2-Deoxyglucose

Clamps were performed according to recent recommendations of the Mouse Metabolic Phenotyping Center Consortium (15 (link)). After surgical implantation of an indwelling catheter in the right jugular vein, the mice were allowed to recover for 1 week prior to clamp experiments. Following an overnight 14-h fast, the mice were infused with 3-[³H]glucose at a rate of 0.05 μCi/min for 120 min to determine basal glucose turnover. Next, a primed infusion of insulin and 3-[³H]glucose was administered at 7.14 milliunits·kg⁻¹·min⁻¹ and 0.24 μCi/min, respectively, for 4 min, after which the rates were reduced to 3 milliunits·kg⁻¹·min⁻¹ insulin and 0.1 μCi/min 3-[³H]glucose for the remainder of the experiment. Blood was collected via tail massage for plasma glucose, insulin, and tracer levels at set time points during the 140-min infusion, and a variable infusion of 20% dextrose was given to maintain euglycemia. Glucose turnover was calculated as the ratio of the 3-[³H]glucose infusion rate to the specific activity of plasma glucose at the end of the basal infusion and during the last 40 min of the hyperinsulinemic-euglycemic clamp study. Hepatic glucose production represents the difference between the glucose infusion rate and the rate of glucose appearance. A 10-μCi bolus injection of [¹⁴C]2-deoxyglucose was given at 90 min to determine tissue-specific glucose uptake, which was calculated from the area under the curve of [¹⁴C]2-deoxyglucose detected in plasma and the tissue content of [¹⁴C]2-deoxyglucose-6-phosphate, as previously described (16 (link)). Following collection of the final blood sample, the mice were anesthetized with an intravenous injection of 150 mg/kg pentobarbital, and tissues were harvested and froze with aluminum forceps in liquid nitrogen. All of the tissues were stored at −80 °C until later use.

After fasting for 6–12 h, the subjects were kept in an air-conditioned room at 19°C with light clothing (usually a T-shirt with underwear) and put their legs on an ice block intermittently (usually for 4 min every 5 min). After 1 h under this cold condition, they were given an intravenous injection of ¹⁸F-fluoro-2-deoxyglucose (FDG) (259 MBq) and kept under the same cold condition. In some cases, the subjects were kept at 26–28°C with standard clothing and without leg icing (warm condition). One hour after the ¹⁸F-FDG injection, whole-body PET/CT scans were performed on a PET/CT system (Aquiduo; Toshiba Medical Systems, Otawara, Tochigi, Japan) in a room at 24°C. With the CT parameters of 120 kv and real-exposure control, unenhanced low-dose spiral axial 2-mm collimated images were obtained. This was used for PET attenuation correction as well as anatomic localization. Subsequently, full-ring PET was performed in six incremental table positions, each ∼15 cm in thickness. The total time of these scans was ∼30 min.
PET and CT images were coregistered and analyzed by a VOX-BASE workstation (J-MAC System, Sapporo, Japan). Two experienced blinded observers assessed the FDG uptake, particularly in both sides of the neck and paravertebral regions, by visually judging the radioactivity greater than background. In parallel, the FDG uptake in the neck region was quantified and expressed as relative to that in the whole brain.

Seahorse XF Cell Mito Stress Test kit (Agilent Technologies, Inc.) was used to determine the O₂ consumption rate (OCR) and Seahorse XF Glycolysis Stress Test kit was used to examine the extracellular acidification rate (ECAR), as previously described (19 (link)). The transfected SAOS-2 cells (1x10⁵) were plated onto a Seahorse XF-96 cell culture microplate. Cells were next equilibrated with XF Base media (Agilent Technologies Deutschland GmbH) at 37˚C for 1 h in an incubator lacking CO₂ and then serum-starved for 1 h in glucose-free media-containing treatments (Invitrogen; Thermo Fisher Scientific, Inc.). A total of 1 mM oligomycin, 1 mM p-trifluoromethoxy carbonyl cyanide phenylhydrazone (MilliporeSigma), 2 mM antimycin A (MilliporeSigma) and 2 mM rotenone (MilliporeSigma) were added to each well at 37˚C overnight to detect the OCR. For the measurement of ECAR, each well contained 10 mM glucose, 1 mM oligomycin (MilliporeSigma) and 80 mM 2-deoxyglucose (MilliporeSigma) at 37˚C overnight. A Seahorse XF-96 analyzer (Agilent Technologies, Inc.) was used to detect the samples and data were assessed using Seahorse XFe24 Wave version 2.2 software (Agilent Technologies, Inc.).

We used a cell-based fluorescently-labeled deoxyglucose analog kit (2-NBDG kit, Cayman Chemical, USA) to evaluate whether compounds 1–11 increased glucose uptake in L6 cells.^49,50 Before the experiment, the differentiated L6 myotube was inoculated into a 96-well plate at a density of 1 × 10⁴–4 × 10⁴ cells/well. After 2 h of starvation in serum-free α-MEM medium, we add 100 μL MEM-α medium, which contained different drugs, to each well, and cells were incubated for 12 h to 100% fusion. After overnight incubation, cells were treated with insulin (100 nM), compounds 1–11 (30 μg mL⁻¹) or normal control (0.1% DMSO) in 100 μL glucose-free α-MEM containing 150 μg mL⁻¹ 2-NBDG. At the end of the treatment, plates were centrifuged for five minutes at 400 g at room temperature. The supernatant was aspirated, and 200 μL of cell-based assay buffer was added to each well. Plates were centrifuged for five minutes at 400 g at room temperature. Then the supernatant was aspirated, and 100 μL of cell-based assay buffer was added to each well. The 2-NBDG taken up by cells was detected with fluorescent filters (excitation/emission = 485/535 nm).