E2 replacement rescues insulin response in POMC neurons from OVX females. (A) In voltage clamp, insulin (20 nM) induced an inward current in POMC neurons from control diet-fed OVX female mice. V_hold = –60 mV. (B) In DIO OVX females, the insulin response was abrogated in POMC neurons. (C) However, the insulin response was rescued in DIO OVX females with E₂ treatment (see “Materials and Methods”). (D) Bar graph summarizing the responses of insulin-induced currents in normal diet and OVX DIO and E₂-treated female Pomc-EGFP mice. Data points represent the mean ± SEM. One-way ANOVA: effect of treatment, F_{(2, 41)} = 17.74, P < 0.0001; Newman-Keuls multiple-comparison post hoc test. **** and ^#### indicate P < 0.001. Cell numbers are indicated. ns, no significant difference.