D u 14c glucose

Transport activity for [³H]riboflavin across the placenta of pregnant Slc52a3+/− mice, which were mated with Slc52a3+/− male mice, at gestation date 16.5 was determined as previously reported36 (link). Pregnant dams were anesthetized with an intraperitoneal administration of sodium pentobarbital. Surgery was performed on each animal lying on a heating pad to maintain constant body temperature. Then, radioisotope-labeled riboflavin (500 nM of [³H]riboflavin, 0.903 TBq/mmol, Moravek Biochemicals) or glucose (40 μM of D-[U-¹⁴C]glucose, 11.5 GBq/mmol, GE Healthcare) was administrated as a bolus via the femoral vein (10 mL/kg). Five minutes after administration, fetuses and placentas were removed and weighted. Fetal tails were collected, and used for genotyping as described above. Fetuses were homogenized in saline, and the samples were placed into Ultima Gold scintillation cocktail (PerkinElmer) followed by solubilization in SOLVABLE (Packard Instrument Co.). The radioactivity was measured by liquid scintillation counting, and the levels of [³H]riboflavin and D-[U-¹⁴C]glucose in each fetus were determined and their transporter activities were calculated.

Fatty acid‐free bovine serum albumin (BSA) and palmitic acid were obtained from Sigma (St. Louis, MO, USA). Glycogen, amyloglucosidase, hexokinase, and glucose‐6‐phosphate dehydrogenase were obtained from Sigma (St. Louis, MO). [1‐¹⁴C] palmitic acid was from American Radiolabeled Chemicals (St. Louis) and D‐[U‐¹⁴C] glucose was from GE Healthcare (Little Chalfont, UK). Protease (cOmplete Ultra Tablets) and phosphatase (PhosStop) inhibitors were from Roche Diagnostics GmbH (Mannheim, Germany). The nonesterified fatty acids (NEFAs) kit was from Wako (Mountain View, CA). Glucose was measured by the glucose oxidase method using a OneTouch Ultra Mini Monitor. The rat glucagon and insulin ELISA kits were from R&D Systems (Minneapolis, MN) and Alpco (Salem, NH), respectively. All antibodies were purchased from Cell Signaling (Danvers, MA), except for P‐ACC and PGC‐1α which were purchased from Millipore (Billerica, MA), and the PEPCK antibody that was purchased from Abcam (Cambridge, MA).

Fatty acid-free bovine serum albumin (FA-free BSA), palmitic acid, triethanolamine hydrochloride (TRA), amyloglucosidase and hexokinase/glucose-6-phosphate dehydrogenase were obtained from Sigma (St. Louis, MO, USA). [1- ¹⁴C] palmitic acid was from Perkin Elmer (Woodbridge, ON, Canada). D-[U- ¹⁴C] glucose was from GE Healthcare (Mississauga, ON, Canada). Protease (cOmplete Ultra Tablets) and phosphatase (PhosSTOP) inhibitors were from Roche Diagnostics GmbH (Mannheim, Germany). Glucose was measured by the glucose oxidase method using a OneTouch Ultra Mini Monitor. The NEFA kit was from Wako (Mountain View, CA, USA) and the rat insulin ELISA kit was from Alpco (Salem, NH, USA). All antibodies were purchased from Cell Signaling (Danvers, MA, USA) except for SLN which was purchased from Millipore (Billerica, MA, USA).