Taraxasterol Inhibits Hepatic Gluconeogenesis and Increases Glycogen Synthesis via the PI3K/Akt Signaling Pathway

Molecular Docking Simulation

T2DM patients are usually accompanied by IR. ¹² Insulin and insulin receptor (INSR) play important roles in the regulation of glucose. ¹³ Therefore, drug interaction with the INSR is one of the first and important step in the drug treatment of diabetes. To investigate the interaction of key targets of taraxasterol in the treatment of diabetes, we performed a docking simulation of taraxasterol with the key target INSR. As seen in Figure 2C and D, taraxasterol showed good interaction with the target INSR based on the parameter scores and the H bond information. To analyze this in more detail, we visualized the docked structure by using Discovery Studio software.

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Figure 2.

Docking simulation. (A) The 2D structure of taraxasterol. (B) The 3D structure of the INSR protein. (C) The 3D structure of taraxasterol binding to INSR. (D) The 2D structure of taraxasterol binding to INSR. Abbreviations: 2D, two-dimensional; 3Dthree-dimensional; INSR, insulin receptor.

The docking results of the taraxasterol ligand and INSR protein revealed that the key amino acid residue involved in interactions was Lys1030 (Figure 2C and D). Conventional hydrogen bonding formed the interactions with Lys1030 and the other interactions exhibited by the INSR were van der Waals interactions with Gly1003, Ser1006, Gln1004, Lys1085, Ser1086, Asp1083, Asn1137, Asp1150, Met1076, and Glu1047, as shown in Table 1. The other interactions exhibited by the taraxasterol were alkyl interactions with Arg1136, Met1139, and Val1010. A molecular docking score greater than 20 indicates that the ligand molecule and the receptor molecule have good binding energy. The docking score of taraxasterol and INSR was 35.531, and it indicated that taraxasterol and INSR had good binding energy.

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Table 1.

The Molecular Docking Arguments.

Interactions	Amino acid residue	-CDOCKER - ENERGY	-CDOCKER - INTERACTION - ENERGY
Conventional hydrogen bonding	Lys1030	−70.6933	36.9538
Van der Waals	Gly1003 Asn1137	−70.7819	37.485
	Gln1004 Asp1150	−72.2767	36.1695
	Ser1006 Met1076	−72.6888	40.7884
	Lys1085 Glu1047	−72.7787	37.9496
	Asp1083	−72.8321	35.531
Alkyl interactions	Arg1136 Met1139	−73.4994	36.7062
	Val1010	−74.0061	35.1434
		−74.9539	37.4603
		−75.9648	36.1634

Taraxasterol Improved Glucose Consumption in Insulin-Resistant Hepatocytes

Firstly, we studied the cytotoxic effect of taraxasterol on hepatocytes, and the cell viability was examined by using a microculture tetrazolium (MTT) assay. Compared to the normal group (NC), the results showed that the concentration of taraxasterol (0.1, 1, 10 µM) had no cytotoxic effect on HepG2 cells. Furthermore, cell viability was significantly reduced at high doses (100 μM taraxasterol). Moreover, the positive drug (metformin, 10⁻⁴ mol/L) had no cytotoxic effect on the HepG2 cells as shown in Figure 3A.

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Figure 3.

Effects of different concentrations of taraxasterol on glucose consumption in HepG2 cells with insulin resistance. (A) MTT assay. (B) Glucose consumption analysis. n = 4 for each group. Values are mean ± SD. *P < 0.05, **P < 0.01 versus IR group (18 mM GlcN stimulated). ^#P < 0.05, ^##P < 0.01 versus normal group. Abbreviations: MTT, microculture tetrazolium; GlcN, glucosamine; IR, insulin resistance.

Then we evaluated the potential roles of taraxasterol on glucose consumption in HepG2 cells. The cellular glucose consumption was significantly decreased in the IR group (18 mM glucosamine [GlcN]). However, glucose consumption was obviously increased in insulin-resistant HepG2 cells when treated with taraxasterol in a dosage-dependent manner in insulin-resistant HepG2 cells (P<0.05; Figure 3B). Furthermore, the effect of taraxasterol at a high dose (10 μM) was stronger than that of the positive drug (metformin, 10⁻⁴ mol/L). These results indicated that taraxasterol could improve glucose consumption in HepG2 cells with IR.

Taraxasterol Increased Glucose Uptake in Hepatocytes with Insulin Resistance

IR in the liver is an important factor causing fasting hyperglycemia and T2DM. To evaluate the potential roles of taraxasterol on HepG2 cells with IR, the cellular glucose uptake was assessed in HepG2 cells treated with GlcN (18 mM) to induce IR. Next, the HepG2 cells were disposed with taraxasterol at different concentrations (0.1, 1, 10 µM), and fluorescent 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-2-deoxy-D-glucose (2-NBDG) was added as fluorescent indicators and incubated cells for 60 min. As seen in Figure 4A, B, and G, compared to the normal group, the fluorescence density and area were significantly decreased when HepG2 cells were treated with GlcN (18 mM), which represents significantly decreased glucose uptake in HepG2 cells with IR. However, taraxasterol (0.1, 1, 10 µM) significantly improved glucose uptake in HepG2 cells with IR, as shown in Figure 4C to E and G. When the concentration of taraxasterol was 10 µM, the glucose uptake was the same as that of the positive drug group (metformin, 10⁻⁴ M). These results indicated that taraxasterol could improve glucose uptake in HepG2 cells with IR.

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Figure 4.

Effects of taraxasterol on glucose uptake in HepG2 cells with insulin resistance. (A) Normal group (NC). (B) Insulin-resistant model group (M). (C to E) Different concentrations of taraxasterol (0.1, 1, and 10 μM). (F) Metformin group (Met, 10⁻⁴ M). (G) Glucose uptake column Chart. 100×magnification. n = 3 for each group. Values are mean ± SD. *P < 0.05, **P < 0.01 versus IR group (18 mM glucosamine [GlcN] stimulated). ^#P < 0.05, ^##P < 0.01 versus normal group.

Taraxasterol Significantly Reduced the Lipid Content in Insulin-Resistant Hepatocytes

To study the lipid regulation of taraxasterol on GlcN-induced insulin-resistant HepG2 cells, oil-red-O staining was used. The number and size of lipid droplets were accumulated in the IR group (Figure 5A, B, and G). After taraxasterol (0.1, 1, 10 µM) treatment, lipid accumulation was decreased in a dose-dependent pattern, and the cells’ state was significantly improved. When the concentration of taraxasterol was 10 μM, the lipid accumulation was less than that of the positive drug group (metformin, 10⁻⁴ M), as shown in Figure 5C to F and G. These results suggested that taraxasterol might reduce lipid accumulation in hepatocytes with IR.

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Figure 5.

Effects of taraxasterol on the lipid profile in HepG2 cells with insulin resistance. (n = 3). (A) Normal group (NC). (B) Insulin-resistant model group(M). (C-E) Different concentrations of taraxasterol (0.1, 1, and 10 μM). (F) Metformin group (Met) (10⁻⁴ M). (G) Lipid accumulation column Chart. 100×magnification. n = 3 for each group. Values are mean ± SD. *P < 0.05, **P < 0.01 versus IR group (18 mM glucosamine [GlcN] stimulated). ^#P <0.05, ^##P <0.01 versus normal group.

Taraxasterol Stimulated Glycogen Synthesis via the PI3K/Akt/GSK-3β Pathway

When INSRs are activated, they usually bind to IRS-1 to activate the downstream glucometabolic-related protein PI3K/Akt. To assess the effect of taraxasterol on the IRS-1/PI3K/Akt/GSK-3β pathway, we examined the expression of PI3K/Akt/GSK3β-related protein in GlcN-treated HepG2 cells. As expected, we found that the expression of phosphorylation of IRS-1 at Ser307 was significantly up-regulated when treated with 18 mM GlcN. However, the expression of phosphorylation of IRS-1 at Ser307 was decreased in a dose-dependent manner when HepG2 cells were treated with taraxasterol (0.1, 1 10 μM) (P < 0.05; Figure 6A). Moreover, the p110α subunits of PI3K were decreased but taraxasterol could improve PI3K expression in GlcN-treated HepG2 cells (P < 0.05; Figure 6B). Furthermore, compared with the normal group, the expression of p-Akt (Ser473) and p-GSK-3β (Ser9) were obviously decreased in GlcN-treated HepG2 cells, while taraxasterol could significantly restore the expression of the phosphorylation levels of Akt and GSK-3β in GlcN-treated HepG2 cells, as shown in Figure 6C and 6D (P < 0.05). At the same time, to explore the potential bio-synergy by which taraxasterol affects glycogen synthesis, we also determined the protein expression of p-GS (Ser645) and GS were also determined. We observed that the protein expression of p-GS (Ser645) was significantly enhanced. Nevertheless, taraxasterol could significantly down-regulate the expression of p-GS (Ser645) and up-regulate the expression of GS (P < 0.05, Figure 6E). These results indicated that taraxasterol could regulate hepatic glycogen synthesis by the PI3K/Akt/GSK-3β signal pathway in HepG2 cells with IR.

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Figure 6.

Effect of taraxasterol on glycogen synthesis in HepG2 cells with insulin resistance. (A) The expression levels of IRS1 and p-IRS1, and the ratios of p-IRS-1/IRS-1. (B) The expression levels of PI3K; GAPDH were used as a loading control. (C) The expression levels of p-Akt, Akt, and the ratios of p-Akt/ Akt. (D) The expression levels of GSK-3β and p-GSK-3β, and the ratios of p-GSK-3β/GSK-3β. (E) The expression levels of GS and p-GS, and the ratios of p-GS/GS. Data are expressed as mean ± SD For each group, n = 3. *P < 0.05, **P < 0.01 versus IR group (18 mM GlcN stimulated). ^#P < 0.05, ^##P < 0.01 versus normal group.

Taraxasterol Inhibited Gluconeogenesis via the PI3K/Akt/FoxO1 Pathway

FoxO1 and its downstream targets, including G6Pase and PEPCK, are important transcription factors in regulating gluconeogenesis. ¹⁴ To further study the effect of taraxasterol to regulate gluconeogenesis in insulin-resistant HepG2 cells, we evaluated the protein FoxO1 and its downstream targets (G6Pase and PEPCK). Compared with the normal group, the p-FoxO1/FoxO1 ratio was down-regulated (P < 0.01; Figure 7A), while the expression of PEPCK and G6Pase were up-regulated in HepG2 cells with IR (P < 0.01; Figure 7B and C). After treatment with taraxastero (0.1, 1, 10 µM) l, the p-FoxO1/FoxO1 ratio was elevated (P < 0.01; Figure 7A), whereas the expression of PEPCK and G6Pase were markedly decreased (P < 0.01; Figure 7B and C). These results suggested that taraxasterol could inhibit gluconeogenesis by regulating the PI3K/Akt/FoxO1 signal pathway in insulin-resistant HepG2 cells.

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Figure 7.

Effect of taraxasterol on gluconeogenesis in cells with insulin resistance. (A) The protein expression levels of FoxO1 and the ratios of p-FoxO1/FoxO1. (B) The protein expression levels of PEPCK and the ratios of PEPCK/GAPDH. (C) The protein expression levels of G6Pase and the ratios of G6Pase/GAPDH. All data are expressed as mean ± SD. For each group, n = 3. *P < 0.05, **P < 0.01 versus IR group (18 mM GlcN stimulated), ^#P < 0.05, ^##P < 0.01, versus normal group.

Materials and Reagents

MTT Assay

An MTT-based assay kit was used to determine the effects of taraxasterol on the viability of HepG2 cells. The cells (1×10⁴ cells/mL) were cultured in a flat-bottomed 96-well plate. After 18 mM of GlcN was added, treatment for 18 h to induce an insulin-resistance model, we removed the medium and treated the cells with taraxasterol (0.1, 1, 10 µM) for 24 h. After incubation, we added 20 μL of MTT solution (5 mg/mL) to each well and culture in an incubator (37 °C, 5% CO₂). After 4 h incubation, to dissolve the formazan crystals, we removed the supernatant and 100 μL of DMSO was added to each well. Then a microplate reader was used to detect the absorbance at 490 nm. We regarded the absorbance of the control group as 100%.

Glucose Consumption Assay

The glucose concentrations of HepG2 cells were measured using a glucose assay kit. The cells were treated in the same way as described above. Taraxasterol at different concentrations was added and incubated for 24 h. On the next day, a glucose reagent was added and incubated at 37 °C for 15 min, and the culture medium was collected. The OD values at 505 nm were measured using a microplate, and glucose concentration in the supernatant was determined by glucose determination kit as shown in Table 2.

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Table 2.

The Materials of This Study.

Material	Company name
Taraxasterol (≥98%, CAS:161009)	Tongtian Biotechnology Co., Ltd
Metformin hydrochloride	Tianan pharmaceutical industry Co., Ltd
HepG2 cells	Cell Bank, Chinese Academy of Sciences
Glucose determined kit	Rong Sheng Biotech Co., Ltd
Glucosamine	Beyotime Biotechnology Co., Ltd
HRP substrate kit	Millipore Corporation
FBS	Gibco Co., Ltd
NBDG	Amgicam Biomedical. Technology Co., Ltd
Oil red O stain kit	Solarbio Science and Technology Co., Ltd
MTT	Solarbio Science and Technology Co., Ltd
DMSO	Solarbio Science and Technology Co., Ltd
BSA	Solarbio Science and Technology Co., Ltd
IRS-1 (1:1000)	Affinity Bioscience
p-IRS-1 (ser307; 1:1000)	Affinity Bioscience
GAPDH (1:1000)	Affinity Bioscience
Akt (1:1000)	Affinity Bioscience
p-Akt (Ser473; 1:1000)	Affinity Bioscience
GSK-3β (1:1000)	Absin Bioscience Inc.
p-GSK-3β (Ser9; 1:1000)	Absin Bioscience Inc.
GS (1:1000)	Absin Bioscience Inc.
p-GS (Ser641; 1:1000)	Absin Bioscience Inc.
FoxO1 (1:1000)	Absin Bioscience Inc.
p-FoxO1 (Ser256; 1:1000)	Absin Bioscience Inc.
PEPCK (1:1000)	Absin Bioscience Inc.
G6Pase (1:1000)	Absin Bioscience Inc.
PI3K (1:1000)	Immunoway Biotechnology Company
RIP Alysis buffer	Cowin Biotech Co., Ltd
BCA protein assay kit	Cowin Biotech Co., Ltd

Abbreviations: NBDG, 2-(N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl) amino)-2-deoxy-D-glucose; HRP, horseradish peroxidase; MTT, microculture tetrazolium; FBS, fetal bovine serum; DMSO, dimethyl sulfoxide; BSA, bovine serum albumin; BCA, bicinchoninic acid.

2-NBDG Glucose Uptake Assay

Cellular glucose uptake levels were assessed using the fluorescent 2-NBDG. In short, HepG2 cells (1×10⁵ cells/well) were cultured in 6-well plates. After reaching 80% confluence, we added GlcN (18 mM) and cultured for 18 h to establish an insulin-resistance model. Next, different concentrations of taraxasterol (0.1, 1, and 10 μM) were added and incubated for 24 h. Afterward, The HepG2 cells were washed 5 times with phosphate-buffered saline (PBS), and 2-NBDG (100 μM) was added to the cells, and the cells were incubated in an incubator (37 °C, 5% CO₂) for 1 h. Then, PBS was used to wash the cells twice to remove the residual probe. Photographs were taken under an inverted fluorescence microscope. "Image J" analysis software was used to quantitatively analyze the results.

Oil red O Staining

After the HepG2 cells were cultured and treated as before, the medium was discarded, and PBS was used to wash the cells twice. Oil red O (ORO) staining solution was added, and the cells were fixed for 20 to 30 min. Then, distilled water was used to wash the cells, which were soaked with 60% isopropyl alcohol for 5 min, and staining with the ORO stain solution (B1:B2 = 3:2) for 10 min. Excess dye was removed by washing, and Mager hematoxylin staining solution was added. After 10 min, we washed the cells 3 times with triple-distilled water. Then, ORO buffer was added for 5 min and the microscope was used to observe the state of the HepG2 cells. "Image J" analysis software was used to quantitatively analyze the results.

Western Blot Assay

The HepG2 cells were cultured and treated as before, metformin (10⁻⁴ M) and different concentrations of taraxasterol (0.1, 1, and 10 μM) were treated for 1 h, RIPA buffer was used to lyse the cells. After 15 min, the cell mixture was collected and centrifuged at 12,000 r/min for 10 min. Then, a BCA assay kit was used to measure the concentrations of proteins according to the specification. Next, 25 µL of RIPA lysis buffer was mixed with 100 µL of total protein and denatured at 80 °C for 8 min. Then all the processed proteins were stored in the refrigerator at −20 °C. Before they were transferred to a polypropylene fluoride membrane, samples containing 30 µg of protein were separated by 10% SDS-PAGE, which was transferred to PVDF membranes. The membranes were washed 3 times, every 5 min, with TBST (0.1% Tween 20 in TBS). Then, they were incubated in BSA (5% bovine serum albumin in TBST) for 2 h and incubated with a specific primary antibody at 4 °C. After 24 h, we washed the membranes 3 times with TBST. Then, an HRP-conjugated secondary antibody was used to incubate these membranes at room temperature for 3 h. Finally, these membranes were treated with chemiluminescence. "Image J" analysis software was used to quantitatively analyze the results.