Animal studies-weight measurements and blood parameters
For the western diet induced obesity and insulin resistance phenotypes, C57BL/6J mice at 8 weeks of age were placed on either a standard laboratory rodent chow or western diet (D12079B, Research Diets) and allowed to eat ad libitum and their weights measured as indicated. Mouse blood serum was collected and measured for glucose, cholesterol and liver function tests using chemical analyzers and insulin and leptin using ELISA at the indicated times.
Glucose tolerance test
Mice were fasted for 6 hours, and glucose (2g/kg) was intraperitoneally injected into the mice and blood collected from the tail vein and glucose concentrations were determined at the 30minutes, 1hr and 2hrs.
Tissue collection and western blotting
Epididymal white adipose tissue, liver, leg and arm muscles were collected from mice at the end of each time point (as indicated) and flash frozen. They were homogenized in SDS lysis buffer (50mM Tris-HCL pH 7.4, 10% Glycerol, 2% SDS) and boiled at 95 C for five minutes. Lysates were then briefly sonicated, boiled again for 5 minutes, before clearing by centrifugation at 14,000rpm for 10 minutes at room temperature. The supernatant was collected, and protein concentration was determined using the BCA kit (Pierce) per manufacturer’s instructions. Protein samples were diluted in SDS sample buffer (final concentration: 62.5mM TrisHCL pH 6.8, 2% SDS, 10% Glycerol, 15.5mg/mL DTT, 0.02mg/mL Bromophenol blue). 25–50 mg of protein was loaded onto each lane of a 4–12% BisTris mini gel or midi gel (Invitrogen) for immunoblotting. Transfer was onto nitrocellulose membranes (0.2 mm, GE Health Care) before blocking for 1h at room temperature and incubating with primary antibodies of the indicated protein targets overnight at 4 C. Membranes were incubated with secondary rabbit antibody (Sigma) or secondary mouse antibody (GE Health Care) for 1h at room temperature. Blots were developed in Perkin-Elmer’s Western Lightning ECL or Millipore’s Immobilon HRP reagents per manufacturer’s instructions.
Cell lines and Antibodies and drugs
BT474, MCF7, MDA-MB-468 cells were acquired from ATCC and cultured in DMEM-F12 and 3T3L-1 was cultured in DMEM. All cell lines were supplemented with 10% Fetal Bovine serum (FBS) and 1% penicillin and streptomycin and 4mM Glutamine.
Antibodies used are PTEN (CST #9559), pAKT T308 (CST #2965), pAKT S473 (CST #4060), pPRAS40 (CST #2997), p4E-BP1 T37/46 (CST#2855), p4E-BPs S65 (CST #9451), IRS1 (CST# 2382), IR-ß (CST #3025), ß-Actin (CST #4970)
Histological analysis
Representative sections of the liver, pancreas, brain, epididymal adipose tissue, retroperitoneal white adipose tissue, skeletal muscle from forelimbs, and skeletal muscle from the hindlimbs were fixed in 10% neutral-buffered formalin, processed in alcohol and xylene, embedded in paraffin, sectioned (5-µm-thick) and stained with hematoxylin and eosin. Oil red O staining was performed on formalin fixed, OCT-embedded frozen sections (5-µm-thick) of liver. For histopathological analysis, hematoxylin–eosin-stained or ORO-stained tissue specimens were evaluated by a board-certified veterinary pathologist (S.E.C.). Liver sections were evaluated and scored, using a semiquantitative histopathology scoring system, with slight modifications, for mouse model of metabolic dysfunction associated fatty liver disease56. Briefly, macrovesicular steatosis, microvesicular steatosis and hepatocellular hypertrophy were separately scored, and the extent and severity of the lesions were graded, into the following categories: 0 (< 5%), 1 (5–10%), 2 (10–25%), 3 (25–75%) and 4 (> 75%). Inflammation was evaluated by counting the number of inflammatory foci per five 100x fields using the following categories: normal (< 0.5 foci), minimal (0.5-1.0 foci), mild (1.0–2.0 foci), moderate (2.0–5.0 foci), severe (> 5.0 foci). An Olympus BX45 light microscope was used to capture images with a DP26 camera using cellSens. Dimension software (v1.16).
Immunohistochemistry
Immunolabeling of PTEN in liver and epididymal white adipose sections was performed at the MSK Biobank and Pathology Core facility. Formalin-fixed, paraffin-embedded sections were stained using an automated staining platform. Briefly, following deparaffinization and heat-induced epitope retrieval, the primary antibody against PTEN (1:200, Cat. No 9559, clone 138G6, Cell Signaling Technologies) 57.
Morphometric analysis of eWAT
Cell size distribution in hematoxylin-eosin (H&E)-stained sections of epididymal white adipose tissue was analyzed from triplicates of 40X images per group and cell size was quantified using Adiposoft Software (Image J)58
Treatment with PTEN inhibitor VO-OHpic
VO-OHpic was suspended in 2% DMSO, 40% PEG 300, 5% Tween-80, ddH2O and administered intraperitonially at a dose of 10mg/kg, every day, once a day. This was done either at the same time as the start of the western diet in mice for 6 weeks or after 2 weeks of western diet feeding for 4 weeks
Treatment with mTORC1 inhibitor RMC-6272 or Rapamycin
RMC-6272 was suspended in 1:1 (v/w) Transcutol/Solutol HS 15 and administered intraperitonially at a dose of 3mg/kg, once a week. This was done either at the same time as the start of the western diet in mice for 6 weeks or after 2 weeks of western diet feeding for 4 weeks. Rapamycin was dissolved in 100% DMSO and administered intraperitonially at a dose of 10mg/kg, three times a week along with western diet.