The herbal composition GGEx18 from Laminaria japonica, Rheum palmatum, and Ephedra sinica inhibits high-fat diet-induced hepatic steatosis via hepatic PPARα activation

Abstract

Context: The activation of peroxisome proliferator-activated receptor α (PPARα) target genes promotes hepatic oxidation of fatty acids. We hypothesized that Gyeongshingangjeehwan 18 (GGEx18), a mixture of three herbs, Laminaria japonica Aresch (Laminariaceae), Rheum palmatum L. (Polygonaceae), and Ephedra sinica Stapf (Ephedraceae), can regulate high-fat diet-induced hepatic steatosis through PPARα activation in the liver.

Objective: To investigate the effects of GGEx18 on obesity-related hepatic steatosis and the responsible mechanism.

Materials and methods: The effects of GGEx18 on hepatic lipid accumulation, serum lipid profiles, and the expression of PPARα target genes were studied in high-fat diet-induced obese mice. The effects of GGEx18 on the expression of the PPARα targets and PPARα reporter gene activation were measured in NMu2Li liver cells.

Results: GGEx18 administration to obese mice for 9 weeks markedly (p < 0.05) decreased hepatic lipid accumulation compared with that in obese control mice. Serum triglyceride and total cholesterol levels were significantly (p <0.05) decreased by GGEx18. GGEx18 treatment increased the messenger RNA levels of PPARα target genes, which are responsible for fatty acid oxidation, in liver tissues. Consistent with the in vivo data, similar activation of genes was observed in GGEx18-treated NMu2Li liver cells. GGEx18 also elevated PPARα reporter gene expression in NMu2Li cells.

Discussion and conclusion: These results suggest that GGEx18 prevents hepatic steatosis and hyperlipidemia in high-fat diet-induced obese mice, and this process may be mediated through PPARα activation in the liver.

Keywords::

• Fatty acid oxidation
• hyperlipidemia
• liver
• obesity
• triglyceride
• UCP2

Introduction

Gyeongshingangjeehwan 18 (GGEx18), an herbal drug composed of three medicinal plants, Rheum palmatum L. (Polygonaceae), Laminaria japonica Aresch (Laminariaceae), and Ephedra sinica Stapf (Ephedraceae), has already been used as an antiobesity drug in local clinics in Korea, although the mechanism of its action remains unknown. These three herbs are reported to have antiobesity, antidiabetic, and lipid-lowering effects (Boozer et al., 2001; You et al., 2009; Huang et al., 2010; Xue et al., 2010). These reports led to the speculation of whether high-fat diet-induced obesity in mice can be regulated more efficiently when treated with a mixture of the above three herbal extracts (GGEx18) at the same time. In our previous study, we found that obesity in mice could be regulated more efficiently when treated with GGEx18 (Yoon et al., 2010), supporting the belief that GGEx18 may regulate obesity-related metabolic disorders including type 2 diabetes, atherosclerosis, hyperlipidemia, and hepatic steatosis.

Steatosis is one of the most common liver diseases and a major contributor to obesity-related morbidity and mortality (Angulo, 2002). The mechanisms underlying liver steatosis are complex and multifunctional. However, a line of evidence suggests that peroxisome proliferator-activated receptor α (PPARα), which regulates lipid metabolism (Yoon, 2009, 2010), is involved in its pathogenesis (Reddy, 2001). Systemic agonists for PPARα have been widely used as lipid-lowering agents; thus, steatosis can be treated via PPARα modulation (Costet et al., 1998; Staels et al., 1998; Basaranoglu et al., 1999). PPARα-null mice develop fatty liver, which is aggravated by a high-fat diet (Kersten et al., 1999). PPARα is highly expressed in the liver, where it regulates a number of enzymes critical for fatty acid catabolism. The target genes of PPARα include those involved in fatty acid uptake, fatty acid binding, and fatty acid oxidation in microsomes, peroxisomes, and mitochondria (Yoon, 2009, 2010). The activation of PPARα target genes, therefore, promotes increased hepatic oxidation of fatty acids, as well as the increased degradation, reduced synthesis, and secretion of triglycerides. We, therefore, hypothesized that GGEx18 can regulate high-fat diet-induced hepatic steatosis through PPARα activation in the liver.

We treated both high-fat diet-induced obese mice and NMu2Li liver cells with GGEx18. We demonstrate that hepatic lipid accumulation and hyperlipidemia were significantly reduced in GGEx18-treated mice compared with the findings in obese controls. Concomitantly, GGEx18 treatment increased the messenger RNA (mRNA) expression of PPARα and its target genes, such as 3-keotacyl-CoA thiolase (thiolase), very long chain acyl-CoA dehydrogenase (VLCAD), and uncoupling protein 2 (UCP2). Similar to the in-vivo data, in addition to increasing the mRNA levels of PPARα target genes, GGEx18 also elevated PPARα reporter gene activation in NMu2Li cells. These data suggest that GGEx18 can inhibit hepatic steatosis and dyslipidemia through PPARα activation in the livers of obese mice.

Materials and methods

Preparation of GGEx18

GGEx18 was prepared from food-grade aqueous extracts of the three herbs (40% L. japonica, 20% E. sinica, and 20% R. palmatum − expressed as % dry weight, Hwalim, Busan, Korea) (Shin et al., 2011). Briefly, three dried herbs with their contents weighed were boiled together in distilled water for 22 h at 95°C. The aqueous extracts were then filtered through a membrane filter (6 μm) and freeze-dried under vacuum.

Animal treatments

Eight-week-old male, wild-type C57BL/6J mice (n = 8/group) were purchased from Central Lab Animal (Seoul, Korea) and randomly divided into five groups. Mice were fed a low-fat diet (normal group; 13% kcal fat, CJ, Incheon, Korea), a high-fat diet (control group; 45% kcal fat, Research Diets, New Brunswick, NJ, USA), or the high-fat diet supplemented with one of three doses of GGEx18 (125, 250, or 500 mg/kg/day) for 9 weeks. At the end of the study, mice were sacrificed under diethyl ether anesthesia, and liver tissues were harvested, weighed, snap-frozen in liquid nitrogen, and stored at −80°C until use. Additional sections of liver were prepared for histological analyses. Blood was collected from the saphenous vein after a 12 h fast, and serum was separated and stored at −80°C until analysis. All animal experiments were approved by the Institutional Animal Care and Use Committee of Dongeui University and conducted according to National Research Council Guidelines.

Blood analysis

Serum concentrations of triglycerides, total cholesterol, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were measured using an automatic blood chemical analyzer (CIBA Corning, Oberlin, OH, USA).

Histological analysis

The right lobe of the liver was fixed in 10% phosphate-buffered formalin for 1 day and processed in a routine manner for paraffin sectioning. Five-micrometer-thick sections were stained with hematoxylin and eosin for microscopic examination.

Reverse transcription-polymerase chain reaction

Total cellular RNA from liver tissues and NMu2Li murine liver cells was prepared using Trizol reagent (Invitrogen, Carlsbad, CA, USA). After 2 μg total RNA were reverse-transcribed using Moloney murine leukemia virus reverse transcriptase (MMLV-RT; Promega, Madison, WI, USA) and an antisense primer, complementary DNA was generated. The RNA was denatured for 5 min at 72°C and immediately placed on ice for 5 min. Denatured RNA was mixed with MMLV-RT, MMLV-RT buffer, and a deoxyribonucleotide triphosphate mixture and incubated for 1 h at 42°C. Synthesized cDNA fragments were amplified by polymerase chain reaction (PCR) in an MJ Research Thermocycler (Waltham, MA, USA). The PCR primers used for gene expression analysis are shown in Table 1. The cDNA was mixed with PCR primers, Taq DNA polymerase (Nanohelix, Daejon, Korea), and a dNTP mixture. The reaction consisted of 28–45 cycles of denaturation for 1 min at 94°C, annealing for 1 min at 56–60°C, and elongation for 1 min at 72°C. The PCR products were analyzed by electrophoresis on a 1% agarose gel. Relative expression levels are presented as a ratio of target gene cDNA vs. β-actin cDNA. PCR products were quantified from agarose gels using GeneGenius (Syngene, Cambridge, UK).

Table 1.  PCR primers and conditions used for cDNA synthesis by RT-PCR.

Gene	GeneBank	Primer sequence	AT (°C)	Cycle	cDNA size (bp)
Thiolase	NM_130864	F: 5′-AAATGGGTCTTATGACATTGGC-3′	56	45	294
		R: 5′-CACTCACCTGACTGGAGTTT-3′
VLCAD	AF017176	F: 5′-CGT CAG AGG TGT ACT TTG ATG G-3′	60	45	268
		R: 5′-CAT GCA CTC AGT CAC ATA CTG C-3′
UCP2	NM_011671	F: 5′-CTG AGC TGG TGA CCT ATG AC-3′	58	40	310
		R: 5′-TGA GCT GCT CAT AGG TGA CA-3′
β-actin	J00691	F: 5′-TGG AAT CCT GTG GCA TCC ATG AAA C-3′	58	28	350
		R: 5′-TAA AAC GCA GCT CAG TAA CAG AAA C-3′

Abbreviations: cDNA, complementary DNA; VLCAD, very long chain acyl-CoA dehydrogenase; UCP2, uncoupling protein 2.

Transient transfection assay

The expression vectors pSG5-mPPARα and PPRE₃-tk-luc reporter gene were generously provided by Dr. Frank J. Gonzalez (National Cancer Institute, NIH, Bethesda, MD, USA). NMu2Li cells were routinely cultured in DMEM containing 10% fetal bovine serum (Invitrogen), penicillin G (100 U/mL), streptomycin sulfate (100 μg/mL), amphotericin B (0.25 μg/mL), and 2-mercaptoethanol (50 μM). Cells were seeded in six-well tissue culture plates (2 × 10⁴ cells/well) 24 h prior to transfection. For all transfections, 200 ng/well of each of the appropriate plasmids were used. Transfections were performed using Lipofectamine (Invitrogen) according to the manufacturer’s instructions. After 6h, the culture medium was changed, and cells were exposed to different concentrations of GGEx18 or fenofibrate for 24 h, after which cells were washed twice with phosphate-buffered saline and assayed for luciferase and β-galactosidase activity using commercial kits according to the manufacturer’s instructions (Promega).

Statistical analysis

All values are expressed as the mean ± standard deviation. Statistical analysis was performed by analysis of variance followed by Turkey’s multiple comparison test. A p value < 0.05 was considered statistically significant.

Results

Inhibition of hepatic lipid accumulation by GGEx18 in nutritionally obese mice

High-fat diet-induced obese controls exhibited considerable hepatic lipid accumulation compared with the accumulation in chow diet-fed normal mice. However, we found that hepatic lipid accumulation was markedly lower in GGEx18-treated obese mice than in obese controls (Figure 1A). When obese mice were treated with GGEx18 at a dose of 250 mg/kg/day, there was a complete inhibition of hepatic lipid accumulation, as indicated by the decreased hepatic lesion scores (Figure 1B).

Figure 1.  Effects of Gyeongshingangjeehwan 18 (GGEx18) on hepatic lipid accumulation in high-fat diet-induced obese mice. Adult male C57BL/6 mice (n = 8/group) were fed a low-fat diet (Normal), a high-fat diet (Control), or the high-fat diet supplemented with 125, 250, or 500 mg/kg/day GGEx18 for 9 weeks. (A) Representative hematoxylin and eosin-stained sections of livers are shown (original magnification, ×100). (B) Histological analyses of hepatic lipid accumulation. Pathological scores of hepatic lipid accumulation are as follows: 0, no lesion; 1, mild; 2, moderate; 3, severe; 4, very severe. All values are expressed as the mean ± standard deviation of four independent experiments. ^#p < 0.05 compared with the normal group, *p < 0.05 compared with the control group.

GGEx18 also significantly decreased liver and pancreas weights that were increased by high-fat diet (Figure 2A). Serum ALT levels were also significantly reduced to the levels of lean normal mice after GGEx18 administration although AST levels were not altered in GGEx18-treated obese mice (Figure 2B and 2C). In addition, organ weights, such as heart, spleen, and kidney, were not changed by GGEx18 in obese mice.

Figure 2.  Effects of Gyeongshingangjeehwan 18 (GGEx18) on organ weights, serum alanine aminotransferase (ALT), and aspartate aminotransferase (AST) levels in high-fat diet-induced obese mice. Adult male C57BL/6 mice (n = 8/group) were fed a low-fat diet (Normal), a high-fat diet (Control), or the high-fat diet supplemented with 125, 250, or 500 mg/kg/day GGEx18 for 9 weeks. (A) Organ weights. (B) Serum ALT levels. (C) Serum AST levels. All values are expressed as the mean ± standard deviation. ^#p < 0.05 compared with the normal group, *p < 0.05 compared with the control group.

Improvement of lipid profiles by GGEx18 in nutritionally obese mice

To determine whether GGEx18 has similar beneficial effects on circulating lipid profiles, its effects on serum triglycerides and total cholesterol were examined. After 9 weeks, obese controls had significantly higher serum levels of triglycerides and total cholesterol than normal mice. However, 44 and 17% reductions of plasma triglycerides and total cholesterol levels, respectively, were observed in the GGEx18 (250 mg/kg/day) group (Figure 3).

Figure 3.  Effects of Gyeongshingangjeehwan 18 (GGEx18) on circulating (A) triglycerides and (B) total cholesterol in high-fat diet-induced obese mice. Adult male C57BL/6 mice (n = 8/group) were fed a low-fat diet (Normal), a high-fat diet (Control), or the high-fat diet supplemented with 125, 250, or 500 mg/kg/day GGEx18 for 9 weeks. Serum concentrations of triglycerides and total cholesterol were measured, and all values are expressed as the mean ± standard deviation. ^#p < 0.05 compared with the normal group, *p <0.05 compared with the control group.

Regulation of hepatic PPARα target gene expression by GGEx18 in nutritionally obese mice

To evaluate whether the effects of GGEx18 on hepatic lipid accumulation and circulating lipid metabolism in obese mice are caused by changes in the expression of genes involved in PPARα activation in the liver, we measured the mRNA levels of PPARα target enzymes, which are involved in fatty acid β-oxidation, in the liver. GGEx18 (250 mg/kg/day) administration to obese mice upregulated the mRNA levels of liver thiolase and VLCAD by 31 and 23%, respectively, compared with their levels in obese controls (Figure 4).

Figure 4.  Effects of Gyeongshingangjeehwan 18 (GGEx18) on PPARα target gene mRNA levels in high-fat diet-induced obese mice. (A)Adult male C57BL/6 mice (n = 8/group) were fed a low-fat diet (Normal), a high-fat diet (Control), or the high-fat diet supplemented with 250 mg/kg/day GGEx18 for 9 weeks. Total cellular RNA was extracted from liver tissue, and mRNA levels were measured using RT-PCR. Data are reported as the mean ± standard deviation of relative density units (R.D.U.) using β-actin as a reference. ^#p < 0.05 compared with the normal group, *p < 0.05 compared with the control group. (B) Representative RT-PCR bands from one of four independent experiments are shown.

We also examined the effects of GGEx18 on hepatic UCP2 gene expression, which is regulated by a selective PPARα activator, fenofibrate that upregulates fatty acid oxidation (Nakatani et al., 2002). GGEx18 administration increased UCP2 mRNA levels by 32% compared with those in control mice (Figure 4).

Regulation of the expression of PPARα target genes by GGEx18 in NMu2Li liver cells

Next, we measured the mRNA expression of PPARα target enzymes in liver cells. Similar to the in-vivo data, treatment of cells with GGEx18 upregulated the mRNA levels of thiolase, VLCAD, and UCP2 at all concentrations used in this study (Figure 5). Maximal levels of thiolase, VLCAD, and UCP2 mRNA expression were achieved at a concentration of 1 μg/mL, which resulted in 18, 69, and 109% increases, respectively, compared with control levels.

Figure 5.  Effects of Gyeongshingangjeehwan 18 (GGEx18) on PPARα target gene mRNA levels in NMu2Li liver cells. (A) NMu2Li cells were transiently transfected with pSG5-mPPARα, the reporter plasmid PPRE-TK-Luc, the β-galactosidase gene, and pBSK. Cells were treated with GGEx18 at concentrations of 0.1, 1, and 10 μg/mL. After incubation for 24 h, cells were harvested. RNA was extracted from cells, and mRNA levels were measured using RT-PCR. Data are reported as the mean ± standard deviation of relative density units (R.D.U.) using β-actin as a reference. ^#p < 0.05 compared with the normal group, *p < 0.05 compared with the control group. (B) Representative RT-PCR bands from one of four independent experiments are shown.

Modulation of PPARα reporter gene expression by GGEx18 in NMu2Li liver cells

To examine the mechanism by which GGEx18 increases the mRNA expression of PPARα target genes, NMu2Li cells were cotransfected with PPARα expression constructs and the luciferase reporter construct PPRE₃-tk-luc, which contains three copies of the PPAR response element (PPRE) from the rat acyl-CoA oxidase (ACOX) gene. The transfected cells were treated with GGEx18 at concentrations found to be nontoxic by Trypan blue exclusion. Treatment of the transfected cells with GGEx18 caused a significant increase in the magnitude of PPARα reporter gene activation (Figure 6). GGEx18 increased PPARα reporter activity by 60–85% at concentrations of 0.1–10 μg/mL compared with control levels. Similarly, the PPARα activator fenofibrate (10 μM) also increased PPARα luciferase activity by 138%.

Figure 6.  Effects of Gyeongshingangjeehwan 18 (GGEx18) on PPARα reporter gene expression in NMu2Li liver cells. NMu2Li cells were transiently transfected with pSG5-mPPARα, the reporter plasmid PPRE-TK-Luc, the β-galactosidase gene, and pBSK. Cells were treated with GGEx18 at concentrations of 0.1, 1, and 10 μg/mL or 10 μM fenofibrate (FF). After incubation for 24 h, cells were harvested and subsequently assayed for luciferase and β-galactosidase activities. Data are expressed as the mean ± standard deviation of relative luciferase units/β-galactosidase activity for three experiments. *p < 0.05 compared with the control group.

Discussion

Nonalcoholic fatty liver disease (NAFLD) is a common hepatic disease that can present as simple steatosis or nonalcoholic steatohepatitis and eventually progress to cirrhosis (Angulo, 2002). The pathogenesis of NAFLD is not well understood, and effective therapy for NAFLD has not been established. As NAFLD is deeply associated with obesity (Fabbrini et al., 2010), this study was undertaken to verify whether hepatic steatosis can be regulated by GGEx18, which decreases body weight gain and adipose tissue mass in high-fat diet-induced obese mice (Shin et al., 2011), and to clarify the molecular mechanism by which GGEx18 exerts these effects.

Our results demonstrate that GGEx18 decreased high-fat diet-induced hepatic steatosis and hyperlipidemia in mice. Excessive intrahepatic triglycerides, or steatosis, has been histologically defined when 5% or more hepatocytes contain visible intracellular triglycerides (Kleiner et al., 2005). In this study, high-fat diet-fed control mice had much higher triglyceride droplet levels than normal low-fat diet-fed mice. However, GGEx18 administration to control mice for 9 weeks significantly reduced lipid droplet levels by 72–96%. Of the three doses of GGEx18, 250 mg/kg/day GGEx18 almost completely prevented obesity-induced hepatic lipid accumulation. This finding is supported by a previous animal study demonstrating that GGEx18 administration (250 mg/kg/day) to obese mice for 9 weeks significantly reduced both body weight gain and total fat mass (Shin et al., 2011). This finding suggests that GGEx18 is beneficial for treating obesity-related hepatic steatosis. In accordance with reduced hepatic lipid accumulation, serum triglyceride and total cholesterol levels were also significantly reduced by GGEx18, indicating a role of GGEx18 in lipid metabolism. This result suggests that GGEx18 can be used to treat obesity-related hepatic steatosis and hyperlipidemia.

To gain insight into the molecular mechanisms underlying the effects described above, we measured the liver mRNA levels of PPARα target enzymes, which are responsible for fatty acid oxidation. PPARα is a ligand-activated transcription factor that regulates the expression of a number of genes critical for lipid and lipoprotein metabolism, thereby leading to lipid homeostasis. PPARα is known to initiate lipid-lowering effects through the transcriptional activation of mitochondrial, peroxisomal, and microsomal fatty acid-metabolizing enzymes (Aoyama et al., 1998; Kroetz et al., 1998; Yan et al., 1998; Jeong et al., 2004). Our previous study demonstrated that the PPARα ligand fenofibrate increases hepatic fatty acid oxidation and thus decreases the levels of plasma triglycerides due to PPARα activation in the liver (Yoon et al., 2003; Jeong et al., 2004). This finding is supported by a report that PPARα-deficient mice exhibited abnormalities in triglyceride and cholesterol metabolism and became obese with age (Costet et al., 1998). Fatty acid oxidative PPARα target genes and PPARα expression levels are suppressed in the adipose tissue and skeletal muscle of obese mice (Kamei et al., 2005). PPARα also upregulates the expression of UCP2, which contains the PPRE in its promoters and participates in fatty acid oxidation (Lentes et al., 1999; Nakatani et al., 2002). In this study, GGEx18 administration to obese mice significantly increased hepatic mRNA levels of peroxisomal and mitochondrial PPARα targets, such as thiolase and VLCAD, respectively. Similar gene activation was observed in GGEx18-treated NMu2Li liver cells. These results are in accordance with our data on the substantial reduction of hepatic lipid droplet, serum triglyceride, and total cholesterol levels by GGEx18. These results suggest that GGEx18 increases PPARα-dependent fatty acid oxidation in the livers of obese mice.

To determine the mechanism by which GGEx18 increases PPARα target gene expression and to examine whether GGEx18-induced PPARα activation prevents hepatic lipid accumulation and hyperlipidemia in obese mice, we examined the effects of GGEx18 on PPARα transactivation using NMu2Li cells. Consistent with the in-vivo data, our reporter assay data also suggest that GGEx18 significantly increased luciferase activity compared with that in control cells, although the luciferase activity was lower in cells treated with GGEx18 than in cells treated with fenofibrate, suggesting that GGEx18 can act as a potential agonist of PPARα and stimulate PPARα-dependent fatty acid catabolism.

Although ephedra-containing dietary supplements and ephedrine have been reported to have toxicity (Haller & Benowitz, 2000; Fleming, 2008), this study showed that significant side effects were not observed in GGEx18-treated mice. Treatment of mice with GGEx18 did not affect organ weights, such as heart, spleen, and kidney. Interestingly, GGEx18 decreased liver and pancreas weights that were increased by high-fat diet. Moreover, GGEx18 also reduced circulating ALT levels to the levels of normal mice. Thus, it seems that GGEx18 may be a safe and effective drug for hepatic steatosis and hyperlipidemia.

In conclusion, these studies demonstrate that GGEx18 may prevent hepatic steatosis and hyperlipidemia, such as hypertriglyceridemia and hypercholesterolemia, in obese mice, by activating hepatic PPARα (Figure 7). Further investigation will be necessary to identify bioactive compounds from GGEx18 to develop natural therapeutic agents or dietary supplements to treat obesity-related metabolic disease.

Figure 7.  Possible mechanism of Gyeongshingangjeehwan 18 (GGEx18) to inhibit hepatic steatosis and hyperlipidemia. GGEx18 may activate PPARα target genes responsible for fatty acid oxidation in the livers of obese mice, leading to decreased hepatic steatosis, hypertriglyceridemia, and hypercholesterolemia.

Declaration of interest

This work was supported by the National Research Foundation of Korea (NRF) Grant funded by the Korea Government (MEST) (NRF-2010-0027498 and NRF-2011-0003703), Korea. The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this paper.