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Korean Society of Life Science (한국생명과학회)
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Protective Effect of Rubus crataegifolius Extracts Against Obesity and Non-alcoholic Fatty Liver Disease via Promotion of AMPK/ACC/CPT-1 Pathway in HFD-induced C57BL/6J Obese Mice

HFD 유도 C57BL/6J 비만 mice에서 AMPK/ACC/CPT-1 경로 촉진을 통한 산딸기 추출물의 비만 및 비알코올성 지방간 질환에 대한 보호 효과

  • Received : 2023.08.28
  • Accepted : 2023.09.27
  • Published : 2023.12.30
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Abstract

Rubus crataegifolius (RC) is a traditional Asian medicinal plant belonging to the Rosaceae family. The fruits of RC are known to prevent adult diseases through antioxidants. In this study, the effects of RC extract (RCex) on obesity and nonalcoholic fatty liver disease (NAFLD) were evaluated in animal models. Twenty-eight male C57BL/6J mice were induced to become obese for 8 weeks and then the extract was orally administered for 8 weeks. RCex reduced body weight, adipose tissue, liver weight. RCex improved biochemical biomarkers including lipid metabolism (alanine aminotransferase (ALT), aspartate aminotransferase (AST), plasma triglyceride (TG), total cholesterol (TC), high-density lipoprotein (HDL) cholesterol and low-density lipoprotein (LDL) cholesterol). The activation of AMP-activated protein kinase (AMPK) reduced the expression of adipogenesis genes (liver × receptor (LXR), sterol regulatory element-binding protein-1c (SREBP-1c), fatty acid synthesis (FAS), acetyl-CoA carboxylase 1 (ACC1) and the effect of enhancing carnitine palmitoyltransferase (CPT) activity by RCex was verified. RCex also influence on plasma production of hormones (adiponectin & leptin) related on energy expenditure and metabolism. In addition, we confirmed that RCex improved glucose intolerance in HFD-induced obese rats. RCex was first demonstrated to have anti-obesity as well as anti-NAFLD effects by regulating fatty acid oxidation and fatty acid synthesis by phosphorylation of AMPK. This suggests that RCex could be a good supplement for the prevention of obesity and related NAFLD.

Rubus crataegifolius (RC)는 장미과에 속하는 전통적인 아시아 약용 식물이다. RC 열매는 항산화 작용을 통해 성인병을 예방하는 것으로 알려져 있다. 본 연구에서는 RC 열매 추출물(RCex)이 비만과 비알코올성 지방간 질환(NAFLD)에 미치는 영향을 동물 모델을 통해 평가하였다. 28마리의 수컷 C57BL/6J 마우스에 8주간 비만을 유도한 후, 추출물을 8주간 경구 투여하였다. 그룹 1은 일반 대조군으로 표준사료를 섭취하였다. 그룹 2는 HFD 대조군으로, 그룹 3에는 심바스타틴(6.5 mg/kg/일)을, 그룹 4에는 RCex (200 mg/kg)을 투여하였다. RCex투여는 실험 마우스의 체중, 지방 조직, 간 무게를 감소시켰으며, 또한 지질 대사(ALT, AST, TC, TG, LDL, HDL)를 포함한 생화학적 바이오마커를 개선하였다. AMPK의 활성화는 지방생성 유전자(LXR, SREBP-1c, FAS, ACC1)의 발현을 감소시켰으며, RCex에 의한 CPT 활성 증진 효과를 검증하였다. RCex는 또한 에너지 소비 및 신진대사와 관련된 호르몬(adiponectin 및 leptin)의 혈장 수준에도 영향을 미쳤다. 또한, RCex가 HFD로 유도된 비만 mice의 포도당 불내성을 개선했음을 확인 하였다. RCex는 AMPK의 인산화를 통해 지방산 산화 및 지방산 합성을 조절함으로써 항비만 및 항NAFLD 효과를 가짐을 처음으로 입증하였다. 이는 R. crataegifolius가 비만 및 관련 NAFLD 예방에 좋은 보충제가 될 수 있음을 시사한다.

Keywords

Introduction

Obesity is closely related to the pathogenesis and severity of NAFLD. Fatty liver disease is common in obese patients, and fatty liver is considered when the triglyceride content in the liver is 5% or more [40]. Because the cause of NAFLD is independent of alcohol, it is classified as non-alcoholic and is considered a metabolic syndrome including obesity [39] 80% of patients with NAFLD are obese with a body mass index (BMI) of 30 or higher. With the increasing incidence of obesity and NAFLD, management of lipid and energy metabolism is important in health, eating habits, and disease states. Therefore, it is important to elucidate the metabolic modulators involved in NAFLD, in which excessive dietary intake leads to NAFLD onset and exacerbation to non-alcoholic steatohepatitis (NASH) leading to obesity.

Obesity-induced free fatty acids and de novo fat formation are known causes of NAFLD [38]. The most important factor inducing liver damage in NAFLD is the accumulation of TG in the liver caused by excessive free fatty acids and inflammation caused by visceral adipose tissue (VAT) [37]. In addition, inflammatory cytokines are released from VAT, and lipid accumulation caused by free fatty acids induces inflammation and insulin resistance [36]. Free fatty acids due to excessive energy supply are introduced into the liver by fatty acid transporters, and free fatty acids are accumulated in the liver in the form of TG. As a result of de novo lipogenesis due to the regulation of SREBP-1c, intrahepatic TG accumulates. Thus, hepatic steatosis is characterized by excessive accumulation of TG [35]. Excessive accumulation of liver lipids causes lipotoxicity, and this lipotoxicity caused by hepatic steatosis is related to the development of insulin resistance, type 2 diabetes, and metabolic syndrome [33, 34].

Obesity and NAFLD are associated with metabolic syndrome, including type 2 diabetes and cardiovascular disease. The severity of fatty liver in obese individuals correlates with impaired blood glucose status [32]. Therefore, it is important to identify regulatory mechanisms driving the progression of obesity and NAFLD and the metabolic underpinnings of NASH.

There are about 250 species of RC in the Rosaceae family worldwide. The unripe fruits of raspberries have been used medicinally in Asia, including Korea and China [31]. In Korea, RC has been used for sperm health management and erectile dysfunction, and has also been used as an adjuvant for gastrointestinal health [29, 30]. Ellagic acid from Rubus protects hepatocytes from damage with its strong antioxidant effect, and also showed effects in CYP3A4 regulation, inhibition of reactive oxygen species production [28], and liver protection from hepatitis virus in animal models [25-27]. Ellagic acid has also been reported to have beneficial effects on cellular lipid metabolism and plasma cholesterol in hyperlipidemic rats [23, 24]. In this study, in obese mice induced by high-fat diet (HFD), when treated with RCex, body weight, adipose tissue weight, hepatic steatosis, expression of lipid metabolism genes related to AMPK, and enzyme activity were investigated, the effects of RCex on obesity and NAFLD were investigated.

Materials and Methods

Plant materials and extract preparation

The unriped fruit of Rubus was purchased from Korea and confirmed on the fruit of Rubus crataegifolius Bunge by Korea Research Institute of Bioscience and Biotechnology (KRIBB). The plant samples were kept in the herbarium of KRIBB. The RCex was prepared using 50% ethanol extract and freeze dried for experiment of Lee’s Biotech Co., Ltd (Yuseong, Daejeon, Korea).

Animal treatment

Male C57BL/6J mice were purchased from Raonbio Corporation (Yongin, Korea) and used in the experiments. Animals were maintained at 22℃, 50±10% relative humidity, and a 12 hr light/dark cycle and allowed to acclimatize for at least one week before being grouped. Animals were fed HFD for 8 weeks, except for normal controls (fed with a normal diet). After 8 weeks of obesity induction period, animals were randomly divided into 4 groups. Group 1 was a normal diet group (n=7, ND) and was fed a standard chow diet. Group 2 served as a high-fat-diet (n=7, HFD) control group, group 3 was given simvastatin (SMV, n=7, 6.5 mg/kg/ day) and group 4 was given RCex extract (n=7, 200 mg/kg). All animals were weighed once a week for 8 weeks. All animals were fasted for 16h and sacrificed under anesthesia. Blood samples were collected for measurement of serum biochemical parameters, fasting blood glucose, prior to animal sacrifice. Liver and adipose tissue were weighed, and for histological analysis, liver tissues were quickly washed, fixed in 10% neutral buffered formaldehyde. Animal experiments were conducted in accordance with the institutional guidelines of Lee's Biotech (Daejeon, Korea) and approved by the Ethics Committee and Animal Use and Care Committee (LS-IACUC-2021-01).

Plasma analysis and hepatic histology

Eight weeks after RCex administration, blood samples were collected using cardiac puncture. The collected blood was centrifuged (10,000 × g, 4℃ for 5 minutes) to separate plasma and stored at -70℃ until analysis. ALT, AST, TG, TC, HDL cholesterol and LDL cholesterol using plasma was measured. Plasma ALT, AST, HDL, and LDL levels were measured using an automated biochemical analyzer. Plasma TG, TC, leptin, and adiponectin levels were directly measured by ELISA method according to the instructions of the manufacturer's assay kit (Bioclinical System Co., Anyang, Korea). Livers were harvested immediately after the mice were sacrificed and fixed in 10% formalin buffer. After making paraffin blocks of the fixed tissue for histological analysis, paraffin sections were sectioned at a thickness of 5 μm, and hematoxylin and eosin staining was performed. The stained slides were subjected to histological analysis using an optical microscope.

Quantitative polymerase chain reaction (qPCR) analysis

Total cellular RNA was prepared from liver using Trizol (Bioneer, Dajeon, Korea) reagent. 2 μg total RNA was reverse transcribed using RT PreMix & Master Mix (Bioneer, Dajeon, Korea) to synthesize cDNA. The cDNA was subjected to PCR using mastermix Taq (Bioneer, Daejeon, Korea) according to the PCR system (Astec, Tokyo, Japan) method. PCR primers used to evaluate mRNA expression of each gene are shown in Table 1. The cycling conditions consisted of 50 cycles of denaturation at 94℃ for 1 min, annealing at 58℃ for 1 min, and elongation at 72℃ for 1 min. Relative expression levels were calculated and presented as the ratio of target gene cDNA to GAPDH cDNA.

Table 1. Nucleotide sequence of primers for qPCR of mRNA

SMGHBM_2023_v33n12_967_t0001.png 이미지

SREBP-1c, sterol regulatory element binding protein-1c; LPL, lipoprotein lipase; LXR, liver X receptor; FAS, fatty acid synthase; ACC1, acety1-CoA carboxylase 1; qPCR, quantitative polymerase chain reaction.

Western blot analysis

To extract liver tissue proteins, liver tissue was homogenized at 4℃ using RIPA lysis buffer supplemented with the protease inhibitor phenylmethylsulfonyl fluoride (PMSF). After centrifugation at 12,000 g and 4℃ for 15 minutes, the protein was separated and the protein concentration was quantified using the BCA method. A 50 mg protein samples were subjected to electrophoresis to separate proteins by size, and a transfer process was performed with a NC membrane. After blocking the transferred membrane with 5% BCA for 1 hour, the primary antibodies of p-AMPK, AMPK, CPT-1, and CPT-2 to be analyzed were incubated overnight at 4℃. After washing the membrane with TBST three times for 10 minutes each, it was incubated with the secondary antibody for 1 hr at room temperature. In addition, bands were identified using an DLx Imaging system (Odyssey, Lincoln, NB, USA). The band density was quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA) and the results are presented.

Fasting blood glucose and oral glucose tolerance test measurement

After 8 weeks of extract treatment, fasting glucose and oral glucose tolerance test measurements were performed to determine glucose tolerance after the animals had been fasted for 8 hr. Mice were orally administered glucose (2 g/kg body weight, dissolved in water). Blood glucose level was measured using a blood glucose meter (Accu-Chek Active, Roche, Germany) at 5 time points (0 min, 30 min, 60 min, 90 min, 120 min) after blood was collected from the tail vein after glucose administration.

Statistical analysis

All values are presented as mean ± standard deviation (S.D.). Significant differences between mean values of each group were analyzed by unpaired Student's t-test using SigmaPlot 2001 (SPSS Inc, Chicago, IL). Statistical significance was defined as a p<0.05 value.

Results

Effects of RCex on body weight, liver and adipose tissue weight in HFD fed mice

A HFD animal model was used to investigate the anti-obesity effects of RCex on body weight, liver, and adipose tissue. Body weight increased over time in HFD groups but decreased significantly in the HFD+RCex groups (Fig. 1A). The HFD-fed group showed increased liver weight levels compared to the ND group, whereas the RCex treatment group showed a decrease in liver weight (Fig. 1B). We also confirmed the effect of RCex on adipose depots in animals (Fig. 2). Widespread adoption of obesity required extensive study on white adipose tissue (WAT), often using the obese C57BL/6J mouse strain as a model. The total adipose tissue is important for the development of metabolic diseases including obesity, NAFLD and insulin resistance, but some adipose depots have been identified to be more associated with risk factors for diseases than others. The major adipose depots (WAT: white adipose tissue) of interest are found in the abdomen and can be divided into subcutaneous adipose tissue (SAT) and VAT [22]. VAT can be further divided into mesenteric, epididymal and retroperitoneal adipose tissues, and the SAT can be further divided into inguinal adipose tissue. The distribution of SAT and VAT varies from person-to-person and depend on several factors such as age, nutrition, sex and energy homeostasis of individual adipose tissue [21]. Indeed, fundamental differences between WAT depots have been reported, including effects on metabolism, endocrine function, and pre-adipocyte specific responses to high fat diet feeding [20]. In this experiment we determined that animals is important for the development of metabolic diseases including obesity, NAFLD and insulin resistance, but some adipose depots have been identified to be more associated with risk factors for diseases than others. The major adipose depots (WAT: white adipose tissue) of interest are found in the abdomen and can be divided into subcutaneous adipose tissue (SAT) and VAT [22]. VAT can be further divided into mesenteric, epididymal and retroperitoneal adipose tissues, and the SAT can be further divided into inguinal adipose tissue. The distribution of SAT and VAT varies from person-to-person and depend on several factors such as age, nutrition, sex and energy homeostasis of individual adipose tissue [21]. Indeed, fundamental differences between WAT depots have been reported, including effects on metabolism, endocrine function, and pre-adipocyte specific responses to high fat diet feeding [20]. In this experiment we determined that animals treated with RCex showed a reduction in total fat found in the abdomen and divided WAT; SAT and VAT. We also checked the presence of VAT storage specific tissue; mesenteric, epididymal, retroperitoneal fat and SAT depot specific tissue; inguinal fat also showed some differences after treatment with RCex in HFD induced obese mice. As shown in Fig. 2, all of HFD-treated groups showed high levels of fat weight, while the RCex treated group showed a decreased in fat weight (Total WAT; SAT&VAT, VAT; mesenteric, epididymal, retroperitoneal depots, SAT; inguinal depot).

SMGHBM_2023_v33n12_967_f0001.png 이미지

Fig. 1. Effects of RCex on body weight and liver weight of HFD-fed C57BL/6J. C57BL/6J mice were fed LFD, HFD, simvastatin (6.5 mg/kg), and RCex (200 mg/kg) for 8 weeks. (A) Body weight. (B) Liver weight. Values are expressed as the mean ± standard deviation (n=7 per each group). # p<0.05 compared with the control (ND). ** p<0.01 compared with HFD treatment.

SMGHBM_2023_v33n12_967_f0002.png 이미지

Fig. 2. Effect of RCex on adipose tissue weight in C57BL/6J fed HFD. C57BL/6J mice were fed LFD, HFD, simvastatin (6.5 mg/kg), and RCex (200 mg/kg) for 8 weeks. (A) Total fat weight, (B) Subcutaneous fat weight, (C) Visceral fat weight, (D) Mesenteric fat weight, (E) Epididymal fat weight, (F) Inguinal fat weight, (G) Retroperitoneal fat weight. Values are expressed as the mean ± standard deviation (n=7 per each group). # p<0.05 and ## p<0.01 compared with the control (ND). * p<0.05 and ** p<0.01 compared with HFD treatment.

SMGHBM_2023_v33n12_967_f0003.png 이미지

Fig. 3. Effects of RCex on biochemical parameters in HFD-fed C57BL/6J. C57BL/6J mice were fed LFD, HFD, simvastatin (6.5 mg/kg), and RCex (200 mg/kg) for 8 weeks. (A) AST, (B) ALT, (C) TG, (D) TC, (E) HDL, (F) LDL. Values are expressed as the mean ± standard deviation (n=7 per each group). # p<0.05 and ## p<0.01 compared with the control (ND). *p<0.05 and **p<0.01 compared with HFD treatment.

SMGHBM_2023_v33n12_967_f0004.png 이미지

Fig. 4. Effects of RCex on hepatic lipid accumulation in HFD-fed C57BL/6J. C57BL/6J mice were fed LFD, HFD, simvastatin (6.5 mg/kg), and RCex (200 mg/kg) for 8 weeks. (A) Macroscopic liver tissue of mice of each group (B) H&E-stained sections of liver tissues (original magnification ×100).

Effects of RCex on biochemical parameters and hepatic fat accumulation in HFD-fed mice

Adipokines are cytokines secreted by visceral adipose tissue. Adipokines and leptin are closely related to the pathogenesis of obesity and NAFLD. Obesity is a state of central and peripheral leptin resistance, and most patients with obesity, NAFLD, and NASH have elevated leptin levels [18, 19]. Leptin functions to regulate energy metabolism and inhibit lipid accumulation in liver tissue [17]. Adiponectin inactivates acetyl-CoA carboxylase, activates AMPK, activates PPAR-α gene to activate hepatic fat oxidation, and inhibits FA synthesis [15, 16]. Plasma leptin and adiponectin levels were evaluated to evaluate the role of RCex on adipokines. Plasma leptin levels were significantly increased in the HFD group compared to the ND group. Leptin concentrations were significantly reduced in the RCex group after 8 weeks of exposure to HFD (Fig. 5A). Conversely, plasma adiponectin levels increased in concentration in the RCex group (Fig. 5B). These results suggest that RCex reduced leptin and increased adipokines in HFD-induced obese and NAFLD mice.

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Fig. 5. Effects of RCex on hormone regulation in HFD-fed C57BL/6J. C57BL/6J mice were fed LFD, HFD, simvastatin (6.5 mg/kg), and RCex (200 mg/kg) for 8 weeks. (A) Leptin, (B) Adiponectin. Values are expressed as the mean ± standard deviation (n=7 per each group). ##p<0.01 compared with the control (ND). *p<0.05 and **p<0.01 compared with HFD treatment.

Effect of RCex on hepatic lipid regulating gene on HFD fed mice

Many studies have shown that excessive lipid accumulation is a major cause of obesity and hepatic steatosis. Therefore, inhibiting systemic and hepatocytes from lipid accumulation is an important strategy for preventing and treating obesity and NAFLD. Thus, promoting β-oxidation is thought to be an effective solution to reduce lipid accumulation in the liver [27]. We investigated the effect of RCex on hepatic lipid regulatory gene expression, protein expression and lipid regulating enzyme activities in HFD induced mice. First, we examined the hepatic expression levels of lipogenesis regulating genes including LXR, SREBP-1c, ACC1, LPL and FAS (Fig. 6). Relative levels of specific mRNA levels were tested using qPCR. In this study, compared to the HFD group, the RCex-treated group significantly reduced the expression of SREBP-1c and LXR mRNA, and the SREBP-1c-regulated adipogenic genes such as FAS, LPL, and ACC were significantly reduced (Fig. 6). By evaluating the activities of AMPK and CPT, which are representative proteins that mediate mitochondrial β-oxidation in the liver, we investigated whether RCex affects the protein expression level of β-oxidation (Fig. 7). Expression of pAMPK, CPT-1 and -2 in the liver was significantly increased in the RCex-fed group compared to the HFD-fed group (p<0.01) (Fig. 7). Phosphorylation of AMPK, which plays an important role in fatty acid oxidation in the liver, activates CPT-1, -2 and promotes fatty acid β-oxidation.

SMGHBM_2023_v33n12_967_f0006.png 이미지

Fig. 6. Effects of RCex on Hepatic expression of lipogenic genes in HFD-fed C57BL/6J. C57BL/6J mice were fed LFD, HFD, simvastatin (6.5 mg/kg), and RCex (200 mg/kg) for 8 weeks. The mRNA expression of LXR, SREBP1c, FAS, ACC1, LPL and GAPDH in liver tissues. Values are expressed as the mean ± standard deviation (n=7 per each group). #p<0.05 and ## p<0.01 compared with the control (ND). *p<0.05 and **p<0.01 compared with HFD treatment.

SMGHBM_2023_v33n12_967_f0007.png 이미지

Fig. 7. Effects of RCex on phosphorylation of AMPK, CPT-1 and CPT-2 in liver tissue in HFD-fed C57BL/6J. C57BL/6J mice were fed LFD, HFD, simvastatin (6.5 mg/kg), and RCex (200 mg/kg) for 8 weeks. Protein levels of phosphorylated AMPK (p-AMPK), CPT-1, CPT-2 were determined by Western blot analysis. Values are expressed as the mean ± standard deviation (n=7 per each group). * p<0.05 and ** p<0.01 compared with HFD treatment.

Effect of RCex on blood sugar levels in HFD-induced obese mice

After 8 weeks of HFD treatment, fasting blood sugar was measured after 8 hr of starvation, and an oral glucose tolerance test (OGTT) was measured every 0, 30, 60, 90, and 120 minutes after 2.0 g/kg glucose administration at 8 hr of fasting. Compared to the normal group (ND), the level was significantly higher (p<0.01) in the HFD group, but the blood sugar level was significantly lower (p<0.01) in HFD-eating animals treated with RCex and SMV (Fig. 8A). The result of Fig. 8B showed that the HFD-fed group showed glucose intolerance, whereas the RCex-fed group showed a significant decrease (p<0.01) in blood sugar at all points after glucose administration. The results shown in Fig. 8 showed that glucose intolerance was improved during RCex treatment.

SMGHBM_2023_v33n12_967_f0008.png 이미지

Fig. 8. Effects of RCex on blood sugar regulation in HFD-fed C57BL/6J. C57BL/6J mice were fed LFD, HFD, simvastatin (6.5 mg/kg), and RCex (200 mg/kg) for 8 weeks. (A) Fasting blood glucose (B) oral glucose tolerance test (OGTT). Values are expressed as the mean ± standard deviation (n=7 per each group). ## p<0.01 compared with the control (ND). * p<0.05 and ** p<0.01 compared with HFD treatment.

Discussion

Obesity has become a global epidemic. The rapid rise of NAFLD over the past decades has greatly increased the number of obese patients and has made it the world's most chronic liver disease. There is growing evidence that obesity-induced adipose tissue dysfunction and the development of NAFLD are closely related [13]. Obesity, especially central obesity, has been identified as major cause of metabolic syndrome, including insulin resistance, type 2 diabetes, hypertension, NAFLD and dyslipidemia, which eventually lead to cardiovascular disease. Among these, NAFLD formation showed that adipose tissue dysfunction and insulin resistance could affect the regulation of de novo lipogenesis and ultimately lead to NAFLD [12].

As adipokines are essential for adipose tissue-hepatic crosstalk, the adiponectin to leptin ratio could be a potential biomarkers for NAFLD formation as well as metabolic disturbances due to obese adipose tissue [11]. In our experimental data (Fig. 5), we showed that adiponectin increased and leptin levels decreased treatment with RCex on HFD treated mice. These results demonstrated that obesity-induced changes in adipokine secretion and adipose tissue-hepatic crosstalk could be corrected by RCex treatment in HFD-induced obese mice. As mentioned above, imbalance in adiponectin and leptin levels are known to causes obesity and the formation NAFLD, and lead to inflammation and insulin resistances too [10]. Adipose tissue dysfunction and insulin resistance can further impair energy balance by promoting de novo biogenesis in the liver that leads to NAFLD formations [9]. For these reasons, measuring the adiponectin to leptin ratio may be useful in reaching obesity induced adipose tissue dysfunction and formation of NAFLD, including IR. The data in Fig. 8 showed that RCex reduced IR in SMV & RCex which showed strong IR status after treating with HFD.

With the rates of obesity and NAFLD increased, understanding how energy metabolism is regulated in healthy and diseased conditions has become a major concern of human societies. Therefore, understanding the regulatory factors to prevention the progression of obesity, NAFLD and NASH is urgently needed in human society. Lipid levels in the adipose tissue or liver are in balanced between lipid synthesis and lipid consumption, and imbalanced lipid metabolism cause lipid accumulation in the body, resulting in hepatic steatosis. Therefore, activating fatty acid oxidation and inhibiting lipid synthesis are considered as a solution to hepatic steatosis.

In mitochondrial fatty acid oxidation, free fatty acids are introduced into the mitochondria in the form of acyl-CoA. Introduced acyl-CoA is oxidized and converted to acetyl-CoA form, followed by β-oxidation through the TCA cycle [8]. In free fatty acid β-oxidation, CPT is involved in transporting free fatty acids into mitochondria. For this reason, activation of CPT1 oxidizes free fatty acids to limit lipid accumulation, thus providing metabolic benefits including prevention of obesity and NAFLD [6, 7]. The fact that mitochondria are involved in FAO and CPT system and transport FFA into mitochondria means that ameliorating mitochondrial dysfunction is an effective strategy to reduce lipid accumulation that causes obesity and NAFLD (Fig. 7).

Besides this, inhibiting de novo lipogenesis in the liver is considered to be able to prevent NAFLD and even NASH [5]. In this process, AMPK plays a central role in regulating lipid metabolism, and phosphorylated AMPK negatively regulates ACC to suppress fatty acid synthesis and increase fatty acid oxidation [4]. ACC converts acetyl-CoA to malonyl-CoA, and malonyl-CoA inhibits the activity of CPT-1, thereby inhibiting fatty acid oxidation. However, phosphorylated AMPK inhibits ACC and induces the activity of CPT-1 to activate fatty acid oxidation (Fig. 9).

AMPK also regulates SREBP-1c, a key regulator of FAS, ACC proteins. In obese and NAFLD mice, the activity of SREBP-1c/FAS pathway was clearly increased while the ACC/CPT-1 pathway was downregulated. Thus, activation of the AMPK/ACC/CPT-1 pathway and inhibition of SREBP-1c/FAS pathway could be the targets for screening drugs to prevent lipid accumulation in obesity & NAFLD [4].

SREBP-1C modulates the response to fat and insulin. It is also a key transcription factor that regulates the expression of enzymes related to lipogenesis and fatty acid desaturation. SREBP-1C is known to be regulated by the insulin signaling pathway and negatively regulated by AMPK [3]. In the NAFLD condition, insulin receptor substrate 1-mediated insulin signaling induces upregulation of SREBP-1c, leading to increased fatty acid synthesis in hepatocytes [2]. LXR is an important factor regulating the biosynthesis of inflammatory cytokines and lipid metabolism. It increases hepatic TG synthesis by increasing the expression of SREBP-1c [1]. In this experiment, we confirmed that RCex ameliorates HFD-induced lipid accumulation in obesity and NAFLD formation. RCex downregulates lipogenesis activitiey (downregulating LXR, SREBP-1c, FAS, ACC gene expression) and enhances CPT enzyme activity, which activates β-oxidation of fatty acids. Our results suggest that RCex treatment is involved in HFD-induced obesity and NAFLD by regulating the AMPK-associated ACC and CPT pathways and the SREBP-1c/FAS pathway through regulation of leptin and adiponectin.

Acknowledgments

This project was supported by Development of Regional Specialized industries +(R&D)/(S3093558) in ministry of SMEs and startups (MSS).

The Conflict of Interest Statement

The authors declare that they have no conflicts of interest with the contents of this article.

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