Nonalcoholic fatty liver disease (NAFLD), or fatty liver, is a chronic liver disease associated with hepatic dysfunction due to excessive triglyceride (TG) accumulation [1
]. NAFLD is closely related to metabolic diseases, such as obesity, insulin resistance, hypertension, and dyslipidemia, and the incidence of NAFLD increases as obesity increases [2
]. Abnormal dietary fat intake directly contributes to a casual role in hepatic lipid accumulation, such as TG, cholesterol, and lipid droplets in the cytoplasm of hepatocytes [3
], and lipotoxicity that is caused by fat accumulation induces hepatic insulin resistance in the pathogenesis of type 2 diabetes [4
The current drugs for treatment of NAFLD have considerable side effects, and their long-term treatment effect is not revealed and unsafe [5
]. Therefore, an effective alternative to pharmacological therapy is needed, such as using natural products, which may be safe and beneficial in the treatment and prevention of NAFLD.
Recently, according to the Food and Agriculture Organization of the United Nations, edible insects have been reported to be possible future human dietary supplements because they are a rich source of unsaturated fatty acids, proteins, minerals, vitamins, and other nutrients [6
]. Various insects have already been used as traditional food or folk remedies in many countries, however, their consumption has been restricted by the lack of scientific proof of positive effects and safety to humans. Allomyrina dichotoma
, also known as “Jangsupungdaeng-i” in Korea, is a species of rhinoceros beetle that is widely used in traditional medicine for its anti-hepatofibrotic, antineoplastic, and antioxidant effects [9
]. The component of pharmacological activity was established in beetle larva [11
]. However, whether A. dichotoma
larva extract (ADLE) can prevent fatty liver or improve hyperglycemia in high-fat diet (HFD)-induced type 2 diabetes has not yet been investigated. Therefore, we investigated the effect of diabetic and liver steatosis of ADLE in diabetic mice induced by HFD-fed mice model, and its molecular mechanisms in palmitate-treated HepG2 cells.
2. Materials and Methods
2.1. Preparation of A. Dichotoma Larva Extract (ADLE)
A. dichotoma larva were purchased from Yechun Bugs land (Yecheon-gun, Gyungsangbuk-do, Korea). The dried A. dichotoma larva were powdered by using a grinder and extracted with ethanol/water (70:30, v/v) at a ratio of 20 mL/g for 3 h at 60 °C thrice, followed by filtration twice through filter paper through No. 2 filter paper (Advantec Toyo Roshi Kaisha, Ltd., Tokyo, Japan). The filtrate was concentrated in a vacuum rotary evaporator (T < 40 °C), lyophilized, and dissolved in deionized water.
2.2. Cell Culture
The HepG2 cells, a human hepatoblastoma cell line, were cultured in Dulbecco’s modified Eagle’s medium (Gibco, Paisley, UK) containing 1% penicillin/streptomycin (Welgene, Daegu, Korea) and 10% fetal bovine serum (Gibco) in an atmosphere of 5% CO2 at 37 °C.
2.3. Preparation of Palmitate and MTT Assay
Sodium palmitate (Sigma, St. Louis, MO, USA) was conjugated with 5% bovine serum albumin (Sigma) at a 1:3 volume ratio to make a 20 mM stock solution [13
]. The HepG2 cells were exposed to 0.5 mM palmitic acid with or without ADLE for 24 h. Cell viability was determined by colorimetry using 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT, thiazolyl blue) (Duchefa Biochemie BV, Haarlem, the Netherlands). Insoluble formazan crystals were dissolved in 2-propanol and detected using a microplate spectrophotometer at 540 nm.
Four-week-old C57BL/6J male mice were obtained from the Korea Research Institute Bioscience & Biotechnology (KRIBB, Daejeon, Korea). The mice were allowed free access to standard chow diet and water for a week. The mice were maintained at an ambient temperature (23 °C) with 12:12 h light–dark cycles with free access to food and water. All animal procedures were approved by the Institution Animal Care and Use Committee at Eulji University (EUIACUC-18-7). After a week of adaptation, the mice were fed a high-fat diet (HFD) of 60% fat (D12492; Research Diets, New Brunswick, NJ, USA) for 6 weeks (n
= 33). Aged-matched control mice were fed a normal-fat diet (NFD) of 4.5% fat (Purina) for the same period (n
= 6). Six weeks after the HFD, blood glucose levels and body weight were checked, and the mice with blood glucose levels >200–250 mg/dL were used for experiments. Diabetic mice were treated orally with ADLE (100 mg/kg/day) or vehicle (distilled water) (daily for 6 weeks) as described previously [14
]. Metformin (Cayman Chemical, MI, USA) was orally administered (100 mg/kg/day) for 6 weeks as a positive control. Body weight and food intake were recorded weekly, and blood glucose was measured every 2 weeks for the 6 week feeding period. The food efficiency ratio (FER) was calculated based on the formula: FER = body weight increased during experimental period (g)/total food intake during the experimental period (g).
2.5. Intraperitoneal Glucose Tolerance Test and Intraperitoneal Insulin Tolerance Test
After fasting for 18 h, blood glucose was measured in whole blood from the tail vein (0 min) by using a one-touch glucometer (Johnson & Johnson, New Brunswick, NJ, USA). Subsequently, the glucose solution dissolved in phosphate-buffered saline (PBS) was injected intraperitoneally (2 g/kg), and blood glucose was detected at 30, 60, and 120 min. For the insulin tolerance test (ITT), the mice were fasted for 4 h, and blood glucose was measured in whole blood from the tail vein (0 min) by using a glucometer, the insulin solution dissolved in PBS was injected intraperitoneally (2 units/kg), and blood glucose was detected at 30, 60, and 120 min. An area under the curve (AUC) trapezoid model from 0 to 120 min after challenge was used to quantitatively evaluate glucose clearance activity. The AUC between any two time points was calculated as follows: (time difference in minutes between sequential reads) × (glucose level 1st time point + glucose level 2nd time point)/2) [16
2.6. Biochemical Analysis in Blood
Serum lipid concentrations were determined with commercially available kits. The total cholesterol (TC), triglyceride (TG), and high-density cholesterol (HDL) levels in serum were measured based on the manufacturer’s instruction (Asan Pharmaceutical Co., Seoul, Korea). The concentration of low-density cholesterol (LDL) was calculated as (total cholesterol−HDL choles tero–[triglyceride/5]) based on the formula of Friedewald et al. [17
]. Aspartate aminotransferase (AST) and alanine transferase (ALT), which are known to be a hepatic function marker, were measured based on the manufacturer’s instruction (Asan Pharmaceutical Co.). The units were expressed as IU/L of serum. All analyses were measured using a UV spectrometer (TECAN Group Ltd, Shanghai, China).
2.7. Assessments of Liver TG and TC
The method for measuring the contents of lipids in the liver was as described by Folch et al. [18
]. Briefly, the liver (20–40 mg) was homogenized in cold PBS. A homogenate of 0.2 mL was extracted with methanol/chloroform (1:2) and centrifuged at 2500× g
for 10 min. An aliquot of the organic phase was collected, dried under nitrogen, and resuspended in Triton X-100/ethanol mixture (1:1, v
). The liver TG and TC levels were determined using its quantification kits (Asan Pharm.). Data were normalized for differences in protein concentration in the liver extracts.
2.8. Western Blot Analysis
The liver tissue and cell lysates were homogenized in mammalian protein extraction buffer (Sigma Chemical Co., St. Louis, MO. USA) in the presence of protease inhibitor cocktail (Sigma) and phenyl methane sulfonyl fluoride (Sigma, PMSF). The lysates were centrifuged at 12,000 rpm at 4 °C for 20 min. The protein contents of the supernatants were determined using protein assay dye reagent concentrate (Bio-Rad Laboratories, Hercules, CA, USA) based on the manufacturer’s instructions. The same concentration of protein was separated electrophoretically by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose blotting membranes (Amersharm, GE Healthcare Life Science, Germany). The antibodies used for western blotting included anti-SREBP-1 (1:1000; Abcam, Cambridge, UK), anti-ACC (1:1000; Cell Signaling Technology), anti-FAS (1:1000; Cell Signaling Technology), anti-AMPKα (1:1000; Cell Signaling Technology), anti-phospho-AMPKα (Thr172) (1:1000; Cell Signaling Technology), and anti-β-actin (1:2500; Abcam). The protein bands were visualized following an enhanced chemiluminescence method using an ELC kit (Millipore, USA). The bands were quantified using Quantity 1 version 4.6.7 software (Bio-Rad Laboratories).
2.9. Quantitative Real-Time Polymerase Chain Reaction
Total RNA was extracted from the mouse liver using Trizol reagent (Invitrogen, Grand Island, NY, USA) and synthesized with PrimescriptTM
1st strand cDNA synthesis kit (Takara Bio Inc., Shiga, Japan) to prepare cDNA. Real-time polymerase chain reaction (PCR) was performed on the ABI real-time PCR system (Applied Biosystem Inc., Forster City, CA, USA) using SYBR Premix Ex Taq II, ROX plus (Takara Bio Inc., Shiga, Japan) based on the manufacturer’s instructions. Amplification was performed as follows: 10 min at 90 °C, 15 s at 95 °C, and 1 min at 60 °C for 40 cycles. Table 1
shows the gene-specific primers. Cyclophilin was used as a reference gene, and all results were normalized to the abundance of cyclophilin mRNA. The relative amounts of mRNAs were calculated using the 2ΔΔCt
2.10. Biochemical Staining
For Oil red O staining assay, parts of the liver were immediately fixed upon dissection in 4% buffered formaldehyde solution (pH = 7.4) for 24 h. Cryosections (10 μm) were incubated with Oil red O solution (Cayman Chemical, Ann Arbor, MI, USA) for 10 min. For Masson’s trichrome staining assay, paraffin embedded liver sections (4 μm), were fixed in acetone and deparaffinized, and stained for Masson’s trichrome (MT). The sections were detected using an Olympus DP70 digital camera (Olympus Co., Tokyo, Japan) by Olympus BX61 microscope (Olympus Co., Tokyo, Japan).
2.11. Statistical Analysis
The results were presented as the mean and standard deviations (SDs). All statistical analyses were performed using SPSS 20.0 software (IBM SPSS V20.0.0 for Windows, IBM Co., Armonk, NY, USA). Significant differences among the groups were analyzed using the LSD comparisons test. Statistical significance was set up at p < 0.05.
The liver is a vital organ that modulates various metabolic processes, including lipid metabolism and glucose homeostasis. Excessive intake of dietary fats leads to lipid accumulation in the liver and can cause type 2 diabetes, chronic diseases, and obesity. NAFLD is reportedly associated with insulin resistance during the development of type 2 diabetes [24
], and these metabolic diseases must be controlled for a heathy life. Although several drugs used to treat NAFLD are currently available, satisfactory outcomes have not been achieved. Natural products have been considered as alternative treatments to prevent NAFLD via various mechanisms, such as antioxidant, anti-inflammation, and antidyslipidemic effects [26
]. Recently, insects have gained attention as a source of an effective bioactive product in many countries, but scientific evidence regarding its safety, biological effects, and molecular mechanisms to be applied as a therapeutic agent is lacking.
Previous studies have shown that ADLE has antihepatofibrotic, antineoplastic, antibiotic, and antioxidant effects, but the antihepatotoxicity effect during the development of type 2 diabetes was not well reported. In this study, we clearly demonstrated that ADLE improved hyperlipidemia and hepatic function in the liver of HFD-fed NAFLD mouse model.
Yoon et al. reported that the administration of ADL powder (3000 mg/kg/day) decreased HFD (35% carbohydrate and 45% fat)-induced body weight gain, and Kim et al. showed that intracerebroventricular cannulation of ADLE (1 μg) into 60% HFD-fed mice significantly reduced body weight by food intake regulation [15
]. In our study, we did not observe food intake, FER, and body weight alteration, but treatment of HFD-fed mice with ADLE ameliorated insulin tolerance. The types of extract, treated concentration and/or duration, and mode of administration might have been responsible for the different effects of ADLE on body weight gain. However, these results suggested that the antidiabetic effect of ADLE was not due to decreased food intake or reduced body weight. Glucose tolerance of diabetic mice treated with ADLE was also significantly improved; the mechanism underlying insulin secretion or anti-apoptotic effects remains to be investigated.
Most of the circulating cholesterol in serum is found in three major lipoproteins such as very-low-density lipoproteins (VLDL), LDL, and HDL, as well, several studies have shown that increased serum TC contributes to the development of NAFLD [29
]. Cholesterol is mainly synthesized in the liver, but hepatic cholesterol can be removed by its conversion to bile acids or by enhanced hepatobiliary secretion, as an essential step in the reverse cholesterol transport process [30
]. In this study, we found that the serum TC levels were not changed by ADLE in contrast to the hepatic TC levels which were significantly decreased in the ADLE-treated HFD group. These results suggested that ADLE might have an effect on the enhanced clearance of cholesterol by increasing bile acid synthesis and excretion in liver. In this study, we did not check bile acid levels in HFD-fed mice and ADLE-treated HFD mice, further studies regarding ADLE and cholesterol catabolism to bile acids will be needed.
The serum lipid profile showed that TG, which is a hepatic insulin resistance marker [31
], was significantly reduced in ADLE-treated HFD mice as compared with that in HFD mice. The serum HDL level was increased as a result of HFD and decreased by the ADLE treatment. This phenomenon may be accompanied by an increase in the TC level during prolonged HFD, and these results were consistent with those of other studies [32
Elevated serum AST and ALT levels are commonly associated with hepatic steatosis and are used as a biomarker for hepatic injury [34
]. In this study, HFD-fed mice showed significantly increased serum AST and ALT levels, but ADLE reversed this effect. Moreover, ADLE decreased HFD-induced hepatic fibrosis and inflammation. Lipotoxicity is a result of an imbalance between lipid uptake and utilization. The abnormal metabolism of FFAs and their derivatives is the main cause of intracellular lipotoxic injury [35
] and the injured steatotic hepatocyte induces inflammation and fibrosis [36
]. In our results, the numbers of lipid vacuoles, TG and TC levels in the liver were reduced, whereas, insulin sensitivity was increased in hyperlipidemic mice fed with ADLE. These results suggested that ADLE has a preventive effect on the development and progression of NAFLD, and the protective effect regarding hepatic lipotoxicity was due to reducing fibrosis and inflammation.
We also found that the accumulation of intracellular TG levels significantly increased in FFA-treated HepG2 cells, and the ADLE treatment of HepG2 cells exhibited an inhibitory effect on FFA-induced hepatic steatosis. At a molecular level, ADLE significantly downregulated the expression of SREBP-1c, FAS, and ACC in the HepG2 cells. SREBP-1c is a transcription factor, which is known to regulate de novo lipogenesis genes, such as ACC, FAS, and stearoyl-CoA desaturase (SCD)-1 [37
]. We found that ADLE attenuated hepatic steatosis not only by altering the expression of genes related to lipogenesis but also by increasing the phosphorylation of AMPK. AMPK is known as a central regulator of multiple metabolic pathways, and its activation leads to the hepatic lipogenesis suppression and enhanced fatty acid oxidation [40
]. Liver specific AMPK activation protects against diet-induced obesity and NAFLD [42
]. Consistent with the in vitro results, the ADLE treatment downregulated the mRNA and protein level of lipogenesis genes and activated AMPK in the liver. Our results suggested that improvement in insulin sensitivity by ADLE is associated with the attenuation of fat storage in the liver via downregulation of lipogenesis genes and activation of AMPK.
We observed that antihyperlipidemia effects and AMPK activity by ADLE was similar to that by Met, which has been suggested as a potential drug for the treatment of NAFLD [43
]. Therefore, our results suggested that ADLE is a potential therapeutic drug for decreasing hyperlipidemia and improving insulin sensitivity in patients with NAFLD and with type 2 diabetes. With regard to the administration of ADLE as a treatment for NAFLD and metabolic disease, quality analysis and its validation of the proper components of ADLE by fractionation is in progress. We found that some fractions showed an antioxidant effect, and therefore reduction of oxidative stress can be one of the mechanisms of amelioration of hepatic insulin resistance.
It has been reported that fat content is higher in the larval stage than in the adult stage, but the protein (Chitin) content was not different during the growth state [44
]. Therefore, the bioactive component changes during the growth stage and is dependent on the nutritional source. Moreover, the nutritional environment including the type of food, health status, and parental size will have an affect, and therefore further studies regarding the amounts of bioactive component in the insects during development will be needed. Moreover, for application of the effect of ADLE on antihepatotoxicity and antihyperglycemia in the human subject, many studies such as toxicity, allergies, and production should be investigated.