Chemicals and reagents

FA-free bovine serum albumin (BSA), insulin (bovine), bicinchoninic acid (BCA), protein assay kit, and secondary antibodies were purchased from Yeasen Biotechnology (Shanghai, China). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Compound C, 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) and STO-609 were purchased from Sellbeck chemicals (Houston, TX, USA). Mem-PER Plus Membrane Protein Extraction Kit and 2-NBDG were purchased from Thermo Fisher Scientific (Sunnyvale, CA, USA). Fetal bovine serum (FBS) was purchased from Bovogen Biologicals (East Keilor, VIC, Australia), and other cell culture materials, including Dulbecco’s modified eagle’s medium (DMEM), horse serum, antibiotic, antimycotic and trypsin solutions were obtained from Gibco (Gaithersburg, MD, USA). Antibodies against AMPKα, phospho-AMPKα (Thr172), acetyl-CoA carboxylase (ACC), phosphorACC (Ser79), phospho-AS160 (Ser588), phospho-p38 MAPK (Thr180/Tyr182), phosphoAKT (Ser473) and phospho-AKT (Thr308), and protein kinase C-θ (PKCθ) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibody against phospho-IRS1 (Tyr632) was obtained from Abcam (Cambridge, UK); and antibodies against AKT, GLUT4, Na+-K+−ATPase, p38 MAPK and β-actin were obtained from Proteintech (Wuhan, China).

Plant material and preparation of the water extract of P. emblica fruits

P. emblica fruits were supplied by the Yimin Agricultural Products Development Co., Ltd (Chuxiong, Yunnan, China), and authenticated by Professor Hong-qing Li, School of Life Science, East China Normal University. A voucher specimen (No.: FPE. Yunnan. 2020) was deposited in the herbarium of East China Normal University (Shanghai, China). The dry powder of P. emblica fruit was extracted with distilled water (solid-liquid ratio 1:10) on a rotary shaker for 24 h under 28–29 ℃, then, was filtered through Whatman qualitative filter paper no. 1 (Sigma-Aldrich). After filtration, the filtrate was concentrated in a vacuum rotary evaporator and freeze-dried to gain the extract (WEPE). The extraction process resulted in a yield of 33.8%, and the extract was stored at drying oven before use.

The stock solution of WEPE (100 mg/mL) was prepared by dissolving the extract in DMSO and filtering it through a sterile syringe filter with a pore diameter of 0.22 μm. Prior to treatment, the stock solution was further diluted to obtain various testing concentrations.

Metabolite profiling and quantitative analysis of phenolic compounds

The metabolic profiling of the WEPE was analyzed using an ACQUITY UHPLC system (Waters Corporation Milford, USA) coupled with an AB SCIEX Triple TOF 5600 System (AB SCIEX, Framingham, MA). Both ESI positive and ESI negative ion modes were employed, with separation achieved on an ACQUITY UPLC BEH C 18 column (100 mm×2.1 mm, 1.7 μm). The binary gradient elution system consisted of (A) water (containing 0.1% formic acid, v/v), and (B) acetonitrile (containing 0.1% formic acid, v/v) and separation was achieved using the following gradient: 0 min, 95% A + 5% B; 2 min, 80% A + 20% B; 4 min, 75% A + 25% B; 9 min, 40% A + 60% B; 14 min, 100% B; 16 min, 100% B; 16.1 min, 95% A + 5% B; 18.1 min, 95% A + 5% B. The flow rate was 0.4 mL/min and column temperature was 45 °C. The injection volume was 5 µL.

Data acquisition was performed in full scan mode (m/z range from 70 to 1000) combined with IDA mode. Parameters of mass spectrometry were as follows: Ion source temperature, 550 °C (+) and 550 °C (−); ion spray voltage, 5500 V (+) and 4500 V (−); curtain gas of 35 PSI; declustering potential, 80 V (+) and − 80 V (−); collision energy, 10 eV (+) and − 10 eV (−); and interface heater temperature, 550 °C (+) and 550 °C (−). For IDA analysis, range of m/z was set as 50-1000, the collision energy was 30 eV. The raw data collected using UNIFI 1.8.1 software underwent preprocessing tasks such as baseline filtering, peak identification, and normalization using Progenesis QI v2.3. Post-processing, compounds were identified using their accurate mass number, secondary fragments, and isotope distribution with databases like HMDB, Lipidmaps, and METLIN for qualitative analysis.

In addition to the LC-MS analysis, phenolic compounds in the extract were quantified using UPLC-ESI-MS/MS. The data gathered from UPLC-ESI-MS/MS was processed using SCIEX OS-MQ Solutions software (Sciex, USA). The regression equations for the quantification are as follows:

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All LC-MS, UPLC-ESI-MS/MS analyses and data processing were performed by Shanghai Luming Biotechnology Co., Ltd.

Cell culture and differentiation

The C2C12 mouse myoblasts were obtained from The National Center for Drug Screening (Shanghai, China). C2C12 mouse myoblasts were cultured in DMEM, supplemented with 10% (v/v) FBS, 100 U/mL streptomycin, and 100 U/mL penicillin, and maintained at 37 °C with 5% CO2. The cells were seeded into cell culture plates at a density of 5 × 104 cells/mL. After reaching approximately 70% confluence (about 24 h), the medium was replaced to DMEM supplemented with 2% (v/v) horse serum and was refreshed after 2, 4 and 6 days of culture. After 6–7 days, the differentiation of C2C12 mouse myoblasts into myotubes was complete, at which point the experiments were initiated.

Palmitate solution preparation

Palmitate was dissolved in 100% ethanol before being diluted in DMEM containing 2% fatty acid-free BSA. The control group was treated with an equivalent volume of ethanol added to the BSA-DMEM solution. All solutions were filtered, aliquoted, and stored at 4 °C for future use.

MTT assay

We utilized an MTT assay to evaluate the effect of WEPE on cell viability. C2C12 myoblasts were cultured in 96-well plates and differentiated into myotubes. These myotubes were incubated in DMEM with 0.2% BSA for 6 h, followed by treatment with varying WEPE concentrations. After incubation, 20 µL of 3 mg/mL MTT was added to each well and treated for 2.5 h at 37 °C. The MTT formazan crystals were dissolved by adding 200 µL of dimethyl sulfoxide to each well and shaking until dissolution. Then, the absorbance of each well was measured at 490 nm using a microplate spectrophotometer and cell viability was calculated using the following formula:

$$\:Cell\:Viability=\frac_}_}\times\:100\%$$

Glucose consumption assay

Glucose consumption was determined using a glucose oxidase assay kit. Briefly, C2C12 myotubes were incubated in DMEM containing 0.2% BSA for 6 h, followed by treatment with different doses of WEPE for 12, 24, and 48 h. Glucose concentrations in the medium were measured post-treatment using a glucose oxidase assay kit according to the manufacturer’s protocol. Glucose consumption was then calculated by subtracting the post-treatment glucose concentration from the initial concentration in the culture medium.

2-NBDG uptake assay

Cell glucose uptake was assessed by measuring the uptake of 2-NBDG. Myotubes were cultured in black 96-well plates and subjected to various concentrations of WEPE for specified durations. An hour prior to cell harvest, the myotubes were rinsed with warm sterile PBS (37 °C) and incubated in glucose-free DMEM supplemented with 0.2% BSA. After an hour, the myotubes were washed again with warm sterile PBS and exposed to a medium containing 80 µM 2-NBDG for 30 min. Following another wash with warm sterile PBS, the fluorescence intensity of each well was measured at an excitation wavelength of 485 nm and an emission wavelength of 520 nm. The formula for calculating cell glucose uptake is as follows:

$$\:\text\text\text\text\text\text\text\:\text\text\text\text\text\text=\frac\text}_\text\text\text\text\text}-\text}_\text\text\text\text}}\text}_\text\text\text\text\text\text}-\text}_\text\text\text\text}}$$

Transfection with small-interfering RNA (siRNA)

C2C12 myotubes were transfected with AMPKα1 siRNA and Negative Control siRNA (60 nM; Shanghai GenePharma) using LipofectamineTM 3000 Reagent (Invitrogen) in DMEM medium, following the manufacturer’s protocol. After 48 h of transfection, the efficiency was evaluated by performing western blotting against the AMPK antibody.

AMPKα1 siRNA:

sense (5’-3’)UUUGAAAGACCAAAGUCGGCU.

antisense (5’-3’)CCGACUUUGGUCUUUCAAACA.

Negative Control siRNA:

sense (5’-3’)UUCUCCGAACGUGUCACGUTT.

antisense (5’-3’)ACGUGACACGUUCGGAGAATT.

Western blotting

Following treatment, cells were rinsed with ice-cold PBS and collected in a lysis buffer (containing 150 mM sodium chloride, 1.0% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), and 50 mmol/L Tris; pH 8.0) supplemented with protease and phosphatase inhibitors. Protein concentrations were quantified using a BCA Protein Assay Kit. Proteins were then separated on 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred onto nitrocellulose membranes. The membranes were blocked with a 5% non-fat dry milk solution for 1 h at room temperature, followed by overnight incubation at 4 °C with primary antibodies. After washing three times with Tris-buffered saline containing 0.1% Tween 20, the membranes were incubated with secondary antibodies for an hour at room temperature. Finally, the blots were washed and visualized using an Odyssey CLx Imaging System, and the results were analyzed with Image-Pro Plus Software.

Statistical analysis

Results are presented as the mean ± standard deviation (SD). Statistically significant differences among experimental groups were determined by one-way analysis of variance followed by Dunnett’s multiple-comparisons tests using SPSS software (IBM, Armonk, New York, USA). P value < 0.05 was considered statistically significant, while P value < 0.01 was deemed extremely significant between groups.