K612 100

Venous blood samples were collected immediately after exercise, and blood analysis was performed using whole blood and serum. To collect the serum, the venous blood samples were allowed to clot at 24–25 °C for 30 min, centrifuged at 2000× g for 15 min at 4 °C, and transferred to new tubes before being stored at −80 °C. Whole blood was used to measure the concentration of blood glucose (ACCU CHEK Performa Glucometer, Roche, Diagnostics, Penzberg, Germany), lactate (Lactate Pro2, LT-1730, ARKRAY, Kyoto, Japan), and TG (Standard LipidoCare Strips–Lipid Profile, 02LA10G, SD Biosensor, Suwon, Korea). Serum was used in ELISA kits for the analysis of the concentration of glycerol (EGLY-200, BioAssay System, Hayward, CA, USA) and FFAs (K612-100, BioVision, Milpitas, CA, USA).

At the end of the experiment (20 weeks old), blood samples from eight mice per group were collected from the facial vein and separated after centrifugation at 1500 g for 10 min. The concentrations of glycerol, free fatty acid and triglyceride in the serum were measured using commercial assay kits (10011725, Cayman Chemical, Ann Arbor, MI, USA; K612-100, Biovision, Milpitas, CA, USA; K622-100, Biovision, Milpitas, CA, USA) according to the manufacturers’ instructions. The concentration of glycerol in the culture medium was measured using commercial assay kits (10011725, Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturers’ instructions.

Cells were seeded at a density of 30,000 cells/well in 100 μL of pre-adipocyte medium in a 96-well plate and incubated for 48 hours in a humidified incubator at 37°C and 5% CO₂. On day 3, differentiation medium containing IBMX plus dexamethasone plus 0.1 μM of rosiglitazone plus insulin was added to the 96-well plate and incubated for 3 days in a humidified incubator at 37°C and 5% CO₂. On day 6, the medium was replaced with adipocyte maintenance medium without dexamethasone and incubated for an additional 3 days in a humidified incubator at 37°C and 5% CO₂. On day 10, minimum essential medium alpha starvation was performed overnight with 1 nM of human insulin (Cat No: I9278, Sigma-Aldrich). Cells were treated with increasing concentrations of the MYL-1501D and reference insulin glargine preparations in Krebs-Ringer bicarbonate-pyruvate medium (Cat No: K612-100, BioVision) containing 1% BSA for 1 hour, followed by stimulation with 3 nM of isoproterenol (Cat No: I5627, Sigma-Aldrich) for 2 hours. Supernatant was collected, a free fatty acid assay was performed (Cat No: K612-100, BioVision), and the plate was read for absorbance at 570 nm. Inhibition of stimulated lipolysis was calculated using the ratio of half-maximal effective concentration values from the test samples vs the working standards in murine 3T3-L1 cells.

Quantification of alanine and pyruvate (K652-100, BioVision), free fatty acids (K612-100, BioVision), glucose and glycogen (K646-100, BioVision), triglyceride (K622-100, BioVision) and branched-chain amino acids (BCAA, K564-100, BioVision) were measured by colorimetric assays using NanoDrop 2000c according to manufacturer’s instructions.

Glucose, total protein, albumin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), alkaline phosphatase (Alk-P), total cholesterol, triglyceride, blood urea nitrogen (BUN), creatinine, uric acid, high-density lipoprotein (HDL), and low-density lipoprotein (LDL) in the serum were quantified using an automatic biochemical analyzer (SIEMENS, Erlangen, Germany). Free fatty acids (BioVision, K612-100, San Francisco, CA, United States) in the serum were quantified using a colorimetric kit and enzyme-linked immunosorbent assay (ELISA) methods.

Three weeks after geniposide treatment, blood was collected from the retroorbital plexus of the mice after 6 h of fasting. Fasting insulin levels were examined by an ELISA kit (Millipore, Billerica, MA, USA). Serum triacylglycerol (TG), glycerol, and nonesterified fatty acid (NEFA) contents were examined using a TG assay kit (E4506, BioVision, California, USA), a free glycerol colorimetric assay kit (K634, BioVision), and a NEFA assay kit (K612-100, BioVision), respectively.

Plasma free fatty acids were analyzed using a kit from BioVision (Catalog# K612-100), and plasma ketone bodies were determined using the procedure as described by Kientsch-Engel and Siess [48 ]. Plasma insulin was determined using an ELISA kit (Mercodia Inc.; USA).