Pgo enzyme

Mice were anesthetized with 2% isoflurane in 95% oxygen/5%carbondioxide air gas. A midline incision to the peritoneal cavity was made, and the major abdominal blood vessels were exposed. Two tied ligatures were placed around the celiac artery and mesenteric artery. One ligature was placed around inferior vena cava at the position right below right renal vein junction, into which heparin (100 units per mouse) was injected. After that, a ligature was placed at the proximal end of the aorta right below the mesenteric artery junction. An open ligature was placed around the aorta right above the celiac artery and tied off after an 18 G blunt end needle was installed for buffer infusion. The perfusion pressure was maintained below 80 mm of mercury at the rate 0.8-1 ml per minute. The buffer temperature was adjusted around 37 °C by an in-line heater (Warner Instrument). Basic composition of the buffer is as follows (mM). NaCl, 129; Hepes, 20; KCl, 4.8; Bicarbonate, 5; CaCl₂, 2.4; MgSO₄, 1; KH₂PO₄, 1; no glucose; pyruvate, 40; BSA, 0.65%; Dextran, 3.6%. The perfusates were collected at 10-min intervals for 100 min. Glucose levels in perfusates were measured by colorimetric assays with PGO enzymes (Sigma #P7119).

Cells were seeded in full media in a 6-well plate (8 × 10⁴ cells in 2 ml) in triplicates. Media was replaced after 24 hours. After 48 hours the media was collected for analysis of L-lactate and glucose and cells were counted for normalization. The concentration of L-lactate was determined using an enzymatic reaction based on the oxidation of L-lactate to pyruvate as previously described^{31 (link)}. The concentration of glucose was determined using a glucose oxidase and peroxidase method, PGO Enzymes (Sigma Aldrich) according to manufacturer’s instructions. Additional information in Supplementary materials and methods.

Cells were seeded in full media in a 6-well plate (8 × 10⁴ cells in 2 mL) in triplicates. Media was replaced after 24 h. After 48 h the media was collected for analysis of L-lactate and glucose and cells were counted for normalization. The concentration of L-lactate was determined using an enzymatic reaction based on the oxidation of L-lactate to pyruvate as previously described [29 (link)]. The concentration of glucose was determined using a glucose oxidase and peroxidase method, PGO Enzymes (Sigma-Aldrich, St. Louis, MI, USA), according to manufacturer’s instructions. Additional information is in Supplementary Materials and Methods.

Tail blood was collected with a Microvette CB300Z (SARSTEDT #16.440.100) for serum and Microvette CB300 K2E (SARSTEDT #16.444.100) was used for plasma. Glucose was measured with a glucose meter or colorimetric assays with PGO enzymes (Sigma #P7119) plus o-dianisidine (Sigma #F5803). Insulin was measured with an ELISA kit (Crystal Chem #90080).

Nicotinamide, o-dianisidine, PGO enzyme and streptozotocin (STZ) were purchased from Sigma Chemical Co. (St Louis, MO, USA); Glibenclamide tablets (Daonil) were purchased from Aventis Pharma Ltd. (Bangkok, Thailand). All other chemicals and reagents were of analytical grade.