Digl 100

The fasting glucose and insulin levels were assessed using commercially available kits formulated specifically for rats (#90010, Crystal Chem, Elk Grove Village, IL, USA and #DIGL-100, BioAssay Systems, Hayward, CA, USA) following each supplier’s instructions. In addition, we calculated the values from the homeostatic model assessment of β-cell function (HOMA-β) for each animal using the previously published equation [30 (link)]: HOMA-β = (Fasting insulin (ng/mL) × 20)/(Fasting glucose (mg/dL) − 3.5). Levels of total cholesterol (CHOL) and triglyceride (TG) serum levels were determined using multi-enzyme-based kits (#MBS726298, MyBioSource, San Diego, CA, USA, and #ECCH-100, BioAssay Systems, Hayward, CA, USA, respectively). Serum and hepatic levels of high-/low-density lipoprotein cholesterol (LDL-c/HDL-c were assayed using a special colorimetric kit (#E2HL-100 BioAssay Systems, CA, USA). Serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase were analyzed using assay kits (#EALT-100 and EASTR-100, BioAssay Systems, CA, USA). All procedures were performed according to the manufacturer’s instructions for each kit (n = 8 samples/group).

Intracellular glucose uptake levels were determined using a glucose assays kit (#DIGL-100, BioAssay Systems, CA), according to the manufacturer's protocol. Briefly, HepG-2 cells (1 × 10⁵) were cultured on a 60 mm culture dish and incubated at 37°C for 24 h, and then the cells were treated with or without GRN A (5 μM) in 1% serum DMEM. After incubation for 0, 6, 12, 24, 48 and 72 h, the cells were harvested and washed with PBS, and then the cells were lysed with RIPA buffer and collected by centrifugation (10000 × g for 20 min). Total protein concentration in the supernatant was determined using BCA protein assay kit (#P0012s, Beyotime, China). To determine the levels of glucose uptake, the culture media (5 μL) were added into 500 μL working solution. After heating for 8 min, the sample was cooled down in cold water bath for 4 min, the mixture samples (200 μL) were transferred into a 96-well plate and the glucose uptake was determined by measuring the absorbance at 630 nm with a micro plate reader (Biotech, power wave, USA).

After the cells were treated for 24 h, 50 μL of the supernatant of the cells to be tested was taken, and the glucose content was determined using a glucose kit (Bioassay Systems DIGL-100). The results were expressed as a percentage of the glucose content of the model group.