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. 2017 Nov 2;18(11):2314.
doi: 10.3390/ijms18112314.

Eburicoic Acid, a Triterpenoid Compound from Antrodia camphorata, Displays Antidiabetic and Antihyperlipidemic Effects in Palmitate-Treated C2C12 Myotubes and in High-Fat Diet-Fed Mice

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Eburicoic Acid, a Triterpenoid Compound from Antrodia camphorata, Displays Antidiabetic and Antihyperlipidemic Effects in Palmitate-Treated C2C12 Myotubes and in High-Fat Diet-Fed Mice

Cheng-Hsiu Lin et al. Int J Mol Sci. .

Abstract

This study was designed to investigate the antidiabetic and antihyperlipidemic effects and mechanisms of eburicoic acid (TRR); one component of Antrodia camphorata in vitro and in an animal model for 14 weeks. Expression levels of membrane glucose transporter type 4 (GLUT4); phospho-5'-adenosine monophosphate-activated protein kinase (AMPK)/total AMPK; and phospho-Akt/total-Akt in insulin-resistant C2C12 myotube cells were significantly decreased by palmitate; and such decrease was prevented and restored by TRR at different concentrations. A group of control (CON) was on low-fat diet over a period of 14 weeks. Diabetic mice; after high-fat-diet (HFD) induction for 10 weeks; were randomly divided into six groups and were given once a day oral gavage doses of either TRR (at three dosage levels); fenofibrate (Feno) (at 0.25 g/kg body weight); metformin (Metf) (at 0.3 g/kg body weight); or vehicle (distilled water) (HF group) over a period of 4 weeks and still on HFD. Levels of glucose; triglyceride; free fatty acid (FFA); insulin; and leptin in blood were increased in 14-week HFD-fed mice as compared to the CON group; and the increases were prevented by TRR, Feno, or Metf as compared to the HF group. Moreover, HFD-induction displayed a decrease in circulating adiponectin levels, and the decrease was prevented by TRR, Feno, or Metf treatment. The overall effect of TRR is to decrease glucose and triglyceride levels and improved peripheral insulin sensitivity. Eburicoic acid, Feno, and Metf displayed both enhanced expression levels of phospho-AMPK and membrane expression levels of GLUT4 in the skeletal muscle of HFD-fed mice to facilitate glucose uptake with consequent enhanced hepatic expression levels of phospho-AMPK in the liver and phosphorylation of the transcription factor forkhead box protein O1 (FOXO1) but decreased messenger RNA (mRNA) of phosphenolpyruvate carboxykinase (PEPCK) to inhibit hepatic glucose production; resulting in lowered blood glucose levels. Moreover; TRR treatment increased hepatic expression levels of the peroxisome proliferator-activated receptor α (PPARα) to enhance fatty acid oxidation; but displayed a reduction in expressions of hepatic fatty acid synthase (FAS) but an increase in fatty acid oxidation PPARα coincident with a decrease in hepatic mRNA levels of sterol response element binding protein-1c (SREBP-1c); resulting in a decrease in blood triglycerides and amelioration of hepatic ballooning degeneration. Eburicoic acid-treated mice reduced adipose expression levels of lipogenic FAS and peroxisome proliferator-activated receptor γ (PPARγ) and led to decreased adipose lipid accumulation. The present findings demonstrated that TRR exhibits a beneficial therapeutic potential in the treatment of type 2 diabetes and hyperlipidemia.

Keywords: Antrodia camphorata; diabetes; eburicoic acid; fatty acid synthase; forkhead box protein O1; peroxisome proliferator-activated receptor α.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Chemical structure of eburicoic acid (TRR).
Figure 2
Figure 2
Effects of eburicoic acid (TRR) on the insulin (Ins)-stimulated expression levels of membrane glucose transporter type 4 (GLUT4), the ratio of phospho-5′-adenosine monophosphate kinase (p-AMPK) to total AMPK (t-AMPK), or phospho-Akt (p-Akt)/total Akt (t-Akt) in insulin-resistant C2C12 myotube cells induced by palmitate (Pal). The symbols “+++”, “###” and “***” represent p < 0.001 as respectively compared to the value of the blank control, positive control (insulin) and negative control (insulin + palmitate) using analysis of variance (ANOVA) and with Dunnett’s tests. (A) Representative image. (BD) Quantification of the membrane GLUT4 expression levels, the ratio of p-AMPK to t-AMPK, or p-Akt/t-Akt expression levels. CON: Blank control; DMSO: Dimethyl sulfoxide, solvent control.
Figure 2
Figure 2
Effects of eburicoic acid (TRR) on the insulin (Ins)-stimulated expression levels of membrane glucose transporter type 4 (GLUT4), the ratio of phospho-5′-adenosine monophosphate kinase (p-AMPK) to total AMPK (t-AMPK), or phospho-Akt (p-Akt)/total Akt (t-Akt) in insulin-resistant C2C12 myotube cells induced by palmitate (Pal). The symbols “+++”, “###” and “***” represent p < 0.001 as respectively compared to the value of the blank control, positive control (insulin) and negative control (insulin + palmitate) using analysis of variance (ANOVA) and with Dunnett’s tests. (A) Representative image. (BD) Quantification of the membrane GLUT4 expression levels, the ratio of p-AMPK to t-AMPK, or p-Akt/t-Akt expression levels. CON: Blank control; DMSO: Dimethyl sulfoxide, solvent control.
Figure 3
Figure 3
Effects of TRR including two parts. (A) Oral glucose tolerance (OGTT) was performed on 12 h fasted ICR mice (n = 5) that were allowed access to 40 and 80 mg/kg TRR or an equivalent amount of vehicle (water), which were given orally 30 min before an oral glucose load (1 g/kg body weight). The control group was given glucose, whereas the normal group was not. Blood samples were collected from the retro-orbital sinus of fasted mice at the time of the glucose administration (0) and every 30 until 120 min after glucose administration and the blood glucose level was monitored. Each point is the mean ± standard error (SE) of five separate mice. ### p < 0.001 compared with the control (CON) group; *** p < 0.001 were significantly different compared with the control group in the same time by ANOVA. (BI) Effects of eburicoic acid (TRR) on (B) body weights, (C) relative tissue weight (%), (D) blood glucose levels, (E) blood triglycerides levels, (F) blood total cholesterol levels, (G) insulin levels, (H) leptin levels, and (I) adiponectin levels at week 14. Mice were fed with 45% high-fat diet (HFD) or low-fat diet (CON) for 14 weeks. After 14 weeks of induction, the HF mice were treated with vehicle, or eburicoic acid (TRR), or fenofibrate (Feno) or metformin (Metf) accompanied with HF diet for 4 weeks. All values are means ± SE (n = 9). ## p < 0.01, ### p < 0.001 compared with the control (CON) group; * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the high-fat-diet (HFD) plus vehicle (distilled water) (HF) group using ANOVA and with Dunnett’s tests. EWAT: Epididymal white adipose tissue; Feno: Fenofibrate (250 mg/kg body weight); Metf: Metformin (300 mg/kg body weight); MWAT: Mesenteric white adipose tissue; RWAT: Retroperioneal white adipose tissue; TRR1, TRR2, or TRR3: 10, 20, or 40 mg/kg body weight eburicoic acid, respectively; Visceral fat: EWAT + RWAT.
Figure 3
Figure 3
Effects of TRR including two parts. (A) Oral glucose tolerance (OGTT) was performed on 12 h fasted ICR mice (n = 5) that were allowed access to 40 and 80 mg/kg TRR or an equivalent amount of vehicle (water), which were given orally 30 min before an oral glucose load (1 g/kg body weight). The control group was given glucose, whereas the normal group was not. Blood samples were collected from the retro-orbital sinus of fasted mice at the time of the glucose administration (0) and every 30 until 120 min after glucose administration and the blood glucose level was monitored. Each point is the mean ± standard error (SE) of five separate mice. ### p < 0.001 compared with the control (CON) group; *** p < 0.001 were significantly different compared with the control group in the same time by ANOVA. (BI) Effects of eburicoic acid (TRR) on (B) body weights, (C) relative tissue weight (%), (D) blood glucose levels, (E) blood triglycerides levels, (F) blood total cholesterol levels, (G) insulin levels, (H) leptin levels, and (I) adiponectin levels at week 14. Mice were fed with 45% high-fat diet (HFD) or low-fat diet (CON) for 14 weeks. After 14 weeks of induction, the HF mice were treated with vehicle, or eburicoic acid (TRR), or fenofibrate (Feno) or metformin (Metf) accompanied with HF diet for 4 weeks. All values are means ± SE (n = 9). ## p < 0.01, ### p < 0.001 compared with the control (CON) group; * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the high-fat-diet (HFD) plus vehicle (distilled water) (HF) group using ANOVA and with Dunnett’s tests. EWAT: Epididymal white adipose tissue; Feno: Fenofibrate (250 mg/kg body weight); Metf: Metformin (300 mg/kg body weight); MWAT: Mesenteric white adipose tissue; RWAT: Retroperioneal white adipose tissue; TRR1, TRR2, or TRR3: 10, 20, or 40 mg/kg body weight eburicoic acid, respectively; Visceral fat: EWAT + RWAT.
Figure 3
Figure 3
Effects of TRR including two parts. (A) Oral glucose tolerance (OGTT) was performed on 12 h fasted ICR mice (n = 5) that were allowed access to 40 and 80 mg/kg TRR or an equivalent amount of vehicle (water), which were given orally 30 min before an oral glucose load (1 g/kg body weight). The control group was given glucose, whereas the normal group was not. Blood samples were collected from the retro-orbital sinus of fasted mice at the time of the glucose administration (0) and every 30 until 120 min after glucose administration and the blood glucose level was monitored. Each point is the mean ± standard error (SE) of five separate mice. ### p < 0.001 compared with the control (CON) group; *** p < 0.001 were significantly different compared with the control group in the same time by ANOVA. (BI) Effects of eburicoic acid (TRR) on (B) body weights, (C) relative tissue weight (%), (D) blood glucose levels, (E) blood triglycerides levels, (F) blood total cholesterol levels, (G) insulin levels, (H) leptin levels, and (I) adiponectin levels at week 14. Mice were fed with 45% high-fat diet (HFD) or low-fat diet (CON) for 14 weeks. After 14 weeks of induction, the HF mice were treated with vehicle, or eburicoic acid (TRR), or fenofibrate (Feno) or metformin (Metf) accompanied with HF diet for 4 weeks. All values are means ± SE (n = 9). ## p < 0.01, ### p < 0.001 compared with the control (CON) group; * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the high-fat-diet (HFD) plus vehicle (distilled water) (HF) group using ANOVA and with Dunnett’s tests. EWAT: Epididymal white adipose tissue; Feno: Fenofibrate (250 mg/kg body weight); Metf: Metformin (300 mg/kg body weight); MWAT: Mesenteric white adipose tissue; RWAT: Retroperioneal white adipose tissue; TRR1, TRR2, or TRR3: 10, 20, or 40 mg/kg body weight eburicoic acid, respectively; Visceral fat: EWAT + RWAT.
Figure 3
Figure 3
Effects of TRR including two parts. (A) Oral glucose tolerance (OGTT) was performed on 12 h fasted ICR mice (n = 5) that were allowed access to 40 and 80 mg/kg TRR or an equivalent amount of vehicle (water), which were given orally 30 min before an oral glucose load (1 g/kg body weight). The control group was given glucose, whereas the normal group was not. Blood samples were collected from the retro-orbital sinus of fasted mice at the time of the glucose administration (0) and every 30 until 120 min after glucose administration and the blood glucose level was monitored. Each point is the mean ± standard error (SE) of five separate mice. ### p < 0.001 compared with the control (CON) group; *** p < 0.001 were significantly different compared with the control group in the same time by ANOVA. (BI) Effects of eburicoic acid (TRR) on (B) body weights, (C) relative tissue weight (%), (D) blood glucose levels, (E) blood triglycerides levels, (F) blood total cholesterol levels, (G) insulin levels, (H) leptin levels, and (I) adiponectin levels at week 14. Mice were fed with 45% high-fat diet (HFD) or low-fat diet (CON) for 14 weeks. After 14 weeks of induction, the HF mice were treated with vehicle, or eburicoic acid (TRR), or fenofibrate (Feno) or metformin (Metf) accompanied with HF diet for 4 weeks. All values are means ± SE (n = 9). ## p < 0.01, ### p < 0.001 compared with the control (CON) group; * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the high-fat-diet (HFD) plus vehicle (distilled water) (HF) group using ANOVA and with Dunnett’s tests. EWAT: Epididymal white adipose tissue; Feno: Fenofibrate (250 mg/kg body weight); Metf: Metformin (300 mg/kg body weight); MWAT: Mesenteric white adipose tissue; RWAT: Retroperioneal white adipose tissue; TRR1, TRR2, or TRR3: 10, 20, or 40 mg/kg body weight eburicoic acid, respectively; Visceral fat: EWAT + RWAT.
Figure 4
Figure 4
Histology of (A) epididymal white adipose tissue and (B) liver tissue of mice in the control (CON), high-fat-diet plus vehicle (distilled water) (HF), HFD + TRR1, HFD + TRR2, HFD + TRR3, HFD + fenofibrate (Feno), or HFD + metformin (Metf) groups by hematoxylin and eosin staining. Arrows indicated the hepatic ballooning degeneration. Each presented is typical and representative of nine mice, and one section per mouse. TRR1, TRR2, or TRR3: 10, 20, or 40 mg/kg body weight eburicoic acid, respectively Feno: Fenofibrate (250 mg/kg body weight); Metf: Metformin (300 mg/kg body weight).
Figure 4
Figure 4
Histology of (A) epididymal white adipose tissue and (B) liver tissue of mice in the control (CON), high-fat-diet plus vehicle (distilled water) (HF), HFD + TRR1, HFD + TRR2, HFD + TRR3, HFD + fenofibrate (Feno), or HFD + metformin (Metf) groups by hematoxylin and eosin staining. Arrows indicated the hepatic ballooning degeneration. Each presented is typical and representative of nine mice, and one section per mouse. TRR1, TRR2, or TRR3: 10, 20, or 40 mg/kg body weight eburicoic acid, respectively Feno: Fenofibrate (250 mg/kg body weight); Metf: Metformin (300 mg/kg body weight).
Figure 5
Figure 5
Semiquantative reverse transcription polymerase chain reaction (RT-PCR) analysis on phosphenolpyruvate carboxykinase (PEPCK), glucose-6-phosphatase (G6Pase), sterol regulatory element-binding protein 1 (SREBP1c), diacylglycerol acyltransferase 2 (DGAT2), glycerol-3-phosphate acyltransferase (GPAT), and sterol regulatory element-binding protein 2 (SREBP2) messenger RNA (mRNA) levels in liver tissue of the mice receiving eburicoic acid (TRR) by oral gavage for 4 weeks. (A) representative image. (B,C) Quantification of the ratio of target gene to GAPDH mRNA expression. All values are means ± SE (n = 9). ### p < 0.001 compared with the control (CON) group; * p < 0.05 and *** p < 0.001 compared with the high-fat diet plus vehicle (HF) group using ANOVA and with Dunnett’s tests. TRR1, TRR2, or TRR3: Eburicoic acid 10, 20, or 40 mg/kg body weight, respectively; Feno: Fenofibrate (250 mg/kg body weight); Metf: Metformin (300 mg/kg body weight). Total RNA (1 μg) isolated from tissue was reverse transcripted by M-MLV reverse transcriptase (M-MLV-RT), 10 μL of reverse transcriptase (RT) products were used as templates for PCR. Signals were quantitated by image analysis; each value was normalized by β-actin.
Figure 5
Figure 5
Semiquantative reverse transcription polymerase chain reaction (RT-PCR) analysis on phosphenolpyruvate carboxykinase (PEPCK), glucose-6-phosphatase (G6Pase), sterol regulatory element-binding protein 1 (SREBP1c), diacylglycerol acyltransferase 2 (DGAT2), glycerol-3-phosphate acyltransferase (GPAT), and sterol regulatory element-binding protein 2 (SREBP2) messenger RNA (mRNA) levels in liver tissue of the mice receiving eburicoic acid (TRR) by oral gavage for 4 weeks. (A) representative image. (B,C) Quantification of the ratio of target gene to GAPDH mRNA expression. All values are means ± SE (n = 9). ### p < 0.001 compared with the control (CON) group; * p < 0.05 and *** p < 0.001 compared with the high-fat diet plus vehicle (HF) group using ANOVA and with Dunnett’s tests. TRR1, TRR2, or TRR3: Eburicoic acid 10, 20, or 40 mg/kg body weight, respectively; Feno: Fenofibrate (250 mg/kg body weight); Metf: Metformin (300 mg/kg body weight). Total RNA (1 μg) isolated from tissue was reverse transcripted by M-MLV reverse transcriptase (M-MLV-RT), 10 μL of reverse transcriptase (RT) products were used as templates for PCR. Signals were quantitated by image analysis; each value was normalized by β-actin.
Figure 6
Figure 6
Membrane GLUT4 protein contents in skeletal muscle, or expression levels of phospho-Akt (p-Akt)/total Akt (t-Akt), phospho-AMPK (p-AMPK) (Thr172)/total AMPK (t-AMPK), or phospho-FOXO1 (p-FOXO1) (Ser256)/total FOXO1 (t-FOXO1) in liver and skeletal muscle of the mice by oral gavage eburicoic acid (TRR). (A,B) Representative images including membrane GLUT4, p-AMPK, t-AMPK, p-Akt, t-Akt, p-FOXO, and t-FOXO1 in different tissues. (C,D) Quantification of the GLUT4 expression levels, the ratio of p-AMPK to t-AMPK, or p-Akt/t-Akt expression levels (mean ± SE, n = 9). Protein was separated by 12% SDS–PAGE detected by Western blot. # p < 0.05, ## p < 0.01, and ### p < 0.001 compared with the control (CON) group; * p < 0.05 and *** p < 0.001 compared with the high-fat-diet plus vehicle (distilled water) (HF) group using ANOVA and with Dunnett’s tests. TRR1, TRR2, or TRR3: Eburicoic acid 10, 20, or 40 mg/kg body weight, respectively; Feno: Fenofibrate (250 mg/kg body weight); Metf: Metformin (300 mg/kg body weight).
Figure 6
Figure 6
Membrane GLUT4 protein contents in skeletal muscle, or expression levels of phospho-Akt (p-Akt)/total Akt (t-Akt), phospho-AMPK (p-AMPK) (Thr172)/total AMPK (t-AMPK), or phospho-FOXO1 (p-FOXO1) (Ser256)/total FOXO1 (t-FOXO1) in liver and skeletal muscle of the mice by oral gavage eburicoic acid (TRR). (A,B) Representative images including membrane GLUT4, p-AMPK, t-AMPK, p-Akt, t-Akt, p-FOXO, and t-FOXO1 in different tissues. (C,D) Quantification of the GLUT4 expression levels, the ratio of p-AMPK to t-AMPK, or p-Akt/t-Akt expression levels (mean ± SE, n = 9). Protein was separated by 12% SDS–PAGE detected by Western blot. # p < 0.05, ## p < 0.01, and ### p < 0.001 compared with the control (CON) group; * p < 0.05 and *** p < 0.001 compared with the high-fat-diet plus vehicle (distilled water) (HF) group using ANOVA and with Dunnett’s tests. TRR1, TRR2, or TRR3: Eburicoic acid 10, 20, or 40 mg/kg body weight, respectively; Feno: Fenofibrate (250 mg/kg body weight); Metf: Metformin (300 mg/kg body weight).
Figure 7
Figure 7
Expression levels of peroxisome proliferator-activated receptor α (PPARα), fatty acid synthase (FAS), and peroxisome proliferator-activated receptor γ (PPARγ) in the liver, and PPARγ and FAS in adipose tissue of mice by oral gavage eburicoic acid (TRR). (A) Representative images including PPARα, FAS, and PPARγ in different tissues. (B,C) Quantification of the expression levels of PPARα, FAS, and PPARγ in the liver and expression levels of FAS and PPARγ in adipose tissue. Protein was separated by 12% SDS–PAGE detected by Western blot. All values are means ± SE (n = 9). # p < 0.05 and ### p < 0.001 compared with the control (CON) group; * p < 0.05 and *** p < 0.001 compared with the HFD plus vehicle (distilled water) (HF) group using ANOVA and with Dunnett’s tests. TRR1, TRR2, or TRR3: 10, 20, or 40 mg/kg body weight eburicoic acid, respectively; Feno: Fenofibrate (250 mg/kg body weight); Metf: Metformin (300 mg/kg body weight).
Figure 7
Figure 7
Expression levels of peroxisome proliferator-activated receptor α (PPARα), fatty acid synthase (FAS), and peroxisome proliferator-activated receptor γ (PPARγ) in the liver, and PPARγ and FAS in adipose tissue of mice by oral gavage eburicoic acid (TRR). (A) Representative images including PPARα, FAS, and PPARγ in different tissues. (B,C) Quantification of the expression levels of PPARα, FAS, and PPARγ in the liver and expression levels of FAS and PPARγ in adipose tissue. Protein was separated by 12% SDS–PAGE detected by Western blot. All values are means ± SE (n = 9). # p < 0.05 and ### p < 0.001 compared with the control (CON) group; * p < 0.05 and *** p < 0.001 compared with the HFD plus vehicle (distilled water) (HF) group using ANOVA and with Dunnett’s tests. TRR1, TRR2, or TRR3: 10, 20, or 40 mg/kg body weight eburicoic acid, respectively; Feno: Fenofibrate (250 mg/kg body weight); Metf: Metformin (300 mg/kg body weight).
Figure 8
Figure 8
A proposed mechanism for TRR to improve diabetes and hyperlipidemia.

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