreased absolute and relative weights of the perirenal fat tissues was observed in the HFLP group compared with the high-fat diet-fed group. The relative weight of the liver in the HFLP group was significantly higher than that in the high-fat diet-fed group (Table 2), and similar to that in the control group.
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700
Body weight (g)
600 500
a
400
a
a
a
a
a
Con
200
HF HFLP
100
30 25
a
300
B Food intake (g)
A
20
b
15 Con 10
HF HFLP
5 0
0
0
1
2
3
4
5
6
7
1
2
3
4
5
6
7
LP treatment periods (weeks) Fig. 2 – Effects of LP treatment on body weight (A) and food intake (B) in rats fed high-fat diet. Values are mean ± SEM (n = 8). a P < 0.05 compared with the control group. b P < 0.05 compared with the high-fat diet-fed group.
Table 2 – Morphological characteristics. High-fat diet
c
Con
HF
± ± ± ± ± ± ± ± ± ± ±
± ± ± ± ± ± ± ± ± ± ±
504 11.90 2.96 1.27 22.85 8.73 23.63 5.88 2.52 28.83 17.28
BW (g) Liver (g) Kidney (g) Heart (g) Perirenal fat tissue Epididymal fat tissue Liver/BW (g/kg) Kidney/BW (g/kg) Heart/BW (g/kg) Perirenal fat tissue/BW (g/kg) Epididymal fat tissue/BW (g/kg)
11 0.35 0.08 0.04 1.57 0.73 0.61 0.11 0.05 2.86 1.29
585 12.97 3.00 1.29 25.49 16.07 22.15 5.14 2.21 43.17 27.24
HFLP a
12 0.42 0.05 0.02 2.22 1.28a 0.50 0.09 0.05 2.98a 1.66a
563 13.75 2.99 1.29 19.80 11.81 24.32 5.31 2.29 34.86 20.88
± ± ± ± ± ± ± ± ± ± ±
23a 0.84 0.14 0.06 1.80 0.87a,b 0.61b 0.14 0.03 2.16 1.08b
Con: control; HF: high-fat diet; HFLP: high-fat diet containing lignophenols; BW: body weight. Values are mean ± SEM (n = 8). a P < 0.05 compared with Con group. b P < 0.05 compared with HF group. c At sacrifice after fasting overnight.
A significant difference in the plasma Tg levels was found between the high-fat diet-fed and HFLP groups (Table 3). No difference in plasma AST was observed between the groups. There was a significant increase of ALT in the HFLP group compared with the high-fat diet-fed group, but no significant difference in AST/ALT ratio was detected between the high-fat diet-fed and HFLP groups.
3.3. Effect of LP treatment on the expression of SREBP-1c mRNA and protein SREBP-1c mRNA expression was significantly higher in the livers of high-fat diet-fed rats than in the control group rats (Fig. 3A). Conversely, the levels of SREBP-1c mRNA in HFLP rats were decreased significantly compared with those in the
Table 3 – Plasma parameters. High-fat diet Con Triglyceride (mg/dL) Total cholesterol (mg/dL) Glucose (mg/dL) Insulin (ng/mL) AST (U/L) ALT (U/L) AST/ALT ratio
67.9 66.4 133.2 1.96 100.0 25.6 3.96
± ± ± ± ± ± ±
10.2 4.5 4.7 0.28 7.3 1.4 0.35
HF 84.4 72.0 133.0 2.02 113.8 32.1 3.65
± ± ± ± ± ± ±
HFLP 8.8 6.6 4.4 0.31 6.6 2.0 0.26
35.9 74.4 131.8 2.54 116.4 41.3 2.83
± ± ± ± ± ± ±
6.9b 7.2 3.3 0.41 11.3 4.0b 0.21a
Con: control; HF: high-fat diet; HFLP: high-fat diet containing lignophenols; AST: aspartate aminotransferase; ALT: alanine aminotransferase. Values are mean ± SEM (n = 7–8). a P < 0.05 compared with Con group. b P < 0.05 compared with HF group.
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Fig. 3 – Effects of LP treatment on the expression of SREBP-1c mRNA (A) and SREBP-1 protein in cytoplasmic extracts (B) of the livers of rats fed a high-fat diet. Values are mean ± SEM (n = 7–8). a P < 0.05 compared with the control group. b P < 0.05 compared with the high-fat diet-fed group.
high-fat diet-fed group, indicating that LP suppressed the mRNA expression of SREBP-1c in the liver. We performed western blot analysis to demonstrate the effect of LP treatment on protein expression in the liver of rats fed high-fat diets. A tendency toward decreased SREBP-1 protein levels in the hepatic cytoplasmic extracts in the HFLP group was observed, as compared with the high-fat diet-fed group (Fig. 3B). However, a statistically significant difference was not achieved. There was no significant difference from the SREBP-1 protein levels in the hepatic nuclear extracts among three groups (data not shown).
3.4. Effect of LP treatment on the expression of ACC protein
In this study, we found that epididymal fat tissue weights and plasma Tg levels were reduced in the HFLP group compared with the high-fat diet-fed group without a change in food intake, suggesting that LP treatment may exert a hypolipidemic effect in rats fed a high-fat diet. Previous studies reported the decrease of plasma parameters such as Tg and T-Cho by plant polyphenolic compounds in animal models of high-fat diet-induced obesity (Lee et al., 2009; Torre-Villalvazo et al., 2008; Wang et al., 2011). For instance, treatment with coffee polyphenols significantly reduced body weight gain, and abdominal and liver fat accumulation in high-fat dietfed mice (Murase et al., 2011). In obese Zucker rats, chronic treatment with quercetin, which is a plant polyphenolic compound, reduced the plasma concentrations of Tg, T-Cho, and free fatty acids (Rivera et al., 2008). Moreover, treatment with
As ACC is one of the target enzymes of SREBP-1c, we performed western blot analysis to measure the expression of ACC protein. Phosphorylated ACC levels in the high-fat dietfed rats were significantly lower than that in the Con group, whereas a significant increase in the abundance of phosphorylated ACC was detected in the HFLP group compared with the high-fat diet-fed group (Fig. 4), indicating that LP treatment was associated with the inactivation of ACC in the liver.
4.
Discussion
LP is a derivative of lignin, which is a component of plant cell walls. LP was produced experimentally using a phaseseparation system, and is composed of a phenol and a concentrated acid, which converts the native lignin into a highly phenolic polymer (Funaoka et al., 1995; Funaoka and Fukatsu, 1996). The chemical structure is similar to that of natural lignin (Fig. 1). Although LP is known to have polyphenolic and antioxidant properties (Fujita et al., 2003), the physiological roles of LP associated with its beneficial effect on diet-induced obesity have not yet been clarified. The major findings of the present study are as follows: (i) the weights of adipose tissues and plasma Tg levels were reduced, (ii) the expression of SREBP-1c mRNA was down-regulated, and (iii) ACC, a target enzyme of SREBP-1c, was inactivated in the livers of rats fed a high-fat diet when lignin-derived LP was provided.
Fig. 4 – Effects of LP treatment on the protein expression of acetyl-CoA carboxylase in the livers of rats fed a high-fat diet. Values are mean ± SEM (n = 7–8). a P < 0.05 compared with the control group. b P < 0.05 compared with the high-fat diet-fed group.
e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 4 ( 2 0 1 2 ) 228–234
EGCG decreased liver weight and liver Tg levels in high-fat diet-fed mice (Bose et al., 2008). Recently, we demonstrated that 1.0% LP treatment lowered the plasma levels of Tg and T-Cho in streptozotocin-induced diabetic rats (Mukai et al., 2011). Therefore, because LP is also a polyphenolic compound, it appears likely that LP treatment suppresses the abdominal fat accumulation in obesity. Although the plasma ALT levels were increased in the HFLP group in this study, there was no significant difference in plasma AST between the groups. In addition, the elevated plasma aminotransferase levels in the HFLP group were not associated with a high AST/ALT ratio. These results suggested that LP treatment does not increase liver damage. Interestingly, LP significantly reduced the expression of SREBP-1c mRNA, and a tendency toward decreased SREBP-1 protein levels was observed in the HFLP group. SREBPs are transcription factors that regulate lipid homeostasis, and are encoded by 2 genes, SREBP-1a and SREBP-1c (Horton et al., 2002). SREBP-1c binds the sterol regulator element in the gene promoters of enzymes involved in lipogenesis, such as ACC and FAS (Jump et al., 2005). When fed an EGCG-containing diet, the mRNA levels of SREBP-1c decreased significantly in the epididymal white adipose tissue of high-fat diet-fed mice (Lee et al., 2009). Furthermore, polyphenol extracted from the bark of the black wattle tree (Acacia meansii) reduced the mRNA expressions of the fatty acid synthesis-related genes SREBP1c, ACC, and FAS in the liver of mice with high-fat diet-induced obesity (Ikarashi et al., 2011). On the other hand, several studies reported that EGCG activated AMP-activated protein kinase (AMPK) in cultured cells (Huang et al., 2009; Murase et al., 2009). AMPK is known to phosphorylate a number of metabolic enzymes associated with lipid metabolism in the liver, such as ACC, which is phosphorylated and inactivated by AMPK (Hardie and Pan, 2002; Saha and Ruderman, 2003). Based on the present findings and previous studies, we hypothesized that LP, which is a polyphenolic compound, may reduce fatty acid synthesis, at least in part by suppressing the expression of SREBP-1c and/or by increasing the phosphorylation of ACC in the liver, and may lead to the decrease of plasma Tg levels. In conclusion, we demonstrated that LP treatment suppresses the weight of adipose tissues, plasma Tg levels, and hepatic expression of SREBP-1c mRNA in rats fed a high-fat diet. Although the mechanism underlying the effects of LP treatment on lipid metabolism in diet-induced obesity should be investigated in future studies, our results may facilitate the formulation of preventive care strategies for obesity.
Conflict of interest statement The authors declare that there are no conflicts of interest.
Role of the funding source This study was supported by Adaptable and Seamless Technology transfer Program through target-driven R&D (AS221Z02004G), Japan Science and Technology Agency research grant.
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Acknowledgement The authors thank Keiko Tamakuma for her technical assistance.
references
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