Figure 5.

DRAM1 Promotes Insulin Resistance through mTOR Activation

(A) Dram1^flox/flox MEFs expressing Cre recombinase (−/−) or a control vector (fl/fl) were grown overnight in serum-depleted DMEM prior to treatment with 5 nM insulin for 15 min. Western blots for phospho-AKT T308, phospho-S6 kinase, phospho-S6 ribosomal protein, and insulin receptor substrate 1 (IRS1) were performed to evaluate activation of the insulin pathway. ERK2 was used as a loading control.

(B) Similar experiments to those in (A) were performed to evaluate the kinetics of insulin pathway activation following different insulin exposure times.

(C) Cells were treated as in (A) except that, prior to insulin treatment, cells were incubated with or without 100 nM rapamycin for 4 h. Western blots for phospho-AKT T308, phospho-S6 kinase, phospho-S6 ribosomal protein, total AKT, total S6 kinase, and total S6 ribosomal protein were performed to evaluate outcomes of mTOR inhibition on cellular insulin sensitivities.

(D) Atg7^flox/floxDram1^−/− or Atg7^flox/floxDram1^+/+ MEFs were treated as in (C) in order to evaluate the role of autophagy in DRAM1-induced insulin resistance.

(E) Quantification of 3 independent experiments described in (D). Phospho-AKT T308 levels were normalized to total AKT levels. Data are mean ± SEM. ^∗∗p < 0.01.

See also Figure S5.