WT and MIF knockout (Mif^–/–) C57BL/6 mice were administered vehicle or 3 mg/kg olanzapine per day for 2 months. (A) Body weight and (B) food intake were monitored. Quantification of (C) MIF transcript levels by qPCR, (D) MIF protein, and (E) AMPK levels (pAMPK: Thr¹⁷² phosphorylated AMPK; T-AMPK: total AMPK) in homogenates of murine hypothalamic tissue after 2 months of treatment. (F) Transcript levels of NPY, AgRP, and POMC measured by qPCR in hypothalamic homogenates from mice treated with or without olanzapine for 2 months. Immunostaining was performed in the hypothalamus from WT mice. The staining for neural cells (NeuN, top panels), microglia (IBA1, middle panels), and astrocytes (GFAP, bottom panels) is red, while the staining for MIF is green (G). Arrow and arrowheads represent costaining of MIF and NeuN in neural cells. Isolated hypothalamic cells were treated with increasing concentrations of olanzapine for 24 hours, after which (H) MIF protein content was measured by Western blot. (I) Total and phospho-AMPK levels in isolated hypothalamic cells were evaluated following 24-hour MIF stimulation in vitro. (J) NPY, AgRP, and POMC gene expression measured in hypothalamic cells following 24 hours of MIF treatment. IgG or anti–MIF monoclonal antibody (2 μg/day) was administered i.c.v. by an osmotic pump to WT mice treated with olanzapine for 2 months. The cumulative food intake (K) and body weight gain (L) were subsequently evaluated. The hypothalamic tissues were collected for AMPK measurements (M). The pAMPK and T-AMPK Western blots are from parallel gels run contemporaneously on identical samples. For each animal group, n = 4–6. A, B, K, and L were analyzed by multivariate (2-way) ANOVA and the rest of the data were analyzed by 2-tailed Student’s t test or 1-way ANOVA. Mean ± SE in A, B, K, and L; mean ± SD in other panels; *P < 0.05 versus vehicle in C–F, versus other groups in A and B, versus control in H–J, versus IgG group in K–M. Olz: olanzapine.