Insulin

β-elemene (95%) was obtained from Yuanda Pharmaceuticals (Dalian, China). Insulin was from Prospec (Israel). Picropodophyllin (PPP) was purchased from Sellek. NVP-AEW541 and Z-VAD were purchased from MedChem Express (Monmouth Junction, NJ, USA).

Primary calvarial OB cultures were established from new born (P4) Rptor^fl/fl mice and Rptor knockout cells generated as previously described.^{14 (link)} For insulin pulse experiments, cells were incubated under serum-free conditions for 4 h then pulsed with 100 nmol·L⁻¹ insulin (ProSpec-Tany TechnoGene Ltd, Ness-Ziona, Israel) for 10 min. RNA and protein was extracted from wild-type and Rptor KO cells, under normal growth and serum starved conditions before or after stimulation with 100 nmol·L⁻¹ insulin, as described above. qRT-PCR and western blotting were performed as described above.

For the glucose tolerance test (GTT), overnight-fasted mice were injected intraperitoneally with glucose at a dose of 2.0 g/kg. For the insulin tolerance test (ITT), 6-h-fasted mice were injected intraperitoneally with 0.75 U/kg insulin (Novolin R; Novo Nordisk AIS, Bagsvaerd, Denmark). Glucose values were converted into natural logarithm (Ln) and the slope of the decline in the blood glucose concentration from 0 to 30 min was calculated using linear regression [time × Ln (glucose)] and multiplied by 100 to obtain the constant rate for glucose disappearance (KITT) 30 (link), 31 (link). Blood samples were collected at regular intervals (0-120 min). Blood glucose was measured by a handheld glucose meter (Accu-Chek Active; Roche, Mannheim, Germany). Serum insulin was analyzed with ELISA kits (Alpco Diagnostics, NH, USA). For insulin signaling experiments, mice were fasted overnight and intraperitoneally injected with 1 U/kg insulin solution (Novolin R). After 10 min, the mice were sacrificed and gastrocnemius SkM were harvested. For the in vitro cultured model, the treated C2C12 myotubes were stimulated with 100 nM insulin (ProSpec-Tany Technogene, Ness Ziona, Israel) for 15 min before harvesting.