Sglt2

Fasted (5 hr) mice were anaesthetised with methoxyflurane at week 12, and liver and white adipose tissues were collected and fixed in 4% formaldehyde overnight before being incubated in 50% ethanol and embedded in paraffin. The tissues were then cut into 4 μm sections and stained with hematoxylin and eosin (CellCentral, UWA) before being visualized and photographed using a Nikon Eclipse TS100 microscope. Additional liver, white and brown adipose tissues, gastrointestinal tract, kidney, and skeletal muscle were collected and snap frozen in liquid nitrogen and stored at −80°C until analysis. White and brown adipose tissue protein expression of AMPK (Cell Signaling, USA), phosphorylated AMPK (Cell Signaling, USA), ACC (Cell Signaling, USA), phosphorylated ACC (Cell Signaling, USA), and UCP-1 (Cell Signaling, USA) were determined using western blot and normalised to actin (Sigma, USA), as previously described [7 (link)]. In a subset of mice (5/group), kidneys were also collected and stained with antisodium glucose cotransporter 2 (SGLT2, Santa Cruz Biotechnology) as previously described [10 (link)].

Frozen tissue was homogenized with Quiagen Tissue Ruptur in 1.5 ml of cold 20 mM HEPES buffer, pH 7.2, containing 1 mM EGTA, 210 mM mannitol, 70 mM sucrose and centrifuged at 1.500×g for 5 min at 4°C. Samples were then analysed by SDS gel electrophoresis (Novex, Life technologies, Australia) and electroblotted to Hybond Nitrocellulose membranes (Amersham Pharmacia Biotech, Bucks, UK). Membranes were then probed with primary antibodies to GLUT1 (ab652, Abcam, Cambridge, UK), SGLT2 (sc98975, Santa Cruz, USA) and actin (Santa Cruz, USA). Proteins were visualized using Luminata Western HRP Substrate (Millipore) in a LAS 4000 image reader (GE Healthcare Life Sciences). Analysis was performed using Image J software (NIH, USA).

Four μm kidney sections were heated at 65 °C for 1 hr and deparaffinized in xylene, followed by rehydration in decreasing concentrations of ethanol (two washes in 100% ethanol, two washes in 95%, one wash in 70%, one wash in 50%, and three wash in TBS). Antigen retrieval was performed for 30 min in citrate buffer (Sigma-Aldrich, St. Louis, MO, C9999, 1:10). Sections were incubated with 3% hydrogen peroxidase (Sigma-Aldrich, St. Louis, MO, H1009, 1:10) for 20 min and incubated with a blocking reagent (Vector Laboratories, Newark, CA, SP-5035) for 1 hr at room temperature. Sections were then incubated with primary antibody SGLT2 (Santa Cruz Biotechnology, Dallas, TX, sc-393350, 1:100) overnight at 4 °C. Incubation with biotin-labeled secondary antibody (Vector Laboratories, Newark, CA, BA-2000, 1:200) was performed at room temperature for 1 hr, followed by incubation with avidin-biotin-peroxidase complex (Vector Laboratories, Newark, CA, PK-6100) and DAB substrate kit (Vector Laboratories, Newark, CA, SK-4100). Counterstain was performed with hematoxylin for 5 min, followed by dehydration in increasing concentrations of ethanol. Sections were examined under a light microscope (Olympus BX41, Tokyo, Japan).