Metabolomics reveals the protective of Dihydromyricetin on glucose homeostasis by enhancing insulin sensitivity

Dihydromyricetin (DMY), an important flavanone found in Ampelopsis grossedentata, possesses antioxidative properties that ameliorate skeletal muscle insulin sensitivity and exert a hepatoprotective effect. However, little is known about the effects of DMY in the context of high-fat diet (HFD)-induced hepatic insulin resistance. Male Sprague-Dawley(SD) rats were fed a HFD(60% fat) supplemented with DMY for 8 weeks. The administration of DMY to the rats with HFD-induced insulin resistance reduces hyperglycemia, plasma levels of insulin, and steatosis in the liver. Furthermore, DMY treatment modulated 24 metabolic pathways, including glucose metabolism, the TCA cycle. DMY significantly enhanced glucose uptake and improved the translocation of glucose transporter 1. The specificity of DMY promoted the phosphorylation of AMP-activated protein kinase (AMPK). In addition, the exposure of HepG2 cells to high glucose concentrations impaired the insulin-stimulated phosphorylation of Akt2 Ser474 and insulin receptor substrate-1 (IRS-1) Ser612, increased GSK-3β phosphorylation, and upregulated G6Pase and PEPCK expression. Collectively, DMY improved glucose-related metabolism while reducing lipid levels in the HFD-fed rats. These data suggest that DMY might be a useful drug for use in type 2 diabetes insulin resistance therapy and for the treatment of hepatic steatosis.


Materials
HepG2 cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Beijing).

Animals and diets
Male Sprague-Dawley (SD) rats weighing 100-140 g were kept in a humidity-controlled and air-conditioned room (22±2°C) with a 12 h light cycle (6:00 a.m.-6:00 p.m.) and food and water available ad libitum for 1 week. The rats were divided randomly into six groups of 10 animals each: a control group that was given deionized distilled water to drink and was fed standard rat chow [2] composed of 60% vegetable starch, 12% fat, and 28% protein; and a model group that was given deionized distilled water and fed a diet of 60% fat, 14% protein and 26% carbohydrate.
The rats in the DMY groups were treated with 100, 200, and 400 mg/kg DMY by oral gavage.
Pioglitazone, a potent insulin sensitizer used in the treatment of type II diabetes, has a known effect on glucose metabolism and was used as a positive control (5 mg/kg) [3,4]. During the 8-week study period, fresh water or flower tea was provided daily at 7:00 PM. After treatment for 4 weeks, blood samples were collected from the ocular vein in heparinized tubes after overnight fasting, and the plasma was separated by centrifugation and stored at -20°C until subsequent use.
After blood sampling, the animals were sacrificed following anesthetization by chloral hydrates (350 mg/kg, i.p.) at the end of the 8-week period. The livers were removed at the time of death and immediately frozen in liquid nitrogen for Western blot or PCR analysis. All serum and hepatic biochemical parameters, with the exception of serum insulin, were measured by the respective kits (Jian Cheng Biotechnology Company, Nanjing, China) according to the manufacturer's instructions. Serum insulin levels were determined using a radioimmunoassay kit (Beijing North Institute of Biological Technology, Beijing, China) according to the manufacturer's instructions.

Histological analysis
Liver tissue specimens were fixed in a 4% buffered neutral formalin solution for at least 24 h, embedded in paraffin wax and sectioned (5 µm thickness) for histopathological evaluation. Liver sections were stained with H&E. The images were observed under a light microscope and photographed at 400× final magnification.

Measurement of insulin tolerance
During the last week of treatment and after a 12 h fasting, the animals were orally gavaged with 2 g/kg body weight glucose dissolved in water, then injected subcutaneously with insulin (0.75 U/kg body weight). Blood samples were taken from the tail vein at 0, 15, 30, 45, 60, 90 and 120 min after glucose loading, and plasma glucose levels were assessed.

Homeostasis model of insulin resistance (HOMA-IR)
Because abnormalities in insulin action are poorly represented by a single measurement of glucose or insulin levels [5], a homeostasis model was used to estimate insulin resistance

Glucose uptake assay
Glucose uptake rates were measured after the addition of the tracer 2-NBDG to the culture medium as previously reported [7]. Cells were cultured in black 96-well plates to 90% confluence and incubated with the respective treatment then washed twice and incubated with 100 μmol/L 2-NBDG in glucose-free culture medium for 20 min. Cells cultured in the absence of 2-NBDG served as a negative control. The cells were washed twice, and fluorescence was detected using a microplate reader (Infinite 1000 M, Tecan, AUSTRIA) with excitation at 488 nm and emission at 520 nm.

Assay of glycogen synthesis
The accumulation of glycogen was determined using a Glycogen Colorimetric/Fluorometric Assay Kit (K646-100, BioVision, USA) as described previously [8]. After the conclusion of experimental treatments, the cells were washed three times with ice-cold PBS. The 106 cells were homogenized in 200 µl dH2O on ice and then boiled for 10 min to inactivate any enzymes. The boiled samples were centrifuged at 18,000×g for 10 min to remove insoluble material, and the supernatant was assayed using a microplate reader (Infinite 1000 M, Tecan, AUSTRIA) with excitation at 488 nm and emission at 520 nm.

siRNA-mediated LKB1 knockdown
HepG2 cells were transfected with negative-control siRNA or LKB1 siRNA according to the manufacturer's protocol. Briefly, cells were seeded into 60-mm dishes. After 24 h, the medium was changed to fresh, antibiotic-free medium, and the cells were cultured for an additional 24 h in the presence or absence of DMY. siRNA plasmids were transfected into HepG2 cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.
The cells were then allowed to express the siRNA for 48 h.

Metabolite profiling
Metabolite profiling was performed according to previous reports [9]. Briefly, rat livers were harvested and lysed in a solution of methanol:chloroform:water (1:2:1) to extract metabolites. All reagents were of chromatographic grade. Metabolite profiling was performed using a TSQ Quantum Access TM triple quadrupole mass spectrometer with an electrospray ionization (ESI) source coupled to a Surveyor auto-sampler, a Surveyor LC pump, and Xcalibur3.0 software for data acquisition and analysis (Thermo Finigan, USA). Mass spectrometry was controlled by XCalibur software Version 3.0 (Thermo Electron Corporation, USA) and was operated in selected reaction monitoring (SRM) mode using electrospray ionization in the negative-ion mode. We used a specific sample as quality control (QC) rather than an internal standard. QC runs were performed at the beginning and end of the sequence. The total ion current and chromatographic patterns were evaluated. Table X shows four representative compounds identified in QC runs.

Quantitative real-time PCR analysis
Total RNA was extracted from liver tissues using the EasyPurfe TM RNA Kit (ER101, TransGen Biotech, Beijing China) according to the manufacturer's instructions. cDNA was synthesized using EasyScript® First-strand Synthesis SuperMix and a PCR System (Eppendorf AG, Germany). The assay was performed according to real-time PCR with TransStart® Top Green qPCR SuperMix (Tli RNaseH Plus) on an iCycler iQ5 thermocycler (BioRad, Hercules, CA) as previously described [10].
The primers used to amplify each gene are listed in Table 1. All samples were analyzed in triplicate, and gene expression levels were normalized to control rat β-actin values. Fold changes between the groups were calculated using the 2 -∆∆Ct method [11].
For Western blotting, total protein was isolated from the livers and cells of different treatment groups using a Protein Extraction Kit. To detect the amount of plasma membrane-localized GLUT2, the plasma membrane was isolated from HepG2 cell lysates according to the protocol of Nishiumi et al. [12]. The assays were performed using standard methods [13,14], and the membrane was incubated overnight at 4°C with the following primary antibodies: phospho-(Thr 172 )-AMPKα2

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Jiang B, Le L, Pan H, Hu K, Xu L, Xiao P. Dihydromyricetin ameliorates the oxidative stress