Estradiol does not increase the activation of insulin signaling in the mediobasal hypothalamus of DIO rats. Male long-evans rats underwent a 1-month prevention of HFD-DIO via cyclic E2 treatment. The MBH were then micropunched, as described [19]. Protein expression and phosphorylation were assessed via Western blotting for phosphorylated (p) and total insulin receptor (IR) (A), insulin receptor substrates 1 and 2 (IRS-1/2) (B), and cyclic AMP response-element binding protein (C) (a distal protein in the insulin signaling cascade). Upper panels display the levels of phosphorylated proteins as quantified by Image J, while lower panels display representative Western blots. No significant differences were observed among groups for any proteins, as assessed with one-way ANOVA, n = 6–7, mean ± SEM.