JOURNAL OF MEDICINAL FOOD J Med Food 17 (5) 2014, 571–581 # Mary Ann Liebert, Inc., and Korean Society of Food Science and Nutrition DOI: 10.1089/jmf.2013.2916

Sasa quelpaertensis Leaf Extract Improves High Fat Diet-Induced Lipid Abnormalities and Regulation of Lipid Metabolism Genes in Rats Jina Kim,1 Yoo-Sun Kim,1 Hyun Ah Lee,1 Ji Ye Lim,1 Mina Kim,1 Oran Kwon,1 Hee-Chul Ko,2 Se-Jae Kim,3 Jae-Ho Shin,4 Yuri Kim1 1

Department of Nutritional Science and Food Management, Ewha Womans University, Seoul, Korea. Jeju Sasa Industry Development Agency and 3Department of Biology, Jeju National University, Jeju-si, Jeju, Korea. 4 Department of Biomedical Laboratory Science, Eulji University, Seongnam-si, Kyeonggi-do, Korea.

2

ABSTRACT Sasa quelpaertensis is a bamboo leaf that is only grown on Jeju Island in South Korea. It is used as a bamboo tea that is consumed for therapeutic purposes, particularly for its anti-diabetic, diuretic, and anti-inflammatory effects. This study investigated the effect of S. quelpaertensis leaf extract (SQE) on high fat-induced lipid abnormalities and regulation of lipid metabolism-related gene expressions in rats. SQE supplementation significantly decreased the levels of plasma triglycerides, total cholesterol, and low-density lipoprotein cholesterol as well as the atherogenic index. SQE restored levels of plasma high-density lipoprotein cholesterol, which were lowered by a high fat diet. Plasma and cardiac resistin levels were also significantly decreased by SQE supplementation. In adipose tissue, mRNA levels of CAAT/enhancer-binding protein b (C/EBPb) were suppressed in the SQE group. SQE supplementation decreased the accumulation of lipid droplets, inflammatory cell infiltrations, levels of triglycerides, and total lipids in the liver and effectively down-regulated expression of sterol regulatory element binding protein-1 (SREBP-1), fatty acid synthetase (FAS), and uncoupling protein 2 (UCP-2). These results suggest that SQE may be a potential treatment for high fat-related disorders by improving lipid profiles and modulating lipid metabolism. KEY WORDS:  gene expression  high-fat diet  lipid metabolism  lipid profile  Sasa quelpaertensis leaf

and lipid homeostasis.9 Adiponectin has been found to decrease with obesity and is associated with metabolic and vascular disorders.10 Resistin is an adipocyte-specific secretory factor that has been found to be present at higher levels in obese rodents. Although the physiological role of resistin in humans remains unclear, it has been reported that resistin-induced insulin resistance and hyperresistinemia contribute to impaired insulin sensitivity in obese rodents.11 Resistin levels also tend to be elevated, while adiponectin levels are decreased, as visceral fat mass is increased in obese individuals.12 Molecular mechanisms that regulate preadipose cell growth, adipose differentiation, and lipogenesis in fat cells have been widely studied. For example, in adipose tissue, peroxisome proliferator activated receptors (PPARs), CAAT/ enhancer-binding proteins (C/EBPs), and sterol regulatorybinding proteins (SREBPs) have been shown to induce the differentiation and activation of adipocyte-specific gene transcription during the early stages of obesity.13–15 In particular, PPAR and C/EBP protein families play a critical role in the induction of differentiation. C/EBPb also induces expression of SREBP-2 to stimulate expression of fatty acid metabolism, including fatty acid synthesis and lipoprotein lipase.14,16 The liver is the primary organ that metabolizes lipids, and the excessive consumption of dietary fats can increase lipid

INTRODUCTION

E

xcessive energy intake results in abnormal lipid metabolism, then an increased prevalence of dyslipidemia, a major risk factor for cardiovascular disease, cholestasis, and overall mortality.1,2 In particular, a high fat diet (HF) is an environmental factor that is considered to be a main cause of obesity and dyslipidemia.3 High fat-induced dyslipidemia is characterized by elevated levels of total cholesterol (TC), triglyceride (TG), and low-density lipoprotein cholesterol (LDL-C), and these factors lead to atherosclerosis and coronary heart disease.4 Excessive accumulation of fat in different organs arises when intake of energy exceeds energy expenditure.5 Visceral fat accumulation can increase the risk of dyslipidemia as well as hypertriglyceridemia and affects the levels of released adipokines.6 Adipocytes secrete adiponectin, leptin, and other hormone-like peptides.7 Leptin is produced by adipose tissue to suppress food intake while stimulating energy expenditure.8 Adiponectin, also known as an adipocyte complement-related protein, plays a role in glucose Manuscript received 17 April 2013. Revision accepted 11 February 2014. Address correspondence to: Yuri Kim, PhD, Department of Nutritional Science and Food Management, Ewha Womans University, 52 Ewhayeodae-gil, Seodaemun-gu, Seoul 120750, Korea, E-mail: [email protected]

571

572

KIM ET AL.

accumulation in the liver and induce obesity. Correspondingly, hepatic fat accumulation is common in obese individuals. In addition, obese individuals generally have a high risk of developing nonalcoholic fatty liver disease (NAFLD).17 Although the pathogenesis is unclear, modulation of transcription factor activity, including that of SREBPs and fatty acid synthetase (FAS), appears to be a key factor for treating NAFLD.11 Recently, there has been a growing interest in natural products that can be used for the treatment and prevention of obesity-related metabolic diseases, including type 2 diabetes mellitus, certain types of cancer, and cardiovascular disease. The genus Sasa (Poaceae), a species of bamboo grasses, is widely cultivated in Korea, China, Japan, and Russia.18 Sasa leaves have been reported to mediate anti-diabetes, antiobesity, anti-oxidative stress, anti-inflammation, and anticancer effects.19–22 Sasa quelpaertensis Nakai is a bamboo grass that is grown only on Jeju Island in South Korea, and several studies have evaluated the health effects of S. quelpaertensis on inflammation and obesity.20,23 However, a limited number of studies have investigated the beneficial health effects of S. quelpaertensis on lipid profiles and adipokines, as well as lipid metabolism-related gene expression in various organs. These factors have been shown to contribute to the complications of obesity-related conditions, including NAFLD, dyslipidemia, and cardiovascular diseases. In the present study, the long-term effects of dietary SQE supplementation on the development of obesity-related metabolic consequences were examined. Fat accumulation and expression of certain genes involved in lipid metabolism were also monitored in various organs, including the liver and adipose tissue during the development of obesity in a rat model. MATERIALS AND METHODS Preparation of the S. quelpaertensis extract S. quelpaertensis leaves were obtained from Mt. Halla on Jeju Island in South Korea. Dried leaves (1 kg) were mixed with 13.3 L water and incubated at 90C for 4 h on a platform shaker. After filtering, S. quelpaertensis extract (SQE) was concentrated at 60C using a rotary evaporator, freezedried, and ground into a powder. The yield of SQE was approximately 11.4% and the extract of SQE was stored at - 20C for further use.

fat), while those in a second group (n = 23) were fed an HF diet (60% of calories are derived from fat) for 16 weeks to induce obesity. After the obesity induction period, the HF group was re-randomized into three groups according to body weight. These three groups were fed experimental diets for 11 weeks and these included: an HF diet (n = 7), an HF diet with SQE 3% supplementation (SQE 3%, n = 8), and an HF diet with SQE 5% supplementation (SQE 5%, n = 8). The animal study protocol was approved by the Institutional Animal Care and Use Committee of Ewha Womans University. Measurement of body weight, food intake, and organ weights Body weights and food intakes were measured twice a week throughout the study period. On the final day of experiments, the rats were sacrificed and livers were resected and weighed. To quantify visceral adipose tissue weight, retroperitoneal, epididymal, and perirenal fat were resected and weighed. The weight was measured to the nearest 0.01 g. Biochemical analysis The rats were sacrificed after 12 h fasting, and blood samples were obtained by cardiac puncture. Plasma concentrations of triglycerides (TG), total cholesterol (TC), and high-density lipoprotein cholesterol (HDL-C) were enzymatically determined using a kit (Asan Pharmaceutical, Seoul, Korea). Levels of low-density lipoprotein cholesterol (LDL-C) were calculated according to the following formula: LDL-C = TC – HDL-C – TG/5 Furthermore, atherogenic index (AI) values were calculated as AI = log (TG/HDL).24 Enzyme-linked immune-sorbent assay (ELISA) kits were used to determine the concentration of plasma adipokines. Plasma adiponectin was determined using a commercial kit (Millipore, Bedford, MA, USA), and resistin was measured using a kit from B-Bridge International (Sunnyvale, CA, USA) according to the manufacturer’s instructions. Hepatic lipid was extracted using the method of Bligh and Dyer,25 and concentrations of TG and TC in the liver were subsequently determined. A commercial kit (Asan Pharma) was used to analyze levels of TG and TC in the liver as well. All procedures were performed according to the manufacturer’s instructions.

Animals and diets Four-week-old male Sprague-Dawley (SD) rats were obtained from Jung-Ang Lab Animal Inc. (Seoul, South Korea) and housed in individual stainless steel wire mesh cages. A uniform temperature (22 – 2C), humidity (50 – 5%), and 12 h/12 h light/dark cycle were maintained. The animals also had free access to food and water. After one week of adaptation, the rats were randomly divided into two groups. The rats in one group (n = 8) were fed a modified AIN-93G diet (NF, 17% of calories are derived from

Histological examination of liver The left lateral lobes of the livers were immediately fixed with 10% phosphate-buffered formalin following necropsy. These livers were then dehydrated through a graded series of ethanol and embedded in paraffin according to standard procedures. Paraffin sections (5 lm) were subsequently cut and deparaffinized in xylene, stained with hematoxylin and eosin (HE), and examined microscopically (Olympus Co., Tokyo, Japan).

573

SASA QUELPAERTENSIS EXTRACT IMPROVES HIGH FAT-INDUCED LIPID ABNORMALITIES

Western blot analysis Liver tissues were homogenized using an ice-cold PROPREP protein extraction solution (Intron Biotechnology, Seoul, Korea). The supernatant was then collected by centrifugation according to the manufacturer’s instructions and proteins were separated using 6% SDS-polyacrylamide gel electrophoresis. Proteins were transferred to polyvinylidene difluoride (PVDF) membranes and probed with antibodies against FAS (Abcam, MA, USA) overnight at 4C. Membranes were washed and then incubated with secondary rabbit IgG-conjugated horseradish peroxidase antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h at room temperature. Visualization of antibody binding was achieved using an enhanced chemiluminescence (ECL) Western blot detection agent (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Detection of b-actin (Santa Cruz Biotech) was used as a loading control. RNA extraction and real-time PCR Total RNA was extracted from liver tissue, adipose tissue, and heart tissue using Trizol reagent (Invitrogen, CA, USA) following the manufacturer’s instruction. cDNA was synthesized from 1 lg total RNA using a RevertAid First Strand cDNA Synthesis Kit (Fermentas, Vilnius, Lithuania). Real-time quantitative PCR was performed using a StepOne-Plus RT-PCR system (Applied Biosystems, Foster City, CA, USA). Relative mRNA expression levels were measured using TaqMan analysis for SREBP-1 [Rn01446560_m1], uncoupling protein-2 (UCP-2) [Rn01754856_m1], C/EBPb [Rn00824635_S1], PPARc [Rn00440940_m1], adipocyte protein 2 (aP2) [Rn00565061_m1], and resistin [Rn00595224_ m1] obtained from Applied Biosystems. Amplifications were performed starting with a 10 min template denaturation step at 95C, followed by 40 cycles at 95C for 15 s and 60C for 1 min. mRNA levels were normalized to that of b-actin [Rn00667869_m1] or GAPDH [Rn01775763_g1]. Quantification was achieved using a comparative CT method. Statistical analysis All results are expressed as the mean – standard error (SE) for each group. Statistical analyses were performed using PASW Statistics 18.0 (SPSS Inc., Chicago, IL, USA). Re-

sults were analyzed using one-way analysis of variance (ANOVA), and differences between groups were analyzed using Duncan’s multiple-comparison test. Differences with a P-value less than.05 were considered statistically significant. RESULTS Effect of SQE on body weight, food intake, and organ weight After the first 16 weeks, body weights for the HF group were significantly higher than those of the NF group. After 11 weeks of SQE 3% or 5% consumption, body weights, and food intakes did not differ among the four groups (Table 1) suggesting that supplementation of an HF diet with SQE 3% or 5% did not affect body weight and food intake. Relative visceral adipose tissue weights (per 100 g body weight), weights for the HF group were found to be significantly higher than those of the NF group (P < .001). However, both SQE supplemented groups tended to have lower visceral adipose tissue weight, although the difference was not statistically significant. Relative liver weights also did not differ among the four groups. Effect of SQE supplementation on plasma lipid levels The effects of SQE on circulating lipid levels were investigated by analyzing levels of plasma TC, TG, HDL-C, LDL-C, and AI values (Fig. 1). Plasma TC and TG levels for the HF group were significantly higher than those of the NF group (Fig. 1A, B; P < .001, P < .01). Furthermore, the HF diet remarkably reduced levels of plasma HDL-C and increased levels of LDL-C (Fig 1C, D; P < .001). Previously, AI values have been used as a significant predictor of atherosclerosis.24,26 Therefore, AI values were also determined for all four groups (Fig. 1E). AI values for the NF group were significantly higher for the NF group compared to the HF group. However, after 11 weeks of SQE treatment, rats receiving SQE 5% exhibited a significant reduction in plasma levels of TC, TG, LDL-C, and AI values. Levels of HDL-C were also increased (P < .01). Similarly, SQE 3% significantly decreased the plasma level of TG and AI values, while it tended to reduce levels of TC, and LDL-C and increase HDL-C levels. However, these differences in levels

Table 1. Effect of Sasa quelpaertensis Extract Supplementation on Body Weight Changes, Food Intakes, Visceral Fat Pad Weight, and Liver Weight of Sprague-Dawley Rats Fed Different Diets for 11 Weeks Group Initial body weight (g) Final body weight (g) Food intakes (g/day) Visceral fat pad (g/100 g bw) Liver (g/100 g bw)

NF

HF

SQE 3%

SQE 5%

507.86 – 16.82a 602.04 – 34.96a 22.16 – 1.58a 3.75 – 0,52a 2.48 – 0.28

559.32 – 41.17b 693.36 – 54.43b 17.36 – 1.18b 5.95 – 1.24b 2.30 – 0.16

560.97 – 41.85b 689.45 – 52.85b 19.09 – 1.23b 5.25 – 1.14b 2.28 – 0.25

559.46 – 41.06b 692.57 – 36.76b 18.66 – 0.66b 5.09 – 0.39b 2.30 – 0.24

Values expressed as mean – standard error. ab Values with different letters in each assay are significantly different from each other between the NF, HF, SQE 3%, and SQE 5% by Duncan’s multiplecomparison test (P < .05). SQE, S. quelpaertensis extract; NF, modified AIN-93G diet; HF, high fat diet; SQE 3%, high fat + SQE 3%; SQE 5%, high fat + SQE 5%; bw, body weight.

574

KIM ET AL.

FIG. 1. Effects of SQE supplementation on plasma lipid profiles. Levels of (A) TC, (B) TG, (C) HDL-C, (D) LDL-C, and (E) AI values in plasma for each group for the experimental periods were analyzed. Data are expressed as the mean – SE. abcdDifferent letters are used to indicate values that significantly differed according to Duncan’s multiple-comparison test (P < .05). AI values were calculated as log (triglycerides/HDL). SQE, Sasa quelpaertensis extract; TC, total cholesterol; TG, triglycerides; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; AI, atherogenic index.

of TC, LDL-C, and HDL-C were not statistically significant compared to the HF group. Effects of SQE on plasma adiponectin and resistin The effects of SQE on plasma adipokine levels are shown in Figure 2. Plasma adiponectin levels were significantly decreased, whereas plasma resistin levels were markedly increased (P < .05, P < .01) by an HF diet. During the 11 weeks of either SQE 3% or 5% supplementation, plasma adiponectin levels appeared to increase, although the difference was not statistically significant (Fig. 2A). However, the increase in levels of plasma resistin induced by an HF diet returned to control levels by SQE 5% supplementation (P < .01; Fig. 2B). To confirm this decrease, mRNA levels of resistin were detected in the heart. Previously, elevated expression of resistin in heart tissue has been associated with an aggravation of cardiovascular metabolic symptoms.27,28 In the HF group, cardiac levels of resistin mRNA were significantly higher compared to the NF group (P < .001). In contrast, levels of resistin mRNA were significantly downregulated following dietary supplementation with SQE 5% (Fig. 2C).

Effects of SQE on lipid metabolism in visceral adipose tissue To evaluate the effects of SQE supplementation on adipogenesis and lipogenesis, mRNA expressions of C/EBPb, PPARc, SREBP-1, and aP2 were detected in visceral adipose tissue (Fig. 3). With an HF diet, C/EBPb expression was found to be up-regulated. In contrast, dietary supplementation with SQE 5% resulted in down-regulation of C/EBPb mRNA expression compared with the HF group (P < .01). In addition, mRNA levels of PPARc, SREBP-1, and aP2 for the HF group were significantly higher compared to the NF group (P < .01 in both cases). However, with SQE supplementation, these levels tended to be down-regulated, although the difference was not statistically significant. Effects of SQE on hepatic morphology In the control rat livers, both lobular architecture and cellular structures were normal (Fig. 4A). In contrast, the livers of HF diet-fed rats exhibited severe mixed micro-/ macrovesicular steatosis (indicated with arrows), swelling of hepatic cells, and focal infiltrations of inflammatory cells (Fig. 4B; indicated with sharp arrows). The livers of HF

SASA QUELPAERTENSIS EXTRACT IMPROVES HIGH FAT-INDUCED LIPID ABNORMALITIES

575

FIG. 2. Effects of SQE supplementation on plasma adipokines. Levels of (A) adiponectin and (B) resistin in plasma were analyzed by ELISA. (C) Levels of resistin mRNA in heart tissues were assessed using real-time quantitative PCR with specific primers and TaqMan probes. Data are expressed as the mean – SE. abcDifferent letters are used to indicate values that significantly differed according to Duncan’s multiple-comparison test (P < .05). ELISA, enzyme-linked immunosorbent assay.

diet-fed rats also contained large fat droplets, while rats treated with SQE 3% (Fig. 4C) or SQE 5% (Fig. 4D) exhibited a dose-dependent decrease in the size of the fat droplets observed. However, mild micro-/macrovesicular steatosis was still observed in the midozonal area of the livers examined. The infiltration of inflammatory cell and swelling of hepatic cells was significantly attenuated by SQE supplementation in a dose-dependent manner, especially in the SQE 5% group. Furthermore, most sinusoids were not obstructed and appeared normal. It was also ob-

served that rats receiving SQE 5% supplementation did not perfectly recover from HF-induced steatosis. Effects of SQE supplementation on liver lipid profile To further characterize the decrease in hepatic fat accumulation in rats supplemented with SQE, liver lipid profiling was performed and analyzed (Fig. 5). An HF diet was found to significantly elevate levels of hepatic TC, TG, and total lipids (P < .01, P < .001, and P < .001, respectively). However,

FIG. 3. Effects of SQE on mRNA expressions of C/EBPb, PPARc, SREBP-1, and aP2 in visceral adipose tissues. Using real-time PCR, mRNA levels of (A) C/EBPb, (B) PPARc, (C) SREBP-1, and (D) aP2 were detected in visceral adipose tissue, with b-actin used as an internal control. Data represent the mean – SE. abDifferent letters are used to indicate values that significantly differed according to Duncan’s multiple-comparison test (P < .05). C/ EBPb, CAAT/enhancer-binding protein b; PPARc, peroxisome proliferator activated receptor c; SREBP-1, sterol regulatory element binding protein-1; aP2, adipocyte protein 2.

576

KIM ET AL.

FIG. 4. Effects of SQE supplementation on liver abnormalities induced by an HF diet. Liver samples from (A) NF, (B) HF, (C) SQE 3%, and (D) SQE 5%. A significant, dose-dependent reduction in the fatty accumulation of rat livers treated with SQE 3% (C) or SQE 5% (D) was observed, compared to HF-treated rats (B). Arrows indicate changes in fatty accumulation; sharp arrows indicate the infiltration of inflammatory cells. Hematoxylin and eosin (HE) staining, scale bar: 100 mm. NF, modified AIN-93G diet; HF, high fat diet; SQE 3%, high fat + SQE 3%; SQE 5%, high fat + SQE 5%. Color images available online at www.liebertpub.com/jmf

SQE 3% or 5% supplementation did not significantly affect TC levels, while both SQE 3% and SQE 5% supplementation significantly lowered levels of TG and total lipid in the liver compared to those of the HF group (P < .001 and P < .01, respectively). In combination with the plasma lipid profiling performed, these data suggest that SQE supplementation can regulate lipid disorders-induced by an HF diet in rats. Effects of SQE on protein and gene expression of FAS, SREBP-1, and UCP-2 in the liver To examine the molecular mechanism(s) that SQE supplementation may mediate in the regulation of lipid me-

tabolism in the liver, expressions of FAS and SREBP-1 were detected at the protein and gene levels. Liver samples of the HF group had elevated protein expressions of FAS. In contrast, liver samples from the SQE 5% group exhibited a significantly lower FAS protein expression (P < .001; Fig. 6A, B). Furthermore, elevated levels of SREBP-1 mRNA were detected in the HF group, but were significantly lower in both the SQE 3% and SQE 5% groups (Fig. 6C). As a well-known thermogenesis regulator, UCP-2 has recently been reported to modulate lipid metabolism.29,30 Significantly up-regulated UCP-2 mRNA expression by an HF diet was lowered by SQE 5% supplementation (P < .01; Fig. 6D).

FIG. 5. Effects of SQE supplementation on liver lipid profiles. Concentrations of (A) TC, (B) TG, and (C) total lipid in liver samples were analyzed. Data represent the mean – SE. ab Different letters are used to indicate values that significantly differed according to Duncan’s multiple-comparison test (P < .05). TC, total cholesterol; TG, triglycerides.

SASA QUELPAERTENSIS EXTRACT IMPROVES HIGH FAT-INDUCED LIPID ABNORMALITIES

577

FIG. 6. Effects of SQE on protein and gene expression of FAS, SREBP1, and UCP-2 in the liver. (A, B) FAS protein levels in the liver of each rat group were assessed by Western blot. Using real-time quantitative PCR, mRNA levels of (C) SREBP-1 and (D) UCP-2 were detected in rat liver samples. Data represent the mean – SE. ab Different letters are used to indicate values that significantly differed according to Duncan’s multiple-comparison test (P < .05). FAS, fatty acid synthetase; SREBP-1, sterol regulatory element binding protein-1; UCP-2, uncoupling protein 2.

DISCUSSION The results of this study indicate that SQE dietary supplementation can improve serum lipid profiles and attenuate lipid accumulation in the liver of rats. These physiological changes were accompanied by changes in gene expression in adipose tissue and liver tissue in rats fed an HF diet. SQE supplementation was associated with the regulation of genes involved in adipocyte differentiation lipogenesis and fatty acid synthesis in the liver. Elevated levels of TC and LDL-C, and low levels of HDL-C, are major contributors to atherosclerosis and cardiovascular disease.31 In this study, SQE supplementation significantly improved the lipid profiles of rats not only by lowering levels of plasma serum TC, TG, and LDL-C, but also by increasing levels of HDL-C compared to the HF group. This result is consistent with a previous study in C57BL/6 mice20 where SQE was shown to significantly restore serum levels of TC and TG to those of the control group. Hepatic lipid profiles were also improved, with a decrease in total lipid and TG levels detected. Furthermore, the atherogenic index of plasma (AIP) indicated that plasma atherogenicity was present, and may be a significant independent predictor of coronary heart disease.24 In the present study, AI values were significantly decreased following SQE supplementation, indicating that S. quelpaertensis leaves can have beneficial effects for treating dyslipidemia and atherosclerosis, without reducing body weight. Body weight and food intake were not found to be affected by SQE supplementation. This result is in contrast with a previous study in C57BL/6 mice where Sasa borealis leaves were found to significantly inhibit weight gain induced by an HF diet.21 This discrepancy might be due to the use of different species of bamboo leaves and a different animal model. A recent study has also demonstrated that orally administered SQE has beneficial effects on obesity,20

and this was demonstrated without an obesity induction period. It is predicted that different administration methods, study purpose, species of plant, and animal models may account for the different outcomes reported. Thus, further studies are needed to confirm the effects of SQE on obesityrelated disorders, and obesity itself, using various conditions and models. Adiponectin and resistin are proteins that have been shown to play key roles in metabolic processes such as glucose regulation32,33 and lipid metabolism.34,35 Furthermore, it has been demonstrated that adiponectin can reduce lipid synthesis in the liver, and can lead to lower concentrations of free fatty acids, and reduce triglyceride production.36 Obesity, atherosclerosis, and diabetes have been associated with lower levels of adiponectin,37 and resistin has been proposed to be a link between obesity, insulin resistance, and type 2 diabetes mellitus. It has also been shown that increased plasma resistin levels in obese animals contribute to insulin resistance by impairing insulin stimulated glucose uptake and insulin action.38 Furthermore, an association has been identified between circulating resistin levels and the development of coronary artery disease, including cardiovascular death and myocardial infarction in humans.32,39 In this study, plasma adiponectin levels of rats receiving SQE supplementation seemed to recover to levels detected for the NF group. However, the changes were not statistically significant. In addition, levels of plasma resistin associated with an HF diet were significantly higher compared to plasma resistin levels of SQE 5% rats which significantly decreased. It has been demonstrated that when expression of resistin is elevated in rat heart tissues, it may aggravate metabolic symptoms, and when it is detected in diabetic hearts, it can promote cardiac hypertrophy.27,28,40 Resistin expression was also increased in response to mechanical stretch in rat neonatal cardiomyocytes in vitro, and may affect cardiac function in animal models.41,42 However,

578

KIM ET AL.

expression of resistin in heart tissues of rats fed an HF diet has not been fully characterized. Therefore, in the present study, mRNA levels of resistin were found to be upregulated in the HF group. In contrast, SQE supplementation effectively suppressed mRNA expression of resistin in rat heart tissues. Further studies are needed to elucidate additional mechanistic detail of SQE effects on resistin-related cardiac gene expression and signaling. To understand the beneficial effects of SQE on lipid metabolism in obesity-related disorders, it is important to investigate, not only plasma lipid profiles, but also novel molecular mechanisms mediated by SQE on lipid metabolism in various organs. This is the first study to reveal the effects of SQE on adipocyte differentiation, lipid synthesis, and lipid metabolism-related gene expression in various organs, including adipose and liver tissues. The transcription factors, C/EBPb and PPARc, are expressed in preadipocytes and their expressions are increased during adipocyte differentiation.43 In a recent study, adipocyte markers, including PPARc and aP2, were found to be overexpressed in the visceral adipose tissue of obese rodents. Furthermore, enhanced expression of C/EBPb led to increases in mRNA levels of PPARc and aP2.44 Yamauchi et al. also demonstrated that PPARc-deficient mice are protected from adipocyte hypertrophy, obesity, and insulin resistance induced by an HF diet or aging.45 Here, adipogenesis-related gene expression in visceral adipose tissue was examined. SQE supplementation appeared to down-regulate mRNA levels of PPARc and aP2. However, the difference was not statistically significant. Furthermore, SQE supplementation was associated with a significant down-regulation C/EBPb mRNA expression, which is induced in very early adipogenesis.15 C/EBPb and C/EBPd are also induced very early during differentiation, thereby, playing an important role in the induction of differentiation.15,46 Moreover, C/EBPb induced expression of SREBP-1, which stimulates fatty acid metabolism including, fatty acid synthesis and lipoprotein lipase.16 Taken together, these results suggest that SQE acts during the early adipogenesis stages rather than during the late stages of adipogenesis, and this has a beneficial effect on the prevention of obesity. A histological analysis of liver tissue revealed that SQE also has a protective effect against the development of nonalcoholic fatty liver. The accumulation of lipid droplets, a typical characteristic of a fatty liver, was examined in the SQE supplemented groups. Compared to the HF group, lower levels of lipid accumulation in the liver were observed. Furthermore, SQE supplementation resulted in lower levels of hepatic TC, TG, and total lipid compared to the HF group. These data confirm the beneficial effects of SQE on lipid profiles in liver and plasma, and are consistent with the lipid levels detected in plasma. Hepatic lipid metabolism is controlled by transcriptional factors such as SREBPs that have been shown to regulate cholesterol metabolism and fatty acid synthesis.47,48 In particular, SREBP-1 is an important regulator of lipogenic enzymes such as acetyl-CoA carboxylase (ACC) and FAS.47 Overexpression of SREBP-1 was found to increase triglyceride accumula-

tion and fatty acid synthesis.49 In the present study, SQE supplementation down-regulated FAS and SREBP-1 expression induced by an HF diet. In addition, a recent study by Sato et al. demonstrated that lignophenols, which are derivatives of bamboo lignin, also down-regulate SREBP-1c and p-ACC in obese rats.50 In the present study body weight, visceral fat pad weight, and liver weight were not affected by SQE supplementation, but lipid metabolism was altered and liver steatosis was suppressed. It has been reported that hepatic statosis is independent of body fat and weight gain. However, in rat models, a short-term HF was found to cause hepatic steatosis whereas visceral and muscle fat content remained unchanged.51–53 It has also been demonstrated that regulation of lipid metabolism is one of the core functions of the liver. Although the pathogenesis of hepatic fat accumulation during liver steatosis induced by an HF diet has not been well-characterized, it is hypothesized that lipoprotein production by the liver, as well as modulation of lipoprotein and lipid clearance, may be mechanisms by which SQE lowers the risk of dyslipidemia and prevents the progression of steatosis. Levels of UCP-2 were also measured. UCP-2 is known as a mitochondrial membrane transporter that may regulate thermogenesis via dissipation of proton gradients and the conversion of fuel to heat.54 In addition, UCP-2 is emerging as a potential regulator of mitochondrial reactive oxygen species (ROS).55 With the liver being the primary organ involved in fatty acid metabolism,56 Uchino et al. demonstrated that ROS generated during lipid metabolism stimulate TNF-a, followed by up-regulation of UCP-2 mRNA expression in hepatocytes.57 UCP-2 expression has also been shown to increase in the liver during metabolic stress, which includes an HF and the onset of obesity.58,59 In this study, expression of UCP-2 in the liver of obese rats was upregulated, and this result is consistent with the results of previous studies conducted in mouse models and SD rats.54,60 Furthermore, this up-regulation was found to be suppressed with SQE supplementation in the present study. This result may indicate that SQE supplementation can suppress liver damage and decrease the protective role of UCP-2 in liver tissue exposed to an excessive fuel supply, by negatively regulating mitochondrial ROS production. The beneficial effects of certain Sasa spp. leaves have previously been reported.20,21 Moreover, SQE has been show to contain various bioactive compounds, including polyphenol p-coumaric acid, which are associated with anti-cancer and anti-oxidant properties.20 For example, p-coumaric acid acts as a direct scavenger of ROS and can reduce serum levels of LDL-C and the oxidation of LDL-C, a major factor in the pathogenesis of atherosclerosis in both rats and humans.61,62 p-Coumaric acid is an efficient antioxidant that scavenges various reactive species, including singlet oxygens, hydroxyl radicals, peroxyl radicals, and peroxynitrite. p-Coumaric acid also inhibits hydrogen peroxide.61,63–66 In addition, p-coumaric acid provides antioxidant protection by scavenging $OH to inhibit LDL peroxidation.61 Tricin has also been found to be a promising

SASA QUELPAERTENSIS EXTRACT IMPROVES HIGH FAT-INDUCED LIPID ABNORMALITIES

antioxidant substance that is derived from bamboo leaves.67 In several studies, it has shown potential as a chemopreventive agent.68–70 In the SQE, p-coumaric acid and tricin concentrations were 23.706 mg/g – 1.766 and, 0.263 mg/g – 0.0087, respectively. These results show that p-coumaric acid and tricin is one of the major bioactive compounds in SQE. However, further studies are necessary to investigate additional bioactive compounds contained in Sasa species leaves that are responsible for improving obesity-related disorders. In conclusion, dietary SQE supplementation significantly improved lipid profiles in both blood plasma and the liver, improved adipokine secretion, and decreased lipid accumulation in the liver by regulating mRNA levels of adipogenesis and lipogenesis-related genes in both adipose tissue and liver tissues. Thus, SQE be beneficial in the regulation of early adipogenesis, and valuable insight has been gained into the possible application of SQE for the prevention of obesity-related metabolic disorders.

13.

ACKNOWLEDGMENT

17.

This work was supported by the Ministry of Knowledge Economy for the Regional Innovation System program in 2010 (No. B0012328).

18.

11.

12.

14.

15.

16.

AUTHOR DISCLOSURE STATEMENT No competing conflicts of interest exist.

19.

REFERENCES 1. Ghosh P, Bitsanis D, Ghebremeskel K, Crawford MA, Poston L: Abnormal aortic fatty acid composition and small artery function in offspring of rats fed a high fat diet in pregnancy. J Physiol 2001;533:815–822. 2. Rizvi F, Iftikhar M, George JP: Beneficial effects of fish liver preparations of sea bass (Lates calcarifer) versus gemfibrozil in high fat diet-induced lipid-intolerant rats. J Med Food 2003;6: 123–128. 3. Bach AC, Ingenbleek Y, Frey A: The usefulness of dietary medium-chain triglycerides in body weight control: fact or fancy? J Lipid Res 1996;37:708–726. 4. Le NA, Walter MF: The role of hypertriglyceridemia in atherosclerosis. Curr Atheroscler Rep 2007;9:110–115. 5. Swinburn B, Ravussin E: Energy balance or fat balance? Am J Clin Nutr 1993;57:766S–770S; discussion 770S–771S. 6. Scaglione R, Di Chiara T, Cariello T, Licata G: Visceral obesity and metabolic syndrome: two faces of the same medal? Intern Emerg Med 2010;5:111–119. 7. Trayhurn P: Endocrine and signalling role of adipose tissue: new perspectives on fat. Acta Physiol Scand 2005;184:285–293. 8. Bjørbaek C, Kahn BB: Leptin signaling in the central nervous system and the periphery. Recent Prog Horm Res 2004;59:305– 331. 9. Berg AH, Combs TP, Du X, Brownlee M, Scherer PE: The adipocyte-secreted protein Acrp30 enhances hepatic insulin action. Nat Med 2001;7:947–953. 10. Kadowaki T, Yamauchi T, Kubota N, Hara K, Ueki K, Tobe K: Adiponectin and adiponectin receptors in insulin resistance, di-

20.

21.

22.

23.

24.

25. 26.

27.

28.

579

abetes, and the metabolic syndrome. J Clin Invest 2006;116: 1784–1792. Shuldiner AR, Yang R, Gong DW: Resistin, obesity and insulin resistance—the emerging role of the adipocyte as an endocrine organ. N Engl J Med 2001;345:1345–1346. Silha JV, Krsek M, Skrha JV, Sucharda P, Nyomba BL, Murphy LJ: Plasma resistin, adiponectin and leptin levels in lean and obese subjects: correlations with insulin resistance. Eur J Endocrinol 2003;149:331–335. Fe`ve B: Adipogenesis: cellular and molecular aspects. Best Pract Res Clin Endocrinol Metab 2005;19:483–499. Rosen ED, Walkey CJ, Puigserver P, Spiegelman BM: Transcriptional regulation of adipogenesis. Genes Dev 2000;14: 1293–1307. Wu Z, Xie Y, Bucher NL, Farmer SR: Conditional ectopic expression of C/EBP beta in NIH-3T3 cells induces PPAR gamma and stimulates adipogenesis. Genes Dev 1995;9:2350–2363. Kim JB, Spiegelman BM: ADD1/SREBP1 promotes adipocyte differentiation and gene expression linked to fatty acid metabolism. Genes Dev 1996;10:1096–1107. Yeon JE, Choi KM, Baik SH, et al.: Reduced expression of peroxisome proliferator-activated receptor-alpha may have an important role in the development of non-alcoholic fatty liver disease. J Gastroenterol Hepatol 2004;19:799–804. Okabe S, Takeuchi K, Takagi K, Shibata M: Stimulatory effect of the water extract of bamboo grass (Folin solution) on gastric acid secretion in pylorus-ligated rats. Jpn J Pharmacol 1975; 25:608–609. Choi YJ, Lim HS, Choi JS, et al.: Blockade of chronic high glucose-induced endothelial apoptosis by Sasa borealis bamboo extract. Exp Biol Med (Maywood) 2008;233:580–591. Kang SI, Shin HS, Kim HM, et al.: Anti-obesity properties of a Sasa quelpaertensis extract in high-fat diet-induced obese mice. Biosci Biotechnol Biochem 2012;76:755–761. Kim EY, Jung EY, Lim HS, Heo YR: The effects of the Sasa borealis leaves extract on plasma adiponectin, resistin, C-reactive protein and homocysteine levels in high fat diet-induced obese C57/BL6J mice. Korean J Nutr 2007;40:303–311. Ren M, Reilly RT, Sacchi N: Sasa health exerts a protective effect on Her2/NeuN mammary tumorigenesis. Anticancer Res 2004;24:2879–2884. Ryou SH, Kang MS, Kim KI, Kang YH, Kang JS: Effects of green tea or Sasa quelpaertensis bamboo leaves on plasma and liver lipids, erythrocyte Na efflux, and platelet aggregation in ovariectomized rats. Nutr Res Pract 2012;6:106–112. Dobiasova M, Frohlich J: The plasma parameter log (TG/HDLC) as an atherogenic index: correlation with lipoprotein particle size and esterification rate in apoB-lipoprotein-depleted plasma (FER(HDL)). Clin Biochem 2001;34:583–588. Bligh EG, Dyer WJ: A rapid method of total lipid extraction and purification. Can J Biochem Physiol 1959;37:911–917. Tan MH, Johns D, Glazer NB: Pioglitazone reduces atherogenic index of plasma in patients with type 2 diabetes. Clin Chem 2004;50:1184–1188. Kim M, Oh JK, Sakata S, et al.: Role of resistin in cardiac contractility and hypertrophy. J Mol Cell Cardiol 2008;45:270– 280. Ding Q, White SP, Ling C, Zhou W: Resistin and cardiovascular disease. Trends Cardiovasc Med 2011;21:20–27.

580

KIM ET AL.

29. Diano S, Horvath TL: Mitochondrial uncoupling protein 2 (UCP2) in glucose and lipid metabolism. Trends Mol Med 2012; 18:52–58. 30. Oh KS, Kim M, Lee J, et al.: Liver PPARalpha and UCP2 are involved in the regulation of obesity and lipid metabolism by swim training in genetically obese db/db mice. Biochem Biophys Res Commun 2006;345:1232–1239. 31. Wallace RB, Anderson RA: Blood lipids, lipid-related measures, and the risk of atherosclerotic cardiovascular diseases. Epidemiol Rev 1987;9:95–119. 32. Krecki R, Krzeminska-Pakula M, Peruga JZ, et al.: Elevated resistin opposed to adiponectin or angiogenin plasma levels as a strong, independent predictive factor for the occurrence of major adverse cardiac and cerebrovascular events in patients with stable multivessel coronary artery disease over 1-year follow-up. Med Sci Monit 2011;17:CR26–CR32. 33. Wang H, Chen DY, Cao J, He ZY, Zhu BP, Long M: High serum resistin level may be an indicator of the severity of coronary disease in acute coronary syndrome. Chin Med Sci J 2009;24:161–166. 34. McTernan PG, Fisher FM, Valsamakis G, et al.: Resistin and type 2 diabetes: regulation of resistin expression by insulin and rosiglitazone and the effects of recombinant resistin on lipid and glucose metabolism in human differentiated adipocytes. J Clin Endocrinol Metab 2003;88:6098–6106. 35. World Health Organization: Obesity: Preventing and Managing the Global Epidemic. Report of a WHO Consultation. World Health Organization Technical Report Series 894. WHO, Geneva, 2000. 36. Xu A, Wang Y, Keshaw H, Xu LY, Lam KS, Cooper GJ: The fat-derived hormone adiponectin alleviates alcoholic and nonalcoholic fatty liver diseases in mice. J Clin Invest 2003;112:91–100. 37. Ahima RS: Adipose tissue as an endocrine organ. Obesity (Silver Spring) 2006;14:242S–249S. 38. Steppan CM, Bailey ST, Bhat S, et al.: The hormone resistin links obesity to diabetes. Nature 2001;409:307–312. 39. Momiyama Y, Ohmori R, Uto-Kondo H, et al.: Serum resistin levels and cardiovascular events in patients undergoing percutaneous coronary intervention. J Atheroscler Thromb 2011;18:108–114. 40. Kang S, Chemaly ER, Hajjar RJ, Lebeche D: Resistin promotes cardiac hypertrophy via the AMP-activated protein kinase/ mammalian target of rapamycin (AMPK/mTOR) and c-Jun Nterminal kinase/insulin receptor substrate 1 ( JNK/IRS1) pathways. J Biol Chem 2011;286:18465–18473. 41. Graveleau C, Zaha VG, Mohajer A, et al.: Mouse and human resistins impair glucose transport in primary mouse cardiomyocytes, and oligomerization is required for this biological action. J Biol Chem 2005;280:31679–31685. 42. Wang BW, Hung HF, Chang H, Kuan P, Shyu KG: Mechanical stretch enhances the expression of resistin gene in cultured cardiomyocytes via tumor necrosis factor-alpha. Am J Physiol Heart Circ Physiol 2007;293:H2305–H2312. 43. Chen N, Bezzina R, Hinch E, et al.: Green tea, black tea, and epigallocatechin modify body composition, improve glucose tolerance, and differentially alter metabolic gene expression in rats fed a high-fat diet. Nutr Res 2009;29:784–793. 44. Shin SS, Jung YS, Yoon KH, et al.: The Korean traditional medicine gyeongshingangjeehwan inhibits adipocyte hypertrophy and visceral adipose tissue accumulation by activating PPARalpha actions in rat white adipose tissues. J Ethnopharmacol 2010;127:47–54.

45. Yamauchi T, Waki H, Kamon J, et al.: Inhibition of RXR and PPARgamma ameliorates diet-induced obesity and type 2 diabetes. J Clin Invest 2001;108:1001–1013. 46. Yeh WC, Cao Z, Classon M, McKnight SL: Cascade regulation of terminal adipocyte differentiation by three members of the C/EBP family of leucine zipper proteins. Genes Dev 1995;9: 168–181. 47. Brown MS, Goldstein JL: The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor. Cell 1997;89:331–340. 48. Horton JD, Goldstein JL, Brown MS: SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver. J Clin Invest 2002;109:1125–1131. 49. You M, Crabb DW: Recent advances in alcoholic liver disease II. Minireview: molecular mechanisms of alcoholic fatty liver. Am J Physiol Gastrointest Liver Physiol 2004;287:G1–G6. 50. Sato S, Mukai Y, Tokuoka Y, Mikame K, Funaoka M, Fujita S: Effect of lignin-derived lignophenols on hepatic lipid metabolism in rats fed a high-fat diet. Environ Toxicol Pharmacol 2012; 34:228–234. 51. Seppa¨la¨-Lindroos A, Vehkavaara S, Ha¨kkinen AM, et al.: Fat accumulation in the liver is associated with defects in insulin suppression of glucose production and serum free fatty acids independent of obesity in normal men. J Clin Endocrinol Metab 2002;87:3023–3028. 52. Jang HH, Park MY, Kim HW, et al.: Black rice (Oryza sativa L.) extract attenuates hepatic steatosis in C57BL/6 J mice fed a highfat diet via fatty acid oxidation. Nutr Metab (London) 2012;9:27. 53. Samuel VT, Liu ZX, Wang A, et al.: Inhibition of protein kinase C(epsilon) prevents hepatic insulin resistance in nonalcoholic fatty liver disease. J Clin Invest 2007;117:739–745. 54. Murase T, Mizuno T, Omachi T, et al.: Dietary diacylglycerol suppresses high fat and high sucrose diet-induced body fat accumulation in C57BL/6J mice. J Lipid Res 2001;42:372–378. 55. Ne`gre-Salvayre A, Hirtz C, Carrera G, et al.: A role for uncoupling protein-2 as a regulator of mitochondrial hydrogen peroxide generation. FASEB J 1997;11:809–815. 56. Pe´gorier JP, Le May C, Girard J: Control of gene expression by fatty acids. J Nutr 2004;134:2444S–2449S. 57. Uchino S, Yamaguchi Y, Furuhashi T, et al.: Steatotic liver allografts up-regulate UCP-2 expression and suffer necrosis in rats. J Surg Res 2004;120:73–82. 58. Chavin KD, Yang S, Lin HZ, et al.: Obesity induces expression of uncoupling protein-2 in hepatocytes and promotes liver ATP depletion. J Biol Chem 1999;274:5692–5700. 59. Memon RA, Hotamisligil GS, Wiesbrock SM, et al.: Upregulation of uncoupling protein 2 mRNA in genetic obesity: lack of an essential role for leptin, hyperphagia, increased tissue lipid content, and TNF-alpha. Biochim Biophys Acta 2000;1484:41–50. 60. Kucera O, Garnol T, Lotkova´ H, et al.: The effect of rat strain, diet composition and feeding period on the development of a nutritional model of non-alcoholic fatty liver disease in rats. Physiol Res 2011;60:317–328. 61. Zang LY, Cosma G, Gardner H, Shi X, Castranova V, Vallyathan V: Effect of antioxidant protection by p-coumaric acid on lowdensity lipoprotein cholesterol oxidation. Am J Physiol Cell Physiol 2000;279:C954–C960. 62. Andreasen MF, Landbo AK, Christensen LP, Hansen A, Meyer AS: Antioxidant effects of phenolic rye (Secale cereale L.) extracts, monomeric hydroxycinnamates, and ferulic acid

SASA QUELPAERTENSIS EXTRACT IMPROVES HIGH FAT-INDUCED LIPID ABNORMALITIES

63.

64.

65.

66.

dehydrodimers on human low-density lipoproteins. J Agric Food Chem 2001;49:4090–4096. Foley S, Navaratnam S, McGarvey DJ, Land EJ, Truscott TG, Rice-Evans CA: Singlet oxygen quenching and the redox properties of hydroxycinnamic acids. Free Radic Biol Med 1999; 26:1202–1208. Castelluccio C, Paganga G, Melikian N, et al.: Antioxidant potential of intermediates in phenylpropanoid metabolism in higher plants. FEBS Lett 1995;368:188–192. Niwa T, Doi U, Kato Y, Osawa T: Inhibitory mechanism of sinapinic acid against peroxynitrite-mediated tyrosine nitration of protein in vitro. FEBS Lett 1999;459:43–46. Swaroop A, Ramasarma T: Inhibition of H2O2 generation in rat liver mitochondria by radical quenchers and phenolic compounds. Biochem J 1981;194:657–665.

581

67. Jiao J, Zhang Y, Liu C, Liu J, Wu X, Zhang Y: Separation and purification of tricin from an antioxidant product derived from bamboo leaves. J Agric Food Chem 2007;55:10086–10092. 68. Cai H, Hudson EA, Mann P, et al.: Growth-inhibitory and cell cycle-arresting properties of the rice bran constituent tricin in human-derived breast cancer cells in vitro and in nude mice in vivo. Br J Cancer 2004;91:1364–1371. 69. Cai H, Al-Fayez M, Tunstall RG, et al.: The rice bran constituent tricin potently inhibits cyclooxygenase enzymes and interferes with intestinal carcinogenesis in ApcMin mice. Mol Cancer Ther 2005;4:1287–1292. 70. Cai H, Boocock DJ, Steward WP, Gescher AJ: Tissue distribution in mice and metabolism in murine and human liver of apigenin and tricin, flavones with putative cancer chemopreventive properties. Cancer Chemother Pharmacol 2007;60:257–266.