Ganoderma lucidum Extract Reduces Insulin Resistance by Enhancing AMPK Activation in High-Fat Diet-Induced Obese Mice

Ganoderma lucidum is used widely in oriental medicine to treat obesity and metabolic diseases. Bioactive substances extracted from G. lucidum have been shown to ameliorate dyslipidemia, insulin resistance, and type 2 diabetes in mice via multiple 5′ AMP-activated protein kinase (AMPK)-mediated mechanisms; however, further studies are required to elucidate the anti-obesity effects of G. lucidum in vivo. In this study, we demonstrated that 3% G. lucidum extract powder (GEP) can be used to prevent obesity and insulin resistance in a mouse model. C57BL/6 mice were provided with a normal diet (ND) or a high-fat diet (HFD) supplemented with 1, 3, or 5% GEP for 12 weeks and the effect of GEP on body weight, liver, adipose tissue, adipokines, insulin and glucose tolerance (ITT and GTT), glucose uptake, glucose-metabolism related proteins, and lipogenesis related genes was examined. GEP administration was found to reduce weight gain in the liver and fat tissues of the mice. In addition, serum parameters were significantly lower in the 3% and 5% GEP mice groups than in those fed a HFD alone, whereas adiponectin levels were significantly higher. We also observed that GEP improved glucose metabolism, reduced lipid accumulation in the liver, and reduced adipocyte size. These effects may have been mediated by enhanced AMPK activation, which attenuated the transcription and translation of lipogenic genes such as fatty acid synthase (FAS), stearoyl-CoA desaturase 1 (SCD1), and sterol regulatory element-binding protein-1c (SREBP1c). Moreover, AMP-activated protein kinase (AMPK) activation increased acetyl-CoA carboxylase (ACC), insulin receptor (IR), IR substrate 1 (IRS1), and Akt protein expression and activation, as well as glucose transporter type 1/4 (GLUT1/4) protein production, thereby improving insulin sensitivity and glucose metabolism. Together, these findings demonstrate that G. lucidum may effectively prevent obesity and suppress obesity-induced insulin resistance via AMPK activation.


Introduction
Obesity is a metabolic disease characterized by excess lipid deposition [1,2] that can increase adipose tissue mass by increasing the number (hyperplasia) or size (hypertrophy) of adipocytes.
anti-obesity effects [18]. G. lucidum has been used for thousands of years to improve health and longevity, and to treat diseases such as hypertension, cancer, and diabetes mellitus [17,19,20], and has been suggested as a potential agent to prevent obesity and metabolic diseases, such as type 2 diabetes, via the regulation of AMPK [9,20,21]. In addition, numerous studies have suggested that G. lucidum exhibits therapeutic properties, including antidiabetic, immunomodulatory, hepatoprotective, antihyperlipidemic, and antihyperglycemic activities [17,20,[22][23][24][25][26]. A dietary supplement containing G. lucidum has been shown to inhibit adipogenesis by suppressing PPARγ, SREBP1c, and C/EBPα (CCAAT-enhancer-binding protein α), and activating AMPK to improve glucose uptake in vitro [9], while ganodermanondiol extracted from G. lucidum has been reported to enhance the activation of AMPK and its upstream kinases to inhibit fatty liver [27]. The polysaccharides present in G. lucidum have also demonstrated hypoglycemic effects mediated by AMPK activation, which enhances phosphoinositide 3-kinase (PI3K) and Akt gene expression and protein synthesis, thus increasing glucose uptake and reducing glycogen synthesis [20,28]. Furthermore, the use of herbal medicines and natural compounds has shown no apparent short nor long term side effects in the human body, making them attractive alternatives to pharmaceutical therapies [17]. Ganoderma lucidum extract powder (GEP) used in this study is in the form of powder which makes it practical to be produced in pill for clinical trial application.
In this study, we verified the effect of G. lucidum extract on obesity in a mouse model, finding that a 3% extract exerted anti-obesity effects in vivo by reducing hyperlipidemia, recovering functional adipokine levels, upregulating glucose metabolism-related protein expression and phosphorylation, and suppressing lipogenesis-related genes. Therefore, our data suggest that 3% G. lucidum extract can be used as a supplement to prevent obesity and associated metabolic diseases.

G. Lucidum Extract Powder
G. lucidum extract powder (ASI7071; GEP) was provided by the National Institute of Horticultural and Herbal Science (Eumseong, Chungcheongbuk-do, Korea). A G. lucidum cultivar was obtained from the National Horticultural Research Institute and cultivated on oakwood before being desiccated using dry heat. Dried samples (0.5 g) were extracted three times with 10 mL of 95% ethanol or 20× volume per weight for 24 h. The extract was filtered under reduced pressure and concentrated by rotary evaporation using a speed-vacuum (Hanil, module 4080C, Gimpo, Gyeonggi-do, Korea). The major bioactive constituents of GEP is stated in Table 1.

Animals
Six-week-old specific pathogen free (SPF) C57BL/6 mice were purchased from RaonBio (Yongin, Gyeonggi-do, Korea). During the experimental period, mice were kept in a SPF environment with a controlled atmosphere (23 ± 2 • C and 55 ± 5% relative humidity) and a 12 h light/dark cycle.

Tissue Weight Measurement
After the 12-week experimental period, the mice were fasted for 12 h and anesthetized with ether. Liver and white adipose tissues (WAT; epididymal fat) were excised, washed with phosphate buffered saline (PBS), dehydrated with filter paper, and weighed.

Histopathological Observation
Epididymal WAT, peritoneal WAT, subcutaneous WAT, and some liver tissues were extracted from the mice, fixed in 10% neutral buffered formalin for 24-48 h, and rinsed with distilled water. The tissues were then dehydrated in stages using 70%, 80%, 90%, 95%, and 100% ethanol and embedded in paraffin. The paraffin block was cut into 4-5 µm thick sections, prepared into slides, washed with xylene, and hydrophilized gradually with 100%, 95%, 90%, 80% and 70% ethanol. The slides were then stained with hematoxylin and eosin, covered with a cover glass, and observed using an optical microscope (Olympus, Tokyo, Japan).

Measurement of Blood Lipid Concentration
All blood analysis was performed after 12 weeks of experimental period (n = 6-7 per group). Blood was collected from the inferior vena cava using a sterile syringe before being allowed to stand in ice for 1 h and centrifuged at 3000 rpm (4 • C) for 10-15 min. The supernatant serum was collected and triglyceride (TG), total cholesterol (TC), high-density lipoprotein-cholesterol (HDL), low-density lipoprotein-cholesterol (LDL), and free fatty acid (FFA) levels were measured using an automated biochemical analyzer (Hitachi 7080; Hitachi Science System Ltd., Chiyoda-ku, Tokyo, Japan).

Measurement of Blood Glucose Concentration
After the 12-week experimental period and 12 h of fasting, blood samples were collected from the tail and glucose was measured using a blood glucose meter (Accu-Check glucometer, Roche, Seoul, Gyeonggi-do, Korea).

Glucose and Insulin Tolerance Tests (GTTs and ITTs)
A separate set of mice (n = 6-7 per group) was used for ITT and GTT measurements after 12 weeks of treatment. HFD-induced obese mice were fasted for 18 h for GTT or 6 h for ITT and intraperitoneally injected with D-glucose (2 g/kg body weight) or insulin (0.75 U/kg body weight in 0.1% bovine serum albumin) [29]. Blood samples were taken from the tail and blood glucose levels were measured using an Accu-Check glucometer (Roche) at the indicated times.

Measurement of Insulin, Leptin, and Adiponectin Levels
Blood insulin, leptin, and adiponectin concentrations were measured using an ELISA reader with corresponding mouse ELISA kits (CrystalChem, Inc., Elk Grove Village, IL, USA). Blood was collected from the inferior vena cava with a sterile syringe after the abdomen had been opened to analyze blood indices. The collected blood was allowed to stand for 1 h on ice and centrifuged at 3000 rpm (4 • C) for 10-15 min. The serum supernatant was diluted 10 times and a 50 µL aliquot was reacted at 4 • C for 12 h before incubation with HRP-anti-mouse insulin, leptin, and adiponectin antibodies at 4 • C for 4 h. Non-specifically bound antibodies were removed by washing with phosphate buffered saline-tween (PBS/T) twice. After treatment with substrate solution for 30 min, absorbance was measured at 450 and 630 nm using the Benchmark Plus microplate reader (BioRad, Hercules, CA, USA).

Measurement of 2-Deoxy-Glucose Uptake Following In Vivo Insulin Stimulation
After the 12-week experimental period, mice were fasted for 16 h, anesthetized with sodium pentobarbital (50 mg/kg; P3761, Sigma), and sacrificed. Epididymal WATs were harvested, preincubated in KBH buffer (8 mM glucose, 5 mM HEPES, 0.1% bovine serum albumin (fraction V), and 2 mM sodium acetate) at 30 • C for 20 min, and then incubated in KBH buffer containing 0.3 µCi [ 14 C] mannitol and 2 µCi/mL 2-[ 3 H]deoxy-glucose without 100 nM human insulin in a shaking water bath at 30 • C for 30 min. The tissues were blotted, rapidly freeze-clamped, and weighed before being dissolved in 0.5 mL of 1 M KOH for 20 min at 70 • C and neutralized with 0.5 mL of 1 M HCl. A 0.3 mL aliquot of each sample was mixed with 6 mL of Biosafe II (Research Products International, Mount Prospect, IL, USA) and radioactivity quantified using a liquid scintillation counter. The [ 14 C] mannitol concentration was measured to quantify extracellular 2-[ 3 H] deoxy-glucose and glucose uptake was calculated by subtracting extracellular 2-[ 3 H] deoxy-glucose from total 2-[ 3 H] deoxy-glucose.

qRT-PCR of Lipogenesis Genes in Adipose and Liver Tissues
Quantitative reverse transcription-PCR (qRT-PCR) was performed to identify changes in the expression of obesity-related genes in adipose and liver tissues. Total RNA was extracted from rapidly-frozen adipose and liver tissues using a RNeasy Lipid Tissue Mini Kit and a RNeasy Mini Kit (Qiagen, Germantown, MD, USA) according to the manufacturer's protocols. The extracted RNA was reverse transcribed using a SuperScript ® IV First-Strand Synthesis System (Invitrogen, Waltham, MA, USA) and the expression of lipogenesis-related genes was measured using real-time PCR with Fast SYBR Green Master Mix (Applied Biosystems, Waltham, MA, USA), 25 µL of real-time PCR reaction mix, 2 µL of cDNA, 12.5 µL of 2X SYBR mix, and 1 µL of forward and reverse primers (Table 3; 10 pmol/µL). PCR amplification was carried out for 40 cycles of pre-denaturation (95 • C) for 15 min, denaturation (95 • C) for 15 s, annealing (55 • C) for 30 s, and extension (72 • C) for 30 s. The expression of 18s ribosomal RNA was used as an internal control to correct relative gene expression. Melting curve analysis was used to confirm primer specificity. Results were analyzed using One-step system software v2.1 (Applied Biosystems).

Statistical Analysis
Data are expressed as the mean ± standard deviation and represent at least three independent experiments. Statistical significance was determined by one-way analysis of variance (ANOVA), followed by Tukey's multiple comparison test using GraphPad Prism software (GraphPad Software, San Diego, CA, USA).

Effect of G. Lucidum Extract on Obesity
To investigate the effects of G. lucidum on obesity, we measured the development of obesity in HFD-induced mice treated with GEP for 12 weeks. As expected, the HFD increased the size and final weight of the mice; however, GEP slightly reduced their size and significantly restored final weight ( Figure 1A). In particular, significant weight gain was observed in the HFD group that was suppressed in dose-dependent manner by GEP treatment ( Figure 1B). Neither the HFD nor GEP affected food intake, indicating that the effect of GEP on HFD-induced obesity may be attributed to metabolic alterations ( Figure 1C). In addition, the feeding efficiency ratio (FER) suggested that GEP treatment reduced the body mass gained per calorie eaten ( Figure 1D), whereas subcutaneous, epidydimal, and mesenteric WAT weight gain followed trends similar to those observed for body weight (Table 4).

Effect of G. Lucidum Extract on Obesity
To investigate the effects of G. lucidum on obesity, we measured the development of obesity in HFD-induced mice treated with GEP for 12 weeks. As expected, the HFD increased the size and final weight of the mice; however, GEP slightly reduced their size and significantly restored final weight ( Figure 1A). In particular, significant weight gain was observed in the HFD group that was suppressed in dose-dependent manner by GEP treatment ( Figure 1B). Neither the HFD nor GEP affected food intake, indicating that the effect of GEP on HFD-induced obesity may be attributed to metabolic alterations ( Figure 1C). In addition, the feeding efficiency ratio (FER) suggested that GEP treatment reduced the body mass gained per calorie eaten ( Figure 1D), whereas subcutaneous, epidydimal, and mesenteric WAT weight gain followed trends similar to those observed for body weight (Table 4).
We also found that 12 weeks of the HFD increased the size of the liver and epididymal WAT compared to the ND (Figure 2A,C) and significantly increased liver and total WAT weight; however, GEP treatment reduced weight in a dose-dependent manner ( Figure 2B,D). These results indicate that GEP can reduce weight gain, which is an obesity parameter. Data represent the mean ± SD of six to seven individual mice. Significant differences vs. the HFD group (control) were determined by one-way ANOVA; * p < 0.1, ** p < 0.01, *** p < 0.001. ND: normal chow diet group (control); HFD: high-fat diet group; HFD + GEP: high-fat diet and Ganoderma lucidum extract powder.
We also found that 12 weeks of the HFD increased the size of the liver and epididymal WAT compared to the ND (Figure 2A,C) and significantly increased liver and total WAT weight; however, GEP treatment reduced weight in a dose-dependent manner ( Figure 2B,D). These results indicate that GEP can reduce weight gain, which is an obesity parameter.

Effect of G. Lucidum Extract on the Liver and Adipose Tissue
Liver and epidydimal WAT were stained to determine the effect of GEP on each tissue. The HFD group displayed greater fat accumulation than the control group, while GEP supplementation, particularly at 3% and 5%, attenuated lipid accumulation in the liver and reduced the size of adipocytes in epidydimal WAT ( Figure 3A,B). Thus, GEP may reduce intracellular fat accumulation in both adipocytes and hepatocytes, which positively correlates with reduced obesity.

Effect of G. Lucidum Extract on the Liver and Adipose Tissue
Liver and epidydimal WAT were stained to determine the effect of GEP on each tissue. The HFD group displayed greater fat accumulation than the control group, while GEP supplementation, particularly at 3% and 5%, attenuated lipid accumulation in the liver and reduced the size of adipocytes in epidydimal WAT ( Figure 3A,B). Thus, GEP may reduce intracellular fat accumulation in both adipocytes and hepatocytes, which positively correlates with reduced obesity.

Effect of G. Lucidum on Serum Lipid Levels
Serum samples were bioanalyzed to determine the effect of GEP on serum TG, TC, HDL, LDL, and FFA levels. Compared to the ND, the HFD significantly increased all serum obesity parameters analyzed, whereas GEP treatment lowered TG, TC, HDL, LDL, and FFA levels ( Table 5). In the ND group, serum analysis detected TG levels of 73.78 ± 1.5 mg/dL, TC levels of 110.71 ± 2.79 mg/dL, HDL levels of 97.29 ± 3.83 mg/dL, LDL levels of 35.92 ± 2.96 mg/dL, and FFA levels of 0.98 ± 0.04 mEq/dL ( Table 5). The HFD increased TG levels to 123.04 ± 3.12, whereas treatment with 1, 3, or 5% GEP reduced TG levels to 118.91 ± 3.04, 104.09 ± 3.66, and 88.83 ± 1.83 mg/dL, respectively. Overall, 1% GEP slightly reduced serum lipid levels while 3% and 5% GEP reduced the levels significantly. A similar pattern was observed for TC, HDL, LDL, and FFA levels, suggesting that GEP alleviates increased cholesterol and lipid concentrations in the blood.

Effect of G. Lucidum Extract on Glucose Uptake, Insulin Tolerance, and Glucose Tolerance
Next, we examined the effects of the HFD and GEP on glucose and insulin. After 12 weeks of treatment, the HFD increased serum glucose and insulin levels compared to the ND, while treatment with 1, 3, and 5% GEP for 12 weeks significantly reduced glucose and insulin levels in a dosedependent manner, with a more pronounced effect on insulin levels ( Figure 4A,B).

Effect of G. Lucidum on Serum Lipid Levels
Serum samples were bioanalyzed to determine the effect of GEP on serum TG, TC, HDL, LDL, and FFA levels. Compared to the ND, the HFD significantly increased all serum obesity parameters analyzed, whereas GEP treatment lowered TG, TC, HDL, LDL, and FFA levels ( Table 5). In the ND group, serum analysis detected TG levels of 73.78 ± 1.5 mg/dL, TC levels of 110.71 ± 2.79 mg/dL, HDL levels of 97.29 ± 3.83 mg/dL, LDL levels of 35.92 ± 2.96 mg/dL, and FFA levels of 0.98 ± 0.04 mEq/dL ( Table 5). The HFD increased TG levels to 123.04 ± 3.12, whereas treatment with 1, 3, or 5% GEP reduced TG levels to 118.91 ± 3.04, 104.09 ± 3.66, and 88.83 ± 1.83 mg/dL, respectively. Overall, 1% GEP slightly reduced serum lipid levels while 3% and 5% GEP reduced the levels significantly. A similar pattern was observed for TC, HDL, LDL, and FFA levels, suggesting that GEP alleviates increased cholesterol and lipid concentrations in the blood.

Effect of G. Lucidum Extract on Glucose Uptake, Insulin Tolerance, and Glucose Tolerance
Next, we examined the effects of the HFD and GEP on glucose and insulin. After 12 weeks of treatment, the HFD increased serum glucose and insulin levels compared to the ND, while treatment with 1, 3, and 5% GEP for 12 weeks significantly reduced glucose and insulin levels in a dose-dependent manner, with a more pronounced effect on insulin levels ( Figure 4A,B).

Effect of G. Lucidum on Adiponectin and Leptin Levels
We also performed GTTs and ITTs to measure the ability of mice to retain circulatory glucose levels over time after glucose and insulin administration ( Figure 4C,D) [32]. In addition, we produced Significant differences compared to the HFD group (control) were determined by one-way ANOVA; * p < 0.1, ** p < 0.01, *** p < 0.001. ND: normal chow diet group; HFD: high-fat diet group; HFD + GEP: high-fat diet and Ganoderma lucidum extract powder.

Effect of G. Lucidum on Adiponectin and Leptin Levels
We also performed GTTs and ITTs to measure the ability of mice to retain circulatory glucose levels over time after glucose and insulin administration ( Figure 4C,D) [32]. In addition, we produced glycemia profiles from the GTT and ITT results using area above the curve (AAC) and area under the curve (AUC) analyses. We found that glucose tolerance, insulin sensitivity, as well as AAC and AUC glycemia profiles were impaired in HFD-induced obese mice compared to the ND group; however, treatment with 3% and 5% GEP significantly improved glucose tolerance and insulin sensitivity in a dose-dependent manner. Similar trends were observed for the GTT AAC and ITT AUC, with the HFD significantly increasing the AAC and reducing the AUC, but GEP treatment gradually restoring these profiles to normal levels as the GEP concentration increased. This suggests that GEP improves insulin regulation and glucose metabolism.
Next, we examined the effect of GEP on leptin and adiponectin in HFD-induced obese mice, observing that their production correlated positively and negatively with adiposity, respectively. GEP significantly increased and decreased leptin and adiponectin levels in a dose-dependent manner, respectively ( Figure 5A,B), indicating that GEP may ameliorate adipokine regulation in obese mice.
Nutrients 2020, 12, x FOR PEER REVIEW 11 of 21 glycemia profiles from the GTT and ITT results using area above the curve (AAC) and area under the curve (AUC) analyses. We found that glucose tolerance, insulin sensitivity, as well as AAC and AUC glycemia profiles were impaired in HFD-induced obese mice compared to the ND group; however, treatment with 3% and 5% GEP significantly improved glucose tolerance and insulin sensitivity in a dose-dependent manner. Similar trends were observed for the GTT AAC and ITT AUC, with the HFD significantly increasing the AAC and reducing the AUC, but GEP treatment gradually restoring these profiles to normal levels as the GEP concentration increased. This suggests that GEP improves insulin regulation and glucose metabolism. Next, we examined the effect of GEP on leptin and adiponectin in HFD-induced obese mice, observing that their production correlated positively and negatively with adiposity, respectively. GEP significantly increased and decreased leptin and adiponectin levels in a dose-dependent manner, respectively ( Figure 5A,B), indicating that GEP may ameliorate adipokine regulation in obese mice.

Effect of G. Lucidum on Glucose Uptake and Glucose Metabolism-Related Proteins
We also investigated GEP effect on glucose metabolism by assessing glucose uptake in the presence and absence of human insulin in WAT from 1%, 3% and 5% GEP treated HFD-induced obese mice, and the expression and activation of proteins involved in insulin signaling. In HFDinduced obese mice administered with insulin, GEP treatment dose-dependently improved glucose uptake ( Figure 6A) and insulin receptor (IR), IR substrate 1 (IRS1), and AKT serine/threonine kinase 1 (AKT1) phosphorylation, thereby increasing glucose transporter type 4 and 1 (GLUT4 and GLUT1) levels as well as AKT1 activation at phosphorylation sites T308 and S473 ( Figure 6B,C). These findings indicate that GEP supplementation may improve glucose metabolism. Data represent the mean ± SD of six to seven individual mice. Significant differences vs. the HFD group (control) were determined by one-way ANOVA; * p < 0.1, ** p < 0.01, *** p < 0.001. ND: normal chow diet group; HFD: high-fat diet group; HFD + GEP: high-fat diet and Ganoderma lucidum extract powder.

Effect of G. Lucidum on Glucose Uptake and Glucose Metabolism-Related Proteins
We also investigated GEP effect on glucose metabolism by assessing glucose uptake in the presence and absence of human insulin in WAT from 1%, 3% and 5% GEP treated HFD-induced obese mice, and the expression and activation of proteins involved in insulin signaling. In HFD-induced obese mice administered with insulin, GEP treatment dose-dependently improved glucose uptake ( Figure 6A) and insulin receptor (IR), IR substrate 1 (IRS1), and AKT serine/threonine kinase 1 (AKT1) phosphorylation, thereby increasing glucose transporter type 4 and 1 (GLUT4 and GLUT1) levels as well as AKT1 activation at phosphorylation sites T308 and S473 ( Figure 6B,C). These findings indicate that GEP supplementation may improve glucose metabolism. The phosphorylation ratio of IR, IRS, and AKT1, and the expression of Glut4 and Glut1 compared to β-actin, presented in folds over ND. Data represent the mean ± SD of six to seven individual mice. Significant differences vs. the HFD group (control) were determined by one-way ANOVA; * p < 0.1, ** p < 0.01, *** p < 0.001. HFD: high-fat diet group; GEP: Ganoderma lucidum extract powder. β-actin was used as a relative density control. The phosphorylation ratio of IR, IRS, and AKT1, and the expression of Glut4 and Glut1 compared to β-actin, presented in folds over ND. Data represent the mean ± SD of six to seven individual mice. Significant differences vs. the HFD group (control) were determined by one-way ANOVA; * p < 0.1, ** p < 0.01, *** p < 0.001. HFD: high-fat diet group; GEP: Ganoderma lucidum extract powder. β-actin was used as a relative density control.

Effect of G. Lucidum Extract on Lipogenesis-Related Genes and Proteins in WAT and the Liver
To confirm the effect of GEP on lipogenesis at the molecular level, we conducted genetic and protein analyses on WAT and the liver and analyzed fatty acid synthase (FAS), stearoyl-CoA desaturase 1 (SCD1), and sterol regulatory element-binding protein-1c (SREBP1c) expression using qRT-PCR. We found that the HFD increased the expression of lipogenesis-related genes (FAS, SCD1, SREBP1c) in both tissues compared to the ND, while 3% and 5% GEP attenuated the expression of all genes tested in a dose-dependent manner ( Figure 7A,B). Together, these results confirm that GEP downregulates the mRNA expression of lipogenesis-related genes, thus inhibiting fat formation.

Effect of G. Lucidum Extract on Lipogenesis-Related Genes and Proteins in WAT and the Liver
To confirm the effect of GEP on lipogenesis at the molecular level, we conducted genetic and protein analyses on WAT and the liver and analyzed fatty acid synthase (FAS), stearoyl-CoA desaturase 1 (SCD1), and sterol regulatory element-binding protein-1c (SREBP1c) expression using qRT-PCR. We found that the HFD increased the expression of lipogenesis-related genes (FAS, SCD1, SREBP1c) in both tissues compared to the ND, while 3% and 5% GEP attenuated the expression of all genes tested in a dose-dependent manner ( Figure 7A,B). Together, these results confirm that GEP downregulates the mRNA expression of lipogenesis-related genes, thus inhibiting fat formation. To investigate the effect of GEP on lipogenic protein expression, we performed western blotting using β-actin as the control ( Figure 8A). Although HFD increased the ratio of lipogenesis-related proteins (FAS, SCD 1, SREBP1c) compared to β-actin levels, 3% and 5% GEP decreased this ratio ( Figure 8B-D), suggesting that GEP inhibits lipogenesis and thus obesity. To investigate the effect of GEP on lipogenic protein expression, we performed western blotting using β-actin as the control ( Figure 8A). Although HFD increased the ratio of lipogenesis-related proteins (FAS, SCD 1, SREBP1c) compared to β-actin levels, 3% and 5% GEP decreased this ratio ( Figure 8B-D), suggesting that GEP inhibits lipogenesis and thus obesity.

Effect of G. Lucidum Extract on AMPK and ACC
GEP supplementation in HFD-induced obese mice enhanced AMPK phosphorylation, leading to the activation of its downstream targets such as ACC ( Figure 9A,B) in a dose-dependent manner; however, GEP did not affect the expression of AMPK or ACC.

Effect of G. Lucidum Extract on AMPK and ACC
GEP supplementation in HFD-induced obese mice enhanced AMPK phosphorylation, leading to the activation of its downstream targets such as ACC ( Figure 9A,B) in a dose-dependent manner; however, GEP did not affect the expression of AMPK or ACC.
1 Figure 9. Effect of GEP on AMPK and ACC protein expression and activation. (A) Western blot analysis and the phosphorylation ratio of (B) AMPK and ACC in the liver and WAT of HFD-induced obese mice stated in fold over ND.

Discussion
G. lucidum has been used to treat metabolic diseases and obesity in oriental medicine; however, there is a lack of biochemical evidence to support its reported anti-obesity activities. In this study, we demonstrated the anti-obesity effects of GEP in an HFD-induced obese C57BL/6 mouse model. The mice were administered varying concentrations of GEP for 12 weeks and then body weight gain, liver and WAT weight, blood parameters, histological features, and the expression of lipogenic genes and proteins were determined.
We observed insignificant improvements in the tested obesity parameters in mice fed a HFD + 1% GEP, while the HFD + 3% and 5% GEP groups displayed significantly lower weight gain and liver and fat tissue weights than the HFD group. Despite no change in food intake, the HFD + 3% and 5% GEP decreased the FER, suggesting that the observed weight loss may be partly attributed to improved energy metabolism. A previous study proposed that GEP concentrations above 4% can effectively improve obesity from the perspective of gut microbiota [33]. Consistently, our study suggests that the consumption of >3% GEP may improve obesity.
Obesity is characterized by adipocyte dysfunction in which lipids can no longer be stored intracellularly and spill over into the blood, causing dyslipidemia [3]. Obesity-induced dyslipidemia is marked by increased TG and LDL, and decreased HDL levels [34]; however, the HFD-induced obese mice treated with GEP displayed lower serum TG, TC, HDL, LDL, and FFA levels, suggesting that GEP may effectively prevent and alleviate hyperlipidemia and cardiovascular diseases induced by obesity. In addition, histological analysis of the liver and WAT revealed that GEP can reduce intracellular fat accumulation in both adipocytes and hepatocytes, which appears to improve obesity. Together, these results indicate that GEP reduces body weight by reducing adipocyte hypertrophy.
Obesity is a major risk factor for metabolic diseases such as type 2 diabetes [3]. In this study, we measured serum glucose and insulin levels, insulin and glucose tolerance, and glycemia AAC and AUC as characteristics of type 2 diabetes. We demonstrated that GEP can lower serum glucose and insulin levels and improve glucose and insulin tolerance in HFD-induced obese mice, suggesting that GEP reduced insulin resistance and improved glucose metabolism in the body. In addition, we demonstrated that GEP improved glucose metabolism by enhancing the phosphorylation of IR, IRS, and Akt, thereby increasing GLUT4 and GLUT1 levels. Akt is a downstream effector in the insulin signaling pathway that, alongside AMPK, inhibits AS160 to downregulate GLUT4 translocation to the plasma membrane [35]. Thus, the activation of IR, IRS, and Akt may increase GLUT4 levels and glucose transport.
The anti-obesity effects of GEP were confirmed by analyzing the mRNA and protein expression of lipogenesis-related genes, revealing that GEP decreased FAS, SCD1, and SREBP1c mRNA and protein expression. SREBP1c is an adipogenic transcription factor that regulates several key genes in adipogenesis such as PPARγ, C/EBPα, FAS, and SCD1 [36]. In addition, SREBP1c mediates the regulation of leptin and adiponectin expression, thus plays an important role in controlling adipogenesis, lipid metabolism, and insulin sensitivity [37]. FAS is a central enzyme in de novo lipogenesis that catalyzes the conversion of malonyl-CoA into palmitate [36], while SCD1 is an enzyme involved in unsaturated fatty acid production that forms a complex with NADH-cytochrome b5 reductase and cytochrome b5 to convert stearoyl-CoA into oleoyl-CoA [38]. The anti-obesity effects of GEP could be attributed to its suppression of these lipogenic genes; however, 1% GEP appeared to increase SREBP1c levels but decreased FAS and SCD1 levels, suggesting that the lipogenesis inhibiting activities of GEP may also be linked to other regulatory mechanisms.
Adipocyte hypertrophy causes dysregulated adipokine secretion that may in turn disrupt metabolism and homeostasis [3]. Leptin is an anorexigenic adipokine that controls food intake by inhibiting appetite. In normal individuals, insulin upregulates leptin production to increase satiety when glucose levels are high; however, obesity induces leptin resistance, resulting in high blood leptin concentrations and high food intake [39]. Adiponectin is an adipokine that stimulates insulin sensitivity, glucose uptake, and fatty acid oxidation, while preventing fatty acid influx into the liver [40]. Consequently, increased adiponectin levels correlate positively with obesity improvements. The effects of both leptin and adiponectin in obesity may be mediated by AMPK since their receptor binding can activate AMPK, which inactivates acetyl-CoA carboxylase (ACC1) [41]. The inactivation of ACC1 reduces the production of malonyl-CoA, a carnitine acyl transferase (CPT1) inhibitor that is important for fatty acid β-oxidation [30]. Thus, increased AMPK activation can enhance β-oxidation and decrease the expression of SREBP1c, a transcription factor involved in lipogenesis and cholesterol synthesis. Adiponectin may also suppress some of the effects of lipid accumulation by activating ceramidase which breaks down ceramide, a molecule that stimulates fatty acid import, leads to lipid accumulation, and disrupts insulin activity. Overall, leptin and adiponectin increase lipid oxidation and decrease fatty acid synthesis and glucose output, thereby enhancing insulin sensitivity [42,43]. In this study, we demonstrated that GEP can reduce leptin and increase adiponectin levels in a dose-dependent manner. GEP restored leptin function, thus reducing hyperleptinemia, and increased adiponectin function to improve lipid and glucose metabolism. These results were consistent with the GEP antiobesity effect observed in other experiments.
Our findings also revealed that treatment with 3% or 5% GEP significantly improved obesity and insulin resistance parameters, as well as the expression and activation of proteins related to insulin signaling pathways. To investigate this further at the molecular level, we assessed AMPK and ACC expression and phosphorylation levels in the liver and WAT of mice treated with 3% or 5% GEP. Notably, GEP enhanced AMPK and ACC activation in a dose-dependent manner but independently of initial protein levels, confirming that the antihyperglycemic and antihyperinsulinemic activities of GEP might be mediated by its ability to increase AMPK phosphorylation [9,27]. Since AMPK can be activated pharmacologically to improve glucose and lipid metabolism in insulin-resistant mice, it could be a promising new target for treating type 2 diabetes [8]. The binding of AMP to the AMPK γ subunit induces conformational changes that allow upstream kinases to phosphorylate AMPK at Thr172 in its α subunit [44]. Activated AMPK can then relay signals to induce catabolism and suppress anabolism, thereby ameliorating obesity and insulin resistance [8,44]. The role of AMPK in metabolic regulation is mediated by various downstream effectors, including ACC, FAS, and GLUT4. ACC converts acetyl-CoA to malonyl-CoA, a fatty acid precursor that downregulates fatty acid oxidation by inhibiting CPT1 [45], thus increasing the availability of FFAs to synthesize other metabolites such as diacylglycerol (DAG) and ceramide [46]. DAG activates PKC which phosphorylates and inhibits insulin receptor kinase (IRK), leading to insulin resistance, while ceramide can inhibit glucose uptake by downregulating GLUT4 at the transcriptional level and inhibiting its localization to the membrane [15,35,47]. Therefore, ACC phosphorylation and inhibition by AMPK may improve GLUT4 localization and insulin sensitivity. A previous study reported that the F31 polysaccharide of G. lucidum might activate AMPK to suppress enzymes that regulate glucose metabolism and inhibit glucose production in the liver, thereby increasing GLUT4 expression and improving insulin resistance [20]. AMPK activation has also been shown to decrease WAT, liver, and body weight by suppressing the expression of adipogenic genes such as FAS and ACC1 [20]. Moreover, β-heteropolysaccharides extracted from G. lucidum have been shown to reduce fasting serum glucose and insulin levels, downregulate the expression of hepatic glucose regulatory enzyme genes, enhance AMPK activation, and increase GLUT4 mRNA and protein levels [20]. F31 polysaccharides and β-heteropolysaccharides contained in G. lucidum may be some of the active substances that exerted the anti-obesity effects via AMPK activation.
The regulation of insulin sensitivity by AMPK might also be linked to the mTOR pathway, which deregulates insulin signaling by activating S6K1, which phosphorylates IRS at multiple serine residues to inhibit its action and reduce glucose uptake [48,49]. In particular, AMPK inhibits mTOR/S6K1 activation, thereby enhancing IR and IRS activation and improving insulin sensitivity [49]. Indeed, studies have shown that G. lucidum may also exert antidiabetic effects by suppressing PEPCK gene expression and inhibiting PTP1B activity, leading to decreased blood glucose levels and improved glucose metabolism [13,50].
GEP used in this study contains myriads of ganoderic acids and glucans (Table 1). Ganoderic acid A has been demonstrated to reduce obesity via SREBP which is a downstream effector of AMPK [51]. Beta 1,3/1,6 glucan serves as immunomodulator in high-cholesterol induced inflammation [52]. Inflammation is one of the factors that contributes to insulin resistance [5]. G. lucidum's glucans ability to alleviate inflammation might help reducing insulin resistance.
Consistent with other works, the findings of this study suggest that G. lucidum extract reduces obesity by targeting AMPK. By activating AMPK, the master regulator of energy homeostasis [46], G. lucidum not only ameliorated obesity, but also improved overall systemic metabolism, including peripheral insulin sensitivity. This study used crude G. lucidum extract which is easier to procure and more practical for clinical application. We also showed that the use of crude G. lucidum extract does not cause adverse effects to C57BL/6 mice. A previous study by Chang, et al. [21] showed G. lucidum ability to significantly reduce obesity using 4% of the extract. Our findings demonstrated that 3% can have significant effects to reduce obesity and insulin resistance, thus minimizing the concentration needed for clinical therapy. Future studies should investigate the anti-obesity effects of each active substances purified from G. lucidum, and GEP application in clinical trial.

Conclusions
This study revealed that G. lucidum could effectively prevent obesity and insulin resistance by attenuating adipocyte hypertrophy and dyslipidemia and improving homeostasis by recovering insulin sensitivity and adipokine function in HFD-induced obese C57BL/6 mice. In particular, G. lucidum exhibited anti-obesity activities by downregulating FAS, SCD1, and SREBP1c, and also displayed antihyperglycemic and antihyperinsulinemic activities by enhancing the activation of AMPK, ACC, IR, IRS, and Akt. Together, these results suggest that G. lucidum extract has the potential to be used as a nutritional supplement to prevent and ameliorate obesity and type 2 diabetes. Future studies should assess the effects of G. lucidum bioactive compounds on other pathways related to obesity in vivo.