Gy105

The liver tissue (0.1 g) were ground in 1 mL of ice-cold Folch solution (chloroform/methanol = 2:1; v/v) and incubated for 30 min at room temperature. The aqueous layer was aspirated and discarded, and the fixed volume of the organic layer was then evaporated to dryness. The dried lipid layer was dissolved with an equal volume of DMSO and then used to determine the TC (BXC0261, Fortress Diagnostics Ltd., Antrim, UK), TG (BXC0271, Fortress Diagnostics Ltd., Antrim, UK), and glycerol (GY105, Randox Laboratories Ltd., Antrim, UK) levels using commercial assay kit. The cholesterol, triglyceride, and glycerol were used as the standards for the standard curve of TC and TG, and glycerol analysis, respectively.

Lipolysis activity of 3T3-L1 cells was determined by measuring the amount of glycerol released into the incubation medium (GY105; Randox, England). The cells were then disrupted by adding 1% Triton X-100 in PBS and the triglyceride content was measured using a TR212 kit (Randox).

Lipolysis rate measurement was determined as previously described [44 (link)]. The 0.2 g of adipose samples were minced and placed into 2 mL of N-tris-(hydroxymethyl)methyl-2-aminoethanesulfonic acid (25 mM) buffer containing isoproterenol (1 μM), and then incubated at 37 °C. After 1, 2, and 3 h of incubation, 0.2 mL of medium was used to analyze the concentrations of glycerol using a commercial reagent (RANDOX GY105, Amtrim, UK), and then the absorbance at 520 nm was recorded by a Hitachi U2800A spectrophotometer. The lipolysis rate was indicated by micromoles of glycerol released per gram of tissue per hour.

Blood samples were taken three times from the antecubital vein into the vacutainer tubes with a silica clot activator before, 15 min, and one hour after the tests. Additionally, in the WAnT group, the blood samples were taken 3 min after the first and second bout for lactate measurement. The samples were centrifuged at 2000g for 10 min at 4 °C. The separated serum and plasma samples were frozen and kept at − 80 °C until later analysis. The tubes containing the samples were number-coded to blind the laboratory personnel regarding the treatment group and the sequence of sample collection.
Serum 25(OH)D₃ concentration was determined by enzyme immunoassay method using 25-OH Vitamin D total ELISA kit (DE1971, Demeditec Diagnostics, Germany), according to the manufacturer’s instructions.
Plasma interleukin 6 (IL-6) concentration was determined by ELISA using a commercial kit (HS600, R&D Systems, USA).
Plasma parathyroid hormone (PTH) was determined using PTH intact ELISA kit (DE 3645, Demeditec Diagnostics, Germany).
Plasma non-esterified fatty acids and glycerol concentrations were determined by direct colorimetric methods using NEFA assay (FA115, Randox, United Kingdom) and Glycerol assay (GY105, Randox, United Kingdom).