Gene expression profiling and inhibition of adipose tissue accumulation of G. bimaculatus extract in rats on high fat diet

Molecular genetic mechanisms underlying the anti-inflammatory effects of ethanol extract (GB) from G. bimaculatus, a type of cricket, are not fully elucidated. G. bimaculatus was reported to be rich in unsaturated fatty acid and to decrease the omega-6/omega-3 fatty acid ratio when fed to chickens. GB may reduce the amount of fat or increase the unsaturated fatty acid ratio. Male Wistar rats fed a high-fat diet (HFD) were orally administered with 5 groups: phosphate buffered saline (PBS, control), GB (100 mg/kg or 200 mg/kg), Pravastatin or Isaria sinclairii (IS) extract, which is reported to have fat-reducing effects, for either 1 or 2 months. GB’s sero-biochemial, hematological and anti-oxidizing hepato-cellular biomarker levels were evaluated to dertermine their antilipidemic, anti-inflammatory, and anti-coagulant effect in rats after 1 or 2 month GB treatments on HFD (fat 60 %) Wistar rat. The abdominal and epididymidal fat weight were measured and the composition of fatty acid was analyzed by GC/MS. Microarray analyses were performed with a rat 28 K cDNA clone set array to identify the gene-expression profiles for the GB exposed high fat dieted Wistar rat. The weight and fatty acid composition of abdominal fat and epididymidal fat, total cholesterol, LDL-cholesterol, and triglyceride in GB treated rats were at lower levels than those of the control group. The anti-oxidant hepato-cellular biomarker levels, protein carbonyl content and malondialdehyde concentration in GB treated rats were significantly decreased. Compared to the control, the GB treated rat group (treated at a dose of 100 and 200 mg/kg), had 190 up-regulated genes including Gpm6a (glycoprotein m6a), Tmem14a (transmembrane protein 14A) and Fasin (fatty acid synthase), with down-regulated 235 genes including Cc121b (chemokine ligand 21b), Glycan1 (glycosylation dependent cell adhesion moleule, Serpinb1a (serine proteinase inhibitor) and Tcrb (T-cell receptor beta chain). The data suggest Fasin-related fatty acid synthesis and adipose differentiation related protein (Adfp), which is related to obesity, were upregulated by GB treatment, indicating their potential therapeutic markers for anti-atheriosclerosis or inflammation.


Background
Obesity is a metabolic disorder and the fundamental cause of other fatal diseases including atherosclerosis, hypertension, diabetes, premature aging and cancer [1]. A high fat diet causes diseases such as obesity and changes the DNA gene expression profile [2][3][4].
Cricket (Gryllus bimaculatus) water extract is used in Oriental medicine as a crude drug for treating fever and hypertension, and crickets are currently reared as food for pets [5]. The main components of G. bimaculatus are protein (52.81 %), ash (minerals) and fat (21.81 %), including rich essential unsaturated conjugated fatty acidssuch as palmitic acid (ω-7, 34.14 %), oleic acid (ω-9, 36.48 %), and linoleic acid (ω-6, 13.58 %) [6]. Water and methanol extracts from crickets were recently found to cause a significant decrease in blood ethanol concentrations by enhancing liver mitochondrial alcohol metabolizing enzymes [7]. The extracts also had protective effects against acute hepatic damage [8].
The fact that G. bimaculatus is abundant in unsaturated fatty acid and decrease of omega-6/omega-3 fatty acid ratio when fed to chickens suggest that G. bimaculatus may reduce fat or increase unsaturated fatty acid ratio in tissues [6]. The antioxidant effect of GB reported in previous studies may relieve the obese state or obese-related disorders. Recent studies report anti-obesity and anti-diabetic effects are Isaria sinclairii (Cicada Dongchunghacho, a fungus cultured on silkworm) [9][10][11]. Pravastatin (a type of statins), lipidlowering drug, especially hydroxymethylglutaryl-CoA reductase inhibitor, is widely used in the treatment and prevention of atherosclerotic diseases [12]. Therefore, we assessed the effects of GB compared its antilipidemic activity with I. sinclarii ethanol extract or Pravastain as positive controls. In this study, the fatty acid composition in abdominal fat tissue and epididymidal tissue of Wistar rats treated with GB was evaluated and compared to Isaria sinclaii [10] extract (IS100) and pravastatin (STA). A high fat diet (HFD) also can cause oxidative stress and was due to lipid peroxidation (malon dialdehyde increase), protein carbonyl content increase, and DNA damage.
We report the sero-biochemical and DNA micro array study of GB in HFD Wistar rats with regard to preventing oxidative stress to proteins, lipids and DNA. This GB holds great promise for use as an anti-obesity drug to decrease fat accumulation in people on high fat diets and prevent changes in liver fat. We demonstrate the potential efficacy of GB in the treatment of anti-lipidemic effect on a HFD rats to be a protective nutraceutical for atherosclerosis disorders, including circulatory disorders, showing gene expression profile with valuable prognostic marker to identify potential therapeutic targets for atherosclerosis and obesity.

Clinical sign and food consumption
No deaths or adverse clinical signs were apparent due to the ingestion of the G. bimaculatus extract or pravastatin. The level of food consumption was similar in all treated groups during the course of the study (Fig. 1). Mean daily food intake was 26.6 g/kg bw/day.

Body weight and adipose fat weight changes
There were no toxicologically significant differences in mean body weight between any of the treatment groups (Fig. 2a). During the 1-month administration period, the body weights of the male Wistar rats in the 2 treatment groups were comparable in the control and trexperimental groups. The mean weekly body weights over time are presented in Fig. 2a. However, about 2 months after the experiment, body weight deviation between the groups increased, especially with the IS100 group. The body weight of the IS100 group continued to decrease for some time. The body weight of the IS100 group was significantly different from that of the control group (p < 0.05). At sacrifice, the abdominal fat tissues and the epididymidal fat tissues were dissected and weighed to investigate adipose tissue changes. The abdominal fat weight of HFD rats was significantly reduced by G. bimaculatus extract (200 mg/kg) (GB200) after 2 months of treatment: Con, 19.16 ± 3.04 g; GB100, 16.37 ± 1.46 (85.4 %); GB 200, 13.03 ± 1.37 g (68.0 %, GB200 vs Con, p < 0.05). Figure 2b also shows the declineof abdominal and epididymidal fat weight in HFD rats treated with GB for 1 month. The relative weight of epididymidal fat tissuedecreased to 75.0 % in GB200 after1 month of treatment. The total fat in GB100 and GB200 decreased by 77.9 and 73 % comparedto the CON (Fig. 2b). The fat-reducing effect of GB extract was better than that of pravastatin (STA) in rats.

Serum biochemical analysis
The changes in serum lipid and lipoprotein profiles after the 1-mo. feeding period are show in Table 1.
The changes in serum lipid and lipoprotein profiles in HFD rats after GB 2 months of treatment are show in Table 2.
The obtained data clearly show that high-fat diet consumption increased the concentration of serum total cholesterol, TG, and LDL-cholesterol in addition to decreasing the HDL-cholesterol concentrations in the experimental rats after 2 months of treatment. Total cholesterol, LDL-cholesterol, TG, and total lipid in the GB-treated group were lower than in the control group. HDL-cholesterol in the GB-treated group was higher than in the control group.
Serum glucose levels were lower in the GB-treated group than in the control (control, 187.67 ± 2.19 mg/dL; GB100, 163.00 ± 18.00 mg/dL; GB200, 192.75 ± 67.45 mg/ dL; STA, 252.00 ± 29.43 mg/dL (STA vs CON, p < 0.05); IS100, 198.75 ± 30.38 mg/dL). In the sera of the GB treated groups, total cholesterol, LDL-cholesterol and triglyceride levels were lower, while HDL-cholesterol was higher than in the control after 2 months. Dosedependent changes were observed in rats on high-fat diets although there were no significant differences compared with the control group (Fig. 3).

Hematology and blood chemical analysis
Some dose-dependent changes were observed between the treated and control groups with respect to the hematological parameters at the end of the experiment. An increase in partial thromboplastin time and thrombin time was observed in the treated groups on high fat diets but without significant differences. Minor changes were found in hematological parameters (hematocrit, MCV, MCHC and factor I) for some GB-treated rat groups. However, the effects of GB were not considered adverse because all changes in hematological data including neutrophil, lymphocyte, monocyte and eosinophil were within the normal physiological range. The platelet count of the IS100 group was increased compared to the CON group (p < 0.05) ( Table 3).

Oxidative protein damage (carbonyl content and catalase) quantitation
Protein oxidative stress was evaluated by measuring protein carbonyl content in the blood (Fig. 4). Catalase activity and carbonyl content were assayed as the biomarkers of protein oxidative damage in the high fat dieted rat model. After 2 months, carbonyl content level was decreased by GB treatment: CON, 12.6 ± 1.1 nmol/mg protein; GB100, 10.4 ± 2.7 nmol/mg protein; GB200 (GB200 vs CON, P < 0.05), 6.8 ± 2.8 nmol/mg protein; STA, 9.8 ± 4.0 nmol/mg protein; IS100, 5.1 ± 1.6 nmol/mg protein (IS100 vs CON, P < 0.05). The protein carbonyl content in the blood was reduced in a dose-dependent manner after extract treatments for 2 months. The protein carbonyl concentration was decreased by 82 and 53 % in GB100 and GB200, respectively (Fig. 4).

DNA microarray
Microarray analysis using a Mouse 28 K cDNA clone set array was performed to identify the geneexpression profiles in the GB treated Wistar rat livers and provided information on potential markers for atherosclerosis. Compared to the control group, the GB treated rats showed 419 (200 mg/kg) and 430 (100 mg/kg) up-regulated genes (15 % increase), and the signal ratio increased from 1.15-fold to 3.28-fold. Gpm6a (glycoprotein m6a), Tmem14a (transmembrane protein 14A) and Fasin (fatty acid synthase) were up-regulated and 421 genes (ratio 0.47~0.85) including Cc121b (chemokine ligand 21b), Glycan1 (glycosylation dependent cell adhesion moleule, Ser-pinb1a (serine proteinase inhibitor) and Tcrb (T-cell receptor beta chain) were down-regulated. The data suggests that Fasin related fatty acid synthesis and Adfp, an adipose differentiation related protein associated with obesity, were upregulated by GB treatment. However, a series of genes involved in signal transduction, fatty acid synthesis, energy metabolism (oxidative metabolism) and cellular defenses were more up-regulated, indicating their    Fig. 4 Anti-oxidative effect of GB on lipids (MDA or IL-6) and proteins (carbonyl or catalase content) after 2-month treatment. Each value represents mean ± S.D. statistically significant from control (*P < 0.05) potential as therapeutic markers for lipid metabolism (Tables 4 and 5).

Fatty acid composition in adipose tissue
The fatty acid profile, as indicated GC-MS, showed a slight dose-dependent increase in arachidonic acid (C20: 4n6, AFA) concentration in the epididymidal of male Wistar rats in the GB-treated groups over a 2-month period compared to the control group ( Table 6). The high fat dieted Wistar rats treated with GB showed increases in the unsaturated fatty acids (FA) ratio, especially single (mono) FA, but had decreases in saturated fatty acid (Tables 6 and 7). In abdominal GB100 or GB200 fat tissue of 1-month treatment period, mono (single) unsaturated fatty acid composition was decreased whereas poly unsaturated fatty acid composition was increased with dose-dependent manner (each group vs CON, p < 0.05). In epididymidal fat tissue of GB100 or GB200 HFD rats of 1-month treatment period, the composition of docosahexaenoic acid in epdidymidal fat tissue showed dose-dependent decrease with significant difference (Gb200 vs CON, p < 0.05). In 1month treatment groups, eicosapentaenoic acid and docosahexaenoic acid composition were increased with statistical significance (GB100 vs CON, p < 0.05; STA vs CON, p < 0.05).
In abdominal fat of 2-month treated HFD rats, poly unsaturated fatty acid were increased, and monounsaturated fatty acid were dose-dependently decreased (each group vs CON, p < 0.05). Stearic acid composition in IS100 group was higher compared with control group (IS100 vs CON, p < 0.05).
In epididymidal fat of 2-month treated rats, arachidonic acid and docosahexaenoic acid composition in GB100 (GB100 vs CON, p < 0.05), and eicosanoic acid composition in IS100 showed significant differences compared with control (IS100 vs CON, p < 0.05).

Discussion
A high-fat diet (HFD) induces obesity and obesity related metabolic complications such as adipose inflammation, hepatic steatosis and hyperlipidemia [13]. Among various animal sources tested [14], cricket extract was found to be a potent functional food; the adipose tissue fat weight of Wistar rats treated with GB over a 2month period was decreased, especially the abdominal fat and epididymidal fat. However, body weight was not significantly different in the GB treated and control groups.
Modulating inflammation in adipose tissue ameliorates obesity-associated metabolic complications [15]. GB extract slightly lowered blood pressure and creatinine phosphokinase or otherwise increased thrombin time, showing anticoagulant activity. Repair of protein oxidative damage caused by a high fat diet, carbonyl content and lipid oxidative damage (malondialdehyde, MDA) were decreased by GB treatment [16]. Hepatic mRNA expression of IL-10 was reported to be increased in obese C57BL6/J mice on high-fat diets [17] and overexpression of IL-10 prevented weight gain in animals on HFD [18].
A high-fat diet decreases energy expenditure and the expression of genes controlling lipid metabolism, mitochondrial function and skeletal system development in the adipose tissue, along with increased expression of extracellular matrix remodeling-and inflammation-related genes [19].
In accordance with the decreased weight of abdominal fat tissue, total cholesterol, LDL-cholesterol, and triglyceride in GB treated rats were lower than those of the control (PBS treated) group. In the sera of rats treated with GB for 1-month, free fatty acid levels were reduced in a dose-dependent manner. In addition, glucose, triglyceride and alkine phosphatase levels were also decreased in the 100 mg/kg GB extract-treated group. The composition of saturated fatty acid was decreased, while unsaturated fatty acid and poly unsaturated fatty acid were increased in the epididymal adipose tissue, suggesting that a diet rich in poly-unsaturated fatty acid decreases adipose tissue mass and suppresses the development of obesity in rats [3,25].

Conclusions
GB demonstrated anti-lipidemic effects in Wistar high fat dieted rats by significantly reducing serum triglyceride and alkaline phosphatase levels. Consequently, GB may be a protective nutraceutical for atherosclerosis disorders, including circulatory disorders. The gene expression profile of high fat dieted Wistar rats treated with GB is a valuable prognostic marker that can be used to identify potential therapeutic targets for atherosclerosis.

Materials
G. bimaculatus was reared in a cricket farm located in Jungsun, Kangwon-Do, South Korea. The cricket was freeze-dried at the Department of Agricultural Biology, National Academy of Agricultural Science, Korea.

Preparation of G. bimaculatus extract (GB)
Dried G. bimaculatus (1 kg) was homogenized and soaked then extracted three times with 70 % ethanol by ultrasonification for 30 min. The samples were filtered through Whatman filter paper and concentrated by evaporation and freeze-drying. The dried powder (ethanol extract, GB) was dissolved in saline prior to use in the test solution. Isaria sinclairii was also extracted with 70 % ethanol through sonication, evaporation and freeze-drying using the same method as for GB.

Animals
Han Tac Sam-WH (Wistar) rats (male), weighing 308.0 ± 11.0 g at 14-weeks of age, were obtained from Samtako Co. Ltd. (Osan, Korea) and divided into five groups of ten rats including the Wistar control group. All procedures were in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals. All experiments were approved by the Laboratory Animals' Ethical Committee of the National Academy of Agricultural Science, RDA and followed national guidelines for the care and use of animals (individual housing). The rats were acclimated for four weeks under normal husbandry conditions (23 ± 2°C, 55 ± 10 %, humidity and 12 h light/dark cycle) and fed a high (60 %) fat diet, D12492 (Research Diet Inc., USA) and water ad libitum. The rats were distributed into the following 5 groups (n = 10) with similar weights: I: control group, PBS treatment, II: treated with 100 mg/ kg G. bimaculatus ethanol extract, III: treated with 200 mg/kg G. bimaculatus ethanol extract, IV: treated with 2 mg/kg Pravastatin (CJ Heathcare CO., Korea), V: 100 mg/kg Isaria sinclairii ethanol extract. Each group (5 sample groups) was maintained for a one month or two month-period (Scheme 1).

Liver homogenate preparation for oxidative damage detection
About five groups named CON, GB100, GB200, STA, IS100, divided 1 month-or 2 month-period subgroup (n = 5), liver tissues were homogenized on ice in a 10fold volume lysis buffer PRO-PREP™ Protein extraction solution (iNtRON, Busan, Korea). The supernatant of the liver homogenate after centrifugation (800 g, 10 min) was assayed for carbonyl content or catalase activity.

Scheme 1 Animal experimental design
Oxidative protein damage (carbonyl content and catalase) quantitation On the supernatant of liver homogenate and blood of five groups named CON, GB100, GB200, STA, IS100, divided 1 month-or 2 month-period subgroup (n = 3), the carbonyl content was determined with an enzymelinked immunoassay according to the manufacturer's protocol for the OxiSelect™ protein carbonyl ELISA kit, Cell Biolabs, Inc. (Sandiego, USA). Catalase activity (CAT, U/mg protein) was measured according to the method based on CAT-mediated decomposition of H 2 O 2 . Potassium phosphate buffer (50 mM, pH 7.0, 0.9 ml) was added to 0.1 ml of the sample followed by H 2 O 2 solution (1 ml, 30 mM). The decrease in the absorbance at 240 nm was measured for 90 s [26].

DNA microarray procedure
After histopathology analysis, microarray hybridization was performed on liver samples (Control, GB 100 and GB200). The total RNA was isolated from the liver using a Qiagen RNeasy Midi Kit (Qiagen, Valencia, USA). A regular microarray was carried out according to the manufacturer's instructions for the FairPlay™ microarray labeling kit (Stratagene, La Jolla, CA). Briefly, 20 μg total RNA from the liver was reverse-transcribed into single stranded cDNA. The cDNA was purified with ethanol precipitation and resuspended in 5 μl of 2x coupling buffer, then coupled with 5 μl of Cy3 or Cy5 dye for 1 h in the dark. The labeled control liver cDNA and the treated liver cDNA were combined and purified. The labeled cDNA was mixed with 1.5 μl of 10 μg/μl salmon DNA, 1.5 μl of 8 μg/μl poly d(A), 1.5 μl of 4 μg/μl yeast tRNA, 4.5 μl of 20x SSC and 0.75 μl of 10 % SDS, heated at 99°C for 2 min, and incubated at 45°C for 15 min. The labeled DNA was loaded onto a microarray chip. A hybridization chamber was assembled with the microarray chip and submerged in a water bath overnight at 60°C. The microarray chip was washed in wash buffer I (2x SSC, 0.1 % SDS) for 15 min, then in wash buffer II (1x SSC) for 5 min and in wash buffer III (0.2x SSC) for 15 min. The slide was dried by centrifuging at 500 g for 15 min and scanned with a BMS Array Scanner, applied precision Array WoRx eBiochip Reader (BioRad, Dallas, USA) using the Cy3 and Cy5 channels [28].

Analysis of fatty acid composition in rat adipose tissue
On five groups named CON, GB100, GB200, STA, IS100, divided 1 month-or 2 month-period subgroup (n = 4), for epididymidal and abdominal fat analysis, the concentrations of free fatty acids and fatty acid composition were analyzed for 29 fatty acids in adipose tissue using gas chromatography-mass spectroscopy (GC-MS). Each cut adipose or epididymidal tissue (0.1 g) was collected and extracted overnight bwith a chloroform: methanol (2:1) mixture. The filtered solution was removed under nitrogen gas. The lipids were then saponified by alkaline hydrolysis of phospholipids at 100°C with 0.5 N methanolic sodium hydroxide and methylated at 100°C with 14 % BF 3 for 15 min. The top layer was transferred to petroleum ether and analyzed by GC/MS (Aglient 6890GC, Aglient 5973 N mass detector, EI mode) with a HP-5 capillary column (Aglient Technolgies, Palto alto, Ca, USA). The inlet temperature was 250°C and the MS transfer line was kept constant at 230°C. The oven temperature was held at 180°C for 20 min, then programmed at 10°C/min to 230°C and held for 10 min. Quantification was achieved using a mixed 37 fatty acid standard: Sigma L9405, 10 ρg/mL (Sigma-Aldrich Inc.). Linoleic acid (C18:2n6) was used as an internal standard.

Statistical analysis
The means and standard error of all parameters studied were determined for each group using the ANOVA test. A Student's t-test was carried out to determine significant differences between control and treated groups. A p value <0.05 was considered significant.